Antibody drug conjugates comprising two glucuronic acid linkers to treat her2 cancers

Abstract

The specification relates to conjugates of Formula (I): and pharmaceutically acceptable salts thereof. The specification also relates the use of the conjugates for the treatment of diseases such as cancer, and intermediates useful for the synthesis of the conjugates.

Description

[0001] ANTIBODY DRUG CONJUGATES COMPRISING TWO GLUCURONIC ACID LINKERS TO TREAT HER2 CANCERS

[0002] Cross-Reference to a Related Patent Application

[0003] This specification claims the benefit of priority to European Patent Application No. 25156373.0 (filed 6 February 2025). The entire text of the above-referenced patent application is incorporated by reference into this specification.

[0004] Field

[0005] This specification relates to certain conjugates comprising cleavable linkers, and to pharmaceutical compositions containing them. This specification also relates to the use of the conjugates in methods of treating diseases such as cancer. This specification further relates to processes and intermediate compounds involved in the preparation of the conjugates.

[0006] Background

[0007] Antibody-drug conjugates (ADCs) are an established method to deliver a drug to a biological target. The drug (or "payload") is released from the ADC at a target site so that the free drug can have a therapeutic effect. If free drug is released before it reaches the target site, then this can lead to off-target toxicity and reduce the therapeutic window of the ADC.

[0008] The antibody and drug portions of an ADC may be conjugated to one another via a linker portion. Release of the free drug may be achieved via cleavage of the linker portion. The linker portion may comprise a trigger that can be selectively cleaved by p-glucuronidase, an enzyme which is broadly upregulated in heme and solid tumors.

[0009] There remains a need for conjugates having p-glucuronidase-cleavable linkers that can selectively deliver a drug to a biological target and that have favourable physicochemical properties, including solubility and lipophilicity. The conjugates of the disclosure may be used for the treatment of diseases such as cancer.

[0010] General Description

[0011] In a first aspect there is provided a conjugate of Formula (I)

[0012] J

[0013] / A-DR

[0014] Ab — GA-BA

[0015]

[0016] or a pharmaceutically acceptable salt thereof, whereinBGL-103-PCT01-NP

[0017] Ab is an antibody or antigen-binding fragment thereof,

[0018] k is an integer from 1 to 10,

[0019] GAis independently a conjugation group conjugated to the antibody or antigen-binding fragment thereof,

[0020] DRis independently a drug comprising a nitrogen atom NR,

[0021] BAis independently a group of Formula (IA)

[0022] O

[0023] , IA)

[0024]

[0025] (JA)

[0026] wherein,

[0027] r is 0, 1, 2, 3, 4, 5, 6, 7, or 8,

[0028] W1is (CH2)n1, wherein n1 is 1, 2, 3, 4, 5, or 6,

[0029] QBis a covalent bond or R1,

[0030] (GA) indicates the point of attachment to GA, and (JA) indicates the point of attachment to JA, JAis independently a group of Formula (IB)

[0031]

[0032] wherein

[0033] E is (CH2)n2, wherein n2 is 0, 1, 2 or 3,

[0034] X1and X2are (CH2)n3, wherein n3 is independently 1, 2, 3 or 4,

[0035] p is 1 or 0,

[0036] q is 0, 1, 2, 3, 4, 5, 6, 7, or 8,

[0037] QJis R1or a covalent bond,

[0038] (NR) indicates the point of attachment to the nitrogen atom NR, and (BA) indicates the point of attachment to BA,

[0039] R1is independently a group of Formula (IC)BGL-103-PCT01-NP

[0040]

[0041] wherein

[0042] m is an integer from 5 to 17,

[0043] R2is Ci-4 alkyl,

[0044] X3is (CH2)n4, wherein n4 is 0, 1, 2 or 3,

[0045] Y is O or NR3, wherein R3is H, Ci-4alkyl or C3-4 cycloalkyl,

[0046] (C=O) indicates the point of attachment to the adjacent C=O group, and (CH2) indicates the point of attachment to the adjacent CH2,

[0047] and wherein at least one of QBand QJis R1.

[0048] In a further aspect there is provided a pharmaceutical composition comprising a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

[0049] In a further aspect there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, for use in therapy.

[0050] In a further aspect there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer.

[0051] In a further aspect there is provided the use of a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament.

[0052] In a further aspect there is provided the use of a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of cancer.

[0053] In a further aspect there is provided a method of treating cancer in a patient in need thereof comprising administering to the patient an effective amount of a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof.

[0054] In a further aspect there is provided a compound of Formula (II)

[0055] JB-D

[0056] / R

[0057] GB-BB(")

[0058]

[0059] XJB-DRBGL-103-PCT01-NP

[0060] or a salt thereof, wherein

[0061] GBis a conjugation group for conjugation to an antibody or antigen-binding fragment thereof, DRis independently a drug comprising a nitrogen atom NR,

[0062] BBis a group of Formula (IIA)

[0063]

[0064] (JB)

[0065] wherein,

[0066] r is 0, 1, 2, 3, 4, 5, 6, 7, or 8,

[0067] W1is (CH2)n1, wherein n1 is 1, 2, 3, 4, 5, or 6,

[0068] QBis a covalent bond or R1,

[0069] (GB) indicates the point of attachment to GB, and (JB) indicates the point of attachment to JB, JBis independently a group of Formula (IIB)

[0070] OH HO,

[0071] ' 'Q' N N q H H

[0072] p

[0073]

[0074] wherein

[0075] E is (CH2)n2, wherein n2 is 0, 1, 2 or 3,

[0076] X1and X2are (CH2)n3, wherein n3 is independently 1, 2, 3 or 4,

[0077] p is 1 or 0,

[0078] q is 0, 1, 2, 3, 4, 5, 6, 7, or 8,

[0079] QJis R1or a covalent bond,

[0080] (NR) indicates the point of attachment to the nitrogen atom NR, and (BB) indicates the point of attachment to BB,

[0081] R1is independently a group of Formula (IC)BGL-103-PCT01-NP

[0082]

[0083] wherein

[0084] m is an integer from 5 to 17,

[0085] R2is Ci-4 alkyl,

[0086] X3is (CH2)n4, wherein n4 is 0, 1, 2 or 3,

[0087] Y is O or NR3, wherein R3is H, Ci-4alkyl or C3-4 cycloalkyl,

[0088] (C=O) indicates the point of attachment to the adjacent C=O group, and (CH2) indicates the point of attachment to the adjacent CH2,

[0089] and wherein at least one of QBand QJis R1.

[0090] In a further aspect there is provided a method of making a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, using a compound of Formula (II), or a salt thereof.

[0091] In a further aspect there is provided intermediates useful for the synthesis of a compound of Formula (II), or a salt thereof.

[0092] A conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, may undergo enzymatic cleavage to release a free drug. Conjugates of Formula (I) may exhibit improved efficacy and / or advantageous physical properties (for example, higher stability, lower lipophilicity, higher aqueous solubility, higher permeability and / or lower plasma protein binding), and / or favourable toxicity profiles (for example reduced off target toxicity), and / or favourable metabolic or pharmacokinetic profiles, in comparison with other conjugates. In embodiments, Conjugates of Formula (I) exhibit improved colloidal stability in comparison with other conjugates. As such, conjugates of Formula (I) may be especially suitable for use in therapy, such as the treatment of cancer.

[0093] A conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, may undergo slower cleavage in the presence of p-glucuronidase relative to other conjugates. Without wishing to be bound by theory, it is believed that slower cleavage of the linker to release a free drug may result in improved tolerability of the conjugate by reducing the proportion of free drug released in off-target tissue.

[0094] DefinitionsBGL-103-PCT01-NP

[0095] So that the present specification may be more readily understood, certain terms are explicitly defined below. In addition, definitions are set forth as appropriate throughout the detailed description. Where examples are provided for a definition, they are not limiting.

[0096] The prefix Cx-y, where x and y are integers, indicates the numerical range of carbon atoms that are present in a group.

[0097] As used herein the term "alkyl" refers to a saturated, linear or branched hydrocarbon radical having the specified number of carbon atoms. Examples of Ci.4alkyl groups include methyl (Me), ethyl (Et), n-propyl (”Pr), i-propyl ('Pr), n-butyl (”Bu), i-butyl ('Bu), s-butyl (sBu), and t-butyl Bu). Examples of Ci-s alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl and n-hexyl.

[0098] As used herein the term "cycloalkyl" refers to a saturated, cyclic hydrocarbon radical having the specified number of carbon atoms. Examples of C3-4 cycloalkyl groups include cyclopropyl and cyclobutyl.

[0099] As used herein the term "conjugation group for conjugation to an antibody, or antigen-binding fragment thereof" refers to an atom or group of atoms capable of forming at least one covalent bond to an antibody, or antigen-binding fragment thereof, through a chemical reaction.

[0100] *

[0101] The use of " ' " in formulas of this specification indicates the point of attachment to the antibody

[0102] or antigen-binding fragment thereof. By way of illustration

[0103]

[0104] indicates that there is a covalent bond connecting the antibody, or antigen-binding fragment thereof, to the carbon atom marked 1.

[0105] For the avoidance of doubt, the use of

[0106]

[0107] in formulas of this specification denotes the point of covalent attachment to a group, where the group is other than the antibody or antigen-binding fragment thereof.

[0108] Certain embodiments of this specification include a group which is said to be "optionally substituted". In further embodiments said group is unsubstituted.

[0109] Units, prefixes, and symbols are denoted in their International System of Units (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range.BGL-103-PCT01-NP

[0110] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. 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 one of skill with a general dictionary of many of the terms used in this disclosure.

[0111] Description of Figures

[0112] Embodiments and experiments illustrating the principles of the disclosure will now be discussed with reference to the accompanying figures in which:

[0113] Figure 1 shows cytotoxicity data for ADC-1 and ADC-2 in a Her2+++ NCI-N87 cell line.

[0114] Figure 2 shows cytotoxicity data for ADC-1 and ADC-2 in a HER2- MDAMB468 cell line.

[0115] Detailed Description

[0116] In one aspect, this specification provides a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, as defined above.

[0117] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein k is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In further embodiments k is 1, 2, 3 or 4. In further embodiments k is 1. In further embodiments k is 2. In further embodiments k is 3. In further embodiments k is 4.

[0118] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein GAis independently selected fromBGL-103-PCT01-NP

[0119]

[0120] wherein RKis H or CH3, RLis C1-6alkyl,

[0121]

[0122] and indicates the point of attachment to the antibody, or antigen-binding fragment thereof.

[0123] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein GAis independently selected from

[0124]

[0125] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein GAis

[0126] O

[0127]

[0128] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein GAisBGL-103-PCT01-NP

[0129] o

[0130]

[0131] JA-DR

[0132] --GA-BA

[0133] In embodiments, for each

[0134]

[0135] one, two or three of QBand QJare R1.

[0136] In embodiments, for each

[0137]

[0138] QBis R1and each QJis a covalent bond.

[0139] In embodiments, for each

[0140]

[0141] QBis R1and each QJis a R1.

[0142] JA-DR

[0143] --GA-BA

[0144] In embodiments, for each

[0145]

[0146] -, QBis a covalent bond, one QJis R1and one QJis a covalent bond.

[0147] JA-DR

[0148] --GA-BA

[0149] In embodiments, for each

[0150]

[0151] -, QBis a covalent bond, and each QJis independently R1. In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein E is independently (CH2)n2, wherein n2 is 0, 1, 2 or 3. In further embodiments E is a covalent bond. In further embodiments E is CH2. In further embodiments E is (CH2)2. In further embodiments E is (CH2)3.

[0152] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein m is independently 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17. In further embodiments m is independently an integer from 6 to 16. In further embodiments m independently is an integer from 7 to 15. In further embodiments m is independently an integer from 8 to 14. In further embodiments m is independently an integer from 9 to 13. In further embodiments m is independently an integer from 10 to 12. In further embodiments m is 11.BGL-103-PCT01-NP

[0153] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein R2is independently Ci_4alkyl. In further embodiments R2is CH3.

[0154] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein p is independently 1 or 0. In further embodiments p is 1.

[0155] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein q is independently 0, 1, 2, 3, 4, 5, 6, 7, or 8. In further embodiments q is independently 1, 2, 3 or 4. In further embodiments q is 2.

[0156] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein r is independently 0, 1, 2, 3, 4, 5, 6, 7, or 8. In further embodiments r is independently 0, 1, 2, 3, 4. In further embodiments r is independently 0, 1 or 2. In further embodiments r is 0.

[0157] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein W1is independently (CH2)n1, wherein n1 is 1, 2, 3, 4, 5 or 6. In further embodiments W1is independently CH2or (CH2)2. In further embodiments W1is CH2. In further embodiments W1is (CH2)2.

[0158] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein X1and X2are independently (CH2)n3, wherein n3 is independently 1, 2, 3 or 4. In further embodiments X1and X2are independently (CH2)n3, wherein n3 is independently 1 or 2. In further embodiments X1is CH2and X2is CH2.

[0159] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein X3is independently (CH2)n4, wherein n4 is 0, 1, 2, or 3. In further embodiments, X3is a covalent bond. In further embodiments X3is CH2. In further embodiments X3is (CH2)2. In further embodiments X3is (CH2)3.

[0160] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein Y is O.

[0161] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein BAis independently a group of Formula (IA1)BGL-103-PCT01-NP

[0162]

[0163] wherein (GA), (JA), R2, m, r, W1, X3, and Y are as defined herein.

[0164] In further embodiments, JAis independently a group of Formula (IB1)

[0165] (BV ^x 11

[0166] (IB1)

[0167]

[0168] wherein (BA), (NR), E, p, q, X1and X2are as defined herein.

[0169] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt

[0170] (IA2)

[0171] thereof, wherein BAis a group of Formula (IA2), or

[0172] (IA2')

[0173]

[0174] and JAis a group of Formula (IB2)BGL-103-PCT01-NP

[0175] OH

[0176]

[0177] wherein ( BA), (GA) and (NR) are as defined herein.

[0178] In embodiments, there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt jA__

[0179] /

[0180] --GA-BA\JA„ thereof, wherein each is

[0181]

[0182] wherein (NR) indicates the point of attachment to the nitrogen atom NR, and

[0183]

[0184] indicates the point of attachment to the antibody, or antigen-binding fragment thereof.

[0185] In embodiments, there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt JA-DR

[0186] /

[0187] GA-BA

[0188] XJA-DR

[0189] thereof, wherein each

[0190]

[0191] isBGL-103-PCT01-NP

[0192] OH

[0193]

[0194] wherein indicates the point of attachment to the antibody, or antigen-binding fragment thereof.

[0195] In embodiments, there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt JA-DR

[0196] /

[0197] --GA-BA

[0198] XJA-DRthereof, wherein eachL

[0199]

[0200] BGL-103-PCT01-NP

[0201] wherein indicates the point of attachment to the antibody, or antigen-binding fragment thereof.

[0202] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein JAis independently a group of Formula (IB3)

[0203] OH HO,

[0204] x2N >< N N q H < H H N p

[0205]

[0206] R2‘Ol

[0207] Jm wherein ( BA), ( NR), E, m, p, q, R2, X1, X2, X3and Y are as defined herein.

[0208] In further embodiments, BAis a group of Formula (IA3)

[0209]

[0210] 'J', wherein (GA), (JA), r and W1are as defined herein. In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt O

[0211] N

[0212]

[0213] thereof, wherein BAis a group of Formula (IA4) (IA4) or Formula (IA4')

[0214] (JA)

[0215]

[0216] (IA41)

[0217] and JAis a group of Formula (IB4)BGL-103-PCT01-NP

[0218] (IB4)

[0219] wherein

[0220]

[0221] In embodiments, there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt JAGA— BA

[0222] thereof, wherein each

[0223]

[0224] is

[0225]

[0226] BGL-103-PCT01-NP

[0227] wherein (NR) indicates the point of attachment to the nitrogen atom NR, and indicates the point of attachment to the antibody, or antigen-binding fragment thereof.

[0228] In embodiments, there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt

[0229]

[0230] and wherein indicates the point of attachment to the antibody, or antigen-binding fragment thereof.

[0231] In embodiments, there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt

[0232] / JA-DR

[0233] --GA-BA

[0234] h r o, h r inXJA-D

[0235] t e e f w e e each

[0236]

[0237] L RBGL-103-PCT01-NP

[0238]

[0239] the point of attachment to the antibody, or antigen-binding fragment thereof.

[0240] In a further aspect there is provided a compound of Formula (II)

[0241] ZJB-DR

[0242] GB— BB(")

[0243]

[0244] XJB-DR

[0245] or a salt thereof, wherein GBis a conjugation group for conjugation to an antibody or antigen-binding fragment thereof,

[0246] BBis a group of Formula (IIA)

[0247] (IIA)

[0248]

[0249] JBis independently a group of Formula (IIB)BGL-103-PCT01-NP

[0250] N E (IIB)

[0251] (BB) H

[0252]

[0253] wherein DR, E, p, QB, QJ, q, R1, r, W1, X1and X2are as defined for any embodiment of a conjugate of Formula (I) disclosed herein, (BB) indicates the point of attachment to BB, (GB) indicates the point of attachment to GB, (JB) indicates the point of attachment to JBand (NR) indicates the point of attachment to the nitrogen atom NR.

[0254] In embodiments there is provided a compound of Formula (II), or a salt thereof, wherein GBis selected from

[0255]

[0256] wherein X° is CH or N, h is 0 or 1, Hal is Cl, Br or I, RKis H or CH3, and RLis C1-6 alkyl.

[0257] In embodiments there is provided a compound of Formula (II), or a salt thereof, wherein GBis selected fromBGL-103-PCT01-NP

[0258]

[0259] In embodiments there is provided a compound of Formula (II), or a salt thereof, wherein GBis

[0260]

[0261] In embodiments there is provided a compound of Formula (II), or a salt thereof, wherein GBis o

[0262]

[0263] In embodiments there is provided a compound of Formula (II), or a salt thereof, wherein BBis a group of Formula (IIA1)

[0264]

[0265] wherein (GB), (JB), R2, m, r, W1, X3, and Y are as defined herein.

[0266] In further embodiments, each JBis a group of Formula (IIB1)

[0267] (IIB1)

[0268]

[0269] BGL-103-PCT01-NP

[0270] wherein (BB), (NR), E, p, q, X1and X2are as defined herein.

[0271] In embodiments there is provided a compound of Formula (II), or a salt thereof, wherein BBis a

[0272] N H

[0273] O

[0274] or Formula (IIA2')

[0275] N H

[0276] H3C'°

[0277]

[0278] and JBis a group of Formula (IIB2)

[0279] HO,

[0280] O O N N H H

[0281] (IIB2)

[0282]

[0283] wherein (BB), (GB), (JB) and (NR) are as defined herein.

[0284] In embodiments there is provided a compound of Formula (II), or a salt thereof, wherein JB- /

[0285] GB— BB

[0286] V

[0287]

[0288] BGL-103-PCT01-NP

[0289]

[0290] wherein (NR) indicates the point of attachment to the nitrogen atom NR.

[0291] In embodiments there is provided a compound of Formula (II), or a salt thereof, that is (2S,2'S,3S,3'S,4S,4'S,5R,5'R,6S,6'S)-6,6'-(((16-(2-((3-((3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanamido)methyl)-l-(2,5,8,ll,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-oyl)azetidin-3-yl)oxy)acetyl)-3,7,25,29-tetraoxo-10,13,19,22-tetraoxa-2,6,16,26,30-pentaazahentriacontane-l,31-diyl)bis(4-((5S,8S,llS,12R)-ll-((S)-sec-butyl)-12-(2-((S)-2-((lR,2R)-3-(((lS,2R)-l-hydroxy-l-phenylpropan-2-yl)amino)-l-methoxy-2-methyl-3-oxopropyl)pyrrolidin-l-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)-2,l-phenylene))bis(oxy))bis(3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid)BGL-103-PCT01-NP

[0292] OH

[0293]

[0294] a salt thereof.

[0295] In embodiments there is provided a compound of Formula (II), or a salt thereof, that is

[0296]

[0297] thereof.

[0298] In embodiments there is provided a compound of Formula (II), or a salt thereof, wherein JBis independently a group of Formula (IIB3)BGL-103-PCT01-NP

[0299] (IIB3)

[0300]

[0301] wherein (BB), (NR), E, m, p, q, R2, X1, X2, X3and Y are as defined herein.

[0302] In further embodiments, BBis a group of Formula ( 11 A3)

[0303] (IIA3)

[0304]

[0305] (JB), wherein (GB), (JB), r and W1are as defined herein. In embodiments there is provided a compound of Formula (II), or a salt thereof, wherein BBis a O O

[0306] (GB)\ N JXX(JB) <GBK A NX

[0307]

[0308] (JB) (JB) group of Formula (IIA4) (IIA4) or Formula (IIA4') (IIA4 )

[0309] and each JBis a group of Formula ( 11 B4)

[0310]

[0311] wherein (BB), (GB), (JB) and (NR) are as defined herein.BGL-103-PCT01-NP

[0312] In embodiments there is provided a compound of Formula (II), or a salt thereof, wherein

[0313] JB

[0314] /

[0315] GB— BB

[0316] V

[0317] is

[0318]

[0319] wherein (NR) indicates the point of attachment to the nitrogen atom NR.

[0320] In embodiments there is provided a compound of Formula (II), or a salt thereof, that isBGL-103-PCT01-NP

[0321]

[0322] In embodiments there is provided a compound of Formula (II), or a salt thereof, that is

[0323]

[0324] BGL-103-PCT01-NP

[0325] The present specification is intended to include all isotopes of atoms occurring in the present compounds and conjugates. Isotopes will be understood to include those atoms having the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium. Isotopes of carbon include13C and14C. Isotopes of nitrogen include15N.

[0326] The compounds disclosed herein may contain one or more chiral centers. Accordingly, if desired, such compounds can be prepared or isolated as pure stereoisomers, i.e. as individual enantiomers, diastereoisomers, or as a stereoisomerically enriched mixture. All such stereoisomer (and enriched) mixtures are included within the scope of the embodiments, unless otherwise stated. Pure stereoisomers (or enriched mixtures) may be prepared using, for example, optically active starting materials or stereoselective reagents well-known in the art. Alternatively, racemic mixtures of such compounds can be separated using, for example, chiral column chromatography, chiral resolving agents and the like.

[0327] Unless stereochemistry is explicitly indicated in a chemical structure or chemical name, the chemical structure or chemical name is intended to embrace all possible stereoisomers, diastereoisomers, conformers, rotamers and tautomers of the compound depicted. For example, a compound containing a chiral carbon atom is intended to embrace both the (R) enantiomer and the (S) enantiomer, as well as mixtures of the enantiomers, including racemic mixtures; and a compound containing two chiral carbons is intended to embrace all enantiomers and diastereoisomers including (R, R), (S, S), (R, S) and (S, R).

[0328] The term "pharmaceutically acceptable salt" is used to specify that a salt is suitable for use in patients. An example list of pharmaceutically acceptable salts and preparation methods can be found in the Handbook of Pharmaceutical Salts: Properties, Selection and Use, P. H. Stahl and C. G. Wermuth, editors, Weinheim / Zurich: Wiley-VCH / VHCA, 2002;

[0329] A suitable pharmaceutically acceptable salt of a conjugate of Formula (I) is, for example, an acid addition salt. In general, an acid addition salt can be prepared using various inorganic or organic acids. Such salts can typically be formed by, for example, mixing the compound with an acid (e.g., a stoichiometric amount of acid) using various methods known in the art.

[0330] A further suitable pharmaceutically acceptable salt of a conjugate of Formula (I) is, for example, a salt formed within a patient's body after administration of a conjugate of Formula (I) to the patient. In a further aspect there is provided a pharmaceutical composition comprising a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.BGL-103-PCT01-NP

[0331] The term "pharmaceutical composition" refers to a preparation which is in such form as to permit the biological activity of the active ingredient, and which contains no additional components which are unacceptably toxic to a patient to which the composition would be administered. Such compositions can be sterile. A pharmaceutical composition according to the present specification will comprise a conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0332] In embodiments there is provided a pharmaceutical composition comprising a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable, non-toxic, sterile carrier. In further embodiments the carrier is a physiological saline, non-toxic buffer, or preservative. Suitable formulations for use in the therapeutic methods disclosed herein are described in Remington's Pharmaceutical Sciences, 22nd ed., Ed. Lloyd V. Allen, Jr. (2012), the contents of which are incorporated by reference.

[0333] Pharmaceutical compositions comprising a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, may be comprised within one or more formulations selected from a capsule, a tablet, an aqueous suspension, a nasal aerosol, or a combination thereof.

[0334] In one aspect there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, for use in therapy.

[0335] In one aspect there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer.

[0336] Where "cancer" is mentioned, this includes both non-metastatic cancer and also metastatic cancer, such that treating cancer involves treatment of both primary tumours and also tumour metastases. In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of HER2 positive cancer.

[0337] In one aspect there is provided the use of a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, as described herein, in the manufacture of a medicament, such as a medicament for the treatment of cancer.

[0338] In one aspect there is provided a method of treating cancer in a patient in need thereof comprising administering to the patient an effective amount of a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof.

[0339] As used herein, the term "treating" or "treatment" refers to alleviating the specified condition, eliminating or reducing one or more symptoms of the condition, slowing or eliminating the

[0340] T1BGL-103-PCT01-NP

[0341] progression of the condition, and delaying the reoccurrence of the condition in a previously afflicted or diagnosed patient or subject.

[0342] As used herein, the term "effective amount" means that amount of a conjugate of Formula (I) that will elicit the biological or medical response of a tissue, system, animal or human this is being sought, for instance, by a researcher or clinician.

[0343] The term "therapeutically effective amount" means any amount which, as compared to a corresponding subject who has not received such amount, results in improved treatment, healing or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder. The term also includes within its scope the amounts effective to enhance normal physiological function.

[0344] The term "patient" refers to any animal (e.g., a mammal), including, but not limited to humans, nonhuman primates, rodents, and the like, which is to be the recipient of a particular treatment. In embodiments the term "patient" refers to a human subject.

[0345] In embodiments there is provided a method of treating cancer in a patient in need thereof, comprising administering to the patient an effective amount of a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein the cancer is a HER2 positive cancer.

[0346] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, and an additional anti-tumour substance for the conjoint treatment of cancer.

[0347] In embodiments there is provided a combination for use in the treatment of cancer comprising a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof and an additional antitumour agent.

[0348] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, in combination with an additional anti-tumour agent.

[0349] Herein, where the term "conjoint treatment" is used in reference to a combination treatment, it is to be understood that this may refer to simultaneous, separate or sequential administration. In one aspect, "conjoint treatment" refers to simultaneous administration. In another aspect, "conjoint treatment" refers to separate administration. In a further aspect, "conjoint treatment" refers to sequential administration.

[0350] In embodiments there is provided a method of treating cancer in a patient in need thereof comprising administering to the patient an effective amount of a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, and simultaneously, separately or sequentiallyBGL-103-PCT01-NP

[0351] administering at least one additional anti-tumour substance to said patient, where the amounts of the conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, and the additional antitumour substance are jointly effective in producing an anti-cancer effect.

[0352] Conjugation or (—

[0353] w

[0354] w

[0355] Examples of GAand GBinclude, but are not limited to, the following, wherein X° is CH or N, h is 0 or 1, RKis H or CH3, Hal is Cl, Br or I, RLis C1-6 alkyl, and

[0356]

[0357] indicates the point of attachment to the 0

[0358] antibody, or antigen-binding fragment thereof. z

[0359] 0

[0360] GAGB

[0361] 0 0

[0362] ^ X)

[0363] _NH2

[0364] 0 p

[0365] ._AN' 7 A

[0366] X)

[0367] 0 0

[0368] 0 0

[0369] 0 0

[0370] K xCk \

[0371] *— <N

[0372] 0

[0373] 0 ^ X)

[0374] RK

[0375] s 7

[0376] sx

[0377] (O2N)h-|j- 0 0

[0378] X. Hak A \

[0379] Nx

[0380] H H O

[0381] Hal^^O^

[0382]

[0383] BGL-103-PCT01-NP

[0384] 0 0

[0385] z

[0386] *

[0387] zN-^Z / \ \H O ^ * \ AHzi H ' z

[0388] \ H.

[0389] Z= z '

[0390] jZ ^

[0391] N'Z

[0392] N\

[0393] *

[0394] HN^I )

[0395] LI

[0396] R ' w

[0397] Z Z- ° o,.

[0398] y^z *— - J " tfl x

[0399] R°' HYN'N > z /

[0400] / / zz- ^'

[0401] I "

[0402] * X- Hal\J\

[0403] N3^

[0404] H \ H2N.0A * O '

[0405] JI J

[0406]

[0407] Antibody or antigen-binding fragment thereofBGL-103-PCT01-NP

[0408] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to, or is immunologically reactive with, a particular antigen.

[0409] In embodiments the antibody is isolated or recombinant. " Isolated", when used herein refers to a polypeptide, e.g., an antibody, that has been identified and separated and / or recovered from a cell or cell culture from which it was expressed. Ordinarily, an isolated antibody will be prepared by at least one purification step. Thus, an "isolated antibody" refers to an antibody which is substantially free of other antibodies having different antigenic specificities.

[0410] In embodiments the antibody comprises at least two "light chains" (LC) and two "heavy chains" (HC). The light chains and heavy chains of such antibodies 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 the heavy chain constant domains CHI, CH2 and CH3 (antibody classes IgA, IgD, and IgG) and optionally the 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").

[0411] In embodiments the antibody is a full-length antibody. An "intact" or "full-length" antibody, as used herein, refers to an antibody having two heavy (H) chain polypeptides and two light (L) chain polypeptides interconnected by disulfide bonds.

[0412] A "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. The variable regions VH and VL can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs) (also known as hypervariable regions), interspersed with regions that are more conserved, termed framework regions (FRs). In embodiments each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The VH or VL chain of the antibody can further include all or part of a heavy or light chain constant region.

[0413] 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) capable of binding to (e.g. being bound by) an antibody or antigen-binding fragment of the disclosure. The CDRs are the main determinants of antigen specificity. There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda Md.)); and (2) an approach based onBGL-103-PCT01-NP

[0414] crystallographic studies of antigen-antibody complexes ( Al-lazikani et al. (1997) J. Molec. Biol.

[0415] 273:927-948)). In addition, combinations of these two approaches are sometimes used in the art to determine CDRs.

[0416] The "constant domains" (or "constant regions") of the heavy chain and of the light chain are not involved directly in binding of an antibody to a target, but exhibit various effector functions. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0417] There are five major classes of heavy chain constant region, classified as IgA, IgG, IgD, IgE and IgM, each with characteristic effector functions designated by isotype. Ig molecules interact with multiple classes of cellular receptors. For example, IgG molecules interact with three classes of Fey receptors (FcyR) specific for the IgG class of antibody, namely FcyRI, FcyRII, and Fey Rl 11. Binding of antibody to Fc receptors on cell surfaces triggers a number of important and diverse biological responses including engulfment and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer and control of immunoglobulin production. The important sequences for the binding of IgG to the FcyR receptors have been reported to be located in the CH2 and CH3 domains.

[0418] In embodiments the antibody or antigen-binding fragment thereof is an IgG isotype. The antibody or antigen-binding fragment thereof can be any IgG subclass, for example IgGl, lgG2, lgG3, or lgG4 isotype. In embodiments the antibody or antigen-binding fragment thereof is based on an IgGl isotype.

[0419] The terms " Fc region", " Fc part" and " Fc" are used interchangeably herein and refer to the portion of a native immunoglobulin that is formed by two Fc chains. Each " Fc chain" comprises a constant domain CH2 and a constant domain CH3. Each Fc chain may also comprise a hinge region. A native Fc region is homodimeric. In embodiments the Fc region may be heterodimeric because it may contain modifications to enforce Fc heterodimerisation. The Fc region contains the carbohydrate moiety and binding sites for complement and Fc receptors (including the FcRn receptor), and has no antigen binding activity. Fc can refer to this region in isolation, or this region in the context of an antibody, antibody fragment, or Fc fusion protein. Polymorphisms have been found in a number of Fc domain sites, including but not limited to EU positions 270, 272, 312, 315, 356, and 358, resulting in minor variations between the sequences described in the instant application and sequences known in the art. As a result, every naturally occurring IgG Fc region is referred to as a "wild type IgG Fc domain"BGL-103-PCT01-NP

[0420] or " WT IgG Fc domain" (i.e., any allele). Human IgGl, lgG2, lgG3, and lgG4 heavy chain sequences can be obtained in a variety of 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.

[0421] In embodiments the antibody of the disclosure is a monoclonal antibody. A "monoclonal antibody" (mAb) refers to a homogeneous antibody population involved in the highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies that typically include different antibodies directed against different antigenic determinants. The term "monoclonal antibody" can encompass both full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single chain (scFv) mutants, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site. Furthermore, "monoclonal antibody" refers to such antibodies made in any number of ways including, but not limited to, hybridoma, phage selection, recombinant expression, and transgenic animals. In embodiments the antibody of the disclosure is an isolated monoclonal antibody. In further embodiments the antibody is a fully human monoclonal antibody.

[0422] In embodiments the antibody of the disclosure is a full-length antibody described above.

[0423] Alternatively, the antibody can be an antigen-binding fragment. The term "antigen-binding fragment" as used herein incudes 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 is capable of binding to the antigen.

[0424] In embodiments the antigen-binding fragment of the disclosure is a Fab fragment. The antibody according to the disclosure can also be a Fab', an Fv, an scFv, an Fd, a V NAR domain, an IgNAR, an intrabody, an IgG CH2, a minibody, a single-domain antibody, an Fcab, an scFv-Fc, F(ab')2, a di-scFv, a bi-specific T-cell engager (BITE), a F(ab')3, a tetrabody, a triabody, a diabody, a DVD-lg, an (scFv)2, a mAb2 or a DARPin.

[0425] The terms " Fab fragment" and " Fab" are used interchangeably herein and contain a single light chain (e.g. a constant domain CL and a VL) and a single heavy chain (e.g. a constant domain CHI and a VH). The heavy chain of a Fab fragment is not capable of forming a disulfide bond with another heavy chain.

[0426] A " Fab1fragment" contains a single light chain and a single heavy chain but in addition to the CHI and the VH, a " Fab1fragment" contains the region of the heavy chain between the CHI and CH2BGL-103-PCT01-NP

[0427] domains that is required for the formation of an inter-chain disulfide bond. Thus, two " Fab1fragments" can associate via the formation of a disulfide bond to form a F(ab')2 molecule.

[0428] A " F(ab')2 fragment" contains two light chains and two heavy chains. Each chain includes a portion of the constant region necessary for the formation of an inter-chain disulfide bond between two heavy chains.

[0429] An " Fv fragment" contains only the variable regions of the heavy and light chain. It contains no constant regions.

[0430] A "single-domain antibody" is an antibody fragment containing a single antibody domain unit (e.g., VH or VL).

[0431] A "single-chain Fv" ("scFv") is antibody fragment containing the VH and VL domain of an antibody, linked together to form a single chain. A polypeptide linker is commonly used to connect the VH and VL domains of the scFv.

[0432] A "tandem scFv", also known as a TandAb, is a single-chain Fv molecule formed by covalent bonding of two scFvs in a tandem orientation with a flexible peptide linker.

[0433] A "bi-specificT cell engager" (BiTE) is a fusion protein consisting of two single-chain variable fragments (scFvs) on a single peptide chain. One of the scFvs binds to T cells via the CD3 receptor, and the other to a tumour cell antigen.

[0434] A "diabody" is a small bivalent and bispecific antibody fragment comprising a heavy chain variable domain (VH) connected to a light chain variable domain (VL) on the same polypeptide chain (VH-VL) connected by a peptide linker that is too short to allow pairing between the two domains on the same chain (Kipriyanov, Int. J. Cancer 77 (1998), 763-772). This forces pairing with the complementary domains of another chain and promotes the assembly of a dimeric molecule with two functional antigen binding sites.

[0435] A " DARPin" is a bispecific ankyrin repeat molecule. DARPins are derived from natural ankyrin proteins, which can be found in the human genome and are one of the most abundant types of binding proteins. A DARPin library module is defined by natural ankyrin repeat protein sequences, using 229 ankyrin repeats for the initial design and another 2200 for subsequent refinement. The modules serve as building blocks for the DARPin libraries. The library modules resemble human genome sequences. A DARPin is composed of 4 to 6 modules. Because each module is approx. 3.5 kDa, the size of an average DARPin is 16-21 kDa. Selection of binders is done by ribosome display,BGL-103-PCT01-NP

[0436] which is completely cell-free and is described in He M. and Taussig MJ., Biochem Soc Trans. 2007, Nov;35(Pt 5):962-5.

[0437] In embodiments the antibody or antigen-binding fragment thereof can be further modified to contain additional chemical moieties not normally part of the protein. Those derivatised moieties can improve the solubility, the biological half-life or absorption of the protein. The moieties can also reduce or eliminate any desirable side effects of the proteins and the like. An overview for those moieties can be found in Remington's Pharmaceutical Sciences, 22nd ed., Ed. Lloyd V. Allen, Jr. (2012).

[0438] Drug (DR)

[0439] As used herein, DRis independently a drug comprising a nitrogen atom NR. For a conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, nitrogen atom NRis covalently attached to JA. For a compound of Formula (II) or a salt thereof, nitrogen atom NRis covalently attached to JB. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, may undergo cleavage to release the drug in its free drug form. DRmay be represented as N(Rm)(Rn), wherein N(Rm)(Rn) is collectively the drug and the nitrogen atom shown is NR. In this representation, the free drug form is HN(Rm)(Rn).

[0440] For a conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, or a compound of Formula (II) or a salt thereof, the nitrogen atom NRmay be the nitrogen atom of a secondary of tertiary carbamate. In other words, the nitrogen atom NRmay, in the free drug form, be the nitrogen atom of a primary or secondary amine.

[0441] In embodiments there is provided a conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, or a compound of Formula (II) or a salt thereof, wherein the nitrogen atom NRin the nitrogen atom of an aliphatic secondary of tertiary carbamate. In other words, the nitrogen atom NR, in the free drug form, is the nitrogen atom of an aliphatic primary or secondary amine.

[0442] In embodiments the drug is a therapeutic agent. In further embodiments the drug is a cytotoxic drug. In further embodiments the drug is a DNA damage response inhibitor, antineoplastic agent and tubulin disrupting agent. In further embodiments the drug is a topoisomerase I inhibitor or microtubule inhibitor (MTI). In further embodiments the drug is a camptothecin drug.

[0443] In embodiments the drug is a diagnostic agent or an imaging agent.BGL-103-PCT01-NP

[0444] In embodiments there is provided a conjugate of Formula (I) or a pharmaceutically acceptable salt

[0445] thereof, or a compound of Formula (II) or a salt thereof, wherein

[0446]

[0447] DRis. In these embodiments the free drug form of the drug is exatecan ((lS,9S)-l-amino-9-ethyl-5-fluoro- 9-hydroxy-4-methyl-l,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b]quinoline-10, 13-dione).

[0448] In embodiments there is provided a conjugate of Formula (I) or a pharmaceutically acceptable salt

[0449] thereof, or a compound of Formula (II) or a salt thereof, wherein

[0450]

[0451] DRis

[0452] . In these embodiments, the free drug form of the drug

[0453]

[0454] is ((lS,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-lH,12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-l-yl)bicyclo[l.l.l]pentane-l-carboxamide (DA). In embodiments there is provided a conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, or a compound of Formula (II) or a salt thereof, wherein DRisBGL-103-PCT01-NP

[0455]

[0456] HO. in these embodiments the free drug form of the drug is MMAE ((S)-N-((3R,4S,5S)-l-((S)-2-((lR,2R)-3-(((lS,2R)-l-hydroxy-l-phenylpropan-2-yl)amino)-l-methoxy-2-methyl-3-oxopropyl)pyrrolidin-l-yl)-3-methoxy-5-methyl-l-oxoheptan-4-yl)- N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamide).

[0457] The released drug may, following release from the conjugate in the biological system, undergo a chemical modification, such as an enzyme-mediated chemical reaction and / or metabolism. As such, in embodiments the drug may be a pro-drug.

[0458] Examples

[0459] The specification will now be illustrated by the following non-limiting Examples.

[0460] General Information

[0461] Flash chromatography was performed using a BIOTAGE ISOLERA and fractions checked for purity using thin-layer chromatography (TLC). TLC was performed using MERCK KIESELGEL 60 F254 silica gel, with fluorescent indicator on aluminium plates. Visualisation of TLC was achieved with UV light. Extraction and chromatography solvents were bought and used without further purification from VWR U. K.

[0462] All fine chemicals were purchased from SIGMA-ALDRICH unless otherwise stated.

[0463] Pegylated reagents were obtained from QUANTA BIODESIGN US via STRATECH UK.

[0464] LC / MS conditions

[0465] Positive mode electrospray mass spectrometry was performed using a WATERS ACQUITY H-CLASS SQD2 using one of the following methods.

[0466] The HPLC (WATERS ALLIANCE 2695) was run using a mobile phase of water (A) (formic acid 0.1%) and acetonitrile (B) (formic acid 0.1%).

[0467] LCMS 3 min: Initial composition 5% B held over 25 seconds, then increased from 5% B to 100% B over a 1 minute 35 seconds' period. The composition was held for 50 seconds at 100% B, thenBGL-103-PCT01-NP

[0468] returned to 5% B in 5 seconds and held there for 5 seconds. The total duration of the gradient run was 3.0 minutes. Flow rate was 0.8 mL / minute. Wavelength detection range: 190 to 800 nm.

[0469] Columns: WATERS ACQUITY UPLC BEH SHIELD RP18 1.7pm 2.1 x 50 mm at 50 °C fitted with WATERS ACQUITY UPLC BEH SHIELD RP18 VANGUARD Pre-column, 130A, 1.7pm, 2.1 mm x 5 mm.

[0470] LCMS 15 min: initial composition 5% B held over 1 min, then increase from 5% B to 100% B over a 9 min period. The composition was held for 2 min at 100% B, then returned to 5% B in 0.10 minutes and hold there for 3 min. Total gradient run time equals 15 min. Flow rate 0.6 mL / min. Wavelength detection range: 190 to 800 nm. Oven temperature: 50°C. Column: WATERS ACQUITY UPLC CSH C18 1.7pm 2.1 x 100mm fitted with WATERS ACQUITY UPLC CSH C18 VANGUARD Pre-column, 1.7pm, 2.1 mm x 5 mm.

[0471] HPLC conditions

[0472] Reverse-phase ultra-fast high-performance liquid chromatography (UFLC) was carried out on a SHIMADZU PROMINENCE machine using a PHENOMENEX GEMINI NX 5p C18 column (at 50 °C) dimensions: 150 x 21.2 mm. Eluents used were solvent A (H2O with 0.1% formic acid) and solvent B (CH3CN with 0.1% formic acid). All UFLC experiments were performed with gradient conditions: Initial composition 13% B increased to 30% B over a 3 minutes period, then increased to 45% B over 8 minutes and again to 100% over 6 minutes before returning to 13% over 2 min and hold for 1 min. The total duration of the gradient run was 20.0 minutes. Flow rate was 20.0 mL / minute and detection was at 254 and 223 nm.

[0473] Abbreviations

[0474] ADC Antibody-drug conjugate

[0475] Boc tert-butyloxycarbonyl

[0476] Cbz benzyloxycarbonyl

[0477] CSH Charged surface hybrid

[0478] DAR Drug to antibody ratio

[0479] DCM Dichloromethane

[0480] DIPEA Di isopropyl ethylamine

[0481] DMF Dimethyl formamide

[0482] DMSO Dimethyl sulfoxide

[0483] ESI Electrospray ionisation

[0484] Fmoc Fluorenylmethoxycarbonyl

[0485] HATU Hexafluorophosphate azabenzotriazole tetramethyl uronium

[0486] IC50Inhibitory concentration 50 %BGL-103-PCT01-NP

[0487] LCMS Liquid chromatography mass spectrometry

[0488] mAb Monoclonal antibody

[0489] ND Not determined

[0490] NMR Nuclear magnetic resonance

[0491] RP Reverse phase

[0492] RPMI Roswell Park Memorial Institute

[0493] RT Retention time

[0494] SEC Size exclusion chromatography

[0495] Su succinimide

[0496] TBS Tert-butyl dimethyl silane

[0497] TEA Triethylamine

[0498] TFA Trifluoro acetic acid

[0499] THF Tetra hydro furan

[0500] TLC Thin layer chromatography

[0501] UHPLC Ultra-high performance liquid chromatography

[0502] UPLC Ultra-performance liquid chromatography

[0503] UV Ultra violet

[0504] Synthesis of Intermediate 1:

[0505] CbzOSu, DIPEA CbzHN i. NaH H2, Pd / C N

[0506] Boc CAS: 1008526-71-3 78%

[0507] i. TFA FmocCI, DIPEA N OSu Boc 85%

[0508] DIPEA

[0509]

[0510] Intermediate 1 tert-butyl 3-(aminomethyl)-3-hydroxyazetidine-l-carboxylate (10 g, 49.44 mmol) was added to N-ethyl- / V-isopropylpropan-2-amine (19.17 g, 148.33 mmol) and / V-(Benzyloxycarbonyloxy)succinimide (14.79 g, 59.33 mmol) in THF (150 mL) under nitrogen. The resulting mixture was stirred at 25 °C for 3 hours. The solvent was removed under reduced pressure._The crude product was purified by flash silica chromatography, elution gradient 0 to 10% MeOH in DCM. Pure fractions were evaporated toBGL-103-PCT01-NP

[0511] dryness to afford tert-butyl 3-((((benzyloxy)carbonyl)amino)methyl)-3-hydroxyazetidine-l-carboxylate (13.00 g, 78 %) as a yellow oil. m / z (ES+), [M+H]+ = 337; TFA, HPLC tR = 0.903 min tert-butyl 3-((((benzyloxy)carbonyl)amino)methyl)-3-hydroxyazetidine-l-carboxylate (13 g, 38.65 mmol) was added to tert-butyl 2-bromoacetate (9.05 g, 46.38 mmol) and sodium hydride (1.855 g, 77.29 mmol) in THF (150 mL) under nitrogen at 0 °C. The resulting mixture was stirred at 25 °C for 2 hours. The reaction mixture was quenched with water (50 ml), extracted with EtOAc (3 x 150 ml), the top layer was dried over Na2SO4, filtered and evaporated to afford yellow oil. The crude product was purified by flash silica chromatography, elution gradient 0 to 50% EtOAc in petroleum ether. Pure fractions were evaporated to dryness to afford tert-butyl 3-((((benzyloxy)carbonyl)amino)methyl)-3-(2-(tert-butoxy)-2-oxoethoxy)azetidine-l-carboxylate (9.00 g, 51.7 %) as a colourless oil. m / z (ES+), [M+H]+ = 451; TFA, HPLC tR = 1.125 min

[0512] tert-butyl 3-((((benzyloxy)carbonyl)amino)methyl)-3-(2-(tert-butoxy)-2-oxoethoxy)azetidine-l-carboxylate (9 g, 19.98 mmol) was added to Royer(R) Palladium Catalyst Powder (6.07 g, 19.98 mmol) in THF (120 mL) under hydrogen. The resulting mixture was stirred at 25 °C for 6 hours. The reaction mixture was filtered through silica and washed with THF (3 x 50 mL). The solvent was removed under reduced pressure to afford tert-butyl 3-(aminomethyl)-3-(2-(tert-butoxy)-2-oxoethoxy)azetidine-l-carboxylate (6.00 g, 85 %) as a yellow oil. m / z (ES+), [M+H]+ = 317; TFA, HPLC tR = 0.882 min tert-butyl 3-(aminomethyl)-3-(2-(tert-butoxy)-2-oxoethoxy)azetidine-l-carboxylate (5 g, 15.80 mmol) was added to 9-Fluorenylmethyl chloroformate (6.13 g, 23.70 mmol) and N, N-Diisopropylethylamine (6.13 g, 47.41 mmol) in THF (50 mL) under nitrogen. The resulting mixture was stirred at 25 °C for 3 hours. The solvent was removed under reduced pressure._The crude product was purified by flash silica chromatography, elution gradient 0 to 50% EtOAc in petroleum ether. Pure fractions were evaporated to dryness to afford tert-butyl 3-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-3-(2-(tert-butoxy)-2-oxoethoxy)azetidine-l-carboxylate (3.66 g, 43.0 %) as a white solid, m / z (ES+), [M+H]+ = 539; TFA, HPLC tR = 1.230 min

[0513] To tert-butyl 3-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-3-(2-(tert-butoxy)-2-oxoethoxy)azetidine-l-carboxylate (615 mg, 1.14 mmol) in DCM (4 mL) was added TFA (4 mL). The reaction mixture was stirred at room temperature for 1.5 h. The reaction mixture was concentrated under reduced pressure to give a crude product which was used without further purification. A solution of mPeg12 NHS ester (708 mg, 1.03 mmol) and DIPEA (0.397 mL, 2.28 mmol) in DMF (5 mL) was added to crude 2-((3-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)azetidin-3-yl)oxy)acetic acid (436 mg, 1.14 mmol). The reaction mixture was stirred at room temperature for 1.5 h. The reaction mixture was purified by reverse phase column chromatography (5-60% MeCN in waterBGL-103-PCT01-NP

[0514] +0.1% FA) to give 2-((3-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-l- (2,5,8,ll,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-oyl)azetidin-3-yl)oxy)acetic acid (Intermediate 1) (654 mg, 66.5 %) as a colourless oil. LCMS (15 min): 6.43 min; 954.7 [M+H]+ Intermediate 2

[0515] TBSCI

[0516] imidazole, DCM

[0517] HO

[0518]

[0519] TBS-CI (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) at 0°C over a period of 2 hours under nitrogen. The resulting mixture was stirred at 0 °C for 2 hours. The reaction mixture was quenched with water (500 mL), extracted with DCM (2 x 300 mL) and the organic layer was dried over Na2SO4, filtered and evaporated to afford 5-(((tert-butyldimethylsilyl)oxy)methyl)-2-hydroxybenzaldehyde (Intermediate 2, 35.0 g, 100 %) as a colourless material, m / z (ES+), [M+Na]+= 289; NH4HCO3, HPLC tR = 1.505 min.

[0520] Intermediate 3

[0521]

[0522] Silver(I) oxide p.a. (52.2 g, 225.22 mmol) was added to 4A Molecular Sieves (30 g), (2R,3R,4S,5S,6S)-2-bromo-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (44.7 g, 112.61 mmol), Intermediate 2 (15 g, 56.30 mmol) in MeCN (500 mL) at 0°C over a period of 2 hours under nitrogen. The resulting mixture was stirred at 25 °C for 16 hours. The mixture was filtered through a Celite pad. The crude product was purified by flash silica chromatography, elution gradient 0 to 30% EtOAc in petroleum ether. Pure fractions were evaporated to dryness to afford (2S,3R,4S,5S,6S)-2-(4-(((tert-butyldimethylsilyl)oxy)methyl)-2-formylphenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (Intermediate 3) as a pale yellow material, m / z (ES+), [M+Na]+ = 605; TFA, HPLC tR = 1.071 min.

[0523] Intermediate 4BGL-103-PCT01-NP

[0524]

[0525] TFA (8 mL) was added to Et₃SiH (32 mL), tert-butyl carbamate (15.28 g, 130.43 mmol) and Intermediate 3 (20 g, 34.32 mmol) in MeCN (200 mL) at 0°C under nitrogen. The resulting mixture was stirred at 0 °C for 16 hours. The reaction mixture was quenched with saturated NaHCO₃ (200 mL), extracted with EtOAc (2 x 300 mL), and the organic layer was dried over Na2SO4, filtered and evaporated to afford a colourless residue. The crude product was purified by flash silica chromatography, elution gradient 0 to 50% THF in petroleum ether. Pure fractions were evaporated to dryness to afford (2S,3R,4S,5S,6S)-2-(2-(((tert-butoxycarbonyl)amino)methyl)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (Intermediate 4, 18.80 g, 96 %) as a pale yellow material, m / z (ES+), [M+Na]+= 592; TFA, HPLC tR = 1.088 min.

[0526] Intermediate 5

[0527]

[0528] Intermediate 4 (10 g, 17.56 mmol) in HCl / 1,4-dioxane (40 mL) / 1,4-dioxane (40 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 1 hour. The solvent was removed under reduced pressure to afford (2S,3R,4S,5S,6S)-2-(2-(aminomethyl)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate hydrochloride (Intermediate 5) as a white material, m / z (ES+), [M+H]+ = 470; TFA, HPLC tR = 0.587 min.

[0529] Intermediate 6

[0530] Fmoc.,

[0531] N

[0532]

[0533] HBGL-103-PCT01-NP

[0534] 1-(3-Dimethylaminopropyl)-3-ethyl-carbodiimide (1.197 g, 7.71 mmol) was added to 2, 3,5,6-tetrafluorophenol (1.280 g, 7.71 mmol) and 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoic acid (2g, 6.42 mmol) in DCM (20 mL) at 25°C. The resulting mixture was stirred at 25 °C for 2 hours. The reaction mixture was quenched with water (5 mL), extracted with DCM (3 x 5 mL), the organic layer was dried over Na2SO4, filtered and evaporated to afford 2,3,5,6-tetrafluorophenyl 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoate (Intermediate 6, 1.500 g, 50.8 %) as white solid. LCMS (3 mins): 1.35 min; 460 [M+H]+

[0535] Intermediate 7

[0536]

[0537] Sodium bicarbonate (1.097 g, 13.06 mmol) was added to Intermediate 6 ( 2 g, 4.35 mmol) and Intermediate 5 (1 g, 2.13 mmol) in THF (2 mL) and water (2 mL) at 25°C. The resulting mixture was stirred at 25 °C for 2 hours. The reaction mixture was acidified with 2M HCI. The solvent was removed under reduced pressure. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 50% MeCN in water. Pure fractions were evaporated to dryness to afford (2S,3R,4S,5S,6S)-2-(2-((3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)methyl)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (Intermediate 7, 2.20 g, 66.3 %) as a white solid, m / z (ES+), [M+H]+ = 763; base, HPLC tR = 1.167 min Intermediate 8

[0538]

[0539] DIEA (0.412 mL, 2.36 mmol) was added to Intermediate 7 (600mg, 0.79 mmol) and bis(4-nitrophenyl) carbonate (239 mg, 0.79 mmol) in THF (5 mL) at 25°C. The resulting mixture was stirred at 25 °C for 16 hours. The reaction mixture was quenched with water (5 mL), extracted with EtOAc (3x5 mL), the organic layer was dried over Na2SO4, filtered and evaporated to afford yellow solid. The crude product was purified by flash silica chromatography, elution gradient 0 to 70% EtOAc in petroleum ether. PureBGL-103-PCT01-NP

[0540] fractions were evaporated to dryness to afford (2S,3R,4S,5S,6S)-2-(2-((3-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)propanamido)methyl)-4-((((4- nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (Intermediate 8, 590 mg, 81 %) as a white solid, m / z (ES+), [M+H]+ = 928; base, HPLC tR = 1.279 min

[0541] Intermediate 9

[0542]

[0543] (5S,8S,llS,12R)-ll-((S)-sec-butyl)-l-(9H-fluoren-9-yl)-5,8-diisopropyl-12-methoxy-4,10-dimethyl- 3,6,9-trioxo-2-oxa-4,7,10-triazatetradecan-14-oic acid (12 g, 18.81 mmol) in trimethylamine (80 mL) was stirred at 70 °C for 3 hours. The solvent was removed under reduced pressure. The precipitate was collected by filtration, washed with petroleum ether (100 mL) and dried under vacuum to afford (3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamido)-3- methoxy-5-methylheptanoic acid (Intermediate 9, 5.80 g, 74.2 %) as a white solid. LCMS (3 mins): No PDA; 416 [M+H]+.

[0544] Intermediate 10

[0545]

[0546] Intermediate 8 (1.8 g, 1.94 mmol) was added to 2-hydroxypyridine-N-oxide (0.237 g, 2.13 mmol), DIEA (1.016 mL, 5.82 mmol) and Intermediate 9 (0.887 g, 2.13 mmol) in DMA (20 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 1 hour. The solvent was removed under reduced pressure. The crude product was purified by preparative Column: Xselect CSH Prep C18 OBD Colum, 30*150nm, 5μm; Mobile Phase A: Water(0.1%FA), Mobile Phase B: acetonitrile; Flow rate: 60 mL / min mL / min; Gradient: 5% B to 5% B in 2 min, 5% B to 55% B in 2.5 min, 55% B to 60% B in 10 min; Wave Length: 254nm / 220nm nm; RTl(min): 11. Fractions containing the desired compound wereBGL-103-PCT01-NP

[0547] evaporated to dryness to afford (5S,8S,llS,12R)-l-(3-((3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)methyl)-4-(((2S,3R,4S,5S,6S)-3,4,5-triacetoxy-6- (methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)-ll-((S)-sec-butyl)-5,8-diisopropyl-12-methoxy-4,10-dimethyl-3,6,9-trioxo-2-oxa-4,7,10-triazatetradecan-14-oic acid (Intermediate 10, 1.180 g, 50.5 %) as a white solid, m / z (ES+), [M+H]+ = 1204; acid, HPLC tR = 1.842 min Intermediate 11

[0548] TFA, DCM

[0549]

[0550] To tert-butyl (S)-2-((lR,2R)-3-(((lS,2R)-l-hydroxy-l-phenylpropan-2-yl)amino)-l-methoxy-2-methyl-3-oxopropyl)pyrrolidine-l-carboxylate (256 mg, 0.61 mmol) in DCM (2 mL) was added TFA (2 mL). The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was concentrated under reduced pressure to give a crude (2R,3R)-N-((lS,2R)-l-hydroxy-l-phenylpropan-2-yl)-3-methoxy-2-methyl-3-((S)-pyrrolidin-2-yl)propanamide (Intermediate 11) which was used without purification. LCMS (3 min): 0.55 min; 322.0 [M+H]+

[0551] Intermediate 12

[0552]

[0553] Intermediate 11 (150 mg, 0.47 mmol) was dissolved in DMF (2 mL) and N-ethyl-N-isopropylpropan-2-amine (1 mL). A solution of Intermediate 10 (175 mg, 0.15 mmol), 2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethylisouronium hexafluorophosphate(V) (148 mg, 0.39 mmol) and N-ethyl-N-isopropylpropan-2-amine (200 uL) in DMF (2 mL) was added to the previous reaction and was left stirring at 25 °C for lh. The reaction was concentrated under reduced pressure and purified by column chromatography (dichloromethane: methanol, from 0 to 5%) to afford (2S,3S,4S,5R,6S)-6-(2-((3-aminopropanamido)methyl)-4-((5S,8S,llS,12R)-ll-((S)-sec-butyl)-12-(2-((S)-2-((lR,2R)-3-(((lS,2R)-l-hydroxy-l-phenylpropan-2-yl)amino)-l-methoxy-2-methyl-3-oxopropyl)pyrrolidin-l-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenoxy)-3,4,5-BGL-103-PCT01-NP

[0554] trihydroxytetrahydro-2H-pyran-2-carboxylic acid (Intermediate 12, 65% yield). LCMS (3 min): 1.00 min; 1508.1 [M+H]+

[0555] Intermediate 13

[0556]

[0557] Piperidine (0.329 mL, 3.32 mmol) was added to Intermediate 12 (5000 mg, 3.32 mmol) in DMF (15 mL). The resulting mixture was stirred at rt for 3 hours. The crude product was purified by preparative HPLC Column: NanoMicro UniSil PFP, 50250mm10μm; Mobile Phase A: Water(0.1% FA), Mobile Phase B: acetonitrile; Flow rate: 80 mL / min mL / min; Gradient: 43% B to 53% B in 14 min, 95% B to 95% B in18min, 43% B to 43% B in 20 min; Wave Length: 220 nm; RTl(min): 14. Fractions containing the desired compound were evaporated to dryness to afford Intermediate 13 (1865 mg, 43.8 %) as a white solid. LCMS (3 min) RT 1.3 min [M+H]+ 1284.85

[0558] Intermediate 14

[0559]

[0560] To a solution of Intermediate 1 (500 mg, 0.52 mmol) and di-tert-butyl 4,7,13,16-tetraoxa-10-azanonadecanedioate (248 mg, 0.55 mmol) in DMF (10 mL) is added HATU (219 mg, 0.58 mmol) and DIPEA (457 uL, 2.62 mmol). After lh, the reaction is concentrated under reduced pressured and the residue is redissolved in DCM (10 mL) and TFA (5 mL). After lh the reaction is purified by reverse phase chromatography (0.1% formic acid, gradient 10 to 40% acetonitrile) to afford 10-(2-((3-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-l-(2,5,8,ll,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-oyl)azetidin-3-yl)oxy)acetyl)-4,7,13,16-tetraoxa-10-BGL-103-PCT01-NP

[0561] azanonadecanedioic acid(Intermediate 14, 650 mg, 97% yield) as a colourless oil. LCMS (3 min) RT 1.6 min [M+H]+1273.9

[0562] Intermediate 15

[0563]

[0564] To a solution of Intermediate 13 (70 mg, 0.05 mmol) and Intermediate 14 (140 mg, 0.11 mmol) in DMF (5 mL) is added HATU (45 mg, 0.12 mmol) and DIPEA (47 uL, 0.27 mmol). After 2h, the reaction is concentrated under reduced pressure and purified by column chromatography (dichloromethane: methanol, gradient from 0 to 10%) to afford Intermediate 15 (200 mg, 97% yield) as a colourless oil. LCMS (3 min) RT 2.0 min [M+H]2+1903.5

[0565] Intermediate 16

[0566]

[0567] To a solution of Intermediate 15 (200 mg, 0.05 mmol) in methanol (5 mL) is added a solution of potassium carbonate (218 mg, 1.58 mmol) in water (2 mL). The mixture is stirred at 25 C overnight and the crude is purified by reverse phase chromatography (0.1% formic acid, gradient 5 to 50% acetonitrile) to afford Intermediate 16 (100 mg, 57% yield) as a colourless oil. LCMS (3 min) RT 1.6 min [M+H]2+1652.3BGL-103-PCT01-NP

[0568] Linker-Payload 1 (LP1)

[0569]

[0570] To a solution of Intermediate 16 (100 mg, 0.03 mmol) in DMF (5 mL) is added 2,5-dioxopyrrolidin-l-yl 3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanoate (12 mg, 0.05 mmol). After lh, the reaction is concentrated under reduced pressure and purified by preparative HPLC (0.1% formic acid, gradient 15 to 75% acetonitrile) to afford (2S,2'S,3S,3'S,4S,4'S,5R,5'R,6S,6'S)-6,6'-(((16-(2-((3-((3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanamido)methyl)-l-(2,5,8,ll,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-oyl)azetidin-3-yl)oxy)acetyl)-3,7,25,29-tetraoxo-10,13,19,22-tetraoxa-2,6,16,26,30-pentaazahentriacontane-l,31-diyl)bis(4-((5S,8S,llS,12R)-ll-((S)-sec-butyl)-12-(2-((S)-2-((lR,2R)-3-(((lS,2R)-l-hydroxy-l-phenylpropan-2-yl)amino)-l-methoxy-2-methyl-3-oxopropyl)pyrrolidin-l-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)-2,l-phenylene))bis(oxy))bis(3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid) LP1 (85 mg, 81% yield). LCMS (15 min) RT 7.3 min [M+H]2+1727.6

[0571] Intermediate 17

[0572]

[0573] To Intermediate 13 (177 mg, 0.138 mmol) in DMF (4 mL) was added Intermediate 1 (132 mg, 0.14 mmol), HATU (105 mg, 0.28 mmol) and DIPEA (0.144 mL, 0.83 mmol). The reaction mixture was stirred at room temperature for 30 mins. The reaction mixture was purified by reverse phase chromatography (25-50% MeCN in water +0.1% FA) to give Intermediate 17 (153 mg, 50.0 %). LCMS (15 min): 8.15 min; 2219.9 [M+H]+

[0574] Intermediate 18BGL-103-PCT01-NP

[0575]

[0576] To Intermediate 17 (153 mg, 0.07 mmol) in methanol (4 mL) and water (2 mL) was added potassium carbonate (39.2 μl, 0.69 mmol). The reaction mixture was stirred at room temperature for 4 h. The reaction mixture was purified by reverse phase chromatography (15-35% MeCN in water +0.1% FA) to give (2S,3S,4S,5R,6S)-6-(2-((3-(2-((3-(aminomethyl)-l-(2,5,8,ll,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-oyl)azetidin-3-yl)oxy)acetamido)propanamido)methyl)-4-((5S,8S,llS,12R)-ll-((S)-sec-butyl)-12-(2-((S)-2-((lR,2R)-3-(((lS,2R)-l-hydroxy-l-phenylpropan-2-yl)amino)-l-methoxy-2-methyl-3-oxopropyl)pyrrolidin-l-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (Intermediate 18, 93 mg, 72.6 %). LCMS (15 min): 5.64 min; 1857.9 [M+H] Linker-Payload 2 (LP2)

[0577] o

[0578]

[0579] To Intermediate 18 (93 mg, 0.05 mmol) in DMF (2 mL) was added a solution of bis(2,5-dioxopyrrolidin-l-yl) 10-(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanoyl)-4,7,13,16-tetraoxa-10-azanonadecanedioate (17.07 mg, 0.025 mmol) in DMF (0.8 mL). DIPEA (0.026 mL, 0.15 mmol) was added and the reaction mixture stirred at room temperature for 3 h. The reaction mixture was purified by reverse phase chromatography (20-40% MeCN in water +0.1% FA) to give LP2 (54.8 mg, 52.6 %). LCMS (15 min): 7.37 min; 2084.0 [M+2H]+, 1390.0 [M+3H]+, 1042.8 [M+4H]+

[0580] Intermediate 20BGL-103-PCT01-NP

[0581] DIPEA, DMF FmocHN 2. Exatecan mesylate

[0582]

[0583] (2S,3R,4S,5S,6S)-2-(2-((3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)methyl)-4-(hydroxymethyl)phenoxy)-6-((allyloxy)carbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (Intermediate 19, 500 mg, 0.63 mmol, disclosed in WO2025036945 ) and bis(4-nitrophenyl) carbonate (212 mg, 0.70 mmol) were dissolved in DMF (3 mL) and N-ethyl-N-isopropylpropan-2-amine (0.442 mL, 2.54 mmol). After 2h, exatecan mesylate (505 mg, 0.95 mmol) was added in one portion. After 16h, the reaction was purified by reverse phase chromatography (10-80% MeCN in water + 0.05% TFA) to afford (2S,3R,4S,5S,6S)-2-(2-((3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)methyl)-4-(((((lS,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-lH,12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-l-yl)carbamoyl)oxy)methyl)phenoxy)-6-((allyloxy)carbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (Intermediate 20, 250 mg, 0.20 mmol, 31%). RT 1.92 min LCMS (ESI) 1252.4 [M+H]+

[0584] Intermediate 21

[0585] Potassium carbonate

[0586] FmocHN Methanol, water

[0587]

[0588] (2S,3R,4S,5S,6S)-2-(2-((3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)methyl)-4- (((((lS,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-lH,12H-BGL-103-PCT01-NP

[0589] benzo[de]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-l-yl)carbamoyl)oxy)methyl)phenoxy)-6-((allyloxy)carbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (Intermediate 20, 250.0 mg, 200 pmol, 1 Eq) was dissolved in MeOH (2 mL) followed by addition of potassium carbonate (276.3 mg, 2 mmol, 10 Eq) in water (200 pL). The reaction was left stirring overnight and then purified by reverse phase chromatography (5-30% MeCN in water + 0.05% TFA) to afford (2S,3S,4S,5R,6S)-6-(2-((3-aminopropanamido)methyl)-4-(((((lS,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-lH,12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-l-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (Intermediate 21, 18 mg, 20.89 pmol, 10%). RT 1.26 min LCMS (ESI) 863.3 [M+H]+

[0590]

[0591] 10-(2-((3-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-l-(2,5,8,ll,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-oyl)azetidin-3-yl)oxy)acetyl)-4,7,13,16-tetraoxa-10-azanonadecanedioic acid (Intermediate 14, 12.08 mg, 9.49 pmol) and 1-(bis(dimethylamino)methylene)-1H-[1,2,3]triazolo[4,5-b]pyridine-1-ium 3-oxide hexafluorophosphate(V) (7.22 mg, 0.02 mmol) were stirred in DMF (2 mL) and N-ethyl-N-isopropylpropan-2-amine (0.017 mL, 0.09 mmol). After 10 minutes, (2S,3S,4S,5R,6S)-6-(2-((3-aminopropanamido)methyl)-4-(((((lS,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-lH,12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-l-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (Intermediate 21, 18 mg, 0.02 mmol) was added in one portion. After 30 mins, the crude was purified by reverse phase chromatography (15-80% MeCN in water + 0.05% TFA) to afford Intermediate 22 (25 mg, 8.45 pmol, 89 %). RT 1.78 min LCMS (ESI) 1480.8 [M+2H]2+

[0592] Intermediate 23BGL-103-PCT01-NP

[0593]

[0594] Intermediate 22 (25 mg, 8.45 pmol) was dissolved in DMF (5 mL) and pyrrolidine (1 mL). After 30 mins, the reaction is concentrated under reduced pressure. The crude reaction was purified by preparative HPLC (15-60% MeCN in water + 0.05% TFA, Sinergy column) to afford Intermediate 23 (5 mg, 1.826 pmol, 22 %). RT 1.45 min LCMS (ESI) 2738.9 [M+H]+

[0595] Linker-Payload 3 (LP3)

[0596]

[0597] Intermediate 23 (5 mg, 1.83 pmol) was dissolved in DMF (1 mL) and N-ethyl-N-isopropylpropan-2-amine (1.591 pl, 9.13 pmol) followed by addition of 2,5-dioxopyrrolidin-l-yl 3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanoate (0.535 mg, 2.01 pmol). After lh, the reaction is concentrated under reduced pressure and the crude was purified by preparative HPLC (15-60% MeCN in water + 0.05% TFA, Sinergy column) to afford LP3 (0.68 mg, 0.235 pmol, 13 %). RT 6.43 min LCMS (ESI) 2887.7 [M-H]-, [M+H]2+ 1445.8

[0598] Synthesis of DA

[0599] Intermediate ABGL-103-PCT01-NP

[0600]

[0601] To a solution of Exatecan mesylate (CAS: 169869-90-3) (100 mg, 0.19 mmol) in DMF (2 mL) were added 3-((tert-butoxycarbonyl)amino)bicyclo[l.l.l]pentane-l-carboxylic acid (47.0 mg, 0.21 mmol), 2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethylisouronium hexafluorophosphate(V) (107 mg, 0.28 mmol) and N-ethyl-N-isopropylpropan-2-amine (0.098 mL, 0.56 mmol). The resulting solution was stirred at 21 °C for 2 hours. The crude product was purified by flash silica chromatography, elution gradient 0 to 10% MeOH in DCM. Pure fractions were evaporated to dryness to afford Intermediate A (161 mg, 133 %) as a pale yellow residue. LCMS (ESI) m / z [M+H]+ 645.7.

[0602] DA

[0603]

[0604] 2, 2, 2-trifl uoroacetic acid (0.6 ml, 7.78 mmol) was added to Intermediate A (135 mg, 0.21 mmol) in DCM (2.4 mL). The resulting solution was stirred at 21 °C for 45 minutes. The reaction was concentrated to afford crude product. The crude product was purified by flash C18 silica chromatography, elution gradient 0 to 50% MeCN+0.01%TFA in water+0.01%TFA. Pure fractions were evaporated to dryness to afford 3-amino-N-((lS,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-lH,12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-l-yl)bicyclo[l.l.l]pentane-l-carboxamide (DA)(107 mg, 77 %) as a yellow solid.1H NMR (400 MHz, DMSO) 68.69 - 8.58 (m, 4H), 7.77 (d, J = 10.9 Hz, 1H), 7.31 (s, 1H), 6.52 (s, 1H), 5.56 (td, J = 8.6, 4.6 Hz, 1H), 5.42 (s, 2H), 5.20 - 5.10 (m, 1H), 5.03 (d, J = 18.6 Hz, 1H), 3.29 - 3.05 (m, 2H), 2.39 (d, J = 1.8 Hz, 3H), 2.34 - 2.26 (m, 7H), 2.25 - 2.04 (m, 1H), 1.94 - 1.78 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H); LCMS (ESI) m / z [M+H]+ 545.6.BGL-103-PCT01-NP

[0605] Payload Potency

[0606] Medium (RPMI supplemented with 10% (v / v) HYCLONE™ Fetal Bovine Serum) from sub-confluent (80-90% confluency) NCI-N87, MDAMB361 and MDAMB468 in a T175 flask was aspirated and the flask rinsed with PBS (about 20 ml) and emptied. TrypLE (4 ml) was added, the flask returned to the 37 °C gassed incubator for 5 minutes, then rapped sharply to dislodge and dissociate cells from the plastic. The cell suspension was transferred to a sterile 50 ml screw-top centrifuge tube, diluted with growth medium and then centrifuged (400g for 10 min). The supernatant was aspirated and the pellet resuspended in fresh culture medium. The cell concentration and viability were measured of trypan blue cell stained cells, using the LUNA II. Respectively 10000 for NCI-N87 / MDAMB361 and 3000 for MDAMB468 (50 pL / well) were seeded into black 96 well flat bottom plates and incubated overnight before use.

[0607] A lOmM payload stock solution was made from the material in DMSO. Then a 2mM solution was prepared by adding 20 pL of the stock solution to 80 pL of DMSO in a glass vial. A set of 8 10-fold dilutions of the previous payload (warhead) solution were made in a glass vial by serial transfer of 10 pL onto 90 pL of DMSO. These dilutions were transferred from the glass vials to a polypropylene plate, were column 1 has the most diluted sample, all the way to column 10, column 11 has DMSO and column 12 is empty. After, in another deep well, media was added to all 96 wells, according to the number of plates to drug, and dilute the payloads in 1:100. The payload (warhead) dilution in media was dispensed (50 pl / well) into 4 rows, either A-D or E-H of the 96-well plate, containing 50 pl cell suspension seeded the previous day. Control wells (column 12) received 50 pl cell culture medium. The 96-well plate containing cells and payloads (warheads) are incubated at 37 °C in a CO2-gassed incubator for 6 days. At the end of the incubation period, plates were equilibrated to room temperature for lOmin before CellTiter-Glo (Promega) was dispensed (100 pl per well) into each well. Plates were placed on an orbital shaker for 2 min before stabilisation at room temperature for 10 min. Well luminescence was measured, and percentage cell survival was calculated from the mean luminescence in the payload- (warhead-) treated wells compared to the mean luminescence in the DMSO control wells (100%).

[0608] Table 1

[0609] IC5o NCI-N87 (nM) IC50MDAMB361 (nM) IC50MDAMB468 (nM)

[0610] DA30.8 25.6 8.2

[0611] 30

[0612] Exatecan 2.9 1.9 0.43

[0613]

[0614] Table 1 show the cytotoxicity data for DA(N=3) and Exatecan (N=3) in NCI-N87, MDAMB361 and MDAMB468 cell lines.BGL-103-PCT01-NP

[0615] The IC50values were determined by fitting data to a sigmoidal dose-response curve using GRAPHPAD PRISM software v9 (GraphPad, San Diego, CA).

[0616] Antibody-drug conjugation

[0617] General Procedure for Herceptin-WT-LPl-DAR2 conjugation (ADC-1)

[0618] A 5 mM solution of Tris(2-carboxyethyl)phosphine (TCEP, Pierce) in phosphate buffered saline pH 7.4 (PBS, Gibco) was added (1.7molar equivalent / antibody, 355 nanomoles, 7.1 pl) to a 6 ml solution of antibody (Herceptin-WT; 30 mg each, 200 nanomoles) in reduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA, Molekula) to a final antibody concentration of 10 mg / mL. The reduction mixture was heated at +37 °C for 2 hours in an incubated orbital shaker with gentle (60 rpm) shaking. After cooling down to room temperature, AZ14414878 was added as a DMSO solution (4 molar equivalent / antibody, 416 nanomoles, in 0.29 ml DMA) to this reduced antibody solution for a 10% (v / v) final DMSO concentration. The solution was mixed for 1 hours at room temperature, then the conjugation was quenched by addition of N-acetyl cysteine (2.1 micromoles, 21 pl at 100 mM) for > 30 minutes at room temperature, sterile filtered, then purified on an AKTA™ Start FPLC using a GE Healthcare HiLoadTM 26 / 600 column packed with Superdex 200 PG, eluting with 2.6 ml / min PBS. Fractions corresponding to ADC monomer peak were pooled, concentrated using a 15 ml Amicon Ultracell 30KDa MWCO spin filter, sterile-filtered and analysed.

[0619] UHPLC-RP analysis on a Shimadzu Prominence system using a 5 Thermo Scientific MAbPac 50 mm x 2.1 mm column eluting with a gradient of water and acetonitrile on a reduced sample of ADC at 214 nm and 254 nm showed a mixture of unconjugated light chains (L0), light chains attached to a single molecule of LP1 (LI), unconjugated heavy chains (HO) and heavy chains attached to up to three molecules of LP1 (Hl, H2, H3), consistent with a drug-per-antibody ratio (DAR) of 2.01 molecules of LP1 per antibody. Free NAC-quenched payload was < LOD.

[0620] UHPLC-SEC analysis on a Shimadzu Prominence system using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 pm 4.6 x 150 mm column (with a 4 pm 3.0 x 20 mm guard column) eluting with 0.3 ml / minute sterile-filtered SEC buffer containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride and 10% isopropanol (v / v) on a sample of ADC at 280 nm showed a monomer purity of > 99.9%.

[0621] UV (Stunner, Unchained Labs) analysis gave a concentration of final ADC at 4.58 mg / mL in 2.4 mL, obtained mass of ADC of 10.99 mg (74 % yield).BGL-103-PCT01-NP

[0622] General Procedure for Herceptin-WT-LP2-DAR2 conjugation (ADC-2)

[0623] A 5 mM solution of Tris(2-carboxyethyl)phosphine (TCEP, Pierce) in phosphate buffered saline pH 7.4 (PBS, Gibco) was added (1.7molar equivalent / antibody, 355 nanomoles, 7.1 pl) to a 6 ml solution of antibody (Herceptin-WT; 30 mg each, 200 nanomoles) in reduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA, Molekula) to a final antibody concentration of 10 mg / mL. The reduction mixture was heated at +37 °C for 2 hours in an incubated orbital shaker with gentle (60 rpm) shaking. After cooling down to room temperature, LP2 was added as a DMSO solution (4 molar equivalent / antibody, 416 nanomoles, in 0.29 ml DMA) to this reduced antibody solution for a 10% (v / v) final DMSO concentration. The solution was mixed for 1 hours at room temperature, then the conjugation was quenched by addition of N-acetyl cysteine (2.1 micromoles, 21 pl at 100 mM) for > 30 minutes at room temperature, sterile filtered, then purified on an AKTA™ Start FPLC using a GE Healthcare HiLoadTM 26 / 600 column packed with Superdex 200 PG, eluting with 2.6 ml / min PBS. Fractions corresponding to ADC monomer peak were pooled, concentrated using a 15 ml Amicon Ultracell 30KDa MWCO spin filter, sterile-filtered and analysed.

[0624] UHPLC-RP analysis on a Shimadzu Prominence system using a 5 Thermo Scientific MAbPac 50 mm x 2.1 mm column eluting with a gradient of water and acetonitrile on a reduced sample of ADC at 214 nm and 254 nm showed a mixture of unconjugated light chains (L0), light chains attached to a single molecule of LP2 (LI), unconjugated heavy chains (HO) and heavy chains attached to up to three molecules of LP2 (Hl, H2, H3), consistent with a drug-per-antibody ratio (DAR) of 2.01 molecules of LP2 per antibody. Free NAC-quenched payload was < LOD.

[0625] UHPLC-SEC analysis on a Shimadzu Prominence system using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 pm 4.6 x 150 mm column (with a 4 pm 3.0 x 20 mm guard column) eluting with 0.3 ml / minute sterile-filtered SEC buffer containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride and 10% isopropanol (v / v) on a sample of ADC at 280 nm showed a monomer purity of > 99.5%.

[0626] UV (Stunner, Unchained Labs) analysis gave a concentration of final ADC at 4.90 mg / mL in 2.7 mL, obtained mass of ADC of 13.23 mg (85 % yield).

[0627] General Procedure of ADC toxicity evaluation on 3D NCI-N87 and MDAMB468

[0628] Medium from sub-confluent (80-90% confluency) NCI-N87 and MDAMB468 in a T175 flask was aspirated and the flask rinsed with PBS (about 20 ml) and emptied. TrypLE (4 ml) was added, the flask returned to the 37 °C gassed incubator for up to about 5 minutes, then rapped sharply to dislodge andBGL-103-PCT01-NP

[0629] dissociate cells from the plastic. The cell suspension was transferred to a sterile 50 ml screw-top centrifuge tube, diluted with growth medium to a final volume of 15 ml, then centrifuged (400g for 5 min). The supernatant was aspirated, and the pellet re-suspended in 10ml culture medium. Repeated pipetting may be necessary to produce monodisperse cell suspensions. The cell concentration and viability are measured of trypan blue cell-stained cells, using the LUNA II.

[0630] For the ADCs, a stock solution (300pL) of Antibody Drug Conjugate (ADC) was made by dilution of filter-sterilised ADC into cell culture medium. A set of 9x 5-fold dilutions of the previous ADC solution were made in a ImL deep 96 well plate by serial transfer of 60 pl onto 240 pl of cell culture medium. ADC dilution was dispensed (50 pl / well) into 3 replicate wells of the 96-well plate, containing 50 pl cell suspension seeded the previous day. Control wells received 50 pl cell culture medium. The 96-well plate containing cells and ADCs / payloads was incubated at 37 °C in a CO2-gassed incubator for 6 days. At the end of the incubation period, plates were equilibrated to room temperature for lOmin before CellTiter-Glo (Promega) was dispensed (100 pl per well) into each well. IC50for ADC-1 and ADC-2 was determined from the dose-response data using GraphPad Prism using the non-linear curve fit algorithm: sigmoidal dose response, log(inhibitor) vs. Response - Variable slope (four parameters), constrain at the bottom = 0.

[0631] Cell growth medium for the cells was RPMI supplemented with 10% Fetal Bovine Serum.

[0632] Table 2

[0633] Asset Number IC5o NCI-N87(pg / mL) IC50MDAMB468 (pg / mL)

[0634] MMAE (reference) 0.003 ND

[0635] ADC-1 0.005 ND

[0636] ADC-2 0.003 ND

[0637]

[0638] Table 2 shows cytotoxicity data ( IC50in pg / mL) for MMAE, ADC-1 and ADC-2 on Her2+++ NCI-N87 and HER2- MDAMB468. The ADC-1 and ADC-2 IC50values in Figure 1 and Figure 2 were determined by fitting data to a sigmoidal dose-response curve using GraphPad Prism software v9 (GraphPad, San Diego, CA).

[0639] The above description of illustrative embodiments is intended only to acquaint others skilled in the art with the Applicant's specification, its principles, and its practical application so that others skilled in the art may readily adapt and apply the specification in its numerous forms, as they may be best suited to the requirements of a particular use. This description and its specific examples, whileBGL-103-PCT01-NP

[0640] indicating embodiments of this specification, are intended for purposes of illustration only. This specification, therefore, is not limited to the illustrative embodiments described in this specification, and may be variously modified. In addition, it is to be appreciated that various features of the specification that are, for clarity reasons, described in the context of separate embodiments, also may be combined to form a single embodiment. Conversely, various features of the specification that are, for brevity reasons, described in the context of a single embodiment, also may be combined to form sub-combinations thereof.

Claims

1. BGL-103-PCT01-NPClaimsA conjugate of Formula (I)JA-DRAb — GA-BAJA-DRor a pharmaceutically acceptable salt thereof, whereinAb is an antibody or antigen-binding fragment thereof,k is an integer from 1 to 10,GAis independently a conjugation group conjugated to the antibody or antigen-binding fragment thereof,DRis independently a drug comprising a nitrogen atom NR,BAis independently a group of Formula (IA)wherein,r is 0, 1, 2, 3, 4, 5, 6, 7, or 8,W1is (CH2)n1, wherein n1 is 1, 2, 3, 4, 5, or 6,QBis a covalent bond or R1,(GA) indicates the point of attachment to GA, and (JA) indicates the point of attachment to JA, JAis independently a group of Formula (IB)HO.' 'Q' N N q H HpwhereinE is (CH2)n2, wherein n2 is 0, 1, 2 or 3,X1and X2are (CH2)n3, wherein n3 is independently 1, 2, 3 or 4,p is 1 or 0,BGL-103-PCT01-NPq is 0, 1, 2, 3, 4, 5, 6, 7, or 8,QJis R1or a covalent bond,(NR) indicates the point of attachment to the nitrogen atom NR, and (BA) indicates the point of attachment to BA,R1is independently a group of Formula (IC)whereinm is an integer from 5 to 17,R2is Ci-4 alkyl,X3is (CH2)n4, wherein n4 is 0, 1, 2 or 3,Y is O or NR3, wherein R3is H, Ci-4alkyl or C3-4 cycloalkyl,(C=O) indicates the point of attachment to the adjacent C=O group, and (CH2) indicates the point of attachment to the adjacent CH2,and wherein at least one of QBand QJis R1.

2. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in claim 1, wherein r is 0.

3. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in claim 1 or claim 2, wherein W1is CH2or (CH2)2.

4. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 3, wherein E is CH2.

5. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 4, wherein p is 1.BGL-103-PCT01-NP6. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 5, wherein q is 1, 2, 3 or 4.

7. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 6, wherein X1is CH2and X2is CH2.

8. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 7, wherein m is 9, 10, 11, 12 or 13.

9. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 8, wherein R2is CH3.

10. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 9, wherein Y is O.

11. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 10, wherein X3is CH2.

12. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in anyone of claims 1 to 11, whereinDRisBGL-103-PCT01-NP13. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in anyone of claims 1 to 11, whereinDRis14. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in anyone of claims 1 to 11, whereinDRis15. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 14, wherein BAis independently a group of Formula (IA1)16. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in claim 15, wherein JAis independently a group of Formula (IB1)BGL-103-PCT01-NP(BA^Kx 1117. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in claim 15 or claim 16, wherein BAis a group of Formula (IA2)and JAis a group of Formula(IB2)OH18. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 14, wherein JAis independently a group of Formula (IB3)BGL-103-PCT01-NP19. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in claim 18, wherein BAis independently a group of Formula (IA3)°U1 A(J } (IA3)(JA)20. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in claim 18 or 19, wherein BAis a group of Formula (IA4)and JAis a group of Formula (IB4)BGL-103-PCT01-NP21. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 20, wherein GAis independently selected fromwherein RKis H or CH3, RLis C1-6alkyl,and indicates the point of attachment to the antibody or antigen-binding fragment thereof.

22. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in claim 21, wherein GAis23. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 22, wherein k is an integer from 1 to 4.

24. A pharmaceutical composition comprising a conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 23, and a pharmaceutically acceptable excipient.BGL-103-PCT01-NP25. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 23, or a pharmaceutical composition as claimed in claim 24, for use in therapy.

26. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 23, or a pharmaceutical composition as claimed in claim 24, for use in the treatment of cancer.

27. A method of treating cancer in a patient in need thereof comprising administering to the patient an effective amount of a conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 23.

28. Use of a conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 23, in the manufacture of a medicament for the treatment of cancer.

29. A compound of Formula (II)JB-DRor a salt thereof, wherein GBis a conjugation group for conjugation to an antibody or antigenbinding fragment thereof,BBis a group of Formula (IIA)JBis independently a group of Formula (IIB)BGL-103-PCT01-NPN E (IIB)(BBHwherein DR, E, p, QB, QJ, q, R1, r, W1, X1and X2are as defined for a conjugate of Formula (I) in any one of claims 1 to 14, (BB) indicates the point of attachment to BB, (GB) indicates the point of attachment to GB, (JB) indicates the point of attachment to JBand (NR) indicates the point of attachment to the nitrogen atom NR.

30. The compound of Formula (II) or a salt thereof, as claimed in claim 29, wherein BBis a group of Formula (IIA1)31. The compound of Formula (II) or a salt thereof, as claimed in claim 30, wherein JBis independently a group of Formula (IIB1)32. The compound of Formula (II) or a salt thereof, as claimed in claim 30 or claim 31, wherein BBis a group of Formula (IIA2)BGL-103-PCT01-NPand JBis a group of Formula(IIB2)33. The compound of Formula (II) or a salt thereof, as claimed in claim 29, wherein JBis independently a group of Formula (IIB3)(IIB3)34. The compound of Formula (II) or a salt thereof, as claimed in claim 33, wherein BBis a group of Formula (IIA3)(JB)BGL-103-PCT01-NP35. The compound of Formula (II) or a salt thereof, as claimed in claim 33 or claim 34, wherein BBis a group of Formula (IIA4)36. The compound of Formula (II) or a salt thereof, as claimed in any one of claims 29 to 35, wherein GBis selected fromwherein X° is CH or N,h is 0 or 1,BGL-103-PCT01-NPHal is Cl, Br or I,RKis H or CH3, andRLis C1-6 alkyl.

37. The compound of Formula (II) or a salt thereof, as claimed in any one of claims 29 to 36, wherein GBisO

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