Conjugates comprising cleavable linkers

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] BGL-102-PCT01-NP

[0002] CONJUGATES COMPRISING CLEAVABLE LINKERS

[0003] Cross-Reference to a Related Patent Application

[0004] This specification claims the benefit of priority to European Patent Application No. 24205979.8 (filed 10 October 2024). The entire text of the above-referenced patent application is incorporated by reference into this specification.

[0005] Field

[0006] 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.

[0007] Background

[0008] Antibody drug conjugates (ADCs) are an established method to deliver a drug to biological target. It is possible to use an enzymatically cleavable linker to release free drug at the target, so that the free drug can have a therapeutic effect. Through transcriptomic and proteomic profiling of various solid tumour cell lines, p-glucuronidase expression has been identified as being upregulated and typically localised to the lysosome. W02007011968 and WO2015182984 disclose certain antibody drug conjugates comprising p-glucuronidase-cleavable linkers.

[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) or a pharmaceutically acceptable salt thereof, wherein Ab is an antibody or antigen-binding fragment thereof, k is an integer from 1 to 10, BGL-102-PCT01-NP

[0012] GAis independently a conjugation group conjugated to the antibody or antigen-binding fragment thereof, each DRis independently a drug comprising a nitrogen atom NR,

[0013] BAis independently a group of Formula (IA) wherein, r is 0, 1, 2, 3, 4, 5, 6, 7, or 8,

[0014] W1is (CHjJni, wherein nl is 1, 2, 3, 4, 5 or 6,

[0015] QBis a covalent bond or R1,

[0016] (GA) indicates the point of attachment to GA, and (JA) indicates the point of attachment to JA, each JAis independently a group of Formula (IB) wherein

[0017] E is (CHjJn?, wherein n2 is independently 0, 1, 2 or 3, each X1and X2are (CHjJna, wherein each n3 is independently 1, 2, 3 or 4, p is 1 or 0, q is 0, 1, 2, 3, 4, 5, 6, 7, or 8,

[0018] QJis R1or a covalent bond,

[0019] (NR) indicates the point of attachment to the nitrogen atom NR, and (BA) indicates the point of attachment to BA, each R1is independently a group of Formula (IC) wherein BGL-102-PCT01-NP

[0020] R2is Ci-4 alkyl,

[0021] Y is (CHjJr , wherein n4 is 0, 1, 2 or 3,

[0022] Z is (CHzJns, wherein n5 is 0, 1, 2, 3 or 4, m is an integer from 5 to 17,

[0023] (N) indicates the point of attachment to the adjacent N atom, and (C) indicates the point of attachment to the adjacent C atom, and wherein at least one of QBand QJis R1.

[0024] 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.

[0025] In a further aspect there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, for use in therapy.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] In a further aspect there is provided a method of treating cancer in a patient comprising administering to the patient an effective amount of a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof.

[0030] In a further aspect there is provided a compound of Formula (II) or a salt thereof, wherein GBis a conjugation group for conjugation to an antibody or antigenbinding fragment thereof, BBis a group of Formula (IIA) each JBis independently a group of Formula ( 11 B) BGL-102-PCT01-NP wherein DR, E, QB, QJ, R1, X1, X2, p, q and r are as defined above for a conjugate of Formula (I), (BB) indicates the point of attachment to BB, (GB) indicates the point of attachment to GB, (JB) indicates the point of attachment to each JBand (NR) indicates the point of attachment to the nitrogen atom NR.

[0031] In a further aspect there is provided intermediates useful for the synthesis of a compound of Formula (ll)7or a salt thereof.

[0032] 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.

[0033] Definitions

[0034] 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.

[0035] The prefix Cx-y, where x and y are integers, indicates the numerical range of carbon atoms that are present in a group.

[0036] 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- BGL-102-PCT01-NP s alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl and n-hexyl.

[0037] 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 a covalent bond to an antibody, or antigen-binding fragment thereof, through a chemical reaction. gig

[0038] The use of " s' " in formulas of this specification indicates the point of attachment to the antibody or antigen-binding fragment thereof. By way of illustration indicates that there is a covalent bond connecting the antibody, or antigen-binding fragment thereof, to the carbon atom marked 1.

[0039] For the avoidance of doubt, the use of 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.

[0040] Certain embodiments of this specification include a group which is said to be "optionally substituted". In further embodiments said group is unsubstituted.

[0041] 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.

[0042] 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.

[0043] Description of Figures

[0044] Embodiments and experiments illustrating the principles of the disclosure will now be discussed with reference to the accompanying figures in which:

[0045] Figure 1 illustrates the cytotoxicity data for ADC2 and ADC3 in a HER2+++ NCI-N87 cell line.

[0046] Figure 2 illustrates the cytotoxicity data for ADC2 and ADC3 in a HER2- MDAMB468 cell line. BGL-102-PCT01-NP

[0047] Detailed Description

[0048] In one aspect, this specification provides a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, as defined above.

[0049] 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.

[0050] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein GAis selected from wherein RKis H or CH3, RLis Ci.g alkyl, indicates the point of attachment to the antibody, or antigen-binding fragment thereof.

[0051] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein GAis selected from BGL-102-PCT01-NP

[0052] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein GAis In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein GAis

[0053] In embodiments, for each , one, two or three of QBand QJare R1.

[0054] In embodiments, for each each QJis a covalent bond.

[0055] In embodiments, for each each QJis a R1.

[0056] In embodiments, for each covalent bond, one QJis R1and one QJis a covalent bond. BGL-102-PCT01-NP

[0057] In embodiments, for each covalent bond, and each QJis independently R1.

[0058] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein W1is independently (CH2)ni, wherein nl is 1, 2, 3, 4, 5 or 6. In further embodiments W is independently CH2or (CH2)2. In further embodiments W is CH2. In further embodiments W is (CH2)2.

[0059] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein E is (CH2)n2, wherein n2 is independently 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.

[0060] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein each X1and X2are (CH2)n3, wherein each n3 is independently 1, 2, 3 or 4. In further embodiments each X1and X2are (CH2)n3, wherein each n3 is independently 1 or 2. In further embodiments each X1is CH2and each X2is CH2.

[0061] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein Y is (CH2)n4, wherein n4 is independently 0, 1, 2 or 3. In further embodiments Y is CH2. In further embodiments Y is (CH2)2. In further embodiments Y is (CH2)3.

[0062] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein Z is (CH2)n5, wherein n4 is independently 0, 1, 2, 3 or 4. In further embodiments Z is a covalent bond. In further embodiments Z is CH2. In further embodiments Z is (CH2)2. In further embodiments Z is (CH2)3. In further embodiments Z is (CH2)4.

[0063] 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.

[0064] 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. BGL-102-PCT01-NP

[0065] 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.

[0066] 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.

[0067] 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 0, 1 or 2. In further embodiments r is 0.

[0068] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein each BAis a group of Formula ( I Al) wherein (GA), (JA), R2, m, r, W1, Y and Z are as defined herein.

[0069] In further embodiments, each JAis a group of Formula ( I Bl) wherein (BA), (NR), E, p, q, X1and X2are as defined herein.

[0070] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein each BAis a group of Formula (IA1') BGL-102-PCT01-NP wherein (GA), (JA), R2, m, r, W1, Y and Z are as defined herein.

[0071] In further embodiments, each JAis a group of Formula ( I Bl) wherein (BA), (NR), E, p, q, X1and X2are as defined herein.

[0072] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein each BAis a group of Formula (IA2) or Formula (IA2') BGL-102-PCT01-NP and each JAis a group of Formula (IB2) wherein ( BA), (GA) and (NR) are as defined herein.

[0073] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein each JAis a group of Formula (IB3) wherein ( BA), ( NR), E, m, p, q, R2, X1, X2, Y and Z are as defined herein.

[0074] In further embodiments, BAis a group of Formula (IA3) , wherein (GA), (JA), r and W1are as defined herein. In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein each JAis a group of Formula (I B3') BGL-102-PCT01-NP wherein (BA), (NR), E, m, p, q, R2, X1, X2, Y and Z are as defined herein.

[0075] In further embodiments, BAis a group of Formula (IA3) , wherein (GA), (JA), r and W1are as defined herein. In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein BAis a group of Formula (IA4) (IA4) or Formula (IA4') and each JAis a group of Formula (IB4)

[0076] BGL-102-PCT01-NP wherein ( BA), (GA), (JA) and (NR) are as defined herein.

[0077] In embodiments, there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein each

[0078] In embodiments, there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein each

[0079] In embodiments, there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein each BGL-102-PCT01-NP

[0080] In embodiments, there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein each DRis independently selected from DR1, DR2and DR3.

[0081] Where DRis DR1, DR2and / or DR3, then (NR) indicates the point of attachment to DR1, DR2and / or DR3respectively. In embodiments, there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein GA, BA, JAand DRare; BGL-102-PCT01-NP BGL-102-PCT01-NP

[0082] In embodiments, there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt wherein indicates the point of attachment to the antibody, or antigen-binding fragment thereof. BGL-102-PCT01-NP

[0083] In embodiments, there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt wherein indicates the point of attachment to the antibody, or antigen-binding fragment thereof.

[0084] In a further aspect there is provided a compound of Formula (II) BGL-102-PCT01-NP or a salt thereof, wherein GBis a conjugation group for conjugation to an antibody or antigenbinding fragment thereof,

[0085] BBis a group of Formula (IIA) each JBis independently a group of Formula ( 11 B) wherein DR, E, QB, QJ, R1, X1, X2, p, q and r are as defined above for a conjugate of Formula (I), (BB) indicates the point of attachment to BB, (GB) indicates the point of attachment to GB, (JB) indicates the point of attachment to each JBand (NR) indicates the point of attachment to the nitrogen atom

[0086] NR.

[0087] In embodiments there is provided a compound of Formula (II), or a salt thereof, wherein GBis selected from

[0088] BGL-102-PCT01-NP wherein X1is CH or N, h is 0 or 1, Hal is Cl, Br or I, RKis H or CH3, and RLis Ci.g alkyl.

[0089] In embodiments there is provided a compound of Formula (II), or a salt thereof, wherein GBis selected from

[0090] In embodiments there is provided a compound of Formula (II), or a salt thereof, wherein GBis

[0091] In embodiments there is provided a compound of Formula (II), or a salt thereof, wherein GBis BGL-102-PCT01-NP

[0092] In embodiments there is provided a compound of Formula (ll)7or a salt thereof, wherein each BBis a group of Formula (I IA1) wherein (GB), (JB), R2, m, r, W1, Y and Z are as defined herein. In further embodiments, each JBis a group of Formula (I I Bl) wherein (BB), (NR), E, p, q, X1and X2are as defined herein.

[0093] In embodiments there is provided a compound of Formula (II), or a salt thereof, wherein each BBis a group of Formula (IIA1') wherein (GB), (JB), R2, m, r, W1, Y and Z are as defined herein.

[0094] In further embodiments, each JBis a group of Formula ( 11 Bl) BGL-102-PCT01-NP wherein (BB), (NR), E, p, q, X1and X2are as defined herein.

[0095] In embodiments there is provided a compound of Formula (ll)7or a salt thereof, wherein each BBis a group of Formula (IIA2) or Formula (I IA2') and each JBis a group of Formula (IIB2) wherein (BB), (GB), (JB) and (NR) are as defined herein. BGL-102-PCT01-NP

[0096] In embodiments there is provided a compound of Formula (II), or a salt thereof, wherein each JBis a group of Formula (IIB3) wherein (BB), (NR), E, m, p, q, R2, X1, X2, Y and Z are as defined herein. In further embodiments, BBis a group of Formula (I I A3) , wherein (GB), (JB), r and W1are as defined herein.

[0097] In embodiments there is a compound of Formula (II), or a salt thereof, wherein each JBis a group of

[0098] Formula (IIB3') wherein (BB), (NR), E, m, p, q, R2, X1, X2, Y and Z are as defined herein.

[0099] In further embodiments, BBis a group of Formula (I IA3) , wherein (GB), (JB), r and W1are as defined herein. BGL-102-PCT01-NP

[0100] In embodiments there is provided a compound of Formula (ll)7or a salt thereof, wherein BBis a group of Formula (I IA4) (IIA4) or Formula (IIA4') (IIA4) and each JBis a group of Formula ( 11 B4) wherein (BB), (GB), (JB) and (NR) are as defined herein.

[0101] In embodiments there is provided a compound of Formula (II), or a salt thereof, wherein GB, BB, JBand DRare; BGL-102-PCT01-NP BGL-102-PCT01-NP

[0102] In embodiments there is provided a compound of Formula (ll)7or a salt thereof, that is

[0103] BGL-102-PCT01-NP

[0104] (2S,2'S,3S,3'S,4S,4'S,5R,5'R,6S,6'S)-6,6'-((((8S,30S)-8,30-bis(4-(((3S,3aR,6S,6aR)-6- (2,5,8,ll,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-amido)hexahydrofuro[3,2-b]furan-3- yl)amino)-4-oxobutyl)-19-(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanoyl)-3,7,10,28,31,35- hexaoxo-13,16,22,25-tetraoxa-2,6,9,19,29,32,36-heptaazaheptatriacontane-l,37-diyl)bis(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)-2,l- phenylene))bis(oxy))bis(3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid), or a salt thereof. BGL-102-PCT01-NP

[0105] In embodiments there is provided a compound of Formula (ll)7or a salt thereof, that is

[0106] (2S,2'S,3S,3'S,4S,4'S,5R,5'R,6S,6'S)-6,6'-(((16-((S)-6-(((3S,3aR,6S,6aR)-6- (2,5,8,ll,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-amido)hexahydrofuro[3,2-b]furan-3- yl)amino)-2-(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanamido)-6-oxohexanoyl)-3,7,25,29- tetraoxo-10,13,19,22-tetraoxa-2,6,16,26,30-pentaazahentriacontane-l,31-diyl)bis(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)-2,l- phenylene))bis(oxy))bis(3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid), or a salt thereof. BGL-102-PCT01-NP

[0107] 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.

[0108] 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.

[0109] 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).

[0110] A suitable pharmaceutically acceptable salt of a conjugate of Formula (I) is, for example, an acid addition salt. An acid addition salt of a conjugate of Formula (I), may be formed by bringing the compound into contact with a suitable inorganic or organic acid under conditions known to the skilled person. An acid addition salt may for example be formed using an inorganic acid selected from hydrochloric acid, hydrobromic acid, sulphuric acid and phosphoric acid. An acid addition salt may also be formed using an organic acid selected from trifluoroacetic acid, citric acid, maleic acid, oxalic acid, acetic acid, formic acid, benzoic acid, fumaric acid, succinic acid, tartaric acid, lactic acid, pyruvic acid, methanesulfonic acid, benzenesulfonic acid and para-toluenesulfonic acid.

[0111] A suitable pharmaceutically acceptable salt of a conjugate of Formula (I) is, for example, a base addition salt. A base addition salt of a conjugate of Formula (I) may be formed by bringing the compound into contact with a suitable inorganic or organic base under conditions known to the skilled person. A base addition salt may for example be an alkali metal salt (such as a sodium, potassium, or lithium salt) or an alkaline earth metal salt (such as a calcium salt), which may be formed using an alkali metal or alkaline earth metal hydroxide or alkoxide (e.g., an ethoxide or BGL-102-PCT01-NP methoxide). A base addition salt may also be formed using a suitably basic organic amine (e.g., a choline or meglumine salt).

[0112] 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.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] In one aspect there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, for use in therapy.

[0118] In one aspect there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer.

[0119] 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.

[0120] The term "therapy" is intended to have its normal meaning of dealing with a disease in order to entirely or partially relieve one, some or all of its symptoms, or to correct or compensate for the underlying pathology. The term "therapy" also includes "prophylaxis" unless there are specific BGL-102-PCT01-NP indications to the contrary. The terms "therapeutic" and "therapeutically" should be interpreted in a corresponding manner.

[0121] The term "prophylaxis" is intended to have its normal meaning and includes primary prophylaxis to prevent the development of the disease and secondary prophylaxis whereby the disease has already developed and the patient is temporarily or permanently protected against exacerbation or worsening of the disease or the development of new symptoms associated with the disease.

[0122] The term "treatment" is used synonymously with "therapy". Similarly the term "treat" can be regarded as "applying therapy" where "therapy" is as defined herein.

[0123] 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.

[0124] 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.

[0125] In one aspect there is provided a method of treating cancer in a patient comprising administering to the patient an effective amount of a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof.

[0126] The term "effective amount" means an amount of an active ingredient which is sufficient enough to significantly and positively modify the symptoms and / or conditions to be treated (e.g., provide a positive clinical response). The effective amount of an active ingredient for use in a pharmaceutical composition will vary with the particular condition being treated, the severity of the condition, the duration of the treatment, the nature of concurrent therapy, the particular active ingredient(s) being employed, the particular pharmaceutically-acceptable excipient(s) / carrier(s) utilized, and like factors within the knowledge and expertise of the attending physician.

[0127] 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.

[0128] In embodiments there is provided a method of treating cancer in a patient comprising administering to the patient an effective amount of a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein the cancer is a HER2 positive cancer.

[0129] 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. BGL-102-PCT01-NP

[0130] 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.

[0131] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, in combination with an additional anti-tumour agent.

[0132] 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.

[0133] In embodiments there is provided a method of treating cancer in a patient comprising administering to the patient an effective amount of a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, and simultaneously, separately or sequentially 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 anti-tumour substance are jointly effective in producing an anti-cancer effect.

[0134] Conjugation

[0135] Examples of GAand GBinclude, but are not limited to, the following, wherein X1is CH or N, h is 0 or 1, RKis H or CH3, Hal is Cl, Br or I, RLis C1-6 alkyl, and indicates the point of attachment to the antibody, or antigen-binding fragment thereof. BGL-102-PCT01-NP BGL-102-PCT01-NP

[0136] Antibody or antigen-binding fragment thereof

[0137] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to, or is immunologically reactive with, a particular antigen.

[0138] 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.

[0139] 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").

[0140] 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. BGL-102-PCT01-NP

[0141] 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.

[0142] 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 on crystallographic studies of antigen-antibody complexes ( Al -lazikan i et al. (1997) J. Molec. Biol. 273:927-948)). In addition, combinations of these two approaches are sometimes used in the art to determine CDRs.

[0143] 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.

[0144] 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 FcyRIIL 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. BGL-102-PCT01-NP

[0145] 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.

[0146] 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, il, 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" 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_HUIVIAN), P01859 (IGHG2_HUIVIAN), P01860 (IGHG3_HUMAN), and P01861 (IGHG4_HUMAN) respectively.

[0147] 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.

[0148] In embodiments the antibody of the disclosure is a full-length antibody described above.

[0149] 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 BGL-102-PCT01-NP 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.

[0150] 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.

[0151] 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.

[0152] 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 CH2 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.

[0153] 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.

[0154] An "Fv fragment" contains only the variable regions of the heavy and light chain. It contains no constant regions.

[0155] A "single-domain antibody" is an antibody fragment containing a single antibody domain unit (e.g., VH or VL).

[0156] 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.

[0157] 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.

[0158] A "bi-specific T 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. BGL-102-PCT01-NP

[0159] 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.

[0160] 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, which is completely cell-free and is described in He M. and Taussig MJ., Biochem Soc Trans. 2007, Nov;35(Pt 5):962-5.

[0161] 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).

[0162] Drug (DR)

[0163] 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.

[0164] 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).

[0165] 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 BGL-102-PCT01-NP tertiary carbamate. In other words, the nitrogen atom NRmay, in the free drug form, be the nitrogen atom of a primary or secondary amine.

[0166] 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.

[0167] 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.

[0168] In embodiments the drug is a diagnostic agent or an imaging agent.

[0169] In embodiments each DRis

[0170] In embodiments each DRis

[0171] In embodiments each DRis BGL-102-PCT01-NP

[0172] In 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).

[0173] In embodiments the free drug form of the drug

[0174] 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.

[0175] In embodiments the free drug form of the drug is MMAE ((S)-N-((3R,4S,5S)-l-((S)-2-((lR,2R)-3-

[0176] (((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-

[0177] (methylamino)butanamido)butanamide).

[0178] 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.

[0179] Examples

[0180] The specification will now be illustrated by the following non-limiting Examples.

[0181] General Information BGL-102-PCT01-NP

[0182] 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.

[0183] Extraction and chromatography solvents were bought and used without further purification from VWR U.K.

[0184] All fine chemicals were purchased from SIGMA-ALDRICH unless otherwise stated.

[0185] Pegylated reagents were obtained from QUANTA BIODESIGN US via STRATECH UK.

[0186] LC / MS conditions

[0187] Positive mode electrospray mass spectrometry was performed using a WATERS ACQUITY H-CLASS SQD2 using one of the following methods.

[0188] 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%).

[0189] 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, then 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. 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.

[0190] 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.

[0191] HPLC conditions

[0192] 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. BGL-102-PCT01-NP

[0193] 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.

[0194] NMR Method

[0195] Proton NMR chemical shift values were measured on the delta scale at 400 MHz using a BRUKER AV400. The following abbreviations have been used: s, singlet; d, doublet; t, triplet; q, quartet; quin, quintet; m, multiplet; br, broad. Coupling constants are reported in Hz.

[0196] Abbreviations

[0197] TLC Thin layer chromatography

[0198] UV Ultra violet

[0199] LCMS Liquid chromatography mass spectrometry

[0200] CSH Charged surface hybrid

[0201] UPLC Ultra-performance liquid chromatography

[0202] RP Reverse phase

[0203] NMR Nuclear magnetic resonance

[0204] DBU l,8-Diazabicyclo[5.4.0]undec-7-ene

[0205] DMF Dimethyl formamide

[0206] DCM Dichloromethane

[0207] TBS Tert-butyl dimethyl silane

[0208] MS Molecular sieves

[0209] TFA Trifluoro acetic acid

[0210] DMSO Dimethyl sulfoxide

[0211] ESI Electrospray ionisation

[0212] DIPEA Di isopropyl ethylamine

[0213] THF Tetra hydro furan

[0214] HATU Hexafluorophosphate azabenzotriazole tetramethyl uronium

[0215] TEA Triethylamine

[0216] HOPO l-Hydroxy-2-pyridone

[0217] RT Retention time

[0218] ADC Antibody-drug conjugate

[0219] UHPLC Ultra-high performance liquid chromatography mAb Monoclonal antibody

[0220] SEC Size exclusion chromatography

[0221] DAR Drug to antibody ratio BGL-102-PCT01-NP

[0222] RPMI Roswell Park Memorial Institute

[0223] ND Not detectable

[0224] IC50 Inhibitory concentration 50 %

[0225] Fmoc Fluorenylmethoxycarbonyl

[0226] Synthesis of LP1

[0227] Intermediate 1

[0228] 1 . Allyl bromide, DBU

[0229] 2. Acetic anhydride, pyridine

[0230] 3. 30% HBr in acetic acid

[0231] 2,3,4,6,7,8,9,10-octahydropyrimido[l,2-a]azepine (26.5 ml, 177.35 mmol) was added dropwise to a 1-L round bottom flask containing (2S,3S,4S,5R,6R)-3,4,5,6-tetrahydroxytetrahydro-2H-pyran-2- carboxylic acid (31.3 g, 161.22 mmol) in DMF (100 ml) at 21 °C. Next, 3-bromoprop-l-ene (16.72 ml, 193.47 mmol) was added to the reaction mixture dropwise over 10 minutes and the reaction was stirred at 21 °C for 24 hours. Reaction mixture was cooled to 0 °C and treated with pyridine (104 mL, 1289.60 mmol). Acetic anhydride (244 mL, 2579.20 mmol) was next added to the reaction mixture. The reaction was warmed up to room temperature and run for 2 hours at 21 °C. Reaction mixture concentrated under reduced vacuum and the remaining pyridine was azeotropically removed with toluene (1 x 100 mL). Crude material was diluted with DCM (65 mL) and cooled to 0 °C. 30% Hydrobromic acid in acetic acid (175 mL, 3226.03 mmol) was next added to the reaction mixture at 0 °C. The reaction was warmed up to room temperature and run for 2 hours 30 minutes at 21 °C. Solvent was evaporated then the compound was purified by normal phase flash column chromatography to afford (2S,3S,4S,5R,6R)-2-((allyloxy)carbonyl)-6-bromotetrahydro-2H-pyran- 3,4,5-triyl triacetate Intermediate 1 (33 g, 48% yield) as a beige translucent material.XH NMR (500 MHz, CDCI3) 66.67 (d, J = 4.0 Hz, 1H), 5.92 (ddt, J = 16.6, 10.3, 6.0 Hz, 1H), 5.64 (t, J = 9.7 Hz, 1H), 5.42 - 5.23 (m, 3H), 4.88 (dd, J = 10.0, 4.0 Hz, 1H), 4.71 - 4.58 (m, 3H), 2.12 (s, 3H), 2.07 (s, 3H), 2.05 (s, 3H); LCMS (ESI) m / z 445.0 (M + Na)+.

[0232] Intermediate 2 BGL-102-PCT01-NP

[0233] TBS-CI (20.80 g, 138.02 mmol) in DCM (25 mL) was added dropwise to lH-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), 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

[0234] Intermediate 3

[0235] To a vacuum-dried 500 mL round-bottom flask was added molecular sieves (4 A beads, 5.0 g), silver oxide (29.2 g, 125.8 mmol) and acetonitrile (150 mL), producing a black slurry. To this slurry was added a solution of Intermediate 1 (10.7 g, 25.2 mmol) in acetonitrile (50 mL) over 20 min followed by the addition of 5-(((tert-butyldimethylsilyl)oxy)methyl)-2-hydroxybenzaldehyde (Intermediate 2, 13.6 g, 51.1 mmol) in acetonitrile (50 mL) in one portion. The resulting mixture was stirred vigorously at 20 °C for 16 h. After 16 h, the reaction mixture was filtered through a 5-cm pad of Celite and rinsed with dichloromethane (3 x 25 mL). Solvent was evaporated then the compound was purified by normal phase flash column chromatography to afford (2S,3S,4S,5R,6S)-2-((allyloxy)carbonyl)-6-(4- (((tert-butyldimethylsilyl)oxy)methyl)-2-formylphenoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate as a white material Intermediate 3 (5.2 g, 34% yield).1H NMR (400 MHz, CDCI3) 6 10.34 (s, 1H), 7.77 (d, J = 1.8 Hz, 1H), 7.58 (dd, J = 8.6, 2.1 Hz, 1H), 7.14 (d, J = 8.6 Hz, 1H), 5.81-5.92 (m, 1H), 5.39-5.35 (m, 4H), 5.28-5.22 (m, 2H), 4.71 (s, 2H), 4.58-4.67 (m, 2H), 4.20-4.28 (m, 1H), 2.073 (s, 3H), 2.069 (s, 3H), 2.04 (s, 3H), 0.94 (s, 9H), 0.11 (s, 6H); LCMS (ESI) m / z 626.3 (M + NH4)+.

[0236] Intermediate 4 BGL-102-PCT01-NP

[0237] To a solution of Intermediate 3 (5.2 g, 8.6 mmol) in acetonitrile (40 mL) was added tert-butyl carbamate (3.8 g, 32.3 mmol), trifluoroacetic acid (2.0 mL, 25.9 mmol), and triethylsilane (4.1 mL, 25.8 mmol). Stirred for 2 h at 20 °C then solvent was evaporated. To the resulting colorless oil was added 1,4-dioxane (8 mL) and HCI (4.0 M in 1,4-dioxane, 50 mL, 200 mmol). The mixture was stirred at 20 °C for 30 min the solvent was evaporated. The resulting white powder was dissolved in DMSO (3 mL) then passed through cation-exchange resin pre-treated with methanol (WATERS PORAPAK CX). The desired compound was eluted off the resin with methanol to afford (2S,3S,4S,5R,6S)-2- ((allyloxy)carbonyl)-6-(2-(aminomethyl)-4-(hydroxymethyl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate as a white material Intermediate 4 (2.5 g, 80% over 2 steps).1H NMR (500 MHz, CDCL) 6 7.26 (d, J = 2.2 Hz, 1H), 7.21 (dd, J = 8.3, 2.2 Hz, 1H), 7.01 (d, J = 8.3 Hz, 1H), 5.90-5.82 (m, 1H), 5.42 - 5.24 (m, 6H), 5.16 (d, J = 7.1 Hz, 1H), 4.64 - 4.55 (m, 4H), 4.19 (d, J = 9.3 Hz, 1H), 3.84 (d, J = 14.0 Hz, 1H), 3.67 (d, J = 14.0 Hz, 1H), 2.32 (s, 3H), 2.09 (s, 3H), 2.07 (s, 3H), 2.03 (s, 3H). LCMS (ESI) m / z 496.5 (M + H)+.

[0238] Intermediate 5

[0239] To a suspension of Intermediate 4 (2.5 g, 5.0 mmol) in dichloromethane (20 mL) was added / V-ethyl- / V-isopropylpropan-2-amine (1.8 mL, 10.1 mmol) and 2,5-dioxopyrrolidin-l-yl 3-((tert- butoxycarbonyl)amino)propanoate (1.3 g, 4.4 mmol). Stirred at 20 °C for 10 minutes then water (50 mL) was added. Organic layer was separated then the aqueous layer was extracted with dichloromethane (3 x 30 mL). Combined organic layers were dried over Na2SO4 the solvent was evaporated. To a solution of (2S,3S,4S,5R,6S)-2-((allyloxy)carbonyl)-6-(2-((3-((tert- butoxycarbonyl)amino)propanamido)methyl)-4-(hydroxymethyl)phenoxy)tetrahydro-2H-pyran- 3,4,5-triyl triacetate (2.8 g, 4.2 mmol) in dichloromethane (20 mL) was added hydrochloric acid (4.0 M in 1,4-dioxane, 2.6 mL, 83.9 mmol). Stirred at 20 °C for 2 h then solvent was evaporated. The compound was purified by reverse phase flash column chromatography to afford (2S,3S,4S,5R,6S)-2- ((allyloxy)carbonyl)-6-(2-((3-aminopropanamido)methyl)-4-(hydroxymethyl)phenoxy)tetrahydro-2H- pyran-3,4,5-triyl triacetate as a colorless material Intermediate 5 (1.3 g, 53% over 2 steps).XH NMR (500 MHz, D2O) 6 7.22 (d, J = 2.2 Hz, 1H), 7.16 (d, J = 8.2 Hz, 1H), 7.09 (d, J = 8.4 Hz, 1H), 5.84 (ddt, J = 16.6, 10.5, 6.0 Hz, 1H), 5.44 (t, J = 9.2 Hz, 1H), 5.38 (dd, J = 7.6, 3.3 Hz, 1H), 5.35 - 5.23 (m, 4H), 4.62 BGL-102-PCT01-NP

[0240] (d, J = 9.8 Hz, 1H), 4.56 (d, J = 6.0 Hz, 2H), 4.51 (s, 2H), 4.26 (q, J = 15.3 Hz, 2H), 3.23 (t, J = 6.8 Hz, 2H), 2.68 (td, J = 6.8, 1.9 Hz, 2H), 2.06 (d, J = 10.4 Hz, 9H). LCMS (ESI) m / z 567.2 (M + H)+.

[0241] Intermediate 7

[0242] 6

[0243] To a 250 mL round bottom flask was added Intermediate 6 (5.0 g, 34.21 mmol)) in dry DCM (100 mL) under nitrogen gas. To the solution was added pyridine (13.84 mL, 171.07 mmol) followed by tosyl- Cl (16.31 g, 85.53 mmol). The reaction mixture was stirred at 20 °C for 16 h. LC-MS analysis showed formation of desired product and completion of reaction. The reaction mixture was diluted with dichloromethane (200). Organic layer was separated, and compound was extracted in 200 mL dichloromethane. Combined organic layer was washed with HCI solution (IM-300 mL), brine (200 mL) and dried over magnesium sulfate. Solvent was removed under reduced pressure to get crude products. The compound was purified via silica gel column to give (3R,3aS,6R,6aS)- hexahydrofuro[3,2-b]furan-3,6-diyl bis(4-methylbenzenesulfonate) Intermediate 7 (14.90 g, 96 %). 1H NMR (500 MHz, CDCI3) 67.90 - 7.76 (m, 4H), 7.45 - 7.33 (m, 4H), 4.94 - 4.80 (m, 2H), 4.55 - 4.44 (m, 2H), 3.94 (dd, J = 9.6, 6.7 Hz, 2H), 3.75 (dd, J = 9.6, 7.6 Hz, 2H), 2.48 (s, 6H). LCMS (ESI) m / z 455.21 (M + H)+.

[0244] Intermediate 8

[0245] 7 8

[0246] To a 50 mL round bottom flask was added Intermediate 7 (6.0 g, 13.20 mmol) in dry DMF (15 mL) under nitrogen gas. To the solution was added sodium azide (2.146 g, 33.00 mmol). The reaction mixture was at 140 °C for 3 hrs. LC-MS analysis showed formation of desired product and completion of reaction. The reaction mixture was diluted with dichloromethane (200 x 2 mL), and organic layer was separated, washed with water (200 mL), brine (200 mL) and dried over magnesium sulfate. Solvent was removed under reduced pressure to get (3S,3aR,6S,6aR)-3,6-diazidohexahydrofuro[3,2- b]furan Intermediate 8 (2.050 g, 79 %).XH NMR (500 MHz, CDCI3) 64.61 (d, J = 1.9 Hz, 2H), 4.05 (d, J = 4.0 Hz, 2H), 3.97 - 3.82 (m, 4H). LCMS (ESI) m / z 197.1 (M + H)+. BGL-102-PCT01-NP

[0247] Intermediate 9

[0248] To a 250 mL round bottom flask was added Intermediate 8 (1 g, 5.10 mmol) in dry THF ( 20 mL) under nitrogen gas. To the solution was added barium palladium^ I ) carbonate (0.618 g, 0.51 mmol). The reaction mixture was flushed with hydrogen (1.028 g, 509.76 mmol) gas and stirred at 23 °C for 3 hrs. under Hz gas. LC-MS analysis showed formation of desired product and completion of reaction. The reaction mixture was diluted with methanol (20 mL) filtered through celite pad. Celite pad was washed with methanol (50 mL). Filtrate was dried over magnesium sulfate. Solvent was removed under reduced pressure to get (3S,3aR,6S,6aR)-hexahydrofuro[3,2-b]furan-3,6-diamine Intermediate 9 (0.590 g, 80 %).TH NMR (500 MHz, DMSO) 6 4.23 (s, 2H), 3.68 (dd, J = 8.7, 4.5 Hz, 2H), 3.41 (dd, J = 8.7, 1.9 Hz, 2H), 3.23 (dd, J = 4.5, 1.9 Hz, 2H), 1.54 (s, 4H). LCMS (ESI) m / z 145.2 (M + H)+.

[0249] Intermediate 11

[0250] To a 250 mL round bottom flask was added Intermediate 9 (1.50 g, 10.40 mmol) in dry THF (25 mL) under nitrogen gas. To the solution was added sodium hydrogen carbonate (1.748 g, 20.81 mmol) and 2,5-dioxopyrrolidin-l-yl 2,5,8,ll,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-oate Intermediate 10 (7.13 g, 10.40 mmol) in portions under nitrogen gas and stirred at 20 °C for 6 hrs. LC-MS analysis showed formation of desired product and completion of reaction. The reaction mixture was quenched by addition of methanol (10 mL). The reaction mixture was diluted with methanol (20 mL) filtered through celite pad. Celite pad was washed with methanol (50 mL). Filtrate was dried over magnesium sulfate. Solvent was removed under reduced pressure to get crude product, the crud product was purified via silica gel column to give / V-((3S,3aR,6S,6aR)-6- aminohexahydrofuro[3,2-b]furan-3-yl)-2,5,8,ll,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan- 38-amide Intermediate 11 (4.00 g, 53.8 %).XH NMR (500 MHz, MeOD) 6 4.64 - 4.59 (m, 1H), 4.45 (dd, J = 4.1, 1.3 Hz, 1H), 4.29 (dt, J = 4.1, 1.9 Hz, 1H), 3.96 (ddd, J = 10.2, 9.3, 4.9 Hz, 2H), 3.81 - 3.74 (m, 3H), 3.73 - 3.62 (m, 45H), 3.61 - 3.56 (m, 2H), 3.45 (dt, J = 3.8, 1.8 Hz, 1H), 3.40 (s, 3H), 2.52 - 2.47 (m, 2H). LCMS (ESI) m / z 715.6 (M + H)+. BGL-102-PCT01-NP

[0251] Intermediate 13

[0252] To a 250 mL round bottom flask was added (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6- (tert-butoxy)-6-oxohexanoic acid Intermediate 12 (5 g, 11.38 mmol) in dry DMF (20 mL) under nitrogen gas. To the solution was added potassium carbonate (3.14 g, 22.75 mmol) and 3- bromoprop-l-ene (1.485 mL, 17.06 mmol) in portions under nitrogen gas and stirred at 20 °C for 16 hrs. LC-MS analysis showed formation of desired product and completion of reaction. The reaction mixture was diluted with water (500 mL) and organic layer was extracted with ethyl acetate (2x 300 mL), washed with water (300 mL), brine (200 mL) and dried over sodium sulfate (20 g). The solvent was removed to get crude product. The crude product was purified via silica gel column to give 1- allyl 6-(tert-butyl) (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)hexanedioate Intermediate 13 (5.10 g, 93 %). NMR (500 MHz, CDCI3) 6 7.79 - 7.73 (m, 2H), 7.61 (q, J = 3.9 Hz, 2H), 7.40 (t, J = 7.5 Hz, 2H), 7.32 (tt, J = 7.4, 1.2 Hz, 2H), 5.91 (ddt, J = 16.5, 10.9, 5.8 Hz, 1H), 5.42 - 5.23 (m, 3H), 4.66 (d, J = 5.8 Hz, 2H), 4.40 (q, J = 4.8 Hz, 3H), 4.23 (t, J = 7.1 Hz, 1H), 2.26 (t, J = 7.2 Hz, 2H), 1.96 - 1.82 (m, 1H), 1.72 (dq, J = 13.5, 6.1 Hz, 3H), 1.60 - 1.47 (m, 1H), 1.45 (s, 9H). LCMS (ESI) m / z 480.2 (M + H)+.

[0253] Intermediate 14

[0254] To a 100 mL round bottom flask was added Intermediate 13 (5 g, 10.43 mmol) in dry THF (20 mL) under nitrogen gas. To the solution was added HCI (13.03 mL, 52.13 mmol), 4 molar in dioxane under nitrogen gas and stirred at 20 °C for 6 hrs. LC-MS analysis showed formation of desired product and completion of reaction. The reaction mixture was diluted with water (200 mL) and organic layer was extracted with dichloromethane (2x 300 mL), washed with brine (200 mL) and dried over sodium sulfate (20 g). The solvent was removed to get (S)-5-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-6-(allyloxy)-6-oxohexanoic acid Intermediate 14 (4.20 g, 95 %).XH NMR (500 MHz, CDCI3) 6 7.75 (dq, J = 7.6, 1.0 Hz, 2H), 7.62 - 7.52 (m, 2H), 7.42 - 7.35 (m, 2H), 7.30 (tt, J = 7.4, 1.2 Hz, 2H), 5.90 (ddt, J = 16.4, 10.8, 5.8 Hz, 1H), 5.48 (d, J = 8.4 Hz, 1H), 5.37 - 5.20 (m, 2H), 4.64 (d, J = 5.8 Hz, 2H), 4.40 (d, J = 7.2 Hz, 3H), 4.22 (t, J = 7.0 Hz, 1H), 2.45 - 2.24 (m, 2H), 1.93 (p, J = 5.6 Hz, 1H), 1.72 (td, J = 13.9, 6.8 Hz, 3H). (ESI) m / z 424.5 (M - H)’.

[0255] Intermediate 15 BGL-102-PCT01-NP

[0256] To a 100 mL round bottom flask was added Intermediate 14 (1.925 g, 4.55 mmol) under nitrogen gas. To the solution was added HATU (1.862 g, 4.90 mmol) followed by DIPEA (1.222 mL, 6.99 mmol). The reaction mixture was stirred at room temperature for 15 min then Intermediate 11 (2.5 g, 3.50 mmol), was added and reaction mixture was stirred at 23 °C for 3 hrs. LC-MS analysis showed formation of desired product and completion of reaction. The reaction mixture was diluted with DCM (300 mL), washed with water (200 mL), organic layer was extracted (2 x 100 mL), washed with Brine (50 mL), dried over sodium sulfate (5 g). Solvent was removed under reduced pressure to get crude product. The crude product was purified via silica gel column to give allyl (S)-6- (((3S,3aR,6S,6aR)-6-(2,5,8,ll,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38- amido)hexahydrofuro[3,2-b]furan-3-yl)amino)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6- oxohexanoate Intermediate 15 (3.40 g, 87 %)XH NMR (500 MHz, MeOD) 6 7.86 (dd, J = 7.6, 1.2 Hz, 2H), 7.74 (t, J = 7.8 Hz, 2H), 7.46 (td, J = 7.5, 1.4 Hz, 2H), 7.38 (tt, J = 7.5, 1.3 Hz, 2H), 6.05 - 5.93 (m, 1H), 5.39 (dq, J = 17.2, 1.6 Hz, 1H), 5.28 (dq, J = 10.5, 1.4 Hz, 1H), 4.73 - 4.65 (m, 2H), 4.58 (qd, J = 4.1, 1.0 Hz, 2H), 4.46 (dd, J = 10.6, 7.0 Hz, 1H), 4.40 (dd, J = 10.6, 7.0 Hz, 1H), 4.36 - 4.31 (m, 2H), 4.31 - 4.24 (m, 2H), 4.01 (ddd, J = 9.6, 5.0, 1.1 Hz, 2H), 3.84 - 3.73 (m, 5H), 3.72 - 3.60 (m, 44H), 3.60 - 3.56 (m, 2H), 3.41 (s, 3H), 2.49 (td, J = 6.0, 1.9 Hz, 2H), 2.31 (hept, J = 7.2 Hz, 2H), 1.96 - 1.85 (m, 1H), 1.85 - 1.68 (m, 3H). LCMS (ESI) m / z 1121.3 (M + H)+.

[0257] Intermediate 16

[0258] To a 50 mL round bottom flask was added Intermediate 15 (4.3 g, 3.84 mmol) in dry DCM (10 mL) under nitrogen gas. To the solution was added triethylamine (0.535 mL, 3.84 mmol) followed by triphenylphosphine (0.101 g, 0.38 mmol). To the reaction mixture was added Pd(PPha)4 (0.444 g, 0.38 mmol) then formic acid (0.147 mL, 3.84 mmol) was added and reaction mixture was stirred at 23 °C for 6 hrs. LC-MS analysis showed formation of desired product and completion of reaction. Solvent was removed under reduced pressure to get crude products. The crude product was purified via silica gel column to give (S)-6-(((3S,3aR,6S,6aR)-6-(2,5,8,ll,14,17,20,23,26,29,32,35- dodecaoxaoctatriacontan-38-amido)hexahydrofuro[3,2-b]furan-3-yl)amino)-2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-6-oxohexanoic acid Intermediate 16 (3.50 g, 84 %).1H NMR (500 MHz, BGL-102-PCT01-NP

[0259] DMSO) 6 8.12 (dd, J = 10.2, 7.0 Hz, 2H), 7.90 (d, J = 7.6 Hz, 2H), 7.74 (d, J = 7.5 Hz, 2H), 7.63 (d, J = 8.1 Hz, 1H), 7.46 - 7.38 (m, 2H), 7.34 (td, J = 7.4, 1.2 Hz, 2H), 4.38 (s, 2H), 4.32 - 4.20 (m, 3H), 4.11 (ddt, J = 7.2, 4.8, 2.1 Hz, 2H), 3.93 (td, J = 8.4, 4.6 Hz, 1H), 3.85 (dd, J = 9.3, 5.1 Hz, 2H), 3.63 - 3.57 (m, 4H), 3.54 - 3.45 (m, 42H), 3.45 - 3.40 (m, 2H), 3.24 (s, 3H), 2.33 (t, J = 6.5 Hz, 2H), 2.09 (s, 3H), 1.68 (d, J = 9.1 Hz, 1H), 1.64 - 1.48 (m, 3H). LCMS (ESI) m / z 1080.6 (M + H)+.

[0260] Intermediate 17

[0261] To a 100 mL round bottom flask was added Intermediate 16 (0.8 g, 0.74 mmol) under nitrogen gas. To the solution was added HATU (0.366 g, 0.96 mmol) followed by DIPEA (0.388 mL, 2.22 mmol). The reaction mixture was stirred at room temperature for 15 min then Intermediate 5 (0.670 g, 1.11 mmol), was added and reaction mixture was stirred at 23 °C for 3 hrs. LC-MS analysis showed formation of desired product and completion of reaction. The reaction mixture was diluted with DCM (100 mL), washed with water (100 mL), organic layer was extracted (2 x 100 mL), washed with Brine (100 mL), dried over sodium sulfate (15 g). Solvent was removed under reduced pressure and purified via silica gel column to give (2S,3R,4S,5S,6S)-2-(2-((S)-5-(4-(((3S,3aR,6S,6aR)-6- (2,5,8,ll,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-amido)hexahydrofuro[3,2-b]furan-3- yl)amino)-4-oxobutyl)-l-(9H-fluoren-9-yl)-3,6,10-trioxo-2-oxa-4,7,ll-triazadodecan-12-yl)-4- (hydroxymethyl)phenoxy)-6-((allyloxy)carbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate Intermediate 17 (0.640 g, 53.1 %). 1H NMR (500 MHz, DMSO) 6 8.21 (t, J = 6.0 Hz, 1H), 8.11 (dd, J = 19.5, 6.9 Hz, 2H), 7.94 (t, J = 5.8 Hz, 1H), 7.89 (d, J = 7.5 Hz, 2H), 7.73 (t, J = 7.0 Hz, 2H), 7.49 - 7.39 (m, 3H), 7.33 (td, J = 7.5, 1.2 Hz, 2H), 7.17 (dd, J = 8.4, 2.2 Hz, 1H), 7.13 (s, 1H), 7.01 (d, J = 8.4 Hz, 1H), 5.89 (ddt, J = 17.3, 10.5, 5.7 Hz, 1H), 5.56 (d, J = 7.9 Hz, 1H), 5.48 (t, J = 9.6 Hz, 1H), 5.33 (dq, J = 17.2, 1.6 Hz, 1H), 5.26 (dq, J = 10.5, 1.4 Hz, 1H), 5.19 - 5.07 (m, 3H), 4.76 (d, J = 10.0 Hz, 1H), 4.62 (ddt, J = 13.3, 5.6, 1.4 Hz, 1H), 4.54 (ddt, J = 13.3, 5.8, 1.4 Hz, 1H), 4.44 - 4.36 (m, 4H), 4.31 - 4.18 (m, 4H), 4.11 (d, J = 5.9 Hz, 3H), 3.93 (d, J = 5.9 Hz, 1H), 3.88 - 3.81 (m, 2H), 3.66 - 3.56 (m, 5H), 3.56 - 3.45 (m, 42H), 3.45 - 3.39 (m, 3H), 3.29 - 3.27 (m, 1H), 3.24 (s, 3H), 3.18 (d, J = 5.0 Hz, 1H), 2.33 (ddt, J = 14.6, 10.0, 7.6 Hz, 4H), 2.10 - 2.05 (m, 2H), 2.04 (s, 3H), 1.99 (d, J = 4.6 Hz, 6H), 1.54 (dt, J = 43.7, 8.9 Hz, 4H). LCMS (ESI) m / z 1629.8 (M + H)+.

[0262] Intermediate 18 BGL-102-PCT01-NP

[0263] To a solution of Intermediate 17 (25.00 mg, 0.02 mmol, 1.0 eq) in DMF was added bis(4-nitrophenyl) carbonate (28.0 mg, 0.09 mmol, 4.5 eq) and DIPEA (0.013 mL, 0.08 mmol, 4.0 eq). The mixture was stirred at 23 °C for 2 hours. Following this time the mixture was concentrated in vacuo and the residue was sonicated in DCM (200 pL) and diethyl ether (2 mL). The resulting suspension was dried on vacuum filter and the process repeated. The residue was dried to give (2S,3R,4S,5S,6S)-2-(2-((S)- 5-(4-(((3S,3aR,6S,6aR)-6-(2,5,8,ll,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38- amido)hexahydrofuro[3,2-b]furan-3-yl)amino)-4-oxobutyl)-l-(9H-fluoren-9-yl)-3,6,10-trioxo-2-oxa- 4,7,ll-triazadodecan-12-yl)-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6- ((allyloxy)carbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate Intermediate 18 (32 mg, 0.02 mmol, 91%) as a yellow material. RT 7.74 min. LCMS (ESI) m / z 1794.7 [M+H]+.

[0264] Intermediate 19 BGL-102-PCT01-NP

[0265] To a solution of exatecan mesylate (7.41 mg, 0.01 mmol, 1.0 eq) in DCM (1 mL) and DMF (1.000 mL) was added DIPEA (7.28 il, 0.04 mmol, 4.0 eq), Intermediate 18 (25.00 mg, 0.01 mmol, 1.0 eq), and HOPO (1.703 mg, 0.02 mmol, 2.0 eq) and the resultant mixture was stirred at 23 °C for 18 hours. Following this time the mixture was concentrated in vacuo and the residue was purified by reverse phase flash column chromatography (C18 BIOTAGE prepacked column, 40-60 % MeCN [0.1 % formic acid] / water [0.1 % formic acid]) to give (2S,3R,4S,5S,6S)-2-(2-((S)-5-(4-(((3S,3aR,6S,6aR)-6- (2,5,8,ll,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-amido)hexahydrofuro[3,2-b]furan-3- yl)amino)-4-oxobutyl)-l-(9H-fluoren-9-yl)-3,6,10-trioxo-2-oxa-4,7,ll-triazadodecan-12-yl)-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 19 (13 mg, 0.01 mmol, 63 %) as a yellow material. RT 7.66 min. LCMS (ESI) 2090.3 [M+H]+.

[0266] Intermediate 20

[0267] To a solution of Intermediate 19 (15.00 mg, 7.18 pmol, 1.0 eq) in DCM (1 mL) was added triethylamine (2.00 pl, 0.01 mmol, 1.4 eq), Pd(PPha)4 (1.00 mg, 0.87 pmol, 12 mol%) and formic acid (0.541 pl, 0.01 mmol, 1.4 eq) and the mixture stirred at 23 °C for 18 hours the mixture. Following this time the reaction mixture was concentrated and the crude residue was dissolved in methanol (0.25 mL) and THF (0.25 mL). To this solution was added potassium carbonate (9.44 mg, 0.07 mmol, 10 eq) in water (0.5 mL) and the mixture was stirred at 23 °C for 3 hours. Following this time the mixture was concentrated in vacuo to remove organics. The remaining aqueous solution was acidified with citric acid (1 N) until pH 4 was reached and the mixture was stirred for 1 hour at 23 °C. Following this time the mixture was filtered and the residue was purified by reverse phase flash column chromatography (C18 BIOTAGE prepacked column, 20-40 % MeCN [0.1 % formic acid] / water [0.1 % formic acid]) to give (2S,3S,4S,5R,6S)-6-(2-((3-((S)-6-(((3S,3aR,6S,6aR)-6- BGL-102-PCT01-NP

[0268] (2,5,8,ll,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-amido)hexahydrofuro[3,2-b]furan-3- yl)amino)-2-amino-6-oxohexanamido)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)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid Intermediate 20 (8.6 mg, 5.03 pmol, 70%) as a yellow material. RT 5.08 min. LCMS (ESI) 1702.6 [M+H]+.

[0269] Linker-payload LP1

[0270] To a solution of Intermediate 21 (8 mg, 0.01 mmol, 1.0 eq.) and Intermediate 20 (41.9 mg, 0.02 mmol, 2.1 eq.) in DMF (2 mL) was added N-ethyl-N-isopropylpropan-2-amine (10.21 pl, 0.06 mmol, 5.0 eq.) and the mixture stirred at 23 °C for 18 hours. Following this time the reaction mixture was concentrated under reduced pressure. The crude residue was purified by by preparative HPLC (Column: Sunfire Prep C18 OBD column, 19*250mm, 5um; Mobile Phase A: Water (0.1% FA), Mobile Phase B: ACN; Flow rate: 25 mL / min; Gradient: 10% B to 55% B in 15 min; Wave Length: 254nm / 220nm; RTl(min): 13.5). Fractions containing the desired compound were freeze dried directly to dryness to afford (2S,2'S,3S,3'S,4S,4'S,5R,5'R,6S,6'S)-6,6'-((((8S,30S)-8,30-bis(4- (((3S,3aR,6S,6aR)-6-(2,5,8,ll,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38- amido)hexahydrofuro[3,2-b]furan-3-yl)amino)-4-oxobutyl)-19-(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l- yl)propanoyl)-3,7,10,28,31,35-hexaoxo-13,16,22,25-tetraoxa-2,6,9,19,29,32,36- heptaazaheptatriacontane-l,37-diyl)bis(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- BGL-102-PCT01-NP yl)carbamoyl)oxy)methyl)-2,l-phenylene))bis(oxy))bis(3,4,5-trihydroxytetrahydro-2H-pyran-2- carboxylic acid) LP1 (16.70 mg, 4.33 pmmol, 37.0 %) as a colourless solid. RT 6.59 min. LCMS (ESI) 1928.3 [M+H]2+

[0271] Linker-Payload LP2

[0272] Intermediate 23

[0273] To a solution of Intermediate 20 (99 mg, 0.06 mmol) and bis(2,5-dioxopyrrolidin-l-yl) 13-(tert- butoxycarbonyl)-4,7,10,16,19,22-hexaoxa-13-azapentacosanedioate (Intermediate 22, 20 mg, 0.03 mmol) in DMF (2 mL) was added N-ethyl-N-isopropylpropan-2-amine (0.024 mL, 0.14 mmol) and the mixture stirred at 23 C for 4h (until the mono-addition by-product is no longer detected by LCMS). Following this time the reaction mixture was concentrated under reduced pressure. The residue was redissolved in DCM (2 mL) and TFA (1 mL) and the mixture stirred at 23 C for 10 minutes. Following this time the reaction mixture was concentrated under reduced pressure and purified by (C18 BIOTAGE prepacked column, 15-40 % MeCN [0.1 % formic acid] / water [0.1 % formic acid]) to give (2S,2'S,3S,3'S,4S,4'S,5R,5'R,6S,6'S)-6,6'-((((8S,36S)-8,36-bis(4-(((3S,3aR,6S,6aR)-6- (2,5,8,ll,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-amido)hexahydrofuro[3,2-b]furan-3- yl)amino)-4-oxobutyl)-3,7,10,34,37,41-hexaoxo-13,16,19,25,28,31-hexaoxa-2,6,9,22,35,38,42- heptaazatritetracontane-l,43-diyl)bis(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)-2,l-phenylene))bis(oxy))bis(3,4,5-trihydroxytetrahydro-2H-pyran-2- carboxylic acid) Intermediate 23 (75 mg, 71.2 %). RT 1.52 min. LCMS (ESI) 1896.8 [M+H]2+ BGL-102-PCT01-NP

[0274] To a solution of Intermediate 23 (40 mg, 10.55 pmol), 2-hydroxypyridine 1-oxide (1.371 pl, 0.01 mmol) and 2,5-dioxopyrrolidin-l-yl 3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanoate (3.65 mg, 0.01 mmol) in DMF (1 mL) was added N-ethyl-N-isopropylpropan-2-amine (9.18 pl, 0.05 mmol) and the mixture stirred at 23 °C for 10 minutes. After 10 minutes the reaction is complete. Following this time the reaction mixture was concentrated under reduced pressure. Fractions containing the desired compound were freeze dried directly to dryness to afford (2S,2'S,3S,3'S,4S,4'S,5R,5'R,6S,6'S)- 6,6'-((((8S,36S)-8,36-bis(4-(((3S,3aR,6S,6aR)-6-(2,5,8,ll,14,17,20,23,26,29,32,35- dodecaoxaoctatriacontan-38-amido)hexahydrofuro[3,2-b]furan-3-yl)amino)-4-oxobutyl)-22-(3-(2,5- dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanoyl)-3,7,10,34,37,41-hexaoxo-13,16,19,25,28,31-hexaoxa- 2,6,9,22,35,38,42-heptaazatritetracontane-l,43-diyl)bis(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)-2,l-phenylene))bis(oxy))bis(3,4,5-trihydroxytetrahydro-2H- pyran-2-carboxylic acid) LP2 (33.0 mg, 8.37 pmmol, 79 %) as a colourless solid. RT 6.46 min. LCMS (ESI) 1973.0 [M+H]2+.

[0275] Synthesis of LP3

[0276] Intermediate 24 BGL-102-PCT01-NP

[0277] N-ethyl-N-isopropylpropan-2-amine (1.619 mL, 9.29 mmol) was added dropwise to a stirred suspension of Intermediate 4 (1.535 g, 3.10 mmol), and 2,5-dioxopyrrolidin-l-yl 3-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)propanoate (1.518 g, 3.72 mmol) in CH2CI2 (15 mL) at 0°C under nitrogen. The resulting solution was stirred at 25 °C for 1 hour. The reaction mixture was diluted with DCM (20 mL), and washed sequentially with water (20 mL), and saturated aqueous NaCI (20 mL). The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure to dryness to afford crude product.

[0278] The resulting residue was purified by flash silica chromatography, elution gradient 0 to 70% EtOAc in hexanes. Product fractions were concentrated under reduced pressure to dryness to afford (2S,3R,4S,5S,6S)-2-(2-((3-((((9H-fluoren-9-yl)methoxy)carbonyl)arnino)propanarnido)rnethyl)-4- (hydroxymethyl)phenoxy)-6-((allyloxy)carbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (Intermediate 24, 0.967 g, 39.6 %) as a colorless foam. RT 1.39 min. LCMS (ESI) 789.3 [M+H]+.

[0279] Intermediate 25

[0280] To a solution of Intermediate 24 (537 mg, 0.68 mmol) in DMF (5 mL) was added bis(4-nitrophenyl) carbonate (414 mg, 1.36 mmol) and DIPEA (0.474 mL, 2.72 mmol). The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was concentrated under reduced pressure and purified by column chromatography (0-100% EtOAc in hexanes) to give (2S,3R,4S,5S,6S)-2-(2-((3- ((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)methyl)-4-((((4- BGL-102-PCT01-NP nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-((allyloxy)carbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (Intermediate 25, 344 mg, 53.0 %) as a yellow solid. RT 2.00 min. LCMS (ESI) 955.3 [M+H]+.

[0281] Intermediate 26 To a solution of Intermediate 25 (344 mg, 0.36 mmol) in DMF (5 mL) was added Exatecan mesylate (192 mg, 0.36 mmol), HOPO (0.036 mL, 0.36 mmol) and DIPEA (0.251 mL, 1.44 mmol). The reaction mixture was stirred at room temperature for 3 h. The reaction mixture was diluted with EtOAc and washed with brine. The organics were dried (MgSO4), filtered and concentrated under reduced pressure to give a crude product. Purification by column chromatography (100% EtOAc, then 0-10% MeOH in DCM) gave (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 26, 488 mg, 108 %) as a yellow oil. RT 1.98 min LCMS (ESI) 1251.4 [M+H]+. Intermediates 27 and 28

[0282] To a solution of Intermediate 26 (488 mg, 0.39 mmol) in MeOH (2 mL) and THF (2 mL) was added a solution of potassium carbonate (0.222 mL, 3.90 mmol) in water (2 mL). The reaction mixture was stirred at room temperature for 1 h. The reaction mixture purified by RP chromatography (25-55% BGL-102-PCT01-NP

[0283] MeCN in water +0.05% FA) to give Intermediate 27 (97 mg, 22.92 %, RT 1.76 min LCMS (ESI) 1085.3 [M+H]+). and Intermediate 28 (116 mg, 24.56 %, RT 1.93 min LCMS (ESI) 1211.4 [M+H]+) as a yellow solid

[0284] Intermediate 29

[0285] To a solution of Intermediate 28 (116 mg, 0.10 mmol) in THF (1 mL) was added a solution of potassium carbonate (0.055 mL, 0.96 mmol) in MeOH:water (1:1, 2 mL). The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was quenched with acetic acid (0.110 mL, 1.92 mmol) and concentrated under reduced pressure. The resulting crude was suspended in DMF (1 mL) and water (1 mL) and diethylamine (1 mL) added. The reaction mixture was stirred at room temperature for 30 mins. The reaction mixture was concentrated under reduced pressure and purified by RP chromatography (5-45% MeCN in water +0.05% TFA) to give (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 29, 50.0 mg, 60.5 %) as a yellow solid. RT 4.63 min LCMS (ESI) 863.3 [M+H]+

[0286] Intermediate 30

[0287] To a solution of Intermediate 16 (228 mg, 0.21 mmol) in DMF (2 mL) was added HATU (80 mg, 0.21 mmol) and DIPEA (0.221 mL, 1.27 mmol). The reaction mixture was stirred at room temperature for 10 mins followed by addition of di-tert-butyl 4,7,13,16-tetraoxa-10-azanonadecanedioate (155 mg, BGL-102-PCT01-NP

[0288] 0.34 mmol) and the reaction mixture stirred at room temperature for 10 mins. The reaction mixture was concentrated under reduced pressure to give di-tert-butyl 10-((S)-6-(((3S,3aR,6S,6aR)-6- (2,5,8,ll,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-amido)hexahydrofuro[3,2-b]furan-3- yl)amino)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-oxohexanoyl)-4,7,13,16-tetraoxa-10- azanonadecanedioate (Intermediate 30, 319 mg, 100 %) which was used without purification. RT 1.92 min LCMS (ESI) 1513.0 [M+H]+

[0289] Intermediate 31

[0290] To a solution of Intermediate 30 (317 mg, 0.21 mmol) in DCM (0.5 mL) was added TFA (0.5 mL). The reaction mixture was stirred at room temperature for 30 mins. The reaction mixture was purified by RP chromatography (25-55% MeCN in water +0.05% TFA) to give 10-((S)-6-(((3S,3aR,6S,6aR)-6- (2,5,8,ll,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-amido)hexahydrofuro[3,2-b]furan-3- yl)amino)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-oxohexanoyl)-4,7,13,16-tetraoxa-10- azanonadecanedioic acid (Intermediate 31, 152 mg, 51.7 %) as a colourless oil. RT 6.27 min LCMS (ESI) 1400.8 [M+H]+

[0291] Intermediate 32

[0292] To a solution of Intermediate 31 (40.6 mg, 0.03 mmol) in DMF (600 uL) was added HATU (22.06 mg, 0.06 mmol) and DIPEA (0.101 mL, 0.58 mmol). The reaction mixture was stirred at room temperature for 5 mins. A solution of Intermediate 29 (40.6 mg, 0.03 mmol) in DMF (500 uL) was BGL-102-PCT01-NP added to the reaction mixture and stirred at room temperature for 10 mins. The reaction was quenched via addition of acetic acid (0.033 mL, 0.58 mmol). The reaction mixture was purified by RP chromatography (20-60% MeCN in water +0.05% TFA) to give Intermediate 32 (58.0 mg, 64.8 %). RT 7.06 min LCMS (ESI) 1544.4 [M+2H]+; 1030 [M+3H]+; 1543.1 [M-2H]- Intermediate 33

[0293] To a solution of Intermediate 32 (58 mg, 0.02 mmol) in DMF (1 mL) was added diethylamine (1 mL).

[0294] The reaction mixture was stirred at room temperature for 30 mins. The reaction mixture was purified by RP chromatography (5-45% MeCN in water +0.05% TFA) to give Intermediate 33 (38.0 mg, 70.6 %) as a white solid. RT 5.57 min LCMS (ESI) 1433.2 [M+2H]+; 956.0 [M+3H]+

[0295] Linker-payload LP3

[0296] To a solution of 2,5-dioxopyrrolidin-l-yl 3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanoate (7.06 mg, 0.03 mmol) in DMF (0.5 mL) was added Intermediate 33 (38 mg, 0.01 mmol) and DIPEA (20 pl, BGL-102-PCT01-NP

[0297] 0.11 mmol). The reaction mixture was stirred at room temperature for 15 mins. The reaction mixture was quenched via addition of acetic acid (10 pl, 0.17 mmol) and concentrated under reduced pressure. The resulting crude was purified by prep HPLC (Synergi hydro, 30-42.7% MeCN in water +0.05% TFA over 9.5 mins) to give Linker-Payload LP3 (4.40 mg, 11.00 %) as yellow solid. RT 6.35 min LCMS (ESI) 1508.9 [M+2H]+; 1006.4 [M+3H]+; 1507.8 [M-2H]-

[0298] Synthesis of LP4

[0299] Intermediate 34

[0300] To Intermediate 30 (1.09 g, 0.66 mmol) in DCM (2 mL) was added diethylamine (2 mL, 19.33 mmol). The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure to give a crude product. The crude product was dissolved in DMF (3 mL) and 2,5-dioxopyrrolidin-l-yl 3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanoate (193 mg, 0.73 mmol) and DIPEA (0.6 mL, 3.44 mmol) were added. The reaction mixture was stirred at room temperature for 20 mins. The reaction mixture was purified by RP chromatography (20-50% MeCN in water +0.05% TFA) to give di-tert-butyl 14-((S)-6-(((3S,3aR,6S,6aR)-6- (2,5,8,ll,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-amido)hexahydrofuro[3,2-b]furan-3- yl)amino)-2-(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanamido)-6-oxohexanoyl)-5,23-dioxo- 8,ll,17,20-tetraoxa-4,14,24-triazaheptacosanedioate (Intermediate 34, 746 mg, 71.4 %) as a colourless oil. LCMS (5 min method): 2.17 min; 1584.9 [M+H]+

[0301] Intermediate 35 BGL-102-PCT01-NP

[0302] To Intermediate 30 (746 mg, 0.47 mmol) in DCM (2 mL) was added TFA (2 mL). The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure to give 14-((S)-6-(((3S,3aR,6S,6aR)-6-(2,5,8,ll,14,17,20,23,26,29,32,35- dodecaoxaoctatriacontan-38-amido)hexahydrofuro[3,2-b]furan-3-yl)amino)-2-(3-(2,5-dioxo-2,5- dihydro-lH-pyrrol-l-yl)propanamido)-6-oxohexanoyl)-5, 23-dioxo-8, 11,17, 20-tetraoxa-4, 14,24- triazaheptacosanedioic acid (Intermediate 35, 663 mg, 96 %) as an orange oil. LCMS (5 min): 1.27 min; 1472.6 [M+H]+

[0303] LP4

[0304] To Intermediate 35 (50 mg, 0.03 mmol) in DMF (0.5 mL) was added HATU (25.9 mg, 0.07 mmol) and DIPEA (100 pl, 0.57 mmol) and stirred at room temperature for 5 mins. Intermediate 36 (obtainable as disclosed in US 2025 / 0057969 Al, 73.0 mg, 0.07 mmol) was added and the reaction mixture stirred at room temperature for 20 mins. The reaction mixture was purified by RP chromatography (30-60% MeCN in water +0.05% TFA) to give LP4 (16.90 mg, 13.88 %) as an off white solid. LCMS (15 min method): 8.18 min; 1792.4 [M+2H]+; 1195.6 [M+3H]+; 897.2 [M+4H]+

[0305] Synthesis of DR2

[0306] Intermediate A BGL-102-PCT01-NP

[0307] 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-[l,2,3]triazolo[4,5-b]pyridin-3-yl)-l,l,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.

[0308] DR2

[0309] 2,2,2-trifluoroacetic 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 (DR2)(107 mg, 77 %) as a yellow solid.1H NMR (400 MHz, DMSO) 6 8.69 - 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 BGL-102-PCT01-NP

[0310] 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.

[0311] Payload Potency

[0312] 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.

[0313] 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 CC -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%).

[0314] Table 1 BGL-102-PCT01-NP

[0315] Table 1 show the cytotoxicity data for DR2(N=3) and Exatecan DR1(N=3) in NCI-N87, MDAMB361 and MDAMB468 cell lines. The IC5o values in each figure were determined by fitting data to a sigmoidal dose-response curve using GRAPHPAD PRISM software v9 (GraphPad, San Diego, CA).

[0316] Antibody-drug conjugation

[0317] General Procedure for Herceptin-WT-LP conjugation

[0318] Linker-payloads as disclosed herein may be conjugated using conjugations techniques known in the art, such as disclosed in WO 2021 / 148500, the contents of which is incorporated by reference. For example, a conjugate may be prepared from a wild type antibody or by site specific conjugation to an antibody comprising one or more engineered cysteines. The average no. of drugs loaded per antibody in preparations of ADCs using the linker-payloads can be characterized by conventional means such as mass spectroscopy and HPLC.

[0319] Herceptin-WT-LP2 conjugation (ADC2)

[0320] LP2 was added as a DMSO solution (25 molar equivalent / antibody, 2.82 pmole, in 0.104 mL DMSO) to 2.46 mL of the Herceptin-S159S-L174S antibody solution in PBS, 1 mM EDTA, pH 7.4 (17.0 mg, 113.0 nanomoles) for a 10% (v / v) final DMSO concentration. The solution left to react at room temperature for 18 hrs with gentle shaking. Then the conjugation was quenched by addition of / V-acetyl cysteine (14.1 micromoles, 140 pL at 100 mM), then purified in in PBS pH 7.4 PrepSEC-AKTA and formulated in 20 mM Histidine / Histidine HCI, 240 m sucrose pH 6.0 using a 15 mL Amicon Ultracell 30 kDa MWCO spin filter, sterile-filtered and analysed.

[0321] UHPLC analysis on a Shimadzu Prominence system using a Agilent PLRP-S RP 1000A 5um 2.1*50mm column eluting with a gradient of water and acetonitrile on a reduced sample of ADC at 214 nm and 330 nm shows a mixture of light chain conjugated to 1 molecule of LP2, and heavy chain conjugated to 1.0 molecules of LP2, consistent with a drug-per-antibody ratio (DAR) of 7.48 molecules of LP2 per antibody.

[0322] UHPLC 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 BGL-102-PCT01-NP 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 shows a monomer purity of 99.09 %.

[0323] LC-MS analysis on a Exactive Plus EMR mass spectrometer connected to Dionex 3000 HPLC equipment using a Thermo Scientific MAbPac 50 mm x 2.1 mm column eluting with a gradient of water and acetonitrile on a de-glycosylated and reduced sample of ADC at 214 nm shows a mixture of light chain conjugated to 1 molecule of LP2 and heavy chain conjugated to 1.0 molecules of LP2, consistent with a drug-per-antibody ratio (DAR) of 7.750 molecules of LP2 per antibody.

[0324] UHPLC analysis on a Shimadzu Prominence system using a Proteomix HIC Butyl-NP5, 5um, non-porous, 4.6x35 mm (Sepax) column eluting with a gradient of 1.5M ammonium sulphate, 25 mM sodium acetate, pH 7.4 and 25 mM sodium acetate, pH 7.4 with 20% acetonitrile (v / v) on a neat sample of ADC at 214 nm shows singly conjugated to LP2 , consistent with a drug-per-antibody ratio (DAR) of 8.0 molecules of LP2 per antibody.

[0325] Herceptin-WT-LP3 conjugation (ADC3)

[0326] LP3 was added as a DMSO solution (18 molar equivalent / antibody, 0.83 pmole, in 0.053 mL DMSO) to 1.1 mL of the Herceptin-S159S-L174S antibody solution in PBS, 1 mM EDTA, pH 7.4 (9.0 mg, 46.30 nanomoles) for a 10% (v / v) final DMSO concentration. The solution left to react at room temperature for 18 hrs with gentle shaking. Then the conjugation was quenched by addition of N- acetyl cysteine (4.16 micromoles, 41.65 0L at 100 mM), then purified in in PBS pH 7.4 PrepSEC-AKTA and formulated in 20 mM Histidine / Histidine HCI, 240 m sucrose pH 6.0 using a 15 mL Amicon Ultracell 30 kDa MWCO spin filter, sterile-filtered and analysed.

[0327] UHPLC analysis on a Shimadzu Prominence system using a PLRP-S RP 1000A 5um 2.1*50mm column eluting with a gradient of water and acetonitrile on a reduced sample of ADC at 214 nm and 330 nm shows a mixture of light chain conjugated to 1 molecule of LP3, and heavy chain conjugated to 1.0 molecules of LP3, consistent with a drug-per-antibody ratio (DAR) of 7.56 molecules of LP3 per antibody.

[0328] UHPLC 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 shows a monomer purity of 99.09 %. BGL-102-PCT01-NP

[0329] LC-MS analysis on a Exactive Plus EMR mass spectrometer connected to Dionex 3000 HPLC equipment using a Thermo Scientific MAbPac 50 mm x 2.1 mm column eluting with a gradient of water and acetonitrile on a de-glycosylated and reduced sample of ADC at 214 nm shows a mixture of light chain conjugated to 1 molecule of LP3 and heavy chain conjugated to 1.0 molecules of LP3, consistent with a drug-per-antibody ratio (DAR) of 8.0 molecules of LP3 per antibody.

[0330] UHPLC analysis on a Shimadzu Prominence system using a Proteomix HIC Butyl-NP5, 5um, non- porous, 4.6x35 mm (Sepax) column eluting with a gradient of 1.5M ammonium sulphate, 25 mM sodium acetate, pH 7.4 and 25 mM sodium acetate, pH 7.4 with 20% acetonitrile (v / v) on a neat sample of ADC at 214 nm shows singly conjugated to LP3 , consistent with a drug-per-antibody ratio (DAR) of 7.76 molecules of LP3 per antibody.

[0331] General Procedure of ADC toxicity evaluation on 3D NCI-N87, JIMT1 and MDAMB468I

[0332] Media from NCI-N87, JIMT1 and MDAMB468 cells at 80-90% confluency in a T175 flask was aspirated and the flask rinsed with PBS (about 10 ml) and emptied. TrypLE (5ml) Express Enzyme (lx) was added, the flask returned to the 37 °C incubator with 5% CO2 for about 5 minutes, then the flask was shaken to detach the cells from the bottom. 10 mL cell media (RPMI 1640 for NCI-N87 and MDAMB468, DMEM for JIMT1, both supplement with 50% Fetal Bovine Serum) was added to the flask and the cell suspension was transferred to a sterile 50 ml falcon tube, then centrifuged (400g for 5 min). The supernatant was aspirated, and the pellet re-suspended in lOmL culture medium. The cell suspension was well pipetted to break possible aggregates and lOpL solution were mixed with lOpL trypan blue cell-stained cells. 20pL mix were then transferred on a cell counting slide and the cell concentration and viability measured using the LUNA II. According to previous experiments that allowed us to determine the best seeding density, NCI-N87, JIMT1 and MDAMB468 cell lines were respectively seeded at 10000,3000 and 3000 cells / well in a CORNING SPHEROID 96-well microplate.

[0333] A stock solution (650pL) 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 2mL deep 96 well plate by serial transfer of 65 pl onto 585 pl of cell culture medium. ADC dilution was dispensed (50 pl / well) into 2 replicate wells of the 96-well plate, containing 50 pl cell suspension seeded 48h before. Control wells received 50 pl cell culture medium. The 96-well plate containing cells and ADCs was incubated at 37 °C in a CC -gassed incubator for 6 days. At the end of the incubation period, plates were equilibrated to room temperature for lOmin before CELLTITER- GLO 3D Cell Viability Assay was dispensed (100 pl per well) into each well. Plates were pipette mixed for 5 minutes after which the plates were incubated for 25 minutes at room temperature. Well BGL-102-PCT01-NP luminescence was measured, and percentage cell survival was calculated from the mean luminescence in the 2 ADC-treated wells compared to the mean luminescence in the 6 control untreated wells (100%). IC5o was determined from the dose-response data using GRAPHPAD PRISM using the non-linear regression (curve fit) algorithm: Sigmoidal, 4PL, X is log(concentration).

[0334] General Procedure for ADC toxicity evaluation on 3D SKOV3 and SKOV3 GUSB KO

[0335] Media from SKOV3 WT and SKOV3 GUSB KO (P-Glucuronidase knockout cell line, generated using CRISPR targeting) cells at 80-90% confluency in a T175 flask was aspirated and the flask rinsed with PBS (about 10 ml) and emptied. TrypLE (5ml) Express Enzyme (lx) was added, the flask returned to the 37 °C incubator with 5% CO2 for about 5 minutes, then the flask was shaken to detach the cells from the bottom. 10 mL cell media (McCoy's 5A supplemented with 50% Fetal Bovine Serum) was added to the flask and the cell suspension was transferred to a sterile 50 ml falcon tube, then centrifuged (400g for 5 min). The supernatant was aspirated, and the pellet re-suspended in lOmL culture medium. The cell suspension was well pipetted to break possible aggregates and lOuL solution were mixed with lOuL trypan blue cell-stained cells. 20uL mix were then transferred on a cell counting slide and the cell concentration and viability measured using the LUNA II. According to previous experiments that allowed us to determine the best seeding density, SKOV3 and SKOV3 GUSB KO were seeded at 3000cells / well in a CORNING SPHEROID 96-well microplate.

[0336] A stock solution (650pL) 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 2mL deep 96 well plate by serial transfer of 65 pl onto 585 pl of cell culture medium. ADC dilution was dispensed (50 pl / well) into 2 replicate wells of the 96-well plate, containing 50 pl cell suspension seeded 48h before. Control wells received 50 pl cell culture medium. The 96-well plate containing cells and ADCs was incubated at 37 °C in a CC -gassed incubator for 6 days. At the end of the incubation period, plates were equilibrated to room temperature for lOmin before CELLTITER- GLO 3D Cell Viability Assay was dispensed (100 pl per well) into each well. Plates were pipette mixed for 5 minutes after which the plates were incubated for 25 minutes at room temperature. Well luminescence was measured, and percentage cell survival was calculated from the mean luminescence in the 2 ADC-treated wells compared to the mean luminescence in the 6 control untreated wells (100%). IC50 was determined from the dose-response data using GraphPad Prism using the non-linear regression (curve fit) algorithm: Sigmoidal, 4PL, X is log(concentration). BGL-102-PCT01-NP

[0337] General Procedure for ADC toxicity evaluation on NCI-N87, MDAMB468, SKOV3 WT and SKOV3

[0338] GUSB KO

[0339] Media from NCI-N87, MDAMB468, SKOV3 WT and SKOV3 GUSB KO cells at 80-90% confluency in a T175 flask was aspirated and the flask rinsed with PBS (about 10 mL) and emptied. TrypLE (5ml) Express Enzyme (lx) was added, the flask returned to the 37 °C incubator with 5% CO2 for about 5 minutes. The flask was then shaken to detach the cells from the bottom. 10 mL RPMI 1640 and McCoy 5A cell media, both supplemented with 50% Fetal Bovine Serum, were added to the flasks and the cell suspensions were transferred to sterile 50 ml falcon tubes, then centrifuged (400g for 5 min). The supernatant was aspirated, and the pellet re-suspended in lOmL culture medium. The cell suspension was well pipetted to break possible aggregates and lOpL solution were mixed with lOpL trypan blue cell-stained cells. 20pL mix were then transferred on a cell counting slide and the cell concentration and viability measured using the LUNA II. According to previous experiments that allowed us to determine the best seeding density, MDAMB468, SKOV3 WT and SKOV3 GUSB KO cell lines were seeded at 3000 cells / wells while the NCI-N87 were seeded at 10000 cells / wells.

[0340] A stock solution (550pL) 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 2mL deep 96 well plate by serial transfer of 110 pl onto 440 pl of cell culture medium. ADC dilution was dispensed (50 pl / well) into 2 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 was incubated at 37 °C in a CC -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 10 min before stabilisation at room temperature for 1 min. Well luminescence was measured, and percentage cell survival was calculated from the mean luminescence in the 2 ADC-treated wells compared to the mean luminescence in the 6 control untreated wells (100%). IC50 was determined from the dose-response data using GRAPHPAD PRISM using the non-linear regression (curve fit) algorithm: Sigmoidal, 4PL, X is log(concentration). in vitro cytotoxicity data for ADC2 and ADC3 (NCI-N87 and MDAMB468)

[0341] Media from NCI-N87 and MDAMB468 cells at 80-90% confluency in a T175 flask was aspirated and the flask rinsed with PBS (about 10 ml) and emptied. TrypLE (5ml) Express Enzyme (lx) was added, the flask returned to the 37 °C incubator with 5% CO2 for about 5 minutes, then the flask was shaken to detach the cells from the bottom. 10 mL RPMI 1640 cell media supplemented with 50% Fetal Bovine Serum was added to the flask and the cell suspension was transferred to a sterile 50 ml BGL-102-PCT01-NP falcon tube, then centrifuged (400g for 5 min). The supernatant was aspirated, and the pellet resuspended in lOmL culture medium. The cell suspension was well pipetted to break possible aggregates and a 200 pL sample of cells was run on a Vi-CELL analyzer a device that uses trypan blue and automated imaging to determine cell concentration and viability. According to previous experiments that allowed us to determine the best seeding density, NCI-N87 and MDAMB468 cell lines were respectively seeded at 750 cell / wells in a 384 well-plate format using a MultiDrop Combi+ instrument.

[0342] A stock solution (300pL) of Antibody Drug Conjugate (ADC) was made by dilution of filter-sterilised ADC into cell culture medium. A set of lOx 10-fold dilutions of the previous ADC solution were made in a 2mL deep 96 well plate by serial transfer of 30 pl onto 270 pl of cell culture medium. ADC dilution was dispensed (10 pl / well) into 4 replicate wells of the 384-well plate, containing 30 pl cell suspension seeded the previous day. Control wells received 10 pl cell culture medium. The 384-well plate containing cells and ADCs was incubated at 37 °C in a CC -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 (40 pl per well) into each well. Plates were placed on an orbital shaker for 10 min before stabilisation at room temperature for 1 min. Well luminescence was measured and data readout exported in a .csv file. qAC5o was determined via GeneData Screener software, running a 384_ADC template and using a Nexus Plate Parser format while graphs were obtained from plotting the normalised values on GraphPad.

[0343] Table 2

[0344] Table 2 shows cytotoxicity data for ADC2 and ADC3 in HER2+++ NCI-N87 and HER2- MDAMB468 cell lines.

[0345] 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, while indicating embodiments of this specification, are intended for purposes of illustration only. This BGL-102-PCT01-NP 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

BGL-102-PCT01-NPClaimsor 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, each 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 (CHjjni, wherein nl 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, each JAis independently a group of Formula (IB)whereinE is (CHjJnz, wherein n2 is independently 0, 1, 2 or 3, each X1and X2are (CHjJna, wherein each n3 is independently 1, 2, 3 or 4, p is 1 or 0,BGL-102-PCT01-NP q 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, each R1is independently a group of Formula (IC)whereinR2is Ci-4 alkyl,Y is (CHjJru, wherein n4 is 0, 1, 2 or 3,Z is (CHzJns, wherein n5 is 0, 1, 2, 3 or 4, m is an integer from 5 to 17,(N) indicates the point of attachment to the adjacent N atom, and (C) indicates the point of attachment to the adjacent C atom, 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 E is CH2.

3. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in claim 1 or claim 2, wherein W1is CH2 or (CFbh-4. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 3, wherein each X1is CH2 and each X2is CH2.

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

6. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 5, wherein Z is (CFbh-BGL-102-PCT01-NP7. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 6, wherein p is 1.

8. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 7, wherein each q is independently 1, 2, 3 or 4.

9. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 8, wherein r is 0.

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

11. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 10, wherein R2is CH3.

12. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 11, wherein eachBGL-102-PCT01-NP13. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 11, wherein each14. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 11, wherein each15. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 14, wherein each BAis a group of Formula ( I Al)16. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in claim15, wherein each JAis a group of Formula ( I Bl)BGL-102-PCT01-NP17. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in claim16, wherein BAis a group of Formula (IA2)and each JAis a group of Formula (IB2)18. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 14, wherein each JAis a group of Formula (IBB)BGL-102-PCT01-NP19. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed claim18, wherein BAis a group of Formula (IA3)20. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in claim18 or 19, wherein BAis a group of Formula (IA3)and each JAis a group of Formula (IB3)BGL-102-PCT01-NP21. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 20, wherein GAis selected fromwherein RKis H or CH3, RLis Ci.g alkyl,indicates the point of attachment to the antibody or antigen-binding fragment thereof.

22. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in claim21, wherein GAis23. A 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.BGL-102-PCT01-NP24. 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.

25. 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 comprising administering to the patient 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)or a salt thereof, wherein GBis a conjugation group for conjugation to an antibody or antigenbinding fragment thereof,BBis a group of Formula (IIA) )each JBis independently a group of Formula ( 11 B)BGL-102-PCT01-NPwherein DR, E, QB, QJ, R1, X1, X2, p, q and r are 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 each JBand (NR) indicates the point of attachment to the nitrogen atom NR.

30. A compound of Formula (II) or a salt thereof, as claimed in claim 29, wherein each BBis a group of Formula (I I Al)31. A compound of Formula (II) or a salt thereof, as claimed in claim 30, wherein each JBis a group of Formula (I I Bl)BGL-102-PCT01-NP32. A compound of Formula (II) or a salt thereof, as claimed in claim 30 or claim 31, wherein BBis a group of Formula (IIA2)and each JBis a group of Formula ( 11 B2)33. A compound of Formula (II) or a salt thereof, as claimed in any claim 29, wherein each JBis a group of Formula (I I B3)34. A compound of Formula (II) or a salt thereof, as claimed claim 33, wherein BBis a group ofFormula (IIA3)BGL-102-PCT01-NP35. A compound of Formula (II) or a salt thereof, as claimed in claim 33 or claim 34, wherein BBis a group of Formula (I IA4)and each JBis a group of Formula ( 11 B4)36. A compound of Formula (II) or a salt thereof, as claimed in any one of claims 29 to 35, wherein GBis selected fromBGL-102-PCT01-NPwherein X1is CH or N, h is 0 or 1, Hal is Cl, Br or I, RKis H or CH3, and RLis C1-6 alkyl.

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

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