Linker-drug conjugate and antibody-drug conjugate both comprising silatecan derivative

Abstract

The present application relates to a linker-drug conjugate comprising a silatecan derivative, and an antibody-drug conjugate comprising same. More specifically, the present invention relates to a novel linker-drug conjugate and antibody-drug conjugate exhibiting excellent anticancer activity by designing, as an ADC payload, a silatecan derivative having a novel structural modification introduced thereinto, in order to overcome the problem that existing silatecan-based drugs have limited efficacy in the ADC form.

Description

Linker-drug conjugates and antibody-drug conjugates comprising silatecan derivatives

[0001] The present invention relates to linker-drug conjugates and antibody-drug conjugates comprising a silatecan derivative.

[0002] In general, camptothecins and homocamptothecins are DNA topoisomerase I inhibitors, useful as anticancer agents, for example. Analogs of the naturally occurring camptothecins are the most important class of compounds useful in the treatment of solid tumors. Topotecan (tpt) and CPT-11 were the first two camptothecins to receive full approval status by the U.S. Food and Drug Administration (FDA) (topotecan in 1996 for second-line therapy of advanced epithelial ovarian cancer and in 1998 for small cell lung cancer, and CPT-11 in 1998 for first-line therapy of colon cancer).

[0003] 7-Silyl camptothecin (2) (sometimes called silatecan and homosilatecan) is an important class of lipophilic camptothecin analogs. For example, most of the interesting silatecans and homosilatecans contain one or more additional substituents (e.g., hydroxy or amino) on the A ring, and combinations of these substituents can provide significant improvements over the corresponding monosubstituted analogs. For example, 7-tert-butyldimethylsilyl-10-hydroxy camptothecin (DB-67) is currently in late preclinical stages. DB-67 and other silatecans and homosilatecans exhibit a variety of interesting properties, including high activity against a broad spectrum of solid tumors, low binding affinity to blood proteins, resistance to lactone ring-opening, high lipophilicity, and other potential oral availability.

[0004] Since most chemotherapy drugs, such as camptothecin, can cause unwanted toxicity due to side effects when used alone, active research and development is currently being conducted to minimize side effects and maximize the effectiveness of the drugs by selectively allowing the drugs to approach tumor cells.

[0005] Antibody-Drug Conjugate (ADC) is a substance that connects an antibody with the ability to specifically bind to a target antigen on the surface of cancer cells and a cytotoxic drug via a linker. Unlike traditional chemotherapy, it has the ability to accurately deliver the drug to tumors that express the target antigen.

[0006] [Prior Art Literature]

[0007] [Non-patent literature]

[0008] Josien, H.; Bom, D.; Curran, D.P.; Zheng, Y.-H.; Chou, T.-C. Bioorg Med. Chem. Lett., 7, 3189 (1997)

[0009] The present invention relates to linker-drug conjugates comprising silatecan derivatives and antibody-drug conjugates comprising the same. More specifically, the present invention relates to novel linker-drug conjugates and antibody-drug conjugates exhibiting superior anticancer activity by designing silatecan derivatives with novel structural modifications as ADC payloads to overcome the problem of limited efficacy of existing silatecan-based drugs in ADC form.

[0010] However, the problems that the present invention seeks to solve are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

[0011] The first aspect of the present invention provides a linker-drug conjugate comprising a silatecan derivative or a pharmaceutically acceptable salt thereof, represented by the following chemical formula 1 or chemical formula 2; and a linker, wherein the linker binds to A of the following chemical formula 1 or B of the following chemical formula 2:

[0012] [Chemical Formula 1]

[0013] ;

[0014] [Chemical Formula 2]

[0015] ;

[0016] In the above chemical formula 1 and chemical formula 2,

[0017] A is A 1 -(C 0-3 alkylene)-A 2 -(C 0-3 alkylene)-A 3 -A 4 - and,

[0018] A 1 is -NH2, -OH, or -O-CH2-NH2,

[0019] A 2 and A 3 are, independently of each other, absent, or -O-, -NH, -N(C 1-3 alkyl group)-, or -S-,

[0020] A 4 is -C(=O)- or -SO2-;

[0021] B is B 1 -(C 0-3 alkylene)-B 2 -B 3 -B 4 -B 5 -(C 0-3 alkylene)-and,

[0022] B 1 is -NH2, -OH, or -O-CH2-NH2,

[0023] B 2 and B3 are, independently of each other, absent, or -O-, -NH, -N(C 1-3 alkyl group)-, or -S-;

[0024] B 4 does not exist, or is -C(=O)- or -SO2-;

[0025] B 5 does not exist, or is -NH- or -O-,

[0026] R a , R b , R c , R d , and R e are, each independently, C 1-10 Alkyl group, C 2-10 Alkenyl group, C 2-10 Alkynyl group, C 4-20 Aryl group, -(CH2) m R 11 Ki, or SiR 12 R 13 R 14 , where m is an integer in the range of 1 to 10, and R 11 Silver hydroxyl group, C 1-10 Alkoxy group, amino group, C 1-10 Alkylamino group, di-C 1-10 -alkylamino group, F, Cl, cyano group, or nitro group, where R 12 , R 13 and R 14 are, each independently, C 1-10 Alkyl group or C 4-10 It is an aryl group;

[0027] X is H, F, Cl, nitro, amino, hydroxy, or cyano,

[0028] Y is -NH-,

[0029] Z is hydrogen, F, Cl, hydroxyl group, nitro group, cyano group, azido group, formyl group, hydrazino group, amino group, C 1-10 Alkyl group, C 2-10 Alkenyl group, C 2-10Alkynyl group, C 1-10 Alkoxy group, C 1-10 Aryloxy group, C 1-10 Acyloxy group, -C(O)-C 1-10 Alkyl group, -C(O)-C 4-10 Aryl group, -OC(O)OC 1-10 Alkyl group, -OC(O)NR 15 R 16 and, here, R 15 and R 16 are, independently, H, C 1-10 Alkyl group or C 4-10 It is an aryl group,

[0030] W is H, F, Cl, nitro, amino, hydroxy, or cyano, or

[0031] Z and W are connected to each other to form C containing one or more heteroatoms. 1-5 It may be a heterocyclic alkyl ring.

[0032] The second aspect of the present invention provides a pharmaceutical composition for preventing or treating a proliferative disease, comprising a linker-drug conjugate comprising a silatecan derivative according to the first aspect or a pharmaceutically acceptable salt thereof; and a linker, wherein the linker is bound to A of the following chemical formula 1 or B of the following chemical formula 2.

[0033] The third aspect of the present invention provides an antibody-drug conjugate comprising an antibody; a linker; and a silatecan derivative according to the first aspect or a pharmaceutically acceptable salt thereof, wherein the linker binds to A of the following chemical formula 1 or B of the following chemical formula 2.

[0034] The antibody-drug conjugates according to the embodiments of the present disclosure may be configured to include a hydrophilic moiety in the linker structure to enhance their physicochemical superiority and drug efficacy, or to include a conjugation moiety that does not cause an adverse reaction to prevent undesirable dissociation of the conjugated drug. This enhances in vivo stability and prevents drug efficacy, thereby simultaneously enhancing stability and therapeutic efficacy. Furthermore, by including a property that reduces sensitivity to genes involved in payload resistance, the antibody-drug conjugates can be developed into drugs that exhibit superior therapeutic efficacy against metastatic and drug-resistant tumors compared to existing commercially available substances.

[0035] Figure 1: A graph showing the results of a two-dimensional high-throughput screening for the cell killing effect of six A-ring conversion derivatives based on silatecan in vitro.

[0036] Figure 2: This is a result showing the results of confirming the cell killing effect of three B ring conversion derivatives based on silatecan in vitro through two-dimensional high-throughput screening.

[0037] Figure 3: Graph showing the evaluation of cell killing efficacy of silatecan parental structure derivatives (AM20002, AM20008, and AM20011) in vitro.

[0038] Figures 4a to 4i: LC-MS data results for DAR confirmation of synthesized antibody-drug conjugates, the target antibody-drug conjugates being: Trastuzumab-AM21603, Trastuzumab-AM21604, Trastuzumab-AM21605, Trastuzumab-AM21607, Trastuzumab-AM21608, Trastuzumab-AM21609, Trastuzumab-AM21610, Trastuzumab-AM21611, and Trastuzumab-AM21613.

[0039] Figure 5: Graph showing the results of cell killing efficacy through three-dimensional high-efficiency or high-content analysis of silatecan A-, B-cyclic conversion derivative conjugated ADC in vitro.

[0040] Figure 6: Graph showing the cytotoxicity efficacy results of a silatecan derivative conjugated ADC in an MCF-7 cell line with extremely low HER2 expression in vitro.

[0041] Figure 7: Graph showing the cell killing efficacy results of ADCs conjugated with various linkers using AM20011 as a payload in HER2-expressing SK-OV-3 cell lines in vitro.

[0042] Figure 8: Graph confirming the specific binding ability of silatecan A-, B- ring conversion derivative conjugated ADC to HER2 protein.

[0043] Figure 9: Results of confirming the specific binding ability of silatecan A-, B-cyclic conversion derivative conjugated ADC to HER2 on the cell surface in vitro.

[0044] Figure 10: Results of internalization confirmation of Trastuzumab-AM21611.

[0045] Figure 11: Shows the results of measuring the amount of extracellular ATP release for inducing immunogenic cell death of a silatecan derivative ADC.

[0046] Figure 12: Shows the results of measuring the translocation of HSP70 protein for inducing immunogenic cell death of silatecan derivative ADC.

[0047] Figure 13: In vitro and in vivo efficacy evaluation results of Trastzumab conjugated ADC and Trastzumab-Deruxtecan ADC using AM20001 and AM20002 as payloads.

[0048] Figure 14: Results of in vivo efficacy evaluation of four types of silatecan derivative conjugated ADCs.

[0049] Figure 15: A graph showing changes in body weight according to in vivo efficacy evaluation of four types of silatecan derivative-conjugated ADCs.

[0050] Figure 16: In vivo efficacy evaluation results of silatecan derivative conjugated Trastzumab ADC and Trastzumab-Deruxtecan conjugated ADC.

[0051] Figure 17: In vitro efficacy evaluation results for multidrug resistance of silatecan derivatives and approved payloads after MDR inhibitor treatment.

[0052] Hereinafter, with reference to the attached drawings, implementation examples and embodiments of the present invention will be described in detail so that those skilled in the art can easily practice the present invention. However, the present invention may be implemented in various different forms and is not limited to the implementation examples and embodiments described herein. In addition, in the drawings, parts irrelevant to the description have been omitted to clearly explain the present invention, and similar parts have been designated with similar drawing reference numerals throughout the specification.

[0053] Throughout this specification, when a part is said to be "connected" to another part, this includes not only cases where it is "directly connected" but also cases where it is "electrically connected" with another element in between.

[0054] Throughout this specification, when it is said that an element is "on" another element, this includes not only cases where the element is in contact with the other element, but also cases where another element exists between the two elements.

[0055] Throughout this specification, whenever a part is said to "include" a component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise specifically stated.

[0056] The terms "about," "substantially," and the like used in this specification are used in a meaning that is at or close to the numerical value when manufacturing and material tolerances inherent in the meanings mentioned are presented, and are used to prevent unscrupulous infringers from unfairly exploiting the disclosure in which exact or absolute values ​​are mentioned to aid understanding of the present application.

[0057] The terms “step of ~” or “step of ~” as used throughout this specification do not mean “step for ~.”

[0058] Throughout this specification, the term "combination(s) thereof" included in the expressions in the Makushi format means one or more mixtures or combinations selected from the group consisting of the components described in the expressions in the Makushi format, and means including one or more selected from the group consisting of said components.

[0059] Throughout this specification, references to “A and / or B” mean “A or B, or A and B.”

[0060] Below, the implementation examples of the present invention are described in detail, but the present invention may not be limited thereto.

[0061] The first aspect of the present invention provides a linker-drug conjugate comprising a silatecan derivative or a pharmaceutically acceptable salt thereof, represented by the following chemical formula 1 or chemical formula 2; and a linker, wherein the linker binds to A of the following chemical formula 1 or B of the following chemical formula 2:

[0062] [Chemical Formula 1]

[0063] ;

[0064] [Chemical Formula 2]

[0065] ;

[0066] In the above chemical formula 1 and chemical formula 2,

[0067] A is A 1 -(C 0-3 alkylene)-A 2 -(C 0-3 alkylene)-A 3 -A 4 - and,

[0068] A 1 is -NH2, -OH, or -O-CH2-NH2,

[0069] A 2 and A 3 are, independently of each other, absent, or -O-, -NH, -N(C 1-3 alkyl group)-, or -S-,

[0070] A 4 is -C(=O)- or -SO2-;

[0071] B is B 1 -(C 0-3 alkylene)-B 2 -B 3 -B 4 -B 5 -(C 0-3 alkylene)-and,

[0072] B 1 is -NH2, -OH, or -O-CH2-NH2,

[0073] B 2 and B 3 are, independently of each other, absent, or -O-, -NH, -N(C 1-3 alkyl group)-, or -S-;

[0074] B 4 does not exist, or is -C(=O)- or -SO2-;

[0075] B 5 does not exist, or is -NH- or -O-,

[0076] R a , R b , R c , R d , and R e are, each independently, C 1-10 Alkyl group, C2-10 Alkenyl group, C 2-10 Alkynyl group, C 4-20 Aryl group, -(CH2) m R 11 Ki, or SiR 12 R 13 R 14 , where m is an integer in the range of 1 to 10, and R 11 Silver hydroxyl group, C 1-10 Alkoxy group, amino group, C 1-10 Alkylamino group, di-C 1-10 -alkylamino group, F, Cl, cyano group, or nitro group, where R 12 , R 13 and R 14 are, each independently, C 1-10 Alkyl group or C 4-10 It is an aryl group;

[0077] X is H, F, Cl, nitro, amino, hydroxy, or cyano,

[0078] Y is -NH-,

[0079] Z is hydrogen, F, Cl, hydroxyl group, nitro group, cyano group, azido group, formyl group, hydrazino group, amino group, C 1-10 Alkyl group, C 2-10 Alkenyl group, C 2-10 Alkynyl group, C 1-10 Alkoxy group, C 1-10 Aryloxy group, C 1-10 Acyloxy group, -C(O)-C 1-10 Alkyl group, -C(O)-C 4-10 Aryl group, -OC(O)OC 1-10 Alkyl group, -OC(O)NR 15 R 16 and, here, R 15 and R 16 are, independently, H, C 1-10 Alkyl group or C 4-10 It is an aryl group,

[0080] W is H, F, Cl, nitro, amino, hydroxy, or cyano, or

[0081] Z and W are connected to each other to form C containing one or more heteroatoms. 1-5 It may be a heterocyclic alkyl ring.

[0082] In one embodiment of the present invention, R a , R b , R c , R d , and R e may each independently be a methyl group, an ethyl group, an n-propyl group, an iso-propyl group, an n-butyl group, an iso-butyl group, or a tert-butyl group.

[0083] In one embodiment of the present invention, A may be selected from the following:

[0084] NH2-CH2-C(=O)-, NH2-C2H4-C(=O)-, NH2-C3H6-C(=O)-, NH2-C4H8-C(=O)-, NH2-C5H 10 -C(=O)-, NH2-C6H 12 -C(=O)-, OH-CH2-C(=O)-, OH-C2H4-C(=O)-, OH-C3H6-C(=O)-, OH-C4H8-C(=O)-, OH-C5H 10 -C(=O)-, OH-C6H 12 -C(=O)-, NH2-CH2-SO2-, NH2-C2H4-SO2-, NH2-C3H6-SO2-, NH2-C4H8-SO2-, NH2-C5H 10 -SO2-, NH2-C6H 12 -SO2-, OH-CH2-SO2-, OH-C2H4-SO2-, OH-C3H6-SO2-, OH-C4H8-SO2-, OH-C5H 10 -SO2-, OH-C6H 12-SO2-, NH2-OC(=O)-, NH2-O-SO2-, NH2-NH-C(=O)-, NH2-NH-SO2-, OH-NH-C(=O)-, OH-NH-SO2-, NH2CH2-OC(=O)-, NH2CH2-O-SO2-, NH2CH2-NH-C(=O)-, NH2CH2-NH-SO2-, NH2C2H4-OC(=O)-, NH2C2H4-O-SO2-, NH2C2H4-NH-C(=O)-, NH2-C2H4-NH-SO2-, NH2C3H6-OC(=O)-, NH2C3H6-O-SO2-, NH2C3H6-NH-C(=O)- NH2C3H6-NH-SO2-, OH-CH2-OC(=O)-, OH-CH2-O-SO2-, OH-CH2-NH-C(=O)-, OH-CH2-NH-SO2-, OH-C2H4-OC(=O)-, OH-C2H4-O-SO2-, OH-C2H4-NH-C(=O)-, OH-C2H4-NH-SO2-, OH-C3H6-OC(=O)-, OH-C3H6-O-SO2-, OH-C3H6-NH-C(=O)-, OH-C3H6-NH-SO2-, NH2-OCH2-C(=O)-, NH2-OCH2-SO2-, NH2-NHCH2-C(=O)- NH2-NHCH2-SO2-, NH2-OC2H4-C(=O)-, NH2-OC2H4-SO2-, NH2-NHC2H4-C(=O)-, NH2-NHC2H4-SO2-, NH2-OC3H6-C(=O)-, NH2-OC3H6-SO2-, NH2-NHC3H6-C(=O)-, NH2-NHC3H6-SO2-, OH-OCH2-C(=O)-, OH-OCH2-SO2-, OH-NHCH2-C(=O)-, OH-NHCH2-SO2-, OH-OC2H4-C(=O)-, OH-OC2H4-SO2-, OH-NHC2H4-C(=O)-, OH-NHC2H4-SO2- OH-OC3H6-C(=O)-, OH-OC3H6-SO2-, OH-NHC3H6-C(=O)-, OH-NHC3H6-SO2-, NH2CH2-OCH2-C(=O)-, NH2CH2-OCH2-SO2-, NH2CH2-NHCH2-C(=O)-, NH2CH2-NHCH2-SO2-, NH2CH2-OC2H4-C(=O)-NH2CH2-OC2H4-SO2-, NH2CH2-NHC2H4-C(=O)-, NH2CH2-NHC2H4-SO2-, NH2CH2-OC3H6-C(=O)-, NH2CH2-OC3H6-SO2-, NH2CH2-NHC3H6-C(=O)-, NH2CH2-NHC3H6-SO2-, NH2CH2-OC4H8-C(=O)-, NH2CH2-OC4H8-SO2-, NH2CH2-OC5H 10 -C(=O)-, NH2CH2-OC5H 10 -SO2-, NH2CH2-OC6H 12 -C(=O)-, NH2CH2-OC6H 12-SO2-, NH2C2H4-OCH2-C(=O)-, NH2C2H4-OCH2-SO2-, NH2C2H4-NHCH2-C(=O)-, NH2C2H4-NHCH2-SO2-, NH2C2H4-OC2H4-C(=O)-, NH2C2H4-OC2H4-SO2-, NH2C2H4-NHC2H4-C(=O)-, NH2C2H4-NHC2H4-SO2-, NH2C2H4-OC3H6-C(=O)-, NH2C2H4-OC3H6-SO2-, NH2C2H4-NHC3H6-C(=O)-, NH2C2H4-NHC3H6-SO2-, NH2C3H6-OCH2-C(=O)-, NH2C3H6-OCH2-SO2-, NH2C3H6-NHCH2-C(=O)-, NH2C3H6-NHCH2-SO2-, NH2C3H6-OC2H4-C(=O)-, NH2C3H6-OC2H4-SO2-, NH2C3H6-NHC2H4-C(=O)-, NH2C3H6-NHC2H4-SO2-, NH2C3H6-OC3H6-C(=O)-, NH2C3H6-OC3H6-SO2-, NH2C3H6-NHC3H6-C(=O)-, NH2C3H6-NHC3H6-SO2-, NH2-CH2-O-CH2-OC(=O)- NH2-C2H4-O-CH2-OC(=O)-, NH2-C3H6-O-CH2-OC(=O)-, NH2-CH2-O-C2H4-OC(=O)-, NH2-CH2-O-C3H6-OC(=O)-, NH2-CH2-O-CH2-O-SO2-, NH2-C2H4-O-CH2-O-SO2-, NH2-C3H6-O-CH2-O-SO2-, NH2-CH2-O-C2H4-O-SO2-, NH2-CH2-O-C3H6-O-SO2-, NH2-CH2-OO-CH2-C(=O)-, NH2-CH2-OO-CH2-SO2-, NH2-CH2-OO-C2H4-C(=O)- NH2-CH2-OO-C2H4-SO2-, NH2-CH2-OO-C3H6-C(=O)-, NH2-CH2-OO-C3H6-SO2-, NH2-CH2-O-NH-C(=O)-, NH2-CH2-O-NH-SO2-, NH2-CH2-O-NH-CH2-C(=O)-, NH2-CH2-O-NH-CH2-SO2-,NH2-CH2-O-NH-C2H4-C(=O)-, NH2-CH2-O-NH-C2H4-SO2-, NH2-CH2-O-NH-C3H6-C(=O)-, NH2-CH2-O-NH-C3H6-SO2-, NH2-CH2-NH-CH2-OC(=O)-, NH2-C2H4-NH-CH2-OC(=O)-, NH2-C3H6-NH-CH2-OC(=O)-, NH2-CH2-NH-C2H4-OC(=O)-, NH2-CH2-NH-C3H6-OC(=O)-, NH2-NH-CH2-NH-C(=O)-, NH2-NH-C3H6-NH-C(=O)-, NH2-CH2-NH-NH-C(=O)-, NH2-C2H4-NH-NH-C(=O)-, NH2-C3H6-NH-NH-C(=O)-, NH2-CH2-NH-CH2-NH-C(=O)-, NH2-C2H4-NH-CH2-NH-C(=O)-, NH2-C3H6-NH-CH2-NH-C(=O)-, NH2-CH2-NH-C2H4-NH-C(=O)-, NH2-CH2-NH-C3H6-NH-C(=O)-, NH2-NH-CH2-NH-C(=O)-, NH2-NH-C2H4-NH-C(=O)-, NH2-NH-C3H6-NH-C(=O)-, NH2-CH2-NH-NH-SO2-, NH2-C2H4-NH-NH-SO2-, NH2-C3H6-NH-NH-SO2-, NH2-CH2-NH-CH2-NH-SO2-, NH2-C2H4-NH-CH2-NH-SO2-, NH2-C3H6-NH-CH2-NH-SO2-, NH2-CH2-NH-C2H4-NH-SO2-, NH2-CH2-NH-C3H6-NH-SO2-, NH2-CH2-O-CH2-NH-C(=O)-, NH2-CH2-O-C2H4-NH-C(=O)-, NH2-CH2-O-C3H6-NH-C(=O)-, NH2-C2H4-O-CH2-NH-C(=O)-, NH2-C3H6-O-CH2-NH-C(=O)-, NH2-CH2-O-CH2-NH-SO2-, NH2-CH2-O-C2H4-NH-SO2-, NH2-CH2-O-C3H6-NH-SO2-, NH2-C2H4-O-CH2-NH-SO2-, NH2-C3H6-O-CH2-NH-SO2-,OH-CH2-O-CH2-O-C(=O)-, OH-C2H4-O-CH2-O-C(=O)-, OH-C3H6-O-CH2-O-C(=O)-, OH-CH2-O-C2H4-O-C(=O)-, OH-CH2-O-C3H6-O-C(=O)-, OH-CH2-O-CH2-O-SO2-, OH-C2H4-O-CH2-O-SO2-, OH-C3H6-O-CH2-O-SO2-, OH-CH2-O-C2H4-O-SO2-, OH-CH2-O-C3H6-O-SO2-, OH-CH2-NH-CH2-O-C(=O)-, OH-C2H4-NH-CH2-O-C(=O)-, OH-C3H6-NH-CH2-O-C(=O)-, OH-CH2-NH-C2H4-O-C(=O)-, OH-CH2-NH-C3H6-O-C(=O)-, OH-NH-CH2-NH-C(=O)-, OH-NH-C2H4-NH-C(=O)-, OH-NH-C3H6-NH-C(=O)-, OH-CH2-NH-NH-C(=O)-, OH-C2H4-NH-NH-C(=O)-, OH-C3H6-NH-NH-C(=O)-, OH-CH2-NH-CH2-NH-C(=O)-, OH-C2H4-NH-CH2-NH-C(=O)-, OH-C3H6-NH-CH2-NH-C(=O)-, OH-CH2-NH-C2H4-NH-C(=O)-, OH-CH2-NH-C3H6-NH-C(=O)-, OH-NH-CH2-NH-C(=O)-, OH-NH-C2H4-NH-C(=O)-, OH-NH-C3H6-NH-C(=O)-, OH-CH2-NH-NH-SO2-, OH-C2H4-NH-NH-SO2-, OH-C3H6-NH-NH-SO2-, OH-CH2-NH-CH2-NH-SO2-, OH-C2H4-NH-CH2-NH-SO2-, OH-C3H6-NH-CH2-NH-SO2-, OH-CH2-NH-C2H4-NH-SO2-, OH-CH2-NH-C3H6-NH-SO2-, OH-CH2-O-CH2-NH-C(=O)-, OH-CH2-O-C2H4-NH-C(=O)-, OH-CH2-O-C3H6-NH-C(=O)-, OH-C2H4-O-CH2-NH-C(=O)-, OH-C3H6-O-CH2-NH-C(=O)-, OH-CH2-O-CH2-NH-SO2-,OH-CH2-O-C2H4-NH-SO2-, OH-CH2-O-C3H6-NH-SO2-, OH-C2H4-O-CH2-NH-SO2-, OH-C3H6-O-CH2-NH-SO2-, NH2-CH2-O-CH2-O-CH2-OC(=O)-, NH2-CH2-O-C2H4-O-CH2-OC(=O)-, NH2-CH2-O-C3H6-O-CH2-OC(=O)-, NH2-CH2-O-CH2-O-C2H4-OC(=O)-, NH2-CH2-O-CH2-O-C3H6-OC(=O)-, NH2-CH2-O-CH2-O-CH2-O-SO2-, NH2-CH2-O-C2H4-O-CH2-O-SO2-, NH2-CH2-O-C3H6-O-CH2-O-SO2-, NH2-CH2-O-CH2-O-C2H4-O-SO2-, NH2-CH2-O-CH2-O-C3H6-O-SO2-, NH2-CH2-O-CH2-NH-CH2-OC(=O)-, NH2-CH2-O-C2H4-NH-CH2-OC(=O)-, NH2-CH2-O-C3H6-NH-CH2-OC(=O)-, NH2-CH2-O-CH2-NH-C2H4-OC(=O)-, NH2-CH2-O-CH2-NH-C3H6-OC(=O)-, NH2-CH2-O-NH-CH2-NH-C(=O)-, NH2-CH2-O-NH-C2H4-NH-C(=O)-, NH2-CH2-O-NH-C3H6-NH-C(=O)-, NH2-CH2-O-CH2-NH-NH-C(=O)-, NH2-CH2-O-C2H4-NH-NH-C(=O)-, NH2-CH2-O-C3H6-NH-NH-C(=O)-, NH2-CH2-O-CH2-NH-CH2-NH-C(=O)-, NH2-CH2-O-C2H4-NH-CH2-NH-C(=O)-, NH2-CH2-O-C3H6-NH-CH2-NH-C(=O)-, NH2-CH2-O-CH2-NH-C2H4-NH-C(=O)-, NH2-CH2-O-CH2-NH-C3H6-NH-C(=O)-, NH2-CH2-O-NH-CH2-NH-C(=O)-, NH2-CH2-O-NH-C2H4-NH-C(=O)-, NH2-CH2-O-NH-C3H6-NH-C(=O)-, NH2-CH2-O-CH2-NH-NH-SO2-,NH2-CH2-O-C2H4-NH-NH-SO2-, NH2-CH2-O-C3H6-NH-NH-SO2-, NH2-CH2-O-CH2-NH-CH2-NH-SO2-, NH2-CH2-O-C2H4-NH-CH2-NH-SO2-, NH2-CH2-O-C3H6-NH-CH2-NH-SO2-, NH2-CH2-O-CH2-NH-C2H4-NH-SO2-, NH2-CH2-O-CH2-NH-C3H6-NH-SO2-, NH2-CH2-O-CH2-O-CH2-NH-C(=O)-, NH2-CH2-O-CH2-O-C2H4-NH-C(=O)- NH2-CH2-O-CH2-O-C3H6-NH-C(=O)-, NH2-CH2-O-C2H4-O-CH2-NH-C(=O)-, NH2-CH2-O-C3H6-O-CH2-NH-C(=O)-, NH2-CH2-O-CH2-O-CH2-NH-SO2-, NH2-CH2-O-CH2-O-C2H4-NH-SO2-, NH2-CH2-O-CH2-O-C3H6-NH-SO2-, NH2-CH2-O-C2H4-O-CH2-NH-SO2-, NH2-CH2-O-C3H6-O-CH2-NH-SO2-, NH2-CH2-O-CH2-C(=O)-, NH2-CH2-O-C2H4-C(=O)-, NH2-CH2-O-C3H6-C(=O)-, NH2-CH2-O-C4H8-C(=O)-, NH2-CH2-O-C5H, 10 -C(=O)-, NH2-CH2-O-C6H 12 -C(=O)-, NH2-CH2-O-CH2-SO2-, NH2-CH2-O-C2H4-SO2-, NH2-CH2-O-C3H6-SO2-, NH2-CH2-O-C4H8-SO2-, NH2-CH2-O-C5H 10 -SO2-, NH2-CH2-O-C6H 12-SO2-, NH2-CH2-O-NH-C(=O)-, NH2-CH2-O-NH-SO2-, NH2-CH2-O-CH2-OC(=O)-, NH2-CH2-O-CH2-O-SO2-, NH2-CH2-O-CH2-NH-C(=O)-, NH2-CH2-O-CH2-NH-SO2-, NH2-CH2-O-C2H4-OC(=O)-, NH2-CH2-O-C2H4-O-SO2-, NH2-CH2-O-C2H4-NH-C(=O)-, NH2-CH2-O-C2H4-NH-SO2-, NH2-CH2-O-C3H6-OC(=O)-, NH2-CH2-O-C3H6-O-SO2-, NH2-CH2-O-C3H6-NH-C(=O)-, and NH2-CH2-O-C3H6-NH-SO2-.

[0085] In one embodiment of the present invention, A may be selected from the following:

[0086]

[0087] In one embodiment of the present invention, the silatecan derivative represented by Chemical Formula 1 may be selected from the following:

[0088]

[0089]

[0090] In one embodiment of the present invention, B may be selected from the following:

[0091] NH2CH2-, NH2C2H4-, NH2C3H6-, NH2C4H8-, NH2C5H 10 -, NH2C6H 12 -, OHCH2-, OHC2H4-, OHC3H6-, OHC4H8-, OHC5H 10 -, OHC6H 12-, NH2C2H4OCH2-, NH2C2H4OC2H4-, NH2C2H4OC3H6-, NH2C3H6OCH2-, NH2C3H6OC2H4-, NH2C3H6OC3H6-, NH2CH2NHCH2-, NH2CH2NHC2H4-, NH2CH2NHC3H6-, NH2C2H4NHCH2-, NH2C2H4NHC2H4-, NH2C2H4NHC3H6-, NH2C3H6NHCH2-, NH2C3H6NHC2H4-, NH2C3H6NHC3H6-, NH2CH2N(CH3)CH2-, NH2CH2N(CH3)C2H4-, NH2CH2N(CH3)C3H6-, NH2C2H4N(CH3)CH2-, NH2C2H4N(CH3)C2H4-, NH2C2H4N(CH3)C3H6-, NH2C3H6N(CH3)CH2-, NH2C3H6N(CH3)C2H4-, NH2C3H6N(CH3)C3H6-, NH2CH2SCH2-, NH2CH2SC2H4-, NH2CH2SC3H6-, NH2C2H4SCH2-, NH2C2H4SC2H4-, NH2C2H4SC3H6-, NH2C3H6SCH2-, NH2C3H6SC2H4-, NH2C3H6SC3H6-, OHC2H4OCH2-, OHC2H4OC2H4-, OHC2H4OC3H6-, OHC3H6OCH2-, OHC3H6OC2H4-, OHC3H6OC3H6-, OHCH2NHCH2-, OHCH2NHC2H4-, OHCH2NHC3H6-, OHC2H4NHCH2-, OHC2H4NHC2H4-, OHC2H4NHC3H6-, OHC3H6NHCH2-, OHC3H6NHC2H4-, OHC3H6NHC3H6-, OHCH2N(CH3)CH2-, OHCH2N(CH3)C2H4-, OHCH2N(CH3)C3H6-, OHC2H4N(CH3)CH2-, OHC2H4N(CH3)C2H4-, OHC2H4N(CH3)C3H6-, OHC3H6N(CH3)CH2-, OHC3H6N(CH3)C2H4-, OHC3H6N(CH3)C3H6-, OHCH2SCH2-, OHCH2SC2H4-, OHCH2SC3H6-, OHC2H4SCH2-, OHC2H4SC2H4-, OHC2H4SC3H6-, OHC3H6SCH2-,OHC3H6SC2H4-, OHC3H6SC3H6-, NH2-C(=O)-NH-CH2-, NH2-CH2-C(=O)-NH-CH2-, NH2-C2H4-C(=O)-NH-CH2-, NH2-C3H6-C(=O)-NH-CH2-, NH2-C(=O)-NH-C2H4-, NH2-CH2-C(=O)-NH-C2H4-, NH2-C2H4-C(=O)-NH-C2H4-, NH2-C3H6-C(=O)-NH-C2H4-, NH2-C(=O)-NH-C3H6-, NH2-CH2-C(=O)-NH-C3H6-, NH2-C2H4-C(=O)-NH-C3H6-, NH2-C3H6-C(=O)-NH-C3H6-, NH2-SO2-NH-CH2-, NH2-CH2-SO2-NH-CH2-, NH2-C2H4-SO2-NH-CH2-, NH2-C3H6-SO2-NH-CH2-, NH2-SO2-NH-C2H4-, NH2-CH2-SO2-NH-C2H4-, NH2-C2H4-SO2-NH-C2H4-, NH2-C3H6-SO2-NH-C2H4-, NH2-SO2-NH-C3H6-, NH2-CH2-SO2-NH-C3H6-, NH2-C2H4-SO2-NH-C3H6-, NH2-C3H6-SO2-NH-C3H6-, OH-C(=O)-NH-CH2-, OH-CH2-C(=O)-NH-CH2-, OH-C2H4-C(=O)-NH-CH2-, OH-C3H6-C(=O)-NH-CH2-, OH-C(=O)-NH-C2H4-, OH-CH2-C(=O)-NH-C2H4-, OH-C2H4-C(=O)-NH-C2H4-, OH-C3H6-C(=O)-NH-C2H4-, OH-C(=O)-NH-C3H6-, OH-CH2-C(=O)-NH-C3H6-, OH-C2H4-C(=O)-NH-C3H6-, OH-C3H6-C(=O)-NH-C3H6-, OH-SO2-NH-CH2-, OH-CH2-SO2-NH-CH2-, OH-C2H4-SO2-NH-CH2-, OH-C3H6-SO2-NH-CH2-, OH-SO2-NH-C2H4-, OH-CH2-SO2-NH-C2H4-, OH-C2H4-SO2-NH-C2H4-, OH-C3H6-SO2-NH-C2H4-OH-SO2-NH-C3H6-, OH-CH2-SO2-NH-C3H6-, OH-C2H4-SO2-NH-C3H6-, OH-C3H6-SO2-NH-C3H6-, NH2-OC(=O)-NH-CH2-, NH2-CH2-OC(=O)-NH-CH2-, NH2-C2H4-OC(=O)-NH-CH2-, NH2-C3H6-OC(=O)-NH-CH2-, NH2-OC(=O)-NH-C2H4-, NH2-CH2-OC(=O)-NH-C2H4-, NH2-C2H4-OC(=O)-NH-C2H4-, NH2-C3H6-OC(=O)-NH-C2H4- NH2-OC(=O)-NH-C3H6-, NH2-CH2-OC(=O)-NH-C3H6-, NH2-C2H4-OC(=O)-NH-C3H6-, NH2-C3H6-OC(=O)-NH-C3H6-, NH2-NH-C(=O)-NH-CH2-, NH2-CH2-NH-C(=O)-NH-CH2-, NH2-C2H4-NH-C(=O)-NH-CH2-, NH2-C3H6-NH-C(=O)-NH-CH2-, NH2-NH-C(=O)-NH-C2H4-, NH2-CH2-NH-C(=O)-NH-C2H4-, NH2-C2H4-NH-C(=O)-NH-C2H4-, NH2-C3H6-NH-C(=O)-NH-C2H4-, NH2-NH-C(=O)-NH-C3H6-, NH2-CH2-NH-C(=O)-NH-C3H6-, NH2-C2H4-NH-C(=O)-NH-C3H6-, NH2-C3H6-NH-C(=O)-NH-C3H6-, NH2-NH-SO2-NH-CH2-, NH2-CH2-NH-SO2-NH-CH2-, NH2-C2H4-NH-SO2-NH-CH2-, NH2-C3H6-NH-SO2-NH-CH2-, NH2-NH-SO2-NH-C2H4-, NH2-CH2-NH-SO2-NH-C2H4-, NH2-C2H4-NH-SO2-NH-C2H4-, NH2-C3H6-NH-SO2-NH-C2H4-, NH2-NH-SO2-NH-C3H6-, NH2-CH2-NH-SO2-NH-C3H6-, NH2-C2H4-NH-SO2-NH-C3H6-, NH2-C3H6-NH-SO2-NH-C3H6-OH-O-C(=O)-NH-CH2-, OH-CH2-O-C(=O)-NH-CH2-, OH-C2H4-O-C(=O)-NH-CH2-, OH-C3H6-O-C(=O)-NH-CH2-, OH-O-C(=O)-NH-C2H4-, OH-CH2-O-C(=O)-NH-C2H4-, OH-C2H4-O-C(=O)-NH-C2H4-, OH-C3H6-O-C(=O)-NH-C2H4-, OH-O-C(=O)-NH-C3H6-, OH-CH2-O-C(=O)-NH-C3H6-, OH-C2H4-O-C(=O)-NH-C3H6-, OH-C3H6-O-C(=O)-NH-C3H6-, OH-NH-C(=O)-NH-CH2-, OH-CH2-NH-C(=O)-NH-CH2-, OH-C2H4-NH-C(=O)-NH-CH2-, OH-C3H6-NH-C(=O)-NH-CH2-, OH-NH-C(=O)-NH-C2H4-, OH-CH2-NH-C(=O)-NH-C2H4-, OH-C2H4-NH-C(=O)-NH-C2H4-, OH-C3H6-NH-C(=O)-NH-C2H4-, OH-NH-C(=O)-NH-C3H6-, OH-CH2-NH-C(=O)-NH-C3H6-, OH-C2H4-NH-C(=O)-NH-C3H6-, OH-C3H6-NH-C(=O)-NH-C3H6-, OH-NH-SO2-NH-CH2-, OH-CH2-NH-SO2-NH-CH2-, OH-C2H4-NH-SO2-NH-CH2-, OH-C3H6-NH-SO2-NH-CH2-, OH-NH-SO2-NH-C2H4-, OH-CH2-NH-SO2-NH-C2H4-, OH-C2H4-NH-SO2-NH-C2H4-, OH-C3H6-NH-SO2-NH-C2H4-, OH-NH-SO2-NH-C3H6-, OH-CH2-NH-SO2-NH-C3H6-, OH-C2H4-NH-SO2-NH-C3H6-, OH-C3H6-NH-SO2-NH-C3H6-, NH2-NH-O-C(=O)-NH-CH2-, NH2-CH2-NH-O-C(=O)-NH-CH2-, NH2-C2H4-NH-O-C(=O)-NH-CH2-, NH2-C3H6-NH-O-C(=O)-NH-CH2-,NH2-NH-OC(=O)-NH-C2H4-, NH2-CH2-NH-OC(=O)-NH-C2H4-, NH2-C2H4-NH-OC(=O)-NH-C2H4-, NH2-C3H6-NH-OC(=O)-NH-C2H4-, NH2-NH-OC(=O)-NH-C3H6-, NH2-CH2-NH-OC(=O)-NH-C3H6-, NH2-C2H4-NH-OC(=O)-NH-C3H6-, NH2-C3H6-NH-OC(=O)-NH-C3H6-, NH2-NH-NH-C(=O)-NH-CH2-, NH2-CH2-NH-NH-C(=O)-NH-CH2-, NH2-C2H4-NH-NH-C(=O)-NH-CH2-, NH2-C3H6-NH-NH-C(=O)-NH-CH2-, NH2-NH-NH-C(=O)-NH-C2H4-, NH2-CH2-NH-NH-C(=O)-NH-C2H4-, NH2-C2H4-NH-NH-C(=O)-NH-C2H4-, NH2-C3H6-NH-NH-C(=O)-NH-C2H4-, NH2-NH-NH-C(=O)-NH-C3H6-, NH2-CH2-NH-NH-C(=O)-NH-C3H6-, NH2-C2H4-NH-NH-C(=O)-NH-C3H6-, NH2-C3H6-NH-NH-C(=O)-NH-C3H6-, NH2-NH-NH-SO2-NH-CH2-, NH2-CH2-NH-NH-SO2-NH-CH2-, NH2-C2H4-NH-NH-SO2-NH-CH2-, NH2-C3H6-NH-NH-SO2-NH-CH2-, NH2-NH-NH-SO2-NH-C2H4-, NH2-CH2-NH-NH-SO2-NH-C2H4-, NH2-C2H4-NH-NH-SO2-NH-C2H4-, NH2-C3H6-NH-NH-SO2-NH-C2H4-, NH2-NH-NH-SO2-NH-C3H6-, NH2-CH2-NH-NH-SO2-NH-C3H6-, NH2-C2H4-NH-NH-SO2-NH-C3H6-, NH2-C3H6-NH-NH-SO2-NH-C3H6-, OH-NH-OC(=O)-NH-CH2-, OH-CH2-NH-OC(=O)-NH-CH2-, OH-C2H4-NH-OC(=O)-NH-CH2-,OH-C3H6-NH-O-C(=O)-NH-CH2-, OH-NH-O-C(=O)-NH-C2H4-, OH-CH2-NH-O-C(=O)-NH-C2H4-, OH-C2H4-NH-O-C(=O)-NH-C2H4-, OH-C3H6-NH-O-C(=O)-NH-C2H4-, OH-NH-O-C(=O)-NH-C3H6-, OH-CH2-NH-O-C(=O)-NH-C3H6-, OH-C2H4-NH-O-C(=O)-NH-C3H6-, OH-C3H6-NH-O-C(=O)-NH-C3H6-, OH-NH-NH-C(=O)-NH-CH2-, OH-CH2-NH-NH-C(=O)-NH-CH2-, OH-C2H4-NH-NH-C(=O)-NH-CH2-, OH-C3H6-NH-NH-C(=O)-NH-CH2-, OH-NH-NH-C(=O)-NH-C2H4-, OH-CH2-NH-NH-C(=O)-NH-C2H4-, OH-C2H4-NH-NH-C(=O)-NH-C2H4-, OH-C3H6-NH-NH-C(=O)-NH-C2H4-, OH-NH-NH-C(=O)-NH-C3H6-, OH-CH2-NH-NH-C(=O)-NH-C3H6-, OH-C2H4-NH-NH-C(=O)-NH-C3H6-, OH-C3H6-NH-NH-C(=O)-NH-C3H6-, OH-NH-NH-SO2-NH-CH2-, OH-CH2-NH-NH-SO2-NH-CH2-, OH-C2H4-NH-NH-SO2-NH-CH2-, OH-C3H6-NH-NH-SO2-NH-CH2-, OH-NH-NH-SO2-NH-C2H4-, OH-CH2-NH-NH-SO2-NH-C2H4-, OH-C2H4-NH-NH-SO2-NH-C2H4-, OH-C3H6-NH-NH-SO2-NH-C2H4-, OH-NH-NH-SO2-NH-C3H6-, OH-CH2-NH-NH-SO2-NH-C3H6-, OH-C2H4-NH-NH-SO2-NH-C3H6-, OH-C3H6-NH-NH-SO2-NH-C3H6-, NH2-CH2-O-CH2-, NH2-CH2-O-C2H4-, NH2-CH2-O-C3H6-, NH2-CH2-O-C4H8-, NH2-CH2-O-C5H, 10-, NH2-CH2-O-C6H 12-, NH2-CH2-O-C2H4OCH2-, NH2-CH2-O-C2H4OC2H4-, NH2-CH2-O-C2H4OC3H6-, NH2-CH2-O-C3H6OCH2-, NH2-CH2-O-C3H6OC2H4-, NH2-CH2-O-C3H6OC3H6-, NH2-CH2-O-CH2NHCH2-, NH2-CH2-O-CH2NHC2H4-, NH2-CH2-O-CH2NHC3H6-, NH2-CH2-O-C2H4NHCH2-, NH2-CH2-O-C2H4NHC2H4-, NH2-CH2-O-C2H4NHC3H6-, NH2-CH2-O-C3H6NHCH2-, NH2-CH2-O-C3H6NHC2H4-, NH2-CH2-O-C3H6NHC3H6-, NH2-CH2-O-CH2N(CH3)CH2-, NH2-CH2-O-CH2N(CH3)C2H4-, NH2-CH2-O-CH2N(CH3)C3H6-, NH2-CH2-O-C2H4N(CH3)CH2-, NH2-CH2-O-C2H4N(CH3)C2H4-, NH2-CH2-O-C2H4N(CH3)C3H6-, NH2-CH2-O-C3H6N(CH3)CH2-, NH2-CH2-O-C3H6N(CH3)C2H4-, NH2-CH2-O-C3H6N(CH3)C3H6-, NH2-CH2-O-CH2SCH2-, NH2-CH2-O-CH2SC2H4-, NH2-CH2-O-CH2SC3H6-, NH2-CH2-O-C2H4SCH2-, NH2-CH2-O-C2H4SC2H4-, NH2-CH2-O-C2H4SC3H6-, NH2-CH2-O-C3H6SCH2-, NH2-CH2-O-C3H6SC2H4-, NH2-CH2-O-C3H6SC3H6-, NH2-CH2-O-C(=O)-NH-CH2-, NH2-CH2-O-CH2-C(=O)-NH-CH2-, NH2-CH2-O-C2H4-C(=O)-NH-CH2-, NH2-CH2-O-C3H6-C(=O)-NH-CH2-, NH2-CH2-O-C(=O)-NH-C2H4-, NH2-CH2-O-CH2-C(=O)-NH-C2H4-, NH2-CH2-O-C2H4-C(=O)-NH-C2H4-, NH2-CH2-O-C3H6-C(=O)-NH-C2H4-,NH2-CH2-OC(=O)-NH-C3H6-, NH2-CH2-O-CH2-C(=O)-NH-C3H6-, NH2-CH2-O-C2H4-C(=O)-NH-C3H6-, NH2-CH2-O-C3H6-C(=O)-NH-C3H6-, NH2-CH2-O-SO2-NH-CH2-, NH2-CH2-O-CH2-SO2-NH-CH2-, NH2-CH2-O-C2H4-SO2-NH-CH2-, NH2-CH2-O-C3H6-SO2-NH-CH2-, NH2-CH2-O-SO2-NH-C2H4-, NH2-CH2-O-CH2-SO2-NH-C2H4-, NH2-CH2-O-C2H4-SO2-NH-C2H4-, NH2-CH2-O-C3H6-SO2-NH-C2H4-, NH2-CH2-O-SO2-NH-C3H6-, NH2-CH2-O-CH2-SO2-NH-C3H6-, NH2-CH2-O-C2H4-SO2-NH-C3H6-, NH2-CH2-O-C3H6-SO2-NH-C3H6-, NH2-CH2-OOC(=O)-NH-CH2-, NH2-CH2-O-CH2-OC(=O)-NH-CH2-, NH2-CH2-O-C2H4-OC(=O)-NH-CH2-, NH2-CH2-O-C3H6-OC(=O)-NH-CH2-, NH2-CH2-OOC(=O)-NH-C2H4-, NH2-CH2-O-CH2-OC(=O)-NH-C2H4-, NH2-CH2-O-C2H4-OC(=O)-NH-C2H4-, NH2-CH2-O-C3H6-OC(=O)-NH-C2H4-, NH2-CH2-OOC(=O)-NH-C3H6-, NH2-CH2-O-CH2-OC(=O)-NH-C3H6-, NH2-CH2-O-C2H4-OC(=O)-NH-C3H6-, NH2-CH2-O-C3H6-OC(=O)-NH-C3H6-, NH2-CH2-O-NH-C(=O)-NH-CH2-, NH2-CH2-O-CH2-NH-C(=O)-NH-CH2-, NH2-CH2-O-C2H4-NH-C(=O)-NH-CH2-, NH2-CH2-O-C3H6-NH-C(=O)-NH-CH2-, NH2-CH2-O-NH-C(=O)-NH-C2H4-,NH2-CH2-O-CH2-NH-C(=O)-NH-C2H4-, NH2-CH2-O-C2H4-NH-C(=O)-NH-C2H4-, NH2-CH2-O-C3H6-NH-C(=O)-NH-C2H4-, NH2-CH2-O-NH-C(=O)-NH-C3H6-, NH2-CH2-O-CH2-NH-C(=O)-NH-C3H6-, NH2-CH2-O-C2H4-NH-C(=O)-NH-C3H6-, NH2-CH2-O-C3H6-NH-C(=O)-NH-C3H6-, NH2-CH2-O-NH-SO2-NH-CH2-, NH2-CH2-O-CH2-NH-SO2-NH-CH2-, NH2-CH2-O-C2H4-NH-SO2-NH-CH2-, NH2-CH2-O-C3H6-NH-SO2-NH-CH2-, NH2-CH2-O-NH-SO2-NH-C2H4-, NH2-CH2-O-CH2-NH-SO2-NH-C2H4-, NH2-CH2-O-C2H4-NH-SO2-NH-C2H4-, NH2-CH2-O-C3H6-NH-SO2-NH-C2H4-, NH2-CH2-O-NH-SO2-NH-C3H6-, NH2-CH2-O-CH2-NH-SO2-NH-C3H6-, NH2-CH2-O-C2H4-NH-SO2-NH-C3H6-, NH2-CH2-O-C3H6-NH-SO2-NH-C3H6-, NH2-CH2-O-NH-OC(=O)-NH-CH2-, NH2-CH2-O-CH2-NH-OC(=O)-NH-CH2-, NH2-CH2-O-C2H4-NH-OC(=O)-NH-CH2-, NH2-CH2-O-C3H6-NH-OC(=O)-NH-CH2-, NH2-CH2-O-NH-OC(=O)-NH-C2H4-, NH2-CH2-O-CH2-NH-OC(=O)-NH-C2H4-, NH2-CH2-O-C2H4-NH-OC(=O)-NH-C2H4-, NH2-CH2-O-C3H6-NH-OC(=O)-NH-C2H4-, NH2-CH2-O-NH-OC(=O)-NH-C3H6-, NH2-CH2-O-CH2-NH-OC(=O)-NH-C3H6-, NH2-CH2-O-C2H4-NH-OC(=O)-NH-C3H6-,NH2-CH2-O-C3H6-NH-OC(=O)-NH-C3H6-, NH2-CH2-O-NH-NH-C(=O)-NH-CH2-, NH2-CH2-O-CH2-NH-NH-C(=O)-NH-CH2-, NH2-CH2-O-C2H4-NH-NH-C(=O)-NH-CH2-, NH2-CH2-O-C3H6-NH-NH-C(=O)-NH-CH2-, NH2-CH2-O-NH-NH-C(=O)-NH-C2H4-, NH2-CH2-O-CH2-NH-NH-C(=O)-NH-C2H4-, NH2-CH2-O-C2H4-NH-NH-C(=O)-NH-C2H4-, NH2-CH2-O-C3H6-NH-NH-C(=O)-NH-C2H4-, NH2-CH2-O-NH-NH-C(=O)-NH-C3H6-, NH2-CH2-O-CH2-NH-NH-C(=O)-NH-C3H6-, NH2-CH2-O-C2H4-NH-NH-C(=O)-NH-C3H6-, NH2-CH2-O-C3H6-NH-NH-C(=O)-NH-C3H6-, NH2-CH2-O-NH-NH-SO2-NH-CH2-, NH2-CH2-O-CH2-NH-NH-SO2-NH-CH2-, NH2-CH2-O-C2H4-NH-NH-SO2-NH-CH2-, NH2-CH2-O-C3H6-NH-NH-SO2-NH-CH2-, NH2-CH2-O-NH-NH-SO2-NH-C2H4-, NH2-CH2-O-CH2-NH-NH-SO2-NH-C2H4-, NH2-CH2-O-C2H4-NH-NH-SO2-NH-C2H4-, NH2-CH2-O-C3H6-NH-NH-SO2-NH-C2H4-, NH2-CH2-O-NH-NH-SO2-NH-C3H6-, NH2-CH2-O-CH2-NH-NH-SO2-NH-C3H6-, NH2-CH2-O-C2H4-NH-NH-SO2-NH-C3H6-, and NH2-CH2-O-C3H6-NH-NH-SO2-NH-C3H6-.,

[0092] In one embodiment of the present invention, Z may be a methyl group, an ethyl group, an n-propyl group, an iso-propyl group, an n-butyl group, an iso-butyl group, or a tert-butyl group.

[0093] In one embodiment of the present invention, Z and W are connected to each other to form C containing one or more heteroatoms (non-limiting examples include N, O, and S). 1-5 It may be a heterocycloalkyl ring. For example, the above C 1-5 The heterocycloalkyl ring may be oxacyclopentane, oxacyclohexane, dioxycyclopentane, or dioxycyclohexane containing a fused carbon of the parent A-ring.

[0094] In one embodiment of the present invention, the silatecan derivative represented by Chemical Formula 2 may be selected from the following:

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102] In one embodiment of the present invention, the linker may include a cleavable linker or a non-cleavable linker.

[0103] A linker is a chemical linkage that attaches an antibody to a drug. It must maintain stability during circulation or distribution within the body, prevent drug release in non-target tissues, and maintain the conjugate inactive and non-toxic while the drug is bound to the antibody (or other conjugate). In other words, the linker must release the drug only when the linker-drug conjugate or antibody-drug conjugate is properly internalized into the target cell. Furthermore, the linker's binding functional group can affect the drug-to-antibody ratio (DAR) of the antibody-drug conjugate.

[0104] The above linker is composed of an attachment-spacer-release, and a new linker can be developed by modifying and / or improving one or more of the attachment, spacer, and release components of a commercially available linker.

[0105] In one embodiment of the present invention, the non-cleavable linker may be a non-cleavable bifunctional linker or a non-cleavable spacer linker. As a non-limiting example, the non-cleavable linker may be a thiol ether linker or the like.

[0106] In one embodiment of the present invention, the cleavable linker may be an enzymatically cleavable linker or a non-enzymatically cleavable linker.

[0107] In one embodiment of the present invention, the enzyme-cleavable linker can be cleaved by an enzyme such as cathepsin B, protease, glycosidase, β-glucuronidase, phosphatase, pyrophosphatase, or sulfatase.

[0108] In one embodiment of the present invention, the enzyme-cleavable linker may include a peptide moiety, a β-glucuronide moiety, a phosphate moiety, a sulfate moiety, a pyrophosphate moiety, and combinations thereof. The peptide moiety may include one or more amino acids and may be a dipeptide or tetrapeptide linker. As a non-limiting example, the peptide moiety may include Ala-Ala, Ala-Ala-Ala, Ala-Leu-Ala-Leu, Asp-Glu-Val-Asp, Gly-Gly, Gly-Gly-Phe-Gly, Gly-Leu, Gly-Phe, Gly-Phe-Leu-Gly, Gly-Val, Leu-Leu, Leu-Leu-Val-Tyr, Phe-Lys, Pro-Leu-Gly-Leu, Val-Ala, or Val-Cit.

[0109] In one embodiment of the present invention, the non-enzymatically cleavable linker may be an oxidation-reduction reaction linker or an acid-labile linker. As a non-limiting example, the oxidation-reduction reaction linker may include a disulfide linker or the like. Furthermore, as a non-limiting example, the acid-labile linker may include a hydrazone linker, a carbonate linker, a silyl ether linker, or the like.

[0110] In one embodiment of the present invention, the linker may be conjugated to the drug by chemical conjugation or enzymatic conjugation, but may not be limited thereto. Specifically, the chemical conjugation may include, but may not be limited to, lysine amide coupling, cysteine ​​coupling, or non-natural amino acid incorporation by genetic engineering. In addition, specifically, the enzymatic conjugation may include, but may not be limited to, transpeptidation using sortase, transpeptidation using microbial transglutaminase, or n-glycan engineering.

[0111] In one embodiment of the present invention, the linker may further comprise one or more additional cleavable or non-cleavable linkers, preferably selected from the group consisting of: a hydrazine linker, a thiourea linker, a self-immolative linker, a succinimidyl trans-4-(maleimidylmethyl)cyclohexane-1-carboxylate (SMCC) linker, a disulfide linker, a selenoether linker, an amide linker, a thioether linker, and / or a maleimide linker.

[0112] In one embodiment of the present invention, those skilled in the art will appreciate that additional linkers may be suitable. Such linkers may be non-cleavable or cleavable by changes in pH, redox potential, or specific intracellular / extracellular enzymes. Cleavable oligopeptide linkers include protease- or matrix metalloprotease-cleavable linkers. It is understood that the linker may include combinations of the above. For example, the linker may be a valine-citrulline PAB linker.

[0113] In one embodiment of the present invention, the linker may include, but is not limited to, those selected from the following:

[0114] N-succinimidyl-4-(2-pyridyldithio)pentanoate [N-succinimidyl-4-(2-pyridyldithio)pentanoate; SPP], N-succinimidyl-4-(2-pyridyldithio)butanoate [N-succinimidyl-4-(2-pyridyldithio)butanoate; SPDB], sulfo-SPDB, 2,5-dioxopyrrolidin-1-yl 4-(pyridin-2-yldisulfaneyl)pentanoate [2,5-dioxopyrrolidin-1-yl 4-(pyridin-2-yldisulfaneyl)pentanoate; MDS], 2,5-dioxopyrrolidin-1-yl 4-methyl-4-(pyridin-2-yldisulfaneyl)pentanoate [2,5-dioxopyrrolidin-1-yl 4-methyl-4-(pyridin-2-yldisulfaneyl)pentanoate; DMDS], 2,5-dioxopyrrolidin-1-yl 4-((2-(pyridin-2-yl)propan-2-yl)disulfaneyl)butanoate [2,5-dioxopyrrolidin-1-yl 4-((2-(pyridin-2-yl)propan-2-yl)disulfaneyl)butanoate; DSDM], 2,5-dioxopyrrolidin-1-yl (E)-4-[4-[1-[2-[3-methyl-3-(pyridin-2-yldisulfaneyl)butanoyl]hydrazineylidene]ethyl]phenoxy]butanoate [2,5-dioxopyrrolidin-1-yl (E)-4-(4-(1-(2-(3-methyl-3-(pyridin-2-yldisulfaneyl)butanoyl)hydrazineylidene)ethyl)phenoxy)butanoate; NDMDS], bis-maleimidopolyethyleneglycol (BMPEO), N-(β-maleimidopropyloxy)succinimide ester [N-(β- maleimidopropyloxy)succinimide ester; BMPS], ε-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS),γ-maleimidobutyric acid N-succinimidyl ester (GMBS), HBVS, N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate [N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate; SMCC], LC-N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate [long chain N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate; long chain SMCC], Mal-alkane linker, m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), 4-(4-N-maleimidophenyl)-butyric acid hydrazide (MPBH), N-succinimidyl 3-(bromoacetamido)propionate (SBAP), N-succinimidyl iodoacetate (SIA), N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIA). SIAB], N-succinimidyl-4-(iodoacetyl)-aminobenzoate [N-succinimidyl-4-(iodoacetyl)-aminobenzoate; SIAB], succinimidyl-6-(maleimidopropionamido)hexanoate [succinimidyl-6-(maleimidopropionamido)hexanoate; SMPH], , N-(e-maleimidocaproyloxy)sulfosuccinimido ester [N-(e-maleimidocaproyloxy)sulfosuccimido ester; sulfo-EMCS],N-(y-maleimidobutryloxy)sulfosuccinimide ester [N-(y-maleimidobutryloxy)sulfosuccinimde ester; sulfo-GMBS], N-(κ-maleimidoundecanoyloxy)sulfosuccinimide ester [N-(κ- maleimidoundecanoyloxy)sulfosuccinimide ester; sulfo-KMUS], m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester; sulfo-MBS), sulfosuccinimidyl(4-iodo-acetyl)aminobenzoate [sulfosuccinimidyl(4-iodo-acetyl)aminobenzoate; sulfo-SIAB], sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate [sulfosuccinimidyl 4-(N-maleimido-methyl)cyclohexane-l-carboxylate; sulfo-SMCC], sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate [sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate; sulfo-SMPB], succinimidyl-(4-vinylsulfone)benzoate (SVSB), dithiobis-maleimidoethane (DTME), 1,4-bis-maleimidobutane (BMB), 1,4-bismaleimidyl-2,3-dihydroxybutane (BMDB), bis-maleimidohexane (BMH), bis-maleimidoethane (BMOE), 1,8-bis-maleimidodiethyleneglycol (1,8-bis-maleimidodiethyleneglycol; BM(PEO)2], 1, 11-bis-maleimidotriethylene glycol [1,11-bis-maleimidotriethyleneglycol; BM(PEO)3], Phe-Lys-PABC, Val-Cit-PABC, Val-Ala-PABC, MHVCBC (valine-citrulline), MHFKBC (phenylalanine-lysine), MHH, GBC (glucuronic acid), GBCDN (glucuronic acid), β-glucuronide linker, Mal-PEG-NHS, N-((S)-7-benzyl-3,6,9,12-tetraoxo-2,5,8,11-tetraazatridecan-13-yl)-6-(3-methylthio-2,5-dioxopyrrolidin-1-yl)hexanamide (MC-GGFG), (2S)-2-(3-methoxypropanamido)-N-((S)-3-methyl-1-(((S)-1-(methylamino)-1-oxopropan-2-yl)amino)-1-oxobutan-2-yl)-6-(3-(3-methylthio-2,5-dioxopyrrolidin-1-yl)propanamido)hexanamide (MP-NPEG-VA), (2S)-N-((S)-7-benzyl-3,6,9,12-tetraoxo-2,5,8,11-tetraazatridecan-13-yl)-2-(3-methoxypropanamido)-6-(3-(3-methylthio-2,5-dioxopyrrolidin-1-yl)propanamido)hexanamide (MP-NPEG-GGFG), (S)-2-(3-methoxypropanamido)-N-((S)-3-methyl-1-(((S)-1-(methylamino)-1-oxopropan-2-yl)amino)-1-oxobutan-2-yl)-6-(3-(2-methylthioacetamido)propanamido)hexanamide (BrAc-NPEG-VA), (S)-N-((S)-7-benzyl-3,6,9,12-tetraoxo-2,5,8,11-tetraazatridecan-13-yl)-2-(3-methoxypropanamido)-6-(3-(2-methylthioacetamido)propanamido)hexanamide (BrAc-NPEG-GGFG), N-((S)-3-methyl-1-(((S)-1-(methylamino)-1-oxopropan-2-yl)amino)-1-oxobutan-2-yl)-6-(3-(3-methylthio-2,5-dioxopyrrolidin-1-yl)propanamido)hexanamide (MP-VA), and (2S,3S,4S,5R,6S)-3,4,5-Trihydroxy-6-{4-[((methyl(2-(N-methylacetamido)ethyl)carbamoyl)oxy)methyl]-2-[18-(3-methylthio-2,5-dioxopyrrolidin-1-yl)-3,7,10,16-tetraoxo-2,6,9,15-tetraazaoctadecyl]phenoxy}tetrahydro-2H-pyran-2-carboxylic acid (MP-b-Glu-DMEDA).

[0115] In one embodiment of the present invention, the linker may be selected from the following:

[0116] ;

[0117] ;

[0118] ;

[0119] ;

[0120] ;

[0121] ; and

[0122] .

[0123] In the above linker, the dashed line represents a binding site (covalent bond to the antibody or the silatecan derivative), and n is a number from about 1 to about 1,000.

[0124] In one embodiment of the present invention, n is a number of about 1 to about 1,000, about 1 to about 900, about 1 to about 800, about 1 to about 700, about 1 to about 600, about 1 to about 500, about 1 to about 400, about 1 to about 300, about 1 to about 200, about 1 to about 100, about 1 to about 50, about 1 to about 40, about 1 to about 30, about 1 to about 20, about 1 to about 15, about 1 to about 10, about 1 to about 5, about 1 to about 3, about 10 to about 1,000, about 10 to about 900, about 10 to about 800, about 10 to about 700, about 10 to about 600, about It may be, but is not limited to, 10 to about 500, about 10 to about 400, about 10 to about 300, about 10 to about 200, about 10 to about 100, about 10 to about 50, about 10 to about 40, about 10 to about 30, about 10 to about 20, or about 10 to about 15. In one embodiment of the present disclosure, n may be a number of about 10 to about 20, about 10 to about 15, about 1 to about 15, about 1 to about 10, about 1 to about 5, or about 1 to about 3.

[0125] In one embodiment of the present invention, by controlling the number n of the polyethylene glycol (PEG) portion of the linker, for example, a silatecan derivative with increased water solubility can be utilized by forming a 4-arm, 8-arm, or 12-arm branched PEG, or by having a molecular weight of about 1,000 or about 2,000, but the present invention may not be limited thereto.

[0126] In one embodiment of the present invention, the linker-drug conjugate may be a combination in which a silatecan derivative selected from the above-described silatecan derivatives, which is a drug, is conjugated to a linker selected from the above-described linkers.

[0127] In one embodiment of the present invention, the linker-drug conjugate may be selected from the following:

[0128] ;

[0129] ;

[0130] ;

[0131] ;

[0132] ;

[0133] ;

[0134] ;

[0135] ;

[0136] ;

[0137] ;

[0138] ;

[0139] ;

[0140] ;

[0141] ; and

[0142] .

[0143] The second aspect of the present invention provides a pharmaceutical composition for preventing or treating a proliferative disease, comprising a linker-drug conjugate comprising a silatecan derivative according to the first aspect or a pharmaceutically acceptable salt thereof; and a linker, wherein the linker is bound to A of the following chemical formula 1 or B of the following chemical formula 2.

[0144] Detailed explanations of parts that overlap with the first aspect of the present application have been omitted, but the contents described in the first aspect of the present application may be equally applied even if the explanation is omitted in the second aspect of the present application.

[0145] In one embodiment of the present invention, the silatecan derivative or a pharmaceutically acceptable salt thereof may act as a prodrug.

[0146] In one embodiment of the present invention, the proliferative disease may include one or more selected from neoplasms, tumors, cancers, psoriasis, bone diseases, fibroproliferative disorders, and atherosclerosis.

[0147] In one embodiment of the present invention, the cancer may be a solid cancer or a blood cancer.

[0148] In one embodiment of the present invention, the cancer may be one or more selected from gastric cancer, colon cancer, intestinal cancer, colorectal cancer, uterine cancer, uterine fibroids, meningioma, lung cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, and melanoma.

[0149] A third aspect of the present invention provides an antibody-drug conjugate comprising an antibody; a linker; and a silatecan derivative according to the first aspect or a pharmaceutically acceptable salt thereof.

[0150] Detailed descriptions of overlapping parts with the first and second aspects of the present application have been omitted, but the contents described for the first and second aspects of the present application may be equally applied even if the description is omitted in the third aspect of the present application.

[0151] In one embodiment of the present invention, the antibody-drug conjugate may act as a prodrug.

[0152] In one embodiment of the present invention, the antibody may comprise an antibody, a modified antibody, or an antigen-binding fragment thereof that can immunospecifically act against a proliferative disease. Here, the antibody, the modified antibody, or the antigen-binding fragment thereof may comprise at least one selected from a monoclonal antibody, a domain antibody (dAb), a single-chain antibody (scAb), a Fab fragment, an F(ab')2 fragment, a single-chain variable fragment (scFv), a scFv-Fc fragment, a single-domain heavy chain antibody, a single-domain light chain antibody, a variant antibody, a multimeric antibody, or a bispecific antibody, and may be a rabbit, mouse, chimeric, humanized, or fully human monoclonal antibody. In addition, the antibody may comprise an antibody or antibody-derived substance selected from an IgG isotype, such as an IgG1 isotype.

[0153] In one embodiment of the present invention, the antibody is alemtuzumab, apolizumab, aselizumab, atlizumab, bapineuzumab, bevacizumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cetuximab, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab (epratuzumab), erlizumab, ertumaxomab, felvizumab, fontolizumab, gemtuzumab, gemtuzumab ozogamicin, ibritumomab tiuxetan, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, nolovizumab (nolovizumab), numavizumab, ocrelizumab, omalizumab, palivizumab, panitumumab, pascolizumab, pecfusituzumab, pectuzumab,Pertuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resyvizumab, rituximab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab It may be selected from tositumomab, trastuzumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab, urtoxazumab, and visilizumab.

[0154] In one embodiment of the present invention, the antibody-drug conjugate of the present invention has an IC against cancer cells. 50 It may be a substance with a very strong anticancer effect in units of nM or pM, but may not be limited thereto.

[0155] Hereinafter, the present invention will be described in more detail using examples. However, the following examples are provided only to help understand the present invention, and the contents of the present invention are not limited to the following examples.

[0156] [Example]

[0157] [Manufacture of Payload and Linker Payload Intermediates]

[0158] (1) Preparation of tert-butyl (2-fluoro-4-nitrophenyl)carbamate

[0159]

[0160] 2-Fluoro-4-nitroaniline (5.00 g, 32.0 mmol, 1.00 eq) was dissolved in DCM (50.0 mL), and (Boc)2O (7.34 g, 33.6 mmol, 7.73 mL, 1.05 eq), TEA (4.86 g, 48.0 mmol, 6.69 mL, 1.50 eq), and DMAP (469 mg, 3.84 mmol, 0.12 eq.) were added. The reaction mixture was stirred at 20 °C for 17 h. TLC (petroleum ether:ethyl acetate = 5:1) showed that 2-fluoro-4-nitroaniline (Rf = 0.10) remained and two new spots (Rf = 0.58, 0.65) were formed. The reaction mixture was diluted with DCM (100 mL). The organic layer was filtered, washed with water (100 mL), 50% citric acid (100 mL), and brine (100 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel column chromatography (SiO2, petroleum ether:ethyl acetate = 1:0 to 100:1, Rf = 0.65) to finally obtain tert-butyl (2-fluoro-4-nitrophenyl)carbamate (5.15 g, 20.0 mmol, 62.6% yield, 99.8% purity) as a yellow solid.

[0161] 1H NMR (400 MHz, CDCl3): δ 8.37 (t, J = 8.8 Hz, 1H), 8.06 (dd, J = 1.2, 9.2 Hz, 1H), 7.98 (dd, J = 2.4, 10.8 Hz, 1H), 7.02 (br s, 1H), 1.55 (s, 9H).

[0162] LCMS analysis results: Rt = 0.63 min, m / z = 256.0 [M+Na] +

[0163] (2) Preparation of tert-butyl (4-amino-2-fluorophenyl)carbamate

[0164]

[0165] Tert-butyl (2-fluoro-4-nitrophenyl)carbamate (5.15 g, 20.0 mmol, 99.8% purity, 1.00 eq) was dissolved in dioxane (100 mL), and then Fe powder (11.2 g, 200 mmol, 10.0 eq), NH4Cl (5.36 g, 100 mmol, 5.00 eq), H2O (5.00 mL), and MeOH (5.00 mL) were added at 20 °C. The reaction mixture was stirred at 60 °C for 26 h. TLC (petroleum ether:ethyl acetate = 5:1) showed that tert-butyl (2-fluoro-4-nitrophenyl)carbamate (Rf = 0.60) remained and two new spots (Rf = 0.13, 0.18) were formed. The reaction mixture was diluted with 100 mL of EtOAc at 0°C and filtered. The filtrate was dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel column chromatography (SiO2, petroleum ether:ethyl acetate = 1:0 to 5:1) to finally obtain tert-butyl (4-amino-2-fluorophenyl)carbamate (3.53 g, 15.0 mmol, 74.9% yield, 96.3% purity) as a brown oil.

[0166] LCMS analysis results: Rt = 0.49 min, m / z = 226.1 [2M+H] + .

[0167] 1H NMR (400 MHz, DMSO-d6): δ 8.27 (br s, 1H), 6.94 (br s, 1H), 6.33 - 6.27 (m, 2H), 5.22 (s, 2H), 1.41 (s, 9H).

[0168] (3) Preparation of tert-butyl (2-fluoro-4-formamidophenyl)carbamate

[0169]

[0170] Tert-butyl (4-amino-2-fluorophenyl)carbamate (3.53 g, 15.0 mmol, 96.3% purity, 1.00 eq) was dissolved in DCM (35.0 mL), and Py (1.57 g, 19.8 mmol, 1.60 mL, 1.32 eq) and DCC (4.03 g, 19.5 mmol, 3.95 mL, 1.30 eq) were added at 0 °C, and the mixture was stirred for 10 min. The reaction mixture was stirred at 25 °C for 2 h. LCMS analysis showed that tert-butyl (4-amino-2-fluorophenyl)carbamate was completely consumed, and a major peak with the desired m / z was detected (Rt = 0.49 min, MS calculated: 254.1, MS observed: [M-56+H]+ = 199.0). The reaction mixture was diluted with DCM (100 mL), filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel column chromatography (SiO2, petroleum ether:ethyl acetate = 1:0 to 2:1) to finally obtain tert-butyl (2-fluoro-4-formamidophenyl)carbamate (3.49 g, 13.4 mmol, 89.5% yield, 98.0% purity) as a yellow oil.

[0171] LCMS analysis results: Rt = 0.49 min, m / z = 199.0 [M-56+H] + .

[0172] 1H NMR (400 MHz, DMSO-d6): δ 10.30 (s, 1H), 8.83 (br s, 1H), 8.25 (d, J = 1.6 Hz, 1H), 7.57 (dd, J = 2.0, 12.8 Hz, 1H), 7.47 - 7.39 (m, 1H), 7.20 (dd, J = 1.6, 8.8 Hz, 1H), 1.44 (s, 9H).

[0173] (4) Preparation of tert-butyl (2-fluoro-4-isocyanophenyl)carbamate

[0174]

[0175] Tert-butyl (2-fluoro-4-formamidophenyl)carbamate (3.49 g, 13.4 mmol, 98.0% purity, 1.00 eq), 4Å MS (1.00 g), and TEA (6.81 g, 67.2 mmol, 9.36 mL, 5.00 eq) were dissolved in DCM (40.0 mL), and POCl3 (2.48 g, 16.1 mmol, 1.50 mL, 1.20 eq) was added at 0 °C. The reaction mixture was stirred at 0 °C for 2 h. LCMS analysis showed that 9.46% of tert-butyl (2-fluoro-4-formamidophenyl)carbamate remained and 81.3% of the desired compound was detected (Rt = 0.63 min, m / z = 236.1, MS observed: [M+H] + = 237.0). The reaction mixture was stopped by adding 100 mL of saturated Na2CO3 aqueous solution at 0℃, and then extracted with 200 mL of DCM. The organic layer was washed with 100 mL of H2O and 100 mL of brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel column chromatography (SiO2, petroleum ether: ethyl acetate = 1:0 to 50:1) to finally obtain tert-butyl (2-fluoro-4-isocyanophenyl)carbamate (2.05 g, 8.57 mmol, 63.7% yield, 98.8% purity) as a pale yellow solid.

[0176] LCMS analysis results: Rt = 0.63 min, m / z = 237.0 [M+H] + .

[0177] 1H NMR (400 MHz, CDCl3): δ 8.20 (br t, J = 8.8 Hz, 1H), 7.18 - 7.10 (m, 2H), 6.79 (br s, 1H), 1.54 (s, 9H).

[0178] (5) Preparation of 2-methoxy-6-(trimethylsilyl)pyridine

[0179]

[0180] 2-Bromo-6-methoxypyridine (60 g, 319.11 mmol) was dissolved in dry tetrahydrofuran (THF, 600 mL) and cooled to -60°C under a nitrogen atmosphere. n-Butyllithium (n-BuLi, 2.5 M, 127.64 mL) was slowly added to the solution. The mixture was stirred at -60°C for 1 h. Trimethylsilane chloride (TMSCl, 48.54 mL) was then slowly added. The reaction mixture was stirred at room temperature for 3 h.

[0181] The target product was confirmed to be detected by liquid chromatography mass spectrometry (LCMS). The reaction mixture was quenched with a saturated aqueous ammonium chloride solution and extracted with ethyl acetate (EA, 500 mL × 3). The organic layers were combined, dried over sodium sulfate (Na2SO4), and the solvent was removed under reduced pressure.

[0182] The raw material was purified by silica gel column chromatography using petroleum ether (PE, 100%), and finally 38 g (yield: 61.01%) of 2-methoxy-6-(trimethylsilyl)pyridine was obtained as a yellow oil.

[0183] LCMS analysis results: Rt = 1.386 min, m / z = 182.4 [M+1] +

[0184] (6) Preparation of 4-iodo-2-methoxy-6-(trimethylsilyl)nicotinaldehyde

[0185]

[0186] 2-Methoxy-6-(trimethylsilyl)pyridine (50 g, 275.77 mmol) was dissolved in dry tetrahydrofuran (THF, 500 mL) and cooled to -30°C under a nitrogen atmosphere. n-Butyllithium (n-BuLi, 2.5 M, 132.37 mL) was slowly added to the solution. The mixture was stirred at -30°C for 1 h. Then, N-(2-(dimethylamino)ethyl)-N-methylformamide (46.03 mL, 330.92 mmol) was slowly added, and the mixture was stirred at -30°C for 1 h.

[0187] Under the same conditions, n-butyllithium (n-BuLi, 2.5 M, 220.62 mL) was slowly added again, and the mixture was stirred at -30°C for 3 h. After the reaction mixture was cooled to -60°C, iodine (I2, 181.98 g, 28.68 mmol) dissolved in dry THF (500 mL) was rapidly added. The reaction mixture was stirred at -60°C for 30 min.

[0188] The target product was confirmed to be detected by liquid chromatography mass spectrometry (LCMS). The reaction mixture was stopped by adding a saturated aqueous sodium thiosulfate (Na2S2O3) solution, and then extracted with ethyl acetate (EA, 1000 mL × 3). The organic layers were combined, dried over sodium sulfate (Na2SO4), and the solvent was removed under reduced pressure.

[0189] The raw material was first purified by silica gel column chromatography using petroleum ether (PE) and then developed using reverse phase column chromatography with acetonitrile (MeCN): 0.1% TFA aqueous solution (5-65%) to obtain 30 g (yield: 30.82%) of 4-iodo-2-methoxy-6-(trimethylsilyl)nicotinaldehyde as a yellow oil.

[0190] 1H NMR (400 MHz, DMSO-d6): δ 9.86 - 9.80 (m, 1H), 7.55 - 7.50 (m, 1H), 3.74 (s, 3H), 0.05 (s, 9H).

[0191] LCMS analysis results: Rt = 1.644 min, m / z = 336.1 [M+1] +

[0192] (7) Preparation of 3-((but-2-en-1-yloxy)methyl)-4-iodo-2-methoxy-6-(trimethylsilyl)pyridine

[0193]

[0194] 4-Iodo-2-methoxy-6-(trimethylsilyl)nicotinaldehyde (60 g, 179 mmol), but-2-en-1-ol (42.59 g, 591 mmol), and triethylsilane (Et3SiH, 41.63 g, 58 mmol) were dissolved in methylene chloride (DCM, 300 mL), and then trifluoroacetic acid (TFA, 86 mL, 1.16 mol) was slowly added to the solution at 0°C under a nitrogen atmosphere. The mixture was stirred at room temperature for 16 h.

[0195] The target product was confirmed to be detected by liquid chromatography mass spectrometry (LCMS). The reaction mixture was diluted with methylene chloride (DCM, 800 mL), then washed with distilled water (500 mL), saturated aqueous sodium bicarbonate solution (300 mL), and brine (300 mL). The organic layer was dried over sodium sulfate (Na2SO4), and the solvent was removed under reduced pressure.

[0196] The crude material was purified by silica gel column chromatography using ethyl acetate / petroleum ether (EA / PE, 0-20%), and finally 3-((but-2-en-1-yloxy)methyl)-4-iodo-2-methoxy-6-(trimethylsilyl)pyridine was obtained as a yellow oil (50 g, yield: 37.46%).

[0197] LCMS analysis results: Rt = 1.867 min, m / z = 392.2 [M+1] +

[0198] (8) Preparation of 4-ethyl-8-methoxy-6-(trimethylsilyl)-1H-pyrano[3,4-c]pyridine

[0199]

[0200] 3-((But-2-en-1-yloxy)methyl)-4-iodo-2-methoxy-6-(trimethylsilyl)pyridine (60 g, 153.33 mmol) was dissolved in dimethylformamide (DMF, 500 mL), and then potassium carbonate (K2CO3, 42.38 mg, 306.65 mmol), tetra-n-butylammonium bromide (Bu4NBr, 9.43 g, 153.33 mmol), and palladium acetate (Pd(OAc)2, 3.44 g, 15.33 mmol) were added to the solution at room temperature. The reaction mixture was stirred at 85°C for 16 h under a nitrogen atmosphere.

[0201] The target product was detected and confirmed by liquid chromatography mass spectrometry (LCMS). The reaction mixture was diluted with ethyl acetate (EA, 2000 mL), washed with water (1000 mL) and brine (1000 mL × 3). The organic layer was dried over sodium sulfate (Na2SO4), filtered, and the filtrate was concentrated under reduced pressure.

[0202] The residue was purified by column chromatography using ethyl acetate / petroleum ether (EtOAc / PE, 0-20%), and 4-ethyl-8-methoxy-6-(trimethylsilyl)-1H-pyrano[3,4-c]pyridine (24 g, yield: 54.90%) was finally obtained as a yellow solid.

[0203] 1H NMR (400 MHz, CDCl3): δ 6.84 (s, 1H), 6.52 (t, J = 1.1 Hz, 1H), 5.02 (s, 2H), .95 (s, 3H), 2.33 (qd, J = 7.4, 1.2 Hz, 2H), 1.13 (t, J = 7.4 Hz, 3H), 0.28 (s, 9H).

[0204] LCMS analysis results: Rt = 6.060 min, m / z = 264.0 [M+1] +

[0205] (9) Preparation of (3R,4S)-4-ethyl-8-methoxy-6-(trimethylsilyl)-3,4-dihydro-1H-pyrano[3,4-c]pyridine-3,4-diol

[0206]

[0207] K3Fe(CN)6 (181.49 g, 551.23 mmol) was dissolved in a mixture of t-butanol (t-BuOH, 400 mL) and water (H2O, 400 mL), and then potassium carbonate (K2CO3, 76.18 g, 551.23 mmol), methylsulfonamide (CH3SO2NH2, 34.95 g, 367.49 mmol), osmium tetroxide (K2OsO4, 555.27 mg, 1.67 mmol), and (DHQD)2-PYR (4.42 g, 5.01 mmol) were added to the solution. The reaction mixture was stirred at 0°C for 1 h.

[0208] Afterwards, 4-ethyl-8-methoxy-6-(trimethylsilyl)-1H-pyrano[3,4-c]pyridine (44 g, 167.04 mmol) was dissolved in a mixture of t-butanol (200 mL) and water (200 mL) and slowly added. The mixture was stirred at room temperature for 16 h. The target product was detected and confirmed by liquid chromatography mass spectrometry (LCMS).

[0209] After stopping the reaction by adding Na2S2O3 to the reaction mixture, the mixture was stirred at room temperature for 30 minutes. After filtering the mixture, the filtrate was extracted with ethyl acetate (EA, 600 mL × 3) and washed with water (600 mL). The organic layer was dried over sodium sulfate (Na2SO4) and filtered, and the filtrate was concentrated under reduced pressure.

[0210] The residue was purified by column chromatography using ethyl acetate / dichloromethane (EA / DCM, 0-10%), and finally (3R,4S)-4-ethyl-8-methoxy-6-(trimethylsilyl)-3,4-dihydro-1H-pyrano[3,4-c]pyridine-3,4-diol (40 g, yield: 71.92%) was obtained as a white solid.

[0211] 1H NMR (400 MHz, CDCl3): δ 7.32 (s, 1H), 5.17 (d, J = 5.4 Hz, 1H), 4.79 (d, J = 16.5 Hz, 1H), 4.62 (d, J = 16.5 Hz, 1H), .99 (s, 3H), 1.84 (qd, J = 7.4, 2.0 Hz, 2H), 0.91 (t, J = 7.5 Hz, 3H), 0.29 (s, 9H).

[0212] LCMS analysis results: Rt = 11.948 min, m / z = 298.1 [M+1] +

[0213] (10) Preparation of (S)-4-ethyl-4-hydroxy-8-methoxy-6-(trimethylsilyl)-1,4-dihydro-3H-pyrano[3,4-c]pyridinone

[0214]

[0215] (S)-4-Ethyl-8-methoxy-6-(trimethylsilyl)-3,4-dihydro-1H-pyrano[3,4-c]pyridine-3,4-diol (40 g, 134.49 mmol) was dissolved in a mixture of methanol (MeOH, 500 mL) and water (H2O, 50 mL), and then iodine (I2, 136.54 g, 537.95 mmol) and calcium carbonate (CaCO3, 6.92 g, 268.97 mmol) were added to the mixture. The mixture was stirred at 40°C for 16 h. The target product was detected and confirmed by liquid chromatography mass spectrometry (LCMS).

[0216] The reaction was stopped by adding a saturated aqueous solution of Na2S2O3 to the reaction mixture, and the mixture was extracted with ethyl acetate (EA, 500 mL × 3). The organic layer was dried over sodium sulfate (Na2SO4) and filtered, and the filtrate was concentrated under reduced pressure.

[0217] The residue was purified by column chromatography using ethyl acetate / petroleum ether (EA / PE, 0-20%), and finally (S)-4-ethyl-4-hydroxy-8-methoxy-6-(trimethylsilyl)-1,4-dihydro-3H-pyrano[3,4-c]pyridinone (18 g, yield: 45.31%) was obtained as a yellow oil.

[0218] 1H NMR (400 MHz, CDCl3): δ 7.35 (s, 1H), 5.56 (d, J = 15.6 Hz, 1H), 5.25 (d, J = 15.6 Hz, 1H), 4.00 (s, H), 1.79 (q, J = 7.4 Hz, 2H), 0.96 (t, J = 7.4 Hz, 3H), 0.30 (s, 9H).

[0219] LCMS analysis results: Rt = 12.593 min, m / z = 296.1 [M+1] +

[0220] (11) Preparation of (S)-4-ethyl-4-hydroxy-6-iodo-8-methoxy-1,4-dihydro-3H-pyrano[3,4-c]pyridinone

[0221]

[0222] (S)-4-Ethyl-4-hydroxy-8-methoxy-6-(trimethylsilyl)-1,4-dihydro-3H-pyrano[3,4-c]pyridin-3-one (18 g, 60.93 mmol) was dissolved in acetic acid (AcOH, 500 mL), and then NCS (97.64 g, 731.19 mmol) and NaI (109.60 g, 731.19 mmol) were added to the compound. The mixture was stirred at 65°C for 16 h. The target product was detected and confirmed by liquid chromatography mass spectrometry (LCMS).

[0223] The reaction mixture was diluted with ethyl acetate (EA, 300 mL × 3), and then washed with water (500 mL). The organic layer was washed sequentially with saturated aqueous Na2S2O3 solution (500 mL × 3) and NaHCO3 (500 mL × 3), dried over sodium sulfate (Na2SO4), and filtered. The filtrate was concentrated under reduced pressure.

[0224] The residue was purified by column chromatography using ethyl acetate / petroleum ether (EA / PE, 0-10%) to obtain (S)-4-ethyl-4-hydroxy-6-iodo-8-methoxy-1,4-dihydro-3H-pyrano[3,4-c]pyridinone (17 g, yield: 74.24%) as a yellow solid.

[0225] 1H NMR (400 MHz, CDCl3): δ 7.61 (s, 1H), 5.49 (d, J = 15.7 Hz, 1H), 5.18 (d, J = 15.7 Hz, 1H), .98 (s, 3H), 1.78 (q, J = 7.4 Hz, 2H), 0.96 (t, J = 7.4 Hz, 3H).

[0226] LCMS analysis results: Rt = 10.586 min, m / z = 350.0 [M+1] +

[0227] (12) Preparation of (S)-4-ethyl-4-hydroxy-6-iodo-1,7-dihydro-3H-pyrano[3,4-c]pyridinine-3,8(4H)-dione

[0228]

[0229] (S)-4-Ethyl-4-hydroxy-6-iodo-8-methoxy-1,4-dihydro-3H-pyrano[3,4-c]pyridin-3-one (17 g, 48.69 mmol) was dissolved in acrylonitrile (ACN, 300 mL), and then NaI (11.68 g, 77.91 mmol) was added. The mixture was cooled to 0°C under a nitrogen atmosphere, and TMSCl (9.88 mL) and water (4 mL) were slowly added. The mixture was stirred at 0°C for 10 min and then reacted at 65°C for 3 h. The target product was detected and confirmed by liquid chromatography mass spectrometry (LCMS).

[0230] The reaction mixture was quenched by adding saturated Na2S2O3 aqueous solution, and then extracted with ethyl acetate (EtOAc, 300 mL × 3). The organic layer was dried over Na2SO4 and concentrated under reduced pressure. Finally, (S)-4-ethyl-4-hydroxy-6-iodo-1,7-dihydro-3H-pyrano[3,4-c]pyridinine-3,8(4H)-dione (15.5 g, yield: 94.99%) was obtained as a yellow solid.

[0231] 1H NMR (400 MHz, DMSO-d6): δ 12.44 (s, 1H), 6.99 (s, 1H), 6.35 (s, 1H), 5.23 (q, J = 15.9 Hz, 2H), 1.74 (dd, J = 13.1, 5.7 Hz, 2H), 0.79 (t, J = 7.3 Hz, 3H).

[0232] LCMS analysis results: Rt = 6.279 min, m / z = 336.0 [M+1] +

[0233] (13) Preparation of 3-(chlorodimethylsilyl)propyl acetate

[0234]

[0235] Chlorodimethylsilane (11.3 g, 119.9 mmol) was slowly added to a mixture of allyl acetate (1 g, 99.9 mmol), [IrCl(cod)]2 (7 mg, 0.01 mmol), and 1,5-cyclooctadiene (2.16 g, 20 mmol) at 70°C over 1 h. The reaction mixture was stirred in a sealed tube at 85°C for 3 h. The reaction mixture was concentrated to give 3-(chlorodimethylsilyl)propyl acetate (13 g, 66.84% yield) as a yellow oil.

[0236] 1H NMR (400 MHz, CDCl3): δ 3.99 (t, J = 6.8 Hz, 2H), 1.99 (d, J = 2.3 Hz, 3H), 1.68 (ddd, J = 13.6, 12.0, 6.8 Hz, 2H), 0.82 - 0.73 (m, 2H), 0.36 (d, J = 2.4 Hz, 6H).

[0237] (14) Preparation of 3-(dimethyl(3-((tetrahydro-2H-pyran-2-yl)oxy)propen-1-yn-1-yl)silyl)propyl acetate

[0238]

[0239] 2-(Propen-2-yn-1-yloxy)tetrahydro-2H-pyran (35 g, 250 mmol) was dissolved in THF (500 mL), and n-BuLi (120 mL, 2.5 M in hexane) was added at -60 °C. The reaction mixture was stirred at -60 °C for 1 h. Then, 3-(chlorodimethylsilyl)propyl acetate (60 g, 308 mmol) was slowly added at -60 °C, and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was poured into H2O (500 mL), and then extracted with EtOAc (500 mL × 3). The organic layers were combined, washed with brine (100 mL), dried over Na2SO4, and filtered. The filtrate was concentrated, and the raw material was purified by column chromatography with EtOAc / PE (0-20%) to obtain 3-(dimethyl(3-((tetrahydro-2H-pyran-2-yl)oxy)propen-1-yn-1-yl)silyl)propyl acetate (60 g, 80.52% yield) as a yellow oil.

[0240] 1H NMR (400 MHz, CDCl3): δ 4.81 (t, J = 3.2 Hz, 1H), 4.33 - 4.16 (m, 2H), 4.04 (t, J = 6.9 Hz, 2H), 3.88 - 3.76 (m, 1H), 3.56 - 3.47 (m, 1H), 2.05 (s, 3H), 1.90 - 1.65 (m, 5H), 1.62 - 1.48 (m, 3H), 0.69 - 0.57 (m, 2H), 0.17 (s, 6H).

[0241] (15) Preparation of 3-((3-bromopropene-1-yn-1-yl)dimethylsilyl)propyl acetate

[0242]

[0243] PPh3 (58 g, 221.14 mmol) was dissolved in DCM (600 mL), and Br2 (32.13 g, 201.04 mmol) was added at 0 °C. The reaction mixture was stirred at 0 °C for 1 h, and then 3-(dimethyl(3-((tetrahydro-2H-pyran-2-yl)oxy)propen-1-yn-1-yl)silyl)propyl acetate (60 g, 201.04 mmol) was added at 0 °C. The reaction mixture was stirred at room temperature for 16 h. The desired product was confirmed by TLC. The reaction mixture was diluted with DCM (1 L), washed with brine (100 mL), and dried over Na2SO4. After filtration, the filtrate was concentrated and the raw material was purified by column chromatography with DCM / PE (0-28%) to obtain 3-((3-bromopropene-1-yn-1-yl)dimethylsilyl)propyl acetate (40 g, 71.77% yield) as a yellow oil.

[0244] 1H NMR (400 MHz, CDCl3): δ 4.05 (t, J = 6.9 Hz, 2H), 3.90 (s, 2H), 2.10 - 2.02 (m, 3H), 1.77 - 1.62 (m, 2H), 0.68 - 0.59 (m, 2H), 0.20 - 0.12 (m, 6H).

[0245] (16) Preparation of N-(benzo[d][1,3]dioxol-5-yl)formamide

[0246]

[0247] Benzo[d][1,3]dioxol-5-amine (50 g, 385 mmol) was dissolved in DCM (500 mL), and then DCC (75 g, 365 mmol) and pyridine (85.43 g, 1.09 mol) were added, and formic acid (HCOOH, 4.03 g, 87.50 mmol) was slowly added at 0°C. The reaction mixture was stirred at room temperature for 3 h. The reaction mixture was filtered through Celite, and the filtrate was added to H2O (200 mL). The resultant was extracted with DCM (500 mL × 3). The organic layers were combined, washed with brine (100 mL), and dried over Na2SO4. After filtration, the filtrate was concentrated and the raw material was purified by column chromatography with EtOAc / PE (0-50%) to obtain N-(benzo[d][1,3]dioxol-5-yl)formamide (42 g, 69.75% yield) as a yellow solid.

[0248] LCMS analysis results: Rt = 0.758 min, m / z = 166.2 [M+1] +

[0249] (17) Preparation of 5-isocyanobenzo[d][1,3]dioxole

[0250]

[0251] N-(Benzo[d][1,3]dioxole-5-yl)formamide (42 g, 254 mmol) and Et3N (77 g, 763 mmol) were dissolved in DCM (500 mL), and POCl3 (47 g, 305 mmol) was slowly added at 0°C. The reaction mixture was stirred at 0°C for 2 h. The reaction mixture was quenched by the addition of saturated sodium hydroxide solution, and then extracted with DCM (300 mL × 3). The organic layers were combined, dried over Na2SO4, and concentrated. The raw material was purified by column chromatography with EtOAc / PE (0–20%) to give 5-isocyanobenzo[d][1,3]dioxole (23 g, 61.47% yield) as a yellow oil.

[0252] 1H NMR (400 MHz, CDCl3): δ 6.94 - 6.87 (m, 1H), 6.83 (d, J = 1.6 Hz, 1H), 6.76 (d, J = 8.2 Hz, 1H), 6.03 (s, 2H).

[0253] (18) Preparation of (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate

[0254]

[0255] (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycine (5.00 g, 14.1 mmol) was dissolved in a mixture of tetrahydrofuran (THF, 150 mL) and toluene (50.0 mL), and then pyridine (1.34 g, 16.9 mmol, 1.37 mL) and lead(IV) tetraacetate (7.51 g, 16.9 mmol) were added. The mixture was stirred at 85 °C for 2.5 h. Liquid chromatography-mass spectrometry confirmed that (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycine was consumed and the target mass (Rt = 0.43 min) was detected.

[0256] The reaction mixture was filtered through Celite, the filtrate was diluted with water (200 mL), and extracted with ethyl acetate (EtOAc, 100 mL × 3). The combined organic layers were washed with water (100 mL × 3) and brine (100 mL), dried over sodium sulfate (Na2SO4), filtered, and concentrated under reduced pressure. The residue was purified using column chromatography (SiO2, petroleum ether: ethyl acetate = 10:1 → 1:1, Rf = 0.20). Finally, (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate (3.54 g, 9.23 mmol, yield: 65.4%, purity: 96.0%) was obtained as a white solid.

[0257] 1H NMR (400 MHz, DMSO-d6): δ 8.92 (br t, J = 6.8 Hz, 1H), 7.89 (d, J = 7.6 Hz, 2H), 7.72 (d, J = 7.2 Hz, 2H), 7.56 (br t, J = 6.0 Hz, 1H), 7.42 (t, J = 7.2 Hz, 2H), 7.37-7.22 (m, 2H), 5.10 (d, J = 6.8 Hz, 2H), 4.39-4.26 (m, 2H), 4.26-4.15 (m, 1H), 3.77-3.61 (m, 2H), 2.07-1.84 (m, 3H).

[0258] LCMS analysis results: Rt = 0.43 min, m / z = 391.1 [M+Na] +

[0259] (19) Preparation of (6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)glycylglycyl-L-phenylalanine

[0260]

[0261] Fmoc-Phe-OH (2.00 g, 5.00 mmol) and dichloromethane (DCM, 60.0 mL) were added to 2-CTC resin (6.00 g, 5.00 mmol, Sub=1.13 mmol / g), and the mixture was purged with nitrogen (N2) for 30 seconds. Diethylamine (DIEA, 4.00 eq) was added dropwise and stirred at 20°C for 2 hours. Then, methanol (MeOH, 6.00 mL) was added and mixed for 0.5 hours. The mixture was filtered to obtain the resin, which was then washed with dimethylformamide (DMF, 60 mL × 5).

[0262] 20% piperidine / DMF (60.0 mL) was added to the resin, and the deprotection reaction was performed by stirring for 10 minutes under a nitrogen purge at 20°C. The resin was washed with DMF (60 mL × 5), and the completion of the reaction was confirmed by the ninhydrin test.

[0263] Fmoc-Gly-OH (3.00 eq), HBTU (2.85 eq), and DIEA (6.00 eq) were dissolved in DMF (60.0 mL) and added to the resin, and the mixture was stirred at 20°C for 2 h under nitrogen purge to perform the coupling reaction. After confirming the completion of the reaction by confirming colorlessness with the ninhydrin test, the resin was washed with DMF (60 mL × 5).

[0264] Subsequently, additional amino acids including Fmoc-Gly-OH and 6-maleimidohexanoic acid were coupled and synthesized using the same method. After the final coupling, the resin was washed with DMF (60 mL × 5) and MeOH (60 mL × 3) and dried under vacuum.

[0265] The chain-protected peptide was placed in a flask, and 20% HFIP / DCM (60.0 mL) was added three times, and the mixture was reacted at room temperature for 10 minutes under a nitrogen purge. The mixture was filtered, the filtrate was collected, and concentrated under reduced pressure to obtain (6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)glycylglycyl-L-phenylalanine (1.61 g, yield: 64.4%, purity: 94.5%) as a yellow solid.

[0266] LCMS analysis results: Rt = 0.30 min, m / z = 473.2 [M+H] +

[0267] (20) Preparation of 2,5-dioxopyrrolidin-1-yl (6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)glycylglycyl-L-phenylalanineate

[0268]

[0269] To a solution of (6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)glycylglycyl-L-phenylalanine (200 mg, 421 μmol) and HOSu (72.7 mg, 632 μmol) dissolved in dichloromethane (DCM, 2.00 mL) was added DCC (130 mg, 632 μmol, 127 μL) dissolved in DCM (2.00 mL) at 0°C. The mixture was stirred at 25°C for 2.5 h. LCMS analysis showed that 4.54% of (6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)glycylglycyl-L-phenylalanine (Rt = 0.37 min) remained, and 91.5% of the target compound (Rt = 0.41 min, MS cal.: 569.21, MS observed: [M+H] = 570.4) was detected.

[0270] The mixture was filtered, and the filter cake was washed with DCM (2.00 mL). The filtrate was concentrated under reduced pressure to obtain 2,5-dioxopyrrolidin-1-yl (6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)glycylglycyl-L-phenylalanineate (111 mg) as a white oil, which was used in the next step without further purification.

[0271] LCMS analysis results: Rt = 0.41 min, m / z = 570.4 [M+H] +

[0272] (21) Preparation of 3-(2-bromoacetamido)propanoic acid

[0273]

[0274] 3-Aminopropanoic acid (110 g, 1.23 mol) was dissolved in water (1100 mL), and then NaHCO3 (103.72 g, 1.23 mol) was added. A solution of 2-bromoacetyl bromide (124.6 g, 617.33 mmol) in DCM (550 mL) was slowly added thereto at room temperature. The reaction mixture was stirred at room temperature for 16 h.

[0275] LCMS analysis confirmed the desired product. The pH of the reaction mixture was adjusted to 3, and then extracted with EtOAc (1000 mL × 3). The organic layers were combined, dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The crude product was triturated with a mixed solvent of isopropyl alcohol / n-hexane to finally obtain 3-(2-bromoacetamido)propanoic acid (53 g, yield: 20.44%) as a white solid.

[0276] 1H NMR (400 MHz, DMSO-d6): δ 12.75 (s, 1H), 8.35 (s, 1H), 4.04 (s, 2H), .27 (dd, J = 12.5, 6.7 Hz, 2H), 2.39 (t, J = 6.8 Hz, 2H).

[0277] (22) Preparation of 2,5-dioxopyrrolidin-1-yl 3-(2-bromoacetamido)propanoate

[0278]

[0279] 3-(2-Bromoacetamido)propanoic acid (53 g, 252.35 mmol) was dissolved in isopropyl alcohol (i-PrOH, 530 mL), and then NHS (31.95 g, 277.58 mmol) and DIC (35.03 g, 277.58 mmol) were added. The reaction mixture was stirred at room temperature for 16 h.

[0280] LCMS analysis confirmed the desired product. The reaction mixture was left at room temperature for 1 hour and then cooled to 4°C overnight. The resulting crystals were collected, washed with isopropyl alcohol (530 mL), and then dissolved in isopropyl alcohol (530 mL) again and refluxed. The solution was cooled to 4°C overnight, and the resulting crystals were collected and washed with isopropyl alcohol and hexane to finally obtain 2,5-dioxopyrrolidin-1-yl 3-(2-bromoacetamido)propanoate (40 g, yield: 51.62%) as a white solid.

[0281] 1H NMR (400 MHz, DMSO-d6): δ 8.49 (s, 1H), 3.87 (s, 2H), 3.40 (dd, J = 12.5, 6.7 Hz, 2H), 2.88 (t, J = 6.8 Hz, 2H), 2.82 (s, 4H).

[0282] (23) tert-butyl N 6 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 2 Preparation of -(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctathriacontan-38-oil)-L-lysinate

[0283]

[0284] SM-1 (24 g, 56.53 mmol) was dissolved in DMF (250 mL), and then SM-2 (24 g, 40.70 mmol), HOBt (8.4 g, 62.18 mmol), DIPEA (29.23 g, 226.13 mmol), and HBTU (23.58 g, 62.18 mmol) were added at room temperature. The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was poured into water (2000 mL) and extracted with EA (1000 mL × 3). The organic layer was washed with brine (1000 mL), dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography (MeOH / DCM, 0-10%) to finally obtain tert-butyl N 6 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 2 -(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctathriacontan-38-oil)-L-lysinate (47 g, yield: 83.54%) was obtained in the form of a yellow oil.

[0285] LCMS analysis results: Rt = 1.719 min, m / z = 995.8 [M+H] + .

[0286] (24) N 6-(((9H-fluoren-9-yl)methoxy)carbonyl)-N 2 - Preparation of (2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctathriacontan-38-oil)-L-lysine

[0287]

[0288] tert-butyl N 6 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 2 -(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctathriacontan-38-oyl)-L-lysinate (8.00 g, 8.04 mmol) was added DCM (80 mL), and TFA (10 mL) was added at room temperature. The reaction mixture was stirred at room temperature for 16 h. LCMS analysis confirmed the formation of the desired compound. After filtering the reaction mixture, the filtrate was concentrated under reduced pressure to give the crude product N as a yellow oil. 6 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 2 -(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctathriacontan-38-oil)-L-lysine (5.20 g, 72.36% yield) was obtained.

[0289] LCMS analysis results: Rt = 0.913 min, m / z = 939.5 [M+H] + .

[0290] (25) Preparation of tert-butyl (2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctathriacontan-38-oil)-L-lysinate

[0291]

[0292] tert-butyl N 6 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 2-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatotriacontane-38-oyl)-L-lysinate (47 g, 47.23 mmol) was dissolved in DMF (300 mL), and then morpholine (41.14 g, 472.26 mmol) was added. The mixture was stirred at room temperature for 16 h. LCMS analysis confirmed the desired product. The reaction mixture was poured into water (300 mL), filtered, and the filtrate was concentrated under reduced pressure to finally obtain tert-butyl (2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatotriacontane-38-oyl)-L-lysinate (36 g, yield: 98.62%) as a yellow oil.

[0293] LCMS analysis results: Rt = 1.080 min, m / z = 774.1 [M+H] + .

[0294] (26) tert-butyl N 6 -(3-(2-bromoacetamido)propanoyl)-N 2 Preparation of -(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctathriacontan-38-oil)-L-lysinate

[0295]

[0296] Tert-butyl (2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctathriacontan-38-oyl)-L-lysinate (30 g, 38.81 mmol) was dissolved in DMF (300 mL), and then 2,5-diisopyrrolidin-1-yl 3-(2-bromoacetamido)propanoate (23.84 g, 77.62 mmol) was added. The mixture was stirred at room temperature for 16 h. LCMS analysis confirmed the desired product. The reaction mixture was concentrated under reduced pressure, and the residue was purified by reverse-phase column chromatography (MeCN: 0.1% FA in H2O = 5-95%) to finally obtain tert-butyl N 6-(3-(2-bromoacetamido)propanoyl)-N 2 -(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctathriacontan-38-oil)-L-lysinate (20 g, yield: 40.05%) was obtained in the form of a yellow oil.

[0297] LCMS analysis results: Rt = 1.405 min, m / z = 966.2 [M+H] + .

[0298] (27) N 6 -(3-(2-bromoacetamido)propanoyl)-N 2 - Preparation of (2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctathriacontan-38-oil)-L-lysine

[0299]

[0300] tert-butyl N 6 -(3-(2-bromoacetamido)propanoyl)-N 2 -(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctathriacontan-38-oyl)-L-lysinate (20 g, 20.73 mmol) was added to a mixture of TFA (60 mL) and DCM (180 mL). The reaction mixture was stirred at room temperature for 16 h. LCMS analysis confirmed the desired product. The reaction mixture was concentrated under reduced pressure, and the residue was purified by reverse-phase column chromatography (MeCN: 0.1% FA in H2O = 5-95%) to give N 6 -(3-(2-bromoacetamido)propanoyl)-N 2 -(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctathriacontan-38-oil)-L-lysine (14 g, yield: 74.32%) was obtained in the form of a yellow oil.

[0301] LCMS analysis results: Rt = 6.898 min, m / z = 910.7 [M+H] + .

[0302] (28) Preparation of (9H-fluoren-9-yl)methyl ((S)-13-benzyl-2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]furano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-2-methyl-9,12,15,18-tetraoxo-6-oxa-8,11,14,17-tetraaza-2-silanonadecan-19-yl)carbamate

[0303]

[0304] (S)-7-ethyl-7-hydroxy-14-((3-hydroxypropyl)dimethylsilyl)-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]furano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (5.0 g, 9.83 mmol) was dissolved in DMF (40 mL), and then (S)-11-benzyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2-oxa-4,7,10,13,16-pentaazaheptadecan-17-yl acetate (5.57 g, 8.85 mmol) was added. The mixture was stirred at 5°C for 10 minutes, after which 4N hydrochloric acid (8 mL in ethyl acetate) was slowly added. The reaction mixture was stirred at room temperature for 16 hours. LCMS analysis confirmed the desired product. The reaction mixture was filtered and concentrated under reduced pressure at 0°C. The residue was purified by reverse phase column chromatography (MeCN / 0.1% FA / H2O = 5-70%) to obtain (9H-fluoren-9-yl)methyl ((S)-13-benzyl-2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]furano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-2-methyl-9,12,15,18-tetraoxo-6-oxa-8,11,14,17-tetraaza-2-silanonadecan-19-yl)carbamate (2.20 g, yield: 0.76%) as a pale yellow solid.

[0305] LCMS analysis results: Rt = 1.656 min, m / z = 1078.3 [M+H] + .

[0306] (29) Preparation of (S)-2-(2-(2-aminoacetamido)acetamido)-N-(2-(((3-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]furano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)dimethylsilyl)propoxy)methyl)amino)-2-oxoethyl)-3-phenylpropionamide

[0307]

[0308] To a solution of (9H-fluoren-9-yl)methyl ((S)-13-benzyl-2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]furano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-2-methyl-9,12,15,18-tetraoxo-6-oxa-8,11,14,17-tetraaza-2-silanonadecan-19-yl)carbamate (6.0 g, 5.56 mmol) in DMF (40 mL) was added morpholine (2.42 g, 27.82 mmol) under a nitrogen atmosphere. The reaction mixture was stirred at room temperature for 3 h. LCMS analysis confirmed the desired product. The reaction mixture was stopped by adding water (20 mL), and then filtered. The filtrate was purified by reverse phase column chromatography (MeCN / 0.1% FA / H2O = 5-50%) to obtain (S)-2-(2-(2-aminoacetamido)acetamido)-N-(2-(((3-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]furano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)dimethylsilyl)propoxy)methyl)amino)-2-oxoethyl)-3-phenylpropionamide (3.1 g, yield: 65.08%) as a yellow solid.

[0309] LCMS analysis results: Rt = 7.91 min, m / z = 856.7 [M+H] + .

[0310] [Payload and Linker-Payload Manufacturing]

[0311] [Comparative Example 1] Manufacturing of Payload AM20001

[0312] (1) Preparation of (S)-4-ethyl-4-hydroxy-6-iodo-7-(3-(trimethylsilyl)prop-2-ynol-1-yl)-1,7-dihydro-3H-pyrano[3,4-c]pyridine-3,8(4H)-dione

[0313]

[0314] (S)-4-Ethyl-4-hydroxy-6-iodo-1,7-dihydro-3H-pyrano[3,4-c]pyridine-3,8(4H)-dione (2.12 g, 6.08 mmol, 96.1% purity, 1.00 eq) was dissolved in a DME:DMF (3:1, 25.0 mL) mixture, and then NaH (267 mg, 6.69 mmol, 60% purity, 1.10 eq) was added at 0 °C under N2 atmosphere. The mixture was stirred at 0 °C for 10 min, and then LiBr (1.06 g, 12.1 mmol, 305 μL, 2.00 eq) was added. The mixture was stirred at 20 °C for 15 min, after which (3-bromoprop-1-ynol-1-yl)trimethylsilane (3.49 g, 18.2 mmol, 2.98 mL, 3.00 eq) was added. The reaction mixture was stirred in the dark at 20 °C for 20 h. LCMS analysis showed that (S)-4-ethyl-4-hydroxy-6-iodo-1,7-dihydro-3H-pyrano[3,4-c]pyridine-3,8(4H)-dione was completely consumed, and 52.5% of the desired compound was detected (Rt = 0.62 min, m / z = 445.0, MS observed: [M+H] = 445.8). The reaction mixture was quenched by adding 100 mL of NH4Cl at 0 °C, and then extracted with 200 mL of EtOAc. The organic layer was washed with 200 mL of brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel column chromatography (SiO2, petroleum ether: ethyl acetate = 1:0 to 5:1) to obtain (S)-4-ethyl-4-hydroxy-6-iodo-7-(3-(trimethylsilyl)prop-2-ynol-1-yl)-1,7-dihydro-3H-pyrano[3,4-c]pyridine-3,8(4H)-dione (2.10 g, 4.58 mmol, 75.3% yield, 97.2% purity) as a yellow solid.

[0315] LCMS analysis results: Rt = 0.62 min, m / z = 445.8 [M+H] + .

[0316] 1H NMR (400 MHz, CDCl3): δ 7.35 (s, 1H), 5.60 (d, J = 16.4 Hz, 1H), 5.27 - 5.10 (m, H), 3.68 (s, 1H), 1.95 - 1.80 (m, 2H), 1.07 (t, J = 7.6 Hz, 3H), 0.25 (s, 9H).

[0317] SFC analysis results: Rt = 0.77 min, ee value: 100%.

[0318] (2) Preparation of tert-butyl (S)-(4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-11-(trimethylsilyl)-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate

[0319]

[0320] (S)-4-Ethyl-4-hydroxy-6-iodo-7-(3-(trimethylsilyl)prop-2-ynol-1-yl)-1,7-dihydro-3H-pyrano[3,4-c]pyridine-3,8(4H)-dione (800 mg, 1.75 mmol, 97.2% purity, 1.00 eq), tert-butyl (2-fluoro-4-isocyanophenyl)carbamate (742 mg, 3.14 mmol, 100% purity, 1.80 eq), 4Å MS (600 mg), and (Me3Sn)2 (858 mg, 2.62 mmol, 543 μL, 1.50 eq) were dissolved in toluene (10.0 mL), and the reaction mixture was irradiated with a UV lamp at 70°C for 17 h. LCMS analysis showed that (S)-4-ethyl-4-hydroxy-6-iodo-7-(3-(trimethylsilyl)prop-2-ynol-1-yl)-1,7-dihydro-3H-pyrano[3,4-c]pyridine-3,8(4H)-dione was completely consumed and 37.1% of the desired compound was detected (Rt = 0.66 min, m / z = 553.2, MS observed: [M+H] = 554.4). The reaction mixture was quenched by the addition of 50 mL of saturated KF solution at 0°C, and then extracted with 50 mL of EtOAc. The organic layer was washed with 50 mL of brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by silica gel column chromatography (SiO2, petroleum ether:ethyl acetate = 1:0 to 2:1). It was confirmed by TLC (petroleum ether:ethyl acetate = 1:1) and reverse phase HPLC (neutral condition). SFC analysis revealed two peaks (Rt = 1.06 min and Rt = 1.28 min). It was then purified by SFC (column: DAICEL CHIRALCEL OJ (250 mm * 30 mm, 10 μm); mobile phase: [Neu - MeOH]; B%: 20% - 20%, C9.8; 147 min) and finally tert-butyl (S)-(4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-11-(trimethylsilyl)-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate (140 mg, 252.87 μmol, 14.48% yield, 100% purity) was obtained as a yellow solid.

[0321] LCMS analysis results: Rt = 0.66 min, m / z = 554.4 [M+H] + .

[0322] 1H NMR (400 MHz, CDCl3): δ 9.15 (d, J = 8.8 Hz, 1H), 7.84 (d, J = 12.4 Hz, 1H), 7.59 (s, 1H), 7.04 (br d, J = .6 Hz, 1H), 5.75 (d, J = 16.0 Hz, 1H), 5.33 - 5.29 (m, 3H), 3.73 (s, 1H), 1.90 (tdd, J = 7.2, 14.0, 18.0 Hz, 2H), 1.04 (t, J = 7.2 Hz, 3H), 0.68 (s, 9H).

[0323] SFC analysis results: Peak 1, Rt = 1.06 min and Peak 2, Rt = 1.28 min.

[0324] (3) Preparation of (S)-9-amino-4-ethyl-8-fluoro-4-hydroxy-11-(trimethylsilyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione

[0325]

[0326] Tert-butyl (S)-(4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-11-(trimethylsilyl)-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate (140 mg, 252 μmol, 100% purity, 1.00 eq) was dissolved in DCM (1.60 mL), and then TFA (0.20 mL) was added. The reaction mixture was stirred at 25 °C for 3 h. LCMS analysis revealed that 4.98% of tert-butyl (S)-(4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-11-(trimethylsilyl)-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate remained, and 93.7% of the desired compound was detected (Rt = 0.55 min, m / z = 453.1, MS observed: [M+H] + = 454.2). The reaction mixture was diluted with 3 mL of DCM and concentrated under reduced pressure to obtain a residue. The residue was dissolved in MeCN: H2O (10 mL, 1:10), and the solvent was lyophilized to obtain (S)-9-amino-4-ethyl-8-fluoro-4-hydroxy-11-(trimethylsilyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (120 mg, 205 μmol, 81.1% yield, 97.0% purity) as a yellow solid.

[0327] LCMS analysis results: Rt = 0.55 min, m / z = 454.2 [M+H] + .

[0328] 1H NMR (400 MHz, CDCl3): δ 7.68 (d, J = 12.0 Hz, 1H), 7.57 (s, 1H), 7.51 (d, J = 9.6 Hz, 1H), 5.58 (d, J = 16.0 Hz, 1H), 5.39 - 5.33 (m, H), 1.96 (dq, J = 4.0, 7.2 Hz, 2H), 1.01 (t, J = 7.2 Hz, 3H), 0.64 (s, 9H).

[0329] SFC analysis results: Rt = 1.53 min, ee value: 100%.

[0330] [Example 1] Manufacturing of payload AM20002

[0331]

[0332] (S)-9-Amino-4-ethyl-8-fluoro-4-hydroxy-11-(trimethylsilyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (10.0 mg, 21.0 μmol, 95.4% purity, 1.00 eq) and DIEA (5.44 mg, 42.0 μmol, 7.33 μL, 2.00 eq) were dissolved in THF (0.30 mL), and then 2-chloro-2-oxoethyl acetate (5.74 mg, 42.0 μmol, 4.52 μL, 2.00 eq) was added. The mixture was stirred at 25°C for 1 h, concentrated under reduced pressure, redissolved in MeOH (0.30 mL), and K2CO3 (5.81 mg, 42.0 μmol, 2.00 eq) was added. The reaction mixture was stirred at 25°C for 3 h, and the completion of the reaction was confirmed by TLC. The pH of the reaction mixture was adjusted to 5 with 1 N HCl and extracted with 20 mL of EtOAc. The organic layer was washed with H2O (10 mL × 2) and brine (20 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue. The crude material was purified by prep-HPLC (TFA conditions) to obtain (S)-2-hydroxyacetamide-N-(4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-11-(trimethylsilyl)-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)(5.26 mg, 9.77 μmol, 46.4% yield, 100% purity) as an off-white solid.

[0333] LCMS analysis results: Rt = 0.49 min, m / z = 512.3 [M+H] + .

[0334] [Example 2] Manufacturing of payload AM20003

[0335] (1) Preparation of tert-butyl (4-((S)-7-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4'::6,7]indolizino[1,2-b]quinolin-20-ylamino)-4-oxobutyl)carbamate

[0336]

[0337] To a solution of (S)-7-ethyl-8-fluoro-4-hydroxy-1,3,12-trihydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14-dione (317 mg, 1.56 mmol, 25.0 eq) and 4 Å molecular sieves (200 mg) in THF (4.00 mL), NMM (157 mg, 1.56 mmol, 171 μL, 25.0 eq) and IBCF (213 mg, 1.56 mmol, 204 μL, 25.0 eq) were added at 0°C and stirred for 2 h. Subsequently, AMB-CPT1 (28.3 mg, 62.4 μmol, 1.00 eq) dissolved in THF (0.50 mL) was added at 0°C and stirred for an additional hour. The reaction mixture was further stirred at 25°C for an additional 30 hours. LCMS analysis revealed that 4.90% of AMB-CPT1 (Rt = 0.40 min) remained, and 51.8% of the desired product was formed (Rt = 0.46 min, m / z = 638.26, MS observed: [M+H] = 639.4). The pH of the reaction mixture was adjusted to 4–5 with acetic acid at 0°C, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by prep-HPLC (formic acid condition) to finally obtain tert-butyl (4-((S)-7-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-20-ylamino)-4-oxobutyl)carbamate (90.0 mg, crude material before purification) as a pale yellow solid, which was used in the next step of the reaction.

[0338] LCMS analysis results: Rt = 0.46 min, m / z = 639.4 [M+H] +

[0339] (2) Preparation of (S)-4-((7-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-20-ylamino)butanamide

[0340]

[0341] Tert-butyl (4-((S)-7-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-20-ylamino)-4-oxobutyl)carbamate (90.0 mg, 140 μmol, 1.00 eq) was dissolved in DCM (0.32 mL), and then TFA (0.04 mL) was added. The reaction mixture was stirred at 25 °C for 0.5 h. LCMS analysis showed that tert-butyl (4-((S)-7-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-20-ylamino)-4-oxobutyl)carbamate was completely consumed, and one major product with the target m / z value was detected (Rt = 0.31 min, m / z = 538.20, MS observed: [M+H] + = 539.2). The reaction mixture was concentrated under reduced pressure to obtain the residue, which was purified by prep-HPLC (TFA condition) to finally obtain (S)-4-((7-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-20-ylamino)butanamide (10.0 mg, 15.0 μmol, 24.1% yield, 98.3% purity, TFA salt) as a yellow solid.

[0342] 1H NMR (400 MHz, DMSO-d6): δ 10.22 (s, 1H), 9.32 (d, J = 8.4 Hz, 1H), 8.04 (d, J = 12.0 Hz, 1H), 7.73 (br s, 3H), 7.31 (s, 1H), 6.52 (s, 1H), 5.43 (s, 2H), 5.38 (s, 2H), 2.91-2.85 (m, 2H), 2.66 (t, J = 7.2 Hz, 2H), 1.95-1.81 (m, 4H), 0.87 (t, J = 7.2 Hz, 3H), 0.61 (s, 9H)

[0343] LCMS analysis results: Rt = 0.31 min, m / z = 539.2 [M+H] +

[0344] [Example 3] Manufacturing of payload AM20004

[0345] (1) Preparation of tert-butyl (2-aminoethyl)((S)-7-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-20-yl)carbamate

[0346]

[0347] A mixture containing (S)-7-ethyl-8-fluoro-4-hydroxy-1,3,12-trihydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14-dion-20-amine (31.7 mg, 198 μmol, 31.2 μL, 3.00 eq) was dissolved in DCM (0.50 mL), and then 2-((tert-butoxycarbonyl)amino)ethylamine (30.0 mg, 66.1 μmol, 1.00 eq) and 4 Å molecular sieves (100 mg) were added to the DCM (0.50 mL) solution, and triphosgene (13.7 mg, 46.3 μmol, 0.70 eq) was added at 0°C under N2 atmosphere. The reaction mixture was stirred at 25°C for 1 hour, then (S)-7-ethyl-8-fluoro-4-hydroxy-1,3,12-trihydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14-dion-20-amine solution was added and stirred at 25°C for an additional 15 hours. LCMS analysis showed that (S)-7-ethyl-8-fluoro-4-hydroxy-1,3,12-trihydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-3,14-dion-20-amine was completely consumed, and the main peak of the target compound was confirmed (Rt = 0.45 min, m / z = 640.3, MS cal.: 639.25, MS observed: [M+H] + = 640.3). The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. This residue was purified by prep-HPLC (formic acid condition) to finally obtain tert-butyl (2-aminoethyl)((S)-7-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-20-yl)carbamate (17 mg, 26.44 μmol, 39.9% yield, 99.6% purity) as a yellow solid.

[0348] LCMS analysis results: Rt = 0.45 min, m / z = 640.3 [M+H] + .

[0349] (2) Preparation of (S)-20-(2-aminoethylamino)-7-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline

[0350]

[0351] Tert-butyl (2-aminoethyl)((S)-7-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-20-yl)carbamate (17.0 mg, 26.4 μmol, 1.00 eq.) was dissolved in DCM (0.16 mL), and then TFA (0.02 mL) was added. The reaction mixture was stirred at 25 °C for 1 h. LCMS analysis revealed that tert-butyl (2-aminoethyl)((S)-7-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-20-yl)carbamate was completely consumed and a new major peak with the desired m / z was formed (Rt = 0.31 min, MS cal.: 539.20, MS observed: [M+H] + = 540.3). The reaction mixture was concentrated under reduced pressure to obtain a residue. The residue was purified by prep-HPLC (TFA conditions) to obtain (S)-20-(2-aminoethylamino)-7-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline (15.0 mg, 22.8 μmol, 86.5% yield, 99.7% purity, TFA salt) as a yellow solid.

[0352] 1H NMR (400 MHz, DMSO-d6): δ 9.29 (d, J = 8.8 Hz, 1H), 9.03 (d, J = 3.2 Hz, 1H), 7.98 (d, J = 12.4 Hz, 1H), 7.79 (br s, 3H), 7.28 (s, 1H), 7.08 (t, J = 6.0 Hz, 1H), 6.52 - 6.50 (m, 1H), 5.42 (s, 2H), 5.35 (s, 2H), 3.42 (q, J = 6.0 Hz, 2H), 2.95 (br d, J = 5.6 Hz, 2H), 1.93 - 1.78 (m, 2H), 0.87 (t, J = 7.2 Hz, 3H), 0.60 (s, 9H).

[0353] LCMS analysis results: Rt = 0.31 min, m / z = 540.3 [M+H] +

[0354] [Example 4] Manufacturing of payload AM20005

[0355] (1) Preparation of tert-butyl ((S)-20-(assisted hydrazino)-7-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate

[0356]

[0357] (S)-7-Ethyl-8-fluoro-4-hydroxy-1,3,12-trihydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14-dion-20-amine (50.0 mg, 110 μmol, 1.00 eq) was dissolved in DCM (0.50 mL) with 4 Å 200 mg), and then triphosgene (22.9 mg, 77.1 μmol, 0.70 eq) was added in DCM (0.50 mL) at 0 °C under N2 atmosphere. The mixture was stirred at 20 °C for 1 h. Afterwards, (tert-butoxycarbonyl)hydrazine (43.7 mg, 330 μmol, 3.00 eq) dissolved in DCM (0.50 mL) was added. The reaction mixture was stirred at 20 °C for 16 h. LCMS analysis revealed that 30.1% (Rt = 0.41 min) of (S)-7-ethyl-8-fluoro-4-hydroxy-1,3,12-trihydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-3,14-dion-20-amine remained, and 65.5% of the desired compound was formed (Rt = 0.43 min, MS cal.: 611.22, MS observed: [M+H] + = 612.4). The reaction mixture was concentrated under reduced pressure to obtain a residue. The residue was dissolved in DMF (1 mL) and then filtered. The solution was purified by prep-HPLC (FA condition) to finally obtain tert-butyl ((S)-20-(assisted hydrazino)-7-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate (20.0 mg, 32.1 μmol, 29.1% yield, 98.4% purity) as a yellow solid.

[0358] LCMS analysis results: Rt = 0.43 min, m / z = 612.4 [M+H] + .

[0359] (2) Preparation of (S)-20-(hydrazino)-7-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline

[0360]

[0361] Tert-butyl ((S)-20-(auxiliary hydrazino)-7-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate (10.0 mg, 16.0 μmol, 1.00 eq) was dissolved in DCM (0.08 mL), and then TFA (0.01 mL) was added. The reaction mixture was stirred at 20 °C for 1 h. LCMS analysis revealed that 3.61% (Rt = 0.43 min) of tert-butyl ((S)-20-(assisted hydrazino)-7-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate remained, and 90.9% of the desired compound was formed (Rt = 0.36 min, MS cal.: 511.17, MS observed: [M+H] + = 512.2). The reaction mixture was concentrated under reduced pressure to obtain a residue. The residue was purified by prep-HPLC (neutral condition) to finally obtain (S)-20-(hydrazino)-7-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline (3.60 mg, 6.57 μmol, 40.8% yield, 93.3% purity) as a yellow solid.

[0362] 1H NMR (400 MHz, DMSO-d6): δ 9.32 (d, J = 8.8 Hz, 1H), 7.99 (br d, J = 12.4 Hz, 1H), 7.28 (s, 1H), 6.49 (s, 1H), 5.42 (s, 2H), 5.35 (s, 2H), 4.87 - 4.58 (m, 1H), 1.89 - 1.82 (m, 2H), 0.87 (br t, J = 7.2 Hz, 3H), 0.59 (s, 9H).

[0363] LCMS analysis results: Rt = 0.36 min, m / z = 512.2 [M+H] + .

[0364] [Example 5] Manufacturing of payload AM20007

[0365] (1) Preparation of benzyl 3-(((S)-7-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-20-yl)amino)propionate

[0366]

[0367] Benzyl 3-hydroxypropionate (100 mg, 514 μmol, 1.00 eq) was dissolved in THF (5.00 mL), and NMM (52.0 mg, 514 μmol, 56.6 μL, 1.00 eq) and IBCF (70.3 mg, 514 μmol, 67.3 μL, 1.00 eq) were added at 0°C and stirred for 30 minutes. Afterwards, (S)-7-ethyl-8-fluoro-4-hydroxy-1,3,12-trihydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-3,14-dion-20-amine (45.0 mg, 97.3 μmol, 0.189 eq) was added to the mixture. The mixture was stirred at 25°C for 48 h. LCMS analysis confirmed that benzyl 3-hydroxypropionate was completely consumed and the desired compound was formed (Rt = 2.69 min, MS cal.: 629.24, MS observed: [M+H] + = 630.3). The pH of the mixture was adjusted to 6 with acetic acid (17.5 M), filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by prep-HPLC (TFA condition) to obtain benzyl 3-(((S)-7-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-20-yl)amino)propionate (53.0 mg, 80.7 μmol, 15.6% yield, 96.0% purity) as a yellow solid.

[0368] LCMS analysis results: Rt = 2.69 min, m / z = 630.3 [M+H] + .

[0369] (2) Preparation of 3-(((S)-7-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-20-yl)amino)propionic acid

[0370]

[0371] Benzyl 3-(((S)-7-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-20-yl)amino)propionate (20.0 mg, 30.4 μmol, 1.00 eq) was dissolved in THF (0.50 mL) and EtOH (2.00 mL), and then Pd / C (20.0 mg, 18.7 μmol, 10.0% purity, 6.16e-1 eq) was added under N2 atmosphere. After that, H2 (61.4 μg, 30.4 μmol, 1.00 eq) was injected, and the mixture was stirred at 25°C for 6.5 h under H2 (15 Psi). LCMS analysis showed that benzyl 3-(((S)-7-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-20-yl)amino)propionate was completely consumed, and the main peak of the desired compound was confirmed (Rt = 0.38 min, m / z = 540.2). The two parallel reactants were combined for work-up. The mixture was filtered and purified under reduced pressure to obtain the residue. The residue was purified by prep-HPLC (TFA conditions) to give 3-(((S)-7-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-20-yl)amino)propionic acid (13.8 mg, 25.0 μmol, 41.0% yield, 96.4% purity) as a yellow solid.

[0372] 1H NMR (400 MHz, DMSO-d6): δ 10.13 (s, 1H), 9.32 (d, J = 8.4 Hz, 1H), 8.00 (d, J = 12.0 Hz, 1H), 7.30 (s, 1H), 6.51 (s, 1H), 5.42 (s, 2H), 5.37 (s, 2H), 4.55 (t, J = 5.2 Hz, 1H), 3.51 - 3.44 (m, 2H), 2.58 (br t, J = 7.6 Hz, 2H), 1.90 - 1.76 (m, 4H), 0.86 (br t, J = 7.2 Hz, 3H), 0.60 (s, 9H).

[0373] LCMS analysis results: Rt = 0.38 min, m / z = 540.2 [M+H] + .

[0374] [Example 6] Manufacturing of payload AM20008

[0375] Preparation of 2-hydroxyethyl (S)-(4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-11-(trimethylsilyl)-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate

[0376]

[0377] (S)-9-Amino-4-ethyl-8-fluoro-4-hydroxy-11-(trimethylsilyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (45.0 mg, 99.2 μmol, 1.00 eq) and 4 Å MS (100 mg) were dissolved in DCM (0.50 mL), and then triphosgene (20.6 mg, 69.4 μmol, 0.70 eq) in DCM (0.50 mL) was added at 0°C under N2 atmosphere. The mixture was stirred at 25 °C for 1 h, and then 2-((tert-butyldimethylsilyl)oxy)ethan-1-ol (52.4 mg, 297 μmol, 3.00 eq) dissolved in DCM (0.50 mL) was added. The reaction mixture was stirred at 25 °C for 15 h. LCMS analysis showed that (S)-9-amino-4-ethyl-8-fluoro-4-hydroxy-11-(trimethylsilyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione was completely consumed, and the major peak with the desired m / z value was confirmed (Rt = 0.57 min, m / z = 541.17, MS observed: [M+TBS+H] + = 656.4). The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by prep-HPLC (FA condition) to finally obtain 2-hydroxyethyl (S)-(4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-11-(trimethylsilyl)-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate (29.0 mg, 53.2 μmol, 53.6% yield, 99.4% purity) as a pale yellow solid.

[0378] 1H NMR (400 MHz, DMSO-d6): δ 9.96 (br s, 1H), 8.92 (d, J = 8.4 Hz, 1H), 7.98 (d, J = 12.0 Hz, 1H), 7.30 (s, 1H), 6.53 - 6.50 (m, 1H), 5.42 (s, 2H), 5.37 (s, 2H), 4.86 (t, J = 5.6 Hz, 1H), 4.22 - 4.20 (m, 2H), 3.67 (q, J = 5.2 Hz, 2H), 1.93 - 1.78 (m, 2H), 0.87 (t, J = 7.2 Hz, 3H), 0.60 (s, 9H).

[0379] LCMS analysis results: Rt = 0.57 min, m / z = 656.4 [M+TBS+H] + .

[0380] [Example 7] Manufacturing of payload AM20009

[0381] (1) Preparation of 2-fluoro-N-diformylbenzeneamine

[0382]

[0383] DCC (21.4 g, 103 mmol, 21.0 mL, 1.30 eq) was dissolved in DCM (100 mL), and HCOOH (4.99 g, 103 mmol, 1.30 eq) was added at 0 °C under N2 atmosphere and stirred for 10 min. Then, 2-fluorobenzenamine (10.0 g, 79.9 mmol, 1.00 eq) was dissolved in DCM (100 mL), and Py (8.34 g, 105 mmol, 8.51 mL, 1.32 eq) and the previously prepared mixture were added at 0 °C under N2 atmosphere. The reaction mixture was stirred at 25 °C for 15 h. TLC (petroleum ether:ethyl acetate = 2:1) showed that 2-fluorobenzenamine (Rf = 0.65) remained and two new spots (Rf = 0.40, 0.45) were formed. The reaction mixture was diluted with DCM (100 mL), filtered, and reduced pressure to obtain the residue. The residue was purified by silica gel column chromatography (SiO2, petroleum ether:ethyl acetate = 1:0 to 0:1, Rf = 0.45) to obtain 2-fluoro-N-diformylbenzenamine (12.7 g, raw material) as a yellow solid.

[0384] 1H NMR (400 MHz, DMSO-d6): δ 10.27 (s, 1H), 8.26 (s, 1H), 7.50 (d, J = 12.4 Hz, 1H), 7.22 - 7.19 (m, 2H), 2.16 (s, 3H).

[0385] (2) Preparation of 2-fluoro-4-methylphenyl isocyanate

[0386]

[0387] N-Formyl-2-fluorobenzenamine (10.0 mg, 16.0 μmol, 1.00 eq) was dissolved in DCM (0.08 mL), and then TFA (0.01 mL) was added. The reaction mixture was stirred at 20°C for 1 h. LCMS analysis revealed that 3.61% (Rt = 0.43 min) of N-formyl-2-fluorobenzenamine remained, and 90.9% of the desired compound was formed (Rt = 0.36 min, MS cal.: 511.17, MS observed: [M+H] + = 512.2). The reaction mixture was concentrated under reduced pressure to obtain a residue. The residue was purified by prep-HPLC (neutral conditions) to finally obtain 2-fluoro-4-methylphenylisocyanate (3.60 mg, 6.57 μmol, 40.8% yield, 93.3% purity) as a yellow solid.

[0388] 1H NMR (400 MHz, DMSO-d6): δ 9.32 (d, J = 8.8 Hz, 1H), 7.99 (br d, J = 12.4 Hz, 1H), 7.28 (s, 1H), 6.49 (s, 1H), 5.42 (s, 2H), 5.35 (s, 2H), 4.87 - 4.58 (m, 1H), 1.89 - 1.82 (m, 2H), 0.87 (br t, J = 7.2 Hz, 3H), 0.59 (s, 9H).

[0389] LCMS analysis results: Rt = 0.36 min, m / z = 512.2 [M+H] + .

[0390] (3) Preparation of 1-[(tetrahydropyran-2-yl)oxy]-4-(trimethylsilyl)butene

[0391]

[0392] 1-(Tetrahydropyran-2-yl)propyn-2-ol (10.0 g, 71.3 mmol, 10.0 mL, 1.00 eq) was dissolved in THF (100 mL), and n-BuLi (2.50 M, 28.5 mL, 1.00 eq) was added at -60 °C under N2 atmosphere and stirred for 0.5 h. Then, 4-chlorobutyltrichlorosilane (12.2 g, 71.3 mmol, 1.00 eq) was added. The reaction mixture was stirred at 25 °C for 2 h. 1H NMR results confirmed that 1-(tetrahydropyran-2-yl)propyn-2-ol was completely consumed and the desired product was formed. The reaction mixture was neutralized by adding 300 mL of water at 0°C, and then extracted with EtOAc (600 mL, 300 mL × 2). The organic layers were combined, washed with water (300 mL) and brine (300 mL), dried over Na2SO4, filtered, and purified by vacuum filtration to obtain 1-[(tetrahydropyran-2-yl)oxy]-4-(trimethylsilyl)butylene (19.4 g, 70.5 mmol, 98.9% yield) as a yellow oil.

[0393] 1H NMR (400 MHz, CDCl3): δ: 4.81 (t, J = 3.2 Hz, 1H), 4.32 - 4.21 (m, 2H), 3.87 - 3.81 (m, 1H), 3.54 (t, J = 6.8 Hz, 3H), 1.90 - 1.61 (m, 8H), 0.75 - 0.71 (m, 2H), 0.18 (s, 6H).

[0394] (4) Preparation of 1-bromo-4-(trimethylsilyl)butene

[0395]

[0396] PPh3 (10.0 g, 38.2 mmol, 1.05 eq) was dissolved in DCM (100 mL), and Br2 (5.81 g, 36.3 mmol, 1.87 mL, 1.00 eq) was added at 0 °C under N2 atmosphere. The mixture was stirred at 0 °C for 0.5 h. Then, 1-[(tetrahydropyran-2-yl)oxy]-4-(trimethylsilyl)butylene (10.0 g, 36.3 mmol, 1.00 eq) in DCM (40.0 mL) was added at 0 °C and stirred for 2 h. The reaction mixture was stirred at 25 °C for 3 h. TLC (petroleum ether: ethyl acetate = 20:1) showed that 1-[(tetrahydropyran-2-yl)oxy]-4-(trimethylsilyl)butylene (Rf = 0.55) remained and two new spots (Rf = 0.85, 0.05) were formed. The reaction mixture was diluted with 200 mL of water at 0°C, and extracted with DCM (300 mL, 150 mL × 2). The organic layers were combined, washed with 200 mL of saturated aqueous NaHCO3 solution and 200 mL of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by silica gel column chromatography (SiO2, petroleum ether: ethyl acetate = 1:0 to 10:1, Rf = 0.85) to finally obtain 1-bromo-4-(trimethylsilyl)butine (6.43 g, 5.3 mmol, 69.6% yield) as a yellow oil.

[0397] 1H NMR (400 MHz, CDCl3): δ: 3.91 (s, 2H), 3.55 (t, J = 6.8 Hz, 2H), 1.90 - 1.82 (m, 2H), 0.77 - 0.73 (m, 2H), 0.19 (s, 6H).

[0398] (5) Preparation of 7-[4-(trimethylsilyl)butylchloro]-1,3-dihydro-3,6,8-trimethyl-4H-pyrrolo[3,4-b]quinoline-4,9-dione

[0399]

[0400] 1,3-Dihydro-7-hydroxy-3,6,8-trimethyl-4H-pyrrolo[3,4-b]quinoline-4,9-dione (5.00 g, 14.8 mmol, 1.00 eq) was dissolved in DME (100 mL) and DMF (25.0 mL), and then NaH (651 mg, 16.3 mmol, 60% purity, 1.10 eq) was added at 0 °C under N2 atmosphere. The mixture was stirred at 0 °C for 15 min, after which LiBr (2.57 g, 29.6 mmol, 743 μL, 2.00 eq) was added. The mixture was stirred at 20°C for 20 min, and then 1-bromo-4-(trimethylsilyl)butylene (7.52 g, 29.6 mmol, 2.00 eq) was added. The reaction mixture was stirred in the dark at 20°C for 2 h. LCMS analysis revealed the desired mass (Rt = 0.47 min). 400 mL of saturated NH4Cl aqueous solution was added to the reaction mixture at 0°C to stop the reaction, and the mixture was extracted with 300 mL of EtOAc (100 mL × 3). The organic layers were combined, washed with 200 mL of brine, dried over Na2SO4, filtered, and evaporated under reduced pressure to obtain the residue. The residue was purified by silica gel column chromatography (SiO2, petroleum ether: ethyl acetate = 1:0 to 3:1, petroleum ether: ethyl acetate = 2:1, Rf = 0.40) to finally obtain 7-[4-(trimethylsilyl)butylchloro]-1,3-dihydro-3,6,8-trimethyl-4H-pyrrolo[3,4-b]quinoline-4,9-dione (3.83 g, 7.14 mmol, 48.2% yield, 94.7% purity) as a yellow oil.

[0401] 1H NMR (400 MHz, CDCl3): δ: 7.19 (s, 1H), 5.52 (d, J = 16.4 Hz, 1H), 5.19 - 5.03 (m, 3H), 3.61 (s, 1H), 3.53 (t, J = 6.8 Hz, 2H), 1.88 - 1.72 (m, 4H), 0.99 (t, J = 7.2 Hz, 3H), 0.75 - 0.71 (m, 2H), 0.17 (s, 6H).

[0402] LCMS analysis results: Rt = 0.47 min, m / z = 508.0 [M+H] + .

[0403] (6) Preparation of (S)-7-[4-(trimethylsilyl)butylchloro]-8-fluoro-N-(4-fluorophenyl)-3,6,8-trimethyl-4H-pyrrolo[3,4-b]quinoline-4,9-dione-1-carboxamide

[0404]

[0405] 4-Fluorophenylisocyanate (856 mg, 6.34 mmol, 1.70 eq), 7-[4-(trimethylsilyl)butylchloro]-1,3-dihydro-3,6,8-trimethyl-4H-pyrrolo[3,4-b]quinoline-4,9-dione (2.00 g, 3.73 mmol, 1.00 eq), 4 Å MS, (Me3Sn)2 (1.93 g, 5.89 mmol, 1.22 mL, 1.58 eq) were dissolved in toluene (40.0 mL), and the solution was degassed and purged three times with N2. The reaction mixture was irradiated under a UV lamp at 70 °C for 48 h under a N2 atmosphere. LCMS analysis revealed that 7-[4-(trimethylsilyl)butylchloro]-1,3-dihydro-3,6,8-trimethyl-4H-pyrrolo[3,4-b]quinoline-4,9-dione (Rt = 1.57 min) remained, and the desired mass was detected (Rt = 1.68 min). The reaction was stopped by adding 200 mL of saturated KF aqueous solution to the reaction mixture at 0 °C, and extracted with 200 mL of EtOAc (100 mL × 2). The organic layers were combined, washed with 200 mL of brine, dried over Na2SO4, filtered, and vacuum-reduced to obtain the residue. The residue was purified by silica gel column chromatography (SiO2, petroleum ether: ethyl acetate = 1:0 to 1:1, petroleum ether: ethyl acetate = 1:1, Rf = 0.37). After that, it was purified by SFC (column: DAICEL CHIRALPAK AS (250mm30mm, 10um); mobile phase: [CO2-MeOH]; B%: 30%, isobaric mode) and finally (S)-7-[4-(trimethylsilyl)butylchloro]-8-fluoro-N-(4-fluorophenyl)-3,6,8-trimethyl-4H-pyrrolo[3,4-b]quinoline-4,9-dione-1-carboxamide (100 mg, 188 μmol, 5.04% yield, 96.9% purity) was obtained as a yellow solid.

[0406] SFC analysis results: Rt = 1.43 min, ee value: 100%.

[0407] 1H NMR (400 MHz, CDCl3): δ: 7.99 (d, J = 8.0 Hz, 1H), 7.81 (d, J = 10.8 Hz, 1H), 7.63 (s, 1H), 5.75 (d, J = 16.4 Hz, 1H), 5.33 - 5.29 (m, 3H), 3.73 (s, 1H), 3.50 (t, J = 6.0 Hz, 2H), 2.55 (s, 3H), 1.94 - 1.73 (m, 4H), 1.23 - 1.19 (m, 2H), 1.05 (t, J = 7.2 Hz, 3H), 0.68 (s, 6H).

[0408] LCMS analysis results: Rt = 0.49 min, m / z = 515.2 [M+H] + .

[0409] (7) Preparation of 4-(2-azobutyl)-7-[(trimethylsilyl)octylchloro]-8-fluoro-1,3-dihydro-3,6,8-trimethyl-4H-pyrrolo[3,4-b]quinoline-4,9-dione

[0410]

[0411] 4-(2-Chloroethyl)-7-[(trimethylsilyl)octylchloro]-8-fluoro-1,3-dihydro-3,6,8-trimethyl-4H-pyrrolo[3,4-b]quinoline-4,9-dione (100 mg, 188 μmol, 1.00 eq) was dissolved in DMF (2.00 mL), and then NaN3 (0.04 g, 615 μmol, 3.27 eq) was added. The mixture was stirred at 80 °C for 3 h. LCMS analysis showed that the starting material was completely consumed, and the desired mass was detected (Rt = 0.49 min). The reaction was quenched by adding 10.0 mL of water to the reaction mixture at 0 °C, and the mixture was extracted with 10.0 mL of EtOAc (5.00 mL × 2). After combining the organic layers, washing with 10.0 mL of brine, drying with Na2SO4, filtering, and vacuum-reducing the solution to obtain the residue. The residue was purified by prep-HPLC (TFA condition) to finally obtain 4-(2-azobutyl)-7-[(trimethylsilyl)octylchloro]-8-fluoro-1,3-dihydro-3,6,8-trimethyl-4H-pyrrolo[3,4-b]quinoline-4,9-dione (45.0 mg, 85.5 μmol, 45.4% yield, 99.2% purity) as a yellow solid.

[0412] LCMS analysis results: Rt = 0.53 min, m / z = 522.2 [M+H] + .

[0413] (8) Preparation of 4-(2-azobutyl)-7-[(trimethylsilyl)octylchloro]-8-fluoro-1,3-dihydro-3,6,8-trimethyl-4H-pyrrolo[3,4-b]quinoline-4,9-dione

[0414]

[0415] 4-(2-Azobutyl)-7-[(trimethylsilyl)octylchloro]-8-fluoro-1,3-dihydro-3,6,8-trimethyl-4H-pyrrolo[3,4-b]quinoline-4,9-dione (22.0 mg, 41.8 μmol, 1.00 eq) was dissolved in THF (1.00 mL) and H2O (0.30 mL), and then PPh3 (21.9 mg, 83.6 μmol, 2.00 eq) was added. The mixture was stirred at 20 °C for 12 h. Then, HCl (12.0 M, 330 μL, 94.6 eq) was added. The mixture was stirred at 20 °C for 12 h. LCMS analysis showed that the starting material was completely consumed, and the desired mass was detected (Rt = 0.34 min). After filtering the reaction mixture, it was purified by prep-HPLC (TFA conditions) to finally obtain 4-(2-aminobutyl)-7-[(trimethylsilyl)octylchloro]-8-fluoro-1,3-dihydro-3,6,8-trimethyl-4H-pyrrolo[3,4-b]quinoline-4,9-dione (5.00 mg, 8.20 μmol, 19.6% yield, 100% purity, TFA salt) as a yellow solid.

[0416] NMR: δ 8.06 (d, J = 8.0 Hz, 1H), 7.93 (d, J = 10.8 Hz, 1H), 7.54 (s, 3H), 7.34 (s, 1H), 6.53 (s, 1H), 5.43 (s, 2H), 5.36 (s, 2H), 2.72 (t, J = 6.8 Hz, 2H), 2.54 (s, 3H), 1.94 - 1.79 (m, 2H), 1.56 - 1.48 (m, 2H), 1.11 - 1.06 (m, 2H), 0.87 (t, J = 7.2 Hz, 3H), 0.65 (s, 6H).

[0417] LCMS analysis results: Rt = 0.34 min, m / z = 496.2 [M+H] + .

[0418] [Example 8] Manufacturing of payload AM20010

[0419] (1) Preparation of (S)-3-((3-(4-ethyl-4-hydroxy-6-iodo-3,8-dioxo-4,8-dihydro-1H-pyrano[3,4-c]pyridin-7(3H)-yl)prop-1-en-1-yl)dimethylsilyl)propyl acetate

[0420]

[0421] (S)-4-Ethyl-4-hydroxy-6-iodo-1,7-dihydro-3H-pyrano[3,4-c]pyridine-3,8(4H)-dione (15.50 g, 46.26 mmol) was dissolved in a mixture of DME (150 mL) and DMF (50 mL), and the mixture was cooled to 0°C under a nitrogen atmosphere. NaH (2.04 g, 50.88 mmol) was slowly added to the solution. The mixture was stirred at 0°C for 10 min, and then LiBr (8.03 g, 92.51 mmol) was added to the solution. The mixture was stirred at room temperature for 15 min.

[0422] A toluene (100 mL) solution of 3-((3-bromoprop-1-en-1-yl)dimethylsilyl)propyl acetate (25.65 g, 92.51 mmol) was added to the mixture. The reaction mixture was stirred at 70°C for 16 h. Detection of the target product was confirmed by LCMS.

[0423] The reaction mixture was quenched by adding water, and then extracted with ethyl acetate (EtOAc, 300 mL × 3). The organic layers were combined, washed with brine (300 mL × 3), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with EA / PE (0-50%) to give (S)-3-((3-(4-ethyl-4-hydroxy-6-iodo-3,8-dioxo-4,8-dihydro-1H-pyrano[3,4-c]pyridin-7(3H)-yl)prop-1-en-1-yl)dimethylsilyl)propyl acetate (16.5 g, 67.13% yield) as a red oil.

[0424] 1H NMR (400 MHz, DMSO-d6): δ 6.90 (s, 1H), 6.27 (s, 1H), 5.09 (q, J = 16.3 Hz, 2H), 4.91 (q, J = 17.7 Hz, 2H), 3.82 (t, J = 6.8 Hz, 2H), 1.86 (s, 3H), 1.62 (dt, J = 9.8, 6.8 Hz, 2H), 1.53 - 1.45 (m, 2H), 0.67 (t, J = 7.4 Hz, 3H), 0.50 - 0.43 (m, 2H), 0.00 (s, 6H).

[0425] LCMS analysis results: Rt = 11.138 min, m / z = 532.1 [M+1] +

[0426] (2) Preparation of 4-(2-acetoxybutyl)-7-[(trimethylsilyl)octylchloro]-8-fluoro-N-(4-fluorophenyl)-1,3-dihydro-3,6,8-trimethyl-4H-pyrrolo[3,4-b]quinoline-4,9-dione

[0427]

[0428] 4-(2-Acetoxybutyl)-7-[(trimethylsilyl)octylchloro]-8-fluoro-1,3-dihydro-3,6,8-trimethyl-4H-pyrrolo[3,4-b]quinoline-4,9-dione (2.00 g, 3.71 mmol, 1.00 eq), 4-fluorophenylisocyanate (853 mg, 6.31 mmol, 1.70 eq), 4 Å MS (47.0 μmol), hexamethyldistanis [(Me3Sn)2] (1.33 g, 4.06 mmol, 841 μL, 1.09 eq) were mixed in toluene (40.0 mL), degassed three times, and purged with nitrogen. The reaction mixture was irradiated with a UV lamp at 70 °C for 48 h under a nitrogen atmosphere. LCMS analysis revealed that the starting material (Rt = 0.44 min) remained, confirming the formation of the desired compound (Rt = 0.47 min). The reaction mixture was quenched by adding 200 mL of saturated aqueous KF solution at 0°C, and extracted with 200 mL of EtOAc (100 mL × 2). The organic layers were combined, washed with 200 mL of brine and 200 mL of water, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by silica gel column chromatography (SiO2, petroleum ether:ethyl acetate = 1:0 to 2:3, petroleum ether:ethyl acetate = 1:2, Rf = 0.37) to finally obtain 4-(2-acetoxybutyl)-7-[(trimethylsilyl)octylchloro]-8-fluoro-N-(4-fluorophenyl)-1,3-dihydro-3,6,8-trimethyl-4H-pyrrolo[3,4-b]quinoline-4,9-dione (160 mg, 288 μmol, 7.76% yield, 97.0% purity) as a yellow solid.

[0429] LCMS analysis results: Rt = 0.47 min, m / z = 539.3 [M+H] + .

[0430] (3) Preparation of 4-(2-hydroxybutyl)-7-[(trimethylsilyl)octylchloro]-8-fluoro-N-(4-fluorophenyl)-1,3-dihydro-3,6,8-trimethyl-4H-pyrrolo[3,4-b]quinoline-4,9-dione

[0431]

[0432] 4-(2-Acetoxybutyl)-7-[(trimethylsilyl)octylchloro]-8-fluoro-N-(4-fluorophenyl)-1,3-dihydro-3,6,8-trimethyl-4H-pyrrolo[3,4-b]quinoline-4,9-dione (10.0 mg, 16.5 μmol, 1.00 eq) was dissolved in HCl (6.00 M, 889 μL, 323 eq) and stirred at 100 °C for 1 h. LCMS analysis confirmed the complete consumption of the starting material and the formation of the desired compound (Rt = 0.43 min). The reaction mixture was filtered and purified by prep-HPLC (TFA conditions) to give 4-(2-hydroxybutyl)-7-[(trimethylsilyl)octylchloro]-8-fluoro-N-(4-fluorophenyl)-1,3-dihydro-3,6,8-trimethyl-4H-pyrrolo[3,4-b]quinoline-4,9-dione (6.00 mg, 11.5 μmol, 69.8% yield, 95.4% purity) as a yellow solid.

[0433] 1H NMR (400 MHz, DMSO-d6): δ 8.09 (d, J = 8.0 Hz, 1H), 7.91 (d, J = 10.8 Hz, 1H), 7.32 (s, 1H), 6.52 (s, 1H), 5.42 (s, 2H), 5.35 (s, 2H), 4.38 (t, J = 5.2 Hz, 1H), 3.30 - 3.29 (m, 2H), 2.52 (s, 3H), 1.92 - 1.80 (m, 2H), 1.47 - 1.39 (m, 2H), 1.06 - 1.02 (m, 2H), 0.86 (t, J = 7.2 Hz, 3H), 0.61 (s, 6H).

[0434] LCMS analysis results: Rt = 0.43 min, m / z = 497.3 [M+H] + .

[0435] [Example 9] Manufacturing of payload AM20011

[0436] (1) Preparation of (S)-3-((7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]diozolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)dimethylsilyl)propyl acetate

[0437]

[0438] (S)-3-((3-(4-Ethyl-4-hydroxy-6-iodo-3,8-dioxo-4,8-dihydro-1H-pyrano[3,4-c]pyridin-7(3H)-yl)prop-1-en-1-yl)dimethylsilyl)propyl acetate (5 g, 9.41 mmol) was dissolved in DMAc (50 mL), and then 5-isocyanobenzo[d][1,3]diozole (6.92 g, 47.04 mmol) and (Me3Sn)2 (4.62 g, 14.11 mmol) were added at room temperature. The reaction mixture was stirred at room temperature for 16 h under blue LED light (450–455 nm). The target product was detected by LCMS.

[0439] The reaction mixture was poured into a saturated aqueous solution of KF, and the resulting mixture was extracted with ethyl acetate (EtOAc, 100 mL × 3). The organic layers were combined, washed with brine (100 mL), and dried over Na2SO4. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with EtOAc / PE (0-90%), and S)-3-((7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]diozolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)dimethylsilyl)propyl acetate (1.5 g, 28.95% yield) was obtained as a yellow solid.

[0440] 1H NMR (400 MHz, DMSO): δ 7.53 (s, 1H), 7.46 (s, 1H), 7.25 (s, 1H), 6.49 (s, 1H), 6.30 (s, 2H), 5.41 (s, 2H), 5.31 (s, 2H), 3.90 (t, J = 6.8 Hz, 2H), 1.92 (s, 3H), 1.88 - 1.76 (m, 2H), 1.61 - 1.54 (m, 2H), 1.03 (dd, J = 10.3, 6.5 Hz, 2H), 0.86 (t, J = 7.0 Hz, 3H), 0.59 (s, 6H).

[0441] LCMS analysis results: Rt = 10.357 min, m / z = 551.4 [M+1] +

[0442] (2) Preparation of (S)-7-ethyl-7-hydroxy-14-((3-hydroxypropyl)dimethylsilyl)-10,13-dihydro-11H-[1,3]diozolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione

[0443]

[0444] (S)-3-((7-Ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]diozolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)dimethylsilyl)propyl acetate (1.5 g, 2.72 mmol) was added to 6 M aqueous hydrogen chloride (HCl) solution (15 mL) at room temperature. The reaction mixture was stirred at 100°C for 1 h. The target product was detected by LCMS.

[0445] The reaction mixture was concentrated under reduced pressure, and the residue was purified by reverse column chromatography, eluted with MeCN / H2O (5-50%). (S)-7-Ethyl-7-hydroxy-14-((3-hydroxypropyl)dimethylsilyl)-10,13-dihydro-11H-[1,3]diozolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione was obtained as a yellow solid (800 mg, 57.74% yield).

[0446] 1H NMR (400 MHz, DMSO-d6): δ 7.54 (s, 1H), 7.46 (s, 1H), 7.25 (s, 1H), 6.48 (s, 1H), 6.31 (s, 2H), 5.41 (s, 2H), 5.30 (s, 2H), 4.38 (s, 1H), 3.30 (s, 2H), 1.86 (tt, J = 14.1, 7.2 Hz, 2H), 1.48 - 1.34 (m, 2H), 1.01 (dd, J = 10.2, 6.6 Hz, 2H), 0.87 (t, J = 7.3 Hz, 3H), 0.59 (s, 6H).

[0447] LCMS analysis results: Rt = 1.515 min, m / z = 509.3 [M+1] +

[0448] [Example 10] Manufacturing of payload AM20013

[0449] (1) Preparation of N-assisted 2-aminoethylmethanesulfonyl chloride

[0450]

[0451] Sulfate chloride (425 mg, 3.15 mmol, 315 μL, 1.01 eq) and TEA (947 mg, 9.36 mmol, 1.30 mL, 3.00 eq) were dissolved in DCM (5.00 mL), and then tert-Butoxycarbonyl (assisted) ethylenediamine (500 mg, 3.12 mmol, 492 μL, 1.00 eq) was dissolved in DCM (5.00 mL) and added at -40 °C under N2 atmosphere. The reaction mixture was stirred at -40 °C for 0.5 h. The resulting tert-butoxycarbonylated 2-aminoethyl methanesulfonyl chloride (807 mg) was used in the next step without further purification as a white liquid suspension.

[0452] (2) Preparation of tert-butoxycarbonylated 2-aminoethyl methanesulfonylated (S)-7-ethyl-8-fluoro-4-hydroxy-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-3,14-dion-20-ylamine

[0453]

[0454] (S)-7-Ethyl-8-fluoro-4-hydroxy-1,3,12-trihydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-3,14-dion-20-ylamine (40.0 mg, 86.5 μmol, 1.00 eq) and 4 Å MS (100 mg) were mixed in DCM (0.50 mL), and then TEA (80.1 mg, 791 μmol, 110 μL, 9.15 eq) was added at 0°C under N2 atmosphere. After that, tert-butoxycarbonylated 2-aminoethyl methanesulfonyl chloride (223 mg, 865 μmol, 10.0 eq) was added at 0°C under N2 atmosphere. The mixture was stirred at 20°C for 0.5 h. LCMS analysis showed that the starting material was completely consumed, and the major peak with the desired m / z was detected (Rt = 0.46 min, MS calculated: 675.22, MS observed: [M+H] = 676.2). The pH of the reaction mixture was adjusted to 4–5 using acetic acid at 0°C, and then concentrated under reduced pressure to obtain a residue. The residue was dissolved in DMF (1.00 mL) and then filtered. The filtrate was purified by prep-HPLC (FA condition) to finally obtain tert-butoxycarbonylated 2-aminoethyl methanesulfonylated (S)-7-ethyl-8-fluoro-4-hydroxy-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-3,14-dion-20-ylamine (32.0 mg, 47.1 μmol, 54.4% yield, 99.5% purity) as a yellow solid.

[0455] LCMS analysis results: Rt = 0.46 min, m / z = 676.2 [M+H] + .

[0456] (3) Preparation of (S)-7-ethyl-8-fluoro-4-hydroxy-N-(2-(methanesulfonyl)acetyl)-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-3,14-dion-20-ylamine

[0457]

[0458] tert-Butoxycarbonylated 2-aminoethyl methanesulfonylated (S)-7-ethyl-8-fluoro-4-hydroxy-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-3,14-dion-20-ylamine (32.0 mg, 47.1 μmol, 1.00 eq) was dissolved in DCM (0.24 mL), and then TFA (0.03 mL) was added. The reaction mixture was stirred at 20 °C for 1 h. LCMS analysis showed that the starting material was completely consumed, and the major peak with the desired m / z was detected (Rt = 0.33 min, m / z = 576.2, [M+H] + ) The reaction mixture was concentrated under reduced pressure to obtain a residue. The residue was dissolved in H2O:ACN (4:1, 3.00 mL) and lyophilized to obtain (S)-7-ethyl-8-fluoro-4-hydroxy-N-(2-(methanesulfonyl)acetyl)-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-3,14-dion-20-ylamine TFA salt (27.0 mg, 38.7 μmol, 82.2% yield, 99.0% purity) as a yellow solid.

[0459] 1H NMR (400 MHz, DMSO-d6): δ 10.23 - 10.17 (m, 1H), 8.34 (d, J = 8.8 Hz, 1H), 8.03 (d, J = 11.2 Hz, 1H), 7.77 - 7.72 (m, 4H), 7.31 (s, 1H), 6.52 (s, 1H), 5.43 (s, 2H), 5.38 (s, 2H), 3.22 - 3.19 (m, 2H), 2.90 (br t, J = 6.8 Hz, 2H), 1.92 - 1.81 (m, 2H), 0.87 (t, J = 7.2 Hz, 3H), 0.59 (s, 9H).

[0460] LCMS analysis results: Rt = 0.33 min, m / z = 576.2 [M+H] + .

[0461] [Example 11] Manufacturing of linker payload AM21603

[0462] Preparation of (S)-7-ethyl-8-fluoro-4-hydroxy-N-(2-(methanesulfonyl)-N-(2-(2-(2-(4-(4-methoxypyridine-2-carbonyl)phenyl)ethoxy)ethoxy)acetyl)acetyl)-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-3,14-dion-20-ylamine

[0463]

[0464] (S)-7-Ethyl-8-fluoro-4-hydroxy-N-(2-(methanesulfonyl)acetyl)-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-3,14-dion-20-ylamine TFA salt (8.00 mg, 12.0 μmol, 1.00 eq., TFA salt) and 4 Å MS (50.0 mg) were dissolved in DCM (0.50 mL), and NMM (2.44 mg, 24.1 μmol, 2.65 μL, 2.00 eq.) was added at 0°C. Then, 2-(2-(2-(4-(4-methoxypyridine-2-carbonyl)phenyl)phenylethoxy)ethoxy)acetic acid N-hydroxysuccinimide ester (18.8 mg, 24.1 μmol, 2.00 eq.) was added, and the reaction mixture was stirred at 20°C for 2 h. LCMS analysis showed that the starting material was completely consumed and the desired compound was produced 46.3% (Rt = 1.87 min, m / z = 1050.5 [M+H] + ) The pH of the reaction mixture was adjusted to 4-5 with acetic acid at 0℃, and then concentrated under reduced pressure to obtain the residue. The residue was dissolved in DMF (1.00 mL), filtered, and purified by prep-HPLC (TFA conditions) to finally obtain (S)-7-ethyl-8-fluoro-4-hydroxy-N-(2-(methanesulfonyl)-N-(2-(2-(2-(4-(4-methoxypyridine-2-carbonyl)phenyl)ethoxy)ethoxy)acetyl)acetyl)-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-3,14-dion-20-ylamine TFA salt (3.00 mg, 2.84 μmol, 23.5% yield, 99.3% purity) as a yellow solid.

[0465] 1H NMR (400 MHz, DMSO-d6): δ 10.13 (s, 1H), 9.33 (d, J = 8.8 Hz, 1H), 8.25 (t, J = 6.0 Hz, 1H), 8.12 (d, J = 8.0 Hz, 1H), 8.07 (t, J = 5.6) Hz, 1H), 8.03-7.98 (m, 2H), 7.72 (t, J = 5.6 Hz, 1H), 7.31 (s, 1H), 7.25-7.22 (m, 4H), 7.19-7.15 (m, 1H), 6.97 (s, 2H), 6.51-6.50 (m, 1H), 5.42 (s, 2H), 5.36 (s, 2H), 4.50-4.45 (m, 1H), 3.77-3.57 (m, 6H), 3.36 (m, 2H), 3.16-3.11 (m, 2H), 3.05 (br dd, J = 4.4, 13.6 Hz, 1H), 2.82 (br dd, J = 9.2, 13.6 Hz, 1H), 2.56-2.54 (m, 2H), 2.10 (t, J = 7.6 Hz, 2H), 1.92-1.76 (m, 4H), 1.50-1.41 (m, 4H), 1.19-1.15 (m, 2H), 0.87 (t, J = 7.2 Hz, 3H), 0.60 (s, 9H).

[0466] LCMS analysis results: Rt = 1.86 min, m / z = 1050.5 [M+H] + .

[0467] [Example 12] Manufacturing of linker payload AM21604

[0468] Preparation of (S)-7-ethyl-8-fluoro-4-hydroxy-N-(2-(methanesulfonyl)-N-(2-(2-(2-(4-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzoyl)phenyl)oxy)ethoxy)acetyl)acetyl)-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-3,14-dion-20-ylamine

[0469]

[0470] (S)-7-Ethyl-8-fluoro-4-hydroxy-N-(2-(methanesulfonyl)acetyl)-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-3,14-dion-20-ylamine TFA salt (12.0 mg, 18.3 μmol, 1.00 eq, TFA salt) and 4 Å MS (50.0 mg) were dissolved in DCM (0.50 mL), and NMM (3.70 mg, 36.6 μmol, 4.02 μL, 2.00 eq) was added at 0 °C. Then, 2-(2-(2-(4-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzoyl)phenyl)oxy)ethoxy)acetic acid N-hydroxysuccinimide ester (22.9 mg, 36.6 μmol, 2.00 eq) was added, and the reaction mixture was stirred at 20 °C for 2 h. LCMS analysis revealed that the starting material was completely consumed and the desired compound was produced 42.0% (Rt = 1.84 min, m / z = 1050.41, MS observed: [M / 2+H] + = 526.4). The pH of the reaction mixture was adjusted to 4-5 with acetic acid at 0℃, and then concentrated under reduced pressure to obtain the residue. The residue was dissolved in DMF (1.00 mL), filtered, and purified by prep-HPLC (TFA conditions) to finally give (S)-7-ethyl-8-fluoro-4-hydroxy-N-(2-(methanesulfonyl)-N-(2-(2-(2-(4-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzoyl)phenyl)oxy)ethoxy)acetyl)acetyl)-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-3,14-dion-20-yl amine TFA salt (4.3 mg, 4.05 μmol, 41.7% yield, 98.9% purity) as a yellow solid.

[0471] 1H NMR (400 MHz, DMSO-d6): δ 9.31 (d, J = 8.8 Hz, 1H), 8.91 (br d, J = 2.8 Hz, 1H), 8.24 (br t, J = 5.6 Hz, 1H), 8.11 (br d, J = 8.0 Hz, 1H), 8.06 (br t, J = 5.6 Hz, 1H), 8.01 (br t, J = 5.6 Hz, 1H), 7.95 (d, J = 12.4 Hz, 1H), 7.86 (br t, J = 5.2 Hz, 1H), 7.27 (s, 1H), 7.25-7.23 (m, 4H), 7.19-7.15 (m, 1H), 6.97 (s, 2H), 6.49 (s, 1H), 5.41 (s, 2H), 5.34 (s, 2H), 4.53-4.47 (m, 1H), 3.77-3.60 (m, 6H), 3.36 (br s, 2H), 3.23-3.20 (m, 4H), 3.05 (br dd, J = 4.8, 14.0 Hz, 1H), 2.81 (br dd, J = 9.6, 14.0 Hz, 1H), 2.10 (br t, J = 7.2 Hz, 2H), 1.89-1.82 (m, 2H), 1.49-1.43 (m, 4H), 1.21-1.15 (m, 2H), 0.87 (t, J = 7.6 Hz, 3H), 0.59 (s, 9H).

[0472] LCMS analysis results: Rt = 1.84 min, m / z = 526.4 [M / 2+H] + .

[0473] [Example 13] Manufacturing of linker payload AM21605

[0474] Preparation of (S)-7-ethyl-8-fluoro-4-hydroxy-N-(2-(methanesulfonyl)-N-(2-(2-(2-(4-(4-(oxazole-5-carboxamido)benzoyl)phenyl)oxy)ethoxy)acetyl)acetyl)-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-3,14-dion-20-ylamine

[0475]

[0476] (S)-7-Ethyl-8-fluoro-4-hydroxy-N-(2-(methanesulfonyl)acetyl)-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-3,14-dion-20-ylamine TFA salt (12.0 mg, 19.1 μmol, 1.00 eq, TFA salt) and 4 Å MS (60.0 mg, 1.60 μmol) were dissolved in DCM (0.50 mL), and NMM (19.4 mg, 191 μmol, 21.0 μL, 10.0 eq) was added at 0 °C. Then, 2-(2-(2-(4-(4-(oxazole-5-carboxyamido)benzoyl)phenyl)oxy)ethoxy)acetic acid N-hydroxysuccinimide ester (24.0 mg, 38.3 μmol, 2.00 eq) was added, and the reaction mixture was stirred at 20 °C for 1.5 h. LCMS analysis revealed that the starting material was completely consumed and the desired compound was produced 27.1% (Rt = 0.42 min, m / z = 1022.38, MS observed: [M+H] + = 1023.5). The pH of the reaction mixture was adjusted to 4-5 with acetic acid at 0℃, and then concentrated under reduced pressure to obtain the residue. The residue was dissolved in DMF (1.00 mL), filtered, and purified by prep-HPLC (neutral condition) to finally give (S)-7-ethyl-8-fluoro-4-hydroxy-N-(2-(methanesulfonyl)-N-(2-(2-(2-(4-(4-(oxazole-5-carboxamido)benzoyl)phenyl)oxy)ethoxy)acetyl)acetyl)-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-3,14-dion-20-yl amine TFA salt (2.64 mg, 2.45 μmol, 12.7% yield, 94.9% purity) as a yellow solid.

[0477] LCMS analysis results: Rt = 0.42 min, m / z = 1023.5 [M+H]+ .

[0478] [Example 14] Manufacturing of linker payload AM21607

[0479] Preparation of (S)-7-ethyl-8-fluoro-4-hydroxy-N-(5-(4-(2-(2-(2-(4-(4-methoxypyridine-2-carbonyl)phenyl)oxy)ethoxy)ethoxy)acetamido)pentanamido)-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-3,14-dion-20-ylamine

[0480]

[0481] (S)-7-Ethyl-8-fluoro-4-hydroxy-N-(5-aminopentanamido)-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-3,14-dion-20-ylamine (15.0 mg, 19.8 μmol, 1.00 equiv) was dissolved in DCM (1.00 mL), and then NMM (4.02 mg, 39.7 μmol, 4.36 μL, 2.00 equiv) and 2-(2-(2-(4-(4-methoxypyridine-2-carbonyl)phenyl)oxy)ethoxy)acetic acid N-hydroxysuccinimide ester (26.5 mg, 39.7 μmol, 2.00 eq) was added. The reaction mixture was stirred at 25°C for 2 h. LCMS analysis showed that the starting material was consumed and the mass of the desired compound was confirmed (Rt = 1.89 min, m / z = 1079.42, MS observed: [M+H] += 1080.3). The pH of the reaction mixture was adjusted to 6 using acetic acid (17.5 M), filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by prep-HPLC (formic acid condition) to finally give (S)-7-ethyl-8-fluoro-4-hydroxy-N-(5-(4-(2-(2-(2-(4-(4-methoxypyridine-2-carbonyl)phenyl)oxy)ethoxy)ethoxy)acetamido)pentanamido)-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-3,14-dion-20-ylamine (2.50 mg, 2.20 μmol, 11.0% yield, 95.0% purity) as a white solid.

[0482] 1H NMR (400 MHz, DMSO-d6): δ 10.14 (s, 1H), 9.31 (br d, J = 9.6 Hz, 1H), 8.53-8.48 (m, 1H), 8.36-8.25 (m, 1H), 8.16-8.11 (m, 1H), 8.10-8.05 (m, 1H), 8.04-7.96 (m, 2H), 7.31 (s, 1H), 7.28-6.95 (m, 7H), 6.52 (s, 1H), 5.42 (s, 2H), 5.36 (s, 2H), 4.55 (br d, J = 6.8 Hz, 2H), 4.52-4.46 (m, 1H), 4.21-4.02 (m, 6H), 3.74-3.68 (m, 2H), 3.68-3.64 (m, 2H), 3.06-3.02 (m, 1H), 2.83-2.77 (m, 1H), 2.60-2.56 (m, 2H), 2.15-2.05 (m, 2H), 1.94-1.78 (m, 4H), 1.52-1.39 (m, 4H), 1.24-1.17 (m, 2H), 0.86 (br t, J = 7.6 Hz, 3H), 0.60 (s, 9H).

[0483] LCMS analysis results: Rt = 1.89 min, m / z = 1080.3 [M+H] + .

[0484] [Example 15] Manufacturing of linker payload AM21608

[0485] (1) Preparation of 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-yl (S)-(4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-11-(trimethylsilyl)-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate

[0486]

[0487] 2-Hydroxyethyl (S)-(4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-11-(trimethylsilyl)-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate (12.5 mg, 22.9 μmol, 1.00 eq) was dissolved in THF (0.20 mL), and then (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate (13.1 mg, 34.4 μmol, 1.50 eq) and 4 Å MS (100 mg) were added. After adding t-BuOK (7.72 mg, 68.8 μmol, 3.00 eq) at 0 °C, the reaction mixture was stirred at 20 °C for 30 min. LCMS analysis showed that 2-hydroxyethyl (S)-(4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-11-(trimethylsilyl)-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate (Rt = 0.40 min) remained at 21.4%, and 30.8% of the desired compound was formed (Rt = 0.42 min, m / z = 849.28, MS observed: [M+H] += 850.2). After combining the two batches, the pH of the reaction mixture was adjusted to 4-5 using acetic acid at 0℃, and concentrated under reduced pressure to obtain the residue. The residue was dissolved in DMF (1.00 mL), filtered, and purified by prep-HPLC (TFA conditions) to finally obtain 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-yl (S)-(4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-11-(trimethylsilyl)-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate (11 mg, 12.94 μmol, 28.2% yield, 100% purity) as a yellow solid.

[0488] LCMS analysis results: Rt = 0.42 min, m / z = 850.2 [M+H] +

[0489] (2) Preparation of 2-((2-aminoacetamido)methoxy)ethyl (S)-(4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-11-(trimethylsilyl)-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate

[0490]

[0491] 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-yl (S)-(4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-11-(trimethylsilyl)-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate (11.0 mg, 12.9 μmol, 1.00 eq) was dissolved in DMF (0.08 mL) and TEA (0.02 mL), and the reaction mixture was stirred at 20°C for 4 h. LCMS analysis showed that 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-yl (S)-(4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-11-(trimethylsilyl)-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate was completely consumed, and 51.1% of the desired compound was formed (Rt = 0.31 min, m / z = 627.22, MS observed: [M+H] + = 628.3). The pH of the reaction mixture was adjusted to 4-5 using acetic acid at 0℃, and then purified by prep-HPLC (TFA conditions) to finally obtain 2-((2-aminoacetamido)methoxy)ethyl (S)-(4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-11-(trimethylsilyl)-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate (6.00 mg, 8.02 μmol, 67.1% yield, 99.2% purity, TFA salt) as a yellow solid.

[0492] LCMS analysis results: Rt = 0.31 min, m / z = 628.3 [M+H] + .

[0493] (3) Preparation of (S)-10-benzyl-23-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-6,9,12,15,18-pentaoxo-3-oxa-5,8,11,14,17-pentaazatricosyl ((S)-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-11-(trimethylsilyl)-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate

[0494]

[0495] 2-((2-Aminoacetamido)methoxy)ethyl (S)-(4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-11-(trimethylsilyl)-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate (4.00 mg, 5.35 μmol, 1.00 eq, TFA salt) was dissolved in DCM (0.20 mL) and 4 Å MS (20.0 mg), and then NMM (1.08 mg, 10.7 μmol, 1.18 μL, 2.00 eq) was added at 0 °C. Subsequently, 2,5-dioxopyrrolidin-1-yl (6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)glycylglycyl-L-phenylalanineate (6.09 mg, 10.7 μmol, 2.00 eq) was added. The reaction mixture was stirred at 20°C for 1 h. LCMS analysis revealed that 3.94% of 2-((2-aminoacetamido)methoxy)ethyl (S)-(4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-11-(trimethylsilyl)-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate (Rt = 0.31 min) remained, and 42.2% of the desired compound was formed (Rt = 0.39 min, m / z = 1081.40, MS observed: [M+H] += 1082.4). The pH of the reaction mixture was adjusted to 4-5 using acetic acid at 0℃, and the residue was obtained by concentrating under reduced pressure. The residue was dissolved in DMF (1.00 mL), filtered, and purified by prep-HPLC (TFA conditions) to finally give (S)-10-benzyl-23-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-6,9,12,15,18-pentaoxo-3-oxa-5,8,11,14,17-pentaazatricosyl ((S)-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-11-(trimethylsilyl)-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbamate (2.00 mg, 1.77 μmol, 18.6% yield, 95.7% Purity) was obtained in the form of a yellow solid.

[0496] LCMS analysis results: Rt = 0.39 min, m / z = 1082.4 [M+H] + .

[0497] [Example 16] Manufacturing of linker payload AM21609

[0498] Preparation of (S)-7-ethyl-8-fluoro-4-hydroxy-N-(2-(2-(2-(4-(4-(4-methoxypyridine-2-carboxamido)benzoyl)phenyl)oxy)ethoxy)acetyl)-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-3,14-dion-20-ylamine

[0499]

[0500] (S)-7-Ethyl-8-fluoro-4-hydroxy-N-(5-aminopentanamido)-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-3,14-dion-20-ylamine TFA salt (15.0 mg, 24.6 μmol, 1.00 eq, TFA salt) and 4 Å molecular sieves were suspended in DCM (0.50 mL), and NMM (4.98 mg, 49.2 μmol, 5.41 μL, 2.00 eq) was added at 0°C. Then, 2-(2-(2-(4-(4-(4-methoxypyridine-2-carboxamido)benzoyl)phenyl)oxy)ethoxy)acetic acid N-hydroxysuccinimide ester (30.8 mg, 49.2 μmol, 2.00 eq) was added. The mixture was stirred at 20°C for 2 h. LCMS analysis showed that the starting material was completely consumed and the desired mass was confirmed (Rt = 0.45 min). The pH of the reaction mixture was adjusted to 4–5 with acetic acid at 0°C, and then concentrated under reduced pressure to obtain the residue. After purification of the residue by prep-HPLC (TFA condition), (S)-7-ethyl-8-fluoro-4-hydroxy-N-(2-(2-(2-(4-(4-(4-methoxypyridine-2-carboxamido)benzoyl)phenyl)oxy)ethoxy)acetyl)-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-3,14-dion-20-yl amine TFA salt (2.08 mg, 2.07 μmol, 8.39% yield, 100% purity) was finally obtained as a yellow solid.

[0501] 1H NMR (400 MHz, DMSO-d6): δ 8.17 (t, J = 5.2 Hz, 1H), 8.09-8.05 (m, 3H), 8.01 (t, J = 5.6 Hz, 1H), 7.91 (d, J = 10.4 Hz, 1H), 7.59 (t, J = 4.8 Hz, 1H), 7.33 (s, 1H), 7.24-7.15 (m, 5H), 7.01-6.94 (m, 2H), 6.55-6.50 (m, 1H), 5.42 (s, 2H), 5.36 (s, 2H), 4.46-4.41 (m, 1H), 3.73-3.58 (m, 6H), 3.55-3.53 (m, 2H), 3.03-2.98 (m, 3H), 2.80-2.74 (m, 1H), 2.54 (s, 3H), 2.09 (t, J = 7.2 Hz, 2H), 1.92-1.80 (m, 2H), 1.50-1.41 (m, 6H), 1.21-1.15 (m, 2H), 1.06-1.02 (m, 2H), 0.87 (t, J = 7.2 Hz, 3H), 0.62 (s, 6H).

[0502] LCMS analysis results: Rt = 0.44 min, m / z = 1007.6 [M+H] + .

[0503] [Example 17] Manufacturing of linker payload AM21610

[0504] Preparation of (S)-7-ethyl-8-fluoro-4-hydroxy-N-(5-(4-(2-(2-(2-(4-(4-methoxypyridine-2-carboxamido)benzoyl)phenyl)oxy)ethoxy)acetoxy)pentanamido)-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-3,14-dion-20-ylamine

[0505]

[0506] (S)-7-Ethyl-8-fluoro-4-hydroxy-N-(5-hydroxypentanamido)-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-3,14-dion-20-ylamine TFA salt (6.00 mg, 8.59 μmol, 1.00 eq, TFA salt) and 4 Å molecular sieves were suspended in DCM (0.20 mL), and NMM (1.74 mg, 17.1 μmol, 1.89 μL, 2.00 eq) was added at 0°C. Next, 2-(2-(2-(4-(4-methoxypyridine-2-carboxamido)benzoyl)phenyl)oxy)ethoxy)acetic acid N-hydroxysuccinimide ester (9.78 mg, 17.1 μmol, 2.00 eq) was added. The mixture was stirred at 20°C for 2 h. LCMS analysis showed complete consumption of the starting material and the desired mass was confirmed (Rt = 0.46 min). The pH of the reaction mixture was adjusted to 4–5 with acetic acid at 0°C, and then concentrated under reduced pressure to obtain the residue. The residue was purified by prep-HPLC (TFA conditions) and finally (S)-7-ethyl-8-fluoro-4-hydroxy-N-(5-(4-(2-(2-(2-(4-(4-methoxypyridine-2-carboxamido)benzoyl)phenyl)oxy)ethoxy)acetoxy)pentanamido)-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-3,14-dion-20-yl amine TFA salt (2.40 mg, 2.22 μmol, 25.8% yield, 95.8% purity) was obtained as a white solid.

[0507] LCMS analysis results: Rt = 0.45 min, m / z = 1037.6 [M+H] + .

[0508] [Example 18] Manufacturing of linker payload AM21611

[0509] (1) Preparation of 2,5-dioxopyrrolidin-1-yl (6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)glycylglycyl-L-phenylalanineate Preparation of (9H-fluoren-9-yl)methyl (S)-(2-(((3-((7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)dimethylsilyl)propoxy)methyl)amino)-2-oxoethyl)carbamate

[0510]

[0511] To a solution of (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate (342 mg, 886 μmol) in tetrahydrofuran (THF, 4.00 mL) were added (S)-7-ethyl-7-hydroxy-14-((3-hydroxypropyl)dimethylsilyl)-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (113 mg, 221 μmol) and TsOH·H2O (4.21 mg, 2.1 μmol) at 0°C. The mixture was stirred at 25°C for 17 h.

[0512] LCMS analysis revealed that 23.8% of (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate (Rt = 0.49 min) remained, and 56.8% of the target compound (Rt = 0.58 min, MS cal.: 816.28, MS observed: [M+H] = 817.4) was detected. The mixture was concentrated under reduced pressure to obtain a residue. The residue was dissolved in DMF (4.00 mL) and filtered. The filtrate was purified by prep-HPLC (FA condition) to obtain (9H-fluoren-9-yl)methyl (S)-(2-(((3-((7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)dimethylsilyl)propoxy)methyl)amino)-2-oxoethyl)carbamate (100 mg, 117 μmol, yield: 53.1%, purity: 96.3%) as an off-white solid.

[0513] LCMS analysis results: Rt = 0.58 min, m / z = 817.4 [M+H] +

[0514] (2) Preparation of (S)-2-amino-N-((3-((7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)dimethylsilyl)propoxy)methyl)acetamide

[0515]

[0516] (9H-Fluoren-9-yl)methyl (S)-(2-(((3-((7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)dimethylsilyl)propoxy)methyl)amino)-2-oxoethyl)carbamate (100 mg, 117 μmol, 1.00 eq.) was dissolved in dimethylformamide (DMF, 1.00 mL), and then morpholine (102 mg, 1.17 mmol, 103 μL, 10.0 eq.) was added. The mixture was stirred at 25°C for 3.5 h. LCMS analysis showed that (9H-fluoren-9-yl)methyl (S)-(2-(((3-((7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)dimethylsilyl)propoxy)methyl)amino)-2-oxoethyl)carbamate was completely consumed, and the target compound (Rt = 0.38 min, MS cal.: 594.21, MS observed: [M+H] + = 595.3) was detected at 47.4%. The pH of the mixture was adjusted to 5 with acetic acid at 0°C. The mixture was diluted with DMF (0.50 mL) and filtered. The solution was purified by preparative HPLC (TFA condition) to obtain (S)-2-amino-N-((3-((7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)dimethylsilyl)propoxy)methyl)acetamide (44.0 mg, 62.1 μmol, yield: 52.7%, purity: 100%, TFA salt) as a yellow solid.

[0517] LCMS analysis results: Rt = 0.38 min, m / z = 595.3 [M+H] +

[0518] (3) Preparation of N-((S)-13-benzyl-2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-2-methyl-9,12,15,18-tetraoxo-6-oxa-8,11,14,17-tetraaza-2-silanonedecan-19-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide

[0519]

[0520] To a solution of (S)-2-amino-N-((3-((7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)dimethylsilyl)propoxy)methyl)acetamide (26.0 mg, 36.7 μmol, 1.00 eq, TFA salt) and 4 Å MS (50.0 mg) in dichloromethane (DCM, 1.00 mL) was added NMM (7.43 mg, 73.4 μmol, 8.07 μL, 2.00 eq.) at 0°C. Then, 2,5-dioxopyrrolidin-1-yl (6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)glycylglycyl-L-phenylalanine (41.8 mg, 73.4 μmol, 2.00 eq.) was added. The mixture was stirred at 25 °C for 1 h. LCMS analysis showed that 2,5-dioxopyrrolidin-1-yl (6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)glycylglycyl-L-phenylalanine ate was completely consumed and 60.1% of the target compound was detected (Rt = 0.50 min, MS cal.: 1048.40, MS observed: [M+H] = 1049.5). The pH of the mixture was adjusted to 5 with acetic acid at 0°C, and the residue was concentrated under reduced pressure to obtain the residue. The residue was dissolved in DMF (1.00 mL) and then filtered. The filtrate was purified by prep-HPLC (TFA conditions) to obtain N-((S)-13-benzyl-2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-2-methyl-9,12,15,18-tetraoxo-6-oxa-8,11,14,17-tetraaza-2-silanonedecan-19-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide (15.24 mg, 12.63 mg). Afterwards, each was mixed with H2O:ACN (5:1, 2.00 mL) and freeze-dried to give N-((S)-13-benzyl-2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-2-methyl-9,12,15,18-tetraoxo-6-oxa-8,11,14,17-tetraaza-2-silanonedecan-19-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide (27.87 mg, 25.9 μmol, 23.4% yield, 97.6% purity) as yellow. A solid was obtained.

[0521] 1H NMR (400 MHz, DMSO-d6): δ 8.41 - 8.39 (m, 1H), 8.26 - 8.23 ​​(m, 1H), 8.10 - 8.04 (m, 2H), 8.00 (t, J = 5.6 Hz, 1H), 7.53 (s, 1H), 7.45 (s, 1H), 7.24 (s, 1H), 7.22 - 7.13 (m, 5H), 6.98 (s, 2H), 6.48 (s, 1H), 6.30 (s, 2H), 5.40 (s, 2H), 5.30 (s, 2H), 4.48 - 4.44 (m, 3H), 3.74 - 3.54 (m, 6H), 3.36 - 3.34 (m, 2H), 3.30 - 3.28 (m, 2H), 3.04 - 3.00 (m, 1H), 2.80 - 2.74 (m, 1H), 2.11 - 2.07 (m, 2H), 1.90 - 1.79 (m, 2H), 1.52 - 1.41 (m, 6H), 1.21-1.13 (m, 2H), 1.02 - 0.98 (m, 2H), 0.86 (t, J = 7.2 Hz, 3H), 0.58 (s, 6H).

[0522] LCMS analysis results: Rt = 0.50 min, m / z = 1049.5 [M+H] +

[0523] [Example 19] Preparation of linker payload AM21613

[0524] (1) Preparation of N-hydroxysuccinimide ester of tert-butoxycarbonyl-L-glutamic acid-N-[(phenylacetyl)glycylglycyl]

[0525]

[0526] To a solution of tert-butoxycarbonyl-L-glutamic acid-N-[(phenylacetyl)glycylglycyl] (100 mg, 229 μmol, 1.00 eq) and HOSu (31.6 mg, 274 μmol, 1.20 eq) in DCM (1.00 mL) was added DCC (52.0 mg, 252 μmol, 50.9 μL, 1.10 eq) dissolved in DCM (1.00 mL) at 0°C. The reaction mixture was stirred at 20°C for 4 h. LCMS analysis revealed that 28.8% of the starting material (Rt = 0.33 min) remained, and 53.8% of the desired compound was produced (Rt = 0.37 min, m / z = 533.21, MS observed: [M-Boc+H]+ = 434.1). The reaction mixture was filtered and concentrated under reduced pressure to obtain N-hydroxysuccinimide ester of tert-butoxycarbonyl-L-glutamic acid-N-[(phenylacetyl)glycylglycyl] (110 mg, crude material) as a yellow solid, which was used in the next step without purification.

[0527] LCMS analysis results: Rt = 0.37 min, m / z = 434.1 [M-Boc+H] + .

[0528] (2) Preparation of (S)-7-ethyl-8-fluoro-4-hydroxy-N-(5-(4-(2-(2-(2-(4-(phenylacetyl)glycylglycylamido)oxy)ethoxy)acetamido)pentanamido)-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-3,14-dion-20-ylamine

[0529]

[0530] To a solution of (S)-7-ethyl-8-fluoro-4-hydroxy-N-(5-aminopentanamido)-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-3,14-dion-20-ylamine TFA salt (18.0 mg, 25.8 μmol, 1.00 eq, TFA salt) and 4 Å molecular sieves (100 mg) in DMF (0.50 mL) was added NMM (26.1 mg, 258 μmol, 28.4 μL, 10.0 eq) at 0°C. Subsequently, N-hydroxysuccinimide ester of tert-butoxycarbonyl-L-glutamic acid-N-[(phenylacetyl)glycylglycyl] (41.3 mg, 77.5 μmol, 3.00 eq) was added. The reaction mixture was stirred at 20°C for 1 h. LCMS analysis showed that the starting material was completely consumed, and one major peak with the target mass was detected (Rt = 0.44 min, m / z = 993.35, MS observed: [M+H] + = 994.5). The pH of the reaction mixture was adjusted to 4–5 with acetic acid and then filtered. The residue was purified by prep-HPLC (FA condition) to finally obtain (S)-7-ethyl-8-fluoro-4-hydroxy-N-(5-(4-(2-(2-(2-(4-(phenylacetyl)glycylglycylamido)oxy)ethoxy)acetamido)pentanamido)-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-3,14-dion-20-ylamine TFA salt (13.0 mg, 12.7 μmol, 39.3% yield, 97.5% purity) as a yellow solid.

[0531] LCMS analysis results: Rt = 0.44 min, m / z = 994.5 [M+H] + .

[0532] (3) Preparation of (S)-7-ethyl-8-fluoro-4-hydroxy-N-(5-(4-(2-(2-(2-(4-(phenylacetyl)glycylglycylamido)oxy)ethoxy)acetamido)pentanamido)-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-3,14-dion-20-ylamine

[0533]

[0534] (S)-7-Ethyl-8-fluoro-4-hydroxy-N-(5-(4-(2-(2-(4-(phenylacetyl)glycylglycylamido)oxy)ethoxy)acetamido)pentanamido)-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-3,14-dion-20-ylamine tert-butoxycarbonyl protected form (10.0 mg, 9.81 μmol, 1.00 eq) was dissolved in DCM (0.16 mL), and then TFA (0.02 mL) was added. The reaction mixture was stirred at 20 °C for 1 h. LCMS analysis showed that the starting material was completely consumed and one major product with the target mass was obtained. A peak was detected (Rt = 0.35 min, m / z = 893.30, MS observed: [M+H] = 894.4). The reaction mixture was concentrated under reduced pressure to obtain the residue, which was obtained as (S)-7-ethyl-8-fluoro-4-hydroxy-N-(5-(4-(2-(2-(2-(4-(phenylacetyl)glycylglycylamido)oxy)ethoxy)acetamido)pentanamido)-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-3,14-dion-20-ylamine TFA salt (9.89 mg, crude material, TFA salt) as a yellow oil, which was used in the next step without purification.

[0535] LCMS analysis results: Rt = 0.35 min, m / z = 894.4 [M+H] + .

[0536] (4) Preparation of (S)-7-ethyl-8-fluoro-4-hydroxy-N-(5-(4-(2-(2-(4-(phenylacetyl)glycylglycylamido)oxy)ethoxy)acetamido)pentanamido)-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-3,14-dion-20-ylamine-N-(4-(N-succinylamino)butyl)formamide

[0537]

[0538] (S)-7-Ethyl-8-fluoro-4-hydroxy-N-(5-(4-(2-(2-(4-(phenylacetyl)glycylglycylamido)oxy)ethoxy)acetamido)pentanamido)-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-3,14-dion-20-yl amine TFA salt (9.89 mg, 9.81 μmol, 1.00 eq) and 4 Å molecular sieves (50.0 mg) were suspended in DMF (0.30 mL), and NMM (9.92 mg, 98.1 μmol, 10.7 μL, 10.0 eq) was added at 0°C. was added. Then, N-hydroxysuccinimide ester of N-(4-aminobutyl)succinimide (6.05 mg, 19.6 μmol, 2.00 eq) was added. The reaction mixture was stirred at 20°C for 2 h. LCMS analysis revealed that 3.12% of the starting material remained and 56.2% of the target compound was produced (Rt = 0.42 min, m / z = 1086.37, MS observed: [M+H] += 1087.5). The pH of the reaction mixture was adjusted to 4-5 using acetic acid at 0℃ and then filtered. The residue was purified by prep-HPLC (TFA condition) to finally give (S)-7-ethyl-8-fluoro-4-hydroxy-N-(5-(4-(2-(2-(2-(4-(phenylacetyl)glycylglycylamido)oxy)ethoxy)acetamido)pentanamido)-1,3,12,14-tetrahydro-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-3,14-dion-20-ylamine-N-(4-(N-succinylamino)butyl)formamide TFA salt (4.42 mg, 4.02 μmol, 28.0% yield, 99.0% purity) as a yellow solid.

[0539] 1H NMR (400 MHz, DMSO-d6): δ 10.13 (s, 1H), 8.33 (d, J = 8.8 Hz, 1H), 8.20 (t, J = 5.2 Hz, 1H), 8.09-8.05 (m, 2H), 8.01-7.98 (m, 2H), 7.75 (t, J = 5.6 Hz, 1H), 7.62 (t, J = 6.0 Hz, 1H), 7.31 (s, 1H), 7.25-7.19 (m, 4H), 7.17-7.12 (m, 1H), 6.97 (s, 2H), 6.51 (s, 1H), 5.42 (s, 2H), 5.36 (s, 2H), 4.48-4.43 (m, 1H), 3.74-3.57 (m, 6H), 3.46-3.44 (m, 2H), 3.18-3.13 (m, 2H), 3.02-2.99 (m, 3H), 2.77 (dd, J = 9.6, 13.6 Hz, 1H), 2.09 (t, J = 7.2 Hz, 2H), 1.91-1.80 (m, 2H), 1.50-1.41 (m, 4H), 1.20-1.15 (m, 2H), 0.86 (t, J = 6.8 Hz, 3H), 0.59 (s, 9H).

[0540] LCMS analysis results: Rt = 0.42 min, m / z = 1087.5 [M+H] + .

[0541] [Example 20] Manufacturing of linker payload AM21811

[0542] (1) (9H-fluoren-9-yl)methyl Preparation of ((S)-40-(((10S,13S)-2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-2,10,14-trimethyl-9,12-dioxo-6-oxa-8,11-diaza-2-silapentadecan-13-yl)carbamoyl)-38-oxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39-azateratetracontan-44-yl)carbamate

[0543]

[0544] To a solution of (S)-2-amino-N-((S)-1-(((3-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)dimethylsilyl)propoxy)methyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (120 mg, 0.170 mmol) in DMF (2 mL) N6-(((9H-fluoren-9-yl)methoxy)carbonyl)-N2-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctathriacontan-38-oyl)-L-lysine (239 mg, 0.254 mmol), DMTMMT (107 mg, 0.339 mmol), NMM (18 mg, 0.170 mmol) were added. The reaction mixture was stirred at room temperature for 1 h. LCMS analysis confirmed the desired product. The residue was purified by reverse phase column, eluted with MeCN: 0.1% TFA containing water (5-95%) to give (9H-fluoren-9-yl)methyl as a yellow solid. ((S)-40-(((10S,13S)-2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-2,10,14-trimethyl-9,12-dioxo-6-oxa-8,11-diaza-2-silapentadecan-13-yl)carbamoyl)-38-oxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39-azateratetracontan-44-yl)carbamate (130 mg, 47.08% yield) was obtained.

[0545] LCMS analysis results: Rt = 1.402 min, m / z = 815.5 [M / 2+1] +

[0546] (2) Preparation of N-((10S,13S,16S)-20-amino-2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-13-isopropyl-2,10-dimethyl-9,12,15-trioxo-6-oxa-8,11,14-triaza-2-silaicosan-16-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctathriacontan-38-amide

[0547]

[0548] (9H-fluoren-9-yl)methyl ((S)-40-(((10S,13S)-2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-2,10,14-trimethyl-9,12-dioxo-6-oxa-8,11-diaza-2-silapentadecan-13-yl)carbamoyl)-38-oxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39-azateratetracontan-44-yl)carbamate (130 mg, 0.080 mmol) After dissolving in DMF (2 mL), morpholine (35 mg, 0.399 mmol) was added at room temperature. The reaction mixture was stirred at room temperature for 1 h.

[0549] LCMS analysis confirmed the desired product. The reaction mixture was purified by reverse phase column and eluted with MeCN / H2O (5-95%) to obtain the product as a yellow solid. N-((10S,13S,16S)-20-amino-2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-13-isopropyl-2,10-dimethyl-9,12,15-trioxo-6-oxa-8,11,14-triaza-2-silaicosan-16-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctathriacontan-38-amide (110 mg, 97.98% yield) was obtained.

[0550] LCMS analysis results: Rt = 1.063 min, m / z = 704.3 [M / 2+1] +

[0551] (3) N-((10S,13S,16S)-24-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2 Preparation of -b]quinolin-14-yl)-13-isopropyl-2,10-dimethyl-9,12,15,22-tetraoxo-6-oxa-8,11,14,21-tetraaza-2-sila tetracosan-16-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctathriacontan-38-amide

[0552]

[0553] N-((10S,13S,16S)-20-amino-2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]diozolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-13-isopropyl-2,10-dimethyl-9,12,15-trioxo-6-oxa-8,11,14-triaza-2-silaicosan-16-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctathriacontan-38-amide (40 mg, 0.03 mmol) was dissolved in DMF (1 mL). After dissolution, 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate (15 mg, 0.057 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour.

[0554] LCMS analysis confirmed the desired product. The reaction mixture was concentrated under reduced pressure and purified by silica gel column chromatography (MeCN: 0.1% formic acid aqueous solution = 5-95%) to obtain N-((10S,13S,16S)-24-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]diozolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2 -b]quinolin-14-yl)-13-isopropyl-2,10-dimethyl-9,12,15,22-tetraoxo-6-oxa-8,11,14,21-tetraaza-2-sila tetracosan-16-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctathriacontan-38-amide (21.48 mg, 48.49% yield) was obtained as a yellow solid.

[0555] 1H NMR (400 MHz, DMSO) δ: 8.55-8.49 (m, 2H), 8.01 (d, J = 7.7 Hz, 2H), 7.88 (d, J = 5.7 Hz, 1H), 7.67 (d, J = 8.9 Hz, 1H), 7.53 (s, 1H), 7.44 (s, 1H), 7.25 (s, 1H), 6.99 (d, J = 2.9 Hz, 2H), 6.50 (s, 1H), 6.30 (s, 2H), 5.41 (s, 2H), 5.30 (s, 2H), 4.49 (dd, J = 10.2, 6.9 Hz, 1H), 4.40 (dd, J = 10.2, 6.4 Hz, 1H), 4.18 (ddd, J = 24.9, 15.9, 6.5 Hz, 3H), 3.58 (t, J = 7.3 Hz, 3H), 3.51-3.47 (m, 44H), 3.42 (dd, J = 5.8, 3.4 Hz, 3H), 3.29 (s, 1H), 3.23 (s, 3H), 2.94 (s, 2H), 2.34 (ddd, J = 28.8, 14.2, 7.1 Hz, 5H), 1.96-1.81 (m, 3H), 1.59 (s, 1H), 1.47 (s, 3H), 1.31 (d, J = 7.5 Hz, 2H), 1.16 (d, J = 7.1 Hz, 3H), 0.99 (dd, J = 10.3, 6.5 Hz, 2H), 0.87 (t, J = 7.3 Hz, 3H), 0.79 (dd, J = 10.9, 6.8 Hz, 6H), 0.58 (s, 6H).

[0556] LCMS analysis results: Rt = 1.186 min, m / z = 788.3 [M / 2+1] + .

[0557] [Example 21] Preparation of linker payload AM21911

[0558] (1) (9H-fluoren-9-yl)methyl Preparation of ((S)-40-(((S)-13-benzyl-2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]diozolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-2-methyl-9,12,15,18-tetraoxo-6-oxa-8,11,14,17-tetraaza-2-silanonedecan-19-yl)carbamoyl)-38-oxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39-azatetratrcontan-44-yl)carbamate

[0559]

[0560] (S)-2-(2-(2-aminoacetamido)acetamido)-N-(2-(((3-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]diozolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)dimethylsilyl)propoxy)methyl)amino)-2-oxoethyl)-3-phenylpropanamide (300 mg, 0.35 mmol) was dissolved in DMF (3 mL), N6-(((9H-fluoren-9-yl)methoxy)carbonyl)-N2-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctathriacontan-38-oyl)-L-lysine (493.70 mg, 0.53 mmol), DMTMM (220.13 mg, 0.70 mmol) and NMMT (17.73 mg, 0.18 mmol) were added. The mixture was stirred at room temperature for 1 h. LCMS analysis confirmed the desired product. The reaction mixture was purified by silica gel column chromatography (MeCN: 0.1% TFA in H2O = 5-95%) to finally obtain (9H-fluoren-9-yl)methyl ((S)-40-(((S)-13-benzyl-2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]diozolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-2-methyl-9,12,15,18-tetraoxo-6-oxa-8,11,14,17-tetraaza-2-silanonedecan-19-yl)carbamoyl)-38-oxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39-azatetratrcontan-44-yl)carbamate (300 mg, yield: 47.92%) was obtained in the form of a yellow solid.

[0561] LCMS analysis results: Rt = 1.352 min, m / z = 897.7 [(M+18) / 2+H] + .

[0562] (2) Preparation of N-((13S,22S)-26-amino-13-benzyl-2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]diozolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-2-methyl-9,12,15,18,21-pentaoxo-6-oxa-8,11,14,17,20-pentaaza-2-silahexacosane-22-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctathriacontan-38-amide

[0563]

[0564] (9H-fluoren-9-yl)methyl ((S)-40-(((S)-13-benzyl-2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]diozolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-2-methyl-9,12,15,18-tetraoxo-6-oxa-8,11,14,17-tetraaza-2-silanonedecan-19-yl)carbamoyl)-38-oxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39-azatetratrcontan-44-yl)carbamate (300 mg, 0.17 mmol) was dissolved in DMF (3 mL), and then morpholine (0.07 mL) was added. The reaction mixture was stirred at room temperature for 1 hour. LCMS analysis confirmed the desired product. The reaction mixture was purified by silica gel column chromatography under the condition of MeCN: 0.1% TFA in HO (5-95%), and finally N-((13S,22S)-26-amino-13-benzyl-2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]diozolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-2-methyl-9,12,15,18,21-pentaoxo-6-oxa-8,11,14,17,20-pentaaza-2-silahexacosan-22-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctathriacontan-38-amide (150 mg, yield: 48.77%) was obtained in the form of a yellow solid.

[0565] LCMS analysis results: Rt = 1.008 min, m / z = 778.3 [M / 2+H] + .

[0566] (3) N-((13S,22S)-13-benzyl-30-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[ Preparation of 1,2-b]quinolin-14-yl)-2-methyl-9,12,15,18,21,28-hexaoxo-6-oxa-8,11,14,17,20,27-hexaaza-2-silatriacontan-22-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctathriacontan-38-amide

[0567]

[0568] N-((13S,22S)-26-amino-13-benzyl-2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]diozolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-2-methyl-9,12,15,18,21-pentaoxo-6-oxa-8,11,14,17,20-pentaaza-2-silahexacosan-22-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctathriacontan-38-amide (40 mg, 0.03 mmol) After dissolving in DMF (1 mL), 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoic acid (13.05 mg, 0.08 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour. LCMS analysis confirmed the desired product. The reaction mixture was concentrated under reduced pressure, and then purified through silica gel column chromatography under the condition of MeCN:0.1% FA in H2O (5-95%), and finally N-((13S,22S)-13-Benzyl-30-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2 -b]quinolin-14-yl)-2-methyl-9,12,15,18,21,28-hexaoxo-6-oxa-8,11,14,17,20,27-hexaaza-2-silatriacontan-22-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctathriacontan-38-amide (12.01 mg, yield: 27.26%) was obtained as a yellow solid.

[0569] 1H NMR (400 MHz, DMSO) δ: 8.41 (t, J = 6.6 Hz, 1H), 8.26 (t, J = 5.6 Hz, 1H), 8.15-8.06 (m, 2H), 8.04-7.94 (m, 2H), 7.88 (t, J = 5.3 Hz, 1H), 7.53 (s, 1H), 7.45 (s, 1H), 7.27-7.19 (m, 5H), 7.18-7.13 (m, 1H), 6.99 (s, 1H), 6.48 (s, 1H), 6.30 (s, 1H), 5.41 (s, 1H), 5.30 (s, 1H), 4.52-4.44 (m, 3H), 4.19 (dd, J = 13.1, 8.1 Hz, 1H), 3.77-3.55 (m, 12H), 3.52-3.40 (m, 46H), 3.23 (s, 3H), 3.05-2.92 (m, 3H), 2.82-2.66 (m, 2H), 2.42-2.28 (m, 4H), 1.86 (tt, J = 14.1, 6.9 Hz, 2H), 1.67-1.58 (m, 1H), 1.54-1.44 (m, 3H), 1.38-1.16 (m, 5H), 1.01 (dd, J) = 10.2, 6.4 Hz, 2H), 0.87 (t, J = 7.3 Hz, 3H), 0.58 (s, 6H).

[0570] LCMS analysis results: Rt = 1.487 min, m / z = 1706.8 [M+H] + .

[0571] [Example 22] Manufacturing of linker payload AM22011

[0572] Preparation of N-((10S,13S,16S)-27-bromo-2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]diozolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-13-isopropyl-2,10-dimethyl-9,12,15,22,26-pentaoxo-6-oxa-8,11,14,21,25-pentaaza-2-silaheptacosan-16-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctathriacontan-38-amide

[0573]

[0574] N-((10S,13S,16S)-20-amino-2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]diozolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-13-isopropyl-2,10-dimethyl-9,12,15-trioxo-6-oxa-8,11,14-triaza-2-silaicosan-16-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctathriacontan-38-amide (50 mg, 0.036 mmol) was dissolved in DMF (1 mL), 2,5-dioxopyrrolidin-1-yl 3-(2-bromoacetamido)propanoate (22 mg, 0.071 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure and purified through silica gel column chromatography under the condition of MeCN:0.1% FA in H2O (5-95%), and finally N-((10S,13S,16S)-27-bromo-2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]diozolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-13-isopropyl-2,10-dimethyl-9,12,15,22,26-pentaoxo-6-oxa-8,11,14,21,25-pentaaza-2-silaheptacosan-16-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctathriacontan-38-amide (7.59 mg, yield: 13.36%) was obtained in the form of a yellow solid.

[0575] 1H NMR (400 MHz, DMSO-d6) δ: 8.51 (s, 1H), 8.28 (s, 1H), 7.99 (d, J = 7.9 Hz, 2H), 7.82 (s, 1H), 7.64 (d, J = 8.9 Hz, 1H), 7.54 (s, 1H), 7.44 (s, 1H), 7.25 (s, 1H), 6.48 (s, 1H), 6.31 (s, 2H), 5.41 (s, 2H), 5.31 (s, 2H), 4.48 (d, J = 6.6 Hz, 1H), 4.39 (d, J = 10.2 Hz, 1H), 4.29 - 4.09 (m, 3H), 3.82 (s, 1H), 3.58 (t, J = 6.9 Hz, 2H), 3.52 - 3.46 (m, 44H), 3.42 (dd, J = 5.7, 3.5 Hz, 2H), 3.24 (d, J = 8.0 Hz, 4H), 2.99 (s, 2H), 2.39 (d, J = 6.9 Hz, 2H), 2.23 (t, J = 7.0 Hz, 2H), 1.95 - 1.83 (m, 4H), 1.61 (s, 1H), 1.47 (s, 4H), 1.35 (s, 2H), 1.23 (s, 2H), 1.16 (d, J = 7.1 Hz, 3H), 1.01 - 0.96 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H), 0.79 (dd, J = 11.2, 6.8 Hz, 6H), 0.58 (s, 6H).

[0576] LCMS analysis results: Rt = 1.188 min, m / z = 1597.5 [M+H] + .

[0577] [Example 23] Manufacturing of linker payload AM22111

[0578] (1) Preparation of (9H-fluoren-9-yl)methyl ((S)-13-benzyl-2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]furano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-2-methyl-9,12,15,18-tetraoxo-6-oxa-8,11,14,17-tetraaza-2-silanonadecan-19-yl)carbamate

[0579]

[0580] (S)-7-ethyl-7-hydroxy-14-((3-hydroxypropyl)dimethylsilyl)-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]furano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (5.0 g, 9.83 mmol) was dissolved in DMF (40 mL), and then (S)-11-benzyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2-oxa-4,7,10,13,16-pentaazaheptadecan-17-yl acetate (5.57 g, 8.85 mmol) was added. The mixture was stirred at 5°C for 10 minutes, after which 4N hydrochloric acid (8 mL in ethyl acetate) was slowly added. The reaction mixture was stirred at room temperature for 16 hours. LCMS analysis confirmed the desired product. The reaction mixture was filtered and concentrated under reduced pressure at 0°C. The residue was purified by reverse phase column chromatography (MeCN / 0.1% FA / H2O = 5-70%) to obtain (9H-fluoren-9-yl)methyl ((S)-13-benzyl-2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]furano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-2-methyl-9,12,15,18-tetraoxo-6-oxa-8,11,14,17-tetraaza-2-silanonadecan-19-yl)carbamate (2.20 g, yield: 20.76%) as a pale yellow solid.

[0581] LCMS analysis results: Rt = 1.656 min, m / z = 1078.3 [M+H] + .

[0582] (2) Preparation of (S)-2-(2-(2-aminoacetamido)acetamido)-N-(2-(((3-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]furano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)dimethylsilyl)propoxy)methyl)amino)-2-oxoethyl)-3-phenylpropionamide

[0583]

[0584] To a solution of (9H-fluoren-9-yl)methyl ((S)-13-benzyl-2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]furano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-2-methyl-9,12,15,18-tetraoxo-6-oxa-8,11,14,17-tetraaza-2-silanonadecan-19-yl)carbamate (6.0 g, 5.56 mmol) in DMF (40 mL) was added morpholine (2.42 g, 27.82 mmol) under a nitrogen atmosphere. The reaction mixture was stirred at room temperature for 3 h. LCMS analysis confirmed the desired product. The reaction mixture was stopped by adding water (20 mL), and then filtered. The filtrate was purified by reverse phase column chromatography (MeCN / 0.1% FA / H2= 5-50%) to obtain (S)-2-(2-(2-aminoacetamido)acetamido)-N-(2-(((3-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]furano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)dimethylsilyl)propoxy)methyl)amino)-2-oxoethyl)-3-phenylpropionamide (3.1 g, yield: 65.08%) as a yellow solid.

[0585] LCMS analysis results: Rt = 7.91 min, m / z = 856.7 [M+H] + .

[0586] (3) N-((13S,22S)-13-benzyl-33-bromo-2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]furano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-2-methyl-9,12,15,18,21,28,32-heptaoxo-6-oxa-8,11,14,17,20,27,31-heptaaza-2-silatritriacontan-22-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctathriacontan-38-amide manufacturing

[0587]

[0588] N6-(3-(2-bromoacetamido)propanoyl)-N2-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctathriacontan-38-oyl)-L-lysine (1.0 g, 1.17 mmol, 10 g total, divided into 1 g portions) was dissolved in DMF (10 mL), and then HoBt (316 mg, 0.34 mmol) and DIC (295 mg, 2.34 mmol) were added. After the mixture was stirred at room temperature for 1 h, a DMF (10 mL) solution of (S)-2-(2-(2-aminoacetamido)acetamido)-N-(2-(((3-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]furano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)dimethylsilyl)propoxy)methyl)amino)-2-oxoethyl)-3-phenylpropionamide (1.06 g, 1.17 mmol) was added. The reaction mixture was stirred at room temperature for 1 h, and LCMS analysis confirmed the desired product. The reaction residue was purified by reverse phase column chromatography (MeCN:0.1% FA in H2O = 5-95%) to finally give N-((13S,22S)-13-benzyl-33-bromo-2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]furano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-2-methoxy-2 ...13S,22S)-13-benzyl-33-bromo-2-((13S,22S)-13-benzyl-33-bromo-2-((13S,22S)-13-benzyl-33-bromo-2-((13S,22S)-13- Til-9,12,15,18,21,28,32-heptaoxo-6-oxa-8,11,14,17,20,27,31-heptaaza-2-silatritriacontan-22-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctathriacontan-38-amide (6.583 g, yield: 30.45%) was obtained as a yellow solid.

[0589] 1H NMR (400 MHz, DMSO-d6): δ 8.42 (t, J = 6.5 Hz, 1H), 8.28 (t, J = 5.4 Hz, 2H), 8.17 - 8.06 (m, 2H), 8.03 (d, J = 7.6 Hz, 1H), 7.98 (t, J = 5.6 Hz, 1H), 7.83 (t, J = 5.3 Hz, 1H), 7.53 (s, 1H), 7.45 (s, 1H), 7.26 - 7.14 (m, 6H), 6.48 (s, 1H), 6.30 (s, 2H), 5.41 (s, 2H), 5.30 (s, 2H), 4.53 - 4.41 (m, 3H), 4.21 (d, J = 5.2 Hz, 1H), 3.86 - 3.56 (m, 11H), 3.51 - 3.41 (m, 44H), 3.30 - 3.23 (m, 6H), 3.02 (dd, J = 16.4, 5.5 Hz, 3H), 2.78 (dd, J = 13.7, 9.5 Hz, 1H), 2.37 (dt, J = 14.6, 7.4 Hz, 2H), 2.23 (t, J = 6.9 Hz, 2H), 1.96 - 1.78 (m, 2H), 1.63 (s, 1H), 1.54 - 1.42 (m, 3H), 1.35 (d, J = 7.2 Hz, 2H), 1.26 (s, 2H), 1.01 (s, 2H), 0.87 (t, J = 7.3 Hz, 3H), 0.59 (s, 6H).

[0590] LCMS analysis results: Rt = 1.186 min, m / z = 1747.2 [M+H] + .

[0591] [Example 24] Manufacturing of linker payload AM22211

[0592] (1) Preparation of (9H-fluoren-9-yl)methyl ((10S,13S)-2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]diosolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-2,10,14-trimethyl-9,12-dioxo-6-oxa-8,11-diaza-2-silapentadecan-13-yl)carbamate

[0593]

[0594] (S)-7-Ethyl-7-hydroxy-14-((3-hydroxypropyl)dimethylsilyl)-10,13-dihydro-11H-[1,3]diosolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (300 mg, 0.590 mmol) was dissolved in DMF (3 mL), and (5S,8S)-1-(9H-fluoren-9-yl)-5-isopropyl-8-methyl-3,6,9-trioxo-2-oxa-4,7,10-triazaundecan-11-yl acetate (284 mg, 0.590 mmol) was added at room temperature. Subsequently, HCl (4 M in EtOAc, 0.74 mL) was added. The reaction mixture was stirred at room temperature for 3 hours.

[0595] The reaction mixture was purified by reverse phase column chromatography (eluent: MeCN / H2O, 5–95%) to obtain (9H-fluoren-9-yl)methyl ((10S,13S)-2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]diosolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-2,10,14-trimethyl-9,12-dioxo-6-oxa-8,11-diaza-2-silapentadecan-13-yl)carbamate (200 mg, yield: 36.46%) as a yellow solid.

[0596] LCMS analysis results: Rt = 1.460 min, m / z = 930.4 [M+H] +

[0597] (2) Preparation of (S)-2-amino-N-((S)-1-(((3-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]diosolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)dimethylsilyl)propoxy)methyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide

[0598]

[0599] (9H-Fluoren-9-yl)methyl ((10S,13S)-2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]diosolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-2,10,14-trimethyl-9,12-dioxo-6-oxa-8,11-diaza-2-silapentadecan-13-yl)carbamate (180 mg, 0.194 mmol) was dissolved in DMF (3 mL), and then morpholine (85 mg, 0.968 mmol) was added at room temperature. The reaction mixture was stirred at room temperature for 1 h.

[0600] The reaction mixture was purified by reverse phase column chromatography (eluent: MeCN / H2O, 5-95%) to obtain (S)-2-amino-N-((S)-1-(((3-(((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]diosolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)dimethylsilyl)propoxy)methyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (MC24-1918-044, 130 mg, yield: 94.90%) as a yellow solid.

[0601] LCMS analysis results: Rt = 0.981 min, m / z = 708.4 [M+H] +

[0602] (3) Preparation of 6-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-N-((10S,13S)-2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]diosolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-2,10,14-trimethyl-9,12-dioxo-6-oxa-8,11-diaza-2-silapentadecan-13-yl)hexanamide

[0603]

[0604] N-((10S,13S,16S)-20-amino-2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]diosolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-13-isopropyl-2,10-dimethyl-9,12,15-trioxo-6-oxa-8,11,14-triaza-2-silaicosan-16-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctathriacontan-38-amide (23 mg, 0.032 mmol) was dissolved in DMF (1 After dissolving in 1 mL of 2,5-dioxopyrrolidin-1-yl 6-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)hexanoate (25 mg, 0.065 mmol) was added. The mixture was stirred at room temperature for 1 hour.

[0605] The residue of the reaction mixture was purified by silica gel column chromatography, using MeCN: 0.1% FA / H2O (5-95%) as the eluent. Finally, 6-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-N-((10S,13S)-2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]diosolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-2,10,14-trimethyl-9,12-dioxo-6-oxa-8,11-diaza-2-silapentadecan-13-yl)hexanamide (9.58 mg, yield: 30.33%) was obtained as a yellow solid.

[0606] 1H NMR (400 MHz, DMSO-d6): δ 8.50 (t, J = 6.8 Hz, 1H), 7.97 (d, J = 7.2 Hz, 1H), 7.87 (s, 1H), 7.75 (d, J = 8.7 Hz, 1H), 7.54 (s, 1H), 7.44 (s, 1H), 7.25 (s, 1H), 7.00 (s, 2H), 6.48 (s, 1H), 6.31 (s, 2H), 5.41 (s, 2H), 5.31 (s, 2H), 4.52-4.45 (m, 1H), 4.43-4.37 (m, 1H), 4.21-4.09 (m, 2H), 3.58 (t, J = 7.3 Hz, 2H), 3.28 (s, 2H), 2.95 (dd, J = 12.8, 6.7 Hz, 2H), 2.66 (d, J = 7.2 Hz, 1H), 2.32 (d, J = 5.5 Hz, 1H), 2.29 (d, J = 7.2 Hz, 1H), 2.12 (dd, J = 19.6, 7.6 Hz, 2H), 1.90-1.83 (m, 2H), 1.45 (d, J = 5.4 Hz, 4H), 1.36-1.29 (m, 2H), 1.17 (t, J = 8.8 Hz, 5H), 1.01-0.96 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H), 0.79 (dd, J = 10.4, 6.8 Hz, 6H), 0.58 (s, 6H).

[0607] LCMS analysis results: Rt = 1.505 min, m / z = 972.7 [M+H] +

[0608] [Example 25] Manufacturing of linker payload AM22311

[0609] (1) Preparation of 5-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)pentanoic acid

[0610]

[0611] 5-Aminopentanoic acid (1 g, 8.54 mmol) was dissolved in DMF (10 mL), and 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate (3.41 g, 12.80 mmol) was added. The mixture was stirred at room temperature for 16 h.

[0612] LCMS analysis confirmed the desired product. The residue of the reaction mixture was purified by silica gel column chromatography using MeCN: 0.1% TFA / H2O (5-95%) as the eluent. Finally, 5-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)pentanoic acid (1.1 g, yield: 48.03%) was obtained as a white solid.

[0613] LCMS analysis results: Rt = 0.634 min, m / z = 269.2 [M+H] +

[0614] (2) Preparation of 2,5-dioxopyrrolidin-1-yl 5-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)pentanoate

[0615]

[0616] 5-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)pentanoic acid (1.1 g, 4.10 mmol) was dissolved in DMF (10 mL), NHS (566.28 mg, 4.9 mmol) was added, and the mixture was stirred at room temperature for 10 minutes. Then, DIC (1.23 mL) was slowly added, and the mixture was further stirred at room temperature for 16 hours.

[0617] LCMS analysis confirmed the desired product. The residue of the reaction mixture was purified by silica gel column chromatography using MeCN: 0.1% FA / H2O (5-95%) as the eluent. Finally, 2,5-dioxopyrrolidin-1-yl 5-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)pentanoate (0.75 g, yield: 9.17%) was obtained as a white solid.

[0618] LCMS analysis results: Rt = 0.761 min, m / z = 366.2 [M+H] +

[0619] (3) Preparation of (S)-3-((7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)dimethylsilyl)propyl (4-nitrophenyl)carbonate

[0620]

[0621] (S)-7-Ethyl-7-hydroxy-14-((3-hydroxypropyl)dimethylsilyl)-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (500 mg, 0.98 mmol) was dissolved in DMF (5 mL), and 4-nitrophenyl carbonochlorideate (237.78 mg, 1.18 mmol) and triethylamine (0.27 mL) were added. The reaction mixture was stirred at room temperature for 2 h. LCMS analysis detected the desired product. After concentrating the reaction mixture, the residue was purified by silica gel column chromatography (MeCN: 0.1% FA in H2O = 5-95%) to give (S)-3-((7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)dimethylsilyl)propyl (4-nitrophenyl)carbonate (250 mg, 0.315 mmol, 32.1% yield) as a yellow solid.

[0622] LCMS analysis results: Rt = 1.387 min, m / z = 674.2 [M+1] + .

[0623] (4) Preparation of 2-chloro-N-(5-formyl-2-hydroxybenzyl)acetamide

[0624]

[0625] 4-Hydroxybenzaldehyde (60 g, 485.69 mmol) was dissolved in acetic acid (AcOH, 40 mL), and then 2-chloro-N-(hydroxymethyl)acetamide (88.97 g, 728.53 mmol) was added. The mixture was stirred at room temperature for 10 minutes, and then sulfuric acid (384 mL) was slowly added. The reaction mixture was stirred at room temperature for 16 hours. LCMS analysis confirmed the desired product. The reaction mixture was poured into ice water (1000 mL) and extracted with EA (1000 mL × 5). The organic layer was dried over Na2SO3 and concentrated under reduced pressure to obtain 2-chloro-N-(5-formyl-2-hydroxybenzyl)acetamide (110 g, 48.01% yield) as a gray solid (crude).

[0626] LCMS analysis results: Rt = 0.751 min, m / z = 228.2 [M+H] + .

[0627] (5) Preparation of tert-butyl (5-formyl-2-hydroxybenzyl)carbamate

[0628]

[0629] 2-Chloro-N-(5-formyl-2-hydroxybenzyl)acetamide (110 g, 483.21 mmol) was dissolved in 1,4-dioxane (480 mL) and hydrogen chloride (HCl, 480 mL), and the mixture was stirred at 100°C for 1 h. The reaction mixture was evaporated to obtain a residue. The residue was dissolved in 1,4-dioxane (300 mL) and water (300 mL). Tertiary ethylamine (Et3N, 108 mL) and Boc2O (120 g, 549.83 mmol) were added to the mixture at room temperature. The reaction mixture was stirred at room temperature for 16 h. LCMS analysis confirmed the desired product. The reaction mixture was extracted with ethyl acetate (EA, 1000 mL × 3) and washed with water (1000 mL). The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography, and eluted with EA / petroleum ether (0-30%) as an eluent to obtain tert-butyl (5-formyl-2-hydroxybenzyl)carbamate (89.6 g, 20.21% yield) as an oil.

[0630] LCMS analysis results: Rt = 1.065 min, m / z = 252.3 [M+1] +

[0631] (6) Preparation of (2S,3R,4S,5S,6S)-2-(2-(((tert-butylcarbonyl)amino)methyl)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate

[0632]

[0633] Tert-butyl (5-formyl-2-hydroxybenzyl)carbamate (89.6 g, 356.57 mmol) was dissolved in acetonitrile (ACN, 1580 mL), and (2R,3R,4S,5S,6S)-2-bromo-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (212.43 g, 534.86 mmol) and Ag2O (150.39 g, 648.96 mmol) were added at room temperature. The reaction mixture was stirred at room temperature for 16 h. The resulting solid was filtered and washed with acetonitrile (420 mL). Isopropanol (i-PrOH, 448 mL) and NaBH4 (13.49 mg, 356.57 mmol) were added to the combined acetonitrile solution. The reaction mixture was stirred at room temperature for 1 h. LCMS analysis confirmed the desired product. The reaction mixture was quenched with water (2000 mL) and extracted with ethyl acetate (EA, 2000 mL × 3). The organic layer was dried over Na2SO4, and the residue was purified by silica gel column chromatography. Using MeCN: 0.1% FA / H2O (5-95%) as eluent, (2S,3R,4S,5S,6S)-2-(2-(((tert-butylcarbonyl)amino)methyl)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (65 g, 15.86% yield) was finally obtained as a yellow oil.

[0634] LCMS analysis results: Rt = 1.202 min, m / z = 570.2 [M+1] +

[0635] (7) Preparation of (2S,3R,4S,5S,6S)-2-(2-(aminomethyl)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate

[0636]

[0637] (2S,3R,4S,5S,6S)-2-(2-(((tert-butoxycarbonyl)amino)methyl)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (30 g, 52.67 mmol) was dissolved in DCM (300 mL), and then TFA (30 mL) was added. The mixture was stirred at room temperature for 3 h. LCMS analysis confirmed the desired product. The reaction mixture was concentrated under reduced pressure and purified by silica gel column chromatography. Using MeCN: 0.1% FA / H2O (5-95%) as eluent, (2S,3R,4S,5S,6S)-2-(2-(aminomethyl)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (14 g, yield: 48.52%) was finally obtained as a white solid.

[0638] LCMS analysis results: Rt = 0.720 min, m / z = 470.2 [M+1] +

[0639] (8) Preparation of (2S,3R,4S,5S,6S)-2-(2-((3-((tert-butoxycarbonyl)amino)propanamido)methyl)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate

[0640]

[0641] (2S,3R,4S,5S,6S)-2-(2-(aminomethyl)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (14 g, 29.82 mmol) was dissolved in DMF (70 mL), and 2,5-dioxopyrrolidin-1-yl 3-((tert-butoxycarbonyl)amino)propanoate (25.61 g, 89.47 mmol) was added. The mixture was stirred at 50 °C for 16 h. LCMS analysis confirmed the desired product. The residue was purified by silica gel column chromatography using MeCN: 0.1% FA / H2O (5-95%) as the eluent. Finally, (2S,3R,4S,5S,6S)-2-(2-((3-((tert-butoxycarbonyl)amino)propanamido)methyl)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (9 g, yield: 42.12%) was obtained as a white solid.

[0642] LCMS analysis results: Rt = 1.452 min, m / z = 641.3 [M+1] +

[0643] (9) Preparation of (2S,3R,4S,5S,6S)-2-(2-((3-aminopropanamido)methyl)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate

[0644]

[0645] (2S,3R,4S,5S,6S)-2-(2-((3-((tert-butoxycarbonyl)amino)propanamido)methyl)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (9 g, 14.05 mmol) was dissolved in DCM (90 mL), and then TFA (9 mL) was added. The mixture was stirred at room temperature for 16 h. LCMS analysis confirmed the desired product. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography. Using MeCN: 0.1% FA / H2O (5-95%) as eluent, (2S,3R,4S,5S,6S)-2-(2-((3-aminopropanamido)methyl)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (7 g, yield: 56.96%) was finally obtained as a white solid.

[0646] LCMS analysis results: Rt = 0.775 min, m / z = 541.2 [M+1] +

[0647] (10) Preparation of (2S,3R,4S,5S,6S)-2-(2-(1-(9H-fluoren-9-yl)-3,6,10-trioxo-2-oxa-4,7,11-triazadodecan-12-yl)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate

[0648]

[0649] (2S,3R,4S,5S,6S)-2-(2-((3-aminopropanamido)methyl)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (7 g, 12.95 mmol) was dissolved in DMF (50 mL), and 2,5-dioxopyrrolidin-1-yl (((9H-fluoren-9-yl)methoxy)carbonyl)glycinate (10.21 g, 25.90 mmol) was added. The mixture was stirred at room temperature for 16 h. LCMS analysis confirmed the desired product. The reaction mixture was purified by silica gel column chromatography, and MeCN: 0.1% FA / H2O (5-95%) was used as the eluent to finally obtain (2S,3R,4S,5S,6S)-2-(2-(1-(9H-fluoren-9-yl)-3,6,10-trioxo-2-oxa-4,7,11-triazadodecan-12-yl)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (10 g, yield: 29.21%) as a brown solid.

[0650] LCMS analysis results: Rt = 1.262 min, m / z = 820.2 [M+1] +

[0651] (11) Preparation of (2S,3R,4S,5S,6S)-2-(2-(1-(9H-fluoren-9-yl)-3,6,10-trioxo-2-oxa-4,7,11-triazadodecan-12-yl)-4-((((4-nitrophenyloxy)carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate

[0652]

[0653] (2S,3R,4S,5S,6S)-2-(2-(1-(9H-fluoren-9-yl)-3,6,10-trioxo-2-oxa-4,7,11-triazadodecan-12-yl)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (10 g, 3.78 mmol) was dissolved in DMF (250 mL), and then bis(4-nitrophenyl)carbonate (2.30 g, 7.57 mmol) and DIEA (1.47 g, 11.35 mmol) were added. The mixture was stirred at room temperature for 16 h. LCMS analysis confirmed the desired product. The reaction mixture was purified by silica gel column chromatography, and MeCN: 0.1% FA / H2O (5-95%) was used as the eluent to finally obtain (2S,3R,4S,5S,6S)-2-(2-(1-(9H-fluoren-9-yl)-3,6,10-trioxo-2-oxa-4,7,11-triazadodecan-12-yl)-4-((((4-nitrophenyloxy)carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (2.70 g, yield: 57.26%) as a yellow oil.

[0654] LCMS analysis results: Rt = 1.423 min, m / z = 985.0 [M+1] +

[0655] (12) Preparation of (2S,3R,4S,5S,6S)-2-(2-(1-(9H-fluoren-9-yl)-3,6,10-trioxo-2-oxa-4,7,11-triazadodecan-12-yl)-4-(4,7,10,10-tetramethyl-3,8-dioxo-2,9-dioxa-4,7-diazaundecan-12-yl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate

[0656]

[0657] (2S,3R,4S,5S,6S)-2-(2-(1-(9H-fluoren-9-yl)-3,6,10-trioxo-2-oxa-4,7,11-triazadodecan-12-yl)-4-((((4-nitrophenyloxy)carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (2.7 g, 0.74 mmol) was dissolved in DMF (25 mL), and tert-butyl methyl(2-(methylamino)ethyl)carbamate (567.73 mg, 3.02 mmol) was added. The mixture was stirred at room temperature for 3 h. LCMS analysis confirmed the desired product. The reaction mixture was purified by silica gel column chromatography, and MeCN: 0.1% FA / H2O (5-95%) was used as the eluent to finally obtain (2S,3R,4S,5S,6S)-2-(2-(1-(9H-fluoren-9-yl)-3,6,10-trioxo-2-oxa-4,7,11-triazadodecan-12-yl)-4-(4,7,10,10-tetramethyl-3,8-dioxo-2,9-diosa-4,7-diazaundecan-12-yl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (1.8 g, yield: 48.21%) as a white solid.

[0658] LCMS analysis results: Rt = 1.404 min, m / z = 1034. [M+1] +

[0659] (13) Preparation of (2S,3R,4S,5S,6S)-2-(2-(1-(9H-fluoren-9-yl)-3,6,10-trioxo-2-oxa-4,7,11-triazadodecan-12-yl)-4-(((methyl(2-(methylamino)ethyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate

[0660]

[0661] (2S,3R,4S,5S,6S)-2-(2-(1-(9H-fluoren-9-yl)-3,6,10-trioxo-2-oxa-4,7,11-triazadodecan-12-yl)-4-(4,7,10,10-tetramethyl-3,8-dioxo-2,9-dioxa-4,7-diazaundecan-12-yl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (800 mg, 0.77 mmol) was dissolved in DCM (4 mL), and TFA (0.4 mL) was added. The mixture was stirred at room temperature for 4 h. LCMS analysis confirmed the desired product. The reaction mixture was purified by silica gel column chromatography, and MeCN: 0.1% FA / H2O (5-95%) was used as the eluent to finally obtain (2S,3R,4S,5S,6S)-2-(2-(1-(9H-fluoren-9-yl)-3,6,10-trioxo-2-oxa-4,7,11-triazadodecan-12-yl)-4-(((methyl(2-(methylamino)ethyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (420 mg, yield: 41.40%) as a yellow solid.

[0662] LCMS analysis results: Rt = 1.072 min, m / z = 934.2 [M+1] + .

[0663] (14) (2S,3R,4S,5S,6S)-2-(2-(1-(9H-fluoren-9-yl)-3,6,10-trioxo-2-oxa-4,7,11-triazadodecan-12-yl)-4-(13-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1, 3] Preparation of dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-4,7,13-trimethyl-3,8-dioxo-2,9-dioxa-4,7-diaza-13-sila tetradecyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate

[0664]

[0665] (2S,3R,4S,5S,6S)-2-(2-(1-(9H-fluoren-9-yl)-3,6,10-trioxo-2-oxa-4,7,11-triazadodecan-12-yl)-4-(((methyl(2-(methylamino)ethyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (320 mg, 0.34 mmol) was dissolved in DMF (3 mL), (S)-3-((7-Ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)dimethylsilyl)propyl(4-nitrophenyl)carbonate (276.99 mg, 0.41 mmol) was added. The mixture was stirred at room temperature for 2 h. LCMS analysis confirmed the desired product. The reaction mixture was purified by silica gel column chromatography, using MeCN: 0.1% FA / H2O (5-95%) as the eluent, and finally (2S,3R,4S,5S,6S)-2-(2-(1-(9H-fluoren-9-yl)-3,6,10-trioxo-2-oxa-4,7,11-triazadodecan-12-yl)-4-(13-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1, 3]Dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-4,7,13-trimethyl-3,8-dioxo-2,9-dioxa-4,7-diaza-13-sila tetradecyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (470 mg, yield: 66.41%) was obtained as a yellow solid.

[0666] LCMS analysis results: Rt = 11.561 min, m / z = 735.1 [M / 2+1] + .

[0667] (15) Preparation of (2S,3R,4S,5S,6S)-2-(2-((3-(2-aminoacetamido)propanamido)methyl)-4-(13-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-4,7,13-trimethyl-3,8-dioxo-2,9-dioxa-4,7-diaza-13-sila tetradecyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate

[0668]

[0669] (2S,3R,4S,5S,6S)-2-(2-(1-(9H-fluoren-9-yl)-3,6,10-trioxo-2-oxa-4,7,11-triazadodecan-12-yl)-4-(13-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1, 3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-4,7,13-trimethyl-3,8-dioxo-2,9-dioxa-4,7-diaza-13-sila tetradecyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (370 mg, 0.25 mmol) was dissolved in DMF (3 mL), and then morpholine (0.11 mL) was added. The mixture was stirred at room temperature for 2 h. LCMS analysis confirmed the desired product. The reaction mixture was purified by silica gel column chromatography, using MeCN: 0.1% FA / H2O (5-95%) as the eluent, and finally (2S,3R,4S,5S,6S)-2-(2-((3-(2-aminoacetamido)propanamido)methyl)-4-(13-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-4,7,13-trimethyl-3,8-dioxo-2,9-dioxa-4,7-diaza-13-sila tetradecyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (160 mg, yield: 31.03%) was obtained as a yellow solid.

[0670] LCMS analysis results: Rt = 8.207 min, m / z = 624.0 [M / 2+1] + .

[0671] (16) Preparation of (2S,3S,4S,5R,6S)-6-(2-((3-(2-aminoacetamido)propanamido)methyl)-4-(13-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-4,7,13-trimethyl-3,8-dioxo-2,9-dioxa-4,7-diaza-13-sila tetradecyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid

[0672]

[0673] (2S,3R,4S,5S,6S)-2-(2-((3-(2-aminoacetamido)propanamido)methyl)-4-(13-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-4,7,13-trimethyl-3,8-dioxo-2,9-dioxa-4,7-diaza-13-sila tetradecyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (140 mg, 0.02 mmol) was dissolved in ACN(7 After dissolving in a mixed solvent of 1 N HCl (0.51 mL) and HO (3.5 mL), NaOH (0.79 mL) was slowly added. The mixture was stirred at room temperature for 3 hours. LCMS analysis confirmed the desired product. The pH of the reaction mixture was adjusted to pH 7 using 1 N HCl (0.51 mL), and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, and finally, using MeCN: 0.1% FA / H2O (5-95%) as the eluent. (2S,3S,4S,5R,6S)-6-(2-((3-(2-aminoacetamido)propanamido)methyl)-4-(13-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-4,7,13-trimethyl-3,8-dioxo-2,9-diosa-4,7-diaza-13-sila tetradecyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (65 mg, yield: 37.68%) was obtained as a yellow solid.

[0674] LCMS analysis results: Rt = 7.189 min, m / z = 553.9 [M / 2+1] + .

[0675] (17) (2S,3S,4S,5R,6S)-6-(2-(18-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,7,10,16-tetraoxo-2,6,9,15-tetraazaoctadecyl)-4-(13-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-1 Preparation of 0H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-4,7,13-trimethyl-3,8-dioxo-2,9-dioxa-4,7-diaza-13-sila tetradecyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid

[0676]

[0677] (2S,3S,4S,5R,6S)-6-(2-((3-(2-aminoacetamido)propanamido)methyl)-4-(13-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-4,7,13-trimethyl-3,8-dioxo-2,9-dioxa-4,7-diaza-13-sila tetradecyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (45 mg, 0.04 mmol) was dissolved in DMF (1 mL), 2,5-Dioxopyrrolidin-1-yl 5-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)pentanoate (22.29 mg, 0.06 mmol) was added. The mixture was stirred at room temperature for 6 h. LCMS analysis confirmed the desired product. The reaction mixture was purified by silica gel column chromatography using MeCN: 0.1% FA / H2O (5-95%) as the eluent, and finally (2S,3S,4S,5R,6S)-6-(2-(18-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,7,10,16-tetraoxo-2,6,9,15-tetraazaoctadecyl)-4-(13-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H -[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-4,7,13-trimethyl-3,8-dioxo-2,9-dioxa-4,7-diaza-13-sila tetradecyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (19.97 mg, yield: 5.05%) was obtained as a yellow solid.

[0678] 1H NMR (400 MHz, DMSO-d6): δ 8.00 (t, J = 5.7 Hz, 1H), 7.94 - 7.82 (m, 2H), 7.52 (s, 1H), 7.46 (s, 1H), 7.25 (s, 1H), 7.20 - 6.96 (m, 5H), 6.48 (s, 1H), 6.28 (s, 2H), 5.50 (s, 1H), 5.41 (s, 2H), 5.31 (s, 2H), 5.20 (s, 1H), 4.83 (dd, J = 31.5, 16.0 Hz, 3H), 4.29 (s, 2H), 3.78 (d, J = 65.8 Hz, 3H), 3.66 - 3.49 (m, 5H), 3.27 - 3.24 (m, 4H), 2.96 (dd, J = 12.6, 6.7 Hz, 2H), 2.71 (d, J = 29.8 Hz, 7H), 2.31 (dd, J = 13.1, 5.8 Hz, 5H), 2.10 (t, J = 7.3 Hz, 2H), 2.04 - 1.75 (m, 3H), 1.54 (s, 2H), 1.43 (dd, J = 15.0, 7.4 Hz, 3H), 1.33 (dd, J = 14.8, 7.0 Hz, 2H), 1.24 (s, 2H), 1.06 (s, 2H), 0.86 (t, J = 7.3 Hz, 3H), 0.59 (s, 6H).

[0679] LCMS analysis results: Rt = 8.153 min, m / z = 679.0 [M / 2+1] + .

[0680] [Experimental Example]

[0681] [Experimental Example 1] Confirmation of the cell killing ability of six silatecan A ring-converted derivatives.

[0682] The purpose of this study was to evaluate the cytotoxic effects of six silatecan A ring-switched derivatives in HER2-expressing SK-BR-3, NCI-N87, and HER2-nonexpressing MCF-7 human cancer cell lines using a two-dimensional high-throughput screening method using a cell proliferation assay.

[0683] A 40 μL aliquot of cell culture medium containing individual cells in the medium was deposited into each well of a 384-well opaque plate. After culturing the cells for 18 h, six types of silatecan A ring-converted derivatives were diluted in cell culture medium to final concentrations of 500,000, 166,700, 55,600, 18,500, 6,200, 2,100, 685.9, 228.6, 76.2, 25.4, 8.5, 2.8, 0.9, and 0.3 pM, respectively, and dispensed into the well plates where the cells were cultured in 10 μL volumes. After culturing for 6 days in a 37°C carbon dioxide incubator, 10 μL of CellTiterGlo (Promega) was added to each well, and the signal was stabilized at room temperature for 10 min before being measured using a high-resolution luminescence spectrometer.

[0684] As a result, AM20008 showed a cell killing effect equivalent to that of Deruxtecan (Dxd), which was used as a control, in HER2-expressing SK-BR-3 and NCI-N87 cell lines (Fig. 1).

[0685] With respect to Figure 1, the following results were obtained:

[0686] Cell lineAM20003AM20004AM20005AM20007AM20008AM20013DXdMCF-7IC50(nM)NDNDNDNDNDNDNDAUC857.9846.1881.4870.7881.5822.1918.9SK-BR-3IC50(nM)231.4>30010. 1410.66.8726.135.73AUC832.3861.8712.0669.3646.8731.7680.9NCI-N87IC50(nM )243.5ND21.1618.1810.9941.9910.51AUC801.7837.2721.9668.9635.6695.8694.8

[0687] [Experimental Example 2] Confirmation of the cell killing ability of three silatecan B ring-converted derivatives.

[0688] The purpose of this study was to evaluate the cytotoxic effects of three silatecan B ring-switched derivatives in HER2-expressing SK-BR-3, NCI-N87, and HER2-nonexpressing MCF-7 human cancer cell lines using a two-dimensional high-throughput screening method using a cell proliferation assay.

[0689] A 40 μL aliquot of cell culture medium containing individual cells in the medium was deposited into each well of a 384-well opaque plate. After culturing the cells for 18 h, the three silatecan B ring-converted derivatives were diluted in the cell culture medium to final concentrations of 500,000, 166,700, 55,600, 18,500, 6,200, 2,100, 685.9, 228.6, 76.2, 25.4, 8.5, 2.8, 0.9, and 0.3 pM, respectively, and dispensed into the well plates where the cells were cultured in 10 μL volumes. After culturing for 6 days in a 37°C carbon dioxide incubator, 10 μL of CellTiterGlo (Promega) was added to each well, and the signal was stabilized at room temperature for 10 min before being measured using a high-resolution luminescence spectrometer.

[0690] As a result, AM20010 and AM20011 showed cell killing effects equivalent to those of Deruxtecan (Dxd), which was used as a control, in HER2-expressing SK-BR-3 and NCI-N87 cell lines (Fig. 2).

[0691] In relation to Fig. 2, the following results were obtained:

[0692] Cell lineAM20009AM20010AM20011DXdMCF-7IC50(nM)NDNDNDNDAUC899.3826.8810.2918.9SK-BR-3IC50(nM)38.7 72.672.665.73AUC739.1641.0651.8680.9NCI-N87IC50(nM)58.129.06.8410.51AUC702.1650.1639.5694.8

[0693] [Experimental Example 3] Comparison of cell killing abilities of AM20002, AM20008, and AM20011

[0694] The aim of this study was to compare the cytotoxic effects of silatecan A- and B-cyclic variants, AM20002, AM20008, and AM20011, in HER2-expressing SK-BR-3 and NCI-N87 human cancer cell lines using a two-dimensional high-throughput screening method using a cell proliferation assay.

[0695] A 40 μL aliquot of cell culture containing each cell in the medium was deposited into each well of a 384-well opaque plate. After culturing the cells for 18 h, the three derivatives and DXd were diluted in the cell culture medium to final concentrations of 500,000, 166,700, 55,600, 18,500, 6,200, 2,100, 685.9, 228.6, 76.2, 25.4, 8.5, 2.8, 0.9, and 0.3 pM, respectively, and dispensed into the well plates containing the cells in a volume of 10 μL. After culturing for 6 days in a 37°C carbon dioxide incubator, 10 μL of CellTiterGlo (Promega) was added to each well, and the signal was stabilized at room temperature for 10 min before being measured using a high-resolution luminescence spectrometer.

[0696] As a result, in HER2-expressing SK-BR-3 and NCI-N87 cell lines, three types of AM20002, AM20008, and AM20011 showed cell killing effects equivalent to or superior to those of Deruxtecan (Dxd), which was used as a control (Fig. 3).

[0697] In relation to Figure 3, the following results were obtained:

[0698] AM20002AM20008AM20011DXdSK-BR-3IC 50 3.183.441.33.1AUC674.8±11.7648±11.5644.5±9.1683.8±12.4NCI-N87IC 506.15.74.36.8AUC690.0±7.5607.7±8.5615.6±4.4701.9±7.8

[0699] [Experimental Example 4] Manufacturing and QC of ADC

[0700] The antibody in a buffer of 20 mM PB, 150 mM NaCl, pH 7.0 was treated with a 200 mM EDTA aqueous solution. 5 mM TCEP aqueous solution was then added to the solution. The solution prepared through the above process was allowed to react at 22°C until the next day. DMSO and linker-drug (to a final DMA ratio of 10%) were then slowly added. The binding solution was allowed to react at 22°C for 1 hour, and the reaction was stopped by adding a 10 mM NAC aqueous solution. The stopping solution was allowed to react at 22°C for 15 minutes, and then separated on a 10 mL zeba column (with a critical point of 40 kDa) and 15 mL Amicon. TM The antibody-drug conjugate was purified via (50 kDa). The final product was sterilized by passing it through a 0.22 μm PVDF disposable filter and analyzed for concentration, aggregates, MS-DAR, endotoxin, and free drug levels. The final product was stored long-term at -80°C.

[0701] In this example, an antibody-drug conjugate was produced for the anti-HER2 antibody Trastuzumab, and a physical property analysis was performed on the final product to determine that the antibody-drug ratio was approximately 7-8, the purity was 95%, the free linker-drug content was less than 1%, and the endogenous toxin content was less than 1 EU / mg (Table 4, Figures 4a to 4i).

[0702] ADCLinker-PayloadMS-DARMonomer(%)Free drug(mol / mol%)Endotoxin(EU / mg)Trastuzumab-AM21603AM216037.9498.460.64<0.123Trastuzumab-AM21604AM216047.9497. 310.37<0.112Trastuzumab-AM21605AM216058.0695.470.94<0.119Trastuzumab-AM21607AM216077.9497.882.32<0.136Trastuz umab-AM21608AM216087.8894.6<0.14<0.132Trastuzumab-AM21609AM216097.9295.850.24<0.146Trastuzumab-AM21610AM21610 7.9598.000.22<0.137Trastuzumab-AM21611AM216117.9497.410.52<0.127Trastuzumab-AM21613AM216137.9798.050.34<0.139

[0703] Meanwhile, an antibody-drug conjugate was produced for the anti-HER2 antibody Trastuzumab using an extended linker structure. Physical property analysis confirmed that the final product had an antibody-to-drug ratio of approximately 7-8 and a purity of approximately 95%. This verified the feasibility of ADC conjugation using the extended linker design (Table 5).

[0704] ADCLinker-PayloadRP-DARMonomer(%)Trastuzumab-AM21911AM219117.5995.63Trastuzumab-AM2211 1AM221117.1197.66Trastuzumab-AM22211AM222117.2194.98Trastuzumab-AM22311AM223117.9197.66

[0705] [Experimental Example 5] Evaluation of the Cell Killing Effect of Silatecan A-, B-Cyclotransfer Derivative Conjugated ADC

[0706] The cytotoxic effects of Trastuzumab-AM21608, -AM21609, -AM21611, -AM21613 ADCs, which were formed by conjugating silatecan A-, B- ring modifiers to Trastuzumab antibody with MC-GGFG linkers, were evaluated in HER2-expressing SK-BR-3, SK-OV-3 human cancer cell lines using 3D high-throughput or 3D high-content screening using cell proliferation assays.

[0707] 30 μL aliquots of cell culture containing individual cells in the medium were spherically suspended in each well of a low-attachment 384-well opaque or transparent plate with a U-shaped bottom. After culturing the cells for 18 h, the four types of silatecan A- or B-cycle conversion derivative-conjugated ADCs were diluted in the cell culture medium to final concentrations of 500,000, 166,700, 55,600, 18,500, 6,200, 2,100, 685.9, 228.6, 76.2, 25.4, 8.5, 2.8, 0.9, and 0.3 pM, respectively, and dispensed into the well plates in which the cells were cultured in a volume of 10 μL. After culturing for 6 days in a 37°C carbon dioxide incubator, 10 μL of CellTiterGlo (Promega) or a final concentration of 2 μM Calcein solution was added to each well, reacted for 30 minutes, and then measured using a high-resolution luminescence analyzer or a 3D high-content analysis device.

[0708] As a result, Trastuzumab-AM21611, Trastuzumab-AM21608, Trastuzumab-AM21609, and Trastuzumab-AM21613 showed excellent cell killing effects in HER2-expressing SK-BR-3 and SK-OV-3 cell lines (Fig. 5).

[0709] With respect to Figure 5, the following results were obtained:

[0710] Cell line(HER2 exp.)Trastuzumab-AM21609Trastuzumab-AM21613Trastuzumab-AM21608Trastuzumab-AM21611Trastuzumab-DeruxtecanSK-BR-3IC50 (nM)0.150.210.210.080.0795% CI0.12-0.180.12-3.130.17-0.250.07-0.090.06-0.09SK-OV-3IC50 (nM)2.320.501.681.010.8895% CI1.37-3.920.39-0.631.22-2.320.74-1.380.61-1.28

[0711] [Experimental Example 6] Evaluation of the cytotoxic effect of silatecan A-, B- ring conversion derivative conjugated ADC

[0712] The cytotoxic effects of Trastuzumab-AM21608, -AM21609, -AM21611, -AM21613 ADCs, which were formed by conjugating silatecan A-, B-cyclic modifiers to the MC-GGFG linker, were evaluated in the HER2-nonexpressing MCF-7 human breast cancer cell line using a three-dimensional high-throughput screening method using a cell proliferation assay.

[0713] 30 μL aliquots of cell culture containing individual cells in the medium were suspended in each well of a low-attachment 384-well opaque plate with a U-shaped bottom. After culturing the cells for 18 h, the four types of silatecan A- or B-ring-conjugated ADCs were diluted in the cell culture medium to final concentrations of 500,000, 166,700, 55,600, 18,500, 6,200, 2,100, 685.9, 228.6, 76.2, 25.4, 8.5, 2.8, 0.9, and 0.3 pM, respectively, and dispensed into the well plates in which the cells were cultured in a volume of 10 μL. After culturing for 6 days in a 37°C carbon dioxide incubator, 10 μL of CellTiterGlo (Promega) was added to each well to stabilize the signal for 30 minutes, and then measured using a high-resolution luminescence analyzer.

[0714] With respect to Fig. 6, the following results were obtained:

[0715] Cell line(HER2 exp.)Trastuzumab-AM21609Trastuzumab-AM21608Trastuzumab-AM21611Trastuzumab-DeruxtecanMCF-7IC50 (nM)NDNDNDND(-)AUC616.4646.8628.6610.8

[0716] In addition, the cytotoxic effect of Trastuzumab-AM21611, -AM21911, -AM22111, -AM22211, -AM22311 ADCs formed by conjugating Trastuzumab antibody to various linker conjugates using AM20011 as a payload was evaluated in the SK-OV-3 human cancer cell line expressing HER2 using 3D high-throughput or 3D high-content screening as a cell proliferation assay experiment. 30 μL aliquots of cell culture containing each cell in medium were suspended in each well of a low-attachment 384-well opaque plate with a U-shaped bottom. After culturing the cells for 18 hours, ADCs and Enhertu grafted with various linker designs using AM20011 payload were diluted in cell culture medium to final concentrations of 500,000, 166,700, 55,600, 18,500, 6,200, 2,100, 685.9, 228.6, 76.2, 25.4, 8.5, 2.8, 0.9, and 0.3 pM, respectively, and dispensed into well plates where cells were cultured in 10 μL volumes. After culturing for 6 days in a 37°C carbon dioxide incubator, 10 μL of CellTiterGlo (Promega) was added to each well to stabilize the signal for 30 minutes and then measured using a high-resolution luminescence spectrometer.

[0717] As a result, cytotoxic effects were confirmed in ADCs with various linker designs, including Trastuzumab-AM21611, -AM21911, AM22111, -AM22211, and AM22311, in the HER2-expressing SKOV3 cell line (Fig. 7).

[0718] With respect to Fig. 7, the following results were obtained:

[0719] Trastuzumab-AM21611Trastuzumab-AM21911Trastuzumab-AM22111Trastuzumab-AM22211Trastuzumab-AM22311Trastuzumab-DeruxtecanIC 50(nM)0.260.380.460.3322.650.20AUC336.2352.2331.3330.2509.6345.1

[0720] [Experimental Example 7] Evaluation of HER2 antigen-specific binding ability of silatecan derivative-conjugated ADC

[0721] The human HER2 antigen binding affinity of nine ADCs conjugated to Trastuzumab, nine silatecan A- and B-cyclic conversion derivatives, and the control substance, the single antibody Trastuzumab, was evaluated using ELISA.

[0722] As a result of confirming the specific binding affinity of nine ADCs of A- and B-ring-converted derivatives based on silatecan to the HER2 antigen protein, all nine types were confirmed to have binding affinity similar to that of the single antibody Trastuzumab (Fig. 8).

[0723] [Experimental Example 8] Evaluation of the specific binding ability of silatecan derivative-conjugated ADCs to HER2-overexpressing cells.

[0724] The HER2-specific binding ability of four ADCs, Trastuzumab-AM21608, -AM21609, -AM21611, and AM21613, which showed excellent efficacy through in vitro cytotoxicity evaluation, was confirmed in the SK-BR-3 cell line with high HER2 expression.

[0725] All antibodies and ADCs were bound to the HER2-overexpressing human cell line SK-BR-3 at 4°C for 1 hour, and then Alexa488 fluorescence-conjugated anti-human IgG1 antibody was conjugated to the antibodies and ADCs bound to the cells.

[0726] As a result, Fig. 9 shows that when compared with Trastuzumab, a single antibody not conjugated with a silatecan-modified derivative, or Trastuzumab-Deruxtecan, a control antibody conjugate, Trastuzumab-AM21608, -AM21609, -AM21611, and -AM21613 all showed cell surface HER2 binding abilities similar to Trastuzumab or Trastuzumab-Deruxtecan.

[0727] [Experimental Example 9] Evaluation of Internalization of Antibody-Drug Conjugates

[0728] Among the trastuzumab-binding ADCs, the cellular internalization of Trastuzumab-AM21611, which showed excellent efficacy in in vitro 3D high-throughput screening and 3D high-content screening cell proliferation assays, as well as the control substance Trastuzumab-Deruxtecan and the anti-HER2 antibody Trastuzumab was evaluated.

[0729] All antibodies and ADCs were bound to the HER2-overexpressing human cell line SK-BR-3 for 30 min at 4°C, and then Alexa488-conjugated anti-human IgG1 antibodies were conjugated to the cells-bound antibodies and ADCs. The reduction in cell surface antibody-drug conjugates was calculated by dividing the average FITC fluorescence intensity immediately after cell binding by the average fluorescence intensity after acclimation at 37°C for 0, 4, 7, and 24 h, and multiplying by 100.

[0730] Figure 10 is a graph showing the results of an analysis of the internalization rate, calculated by subtracting the surface antibody-drug conjugate reduction amount for each time point from 100 (%). According to the analysis, both trastuzumab and the control substances, trastuzumab-deruxtecan and trastuzumab-AM21611, showed comparable internalization rates at the same time points (Figure 9).

[0731] [Experimental Example 10] Evaluation of Extracellular ATP Release for Inducing Immunogenic Cell Death by Trastuzumab-AM21611

[0732] In addition, to measure the amount of extracellular release for inducing immunogenic cell death of silatecan derivative ADC, ATP protein released extracellularly was measured in SK-BR-3 cell line to confirm the immunogenic cell death induction effect of Trastuzumab-AM21611 ADC.

[0733] A 108 μL aliquot of cell culture containing approximately 3500 SK-BR-3 cells in media was deposited into each well of a 96-well opaque plate. After culturing the cells for 1 day, they were treated with Trastuzumab-AM21611 or the control substance Trastuzumab-Deruxtecan at a final concentration of 500 μM and MMAE 10 μM. MMAE served as a positive control, which is well known to induce immunogenic cell death. After 72 h of incubation in a 37°C carbon dioxide incubator, 30 μL of cell culture from each well was transferred to a new 96-well opaque plate. An equal volume of CellTiterGlo (Promega) was added, and the signal was stabilized for 3 minutes before being measured using a high-resolution luminescence spectrometer.

[0734] Figure 11 shows that AM20011, a modified derivative of silatecan, and Trastuzumab-AM21611, an ADC, released extracellular ATP equivalent to or greater than that of their respective control substances, Dxd or Trastuzumab-Deruxtecan.

[0735] [Experimental Example 11] Evaluation of the Cell Membrane Potential of HSP70 for Inducing Immunogenic Apoptosis by Trastuzumab-AM21611

[0736] To confirm the immunogenic cell death-inducing effect of Trastuzumab-AM21611 ADC, HSP70 protein translocated to the cell surface was measured.

[0737] A 108 μL aliquot of cell culture containing approximately 3500 SK-BR-3 cells in medium was deposited into each well of a 96-well opaque plate. After culturing the cells for 1 day, they were treated with Trastuzumab-AM21611 or the control substance Trastuzumab-Deruxtecan at a final concentration of 500 μM and MMAE at 10 μM. MMAE was used as a positive control, which is well known to induce immunogenic cell death. After culturing for 24 or 48 h in a carbon dioxide incubator at 37°C, the amount of HMGB1 released into the cell culture was measured using an HMGB1 ELISA (Promega).

[0738] Figure 12 shows that Trastuzumab-AM21611, conjugated with AM20011, a modified silatecan derivative, translocated more than twice as much HSP70 protein to the cell surface as the control substance, Trastuzumab-Deruxtecan.

[0739] [Experimental Example 12] Evaluation of the efficacy of antibody-drug conjugates for tumor growth inhibition in xenograft mice with HER2 high-expressing cells (Figure 13)

[0740] To evaluate the efficacy of AMB-CPT1(AM20001) / AMB-CPT1D(AM20002) conjugated ADCs, in vitro and in vivo evaluations were performed. In the in vitro experiment, the cytotoxic effects of various ADCs conjugated to Trastuzumab with AM20001, namely Trastuzumab-mc-PEG4-AA(ala-ala)-AM20001, Trastuzumab-mc-PEG4-AAA(ala-ala-ala)-AM20001, Trastuzumab-mc-AA-PEG4-PABC-AM20001, Trastuzumab-mal-GGFG-AM20001, and Trastuzumab-mal-GGFG-AM20002, were compared and evaluated using the NCI-N87 human cancer cell line with high HER2 expression.

[0741]

[0742]

[0743] As a result of the in vitro evaluation, as shown in Figure 13 and Table 9 below, Trastuzumab-based ADCs conjugated with AM20001 and AM20002 as payloads exhibited relatively lower cytotoxicity compared to Trastuzumab-Deruxtecan.

[0744] Trastuzumab-mc-PEG4-AA-AM20001Trastuzumab-mc-PEG4-AAA-AM20001Trastuzumab-mc-AA-PEG4-PABC-A M20001Trastuzumab-GGFG-AM20001(AM21601)Trastuzumab-mal-GGFG-AM20002(AM21602)T-DXdNCI-N87IC 50 (nM)>50021.8726.92>500282.50.52NDND15.68 to 46.23NDND0.34 to 0.81

[0745] In addition, the same NCI-N87 cell line was transplanted into Balb / c nude mice, and then Trastuzumab-mc-AA-PEG4-PABC-AM20001, Trastuzumab-mal-GGFG-AM20002, or the control substance Trastuzumab-Deruxtecan ADC was administered as a single dose of 2 mg / kg, respectively, to evaluate the in vivo efficacy according to tumor size reduction. Referring to Figure 13, a significant tumor size reduction was observed in the group administered Trastuzumab-Deruxtecan (Enhertu), but in the group administered Trastuzumab-mc-AA-PEG4-PABC-AM20001 or Trastuzumab-mal-GGFG-AM20002, the tumor size continued to grow, and the tumor growth inhibition effect was less than 10%.

[0746] The above results demonstrate that, although AM20001 and AM20002 themselves exhibit excellent cytotoxic effects, when AM20001 and AM20002 were conjugated to Trastuzumab to form ADCs, no significant tumor suppression effect was observed in the ADC form. This suggests that the core structure of the Silatecan family has the characteristic of low conjugability as an ADC payload itself, and that linker and structural optimization are essential to overcome this limitation. When conjugated in an optimized form, Silatecan derivatives can be effective as ADC payloads.

[0747] On the other hand, when the silatecan core structure and its derivatives, AM20011 and AM20008, were conjugated to an antibody-linker to form an ADC, it was confirmed that the in vivo anticancer effect as an ADC was significantly improved (Figs. 14 and 15).

[0748] Therefore, compared to AM20001, it was confirmed that additional modification of the structure and spacer design can significantly affect the applicability of ADC and the potency of ADC.

[0749] [Experimental Example 13] Evaluation of the efficacy of silatecan A-, B-ring-modified derivative conjugated ADC for tumor growth inhibition in xenograft mice with HER2-high expression cells.

[0750] To evaluate the minimum effective dose in vivo of silatecan derivative-conjugated ADCs, NCI-N87 and Calu-3 human cancer cell lines with high HER2 expression were transplanted into Balb / c mice, and Trastuzumab-AM21608, -AM21609, -AM21611, and -AM21613 ADCs were administered as a single dose of 10 mg / kg each.

[0751] Figure 14 (Changes in tumor size according to in vivo efficacy evaluation of silatecan derivative-conjugated ADC): These are the results of in vivo efficacy evaluation of silatecan derivative-conjugated ADC. In vivo efficacy evaluation of Trastuzumab-AM21608, -AM21609, -AM21611, -AM21613 ADCs was conducted. NCI-N87 and Calu-3 human cancer cell lines with high HER2 expression were transplanted into Balb / c mice, and each ADC was administered as a single dose of 10 mg / kg. Changes in tumor size were observed for more than 50 days after drug administration, and complete tumor remission was observed for Trastuzumab-AM21611 and -AM21608, comparable to Trastuzumab-Deruxtecan. In contrast, Trastuzumab-AM21609 and -AM21613 showed tumor shrinkage until day 10.

[0752] Figure 15 (Weight changes following in vivo efficacy evaluation of silatecan derivative-conjugated ADCs): HER2-expressing high-risk human cancer cell lines, NCI-N87 and Calu-3, were transplanted into Balb / c mice, and each of the ADCs, Trastuzumab-AM21608, -AM21609, -AM21611, and AM21613, was administered as a single dose of 10 mg / kg. Body weight changes were observed for more than 50 days after drug administration, and no significant weight changes were observed in any group.

[0753] Figure 16 (In vivo efficacy evaluation of silatecan derivative-conjugated Trastzumab ADC and Trastzumab-Deruxtecan conjugated ADC): HER2-high human cancer cell lines NCI-N87 and Calu-3 were transplanted into Balb / c mice, and Trastuzumab-AM21611 and Trastuzumab-Deruxtecan were administered as a single dose of 1 mg / kg and 2 mg / kg, respectively. In addition, the JIMT-1 cell line with Trastuzumab-resistant characteristics was transplanted into the same mouse model, and the two ADCs were administered as a single dose of 10 mg / kg, respectively. As a result of observing the change in tumor size for more than 35 days after drug administration, superior antitumor activity of Trastuzumab-AM21611 was confirmed compared to Trastuzumab-Deruxtecan in the JIMT-1 model, while similar antitumor efficacy was observed between the two ADCs in the NCI-N87 and Calu-3 models. Therefore, Trastuzumab-AM21611 is judged to exhibit excellent antitumor activity in various HER2-positive tumors, including Trastuzumab-resistant models.

[0754] [Experimental Example 14] Evaluation of the payload's sensitivity to multidrug resistance

[0755] Figure 17 (In vitro efficacy evaluation of silatecan derivatives and approved or clinically approved payloads on multidrug resistance after treatment with MDR inhibitors): The cytotoxicity of silatecan derivatives and payloads under clinical development was comparatively evaluated using human-derived ASPC1 cell lines overexpressing ABCG2 and HCT15 cell lines overexpressing P-gp. In addition, to evaluate the sensitivity of each payload to multidrug resistance (MDR), experiments were performed by treating them with the ABCG2 inhibitor YHO3177 (2 μM) and the P-gp inhibitor Tariquidar (1.25 μM), respectively. Each cell line was prepared as a 30 μL cell suspension and dispensed into each well of a low-attachment 384-well opaque plate with a U-shaped bottom. After culturing the cells for 18 hours, 10 μL of silatecan derivatives and approved payloads were diluted in 13 concentrations ranging from a final concentration of 1 μM to 0.6 pM, and treated per well. After treatment, the cells were cultured in an incubator at 37°C and 5% CO2 for 5 days. After the incubation was completed, 10 μL of CellTiter-Glo (Promega) was added to each well, and the reaction was stabilized for 30 minutes, and then cell viability was measured using a high-resolution luminescence spectrometer. The AM20011 payload exhibited superior cytotoxic activity in the ASPC-1 cell line compared to clinically approved TOPO I inhibitors DXd and SN-38. In addition, when treated with YHO13177, an ABCG2 inhibitor, the cytotoxicity change was less than that of DXd and SN-38, confirming that AM20011 has low sensitivity to ABCG2. In the P-gp-overexpressing HCT-15 cell line, superior cytotoxicity was observed compared to DXd, SN-38, and MMAE, and lower P-gp sensitivity was ultimately confirmed compared to MMAE and DXd through experiments using the P-gp inhibitor Tariquidar. These results suggest that AM20011 is a payload with low sensitivity to multidrug resistance mechanisms associated with drug resistance.

[0756] In relation to Fig. 17, the following results were obtained:

[0757] ASPC-1(nM) (ABCG2 high)HCT-15 (p-gp high)NoneYHO13177Fold changeNoneTariquidarFold changeAM2001135.8638.450.931.00.42.5Dxd120.5050.542.384.91.43.5SN38186.283.892.224.92.81.75MMAE0.871.430.6135.40.3118

[0758] The above description of the present invention is for illustrative purposes only, and those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single entity may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined manner.

[0759] The scope of the present invention is indicated by the claims described below rather than the detailed description above, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims

A silatecan derivative or a pharmaceutically acceptable salt thereof, represented by the following chemical formula 1 or chemical formula 2; and Linker As a linker-drug conjugate comprising, A linker-drug conjugate wherein the linker is bound to A of the following chemical formula 1 or B of the following chemical formula 2: [Chemical Formula 1] ; [Chemical Formula 2] ; In the above chemical formula 1 and chemical formula 2, A is A 1 -(C 0-3 alkylene)-A 2 -(C 0-3 alkylene)-A 3 -A 4 - and, A 1 is -NH2, -OH, or -O-CH2-NH2, A 2 and A 3 are, independently of each other, absent, or -O-, -NH, -N(C 1-3 alkyl group)-, or -S-, A 4 is -C(=O)- or -SO2-; B is B 1 -(C 0-3 alkylene)-B 2 -B 3 -B 4 -B 5 -(C 0-3 alkylene)-and, B 1 is -NH2, -OH, or -O-CH2-NH2, B 2 and B 3 are, independently of each other, absent, or -O-, -NH, -N(C 1-3 alkyl group)-, or -S-; B 4 does not exist, or is -C(=O)- or -SO2-; B 5 does not exist, or is -NH- or -O-, R a , R b , R c , R d , and R e are, each independently, C 1-10 Alkyl group, C 2-10 Alkenyl group, C 2-10 Alkynyl group, C 4-20 Aryl group, -(CH2) m R 11 Ki, or SiR 12 R 13 R 14 , where m is an integer in the range of 1 to 10, and R 11 Silver hydroxyl group, C 1-10 Alkoxy group, amino group, C 1-10 Alkylamino group, di-C 1-10 -alkylamino group, F, Cl, cyano group, or nitro group, where R 12 , R 13 and R 14 are, each independently, C 1-10 Alkyl group or C 4-10 It is an aryl group; X is H, F, Cl, nitro, amino, hydroxy, or cyano, Y is -NH-, Z is hydrogen, F, Cl, hydroxyl group, nitro group, cyano group, azido group, formyl group, hydrazino group, amino group, C 1-10 Alkyl group, C 2-10 Alkenyl group, C 2-10 Alkynyl group, C 1-10 Alkoxy group, C 1-10 Aryloxy group, C 1-10 Acyloxy group, -C(O)-C 1-10 Alkyl group, -C(O)-C 4-10 Aryl group, -OC(O)OC 1-10 Alkyl group, -OC(O)NR 15 R 16 and, here, R 15 and R 16 are, independently, H, C 1-10 Alkyl group or C 4-10 It is an aryl group, W is H, F, Cl, nitro, amino, hydroxy, or cyano, or Z and W are connected to each other to form C containing one or more heteroatoms. 1-5 It is a heterocyclic alkyl ring. In the first paragraph, A linker-drug conjugate wherein A is selected from the following: NH2-CH2-C(=O)-, NH2-C2H4-C(=O)-, NH2-C3H6-C(=O)-, NH2-C4H8-C(=O)-, NH2-C5H 10 -C(=O)-, NH2-C6H 12 -C(=O)-, OH-CH2-C(=O)-, OH-C2H4-C(=O)-, OH-C3H6-C(=O)-, OH-C4H8-C(=O)-, OH-C5H 10 -C(=O)-, OH-C6H 12 -C(=O)-, NH2-CH2-SO2-, NH2-C2H4-SO2-, NH2-C3H6-SO2-, NH2-C4H8-SO2-, NH2-C5H 10 -SO2-, NH2-C6H 12 -SO2-, OH-CH2-SO2-, OH-C2H4-SO2-, OH-C3H6-SO2-, OH-C4H8-SO2-, OH-C5H 10 -SO2-, OH-C6H 12 -SO2-, NH2-OC(=O)-, NH2-O-SO2-, NH2-NH-C(=O)-, NH2-NH-SO2-, OH-NH-C(=O)-, OH-NH-SO2-, NH2CH2-OC(=O)-, NH2CH2-O-SO2-, NH2CH2-NH-C(=O)-, NH2CH2-NH-SO2-, NH2C2H4-OC(=O)-, NH2C2H4-O-SO2-, NH2C2H4-NH-C(=O)-, NH2-C2H4-NH-SO2-, NH2C3H6-OC(=O)-, NH2C3H6-O-SO2-, NH2C3H6-NH-C(=O)- NH2C3H6-NH-SO2-, OH-CH2-OC(=O)-, OH-CH2-O-SO2-, OH-CH2-NH-C(=O)-, OH-CH2-NH-SO2-, OH-C2H4-OC(=O)-, OH-C2H4-O-SO2-, OH-C2H4-NH-C(=O)-, OH-C2H4-NH-SO2-, OH-C3H6-OC(=O)-, OH-C3H6-O-SO2-, OH-C3H6-NH-C(=O)-, OH-C3H6-NH-SO2-, NH2-OCH2-C(=O)-, NH2-OCH2-SO2-, NH2-NHCH2-C(=O)- NH2-NHCH2-SO2-, NH2-OC2H4-C(=O)-, NH2-OC2H4-SO2-, NH2-NHC2H4-C(=O)-, NH2-NHC2H4-SO2-, NH2-OC3H6-C(=O)-, NH2-OC3H6-SO2-, NH2-NHC3H6-C(=O)-, NH2-NHC3H6-SO2-, OH-OCH2-C(=O)-, OH-OCH2-SO2-, OH-NHCH2-C(=O)-, OH-NHCH2-SO2-, OH-OC2H4-C(=O)-, OH-OC2H4-SO2-, OH-NHC2H4-C(=O)-, OH-NHC2H4-SO2- OH-OC3H6-C(=O)-, OH-OC3H6-SO2-, OH-NHC3H6-C(=O)-, OH-NHC3H6-SO2-, NH2CH2-OCH2-C(=O)-, NH2CH2-OCH2-SO2-, NH2CH2-NHCH2-C(=O)-, NH2CH2-NHCH2-SO2-, NH2CH2-OC2H4-C(=O)-NH2CH2-OC2H4-SO2-, NH2CH2-NHC2H4-C(=O)-, NH2CH2-NHC2H4-SO2-, NH2CH2-OC3H6-C(=O)-, NH2CH2-OC3H6-SO2-, NH2CH2-NHC3H6-C(=O)-, NH2CH2-NHC3H6-SO2-, NH2CH2-OC4H8-C(=O)-, NH2CH2-OC4H8-SO2-, NH2CH2-OC5H 10 -C(=O)-, NH2CH2-OC5H 10 -SO2-, NH2CH2-OC6H 12 -C(=O)-, NH2CH2-OC6H 12 -SO2-, NH2C2H4-OCH2-C(=O)-, NH2C2H4-OCH2-SO2-, NH2C2H4-NHCH2-C(=O)-, NH2C2H4-NHCH2-SO2-, NH2C2H4-OC2H4-C(=O)-, NH2C2H4-OC2H4-SO2-, NH2C2H4-NHC2H4-C(=O)-, NH2C2H4-NHC2H4-SO2-, NH2C2H4-OC3H6-C(=O)-, NH2C2H4-OC3H6-SO2-, NH2C2H4-NHC3H6-C(=O)-, NH2C2H4-NHC3H6-SO2-, NH2C3H6-OCH2-C(=O)-, NH2C3H6-OCH2-SO2-, NH2C3H6-NHCH2-C(=O)-, NH2C3H6-NHCH2-SO2-, NH2C3H6-OC2H4-C(=O)-, NH2C3H6-OC2H4-SO2-, NH2C3H6-NHC2H4-C(=O)-, NH2C3H6-NHC2H4-SO2-, NH2C3H6-OC3H6-C(=O)-, NH2C3H6-OC3H6-SO2-, NH2C3H6-NHC3H6-C(=O)-, NH2C3H6-NHC3H6-SO2-, NH2-CH2-O-CH2-OC(=O)- NH2-C2H4-O-CH2-OC(=O)-, NH2-C3H6-O-CH2-OC(=O)-, NH2-CH2-O-C2H4-OC(=O)-, NH2-CH2-O-C3H6-OC(=O)-, NH2-CH2-O-CH2-O-SO2-, NH2-C2H4-O-CH2-O-SO2-, NH2-C3H6-O-CH2-O-SO2-, NH2-CH2-O-C2H4-O-SO2-, NH2-CH2-O-C3H6-O-SO2-, NH2-CH2-OO-CH2-C(=O)-, NH2-CH2-OO-CH2-SO2-, NH2-CH2-OO-C2H4-C(=O)- NH2-CH2-OO-C2H4-SO2-, NH2-CH2-OO-C3H6-C(=O)-, NH2-CH2-OO-C3H6-SO2-, NH2-CH2-O-NH-C(=O)-, NH2-CH2-O-NH-SO2-, NH2-CH2-O-NH-CH2-C(=O)-, NH2-CH2-O-NH-CH2-SO2-,NH2-CH2-O-NH-C2H4-C(=O)-, NH2-CH2-O-NH-C2H4-SO2-, NH2-CH2-O-NH-C3H6-C(=O)-, NH2-CH2-O-NH-C3H6-SO2-, NH2-CH2-NH-CH2-OC(=O)-, NH2-C2H4-NH-CH2-OC(=O)-, NH2-C3H6-NH-CH2-OC(=O)-, NH2-CH2-NH-C2H4-OC(=O)-, NH2-CH2-NH-C3H6-OC(=O)-, NH2-NH-CH2-NH-C(=O)-, NH2-NH-C3H6-NH-C(=O)-, NH2-CH2-NH-NH-C(=O)-, NH2-C2H4-NH-NH-C(=O)-, NH2-C3H6-NH-NH-C(=O)-, NH2-CH2-NH-CH2-NH-C(=O)-, NH2-C2H4-NH-CH2-NH-C(=O)-, NH2-C3H6-NH-CH2-NH-C(=O)-, NH2-CH2-NH-C2H4-NH-C(=O)-, NH2-CH2-NH-C3H6-NH-C(=O)-, NH2-NH-CH2-NH-C(=O)-, NH2-NH-C2H4-NH-C(=O)-, NH2-NH-C3H6-NH-C(=O)-, NH2-CH2-NH-NH-SO2-, NH2-C2H4-NH-NH-SO2-, NH2-C3H6-NH-NH-SO2-, NH2-CH2-NH-CH2-NH-SO2-, NH2-C2H4-NH-CH2-NH-SO2-, NH2-C3H6-NH-CH2-NH-SO2-, NH2-CH2-NH-C2H4-NH-SO2-, NH2-CH2-NH-C3H6-NH-SO2-, NH2-CH2-O-CH2-NH-C(=O)-, NH2-CH2-O-C2H4-NH-C(=O)-, NH2-CH2-O-C3H6-NH-C(=O)-, NH2-C2H4-O-CH2-NH-C(=O)-, NH2-C3H6-O-CH2-NH-C(=O)-, NH2-CH2-O-CH2-NH-SO2-, NH2-CH2-O-C2H4-NH-SO2-, NH2-CH2-O-C3H6-NH-SO2-, NH2-C2H4-O-CH2-NH-SO2-, NH2-C3H6-O-CH2-NH-SO2-,OH-CH2-O-CH2-O-C(=O)-, OH-C2H4-O-CH2-O-C(=O)-, OH-C3H6-O-CH2-O-C(=O)-, OH-CH2-O-C2H4-O-C(=O)-, OH-CH2-O-C3H6-O-C(=O)-, OH-CH2-O-CH2-O-SO2-, OH-C2H4-O-CH2-O-SO2-, OH-C3H6-O-CH2-O-SO2-, OH-CH2-O-C2H4-O-SO2-, OH-CH2-O-C3H6-O-SO2-, OH-CH2-NH-CH2-O-C(=O)-, OH-C2H4-NH-CH2-O-C(=O)-, OH-C3H6-NH-CH2-O-C(=O)-, OH-CH2-NH-C2H4-O-C(=O)-, OH-CH2-NH-C3H6-O-C(=O)-, OH-NH-CH2-NH-C(=O)-, OH-NH-C2H4-NH-C(=O)-, OH-NH-C3H6-NH-C(=O)-, OH-CH2-NH-NH-C(=O)-, OH-C2H4-NH-NH-C(=O)-, OH-C3H6-NH-NH-C(=O)-, OH-CH2-NH-CH2-NH-C(=O)-, OH-C2H4-NH-CH2-NH-C(=O)-, OH-C3H6-NH-CH2-NH-C(=O)-, OH-CH2-NH-C2H4-NH-C(=O)-, OH-CH2-NH-C3H6-NH-C(=O)-, OH-NH-CH2-NH-C(=O)-, OH-NH-C2H4-NH-C(=O)-, OH-NH-C3H6-NH-C(=O)-, OH-CH2-NH-NH-SO2-, OH-C2H4-NH-NH-SO2-, OH-C3H6-NH-NH-SO2-, OH-CH2-NH-CH2-NH-SO2-, OH-C2H4-NH-CH2-NH-SO2-, OH-C3H6-NH-CH2-NH-SO2-, OH-CH2-NH-C2H4-NH-SO2-, OH-CH2-NH-C3H6-NH-SO2-, OH-CH2-O-CH2-NH-C(=O)-, OH-CH2-O-C2H4-NH-C(=O)-, OH-CH2-O-C3H6-NH-C(=O)-, OH-C2H4-O-CH2-NH-C(=O)-, OH-C3H6-O-CH2-NH-C(=O)-, OH-CH2-O-CH2-NH-SO2-,OH-CH2-O-C2H4-NH-SO2-, OH-CH2-O-C3H6-NH-SO2-, OH-C2H4-O-CH2-NH-SO2-, OH-C3H6-O-CH2-NH-SO2-, NH2-CH2-O-CH2-O-CH2-OC(=O)-, NH2-CH2-O-C2H4-O-CH2-OC(=O)-, NH2-CH2-O-C3H6-O-CH2-OC(=O)-, NH2-CH2-O-CH2-O-C2H4-OC(=O)-, NH2-CH2-O-CH2-O-C3H6-OC(=O)-, NH2-CH2-O-CH2-O-CH2-O-SO2-, NH2-CH2-O-C2H4-O-CH2-O-SO2-, NH2-CH2-O-C3H6-O-CH2-O-SO2-, NH2-CH2-O-CH2-O-C2H4-O-SO2-, NH2-CH2-O-CH2-O-C3H6-O-SO2-, NH2-CH2-O-CH2-NH-CH2-OC(=O)-, NH2-CH2-O-C2H4-NH-CH2-OC(=O)-, NH2-CH2-O-C3H6-NH-CH2-OC(=O)-, NH2-CH2-O-CH2-NH-C2H4-OC(=O)-, NH2-CH2-O-CH2-NH-C3H6-OC(=O)-, NH2-CH2-O-NH-CH2-NH-C(=O)-, NH2-CH2-O-NH-C2H4-NH-C(=O)-, NH2-CH2-O-NH-C3H6-NH-C(=O)-, NH2-CH2-O-CH2-NH-NH-C(=O)-, NH2-CH2-O-C2H4-NH-NH-C(=O)-, NH2-CH2-O-C3H6-NH-NH-C(=O)-, NH2-CH2-O-CH2-NH-CH2-NH-C(=O)-, NH2-CH2-O-C2H4-NH-CH2-NH-C(=O)-, NH2-CH2-O-C3H6-NH-CH2-NH-C(=O)-, NH2-CH2-O-CH2-NH-C2H4-NH-C(=O)-, NH2-CH2-O-CH2-NH-C3H6-NH-C(=O)-, NH2-CH2-O-NH-CH2-NH-C(=O)-, NH2-CH2-O-NH-C2H4-NH-C(=O)-, NH2-CH2-O-NH-C3H6-NH-C(=O)-, NH2-CH2-O-CH2-NH-NH-SO2-,NH2-CH2-O-C2H4-NH-NH-SO2-, NH2-CH2-O-C3H6-NH-NH-SO2-, NH2-CH2-O-CH2-NH-CH2-NH-SO2-, NH2-CH2-O-C2H4-NH-CH2-NH-SO2-, NH2-CH2-O-C3H6-NH-CH2-NH-SO2-, NH2-CH2-O-CH2-NH-C2H4-NH-SO2-, NH2-CH2-O-CH2-NH-C3H6-NH-SO2-, NH2-CH2-O-CH2-O-CH2-NH-C(=O)-, NH2-CH2-O-CH2-O-C2H4-NH-C(=O)- NH2-CH2-O-CH2-O-C3H6-NH-C(=O)-, NH2-CH2-O-C2H4-O-CH2-NH-C(=O)-, NH2-CH2-O-C3H6-O-CH2-NH-C(=O)-, NH2-CH2-O-CH2-O-CH2-NH-SO2-, NH2-CH2-O-CH2-O-C2H4-NH-SO2-, NH2-CH2-O-CH2-O-C3H6-NH-SO2-, NH2-CH2-O-C2H4-O-CH2-NH-SO2-, NH2-CH2-O-C3H6-O-CH2-NH-SO2-, NH2-CH2-O-CH2-C(=O)-, NH2-CH2-O-C2H4-C(=O)-, NH2-CH2-O-C3H6-C(=O)-, NH2-CH2-O-C4H8-C(=O)-, NH2-CH2-O-C5H, 10 -C(=O)-, NH2-CH2-O-C6H 12 -C(=O)-, NH2-CH2-O-CH2-SO2-, NH2-CH2-O-C2H4-SO2-, NH2-CH2-O-C3H6-SO2-, NH2-CH2-O-C4H8-SO2-, NH2-CH2-O-C5H 10 -SO2-, NH2-CH2-O-C6H 12 -SO2-, NH2-CH2-O-NH-C(=O)-, NH2-CH2-O-NH-SO2-, NH2-CH2-O-CH2-OC(=O)-, NH2-CH2-O-CH2-O-SO2-, NH2-CH2-O-CH2-NH-C(=O)-, NH2-CH2-O-CH2-NH-SO2-, NH2-CH2-O-C2H4-OC(=O)-, NH2-CH2-O-C2H4-O-SO2-, NH2-CH2-O-C2H4-NH-C(=O)-, NH2-CH2-O-C2H4-NH-SO2-, NH2-CH2-O-C3H6-OC(=O)-, NH2-CH2-O-C3H6-O-SO2- NH2-CH2-O-C3H6-NH-C(=O)-, 및 NH2-CH2-O-C3H6-NH-SO2-. In the first paragraph, A linker-drug conjugate wherein A is selected from the following: In the first paragraph, The above B is a linker-drug conjugate selected from the following: NH2CH2-, NH2C2H4-, NH2C3H6-, NH2C4H8-, NH2C5H 10 -, NH2C6H 12 -, OHCH2-, OHC2H4-, OHC3H6-, OHC4H8-, OHC5H 10 -, OHC6H 12 -, NH2C2H4OCH2-, NH2C2H4OC2H4-, NH2C2H4OC3H6-, NH2C3H6OCH2-, NH2C3H6OC2H4-, NH2C3H6OC3H6-, NH2CH2NHCH2-, NH2CH2NHC2H4-, NH2CH2NHC3H6-, NH2C2H4NHCH2-, NH2C2H4NHC2H4-, NH2C2H4NHC3H6-, NH2C3H6NHCH2-, NH2C3H6NHC2H4-, NH2C3H6NHC3H6-, NH2CH2N(CH3)CH2-, NH2CH2N(CH3)C2H4-, NH2CH2N(CH3)C3H6-, NH2C2H4N(CH3)CH2-, NH2C2H4N(CH3)C2H4-, NH2C2H4N(CH3)C3H6-, NH2C3H6N(CH3)CH2-, NH2C3H6N(CH3)C2H4-, NH2C3H6N(CH3)C3H6-, NH2CH2SCH2-, NH2CH2SC2H4-, NH2CH2SC3H6-, NH2C2H4SCH2-, NH2C2H4SC2H4-, NH2C2H4SC3H6-, NH2C3H6SCH2-, NH2C3H6SC2H4-, NH2C3H6SC3H6-, OHC2H4OCH2-, OHC2H4OC2H4-, OHC2H4OC3H6-, OHC3H6OCH2-, OHC3H6OC2H4-, OHC3H6OC3H6-, OHCH2NHCH2-, OHCH2NHC2H4-, OHCH2NHC3H6-, OHC2H4NHCH2-, OHC2H4NHC2H4-, OHC2H4NHC3H6-, OHC3H6NHCH2-, OHC3H6NHC2H4-, OHC3H6NHC3H6-, OHCH2N(CH3)CH2-, OHCH2N(CH3)C2H4-, OHCH2N(CH3)C3H6-, OHC2H4N(CH3)CH2-, OHC2H4N(CH3)C2H4-, OHC2H4N(CH3)C3H6-, OHC3H6N(CH3)CH2-, OHC3H6N(CH3)C2H4-, OHC3H6N(CH3)C3H6-, OHCH2SCH2-, OHCH2SC2H4-, OHCH2SC3H6-, OHC2H4SCH2-, OHC2H4SC2H4-, OHC2H4SC3H6-, OHC3H6SCH2-,OHC3H6SC2H4-, OHC3H6SC3H6-, NH2-C(=O)-NH-CH2-, NH2-CH2-C(=O)-NH-CH2-, NH2-C2H4-C(=O)-NH-CH2-, NH2-C3H6-C(=O)-NH-CH2-, NH2-C(=O)-NH-C2H4-, NH2-CH2-C(=O)-NH-C2H4-, NH2-C2H4-C(=O)-NH-C2H4-, NH2-C3H6-C(=O)-NH-C2H4-, NH2-C(=O)-NH-C3H6-, NH2-CH2-C(=O)-NH-C3H6-, NH2-C2H4-C(=O)-NH-C3H6-, NH2-C3H6-C(=O)-NH-C3H6-, NH2-SO2-NH-CH2-, NH2-CH2-SO2-NH-CH2-, NH2-C2H4-SO2-NH-CH2-, NH2-C3H6-SO2-NH-CH2-, NH2-SO2-NH-C2H4-, NH2-CH2-SO2-NH-C2H4-, NH2-C2H4-SO2-NH-C2H4-, NH2-C3H6-SO2-NH-C2H4-, NH2-SO2-NH-C3H6-, NH2-CH2-SO2-NH-C3H6-, NH2-C2H4-SO2-NH-C3H6-, NH2-C3H6-SO2-NH-C3H6-, OH-C(=O)-NH-CH2-, OH-CH2-C(=O)-NH-CH2-, OH-C2H4-C(=O)-NH-CH2-, OH-C3H6-C(=O)-NH-CH2-, OH-C(=O)-NH-C2H4-, OH-CH2-C(=O)-NH-C2H4-, OH-C2H4-C(=O)-NH-C2H4-, OH-C3H6-C(=O)-NH-C2H4-, OH-C(=O)-NH-C3H6-, OH-CH2-C(=O)-NH-C3H6-, OH-C2H4-C(=O)-NH-C3H6-, OH-C3H6-C(=O)-NH-C3H6-, OH-SO2-NH-CH2-, OH-CH2-SO2-NH-CH2-, OH-C2H4-SO2-NH-CH2-, OH-C3H6-SO2-NH-CH2-, OH-SO2-NH-C2H4-, OH-CH2-SO2-NH-C2H4-, OH-C2H4-SO2-NH-C2H4-, OH-C3H6-SO2-NH-C2H4-OH-SO2-NH-C3H6-, OH-CH2-SO2-NH-C3H6-, OH-C2H4-SO2-NH-C3H6-, OH-C3H6-SO2-NH-C3H6-, NH2-OC(=O)-NH-CH2-, NH2-CH2-OC(=O)-NH-CH2-, NH2-C2H4-OC(=O)-NH-CH2-, NH2-C3H6-OC(=O)-NH-CH2-, NH2-OC(=O)-NH-C2H4-, NH2-CH2-OC(=O)-NH-C2H4-, NH2-C2H4-OC(=O)-NH-C2H4-, NH2-C3H6-OC(=O)-NH-C2H4- NH2-OC(=O)-NH-C3H6-, NH2-CH2-OC(=O)-NH-C3H6-, NH2-C2H4-OC(=O)-NH-C3H6-, NH2-C3H6-OC(=O)-NH-C3H6-, NH2-NH-C(=O)-NH-CH2-, NH2-CH2-NH-C(=O)-NH-CH2-, NH2-C2H4-NH-C(=O)-NH-CH2-, NH2-C3H6-NH-C(=O)-NH-CH2-, NH2-NH-C(=O)-NH-C2H4-, NH2-CH2-NH-C(=O)-NH-C2H4-, NH2-C2H4-NH-C(=O)-NH-C2H4-, NH2-C3H6-NH-C(=O)-NH-C2H4-, NH2-NH-C(=O)-NH-C3H6-, NH2-CH2-NH-C(=O)-NH-C3H6-, NH2-C2H4-NH-C(=O)-NH-C3H6-, NH2-C3H6-NH-C(=O)-NH-C3H6-, NH2-NH-SO2-NH-CH2-, NH2-CH2-NH-SO2-NH-CH2-, NH2-C2H4-NH-SO2-NH-CH2-, NH2-C3H6-NH-SO2-NH-CH2-, NH2-NH-SO2-NH-C2H4-, NH2-CH2-NH-SO2-NH-C2H4-, NH2-C2H4-NH-SO2-NH-C2H4-, NH2-C3H6-NH-SO2-NH-C2H4-, NH2-NH-SO2-NH-C3H6-, NH2-CH2-NH-SO2-NH-C3H6-, NH2-C2H4-NH-SO2-NH-C3H6-, NH2-C3H6-NH-SO2-NH-C3H6-OH-O-C(=O)-NH-CH2-, OH-CH2-O-C(=O)-NH-CH2-, OH-C2H4-O-C(=O)-NH-CH2-, OH-C3H6-O-C(=O)-NH-CH2-, OH-O-C(=O)-NH-C2H4-, OH-CH2-O-C(=O)-NH-C2H4-, OH-C2H4-O-C(=O)-NH-C2H4-, OH-C3H6-O-C(=O)-NH-C2H4-, OH-O-C(=O)-NH-C3H6-, OH-CH2-O-C(=O)-NH-C3H6-, OH-C2H4-O-C(=O)-NH-C3H6-, OH-C3H6-O-C(=O)-NH-C3H6-, OH-NH-C(=O)-NH-CH2-, OH-CH2-NH-C(=O)-NH-CH2-, OH-C2H4-NH-C(=O)-NH-CH2-, OH-C3H6-NH-C(=O)-NH-CH2-, OH-NH-C(=O)-NH-C2H4-, OH-CH2-NH-C(=O)-NH-C2H4-, OH-C2H4-NH-C(=O)-NH-C2H4-, OH-C3H6-NH-C(=O)-NH-C2H4-, OH-NH-C(=O)-NH-C3H6-, OH-CH2-NH-C(=O)-NH-C3H6-, OH-C2H4-NH-C(=O)-NH-C3H6-, OH-C3H6-NH-C(=O)-NH-C3H6-, OH-NH-SO2-NH-CH2-, OH-CH2-NH-SO2-NH-CH2-, OH-C2H4-NH-SO2-NH-CH2-, OH-C3H6-NH-SO2-NH-CH2-, OH-NH-SO2-NH-C2H4-, OH-CH2-NH-SO2-NH-C2H4-, OH-C2H4-NH-SO2-NH-C2H4-, OH-C3H6-NH-SO2-NH-C2H4-, OH-NH-SO2-NH-C3H6-, OH-CH2-NH-SO2-NH-C3H6-, OH-C2H4-NH-SO2-NH-C3H6-, OH-C3H6-NH-SO2-NH-C3H6-, NH2-NH-O-C(=O)-NH-CH2-, NH2-CH2-NH-O-C(=O)-NH-CH2-, NH2-C2H4-NH-O-C(=O)-NH-CH2-, NH2-C3H6-NH-O-C(=O)-NH-CH2-,NH2-NH-OC(=O)-NH-C2H4-, NH2-CH2-NH-OC(=O)-NH-C2H4-, NH2-C2H4-NH-OC(=O)-NH-C2H4-, NH2-C3H6-NH-OC(=O)-NH-C2H4-, NH2-NH-OC(=O)-NH-C3H6-, NH2-CH2-NH-OC(=O)-NH-C3H6-, NH2-C2H4-NH-OC(=O)-NH-C3H6-, NH2-C3H6-NH-OC(=O)-NH-C3H6-, NH2-NH-NH-C(=O)-NH-CH2-, NH2-CH2-NH-NH-C(=O)-NH-CH2-, NH2-C2H4-NH-NH-C(=O)-NH-CH2-, NH2-C3H6-NH-NH-C(=O)-NH-CH2-, NH2-NH-NH-C(=O)-NH-C2H4-, NH2-CH2-NH-NH-C(=O)-NH-C2H4-, NH2-C2H4-NH-NH-C(=O)-NH-C2H4-, NH2-C3H6-NH-NH-C(=O)-NH-C2H4-, NH2-NH-NH-C(=O)-NH-C3H6-, NH2-CH2-NH-NH-C(=O)-NH-C3H6-, NH2-C2H4-NH-NH-C(=O)-NH-C3H6-, NH2-C3H6-NH-NH-C(=O)-NH-C3H6-, NH2-NH-NH-SO2-NH-CH2-, NH2-CH2-NH-NH-SO2-NH-CH2-, NH2-C2H4-NH-NH-SO2-NH-CH2-, NH2-C3H6-NH-NH-SO2-NH-CH2-, NH2-NH-NH-SO2-NH-C2H4-, NH2-CH2-NH-NH-SO2-NH-C2H4-, NH2-C2H4-NH-NH-SO2-NH-C2H4-, NH2-C3H6-NH-NH-SO2-NH-C2H4-, NH2-NH-NH-SO2-NH-C3H6-, NH2-CH2-NH-NH-SO2-NH-C3H6-, NH2-C2H4-NH-NH-SO2-NH-C3H6-, NH2-C3H6-NH-NH-SO2-NH-C3H6-, OH-NH-OC(=O)-NH-CH2-, OH-CH2-NH-OC(=O)-NH-CH2-, OH-C2H4-NH-OC(=O)-NH-CH2-,OH-C3H6-NH-O-C(=O)-NH-CH2-, OH-NH-O-C(=O)-NH-C2H4-, OH-CH2-NH-O-C(=O)-NH-C2H4-, OH-C2H4-NH-O-C(=O)-NH-C2H4-, OH-C3H6-NH-O-C(=O)-NH-C2H4-, OH-NH-O-C(=O)-NH-C3H6-, OH-CH2-NH-O-C(=O)-NH-C3H6-, OH-C2H4-NH-O-C(=O)-NH-C3H6-, OH-C3H6-NH-O-C(=O)-NH-C3H6-, OH-NH-NH-C(=O)-NH-CH2-, OH-CH2-NH-NH-C(=O)-NH-CH2-, OH-C2H4-NH-NH-C(=O)-NH-CH2-, OH-C3H6-NH-NH-C(=O)-NH-CH2-, OH-NH-NH-C(=O)-NH-C2H4-, OH-CH2-NH-NH-C(=O)-NH-C2H4-, OH-C2H4-NH-NH-C(=O)-NH-C2H4-, OH-C3H6-NH-NH-C(=O)-NH-C2H4-, OH-NH-NH-C(=O)-NH-C3H6-, OH-CH2-NH-NH-C(=O)-NH-C3H6-, OH-C2H4-NH-NH-C(=O)-NH-C3H6-, OH-C3H6-NH-NH-C(=O)-NH-C3H6-, OH-NH-NH-SO2-NH-CH2-, OH-CH2-NH-NH-SO2-NH-CH2-, OH-C2H4-NH-NH-SO2-NH-CH2-, OH-C3H6-NH-NH-SO2-NH-CH2-, OH-NH-NH-SO2-NH-C2H4-, OH-CH2-NH-NH-SO2-NH-C2H4-, OH-C2H4-NH-NH-SO2-NH-C2H4-, OH-C3H6-NH-NH-SO2-NH-C2H4-, OH-NH-NH-SO2-NH-C3H6-, OH-CH2-NH-NH-SO2-NH-C3H6-, OH-C2H4-NH-NH-SO2-NH-C3H6-, OH-C3H6-NH-NH-SO2-NH-C3H6-, NH2-CH2-O-CH2-, NH2-CH2-O-C2H4-, NH2-CH2-O-C3H6-, NH2-CH2-O-C4H8-, NH2-CH2-O-C5H, 10 -, NH2-CH2-O-C6H 12 -, NH2-CH2-O-C2H4OCH2-, NH2-CH2-O-C2H4OC2H4-, NH2-CH2-O-C2H4OC3H6-, NH2-CH2-O-C3H6OCH2-, NH2-CH2-O-C3H6OC2H4-, NH2-CH2-O-C3H6OC3H6-, NH2-CH2-O-CH2NHCH2-, NH2-CH2-O-CH2NHC2H4-, NH2-CH2-O-CH2NHC3H6-, NH2-CH2-O-C2H4NHCH2-, NH2-CH2-O-C2H4NHC2H4-, NH2-CH2-O-C2H4NHC3H6-, NH2-CH2-O-C3H6NHCH2-, NH2-CH2-O-C3H6NHC2H4-, NH2-CH2-O-C3H6NHC3H6-, NH2-CH2-O-CH2N(CH3)CH2-, NH2-CH2-O-CH2N(CH3)C2H4-, NH2-CH2-O-CH2N(CH3)C3H6-, NH2-CH2-O-C2H4N(CH3)CH2-, NH2-CH2-O-C2H4N(CH3)C2H4-, NH2-CH2-O-C2H4N(CH3)C3H6-, NH2-CH2-O-C3H6N(CH3)CH2-, NH2-CH2-O-C3H6N(CH3)C2H4-, NH2-CH2-O-C3H6N(CH3)C3H6-, NH2-CH2-O-CH2SCH2-, NH2-CH2-O-CH2SC2H4-, NH2-CH2-O-CH2SC3H6-, NH2-CH2-O-C2H4SCH2-, NH2-CH2-O-C2H4SC2H4-, NH2-CH2-O-C2H4SC3H6-, NH2-CH2-O-C3H6SCH2-, NH2-CH2-O-C3H6SC2H4-, NH2-CH2-O-C3H6SC3H6-, NH2-CH2-O-C(=O)-NH-CH2-, NH2-CH2-O-CH2-C(=O)-NH-CH2-, NH2-CH2-O-C2H4-C(=O)-NH-CH2-, NH2-CH2-O-C3H6-C(=O)-NH-CH2-, NH2-CH2-O-C(=O)-NH-C2H4-, NH2-CH2-O-CH2-C(=O)-NH-C2H4-, NH2-CH2-O-C2H4-C(=O)-NH-C2H4-, NH2-CH2-O-C3H6-C(=O)-NH-C2H4-,NH2-CH2-OC(=O)-NH-C3H6-, NH2-CH2-O-CH2-C(=O)-NH-C3H6-, NH2-CH2-O-C2H4-C(=O)-NH-C3H6-, NH2-CH2-O-C3H6-C(=O)-NH-C3H6-, NH2-CH2-O-SO2-NH-CH2-, NH2-CH2-O-CH2-SO2-NH-CH2-, NH2-CH2-O-C2H4-SO2-NH-CH2-, NH2-CH2-O-C3H6-SO2-NH-CH2-, NH2-CH2-O-SO2-NH-C2H4-, NH2-CH2-O-CH2-SO2-NH-C2H4-, NH2-CH2-O-C2H4-SO2-NH-C2H4-, NH2-CH2-O-C3H6-SO2-NH-C2H4-, NH2-CH2-O-SO2-NH-C3H6-, NH2-CH2-O-CH2-SO2-NH-C3H6-, NH2-CH2-O-C2H4-SO2-NH-C3H6-, NH2-CH2-O-C3H6-SO2-NH-C3H6-, NH2-CH2-OOC(=O)-NH-CH2-, NH2-CH2-O-CH2-OC(=O)-NH-CH2-, NH2-CH2-O-C2H4-OC(=O)-NH-CH2-, NH2-CH2-O-C3H6-OC(=O)-NH-CH2-, NH2-CH2-OOC(=O)-NH-C2H4-, NH2-CH2-O-CH2-OC(=O)-NH-C2H4-, NH2-CH2-O-C2H4-OC(=O)-NH-C2H4-, NH2-CH2-O-C3H6-OC(=O)-NH-C2H4-, NH2-CH2-OOC(=O)-NH-C3H6-, NH2-CH2-O-CH2-OC(=O)-NH-C3H6-, NH2-CH2-O-C2H4-OC(=O)-NH-C3H6-, NH2-CH2-O-C3H6-OC(=O)-NH-C3H6-, NH2-CH2-O-NH-C(=O)-NH-CH2-, NH2-CH2-O-CH2-NH-C(=O)-NH-CH2-, NH2-CH2-O-C2H4-NH-C(=O)-NH-CH2-, NH2-CH2-O-C3H6-NH-C(=O)-NH-CH2-, NH2-CH2-O-NH-C(=O)-NH-C2H4-,NH2-CH2-O-CH2-NH-C(=O)-NH-C2H4-, NH2-CH2-O-C2H4-NH-C(=O)-NH-C2H4-, NH2-CH2-O-C3H6-NH-C(=O)-NH-C2H4-, NH2-CH2-O-NH-C(=O)-NH-C3H6-, NH2-CH2-O-CH2-NH-C(=O)-NH-C3H6-, NH2-CH2-O-C2H4-NH-C(=O)-NH-C3H6-, NH2-CH2-O-C3H6-NH-C(=O)-NH-C3H6-, NH2-CH2-O-NH-SO2-NH-CH2-, NH2-CH2-O-CH2-NH-SO2-NH-CH2-, NH2-CH2-O-C2H4-NH-SO2-NH-CH2-, NH2-CH2-O-C3H6-NH-SO2-NH-CH2-, NH2-CH2-O-NH-SO2-NH-C2H4-, NH2-CH2-O-CH2-NH-SO2-NH-C2H4-, NH2-CH2-O-C2H4-NH-SO2-NH-C2H4-, NH2-CH2-O-C3H6-NH-SO2-NH-C2H4-, NH2-CH2-O-NH-SO2-NH-C3H6-, NH2-CH2-O-CH2-NH-SO2-NH-C3H6-, NH2-CH2-O-C2H4-NH-SO2-NH-C3H6-, NH2-CH2-O-C3H6-NH-SO2-NH-C3H6-, NH2-CH2-O-NH-OC(=O)-NH-CH2-, NH2-CH2-O-CH2-NH-OC(=O)-NH-CH2-, NH2-CH2-O-C2H4-NH-OC(=O)-NH-CH2-, NH2-CH2-O-C3H6-NH-OC(=O)-NH-CH2-, NH2-CH2-O-NH-OC(=O)-NH-C2H4-, NH2-CH2-O-CH2-NH-OC(=O)-NH-C2H4-, NH2-CH2-O-C2H4-NH-OC(=O)-NH-C2H4-, NH2-CH2-O-C3H6-NH-OC(=O)-NH-C2H4-, NH2-CH2-O-NH-OC(=O)-NH-C3H6-, NH2-CH2-O-CH2-NH-OC(=O)-NH-C3H6-, NH2-CH2-O-C2H4-NH-OC(=O)-NH-C3H6-,NH2-CH2-O-C3H6-NH-OC(=O)-NH-C3H6-, NH2-CH2-O-NH-NH-C(=O)-NH-CH2-, NH2-CH2-O-CH2-NH-NH-C(=O)-NH-CH2-, NH2-CH2-O-C2H4-NH-NH-C(=O)-NH-CH2-, NH2-CH2-O-C3H6-NH-NH-C(=O)-NH-CH2-, NH2-CH2-O-NH-NH-C(=O)-NH-C2H4-, NH2-CH2-O-CH2-NH-NH-C(=O)-NH-C2H4-, NH2-CH2-O-C2H4-NH-NH-C(=O)-NH-C2H4-, NH2-CH2-O-C3H6-NH-NH-C(=O)-NH-C2H4-, NH2-CH2-O-NH-NH-C(=O)-NH-C3H6-, NH2-CH2-O-CH2-NH-NH-C(=O)-NH-C3H6-, NH2-CH2-O-C2H4-NH-NH-C(=O)-NH-C3H6-, NH2-CH2-O-C3H6-NH-NH-C(=O)-NH-C3H6-, NH2-CH2-O-NH-NH-SO2-NH-CH2-, NH2-CH2-O-CH2-NH-NH-SO2-NH-CH2-, NH2-CH2-O-C2H4-NH-NH-SO2-NH-CH2-, NH2-CH2-O-C3H6-NH-NH-SO2-NH-CH2-, NH2-CH2-O-NH-NH-SO2-NH-C2H4-, NH2-CH2-O-CH2-NH-NH-SO2-NH-C2H4-, NH2-CH2-O-C2H4-NH-NH-SO2-NH-C2H4-, NH2-CH2-O-C3H6-NH-NH-SO2-NH-C2H4-, NH2-CH2-O-NH-NH-SO2-NH-C3H6-, NH2-CH2-O-CH2-NH-NH-SO2-NH-C3H6-, NH2-CH2-O-C2H4-NH-NH-SO2-NH-C3H6-, 및 NH2-CH2-O-C3H6-NH-NH-SO2-NH-C3H6-., In the first paragraph, A linker-drug conjugate, wherein the linker is a non-cleavable linker or a cleavable linker. In the first paragraph, A linker-drug conjugate, wherein the cleavable linker is an enzyme-cleavable linker. In the first paragraph, A linker-drug conjugate wherein the linker is selected from the following: N-succinimidyl-4-(2-pyridyldithio)pentanoate [N-succinimidyl-4-(2-pyridyldithio)pentanoate; SPP], N-succinimidyl-4-(2-pyridyldithio)butanoate [N-succinimidyl-4-(2-pyridyldithio)butanoate; SPDB], sulfo-SPDB, 2,5-dioxopyrrolidin-1-yl 4-(pyridin-2-yldisulfaneyl)pentanoate [2,5-dioxopyrrolidin-1-yl 4-(pyridin-2-yldisulfaneyl)pentanoate; MDS], 2,5-dioxopyrrolidin-1-yl 4-methyl-4-(pyridin-2-yldisulfaneyl)pentanoate [2,5-dioxopyrrolidin-1-yl 4-methyl-4-(pyridin-2-yldisulfaneyl)pentanoate; DMDS], 2,5-dioxopyrrolidin-1-yl 4-((2-(pyridin-2-yl)propan-2-yl)disulfaneyl)butanoate [2,5-dioxopyrrolidin-1-yl 4-((2-(pyridin-2-yl)propan-2-yl)disulfaneyl)butanoate; DSDM], 2,5-dioxopyrrolidin-1-yl (E)-4-[4-[1-[2-[3-methyl-3-(pyridin-2-yldisulfaneyl)butanoyl]hydrazineylidene]ethyl]phenoxy]butanoate [2,5-dioxopyrrolidin-1-yl (E)-4-(4-(1-(2-(3-methyl-3-(pyridin-2-yldisulfaneyl)butanoyl)hydrazineylidene)ethyl)phenoxy)butanoate; NDMDS], bis-maleimidopolyethyleneglycol (BMPEO), N-(β-maleimidopropyloxy)succinimide ester [N-(β- maleimidopropyloxy)succinimide ester; BMPS], ε-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS),γ-maleimidobutyric acid N-succinimidyl ester (GMBS), HBVS, N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate [N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate; SMCC], LC-N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate [long chain N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate; long chain SMCC], Mal-alkane linker, m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), 4-(4-N-maleimidophenyl)-butyric acid hydrazide [4-(4-N-maleimidophenyl)-butyric acid hydrazide; MPBH], N-succinimidyl 3-(bromoacetamido)propionate [N-succinimidyl 3-(bromoacetamido)propionate; SBAP], N-succinimidyl iodoacetate (SIA), N-succinimidyl-4-(iodoacetyl)-aminobenzoate [N-succinimidyl-4-(iodoacetyl)-aminobenzoate; SIAB], N-succinimidyl-4-(iodoacetyl)-aminobenzoate [N-succinimidyl-4-(iodoacetyl)-aminobenzoate; SIAB], succinimidyl-6-(maleimidopropionamido)hexanoate [succinimidyl-6-(maleimidopropionamido)hexanoate; SMPH], , N-(e-maleimidocaproyloxy)sulfosuccinimido ester [N-(e-maleimidocaproyloxy)sulfosuccimido ester; sulfo-EMCS],N-(y-maleimidobutryloxy)sulfosuccinimide ester [N-(y-maleimidobutryloxy)sulfosuccinimde ester; sulfo-GMBS], N-(κ-maleimidoundecanoyloxy)sulfosuccinimide ester [N-(κ- maleimidoundecanoyloxy)sulfosuccinimide ester; sulfo-KMUS], m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS), sulfosuccinimidyl(4-iodo-acetyl)aminobenzoate [sulfosuccinimidyl(4-iodo-acetyl)aminobenzoate; sulfo-SIAB], sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate [sulfosuccinimidyl 4-(N-maleimido-methyl)cyclohexane-l-carboxylate; sulfo-SMCC], sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate [sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate; sulfo-SMPB], succinimidyl-(4-vinylsulfone)benzoate (SVSB), dithiobis-maleimidoethane (DTME), 1,4-bis-maleimidobutane (BMB), 1,4-bismaleimidyl-2,3-dihydroxybutane (BMDB), bis-maleimidohexane (BMH), bis-maleimidoethane (BMOE), 1,8-bis-maleimidodiethyleneglycol (1,8-bis-maleimidodiethyleneglycol; BM(PEO)2], 1, 11-bis-maleimidotriethylene glycol [1,11-bis-maleimidotriethyleneglycol; BM(PEO)3], Phe-Lys-PABC, Val-Cit-PABC, Val-Ala-PABC, MHVCBC (valine-citrulline), MHFKBC (phenylalanine-lysine), MHH, GBC (glucuronic acid), GBCDN (glucuronic acid), β-glucuronide linker, Mal-PEG-NHS, N-((S)-7-benzyl-3,6,9,12-tetraoxo-2,5,8,11-tetraazatridecan-13-yl)-6-(3-methylthio-2,5-dioxopyrrolidin-1-yl)hexanamide (MC-GGFG), (2S)-2-(3-methoxypropanamido)-N-((S)-3-methyl-1-(((S)-1-(methylamino)-1-oxopropan-2-yl)amino)-1-oxobutan-2-yl)-6-(3-(3-methylthio-2,5-dioxopyrrolidin-1-yl)propanamido)hexanamide (MP-NPEG-VA), (2S)-N-((S)-7-benzyl-3,6,9,12-tetraoxo-2,5,8,11-tetraazatridecan-13-yl)-2-(3-methoxypropanamido)-6-(3-(3-methylthio-2,5-dioxopyrrolidin-1-yl)propanamido)hexanamide (MP-NPEG-GGFG), (S)-2-(3-methoxypropanamido)-N-((S)-3-methyl-1-(((S)-1-(methylamino)-1-oxopropan-2-yl)amino)-1-oxobutan-2-yl)-6-(3-(2-methylthioacetamido)propanamido)hexanamide (BrAc-NPEG-VA), (S)-N-((S)-7-benzyl-3,6,9,12-tetraoxo-2,5,8,11-tetraazatridecan-13-yl)-2-(3-methoxypropanamido)-6-(3-(2-methylthioacetamido)propanamido)hexanamide (BrAc-NPEG-GGFG), N-((S)-3-methyl-1-(((S)-1-(methylamino)-1-oxopropan-2-yl)amino)-1-oxobutan-2-yl)-6-(3-(3-methylthio-2,5-dioxopyrrolidin-1-yl)propanamido)hexanamide (MP-VA), and (2S,3S,4S,5R,6S)-3,4,5-Trihydroxy-6-{4-[((methyl(2-(N-methylacetamido)ethyl)carbamoyl)oxy)methyl]-2-[18-(3-methylthio-2,5-dioxopyrrolidin-1-yl)-3,7,10,16-tetraoxo-2,6,9,15-tetraazaoctadecyl]phenoxy}tetrahydro-2H-pyran-2-carboxylic acid (MP-b-Glu-DMEDA). In the first paragraph, A linker-drug conjugate wherein the linker is selected from the following: ; ; ; ; ; ; and , In the above linker, the broken line represents a binding site, and n is a number from about 1 to about 1,000. In the first paragraph, The above linker-drug conjugate is selected from the following linker-drug conjugates: ; ; ; ; ; ; ; ; ; ; ; ; ; ; and . A linker-drug conjugate comprising a silatecan derivative represented by the following chemical formula 1 or 2 or a pharmaceutically acceptable salt thereof; and a linker. Includes, The above linker is bonded to A of the following chemical formula 1 or B of the following chemical formula 2. Pharmaceutical composition for the prevention or treatment of proliferative diseases: [Chemical Formula 1] ; [Chemical Formula 2] ; In the above chemical formula 1 and chemical formula 2, A is A 1 -(C 0-3 alkylene)-A 2 -(C 0-3 alkylene)-A 3 -A 4 - and, A 1 is -NH2, -OH, or -O-CH2-NH2, A 2 and A 3 are, independently of each other, absent, or -O-, -NH, -N(C 1-3 alkyl group)-, or -S-, A 4 is -C(=O)- or -SO2-; B is B 1 -(C 0-3 alkylene)-B 2 -B 3 -B 4 -B 5 -(C 0-3 alkylene)-, B 1 is -NH2, -OH, or -O-CH2-NH2, B 2 and B 3 are, independently of each other, absent, or -O-, -NH, -N(C 1-3 alkyl group)-, or -S-; B 4 does not exist, or is -C(=O)- or -SO2-; B 5 does not exist, or is -NH- or -O-, R a , R b , R c , R d , and R e are, each independently, C 1-10 Alkyl group, C 2-10 Alkenyl group, C 2-10 Alkynyl group, C 4-20 Aryl group, -(CH2) m R 11 Ki, or SiR 12 R 13 R 14 , where m is an integer in the range of 1 to 10, and R 11 Silver hydroxyl group, C 1-10 Alkoxy group, amino group, C 1-10 Alkylamino group, di-C 1-10 -alkylamino group, F, Cl, cyano group, or nitro group, where R 12 , R 13 and R 14 are, each independently, C 1-10 Alkyl group or C 4-10 It is an aryl group; X is H, F, Cl, nitro, amino, hydroxy, or cyano, Y is -NH-, Z is hydrogen, F, Cl, hydroxyl group, nitro group, cyano group, azido group, formyl group, hydrazino group, amino group, C 1-10 Alkyl group, C 2-10 Alkenyl group, C 2-10 Alkynyl group, C 1-10 Alkoxy group, C 1-10 Aryloxy group, C 1-10 Acyloxy group, -C(O)-C 1-10 Alkyl group, -C(O)-C 4-10 Aryl group, -OC(O)OC 1-10 Alkyl group, -OC(O)NR 15 R 16 and, here, R 15 and R 16 are, independently, H, C 1-10 Alkyl group or C 4-10 It is an aryl group, W is H, F, Cl, nitro, amino, hydroxy, or cyano, or Z and W are connected to each other to form C containing one or more heteroatoms. 1-5 It is a heterocyclic alkyl ring. In paragraph 10, A pharmaceutical composition wherein the above silatecan derivative or a pharmaceutically acceptable salt thereof acts as a prodrug. In paragraph 10, A pharmaceutical composition, wherein the proliferative disease comprises at least one selected from neoplasms, tumors, cancer, psoriasis, bone diseases, fibroproliferative disorders, and atherosclerosis. In paragraph 12, A pharmaceutical composition wherein the cancer is a solid cancer or a blood cancer. In paragraph 12, A pharmaceutical composition, wherein the cancer is at least one selected from gastric cancer, colon cancer, intestinal cancer, colorectal cancer, uterine cancer, uterine fibroids, meningioma, lung cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, and melanoma. In paragraph 10, The above linker-drug conjugate is selected from the following linker-drug conjugates: ; ; ; ; ; ; ; ; ; ; ; ; ; ; and . antibody; linker; and Silatecan derivative represented by the following chemical formula 1 or chemical formula 2 or a pharmaceutically acceptable salt thereof As an antibody-drug conjugate comprising: An antibody-drug conjugate wherein the linker binds to A of the following chemical formula 1 or B of the following chemical formula 2: [Chemical Formula 1] ; [Chemical Formula 2] ; In the above chemical formula 1 and chemical formula 2, A is A 1 -(C 0-3 alkylene)-A 2 -(C 0-3 alkylene)-A 3 -A 4 - and, A 1 is -NH2, -OH, or -O-CH2-NH2, A 2 and A 3 are, independently of each other, absent, or -O-, -NH, -N(C 1-3 alkyl group)-, or -S-, A 4 is -C(=O)- or -SO2-; B is B 1 -(C 0-3 alkylene)-B 2 -B 3 -B 4 -B 5 -(C 0-3 alkylene)-, B 1 is -NH2, -OH, or -O-CH2-NH2, B 2 and B 3 are, independently of each other, absent, or -O-, -NH, -N(C 1-3 alkyl group)-, or -S-; B 4 does not exist, or is -C(=O)- or -SO2-; B 5 does not exist, or is -NH- or -O-, R a , R b , R c , R d , and R e are, each independently, C 1-10 Alkyl group, C 2-10 Alkenyl group, C 2-10 Alkynyl group, C 4-20 Aryl group, -(CH2) m R 11 Ki, or SiR 12 R 13 R 14 , where m is an integer in the range of 1 to 10, and R 11 Silver hydroxyl group, C 1-10 Alkoxy group, amino group, C 1-10 Alkylamino group, di-C 1-10 -alkylamino group, F, Cl, cyano group, or nitro group, where R 12 , R 13 and R 14 are, each independently, C 1-10 Alkyl group or C 4-10 It is an aryl group; X is H, F, Cl, nitro, amino, hydroxy, or cyano, Y is -NH-, Z is hydrogen, F, Cl, hydroxyl group, nitro group, cyano group, azido group, formyl group, hydrazino group, amino group, C 1-10 Alkyl group, C 2-10 Alkenyl group, C 2-10 Alkynyl group, C 1-10 Alkoxy group, C 1-10 Aryloxy group, C 1-10 Acyloxy group, -C(O)-C 1-10 Alkyl group, -C(O)-C 4-10 Aryl group, -OC(O)OC 1-10 Alkyl group, -OC(O)NR 15 R 16 and, here, R 15 and R 16 are, independently, H, C 1-10 Alkyl group or C 4-10 It is an aryl group, W is H, F, Cl, nitro, amino, hydroxy, or cyano, or Z and W are connected to each other to form C containing one or more heteroatoms. 1-5 It is a heterocyclic alkyl ring. In paragraph 16, An antibody-drug conjugate, wherein the antibody-drug conjugate acts as a prodrug. In paragraph 16, An antibody-drug conjugate comprising an antibody, a modified antibody, or an antigen-binding fragment thereof capable of immunospecifically acting against a proliferative disease. In paragraph 16, The antibodies include alemtuzumab, apolizumab, aselizumab, atlizumab, bapineuzumab, bevacizumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cetuximab, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, and erlizumab. (erlizumab), ertumaxomab, felvizumab, fontolizumab, gemtuzumab, gemtuzumab ozogamicin, ibritumomab tiuxetan, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, nolovizumab, Numavizumab, ocrelizumab, omalizumab, palivizumab, panitumumab, pascolizumab, pecfusituzumab, pectuzumab, pertuzumab,pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resyvizumab, rituximab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, tositumomab, trastuzumab An antibody-drug conjugate selected from (trastuzumab), tucotuzumab celmoleukin, tucusituzumab, umavizumab, urtoxazumab, and visilizumab. In paragraph 16, The antibody-drug conjugate comprises an antibody-drug conjugate selected from the following linker-drug conjugates: ; ; ; ; ; ; ; ; ; ; ; ; ; ; and .

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