Antibody drug conjugate platform

Abstract

The present disclosure provides antibody drug conjugate platforms, and antibody drug conjugates comprising an antibody or antigen binding fragment thereof and a linker-payload derived from the platforms, and pharmaceutical compositions comprising the antibody drug conjugates.

Description

Cross-references to related applications

[0001] This application claims priority to International Application No. PCT / CN2023 / 138423, filed on December 13, 2023, the disclosure of which is hereby incorporated by reference in its entirety.

[0002] sequence list This application contains a sequence list, which has been submitted electronically in XML format. The XML file is named “01368-0059-00PCT.xml”, created on November 4, 2024, and is 3,422 bytes in size. The sequence list is incorporated herein by reference in its entirety. Technical Field

[0003] This document discloses an antibody-drug conjugate platform and an antibody-drug conjugate (ADC) comprising the platform and an antibody or an antigen-binding fragment thereof, as well as the uses of the ADC platform and the ADC. Background Technology

[0004] Chemotherapy, along with surgery, radiation therapy, and targeted therapy, remains one of the most important anti-cancer treatments. Despite the existence of various types of highly effective cytotoxic agents, the minimal difference between tumor cells and normal cells limits the widespread clinical application of these anti-cancer compounds due to their toxic side effects. Antibody drugs have become frontline drugs in anti-cancer therapy due to their specificity against tumor cell surface antigens. However, when antibodies are used alone as anti-cancer drugs, their efficacy is often unsatisfactory.

[0005] Antibody-drug conjugates (ADCs) combine antibodies or antibody fragments with bioactive cytotoxins via chemically stable linkers. This allows them to fully utilize the specificity of antibodies in binding to antigens on the surface of normal or tumor cells and the high efficiency of cytotoxins, while avoiding the low efficacy of antibodies and the toxic side effects of cytotoxins. This means that, compared to conventional chemotherapy drugs, ADCs can specifically bind to tumor cells and reduce their effect on normal cells.

[0006] Therefore, the industry needs ADCs that are effective in treating cancer. Summary of the Invention

[0007] This article provides an antibody-drug conjugate platform and antibody-drug conjugates (ADCs). It also provides uses of the said ADC platform for preparing ADCs, such as for therapeutic applications.

[0008] In some implementations, compounds of formula (I) are provided herein: , Or a pharmaceutically acceptable salt, tautomer, solvate, or stereoisomer thereof, wherein the values ​​of the variables (e.g., Z, Y, X, W, V, U, T, P, s, v', w', x', y') are as set forth herein.

[0009] In other embodiments, this document provides antibody-drug conjugates of formula (II): , Or a pharmaceutically acceptable salt, tautomer, solvate, or stereoisomer thereof, wherein the values ​​of the variables (e.g., BA, Z', Y, X, W, V, U, T, P, n, s, v', w', x', y') are as described herein.

[0010] In other embodiments, this document provides pharmaceutical compositions comprising the ADCs described herein, such as compounds of formula (II) or pharmaceutically acceptable salts, tautomers, solvates or stereoisomers thereof, and pharmaceutically acceptable excipients.

[0011] Other objectives and advantages will be set forth in part in the description which follows, and in part will be understood from the description or may be learned by practice. These objectives and advantages will be realized and obtained by means of the elements and combinations particularly pointed out in the appended claims.

[0012] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only, and do not limit the claims.

[0013] The accompanying drawings, which are incorporated in and form part of this specification, and together with the description, serve to explain the principles set forth herein. Attached Figure Description

[0014] Figure 1 This demonstrates the killing effect on payload cells in the A375 cell line.

[0015] Figure 2 This demonstrates effective payload cell killing in the Calu-6 cell line.

[0016] Figure 3 This demonstrates the killing effect on payload cells in the A375 cell line.

[0017] Figure 4 This demonstrates effective payload cell killing in the Calu-6 cell line.

[0018] Figure 5 The study demonstrated the killing effect of ADC cells on the NCI-H358 cell line.

[0019] Figure 6 The study showed that the ADC cells in the CAPAN-1 cell line were killed.

[0020] Figure 7 The study demonstrated the killing effect of ADC cells on the MDA-MB-453 cell line.

[0021] Figure 8 The study demonstrated the killing effect of ADC cells on the NCI-H358 cell line.

[0022] Figure 9 The study showed that the ADC cells in the CAPAN-1 cell line were killed.

[0023] Figure 10 The study demonstrated the killing effect of ADC cells on the MDA-MB-453 cell line.

[0024] Figure 11 The study demonstrated the killing effect of ADC cells on the NCI-358 cell line.

[0025] Figure 12 The study showed that the ADC cells in the CAPAN-1 cell line were killed.

[0026] Figure 13 The study demonstrated the killing effect of ADC cells on the MDA-MB-453 cell line.

[0027] Figure 14 The study demonstrated the killing effect of ADC cells on the NCI-H358 cell line.

[0028] Figure 15 The study showed that the ADC cells in the CAPAN-1 cell line were killed.

[0029] Figure 16 The study demonstrated the killing effect of ADC cells on the MDA-MB-453 cell line.

[0030] Figure 17 The study demonstrated the killing effect of ADC cells on the NCI-H358 cell line.

[0031] Figure 18 The study showed that the ADC cells in the CAPAN-1 cell line were killed.

[0032] Figure 19 The study demonstrated the killing effect of ADC cells on the MDA-MB-453 cell line.

[0033] Figure 20 The study demonstrated the killing effect of ADC cells on the NCI-H358 cell line.

[0034] Figure 21 The study showed that the ADC cells in the CAPAN-1 cell line were killed.

[0035] Figure 22 The study demonstrated the killing effect of ADC cells on the MDA-MB-453 cell line.

[0036] Figure 23 The study demonstrated the killing effect of ADC cells on the NCI-H358 cell line.

[0037] Figure 24 The study showed that the ADC cells in the CAPAN-1 cell line were killed.

[0038] Figure 25 The study demonstrated the killing effect of ADC cells on the MDA-MB-453 cell line.

[0039] Figure 26 Displays ADC bystander kill assay.

[0040] Figure 27 Displays ADC bystander kill assay.

[0041] Figure 28 Displays ADC bystander kill assay.

[0042] Figure 29 Displays ADC bystander kill assay.

[0043] Figure 30 Displays ADC bystander kill assay.

[0044] Figure 31 Displays ADC bystander kill assay.

[0045] Figure 32 Displays ADC bystander kill assay.

[0046] Figure 33 Displays ADC bystander kill assay.

[0047] Figure 34 Displays ADC bystander kill assay.

[0048] Figure 35 Displays ADC bystander kill assay.

[0049] Figure 36 Displays ADC bystander kill assay.

[0050] Figure 37 Displays ADC bystander kill assay.

[0051] Figure 38 The study demonstrated the killing effect of ADC cells on the NCI-H358 cell line.

[0052] Figure 39 The study showed that the ADC cells in the CAPAN-1 cell line were killed.

[0053] Figure 40 The study demonstrated the killing effect of ADC cells on the MDA-MB-453 cell line.

[0054] Figure 41 Displays ADC bystander kill assay.

[0055] Figure 42 Displays ADC bystander kill assay. Detailed Implementation

[0056] definition Unless explicitly defined below or elsewhere in this document, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art.

[0057] Unless the context clearly indicates otherwise, as used herein (including the appended claims), the singular forms of words such as “a,” “an,” and “the” include their corresponding plural forms.

[0058] Unless explicitly stated or obvious from the context, the term "about" as used herein means, as determined by one of ordinary skill in the art, that a value or composition is within an acceptable range of error for a particular value or composition, the range of error being partly dependent on the method of measurement or determination of the value or composition, i.e., the limitations of the measurement system. For example, "about" may mean within one or more standard deviations according to practice in the art. "About" may mean a range of up to 10% (i.e., ±10%). Thus, "about" can be understood as being within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% of the stated value, whether greater or less. For example, about 5 mg may include any amount between 4.5 mg and 5.5 mg. Furthermore, particularly for biological systems or processes, the term may mean within an order of magnitude of a value or up to 5 times it. When a particular value or composition is provided in this disclosure, unless otherwise stated, it should be assumed that the meaning of "about" is within an acceptable range of error for that particular value or composition.

[0059] Unless the context clearly indicates otherwise, the term “or” is used to mean the term “and / or” and is used interchangeably with the term “and / or”.

[0060] As used herein, the terms "administration" and "administering," when applied to animals, humans, subjects, cells, tissues, organs, or biological fluids, mean contacting an exogenous pharmaceutical agent, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. Cell treatment encompasses contacting the cells with an agent, as well as contacting the agent with a fluid, wherein the fluid contacts the cells.

[0061] The term "subject" in this document includes any living organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit, primate) and most preferably a human (e.g., a patient who has the condition described herein or is at risk of having said condition).

[0062] "Treatment" of any disease or condition can mean, on one hand, improving the disease or condition (i.e., slowing, stopping, or reducing the development of the disease or at least one of its clinical symptoms). On the other hand, "treatment" can mean alleviating or improving at least one bodily parameter, including those that the patient may not be aware of. Furthermore, "treatment" can mean regulating a disease or condition physically (e.g., stabilizing perceptible symptoms), physiologically (e.g., stabilizing bodily parameters), or both.

[0063] The terms “improvement,” “increase,” “suppression,” and “reduction” indicate values ​​relative to a baseline or other reference measurement. In some embodiments, an appropriate reference measurement may be included in a system (e.g., in a single individual) under conditions where the agent or treatment is absent (e.g., before and / or after) or where a suitable comparable reference agent is otherwise equivalent. In some embodiments, an appropriate reference measurement may be included in a comparable system that is known or expected to react in a comparable manner in the presence of a relevant agent or treatment.

[0064] In some aspects, this disclosure provides compositions, such as pharmaceutically acceptable compositions, formulated together with at least one pharmaceutically acceptable excipient. As used herein, the term "pharmaceuticalally acceptable excipient" includes any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents, etc. The excipient may be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, or epidermal administration (e.g., by injection or infusion).

[0065] As used herein, the term "therapeutic effective amount" or "effective amount" refers to an amount of agent sufficient to achieve such treatment for a disease or at least one clinical symptom of a disease or condition when administered to a subject. "Therapeutic effective amount" may vary depending on: the amount of agent; the disease, condition, and / or the symptoms of the disease or condition; the severity of the disease, condition, and / or the symptoms of the disease or condition; the age of the subject to be treated; and / or the weight of the subject to be treated. An appropriate amount in any given situation may be obvious to those skilled in the art or may be determined by routine experimentation. In the case of combination therapy, "therapeutic effective amount" refers to the total amount of the combined components.

[0066] The terms “toxin,” “payload,” or “cytotoxic agent” are used herein to refer to molecules that inhibit or reduce the expression of molecules in cells, suppress or reduce cellular function, induce apoptosis, and / or cause cell death. These terms include radioactive isotopes, chemotherapeutic agents, and toxins such as small molecule toxins or enzyme-active toxins of bacterial, fungal, plant, or animal origin, including fragments and / or variants thereof. Examples of cytotoxic agents include (but are not limited to) auristatin (e.g., auristatin E, auristatin F, MMAE, and MMAF), auromycin, maytansinoid, pyrrolobenzodiazepine (PBD), ricin, ricin A chain, combrestatin, duocarmycin, dolastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, anthraxin-2, actinomycin, diphtheria toxin, and Pseudomonas exotoxin (PE). A, PE40, abrinogen, abrinogen A chain, modeccin A chain, α-Dacococcus, leucinogen, mitogen, retstrictocin, phenolmycin, enomycin, curicin, crotonin, and calicheamicin, as well as radioactive isotopes such as At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212 or 213, P32, and Lu177.

[0067] "Alkyl" is a saturated 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 5, 1 to 4, or 2 to 6 carbon atoms. Representative alkyl groups include -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, and -n-hexyl; while saturated branched alkyl groups include -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc. Alkyl groups may be substituted or unsubstituted. In some embodiments, when the alkyl group described herein is allegedly “substituted”, it may be substituted by any one or more substituents as seen in the exemplary compounds and embodiments disclosed herein, 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.

[0068] "Cycloalkyl" refers to a saturated or partially saturated cyclic alkyl group having 3 to 10 carbon atoms, having a single ring or multiple condensed or bridged rings optionally substituted by 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, etc. Examples of unsaturated cycloalkyl groups include cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, hexadienyl, etc. Cycloalkyl groups may be substituted or unsubstituted. Such substituted cycloalkyl groups include, for example, cyclohexanone, etc.

[0069] "Aryl" is an aromatic carbocyclic group having 6 to 14 carbon atoms, comprising a monocyclic ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthracene). In some embodiments, the aryl group contains 6-14 carbons in the ring moiety 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., dihydroindenyl, tetrahydronaphthyl, etc.).

[0070] A "heteroaryl" is an aryl ring system having one to four heteroatoms as ring atoms in a heteroaryl ring system, 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 even 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) groups such as: pyrrolo, pyrazol, imidazolyl, triazol, tetrazol, oxazol, isoxazol, thiazolyl, pyrrolo, pyridinyl, pyridazinyl, pyrimidinyl, thiophene, benzothiophene, furanyl, benzofuranyl (e.g., isobenzofuran-1,3-diimine), indole, azaindole (e.g., pyrrolopyridinyl or 1H-pyrrolo[2,3-b]pyridinyl), inzol, 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, thianaphthyl, purine, xanthine, adenine, guanine, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl and quinazolinyl.

[0071] "Heterocyclic group" or "heterocycle" is a non-aromatic cycloalkyl or aryl group in which one to four ring carbon atoms are independently replaced by heteroatoms independently selected 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. In some embodiments, the heterocyclic group is aromatic. In some embodiments, the heterocyclic group is non-aromatic. 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 imidazolyl, imidazolinyl, and imidazoalkyl. The phrase heterocyclic group includes fused ring types, including those containing fused aromatic and non-aromatic groups, such as benzotriazolyl, 2,3-dihydrobenzo[1,4]dioxane-hexenyl, and benzo[1,3]dioxane-pentenyl. The phrase also includes bridging polycyclic systems containing heteroatoms, such as (but not limited to) quinine cyclic groups. Representative examples of heterocyclic groups include (but are not limited to) aziridinyl, aziridine, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, tetrahydrothiophene, tetrahydrofuranyl, dioxacyclopentenyl, furanyl, thiophene, pyrrolyl, pyrrololinyl, imidazolyl, imidazolinyl, pyrazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolinyl, isothiazolyl, thiazolinyl, isothiazolyl, thiadiazolyl, oxadiazolyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetra Hydropyranyl (e.g., tetrahydro-2H-pyranyl), tetrahydrothiaranyl, oxothiacyclohexane, dioxy, dithiaalkyl, pyranyl, pyridyl, pyrimidinyl, pyrazinyl, triazinyl, dihydropyridinyl, dihydrodithiacyclohexadienyl, dihydrodithionyl, peripetaninyl, quininecycloyl, indoleyl, indolinyl, isoindoleyl, azaindoleyl (pyrrolopyridinyl), indazoleyl, inazinyl, benzotriazolyl, benzimidazolyl, benzofuranyl, benzothiophenyl, benzothiazole Benzoxadiazolyl, benzoxazinyl, benzodithiacyclohexadienyl, benzooxazinyl, benzothiazinyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[1,3]dioxacyclopentenyl, pyrazolopyridyl, imidazopyridyl (azabenzimidazolyl; e.g., 1H-imidazo[4,5-b]pyridyl or 1H-imidazo[4,5-b]pyridin-2(3H)-keto), triazolopyridyl, isoxazolopyridyl, purine, Xanthine, adenine, guanine, quinolinyl, isoquinolinyl, quinazinyl, quinoxalinyl, quinazolinyl, cyclophosphinyl, phthalazinyl, naphthidyl, pteridyl, thianaphthyl, dihydrobenzothiazinyl, dihydrobenzofuranyl, dihydroindolyl, dihydrobenzodioxane-hexenyl, tetrahydroindolyl, tetrahydroindolyl, tetrahydrobenzimidazolyl, tetrahydrobenzotriazolyl, tetrahydropyrrolopyridyl, tetrahydropyrazolopyridyl, tetrahydroimidazopyridyl, tetrahydrotriazolopyridyl, and tetrahydroquinolinyl.The representative substituted heterocyclic group may be monosubstituted or substituted more than once, for example (but not limited to) pyridyl or morpholino groups substituted or disubstituted by various substituents (such as those listed below) 2-, 3-, 4-, 5- or 6-substituted.

[0072] "Cycloalkylalkyl" is a group of the formula -alkyl-cycloalkyl, wherein the alkyl and cycloalkyl groups are defined above. The substituted cycloalkylalkyl group may be substituted at either the alkyl, cycloalkyl, or both alkyl and cycloalkyl moieties of the group. Representative cycloalkylalkyl groups include (but are not limited to) cyclopentylmethyl, cyclopentylethyl, cyclohexylmethyl, cyclohexylethyl, and cyclohexylpropyl. Representative substituted cycloalkylalkyl groups may be monosubstituted or substituted more than once.

[0073] "Aryl" is a group of the formula -alkyl-aryl, wherein the alkyl and aryl groups are defined above. A substituted aryl group may be substituted at either the alkyl, aryl, or both alkyl and aryl moieties 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-dihydroindenyl.

[0074] "Heterocyclic alkyl" is a group of the formula -alkyl-heterocyclic, wherein the alkyl and heterocyclic groups are defined above. The substituted heterocyclic alkyl group may be substituted at the alkyl, heterocyclic, or both alkyl and heterocyclic moieties of the group. Representative heterocyclic alkyl groups include (but are not limited to) 4-ethylmorpholino, 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.

[0075] "Halogen" refers to chlorine, iodine, bromine, or fluorine.

[0076] "alkoxy (alkoxy or alkoxyl)" is -O (alkyl), where alkyl is defined above.

[0077] "Alkoxyalkyl" is -(alkyl)O(alkyl), wherein each alkyl group is independently as defined above.

[0078] As used herein, “aryloxy” 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 with an oxygen atom, i.e., the aromatic compound includes a single bond with an oxygen atom and wherein said group is located on the oxygen atom, for example, C6H5-O- for phenoxy. The aryloxy substituent is bonded to the compound in which it is substituted with that oxygen atom. The aryloxy group is optionally substituted. The aryloxy group includes (but is not limited to) those groups having a ring of carbon atoms of 6 to 20, i.e., C6H5-O-. 6-20Aryloxy group; 6 to 15 ring carbon atoms, i.e., C 6-15 aryloxy group, 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.

[0079] The "amine" group is a group with the formula -NH2.

[0080] The "hydroxylamine" group is represented by the formula N(R) # )OH or NHOH groups, wherein R # The substituted or unsubstituted alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclic or heterocyclic alkyl groups are defined as herein.

[0081] The "alkoxyamine" group has the formula -N(R) # )O-alkyl or -NHO-alkyl groups, wherein R # As defined above.

[0082] The "arylalkoxyamine" group is of the formula N(R) # )O-aryl or NHO-aryl groups, wherein R # As defined above.

[0083] The "alkylamine" group is a group of the formula NHalkyl or N(alkyl)2, wherein each alkyl group is independently as defined above.

[0084] The "amino carbonyl" group is represented by the formula -C(=O)N(R). # )2、-C(=O)NH(R # ) or C(=O)NH2 groups, wherein each R # As defined above.

[0085] "Acylamino" is represented by the formula NHC(=O)(R # ) or N(alkyl)C(=O)(R # ) groups, wherein each alkyl group and R # Independently as defined above.

[0086] "O(alkyl)aminocarbonyl" is a compound with the formula -O(alkyl)C(=O)N(R) # )2、-O(alkyl)C(=O)NH(R) # ) or -O(alkyl)C(=O)NH2 groups, wherein each R # Independently as defined above.

[0087] The "N-oxide" group has the formula -N + -O - . group.

[0088] "Carboxyl group" is a group with the formula C(=O)OH.

[0089] The ketone group has the formula C(=O)(R) # ) groups, wherein R # As defined above.

[0090] The "aldehyde" group is a group with the formula -CH (=O).

[0091] The ester group has the formula C(=O)O(R) # ) or OC(=O)(R # ) groups, wherein R # As defined above.

[0092] The urea group has the 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 # The groups, wherein each alkyl group and R # Independently as defined above.

[0093] The "imine" group is represented by the formula -N=C(R). # )2 or -C(R # )=N(R # ) groups, wherein each R # Independently as defined above.

[0094] The "imide" group has the formula -C(=O)N(R#)C(=O)(R # ) or N((C=O)(R # ))2 groups, wherein each R # Independently as defined above.

[0095] The "carbamate" group has the formula -OC(=O)N(R) # )2、-OC(=O)NH(R # ), -N(R # )C(=O)O(R # ) or -NHC(=O)O(R # ) groups, wherein each R # Independently as defined above.

[0096] The "ammonia" base is represented by the 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 group, where each R # Independently as defined above.

[0097] The guanidine group is of the 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 # The group consisting of ))2 or -N=C(NH2)2, wherein each R # Independently as defined above.

[0098] The "enamine" group has the 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) group, wherein each R # Independently as defined above.

[0099] The oxime base is given by the formula -C(=NO(R) # ))(R # -C(=NOH)(R) # -CH(=NO(R) # The group is either -CH or -NOH, where each R # Independently as defined above.

[0100] The "acylhydrazine" group has the 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 # The group consisting of )2 or -C(=O)NHNH2, wherein each R # Independently as defined above.

[0101] The hydrazine group is represented by the formula -N(R) # )N(R # )2、-NHN(R # )2、-N(R # )NH(R # ), -N(R # )NH2、-NHNH(R # The group consisting of )2 or -NHNH2, wherein each R # Independently as defined above.

[0102] The "hydrazone" base is represented by the 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) groups, wherein each R # Independently as defined above.

[0103] The "azide" group is a group with the formula -N3.

[0104] The "isocyanate" group is a group with the formula N=C=O.

[0105] The "isothiocyanate" group is a group with the formula N=C=O.

[0106] The "cyanate ester" group is a group of the formula OCN.

[0107] The "thiocyanate" group is a group of the formula SCN.

[0108] The sulfide group has the formula -S(R) # ) groups, wherein R # As defined above.

[0109] The "thiocarbonyl" group is represented by the formula -C(=S)(R # ) groups, wherein R # As defined above.

[0110] The "sulfinyl group" is represented by the formula -S(=O)(R # ) groups, wherein R # As defined above.

[0111] The sulfone group is represented by the formula -S(=O)2(R). # ) groups, wherein R # As defined above.

[0112] "Sulfonylamino" is represented by the formula -NHSO2(R # ) or -N(alkyl)SO2(R # ) groups, wherein each alkyl group and R # Defined above.

[0113] The sulfonamide group has the formula -S(=O)2N(R) # )2 or -S(=O)2NH(R # The group consisting of ) or -S(=O)2NH2, wherein each R # Independently as defined above.

[0114] The "phosphonate" group has the formula -P(=O)(O(R) # ))2, -P(=O)(OH)2, -OP(=O)(O(R # ))(R # ) or -OP(=O)(OH)(R # ) groups, wherein each R # Independently as defined above.

[0115] The phosphine group is represented by the formula -P(R) # )2 groups, wherein each R # Independently as defined above.

[0116] When a group described herein (other than alkyl) is referred to as “substituted,” it may be substituted by any one or more suitable substituents. Illustrative examples of substituents are those found in the exemplary 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; amide; phosphonate; phosphine; thiocarbonyl; sulfinyl; sulfonyl; sulfonamide; ketone; aldehyde; ester; urea; carbamate; oxime; hydroxylamine; alkoxyamine; arylalkoxyamine; N-oxide; hydrazine; acylhydrazine; hydrazone; azide; isocyanate; isothiocyanate; cyanate; thiocyanate; oxygen (=O); B(OH)2, O(alkyl)aminocarbonyl ; cycloalkyl, which may be monocyclic or fused or unfused polycyclic (e.g., cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl), or heterocyclic, which may be monocyclic or fused or unfused polycyclic (e.g., pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl or thiazinyl); monocyclic or fused or unfused polycyclic aryl or heteroaryl (e.g., phenyl, naphthyl, pyrrolidinyl, indolyl, furanyl, thiophene, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, triazolyl, tetrazolyl, pyrazolyl, pyridinyl, quinolinyl, isoquinolinyl, acridinel, pyrazinyl, pyridazinyl, pyrimidinyl, benzimidazolyl, benzothiophene or benzofuranyl) aryloxy; arylalkoxy; heterocyclic oxy; and heterocyclic alkoxy.

[0117] As used herein, the term "pharmaceutically acceptable salt" refers to a salt prepared from a pharmaceutically acceptable, non-toxic acid or base (including inorganic acids and bases as well as organic acids and bases).

[0118] As used herein and unless otherwise indicated, the term "solvent" means a compound or salt thereof that also includes stoichiometric or non-stoichiometric solvents bound by non-covalent intermolecular forces. In one embodiment, the solvate is a hydrate.

[0119] As used herein and unless otherwise indicated, the term "hydrate" means a compound or salt thereof that also includes stoichiometric or non-stoichiometric amounts of water bound together by non-covalent intermolecular forces.

[0120] All pharmaceutically acceptable salts, solvates, and / or hydrates of the compounds illustrated herein are within the scope of this disclosure.

[0121] 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 ureas, 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 are conveniently formed by esterifying any carboxylic acid moiety present on the molecule. Prodrugs are generally prepared using well-known methods, such as... Burger's Medicinal Chemistry and Drug Discovery The 6th edition (edited by Donald J. Abraham, 2001, Wiley) and Design and Application of Prodrugs The methods described in (edited by H. Bundgaard, 1985, Harwood Academic Publishers Gmfh).

[0122] 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 be substantially free of its opposite enantiomers. A stereoisomer-pure compound having two chiral centers will be substantially free of other diastereomers of the compound. Typical stereoisomer-pure compounds contain 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. The compound may have a chiral center and may exist as a racemic compound, individual enantiomers or diastereomers, and mixtures thereof. All such isomers are included within the embodiments disclosed herein, including mixtures thereof. The embodiments disclosed herein cover the use of the stereoisomeric pure forms of such compounds and the use of mixtures of those forms. 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 synthesized asymmetrically or resolved 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 (edited by EL Eliel, Univ. of Notre Dame Press, Notre Dame, IN, 1972).

[0123] 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 in either cis or trans isomer form. In other embodiments, the compound is a mixture of cis and trans isomers.

[0124] "Tautomers" refer to the equilibrium isomers of a compound. The concentration of these isomers will depend on the environment in which the compound exists and can vary depending on, for example, whether the compound is a solid or in an organic or aqueous solution. For example, in aqueous solution, pyrazole can exhibit the following isomers, which are called tautomers of each other: .

[0125] As will be readily understood by those skilled in the art, a wide variety of functional groups and other structures can exhibit tautomerism, and all tautomers of the compound are within the scope of this disclosure.

[0126] It should also be noted that the compound may contain atomic isotopes in non-natural proportions at one or more atoms. For example, the compound may use radioactive isotopes, such as tritium ( 3 H), Iodine-125 ( 125 I), sulfur-35 ( 35 S) or carbon-14 ( 14 C) Radiolabeling, or isotopic enrichment with, for example, deuterium ( 2 H), carbon-13 ( 13 C) or nitrogen-15 ( 15 As used herein, an "isotope configuration" is an isotopically enriched compound. The term "isotope enrichment" means that the isotopic composition of an atom differs from the natural isotopic composition of said atom. "Isotope enrichment" can also refer to a compound containing at least one atom whose isotopic composition differs from the natural isotopic composition of said atom. 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 assay reagents), and diagnostic agents (e.g., in vivo imaging agents). All isotopic variations of the compounds described herein, whether or not radioactive, are intended to be covered within the scope of the embodiments provided herein. In some embodiments, isotope configurations of the compound are provided, for example, isotope configurations of compounds that are deuterium-rich, carbon-13-rich, or nitrogen-15-rich.

[0127] It should be noted that if there is a difference between the drawn structure and the name of the structure, the drawn structure shall prevail.

[0128] As used herein, the term "residue" refers to a chemical portion of a compound that is retained after a chemical reaction. For example, the terms "amino acid residue" or "N-alkyl amino acid residue" refer to a product of an amino acid or N-alkyl amino acid coupled with an amide or peptide of a suitable coupling partner; wherein, for example, water molecules are expelled after coupling of the amino acid or N-alkyl amino acid with an amide or peptide, resulting in a product incorporating an amino acid residue or N-alkyl amino acid residue.

[0129] As used herein, "sugar," "glycosyl," or "sugar residue" refers to a carbohydrate moiety that may contain 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" contains 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" contains 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-glucosol) that are linked via their amino groups to the remainder of the molecule to form an amide bond (i.e., glucosamide) with the remainder of the molecule.

[0130] Certain groups, parts, substituents, and atoms are drawn with wobbly lines, such as those intersecting with one or more bonds or extending from the atom, to indicate the atoms through which the group, part, substituent, or atom is bonded. For example, a phenyl group substituted with a propyl group is drawn as follows: or or It has the following structure: .

[0131] As used herein, unless otherwise specified, illustrations showing substituents bonded to cyclic groups (e.g., aromatic, heteroaromatic, fused rings, and saturated or unsaturated cycloalkyl or heterocycloalkyl) via inter-ring bonds are intended to indicate that, according to the techniques set forth herein or known in the art to which this disclosure pertains, the cyclic group may be substituted by the substituent at any ring position in the cyclic group or on any ring in the fused ring group.

[0132] Unless otherwise specified, illustrations showing substituents bonded to acyclic groups via bonds between two atoms are intended to indicate that, according to techniques set forth herein or known in the art to which this disclosure pertains, the substituent may bond to any atom of the bond through which the substituent bond passes. Thus, for example, Coverage and .

[0133] Payload This article provides exatecan analogues, for example, for use in the platforms and / or ADCs described herein.

[0134] One embodiment provides a compound of any of the following formulas, or a pharmaceutically acceptable salt and / or solvate thereof:

[0135] In one implementation, the compound is Or its pharmaceutically acceptable salts, stereoisomers, and / or solvates. In one embodiment, the compound is... Or its pharmaceutically acceptable salts, stereoisomers and / or solvates.

[0136] The residues of the payload described herein may be combined with any linker described herein to form a platform, or the residues of the payload described herein may be combined with any linker described herein and any antibody or its antigen-binding fragment described herein to form an ADC.

[0137] covalent connector This article also provides covalent connectors, for example, for use in the platforms and / or ADCs described herein.

[0138] In some implementations (e.g., for connectors in a platform), the connector is: , in: The bond that indicates T is attached to the payload residue or drug unit (P); Z is a heterocycle, which is optionally substituted with at least one carbonyl or methanesulfonyl group; Y is either aryl or -C≡C-(CH2). a -; The subscript 'a' is 1, 2, 3, or 4; The subscript y' is 0 or 1; X represents C that has been substituted or not substituted. 1-5 alkyl; The subscript x' is 0 or 1; W stands for oxygen; The subscript w' is 0 or 1; V is C 1-4 Alkyl or -C(=O)-NH-(CH2) b Wherein N is optionally C 1-5 Alkyl substitution and subscript b is 1 or 2; The subscript v' is 0 or 1; U is a hydrophilic group; T represents oxygen or a bond; and The subscript s is 0 or 1.

[0139] In some implementations (e.g., for connectors used in ADCs), the connector is: , in: The bond that indicates T is attached to the payload residue or drug unit (P); Z' is a heterocycle, which is optionally substituted with at least one carbonyl or methanesulfonyl group; Y is either aryl or -C≡C-(CH2). a -; The subscript 'a' is 1, 2, 3, or 4; The subscript y' is 0 or 1; X represents C that has been substituted or not substituted. 1-5 alkyl; The subscript x' is 0 or 1; W stands for oxygen; The subscript w' is 0 or 1; V is C 1-4 Alkyl or -C(=O)-NH-(CH2) b Wherein N is optionally C 1-5 Alkyl substitution and subscript b is 1 or 2; The subscript v' is 0 or 1; U is a hydrophilic group; T represents oxygen or a bond; and The subscript s is 0 or 1.

[0140] In some implementation schemes, Z is , or .

[0141] In some implementation schemes, Z' is , , , or ,and Indicates the key to which Z is connected (e.g., Ab).

[0142] In some embodiments, Y is phenylene or -C≡C-(CH2)3-. In other embodiments, Y is phenylene or -C≡C-(CH2)3-, and the subscript y' is 1.

[0143] In some implementations, the subscript y' is 1. In other implementations, the subscript y' is 0.

[0144] In some embodiments, X is -(CH2)5-, -CH(CH2NH2)(CH2)4-, -CH2CH(NH2)CH2-, -CH(CH2NH2)CH2-, or -CH(CH2NH2)(CH2)2-. In other embodiments, X is -(CH2)5-, -CH(CH2NH2)(CH2)4-, -CH2CH(NH2)CH2-, -CH(CH2NH2)CH2-, or -CH(CH2NH2)(CH2)2-, and the subscript x' is 1.

[0145] In some implementations, the subscript x' is 1. In other implementations, the subscript x' is 0.

[0146] In some implementations, the subscript w' is 1. In other implementations, the subscript w' is 0.

[0147] In some embodiments, V is ethyl or -C(=O)-NH-(CH2). b Where N is arbitrarily divided by C 1-3 Alkyl substitution, and the subscript b is 1 or 2. In other embodiments, V is ethyl or -C(=O)-NH-(CH2). b Where N is arbitrarily divided by C 1-3 Alkyl substitution, and the subscript b is 1 or 2; and the subscript v' is 1. In some embodiments, V is... , or In other implementations, V is... , or And the subscript v' is 1.

[0148] In some implementations, the subscript v' is 1. In other implementations, the subscript v' is 0.

[0149] In some embodiments, U is a sugar, phosphate ester, sulfate ester, phosphate diester, or phosphonate. In other embodiments, U is a sugar, and said sugar is β-D-galactose, N-acetyl-PD-galactosamine, N-acetyl-aD-galactosamine, N-acetyl-PD-glucosamine, β-D-glucuronic acid, aL-iduronic acid, aD-galactose, aD-glucose, β-D-glucose, aD-mannose, β-D-mannose, aL-fucose, β-D-xylose, or neuraminic acid; modified with sulfate, phosphate, carboxyl, amino, or O-acetyl groups.

[0150] In some implementations, U is or In some implementations, U is... or .

[0151] In some implementations, T represents oxygen. In some implementations, T represents a bond.

[0152] In some implementations, the subscript s is 1. In other implementations, the subscript s is 0.

[0153] Connector and payload (platform) This article also provides platforms, such as those for fabricating ADCs, like the ADC described in this article.

[0154] One embodiment is a compound of formula (I): , Or its pharmaceutically acceptable salts, tautomers, solvates or stereoisomers, wherein: Z is a heterocycle, which is optionally substituted with at least one carbonyl or methanesulfonyl group; Y is either aryl or -C≡C-(CH2). a - The subscript 'a' is 1, 2, 3, or 4; The subscript y' is 0 or 1; X represents C that has been substituted or not substituted. 1-5 alkyl; The subscript x' is 0 or 1; W stands for oxygen; The subscript w' is 0 or 1; V is C 1-4 Alkyl or -C(=O)-NH-(CH2) b Wherein N is optionally C 1-5 Alkyl substitution and subscript b is 1 or 2; The subscript v' is 0 or 1; U is a hydrophilic group; T represents oxygen or a bond; P represents the payload residue or drug unit; and The subscript s is 0 or 1.

[0155] In some embodiments, the compound of formula (I) is represented by formula (Ia): , Or a pharmaceutically acceptable salt, tautomer, solvate or stereoisomer thereof, wherein the values ​​of variables (e.g. Z, Y, y', X, x', W, w', V, v', U, s, T, P) are as set forth herein, for example with respect to compounds of formula (I).

[0156] In some implementations, P is: , , , , , , , , , or .

[0157] Alternative values ​​for variables (e.g., Z, Y, y', X, x', W, w', V, v', U, s, T) are as described herein, for example, regarding covalent joints.

[0158] Specific examples of platform compounds are illustrated in the embodiments. In some embodiments, the compound is selected from any of the following, or pharmaceutically acceptable salts, solvates, and / or stereoisomers thereof:

[0159] Binding agents, including antibodies In some implementations, BA is an antibody or its antigen-binding fragment, such as a humanized, chimeric, or human antibody or its antigen-binding fragment.

[0160] In some embodiments, the antibody or its antigen-binding fragment specifically binds to human B7H3. In some embodiments, the antibody or its antigen-binding fragment is ifinatimab.

[0161] In some embodiments, the antibody or its antigen-binding fragment specifically binds to human B7H3 and comprises (i) a heavy chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 2; and a light chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 1; optionally wherein the CDR is identical to those CDRs of ifenamarumab. In some embodiments, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 2; and the amino acid sequence of the light chain variable region is SEQ ID NO: 1.

[0162] In some embodiments, one, two, three, four, five, six, seven, eight, nine, or ten amino acids are further incorporated (inserted, deleted, or substituted) into the framework region of the antibody, for example, SEQ ID NO: 1 or SEQ ID NO: 2.

[0163] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain constant region of the IgG1, IgG2, IgG3 or IgG4 subclass, and / or a light chain constant region of the κ or λ type.

[0164] In some embodiments, the antibody or its antigen-binding fragment is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human engineered antibody, a single-chain antibody (scFv), a Fab fragment, a Fab' fragment, or an F(ab')2 fragment.

[0165] In some embodiments, the antibody or its antigen-binding fragment has antibody-dependent cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

[0166] In some implementations, the Fc domain is IgG1 with reduced effector function.

[0167] Antibody-drug conjugates (ADCs) This article also provides ADCs, for example, for use in therapies such as cancer treatments.

[0168] An ADC with implementation of formula (II): , Or its pharmaceutically acceptable salts, tautomers, solvates or stereoisomers, wherein: BA is a conjugate selected from humanized, chimeric, or human antibodies or their antigen-binding fragments; Z' is a heterocycle, which is optionally substituted with at least one carbonyl or methanesulfonyl group; Y is either aryl or -C≡C-(CH2). a -; The subscript 'a' is 1, 2, 3, or 4; The subscript y' is 0 or 1; X represents C that has been substituted or not substituted. 1-5 alkyl; The subscript x' is 0 or 1; W stands for oxygen; The subscript w' is 0 or 1; V is C 1-4 Alkyl or -C(=O)-NH-(CH2) b Wherein N is optionally C 1-5 Alkyl substitution and subscript b is 1 or 2; The subscript v' is 0 or 1; U is a hydrophilic group; T represents oxygen or a bond; P represents the payload residue or drug unit; The subscript s is 0 or 1; and The subscript n is from 1 to 15.

[0169] In some implementations, the ADC of equation (II) is represented by equation (IIa): , Or a pharmaceutically acceptable salt, tautomer, solvate or stereoisomer thereof, wherein the values ​​of the variables (e.g. BA, Z', Y, y', X, x', W, w', V, v', U, s, T, P, n) are as set forth herein, for example with respect to compounds of formula (II).

[0170] In some implementations, the subscript n is 3 to 12, for example 4 to 11, 5 to 10. In some implementations, the subscript n is 7 to 9. In some implementations, the subscript n is approximately 8.

[0171] Alternative values ​​for variables (e.g., BA, Z', Y, y', X, x', W, w', V, v', U, s, T, P, n) are as described herein. For example, alternative values ​​for variables Z', Y, y', X, x', W, w', V, v', U, s, and T are as described herein regarding covalent linkers. For example, alternative values ​​for variable P are as described herein regarding payloads and / or platforms. For example, alternative values ​​for variable BA are as described herein, for example, regarding binders (including antibodies).

[0172] In some implementations, the ADC is represented by one of the following formulas, or a pharmaceutically acceptable salt, solvate, and / or stereoisomer thereof:

[0173] Where Ab is a humanized, chimeric, or human antibody or its antigen-binding fragment, and the subscript n is from 1 to 15. Alternative values ​​for the variable subscript n are as described herein, for example, with respect to compounds of formula (II) and (IIa).

[0174] In some implementations, Ab is ifenamarab. Alternative values ​​for Ab are as described herein, for example, regarding conjugates (including antibodies).

[0175] The antibody-drug conjugates disclosed herein can be produced by any method known in the art. In one embodiment, host cells transformed with isolated nucleic acids containing sequences encoding antibodies or antigen-binding fragments thereof are cultured under suitable culture conditions. The antibody or antigen-binding fragment thereof is thus expressed and can be recovered from the cell culture. A payload (e.g., a payload described herein) is conjugated to the antibody or antigen-binding fragment using a adapter (e.g., an adapter described herein) to produce an antibody-drug conjugate. In some embodiments, an ADC platform (e.g., a platform described herein) is conjugated to the antibody or antigen-binding fragment to produce an antibody-drug conjugate.

[0176] The specific methods for generating the antibody-drug conjugates of this disclosure are described in the examples.

[0177] Compositions and Formulations 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.

[0178] 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 (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)), in the form of a lyophilized formulation or an aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic to recipients at the doses and concentrations employed 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 ammonium 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 residuals) This includes: 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., zinc-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 neutral active hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, 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 (e.g., chondroitinase).

[0179] Exemplary lyophilized formulations are described in U.S. Patent No. 6,267,958. Aqueous formulations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter comprising histidine-acetate buffer.

[0180] Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include a semi-permeable matrix of a solid hydrophobic polymer containing an antibody-drug conjugate, said matrix being in the form of a shaped article, such as a membrane or microcapsule.

[0181] Preparations intended for in vivo administration are typically sterile. Sterility can be readily achieved, for example, through filtration via a sterile filter membrane.

[0182] Methods using ADC In some implementations, this document describes a method for treating cancer in a subject (e.g., a patient) of need, the method comprising administering to the subject an effective amount of the ADC or a pharmaceutical composition thereof described herein.

[0183] The antibody-drug conjugates disclosed herein can be administered by any suitable route, including parenteral, intrapulmonary, and intranasal administration, and, if intended for local treatment, intralesional administration may be used. Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Administration may be carried out via any suitable route, such as by injection, for example, intravenous or subcutaneous injection, depending in part on whether the administration is transient or long-term. Various dosing regimens are considered herein, including (but not limited to) single or multiple administrations at different time points, concentrated administration, and pulsatile infusion.

[0184] The antibody-drug 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 of the drug, the method of administration, the timing of administration, and other factors known to the practicing physician.

[0185] Example List of abbreviations

[0186] UPLC analysis method Method A: Mobile phase A: 0.1% FA aqueous solution, B: MeCN; Gradient: 10% B maintained for 0.2 min, 10%-95% B for 5.8 min, 95% B maintained for 0.5 min; Flow rate: 0.6 mL / min; Column: ACQUITY UPLC® BEH C18 1.7 μm.

[0187] Method B: Mobile phase A: 0.1% FA aqueous solution, B: MeCN; Gradient: 10% B maintained for 0.5 min, 10%-90% B, 2.5 min, 90% B maintained for 0.2 min; Flow rate: 0.6 mL / min; Column: ACQUITY UPLC® BEH C18 1.7 μm.

[0188] Method C: Mobile phase A: 0.1% FA aqueous solution, B: MeCN; Gradient: 10% B maintained for 0.2 min, 10%-90% B, 1.3 min, 90% B maintained for 0.3 min; Flow rate: 0.6 mL / min; Column: ACQUITY UPLC® BEH C18 1.7 μm.

[0189] Example 1 Step 1: N-((1) S 9 S )-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1 H ,12 H -Benz[de]pyrano[3',4':6,7]inzizo[1,2- b Quinoline-1-yl)-3-hydroxy-2,2-dimethylpropionamide (P1). DIPEA (21 μL, 16 mg, 0.13 mmol) and ethatecan mesylate (23 mg, 0.043 mmol) were added to a mixture of 1a (5 mg, 0.042 mmol) and HATU (16 mg, 0.042 mmol) in DMF (1 mL). The resulting brown mixture was stirred at room temperature for 2 h. After the reaction was complete, the sample was analyzed by preparative HPLC (TFA) (method: column: XBridge Prep C18 OBD 5 μm 19). The mixture was purified by 150 mm; mobile phase: A-water (0.1% TFA): B-acetonitrile; flow rate: 20 mL / min, and the fractions were lyophilized to give P1 (15 mg, 65.5% yield) as a white powder. 1 H NMR (400 MHz, DMSO-d6): δ 8.00 (d, J = 8.4 Hz, 1H), 7.79 (d, J = 11.2 Hz, 1H), 7.31 (s,1H), 6.52 (s, 1H), 5.59-5.54 (m, 1H), 5.42 (s, 2H), 5.18 (q, J = 19.2 Hz, 2H), 4.87 (t, J = 5.2 Hz, 1H), 3.45 (dd, J= 10.2, 4.8 Hz, 1H), 3.41-3.28 (m, 1H),3.15 (t, J = 5.6 Hz, 2H), 2.40 (s, 3H), 2.24-2.07 (m, 2H), 1.92-1.80 (m, 2H),1.11 (d, J = 7.6 Hz, 6H), 0.87 (t, J = 7.2 Hz, 3H). MS (ESI) m / z: 536.4 [M+H] + .

[0190] Example 2 Step 1: N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinolin-1-yl)-2-fluoro-3-hydroxy-2-methylpropionamide (P2). Exatecan mesylate (50 mg, 0.094 mmol), HATU (54 mg, 141 mmol), and DIEA (36 mg, 0.28 mmol) were added to a solution of compound 2a (23 mg, 0.19 mmol) in DMF (2 mL). The mixture was stirred at room temperature for 1 h. The sample was analyzed by preparative HPLC (FA) (method: column: XBridge Prep C18 OBD 5 μm 19). The mixture was purified by 150 mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min, and the fractions were lyophilized to give P2 (8.4 mg, 16.6% yield) as a white solid. UPLC-MS, RT = 3.86 min. 1 H NMR (400MHz, DMSO- d 6 ) δ 8.72 (dd, J = 8.4, 2.4 Hz, 1H), 7.78 (d, J = 11.2 Hz, 1H), 7.31(s, 1H), 6.52 (s, 1H), 5.58 (d, J = 8.0 Hz, 1H), 5.42 (s, 2H), 5.32 - 5.05 (m,3H), 3.83 (dd, J= 26.8, 12.0 Hz, 1H), 3.61 (dd, J = 21.6, 12.0 Hz, 1H), 3.22-3.07 (m, 2H), 2.46-2.30 (m, 3H), 2.28-2.05 (m, 2H), 2.02-1.74 (m, 2H), 1.45(d, J = 21.4 Hz, 3H), 0.87 (t, J = 7.2 Hz, 3H). MS (ESI) m / z: 540.3 [M+H] + .

[0191] Example 3 Step 1: N-(3-fluoro-7-(3-methoxypropyl)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (3c). 3a (300.00 mg, 1.28 mmol) was added dropwise to a solution of LDA (1.40 mL, 2.81 mmol, 2 M in THF) in 19 mL of THF at -78 °C, and the mixture was stirred at the same temperature for 2 h. Then, a solution of 3b (0.38 g, 1.91 mmol) in 1 mL of THF was added dropwise. The reaction mixture was then slowly heated to 0 °C and stirred continuously for 4 h. The reaction mixture was subsequently quenched with a saturated aqueous solution of NH4Cl (50 mL) and quenched with EtOAc (30 mL). 3) Extraction of organic materials. The combined organic layers were washed with brine (50 mL) and dried over MgSO4, then the combined extracts were concentrated under vacuum. The crude residue was purified by rapid column chromatography (silica gel, hexane:EtOAc = 97:3) to give 3c (80.00 mg, 20.41% yield) as a colorless oil. MS (ESI) m / z: 308.2 [M+H] + .

[0192] Step 2: 8-Amino-6-fluoro-2-(3-methoxypropyl)-5-methyl-3,4-dihydronaphthyl-1(2H)-one (3d). Compound 3d (69.06 mg, crude) was synthesized according to the synthetic procedure of Example 5-1 in EP3677568A1. MS (ESI) m / z: 266.2 [M+H] + .

[0193] Step 3: (9S)-9-ethyl-5-fluoro-9-hydroxy-1-(3-methoxypropyl)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-10,13-dione (3f). Compound 3f (80.00 mg, 62.40% yield) was synthesized according to the synthetic procedure of Example 6-1 in EP3677568A1. MS (ESI) m / z: 493.1 [M+H] + .

[0194] Step 4: (9S)-9-ethyl-5-fluoro-9-hydroxy-1-(3-hydroxypropyl)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-10,13-dione (P3). BBr3 (71.26 mg, 0.28 mmol) was added to a solution of 3f (70.00 mg, 0.14 mmol) in CH2Cl2 (20 mL) at -0 °C, and the mixture was stirred at the same temperature for 1 h. The reaction mixture was then slowly heated to 25 °C and stirred continuously for 3 h. The reactants were subsequently quenched with a saturated aqueous solution of NaHCO3, and the organic material was quenched with EtOAc (30 mL). 3) Extraction was performed three times. The combined organic layers were washed with brine (50 mL) and dried over MgSO4, then the combined extracts were concentrated under vacuum. The crude residue was purified by rapid column chromatography (silica gel, CH2Cl2:MeOH = 90:10) to give a mixture P3 (4.20 mg, 6.18% yield) as a gray solid. MS (ESI) m / z: 479.4 [M+H] + .

[0195] UPLC analysis: P3-1, peak 1, retention time = 4.49 min; P3-2, peak 2, retention time = 4.65 min (Mobile phase A: 0.1% FA aqueous solution, B: MeCN; gradient: 15% B for 1 min, 15%-95% B for 9 min, 95% B for 2 min; flow rate: 0.6 mL / min; column: ACQUITY UPLC® BEH C18 1.7 μm).

[0196] P3-1: 1 H NMR (400 MHz, DMSO-d6) δ 7.74 (d, J= 11.1 Hz, 1H), 7.30 (s,1H), 6.51 (s, 1H), 5.43 (s, 2H), 5.36 (d, J = 18.7 Hz, 1H), 5.22 (d, J = 18.7 Hz,1H), 3.52 - 3.39 (m, 2H), 3.18 - 2.97 (m, 2H), 2.38 (s, 3H), 2.34 - 2.25 (m,2H), 2.01 - 1.78 (m, 3H), 1.78 - 1.51 (m, 4H), 0.87 (t, J = 7.3 Hz, 3H)。

[0197] P3-2: 1 H NMR (400 MHz, DMSO-d6) δ 7.74 (d, J = 11.1 Hz, 1H), 7.30 (s,1H), 6.52 (s, 1H), 5.43 (s, 2H), 5.37 (d, J = 18.8 Hz, 1H), 5.23 (d, J = 18.7 Hz,1H), 3.51 - 3.40 (m, 2H), 3.16 - 2.98 (m, 2H), 2.38 (s, 3H), 2.35 - 2.24 (m,2H), 1.99 - 1.81 (m, 3H), 1.78 - 1.53 (m, 4H), 0.87 (t, J = 7.3 Hz, 3H)。

[0198] Example 4 Step 1: (9S)-1-(3-(1,3-dioxoisoindoline-2-yl)propyl)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-10,13-dione (4a). A mixture of PPh3 (49.3 mg, 0.188 mmol) and DIAD (38.0 mg, 0.188 mmol) in THF (1 mL) was stirred at room temperature for 5 min, and then added to a mixture of compound P3 (60 mg, 0.125 mmol) and phthalimide (20.3 mg, 0.138 mmol) in THF (1 mL). The resulting yellow mixture was stirred at room temperature for 1 h. The reactants were quenched by adding water (10 mL) and EtOAc (10 mL) 2) 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. The residue was purified by silica gel chromatography (SiO₂ 5 g, EtOAc / petroleum ether = 20%–70%), and the fraction was concentrated under vacuum to give a crude product (130 mg, crude product) as a yellow solid. MS (ESI) m / z: 608.5 [M+H] + .

[0199] Step 2: (9S)-1-(3-aminopropyl)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]inzazo[1,2-b]quinoline-10,13-dione (P4) formate. N2H4·H2O (80%, 20 μL) was added to a solution of 4a (130 mg, crude) in EtOH (2 mL). The mixture was stirred at 60 °C for 60 min. The mixture was acidified to pH 2 with 3N HCl and then purified by preparative HPLC (FA). The fraction was lyophilized to give the target product P4 (12.2 mg, 99.68% purity) as a white solid. MS (ESI) m / z: 478.4 [M+H] + . 1H NMR (400 MHz, DMSO-d6) δ 7.75 (d, J = 10.8 Hz, 1H), 7.31 (s, 1H), 5.44(s, 2H), 5.34 (q, J = 18.8 Hz, 3H), 3.10 (m, 1H), 2.76 (s, 2H), 2.38 (s, 3H), 2.27 (d, J = 12.8 Hz, 2H), 1.91 (m, 6H), 1.62 (m, 3H), 0.87 (t, J = 7.4 Hz, 4H).

[0200] Example 5 Step 1: tert-Butyl(2-iodoethoxy)dimethylsilane (5b). A solution of compound 5a (3000 mg, 17.44 mmol) in CH₂Cl₂ (40 mL) was cooled to 0 °C, and then imidazole (1779 mg, 26.16 mmol) and TBSCl (3139 mg, 20.93 mmol) were added sequentially at 0 °C. The mixture was stirred at 20 °C for 16 h. TLC (SiO₂, petroleum ether) showed the reaction was complete. The reactants were poured into water (40 mL) and stirred with CH₂Cl₂ (50 mL). 3) Extraction. The combined organic layers were dried over Na2SO4 and concentrated to obtain a residue. The residue was purified by silica gel column chromatography (eluent: petroleum ether = 100) to give 5b (3.5 g, 70.2% yield) as a colorless oil.

[0201] Step 2: N-(7-(2-((tert-butyldimethylsilyl)oxy)ethyl)-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (5c). KO was added dropwise to a solution of compound 3a (2350 mg, 10 mmol) in THF (40 mL) at 0 °C. t- Bu (1 M in THF) (22 mL, 22 mmol), and the internal temperature was maintained at 0°C. The mixture was stirred at 0°C for 30 min, and then 5b (3432 mg, 12 mmol) was slowly added. LCMS showed that the reaction was complete. 1 N HCl was added dropwise to the mixture to adjust the pH to 2. The mixture was poured into a saturated NaHCO3 aqueous solution (50 mL), and EtOAc (60 mL) was added. 3) Extraction. The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (elution: petroleum ether / EtOAc = 100 / 0 to 83 / 17) to give 5c (700 mg, 17.8% yield) as a yellow solid. MS (ESI) m / z: 394.2 [M+H] + . 1 H NMR (400 MHz, DMSO- d6 ) δ =7.40 (s, 2H), 6.34 (d, J =12.6, 1H), 6.34 (d, J =12.6, 1H), 4.45 (t, J =5.2, 1H), 4.45 (t, J =5.2, 1H), 3.65 - 3.35 (m, 2H), 3.55 - 3.45 (m, 2H), 2.83 (s, 1H), 2.83 (s, 1H), 2.72 - 2.61 (m, 1H), 2.46 (d, J =5.0, 1H), 2.11 - 1.99 (m, 3H),1.97 (d, J =1.0, 3H), 1.72 - 1.60 (m, 1H), 1.43 (d, J =6.2, 1H).

[0202] Step 3: 8-Amino-6-fluoro-2-(2-hydroxyethyl)-5-methyl-3,4-dihydronaphthyl-1(2H)-one (5d). 2 N HCl (3 mL) was added to a solution of compound 5c (700 mg, 1.78 mmol) in MeOH (3 mL). The mixture was stirred at 60 °C for 16 h. LCMS showed the reaction was complete. The mixture was poured into a saturated NaHCO3 aqueous solution (20 mL) and treated with EtOAc (30 mL). 3) Extraction. The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (elution: petroleum ether / EtOAc = 100 / 0 to 76 / 24) to give 5d (200 mg, 47% yield) as a grayish-white solid. MS (ESI) m / z: 238.2 [M+H] + .

[0203] Step 4: (9S)-9-ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethyl)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-10,13-dione (P5). 3e (110 mg, 0.42 mmol) and PPTS (16 mg, 0.06 mmol) were added to a solution of compound 5d (100 mg, 0.42 mmol) in toluene (6 mL) and o-cresol (0.35 mL). The mixture was refluxed at 140 °C for 5 h. LCMS showed the reaction was complete. The solvent was evaporated, and the residue was purified by silica gel column chromatography (elution buffer: CH2Cl2 / MeOH = 100 / 0 to 92 / 8) to give P5 (80 mg, 41% yield) as an orange solid. MS (ESI) m / z: 465.2 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ = 7.74 (d, J =11.0, 1H), 7.31 (s, 1H), 6.53 (d, J =1.9, 1H), 5.44 (s, 2H),5.30 (s, 2H), 4.77 (t, J =4.8, 1H), 3.69 - 3.56 (m, 4H), 3.10 (t, J =18.6, 2H),2.38 (s, 3H), 2.29 (d, J =9.7, 1H), 1.99 - 1.81 (m, 3H), 1.73 (d, J =5.4, 2H),0.93 - 0.81 (m, 3H).

[0204] P5 (22 mg) was separated by SFC (mobile phase A: CO2, mobile phase B: MeOH, gradient: 5%-40% B for 5 min, 40% for 2 min; flow rate: 2 mL / min; column: Daicel_ChiralPAK-IBN_100x3.0 mm_3 μm; column temperature: 35℃) to obtain P5-1 (6 mg, peak 1, retention time = 4.806 min) and P5-2 (8.8 mg, peak 2, retention time = 5.363 min), which are isomers at the ethanol substituents.

[0205] Example 6 Step 1: 2-((tert-butyldiphenylsilyl)oxy)ethyl carbonate (4-nitrophenyl) ester (6b). Add DIEA (176 μL, 1.0 mmol) and DMAP (4.1 mg, 0.033 mmol) to a solution of 6a (100 mg, 0.33 mmol) and bis(4-nitrophenyl) carbonate (123 mg, 0.40 mmol) in anhydrous CH2Cl2 (2 mL), and stir overnight at room temperature. Pour the solution into 1 N HCl (2 mL), and stir with CH2Cl2 (2 mL). 3) Extraction. The organic phase was concentrated and purified by rapid column chromatography (silica gel, petroleum ether / ethyl acetate = 5:1) to give compound 6b (148 mg, 95.5% yield) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 8.27 (d, J = 9.1 Hz, 2H), 7.69 (d, J = 6.5Hz, 4H), 7.50 - 7.31 (m, 8H), 4.47 - 4.36 (m, 2H), 3.99 - 3.89 (m, 2H), 1.07 (s, 9H).

[0206] Step 2: 2-((tert-butyldiphenylsilyl)oxy)ethyl carbamate (6c) was added to a solution of ethanotecan mesylate (50 mg, 0.094 mmol), 6b (53 mg, 0.11 mmol), and HOBt (1.3 mg, 0.009 mmol) in anhydrous DMF (1 mL), and stirred overnight at room temperature. The solution was poured into saturated NH4Cl (5 mL) and thiocarbamate (5 mL). 3) Extraction. The organic phase was concentrated and purified by rapid column chromatography (silica gel, petroleum ether / ethyl acetate = 1:3) to give compound 6c (68 mg, 94.9% yield) as a yellow solid. MS (ESI) m / z: 762.4 [M+H] + . 1H NMR (400MHz, CDCl3) δ 7.70 - 7.60 (m, 5H), 7.59 - 7.52 (m, 1H), 7.36 - 7.27 (m, 7H), 5.68 (d, J = 16.2 Hz, 1H), 5.33 (d, J = 16.3 Hz, 1H), 5.25 - 5.15 (m, 1H), 5.09 -4.96 (m, 2H), 4.56 - 4.44 (m, 1H), 4.33 - 4.21 (m, 1H), 4.04 - 3.89 (m, 2H), 3.70 (s, 1H), 3.17 - 2.99 (m, 2H), 2.47 - 2.34 (m, 4H), 2.19 - 2.07 (m, 1H), 2.05 - 1.88 (m, 2H), 1.08 (t, J = 7.3 Hz, 3H), 1.04 (s, 9H).

[0207] Step 3: 2-Hydroxyethyl carbamate (P6) of ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)carbamate (P6). At 0°C, add 1 M TBAF in THF (102 μL, 0.102 mmol) to a solution of P6 (65 mg, 0.085 mmol) in anhydrous THF (2 mL) and stir for 40 min at room temperature. Pour the solution into saturated NH4Cl (5 mL) and add CH2Cl2 / MeOH (5 / 1, 6 mL). 3) Extraction. The organic phase was concentrated and purified by rapid column chromatography (silica gel, CH2Cl2 / MeOH = 10:1) to give compound P6 as a pale yellow solid (39 mg, 87.3% yield). MS (ESI) m / z: 524.4 [M+H] + . 1 H NMR (400 MHz, DMSO- d 6) δ 7.98 (d, J = 8.8 Hz, 1H), 7.77 (d, J=10.9 Hz, 1H), 7.31 (s, 1H), 6.52 (s, 1H), 5.43 (s, 2H), 5.33 - 5.17 (m, 3H), 4.78 (t, J = 5.4 Hz, 1H), 4.19 - 4.01 (m, 2H), 3.68 - 3.57 (m, 2H), 3.30 - 3.20(m, 1H), 3.17 - 3.06 (m, 1H), 2.38 (s, 3H), 2.26 - 2.07 (m, 2H), 1.95 - 1.78(m, 2H), 0.87 (t, J = 7.4 Hz, 3H).

[0208] Example 7 Step 1: N-(benzo[d][1,3]dioxacyclopenten-5-yl)acetamide (7b). Ac₂O (27.85 g, 273 mmol) and Et₃N (36.76 g, 364 mmol) were added to a solution of 7a (25.00 g, 182 mmol) in CH₂Cl₂ (200 mL) at 0 °C. The reaction mixture was allowed to react at 20 °C for 3 h. LC-MS showed the reaction was complete. The reaction mixture was concentrated to 50 mL. EtOAc (200 mL) was added to the residue, followed by saturated NaHCO₃ (200 mL). 3) Washing. The organic layer was dried over Na₂SO₄ and filtered. The filtrate was concentrated under vacuum, and the residue was used directly in the next step without further post-treatment or purification (28.25 g, crude product). MS (ESI) m / z: 180.1 [M+H] + Step 2: N-(6-bromobenzo[d][1,3]dioxacyclopenten-5-yl)acetamide (7c). A solution of 7b (28.25 g, crude) and sodium acetate (15.4 g, 188.3 mmol) in acetic acid (100 mL) was heated to 60 °C, and then a mixture of bromine (30.1 g, 188.3 mmol) and acetic acid (60 mL) was added dropwise to the reaction solution. The reaction temperature was raised to 80 °C and stirred at 80 °C for 2 h. LCMS showed that the reaction was complete, and the reaction solution was poured into ice water, producing a yellow solid. The solid was filtered and washed three times with water to obtain the crude product, which was recrystallized from EtOH to give 7c (17.1 g, 42% yield). MS (ESI) m / z: 258.1 / 260.1 [M+H]+ . 1H NMR (400 MHz, DMSO) δ 9.36 (s, 1H), 7.22 (s,1H), 7.08 (d, J = 3.7 Hz, 1H), 6.07 (s, 2H), 2.02 (s, 3H).

[0209] Step 3: N-(6-(1-hydroxycyclobutyl)benzo[d][1,3]dioxanepenten-5-yl)acetamide (7d). A solution of compound 7c (8.5 g, 33.01 mmol) in THF (200 mL) was cooled to -90 °C and n-BuLi (15.84 mL, 39.60 mmol) was added to the mixture under N2 protection for 2 h. The internal temperature was maintained below -85 °C. The mixture was stirred at -85 °C for 20 min. A solution of cyclobutanone (2.7 g, 39.60 mmol) in THF (50 mL) was added dropwise to the mixture. The mixture was stirred at -85 °C for 30 min and then heated to room temperature for 1 h. The mixture was quenched with saturated NH4Cl solution (200 mL) and treated with EtOAc (150 mL). 3) Extraction. The combined organic layers were dried over Na₂SO₄. After filtration and evaporation, the extract was further processed using MTBE (5 mL). 3) The residue was washed and recrystallized from EtOH to give 7d (3.1 g, 37.5% yield). MS (ESI) m / z: 250.1 [M+H] + .

[0210] Step 4: N-(6-oxo-6,7,8,9-tetrahydronaphtho[1,2-d][1,3]dioxacyclopenten-5-yl)acetamide (7e). AgNO3 (12.4 mL, 6.22 mmol) and K2S2O8 (8.4 g, 31.1 mmol) were added to a solution of compound 7d (3.1 g, 12.44 mmol) in CH2Cl2 (20 mL) and H2O (20 mL). The mixture was stirred at 20 °C for 16 h. The mixture was filtered through diatomaceous earth, and the filtration residue was treated with CH2Cl2:MeOH = 1:1 (50 mL). 3) Washing. Pour the filtrate into water and wash with CH2Cl2 (300 mL). 3) Extraction. The combined organic layers were dried over Na₂SO₄. After filtration and evaporation, the residue was purified by silica gel column chromatography (eluent: petroleum ether / CH₂Cl₂ = 100 / 0 to 0 / 100) to give 7e (1.9 g, 61.8% yield) as a pale yellow solid. MS (ESI) m / z: 248.1 [M+H] + Step 5: (Z)-N-(7-(hydroxyimino)-6-oxo-6,7,8,9-tetrahydronaphtho[1,2-d][1,3]dioxacyclopenten-5-yl)acetamide (7f). t-BuOK (1 M in THF, 9.28 mL, 9.28 mmol) was added dropwise to a solution of compound 7e (1.9 g, 7.69 mmol) in THF (20 mL) and t-BuOH (5 mL) at 0 °C. The atmosphere was purged with N2, and the mixture was cooled to 0 °C using an ice-water bath. After 5 min, amyl nitrite (1.09 g, 9.28 mmol) was added to the stirred mixture. LCMS showed the reaction was complete, and the reaction mixture was warmed to room temperature. 1 N HCl was added to adjust the pH to 1. The reaction was then carried out using CH2Cl2: THF (2:1, v / v, 50 mL). 3) Extraction of the aqueous layer. The combined organic layers were washed with brine (100 mL) and dried over Na₂SO₄. After filtration and evaporation, the residue was mixed with MTBE (20 mL). 2) Grind together to obtain 7f (1.5 g, 70.6% yield). MS (ESI) m / z: 277.1 [M+H] + Step 6: N,N'-(6-oxo-6,7,8,9-tetrahydronaphtho[1,2-d][1,3]dioxacyclopenten-5,7-diyl)diacetamide (7 g). PtO2 (150 mg) was added to a solution of compound 7f (1.5 g, 5.43 mmol) in Ac2O (5 mL). The mixture was stirred at 20 °C for 16 h under H2 (15 psi). LCMS showed the reaction was complete. The mixture was filtered through a diatomaceous earth mat. The filtrate was diluted with EtOAc (100 mL) and saturated with NaHCO3 (100 mL). 3) Washing. The organic layer was dried over Na₂SO₄. After filtration and evaporation, the residue (7 g, 1.3 g, crude product) was used in the next step without further processing or purification. MS (ESI) m / z: 305.2 [M+H] + Step 7: N-(5-amino-6-oxo-6,7,8,9-tetrahydronaphtho[1,2-d][1,3]dioxacyclopenten-7-yl)acetamide (7h). HCl (2 N, 10 mL) was added to a solution of 7 g (1.3 g, crude) of the compound in MeOH (10 mL). The mixture was stirred at 60 °C for 3 h. The mixture was cooled to room temperature, and the pH was adjusted to 8 using saturated NaHCO3. EtOAc (50 mL) was then added. 3) Extract the mixture. Dry and concentrate the organic layer. Purify the residue by silica gel column chromatography (elution: CH₂Cl₂MeOH = 100 / 0 to 90 / 10) to give the compound as a grayish-white solid for 7 h (670 mg, 59.8% yield). MS (ESI) m / z: 263.1 [M+H] + Step 8: N-((10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)acetamide (7i). Add 3e (479 mg, 1.82 mmol) and PPTS (38 mg, 0.15 mmol) to a solution of compound 7h (400 mg, 1.52 mmol) in toluene (40 mL) and o-cresol (0.5 mL). Reflux the mixture at 140 °C for 24 h. LCMS showed the reaction was complete, and a black solid precipitated. After filtration, rinse with acetone (10 mL). 3) The filter cake was washed to obtain 7i (580 mg, crude product), a dark brown solid, which was used in the next step without further processing or purification. MS (ESI) m / z: 490.3 [M+H] + Step 9: (1S,10S)-1-amino-10-ethyl-10-hydroxy-1,2,3,10,13,16-hexahydro-11H,14H-benzo[de][1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-11,14-dione (P7). A solution of compound 7i (580 mg, crude) in MsOH (10 mL) and H2O (10 mL) was refluxed at 110 °C for 5 h. LCMS showed the reaction was complete. The mixture was filtered and passed by preparative HPLC (method: column: XBridge Prep C18OBD 5 μm 19). Purification was performed using a mobile phase of 150 mL / min (A-water (0.1% TFA):B-acetonitrile; flow rate: 20 mL / min). The fractions were lyophilized to give compound P7 as a yellow solid (110 mg, 20.8% yield). MS (ESI) m / z: 448.2 [M+H] + Retention time (0.82 min).

[0211] Example 8 Step 1: N-((1S,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)-3-hydroxy-2,2-dimethylpropionamide (P8). Compound P8 (1.8 mg, 17.9% yield) was synthesized according to the synthetic procedure of Step 1 in Example 1. MS (ESI) m / z: 548.4 [M+ H] + .

[0212] Example 9 Step 1: N-((1S,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)-2-fluoro-3-hydroxy-2-methylpropionamide (P9). HATU (25 mg, 0.066 mmol) and DIPEA (0.02 mL, 0.11 mmol) were added to solutions of 2a (17 mg, 0.14 mmol) and P7 (30 mg, 0.055 mmol), and stirred for 10 min at room temperature. Preparative HPLC was performed (method: column XBridge Prep C18 OBD 5 μm 19). The solution was purified using a mobile phase of 150 mm; A-water (0.1% TFA): B-acetonitrile; flow rate: 20 mL / min, and the fractions were lyophilized to give P9 as a yellow solid (14.5 mg, 47.6% yield). MS (ESI) m / z: 552.5 [M+H] + .

[0213] Example 10 Step 1: 2-((tert-butyldimethylsilyl)oxy)ethyl carbonate (4-nitrophenyl) ester (10b). NPC (2092 mg, 6.81 mmol) and DIPEA (1.5 mL, 8.51 mmol) were added to a solution of 10a (1000 mg, 5.67 mmol) in anhydrous DCM (10 mL) at 0 °C, and the solution was stirred at room temperature for 1.5 h. The solution was quenched with 1 N HCl (5 mL) and then quenched with CH2Cl2 (50 mL). 3) Extraction and purification by silica gel column chromatography (petroleum ether / ethyl acetate = 85 / 15) yielded 10b (1890 mg, 97.6% yield) as a yellow oil. MS (ESI) m / z: 342.4 [M+H] + Step 2: 2-((1S,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)carbamate (10c). HOBt (10.2 mg, 0.075 mmol) and DIPEA (0.13 mL, 0.75 mmol) were added to a solution of 10b (103 mg, 0.3 mmol) and P7 (135 mg, 0.25 mmol) in anhydrous DMF (2 mL), and the mixture was stirred at room temperature for 5 h. The resulting solution was concentrated and purified by silica gel column chromatography (CH₂Cl₂ / ethyl acetate = 50 / 50) to give 10c (148 mg, 91.7% yield) as a white solid. MS (ESI) m / z: 650.4 [M+H] + .

[0214] Step 3: 2-Hydroxyethyl carbamate (P10) of ((1S,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)carbamate (P10). Add 2N HCl (2 mL) to a solution of 10c (140 mg, 0.22 mmol) in MeOH (2 mL) and stir at room temperature for 2 h. Then, use CH2Cl2 (20 mL) to... 3) Extraction solution. The organic phase was concentrated and purified by silica gel column chromatography (MeOH / CH2Cl2 = 90 / 10) to give P10 as a yellow solid (60 mg, 52% yield). MS (ESI) m / z: 536.4 [M+H] + .

[0215] Example 11 Step 1: (9S)-9-ethyl-5-fluoro-9-hydroxy-1-(((2-hydroxyethyl)(methyl)amino)methyl)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-10,13-dione (P11). 37% formaldehyde solution (2.8 μL, 0.04 mmol) was added to a solution of 11a (10 mg, 0.02 mmol) in MeOH (1 mL). The solution was then cooled to 0°C, and NaBH3CN (2 mg, 0.03 mmol) was added in a single batch. The solution was then heated to room temperature and stirred for 2 h. The mixture was then quenched with H2O and analyzed by preparative HPLC (FA) (Method: Column: XBridge Prep C18 OBD 5 μm 19). Purification was performed using a mobile phase of 150 mL / min (A-water (0.1% FA):B-acetonitrile; flow rate: 20 mL / min). The fractions were lyophilized to give P11 as a white solid (5.5 mg, 54.2% yield). MS (ESI) m / z: 508.5 [M+H] + .

[0216] Example 12 Step 1: 2-(3-iodopropoxy)tetrahydro-2 H -Pyran (12b). NaI (4.0 g, 27 mmol) was added to a solution of compound 12a (2.0 g, 9.01 mmol) in acetone (20 mL). The mixture was stirred at 60 °C for 3 h. The mixture was diluted with hexane (40 mL) and washed with water (40 mL) and brine (40 mL). The combined organic layers were dried over Na₂SO₄ and concentrated to give 12b (1.3 g, 54% yield) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 4.61 (dd, J= 4.4, 2.8 Hz, 1H), 3.91 - 3.83 (m, 1H), 3.83 - 3.77 (m, 1H), 3.56 - 3.49 (m,1H), 3.45 (dt, J = 10.0, 5.9 Hz, 1H), 3.30 (td, J = 6.8, 1.0 Hz, 2H), 2.10 (ddd, J = 12.7, 6.8, 5.9 Hz, 2H), 1.90 - 1.63 (m, 3H), 1.61 - 1.48 (m, 5H).

[0217] Step 2: N -(6-oxo-7-(3-((tetrahydro-2H-pyran-2-yl)oxy)propyl)-6,7,8,9-tetrahydronaphtho[1,2-] d [1,3]dioxacyclopenten-5-yl)acetamide (12c). Compound 7e (100 mg, 3.52 mmol) was added dropwise to a solution of THF (5 mL) at -40 °C under N2. t -BuOK (1.2 mL, 1.212 mmol, 1 M in THF). The mixture was stirred at -40 °C for 30 min under N2. Then, compound 12b (164 mg, 0.606 mmol, dissolved in 0.2 mL THF) was added dropwise to the mixture. The mixture was stirred for 16 h and gradually heated to room temperature under N2. The mixture was quenched with saturated NH4Cl (3 mL) and treated with EtOAc (3 mL). 3) Extraction. The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated to obtain a residue. The residue was purified by silica gel column chromatography (petroleum ether / EtOAc = 80:20) to give compound 12c as a yellow solid (20 mg, 12.7% yield). MS (ESI) m / z: 248.1 [M+H] + . 1H NMR (400 MHz, CDCl3) δ 12.44 (s, 1H), 8.26 (s, 1H), 6.01 - 5.98 (m, 2H), 4.59 - 4.53 (m,1H), 3.90 - 3.72 (m, 2H), 3.53 - 3.36 (m, 2H), 3.00 - 2.88 (m, 1H), 2.79 -2.67 (m, 1H), 2.55 - 2.44 (m, 1H), 2.19 (s, 3H), 2.17 - 2.09 (m, 1H), 2.06 -1.86 (m, 2H), 1.86 - 1.65 (m, 6H), 1.60 - 1.54 (m, 2H).

[0218] Step 3: 5-Amino-7-(3-hydroxypropyl)-8,9-dihydronaphtho[1,2- d [1,3]dioxacyclopentene-6(7) H )-Ketone (12d). 2 N HCl (5 mL) was added to a solution of compound 12c (20 mg, 0.05 mmol) in MeOH (5 mL). The mixture was stirred at 60 °C for 3 h. The mixture was concentrated under vacuum to remove MeOH. The mixture was then adjusted to pH 8 using saturated Na₂CO₃ and treated with DCM (5 mL). 3) Extraction. The combined organic phases were concentrated under vacuum to obtain a residue, which was purified by silica gel column chromatography (elution: petroleum ether / EtOAc = 0 / 100) to give 12d (10 mg, 74% yield) as a yellow oil. MS (ESI) m / z: 264.2 [M+H] + .

[0219] Step 4: (1) R 10 S )-10-ethyl-10-hydroxy-1-(3-hydroxypropyl)-1,2,3,10,13,16-hexahydro-11 H ,14 H -Benz[de][1,3]dioxacyclopenten[4,5-] g ]pyrano[3',4':6,7]inzazo[1,2- b Quinoline-11,14-dione (P12-1) and (1 S 10 S )-10-ethyl-10-hydroxy-1-(3-hydroxypropyl)-1,2,3,10,13,16-hexahydro-11 H ,14 H-Benz[de][1,3]dioxacyclopenten[4,5-] g ]pyrano[3',4':6,7]inzazo[1,2- b Quinoline-11,14-dione (P12-2). A solution of compound 12d (80 mg, 0.304 mmol) in toluene (14 mL) and o-cresol (2 mL) was added with 3e (88 mg, 0.334 mmol) and PPTS (23 mg, 0.091 mmol). The mixture was refluxed at 140 °C for 16 h. The mixture was concentrated under vacuum to remove toluene and purified to crude product (a mixture of P12-1 and P12-2) by silica gel column chromatography (elution: DCM / MeOH = 90 / 10), followed by preparative HPLC (FA) (method: column: XBridge Prep C18 OBD 5 μm 19). Purification was performed using a mobile phase of 150 mm (A-water (0.1% formic acid):B-acetonitrile; flow rate: 20 mL / min). The fractions were lyophilized to give P12-1 (15 mg, 710% yield) and P12-2 (20 mg, 13% yield) as white solids.

[0220] Compound P12-1. MS (ESI) m / z: 491.5 [M+H] + . 1 H NMR (400 MHz, DMSO- d 6) δ 7.37(s, 1H), 7.23 (s, 1H), 6.48 (s, 1H), 6.27 (d, J = 2.9 Hz, 2H), 5.42 (s, 2H), 5.31 (d, J = 18.9 Hz, 1H), 5.19 (d, J = 18.8 Hz, 1H), 4.42 (t, J = 5.1 Hz, 1H),3.48 - 3.42 (m, 2H), 3.01 - 2.91 (m, 2H), 2.22 (d, J = 13.4 Hz, 1H), 1.93 -1.75 (m, 4H), 1.73 - 1.63 (m, 2H), 1.61 - 1.54 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H).

[0221] Compound P12-2. MS (ESI) m / z: 491.5 [M+H] + . 1 H NMR (400 MHz, DMSO- d 6) δ 7.37(s, 1H), 7.23 (s, 1H), 6.49 (s, 1H), 6.27 (s, 2H), 5.42 (s, 2H), 5.31 (d, J =18.9 Hz, 1H), 5.20 (d, J = 18.9 Hz, 1H), 4.43 (t, J = 5.2 Hz, 1H), 3.47 - 3.43(m, 2H), 3.02 - 2.86 (m, 2H), 2.22 (d, J = 13.3 Hz, 1H), 1.94 - 1.78 (m, 3H), 1.73 - 1.63 (m, 2H), 1.63 - 1.53 (m, 3H), 0.87 (t, J = 7.3 Hz, 3H).

[0222] Example 1-1 Step 1: 1-(9H-fluorene-9-yl)-11,11-dimethyl-3,6-dioxo-2,9-dioxa-4,7-diazadodecane-12-acid benzyl ester (1-1c). Scandium trifluoromethanesulfonate (2.0 g, 2.87 mmol) was added to a mixture of 1-1a (1000 mg, 2.71 mmol), 1-1b (1580 mg, 813 mmol), and dried 4 Å molecular sieve (3.0 g) in anhydrous THF (40 mL), and stirred overnight at room temperature under N2 atmosphere. The solution was filtered through diatomaceous earth, diluted with EtOAc (200 mL), and then diluted with saturated NaHCO3 (50 mL). 3) Washing. The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated. The residue was purified by silica gel column chromatography (eluent: petroleum ether / EtOAc = 100 / 0 to 50 / 50) to give compound 1-1c as a white solid (950 mg, 67.9% yield). MS (ESI) m / z: 539.4 [M+Na] + .

[0223] Step 2: Benzyl 3-((2-aminoacetamido)methoxy)-2,2-dimethylpropionate (1-1d). Acetyl-L-cysteine ​​(450 mg, 2.76 mmol) was added to a solution of compound 1-1c (950 mg, 1.84 mmol) in DMF (6 mL). Then Et2NH (2.69 g, 36.8 mmol) was added. The mixture was stirred at room temperature for 30 min. The mixture was concentrated under high vacuum and analyzed by preparative HPLC (method: column: XBridge Prep C18 OBD 5 μm 19). Purification was performed using a mobile phase of 150 mL / min (A-water (neutral conditions): B-acetonitrile; flow rate: 20 mL / min). The fractions were lyophilized to give compound 1-1d (340 mg, 62.6%) as a colorless oil. MS (ESI) m / z: 317.3 [M+Na]+.

[0224] Step 3: (2R,3R,4S,5S,6R)-triacetic acid 2-((S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-(benzyloxy)-3-oxopropoxy)-6-(acetoxymethyl)tetrahydro-2H-pyran-3,4,5-trimethyl ester (1-1 g). Add 1-1f (3350 mg, 8.14 mmol) and 4A molecular sieve (4.0 g) to a solution of compound 1-1e (2.0 g, 4.79 mmol) in CH2Cl2 (30 mL), and stir the mixture at room temperature for 30 min under N2 atmosphere. Then add AgOTf (1.8 g, 7.19 mmol) to the mixture. Stir the mixture at room temperature for 16 h. Filter the mixture, dilute the filtrate with EtOAc (300 mL), and stir with saturated NaHCO3 (100 mL). 3) and saline (100 mL) 3) Wash the organic layer, dry it with anhydrous Na₂SO₄, filter and concentrate. Purify the residue by silica gel column chromatography (elution: petroleum ether / EtOAc = 100 / 0 to 40 / 60) to give 1–1 g (1300 mg, 36.3% yield). MS (ESI) m / z: 770.4 [M+Na] + .

[0225] Step 4: (2R,3S,4S,5R,6R)-triacetic acid 2-(acetoxymethyl)-6-((S)-2-amino-3-(benzyloxy)-3-oxopropoxy)tetrahydro-2H-pyran-3,4,5-trimethyl ester (1-1h). Et2NH (2.5 g, 34.77 mmol) was added to a solution of 1-1 g (1.3 g, 1.74 mmol) of the compound in DMF (10 mL). The mixture was stirred at room temperature for 30 min. The mixture was concentrated under high vacuum and reacted with toluene (5 mL). 3) Co-evaporation yielded a brown solid of the compound for 1–1 h (914 mg, theoretical yield). MS (ESI) m / z: 526.4 [M+H] + .

[0226] Step 5: (2R,3R,4S,5S,6R)-triacetic acid 2-((S)-2-((S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-3-(benzyloxy)-3-oxopropoxy)-6-(acetoxymethyl)tetrahydro-2H-pyran-3,4,5-triyl ester (1-1j). Compound 1-1h (708 mg, 2.086 mmol), HATU (992 mg, 2.61 mmol), and DIEA (450 mg, 3.48 mmol) were added to a solution of compound 1-1h (914 mg, 1.739 mmol) in DMF (8 mL). The mixture was stirred at room temperature for 30 min. The mixture was concentrated and purified by rapid column chromatography (eluting with petroleum ether / EtOAc = 100 / 0 to 50 / 50). Compound 1-1j (1210 mg, 82.3%) was obtained as a white solid. MS (ESI) m / z: 869.5 [M+Na] + .

[0227] Step 6: N-((((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine)-O-((2R,3R,4S,5S,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)-L-serine (1-1k). Pd / C (10%, 242 mg) was added to a solution of compound 1-1j (1210 mg, 1.43 mmol) in MeOH (10 mL). The mixture was stirred at room temperature under H2 for 2 h. The mixture was filtered and concentrated to give crude product 1-1k (784 mg, crude product) as a white solid. MS (ESI) m / z: 779.5 [M+Na] + .

[0228] Step 7: (2R,3R,4S,5S,6R)-triacetic acid 2-(((S)-13-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-4,4-dimethyl-3,9,12-trioxo-1-phenyl-2,6-dioxa-8,11-diazatetradecane-14-yl)oxy)-6-(acetoxymethyl)tetrahydro-2H-pyran-3,4,5-triyl ester (1-1l). Compound 1-1d (117 mg, 0.396 mmol), HATU (188 mg, 0.496 mmol), and DIEA (85 mg, 0.661 mmol) were added to a solution of compound 1-1k (250 mg, 0.33 mmol) in DMF (4 mL). The mixture was stirred at room temperature for 30 min. The mixture was concentrated and analyzed by preparative HPLC (FA) (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). The fractions were lyophilized to give compound 1-1L (248 mg, 84.8% yield) as a grayish-white solid. MS (ESI) m / z: 1056.7 [M+Na] + .

[0229] Step 8: (5S,8S)-1-(9H-fluorene-9-yl)-5-isopropyl-17,17-dimethyl-3,6,9,12-tetraoxo-8-((((2R,3R,4S,5S,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)methyl)-2,15-dioxa-4,7,10,13-tetraazaoctadecane-18-acid (1-1m). Compound 1-1m (264 mg, crude product) was synthesized according to the synthetic procedure of Step 3 in Example 1-1.

[0230] Step 9: (2R,3R,4S,5S,6R)-triacetic acid 2-((S)-2-((S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-3-((2-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,1) 3,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-2-oxoethyl)amino)-3-oxopropoxy)-6-(acetoxymethyl)tetrahydro-2H-pyran-3,4,5-triyl ester (1-1n). Exatecan mesylate (156 mg, 0.29 mmol), HATU (160 mg, 0.42 mmol), and DIEA (109 mg, 0.84 mmol) were added to a solution of compound 1-1m (264 mg, 0.28 mmol) in DMF (6 mL). The mixture was stirred at room temperature for 30 min. Preparative HPLC (FA) was performed using a column of XBridge Prep C18 OBD 5 μm 19 μm. The mobile phase was 150 mm; flow rate: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min. The purified mixture was lyophilized to give compound 1-1n as a grayish-white solid (248 mg, 69.3% yield). MS (ESI) m / z: 1260.7 [M+H] + .

[0231] Step 10: (2R,3S,4S,5R,6R)-triacetic acid 2-(acetoxymethyl)-6-((S)-2-((S)-2-amino-3-methylbutamido)-3-((2-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-2-oxoethyl)amino)-3-oxopropoxy)tetrahydro-2H-pyran-3,4,5-triyl ester (1-1o). Et₂NH (133 mg, 1.82 mmol) was added to a solution of compound 1-1n (248 mg, 0.18 mmol) in DMF (6 mL). The mixture was stirred at room temperature for 30 min. The mixture was concentrated under high vacuum to give compound 1-1o (207 mg, crude product) as a grayish-white solid, which was used directly without further purification. MS (ESI) m / z: 1138.7 [M+H] + .

[0232] Step 11: (S)-2-amino-N-((S)-1-((2-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-2-oxoethyl)amino)-1-oxo-3-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)prop-2-yl)-3-methylbutyramide carboxylate (1-1p). K₂CO₃ (50 mg, 0.36 mmol) was added to a solution of compound 1-1o (207 mg, crude) in MeOH (6 mL). The mixture was stirred at room temperature for 60 min. Preparative HPLC (FA) was performed (method: column: XBridge Prep C18 OBD 5 μm 19). The mobile phase was 150 mm; flow rate: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min. The purified mixture was lyophilized to give compound 1-1p (105 mg, 56.8% yield) as a white solid. MS (ESI) m / z: 970.7 [M+H] + .

[0233] Step 12: 6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-N-((11S,14S)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo [1,2-b]quinolin-1-yl)amino)-2,2,15-trimethyl-1,7,10,13-tetraoxo-11-((((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)methyl)-4-oxa-6,9,12-triazahexadecane-14-yl)hexamethylene (1-1). Compound 1-1q (13 mg, 0.062 mmol), HATU (24 mg, 0.062 mmol), and DIEA (11 mg, 0.082 mmol) were added to a solution of compound 1-1p (40 mg, 0.041 mmol) in DMF (2 mL). The mixture was stirred at room temperature for 30 min. Preparative HPLC (FA) method (Column: XBridge Prep C18 OBD 5 μm 19) The mobile phase was 150 mm; flow rate: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min. The purified mixture was lyophilized to give compound 1-1 as a white solid (18.5 mg, 33.7% yield). MS (ESI) m / z: 1185.8 [M+Na] + .

[0234] Examples 1-2 Step 1: N-((((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine)-O-((2R,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)-L-serine (1-2b). Wet Pd / C (400 mg, 10% purity) was added to a mixture of 1-2a (4.1 g, 4.92 mmol, purchased from MCE) in MeOH (50 mL), THF (10 mL), and DCM (20 mL). The black suspension was purged three times with an H2 gasbag and then stirred at room temperature for 1 h. The black suspension was filtered through a diatomaceous earth pad and washed with MeOH (200 mL). The combined organic layers were concentrated under vacuum to give 1-2b (3650 mg, 99.8% yield) as a grayish-white solid. MS (ESI) m / z: 743.6 [M+H]+ .

[0235] Step 2: (2R,3R,4S,5S,6S)-triacetic acid 2-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-3-((2-(benzyloxy)-2-oxoethyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-trimethyl ester (1-2d). HATU (1.87 g, 4.92 mmol) and DIPEA (1.59 g, 12.29 mmol) were added to solutions of 1-2b (3.65 g, 4.92 mmol) and 1-2c (1.66 g, 4.92 mmol) in DMF (50 mL). The mixture was stirred at room temperature for 30 min. The mixture was purified by rapid column chromatography (MeOH / DCM = 0–10%), and the fractions were concentrated under vacuum to give 1–2d (3.8 g, 86.9% yield) as a grayish-white, foamy solid. MS (ESI) m / z: 890.7 [M+H] + .

[0236] Step 3: N-((((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine)-O-((2R,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)-L-serylglycine (1-2e). Wet Pd / C (400 mg, 10% purity) was added to a mixture of 1-2e (3.8 g, 4.27 mmol) in MeOH (150 mL) and DCM (50 mL). The black suspension was purged three times with an H2 gasbag and then stirred at room temperature for 40 min. The black suspension was filtered through a diatomaceous earth pad and washed with MeOH (150 mL). The organic layers were combined and concentrated under vacuum to give 1-2e (3.3 g, 96.6% yield) as a grayish-white solid. MS (ESI) m / z: 800.7 [M+H] + .

[0237] Step 4: (2R,3R,4S,5S,6S)-triacetic acid 2-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-3-((acetoxymethyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-trimethyl ester (1-2f). Add Pb(OAc)4 (2.74 g, 6.19 mmol), Cu(OAc)2 (74.9 mg, 0.41 mmol), and HOAc (247.8 mg, 4.13 mmol) to a solution of 1-2e (3.3 g, 4.13 mmol) in DMF (30 mL). Pb(OAc)4 (2.74 g, 6.19 mmol), Cu(OAc)2 (74.9 mg, 0.41 mmol), and HOAc (247.8 mg, 4.13 mmol) to the solution. The resulting dark mixture was purged three times with an N2 gas bag and then stirred at 65 °C for 40 min, and the mixture turned dark blue. Dilute the mixture with EtOAc (300 mL), and with saline (100 mL). 3) The sample was washed, dried over Na₂SO₄, filtered, and concentrated under vacuum to obtain the residue. The residue was purified by rapid column chromatography (MeOH / DCM = 0 / 100~10 / 90), and the fraction was concentrated under vacuum to give 1-2 f (2.8 g, 83.4% yield) as a pale yellow solid. MS (ESI) m / z: 836.6 [M+Na] + .

[0238] Step 5: (2R,3R,4S,5S,6S)-triacetic acid 2-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-3-(((3-(benzyloxy)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (1-2 g). A white suspension mixture of 1-1b (300 mg, 0.37 mmol), 1-2f (154 mg, 0.74 mmol), and 4 Å molecular sieve (200 mg) in anhydrous THF (10 mL) was stirred at room temperature for 10 min. Sc(OTf)3 (217.9 mg, 0.44 mmol) was added, and the resulting yellow suspension was stirred at room temperature for 4 h. The mixture of yellow suspensions was filtered through a diatomaceous earth mat and washed with EtOAc (30 mL). The combined organic layers were washed with saturated NaHCO3 (30 mL) and brine (30 mL), dried over Na2SO4, filtered, and the filtrate was concentrated under vacuum to give a residue. The residue was purified by silica gel column chromatography (MeOH / DCM = 0 / 100–5 / 95), and the fraction was concentrated under vacuum to give 1–2 g (275 mg, 77.5% yield) of a white foamy solid. MS (ESI) m / z: 984.8 [M+Na] + .

[0239] Step 6: (5S,8S)-1-(9H-fluorene-9-yl)-5-isopropyl-14,14-dimethyl-3,6,9-trioxo-8-((((2R,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)methyl)-2,12-dioxa-4,7,10-triazapentadecan-15-acid (1-2 h). Add wet Pd / C (55 mg, 10% purity) to a solution of 1-2 g (275 mg, 0.29 mmol) in MeOH (10 mL). Purge the black suspension three times with an H2 gasbag, then stir at room temperature for 2 h. The mixture was filtered through a syringe tip and washed with MeOH (15 mL), then concentrated under vacuum to give a white, foamy solid (230 mg, crude product) over 1–2 hours. MS (ESI) m / z: 894.6 [M+Na] + .

[0240] Step 7: (2R,3R,4S,5S,6S)-triacetic acid 2-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-3-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (1-2i). Add DIPEA (102 mg, 0.79 mmol) to a mixture of 1–2 h (230 mg, crude), ethatecan mesylate (140 mg, 0.26 mmol), and HATU (100.3 mg, 0.26 mmol) in DMF (5 mL). Stir the resulting brown mixture at room temperature for 1 h. Dilute the mixture with EtOAc (20 mL) and saline (20 mL). 3) Wash, dry with Na2SO4, filter, and concentrate under vacuum to obtain the residue. Purify the residue by rapid column chromatography (MeOH / DCM = 0%–3%), concentrate under vacuum to give 1–2i (325 mg, 95.6% yield) as a grayish-white foamy solid. MS (ESI) m / z: 1289.9 [M+H] + .

[0241] Step 8: (2S,3S,4S,5R,6R)-6-((S)-2-((S)-2-amino-3-methylbutamido)-3-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-2j). Et₂NH (523.2 mg, 5.06 mmol) was added to a solution of 1-2i (325 mg, 0.25 mmol) in DMF (5 mL). The mixture was stirred at room temperature for 20 min. The mixture was then concentrated under vacuum to obtain a crude product. The product was dissolved in MeOH (6 mL), K₂CO₃ (174.7 mg, 1.26 mmol) was added, and the mixture was stirred at room temperature for 10 min. Then, H₂O (2 mL) was added to the mixture, and the mixture was stirred at room temperature for 30 min. The mixture was acidified to pH 3 with saturated KHSO₄ at 0 °C, filtered, and purified by preparative HPLC (0.1% FA). The fractions were lyophilized to give 1-2j (140 mg, 59.7% yield) as a pale yellow solid. MS (ESI) m / z: 927.4 [M+H] + . 1 H NMR (400 MHz, DMSO) δ 9.56 (s, 1H), 8.39 (s, 1H), 8.07 (d, J = 8.4 Hz, 1H), 7.77 (d, J = 11.2 Hz, 1H), 7.31 (s, 1H), 6.52 (s, 1H), 5.54 (dd, J = 13.2, 7.2 Hz, 1H), 5.43 (s, 2H), 5.18 (dd, J = 41.6, 18.8 Hz, 2H),5.09 - 5.02 (m, 1H), 4.96 (s, 1H), 4.62 (dd, J = 10.0, 6.8 Hz, 1H), 4.56 - 4.44(m, 2H), 4.19 (d, J = 7.6 Hz, 1H), 3.82 (dd, J = 10.8, 6.8 Hz, 1H), 3.61 (dd,J =11.6, 6.4 Hz, 2H), 3.17-3.05 (m, 4H), 2.94 (t, J = 8.0 Hz, 1H), 2.39 (s, 3H), 2.11 (dt, J = 21.3, 7.6 Hz, 2H), 2.03 - 1.93 (m, 2H), 1.92 - 1.78 (m, 3H), 1.12(d, J = 8.0 Hz, 6H), 0.87 (dd, J = 13.0, 6.6 Hz, 9H).

[0242] Step 9: 6-(2-(methylthio)pyrimidin-5-yl)hex-5-alkynyl acid (1-2m). CuI (185.7 mg, 0.975 mmol), Pd(PPh3)Cl2 (684.4 mg, 0.975 mmol), and TEA (4.1 mL, 2.960 g, 29.254 mmol) were added to a mixture of 1-2k (2.0 g, 9.751 mmol) and 1-2l (1.31 g, 11.702 mmol) in DMF (30 mL). The mixture was degassed three times under a nitrogen atmosphere and then stirred at 95 °C for 2 h. The mixture was diluted with EtOAc (30 mL) and then with saline (30 mL). 2) The sample was washed, dried over Na₂SO₄, filtered, and concentrated under vacuum to obtain the residue. The residue was purified by rapid column chromatography (MeOH / DCM = 0 / 100 ~ 3 / 97), and the fraction was concentrated under vacuum to give a 1-2 m (1.6 g, approximately 65% ​​yield) yellow solid. MS (ESI) m / z: 237.1 [M+H] + .

[0243] Step 10: 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-alkynyl acid (1-2n). Add m-CPBA (1113.4 mg, 6.452 mmol) to a solution of 1-2m (520 mg, 1.613 mmol) in DCM (10 mL). Stir the resulting yellow suspension at room temperature for 1.5 h. Dilute the mixture with EtOAc (50 mL) and quench by adding saturated Na2S2O3 (30 mL), stirring for 10 min at room temperature, and then add DCM (30 mL). 3) Extraction. The combined organic layers were washed with brine (50 mL), dried over Na₂SO₄, filtered, and concentrated under vacuum to obtain a crude product as a yellow solid. The crude product was purified by preparative HPLC (FA), and the fractions were freeze-dried to give 1–2n (78.0 mg, 18% yield) as a white solid. MS (ESI) m / z: 269.1 [M+H] + . 1 H NMR (400 MHz, DMSO) δ 12.17 (s, 1H), 9.12 (s, 2H), 3.41 (s, 3H), 2.60 (t, J = 7.2 Hz, 2H), 2.41 (t, J = 7.2 Hz, 2H), 1.82 (p, J = 6.8 Hz, 2H).

[0244] Step 11: (2S,3S,4S,5R,6R)-6-((S)-3-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamidamido)butamido)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-2). HATU (8.6 mg, 0.024 mmol) and DIPEA (7 μL, 5.2 mg, 0.04 mmol) were added to solutions of 1-2n (5.2 mg, 0.019 mmol) and 1-2j (15 mg, 0.016 mmol) in DMF (2 mL). The mixture was stirred at room temperature for 30 min. The mixture was purified by preparative HPLC (FA 0.1%), and the fractions were lyophilized to give 1-2 (7.1 mg, 37.3% yield) as a white solid. MS (ESI) m / z: 1177.9 [M+H] + .

[0245] Examples 1-3 Step 1: (2S,3S,4S,5R,6R)-6-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexamido)-3-methylbutamido)-3-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-3). Synthesized 1-3 (55 mg, 52% yield) according to the same procedure as step 12 of Example 1-1. MS (ESI) m / z: 1120.9 [M+H] + .

[0246] Examples 1-4 Steps 1 to 4: (2S,3S,4S,5R,6R)-6-((2S)-2-((S)-2-amino-3-methylbutamido)-3-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)amino)-2-fluoro-2-methyl-3-oxopropoxy)methyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-4e). 1-4e (94 mg, 54.6% yield) was synthesized according to a similar procedure to 1-2j. MS (ESI) m / z: 931.7 [M+H] + .

[0247] Step 5: (2S,3S,4S,5R,6R)-6-((2S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexamido)-3-methylbutamido)-3-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)amino)-2-fluoro-2-methyl-3-oxopropoxy)methyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-4). Synthesized 1-4 (17.2 mg, 53.7% yield) according to the same procedure as step 12 of Example 1-1. MS (ESI) m / z: 1124.9 [M+H] + .

[0248] Examples 1-5 Step 1: (2R,3R,4S,5S,6S)-triacetic acid 2-(((S)-13-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-4,4-dimethyl-3,9,12-trioxo-1-phenyl-2,6-dioxa-8,11-diazatetradecane-14-yl)oxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (1-5a). Compounds 1-1d (58.8 mg, 0.2 mmol), HATU (70.6 mg, 0.496 mmol), and DIEA (49.2 mg, 0.38 mmol) were added to a solution of compounds 1-2b (150 mg, 0.19 mmol) in DMF (4 mL). Stir the mixture at room temperature for 30 min. Dilute the reaction mixture with EtOAc (20 mL) and with saline (30 mL). 3) Washing. The organic layer was dried over Na2SO4. After filtration and concentration, the residue was purified by silica gel column chromatography (eluent: petroleum ether / EtOAc = 100 / 0 to 30 / 70) to give 1-5a as a white solid (140 mg, 72.4% yield). MS (ESI) m / z: 1019.8 [M+H] + .

[0249] Step 2: (5S,8S)-1-(9H-fluorene-9-yl)-5-isopropyl-17,17-dimethyl-3,6,9,12-tetraoxo-8-((((2R,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)methyl)-2,15-dioxa-4,7,10,13-tetraazaoctadecane-18-acid (1-5b). Pd / C (10%, 14 mg) was added to a solution of compound 1-5a (140 mg, 0.14 mmol) in MeOH (10 mL) and THF (5 mL). The mixture was stirred at room temperature under H2 atmosphere for 1 h. The mixture was filtered and concentrated to give crude product 1-5b (125 mg, 92.9% yield) as a white solid. MS (ESI) m / z: 929.4 [M+H] + .

[0250] Step 3: (2R,3R,4S,5S,6S)-triacetic acid 2-((S)-2-((S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-3-((2-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10, 13,15-Hexahydro-1H,12H-Benzopyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-2-oxoethyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-trimethyl ester (1-5c). Exatecan mesylate (79 mg, 0.15 mmol), HATU (54.4 mg, 0.16 mmol), and DIEA (33.54 mg, 0.26 mmol) were added to a solution of compounds 1-5b (125 mg, 0.13 mmol) in DMF (3 mL). The mixture was stirred at room temperature for 30 min. The reaction mixture was diluted with EtOAc (20 mL) and then with saline (30 mL). 3) Washing. The organic layer was dried over Na₂SO₄. After filtration and concentration, the residue was purified by silica gel column chromatography (eluent: petroleum ether / EtOAc = 100 / 0 to 100 / 0) to give a white solid, 1–5c (130 mg, 74.4% yield). MS (ESI) m / z: 1346.5 [M+H] + .

[0251] Step 4: (2R,3R,4S,5S,6S)-triacetic acid 2-((S)-2-((S)-2-amino-3-methylbutamido)-3-((2-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-2-oxoethyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (1-5d). Et₂NH (142 mg, 2.0 mmol) was added to a solution of compound 1-5c (130 mg, 0.10 mmol) in DMF (3 mL). The mixture was stirred at room temperature for 1 h. The mixture was concentrated under high vacuum to give compound 1-5d (110 mg, crude product) as a grayish-white solid, which was used directly without further purification. MS (ESI) m / z: 1124.8 [M+H] + .

[0252] Step 5: (2S,3S,4S,5R,6R)-6-((S)-2-((S)-2-amino-3-methylbutamido)-3-((2-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-2-oxoethyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-5e). Na₂CO₃ (74 mg 0.7 mmol) was added to a solution of compounds 1–5d (110 mg, 0.1 mmol) in MeOH (10 mL) and H₂O (3 mL). The mixture was stirred at room temperature for 2 h. The mixture was acidified with HOAc and the pH was adjusted to 6. After filtration, the solution was analyzed by preparative HPLC (FA) (method: column: XBridge Prep C18 OBD 5 μm 19). The mobile phase was 150 mm; flow rate: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min. The purified mixture was lyophilized to give compound 1-5e as a grayish-white solid (80 mg, 80.2% yield). MS (ESI) m / z: 985.3 [M+H] + .

[0253] Step 6: (2S,3S,4S,5R,6R)-6-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexamido)-3-methylbutamido)-3-((2-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2, 3,9,10,13,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-2-oxoethyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-5). Compounds 1-1q (5.8 mg, 0.028 mmol), HATU (8.45 mg, 0.025 mmol), and DIEA (1.45 mg, 0.05 mmol) were added to a solution of compounds 1-5e (25 mg, 0.025 mmol) in DMF (2 mL). The mixture was stirred at room temperature for 30 min. Preparative HPLC (FA) method (Column: XBridge Prep C18 OBD 5 μm 19) The mobile phase was 150 mm; flow rate: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min. The purified mixture was lyophilized to give compounds 1-5 as grayish-white solids (15 mg, 50.9% yield). MS (ESI) m / z: 1178.2 [M+H] + .

[0254] Examples 1-6 Step 1: Ethyl (S)-3-(3-(((benzyloxy)carbonyl)amino)-2-((tert-butoxycarbonyl)amino)propoxy)propionate (1-6c). Add dropwise 50% wt NaOH aqueous solution (7.2 g, 77.07 mmol) to a mixture of 1-6a (5.0 g, 15.41 mmol) (purchased from WUXI), TBAB (496.9 mg, 1.54 mmol), and 1-6b (14.0 g, 77.07 mmol) in DCM (50 mL). Stir the mixture overnight at room temperature. Dilute the mixture with DCM (100 mL) and then with DCM (50 mL). 2) Extraction. The combined organic layers were rinsed with 50 mL of brine. 2) The sample was washed, dried over Na₂SO₄, filtered, and concentrated under vacuum to obtain the residue. The residue was purified by silica gel column chromatography (petroleum ether / EtOAc = 0%–30%), and the fraction was concentrated under vacuum to give 1–6c (3.06 g, approximately 46.8% purity), a colorless, transparent oil. MS (ESI) m / z: 447.4 [M+Na] + . 1 H NMR (400 MHz, cdcl3) δ 7.32 (m, 5H), 5.35 (s, 1H), 5.18 - 5.04 (m, 2H), 4.19 - 4.09 (dq, J = 6.8, 1.6 Hz 2H), 3.82 (m, 1H), 3.69 (m, 2H), 3.56 (dd, J =9.2, 3.6 Hz, 1H), 3.47 (dd, J = 9.6, 5.2 Hz, 1H), 3.45 - 3.37 (m, 1H), 3.36~3.25 (m, 1H), 2.55 (t, J = 6.0 Hz, 2H), 1.43 (s, 9H), 1.24 (t, J = 7.2 Hz, 3H).

[0255] Step 2: (S)-3-(3-(((benzyloxy)carbonyl)amino)-2-((tert-butoxycarbonyl)amino)propoxy)propionic acid (1-6d). Add 4 N NaOH (2.7 mL, 10.8 mmol) to a solution of 1-6c (3.06 g, 7.21 mmol) in MeOH (30 mL), and stir for 12 h at room temperature. Dilute the mixture with water (30 mL) and acidify to pH = 3 with 1 NKHSO4 in an ice bath, then use EtOAc (50 mL). 4) Extraction. The combined organic layers were rinsed with brine (50 mL). 3) The solution was washed, dried over Na₂SO₄, filtered, and the filtrate was concentrated under vacuum to obtain a colorless, transparent oily substance, 1-6d (2.8 g, 98% yield). MS (ESI) m / z: 419.3 [M+Na] + .

[0256] Step 3: (S)-3-(3-amino-2-((tert-butoxycarbonyl)amino)propoxy)propionic acid (1-6e). Wet Pd / C (250 mg) was added to a solution of 1-6e (2.8 g, 7.1 mmol) in MeOH (30 mL), and the mixture was stirred at room temperature for 30 min. The black suspension was filtered through a syringe tip, washed with H₂O, and then concentrated under vacuum to give 1-6e (1.85 g) as a white solid. MS (ESI) m / z: 263.2 [M+H] + . 1 H NMR (400 MHz, d2o) δ 4.1~3.9 (m,1H), 3.73 (t, J = 6.0 Hz, 2H), 3.64 (dd, J = 10.4, 4.4 Hz, 1H), 3.57 (dd, J = 10.4, 5.2 Hz, 1H), 3.22 (dd, J = 13.2, 4.4 Hz, 1H), 3.08 (dd, J = 13.2, 8.0 Hz, 1H), 2.45 (t, J = 6.0 Hz, 2H), 1.44 (s, 9H).

[0257] Step 4: (S)-3-(2-((tert-butoxycarbonyl)amino)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)propoxy)propionic acid (1-6 g). Add 1-6f (397.7 mg, 2.86 mmol) to a solution of 1-6e (500 mg, 1.91 mmol) in 1 N NaHCO3 (10 mL). Stir the mixture at 0 °C for 30 min, then slowly heat to room temperature for 2 h. Acidify the mixture to pH = 3 with 1 N KHSO4 in an ice bath, and then use EtOAc (20 mL). 2) Extraction. The combined organic layers were washed with brine (20 mL), dried over Na₂SO₄, filtered, and the filtrate was concentrated under vacuum to give 1–6 g (560 mg, 85.8% yield) of a white solid. MS (ESI) m / z: 365.3 [M+Na] + . 1 H NMR (400 MHz, cdcl3) δ 6.69(s, 2H), 5.06 (d, J= 9.2 Hz, 1H), 4.08~3.97 (m, 1H), 3.82~3.68 (m, 3H), 3.59(dd, J = 14.0, 3.6 Hz, 2H), 3.51 (dd, J = 9.6, 4.4 Hz, 1H), 2.64 (t, J = 6.2 Hz, 1H), 1.38 (s, 9H).

[0258] Step 5: (2S,3S,4S,5R,6R)-6-(((6S,13S,16S)-6-((2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)methyl)-16-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de [Pyrano[3',4':6,7]inzazo[1,2-b]quinoline-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)carbamoyl)-13-isopropyl-2,2-dimethyl-4,11,14-trioxo-3,8-dioxa-5,12,15-triazaheptadecane-17-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-6 h). HATU (8.6 mg, 0.02 mmol) and DIPEA (5.2 mg, 0.04 mmol) were added to a solution of 1-6 g (6.7 mg, 0.02 mmol) in DMF (2 mL). The mixture was stirred at room temperature for 10 min. 1-2 g (15 mg, 0.02 mmol) was added, and the mixture was stirred at room temperature for 30 min. The mixture was purified by preparative HPLC (FA) to obtain a grayish-white solid over 1–6 h (9.1 mg, 44.4% yield). MS (ESI) m / z: 1252.1 [M+H] + .

[0259] Step 6: (2S,3S,4S,5R,6R)-6-((S)-2-((S)-2-(3-((S)-2-amino-3-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)propoxy)propamido)-3-methylbutamido)-3-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1 0,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-6). ZnBr2 (32.4 mg, 0.14 mmol) was added to the suspension in DCM (1.5 mL) for 1-6 h (9.1 mg, 0.007 mmol), and the mixture was stirred at 45 °C for 8 h. The mixture was concentrated under vacuum and dissolved in DMSO, and then analyzed by preparative HPLC (0.1% FA) (Method: Column: XBridge Prep C18 OBD 5 μm 19). Purification was performed using a mobile phase of 150 mL / min (A-water (0.1% FA):B-acetonitrile; flow rate: 20 mL / min). The fractions were lyophilized to give 1-6 (7.1 mg, 85.7% yield) as a white solid. MS (ESI) m / z: 1151.9 [M+H] + .

[0260] Examples 1-7 Step 1: 3-(2-(methylthio)pyrimidin-5-carbamate)tert-butyl propionate (1-7c). Add HATU (1622 mg, 4.27 mmol) and DIPEA (1.9 mL, 1504 mg, 11.63 mmol) to the mixture of 1-7a (600 mg, 3.53 mmol) in DMF (15 mL). Stir the mixture at 45 °C for 10 min, add 1-7b (704.6 mg, 3.878 mmol), and stir at 45 °C for 40 min. Dilute the mixture with EtOAc (50 mL) and then with saline (30 mL). 3) The residue was washed, dried over Na₂SO₄, filtered, and concentrated under vacuum to obtain the residue. The residue was purified by rapid column chromatography (MeOH / DCM = 0 / 100 ~ 10 / 90) to give 1-7c (1.3 g crude product) as a brown oil. MS (ESI) m / z: 298.4 [M+H] + .

[0261] Step 2: 3-(2-(methanesulfonyl)pyrimidin-5-carbamate)tert-butyl propionate (d1-7). Add m-CPBA (1160.5 mg, 6.725 mmol) to a solution of d1-7 (500 mg, crude) in DCM (10 mL). Stir the white suspension at room temperature for 1 h. Dilute the mixture with EtOAc (50 mL) and quench by adding saturated Na2S2O3 (30 mL), stirring for 10 min at room temperature, then add DCM (30 mL). 3) Extraction. The combined organic layers were washed with brine (50 mL), dried over Na₂SO₄, filtered, and concentrated under vacuum to obtain a crude product as a yellow solid. The crude product was purified by rapid column chromatography (MeOH / DCM = 0 / 100 ~ 10 / 90), and the fractions were concentrated under vacuum to obtain 1-7d as a white solid (303 mg, approximately 50% yield). MS (ESI) m / z: 330.3 [M+H] + . 1 H NMR (400 MHz, CDCl3) δ 9.27 (s, 2H), 7.38 (s, 1H), 3.74 (dd, J = 11.6, 6.0Hz, 1H), 3.40 (s, 2H), 2.60 (t, 1H), 1.47(s, 5H).

[0262] Step 3: 3-(2-(methanesulfonyl)pyrimidin-5-carbamate)propionic acid (1-7e). A mixture of 1-7e (303.0 mg, 0.92 mmol) in TFA (1 mL) and DCM (2 mL) was stirred at room temperature for 1.5 h. The mixture was concentrated under vacuum and co-evaporated three times with toluene to give 1-7e (255 mg) as a grayish-white solid. MS (ESI) m / z: 274.1 [M+H] + .

[0263] Step 4: (2S,3S,4S,5R,6R)-6-((S)-3-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-2-((S)-3-methyl-2-(3-(2-(methanesulfonyl)pyrimidin-5-carbamoyl)propamido)butamido)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-7). HATU (11.5 mg, 0.030 mmol) and DIPEA (9 μL, 7.0 mg, 0.054 mmol) were added to solutions of 1-7e (7.1 mg, 0.026 mmol) and 1-2j (20.0 mg, 0.022 mmol) in DMF (2 mL). The mixture was stirred at room temperature for 30 min. The results were analyzed by preparative HPLC (FA 0.1%) (method: column: XBridge Prep C18 OBD 5 μm 19). The mobile phase was 150 mm; flow rate: A-water (0.1% FA): B-acetonitrile; flow rate: 20 mL / min. The purified mixture was lyophilized to give 1-7 (13.4 mg, approximately 52.5% yield) as a white solid. MS (ESI) m / z: 1182.8 [M+H] + .

[0264] Examples 1-8 Step 1: 3-(isopropylamino)propionate tert-butyl ester (1-8a). NaBH3CN (1.28 g, 20.0 mmol) was added to a solution of 1-7b (908 mg, 5.0 mmol) and acetone (580 mg, 10.0 mmol) in MeOH-H2O (1:1, 20 mL) at room temperature. The reaction mixture was stirred at room temperature for 18 h. The mixture was concentrated under vacuum, and the residue was redissolved in DCM (20 mL) and washed with saturated NaHCO3 (10 mL), H2O (10 mL), and brine (10 mL). The residue was dried over Na2SO4, filtered, and the filtrate was concentrated under vacuum to give crude target product 1-8a (1.21 g) as a colorless oil, which was used directly in the next step. MS (ESI) m / z: 188.2 [M+H] + .

[0265] Step 2: 3-(Isopropylamino)propionic acid (1-8b). A solution of 1-8a (600 mg, 2.5 mmol) in TFA / DCM (5:2, 7 mL) was stirred overnight at room temperature. The mixture was concentrated under vacuum, and the residue was redissolved in water. The aqueous layer was washed with EtOAc and lyophilized to give 1-8b (444.4 mg, 77.9% yield) as a white solid. MS (ESI) m / z: 132.1 [M+H] + .

[0266] Step 3: ( S )-7-(2,5-dioxo-2,5-dihydro-1 H (-pyrrolo-1-yl)-11-isopropyl-2,2-dimethyl-4,10-dioxo-3,9-dioxa-5,11-diazatetradecane-14-acid (1-8d). DIPEA (30 mg, 0.23 mmol) and HOBt (2 mg, 0.014 mmol) were added to solutions of 1-8b (12.3 mg, 0.094 mmol) and 1-8c (20 mg, 0.046 mmol) in DMF (1 mL) at room temperature. The reaction mixture was stirred at room temperature for 16 min. The reaction mixture was diluted with DMF and 0.1 N Acidified with HCl. The mixture was purified by preparative HPLC (FA), and the fractions were lyophilized to give 1–8 days (6.8 mg, 34.6% yield) as a pale yellow solid. MS (ESI) m / z: 450.48 [M+Na] + .

[0267] Step 4: (2) S ,3 S 4 S 5 R 6 R )-6-(((7 S ,16 S ,19 S )-7-(2,5-dioxo-2,5-dihydro-1 H -pyrrole-1-yl)-19-(((3-(((1) S 9 S )-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1 H ,12 H-Benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)carbamoyl)-11,16-diisopropyl-2,2-dimethyl-4,10,14,17-tetraoxo-3,9-dioxa-5,11,15,18-tetraazaeicosano-20-yl)oxy)-3,4,5-trihydroxytetrahydro-2 H 1-Pyran-2-carboxylic acid (1-8e). HATU (6.0 mg, 0.0159 mmol), DIPEA (4.7 mg, 0.0363 mmol), and 1-2j (13.4 mg, 0.0145 mmol) were added to a solution of 1-8d (6.8 mg, 0.0159 mmol) in DMF (0.5 mL). The reaction mixture was stirred at room temperature for 30 min. The mixture was diluted with DMF (1.5 mL) and acidified with HOAc. Purification was performed by preparative HPLC (FA), and the fractions were lyophilized to give 1-8e (5.1 mg, 26.4% yield) as a white solid. MS (ESI) m / z: 1358.9 [M+Na] + .

[0268] Step 5: (2) S ,3 S 4 S 5 R 6 R )-6-(((2 S 5 S ,14 S )-15-amino-14-(2,5-dioxo-2,5-dihydro-1 H -pyrrole-1-yl)-2-(((3-(((1) S 9 S )-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1 H ,12 H -Benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)carbamoyl)-5,10-diisopropyl-4,7,11-trioxo-12-oxa-3,6,10-triazapentadecanyl)oxy)-3,4,5-trihydroxytetrahydro-2 H1-Pyran-2-carboxylic acid (1-8). ZnBr2 (32.4 mg, 0.14 mmol) was added to a suspension of 1-8e (5.1 mg, 0.007 mmol) in DCM (1.5 mL), and the mixture was stirred at 45 °C for 21 h. The mixture was concentrated under vacuum and dissolved in 0.1% FA aqueous solution / ACN (80:20, 1 mL), and then purified by preparative HPLC (0.1% TFA). The fractions were lyophilized to give 1-8TFA (2.2 mg, 46.6% yield) as a white solid. MS (ESI) m / z: 1236.8 [M+H] + .

[0269] Examples 1-9 Step 1: (3-oxopropane-1,2-diyl)(S)-benzyl dicarboxylate tert-butyl ester (1-9b). Cool a solution of (COCl)₂ (3.9 g, 30.83 mmol) in DCM (50 mL) to -70 °C, and add DMSO (3.6 g, 46.24 mmol) dropwise. Then stir at -70 °C for 30 min. Add 1-9a (5.0 g, 15.41 mmol) dissolved in DMSO (10 mL) via syringe, and stir at -70 °C for 30 min. Add TEA (7.8 g, 77.1 mmol) dropwise, and then slowly heat to room temperature. Pour the mixture into water (100 mL), and use EtOAc (50 mL) to... 2) Extraction. The combined organic layers were rinsed with brine (100 mL) 2) Wash, dry with Na2SO4, filter, and concentrate the filtrate under vacuum to obtain the residue. Purify the residue by rapid column chromatography (using EtOAc / petroleum ether = 1 / 99~50 / 50 as the mobile phase), and concentrate the fraction under vacuum to give 1-9b (4.86 g, 97.8% yield) as a yellow oil. 1 H NMR (400 MHz, cdcl3) δ 9.64 (s, 1H),7.39 - 7.30 (m, 5H), 5.93 (s, 1H), 4.86 (s, 1H), 4.35-4.22(m, 1H), 3.77 -3.64 (m, 1H), 3.56 (dt, J = 14.8, 4.8 Hz, 1H), 1.40 (s, 9H).

[0270] Step 2: Ethyl (R)-6-(((benzyloxy)carbonyl)amino)-7-((tert-butoxycarbonyl)amino)hept-4-enoate (1-9d). Add dropwise to a suspension of 1-9b (6.5 g, 14.3 mmol) in toluene (30 mL) at 0 °C. t- Buok (1 M solution in THF, 14.5 mL, 14.5 mmol), the mixture turned dark within 30 min. 1-9c (2.0 g, 6.2 mmol) was added dropwise to toluene (20 mL) at -70 °C, followed by slow warming to room temperature. The mixture was quenched by adding saturated NH4Cl (50 mL). After separation, the toluene phase was rinsed with brine (30 mL). 2) The solution was washed, dried over Na₂SO₄, filtered, and the filtrate was concentrated under vacuum to obtain the residue. The residue was purified by rapid column chromatography (EtOAc / petroleum ether = 0 / 100~30 / 70~50 / 50) to give 1-9d (808 mg, 30.8% yield) as a white solid. MS (ESI) m / z: 443.4 [M+Na] + . 1 H NMR (400 MHz, cdcl3) δ 7.40 - 7.27 (m, 5H), 5.72~5.60 (m, 0.2 H), 5.60 -5.46 (m, 0.8H), 5.40 (dd, J = 16.0 Hz, 6.4 Hz, 0.2H), 5.34~5.20 (m, 1.8 H),4.95~4.70 (m, 1 H), 5.16~5.01 (m, 2H), 4.60~4.40 (m, 1 H),4.12 (q, J = 7.2Hz, 2H), 3.39 - 3.08 (m, 2H), 2.6~2.2 (m, 4H), 1.42 (s, 9H), 1.24 (t, J = 7.2Hz, 1H).

[0271] Step 3: (R)-6-(((benzyloxy)carbonyl)amino)-7-((tert-butoxycarbonyl)amino)hept-4-enoic acid (1-9e). Add 4 N NaOH (1.92 mL, 7.69 mmol) to a solution of 1-9d (808.0 mg, 1.92 mmol) in MeOH (10 mL). Stir the mixture at room temperature for 3.5 h. Dilute the mixture with water (20 mL), acidify to pH = 3 with saturated KHSO4, and then with EtOAc (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 give 1-9e (781 mg) as a white solid. MS (ESI) m / z: 415.4 [M+Na] + .

[0272] Step 4: (R)-6-amino-7-((tert-butoxycarbonyl)amino)heptanic acid (1-9f). Wet Pd / C (67 mg) was added to a solution of 1-9e (781.0 mg, 1.99 mmol) in MeOH (8 mL), and the mixture was stirred at room temperature for 24 h. The black suspension was filtered through a syringe tip, washed with MeOH / H₂O (5:1, 30 mL), and then concentrated under vacuum to give 1-9f as a white solid (510 mg, 98.5% yield). MS (ESI) m / z: 261.3 [M+H] + .

[0273] Step 5: (R)-7-((tert-butoxycarbonyl)amino)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)heptanic acid (1-9 g). Add 1-6 e (455.8 mg, 2.94 mmol) to a mixture of 1-9 f (510 mg, 1.96 mmol) in 1 N NaHCO3 (10 mL) at 0 °C, then stir at 0 °C for 30 min and allow to warm to room temperature for 3 h. Acidify the reactants to pH = 3 with saturated KHSO4, and use EtOAc (30 mL) 3) Extraction. The combined organic layers were washed with brine (50 mL), dried over Na₂SO₄, filtered, and the filtrate was concentrated to obtain the residue. The residue was purified by rapid column chromatography (MeOH / DCM = 0 / 100~5 / 95), and the fractions were concentrated under vacuum to give 1-9 g (590 mg, 85% yield) of a pale yellow oil. MS (ESI) m / z: 363.3 [M+Na] + .

[0274] Step 7: (2S,3S,4S,5R,6R)-6-((S)-2-((S)-2-((R)-7-amino-6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)heptanamido)-3-methylbutamido)-3-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,1) 3-Dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-9). 1-9 were synthesized as a white solid (5.8 mg, 45% yield) according to the synthetic procedure of Examples 1-6.

[0275] Examples 1-10 Step 1: (2) S ,3 S 4 S 5 R 6 R )-6-(((8 S ,11 S )-11-(((3-(((1 S 9 S )-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1 H ,12 H -benzo[ de ]pyrano[3',4':6,7]inzazo[1,2-b]quinoline-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)carbamoyl)-1-(9 H -fluorene-9-yl)-8-isopropyl-4-methyl-3,6,9-trioxo-2-oxa-4,7,10-triazadodecane-12-yl)oxy)-3,4,5-trihydroxytetrahydro-2 H2-Pyrano-2-carboxylic acid (1-10b). HATU (13.5 mg, 0.0356 mmol), DIPEA (10.5 mg, 0.081 mmol), and 1-2j (30 mg, 0.0323 mmol) were added to a solution of 1-10a (11.1 mg, 0.0356 mmol) in DMF (1 mL). The reaction mixture was stirred at room temperature for 30 min. The mixture was diluted with DMF (1.5 mL) and acidified with HOAc. Purification was performed by preparative HPLC (FA), and the fractions were lyophilized to give 1-10b (14.6 mg, 36.9% yield) as a pale yellow solid. MS (ESI) m / z: 1221.0 [M+H] + .

[0276] Step 2: (2S,3S,4S,5R,6R)-6-((S)-3-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-2-((S)-3-methyl-2-(2-(methylamino)acetamyl)butamido)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-10c). Add Et₂NH (8.8 mg, 0.12 mmol) to a solution of 1-10b (14.6 mg, 0.012 mmol) in DMF (0.3 mL). Stir the mixture at room temperature for 20 min. Concentrate the reaction mixture under vacuum and co-evaporate twice with toluene to give 1-10c as a brown solid, which is used directly in the next step.

[0277] Step 3: (2S,3S,4S,5R,6R)-6-(((7S,15S,18S)-7-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-18-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3'] [4':6,7]Indazino[1,2-b]quinoline-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)carbamoyl)-15-isopropyl-2,2,11-trimethyl-4,10,13,16-tetraoxo-3,9-dioxa-5,11,14,17-tetraazanonadecan-19-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-10e). DSC (3.5 mg, 0.0136 mmol) and DIPEA (2.3 mg, 0.018 mmol) were added to a mixture of 1-10d (3.6 mg, 0.0132 mmol) in DMF (0.2 mL). The mixture was stirred at room temperature for 3 h. Then, crude solid 1-10c was added, and the mixture was stirred again at room temperature for 30 min. The mixture was diluted with DMF (1.5 mL) and acidified with HOAc. Purification by preparative HPLC (FA) yielded 1-10e as a pale yellow solid (5.4 mg, 34.8% yield). MS (ESI) m / z: 1294.9 [M+H] + .

[0278] Step 4: (2S,3S,4S,5R,6R)-6-(((2S,5S,13S)-14-amino-13-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-2-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12) H-benzo[de]pyrano[3',4':6,7]inzazo[1,2-b]quinoline-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)carbamoyl)-5-isopropyl-9-methyl-4,7,10-trioxo-11-oxa-3,6,9-triazatetradecyl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-10). ZnBr2 (24 mg, 0.11 mmol) was added to a suspension of 1-10e (5.4 mg, 0.0042 mmol) in DCM (0.5 mL), and the mixture was stirred at 45 °C for 24 h. The mixture was concentrated under vacuum and dissolved in 0.1% FA aqueous solution / ACN (80:20, 1 mL), then purified by preparative HPLC (0.1% TFA). The fractions were lyophilized to give 1-10 (1.3 mg, 26% yield) as a pale yellow solid. MS (ESI) m / z: 1194.9 [M+H] + .

[0279] Examples 1-11 Step 1: (4-Hydroxybutane-1,2-diyl)(R)-benzyl dicarboxylate tert-butyl ester (1-11b). 1-11a (2.00 g, 5.68 mmol) and K₂CO₃ (863 mg, 6.24 mmol) were added to DMF (10 mL), followed by dropwise addition of CH₃I (1.61 g, 11.35 mmol) at 0 °C. The resulting mixture was stirred at 0 °C for 20 min, then heated to 25 °C and stirred further at 25 °C for 60 min. The reaction was monitored by TLC (petroleum ether / EtOAc) and LCMS. After complete reaction, the reaction mixture was diluted with EtOAc (80 mL) and diluted with brine (30 mL). 3) and H2O (30 mL) 2) Washing. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain methyl ester (2.08 g, quantitative) as a light yellow solid.

[0280] Methyl ester (1.00 g, 2.73 mmol) was dissolved in MeOH (15 mL), followed by the addition of LiBH4 (6.80 mL, 2 M stock solution in THF) at 0 °C. The resulting mixture was stirred at 25 °C for 2 h. The reaction was monitored by LCMS and TLC. After complete reaction, saturated NH4Cl aqueous solution (10 mL) was added to quench the reaction. The reaction mixture was diluted with H2O (80 mL) and treated with EtOAc (50 mL). 3) Extraction. The combined organic layers were rinsed with brine (40 mL). 2) and water (40 mL) 2) The sample was washed, dried over anhydrous Na₂SO₄, filtered, concentrated under reduced pressure, and further purified by rapid column chromatography (petroleum ether / EtOAc) to give 1-11b as a white solid (760 mg, 82.3% yield). MS (ESI) m / z: 239.2 [M-Boc+H] + .

[0281] Step 2: (4-(((4-nitrophenoxy)carbonyl)oxy)butane-1,2-diyl)(R)-benzyl dicarboxylate tert-butyl ester (1-11c). 1-11b (300 mg, 0.89 mmol) and bis(4-nitrophenyl) carbonate (405 mg, 1.33 mmol) were dissolved in DMF (5 mL), followed by the addition of DIEA (229 mg, 1.77 mmol). The resulting mixture was stirred at 25 °C for 3 h. After complete reaction, the reaction mixture was diluted with EtOAc (100 mL) and diluted with brine (35 mL). 3) and water (35 mL) 3) Washing. The organic layer was dried over anhydrous Na₂SO₄, filtered, concentrated under reduced pressure, and further purified by rapid column chromatography (petroleum ether / EtOAc) to give 1-11c as a white solid (371 mg, 83.1% yield). MS (ESI) m / z: 404.4 [M-Boc+H] + .

[0282] Step 3: (R)-7-(((benzyloxy)carbonyl)amino)-2,2-dimethyl-4,11-dioxo-3,10-dioxa-5,12-diazapentadecan-15-acid (1-11e). 1-11c (420 mg, 0.83 mmol) and 1-11d (149 mg, 1.67 mmol) were dissolved in DMF (5 mL), followed by the addition of an aqueous solution of NaHCO3 (1 M, 5 mL). The resulting mixture was stirred at 25 °C for 2 h. The reaction was monitored by LCMS. After complete reaction, the reaction mixture was concentrated and purified by rapid column chromatography (DCM:MeOH) to give 1-11e as a pale yellow solid (339 mg, 89.6% yield). MS (ESI) m / z: 354.4 [M-Boc+H] + .

[0283] Step 4: (R)-7-amino-2,2-dimethyl-4,11-dioxo-3,10-dioxa-5,12-diazapentadecan-15-acid (1-11f). 1-11e (360 mg, 0.79 mmol) was dissolved in MeOH (18 mL), followed by the addition of Pd / C (wet matrix, 108 mg) and NH3-MeOH (7 M, 2 drops). The resulting mixture was stirred at room temperature under H2 (15 psi) for 2.5 h. After complete reaction, the reaction mixture was filtered and concentrated under reduced pressure to give 1-11f (252 mg, quantitative) as a clear syrup. The crude product was used directly in the next step without purification. MS (ESI) m / z: 320.3 [M+H] + .

[0284] Step 5: (R)-7-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-2,2-dimethyl-4,11-dioxo-3,10-dioxa-5,12-diazapentadecan-15-acid (1-11 g). 1-11 f (250 mg, 0.78 mmol) and 1-6 f (243 mg, 1.57 mmol) were dissolved at 0 °C in a mixed solvent of ACN (8 mL) and NaHCO3 aqueous solution (1 M, 16 mL). The resulting mixture was stirred at 0 °C for 0.5 h and then further stirred at 25 °C until the reaction was complete. The reaction mixture was then acidified to pH ~3 with KHSO4 aqueous solution (1 M) and treated with EtOAc (40 mL). 3) Extraction. The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to obtain a yellow oily crude product. The crude product was purified by rapid column chromatography (DCM:MeOH) to give 1-11 g (280 mg, 89.6% yield) of a white solid. MS (ESI) m / z: 422.3 [M+Na] + .

[0285] Step 6: (2S,3S,4S,5R,6R)-6-(((7R,17S,20S)-7-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-20-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3', [4':6,7]Indazino[1,2-b]XXXquinoline-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)carbamoyl)-17-isopropyl-2,2-dimethyl-4,11,15,18-tetraoxo-3,10-dioxa-5,12,16,19-tetraazacotetra-21-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-11h). HATU (200 mg, 0.53 mmol) and DIEA (186 mg, 1.43 mmol) were added to a solution of compound 1-11 g (230 mg, 0.58 mmol) in DMF (6 mL). The mixture was stirred at room temperature for 10 min. Compound 1-2j (500 mg, 0.48 mmol) was added to the mixture, and the mixture was stirred at room temperature for 1 h. Pour the reaction mixture into MTBE (50 ml) and i In 5 mL of PrOH, stir for 10 min, filter, and dry. The crude product (628 mg, theoretical yield) was used in the next step without further purification after 1–11 h. MS (ESI) m / z: 1331.0 [M+Na] + .

[0286] Step 7: (2S,3S,4S,5R,6R)-6-(((2S,5S,15R)-16-amino-15-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-2-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-Benzo[de]pyrano[3',4':6,7]inzazido[1,2-b]quinoline-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)carbamoyl)-5-isopropyl-4,7,11-trioxo-12-oxa-3,6,10-triazahexadecyl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-11). ZnBr2 (5.4 g, 24 mmol) was added to a solution of compound 1-11h (628 mg, 0.48 mmol) in DCM (10 mL). The mixture was stirred at 39-40 °C for 16 h. The mixture was concentrated and dissolved in H2O (0.1% FA, 9 mL). Preparative HPLC was performed (method: column: XBridge Prep C18 OBD 5 μm 19). The solution was purified using a mobile phase of 150 mm; A-water (0.1% TFA): B-acetonitrile; flow rate: 20 mL / min, yielding compound 1-11 (TFA salt, 240 mg, 38.2% yield in 2 steps) as a pale yellow solid. MS (ESI) m / z: 1208.9 [M+H] + .

[0287] Examples 1-12 Steps 1 and 2: (2S,3S,4S,5R,6R)-6-((S)-2-((S)-2-(3-((R)-4-amino-3-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)butoxy)propamido)-3-methylbutamido)-3-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl- 10,13-Dioxo-2,3,9,10,13,15-Hexahydro-1H,12H-Benzopyrano[3',4':6,7]inzazino[1,2-b]quinoline-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-12). 1-12 was synthesized as a white solid (13.4 mg, 79.1% yield) according to the synthetic procedure of Examples 1-6. MS (ESI) m / z: 1165.7 [M+H]+ .

[0288] Examples 1-13 Step 1: 3-Bromo-5-(methylthio)-1,2,4-thiadiazole (1-13b). Br2 (5.2 g, 32.9 mmol) was added dropwise to a solution of compound 1-13a (5 g, 29.4 mmol) in dichloromethane (100 mL) at 0 °C. After addition, the resulting mixture was stirred overnight at room temperature. Excess Na2SO3 and water (50 mL) were added to the reaction mixture to decompose the excess Br2. The mixture was separated, and the separated organic layer was washed with brine (50 mL × 3), dried over Na2SO4, and concentrated under vacuum. The residue was purified by rapid column chromatography (eluting with petroleum ether: EtOAc = 0%–40%) to give compound 1-13b (4.2 g, 68.2% yield) as a pale yellow solid. MS (ESI) m / z: 212.9 [M+H] + .

[0289] Step 2: Methyl 4-(5-(methylthio)-1,2,4-thiadiazol-3-yl)benzoate (1-13d). Compound 1-13c (109.72 mg, 0.568 mmol), K2CO3 (168 mg, 0.947 mmol), and Pd(dppf)Cl2.DCM (34.6 mg, 0.047 mmol) were added to a solution of compound 1-13b (100 mg, 0.47 mmol) in toluene (4 mL) and H2O (1 mL). The mixture was stirred at 110 °C for 3 h under a N2 atmosphere. 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%). Compound 1-13d (56 mg, 44.4% yield) was given as a grayish-white solid. MS (ESI) m / z: 267.1 [M+H] + .

[0290] Step 3: 4-(5-(methylthio)-1,2,4-thiadiazol-3-yl)benzoic acid (1-13e). LiOH (17 mg, 0.41 mmol) was added to a solution of compound 1-13e (54 mg, 0.20 mmol) in MeOH (3 mL) and H₂O (1 mL). The mixture was stirred at room temperature for 2 h. The mixture was adjusted to pH 7 and purified by preparative HPLC (FA conditions) to give compound 1-13e (36 mg, 70.3% yield) as a white solid. MS (ESI) m / z: 253.1 [M+H] + .

[0291] Step 4: 4-(5-(methanesulfonyl)-1,2,4-thiadiazol-3-yl)benzoic acid (1-13f). m-CPBA (96 mg, 0.55 mmol) was added to a solution of compound 1-13e (35 mg, 0.14 mmol) in DCM (3 mL) and THF (3 mL). The mixture was stirred at room temperature for 16 h. The mixture was concentrated and analyzed by preparative HPLC (method: column: XBridgePrep 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. Compound 1-13f (12 mg, 99% purity) was obtained as a white solid.

[0292] Step 5: (2S,3S,4S,5R,6R)-6-((S)-3-(((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-2-((S)-3-methyl-2-(4-(5-(methanesulfonyl)-1,2,4-thiadiazol-3-yl)benzamido)butamido)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-13). HATU (9.02 mg, 0.024 mmol) and DIEA (5.58 mg, 0.043 mmol) were added to a solution of compound 1-13f (7.36 mg, 0.026 mmol) in DMF (2 mL). The mixture was stirred at room temperature for 30 min. Compound 1-2j (20 mg, 0.022 mmol) was added to the mixture, and the mixture was stirred at room temperature for 15 min. The solution was analyzed by preparative HPLC (method: column: XBridge Prep C18 OBD 5 μm 19). The mobile phase was 150 mm; flow rate: A-water (0.1% TFA): B-acetonitrile; flow rate: 20 mL / min. The reactants were purified to give compounds 1-13 as a white solid (7.6 mg, 29.5% yield). MS (ESI) m / z: 1193.5 [M+H] + .

[0293] Examples 1-14 Step 1: (2-(((3-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)propoxy)methyl)amino)-2-oxoethyl)carbamate (9H-fluorene-9-yl)methyl ester (1-14a). 1-14a (85 mg, 79% yield) was synthesized according to the same procedure as Step 1 in Example 1-1. MS (ESI) m / z: 787.5 [M+H] + .

[0294] Step 2: 2-Amino-N-((3-(((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)propoxy)methyl)acetamide (1-14b). 1-14b was synthesized according to the same procedure as in Step 10 of Example 1-1. MS (ESI) m / z: 565.3 [M+H] + .

[0295] Step 3: (2R,3R,4S,5S,6R)-triacetic acid 2-((2S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-3-((2-(((3-(((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)propoxy)methyl)amino)-2-oxoethyl)amino)-3-oxopropoxy)-6-(acetoxymethyl)tetrahydro-2H-pyran-3,4,5-triyl ester (1-14c). Synthesized 1-14c (45 mg, 32% yield, two steps) according to the same procedure as step 12 of Example 1-1. MS (ESI) m / z: 1304.7 [M+H] + .

[0296] Step 4: (2R,3S,4S,5R,6R)-triacetic acid 2-(acetoxymethyl)-6-((2S)-2-((S)-2-amino-3-methylbutamido)-3-((2-(((3-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)propoxy)methyl)amino)-2-oxoethyl)amino)-3-oxopropoxy)tetrahydro-2H-pyran-3,4,5-triyl ester (1-14d). Synthesized for 1-14d according to the same procedure as Step 10 of Example 1-1. MS (ESI) m / z: 1081.6 [M+H] + .

[0297] Step 5: (2S)-2-amino-N-((2S)-1-((2-(((3-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)propoxy)methyl)amino)-2-oxoethyl)amino)-1-oxo-3-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)prop-2-yl)-3-methylbutyramide (1-14e). 1-14e (19.2 mg, 60% yield, two steps) was synthesized according to the same procedure as step 11 of Example 1-1. MS (ESI) m / z: 913.5 [M+H] + .

[0298] Step 6: 6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-N-((11S,14S)-1-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indazine [1,2-b]quinolin-1-yl)-15-methyl-7,10,13-trioxo-11-((((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)methyl)-4-oxa-6,9,12-triazahexadecane-14-yl)hexanoamide (1-14f). 1-14f (6.4 mg, 28% yield) was synthesized according to the same procedure as step 12 of Example 1-1. MS (ESI) m / z: 1106.7 [M+H] + .

[0299] Examples 1-15 Step 1: (2R,3R,4S,5S,6S)-triacetic acid 2-((2S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-3-((2-(((3-(((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)propoxy)methyl)amino)-2-oxoethyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (1-15a). Compound 1-15a (45 mg, 36.5% yield) was synthesized according to the synthetic procedure in step 9 of Example 1-1. MS (ESI) m / z: 1290.9 [M+H] + .

[0300] Step 2: (2R,3R,4S,5S,6S)-triacetic acid 2-((2S)-2-((S)-2-amino-3-methylbutamido)-3-((2-(((3-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)propoxy)methyl)amino)-2-oxoethyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (1-15b). Compound 1-15b (37 mg, crude) was synthesized according to the synthetic procedure of Step 10 in Example 1-1. MS (ESI) m / z: 1067.7 [M+H] + .

[0301] Step 3: (2S,3S,4S,5R,6R)-6-((2S)-2-((S)-2-amino-3-methylbutamido)-3-((2-(((3-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)propoxy)methyl)amino)-2-oxoethyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-15c). Compound 1-15c (18 mg, 56.0% yield) was synthesized according to the synthetic procedure of Step 11 in Example 1-1. MS (ESI) m / z: 927.7 [M+H] + .

[0302] Step 4: (2S,3S,4S,5R,6R)-6-((2S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexamido)-3-methylbutamido)-3-((2-(((3-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)propoxy)methyl)amino)-2-oxoethyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-15). Compounds 1-15 (3.2 mg, 14.7% yield) were synthesized according to the synthetic procedure in step 5 of Examples 1-13. MS (ESI) m / z: 1120.8 [M+H] + .

[0303] Examples 1-16 Step 1: (2R,3R,4S,5S,6R)-triacetic acid 2-((2S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-3-((3-(((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)propyl)amino)-3-oxopropoxy)-6-(acetoxymethyl)tetrahydro-2H-pyran-3,4,5-triyl ester (1-16a). DIPEA (14 μL, 0.082 mmol) was added to a mixture of 1-1kJ (27 mg, 0.035 mmol) and TSTU (11 mg, 0.035 mmol) in DMF (1 mL). The mixture was stirred at room temperature for 10 min, and then P4 (13 mg, 0.027 mmol) was added. The mixture was stirred at room temperature for 30 min. The sample was then analyzed by preparative HPLC (FA) (method: column: XBridgePrep C18 OBD 5 μm 19). The mixture was purified using a mobile phase of 150 mm; mobile phase: A-water (0.1% FA): B-acetonitrile; flow rate: 20 mL / min. The fractions were lyophilized to give 1-16a (20 mg, 60% yield) as a white solid. MS (ESI) m / z: 1216.9 [M+H] + .

[0304] Step 2: (2S)-2-amino-N-((2S)-1-((3-(((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)propyl)amino)-1-oxo-3-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)propyl-2-yl)-3-methylbutyramide (1-16b). Add Et₂NH (34 μL, 0.33 mmol) to a solution of 1-16a (20 mg, 0.016 mmol) in DMF (1 mL). Stir the mixture at room temperature for 20 min. Concentrate the reaction mixture under vacuum and co-evaporate twice with toluene, then dissolve it in MeOH (1 mL) and add K₂CO₃ (6 mg, 0.04 mmol). Stir the mixture at room temperature for 40 min. Cool the mixture to 0 °C using an ice bath, then acidify it to pH 3 with 3 NHCl, filter, and pass it through preparative HPLC (FA) (method: column: XBridge Prep C18 OBD 5 μm 19). Purification was performed using a mobile phase of 150 mL / min (A-water (0.1% TFA):B-acetonitrile; flow rate: 20 mL / min). The fractions were lyophilized to give 1-16b (10 mg, 75% yield) as a white solid. MS (ESI) m / z: 826.7 [M+H] + .

[0305] Step 3: 6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-N-((2S)-1-(((2S)-1-((3-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)propyl)amino)-1-oxo-3-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyrano-2-yl)oxy)propyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)hexamamide (1-16). Add HATU (5 mg, 0.012 mmol) and DIPEA (2 μL, 0.013 mmol) to a solution of 1-1q (3 mg, 0.012 mmol) in DMF (1 mL). Stir the mixture at room temperature for 10 min, then add 1-16b (32 mg, 0.035 mmol). Stir the resulting yellow mixture at room temperature for 15 min. Analyze the result by preparative HPLC (FA) (method: column: XBridge Prep C18 OBD 5 μm 19). The reaction mixture was purified by lyophilizing the fractions to give 1-16 (8 mg, 50% yield) as a white solid. The mobile phase was: A-water (0.1% TFA); B-acetonitrile; flow rate: 20 mL / min. MS (ESI) m / z: 1019.8 [M+H] + .

[0306] Examples 1-17 Step 1: (2R,3R,4S,5S,6S)-triacetic acid 2-((2S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-3-(((2-(((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)ethoxy)methyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (1-17a). 1-17a (110 mg, 69.2% yield) was synthesized according to the same procedure as step 1 of Example 1-1. MS (ESI) m / z: 1218.5 [M+H] + .

[0307] Step 2: (2R,3R,4S,5S,6S)-triacetic acid 2-((2S)-2-((S)-2-amino-3-methylbutyramido)-3-(((2-(((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)ethoxy)methyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (1-17b). 1-17b (90 mg, crude product) was synthesized according to the same procedure as Step 4 in Examples 1-5. MS (ESI) m / z: 997.1 [M+H] + .

[0308] Step 3: (2S,3S,4S,5R,6R)-6-((2S)-2-((S)-2-amino-3-methylbutamido)-3-(((2-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)ethoxy)methyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-17c). 1-17c (50 mg, 65.0%) was synthesized according to the same procedure as Step 5 in Examples 1-5. MS (ESI) m / z: 856.3 [M+H] + .

[0309] Step 4: (2S,3S,4S,5R,6R)-6-((2S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexamido)-3-methylbutamido)-3-(((2-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)ethoxy)methyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-17). Synthesized 1-17 (25 mg, 41.4%) according to the same procedure as step 6 of Examples 1-5. MS (ESI) m / z: 1050.1 [M+H] + .

[0310] Examples 1-18 Step 1: (2R,3R,4S,5S,6S)-triacetic acid 2-((2S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-3-(((3-(((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)propoxy)methyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (1-18a). A white suspension of 1-2f (102 mg, 0.13 mmol), P3 (60 mg, 0.13 mmol), and 4 Å molecular sieve (50 mg) in anhydrous THF (3 mL) was stirred at room temperature for 10 min. Sc(OTf)3 (80 mg, 0.16 mmol) was added, and the resulting yellow suspension was stirred at room temperature for 4 h. The yellow suspension mixture was filtered through a diatomaceous earth mat and washed with EtOAc (20 mL). The combined organic layers were washed with saturated NaHCO3 (30 mL) and brine (10 mL), dried over Na2SO4, filtered, and the filtrate was concentrated under vacuum to give a residue. The residue was purified by silica gel column chromatography (MeOH / DCM = 0%–5%), and the fraction was concentrated under vacuum to give 1-18a (147 mg, 95% yield) as a white foamy solid. MS (ESI) m / z: 1232.9 [M+H] + .

[0311] Step 2: (2S,3S,4S,5R,6R)-6-((2S)-2-((S)-2-amino-3-methylbutamido)-3-(((3-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)propoxy)methyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-18b). Add Et2NH (197 μL, 1.9 mmol) to the solution of 1-18a (147 mg, 0.12 mmol) in DMF (3 mL). The mixture was stirred at room temperature for 20 min. The reaction mixture was concentrated under vacuum and co-evaporated twice with toluene, then dissolved in MeOH (3 mL) and H₂O (0.5 mL), with K₂CO₃ (50 mg, 0.36 mmol) added. The mixture was stirred at room temperature for 30 min. The mixture was cooled to 0 °C using an ice bath, then acidified to pH 3 with 3 N HCl, filtered, and passed through preparative HPLC (FA) (method: column: XBridge Prep C18OBD 5 μm 19). Purification was performed using a mobile phase of 150 mL / min (A-water (0.1% FA):B-acetonitrile; flow rate: 20 mL / min). The fractions were lyophilized to give 1-18b (65 mg, 61% yield) as a white solid. MS (ESI) m / z: 870.7 [M+H] + .

[0312] Step 3: (2S,3S,4S,5R,6R)-6-(((7S,12S,15S)-7-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-15-(((3-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo) [de]pyrano[3',4':6,7]inzazido[1,2-b]quinoline-1-yl)propoxy)methyl)carbamoyl)-12-isopropyl-2,2-dimethyl-4,10,13-trioxo-3,9-dioxa-5,11,14-triazahexadecane-16-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-18c). DSC (14 mg, 0.052 mmol) and DIPEA (18 uL, 0.11 mmol) were added to a solution of 1-10d (14 mg, 0.052 mmol) in DMF (1 mL). The mixture was stirred overnight at room temperature. 1-18b (32 mg, 0.035 mmol) was added, followed by stirring for 10 min at room temperature. Preparative HPLC (FA) method (Column: XBridge Prep C18 OBD 5 μm 19) The mobile phase was 150 mm; flow rate: A-water (0.1% FA): B-acetonitrile; flow rate: 20 mL / min. The purified mixture was lyophilized to give 1-18c (18 mg, 44% yield) as a pale yellow solid. MS (ESI) m / z: 1166.9 [M+H] + .

[0313] Step 4: (2S,3S,4S,5R,6R)-6-((2S)-2-((S)-2-((((S)-3-amino-2-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)propoxy)carbonyl)amino)-3-methylbutamido)-3-(((3-(((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)propoxy)methyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-18). ZnBr2 (79 mg, 0.35 mmol) was added to a mixture of 1-18c (10 mg, 0.009 mmol) in DCM (1 mL), and the mixture was stirred at 40 °C for 2 h. The mixture was concentrated under vacuum to remove DCM, then dissolved in DMSO, and analyzed by preparative HPLC (FA) (method: column: XBridge Prep C18 OBD 5 μm 19). Purification was performed using a mobile phase of 150 mL / min (A-water (0.1% FA):B-acetonitrile; flow rate: 20 mL / min). The fractions were lyophilized to give 1-18 (4.7 mg, 50% yield) as a white solid. MS (ESI) m / z: 1066.8 [M+H] + .

[0314] Examples 1-19 Step 1: (2S,3S,4S,5R,6R)-6-((2S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexamido)-3-methylbutamido)-3-(((3-(((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)propoxy)methyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-19). Add HATU (14 mg, 0.037 mmol) and DIPEA (17 μL, 0.11 mmol) to a solution of 1-1q (8 mg, 0.038 mmol) in DMF (1.5 mL). Stir the mixture at room temperature for 10 min, then add 1-18b (32 mg, 0.035 mmol). Stir the resulting yellow mixture at room temperature for 15 min. Analyze the result by preparative HPLC (FA) (method: column: XBridge Prep C18 OBD 5 μm 19). The reaction mixture was purified by lyophilizing the fractions to give 1-19 (17.9 mg, 48% yield) as a white solid. The mobile phase was: A-water (0.1% TFA): B-acetonitrile; flow rate: 20 mL / min. MS (ESI) m / z: 1063.8 [M+H] + Examples 1-20 Examples 1-20 (25 mg, 41.4%) were synthesized according to the same procedure as Examples 1-17.

[0315] Examples 1-21 Step 1: (2R,3R,4S,5S,6S)-triacetic acid 2-(((S)-12-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)amino)-1,8,11-trioxo-2,5-dioxa-7,10-diazatriadecan-13-yl)oxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (1-21b). Sc(OTf)3 (62 mg, 0.12 mmol) was added to a solution of 1-21a (90 mg, 0.1 mmol), P6 (56 mg, 0.1 mmol), and 4 Å MS (200 mg) in anhydrous THF (2 mL) under a nitrogen atmosphere, and the mixture was stirred at room temperature for 16 h. The solution was filtered and washed with THF (50 mL). The pH of the filtrate was adjusted to 8–9 using a NaHCO3 aqueous solution (10 mL), and then washed with CH2Cl2 (20 mL). 3) Extraction. The organic phase was concentrated and purified by silica gel column chromatography (CH2Cl2 / MeOH = 90 / 10) to give 1-21b as a yellow solid (100 mg, 72.5% yield). MS (ESI) m / z: 1335.3 [M+H] + .

[0316] Step 2: (2R,3R,4S,5S,6S)-triacetic acid 2-(((S)-12-((S)-2-amino-3-methylbutamido)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)amino)-1,8,11-trioxo-2,5-dioxa-7,10-diazatriadecan-13-yl)oxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (1-21c). Et₂NH (111 mg, 1.5 mmol) was added to a solution of 1-21b (100 mg, 0.075 mmol) in anhydrous DMF (3 mL), and the mixture was stirred for 20 min at room temperature. 1-21c (80 mg, crude product) was co-evaporated five times with toluene to remove H₂O. MS (ESI) m / z: 1113.1 [M+H] + .

[0317] Step 3: (2S,3S,4S,5R,6R)-6-(((S)-12-((S)-2-amino-3-methylbutamido)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)amino)-1,8,11-trioxo-2,5-dioxa-7,10-diazatriadecan-13-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-21d). K₂CO₃ (50.2 mg, 0.360 mmol) was added to a solution of 1-21c (80 mg, 0.072 mmol) in MeOH (2 mL) and water (2 mL), and stirred at room temperature for 30 min. The pH was adjusted to approximately 4 by gradually adding 20% ​​HOAc aqueous solution. Preparative HPLC was performed (method: column XBridge Prep C18 OBD 5 μm 19). 150 mm; Mobile phase: A-water (0.1% FA): B-acetonitrile; Flow rate: 20 mL / min) The purified solution was lyophilized to give 1-21d (25 mg, 35.8%) as a white solid. MS (ESI) m / z: 973.5 [M+H] + .

[0318] Step 4: (2S,3S,4S,5R,6R)-6-(((S)-12-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexamido)-3-methylbutamido)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9 ,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)amino)-1,8,11-trioxo-2,5-dioxa-7,10-diazatriadecan-13-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-21). HATU (12 mg, 0.031 mmol) and DIPEA (0.009 mL, 0.051 mmol) were added to a solution of 1-1q (5.8 mg, 0.027 mmol) in anhydrous DMF (1.0 mL), and the mixture was stirred for 10 min at room temperature. 1-21d (25 mg, 0.026 mmol) was added to the above mixture, and the mixture was stirred for 10 min at room temperature. Preparative HPLC (method: XBridge Prep C18 OBD column 5 μm 19) The purified solution was purified by lyophilization (150 mm; mobile phase: A-water (0.1% FA): B-acetonitrile; flow rate: 20 mL / min), and the fractions were lyophilized to give 1-21 (7.4 mg, 24.7% yield) as a white solid. MS (ESI) m / z: 1187.8 [M+Na] + .

[0319] Examples 1-22 Step 1: (2R,3R,4S,5R,6R)-triacetic acid 2-(((S)-12-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)amino)-1,8,11-trioxo-2,5-dioxa-7,10-diazatriadecan-13-yl)oxy)-6-(acetoxymethyl)tetrahydro-2H-pyran-3,4,5-triyl ester (1-22b). Sc(OTf)3 (40 mg, 0.081 mmol) was added to a solution of 1-22a (60 mg, 0.068 mmol), P6 (36 mg, 0.068 mmol), and 4 Å MS (150 mg) in anhydrous THF (1 mL) under a nitrogen atmosphere, and the solution was stirred at room temperature for 16 h. The solution was filtered and washed with THF (50 mL). The pH of the filtrate was adjusted to 8–9 using a NaHCO3 aqueous solution (10 mL), and then washed with CH2Cl2 (20 mL). 3) Extraction. The organic phase was concentrated and purified by silica gel column chromatography (CH2Cl2 / MeOH = 90 / 10) to give 1-22b as a yellow solid (45 mg, 49.2% yield). MS (ESI) m / z: 1348.9 [M+H] + .

[0320] Step 2: (2R,3R,4S,5R,6R)-triacetic acid 2-(acetoxymethyl)-6-(((S)-12-((S)-2-amino-3-methylbutamido)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)amino)-1,8,11-trioxo-2,5-dioxa-7,10-diazatriadecan-13-yl)oxy)tetrahydro-2H-pyran-3,4,5-triyl ester (1-22c). Et₂NH (49 mg, 0.67 mmol) was added to a solution of 1-22b (45 mg, 0.033 mmol) in anhydrous DMF (1 mL) at room temperature, and the mixture was stirred for 20 min at room temperature. 1-22c (35 mg, crude product) was co-evaporated five times with toluene to remove H₂O. MS (ESI) m / z: 1127.1 [M+H] + .

[0321] Step 3: (2S,3S,4S,5R,6R)-6-(((S)-12-((S)-2-amino-3-methylbutamido)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)amino)-1,8,11-trioxo-2,5-dioxa-7,10-diazatriadecan-13-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-21d). K₂CO₃ (22 mg, 0.16 mmol) was added to a solution of 1-22°C (35 mg, 0.031 mmol) in MeOH (1 mL) and water (1 mL) at 0 °C, and stirred for 30 min at room temperature. The pH was adjusted to approximately 4 by gradually adding 20% ​​HOAc aqueous solution. Preparative HPLC was performed (method: column XBridge Prep C18 OBD 5 μm 19). 150 mm; Mobile phase: A-water (0.1% FA): B-acetonitrile; Flow rate: 20 mL / min) The purified solution was lyophilized to give 1-22d (9 mg, 30.2%) as a white solid. MS (ESI) m / z: 958.7 [M+H] + .

[0322] Step 4: ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)carbamic acid(10S,13S)-20-(2,5-dioxo- 2,5-Dihydro-1H-pyrrolo-1-yl)-13-isopropyl-6,9,12,15-tetraoxo-10-((((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)methyl)-3-oxa-5,8,11,14-tetraazaeicosyl ester (1-22). HATU (0.011 mg, 0.011 mmol) and DIPEA (0.003 mL, 0.011 mmol) were added to a solution of 1-1q (2.1 mg, 0.009 mmol) in anhydrous DMF (1.0 mL), and the mixture was stirred for 10 min at room temperature. 1-22d (9.0 mg, 0.009 mmol) was added to the above mixture, and the mixture was stirred for 10 min at room temperature. Preparative HPLC (method: XBridge Prep C18 OBD column 5 μm 19) The purified solution was purified by lyophilization (150 mm; mobile phase: A-water (0.1% FA): B-acetonitrile; flow rate: 20 mL / min), and the fractions were lyophilized to give 1-22 (2.8 mg, 25.9% yield) as a white solid. MS (ESI) m / z: 1152.2 [M+H] + .

[0323] Examples 1-23 Step 1: (2R,3R,4S,5S,6S)-triacetic acid 2-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-3-(((2-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)carbamoyl)oxy)ethoxy)methyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (1-23a). Add 4 Å MS (840 mg) to a solution of 1-2f (153 mg, 0.18 mmol) and P6 (150 mg, 0.18 mmol) in anhydrous THF (2 mL) and stir for 20 min. Add Sc(OTf)3 (120 mg, 0.24 mmol), stir at 35 °C for 14 h and then at 50 °C for 24 h. Filter the mixture and wash the filtrate with saturated NaHCO3 (5 mL). Use DCM / MeOH (10:1, 5.5 mL) 3) Extraction of the aqueous phase. The organic phase was concentrated and purified by rapid column chromatography (petroleum ether / EtOAc = 70 / 30 to 0 / 100) to give product 1-23a (165 mg, 70% yield, 95% purity) and rSMP6 (60 mg) as yellow solids. LCMS (ESI): 1299.8 [M+Na] + .

[0324] Step 2: (2R,3R,4S,5S,6S)-triacetic acid 2-((S)-2-((S)-2-amino-3-methylbutamido)-3-(((2-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)carbamoyl)oxy)ethoxy)methyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-trimethyl ester (1-23b). Add Et₂NH (0.305 mL, 2.93 mmol) to a solution of 1-23a (160 mg, 0.15 mmol) in anhydrous DMF (2 mL), and stir for 15 min at room temperature. Dilute the solution with toluene, concentrate, and use directly without further purification. LCMS (ESI): 893.6 [M + Na] + .

[0325] Step 3: (2S,3S,4S,5R,6R)-6-((S)-2-((S)-2-amino-3-methylbutamido)-3-(((2-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)carbamoyl)oxy)ethoxy)methyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-23c). Add Na₂CO₃ (42 mg, 0.39 mmol) to a solution of 1-23b (136 mg, 0.13 mmol) in MeOH / H₂O (1 / 0.5 mL) and stir for 3 h at room temperature. Add K₂CO₃ (18 mg) and stir for 1 h. Add water (1 mL) and adjust the pH of the solution to 4 with HOAc. Analyze the solution by preparative HPLC (mobile phase A: 0.1% FA aqueous solution, B: MeCN; gradient: 20%-32%-35%-90% B; flow rate: 20 mL / min; column: Xbridge Prep C18 OBD 5 μm, 19... The purified solution (150 mm) was lyophilized to give the title compound 1-23c (37 mg, 31.4% yield, 93% purity) as a yellow solid. LCMS (ESI): 915.7 [M+H] + .

[0326] Step 4: (2S,3S,4S,5R,6R)-6-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexamido)-3-methylbutamido)-3-(((2-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)carbamoyl)oxy)ethoxy)methyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-23). DIEA (14 μL, 0.077 mmol) was added to a solution of 1-1q (10 mg, 0.046 mmol) and HATU (16.4 mg, 0.042 mmol) in anhydrous DMF (0.5 mL), and the mixture was stirred for 5 min at room temperature. The reaction solution was then added to a solution of 1-23c (37 mg, 0.039 mmol) in anhydrous DMF (0.5 mL), and the mixture was stirred for 10 min at room temperature. The reaction was then analyzed by preparative HPLC (mobile phase A: 0.1% FA aqueous solution, B: MeCN; gradient: 32%-43%-95% B; flow rate: 20 mL / min; column: Xbridge Prep C18 OBD 5 μm, 19). The purified solution (150 mm) was lyophilized to give the title compound 1-23 as a pink solid (17 mg, 60% yield, 98% purity). LCMS (ESI): 1108.9 [M+H] + .

[0327] Examples 1-24 Step 1: (2R,3R,4S,5S,6S)-triacetic acid 2-((2S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-3-(((3-(((1S,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro- 1H,13H-benzo[de][1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)amino)-2-fluoro-2-methyl-3-oxopropoxy)methyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-trimethyl ester (1-24a). HATU (95 mg, 0.25 mmol) and DIPEA (0.11 mL, 0.62 mmol) were added to a solution of 1-4c (180 mg, 0.21 mmol) and P7 (132 mg, 0.25 mmol) in anhydrous DMF (3 mL), and the mixture was stirred at room temperature for 30 min. The solution was concentrated and purified by silica gel column chromatography (MeOH / CH2Cl2 = 5:95) to give 1-24a as a yellow solid (210 mg, 78.3% yield). MS (ESI) m / z: 1305.8 [M+H] + .

[0328] Step 2: (2R,3R,4S,5S,6S)-triacetic acid 2-((2S)-2-((S)-2-amino-3-methylbutamido)-3-(((3-(((1S,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)amino)-2-fluoro-2-methyl-3-oxopropoxy)methyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (1-24b). Et₂NH (0.34 mL, 3.22 mmol) was added to a solution of 1-24a (210 mg, 0.16 mmol) in anhydrous DMF (3 mL), and the mixture was stirred at room temperature for 30 min. The resulting solution was concentrated to give 1-24b (170 mg, 97.6% yield), which was used directly in the next step. MS (ESI) m / z: 1084.5 [M+H] + .

[0329] Step 3: (2S,3S,4S,5R,6R)-6-((2S)-2-((S)-2-amino-3-methylbutamido)-3-(((3-(((1S,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)amino)-2-fluoro-2-methyl-3-oxopropoxy)methyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-24c)trifluoroacetate. To a solution of 1-24b (170 mg, 0.16 mmol) in MeOH (2 mL) and water (1 mL), 1 N K₂CO₃ (0.55 mL, 0.55 mmol) was added, and the mixture was stirred at room temperature for 30 min. The resulting solution was quenched with HOAc, the MeOH was removed by concentration, and the solution was analyzed by preparative HPLC (method: XBridge Prep C18 OBD column 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 fractions were lyophilized to give 1-24c (trifluoroacetate) as a white solid (90 mg, 54.2% yield). MS (ESI) m / z: 943.5 [M+H] + .

[0330] Step 4: (2S,3S,4S,5R,6R)-6-((2S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexamido)-3-methylbutamido)-3-(((3-(((1S,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14) ,16-hexahydro-1H,13H-benzo[de][1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)amino)-2-fluoro-2-methyl-3-oxopropoxy)methyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-24). HATU (4.3 mg, 0.011 mmol) and DIPEA (0.005 mL, 0.028 mmol) were added to a solution of 1-1q (2.2 mg, 0.01 mmol) in anhydrous DMF (1.0 mL), and the mixture was stirred for 10 min at room temperature. 1-24c (10 mg, 0.01 mmol) was added to the above mixture, and the mixture was stirred for 10 min at room temperature. Preparative HPLC (method: XBridge Prep C18 OBD column 5 μm 19) The purified solution was purified by lyophilization to give 1-24 (5 mg, 46.5% yield) as a white solid. The mobile phase was: A-water (0.1% FA); B-acetonitrile; flow rate: 20 mL / min. MS (ESI) m / z: 1137.6 [M+H] + .

[0331] Examples 1-25 Step 1: (2R,3R,4S,5S,6S)-triacetic acid 2-((S)-2-((S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-3-(((2-((((1S,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)carbamoyl)oxy)ethoxy)methyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (1-25a). Add 4 Å MS (200 mg) to a solution of P10 (60 mg, 0.11 mmol) and 1-2f (111 mg, 0.13 mmol) in anhydrous THF (3 mL), and stir at room temperature under N2 for 20 min. Add Sc(OTf)3 (73 mg, 0.15 mmol) to the mixture under N2 atmosphere, and stir at 60 °C for 16 h. Filter the solution and wash with THF (50 mL). Adjust the pH of the filtrate to 8–9 using saturated NaHCO3 (20 mL), and wash with CH2Cl2 (20 mL). 3) Extraction. The organic phase was concentrated and purified by silica gel column chromatography (ethyl acetate / CH2Cl2 = 50 / 50) to give 1-25a (30 mg, 20.8% yield) as a yellow solid. MS (ESI) m / z: 1289.7 [M+Na] + .

[0332] Step 2: (2R,3R,4S,5S,6S)-triacetic acid 2-((S)-2-((S)-2-amino-3-methylbutamido)-3-(((2-((((1S,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)carbamoyl)oxy)ethoxy)methyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-trimethyl ester (1-25b). Et₂NH (0.049 mL, 0.47 mmol) was added to a solution of 1-25a (30 mg, 0.023 mmol) in anhydrous DMF (1 mL), and the mixture was stirred at room temperature for 30 min. The resulting solution was concentrated to give 1-25b (24 mg, 96.7% yield), which was used directly in the next step. MS (ESI) m / z: 1067.5 [M+H] + .

[0333] Step 3: (2S,3S,4S,5R,6R)-6-((S)-2-((S)-2-amino-3-methylbutamido)-3-(((2-((((1S,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)carbamoyl)oxy)ethoxy)methyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-25c)trifluoroacetate. 1 NK₂CO₃ (0.079 mL, 0.079 mmol) was added to a solution of 1-25b (24 mg, 0.022 mmol) in MeOH (0.5 mL) and water (0.2 mL), and stirred for 30 min at room temperature. The resulting solution was quenched with HOAc, concentrated to remove MeOH, and analyzed by preparative HPLC (method: XBridge Prep C18 OBD column 5 μm 19). Purification was performed using a mobile phase of 150 mL / min (A-water (0.1% TFA):B-acetonitrile; flow rate: 20 mL / min). The fractions were lyophilized to give 1-25c (trifluoroacetate) as a white solid (12 mg, 51.3% yield). MS (ESI) m / z: 927.5 [M+H] + .

[0334] Step 4: (2S,3S,4S,5R,6R)-6-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexamido)-3-methylbutamido)-3-(((2-((((1S,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,1) 1,14,16-Hexahydro-1H,13H-benzo[de][1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)carbamoyl)oxy)ethoxy)methyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-25). HATU (4.4 mg, 0.012 mmol) and DIPEA (0.005 mL, 0.029 mmol) were added to a solution of 1-1q (2.3 mg, 0.01 mmol) in anhydrous DMF (1 mL), and the mixture was stirred for 10 min at room temperature. 1-25c (10 mg, 0.01 mmol) was added to the above mixture, and the mixture was stirred for 10 min at room temperature. Preparative HPLC (method: XBridge Prep C18 OBD column 5 μm 19) The purified solution was purified by lyophilization (150 mL / min; mobile phase: A-water (0.1% FA): B-acetonitrile; flow rate: 20 mL / min), and the fractions were lyophilized to give 1-25 (3.7 mg, 34.4% yield) as a white solid. MS (ESI) m / z: 1121.5 [M+H] + .

[0335] Examples 1-26 Step 1: (2S,3S,4S,5R,6R)-6-(((7R,17S,20S)-7-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-20-(((3-(((1S,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxanepenten[4,5-g ]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)amino)-2-fluoro-2-methyl-3-oxopropoxy)methyl)carbamoyl)-17-isopropyl-2,2-dimethyl-4,11,15,18-tetraoxo-3,10-dioxa-5,12,16,19-tetraazacotetra-21-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-26a). HATU (8.7 mg, 0.023 mmol) and DIPEA (0.007 mL, 0.038 mmol) were added to a solution of 1-11 g (9.2 mg, 0.023 mmol) in anhydrous DMF (1 mL), and stirred for 10 min at room temperature. Then, 1-24c (20 mg, 0.019 mmol) was added to the above mixture, and the mixture was stirred at room temperature for 10 min. The sample was then analyzed by preparative HPLC (method: XBridge Prep C18 OBD column 5 μm 19). The purified solution was purified by lyophilization at 150 mm; mobile phase: A-water (0.1% FA): B-acetonitrile; flow rate: 20 mL / min. The fractions were then lyophilized to give 1-26a (18 mg, 71.8% yield) as a white solid. MS (ESI) m / z: 1324.8 [M+H] + .

[0336] Step 2: (2S,3S,4S,5R,6R)-6-(((2S,5S,15R)-16-amino-15-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-2-(((3-(((1S,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1, 3] Dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]inzazido[1,2-b]quinoline-1-yl)amino)-2-fluoro-2-methyl-3-oxopropoxy)methyl)carbamoyl)-5-isopropyl-4,7,11-trioxo-12-oxa-3,6,10-triazahexadecyl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-26). ZnBr2 (124 mg, 0.54 mmol) was added to a solution of 1-26a (18 mg, 0.014 mmol) in DCM (0.4 mL), and the solution was stirred under reflux for 16 h. The resulting solution was quenched with 0.1% FA aqueous solution at 0 °C. Preparative HPLC was performed (method: column XBridge Prep C18 OBD 5 μm 19). The purified solution was purified by lyophilization to give 1-26 (12 mg, 72.1% yield) as a white solid. The mobile phase was: A-water (0.1% FA); B-acetonitrile; flow rate: 20 mL / min. MS (ESI) m / z: 1224.6 [M+H] + .

[0337] Examples 1-27 Step 1: (2S,3S,4S,5R,6R)-6-(((7R,17S,20S)-7-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-20-(((2-((((1S,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxanepenten[ [4,5-g]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)carbamoyl)oxy)ethoxy)methyl)carbamoyl)-17-isopropyl-2,2-dimethyl-4,11,15,18-tetraoxo-3,10-dioxa-5,12,16,19-tetraazacotetra-21-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-27a). HATU (4.4 mg, 0.012 mmol) and DIPEA (0.003 mL, 0.019 mmol) were added to a solution of 1-11 g (4.7 mg, 0.012 mmol) in anhydrous DMF (0.5 mL), and the mixture was stirred for 10 min at room temperature. Then add 1-25 c (10 mg, 0.01 mmol) to the above mixture and stir for 10 min at room temperature. Analyze by preparative HPLC (method: XBridge Prep C18 OBD column 5 μm 19). The purified solution was purified by lyophilization at a flow rate of 150 mL / min. The fractions were then lyophilized to give 1-27a (7.3 mg, 58.1% yield) as a white solid. MS (ESI) m / z: 1308.6 [M+H] + .

[0338] Step 2: (2S,3S,4S,5R,6R)-6-(((2S,5S,15R)-16-amino-15-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-2-(((2-((((1S,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[d] [e][1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]inzazo[1,2-b]quinoline-1-yl)carbamoyl)oxy)ethoxy)methyl)carbamoyl)-5-isopropyl-4,7,11-trioxo-12-oxa-3,6,10-triazahexadecyl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-27). ZnBr2 (51 mg, 0.22 mmol) was added to a solution of 1-27a (7.3 mg, 0.006 mmol) in DCM (0.2 mL), and the solution was stirred under reflux for 16 h. The resulting solution was quenched with 0.1% FA aqueous solution at 0 °C. Preparative HPLC was performed (method: column XBridge Prep C18 OBD 5 μm 19). The purified solution was purified by lyophilization to give 1-27 (4.2 mg, 62.3% yield) as a white solid. The mobile phase was: A-water (0.1% FA); B-acetonitrile; flow rate: 20 mL / min. MS (ESI) m / z: 1208.7 [M+H] + .

[0339] Examples 1-28 Step 1: (2R,3R,4S,5S,6S)-triacetic acid 2-((S)-2-((S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-3-(((3-(((1S,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro- 1H,13H-benzo[de][1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-trimethyl ester (1-28a). HATU (238 mg, 0.62 mmol) and DIPEA (0.28 mL, 1.55 mmol) were added to a solution of 1-2 h (450 mg, 0.52 mmol) and P7 (340 mg, 0.62 mmol) in anhydrous DMF (10 mL), and stirred for 30 min at room temperature. The solution was concentrated and purified by silica gel column chromatography (MeOH / CH2Cl2 = 5 / 95) to give 1-28a as a yellow solid (440 mg, 65.5% yield). MS (ESI) m / z: 1301.6 [M+H] + .

[0340] Step 2: (2R,3R,4S,5S,6S)-triacetic acid 2-((S)-2-((S)-2-amino-3-methylbutamido)-3-(((3-(((1S,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (1-28b). Et₂NH (0.71 mL, 6.76 mmol) was added to a solution of 1-28a (440 mg, 0.34 mmol) in anhydrous DMF (5 mL), and the mixture was stirred at room temperature for 30 min. The resulting solution was concentrated to give 1-28b (350 mg, 95.9% yield), which was used directly in the next step. MS (ESI) m / z: 1079.5 [M+H] + .

[0341] Step 3: (2S,3S,4S,5R,6R)-6-((S)-2-((S)-2-amino-3-methylbutamido)-3-(((3-(((1S,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-28c)trifluoroacetate. 1N K₂CO₃ (1.14 mL, 1.14 mmol) was added to a solution of 1-28b (350 mg, 0.32 mmol) in MeOH (5 mL) and water (2 mL) at 0 °C, and the mixture was stirred for 30 min at room temperature. The resulting solution was quenched with HOAc, the MeOH was removed by concentration, and the solution was analyzed by preparative 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% TFA):B-acetonitrile; flow rate: 20 mL / min). The fractions were lyophilized to give 1-28c (trifluoroacetate) as a white solid (130 mg, 38.1% yield). MS (ESI) m / z: 939.5 [M+H] + .

[0342] Step 4: (2S,3S,4S,5R,6R)-6-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexamido)-3-methylbutamido)-3-(((3-(((1S,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14) ,16-hexahydro-1H,13H-benzo[de][1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-28). HATU (14.7 mg, 0.038 mmol) and DIPEA (0.011 mL, 0.064 mmol) were added to a solution of 1-1q (7.2 mg, 0.034 mmol) in anhydrous DMF (1.0 mL), and the mixture was stirred for 10 min at room temperature. 1-28c (30.0 mg, 0.032 mmol) was added to the above mixture, and the mixture was stirred for 10 min at room temperature. Preparative HPLC (method: XBridge Prep C18 OBD column 5 μm 19) The purified solution was purified by lyophilization to give 1-28 (15.0 mg, 41.5% yield) as a white solid. The mobile phase was: A-water (0.1% TFA); B-acetonitrile; flow rate: 20 mL / min. MS (ESI) m / z: 1132.8 [M+H] + .

[0343] Examples 1-29 Step 1: (2S,3S,4S,5R,6R)-6-((S)-3-(((3-(((1S,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]in Zizazo[1,2-b]quinoline-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-2-((S)-3-methyl-2-(4-(5-(methanesulfonyl)-1,2,4-thiadiazol-3-yl)benzamido)butamido)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-29). HATU (7.9 mg, 0.021 mmol) and DIPEA (0.008 mL, 0.043 mmol) were added to a solution of 1-13f (5.9 mg, 0.021 mmol) in anhydrous DMF (0.5 mL), and the mixture was stirred for 15 min at room temperature. Then, 1-28c (trifluoroacetate) (18.0 mg, 0.017 mmol) was added to the mixture, and the mixture was stirred for 10 min at room temperature. Preparative HPLC (method: XBridge Prep C18OBD column, 5 μm, 19) The purified solution was purified by lyophilization to give 1-29 (5.5 mg, 26.7% yield) as a yellow solid. The mobile phase was: A-water (0.1% TFA); B-acetonitrile; flow rate: 20 mL / min. MS (ESI) m / z: 1206.0 [M+H] + .

[0344] Examples 1-30 Step 1: (2S,3S,4S,5R,6R)-6-(((7R,17S,20S)-7-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-20-(((3-(((1S,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxanepenten[4,5-g ]pyrano[3',4':6,7]inzazo[1,2-b]quinoline-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)carbamoyl)-17-isopropyl-2,2-dimethyl-4,11,15,18-tetraoxo-3,10-dioxa-5,12,16,19-tetraazacotetra-21-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-30a). HATU (7.9 mg, 0.021 mmol) and DIPEA (0.008 mL, 0.043 mmol) were added to a solution of 1-11 g (5.9 mg, 0.021 mmol) in anhydrous DMF (0.5 mL), and stirred for 15 min at room temperature. Then, 1-28c (trifluoroacetate) (18.0 mg, 0.017 mmol) was added to the above mixture, and the mixture was stirred at room temperature for 10 min. The sample was analyzed by preparative HPLC (method: XBridge Prep C18 OBD column 5 μm 19). The purified solution was purified by lyophilization (150 mm; mobile phase: A-water (0.1% TFA): B-acetonitrile; flow rate: 20 mL / min), and the fractions were lyophilized to give 1-30a (5.5 mg, 26.7% yield) as a yellow solid. MS (ESI) m / z: 1320.8 [M+H] + .

[0345] Step 2: (2S,3S,4S,5R,6R)-6-(((2S,5S,15R)-16-amino-15-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-2-(((3-(((1S,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1] [3] Dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]inzazido[1,2-b]quinoline-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)carbamoyl)-5-isopropyl-4,7,11-trioxo-12-oxa-3,6,10-triazahexadecyl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-30). ZnBr2 (110 mg, 0.49 mmol) was added to a solution of 1-30a (16 mg, 0.012 mmol) in DCM (0.4 mL), and the mixture was stirred under reflux for 16 h. The resulting solution was quenched with 0.1% FA aqueous solution at 0 °C. Preparative HPLC was performed (method: column XBridge Prep C18 OBD 5 μm 19). The purified solution was purified by lyophilization to give 1-30 (12 mg, 72.1% yield) as a white solid. The mobile phase was: A-water (0.1% FA); B-acetonitrile; flow rate: 20 mL / min. MS (ESI) m / z: 1220.6 [M+H] + .

[0346] Examples 1-31 Step 1: (2R,3R,4S,5S,6S)-triacetic acid 2-((2S)-2-((S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-3-(((2-((((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)methyl)(methyl)amino)ethoxy)methyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (1-31a). Add 4 Å MS (200 mg) to a solution of 1-2f (55 mg, 0.066 mmol) and P11 (45 mg, 0.055 mmol) in anhydrous THF (2 mL) and stir for 20 min. Add Sc(OTf)3 (41.7 mg, 0.083 mmol) and stir the solution at 40 °C for 15 h. Add 1-2f (23 mg) and stir the solution at 40 °C for 8 h. Add 4 Å MS (100 mg) and Sc(OTf)3 (15 mg) and then stir at 40 °C for another 14 h. Filter the mixture and wash the filtrate with saturated NaHCO3 (5 mL). Use DCM / MeOH (10:1, 5.5 mL) 3) Extraction of the aqueous phase. The organic phase was concentrated and purified by rapid column chromatography (DCM / MeOH = 100 / 0 to 90 / 10) to obtain crude product 1-31a as a yellow solid (84 mg, theoretical 55 mg, 78.9% yield). LCMS (ESI): 1283.9 [M+H] + .

[0347] Step 2: (2R,3R,4S,5S,6S)-triacetic acid 2-((2S)-2-((S)-2-amino-3-methylbutamido)-3-(((2-((((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)methyl)(methyl)amino)ethoxy)methyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (1-31b). Add Et₂NH₃ (91 μL, 0.87 mmol) to a solution of 1-31a (84 mg crude product, theoretical 55 mg, 0.044 mmol) in anhydrous DMF (1 mL), and stir the solution at room temperature for 10 min. Dilute the solution with toluene, concentrate, and use directly without further purification. LCMS (ESI): 1061.8 [M+Na] + .

[0348] Step 3: (2S,3S,4S,5R,6R)-6-((2S)-2-((S)-2-amino-3-methylbutamido)-3-(((2-((((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)methyl)(methyl)amino)ethoxy)methyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-31c). K₂CO₃ (18 mg, 0.13 mmol) was added to a solution of 1-31b (45 mg, crude, 0.043 mmol) in MeOH / H₂O (1 / 0.5 mL), and the mixture was stirred at room temperature for 2 h. Water (1 mL) was added, and the solution was adjusted to pH 6 with 1 N HCl. Preparative HPLC was performed (mobile phase A: 0.1% FA aqueous solution, B: MeCN; gradient: 20%-28%-32% B; flow rate: 20 mL / min; column: XbridgePrep C18 OBD 5 μm, 19...). The purified solution (150 mm) was lyophilized to give the title compound 1-31c (11 mg, 28.3% yield, 89% purity) as a white solid. LCMS (ESI): 899.8 [M+H] + .

[0349] Step 4: (2S,3S,4S,5R,6R)-6-((2S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexamido)-3-methylbutamido)-3-(((2-((((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)methyl)(methyl)amino)ethoxy)methyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-31). DIEA (4 μL, 0.024 mmol) was added to a solution of 1-1q (3.2 mg, 0.015 mmol) and HATU (5.2 mg, 0.013 mmol) in anhydrous DMF (0.5 mL), and the mixture was stirred for 5 min at room temperature. The reaction solution was then added to a solution of 1-31c (11 mg, 0.012 mmol) in anhydrous DMF (0.5 mL), and the mixture was stirred for 15 min at room temperature. The reaction was then analyzed by preparative HPLC (mobile phase A: 0.1% FA aqueous solution, B: MeCN; gradient: 25%-40%-95% B; flow rate: 20 mL / min; column: Xbridge Prep C18 OBD 5 μm, 19). The purified solution (150 mm) was lyophilized to give the title compound 1-31 (4.3 mg, 32% yield, 96% purity) as a grayish-white solid. LCMS (ESI): 1092.8 [M+H] + .

[0350] Examples 1-32 Step 1: (2R,3R,4S,5S,6S)-triacetic acid 2-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-3-(((3-(((1R,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)propoxy)methyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (1-32a). Add 4 Å MS (50 mg) to a solution of P12-1 (50 mg, 0.10 mmol) and 1-2f (99 mg, 0.12 mmol) in anhydrous THF (2 mL) and stir for 20 min. Add Sc(OTf)3 (74 mg, 0.15 mmol) and stir at 35 °C for 14 h and then at 50 °C for 16 h. Filter the mixture and wash the filtrate with saturated NaHCO3 (5 mL). Use DCM / MeOH (10:1, 5.5 mL) 3) Extraction of the aqueous phase. The organic phase was concentrated and purified by rapid column chromatography (petroleum ether / EtOAc = 70 / 30 to 0 / 100) to give compound 1-32a as a white solid (50 mg, 39.4% yield). MS (ESI) m / z: 1245.0 [M+H] + .

[0351] Step 2: (2R,3R,4S,5S,6S)-triacetic acid 2-((S)-2-((S)-2-amino-3-methylbutamido)-3-(((3-((1R,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)propoxy)methyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (1-32b). Add Et₂NH (0.083 mL, 0.81 mmol) to a solution of 1-32a (50 mg, 0.040 mmol) in anhydrous DMF (2 mL), and stir for 15 min at room temperature. Dilute the solution with toluene, concentrate, and use directly without further purification. MS (ESI) m / z: 1022.8 [M+H] + .

[0352] Step 3: (2S,3S,4S,5R,6R)-6-((S)-2-((S)-2-amino-3-methylbutamido)-3-(((3-((1R,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)propoxy)methyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-32c). Add Na₂CO₃ (36 mg, 0.34 mmol) to a solution of 1-32b (50 mg, 0.049 mmol) in MeOH / H₂O (5 / 2 mL) and stir for 1 h at room temperature. Adjust the pH of the solution to 4 with HOAc. Analyze the solution by preparative HPLC (mobile phase A: 0.1% FA aqueous solution, B: MeCN; gradient: 20%-32%-35%-90% B; flow rate: 20 mL / min; column: Xbridge Prep C18 OBD 5 μm, 19... The purified solution (150 mm) was lyophilized to give the title compound 1-32c (20 mg, 47.6% yield) as a yellow solid. MS (ESI) m / z: 882.7 [M+H] + .

[0353] Step 4: (2S,3S,4S,5R,6R)-6-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexamido)-3-methylbutamido)-3-(((3-((1R,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-1-yl)propoxy)methyl)amino)-3-oxopropoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (1-32). Add DIEA (33 μL, 0.12 mmol) to a solution of 1-1q (26 mg, 0.12 mmol) and HATU (35 mg, 0.093 mmol) in anhydrous DMF (3 mL), and stir for 5 min at room temperature. Add the reaction solution to a solution of 1-32c (55 mg, 0.062 mmol) in anhydrous DMF (2 mL), and stir for 10 min at room temperature. Analyze the reaction solution by preparative HPLC (mobile phase A: 0.1% FA aqueous solution, B: MeCN; gradient: 32%-43%-95% B; flow rate: 20 mL / min; column: Xbridge Prep C18 OBD 5 μm, 19). The purified solution (150 mm) was lyophilized to give the title compound 1-32 (9.4 mg, 38.5%) as a yellow solid. MS (ESI) m / z: 1075.9 [M+H] + .

[0354] Table 1:

[0355] ADC preparation and characterization Preparation of DAR8 antibody-drug conjugates. Antibody (e.g., ifenamarab) in conjugation buffer (concentration 0.5-25 mg / mL, PBS buffer pH 6.0-8.5) was incubated at reduction temperature (0℃-40℃) for 10 min. 8-15 equivalents of TECP solution (5 mM stock solution in PBS buffer) were added to the reaction mixture, and the reduction reaction was allowed to proceed at reduction temperature for 1-8 h. After cooling the reduction mixture to 0-25℃, organic solvents (e.g., DMSO, DMF, DMA, PG, acetonitrile, 0-25% v / v) and adapter-loador stock solution (10-25 equivalents, 10 mM stock solution in organic solvent) were added incrementally. The conjugation solution was incubated at 0-25℃ for 1-3 h, and the reaction mixture was quenched with N-acetylcysteine ​​(1 mM stock solution). The solution is buffer-exchanged (rotary desalting column, ultrafiltration, and dialysis) to 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).

[0356] ADC characterization. The following analytical methods were used to characterize the ADCs. The drug-to-antibody ratio (DAR) of the ADCs was determined by LCMS or HIC methods. The SEC purity of the ADCs was >95%.

[0357] 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) Injection 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.

[0358] HIC method: 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) Injection 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 programs: 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.

[0359] SEC approach: 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 μm 4.6×150 mm, Waters Column temperature: room temperature Mobile phase A: 200 mM phosphate buffer, 250 mM potassium chloride, 15% isopropanol, pH 7.0 Gradient program: 0.3 mL / min flow rate during 10 min isocratic elution. Injection sample volume: 20 μg Evaluation of ADC hydrophobicity. According to HIC (hydrophobic interaction column) chromatography, ADCs with higher hydrophobic properties will appear with a later retention time.

[0360] Table 2.

[0361] Antibody information Ifenamic acid antibody (anti-B7H3 antibody) Light chain sequence (SEQ ID NO:1) EIVLTQSPATLSLSPGERATLSCRASSRLIYMHWYQQKPGQAPRPLIYATSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWNSNPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Heavy chain sequence (SEQ ID NO:2) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYVMHWVRQAPGQGLEWMGYINPYNDDVKYNEKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARWGYYGSPLYYFDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK cell lines A375 (ATCC, CRL-1619). A-375 is a cell line exhibiting epithelial morphology, isolated from the skin of a 54-year-old female patient with malignant melanoma, and purchased from ATCC. The basal medium for A375 was DMEM, high glucose, and GlutaMAX™ supplement (Gibco, 10566024). To prepare complete growth medium, 10% fetal bovine serum (Gibco, 10099-141C) was added to the basal medium. The cell line was grown at 37°C in a humidified 5% CO2 atmosphere, and the presence of Mycoplasma was periodically detected using the MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza, LT07-710).

[0362] Calu-6 (ATCC, HTB-56). Calu-6 is an epithelial cell line isolated from undifferentiated carcinoma, and was purchased from ATCC. The basal medium for Calu-6 was Eagle's Minimum Essential Medium (ATCC, 30-2003). To prepare complete growth medium, 10% fetal bovine serum (Gibco, 10099-141C) was added to the basal medium. The cell line was grown at 37°C in a humidified 5% CO2 atmosphere, and the presence of mycoplasma was periodically tested using the MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza, LT07-710).

[0363] NCI-H358 (ATCC, CRL-5807). NCI-H358 cells were epithelioid cells isolated from the bronchioles of male patients with bronchoalveolar carcinoma, and were purchased from ATCC. The basal medium for NCI-H358 was ATCC 30-2001 RPMI-1640 medium. To prepare complete growth medium, fetal bovine serum (Gibco, 10099-141C) was added to the basal medium to a final concentration of 10%. The cell line was grown at 37°C in a humidified 5% CO2 atmosphere, and the presence of mycoplasma was periodically tested using the MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza, LT07-710).

[0364] NCI-H1650 (ATCC, CRL-5883). NCI-H1650 is a cell line exhibiting epithelial morphology, isolated in 1987 from the lung tissue of a 27-year-old male smoker with stage 3 bronchoalveolar carcinoma. NCI-H1650 was purchased from ATCC. The basal medium for NCI-H1650 was ATCC RPMI-1640 medium ATCC 30-2001. To prepare complete growth medium, 10% fetal bovine serum (Gibco, 10099-141C) was added to the basal medium. The cell line was grown at 37°C in a humidified 5% CO2 atmosphere, and the presence of mycoplasma was periodically tested using the MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza, LT07-710).

[0365] NCI-H1048 (ATCC, CRL-5853). NCI-H1048 is a cell line exhibiting epithelial morphology, and was purchased from ATCC. The basal medium for NCI-H1048 was ATCC DMEM:F12 medium, catalog number 30-2006. To prepare complete growth medium, fetal bovine serum (Gibco, 10099-141C) was added to the basal medium to a final concentration of 10%. The cell line was grown at 37°C in a humidified 5% CO2 atmosphere, and the presence of mycoplasma was periodically tested using the MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza, LT07-710).

[0366] Capan-1 (ATCC, HTB-79). Capan-1 is an epithelial cell line isolated from the pancreas of a 40-year-old Caucasian male with pancreatic adenocarcinoma, and was purchased from ATCC. The basal medium for Capan-1 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 the basal medium to a final concentration of 20%. The cell line was grown at 37°C in a humidified 5% CO2 atmosphere, and the presence of mycoplasma was periodically detected using the MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza, LT07-710).

[0367] MDA-MB-453 (SIBS). MDA-MB-453 was derived from exudate from a 48-year-old female patient with metastatic breast cancer (involving lymph nodes, brain, pleural cavity, and pericardial cavity), and was purchased from SIBS. The basal medium for MDA-MB-453 was RPMI 1640 HEPES medium (Gibco, 22400105). To prepare complete growth medium, 10% fetal bovine serum (Gibco, 10099-141C) was added to the basal medium. The cell line was grown at 37°C in a humidified 5% CO2 atmosphere, and the presence of mycoplasma was periodically tested using the MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza, LT07-710).

[0368] The binding affinity of antibodies to various cell lines was determined using the Quantum™ Simply Cellular® (“QSC”) microsphere kit, according to the manufacturer’s protocol (Bangs Laboratories, Inc.). Data are reported in Table 3.

[0369] Table 3: B7H3 expression levels

[0370] Payload cell killing in A375 and Calu-6 cancer lines Cell killing efficacy of payload cells was evaluated in A375 and Calu-6 cancer lines. Cells were seeded at 100 μl / well (A375 (1E3 / well) or Calu-6 (2E3 / well)) into 96-well plates (Greiner: 655090) and incubated overnight at 37°C and 5% CO2. 50 μl / well of fresh growth medium containing different concentrations of the compound was added, and the plates were incubated at 37°C and 5% CO2 for 6 days. Cell viability was assessed using Cell Titer-Glo (Promega, G7573). 70 μL of Cell Titer-Glo® reagent was added to each well. The plates were incubated at room temperature for 10 minutes to stabilize the luminescence signal. The plates were analyzed using a microplate reader.

[0371] The data is summarized in Tables 4A and 4B, and Figures 1 to 4 middle.

[0372] Table 4A.

[0373] Table 4B.

[0374] ADCs directly kill NCI-H358, Capan-1, and MDA-MB-453 cancer cell lines. Direct ADC killing was evaluated in NCI-H358, Capan-1, and MDA-MB-453 cancer lines. Cells were seeded at 80 μl / well (NCI-H358 or MDA-MB-453 (2E3 / well) or Capan-1 (4E3 / well)) into 3D 96-well plates (Corning: 4520) and incubated overnight at 37°C and 5% CO2. Fresh growth medium containing different concentrations of ADC was added to each well, and the plates were incubated at 37°C and 5% CO2 for 6 days. Cell viability was assessed using 100 μl / well of 3D reagent (Promega, G9683). 3D plates were incubated at room temperature for 30 minutes to stabilize the luminescence signal. The plates were analyzed using a microplate reader.

[0375] Data is summarized in Tables 5A to 5H and Figures 5 to 25 and Figures 38 to 40 In the study, the isotype ADC was an antiviral Ab that did not recognize B7H3 and was used as a negative control.

[0376] Table 5A.

[0377] Table 5B.

[0378] Table 5C.

[0379] Table 5D.

[0380] Table 5E.

[0381] Table 5F.

[0382] Table 5G.

[0383] Table 5H.

[0384] ADC Bystander Kill: Co-cultivation of NCI-H358 and MDA-MB-453-nanoLuc MDA-MB-453-nanoLuc cell line construction. PT67-nanoLuc cells were cultured, and then the cell-medium (containing the virus / nano-Luc gene) was collected and filtered. MDA-MB-453 cells were seeded at 1E5 cells / well in 6-well plates. The plates were incubated overnight at 37°C and 5% CO2. PT67-nanoLuc cell culture medium and 8 μg / ml polybrene were added. Infection was repeated daily for three days. MDA-MB-453-nanoLuc cells were cultured for 5 days with the addition of 1 mg / ml geneticin. MDA-MB-453-nanoLuc cells were collected, and Nano-Glo reagent (Promega: N1120) was added to test nano-Luc transfection efficiency.

[0385] ADC bystander killing. NCI-H358 cells and MDA-MB-453-nanoLuc (10:1) or MDA-MB-453-nanoLuc cells alone were co-cultured or cultured at 80 μl / well in 3D 96-well plates (Corning: 4520). Cells were incubated overnight at 37°C and 5% CO2. Fresh growth medium containing different concentrations of ADC was added at 40 μl / well. Cells were incubated at 37°C and 5% CO2 for 6 days. The 3D plates were centrifuged at 1500 rpm at 25°C for 5 min, and the supernatant was discarded. Calu-6-nanoLuc cell viability was detected using 150 μl / well of Nano-Glo reagent (Promega: N1120). The 3D plates were incubated at room temperature for 10 min to stabilize the luminescence signal. The plates were analyzed using a microplate reader.

[0386] Data is summarized in Tables 6A to 6G and Figures 26 to 37 , Figure 41 and Figure 42 middle.

[0387] Table 6A.

[0388] Table 6B.

[0389] Table 6C.

[0390] Table 6D.

[0391] Table 6E.

[0392] Table 6F.

[0393] Table 6G.

[0394] In vivo efficacy study of ADC Subcutaneous implantation of 3 × 10 PBS / Matrix gel per 200 μL in the right abdomen of female BALB / c nude mice 6 H1650 / capan-1 / H441 / H1048 / HCC827 / H1975 cells were inoculated. After inoculation, tumor volume was measured twice weekly in two dimensions using a diameter gauge, and the formula V = 0.5(a × b) was used. 2 ) in mm 3This indicates that a and b are the long and short diameters of the tumor, respectively. When the tumor size reaches an average volume of approximately 200 mm... 3 Mice were randomly assigned to three groups of eight animals each, and on day 1, they were treated intravenously with a medium or ADC at doses of 1 mpk, 3 mpk, or 10 mpk. Partial regression (PR) was defined as tumor volume less than 50% of the initial tumor volume on day 1 in three consecutive measurements, and complete regression (CR) was defined as tumor volume less than 14 mm in three consecutive measurements. 3 Data are presented as mean tumor volume ± standard error of the mean (SEM). Tumor growth inhibition (TGI) was calculated using the following formula: Tumor volume after treatment at time t = time t Tumor volume after treatment at time t0 = 0 Placebo tumor volume at time t = placebo t Placebo t0 = Placebo tumor volume at time 0 ADC plasma stability ADC is incubated together with plasma: 1. Dilute the ADC in mouse or human plasma to produce a final solution of 100 μg / mL ADC in plasma. 2. Incubate the sample at 37°C. 3. Take out equal aliquots (100 μL) at four time points (0 h, 4 h, 24 h, 72 h, 96 h or 168 h). 4. Freeze the sample at -80°C until analysis.

[0395] Plasma payload concentration was measured under the following conditions: Instrument: LC-MS / MS (Triple Quad 5500) Monitor: MRM Column: Advanced Materials Technology, HALO AQ-C18 2.7 μm 90Å, 50 2.1 mm Column temperature: 40℃ Mobile phase A: H2O - 0.1% FA Mobile phase B: ACN - 0.1% FA MMAE gradient programs: 20% B-20% B (0 min-0.2 min), 20% B-80% B (0.2 min-1.5 min), 80% B-80% B (1.5 min-2.2 min), 80% B-20% B (2.20 min-2.21 min), 20% B-20% B (2.21 min-3.0 min); Dxd gradient programs: 2% B-2% B (0 min-0.2 min), 2% B-98% B (0.2 min-1.2 min), 98% B-98% B (1.2 min-2.0 min), 98% B-2% B (2.0 min-2.01 min), 2% B-2% B (2.01 min-4.0 min); Injection sample volume: 10 μL (DXd or DXd analogue) Plasma ADC and total Ab (Tab) concentrations were measured under the following conditions: Assay: Ligand binding assay (ELISA) Capture reagent: B7H3 ECD Detection reagents: For ADC, the reagents are anti-payload Abs, and for total Abs, the reagents are anti-human IgG polyclonal Abs.

[0396] In the mouse PK study, blood samples were collected from mice carrying H1650 / capan-1 / H441 / H1048 / HCC827 / H1975 tumors or non-tumor-carrying mice at 0 h, 2 h, 4 h, 8 h, 24 h, 72 h, and 168 h after intravenous administration of a single dose of ADC. Plasma was then separated by centrifugation (4 °C, 3000 × g, 7 min). ADC and total Ab concentrations were measured using an internally developed Meso Scale Discovery (MSD) ligand binding method. In short, a His-tagged B7H3 extracellular domain fusion protein was used as the capture reagent, and biotin-labeled anti-payload Abs or goat anti-human κAbs were used as detection reagents for ADC or total Ab measurements, respectively.

[0397] Equivalent content The foregoing written description is considered sufficient to enable those skilled in the art to practice the embodiments. The foregoing description and examples detail certain embodiments and illustrate the best mode considered by the inventors. However, it will be understood that, however detailed the foregoing may be presented in the text, the embodiments can be practiced in many ways and should be interpreted in accordance with the appended claims and any equivalents thereof.

Claims

1. A compound of formula (I): ， Or its pharmaceutically acceptable salts, tautomers, solvates or stereoisomers, wherein: Z is a heterocycle, which is optionally substituted with at least one carbonyl or methanesulfonyl group; Y is either aryl or -C≡C-(CH2). a - The subscript 'a' is 1, 2, 3, or 4; The subscript y' is 0 or 1; X represents C that has been substituted or not substituted. 1-5 alkyl; The subscript x' is 0 or 1; W stands for oxygen; The subscript w' is 0 or 1; V is C 1-4 Alkyl or -C(=O)-NH-(CH2) b Wherein N is optionally C 1-5 Alkyl substitution and subscript b is 1 or 2; The subscript v' is 0 or 1; U is a hydrophilic group; T represents oxygen or a bond; P represents the payload residue or drug unit; and The subscript s is 0 or 1.

2. The compound of claim 1, wherein Z is , or .

3. The compound of claim 1 or 2, wherein Y is phenylene or -C≡C-(CH2)3-.

4. The compound according to any one of claims 1-3, wherein X is -(CH2)5-, -CH(CH2NH2)(CH2)4-, -CH2CH(NH2)CH2-, -CH(CH2NH2)CH2- or -CH(CH2NH2)(CH2)2-.

5. The compound according to any one of claims 1-4, wherein V is ethyl or -C(=O)-NH-(CH2). b Wherein N is optionally C 1-3 Alkyl substitution and subscript b is 1 or 2.

6. The compound of claim 5, wherein V is , or .

7. The compound according to any one of claims 1-6, wherein U is a sugar, phosphate ester, sulfate ester, phosphate diester or phosphonate.

8. The compound of claim 7, wherein U is a sugar, and the sugar is β-D-galactose, N-acetyl-PD-galactosamine, N-acetyl-aD-galactosamine, N-acetyl-PD-glucosamine, β-D-glucuronic acid, aL-iduronic acid, aD-galactose, aD-glucose, β-D-glucose, aD-mannose, β-D-mannose, aL-fucose, β-D-xylose, neuraminic acid; and modified with sulfate, phosphate, carboxyl, amino, or O-acetyl groups.

9. The compound of claim 8, wherein U is or .

10. The compound of claim 9, wherein U is or .

11. The compound according to any one of claims 1-10, wherein T is oxygen.

12. The compound according to any one of claims 1-11, wherein P is: , , , , , , , , , or .

13. The compound of claim 1, wherein the compound is selected from any of the following, or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotopic conformation, or prodrug: 。 14. An antibody-drug conjugate of formula (II): ， Or its pharmaceutically acceptable salts, tautomers, solvates or stereoisomers, wherein: BA is a conjugate selected from humanized, chimeric, or human antibodies or their antigen-binding fragments; Z' is a heterocycle, which is optionally substituted with at least one carbonyl or methanesulfonyl group; Y is either aryl or -C≡C-(CH2). a -; The subscript 'a' is 1, 2, 3, or 4; The subscript y' is 0 or 1; X represents C that has been substituted or not substituted. 1-5 alkyl; The subscript x' is 0 or 1; W stands for oxygen; The subscript w' is 0 or 1; V is C 1-4 Alkyl or -C(=O)-NH-(CH2) b Wherein N is optionally C 1-5 Alkyl substitution and subscript b is 1 or 2; The subscript v' is 0 or 1; U is a hydrophilic group; T represents oxygen or a bond; P represents the payload residue or drug unit; The subscript s is 0 or 1; and The subscript n is from 1 to 15.

15. The antibody-drug conjugate of claim 14, wherein Z' is , , , or ,and Indicates that Z' is connected to the key BA.

16. The antibody-drug conjugate of claim 14 or 15, wherein Y is phenylene or -C≡C-(CH2)3-.

17. The antibody-drug conjugate according to any one of claims 14-16, wherein X is -(CH2)5-, -CH(CH2NH2)(CH2)4-, -CH2CH(NH2)CH2-, -CH(CH2NH2)CH2-, or -CH(CH2NH2)(CH2)2-.

18. The antibody-drug conjugate according to any one of claims 14-17, wherein V is ethyl or -C(=O)-NH-(CH2). b Wherein N is optionally C 1-3 Alkyl substitution and subscript b is 1 or 2.

19. The antibody-drug conjugate of claim 18, wherein V is... , or .

20. The antibody-drug conjugate according to any one of claims 14-19, wherein U is a sugar, phosphate ester, sulfate ester, phosphate diester, or phosphonate.

21. The antibody-drug conjugate of claim 20, wherein U is a sugar, and said sugar is β-D-galactose, N-acetyl-PD-galactosamine, N-acetyl-aD-galactosamine, N-acetyl-PD-glucosamine, β-D-glucuronic acid, aL-iduronic acid, aD-galactose, aD-glucose, β-D-glucose, aD-mannose, β-D-mannose, aL-fucose, β-D-xylose, or neuraminic acid; and modified with sulfate, phosphate, carboxyl, amino, or O-acetyl groups.

22. The antibody-drug conjugate of claim 21, wherein U is or .

23. The antibody-drug conjugate of claim 22, wherein U is or .

24. The antibody-drug conjugate according to any one of claims 14-23, wherein T is oxygen.

25. The antibody-drug conjugate according to any one of claims 14-24, wherein P is: , , , , , , , , , or .

26. The antibody-drug conjugate according to any one of claims 14-25, wherein BA is ifenamarab.

27. An antibody-drug conjugate or a pharmaceutically acceptable salt, solvate, or hydrate thereof, any of the following: Where Ab is a humanized, chimeric, or human antibody or its antigen-binding fragment, and the subscript n is from 1 to 15.

28. The antibody-drug conjugate of claim 27, wherein Ab is ifenamarab.

29. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 14-28 or a pharmaceutically acceptable salt, tautomer, solvate or stereoisomer thereof, and a pharmaceutically acceptable excipient.

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