Conjugates comprising cleavable linkers

Abstract

The present specification relates to conjugates comprising a linker of formula (ICA): (ICA) and their pharmaceutically acceptable salts. The specification also relates to the use of these conjugates for the treatment of diseases such as cancer, as well as intermediates useful in the synthesis of these conjugates.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480026170.9 (22) Application Date 2024.04.09 (30) Priority Data 63 / 496,709 2023.04.18 US (85) PCT International Application Entering National Phase Date 2025.10.16 (86) PCT International Application Application Data PCT / EP2024 / 059544 2024.04.09 (87) PCT International Application Publication Data WO2024 / 217922 EN 2024.10.24 (71) Applicant: AstraZeneca (Sweden) Ltd. Address: Södertälje, Sweden (72) Inventors: A.H. Khan, A.L.S. Maderna, M.T.W. Lee, N.D. Bartolo, W.R.F. Gundre (74) Patent Agency: Beijing Tongli Juncheng Intellectual Property Agency Co., Ltd., 11205 Patent Attorney: Yu Na (51) Int.Cl. A61K 47 / 68 (2006.01) A61P 35 / 00 (2006.01) (54) Invention Title: Conjugates Containing a Pyrolytic Connector (57) Abstract: This specification relates to conjugates containing a connector of the formula (ICA): (ICA) and their pharmaceutically acceptable salts. This specification also relates to the use of these conjugates for the treatment of diseases such as cancer, and intermediates that can be used to synthesize these conjugates. Claims 6 pages, Description 55 pages, Drawings 5 ​​pages, CN 121175073 A 2025.12.19 CN 1 21 17 50 73 A 1. A conjugate of formula (IC) or a pharmaceutically acceptable salt thereof, wherein Ab is an antibody or an antigen-binding fragment thereof, k is an integer from 1 to 10, each GA is independently a conjugate group conjugated to said antibody or an antigen-binding fragment thereof, each DC is, each JA is independently a group of formula (ICA), E is (CH2)n1, wherein n1 is 0, 1, 2 or 3, Q is, R1 is a C1-4 alkyl group, X is (CH2)n2, wherein n2 is 0, 1, 2 or 3, Y is (CH2)n3, wherein n3 is 0, 1, 2, 3 or 4, Z is (CH2)n4, wherein n4 is 1, 2, 3, 4 or 5, m is an integer from 5 to 17, p is 1 or 0, (GA) indicates the attachment point to GA, and (DC) indicates the attachment point to DC. 2. The conjugate of formula (IC) according to claim 1, or a pharmaceutically acceptable salt thereof, wherein Q is the conjugate of claim 1 / 6, page 2, CN 121175073 A. 3. The conjugate of formula (IC) according to claim 1 or claim 2, or a pharmaceutically acceptable salt thereof, wherein m9, 10, 11, 12, or 13. 4. A conjugate of formula (IC) according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein R1 is CH3. 5. A conjugate of formula (IC) according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, wherein E is CH2. 6. A conjugate of formula (IC) according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, wherein X is CH2. 7. A conjugate of formula (IC) according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, wherein Y is (CH2)2. 8. A conjugate of formula (IC) according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, wherein Z is (CH2)2. 9. A conjugate of formula (IC) according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, wherein p is 1. 10. A conjugate of formula (IC) according to claim 1, or a pharmaceutically acceptable salt thereof, wherein each JA is a group (ICB) of formula (ICB). 11. A conjugate of formula (IC) according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof, wherein GA is selected from claims 2 / 6, page 3, CN 121175073 A, wherein RK is H or CH3, RL is C1-6 alkyl, and indicates an attachment site with said antibody or its antigen-binding fragment. 12. A conjugate of formula (IC) according to claim 11, or a pharmaceutically acceptable salt thereof, wherein GA is... 13. A conjugate of formula (IC) according to claim 11, or a pharmaceutically acceptable salt thereof, wherein GA is... 14. A conjugate of formula (IC) according to any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, wherein k is an integer from 2 to 8. 15. A pharmaceutical composition comprising a conjugate of formula (IC) according to any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. 16. A conjugate of formula (IC) according to any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof, for use in a therapeutic manner. 17. A conjugate of formula (IC) according to any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer. 18. A method of treating a patient with cancer, the method comprising administering to the patient a conjugate of formula (IC) according to any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof. 19. Use of a conjugate of formula (IC) according to any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating cancer. 20. A compound of formula (IIC) or a salt thereof, wherein GB is a conjugating group for conjugation with an antibody or an antigen-binding fragment thereof. (Claims 3 / 6)Page 4 CN 121175073 A JB is a group (IICA) of formula (IICA), wherein DC, E, Q, R1, X, Y, Z, m and p are as defined for the conjugate of formula (IC) in any one of claims 1 to 9, (GB) indicates the attachment point with GB, and (DC) indicates the attachment point with DC. 21. A compound of formula (IIC) or a salt thereof according to claim 20, wherein GB is selected from and , wherein X1 is CH or N, h is O or 1, Hal is Cl, Br or I, RK is H or CH3, and RL is C1-6 alkyl. 22. A compound of formula (IIC) or a salt thereof according to claim 21, wherein GB is . 23. A compound of formula (IIC) or a salt thereof according to claim 21, wherein GB is . Page 4 / 6 CN 121175073 A. 24. The compound of formula (IIC) according to claim 20, or a salt thereof, wherein the compound is (2S,3S,4S,5R,6S)-6-(2-((3-((S)-6-(((3S,3aR,6S,6aR)-6-(2,5,8,11,14,17,20, 23,26,29,32,35-dodecoxaoctane-38-amido)hexahydrofurano[3,2-b]furan-3-yl)amino)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)propamido)-6-oxohexamido)propamido)methyl)- 4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid or a salt thereof. 25. The compound of formula (IIC) according to claim 20, or a salt thereof, wherein the compound is (2S,3S,4S,5R,6S)-6-(2-((S)-8-(4-(((3S,3aR,6S,6aR)-6-(2,5,8,11,14,17,20,23,26,29,32,35-dodecanooctane-38-amido)hexahydrofurano[3,2-b]furan-3-yl)amino)-4- (O-butyl)-15-bromo-3,7,10,14-tetraoxo-2,6,9,13-tetraazapentadecanyl)-4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]Indolezido[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid or a salt thereof. 26. A compound of formula (IXC) as claimed on pages 5 / 6 of CN 121175073 A (IXC) or a salt thereof, wherein GB is a conjugating group for conjugation with an antibody or an antigen-binding fragment thereof, and E, Y, Z and p are as defined for a conjugate of formula (IC) in any one of claims 1, 5, 7, 8 or 9, RQ1 is H or RP1, each RQ2 is independently H or RP2, and RQ3 is H or RP3, wherein RP1 is a carboxylic acid protecting group, each RP2 is independently an alcohol protecting group and RP3 is an amine protecting group. Claims 6 / 6 Page 7 CN 121175073 A Conjugates Containing a Cleavable Linker

[0001] Cross-Reference to Related Patent Applications

[0002] This specification claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 496,709 (filed April 18, 2023). The entire text of the above patent application is incorporated herein by reference.

[0003] This specification relates to certain conjugates containing a cleavable linker and a topoisomerase I inhibitor (TOPO1i), and to pharmaceutical compositions containing them. This specification also relates to the use of the conjugates in methods of treating diseases such as cancer. This specification further relates to methods and intermediate compounds involved in the preparation of the conjugates. Background Art

[0004] Antibody-drug conjugates (ADCs) are a well-established method for delivering drugs to biological targets. Enzymatically cleavable linkers can be used to release free drugs at the target, such that the free drugs can have therapeutic effects. Transcriptomic and proteomic profiling analyses of various solid tumor cell lines have identified β-glucuronidase expression as upregulated and typically localized to lysosomes. WO2007011968 and WO2015182984 disclose certain antibody-drug conjugates containing β-glucuronidase-cleavable linkers.

[0005] Topoisomerase inhibitors are compounds that block the action of topoisomerases (topoisomerase I and II), enzymes that control changes in DNA structure by catalyzing the breaking and rejoining of the phosphodiester backbone of the DNA strand during the normal cell cycle. An example of a topoisomerase I inhibitor is essanotecan.

[0006] There remains a need for conjugates having β-glucuronidase-cleavable linkers that can selectively deliver drugs to biological targets and possess favorable physicochemical properties, including solubility and lipophilicity. The conjugates disclosed herein could be used to treat diseases such as cancer.

[0007] In a first aspect, a conjugate of formula (IC) is provided.

[0008]

[0009] or a pharmaceutically acceptable salt thereof, wherein

[0010] Ab is an antibody or an antigen-binding fragment thereof,

[0011] k is an integer from 1 to 10,

[0012] each GA is independently a conjugated group conjugated to the antibody or an antigen-binding fragment thereof,

[0013] each DC is

[0014] ,

[0015] each JA is independently a group of formula (ICA)

[0016]

[0017] (ICA),

[0018] E is (CH2)n1, wherein n1 is 0, 1, 2 or 3,

[0019] Q is or,

[0020] wherein ring F1 is a saturated bicycle having 6, 7 or 8 carbon atoms and optionally 1 or 2 oxygen atoms, ring F2 is a saturated bicycle having 2 A saturated bicyclic ring having one nitrogen atom, 4, 5, 6, 7 or 8 carbon atoms and optionally 1 oxygen atom, and ring F3 is a saturated bicyclic ring having one nitrogen atom, 5, 6, 7 or 8 carbon atoms and optionally 1 oxygen atom,

[0021] R1 is a C1-4 alkyl group,

[0022] X is (CH2)n2, where n2 is 0, 1, 2 or 3,

[0023] Y is (CH2)n3, where n3 is 0, 1, 2, 3 or 4,

[0024] Z is (CH2)n4, where n4 is 1, 2, 3, 4 or 5,

[0025] m is an integer from 5 to 17,

[0026] p is 1 or 0,

[0027] (GA) indicates the attachment point to GA, and

[0028] (DC) indicates the attachment point to DC.

[0029] In another aspect, a pharmaceutical composition is provided comprising a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

[0030] In another aspect, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided for use in a therapy.

[0031] In another aspect, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided for use in the treatment of cancer.

[0032] In another aspect, use of a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided in the manufacture of a medicament.

[0033] In another aspect, use of a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided in the manufacture of a medicament for treating cancer.

[0034] In another aspect, a method of treating a patient with cancer is provided, the method comprising administering to the patient an effective amount of a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof.

[0035] In another aspect, a compound of formula (IIC) is provided.

[0036] Specification 2 / 55 page 9 CN 121175073 A

[0037] Or a salt thereof, wherein GB is a conjugating group for conjugation with an antibody or an antigen-binding fragment thereof,

[0038] JB is a group of formula (IICA)

[0039]

[0040] (IICA),

[0041] wherein DC, E, Q, R1, X, Y, Z, m and p are as defined above for conjugates of formula (IC), (GB) indicates an attachment site with GB, and (DC) indicates an attachment site with DC.

[0042] In another aspect, intermediates for the synthesis of compounds of formula (IIC) or salts thereof are provided.

[0043] Conjugates of formula (IC) or pharmaceutically acceptable salts thereof are subject to enzymatic cleavage to release free drug (exatecan). Conjugates of formula (IC) may exhibit improved efficacy and / or favorable physical properties (e.g., higher stability, lower lipophilicity, higher water solubility, higher permeability and / or lower plasma protein binding) and / or favorable toxicological characteristics (e.g., reduced off-target toxicity) and / or favorable metabolic or pharmacokinetic characteristics compared to other conjugates. In embodiments, conjugates of formula (I), (IC), (IM), or (IT) exhibit improved colloidal stability compared to other conjugates. Therefore, conjugates of formula (IC) may be particularly suitable for use in therapies such as cancer treatment.

[0044] Definitions

[0045] Certain terms are explicitly defined below to facilitate a better understanding of this specification. Furthermore, definitions are appropriately set forth throughout the specific embodiments. Where examples are provided for the definitions, they are not limiting.

[0046] Prefix Cx-y, where x and y are integers, indicates a numerical range of carbon atoms present in the group.

[0047] As used herein, the term "alkyl" refers to a saturated straight-chain or branched hydrocarbon group having a specified number of carbon atoms. Examples of C1-4 alkyl groups include methyl (Me), ethyl (Et), n-propyl (nPr), isopropyl (iPr), n-butyl (nBu), isobutyl (iBu), sec-butyl (sBu), and tert-butyl (tBu). Examples of C1-6 alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, and n-hexyl.

[0048] As used herein, the term "bicyclic" refers to a fused, bridged, or spirocyclic bicyclic ring.

[0049] As used herein, the term "conjugating group for conjugation with an antibody or its antigen-binding fragment" refers to an atom or atomic group capable of forming a covalent bond with an antibody or its antigen-binding fragment by a chemical reaction.

[0050] The use of " " in the formulas of this specification indicates the attachment point with the antibody or its antigen-binding fragment. For illustration, " " indicates the presence of a covalent bond connecting the antibody or its antigen-binding fragment to a carbon atom labeled 1.

[0051] To avoid any doubt, the use of “ " in the formulas of this specification indicates a covalent attachment point with a group, whereinThis group is not an antibody or its antigen-binding fragment. Specification 3 / 55 pages 10 CN 121175073 A

[0052] Some embodiments of this specification include a group referred to as “optionally substituted”. In other embodiments, the group is unsubstituted.

[0053] As used herein, is ring F1, is ring F2, and is ring F3.

[0054] Units, prefixes, and symbols are expressed in their internationally recognized (SI) form. Numerical ranges include numerical values ​​that define the range.

[0055] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure relates. For example, *Concise Dictionary of Biomedicine and Molecular Biology*, Juo, Pei-Show, 2nd edition, 2002, CRC Press; *The Dictionary of Cell and Molecular Biology*, 3rd edition, 1999, Academic Press; and *Oxford Dictionary of Biochemistry and Molecular Biology*, revised edition, 2000, Oxford University Press provide a general dictionary for those skilled in the art of the use of many terms used in this disclosure. Brief Description of the Drawings

[0056] Embodiments and experiments illustrating the principles of this disclosure will now be discussed with reference to the accompanying drawings, in which:

[0057] Figure 1A shows cytotoxicity data of ADC-1 and control ADC-1 in the Her2+++ NCI-N87 cell line.

[0058] Figure 1B shows cytotoxicity data of ADC-1 and control ADC-1 in the HER2++ JIMT1 cell line.

[0059] Figure 2A shows cytotoxicity data of ADC-1 and control ADC-1 in the HER2- MDAMB468 cell line.

[0060] Figure 2B shows the cytotoxicity data of ADC-1 and control ADC-1 in the Her2+++ SKOV3 cell line.

[0061] Figure 3 shows the cytotoxicity data of ADC-1 and control ADC-1 in the SKOV3 GUSB KO cell line.

[0062] Figure 4A shows the cytotoxicity data of ADC-3, ADC-2, and ADC-1 in the Her2+++ NCI-N87 cell line.

[0063] Figure 4B shows the cytotoxicity data of ADC-3, ADC-2, and ADC-1 in the HER2+++ / GUSB+++ SKOV3 WT cell line.

[0064] Figure 4C shows cytotoxicity data for ADC-3, ADC-2, and ADC-1 in the HER2+++ / GUSB-SKOV3 GUSB KO cell line.

[0065] Figure 4D shows cytotoxicity data for ADC-3, ADC-2, and ADC-1 in the HER2-MDAMB468 cell line.

[0066] Figure 5 shows colloidal stability data for ADC-3, ADC-2, and ADC-1. Detailed Description

[0067] In one aspect, this specification provides conjugates of formula (IC) as defined above, or pharmaceutically acceptable salts thereof.

[0068] In an embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, wherein k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, k is an integer from 2 to 10. In another embodiment, k is an integer from 2 to 8. In another embodiment, k is 4. In another embodiment, k is 8.

[0069] In an embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, wherein GA is selected from CN 121175073 A, page 4 / 55 of the specification

[0070]

[0071] and,

[0072] wherein RK is H or CH3, RL is C1-6 alkyl, and indicates an attachment site with an antibody or an antigen-binding fragment thereof.

[0073] In an embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, wherein GA is selected from

[0074]

[0075] and.

[0076] In an embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, wherein GA is

[0077] .

[0078] In an embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, wherein GA is

[0079] .

[0080] In an embodiment, a conjugate of formula (I), (IC), (IM), or (IT) or a pharmaceutically acceptable salt thereof is provided, wherein GA is

[0081] . Specification 5 / 55 pages 12 CN 121175073 A

[0082] In an embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, wherein Q is, and wherein ring F1 is a saturated bicyclic having 6, 7, or 8 carbon atoms and optionally 1 or 2 oxygen atoms. In another embodiment, the bicyclic is a fused bicyclic.

[0083] In an embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, wherein Q is, and ring F1 is a saturated bicyclic having 6, 7, or 8 carbon atoms and 1 or 2 oxygen atoms. In another embodiment, the bicyclic is a fused bicyclic.

[0084] In an embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, wherein Q is.

[0085] In an embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, wherein Q is.

[0086] In an embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, wherein Q is, and wherein ring F2 is a saturated bicyclic having 2 nitrogen atoms, 4, 5, 6, 7 or 8 carbon atoms and optionally 1 oxygen atom. In another embodiment, the bicyclic is a spirocyclic bicyclic. In another embodiment, the bicyclic is a bridging bicyclic.

[0087] In an embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, wherein Q is, and wherein ring F2 is a saturated bicyclic having 2 nitrogen atoms and 4, 5, 6, 7 or 8 carbon atoms. In another embodiment, the bicyclic is a spirocyclic bicyclic. In another embodiment, the bicyclic is a bridging bicyclic.

[0088] In an embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, wherein Q is.

[0089] In an embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, wherein Q is.

[0090] In an embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, wherein Q is specified in specification 6 / 55 page 13 CN 121175073 A.

[0091] In an embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, wherein Q is.

[0092] In an embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, wherein Q is, wherein ring F3 is a saturated bicycle having 1 nitrogen atom, 5, 6, 7 or 8 carbon atoms and optionally 1 oxygen atom. In another embodiment, the bicycle is a spirobicycle. In another embodiment, the bicycle is a bridging bicycle.

[0093] In an embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, wherein Q is, wherein ring F3 is a saturated bicycle having 1 nitrogen atom and 5, 6, 7 or 8 carbon atoms. In another embodiment, the bicycle is a spirocyclic bicycle. In another embodiment, the bicycle is a bridged bicycle.

[0094] In an embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, wherein E is (CH2)n1, wherein n1 is 0, 1, 2 or 3. In another embodiment, E is a covalent bond. In another embodiment, E is CH2. In another embodiment, E is (CH2)2. In another embodiment, E is (CH2)3.

[0095] In an embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, wherein X is(CH2)n2, wherein n2 is 0, 1, 2, or 3. In another embodiment, X is a covalent bond. In another embodiment, X is CH2. In another embodiment, X is (CH2)2. In another embodiment, X is (CH2)3.

[0096] In an embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, wherein Y is (CH2)n3, wherein n3 is 0, 1, 2, 3, or 4. In another embodiment, Y is a covalent bond. In another embodiment, Y is CH2. In another embodiment, Y is (CH2)2. In another embodiment, Y is (CH2)3. In another embodiment, Y is (CH2)4.

[0097] In an embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, wherein Z is (CH2)n4, wherein n4 is 1, 2, 3, 4, or 5. In another embodiment, Z is CH2. In another embodiment, Z is (CH2)2. In another embodiment, Z is (CH2)3. In another embodiment, Z is (CH2)4. In another embodiment, Z is (CH2)5.

[0098] In an embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, wherein m is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17. In another embodiment, m is an integer from 6 to 16. In another embodiment, m is an integer from 7 to 15. In another embodiment, m is an integer from 8 to 14. In another embodiment, m is an integer from 9 to 13. In another embodiment, m is an integer from 10 to 12. In another embodiment, m is 11.

[0099] In an embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, wherein R1 is a C1-4 alkyl group. In another embodiment, R1 is CH3.

[0100] In an embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, wherein p is 1.

[0101] In an embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, wherein each JA is a group of formula (ICB)

[0102]

[0103] (ICB).

[0104] In an embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, wherein each JA is a group of formula (ICB')

[0105]

[0106] (ICB').

[0107] In an embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, wherein each JA is a group of formula (ICB2)

[0108]

[0109] (ICB2).

[0110] In another aspect, a compound of formula (IIC) is provided

[0111]

[0112] or a salt thereof, wherein GB is a conjugating group for conjugation with an antibody or an antigen-binding fragment thereof,

[0113] JB is a group of formula (IICA)

[0114]

[0115] (IICA),

[0116] wherein DC, E, Q, R1, X, Y, Z, m and p are defined in any embodiment of the conjugate of formula (IC) disclosed herein, (GB) indicates an attachment point with GB, and (DC) indicates an attachment point with DC.

[0117] In an embodiment, a compound of formula (IIC) or a salt thereof is provided, wherein GB is selected from

[0118]

[0119] and,

[0120] wherein X1 is CH or N, h is O or 1, Hal is Cl, Br or I, RK is H or CH3, and RL is C1-6 alkyl.

[0121] In an embodiment, a compound of formula (IIC) or a salt thereof is provided, wherein GB is selected from

[0122]

[0123] and.

[0124] In an embodiment, a compound of formula (IIC) or a salt thereof is provided, wherein GB is

[0125] .

[0126] In an embodiment, a compound of formula (IIC) or a salt thereof is provided, wherein GB is

[0127] .

[0128] In an embodiment, a compound of formula (II), (IIC), (IIM) or (IIT) or a salt thereof is provided, wherein GB is.

[0129] In an embodiment, a compound of formula (IIC) or a salt thereof is provided, wherein JB is a group of formula (IICB)

[0130]

[0131] (IICB).

[0132] In an embodiment, a compound of formula (II) or a salt thereof is provided, wherein JB is a group of formula (IICB2)

[0133]

[0134] (IICB2).

[0135] In an embodiment, a compound of formula (IIC) is provided, the compound being selected from; Specification 10 / 55 pages 17 CN 121175073 A

[0136]

[0137] (2S,3S,4S,5R,6S)-6-(2-((3-((S)-6-(((3S,3aR,6S,6aR)-6-(2,5,8,11,14,17, 20,23,26,29,32,35-dodecoxaoctane-38-amido)hexahydrofurano[3,2-b]furan-3-yl)amino(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)propamido)-6-oxohexanoamido)propamido)methyl)-4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indoleazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid, and

[0138]

[0139] (2S,3S,4S,5R,6S)-6-(2-((3-((S)-6-((1S,4S)-5-(2,5,8,11,14,17,20,23,26, 29,32,35-dodecoxaoctane-38-acyl)-2,5-diazabicyclo[2.2.1]hept-2-yl)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)propamido)-6-oxohexamido)propamido)methyl)-4-(((((1S, 9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H) ,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid,

[0140] or a salt thereof. Specification 11 / 55 pages 18 CN 121175073 A

[0141] In an embodiment, a compound of formula (IIC) is provided, namely

[0142]

[0143] (2S,3S,4S,5R,6S)-6-(2-((S)-8-(4-(((3S,3aR,6S,6aR)-6-(2,5,8,11,14,17, 20,23,26,29,32,35-dodecoxaoctane-38-amido)hexahydrofurano[3,2-b]furan-3-yl)amino)-4-oxobutyl)-15-bromo-3,7,10,14-tetraoxo-2,6,9,13-tetraazapentadecanyl)-4-((((1S, 9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid or a salt thereof.

[0144] In another aspect, a compound of formula (IXC)

[0145]

[0146] (IXC)

[0147] or a salt thereof is provided, wherein GB is a conjugating group for conjugating an antibody or an antigen-binding fragment thereof, and E, Y, Z and p are defined in any embodiment of the conjugate of formula (IC), RQ1 is H or RP1, each RQ2 is independently H or RP2, and RQ3 is H or RP3, wherein RP1 is a carboxylic acid protecting group, each RP2 is independently an alcohol protecting group and RP3 is an amine protecting group. Specification 12 / 55 pages 19 CN 121175073 A

[0148] In an embodiment, a compound of formula (IXC) or a salt thereof is provided, wherein RQ1 is H, each RQ2 is H, and RQ3 is H. In another embodiment, GB is or.

[0149] In an embodiment, a compound of formula (IXC) or a salt thereof is provided, wherein RQ1 is H, each RQ2 is H, and RQ3 is RP3. In another embodiment, RQ1 is H, each RQ2 is H, and RQ3 is tert-butoxycarbonyl (Boc). In another embodiment, GB is or.

[0150] In an embodiment, a compound of formula (IXC) or a salt thereof is provided, wherein RP1 is selected from benzyl, allyl, and C1-4 alkyl. In another embodiment, RP1 is allyl. In another embodiment, RP1 is methyl.

[0151] In an embodiment, a compound of formula (IXC) or a salt thereof is provided, wherein each RP2 is independently selected from benzyl, C(O)OCH2CH=CH2, and acetyl. In another embodiment, each RP2 is acetyl.

[0152] In one embodiment, a compound of formula (IXC) or a salt thereof is provided, wherein RP3 is selected from trifluoroacetyl, tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), and fluorenylmethoxycarbonyl (Fmoc). In another embodiment, RP3 is fluorenylmethoxycarbonyl.

[0153] In an embodiment, a compound of formula (IXC) is provided, namely (2S,3S,4S,5R,6S)-6-(2-((3-((S)-6-(((3S,3aR,6S,6aR)-6-((tert-butoxycarbonyl)amino)hexahydrofurano[3,2-b]furan-3-yl)amino)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)propamido)-6-oxohexamido)propamido)methyl)-4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15- ...3,9,10,13,15-yl)amino)-2-(3,2-b]furano[3,2-b]furano[3,2-b]furano[3,2-b]furano[3,2-b]furano[3,2-b]furano[3,2-b]furano[3,2-b]furano[3,2-b]furano[3,2-b]furano[3,Hydrogen-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid or a salt thereof.

[0154] In an embodiment, a compound of formula (IXC) is provided, namely (2S,3S,4S,5R,6S)-6-[2-[[3-[[(2S)-6-[[(3S,3aR,6S,6aR)-3-amino-2,3,3a,5,6,6a-hexahydrofurano[3,2-b]furan-6-yl]amino]-2-[3-(2 ,5-dioxopyrrolo-1-yl)propionylamino]-6-oxo-hexano]amino]propionylamino]methyl]-4-[[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexane[14.7.1.02,14.04,13.06,11.020,24]tetraco-1,6(11),12,14,16(24),17,19-hepten-23-yl]carbamoyloxymethyl]phenoxy]-3,4,5-trihydroxy-tetrahydropyran-2-carboxylic acid or a salt thereof.

[0155] This specification is intended to include all isotopes of atoms present in the compounds and conjugates of the present invention. Isotopes will be understood to include those atoms having the same atomic number but different mass numbers. For example, hydrogen isotopes include tritium and deuterium. Carbon isotopes include 13C and 14C. Nitrogen isotopes include 15N.

[0156] The compounds disclosed herein may contain one or more chiral centers. Thus, if desired, such compounds can be prepared or separated as pure stereoisomers, i.e., as individual enantiomers, diastereomers, or as mixtures enriched with stereoisomers. Unless otherwise stated, all such stereoisomers (and enriched mixtures) are included within the scope of the embodiments. Pure stereoisomers (or enriched mixtures) can be prepared using, for example, optically active starting materials or stereoselective reagents well known in the art. Alternatively, racemic mixtures of such compounds can be separated using, for example, chiral column chromatography, chiral resolving agents, etc.

[0157] Unless the stereochemistry is explicitly indicated in the chemical structure or chemical name, the chemical structure or chemical name is intended to cover all possible stereoisomers, diastereomers, conformational isomers, rotational isomers, and tautomers of the described compound. For example, compounds containing a chiral carbon atom are intended to cover both (R) enantiomers and (S) enantiomers, as well as mixtures of enantiomers, including racemic mixtures; and compounds containing two chiral carbons are intended to cover all enantiomers and diastereomers.The conjugates include (R,R), (S,S), (R,S), and (S,R).

[0158] Suitable pharmaceutically acceptable salts of the conjugates of formula (IC) are, for example, acid addition salts. Acid addition salts of the conjugates of formula (IC) can be formed by contacting the compound with a suitable inorganic or organic acid under conditions known to a person skilled in the art. Acid addition salts can be formed, for example, using inorganic acids selected from hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid. Acid addition salts can also be formed using organic acids selected from trifluoroacetic acid, citric acid, maleic acid, oxalic acid, acetic acid, formic acid, benzoic acid, fumaric acid, succinic acid, tartaric acid, lactic acid, pyruvic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.

[0159] Suitable pharmaceutically acceptable salts of the conjugates of formula (IC) are, for example, base addition salts. Base addition salts of the conjugates of formula (IC) can be formed by contacting the compound with a suitable inorganic or organic base under conditions known to a person skilled in the art. Alkali addition salts can be, for example, alkali metal salts (such as sodium, potassium, or lithium salts) or alkaline earth metal salts (such as calcium salts), which can be formed using alkali metal or alkaline earth metal hydroxides or alkoxides (e.g., ethanol or methanol salts). Alkali addition salts can also be formed using suitable basic organic amines (e.g., choline or meglumine salts).

[0160] Another suitable pharmaceutically acceptable salt of the conjugate of formula (IC) is, for example, a salt formed in the body of a patient after administration of the conjugate of formula (IC).

[0161] In another aspect, a pharmaceutical composition is provided comprising a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0162] The term "pharmaceutical composition" refers to an formulation in a form that allows for the biological activity of the active ingredient and is free of additional components that would have unacceptable toxicity to a patient to whom the composition will be administered. Such compositions can be sterile. The pharmaceutical compositions according to this specification comprise a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0163] In an embodiment, a pharmaceutical composition is provided comprising a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, carrier, buffer, or stabilizer. Such substances shall be nontoxic and shall not interfere with the efficacy of the active ingredient. The specific nature of the carrier or other material will depend on the route of administration, which may be oral or by injection, such as intradermal, subcutaneous, or intravenous injection.

[0164] In an embodiment, a pharmaceutical composition is provided comprising a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable nontoxic sterile carrier. In another embodiment, the carrier is physiological saline, a nontoxic buffer, or a preservative. Formulations suitable for the treatment methods disclosed herein are available at Remington's Pharmaceutical.The contents of this paper are described in Sciences, 22nd edition, edited by Lloyd V. Allen, Jr. (2012), the contents of which are incorporated herein by reference.

[0165] Examples of suitable excipients include one or more of water, saline, phosphate-buffered saline, dextran, glycerol, and ethanol, and any combination thereof. In an embodiment, a pharmaceutical composition is provided comprising a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof, and one or more isotonic agents. In another embodiment, one or more isotonic agents are selected from sugars, polyols, and sodium chloride.

[0166] In an embodiment, a pharmaceutical composition is provided comprising a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof, which is contained in one or more formulations selected from capsules, tablets, aqueous suspensions, solutions, nasal aerosols, and lyophilized powders that can be rehydrated prior to use to prepare suspensions or solutions.

[0167] In one embodiment, a pharmaceutical composition is provided comprising a conjugate of formula (IC) or a pharmaceutically acceptable salt, buffer, surfactant, and / or stabilizer as described in page 14 / 55 of CN 121175073 A. In another embodiment, the buffer is an acetate, phosphate, or citrate buffer. In another embodiment, the surfactant is polysorbate. In another embodiment, the stabilizer is human albumin.

[0168] Pharmaceutical compositions comprising a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof may be administered to a patient via any suitable systemic or local route of administration. For example, administration may be oral, oral, sublingual, ocular, intranasal, intratracheal, pulmonary, local, percutaneous, genitourinary, rectal, subcutaneous, intravenous, intraarterial, intraperitoneal, intramuscular, intracranial, intrathecal, epidural, intraventricular, or intratumoral.

[0169] Pharmaceutical compositions of the subtotal (IC) conjugate or a pharmaceutically acceptable salt thereof may be formulated for administration by any suitable means, such as via an epidermal or transdermal patch, ointment, lotion, cream, or gel; via a nebulizer, vaporizer, or inhaler; via injection or infusion; or in the form of capsules, tablets, liquid solutions or suspensions in aqueous or non-aqueous media, drops, suppositories, enemas, sprays, or powders. The most suitable route of administration in any given case will depend on the physical and mental condition of the subject, the nature and severity of the disease, and the desired characteristics of the formulation.

[0170] Pharmaceutical compositions of the subtotal (IC) conjugate or a pharmaceutically acceptable salt thereof for oral administration may be in tablet, capsule, powder, or liquid form. Tablets may contain a solid carrier or adjuvant. Liquid pharmaceutical compositions typically contain a liquid carrier, such as water, petroleum, animal or vegetable oil, mineral oil, or synthetic oil. Physiological saline solutions, glucose solutions, etc., may be included.Sugar or other sugar solutions or glycols, such as ethylene glycol, propylene glycol, or polyethylene glycol. The capsule may contain a solid carrier, such as gelatin.

[0171] For intravenous, skin, or subcutaneous injection, or injection at the site of infection, the conjugate of formula (IC) or a pharmaceutically acceptable salt thereof will be in the form of a pyrogen-free, parenteral-acceptable aqueous solution having suitable pH, isotonicity, and stability. Those skilled in the art are fully capable of preparing suitable solutions using, for example, isotonic mediators (such as sodium chloride injection, Ringer's solution, lactated Ringer's solution). Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed.

[0172] In one aspect, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided for use in a therapeutic context.

[0173] In one aspect, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided for use in the treatment of cancer.

[0174] When referring to “cancer,” cancer includes both non-metastatic and metastatic cancer, such that treating cancer involves treating both the primary tumor and the metastatic tumor.

[0175] The term “therapy” is intended to have its normal meaning of treating a disease to completely or partially alleviate one, some, or all of its symptoms, or to correct or compensate for an underlying pathology. The term “therapy” also includes “prevention,” unless specifically indicated otherwise. The terms “treatment” and “in treatment” should be interpreted in a corresponding manner.

[0176] The term “prevention” is intended to have its normal meaning and includes primary prevention of disease progression and secondary prevention where the disease has progressed and temporarily or permanently protects the patient against the aggravation or worsening of the disease or the development of new symptoms associated with the disease.

[0177] The term “treatment” is used synonymously with “therapeutic treatment.” Similarly, the term “treatment” can be considered as “applied therapy,” where “therapeutic treatment” is as defined herein.

[0178] In an embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided for use in the treatment of HER2-positive cancer.

[0179] In one aspect, use is provided of a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof as described herein in a pharmaceutical product, such as a medicament for treating cancer.

[0180] In one aspect, a method of treating a patient with cancer is provided, the method comprising administering to the patient an effective amount of a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof.

[0181] The term “treatment” as used herein refers to (1) a therapeutic measure that cures, slows, alleviates, reduces the symptoms of a diagnosed pathological condition or symptom and / or stops its development, and (2) a preventive measure that prevents and / or slows the development of a targeted pathological condition or symptom.(prophylactic / preventative) measures. Therefore, those who need treatment include those who already have the condition; those who are susceptible to the condition; and those who need to prevent the condition. In some respects, the patient’s cancer is successfully “treated” according to the method of this disclosure if the patient shows, for example, complete, partial or transient remission of a certain type of cancer.

[0182] The term “effective amount” means an amount of active ingredient sufficient to significantly and positively alter the symptoms and / or condition to be treated (e.g., provide a positive clinical response). The effective amount of active ingredient used in a pharmaceutical composition will vary depending on the specific condition being treated, the severity of the condition, the duration of treatment, the nature of concurrent therapies, the specific active ingredient used, the specific pharmaceutically acceptable excipient / carrier utilized, and similar factors within the knowledge and expertise of the attending physician.

[0183] The term “patient” means any animal (e.g., mammal), including but not limited to humans, non-human primates, rodents, etc., who will be the recipient of the specific treatment. In embodiments, the term “patient” means a human subject.

[0184] In an embodiment, a method of treating a patient with cancer is provided, the method comprising administering to the patient an effective amount of a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof, wherein the cancer is HER2-positive cancer.

[0185] In an embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, along with additional antitumor substances for combination therapy of cancer.

[0186] In an embodiment, a combination for use in the treatment of cancer is provided, the combination comprising a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof, along with additional antitumor agents.

[0187] In an embodiment, a combination of a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof and additional antitumor agents is provided.

[0188] In this document, when the term “combination therapy” is used to refer to combination therapy, it should be understood that this can mean simultaneous, separate, or sequential administration. In one aspect, “combination therapy” means simultaneous administration. In another aspect, “combination therapy” means separate administration. In yet another aspect, “combination therapy” means sequential administration.

[0189] In an embodiment, a method of treating a patient with cancer is provided, the method comprising administering to the patient an effective amount of a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof, and administering to the patient simultaneously, separately or sequentially at least one additional antitumor substance, wherein the amounts of the conjugate of formula (IC) or a pharmaceutically acceptable salt thereof and the additional antitumor substance are jointly effective in producing an anticancer effect.

[0190] Conjugate

[0191] Examples of GA and GB include, but are not limited to, the following groups, wherein X1 is CH or N, h is O or 1, RK is H or CH3, Hal isCl, Br, or I, RL is a C1-6 alkyl group and indicates the attachment site to the antibody or its antigen-binding fragment. Specification 16 / 55 page 23 CN 121175073 A

[0192] Specification 17 / 55 page 24 CN 121175073 A

[0193]

[0194] Antibody or its antigen-binding fragment

[0195] As used herein, the term “antibody” refers to an immunoglobulin molecule that specifically binds to or reacts immunologically with a particular antigen.

[0196] In embodiments, the antibody is isolated or recombinant. “Isolated” as used herein means a polypeptide, such as an antibody, that has been identified and isolated and / or recovered from the cell or cell culture in which it is expressed. Isolated antibodies are typically prepared by at least one purification step. Thus, “isolated antibody” refers to an antibody that is substantially free of other antibodies with different antigen specificities.

[0197] In embodiments, the antibody comprises at least two “light chains” (LC) and two “heavy chains” (HC). The light and heavy chains of this type of antibody are polypeptides composed of several domains. Each heavy chain contains a heavy chain variable region (hereinafter abbreviated as "VH") and a heavy chain constant region (hereinafter abbreviated as "CH"). The heavy chain constant region contains heavy chain constant domains CH1, CH2, and CH3 (antibody classes IgA, IgD, and IgG), and optionally a heavy chain constant domain CH4 (antibody classes IgE and IgM). Each light chain contains a light chain variable domain (hereinafter abbreviated as "VL") and a light chain constant domain (hereinafter abbreviated as "CL").

[0198] In embodiments, the antibody is a full-length antibody. As used herein, a "full-length" or "complete" antibody means an antibody having two heavy (H) chain polypeptides and two light (L) chain polypeptides linked by disulfide bonds.

[0199] The "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. The variable regions VH and VL can be further subdivided into hypervariable regions (called complementarity-determining regions (CDRs)) (also called hypervariable regions), interspersed with more conserved regions (called frame regions (FRs)). In embodiments, each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The VH or VL chain of the antibody may also include all or part of the heavy chain or light chain constant regions.

[0200] Binding between the antibody and its target antigen or epitope is mediated by CDRs. The term "epitaxy" refers to a target protein region (e.g., a polypeptide) capable of binding (e.g., being bound by) the antibody or antigen-binding fragment of this disclosure. CDRs are antigen-specificKey determinants. At least two techniques are used to determine CDRs: (1) methods based on cross-species sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest (5th edition, 1991, National Institutes of Health, Bethesda Md.)); and (2) methods based on the crystallographic study of antigen-antibody complexes (Al-lazikani et al., (1997) J. Molec. Biol. 273: 927-948). In addition, these two methods are sometimes used in combination in the art to determine CDRs.

[0201] The “constant structural domains” (or “constant regions”) of the heavy and light chains do not directly participate in the binding of antibodies to targets, but exhibit a variety of effector functions. The constant regions of antibodies can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0202] The heavy chain constant region is mainly divided into five classes: IgA, IgG, IgD, IgE, and IgM, each with a characteristic effector function determined by the isotype. Ig molecules interact with multiple types of cell receptors. For example, IgG molecules interact with three classes of Fcγ receptors (FcγR) specifically targeting IgG antibodies (i.e., FcγRI, FcγRII, and FcγRIIII). The binding of antibodies to Fc receptors on the cell surface triggers many important and diverse biological responses, including engulfing and destroying antibody-coated particles, clearing immune complexes, killing cells and lysing antibody-coated target cells (known as antibody-dependent cell-mediated cytotoxicity, or ADCC), releasing inflammatory mediators, placental transfer, and controlling the production of immunoglobulins. It has been reported that the important sequences for IgG binding to FcγR receptors are located in the CH2 and CH3 domains.

[0203] In embodiments, the antibody or its antigen-binding fragment is an IgG isotype. The antibody or its antigen-binding fragment can be any IgG subclass, such as IgG1, IgG2, IgG3, or IgG4 isotypes. In embodiments, the antibody or its antigen-binding fragment is based on the IgG1 isotype.

[0204] The terms “Fc region,” “Fc moiety,” and “Fc” are used interchangeably herein and refer to a portion of a natural immunoglobulin formed by two Fc chains. Each “Fc chain” contains a constant structural domain CH2 and a constant structural domain CH3. Each Fc chain may also contain a hinge region. The natural Fc region is a homodimer. In embodiments, the Fc region can be a heterodimer, as it may contain modifications that enhance Fc heterodimerization. The Fc region contains a carbohydrate moiety as well as complement and an Fc receptor (packed with...).The Fc region contains a binding site for the FcRn receptor and has no antigen-binding activity. Fc can refer to the region in its independent form or in its form within an antibody, antibody fragment, or Fc fusion protein. Polymorphisms have been found at many Fc domain sites, including but not limited to EU positions 270, 272, 312, 315, 356, and 358, resulting in minor differences between the sequences described herein and sequences known in the art. Therefore, each naturally occurring IgG Fc region is referred to as a “wild-type IgG Fc domain” or “WT IgG Fc domain” (i.e., any allele). Human IgG1, IgG2, IgG3, and IgG4 heavy chain sequences are available in various sequence databases, including the UniProt database (www.uniprot.org), with accession numbers P01857 (IGHG1_HUMAN), P01859 (IGHG2_HUMAN), P01860 (IGHG3_HUMAN), and P01861 (IGHG4_HUMAN), respectively.

[0205] In embodiments, the antibodies of this disclosure are monoclonal antibodies. A “monoclonal antibody” (mAb) refers to a homogeneous group of antibodies that participate in the highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies, which typically include different antibodies targeting different antigenic determinants. The term "monoclonal antibody" can include full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single-chain (scFv) mutants, fusion proteins containing antibody moieties, and any other modified immunoglobulin molecules containing antigen recognition sites. Furthermore, "monoclonal antibody" refers to such antibodies prepared in any manner, including but not limited to hybridomas, phage selection, recombinant expression, and transgenic animals. In embodiments, the antibody of this disclosure is an isolated monoclonal antibody. In another embodiment, the antibody is a fully human monoclonal antibody.

[0206] In embodiments, the antibody of this disclosure is the full-length antibody described above. Alternatively, the antibody can be an antigen-binding fragment. As used herein, the term "antigen-binding fragment" includes any naturally occurring or artificially constructed antigen-binding polypeptide configuration comprising one, two, or three light chain CDRs and / or one, two, or three heavy chain CDRs, wherein the polypeptide is capable of binding to an antigen.

[0207] In embodiments, the antigen-binding fragment of this disclosure is a Fab fragment. According to this disclosure, antibodies can also be Fab', Fv, scFv, Fd, V NAR domains, IgNAR, intracellular antibodies, IgG CH2, microantibodies, single-domain antibodies, etc.Fcab, scFv-Fc, F(ab')2, di-scFv, bispecific T-cell connector (BITE), F(ab')3, tetravalent antibody, trivalent antibody, bivalent antibody, DVD-Ig, (scFv)2, mAb2, or DARPin.

[0208] The terms “Fab fragment” and “Fab” are used interchangeably herein and comprise a single light chain (e.g., a constant domain CL and a VL) and a single heavy chain (e.g., a constant domain CH1 and a VH). The heavy chain of a Fab fragment cannot form a disulfide bond with another heavy chain.

[0209] A “Fab' fragment” contains a single light chain and a single heavy chain, but in addition to CH1 and VH, a “Fab' fragment” also contains a heavy chain region between the CH1 and CH2 domains, which is necessary for the formation of interchain disulfide bonds. Therefore, two “Fab' fragments” can associate to form an F(ab')2 molecule via the formation of disulfide bonds.

[0210] The “F(ab')2 fragment” comprises two light chains and two heavy chains. Each chain includes a portion of a constant region necessary for the formation of an interchain disulfide bond between the two heavy chains.

[0211] The “Fv fragment” comprises only the variable regions of the heavy and light chains. It does not contain a constant region.

[0212] A “single-domain antibody” is an antibody fragment containing a single antibody domain unit (e.g., VH or VL).

[0213] A “single-chain Fv” (“scFv”) is an antibody fragment containing both the antibody VH and VL domains linked together to form a single chain. A peptide linker is typically used to link the VH and VL domains of the scFv.

[0214] A “tandem scFv,” also known as a TandAb, is a single-chain Fv molecule formed by covalently binding two scFvs in a tandem orientation to a flexible peptide linker.

[0215] A “bispecific T-cell adaptor” (BiTE) is a fusion protein consisting of two single-chain variable fragments (scFvs) on a single peptide chain. One scFv binds to T cells via the CD3 receptor, while the other binds to tumor cell antigens.

[0216] A “dimeric antibody” is a small, bivalent, bispecific antibody fragment composed of a heavy chain variable domain (VH) and a light chain variable domain (VL) linked together on the same polypeptide chain (VH-VL), with a peptide linker too short to allow the two domains on the same chain to pair with each other (Kipriyanov, Int. J. Cancer 77 (1998), 763-772). This forces pairing with the complementary domain of the other chain and promotes assembly into a dimer molecule with two functional antigen-binding sites.

[0217] “DARPin” is a bispecific ankyrin repeat molecule. DARPin is derived from natural ankyrin, which can be found in the human genome and is one of the most abundant types of binding proteins. The DARPin library module consists of natural ankyrin repeat proteins.The white sequence was defined using 229 ankyrin repeat sequences for initial design, and an additional 2200 repeat sequences were used for subsequent improvements as described in the specification 20 / 55, page 27, CN 121175073 A. These modules serve as building blocks for the DARPin library. The library modules are similar to human genome sequences. DARPin consists of 4 to 6 modules. Because each module is approximately 3.5 kDa, the average size of DARPin is 16 to 21 kDa. Selection of the binding sites was accomplished via ribosome display, a completely cell-free technique described in He M. and Taussig MJ., Biochem Soc Trans. 2007, November, 35(Pt 5):962-5.

[0218] In embodiments, the antibody or its antigen-binding fragment may be further modified to include additional chemical moieties that are not normally part of the protein. Those derived moieties may improve the protein's solubility, biological half-life, or absorption. These moieties may also reduce or eliminate any desired side effects of the protein, etc. An overview of these sections can be described in Remington's Pharmaceutical Sciences, 22nd edition, edited by Lloyd V. Allen, Jr. (2012).

[0219] Examples

[0220] This specification will now be illustrated by the following non-limiting examples.

[0221] General Information

[0222] Rapid chromatography was performed using BIOTAGE ISOLERA, and the purity of the fractions was checked using thin-layer chromatography (TLC). TLC was performed on aluminum plates with a fluorescent indicator using MERCK KIESELGEL 60 F254 silica gel. Visualization of the TLC was achieved using UV light.

[0223] Extraction and chromatographic solvents were purchased from VWR UK and used without further purification.

[0224] Unless otherwise stated, all fine chemicals were purchased from SIGMA-ALDRICH.

[0225] Polyethylene glycolation reagents were obtained from QUANTA BIODESIGN US via STRATECH UK.

[0226] LC / MS Conditions

[0227] Positive mode electrospray mass spectrometry was performed using a WATERS ACQUITY H-CLASS SQD2 with one of the following methods.

[0228] (a) HPLC was run using a mobile phase of water (A) (formic acid 0.1%) and acetonitrile (B) (formic acid 0.1%).

[0229] LCMS 3 min: The initial composition of 5% B was maintained for 25 seconds, and then increased from 5% B to 100% B in a period of 1 minute and 35 seconds. The composition was then increased to 100%.The initial composition was held at 5% B for 50 seconds, then returned to 5% B within 5 seconds and held for 5 seconds. The total duration of the gradient run was 3.0 minutes. The flow rate was 0.8 mL / min. Wavelength detection range: 190 nm to 800 nm. Column: WATERS ACQUITY UPLC BEH SHIELD RP18 1.7 µm 2.1 × 50 mm at 50 °C, with a WATERS ACQUITY UPLC BEH SHIELD RP18 VANGUARD pre-column, 130A, 1.7 µm, 2.1 mm × 5 mm.

[0230] LCMS 15 minutes: The initial composition of 5% B was held for 1 minute, then increased from 5% B to 100% B over a 9-minute period. The composition was held at 100% B for 2 minutes, then returned to 5% B within 0.10 minutes and held for 3 minutes. The total gradient run time was 15 minutes. The flow rate was 0.6 mL / min. Wavelength detection range: 190nm to 800nm. Oven temperature: 50℃. Column: WATERS ACQUITY UPLC CSH C18 1.7µm 2.1×100mm, equipped with WATERS ACQUITY UPLC CSH C18 VANGUARD pre-column, 1.7µm, 2.1mm×5mm.

[0231] (b) HPLC (Agilent 1290) was run using a mobile phase of water (A) (TFA 0.03%) and acetonitrile (B) (0.03% TFA) or water (A) (TFA 0.05%) and acetonitrile (B) (0.05% TFA). The initial composition is (a) 100% A held for 2-4 minutes, then increased to 90% B over 2-5 minutes, or (b) 5%-20% B increased to 90%-98% B over 3-17 minutes. The flow rate is 0.3 mL / min-1.5 mL / min. The columns are (1) ATLANTIS T3 3μm 4.6 150mm, at 40 ℃ (detector ELSD or wavelength detection range: 210nm), (2) ACQUITY UPLC BEH C18 2.1 100mm 1.7μm, at 40 ℃ (wavelength detection range: 210nm or 220nm), (3) UPLC BEH C18 1.7μm, 2.1 100mm, at 40 ℃ (wavelength detection range: 223nm), (4) XBRIDGE C18 (4.6 150, 3.5μm), at 40 ℃, (5) ACQUITY UPLC HSS PFP 2.1 150mm 1.8μm, at 40 ℃ (wavelength detection range: 220nm), (6) UPLCBEH Phenyl 1.7μm, 2.1 150mm, at 40℃ (wavelength detection range: 210nm), (7) EC-C18 2.7μm, 3.0 50mm, at 40℃ (wavelength detection range: 210nm) or (8) YMC-Triart C18 50 3.0mm S-3μm, 12nm, at 45℃ (detector ELSD). Injection volume 2μL.

[0232] HPLC conditions

[0233] Reversed-phase ultra-fast high-performance liquid chromatography (UFLC) was performed on a SHIMADZU PROMINENCE machine using a PHENOMENEX GEMINI NX 5µ C18 column (at 50℃) with dimensions of 150×21.2mm. The eluents used were solvent A (H2O containing 0.1% formic acid) and solvent B (CH3CN containing 0.1% formic acid). All UFLC experiments were performed under gradient conditions: the initial composition of 13% B was increased to 30% B over a 3-minute interval, then to 45% B over an 8-minute interval, and then to 100% B over a 6-minute interval, before returning to 13% and holding for 1 minute over a 2-minute interval. The total duration of the gradient run was 20.0 minutes. The flow rate was 20.0 mL / min and detection was performed at 254 nm and 223 nm.

[0234] NMR Methods

[0235] Proton NMR chemical shift values ​​were measured at 400 MHz on a delta scale using a BRUKER AV400. The following abbreviations have been used: s, singlet; d, doublet; t, triplet; q, quartet; quin, quintet; m, multiplet; br, broad peak. Coupling constants are reported in Hz. Instructions for Use, Page 22 / 55, 29, CN 121175073 A

[0236]

[0237] Intermediate 1

[0238]

[0239] At 21°C, 2,3,4,6,7,8,9,10-octahydropyrimidine[1,2-a]azapyridine (26.5 ml, 177.35 mmol) was added dropwise to a 1-L round-bottom flask containing a solution of (2S,3S,4S,5R,6R)-3,4,5,6-tetrahydroxytetrahydro-2H-pyran-2-carboxylic acid (31.3 g, 161.22 mmol) in DMF (100 ml). Next, 3-bromoprop-1-ene (16.72 mL, 193.47 mmol) was added dropwise to the reaction mixture over 10 minutes, and the reaction mixture was stirred at 21 °C for 24 hours. The reaction mixture was cooled to 0 °C and treated with pyridine (104 mL, 1289.60 mmol). Acetic anhydride (244 mL, 2579.20 mmol) was then added to the reaction mixture. The reaction mixture was warmed to room temperature and run at 21 °C for 2 hours. The reaction mixture was then...The compound was concentrated under reduced pressure, and the remaining pyridine was removed by azeotropic mixing with toluene (1 × 100 mL). The crude compound was diluted with DCM (65 mL) and cooled to 0 °C. Next, a 30% hydrobromic acid solution in acetic acid (175 mL, 3226.03 mmol) was added to the reaction mixture at 0 °C. The reaction mixture was warmed to room temperature and run at 21 °C for 2 hours and 30 minutes. The solvent was evaporated, and the compound was then purified by normal-phase rapid column chromatography to give intermediate 1 (33 g, 48% yield) of triacetic acid (2S,3S,4S,5R,6R)-2-((allyloxy)carbonyl)-6-bromotetrahydro-2H-pyran-3,4,5-trimethyl ester, which was a beige translucent substance. 1H NMR (500MHz, CDCl3) δ 6.67 (d , J = 4.0Hz, 1H) , 5.92 (ddt, J = 16.6, 10.3, 6.0Hz, 1H) , 5.64 (t, J = 9.7Hz, 1H) , 5.42 – 5.23 (m, 3H) , 4.88 (dd, J = 10.0, 4.0Hz, 1H), 4.71 – 4.58 (m, 3H), 2.12 (s, 3H), 2.07 (s, 3H), 2.05 (s, 3H); LCMS (ESI) m / z 445.0 (M + Na)+.

[0240] Alternative Synthesis of Intermediate 1

[0241]

[0242] Iodine (1.19 kg, 4.69 mol) was added to acetic anhydride (3500 mL) stirred under nitrogen at 0-10 °C. The resulting mixture was adjusted to 20-30 °C, and glucuronic acid (7 kg, 36.06 mol) was added in portions, maintaining the temperature at 25-30 °C. The reactants were stirred at this temperature under nitrogen for 1 hour, and then cooled to 0 °C. A solution of sodium thiosulfate pentahydrate (2.33 kg) in water (35.2 L) was added to the stirred mixture at 0-10 °C, and then stirred at 20-30 °C for 2 hours. Water (35.2 L) was added to the stirred mixture, and the mixture was extracted with isopropyl acetate (3 × 35.2 L). The organic layer was concentrated to dryness to give crude 1,2,3,4-tetra-O-acetyl-β-D-glucuronic acid (16.08 kg, 61% w / w determination, 75%). LCMS m / z (ES+), [M+Na]+ = 384.6

[0243] Crude 1,2,3,4-tetra-O-acetyl-β-D-glucuronic acid, 61% w / w (16 kg; 27.05 mol), was dissolved in acetyl acetate.The mixture was stirred in isopropyl acetate (42.83 kg) at 20-30°C. N,N-diisopropylethylamine (12.25 kg, 94.68 mol) was added to the reactants over 11 minutes at 20-30°C, followed by dropwise addition of 3-bromopropene (9.8 kg, 81.15 mol) over 5 minutes at 20-30°C. The resulting mixture was stirred at 20-30°C for 48 hours. Isopropyl acetate (42.83 kg) and water (49 kg) were added to the stirred mixture. The organic layer was separated and the pH was adjusted to 4-5 by adding aqueous hydrochloric acid (0.6 N, 43.71 kg) at 20-30°C. The separated organic layer was washed with brine (25% aqueous solution, 49 L) and concentrated to dryness to obtain crude 1,2,3,4-tetra-O-acetyl-β-D-glucuronide (12.0 kg, 87.5% w / w determination, 86.6%) as a brown solid. LCMS m / z (ES+), [M+Na]+ = 424.833%. At 0 °C, a hydrobromic acid solution in acetic acid (534 mL, 2.982 mol) was added dropwise to a stirred mixture of crude 1,2,3,4-tetra-O-acetyl-β-D-glucuronide (200 g, 0.497 mol) in isopropyl acetate (500 mL). The reaction mixture was adjusted to 20-30 °C and stirred for 8 hours. The reaction mixture was extracted with isopropyl acetate (2400 mL), the extract was washed with brine (25% aqueous solution, 3 × 2000 mL) and concentrated to dryness to give a crude product (231.3 g, 80.5%) as a black oil. LCMS (ES+), [M+Na]+ = 445.2 & 447, 1H NMR (300MHz, CDCl3) δ 6.65 (d, J = 4.2Hz, 1H), 5.59–5.84 (m, 1H), 5.62 (t, J = 9.6Hz, 1H), 5.40–5.23 (m, 3H), 4.87 (dd, J = 9.9, 3.9Hz, 1H), 4.66–4.59 (m, 3H), 2.21–2.03 (m, 9H)

[0244] Intermediate 2

[0245] Specification 24 / 55 pages 31 CN 121175073 A

[0246] A solution of TBS-Cl (20.80 g, 138.02 mmol) in DCM (25 mL) was added dropwise to 1H-imidazole (17.90 g, 262.90 mmol) and 2-hydroxy-5-(hydroxymethyl)benzaldehyde (20 g, 131.45 mmol) over a 2-hour period at 0°C under nitrogen atmosphere.The mixture was stirred at 0°C for 2 hours. The reaction mixture was quenched with water (500 mL), extracted with DCM (2 × 300 mL), and the organic layer was dried over Na2SO4, filtered and evaporated to give intermediate 2 (35.0 g, 100%) of 5-(((tert-butyldimethylsilyl)oxy)methyl)-2-hydroxybenzaldehyde as a colorless substance. m / z (ES+), [M+ Na]+ = 289; NH4HCO3, HPLC tR = 1.505 min

[0247] Intermediate 3

[0248]

[0249] Molecular sieve (4 Å beads, 5.0 g), silver oxide (29.2 g, 125.8 mmol) and acetonitrile (150 mL) were added to a vacuum-dried 500 mL round-bottom flask to produce a black slurry. Over 20 minutes, a solution of intermediate 1 (10.7 g, 25.2 mmol) in acetonitrile (50 mL) was added to the slurry, followed by a single addition of a solution of 5-(((tert-butyldimethylsilyl)oxy)methyl)-2-hydroxybenzaldehyde (intermediate 2, 13.6 g, 51.1 mmol) in acetonitrile (50 mL). The resulting mixture was vigorously stirred at 20 °C for 16 hours. After 16 hours, the reaction mixture was filtered through a 5-cm diatomaceous earth mat and washed with dichloromethane (3 × 25 mL). The solvent was evaporated, and the compound was then purified by normal-phase rapid column chromatography to give triacetic acid (2S,3S,4S,5R,6S)-2-((allyloxy)carbonyl)-6-(4-(((tert-butyldimethylsilyl)oxy)methyl)-2-formylphenoxy)tetrahydro-2H-pyran-3,4,5-trimethyl ester intermediate 3 (5.2 g, 34% yield), which was a white substance. 1H NMR (400MHz, CDCl3) δ 10.34 (s, 1H), 7.77 (d, J = 1.8Hz, 1H), 7.58 (dd, J = 8.6, 2.1Hz, 1H), 7.14 (d, J = 8.6Hz, 1H), 5.81‑5.92 (m, 1H) , 5.39‑5.35 (m, 4H) , 5.28‑5.22 (m, 2H) , 4.71 (s, 2H) , 4.58‑4.67 (m, 2H) , 4.20‑4.28 (m, 1H) , 2.073 (s, 3H) , 2.069 (s, 3H), 2.04 (s, 3H), 0.94 (s, 9H), 0.11 (s, 6H); LCMS (ESI) m / z 626.3 (M + NH4)+.

[0250] Intermediate 4

[0251]

[0252] Tert-butyl carbamate (3.8 g, 32.3 mmol), trifluoroacetic acid (2.0 mL, 25.9 mmol), and triethylsilane (4.1 mL, 25.8 mmol) were added to a solution of intermediate 3 (5.2 g, 8.6 mmol) in acetonitrile (40 mL). The mixture was stirred at 20 °C for 2 hours, and then the solvent was evaporated. 1,4-dioxane (8 mL) and HCl (4.0 M 1,4-dioxane solution, 50 mL, 200 mmol) were added to the resulting colorless oil. The mixture was stirred at 20 °C for 30 minutes, and the solvent was evaporated. The resulting white powder was dissolved in DMSO (3 mL) and then passed through a cation exchange resin (WATERS PORAPAK CX) pretreated with methanol. The desired compound was eluted from the resin with methanol to give a white substance, triacetic acid (2S,3S,4S,5R,6S)-2-((allyloxy)carbonyl)-6-(2-(aminomethyl)-4-(hydroxymethyl)phenoxy)tetrahydro-2H-pyran-3,4,5-trimethyl ester intermediate 4 (2.5 g, 80%, by 2 steps). 1H NMR (500MHz, CDCl3) δ 7.26 (d, J = 2.2Hz, 1H), 7.21 (dd, J = 8.3, 2.2Hz, 1H), 7.01 (d, J = 8.3Hz, 1H), 5.90–5.82 (m, 1H), 5.42–5.24 (m, 6H), 5.16 (d, J = 7.1Hz, 1H), 4.64–4.55 (m, 4H), 4.19 (d, J = 9.3Hz, 1H), 3.84 (d, J = 14.0Hz, 1H), 3.67 (d, J = 14.0Hz, 1H), 2.32 (s, 3H), 2.09 (s, 3H), 2.07 (s, 3H), 2.03 (s, 3H). LCMS (ESI) m / z 496.5 (M + H)+.

[0253] Intermediate 5

[0254]

[0255] N-ethyl-N-isopropylpropyl-2-amine (1.8mL, 10.1mmol) and 2,5-dioxopyrrolidine-1-yl ester of 3-((tert-butoxycarbonyl)amino)propionic acid (1.3g, 4.4mmol) were added to intermediate 4 (2.5g, 5.0mmol) in a suspension of dichloromethane (20mL). The mixture was stirred at 20°C for 10 minutes, and then water (50mL) was added. The organic layer was separated, and then the water layer was used...Extracted with dichloromethane (3 × 30 mL). The combined organic layers were dried over Na₂SO₄ and the solvent was evaporated. Hydrochloric acid (2.6 mL, 83.9 mmol) was added to a solution of triacetic acid (2S,3S,4S,5R,6S)-2-((allyloxy)carbonyl)-6-(2-((3-((tert-butoxycarbonyl)amino)propamido)methyl)-4-(hydroxymethyl)phenoxy)tetrahydro-2H-pyran-3,4,5-trimethyl ester (2.8 g, 4.2 mmol) in dichloromethane (20 mL). The mixture was stirred at 20 °C for 2 hours, and then the solvent was evaporated. The compound was purified by reversed-phase rapid column chromatography to obtain triacetic acid (2S,3S,4S,5R,6S)-2-((allyloxy)carbonyl)-6-(2-((3-aminopropamido)methyl)-4-(hydroxymethyl)phenoxy)tetrahydro-2H-pyran-3,4,5-trimethyl ester intermediate 5 (1.3 g, 53%, in 2 steps) as a colorless substance. 1H NMR (500MHz, D2O) δ 7.22 (d, J = 2.2Hz, 1H), 7.16 (d, J = 8.2Hz, 1H), 7.09 (d, J = 8.4Hz, 1H), 5.84 (ddt, J = 16.6, 10.5, 6.0Hz, 1H) , 5.44 (t, J = 9.2Hz, 1H) , 5.38 (dd , J = 7.6, 3.3Hz, 1H) , 5.35 – 5.23 (m, 4H) , 4.62 (d , J = 9.8Hz, 1H) , 4.56 (d , J = 6.0Hz, 2H) , 4.51 (s, 2H), 4.26 (q, J = 15.3Hz, 2H), 3.23 (t, J = 6.8Hz, 2H), 2.68 (td, J = 6.8, 1.9Hz, 2H), 2.06 (d, J = 10.4Hz, 9H). LCMS (ESI) m / z 567.2 (M + H)+.

[0256] Alternative synthesis of intermediate 5

[0257]

[0258] Fmoc-β-alanine (1.11kg, 3.57mol) was added to a stirred reactor containing intermediate 4 (2.1kg, 90.5% w / w, 3.57mol) and acetonitrile (19L). The stirred mixture was cooled to 0°C. Add 1.36 kg (3.57 mol) of aziridine triazole hexafluorophosphate and 0.92 kg (7.14 mol) of N,N-diisopropylethylamine to the mixture and stir for 4 minutes.For hours, the temperature was maintained at 0°C. Water (19 L) and ethyl acetate (19 L) were added to the stirred mixture. The organic phase was separated and concentrated to approximately 19 L under vacuum. Ethyl acetate (28.5 L) was added to the concentrated solution and stirred at 20°C–25°C for 18 hours. The resulting suspension was filtered, the filter cake was washed with ethyl acetate (3.87 L) and dried under vacuum to give triacetic acid (2S,3R,4S,5S,6S)-2-(2-((3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)methyl)-4-(hydroxymethyl)phenoxy)-6-((allyloxy)carbonyl)tetrahydro-2H-pyran-3,4,5-trimethyl ester (1.6 kg, 99% w / w, 56%). LCMS m / z 789 [M+H]+

[0259] 2,3,4,6,7,8,9,10-octahydropyrimido[1,2-a]azapyridine (385.98 g, 2.54 mol) was added to a stirred reactor containing triacetic acid (2S,3R,4S,5S,6S)-2-(2-((3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)methyl)-4-(hydroxymethyl)phenoxy)-6-((allyloxy)carbonyl)tetrahydro-2H-pyran-3,4,5-trimethyl ester (1 kg, 1.27 mol) and tetrahydrofuran (10 L) at -45 °C under nitrogen atmosphere. The mixture was stirred at -45°C for four hours, then diluted with acetonitrile (5 L) and quenched with a solution of tert-butyl methyl ether (2.54 L, 2.0 M, 5.07 mol) with added hydrogen chloride. The mixture was concentrated under vacuum to about 5 L and diluted with n-heptane (5 L). The acetonitrile layer containing intermediate 8 was collected (3.88 kg of MeCN solution, 89.97% area, assumed to be 100%). LCMS m / z 566.6 [M+H]+

[0260] Intermediate 7

[0261]

[0262] Anhydrous DCM (100 mL) solution of intermediate 6 (5.0 g, 34.21 mmol) was added to a 250 mL round-bottom flask under nitrogen. Pyridine (13.84 mL, 171.07 mmol) was added to the solution, followed by toluenesulfonyl-Cl (16.31 g, 85.53 mmol). The reaction mixture was stirred at 20 °C for 16 hours. LC-MS analysis showed the formation of the desired product and completion of the reaction. The reaction mixture was diluted with dichloromethane (200 mL). The organic layers were separated, and the compound was extracted in 200 mL of dichloromethane. The combined organic layers were washed with HCl solution (1 M - 300 mL), brine (200 mL), and dried over magnesium sulfate.The solvent was removed to obtain the crude product. The compound was purified by silica gel column chromatography to give (3R,3aS,6R,6aS)-hexahydrofurano[3,2-b]furan-3,6-dimethylbis(4-methylbenzenesulfonate) intermediate 7 (14.90 g, 96%). ¹H NMR (500 MHz, CDCl₃) δ 7.90 – 7.76 (m, 4H), 7.45 – 7.33 (m, 4H), 4.94 – 4.80 (m, 2H), 4.55 – 4.44 (m, 2H), 3.94 (dd, J = 9.6, 6.7 Hz, 2H), 3.75 (dd, J = 9.6, 7.6 Hz, 2H), 2.48 (s, 6H). LCMS (ESI) m / z 455.21 (M + H)+.

[0263] Intermediate 8

[0264]

[0265] Anhydrous DMF (15 mL) solution of intermediate 7 (6.0 g, 13.20 mmol) was added to a 50 mL round-bottom flask under nitrogen. Sodium azide (2.146 g, 33.00 mmol) was added to the solution. The reaction mixture was incubated at 140 °C for 3 hours. LC-MS analysis showed that the desired product was formed and the reaction was complete. The reaction mixture was diluted with dichloromethane (200 × 2 mL) and the organic layer was separated, washed with water (200 mL), brine (200 mL), and dried over magnesium sulfate. The solvent was removed under reduced pressure to give (3S, 3aR, 6S, 6aR)-3,6-diazidohexahydrofurano[3,2-b]furan intermediate 8 (2.050 g, 79%). 1H NMR (500MHz, CDCl3) δ 4.61 (d, J = 1.9Hz, 2H), 4.05 (d, J = 4.0Hz, 2H), 3.97 – 3.82 (m, 4H). LCMS (ESI) m / z 197.1 (M + H)+.

[0266] Intermediate 9 Specification 27 / 55 pages 34 CN 121175073 A

[0267]

[0268] An anhydrous THF (20mL) solution of intermediate 8 (1g, 5.10mmol) was added to a 250mL round-bottom flask under nitrogen atmosphere. Barium palladium(II) carbonate (0.618g, 0.51mmol) was added to the solution. The reaction mixture was purged with hydrogen gas (1.028 g, 509.76 mmol) and stirred at 23 °C for 3 hours under H2 atmosphere. LC-MS analysis showed the formation of the desired product and completion of the reaction. The reaction mixture was diluted with methanol (20 mL) and filtered through a diatomaceous earth filter. The diatomaceous earth filter was then diluted with methanol (50 mL).Wash. Dry the filtrate with magnesium sulfate. Remove the solvent under reduced pressure to give (3S,3aR,6S,6aR)-hexahydrofurano[3,2-b]furan-3,6-diamine intermediate 9 (0.590 g, 80%). ¹H NMR (500 MHz, DMSO) δ 4.23 (s, 2H), 3.68 (dd, J = 8.7, 4.5 Hz, 2H), 3.41 (dd, J = 8.7, 1.9 Hz, 2H), 3.23 (dd, J = 4.5, 1.9 Hz, 2H), 1.54 (s, 4H). LCMS (ESI) m / z 145.2 (M + H)+.

[0269] Alternative Synthesis of Intermediate 9

[0270]

[0271] Intermediate 6 (4 kg, 8.8 mol) and benzylamine (12 L) were added to a reactor under nitrogen atmosphere. The stirred mixture was heated to 160 °C and maintained for 24 hours, then cooled to 20 °C–25 °C, diluted with tert-butyl methyl ether (80 L), and further cooled to 10 °C. p-Toluenesulfonic acid (12.11 kg, 70.44 mol) was added to the mixture, which was stirred at 20 °C–25 °C for 2.5 hours and then filtered. The filter cake was washed with tert-butyl methyl ether (8 L) and the combined filtrates were washed with a saturated aqueous solution of sodium bicarbonate (20 L). The organic phase was evaporated to dryness, dissolved in ethanol (20 L) and evaporated to dryness to give crude intermediate 7 (3.05 kg, 80.5% w / w, 85.9%) LCMS m / z (ES+), [M+H]+ = 325.1

[0272] Intermediate 7 (1.5 kg, 3.08 mol) and ethanol (12.12 L) were added to the reactor under a nitrogen atmosphere and on dryness. 10% by weight of carbon-supported palladium (120.8, 10% w / w) was added to the solution. The reaction mixture was purged with hydrogen and stirred at 80 °C for 16 hours under hydrogen. The mixture was cooled to 20 °C–25 °C and filtered through cellulose (2.42 kg). The filter cake was washed with ethanol (2.44 L) and the combined filtrates were concentrated to dryness. The residue was dissolved in acetonitrile (6.04 L) and concentrated to dryness to give intermediate 9 (509 g, 84% w / w, 80.2%). LCMS m / z (ES+), [M+H]+ = 145

[0273] Intermediate 11

[0274]

[0275] Anhydrous THF (25 mL) solution of intermediate 9 (1.50 g, 10.40 mmol) was added to a 250 mL round-bottom flask under nitrogen. Sodium bicarbonate (1.748 g, 20.81 mmol) and 2,5,8,11,14,17,20, were added to the solution in portions under nitrogen.23,26,29,32,35-dodecoxaoctanetriane-38-acid 2,5-dioxopyrrolidine-1-yl ester intermediate 10 (7.13 g, 10.40 mmol) was stirred at 20 °C for 6 hours. LC-MS analysis showed the formation of the desired product and the completion of the reaction. The reaction mixture was quenched by adding methanol (10 mL). The reaction mixture was diluted with methanol (20 mL) and filtered through a diatomaceous earth mat. The diatomaceous earth mat was washed with methanol (50 mL). The filtrate was dried over magnesium sulfate. The solvent was removed under reduced pressure to give the crude product. The crude product was purified by silica gel column chromatography to obtain N-((3S,3aR,6S,6aR)-6-aminohexahydrofurano[3,2-b]furan-3-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecoxaoctane-38-amide intermediate 11 (4.00 g, 53.8%). 1H NMR (500MHz, MeOD) δ 4.64 – 4.59 (m, 1H) , 4.45 (dd, J = 4.1, 1.3Hz, 1H) , 4.29 (dt, J = 4.1, 1.9Hz, 1H), 3.96 (ddd, J = 10.2 , 9.3 , 4.9Hz , 2H) , 3.81 – 3.74 (m, 3H) , 3.73 – 3.62 (m, 45H) , 3.61 – 3.56 (m, 2H) , 3.45 (dt, J = 3.8, 1.8Hz, 1H) , 3.40 (s, 3H) , 2.52 – 2.47 (m, 2H). LCMS (ESI) m / z 715.6 (M + H)+.

[0276] Intermediate 13

[0277]

[0278] An anhydrous DMF (20 mL) solution of (S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-6-(tert-butoxy)-6-oxohexanoic acid intermediate 12 (5 g, 11.38 mmol) was added to a 250 mL round-bottom flask under nitrogen. Potassium carbonate (3.14 g, 22.75 mmol) and 3-bromoprop-1-ene (1.485 mL, 17.06 mmol) were added in portions to the solution under nitrogen and the mixture was stirred at 20 °C for 16 hours. LC-MS analysis showed that the desired product was formed and the reaction was complete. The reaction mixture was diluted with water (500 mL), and the organic layer was extracted with ethyl acetate (2 × 300 mL), washed with water (300 mL), brine (200 mL), and subjected to sodium sulfate.(20 g) Dry. Remove the solvent to obtain the crude product. Purify the crude product by silica gel column chromatography to obtain (S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)adipic acid 1-allyl 6-(tert-butyl) intermediate 13 (5.10 g, 93%). 1H NMR (500MHz, CDCl3) δ 7.79 – 7.73 (m, 2H) , 7.61 (q, J = 3.9Hz, 2H) , 7.40 (t, J = 7.5Hz, 2H) , 7.32 (tt, J = 7.4, 1.2Hz, 2H) , 5.91 , 2.26 (t, J = 7.2 Hz, 2H), 1.96 – 1.82 (m, 1H), 1.72 (dq, J = 13.5, 6.1 Hz, 3H), 1.60 – 1.47 (m, 1H), 1.45 (s, 9H). LCMS (ESI) m / z 480.2 (M + H)+.

[0279] Intermediate 14

[0280]

[0281] An anhydrous THF (20 mL) solution of intermediate 13 (5 g, 10.43 mmol) was added to a 100 mL round-bottom flask under nitrogen. HCl (13.03 mL, 52.13 mmol) (4 mol dioxane solution) was added to the solution under nitrogen and stirred at 20 °C for 6 hours. LC-MS analysis showed that the desired product was formed and the reaction was complete. The reaction mixture was diluted with water (200 mL), and the organic layer was extracted with dichloromethane (2 × 300 mL), washed with brine (200 mL), and dried over sodium sulfate (20 g). The solvent was removed to give intermediate 14 (4.20 g, 95%) of (S)-5-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-6-(allyloxy)-6-oxohexanoic acid. 1H NMR (500MHz, CDCl3) δ 7.75 (dq, J = 7.6, 1.0Hz, 2H) , 7.62 – 7.52 (m, 2H) , 7.42 – 7.35 (m, 2H) , 7.30 (tt, J = 7.4, 1.2Hz, 2H) , 5.90 (ddt, J = 16.4, 10.8, 5.8Hz, 1H), 5.48(d, J = 8.4Hz, 1H), 5.37 – 5.20 (m, 2H), 4.64 (d, J = 5.8Hz, 2H), 4.40 (d, J = 7.2Hz, 3H), 4.22 (t, J = 7.0Hz, 1H), 2.45 – 2.24 (m, 2H), 1.93 (p, J = 5.6Hz, 1H), 1.72 (td, J = 13.9, 6.8Hz, 3H). (ESI) m / z 424.5 (M‑H)‑.

[0282] Intermediate 15 Specification 29 / 55 pages 36 CN 121175073 A

[0283]

[0284] Intermediate 14 (1.925 g, 4.55 mmol) was added to a 100 mL round-bottom flask under nitrogen atmosphere. HATU (1.862 g, 4.90 mmol) was added to the solution, followed by DIPEA (1.222 mL, 6.99 mmol). The reaction mixture was stirred at room temperature for 15 minutes, then intermediate 11 (2.5 g, 3.50 mmol) was added, and the reaction mixture was stirred at 23 °C for 3 hours. LC-MS analysis showed that the desired product was formed and the reaction was complete. The reaction mixture was diluted with DCM (300 mL), washed with water (200 mL), and the organic layer was extracted (2 × 100 mL), washed with brine (50 mL), and dried over sodium sulfate (5 g). The solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain (S)-6-(((3S,3aR,6S,6aR)-6-(2,5,8,11,14,17,20,23,26,29,32,35-dodecanooctane-38-amido)hexahydrofurano[3,2-b]furan-3-yl)amino)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-6-oxohexanoic acid allyl ester intermediate 15 (3.40 g, 87%) 1H NMR (500 MHz, MeOD) δ 7.86 (dd, J = 7.6, 1.2 Hz, 2H), 7.74 (t, J = 7.8 Hz, 2H), 7.46 (td, J = 7.5, 1.4Hz, 2H), 7.38 (tt, J = 7.5, 1.3Hz, 2H), 6.05 – 5.93 (m, 1H), 5.39 (dq, J = 17.2, 1.6Hz, 1H), 5.28 (dq, J = 10.5 , 1.4Hz, 1H) , 4.73 – 4.65(m, 2H) , 4.58 (qd, J = 4.1, 1.0Hz, 2H) , 4.46 (dd, J = 10.6, 7.0Hz, 1H) , 4.40 (dd, J = 10.6, 7.0Hz, 1H) , 4.36 – 4.31 (m, 2H) , 4.31 – 4.24 (m, 2H) , 4.01 (ddd , J = 9.6, 5.0, 1.1Hz, 2H) , 3.84 – 3.73 (m, 5H) , 3.72 – 3.60 (m, 44H) , 3.60 – 3.56 (m, 2H) , 3.41 (s, 3H) , 2.49 (td, J = 6.0, 1.9Hz, 2H), 2.31 (hept, J = 7.2Hz, 2H), 1.96 – 1.85 (m, 1H), 1.85 – 1.68 (m, 3H). LCMS (ESI) m / z 1121.3 (M + H)+.

[0285] Intermediate 16

[0286]

[0287] Anhydrous DCM (10 mL) solution of intermediate 15 (4.3 g, 3.84 mmol) was added to a 50 mL round-bottom flask under nitrogen atmosphere. Triethylamine (0.535 mL, 3.84 mmol) was added to the solution, followed by triphenylphosphine (0.101 g, 0.38 mmol). Pd(PPh3)4 (0.444 g, 0.38 mmol) was added to the reaction mixture, followed by formic acid (0.147 mL, 3.84 mmol), and the reaction mixture was stirred at 23 °C for 6 hours. LC-MS analysis showed the formation of the desired product and completion of the reaction. The solvent was removed under reduced pressure to give the crude product. The crude product was purified by silica gel column chromatography to give (S)-6-(((3S,3aR,6S,6aR)-6-(2,5,8,11,14,17,20,23,26,29,32,35-dodecoxaoctane-38-amido)hexahydrofurano[3,2-b]furan-3-yl)amino)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-6-oxohexanoic acid intermediate 16 (3.50 g, 84%). 1H NMR (500MHz, DMSO) δ 8.12 (dd , J = 10.2, 7.0Hz, 2H) , 7.90 (d , J = 7.6Hz, 2H) , 7.74 (d, J = 7.5Hz, 2H) , 7.63 (d, J = 8.1Hz, 1H) , 7.46 – 7.38 (m , 2H) , 7.34 (td, J = 7.4, 1.2Hz, 2H), 4.38 (s, 2H), 4.32 – 4.20 (m, 3H), 4.11 (ddt, J = 7.2, 4.8, 2.1Hz, 2H), 3.93 (td, J = 8.4, 4.6Hz, 1H) , 3.85 (dd, J = 9.3, 5.1Hz, 2H) , 3.63 – 3.57 (m, 4H) , 3.54 – 3.45 (m, 42H) , 3.45 – 3.40 (m , 2H) , 3.24 (s , 3H) , 2.33 (t, J = 6.5 Hz, 2H), 2.09 (s, 3H), 1.68 (d, J = 9.1 Hz, 1H), 1.64 – 1.48 (m, 3H). LCMS (ESI) m / z 1080.6 (M + H)+.

[0288] Intermediate 17 Specification 30 / 55 pages 37 CN 121175073 A

[0289]

[0290] Intermediate 16 (0.8 g, 0.74 mmol) was added to a 100 mL round-bottom flask under nitrogen atmosphere. HATU (0.366 g, 0.96 mmol) was added to the solution, followed by DIPEA (0.388 mL, 2.22 mmol). The reaction mixture was stirred at room temperature for 15 minutes, then intermediate 5 (0.670 g, 1.11 mmol) was added, and the reaction mixture was stirred at 23 °C for 3 hours. LC-MS analysis showed that the desired product was formed and the reaction was complete. The reaction mixture was diluted with DCM (100 mL), washed with water (100 mL), extracted with organic layers (2 × 100 mL), washed with brine (100 mL), and dried over sodium sulfate (15 g). The solvent was removed under reduced pressure and purified by silica gel column chromatography to obtain triacetic acid (2S,3R,4S,5S,6S)-2-(2-((S)-5-(4-(((3S,3aR,6S,6aR)-6-(2,5,8,11,14,17,20,23,26,29,32,35-dodecanoic acid-38-amido)hexahydrofurano[3,2-b]furan-3-yl)amino)-4-oxobutyl)-1-(9H-fluorene-9-yl)-3,6,10-trioxo-2-oxa-4,7,11-triazadodecanoic acid-12-yl)-4-(hydroxymethyl)phenoxy)-6-((allyloxy)carbonyl)tetrahydro-2H-pyran-3,4, 5-Trimethylester intermediate 17 (0.640 g, 53.1%). ¹H NMR (500 MHz, DMSO) δ 8.21(t, J = 6.0Hz, 1H), 8.11 (dd, J = 19.5, 6.9Hz, 2H), 7.94 (t, J = 5.8Hz, 1H), 7.89 (d, J = 7.5Hz, 2H), 7.73 (t, J = 7.0Hz, 2H), 7.49 – 7.39 (m, 3H), 7.33 (td, J = 7.5, 1.2Hz, 2H), 7.17 (dd, J = 8.4, 2.2Hz, 1H), 7.13 (s, 1H), 7.01 (d, J = 8.4Hz, 1H), 5.89 (ddt, J = 17.3, 10.5, 5.7Hz, 1H), 5.56 (d, J = 7.9Hz, 1H), 5.48 (t, J = 9.6Hz, 1H), 5.33 (dq, J = 17.2, 1.6Hz, 1H), 5.26 (dq, J = 10.5, 1.4Hz, 1H), 5.19 – 5.07 (m, 3H), 4.76 (d, J = 10.0Hz, 1H), 4.62 (ddt, J = 13.3, 5.6, 1.4Hz, 1H), 4.54 (ddt, J = 13.3, 5.8, 1.4Hz, 1H), 4.44 – 4.36 (m, 4H), 4.31 – 4.18 (m, 4H), 4.11 (d, J = 5.9Hz, 3H), 3.93 (d, J = 5.9Hz, 1H), 3.88 – 3.81 (m, 2H), 3.66 – 3.56 (m, 5H), 3.56 – 3.45 (m, 42H), 3.45 – 3.39 (m, 3H), 3.29 – 3.27 (m, 1H), 3.24 (s, 3H), 3.18 (d, J = 5.0Hz, 1H), 2.33 (ddt, J = 14.6, 10.0, 7.6Hz, 4H), 2.10 – 2.05 (m, 2H), 2.04 (s, 3H), 1.99 (d, J = 4.6Hz, 6H), 1.54 (dt, J = 43.7, 8.9Hz, 4H). LCMS (ESI) m / z 1629.8 (M + H)+.

[0291] Intermediate 18

[0292] DescriptionPage 31 / 55 38 CN 121175073 A

[0293] Bis(4-nitrobenzene carbonate) (28.0 mg, 0.09 mmol, 4.5 equivalents) and DIPEA (0.013 mL, 0.08 mmol, 4.0 equivalents) were added to a solution of intermediate 17 (25.00 mg, 0.02 mmol, 1.0 equivalents) in DMF. The mixture was stirred at 23 °C for 2 hours. After this time, the mixture was concentrated under vacuum, and the residue was sonicated in DCM (200 µL) and diethyl ether (2 mL). The resulting suspension was dried on a vacuum filter and the process was repeated. The residue was dried to obtain a yellow substance, triacetic acid (2S,3R,4S,5S,6S)-2-(2-((S)-5-(4-(((3S,3aR,6S,6aR)-6-(2,5,8,11, 14,17,20,23,26,29,32,35-dodecanoic acid-38-amido)hexahydrofurano[3,2-b]furan-3-yl)amino)-4-oxobutyl)-1-(9H-fluorene-9-yl)-3,6,10-trioxo-2-oxa-4,7,11-triazadodecane- 12-yl)-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-((allyloxy)carbonyl)tetrahydro-2H-pyran-3,4,5-trimethyl ester intermediate 18 (32 mg, 0.02 mmol, 91%). RT 7.74 min. LCMS (ESI) m / z 1794.7 [M+H]+.

[0294] Intermediate 19

[0295]

[0296] DIPEA (7.28µl, 0.04mmol, 4.0 equivalent), intermediate 18 (25.00mg, 0.01mmol, 1.0 equivalent), and HOPO (1.703mg, 0.02mmol, 2.0 equivalent) were added to a solution of ethathecan mesylate (7.41mg, 0.01mmol, 1.0 equivalent) in DCM (1mL) and DMF (1.000mL), and the resulting mixture was stirred at 23°C for 18 hours. After this time, the mixture was concentrated under vacuum, and the residue was purified by reversed-phase rapid column chromatography (C18 BIOTAGE pre-packed column, 40%–60% MeCN [0.1% formic acid] / water [0.1% formic acid]) to give a yellow substance of triacetic acid (2S,3R,4S,5S,6S)-2-(2-((S)-5-(4-(((3S,3aR,6S,6aR)-6-(2,5,8,11,14,17,20,23,26,29,32,35-dodecoxaoctane-38-amido)hexahydrofurano[3,2-b]furan-3-yl)amino)-4-oxobutyl)-1-(9H-fluorene-9-yl)-3,6,10-Trioxo-2-oxa-4,7,11-triazadodecane-12-yl)-4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenoxy)-6-((allyloxy)carbonyl)tetrahydro-2H-pyran-3,4,5-trimethyl ester intermediate 19 (13 mg, 0.01 mmol, 63%). RT 7.66 min. LCMS (ESI) 2090.3 [M+H]+.

[0297] Intermediate 20 Specification 32 / 55 pages 39 CN 121175073 A

[0298]

[0299] Triethylamine (2.00µl, 0.01mmol, 1.4 equivalent), Pd(PPh3)4 (1.00mg, 0.87µmol, 12mol%) and formic acid (0.541µl, 0.01mmol, 1.4 equivalent) were added to a solution of intermediate 19 (15.00mg, 7.18µmol, 1.0 equivalent) in DCM (1mL), and the mixture was stirred at 23°C for 18 hours. After this time, the reaction mixture was concentrated, and the crude residue was dissolved in methanol (0.25mL) and THF (0.25mL). Add a 0.5 mL solution of potassium carbonate (9.44 mg, 0.07 mmol, 10 equivalents) in water to the solution and stir the mixture at 23°C for 3 hours. After this time, concentrate the mixture under vacuum to remove organic matter. Acidify the remaining aqueous solution with citric acid (1N) until pH 4 is reached, and stir the mixture at 23°C for 1 hour. After this time, the mixture was filtered, and the residue was purified by reversed-phase rapid column chromatography (C18 BIOTAGE pre-packed column, 20%–40% MeCN [0.1% formic acid] / water [0.1% formic acid]) to obtain the yellow substance (2S,3S,4S,5R,6S)-6-(2-((3-((S)-6-(((3S,3aR,6S,6aR)-6-(2,5,8,11,14,17,20,23,26,29,32,35-dodecanooctane-38-amido)hexahydrofurano[3 [2-b]furan-3-yl)amino)-2-amino-6-oxohexamido)propamido)methyl)-4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indoleazine[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid intermediate 20 (8.6 mg, 5.03 µmol, 70%). RT 5.08 min. LCMS (ESI) 1702.6 [M+H]+.

[0300] Connector-Load Capacity LP-1

[0301]

[0302] Pyridine (0.7 µl, 5.5 µmol, 1.5 equivalent) and 2,5-dioxopyrrolidine-1-yl ester of 3-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)propionic acid (0.970 mg, 3.64 µmol, 1.0 equivalent) were added to a solution of intermediate 20 (6.20 mg, 3.64 µmol, 1.0 equivalent) in DMF (0.5 mL). The mixture was stirred at 23 °C for 3 hours. After this time, the reaction mixture was concentrated under vacuum, and the residue was purified by reversed-phase HPLC (CSH 35% MeCN [0.1% formic acid] / water [0.1% formic acid], for 7 minutes) to give a white substance (2S,3S,4S,5R,6S)-6-(2-((3-((S)-6-(((3S, 3S, 4S)-6-( ...6S)-6-(((3S)-6-(6S)-6-(((3S)-6-(6S)-6-(6S)-6-(6S)-6-(6S)-6-(6S)-6-(6S)-6-(6S)-6-(6S)-6-(6S)-6-(6S)-6-(6S 3aR,6S,6aR)-6-(2,5,8,11,14,17,20,23,26,29,32,35-dodecanooctane-38-amido)hexahydrofurano[3,2-b]furan-3-yl)amino)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)propamido)-6-oxohexano)propamido)methyl)-4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazido[1,2-b] Quinolino-1-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid LP-1 (2.7 mg, 1.46 µmol, 40%). RT 5.87 min. LCMS (ESI) 1853.5 [M+H]+.

[0303] Alternative synthesis of LP-1

[0304] Intermediate A

[0305]

[0306] Boc anhydride (83.3 g, 381.5 mmol, 2.5 equivalents) was added to a methanol (440 mL) solution of intermediate 9 (22.0 g, 152.6 mmol), and the reaction mixture was stirred at 20 °C for 2 h. ELSD analysis showed that the desired product was formed and completelyThe reaction proceeded. The solvent was removed under reduced pressure to obtain a crude product. The crude product was slurried with MTBE (200 mL), filtered, washed with MTBE (40 mL), and dried to give N-[(3S,3aR,6S,6aR)-6-(tert-butoxycarbonylamino)-2,3,3a,5,6,6a-hexahydrofurano[3,2-b]furan-3-yl]carbamate intermediate A (44.0 g, 84.3%). LCMS m / z 366.8 [M+Na]+

[0307] Intermediate B

[0308]

[0309] An ethyl acetate solution (4 M, 8.0 mL) of HCl was added to an ethyl acetate solution (4 mmol, 1.0 equivalent) of intermediate A (2.0 g, 5.81 mmol, 1.0 equivalent), and the reaction mixture was stirred at 20 °C for 6 hours. ELSD analysis showed that the desired product was formed and the reaction was complete. The reactants were quenched with potassium phosphate solution (2M, 20 mL), the layers were separated, and the organic layer was retained. The aqueous layer was extracted with ethyl acetate (10 mL), and the organic layers were combined. The solvent was removed under reduced pressure to give intermediate B (800 mg, 57.1%) of N-[(3S,3aR,6S,6aR)-3-amino-2,3,3a,5,6,6a-hexahydrofurano[3,2-b]furan-6-yl]carbamate tert-butyl ester. ¹H NMR (400 MHz, DMSO-d6) δ 4.32 (m, 1H), 3.93 (m, 1H), 3.85 (m, 2H), 3.6 (m, 2H), 3.28 (m, 1H), 1.47 (s, 9H). LCMS m / z 145 [M+H-Boc]+

[0310] Intermediate C

[0311]

[0312] HATU (16.8 g, 44.2 mmol, 1.2 equivalent) and DIPEA (10.9 mL, 62.6 mmol, 1.7 equivalent) were added to a solution of intermediate 14 (17.16 g, 40.5 mmol, 1.1 equivalent) in DCM (180 mL) at 25 °C. The reaction mixture was stirred for 15 min. A solution of intermediate B (9.0 g, 36.8 mmol, 1.0 equivalent) in DCM (90 mL) was added to the reaction solution. The reaction mixture was stirred for 2 h. LC-MS analysis showed that the desired product was formed and the reaction was complete. The reaction mixture was washed twice with NaCl (15% aqueous solution, 90 mL) and the aqueous layer was discarded. The solvent was removed under reduced pressure to obtain (2S)-6-[[(3S,3aR,6S,6aR)-6-(tert-butoxycarbonylamino)-2,3,3a,5,6,6a-hexahydrofurano[3,2-b]furan-3-yl]amino]-2-(9H-fluorene-9-ylmethoxycarbonylamino)-6-oxo-hexanoate allyl ester intermediate C (9.0 g, 65.2%). LCMS m / z 671.6 [M+Na]+ Specification 34 / 55 pages 41 CN 121175073 A

[0313] Intermediate D

[0314]

[0315] Formic acid (1.05 mL, 27.7 mmol, 2.0 equivalent), triethylamine (5.79 mL, 41.6 mmol, 3.0 equivalent) and Pd(PPh3)4 (1.6 g, 1.39 mmol, 0.1 equivalent) were added to a solution of intermediate C (9.0 g, 13.9 mmol, 1.0 equivalent) in DCM / MeOH (20:1, 128.6 mL: 6.4 mL). The reaction mixture was stirred at 20°C–25°C for 3 hours. LC-MS analysis showed the formation of the desired product and completion of the reaction. The solvent was removed under reduced pressure to give the crude product. The crude product was purified by silica gel column chromatography (DCM to DCM:MeOH 4:1) to give intermediate D (2S)-6-[[(3S,3aR,6S,6aR)-6-(tert-butoxycarbonylamino)-2,3,3a,5,6,6a-hexahydrofurano[3,2-b]furan-3-yl]amino]-2-(9H-fluorene-9-ylmethoxycarbonylamino)-6-oxohexanoic acid (7.0 g, 82.9%). LCMS m / z 610.1 [M+H]+

[0316] Intermediate E

[0317]

[0318] Intermediate 5 (11.2 g, 19.7 mmol, 3.0 equivalent), HATU (3.74 g, 9.84 mmol, 1.5 equivalent), and DIPEA (3.43 mL, 19.7 mmol, 3.0 equivalent) were added to a solution of intermediate D (4.0 g, 6.56 mmol, 1.0 equivalent) in DMF (80 mL) at 0 °C–5 °C. The reaction mixture was stirred at 0 °C–5 °C for 1 hour. LC-MS analysis showed that the desired product was formed and the reaction was complete. The reaction mixture was diluted with ethyl acetate (80 mL) and washed twice with NaCl (15% aqueous solution, 40 mL). The aqueous layer was discarded, and the solvent was removed from the organic layer under reduced pressure to give the crude product. The crude product was purified by silica gel column chromatography (EtOAC to 20% MeOH) to obtain (2S,3S,4S,5R,6S)-6-[2-[[3-[[(2S)-6-[[(3S,3aR,6S,6aR)-6-(tert-butoxycarbonylamino)-2,3,3a,5,6,6a-hexahydrofurano[3,2-b]furan-3-yl]amino]-2-(9H-fluorene-9-ylmethoxycarbonylamino)-6-oxo-hexanoyl]amino]propionylamino]methyl]-4-(hydroxymethyl)phenoxy[M+H]+ 3,4,5-Triacetoxy-tetrahydropyran-2-carboxylic acid allyl ester intermediate E (3.6 g, 47.4%) LCMS m / z 1158.2 [M+H]+

[0319] Intermediate F

[0320] Specification 35 / 55 pages 42 CN 121175073 A

[0321] Bis(4-nitrobenzene carbonate) (840 mg, 2.76 mmol, 4.0 equivalent) and DIPEA (481 μL, 2.76 mmol, 4.0 equivalent) were added to a solution of intermediate E (800 mg, 691 μmol, 1.0 equivalent) in DMF (8 mL). The mixture was stirred at 20 °C–25 °C for 2 hours. LC-MS analysis showed that the desired product was formed and the reaction was complete. The solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (5%–50% aqueous MeCN solution, 0.1% formic acid) to obtain intermediate F (400 mg, 43.8%) of (2S,3S,4S,5R, 6S)-6-[2-[[3-[[(2S)-6-[[(3S,3aR,6S,6aR)-6-(tert-butoxycarbonylamino)-2,3,3a,5,6,6a-hexahydrofurano[3,2-b]furan-3-yl]amino]-2-(9H-fluorene-9-ylmethoxycarbonylamino)-6-oxo-hexanoyl]amino]propionylamino]methyl]-4-[(4-nitrophenoxy)carbonyloxymethyl]phenoxy]-3,4,5-triacetoxy-tetrahydropyran-2-carboxylic acid allyl ester (400 mg, 43.8%) ¹H NMR (300 MHz, DMSO-d6) δ ppm 1.14 ‑ 1 .29 (m , 3 H) 1 .38 (s , 9 H) 1 .53 (br dd , J=18.52, 8.25Hz, 4 H) 1 .98 ‑ 2.09 (m , 12 H) 2.13 (d , J=2.93Hz, 1 H) 2.32 - 2.45 (m, 2 H) 3.35 (s, 11 H) 3.58 (br d, J=6.42Hz, 2 H) 3.71 (s, 12 H) 3.78 - 3.88 (m, 3 H) 3.90 - 4.17 (m, 4 H) 4.18 - 4.42 (m, 6 H) 4.50‑ 4.67 (m, 2 H) 4.80 (d , J=10.09Hz, 1 H) 5.09 ‑ 5.38 (m, 6 H) 5.47 ‑ 5.55 (m, 1 H) 5.65 (d , J=7.89Hz, 1 H) 5.82 ‑ 5.96 (m, 1 H) 6.10 (s , 4 H) 7 .11 (d ,J=8.44Hz, 1 H) 7 .21 (br d , J=5.50Hz, 1 H) 7 .28 ‑ 7.37 (m, 4 H) 7.37 ‑ 7.44 (m, 3 H) 7.46 ‑ 7.61 (m, 3 H) 7.65 ‑ 7.76 (m, 2 H) 7 .89 (d , J=7 .34Hz, 2 H) 7 .99 (br t, J=5.50Hz, 1 H) 8.09 (d , J=6.97Hz, 1 H) 8.27 ‑ 8.34 (m, 3 H) LCMS m / z 1323.1 [M+H]+

[0322] Intermediate G

[0323]

[0324] DIPEA (369 µl, 2.12 mmol, 4.0 equivalent), intermediate F (700 mg, 529 μmol, 1.0 equivalent), and HOPO (117.5 mg, 1.06 mmol, 2.0 equivalent) were added to a solution of ethathecan mesylate (1.12 g, 2.12 mmol, 4.0 equivalent) in DMF (7 mL), and the resulting mixture was stirred at 20 °C–25 °C for 1 h. LC-MS analysis showed the formation of the desired product and the completion of the reaction. The reaction mixture was diluted with ethyl acetate (9 mL) and washed with NaCl (15% aqueous solution, 12 mL). The solvent was removed under reduced pressure to give the crude product. The crude product was slurried with ethyl acetate / MTBE (1:1, 5 mL), filtered, and dried to give (2S,3S,4S,5R,6S)-6-[2-[[3-[[(2S)-6-[[(3S,3aR,6S,6aR)-6-(tert-butoxycarbonylamino)-2,3,3a ,5,6,6a-hexahydrofurano[3,2-b]furan-3-yl]amino]-2-(9H-fluorene-9-ylmethoxycarbonylamino)-6-oxo-hexanoyl]amino]propionylamino]methyl]-4-[[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexane[14.7.1.02,14.04,13.06,11.020,24] teicocarb-1,6(11) ,12,14,16(24) ,17,19-hepten-23-yl]carbamoyloxymethyl]phenoxy]-3, 4 5-Triacetoxy-tetrahydropyran-2-carboxylic acid allyl ester intermediate G (360 mg, 73.5%). ¹H NMR (400 MHz, DMSO-d6) δ ppm 0.81–0.94 (m, 2H) 1.11–1.26 (m, 3H) 1.37 (s, 6H) 1.42–1.59 (m,3 H) 1.81 - 1.92 (m, 1 H) 1.95 - 2.15 (m, 9 H) 2.29 - 2.44 (m, 3 H) 2.73 (s, 1 H) 2.89 (s, 1 H) 3.49 - 3.61 (m, 2 H) 3.71 (s, 16 H) 3.81 (br d, J =6.36Hz, 2 H) 3.85 - 3.95 (m, 1 H) 3.99 - 4.12 (m, 2 H) 4.12 - 4.28 (m, 2 H) Instruction manual 36 / 55 pages 43 CN 121175073 A 4.33 (br d , J=3.91Hz, 1 H) 4.39 (br d , J=4.16Hz, 1 H) 4.48 ‑ 4.64 (m , 1 H) 4.78 (br d , J=10.27Hz, 1 H) 5.06 ‑ 5.34 (m , 5 H) 5.41 ‑ 5.53 (m , 2 H) 5.60 (br d, J=7.58Hz, 1 H) 5.81 ‑ 5.93 (m, 1 H) 6.09 (s, 5 H) 6.92 (d, J=8.26Hz, 2 H) 7.09 (br d , J=8.56Hz, 1 H) 7.17 ‑ 7.42 (m, 5 H) 7.64 ‑ 7.79 (m, 2 H) 7.80 - 7.90 (m, 1 H) 7.93 - 7.99 (m, 1 H) 8.00 - 8.14 (m, 3 H) LCMS m / z 810.3 [M+ 2H]2+

[0325] Intermediate H

[0326]

[0327] K2CO3 (1.02 g, 7.41 mmol, 10.0 equivalent) and water (260 μL) were added to a solution of intermediate G (1.5 g 80%, 741 μmol, 1.0 equivalent) in MeOH (2.6 mL). The resulting mixture was stirred at 20 °C–25 °C for 1 hour. LC-MS analysis showed that the desired product was formed and the reaction was complete. The solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (5%–50% aqueous solution of MeCN, 0.1% formic acid) to obtain (2S,3S,4S,5R,6S)-6-[2-[[3-[[(2S)-6-[[(3S,3aR,6S,6aR)-6-(tert-butoxycarbonylamino)-2,3,3a,5,6,6a-hexahydrofurano[3,2-b]furan-[3-yl]amino]-2-amino-6-oxo-hexanoyl]amino]propionylamino]methyl]-4-[[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexane[14.7.1.02,14.04,13.06,11.020,24]tetracarbon-1,6(11),12,14,16(24),17,19-hepten-23-yl]carbamoyloxymethyl]phenoxy]-3,4,5-trihydroxy-tetrahydropyran-2-carboxylic acid intermediate H (450 mg, 67%) 1H NMR (500 MHz, DMSO-d6) δ ppm 0.84 0.92 (m, 3 H) 1.24 (br s, 4 H) 1.29 (br d , J=7.32Hz, 1 H) 1.38 (s, 8 H) 1.43 - 1.53 (m, 2 H) 1.60 (br s, 2 H) 1.81 - 2.11 (m, 5 H) 2.14 ‑ 2.24 (m, 2 H) 2.29 ‑ 2.36 (m, 1 H) 2.39 (s, 3 H) 2.40‑ 2.44 (m, 1 H) 3.13‑ 3.23 (m, 7 H) 3.23‑ 3.32 (m, 14 H) 3.35 (br s, 12H) 3.50‑ 3.60 (m, 8 H) 3.77 ‑ 3.85 (m, 3 H) 4.05 (br s, 1 H) 4.17 (br dd, J=13.43, 3.97Hz, 1 H) 4 .34 (br d , J=3 .36Hz , 1 H) 4 .39 (d , J=3 .97Hz, 1 H) 4.49 (br dd, J=13 .28, 7.78Hz, 1 H) 4.60 (br d, J=6.71Hz, 1 H) 5.03 (br d, J=12.21Hz, 1 H) 5.10 (br d, J=12.21Hz, 1 H) 5.28 (br s, 3 H) 5.45 (s, 2 H) 6.52 (br s, 1 H) 7.12 (d, J =8.24Hz, 1 H) 7.20 (br s, 1 H) 7.32 (s, 1 H) 7.32 ‑ 7.34 (m, 1 H) 7.40 (s, 1 H) 7.78 (d, J=10.68Hz, 1 H) 8.03-8.15 (m, 1H) 8.17 (s, 2 H) 8.24 (br d, J= 6.10Hz, 1 H) 8.47 (br s, 1 H) 9.30 (br s, 1 H). LCMS m / z 616.2 [M+2H]2+

[0328] Intermediate I Specification 37 / 55 pages 44 CN 121175073 A

[0329]

[0330] Pyridine (225μL) and N-succinimide 3-maleimide propionate (195mg, 731μmol, 2.0 equivalent) were added to a solution of intermediate H (450mg, 365μmol, 1.0 equivalent) in DMF (4.5mL) at 20℃-25℃, and the reaction mixture was stirred for 1 hour. LC-MS analysis showed that the desired product was formed and the reaction was complete. The solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (5%–50% aqueous solution of MeCN, 0.1% formic acid) to obtain (2S, 3S, ... 4S,5R,6S)-6-(2-((3-((S)-6-(((3S,3aR,6S,6aR)-6-((tert-butoxycarbonyl)amino)hexahydrofurano[3,2-b]furan-3-yl)amino)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)propamido)-6-oxohexamido)propamido)methyl)-4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1- Intermediate I of (230 mg, 52.4%) carbamoyloxymethylphenoxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (230 mg, 52.4%) ¹H NMR (500 MHz, DMSO-d6) δ ppm 0.84–0.91 (m, 2H) 1.23 (br d, J = 9.77 Hz, 5H) 1.38 (s, 6H) 1.81–1.94 (m, 1H) 1.96–2.07 (m, 2H) 2.13–2.24 (m, 1H) 2.25–2.36 (m, 1H) 2.50–2.52 (m, 7H) 3.05–3.18 (m, 1 H) 3.24 (br d, J=6.10Hz, 2 H) 3.29 (br s, 3 H) 3.34 (br s, 24 H) 3.51 - 3.63 (m, 4 H) 3.71 (s, 9 H) 3.75 -3.88 (m, 3 H) 4.01 ‑ 4.15 (m, 2 H) 4.21 (br d , J= 9.46Hz, 1 H) 4.34 (d , J=3.66Hz, 1 H) 4.36 ‑ 4.46 (m , 1 H) 4.72 (br s , 1 H) 5.06 - 5.15 (m, 1 H) 5.27 (br s, 2 H) 5.45 (s, 2 H) 6.09 (s, 3 H) 6.51 (s, 1 H) 6.97 (s, 1 H) 7.10 (d , J=8.24Hz, 1 H) 7.19 (br d , J=5.80Hz, 1 H) 7.31 (s, 1 H) 7.33 (s, 1 H) 7.77 (d, J=10.99Hz, 1 H) 7.94 (br t, J=5.34Hz, 1 H) 8.06 (br d, J=8.55Hz, 1 H) 8.12 (br d, J=6.71Hz, 1 H) 8.20 (br d, J=8.24Hz, 1 H) LCMS m / z 1382.1 [M+H]+

[0331] Intermediate J

[0332]

[0333] TFA (1.3 mL) was added to a solution of intermediate I (230 mg, 166 μmol, 1.0 equivalent) in DCM (2.6 mL), and the reaction mixture was stirred at 25 °C for 1 hour. LC-MS analysis showed that the desired product was formed and the reaction was complete. The solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (5%–50% aqueous solution of MeCN, 0.1% formic acid), [Instructions for Use, pages 38 / 55, CN 121175073 A] to obtain (2S,3S,4S,5R,6S)-6-[2-[[3-[[(2S)-6-[[(3S,3aR,6S,6aR)-3-amino-2,3,3a,5,6,6a-hexahydrofurano[3,2-b]furan-6-yl]amino]-2-[3-(2,5-dioxopyrrolo-1-yl)propionylamino]-6- [Oxy-hexanoyl]amino]propionylamino]methyl]-4-[[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexane[14.7.1 .02,14.04,13.06,11 .020,24]tetraco-1,6(11),12,14,16(24),17,19-hepten-23-yl]carbamoyloxymethyl]phenoxy]-3,4,5-trihydroxyIntermediate J (120 mg, 54.5%) of tetrahydropyran-2-carboxylic acid. LCMS m / z 641.5 [M+H]+

[0334] Connector-load LP-1

[0335]

[0336] NaHCO3 (13.1 mg, 156 μmol, 2.0 equivalent) and 2,5,8,11,14,17,20,23,26,29,32,35-dodecoxaoctane-38-acid 2,5-dioxopyrrolidine-1-yl ester intermediate 10 (64.2 mg, 93.6 μmol, 1.2 equivalent) were added to a solution of intermediate J (100 mg, 78.0 μmol, 1.0 equivalent) in THF (1 mL). The reaction mixture was stirred at 20 °C–25 °C for 16 hours. LC-MS analysis showed that the desired product was formed and the reaction was complete. The solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (5%–50% aqueous solution of MeCN, 0.1% formic acid) to obtain (2S,3S,4S,5R,6S)-6-(2-((3-((S)-6-(((3S,3aR,6S,6aR)-6-(2,5,8,11,14,17,20,23,26,29,32,35-dodecoxaoctane-38-amido)hexahydrofurano[3,2-b]furan-3-yl)amino)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)propamido)-6-oxohexamido)propamido)methyl)-4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3, 9,10,13,15-Hexahydro-1H,12H-Benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid LP-1 (60 mg, 40%). LCMS m / z 927.2 [M+2H]2+

[0337] Synthesis of LP-2

[0338] Intermediate 38

[0339]

[0340] HATU (1.0 g, 2.63 mmol) and DIPEA (1.06 mL, 6.07 mmol) were added to a solution of (S)-5-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-6-((3-(allyloxy)-3-oxopropyl)amino)-6-oxohexanoic acid (1.0 g, 2.02 mmol) in DCM (20 mL). The reaction mixture was stirred at 20 °C for 15 minutes, and then (1S,4S)-2,5-dioxopropyl) was added.The reaction mixture was prepared with tert-butyl heptane-2-carboxylate (0.481 g, 2.43 mmol) and stirred for another 2 hours. The reaction mixture was diluted with EtOAc (2 × 300 mL), and the organic layer was washed with water (1 × 300 mL), followed by washing with salt water (1 × 200 mL). The mixture was dried over magnesium sulfate and then the solvent was removed under reduced pressure. Purified by rapid column chromatography (80%–100% EtOAc / hexane) to provide (1S,4S)-5-((S)-5-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-6-((3-(allyloxy)-3-oxopropyl)amino)-6-oxohexanoyl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid tert-butyl ester intermediate 38 (1.250 g, 1.85 mmol, 92% yield). 1H NMR (500MHz, CDCl3) δ 7.76 (d, J = 7.6Hz, 2H), 7.62 (d, J = 7.5Hz, 2H), 7.40 (t, J = 7.5Hz, 2H), 7.31 (t, J = 7.5Hz, 2H), 6.00 (d, J = 7.8Hz, 1H), 5.89 (m, 1H), 5.30–5.18 (m, 2H), 4.91 (d, J = 10.9Hz, 1H), 4.65–4.47 (m, 3H), 4.46–4.31 (m, 3H), 4.22 (t , J = 7.2Hz, 1H), 4.10 (d, J = 9.9Hz, 1H), 3.58–3.32 (m, 6H), 2.60 (t, J = 6.9Hz, 2H), 2.38–2.16 (m, 2H), 1.94–1.56 (m, 6H), 1.46 (q, J = 6.2Hz, 9H); LCMS (ESI) m / z [M+H]+ 675.4.

[0341] Intermediate 39

[0342]

[0343] Triphenylphosphine (0.039 g, 0.15 mmol) was added to a solution of intermediate 38 (1.0 g, 1.48 mmol) in DCM (20 mL), followed by the addition of triethylamine (0.310 mL, 2.22 mmol), and then tetrakis(triphenylphosphine)palladium(O) (0.171 g, 0.15 mmol). The reaction mixture was stirred at 20 °C for 5 hours. The reaction mixture was then analyzed by rapid column chromatography (0%–15%).MeOH:DCM) was purified to provide 39 intermediates of 3-((S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-6-((1S,4S)-5-(tert-butoxycarbonyl)-2,5-diazabicyclo[2.2.1]hept-2-yl)-6-oxohexamido)propionic acid as a white solid (0.8 g, 1.26 mmol, 85% yield). 1H NMR (500MHz, CDCl3) δ 7.83 (d, J = 7.5Hz, 2H) , 7.70 (t, J = 9.1Hz, 2H) , 7.42 (t, J = 7.5Hz, 2H) , 7.40–7.27 (m, 3H) , 5.32–5.14 (m, 1H) , 4.73–4.63 (m , 1H) , 4.55 (d , J = 26.8Hz, 1H) , 4.42 (d , J = 8.4Hz, 2H) , 4.26 (t, J = 6.9Hz, 1H) , 4.09 (d , J = 8.7Hz, 1H) , 3.60 (d , J = 9.7Hz, 1H), 3.54–3.40 (m, 5H), 3.33–3.18 (m, 2H), 2.53 (m, 3H), 2.38 (dt, J = 15.5, 7.2Hz, 1H), 2.29 (d, J = 7.4Hz, 1H), 2.01–1.55 (m, 6H), 1.55–1.42 (m, 9H). LCMS (ESI) m / z [M+H]+ 635.3

[0344] Intermediate 40

[0345]

[0346] Trifluoroacetic acid (0.12 mL, 1.58 mmol) was added to a solution of intermediate 39 (1.0 g, 1.58 mmol) in DCM (20 mL). The reaction mixture was stirred at 20 °C for 5 hours. The mixture was purified by rapid column chromatography (0%-15% MeOH:DCM) to provide 3-((S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-6-((1S,4S)-2,5-diazabicyclo[2.2.1]hept-2-yl)-6-oxohexamido)propionic acid intermediate 40 (0.76 g, 1.42 mmol, 90% yield) as a colorless gel. ¹H NMR (500MHz, MeOD) δ 7.82 (d, J = 7.5Hz, 2H), 7.69 (dd, J = 10.0, [Instructions for use] 40 / 55 pages 47 CN]121175073 A 7.4Hz, 2H) , 7.41 (t, J = 7.5Hz, 2H) , 7.33 (tt, J = 7.5, 1.6Hz, 2H) , 5.70–5.61 (m, 1H) , 4.82 (s, 1H) , 4.50 (d , J = 16.9Hz, 1H), 4.46–4.35 (m, 2H), 4.24 (t, J = 6.8Hz, 1H), 4.06 (ddd, J = 13.0, 8.2, 5.2Hz, 1H), 3.89 (d, J = 6.7Hz, 1H) , 3.78–3.65 (m, 1H) , 3.61–3.52 (m, 2H) , 3.53–3.36 (m, 4H) , 3.34 (s, 1H) , 2.64–2.47 (m, 3H) , 2.46–2.33 (m, 1H) , 2.22–2.11 (m, 1H) , 2.01 (dt, J = 13.3, 9.5Hz, 2H) , 1.84 – 1.70 (m, 2H) , 1.66 (h, J = 6.3Hz, 2H). LCMS (ESI) m / z [M+H] + 535.8.

[0347] Intermediate 41

[0348]

[0349] Triethylamine (0.645 mL, 4.63 mmol) was added to a solution of intermediate 40 (1.0 g, 1.54 mmol) in DCM (20 mL), followed by the addition of 2,5,8,11,14,17,20,23,26,29,32,35-dodecanoic acid 2,5-dioxopyrrolidine-1-yl ester (1.269 g, 1.85 mmol). The reaction mixture was stirred at 20 °C for 4 hours. Purified by rapid column chromatography (0%-15% MeOH:DCM) to provide 3-((S)-6-((1S,4S)-5-(2,5,8,11,14,17,20,23,26,29,32,35-dodecanoic acid-38-acyl)-2,5-diazabicyclo[2.2.1]hept-2-yl)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-6-oxohexamido)propionic acid intermediate 41 (1.12 g, 1.01 mmol, 95% yield). LCMS (ESI) m / z (M+NH4)+ 1122.7.

[0350] Intermediate 42

[0351]

[0352] To intermediate 41 (0.4 g, 0HATU (0.186 g, 0.49 mmol) and DIPEA (0.2 mL, 1.15 mmol) were added to a solution in DCM (10 mL). The mixture was stirred at 20 °C for 15 min, and then triacetic acid (2S, 3S, 4S, 5R, 6S)-2-((allyloxy)carbonyl)-6-(2-(aminomethyl)-4-(hydroxymethyl)phenoxy)tetrahydro-2H-pyran-3,4,5-trimethyl ester hydrochloride (intermediate 4.HCl) (0.198 g, 0.37 mmol) was added in a single batch. The mixture was stirred for another 30 min. Water (20 mL) was added and the mixture was stirred for another 30 min. The layers were separated, and the aqueous layer was extracted with DCM (3 × 20 mL). The combined organic layers were washed with brine (1 × 50 mL). The mixture was dried over Na2SO4, and then the solvent was evaporated under vacuum. The triacetic acid (2S,3R,4S,5S,6S)-2-(2-((S)-5-(4-((1S,4S)-5-(2,5,8,11,14,17,20,23,26,29,32,35-dodecanoic acid-38-acyl)-2,5-diazabicyclo[2.2.1]hept-2-yl)-4-oxobutyl)-1-(9H-fluorene-9-yl)-3,6,10-trioxo-2-oxa-4,7,11-triazadodecane- 12-yl)-4-(hydroxymethyl)phenoxy)-6-((allyloxy)carbonyl)tetrahydro-2H-pyran-3,4,5-trimethyl ester intermediate 42 Instructions for use 41 / 55 pages 48 CN 121175073 A (300.9 mg, 0.19 mmol, 52.5% yield). 1H NMR (DMSO‑d6) δ: 8.19 (br s, 1H) , 7.96 (br d , J=5.5Hz, 1H) , 7.88 (d , J=7.5Hz, 2H) , 7.72 (t, J=6.4Hz, 2H) , 7.47 (br d , J= 10.1Hz, 1H), 7.41 (t, J=7.5Hz, 2H), 7.32 (t, J=7.5Hz, 2H), 7.16 (br d, J= 8.3Hz, 1H), 7.12 (s, 1H), 7.00 (d, J=8.3Hz, 1H) , 5.83‑5.93 (m, 1H) , 5.45‑5.56 (m, 2H) , 5.22‑5.35 (m, 2H) , 5.07‑5.17 (m, 3H) , 4.65‑4.77 (m, 3H) ,4.50‑4.65 (m , 3H) , 4.41 (d , J=5.6Hz, 2H) , 4.16‑4.29 (m , 4H) , 4.03‑4.13 (m , 1H) , 3.93 (br d , J=8.3Hz , 1H) , 3.56‑3 .65 (m, 2H), 3.46-3.53 (m, 43H), 3.39-3.44 (m, 3H), 3.33-3.34 (m, 1H), 3.21-3.25 (m, 5H), 3.11-3.20 (m, 2H), 2.65‑2.68 (m, 1H) , 2.52‑2.55 (m, 2H) , 2.29‑2.46 (m, 6H) , 2.22 (br d , J=14.9Hz, 1H) , 2.06‑ 2.15 (m, 2H) , 1.94‑2.05 (m, 9H) , 1.85 (br s, 1H) , 1.77 (br s, 1H) , 1.71 (br s, 2H) , 1.52 (br d , J=7.6Hz, 4H) , 1.23 (s, 1H) , 1.14 (s, 1H). LCMS (ESI) m / z [M+H]+ 1581.7.

[0353] Intermediate 43

[0354]

[0355] Bis(4-nitrobenzene carbonate) (115 mg, 0.38 mmol) was added to a solution of intermediate 42 (100 mg, 0.06 mmol) in DMF (1.0 mL) and DCM (1.0 mL) at 25 °C, followed by the addition of DIPEA (0.055 mL, 0.32 mmol). The reaction mixture was then stirred at 21 °C for 1 hour. After this time, the reaction mixture was concentrated under vacuum, and the residue was wet-milled in diethyl ether (5 mL). The resulting solid was then dissolved in DCM (2 mL), and diethyl ether (10 mL) was added to the resulting solution. The mixture was sonicated and centrifuged, and the supernatant was removed. This process was repeated twice, recovering the resulting solid after each cycle, until no further precipitate formed. This yields a white solid triacetic acid (2S,3R,4S,5S,6S)-2-(2-((S)-5-(4-((1S,4S)-5-(2,5,8,11,14,17,20,23,26,29,32,35-dodecoxaoctane-38-acyl)-2,5-diazabicyclo[2.2.1]hept-2-yl)-4-oxobutyl)-1-(9H-fluorene-9-yl)-3,6,10-trioxo-2-Oxa-4,7,11-triazadodecane-12-yl)-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-((allyloxy)carbonyl)tetrahydro-2H-pyran-3,4,5-trimethyl ester intermediate 43 (100 mg, 0.06 mmol, 91% yield), was used directly in the next step without further purification. LCMS (ESI) m / z [M+H]+ 1748.5.

[0356] Intermediate 44 Specification 42 / 55 pages 49 CN 121175073 A

[0357]

[0358] To a solution of intermediate 43 (75 mg, 0.04 mmol) in DCM (1 mL), a solution of 2-hydroxypyridine 1-oxide (5.24 mg, 0.05 mmol) and ethatecan mesylate (23.95 mg, 0.05 mmol) in DMF (1 mL) and DIPEA (0.022 mL, 0.13 mmol) was added. The resulting mixture was stirred at 21 °C for 18 hours. After this time, the reaction mixture was concentrated under vacuum, and the residue was purified by reversed-phase rapid column chromatography (30%–50% MeCN / water [0.1% formic acid]) to give triacetic acid (2S,3R,4S,5S,6S)-2-(2-((S)-5-(4-((1S,4S)-5-(2,5,8,11,14,17,20,23, 26,29,32,35-dodecanoic acid-38-acyl)-2,5-diazabicyclo[2.2.1]hept-2-yl)-4-oxobutyl)-1-(9H-fluorene-9-yl)-3,6,10-trioxo-2-oxa-4,7,11-triazadodecanoic acid-12-yl)-4-(((((1S, 9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenoxy)-6-((allyloxy)carbonyl)tetrahydro-2H-pyran-3,4,5-trimethyl ester intermediate 44 (43.0 mg, 0.021 mmol, 49%). LCMS (ESI) m / z [M+H]+ 2044.2.

[0359] Intermediate 45

[0360]

[0361] Formic acid (1.587 µl, 0.04 mmol), triethylamine (5.86 µl, 0.04 mmol), and Pd(Ph3P)4 (2.431 mg, 2.10 µmol) were added to a solution of intermediate 44 (43.0 mg, 0.02 mmol) in DCM (2 mL). The reaction mixture was then stirred.The mixture was stirred at 21°C for 18 hours. After this time, the reaction mixture was concentrated under vacuum to obtain (2S,3S,4S,5R,6S)-6-(2-((S)-5-(4-((1S,4S)-5-(2,5,8,11,14,17,20,23,26,29,32,35-dodecanoic acid-38-acyl)-2,5-diazabicyclo[2.2.1]hept-2-yl)-4-oxobutyl)-1-(9H-fluorene-9-yl)-3,6,10-trioxo-2-oxa-4,7,11-triazadodecanoic acid-12-yl)-4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl- 10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-triacetoxytetrahydro-2H-pyran-2-carboxylic acid intermediate 45 (37.0 mg, 0.02 mmol, 88%) was used in the next step without further purification. LCMS (ESI) m / z [M+H]+ 2005.8.

[0362] Intermediate 46

[0363]

[0364] To a solution of intermediate 45 (37.0 mg, 0.02 mmol) in THF (0.5 mL), an aqueous solution of potassium carbonate (10.51 µl, 0.18 mmol) and water (0.5 mL) were added. The resulting reaction mixture was stirred at 21 °C for 36 hours. After this time, an aqueous solution of citric acid (1 M) was added until the pH was less than 7.0. The reaction mixture was directly purified by reverse-phase fast column chromatography (20%–40% MeCN / water [0.1% formic acid]) to give a colorless gel-like substance (2S,3S,4S,5R,6S)-6-(2-((3-((S)-6-((1S,4S)-5-(2,5,8,11,14,17,20,23,26,29,32,35-dodecoxaoctane-38-acyl)-2,5-diazabicyclo[2.2.1]hept-2-yl)-2-amino-6-oxohexamido)propamido)methyl)-4-(((((1S,9S)-9- Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxyIntermediate 46 of tetrahydro-2H-pyran-2-carboxylic acid (10 mg, 6.04 µmol, 32.7% yield). LCMS (ESI) m / z [M+H]+ 1656.7.

[0365] LP-2

[0366]

[0367] A solution of sodium bicarbonate (0.761 mg, 9.06 µmol) in water (1 mL) was added to a solution of intermediate 46 (10 mg, 6.04 µmol) and 2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)propionic acid 2,5-dioxopyrrolo-1-yl ester (1.929 mg, 7.25 µmol) in THF (1 mL), and the reaction mixture was stirred at 21 °C for 3 hours. After this time, the reaction mixture was concentrated under vacuum to remove THF, and the remaining aqueous solution was directly purified by reversed-phase rapid column chromatography (30%–40% MeCN / water [0.1% formic acid]) to give (2S,3S,4S,5R,6S)-6-(2-((S)-6-((1S,4S)-5-(2,5,8,11,14,17,20,23,26,29,32,35-dodecoxaoctane-38-) as a white solid. Acyl)-2,5-diazabicyclo[2.2.1]hept-2-yl)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)propamido)-6-oxohexamido)propamido)methyl)-4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid LP-2 (7.5 mg, 4.15 µmol, 68.7%). LCMS (ESI) m / z [M-H] - 1806.6.

[0368] Synthesis of LP-3

[0369]

[0370] The reference connector-load LP-3 was prepared by a synthesis procedure similar to that disclosed above.

[0371] Synthesis of LP-4

[0372]

[0373] 2,5-dioxopyrrolidine-1-yl ester of 3-(2-bromoacetamyl)propionic acid (65.0 mg, 0.21 mmol) was added to a solution of intermediate 20 (300 mg, 0.18 mmol) and DIEA (0.031 mL, 0.18 mmol) in DMA (5 mL) under nitrogen at 25 °C. The resulting mixture was stirred at 25 °C for 2 hours.

[0374] The crude mixture was placed directly onto a C18 rapid chromatograph for the first purification: the elution gradient was 0% to 100% MeCN / water (0.1% FA). The pure fraction was evaporated to dryness to obtain the crude product. The crude product was purified by preparative HPLC (column: Sunfire Prep C18 OBD column, 19 × 250 mm, 5 μm; mobile phase A: water (0.1% FA), mobile phase B: ACN; flow rate: 25 mL / min; gradient: 32% B to 36% B over 15 min; wavelength: 254 nm / 220 nm; RT1 (min): 13.72) with MeCN as the eluent. The fraction containing the desired compound was directly freeze-dried to dryness to obtain a yellow solid: (2S,3S,4S,5R,6S)-6-(2-((S)-8-(4-(((3S,3aR,6S,6aR)-6-(2,5,8,11,14,17,20,23,26, 29,32,35-dodecanooctane-38-amido)hexahydrofurano[3,2-b]furan-3-yl)amino)-4-oxobutyl)-15-bromo-3,7,10,14-tetraoxo-2,6,9,13-tetraazapentadecanyl)-4-(((((1S,9S)-9-ethyl-5- Fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3', 4':6,7]indoleazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydrogen-2H-pyrano-2-carboxylic acid (LP-4) (47.0 mg, 14.08%). Specification page 45 / 55, CN 121175073 A. 1H NMR (400MHz, DMSO) δ 0.88 (t, J = 7.2Hz, 3H), 1.38 – 1.60 (m, 4H), 1.79 – 1.95 (m, 2H), 2.03 (d, J = 7.2Hz, 2H), 2.18 (s, 2H) , 2.31 (d, J = 8.0Hz, 6H) , 2.38 (d, J = 2.0Hz, 3H) , 3.14 (s, 1H) , 3.25 – 3.32 (br, 7H) , 3.31 (s, 3H) , 3.40 – 3.44 (m, 3H) , 3.46 – 3.48 (m, 3H) , 3.50 – 3.53 (m, 37H) , 3.55 – 3.62 (m, 5H) , 3.82 (d, J = 6.0Hz, 5H), 4.01 – 4.11 (m, 3H) , 4.17 (d , J = 6.8Hz, 1H) , 4.30 (s, 2H) , 4.36 (s, 3H) , 4.94 – 5.15 (m, 3H) , 5.29 (s, 4H) , 5.46 (s, 3H) , 5.56 (d , J = 4.0Hz, 1H) , 6.55 (s, 1H) , 7.09 (d , J = 8.0Hz, 1H) , 7.18 – 7.26 (m, 1H) , 7.26 – 7.35 (m, 2H) , 7.75 – 7.81 (m , 1H) , 7 .92 – 7 .96 (br, 1H) , 8.06 (s , 1H) , 8.12 (s , 1H) , 8.17 (s , 1H) , 8.25 – 8.31 (br, 2H) (exchange one proton) ES+(M+1 =1894)

[0375] Antibody-Drug Conjugation

[0376] ADC-1: Herceptin-WT-LP-1 (DAR 8)

[0377] LP-1 was added as a DMSO solution (16 molar equivalents / antibody, 0.64 μmol, in 0.128 mL DMSO) to 1.82 mL of Herceptin-wild-type antibody in PBS, 1 mM EDTA (pH 7.4) (6.0 mg, 40.0 nanomoles), with a final DMSO concentration of 10% (v / v). The solution was allowed to react at room temperature for 1 hour with gentle shaking. The conjugation was then quenched by adding N-acetylcysteine ​​(3.2 μmol, 32.03 μL, 100 mM), and then purified and prepared in PBS pH 7.4 by rotary filtration using a 15 mL AMICON ULTRACELL 30 kDa MWCO rotary filter, aseptically filtered, and analyzed.

[0378] UHPLC analysis of the reduced ADC sample at 214 nm and 330 nm using a THERMO SCIENTIFIC MAbPac 50 mm × 2.1 mm column on a SHIMADZU PROMINENCE system with gradient elution of water and acetonitrile showed a mixture of light chains conjugated with 1 LP-1 molecule and heavy chains conjugated with 3.0 LP-1 molecules, consistent with a drug / antibody ratio (DAR) of 7.9 LP-1 molecules / antibody.

[0379] A Toosoh BIOSCIENCE TSKgel SuperSW mAb HTP 4µm 4.6×150mm column (with 4µm 3) was used on the SHIMADZU PROMINENCE system.A 0×20 mm guard column was used, and the sample was eluted with SEC buffer containing 200 mM potassium phosphate (pH 6.95), 250 mM potassium chloride, and 10% isopropanol (v / v) filtered aseptically at 0.3 mL / min. UHPLC analysis of the ADC sample at 280 nm showed a monomer purity of 97.9%. UHPLC SEC analysis yielded a final ADC concentration of 2.86 mg / mL in 1.6 mL, with a yield of 4.5 mg (75% yield).

[0380] LC-MS analysis of the deglycosylated and reduced ADC samples at 214 nm using a THERMO SCIENTIFIC MAbPac 50 mm × 2.1 mm column on an EXACTIVE PLUS EMR mass spectrometer connected to a DIONX 3000 HPLC system with gradient elution of water and acetonitrile showed a mixture of light chains conjugated with 1 LP-1 molecule and heavy chains conjugated with 3.0 LP-1 molecules, consistent with a drug / antibody ratio (DAR) of 7.9 LP-1 molecules / antibody.

[0381] Using a PROTEOMIX HIC Butyl-NP5, 5 μm, non-porous, 4.6 × 35 mm (SEPAX) column on the SHIMADZU PROMINENCE system, eluted with a gradient of 1.5 M ammonium sulfate, 25 mM sodium acetate pH 7.4 and 25 mM sodium acetate pH 7.4 with 20% acetonitrile (v / v), UHPLC analysis of the pure ADC sample at 214 nm showed a single conjugated LP-1, consistent with the drug / antibody ratio (DAR) of 8 LP-1 molecules / antibody.

[0382] ADC-2: Herceptin-WT-LP-2 (DAR 8)

[0383] LP-2 was added as a DMSO solution (13 molar equivalents / antibody, 1.3 μmol, in 0.130 mL DMSO) to 3.0 mL of Herceptin-wild-type antibody in PBS, 1 mM EDTA (pH 7.4) (15.0 mg, 100.0 nanomolars), resulting in a final DMSO concentration of 10% (v / v). The solution was allowed to react at room temperature for 1 hour with gentle shaking. The conjugation was then quenched by adding N-acetylcysteine ​​(6.5 μmol, 65.0 µL, 100 mM), and then purified by SECprep-AKTA in PBS pH 7.4 (see instructions on pages 46 / 55, CN 121175073 A). The solution was prepared by rotary filtration using a 15 mL AMICON ULTRACELL 30 kDa MWCO rotary filter in 20 mM His / His HCl, 240 mM sucrose pH 6.0, aseptically filtered, and analyzed.

[0384] In SHIMADZUUHPLC analysis of the reduced ADC sample at 214 nm and 330 nm using a THERMO SCIENTIFIC MAbPac 50 mm × 2.1 mm column on the PROMINENCE system with gradient elution of water and acetonitrile showed a mixture of light chains conjugated to 1 LP-2 molecule and heavy chains conjugated to 3.0 LP-2 molecules, consistent with a drug / antibody ratio (DAR) of 7.98 LP-2 molecules / antibody.

[0385] Using a TOSOH BIOSCIENCE TSKgel SuperSW mAb HTP 4µm 4.6×150mm column (with a 4µm 3.0×20mm guard column) on a SHIMADZU PROMINENCE system, the sample of ADC was eluted with SEC buffer containing 200mM potassium phosphate pH 6.95, 250mM potassium chloride and 10% isopropanol (v / v) filtered aseptically at 0.3 mL / min. UHPLC analysis of the sample at 280 nm showed a monomer purity of 99.23%.

[0386] LC-MS analysis of the deglycosylated and reduced ADC samples at 214 nm using a THERMO SCIENTIFIC MAbPac 50 mm × 2.1 mm column on an EXACTIVE PLUS EMR mass spectrometer connected to a DIONX 3000 HPLC system with gradient elution of water and acetonitrile showed a mixture of light chains conjugated with 1 LP-2 molecule and heavy chains conjugated with 3.0 LP-2 molecules, consistent with a drug / antibody ratio (DAR) of 8.0 LP-2 molecules / antibody.

[0387] Using a PROTEOMIX HIC Butyl-NP5, 5 μm, non-porous, 4.6 × 35 mm (Sepax) column on the SHIMADZU PROMINENCE system, eluted with a gradient of 1.5 M ammonium sulfate, 25 mM sodium acetate pH 7.4 and 25 mM sodium acetate pH 7.4 with 20% acetonitrile (v / v), UHPLC analysis of the pure ADC sample at 214 nm showed a single conjugation to LP-2, consistent with the drug / antibody ratio (DAR) of 8 LP-2 molecules / antibody.

[0388] ADC-3: Herceptin-WT-LP-3 (DAR 8) (Reference)

[0389] LP-3 was added as a DMSO solution (14 mol equivalents / antibody, 1.4 μmol in 0.39 mL DMSO) to 6.0 mL of Herceptin-wild-type antibody in PBS, 1 mM EDTA (pH 7.4) (30.0 mg, 200.0 nanomoles), resulting in a final DMSO concentration of 10% (v / v). The solution was allowed to react at room temperature for 1 hour with gentle shaking. Conjugation was then performed by adding...N-acetylcysteine ​​(14 μmol, 130.0 µL, 100 mM) was used to quench the ADC, which was then purified in PBS pH 7.4 by SECprep-AKTA and prepared by rotary filtration in 20 mM His / His HCl and 240 mM sucrose pH 6.0 using a 15 mL AMICON ULTRACELL 30 kDa MWCO rotary filter. The ADC was aseptically filtered and analyzed.

[0390] UHPLC analysis of the reduced ADC sample at 214 nm and 330 nm using a THERMO SCIENTIFIC MAbPac 50 mm × 2.1 mm column on a SHIMADZU PROMINENCE system with gradient elution of water and acetonitrile showed a mixture of light chain conjugated with 1 LP-3 molecule and heavy chain conjugated with 3.0 LP-3 molecules, consistent with a drug / antibody ratio (DAR) of 7.89 LP-3 molecules / antibody.

[0391] Using a TOSOH BIOSCIENCE TSKgel SuperSW mAb HTP 4µm 4.6×150mm column (with a 4µm 3.0×20mm guard column) on a SHIMADZU PROMINENCE system, the sample of ADC was eluted with SEC buffer containing 200mM potassium phosphate pH 6.95, 250mM potassium chloride and 10% isopropanol (v / v) filtered aseptically at 0.3 mL / min. UHPLC analysis of the sample at 280 nm showed a monomer purity of 98.67%.

[0392] LC-MS analysis of the deglycosylated and reduced ADC samples at 214 nm using a THERMO SCIENTIFIC MAbPac 50 mm × 2.1 mm column on an EXACTIVE PLUS EMR mass spectrometer connected to a DIONX 3000 HPLC system with gradient elution of water and acetonitrile showed a mixture of light chains conjugated with 1 LP-3 molecule and heavy chains conjugated with 3.0 LP-3 molecules, consistent with a drug / antibody ratio (DAR) of 8.0 LP-3 molecules / antibody.

[0393] Using a PROTEOMIX HIC Butyl-NP5, 5 μm, non-porous, 4.6 × 35 mm (Sepax) column on the SHIMADZU PROMINENCE system, eluted with a gradient of 1.5 M ammonium sulfate, 25 mM sodium acetate pH 7.4, and 25 mM sodium acetate pH 7.4 with 20% acetonitrile (v / v) (see page 47 / 55 of the instruction manual, CN 121175073 A), UHPLC analysis of the pure ADC sample at 214 nm showed a single conjugation to LP-3, consistent with the drug / antibody ratio (DAR) of 8 LP-3 molecules / antibody.

[0394] ADC-4: Herceptin-WT-LP-4 (DAR 8)

[0395] LP-4 was added as a DMSO solution (25 molar equivalents / antibody, 4.0 μmol, in 0.166 mL DMSO) to 2.0 mL of Herceptin-wild-type antibody in PBS, 1 mM EDTA, 15% borate buffer pH 8.3 (20.0 mg, 133.0 nanomolars), resulting in a final DMSO concentration of 10% (v / v). The solution was allowed to react at room temperature for 1 hour with gentle shaking. Purification was performed in PBS pH 7.4 using AKTA-SECPrep and aseptically filtered and analyzed using a 15 mL Amicon Ultracell 30 kDa MWCO rotary filter in 20 mM histidine / histidine HCl, 240 mM sucrose pH 6.0 via rotary filtration.

[0396] Using a Thermo Scientific MAbPac 50mm × 2.1mm column on a Shimadzu Prominence system, with gradient elution of water and acetonitrile, UHPLC analysis of the reduced ADC sample at 214nm and 330nm showed a mixture of light chains conjugated with 1 LP-4 molecule and heavy chains conjugated with 3.0 LP-4 molecules, consistent with a drug / antibody ratio (DAR) of 7.87 LP-4 molecules / antibody.

[0397] Using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4µm 4.6×150mm column (with a 4µm 3.0×20mm guard column) on a Shimadzu Prominence system, the sample was eluted with SEC buffer containing 200mM potassium phosphate (pH 6.95), 250mM potassium chloride, and 10% isopropanol (v / v) filtered aseptically at 0.3 mL / min. UHPLC analysis of the ADC sample at 280 nm showed a monomer purity of 99.68%. UHPLC SEC analysis yielded a final ADC concentration of 4.92 mg / mL in 3.5 mL, with a yield of 17.2 mg (86% yield).

[0398] LC-MS analysis of the deglycosylated and reduced ADC samples at 214 nm using a Thermo Scientific MAbPac 50 mm × 2.1 mm column on an Exactive Plus EMR mass spectrometer connected to a Dionex 3000 HPLC system with gradient elution of water and acetonitrile showed a mixture of light chains conjugated to 1 LP-4 molecule and heavy chains conjugated to 3.0 LP-4 molecules, consistent with a drug / antibody ratio (DAR) of 8.0 LP-4 molecules / antibody.

[0399] In ShimadzuUHPLC analysis of the pure ADC sample at 214 nm on a Proteomix HIC Butyl-NP5, 5 μm, non-porous, 4.6 × 35 mm (Sepax) column using a Proteomix HIC Butyl-NP5 column with gradient elution of 1.5 M ammonium sulfate, 25 mM sodium acetate pH 7.4 and 25 mM sodium acetate pH 7.4 with 20% acetonitrile (v / v) showed single conjugation to LP-4, consistent with a drug / antibody ratio (DAR) of 7.96 LP-4 molecules / antibody.

[0400] Control ADC-1

[0401] The DAR 8 negative control ADC (Control ADC-1) was prepared by conjugating the negative control antibody NIP 2 2 8 (as disclosed in WO2015127273A1, the contents of which are incorporated herein by reference) and LP-1 by a conjugation method similar to that described for ADC-1. NIP228 is an IgG1 that binds complementary to transferrin, a transmembrane protein, which is expressed at low levels in the cell lines being tested, making it a suitable negative control.

[0402] Evaluation of ADC toxicity for 3D NCI-N87, JIMT1, and MDAMB468

[0403] The culture medium for NCI-N87, JIMT1, and MDAMB468 cells at 80%–90% confluence was aspirated from the T175 flasks, and the flasks were rinsed with PBS (approximately 10 ml) and emptied. TrypLE (5 ml) of Express Enzyme (1×) was added, and the flasks were returned to a 37°C incubator containing 5% CO2 for approximately 5 minutes. The flasks were then shaken to detach the cells from the bottom. Add 10 mL of cell culture medium (RPMI 1640 for NCI-N87 and MDAMB468, and DMEM for JIMT1, both supplemented with 50% fetal bovine serum) to a flask, and transfer the cell suspension to a sterile 50 mL Falcon tube, then centrifuge (400 g, 5 min). Aspirate the supernatant and resuspend the pellet in 10 mL of culture medium. Pipette the cell suspension thoroughly to disintegrate any possible aggregates, and mix 10 μL of the solution with 10 μL of trypan blue-stained cells. Then transfer 20 μL of the mixture to a cell counting slide, and measure cell concentration and viability using LUNA II. Based on previous experiments that allowed us to determine the optimal seeding density, NCI-N87, JIMT1, and MDAMB468 cell lines were seeded in CORNING SPHEROID 96-well microplates at 10,000, 3,000, and 3,000 cells / well, respectively.

[0404] A stock solution (650 µL) of antibody-drug conjugates (ADCs) was prepared by diluting filtered, sterile ADC into cell culture medium. A series of 9×5-fold dilutions of the previous ADC solution were prepared in 2 mL deep 96-well plates by serially transferring 65 µL to 585 µL of cell culture medium. ADC dilutions (50 µL / well) were aliquoted into two replicate wells of the 96-well plate containing 50 µL of cell suspension seeded 48 hours prior. Control wells received 50 µL of cell culture medium. The 96-well plates containing cells and ADC were incubated at 37 °C in a CO2-filled incubator for 6 days. At the end of the incubation period, the plates were equilibrated to room temperature for 10 minutes, and then CELLTITER-GLO 3D Cell Viability Assay (100 µL / well) was aliquoted into each well. The plates were mixed with a pipette for 5 minutes and then incubated at room temperature for 25 minutes. The luminescence of the wells was measured, and the percentage of cell viability (100%) was calculated by comparing the average luminescence in 2 wells treated with the average luminescence in 6 untreated control wells. IC50 was determined from the dose-response data using GRAPHPAD PRISM with a nonlinear regression (curve fitting) algorithm: sigmoid, 4PL, where X is log(concentration).

[0405] Table 1

[0406]

[0407] Evaluation of ADC toxicity for 3D SKOV3 and SKOV3 GUSB KO

[0408] The culture medium of 80%–90% confluenced SKOV3 WT and SKOV3 GUSB KO (glucuronidase knockout cell line, generated using CRISPR targeting) cells was aspirated from T175 flasks, and the flasks were rinsed with PBS (approximately 10 ml) and emptied. Add TrypLE (5 ml) Express Enzyme (1×), return the flask to a 37°C incubator containing 5% CO2 and incubate for approximately 5 minutes, then shake the flask to detach the cells from the bottom. Add 10 mL of cell culture medium (McCoy's 5A supplemented with 50% fetal bovine serum) to the flask and transfer the cell suspension to a sterile 50 mL Falcon tube, then centrifuge (400 g, 5 min). Aspirate the supernatant and resuspend the pellet in 10 mL of culture medium. Pipette the cell suspension thoroughly to disrupt any possible aggregates, and mix 10 μL of the solution with 10 μL of trypan blue-stained cells. Then transfer 20 μL of the mixture to a cell counting slide and measure cell concentration and viability using LUNA II. Based on previous experiments that allowed us to determine the optimal seeding density, seed SKOV3 and SKOV3 GUSB KO at 3000 cells / well in CORNING SPHEROID 96-well microplates.

[0409] A stock solution (650 µL) of antibody-drug conjugates (ADCs) was prepared by diluting filtered, sterile ADC into cell culture medium. A series of 9×5-fold dilutions of the previous ADC solution were prepared in 2 mL deep 96-well plates by serially transferring 65 µL to 585 µL of cell culture medium. ADC dilutions (50 µL / well) were aliquoted into two replicate wells of the 96-well plate containing 50 µL of cell suspension seeded 48 hours prior. Control wells received 50 µL of cell culture medium. The 96-well plates containing cells and ADC were incubated at 37 °C in a CO2-filled incubator for 6 days. At the end of the incubation period, the plates were equilibrated to room temperature for 10 minutes, and then CELLTITER-GLO 3D Cell Viability Assay (100 µL / well) was aliquoted into each well. The plates were mixed with a pipette for 5 minutes and then incubated at room temperature for 25 minutes. The luminescence of the wells was measured, and the percentage of cell viability (100%) was calculated by comparing the average luminescence in 2 wells treated with the average luminescence in 6 untreated control wells. The IC50 was determined from the dose-response data using a nonlinear regression (curve fitting) algorithm with GraphPad Prism: S-shape, 4PL, X is log(concentration).

[0410] Table 2 Specification 49 / 55 pages 56 CN 121175073 A

[0411]

[0412] Evaluation of ADC toxicity for NCI-N87, MDAMB468, SKOV3 WT and SKOV3 GUSB KO

[0413] The culture medium of NCI-N87, MDAMB468, SKOV3 WT and SKOV3 GUSB KO cells with 80%-90% confluence was aspirated from the T175 flasks, and the flasks were rinsed with PBS (about 10 mL) and emptied. Add TrypLE (5 ml) Express Enzyme (1×), and return the flask to a 37°C incubator containing 5% CO2 for approximately 5 minutes. Then shake the flask to detach the cells from the bottom. Add 10 mL of RPMI 1640 and McCoy 5A cell culture medium, both supplemented with 50% fetal bovine serum, to the flask, and transfer the cell suspension to a sterile 50 mL Falcon tube. Centrifuge (400 g, 5 min). Aspirate the supernatant and resuspend the pellet in 10 mL of culture medium. Pipette the cell suspension thoroughly to disintegrate any possible aggregates, and mix 10 µL of the solution with 10 µL of trypan blue-stained cells. Then transfer 20 µL of the mixture to a cell counting slide and measure cell concentration and viability using LUNA II. Based on previous experiments that allowed us to determine the optimal seeding density, MDAMB468, SKOV3 WT, and SKOV3 GUSB were used.KO cell lines were seeded at 3000 cells / well, while NCI-N87 was seeded at 10000 cells / well.

[0414] Stock solutions (550 µL) of antibody-drug conjugates (ADCs) were prepared by diluting filtered, sterile ADCs into cell culture medium. A series of 9×5-fold dilutions of the previous ADC solution were prepared in 2 mL deep 96-well plates by sequentially transferring 110 µL onto 440 µL of cell culture medium. The ADC dilution (50 µL / well) was dispensed into two replicate wells of the 96-well plate containing 50 µL of cell suspension seeded the previous day. Control wells received 50 µL of cell culture medium. The 96-well plates containing cells and ADCs were incubated at 37 °C in a CO2-filled incubator for 6 days. At the end of the incubation period, the plates were equilibrated to room temperature for 10 minutes, and CellTiter-Glo (Promega) (100 µL / well) was dispensed into each well. The plate was placed on a fixed-track shaker for 10 minutes and then stabilized at room temperature for 1 minute. The luminescence of the wells was measured, and the percentage of cell viability (100%) was calculated by comparing the average luminescence in two ADC-treated wells with the average luminescence in six untreated control wells. IC50 was determined from the dose-response data using a nonlinear regression (curve fitting) algorithm with GRAPHPAD PRISM: sigmoid, 4PL, X is log(concentration).

[0415] Table 3

[0416]

[0417] kD colloidal stability data for ADC-2, ADC-3, and ADC-1

[0418] Colloidal stability describes the tendency of proteins to form aggregates and precipitate from solution. The kD values ​​below represent the rate constant of this precipitation process; the lower the value (the more negative), the higher the tendency of the conjugate to precipitate. All ADCs and native mAbs were concentrated to approximately 10 mg / mL in 20 mM His / His HCl, 240 mM sucrose, pH 6.0. Serial dilutions were performed in formulation buffer. Each sample was tested three times in triplicate. The Kd value is the average of each triplicate. Outlier results (marked as " ") are omitted from the average calculation. The results are shown in Table 4 and Figure 5, which show that the precipitation tendency of ADC-1 is statistically significantly reduced compared to ADC-2 and ADC-3.

[0419] Table 4 Specification 50 / 55 pages 57 CN 121175073 A

[0420]

[0421] The above description of the exemplary embodiments is intended only to familiarize others skilled in the art with the applicant's specification, its principles and practical applications, so that others skilled in the art can easily adapt and apply the specification in its various forms as they may be best suited to the requirements of a particular purpose. The specific embodiments and examples thereto are intended for illustrative purposes only when indicating embodiments of this specification. Therefore, this specification is not limited to those described herein.These are exemplary embodiments, and various modifications can be made. Furthermore, it should be understood that, for clarity, the various features of the specification described in the context of a single embodiment can also be combined to form a single embodiment. Conversely, for brevity, the various features of the specification described in the context of a single embodiment can also be combined to form its sub-combinations.

[0422] Statement of Disclosure C

[0423] 1C. A conjugate of formula (IC)

[0424]

[0425] or a pharmaceutically acceptable salt thereof, wherein

[0426] Ab is an antibody or an antigen-binding fragment thereof,

[0427] k is an integer from 1 to 10,

[0428] each GA is independently a conjugated group conjugated to said antibody or an antigen-binding fragment thereof,

[0429] each DC is

[0430] ,

[0431] each JA is independently a group of formula (ICA)

[0432]

[0433] (ICA),

[0434] E is (CH2)n1, wherein n1 is 0, 1, 2 or 3, Specification 51 / 55 pages 58 CN 121175073 A

[0435] Q is or,

[0436] Wherein ring F1 is a saturated bicyclic ring having 6, 7, or 8 carbon atoms and optionally 1 or 2 oxygen atoms, ring F2 is a saturated bicyclic ring having 2 nitrogen atoms as shown, 4, 5, 6, 7, or 8 carbon atoms and optionally 1 oxygen atom, and ring F3 is a saturated bicyclic ring having 1 nitrogen atom as shown, 5, 6, 7, or 8 carbon atoms and optionally 1 oxygen atom,

[0437] R1 is a C1-4 alkyl group,

[0438] X is (CH2)n2, where n2 is 0, 1, 2, or 3,

[0439] Y is (CH2)n3, where n3 is 0, 1, 2, 3, or 4,

[0440] Z is (CH2)n4, where n4 is 1, 2, 3, 4, or 5,

[0441] m is an integer from 5 to 17,

[0442] p is 1 or 0,

[0443] (GA) indicates the attachment point to GA, and

[0444] (DC) indicates the attachment point to DC.

[0445] 2C. A conjugate of formula (IC) according to statement 1C or a pharmaceutically acceptable salt thereof, wherein Q is

[0446] or.

[0447] 3C. A conjugate of formula (IC) according to statement 1C or a pharmaceutically acceptable salt thereof, wherein Q is

[0448] .

[0449] 4C. A conjugate of formula (IC) according to any one of statements 1C to 3C or a pharmaceutically acceptable salt thereof, wherein m is 9, 10, 11, 12 or 13.

[0450] 5C. A conjugate of formula (IC) according to any one of statements 1C to 4C or a pharmaceutically acceptable salt thereof, wherein R1 is CH3.

[0451] 6C. A conjugate of formula (IC) according to any one of statements 1C to 5C, or a pharmaceutically acceptable salt thereof, wherein E is CH2.

[0452] 7C. A conjugate of formula (IC) according to any one of statements 1C to 6C, or a pharmaceutically acceptable salt thereof, wherein X is CH2.

[0453] 8C. A conjugate of formula (IC) according to any one of statements 1C to 7C, or a pharmaceutically acceptable salt thereof, wherein Y is (CH2)2.

[0454] 9C. A conjugate of formula (IC) according to any one of statements 1C to 8C, or a pharmaceutically acceptable salt thereof, wherein Z is (CH2)2.

[0455] 10C. A conjugate of formula (IC) according to any one of statements 1C to 9C, or a pharmaceutically acceptable salt thereof, wherein p is 1.

[0456] 11C. A conjugate of formula (IC) according to statement 1C or a pharmaceutically acceptable salt thereof, wherein each JA is a group of formula (ICB).

[0457]

[0458] (ICB).

[0459] 12C. A conjugate of formula (IC) according to any one of statements 1C to 11C or a pharmaceutically acceptable salt thereof, wherein GA is selected from

[0460]

[0461] and,

[0462] wherein RK is H or CH3, RL is C1-6 alkyl, and indicates the attachment site with said antibody or its antigen-binding fragment.

[0463] 13C. A conjugate of formula (IC) according to statement 12C or a pharmaceutically acceptable salt thereof, wherein GA is

[0464] .

[0465] 14C. A conjugate of formula (I) according to any one of statements 1C to 13C, or a pharmaceutically acceptable salt thereof, wherein k is an integer from 2 to 8.

[0466] 15C. A pharmaceutical composition comprising a conjugate of formula (I) according to any one of statements 1C to 14C, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0467] 16C. A conjugate of formula (I) according to any one of statements 1C to 14C, or a pharmaceutically acceptable salt thereof, for use in a therapeutic manner.

[0468] 17C. A conjugate of formula (I) according to any one of statements 1C to 14C, or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer.

[0469] 18C. A method of treating a patient with cancer, the method comprising administering to the patient a conjugate of formula (I) according to any one of statements 1C to 14C, or a pharmaceutically acceptable salt thereof. Instruction manual, pages 53 / 55, 60 CN 121175073 A

[0470] 19C. Use of a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof according to any one of statements 1C to 14C in the manufacture of a medicament for treating cancer.

[0471] 20C. A compound of formula (IIC)

[0472]

[0473] or a salt thereof, wherein GB is a conjugate group for conjugation with an antibody or an antigen-binding fragment thereof,

[0474] JB is a group of formula (IICA)

[0475]

[0476] (IICA),

[0477] wherein DC, E, Q, R1, X, Y, Z, m and p are as defined for a conjugate of formula (IC) in any one of statements 1C to 10C, (GB) indicates an attachment point with GB, and (DC) indicates an attachment point with DC.

[0478] 21C. A compound of formula (IIC) according to statement 20C, or a salt thereof, wherein GB is selected from

[0479]

[0480] and,

[0481] wherein X1 is CH or N,

[0482] h is O or 1,

[0483] Hal is Cl, Br or I,

[0484] RK is H or CH3, and

[0485] RL is C1-6 alkyl.

[0486] 22C. A compound of formula (IIC) according to statement 21C, or a salt thereof, wherein GB is

[0487] . Specification page 54 / 55 61 CN 121175073 A

[0488] 23C. A compound of formula (IIC) or a salt thereof according to statement 20C, which is

[0489]

[0490] (2S,3S,4S,5R,6S)-6-(2-((3-((S)-6-(((3S,3aR,6S,6aR)-6-(2,5,8,11,14,17, 20,23,26,29,32,35-dodecoxaoctane-38-amido)hexahydrofurano[3,2-b]furan-3-yl)amino)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)propamido)-6-oxohexamido)propamido) Methyl)-4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid or its salts. Specification 55 / 55 pages 62 CN 121175073 A Figure 1A Figure 1B Specification Figures 1 / 5 pages 63 CN 121175073 A Figure 2A Figure 2B Specification Figures 2 / 5 pages 64 CN121175073 A Figure 3 Figure 4A Instruction Manual Appendix 3 / 5 Page 65 CN 121175073 A Figure 4B Figure 4C Instruction Manual Appendix 4 / 5 Page 66 CN 121175073 A Figure 4D Figure 5 Instruction Manual Appendix 5 / 5 Page 67 CN 121175073 A

Claims

1. A conjugate of formula (IC) Or its pharmaceutically acceptable salt, wherein Ab is an antibody or its antigen-binding fragment. k is an integer from 1 to 10. Each G A Independently, it is a conjugation group that is conjugated to the antibody or its antigen-binding fragment. Each D C yes ， Each J A Independently a group of formula (ICA) (ICA), E is (CH2) n1 Where n1 is 0, 1, 2 or 3, Q is , R 1 It is C 1-4 alkyl, X is (CH2) n2 Where n2 is 0, 1, 2 or 3. Y is (CH2) n3 Where n3 is 0, 1, 2, 3 or 4. Z is (CH2) n4 Where n4 is 1, 2, 3, 4, or 5. m is an integer between 5 and 17. p is 1 or 0. (G A Instructions and G A The attachment point, and (D C Instructions and D C Attachment point.

2. The conjugate of formula (IC) according to claim 1, or a pharmaceutically acceptable salt thereof, wherein Q is... 。 3. A conjugate of formula (IC) according to claim 1 or claim 2, or a pharmaceutically acceptable salt thereof, wherein m is 9, 10, 11, 12 or 13.

4. A conjugate of formula (IC) according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein R 1 It is CH3.

5. A conjugate of formula (IC) according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, wherein E is CH2.

6. A conjugate of formula (IC) according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, wherein X is CH2.

7. A conjugate of formula (IC) according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, wherein Y is (CH2)2.

8. A conjugate of formula (IC) according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, wherein Z is (CH2)2.

9. A conjugate of formula (IC) according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, wherein p is 1.

10. The conjugate of formula (IC) according to claim 1, or a pharmaceutically acceptable salt thereof, wherein each J A It is a group of formula (ICB) (ICB).

11. A conjugate of formula (IC) according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof, wherein G A Selected from and , Where R K It is H or CH3, R L It is C 1-6 Alkyl, and Indicates the attachment site to the antibody or its antigen-binding fragment.

12. The conjugate of formula (IC) according to claim 11 or a pharmaceutically acceptable salt thereof, wherein G A yes 。 13. The conjugate of formula (IC) according to claim 11 or a pharmaceutically acceptable salt thereof, wherein G A yes 。 14. A conjugate of formula (IC) according to any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof, wherein k is an integer from 2 to 8.

15. A pharmaceutical composition comprising a conjugate of formula (IC) according to any one of claims 1 to 14 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

16. A conjugate of formula (IC) according to any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof, for use in a therapeutic manner.

17. A conjugate of formula (IC) according to any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer.

18. A method of treating a patient with cancer, the method comprising administering to the patient a conjugate of formula (IC) according to any one of claims 1 to 14 or a pharmaceutically acceptable salt thereof.

19. Use of a conjugate of formula (IC) according to any one of claims 1 to 14 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating cancer.

20. A compound of formula (IIC) or its salt, wherein G B It is a conjugating group used to conjugate with antibodies or their antigen-binding fragments. J B It is a group of formula (IICA) (IICA), Where D C E, Q, R 1 X, Y, Z, m, and p are as defined for the conjugate of formula (IC) in any one of claims 1 to 9, (G B Instructions and G B The attachment point, and (D C Instructions and D C Attachment point.

21. The compound of formula (IIC) according to claim 20, or a salt thereof, wherein G B Selected from and , Where X 1 Is it CH or N? h is 0 or 1. Hal is Cl, Br, or I. R K It is H or CH3, and R L It is C 1-6 alkyl.

22. The compound of formula (IIC) according to claim 21, or a salt thereof, wherein G B yes 。 23. The compound of formula (IIC) according to claim 21, or a salt thereof, wherein G B yes 。 24. The compound of formula (IIC) according to claim 20, or a salt thereof, is (2S,3S,4S,5R,6S)-6-(2-((3-((S)-6-(((3S,3aR,6S,6aR)-6-(2,5,8,11,14,17,20,23,26,29,32,35-dodecoxaoctane-38-amido)hexahydrofurano[3,2-b]furan-3-yl)amino)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)propionamido)-6- 4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid or a salt thereof.

25. The compound of formula (IIC) according to claim 20, or a salt thereof, is (2S,3S,4S,5R,6S)-6-(2-((S)-8-(4-(((3S,3aR,6S,6aR)-6-(2,5,8,11,14,17,20,23,26,29,32,35-dodecoxaoctane-38-amido)hexahydrofurano[3,2-b]furan-3-yl)amino)-4-oxobutyl)-15-bromo-3,7,10,14-tetraoxo-2,6,9,1 3-Tetraazapentadecanyl)-4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid or a salt thereof.

26. A compound of formula (IXC) (IXC) or its salt, wherein G B It is a conjugating group for conjugating an antibody or its antigen-binding fragment, and E, Y, Z, and p are as defined for conjugates of formula (IC) in any one of claims 1, 5, 7, 8, or 9, R Q1 Is it H or R? P1 Each R Q2 Independently, it is H or R P2 And R Q3 Is it H or R? P3 , where R P1 It is a carboxylic acid protecting group, each R P2 Independently, it is an alcohol protecting group and R P3 It is an amine protecting group.

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