Conjugate including a severable linker

Abstract

This specification defines a conjugate including a linker of formula (ICA) as defined herein. [Case 1] This specification relates to JPEG2026515801000128.jpg56128 and their pharmaceutically acceptable salts. This specification also relates to the use of conjugates for the treatment of diseases such as cancer, and intermediates useful for the synthesis of conjugates.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This specification claims priority to U.S. Provisional Patent Application No. 63 / 496,709 (filed April 18, 2023). The full text of the patent application referenced above is incorporated herein by reference.

[0002] This specification relates to certain conjugates comprising a cleavable linker and a topoisomerase I inhibitor (TOPO1i), and to pharmaceutical compositions comprising them. This specification also relates to the use of conjugates in methods for treating diseases such as cancer. This specification further relates to processes and intermediate compounds involved in the preparation of conjugates. [Background technology]

[0003] Antibody drug conjugates (ADCs) are an established method for delivering drugs to biological targets. It is possible to use an enzymatically cleavable linker to release the free drug at the target so that the free drug may have therapeutic effects. Through transcriptome and proteome profiling of various solid tumor cell lines, β-glucuronidase expression has been identified as upregulated and typically localized to lysosomes. International Publications 2007011968 and 2015182984 disclose certain antibody drug conjugates containing β-glucuronidase-cleavable linkers.

[0004] Topoisomerase inhibitors are chemical compounds that block the action of topoisomerases (topoisomerase I and II), which are enzymes that regulate changes in DNA structure by catalyzing the cleavage and recombination of the phosphodiester backbone of DNA strands during the normal cell cycle. Exatecan is an example of a topoisomerase I inhibitor.

[0005] There is still a need for conjugates having a β-glucuronidase-cleavable linker that can selectively deliver a drug to a biological target and have favorable physicochemical properties including solubility and lipophilicity. The conjugates of the present disclosure can be used for the treatment of diseases such as cancer. SUMMARY OF THE INVENTION

[0006] In a first aspect, a conjugate of formula (IC) Ab-(G A -J A -D C ) k (IC) or a pharmaceutically acceptable salt thereof is provided, wherein Ab is an antibody or an antigen-binding fragment thereof, k is an integer from 1 to 10, each G A is independently a conjugation group conjugated to an antibody or an antigen-binding fragment thereof, each D C is

[0007]

Chemical formula

[0008] ]]

Chemical formula

[0009]

Chemical formula

[0010] In a further embodiment, a pharmaceutical composition is provided comprising a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0011] In a further embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided for use in therapy.

[0012] In a further embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided for use in the treatment of cancer.

[0013] In a further embodiment, the use of a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof in the manufacture of a pharmaceutical product is provided.

[0014] In a further embodiment, the use of a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof in the manufacture of a pharmaceutical product for the treatment of cancer is provided.

[0015] In a further embodiment, a method is provided for treating cancer in a patient, comprising administering to the patient an effective amount of a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof.

[0016] In a further embodiment, the compound of formula (IIC) G B -J B -D C (IIC) Or a salt thereof is provided, in the formula, G B However, it is a conjugation group for conjugation to an antibody or its antigen-binding fragment, J B However, this is the basis of equation (IICA),

[0017] [ka] In the formula, D C E, Q, R 1 X, Y, Z, m, and p are as defined above for the conjugate of equation (IC), and (G B ) but, G B The connection point to (D C ) but, D C This indicates the connection point to [the specified location].

[0018] In a further embodiment, intermediates useful for the synthesis of compounds of formula (IIC) or salts thereof are provided.

[0019] The conjugate of formula (IC), or a pharmaceutically acceptable salt thereof, may undergo enzymatic cleavage to release the free drug (exatecan). Compared to other conjugates, the conjugate 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 a preferred toxicity profile (e.g., reduced off-target toxicity), and / or a preferred metabolic or pharmacokinetic profile. In embodiments, the conjugates of formula (I), (IC), (IM), or (IT) exhibit improved colloidal stability compared to other conjugates. Therefore, the conjugate of formula (IC) may be particularly suitable for use in therapies such as cancer treatment.

[0020] definition To make this specification easier to understand, certain terms are explicitly defined below. In addition, definitions are provided as needed throughout the detailed description. Where examples are provided for definitions, they are not limiting.

[0021] x and y are integers, prefix C x~y This indicates the numerical range of carbon atoms present in the base.

[0022] As used herein, the term "alkyl" refers to a saturated, linear, or branched hydrocarbon radical having a specific number of carbon atoms. 1~4 Examples of alkyl groups include methyl (Me), ethyl (Et), n-propyl ( n Pr), i-propyl( i Pr), n-butyl ( n Bu), i-butyl ( i Bu), s-butyl ( s Bu), and t-butyl ( t Bu) is one example. 1~6 Examples of alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, and n-hexyl.

[0023] As used herein, the term “bicyclic ring” refers to a condensed, cross-linked, or spirocyclic bicyclic ring.

[0024] As used herein, the term “conjugation group for conjugation to an antibody or its antigen-binding fragment” refers to an atom or group of atoms that can form a covalent bond to an antibody or its antigen-binding fragment through a chemical reaction.

[0025] In the formulas of this specification

[0026] [ka] The use of indicates a binding site to the antibody or its antigen-binding fragment. For example,

[0027] [ka] This indicates the presence of a covalent bond connecting the antibody, or its antigen-binding fragment, to the carbon atom marked with 1.

[0028] To avoid any ambiguity, in the formulas of this specification

[0029] [ka] The use of indicates a covalent bond site to a base, and the base is other than an antibody or its antigen-binding fragment.

[0030] Certain embodiments of this specification include a group referred to as "optionally substituted." In further embodiments, the group is unsubstituted.

[0031] When used in this specification,

[0032] [ka] is ring F 1 And,

[0033] [ka] is ring F 2 And,

[0034] [ka] is ring F 3 That is the case.

[0035] Units, prefixes, and symbols are shown in the format recognized by the International System of Units (SI). Numerical ranges include the number that defines the range.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art relating to this disclosure. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press provide many common dictionaries of the terms used herein. [Brief explanation of the drawing]

[0037] Next, embodiments and experiments illustrating the principles of this disclosure will be described with reference to the attached drawings. [Figure 1A] This shows cytotoxicity data for ADC-1 and control ADC-1 in the Her2+++ NCI-N87 cell line. [Figure 1B]This shows cytotoxicity data for ADC-1 and control ADC-1 in the HER2++ JIMT1 cell line. [Figure 2A] The cytotoxicity data for ADC-1 and control ADC-1 in the HER2-MDAMB468 cell line are shown. [Figure 2B] This shows cytotoxicity data for ADC-1 and control ADC-1 in the Her2+++SKOV3 cell line. [Figure 3] This document presents cytotoxicity data for ADC-1 and control ADC-1 in the SKOV3 GUSB KO cell line. [Figure 4A] This shows cytotoxicity data for ADC-3, ADC-2, and ADC-1 in the Her2+++ NCI-N87 cell line. [Figure 4B] This shows cytotoxicity data for ADC-3, ADC-2, and ADC-1 in HER2+++ / GUSB+++SKOV3 WT cell lines. [Figure 4C] This shows cytotoxicity data for ADC-3, ADC-2, and ADC-1 in the HER2+++ / GUSB-SKOV3 GUSB KO cell line. [Figure 4D] The cytotoxicity data for ADC-3, ADC-2, and ADC-1 in the HER2-MDAMB468 cell line are shown. [Figure 5] Colloidal stability data for ADC-3, ADC-2, and ADC-1 are shown. [Modes for carrying out the invention]

[0038] In one embodiment, this specification provides a conjugate of formula (IC) as defined above, or a pharmaceutically acceptable salt thereof.

[0039] In embodiments, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, where k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In further embodiments, k is an integer from 2 to 10. In further embodiments, k is an integer from 2 to 8. In further embodiments, k is 4. In further embodiments, k is 8.

[0040] In embodiments, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, wherein G A but,

[0041] [ka] Selected from, in the formula, R K However, it is H or CH3, and R L However, C 1~6 It is alkyl,

[0042] [ka] However, it indicates a binding site to the antibody or its antigen-binding fragment.

[0043] In embodiments, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, wherein G A but,

[0044] [ka] Selected from.

[0045] In embodiments, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, wherein G A but,

[0046] [ka] That is the case.

[0047] In embodiments, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, wherein G A but,

[0048] [ka] That is the case.

[0049] In embodiments, conjugates of formula (I), (IC), (IM), or (IT), or pharmaceutically acceptable salts thereof are provided, where G A but,

[0050] [ka] That is the case.

[0051] In the embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, where Q is

[0052] [ka] And in the formula, ring F 1 However, the ring is a saturated bicyclic ring having 6, 7, or 8 carbon atoms and optionally 1 or 2 oxygen atoms. In further embodiments, the bicyclic ring is a fused bicyclic ring.

[0053] In the embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, where Q is

[0054] [ka] And ring F 1 However, the ring is a saturated bicyclic ring having 6, 7, or 8 carbon atoms and 1 or 2 oxygen atoms. In further embodiments, the bicyclic ring is a fused bicyclic ring.

[0055] In the embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, where Q is

[0056] [ka] That is the case.

[0057] In the embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, where Q is

[0058] [ka] That is the case.

[0059] In the embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, where Q is

[0060] [ka] And in the formula, ring F 2 The resulting ring is a saturated bicyclic ring having two nitrogen atoms, four, five, six, seven, or eight carbon atoms, and optionally one oxygen atom. In further embodiments, the bicyclic ring is a spirocyclic bicyclic ring. In further embodiments, the bicyclic ring is a bridging bicyclic ring.

[0061] In the embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, where Q is

[0062] [ka] And in the formula, ring F 2 The ring is a saturated bicyclic ring having two nitrogen atoms and four, five, six, seven, or eight carbon atoms. In a further embodiment, the bicyclic ring is a spirocyclic bicyclic ring. In a further embodiment, the bicyclic ring is a bridging bicyclic ring.

[0063] In the embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, where Q is

[0064] [ka] That is the case.

[0065] In the embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, where Q is

[0066] [ka] That is the case.

[0067] In the embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, where Q is

[0068] [ka] That is the case.

[0069] In the embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, where Q is

[0070] [ka] That is the case.

[0071] In the embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, where Q is

[0072] [ka] And in the formula, ring F 3 The resulting ring is a saturated bicyclic ring having one nitrogen atom, five, six, seven, or eight carbon atoms, and optionally one oxygen atom. In further embodiments, the bicyclic ring is a spirocyclic bicyclic ring. In further embodiments, the bicyclic ring is a bridging bicyclic ring.

[0073] In the embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, where Q is

[0074] [ka] And in the formula, ring F 3 The resulting ring is a saturated bicyclic ring having one nitrogen atom and five, six, seven, or eight carbon atoms. In further embodiments, the bicyclic ring is a spirocyclic bicyclic ring. In further embodiments, the bicyclic ring is a bridging bicyclic ring.

[0075] In embodiments, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, where E is (CH2) n1 In the formula, n1 is 0, 1, 2, or 3. In a further embodiment, E is a covalent bond. In a further embodiment, E is CH2. In a further embodiment, E is (CH2)2. In a further embodiment, E is (CH2)3.

[0076] In embodiments, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, where X is (CH2) n2 In the formula, n2 is 0, 1, 2, or 3. In a further embodiment, X is a covalent bond. In a further embodiment, X is CH2. In a further embodiment, X is (CH2)2. In a further embodiment, X is (CH2)3.

[0077] In embodiments, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, where Y is (CH2) n3 In the formula, n3 is 0, 1, 2, 3, or 4. In a further embodiment, Y is a covalent bond. In a further embodiment, Y is CH2. In a further embodiment, Y is (CH2)2. In a further embodiment, Y is (CH2)3. In a further embodiment, Y is (CH2)4.

[0078] In embodiments, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, where Z is (CH2) n4 In the formula, n4 is 1, 2, 3, 4, or 5. In a further embodiment, Z is CH2. In a further embodiment, Z is (CH2)2. In a further embodiment, Z is (CH2)3. In a further embodiment, Z is (CH2)4. In a further embodiment, Z is (CH2)5.

[0079] In embodiments, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, where m is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17. In further embodiments, m is an integer from 6 to 16. In further embodiments, m is an integer from 7 to 15. In further embodiments, m is an integer from 8 to 14. In further embodiments, m is an integer from 9 to 13. In further embodiments, m is an integer from 10 to 12. In further embodiments, m is 11.

[0080] In embodiments, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, where R 1 However, C 1~4 It is alkyl. In further embodiments, R 1 This is CH3.

[0081] In the embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, where p is 1.

[0082] In the embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, where each J A However, this is the basis of equation (ICB).

[0083] [ka]

[0084] In the embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, where each J A However, this is the basis of equation (ICB').

[0085] [ka]

[0086] In the embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, where each J A However, this is the basis of equation (ICB2).

[0087] [ka]

[0088] In a further embodiment, the compound of formula (IIC) G B -J B -D C (IIC) Or a salt thereof is provided, in the formula, G B However, it is a conjugation group for conjugation to an antibody or its antigen-binding fragment, J B However, this is the basis of equation (IICA),

[0089] [ka] In the formula, D C E, Q, R 1 X, Y, Z, m, and p are as defined in any embodiment of the conjugate of formula (IC) disclosed herein, and (G B ) but, G B The connection point to (D C ) but, D C This indicates the connection point to [the specified location].

[0090] In the embodiment, a compound of formula (IIC) or a salt thereof is provided, where G B but,

[0091] [ka] Selected from, X 1 However, CH is or N, h is 0 or 1, Hal is Cl, Br, or I, and R K However, it is H or CH3, and R L However, C 1~6 It is alkyl.

[0092] In the embodiment, a compound of formula (IIC) or a salt thereof is provided, where G B but,

[0093] [ka] Selected from.

[0094] In the embodiment, a compound of formula (IIC) or a salt thereof is provided, where G B but,

[0095] [ka] That is the case.

[0096] In the embodiment, a compound of formula (IIC) or a salt thereof is provided, where G B but,

[0097] [ka] That is the case.

[0098] In the embodiment, compounds of formula (II), (IIC), (IIM), or (IIT), or salts thereof are provided, where G B but,

[0099] [ka] That is the case.

[0100] In the embodiment, a compound of formula (IIC) or a salt thereof is provided, where J B However, this is the basis of equation (IICB).

[0101] [ka]

[0102] In the embodiment, a compound of formula (II) or a salt thereof is provided, where J B However, this is the basis of equation (IICB2).

[0103] [ka]

[0104] In the embodiment,

[0105] [ka] (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-dodecaoxaoctatricontan-38-amide)hexahydrofl[3,2-b]furan-3-yl)amino)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)propanamide)-6-oxohexamide )propanamide)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]indolidino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid, and

[0106] [ka] (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-dodecaoxaoctatricontan-38-oil)-2,5-diazabicyclo[2.2.1]heptan-2-yl)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)propanamide)-6-oxohexanamide)propanamide)meth Compounds of formula (IIC) selected from (I)-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]indolidino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid, Alternatively, the salt may be provided.

[0107] In the embodiment,

[0108] [ka] (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-dodecaoxaoctatricontan-38-amide)hexahydrofl[3,2-b]furan-3-yl)amino)-4-oxobutyl)-15-bromo-3,7,10,14-tetraoxo-2,6,9,13-tetraazapentadecyl)-4-((((( A compound of formula (IIC), or a salt thereof, is provided, which is 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]indolidino-[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid.

[0109] In a further aspect, a compound of formula (IXC)

[0110]

Chemical formula

[0111] In an embodiment, a compound of formula (IXC), or a salt thereof is provided, wherein R Q1 is H, and each R Q2 is H, and R Q3 is H. In a further embodiment, G B is

[0112]

Chemical formula

[0113] In an embodiment, a compound of formula (IXC), or a salt thereof is provided, wherein R Q1 is H, and each R Q2 is H, and R Q3 is R P3 In a further embodiment, R Q1 is H, and each R Q2 is H, and R Q3 is tert-butyloxycarbonyl (Boc). In a further embodiment, G[[ID= seventy]] B is

[0114] [ka] That is the case.

[0115] In the embodiment, a compound of formula (IXC) or a salt thereof is provided, where R P1 However, benzyl, allyl, and C 1~4 Selected from alkyl. In further embodiments, R P1 is an allele. In further embodiments, R P1 It is methyl.

[0116] In the embodiment, a compound of formula (IXC) or a salt thereof is provided, where each R P2 However, these are independently selected from benzyl, C(O)OCH2CH=CH2, and acetyl. In further embodiments, each R P2 It is acetyl.

[0117] In the embodiment, a compound of formula (IXC) or a salt thereof is provided, where R P3 However, R is selected from trifluoroacetyl, tert-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), and fluorenylmethoxycarbonyl (Fmoc). In further embodiments, R P3 It is fluorenylmethoxycarbonyl.

[0118] In this embodiment, (2S,3S,4S,5R,6S)-6-(2-((3-((S)-6-(((3S,3aR,6S,6aR)-6-((tert-butoxycarbonyl)amino)hexahydrofl[3,2-b]furan-3-yl)amino)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)propanamide)-6-oxohexanamide)propanamide)methyl)-4-(((((1S,9S) A compound of formula (IXC), or a salt thereof, is provided, which is -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]indolidino-[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid.

[0119] In this embodiment, (2S,3S,4S,5R,6S)-6-[2-[[3-[[(2S)-6-[[(3S,3aR,6S,6aR)-3-amino-2,3,3a,5,6,6a-hexahydrofluoro[3,2-b]furan-6-yl]amino]-2-[3-(2,5-dioxopyrrole-1-yl)propanoylamino]-6-oxo-hexanoyl]amino]propanoylamino]methyl]-4-[[(10S,23S)-10-ethyl-18-fluor A compound of formula (IXC), or a salt thereof, is provided, which is lo-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamoyloxymethyl]phenoxy]-3,4,5-trihydroxytetrahydropyran-2-carboxylic acid.

[0120] This specification is intended to include all isotopes of atoms that occur in the compound and conjugate. It will be understood that isotopes include atoms that have the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium. Isotopes of carbon include,13 C and 14 It contains C. The isotopes of nitrogen are: 15 Includes N

[0121] The compounds disclosed herein may contain one or more chiral centers. Therefore, if desired, such compounds may be prepared or isolated as pure stereoisomers, i.e., as individual enantiomers, diastereoisomers, or as stereoisomerically concentrated mixtures. All such stereoisomer (and concentrated) mixtures are included within the scope of the embodiments unless otherwise specified. Pure stereoisomers (or concentrated mixtures) may be prepared, for example, using optically active starting materials or stereoselective reagents well known in the art. Alternatively, racemic mixtures of such compounds may be separated, for example, using chiral column chromatography, chiral resolving agents, etc.

[0122] Unless stereochemistry is explicitly specified in the chemical structure or chemical name, the chemical structure or chemical name is intended to encompass all possible stereoisomers, diastereoisomers, conformational isomers, rotational isomers, and tautomers of the compound shown. For example, a compound containing a chiral carbon atom is intended to encompass both the (R) and (S) enantiomers, as well as mixtures of enantiomers, including racemic mixtures, and a compound containing two chiral carbon atoms is intended to encompass all enantiomers and diastereoisomers, including (R,R), (S,S), (R,S), and (S,R).

[0123] A suitable pharmaceutically acceptable salt of the conjugate of formula (IC) is, for example, an acid addition salt. Acid addition salts of the conjugate of formula (IC) can be formed by contacting the compound with a suitable inorganic or organic acid under conditions known to those skilled in the art. Acid addition salts can be formed using, for example, an inorganic acid selected from hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid. Acid addition salts can also be formed using an organic acid 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.

[0124] A suitable pharmaceutically acceptable salt of the conjugate of formula (IC) is, for example, a base addition salt. A base addition salt of the conjugate of formula (IC) can be formed by contacting the compound with a suitable inorganic or organic base under conditions known to those skilled in the art. The base addition salt may be, for example, an alkali metal salt (such as sodium, potassium, or lithium salt) or an alkaline earth metal salt (such as calcium salt), which can be formed using an alkali metal or alkaline earth metal hydroxide or alkoxide (e.g., ethoxide or methoxide). The base addition salt may also be formed using a preferably basic organic amine (e.g., choline or meglumine salt).

[0125] Further preferred pharmaceutically acceptable salts of the conjugate of formula (IC) are, for example, salts formed in the patient's body after administration of the conjugate of formula (IC) to the patient.

[0126] In a further embodiment, a pharmaceutical composition is provided comprising a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0127] The term "pharmaceutical composition" refers to a preparation in which the active ingredient is enabled to have biological activity and which does not contain additional ingredients that are unacceptably toxic to the patient to whom the composition is administered. Such a composition may be sterile. Pharmaceutical compositions according to this specification will include a conjugate of formula (IC), or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0128] In embodiments, pharmaceutical compositions are provided comprising a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, carrier, buffer, or stabilizer. Such substances should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact properties of the carrier or other substance will vary depending on the route of administration (which may be oral or by injection (e.g., cutaneous, subcutaneous, or intravenous)).

[0129] In embodiments, a pharmaceutical composition is provided comprising a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable, non-toxic, sterile carrier. In further embodiments, the carrier is physiological saline, a non-toxic buffer, or a preservative. Formulations suitable for use in the therapeutic methods disclosed herein are described in Remington's Pharmaceutical Sciences, 22nd ed., Ed. Lloyd V. Allen, Jr. (2012), which are incorporated by reference.

[0130] Examples of suitable excipients include one or more of water, physiological saline, phosphate-buffered saline, dextrose, glycerol, and ethanol, as well as any combination thereof. In embodiments, pharmaceutical compositions are provided comprising a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof, and one or more isotonic agents. In further embodiments, one or more isotonic agents are selected from sugars, polyalcohols, and sodium chloride.

[0131] In embodiments, pharmaceutical compositions are provided that contain a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof, contained within one or more formulations selected from capsules, tablets, aqueous suspensions, solutions, nasal aerosols, and lyophilized powders that can be reconstituted before use to prepare a suspension or solution.

[0132] In embodiments, a pharmaceutical composition is provided comprising a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof, a buffer, a surfactant, and / or a stabilizer. In further embodiments, the buffer is acetic acid, phosphoric acid, or citrate buffer. In further embodiments, the surfactant is polysorbate. In further embodiments, the stabilizer is human albumin.

[0133] Pharmaceutical compositions comprising a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof may be administered to a patient by any suitable systemic or topical route of administration. For example, administration may be oral, oral cavity, sublingual, ocular, intranasal, tracheal, pulmonary, topical, percutaneous, urogenital, rectal, subcutaneous, intravenous, intra-arterial, intraperitoneal, intramuscular, intracranial, intrathecal, epidural, intraventricular, or intratumoral.

[0134] Pharmaceutical compositions comprising a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof may be formulated for administration by any suitable means, for example, by an epidermal or transdermal patch, ointment, lotion, cream, or gel, by a sprayer, vaporizer, or inhaler, by injection or infusion, or in the form of capsules, tablets, liquid solutions or suspensions in water or a non-aqueous medium, drops, suppositories, enemas, sprays, or powders. The most appropriate route for administration in any given case depends on the physical and mental condition of the subject, the nature and severity of the disease, and the desired characteristics of the formulation.

[0135] A pharmaceutical composition for oral administration containing a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof may be in the form of a tablet, capsule, powder, or liquid. Tablets may contain a solid carrier or adjuvant. Liquid pharmaceutical compositions generally contain a liquid carrier such as water, petroleum, animal or vegetable oil, mineral oil, or synthetic oil. They may also contain saline solution, dextrose, or other sugar solutions, or glycols, such as ethylene glycol, propylene glycol, or polyethylene glycol. Capsules may contain a solid carrier such as gelatin.

[0136] For intravenous, cutaneous, or subcutaneous injection, or injection at a site of pain, the conjugate of formula (IC), or a pharmaceutically acceptable salt thereof, would be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has a suitable pH, isotonicity, and stability. Those skilled in the art can readily prepare a suitable solution using an isotonic vehicle such as sodium chloride injection, Ringer's injection, or lactated Ringer's injection. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed.

[0137] In one embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided for use in therapy.

[0138] In one embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided for use in the treatment of cancer.

[0139] When "cancer" is mentioned, it includes both non-metastatic and metastatic cancers, and consequently, treating cancer includes treating both the primary tumor and tumor metastases.

[0140] The term "therapy" is intended to have its ordinary meaning of addressing a disease in order to completely or partially alleviate one, several, or all of the symptoms of the disease, or to correct or compensate for the underlying condition. The term "therapy" also includes "prevention" unless a specific contrary indication is given. The terms "therapeutic" and "therapeutically" should be interpreted in accordance with the corresponding means.

[0141] The term "prevent" is intended to have its ordinary meaning and can include primary prevention to prevent the onset of a disease, and secondary prevention where the disease has already occurred and the patient is protected temporarily or permanently from disease progression or worsening or the onset of new symptoms associated with the disease.

[0142] The term "treatment" is used synonymously with "therapy". Similarly, the term "treat" can be considered to mean "apply therapy", where "therapy" is as defined herein.

[0143] In an embodiment, there is provided a conjugate of formula (IC), or a pharmaceutically acceptable salt thereof, for use in the treatment of HER2-positive cancer.

[0144] In one aspect, there is provided the use of a conjugate of formula (IC), or a pharmaceutically acceptable salt thereof, as described herein, in the manufacture of a medicament, such as a medicament for the treatment of cancer.

[0145] In one aspect, there is provided a method of treating cancer in a patient, comprising administering to the patient an effective amount of a conjugate of formula (IC), or a pharmaceutically acceptable salt thereof.

[0146] The terms "treating" or "treatment", etc., refer to both (1) therapeutic means that cure, slow down, alleviate, and / or halt the progression of the symptoms of a diagnosed pathological condition or disorder, and (2) prophylactic or preventive means that prevent and / or delay the onset of the targeted pathological condition or disorder. Thus, those who need treatment include those who already have a disorder; those who tend to have a disorder; and those in whom the disorder should be prevented. In certain embodiments, when a patient shows a complete, partial, or transient remission of a particular type of cancer, for example, the patient is successfully "treated" for the cancer according to the methods of the present disclosure.

[0147] The term "effective amount" means an amount of an active ingredient sufficient to significantly and positively modify the symptoms and / or condition being treated (e.g., provide a positive clinical response). The effective amount of an active ingredient for use in a pharmaceutical composition varies depending on the particular condition being treated, the severity of the condition, the duration of treatment, the nature of combination therapies, the particular active ingredient used, the particular pharmaceutically acceptable excipient / carrier utilized, and similar factors within the knowledge and expertise of the attending physician.

[0148] The term "patient" refers to any animal (e.g., a mammal) including, but not limited to, humans, non-human primates, rodents, etc., that is the recipient of a particular treatment. In embodiments, the term "patient" refers to a human subject.

[0149] In embodiments, there is provided a method of treating cancer in a patient, comprising administering to the patient an effective amount of a conjugate of formula (IC), or a pharmaceutically acceptable salt thereof, wherein the cancer is a HER2-positive cancer.

[0150] In embodiments, there is provided a conjugate of formula (IC), or a pharmaceutically acceptable salt thereof, and an additional anti-tumor agent for the combination treatment of cancer.

[0151] In embodiments, combinations for use in the treatment of cancer are provided, comprising a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof, and an additional antitumor agent.

[0152] In embodiments, a conjugate of formula (IC), or a pharmaceutically acceptable salt thereof, is provided in combination with an additional antitumor agent.

[0153] In this specification, when the term “combined therapy” is used in relation to combination therapy, it should be understood that it may refer to simultaneous, separate, or sequential administration. In one embodiment, “combined therapy” refers to simultaneous administration. In another embodiment, “combined therapy” refers to separate administration. In yet another embodiment, “combined therapy” refers to sequential administration.

[0154] In embodiments, a method is provided for treating cancer in a patient, 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 at least one additional antitumor substance simultaneously, separately, or sequentially, wherein the amounts of the conjugate of formula (IC) or a pharmaceutically acceptable salt thereof and the additional antitumor substance are collectively effective in producing an anticancer effect.

[0155] Conjugation G A and G B Examples, though not limited to these, include the following, where X 1 is CH or N, h is 0 or 1, R K is H or CH3, Hal is Cl, Br, or I, and R L C 1~6 It is alkyl,

[0156] [ka] This indicates a binding site to the antibody or its antigen-binding fragment.

[0157] [Table 1-1]

[0158] [Table 1-2]

[0159] Antibodies or their antigen-binding fragments As used herein, the term “antibody” refers to an immunoglobulin molecule that is specifically bound to or immunologically reactive to a particular antigen.

[0160] In embodiments, the antibody is either isolated or recombinant. “Isolated,” as used herein, means a polypeptide, e.g., an antibody, identified, isolated, and / or recovered from the cells or cell culture on which it is expressed. Typically, isolated antibodies are prepared by at least one purification step. Therefore, “isolated antibody” means an antibody that substantially does not contain other antibodies with different antigen specificities.

[0161] In embodiments, the antibody comprises at least two "light chains" (LC) and two "heavy chains" (HC). The light and heavy chains of such an antibody are polypeptides consisting of several domains. Each heavy chain comprises a heavy chain variable region (abbreviated herein as "VH") and a heavy chain constant region (abbreviated herein as "CH"). The heavy chain constant region comprises 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 comprises a light chain variable domain (abbreviated herein as "VL") and a light chain constant domain (abbreviated herein as "CL").

[0162] In the embodiments, the antibody is a full-length antibody. As used herein, “intact” or “full-length” antibody refers to an antibody having two heavy (H) chain polypeptides and two light (L) chain polypeptides interconnected by disulfide bonds.

[0163] The "variable region" of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, either alone or in combination. The variable regions VH and VL can be further subdivided into regions with hypervariable regions, also called complementarity-determining regions (CDRs) (or hypervariable regions), and more conserved regions called framework regions (FRs). In embodiments, each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The VH or VL chain of an antibody can further include all or part of a heavy chain or light chain constant region.

[0164] The binding between an antibody and its target antigen or epitope is mediated by the CDRs. The term "epitope" refers to a target protein region (e.g., polypeptide) that can bind to (e.g., be bound by) an antibody or antigen-binding fragment of the present disclosure. CDRs are the major determinants of antigen specificity. There are at least two techniques for determining CDRs: (1) an approach based on interspecies sequence variability (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda Md.)), (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al. (1997) J. Molec. Biol. 273:927-948). Further, combinations of these two approaches are sometimes used in the art to determine CDRs.

[0165] The "constant domains" (or "constant regions") of the heavy and light chains do not directly participate in the binding of the antibody to its target but exhibit various effector functions. The constant region of an antibody 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 of the classical complement system (Clq).

[0166] The heavy chain constant region has five major classes, classified as IgA, IgG, IgD, IgE, and IgM, each possessing characteristic effector functions designated by its isotype. Ig molecules interact with multiple classes of cell receptors. For example, IgG molecules interact with three classes of Fcγ receptors (FcγR) specific to the IgG class of antibodies: FcγRI, FcγRII, and FcγRIII. Antibody binding to Fc receptors on the cell surface triggers several important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (known as antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer, and regulation of immunoglobulin production. Sequences crucial for IgG binding to FcγR receptors have been reported to be located in the CH2 and CH3 domains.

[0167] In some embodiments, the antibody or its antigen-binding fragment is an IgG isotype. The antibody or its antigen-binding fragment may be any IgG subclass, for example, IgG1, IgG2, IgG3, or IgG4 isotype. In embodiments, the antibody or its antigen-binding fragment is based on the IgG1 isotype.

[0168] The terms “Fc region,” “Fc moiety,” and “Fc” are used interchangeably herein and refer to a portion of innate immunoglobulin formed by two Fc chains. Each “Fc chain” contains a constant domain CH2 and a constant domain CH3. Each Fc chain may also contain a hinge region. The innate Fc region is homodimer. In embodiments, the Fc region may include modifications that force Fc heterodimerization and thus may be heterodimer. The Fc region contains a carbohydrate moiety, as well as complement and Fc receptor (including FcRn receptor) binding sites and does not have antigen-binding activity. Fc may refer to this isolated region or this region in the context of an antibody, antibody fragment, or Fc fusion protein. Polymorphisms have been found in several Fc domain sites, including but not limited to positions EU270, 272, 312, 315, 356, and 358, resulting in slight variations between the sequences described herein and sequences known in the art. As a result, all naturally occurring IgG Fc regions are called "wild-type IgG Fc domains" or "WT IgG Fc domains" (i.e., any allele). Human IgG1, IgG2, IgG3, and IgG4 heavy-chain sequences can be obtained in various sequence databases, including the UniProt database (www.uniprot.org), under accession numbers P01857 (IGHG1_HUMAN), P01859 (IGHG2_HUMAN), P01860 (IGHG3_HUMAN), and P01861 (IGHG4_HUMAN), respectively.

[0169] In embodiments, the antibodies of this disclosure are monoclonal antibodies. A “monoclonal antibody” (mAb) refers to a homogeneous population of antibodies involved in the highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies, which typically contain different antibodies against different antigenic determinants. The term “monoclonal antibody” can encompass not only full-length monoclonal antibodies but also antibody fragments (Fab, Fab', F(ab')2, Fv, etc.), single-chain (scFv) variants, fusion proteins containing antibody moieties, and other modified immunoglobulin molecules containing antigen recognition sites. Furthermore, “monoclonal antibody” refers to antibodies created by various methods, such as hybridomas, phage selection, recombinant expression, and transgenic animals. In embodiments, the antibodies of this disclosure are isolated monoclonal antibodies. In further embodiments, the antibodies are fully human monoclonal antibodies.

[0170] In embodiments, the antibody of this disclosure is the full-length antibody described above. Alternatively, the antibody may be an antigen-binding fragment. As used herein, the term “antigen-binding fragment” includes any naturally occurring or artificially constructed configuration of an antigen-binding polypeptide comprising one, two, or three light chain CDRs and / or one, two, or three heavy chain CDRs, wherein the polypeptide can bind to an antigen.

[0171] In embodiments, the antigen-binding fragment of the Disclosure is a Fab fragment. The antibodies according to the Disclosure may also be Fab', Fv, scFv, Fd, V NAR domain, IgNAR, intrabody, IgG CH2, minibody, single-domain antibody, Fcab, scFv-Fc, F(ab')2, di-scFv, bi-specific T-cell engager (BITE), F(ab')3, tetrabody, triabody, diabody, DVD-Ig, (scFv)2, mAb2, or DARPin.

[0172] The terms “Fab fragment” and “Fab” are used interchangeably herein and include a single light chain (e.g., constant domains CL and VL) and a single heavy chain (e.g., constant domains CH1 and VH). The heavy chain of a Fab fragment cannot form disulfide bonds with another heavy chain.

[0173] A "Fab' fragment" contains a single light chain and a single heavy chain, but in addition to CH1 and VH, it also contains a heavy chain region between the CH1 and CH2 domains required for the formation of an interchain disulfide bond. Therefore, two "Fab' fragments" can associate via the formation of a disulfide bond to form an F(ab')2 molecule.

[0174] The "F(ab')2 fragment" contains two light chains and two heavy chains. Each chain contains a portion of the constant region necessary for the formation of interchain disulfide bonds between the two heavy chains.

[0175] The "Fv fragment" contains only the variable regions of the heavy and light chains. It does not contain the steady region.

[0176] A "single-domain antibody" is an antibody fragment containing a single antibody domain unit (e.g., VH or VL).

[0177] A "single-chain Fv" ("scFv") is an antibody fragment containing the VH and VL domains of an antibody, which are linked together to form a single chain. Polypeptide linkers are commonly used to connect the VH and VL domains of scFv.

[0178] "TandAb," also known as "tandem scFv," is a single-chain Fv molecule formed by the covalent bonding of two scFv molecules to a flexible peptide linker in a tandem orientation.

[0179] A "bispecific T cell engager" (BiTE) is a fusion protein consisting of two single-stranded variable fragments (scFv) on a single peptide chain. One scFv binds to T cells via the CD3 receptor, while the other binds to tumor cell antigens.

[0180] A "diabody" is a small, bivalent, bispecific antibody fragment containing a heavy chain variable domain (VH) connected to a light chain variable domain (VL) on the same polypeptide chain (VH-VL), linked by a peptide linker that is too short to allow pairing between two domains on the same chain (Kipriyanov, Int. J. Cancer 77(1998), 763-772). This forces pairing with a complementary domain on another chain, promoting the assembly of a dimeric molecule with two functional antigen-binding sites.

[0181] DARPin is a bispecific ankyrin repeat molecule. DARPin is derived from the native ankyrin protein, one of the most abundant types of binding proteins found in the human genome. DARPin library modules are defined by the native ankyrin repeat protein sequence, using 229 ankyrin repeats for the initial design and another 2200 for subsequent refinement. The modules serve as building blocks for the DARPin library. The library modules are similar to the human genome sequence. DARPin consists of 4 to 6 modules. Each module is approximately 3.5 kDa, so the average size of DARPin is 16 to 21 kDa. Binding agent selection was performed by ribosome display, completely cell-free, as described in He M. and Taussig MJ., Biochem Soc Trans. 2007, Nov;35(Pt 5):962-5.

[0182] In embodiments, antibodies or their antigen-binding fragments may be further modified to include additional chemical moieties that are not typically part of a protein. These derivatized moieties can improve the protein's solubility, biological half-life, or absorption. The moieties may also reduce or eliminate any desired side effects, such as those of the protein. An overview of these moieties is provided in Remington's Pharmaceutical Sciences, 22nd ed., Ed. Lloyd V. Allen, Jr. (2012). [Examples]

[0183] This specification is illustrated here by the following non-limiting embodiments.

[0184] General information Flash chromatography was performed using a Biotage ISOLERA, and the purity of the fractions was confirmed using thin-layer chromatography (TLC). TLC was performed using MERCK KIESELGEL 60 F254 silica gel with a fluorescent indicator on an aluminum plate. Visualization of the TLC was achieved using UV light.

[0185] Extraction and chromatography solvents were purchased from VWR UK and used without further purification.

[0186] All fine chemicals were purchased from SIGMA-ALDRICH unless otherwise noted.

[0187] The pegylation reagent was obtained from QUANTA BIODESIGN US via STRATECH UK.

[0188] LC / MS conditions Positive-mode electrospray mass spectrometry was performed using a WATERS ACQUITY H-CLASS SQD2 with one of the following methods. (a) HPLC (WATERS ALLIANCE 2695) was performed using water (A) (0.1% formic acid) and acetonitrile (B) (0.1% formic acid) as mobile phases. LCMS 3 minutes: The initial composition of 5%B was held for 25 seconds, then increased from 5%B to 100%B over 1 minute and 35 seconds. The composition was held at 100%B for 50 seconds, then returned to 5%B over 5 seconds, and held there 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: 190~800 nm. Column: WATERS ACQUITY UPLC BEH SHIELD RP18 VANGUARD pre-column, 130A, 1.7 μm, 2.1 mm × 5 mm mounted on a WATERS ACQUITY UPLC BEH SHIELD RP18 1.7 μm 2.1 × 50 mm column at 50°C. LCMS 15 min: Initial composition 5%B held for 1 minute, then increased from 5%B to 100%B over 9 minutes. Composition held at 100%B for 2 minutes, then returned to 5%B over 0.10 minutes, held there for 3 minutes. Total gradient run time equals 15 minutes. Flow rate 0.6 mL / min. Wavelength detection range: 190~800 nm. Oven temperature: 50°C. Column: WATERS ACQUITY UPLC CSH C18 1.7 μm 2.1 × 100 mm column fitted with WATERS ACQUITY UPLC CSH C18 VANGUARD pre-column, 1.7 μm, 2.1 mm × 5 mm. (b) HPLC (Agilent 1290) was performed using either water (A) (TFA 0.03%) and acetonitrile (B) (0.03% TFA) or water (A) (TFA 0.05%) and acetonitrile (B) (0.05% TFA) as the mobile phase. The initial composition was either (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 was 0.3-1.5 mL / min. The column used was (1) ATLANTIS T3 3 μm 4.6 * 150 mm, 40°C (Detector ELSD or wavelength detection range: 210 nm), (2) ACQUITY UPLC BEH C18 2.1 *100 mm 1.7 μm, 40℃ (wavelength detection range: 210 nm or 220 nm), (3) ULC BEH C18 1.7 μm, 2.1 * 100mm, 40℃ (wavelength detection range: 223nm), (4)XBRIDGE C18 (4.6 * 150, 3.5μm), 40℃, (5)ACQUITY UPLC HSS PFP 2.1 * 150 mm 1.8 μm, 40℃ (wavelength detection range: 220 nm), (6) ULC BEH phenyl 1.7 μm, 2.1 * 150mm, 40℃ (wavelength detection range: 210nm), (7)EC-C18 2.7μm, 3.0 * 50mm, 40℃ (wavelength detection range: 210nm), or (8) YMC-Triart C18 50 * The sample size was 3.0 mm, S-3 μm, 12 nm, and the temperature was 45°C (detector: ELSD). The injection volume was 2 μL.

[0189] HPLC conditions Reverse-phase ultra-fast high-performance liquid chromatography (UFLC) was performed on a SHIMADZU PROMINENCE instrument using a PHENOMENEX GEMINI NX 5μ C18 column (50°C) with dimensions of 150 × 21.2 mm. 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 the following gradient conditions: the initial composition of 13% B was increased to 30% B over 3 minutes, then to 45% B over 8 minutes, then again to 100% over 6 minutes, and then back down to 13% over 2 minutes, held for 1 minute. 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 and 223 nm.

[0190] NMR method Proton NMR chemical shift values ​​were measured on a delta scale at 400 MHz using a BRUKER AV400. The following abbreviations were used: s, singlet; d, doublet; t, triplet; q, quartet; quin, quintet; m, multiplet; br, broad. Coupling constants are reported in Hz.

[0191] [Table 2]

[0192] Intermediate 1

[0193] [ka] 2,3,4,6,7,8,9,10-Octahydropyrimide[1,2-a]azepine (26.5 mL, 177.35 mmol) was added dropwise at 21°C to a 1 L round-bottom flask containing (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-bromopropa-1-ene (16.72 mL, 193.47 mmol) was added dropwise to the reaction mixture over 10 minutes, and the reaction 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). Next, acetic anhydride (244 mL, 2579.20 mmol) was added to the reaction mixture. The reaction mixture was warmed to room temperature and carried out at 21°C for 2 hours. The reaction mixture was concentrated under reduced pressure, and residual pyridine was removed azeotropically with toluene (1 × 100 mL). The crude product was diluted with DCM (65 mL) and cooled to 0°C. Next, 30% hydrobromic acid 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 carried out at 21°C for 2 hours and 30 minutes. The solvent was evaporated, and the compound was then purified by normal-phase flash column chromatography to obtain triacetic acid (2S,3S,4S,5R,6R)-2-((allyloxy)carbonyl)-6-bromotetrahydro-2H-pyran-3,4,5-triyl intermediate 1 (33 g, 48% yield) as a beige translucent substance.1 H 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)+.

[0194] Alternative synthesis of intermediate 1

[0195] [ka] 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 gradually while maintaining the temperature at 25-30°C. The reaction mixture was stirred under nitrogen at this temperature 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, extracted with isopropyl acetate (3 × 35.2 L), and the organic layer was concentrated to dryness to obtain crude 1,2,3,4-tetra-O-acetyl-β-D-glucuronic acid (16.08 kg, 61% w / w assay, 75%). LCMS m / z(ES+),[M+Na] + =384.6

[0196] Crude 1,2,3,4-tetra-O-acetyl-β-D-glucuronic acid, 61% w / w (16 kg, 27.05 mol) was dissolved in isopropyl acetate (42.83 kg) and stirred at 20-30°C. N,N-diisopropylethylamine (12.25 kg, 94.68 mol) was added to the reaction mixture over 11 minutes at 20-30°C, followed by the 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 at 20-30°C by adding aqueous hydrochloric acid solution (0.6 N, 43.71 kg). The separated organic layer was washed with brine (25% aqueous solution, 49 L), concentrated to dryness, and crude 1,2,3,4-tetra-O-acetyl-β-D-glucuronate allyl ester was obtained as a brown solid (12.0 kg, 87.5% w / w assay, 86.6%). LCMS m / z(ES+), [M+Na] + =424.8 33% hydrobromic acid (534 mL, 2.982 mol) in acetic acid was added dropwise at 0°C to a stirred mixture of crude 1,2,3,4-tetra-O-acetyl-β-D-glucuronic acid allyl ester (200 g, 0.497 mol) in isopropyl acetate (500 mL). The 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 obtain the crude product as black oil (231.3 g, 80.5%). LCMS (ES+),[M+Na] + =445.2 & 447, 1 H NMR(300MHz,CDCl3)δ 6.65(d,J=4.2Hz,1H),5.59-5.84(m,1H),5.62(t,J=9.6Hz,1H),5.40-5.2 3(m,3H),4.87(dd,J=9.9,3.9Hz,1H),4.66-4.59(m,3H),2.21-2.03(m,9H)

[0197] Intermediate 2

[0198] [ka]

[0199] 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) in DCM (500 mL) under nitrogen at 0°C for 2 hours. The resulting mixture was stirred at 0°C for 2 hours. The reaction mixture was quenched with water (500 mL), extracted with DCM (2 × 300 mL), the organic layer was dried over Na₂SO₄, filtered, and evaporated to obtain 5-(((tert-butyldimethylsilyl)oxy)methyl)-2-hydroxybenzaldehyde intermediate 2 (35.0 g, 100%) as a colorless substance. m / z(ES+), [M+Na] + =289, NH4HCO3, HPLC tR=1.505 min

[0200] Intermediate 3

[0201] [ka]

[0202] A black slurry was prepared by adding molecular sieves (4 Å beads, 5.0 g), silver oxide (29.2 g, 125.8 mmol), and acetonitrile (150 mL) to a vacuum-dried 500 mL round-bottom flask. To this slurry, a solution of intermediate 1 (10.7 g, 25.2 mmol) in acetonitrile (50 mL) was added over 20 minutes, followed by the single addition 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 Celite pad and rinsed with dichloromethane (3 × 25 mL). The solvent was evaporated, and the compound was then purified by normal-phase flash column chromatography to obtain (2S,3S,4S,5R,6S)-2-((allyloxy)carbonyl)-6-(4-(((tert-butyldimethylsilyl)oxy)methyl)-2-formylphenoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetic acid as white substance intermediate 3 (5.2 g, 34% yield). 1 H 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)+.

[0203] Intermediate 4

[0204] [ka]

[0205] To a solution of intermediate 3 (5.2 g, 8.6 mmol) in acetonitrile (40 mL), 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. The mixture was stirred at 20°C for 2 hours, and then the solvent was evaporated. To the resulting colorless oil, 1,4-dioxane (8 mL) and HCl (4.0 M in 1,4-dioxane, 50 mL, 200 mmol) were added. 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) pre-treated with methanol. The desired compound was eluted from the resin with methanol to obtain (2S,3S,4S,5R,6S)-2-((allyloxy)carbonyl)-6-(2-(aminomethyl)-4-(hydroxymethyl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetic acid as white intermediate 4 (2.5 g, 80%) over two steps. 1 H 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)+.

[0206] Intermediate 5

[0207] [ka]

[0208] To a suspension of intermediate 4 (2.5 g, 5.0 mmol) in dichloromethane (20 mL), N-ethyl-N-isopropylpropan-2-amine (1.8 mL, 10.1 mmol) and 3-(tert-butoxycarbonyl)amino)propanoic acid 2,5-dioxopyrrolidine-1-yl (1.3 g, 4.4 mmol) were added. The mixture was stirred at 20°C for 10 minutes, and then water (50 mL) was added. The organic layer was separated, and the aqueous layer was extracted with dichloromethane (3 × 30 mL). The combined organic layers were dried over Na₂SO₄, and the solvent was evaporated. To a solution of (2S,3S,4S,5R,6S)-2-((allyloxy)carbonyl)-6-(2-((3-((tert-butoxycarbonyl)amino)propanamide)methyl)-4-(hydroxymethyl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyl (2.8 g, 4.2 mmol) triacetic acid (4.0 M in 1,4-dioxane, 2.6 mL, 83.9 mmol) was added. The mixture was stirred at 20°C for 2 hours, and then the solvent was evaporated. The compound was purified by reverse-phase flash column chromatography to obtain (2S,3S,4S,5R,6S)-2-((allyloxy)carbonyl)-6-(2-((3-aminopropanamide)methyl)-4-(hydroxymethyl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate as colorless intermediate 5 (1.3 g, 53%) after two steps. 1 H 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)+.

[0209] Alternative synthesis of intermediate 5

[0210] [ka]

[0211] Fmoc-β-alanine (1.11 kg, 3.57 mol) was added to a stirred reactor containing intermediate 4 (2.1 kg, 90.5% w / w, 3.57 mol) and acetonitrile (19 L). The stirred mixture was cooled to 0°C. Hexafluorophosphate azabenzotriazole tetramethyluronium (1.36 kg, 3.57 mol) and N,N-diisopropylethylamine (0.92 kg, 7.14 mol) were added, and the mixture was stirred for 4 hours while maintaining the temperature 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 the mixture was stirred at 20-25°C for 18 hours. The resulting suspension was filtered, the cake was washed with ethyl acetate (3.87 L), and dried under vacuum to obtain (2S,3R,4S,5S,6S)-2-(2-((3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamide)methyl)-4-(hydroxymethyl)phenoxy)-6-((allyloxy)carbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetic acid (1.6 kg, 99% w / w, 56%). LCMS m / z 789[M+H] +

[0212] Under nitrogen at -45°C, 2,3,4,6,7,8,9,10-octahydropyrimide[1,2-a]azepine (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)propanamide)methyl)-4-(hydroxymethyl)phenoxy)-6-((allyloxy)carbonyl)tetrahydro-2H-pyran-3,4,5-triyl (1 kg, 1.27 mol) and tetrahydrofuran (10 L) at -45°C. The mixture was stirred at -45°C for 4 hours, then diluted with acetonitrile (5 L), and quenched by adding hydrogen chloride solution in tert-butyl methyl ether (2.54 L, 2.0 M, 5.07 mol). The mixture was concentrated to approximately 5 L under vacuum and diluted with n-heptane (5 L). The acetonitrile layer containing intermediate 8 was recovered (3.88 kg of MeCN solution, 89.97% area, assumed to be 100%). LCMS m / z 566.6[M+H] +

[0213] Intermediate 7

[0214] [ka]

[0215] Intermediate 6 (5.0 g, 34.21 mmol) in dry DCM (100 mL) was added to a 250 mL round-bottom flask under nitrogen gas. Pyridine (13.84 mL, 171.07 mmol), followed by tosyl-Cl (16.31 g, 85.53 mmol) was added to the solution. 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 layer was separated, and the compound was extracted in 200 mL of dichloromethane. The combined organic layers were washed with HCl solution (1 M - 300 mL) and brine (200 mL), and dried over magnesium sulfate. The solvent was removed under reduced pressure to obtain the crude product. The compound was purified via silica gel column chromatography to obtain bis(4-methylbenzenesulfonic acid)(3R,3aS,6R,6aS)-hexahydroflu[3,2-b]furan-3,6-diyl 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). LC-MS (ESI) m / z 455.21 (M+H) + .

[0216] Intermediate 8

[0217] [ka]

[0218] Intermediate 7 (6.0 g, 13.20 mmol) in dry DMF (15 mL) was added to a 50 mL round-bottom flask under nitrogen gas. 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 the formation of the desired product and completion of the reaction. The reaction mixture was diluted with dichloromethane (200 × 2 mL), the organic layer was separated, washed with water (200 mL) and brine (200 mL), and dried over magnesium sulfate. The solvent was removed under reduced pressure to obtain (3S,3aR,6S,6aR)-3,6-diazidohexahydroflu[3,2-b]furan intermediate 8 (2.050 g, 79%). 1 H 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) + .

[0219] Intermediate 9

[0220] [ka]

[0221] Intermediate 8 (1 g, 5.10 mmol) in dry THF (20 mL) was added to a 250 mL round-bottom flask under nitrogen gas. Barium palladium(II) carbonate (0.618 g, 0.51 mmol) was added to the solution. The reaction mixture was flushed with hydrogen gas (1.028 g, 509.76 mmol) and stirred at 23 °C for 3 hours under H2 gas. 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 Celite pad. The Celite pad was washed with methanol (50 mL). The filtrate was dried over magnesium sulfate. The solvent was removed under reduced pressure to obtain (3S,3aR,6S,6aR)-hexahydroflu[3,2-b]furan-3,6-diamine intermediate 9 (0.590 g, 80%). 1H NMR(500MHz,DMSO)δ 4.23(s,2H),3.68(dd,J=8.7,4.5Hz,2H),3.41(dd,J=8.7,1.9Hz,2H),3.23(dd,J=4.5,1.9Hz,2H),1.54(s,4H).LCMS(ESI)m / z 145.2(M+H) + .

[0222] Alternative synthesis of intermediate 9

[0223] [ka]

[0224] Intermediate 6 (4 kg, 8.8 mol) and benzylamine (12 L) were added to the reactor under nitrogen. The stirred mixture was heated to 160°C for 24 hours, then cooled to 20-25°C, diluted with tert-butyl methyl ether (80 L), and further cooled to 10°C. Para-toluenesulfonic acid (12.11 kg, 70.44 mol) was added, and the mixture was stirred at 20-25°C for 2.5 hours, then filtered. The cake was washed with tert-butyl methyl ether (8 L), and the combined filtrate was washed with saturated sodium bicarbonate aqueous solution (20 L). The organic phase was evaporated to dryness, dissolved in ethanol (20 L), and evaporated to dryness to obtain crude intermediate 7 (3.05 kg, 80.5% w / w, 85.9%). LCMS m / z(ES+), [M+H] + =325.1

[0225] Intermediate 7 (1.5 kg, 3.08 mol) and ethanol (12.12 L) were added to the reactor under a dry nitrogen atmosphere. 10 wt% palladium-carbon (120.8, 10% w / w) was added to the solution. The reaction mixture was flushed with hydrogen gas and stirred under hydrogen at 80°C for 16 hours. The mixture was cooled to 20-25°C and filtered through cellulose (2.42 kg). The cake was washed with ethanol (2.44 L), and the combined filtrate was concentrated to dryness. The residue was dissolved in acetonitrile (6.04 L), concentrated to dryness, and intermediate 9 (509 g, 84% w / w, 80.2%) was obtained. LCMS m / z(ES+),[M+H] +=145

[0226] Intermediate 11

[0227] [ka]

[0228] Intermediate 9 (1.50 g, 10.40 mmol) in dry THF (25 mL) was added to a 250 mL round-bottom flask under nitrogen gas. Sodium bicarbonate (1.748 g, 20.81 mmol) and 2,5-dioxopyrrolidine-1-yl 2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatricontan-38-oate intermediate 10 (7.13 g, 10.40 mmol) were gradually added to the solution under nitrogen gas, and the mixture was stirred at 20°C for 6 hours. LC-MS analysis showed the formation of the desired product and 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 Celite pad. The Celite pad was washed with methanol (50 mL). The filtrate was dried over magnesium sulfate. The solvent was removed under reduced pressure to obtain the crude product. The crude product was purified via silica gel column to obtain N-((3S,3aR,6S,6aR)-6-aminohexahydrofl[3,2-b]furan-3-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxatricatriacontane-38-amide intermediate 11 (4.00 g, 53.8%). 1 H 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) + .

[0229] Intermediate 13

[0230] [ka]

[0231] In a 250 mL round-bottom flask, under nitrogen gas, (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-(tert-butoxy)-6-oxohexanoic acid intermediate 12 (5 g, 11.38 mmol) in dry DMF (20 mL) was added. To the solution, under nitrogen gas, potassium carbonate (3.14 g, 22.75 mmol) and 3-bromopropa-1-ene (1.485 mL, 17.06 mmol) were added little by little, and the 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 water (500 mL), the organic layer was extracted with ethyl acetate (2 × 300 mL), washed with water (300 mL) and brine (200 mL), and dried over sodium sulfate (20 g). The solvent was removed to obtain the crude product. The crude product was purified via silica gel column to obtain (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)hexanedionic acid 6-(tert-butyl)1-allyl intermediate 13 (5.10 g, 93%). 1 H 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(ddt,J=16.5,10.9,5.8Hz,1H),5.42-5.23(m,3H),4.66(d,J=5.8Hz, 2H),4.40(q,J=4.8Hz,3H),4.23(t,J=7.1Hz,1H),2.26(t,J=7.2Hz,2H),1.96-1.82 (m,1H),1.72(dq,J=13.5,6.1Hz,3H),1.60-1.47(m,1H),1.45(s,9H).LCMS(ESI)m / z 480.2(M+H) + .

[0232] Intermediate 14

[0233] [ka]

[0234] Intermediate 13 (5 g, 10.43 mmol) in dry THF (20 mL) was added to a 100 mL round-bottom flask under nitrogen gas. To the solution, 4 moles of HCl in dioxane (13.03 mL, 52.13 mmol) were added under nitrogen gas, and the mixture was stirred at 20°C for 6 hours. LC-MS analysis showed the formation of the desired product and completion of the reaction. The reaction mixture was diluted with water (200 mL), the organic layer was extracted with dichloromethane (2 × 300 mL), washed with brine (200 mL), and dried over sodium sulfate (20 g). After removing the solvent, (S)-5-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-(allyloxy)-6-oxohexanoic acid intermediate 14 (4.20 g, 95%) was obtained. 1 H 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(MH) - .

[0235] Intermediate 15

[0236] [ka]

[0237] Intermediate 14 (1.925 g, 4.55 mmol) was added to a 100 mL round-bottom flask under nitrogen gas. HATU (1.862 g, 4.90 mmol), followed by DIPEA (1.222 mL, 6.99 mmol), was added to the solution. 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 the formation of the desired product and completion of the reaction. The reaction mixture was diluted with DCM (300 mL), washed with water (200 mL), 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 via silica gel column to obtain (S)-6-(((3S,3aR,6S,6aR)-6-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatricontan-38-amide)hexahydrofl[3,2-b]furan-3-yl)amino)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)6-oxohexanoate allyl intermediate 15 (3.40 g, 87%). 11H 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, 1.4 Hz, 2H), 7.38 (tt, J = 7.5, 1.3 Hz, 2H), 6.05 - 5.93 (m, 1H), 5.39 (dq, J = 17.2, 1.6 Hz, 1H), 5.28 (dq, J = 10.5, 1.4 Hz, 1H), 4.73 - 4.65 (m, 2H), 4.58 (qd, J = 4.1, 1.0 Hz, 2H), 4.46 (dd, J = 10.6, 7.0 Hz, 1H), 4.40 (dd, J = 10.6, 7.0 Hz, 1H), 4.36 - 4.31 (m, 2H), 4.31 - 4.24 (m, 2H), 4.01 (ddd, J = 9.6, 5.0, 1.1 Hz, 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, 1.9 Hz, 2H), 2.31 (hept, J = 7.2 Hz, 2H), 1.96 - 1.85 (m, 1H), 1.85 - 1.68 (m, 3H). LCMS (ESI) m / z 1121.3 (M + H) + .

[0238] Intermediate 16

[0239] [Chemical Structure]

[0240] Intermediate 15 (4.3 g, 3.84 mmol) in dry DCM (10 mL) was added to a 50 mL round-bottom flask under nitrogen gas. Triethylamine (0.535 mL, 3.84 mmol), followed by triphenylphosphine (0.101 g, 0.38 mmol), was added to the solution. 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 obtain the crude product. The crude product was purified via silica gel column to obtain (S)-6-(((3S,3aR,6S,6aR)-6-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatricontan-38-amide)hexahydrofl[3,2-b]furan-3-yl)amino)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-oxohexanoic acid intermediate 16 (3.50 g, 84%). 1 H 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),7.46-7.3 8(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),3.9 3(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,2 H),3.24(s,3H),2.33(t,J=6.5Hz,2H),2.09(s,3H),1.68(d,J=9.1Hz,1H),1.64-1.48(m,3H).LCMS(ESI)m / z 1080.6(M+H) + .

[0241] Intermediate 17

[0242] [ka]

[0243] Intermediate 16 (0.8 g, 0.74 mmol) was added to a 100 mL round-bottom flask under nitrogen gas. HATU (0.366 g, 0.96 mmol), followed by DIPEA (0.388 mL, 2.22 mmol), was added to the solution. 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 the formation of the desired product and completion of the reaction. The reaction mixture was diluted with DCM (100 mL), washed with water (100 mL), the organic layer was extracted (2 × 100 mL), washed with brine (100 mL), and dried over sodium sulfate (15 g). The solvent was removed under reduced pressure and the solution was purified via a silica gel column 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-dodecaoxaoctatricontane-38-amide)hexahydroflo[3,2-b]furan-3 Intermediate 17 (0.640 g, 53.1%) was obtained from (-yl)amino)-4-oxobutyl)-1-(9H-fluoren-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-triyl.1H NMR (500 MHz, DMSO) δ 8.21 (t, J = 6.0 Hz, 1H), 8.11 (dd, J = 19.5, 6.9 Hz, 2H), 7.94 (t, J = 5.8 Hz, 1H), 7.89 (d, J = 7.5 Hz, 2H), 7.73 (t, J = 7.0 Hz, 2H), 7.49 - 7.39 (m, 3H), 7.33 (td, J = 7.5, 1.2 Hz, 2H), 7.17 (dd, J = 8.4, 2.2 Hz, 1H), 7.13 (s, 1H), 7.01 (d, J = 8.4 Hz, 1H), 5.89 (ddt, J = 17.3, 10.5, 5.7 Hz, 1H), 5.56 (d, J = 7.9 Hz, 1H), 5.48 (t, J = 9.6 Hz, 1H), 5.33 (dq, J = 17.2, 1.6 Hz, 1H), 5.26 (dq, J = 10.5, 1.4 Hz, 1H), 5.19 - 5.07 (m, 3H), 4.76 (d, J = 10.0 Hz, 1H), 4.62 (ddt, J = 13.3, 5.6, 1.4 Hz, 1H), 4.54 (ddt, J = 13.3, 5.8, 1.4 Hz, 1H), 4.44 - 4.36 (m, 4H), 4.31 - 4.18 (m, 4H), 4.11 (d, J = 5.9 Hz, 3H), 3.93 (d, J = 5.9 Hz, 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 = 5.0 Hz, 1H), 2.33 (ddt, J = 14.6, 10.0, 7.6 Hz, 4H), 2.10 - 2.05 (m, 2H), 2.04 (s, 3H), 1.99 (d, J = 4.6 Hz, 6H), 1.54 (dt, J = 43.7, 8.9 Hz, 4H). LCMS (ESI) m / z 1629.8 (M + H). + .

[0244] Intermediate 18

[0245]

Chem.

[0246] To a solution of intermediate 17 (25.00 mg, 0.02 mmol, 1.0 equivalent) in DMF, bis(4-nitrophenyl) carbonate (28.0 mg, 0.09 mmol, 4.5 equivalents) and DIPEA (0.013 mL, 0.08 mmol, 4.0 equivalents) were added. 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 is dried and 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-dodecaoxaoctatricontan-38-amide)hexahydrofl[3,2-b]furan-3-yl)amino)-4-oxobutyl)-1-(9H- Fluoren-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-triyl intermediate 18 (32 mg, 0.02 mmol, 91%) was obtained as a yellow substance. RT 7.74 min. LC-MS (ESI) m / z 1794.7[M+H]+.

[0247] Intermediate 19

[0248] [ka]

[0249] To a solution of exatecan mesylate (7.41 mg, 0.01 mmol, 1.0 equivalent) in DCM (1 mL) and DMF (1.000 mL), DIPEA (7.28 μL, 0.04 mmol, 4.0 equivalent), intermediate 18 (25.00 mg, 0.01 mmol, 1.0 equivalent), and HOPO (1.703 mg, 0.02 mmol, 2.0 equivalent) were added, and the resulting mixture was stirred at 23°C for 18 hours. After this, the mixture was vacuum concentrated, and the residue was subjected to reverse-phase flash column chromatography (C18). Purified by BIOTAGE prepack column and 40-60% MeCN [0.1% formic acid] / water [0.1% formic acid], 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-dodecaoxaoctatricontan-38-amide)hexahydrofl[3,2-b]furan-3-yl)amino)-4-oxobutyl)-1-(9H-fluoren-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]indolidino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenoxy)-6-((allyloxy)carbonyl)tetrahydro-2H-pyran-3,4,5-triyl intermediate 19 (13 mg, 0.01 mmol, 63%) was obtained as a yellow substance. RT 7.66 min. LCMS (ESI) 2090.3[M+H]+.

[0250] Intermediate 20

[0251] [ka]

[0252] To a solution of intermediate 19 (15.00 mg, 7.18 μmol, 1.0 equivalent) in DCM (1 mL), triethylamine (2.00 μL, 0.01 mmol, 1.4 equivalents), Pd(PPh3)4 (1.00 mg, 0.87 μmol, 12 mol%), and formic acid (0.541 μL, 0.01 mmol, 1.4 equivalents) were added, 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 (0.25 mL) and THF (0.25 mL). To this solution, potassium carbonate (9.44 mg, 0.07 mmol, 10 equivalents) in water (0.5 mL) was added, and the mixture was stirred at 23°C for 3 hours. After this time, the mixture was vacuum concentrated to remove organic matter. The remaining aqueous solution was acidified with citric acid (1N) to pH 4, and the mixture was stirred at 23°C for 1 hour. After this, the mixture is filtered, and the residue is purified by reverse-phase flash column chromatography (C18 BIOTAGE pre-packed column, 20-40% MeCN [0.1% formic acid] / water [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-dodecaoxaoctatricontan-38-amide)hexahydrofl[3,2-b]furan-3-yl)amino)-2-amino-6-oxohexaamide)propanamide)Me Intermediate 20 (8.6 mg, 5.03 μmol, 70%) of 20 trihydroxytetrahydro-2H-pyran-2-carboxylic acid was obtained as a yellow substance. RT 5.08 min. LC-MS (ESI) 170 2.6[M+H]+.

[0253] Linker-Payload LP-1

[0254] [ka]

[0255] To a solution of intermediate 20 (6.20 mg, 3.64 μmol, 1.0 equivalent) in DMF (0.5 mL), pyridine (0.7 μL, 5.5 μmol, 1.5 equivalent) and 3-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)propanoate 2,5-dioxopyrrolidine-1-yl (0.970 mg, 3.64 μmol, 1.0 equivalent) were added. The mixture was stirred at 23°C for 3 hours. After this, the reaction mixture was concentrated under vacuum, and the residue was subjected to reverse-phase HPLC (CSH over 7 minutes). Purified by 35% MeCN [0.1% formic acid] / water [0.1% formic acid], (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-dodecaoxaoctatricontan-38-amide)hexahydrofloxac (2.7 mg, 1.46 μmol, 40%) of (mid)propanamide)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]indolidino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid LP-1 (2.7 mg, 1.46 μmol, 40%) was obtained as a white substance. RT 5.87 min. LC-MS (ESI) 1853.5[M+H]+.

[0256] Alternative synthesis of LP-1 Intermediate A

[0257] [ka]

[0258] Intermediate 9 (22.0 g, 152.6 mmol) in methanol (440 mL) was mixed with boc anhydrous (83.3 g, 381.5 mmol, 2.5 equivalents), and the reaction mixture was stirred at 20°C for 2 hours. ELSD analysis indicated the formation of the desired product and completion of the reaction. The solvent was removed under reduced pressure to obtain the crude product. The crude product was slurryed with MTBE (200 mL), filtered, washed with MTBE (40 mL), and dried to obtain N-[(3S,3aR,6S,6aR)-6-(tert-butoxycarbonylamino)-2,3,3a,5,6,6a-hexahydrofluoro[3,2-b]furan-3-yl]carbamate tert-butyl intermediate A (44.0 g, 84.3%). LCMS m / z 366.8[M+Na] +

[0259] Intermediate B

[0260] [ka]

[0261] Intermediate A (2.0 g, 5.81 mmol, 1.0 equivalent) in ethyl acetate (40 mL) was mixed with HCl (4 M, 8.0 mL) in ethyl acetate, and the reaction mixture was stirred at 20°C for 6 hours. ELSD analysis indicated the formation of the desired product and completion of the reaction. The reaction mixture was quenched with potassium phosphate solution (2 M, 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 obtain N-[(3S,3aR,6S,6aR)-3-amino-2,3,3a,5,6,6a-hexahydrofluoro[3,2-b]furan-6-yl]carbamate tert-butyl intermediate B (800 mg, 57.1%). 1 H NMR(400MHz,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] +

[0262] Intermediate C

[0263] [ka]

[0264] To a solution of intermediate 14 (17.16 g, 40.5 mmol, 1.1 equivalents) in 180 mL of DCM at 25°C, HATU (16.8 g, 44.2 mmol, 1.2 equivalents) and DIPEA (10.9 mL, 62.6 mmol, 1.7 equivalents) were added. The reaction mixture was stirred for 15 minutes. Intermediate B (9.0 g, 36.8 mmol, 1.0 equivalent) in 90 mL of DCM was added to the reaction solution. The reaction mixture was stirred for 2 hours. LC-MS analysis showed the formation of the desired product and completion of the reaction. 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-hexahydrofluor[3,2-b]furan-3-yl]amino]-2-(9H-fluoren-9-ylmethoxycarbonylamino)-6-oxohexanoate allyl intermediate C (9.0 g, 65.2%). LCMS m / z 671.6[M+Na] +

[0265] Intermediate D

[0266] [ka]

[0267] 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), formic acid (1.05 mL, 27.7 mmol, 2.0 equivalents), triethylamine (5.79 mL, 41.6 mmol, 3.0 equivalents), and Pd(PPh3)4 (1.6 g, 1.39 mmol, 0.1 equivalent) were added. The reaction mixture was stirred at 20-25°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 obtain the crude product. The crude product was purified via silica gel column (DCM vs. DCM:MeOH 4:1) to obtain (2S)-6-[[(3S,3aR,6S,6aR)-6-(tert-butoxycarbonylamino)-2,3,3a,5,6,6a-hexahydrofluor[3,2-b]furan-3-yl]amino]-2-(9H-fluoren-9-ylmethoxycarbonylamino)-6-oxohexanoic acid intermediate D (7.0 g, 82.9%). LCMS m / z 610.1[M+H] +

[0268] Intermediate E

[0269] [ka]

[0270] To a solution of intermediate D (4.0 g, 6.56 mmol, 1.0 equivalent) in DMF (80 mL) at 0-5°C, 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. The reaction mixture was stirred at 0-5°C for 1 hour. LC-MS analysis showed the formation of the desired product and completion of the reaction. 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 obtain the crude product. The crude product was purified via silica gel column (EtOAC ~ 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-hexahydrofluor[3,2-b]furan-3-yl]amino]-2-(9H-fluoren-9-ylmethoxycarbonylamino)-6-oxo-hexanoyl]amino]propanoylamino]methyl]-4-(hydroxymethyl)phenoxy]-3,4,5-triacetoxy-tetrahydropyran-2-carboxylic acid allyl intermediate E (3.6g, 47.4%). LCMS m / z 1158.2[M+H] +

[0271] Intermediate F

[0272] [ka]

[0273] To a solution of intermediate E (800 mg, 691 μmol, 1.0 equivalent) in DMF (8 mL), bis(4-nitrophenyl) carbonate (840 mg, 2.76 mmol, 4.0 equivalents) and DIPEA (481 μL, 2.76 mmol, 4.0 equivalents) were added. The mixture was stirred at 20-25°C for 2 hours. LC-MS analysis showed the formation of the desired product and completion of the reaction. The solvent was removed under reduced pressure to obtain the crude product.The crude product was purified via silica gel column (5%~50% MeCN in water, 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-hexahydrofluor[3,2-b]furan-3-yl]amino]-2-(9H-fluoren-9-ylmethoxycarbonylamino)-6-oxo-hexanoyl]amino]propanoylamino]methyl]-4-[(4-nitrophenoxy)carbonyloxymethyl]phenoxy]-3,4,5-triacetoxy-tetrahydropyran-2-carboxylic acid allyl intermediate F (400 mg, 43.8%). NMR(300MHz,DMSO-d6)δ ppm 1.14-1.29(m,3H)1.38(s,9H)1.53(br dd,J=18.52,8.25Hz,4H)1.98-2.09(m,12H)2.13(d,J=2.93Hz,1H)2.32-2.45(m,2H)3.35(s,11H)3.58(br d,J=6.42Hz,2H)3.71(s,12H)3.78-3.88(m,3H)3.90-4.17(m,4H)4.18-4.42(m,6H)4.50-4.67(m,2H)4.80(d,J=10.09Hz, 1H)5.09-5.38(m,6H)5.47-5.55(m,1H)5.65(d,J=7.89Hz,1H)5.82-5.96(m,1H)6.10(s,4H)7.11(d,J=8.44Hz,1H)7.21(br d,J=5.50Hz,1H)7.28-7.37(m,4H)7.37-7.44(m,3H)7.46-7.61(m,3H)7.65-7.76(m,2H)7.89(d,J=7.34Hz,2H)7.99(br t,J=5.50Hz,1H)8.09(d,J=6.97Hz,1H)8.27-8.34(m,3H)LCMS m / z 1323.1[M+H]. +

[0274] intermediate G

[0275] [ka]

[0276] To a solution of exatecan mesylate (1.12 g, 2.12 mmol, 4.0 equivalents) in DMF (7 mL), DIPEA (369 μL, 2.12 mmol, 4.0 equivalents), intermediate F (700 mg, 529 μmol, 1.0 equivalent), and HOPO (117.5 mg, 1.06 mmol, 2.0 equivalents) were added, and the resulting mixture was stirred at 20-25°C for 1 hour. LC-MS analysis showed the formation of the desired product and 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 obtain the crude product. The crude product is slurryed with ethyl acetate / MTBE (1:1, 5 mL), filtered, and dried 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-hexahydrofluor[3,2-b]furan-3-yl]amino]-2-(9H-fluoren-9-ylmethoxycarbonylamino)-6-oxo-hexanoyl]amino]propanoylamino]methyl]-4-[[( We obtained 10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-dialkyl-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamoyloxymethyl]phenoxy]-3,4,5-triacetoxy-tetrahydropyran-2-carboxylic acid allyl 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, 3H) 1.81 - 1.92 (m, 1H) 1.95 - 2.15 (m, 9H) 2.29 - 2.44 (m, 3H) 2.73 (s, 1H) 2.89 (s, 1H) 3.49 - 3.61 (m, 2H) 3.71 (s, 16H) 3.81 (br d, J = 6.36 Hz, 2H) 3.85 - 3.95 (m, 1H) 3.99 - 4.12 (m, 2H) 4.12 - 4.28 (m, 2H) 4.33 (br d, J = 3.91 Hz, 1H) 4.39 (br d, J = 4.16 Hz, 1H) 4.48 - 4.64 (m, 1H) 4.78 (br d, J = 10.27 Hz, 1H) 5.06 - 5.34 (m, 5H) 5.41 - 5.53 (m, 2H) 5.60 (br d, J = 7.58 Hz, 1H) 5.81 - 5.93 (m, 1H) 6.09 (s, 5H) 6.92 (d, J = 8.26 Hz, 2H) 7.09 (br d, J = 8.56 Hz, 1H) 7.17 - 7.42 (m, 5H) 7.64 - 7.79 (m, 2H) 7.80 - 7.90 (m, 1H) 7.93 - 7.99 (m, 1H) 8.00 - 8.14 (m, 3H) LCMS m / z 810.3 [M + 2H]. 2+

[0277] Intermediate H

[0278]

Chem.

[0279] To a solution of intermediate G (1.5 g 80%, 741 μmol, 1.0 equivalent) in MeOH (2.6 mL), K2CO3 (1.02 g, 7.41 mmol, 10.0 equivalents) and water (260 μL) were added. The resulting mixture was stirred at 20-25°C for 1 hour. LC-MS analysis showed the formation of the desired product and completion of the reaction. The solvent was removed under reduced pressure to obtain the crude product. The crude product was purified via silica gel column (5%~50% MeCN in water, 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-hexahydrofluoro[3,2-b]furan-3-yl]amino]-2-amino-6-oxo-hexanoyl]amino]propanoylamino]methyl]-4-[[(10S,2 3S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptan-23-yl]carbamoyloxymethyl]phenoxy]-3,4,5-trihydroxytetrahydropyran-2-carboxylic acid intermediate (450 mg, 67%) was obtained by 1H NMR(500MHz,DMSO-d6)δ ppm 0.84-0.92(m,3H)1.24(br s,4H)1.29(br d,J=7.32Hz,1H)1.38(s,8H)1.43-1.53(m,2H)1.60(br s,2H)1.81-2.11(m,5H)2.14-2.24(m,2H)2.29-2.36(m,1H)2.39(s,3H)2.40-2.44(m,1H)3.13-3.23(m,7H)3.23-3.32(m,14H)3.35(br s,12H)3.50-3.60(m,8H)3.77-3.85(m,3H)4.05(br s,1H)4.17(br dd,J=13.43,3.97Hz,1H)4.34(br d,J=3.36Hz,1H)4.39(d,J=3.97Hz,1H)4.49(br dd,J=13.28,7.78Hz,1H)4.60(br d,J=6.71Hz,1H)5.03(br d,J=12.(21 Hz, 1H), 5.10 (broad d, J = 12.21 Hz, 1H), 5.28 (broad s, 3H), 5.45 (s, 2H), 6.52 (broad s, 1H), 7.12 (d, J = 8.24 Hz, 1H), 7.20 (broad s, 1H), 7.32 (s, 1H), 7.32 - 7.34 (m, 1H), 7.40 (s, 1H), 7.78 (d, J = 10.68 Hz, 1H), 8.03 - 8.15 (m, 1H), 8.17 (s, 2H), 8.24 (broad d, J = 6.10 Hz, 1H), 8.47 (broad s, 1H), 9.30 (broad s, 1H). LCMS m / z 616.2 [M + 2H]. 2+

[0280] Intermediate I

[0281]

Chem.

[0282] To a solution of intermediate H (450 mg, 365 μmol, 1.0 equivalent) in DMF (4.5 mL) at 20-25°C, pyridine (225 μL) and N-succinimidyl 3-maleimidiopropionic acid (195 mg, 731 μmol, 2.0 equivalents) were added, and the reaction mixture was stirred for 1 hour. LC-MS analysis showed the formation of the desired product and completion of the reaction. The solvent was removed under reduced pressure to obtain the crude product. The crude product was purified via silica gel column (5%~50% MeCN in water, 0.1% formic acid) to obtain (2S,3S,4S,5R,6S)-6-(2-((3-((S)-6-(((3S,3aR,6S,6aR)-6-((tert-butoxycarbonyl)amino)hexahydrofl[3,2-b]furan-3-yl)amino)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)propanamide)-6-oxohexanamide)propanamide Intermediate I (230 mg, 52.4%) was obtained from 1H)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]indolidino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid intermediate I (230 mg, 52.4%). NMR(500MHz,DMSO-d6)δ ppm 0.84-0.91(m,2H)1.23(br d,J=9.77Hz,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,1H)3.24(br d,J=6.10Hz,2H)3.29(br s,3H)3.34(br s,24H)3.51-3.63(m,4H)3.71(s,9H)3.75-3.88(m,3H)4.01-4.15(m,2H)4.21(br d,J=9.46Hz,1H)4.34(d,J=3.66Hz,1H)4.36-4.46(m,1H)4.72(br s,1H)5.06-5.15(m,1H)5.27(br s,2H)5.45(s,2H)6.09(s,3H)6.51(s,1H)6.97(s,1H)7.10(d,J=8.24Hz,1H)7.19(br d,J=5.80Hz,1H)7.31(s,1H)7.33(s,1H)7.77(d,J=10.99Hz,1H)7.94(br t,J=5.34Hz,1H)8.06(br d,J=8.55Hz,1H)8.12(br d,J=6.71Hz,1H)8.20(br d,J=8.24Hz,1H)LCMS m / z 1382.1[M+H]. +

[0283] Intermediate J

[0284] [ka]

[0285] To a solution of intermediate I (230 mg, 166 μmol, 1.0 equivalent) in DCM (2.6 mL), TFA (1.3 mL) was added, and the reaction mixture was stirred at 25°C for 1 hour. LC-MS analysis showed the formation of the desired product and completion of the reaction. The solvent was removed under reduced pressure to obtain the crude product. The crude product was purified via silica gel column (5%~50% MeCN in water, 0.1% formic acid) to obtain (2S,3S,4S,5R,6S)-6-[2-[[3-[[(2S)-6-[[(3S,3aR,6S,6aR)-3-amino-2,3,3a,5,6,6a-hexahydrofloxacin[3,2-b]furan-6-yl]amino]-2-[3-(2,5-dioxopyrrole-1-yl)propanoylamino]-6-oxo-hexanoyl]amino]propanoylamino]methyl]-4-[[(1 Intermediate J (120 mg, 54.5%) of (0S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamoyloxymethyl]phenoxy]-3,4,5-trihydroxytetrahydropyran-2-carboxylic acid was obtained. LCMS m / z 641.5[M+H] +

[0286] Linker-Payload LP-1

[0287] [ka]

[0288] To a solution of intermediate J (100 mg, 78.0 μmol, 1.0 equivalent) in THF (1 mL), NaHCO3 (13.1 mg, 156 μmol, 2.0 equivalents) and 2,5-dioxopyrrolidine-1-yl 2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatricontan-38-oate intermediate 10 (64.2 mg, 93.6 μmol, 1.2 equivalents) were added. The reaction mixture was stirred at 20-25°C for 16 hours. LC-MS analysis showed the formation of the desired product and completion of the reaction. The solvent was removed under reduced pressure to obtain the crude product. The crude product was purified via silica gel column (5%~50% MeCN in water, 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-dodecaoxaoctatricontan-38-amide)hexahydrofloxac We obtained LP-1 (60 mg, 40%) of (de)-6-oxohexanamide)propanamide)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]indolidino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid. LCMS m / z 927.2[M+2H] 2+

[0289] LP-2 synthesis Intermediate 38

[0290] [ka]

[0291] To a solution of (S)-5-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((3-(allyloxy)-3-oxopropyl)amino)-6-oxohexanoic acid (1.0 g, 2.02 mmol) in DCM (20 mL), HATU (1.0 g, 2.63 mmol) and DIPEA (1.06 mL, 6.07 mmol) were added. The reaction mixture was stirred at 20°C for 15 minutes, then (1S,4S)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate tert-butyl (0.481 g, 2.43 mmol) was added, and the reaction mixture was stirred for a further 2 hours. The reaction mixture was diluted with ELISA (2 × 300 mL), and the organic layer was washed with water (1 × 300 mL), followed by brine (1 × 200 mL). The mixture was dried over magnesium sulfate, and then the solvent was removed under reduced pressure. Purification by flash column chromatography (80-100% ethyl acetate / hexane) yielded (1S,4S)-5-((S)-5-((((9H-fluoren-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 intermediate 38 (1.250 g, 1.85 mmol, 92% yield) as a white solid. 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),4.4 6-4.31(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=6.9H z,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.

[0292] Intermediate 39

[0293] [ka]

[0294] To a solution of intermediate 38 (1.0 g, 1.48 mmol) in DCM (20 mL), triphenylphosphine (0.039 g, 0.15 mmol), followed by triethylamine (0.310 mL, 2.22 mmol), and then tetrakis(triphenylphosphine)palladium(0) (0.171 g, 0.15 mmol) were added. The reaction mixture was stirred at 20°C for 5 hours. The intermediate 3-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((1S,4S)-5-(tert-butoxycarbonyl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)-6-oxohexanamide)propanoic acid intermediate 39 (0.8 g, 1.26 mmol, 85% yield) was provided as a white solid. 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

[0295] Intermediate 40

[0296] [ka]

[0297] To a solution of intermediate 39 (1.0 g, 1.58 mmol) in DCM (20 mL), trifluoroacetic acid (0.12 mL, 1.58 mmol) was added. The reaction mixture was stirred at 20°C for 5 hours. The mixture was purified by flash column chromatography (0-15% MeOH:DCM) to obtain 3-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((1S,4S)-2,5-diazabicyclo[2.2.1]heptan-2-yl)-6-oxohexanamide)propanoic acid intermediate 40 (0.76 g, 1.42 mmol, 90% yield) as a colorless gum. 11H NMR (500 MHz, MeOD) δ 7.82 (d, J = 7.5 Hz, 2H), 7.69 (dd, J = 10.0, 7.4 Hz, 2H), 7.41 (t, J = 7.5 Hz, 2H), 7.33 (tt, J = 7.5, 1.6 Hz, 2H), 5.70 - 5.61 (m, 1H), 4.82 (s, 1H), 4.50 (d, J = 16.9 Hz, 1H), 4.46 - 4.35 (m, 2H), 4.24 (t, J = 6.8 Hz, 1H), 4.06 (ddd, J = 13.0, 8.2, 5.2 Hz, 1H), 3.89 (d, J = 6.7 Hz, 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, 9.5 Hz, 2H), 1.84 - 1.70 (m, 2H), 1.66 (h, J = 6.3 Hz, 2H). LCMS (ESI) m / z [M + H]+ 535.8。

[0298] Intermediate 41

[0299]

Chem.

[0300] To a solution of intermediate 40 (1.0 g, 1.54 mmol) in DCM (20 mL), triethylamine (0.645 mL, 4.63 mmol) was added, followed by 2,5-dioxopyrrolidine-1-yl 2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatricontane-38-oate (1.269 g, 1.85 mmol). The reaction mixture was stirred at 20°C for 4 hours. Purification by flash column chromatography (0-15% MeOH:DCM) yielded 3-((S)-6-((1S,4S)-5-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatricontan-38-oil)-2,5-diazabicyclo[2.2.1]heptan-2-yl)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-oxohexanamide)propanoic acid intermediate 41 (1.12 g, 1.01 mmol, 95% yield) as a colorless gum. LCMS(ESI)m / z(M+NH4)+1122.7.

[0301] Intermediate 42

[0302] [ka]

[0303] To a solution of intermediate 41 (0.4 g, 0.36 mmol) in DCM (10 mL), HATU (0.186 g, 0.49 mmol) and DIPEA (0.2 mL, 1.15 mmol) were added. The mixture was stirred at 20°C for 15 minutes, and then triacetic acid (2S,3S,4S,5R,6S)-2-((allyloxy)carbonyl)-6-(2-(aminomethyl)-4-(hydroxymethyl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyl hydrochloride (intermediate 4.HCl) (0.198 g, 0.37 mmol) was added all at once. The mixture was stirred for a further 30 minutes. Water (20 mL) was added and the mixture was stirred for a further 30 minutes. 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 solution was dried with Na2SO4, then the solvent was evaporated under vacuum. It was purified by flash column chromatography (5-40% MeOH:DCM) to obtain 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-dodecaoxaoctatricontan-38-oil)-2,5-diazabicyclo[2.2.1]heptan-2-yl) Intermediate 42 (300.9 mg, 0.19 mmol, 52.5% yield) of -4-oxobutyl)-1-(9H-fluoren-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-triyl was provided as a colorless gum. 11H NMR (DMSO-d6) δ: 8.19 (broad singlet, 1H), 7.96 (broad doublet, J = 5.5 Hz, 1H), 7.88 (doublet, J = 7.5 Hz, 2H), 7.72 (triplet, J = 6.4 Hz, 2H), 7.47 (broad doublet, J = 10.1 Hz, 1H), 7.41 (triplet, J = 7.5 Hz, 2H), 7.32 (triplet, J = 7.5 Hz, 2H), 7.16 (broad doublet, J = 8.3 Hz, 1H), 7.12 (singlet, 1H), 7.00 (doublet, J = 8.3 Hz, 1H), 5.83 - 5.93 (multiplet, 1H), 5.45 - 5.56 (multiplet, 2H), 5.22 - 5.35 (multiplet, 2H), 5.07 - 5.17 (multiplet, 3H), 4.65 - 4.77 (multiplet, 3H), 4.50 - 4.65 (multiplet, 3H), 4.41 (doublet, J = 5.6 Hz, 2H), 4.16 - 4.29 (multiplet, 4H), 4.03 - 4.13 (multiplet, 1H), 3.93 (broad doublet, J = 8.3 Hz, 1H), 3.56 - 3.65 (multiplet, 2H), 3.46 - 3.53 (multiplet, 43H), 3.39 - 3.44 (multiplet, 3H), 3.33 - 3.34 (multiplet, 1H), 3.21 -​​​​​​​​​​​​​​​To a solution of Intermediate 42 (100 mg, 0.06 mmol) in DMF (1.0 mL) and DCM (1.0 mL) at 25°C, bis(4-nitrophenyl) carbonate (115 mg, 0.38 mmol), followed by DIPEA (0.055 mL, 0.32 mmol), was added. The reaction mixture was then stirred at 21°C for 1 hour. After this, the reaction mixture was concentrated under vacuum, and the residue was ground in ether (5 mL). The resulting solid was then dissolved in DCM (2 mL), and ether (10 mL) was added to the resulting solution. The mixture was sonicated, centrifuged, and the supernatant was removed. This process was repeated twice, and the solid obtained after each cycle was collected until no further precipitate formed. This is triacetate (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-dodecaoxaoctatricontan-38-oil)-2,5-diazabicyclo[2.2.1]heptan-2-yl)-4-oxobutyl)-1-(9H-fluoren-9-yl)-3,6,10-to The intermediate 43 (100 mg, 0.06 mmol, 91% yield) of lyoxo-2-oxa-4,7,11-triazadodecane-12-yl)-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-((allyloxy)carbonyl)tetrahydro-2H-pyran-3,4,5-triyl was obtained as a white solid and proceeded to the next step without further purification. LCMS(ESI) m / z[M+H]+1748.5.

[0307] Intermediate 44

[0308] [ka]

[0309] To a solution of intermediate 43 (75 mg, 0.04 mmol) in DCM (1 mL), 2-hydroxypyridine 1-oxide (5.24 mg, 0.05 mmol), exatecan mesylate (23.95 mg, 0.05 mmol), and DIPEA (0.022 mL, 0.13 mmol) in DMF (1 mL) were added. The resulting mixture was stirred at 21°C for 18 hours. Afterward, the reaction mixture was vacuum concentrated, and the residue was purified by reverse-phase flash column chromatography (30-50% MeCN / water [0.1% formic acid]) to obtain triacetate (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-dodecaoxaoctatricontan-38-oil)-2,5-diazabicyclo[2.2.1]heptan-2-yl)-4-oxobutyl)-1-(9H-fluoren-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]indolidino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenoxy)-6-((allyloxy)carbonyl)tetrahydro-2H-pyran-3,4,5-triyl intermediate 44 (43.0 mg, 0.021 mmol, 49%) was obtained as a white solid. LCMS(ESI)m / z[M+H]+2044.2.

[0310] Intermediate 45

[0311] [ka]

[0312] To a solution of intermediate 44 (43.0 mg, 0.02 mmol) in DCM (2 mL), 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. Next, the reaction mixture was stirred at 21°C for 18 hours, after which the reaction mixture was vacuum concentrated 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-dodecaoxaoctatricontan-38-oil)-2,5-diazabicyclo[2.2.1]heptan-2-yl)-4-oxobutyl)-1-(9H-fluoren-9-yl)-3,6,10-trioxo-2-oxa-4,7,11-triazadodecane-12-yl Intermediate 45 (37.0 mg, 0.02 mmol, 88%) of 45-(((((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]indolidino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-triacetoxytetrahydro-2H-pyran-2-carboxylic acid was obtained and used in the next step without further purification. LCMS(ESI)m / z[M+H]+2005.8.

[0313] Intermediate 46

[0314] [ka]

[0315] To a solution of intermediate 45 (37.0 mg, 0.02 mmol) in THF (0.5 mL), potassium carbonate aqueous solution (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, citric acid aqueous solution (1 M) was added until the pH fell below 7.0. The reaction mixture was directly purified by reverse-phase flash column (20-40% MeCN / water [0.1% formic acid]) to (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-dodecaoxaoctatricontan-38-oil)-2,5-diazabicyclo[2.2.1]heptan-2-yl)-2-amino-6-oxohexanamide)propanamide)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]indolidino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid intermediate 46 (10 mg, 6.04 μmol, 32.7% yield) was obtained as a colorless gum. LCMS(ESI)m / z[M+H]+1656.7.

[0316] LP-2

[0317] [ka]

[0318] To a solution of intermediate 46 (10 mg, 6.04 μmol) and 3-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)propanoate 2,5-dioxopyrrolidine-1-yl (1.929 mg, 7.25 μmol) in THF (1 mL), a solution of sodium bicarbonate (0.761 mg, 9.06 μmol) in water (1 mL) was added, and the reaction mixture was stirred at 21 °C for 3 hours. After this, the reaction mixture is vacuum concentrated to remove THF, and the remaining aqueous solution is directly purified by reverse-phase flash column chromatography (30-40% MeCN / water [0.1% formic acid]) to obtain (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-dodecaoxaoctatricontan-38-oil)-2,5-diazabicyclo[2.2.1]heptan-2-yl)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl) Propanamide)-6-oxohexanamide)propanamide)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]indolidino[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%) was obtained as a white solid. LCMS(ESI)m / z[MH]-1806.6.

[0319] LP-3 synthesis

[0320] [ka]

[0321] The reference linker-payload LP-3 was prepared by a synthesis procedure similar to that disclosed above.

[0322] LP-4 synthesis

[0323] [ka] 3-(2-bromoacetamide)propanoic acid 2,5-dioxopyrrolidine-1-yl (65.0 mg, 0.21 mmol) was added to 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.

[0324] The crude mixture was subjected to first purification: direct C18 flash chromatography for an elution gradient of 0-100% MeCN in water (0.1% FA). The pure fraction was evaporated to dryness to obtain the crude product. The crude product was preparatively HPLC (column: Sunfire Prep C18 OBD column, 19 * MeCN was purified using the following method: 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 minutes; wavelength: 254 nm / 220 nm; RT1 (min): 13.72). The fraction containing the desired compound is directly freeze-dried to obtain (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-dodecaoxaoctatricontan-38-amide)hexahydrofl[3,2-b]furan-3-yl)amino)-4-oxobutyl)-15-bromo-3,7,10,14-tetraoxo-2,6,9,13-tetraazapentadecyl )-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]indolidino-[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (LP-4) (47.0 mg, 14.08%) was obtained as a yellow solid. 11H 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=7.2H z,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),3.5 0-3.53(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) (one proton was exchanged) ES + (M+1=1894)

[0325] Antibody-drug conjugation ADC-1: Herceptin-WT-LP-1 (DAR 8) LP-1 was added as DMSO solution (16 molar equivalents / antibody, 0.64 μmol in 0.128 mL of DMSO) to 1.82 mL (6.0 mg, 40.0 nanomoles) of Herceptin-wt antibody solution in PBS, 1 mM EDTA, pH 7.4, to achieve 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 micromoles, 32.03 μL at 100 mM), and subsequently purified in PBS pH 7.4 by spin filtration using a 15 mL AMICON ULTRACELL 30 kDa MWCO spin filter, formulated, filtered sterile, and analyzed.

[0326] UHPLC analysis using a SHIMADZU PROMINENCE system with a THERMO SCIENTIFIC MAbPac 50mm×2.1mm column eluting ADC reduction samples at 214nm and 330nm with water and acetonitrile gradients revealed a mixture of one LP-1 conjugated light chain and three LP-1 conjugated heavy chain, consistent with a drug-to-antibody ratio (DAR) of 7.9 LP-1 molecules per antibody.

[0327] UHPLC analysis of ADC samples at 280 nm using a SHIMADZU PROMINENCE system with a TOSOH BIOSCIENCE TSKgel SuperSW mAb HTP 4μm 4.6×150mm column (along with a 4μm 3.0×20mm guard column) and eluting with 0.3 mL / min sterile filtered SEC buffer containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride, and 10% isopropanol (v / v), showed a monomer purity of 97.9%. UHPLC SEC analysis showed a final ADC concentration of 2.86 mg / mL in 1.6 mL, and the obtained mass was 4.5 mg (75% yield).

[0328] LC-MS analysis performed on an EXACTIVE PLUS EMR mass spectrometer connected to a DIONEX 3000 HPLC instrument using a THERMO SCIENTIFIC MAbPac 50 mm × 2.1 mm column that elutes ADC deglycosylation and reduction samples with a water and acetonitrile gradient at 214 nm showed a mixture of 1 LP-1 conjugated light chain and 3.0 LP-1 conjugated heavy chain, consistent with a drug-to-antibody ratio (DAR) of 7.9 LP-1 molecules per antibody.

[0329] UHPLC analysis using a SHIMADZU PROMINENCE system with an A PROTEOMIX HIC Butyl-NP5, 5µm, non-porous, 4.6×35mm (SEPAX) column eluting neat ADC samples at 214nm with gradients of 1.5M ammonium sulfate, 25mM sodium acetate, pH 7.4, and 20% acetonitrile (v / v), showed a single conjugated LP-1, consistent with a drug-to-antibody ratio (DAR) of 8 molecules of LP-1 per antibody.

[0330] ADC-2: Herceptin-WT-LP-2 (DAR 8) LP-2 was added as DMSO solution (13 molar equivalents / antibody, 1.3 μmol in 0.130 mL of DMSO) to 3.0 mL (15.0 mg, 100.0 nanomoles) of Herceptin-wt antibody solution in PBS, 1 mM EDTA, pH 7.4 to achieve 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 micromoles, 65.0 μL at 100 mM), purified in PBS pH 7.4 using SECprep-AKTA, formulated in 20 mM His\His HCl, 240 mM sucrose pH 6.0 by spin filtration using a 15 mL AMICON ULTRACELL 30 kDa MWCO spin filter, filtered sterile, and analyzed.

[0331] UHPLC analysis using a SHIMADZU PROMINENCE system with a THERMO SCIENTIFIC MAbPac 50mm×2.1mm column eluting ADC reduction samples at 214nm and 330nm with water and acetonitrile gradients revealed a mixture of one LP-2 conjugated light chain and three LP-2 conjugated heavy chain, consistent with a drug-to-antibody ratio (DAR) of 7.98 LP-2 molecules per antibody.

[0332] UHPLC analysis of ADC samples at 280 nm using a SHIMADZU PROMINENCE system with a TOSOH BIOSCIENCE TSKgel SuperSW mAb HTP 4μm 4.6×150mm column (along with a 4μm 3.0×20mm guard column) and eluted with 0.3 mL / min sterile filtered SEC buffer containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride, and 10% isopropanol (v / v), showed a monomer purity of 99.23%.

[0333] LC-MS analysis performed on an EXACTIVE PLUS EMR mass spectrometer connected to a DIONEX 3000 HPLC instrument using a THERMO SCIENTIFIC MAbPac 50 mm × 2.1 mm column that elutes ADC deglycosylation and reduction samples with a water and acetonitrile gradient at 214 nm showed a mixture of one LP-2 conjugated light chain and three LP-2 conjugated heavy chain, consistent with a drug-to-antibody ratio (DAR) of 8.0 LP-2 molecules per antibody.

[0334] UHPLC analysis using a SHIMADZU PROMINENCE system with a PROTEOMIX HIC Butyl-NP5, 5um, non-porous, 4.6 × 35 mm (Sepax) column eluting neat ADC samples at 214 nm with 1.5 M ammonium sulfate, 25 mM sodium acetate, pH 7.4, and 20% acetonitrile (v / v) gradients, showed that LP-2 was single-unit conjugated, consistent with a drug-to-antibody ratio (DAR) of 8 molecules of LP-2 per antibody.

[0335] ADC-3: Herceptin-WT-LP-3 (DAR 8) (see reference) LP-3 was added as DMSO solution (14 molar equivalents / antibody, 1.4 μmol in 0.39 mL of DMSO) to 6.0 mL (30.0 mg, 200.0 nanomoles) of Herceptin-wt antibody solution in PBS, 1 mM EDTA, pH 7.4, to achieve 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 ​​(14 micromoles, 130.0 μL at 100 mM), purified in PBS pH 7.4 using SECprep-AKTA, formulated in 20 mM His\His HCl, 240 mM sucrose pH 6.0 by spin filtration using a 15 mL AMICON ULTRACELL 30 kDa MWCO spin filter, filtered sterile, and analyzed.

[0336] UHPLC analysis using a SHIMADZU PROMINENCE system with a THERMO SCIENTIFIC MAbPac 50mm×2.1mm column eluting ADC reduction samples at 214nm and 330nm with water and acetonitrile gradients revealed a mixture of one LP-3 conjugated light chain and three LP-3 conjugated heavy chain, consistent with a drug-to-antibody ratio (DAR) of 7.89 LP-3 molecules per antibody.

[0337] UHPLC analysis of ADC samples at 280 nm using a SHIMADZU PROMINENCE system with a TOSOH BIOSCIENCE TSKgel SuperSW mAb HTP 4μm 4.6×150mm column (along with a 4μm 3.0×20mm guard column) and eluted with 0.3 mL / min sterile filtered SEC buffer containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride, and 10% isopropanol (v / v), showed monomer purity of 98.67%.

[0338] LC-MS analysis performed on an EXACTIVE PLUS EMR mass spectrometer connected to a DIONEX 3000 HPLC instrument using a THERMO SCIENTIFIC MAbPac 50 mm × 2.1 mm column that elutes ADC deglycosylation and reduction samples with a water and acetonitrile gradient at 214 nm showed a mixture of one LP-3 conjugated light chain and three LP-3 conjugated heavy chain, consistent with a drug-to-antibody ratio (DAR) of 8.0 LP-3 molecules per antibody.

[0339] UHPLC analysis using a SHIMADZU PROMINENCE system with a PROTEOMIX HIC Butyl-NP5, 5um, non-porous, 4.6 × 35 mm (Sepax) column eluting neat ADC samples at 214 nm with 1.5 M ammonium sulfate, 25 mM sodium acetate, pH 7.4, and 20% acetonitrile (v / v) gradients, showed that LP-3 was single-unit conjugated, consistent with a drug-to-antibody ratio (DAR) of 8 molecules of LP-3 per antibody.

[0340] ADC-4: Herceptin-WT-LP-4 (DAR 8) LP-4 was added as DMSO solution (25 molar equivalents / antibody, 4.0 μmol in 0.166 mL of DMSO) to 2.0 mL (20.0 mg, 133.0 nanomoles) of Herceptin-wt antibody solution in PBS, 1 mM EDTA, and 15% borate buffer, pH 8.3, to achieve a 10% (v / v) final DMSO concentration. The solution was reacted at room temperature for 1 hour with gentle shaking. The solution was purified in PBS pH 7.4 using AKTA-SECPrep, formulated in 20 mM histidine / histidine HCl, 240 mM sucrose, pH 6.0 by spin filtration using a 15 mL Amicon Ultracell 30 kDa MWCO spin filter, filtered sterile, and analyzed.

[0341] UHPLC analysis using a Shimadzu Prominence system with a Thermo Scientific MAbPac 50mm × 2.1mm column eluting ADC reduction samples at 214nm and 330nm with water and acetonitrile gradients revealed a mixture of one LP-4 conjugated light chain and 3.0 LP-4 conjugated heavy chain, consistent with a drug-to-antibody ratio (DAR) of 7.87 LP-4 molecules per antibody.

[0342] UHPLC analysis of ADC samples at 280 nm using a Shimadzu Prominence system with a Tosoh Bioscience TSKgel SuperSW mAb HTP 4μm 4.6×150 mm column (along with a 4μm 3.0×20 mm guard column) and eluted with 0.3 mL / min sterile filtered SEC buffer containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride, and 10% isopropanol (v / v), showed a monomer purity of 99.68%. UHPLC SEC analysis showed a final ADC concentration of 4.92 mg / mL in 3.5 mL, with a mass of 17.2 mg (86% yield).

[0343] LC-MS analysis performed on an Exactive Plus EMR mass spectrometer connected to a Dionex 3000 HPLC instrument using a Thermo Scientific MAbPac 50 mm × 2.1 mm column that elutes ADC deglycosylation and reduction samples with a water and acetonitrile gradient at 214 nm showed a mixture of one LP-4 conjugated light chain and three LP-4 conjugated heavy chain, consistent with a drug-to-antibody ratio (DAR) of 8.0 LP-4 molecules per antibody.

[0344] UHPLC analysis on a Shimadzu Prominence system using a Proteomix HIC Butyl-NP5, 5um, non-porous, 4.6 × 35 mm (Sepax) column eluting neat ADC samples at 214 nm with gradients of 1.5 M ammonium sulfate, 25 mM sodium acetate, pH 7.4, and 20% acetonitrile (v / v), showed that LP-4 was conjugated singly, consistent with a drug-to-antibody ratio (DAR) of 7.96 molecules of LP-4 per antibody.

[0345] Control ADC-1 The DAR 8 negative control ADC (control ADC-1) was prepared by conjugating the negative control antibody NIP228 (whose contents are incorporated by reference in WO2015127273A1) and LP-1 using a conjugation process similar to that described for ADC-1. NIP228 is IgG1 with complementary binding to transferrin, a transmembrane protein with low expression on the tested cell lines, and therefore serves as a suitable negative control.

[0346] ADC toxicity evaluated using 3D NCI-N87, JIMT1, and MDAMB468 The culture medium from 80-90% confluence NCI-N87, JIMT1, and MDAMB468 cells was aspirated from a T175 flask, and the flask was rinsed with PBS (approximately 10 mL) and emptied. TrypLE (5 mL) Express Enzyme (1 ×) was added, and the flask was returned to a 37°C incubator with 5% CO2 for approximately 5 minutes, then the flask was shaken to detach the cells from the bottom. 10 mL of cell medium (RPMI 1640 for NCI-N87 and MDAMB468, DMEM for JIMT1, both supplemented with 50% fetal bovine serum) was added to the flask, and the cell suspension was transferred to a sterile 50 mL Falcon tube and then centrifuged (400 g, 5 min). The supernatant was aspirated, and the pellet was resuspended in 10 mL of culture medium. The cell suspension was thoroughly pipetted to break up any aggregates, and 10 μL of the solution was mixed with 10 μL of trypan blue-stained cells. Next, 20 μL of the mixture was transferred to a cell counting slide, and cell concentration and viability were measured using LUNA II. Following previous experiments that allowed for the determination of the best seeding density, NCI-N87, JIMT1, and MDAMB468 cell lines were seeded at 10,000, 3,000, and 3,000 cells / well in Corning Spheroid 96-well microplates, respectively.

[0347] A stock solution (650 μL) of antibody-drug conjugate (ADC) was prepared by diluting the ADC in filter-sterilized cell culture medium. A set of 9 × 5-fold dilutions of the previous ADC solution was prepared by transferring 65 μL of each solution onto 585 μL of cell culture medium in a 2 mL deep 96-well plate. The ADC dilutions were dispensed (50 μL / well) into two replication wells of the 96-well plate containing 50 μL of cell suspension seeded 48 hours prior. 50 μL of cell culture medium was placed in the control well. The 96-well plate containing cells and ADC was incubated at 37°C for 6 days in a CO2-supplied incubator. At the end of the incubation period, the plate was equilibrated at room temperature for 10 minutes, and then the CELLTITER-GLO 3D Cell Viability Assay was dispensed into each well (100 μL per well). The plates were pipetted together for 5 minutes, and then incubated at room temperature for 25 minutes. Well luminescence was measured, and the cell viability percentage was calculated from the average luminescence in two ADC-treated wells compared to the average luminescence (100%) in six control untreated wells. IC 50 The sigmoid function was determined from dose-response data using GRAPHPAD PRISM, which employs a nonlinear regression (curve fitting) algorithm: sigmoid, 4PL, where X is log(concentration).

[0348] [Table 3]

[0349] ADC toxicity evaluated with 3D SKOV3 and SKOV3 GUSB KO The culture medium was aspirated from SKOV3 WT and SKOV3 GUSB KO (β-glucuronidase knockout cell line generated using CRISPR targeting) cells at 80–90% confluence in a T175 flask, and the flask was rinsed with PBS (approximately 10 mL) and emptied. TrypLE (5 mL) Express Enzyme (1 ×) was added, and the flask was returned to a 37°C incubator with 5% CO2 for approximately 5 minutes, then the flask was shaken to detach the cells from the bottom. 10 mL of cell medium (McCoy's 5A supplemented with 50% fetal bovine serum) was added to the flask, and the cell suspension was transferred to a sterile 50 mL Falcon tube and then centrifuged (400 g, 5 min). The supernatant was aspirated, and the pellet was resuspended in 10 mL of culture medium. The cell suspension was thoroughly pipetted to break up any aggregates, and 10 μL of the solution was mixed with 10 μL of trypan blue-stained cells. Next, a 20 μL mixture was transferred to a cell counting slide, and cell concentration and viability were measured using LUNA II. Following previous experiments that allowed for the determination of the optimal seeding density, SKOV3 and SKOV3 GUSB KO were seeded at 3000 cells / well in a Corning Spheroid 96-well microplate.

[0350] A stock solution (650 μL) of antibody-drug conjugate (ADC) was prepared by diluting the ADC in filter-sterilized cell culture medium. A set of 9 × 5-fold dilutions of the previous ADC solution was prepared by transferring 65 μL of each solution onto 585 μL of cell culture medium in a 2 mL deep 96-well plate. The ADC dilutions were dispensed (50 μL / well) into two replication wells of the 96-well plate containing 50 μL of cell suspension seeded 48 hours prior. 50 μL of cell culture medium was placed in the control well. The 96-well plate containing cells and ADC was incubated at 37°C for 6 days in a CO2-supplied incubator. At the end of the incubation period, the plate was equilibrated at room temperature for 10 minutes, and then the CELLTITER-GLO 3D Cell Viability Assay was dispensed into each well (100 μL per well). The plates were pipetted together for 5 minutes, and then incubated at room temperature for 25 minutes. Well luminescence was measured, and the cell viability percentage was calculated from the average luminescence in two ADC-treated wells compared to the average luminescence (100%) in six control untreated wells. IC 50 The sigmoid function was determined from dose-response data using GraphPad Prism, which employs a nonlinear regression (curve fitting) algorithm: sigmoid, 4PL, where X is log(concentration).

[0351] [Table 4]

[0352] ADC toxicity evaluated with NCI-N87, MDAMB468, SKOV3 WT, and SKOV3 GUSB KO The culture medium from 80-90% confluence NCI-N87, MDAMB468, SKOV3 WT, and SKOV3 GUSB KO cells was aspirated from a T175 flask, and the flask was rinsed with PBS (approximately 10 mL) and emptied. TrypLE (5 mL) Express Enzyme (1 ×) was added, and the flask was returned to a 37°C incubator with 5% CO2 for approximately 5 minutes. The flask was then shaken to detach the cells from the bottom. 10 mL of RPMI 1640 and McCoy 5A cell medium, both supplemented with 50% fetal bovine serum, was added to the flask, and the cell suspension was transferred to a sterile 50 mL Falcon tube and then centrifuged (400 g, 5 min). The supernatant was aspirated, and the pellet was resuspended in 10 mL of culture medium. The cell suspension was thoroughly pipetted to break up any aggregates, and 10 μL of the solution was mixed with 10 μL of trypan blue-stained cells. Next, 20 μL of the mixture was transferred to a cell counting slide, and cell concentration and viability were measured using LUNA II. Following previous experiments that allowed for the determination of the best seeding density, MDAMB468, SKOV3 WT, and SKOV3 GUSB KO cell lines were seeded at 3000 cells / well, while NCI-N87 was seeded at 10000 cells / well.

[0353] A stock solution (550 μL) of antibody-drug conjugate (ADC) was prepared by diluting the ADC in filter-sterilized cell culture medium. A set of 9 × 5-fold dilutions of the previous ADC solution was prepared by transferring 110 μL of each solution onto 440 μL of cell culture medium in a 2 mL deep 96-well plate. The ADC dilutions were dispensed (50 μL / well) into two replication wells of the 96-well plate containing 50 μL of cell suspension seeded the previous day. 50 μL of cell culture medium was placed in the control well. The 96-well plate containing cells and ADC was incubated at 37°C for 6 days in a CO2-supplied incubator. At the end of the incubation period, the plate was equilibrated at room temperature for 10 minutes, and then CellTiter-Glo (Promega) was dispensed into each well (100 μL per well). The plate was placed on an orbital shaker for 10 minutes before stabilization at room temperature for 1 minute. Well luminescence was measured, and the cell survival percentage was calculated from the average luminescence in two ADC-treated wells compared to the average luminescence (100%) in six control, untreated wells. 50 The sigmoid function was determined from dose-response data using GRAPHPAD PRISM, which employs a nonlinear regression (curve fitting) algorithm: sigmoid, 4PL, where X is log(concentration).

[0354] [Table 5]

[0355] kD colloidal stability data for ADC-2, ADC-3, and ADC-1 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; a lower value (more negative) indicates a higher tendency of the conjugate to precipitate. All ADCs and natural 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 in three ways. The Kd values ​​are the average of each of the three tests. Outlier results (" *Those marked as '' were excluded from the average calculation. The results are shown in Table 4 and Figure 5, which demonstrate a statistically significant reduced precipitation tendency for ADC-1 compared to ADC-2 and ADC-3.

[0356] [Table 6]

[0357] The above description of exemplary embodiments is intended solely to inform those skilled in the art of the applicant's specification, its principles, and its practical applications, so that they may readily adapt and apply this specification in its many forms to best suit the requirements of their particular use. This description and its specific examples illustrate embodiments of this specification and are for illustrative purposes only. Thus, this specification is not limited to the exemplary embodiments described herein and can be modified in various ways. In addition, for reasons of clarity, various features of this specification described in the context of separate embodiments can also be combined to form a single embodiment. Conversely, for reasons of brevity, various features of this specification described in the context of a single embodiment can also be combined to form partial combinations thereof.

[0358] Contact C of this disclosure 1C. Conjugate of formula (IC) Ab-(G A -J A -D C ) k (I C) or a pharmaceutically acceptable salt thereof, in the formula, Ab is an antibody or its antigen-binding fragment. k is an integer between 1 and 10. each G A However, independently, it is a conjugation group conjugated to an antibody or its antigen-binding fragment. Each D C but,

[0359] [ka] And, each J A However, independently, it is the basis of formula (ICA),

[0360] [ka] E is (CH2) n1 And in the formula, n1 is 0, 1, 2, or 3. Q is

[0361] [ka] And, In the formula, ring F 1 However, it is a saturated bicyclic ring having 6, 7, or 8 carbon atoms and optionally 1 or 2 oxygen atoms, and ring F 2 However, the ring F is a saturated bicyclic ring having two nitrogen atoms, four, five, six, seven, or eight carbon atoms, and optionally one oxygen atom. 3 However, the expressed saturated bicyclic ring has one nitrogen atom, five, six, seven, or eight carbon atoms, and optionally one oxygen atom. R 1 However, C 1~4 It is alkyl, X is (CH2) n2 And in the formula, n2 is 0, 1, 2, or 3. Y is (CH2) n3 In the formula, n3 is 0, 1, 2, 3, or 4. Z is (CH2) n4 And in the formula, n4 is 1, 2, 3, 4, or 5. m is an integer between 5 and 17. p is 1 or 0, (G A ) but, G A It shows the connection point to, (D C ) but, D C This indicates the connection point to [the specified location].

[0362] 2C.Q is,

[0363] [ka] A conjugate of formula (IC) disclosed in Statement 1C or a pharmaceutically acceptable salt thereof.

[0364] 3C.Q,

[0365] [ka] A conjugate of formula (IC) disclosed in Statement 1C or a pharmaceutically acceptable salt thereof.

[0366] A conjugate of formula (IC) disclosed in any one of statements 1C to 3C, or a pharmaceutically acceptable salt thereof, wherein 4C.m is 9, 10, 11, 12, or 13.

[0367] 5C.R 1 However, a conjugate of formula (IC) disclosed in any one of statements 1C to 4C, which is CH3, or a pharmaceutically acceptable salt thereof.

[0368] A conjugate of formula (IC) disclosed in any one of statements 1C to 5C, or a pharmaceutically acceptable salt thereof, wherein 6C.E is CH2.

[0369] A conjugate of formula (IC) disclosed in any one of statements 1C to 6C, or a pharmaceutically acceptable salt thereof, wherein 7C.X is CH2.

[0370] A conjugate of formula (IC) disclosed in any one of statements 1C to 7C, where 8C.Y is (CH2)2, or a pharmaceutically acceptable salt thereof.

[0371] A conjugate of formula (IC) disclosed in any one of statements 1C to 8C, or a pharmaceutically acceptable salt thereof, wherein 9C.Z is (CH2)2.

[0372] A conjugate of formula (IC) disclosed in any one of statements 1C to 9C, or a pharmaceutically acceptable salt thereof, wherein 10C.p is 1.

[0373] 11C.Each J A However, the conjugate of formula (IC) disclosed in Statement 1C, or a pharmaceutically acceptable salt thereof, which is the basis of formula (ICB).

[0374] [ka]

[0375] 12C.G A but,

[0376] [ka] Selected from, in the formula, R K However, it is H or CH3, and R L However, C 1~6 It is alkyl,

[0377] [ka] A conjugate of formula (IC) disclosed in any one of statements 1C to 11C, or a pharmaceutically acceptable salt thereof, which indicates a binding site to an antibody or its antigen-binding fragment.

[0378] 13C.G A but,

[0379] [ka] A conjugate of formula (IC) disclosed in Statement 12C or a pharmaceutically acceptable salt thereof.

[0380] A conjugate of formula (IC) disclosed in one of statements 1C to 13C, where 14C.k is an integer between 2 and 8, or a pharmaceutically acceptable salt thereof.

[0381] 15C. A pharmaceutical composition comprising a conjugate of formula (I) disclosed in any one of statements 1C to 14C, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0382] 16C. Conjugates of formulas (IC) disclosed in any one of statements 1C to 14C, or pharmaceutically acceptable salts thereof, for use in therapy.

[0383] 17C. Conjugates of formula (IC) disclosed in any one of statements 1C to 14C, or pharmaceutically acceptable salts thereof, for use in the treatment of cancer.

[0384] 18C. A method for treating cancer in a patient, comprising administering to the patient a conjugate of formula (IC) disclosed in any one of statements 1C to 14C, or a pharmaceutically acceptable salt thereof.

[0385] 19C. Use of a conjugate of formula (IC) disclosed in any one of statements 1C to 14C, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicinal product for the treatment of cancer.

[0386] 20C. Compounds of formula (IIC) G B -J B -D C (IIC) or a salt thereof, in the formula G B However, it is a conjugation group for conjugation to an antibody or its antigen-binding fragment, J B However, this is the basis of equation (IICA),

[0387] [ka] In the formula, D C E, Q, R 1X, Y, Z, m, and p are defined for the conjugate of expression (IC) in any one of statements 1C to 10C, and (G B ) but, G B The connection point to (D C ) but, D C A compound of formula (IIC) or a salt thereof that exhibits a bonding site to [a specific element].

[0388] 21C.G B but,

[0389] [ka] Selected from, in the formula, X 1 However, it is CH or N, h is 0 or 1, Hal is Cl, Br, or I. R K However, it is either H or CH3. R L However, C 1~6 A compound or salt thereof of formula (IIC) disclosed in Statement 20C, which is alkyl.

[0390] 22C.G B but,

[0391] [ka] A compound of formula (IIC) or a salt thereof disclosed in Statement 21C.

[0392] 23C.

[0393] [ka] (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-dodecaoxaoctatricontan-38-amide)hexahydrofl[3,2-b]furan-3-yl)amino)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)propanamide)-6-oxohexanamide)propanamide)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]indolidino-[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid, a compound of formula (IIC) disclosed in Statement 20C or a salt thereof.

Claims

1. Formula (IC): Ab-(G Ａ -J Ａ -D Ｃ ) ｋ (IC) [During the ceremony, Ab is an antibody or its antigen-binding fragment. k is an integer between 1 and 10. Each G Ａ This is independently a conjugation group conjugated to the antibody or its antigen-binding fragment, Each D Ｃ teeth, 【Chemistry 1】 And, Each J Ａ Independently, formula (ICA): 【Chemistry 2】 It is the basis of, E is (CH ２ ) ｎ１ where n1 is 0, 1, 2, or 3, Q is, 【Transformation 3】 And, R １ C １～４ It is alkyl, X is (CH ２ ) ｎ２ In the formula, n² is 0, 1, 2, or 3. Y is (CH ２ ) ｎ３ In the formula, n3 is 0, 1, 2, 3, or 4. Z is (CH ２ ) ｎ４ In the formula, n4 is 1, 2, 3, 4, or 5. m is an integer between 5 and 17. p is either 1 or 0, (G Ａ ) is G Ａ It shows the connection point to, (D Ｃ ) is D Ｃ [Indicates the connection point to] The conjugate of or a pharmaceutically acceptable salt thereof.

2. Q is, 【Chemistry 4】 A conjugate of formula (IC) or a pharmaceutically acceptable salt thereof as described in claim 1.

3. m is 9, 10, 11, 12, or 13, and / or R1 is CH3. A conjugate of formula (IC) as described in claim 1, or a pharmaceutically acceptable salt thereof.

4. E is CH ２ is, and / or X is CH2. A conjugate of formula (IC) as described in claim 1, or a pharmaceutically acceptable salt thereof.

5. Y is (CH ２ ) ２ is, and / or Z is CH₂ or (CH₂)₂. A conjugate of formula (IC) as described in claim 1, or a pharmaceutically acceptable salt thereof.

6. A conjugate of formula (IC) according to claim 1 or a pharmaceutically acceptable salt thereof, wherein p is 1.

7. Each J Ａ However, formula (ICB): 【Transformation 5】 A conjugate of formula (IC) as described in claim 1, or a pharmaceutically acceptable salt thereof, which is the base of the formula.

8. G Ａ but, 【Transformation 6】 [During the ceremony, R Ｋ is H or CH ３ And R Ｌ C １～６ It is alkyl, 【Transformation 7】 [This indicates a binding site to the antibody or its antigen-binding fragment.] A conjugate of formula (IC) according to claim 1 or a pharmaceutically acceptable salt thereof, selected from the above.

9. G Ａ but, 【Transformation 8】 A conjugate of formula (IC) or a pharmaceutically acceptable salt thereof as described in claim 8.

10. G Ａ but, 【Chemistry 9】 A conjugate of formula (IC) or a pharmaceutically acceptable salt thereof as described in claim 8.

11. A conjugate of formula (IC) according to claim 1 or a pharmaceutically acceptable salt thereof, wherein k is an integer from 2 to 8.

12. A pharmaceutical composition comprising a conjugate of formula (IC) according to any one of claims 1 to 11 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

13. A pharmaceutical product for (i) therapy or (ii) treatment of cancer, comprising a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 11.

14. Formula (IIC): G Ｂ -J Ｂ -D Ｃ (IIC) [During the ceremony, G Ｂ This is a conjugation group for conjugation to an antibody or its antigen-binding fragment, J Ｂ The formula is (IICA): 【Chemistry 10】 It is the basis of, and in the formula, D Ｃ E, Q, R １ X, Y, Z, m, and p are defined as for the conjugate of formula (IC) described in any one of claims 1 to 6, and (G Ｂ ) is G Ｂ The connection point to (D Ｃ ) is D Ｃ [Indicates the connection point to] A compound or salt thereof.

15. G Ｂ but, 【Chemistry 11】 [During the ceremony, X １ is CH or N, h is either 0 or 1, Hal is Cl, Br, or I. R Ｋ is H or CH ３ And, R Ｌ C １～６ It is alkyl. A compound of formula (IIC) or a salt thereof according to claim 14, selected from the above.

16. G Ｂ but, 【Chemistry 12】 or 【Chemistry 13】 The compound of formula (IIC) or a salt thereof according to claim 15. 【Request Item 17】 【Chemistry 14】 (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-dodecaoxaoctatricontan-38-amide)hexahydrofloxac -(((((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]indolidino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid or a salt thereof, which is a compound of formula (IIC) or a salt thereof according to claim 14. 【Request Item 18】 【Chemistry 15】 (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-dodecaoxaoctatricontane-38-amide)hexahydrofloxac[3,2-b]furan-3-yl)amino)-4-oxobutyl)-15-bromo-3,7,10,14-tetraoxo-2,6,9,13-tetraazapentadecyl)-4-(((((1S, A compound of formula (IIC) or a salt thereof according to claim 14, which is 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]indolidino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid or a salt thereof.

19. Formula (IXC): 【Chemistry 16】 [In the ceremony, G Ｂ is a conjugation group for conjugation to an antibody or its antigen-binding fragment, where E, Y, Z and p are as defined for the conjugate of formula (IC) described in any one of claims 1, 4, 5, or 6, and R Ｑ１ H or R Ｐ１ And each R Ｑ２ H or R Ｐ２ And R Ｑ３ H or R Ｐ３ And R Ｐ１ is a carboxylic acid protecting group, and each R Ｐ２ R is independently an alcohol protecting group. Ｐ３ [This is an amine protecting group.] A compound or salt thereof.

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