Conjugates comprising a topoisomerase i inhibitor

Abstract

The specification relates to conjugates comprising one or more Dc 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] TOP-201-PCT01-NP CONJUGATES COMPRISING A TOPOISOMERASE I INHIBITOR Cross‑Reference to Related Patent Application This specification claims the benefit of priority to European Patent Application EP 24175179.1 (filed 10 May 2024) and European Patent Application EP 24205697.6 (filed 9 October 2024). The entire text of the above-referenced patent applications are incorporated by reference into this specification. Field This specification relates to certain conjugates comprising cleavable linkers and a topoisomerase I inhibitor (TOPO1i), and to pharmaceutical compositions containing them. This specification also relates to the use of the conjugates in methods of treating diseases such as cancer. This specification further relates to processes and intermediate compounds involved in the preparation of the conjugates. Background Topoisomerase inhibitors are chemical compounds that block the action of topoisomerase (topoisomerase I and II), which is a type of enzyme that controls the changes in DNA structure by catalyzing the breaking and re-joining of the phosphodiester backbone of DNA strands during the normal cell cycle. Exatecan is an example of a topoisomerase I inhibitor. Antibody drug conjugates (ADCs) are an established method to deliver topoisomerase I inhibitors, such as exatecan, to a biological target. There remains a need for conjugates comprising a topoisomerase I inhibitor having a favourable therapeutic index and favourable physicochemical properties. The conjugates of the disclosure may be used for the treatment of diseases such as cancer. General Description In a first aspect there is provided a conjugate of Formula (I) Ab – (GA–JA–DC)k (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, each GAis independently a conjugation group conjugated to the antibody or antigen-binding fragment thereof, TOP-201-PCT01-NP each DCis independently , wherein b is 0, 1, 2 or 3, each JAis independently a group of Formula (IA) R1is C1-4alkyl, X is (CH2)n2, wherein n2 is 0, 1, 2 or 3, Y is (CH2)n3, wherein n3 is 0, 1, 2, 3 or 4, Z is (CH2)n4, wherein n4 is 0, 1, 2, 3, 4 or 5, m is an integer from 5 to 17, p is 1 or 0, (GA) indicates the point of attachment to GA, and (DC) indicates the point of attachment to DC. TOP-201-PCT01-NP In embodiments, each DCis independently . 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. In a further aspect there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, for use in therapy. 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. 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. 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. 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. In a further aspect there is provided a compound of Formula (II) GB–JB–DC(II) or a salt thereof, wherein GBis a conjugation group for conjugation to an antibody or antigen- binding fragment thereof, JBis a group of Formula (IIA) TOP-201-PCT01-NP wherein DC, E, Q, R1, X, Y, Z, m and p are as defined above for a conjugate of Formula (I), (GB) indicates the point of attachment to GB, and (DC) indicates the point of attachment to DC. In a further aspect there is provided intermediates useful for the synthesis of a compound of Formula (II) or a salt thereof. In a further aspect there is provided intermediates useful for the synthesis of a compound of Formula (II) or a salt thereof, including a compound of Formula (IIIA) In a further aspect there is provided intermediates useful for the synthesis of a compound of Formula (II) or a salt thereof, including a compound of Formula (III) , wherein n is 0, 1, 2, or 3, or a salt thereof. TOP-201-PCT01-NP A conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, may undergo enzymatic cleavage to release the compound of Formula (IIIA) or (III). The compound of Formula (IIIA) or (III) provides an anti-cancer effect by, as a minimum, acting as a topoisomerase inhibitor. The compound of Formula (IIIA) or (III) may also exhibit advantageous physical properties (for example, lower lipophilicity, higher aqueous solubility, higher permeability and / or lower plasma protein binding), and / or favourable toxicity profiles (for example a decreased activity at hERG), and / or favourable metabolic or pharmacokinetic profiles, in comparison with other known topoisomerase inhibitors. Further, conjugates of Formula (I) may exhibit improved efficacy and / or advantageous physical properties (for example, higher colloidal stability, higher chemical 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. As such, conjugates of Formula (I) and the compound of Formula (IIIA) or (III) may be especially suitable as therapeutic agents, such as for the treatment of cancer. Definitions 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. The prefix Cx-y, where x and y are integers, indicates the numerical range of carbon atoms that are present in a group. As used herein the term “alkyl” refers to a saturated, linear or branched hydrocarbon radical having the specified number of carbon atoms. Examples of C1-4 alkyl groups include methyl (Me), ethyl (Et), n-propyl (nPr), i-propyl (iPr), n-butyl (nBu), i-butyl (iBu), s-butyl (sBu), and t-butyl (tBu). Examples of C1- 6 alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl and n-hexyl. 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.The use of “ ” in formulas of this specification indicates the point of attachment to the antibodyor antigen-binding fragment thereof. By way of illustration indicates that there is a TOP-201-PCT01-NP covalent bond connecting the antibody, or antigen-binding fragment thereof, to the carbon atom marked 1.For the avoidance of doubt, the use of “ ” in formulas of this specification denotes the point ofcovalent attachment to a group, where the group is other than the antibody or antigen-binding fragment thereof. Certain embodiments of this specification include a group which is said to be “optionally substituted”. In further embodiments said group is unsubstituted. 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. 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. Description of Figures Embodiments and experiments illustrating the principles of the disclosure will now be discussed with reference to the accompanying figures in which: Figure 1A illustrates cytotoxicity data for payload (warhead) P-1 and Exatecan (Reference) in an NCI- N87 cell line. Figure 1B illustrates cytotoxicity data for payload (warhead) P-1 and Exatecan (Reference) in an MDAMB361 cell line. Figure 1C illustrates cytotoxicity data for payload (warhead) P-1 and Exatecan (Reference) in an MDAMB468 cell line. Figure 2A illustrates cytotoxicity data for ADC-1 and ADC-2 (Reference) in a HER2- MDAMB468 cell line. Figure 2B illustrates cytotoxicity data for ADC-1 and ADC-2 (Reference) in a Her2+++ / GUSB+++ SKOV3 WT cell line. Figure 2C illustrates cytotoxicity data for ADC-1 and ADC-2 (Reference) in a HER2+++ / GUSB- SKOV3 GUSB KO cell line. TOP-201-PCT01-NP Figure 3A illustrates cytotoxicity data for payload (warhead) P-1, P-2 and P-3 in a Her2+++ NCI-N87 cell line. Figure 3B illustrates cytotoxicity data for payload (warhead) P-1, P-2 and P-3 in a HER2- MDAMB468 cell line. Figure 4A illustrates the cytotoxicity data for ADC-1, ADC-2 (Reference), ADC-3 and ADC-4 in a Her2+++ NCI-N87 cell line. Figure 4B illustrates the cytotoxicity data for ADC-1, ADC-2 (Reference), ADC-3 and ADC-4 a HER2- MDAMB468 cell line. Figure 4C illustrates the cytotoxicity data for ADC-1, ADC-2 (Reference), ADC-3 and ADC-4 in a HER2+++ / GUSB+++ SKOV3 WT cell line. Figure 4D illustrates the cytotoxicity data for ADC-1, ADC-2 (Reference), ADC-3 and ADC-4 in a HER2+++ / GUSB- SKOV3 GUSB KO cell line. Detailed Description In one aspect, this specification provides a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, as defined above. 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 an integer from 2 to 10. In further embodiments k is an integer from 2 to 8. In further embodiments k is 4. In further embodiments k is 8. In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein GAis selected from

[0002] TOP-201-PCT01-NP wherein RKis H or CH3, RLis C1-6alkyl, and indicates the point of attachment to the antibody, or antigen-binding fragment thereof. In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein GAis selected from . 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 TOP-201-PCT01-NP . In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable saltthereof, wherein In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein E is (CH2)n1, wherein n1 is 0, 1, 2 or 3. In further embodiments E is a covalent bond. In further embodiments E is CH2. In further embodiments E is (CH2)2. In further embodiments E is (CH2)3. In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein X is (CH2)n2, wherein n2 is 0, 1, 2 or 3. In further embodiments X is a covalent bond. In further embodiments X is CH2. In further embodiments X is (CH2)2. In further embodiments X is (CH2)3. In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein Y is (CH2)n3, wherein n3 is 0, 1, 2, 3 or 4. In further embodiments Y is a covalent bond. In further embodiments Y is CH2. In further embodiments Y is (CH2)2. In further embodiments Y is (CH2)3. In further embodiments Y is (CH2)4. In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein Z is (CH2)n4, wherein n4 is 0, 1, 2, 3, 4 or 5. In further embodiments Z is a covalent bond. In further embodiments Z is (CH2)n4, wherein n4 is 1, 2, 3, 4 or 5. 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. In further embodiments Z is (CH2)5. In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein m is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17. In further embodiments m is an integer from 6 to 16. In further embodiments m is an integer from 7 to 15. In further embodiments m is an integer from 8 to 14. In further embodiments m is an integer from 9 to 13. In further embodiments m is an integer from 10 to 12. In further embodiments m is 11. In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein R1is C1-4 alkyl. In further embodiments R1is CH3. TOP-201-PCT01-NP In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein p is 1. In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein each JAis a group of Formula (IB) (IB). In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein each JAis a group of Formula (IB’) In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein each JAis a group of Formula (IB1) TOP-201-PCT01-NP (IB1). In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein each JAis a group of Formula (IB1’) In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein each Dcis . TOP-201-PCT01-NP In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein each Dcis . In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein each Dcis . In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein each Dcis . TOP-201-PCT01-NP In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein each Dcis . In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein each (GA–JA–DC) is . In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein each (GA–JA–DC) is TOP-201-PCT01-NP . In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein each (GA–JA–DC) is . In a further aspect there is provided a compound of Formula (II) GB–JB–DC(II) TOP-201-PCT01-NP or a salt thereof, wherein GBis a conjugation group for conjugation to an antibody or antigen- binding fragment thereof, JBis a group of Formula (IIA) wherein DC, E, Q, R1, X, Y, Z, m and p are as defined in any embodiment of a conjugate of Formula (I) disclosed herein, (GB) indicates the point of attachment to GB, and (DC) indicates the point of attachment to DC. In embodiments there is provided a compound of Formula (II), or a salt thereof, wherein GBis selected from wherein X1is CH or N, h is 0 or 1, Hal is Cl, Br or I, RKis H or CH3, and RLis C1-6 alkyl. In embodiments there is provided a compound of Formula (II), or a salt thereof, wherein GBis selected from TOP-201-PCT01-NP . In embodiments there is provided a compound of Formula (II), or a salt thereof, wherein GBis . In embodiments there is provided a compound of Formula (II), or a salt thereof, wherein GBis . In embodiments there is provided a compound of Formula (II) or a salt thereof, wherein JBis a group of Formula (IIB) In embodiments there is provided a compound of Formula (II) or a salt thereof, wherein JBis a group of Formula (IIB’) TOP-201-PCT01-NP In embodiments there is provided a compound of Formula (II) or a salt thereof, wherein JBis a group of Formula (IIB1) In embodiments there is provided a compound of Formula (II) or a salt thereof, wherein JBis a group of Formula (IIB1’) (IIB1’). In embodiments there is provided a compound of Formula (II) that is TOP-201-PCT01-NP (2S,3S,4S,5R,6S)-6-(2-((3-((S)-6-(((3S,3aR,6S,6aR)-6-(2,5,8,11,14,17,20,23,26,29,32,35- dodecaoxaoctatriacontan-38-amido)hexahydrofuro[3,2-b]furan-3-yl)amino)-2-(3-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)propanamido)-6-oxohexanamido)propanamido)methyl)-4-((((3-(((1S,9S)-9- ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H- benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)bicyclo[1.1.1]pentan-1- yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid, or a salt thereof. In embodiments there is provided a compound of Formula (II) that is (2S,3S,4S,5R,6S)-6-(2-((3-((S)-6-(((3S,3aR,6S,6aR)-6-(2,5,8,11,14,17,20,23,26,29,32,35- dodecaoxaoctatriacontan-38-amido)hexahydrofuro[3,2-b]furan-3-yl)amino)-2-(2-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)acetamido)-6-oxohexanamido)propanamido)methyl)-4-((((3-(((1S,9S)-9-ethyl- TOP-201-PCT01-NP 5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H- benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)bicyclo[1.1.1]pentan-1- yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid, or a salt thereof. In embodiments there is provided a compound of Formula (II) that is (2S,3S,4S,5R,6S)-6-(2-((S)-8-(4-(((3S,3aR,6S,6aR)-6-(2,5,8,11,14,17,20,23,26,29,32,35- dodecaoxaoctatriacontan-38-amido)hexahydrofuro[3,2-b]furan-3-yl)amino)-4-oxobutyl)-15-bromo- 3,7,10,14-tetraoxo-2,6,9,13-tetraazapentadecyl)-4-((((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4- methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2- b]quinolin-1-yl)carbamoyl)bicyclo[1.1.1]pentan-1-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5- trihydroxytetrahydro-2H-pyran-2-carboxylic acid, or a salt thereof. In a further aspect there is provided a compound of Formula (IIIA) or a salt thereof, wherein b is 0, 1, 2 or 3. In embodiments, b is 0, 1 or 2. In embodiments, b is 0 or 1. TOP-201-PCT01-NP In embodiments, there is provided a compound of Formula (IIIA), or a salt thereof, wherein the salt is a pharmaceutically acceptable salt. In a further aspect there is provided a compound of Formula (III) or a salt thereof, wherein b is 0, 1, 2 or 3. In embodiments, b is 0, 1 or 2. In embodiments, b is 0 or 1. In embodiments, there is provided a compound of Formula (III), or a salt thereof, wherein the salt is a pharmaceutically acceptable salt. In embodiments there is provided a compound of Formula (IIIA) or (III) that is amino-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1- yl)bicyclo[1.1.1]pentane-1-carboxamide, or a salt thereof. In further embodiments, the salt is a pharmaceutically acceptable salt. In embodiments there is provided a compound of Formula (IIIA) that is TOP-201-PCT01-NP amino-N-((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1- yl)bicyclo[1.1.1]pentane-1-carboxamide, or a salt thereof. In further embodiments, the salt is a pharmaceutically acceptable salt. In embodiments there is provided a compound of Formula (IIIA) or (III) that is amino-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2,2- difluorobicyclo[1.1.1]pentane-1-carboxamide, or a salt thereof. In further embodiments, the salt is a pharmaceutically acceptable salt. 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. 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 TOP-201-PCT01-NP 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. 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). A suitable pharmaceutically acceptable salt of a conjugate of Formula (I) or a compound of Formula (IIIA) or (III) is, for example, an acid addition salt. An acid addition salt of a conjugate of Formula (I) or a compound of Formula (IIIA) or (III) 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. 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 methoxide). A base addition salt may also be formed using a suitably basic organic amine (e.g., a choline or meglumine salt). A further suitable pharmaceutically acceptable salt of a conjugate of Formula (I) or a compound of Formula (IIIA) or (III) is, for example, a salt formed within a patient’s body after administration of a conjugate of Formula (I) to the patient. TOP-201-PCT01-NP 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. 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. In embodiments there is provided a pharmaceutical composition comprising a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, carrier, buffer or stabiliser. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous, or intravenous. 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. Examples of suitable excipients include one or more of water, saline, phosphate buffered saline, dextrose, glycerol and ethanol, as well as any combination thereof. In embodiments there is provided a pharmaceutical composition comprising a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, and one or more isotonic agents. In further embodiments the one or more isotonic agents are selected from a sugar, a polyalcohol and sodium chloride. In embodiments there is provided a pharmaceutical composition comprising a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, contained within one or more formulations selected from a capsule, a tablet, an aqueous suspension, a solution, a nasal aerosol, and a lyophilised powder which can be reconstituted to make a suspension or solution before use. In embodiments there is provided a pharmaceutical composition comprising a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, a buffer, a surfactant and / or a stabiliser agent. In TOP-201-PCT01-NP further embodiments the buffer is an acetate, phosphate or citrate buffer. In further embodiments the surfactant is polysorbate. In further embodiments the stabiliser agent is human albumin. The pharmaceutical composition comprising a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, can be administered to a patient by any appropriate systemic or local route of administration. For example, administration may be oral, buccal, sublingual, ophthalmic, intranasal, intratracheal, pulmonary, topical, transdermal, urogenital, rectal, subcutaneous, intravenous, intra-arterial, intraperitoneal, intramuscular, intracranial, intrathecal, epidural, intraventricular or intratumoural. The pharmaceutical composition comprising a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, can be formulated for administration by any appropriate means, for example by epidermal or transdermal patches, ointments, lotions, creams, or gels; by nebulisers, vaporisers, or inhalers; by injection or infusion; or in the form of capsules, tablets, liquid solutions or suspensions in water or non-aqueous media, drops, suppositories, enemas, sprays, or powders. The most suitable route for administration in any given case will depend on the physical and mental condition of the subject, the nature and severity of the disease, and the desired properties of the formulation. Pharmaceutical compositions comprising a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. A capsule may comprise a solid carrier such a gelatin. For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and / or other additives may be included, as required. In one aspect there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, for use in therapy. TOP-201-PCT01-NP In one aspect there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer. 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. 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 indications to the contrary. The terms "therapeutic" and "therapeutically" should be interpreted in a corresponding manner. 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. The term “treatment” is used synonymously with “therapy”. Similarly the term “treat” can be regarded as “applying therapy” where “therapy” is as defined herein. 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. 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. 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. Terms such as “treating” or “treatment” refer to both (1) therapeutic measures that cure, slow down, lessen symptoms of, and / or halt progression of a diagnosed pathologic condition or disorder and (2) prophylactic or preventative measures that prevent and / or slow the development of a targeted pathologic condition or disorder. Thus, those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented. In certain aspects, a patient is successfully "treated" for cancer according to the methods of the present disclosure if the patient shows, e.g., total, partial, or transient remission of a certain type of cancer. TOP-201-PCT01-NP 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. The term “patient” refers to any animal (e.g., a mammal), including, but not limited to humans, non- human 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. 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. 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. In embodiments there is provided a combination for use in the treatment of cancer comprising a conjugate of the Formula (I), or a pharmaceutically acceptable salt thereof and an additional anti- tumour agent. In embodiments there is provided a conjugate of the Formula (I), or a pharmaceutically acceptable salt thereof, in combination with an additional anti-tumour agent. 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. 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. TOP-201-PCT01-NP Conjugation 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 C1-6alkyl, and indicates the point of attachment to the antibody, or antigen-binding fragment thereof. GAGB

[0003] TOP-201-PCT01-NP Antibody or antigen-binding fragment thereof As used herein, the term “antibody” refers to an immunoglobulin molecule that specifically binds to, or is immunologically reactive with, a particular antigen. TOP-201-PCT01-NP 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. 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 CH1, 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”). 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. 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. 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-lazikani 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. TOP-201-PCT01-NP 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. 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 Fcγ receptors (FcγR) specific for the IgG class of antibody, namely FcγRI, FcγRII, and FcγRIII. 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 FcγR receptors have been reported to be located in the CH2 and CH3 domains. 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 IgG1, IgG2, IgG3, or IgG4 isotype. In embodiments the antibody or antigen-binding fragment thereof is based on an IgG1 isotype. The terms “Fc region”, “Fc part” and “Fc” are used interchangeably herein and refer to the portion of a native immunoglobulin that is formed by two Fc chains. Each “Fc chain” comprises a constant domain CH2 and a constant domain CH3. Each Fc chain may also comprise a hinge region. A native Fc region is homodimeric. In embodiments the Fc region may be heterodimeric because it may contain modifications to enforce Fc heterodimerisation. The Fc region contains the carbohydrate moiety and binding sites for complement and Fc receptors (including the FcRn receptor), and has no antigen binding activity. Fc can refer to this region in isolation, or this region in the context of an antibody, antibody fragment, or Fc fusion protein. Polymorphisms have been found in a number of Fc domain sites, including but not limited to EU positions 270, 272, 312, 315, 356, and 358, resulting in minor variations between the sequences described in the instant application and sequences known in the art. As a result, every naturally occurring IgG Fc region is referred to as a “wild type IgG Fc domain” or “WT IgG Fc domain” (i.e., any allele). Human IgG1, IgG2, IgG3, and IgG4 heavy chain sequences can be obtained in a variety of sequence databases, including the UniProt database TOP-201-PCT01-NP (www.uniprot.org) under accession numbers P01857 (IGHG1_HUMAN), P01859 (IGHG2_HUMAN), P01860 (IGHG3_HUMAN), and P01861 (IGHG4_HUMAN) respectively. 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. In embodiments the antibody of the disclosure is a full-length antibody described above. Alternatively, the antibody can be an antigen-binding fragment. The term “antigen-binding fragment” as used herein incudes any naturally-occurring or artificially-constructed configuration of an antigen-binding polypeptide comprising one, two or three light chain CDRs, and / or one, two or three heavy chain CDRs, wherein the polypeptide is capable of binding to the antigen. 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-Ig, an (scFv)2, a mAb2 or a DARPin. 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 CH1 and a VH). The heavy chain of a Fab fragment is not capable of forming a disulfide bond with another heavy chain. A “Fab' fragment” contains a single light chain and a single heavy chain but in addition to the CH1 and the VH, a “Fab' fragment” contains the region of the heavy chain between the CH1 and CH2 domains that is required for the formation of an inter-chain disulfide bond. Thus, two “Fab' fragments” can associate via the formation of a disulfide bond to form a F(ab')2 molecule. TOP-201-PCT01-NP 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. An “Fv fragment” contains only the variable regions of the heavy and light chain. It contains no constant regions. A “single-domain antibody” is an antibody fragment containing a single antibody domain unit (e.g., VH or VL). 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. 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. 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. 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. 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. TOP-201-PCT01-NP 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). Examples The specification will now be illustrated by the following non-limiting Examples. General Information Flash chromatography was performed using a BIOTAGE ISOLERA and fractions checked for purity using thin-layer chromatography (TLC). TLC was performed using MERCK KIESELGEL 60 F254 silica gel, with fluorescent indicator on aluminium plates. Visualisation of TLC was achieved with UV light. Extraction and chromatography solvents were bought and used without further purification from VWR U.K. All fine chemicals were purchased from SIGMA-ALDRICH unless otherwise stated. Pegylated reagents were obtained from QUANTA BIODESIGN US via STRATECH UK. LC / MS conditions Positive mode electrospray mass spectrometry was performed using a WATERS ACQUITY H-CLASS SQD2. 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%). 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 RP181.7µm 2.1 x 50 mm at 50 °C fitted with WATERS ACQUITY UPLC BEH SHIELD RP18 VANGUARD Pre-column, 130A, 1.7µm, 2.1 mm x 5 mm. 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 TOP-201-PCT01-NP 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 1.7µm 2.1 x 100mm fitted with WATERS ACQUITY UPLC CSH C18 VANGUARD Pre-column, 1.7µm, 2.1 mm x 5 mm. HPLC conditions Reverse-phase ultra-fast high-performance liquid chromatography (UFLC) was carried out on a SHIMADZU PROMINENCE machine using a PHENOMENEX GEMINI NX 5µ C18 column (at 50 °C) dimensions: 150 x 21.2 mm. Eluents used were solvent A (H2O with 0.1% formic acid) and solvent B (CH3CN with 0.1% formic acid). All UFLC experiments were performed with gradient conditions: Initial composition 13% B increased to 30% B over a 3 minutes period, then increased to 45% B over 8 minutes and again to 100% over 6 minutes before returning to 13% over 2 min and hold for 1 min. The total duration of the gradient run was 20.0 minutes. Flow rate was 20.0 mL / minute and detection was at 254 and 223 nm. NMR Method 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. Abbreviations UV Ultra violet LCMS Liquid chromatography mass spectrometry CSH Charged surface hybrid UPLC Ultra-performance liquid chromatography RP Reverse phase NMR Nuclear magnetic resonance DMF Dimethyl formamide DCM Dichloromethane TFA Trifluoro acetic acid DMSO Dimethyl sulfoxide ESI Electrospray ionisation DIPEA Di isopropyl ethylamine THF Tetra hydro furan HATU Hexafluorophosphate azabenzotriazole tetramethyl uronium TOP-201-PCT01-NP HOPO 2-hydroxypyridine 1-oxide RT Retention time ADC Antibody-drug conjugate UHPLC Ultra-high performance liquid chromatography mAb Monoclonal antibody SEC Size exclusion chromatography DAR Drug to antibody ratio RPMI Roswell Park Memorial Institute ND Not detectable IC50 Inhibitory concentration 50 % Fmoc fluorenylmethoxycarbonyl TBME tertiary-butyl methyl ether Intermediate 1 To a 250 mL round bottom flask was added 1,4:3,6-dianhydro-D-mannitol (641-74-7) (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 water (200 mL). The organic layer was separated, and compound was extracted in 200 mL dichloromethane. Combined organic layer was washed with HCl solution (1M-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 1 (14.90 g, 96 %).1H NMR (500 MHz, CDCl3) δ 7.90 – 7.76 (m, 4H), 7.45 – 7.33 (m, 4H), 4.94 – 4.80 (m, 2H), 4.55 – 4.44 (m, 2H), 3.94 (dd, J = 9.6, 6.7 Hz, 2H), 3.75 (dd, J = 9.6, 7.6 Hz, 2H), 2.48 (s, 6H). LCMS (ESI) m / z 455.21 (M + H)+. Intermediate 2 TOP-201-PCT01-NP To a 50 mL round bottom flask was added Intermediate 1 (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 2 (2.050 g, 79 %).1H NMR (500 MHz, CDCl3) δ 4.61 (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)+. Intermediate 3 To a 250 mL round bottom flask was added Intermediate 2 (1 g, 5.10 mmol) in dry THF (20 mL) under nitrogen gas. To the solution was added barium palladium(II) 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 H2gas. 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 3 (0.590 g, 80 %).1H NMR (500 MHz, DMSO) δ 4.23 (s, 2H), 3.68 (dd, J = 8.7, 4.5 Hz, 2H), 3.41 (dd, J = 8.7, 1.9 Hz, 2H), 3.23 (dd, J = 4.5, 1.9 Hz, 2H), 1.54 (s, 4H). LCMS (ESI) m / z 145.2 (M + H)+. Intermediate 4 TOP-201-PCT01-NP To a 250 mL round bottom flask was added Intermediate 3 (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 methyl-PEG-11 NHS ester (756525-94-7) (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 N-((3S,3aR,6S,6aR)-6- aminohexahydrofuro[3,2-b]furan-3-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan- To a 250 mL round bottom flask was added Fmoc-Aad(O-t-Bu)-OH (159751-47-0) (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-1-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 (2 x 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 5 (5.10 g, 93 %).1H NMR (500 MHz, CDCl3) δ 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)+. Intermediate 6 TOP-201-PCT01-NP To a 100 mL round bottom flask was added Intermediate 5 (5 g, 10.43 mmol) in dry THF (20 mL) under nitrogen gas. To the solution was added HCl (13.03 mL, 52.13 mmol), 4M 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 6 (4.20 g, 95 %).1H NMR (500 MHz, CDCl3) δ 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)-. Intermediate 7 To a 100 mL round bottom flask was added Intermediate 6 (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 21 °C for 15 min then Intermediate 4 (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- (2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-amido)hexahydrofuro[3,2-b]furan-3- yl)amino)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-oxohexanoate Intermediate 7 (3.40 g, 87 %)1H NMR (500 MHz, MeOD) δ 7.86 (dd, J = 7.6, 1.2 Hz, 2H), 7.74 (t, J = 7.8 Hz, 2H), 7.46 (td, J = 7.5, 1.4 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)+. Intermediate 8 TOP-201-PCT01-NP To a 50 mL round bottom flask was added Intermediate 7 (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(PPh3)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-(2,5,8,11,14,17,20,23,26,29,32,35- dodecaoxaoctatriacontan-38-amido)hexahydrofuro[3,2-b]furan-3-yl)amino)-2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-6-oxohexanoic acid Intermediate 8 (3.50 g, 84 %).1H NMR (500 MHz, DMSO) δ 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)+. Intermediate 9 To Intermediate 8 (0.410 g, 0.38 mmol) in DCM (8 mL) was added tert-butyl 3-aminopropanoate hydrochloride (0.138 g, 0.76 mmol), HATU (0.289 g, 0.76 mmol) and DIPEA (0.265 mL, 1.52 mmol). DMF (2 mL) was added and the reaction mixture stirred at 21 °C for 2 h. The reaction mixture was diluted with EtOAc. The organics were washed with sat. NaHCO3, 1 M citric acid, and brine. The organics were dried and concentrated under reduced pressure to give 600 mg of crude tert-butyl 3- ((S)-6-(((3S,3aR,6S,6aR)-6-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38- amido)hexahydrofuro[3,2-b]furan-3-yl)amino)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6- oxohexanamido)propanoate Intermediate 9 which was used without further purification. LCMS (3 min): 1.78 min; 1208.6 [M+H]+. Intermediate 10 TOP-201-PCT01-NP Intermediate 9 (400 mg, 0.33 mmol) was added to HCl in 1,4-dioxane 4N (5 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 2 hours. The solvent was removed under reduced pressure afford Intermediate 10 (370 mg, 97 %) as a pale yellow solid. LCMS (ESI) m / z [M+Na]+ 1174.2 Intermediate 11 To a solution of Intermediate 10 (1.3 g, 1.13 mmol) in THF (20 mL) was added HATU (0.644 g, 1.69 mmol) at 21 °C. DIPEA (0.592 mL, 3.39 mmol) and (2S,3R,4S,5S,6S)-2-(2-(aminomethyl)-4- (hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (0.636 g, 1.35 mmol) was added after the reaction had been stirred for 5 minutes. The resulting mixture was stirred at 21 °C for 2 hours. The reaction mixture was evaporated to dryness and redissolved in EtOAc (20 mL), and washed sequentially with water (20 mL x 3). The organic layer was dried over Na2SO4, filtered and evaporated to afford crude product. The crude product was triturated with TBME. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 100 % MeCN in water. Pure fractions were evaporated to dryness to afford Intermediate 11 (1.4 g, 77 %) as a yellow solid. LCMS (ESI) m / z [M+H]+ 1602.9. Intermediate 12 TOP-201-PCT01-NP Intermediate 11 (1.4 g, 0.87 mmol) and bis(4-nitrophenyl) carbonate (0.399 g, 1.31 mmol) in DMF (20 mL) was added DIEA (0.458 mL, 2.62 mmol) at 21 °C. The resulting mixture was stirred at 21 °C for 6 hours. The reaction mixture was evaporated to dryness and redissolved in EtOAc (20 mL), and washed sequentially with water (20 mL x 3). The organic layer was dried over Na2SO4, filtered and evaporated to afford crude product. The crude product was triturated with TBME. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 100% MeCN in water. Pure fractions were evaporated to dryness to afford Intermediate 12 (0.761 g, 49.3 %) as a yellow solid. LCMS (ESI) m / z [M+Na]+ 1789.8. Intermediate 13 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[1.1.1]pentane-1-carboxylic acid (47.0 mg, 0.21 mmol), 2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethylisouronium hexafluorophosphate(V) (107 mg, 0.28 mmol) and N-ethyl-N-isopropylpropan-2-amine (0.098 mL, 0.56 mmol). The resulting solution was stirred at 21 °C for 2 hours. The crude product was purified by flash silica chromatography, elution gradient 0 to 10% MeOH in DCM. Pure fractions were evaporated to dryness to afford Intermediate 13 (161 mg, 133 %) as a pale yellow residue. LCMS (ESI) m / z [M+H]+ 645.7. TOP-201-PCT01-NP P-1 2,2,2-trifluoroacetic acid (0.6 ml, 7.78 mmol) was added to Intermediate 13 (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-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl- 10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin- 1-yl)bicyclo[1.1.1]pentane-1-carboxamide (P-1)(107 mg, 77 %) as a yellow solid.1H NMR (400 MHz, DMSO) δ 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 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. Intermediate 14 N-ethyl-N-isopropylpropan-2-amine (0.079 mL, 0.46 mmol) was added to Intermediate 12 (268 mg, 0.15 mmol), P-1 (100 mg, 0.15 mmol) and 2-hydroxypyridine 1-oxide (18.56 mg, 0.17 mmol) in DMF TOP-201-PCT01-NP (1 mL) and DCM (1 mL). The resulting solution was stirred at 21 °C for 1 hour. The reaction was concentrated to afford crude product. The crude product was purified by flash C18 silica chromatography, elution gradient 5 to 60% MeCN+0.1%FA in water+0.1%FA. Pure fractions were evaporated to dryness to afford Intermediate 14 (247 mg, 74.9 %) as a pale yellow solid. LCMS (ESI) m / z [M+2H]2+ 1087.6. Intermediate 15 A solution of potassium carbonate (63.6 mg, 0.46 mmol) in water (2 mL) was added to a stirred solution of Intermediate 14 (100 mg, 0.05 mmol) in MeOH (2 mL) and THF (2 mL). The resulting solution was stirred at 21 °C for 24 hours. The reaction mixture was neutralised with citric acid and then purified by flash C18 silica chromatography, elution gradient 5 to 40% MeCN+0.1%FA in water+0.1%FA. Product-containing fractions were evaporated to dryness to afford Intermediate 15 (77 mg, 93 %) with ~25% epimerisation as a pale yellow solid. LCMS (ESI) m / z [M+H]+ 1811.7. LP-1 TOP-201-PCT01-NP 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate (16.98 mg, 0.06 mmol) was added to Intermediate 15 (77 mg, 0.04 mmol) and N-ethyl-N-isopropylpropan-2-amine (0.022 mL, 0.13 mmol) in DCM (1 mL) and DMF (1 mL). The resulting solution was stirred at 21 °C for 2 hours. The reaction mixture was acidified with formic acid and then concentrated to afford crude product. The crude product was purified by preparative HPLC (ACCQprep), elution gradient 30 to 45% MeCN+0.1%FA in water+0.1%FA. Pure fractions were evaporated to dryness to afford (2S,3S,4S,5R,6S)-6-(2-((3-((S)-6-(((3S,3aR,6S,6aR)-6-(2,5,8,11,14,17,20,23,26,29,32,35- dodecaoxaoctatriacontan-38-amido)hexahydrofuro[3,2-b]furan-3-yl)amino)-2-(3-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)propanamido)-6-oxohexanamido)propanamido)methyl)-4-((((3-(((1S,9S)-9- ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H- benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)bicyclo[1.1.1]pentan-1- yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (LP-1) (29.0 mg, 34.8 %) as a white solid.1H NMR (400 MHz, DMSO) δ 8.48 (d, J = 8.8 Hz, 1H), 8.33 – 8.21 (m, 1H), 8.13 (d, J = 6.9 Hz, 1H), 8.09 (t, J = 7.9 Hz, 2H), 8.01 – 7.95 (m, 1H), 7.94 – 7.87 (m, 1H), 7.75 (d, J = 10.9 Hz, 1H), 7.30 (s, 1H), 7.24 – 7.16 (m, 2H), 7.06 (d, J = 8.4 Hz, 1H), 6.98 (s, 2H), 6.50 (s, 1H), 5.59 – 5.49 (m, 2H), 5.42 (s, 2H), 5.24 (d, J = 4.0 Hz, 1H), 5.14 (d, J = 18.9 Hz, 1H), 5.01 (d, J = 18.7 Hz, 1H), 4.96 – 4.85 (m, 3H), 4.37 (s, 2H), 4.34 – 4.26 (m, 2H), 4.15 – 4.05 (m, 3H), 3.90 – 3.79 (m, 3H), 3.63 – 3.55 (m, 7H), 3.53 – 3.49 (m, 38H), 3.49 – 3.46 (m, 3H), 3.45 – 3.40 (m, 2H), 3.24 (s, 3H), 3.23 – 3.02 (m, 2H), 2.44 – 2.35 (m, 5H), 2.35 – 2.28 (m, 4H), 2.23 (s, 6H), 2.19 – 2.07 (m, 2H), 2.06 – 1.99 (m, 2H), 1.94 – 1.79 (m, 2H), 1.61 – 1.32 (m, 5H), 0.88 (t, J = 7.3 Hz, 3H); LCMS (ESI) m / z [M+H]+ 1962.8. LP-2 (Reference) Reference linker-payload LP-2 may be prepared by an analogous synthetic procedure to that disclosed above. Intermediate 16 TOP-201-PCT01-NP 3-((tert-butoxycarbonyl)amino)-2,2-difluorobicyclo[1.1.1]pentane-1-carboxylic acid (91 mg, 0.34 mmol) was added to Exatecan mesylate (CAS: 169869-90-3) (150 mg, 0.34 mmol), HATU (196 mg, 0.52 mmol) and DIEA (0.180 mL, 1.03 mmol) in DMA (5 mL) under nitrogen. The resulting mixture was stirred at 15 °C for 2 hours. The reaction mixture was purified by flash C18-flash chromatography, elution gradient 0 to 100% MeCN in water (0.1% FA). Pure fractions were evaporated to dryness to afford intermediate 16 tert-butyl (3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4- methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2- b]quinolin-1-yl)carbamoyl)-2,2-difluorobicyclo[1.1.1]pentan-1-yl)carbamate (160 mg, 68.2 %) as a yellow solid.1H NMR (400 MHz, DMSO) δ 0.88 (t, J = 7.3 Hz, 3H), 1.39 (s, 10H), 1.86 (h, J = 7.0 Hz, 2H), 2.00 – 2.20 (m, 3H), 2.43 (d, J = 7.3 Hz, 5H), 3.18 (d, J = 6.5 Hz, 2H), 5.03 – 5.24 (m, 2H), 5.43 (s, 2H), 5.58 (d, J = 7.3 Hz, 1H), 6.54 (s, 1H), 7.32 (s, 1H), 7.81 (d, J = 10.9 Hz, 1H), 8.10 (d, J = 33.6 Hz, 1H), 8.74 (d, J = 8.7 Hz, 1H). LCMS (ESI) m / z (M+H)+ 681 P-2 4 M HCl in dioxane (3 mL, 12.00 mmol) was added to intermediate 16 (100 mg, 0.15 mmol) in dioxane (3 mL) under nitrogen. The resulting mixture was stirred at 15 °C for 1 hour. The solvent was TOP-201-PCT01-NP removed under reduced pressure. The crude product was purified by preparative HPLC (Column: Kinetex EVO C18 Column, 21.2x250mm, 5 µm; Mobile Phase A: Water(0.05% TFA), Mobile Phase B: ACN; Flow rate: 25 mL / min; Gradient: 18%B to 31% B in 10 min; Wave Length: 254nm / 220nm ; RT1(min): 8.37).The factions were removed by N2 flow, instead of evaporating with a rotary evaporator.Fractions containing the desired compound were freeze dried directly to dryness to afford P-23-amino-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15- hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2,2- difluorobicyclo[1.1.1]pentane-1-carboxamide (63.0 mg, 63.7 %) as a yellow solid.1H NMR (400 MHz, DMSO) δ 0.81 – 0.89 (m, 3H), 1.85 – 1.91 (m, 2H), 2.20 – 2.29 (m, 4H), 2.39 – 2.49 (m, 5H), 3.18 (d, J = 6.4 Hz, 2H), 5.15 (d, J = 8.6 Hz, 2H), 5.43 (s, 2H), 5.60 – 5.69 (m, 1H), 6.55 (s, 1H), 7.33 (s, 1H), 7.82 (d, J = 10.9 Hz, 1H), 8.47 (s, 3H), 8.82 – 8.89 (m, 1H), one exchangeable proton not seen. LC-MS (ESI) m / z (M+H)+ 581.4 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[1.1.1]pentane-1-carboxylic acid (0.470 g, 2.07 mmol), 2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethylisouronium hexafluorophosphate(V) (107 mg, 0.28 mmol) and N-ethyl-N-isopropylpropan-2-amine (0.098 mL, 0.56 mmol). The resulting solution was stirred at 21 °C for 2 hours. The crude product was purified by flash silica chromatography, elution gradient 0 to 10% MeOH in DCM. Pure fractions were evaporated to dryness to afford intermediate 17 tert-butyl (3-(((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13- dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1- yl)carbamoyl)bicyclo[1.1.1]pentan-1-yl)carbamate (0.679 g, 56.0 %) as a pale yellow residue. The sample was found to be ca.35% epimerised - taken through as a mixture. LCMS (ESI) m / z 645 (M+H)+ (S,S) diastereomer elutes first, (R,S) diastereomer elutes second on reverse phase (C18) HPLC. TOP-201-PCT01-NP 2,2,2-trifluoroacetic acid (0.6 ml, 7.78 mmol) was added to intermediate 17 (639 mg, 0.99 mmol) in DCM (9.4 mL). The resulting solution was stirred at room temperature for 45 minutes. The reaction was concentrated to afford crude product. The crude product was purified by reverse phase HPLC (20-40 % MeCN:Water [0.05% TFA]). Pure fractions were evaporated to dryness to afford P-13- amino-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro- 1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)bicyclo[1.1.1]pentane-1- carboxamide (100 mg, 15.34 %) as a yellow solid and P-33-amino-N-((1R,9S)-9-ethyl-5-fluoro-9- hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H- benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)bicyclo[1.1.1]pentane-1-carboxamide (50.0 mg, 7.67 %) as a pale yellow solid . R enantiomer 1H NMR (400 MHz, DMSO) δ 8.67 (d, J = 8.8 Hz, 1H), 8.61 (s, 4H), 7.80 (d, J = 10.9 Hz, 1H), 7.33 (s, 1H), 6.53 (s, 1H), 5.61 – 5.51 (m, 1H), 5.46 – 5.41 (m, 2H), 5.17 (d, J = 18.8 Hz, 1H), 5.06 (d, J = 18.6 Hz, 1H), 3.28 – 3.09 (m, 3H), 2.35 (d, J = 37.9 Hz, 10H), 2.22 – 2.05 (m, 1H), 1.98 – 1.79 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H). LC-MS (ESI) m / z [M+H]+ 545.8 Intermediate 18 DIC (6.03 mL, 38.68 mmol) was added to 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetic acid (3 g, 19.34 mmol) and 2,3,5,6-tetrafluorophenol (3.85 g, 23.21 mmol) in MeCN (20 mL) under nitrogen. The resulting mixture was stirred at 15 °C for 2 hours. The reaction mixture was filtered through celite. The solvent was removed under reduced pressure. The crude product intermediate 18 2,3,5,6-tetrafluorophenyl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (4.00 g, 68.2 %) presented TOP-201-PCT01-NP as a yellow solid and was used in the next step directly without further purification.1H NMR (400 MHz, DMSO) δ 4.14 (s, 2H), 7.14 (s, 2H), 8.00 - 8.11 (m, 1H). LC-MS (ESI) m / z [M+H]+ 304 LP-3 DIEA (0.028 mL, 0.16 mmol) was added to intermediate 15 (290 mg, 0.16 mmol) and intermediate 18 (48.5 mg, 0.16 mmol) in MeCN (5 mL) under nitrogen. The resulting mixture was stirred at 15 °C for 2 hours. Following this time, the solvent was removed under reduced pressure. The residue was purified by flash C18-flash chromatography, elution gradient 10 to 60% MeCN in water (0.1% FA). Pure fractions were evaporated to dryness to afford crude product. The crude product was purified by preparative HPLC (Column: Kinetex EVO C18 Column, 21.2x250mm, 5µm; Mobile Phase A: Water(0.1% FA), Mobile Phase B: ACN; Flow rate: 25 mL / min; Gradient: 31% B to 34% B in 10 min; Wave Length: 254nm / 220nm; RT1(min): 9.13). Fractions containing the desired compound were freeze dried directly to dryness to afford LP-3 (2S,3S,4S,5R,6S)-6-(2-((3-((S)-6-(((3S,3aR,6S,6aR)-6- (2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-amido)hexahydrofuro[3,2-b]furan-3- yl)amino)-2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-6- oxohexanamido)propanamido)methyl)-4-((((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13- dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1- yl)carbamoyl)bicyclo[1.1.1]pentan-1-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro- 2H-pyran-2-carboxylic acid (22.00 mg, 7.05 %) as a yellow solid.1H NMR (400 MHz, DMSO) δ 0.81 – 0.90 (m, 3H), 1.23 (s, 2H), 1.44 – 1.56 (m, 4H), 1.80 – 1.94 (m, 2H), 2.21 (s, 4H), 2.23 – 2.29 (m, 7H), 2.30 – 2.39 (m, 4H), 2.39 (d, J = 1.8 Hz, 4H), 3.12 (s, 1H), 3.15 – 3.20 (m, 5H), 3.23 (s, 3H), 3.24 – 3.26 (m, 2H), 3.26 – 3.29 (m, 3H), 3.30 – 3.31 (m, 4H), 3.38 – 3.52 (m, 38H), 3.53 – 3.61 (m, 5H), 3.81 (d, J = 5.1, 9.4 Hz, 2H), 4.07 – 4.36 (m, 6H), 4.53 – 4.58 (m, 2H), 4.85 – 4.88 (m, 1H), 4.90 (s, 2H), 5.12 (d, J TOP-201-PCT01-NP = 7.9 Hz, 2H), 5.42 (s, 3H), 5.52 (s, 1H), 6.53 (d, J = 2.5 Hz, 1H), 7.02 – 7.11 (m, 2H), 7.07 (s, 1H), 7.22 (d, J = 8.8 Hz, 2H), 7.30 (s, 1H), 7.79 – 8.03 (m, 2H), 8.12 – 8.19 (m, 2H), 8.51 (d, J = 9.0 Hz, 2H). LCMS (ESI) m / z (M+2H)2+ 947.7 LP-4 2,5-dioxopyrrolidin-1-yl 3-(2-bromoacetamido)propanoate (50.9 mg, 0.17 mmol) was added to intermediate 15 (300 mg, 0.17 mmol) in DMSO (2 mL) under nitrogen. The resulting mixture was stirred at 15 °C for 2 hours. The reaction mixture was purified by preparative HPLC (Column: Xbridge Prep Shield RP18 OBD, 19x250mm, 5 µm; Mobile Phase A: Water(0.1% FA), Mobile Phase B: ACN; Flow rate: 25 mL / min; Gradient (B%): 32% B to 24% B in 10 min; Wave Length: 254nm / 220nm; RT1(min): 8.28 / 9.88). Fractions containing the desired compound were freeze dried directly to dryness to afford (2S,3S,4S,5R,6S)-6-(2-((S)-8-(4-(((3S,3aR,6S,6aR)-6- (2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-amido)hexahydrofuro[3,2-b]furan-3- yl)amino)-4-oxobutyl)-15-bromo-3,7,10,14-tetraoxo-2,6,9,13-tetraazapentadecyl)-4-((((3-(((1S,9S)-9- ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H- benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)bicyclo[1.1.1]pentan-1- yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid LP-4 (57.0 mg, 17.18 %) as a yellow solid.1H NMR (400 MHz, DMSO) δ 0.80 – 0.89 (m, 3H), 1.43 – 1.58 (s, 3H), 1.86 (d, J = 7.2 Hz, 2H), 2.05 (s, 2H), 2.22 (s, 6H), 2.31 (d, J = 6.9 Hz, 6H), 2.31 – 2.39 (m, 3H),2.40 (s, 3H), 3.14 – 3.24 (m, 8H), 3.39 – 3.50 (m, 55H), 3.59 (d, J = 8.1 Hz, 3H), 3.83 – 4.08 (m, 5H), 4.18 – 4.29 (m, 3H), 4.90 – 4.99 (m, 3H), 5.10 (s, 2H), 5.15 (s, 1H), 5.27 (s, 1H), 5.37 (s, 2H), 5.43 – 5.54 (m, 2H), 6.52 (s, 1H), 7.06 (d, J = 8.5 Hz, 1H), 7.19 (d, J = 5.8 Hz, 1H), 7.21 (s, 1H), 7.31 (s, 1H), 7.80 (d, J = 10.8 Hz, 1H), 7.94 – 7.99 (m, 2H), 8.03 (d, J = 8.1 Hz, 1H), 8.12 (dd, J = 6.8, 9.2 Hz, 1H), 8.21 (s, 1H), 8.28 (s, 1H), 8.51 (d, J = 8.9 Hz, 1H); LCMS (ESI) m / z (M79Br+2H)2+ 1002.1 Payload Potency Part 1 TOP-201-PCT01-NP 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 re- suspended 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 µL / well) were seeded into black 96 well flat bottom plates and incubated overnight before use. A 10mM payload stock solution was made from the material in DMSO. Then a 2mM solution was prepared by adding 20 µL of the stock solution to 80 µL of DMSO in a glass vial. A set of 810-fold dilutions of the previous payload (warhead) solution were made in a glass vial by serial transfer of 10 µL onto 90 µL 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 µl / well) into 4 rows, either A-D or E-H of the 96-well plate, containing 50 µl cell suspension seeded the previous day. Control wells (column 12) received 50 µl cell culture medium. The 96-well plate containing cells and payloads (warheads) are incubated at 37 °C in a CO2-gassed incubator for 6 days. At the end of the incubation period, plates were equilibrated to room temperature for 10min before CellTiter-Glo (Promega) was dispensed (100 µl 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%). Table 1 IC50NCI-N87 (nM) IC50MDAMB361 (nM) IC50MDAMB468 (nM) P-1 30.8 25.6 8.2 Exatecan (reference) 2.9 1.9 0.43 30 TOP-201-PCT01-NP Table 1 and Figure 1a, 1b and 1c show the cytotoxicity data for payload (warhead) P-1 (N=3) and Exatecan (N=3) (Reference) in NCI-N87, MDAMB361 and MDAMB468 cell lines. The IC50 values in each figure were determined by fitting data to a sigmoidal dose-response curve using GRAPHPAD PRISM software v9 (GraphPad, San Diego, CA). Antibody-drug conjugation ADC-1: Herceptin-WT-LP-1 (DAR 8) LP-1 was added as a DMSO solution (14 molar equivalent / antibody, 2.33 µmole, in 0.505mL DMSO) to 4.55 mL of the Trastuzumab antibody solution in PBS, 1 mM EDTA, pH 7.4 (25.0 mg, 167.0 nanomoles) for a 10% (v / v) final DMSO concentration. The solution left to react at room temperature for 1 hours with gentle shaking. Then the conjugation was quenched by addition of N- acetyl cysteine (11.7 micromoles, 116.67 ^L at 100 mM), then purified in PBS using AKTA PrepSEC and formulated in 20 mM Histidine / Histidine HCL, 240 mM sucrose pH 6.0 by spin filtration using a 15 mL AMICON ULTRACELL 30 kDa MWCO spin filter, sterile-filtered and analysed. UHPLC analysis on a SHIMADZU PROMINENCE system using a THERMO SCIENTIFIC MAbPac 50 mm x 2.1 mm column eluting with a gradient of water and acetonitrile on a reduced sample of ADC at 214 nm and 360 nm shows a mixture of light chain conjugated to 1 molecule of LP-1, and heavy chain conjugated to 3.0 molecules of LP-1, consistent with a drug-per-antibody ratio (DAR) of 7.99 molecules of LP-1 per antibody. UHPLC analysis on a SHIMADZU PROMINENCE system using a TOSOH BIOSCIENCE TSKgel SuperSW mAb HTP 4 µm 4.6 x 150 mm column (with a 4 µm 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 99.79%. 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 LP-1, and heavy chain conjugated to 3.0 molecules of LP-1, consistent with a drug-per-antibody ratio (DAR) of 8.0 molecules of LP-1 per antibody. 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 TOP-201-PCT01-NP sample of ADC at 214 nm shows singly conjugated to LP-1 , consistent with a drug-per-antibody ratio (DAR) of 7.96 molecules of LP-1 per antibody. ADC-2: Herceptin-WT-LP-2 (DAR 8) (Reference) Reference ADC-2 may be prepared by an analogous method to that described for ADC-1. ADC-3 synthesis: Herceptin-WT-LP-3 (DAR 8) LP-3 was added as a DMSO solution (17 molar equivalent / antibody, 2.83 µmole, in 0.116 mL DMSO) to 5.5 mL of the Herceptin-wt antibody solution in PBS, 1 mM EDTA, pH 7.4 (25.0 mg, 167.0 nanomoles) for a 10% (v / v) final DMSO concentration. The solution left to react at room temperature for 1 hours with gentle shaking. Then the conjugation was quenched by addition of N-acetyl cysteine (14.2 micromoles, 141.65 µL at 100 mM), then purified in PBS pH 7.4 by AKTAPrep-SEC followed by formulation in 20 mM His / His HCl, 240 mM sucrose pH 6.0 using 15 mL Amicon Ultracell 30 kDa MWCO spin filter, sterile-filtered and analysed. ADC-4 synthesis: Herceptin-WT-LP-4 (DAR 8) LP-4 was added as a DMSO solution (25 molar equivalent / antibody, 4.13 µmole, in 0.206 mL DMSO) to 5.5 mL of the Herceptin-wt antibody solution in PBS, 1 mM EDTA, pH 7.4 (25.0 mg, 167.0 nanomoles) for a 10% (v / v) final DMSO concentration. The solution left to react at room temperature for 24 hours with gentle shaking. Then the conjugation was purified in PBS pH 7.4 by AKTAPrep-SEC followed by formulation in 20 mM His / His HCl, 240 mM sucrose pH 6.0 using 15 mL Amicon Ultracell 30 kDa MWCO spin filter, sterile-filtered and analysed. ADC-3 / ADC-4 analysis UHPLC analysis on a Shimadzu Prominence system using a Thermo Scientific MAbPac 50 mm x 2.1 mm column eluting with a gradient of water and acetonitrile on a reduced sample of ADC at 214 nm and 330 nm shows; for ADC-3 a mixture of light chain conjugated to 1 molecule of LP-3, and heavy chain conjugated to 3.0 molecules of LP-3, consistent with a drug-per-antibody ratio (DAR) of 7.69 molecules of LP-3 per antibody; for ADC-4 a mixture of light chain conjugated to 1 molecule of LP-4, and heavy chain conjugated to 3.0 molecules of LP-4, consistent with a drug-per-antibody ratio (DAR) of 8.01 molecules of LP-4 per antibody. UHPLC analysis on a Shimadzu Prominence system using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 µm 4.6 x 150 mm column (with a 4 µm 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 TOP-201-PCT01-NP chloride and 10% isopropanol (v / v) on a sample of ADC at 280 nm shows; for ADC-3, a monomer purity of 99.27%; for ADC-4 a monomer purity of 99.79%. 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; for ADC-3, shows a mixture of light chain conjugated to 1 molecule of LP-3, and heavy chain conjugated to 3.0 molecules of LP-3, consistent with a drug-per-antibody ratio (DAR) of 8.0 molecules of LP-3 per antibody; for ADC-4, shows a mixture of light chain conjugated to 1 molecule of LP-4, and heavy chain conjugated to 3.0 molecules of LP-4, consistent with a drug-per-antibody ratio (DAR) of 8.0 molecules of LP-4 per antibody. 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; ADC-3 as being Herceptin solely conjugated to LP-3 , consistent with a drug- per-antibody ratio (DAR) of 8.0 molecules of LP-3 per antibody; ADC-4 as being Herceptin solely conjugated to LP-4 , consistent with a drug-per-antibody ratio (DAR) of 8 molecules of LP-4 per antibody. ADC Cytotoxicity Data Part 1 Media from 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 (1x) 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 10mL culture medium. The cell suspension was well pipetted to break possible aggregates and 10µL solution were mixed with 10µL trypan blue cell-stained cells. 20µL 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. TOP-201-PCT01-NP A stock solution (550µL) 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 µl onto 440 µl of cell culture medium. ADC dilution was dispensed (50 µl / well) into 2 replicate wells of the 96-well plate, containing 50 µl cell suspension seeded the previous day. Control wells received 50 µl cell culture medium. The 96-well plate containing cells and ADCs was incubated at 37 °C in a CO2-gassed incubator for 6 days. At the end of the incubation period, plates were equilibrated to room temperature for 10min before CellTiter-Glo (Promega) was dispensed (100 µl 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). Table 2 IC50 MDAMB468 IC50 SKOV3 WT IC50 SKOV3 GUSB KO 3D (µg / mL) 3D (µg / mL) (µg / mL) ADC-1 ND 0.06 ND ADC-2 (Reference) ND 0.01 ND Table 2 and Figure 2a, 2b and 2c show cytotoxicity data for ADC-1 (N=3) and ADC-2 (N=3) in HER2- MDAMB468, Her2+++ / GUSB+++ SKOV3 WT and HER2+++ / GUSB- SKOV3 GUSB KO cell lines. The IC50 values in each figure were determined by fitting data to a sigmoidal dose-response curve using GraphPad Prism software v9 (GraphPad, San Diego, CA). Payload and ADC Cytotoxicity Data Part 2 Medium from sub-confluent (80-90% confluency) NCI-N87, MDAMB468, SKOV3 and SKOV3 GUSB KO in a T175 flask was aspirated and the flask rinsed with PBS (about 20 ml) and emptied. TrypLE (4 ml) was added, the flask returned to the 37 °C gassed incubator for up to about 5 minutes, then rapped sharply to dislodge and dissociate cells from the plastic. The cell suspension was transferred to a sterile 50 ml screw-top centrifuge tube, diluted with growth medium to a final volume of 15 ml, then centrifuged (400g for 5 min). The supernatant was aspirated, and the pellet re-suspended in 10ml culture medium. Repeated pipetting may be necessary to produce monodisperse cell suspensions. The cell concentration and viability are measured of trypan blue cell-stained cells, using the LUNA II. TOP-201-PCT01-NP A 10mM payload stock solution was made from the material in DMSO. Then a 2mM solution was prepared by adding 20 µL of the stock solution to 80 µL of DMSO in a glass vial. A set of 910-fold dilutions of the previous warhead solution were made in a glass vials by serial transfer of 10 µL onto 90 µL 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, add media to all 96 wells, according to the number of plates to drug, and dilute the warheads in 1:50. The warhead dilution in media was dispensed (10 µl / well) into 4 rows, either A-D or E-H of the 96-well plate, containing 30 µl cell suspension seeded the previous day. Control wells (column 12) received 10 µl cell culture medium. For the ADCs, a stock solution (300µL) 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 30 µl onto 270 µl of cell culture medium. ADC dilution was dispensed (10 µl / well) into 4 replicate wells of the 384-well plate, containing 30 µl cell suspension seeded the previous day. Control wells received 10 µl cell culture medium. The 384-well plate containing cells and ADCs / payloads was incubated at 37 °C in a CO2-gassed incubator for 6 days. At the end of the incubation period, plates were equilibrated to room temperature for 10min before CellTiter-Glo (Promega) was dispensed (40 µl per well) into each well. IC50 was determined from the dose-response data using GraphPad Prism using the non-linear curve fit algorithm: sigmoidal dose response, log(inhibitor) vs. Response – Variable slope (four parameters), constrain at the bottom = 0. Cell growth medium for NCI-N87 and MDAMB468 cells was RPMI supplemented with 10% Fetal Bovine Serum while for SKOV3 WT and SKOV3 GUSB KO was McCoy`s 5A supplemented with 10% Fetal Bovine Serum. Table 3 IC50 NCI-N87(nM) IC50 MDAMB468 (nM) Exatecan (N=5) 3.2 0.6 (reference) P-1 (N=5) 30.0 5.1 P-2 (N=2) 4.6 5.1 P-3 (N=2) 31.1 6.5 Table 3 and Figures 3A and 3B show the cytotoxicity data for Exatecan (N=5), P-1 (N=5) , P-2 (N=2) and P-3 (N=2) in Her2+++ NCI-N87 and HER2- MDAMB468 cell lines. The IC50values in each figure were TOP-201-PCT01-NP determined by fitting data to a sigmoidal dose-response curve using GraphPad Prism software v9 (GraphPad, San Diego, CA). Table 4 IC50 NCI-N87 IC50 MDAMB468 IC50 SKOV33D IC50 SKOV3 KO (µg / mL) (µg / mL) (µg / mL) (µg / mL) ADC-1 0.03 ND 0.01 ND ADC-2 (Reference) 0.04 ND 0.01 ND ADC-3 0.07 ND 0.05 ND ADC-4 0.05 ND 0.03 ND Table 4 and Figures 4A, 4B, 4C and 4D show the cytotoxicity data for ADC-1 (N=2), ADC-2 (N=2), ADC- 3 (N=2) and ADC-4 (N=2) in Her2+++ NCI-N87, HER2+++ / GUSB+++ SKOV3 WT, HER2+++ / GUSB- SKOV3 GUSB KO and HER2- MDAMB468 cell lines. The IC50values in each figure were determined by fitting data to a sigmoidal dose-response curve using GraphPad Prism software v9 (GraphPad, San Diego, CA). Passive Permeability The bidirectional permeability of Exatecan (reference) and P-1 were evaluated using MDCKII (MDR-1 knockout) cell monolayers. Cells were seeded on Transwell inserts and cultured for 4-8 days. Monolayer integrity was assessed by measuring transepithelial electrical resistance (TEER). Compounds were tested at 1 μM, with metoprolol, digoxin, acyclovir and enalapril as control compounds. Compounds were incubated with the cell monolayers for 2 hours in the apical-to-basolateral (A-B) and basolateral-to-apical (B-A) directions. Samples from the donor and receiver compartments were collected, quenched, and analysed by LC-MS / MS. The apparent permeability (Papp) was calculated. Lucifer yellow was used to monitor monolayer integrity, with a leakage cutoff of 1%. Data processing rules were applied, including using the lower limit of detection (LLOD) for samples below the signal- to-noise ratio threshold, and applying criteria for internal standard and carryover. Table 5 TOP-201-PCT01-NP A-B Papp(1x10-6cm. s-1) B-A Papp(1x10-6cm. s-1) P-1 0.45 0.64 Exatecan (reference) 2.81 8.37 The data in Table 5 shows lower passive permeability for P1 relative to exatecan in both apical-to- basolateral (A-B) and basolateral-to-apical (B-A) directions. 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 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.

[0004] TOP-201-PCT01-NP Statements of Disclosure P 1P. A conjugate of Formula (I) Ab – (GA–JA–DC)k (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, each GAis independently a conjugation group conjugated to the antibody or antigen-binding fragment thereof, each DCis independently , wherein b is 0, 1, 2 or 3, each JAis independently a group of Formula (IA) R1is C1-4alkyl, X is (CH2)n2, wherein n2 is 0, 1, 2 or 3, TOP-201-PCT01-NP Y is (CH2)n3, wherein n3 is 0, 1, 2, 3 or 4, Z is (CH2)n4, wherein n4 is 1, 2, 3, 4 or 5, m is an integer from 5 to 17, p is 1 or 0, (GA) indicates the point of attachment to GA, and (DC) indicates the point of attachment to DC. 2P. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as disclosed in statement 1P, wherein Q is . 3P. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as disclosed in statement 1P or statement 2P, wherein m is 9, 10, 11, 12 or 13. 4P. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as disclosed in any one of statements 1P to 3P, wherein R1is CH3. 5P. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as disclosed in any one of statements 1P to 4P, wherein E is CH2. 6P. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as disclosed in any one of statements 1P to 5P, wherein X is CH2. 7P. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as disclosed in any one of statements 1P to 6P, wherein Y is (CH2)2. TOP-201-PCT01-NP 8P. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as disclosed in any one of statements 1P to 7P, wherein Z is (CH2)2. 9P. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as disclosed in any one of statements 1P to 8P, wherein p is 1. 10P. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as disclosed in statement 1P, wherein each JAis a group of Formula (IB) (IB). 11P. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as disclosed in any one of statements 1P to 10P, wherein each b is 0. 12P. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as disclosed in any one of statements 1P to 11P, wherein GAis selected from

[0005] TOP-201-PCT01-NP wherein RKis H or CH3, RLis C1-6alkyl, and indicates the point of attachment to the antibody or antigen-binding fragment thereof. 13P. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as disclosed in statement 12P, wherein GAis 14P. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as disclosed in statement 1P, wherein each (GA–JA–DC) is

[0006] TOP-201-PCT01-NP . 15P. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as disclosed in any one of statements 1P to 14P, wherein k is an integer from 2 to 8. 16P. A pharmaceutical composition comprising a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, as disclosed in any one of statements 1P to 15P, and a pharmaceutically acceptable excipient. 17P. A conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, as disclosed in any one of statements 1P to 15P, or a pharmaceutical composition as disclosed in statement 16P, for use in therapy. 18P. A conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, as disclosed in any one of statements 1P to 15P, or a pharmaceutical composition as disclosed in statement 16P, for use in the treatment of cancer. TOP-201-PCT01-NP 19P. 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 disclosed in any one of statements 1P to 15P, or a pharmaceutical composition as disclosed in statement 16. 20P. Use of a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, as disclosed in any one of statements 1P to 15P, in the manufacture of a medicament for the treatment of cancer. 21P. A compound of Formula (II) GB–JB–DC(II) or a salt thereof, wherein GBis a conjugation group for conjugation to an antibody or antigen- binding fragment thereof, JBis a group of Formula (IIA) (IIA), wherein DC, E, Q, R1, X, Y, Z, m and p are as defined for a conjugate of Formula (I) in any one of statements 1P to 11P, (GB) indicates the point of attachment to GB, and (DC) indicates the point of attachment to DC. 22P. A compound of Formula (II) or a salt thereof, as disclosed in statement 21P, wherein GBis selected from TOP-201-PCT01-NP wherein X1is CH or N, h is 0 or 1, Hal is Cl, Br or I, RKis H or CH3, and RLis C1-6alkyl. 23P. A compound of Formula (II) or a salt thereof, as disclosed in statement 22P, wherein GBis 24P. A compound of Formula (II) or a salt thereof, as disclosed in statement 21P, that is (2S,3S,4S,5R,6S)-6-(2-((3-((S)-6-(((3S,3aR,6S,6aR)-6-(2,5,8,11,14,17,20,23,26,29,32,35- dodecaoxaoctatriacontan-38-amido)hexahydrofuro[3,2-b]furan-3-yl)amino)-2-(3-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)propanamido)-6-oxohexanamido)propanamido)methyl)-4-((((3-(((1S,9S)-9- ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H- benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)bicyclo[1.1.1]pentan-1- yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid, or a salt thereof. TOP-201-PCT01-NP 25P. A compound of Formula (III) , wherein n is 0, 1, 2, or 3, or a salt thereof. 26P. A compound of Formula (III), as disclosed in statement 25P, that is , or a salt thereof.

Claims

TOP-201-PCT01-NP Claims 1. A conjugate of Formula (I) Ab – (GA–JA–DC)k (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, each GAis independently a conjugation group conjugated to the antibody or antigen-binding fragment thereof, each DCis independently, wherein b is 0, 1, 2 or 3, each JAis independently a group of Formula (IA)R1is C1-4 alkyl,TOP-201-PCT01-NP X is (CH2)n2, wherein n2 is 0, 1, 2 or 3, Y is (CH2)n3, wherein n3 is 0, 1, 2, 3 or 4, Z is (CH2)n4, wherein n4 is 0, 1, 2, 3, 4 or 5, m is an integer from 5 to 17, p is 1 or 0, (GA) indicates the point of attachment to GA, and (DC) indicates the point of attachment to DC.

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

3. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in claim 1 or claim 2, wherein m is 9, 10, 11, 12 or 13.

4. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 3, wherein R1is CH3.

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

7. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 6, wherein Y is (CH2)2.TOP-201-PCT01-NP 8. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 7, wherein Z is CH2 or (CH2)2.

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

10. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in claim 1, wherein each JAis a group of Formula (IB)(IB).

11. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in claim 1, wherein each JAis a group of Formula (IB1)(IB1).

12. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 11, wherein each b is 0.TOP-201-PCT01-NP 13. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 12, wherein each DCis independently.

14. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 13, wherein GAis selected fromwherein RKis H or CH3, RLis C1-6 alkyl, andindicates the point of attachment to the antibody or antigen-binding fragment thereof.

15. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in claim 14, wherein GAisTOP-201-PCT01-NP16. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in claim 1, wherein each (GA–JA–DC) is17. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in claim 1, wherein each (GA–JA–DC) isTOP-201-PCT01-NP.

18. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in claim 1, wherein each (GA–JA–DC) is.

19. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 18, wherein k is an integer from 2 to 8.TOP-201-PCT01-NP 20. A pharmaceutical composition comprising a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 19, and a pharmaceutically acceptable excipient.

21. A conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 19, or a pharmaceutical composition as claimed in claim 20, for use in therapy.

22. A conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 19, or a pharmaceutical composition as claimed in claim 20, for use in the treatment of cancer.

23. 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 19, or a pharmaceutical composition as claimed in claim 20.

24. Use of a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 19, in the manufacture of a medicament for the treatment of cancer.

25. A compound of Formula (II) GB–JB–DC(II) or a salt thereof, wherein GBis a conjugation group for conjugation to an antibody or antigen- binding fragment thereof, JBis a group of Formula (IIA)TOP-201-PCT01-NPwherein DC, E, Q, R1, X, Y, Z, m and p are as defined for a conjugate of Formula (I) in any one of claims 1 to 13, (GB) indicates the point of attachment to GB, and (DC) indicates the point of attachment to DC.

26. A compound of Formula (II) or a salt thereof, as claimed in claim 25, wherein GBis selected fromwherein X1is CH or N, h is 0 or 1, Hal is Cl, Br or I, RKis H or CH3, and RLis C1-6alkyl.TOP-201-PCT01-NP 27. A compound of Formula (II) or a salt thereof, as claimed in claim 26, wherein GBis28. A compound of Formula (II) or a salt thereof, as claimed in claim 25, that is (2S,3S,4S,5R,6S)- 6-(2-((3-((S)-6-(((3S,3aR,6S,6aR)-6-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38- amido)hexahydrofuro[3,2-b]furan-3-yl)amino)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)propanamido)-6-oxohexanamido)propanamido)methyl)-4-((((3-(((1S,9S)-9-ethyl-5-fluoro-9- hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H- benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)bicyclo[1.1.1]pentan-1- yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid, or a salt thereof.

29. A compound of Formula (II) or a salt thereof, as claimed in claim 25, that is (2S,3S,4S,5R,6S)- 6-(2-((3-((S)-6-(((3S,3aR,6S,6aR)-6-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38- amido)hexahydrofuro[3,2-b]furan-3-yl)amino)-2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)acetamido)-6-oxohexanamido)propanamido)methyl)-4-((((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy- 4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2- b]quinolin-1-yl)carbamoyl)bicyclo[1.1.1]pentan-1-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5- trihydroxytetrahydro-2H-pyran-2-carboxylic acid, or a salt thereof.

30. A compound of Formula (II) or a salt thereof, as claimed in claim 25, that is (2S,3S,4S,5R,6S)- 6-(2-((S)-8-(4-(((3S,3aR,6S,6aR)-6-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38- amido)hexahydrofuro[3,2-b]furan-3-yl)amino)-4-oxobutyl)-15-bromo-3,7,10,14-tetraoxo-2,6,9,13- tetraazapentadecyl)-4-((((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1- yl)carbamoyl)bicyclo[1.1.1]pentan-1-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro- 2H-pyran-2-carboxylic acid, or a salt thereof.TOP-201-PCT01-NP 31. A compound of Formula (IIIA), wherein n is 0, 1, 2, or 3, or a salt thereof.

32. A compound of Formula (IIIA) as claimed in claim 31, that is a compound of Formula (III), wherein n is 0, 1, 2, or 3, or a salt thereof.

33. A compound of Formula (IIIA) or Formula (III), as claimed in claim 31 or claim 32, that is, or a salt thereof.

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