Auristatin f analogue, antibody drug conjugate and use thereof

Abstract

The invention belongs to the field of biomedicine, and specifically relates to an auristatin F analogue, and an antibody drug conjugate and a use thereof. Specifically, provided are an auristatin F analogue, an antibody drug conjugate thereof, a pharmaceutical composition containing same, a use thereof in preparation of a drug, a preparation method therefor, and an antibody drug conjugate thereof. By means of optimizing the structure of the compound, the tumor cell killing effect of the ADC can be improved, and the therapeutic efficacy and safety of the drug can also be improved.

Description

Auristatin F analogs and their antibody-drug conjugates and their applications Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to an auristatin F analogue and an antibody-drug conjugate thereof and applications thereof. Background Art

[0002] The concept of antibody-drug conjugates (ADCs) was proposed in 1900 and was dubbed the "magic bullet." With technological advancements, this technology has gradually been realized and applied to cancer treatment. ADCs are complexes composed of a monoclonal antibody (antibody) that specifically targets tumor-associated antigens and a small molecule toxin (payload) coupled via a linker. Once in the body, the ADC molecules bind to antigens on the surface of target cells through the guidance of the monoclonal antibody, leading to internalization. Once inside the cells, the ADC molecules can release effector molecules through chemical or enzymatic reactions to eliminate the target cells. Due to their targeted nature and intracellular drug release, ADCs can increase local drug concentrations, reduce toxic side effects on normal cells, and expand the therapeutic window. They combine the powerful killing effects of traditional small molecule chemotherapy with the tumor-targeting properties of antibody drugs. This technology has broad application prospects in cancer treatment and has achieved some encouraging results in clinical studies.

[0003] Linkers in ADCs are categorized as cleavable and non-cleavable. Cleavable linkers can be cleaved by specific enzymes or other mechanisms within the cellular environment after the ADC enters the cell; non-cleavable linkers, on the other hand, cannot be cleaved by enzymes or other mechanisms within the cell. The bystander effect of ADC drugs refers to the ability of ADCs to kill target antigen-positive tumor cells while also killing surrounding tumor cells that are negative or low in target antigen expression. This phenomenon is crucial for treating heterogeneous tumor expression, as tumor tissue may contain cells that express, underexpress, or even no target antigen at all.

[0004] Auristatins are decapeptides isolated from the Indian Ocean sea hare. They inhibit tumor growth primarily by targeting tubulin, with activity approximately 1,000 times greater than that of traditional chemotherapy drugs. MMAE (marigoldenin E) and MMAF (methylmarigoldenin F) are the two most commonly used auristatin compounds in ADC development. Currently, five marketed drugs (Adcetris, Polivy, Padcev, Blenrep, and Vedicizumab) use MMAE / MMAF as payloads. Over 20 ADCs using auristatins as payloads are in clinical trials, accounting for over 50% of the total ADC pipeline.

[0005] Summary of the Invention

[0006] The first object of the present invention is to provide a compound (i.e., an auristatin F analog), or an isotope-labeled compound, optical isomer, geometric isomer, tautomer or isomer mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein the compound comprises the structure represented by formula (I):

[0007] Wherein, W is selected from a single bond, -X-(CH2) m1 -CR3R4-NR5-, -X-CR3R4-(CH2) m1 -NR5-, -X-(CH2O) m2 -CR3R4-NR5-, -X-CR3R4-(CH2O) m2 -NR5-, -X-(CH2CH2O) m3 -CR3R4-NR5-、-X-(CH2CH2O) m3 -(CH2) m1 -NR5-, -X-CR3R4-(CH2CH2O) m3 -NR5-, -X-(CH2O) m2 -R3R4N-、-X-(CH2) m1 -R3R4N-、-XR5-(CH2) m1 -R3R4N-、-X-(CH2) m1 -(CH2O) m2 -(CH2) m4 -CR3R4-NR5- or -X-CR3R4-(CH2) m1 -(CH2O) m2 -(CH2) m4 -NR5-;

[0008] X is selected from an alkylene group, an imino group, a nitrogen atom, an oxygen atom or a sulfur atom;

[0009] R3, R4, and R5 are each independently selected from a hydrogen atom, a deuterium atom, a halogen, a hydroxyl group, an amino group, a cyano group, a nitro group, a sulfone group, a C1-C6 alkyl group, or a C3-C6 cycloalkyl group; or R3 and R4, together with the carbon atom to which they are attached, form a C3-C6 cycloalkyl group; or R3 and R4, together with the carbon atom to which they are attached and a nitrogen atom, form a 3-6 membered heterocyclic group; in R3, R4, and R5, one or more hydrogen atoms in the C1-C6 alkyl group or C3-C6 cycloalkyl group are optionally substituted with a deuterium atom, a halogen, a hydroxyl group, a cyano group, an amino group, a C1-C3 alkyl group, or a phenyl group;

[0010] m1, m2, m3, and m4 are each independently selected from any integer from 0 to 6;

[0011] The wavy line to which W in Formula (I) is connected represents a hydrogen atom, or is covalently linked to the structure of the Linker unit and / or the linked antibody or antigen-binding fragment thereof.

[0012] As a preferred embodiment, W is selected from -X-(CH2) m1 -CR3R4-NR5-, -X-CR3R4-(CH2) m1 -NR5-, -X-(CH2CH2O) m3 -(CH2) m1 -NR5-, -X-(CH2) m1 -R3R4N-、-XR5-(CH2) m1 -R3R4N-、-X-(CH2) m1 -(CH2O) m2 -(CH2) m4 -CR3R4-NR5- or -X-CR3R4-(CH2) m1 -(CH2O) m2 -(CH2) m4 -NR5-;

[0013] X is selected from an imino group, a nitrogen atom or an oxygen atom;

[0014] R3 and R4 are each independently selected from a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl group, or a C3-C6 cycloalkyl group; or R3 and R4, together with the carbon atom to which they are attached, form a C3-C6 cycloalkyl group; or R3 and R4, together with the carbon atom to which they are attached and a nitrogen atom, form a 3-6 membered heterocyclic group; in R3 and R4, one or more hydrogen atoms in the C1-C6 alkyl group or C3-C6 cycloalkyl group are optionally substituted with a deuterium atom, a halogen, a C1-C3 alkyl group, or a phenyl group;

[0015] R5 is selected from a hydrogen atom, a deuterium atom or a halogen;

[0016] m1, m2, and m4 are each independently selected from any integer from 1 to 4.

[0017] As a preferred embodiment, W is selected from any of the following structures:

[0018] As a preferred embodiment, the compound contains the structure shown in formula (II):

[0019] Wherein, W refers to the same as described in any of the previous schemes;

[0020] L is selected from the following groups: NH-Val-Cit- C＝O 、 NH -Val-Ala- C＝O 、 NH -Val-Lys- C＝O 、 NH -Val-Lys(Ac)- C＝O 、 NH -Phe-Lys- C＝O 、 NH -Phe-Lys(Ac)- C＝O 、 NH -D-Val-Leu-Lys- C＝O 、 NH -Gly-Gly-Arg- C＝O 、 NH -Ala-Ala-Asru- C＝O 、 NH -Gly-Gly-Gly- C＝O 、 NH -Gly-Gly-Gly-Gly-Gly- C＝O , or L1 or L2 as shown below; preferably L1 or L2;

[0021] In L1-L2, R1 is a single bond or is selected from the following structures:

[0022] wherein n3 and n4 are each independently an integer of 0 to 6, and R2 is a hydrophilic group or a structure having a hydrophilic group;

[0023] The wavy line to which L is connected in formula (II) represents a hydrogen atom, or is covalently linked to a structure that is linked to an antibody or antigen-binding fragment thereof.

[0024] As a preferred embodiment, R2 is a hydroxyl group, or a structure having at least three oxygen-containing hydrophilic groups; preferably, the oxygen-containing hydrophilic groups are the same or different and are independently selected from -O- or a hydroxyl group.

[0025] Preferably, R2 is any of the following structures:

[0026] As a preferred embodiment, R1 is a single bond or is selected from the following structures:

[0027] As a preferred embodiment, when L is selected from L1, X in W is an oxygen atom; in other words, the linking group at the junction of L1 and W is -CH2O-; when L is selected from L2, X in W is an imino group; in other words, the linking group at the junction of L2 and W is -CONH-. In specific implementations, those skilled in the art can select a matching W structure within the scope of the present invention in combination with the description herein.

[0028] As a preferred embodiment, the compound contains the structure shown in formula (III):

[0029] Wherein, P is a structure connecting an antibody or an antigen-binding fragment thereof;

[0030] The references of L and W are the same as those described in any of the above solutions.

[0031] As a preferred embodiment, P is selected from P1, P2 or P3:

[0032] wherein n1 is an integer from 1 to 10, preferably an integer from 2 to 6;

[0033] n2 is an integer from 1 to 12, preferably an integer from 5 to 9;

[0034] Z is selected from a bond, a C1-C6 alkylene group, an imino group, an ester group, an amide group, a sulfone group, a sulfonamide group, a ketone group, a urea group or a phosphate group.

[0035] As a preferred embodiment, the structure of the compound is shown as any one of the following:

[0036] The second object of the present invention is to provide an antibody-drug conjugate comprising:

[0037] i) an antibody or an antigen-binding fragment thereof; and,

[0038] ii) the compound or its isotope-labeled compound, optical isomer, geometric isomer, tautomer or isomer mixture, or a pharmaceutically acceptable salt or solvate thereof.

[0039] As a preferred embodiment, the structure of the antibody-drug conjugate is shown in formula (IV):

[0040] Wherein, Ab is an antibody or an antigen-binding fragment thereof;

[0041] n6 is an integer or decimal selected from 1 to 10.

[0042] As a preferred embodiment, n6 is selected from an integer or decimal between 1 and 8, and preferably n6 is selected from an integer or decimal between 6 and 8.

[0043] As a preferred embodiment, the antibody targets HER2, HER3, B7H3, B7H4, EGFR, Claudin18.2, c-MET, Nectin-4, ROR1, GPNMB, CD56, TACSTD2 (TROP2), CEACAM5, folate receptor-a, mesothelin, ENPP3, guanylate cyclase C, SLC44A4, NaPi2b, CD70, mucin 1, STEAP1, connexin 4, 5T4, SLTRK6, SC-16, LIV-1, P-cadherin, PSMA, fibronectin extradomain B, endothelin receptor ETB, tenascin c, collagen IV, VEGFR2, periostin, CD30, CD79b, CD19, CD22, CD138, CD37, CD33, CD74, CD19 and CD98.

[0044] As a preferred embodiment, the antibody is a monoclonal antibody.

[0045] The third object of the present invention is to provide a pharmaceutical composition comprising: the aforementioned compound or its isotope-labeled compound, or its optical isomers, geometric isomers, tautomers or isomer mixtures, or pharmaceutically acceptable salts thereof, or the aforementioned antibody-drug conjugate; and one or more pharmaceutically acceptable excipients.

[0046] The fourth object of the present invention is to provide the use of the aforementioned compound or its isotope-labeled compound, or its optical isomers, geometric isomers, tautomers or isomer mixtures, or its pharmaceutically acceptable salts, or the aforementioned antibody-drug conjugate in the preparation of drugs.

[0047] As a preferred embodiment, the drug is used for preventing or treating tumors.

[0048] Preferably, the tumor is a cancer associated with the expression of one or more of the following: HER2, HER3, B7H3, B7H4, EGFR, Claudin18.2, c-MET, Nectin-4, ROR1, GPNMB, CD56, TACSTD2 (TROP2), CEACAM5, folate receptor-a, mesothelin, ENPP3, guanylate cyclase C, SLC44A4, NaPi2b, CD70, mucin 1, STEAP1, connexin 4, 5T4, SLTRK6, SC-16, LIV-1, P-cadherin, PSMA, fibronectin extradomain B, endothelin receptor ETB, tenascin c, collagen IV, VEGFR2, periostin, CD30, CD79b, CD19, CD22, CD138, CD37, CD33, CD74, CD19 and CD98.

[0049] More preferably, the tumor is selected from one or more of breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer, colon cancer, rectal cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer and lymphoma.

[0050] The fifth object of the present invention is to provide a method for preventing or treating tumors, comprising: administering an effective amount of the aforementioned compound or its isotope-labeled compound, or its optical isomers, geometric isomers, tautomers or isomer mixtures, or their pharmaceutically acceptable salts, or the aforementioned antibody-drug conjugate, or the aforementioned pharmaceutical composition.

[0051] As a preferred embodiment, the tumor is a cancer associated with the expression of one or more of the following: HER2, HER3, B7H3, B7H4, EGFR, Claudin18.2, c-MET, Nectin-4, ROR1, GPNMB, CD56, TACSTD2 (TROP2), CEACAM5, folate receptor-a, mesothelin, ENPP3, guanylate cyclase C, SLC44A4, NaPi2b, CD70, mucin 1, STEAP1, connexin 4, 5T4, SLTRK6, SC-16, LIV-1, P-cadherin, PSMA, fibronectin extradomain B, endothelin receptor ETB, tenascin c, collagen IV, VEGFR2, periostin, CD30, CD79b, CD19, CD22, CD138, CD37, CD33, CD74, CD19 and CD98.

[0052] More preferably, the tumor is selected from one or more of breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer, colon cancer, rectal cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer and lymphoma.

[0053] The sixth object of the present invention is to provide a method for preparing the aforementioned compound, wherein the reaction raw materials include: a compound containing a structure represented by formula (I).

[0054] As a preferred embodiment, the preparation method comprises: reacting a compound containing the structure L and the structure represented by formula (I) with a compound containing the structure P to obtain the compound;

[0055] Alternatively, the method comprises reacting a compound containing a P structure and an L structure with a compound containing a structure represented by formula (I) to obtain the compound.

[0056] As a preferred embodiment, the method for preparing the compound comprises: reacting a compound containing an L structure with a compound containing a structure represented by formula (I), and then reacting the compound containing an L structure and a structure represented by formula (I) with a compound containing a P structure to obtain the compound;

[0057] Alternatively, the method comprises reacting a compound containing a P structure with a compound containing an L structure, and then reacting the compound containing the P structure and the L structure with a compound containing the structure represented by formula (I) to obtain the compound.

[0058] The seventh object of the present invention is to provide a method for preparing the aforementioned antibody-drug conjugate, comprising: coupling the following i) and ii):

[0059] i) an antibody or an antigen-binding fragment thereof; and,

[0060] ii) the aforementioned compound or its isotope-labeled compound, optical isomer, geometric isomer, tautomer or isomer mixture, or a pharmaceutically acceptable salt or solvate thereof.

[0061] As a preferred embodiment, the method for preparing the antibody-drug conjugate comprises: reducing the interchain disulfide bonds of the antibody or antigen-binding fragment thereof to free thiol groups, and then coupling with the P-terminus of the compound or its isotope-labeled compound, optical isomer, geometric isomer, tautomer or isomer mixture, or pharmaceutically acceptable salt or solvate thereof.

[0062] By optimizing the compound structure, the present invention can enhance the tumor cell killing effect of ADC and simultaneously improve the efficacy and safety of the drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0064] FIG1 is the efficacy evaluation result of the ADC drug in Experimental Example 3 of the present invention in the JIMT-1 human breast cancer model.

[0065] FIG2 is the safety evaluation results of the ADC drug in Experimental Example 3 of the present invention in the JIMT-1 human breast cancer model. DETAILED DESCRIPTION

[0066] The following describes specific embodiments of the present invention in detail. It should be understood that the specific embodiments described herein are intended only to illustrate and explain the present invention and are not intended to limit the present invention. Those skilled in the art may make various modifications and variations to the present invention without departing from the scope or spirit of the present invention. For example, features illustrated or described as part of one embodiment may be used in another embodiment to produce further embodiments.

[0067] Unless otherwise indicated, all terms (including technical and scientific terms) used to disclose the present invention have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. By way of further guidance, the following definitions are provided to better understand the teachings of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0068] The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the any and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND" and technical solutions connected by "logical OR". For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution of all being connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution of all being connected by "logical AND").

[0069] As used herein, the terms "comprising," "including," and "comprising" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps.

[0070] The recitation of numerical ranges herein by endpoints includes all numbers and fractions subsumed within the range, as well as the recited endpoints.

[0071] Concentration values ​​used in this invention include fluctuations within a certain range. For example, fluctuations within a certain precision range are permitted. For example, for 2%, fluctuations within ±0.1% are permitted. For larger values ​​or values ​​that do not require overly precise control, greater fluctuations are permitted. For example, for 100 mM, fluctuations within ±1%, ±2%, ±5%, etc. are permitted. Regarding molecular weight, fluctuations within ±10% are permitted.

[0072] In the present invention, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.

[0073] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0074] In the present invention, the terms "preferably," "better," "more preferably," and "suitably" are merely used to describe preferred implementations or examples and should be understood not to limit the scope of protection of the present invention. In the present invention, the terms "optionally," "optional," and "optional" are intended to be optional, i.e., to select from either of the two parallel options of "with" or "without." If multiple "options" appear in a technical solution, unless otherwise specified and without conflict or mutual constraints, each "optional" is considered independent.

[0075] As used herein, the term "antibody" refers to an immunoglobulin, which is a tetrapeptide structure consisting of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. Immunoglobulins differ in their heavy chain constant region amino acid composition and sequence, resulting in different antigenicity.

[0076] In the present invention, the term "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that fragments of full-length antibodies can be used to perform the antigen-binding function of an antibody. Examples of binding fragments included in "antigen-binding fragments" include: (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CHI domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments connected by a disulfide bridge on the hinge region; (iii) F fragments consisting of VH and CHI domains; (iv) Fv fragments consisting of the VH and VL domains of a single arm of an antibody; (v) single domain or dAb fragments, which consist of a VH domain; and (vi) isolated complementary determining regions (CDRs) or (vii) a combination of two or more isolated CDRs, optionally connected by a synthetic linker. In addition, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be connected by a synthetic linker using recombinant methods, thereby enabling them to be produced as a single protein chain (called single-chain Fv (scFv)) in which the VL and VH regions are paired to form a monovalent molecule. Such single-chain antibodies are also included in the term "antigen-binding fragment" of an antibody.

[0077] In the present invention, "pharmaceutically acceptable salts" include acid addition salts and base addition salts thereof. Among them, suitable acid addition salts are formed from acids that form pharmaceutically acceptable salts. Examples include aspartate, benzoate, bicarbonate / carbonate, glucoheptonate, gluconate, nitrate, orotate, palmitate and other similar salts. Suitable base addition salts are formed from bases that form pharmaceutically acceptable salts. Examples include aluminum salts, arginine salts, choline salts, magnesium salts and other similar salts. For a review of suitable salts, see "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" by Stahl and Wermuth (Wiley-YCH, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds of the present invention are known to those skilled in the art.

[0078] In the present invention, "isomer" includes stereoisomers, which means isomers formed due to at least one asymmetric center. In compounds with one or more (e.g., one, two, three, or four) asymmetric centers, racemic mixtures, single enantiomers, diastereomeric mixtures, and individual diastereomers can be produced. Specific individual molecules can also exist as geometric isomers (cis / trans). Similarly, the compounds of the present invention can exist as mixtures of two or more structurally different forms in rapid equilibrium (commonly referred to as tautomers). Representative examples of tautomers include keto-enol tautomers, phenol-ketone tautomers, nitroso-oxime tautomers, imine-enamine tautomers, etc. The scope of this application covers all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

[0079] Solid lines, solid wedges, or dashed wedges may be used herein to depict carbon-carbon bonds of the compounds of the present invention. The use of solid lines to depict bonds to asymmetric carbon atoms is intended to indicate that all possible stereoisomers at that carbon atom are included (e.g., specific enantiomers, racemic mixtures, etc.). The use of solid or dashed wedges to depict bonds to asymmetric carbon atoms indicates the presence of the indicated stereoisomers. When present in a racemic mixture, solid and dashed wedges are used to define relative stereochemistry, not absolute stereochemistry. Unless otherwise indicated, the compounds of the present invention may exist as stereoisomers, including cis and trans isomers, optical isomers (e.g., R and S enantiomers), diastereomers, geometric isomers, rotational isomers, conformational isomers, atropisomers, and mixtures thereof. The compounds of the present invention may exhibit more than one type of isomerism and consist of mixtures thereof (e.g., racemic mixtures and diastereomeric pairs).

[0080] In the present invention, "solvate" refers to a pharmaceutically acceptable solvate formed between the ligand-drug conjugate compound of the present invention and one or more solvent molecules. Non-limiting examples of solvent molecules include water, ethanol, acetonitrile, isopropanol, DMSO, and ethyl acetate.

[0081] Also included within the scope of the present invention are pharmaceutically acceptable isotopically labeled compounds which are identical to the compounds of the present invention except that one or more atoms are replaced by an atom having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number prevalent in nature. Examples of suitable isotopes for inclusion in the compounds of the present invention include, but are not limited to, isotopes of hydrogen (e.g., deuterium 2 H), tritium ( 3 H)); carbon isotopes (e.g. 11 C. 13 C and 14 C); isotopes of chlorine (e.g. 36 Cl); isotopes of fluorine (e.g. 18 F); isotopes of iodine (such as 123 I and 125 I); isotopes of nitrogen (e.g. 13 N and 15 N); oxygen isotopes (e.g. 15 O. 17 O and 18 O); isotopes of phosphorus (such as 32 P); and sulfur isotopes (e.g. 35 S).

[0082] Also within the scope of the present invention are esters of the compounds of the invention, including physiologically hydrolyzable esters (which can be hydrolyzed under physiological conditions to release the compound of the invention in either free acid or alcohol form).

[0083] Also included within the scope of the present invention are metabolites of the compounds of the invention, i.e., substances formed in vivo upon administration of the compounds of the invention. Such products may be produced, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, enzymatic hydrolysis, etc. of the administered compound. Thus, the present invention includes metabolites of the compounds of the invention, including compounds produced by contacting a compound of the invention with a mammal for a period of time sufficient to produce a metabolic product thereof.

[0084] Also included within the scope of the present invention are prodrugs of the compounds of the present invention, which are certain derivatives of the compounds of the present invention that may themselves have little or no pharmacological activity but, when administered to or on the body, are converted, for example, by hydrolytic cleavage, into compounds of the present invention having the desired activity. Typically, such prodrugs will be functional group derivatives of the compounds that are readily converted in vivo into the desired therapeutically active compound.

[0085] The present invention also encompasses compounds of the present invention that contain protecting groups. During any process for preparing the compounds of the present invention, it may be necessary and / or desirable to protect sensitive or reactive groups on any of the molecules involved, thereby forming a chemically protected form of the compounds of the present invention. This can be achieved using conventional protecting groups, for example, those described in TW Greene & 20 P. G. M. Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991, which references are incorporated herein by reference. Protecting groups can be removed at an appropriate subsequent stage using methods known in the art.

[0086] The term "alkyl" refers to a saturated aliphatic hydrocarbon group including straight-chain and branched-chain groups.

[0087] The term "alkoxy" refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl or cycloalkyl is as defined above.

[0088] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent.

[0089] The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 6 ring atoms, one or more of which is selected from nitrogen, oxygen or S(O) m (wherein m is an integer from 0 to 2) heteroatoms, excluding the ring portion of -OO-, -OS- or -SS-, the remaining ring atoms being carbon. Preferably, 1 to 4 of them are heteroatoms. The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0090] The term "hydroxy" refers to -OH.

[0091] The term "cyano" refers to -CN.

[0092] The term "amino" refers to -NH2.

[0093] The term "phenyl" refers to

[0094] The term "comprising a maleimide linker" means comprising a maleimide group.

[0095] As used herein, the term "substituted" refers to the replacement of one or more (e.g., one, two, three, or four) hydrogen atoms on a designated atom with a selected group selected from the indicated group, provided that the designated atom's normal valence in the present context is not exceeded and that the substitution results in a stable compound. Combinations of substituents and / or variables are permissible only if such combinations result in stable compounds.

[0096] In the present invention, if a substituent is described as being "optionally substituted with...", the substituent may be unsubstituted or substituted. If a carbon of a substituent is described as being optionally substituted with one or more of the substituents listed, one or more hydrogens on the carbon (to the extent of any hydrogens present) may be replaced individually and / or collectively with independently selected optional substituents. If a nitrogen of a substituent is described as being optionally substituted with one or more of the substituents listed, one or more hydrogens on the nitrogen (to the extent of any hydrogens present) may each be replaced with an independently selected optional substituent.

[0097] In the present invention, if a substituent is described as being "independently selected" from a group of groups, each substituent is selected independently of the other. Thus, each substituent may be the same as or different from another (other) substituent.

[0098] The present invention first provides a compound (i.e., an auristatin F analog), or an isotope-labeled compound, optical isomer, geometric isomer, tautomer or isomer mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein the compound comprises the structure represented by formula (I):

[0099] Wherein, W is selected from a single bond, -X-(CH2) m1 -CR3R4-NR5-, -X-CR3R4-(CH2) m1 -NR5-, -X-(CH2O) m2 -CR3R4-NR5-, -X-CR3R4-(CH2O) m2 -NR5-, -X-(CH2CH2O) m3 -CR3R4-NR5-、-X-(CH2CH2O) m3 -(CH2) m1 -NR5-, -X-CR3R4-(CH2CH2O) m3 -NR5-, -X-(CH2O) m2 -R3R4N-、-X-(CH2) m1 -R3R4N-、-XR5-(CH2) m1 -R3R4N-、-X-(CH2) m1 -(CH2O) m2 -(CH2) m4 -CR3R4-NR5- or -X-CR3R4-(CH2) m1 -(CH2O) m2 -(CH2) m4 -NR5-;

[0100] X is selected from an alkylene group, an imino group, a nitrogen atom, an oxygen atom or a sulfur atom;

[0101] R3, R4, and R5 are each independently selected from a hydrogen atom, a deuterium atom, a halogen, a hydroxyl group, an amino group, a cyano group, a nitro group, a sulfone group, a C1-C6 alkyl group, or a C3-C6 cycloalkyl group; or R3 and R4, together with the carbon atom to which they are attached, form a C3-C6 cycloalkyl group; or R3 and R4, together with the carbon atom to which they are attached and the nitrogen atom, form a 3-6 membered heterocyclic group; in R3, R4, and R5, one or more hydrogen atoms in the C1-C6 alkyl group or the C3-C6 cycloalkyl group are optionally substituted with a deuterium atom, a halogen, a hydroxyl group, a cyano group, an amino group, a C1-C3 alkyl group, or a phenyl group;

[0102] m1, m2, m3, and m4 are each independently selected from any integer from 0 to 6;

[0103] The wavy line to which W in Formula (I) is connected represents a hydrogen atom, or is covalently linked to the structure of the Linker unit and / or the linked antibody or antigen-binding fragment thereof.

[0104] In some embodiments, W is selected from -X-(CH2) m1 -CR3R4-NR5-, -X-CR3R4-(CH2) m1 -NR5-, -X-(CH2CH2O) m3 -(CH2) m1 -NR5-, -X-(CH2) m1 -R3R4N-、-XR5-(CH2) m1 -R3R4N-、-X-(CH2) m1 -(CH2O) m2 -(CH2) m4 -CR3R4-NR5- or -X-CR3R4-(CH2) m1 -(CH2O) m2 -(CH2) m4 -NR5-;

[0105] X is selected from an imino group, a nitrogen atom or an oxygen atom;

[0106] R3 and R4 are each independently selected from a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl group, or a C3-C6 cycloalkyl group; or R3 and R4, together with the carbon atom to which they are attached, form a C3-C6 cycloalkyl group; or R3 and R4, together with the carbon atom to which they are attached and a nitrogen atom, form a 3-6 membered heterocyclic group; in R3 and R4, one or more hydrogen atoms in the C1-C6 alkyl group or C3-C6 cycloalkyl group are optionally substituted with a deuterium atom, a halogen, a C1-C3 alkyl group, or a phenyl group;

[0107] R5 is selected from a hydrogen atom, a deuterium atom or a halogen;

[0108] m1, m2, and m4 are each independently selected from any integer from 1 to 4.

[0109] In some embodiments, W is selected from any of the following structures:

[0110] In some embodiments, the compound comprises a structure as shown in formula (II):

[0111] Wherein, W refers to the same as described in any of the previous schemes;

[0112] L is selected from the following groups: NH -Val-Cit- C＝O 、 NH -Val-Ala- C＝O 、 NH -Val-Lys- C＝O 、 NH -Val-Lys(Ac)- C＝O 、 NH -Phe-Lys- C＝O 、 NH -Phe-Lys(Ac)- C＝O 、 NH -D-Val-Leu-Lys- C＝O 、 NH -Gly-Gly-Arg- C＝O 、 NH -Ala-Ala-Asru- C＝O 、 NH -Gly-Gly-Gly- C＝O 、 NH -Gly-Gly-Gly-Gly-Gly- C＝O , or L1 or L2 as shown below; preferably L1 or L2;

[0113] In L1-L2, R1 is a single bond or is selected from the following structures:

[0114] wherein n3 and n4 are each independently an integer of 0 to 6, and R2 is a hydrophilic group or a structure having a hydrophilic group;

[0115] The wavy line to which L is connected in formula (II) represents a hydrogen atom, or is covalently linked to a structure that is linked to an antibody or antigen-binding fragment thereof.

[0116] In some embodiments, R2 is a hydroxyl group, or a structure having at least three oxygen-containing hydrophilic groups; preferably, the oxygen-containing hydrophilic groups are the same or different, and are each independently selected from -O- or a hydroxyl group.

[0117] Preferably, R2 is any of the following structures:

[0118] In some embodiments, R1 is a single bond or is selected from the following structures:

[0119] In some embodiments, when L is selected from L1, X in W is an oxygen atom; when L is selected from L2, X in W is an imino group.

[0120] In some embodiments, the compound comprises a structure as shown in formula (III):

[0121] Wherein, P is a structure connecting an antibody or an antigen-binding fragment thereof;

[0122] The references of L and W are the same as those described in any of the above solutions.

[0123] In some embodiments, P is a group comprising a maleimide linker. For example, in some embodiments, P is selected from P1 or P2:

[0124] In some embodiments, P is selected from P3:

[0125] wherein n1 is an integer from 1 to 10, preferably an integer from 2 to 6;

[0126] n2 is an integer from 1 to 12, preferably an integer from 5 to 9;

[0127] Z is selected from a bond, a C1-C6 alkylene group, an imino group, an ester group, an amide group, a sulfone group, a sulfonamide group, a ketone group, a urea group or a phosphate group.

[0128] In some embodiments, the structure of the compound is any one of the following:

[0129] The second object of the present invention is to provide an antibody-drug conjugate comprising:

[0130] i) an antibody or an antigen-binding fragment thereof; and,

[0131] ii) the compound or its isotope-labeled compound, optical isomer, geometric isomer, tautomer or isomer mixture, or a pharmaceutically acceptable salt or solvate thereof.

[0132] In some embodiments, the structure of the antibody drug conjugate is shown in Formula (IV):

[0133] Wherein, Ab is an antibody or an antigen-binding fragment thereof;

[0134] n6 is an integer or decimal selected from 1 to 10.

[0135] In some embodiments, n6 is selected from an integer or decimal between 1 and 8, and preferably n6 is selected from an integer or decimal between 6 and 8.

[0136] As an example, in a specific embodiment, n6 can be 1, 2, 3, 4, 5, 6, 6.5, 7, 7.5, 8, 9 or 10.

[0137] In some embodiments, the antibody targets one or more of HER2, HER3, B7H3, B7H4, EGFR, Claudin18.2, c-MET, Nectin-4, ROR1, GPNMB, CD56, TACSTD2 (TROP2), CEACAM5, folate receptor-a, mesothelin, ENPP3, guanylate cyclase C, SLC44A4, NaPi2b, CD70, mucin 1, STEAP1, connexin 4, 5T4, SLTRK6, SC-16, LIV-1, P-cadherin, PSMA, fibronectin extradomain B, endothelin receptor ETB, tenascin c, collagen IV, VEGFR2, periostin, CD30, CD79b, CD19, CD22, CD138, CD37, CD33, CD74, CD19, and CD98.

[0138] In some embodiments, the antibody is a monoclonal antibody.

[0139] The antibodies of the present invention include murine antibodies, chimeric antibodies, humanized antibodies and fully human antibodies, with humanized antibodies and fully human antibodies being preferred.

[0140] In a specific embodiment, the antibody or antigen-binding fragment thereof is Trastuzumab, Pertuzumab, Nimotuzumab, Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96, Glematumamab or an antigen-binding fragment thereof.

[0141] The third object of the present invention is to provide a pharmaceutical composition comprising: the aforementioned compound or its isotope-labeled compound, or its optical isomers, geometric isomers, tautomers or isomer mixtures, or pharmaceutically acceptable salts thereof, or the aforementioned antibody-drug conjugate; and one or more pharmaceutically acceptable excipients.

[0142] In some embodiments, the dosage form of the pharmaceutical composition is selected from tablets, capsules, granules, powders, syrups, inhalants or injections.

[0143] In some embodiments, the excipient is selected from one or more of a carrier, an excipient, and a diluent. To improve the efficacy of the drug administration, those skilled in the art can select and / or combine the excipients and adjust their dosages based on common sense (e.g., pharmacopoeias).

[0144] The fourth object of the present invention is to provide the use of the aforementioned compound or its isotope-labeled compound, or its optical isomers, geometric isomers, tautomers or isomer mixtures, or its pharmaceutically acceptable salts, or the aforementioned antibody-drug conjugate in the preparation of drugs.

[0145] In some embodiments, the drug is used to prevent or treat tumors; preferably, the tumor is a tumor that is selected from HER2, HER3, B7H3, B7H4, EGFR, Claudin18.2, c-MET, Nectin-4, ROR1, GPNMB, CD56, TACSTD2 (TROP2), CEACAM5, folate receptor-a, mesothelin, ENPP3, guanylate cyclase C, SLC44A4, NaPi2b, CD70, mucin 1, STEAP1, connexin 4, 5T4, SLTRK6, SC-16, LIV-1, P-cadherin, PSMA, fibronectin extracellular structure. Domain B, endothelin receptor ETB, tenascin c, collagen IV, VEGFR2, periostin, CD30, CD79b, CD19, CD22, CD138, CD37, CD33, CD74, CD19 and CD98 related to the expression of one or more cancers; more preferably, the tumor is selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer, colon cancer, rectal cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer and one or more lymphoma.

[0146] The fifth object of the present invention is to provide a method for preventing or treating tumors, comprising: administering an effective amount of the aforementioned compound or its isotope-labeled compound, or its optical isomers, geometric isomers, tautomers or isomer mixtures, or their pharmaceutically acceptable salts, or the aforementioned antibody-drug conjugate, or the aforementioned pharmaceutical composition.

[0147] In some embodiments, the tumor is a cancer associated with the expression of one or more of HER2, HER3, B7H3, B7H4, EGFR, Claudin18.2, c-MET, Nectin-4, ROR1, GPNMB, CD56, TACSTD2 (TROP2), CEACAM5, folate receptor-a, mesothelin, ENPP3, guanylate cyclase C, SLC44A4, NaPi2b, CD70, mucin 1, STEAP1, connexin 4, 5T4, SLTRK6, SC-16, LIV-1, P-cadherin, PSMA, fibronectin extradomain B, endothelin receptor ETB, tenascin c, collagen IV, VEGFR2, periostin, CD30, CD79b, CD19, CD22, CD138, CD37, CD33, CD74, CD19, and CD98.

[0148] More preferably, the tumor is selected from one or more of breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer, colon cancer, rectal cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer and lymphoma.

[0149] The sixth object of the present invention is to provide a method for preparing the aforementioned compound, wherein the reaction raw materials include: a compound containing a structure represented by formula (I).

[0150] In some embodiments, the preparation method comprises: reacting a compound containing the structure L and the structure represented by formula (I) with a compound containing the structure P to obtain the compound;

[0151] Alternatively, the method comprises reacting a compound containing a P structure and an L structure with a compound containing a structure represented by formula (I) to obtain the compound.

[0152] In some embodiments, the method for preparing the compound comprises: reacting a compound containing an L structure with a compound containing a structure represented by formula (I), and then reacting the compound containing the L structure and the structure represented by formula (I) with a compound containing a P structure to obtain the compound;

[0153] Alternatively, the method comprises reacting a compound containing a P structure with a compound containing an L structure, and then reacting the compound containing the P structure and the L structure with a compound containing the structure represented by formula (I) to obtain the compound.

[0154] In some embodiments, those skilled in the art can select specific compounds based on common sense. In some embodiments, protecting groups can be added or removed at the position of the active group in the compound according to the reaction requirements. In some embodiments, the reaction reagents and reaction conditions (including solvent, temperature, pressure, etc.) of each step can be optimized based on considerations of the reaction effect. No particular limitation is imposed herein.

[0155] The seventh object of the present invention is to provide a method for preparing the aforementioned antibody-drug conjugate, comprising: coupling the following i) and ii):

[0156] i) an antibody or an antigen-binding fragment thereof; and,

[0157] ii) the aforementioned compound or its isotope-labeled compound, optical isomer, geometric isomer, tautomer or isomer mixture, or a pharmaceutically acceptable salt or solvate thereof.

[0158] In some embodiments, the method for preparing the antibody-drug conjugate comprises reducing the interchain disulfide bonds of the antibody or antigen-binding fragment thereof to free sulfhydryl groups, and then coupling with the P-terminus of the compound or its isotope-labeled compound, optical isomer, geometric isomer, tautomer or isomer mixture, or pharmaceutically acceptable salt or solvate thereof.

[0159] In some embodiments, those skilled in the art can set the dosage ratio of Ab and the compound in combination with common sense. In some embodiments, the reaction reagents and reaction conditions (including solvent, temperature, pressure, etc.) of each step can be optimized based on consideration of the reaction effect. No particular limitation is made herein.

[0160] The embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. For experimental methods in the following examples where specific conditions are not specified, reference is made to the guidance provided in the present invention, and may also be made to experimental manuals or conventional conditions in the art, other experimental methods known in the art, or conditions recommended by the manufacturer.

[0161] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0162] In each ADC preparation example, the ADC sample analysis and detection method is as follows:

[0163] 1. Reversed-phase chromatography detection

[0164] Prepare mobile phase A: 0.5‰ TFA in acetonitrile, mobile phase B: 0.5‰ TFA in water. Add 1μL of DTT (10mM) to 20μL of the sample to be tested (1mg / mL) and allow to react at room temperature for 30 minutes for reduction. Use an Aglient PLRP-S 1000A 5μm column (50 x 2.1mm) and perform gradient elution (mobile phase A: 0-100%) at 70°C.

[0165] 2.SEC detection

[0166] Take an appropriate amount of this product, dissolve and dilute it with solvent to make about 1-2 mg of sample per 1 ml, use diol-bonded gel as the filler (Waters Xbridge Premier Protein SEC 7.8*300mm or equivalent performance column); use 20mM phosphate buffer and 150mM sodium chloride solution as the mobile phase; the detection wavelength is 280nm; the column temperature is room temperature; the flow rate is 0.8ml per minute; the injection volume is 10μL.

[0167] The specific designations of the abbreviations involved in the following embodiments are shown in Table 1 below.

[0168] Table 1

[0169] Example 1 Preparation of Compound DL-1

[0170] Step 1: Synthesis of tert-butyl ((3R, 4R, 7S)-7-benzyl-3-((S)-1-(3R, 4S, 5S)-4-(S)-2-[(S)-2-(dimethylamino)-3-methylbutanamido)-N, 3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-4-methyl-5,8-dioxo-2,12-dioxy-6,9-diazatetradec-14yl)carbamate

[0171] Compound 1 (110 mg, 0.147 mmol) and HATU (62 mg, 0.163 mmol) were added to N,N-dimethylformamide (5 mL) and stirred at room temperature for 10 minutes. DIEA (70 mg, 0.543 mmol) was then added and stirred at room temperature for 10 minutes. Finally, N-BOC-2-(2-aminoethoxy)ethylamine (30 mg, 0.147 mmol) was added and stirred at room temperature for 3 hours. The reaction was considered complete when LCMS showed less than 3% of compound 1 in the reaction solution. The solvent was evaporated under reduced pressure, and the crude product was purified by preparative liquid phase separation using acetonitrile / water. Compound 2 (136 mg, 98% yield) was obtained. MS: 933.26 [M+H] + .

[0172] Step 2: Synthesis of (S)-N-((3R,4S,5S)-1-((S)-2-((3R,4R,7S)-14-amino-7-benzyl-4-methyl-5,8-dioxo-2,12-dioxo-6,9-diazatetradec-3-yl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)-2-(S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamide

[0173] Compound 2 (136 mg, 0.146 mmol) was placed in a 25 mL single-necked flask. Dichloromethane (3 mL) and HCl / 1,4-dioxane solution (1 mL, 4 M) were added. The mixture was allowed to react at room temperature for 1 hour. LCMS confirmed the complete reaction of compound 2. After spin drying, the crude product, compound 3 (150 mg, 123.6% yield), was obtained. MS: 833.14 [M+H] + .

[0174] Step 3: Synthesis of 4-(((S)-2-[(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl((3R,4R,7S)-7-benzyl-3-((S)-1-((3R,4S,5S)-4-(S)-2-[(S)-2-(dimethylamino)-3-methylbutanamido]-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-4-methyl-5,8-dioxo-2,12-dioxo-6,9-diazatetradec-14yl)carbamate

[0175] Compound 3 (100 mg, 0.12 mmol) and compound 4 (92 mg, 0.12 mmol) were added to N,N-dimethylformamide (5 mL) and stirred at room temperature for 10 minutes. DIEA (62 mg, 0.48 mmol) was then added and stirred at room temperature for 2 hours. The reaction was considered complete when LCMS showed less than 3% of compound 3 in the reaction solution. The solvent was evaporated under reduced pressure, and the crude product was purified by preparative liquid phase separation using acetonitrile / water. Compound 5 (white solid, 182 mg, yield 103.9%) was obtained. MS: 731 [M+2H] 2+ .

[0176] Step 4: Synthesis of 4-(((S)-2-[(S)-2-amino-3-methylbutanamido)-5-ureidopentanamido]benzyl((3R,4R,7S)-7-benzyl-3-(S)-1-((3R,4S,5S)-4-((S)-2-(S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-4-methyl-5,8-dioxo-2,12-dioxo-6,9-diazatetradec-14yl)carbamate

[0177] Compound 5 (182 mg, 0.125 mmol) was placed in a 25 mL single-necked flask, and N,N-dimethylformamide (3 mL) and morpholine (2 mL) were added. The reaction was allowed to proceed at room temperature for 30 min. LCMS confirmed the complete reaction of compound 5. The reaction solution was spin-dried, and the crude product was purified by preparative liquid phase separation using acetonitrile / water. Compound 6 (white solid, 130 mg, yield 84.2%) was obtained. MS: 1238.6 [M+H] + .

[0178] Step 5: Synthesis of 3-((tert-Butoxycarbonyl)amino)-5-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-5-oxopentanoic acid

[0179] Compound 7 (50 mg, 0.202 mmol) and HATU (76 mg, 0.202 mmol) were added to N,N-dimethylformamide (3 mL) and stirred at room temperature for 10 minutes. N-methylglucamine (39 mg, 0.202 mmol) was then added and stirred at room temperature for 10 minutes. Finally, DIEA (104 mg, 0.808 mmol) was added and stirred at room temperature for 1 hour. LCMS showed that the reaction solution contained less than 3% of compound 7, which was considered complete. The reaction solution was evaporated to dryness under reduced pressure, and the crude product was purified by preparative liquid phase separation using acetonitrile / water. Compound 8 (white solid, 70 mg, yield 81.5%) was obtained. MS: 425.45 [M+H] +.

[0180] Step 6: Synthesis of 3-amino-5-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-5-oxopentanoic acid

[0181] Compound 8 (30 mg, 0.071 mmol) was placed in a 25 mL single-necked flask. Dichloromethane (2 mL) and HCl / 1,4-dioxane solution (1 mL, 4 M) were added and allowed to react at room temperature for 30 min. LCMS analysis confirmed the complete reaction of compound 10. The reaction solution was then dried to afford crude compound 9 (white solid, 20 mg, 86.7% yield). MS: 325.33 [M+H] + .

[0182] Step 7: Synthesis of 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-33-(2-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-2-oxoethyl)-3,31-dioxo-7,10,16,19,22,25,28-octaoxa-4,32-diazapentadecane-35-enoic acid

[0183] Compound 10 (36 mg, 0.062 mmol) and HATU (28 mg, 0.062 mmol) were added to N,N-dimethylformamide (5 mL) and stirred at room temperature for 10 minutes. DIEA (32 mg, 0.248 mmol) was then added and stirred at room temperature for 10 minutes. Finally, compound 9 (20 mg, 0.062 mmol) was added and stirred at room temperature for 3 hours. The reaction was considered complete when LCMS showed that the reaction solution contained less than 3% of compound 10. The solvent was evaporated under reduced pressure, and the crude product was purified by preparative liquid phase separation using acetonitrile / water. Compound 11 (8 mg, 14.3% yield) was obtained. MS: 899.95 [M+H] + .

[0184] Step 8: Synthesis of 4-((2S,5S)-4-(2,5-dioxo-2,5-dihydro-1-dihydro-1-dihydro-1-pyranyl-1-yl)-5-isopropyl-5-isopropyl-9-(2-(methyl-(2S,3R,4R,4R,5R,5R)-2,3,4,4,5,5,6-pentahydroxy-hydrogen R,4R,7S)-7-benzyl-3-((S)-1-((3R,4S,5S)-4-(S)-2-((dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-4-methyl-5,8-dioxo-2,12-dioxo-6,9-diazatetradec-14yl)carbamate

[0185] Compound 11 (8 mg, 0.0089 mmol) and HATU (4 mg, 0.011 mmol) were added to N,N-dimethylformamide (3 mL) and stirred at room temperature for 10 minutes. Compound 6 (11 mg, 0.0089 mmol) was then added and stirred until uniform. DIEA (4.6 mg, 0.036 mmol) was then added and stirred at room temperature for 3 hours. LCMS showed that the reaction solution contained less than 3% of compound 11, which was considered complete. The reaction solution was evaporated to dryness under reduced pressure, and the crude product was purified by preparative liquid phase separation using acetonitrile / water. Freeze-dried to obtain compound DL-1 (white solid, 1.3 mg, 6.9% yield). MS: 2119.54 [M+H] + .

[0186] 1 H NMR (400MHz, DMSO-d6) δ8.36–8.11(m,1H),8.01(m,2H),7.80(s,1H),7.59(s,1H),7.34–7.11(m,4H),6.98( m,1H),5.98(s,1H),5.41(s,1H),4.93(s,1H),4.63(m,2H),4.53(m,1H),4.37(s,2H),4.20(s,1H),3.97(s, 1H),3.62–3.47(m,20H),3.31(m,87H),3.23–3.11(m,5H),3.03(m,3H),2.84–2.70(m,1H),2.70–2.60(m,1H),2.3 3(m,1H),2.27(m,1H),2.20(m,3H),1.94(m,1H),1.74(s,2H),1.45(s,2H),1.07–0.80(m,12H),0.78–0.69(m,3H).

[0187] Example 2: Preparation of compound DL-2

[0188] Step 1: Synthesis of tert-butyl ((3R, 4R, 7S)-7-benzyl-3-((S)-1-(3R, 4S, 5S)-4-(S)-2-[(S)-2-(dimethylamino)-3-methylbutanamido)-N, 3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-4-methyl-5,8-dioxo-2,12-dioxy-6,9-diazatetradec-14yl)carbamate

[0189] Compound 1 (110 mg, 0.147 mmol) and HATU (62 mg, 0.163 mmol) were added to N,N-dimethylformamide (5 mL) and stirred at room temperature for 10 minutes. DIEA (70 mg, 0.543 mmol) was then added and stirred at room temperature for 10 minutes. Finally, N-BOC-2-(2-aminoethoxy)ethylamine (30 mg, 0.147 mmol) was added and stirred at room temperature for 3 hours. The reaction was considered complete when LCMS showed less than 3% of compound 1 in the reaction solution. The solvent was evaporated under reduced pressure, and the crude product was purified by preparative liquid phase separation using acetonitrile / water. Compound 2 (136 mg, 98% yield) was obtained. MS: 933.26 [M+H] + .

[0190] Step 2: Synthesis of (S)-N-((3R,4S,5S)-1-((S)-2-((3R,4R,7S)-14-amino-7-benzyl-4-methyl-5,8-dioxo-2,12-dioxo-6,9-diazatetradec-3-yl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)-2-(S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamide

[0191] Compound 2 (136 mg, 0.146 mmol) was placed in a 25 mL single-necked flask. Dichloromethane (3 mL) and HCl / 1,4-dioxane solution (1 mL, 4 M) were added. The mixture was allowed to react at room temperature for 1 hour. LCMS confirmed the complete reaction of compound 2. After spin drying, the crude product, compound 3 (150 mg, 123.6% yield), was obtained. MS: 833.14 [M+H] + .

[0192] Step 3: Synthesis of 4-(((S)-2-[(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl((3R,4R,7S)-7-benzyl-3-((S)-1-((3R,4S,5S)-4-(S)-2-[(S)-2-(dimethylamino)-3-methylbutanamido]-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-4-methyl-5,8-dioxo-2,12-dioxo-6,9-diazatetradec-14yl)carbamate

[0193] Compound 3 (100 mg, 0.12 mmol) and compound 4 (92 mg, 0.12 mmol) were added to N,N-dimethylformamide (5 mL) and stirred at room temperature for 10 minutes. DIEA (62 mg, 0.48 mmol) was then added and stirred at room temperature for 2 hours. The reaction was considered complete when LCMS showed less than 3% of compound 3 in the reaction solution. The solvent was evaporated under reduced pressure, and the crude product was purified by preparative liquid phase separation using acetonitrile / water. Compound 5 (white solid, 182 mg, yield 103.9%) was obtained. MS: 731 [M+2H] 2+ .

[0194] Step 4: Synthesis of 4-(((S)-2-[(S)-2-amino-3-methylbutanamido)-5-ureidopentanamido]benzyl((3R,4R,7S)-7-benzyl-3-(S)-1-((3R,4S,5S)-4-((S)-2-(S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-4-methyl-5,8-dioxo-2,12-dioxo-6,9-diazatetradec-14yl)carbamate

[0195] Compound 5 (182 mg, 0.125 mmol) was placed in a 25 mL single-necked flask, and N,N-dimethylformamide (3 mL) and morpholine (2 mL) were added. The reaction was allowed to proceed at room temperature for 30 min. LCMS confirmed the complete reaction of compound 5. The reaction solution was spin-dried, and the crude product was purified by preparative liquid phase separation using acetonitrile / water. Compound 6 (white solid, 130 mg, yield 84.2%) was obtained. MS: 1238.6 [M+H] + .

[0196] Step 5: 4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-4,7,35-trioxo-2-(3-ureidopropyl)-10,13,16,19,22,25,28,31-octyloxy-3,6,34-triazaheptanecarboxamido)benzyl ((3R,4R,7S)-7-benzyl Synthesis of 3-((S)-1-(3R, 4S, 5S)-4-(S)-2-[(S)-2-(dimethylamino)-3-methylbutyramido)-N, 3-dimethylbutyramido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-4-methyl-5,8-dioxo-2,12-dioxo-6,9-diazatetradec-14-yl)carbamate

[0197] Compound 10 (17 mg, 0.028 mmol) and HATU (13 mg, 0.034 mmol) were added to N,N-dimethylformamide (3 mL) and stirred at room temperature for 10 minutes. Compound 6 (35 mg, 0.028 mmol) and DIEA (14 mg, 0.112 mmol) were then added and stirred at room temperature for 3 hours. The reaction was considered complete when LCMS showed less than 3% of compound 6 in the reaction solution. The solvent was evaporated under reduced pressure, and the crude product was purified by preparative liquid phase separation using acetonitrile / water. Compound DL-2 was obtained (white solid, 5 mg, 9.9% yield). MS: 1813.22 [M+H] + .

[0198] 1 H NMR(400MHz,DMSO-d6)δ8.12(m,1H),8.08–7.97(m,3H),7.93(m,1H),7.86(m,1H),7.58(m,2H),7.29–7.16(m,7H),7.00(s,2H),5 .99(m,1H),5.41(s,2H),4.93(s,2H),4.72–4.56(m,2H),4.56–4.47(m,1H),4.38(m,1H),4.29–4.18(m,1H),3.97(s,1H),3.78(m, 1H),3.61(m,4H),3.53–3.46(m,26H),3.37(m,8H),3.25(s,1H),3.24–3.10(m,12H),3.07–2.88(m,7H),2.76(m,1H),2.69–2.59( m,1H),2.46–2.25(m,6H),2.20(m,6H),2.04–1.84(m,5H),1.81–1.54(m,5H),1.35(m,6H),1.05–0.80(m,22H),0.77–0.66(m,6H).

[0199] Example 3: Preparation of Compound DL-3

[0200] Step 1: Synthesis of ((9H-fluoren-9-yl)methoxycarbonyl)glycylglycyl-L-phenylalanine tert-butyl ester

[0201] Compound 12 (100.0 mg, 0.282 mmol) and HATU (107.3 mg, 0.282 mmol) were added to N,N-dimethylformamide (3 mL) and stirred at room temperature for 10 minutes. DIEA (140 μL, 0.846 mmol) and tert-butyl phenylalaninate (62.5 mg, 0.282 mmol) were then added and stirred at room temperature for 1 hour. LCMS showed that the reaction solution contained less than 3% of compound 12, which was considered complete. The reaction solution was diluted 2-fold and washed with water (15 mL) and saturated brine (15 mL) in sequence. The organic phases were combined, extracted twice, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The product was purified by preparative liquid separation using acetonitrile / water. Compound 13 (white solid, 157.0 mg, 99.8% yield) was obtained. MS: 580.17 [M+Na] + .

[0202] Step 2: Synthesis of (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalanine

[0203] Compound 13 (157.00 mg, 0.282 mmol) was added to DCM (2 mL) and HCl / 1,4-dioxane solution (1 ml, 4 M) and allowed to react at room temperature for 60 min. LCMS monitored the complete reaction of compound 13. The reaction solution was distilled under reduced pressure to remove the solvent, and the product was purified by preparative liquid phase separation using acetonitrile / water. Compound 14 (white solid, 124.1 mg, yield 87.9%) was obtained. MS: 502.11 [M+H] + .

[0204] Step 3: Synthesis of (9H-fluoren-9-yl)methyl((3R,4R,7S)-7-benzyl-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-4-methyl-5,8,18-trioxy-2,12,15-trioxy-6,9,17-triazacyclononadecyl)carbamate

[0205] Compound 15 (408.0 mg, 0.489 mmol), compound 16 (90.2 mg, 0.245 mmol) and p-toluenesulfonic acid (126.5 mg, 0.735 mmol) were stirred at room temperature for 10 hours. LCMS showed that the reaction solution contained less than 3% of compound 16, which was considered to be the end of the reaction. The reaction solution was diluted 2-fold and washed with water (15 mL) and saturated brine (15 mL) in sequence. The organic phases were combined and extracted twice, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The product was purified by preparative liquid separation using acetonitrile / water. Compound 17 (white solid, 162.2 mg, yield 58.0%) was prepared. MS: 1141.59 [M+H] + .

[0206] Step 4: Synthesis of (S)-N-((3R,4S,5S)-1-((S)-2-((3R,4R,7S)-19-amino-7-benzyl-4-methyl-5,8,18-trioxy-2,12,15-trioxy-6,9,17-triazetidin-3-yl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)-2-(S)-2-(dimethylamino)-3-methylbutanamide)-N,3-dimethylbutanamide

[0207] Compound 17 (162.2 mg, 0.282 mmol) was added to N,N-dimethylformamide (2 mL) and morpholine (1 ml) and allowed to react at room temperature for 60 min. LCMS monitored the complete reaction of compound 17. The reaction solution was distilled under reduced pressure to remove the solvent, and the product was purified by preparative liquid phase separation using acetonitrile / water. Compound 18 (white solid, 86.4 mg, yield 66.2%) was obtained. MS: 919.65 [M+H] + .

[0208] Step 5: Synthesis of (9H-fluoren-9-yl)methyl((3R,4R,7S,22S)-7,22-dibenzyl-3-((S)-1-(3R,4S,5S)-4-((S,2-(S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-4-methyl-5,8,18,21,24,27-hexaoxo-2,12,15-trioxy-6,9,17,20,23,26-hexaazaoctadecane-28-yl)carbamate

[0209] Compound 14 (47.1 mg, 0.094 mmol) and HATU (35.7 mg, 0.094 mmol) were added to N,N-dimethylformamide (3 mL) and stirred at room temperature for 10 minutes. DIEA (47 μL, 0.282 mmol) and compound 18 (86.4 mg, 0.094 mmol) were then added and stirred at room temperature for 1 hour. LCMS showed that the reaction was complete when compound 14 was less than 3% in the reaction solution. The reaction solution was diluted 2-fold and washed with water (15 mL) and saturated brine (15 mL) in sequence. The organic phases were combined and extracted twice, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The product was purified by preparative liquid separation using acetonitrile / water. Compound 19 (white solid, 64.2 mg, 48.7% yield) was obtained. MS: 702.19 [M+2H] 2+ .

[0210] Step 6: Synthesis of (S)-N-((3R,4S,5S)-1-((S)-2-((3R,4R,7S,22S)-28-amino-7,22-dibenzyl-4-methyl-5,8,18,21,24,27-hexaoxo-2,12,15-trioxa-6,9,17,20,23,26-hexaazaoctan-3-yl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)-2-(S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamide

[0211] Compound 19 (64.2 mg, 0.046 mmol) was added to N,N-dimethylformamide (2 mL) and morpholine (1 ml) and allowed to react at room temperature for 60 min. LCMS monitored the complete reaction of compound 19. The reaction solution was distilled under reduced pressure to remove the solvent, and the product was purified by preparative liquid phase separation using acetonitrile / water. Compound 20 (white solid, 22.1 mg, yield 40.9%) was obtained. MS: 1180.73 [M+H] + .

[0212] Step 7: Synthesis of N-((3R, 4R, 7S, 22S)-7,22-dibenzyl-3-((S)-1-(3R, 4S, 5S)-4-((S-pyrrolidin-2-yl)-4-methyl-5,8,18,21,24,27-hexaoxo-2,12,15-trioxo-6,9,17,20,23,26-hexaazaoct-28-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide

[0213] Compound 20 (10.0 mg, 0.0085 mmol) was added to N,N-dimethylformamide (2 mL) and DIEA (5 μL, 0.025 mmol) and allowed to react at room temperature for 2 hours. LCMS showed that the reaction solution contained less than 3% of compound 20, which was considered complete. The reaction solution was diluted 2-fold and washed with water (15 mL) and saturated brine (15 mL) in sequence. The organic phases were combined and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and purified by preparative liquid phase separation using acetonitrile / water. Compound DL-3 was obtained (white solid, 9.2 mg, yield 79.1%). MS: 1373.69 [M+H] + .

[0214] 1 H NMR(400MHz,DMSO-d6)δ8.48(m,1H),8.18–8.10(m,1H),8.07(m,1H),8.01(m,2H),7.95(m,2H),7.31– 7.10(m,10H),6.99(s,2H),4.61–4.39(m,5H),3.86–3.64(m,7H),3.59(m,2H),3.54–3.43(m,6H),3.26 –3.09(m,11H),3.09–2.92(m,6H),2.78(m,3H),2.69–2.57(m,2H),2.39–2.23(m,3H),2.20(m,6H),2. 11(m,2H),1.89(s,7H),1.47(m,5H),1.30(s,1H),1.20(m,3H),1.00–0.79(m,16H),0.79–0.63(m,6H).

[0215] Example 4: Preparation of Compound DL-4

[0216] Step 1: Synthesis of ((9H-fluoren-9-yl)methoxycarbonyl)glycylglycyl-L-phenylalanine tert-butyl ester

[0217] Compound 12 (100.0 mg, 0.282 mmol) and HATU (107.3 mg, 0.282 mmol) were added to N,N-dimethylformamide (3 mL) and stirred at room temperature for 10 minutes. DIEA (140 μL, 0.846 mmol) and tert-butyl phenylalaninate (62.5 mg, 0.282 mmol) were then added and stirred at room temperature for 1 hour. LCMS showed that the reaction solution contained less than 3% of compound 12, which was considered complete. The reaction solution was diluted 2-fold and washed with water (15 mL) and saturated brine (15 mL) in sequence. The organic phases were combined, extracted twice, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The product was purified by preparative liquid separation using acetonitrile / water. Compound 13 (white solid, 157.0 mg, 99.8% yield) was obtained. MS: 580.17 [M+Na] + .

[0218] Step 2: Synthesis of (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalanine

[0219] Compound 13 (157.00 mg, 0.282 mmol) was added to DCM (2 mL) and HCl / 1,4-dioxane solution (1 ml, 4 M). The reaction was allowed to proceed at room temperature for 60 min. LCMS monitored the complete reaction of compound 13. The solvent was removed by distillation under reduced pressure, and the mixture was purified by preparative liquid phase separation using acetonitrile / water. Compound 14 (white solid, 124.1 mg, yield 87.9%) was obtained. MS: 502.11 [M+H] + .

[0220] Step 3: Synthesis of (9H-fluoren-9-yl)methyl((3R,4R,7S)-7-benzyl-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-4-methyl-5,8,18-trioxy-2,12,15-trioxy-6,9,17-triazacyclononadecyl)carbamate

[0221] Compound 15 (408.0 mg, 0.489 mmol), compound 16 (90.2 mg, 0.245 mmol) and p-toluenesulfonic acid (126.5 mg, 0.735 mmol) were stirred at room temperature for 10 hours. LCMS showed that the reaction solution contained less than 3% of compound 16, which was considered to be the end of the reaction. The reaction solution was diluted 2-fold and washed with water (15 mL) and saturated brine (15 mL) in sequence. The organic phases were combined and extracted twice, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The product was purified by preparative liquid separation using acetonitrile / water. Compound 17 (white solid, 162.2 mg, yield 58.0%) was prepared. MS: 1141.59 [M+H] + .

[0222] Step 4: Synthesis of (S)-N-((3R,4S,5S)-1-((S)-2-((3R,4R,7S)-19-amino-7-benzyl-4-methyl-5,8,18-trioxy-2,12,15-trioxy-6,9,17-triazetidin-3-yl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)-2-(S)-2-(dimethylamino)-3-methylbutanamide)-N,3-dimethylbutanamide

[0223] Compound 17 (162.2 mg, 0.282 mmol) was added to N,N-dimethylformamide (2 mL) and morpholine (1 ml) and allowed to react at room temperature for 60 min. LCMS monitored the complete reaction of compound 17. The reaction solution was distilled under reduced pressure to remove the solvent, and the product was purified by preparative liquid phase separation using acetonitrile / water. Compound 18 (white solid, 86.4 mg, yield 66.2%) was obtained. MS: 919.65 [M+H] + .

[0224] Step 5: Synthesis of (9H-fluoren-9-yl)methyl((3R,4R,7S,22S)-7,22-dibenzyl-3-((S)-1-(3R,4S,5S)-4-((S,2-(S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-4-methyl-5,8,18,21,24,27-hexaoxo-2,12,15-trioxy-6,9,17,20,23,26-hexaazaoctadecane-28-yl)carbamate

[0225] Compound 14 (47.1 mg, 0.094 mmol) and HATU (35.7 mg, 0.094 mmol) were added to N,N-dimethylformamide (3 mL) and stirred at room temperature for 10 minutes. DIEA (47 μL, 0.282 mmol) and compound 18 (86.4 mg, 0.094 mmol) were then added and stirred at room temperature for 1 hour. LCMS showed that the reaction was complete when compound 14 was less than 3% in the reaction solution. The reaction solution was diluted 2-fold and washed with water (15 mL) and saturated brine (15 mL) in sequence. The organic phases were combined and extracted twice, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The product was purified by preparative liquid separation using acetonitrile / water. Compound 19 (white solid, 64.2 mg, 48.7% yield) was obtained. MS: 702.19 [M+2H] 2+ .

[0226] Step 6: Synthesis of (S)-N-((3R,4S,5S)-1-((S)-2-((3R,4R,7S,22S)-28-amino-7,22-dibenzyl-4-methyl-5,8,18,21,24,27-hexaoxo-2,12,15-trioxa-6,9,17,20,23,26-hexaazaoctan-3-yl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)-2-(S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamide

[0227] Compound 19 (64.2 mg, 0.046 mmol) was added to N,N-dimethylformamide (2 mL) and morpholine (1 ml) and allowed to react at room temperature for 60 min. LCMS monitored the complete reaction of compound 19. The reaction solution was distilled under reduced pressure to remove the solvent, and the product was purified by preparative liquid phase separation using acetonitrile / water. Compound 20 (white solid, 22.1 mg, yield 40.9%) was obtained. MS: 1180.73 [M+H] + .

[0228] Step 7: Synthesis of N-((3R,4R,7S,22S)-7,22-dibenzyl-3-((S)-1-(3R,4S,5S)-4-((S-methylheptanoyl)pyrrolidin-2-yl)-4-methyl-5,8,18,21,24,27-hexaoxo-2,12,15-trioxy-6,9,17,20,23,26-hexaazaheptane-28-yl)-1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propionamido)-3,6,9,12,15,18,21,124-octaoxaheptane-27-amide

[0229] Compound 10 (5.1 mg, 0.0085 mmol) and HATU (3.5 mg, 0.0085 mmol) were added to N,N-dimethylformamide (3 mL) and stirred at room temperature for 10 minutes. DIEA (5 μL, 0.025 mmol) and compound 20 (10.0 mg, 0.094 mmol) were then added and stirred at room temperature for 1 hour. LCMS showed that the reaction was complete when compound 10 was less than 3% in the reaction solution. The reaction solution was diluted 2-fold and washed with water (15 mL) and saturated brine (15 mL) in sequence. The organic phases were combined, extracted twice, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The product was purified by preparative liquid separation using acetonitrile / water. Compound 12 (white solid, 6.4 mg, 43.1% yield) was obtained. MS: 1755.12 [M+H] + .

[0230] 1 H NMR (400MHz, DMSO-d6) δ8.35–8.22(m,2H),8.19–8.08(m,2H),8.07–7.90(m,4H),7.32–7.09(m, 10H),7.00(s,2H),4.53(m,5H),3.84–3.64(m,7H),3.60(m,6H),3.56–3.45(m,31H),3.25(m,2H ),3.24–3.18(m,6H),3.18–3.10(m,6H),3.07(m,2H),3.05–2.94(m,5H),2.78(m,3H),2.43–2.2 4(m,6H),2.20(m,7H),2.01(s,3H),1.30(s,4H),1.04(m,1H),0.90(m,15H),0.79–0.62(m,6H).

[0231] Example 5: Preparation of Compound DL-5

[0232] Step 1: Synthesis of ((9H-fluoren-9-yl)methoxycarbonyl)glycylglycyl-L-phenylalanine tert-butyl ester

[0233] Compound 12 (100.0 mg, 0.282 mmol) and HATU (107.3 mg, 0.282 mmol) were added to N,N-dimethylformamide (3 mL) and stirred at room temperature for 10 minutes. DIEA (140 μL, 0.846 mmol) and tert-butyl phenylalaninate (62.5 mg, 0.282 mmol) were then added and stirred at room temperature for 1 hour. LCMS showed that the reaction solution contained less than 3% of compound 12, which was considered complete. The reaction solution was diluted 2-fold and washed with water (15 mL) and saturated brine (15 mL) in sequence. The organic phases were combined, extracted twice, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The product was purified by preparative liquid separation using acetonitrile / water. Compound 13 (white solid, 157.0 mg, 99.8% yield) was obtained. MS: 580.17 [M+Na] + .

[0234] Step 2: Synthesis of (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalanine

[0235] Compound 13 (157.00 mg, 0.282 mmol) was added to DCM (2 mL) and HCl / 1,4-dioxane solution (1 ml, 4 M). The reaction was allowed to proceed at room temperature for 60 min. LCMS monitored the complete reaction of compound 13. The solvent was removed by distillation under reduced pressure, and the mixture was purified by preparative liquid phase separation using acetonitrile / water. Compound 14 (white solid, 124.1 mg, yield 87.9%) was obtained. MS: 502.11 [M+H] + .

[0236] Step 3: Synthesis of (9H-fluoren-9-yl)methyl((3R,4R,7S)-7-benzyl-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-4-methyl-5,8,18-trioxy-2,12,15-trioxy-6,9,17-triazacyclononadecyl)carbamate

[0237] Compound 15 (408.0 mg, 0.489 mmol), compound 16 (90.2 mg, 0.245 mmol) and p-toluenesulfonic acid (126.5 mg, 0.735 mmol) were stirred at room temperature for 10 hours. LCMS showed that the reaction solution contained less than 3% of compound 16, which was considered to be the end of the reaction. The reaction solution was diluted 2-fold and washed with water (15 mL) and saturated brine (15 mL) in sequence. The organic phases were combined and extracted twice, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The product was purified by preparative liquid separation using acetonitrile / water. Compound 17 (white solid, 162.2 mg, yield 58.0%) was prepared. MS: 1141.59 [M+H] + .

[0238] Step 4: Synthesis of (S)-N-((3R,4S,5S)-1-((S)-2-((3R,4R,7S)-19-amino-7-benzyl-4-methyl-5,8,18-trioxy-2,12,15-trioxy-6,9,17-triazetidin-3-yl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)-2-(S)-2-(dimethylamino)-3-methylbutanamide)-N,3-dimethylbutanamide

[0239] Compound 17 (162.2 mg, 0.282 mmol) was added to N,N-dimethylformamide (2 mL) and morpholine (1 ml) and allowed to react at room temperature for 60 min. LCMS monitored the complete reaction of compound 17. The reaction solution was distilled under reduced pressure to remove the solvent, and the product was purified by preparative liquid phase separation using acetonitrile / water. Compound 18 (white solid, 86.4 mg, yield 66.2%) was obtained. MS: 919.65 [M+H] + .

[0240] Step 5: Synthesis of (9H-fluoren-9-yl)methyl((3R,4R,7S,22S)-7,22-dibenzyl-3-((S)-1-(3R,4S,5S)-4-((S,2-(S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-4-methyl-5,8,18,21,24,27-hexaoxo-2,12,15-trioxy-6,9,17,20,23,26-hexaazaoctadecane-28-yl)carbamate

[0241] Compound 14 (47.1 mg, 0.094 mmol) and HATU (35.7 mg, 0.094 mmol) were added to N,N-dimethylformamide (3 mL) and stirred at room temperature for 10 minutes. DIEA (47 μL, 0.282 mmol) and compound 18 (86.4 mg, 0.094 mmol) were then added and stirred at room temperature for 1 hour. LCMS showed that the reaction was complete when compound 14 was less than 3% in the reaction solution. The reaction solution was diluted 2-fold and washed with water (15 mL) and saturated brine (15 mL) in sequence. The organic phases were combined and extracted twice, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The product was purified by preparative liquid separation using acetonitrile / water. Compound 19 (white solid, 64.2 mg, 48.7% yield) was obtained. MS: 702.19 [M+2H] 2+ .

[0242] Step 6: Synthesis of (S)-N-((3R,4S,5S)-1-((S)-2-((3R,4R,7S,22S)-28-amino-7,22-dibenzyl-4-methyl-5,8,18,21,24,27-hexaoxo-2,12,15-trioxa-6,9,17,20,23,26-hexaazaoctan-3-yl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)-2-(S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamide

[0243] Compound 19 (64.2 mg, 0.046 mmol) was added to N,N-dimethylformamide (2 mL) and morpholine (1 ml) and allowed to react at room temperature for 60 min. LCMS monitored the complete reaction of compound 19. The reaction solution was distilled under reduced pressure to remove the solvent, and the product was purified by preparative liquid phase separation using acetonitrile / water. Compound 20 (white solid, 22.1 mg, yield 40.9%) was obtained. MS: 1180.73 [M+H] + .

[0244] Step 7: N-((3R,4R,7S,22S)-7,22-dibenzyl-3-((S)-1-(3R,4S,5S)-4-((S-methylheptanoyl)pyrrolidin-2-yl)-4-methyl-5,8,18,21,24,27-hexaoxo-2,12,15-trioxa-6,9,17,20,23,26-hexahydro Synthesis of 2-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-oxo-7,10,13,16,19,25,28-octyloxy-4-azabenzenetricarbonyl-31-amino)-N5-methyl-N5-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl) glutaramide

[0245] Compound 11 (4.2 mg, 0.0085 mmol) and HATU (4 mg, 0.011 mmol) were added to N,N-dimethylformamide (3 mL) and stirred at room temperature for 10 minutes. Compound 20 (5.0 mg, 0.0085 mmol) was then added and stirred until uniform. DIEA (4.6 mg, 0.036 mmol) was then added and stirred at room temperature for 3 hours. LCMS showed that the reaction solution contained less than 3% of compound 11, which was considered complete. The reaction solution was evaporated to dryness under reduced pressure, and the crude product was purified by preparative liquid phase separation using acetonitrile / water. Freeze-dried to obtain compound DL-5 (white solid, 1.5 mg, 8.5% yield). MS: 1042.84 [M+2H] 2+ .

[0246] 1 H NMR (400MHz, DMSO-d6) δ8.53–8.43(m,1H),8.19–7.89(m,7H),7.30–7.07(m,10H),6.99(d,J=1.5Hz ,2H),4.59(s,3H),4.52(m,6H),4.37(s,3H),3.96(s,2H),3.88–3.65(m,8H),3.64–3.54(m,7H),3. 54–3.43(m,31H),3.26–3.19(m,10H),3.18–3.12(m,6H),3.09–2.93(m,8H),2.87–2.70(m,5H),2.6 9–2.60(m,2H),2.30(m,9H),2.24–2.11(m,7H),1.09–0.95(m,2H),0.94–0.80(m,14H),0.72(m,6H).

[0247] Example 6: Preparation of Compound DL-6

[0248] Step 1: Synthesis of 4-((S)-2-(S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl((3R,4R,7S)-7-benzyl-3-(S)-1-(3R,4S,5S)-4-(S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-4-methyl-5,8-dioxo-2,12-dioxa-6,9-diazatetradec-14yl)carbamate

[0249] Compound 21 (4.4 mg, 0.006 mmol) and compound 3 (5 mg, 0.006 mmol) were added to N,N-dimethylformamide (3 mL), followed by DIEA (3 mg, 0.024 mmol). The reaction was stirred at room temperature for 1 hour. LCMS showed that the reaction solution contained less than 3% of compound 2, which was considered complete. The reaction solution was evaporated to dryness under reduced pressure, and the crude product was purified by preparative liquid phase separation using acetonitrile / water. Compound DL-6 (white solid, 6 mg, 69.7% yield) was obtained by lyophilization. MS: 1431.8 [M+H] + .

[0250] 1 H NMR(400MHz,DMSO-d6)δ9.97(s,1H),8.10–7.98(m,3H),7.94(m,1H),7.79(m,1H),7.57(m,2H),7.23(m,6H),7.14(m,1H),7.00(s,2H),5.9 7(m,1H),5.37(m,2H),4.93(s,2H),4.76–4.57(m,2H),4.55–4.46(m, 1H),4.38(m,1H),4.23–4.14(m,1H),3.96(s,1H),3.78(m,1H),3.50( m,1H),3.37(m,7H),3.23–3.15(m,7H),3.08–2.89(m,7H),2.75(m,1H),2.68–2.60(m,1H),2.45–2.35(m,1H),2.34–2.24(m,2H),2.23–2.0 6(m,9H),2.03–1.84(m,4H),1.82–1.63(m,4H),1.53–1.35(m,7H),1. 30–1.13(m,4H),1.03(m,1H),1.00–0.79(m,20H),0.78–0.65(m,6H).

[0251] Example 7: Preparation of Compound DL-7

[0252] Step 1: Synthesis of (S)-N-((3R,4S,5S)-1-(S)-2-((1R,2R)-3-(S)-1-((5-aminopentyl)amino)-1-oxo-3-phenylpropan-2-yl)amino]-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamide

[0253] Compound 1 (50 mg, 0.067 mmol) and HATU (31 mg, 0.08 mmol) were added to N,N-dimethylformamide (3 mL) and stirred at room temperature for 10 minutes. 1,5-Pentanediamine (14 mg, 0.137 mmol) was then added and stirred at room temperature for 10 minutes. Finally, DIEA (35 mg, 0.268 mmol) was added and stirred at room temperature for 3 hours. LCMS showed that the reaction solution contained less than 3% of compound 1, indicating that the reaction was complete. The reaction solution was evaporated to dryness under reduced pressure, and the crude product was purified by preparative liquid phase separation using acetonitrile / water. Compound 22 (white solid, 40 mg, 71.9% yield) was obtained. MS: 831.17 [M+H] + .

[0254] Step 2: Synthesis of 4-((S)-2-(S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(5-(S)-2-((2R,3R)-3-(S)-1-((3R,4S,5S)-4-(S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-ethoxy-2-methylpropanamido)3-phenylpropanamido(pentyl)carbamate

[0255] Compound 21 (10 mg, 0.012 mmol) and compound 22 (9 mg, 0.012 mmol) were added to N,N-dimethylformamide (3 mL), followed by DIEA (6 mg, 0.048 mmol). The reaction was stirred at room temperature for 1 hour. LCMS showed that the reaction solution contained less than 3% of compound 21, which was considered complete. The reaction solution was evaporated to dryness under reduced pressure, and the crude product was purified by preparative liquid phase separation using acetonitrile / water. Compound DL-7 (white solid, 13 mg, 76.5% yield) was prepared and lyophilized. MS: 1429.83 [M+H] + .

[0256] 1H NMR(400MHz, DMSO-d6)δ9.96(s,1H),8.31–8.21(m,1H),8.10–7.97(m,3H),7.86(m,1H),7.79(m,1H),7.58(m,2H),7.30–7.18(m,5H),7.15(m, 2H),7.00(s,2H),5.96(m,1H),5.40(s,2H),4.92(s,2H),4.61(m,2H), 4.55–4.42(m,2H),4.38(m,1H),4.23–4.15(m,1H),3.97(s,1H),3.79(m ,1H),3.52(m,2H),3.36(m,3H),3.24(m,3H),3.17(m,3H),3.10–2.89( m,9H),2.81–2.71(m,1H),2.68–2.60(m,1H),2.33(m,4H),2.23–2.07(m ,8H),1.95(m,4H),1.82–1.65(m,4H),1.58(m,1H),1.49(m,5H),1.36(m ,5H),1.26–1.12(m,5H),1.04(m,1H),0.97–0.79(m,17H),0.73(m,6H).

[0257] Example 8: Preparation of Compound DL-8

[0258] Step 1: Synthesis of 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentaamido)benzyl(5-((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido)pentyl)carbamate

[0259] Compound 22 (30 mg, 0.036 mmol) and compound 4 (28 mg, 0.036 mmol) were added to N,N-dimethylformamide (5 mL) and stirred at room temperature for 10 minutes. DIEA (18 mg, 0.144 mmol) was then added and stirred at room temperature for 2 hours. The reaction was considered complete when LCMS showed less than 3% of compound 22 in the reaction solution. The solvent was evaporated under reduced pressure, and the crude product was purified by preparative liquid phase separation using acetonitrile / water. Compound 23 was obtained (white solid, 51 mg, 97.2% yield). MS: 729.94 [M+2H]2+

[0260] Step 2: Preparation of 4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl(5-((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido)pentyl)carbamate

[0261] Compound 23 (51 mg, 0.035 mmol) was placed in a 25 mL single-necked flask, and N,N-dimethylformamide (5 mL) and morpholine (2 mL) were added. The reaction was allowed to proceed at room temperature for 30 min. LCMS monitored the complete reaction of compound 23. The reaction solution was spin-dried, and the crude product was purified by preparative liquid phase separation using acetonitrile / water. Compound 24 (white solid, 13 mg, yield 30.2%) was obtained. MS: 618.81 [M+2H] 2+

[0262] Step 3: 4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-4,7,35-trioxo-2-(3-ureidopropyl)-10,13,16,19,22,25,28,31-octaoxo-3,6,34-triazaheptantriamido)benzyl (5-((S)-2-((2 Synthesis of (R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyramido)-N,3-dimethylbutyramido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido)pentyl)carbamate

[0263] Compound 10 (6 mg, 0.01 mmol) and HATU (5 mg, 0.012 mmol) were added to N,N-dimethylformamide (3 mL) and stirred at room temperature for 10 minutes. Compound 24 (13 mg, 0.01 mmol) and DIEA (5 mg, 0.04 mmol) were then added and stirred at room temperature for 3 hours. The reaction was considered complete when LCMS showed less than 3% of compound 24 in the reaction solution. The solvent was evaporated under reduced pressure, and the crude product was purified by preparative liquid phase separation using acetonitrile / water. Compound DL-8 (white solid, 9 mg, 49.7% yield) was prepared and lyophilized. MS: 1811.25 [M+H] +

[0264] 1 H NMR(400MHz,DMSO-d6)δ9.96(s,1H),8.27–8.11(m,1H),8.12–7.95(m,3H),7.83(m ,1H),7.58(m,2H),7.26(m,3H),7.24–7.19(m,3H),7.18–7.12(m,1H),7.00(m,1H) ,5.96(m,1H),5.36(m,2H),4.92(s,2H),4.74–4.56(m,1H),4.53–4.41(m,1H),4.4 3–4.32(m,1H),4.23(m,1H),3.97(s,1H),3.79(m,1H),3.67–3.55(m,4H),3.56–3. 41(m,27H),3.36(m,3H),3.24(m,4H),3.15(m,5H),3.08(s,1H),3.06–2.88(m,9H) ,2.81–2.72(m,1H),2.70–2.62(m,1H),2.45(m,1H),2.40(m,1H),2.36–2.28(m,3H ),2.26–2.12(m,7H),2.04–1.84(m,4H),1.83–1.65(m,3H),1.56(m,1H),1.41–1.2 6(m,7H),1.22(m,2H),1.21–1.12(m,2H),1.02(m,1H),0.89(m,21H),0.74(m,6H).

[0265] Example 9: Preparation of Compound DL-9

[0266] Step 1: Synthesis of (S)-N-((3R,4S,5S)-1-(S)-2-((1R,2R)-3-((S)-1-((4-(aminomethyl)cyclohexyl)methyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)-2-(((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamide

[0267] Compound 1 (20 mg, 0.027 mmol) and HATU (13 mg, 0.032 mmol) were added to N,N-dimethylformamide (3 mL) and stirred at room temperature for 10 minutes. 1,4-Cyclohexanedimethylamine (8 mg, 0.054 mmol) was then added and stirred at room temperature for 10 minutes. Finally, DIEA (14 mg, 0.108 mmol) was added and stirred at room temperature for 3 hours. The reaction was considered complete when LCMS showed that the reaction solution contained less than 3% of compound 1. The solvent was evaporated under reduced pressure, and the crude product was purified by preparative liquid phase separation using acetonitrile / water. Compound 25 (white solid, 17 mg, 72.3% yield) was obtained. MS: 871.23 [M+H] +

[0268] Step 2: Synthesis of 4-((S)-2-(S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl((4-(((S)-2-((2R,3R)-3-(S)-1-((3R,4S,5S)-4-(S)-2-(S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanamido)methyl)cyclohexyl)methyl)carbamate

[0269] Compound 25 (17 mg, 0.02 mmol) and compound 21 (14 mg, 0.02 mmol) were added to N,N-dimethylformamide (3 mL), followed by DIEA (10 mg, 0.08 mmol). The reaction was stirred at room temperature for 1 hour. LCMS showed that the reaction solution contained less than 3% of compound 25, which was considered complete. The reaction solution was evaporated to dryness under reduced pressure, and the crude product was purified by preparative liquid phase separation using acetonitrile / water. Compound DL-9 (white solid, 7.4 mg, 25.2% yield) was obtained by lyophilization. MS: 1469.89 [M+H] +

[0270] 1H NMR(400MHz,DMSO-d6)δ9.97(s,1H),8.08(m,1H),8.05–8.01(m,1H),7.80(m,1H),7.57(m,2H),7.32–7.11(m,8H),6.99(s,2H),6.73(m ,1H),6.05–5.95(m,1H),5.41(s,2H),4.93(s,2H),4.63(m,1H),4.52(m,1H),4.38(m,1H),4.24–4.11(m,1H),3.97(s,1H),3.81(m,2H), 3.23(m,6H),3.18(m,5H),3.11–2.86(m,9H),2.86–2.71(m,2H),2.69–2.59(m,1H),2.26–2.06(m,8H),2.05–1.85(m,4H),1.80–1.70(m, 2H),1.66(m,1H),1.63–1.57(m,1H),1.48(m,7H),1.37–1.14(m,15H),1.04(m,1H),0.96(m,1H),0.94–0.79(m,20H),0.78–0.68(m,7H).

[0271] Example 10: Preparation of Compound DL-10

[0272] Step 1: Synthesis of (9H-fluoren-9-yl)methyl (R)-(12,12-dimethyl-2,10-dioxo-8-phenyl-5,11-dioxo-3,9-diazatridecyl)carbamate

[0273] Compound 16 (100 mg, 0.272 mmol), compound 26 (200 mg, 0.797 mmol), and TsOH (137 mg, 0.797 mmol) were added to dichloromethane (3 mL) and stirred at room temperature for 3 hours. The reaction was considered complete when LCMS showed less than 3% of compound 26 in the reaction solution. The solvent was evaporated under reduced pressure, and the crude product was purified by preparative liquid phase separation using acetonitrile / water. Compound 27 (white solid, 71 mg, 46.6% yield) was obtained. MS: 560.66 [M+H] +

[0274] Step 2: Synthesis of (9H-fluoren-9-yl)methyl (R)-(2-(((3-amino-3-phenylpropoxy)methyl)amino)-2-oxoethyl)carbamate

[0275] Compound 27 (71 mg, 0.127 mmol) was placed in a 25 mL single-necked flask, and dichloromethane (3 mL) and HCl-1,4-dioxane solution (1 mL, 4 M) were added. The reaction was allowed to proceed at room temperature for 1 hour. LCMS confirmed the complete reaction of compound 27. The crude product was spin-dried to dryness and purified by preparative liquid phase separation using acetonitrile / water to obtain compound 28 (28 mg, 47.9% yield). MS: 460.55 [M+H] +

[0276] Step 3: Synthesis of (9H-fluoren-9-yl)methyl((3R,4R,7S,10R)-7-benzyl-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-4-methyl-5,8,16-trioxy-10-phenyl-2,13-dioxa-6,9,15-triazaheptane-17-yl)carbamate

[0277] Compound 1 (45 mg, 0.061 mmol) and HATU (28 mg, 0.073 mmol) were added to N,N-dimethylformamide (5 mL) and stirred at room temperature for 10 minutes. DIEA (31 mg, 0.244 mmol) was then added and stirred at room temperature for 10 minutes. Finally, compound 28 (28 mg, 0.061 mmol) was added and stirred at room temperature for 3 hours. The reaction was considered complete when LCMS showed less than 3% of compound 1 in the reaction solution. The solvent was evaporated under reduced pressure, and the crude product was purified by preparative liquid phase separation using acetonitrile / water. Compound 29 (16 mg, 22.1% yield) was obtained. MS: 1188.53 [M+H] +

[0278] Step 4: Synthesis of (S)-N-((3R,4S,5S)-1-((S)-2-((3R,4R,7S,10R)-17-amino-7-benzyl-4-methyl-5,8,16-trioxo-10-phenyl-2,13-dioxa-6,9,15-triazaheptan-3-yl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamide

[0279] Compound 29 (16 mg, 0.013 mmol) was placed in a 25 mL single-necked flask, and N,N-dimethylformamide (3 mL) and morpholine (1 mL) were added. The reaction was allowed to proceed at room temperature for 30 min. LCMS monitored the complete reaction of compound 29. The reaction solution was spin-dried, and the crude product was purified by preparative liquid phase separation using acetonitrile / water. Compound 30 was obtained as a white solid, 6 mg, yield 46.2%. MS: 966.29 [M+H] +

[0280] Step 5: Synthesis of N-((3R,4R,7S,10R,20S)-7,20-dibenzyl-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-4-methyl-5,8,16,19,22,25-hexaoxy-10-phenyl-2,13-dioxacyclo-6,9,15,18,21,24-hexaazahexadecane-26-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide

[0281] Compound 31 (3 mg, 0.006 mmol) and HATU (2.7 mg, 0.007 mmol) were added to N,N-dimethylformamide (3 mL) and stirred at room temperature for 10 minutes. DIEA (3.1 mg, 0.024 mmol) was then added and stirred at room temperature for 10 minutes. Finally, compound 30 (6 mg, 0.006 mmol) was added and stirred at room temperature for 3 hours. The reaction was considered complete when LCMS showed less than 3% of compound 31 in the reaction solution. The solvent was evaporated under reduced pressure, and the crude product was purified by preparative liquid phase separation using acetonitrile / water. Freeze-dried to obtain compound DL-10 (5 mg, 58.7% yield). MS: 1420.77 [M+H] +

[0282] 1H NMR(400MHz,DMSO)δ8.16–8.03(m,2H),7.97(m,1H),7.74(m,1H),7.68(m,1H),7.27–7.1 4(m,15H),7.00–6.95(m,2H),6.64(s,1H),6.51(s,1H),5.32(m,1H),4.52(m,3H),3.74– 3.63(m,5H),3.25–3.13(m,9H),2.98(m,5H),2.84–2.69(m,8H),2.32(m,3H),2.10(m,2H ),2.00(m,7H),1.85(s,4H),1.47(m,10H),1.30(m,5H),0.98–0.81(m,21H),0.76(m,2H).

[0283] Example 11: Preparation of Compound DL-11

[0284] Step 1: Synthesis of (9H-fluoren-9-yl)methyl(14,14-dimethyl-2,12-dioxo-5,13-dioxo-3,11-diazapentadecanyl)carbamate

[0285] Compound 16 (60.0 mg, 0.163 mmol), compound 5-Boc-amino-1-pentanol (99.33 mg, 0.489 mmol) and p-toluenesulfonic acid (84.21 mg, 0.489 mmol) were added to dichloromethane (3 mL) and stirred at room temperature for 2 hours. LCMS showed that the reaction solution contained less than 3% of compound 16, which was considered complete. The reaction solution was diluted 2-fold and washed with water (15 mL) and saturated brine (15 mL) in sequence. The organic phases were combined and extracted twice, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The product was purified by preparative liquid separation using acetonitrile / water. Compound 32 (white solid, 32.0 mg, 46% yield) was obtained. MS: 534.29 [M+Na] + .

[0286] Step 2: Synthesis of (9H-fluoren-9-yl)methyl (2-((((5-aminopentyl)oxy)methyl)amino)-2-oxoethyl)carbamate

[0287] Compound 32 (32.00 mg, 0.063 mmol) was added to dichloromethane (2 mL) and HCl (4M dioxane, 1 mL) and allowed to react at room temperature for 30 minutes. LCMS monitored the complete reaction of compound 32. The reaction solution was distilled under reduced pressure to remove the solvent, and the product was purified by preparative liquid phase separation using acetonitrile / water. Compound 33 (white solid, 21.0 mg, yield 81.7%) was obtained. MS: 412.25 [M+H] + .

[0288] Step 3: Synthesis of (9H-fluoren-9-yl)methyl((3R,4R,7S)-7-benzyl-3-((S)-1-(3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-4-methyl-5,8,18-trioxy-2,15-dioxy-6,9,17-triazacyclononadecyl)carbamate

[0289] Compound 1 (38.1 mg, 0.051 mmol) and HATU (19.4 mg, 0.051 mmol) were added to N,N-dimethylformamide (3 mL) and stirred at room temperature for 10 minutes. DIEA (26 μL, 0.153 mmol) and compound 33 (21.0 mg, 0.051 mmol) were then added and stirred at room temperature for 1 hour. The reaction was considered complete when LCMS showed less than 3% of compound 1 in the reaction solution. The solvent was evaporated under reduced pressure, and the product was purified by preparative liquid phase separation using acetonitrile / water. Compound 34 (white solid, 22.0 mg, 37.9% yield) was obtained. MS: 1139.99 [M+H] + .

[0290] Step 4: Synthesis of (S)-N-((3R,4S,5S)-1-((S)-2-((3R,4R,7S)-19-amino-7-benzyl-4-methyl-5,8,18-trioxo-2,15-dioxa-6,9,17-triazetidin-3-yl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)-2-(S)-2-(dimethylamino)-3-methylbutanamide)-N,3-dimethylbutanamide

[0291] Compound 34 (22.0 mg, 0.019 mmol) was added to N,N-dimethylformamide (2 mL) and morpholine (1 ml) and allowed to react at room temperature for 30 minutes. LCMS monitored the complete reaction of compound 34. The reaction solution was distilled under reduced pressure to remove the solvent, and the product was purified by preparative liquid phase separation using acetonitrile / water. Compound 35 (white solid, 12.0 mg, yield 67.8%) was obtained. MS: 917.79 [M+H] + .

[0292] Step 5: Synthesis of N-((3R, 4R, 7S, 22S)-7,22-dibenzyl-3-((S)-1-(3R, 4S, 5S)-4-((S-pyrrolidin-2-yl)-4-methyl-5,8,18,21,24,27-hexaoxo-2,15-dioxo-6,9,17,20,23,26-hexaazaoct-28-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide

[0293] Compound 31 (6.2 mg, 0.013 mmol) and HATU (5.0 mg, 0.013 mmol) were added to N,N-dimethylformamide (2 mL) and stirred at room temperature for 10 minutes. DIEA (7 μL, 0.039 mmol) and compound 35 (12.0 mg, 0.013 mmol) were then added and stirred at room temperature for 1 hour. The reaction was considered complete when LCMS showed less than 3% of compound 31 in the reaction solution. The solvent was evaporated under reduced pressure and purified by preparative liquid phase separation using acetonitrile / water. Compound DL-11 was obtained (white solid, 2.5 mg, yield 14.0%). MS: 1371.70 [M+H] + .

[0294] 1H NMR(400MHz,DMSO-d6)δ8.43(s,3H),8.27(d,J=7.3Hz,2H),8.18–7.97(m,5H),7.95(s,1H),7.30–7.14(m,10H) ,6.99(s,2H),6.75(s,2H),4.63(m,3H),4.50(m,5H),3.96(s,2H),3.85–3.60(m,9H),3.56(s,3H),3.22(m,4H) ,3.20–3.14(m,4H),3.11–2.93(m,9H),2.89(s,2H),2.80(m,3H),2.73(s,2H),2.64(m,2H),2.20(m,7H),2.11( m,3H),1.92(m,4H),1.76(m,5H),1.60–1.31(m,16H),1.30–1.11(m,9H),1.00–0.80(m,16H),0.80–0.64(m,7H).

[0295] Example 12: Preparation of Compound DL-12

[0296] Step 1: Synthesis of tert-butyl (R)-3-(10-(9H-fluoren-9-yl)-5,8-dioxo-2,9-dioxo-4,7-diazadecyl)pyrrolidine-1-carboxylate

[0297] Compound 16 (50.0 mg, 0.135 mmol), compound (R)-1-BOC-3-hydroxymethylpyrrolidine (54.3 mg, 0.270 mmol) and p-toluenesulfonic acid (69.8 mg, 0.405 mmol) were added to dichloromethane (3 mL) and stirred at room temperature for 2 hours. LCMS showed that the reaction solution contained less than 3% of compound 16, which was considered to be the end of the reaction. The reaction solution was diluted 2-fold and washed with water (15 mL) and saturated brine (15 mL) in sequence. The organic phases were combined and extracted twice, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The product was purified by preparative liquid separation using acetonitrile / water. Compound 36 (white solid, 23.0 mg, yield 33.2%) was prepared. MS: 510.29 [M+H] + .

[0298] Step 2: Synthesis of (9H-fluoren-9-yl)methyl (R)-(2-oxo-2-(((pyrrolidin-3-ylmethoxy)methyl)amino)ethyl)carbamate

[0299] Compound 36 (32.00 mg, 0.063 mmol) was added to dichloromethane (2 mL) and HCl (4M dioxane, 1 mL) and allowed to react at room temperature for 30 minutes. LCMS monitored the complete reaction of compound 36. The reaction solution was distilled under reduced pressure to remove the solvent, and then purified by preparative liquid phase separation using acetonitrile / water. Compound 37 (white solid, 21.0 mg, yield 81.7%) was obtained. MS: 410.29 [M+H] + .

[0300] Step 3: Synthesis of (9H-fluoren-9-ylmethyl-2-(R)-1-((((2R,3R,3R)-3-(((S)-1-(3R,4S,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-ethoxy-2-methylpropanoyl)-L-phenylalanyl)pyrrolidin-3-yl)methoxy)methyl)amino)-2-oxoethyl)carbamate

[0301] Compound 1 (38.1 mg, 0.051 mmol) and HATU (19.4 mg, 0.051 mmol) were added to N,N-dimethylformamide (3 mL) and stirred at room temperature for 10 minutes. DIEA (26 μL, 0.153 mmol) and compound 37 (21.0 mg, 0.051 mmol) were then added and stirred at room temperature for 1 hour. The reaction was considered complete when LCMS showed less than 3% of compound 37 in the reaction solution. The solvent was evaporated under reduced pressure and purified by preparative liquid phase separation using acetonitrile / water. Compound 38 (white solid, 22.0 mg, 37.9% yield) was obtained. MS: 1137.98 [M+H] + .

[0302] Step 4: Synthesis of (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-((R)-3-(((2-aminoacetamido)methoxy)methyl)pyrrolidin-1-yl)-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamide)-N,3-dimethylbutanamide

[0303] Compound 38 (22.0 mg, 0.019 mmol) was added to N,N-dimethylformamide (2 mL) and morpholine (1 ml) and allowed to react at room temperature for 30 minutes. LCMS monitored the complete reaction of compound 38. The reaction solution was distilled under reduced pressure to remove the solvent, and the product was purified by preparative liquid phase separation using acetonitrile / water. Compound 39 was obtained (white solid, 12.0 mg, yield 67.8%). MS: 915.83 [M+H] + .

[0304] Step 5: Synthesis of N-((S)-9-benzyl-1-((R)-1-(((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamide)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanyl)pyrrolidin-3-yl)-5,8,11,14-tetraoxo-2-oxy-4,7,10,13-tetraazapentadecan-15-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide

[0305] Compound 31 (6.2 mg, 0.013 mmol) and HATU (5.0 mg, 0.013 mmol) were added to N,N-dimethylformamide (3 mL) and stirred at room temperature for 10 minutes. DIEA (7 μL, 0.039 mmol) and compound 39 (12.0 mg, 0.013 mmol) were then added and stirred at room temperature for 1 hour. The reaction was considered complete when LCMS showed less than 3% of compound 31 in the reaction solution. The solvent was evaporated under reduced pressure and purified by preparative liquid phase separation using acetonitrile / water. Compound DL-12 was obtained (white solid, 6.4 mg, 35.6% yield). MS: 1369.69 [M+H] + .

[0306] 1H NMR(400MHz, DMSO-d6)δ9.50(s,2H),8.92(m,2H),8.49(m,3H),8.27(m,3H),8.17–7.92(m,5H),7.41–7.31(m,2H),7. 27–7.17(m,10H),6.99(s,2H),4.73–4.44(m,8H),3.98(s,2H),3.83(m,1H),3.80–3.64(m,9H),3.65–3.59(m,2H),3.5 8–3.54(m,2H),3.28–3.21(m,6H),3.21–3.12(m,7H),3.12–3.03(m,4H),3.00(s,4H),2.86–2.71(m,10H),2.70(s,1H) ,2.39–2.19(m,7H),2.18–1.98(m,6H),1.73(m,6H),1.48(m,9H),1.39–1.09(m,8H),1.10–0.81(m,22H),0.77(m,4H).

[0307] Example 13: Preparation of Compound DL-13

[0308] Step 1: Synthesis of 9H-fluoren-9-yl)methyl (8,8,12,12-tetramethyl-2,10-dioxo-5,11-dioxo-3,9-diazatridecyl)carbamate

[0309] Compound 16 (60 mg, 0.163 mmol) and compound 40 (66.67 mg, 0.326 mmol) were placed in a 10 mL single-necked flask. Dichloromethane (3 mL) and TsOH (2.8 mg, 0.0163 mmol) were added and allowed to react at room temperature for 2 h. LCMS monitored the complete reaction of compound 16. The crude product was spin-dried to dryness and purified by preparative liquid phase separation using acetonitrile / water. Compound 41 (white solid, 38 mg, yield 56.7%) was obtained. MS: 512.26 [M+H] +

[0310] Step 2: Synthesis of (9H-fluoren-9-yl)methyl (2-(((3-amino-3-methylbutoxy)methyl)amino)-2-oxoethyl)carbamate

[0311] Compound 41 (38 mg, 0.074 mmol) was added to 5 mL of HCl / dioxane (4 M) solution and stirred at room temperature for 20 minutes. LCMS showed that compound 41 was completely consumed in the reaction solution. The solvent was evaporated under reduced pressure, and the crude product was purified by preparative liquid phase separation using acetonitrile / water to obtain compound 42 (30 mg, 97.2% yield). MS: 412.14 [M+H] +

[0312] Step 3: Synthesis of (9H-fluoren-9-yl)methyl((3R,4R,7R)-7-benzyl-3-((S)-1-(3R,4S,5S)-4-((S,-2-(S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-4,10,10-trimethyl-5,8,16-trioxo-2,13-dioxo-6,9,15-triazaheptadecan-17-yl)carbamate

[0313] Compound 1 (30 mg, 0.072 mmol), HATU (54.72 mg, 0.144 mmol), and compound 42 (53 mg, 0.072 mmol) were added to N,N-dimethylformamide (3 mL) and stirred at room temperature for 10 minutes. Finally, DIEA (46.44 mg, 0.36 mmol) was added and stirred at room temperature for 1 hour. The reaction was considered complete when LCMS showed less than 3% of compound 1 in the reaction solution. The solvent was evaporated under reduced pressure, and the crude product was purified by preparative liquid phase separation using acetonitrile / water. Compound 43 (30 mg, 36.5% yield) was obtained. MS: 580.74 [M+H+Na] 2+

[0314] Step 4: Synthesis of (S)-N-(3R,4S,5S)-1-(S)-2-((3R,4R,7R)-17-amino-7-benzyl-4,10-trimethyl-5,8,16-trioxy-2,13-dioxy-6,9,15-triazaheptadecan-3-yl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)-2-(((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamide

[0315] Compound 43 (30 mg, 0.0263 mmol) was placed in a 25 mL single-necked flask, and N,N-dimethylformamide (3 mL) and morpholine (2 mL) were added. The reaction was allowed to proceed at room temperature for 30 min. LCMS monitored the complete reaction of compound 43. The reaction solution was spin-dried, and the crude product was purified by preparative liquid phase separation using acetonitrile / water. Compound 44 was obtained as a white solid, 18 mg, yield 74.5%. MS: 917.25 [M+H] +

[0316] Step 5: N-((3R,4R,7R,20S)-7,20-dibenzyl-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S,-2-(dimethylamino)-3-methylbutanamide)-((3R,4S,5S)-4-((S)-2-((S,-2-(dimethylamino)-3-methylbutanamide)-N, Synthesis of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide (3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-(3-dimethylbutyramide)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-4,10,10-trimethyl-5,8,16,19,22,5-hexaoxo-2,13-dioxo-6,9,15,18,21,24-hexaazahexacan-26-yl)

[0317] Compound 31 (9.25 mg, 0.0196 mmol), HATU (14.89 mg, 0.0392 mmol), and compound 44 (18 mg, 0.0196 mmol) were added to N,N-dimethylformamide (3 mL). DIEA (12.64 mg, 0.098 mmol) was added and stirred at room temperature for 1 hour. LCMS showed that the reaction solution contained less than 3% of compound 44, which was considered complete. The reaction solution was evaporated to dryness under reduced pressure, and the crude product was purified by preparative liquid phase separation using acetonitrile / water. Compound DL-12 was obtained (white solid, 2.8 mg, 10.4% yield). MS: 1371.7 [M+H] +

[0318] 1 H NMR (400 MHz, DMSO-d 6)δ9.52(s,1H),8.90(s,1H),8.79(s,1H),8.46(m,1H),8.33–8.23(m,1H),8.09(m,2H),7.98(m,2H),7.29–7.11(m,10H),6.98(s,2 H),4.72–4.55(m,3H),4.47(m,4H),3.97(s,2H),3.82(m,1H),3.79–3.69(m,4H),3.66(m,2H),3.59(m,2H),3.32–3.28(m,3H),3.23 (s,2H),3.22–3.14(m,5H),3.06(m,3H),2.98(m,2H),2.76(m,6H),2.36–2.18(m,4H),2.11(m,2H),2.03(m,2H),1.82(m,5H),1.61 (m,1H),1.46(m,5H),1.25–1.16(m,6H),1.15(s,2H),1.09–1.04(m,1H),1.04–0.99(m,2H),0.98–0.81(m,15H),0.80–0.70(m,3H).

[0319] Example 14: Preparation of Compound DL-14

[0320] Step 1: Synthesis of (9H-fluoren-9-yl)methyl (2-((((1-(tert-butoxycarbonyl)amino)cyclopropyl)methoxy)methyl)amino)-2-oxoethyl)carbamate

[0321] Compound 16 (60.0 mg, 0.163 mmol), compound 45 (91.6 mg, 0.489 mmol) and p-toluenesulfonic acid (2.8 mg, 0.016 mmol) were added to dichloromethane (3 mL) and stirred at room temperature for 2 hours. LCMS showed that the reaction solution contained less than 3% of compound 16, which was considered complete. The reaction solution was diluted 2-fold and washed with water (15 mL) and saturated brine (15 mL) in sequence. The organic phases were combined and extracted twice, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The product was purified by preparative liquid separation using acetonitrile / water. Compound 46 (white solid, 53.0 mg, yield 65.6%) was obtained. MS: 496.45 [M+H] + .

[0322] Step 2: Synthesis of (9H-fluoren-9-yl)methyl (2-((((1-aminocyclopropyl)methoxy)methyl)amino)-2-oxoethyl)carbamate

[0323] Compound 46 (53.0 mg, 0.107 mmol) was added to dichloromethane (2 mL) and TFA (0.5 ml) and allowed to react at room temperature for 30 minutes. LCMS monitored the complete reaction of compound 46. The reaction solution was distilled under reduced pressure to remove the solvent, and the product was purified by preparative liquid phase separation using acetonitrile / water. Compound 47 (white solid, 15.1 mg, yield 35.5%) was obtained. MS: 396.58 [M+H] + .

[0324] Step 3: Synthesis of (9H-fluoren-9-ylmethylmethyl-2-(2R,3R)-3-(S)-1-(3R,4S,4S,5S)-4-(S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-3-methoxymethylbutyryl-2-oxoethyl)carbamate

[0325] Compound 1 (28.0 mg, 0.038 mmol), HOBT (5.1 mg, 0.038 mmol), and EDCI (7.2 mg, 0.038 mmol) were added to dichloromethane (3 mL) and stirred at room temperature for 20 minutes. DIEA (19 μL, 0.114 mmol) and compound 47 (15.0 mg, 0.038 mmol) were then added and stirred at room temperature for 1 hour. LCMS showed that the reaction solution contained less than 3% of compound 47, which was considered complete. The reaction solution was diluted 2-fold and washed sequentially with water (15 mL) and saturated brine (15 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The product was purified by preparative liquid separation using acetonitrile / water. Compound 48 (white solid, 23.6 mg, 55.3% yield) was obtained. MS: 1124.67 [M+H] + .

[0326] Step 4: Synthesis of (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-((1-(((2-aminoacetamido)methoxy)methyl)cyclopropyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamide)-N,3-dimethylbutanamide

[0327] Compound 48 (23.6 mg, 0.021 mmol) was added to N,N-dimethylformamide (2 mL) and morpholine (1 ml) and allowed to react at room temperature for 30 minutes. LCMS monitored the complete reaction of compound 48. The reaction solution was distilled under reduced pressure to remove the solvent, and the product was purified by preparative liquid phase separation using acetonitrile / water. Compound 49 (white solid, 13.2 mg, yield 71.4%) was obtained. MS: 902.37 [M+H] + .

[0328] Step 5: N-((S)-9-benzyl-1-(1-((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamide)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-ethoxy-2-methylpropionamido)3-phenylpropionamidocyclopropyl)-5,8,11,14-tetraoxo-2-oxy-4,7,10,13-tetraazapentadecan-15-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide

[0329] Compound 31 (7.1 mg, 0.015 mmol) and HATU (5.7 mg, 0.015 mmol) were added to N,N-dimethylformamide (3 mL) and stirred at room temperature for 10 minutes. DIEA (8 μL, 0.045 mmol) and compound 49 (13.2 mg, 0.015 mmol) were then added and stirred at room temperature for 1 hour. The reaction was considered complete when LCMS showed less than 3% of compound 49 in the reaction solution. The solvent was evaporated under reduced pressure and purified by preparative liquid phase separation using acetonitrile / water. Compound DL-14 was obtained (white solid, 7.8 mg, 38.3% yield). MS: 689.56 [M+Na+H] 2+ .

[0330] 1H NMR(400MHz,DMSO-d6)δ9.57(s,1H),8.44(m,1H),8.29–8.17(m,2H),8.10(m,2H),8.07–7.92(m,3H),7.30–7.16(m,10H), 7.13(m,1H),6.99(s,2H),4.65(m,1H),4.62–4.45(m,5H),3.79–3.69(m,6H),3.69–3.46(m,8H),3.43–3.33(m,5H),3.33–3 .14(m,9H),3.10–2.97(m,5H),2.85–2.67(m,9H),2.33–2.19(m,3H),2.15–1.98(m,4H),1.90–1.70(m,0H),1.65(m,3H),1 .48(m,6H),1.35–1.27(m,2H),1.19(m,3H),1.03(m,2H),1.00–0.85(m,17H),0.85–0.71(m,6H),0.65(m,2H),0.54(m,2H).

[0331] Example 15: Preparation of Compound DL-15

[0332] Step 1: Synthesis of tert-butyl (1-(9H-fluoren-9-yl)-3,6-dioxo-2,9,12-trioxo-4,7-diazatetradec-14-yl)(methyl)carbamate

[0333] Compound 16 (67 mg, 0.1825 mmol) and compound 50 (100 mg, 0.456 mmol) were placed in a 10 mL single-necked vial. Dichloromethane (3 mL) and TsOH (2.8 mg, 0.0163 mmol) were added and reacted at room temperature for 2 h. LCMS confirmed the complete reaction of compound 16. After spin drying, the crude product, compound 51, was obtained and used directly as the starting material for the next step. MS: 528.35 [M+H] +

[0334] Step 2: Synthesis of (9H-fluoren-9-yl)methyl (11-oxo-5,8-dioxo-2,10-diazadodec-12-yl)carbamate

[0335] Crude compound 51 was added to 5 mL of 4M HCl / dioxane solution and stirred at room temperature for 20 minutes. LCMS showed that compound 51 was completely consumed in the reaction solution. The solvent was evaporated under reduced pressure, and the crude product was purified by preparative liquid phase separation using acetonitrile / water to obtain compound 52 (17 mg, 97.2% yield). MS: 428.29 [M+H] +

[0336] Step 3: (9H-fluoren-9-yl)methyl((3R,4R,7R)-7-benzyl-3-((S)-1-(3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-4,9-dimethyl-5,8,18-trioxy-2,12,15-trioxy-6,9,17-triazapentadecan-19-yl)carbamate

[0337] Compound 52 (17 mg, 0.039 mmol), HATU (17.78 mg, 0.0468 mmol), and compound 1 (32 mg, 0.0429 mmol) were added to N,N-dimethylformamide (3 mL) and stirred at room temperature for 10 minutes. Finally, DIEA (25.155 mg, 0.195 mmol) was added and stirred at room temperature for 1 hour. The reaction was considered complete when LCMS showed less than 3% of compound 52 in the reaction solution. The solvent was evaporated under reduced pressure, and the crude product was purified by preparative liquid phase separation using acetonitrile / water. Compound 53 (32 mg, 71.0% yield) was obtained. MS: 1155.99 [M+H] +

[0338] Step 4: Synthesis of (S)-N-((3R,4S,5S)-1-((S)-2-((3R,4R,7R)-19-amino-7-benzyl-4,9-dimethyl-5,8,18-trioxy-2,12,15-trioxa-6,9,17-triazapentadecan-3-yl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamide

[0339] Compound 53 (32 mg, 0.0277 mmol) was placed in a 25 mL single-necked flask, and N,N-dimethylformamide (3 mL) and morpholine (2 mL) were added. The reaction was allowed to proceed at room temperature for 30 min. LCMS confirmed the complete reaction of compound 53. The reaction solution was dried by spin drying, and the crude product was purified by preparative liquid phase separation using acetonitrile / water. Compound 54 was obtained as a white solid, 16.5 mg, yield 63.89%. MS: 933.82 [M+H] +

[0340] Step 5: Synthesis of N-((3S,4S,7S,22R)-7,22-dibenzyl-3-((R)-1-((3S,4R,5R)-4-((R,(R)-2-(dimethylamino)-3-methylbutanamide)-3-methylbutanamide)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-4,9-dimethyl-5,8,18,21,24,27-hexaoxo-2,12,15-trioxo-6,9,17,20,23,26-hexaazaoctadecane-28-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide

[0341] Compound 31 (16.5 mg, 0.0177 mmol), HATU (8.11 mg, 0.0213 mmol), and compound 54 (9.23 mg, 0.0195 mmol) were added to N,N-dimethylformamide (3 mL), and finally DIEA (11.47 mg, 0.0889 mmol) was added. The reaction was stirred at room temperature for 4 hours. LCMS showed that the reaction solution contained less than 3% of compound 54, which was considered complete. The reaction solution was evaporated to dryness under reduced pressure, and the crude product was purified by preparative liquid phase separation using acetonitrile / water. Compound DL-15 was obtained (white solid, 13 mg, 10.4% yield). MS: 705.8 [M+H+Na] 2+

[0342] 1 H NMR(400MHz,DMSO-d6)δ9.51(s,1H),8.96–8.86(m,1H),8.54–8.38(m,2H),8.24(m,2H),8.08(m,2H),8.03–7.94(m,1H),7.28– 7.11(m,9H),6.98(m,2H),5.05(m,1H),4.89(m,1H),4.70(s,1H),4.66–4.57(m,2H),4.56–4.38(m,4H),3.71(m,8H),3.63–3.53 (m,3H),3.40–3.32(m,3H),3.31–3.13(m,8H),3.03(m,7H),2.92–2.86(m,1H),2.86–2.70(m,8H),2.30(m,4H),2.11(m,3H),2. 06–1.97(m,1H),1.75(m,4H),1.47(m,5H),1.32–1.24(m,2H),1.18(m,3H),1.07–0.91(m,11H),0.90–0.81(m,6H),0.77(m,3H).

[0343] Example 16: Preparation of Compound DL-16

[0344] Step 1: Synthesis of (9H-fluoren-9-yl)methyl (S)-(6,11,11-trimethyl-2,9-dioxo-5,10-dioxa-3,8-diazadodecyl)carbamate

[0345] Compound 16 (143 mg, 0.815 mmol), compound 55 (100 mg, 0.272 mmol), and TsOH (5 mg, 0.027 mmol) were added to dichloromethane (4 mL) and stirred at room temperature for 3 hours. LCMS showed that the reaction solution contained less than 3% of compound 16, which was considered complete. The reaction solution was evaporated to dryness under reduced pressure, and the crude product was purified by preparative liquid phase separation using acetonitrile / water. Compound 56 (white solid, 86 mg, 65.4% yield) was obtained. MS: 484.57 [M+H] +

[0346] Step 2: Synthesis of (9H-fluoren-9-yl)methyl (S)-(2-((((1-aminopropan-2-yl)oxy)methyl)amino)-2-oxoethyl)carbamate

[0347] Compound 56 (86 mg, 0.178 mmol) was placed in a 25 mL single-necked flask, and dichloromethane (4 mL) and trifluoroacetic acid (2 mL) were added. The reaction was allowed to proceed at room temperature for 1 h. LCMS confirmed the complete reaction of compound 56. The crude product was spin-dried to dryness and purified by preparative liquid phase separation using acetonitrile / water to obtain compound 57 (50 mg, 73.3% yield). MS: 384.45 [M+H] +

[0348] Step 3: Synthesis of (9H-fluoren-9-yl)methyl((3R,4R,7S,11S)-7-benzyl-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-4,11-dimethyl-5,8,15-trioxy-2,12-dioxa-6,9,14-triazahexadec-16-yl)carbamate

[0349] Compound 1 (78 mg, 0.104 mmol), HOBT (24 mg, 0.177 mmol), and EDCI (30 mg, 0.156 mmol) were added to dichloromethane (5 mL) and stirred at room temperature for 10 minutes. Finally, compound 57 (40 mg, 0.104 mmol) was added and stirred at room temperature for 3 hours. The reaction was considered complete when LCMS showed less than 3% of compound 57 in the reaction solution. The solvent was evaporated under reduced pressure, and the crude product was purified by preparative liquid phase separation using acetonitrile / water. Compound 58 (39 mg, 32.8% yield) was obtained. MS: 1112.44 [M+H] +

[0350] Step 4: Synthesis of (S)-N-((3R,4S,5S)-1-((S)-2-((3R,4R,7S,11S)-16-amino-7-benzyl-4,11-dimethyl-5,8,15-trioxo-2,12-dioxa-6,9,14-triazahexadec-3-yl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamide

[0351] Compound 58 (39 mg, 0.035 mmol) was placed in a 25 mL single-necked flask, and N,N-dimethylformamide (3 mL) and morpholine (1 mL) were added. The reaction was allowed to proceed at room temperature for 30 min. LCMS monitored the complete reaction of compound 58. The reaction solution was spin-dried, and the crude product was purified by preparative liquid phase separation using acetonitrile / water. Compound 59 was obtained as a white solid, 26 mg, yield 83.5%. MS: 890.19 [M+H] +

[0352] Step 5: Synthesis of N-((3R,4R,7S,11S,19S)-7,19-dibenzyl-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-4,11-dimethyl-5,8,15,18,21,24-hexaoxo-2,12-dioxa-6,9,14,17,20,23-hexaazacyclopentan-25-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide

[0353] Compound 31 (14 mg, 0.029 mmol) and HATU (13 mg, 0.035 mmol) were added to N,N-dimethylformamide (3 mL) and stirred at room temperature for 10 minutes. Compound 59 (26 mg, 0.029 mmol) and DIEA (15 mg, 0.116 mmol) were then added and stirred at room temperature for 3 hours. The reaction was considered complete when LCMS showed less than 3% of compound 59 in the reaction solution. The solvent was evaporated under reduced pressure, and the crude product was purified by preparative liquid phase separation using acetonitrile / water. Freeze-dried to obtain compound DL-16 (19 mg, 48.7% yield). MS: 1344.68 [M+H] +

[0354] 1 H NMR(400MHz,DMSO-d6)δ9.54(s,1H),8.88(m,1H),8.45m,1H),8.31–8.24(m,1H),8.13–8.05(m,2H),7.99(m ,1H),7.27–7.21(m,8H),7.19(m,2H),6.99(s,2H),4.71–4.44(m,6H),3.84–3.64(m,10H),3.29–3.12(m,8H) ,3.09–2.95(m,6H),2.77m,8H),2.37–2.21(m,3H),2.11(t,J=7.4Hz,3H),2.04–1.95(m,2H),1.91–1.73(m, 3H),1.63(m,1H),1.47(m,5H),1.32–1.14(m,8H),1.07–0.90(m,13H),0.90–0.81(m,9H),0.80–0.72(m,3H).

[0355] Example 17: Preparation of Compound DL-17

[0356] Step 1: Synthesis of (9H-fluoren-9-yl)methyl (2-oxo-5,8,11,14-tetraoxa-3-azahexadecane-1,16-diyl) dicarbamate

[0357] Compound 16 (200.0 mg, 0.543 mmol), compound 60 (637.1 mg, 2.172 mmol) and p-toluenesulfonic acid (18.7 mg, 0.109 mmol) were added to dichloromethane (3 mL) and stirred at room temperature for 2 hours. LCMS showed that the reaction solution contained less than 3% of compound 16, which was considered complete. The reaction solution was diluted 2-fold and washed with water (15 mL) and saturated brine (15 mL) in sequence. The organic phases were combined and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and purified by preparative liquid separation using acetonitrile / water. Compound 61 (white solid, 163.3 mg, 50.0% yield) was obtained. MS: 602.71 [M+H] + .

[0358] Step 2: Synthesis of (9H-fluoren-9-yl)methyl (16-amino-2-oxo-5,8,11,14-tetraoxa-3-azahexadecyl)carbamate

[0359] Compound 61 (163.3 mg, 0.271 mmol) was added to dichloromethane (2 mL) and TFA (0.5 ml) and reacted at 4°C for 1 hour. LCMS monitored the complete reaction of compound 61. The reaction solution was distilled under reduced pressure to remove the solvent, and the product was purified by preparative liquid phase separation using acetonitrile / water. Compound 62 (white solid, 92.2 mg, yield 67.7%) was obtained. MS: 502.64 [M+H] + .

[0360] Step 3: Synthesis of (9H-fluoren-9-yl)methyl((3R,4R,7S)-7-benzyl-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-4-methyl-5,8,24-trioxo-2,12,15,18,21-pentaoxa-6,9,23-triazapentaicosan-25-yl)carbamate

[0361] Compound 1 (86.9 mg, 0.184 mmol), HOBT (49.7 mg, 0.368 mmol), EDCI (70.5 mg, 0.368 mmol), and compound 62 (92.2 mg, 0.184 mmol) were added to N,N-dimethylformamide (3 mL) and stirred at room temperature for 1 hour. LCMS showed that the reaction solution contained less than 3% of compound 62, which was considered complete. The reaction solution was diluted 2-fold and washed with water (15 mL) and saturated brine (15 mL) in sequence. The organic phases were extracted twice and combined, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The product was purified by preparative liquid separation using acetonitrile / water. Compound 63 (white solid, 55.3 mg, yield 24.5%) was obtained. MS: 626.58 [M+Na+H] 2+ .

[0362] Step 4: Synthesis of (S)-N-((3R,4S,5S)-1-((S)-2-((3R,4R,7S)-25-amino-7-benzyl-4-methyl-5,8,24-trioxo-2,12,15,18,21-pentaoxa-6,9,23-triazapentaicostan-3-yl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamide)-N,3-dimethylbutanamide

[0363] Compound 63 (55.3 mg, 0.0450 mmol) was added to N,N-dimethylformamide (2 mL) and triethylamine (1 mL) and allowed to react at room temperature for 3 hours. LCMS monitored the complete reaction of compound 63. The reaction solution was distilled under reduced pressure to remove the solvent, and then purified by preparative liquid phase separation using acetonitrile / water to obtain compound 64 (white solid, 40.2 mg, 88.7% yield).

[0364] Step 5: Synthesis of N-((3R,4R,7S,28S)-7,28-dibenzyl-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-4-methyl-5,8,24,27,30,33-hexaoxo-2,12,15,18,21-pentaoxa-6,9,23,26,29,32-hexaazatetratrioxan-34-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide

[0365] Compound 31 (18.8 mg, 0.0397 mmol), HOBT (10.7 mg, 0.0794 mmol), EDCI (15.2 mg, 0.0794 mmol), and compound 64 (40.2 mg, 0.0397 mmol) were added to N,N-dimethylformamide (3 mL) and stirred at room temperature for 1 hour. LCMS showed that the reaction solution contained less than 3% of compound 64, which was considered complete. The reaction solution was diluted 2-fold and washed with water (15 mL) and saturated brine (15 mL) in sequence. The organic phases were extracted twice, combined, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The product was purified by preparative liquid separation using acetonitrile / water. Compound DL-17 (white solid, 19.1 mg, 32.7% yield) was obtained. MS: 742.48 [M+Na+H] 2+ .

[0366] 1 H NMR(400MHz,DMSO-d6)δ8.47(d,1H),8.31–8.24(m,2H),8.11(d,1H),8.08–8.02(m,3H),8.00(d,2H),7.96(t,1H),7.2 9–7.13(m,10H),7.00(d,2H),4.67(d,3H),4.58–4.48(m,4H),3.98(s,2H),3.78(t,2H),3.70(dd,5H),3.62(d,1H),3. 50(dd,11H),3.23–3.16(m,8H),3.08(s,2H),3.05(d,2H),3.00(s,3H),2.70–2.62(m,3H),2.36–2.31(m,2H),2.21(d, 6H),2.12(t,3H),1.92(s,4H),1.75(s,4H),1.48(q,6H),1.21(q,4H),1.04(d,2H),1.00–0.81(m,14H),0.74(dt,6H).

[0367] Example 18: Preparation of Compound DL-18

[0368] Step 1: Synthesis of tert-butyl (1-(10-(9H-fluoren-9-yl)-5,8-dioxo-2,9-dioxo-4,7-diazadecyl)cyclobutyl)carbamate

[0369] Compound 16 (50 mg, 0.136 mmol), compound 65 (80 mg, 0.398 mmol), and TsOH (5 mg, 0.027 mmol) were added to dichloromethane (4 mL) and stirred at room temperature for 3 hours. The reaction was considered complete when LCMS showed less than 3% of compound 65 in the reaction solution. The solvent was evaporated under reduced pressure, and the crude product was purified by preparative liquid phase separation using acetonitrile / water. Compound 66 (white solid, 22 mg, 31.7% yield) was obtained. MS: 510.60 [M+H] + .

[0370] Step 2: Synthesis of (9H-fluoren-9-yl)methyl (2-((((1-aminocyclobutyl)methoxy)methyl)amino)-2-oxoethyl)carbamate

[0371] Compound 66 (22 mg, 0.043 mmol) was placed in a 25 mL single-necked flask, and dichloromethane (4 mL) and trifluoroacetic acid (2 mL) were added. The reaction was allowed to proceed at room temperature for 1 h. LCMS confirmed the complete reaction of compound 66. The crude product was spin-dried to dryness and purified by preparative liquid phase separation using acetonitrile / water to obtain compound 67 (10 mg, 56.7% yield). MS: 410.49 [M+H] + .

[0372] Step 3: Synthesis of (9H-fluoren-9-yl)methyl(2-((1-(S)-2-((2R,3R)-3-(S)-1-((3R,4S,5S)-4-(S)-2-((S)-2(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methyl-2-methylpropionamido)3-phenylpropionamido(cyclobutyl)methoxy)methyl)amino)-2-oxoethyl)carbamate

[0373] Compound 1 (14 mg, 0.018 mmol) and HATU (8.2 mg, 0.022 mmol) were added to dichloromethane (5 mL) and stirred at room temperature for 10 minutes. Compound 67 (7.5 mg, 0.018 mmol) and DIEA (9.3 mg, 0.072 mmol) were then added and stirred at room temperature for 3 hours. The reaction was considered complete when LCMS showed less than 3% of compound 67 in the reaction solution. The solvent was evaporated under reduced pressure, and the crude product was purified by preparative liquid phase separation using acetonitrile / water. Compound 68 (10 mg, 48.9% yield) was obtained. MS: 1138.47 [M+H] + .

[0374] Step 4: Synthesis of (S)-N-((3R,4S,5S)-1-(S)-2-((1R,2R)-3-((S)-1-(((2-aminoacetamido)methoxy)methyl)cyclobutyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamide)-N,3-dimethylbutanamide

[0375] Compound 68 (10 mg, 0.035 mmol) was placed in a 25 mL single-necked vial. N,N-dimethylformamide (3 mL) and TEA (1 mL) were added and allowed to react at room temperature for 3 h. LCMS monitoring confirmed the complete reaction of compound 68. The reaction solution was then dried to give crude compound 69. MS: 916.23 [M+H] + .

[0376] Step 5: Synthesis of N-((S)-9-benzyl-1-(1-(S)-2-((2R,3R)-3-(S)-1-(3R,4S,5S)-4-(S)-2-(S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropylamino)-3-phenylpropanamido)cyclobutyl)-5,8,11,14-tetraoxo-2-oxa-4,7,10,13-tetraazapentadecan-15-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide

[0377] Compound 31 (4 mg, 0.0088 mmol) and HATU (4 mg, 0.0106 mmol) were added to N,N-dimethylformamide (3 mL) and stirred at room temperature for 10 minutes. Compound 69 (0.0088 mmol) and DIEA (4.5 mg, 0.0352 mmol) were then added and stirred at room temperature for 3 hours. The reaction was considered complete when LCMS showed less than 3% of compound 69 in the reaction solution. The solvent was evaporated under reduced pressure, and the crude product was purified by preparative liquid phase separation using acetonitrile / water. Freeze-dried to obtain compound DL-18 (2.7 mg, 22.5% yield). MS: 1370.71 [M+H] + .

[0378] 1H NMR(400MHz,DMSO)δ8.49(d,1H),8.33–8.20(m,1H),8.04(ddd,4H),7.87(s,1H),7.38–7.08(m,10H),6.98(s,1H),4.66(d,1H),4.64 –4.57(m,1H),4.57–4.44(m,4H),4.33(d,1H),4.28–4.19(m,1H),4.06(dd,1H),3.96(s,2H),3.82–3.68(m,5H),3.66(d,2H),3.61(d ,1H),3.55(dd,4H),3.46(s,4H),3.18(dd,7H),3.05(dd,2H),2.98(s,2H),2.84–2.70(m,1H),2.67–2.60(m,1H),2.32(dd,1H),2.19 (d,7H),2.09(dd,3H),2.01(dd,3H),1.74(s,5H),1.46(dd,4H),1.33–1.11(m,4H),1.01(dd,3H),0.95–0.80(m,14H),0.72(dt,6H).

[0379] Example 19: Preparation of DL-19

[0380] Step 1: Synthesis of tert-butyl (1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundec-11-yl)carbamate

[0381] Compound 16 (200.0 mg, 0.543 mmol), compound 70 (350.1 mg, 2.172 mmol) and p-toluenesulfonic acid (18.7 mg, 0.109 mmol) were added to dichloromethane (3 mL) and stirred at room temperature for 2 hours. LCMS showed that the reaction solution contained less than 3% of compound 16, which was considered complete. The reaction solution was diluted 2-fold and washed with water (15 mL) and saturated brine (15 mL) in sequence. The organic phases were combined and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and purified by preparative liquid separation using acetonitrile / water. Compound 71 (white solid, 115.2 mg, yield 45.2%) was prepared. MS: 470.46 [M+H] + .

[0382] Step 2: Synthesis of (9H-fluoren-9-yl)methyl (2-(((2-aminoethoxy)methyl)amino)-2-oxoethyl)carbamate

[0383] Compound 71 (115.2 mg, 0.245 mmol) was added to dichloromethane (2 mL) and TFA (0.5 ml) and allowed to react at room temperature for 30 minutes. LCMS monitored the complete reaction of compound 71. The reaction solution was distilled under reduced pressure to remove the solvent, and then purified by preparative liquid phase separation using acetonitrile / water. Compound 72 (white solid, 60.3 mg, yield 66.5%) was obtained. MS: 370.48 [M+H] + .

[0384] Step 3: Synthesis of (9H-fluoren-9-yl)methyl((3R,4R,7S)-7-benzyl-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-4-methyl-5,8,15-trioxo-2,12-dioxa-6,9,14-triazahexadec-16-yl)carbamate

[0385] Compound 1 (121.8 mg, 0.163 mmol), HOBT (44.1 mg, 0.327 mmol), EDCI (62.6 mg, 0.327 mmol), and compound 72 (60.3 mg, 0.163 mmol) were added to N,N-dimethylformamide (3 mL) and stirred at room temperature for 1 hour. LCMS showed that the reaction solution contained less than 3% of compound 72, which was considered complete. The reaction solution was diluted 2-fold and washed with water (15 mL) and saturated brine (15 mL) in sequence. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The product was purified by preparative liquid separation using acetonitrile / water. Compound 73 (white solid, 47.8 mg, yield 26.7%) was obtained. MS: 1098.62 [M+H] + .

[0386] Step 4: Synthesis of (S)-N-((3R,4S,5S)-1-((S)-2-((3R,4R,7S)-16-amino-7-benzyl-4-methyl-5,8,15-trioxo-2,12-dioxa-6,9,14-triazahexadec-3-yl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheterocyclopentane-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamide)-N,3-dimethylbutanamide

[0387] Compound 73 (47.8 mg, 0.0436 mmol) was added to N,N-dimethylformamide (2 mL) and triethylamine (1 mL) and allowed to react at room temperature for 3 hours. LCMS monitored the complete reaction of compound 73. The reaction solution was distilled under reduced pressure to remove the solvent and purified by preparative liquid phase separation using acetonitrile / water. Compound 74 (white solid, 37.0 mg, yield 97.1%) was obtained. MS: 876.24 [M+H] + .

[0388] Step 5: Synthesis of N-((3R,4R,7S,19S)-7,19-dibenzyl-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-4-methyl-5,8,15,18,21,24-hexaoxa-2,12-dioxa-6,9,14,17,20,23-hexaazapentaicosan-25-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide

[0389] Compound 31 (20.0 mg, 0.0846 mmol), HOBT (11.4 mg, 0.0846 mmol), EDCI (16.2 mg, 0.0846 mmol), and compound 74 (37.0 mg, 0.0423 mmol) were added to N,N-dimethylformamide (3 mL) and stirred at room temperature for 1 hour. LCMS showed that the reaction solution contained less than 3% of compound 74, which was considered complete. The reaction solution was diluted 2-fold and washed with water (15 mL) and saturated brine (15 mL) in sequence. The organic phases were extracted twice and combined, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The product was purified by preparative liquid separation using acetonitrile / water. Compound DL-19 was prepared (white solid, 17.2 mg, yield 30.6%). MS: MS: 676.46 [M+Na+H] 2+ .

[0390] 1H NMR(400MHz,DMSO-d6)δ8.27(dd,2H),8.15–7.94(m,6H),7.31–7.08(m,10H),7.00(d,2H),4.76–4.60(m,3H),4.54(q,5H),3.70(dd,6H),3.24–3.1 9(m,5H),3.18(s,2H),3.08(s,3H),3.00(s,2H),2.21(d,5H),2.12(t,3H ),1.77(s,4H),1.48(h,7H),1.30(s,2H),1.21(q,4H),1.04(d,2H),1.01– 0.81(m,12H),0.74(dt,6H).

[0391] Example 20: Preparation of DL-20

[0392] Step 1: Synthesis of (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)glycerylglyceryl-L-phenylalanine

[0393] Compound 75 (20 mg, 0.075 mmol) and HATU (28 mg, 0.075 mmol) were added to N,N-dimethylformamide (3 mL) and stirred at room temperature for 10 minutes. Compound 76 (21 mg, 0.075 mmol) and DIEA (38 mg, 0.3 mmol) were then added and stirred at room temperature for 3 hours. The reaction was considered complete when LCMS showed less than 3% of compound 75 in the reaction solution. The solvent was evaporated under reduced pressure, and the crude product was purified by preparative liquid phase separation using acetonitrile / water. Compound 77 (14 mg, 35.2% yield) was obtained. MS: 530.57 [M+H] + .

[0394] Step 2: Synthesis of N-(3R,4R,7S,22S)-7,22-dibenzyl-3-(S)-1-(3R,4S,5S)-4-(S)-2-(S)-2-(dimethylamino)-3-methylbutanamide)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-4-methyl-5,8,18,21,24,27-hexaoxo-2,12,15-trioxa-6,9,17,20,23,26-hexaazaoctacosan-28-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexane-5-imide

[0395] Compound 77 (5 mg, 0.009 mmol), HOBT (1.8 mg, 0.014 mmol), and EDCI (2.6 mg, 0.014 mmol) were added to N,N-dimethylformamide (3 mL) and stirred at room temperature for 3 minutes. Compound 18 (8.7 mg, 0.009 mmol) was then added and stirred at room temperature for 3 hours. The reaction was considered complete when LCMS showed less than 3% of compound 18 in the reaction solution. The solvent was evaporated under reduced pressure, and the crude product was purified by preparative liquid phase separation using acetonitrile / water. Freeze-dried to obtain compound DL-20 (5.7 mg, 44.3% yield). MS: 1431.77 [M+H] + .

[0396] 1H NMR(400MHz,DMSO)δ9.10(s,2H),8.55–7.92(m,9H),7.30–7.10(m,11H),4.66(dd,1H),4.58–4.47(m, 4H),3.97(s,1H),3.80–3.68(m,6H),3.60(dd,1H),3.49(d,7H),3.26–3.14(m,11H),3.04(d,6H),2.8 5–2.71(m,2H),2.69–2.60(m,1H),2.58(t,2H),2.33(dd,3H),2.19(t,7H),1.96–1.88(m,2H),1.83(d t,3H),1.77–1.73(m,1H),1.46(dd,1H),1.24(s,1H),1.04(d,1H),1.00–0.81(m,17H),0.74(dt,7H).

[0397] Example 21: Preparation of DL-21

[0398] Step 1: Synthesis of (1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-oxo-7,10,13,16,19,22,25,28-octaoxa-4-azatrioxan-31-oleyl)glycylglycyl-L-phenylalanine

[0399] Compound 10 (300 mg, 0.507 mmol), NHS (90 mg, 0.783 mmol), and EDCI (150 mg, 0.781 mmol) were added to water (10 mL) and stirred at room temperature for 30 minutes. EDCI (200 mg, 0.781 mmol) was added and the reaction was continued at room temperature for 30 minutes. Compound 76 (300 mg, 0.717 mmol) and DIEA (74.2 mg, 0.575 mmol) were added and the reaction was continued at room temperature for 30 minutes. The reaction was considered complete when LCMS showed that the reaction solution contained less than 3% of compound 10. The solvent was evaporated under reduced pressure, and the crude product was purified by preparative liquid phase separation using acetonitrile / water. Compound 78 (white solid, 150 mg, 34.6% yield) was obtained. MS: 853.40 [M+H] +

[0400] Step 2: N-((3R,4R,7S,20S)-7,20-dibenzyl-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-4,10,10-trimethyl Synthesis of 1-(5,8,16,19,22,25-hexaoxo-2,13-dioxa-6,9,15,18,21,24-hexaazacosano-26-yl)-1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propionamido)-3,6,9,12,15,18,21,24-octaoxadocosano-27-amide

[0401] Compound 78 (22.4 mg, 0.026 mmol) and HATU (12.4 mg, 0.033 mmol) were added to N,N-dimethylformamide (3 mL) and stirred at room temperature for 10 minutes. Compound 44 (20 mg, 0.022 mmol) and DIEA (3.7 mg, 0.028 mmol) were then added and stirred at room temperature for 3 hours. The reaction was considered complete when LCMS showed less than 3% of compound 44 in the reaction solution. The solvent was evaporated under reduced pressure, and the crude product was purified by preparative liquid phase separation using acetonitrile / water. Freeze-dried to obtain compound DL-21 (5.4 mg, 14.0% yield). MS: 1752.01 [M+H] +

[0402] 1H NMR (400MHz, DMSO) δ8.48(m,1H),8.30(m,1H),8.16(m,1H),8.13–8.06(m,1H),7.98(m,2H),7.29–7.18(m,5H),7.15(m,1H),7.0 1(s,1H),4.71–4.57(m,2H),4.57–4.41(m,3H),3.79–3.66(m,3H),3.63–3.58(m,2H),3.53–3.47(m,14H),3.37–3.27(m,53H),3 .24–3.13(m,6H),3.11–2.91(m,5H),2.85–2.71(m,2H),2.70–2.60(m,2H),2.40(m,2H),2.36–2.29(m,2H),2.21(m,5H),2.01–1 .81(m,4H),1.76(s,2H),1.63(m,1H),1.46(s,1H),1.32(s,2H),1.26–1.17(m,4H),1.04(m,2H),0.97–0.82(m,8H),0.74(m,4H).

[0403] Example 22: Preparation of ADC-1

[0404] To a PBS buffer solution of trastuzumab (0.05 M PBS aqueous solution at pH 7.0; 2 mg, 0.014 μmol) was added a prepared aqueous solution of the reducing agent tris(2-carboxyethyl)phosphine (4-10 eq). Under inert gas protection, the mixture was placed in a water bath or metal bath and shaken at 25-37° C. for 0.5-2.0 hours. After stopping the reaction, the reaction solution was cooled to room temperature for use in the coupling reaction in the subsequent ADC preparation examples.

[0405] An aqueous solution of compound DL-1 (0.106 mL, 4.0 mg / mL, 0.2 μmol) was added to the above 0.208 mL of the antibody reaction solution, the mixture was aerated with Ar gas, and the mixture was placed on a shaker for 1.5 hours at 25°C. The reaction was then stopped. An aqueous solution of compound NAC (0.004 mL, 100 mM, 0.4 μmol) was added to the reaction solution, the mixture was aerated with Ar gas, and the mixture was agitated at 25°C for 0.3 hours. The reaction was then stopped. The reaction solution was ultrafiltered using an Amicon Ultra-15 30 kD ultrafiltration tube with 0.05 M PBS buffer (pH 7.0) to obtain a PBS buffer solution of the conjugated product ADC-1 (4.32 mg / mL, 0.43 mL), which was stored frozen at -80°C.

[0406] The average DAR value (Drug-to-Antibody Ratio) calculated by the reduction-reverse phase chromatography method was n=6.23.

[0407] Example 23: Preparation of ADC-2

[0408] An aqueous solution of compound DL-2 (0.091 mL, 4.0 mg / mL, 0.2 μmol) was added to the above 0.208 mL antibody reaction solution, the mixture was aerated with Ar gas, and the mixture was placed on a shaker for 1.5 hours at 25°C. The reaction was then stopped. An aqueous solution of compound NAC (0.004 mL, 100 mM, 0.4 μmol) was added to the reaction solution, the mixture was aerated with Ar gas, and the mixture was agitated at 25°C for 0.3 hours. The reaction was then stopped. The reaction solution was ultrafiltered using an Amicon Ultra-15 30 kD ultrafiltration tube with 0.05 M PBS buffer (pH 7.0) to obtain a PBS buffer solution of the conjugated product ADC-2 (4.54 mg / mL, 0.4 mL), which was stored frozen at -80°C.

[0409] The average DAR value calculated by reduction-reverse phase chromatography method was n=7.22.

[0410] Example 24: Preparation of ADC-3

[0411] A 10% DMF / water solution of compound DL-3 (0.091 mL, 4.0 mg / mL, 0.2 μmol) was added to the above 0.208 mL antibody reaction solution, purged with Ar gas, and placed on a shaker for 1.5 hours at 25°C. The reaction was then stopped. An aqueous solution of compound NAC (0.004 mL, 100 mM, 0.4 μmol) was added to the reaction solution, purged with Ar gas, and placed on a shaker for 0.3 hours at 25°C. The reaction was then stopped. The reaction solution was ultrafiltered using an Amicon Ultra-15 30 kD ultrafiltration tube with 0.05 M PBS buffer (pH 7.0) to obtain a PBS buffer solution of the conjugated product ADC-3 (5.7 mg / mL, 0.32 mL), which was stored frozen at -80°C.

[0412] The average DAR value calculated by reduction-reverse phase chromatography method was n=6.38.

[0413] Example 25: Preparation of ADC-4

[0414] An aqueous solution of compound DL-4 (0.088 mL, 4.0 mg / mL, 0.2 μmol) was added to the above 0.208 mL antibody reaction solution, the mixture was aerated with Ar gas, and the mixture was placed on a shaker for 1.5 hours at 25°C. The reaction was then stopped. An aqueous solution of compound NAC (0.004 mL, 100 mM, 0.4 μmol) was added to the reaction solution, the mixture was aerated with Ar gas, and the mixture was agitated at 25°C for 0.3 hours. The reaction was then stopped. The reaction solution was ultrafiltered using an Amicon Ultra-15 30 kD ultrafiltration tube with 0.05 M PBS buffer (pH 7.0) to obtain a PBS buffer solution of the conjugated product ADC-4 (5.0 mg / mL, 0.37 mL), which was stored frozen at -80°C.

[0415] The average DAR value calculated by reduction-reverse phase chromatography method was n=6.06.

[0416] Example 26: Preparation of ADC-5

[0417] An aqueous solution of compound DL-5 (0.103 mL, 4.0 mg / mL, 0.2 μmol) was added to the above 0.208 mL of the antibody reaction solution, the mixture was aerated with Ar gas, and the mixture was placed on a shaker for 1.5 hours at 25°C. The reaction was then stopped. An aqueous solution of compound NAC (0.004 mL, 100 mM, 0.4 μmol) was added to the reaction solution, the mixture was aerated with Ar gas, and the mixture was agitated at 25°C for 0.3 hours. The reaction was then stopped. The reaction solution was ultrafiltered using an Amicon Ultra-15 30 kD ultrafiltration tube with 0.05 M PBS buffer (pH 7.0) to obtain a PBS buffer solution of the conjugated product ADC-5 (4.88 mg / mL, 0.38 mL), which was stored frozen at -80°C.

[0418] The average DAR value calculated by reduction-reverse phase chromatography method was n=7.35.

[0419] Example 27: Preparation of ADC-6

[0420] An aqueous solution of compound DL-6 (0.0715 mL, 4.0 mg / mL, 0.2 μmol) was added to the above 0.208 mL of the antibody reaction solution, the mixture was aerated with Ar gas, and the mixture was placed on a shaker for 1.5 hours at 25°C. The reaction was then stopped. An aqueous solution of compound NAC (0.004 mL, 100 mM, 0.4 μmol) was added to the reaction solution, the mixture was aerated with Ar gas, and the mixture was agitated at 25°C for 0.3 hours. The reaction was then stopped. The reaction solution was ultrafiltered using an Amicon Ultra-15 30 kD ultrafiltration tube with 0.05 M PBS buffer (pH 7.0) to obtain a PBS buffer solution of the conjugated product ADC-6 (4.34 mg / mL, 0.43 mL), which was stored frozen at -80°C.

[0421] The average DAR value calculated by reduction-reverse phase chromatography method was n=7.38.

[0422] Example 28: Preparation of ADC-7

[0423] An aqueous solution of compound DL-7 (0.0953 mL, 3.0 mg / mL, 0.2 μmol) was added to the above 0.208 mL antibody reaction solution, purged with Ar gas, and placed in a shaker for 1.5 hours at 25°C. The reaction was then stopped. An aqueous solution of compound NAC (0.004 mL, 100 mM, 0.4 μmol) was added to the reaction solution, purged with Ar gas, and placed in a shaker for 0.3 hours at 25°C. The reaction was then stopped. The reaction solution was ultrafiltered using an Amicon Ultra-15 30 kD ultrafiltration tube with 0.05 M PBS buffer (pH 7.0) to obtain a PBS buffer solution of the conjugated product ADC-7 (5.09 mg / mL, 0.36 mL), which was stored frozen at -80°C.

[0424] The average DAR value calculated by reduction-reverse phase chromatography method was n=7.71.

[0425] Example 29: Preparation of ADC-8

[0426] An aqueous solution of compound DL-8 (0.0905 mL, 4.0 mg / mL, 0.2 μmol) was added to the above 0.208 mL of the antibody reaction solution, the mixture was aerated with Ar gas, and the mixture was placed on a shaker for 1.5 hours at 25°C. The reaction was then stopped. An aqueous solution of compound NAC (0.004 mL, 100 mM, 0.4 μmol) was added to the reaction solution, the mixture was aerated with Ar gas, and the mixture was agitated at 25°C for 0.3 hours. The reaction was then stopped. The reaction solution was ultrafiltered using an Amicon Ultra-15 30 kD ultrafiltration tube with 0.05 M PBS buffer (pH 7.0) to obtain a PBS buffer solution of the conjugated product ADC-8 (3.69 mg / mL, 0.475 mL), which was stored frozen at -80°C.

[0427] The average DAR value calculated by reduction-reverse phase chromatography method was n=7.35.

[0428] Example 30: Preparation of ADC-9

[0429] A 20% DMF / water solution of compound DL-9 (0.074 mL, 4.0 mg / mL, 0.2 μmol) was added to the above 0.208 mL of the antibody reaction solution, purged with Ar gas, placed in a shaker, and allowed to react at 25°C for 1.5 hours. The reaction was then stopped. An aqueous solution of compound NAC (0.004 mL, 100 mM, 0.4 μmol) was added to the reaction solution, purged with Ar gas, placed in a shaker, and allowed to react at 25°C for 0.3 hours. The reaction was then stopped. The reaction solution was ultrafiltered using an Amicon Ultra-15 30 kD ultrafiltration tube using 0.05 M PBS buffer (pH 7.0) to obtain a PBS buffer solution of compound ADC-9 (4.56 mg / mL, 0.4 mL), which was stored frozen at -80°C.

[0430] The average DAR value calculated by reduction-reverse phase chromatography method was n=7.89.

[0431] Example 31: Preparation of ADC-10

[0432] A 20% DMF / water solution of compound DL-10 (0.071 mL, 4.0 mg / mL, 0.2 μmol) was added to the above 0.208 mL antibody reaction solution, purged with Ar gas, and placed on a shaker for 1.5 hours at 25°C. The reaction was then stopped. An aqueous solution of compound NAC (0.004 mL, 100 mM, 0.4 μmol) was added to the reaction solution, purged with Ar gas, and placed on a shaker for 0.3 hours at 25°C. The reaction was then stopped. The reaction solution was ultrafiltered using an Amicon Ultra-15 30 kD ultrafiltration tube with 0.05 M PBS buffer (pH 7.0) to obtain a PBS buffer solution of the conjugated product ADC-10 (3.1 mg / mL, 0.41 mL), which was stored frozen at -80°C.

[0433] The average DAR value calculated by reduction-reverse phase chromatography method was n=7.81.

[0434] Example 32: Preparation of ADC-11

[0435] A 20% DMF / water solution of compound DL-11 (0.069 mL, 4.0 mg / mL, 0.2 μmol) was added to the above 0.208 mL antibody reaction solution, purged with Ar gas, placed in a shaker, and allowed to react at 25°C for 1.5 hours. The reaction was then stopped. An aqueous solution of compound NAC (0.004 mL, 100 mM, 0.4 μmol) was added to the reaction solution, purged with Ar gas, placed in a shaker, and allowed to react at 25°C for 0.3 hours. The reaction was then stopped. The reaction solution was ultrafiltered using an Amicon Ultra-15 30 kD ultrafiltration tube with 0.05 M PBS buffer (pH = 7.0) to obtain a PBS buffer solution of the conjugated product ADC-11 (5.5 mg / mL, 0.336 mL), which was stored frozen at -80°C.

[0436] The average DAR value calculated by reduction-reverse phase chromatography method was n=6.2.

[0437] Example 33: Preparation of ADC-12

[0438] A 20% DMF / water solution of compound DL-12 (0.069 mL, 4.0 mg / mL, 0.2 μmol) was added to the above 0.208 mL antibody reaction solution, purged with Ar gas, placed in a shaker, and allowed to react at 25°C for 1.5 hours. The reaction was then stopped. An aqueous solution of compound NAC (0.004 mL, 100 mM, 0.4 μmol) was added to the reaction solution, purged with Ar gas, placed in a shaker, and allowed to react at 25°C for 0.3 hours. The reaction was then stopped. The reaction solution was ultrafiltered using an Amicon Ultra-15 30 kD ultrafiltration tube with 0.05 M PBS buffer (pH 7.0) to obtain a PBS buffer solution of the conjugated product ADC-12 (5.14 mg / mL, 0.356 mL), which was stored frozen at -80°C.

[0439] The average DAR value calculated by reduction-reverse phase chromatography method was n=6.5.

[0440] Example 34: Preparation of ADC-13

[0441] A 20% DMF / water solution of compound DL-13 (0.068 mL, 4.0 mg / mL, 0.2 μmol) was added to the above 0.208 mL antibody reaction solution, purged with Ar gas, and placed on a shaker for 1.5 hours at 25°C. The reaction was then stopped. An aqueous solution of compound NAC (0.004 mL, 100 mM, 0.4 μmol) was added to the reaction solution, purged with Ar gas, and placed on a shaker for 0.3 hours at 25°C. The reaction was then stopped. The reaction solution was ultrafiltered using an Amicon Ultra-15 30 kD ultrafiltration tube to obtain a PBS buffer solution of the conjugated product ADC-13 (4.55 mg / mL, 0.4 mL), which was stored frozen at -80°C.

[0442] The average DAR value calculated by reduction-reverse phase chromatography method was n=7.21.

[0443] Example 35: Preparation of ADC-14

[0444] A 20% DMF / water solution of compound DL-14 (0.068 mL, 4.0 mg / mL, 0.2 μmol) was added to the above 0.208 mL of the antibody reaction solution, purged with Ar gas, and placed on a shaker for 1.5 hours at 25°C. The reaction was then stopped. An aqueous solution of compound NAC (0.004 mL, 100 mM, 0.4 μmol) was added to the reaction solution, purged with Ar gas, and placed on a shaker for 0.3 hours at 25°C. The reaction was then stopped. The reaction solution was ultrafiltered using an Amicon Ultra-15 30 kD ultrafiltration tube to obtain a PBS buffer solution of the conjugated product ADC-14 (5.29 mg / mL, 0.35 mL), which was stored frozen at -80°C.

[0445] The average DAR value calculated by reduction-reverse phase chromatography method was n=7.62.

[0446] Example 36: Preparation of ADC-15

[0447] A 20% DMF / water solution of compound DL-15 (0.07 mL, 4.0 mg / mL, 0.2 μmol) was added to the above 0.208 mL of the antibody reaction solution, purged with Ar gas, and placed on a shaker for 1.5 hours at 25°C. The reaction was then stopped. An aqueous solution of compound NAC (0.004 mL, 100 mM, 0.4 μmol) was added to the reaction solution, purged with Ar gas, and placed on a shaker for 0.3 hours at 25°C. The reaction was then stopped. The reaction solution was ultrafiltered using an Amicon Ultra-15 30 kD ultrafiltration tube to obtain a PBS buffer solution of the conjugated product ADC-15 (5.66 mg / mL, 0.34 mL), which was stored frozen at -80°C.

[0448] The average DAR value calculated by reduction-reverse phase chromatography method was n=7.52.

[0449] Example 37: Preparation of ADC-16

[0450] A 20% DMF / water solution of compound DL-16 (0.07 mL, 4.0 mg / mL, 0.2 μmol) was added to the above 0.208 mL of the antibody reaction solution, and the mixture was aerated with Ar gas and placed on a shaker for 1.5 hours at 25°C. The reaction was then stopped. An aqueous solution of compound NAC (0.004 mL, 100 mM, 0.4 μmol) was added to the reaction solution, and the mixture was aerated with Ar gas and placed on a shaker for 0.3 hours at 25°C. The reaction was then stopped. The reaction solution was ultrafiltered using an Amicon Ultra-15 30 kD ultrafiltration tube to obtain a PBS buffer solution of the conjugated product ADC-16 (4.68 mg / mL, 0.4 mL), which was stored frozen at -80°C.

[0451] The average DAR value calculated by reduction-reverse phase chromatography method was n=7.13.

[0452] Example 38: Preparation of ADC-17

[0453] A 20% DMF / water solution of compound DL-17 (0.076 mL, 4.0 mg / mL, 0.2 μmol) was added to the above 0.208 mL of the antibody reaction solution, and the mixture was aerated with Ar gas and placed on a shaker for 1.5 hours at 25°C. The reaction was then stopped. An aqueous solution of compound NAC (0.004 mL, 100 mM, 0.4 μmol) was added to the reaction solution, aerated with Ar gas, and placed on a shaker for 0.3 hours at 25°C. The reaction was then stopped. The reaction solution was ultrafiltered using an Amicon Ultra-15 30 kD ultrafiltration tube to obtain a PBS buffer solution of the conjugated product ADC-17 (3.55 mg / mL, 0.55 mL), which was stored frozen at -80°C.

[0454] The average DAR value calculated by reduction-reverse phase chromatography method was n=6.30.

[0455] Example 39: Preparation of ADC-18

[0456] A 20% DMF / water solution of compound DL-18 (0.071 mL, 4.0 mg / mL, 0.2 μmol) was added to the above 0.208 mL of the antibody reaction solution, purged with Ar gas, and placed on a shaker for 1.5 hours at 25°C. The reaction was then stopped. An aqueous solution of compound NAC (0.004 mL, 100 mM, 0.4 μmol) was added to the reaction solution, purged with Ar gas, and placed on a shaker for 0.3 hours at 25°C. The reaction was then stopped. The reaction solution was ultrafiltered using an Amicon Ultra-15 30 kD ultrafiltration tube to obtain a PBS buffer solution of the conjugated product ADC-18 (3.24 mg / mL, 0.52 mL), which was stored frozen at -80°C.

[0457] The average DAR value calculated by reduction-reverse phase chromatography method was n=5.90.

[0458] Example 40: Preparation of ADC-19

[0459] A 20% DMF / water solution of compound DL-19 (0.074 mL, 4.0 mg / mL, 0.2 μmol) was added to the above 0.208 mL of the antibody reaction solution, purged with Ar gas, and placed on a shaker for 1.5 hours at 25°C. The reaction was then stopped. An aqueous solution of compound NAC (0.004 mL, 100 mM, 0.4 μmol) was added to the reaction solution, purged with Ar gas, and placed on a shaker for 0.3 hours at 25°C. The reaction was then stopped. The reaction solution was ultrafiltered through an Amicon Ultra-15 30 kD ultrafiltration tube to obtain a PBS buffer solution of the conjugated product ADC-19 (3.3 mg / mL, 0.5 mL), which was stored frozen at -80°C.

[0460] The average DAR value calculated by reduction-reverse phase chromatography method was n=6.70.

[0461] Example 41: Preparation of ADC-20

[0462] A 20% DMF / water solution of compound DL-20 (0.074 mL, 4.0 mg / mL, 0.2 μmol) was added to the above 0.208 mL of the antibody reaction solution, purged with Ar gas, and placed on a shaker for 1.5 hours at 25°C. The reaction was then stopped. An aqueous solution of compound NAC (0.004 mL, 100 mM, 0.4 μmol) was added to the reaction solution, purged with Ar gas, and placed on a shaker for 0.3 hours at 25°C. The reaction was then stopped. The reaction solution was ultrafiltered using an Amicon Ultra-15 30 kD ultrafiltration tube to obtain a PBS buffer solution of the conjugated product ADC-20 (3.85 mg / mL, 0.49 mL), which was stored frozen at -80°C.

[0463] The average DAR value calculated by reduction-reverse phase chromatography method was n=7.51.

[0464] Example 42: Preparation of ADC-21

[0465] At 37°C, a PBS buffer solution of the antibody Pertuzumab (0.05 M PBS solution at pH 7.0; 0.092 mL, 21.64 mg / mL, 0.014 μmol) was added with a prepared aqueous solution of the reducing agent tris(2-carboxyethyl)phosphine (10 mM, 0.014 mL, 0.14 μmol). The solution was filled with Ar gas and placed in a metal bath oscillator for oscillation at 37°C for 1.5 hours. After stopping the reaction, the reaction solution was naturally cooled to room temperature (25°C) for subsequent reactions. A 20% DMF / water solution of compound DL-20 (0.1 mL, 4.0 mg / mL, 0.28 μmol) was added to the above 0.106 mL antibody reaction solution, purged with Ar gas, and placed on a shaker for 2.0 hours at 25°C to terminate the reaction. An aqueous solution of compound NAC (0.004 mL, 100 mM, 0.4 μmol) was added to the reaction solution, placed on a shaker, and allowed to shake at 25°C for 0.3 hours to terminate the reaction. The reaction solution was ultrafiltered using an Amicon Ultra-15 30 kD ultrafiltration tube using 0.05 M PBS buffer (pH 7.0) to obtain a PBS buffer solution of the Pertuzu mAB conjugate ADC-21 (3.69 mg / mL, 0.54 mL), which was stored frozen at -80°C.

[0466] The average DAR value calculated by reduction-reverse phase chromatography method was n=7.05.

[0467] Example 43: Preparation of ADC-22

[0468] At 37°C, a prepared reducing agent aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.008 mL, 0.084 μmol) was added to a PBS buffered aqueous solution of the antibody Rituximab (0.05 M PBS aqueous solution at pH 7.0; 0.2 mL, 10 mg / mL, 0.014 μmol), the mixture was filled with Ar gas, placed in a metal bath oscillator, and oscillated at 37°C for 1.5 hours. After stopping the reaction, the reaction solution was naturally cooled to room temperature (25°C) for subsequent reactions. A 20% DMF / water solution of compound DL-4 (0.117 mL, 4.0 mg / mL, 0.28 μmol) was added to the above 0.208 mL antibody reaction solution, purged with Ar gas, and placed on a shaker for 2.0 hours at 25°C to terminate the reaction. An aqueous solution of compound NAC (0.004 mL, 100 mM, 0.4 μmol) was added to the reaction solution, placed on a shaker, and allowed to shake at 25°C for 0.3 hours to terminate the reaction. The reaction solution was ultrafiltered using an Amicon Ultra-15 30 kD ultrafiltration tube with 0.05 M PBS buffer (pH 7.0) to obtain a PBS buffer solution of the Rituzu mAB conjugate ADC-22 (5.34 mg / mL, 0.36 mL), which was stored frozen at -80°C.

[0469] The average DAR value calculated by reduction-reverse phase chromatography method was n=7.29.

[0470] Example 44: Preparation of ADC-23

[0471] At 37°C, a PBS buffer solution of the B7H4-targeting antibody mAb-1 (0.05 M PBS solution at pH 7.0; 0.709 mL, 2.82 mg / mL, 0.014 μmol) was added with a prepared reducing agent aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.012 mL, 0.14 μmol). The mixture was filled with Ar gas and placed in a metal bath oscillator for oscillation at 37°C for 1.5 hours. After stopping the reaction, the reaction solution was naturally cooled to room temperature (25°C) for subsequent reactions. A 20% DMF / water solution of compound DL-4 (0.117 mL, 4.0 mg / mL, 0.28 μmol) was added to the above 0.721 mL antibody reaction solution, purged with Ar gas, and placed on a shaker for 2.0 hours at 25°C to terminate the reaction. An aqueous solution of compound NAC (0.004 mL, 100 mM, 0.4 μmol) was added to the reaction solution, placed on a shaker, and allowed to shake at 25°C for 0.3 hours to terminate the reaction. The reaction solution was ultrafiltered using an Amicon Ultra-15 30 kD ultrafiltration tube using 0.05 M PBS buffer (pH 7.0) to obtain a PBS buffer solution of the AZmAB conjugate product ADC-23 (4.49 mg / mL, 0.45 mL), which was stored frozen at -80°C.

[0472] The average DAR value calculated by reduction-reverse phase chromatography method was n=7.28.

[0473] Example 45: Preparation of ADC-24

[0474] At 37°C, a PBS buffer solution of the antibody Cetuximab (0.05 M PBS solution at pH 7.0; 0.25 mL, 8.0 mg / mL, 0.014 μmol) was added with a prepared aqueous solution of the reducing agent tris(2-carboxyethyl)phosphine (10 mM, 0.016 mL, 0.168 μmol), filled with Ar gas, and placed in a metal bath oscillator for oscillation at 37°C for 1.5 hours. After stopping the reaction, the reaction solution was naturally cooled to room temperature (25°C) for subsequent reactions. A 20% DMF / water solution of compound DL-4 (0.117 mL, 4.0 mg / mL, 0.28 μmol) was added to the above 0.266 mL antibody reaction solution, filled with Ar gas, placed in a shaker, and shaken at 25°C for 2.0 hours to stop the reaction. An aqueous solution of compound NAC (0.004 mL, 100 mM, 0.4 μmol) was added to the reaction solution, placed in a shaker, and shaken at 25°C for 0.3 hours to stop the reaction. The reaction solution was ultrafiltered using an Amicon Ultra-15 30 kD ultrafiltration tube using 0.05 M PBS buffer at pH = 7.0 to obtain a PBS buffer solution of the Cetuxi mAB conjugate product ADC-24 (4.04 mg / mL, 0.51 mL), which was stored frozen at -80°C.

[0475] The average DAR value calculated by reduction-reverse phase chromatography method was n=7.79.

[0476] Example 46: Preparation of ADC-25

[0477] At 37°C, a PBS buffer solution of the antibody Pertuzumab (0.05 M PBS solution at pH 7.0; 0.092 mL, 21.64 mg / mL, 0.014 μmol) was added with a prepared aqueous solution of the reducing agent tris(2-carboxyethyl)phosphine (10 mM, 0.014 mL, 0.14 μmol). The solution was filled with Ar gas and placed in a metal bath oscillator for oscillation at 37°C for 1.5 hours. After stopping the reaction, the reaction solution was naturally cooled to room temperature (25°C) for subsequent reactions. A 20% DMF / water solution of compound DL-4 (0.117 mL, 4.0 mg / mL, 0.28 μmol) was added to the above 0.106 mL antibody reaction solution, purged with Ar gas, and placed on a shaker for 2.0 hours at 25°C to terminate the reaction. An aqueous solution of compound NAC (0.004 mL, 100 mM, 0.4 μmol) was added to the reaction solution, placed on a shaker, and allowed to react at 25°C for 0.3 hours to terminate the reaction. The reaction solution was ultrafiltered using an Amicon Ultra-15 30 kD ultrafiltration tube using 0.05 M PBS buffer (pH 7.0) to obtain a PBS buffer solution of the Pertuzu mAB conjugate ADC-25 (3.85 mg / mL, 0.51 mL) which was stored frozen at -80°C.

[0478] The average DAR value calculated by reduction-reverse phase chromatography method was n=4.42.

[0479] Example 47: Preparation of ADC-26

[0480] An aqueous solution of compound DL-21 (0.088 mL, 4.0 mg / mL, 0.2 μmol) was added to the above 0.208 mL antibody reaction solution, filled with Ar gas, placed in a shaker, and reacted at 25°C for 1.5 hours. The reaction was then stopped. An aqueous solution of compound NAC (0.004 mL, 100 mM, 0.4 μmol) was added to the reaction solution, filled with Ar gas, placed in a shaker, and reacted at 25°C for 0.3 hours. The reaction was then stopped. The reaction solution was ultrafiltered using an Amicon Ultra-15 30 kD ultrafiltration tube using 0.05 M PBS buffer at pH 7.0 to obtain a PBS buffer solution of the conjugated product ADC-26 (5.0 mg / mL, 0.42 mL), which was stored frozen at -80°C.

[0481] The average DAR value calculated by reduction-reverse phase chromatography method was n=6.5.

[0482] Biological evaluation

[0483] Test Example 1: In vitro inhibition test of tumor cell proliferation by ADC drugs

[0484] 1. Test Purpose

[0485] The purpose of this test example is to detect the in vitro proliferation inhibitory activity of some ADC drugs of the present invention on tumor cells with different expression levels. After treating cells with different concentrations of ADC drugs in vitro and culturing for 6 days, the cell viability was detected using CTG (CellTiter-Glo Luminescent Cell Viability Assay, Promega, Product No. G7571) reagent. The drug concentration was used as the horizontal axis and the response value was used as the vertical axis. The four-parameter curve was fitted according to the IC 50 The values ​​were used to evaluate the inhibitory effect of ADC drugs on the proliferation of different tumor cells.

[0486] 2. Test methods

[0487] 1. Cell culture

[0488] NCI-N87 cells (Cell Bank of the Chinese Academy of Sciences, cell number TCHu130) were cultured with 10% FBS 1640 complete medium, JIMT-1 cells (Nanjing Kebai Biological, cell number CBP60378) were cultured with 10% FBS DMEM complete medium, MDA-MB-468 cells (ATCC, cell number HTB-132) were cultured with 10% FBS DMEM complete medium, SK-BR-3 cells (Cell Bank of the Chinese Academy of Sciences, cell number TcHu225) were cultured with 10% FBS McCoy'5A complete medium was used to culture the MDA-MB-231 cells (National Laboratory Cell Resource Sharing Platform, cell number 1101HUM-PUMC000014) with 10% FBS 1640 complete medium, NCI-H2170 cells (Cell Bank of the Chinese Academy of Sciences, cell number SCSP-5098) with 10% FBS 1640 complete medium, and A431 cells (Nanjing Kebai Biological, cell number CBP60330) with 10% FBS DMEM complete medium.

[0489] 2. Cell Preparation

[0490] Take stable cells and wash them twice with 5-10 mL of DPBS. Then, add 1-2 mL of 0.25% Trypsin-EDTA to digest them. Once digestion is complete, add 2.5-3 times the volume of trypsin-containing complete medium to the culture flask to terminate digestion. Transfer the digested cells to a centrifuge tube and centrifuge at 1000 rpm for 5 minutes. After centrifugation, discard the supernatant and resuspend the cells in complete medium. Gently pipette to evenly distribute the cells before counting.

[0491] 3. Laying planks

[0492] Tumor cells were diluted to 5.55 × 10 4 cells / mL, add 90 μL per well to the predetermined position of a 96-well white opaque plate, and fill the edge wells with sterile DPBS at a filling volume of 200 μL / well. Place in a 37°C, 5% CO2 incubator for overnight adherent culture;

[0493] 4. Sample preparation

[0494] Pre-dilute the ADC samples to 1350 nM in complete medium and perform a 5-fold serial dilution for a total of nine steps. Add 10 μL of diluted ADC sample per well to the designated positions of a 96-well plate, with two replicates for each concentration. Add complete medium to the cells in column 11 to serve as a control for normal cell growth.

[0495] 5. Cultivation

[0496] After adding, tap the plate several times around the sides to mix the sample and culture medium evenly, and then place the plate in a 37°C, 5% CO2 incubator for 6 days.

[0497] 6. Cell Viability Assay

[0498] After culturing for 6 days, After the reagent is thawed and the cell culture plate is placed at room temperature for 30 minutes, 50 μL is added to each well. The cell plate is shaken on a shaker for 2 minutes and then incubated at room temperature for 10 minutes to stabilize the luminescent signal.

[0499] 7. Read the plate

[0500] Luminescence chemiluminescence values ​​were read using a BMG Omega microplate reader.

[0501] 8. Analysis Methods

[0502] The IC was calculated by fitting a four-parameter curve with the final ADC concentration in nM as the horizontal axis and the luminescence value as the vertical axis. 50 The analysis software was Omega-Data Analysis.

[0503] 3. Data Analysis

[0504] The results are shown in Tables 2, 3 and 4 below.

[0505] Table 2 Inhibitory activity of HER2 ADC drugs on tumor cell proliferation in vitro

[0506] Conclusion: The ADC drug conjugated with trastuzumab in the present invention has obvious proliferation inhibitory activity against the above NCI-N87 cells and JIMT-1 cells, but weak proliferation inhibitory effect on HER2-negative cells MDA-MB-468; the negative antibody conjugated with ADC-22 has no killing effect on NCI-N87, reflecting safety.

[0507] Table 3 Inhibitory activity of B7H4 ADC drugs on tumor cell proliferation in vitro

[0508] Table 4 Inhibitory activity of EGFR ADC drugs on tumor cell proliferation in vitro

[0509] Conclusion: ADCs coupled with antibodies for various targets have good killing effects on both high- and low-expressing cells, but weak killing effects on negative cells.

[0510] Experimental Example 2: Evaluation of the bystander killing activity of ADC drugs

[0511] 1. Purpose of the test:

[0512] The bystander effect of ADC-4 on HER2-negative human breast cancer cells MDA-MB-468 was studied by co-culturing HER2-high-expressing human gastric cancer cells NCI-N87 and HER2-negative human breast cancer cells MDA-MB-468.

[0513] 2. Test methods:

[0514] NCI-N87 cells and MDA-MB-468 cells were plated at a ratio of 3:1 to form a co-culture group; NCI-N87 cells were plated alone to form an NCI-N87 cell group; and MDA-MB-468 cells were plated alone to form an MDA-MB-468 cell group. After cells in each group adhered, 1.08 nM ADC-4 was added. A control group (Untreated) without drug was also included in each group. After 6 days of incubation, cells were harvested by trypsinization and the total number of viable cells was counted using a cell counter. 5 μl / well of labeled HER2 / ERBB2 Antibody (PE) (Sino Biological, Cat. No. 10004-MM07T-P) was added and incubated at 4°C for 20 minutes. After centrifugation and washing, the cells were resuspended in DPBS and analyzed by flow cytometry for the ratio of the two cell types. Scatter plots were generated using FlowJo software (FlowJo_v10.8.1_CL). The killing rate of the ADC against NCI-N87 and MDA-MB-468 cells was calculated using the following formulas I and II. The data from each group were compared to evaluate the bystander effect of the ADC.

[0515] 3. Test results

[0516] Table 5 Bystander killing effect test results

[0517] The results in Table 5 show that ADC-4-conjugated toxins have excellent transmembrane ability and easily penetrate cell membranes to exert a bystander killing effect. In this study, ADC-4 alone at 1.08 nM had almost no killing effect on HER2-negative MDA-MB-468 cells. However, when HER2-high-expressing NCI-N87 cells and HER2-negative MDA-MB-468 cells were mixed in a 3:1 ratio, 1.08 nM ADC-4 killed NCI-N87 cells while also having a killing rate of 68% against MDA-MB-468. This indicates that ADC-4 internalizes into NCI-N87 cells, lyses them, and releases the toxin, which then enters adjacent MDA-MB-468 cells through a bystander effect to exert its efficacy, and this killing effect is independent of target cell antigen expression.

[0518] Experimental Example 3: Evaluation of the efficacy of ADC drugs in JIMT-1 tumor-bearing mice

[0519] 1. Objective: To evaluate the anti-tumor effect of ADC drugs in the female NOD / SCID mouse model with subcutaneous xenografts of human breast cancer JIMT-1 cells.

[0520] 2. Test Materials

[0521] 1. Test drug

[0522] ADC-3: 3 mg / kg

[0523] ADC-3: 5 mg / kg

[0524] Blank control: physiological saline

[0525] 2. Preparation method: dilute with normal saline.

[0526] 3. Experimental animals: female NOD / SCID mice.

[0527] 3. Test methods

[0528] NOD / SCID mice were subcutaneously inoculated with 5×10 JIMT-1 cells on the right back. 6 (0.1 mL / mouse), and observe the tumor growth regularly. 3Mice were randomly divided into groups based on tumor size and body weight for dosing. The trial consisted of a blank control group, an ADC-3 3mg / kg group, and an ADC-3 5mg / kg group, with four mice in each group. Mice were dosed via tail vein injection on the day of grouping (designated day 0), with a single dose administered for a total of 28 days. Efficacy was evaluated based on relative tumor inhibition (TGI), and safety was assessed based on changes in animal body weight and mortality.

[0529] Following tumor cell inoculation, routine monitoring included tumor growth and the effects of treatment on the animals' normal behavior. Specifically, monitoring included activity, food and water intake, weight gain or loss, and any abnormalities of the eyes, coat, or other areas. Any clinical symptoms observed during the trial were recorded in the raw data. Following the initiation of dosing, body weight and tumor size were measured twice weekly.

[0530] Calculation formula:

[0531] Tumor volume (mm 3 )=1 / 2×(a×b 2 ) (where a represents the major diameter and b represents the minor diameter); relative tumor inhibition rate (%) = (1-T / C) × 100% (where T / C% is the tumor proliferation rate, which is the percentage value of the relative tumor volume (or body weight) of the treatment group and the control group at a certain time point).

[0532] StudyDirector™ (version 3.1.399.19, supplied by Studylog System, Inc.) software was used to collect data, including tumor diameter measurements and animal body weights. Raw data, measured using a balance and vernier calipers, were directly imported into the software, and any changes in the data were recorded. All procedures, including dosing, tumor measurement, and body weight, were performed in a biosafety cabinet or clean bench.

[0533] 4. Test results:

[0534] 1. Evaluation of the efficacy of the test drug in the JIMT-1 human breast cancer model:

[0535] The results are shown in Figure 1. The study was completed on day 28 after a single tail vein administration. ADC-3 (3 mg / kg) demonstrated an 84.07% tumor inhibition rate, while ADC-3 (5 mg / kg) demonstrated an inhibition rate of 99.32%, with 50% of tumors completely regressing (2 / 4). Both treatment groups demonstrated excellent tumor suppression.

[0536] 2. Safety evaluation of the test drug in the JIMT-1 human breast cancer model:

[0537] As shown in FIG2 , none of the test drug groups caused weight loss in mice, and no animals died in any treatment group, indicating that the treatment was well tolerated.

[0538] The above-described embodiments merely illustrate several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, and all such variations and improvements fall within the scope of protection of the present invention.

Claims

1. A compound, or an isotope-labeled compound, optical isomer, geometric isomer, tautomer or isomer mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein: The compound comprises the structure shown in formula (I): Wherein W is selected from a single bond, -X-(CH2) m1 -CR3R4-NR5-, -X-CR3R4-(CH2) m1 -NR5-, -X-(CH2O) m2 -CR3R4-NR5-, -X-CR3R4-(CH2O) m2 -NR5-, -X-(CH2CH2O) m3 -CR3R4-NR5-, -X-(CH2CH2O) m3 -(CH2) m1 -NR5-, -X-CR3R4-(CH2CH2O) m3 -NR5-, -X-(CH2O) m2 -R3R4N-、-X-(CH2) m1 -R3R4N-、-XR5-(CH2) m1 -R3R4N-、-X-(CH2) m1 -(CH2O) m2 -(CH2) m4 -CR3R4-NR5- or -X-CR3R4-(CH2) m1 -(CH2O) m2 -(CH2) m4 -NR5-; X is selected from an alkylene group, an imino group, a nitrogen atom, an oxygen atom or a sulfur atom; R3, R4, and R5 are each independently selected from a hydrogen atom, a deuterium atom, a halogen, a hydroxyl, an amino group, a cyano group, a nitro group, a sulfone group, a C1-C6 alkyl group, or a C3-C6 cycloalkyl group; or, R3 and R4 together with the carbon atom to which they are connected form a C3-C6 cycloalkyl group; or, R3 and R4 together with the carbon atom to which they are connected and a nitrogen atom form a 3-6-membered heterocyclic group; in R3, R4, and R5, one or more hydrogen atoms in the C1-C6 alkyl group or the C3-C6 cycloalkyl group are optionally substituted by a deuterium atom, a halogen, a hydroxyl, a cyano group, an amino group, a C1-C3 alkyl group, or a phenyl group; m1, m2, m3, and m4 are each independently selected from any integer from 0 to 6; The wavy line to which W in formula (I) is connected represents a hydrogen atom, or is covalently connected to the structure of the linker unit and / or the linked antibody or antigen-binding fragment thereof.

2. The compound according to claim 1, wherein W is selected from -X-(CH2) m1 -CR3R4-NR5-, -X-CR3R4-(CH2) m1 -NR5-, -X-(CH2CH2O) m3 -(CH2) m1 -NR5-, -X-(CH2) m1 -R3R4N-、-XR5-(CH2) m1 -R3R4N-、-X-(CH2) m1 -(CH2O) m2 -(CH2) m4 -CR3R4-NR5- or -X-CR3R4-(CH2) m1 -(CH2O) m2 -(CH2) m4 -NR5-; X is selected from an imino group, a nitrogen atom or an oxygen atom; R3 and R4 are each independently selected from a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl group, a C3-C6 cycloalkyl group, or R3 and R4 together with the carbon atom to which they are connected form a C3-C6 cycloalkyl group, or R3 and R4 together with the carbon atom to which they are connected and a nitrogen atom form a 3-6 membered heterocyclic group; in R3 and R4, one or more hydrogen atoms in the C1-C6 alkyl group or the C3-C6 cycloalkyl group are optionally substituted by a deuterium atom, a halogen, a C1-C3 alkyl group or a phenyl group; R5 is selected from a hydrogen atom, a deuterium atom or a halogen; m1, m2, and m4 are each independently selected from any integer between 1 and 4.

3. The compound according to claim 1, wherein W is selected from any of the following structures:

4. The compound according to any one of claims 1 to 3, wherein The compound contains the structure shown in formula (II): Wherein, W refers to any one of claims 1 to 3; L is selected from the following groups: NH -Val-Cit- C＝O , NH -Val-Ala- C＝O , NH -Val-Lys- C＝O , NH -Val-Lys(Ac)- C＝O , NH -Phe-Lys- C＝O , NH -Phe-Lys(Ac)- C＝O , NH -D-Val-Leu-Lys- C＝O , NH -Gly-Gly-Arg- C＝O , NH -Ala-Ala-Asru- C＝O , NH -Gly-Gly-Gly- C＝O , NH -Gly-Gly-Gly-Gly-Gly- C＝O , or L1 or L2 as shown below; preferably L1 or L2; In L1-L2, R1 is a single bond or is selected from the following structures: wherein n3 and n4 are each independently an integer of 0 to 6, and R2 is a hydrophilic group or a structure with a hydrophilic group; The wavy line to which L in formula (II) is connected represents a hydrogen atom, or is covalently connected to a structure that is connected to an antibody or an antigen-binding fragment thereof.

5. The compound according to claim 4, wherein R2 is a hydroxyl group, or a structure having at least three oxygen-containing hydrophilic groups; preferably, the oxygen-containing hydrophilic groups are the same or different, and each is independently selected from -O- or a hydroxyl group; Preferably, R2 is any of the following structures:

6. The compound according to claim 4, wherein R1 is a single bond or is selected from the following structures:

7. The compound according to any one of claims 4 to 6, wherein When L is selected from L1, X in W is an oxygen atom; when L is selected from L2, X in W is an imino group.

8. The compound according to any one of claims 4 to 7, wherein The compound contains the structure shown in formula (III): Wherein, P is a structure connecting an antibody or an antigen-binding fragment thereof; The references of L and W are the same as those described in any one of claims 4 to 7.

9. The compound according to claim 8, wherein P is selected from P1, P2 or P3: Wherein, n1 is an integer of 1 to 10, preferably an integer of 2 to 6; n2 is an integer from 1 to 12, preferably an integer from 5 to 9; Z is selected from a bond, a C1-C6 alkylene group, an imino group, an ester group, an amide group, a sulfone group, a sulfonamide group, a ketone group, a urea group or a phosphate group.

10. The compound according to claim 9, wherein The structure of the compound is shown as follows:

11. An antibody-drug conjugate comprising: i) an antibody or an antigen-binding fragment thereof; and, ii) The compound according to any one of claims 1 to 10, or an isotope-labeled compound thereof, an optical isomer, a geometric isomer, a tautomer or a mixture of isomers, or a pharmaceutically acceptable salt or solvate thereof.

12. The antibody-drug conjugate according to claim 11, wherein: The structure of the antibody-drug conjugate is shown in formula (IV): Wherein, Ab is an antibody or an antigen-binding fragment thereof; n6 is an integer or decimal selected from 1 to 10.

13. The antibody-drug conjugate according to claim 12, wherein: n6 is selected from an integer or decimal between 1 and 8, and preferably n6 is selected from an integer or decimal between 6 and 8.

14. The antibody-drug conjugate according to any one of claims 11 to 13, wherein The antibody targets one or more of HER2, HER3, B7H3, B7H4, EGFR, Claudin18.2, c-MET, Nectin-4, ROR1, GPNMB, CD56, TACSTD2 (TROP2), CEACAM5, folate receptor-a, mesothelin, ENPP3, guanylate cyclase C, SLC44A4, NaPi2b, CD70, mucin 1, STEAP1, connexin 4, 5T4, SLTRK6, SC-16, LIV-1, P-cadherin, PSMA, fibronectin extradomain B, endothelin receptor ETB, tenascin c, collagen IV, VEGFR2, periostin, CD30, CD79b, CD19, CD22, CD138, CD37, CD33, CD74, CD19 and CD98; Preferably, the antibody is a monoclonal antibody.

15. A pharmaceutical composition comprising: a compound according to any one of claims 1 to 10 or an isotope-labeled compound thereof, or an optical isomer, geometric isomer, tautomer or isomer mixture thereof, or a pharmaceutically acceptable salt thereof, or an antibody-drug conjugate according to any one of claims 11 to 14; and one or more pharmaceutically acceptable excipients.

16. Use of the compound according to any one of claims 1 to 10 or its isotope-labeled compound, or its optical isomer, geometric isomer, tautomer or isomer mixture, or its pharmaceutically acceptable salt, or the antibody-drug conjugate according to any one of claims 11 to 14 in the preparation of a drug.

17. The use according to claim 16, wherein: The drug is used to prevent or treat tumors; preferably, the tumor is a tumor selected from HER2, HER3, B7H3, B7H4, EGFR, Claudin18.2, c-MET, Nectin-4, ROR1, GPNMB, CD56, TACSTD2 (TROP2), CEACAM5, folate receptor-a, mesothelin, ENPP3, guanylate cyclase C, SLC44A4, NaPi2b, CD70, mucin 1, STEAP1, connexin 4, 5T4, SLTRK6, SC-16, LIV-1, P-cadherin, PSMA, fibronectin extradomain B, endothelin receptor ETB, tenascin c, collagen IV, VEGFR2, periostin, CD30, CD79b, CD19, CD22, CD138, CD37, CD33, CD74, CD19 and CD98 are related to the expression of one or more cancers; more preferably, the tumor is selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer, colon cancer, rectal cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer and lymphoma.

18. A method for preventing or treating tumors, comprising: Administering an effective amount of the compound according to any one of claims 1 to 10 or its isotope-labeled compound, or its optical isomer, geometric isomer, tautomer or isomer mixture, or its pharmaceutically acceptable salt, or the antibody drug conjugate according to any one of claims 11 to 14, or the pharmaceutical composition according to claim 15.

19. The method for preventing or treating tumors according to claim 18, wherein: The tumor is a tumor with a gene selected from HER2, HER3, B7H3, B7H4, EGFR, Claudin18.2, c-MET, Nectin-4, ROR1, GPNMB, CD56, TACSTD2 (TROP2), CEACAM5, folate receptor-a, mesothelin, ENPP3, guanylate cyclase C, SLC44A4, NaPi2b, CD70, mucin 1, STEAP1, connexin 4, 5T4, SLTRK6, SC-16, LIV-1, P-cadherin, PSMA, fibronectin extracellular domain B, endothelin receptor ETB, Cancers associated with the expression of one or more of tenascin c, collagen IV, VEGFR2, periostin, CD30, CD79b, CD19, CD22, CD138, CD37, CD33, CD74, CD19 and CD98; preferably, the tumor is selected from one or more of breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer, colon cancer, rectal cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer and lymphoma.

20. The method for producing a compound according to any one of claims 1 to 10, wherein The reaction raw materials include: a compound having a structure represented by formula (I); Preferably, the preparation method comprises: reacting a compound containing the structure L and the structure represented by formula (I) with a compound containing the structure P to obtain the compound; Or it includes: reacting a compound containing a P structure and an L structure with a compound containing a structure represented by formula (I) to obtain the compound.

21. A method for preparing a compound according to claim 20, comprising: Reacting a compound containing an L structure with a compound containing a structure represented by formula (I), and then reacting the compound containing an L structure and a structure represented by formula (I) with a compound containing a P structure to obtain the compound; Or it includes: reacting a compound containing a P structure with a compound containing an L structure, and then reacting the compound containing the P structure and the L structure with a compound containing the structure represented by formula (I) to obtain the compound.

22. The method for preparing an antibody drug conjugate according to any one of claims 11 to 14, comprising: The following i) and ii) are coupled: i) antibodies or antigen-binding fragments thereof; as well as, ii) The compound according to any one of claims 1 to 10, or an isotope-labeled compound thereof, an optical isomer, a geometric isomer, a tautomer or a mixture of isomers, or a pharmaceutically acceptable salt or solvate thereof.

23. The method for preparing an antibody-drug conjugate according to claim 22, comprising: The interchain disulfide bonds of the antibody or its antigen-binding fragment are reduced to free thiol groups, and then coupled with the P-terminus of the compound or its isotope-labeled compound, optical isomer, geometric isomer, tautomer or isomer mixture, or its pharmaceutically acceptable salt or solvate.

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