Bioactive conjugates, methods of making and uses thereof

Abstract

Provided herein are compounds comprising dimers based on pyrrolobenzodiazepine (PBD), and conjugates thereof. In some embodiments, the compounds have formula (I) or are pharmaceutically acceptable salts, tautomers, solvates, stereoisomers, or isotopic bodies thereof. (I)

Description

Cross-reference to related applications

[0001] This application claims priority to International Application No. PCT / CN2023 / 112181, filed on August 10, 2023, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0002] This disclosure relates to antibody-drug conjugate platform compounds comprising pyrrolobenzodiazepine (PBD)-based dimers and antibody-drug conjugates (ADCs) comprising the platform and an antibody or an antigen-binding fragment thereof, as well as the use of the ADC platform and the ADC. sequence list This application contains a sequence list, which has been electronically submitted in XML format. The XML file is named "01368-0077-00PCT-ST26.xml", created on August 7, 2024, and is 5,839 bytes in size. The sequence list is incorporated herein by reference in its entirety. Background Technology

[0003] Some pyrrolobenzodiazepines (PBDs) possess the ability to recognize and bind to specific DNA sequences; the preferred sequence is PuGPu. The first PBD antitumor antibiotic, amiodarone, was discovered in 1965 (Leimgruber et al., J. Am. Chem. Soc., 87, 5793-5795 (1965); Leimgruber et al., J. Am. Chem. Soc., 87, 5791-5793 (1965)). Since then, numerous naturally occurring PBDs have been reported, and more than a dozen synthetic routes have been developed to synthesize various analogs (Thurston et al., Chem. Rev. 1994, 433-465 (1994)). Family members include abenomycin (Hochlowski et al., J. Antibiotics, 40, 145-148 (1987)), chezamycin (Konishi et al., J. Antibiotics, 37, 200-206 (1984)), DC-81 (Japanese Patent 58-180 487; Thurston et al., Chem. Brit., 26, 767-772 (1990); Bose et al., Tetrahedron, 48, 751-758 (1992)), methylanisoxamycin (Kuminoto et al., J. Antibiotics, 33, 665-667 (1980)), neoanisoxamycin A and B (Takeuchi et al., J. Antibiotics, 29, 93-96 (1976)), and polomycin (Tsunakawa et al., J. Antibiotics, 41, ). 1366-1373 (1988)), pravastatin (Shimizu et al., J. Antibiotics, 29, 2492-2503 (1982); Langley and Thurston, J. Org. Chem., 52, 91-97 (1987)), sibanamicin (DC-102) (Hara et al., J. Antibiotics, 41, 702-704 (1988); Itoh et al., J. Antibiotics, 41, 1281-1284 (1988)), siberiamycin (Leber et al., J. Am. Chem. Soc., 110, 2992-2993 (1988)), and tomatine (Arima et al., J. Antibiotics, 25, 437-444 (1972)). PBD has the following general structure: .

[0004] The number, type, and position of substituents on the aromatic A ring and pyrrolo C ring of PBD, as well as the saturation of the C ring, vary. The N10-C11 positions of the B ring contain imine (N═C), methylamine (NH—CH(OH)), or methylamine methyl ether (NH—CH(OMe)), which are electrophilic centers responsible for alkylating DNA. All known natural products have an (S) configuration at the chiral C11a position, which gives them a right-handed twist when viewed from the C ring to the A ring. This endows them with an appropriate three-dimensional shape with isohelicity in the minor groove of type B DNA, resulting in a tight fit at the binding site (Kohn, Antibiotics III. Springer-Verlag, New York, pp. 3-11 (1975); Hurley and Needham-VanDevanter, Acc. Chem. Res., 19, 230-237 (1986)). PBDs can form adducts in minor grooves, which allows them to interfere with DNA processing and thus can be used as antitumor agents.

[0005] The biological activity of these molecules can be enhanced by linking two PBD units together via their C8 / C′-hydroxyl functional groups through flexible alkylene linkers (Bose, DS et al., J. Am. Chem. Soc., 114, 4939-4941 (1992); Thurston, DE et al., J. Org. Chem., 61, 8141-8147 (1996)). PBD dimers are believed to form sequence-selective DNA damage, such as palindromic 5′-Pu-GATC-Py-3′ interstrand crosslinks (Smellie, M. et al., Biochemistry, 42, 8232-8239 (2003); Martin, C. et al., Biochemistry, 44, 4135-4147), which is considered a major reason for their biological activity. An example of a PBD dimer is SG2000 (SJG-136): (Gregson, S. et al., J. Med. Chem., 44, 737-748 (2001); Alley, MC et al., Cancer Research, 64, 6700-6706 (2004); Hartley, JA et al., Cancer Research, 64, 6693-6699 (2004)).

[0006] Previous PBD dimers were prepared symmetrically, meaning the two monomers in the dimer were identical, due to the way these highly efficient compounds crosslink DNA. This synthetic route offers direct synthesis, either by simultaneously constructing the PBD dimer moiety already formed with dimer linkages, or by reacting the constructed PBD monomer moiety with the dimer linker group. These synthetic methods limit the options for preparing PBD-containing targeted conjugates.

[0007] Antibody-drug conjugates (ADCs) comprise antibodies operatively linked to a small, biologically active molecule (also known as a toxin or payload). ADCs selectively deliver potent payloads to target-expressing cells, potentially reducing off-target side effects and / or toxicity and improving therapeutic efficacy. The lipophilicity of many payloads adversely affects the properties of ADCs, preventing efficient delivery to target cells. The low bioavailability of lipophilic payloads narrows the therapeutic window for ADC therapy. Furthermore, the hydrophobicity of payloads can challenge their conjugation with antibodies, a reaction that occurs under aqueous conditions.

[0008] Therefore, there is a continuous need to develop hydrophilic linkers for protein conjugates (e.g., ADCs), which will allow for improved conjugation of lipophilic payloads, improved regulation of biological targets, increased bioavailability, and improved therapeutic windows. Currently, there is a continued need for PBD dimers (including lipophilic PBD dimers and / or asymmetric PBD dimers) as payloads for ADCs. Summary of the Invention

[0009] This article provides an antibody-drug conjugate platform and antibody-drug conjugates (ADCs). Furthermore, it describes the uses of the ADC platform for the preparation of ADCs.

[0010] In some implementations, compounds of formula (I) are provided herein: (I) Or its pharmaceutically acceptable salts, tautomers, solvates, stereoisomers, or isotopes, wherein: Each of rings A and B is independently one of equations (IIa), (IIb), (IIc), (IId), (IIe), (IIIf), or (IIg): , , , , , or ;in: Each ring C is independently a cyclopropyl ring or a cyclobutyl ring; Each R 1 Independently H, OH, C 1-4 Alkyl, C 1-4 Alkyl group or -O-(CH2CH2O) t1 -CH3; Each R 2 Independently H or C 1-4 alkyl; Each R 3 and R 4 Independently for H, NR a1 R b1 OH, C 1-4 Alkyl, C 1-4 alkoxy or aryl; Each R a1 and R b1 Independently H or C 1-4 alkyl; Each R 5 Independently for H and C 1-4 Alkyl, C 1-4 alkoxy or aryl; Each m1, n1, and o1 is independently 1 or 2; t1 can be 1, 2, 3, 4, 5, 6, 7 or 8; The bonds marked by ring A being linked to a cleavable 1 when s1 is 1 or to -H when s1 is 0, or the bonds marked by ring B being linked to a cleavable 2, and The key is marked when ring A or ring B is connected to the connector; The connector is –(CH2). r -、-(CH2) p -X-(CH2) q -or-(CH2) p -CH=CH-(CH2) q -;in: Each r, p, and q is independently 1, 2, 3, 4, 5, 6, 7, or 8; The sum of p and q is 1, 2, 3, 4, 5, 6, 7 or 8; X is NR 6 , NHC(=O), C(=O)NH, O, SO2, substituted or unsubstituted aryl rings, substituted or unsubstituted heteroaryl rings, substituted or unsubstituted heterocycles, or substituted or unsubstituted rings; and R 6 For H or C 1-4 alkyl; Decomposable 1, when present, has formula (VIa) or (VIc): or ,in: Su refers to the sugar portion; Each R 11 Independently hydrogen, halogen, substituted or unsubstituted C 1-4 Alkyl, -CN, or -NO2; and # Marks the bonds that can be broken down when linked to ring A; s1 is 0 or 1; The pyrolytic 2 has the formula (VIIa1), (VIIc1), (VIId1), or (VIIf1): , , or ;in: Su refers to the sugar portion; Each R 9 Independently hydrogen, halogen, substituted or unsubstituted C 1-4 Alkyl, -CN, or -NO2; # The bond in the case where the 2-linked ring B can be broken; and ## Marks bonds that can be split into 2-linked spacers; Spacers are bonds, ### -NH-(CH2CH2O) m2 -CH2CH2-C(═O)-、 ### -NH-(CH2CH2O) m2 -CH2-C(═O)-、 ### -(CH2) m2 -C(═O)-、 ### -CH2-C(═O)-NH-(CH2) m2 -C(═O)-、 ### -(CH2CH2O) m2 -CH2CH2-C(═O)-、 ### -CH[-(CH2) m2 -COOH]-C(═O)-、 ### -CH2-C(═O)-NH-(CH2) m2 -C(═O)-NH-(CH2) m2 -C(═O)-、 ### -C(═O)-(CH2) m2 -C(═O)- or ### -NH-(CH2) m2-C(═O)-; where: Each m² is independently 1, 2, 3, 4, 6, 7, or 8; and ### The key in the case where the spacer is linked to the concatenation; and The conjoint has formula (III), (IV) or (V): , or ,in: U2 is a bond, a heteroaryl or a aryl group; V2 represents a bond or -C≡C-(CH2) n2 -; n2 is an integer from 0 to 10, inclusive. W2 is -C(=O)-, -NH-, or -O-; RG3 is or ; RS3 is -NR a2 R b2 ; R a2 and R b2 Each of them is independently H or substituted or unsubstituted C. 1-4 alkyl; RE3 is a key, -O-, -OC(=O-), -OC(=O)NR 7 -、-NHC(=O)NR 7 -、-OS(=O)2NR 7 -、-NHS(=O)2NR 7 -or-OC(=O)NHS(=O)2NR 7 -; R 7 H or substituted or unsubstituted C 1-4 alkyl; W3 is -C(=O)-, -NH-, or -O-; t3 is 1 or 2; s3 is 0, 1, or 2; RG4 is , , , or ; RE4 is a key, -O-, -OC(=O)-, -OC(=O)NR 8 -、-NHC(=O)NR 8 -、-OS(=O)2NR 8 -、-NHS(=O)2NR8 -or-OC(=O)NHS(=O)2NR 8 -; R 8 H or substituted or unsubstituted C 1-4 alkyl; W4 is -C(=O)-, -NH-, or -O-; t4 is 1, 2, 3, 4, 5, 6, 7, or 8; and s4 can be 0, 1, or 2.

[0011] In some implementations, compounds of formula (IA) are provided herein: (IA), Or its pharmaceutically acceptable salts, tautomers, solvates, stereoisomers, or isotopes, wherein: Each of rings A and B is independently one of equations (IIa), (IIb), (IIc), (IId), (IIe), (IIIf), or (IIg): , , , , , or ;in: Each ring C is independently a cyclopropyl ring or a cyclobutyl ring; Each R 1 Independently H, OH, C 1-4 Alkyl, C 1-4 Alkyl group or -O-(CH2CH2O) t1 -CH3; Each R 2 Independently H or C 1-4 alkyl; Each R 3 and R 4 Independently for H, NR a1 R b1 OH, C 1-4 Alkyl, C 1-4 alkoxy or aryl; Each R a1 and R b1 Independently H or C 1-4 alkyl; Each R 5 Independently for H and C 1-4 Alkyl, C 1-4 alkoxy or aryl; Each m1, n1, and o1 is independently 1 or 2; t1 can be 1, 2, 3, 4, 5, 6, 7 or 8; The bonds marked by ring A being linked to a splittable 1 when s1 is 1 or to -H when s1 is 0, or the bonds marked by ring B being linked to a splittable 2; and The key is marked when ring A or ring B is connected to the connector; The connector is –(CH2). r -、-(CH2) p -X-(CH2) q -or-(CH2) p -CH=CH-(CH2) q -;in: Each r, p, and q is independently 1, 2, 3, 4, 5, 6, 7, or; The sum of p and q is 1, 2, 3, 4, 5, 6, 7 or; X is NR 6 , NHC(=O), C(=O)NH, O, SO2, substituted or unsubstituted aryl rings, substituted or unsubstituted heteroaryl rings, substituted or unsubstituted heterocycles, or substituted or unsubstituted rings; and R 6 For H or C 1-4 alkyl; Decomposable 1 has the following formula when it exists: ,in: Su refers to the sugar portion; Each R 11 Independently hydrogen, halogen, substituted or unsubstituted C 1-4 Alkyl, -CN, or -NO2; and # Marks the bonds that can be broken down when linked to ring A; s1 is 0 or 1; The pyrolytic 2 has formula (VIIa1) or (VIId1): or ,in: Su refers to the sugar portion; Each R 9 Independently hydrogen or C 1-4 alkyl; # The bond in the case where the 2-linked ring B can be broken; and ## Marks bonds that can be split into 2-linked spacers; Spacers are bonds, ### -NH-(CH2CH2O) m2-CH2CH2-C(═O-) or ### -NH-(CH2CH2O) m2 -CH2-C(═O)-; where: Each m² is independently 1, 2, 3, 4, 6, 7, or 8; and ### The marker spacer is linked to the key in the case of -C (=O)-; RE4 is the key or -O-; and t4 can be 1, 2, 3, 4, 5, 6, 7 or 8. Attached Figure Description

[0012] Figure 1A and Figure 1B Line graphs showing the killing effect of the conjugates on NOMO-1 (1A) and K562 (1B) cells are presented.

[0013] Figure 2A and Figure 2B Line graphs showing the killing effect of the conjugates on NOMO-1 (2A) and K562 (2B) cells are presented.

[0014] Figure 3A and Figure 3B Line graphs showing the killing effect of the conjugates on NOMO-1 (3A) and K562 (3B) cells are presented.

[0015] Figure 4A and Figure 4B Line graphs showing the killing effect of the conjugates on NOMO-1 (4A) and K562 (4B) cells are presented.

[0016] Figure 5A and Figure 5B Line graphs showing the killing effect of the conjugates on NOMO-1 (5A) and K562 (5B) cells.

[0017] Figure 6A and Figure 6B Line graphs showing the killing effect of the conjugates on NOMO-1 (6A) and K562 (6B) cells. Detailed Implementation

[0018] This document provides antibody-drug conjugate platform compounds comprising pyrrolobenzodiazepine (PBD)-based dimers and antibody-drug conjugates (ADCs) comprising said platform and an antibody or an antigen-binding fragment thereof. ADCs can be used to treat diseases or conditions, such as cancer, for example by providing compositions comprising ADCs. The conjugates contain a β-glucuronide-based linker containing a site that can be cleaved by an enzyme having β-glucuronidase activity.

[0019] In this disclosure, it should be understood that this disclosure is not limited to the specific methods and / or experimental conditions described, as such methods and conditions may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting.

[0020] While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the invention, preferred methods and materials are described hereafter. All patents, applications, and non-patent publications referenced in this specification are incorporated herein by reference in their entirety.

[0021] definition In this disclosure, unless otherwise indicated, the following terms have the following meanings. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. If multiple definitions are provided for a term herein, those definitions shall prevail unless otherwise stated.

[0022] When a trade name is used in this document, unless the context otherwise indicates, reference to the trade name also refers to the product formulation, generic drug, and active pharmaceutical ingredient of the product under that trade name.

[0023] The term "antibody" is used in the broadest sense and specifically encompasses intact monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments exhibiting desired biological activity. An intact antibody primarily has two regions: a variable region and a constant region. The variable region binds to and interacts with the target antigen. The variable region includes a complementarity-determining region (CDR) that recognizes and binds to a specific binding site on a particular antigen. The constant region is recognized by and interacts with the immune system (see, for example, Janeway et al., 2001, Immunol. Biology, 5th edition, Garland Publishing, New York). Antibodies can belong to any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of any of these. Antibodies can be derived from any suitable species. In some embodiments, the antibody has a human or mouse origin. Antibodies can be, for example, human, humanized, or chimeric.

[0024] The term "humanized" or "humanized antibody" refers to an antibody form containing sequences derived from non-human (e.g., rodent) antibodies as well as human antibodies. Such antibodies contain minimal sequences derived from non-human immunoglobulins. Generally, humanized antibodies will contain at least one and usually two variable domains, where all or almost all hypervariable loops correspond to hypervariable loops of non-human immunoglobulins, and all or almost all FR regions are FR regions of human immunoglobulin sequences. Humanized antibodies will optionally also contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the immunoglobulin constant region (Fc) of human immunoglobulins. When it is necessary to distinguish humanized antibodies from parental rodent antibodies, the prefix "hum," "hu," "Hu," or "h" is added to the antibody clone name. Humanized forms of rodent antibodies will generally contain the same CDR sequence as parental rodent antibodies, but may include certain amino acid substitutions to increase affinity, increase the stability of the humanized antibody, remove post-translational modifications, or for other reasons. As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, meaning that the individual antibodies constituting said population are identical except for trace amounts of possibly naturally occurring mutations. Monoclonal antibodies are highly specific (targeting a single antigenic site). The modifier "monoclonal" should not be interpreted as requiring the production of the antibody through any particular method.

[0025] A “complete antibody” is an antibody that contains an antigen-binding variable region and light chain constant domains (CL) and heavy chain constant domains CH1, CH2, CH3, and CH4, depending on the antibody class. The constant domains can be natural sequence constant domains (e.g., human natural sequence constant domains) or amino acid sequence variants thereof.

[0026] An "antibody fragment" comprises a portion of a complete antibody, including its antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments, bivalent antibodies, trivalent antibodies, tetravalent antibodies, linear antibodies, single-chain antibody molecules, scFv, scFv-Fc, multispecific antibody fragments formed from antibody fragments, fragments generated from Fab expression libraries, or epitope-binding fragments of any of the above that are immune-specifically bound to a target antigen (e.g., cancer cell antigen, viral antigen, or microbial antigen).

[0027] An "antigen" is an entity that an antibody specifically binds to.

[0028] The terms "specific binding" and "specifically binds" mean that an antibody or antibody derivative will bind to its corresponding target antigen in a highly selective manner, rather than to a multitude of other antigens. Typically, antibodies or antibody derivatives bind at least about 1 × 10⁻⁶. -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 The affinity of M for binding is at least twice that for binding to a predetermined antigen (e.g., BSA, casein) other than the predetermined antigen or closely related antigens.

[0029] The terms “inhibit” or “inhibition of” mean to reduce a measurable amount or to completely prevent it.

[0030] The term "therapeutic effective dose" refers to the amount of a drug that is effective in treating a disease or symptom in a mammal. In the case of cancer, a therapeutically effective dose of a drug can reduce the number of cancer cells; reduce tumor size; inhibit (i.e., to some extent slow down or stop) the infiltration of cancer cells into surrounding organs; inhibit (i.e., to some extent slow down or stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate one or more symptoms associated with cancer to some extent. In terms of the drug's ability to inhibit growth and / or kill existing cancer cells, it can be cytoseptic and / or cytotoxic. For cancer therapies, efficacy can be measured, for example, by assessing time to progression (TTP) and / or determining response rate (RR).

[0031] The term “substantial” or “substantially” means the majority of a mixture or sample, i.e., >50% of the population, preferably greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the population.

[0032] The terms "intracellular cleavage" and "intracellular breakdown" refer to the metabolic process or reaction of a ligand-drug conjugate (e.g., an antibody-drug conjugate (ADC)) within a cell, in which the covalent link (e.g., a linker) between the drug moiety (D) and the ligand unit (e.g., an antibody (BA or Ab)) is disrupted, resulting in the dissociation of the free drug or another metabolite of the conjugate from the antibody within the cell. Therefore, the cleavage portion of a drug-linker-ligand conjugate is an intracellular metabolite.

[0033] The term "cytotoxic activity" refers to the cytotoxic, cell-inhibiting, or antiproliferative effects of a drug-linker-ligand conjugate compound or its intracellular metabolite. Cytotoxic activity can be expressed as the IC50 value, which is the concentration (moles or mass) per unit volume when half of the cells are viable.

[0034] As used herein, the term "cytotoxic agent" refers to a substance that inhibits cell function and / or causes cell damage. The term is intended to include radioisotopes (e.g., radioisotopes of 211At, 131I, 125I, 90Y, 186Re, 188Re, 153Sm, 212Bi, 32P, 60C, and Lu), chemotherapeutic agents, and toxins (e.g., small molecule toxins or enzyme-active toxins of bacterial, fungal, plant, or animal origin), including their synthetic analogues and derivatives.

[0035] The terms “cancer” and “cancerous” refer to or describe a physiological disorder or ailment in mammals that is typically characterized by dysregulation of cell growth. A “tumor” contains one or more cancerous cells.

[0036] In this article, "autoimmune disease" refers to a disease or condition that originates from and targets an individual's own tissues or proteins.

[0037] Examples of “patient” or “subject” include, but are not limited to, mammals such as humans, rats, mice, guinea pigs, monkeys, pigs, goats, cattle, horses, dogs, or cats, as well as birds or poultry. In this implementation, the patient is a human.

[0038] Unless the context otherwise indicates, the term "treat" or "treatment" refers to therapeutic treatments and preventative measures intended to prevent recurrence, wherein the aim is to suppress or slow (alleviate) unwanted physiological changes or conditions, such as the development or spread of cancer. For the purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to, symptom relief, reduction in disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. "Treatment" may also mean, for example, prolonged survival compared to expected survival without treatment. Those requiring treatment include those who already have the disease or condition and those who are susceptible to it.

[0039] In the context of cancer, the term "treatment" includes any or all of the following: inhibiting the growth of tumor cells, cancer cells, or tumors; inhibiting the replication of tumor cells or cancer cells; reducing the overall tumor burden or the number of cancer cells; and improving one or more symptoms associated with the disease.

[0040] In the context of autoimmune diseases, the term "treatment" includes any or all of the following: inhibiting the replication of cells associated with an autoimmune disease state (including, but not limited to, cells that produce autoimmune antibodies), reducing the autoimmune antibody load, and improving one or more symptoms of the autoimmune disease.

[0041] As used herein, in this specification, and in the appended claims, the indefinite article “a / an” and the definite article “the” include both plural and single indicators, unless the context clearly indicates otherwise.

[0042] As used herein and unless otherwise stated, the terms “about” and “approximately”, when used in conjunction with the amount or weight percentage of a component of a composition, mean an amount or weight percentage that is generally recognized by those skilled in the art to provide a pharmacological effect equivalent to that obtained from the specified amount or weight percentage. In some embodiments, the terms “about” and “approximately”, when used in this context, cover an amount or weight percentage of up to 30%, 20%, 15%, 10%, or 5% of the specified amount or weight percentage.

[0043] As used herein and unless otherwise stated, the terms “about” and “approximately” when used in conjunction with numerical values ​​or ranges of values ​​provided to characterize a particular solid form (e.g., a particular temperature or temperature range, such as describing melting, dehydration, desolventizing, or glass transition temperatures; mass changes, such as mass changes with temperature or humidity; solvent or water content, expressed, for example, by mass or percentage; or peak positions, such as in analyses performed, for example, by IR or Raman spectroscopy or XRPD) indicate that the value or range of values ​​may deviate to a degree that would be reasonable to a person skilled in the art, while still describing the solid form. Techniques used to characterize crystalline forms and amorphous solids include, but are not limited to, thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), X-ray powder diffraction (XRPD), single-crystal X-ray diffraction, vibrational spectroscopy (e.g., infrared (IR) and Raman spectroscopy), solid-state and solution nuclear magnetic resonance (NMR) spectroscopy, optical microscopy, hot-stage optical microscopy, scanning electron microscopy (SEM), electron crystallography and quantitative analysis, particle size analysis (PSA), surface area analysis, solubility studies, and dissolution studies. In some embodiments, the terms "about" and "approximately" used in this context indicate that the numerical value or range of values ​​may vary within 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1.5%, 1%, 0.5%, or 0.25% of the stated value or range. For example, in some embodiments, the values ​​for XRPD peak positions may vary by up to ±0.2° 2θ while still describing a specific XRPD peak.

[0044] As used in this article, the term "inclusive" includes the endpoints of the range when referring to a range. For example, if n is an integer from 0 to 4, then n can be any one of 0, 1, 2, 3, or 4.

[0045] "Alkyl" is a saturated, partially saturated, or unsaturated straight-chain or branched acyclic hydrocarbon having 1 to 10 carbon atoms, typically 1 to 8 carbon atoms, or in some embodiments 1 to 6, 1 to 4, or 2 to 6 carbon atoms. Representative alkyl groups include -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, and -n-hexyl; saturated branched alkyl groups include -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, allyl, CH=CH(CH3), -CH=C(CH3)2, -C(CH3)=CH2, -C(CH3)=CH(CH3), C(CH2CH3)=CH2, C≡CH, -C≡C(CH3), -C≡C(CH2CH3), -CH2C≡CH, -CH2C≡C(CH3), and CH2C≡C(CH2CH3). Alkyl groups may be substituted or unsubstituted. In some embodiments, when the alkyl groups described herein are referred to as “substituted,” they may be substituted with any one or more substituents, such as those found in the compounds disclosed herein and in the embodiments thereof; and halogens (chlorine, iodine, bromine, or fluorine); hydroxyl; alkoxy; alkoxyalkyl; amino; alkylamino; carboxyl; nitro; cyano; thiol; thioether; imine; imide; amidine; guanidine; enamine; aminocarbonyl; acylamino; phosphonate; phosphine; thiocarbonyl; sulfonyl; sulfonamide; ketone; aldehyde; ester; urea; carbamate; oxime; hydroxylamine; alkoxyamine; arylalkoxyamine; N-oxide; hydrazine; acylhydrazine; hydrazone; azide; isocyanate; isothiocyanate; cyanate; thiocyanate; B(OH)2; or O(alkyl)aminocarbonyl.

[0046] "Alkenyl" is a straight-chain or branched acyclic hydrocarbon having 2 to 10 carbon atoms, typically 2 to 8 carbon atoms, and including at least one carbon-carbon double bond. Representative straight-chain and branched (C2-C8) alkenyl groups include -vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, -1-hexenyl, 2-hexenyl, -3-hexenyl, -1-heptenyl, -2-heptenyl, -3-heptenyl, -1-octenyl, -2-octenyl, 3-octenyl, etc. The double bond of an alkenyl group can be non-conjugated or conjugated with another unsaturated group. The alkyl group can be unsubstituted or substituted.

[0047] "Cycloalkyl" is a saturated or partially saturated cyclic alkyl group having 3 to 10 carbon atoms, having a single cyclic ring or multiple fused or bridged rings that may optionally be substituted with 1 to 3 alkyl groups. In some embodiments, the cycloalkyl group has 3 to 8 ring members, while in other embodiments, the number of ring carbon atoms ranges from 3 to 5, 3 to 6, or 3 to 7. Such cycloalkyl groups include, for example, monocyclic structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 1-methylcyclopropyl, 2-methylcyclopentyl, 2-methylcyclooctyl, etc., or polycyclic or bridged ring structures such as adamantyl. Examples of unsaturated cycloalkyl groups include cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, and hexadienyl, etc. Cycloalkyl groups may be substituted or unsubstituted. Such substituted cycloalkyl groups include, for example, cyclohexanone.

[0048] "Aryl" is an aromatic carbocyclic group having 6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiple fused rings (e.g., naphthyl or anthracene). In some embodiments, the aryl group contains 6 to 14 carbon atoms in the ring portion of the group, and in other embodiments, it contains 6 to 12 or even 6 to 10 carbon atoms. Specific aryl groups include phenyl, biphenyl, naphthyl, etc. Aryl groups may be substituted or unsubstituted. The phrase "aryl" also includes groups containing fused rings, such as fused aromatic-aliphatic ring systems (e.g., indanyl, tetrahydronaphthyl, etc.).

[0049] "Aneryl" is a divalent aryl group as defined in this article.

[0050] "Heteroaryl" is an aryl ring system in a heteroaromatic ring system having one to four heteroatoms as ring atoms, wherein the remaining atoms are carbon atoms. In some embodiments, the heteroaryl contains 5 to 6 ring atoms in the ring portion of the group, and in other embodiments, it contains 6 to 9 or 6 to 10 atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen. In some embodiments, the heteroaryl ring system is monocyclic or bicyclic. Non-limiting examples include, but are not limited to, the following groups: such as pyrroloyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyrroloyl, pyridinyl, pyrazinyl, thiophene, benzothiophene, furanyl, benzofuranyl (e.g., isobenzofuran-1,3-diimine), indoleyl, azaindoleyl (e.g., pyrrolopyridinyl or 1H-pyrrolo[2,3-b]pyridinyl), indazoleyl, benzimidazolyl (e.g., 1H-benzo[d]imidazolyl), imidazopyridyl (e.g., azabenzimidazolyl, 3H-imidazo[4,5-b]pyridyl or 1H-imidazo[4,5-b]pyridyl), pyrazolopyridyl, triazolopyridyl, benzotriazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, isoxazolopyridyl, thionyl, purine, xanthine, adenine, guanine, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl and quinazolinyl.

[0051] "Anearyl" is a divalent heteroaryl group as defined in this article.

[0052] A "heterocyclic group" is an aromatic (also called a heteroaryl) or non-aromatic cycloalkyl group in which one to four ring carbon atoms are independently replaced by heteroatoms from the group consisting of O, S, and N. In some embodiments, the heterocyclic group comprises 3 to 10 ring members, while other such groups have 3 to 5, 3 to 6, or 3 to 8 ring members. The heterocyclic group may also be bonded to other groups at any ring atom (i.e., at any carbon atom or heteroatom of the heterocycle). The heterocyclic group may be substituted or unsubstituted. Heterocyclic groups encompass unsaturated, partially saturated, and saturated ring systems, such as, for example, imidazolyl, imidazolinyl, and imidazoalkyl. The term "heterocyclic group" includes fused ring types, including those containing fused aromatic and non-aromatic groups, such as, for example, benzotriazolyl, 2,3-dihydrobenzo[1,4]dioxane-hexenyl, and benzo[1,3]dioxane-pentenyl. The term also includes bridged polycyclic systems containing heteroatoms, such as, but not limited to, quinine cycloalkanes. Representative examples of heterocyclic groups include, but are not limited to, aziridinyl, aziridine, pyrrolyl, imidazoyl, pyrazolyl, thiazoyl, tetrahydrothiophene, tetrahydrofuranyl, dioxacyclopentenyl, furanyl, thiophene, pyrrolyl, pyrrololinyl, imidazolyl, imidazolinyl, pyrazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolinyl, thiazolinyl, isothiazolyl, thiazolyl, thiadiazolyl, oxadiazolyl, piperidinyl, piperazinyl, morpholinyl, and thiomorphoyl. Phinyl, tetrahydropyranyl (e.g., tetrahydro-2H-pyranyl), tetrahydrothiaranyl, oxothiacyclohexane, dioxacyclohexyl, dithiaranyl, pyridyl, pyrimidinyl, pyrazinyl, triazinyl, dihydropyridinyl, dihydrodithiazinyl, dihydrodithionyl, peripetaazinyl, quininecycloyl, indoleyl, indolinyl, isoindoleyl, azaindoleyl (pyrrolopyridyl), indazoleyl, inazinyl, benzotriazolyl, benzimidazolyl, benzofuranyl, benzo[] Thiophene, benzothiazolyl, benzoxadiazolyl, benzoxazinyl, benzodithiazinyl, benzoxoxazinyl, benzothiazinyl, benzoxazolyl, benzothiazolyl, benzothiazolyl, benzo[1,3]dioxacyclopentenyl, pyrazolopyridyl, imidazopyridyl (azabenzimidazolyl; e.g., 1H-imidazo[4,5-b]pyridyl or 1H-imidazo[4,5-b]pyridyl), triazolopyridyl, isoxazolopyridyl, purine, xanthine The following are listed: adenine, guanine, quinolinyl, isoquinolinyl, quinazinyl, quinoxalinyl, quinazolinyl, cinolinyl, phthalazinyl, naphthidyl, pteridinyl, thionyl, dihydrobenzothiazinyl, dihydrobenzofuranyl, dihydroindolyl, dihydrobenzodioxane-hexenyl, tetrahydroindolyl, tetrahydroindazoleyl, tetrahydrobenzimidazolyl, tetrahydrobenzotriazolyl, tetrahydropyrrolopyridyl, tetrahydropyrazolopyridyl, tetrahydroimidazopyridyl, tetrahydrotriazolopyridyl, and tetrahydroquinolinyl.Representative substituted heterocyclic groups can be monosubstituted or substituted more than once, such as, but not limited to, pyridyl or morpholinyl, which are 2-substituted, 3-substituted, 4-substituted, 5-substituted, or 6-substituted, or disubstituted with various substituents (such as those listed below). "Cycloalkylalkyl" is a group of the formula -alkyl-cycloalkyl, wherein the alkyl and cycloalkyl groups are as defined above. Substituted cycloalkylalkyl groups can be substituted at the alkyl, cycloalkyl, or both alkyl and cycloalkyl portions of the group. Representative cycloalkylalkyl groups include, but are not limited to, cyclopentylmethyl, cyclopentylethyl, cyclohexylmethyl, cyclohexylethyl, and cyclohexylpropyl. Representative substituted cycloalkylalkyl groups can be monosubstituted or substituted more than once.

[0053] "Aryl" is a group of the formula -alkyl-aryl, wherein the alkyl and aryl groups are as defined above. A substituted aryl group may be substituted at the alkyl, aryl, or both alkyl and aryl portions of the group. Representative aryl groups include, but are not limited to, benzyl and phenethyl, as well as fused (cycloalkylaryl)alkyl groups, such as 4-ethyl-indenyl.

[0054] "Heterocyclic alkyl" is a group of the formula -alkyl-heterocyclic, wherein the alkyl and heterocyclic groups are as defined above. A substituted heterocyclic alkyl group may be substituted at the alkyl, heterocyclic, or both alkyl and heterocyclic portions of the group. Representative heterocyclic alkyl groups include, but are not limited to, 4-ethyl-morpholino, 4-propylmorpholino, furan-2-ylmethyl, furan-3-ylmethyl, pyridin-3-ylmethyl, (tetrahydro-2H-pyran-4-yl)methyl, (tetrahydro-2H-pyran-4-yl)ethyl, tetrahydrofuran-2-ylmethyl, tetrahydrofuran-2-ylethyl, and indole-2-ylpropyl.

[0055] "Halogen" refers to chlorine, iodine, bromine, or fluorine.

[0056] "Hydroxyalkyl" is an alkyl group as described above that has been replaced by one or more hydroxyl groups.

[0057] "Alkoxy" is O (alkyl), where alkyl is as defined above.

[0058] "Alkoxyalkyl" is (alkyl)O(alkyl), where alkyl is as defined above.

[0059] As used herein, "alkynyl" refers to a monovalent hydrocarbon moiety containing at least two carbon atoms and one or more carbon-carbon triple bonds. The alkynyl group may optionally be substituted and may be straight-chain, branched-chain, or cyclic. Alynyl groups include, but are not limited to, those having 2-20 carbon atoms, i.e., C1... 2-20 Alkynyl group; 2-12 carbon atoms, i.e., C 2-12 Alkynyl group; 2-8 carbon atoms, i.e., C64. 2-8 Alkynyl group; 2-6 carbon atoms, i.e., C64. 2-6The alkynyl group; and 2-4 carbon atoms, i.e., C 2-4 Alkynyl group. Examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, and butynyl.

[0060] As used herein, “haloalkyl” means an alkyl group as defined above, wherein the alkyl group includes at least one substituent selected from halogens (e.g., fluorine (F), chlorine (Cl), bromine (Br), or iodine (I)). Examples of haloalkyl groups include, but are not limited to, -CF3, -CH2CF3, -CCl2F, and -CCl3.

[0061] As used herein, “haloalkoxy” means an alkoxy group as defined above, wherein the alkoxy group includes at least one substituent selected from halogens (e.g., F, Cl, Br or I).

[0062] As used herein, “arylalkyl” refers to the monovalent portion of a group in an alkyl compound, wherein the alkyl compound is substituted with an aromatic substituent, i.e., the aromatic compound includes a single bond with an alkyl group, and wherein the group is located on the alkyl group. The arylalkyl group is bonded to the chemical structure shown by the alkyl group. The arylalkyl group can be represented by, for example, the following structures: B-CH2-, B-CH2-CH2-, B-CH2-CH2-CH2-, B-CH2-CH2-CH2-CH2-, B-CH(CH3)-CH2-CH2-, B-CH2-CH(CH3)-CH2-, where B is an aromatic moiety, such as phenyl. The arylalkyl group may optionally be substituted, i.e., the aryl and / or alkyl groups may be substituted as disclosed herein. Examples of arylalkyl groups include, but are not limited to, benzyl.

[0063] As used herein, “alkylaryl” refers to the monovalent portion of a group in an aryl compound, wherein the aryl compound is substituted with an alkyl substituent, i.e., the aryl compound comprises a single bond with an alkyl group, and wherein the group is located on an aryl group. Alkylaryl groups are bonded to the illustrated chemical structure via the aryl group. Alkylaryl groups can be represented by, for example, the following structures: -B-CH3, -B-CH2-CH3, -B-CH2-CH2-CH3, -B-CH2-CH2-CH2-CH3, -B-CH(CH3)-CH2-CH3, -B-CH2-CH(CH3)-CH3, where B is an aromatic moiety, such as a phenyl group. Alkylaryl groups are optionally substituted, i.e., the aryl group and / or the alkyl group may be substituted as disclosed herein. Examples of alkylaryl groups include, but are not limited to, tolueneyl groups.

[0064] As used herein, “aryloxy group” refers to the monovalent portion of a group in an aromatic compound, wherein the ring atom is a carbon atom and wherein the ring is substituted by an oxygen group, i.e., the aromatic compound comprises a single bond with an oxygen atom, and wherein the group is located on the oxygen atom, for example, C6H5-O- for phenoxy groups. The aryloxy substituents are bonded to the compound they substituted through this oxygen atom. The aryloxy group is optionally substituted. Aryloxy groups include, but are not limited to, those having 6 to 20 ring carbon atoms, i.e., C6H5-O-. 6-20 Aryloxy group; 6 to 15 ring carbon atoms, i.e., C 6-15 Aryloxy groups; and 6 to 10 ring carbon atoms, i.e., C 6-10 Aryloxy group. Examples of aryloxy groups include, but are not limited to, phenoxy, naphthoxy, and anthraceneoxy groups.

[0065] "Amino" is a group with the formula NH2.

[0066] The "hydroxylamine" group is of the formula N(R) # )OH or NHOH groups, wherein R # It is a substituted or unsubstituted alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclic or heterocyclic alkyl group as defined herein.

[0067] The "alkoxyamine" group is of the formula -N(R # )O-alkyl or -NHO-alkyl groups, wherein R # As defined above.

[0068] The "arylalkoxyamine" group is of the formula N(R) # )O-aryl or NHO-aryl groups, wherein R # As defined above.

[0069] "Amino carbonyl" is a group with the following formula: -C(=O)N(R # )2、-C(=O)NH(R # ) or C(=O)NH2, where each R # As defined above.

[0070] "Acylamino" is a group of the following formula: NHC(=O)(R # ) or N(alkyl)C(=O)(R # ), wherein each alkyl group and R # Independently as defined above.

[0071] "O(alkyl)aminocarbonyl" is a group with the following formula: -O(alkyl)C(=O)N(R) # )2、-O(alkyl)C(=O)NH(R) # ) or -O(alkyl)C(=O)NH2, where each R #Independently as defined above.

[0072] The "N-oxide" group is of the formula -N + -O - . group.

[0073] The "carboxyl group" is a group with the formula C(=O)OH.

[0074] The ketone group is represented by the formula C(=O)(R). # ) groups, wherein R # As defined above.

[0075] The "aldehyde" group is a group with the formula -CH (=O).

[0076] The "ester" group is a group with the following formula: C(=O)O(R) # ) or OC(=O)(R # ), where R # As defined above.

[0077] The "urea" group is a group with the following formula: -N(alkyl)C(=O)N(R) # )2、-N(alkyl)C(=O)NH(R # -N(alkyl)C(=O)NH2, -NHC(=O)N(R) # )2、-NHC(=O)NH(R # ) or NHC(=O)NH2 # Each alkyl group and R # Independently as defined above.

[0078] The "imine" group is a group with the following formula: -N=C(R # )2 or -C(R # )=N(R # ), where each R # Independently as defined above.

[0079] "Imine" is a group with the following formula: -C(=O)N(R#)C(=O)(R # ) or N((C=O)(R # ))2, where each R # Independently as defined above.

[0080] The "carbamate" group is a group with the following formula: -OC(=O)N(R) # )2、-OC(=O)NH(R # ), -N(R # )C(=O)O(R # ) or -NHC(=O)O(R # ), where each R# Independently as defined above.

[0081] The "midamine" group is a group with the following formula: -C(=N(R # ))N(R # )2、-C(=N(R # ))NH(R # -C(=N(R) # ))NH2、-C(=NH)N(R # )2、-C(=NH)NH(R # -C(=NH)NH2, -N=C(R)NH2 # )N(R # 2. -N=C(R) # )NH(R # -N=C(R) # )NH2、-N(R # )C(R # )=N(R # ),-NHC(R # )=N(R # ), -N(R # )C(R # )=NH or -NHC(R # )=NH, where each R # Independently as defined above.

[0082] The "guanidine" group is a group with the following formula: -N(R # )C(=N(R # ))N(R # )2、-NHC(=N(R # ))N(R # )2、-N(R # )C(=NH)N(R # )2、-N(R # )C(=N(R # ))NH(R # ), -N(R # )C(=N(R # ))NH2、-NHC(=NH)N(R # )2、-NHC(=N(R # ))NH(R # -NHC(=N(R) # ))NH2、-NHC(=NH)NH(R # -NHC(=NH)NH2, -N=C(N(R) # )2)2、-N=C(NH(R #))2 or -N=C(NH2)2, where each R # Independently as defined above.

[0083] The "enamine" group is a group with the following formula: -N(R # )C(R # )=C(R # )2、-NHC(R # )=C(R # )2、-C(N(R # )2)=C(R # )2、-C(NH(R # ))=C(R # 2. -C(NH2)=C(R) # )2、-C(R # )=C(R # )(N(R # )2) C(R) # )=C(R # )(NH(R # )) or -C(R # )=C(R # (NH2), where each R # Independently as defined above.

[0084] The oxime group is a group with the following formula: -C(=NO(R) # ))(R # -C(=NOH)(R) # -CH(=NO(R) # )) or -CH (=NOH), where each R # Independently as defined above.

[0085] The "acylhydrazine" group is a group with the following formula: -C(=O)N(R) # )N(R # )2、-C(=O)NHN(R # )2、-C(=O)N(R # )NH(R # -C(=O)N(R) # )NH2、-C(=O)NHNH(R # )2 or -C(=O)NHNH2, where each R # Independently as defined above.

[0086] The hydrazine group is a group with the following formula: -N(R # )N(R # )2、-NHN(R # )2、-N(R # )NH(R# ), -N(R # )NH2、-NHNH(R # )2 or -NHNH2, where each R # Independently as defined above.

[0087] The "hydrazone" group is a group with the following formula: -C(=NN(R) # )2)(R # )2、-C(=NNH(R # ))(R # )2、-C(=N-NH2)(R # )2、-N(R # (N=C(R)) # )2) or -NH(N=C(R # )2), where each R # Independently as defined above.

[0088] The "azide" group is a group of the formula -N3.

[0089] The "isocyanate" group is a group with the formula N=C=O.

[0090] The "isothiocyanate" group is a group with the formula N=C=S.

[0091] The "cyanate ester" group is a group of the formula OCN.

[0092] The "thiocyanate" group is a group of the formula SCN.

[0093] The "thioether" group is a group with the following formula: -S(R # ), where R # As defined above.

[0094] The "thiocarbonyl" group is a group with the following formula: -C(=S)(R # ), where R # As defined above.

[0095] "Sylenyl group" is a group with the following formula: -S(=O)(R # ), where R # As defined above.

[0096] The sulfone group is a group with the following formula: -S(=O)2(R # ), where R # As defined above.

[0097] "Sulfonylamino" is a group with the following formula: -NHSO2(R # ) or -N(alkyl)SO2(R # ), wherein each alkyl group and R# As defined above.

[0098] The "sulfonamide" group is a group with the following formula: -S(=O)2N(R) # )2 or -S(=O)2NH(R # ) or -S(=O)2NH2, where each R # Independently as defined above.

[0099] The "phosphonate" group is a group with the following formula: -P(=O)(O(R) # ))2, -P(=O)(OH)2, -OP(=O)(O(R # ))(R # ) or -OP(=O)(OH)(R # ), where each R # Independently as defined above.

[0100] The "phosphine" group is a group with the following formula: -P(R # )2, where each R # Independently as defined above.

[0101] When groups described herein (other than alkyl groups) are referred to as “substituted,” they may be substituted with any one or more suitable substituents. Illustrative examples of substituents are those found in the compounds and embodiments disclosed herein, as well as halogens (chlorine, iodine, bromine, or fluorine); alkyl; hydroxyl; alkoxy; alkoxyalkyl; amino; alkylamino; carboxyl; nitro; cyano; thiol; thioether; imine; imide; amidine; guanidine; enamine; aminocarbonyl; acylamino; phosphonate; phosphine; thiocarbonyl; sulfinyl; sulfonyl; sulfonamide; ketone; aldehyde; ester; urea; carbamate; oxime; hydroxyamine; alkoxyamine; arylalkoxyamine; N-oxide; hydrazine; acylhydrazine; hydrazone; azide; isocyanate; isothiocyanate; cyanate; thiocyanate; oxygen (═O); B(OH)₂; O(alkyl)aminocarbonyl; cycloalkyl, which may It can be a monocyclic or fused or non-fused polycyclic (e.g., cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl); or a heterocyclic group, which can be a monocyclic or fused or non-fused polycyclic (e.g., pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, or thiazinyl); a monocyclic or fused or non-fused polycyclic aryl or heteroaryl (e.g., phenyl, naphthyl, pyrrolidinyl, indolyl, furanyl, thiophene, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, triazolyl, tetrazolyl, pyrazolyl, pyridinyl, quinolinyl, isoquinolinyl, acridineyl, pyrazinyl, pyridazinyl, pyrimidinyl, benzimidazolyl, benzothiophene, or benzofuranyl); aryloxy; arylalkoxy; heterocyclic oxy; and heterocyclic alkoxy.

[0102] As used herein, “pharmaceuticalally acceptable salt” refers to a salt prepared from a pharmaceutically acceptable, non-toxic acid or base (including inorganic acids or bases and organic acids or bases).

[0103] As used herein and unless otherwise indicated, the term "solvent" means a compound or a salt thereof that also includes a stoichiometric or non-stoichiometric amount of solvent bound by non-covalent intermolecular forces. In one embodiment, the solvate is a hydrate.

[0104] As used herein and unless otherwise indicated, the term "hydrate" means a compound or its salt that also includes stoichiometric or nonstoichiometric amounts of water bound together by noncovalent intermolecular forces.

[0105] As used herein and unless otherwise indicated, the term "prodrug" means a compound derivative that can be hydrolyzed, oxidized, or otherwise reacted under biological conditions (in vitro or in vivo) to provide an active compound. Examples of prodrugs include, but are not limited to, derivatives and metabolites of compounds that include biohydrolyzable moieties, such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable acylureas, and biohydrolyzable phosphate ester analogs. In some embodiments, the prodrug of a compound having a carboxyl functional group is a lower alkyl ester of a carboxylic acid. Carboxylic acid esters can be formed by esterification of any carboxylic acid moiety present on the molecule. Prodrugs are generally prepared using well-known methods, such as by... Burger's Medicinal Chemistry and Drug Discovery 6th edition (edited by Donald J. Abraham, 2001, Wiley) and Design and Application of Prodrugs The methods described in (H. Bundgaard, ed., 1985, Harwood Academic Publishers Gmfh).

[0106] As used herein and unless otherwise indicated, the terms "stereoisomer" or "stereoisomer-pure" mean that one stereoisomer of a compound is substantially free of other stereoisomers of the compound. For example, a stereoisomer-pure compound having one chiral center will substantially free of its opposite enantiomers. A stereoisomer-pure compound having two chiral centers will substantially free of other diastereomers of the compound. A typical stereoisomer-pure compound contains more than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of other stereoisomers of the compound, more than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of other stereoisomers of the compound, more than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of other stereoisomers of the compound, or more than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of other stereoisomers of the compound. Compounds may have a chiral center and may occur as racemates, individual enantiomers, or diastereomers and mixtures thereof. All such isomeric forms are included within the embodiments disclosed herein, including mixtures thereof. The use of stereoisomeric pure forms of such compounds and mixtures thereof is covered in the embodiments disclosed herein. For example, mixtures comprising equal or unequal amounts of enantiomers of a particular compound may be used in the methods and compositions disclosed herein. These isomers may be resolved by asymmetric synthesis or using standard techniques such as chiral columns or chiral resolving agents. See, for example, Jacques, J. et al. Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen, SH et al., Tetrahedron 33:2725 (1977); Eliel, EL, Stereochemistry of Carbon Compounds (McGrawHill, NY, 1962); and Wilen, SH, Tables of Resolving Agents and Optical Resolutions Page 268 (EL Eliel, ed., Univ. of Notre Dame Press, Notre Dame, IN, 1972).

[0107] It should also be noted that the compound may include E and Z isomers, or mixtures thereof, as well as cis and trans isomers, or mixtures thereof. In some embodiments, the compound is isolated into cis or trans isomers. In other embodiments, the compound is a mixture of cis and trans isomers.

[0108] "Tautomers" refer to the isomers of a compound that are in equilibrium with each other. The concentration of the isomers will depend on the environment in which the compound is present and may vary depending on, for example, whether the compound is a solid or in an organic or aqueous solution. For example, in aqueous solution, pyrazole may exhibit the following isomers, which are referred to as tautomers of each other: .

[0109] Those skilled in the art will readily understand that various functional groups and other structures may exhibit tautomerism, and all tautomers of the compound are within the scope of this disclosure.

[0110] It should also be noted that one or more atoms in a compound may contain atomic isotopes in non-natural proportions. For example, the compound may contain atomic isotopes such as tritium (…). 3 H), Iodine-125 ( 125 I), sulfur-35 ( 35 S) or carbon-14 ( 14 Radiolabeling can be performed using radioactive isotopes such as C, or deuterium (C). 2 H), carbon-13 ( 13 C) or nitrogen-15 ( 15 N) Isotope enrichment. As used herein, “isotope body” refers to an isotopically enriched compound. The term “isotope enrichment” means that the atoms have an isotopic composition other than the natural isotopic composition of the atoms. “Isotope enrichment” can also refer to a compound in which at least one atom has an isotopic composition different from the natural isotopic composition of the atoms. The term “isotopic composition” refers to the amount of each isotope present in a given atom. Radiolabeled and isotopically enriched compounds can be used as therapeutic agents (e.g., cancer and inflammation treatment agents), research reagents (e.g., binding analytical reagents), and diagnostic agents (e.g., in vivo imaging agents). All isotopic variations (whether radioactive or not) of the compounds described herein are intended to be covered within the scope of the embodiments provided herein. In some embodiments, isotopes of the compounds are provided, for example, isotopes enriched in deuterium, carbon-13, or nitrogen-15.

[0111] It should be noted that if there is an inconsistency between the described structure and the name of the structure, the described structure should be given higher weight.

[0112] As used herein, the term "residue" refers to the chemical portion remaining after an intrinsic chemical reaction of a compound. For example, the terms "amino acid residue" or "N-alkyl amino acid residue" refer to the product of amide or peptide coupling of an amino acid or N-alkyl amino acid with a suitable coupling agent; wherein, for example, amide or peptide coupling of an amino acid or N-alkyl amino acid results in the expulsion of a water molecule, thereby producing a product containing an amino acid residue or N-alkyl amino acid residue.

[0113] As used herein, "sugar," "glycosyl," or "sugar residue" refers to a carbohydrate moiety that may comprise a 3-carbon (triose) unit, a 4-carbon (teuose) unit, a 5-carbon (pentose) unit, a 6-carbon (hexose) unit, a 7-carbon (heptaose) unit, or a combination thereof, and may be a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, pentasaccharide, oligosaccharide, or any other polysaccharide. In some cases, "sugar," "glycosyl," or "sugar residue" comprises furanose (e.g., furanose ribose, furanose fructose) or pyranose (e.g., pyranose glucose, pyranose galactose) or a combination thereof. In some cases, "sugar," "glycosyl," or "sugar residue" comprises aldose or ketose, or a combination thereof. Non-limiting examples of monosaccharides include ribose, deoxyribose, xylose, arabinose, glucose, mannose, galactose, and fructose. Non-limiting examples of disaccharides include sucrose, maltose, lactose, lactulose, and trehalose. Other “sugars” or “glycosyl” or “sugar residues” include polysaccharides and / or oligosaccharides, including but not limited to amylose, amylopectin, glycogen, inulin, and cellulose. In some cases, “sugars” or “glycosyl” or “sugar residues” are amino-sugars. In some cases, “sugars” or “glycosyl” or “sugar residues” are glucosamine residues (1-amino-1-deoxy-D-glucol), which are linked to the rest of the molecule via their amino groups, thereby forming an amide bond with the rest of the molecule (i.e., glucosamide).

[0114] As used herein, “binding agent” means any molecule capable of specifically binding to a given binding agent (e.g., antigen), such as an antibody.

[0115] As used herein, the term "amino acid" refers to an organic compound containing an amino (-NH2) and a carboxyl (-COOH) functional group, as well as a side chain (R group), the side chain being specific for each amino acid. Amino acids can be proteogenous or non-proteogenous. "Proteogenous" means that an amino acid is one of the twenty naturally occurring amino acids found in proteins. Proteogenous amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. "Non-proteogenous" means that an amino acid is not naturally found in proteins or is not directly produced by cellular mechanisms (e.g., it is a product of post-translational modification). Non-limiting examples of non-proteogenous amino acids include γ-aminobutyric acid (GABA), taurine (2-aminoethanesulfonic acid), theanine (L-γ-glutamylethylamide), hydroxyproline, β-alanine, ornithine, and citrulline.

[0116] As used herein, the term "peptide," in its broadest sense across its various grammatical forms, refers to a compound consisting of two or more subunit amino acids, amino acid analogs, or other peptide mimics. These subunits may be linked by peptide bonds or other bonds such as ester bonds, ether bonds, etc. The term "amino acid," as used herein, refers to natural and / or non-natural, proteogenic or non-proteogenic, or synthetic amino acids, including glycine and its D or L optical isomers, as well as amino acid analogs and peptide mimics. If the peptide chain is short, such as two, three, or more amino acids, it is generally called an oligopeptide. If the peptide chain is long, the peptide is generally called a polypeptide or protein. The definition encompasses full-length proteins, their analogs, mutants, and fragments. The term also includes post-expression modifications of polypeptides, such as glycosylation, acetylation, phosphorylation, etc. Furthermore, due to the presence of ionizable amino and carboxyl groups in the molecule, certain peptides can be obtained in acidic or basic salt or neutral forms. Peptides can be obtained directly from the source organism or can be produced recombinantly or synthetically.

[0117] The amino acid sequences of antibodies can be numbered using any known numbering scheme, including those described below: Kabat et al. (“Kabat” numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 (“Chothia” numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 (“Contact” numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 (“IMGT” numbering scheme); and Honegge and Pluckthun, J. Mol. Biol., 2001, 309:657-70 (“AHo” numbering scheme). Unless otherwise stated, the numbering scheme used herein is the Kabat numbering scheme. However, the choice of numbering scheme does not imply the absence of differences in the sequence, and those skilled in the art can readily confirm the sequence positions by examining the amino acid sequences of one or more antibodies. Unless otherwise stated, when referring to residues in the constant region of the antibody heavy chain, the “EU numbering scheme” is generally used (e.g., as reported by Kabat et al., ibid.).

[0118] As used in this article, “cytotoxic activity” refers to the activity that reduces or diminishes the viability of the tested cell lines.

[0119] In the following claims and in the preceding description, unless the context requires otherwise due to the language of expression or necessary implication, the word “comprise” or variations thereof (such as “comprises / comprising”) are used to include the meaning that specifies the presence of the described feature in the various embodiments rather than excluding the presence of additional features or adding additional features.

[0120] Aspect 1 In some implementations, the platform is a conjugate-connector-payload compound of formula (I): (I) Or its pharmaceutically acceptable salts, tautomers, solvates, stereoisomers, or isotopes, wherein: Each of rings A and B is independently one of equations (IIa), (IIb), (IIc), (IId), (IIe), (IIIf), or (IIg): , , , , , or ;in: Each ring C is independently a cyclopropyl ring or a cyclobutyl ring; Each R 1 Independently H, OH, C 1-4 Alkyl, C 1-4 Alkyl group or -O-(CH2CH2O) t1 -CH3; Each R 2 Independently H or C 1-4 alkyl; Each R 3 and R 4 Independently for H, NR a1 R b1 OH, C 1-4 Alkyl, C 1-4 alkoxy or aryl; Each R a1 and R b1 Independently H or C 1-4 alkyl; Each R 5 Independently for H and C 1-4 Alkyl, C 1-4 alkoxy or aryl; Each m1, n1, and o1 is independently 1 or 2; t1 can be 1, 2, 3, 4, 5, 6, 7 or 8; The bonds marked by ring A being linked to a splittable 1 when s1 is 1 or to -H when s1 is 0, or the bonds marked by ring B being linked to a splittable 2; and The key is marked when ring A or ring B is connected to the connector; The connector is –(CH2). r -、-(CH2) p -X-(CH2) q -or-(CH2) p -CH=CH-(CH2) q -;in: Each r, p, and q is independently 1, 2, 3, 4, 5, 6, 7, or 8; The sum of p and q is 1, 2, 3, 4, 5, 6, 7 or 8; X is NR 6, NHC(=O), C(=O)NH, O, SO2, substituted or unsubstituted divalent aryl rings, substituted or unsubstituted divalent heteroaryl rings, substituted or unsubstituted divalent heterocycles, or substituted or unsubstituted divalent rings; and R 6 For H or C 1-4 alkyl; Decomposable 1, when present, has formula (VIa) or (VIc): or ,in: Su refers to the sugar portion; Each R 11 Independently hydrogen, halogen, substituted or unsubstituted C 1-4 Alkyl, -CN, or -NO2; and # Marks the bonds that can be broken down when linked to ring A; s1 is 0 or 1; The pyrolytic 2 has the formula (VIIa1), (VIIc1), (VIId1), or (VIIf1): , , or ;in: Su refers to the sugar portion; Each R 9 Independently hydrogen, halogen, substituted or unsubstituted C 1-4 Alkyl, -CN, or -NO2; # The bond in the case where the 2-linked ring B can be broken; and ## Marks bonds that can be split into 2-linked spacers; Spacers are bonds, ### -NH-(CH2CH2O) m2 -CH2CH2-C(═O)-、 ### -NH-(CH2CH2O) m2 -CH2-C(═O)-、 ### -(CH2) m2 -C(═O)-、 ### -CH2-C(═O)-NH-(CH2) m2 -C(═O)-、 ### -(CH2CH2O) m2 -CH2CH2-C(═O)-、 ### -CH[-(CH2) m2 -COOH]-C(═O)-、 ###-CH2-C(═O)-NH-(CH2) m2 -C(═O)-NH-(CH2) m2 -C(═O)-、 ### -C(═O)-(CH2) m2 -C(═O)- or ### -NH-(CH2) m2 -C(═O)-; where: Each m² is independently 1, 2, 3, 4, 6, 7, or 8; and ### The key in the case where the spacer is linked to the concatenation; and The conjoint has formula (III), (IV) or (V): , or ,in: U2 is a bond, a heteroaryl or a aryl group; V2 represents a bond or -C≡C-(CH2) n2 -; n2 is an integer from 0 to 10, inclusive. W2 is -C(=O)-, -NH-, or -O-; RG3 is or ; RS3 is -NR a2 R b2 ; R a2 and R b2 Each of them is independently H or substituted or unsubstituted C. 1-4 alkyl; RE3 is a key, -O-, -OC(=O-), -OC(=O)NR 7 -、-NHC(=O)NR 7 -、-OS(=O)2NR 7 -、-NHS(=O)2NR 7 -or-OC(=O)NHS(=O)2NR 7 -; R 7 H or substituted or unsubstituted C 1-4 alkyl; W3 is -C(=O)-, -NH-, or -O-; t3 is 1 or 2; s3 is 0, 1, or 2; RG4 is , , , or ; RE4 is a key, -O-, -OC(=O)-, -OC(=O)NR 8 -、-NHC(=O)NR 8 -、-OS(=O)2NR 8 -、-NHS(=O)2NR 8 -or-OC(=O)NHS(=O)2NR 8 -; R 8 H or substituted or unsubstituted C 1-4 alkyl; W4 is -C(=O)-, -NH-, or -O-; t4 is 1, 2, 3, 4, 5, 6, 7, or 8; and s4 can be 0, 1, or 2.

[0121] In some implementations, the connector is -(CH2). r Or -(CH2) p -X-(CH2) q -; Ring A has formula (IIa), (IIb), (IIc), (IId) or (IIg); and ring B has formula (IIa), (IIb), (IId) or (IIg).

[0122] In some implementations, the connector is -(CH2). r - Ring A has formula (IIg), and ring B has formula (IIg).

[0123] In some implementations, the connector is -(CH2). r - Ring A has equation (IId), and ring B has equation (IId).

[0124] In some implementations, the connector is -(CH2). r - Ring A has equation (IIb), and ring B has equation (IIa).

[0125] In some implementations, the connector is -(CH2). r - Ring A has equation (IIa), and ring B has equation (IIb).

[0126] In some implementations, the connector is -(CH2). r - Ring A has equation (IIa), and ring B has equation (IIa).

[0127] In some implementations, the connector is -(CH2). r - Ring A has equation (IIc), and ring B has equation (IIa).

[0128] In some implementations, the connector is -(CH2). p -X-(CH2) q - Ring A has equation (IIa), and ring B has equation (IIa).

[0129] In some implementations, the connector is -(CH2). r - Ring A has equation (IIc), and ring B has equation (IIb).

[0130] In some implementations, r is 3 or 5.

[0131] In some implementations, the connector is -(CH2). r -And R is 5.

[0132] In some implementations, the connector is -(CH2). p -X-(CH2) q - where X is an unsubstituted aryl ring, p is 1, and q is 1. In some embodiments, X is a phenylene oxide.

[0133] In some implementations, formula (IIa) is (IIa1).

[0134] In some implementations, formula (IId) is (IId1).

[0135] In some implementations, formula (IIg) is (IIg1).

[0136] In some implementations, formula (IIc) is (IIc1) (IIc2) or (IIc3).

[0137] In some implementations, formula (IIc) is (IIc3).

[0138] In some implementations, ring A is , , , , , , , , , or .

[0139] In some implementation schemes, ring B is , , , , , , , or .

[0140] In some implementations, when the connector is -(CH2) p -X-(CH2) q -or-(CH2) p -CH=CH-(CH2) q When -, ring A has equation (IIa), m in ring A is 2, and ring B has equations (IIa), (IIb), (IIc), (IId), (IIe), (IIIf) or (IIg).

[0141] In some implementations, when the connector is -(CH2) p -X-(CH2) q -or-(CH2) p -CH=CH-(CH2) q When -, ring A has equation (IIa), m in ring A is 1, and ring B has equations (IIa), (IIb), (IIc), (IId), (IIe), (IIIf) or (IIg).

[0142] In some implementations, the connector is –(CH2). r -、-(CH2) p -X-(CH2) q -or–(CH2) p -CH=CH-(CH2) q -; and X is O, NR 6 ,NHC(=O),-( m -C6H4)-、 or .

[0143] In some implementations, the connector is –(CH2). r -

[0144] In some implementations, the connector is –(CH2). p -O-(CH2) q -or–(CH2) p -NH-(CH2) q -

[0145] In some implementations, the sum of p and q is 4.

[0146] In some implementations, the connector is , , , or , , , , , , , , , , or .

[0147] In some implementations, the sum of p and q is 2.

[0148] In some implementations, the connector is .

[0149] In some implementations, the connector is –(CH2). p -CH=CH-(CH2) q -

[0150] In some implementations, the sum of p and q is 3.

[0151] In some implementations, each Su is independently for , , or , or its stereoisomers; each m is independently 0 or 1; and Indicates the connection point with either splittable 1 or splittable 2.

[0152] In some implementations, each Su is independently for or , or its stereoisomers.

[0153] In some implementations, each Su is .

[0154] In some implementations, s1 is 1.

[0155] In alternative embodiments, the pyrolytic 1 has formulas (VIa), (VIb), (VIc), (VId), (VIe), (VIIf), (VIg), (VIh), or (VIi): (VIa) (VIb) (VIc) (VId) (VIe) (VIf) (VIg) (VIh) or (VIi)

[0156] In some implementations, the pyrolytic 1 has formula (VIa).

[0157] In some implementations, 1 can be split into or .

[0158] In some implementations, s1 is 0.

[0159] In an alternative implementation, when s1 is 0, ring A has equations (IIa'), (IIb'), (IIc'), (IId'), (IIe'), (IIIf'), or (IIg'): , where R 10 Independently H, OH or C 1-4 Alkyl groups, and other variables (R) 1 R 2 R 3 R 4 R 5 m1, n1, o1) are as defined above regarding aspect 1.

[0160] In an alternative embodiment, the pyrolytic 2 has the following formulas: (VIIa1), (VIIb1), (VIIc1), (VIId1), (VIIe1), (VIIf1), (VIIa2), (VIIb2), (VIIc2), (VIId2), (VIIe2), (VIIf2), (VIIa3), (VIIb3), (VIIc3), (VIId3), (VIIe3), or (VIIf3): .

[0161] In some implementations, s1 is 1 and the decomposable 2 has one of the following: (VIIa1) (VIId1) (VIIa2) (VIId2) (VIIa3) or (VIId3).

[0162] In some embodiments, s1 is 1 and s2 is divisible by formula (VIIa1) or (VIId1).

[0163] In some embodiments, the pyrolytic 2 has formula (VIIa1) or (VIId1).

[0164] In some implementation schemes, each R 9 It is hydrogen.

[0165] In some implementations, it can be split into 2. or .

[0166] In some implementations, the spacer is ### -NH-(CH2CH2O) m2 -CH2CH2-C(═O-) or ### -NH-(CH2CH2O) m2 -CH2-C(═O)-.

[0167] In some implementations, the spacer is ### -NH-(CH2CH2O)4-CH2CH2-C(═O)- or ### -NH-(CH2CH2O)4-CH2-C(═O)-.

[0168] In some implementations, the spacer is ### -NH-(CH2CH2O)4-CH2CH2-C(═O)-.

[0169] In some implementations, the fascicle has formula (III).

[0170] In some implementations, U2 is an aryl group.

[0171] In some implementations, U2 is phenylene.

[0172] In some implementations, U2 is .

[0173] In some implementations, V2 is the key.

[0174] In some implementations, V2 is -C≡C-(CH2). n -

[0175] In some implementations, V2 is -C≡C-(CH2)3-.

[0176] In some implementations, U2 is a heteroaryl group.

[0177] In some implementations, U2 is a divalent pyrimidine ring.

[0178] In some implementations, U2 is or .

[0179] In some implementations, U2 is the key.

[0180] In some implementations, W2 is -C(=O)-.

[0181] In some implementations, the conjugate is .

[0182] In some implementations, the fascicle has formula (V).

[0183] In some implementations, RG4 is , , , or .

[0184] In some implementations, RG4 is or .

[0185] In some implementations, RG4 is .

[0186] In some implementations, t4 is 1, 2, 3, 4, 5, or 6.

[0187] In some implementations, t4 is 1, 2, 3, 4, or 5.

[0188] In some implementations, s4 is 0.

[0189] In some implementations, the sum of s4 and t4 is 1 or 5. In some implementations, RE4 is a key or -O-.

[0190] In some implementations, W4 is -C(=O)-.

[0191] In some implementations, the conjugate is or .

[0192] In some implementations, the fascicle has formula (IV).

[0193] In some implementations, RG3 is or .

[0194] In some implementations, RG3 is .

[0195] In some implementations, RS3 is NH2 or -N(CH3)2.

[0196] In some implementations, RS3 is NH2.

[0197] In some implementations, RE3 is the key.

[0198] In some implementations, t3 is 2.

[0199] In some implementations, RE3 is -O-.

[0200] In some implementations, s3 is 0.

[0201] In some implementations, W3 is -C(=O)-.

[0202] In some implementations, the conjugate is .

[0203] In some embodiments, the compound is one of the following: , , , , , , , , , , , , , , , , , or .

[0204] Aspect 2 In some implementations, the platform is a conjugate-connector-payload compound of formula (IA): (IA), Or its pharmaceutically acceptable salts, tautomers, solvates, stereoisomers, or isotopes, wherein: Each of rings A and B is independently one of equations (IIa), (IIb), (IIc), (IId), (IIe), (IIIf), or (IIg): in: Each ring C is independently a cyclopropyl ring or a cyclobutyl ring; Each R 1 Independently H, OH, C 1-4 Alkyl, C 1-4 Alkyl group or -O-(CH2CH2O) t1 -CH3; Each R 2 Independently H or C 1-4 alkyl; Each R 3 and R 4 Independently for H, NR a1 R b1 OH, C 1-4 Alkyl, C 1-4 alkoxy or aryl; R a1 and R b1 Each of them is independently H or C. 1-4 alkyl; Each R 5 Independently for H and C 1-4 Alkyl, C 1-4 alkoxy or aryl; Each m1, n1, and o1 is independently 1 or 2; t1 can be 1, 2, 3, 4, 5, 6, 7 or 8; The bonds marked by ring A being linked to a cleavable 1 when s1 is 1 or to -H when s1 is 0, or the bonds marked by ring B being linked to a cleavable 2, and The key is marked when ring A or ring B is connected to the connector; The connector is –(CH2). r -、-(CH2) p -X-(CH2) q -or-(CH2) p -CH=CH-(CH2) q -;in: Each r, p, and q is independently 1, 2, 3, 4, 5, 6, 7, or 8; The sum of p and q is 1, 2, 3, 4, 5, 6, 7 or 8; X is NR 6 , NHC(=O), C(=O)NH, O, SO2, substituted or unsubstituted divalent aryl rings, substituted or unsubstituted divalent heteroaryl rings, substituted or unsubstituted divalent heterocycles, or substituted or unsubstituted divalent rings; and R 6 For H or C 1-4 alkyl; Decomposable 1 has the following formula when it exists: ,in: Su refers to the sugar portion; Each R 11 Independently hydrogen, halogen, substituted or unsubstituted C 1-4Alkyl, -CN, or -NO2; and # Marks the bonds that can be broken down when linked to ring A; s1 is 0 or 1; The pyrolytic 2 has formula (VIIa1) or (VIId1): or ,in: Su refers to the sugar portion; Each R 9 Independently hydrogen or C 1-4 alkyl; # The bond in the case where the 2-linked ring B can be broken; and ## Marks bonds that can be split into 2-linked spacers; Spacers are bonds, ### -NH-(CH2CH2O) m2 -CH2CH2-C(═O-) or ### -NH-(CH2CH2O) m2 -CH2-C(═O)-; where: Each m² is independently 1, 2, 3, 4, 6, 7, or 8; and ### The marker spacer is linked to the key in the case of -C (=O)-; RE4 is the key or -O-; and t4 can be 1, 2, 3, 4, 5, 6, 7 or 8.

[0205] In some implementations, s1 is 0.

[0206] In some implementations, the connector is -(CH2). r Or -(CH2) p -X-(CH2) q - Ring A has formula (IIa), (IIb), (IIc), (IId) or (IIg), and ring B has formula (IIa), (IIb), (IId) or (IIg).

[0207] In some implementations, the connector is -(CH2). r - Ring A has formula (IIg), and ring B has formula (IIg).

[0208] In some implementations, the connector is -(CH2). r - Ring A has equation (IId), and ring B has equation (IId).

[0209] In some implementations, the connector is -(CH2). r- Ring A has equation (IIb), and ring B has equation (IIa).

[0210] In some implementations, the connector is -(CH2). r - Ring A has equation (IIa), and ring B has equation (IIb).

[0211] In some implementations, the connector is -(CH2). r - Ring A has equation (IIa), and ring B has equation (IIa).

[0212] In some implementations, the connector is -(CH2). r - Ring A has equation (IIc), and ring B has equation (IIa). In some implementations, the connector is -(CH2). p -X-(CH2) q - Ring A has equation (IIa), and ring B has equation (IIa).

[0213] In some implementations, the connector is -(CH2). r - Ring A has equation (IIc), and ring B has equation (IIb).

[0214] In some implementations, r is 3 or 5.

[0215] In some implementations, the connector is -(CH2). r -And R is 5.

[0216] In some implementations, the connector is .

[0217] In some implementations, the sum of p and q is 2.

[0218] In some implementations, the connector is -(CH2). p -X-(CH2) q - where X is an unsubstituted aryl ring, p is 1, and q is 1. In some embodiments, X is a phenylene oxide.

[0219] In some implementations, formula (IIa) is (IIa1).

[0220] In some implementations, formula (IId) is (IId1).

[0221] In some implementations, formula (IIg) is (IIg1).

[0222] In some implementations, formula (IIc) is (IIc1) (IIc2) or (IIc3).

[0223] In some implementations, formula (IIc) is (IIc3).

[0224] In some implementations, ring A is , , , , , , , , , or .

[0225] In some implementation schemes, ring B is , , , , , , , or .

[0226] In some implementations, when the connector is -(CH2) p -X-(CH2) q -or-(CH2) p -CH=CH-(CH2) q When -, ring A has equation (IIa), m in ring A is 2, and ring B has equations (IIa), (IIb), (IIc), (IId), (IIe), (IIIf) or (IIg).

[0227] In some implementations, when the connector is -(CH2) p -X-(CH2) q -or-(CH2) p -CH=CH-(CH2) q When -, ring A has equation (IIa), m in ring A is 1, and ring B has equations (IIa), (IIb), (IIc), (IId), (IIe), (IIIf) or (IIg).

[0228] In some implementations, each Su is independently for , , or , or its stereoisomers; each m is independently 0 or 1; and Indicates the connection point with either splittable 1 or splittable 2.

[0229] In some implementations, each Su is independently for or , or its stereoisomers.

[0230] In some implementations, each Su is .

[0231] In some implementations, 1 can be split into or .

[0232] In some implementations, s1 is 0.

[0233] In an alternative implementation, when s1 is 0, ring A has equations (IIa'), (IIb'), (IIc'), (IId'), (IIe'), (IIIf'), or (IIg'): , where R 10 Independently H, OH or C 1-4 Alkyl groups, and other variables (R) 1 R 2 R 3 R 4 R 5 m1, n1, o1) are as defined above regarding aspect 2.

[0234] In some implementations, s1 is 1.

[0235] In some implementations, the pyrolytic 1 has formula (VIa).

[0236] In some implementations, the pyrolytic 2 has one of the following formulas: In some embodiments, the pyrolytic 2 has formula (VIIa1) or (VIId1).

[0237] In some implementation schemes, each R 9 It is hydrogen.

[0238] In some implementations, it can be split into 2. or .

[0239] In some implementations, the spacer is ### -NH-(CH2CH2O) m2 -CH2CH2-C(═O-) or ### -NH-(CH2CH2O) m2-CH2-C(═O)-.

[0240] In some implementations, the spacer is ### -NH-(CH2CH2O)4-CH2CH2-C(═O)- or ### -NH-(CH2CH2O)4-CH2-C(═O)-.

[0241] In some implementations, the spacer is ### -NH-(CH2CH2O)4-CH2CH2-C(═O)-.

[0242] In some implementation schemes, for .

[0243] In some embodiments, the compound is one of the following: or .

[0244] Aspect 3 This document provides ligand-drug conjugates or pharmaceutically acceptable salts or solvates thereof, wherein the conjugates comprise via a conjugate or Ligands conjugated to compounds provided herein, wherein the conjugates have one of the following formulas: Ligands - [Formula (I)] x Or ligand - [Formula (IA)] x , Where x is about 1 to about 12, and where equation (I) is as described above with respect to aspect 1 and equation (IA) is as described above with respect to aspect 2.

[0245] In one embodiment, x is about 1 to about 10. In one embodiment, x is about 1.5 to about 10. In one embodiment, x is about 1.5 to about 8. In one embodiment, x is about 1.5 to about 6. In one embodiment, x is about 1.5 to about 4. In one embodiment, x is about 1.8 to about 4. In one embodiment, x is about 1.8 to about 2.5. In one embodiment, x is about 2.

[0246] In some implementations, the ligand is a humanized, monoclonal, chimeric, or human antibody or its antigen-binding fragment.

[0247] In some implementations, the ligand passes through the compound's Partially conjugated to the compounds provided herein.

[0248] In some implementations, the conjugate has the following formula: Where Ab is a humanized, monoclonal, chimeric, or human antibody or its antigen-binding fragment, and the values ​​of the remaining variables (e.g., cleavable 1, s1, loop A, linker, loop B, cleavable 2, spacer, RE4, t4) are as described herein.

[0249] In some implementations, the ligand or Ab binds to one or more receptors selected from the following: B7-H3, cytokeratin 15, PTK7, HER3, HER2, CD7, CD19, CD20, CD22, CD25, CD27, CD30, CD33, CD37, CD38, CD46, CD70, CD71, CD74, CD79b, CD123, CD138, CD142, CD166, CD205, CD228, CCR2, CA6, p-cadherin, CEA, CE ACAM5, C4.4a, DLL3, EGFR, EGFRVIII, ENPP3, EphA2, EphrinA, FLOR1, FGFR2, GCC, cKIT, LIV1, LY6E, MSLN, MUC16, NaP i2b, Nectin4, gpNMB, PSMA, SLITRK6, STEAP1, TROP2, 5T4, SSEA4, GloboH, Gb5, STn, Tn, B7H3, BCMA, MUC1, cMet, ROR1 MSLN, FRa, CLDN18.2, CLDN6, PTK7, Axl, FGFR2b, CLL1, CCR7, GPC1, GPC3, ISAC, CDCP1, ITGB6, ADAM9 or CD45-iADC.

[0250] In one embodiment, Ab is an anti-CD74 antibody. In another embodiment, Ab is the anti-CD74 antibody STRO-001.

[0251] In some implementations, the conjugate is:

[0252] In some implementations, the conjugate is

[0253] Methods for preparing conjugates This document provides a method for preparing conjugates by contacting the conjugate (BA) with a conjugate-connector-loador compound (platform) under conditions suitable for forming a bond between the conjugate (BA) and the conjugate-connector-loador compound. The reaction conditions can be any suitable reaction conditions known in the art. The conjugate can be an antibody, and the bond can form an antibody-drug conjugate.

[0254] Examples of such reactions are provided in the embodiments below.

[0255] In some embodiments, the method of preparing the conjugate includes treating the compound with a binder under coupling conditions or contacting the compound with a binder. The compound may include a reactive linker bonded to at least one payload. The compound may be any of the linker or platform compounds disclosed herein.

[0256] Pharmaceutical Composition This document also provides compositions comprising the ADCs described herein, including pharmaceutical compositions. In some embodiments, the compositions (e.g., pharmaceutical compositions) further comprise pharmaceutically acceptable excipients.

[0257] The pharmaceutical compositions according to this disclosure can be prepared by mixing an antibody-drug conjugate having the desired purity with one or more optional pharmaceutically acceptable carriers in the form of a lyophilized formulation or an aqueous solution (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the doses and concentrations used and include, but are not limited to, buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexamethyl diammonium chloride; benzalkonium chloride; benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) (Based on) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers described herein also include interstitial drug dispersants, such as soluble, neutrally active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary embodiments of sHASEGP (including rHuPH20) and methods of use are described in U.S. Patent Nos. 7,871,607 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycans, such as chondroitinase.

[0258] Using conjugates In some embodiments, this document describes a method for treating a disease or condition (e.g., a proliferative disease, cancer, metabolic disease, inflammatory disease, or neurodegenerative disease) in a subject of need, the method comprising administering to the patient a therapeutically effective amount of the conjugate or pharmaceutical composition disclosed herein.

[0259] The conjugates disclosed herein may be administered by any suitable route, including parenteral, intrapulmonary, and intranasal administration, and, if local treatment is required, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Administration may be carried out by any suitable route, such as by injection (e.g., intravenous or subcutaneous), depending in part on whether the administration is transient or prolonged. This document considers various dosing schedules, including but not limited to single or multiple administrations at various time points, bolus administration, and pulsatile infusions.

[0260] The conjugates disclosed herein can be formulated, administered, and applied in accordance with good medical practice. Factors to be considered in this context include the specific disease being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the disease, the site of delivery, the method of administration, the schedule of administration, and other factors known to the medical practitioner.

[0261] Example The examples below are intended to be illustrative and should not be considered as limiting in any way. Unless otherwise stated, the experimental methods described in the examples below are conventional methods. Unless otherwise stated, reagents and materials are commercially available. All solvents and chemicals used are analytical grade or chemically pure. Solvents were redistilled before use. Anhydrous solvents were prepared according to standard or reference methods. Silica gel (100-200 mesh) for column chromatography and silica gel (GF254) for thin-layer chromatography (TLC) were available from Tsingdao Haiyang Chemical Co., Ltd. or Yantai Chemical Co., Ltd. in China; unless otherwise stated, elution was performed with petroleum ether (60-90°C) / ethyl acetate (v / v) and visualization was achieved by a solution of iodine or phosphomolybdic acid in ethanol. Unless otherwise stated, all extraction solvents were dried over anhydrous Na2SO4. Recordings were performed on a Bruck-400, Varian 400MR NMR spectrometer. 1 1H NMR spectra, with TMS (tetramethylsilane) as an internal standard. Coupling constants are given in Hertz. Peaks are reported as singlets (s), doublets (d), triplets (t), quartets (q), quintets (p), sextets (h), heptets (hept), multiplets (m), or combinations thereof; br represents a broad peak. LC / MS data were recorded using an Agilent 1100 or 1200 high-performance liquid chromatography-ion trap mass spectrometer (LC-MSD trap) equipped with a diode array detector (DAD) and an ion trap (ESI source) for detection at 214 nm and 254 nm. All compound names except those of the reagents mentioned are obtained via ChemDraw. ® Version 18.0 was generated.

[0262] In the following embodiments, the following abbreviations are used:

[0263] UPLC analysis method Method A: Mobile phase A: 0.1% FA aqueous solution, B: MeCN; Gradient: 10% B for 0.2 min, 10% - 95% B for 5.8 min, 95% B for 0.5 min; Flow rate: 0.6 mL / min; Column: ACQUITY UPLC® BEH C18 1.7µm.

[0264] Method B: Mobile phase A: 0.1% FA aqueous solution, B: MeCN; Gradient: 10% B for 0.5 min, 10% - 90% B for 2.5 min, 90% B for 0.2 min; Flow rate: 0.6 mL / min; Column: ACQUITY UPLC® BEH C18 1.7µm.

[0265] Method C: Mobile phase A: 0.1% FA aqueous solution, B: MeCN; Gradient: 10% B for 0.2 min, 10% - 90% B for 1.3 min, 90% B for 0.3 min; Flow rate: 0.6 mL / min; Column: ACQUITY UPLC® BEH C18 1.7µm.

[0266] Example Int-2 Step 1: ((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)carbamate (Int-2b) EEDQ (4.77 g, 19.3 mmol) was added to a solution of Int-2a (5 g, 18.4 mmol) and (4-aminophenyl)methanol (2.37 g, 19.3 mmol) in 100 mL of anhydrous THF. The mixture was stirred at rt for 40 h. The mixture was concentrated. The residue was liquefied with MTBE (30 V) slurry and stirred for 2 h. The solids were separated by filtration under vacuum for 3 h to give Int-2b (5.16 g, 74% yield). MS (ESI) m / z: 378.4 [M+H] + .

[0267] Step 2: (S)-2-amino-N-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropyl-2-yl)-3-methylbutyramide (Int-2c) Pd(PPh3)4 (76.6 mg, 0.066 mmol) was added to a solution of Int-2b (500 mg, 1.33 mmol) in CH2Cl2 (10 mL) and pyrrolidine (270.8 μL, 3.31 mmol) under rt and N2 conditions. The reaction mixture was stirred under rt for 0.5 h. The reaction was concentrated and purified by silica gel column chromatography (CH2Cl2 / MeOH = 92 / 8) to give Int-2c (370 mg, 95% yield) as a white solid. MS (ESI) m / z: 294.3 [M+H] + .

[0268] Step 3: ((17S,20S)-21-((4-(hydroxymethyl)phenyl)amino)-17-isopropyl-20-methyl-15,18,21-trioxo-3,6,9,12-tetraoxa-16,19-diazaeicosyl)carbamate (9H-fluorene-9-yl)methyl ester (Int-2) DIEA (326 mg, 2.52 mmol) was added to a solution of Int-2c (370 mg, 1.26 mmol) and HATU (575.8 mg, 1.51 mmol) in 4 mL of anhydrous DMF. The mixture was stirred at rt for 10 min. Then, a solution of Int-2d (645 mg, 1.33 mmol) in DMF (4 mL) was added to the mixture. The reaction was stirred for 1 h. The mixture was concentrated and purified by silica gel column chromatography (CH2Cl2 / MeOH = 95 / 5) to give a light brown solid Int-2 (680 mg, 71% yield). MS (ESI) m / z: 763.4 [M+H] + .

[0269] Example Int-7 Step 1: (2S,3R,4S,5S,6S)-triacetic acid 2-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nonadecano-19-amido)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (Int-7) A mixture of Int-7a (400 mg, 0.88 mmol; purchased from Bide Pharmacy), Int-2d (428 mg, 0.88 mmol; purchased from MCE), and EEDQ (282 mg, 1.14 mmol) in CH2Cl2 (5 mL) was stirred overnight at rt. The mixture was concentrated under vacuum to obtain a residue, which was purified by silica gel column chromatography (MeOH / CH2Cl2 = 0 / 100~1 / 99) to give Int-7 (364 mg, 50.8% yield) as a grayish-white solid.

[0270] Example Int-9 Step 1: (S)-(3-(hydroxymethyl)-3,4-dihydroisoquinoline-2(1H)-yl)(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)phenyl)methyl ketone (Int-9b) DECI (780 mg, 4.07 mmol) was added to a mixture of Int-9a (1.0 g, 2.71 mmol), HOPO (452 ​​mg, 4.07 mmol), and CH2Cl2 (15 mL). The mixture was stirred at rt for 10 min. Then, a mixture of (S)-(1,2,3,4-tetrahydroisoquinoline-3-yl)methanol (442 mg, 2.71 mmol), Et3N (943 μL, 6.78 mmol), and CH2Cl2 (5 mL) was added to the reaction mixture, and stirring was continued at rt for 1 hour. The mixture was then diluted with water (20 mL). 2) Washing. The organic layer was dried over Na₂SO₄ and filtered. The filtrate was concentrated under vacuum to obtain the residue, which was purified by silica gel column chromatography (petroleum ether / EtOAc = 50 / 50) to give a yellow solid Int-9b (1.1 g, 79.1% yield). MS (ESI) m / z: 515.4 [M+H] + .

[0271] Step 2: (S)-(3-(((tert-butyldimethylsilyl)oxy)methyl)-3,4-dihydroisoquinoline-2(1H)-yl)(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)phenyl)methyl ketone (Int-9c) Anhydrous TBSCl (484 mg, 3.21 mmol) was added to a solution of Int-9b (1.1 g, 2.14 mmol), imidazole (291 mg, 4.28 mmol), and CH2Cl2 (20 mL). The mixture was stirred at rt for 16 h. The mixture was filtered, and the filtered organic solvent was concentrated under vacuum to obtain a residue, which was purified by silica gel column chromatography (petroleum ether / EtOAc = 70 / 30) to give a yellow solid Int-9c (976 mg, 81.1% yield). MS (ESI) m / z: 629.4 [M+H] + .

[0272] Step 3: (S)-(3-(((tert-butyldimethylsilyl)oxy)methyl)-3,4-dihydroisoquinoline-2(1H)-yl)(4-hydroxy-5-methoxy-2-nitrophenyl) methyl ketone (Int-9d) LiOAc (80 mg, 1.21 mmol) was added to a solution of Int-9c (780 mg, 1.21 mmol), DMF (10 mL), and H2O (380 μL). The mixture was stirred at 40 °C for 2 h. After the reaction was complete, the mixture was diluted with water (20 mL) and then diluted with EtOAc (20 mL). 2) Extraction. The combined organic solvents were washed with brine (30 mL), dried over Na₂SO₄, filtered, and the filtrate was concentrated under vacuum to obtain the residue. This residue was purified by silica gel column chromatography (petroleum ether / EtOAc = 40 / 60) to give a yellow oil, Int-9d (571 mg, 99.5% yield). MS (ESI) m / z: 473.4 [MH] + .

[0273] Step 4: (S)-(3-(((tert-butyldimethylsilyl)oxy)methyl)-3,4-dihydroisoquinoline-2(1H)-yl)(4-((5-iodopentyl)oxy)-5-methoxy-2-nitrophenyl) ketone (Int-9) Add K₂CO₃ (523 mg, 3.78 mmol) to a solution of Int-9d (594 mg, 1.26 mmol), 1,5-diiodopentane (936 μL, 6.29 mmol), and DMF (5 mL). Stir the mixture overnight at rt. After the reaction is complete, dilute the mixture with water (10 mL) and EtOAc (10 mL). 2) Extraction. The combined organic compounds were washed with brine (20 mL), dried over Na₂SO₄, filtered, and the filtrate was concentrated under vacuum to obtain the residue. This residue was purified by silica gel column chromatography (petroleum ether / EtOAc = 70 / 30) to give a yellow oily Int-9 (642 mg, 76.2% yield). MS (ESI) m / z: 669.3 [M+H] + .

[0274] Example Int-10 Step 1: (S)-(2-(6-(((tert-butyldimethylsilyl)oxy)methyl)-5-azaspiro[2.4]heptane-5-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamate (Int-10b) Pyridine (158 μL, 1.96 mmol) was added to a 0°C solution of Int-10a (1.0 g, 1.78 mmol, prepared according to the procedure described in CN111164208A) and CH2Cl2 (15 mL). Then, AllocCl (227 μL, 2.14 mmol) was added to the mixture. The mixture was stirred at 0°C under N2 for 30 min. The mixture was then diluted with water (15 mL). 2) Washing. The organic layer was dried over Na2SO4 and filtered. The filtrate was concentrated under vacuum to obtain the residue, which was purified by silica gel column chromatography (petroleum ether / EtOAc = 70 / 30) to give a yellow solid Int-10b (885 mg, 79.1% yield). MS (ESI) m / z: 647.3 [M+H] + .

[0275] Step 2: (S)-(2-(6-(((tert-butyldimethylsilyl)oxy)methyl)-5-azaspiro[2.4]heptane-5-carbonyl)-5-hydroxy-4-methoxyphenyl)carbamate (Int-10c) Compound Int-10c (490 mg, 58.8% yield) was synthesized according to the synthetic procedure of step 3 in Example Int-9. MS (ESI) m / z: 491.4 [M+H] + .

[0276] Step 3: (5-((5-(4-((S)-3-(((tert-butyldimethylsilyl)oxy)methyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-2-methoxy-5-nitrophenoxy)pentyl)oxy)-2-((S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-5-azaspiro[2.4]heptane-5-carbonyl)-4-methoxyphenyl)carbamate (Int-10d) Add K₂CO₃ (398 mg, 2.88 mmol) to a solution of Int-9 (642 mg, 0.961 mmol), Int-10c (471 mg, 0.961 mmol), and DMF (15 mL). Stir the mixture overnight at rt. After the reaction is complete, dilute the mixture with water (30 mL) and EtOAc (30 mL). 2) Extraction. The combined organic compounds were washed with brine (40 mL), dried over Na₂SO₄, filtered, and the filtrate was concentrated under vacuum to obtain the residue. This residue was purified by silica gel column chromatography (petroleum ether / EtOAc = 70 / 30) to give a white solid Int-10d (874 mg, 88.3% yield). MS (ESI) m / z: 1031.8 [MH] + .

[0277] Step 4: (5-((5-(5-amino-4-((S)-3-(((tert-butyldimethylsilyl)oxy)methyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-2-methoxyphenoxy)pentyl)oxy)-2-((S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-5-azaspiro[2.4]heptane-5-carbonyl)-4-methoxyphenyl)carbamate (Int-10) A mixed solution of Zn (63 mg, 0.97 mmol), EtOH (2 mL), AcOH (150 μL), and H₂O (150 μL) was stirred at rt for 10 min. Then, a solution of Int-10 in EtOH (1 mL) was added to the reaction mixture. The mixture was stirred at rt for 10 min. After the reaction was complete, the mixture was filtered, and the filtrate was concentrated under vacuum to obtain a residue, which was purified by silica gel column chromatography (petroleum ether / EtOAc = 40 / 20) to give a yellow oily Int-10 (74 mg, 76.2% yield). MS (ESI) m / z: 1002.3 [M+H] + .

[0278] Example Int-12 Step 1: (S)-(2-(6-(hydroxymethyl)-5-azaspiro[2.4]heptane-5-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamate (Int-12a) p-Toluenesulfonic acid hydrate (417 mg, 2.19 mmol) was added to a solution of Int 10b (2.36 g, 3.65 mmol) in THF (40 mL) and water (2 mL). The reaction mixture was stirred at 22 °C for 3 h. The mixture was diluted with EtOAc (80 mL) and washed with water and brine. The organic phase was concentrated and purified by silica gel column chromatography (petroleum ether / EtOAc = 35 / 65) to give Int-12a (1.69 g, 87% yield). MS (ESI) m / z: 533.3 [M+H] + .

[0279] Step 2: (S)-7-methoxy-5-oxo-8-((triisopropylsilyl)oxy)-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-10(5H)-formate allyl ester (Int-12b) DIAD (1.14 mL, 5.75 mmol) was added to a solution of Int-12a (1.53 g, 2.87 mmol) and PPh3 (2.26 g, 8.62 mmol) in THF (30 mL). The reaction mixture was stirred at 40 °C under N2 for 1 h. The solvent was removed under vacuum, and the residue was purified by silica gel column chromatography (petroleum ether / EtOAc = 35 / 65) to give Int-12b (940 mg, 64% yield). MS (ESI) m / z: 515.3 [M+H] + .

[0280] Step 3: (S)-8-hydroxy-7-methoxy-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-10(5H)-formate allyl ester (Int-12) LiOAc (121 mg, 1.83 mmol) was added to a solution of Int-12b (940 mg, 1.83 mmol) in wet DMF (15 mL, 4g / 1 DMF / water). The reaction was continued at 25°C for 2 h. The mixture was diluted with EtOAc and washed twice with H2O and brine. The organic phase was concentrated and purified by silica gel column chromatography (petroleum ether / EtOAc = 35 / 65) to give Int-12 (605 mg, 92% yield). MS (ESI) m / z: 359.2 [M+H] + .

[0281] Example Int-13 Step 1: (S)-8-((5-(4-((S)-3-(((tert-butyldimethylsilyl)oxy)methyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-2-methoxy-5-nitrophenoxy)pentyl)oxy)-7-methoxy-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-10(5H)-formate allyl ester (Int-13b) Int-12 (450 mg, 1.26 mmol) and K₂CO₃ (226 mg, 1.63 mmol) were added to a solution of Int-13a (818 mg, 1.32 mmol) in 1 mL of DMF. The mixture was stirred at rt for 24 h. The mixture was diluted with EtOAc and washed with H₂O and brine. The organic phase was concentrated, and the crude product was purified by silica gel column chromatography (petroleum ether / EtOAc = 35 / 65) to give Int-13b (930 mg, 82% yield). MS (ESI) m / z: 899.5 [M+H] + .

[0282] Step 2: (S)-8-((5-(5-amino-4-((S)-3-(((tert-butyldimethylsilyl)oxy)methyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-2-methoxyphenoxy)pentyl)oxy)-7-methoxy-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-10(5H)-formate allyl ester (Int-13) Zinc powder (2.65 g, 38.3 mmol) was added to a mixture of EtOH (10 mL), water (0.625 mL), and AcOH (0.625 mL) at 0 °C. The reaction mixture was stirred at 5 °C for 30 min. Int-13b (930 mg, 1.04 mmol) was added dropwise to a solution of EtOH (6 mL) at 5 °C. The reaction was continued at 5 °C for 50 min. The reaction solution was filtered. The filtrate was diluted with EtOAc and washed with water, saturated aqueous NaHCO3, and brine. The organic phase was dried over Na2SO4, filtered, and the solvent was removed by rotary evaporation under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography (petroleum ether / EtOAc = 80 / 20) to give a yellow solid Int-13 (806 mg, 90% yield). MS (ESI) m / z: 869.5 [M+H] + .

[0283] Example Int-14 (S)-3-((5-(5-amino-4-((S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-5-azaspiro[2.4]heptane-5-carbonyl)-2-methoxyphenoxy)pentyl)oxy)-2-methoxy-14-oxo-6,6a,7,12-tetrahydrobenzo[5,6][1,4]diazazo[1,2-b]isoquinoline-5(14H)-formate allyl ester (Int-14) Int-14 (440 mg) was prepared according to a similar procedure to that described in the synthesis of Int-13. MS (ESI) m / z: 869.6 [M+H] + .

[0284] Example Int-17 Step 1: (S)-(5-((3-(bromomethyl)benzyl)oxy)-2-(6-(((tert-butyldimethylsilyl)oxy)methyl)-5-azaspiro[2.4]heptane-5-carbonyl)-4-methoxyphenyl)carbamate (Int-17a) Add K₂CO₃ (371.8 mg, 2.690 mmol) to a mixture of Int-12 (1.20 g, 2.4 mmol) and 1,3-bis(bromomethyl)benzene (3.2 g, 12.2 mmol) in DMF (10 mL). Stir the mixture at rt for 4 hr. Dilute the mixture with EtOAc (20 mL) and with saline (20 mL). 3) Wash, dry with Na2SO4, filter, and concentrate the filtrate under vacuum to obtain the residue. Purify by silica gel column chromatography (EtOAc / petroleum ether = 0 / 100~15 / 85), concentrate and fractionate under vacuum to give a colorless oily Int-17a (787.8 mg, 47.8% yield). MS (ESI) m / z: 673.4 [M+H] + .

[0285] Step 2: (S)-(2-(6-(((tert-butyldimethylsilyl)oxy)methyl)-5-azaspiro[2.4]heptane-5-carbonyl)-5-hydroxy-4-methoxyphenyl)tert-butyl carbamate Int-17b A mixture of Int-14b (447 mg, 1.10 mmol) and Boc2O (249.1 mg, 1.43 mmol) in CH2Cl2 (5 mL) was stirred at 70 °C for 20 hr. The mixture was concentrated under vacuum and purified by silica gel column chromatography (EtOAc / petroleum ether = 0 / 100~20 / 80) to give a colorless oil of Int-17b (480 mg, 86.1% yield). MS (ESI) m / z: 507.5 [M+H] + .

[0286] 1H NMR (400 MHz, DMSO- d6 ) δ 9.57 (s, 1H), 8.95 (s, 1H), 7.25 (s, 1H), 6.78 (s, 1H), 4.30 (s, 1H), 3.77 (d, J = 42.4 Hz, 5H), 3.49 (s, 1H), 3.17 (s,1H), 1.99 (m, 4H), 1.44 (s, 9H), 0.88 (s, 9H), 0.64 – 0.42 (m, 4H), 0.05 (s, 6H).

[0287] Step 3: (5-((3-((5-((tert-butoxycarbonyl)amino)-4-((S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-5-azaspiro[2.4]heptane-5-carbonyl)-2-methoxyphenoxy)methyl)benzyl)oxy)-2-((S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-5-azaspiro[2.4]heptane-5-carbonyl)-4-methoxyphenyl)carbamate allyl ester (Int-17c) Add K₂CO₃ (157 mg, 1.14 mmol) to a mixture of Int-17b (480 mg, 0.95 mmol) and Int-17a (637 mg, 0.95 mmol) in DMF (10 mL). Stir the mixture overnight at rt. Dilute the mixture with EtOAc (30 mL) and with saline (20 mL). 3) Wash, dry with Na2SO4, filter, and concentrate the filtrate under vacuum to obtain the residue. Purify by silica gel column chromatography (EtOAc / petroleum ether = 0 / 100~30 / 70), concentrate and fractionate under vacuum to give a colorless oily Int-17c (721 mg, 69.2% yield). MS (ESI) m / z: 1121.9 [M+Na] + ] 1H NMR (400 MHz, DMSO- d6 ) δ 8.88 (s, 1H), 8.56 (s, 1H), 7.96 (s, 3H),7.57 (s, 1H), 7.51 (s, 1H), 7.44 (d, J = 8.2 Hz, 4H), 6.90 (d, J = 5.6 Hz,2H), 6.05 – 5.85 (m, 1H), 5.69 (s, 3H), 5.33 (dq, J = 17.2, 1.6 Hz, 1H), 5.26– 5.18 (m, 1H), 5.11 (s, 4H), 4.57 (dt, J = 5.4, 1.4 Hz, 2H), 4.29 (s, 2H),3.90 – 3.62 (m, 11H), 3.49 – 3.33 (m, 3H), 3.29 (s, 2H), 2.90 (s, 10H), 2.74(d, J = 8.4 Hz, 9H), 2.50 (dt, J = 3.8, 1.9 Hz, 12H), 2.09 – 1.77 (m, 5H), 1.45 (s, 9H), 0.88 (s, 18H), 0.56 (ddd, J = 18.4, 11.9, 5.9 Hz, 8H), 0.02 (d,J = 4.1 Hz, 12H).

[0288] Step 4: (5-((3-((5-amino-4-((S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-5-azaspiro[2.4]heptane-5-carbonyl)-2-methoxyphenoxy)methyl)benzyl)oxy)-2-((S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-5-azaspiro[2.4]heptane-5-carbonyl)-4-methoxyphenyl)carbamate allyl ester (Int-17) TBS-OTf (312 μL, 359.0 mg, 1.36 mmol) was added to a solution of Int-17c (711 mg, 0.647 mmol) in CH2Cl2 (10 mL) at 0 °C, followed by stirring at the same temperature for 1 h. Then, 2,6-dimethylpyridine (395 μL, 364 mg, 3.40 mmol) was added. The reaction was stirred at 0 °C for 10 min. The mixture was quenched with water (30 mL) and then quenched with CH2Cl2 (30 mL). 3) Extraction. The combined organic layers were washed with brine (50 mL), dried over Na₂SO₄, filtered, and the filtrate was concentrated under vacuum to obtain the residue. This residue was purified by silica gel column chromatography (EtOAc / petroleum ether = 0 / 100–50 / 50) to give a white solid Int-17 (351.2 mg, 54.3% yield). MS (ESI) m / z: 999.9 [M+H] + .

[0289] Example Int-18 Step 1: (S)-4-methylenepiperidine-1,2-dicarboxylic acid 1-(tert-butyl) ester 2-methyl ester (Int-18b) In a N2 atmosphere at 0°C, a solution of MePh3PBr (8.26 g, 22.7 mmol) in anhydrous THF (70 mL) was added. t BuOK (1M, 22.7 mL, 22.7 mmol) was stirred at 0 °C for 1 h. Under a N2 atmosphere at 0 °C, a solution of Int-18a (5.3 g, 20.6 mmol) in anhydrous THF (30 mL) was added to the reaction solution, and the mixture was stirred for 1 h. Water (50 mL) and saturated NH4Cl (50 mL) were added to the solution. The organic phase was separated, and the aqueous phase was treated with EtOAc (50 mL). 3) Extraction. The combined organic phases were concentrated and purified by rapid column chromatography (petroleum ether / EtOAc = 90 / 10) to give a colorless oil of Int-18b (3.7 g, 70% yield). MS (ESI) m / z: 156.1 [M+H-Boc] + .

[0290] 1 H NMR (400 MHz, CDCl3) δ 5.08 – 4.97 (m, 0.5H), 4.87 – 4.78 (m,0.5H), 4.79 (s, 2H), 4.20 – 3.97 (m, 1H), 3.71 (s, 3H), 3.12 – 2.90 (m, 1H), 2.81 – 2.67 (m, 1H), 2.49 – 2.37 (m, 1H), 2218 – 2.10 (m, 2H), 1.47 (s, 9H).

[0291] Step 2: (S)-4-methylenepiperidine-1,2-dicarboxylic acid 1-benzyl ester 2-methyl ester (Int-18c) In a N2 atmosphere at 0°C, 4 M HCl in 20 mL of EtOAc was added to a solution of Int-18b (3.7 g, 14.5 mmol) in 25 mL of CH2Cl2, and the mixture was stirred at 100 mL for 30 min. The solution was concentrated and redissolved in 100 mL of CH2Cl2. Cb2Cl (2.5 mL, 17.4 mmol) and Et3N (4.1 mL, 29.1 mmol) were added to the solution at 0°C, and the mixture was stirred at 100 mL for another 1 h. The solution was then poured into 50 mL of 0.5 N HCl and 50 mL of water, and then dissolved in 50 mL of CH2Cl2. 2) Extraction. The combined organic phases were concentrated and purified by rapid column chromatography (petroleum ether / EtOAc = 90 / 10) to give a colorless oil of Int-18c (3.58 g, 85% yield). MS (ESI) m / z: 312.2 [M+Na] + .

[0292] 1H NMR (400 MHz, CDCl3) δ 7.45 – 7.24 (m, 5H), 5.17 (s, 2H), 5.14 –4.90 (m, 1H), 4.81 (s, 2H), 4.28 – 4.08 (m, 1H), 3.69 (d, J = 18.8 Hz, 3H), 3.23 – 3.01 (m, 1H), 2.77 (t, J = 15.7 Hz, 1H), 2.50 – 2.39 (m, 1H), 2.30 –2.12 (m, 2H).

[0293] Step 3: (S)-6-azaspiro[2.5]octane-5,6-dicarboxylic acid 6-benzyl ester 5-methyl ester (Int-18d) ZnEt2 (2 M in hexane, 24.7 mL, 49.5 mmol) was added to anhydrous CH2Cl2 (70 mL) under N2 atmosphere at 0 °C, and the mixture was stirred at 0 °C for 10 min. Anhydrous TFA (3.9 mL, 49.5 mmol) was slowly added, and the mixture was stirred at 0 °C for 1 h. Diiodomethane (4.1 mL, 49.5 mmol) was slowly added, and the mixture was stirred at 0 °C for 1 h. Int-18c (3.58 g, 12.4 mmol) was slowly added to a solution of anhydrous CH2Cl2 (30 mL), and the mixture was stirred at 0 °C for 30 min, followed by stirring at rt for 14 h. The solution was filtered through Celite. The filtrate was washed with saturated NH4Cl (50 mL) and water (50 mL). The aqueous phase was rinsed with CH2Cl2 (50 mL) 2) Extraction. The organic phase was concentrated and purified by rapid column chromatography (petroleum ether / EtOAc = 90 / 10) to give a colorless oil, Int-18d (2.97 g, 79% yield). MS (ESI) m / z: 326.3 [M+Na] + .

[0294] 1H NMR (400 MHz, CDCl3) δ 7.45 – 7.25 (m, 5H), 5.24 – 5.08 (m, 2H), 5.04 – 4.84 (m, 1H), 4.21 – 4.02 (m, 1H), 3.72 (d, J = 6 Hz, 3H), 3.36 – 3.14(m, 1H), 2.24 – 2.12 (m, 1H), 2.00 – 1.85 (m, 1H), 1.62 – 1.54 (m, 1H), 0.91 – 0.72 (m, 1H), 0.42 – 0.22 (m, 4H).

[0295] Step 4: (S)-5-(hydroxymethyl)-6-azaspiro[2.5]octane-6-carboxylic acid benzyl ester (Int-18e) LiBHEt3 (1 M in THF, 24.5 mL, 24.5 mmol) was added to a solution of Int-18d (2.97 g, 9.79 mmol) in anhydrous THF (50 mL) under N2 atmosphere at 0 °C, and the mixture was stirred at 0 °C for 2 h. Water (2 mL) was added dropwise to the solution, which was then washed with saline (15 mL) and treated with EtOAc (20 mL). 3) Extraction. The organic phase was concentrated and purified by rapid column chromatography (petroleum ether / EtOAc = 50 / 50) to give a colorless oil of Int-18e (2.46 g, 91% yield). MS (ESI) m / z: 276.3 [M+H] + .

[0296] 1 H NMR (400 MHz, CDCl3) δ 7.40 – 7.29 (m, 5H), 5.22 – 5.09 (m, 2H), 4.53 – 4.39 (m, 1H), 4.20 – 4.06 (m, 1H), 4.02 (dd, J = 11.0, 9.5 Hz, 1H),3.67 (dd, J = 11.1, 5.6 Hz, 1H), 3.19 – 3.05 (m, 1H), 2.08 – 1.97 (m, 1H),1.95 – 1.81 (m, 1H), 1.08 – 0.97 (m, 1H), 0.91 – 0.79 (m, 2H), 0.47 – 0.38(m, 1H), 0.35 – 0.23 (m, 3H).

[0297] Step 5: (S)-(6-azaspiro[2.5]oct-5-yl)methanol (Int-18f) A solution of Int-18e (2.46 g, 8.95 mmol) in MeOH (40 mL) was added to a solution of Int-18e in MeOH (8 mL) under a nitrogen atmosphere, and the mixture was stirred for 3 h under a hydrogen atmosphere. The solution was filtered and concentrated to give a yellow oily Int-18f (1.3 g, quantitative), which was used directly in the next step without further purification. MS (ESI) m / z: 142.1 [M+H] + .

[0298] 1 H NMR (400 MHz, CDCl3) δ 3.61 (dd, J = 10.9, 3.7 Hz, 1H), 3.48 (dd, J =10.9, 7.7 Hz, 1H), 3.14 (ddd, J = 12.0, 4.1, 2.6 Hz, 1H), 2.89 (ddd, J = 11.1,6.9, 3.3 Hz, 1H), 2.81 (td, J = 12.1, 3.0 Hz, 1H), 1.89 (td, J = 12.7, 4.2 Hz, 1H), 1.65 (t, J = 12.1 Hz, 1H), 0.90 – 0.77 (m, 2H), 0.39 – 0.32 (m, 2H), 0.32 – 0.23 (m, 2H).

[0299] Step 6: (S)-(4-(benzyloxy)-5-methoxy-2-nitrophenyl)(5-(hydroxymethyl)-6-azaspiro[2.5]oct-6-yl)methyl ketone (Int-18g) Oxaloyl chloride (2.3 mL, 26.7 mmol) was added to a solution of 4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid (2.7 g, 8.9 mmol) in CH2Cl2 (50 mL) at 0 °C under a N2 atmosphere, followed by the addition of 3 drops of DMF and stirring for 1 h. The turbid solution became clear and no gas elution occurred. The solution was concentrated to remove excess oxalyl chloride. The residue was dissolved in anhydrous CH2Cl2 (20 mL) and added at 0 °C to a solution of Int-18f (1.28 g, 8.9 mmol) and DIEA (6.3 mL, 35.6 mmol) in anhydrous CH2Cl2 (30 mL), and stirred for 20 min. The solution was washed with 0.5 N HCl (30 mL) and then with CH2Cl2 (20 mL). 2) Extraction. The organic phase was concentrated and purified by rapid column chromatography (petroleum ether / EtOAc = 20 / 80) to give the title compound Int-18 g (3 g, 79% yield) as a grayish-white solid. MS (ESI) m / z: 427.4 [M+H] + .

[0300] Step 7: (S)-(2-amino-4-(benzyloxy)-5-methoxyphenyl)(5-(hydroxymethyl)-6-azaspiro[2.5]oct-6-yl)methyl ketone (Int-18h) TBSCl (216 mg, 1.4 mmol) was added to a solution of Int-18 g (500 mg, 1.17 mmol) and imidazole (163 mg, 6.16 mmol) in anhydrous DMF (10 mL) under N2 atmosphere at 0 °C, and the mixture was stirred at rt for 1 h. The solution was then added to water (30 mL) and diluted with EtOAc (15 mL). 3) Extraction, concentration, and purification by rapid column chromatography (petroleum ether / EtOAc = 75 / 25) yielded a grayish-white solid, Int-18h (620 mg, 97.8% yield). MS (ESI) m / z: 541.7 [M+H] + .

[0301] Step 8: (S)-(2-amino-4-hydroxy-5-methoxyphenyl)(5-(((tert-butyldimethylsilyl)oxy)methyl)-6-azaspiro[2.5]oct-6-yl)methyl ketone (Int-18) 10% Pd / C (44 mg) was added to a solution of Int-18h (440 mg, 0.81 mmol) in MeOH (8 mL), and the mixture was stirred for 7 h under H2 atmosphere. The solution was filtered and concentrated to give a pink solid, the title compound Int-18 (342 mg, quantified). MS (ESI) m / z: 421.4 [M+H] + .

[0302] 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.04 (s, 1H), 6.60 (s, 1H), 6.19 (s, 1H), 4.77 (s, 2H), 3.92 – 3.82 (m, 1H), 3.81 – 3.71 (m, 1H), 3.63 (s, 3H), 3.05(s, 1H), 1.97 (dd, J = 13.6, 5.4 Hz, 1H), 1.84 (t, J = 10.8 Hz, 1H), 1.07 (d, J =13.6 Hz, 1H), 0.84 (s, 9H), 0.45 (d, J = 5.5 Hz, 1H), 0.32 (d, J = 6.0 Hz, 1H), 0.27 – 0.18 (m, 2H), 0.03 (s, 3H), 0.01 (s, 3H).

[0303] Example Int-21 Step 1: ((17S,20S)-17-isopropyl-20-methyl-21-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-15,18,21-trioxo-3,6,9,12-tetraoxa-16,19-diazatetramonyl)carbamate (9H-fluorene-9-yl)methyl ester (Int-21) DIEA (233 μL, 1.31 mmol) was added to a mixture of Int-2 (500 mg, 0.656 mmol), bis(4-nitrophenyl) carbonate (300 mg, 0.981 mmol), and DMF (10 mL). The mixture was stirred overnight at rt. The mixture was concentrated under vacuum to obtain a residue, which was purified by silica gel column chromatography (petroleum ether / EtOAc = 0 / 100) to give Int-21 (386 mg, 63.5% yield) as a white solid. MS (ESI) m / z: 928.7 [M+H] + .

[0304] Example Int-22 Int-22 was synthesized according to the improved synthesis procedure described in reference (Tetrahedron Letters 54 (2013) 349-3495).

[0305] Example Int-23 Step 1: Methyl 4-(5-(methylthio)-1,2,4-thiadiazol-3-yl)benzoate (Int-23c) Compounds Int-23b (109.72 mg, 0.568 mmol), K2CO3 (168 mg, 0.947 mmol), and Pd(dppf)Cl2.CH2Cl2 (34.6 mg, 0.047 mmol) were added to a solution of compound Int-23a (100 mg, 0.47 mmol) in toluene (4 mL) and H2O (1 mL). The mixture was stirred at 110 °C under a N2 atmosphere for 3 h. The mixture was filtered through a diatomaceous earth mat, diluted with EtOAc (100 mL), and then diluted with saline (50 mL). 4) Washing. The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated. The crude product was purified by rapid column chromatography (eluting with petroleum ether / EtOAc = 0-40%). A grayish-white solid, Int-23c (56 mg, 44.4% yield), was obtained. MS (ESI) m / z: 267.1 [M+H] + .

[0306] Step 2: 4-(5-(methylthio)-1,2,4-thiadiazol-3-yl)benzoic acid (Int-23d) LiOH (17 mg, 0.41 mmol) was added to a solution of compound Int-23c (54 mg, 0.20 mmol) in MeOH (3 mL) and H₂O (1 mL). The mixture was stirred at rt for 2 h. The mixture was adjusted to pH 7 and purified by prep-HPLC (FA conditions) to give compound Int-23d (36 mg, 70.3% yield) as a white solid. MS (ESI) m / z: 253.1 [M+H] + .

[0307] Step 3: 4-(5-(methanesulfonyl)-1,2,4-thiadiazol-3-yl)benzoic acid (Int-23) m-CPBA (96 mg, 0.55 mmol) was added to a solution of compound Int-23d (35 mg, 0.14 mmol) in CH2Cl2 (3 mL) and THF (3 mL). The mixture was stirred at room temperature for 16 h. The mixture was concentrated and analyzed by prep-HPLC (method: column: XBridge Prep C18 OBD 5 μm 19). Purification was performed using a mobile phase of 150 mm; A-water (0.1% TFA): B-acetonitrile; flow rate: 20 mL / min. The compound Int-23 (12 mg, 99% purity) was obtained as a white solid. MS (ESI) m / z: 284.8 [M+H] + .

[0308] Example Int-25 Step 1 (2S,3R,4S,5S,6S)-triacetic acid 2-(2-azido-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (Int-25) Int-7b (450 mg, 0.99 mmol) was dissolved in anhydrous THF (2 mL). The mixture was cooled to 0 °C, and then imidazole-1-sulfonyl azide (414.2 mg, 1.98 mmol) was added to 1 mL MeOH, followed by K2CO3 and a catalytic amount of CuSO4 in 1 mL H2O. The mixture was stirred at rt under a nitrogen atmosphere for 2 h. EtOAc (5 mL) was added, the organic phase was collected, dried over Na2SO4, concentrated, and then purified by column chromatography (petroleum ether / EtOAc = 50 / 50) to give a white solid Int-25 (350 mg, 87.2%). MS (ESI) m / z: 504.2 [M+Na] + .

[0309] 1 H NMR (400 MHz, CDCl3) δ 7.17 – 7.03 (m, 3H), 5.44 – 5.26 (m, 3H), 5.07 (d, J = 7.1 Hz, 1H), 4.64 (d, J = 2.2 Hz, 2H), 4.13 (dd, J = 6.8, 2.8 Hz, 1H), 3.83 – 3.71 (m, 3H), 2.17 – 2.00 (m, 9H).

[0310] Example Int-26 Step 1: (15-oxo-3,6,9,12-tetraoxa-16-aza-nonadenylon-18-yn-1-yl)carbamate (9H-fluorene-9-yl) methyl ester (Int-26a) HATU (312 mg, 0.82 mmol) and DIPEA (224 μL, 1.23 mmol) were added to a solution of Int-2d (200 mg, 0.41 mmol) in 2 mL of DMF at 0 °C. The mixture was stirred at 0 °C for 10 min. Propylamine (25 μL, 0.45 mmol) was added. The mixture was stirred at rt under a nitrogen atmosphere for 1 h. The solvent was then removed by evaporation, and the crude product was purified by column chromatography (MeOH / CH2Cl2 = 5 / 95) to give a brown oily Int-26a (200 mg, 93% yield). MS (ESI) m / z: 525.8 [M+H] + .

[0311] Step 2: 1-Amino-N-(prop-2-yn-1-yl)-3,6,9,12-tetraoxapentadecan-15-amide (Int-26) Diethylamine (0.98 mL, 9.5 mmol) was added to a solution of Int-26a (500 mg, 0.95 mmol) in 2 mL of DMF at 0 °C. The mixture was stirred at 0 °C for 30 min. The solvent was then removed by evaporation, and the crude product was purified by column chromatography (MeOH / CH2Cl2 = 5 / 95) to obtain a pale yellow solid of Int-26 (250 mg, 87% yield). MS (ESI) m / z: 303.3 [M+H] + .

[0312] Example 1: Synthetic Fissure - Connector - Payload Example Ref-2-1 According to the procedure synthesis Ref-2-1a described in US2015283262A1 and Org. Process Res. Dev. 2018, 22, 1241-1256.

[0313] Step 1: (11S,11aS)-11-hydroxy-7-methoxy-8-((5-(((S)-7-methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a [1,4]diaza-10(5H)-formic acid 4-((21S,24S)-1-((1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-yl)-21-isopropyl-24-methyl-3,19,22-trioxo-2,7,10,13,16-pentaoxa-4,20,23-triazapenta-25-amido)benzyl ester (Ref-2-1) HATU (5.0 mg, 0.01 mmol) and DIEA (4 μL, 0.02 mmol) were added to a solution of Int-1 (5.3 mg, 0.01 mmol, prepared according to the procedures described in WO2022120132 A1 and WO2022079211 A1) in 0.5 mL DMF at 0 °C. The mixture was stirred at rt for 15 min. Ref-2-1a (10 mg, 0.01 mmol) in 0.5 mL DMF was added to the mixture at 0 °C and the reaction was stirred at rt for 2 h. The mixture was then analyzed by prep-HPLC (method: column: XBridge Prep C18 OBD 5μm 19). 150 mm; Mobile phase: A-water (formic acid-free): B-acetonitrile; Flow rate: 20 mL / min. Purification was performed by lyophilizing the fractions to give Ref-2-1 as a white solid (2.5 mg, 17.1% yield). MS (ESI) m / z: 1345.9 [M+H] + .

[0314] Example Ref-2-2 Int-3 is synthesized according to the procedure described in WO 2017059289 A1 and Org. Process Res. Dev. 2022, 26, 2155-2175.

[0315] Step 1: (5-((5-(5-((((4-((21S,24S)-1-(9H-fluorene-9-yl)-21-isopropyl-24-methyl-3,19,22-trioxo-2,7,10,13,16-pentaoxa-4,20,23-triazapenta-25-amido)benzyl)oxy)carbonyl)amino)-4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxyphenoxy)pentyl)oxy)-2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-4-methoxyphenyl)carbamate (9H-fluorene-9-yl)methyl tert-butyl ester (Ref-2-2a) Triphosgene (24.9 mg, 0.084 mmol) was added to a solution of Int-3 (200 mg, 0.1210 mmol) and 200 mg of 4Å MS in anhydrous THF (2.5 mL), followed by the addition of Et3N (64 μL, 0.462 mmol) at 0 °C under N2. The mixture was stirred at 0 °C under N2 for 10 min. Isocyanate formation was monitored by LCMS analysis with MeOH quenching. A solution of Int-2 (176 mg, 0.231 mmol), dibutyltin dilaurate (13.3 mg, 0.021 mmol), and Et3N (43.7 μL, 0.315 mmol) in anhydrous THF (2.5 mL) was added to the mixture. The mixture was stirred at rt for 3 h. The mixture was filtered, and the filtrate was concentrated. The residue was purified by silica gel column chromatography (CH2Cl2 / MeOH = 4 / 96) to give Ref-2-2a as a white solid (304 mg, 83% yield). MS (ESI) m / z: 1742.1 [M+H] + .

[0316] Step 2: (5-((5-(5-((((4-((21S,24S)-1-(9H-fluorene-9-yl)-21-isopropyl-24-methyl-3,19,22-trioxo-2,7,10,13,16-pentaoxa-4,20,23-triazapenta-25-amido)benzyl)oxy)carbonyl)amino)-4-((S)-2-(hydroxymethyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxyphenoxy)pentyl)oxy)-2-((S)-2-(hydroxymethyl)-4-methylenepyrrolidine-1-carbonyl)-4-methoxyphenyl)tert-butyl carbamate (Ref-2-2b) p-Toluenesulfonic acid hydrate (40 mg, 0.21 mmol) was added to a solution of Ref-2-2a (304 mg, 0.17 mmol) in THF (3 mL) and water (0.15 mL). The reaction mixture was stirred at 22 °C for 4 h. The mixture was diluted with EtOAc (20 mL) and washed with water, saturated NaHCO3, and brine. The organic phase was concentrated and purified by silica gel column chromatography (CH2Cl2 / MeOH = 5 / 95) to give Ref-2-2b (213 mg, 81% yield) as a white solid. MS (ESI) m / z: 1513.9 [M+H] + .

[0317] Step 3: (11S,11aS)-8-((5-(((11S,11aS)-10-(tert-butoxycarbonyl)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-11-hydroxy-7-methoxy-2-methylene-5-oxo- 2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid 4-((21S,24S)-1-(9H-fluorene-9-yl)-21-isopropyl-24-methyl-3,19,22-trioxo-2,7,10,13,16-pentaoxa-4,20,23-triazapenta-25-amido)benzyl ester (Ref-2-2c) DMP (58.9 mg, 0.139 mmol) was slowly added fractionally to a solution of Ref-2-2b (100 mg) in anhydrous CH2Cl2 (2 mL) at 0 °C. The reaction was then heated to rt and stirred overnight. The reaction was quenched with saturated Na2S2O3, followed by the addition of saturated NaHCO3 and water. The layers were separated, and the organic layer was washed with saturated Na2S2O3, saturated NaHCO3, and brine, and dried over Na2SO4. The crude product was purified by silica gel column chromatography (CH2Cl2 / MeOH = 5 / 95) to give Ref-2-2c (80 mg, 80% yield) as a white solid. MS (ESI) m / z: 1509.8 [M+H] + .

[0318] Step 4: (11S,11aS)-11-hydroxy-7-methoxy-8-((5-(((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H -Benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid 4-((21S,24S)-1-(9H-fluorene-9-yl)-21-isopropyl-24-methyl-3,19,22-trioxo-2,7,10,13,16-pentaoxa-4,20,23-triazapenta-25-amido)benzyl ester (Ref-2-2d) Ref-2-2c (20 mg, 0.013 mmol) was cooled to -3 °C. Separately, a solution of 95% TFA in H₂O (0.5 mL) was cooled to -3 °C and then added to Ref-2-2c. The reaction mixture was stirred at -3 °C for 30 min, then poured into a 1:1 solution of CHCl₃: saturated NaHCO₃ (16 mL) at 0 °C. The organic layer was separated, dried on Na₂SO₄, filtered, and then removed under vacuum. The crude material was used directly for the next step without further purification. MS (ESI) m / z: 1391.7 [M+H] + .

[0319] Step 5: (11S,11aS)-11-hydroxy-7-methoxy-8-((5-(((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-2-methylene-5-oxo-2,3,11,1 1a-Tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid 4-((17S,20S)-1-amino-17-isopropyl-20-methyl-15,18-dioxo-3,6,9,12-tetraoxa-16,19-diaza-21-amido)benzyl ester (Ref-2-2e) Et₂NH (14 μL, 0.129 mmol) was added to a solution of crude Ref-2-2d (18 mg, 0.013 mmol) in 0.5 mL DMF. The mixture was stirred at rt for 0.5 h. After the reaction was complete, the mixtures were combined and analyzed by prep-HPLC (method: column: XBridge Prep C18 OBD 5μm 19). Purification was performed using a mobile phase of 150 mm (A-water (0.1% formic acid):B-acetonitrile; flow rate: 20 mL / min) to give Ref-2-2e (5 mg, 33% yield). MS (ESI) m / z: 1169.7 [M+H] + .

[0320] Step 6: (11S,11aS)-11-hydroxy-7-methoxy-8-((5-(((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[ 1,2-a][1,4]diaza-10(5H)-formic acid 4-((21S,24S)-1-((1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-yl)-21-isopropyl-24-methyl-3,19,22-trioxo-2,7,10,13,16-pentaoxa-4,20,23-triazapenta-25-amido)benzyl ester (Ref-2-2) DIEA (8.6 μL, 0.05 mmol) was added to Ref-2-2e (14.5 mg, 0.012 mmol) and Int 4 (10.8 mg, 0.037 mmol, as described in the table below). J. Am. Chem. Soc. Prepared according to the procedure in 2020, 142, 20, 9285–9301) in 1 mL of DMF solution. Stir the mixture at rt for 20 min. Pass the mixture through prep-HPLC (method: column: XBridge Prep C18 OBD 5μm 19). Purification was performed using a mobile phase of 150 mL / min (A-water (formic acid-free):B-acetonitrile; flow rate: 20 mL / min) to obtain a light gray solid Ref-2-2 (7.9 mg, 46% yield). MS (ESI) m / z: 1345.7 [M+H] + .

[0321] Example 2-1 Step 1: (5-((5-(5-amino-4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidine-1-carbonyl)-2-methoxyphenoxy)pentyl)oxy)-2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidine-1-carbonyl)-4-hydroxyphenyl)carbamate tert-butyl ester (2-1c) Compound 2-1c (2.66 g, 66.3%) was synthesized according to a similar procedure described below: WO 2017059289A1 and Org. Process Res. Dev. 2022, 26, 2155-2175. MS (ESI) m / z: 930.1 [M+H] + .

[0322] Step 2: (2S,3S,4S,5R,6S)-4,5-diacetoxy-6-(4-((((5-((5-(5-((tert-butoxycarbonyl)amino)-4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidine-1-carbonyl)-2-hydroxyphenoxy)pentyl)oxy)-2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidine-1-carbonyl)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenoxy)-2-(methoxycarbonyl)tetrahydro-2H-pyran-3-carboxylic acid (2-1d) Compound 2-1d (2.17 g, 67.4%) was synthesized according to the synthetic procedure of step 1 in Example Ref-2-2. MS (ESI) m / z: 1395.8 [M+H] + .

[0323] Step 3: (2S,3R,4S,5S,6S)-triacetic acid 2-(4-((((5-((5-(5-amino-4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidine-1-carbonyl)-2-methoxyphenoxy)pentyl)oxy)-2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidine-1-carbonyl)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-1e) TBSOTf (162 mg, 0.61 mmol) was added to a solution of 2-1d (0.57 g, 0.41 mmol) in CH2Cl2 (6 mL) on an ice bath. After addition, the reaction mixture was stirred overnight at rt, followed by the addition of 2,6-dimethylpyridine and stirring overnight. After the reaction was complete, the mixture was... saturation Quenching with 10 mL of NH4Cl aqueous solution, followed by 10 mL of CH2Cl2. 3) Extraction. The combined organic layers were washed with H₂O (15 mL) and brine (15 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum to obtain the residue. This residue was purified by silica gel column chromatography (A-CH₂Cl₂; B-MeOH) to give a pale yellow solid 2-1e (439 mg, 83.1%). MS (ESI) m / z: 1295.2 [M+H] + .

[0324] Step 4: (2S,3S,4S,5R,6S)-6-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nonadecano-19-amido)-4-((((2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidine-1-carbonyl)-5-((5-(4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)) Pyrrolidine-1-carbonyl)-2-methoxy-5-((((4-(((2S,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)amino)phenoxy)pentyl)oxy)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-1f) Compound 2-1f (205 mg, 91.1%) was synthesized according to the synthetic procedure of step 1 in Example Ref-2-2. MS (ESI) m / z: 2246.0 [M+H] + .

[0325] Step 5: (2S,3S,4S,5R,6S)-6-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nonadecano-19-amido)-4-((((2-((S)-2-(hydroxymethyl)pyrrolidine-1-carbonyl)-5-((5-(4-((S)-2-(hydroxymethyl)pyrrolidine-1-carbonyl)-2- Methoxy-5-((((4-(((2S,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)amino)phenoxy)pentyl)oxy)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-1g) Compound 2-1 g (162 mg, 88.1%) was synthesized according to the synthetic procedure in step 2 of Example Ref-2-2. MS (ESI) m / z: 2018.5 [M+H] + .

[0326] Step 6: (2S,3S,4S,5R,6S)-6-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nine-19-amido)-4-((((11S,11aS)-11-hydroxy-8-((5-(((11S,11aS)-11-hydroxy-7-methoxy-5-oxo-10-(((4-(((2S,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro- 2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-1h) Add 2-1 g (152 mg, 0.075 mmol) of Des Martin periodane (130 mg, 0.30 mmol) to a solution of CH2Cl2 (10 mL) on an ice bath. After addition, react the reaction mixture at rt for 3 h. After the reaction is complete, dilute the mixture with CH2Cl2 (15 mL) and... saturation Na2S2O3 aqueous solution and saturation Quenching was performed with a mixture of NaHCO3 aqueous solution (1:1, 15 mL). The aqueous phase was then quenched with CH2Cl2 (15 mL). 3) Extraction. The combined organic layers were washed with H₂O (30 mL) and brine (30 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum to obtain the residue. This residue was purified by silica gel column chromatography (A-CH₂Cl₂; B-MeOH) to give a pale yellow solid (136 mg, 89.3%) over 2–1 h. MS (ESI) m / z: 2014.4 [M+H] + .

[0327] Step 7: (2S,3S,4S,5R,6S)-6-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nine-19-amido)-4-((((11S,11aS)-8-((5-(((11S,11aS)-10-(((4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)-11-hydroxy 7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-11-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-1i) LiBr (217 mg, 2.50 mmol) was added to MeCN / H2O (9 / 1, 1) under rt conditions. v / v Add 50 mg, 0.025 mmol) and Et3N (25 mg, 0.25 mmol) to a pre-dissolved solution in 0.5 mL for 2–1 h. Stir the resulting suspension at rt for 1.5 h. Acidify the mixture with AcOH (15 mg, 0.25 mmol) at 0 °C and then concentrate under vacuum. Recycle the residue with toluene (10 mL) 3) The mixture was co-evaporated to obtain a yellow, oily crude product, which was used in the next step without further purification. MS (ESI) m / z: 1733.4 [M+H] + .

[0328] Step 8: (2S,3S,4S,5R,6S)-6-(2-(1-amino-3,6,9,12-tetraoxapentadecano-15-amido)-4-((((11S,11aS)-8-((5-(((11S,11aS)-10-(((4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)-11-hydroxy-7-methoxy-5-oxo 2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-11-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-1j) Et₂NH (37 mg, 0.25 mmol) was added to a solution of 2-1i (crude product) in DMF (1 mL) at rt. The resulting solution was reacted at rt for 2 h, and the reaction was observed to be complete by LCMS analysis. The mixture was diluted with DMF (1 mL) and acidified with AcOH (15 mg, 0.25 mmol) at 0 °C. The mixture was then subjected to prep-HPLC (method: column: XBridge Prep C18 OBD 5 μm 19). Purification was performed using a mobile phase of 150 mL / min (A-water (0.1% formic acid):B-acetonitrile; flow rate: 20 mL / min) to give a white solid 2-1j (20 mg, 53.3% yield). MS (ESI) m / z: 1512.0 [M+H] + .

[0329] Step 9: (2S,3S,4S,5R,6S)-6-(2-(1-((1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nonadecano-19-amido)-4-((((11S,11aS)-8-((5-(((11S,11aS)-10-(((4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl) oxy)carbonyl)-11-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-11-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-1) DIPEA (8.6 mg, 0.066 mmol) was added to a mixed solution of 2-1j (20 mg, 0.013 mmol) and Int-4 (20 mg, 0.066 mmol) in DMF (0.5 mL) under rt. The resulting solution was reacted under rt for 0.5 h, and the reaction was observed to be complete by LCMS analysis. The mixture was diluted with DMF (1 mL) and acidified with AcOH (4.0 mg, 0.066 mmol) at 0 °C. Prep-HPLC was used (method: column: XBridge Prep C18 OBD 5 μm 19). The mixture was purified to a white solid, 2-1 (15.6 mg, 69.9% yield), using a mobile phase of A-water (0.1% formic acid) and B-acetonitrile (flow rate: 20 mL / min). MS (ESI) m / z: 1687.4 [M+H] + .

[0330] Example 2-2 Step 1: (2S,3R,4S,5S,6S)-triacetic acid 2-(4-((((5-((5-(5-((5-((tert-butoxycarbonyl)amino)-4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxyphenoxy)pentyl)oxy)-2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-2a) Triphosgene (80 mg, 0.27 mmol) and Et3N (224 μL, 163 mg, 1.62 mmol) were added to a mixture of Int-3 (700 mg, 0.73 mmol) (synthesized according to the procedure described in Org. Process Res. Dev. 2022, 26, 2155-2175) and 4 Å molecular sieve (200 mg) in anhydrous THF (8 mL) at 0 °C, followed by stirring on ice for 10 min. Then, a mixture of Int-6 (355 mg, 0.81 mmol), dibutyltin dilaurate (46 mg, 0.073 mmol), and Et3N (153 μL, 111 mg, 1.10 mmol) in THF (4 mL) was added. The mixture was stirred at rt for 1.5 h. The solvent was removed by evaporation, and the crude product was purified by silica gel column chromatography (10 g, EtOAc / petroleum = 0 / 100~30 / 70~100 / 0). The product was concentrated under vacuum to obtain a crude fraction, which was further purified by reversed-phase column chromatography (60 g C18, MeCN / H2O = 30 / 70~100 / 0) to give a white solid 2-2a (807 mg, 77% yield). MS (ESI) m / z: 1420.4 [M+H] + .

[0331] Step 2: (2S,3R,4S,5S,6S)-triacetic acid 2-(4-((((5-((5-(5-amino-4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxyphenoxy)pentyl)oxy)-2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-2b) The solution of 2-2a (784 mg, 0.55 mmol) in hexafluoroisopropanol (8 mL) in a sealed tube was heated to 80 °C and maintained for 4 days. The mixture was concentrated under vacuum to obtain a crude product, which was then purified by silica gel column chromatography (10 g, MeOH / CH2Cl2 = 0 / 100~3 / 97) to give a white solid 2-2b (516 mg, 71% yield). MS (ESI) m / z: 1319.9 [M+H] + .

[0332] Step 3: (2S,3R,4S,5S,6S)-triacetic acid 2-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nonadecano-19-amido)-4-((((2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-5-((5-(4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)- 4-Methylenepyrrolidine-1-carbonyl)-2-methoxy-5-((((4-(((2S,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)amino)phenoxy)pentyl)oxy)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-2c) Triphosgene (42 mg, 0.14 mmol) and Et3N (116 μL, 84 mg, 0.42 mmol) were added to a mixture of 2-2b (500 mg, 0.38 mmol) and 4Å molecular sieve (100 mg) in anhydrous THF (5 mL) at 0 °C, followed by stirring on an ice bath for 10 min. LCMS (quenched with MeOH) showed complete consumption of 2-2b. Then, a mixture of Int-7 (357 mg, 0.39 mmol), dibutyltin dilaurate (12 mg, 0.019 mmol), and Et3N (80 μL, 57 mg, 0.28 mmol) in THF (5 mL) was added. The mixture was stirred at rt for 1.5 h. After the reaction was complete, the product was purified by silica gel column chromatography (10 g, MeOH / CH2Cl2 = 0 / 100~1 / 99), and concentrated under vacuum to give a yellow foamy solid 2-2c (857 mg, 99% yield). MS (ESI) m / z: 2293.4 [M+Na]+ ] Step 4: (2S,3R,4S,5S,6S)-triacetic acid 2-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nine-19-amido)-4-((((2-((S)-2-(hydroxymethyl)-4-methylenepyrrolidine-1-carbonyl)-5-((5-(4-(((S)-2-(hydroxymethyl)-4-methylenepyrrolidine-1-carbonyl) 2-Methoxy-5-((((4-(((2S,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)amino)phenoxy)pentyl)oxy)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-2d) Add PTSA (36 mg, 0.21 mmol) to a solution of 2-2c (200 mg, 0.088 mmol) in THF / H2O (2 mL / 0.1 mL). Stir the mixture at rt for 1.5 h. Quench the mixture with saturated NaHCO3 (10 mL) and EtOAc (20 mL). 3) Extraction. The combined organic layers were washed with brine (10 mL), dried over Na₂SO₄, filtered, and concentrated under vacuum to obtain the residue. This residue was then purified by silica gel column chromatography (10 g, MeOH / CH₂Cl₂ = 0 / 100~10 / 90) to give a yellow foamy solid (2-2 d, 165 mg, 92% yield). MS (ESI) m / z: 2064.4 [M+Na + ] Step 5: (2S,3R,4S,5S,6S)-triacetic acid 2-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nine-19-amido)-4-((((11S,11aS)-11-hydroxy-8-((5-(((11S,11aS)-11-hydroxy-7-methoxy-2-methylene-5-oxo-10-(((4-(((2S,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2 H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10-carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-2e) A mixture of 2-2d (165 mg, 0.080 mmol), BAIB (55 mg, 0.17 mmol), and TEMPO (1.3 mg, 0.008 mmol) in CH2Cl2 (3 mL) was stirred at rt for 30 h. The mixture was quenched by adding 10% Na2S2O3 solution (10 mL) and then stirred with CH2Cl2 (10 mL). 3) Extraction. The combined organic layers were washed with brine (10 mL), dried over Na₂SO₄, filtered, and the filtrate was concentrated under vacuum to obtain the residue. This residue was purified by silica gel column chromatography (5 g, EtOAc / petroleum ether = 30 / 80~100 / 0 and MeOH / CH₂Cl₂ = 0 / 100~1 / 99) to give a brown foamy solid 2-2e (123 mg, 45% yield). MS (ESI) m / z: 2060.5 [M+Na + ] Step 6: (2S,3S,4S,5R,6S)-6-(2-(1-amino-3,6,9,12-tetraoxapentadecano-15-amido)-4-((((11S,11aS)-8-((5-(((11S,11aS)-10-(((4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)-11-hydroxy-7-methoxy-2-methylene-5-oxo 2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-2f) LiOH (10.1 mg, 0.42 mmol) was added to a solution of 2-2e (123 mg, 0.06 mmol) in THF / H2O (3 mL / 1 mL) on an ice bath, followed by stirring at the same temperature for 1 h and then heating to rt for 1 h. The mixture was diluted with H2O (3 mL) and washed with EtOAc (10 mL). The aqueous phase was acidified to pH 6 with 1N KHSO4 and then analyzed by prep-HPLC (0.1% FA) (Method: Column: XBridge Prep C18 OBD 5μm 19). Purification was performed using a mobile phase of 150 mL / min (A-water (0.1% formic acid):B-acetonitrile; flow rate: 20 mL / min) to give a white solid 2-2f (21.7 mg, 23.3% yield). MS (ESI) m / z: 1535.8 [M+H] + .

[0333] Step 7: (2S,3S,4S,5R,6S)-6-(2-(1-((1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nonadecano-19-amido)-4-((((11S,11aS)-8-((5-(((11S,11aS)-10-(((4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl) -11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-2) DIPEA (4.7 mg, 0.036 mmol) was added to a mixture of 2-2f (18.7 mg, 0.012 mmol) and Int-4 (10.5 mg, 0.036 mmol) in DMF (2 mL). The mixture was stirred at rt for 30 min. The mixture was then passed through prep-HPLC (0.1% FA) (method: column: XBridge Prep C18 OBD 5μm 19). Purification was performed using a mobile phase of 150 mL / min (A-water (0.1% formic acid):B-acetonitrile; flow rate: 20 mL / min) to give a white solid 2-2 (11.9 mg, 57.9% yield). MS (ESI) m / z: 1733.8 [M+Na] + .

[0334] Examples 2-4 Step 1: (2S,3R,4S,5S,6S)-triacetic acid 2-(4-((((5-((5-(5-((((allyloxy)carbonyl)amino)-4-((S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-5-azaspiro[2.4]heptane-5-carbonyl)-2-methoxyphenoxy)pentyl)oxy)-2-((S)-3-(((tert-butyldimethylsilyl)oxy)methyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-4a) Triphosgene (12 mg, 0.040 mmol) and Et3N (31 μL, 0.22 mmol) were added to a solution of Int-10 (100 mg, 0.100 mmol), 4Å molecular sieve (100 mg), and THF (3 mL) at 0 °C. The mixture was stirred at 0 °C under N2 for 10 min. A solution of Int-6 (57 mg, 0.13 mmol), Et3N (21 μL, 0.15 mmol), and dibutyltin dilaurate (6.3 mg, 0.010 mmol) in THF (1 mL) was added to the above reaction mixture. The mixture was stirred at 0 °C under N2 overnight. The mixture was filtered. The filtrate was concentrated under vacuum to obtain a residue, which was purified by silica gel column chromatography (petroleum ether / EtOAc = 20 / 80) to give 2-4a (140 mg, 94.0% yield) as a white solid. MS (ESI) m / z: 1490.2 [M+H] + .

[0335] Step 2: (2S,3R,4S,5S,6S)-triacetic acid 2-(4-((((5-((5-(5-amino-4-((S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-5-azaspiro[2.4]heptane-5-carbonyl)-2-methoxyphenoxy)pentyl)oxy)-2-((S)-3-(((tert-butyldimethylsilyl)oxy)methyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-4b) Pd(PPh3)4 (11 mg, 0.0095 mmol) was added to a solution of 2-4a (140 mg, 0.095 mmol), pyrrolidine (20 μL, 0.24 mmol), and CH2Cl2 (5 mL) under N2. The mixture was stirred under N2 at rt for 1 h. The mixture was concentrated under vacuum to obtain a residue, which was purified by silica gel column chromatography (CH2Cl2 / MeOH = 95 / 5) to give 2-4b (98 mg, 74.8% yield) as a white solid. MS (ESI) m / z: 1383.9 [M+H] + .

[0336] Step 3: (2S,3R,4S,5S,6S)-triacetic acid 2-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nineta-19-amido)-4-((((5-((5-(4-((S)-3-(((tert-butyldimethylsilyl)oxy)methyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-2-methoxy-5-((((4-(((2S,3R,4S,5S) ,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)amino)phenoxy)pentyl)oxy)-2-((S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-5-azaspiro[2.4]heptane-5-carbonyl)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-4c) Compound 2-4c (210 mg, 75.0% yield) was synthesized according to the synthetic procedure in step 1 of Examples 2-4. MS (ESI) m / z: 2334.3 [M+H] + .

[0337] Step 4: (2S,3R,4S,5S,6S)-triacetic acid 2-(4-((((5-((5-(5-amino-4-((S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-5-azaspiro[2.4]heptane-5-carbonyl)-2-methoxyphenoxy)pentyl)oxy)-2-((S)-3-(((tert-butyldimethylsilyl)oxy)methyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-4d) PTSA (41 mg, 0.22 mmol) was added to a mixture of 2-4c (210 mg, 0.0900 mmol) in THF (5 mL) and H₂O (250 μL). The mixture was stirred at rt for 3 h. The mixture was concentrated under vacuum to obtain a residue, which was purified by silica gel column chromatography (CH₂Cl₂ / MeOH = 95 / 5) to give 2-4d (110 mg, 58.2% yield) as a white solid. MS (ESI) m / z: 2106.3 [M+H] + .

[0338] Step 5: (2S,3R,4S,5S,6S)-triacetic acid 2-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nine-19-amido)-4-((((11S)-11-hydroxy-8-((5-(((6S)-6-hydroxy-2-methoxy-14-oxo-5-(((4-(((2S,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy) )benzyl)oxy)carbonyl)-5,6,6a,7,12,14-hexahydrobenzo[5,6][1,4]diazazo[1,2-b]isoquinoline-3-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazazo-2,1'-cyclopropane]-10-carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester(2-4e) Add 55 mg of Desmond-Martin periodane (0.13 mmol) to a solution of 2-4d (110 mg, 0.0052 mmol) in CH2Cl2 (5 mL) at 0 °C. Stir the mixture under N2 at rt for 2 h. Filter the mixture and wash the filtrate with 3 M Na2S2O3 and 3 M NaHCO3. Dry the organic phase over Na2SO4, filter, and concentrate the filtrate under vacuum to give a white solid 2-4e (107 mg, 99.2% yield), which was used in the next step without further purification. MS (ESI) m / z: 2102.7 [M+H] + .

[0339] Step 6: (2S,3S,4S,5R,6S)-6-(2-(1-amino-3,6,9,12-tetraoxapentadecano-15-amido)-4-((((11S,11aS)-8-((5-(((6S,6aS)-5-(((4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)-6-hydroxy-2-methoxy-14-oxo-5,6, 6a,7,12,14-Hexahydrobenzo[5,6][1,4]diazazo[1,2-b]isoquinoline-3-yl)oxy)pentyl)oxy)-11-hydroxy-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazazo-2,1'-cyclopropane]-10-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-4f) Et3N (398 μL, 2.86 mmol) was added to a mixture of 2-4e (660 mg, 0.286 mmol) and saturated LiBr (CH3CN / H2O = 10:1, 10 mL). The mixture was stirred at rt for 1 h. The mixture was adjusted to pH 7 using AcOH and concentrated under vacuum to obtain a crude product. The resulting white form was redissolved in DMF (10 mL), followed by the addition of 4Å molecular sieve (600 mg) and Et2NH (592 μL, 5.72 mmol). The mixture was stirred at rt for 1 h. The mixture was adjusted to pH 7 using AcOH and filtered, and then subjected to prep-HPLC (method: column: XBridge Prep C18 OBD 5μm 19). Purification was performed using a mobile phase of 150 mL / min (A-water (0.1% formic acid):B-acetonitrile; flow rate: 20 mL / min) to obtain a white solid 2–4 f (120 mg, 26.2% yield). MS (ESI) m / z: 1600.4 [M+H] + .

[0340] Step 7: (2S,3S,4S,5R,6S)-6-(2-(1-((1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nonadecano-19-amido)-4-((((11S,11aS)-8-((5-(((6S,6aS)-5-(((4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl) -6-Hydroxy-2-methoxy-14-oxo-5,6,6a,7,12,14-hexahydrobenzo[5,6][1,4]diazazo[1,2-b]isoquinoline-3-yl)oxy)pentyl)oxy)-11-hydroxy-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazazo-2,1'-cyclopropane]-10-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-4) DIEA (7.7 μL, 0.039 mmol) was added to a mixture of 2-4f (10 mg, 0.0063 mmol) and Int-4 (3.6 mg, 0.013 mmol) in DMF (2 mL). The mixture was stirred at rt for 1 h. The mixture was adjusted to pH 7 using AcOH and filtered, and then analyzed by prep-HPLC (method: column: XBridge Prep C18 OBD 5μm 19). Purification was performed using a mobile phase of 150 mL / min (A-water (0.1% formic acid):B-acetonitrile; flow rate: 20 mL / min) to give a white solid 2-4 (4.2 mg, 37.2% yield). MS (ESI) m / z: 1797.8 [M+H] + .

[0341] Examples 2-6 Step 1: (2S,3R,4S,5S,6S)-triacetic acid 2-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nine-19-amido)-4-((((11S,11aS)-8-((5-(((6S,6aS)-2,6-dimethoxy-14-oxo-5-(((4-(((2S,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy) Benzyl)oxy)carbonyl)-5,6,6a,7,12,14-hexahydrobenzo[5,6][1,4]diazazo[1,2-b]isoquinoline-3-yl)oxy)pentyl)oxy)-7,11-dimethoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazazo-2,1'-cyclopropane]-10-carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-6a) TMSCl (18 μL, 0.14 mmol) was added to a mixed solution of 2-4e (30 mg, 0.014 mmol) in MeOH (20 μL, 0.50 mmol) and THF (0.5 mL). The mixture was stirred at 65 °C for 2 h. The mixture was concentrated under vacuum to obtain a crude product, which was purified by silica gel column chromatography (CH2Cl2 / MeOH = 95 / 5) to give 2-6a (25 mg, 83.3% yield) as a white solid. MS (ESI) m / z: 2130.4 [M+H] + .

[0342] Step 2: (2S,3S,4S,5R,6S)-6-(2-(1-amino-3,6,9,12-tetraoxapentadecano-15-amido)-4-((((11S,11aS)-8-((5-(((6S,6aS)-5-(((4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)-2,6-dimethoxy-14-oxo-5,6, 6a,7,12,14-Hexahydrobenzo[5,6][1,4]diazazo[1,2-b]isoquinoline-3-yl)oxy)pentyl)oxy)-7,11-dimethoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazazo-2,1'-cyclopropane]-10-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-6b) LiOH (0.6 mg, 0.023 mmol) was added to a solution of 2-6a (5 mg, 0.0023 mmol) in H₂O (0.2 mL) and THF (0.2 mL). The mixture was stirred at rt for 1 h. The mixture was adjusted to pH 7 using AcOH and concentrated under vacuum to obtain a residue, which was analyzed using prep-HPLC (method: column: XBridge Prep C18 OBD 5μm 19). Purification was performed using a mobile phase of 150 mL / min (A-water (0.1% formic acid):B-acetonitrile; flow rate: 20 mL / min) to give a white solid 2-6b (2 mg, 54.1% yield). MS (ESI) m / z: 1628.2 [M+H] + .

[0343] Step 3: (2S,3S,4S,5R,6S)-6-(2-(1-((1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nonadecano-19-amido)-4-((((11S,11aS)-8-((5-(((6S,6aS)-5-(((4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl) 2,6-dimethoxy-14-oxo-5,6,6a,7,12,14-hexahydrobenzo[5,6][1,4]diazazo[1,2-b]isoquinoline-3-yl)oxy)pentyl)oxy)-7,11-dimethoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazazo-2,1'-cyclopropane]-10-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-6) Synthesized 2-6 (2.7 mg, 53.6% yield) according to step 7 of Examples 2-4. MS (ESI) m / z: 1803.9 [M+Na] + .

[0344] Examples 2-7 Step 1: (2S,3R,4S,5S,6S)-triacetic acid 2-(4-((((5-((5-(5-((((allyloxy)carbonyl)amino)-4-((S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-5-azaspiro[2.4]heptane-5-carbonyl)-2-methoxyphenoxy)pentyl)oxy)-2-((S)-3-(((tert-butyldimethylsilyl)oxy)methyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-4-methoxyphenyl)carbamoyl)oxy)methyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-7a) Compound 2-7a (340 mg, 86.5% yield) was synthesized according to the synthetic procedure of step 1 in Examples 2-4. MS (ESI) m / z: 1513.1 [M+Na] + .

[0345] Step 2: (2S,3R,4S,5S,6S)-triacetic acid 2-(4-((((5-((5-(5-amino-4-((S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-5-azaspiro[2.4]heptane-5-carbonyl)-2-methoxyphenoxy)pentyl)oxy)-2-((S)-3-(((tert-butyldimethylsilyl)oxy)methyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-4-methoxyphenyl)carbamoyl)oxy)methyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-7b) Compound 2-7b (127 mg, 55.0% yield) was synthesized according to the synthetic procedure in step 2 of Examples 2-4. MS (ESI) m / z: 1428.9 [M+H] + .

[0346] Step 3: (2S,3R,4S,5S,6S)-triacetic acid 2-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nonadecano-19-amido)-4-((((5-((5-(4-((S)-3-(((tert-butyldimethylsilyl)oxy)methyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-2-methoxy-5-((((3-nitro-4-(((2S,3R,4S) ,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)amino)phenoxy)pentyl)oxy)-2-((S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-5-azaspiro[2.4]heptane-5-carbonyl)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-7c) Compound 2-7c (200 mg, 94.3% yield) was synthesized according to the synthetic procedure in step 3 of Examples 2-4. MS (ESI) m / z: 2379.3 [M+H] + .

[0347] Step 4: (2S,3R,4S,5S,6S)-triacetic acid 2-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nine-19-amido)-4-((((5-((5-(4-((S)-3-(hydroxymethyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-2-methoxy-5-((((3-nitro-4-(((2S,3R,4S) ,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)amino)phenoxy)pentyl)oxy)-2-((S)-6-(hydroxymethyl)-5-azaspiro[2.4]heptane-5-carbonyl)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-7d) Compounds were synthesized 2-7 days (160 mg, 89.2% yield) according to the synthetic procedure in step 4 of Examples 2-4. MS (ESI) m / z: 2150.8 [M+H] + .

[0348] Step 5: (2S,3R,4S,5S,6S)-triacetic acid 2-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nine-19-amido)-4-((((11S)-11-hydroxy-8-((5-(((6S)-6-hydroxy-2-methoxy-5-(((3-nitro-4-(((2S,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl) )oxy)carbonyl)-14-oxo-5,6,6a,7,12,14-hexahydrobenzo[5,6][1,4]diazazo[1,2-b]isoquinoline-3-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazazo-2,1'-cyclopropane]-10-carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester(2-7e) Compound (2-7e, 156 mg, 98.2% yield) was synthesized according to the synthetic procedure in step 5 of Examples 2-4. MS (ESI) m / z: 2329.3 [M+H] + .

[0349] Step 6: (2S,3S,4S,5R,6S)-6-(2-(1-amino-3,6,9,12-tetraoxapentadecano-15-amido)-4-((((11S,11aS)-8-((5-(((6S,6aS)-5-(((4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-3-nitrobenzyl)oxy)carbonyl)-6-hydroxy-2-methoxy-14-oxo-5 ,6,6a,7,12,14-Hexahydrobenzo[5,6][1,4]diazazo[1,2-b]isoquinoline-3-yl)oxy)pentyl)oxy)-11-hydroxy-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazazo-2,1'-cyclopropane]-10-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-7f) Compounds 2-7f (20 mg, 16.3% yield) were synthesized according to the synthetic procedure in step 6 of Examples 2-4. MS (ESI) m / z: 1645.3 [M+H] + .

[0350] Step 7: (2S,3S,4S,5R,6S)-6-(2-(1-((1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nonadecano-19-amido)-4-((((11S,11aS)-8-((5-(((6S,6aS)-5-(((4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-3-nitrobenzyl)oxy) Carbonyl)-6-hydroxy-2-methoxy-14-oxo-5,6,6a,7,12,14-hexahydrobenzo[5,6][1,4]diazazo[1,2-b]isoquinoline-3-yl)oxy)pentyl)oxy)-11-hydroxy-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazazo-2,1'-cyclopropane]-10-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-7) Compounds 2-7 were synthesized according to the synthetic procedure in step 6 of Examples 2-4 (11.1 mg, 48.7% yield). MS (ESI) m / z: 1843.2 [M+Na] + .

[0351] Examples 2-8 Step 1: (2S,3R,4S,5S,6S)-triacetic acid 2-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nonadecano-19-amido)-4-((((5-((5-(((S)-10-((allyloxy)carbonyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[ e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)pentyl)oxy)-2-((S)-3-(((tert-butyldimethylsilyl)oxy)methyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-8a) Compound 2-8a (367 mg, 87% yield) was synthesized according to the synthetic procedure in step 1 of Ref-2-2. MS (ESI) m / z: 1819.8 [M+H] + .

[0352] Step 2: (2S,3R,4S,5S,6S)-triacetic acid 2-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nine-19-amido)-4-((((5-((5-((((S)-10-((allyloxy)carbonyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H) -spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)pentyl)oxy)-2-((S)-3-(hydroxymethyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-8b) Compound 2-8b (268 mg, 72% yield) was synthesized according to the synthetic procedure in step 2 of Ref-2-2. MS (ESI) m / z: 1705.8 [M+H] + .

[0353] Step 3: (2S,3R,4S,5S,6S)-triacetic acid 2-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nineta-19-amido)-4-((((6S,6aS)-3-((5-(((S)-10-((allyloxy)carbonyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[]]) [e]pyrrolo[1,2-a][1,4]diazazo-2,1'-cyclopropane]-8-yl)oxy)pentyl)oxy)-6-hydroxy-2-methoxy-14-oxo-5,6,6a,7,12,14-hexahydrobenzo[5,6][1,4]diazazo[1,2-b]isoquinoline-5-carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-8c) Des Martin periodane (70 mg, 0.16 mmol) was slowly added fractionally to 2-8b (267 mg, 0.16 mmol) in anhydrous CH2Cl2 (5 mL) at 0 °C. The reaction was then heated to rt and stirred for 2 h. The reaction was quenched with saturated Na2S2O3, followed by the addition of saturated NaHCO3 and water. The layers were separated, and the organic layer was washed with brine and dried over Na2SO4. The crude product was purified by silica gel column chromatography (CH2Cl2 / EtOAc = 93 / 7) to give 2-8c (218 mg, 82% yield). MS (ESI) m / z: 1703.8 [M+H] + .

[0354] Step 4: (2S,3S,4S,5R,6S)-6-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nonadecano-19-amido)-4-((((6S,6aS)-3-((5-(((S)-10-((allyloxy)carbonyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzene]) [e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)pentyl)oxy)-6-hydroxy-2-methoxy-14-oxo-5,6,6a,7,12,14-hexahydrobenzo[5,6][1,4]diaza-2,2-b]isoquinoline-5-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-8d) LiBr (306 mg, 3.52 mmol) was dissolved in MeCN / H₂O (0.8 / 0.08 mL), followed by the addition of 2-8c (60 mg, 0.04 mmol) to the mixture. Et₃N (49 μL, 0.35 mmol) was added to the reaction mixture at set time, and the mixture was stirred at set time for 1 h. The reaction was deemed complete by LC-MS, and the mixture was neutralized and concentrated with AcOH (21 μL, 0.35 mmol). The residue was used directly in the next step without further purification. MS (ESI) m / z: 1563.7 [M+H] + .

[0355] Step 5: (2S,3S,4S,5R,6S)-6-(4-((((6S,6aS)-3-((5-(((S)-10-((allyloxy)carbonyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)pentyl 6-hydroxy-2-methoxy-14-oxo-5,6,6a,7,12,14-hexahydrobenzo[5,6][1,4]diazazo[1,2-b]isoquinoline-5-carbonyl)oxy)methyl)-2-(1-amino-3,6,9,12-tetraoxapentadecano-15-amido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-8e) Et₂NH (67 μL, 0.65 mmol) was added to a solution of crude 2–8 d (55 mg, 0.032 mmol) in 2 mL DMF. The mixture was stirred at rt for 0.5 h. The mixture was concentrated to obtain the crude product, which was then subjected to prep-HPLC (method: column: XBridge Prep C18 OBD 5μm 19). Purification was performed using a mobile phase of 150 mL / min (A-water (0.1% formic acid):B-acetonitrile; flow rate: 20 mL / min) to give a white solid 2-8e (28 mg, 37% yield). MS (ESI) m / z: 1341.7 [M+H] + .

[0356] Step 6: (2S,3S,4S,5R,6S)-6-(2-(1-amino-3,6,9,12-tetraoxapentadecano-15-amido)-4-((((6S,6aS)-6-hydroxy-2-methoxy-3-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[ 1,2-a][1,4]diazazo-2,1'-cyclopropane]-8-yl)oxy)pentyl)oxy)-14-oxo-5,6,6a,7,12,14-hexahydrobenzo[5,6][1,4]diazazo[1,2-b]isoquinoline-5-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-8f) Pd(PPh3)4 (1.2 mg, 0.021 mmol) was added to a solution of 2-8e (28 mg, 0.021 mmol) and pyrrolidine (1.9 μL, 0.023 mmol) in CH2Cl2 (1 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 1 h. The reaction was neutralized and concentrated with AcOH (1.3 μL, 0.023 mmol). The crude product was subjected to prep-HPLC (method: column: XBridge Prep C18 OBD 5μm 19). Purification was performed using a mobile phase of 150 mL / min (A-water (0.1% formic acid):B-acetonitrile; flow rate: 20 mL / min) to give a white solid 2–8 f (16 mg, 61% yield). MS (ESI) m / z: 1257.7 [M+H] + .

[0357] Step 7: (2S,3S,4S,5R,6S)-6-(2-(1-((1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-yl)-3-oxo-2,7,10,13,16-pentaoxa-4-aza-nonadecano-19-amido)-4-((((6S,6aS)-6-hydroxy-2-methoxy-3-((5-((((S)-7-methoxy-5-oxo-5,10,11,11) α-Tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)pentyl)oxy)-14-oxo-5,6,6a,7,12,14-hexahydrobenzo[5,6][1,4]diaza-1,2-b]isoquinoline-5-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-8) DIEA (11.1 μL, 0.064 mmol) was added to a solution of 2-8f (16 mg, 0.013 mmol) and Int-4 (11.1 mmol, 0.038 mmol) in 1 mL of DMF. The mixture was stirred at rt for 20 min. The mixture was then passed through prep-HPLC (method: column: XBridge Prep C18 OBD 5μm 19). Purification was performed using a mobile phase of 150 mL / min (A-water (0.1% formic acid):B-acetonitrile; flow rate: 20 mL / min) to give a white solid 2-8 (5.1 mg, 28% yield). MS (ESI) m / z: 1433.8 [M+H] + .

[0358] Example 2-10 Step 1 (2S,3R,4S,5S,6S)-triacetic acid 2-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16-pentaoxa-4-aza-nonadecano-19-amido)-4-((((5-((5-(((S)-5-((((S)-5-((allyloxy)carbonyl)-2-methoxy-14-oxo-5,6,6a,7,12,14-hexahydrobenzo[5, 6][1,4]diazazo[1,2-b]isoquinoline-3-yl)oxy)pentyl)oxy)-2-((S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-5-azaspiro[2,4]heptane-5-carbonyl)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-10a) Int-14 (200 mg, 0.23 mmol) was dissolved in anhydrous THF (2 mL), and 4 Å molecular sieve (200 mg) was added. The mixture was cooled to 0 °C, and then triphosgene (24.6 mg, 0.083 mmol) was added, followed by Et3N (71.1 μL, 0.5 mmol). The mixture was stirred at 0 °C under a nitrogen atmosphere for 10 min. Int-7 (234.1 mg, 0.25 mmol), Et3N (48.5 μL, 0.345 mmol), and dibutyltin dilaurate (27.3 μL, 0.046 mmol) were mixed in anhydrous THF (1 mL), and this solution was then gradually added to the above reaction solution at 0 °C. The reaction solution was heated to rt and stirred for 2 h. The solvent was concentrated, and then purified by column chromatography (MeOH / CH2Cl2 = 5 / 95) to give a pale yellow solid 2-10a (365 mg, 87.2% yield). MS (ESI) m / z: 1821.1 [M+H] + .

[0359] Step 2 (2S,3R,4S,5S,6S)-triacetic acid 2-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nonadecano-19-amido)-4-((((5-((5-((((S)-5-(((allyloxy)carbonyl)-2-methoxy-14-oxo-5,6,6a,7,12,14-hexadecyl) Hydrobenzo[5,6][1,4]diazazo[1,2-b]isoquinoline-3-yl)oxy)pentyl)oxy)-2-((S)-6-(hydroxymethyl)-5-azaspiro[2,4]heptane-5-carbonyl)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-10b) Dissolve 2-10a (365 mg, 0.2 mmol) in THF / H2O (3 mL / 60 μL, 50 / 1), and add TsOH. . H2O (22.9 mg, 0.12 mmol) was added, and the mixture was stirred at rt for 2 h. The solvent was then removed by evaporation, and the residue was purified by column chromatography (MeOH / EtOAc = 2 / 98) to give a pale yellow solid, 2-10b (300 mg, 87.7% yield). MS (ESI) m / z: 1705.7 [M+H] + .

[0360] Step 3: (2S,3R,4S,5S,6S)-triacetic acid 2-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nine-19-amido)-4-((((11S,11aS)-8-((5-(((S)-5-((allyloxy)carbonyl)-2-methoxy-14-oxo-5,6,6a,7,12,14-hexahydrobenzo[5,6][ [1,4]diazazo[1,2-b]isoquinoline-3-yl)oxy)pentyl)oxy)-11-hydroxy-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazazo-2,1'-cyclopropane]-10-carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-10c) Des Martin periodane (82 mg, 0.19 mmol) was added to a solution of 2-10b (300 mg, 0.18 mmol) in CH2Cl2 (1 mL) under rt, and the mixture was stirred under nitrogen atmosphere for 3 h under rt. The reaction solution was then diluted with CH2Cl2 (10 mL) and washed with saturated Na2S2O3 (5 mL) and brine (5 mL). The organic phase was collected, dried over anhydrous Na2SO4, filtered, and concentrated under vacuum to give a crude product, which was purified by silica gel column chromatography (MeOH / CH2Cl2 = 5 / 95) to give a yellow solid 2-10c (270 mg, 90.1% yield). MS (ESI) m / z: 1726.2 [M+Na] + .

[0361] Step 4: (2S,3S,4S,5R,6S)-6-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nonadecano-19-amido)-4-((((11S,11aS)-8-((5-(((S)-5-((allyloxy)carbonyl)-2-methoxy-14-oxo-5,6,6a,7,12,14-hexahydrobenzo[5,6]) [1,4]diazazo[1,2-b]isoquinoline-3-yl)oxy)pentyl)oxy)-11-hydroxy-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazazo-2,1'-cyclopropane]-10-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-10d). 2-10c (50 mg, 0.032 mmol) was added to a solution of LiBr (277.6 mg, 3.2 mmol) in CH3CN / H2O (0.5 mL / 50 μL) under reflux, followed by the addition of triethylamine (44.3 μL, 0.32 mmol). The reaction mixture was stirred under nitrogen atmosphere for 1 h under reflux. The solvent was removed by evaporation to give a yellow oily crude product 2-10d, which was used directly in the next step without further purification. MS (ESI) m / z: 1563.7 [M+H] + .

[0362] Step 5: (2S,3S,4S,5R,6S)-6-(4-((((11S,11aS)-8-((5-(((S)-5-((allyloxy)carbonyl)-2-methoxy-14-oxo-5,6,6a,7,12,14-hexahydrobenzo[5,6][1,4]diazazo[1,2-b]isoquinoline-3-yl)oxy)pentyl)oxy)-11-hydroxy-7 -Methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-10-carbonyl)oxy)methyl)-2-(1-amino-3,6,9,12-tetraoxapentadecano-15-amido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid(2-10e) Diethylamine (0.32 mmol, 33 μL) was added to the crude product in 1 mL DMF at 0 °C for 2–10 days. The reaction solution was heated to rt and stirred for 1 h. The reaction solution was neutralized with AcOH and then subjected to prep-HPLC (method: column: XBridge Prep C18 OBD 5μm 19). Purification was performed using a mobile phase of 150 mm (A-water (0.1% formic acid):B-acetonitrile; flow rate: 20 mL / min), followed by lyophilization to obtain a white solid, 2-10e (25 mg, 58% yield). MS (ESI) m / z: 1342.3 [M+H] + .

[0363] Step 6: (2S,3S,4S,5R,6S)-6-(2-(1-amino-3,6,9,12-tetraoxapentadecano-15-amido)-4-((((11S,11aS)-11-hydroxy-7-methoxy-8-((5-(((S)-2-methoxy-14-oxo-5,6,6a,7,12,14-hexahydrobenzo[5,6][1,4]di) Azazo[1,2-b]isoquinoline-3-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazazo-2,1'-cyclopropane]-10-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-10f) Pd(PPh3)4 (1 mg, cat.) and diethylamine (3.87 μL, 0.037 mmol) were added to a solution of 2-10e in 1 mL of CH2Cl2 at 0 °C. The reaction solution was stirred at 0 °C for 1 h. The reaction solution was neutralized with AcOH and then subjected to prep-HPLC (method: column: XBridge Prep C18 OBD 5μm 19). Purification was performed using a mobile phase of 150 mL / min (A-water (0.1% formic acid):B-acetonitrile; flow rate: 20 mL / min). The purified product was lyophilized to give 2–10 f (15 mg, 80% yield) of a white solid. MS (ESI) m / z: 1258.2 [M+H] + .

[0364] Step 7: (2S,3S,4S,5R,6S)-6-(2-(1-((1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nonadecano-19-amido)-4-((((11S,11aS)-11-hydroxy-7-methoxy-8-((5-((((S)-2-methoxy-14-oxo-5,6,6a,7 ,12,14-Hexahydrobenzo[5,6][1,4]diazazo[1,2-b]isoquinoline-3-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazazo-2,1'-cyclopropane]-10-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-10) Int-4 (7 mg, 0.024 mmol) and DIPEA (11.4 μL, 0.06 mmol) were added to a solution of 2-10f (15 mg, 0.012 mmol) in 0.5 mL of DMF at 0 °C. The reaction solution was stirred at 0 °C for 0.5 h. The reaction solution was neutralized with AcOH and then subjected to prep-HPLC (method: column: XBridge Prep C18 OBD 5μm 19). 150 mm; Mobile phase: A-water (0.1% formic acid): B-acetonitrile; Flow rate: 20 mL / min) Purification, lyophilized to give a white solid 2-10 (6.3 mg, 36.8% yield). MS (ESI) m / z: 1434.2 [M+H] + .

[0365] Example 2-12 Step 1: (S)-7-methoxy-8-((triisopropylsilyl)oxy)-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (2-12a) Pyrrolidine (0.3 mL, 3.65 mmol) and Pd(PPh3)4 (168.52 mg, 0.15 mmol) were added to a solution of Int-12b (750 mg, 1.46 mmol) in CH2Cl2 (6 mL) at rt. The mixture was stirred at rt under N2 for 30 min. The solvent was evaporated and the mixture was purified by silica gel column chromatography (A-petroleum ether; B-EtOAc) to give a white solid product 2-12a (580 mg, 87.8% yield). MS (ESI) m / z: 431.2 [M+H] + .

[0366] Step 2: (S)-8-hydroxy-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (2-12b) LiOAc (133 mg, 2.02 mmol) was added to a mixed solution of compound 2-12a (580 mg, 1.35 mmol) in DMF (3 mL) and water (0.15 mL). The mixture was stirred at 20 °C for 1 h. The reaction mixture was diluted with EtOAc (20 mL) and then diluted with saline (15 mL). 3) Washing. The organic layer was dried over Na2SO4. After filtration and evaporation, the residue was purified by silica gel column chromatography (eluent: A-CH2Cl2; B-MeOH) to give a white solid 2-12b (350 mg, 89.98% yield). MS (ESI) m / z: 275.2 [M+H] + .

[0367] Step 3: (S)-2-methoxy-3-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazazo-2,1'-cyclopropane]-8-yl)oxy)pentyl)oxy)-14-oxo-6,6a,7,12-tetrahydrobenzo[5,6][1,4]diazazo[1,2-b]isoquinoline-5(14H)-formic acid allyl ester (2-12c) K₂CO₃ (91 mg, 0.66 mmol) was added to a solution of 2-12b (90 mg, 0.33 mmol) and Int-15 (195 mg, 0.33 mmol) in DMF (2 mL) under rt. The mixture was stirred under rt for 20 h. The reaction mixture was diluted with EtOAc (20 mL) and then diluted with saline (10 mL). 3) Washing. The organic layer was dried over Na2SO4, filtered, and concentrated to obtain the residue. This residue was purified by silica gel column chromatography (A-petroleum ether; B-EtOAc) to give a grayish-white solid 2-12c (150 mg, 59.0% yield). MS (ESI) m / z: 737.4 [M+H] + .

[0368] Step 4: (2S,3R,4S,5S,6S)-triacetic acid 2-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nine-19-amido)-4-((((S)-8-((5-((((S)-5-(((allyloxy)carbonyl)-2-methoxy-14-oxo-5,6,6a,7,12,14-hexahydrobenzo[5,6][1]) [1,4]diazazo[1,2-b]isoquinoline-3-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazazo-2,1'-cyclopropane]-10-carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-12d) A solution of Int-7 (151 mg, 0.16 mmol) in CH2Cl2 (6 mL) was cooled to -90 °C. Triphosgene (12 mg, 0.04 mmol) was added to CH2Cl2 (2 mL) and pyridine (0.087 mL, 1.09 mmol) under N2 protection at -90 °C, while maintaining the temperature below -85 °C. The mixture was stirred at -90 °C for 20 min, followed by the addition of 2-12c (100 mg, 0.14 mmol) to CH2Cl2 (2 mL). The reaction was heated to 0 °C and stirred at 0 °C for 2 h. The reaction mixture was quenched with water (20 mL) and then quenched with CH2Cl2 (30 mL). 3) Extraction. The organic layer was dried over Na2SO4 and concentrated to obtain a residue, which was purified by silica gel column chromatography (A-petroleum ether; B-EtOAc) to give a grayish-white solid, 2–12 d (150 mg, 49.1% yield). MS (ESI) m / z: 1688.3 [M+H] + .

[0369] Step 5: (2S,3S,4S,5R,6S)-6-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nine-19-amido)-4-((((S)-8-((5-((((S)-5-((allyloxy)carbonyl)-2-methoxy-14-oxo-5,6,6a,7,12,14-hexahydrobenzo[5,6][ [1,4]diazazo[1,2-b]isoquinoline-3-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazazo-2,1'-cyclopropane]-10-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-12e) LiBr (773 mg, 8.89 mmol) and Et3N (0.12 mL, 0.89 mmol) were added to a solution of 12-2d (150 mg, 0.09 mmol) in MeCN (10 mL) and water (1 mL) under reflux. The mixture was stirred under reflux for 2 h. The reaction mixture was acidified with AcOH (0.15 mL) and the solvent was evaporated to give residue 2-12e (crude product), which was used for the next step without further purification. MS (ESI) m / z: 1548.2 [M+H] + .

[0370] Step 6: (2S,3S,4S,5R,6S)-6-(4-((((S)-8-((5-(((S)-5-(((allyloxy)carbonyl)-2-methoxy-14-oxo-5,6,6a,7,12,14-hexahydrobenzo[5,6][1,4]diazazo[1,2-b]isoquinoline-3-yl)oxy)pentyl)oxy)-7-methoxy-5- oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-10-carbonyl)oxy)methyl)-2-(1-amino-3,6,9,12-tetraoxapentadecano-15-amido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-12f). Diethylamine (0.09 mL, 0.89 mmol) was added to a solution of 2-12e (crude product) in DMF (5 mL) under rt. The reaction mixture was stirred under rt for 1 h. LCMS showed that the reaction was complete. The reaction mixture was acidified with AcOH (0.10 mL) and analyzed by prep-HPLC (method: column: XBridge Prep C18 OBD 5μm 19). Purification was performed using a mobile phase of 150 mL / min (A-water (0.1% formic acid):B-acetonitrile; flow rate: 20 mL / min) to give a white solid 2-12 f (50 mg, 40.3% yield). MS (ESI) m / z: 1324.6 [M+H] + .

[0371] Step 7: (2S,3S,4S,5R,6S)-6-(2-(1-amino-3,6,9,12-tetraoxapentadecano-15-amido)-4-((((S)-7-methoxy-8-((5-((((S)-2-methoxy-14-oxo-5,6,6a,7,12,14-hexahydrobenzo[5,6][1,4]diazazo[1, 2-b]Isoquinoline-3-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-10-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-12g) Dimethyl ketone (10 mg, 0.08 mmol) and Pd(PPh3)4 (4.36 mg, 0.04 mmol) were added to a solution of 2-12f (50 mg, 0.04 mmol) in THF (3 mL) under rt. The mixture was stirred under N2 for 1 h under rt. The solvent was evaporated, and the mixture was passed through a prep-HPLC system (method: column: XBridge Prep C18 OBD 5μm 19). Purification was performed using a mobile phase of 150 mL / min (A-water (0.1% formic acid):B-acetonitrile; flow rate: 20 mL / min) to give 2–12 g (30 mg, 60.1% yield) of a white solid. MS (ESI) m / z: 1241.5 [M+H] + .

[0372] Step 8: (2S,3S,4S,5R,6S)-6-(2-(1-((1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nonadecano-19-amido)-4-((((S)-7-methoxy-8-((5-((((S)-2-methoxy-14-oxo-5,6,6a,7,12,14- Hexahydrobenzo[5,6][1,4]diazazo[1,2-b]isoquinoline-3-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazazo-2,1'-cyclopropane]-10-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-12) Int-4 (7.39 mg, 0.03 mmol) and DIPEA (0.0082 mL, 0.05 mmol) were added to a solution of 2-12 g (30 mg, 0.02 mmol) of compound in DMF (2 mL) under rt. The mixture was stirred under rt for 2 h. The reaction mixture was then subjected to prep-HPLC (method: column: XBridge Prep C18 OBD 5 μm 19). Purification was performed using a mobile phase of 150 mL / min (A-water (0.1% formic acid):B-acetonitrile; flow rate: 20 mL / min) to give a white solid 2-12 (16.8 mg, 48.1% yield). MS (ESI) m / z: 1418.3 [M+H] + Example 2-14 Step 1: (2S,3R,4S,5S,6S)-triacetic acid 2-(4-((((5-((3-((5-((((allyloxy)carbonyl)amino)-4-((S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-5-azaspiro[2.4]heptane-5-carbonyl)-2-methoxyphenoxy)methyl)benzyl)oxy)-2-((S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-5-azaspiro[2.4]heptane-5-carbonyl)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-14a) Triphosgene (38 mg, 0.13 mmol) and Et3N (236 μL, 172 mg, 1.70 mmol) were added to a mixture of Int-17 (340 mg, 0.34 mmol) and 4Å molecular sieve (100 mg) in anhydrous THF (4 mL) at 0 °C, followed by stirring on ice for 10 min. A mixture of Int-6 (180 mg, 0.41 mmol) and dibutyltin dilaurate (21 mg, 0.034 mmol) in THF (4 mL) was then added. The mixture was stirred overnight at rt. After the reaction was complete, the mixture was purified by silica gel column chromatography (10 g, EtOAc / petroleum = 0 / 100–50 / 50), and the fraction was concentrated under vacuum to give 2-14a as a white solid (435.6 mg, 87.4% yield). MS (ESI) m / z: 1466.4 [M+H] + .

[0373] Step 2: (2S,3R,4S,5S,6S)-triacetic acid 2-(4-((((5-((3-((5-amino-4-((S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-5-azaspiro[2.4]heptane-5-carbonyl)-2-methoxyphenoxy)methyl)benzyl)oxy)-2-((S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-5-azaspiro[2.4]heptane-5-carbonyl)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-14b) Dimethyl ketone (83 mg, 0.59 mmol) and Pd(PPh3)4 (34 mg, 0.030 mmol) were added to a mixture of 2-14a (435.6 mg, 0.30 mmol) and THF (5.0 mL). The pale yellow mixture was degassed three times with an N2 balloon, followed by stirring under nitrogen atmosphere at rt for 2 h. The mixture was filtered, concentrated under vacuum, and purified by reversed-phase chromatography (C18, MeCN / H2O = 20 / 80~100 / 0). The fraction was concentrated under vacuum to give 2-14b (375 mg, 91.3% yield) as a white solid. MS (ESI) m / z: 1382.4 [M+H] + .

[0374] Step 3: ((2S,3R,4S,5S,6S)-triacetic acid 2-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nonadecano-19-amido)-4-((((5-((3-((4-((S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-5-azaspiro[2.4]heptane-5-carbonyl)-2-methoxy-5-((((4-((2S,3R,4S,5S, 6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)amino)phenoxy)methyl)benzyl)oxy)-2-((S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-5-azaspiro[2.4]heptane-5-carbonyl)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-14c) Triphosgene (18 mg, 0.062 mmol) and Et3N (113 μL, 82 mg, 0.81 mmol) were added to a mixture of 2-14b (224 mg, 0.16 mmol) and 4Å molecular sieve (100 mg) in anhydrous THF (3 mL) at 0 °C, followed by stirring on ice for 10 min. A mixture of Int-7 (180 mg, 0.20 mmol) and dibutyltin dilaurate (10 mg, 0.016 mmol) in THF (2 mL) was added to the above solution. The mixture was stirred overnight at rt. The mixture was filtered, concentrated under vacuum, and the crude product was purified by reversed-phase chromatography (C18, MeCN / H2O = 20 / 80~100 / 0), followed by concentration fractionation under vacuum to give 2-14c (311 mg, 82.3% yield) as a white solid. MS (ESI) m / z: 2332.5 [M+H]+ .

[0375] Step 4: (2S,3R,4S,5S,6S)-triacetic acid 2-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nonadecano-19-amido)-4-((((5-((3-((4-((S)-6-(hydroxymethyl)-5-azaspiro[2.4]heptane-5-carbonyl)-2-methoxy-5-((((4-(((2S,3R,4S,5S,6S) ... S)-3,4,5-Triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)amino)phenoxy)methyl)benzyl)oxy)-2-((S)-6-(hydroxymethyl)-5-azaspiro[2.4]heptane-5-carbonyl)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-14d) A solution of 2-14d (102.0 mg, 0.044 mmol) in AcOH / THF / H2O (1 mL / 0.6 mL / 0.3 mL) was stirred overnight at rt. The mixture was diluted with EtOAc (30 mL), washed with brine (20 mL), dried over Na2SO4, filtered, and the filtrate was concentrated under vacuum to obtain a residue. This residue was then purified by silica gel column chromatography (MeOH / CH2Cl2 = 0 / 100~3 / 97), and the fraction was concentrated under vacuum to give a white solid 2-14d (90 mg, 97.8% yield). MS (ESI) m / z: 2104.6 [M+H] + .

[0376] Step 5: (2S,3R,4S,5S,6S)-triacetic acid 2-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nine-19-amido)-4-((((11S,11aS)-11-hydroxy-8-((3-((((11S,11aS)-11-hydroxy-7-methoxy-5-oxo-10-(((4-(((2S,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl) 5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)methyl)benzyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-10-carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-14e) Add Desmond-Martin periodane (42 mg, 0.098 mmol) to a solution of 2–14 d (90 mg, 0.043 mmol) in CH2Cl2 (3 mL), then stir at rt for 2 h. Quench the reaction mixture with saturated Na2S2O3 / saturated NaHCO3 / H2O (1:1:1, 15 mL) and quench with CH2Cl2 (20 mL). 3) Extraction. The combined organic layers were rinsed with 20 mL of brine. 3) Wash, dry with Na₂SO₄, filter, and concentrate the filtrate under vacuum to obtain a yellow solid 2-14e (87.0 mg, crude product). MS (ESI) m / z: 2100.6 [M+H] + .

[0377] Step 6: (2S,3S,4S,5R,6S)-6-(2-(1-amino-3,6,9,12-tetraoxapentadecano-15-amido)-4-((((11S,11aS)-8-((3-((((11S,11aS)-10-(((4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)-11-hydroxy-7-methoxy-5-oxo-5,10,11,11 α-Tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)methyl)benzyl)oxy)-11-hydroxy-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-10-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-14f). Add a mixture of LiBr (360.0 mg, 4.143 mmol) in MeCN / H2O (0.9 mL / 0.3 mL) and Et3N (31 μL, 23.06 mmol) to a solution of 2-14e (87 mg, crude product). Stir the mixture at rt for 5 h. Acidify the mixture to pH 5 with AcOH. Concentrate the mixture under vacuum to obtain the crude product, which is then redissolved in DMF (2 mL). Add diethylamine (214 μL, 2.071 mmol) and stir the mixture at rt for 1 h. Acidify the mixture with AcOH, filter, and pass through prep-HPLC (0.1% FA) (Method: Column: XBridge Prep C18 OBD 5 μm 19). 150 mm; Mobile phase: A-water (0.1% formic acid): B-acetonitrile; Flow rate: 20 mL / min) Purification, concentrated and fractionated under vacuum, yielded a white solid 2–14 f (45 mg, 68.0%). MS (ESI) m / z: 1598.3 [M+H] + .

[0378] Step 7: (2S,3S,4S,5R,6S)-6-(2-(1-((1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nonadecano-19-amido)-4-((((11S,11aS)-8-((3-((((11S,11aS)-10-(((4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)-11-hydroxy -7-Methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)methyl)benzyl)oxy)-11-hydroxy-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-10-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-14) DIPEA (5 μL, 4 mg, 0.028 mmol) was added to a mixture of 2-14f (45 mg, 0.028 mmol) and Int-4 (12 mg, 0.042 mmol) in DMF (1.5 mL). The mixture was stirred at rt for 15 min. After 2-14f was completely consumed, the mixture was subjected to prep-HPLC (0.1% FA) (method: column: XBridge Prep C18 OBD 5μm 19). Purification was performed using a mobile phase of 150 mL / min (A-water (0.1% formic acid):B-acetonitrile; flow rate: 20 mL / min) to give a white solid 2-14 (15.3 mg, 30.6% yield). MS (ESI) m / z: 1774.8 [M+H] + .

[0379] Example 2-16 Step 1: (S)-8-((3-(bromomethyl)benzyl)oxy)-7-methoxy-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-10(5H)-formate allyl ester (2-16a) K₂CO₃ (94 mg, 0.68 mmol) was added to a solution of Int-12 (162 mg, 0.45 mmol) and 1,3-bis(bromomethyl)benzene (596 mg, 2.26 mmol) in DMF (1.5 mL) under reflux. After addition, the reaction mixture was reacted under reflux for 2 h. The mixture was diluted with EtOAc (15 mL). The organic layer was then... saturation The sample was washed with 15 mL of NH4Cl aqueous solution, 15 mL of H2O, and 15 mL of brine. It was dried over anhydrous Na2SO4, filtered, and concentrated under vacuum to obtain a residue. This residue was purified by silica gel column chromatography (A-petroleum ether; B-EtOAc) to give a white solid 2-16a (195 mg, 79.5% yield). MS (ESI) m / z: 541.3 [M+H] + .

[0380] Step 2: (S)-8-((3-((5-amino-4-((S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-5-azaspiro[2.4]heptane-5-carbonyl)-2-methoxyphenoxy)methyl)benzyl)oxy)-7-methoxy-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-10(5H)-formate allyl ester (2-16b) Add Int-14b (243 mg, 0.60 mmol) to a solution of DMF (6.5 mL) on an ice bath. t -BuOK (69 mg, 0.60 mmol) was added and stirred for 0.5 h. Then, 2-16a (295 mg, 0.54 mmol) was added at the same temperature and stirred for another 0.5 h. The mixture was then... saturation Quench with 10 mL of NH4Cl aqueous solution. Remove the aqueous layer with 15 mL of EtOAc. 3) Extraction. The combined organic layers were washed with H₂O (20 mL) and brine (20 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum to obtain the residue. This residue was purified by silica gel column chromatography (A-petroleum ether; B-EtOAc) to give a pale yellow solid 2-16b (376.3 mg, 79.6% yield). MS (ESI) m / z: 868.1 [M+H] + .

[0381] Step 3: (2R,3S,4R,5R,6R)-triacetic acid 2-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nine-19-amido)-4-((((5-((3-((((S)-10-((allyloxy)carbonyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]) ]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)methyl)benzyl)oxy)-2-((S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-5-azaspiro[2,4]heptane-5-carbonyl)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-16c) Compound 2-16c (625 mg, 79.2% yield) was synthesized according to the synthetic procedure of step 1 in Example Ref-2-2. MS (ESI) m / z: 1818.0 [M+H] + .

[0382] Step 4: (2R,3S,4R,5R,6R)-triacetic acid 2-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nine-19-amido)-4-((((5-((3-((((S)-10-((allyloxy)carbonyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H- Spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)methyl)benzyl)oxy)-2-((S)-6-(hydroxymethyl)-5-azaspiro[2,4]heptane-5-carbonyl)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-16d) Compound 2-16d (568 mg, 96.9% yield) was synthesized according to the synthetic procedure of step 2 in Examples 2-8. MS (ESI) m / z: 1703.8 [M+H] + .

[0383] Step 5: (2R,3S,4R,5R,6R)-triacetic acid 2-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nonadecano-19-amido)-4-((((11S,11aS)-8-((3-((((S)-10-((allyloxy)carbonyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1] ,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)methyl)benzyl)oxy)-11-hydroxy-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-10-carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-16e) Compound 2-16e (200 mg, 99.9% yield) was synthesized according to the synthetic procedure in step 3 of Examples 2-8. MS (ESI) m / z: 1702.1 [M+H] + .

[0384] Step 6: (2R,3R,4R,5S,6R)-6-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nonadecano-19-amido)-4-((((11S,11aS)-8-((3-((((S)-10-((allyloxy)carbonyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[] 1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)methyl)benzyl)oxy)-11-hydroxy-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-10-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-16f) Compound 2-16f (crude, 0.12 mmol) was synthesized according to the synthetic procedure in step 4 of Examples 2-8. MS (ESI) m / z: 1562.4 [M+H] + .

[0385] Step 7: (2R,3R,4R,5S,6R)-6-(4-((((11S,11aS)-8-((3-((((S)-10-((allyloxy)carbonyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)methyl)benzyl)oxy) -11-Hydroxy-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-10-carbonyl)oxy)methyl)-2-(1-amino-3,6,9,12-tetraoxapentadecano-15-amido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-16g) Compound 2-16 g (85 mg, 54.1%, obtained via two steps) was synthesized according to the synthetic procedure in step 5 of Examples 2-8. MS (ESI) m / z: 1339.8 [M+H] + .

[0386] Step 8: (2R,3R,4R,5S,6R)-6-(2-(1-amino-3,6,9,12-tetraoxapentadecano-15-amido)-4-((((11S,11aS)-11-hydroxy-7-methoxy-8-((3-((((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a]) [1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)methyl)benzyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-10-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-16h) Compounds were synthesized for 2-16 hours (41 mg, 51.1%) according to the synthetic procedure in step 6 of Examples 2-8. MS (ESI) m / z: 1256.1 [M+H] + .

[0387] Step 9: (2R,3R,4R,5S,6R)-6-(2-(1-((1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nonadecano-19-amido)-4-((((11S,11aS)-11-hydroxy-7-methoxy-8-((3-((((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1 H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)methyl)benzyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-10-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-16) Compound 2-16 (17.1 mg, 50.0%) was synthesized according to the synthetic procedure of step 6 in Example Ref-2-2. MS (ESI) m / z: 1432.1 [M+H] + .

[0388] Example 2-18 Step 1: (S)-8-((5-(5-amino-4-((S)-5-(((tert-butyldimethylsilyl)oxy)methyl)-6-azaspiro[2.5]octane-6-carbonyl)-2-methoxyphenoxy)pentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-formic acid allyl ester (2-18a) K₂CO₃ (100 mg, 0.71 mmol) was added to a solution of Int-18 (150 mg, 0.36 mmol) and Int-19 (213 mg, 0.36 mmol) in DMSO (3 mL) under a N₂ atmosphere, and the mixture was stirred at 80 °C for 20 min. The solution was then added to water (50 mL) and diluted with EtOAc (20 mL). 3) Extraction, using saline (20 mL) 2) Washing. The organic phase was concentrated and purified by rapid column chromatography (petroleum ether / EtOAc = 25 / 75) to give a grayish-white solid 2-18a (277 mg, 84% yield). MS (ESI) m / z: 925.7 [M+H] + .

[0389] Step 2: (2S,3R,4S,5S,6S)-triacetic acid 2-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nonadecano-19-amido)-4-((((5-((5-((((S)-10-((allyloxy)carbonyl)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetra- Hydrogen-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-2-((S)-5-(((tert-butyldimethylsilyl)oxy)methyl)-6-azaspiro[2.5]octane-6-carbonyl)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-18b) A solution of triphosgene (22.5 mg, 0.07 mmol) in anhydrous THF (300 μL) was added to a solution of 2-18a (186 mg, 0.2 mmol) in anhydrous THF (2 mL) at 0 °C under a N2 atmosphere, followed by the addition of Et3N (43 μL, 0.3 mmol), and the mixture was stirred at rt for 10 min. Then, a solution of Int-7 (199 mg, 0.2 mmol), dibutyltin dilaurate (12 μL, 0.02 mmol), and Et3N (43 μL, 0.3 mmol) in anhydrous THF (1 mL) was added to this solution, dried using 4 Å MS, and stirred at rt for 1 h. The solution was filtered and washed with saturated NH4Cl (5 mL) and water (5 mL), and then irrigated with EtOAc (10 mL). 3) Extraction, concentration, and purification by rapid column chromatography (10% MeOH in CH2Cl2 / CH2Cl2 = 30 / 70) yielded a yellow solid, title compound 2-18b (343 mg, 91% yield). MS (ESI) m / z: 1876.4 [M+H] + .

[0390] Step 3: (2S,3R,4S,5S,6S)-triacetic acid 2-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nine-19-amido)-4-((((5-((5-((((S)-10-((allyloxy)carbonyl)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11 ,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-2-((S)-5-(hydroxymethyl)-6-azaspiro[2.5]octane-6-carbonyl)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-18c) PTSA (35.5 mg, 0.18 mmol) was added to a solution of 2-18b (343 mg, 0.18 mmol) in THF / H2O (2.1 / 0.7 mL) under N2 atmosphere, and the mixture was stirred at rt for 5 h. The solution was then added to saturated NaHCO3 (10 mL) and diluted with EtOAc (10 mL). 3) Extraction, concentration, and purification by rapid column chromatography (10% MeOH in CH2Cl2 / CH2Cl2 = 30 / 70) yielded a grayish-white solid, 2-18c (262 mg, 81.3% yield). MS (ESI) m / z: 1762.3 [M+H] + .

[0391] Step 4: (2S,3R,4S,5S,6S)-triacetic acid 2-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nonadecano-19-amido)-4-((((6S,6aS)-3-((5-(((S)-10-((allyloxy)carbonyl)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-benzene) [e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-6-hydroxy-2-methoxy-12-oxo-5,6a,7,9,10,12-hexahydro-6H-spiro[benzo[e]pyrido[1,2-a][1,4]diaza-8,1'-cyclopropane]-5-carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-18d) Add 70 mg, 0.16 mmol of Des Martin periodane to a solution of 2-18°C (260 mg, 0.15 mmol) in CH₂Cl₂ (2 mL) at 0 °C and stir at rt for 1.5 h. Add 70 mg, 0.16 mmol of Des Martin periodane again at 0 °C and stir at rt for another 30 min. Add 5 mL of saturated Na₂S₂O₃ and 5 mL of saturated NaHCO₃ to the solution and stir with EtOAc (5 mL). 3) Extraction, concentration, and purification by rapid column chromatography (10% MeOH in CH2Cl2 / CH2Cl2 = 30 / 70) yielded a mixture (aldehyde and cyclic form of the product, 256 mg). AcOH (170 μL, 2.9 mmol) was added to the mixture (256 mg, 0.14 mmol) in anhydrous CH2Cl2 (4 mL) under N2 atmosphere, and the mixture was stirred at rt for 16 h. The solution was concentrated and purified by rapid column chromatography (10% MeOH in CH2Cl2 / CH2Cl2 = 30 / 70) to give a pale yellow solid for 2–18 days (180 mg, 69% yield). MS (ESI) m / z: 1760.3 [M+H] + .

[0392] Step 5: (2S,3R,4S,5S,6S)-triacetic acid 2-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nonadecano-19-amido)-4-((((6S,6aS)-3-((5-(((S)-10-((allyloxy)carbonyl)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H- Benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-2,6-dimethoxy-12-oxo-5,6a,7,9,10,12-hexahydro-6H-spiro[benzo[e]pyridino[1,2-a][1,4]diaza-8,1'-cyclopropane]-5-carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-18e) TMSCl (74 μL, 0.57 mmol) and anhydrous MeOH (82 μL, 1.99 mmol) were added to a solution of 2-18d (100 mg, 0.056 mmol) in anhydrous THF (1 mL) under N2 atmosphere. The mixture was stirred at rt for 30 min, followed by stirring at 50 °C for 90 min. The concentrated solution was purified by rapid column chromatography (10% MeOH in CH2Cl2 / CH2Cl2 = 30 / 70) to give a yellow solid of 2-18e (37.5 mg, 37% yield). MS (ESI) m / z: 1774.3 [M+H] + .

[0393] Step 6: (2S,3R,4S,5S,6S)-triacetic acid 2-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nine-19-amido)-4-((((6S,6aS)-2,6-dimethoxy-3-((5-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1 H-Benz[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-12-oxo-5,6a,7,9,10,12-hexahydro-6H-spiro[benzo[e]pyridino[1,2-a][1,4]diaza-8,1'-cyclopropane]-5-carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-18f) Anhydrous THF (0.6 mL) and anhydrous MeOH (0.2 mL) were added to a mixture of 2-18e (49 mg, 0.028 mmol), dimethyl ketone (4.1 mg, 0.029 mmol), and Pd(PPh3)4 (0.8 mg, 0.7 μmol) under a nitrogen atmosphere, and the mixture was stirred at rt for 30 min. The concentrated solution was purified by rapid column chromatography (10% MeOH in CH2Cl2 / CH2Cl2 = 30 / 70) to give a yellow solid 2-18f (28 mg, 60% yield). MS (ESI) m / z: 1690.3 [M+H] + .

[0394] Step 7: (2S,3S,4S,5R,6S)-6-(2-(1-amino-3,6,9,12-tetraoxapentadecano-15-amido)-4-((((6S,6aS)-2,6-dimethoxy-3-((5-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo) [1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-12-oxo-5,6a,7,9,10,12-hexahydro-6H-spiro[benzo[e]pyrido[1,2-a][1,4]diaza-8,1'-cyclopropane]-5-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-18g) An aqueous solution of LiOH (4 mg, 0.17 mmol, 2 M, 83 μL) was added to a solution of 2-18f (28 mg, 0.017 mmol) in THF (0.3 mL) at 0 °C. The mixture was stirred at 0 °C for 30 min, followed by stirring at rt for 2 h. AcOH (20 μL) was then added to the solution. The solution was then passed through a prep-HPLC system (column: Sunfire Prep C18 OBD™ 5 μm, 19...). Purification was performed using a mobile phase of 150 mm; mobile phase A: 0.1% FA aqueous solution, mobile phase B: MeCN; gradient: 20% - 55% B; flow rate: 20 mL / min. The solution was then lyophilized to give 2-18 g (9 mg, 41% yield) of a white solid. MS (ESI) m / z: 1328.0 [M+H] + .

[0395] Step 8: (2S,3S,4S,5R,6S)-6-(2-(1-((1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-yl)-3-oxo-2,7,10,13,16-pentaoxa-4-aza-nonadecano-19-amido)-4-((((6S,6aS)-2,6-dimethoxy-3-((5-((((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10 ,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-12-oxo-5,6a,7,9,10,12-hexahydro-6H-spiro[benzo[e]pyrido[1,2-a][1,4]diaza-8,1'-cyclopropane]-5-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-18) Add DIEA (4 μL, 20 μmol) to a solution of 2-18 g (9 mg, 7 μmol) and Int-4 (2 mg, 7 μmol) in anhydrous THF (0.5 mL), and stir at rt for 10 min. Pass the solution through a prep-HPLC (column: SunfirePrep C18 OBD™ 5 μm, 19...). 150 mm; Mobile phase A: 0.1% FA aqueous solution, B: MeCN; Gradient: 50% - 80% - 95% B; Flow rate: 20 mL / min) Purified, lyophilized, to give compound 2-18 (5.8 mg, 56.9% yield) as a grayish-white solid. MS (ESI) m / z: 1504.1 [M+H] + .

[0396] Example 2-20 Step 1: (2S,3R,4S,5S,6S)-triacetic acid 2-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nine-19-amido)-4-((((6S,6aS)-6-hydroxy-2-methoxy-3-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro) [benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)pentyl)oxy)-14-oxo-5,6,6a,7,12,14-hexahydrobenzo[5,6][1,4]diaza-1,2-b]isoquinoline-5-carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-20a) Pd(PPh3)4 (1 mg, cat.) and dimethyl ketone (13 mg, 0.094 mmol) were added to a solution of 2-8c (80 mg, 0.048 mmol) in 2 mL of CH2Cl2 at 0 °C. The reaction solution was stirred at rt for 2 h. The organic solvent was removed by evaporation. The crude product was purified by silica gel column chromatography (MeOH / CH2Cl2 = 10 / 90) to give a white solid 2-20a (60 mg, 79% yield). MS (ESI) m / z: 1260.0 [M+H] + .

[0397] Step 2: (2S,3R,4S,5S,6S)-triacetic acid 2-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nine-19-amido)-4-((((6S,6aS)-6-hydroxy-2-methoxy-3-((5-(((S)-7-methoxy-5-oxo-10-(((4-(((2S,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2- 5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)pentyl)oxy)-14-oxo-5,6,6a,7,12,14-hexahydrobenzo[5,6][1,4]diaza-1,2-b]isoquinoline-5-carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-20b) Int-6 (60 mg, 0.04 mmol) was dissolved in anhydrous CH2Cl2 (1 mL), followed by the addition of 4 Å molecular sieve (100 mg) and then anhydrous pyridine (16.1 μL, 0.2 mmol). The mixture was cooled to -78 °C, and then triphosgene (4.3 mg, 0.014 mmol) was added dropwise to 0.5 mL of CH2Cl2. The mixture was stirred at -78 °C under a nitrogen atmosphere for 15 min. 2-20a (17.6 mg, 0.04 mmol) was added to anhydrous CH2Cl2 (1 mL). The reaction solution was stirred at 0 °C for 1 h. The organic solvent was removed by evaporation. The crude product was purified by silica gel column chromatography (MeOH / CH2Cl2 = 15 / 85) to give 2-20b (79 mg, 95% yield) as a pale yellow solid. MS (ESI) m / z: 2086.8 [M+H] + .

[0398] Step 3: (2S,3S,4S,5R,6S)-6-(2-(1-amino-3,6,9,12-tetraoxapentadecano-15-amido)-4-((((6S,6aS)-3-((5-(((S)-10-(((4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)-7-methoxy-5-oxo-5,10,11,11 α-Tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)pentyl)oxy)-6-hydroxy-2-methoxy-14-oxo-5,6,6a,7,12,14-hexahydrobenzo[5,6][1,4]diaza-1,2-b]isoquinoline-5-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-20c). Add saturated LiBr (1 mL) to CH3CN / H2O (10 / 1, v / v 2-20b (80 mg, 0.04 mmol) was added to a solution of triethylamine (0.2 mmol, 27 μL). The reaction mixture was stirred at rt under N2 atmosphere for 4 h. The solvent was removed by evaporation to give a crude product as a yellow solid, which was used directly in the next step without further purification.

[0399] Diethylamine (0.38 mmol, 40 μL) was added to 1 mL of the crude product in DMF at 0 °C. The reaction solution was stirred at rt for 1 h. The reaction solution was neutralized with AcOH and then subjected to prep-HPLC (method: column: XBridge Prep C18 OBD 5μm 19). Purification was performed using a mobile phase of 150 mL / min (A-water (0.1% formic acid):B-acetonitrile; flow rate: 20 mL / min). The purified product was lyophilized to give a white solid at 2-20°C (30 mg, 50% yield). MS (ESI) m / z: 1584.1 [M+H] + .

[0400] Step 4: (2S,3S,4S,5R,6S)-6-(2-(1-((1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nonadecano-19-amido)-4-((((6S,6aS)-3-((5-(((S)-10-(((4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl) -7-Methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazazo-2,1'-cyclopropane]-8-yl)oxy)pentyl)oxy)-6-hydroxy-2-methoxy-14-oxo-5,6,6a,7,12,14-hexahydrobenzo[5,6][1,4]diazazo[1,2-b]isoquinoline-5-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-20) Int-4 (11 mg, 0.038 mmol) and DIPEA (18.2 μL, 0.095 mmol) were added to a solution of 2-20c (30 mg, 0.019 mmol) in 1 mL of DMF at 0 °C. The reaction solution was stirred at 0 °C for 1 h. The reaction solution was neutralized with AcOH and then subjected to prep-HPLC (method: column: XBridge Prep C18 OBD 5μm 19). Purification was performed using a mobile phase of 150 mL / min (A-water (0.1% formic acid):B-acetonitrile; flow rate: 20 mL / min). The purified product was lyophilized to give a white solid, 2-20 (14.7 mg, 42.6% yield). MS (ESI) m / z: 1759.94 [M+H] + .

[0401] Example 2-22 Step 1: (2S,3S,4S,5R,6S)-6-(4-((((6S,6aS)-3-((5-(((11S,11aS)-10-(((4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-3-(22-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-aza-eicosicosamido)benzyl)oxy)carbonyl)-11 -Hydroxy-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazazo-2,1'-cyclopropane]-8-yl)oxy)pentyl)oxy)-6-hydroxy-2-methoxy-14-oxo-5,6,6a,7,12,14-hexahydrobenzo[5,6][1,4]diazazo[1,2-b]isoquinoline-5-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-22) DIEA (8.9 μL, 0.05 mmol) was added to a mixture of 6-maleimide hexanoic acid (5.3 mg, 0.025 mmol), HATU (7.1 mg, 0.019 mmol), and DMF (0.5 mL) under rt. After stirring for 10 min, 2-4f (20 mg, 0.0125 mmol) was added to the mixture. The mixture was stirred under rt for 1 h. The mixture was adjusted to pH 7 using AcOH and filtered, and then analyzed using prep-HPLC (method: column: XBridge Prep C18 OBD 5μm 19). 150 mm; Mobile phase: A-water (0.1% formic acid): B-acetonitrile; Flow rate: 20 mL / min) The filtrate was purified to give a white solid 2-22 (13.6 mg, 60.7% yield). MS (ESI) m / z: 1792.7 [M+H] + .

[0402] Example 2-23 Step 1: (2S,3S,4S,5R,6S)-6-(4-((((6S,6aS)-3-((5-(((11S,11aS)-10-(((4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-3-((R)-7-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-2,2-dimethyl-4,11-dioxo-3,10,15,18,21,24-hexaoxa-5,12-diazahepta-27-amide) )benzyl)oxy)carbonyl)-11-hydroxy-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazazo-2,1'-cyclopropane]-8-yl)oxy)pentyl)oxy)-6-hydroxy-2-methoxy-14-oxo-5,6,6a,7,12,14-hexahydrobenzo[5,6][1,4]diazazo[1,2-b]isoquinoline-5-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-23a) DIEA (16 μL, 0.089 mmol) was added to a mixture of 2-4f (47 mg, 0.030 mmol) and Int-22 (16 mg, 0.037 mmol) in DMF (1 mL). The mixture was stirred at rt for 3 h. The mixture was adjusted to pH 7 using AcOH and filtered, and then analyzed using prep-HPLC (method: column: XBridge Prep C18 OBD 5μm 19). 150 mm; Mobile phase: A-water (0.1% formic acid): B-acetonitrile; Flow rate: 20 mL / min) The filtrate was purified to give a white solid 2-23a (35 mg, 62.5% yield). MS (ESI) m / z: 1910.0 [M+H] + .

[0403] Step 2: (2S,3S,4S,5R,6S)-6-(2-((R)-22-amino-21-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-17-oxo-4,7,10,13,18-pentaoxa-16-aza-eicosicosamido)-4-((((11S,11aS)-8-((5-(((6S,6aS)-5-(((4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl) 6-hydroxy-2-methoxy-14-oxo-5,6,6a,7,12,14-hexahydrobenzo[5,6][1,4]diazazo[1,2-b]isoquinoline-3-yl)oxy)pentyl)oxy)-11-hydroxy-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazazo-2,1'-cyclopropane]-10-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-23) TFA (1 mL) was added to a mixture of 2-23a (20 mg, 0.010 mmol) in CH2Cl2 (2 mL) and H2O (200 μL) at 0 °C. The mixture was stirred at 0 °C for 2 h. The mixture was concentrated under vacuum to obtain a residue, which was analyzed by prep-HPLC (method: column: XBridge Prep C18 OBD 5 μm 19). Purification was performed using a mobile phase of 150 mL / min (A-water (0.1% formic acid):B-acetonitrile; flow rate: 20 mL / min) to give a white solid 2-23 (8.9 mg, 47.1% yield). MS (ESI) m / z: 1810.2 [M+H] + .

[0404] Example 2-24 Step 1: (2S,3S,4S,5R,6S)-6-(4-((((6S,6aS)-3-((5-(((11S,11aS)-10-(((4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-3-(1-(4-(5-(methanesulfonyl)-1,2,4-thiadiazol-3-yl)phenyl)-1-oxo-5,8,11,14-tetraoxa-2-azahepta-17-amido)benzyl)oxy)carbonyl)- 11-Hydroxy-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazazo-2,1'-cyclopropane]-8-yl)oxy)pentyl)oxy)-6-hydroxy-2-methoxy-14-oxo-5,6,6a,7,12,14-hexahydrobenzo[5,6][1,4]diazazo[1,2-b]isoquinoline-5-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-24) Compounds 2-24 (7.4 mg, 31.8% yield) were synthesized according to the synthetic procedure of step 1 in Examples 2-22. MS (ESI) m / z: 1865.5 [M+H] + .

[0405] Example 2-25 Step 1: (2S,3R,4S,5S,6S)-triacetic acid 2-(4-((((11S,11aS)-11-((2,5,8,11-tetraoxatridecane-13-yl)oxy)-8-((5-(((11S,11aS)-11-((2,5,8,11-tetraoxatridecane-13-yl)oxy)-10-(((3-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16-pentaoxa-4-aza-nineta-19-amido)-4-(((2S,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)) Tetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10-carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-25a) TMSCl (48 mg, 0.44 mmol) was added to a solution of 2-2e (90 mg, 0.044 mmol) and 2,5,8,11-tetraoxatridecane-13-ol (322 mg, 1.55 mmol) in THF (0.9 mL) on an ice bath. The reaction was then stirred at 65 °C for 5 h. The mixture was quenched with H₂O (5 mL). The aqueous layer was then treated with EtOAc (10 mL). 3) Extraction. The combined organic layers were washed with brine (20 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum to obtain the residue. This residue was purified by silica gel column chromatography (A-CH₂Cl₂; B-MeOH) to give a pale yellow oil, 2-25a (86.7 mg, 81.2%). MS (ESI) m / z: 2418.1 [M+H] + .

[0406] Step 2: (2S,3S,4S,5R,6S)-6-(4-((((11S,11aS)-11-((2,5,8,11-tetraoxatridecane-13-yl)oxy)-8-((5-(((11S,11aS)-11-((2,5,8,11-tetraoxatridecane-13-yl)oxy)-10-(((3-(1-amino-3,6,9,12-tetraoxapentadecan-15-amido)-4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2- 7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-25b) LiOH (8.6 mg, 0.36 mmol) was added to a mixed solution of 2-25a (86.7 mg, 0.036 mmol) in THF-H2O (1:1, 6 mL) at rt. The reaction was stirred at this temperature for 2 h after addition. After the reaction was complete, the mixture was quenched with AcOH (22 mg, 0.36 mmol) and concentrated under vacuum to obtain a residue, which was diluted with DMF (3 mL). Prep-HPLC was performed (method: column: XBridge Prep C18 OBD 5μm 19). The crude product was purified using a mobile phase of 150 mL / min (A-water (0.1% formic acid):B-acetonitrile; flow rate: 20 mL / min) to give a white solid 2-25b (17 mg, 25.3% yield). MS (ESI) m / z: 1916.2 [M+H] + .

[0407] Step 3: (2S,3S,4S,5R,6S)-6-(4-((((11S,11aS)-11-((2,5,8,11-tetraoxatridecane-13-yl)oxy)-8-((5-(((11S,11aS)-11-((2,5,8,11-tetraoxatridecane-13-yl)oxy)-10-(((3-(1-((1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-yl)-3-oxo-2,7,10,13,16-pentaoxa-4-aza-nonadecano-19-amido)-4-(((2S,3R,4S,5S,6S)-6-carboxylic acid) 3,4,5-Trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-25) Compound 2-25 (13 mg, 71.6%) was synthesized according to the synthetic procedure of step 6 in Example Ref-2-2. MS (ESI) m / z: 2092.4 [M+H] + .

[0408] Example 2-29 Step 1: (2S,3S,4S,5R,6S)-6-(4-((((11S,11aS)-8-((5-(((11S,11aS)-10-(((4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-3-((R)-7-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-2,2-dimethyl-4,11-dioxo-3,10,15,18,21,24-hexaoxa-5,12-diazahepta-27-amido) Benzyl)oxy)carbonyl)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-29a) Add DIEA (6 μL, 0.03 mmol) to a mixture of 2-2f (15 mg, 0.0099 mmol), Int-22 (9 mg, 0.02 mmol), and DMF (2 mL). Stir the mixture at rt for 3 h. Adjust the pH of the mixture to 7 using AcOH and filter. Perform prep-HPLC (method: column: XBridge Prep C18 OBD 5μm 19). 150 mm; Mobile phase: A-water (0.1% formic acid): B-acetonitrile; Flow rate: 20 mL / min) The filtrate was purified to give a white solid 2-29a (12 mg, 66.7% yield). MS (ESI) m / z: 1846.4 [M+H] + .

[0409] Step 2: (2S,3S,4S,5R,6S)-6-(2-((R)-22-amino-21-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-17-oxo-4,7,10,13,18-pentaoxa-16-aza-eicosicosamido)-4-((((11S,11aS)-8-((5-(((11S,11aS)-10-(((4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl) 11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-29) TFA (400 μL) was added to a mixture of 2-29a (12 mg, 0.0065 mmol) in CH2Cl2 (2 mL) and H2O (200 μL) at 0 °C. The mixture was stirred at rt for 1 h. The mixture was concentrated under vacuum to obtain a residue, which was analyzed by prep-HPLC (method: column: XBridge Prep C18 OBD 5 μm 19). Purification was performed using a mobile phase of 150 mL / min (A-water (0.1% formic acid):B-acetonitrile; flow rate: 20 mL / min) to give a white solid 2-29 (7.0 mg, 61.9% yield). MS (ESI) m / z: 1745.6 [M+H] + .

[0410] Examples 2-34 Step 1: (2S,3R,4S,5S,6S)-triacetic acid 2-(2-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nine-19-amido)-4-((((11S,11aS)-8-((5-(((11S,11aS)-7,11-dimethoxy-2-methylene-5-oxo-10-(((4-(((2S,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2- 2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7,11-dimethoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10-carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-34a) TMSCl (31 μL, 0.25 mmol) was added to a solution of 2-2e (50 mg, 0.025 mmol), MeOH (35 μL, 0.86 mmol), and THF (5 mL). The mixture was stirred at 65 °C for 4 h. The mixture was concentrated under vacuum to obtain a residue, which was purified by silica gel column chromatography (petroleum ether / EtOAc = 30 / 70) to give a yellow solid 2-34a (50 mg, 98.0% yield). MS (ESI) m / z: 2066.6 [M+H] + .

[0411] Step 2: (2S,3S,4S,5R,6S)-6-(2-(1-amino-3,6,9,12-tetraoxapentadecano-15-amido)-4-((((11S,11aS)-8-((5-(((11S,11aS)-10-(((4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)-7,11-dimethoxy-2-methylene-5-oxo 2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7,11-dimethoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-34b) LiOH (8 mg, 0.34 mmol) was added to a solution of 2-34a (50 mg, 0.0024 mmol), H₂O (2 mL), and THF (2 mL). The mixture was stirred at rt for 1 h. The mixture was adjusted to pH 7 using AcOH and concentrated under vacuum to obtain a residue, which was analyzed by prep-HPLC (method: column: XBridge Prep C18 OBD 5μm 19). Purification was performed using a mobile phase of 150 mL / min (A-water (0.1% formic acid):B-acetonitrile; flow rate: 20 mL / min) to give a white solid 2-34b (10 mg, 27.0% yield). MS (ESI) m / z: 1563.9 [M+H] + .

[0412] Step 3: (2S,3S,4S,5R,6S)-6-(2-(1-((1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-yl)-3-oxo-2,7,10,13,16-penta-4-aza-nonadecano-19-amido)-4-((((11S,11aS)-8-((5-(((11S,11aS)-10-(((4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl) 7,11-dimethoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7,11-dimethoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-34) Add DIEA (6 μL, 0.03 mmol) to a mixture of 2-34b (10 mg, 0.0064 mmol), Int-4 (4 mg, 0.013 mmol), and DMF (2 mL). Stir the mixture at rt for 1 h. Adjust the pH to 7 using AcOH, filter, and perform prep-HPLC (method: column: XBridge Prep C18 OBD 5μm 19). 150 mm; Mobile phase: A-water (0.1% formic acid): B-acetonitrile; Flow rate: 20 mL / min) The filtrate was purified to give a white solid 2-34 (6.0 mg, 54.1% yield). MS (ESI) m / z: 1740.1 [M+H] + .

[0413] Example 2-35 Step 1: (2S,3R,4S,5S,6S)-triacetic acid 2-(2-azido-4-((((2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-5-((5-(4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxy-5-( (((4-(((2S,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)amino)phenoxy)pentyl)oxy)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-35a) 2-2b (100 mg, 0.076 mmol) was dissolved in anhydrous THF (1 mL), and 100 mg of 4 Å molecular sieve was added. The mixture was cooled to 0 °C, and then triphosgene (8.11 mg, 0.027 mmol) was added, followed by triethylamine (23.43 μL, 0.167 mmol). The mixture was stirred at 0 °C under a nitrogen atmosphere for 15 min. Int-25 (38.3 mg, 0.08 mmol), triethylamine (16 μL, 0.114 mmol), and dibutyltin dilaurate (9 μL, 0.015 mmol) were mixed in anhydrous THF (1 mL), and this solution was then gradually added to the above reaction solution at 0 °C. The reaction solution was heated to rt and stirred for 2 h. The reaction solution was concentrated and then purified by column chromatography (petroleum ether / EtOAc = 40 / 60) to give a white solid 2-35a (135 mg, 97% yield). MS (ESI) m / z: 1827.4 [M+H] + .

[0414] Step 2: (2S,3R,4S,5S,6S)-triacetic acid 2-(2-azido-4-((((2-((S)-2-(hydroxymethyl)-4-methylenepyrrolidine-1-carbonyl)-5-((5-(4-(((S)-2-(hydroxymethyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxy-5-((((4-(((2S,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)amino)phenoxy)pentyl)oxy)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-35b) Compound 2-35a (135 mg, 0.074 mmol) was dissolved in THF / H2O (1 mL / 20 μL, 50 / 1), and TsOH was added. . H2O (28 mg, 0.148 mmol) was added, and the mixture was stirred at rt for 12 h. The solvent was then removed by evaporation, and the residue was purified by column chromatography (MeOH / CH2Cl2 = 6 / 94) to give a white solid 2-35b (83 mg, 70.26% yield). MS (ESI) m / z: 1599.3 [M+H] + .

[0415] Step 3: (2S,3R,4S,5S,6S)-triacetic acid 2-(2-azido-4-((((11S,11aS)-11-hydroxy-8-((5-(((11S,11aS)-11-hydroxy-7-methoxy-2-methylene-5-oxo-10-(((4-(((2S,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)-2, 3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10-carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-35c) Des Martin periodane (51 mg, 0.12 mmol) was added to a solution of 2-35b (80 mg, 0.05 mmol) in CH2Cl2 (1 mL) under rt, and the mixture was stirred under nitrogen atmosphere for 12 h under rt. The reaction solution was then diluted with CH2Cl2 (10 mL) and washed with saturated Na2S2O3 (1 mL) and brine (5 mL). The organic phase was collected, dried over anhydrous Na2SO4, filtered, and concentrated under vacuum to obtain a residue, which was purified by silica gel column chromatography (MeOH / CH2Cl2 = 6 / 94) to give 2-35c (65 mg, 81% yield) as a white solid. MS (ESI) m / z: 1595.2 [M+H] + .

[0416] Step 4: (2S,3R,4S,5S,6S)-triacetic acid 2-(2-(4-(17-amino-3-oxo-6,9,12,15-tetraoxa-2-azaheptadecyl)-1H-1,2,3-triazol-1-yl)-4-((((11S,11aS)-11-hydroxy-8-((5-(((11S,11aS)-11-hydroxy-7-methoxy-2-methylene-5-oxo-10-(((4-(((2S,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro) -2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10-carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-35d) DIPEA (15 μL, 0.082 mmol) was added to a solution of 2-35c (65 mg, 0.041 mmol) and Int-26 (13.6 mg, 0.045 mmol) in 1 mL DMF at 0 °C, followed by the addition of a catalytic amount of CuI. The mixture was degassed with N2 for 10 min and then stirred under a nitrogen atmosphere for 1 h. The solvent was then removed by evaporation, and the residue was purified by column chromatography (MeOH / CH2Cl2 = 10 / 90) to give 2-35d (70 mg, 90.5% yield) as a white solid. MS (ESI) m / z: 1897.1 [M+H] + .

[0417] Step 5: (2S,3S,4S,5R,6S)-6-(2-(4-(17-amino-3-oxo-6,9,12,15-tetraoxa-2-azaheptadecyl)-1H-1,2,3-triazol-1-yl)-4-((((11S,11aS)-8-((5-(((11S,11aS)-10-(((4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)-11-hydroxy-7- Methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-35e) Add saturated LiBr (1 mL) to CH3CN / H2O (10 / 1, v / v Add 2–35 mg (30 mg, 0.016 mmol) to a solution of triethylamine (0.32 mmol, 45 μL). Stir the reaction mixture under nitrogen atmosphere for 1 h at rt. Remove the solvent by evaporation to obtain a crude product as a yellow solid, which is then analyzed by prep-HPLC (method: column: XBridge PrepC18 OBD 5 μm 19). Purification was performed using a mobile phase of 150 mL / min (A-water (0.1% formic acid):B-acetonitrile; flow rate: 20 mL / min). The purified product was lyophilized to give a white solid at 2-35°C (9 mg, 35% yield). MS (ESI) m / z: 1617.4 [M+H] + .

[0418] Step 6: (2S,3S,4S,5R,6S)-6-(2-(4-(1-((1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-yl)-3,19-dioxo-2,7,10,13,16-pentaoxa-4,20-diaza-eicosano-21-yl)-1H-1,2,3-triazol-1-yl)-4-((((11S,11aS)-8-((5-(((11S,11aS)-10-(((4-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2- 11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2-35) Int-4 (3.3 mg, 0.011 mmol) and DIPEA (5.4 μL, 0.028 mmol) were added to a solution of 2-35e (9 mg, 0.003 mmol) in 0.5 mL of DMF at 0 °C. The reaction solution was stirred at 0 °C for 1 h. The reaction solution was neutralized with AcOH and then subjected to prep-HPLC (method: column: XBridge Prep C18 OBD 5μm 19). Purification was performed using a mobile phase of 150 mL / min (A-water (0.1% formic acid):B-acetonitrile; flow rate: 20 mL / min). The purified product was lyophilized to give a white solid at 2-35 μL (5.3 mg, 53% yield). MS (ESI) m / z: 1792.9 [M+H] + .

[0419] The aforementioned conjugate-connector-payload compounds are summarized in Table 1 below.

[0420] Table 1: Fittings - Joints - Effective Load

[0421] Example 2: Preparation and Characterization of Antibody-Drug Conjugates Preparation of DAR2 antibody-drug conjugates. Anti-CD74 antibody STRO-001 or allotype antibody CB6 (or "mAb1") was incubated with 1 / 2000-1 / 500 w / w (EndoS2 / mAb weight ratio) endoS2 in reaction buffer (0.5-25 mg / mL, 50 mM Tris-HCl buffer, pH 7.0-8.5) at a reaction temperature (0-40°C) for 1-24 h. 2-40 eq. UDP-GalNAz (20 mM) and 0.1 w / w%-10 w / w% (GalT / mAb weight ratio) β1,4-GalT were added to the reaction mixture and incubated in reaction buffer (50 mM Tris-HCl buffer, pH 7.0-8.5, 20 mM MnCl2) at a reaction temperature (0-40°C) for 8-24 h. The reaction mixture was purified with protein A resin to obtain mAb1-GalNAz.

[0422] At 0–25 °C, for 0.5–24 h, organic solvents (e.g., DMSO, DMF, DMA, PG, acetonitrile, 0–25% v / v) and adapter-loador stock solution (10–25 eq., 10 mM in organic solvent) are added progressively to a reaction buffer (PBS buffer, pH 7.0–8.5) containing mAb1-GalNAz (1–20 mg / mL). The buffer solution is then replaced (by centrifugation, desalting column, ultrafiltration, and dialysis) with a storage buffer (e.g., pH 5.5–6.5 histidine acetate buffer with optional additives such as sucrose, trehalose, Tween 20, 60, or 80).

[0423] The synthesized ADCs are shown in Table 2 below. The Ab for all ADCs is STRO-001, except for the same type of ADC, where the Ab is CB6.

[0424] ADC characterization. pass The following analytical methods were used to characterize the ADCs. All ADCs had a SEC purity >95%.

[0425] Drug:Antibody Ratio (DAR) Measurement by LCMS or HIC Methods LCMS method: LC-MS analysis was performed under the following measurement conditions: LC-MS system: Vanquish Flex UHPLC and Orbitrap Exploris 240 mass spectrometer Column: MAbPac™ RP, 2.1 50 mm, 4 μm, 1,500 Å, Thermo Scientific™ Column temperature: 80℃ Mobile phase A: 0.1% formic acid (FA) aqueous solution Mobile phase B: Acetonitrile solution containing 0.1% formic acid (FA) Gradient program: 25 %B-25 %B (0 min-2 min), 25 %B-50 %B (2 min-18 min), 50 %B-90 %B (18 min-18.1 min), 90 %B-90 %B (18.1 min-20 min), 90 %B-25 %B (20 min-20.1 min), 25 %B-25 %B (20.1 min-25 min) Injected sample volume: 1 μg MS parameters: Full and denatured MS data were acquired in HMR mode with R=15k, and deconvolution was performed using the ReSpect™ algorithm and sliding window integration in ThermoScientific™ BioPharma Finder™ 4.0 software.

[0426] HIC method: HPLC analysis was performed under the following measurement conditions: HPLC System: Waters ACQUITY ARC HPLC System Detector: Measurement wavelength: 280 nm Column: Tosoh Bioscience 4.6 μm ID×3.5 cm, 2.5 μm butyl nonporous resin column Column temperature: 25℃ Mobile phase A: 1.5 M ammonium sulfate, 50 mM phosphate buffer, pH 7.0 Mobile phase B: 50 mM phosphate buffer, 25% (v / v) isopropanol, pH 7.0 Gradient program: 0%B-0%B (0 min-2 min), 0%B-100%B (2 min-15 min), 100%B-100%B (15 min-16 min), 100%B-0%B (16 min-17 min), 0%B-0%B (17 min-20 min) Injected sample volume: 20 μg SEC method for determining ADC purity HPLC analysis was performed under the following measurement conditions: HPLC system: Waters H-Class UPLC system Detector: Measurement wavelength: 280 nm Column: ACQUITY UPLC BEH200 SEC 1.7 um 4.6x150 mm, Waters Column temperature: room temperature Mobile phase A: 200 mM phosphate buffer, 250 mM potassium chloride, 15% isopropanol, pH 7.0 Gradient program: 10 min isocratic elution at a flow rate of 0.3 mL / min Injected sample volume: 20 μg Hydrophobicity assessment of ADC using the HIC method: ADCs with higher hydrophobic properties will appear as the retention time of HIC (hydrophobic interaction column) chromatography increases. The DAR2 peak is used as a reference.

[0427] HPLC analysis was performed under the following measurement conditions: Method 1 HPLC System: Waters ACQUITY ARC HPLC System Detector: Measurement wavelength: 280 nm Column: Tosoh Bioscience 4.6 μm ID×3.5 cm, 2.5 μm butyl nonporous resin column Column temperature: 25℃ Mobile phase A: 1.5 M ammonium sulfate, 50 mM phosphate buffer, pH 7.0 Mobile phase B: 50 mM phosphate buffer, 25% (v / v) isopropanol, pH 7.0 Gradient program: 0%B-0%B (0 min-2 min), 0%B-100%B (2 min-15 min), 100%B-100%B (15 min-16 min), 100%B-0%B (16 min-17 min), 0%B-0%B (17 min-20 min) Injected sample volume: 20 μg Method 2 HPLC System: Waters ACQUITY ARC HPLC System Detector: Measurement wavelength: 280 nm Column: MABPac HIC-10, 5 μm, 4.6×10 mm (Thermo) Column temperature: 25℃ Mobile phase A: 1.5 M ammonium sulfate, 50 mM sodium phosphate, pH 7.0 Mobile phase B: 50 mM sodium phosphate, pH 7.0 Gradient program: 20%B-20%B (0 min-1 min), 0%B-0%B (1 min-35 min), 20%B-20%B (35 min-40 min) Flow rate: 0.5 mL / min Sample preparation: Dilute the sample to 0.5 mg / mL with the initial mobile phase.

[0428] Table 2: ADC Structure

[0429] Anti-CD74 antibody STRO-001 Light chain sequence (SEQ ID NO: 1) DIQMTQSPSSVSASVGDRVTITCRASQGIGSWLAWYQQKPGKAPKLLIYAADRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYHTYPLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Heavy chain sequence (SEQ ID NO: 2) QVQLVESGGGVVQPGRSLRLSCAASGFNFSDYGMHWVRQAPGKGLEWVAVIWYDGSISYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGTVEHGAVYGTDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Anti-SARS-CoV-2 antibody CB6 (isotype antibody) Light chain sequence (SEQ ID NO: 3) DIVMTQSPSSLSASVGDRVTITCRASQSISRYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPEYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Heavy chain sequence (SEQ ID NO: 4) EVQLVESGGGLVQPGGSLRLSCAASGFTVSSNYMSWVRQAPGKGLEWVSVIYSGGSTFYADSVKGRFTISRDNSMNTLFLQMNSLRAEDTAVYYCARVLPMYGDYLDYWGQG TLVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPPAAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Example 3: ADC directly kills NOMO-1 and K562 cancer cell lines cell lines NOMO-1 (JCRB, IFO50474). NOMO-1 (purchased from JCRB) is a cell line exhibiting hematopoietic lymphocyte morphology. The basal medium was RPMI-1640 medium prepared by ATCC, catalog number ATCC 30-2001. To prepare complete growth medium, fetal bovine serum (Gibco, 10099-141C) was added to a final concentration of 10%. The cell line was grown at 37°C in a humid 5% CO2 atmosphere, and the presence of mycoplasma was periodically tested using the MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza, LT07-710). NOMO-1 is a CD74-positive cell line (Table 3).

[0430] K562 (ATCC, CCL-243). K562 (purchased from ATCC) is a cell line exhibiting lymphoblastic morphology. The basal medium for K562 was Iscove's Modified Dulbecco's Medium (catalog number 30-2005) prepared by ATCC. To prepare complete growth medium, fetal bovine serum (Gibco, 10099-141C) was added to a final concentration of 10%. The cell line was grown at 37°C in a humid 5% CO2 atmosphere, and the presence of mycoplasma was periodically tested using the MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza, LT07-710). K562 is a CD74-negative cell line (Table 3).

[0431] Table 3: CD74 expression levels

[0432] ADC direct kill. In NOMO-1 (CD74) + ) and K562 (CD74) - The direct killing effect of ADCs was evaluated in cancer cell lines. Cells were seeded (6E3 / well for NOMO-1, 3E3 / well for K562) into 96-well plates (Greiner: 655090), 100 µl per well (containing 100 µg / ml Fc inhibitor for NOMO-1 cells) and incubated at 37°C, 5% CO2 for 2 h. Fresh growth medium containing different concentrations of ADC was added, 50 μL per well, and incubated at 37°C, 5% CO2 for 6 days. Cell viability was assessed using CellTiter-Glo (Promega, G7573), 70 μL per well. The plates were incubated at room temperature for 10 min to stabilize the luminescence signal. The plates were analyzed using a microplate reader.

[0433] Data on direct cell killing by ADCs are shown in Tables 4 to 9. Figures 1A to 6B .

[0434] Table 4: Direct cell-killing effects of ADCs

[0435] Table 5: Direct Cell Killing by ADCs

[0436] Table 6: Direct Cell Killing by ADCs

[0437] Table 7: Direct Cell Killing by ADCs

[0438] Table 8: Direct Cell Killing by ADCs

[0439] Table 9: Direct Cell Killing by ADCs

[0440] Although the foregoing disclosure has been described in considerable detail by way of illustration and example for purposes of clarity, it will be apparent to those skilled in the art that minor changes and modifications can be made. Therefore, the descriptions and embodiments described should not be considered limiting.

[0441] It should be understood that if any publication is mentioned in this document, such mention does not constitute an acknowledgment that the publication constitutes common knowledge in its field in any country.

[0442] All non-patent publications, patents, patent applications, and published patent applications mentioned herein are incorporated herein by reference in their entirety.

Claims

1. A compound of formula (I): (I)， Or its pharmaceutically acceptable salts, tautomers, solvates, stereoisomers, or isotopes, wherein: Each of rings A and B is independently one of equations (IIa), (IIb), (IIc), (IId), (IIe), (IIIf), or (IIg): 、 、 、 , , or ;in: Each ring C is independently a cyclopropyl ring or a cyclobutyl ring; each R is independently H, OH, C 1 independently H, OH, C 1-4 alkyl, C 1-4 alkoxy or -0-(CH2CH20) t1 -CH3; each R is independently H or C1-6alkyl; 2 independently H or C1-6alkyl; 1-4 C1-6alkyl; each R 3 and R 4 independently H, NR a1 R b1 , OH, C 1-4 alkyl, C 1-4 alkoxy or aryl; R a1 and each of R b1 is independently H or C 1-4 alkyl; Each R 5 Independently for H and C 1-4 Alkyl, C 1-4 alkoxy or aryl; Each m1, n1, and o1 is independently 1 or 2; t1 can be 1, 2, 3, 4, 5, 6, 7 or 8; The bonds marked by ring A being linked to a splittable 1 when s1 is 1 or to -H when s1 is 0, or the bonds marked by ring B being linked to a splittable 2; and The key is marked when ring A or ring B is connected to the connector; The connector is –(CH2). r -、-(CH2) p -X-(CH2) q -or-(CH2) p -CH=CH-(CH2) q -;in: Each r, p, and q is independently 1, 2, 3, 4, 5, 6, 7, or 8; The sum of p and q is 2, 3, 4, 5, 6, 7 or 8; X is NR 6 , NHC(=O), C(=O)NH, O, SO2, substituted or unsubstituted aryl rings, substituted or unsubstituted heteroaryl rings, substituted or unsubstituted heterocycles, or substituted or unsubstituted rings; and R 6 is H or C 1-4 alkyl; Decomposable 1, when present, has formula (VIa) or (VIc): or ,in: Su refers to the sugar portion; Each R 11 Independently hydrogen, halogen, substituted or unsubstituted C 1-4 Alkyl, -CN, or -NO2; and # Marks the bonds that can be broken down when linked to ring A; s1 is 0 or 1; The pyrolytic 2 has the formula (VIIa1), (VIIc1), (VIId1), or (VIIf1): , , or ;in: Su refers to the sugar portion; each R is independently hydrogen, halogen, substituted or unsubstituted C 9 is independently hydrogen, halogen, substituted or unsubstituted C 1-4 alkyl, -CN, or -NO2; # The bond in the case where 2 links to ring B can be broken; and ## Marks bonds that can be split into 2-linked spacers; Spacers are bonds, ### -NH-(CH2CH2O) m2 -CH2CH2-C(═O)-、 ### -NH-(CH2CH2O) m2 -CH2-C(═O)-、 ### -(CH2) m2 -C(═O)-、 ### -CH2-C(═O)-NH-(CH2) m2 -C(═O)-、 ### -(CH2CH2O) m2 -CH2CH2-C(═O)-、 ### -CH[-(CH2) m2 -COOH]-C(═O)-、 ### -CH2-C(═O)-NH-(CH2) m2 -C(═O)-NH-(CH2) m2 -C(═O)-、 ### -C(═O)-(CH2) m2 -C(═O)- or ### -NH-(CH2) m2 -C(═O)-; where: Each m² is independently 1, 2, 3, 4, 6, 7, or 8; and ### the bond in the case where the label spacer is linked to the conjugate; and The conjoint has formula (III), (IV) or (V): , or ,in: U2 is a bond, a heteroaryl or a aryl group; V2is a bond or -C=C-(CH2) n2 -; n2 is an integer from 0 to 10, inclusive. W2 is -C(=O)-, -NH-, or -O-; RG3 is or ; RS3 is -NR a2 R b2 ; R a2 and R b2 Each of them is independently H or substituted or unsubstituted C. 1-4 alkyl; RE3 is a key, -O-, -OC(=O-), -OC(=O)NR 7 -、-NHC(=O)NR 7 -、-OS(=O)2NR 7 -、-NHS(=O)2NR 7 -or-OC(=O)NHS(=O)2NR 7 ; R 7 H or substituted or unsubstituted C 1-4 alkyl; W3 is -C(=O)-, -NH-, or -O-; t3 is 1 or 2; s3 is 0, 1, or 2; RG4 is , , , or ; RE4 is a key, -O-, -OC(=O)-, -OC(=O)NR 8 -、-NHC(=O)NR 8 -、-OS(=O)2NR 8 -、-NHS(=O)2NR 8 -or-OC(=O)NHS(=O)2NR 8 -; R 8 H or substituted or unsubstituted C 1-4 alkyl; W4 is -C(=O)-, -NH-, or -O-; t4 is 1, 2, 3, 4, 5, 6, 7, or 8; and s4 can be 0, 1, or 2.

2. The compound of claim 1, wherein... The connector is -(CH2). r Or -(CH2) p -X-(CH2) q -; Ring A has formulas (IIa), (IIb), (IIc), (IId), or (IIg); and Ring B has formulas (IIa), (IIb), (IId), or (IIg).

3. The compound of claim 1 or claim 2, wherein... the linker is -(CH2) r - ring A has the formula (IIg), and ring B has the formula (IIg); the linker is -(CH2) r - ring A has the formula (HIa), and ring B has the formula (HIa); the linker is -(CH2) r - ring A has formula (IIb), and ring B has formula (IIa); the linker is -(CH2) r - ring A has the formula (IIa), and ring B has the formula (IIb); the linker is -(CH2) r - ring A has the formula (IIa), and ring B has the formula (IIa); the linker is -(CH2) r - ring A has the formula (lie), and ring B has the formula (Ha); or The connector is -(CH2). p -X-(CH2) q - Ring A has equation (IIa), and ring B has equation (IIa).

4. The compound of any one of claims 1-3, wherein the linker is -(CH2) r and r is 5.

5. The compound of any one of claims 1-3, wherein the linker is -(CH2) p - X-(CH2) q -, X is an unsubstituted aryl ring, p is 1, and q is 1.

6. The compound of claim 5, wherein X is a phenylene oxide.

7. The compound according to any one of claims 1-6, wherein Equation (IIa) is (IIa1); Equation (IId) is (IId1); Formula (IIg) is (IIg1); and / or Equation (IIc) is (IIc1) (IIc2) or (IIc3).

8. The compound according to any one of claims 1-7, wherein ring A is , , , , , , , , , or .

9. The compound according to any one of claims 1-8, wherein ring B is , , , , , , , or .

10. The compound according to any one of claims 1-9, wherein Each Su is independent for , , or , or its stereoisomers; Each m is independently 0 or 1; and Indicates the connection point with either splittable 1 or splittable 2.

11. The compound of claim 10, wherein each Su is independently... or , or its stereoisomers.

12. The compound of claim 11, wherein each Su is .

13. The compound according to any one of claims 1-12, wherein s1 is 1.

14. The compound of claim 13, wherein the cleavable 1 has formula (VIa).

15. The compound of claim 14, wherein it is cleavable into 1... or .

16. The compound according to any one of claims 1-12, wherein s1 is 0.

17. The compound according to any one of claims 1-16, wherein the cleavable 2 has the formula (VIIa1) or (VIId1): or .

18. The compound of claim 17, wherein it is cleavable into 2... or .

19. The compound of any one of claims 1-18, wherein the spacer is ### -NH-(CH2CH2O) m2 -CH2CH2-C(=0)- or ### -NH-(CH2CH2O) m2 -CH2-C(=0)-.

20. The compound of any one of claims 1-19, wherein the spacer is ### -NH-(CH2CH20)4-CH2CH2-C(=0)-.

21. The compound according to any one of claims 1-20, wherein the conjugate has formula (III).

22. The compound of claim 21, wherein U2 is an arylene.

23. The compound of claim 22, wherein U2 is .

24. The compound according to any one of claims 21-23, wherein V2 is a bond.

25. The compound according to any one of claims 21-24, wherein W2 is -C(=O)-.

26. The compound according to any one of claims 21-25, wherein the conjugate is .

27. The compound according to any one of claims 1-20, wherein the conjugate has the formula (V).

28. The compound of claim 27, wherein RG4 is or .

29. The compound of claim 27 or 28, wherein t4 is 1, 2, 3, 4 or 5.

30. The compound according to any one of claims 27-29, wherein s4 is 0.

31. The compound according to any one of claims 27-30, wherein the sum of s4 and t4 is 1 or 5.

32. The compound according to any one of claims 27-31, wherein RE4 is a bond or -O-.

33. The compound according to any one of claims 27-32, wherein W4 is -C(=O)-.

34. The compound according to any one of claims 27-33, wherein the conjugate is or .

35. The compound according to any one of claims 1-20, wherein the conjugate has formula (IV).

36. The compound of claim 35, wherein RG3 is .

37. The compound of claim 35 or 36, wherein RS3 is NH2.

38. The compound according to any one of claims 35-37, wherein t3 is 2.

39. The compound according to any one of claims 35-38, wherein RE3 is -O-.

40. The compound according to any one of claims 35-39, wherein s3 is 0.

41. The compound according to any one of claims 35-40, wherein W3 is -C(=O)-.

42. The compound according to any one of claims 35-41, wherein the conjugate is .

43. The compound of claim 1, wherein the compound is , , , , , , , , , , , , , , , , , or , Or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, or isotope thereof.

44. A compound of formula (IA): (IA), Or its pharmaceutically acceptable salts, tautomers, solvates, stereoisomers, or isotopes, wherein: Each of rings A and B is independently one of equations (IIa), (IIb), (IIc), (IId), (IIe), (IIIf), or (IIg): 、 、 、 , , or ;in: Each ring C is independently a cyclopropyl ring or a cyclobutyl ring; Each R 1 Independently H, OH, C 1-4 Alkyl, C 1-4 Alkyl group or -O-(CH2CH2O) t1 -CH3; Each R 2 Independently H or C 1-4 alkyl; Each R 3 and R 4 Independently for H, NR a1 R b1 OH, C 1-4 Alkyl, C 1-4 alkoxy or aryl; R a1 and each of R b1 is independently H or C 1-4 alkyl; Each R 5 Independently for H and C 1-4 Alkyl, C 1-4 alkoxy or aryl; Each m1, n1, and o1 is independently 1 or 2; t1 can be 1, 2, 3, 4, 5, 6, 7 or 8; The bonds marked by ring A being linked to a splittable 1 when s1 is 1 or to -H when s1 is 0, or the bonds marked by ring B being linked to a splittable 2; and The key is marked when ring A or ring B is connected to the connector; -(CH2) r -CH2- p -(CH2) q -CH2- p -CH2- q -; wherein: Each r, p, and q is independently 1, 2, 3, 4, 5, 6, 7, or 8; The sum of p and q is 1, 2, 3, 4, 5, 6, 7 or 8; X is NR 6 , NHC(=0), C(=0)NH, O, S02, a substituted or unsubstituted aryl ring, a substituted or unsubstituted heteroaryl ring, a substituted or unsubstituted heterocycle, or a substituted or unsubstituted ring; and R 6 For H or C 1-4 alkyl; Decomposable 1 has the following formula when it exists: ,in: Su refers to the sugar portion; Each R 11 Independently hydrogen, halogen, substituted or unsubstituted C 1-4 Alkyl, -CN, or -NO2; and # Marks the bonds that can be broken down when linked to ring A; s1 is 0 or 1; The pyrolytic 2 has formula (VIIa1) or (VIId1): or ,in: Su refers to the sugar portion; Each R 9 Independently hydrogen or C 1-4 alkyl; # The bond in the case where 2 links to ring B can be broken; and ## Marks bonds that can be split into 2-linked spacers; Spacers are bonds, ### -NH-(CH2CH2O) m2 -CH2CH2-C(═O-) or ### -NH-(CH2CH2O) m2 -CH2-C(═O)-; where: Each m² is independently 1, 2, 3, 4, 6, 7, or 8; and ### Mark the key when the spacer is linked to -C (=O)-; RE4 is a key or -O-; and t4 can be 1, 2, 3, 4, 5, 6, 7 or 8.

45. The compound of claim 44, wherein... The connector is -(CH2). r Or -(CH2) p -X-(CH2) q -; Ring A has formulas (IIa), (IIb), (IIc), (IId), or (IIg); and Ring B has formulas (IIa), (IIb), (IId), or (IIg).

46. ​​The compound of claim 44 or claim 45, wherein... The connector is -(CH2). r - Ring A has formula (IIg), and ring B has formula (IIg); The connector is -(CH2). r - Ring A has equation (IId), and ring B has equation (IId); The connector is -(CH2). r - Ring A has equation (IIb), and ring B has equation (IIa); The connector is -(CH2). r - Ring A has equation (IIa), and ring B has equation (IIb); The connector is -(CH2). r - Ring A has equation (IIa), and ring B has equation (IIa); The connector is -(CH2). r - Ring A has equation (IIc), and ring B has equation (IIa); or The connector is -(CH2). p -X-(CH2) q - Ring A has equation (IIa), and ring B has equation (IIa).

47. The compound according to any one of claims 44-46, wherein the linker is -(CH2). r - And r is 5.

48. The compound according to any one of claims 44-46, wherein the linker is -(CH2). p -X-(CH2) q - X is an unsubstituted aryl ring, p is 1, and q is 1.

49. The compound of claim 48, wherein X is a phenylene oxide.

50. The compound according to any one of claims 44-49, wherein Equation (IIa) is (IIa1); Equation (IId) is (IId1); Formula (IIg) is (IIg1); and / or Equation (IIc) is (IIc1) (IIc2) or (IIc3).

51. The compound according to any one of claims 44-50, wherein ring A is , , , , , , , , , or .

52. The compound according to any one of claims 44-51, wherein ring B is , , , , , , , or .

53. The compound according to any one of claims 44-52, wherein Each Su is independent for , , or , or its stereoisomers; Each m is independently 0 or 1; and Indicates the connection point with either splittable 1 or splittable 2.

54. The compound of claim 53, wherein each Su is independently... or , or its stereoisomers.

55. The compound of claim 54, wherein each Su is .

56. The compound according to any one of claims 44-55, wherein s1 is 1.

57. The compound of claim 56, wherein each R 11 It can be hydrogen or -NO2 independently.

58. The compound of claim 57, wherein the cleavage capability is 1... or .

59. The compound according to any one of claims 44-55, wherein s1 is 0.

60. The compound according to any one of claims 44-59, wherein it is cleavable into 2... or .

61. The compound according to any one of claims 44-60, wherein the spacer is ### -NH-(CH2CH2O) m2 -CH2CH2-C(═O-) or ### -NH-(CH2CH2O) m2 -CH2-C(═O)-.

62. The compound according to any one of claims 44-61, wherein the spacer is ### -NH-(CH2CH2O)4-CH2CH2-C(═O)-.

63. The compound according to any one of claims 44-62, wherein... for .

64. The compound of claim 44, wherein the compound is 、 、 、 ； 、 、 、 、 、 、 、 、 、 or ， Or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, or isotope thereof.

65. A ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein the conjugate comprises a drug via a conjugate or A ligand conjugated to the compound of any one of claims 1-64, wherein the conjugate has the following formula: Ligand - [Formula (I or Ia)] x , Where x is approximately 1 to approximately 10.

66. The conjugate of claim 65, wherein the ligand is a humanized, monoclonal, chimeric, or human antibody or an antigen-binding fragment thereof.

67. The conjugate as claimed in claim 65 or 66, wherein the conjugate has the following formula: Among them, Ab is a humanized, monoclonal, chimeric, or human antibody or its antigen-binding fragment.

68. The conjugate of claim 66 or 67, wherein the ligand or Ab binds to one or more receptors selected from: B7-H3, cytokeratin 15, PTK7, HER3, HER2, CD7, CD19, CD20, CD22, CD25, CD27, CD30, CD33, CD37, CD38, CD46, CD70, CD71, CD74, CD79b, CD123, CD138, CD142, CD166, CD205, CD228, CCR2, CA6, p-cadherin, CEA, CEACAM5, C4.4a, DLL3, EGFR, EGFRVIII, ENPP3, EphA2, EphrinA, FLOR1, FGFR2, GCC, cKIT, LIV1, LY6E, MSLN, MUC16, NaPi2b, Nectin4, gpNMB, PSMA, SLITRK6, STEAP1, TROP2, 5T4, SSEA4, GloboH, Gb5, STn, Tn, B7H3, BCMA, MUC1, cMet, ROR1 MSLN, FRa, CLDN18.2, CLDN6, PTK7, Axl, FGFR2b, CLL1, CCR7, GPC1, GPC3, ISAC, CDCP1, ITGB6, ADAM9, or CD45.

69. The conjugate or pharmaceutically acceptable salt or solvate thereof as described in any one of claims 65-68, wherein the conjugate is selected from: 。 70. The conjugate of claim 69, wherein x is about 1.8 to about 2.

5.

71. The conjugate as claimed in claim 69 or 70, wherein Ab is STRO-001.

72. A pharmaceutical composition comprising the conjugate or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer or isotope of any one of claims 65-71 and a pharmaceutically acceptable excipient.

73. A method for treating a subject with a proliferative disease, metabolic disease, inflammatory disease, or neurodegenerative disease, the method comprising administering to the subject an effective amount of any one of claims 65-71, or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, or isotope thereof, or the pharmaceutical composition of claim 72.

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