WO2025262641A1 - Antibody drug conjugates that bind cdcp1 and uses thereof - Google Patents

Abstract

The present disclosure provides antibody-drug conjugates comprising an antibody or binding fragment thereof that binds CUB Domain-Containing Protein-1 linked to a pyridinobenzodiazepine useful for treating cancer.

Description

133186-5030-WO ANTIBODY DRUG CONJUGATES THAT BIND CDCP1 AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS T_{his application claims priority to U.S. Provisional Application No. 63/661,736, filed June 19,} 2024, which is incorporated by reference herein in its entirety. 5 SEQUENCE LISTING The instant application contains a Sequence Listing that has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. The Sequence Listing for this application is labeled “133186-5030-WO Sequence Listing.xml”, which was created on June 17, 2025, and is 344,064 bytes in size. 10 FIELD The disclosure relates to DNA-alkylating units comprising alkylating binding units. In particular it relates to antibody-drug conjugates comprising a pyrridinobenzodiazepine (PDD), and to pharmaceutically acceptable salts thereof, which are useful as medicaments, in particular as anti-proliferative agents. 15 BACKGROUND The pyrrolobenzodiazepines (PBDs) are a group of compounds some of which have been shown to be sequence-selective DNA minor-groove binding agents. The PBDs were originally discovered in Streptomyces species. ^[1] They are tricyclic in nature, and are comprised of fused 6-7-5–membered rings and can be identified as an anthranilate (A ring), a diazepine (B ring) 20 and a pyrrolidine (C ring) ^[1c] They are characterized by an electrophilic N10=C11 imine group (as shown below) or the hydrated equivalent, a carbinolamine [NH-CH(OH)], or a carbinolamine alkyl ether ([NH-CH(OR, where R = alkyl)] which can form a covalent bond to a C2-amino group of guanine in DNA to form a DNA adduct^[2]. The natural products interact in the minor groove of the DNA helix with excellent fit (i.e., good “isohelicity”) due to a right-25 handed longitudinal twist induced by a stereogenic C11a-position which has the (S)- configuration ^[3]. DB1/ 159693888.5 1

133186-5030-WO y_{l ether} Scheme 1: Interconvertible forms of the PBD The DNA adduct has been reported to inhibit a number of biological processes including the binding of transcription factors ^{[4] [5]} and the function of enzymes such as endonucleases ^[6] and 5 RNA polymerase ^[7]. PBD monomers (e.g., anthramycin) have been shown by footprinting ^[3], NMR ^[8], molecular modelling ^[9] and X-ray crystallography ^[10] to span three base pairs and to have a thermodynamic preference for the sequence 5’-Pu-G-Pu-3’ (where Pu = purine, and G is the reacting guanine) ^[11] and a kinetic preference for the sequence 5’-Py-G-Py-3’ (where Py is pyridine). 10 PBDs are thought to interact with DNA by first locating at a low-energy binding sequence (i.e., a 5’-Pu-G-Pu-3’ triplet) through Van der Waals, hydrogen bonding and electrostatic interactions [^4]. Then, once in place, a nucleophilic attack by the exocyclic C2-amino group of the central guanine occurs to form the covalent adduct ^[4]. Once bound, the PBD remains anchored in the DNA minor groove, avoiding DNA repair by causing negligible distortion of the DNA helix ^[10]. 15 The ability of PBDs to form an adduct in the minor groove and for PBD dimers to crosslink DNA enables them to interfere with DNA processing and, hence, their potential for use as antiproliferative agents. WO-A-2017/032983, WO-A-2013/164592, WO-A-2017/223275, WO-A-2021/137646, WO-A- 2019/126691, WO-A-2019/104289, US 10526294, and US 10143695 disclose PBD (6-7-5)20 and pyridinobenzodiazepine (PDD; 6-7-6) monomers linked to heterocyclic chains via their A- rings, all of which have been shown to act as cytotoxic agents in vitro and as anti-tumour agents in vivo in animal tumour models. Furthermore, the C8’-linked PBD dimer SJG-136 ^[12] has completed Phase I clinical trials for leukaemia and ovarian cancer ^[13] and a number of PBD dimer-based antibody-drug conjugates (ADCs) have been devised and some are in various 25 stages of clinical trials. DB1/ 159693888.5 2

133186-5030-WO In addition to this, the indolinobenzodiazepine-based ADCs IMGN779 and IMGN632 ^[14] have both progressed to Phase II and III studies respectively. 5 US-A-2019/151465 (LegoChem) discloses antibody-drug conjugates (ADCs) wherein a plurality of active agents is conjugated to an antibody through at least one branched linker. WO- A-2020/222573 (LegoChem) discloses a tris structured linker. WO-A-2018/234636 (Glykos) discloses hydrophilic linkers and conjugates. The natural PBD monomer Sibiromycin ^[15] is one of the most potent naturally-occurring PBDs 10 reported (sub-micromolar cytotoxicity) and has a sibirosamine sugar on the C7 position of the molecule. The potent cytotoxicity is thought to relate to its DNA-binding profile and potential ability to inhibit transcription factor binding ^[16]. Carbohydrate moieties have previously been incorporated into PBD-based monomers developed by Lown and colleagues ^[17]. It was observed that addition of a sugar moiety to certain positions 15 on the polyamide chain enhanced cytotoxicity in some cell-lines. It is known that glucuronide and glucose are cleaved by glycosidases in vivo and they have been incorporated into both amine-containing and phenol-containing linker-payloads of various families as part of the linker construct ^[18]. The incorporation of glucose in this context enhances PK properties of the resultant ADC, and the payload is liberated from the linker payload 20 construct through cleavage of the glucuronide moiety. A glucose moiety has also been incorporated into Auristatin (e.g., monomethyl auristatin E, “MMAE”) -based pro-drugs ^[19], enhancing efficacy, tolerability and solubility when compared to the parent unsubstituted MMAE molecule. DB1/ 159693888.5 3

133186-5030-WO Other sequence-selective DNA minor-groove binding agents are known, including the duocarmycins or CXIs (for example, cyclopropapyrroloindole (CPI), cyclopropabenzindole (CBI) or cyclopropathienoindole (CTI) moieties).¹ Many CXI analogues have since been developed, including bisalkylating dimers (A-A inter- 5 strand cross-linkers) consisting of two CXI units, such as bizelesin. C_{l Cl HN NH H} Bizelesin is an example of the seco-CPI (open form, with a chloromethyl functionality), rather than the spiro (closed, cyclopropyl) form, found in CC-1065.⁵ 10 This seco modification was found to be a prodrug of the CPI, and was more stable but had equivalent activity to the CPI dimer U-77809 (the corresponding parent drug form). Prodrug (seco) CXI forms are known to spirocyclise via a Winstein-Baird mechanism (Scheme 5) to the cyclopropane active, spiro form. 15 Scheme 5: Interconvertible forms of the CXI WO-A-2015/104373 and WO-A-2015/104386 (to Synthon) WO-A-2017/012924 (to Nerviano) and WO-A-2003/0022806 (to Boger) also disclose CXI moieties. The exceptional potency of PBD (6-7-5) and PDD (6-7-6) compounds and CXI compounds make them attractive candidates for targeted delivery via an Antibody-Drug Conjugate (ADC). 20 The present disclosure seeks to address this need and to overcome problem(s) associated with the prior art. DB1/ 159693888.5 4

133186-5030-WO SUMMARY In aspects, the disclosure provides an antibody-drug conjugate having formula (I): Ab-[L–D]_n formula (I) 5 wherein in formula (I): Ab is an antibody or binding fragment thereof that binds CUB Domain-Containing Protein-1 (CDCP1); D is a drug moiety comprising a pyrridinobenzodiazepine (PDD); L is a linker; 10 wherein D and/or L comprise at least one carbohydrate substituent R_S. In embodiments, RS is a univalent saccharide substituent, preferably RS is glycosyl or O- glycosyl. 15 In embodiments, the antibody or binding fragment thereof comprises: (i) a heavy chain variable region (VH) that comprises: (a) a VH complementarity determining region one (CDRH1) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 2, (b) a VH complementarity determining region two (CDRH2) comprising and/or 20 consisting of the amino acid sequence of SEQ ID NO: 3, and (c) a VH complementarity determining region three (CDRH3) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 4, and (ii) a light chain variable region (VL) that comprises: (a) a VL complementarity determining region one (CDRL1) comprising and/or 25 consisting of the amino acid sequence of SEQ ID NO: 6, (b) a VL complementarity determining region two (CDRL2) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 7, and (c) a VL complementarity determining region three (CDRL3) comprising and/or consisting of the amino acid sequence of SEQ ID NO:8. 30 In embodiments, the antibody or binding fragment thereof comprises a VH that comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid DB1/ 159693888.5 5

133186-5030-WO sequence of SEQ ID NO: 1, and/or a VL that comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 5. In embodiments, the antibody or binding fragment thereof comprises a heavy chain that comprises 5 the amino acid sequence of SEQ ID NO: 1 and/or a light chain that comprises the amino acid sequence of SEQ ID NO: 5. In embodiments, the antibody or binding fragment thereof comprises: (i) a heavy chain variable region (VH) that comprises: (a) a VH complementarity determining region one (CDRH1) comprising and/or 10 consisting of the amino acid sequence of SEQ ID NO: 16, (b) a VH complementarity determining region two (CDRH2) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 17, and (c) a VH complementarity determining region three (CDRH3) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 18, 15 and (ii) a light chain variable region (VL) that comprises: (a) a VL complementarity determining region one (CDRL1) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 20, (b) a VL complementarity determining region two (CDRL2) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 21, and 20 (c) a VL complementarity determining region three (CDRL3) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 22. In embodiments, the antibody or binding fragment thereof comprises a VH that comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 25 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 15, and/or a VL that comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 19. In embodiments, the antibody or binding fragment thereof comprises a heavy chain that comprises 30 the amino acid sequence of SEQ ID NO: 15 and/or a light chain that comprises the amino acid sequence of SEQ ID NO: 19. In embodiments, the antibody or binding fragment thereof comprises a VH that comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least DB1/ 159693888.5 6

133186-5030-WO 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from SEQ ID NOs: 23-176 and/or a VL that comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence 5 selected from SEQ ID NOs: 177-326. In embodiments, the antibody or binding fragment thereof comprises a VH that comprises an amino acid sequence selected from SEQ ID NOs: 23-176 and/or a VL that comprises an amino acid sequence selected from SEQ ID NOs: 177-326 In another aspect, the disclosure provides a pharmaceutical composition comprising an antibody-drug conjugate of formula (I), and a pharmaceutically acceptable carrier. 10 In another aspect, the disclosure provides a method of treating a cancer comprising administering to a subject in need thereof a therapeutically effective amount of an antibody-drug conjugate of formula (I). In embodiments, the cancer in triple-negative breast cancer (TNBC). BRIEF DESCRIPTION OF THE DRAWINGS Embodiments of the present disclosure will now be described further, with reference to the 15 accompanying drawings, in which: Figure 1 illustrates the results of the β-Glucosidase assay at 5 min. Aliquots of 100 µL were taken and analysed by LC-MS at 5 min, 30 min, 1 h, 2 h, 18 h, 36 h, 60 h, 84 h and 132 h. Figure 2 illustrates the results of the β-Glucosidase assay at 30 min. Aliquots of 100 µL were taken and analysed by LC-MS at 5 min, 30 min, 1 h, 2 h, 18 h, 36 h, 60 h, 84 h and 132 h. 20 Figure 3 illustrates the results of the β-Glucosidase assay at 1 h. Aliquots of 100 µL were taken and analysed by LC-MS at 5 min, 30 min, 1 h, 2 h, 18 h, 36 h, 60 h, 84 h and 132 h. Figure 4 illustrates the results of the β-Glucosidase assay at 2 h. Aliquots of 100 µL were taken and analysed by LC-MS at 5 min, 30 min, 1 h, 2 h, 18 h, 36 h, 60 h, 84 h and 132 h. Figure 5 illustrates the results of the β-Glucosidase assay at 18 h. Aliquots of 100 µL were 25 taken and analysed by LC-MS at 5 min, 30 min, 1 h, 2 h, 18 h, 36 h, 60 h, 84 h and 132 h. Figure 6 illustrates the results of the β-Glucosidase assay at 36 h. Aliquots of 100 µL were taken and analysed by LC-MS at 5 min, 30 min, 1 h, 2 h, 18 h, 36 h, 60 h, 84 h and 132 h. Figure 7 illustrates the results of the β-Glucosidase assay at 60 h. Aliquots of 100 µL were taken and analysed by LC-MS at 5 min, 30 min, 1 h, 2 h, 18 h, 36 h, 60 h, 84 h and 132 h. DB1/ 159693888.5 7

133186-5030-WO Figure 8 illustrates the results of the β-Glucosidase assay at 84 h. Aliquots of 100 µL were taken and analysed by LC-MS at 5 min, 30 min, 1 h, 2 h, 18 h, 36 h, 60 h, 84 h and 132 h. Figure 9 illustrates the results of the β-Glucosidase assay at 132 h. Aliquots of 100 µL were taken and analysed by LC-MS at 5 min, 30 min, 1 h, 2 h, 18 h, 36 h, 60 h, 84 h and 132 h. 5 Figures 10A- 10F illustrates the results of the β-Galactosidase assay. Aliquots of 100 µL were taken and analysed by LC-MS at 0 min, 5 min, 30 min, 90 min, 4.5 h, 7.5 h and 20 h. Figures 11A- 11D illustrates the results of the β-Glucuronidase assay with (2S,3S,4S,5R,6S)-6- (((S)-3-(4-((5-((4-aminophenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12- oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-2-yl)oxy)-3,4,5- 10 trihydroxytetrahydro-2H-pyran-2-carboxylic acid (88). Aliquots of 100 µL were taken and analysed by LC-MS at 0 min, 5 min, 30 min and 90 min. Figures 12A- 12E illustrates the results of the β-Glucuronidase assay with (2S,3S,4S,5R,6S)-6- (4-((((6aS)-3-(4-((5-((4-aminophenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4- oxobutoxy)-6-hydroxy-2-methoxy-12-oxo-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-15 a][1,4]diazepine-5-carbonyl)oxy)methyl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H- pyran-2-carboxylic acid (138). Aliquots of 100 µL were taken and analysed by LC-MS at 0 min, 5 min, 30 min, 90 min, and 150 min. Figures 13A- 13E illustrates the results of the β-Glucuronidase assay with (2S,3S,4S,5R,6S)-6- (4-((((6aS)-3-(4-((5-((4-(5-((4-aminophenyl)carbamoyl)-1-methyl-1H-pyrrol-3-20 yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-6-hydroxy-2-methoxy- 12-oxo-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5- carbonyl)oxy)methyl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (146). Aliquots of 100 µL were taken and analysed by LC-MS at 0 min, 5 min, 30 min, 90 min, and 150 min. 25 Figures 14A- 14E illustrates the results of the β-Glucuronidase assay with (2S,3S,4S,5R,6S)-6- (4-((((5-(3-carboxypropoxy)-4-methoxy-2-((S)-2-((methoxyimino)methyl)piperidine-1- carbonyl)phenyl)carbamoyl)oxy)methyl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H- pyran-2-carboxylic acid (150). Aliquots of 100 µL were taken and analysed by LC-MS at 0 min, 5 min, 30 min, 90 min, and 150 min. 30 Figure 15 illustrates the HIC profile of Trastuzumab. DB1/ 159693888.5 8

133186-5030-WO F_{igure 16 illustrates the PLRP trace of Trastuzumab. Heavy (left peak) and light (right peak)} chain peaks as indicated. Figure 17 illustrates the HIC profile of Trastuzumab-91. Average DAR calculated as 8. The conjugation process caused no significant aggregation compared to the starting antibody, with 5 93.1% monomer formed. Figure 18 illustrates the SEC profile of Trastuzumab-91; 93.1% monomer. No free toxin linker could be detected in the ADC sample. Figure 19 is a graph illustrating dose tolerability of Trastuzumab-91 (DAR 8) in non-tumour bearing CD1 mice. 10 Figure 20 is a graph illustrating in vivo efficacy of Trastuzumab-91 (DAR 8) at a single dose of 5 mg/kg. Figure 21 illustrates the protection of the N11-C12-alkylating imine positions or the masking of the C8-positions on G-alkylating PDD-constructs using with sugar-based moieties. Figure 22 is a scheme illustrating PDD-pro-drug compounds before and after cleavage of the 15 sugar group. Figure 23 is a graph illustrating in vivo efficacy of compound 180 at a single dose of 5 mg/kg. Figure 24A depicts an overview of a non-limiting example of a synthesis scheme for the intermediate compound 1.13. Figures 24B, 24C, 24D, 24E, 24F, 24G, 24H, 24I, and 24J collectively illustrate a non-limiting 20 example of synthesis steps to produce 1.2 (FIG.24B); 1.4 (FIG.24C); 1.5 (FIG.24D); 1.7 (FIG.24E); 1.8 (FIG.24F); 1.10 (FIG.24G); 1.11 (FIG.24H); 1.12 (FIG.24I); and 1.13 (FIG.24J). Figure 25A depicts an overview of a non-limiting example of a synthesis scheme for the subject compound 180. 25 Figures 25B, 25C, 25D, 25E, 25F, 25G, and 25H collectively illustrate a non-limiting example of synthesis steps to produce 1.14 (FIG.25B); 1.15 (FIG.25C); 1.16 (FIG.25D); 1.17 (FIG. 25E); 1.18 and 1.19 (FIG.25F); 1.20 (FIG.25G); and compound 180 (FIG.25H). DB1/ 159693888.5 9

133186-5030-WO Figure 26A illustrates the size exclusion chromatography (SEC) profile of the exemplary monoclonal antibody. Figure 26B illustrates the hydrophobic interaction chromatography (HIC) profile of the selected monoclonal antibody. 5 Figure 27A shows the hydrophobic interaction chromatography (HIC) profile of the exemplary antibody-drug conjugate (ADC). The average drug-to-antibody ratio (DAR) was calculated to be 4.2, and each DAR species assigned as indicated (i.e. DAR 0, DAR 2, DAR 4, DAR 6, or DAR 8). Figure 27B shows the size exclusion chromatography (SEC) profile of the exemplary ADC 10 sample. Figure 28 depicts the mean tumor volume in a CDX mouse-tumor xenograft prostate cancer model as a function of time after administration of the exemplary ADC targeting CDCP1 on days 1, 8, and 15. A dose-dependent regression in tumor size was observed. DETAILED DESCRIPTION 15 Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this disclosure belongs. All patents and publications referred to herein are incorporated by reference in their entireties. Definitions The following abbreviations are used throughout the specification: Ac acetyl; Alloc 20 allyloxycarbonyl; Boc tert-butyloxycarbonyl; DHP dihydropyran; DMAP 4- dimethylaminopyridine; DMF dimethylformamide; EDCI 1-ethyl-3-(3-dimethylamino- propyl)carbodiimide; Et ethyl; Me methyl; Ph phenyl; Tf trifluoromethanesulfonate; TFA trifluoroacetic acid; THF tetrahydrofuran and THP tetrahydropyranyl. “Substituted”, when used in connection with a chemical substituent or moiety (e.g., an alkyl 25 group), means that one or more hydrogen atoms of the substituent or moiety have been replaced with one or more non-hydrogen atoms or groups, provided that valence requirements are met and that a chemically stable compound results from the substitution. “Optionally substituted” refers to a parent group which may be unsubstituted or which may be substituted with one or more substituents. Suitably, unless otherwise specified, when optional 30 substituents are present, the optional substituted parent group comprises from one to three optional substituents. Where a group may be “optionally substituted with 1, 2 or 3 groups”, this DB1/ 159693888.5 10

133186-5030-WO means that the group may be substituted with 0, 1, 2 or 3 of the optional substituents. Suitably, the group is substituted with 1, 2 or 3 of the optional substituents. Where a group is “optionally substituted with one or two optional substituents”, this means that the group may be substituted with 0, 1 or 2 of the optional substituents. Suitably, the group may be optionally substituted 5 with 0 or 1 optional substituents. In some aspects, suitably the group is not optionally substituted. In other aspects, suitably the group is substituted with 1 of the optional substituents. Optional substituents may be selected from C1-8 alkyl, C2-7 alkenyl, C2-7 alkynyl, C1-12 alkoxy, C_5-20 aryl, C_3-10 cycloalkyl, C_3-10 cycloalkenyl, C_3-10 cycloalkynyl, C_3-20 heterocyclyl, C_3-20 10 heteroaryl, acetal, acyl, acylamido, acyloxy, amidino, amido, amino, aminocarbonyloxy, azido, carboxy, cyano, ether, formyl, guanidino, halo, hemiacetal, hemiketal, hydroxamic acid, hydroxyl, imidic acid, imino, ketal, nitro, nitroso, oxo, oxycarbonyl, oxycarboyloxy, sulfamino, sulfamyl, sulfate, sulfhydryl, sulfinamino, sulfinate, sulfino, sulfinyl, sulfinyloxy, sulfo, sulfonamido, sulfonamino, sulfonate, sulfonyl, sulfonyloxy, uredio groups. In some aspects, the15 optional substituents are 1, 2 or 3 optional substituents independently selected from OH, C_1-8 alkyl, OC_1-12 alkyl, and halogen. More suitably, the optional substituents are selected from OH, C_1-8 alkyl and OC_1-12 alkyl; more suitably, the optional substituents are selected from C_1-8 alkyl and OC1-12 alkyl. “Independently” or “Independently selected” is used in the context of statement that, for 20 example, “each R', R'', is independently H, C1-8 alkyl...” and means that each instance of the functional group, e.g., R', is selected from the listed options independently of any other instance of R' or R'' in the compound. Hence, for example, H may be selected for the first instance of R' in the compound; methyl may be selected for the next instance of R' in the compound; and ethyl may be selected for the first instance of R'' in the compound. 25 “C1-8 alkyl”: refers to straight chain and branched saturated hydrocarbon groups, generally having from 1 to 8 carbon atoms; suitably a C1-7 alkyl; suitably a C1-6 alkyl; suitably a C1-5 alkyl; more suitably a C_1-4 alkyl; more suitably a C_1-3 alkyl. Examples of alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-butyl, pent-1-yl, pent-2-yl, pent-3-yl, 3- methylbut-1-yl, 3-methylbut-2-yl, 2-methylbut-2-yl, 2,2,2-trimethyleth-1-yl, n-hexyl, n-heptyl, 30 n-octyl and the like. “Alkylene” refers to a divalent radical derived from an alkane which may be a straight chain or branched, as exemplified by –CH₂CH₂CH₂CH₂-. The alkylene may have the number of carbons as discussed above for alkyl groups. DB1/ 159693888.5 11

133186-5030-WO The term “amino acid” refers to naturally occurring (or “canonical”) α-amino acids and their stereoisomers, unnatural (or “non-canonical”) amino acids and their stereoisomers, and modified or synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally-occurring amino acids. “Stereoisomers” of amino 5 acids refers to mirror image isomers of the amino acids, such as L-amino acids or D-amino acids. For example, a stereoisomer of a naturally-occurring amino acid refers to the mirror image isomer of the naturally-occurring amino acid, i.e., the D-amino acid. Naturally-occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. 10 Naturally-occurring α-amino acids include, without limitation, they are amino acids selected from alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val). Stereoisomers of naturally-occurring 15 α-amino acids include, without limitation, D-alanine (D-Ala), D-arginine (D-Arg), D-asparagine (D-Asn), D-aspartic acid (D-Asp), D-cysteine (D-Cys), D-glutamine (D-Gln), D-glutamic acid (D-Glu), D-glycine (D-Gly), D-histidine (D-His), D-isoleucine (D-Ile), D-leucine (D-Leu), D- lysine (D-Lys), D-methionine (D-Met), D-phenylalanine (D-Phe), D-proline (D-Pro), D-serine (D-Ser), D-threonine (Thr), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and D-valine (D-Val). 20 Unnatural amino acids include, without limitation, amino acid analogs, amino acid mimetics, and synthetic amino acids in either the L- or D-configuration that function in a manner similar to the naturally-occurring amino acids. For example, “amino acid analogs” are unnatural amino acids that have the same basic chemical structure as naturally-occurring amino acids, i.e., an α- carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., 25 homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Non-limiting examples of unnatural amino acids include 1-aminocyclopentane-1-carboxylic acid (Acp), 1-aminocyclobutane-1-carboxylic acid (Acb), 1-aminocyclopropane-1-carboxylic30 acid (Acpc), citrulline (Cit), homocitrulline (HoCit), α-aminohexanedioic acid (Aad), 3-(4- pyridyl)alanine (4-Pal), 3-(3-pyridyl)alanine (3-Pal), propargylglycine (Pra), α-aminoisobutyric acid (Aib), α-aminobutyric acid (Abu), norvaline (Nva), α,β-diaminopropionic acid (Dpr), α,γ- diaminobutyric acid (Dbu), α-tert-butylglycine (Bug), 3,5-dinitrotyrosine (Tyr(3,5-di NO2)), norleucine (Nle), 3-(2-naphthyl)alanine (Nal-2), 3-(1-naphthyl)alanine (Nal-1), 35 cyclohexylalanine (Cha), di-n-propylglycine (Dpg), cyclopropylalanine (Cpa), homoleucine DB1/ 159693888.5 12

133186-5030-WO (Hle), homoserine (HoSer), homoarginine (Har), homocysteine (Hcy), methionine sulfoxide (Met(O)), methionine methylsulfonium (Met (S-Me)), α-cyclohexylglycine (Chg), 3-benzo- thienylalanine (Bta), taurine (Tau), hydroxyproline (Hyp), O-benzyl-hydroxyproline (Hyp(Bzl)), homoproline (HoPro), β-homoproline (βHoPro), thiazolidine-4-carboxylic acid 5 (Thz), nipecotic acid (Nip), isonipecotic acid (IsoNip), 3-carboxymethyl-1-phenyl-1,3,8- triazaspiro[4,5]decan-4-one (Cptd), tetrahydro-isoquinoline-3-carboxylic acid (3-Tic), 5H- thiazolo [3,2-a]pyridine-3-carboxylic acid (Btd), 3-aminobenzoic acid (3-Abz), 3-(2- thienyl)alanine (2-Thi), 3-(3-thienyl)alanine (3-Thi), α-aminooctanedioc acid (Asu), diethylglycine (Deg), 4-amino-4-carboxy-1,1-dioxo-tetrahydrothiopyran (Acdt), 1-amino-1-(4- 10 hydroxycyclohexyl) carboxylic acid (Ahch), 1-amino-1-(4-ketocyclohexyl)carboxylic acid (Akch), 4-amino-4-carboxytetrahydropyran (Actp), 3-nitrotyrosine (Tyr(3-NO2)), 1-amino-1- cyclohexane carboxylic acid (Ach), 1-amino-1-(3-piperidinyl)carboxylic acid (3-Apc), 1-amino- 1-(4-piperidinyl)carboxylic acid (4-Apc), 2-amino-3-(4-piperidinyl) propionic acid (4-App), 2- aminoindane-2-carboxylic acid (Aic), 2-amino-2-naphthylacetic acid (Ana), (2S, 5R)-5- 15 phenylpyrrolidine-2-carboxylic acid (Ppca), 4-thiazoylalanine (Tha), 2-aminooctanoic acid (Aoa), 2-aminoheptanoic acid (Aha), ornithine (Orn), azetidine-2-carboxylic acid (Aca), α- amino-3-chloro-4,5-dihydro-5-isoazoleacetic acid (Acdi), thiazolidine-2-carboxylic acid (Thz(2- COOH)), allylglycine (Agl), 4-cyano-2-aminobutyric acid (Cab), 2-pyridylalanine (2-Pal), 2- quinoylalanine (2-Qal), cyclobutylalanine (Cba), a phenylalanine analog, derivatives of lysine, 20 ornithine (Orn) and α,γ-diaminobutyric acid (Dbu), stereoisomers thereof, and combinations thereof (see, e.g., Liu et al., Anal. Biochem., 295:9-16 (2001)). As such, the unnatural α-amino acids are present either as unnatural L-α-amino acids, unnatural D-α-amino acids, or combinations thereof. “Amino acid mimetics” are chemical compounds that have a structure that is different from the25 general chemical structure of an amino acid, but that function in a manner similar to a naturally- occurring amino acid. Suitable amino acid mimetics include, without limitation, β-amino acids and γ-amino acids. In β-amino acids, the amino group is bonded to the β-carbon atom of the carboxyl group such that there are two carbon atoms between the amino and carboxyl groups. In γ-amino acids, the amino group is bonded to the γ-carbon atom of the carboxyl group such that 30 there are three carbon atoms between the amino and carboxyl groups. Suitable R groups for β- or γ-amino acids include, but are not limited to, side-chains present in naturally-occurring amino acids and unnatural amino acids. “C6-26 aralkyl” refers to an arylalkyl group having 6 to 26 carbon atoms and comprising an alkyl group substituted with an aryl group. Suitably the alkyl group is a C1-6 alkyl group and the aryl35 group is phenyl. Examples of C6-26 aralkyl include benzyl and phenethyl. In some cases, the C6- DB1/ 159693888.5 13

133186-5030-WO 2₆ aralkyl group may be optionally substituted, and an example of an optionally substituted C_6-26 aralkyl group is 4-methoxylbenzyl. “C_5-20 Aryl”: refers to fully unsaturated monocyclic, bicyclic and polycyclic aromatic hydrocarbons having at least one aromatic ring and having a specified number of carbon atoms 5 a comprise their ring members (e.g., C5-20 aryl refers to an aryl group having from 5 to 20 carbon atoms as ring members). The aryl group may be attached to a parent group or to a substrate at any ring atom and may include one or more non-hydrogen substituents unless such attachment or substitution would violate valence requirements. Suitably, a C6-14 aryl is selected from a C_6-12 aryl, more suitably, a C_6-10 aryl. Examples of aryl groups include phenyl. 10 “Arylene” refers to a divalent radical derived from an aryl group, e.g., –C6H4- which is the arylene derived from phenyl (phenylene). “C3-8 cycloalkyl” or “3- to 8-membered cycloalkyl” means a closed ring of carbon atoms having 3 to 8 carbon atoms, preferably 3 to 7 carbon atoms, more preferably 3 to 6 carbon atoms and encompasses, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and 15 cyclooctyl. “C3-8 cycloalkylene” or “3- to 8-membered cycloalkylene” refers to a divalent radical derived from a cycloalkyl group, e.g., –C6H10-. “C_3-8 cycloalkenylene” refers to a divalent radical derived from a cycloalkenyl group, that is a carbocyclic group with one or more C=C, e.g., –C6H8-. 20 Halogen or halo: refers to a group selected from F, Cl, Br, and I. Suitably, the halogen or halo is F or Cl. In some aspects, suitably, the halogen is F. In other aspects, suitably the halogen is Cl. “C_5-10 heteroaryl” or “5- to 10-membered heteroaryl”: refers to unsaturated monocyclic or clic aromatic groups comprising from 5 to 10 ring atoms, whether carbon or heteroatoms, of 25 which from 1 to 5 are ring heteroatoms. Suitably, any monocyclic heteroaryl ring has from 5 to 6 ring atoms and from 1 to 3 ring heteroatoms. Suitably each ring heteroatom is independently selected from nitrogen, oxygen, and sulfur. The bicyclic rings include fused ring systems and, in particular, include bicyclic groups in which a monocyclic heterocycle comprising 5 ring atoms is fused to a benzene ring. The heteroaryl group may be attached to a parent group or to a 30 substrate at any ring atom and may include one or more non-hydrogen substituents unless such attachment or substitution would violate valence requirements or result in a chemically unstable compound. DB1/ 159693888.5 14

133186-5030-WO Examples of monocyclic heteroaryl groups include, but are not limited to, those derived from: N1: pyrrole, pyridine; O1: furan; S1: thiophene; 5 N₁O₁: oxazole, isoxazole, isoxazine; N₂O₁: oxadiazole (e.g., 1-oxa-2,3-diazolyl, 1-oxa-2,4-diazolyl, 1-oxa-2,5-diazolyl, 1-oxa-3,4- diazolyl); N₃O₁: oxatriazole; N1S1: thiazole, isothiazole; 10 N2: imidazole, pyrazole, pyridazine, pyrimidine, pyrazine; N3: triazole, triazine; and, N₄: tetrazole. Examples of heteroaryl which comprise fused rings, include, but are not limited to, those derived from: 15 O₁: benzofuran, isobenzofuran; N₁: indole, isoindole, indolizine, isoindoline; S₁: benzothiofuran; N1O1: benzoxazole, benzisoxazole; N1S1: benzothiazole; 20 N2: benzimidazole, indazole; O₂: benzodioxole; N₂O₁: benzofurazan; N2S1: benzothiadiazole; N3: benzotriazole; and 25 N4: purine (e.g., adenine, guanine), pteridine; “Heteroarylene” refers to a divalent radical derived from a heteroaryl group (such as those described above) as exemplified by pyridinyl –[C5H3N]-. Heteroarylenes may be monocyclic, bicyclic, or tricyclic ring systems. Representative heteroarylenes, are not limited to, but may be selected from triazolylene, tetrazolylene, oxadiazolylene, pyridylene, furylene, benzofuranylene, 30 thiophenylene, benzothiophenylene, quinolinylene, pyrrolylene, indolylene, oxazolylene, benzoxazolylene, imidazolylene, benzimidazolylene, thiazolylene, benzothiazolylene, isoxazolylene, pyrazolylene, isothiazolylene, pyridazinylene, pyrimidinylene, pyrazinylene, triazinylene, cinnolinylene, phthalazinylene, quinazolinylene, pyrimidylene, azepinylene, oxepinylene, and quinoxalinylene. Heteroarylenes are optionally substituted. DB1/ 159693888.5 15

133186-5030-WO “C_6-16 heteroarylalkyl” refers to an alkyl group substituted with a heteroaryl group. Suitably the alkyl is a C1-6 alkyl group and the heteroaryl group is C5-10 heteroaryl as defined above. Examples of C6-16 heteroarylalkyl groups include pyrrol-2-ylmethyl, pyrrol-3-ylmethyl, pyrrol- 4-ylmethyl, pyrrol-3-ylethyl, pyrrol-4-ylethyl, imidazol-2-ylmethyl, imidazol-4-ylmethyl, 5 imidazol-4-ylethyl, thiophen-3-ylmethyl, furan-3-ylmethyl, pyridin-2-ylmethyl, pyridin-2- ylethyl, thiazol-2-ylmethyl, thiazol-4-ylmethyl, thiazol-2-ylethyl, pyrimidin-2-ylpropyl, and the like. “C3-20 heterocyclyl”: refers to saturated or partially unsaturated monocyclic, bicyclic or polycyclic groups having ring atoms composed of 3 to 20 ring atoms, whether carbon atoms or 10 heteroatoms, of which from 1 to 10 are ring heteroatoms. Suitably, each ring has from 3 to 8 ring atoms and from 1 to 4 ring heteroatoms (e.g., suitably C3-5 heterocyclyl refers to a heterocyclyl group having 3 to 5 ring atoms and 1 to 4 heteroatoms as ring members). The ring heteroatoms are independently selected from nitrogen, oxygen, and sulphur. As with bicyclic cycloalkyl groups, bicyclic heterocyclyl groups may include isolated rings, 15 spiro rings, fused rings, and bridged rings. The heterocyclyl group may be attached to a parent group or to a substrate at any ring atom and may include one or more non-hydrogen substituents unless such attachment or substitution would violate valence requirements or result in a chemically unstable compound. Examples of monocyclic heterocyclyl groups include, but are not limited to, those derived from: 20 N1 : aziridine, azetidine, pyrrolidine, pyrroline, 2H-pyrrole or 3H-pyrrole, piperidine, dihydropyridine, tetrahydropyridine, azepine; O₁: oxirane, oxetane, tetrahydrofuran, dihydrofuran, tetrahydropyran, dihydropyran, pyran, oxepin; S1: thiirane, thietane, tetrahydrothiophene, tetrahydrothiopyran, thiepane; 25 O2: dioxoiane, dioxane, and dioxepane; O3: trioxane; N₂: imidazoiidine, pyrazolidine, imidazoline, pyrazoline, piperazine: N₁O₁: tetrahydrooxazole, dihydrooxazole, tetrahydroisoxazole, dihydroisoxazole, morpholine, tetrahydrooxazine, dihydrooxazine, oxazine; 30 N1S1: thiazoline, thiazolidine, thiomorpholine; N2O1: oxadiazine; O1S1: oxathiole and oxathiane (thioxane); and N₁O₁S₁: oxathiazine. DB1/ 159693888.5 16

133186-5030-WO Examples of substituted monocyclic heterocyclyl groups include those derived from saccharides, in cyclic form, for example, furanoses, such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse, and pyranoses, such as aliopyranose, altropyranose, glucopyranose, mannopyranose, gulopyranose, idopyranose, galactopyranose, and talopyranose. 5 “Nucleic acid”, refers to a linear polymer of nucleosides (including deoxyribo-nucleosides, ribonucleosides, or analogs thereof) joined by inter-nucleosidic linkages. Nucleic acid may encompass the term “polynucleotide” as well as “oligonucleotide”. The linear polymer may be represented by a sequence of letters, such as “ATGCCTG,” where it will be understood that the nucleotides are in 5' to 3' order from left to right and that “A” denotes deoxyadenosine, “C” 10 denotes deoxycytidine, “G” denotes deoxyguanosine, and “T” denotes deoxythymidine, unless otherwise noted. Another natural nucleotide is “U”, denoting uridine. The letters A, C, G, T and U can be used to refer to the bases themselves, to nucleosides, or to nucleotides comprising the bases, as is standard in the art. In naturally occurring nucleic acids, the inter-nucleoside linkage is typically a phosphodiester bond, and the subunits are referred to as “nucleotides.” 15 Nucleic acids may also include other inter-nucleoside linkages, such as phosphoro-thioate linkages, and the like. Such analogs of nucleotides that do not include a phosphate group are considered to fall within the scope of the term “nucleotide”" as used herein, and nucleic acids comprising one or more inter-nucleoside linkages that are not phosphodiester linkages are still referred to as "polynucleotides”, “oligonucleotides”, etc. 20 Nitrogen protecting groups Nitrogen protecting groups are well known in the art and are groups that block or protect the nitrogen groups from further reaction. Nitrogen protecting groups are exemplified by carbamates, such as methyl or ethyl carbamate, 9-fluorenylmethyloxy-carbonyl (Fmoc), substituted ethyl carbamates, carbamates cleaved by 1,6-beta-elimination, ureas, amides, 25 peptides, alkyl and aryl derivatives. Carbamate protecting groups have the general formula: . In this specification a zig-zag line (or wavy line ) indicates the point of attachment of the shown group (e.g., the protecting group above) to the rest of the compound (e.g. a compound of formula (I)). Suitable nitrogen protecting groups may be selected from acetyl, trifluoroacetyl, t- 30 butyloxy-carbonyl (BOC), benzyloxycarbonyl (Cbz) and 9-fluorenylmethyloxy-carbonyl (Fmoc). DB1/ 159693888.5 17

133186-5030-WO A large number of possible carbamate nitrogen protecting groups are listed on pages 706 to 771 of Wuts, P.G.M. and Greene, T.W., Protective Groups in Organic Synthesis, 4^th Edition, Wiley- lnterscience, 2007, and in P. Kocienski, Protective Groups, 3rd Edition (2005) which are incorporated herein by reference. 5 Particularly preferred protecting groups include Alloc (allyloxycarbonyl), Troc (2,2,2- Trichloroethyl carbonate), Teoc [2-(Trimethylsilyl)ethoxycarbony], BOC (tert- butyloxycarbonyl), Doc (2,4-dimethylpent-3-yloxycarbonyl), Hoc (cyclohexyloxy-carbonyl), TcBOC (2,2,2-trichloro-tert-butyloxycarbonyl), Fmoc (9-fluorenylmethyloxycarbonyl), 1-Adoc (1-Adamantyloxycarbonyl) and 2-Adoc (2-adamantyloxycarbonyl). 10 Hydroxyl protecting groups Hydroxyl protecting groups are well known in the art, a large number of suitable groups are described on pages 16 to 366 of Wuts, P.G.M. and Greene, T.W., Protective Groups in Organic Synthesis, 4^th Edition, Wiley-lnterscience, 2007, and in P. Kocienski, Protective Groups, 3rd Edition (2005) which are incorporated herein by reference. 15 Classes of particular interest include silyl ethers, methyl ethers, alkyl ethers, benzyl ethers, esters, benzoates, carbonates, and sulfonates. Suitable protecting groups include THP (tetrahydropyranyl ether). Hydroxyls may also be protected as N methyl piperazine carbamate, especially the hydroxyl group of a phenol. The term “antibody” herein is used in the broadest sense and encompasses various antibody 20 structures, including but not limited to monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity. Antibodies may be murine, human, humanized, chimeric, or derived from other species. An antibody is a protein generated by the immune system that is capable of recognizing and binding to a specific antigen. (Janeway, C., Travers, 25 P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York). A target antigen generally has numerous binding sites, also called epitopes, recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody. An antibody includes a full-length immunoglobulin molecule or an immunologically active portion 30 of a full-length immunoglobulin molecule, i.e., a molecule that contains an antigen-binding site that immunospecifically binds an antigen of a target of interest or part thereof, such targets including but not limited to, cancer cell or cells that produce autoimmune antibodies associated with an autoimmune disease. DB1/ 159693888.5 18

133186-5030-WO An exemplary antibody such as an IgG comprises two heavy chains and two light chains. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The VH and VL regions can be 5 further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). 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. 10 The hypervariable region generally encompasses amino acid residues from about amino acid residues 24-34 (LCDR1; “L” denotes light chain), 50-56 (LCDR2) and 89-97 (LCDR3) in the light chain variable region and around about 31-35 (HCDR1; “H” denotes heavy chain), 50-65 (HCDR2), and 95-102 (HCDR3) in the heavy chain variable region; Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service, National 15 Institutes of Health, Bethesda, Md. (1991) and/or those residues forming a hypervariable loop (e.g. residues 26-32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3) in the light chain variable region and 26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chain variable region; Chothia and Lesk (1987) J. Mol. Biol.196:901-917. The term “monoclonal antibody” as used herein refers to an antibody obtained from a 20 population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against 25 different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any method. For example, the monoclonal antibodies to be used in accordance with 30 the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein. The terms “nanobody,” “single-domain antibody (sdAb)”, “variable domain of a heavy chain- 35 only antibody (VHH),” or “single domain-based VHH,” as used herein are used interchangeably DB1/ 159693888.5 19

133186-5030-WO and refer to single-domain heavy chain-only antibody derived from the Camelidae family. In some embodiments, a nanobody is an antibody fragment derived from the Camelidae heavy- chain only IgG antibody. The term “diabodies” refers to small antibody fragments with two antigen-binding sites, which 5 fragments comprise a variable heavy domain (VH) connected to a variable light domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. The term “chimeric” antibody refers to a recombinant antibody in which a portion of the heavy 10 and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species. A “human antibody” is an antibody that possesses an amino-acid sequence corresponding to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies known to one of skill in the art. This definition of a human antibody 15 specifically excludes a humanized antibody comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including methods described in Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al, J. Immunol, 147(I):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol, 5: 368-74 (2001). Human antibodies can be prepared by 20 administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immunized HuMab mice (see, e.g., Nils Lonberg et al., 1994, Nature 368:856-859, WO 98/24884, WO 94/25585, WO 93/1227, WO 92/22645, WO 92/03918 and WO 01/09187 regarding HuMab mice), xenomice (see, e.g., U.S. Pat. Nos.6,075,181 and 6,150,584 regarding 25 XENOMOUSE™ technology) or Trianni mice (see, e.g., WO 2013/063391, WO 2017/035252 and WO 2017/136734). The term “humanized antibody” refers to an antibody that has been engineered to comprise one or more human framework regions in the variable region together with non-human (e.g., mouse, rat, or hamster) complementarity-determining regions (CDRs) of the heavy and/or light chain. 30 In certain embodiments, a humanized antibody comprises sequences that are entirely human except for the CDR regions. Humanized antibodies are typically less immunogenic to humans, relative to non-humanized antibodies, and thus offer therapeutic benefits in certain situations. Those skilled in the art will be aware of humanized antibodies and will also be aware of suitable techniques for their generation. See for example, Hwang, W. Y. K., et al., Methods 36:35, 2005; DB1/ 159693888.5 20

133186-5030-WO Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033, 1989; Jones et al., Nature, 321:522- 25, 1986; Riechmann et al., Nature, 332:323-27, 1988; Verhoeyen et al., Science, 239:1534-36, 1988; Orlandi et al., Proc. Natl. Acad. Sci. USA, 86:3833-37, 1989; U.S. Pat. Nos.5,225,539; 5,530,101; 5,585,089; 5,693,761; 5,693,762; 6,180,370; and Selick et al., WO 90/07861, each of 5 which is incorporated herein by reference in its entirety. The “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of 10 immunoglobulins are called α, δ, ε, γ, and μ, respectively. The immunoglobulin disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. The immunoglobulins can be derived from any species. In one aspect, however, the immunoglobulin is of human, murine, or rabbit origin. 15 A “binding fragment thereof” refers to a fragment of an antibody that retains the ability to specifically bind to an antigen (preferably with substantially the same binding affinity). Such fragment is sometimes referred to as “antigen-binding domain” of an antibody (or simply “binding domain” ). Examples of an binding fragment thereof includes (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a 20 bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., 1989 Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR), disulfide-linked Fvs (dsFv), diabodies; linear 25 antibodies; single-chain antibody molecules (e.g., ScFv); nanobodies; anti-idiotypic (anti-Id) antibodies and intrabodies. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they may be joined, using recombinant methods (e.g., by a synthetic linker) thus enabling them to be produced as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (ScFv)); see e.g., 30 Bird et al., Science 242:423-426 (1988) and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of 35 another chain and creating two antigen-binding sites (see e.g., Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak et al., 1994, Structure 2:1121-1123). DB1/ 159693888.5 21

133186-5030-WO An antibody “variable domain” refers to the variable region of the antibody light chain (VL) or the variable region of the antibody heavy chain (VH), either alone or in combination. As known in the art, the variable regions of the heavy and light chains each consist of four framework regions (FR) connected by three complementarity determining regions (CDRs), and contribute 5 to the formation of the antigen-binding site of antibodies. “Complementarity determining region” or “CDR” as the terms are used herein refer to short polypeptide sequences within the variable region of both heavy and light chain polypeptides that are primarily responsible for mediating specific antigen recognition. There are three CDRs (termed CDR1, CDR2, and CDR3) within each VL and each VH. 10 As will be appreciated by those in the art, the exact numbering and placement of the CDRs can be different among different numbering systems. However, it should be understood that the disclosure of a variable heavy and/or variable light sequence includes the disclosure of the associated CDRs. Accordingly, the disclosure of each variable heavy region is a disclosure of the vhCDRs (e.g., vhCDR1, vhCDR2 and vhCDR3) and the disclosure of each variable light 15 region is a disclosure of the vlCDRs (e.g., vlCDR1, vlCDR2 and vlCDR3). In certain embodiments, the CDRs of an antibody can be determined according to the IMGT numbering system as described in Lefranc M-P, (1999) The Immunologist 7: 132- 136 and Lefranc M-P et al, (1999) Nucleic Acids Res 27: 209-212. Unless stated otherwise herein, references to residue numbers in the variable domain of antibodies means residue numbering by 20 the IMGT numbering system. In other embodiments, the CDRs of an antibody can be determined according to MacCallum RM et al, (1996) J Mol Biol 262: 732-745. See also, e.g., Martin A. “Protein Sequence and Structure Analysis of Antibody Variable Domains,” in Antibody Engineering, Kontermann and Diibel, eds., Chapter 31, pp.422-439, Springer-Verlag, Berlin (2001). In other embodiments, 25 the CDRs of an antibody can be determined according to the AbM numbering scheme, which refers to AbM hypervariable regions, which represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.). Residues in a variable domain are numbered according Kabat, which is a numbering system used for heavy chain variable domains or light 30 chain variable domains of the compilation of antibodies. See, Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain may include a single amino DB1/ 159693888.5 22

133186-5030-WO acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g., residues 82a, 82b, and 82c, according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence. Various 5 algorithms for assigning Kabat numbering are available. The algorithm implemented in the version 2.3.3 release of Abysis (abysis.org) is used herein to assign Kabat numbering to variable regions CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, and CDRH3. “Framework” or “framework region” or “FR” refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four 10 FR domains: FR1, FR2, FR3, and FR4. A “human consensus framework” is a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as 15 in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), Vols.1-3. In one embodiment, for the VL, the subgroup is subgroup kappa I as in Kabat et al., supra. In one embodiment, for the VH, the subgroup is subgroup Ill as in Kabat et al., supra. The “hinge region” is generally defined as stretching from 216-238 (EU numbering) or 226-251 20 (Kabat numbering) of human IgG1. The hinge can be further divided into three distinct regions, the upper, middle (e.g., core), and lower hinge. The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region 25 extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National 30 Institutes of Health, Bethesda, Md. (1991). A “blocking” antibody or an “antagonist” antibody is one which inhibits or reduces biological activity of the antigen it binds. Certain blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen. DB1/ 159693888.5 23

133186-5030-WO An “epitope” is a term of art that indicates the site or sites of interaction between an antibody and its antigen(s). As described by (Janeway, C, Jr., P. Travers, et al. (2001). Immunobiology: the immune system in health and disease. Part II, Section 3- 8. New York, Garland Publishing, Inc.): “An antibody generally recognizes only a small region on the surface of a large molecule 5 such as a protein... [Certain epitopes] are likely to be composed of amino acids from different parts of the [antigen] polypeptide chain that have been brought together by protein folding. Antigenic determinants of this kind are known as conformational or discontinuous epitopes because the structure recognized is composed of segments of the protein that are discontinuous in the amino acid sequence of the antigen but are brought together in the three-dimensional 10 structure. In contrast, an epitope composed of a single segment of polypeptide chain is termed a continuous or linear epitope” (Janeway, C. Jr., P. Travers, et al. (2001). Immunobiology: the immune system in health and disease. Part II, Section 3-8. New York, Garland Publishing, Inc.). The epitope/paratope residue can be defined by a specific criterion, e.g., distance between atoms in the Ab and the Ag (e.g., a distance of equal to or less than about 4 Å from a heavy atom of 15 the cognate antibody and a heavy atom of the antigen). In another aspect, an epitope/paratope residue can be characterized as participating in a hydrogen bond interaction with the cognate antibody/antigen, or with a water molecule that is also hydrogen bonded to the cognate antibody/antigen (water-mediated hydrogen bonding). In another aspect, an epitope/paratope residue can be characterized as forming a salt bridge with a residue of the cognate 20 antibody/antigen. In yet another aspect, an epitope/paratope residue can be characterized as a residue having a non-zero change in buried surface area (BSA) due to interaction with the cognate antibody/antigen. At a less detailed level, epitope/paratope can be characterized through function, e.g., by competition binding with other Abs. The epitope/paratope can also be defined more generically as comprising amino acid residues for which substitution by another amino 25 acid will alter the characteristics of the interaction between the Ab and Ag (e.g., alanine scanning). An “antibody that binds to the same epitope” as a reference antibody refers to an antibody that contacts an overlapping set of amino acid residues of the antigen as compared to the reference antibody or blocks binding of the reference antibody to its antigen in a competition assay by 30 50% or more. The amino acid residues of an antibody that contact an antigen can be determined, for example, by determining the crystal structure of the antibody in complex with the antigen or by performing hydrogen/deuterium exchange. In some embodiments, residues of an antibody that are within 5 Å the antigen are considered to contact the antigen. In some embodiments, an antibody that binds to the same epitope as a reference antibody blocks binding of the reference 35 antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay by 50% or more. DB1/ 159693888.5 24

133186-5030-WO “Fv” consists of a dimer of one heavy- and one light-chain variable region domain in tight, non- covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. 5 “Single-chain Fv” also abbreviated as “sFv” or “ScFv” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Plückthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore 10 eds., Springer-Verlag, New York, pp.269-315 (1994). The term an “isolated antibody” when used to describe the various antibodies disclosed herein, means an antibody that has been identified and separated and/or recovered from a cell or cell culture from which it was expressed. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, 15 and can include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) approaches. For a review of methods for assessment of antibody purity, see, for example, Flatman et al., J. 20 Chromatogr. B 848:79-87 (2007). In an embodiment, the antibody will be purified (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. An antibody that “preferentially binds” or “specifically binds” (used interchangeably herein) to 25 an epitope is a term well understood in the art, and methods to determine such specific or preferential binding are also well known in the art. A molecule is said to exhibit “specific binding” or “preferential binding” if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular cell or substance than it does with alternative cells or substances. An antibody “specifically binds” or “preferentially binds” to a 30 target if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances. In a non-limiting example, an antibody that specifically or preferentially binds to a CDCP1 epitope is an antibody that binds this epitope with greater affinity, avidity, more readily, and/or with greater duration than it binds to other CDCP1 epitopes or non-CDCP1 epitopes. It is also understood by reading this definition that, for 35 example, an antibody (or moiety, or epitope) which specifically or preferentially binds to a first DB1/ 159693888.5 25

133186-5030-WO target may or may not specifically or preferentially bind to a second target. As such, “specific binding” or “preferential binding” does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding. 5 “Specific binding” or “preferential binding” includes a compound, e.g., a protein, a nucleic acid, an antibody, and the like, which recognizes and binds to a specific molecule, but does not substantially recognize or bind other molecules in a sample. For instance, an antibody which recognizes and binds to its cognate antigen in a sample, but does not substantially recognize or bind other molecules in the sample, specifically binds to that cognate antigen. Thus, under10 designated assay conditions, the specified binding moiety (e.g., an antibody or an antigen- binding portion thereof) binds preferentially to a particular target molecule and does not bind in a significant amount to other components present in a test sample. The term “specific binding” or “specifically binds to” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide target as used herein can be exhibited, for 15 example, by a molecule having a Kd for the target of 10⁻⁴ M or lower, alternatively 10⁻⁵ M or lower, alternatively 10⁻⁶ M or lower, alternatively 10⁻⁷ M or lower, alternatively 10⁻⁸ M or lower, alternatively 10⁻⁹ M or lower, alternatively 10^-10 M or lower, alternatively 10⁻¹¹ M or lower, alternatively 10⁻¹² M or lower or a Kd in the range of 10⁻⁴ M to 10⁻⁶ M or 10⁻⁶ M to 10⁻¹⁰ M or 10⁻⁷ M to 10⁻⁹ M. As will be appreciated by the skilled artisan, affinity and KD values are 20 inversely related. A high affinity for an antigen is measured by a low KD value. In one embodiment, the term “specific binding” refers to binding where a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope. As used herein the terms “specific binding,” “specifically binds,” and “selectively binds,” refer to antibody binding to an epitope of CDCP1. 25 A variety of assays may be used to select an antibody or peptide that specifically binds a molecule of interest. For example, solid-phase ELISA immunoassay, immunoprecipitation, BIAcore™ (GE Healthcare, Piscataway, NJ), fluorescence-activated cell sorting (FACS), Octet™ (FortéBio, Inc., Menlo Park, CA) and Western blot analysis are among many assays that may be used to identify an antibody that specifically reacts with an antigen or a receptor, or 30 ligand binding portion thereof, that specifically binds with a cognate ligand or binding partner. Typically, a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 times background, even more specifically, an antibody is said to “specifically bind” an antigen when the equilibrium dissociation constant (K_D) value is less or equal to 1 µM, such as less or equal to 100 nM, less or equal to 10 nM, less or equal to 100 pM, 35 less or equal to 10 pM, or less or equal to 1 pM. DB1/ 159693888.5 26

133186-5030-WO The term “affinity,” as used herein, means the strength of the binding of an antibody to an epitope. The affinity of an antibody is given by the dissociation constant Kd, defined as [Ab]×[Ag]/[Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of the unbound antibody and [Ag] is the molar 5 concentration of the unbound antigen. The affinity constant Ka is defined by 1/Kd. Methods for determining the affinity of mAbs can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol.92:589-601 (1983), which references are entirely 10 incorporated herein by reference. One standard method well known in the art for determining the affinity of mAbs is the use of surface plasmon resonance (SPR) screening (such as by analysis with a BIAcore™ SPR analytical device). The term “compete”, as used herein with regard to an antibody, means that binding of a first antibody, or an antigen-binding portion thereof, to an antigen reduces the subsequent binding of 15 the same antigen by a second antibody or an antigen-binding portion thereof. In general, the binding a first antibody creates steric hindrance, conformational change, or binding to a common epitope (or portion thereof), such that the binding of the second antibody to the same antigen is reduced. Standard competition assays may be used to determine whether two antibodies compete with each other. One suitable assay for antibody competition involves the 20 use of the Biacore technology, which can measure the extent of interactions using surface plasmon resonance (SPR) technology, typically using a biosensor system (such as a BIACORE® system). For example, SPR can be used in an in vitro competitive binding inhibition assay to determine the ability of one antibody to inhibit the binding of a second antibody. Another assay for measuring antibody competition uses an ELISA-based approach. 25 The term “KD”, as used herein, is intended to refer to the dissociation constant of a particular antibody-antigen interaction. It is calculated by the formula: Koff/Kon=KD. The term “IC50”, as used herein, is intended to refer to the effective concentration of antibody of the present invention needed to neutralize 50% of the bioactivity of IL-23 on human lymphoma DB cells in the bioassay described in Example 5: Inhibition of STAT3 activation in 30 human DB cell Assay. “EC50” with respect to an agent and a particular activity (e.g., binding to a cell, inhibition of enzymatic activity, activation or inhibition of an immune cell), refers to the efficient concentration of the agent which produces 50% of its maximum response or effect with respect to such activity. “EC100” with respect to an agent and a particular activity refers to the efficient DB1/ 159693888.5 27

133186-5030-WO concentration of the agent which produces its substantially maximum response with respect to such activity. In one aspect, the inventors have surprisingly discovered antibody-drug conjugates (ADCs), wherein carbohydrate (e.g., sugar) moieties can be successfully incorporated on to specific 5 positions of a pyrridinobenzodiazepine (PDD) portion of the ADCs, thereby enhancing hydrophilicity of the payload itself. The specific locations selected allow the addition of a sugar group to the molecule, without adversely interfering with the ability of the released agent to kill cancer cells. This is advantageous as substitutions on certain positions of the heterocyclic chain on a payload can alter DNA binding and cytotoxicity. In a non-limiting example, Fig.21 shows 10 non-limiting examples of second generation linker payloads with sugar moieties at specific positions of the PDDs. In the case of the subject material, the liberated parent can bind effectively to DNA resulting in potent cytotoxicity. The glycosylated substituent may act as a prodrug moiety that is selectively cleaved at the tumour site, so an ADC containing the glycosylated payload produces potent in vivo efficacy (similar to the unsubstituted parent 15 molecule) but has a substantially increased tolerability profile, thereby widening the therapeutic window substantially. In some embodiments, the compounds and/or conjugates thereof described herein that comprise at least one glycosylated substituent have enhanced or increased hydrophilicity compared to said compounds that do not comprise at least one glycosylated substituent. In some embodiments, the compounds and/or conjugates thereof described herein 20 that comprise at least one glycosylated substituent have enhanced or increased tolerability compared to said compounds that do not comprise at least one glycosylated substituent. When combined, these traits result in highly effective compounds. Thus, the inventors have discovered in part that the incorporation of sugar moieties into the structure of a PDD provides favourable properties for efficient conjugation, efficacy and tolerability. 25 There is nevertheless a need for improved cytotoxic payloads especially those that may be useful in ADCs. For example, 50 mg/kg is an established single dose ‘platform’ MTD for linker-payload N-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3- methylbutanamido)propanamido)phenyl)-4-(4-(((S)-2-methoxy-12-oxo-6a,7,8,9,10,12- hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2- 30 carboxamide (i.e., control 3) when conjugated to multiple antibodies at DAR 4, and the effective dose is 10 mg/kg with regressions observed at 5 mg/kg. Given the hydrophobic nature of the construct, it is not possible to produce ADCs at higher DARs (which are thought to have better bystander effect than DAR 4) with control 3. ANTIBODY-DRUG CONJUGATES DB1/ 159693888.5 28

133186-5030-WO In aspects, the disclosure provides antibody-drug conjugates (ADCs). In embodiments, ADCs comprise an antibody, linker and payload. In embodiments, linker payload complexes contain hydrophilic groups (e.g. in the form of extended PEG) in order to reduce hydrophobicity and promote efficient conjugation of the structure to the antibody. In embodiments, DNA-interactive 5 agents are usually particularly hydrophobic in nature, leading to significant issues with PK and during the conjugation process. As disclosed herein, the use of sugar groups in the payload structure itself as masking agents, surprisingly reduces hydrophobicity, enhances PK properties and increases the tolerability of the ADC. This is particularly advantageous because it may result in (a) more efficient conjugation than other classes of payloads, (b) similar cytotoxicity to the 10 unsubstituted form of the payload with significantly improved tolerability and conjugation properties compared to molecules in the prior art, and (c) enhanced PK profile compared to unsubstituted agents. In one aspect, the disclosure provides an antibody-drug conjugate of formula (I): Ab-[L–D]n 15 formula (I) wherein in formula (I): Ab is an antibody or binding fragment thereof that binds CUB Domain-Containing Protein-1 (CDCP1); D is a drug moiety comprising a pyrridinobenzodiazepine (PDD); 20 L is a linker; wherein D and/or L comprise at least one carbohydrate substituent R_S. In embodiments, the carbohydrate substituent RS is a univalent saccharide substituent. In embodiments RS is glycosyl or O-glycosyl. In embodiments, D comprises a moiety of formula (IIa) 25 DB1/ 159693888.5 29

133186-5030-WO wherein: the dotted line indicates the optional presence of a double bond between one or more of C1 and C2, C2 and C3, and C3 and C4; the wavy line indicates the point of attachment to L; 5 m is 0 or 1; R₁, R₃ and R₄ are independently selected from H and R₂₉; R₂ is selected from H, R_S, L₂-R_58, R₂₉, and –L_S-R_S, or one of R₁ and R₂, R₂ and R₃, or R₃ and R₄, together with the carbon atoms to which they are attached form a 6-membered aryl, or a 5- or 6-membered cyclic, heterocyclic, or heteroaryl ring optionally substituted with 1, 2 or 3 independently10 selected optional R20 groups; R5 and R6 are selected such that either (i) R5 is selected from H, OH and OC1-6 alkyl; and R6 is selected from H, SO₃H, –L_S-R_S, nitrogen protecting groups, –L₂-R₅₈ and R_A; (ii) R₅ is oxo or H, and R₆ is H or C_1-6 alkyl; or (iii) R₅ and R₆ together form a double bond; R_{7 and R9 are independently selected from H and R20;} 15 R8 is selected from H, SR24, SCH2Ph, R20, L2-R58, and –LS-RS; RA is selected from (CH2)j-OH, (CH2)j-CO2R26, C(=O)-O-(CH2)k-NR26R27, (CH2)jNR26R27, C(=O)-NH-(CH2)j-NR26R27 and C(=O)-NH-(CH2)k-C(=NH)NR26R27; L₂ is a bond or a lin P, O, S or halogen, and optionally incorporates ether, oxo, carboxamidyl, urethanyl, branched,20 cyclic, unsaturated, heterocyclyl, aryl or heteroaryl moieties; and R58 is RA, O-(CH2)k-NR26R26, or NHNH2; each R29 is independently selected from R20, R21, =CH2, =CH-(CH2)s-CH3, =CH-(CH2)s-R21, =O, (CH2)s-OR21, (CH2)s-CO2R21, (CH2)s-NR21R24, O-(CH2)t-NR21R24, NH-C(O)-R21, O- (CH₂)_t-NH-C(O)-R₂₁, O-(CH₂)_t-C(O)-NH-R₂₁, (CH₂)_s-SO₂R₂₁, O-SO₂R₂₁, (CH₂)_s-C(O)R₂₁25 and (CH₂)_s-C(O)NR₂₁R₂₄; each R₂₀ is independently selected from F, Cl, Br, (CH₂)_j-OH, C_1-6 alkyl, OC_1-6 alkyl, R_S, OCH2Ph, (CH2)j-CO2R26, O-(CH2)k-NR26R27, C(=O)-O-(CH2)k-NR26R27, C(=O)-NR26R27, (CH2)j-NR26R27, NR26NH2, C(=O)-NH-(CH2)j-NR26R27, C(=O)-NH-C6H4-(CH2)j-R26, C(=O)-NH-(CH2)k-C(=NH)NR26R27, –L2-R58, S(O)2-(C1-6 alkyl), O-(CH2)k-O-(C1-6 alkyl), 30 (CH₂)_j-S(O)₂-NR₂₆R₂₇, C(=NH)-O-(C_1-6 alkyl), (CH₂)_k-O-(C_1-6 alkyl), CN, NCO, Cy, nd , , , , , or 6; each k and t is independently selected from 1, 2, 3, 4, 5 or 6; DB1/ 159693888.5 30

133186-5030-WO each R₂₁ is independently selected from H, C_1-12 alkyl, C_5-6 heterocyclyl, C_5-9 heteroaryl, C_6-15 heteroarylalkyl, phenyl and C7-12 aralkyl groups; wherein the heterocyclyl, heteroaryl, heteroarylalkyl, phenyl and aralkyl groups are optionally substituted with 1, 2 or 3 independently selected optional R20 groups; 5 each R24, R26 and R27 is independently selected from H and C1-12 alkyl; each Cy is independently selected from a C_5-6 heterocyclyl or C_5-6 heteroaryl group, wherein the heterocyclyl or heteroaryl groups are optionally substituted with 1 or 2 R₂₀ groups; L_S is a bond, an amino acid, a peptide chain having from 2 to 6 amino acids, an alkylene chain containing from 1 to 12 carbon atoms which may contain one or more carbon-carbon 10 double or triple bonds, a paraformaldehyde chain –(OCH2)1-12-, a polyethylene glycol chain -(OCH2CH2)1-6-, which chains may be interrupted by or optionally incorporates one o_{r more of P, O, S, NH, C5-9 heteroarylene, phenylene, heterocyclyl, cycloalkyl, ether,} oxo, carboxamidyl, and/or urethanyl moieties wherein the C_5-9 heteroarylene, phenylene, heterocyclyl, and/or cycloalkyl moieties are optionally substituted, optionally LS is: L_{C O} HN _O 15 ; m an amino acid, an amino acid derivative, a peptide chain having from 2 to 6 amino acids or amino acid derivatives, an alkylene chain containing from 1 to 12 carbon atoms which may contain one or more carbon-carbon double or triple bonds, a paraformaldehyde chain –(OCH2)1-12-, a polyethylene glycol 20 chain -(OCH₂CH₂)_1-8-, which chains may be interrupted by one or more P, O, S and/or NH g_{roups and/or C5-9 heteroarylene and/or phenylene, wherein each C5-9 heteroarylene group} and/or each phenylene group is optionally substituted; and RS is a univalent saccharide substituent, preferably glycosyl or O-glycosyl. In one aspect, the disclosure provides an antibody-drug conjugate of formula (I): 25 Ab-[L–D]_n formula (I) wherein in formula (I): Ab is a means for binding CUB Domain-Containing Protein-1 (CDCP1) (e.g., an antibody comprising a heavy chain variable region sequence as set forth in SEQ ID NO: 1, 15,30 or 23-158 and a light chain variable region sequence as set forth in SEQ ID NO: 5, 19, or 159- 295, respectively); D is a drug moiety comprising a pyrridinobenzodiazepine (PDD); L is a linker; wherein D and/or L comprise at least one carbohydrate substituent RS. DB1/ 159693888.5 31

133186-5030-WO In embodiments, the carbohydrate substituent R_S is a univalent saccharide substituent. In embodiments RS is glycosyl or O-glycosyl. In embodiments, D comprises a moiety of formula (IIa) 5 wherein: the dotted line indicates the optional presence of a double bond between one or more of C1 and C2, C2 and C3, and C3 and C4; the wavy line indicates the point of attachment to L; 10 m is 0 or 1; R1, R3 and R4 are independently selected from H and R29; R2 is selected from H, RS, L2-R58, R29, and –LS-RS, or one of R1 and R2, R2 and R3, or R3 and R4, together with the carbon atoms to which they are attached form a 6-membered aryl, or a 5- or 6-membered cyclic, heterocyclic, or heteroaryl ring optionally substituted with 1, 2 or 3 independently15 selected optional R₂₀ groups; R₅ and R₆ are selected such that either (i) R₅ is selected from H, OH and OC_1-6 alkyl; and R₆ is selected from H, SO3H, –LS-RS, nitrogen protecting groups, –L2-R58 and RA; (ii) R5 is oxo or H, and R6 is H or C1-6 alkyl; or (iii) R5 and R6 together form a double bond; R_{7 and R9 are independently selected from H and R20;} 20 R8 is selected from H, SR24, SCH2Ph, R20, L2-R58, and –LS-RS; R_A is selected from (CH₂)_j-OH, (CH₂)_j-CO₂R₂₆, C(=O)-O-(CH₂)_k-NR₂₆R₂₇, (CH₂)_jNR₂₆R₂₇, C(=O)-NH-( L2 is a bond or a lin P, O, S or halogen, and optionally incorporates ether, oxo, carboxamidyl, urethanyl, branched,25 cyclic, unsaturated, heterocyclyl, aryl or heteroaryl moieties; and R58 is RA, O-(CH2)k-NR26R26, or NHNH2; each R₂₉ is independently selected from R₂₀, R₂₁, =CH₂, =CH-(CH₂)_s-CH₃, =CH-(CH₂)_s-R₂₁, =O, (CH₂)_s-OR₂₁, (CH₂)_s-CO₂R₂₁, (CH₂)_s-NR₂₁R₂₄, O-(CH₂)_t-NR₂₁R₂₄, NH-C(O)-R₂₁, O- DB1/ 159693888.5 32

133186-5030-WO (CH₂)_t-NH-C(O)-R₂₁, O-(CH₂)_t-C(O)-NH-R₂₁, (CH₂)_s-SO₂R₂₁, O-SO₂R₂₁, (CH₂)_s-C(O)R₂₁ and (CH2)s-C(O)NR21R24; each R20 is independently selected from F, Cl, Br, (CH2)j-OH, C1-6 alkyl, OC1-6 alkyl, RS, OCH2Ph, (CH2)j-CO2R26, O-(CH2)k-NR26R27, C(=O)-O-(CH2)k-NR26R27, C(=O)-NR26R27, 5 (CH2)j-NR26R27, NR26NH2, C(=O)-NH-(CH2)j-NR26R27, C(=O)-NH-C6H4-(CH2)j-R26, C(=O)-NH-(CH₂)_k-C(=NH)NR₂₆R₂₇, –L₂-R₅₈, S(O)₂-(C_1-6 alkyl), O-(CH₂)_k-O-(C_1-6 alkyl), (CH₂)_j-S(O)₂-NR₂₆R₂₇, C(=NH)-O-(C_1-6 alkyl), (CH₂)_k-O-(C_1-6 alkyl), CN, NCO, Cy, C(O)-NH-(CH₂)_j-Cy, C(O)-Cy, NH-C(O)-NR₂₆R₂₇ and 10 or 6; each k and t is independently selected from 1, 2, 3, 4, 5 or 6; each R₂₁ is independently selected from H, C_1-12 alkyl, C_5-6 heterocyclyl, C_5-9 heteroaryl, C_6-15 heteroarylalkyl, phenyl and C_7-12 aralkyl groups; wherein the heterocyclyl, heteroaryl, heteroarylalkyl, phenyl and aralkyl groups are optionally substituted with 1, 2 or 3 15 independently selected optional R20 groups; each R24, R26 and R27 is independently selected from H and C1-12 alkyl; each Cy is independently selected from a C5-6 heterocyclyl or C5-6 heteroaryl group, wherein the heterocyclyl or heteroaryl groups are optionally substituted with 1 or 2 R₂₀ groups; L_S is a bond, an amino acid, a peptide chain having from 2 to 6 amino acids, an alkylene chain 20 containing from 1 to 12 carbon atoms which may contain one or more carbon-carbon double or triple bonds, a paraformaldehyde chain –(OCH2)1-12-, a polyethylene glycol chain -(OCH2CH2)1-6-, which chains may be interrupted by or optionally incorporates one o_{r more of P, O, S, NH, C5-9 heteroarylene, phenylene, heterocyclyl, cycloalkyl, ether,} oxo, carboxamidyl, and/or urethanyl moieties wherein the C_5-9 heteroarylene, phenylene,25 heterocyclyl, and/or cycloalkyl moieties are optionally substituted, optionally L_S is: L_{C O O} ; m an amino acid, an amino acid derivative, a peptide chain having from 2 to 6 amino acids or amino acid derivatives, an alkylene chain containing from 1 to 12 carbon atoms which may contain one or more carbon-carbon 30 double or triple bonds, a paraformaldehyde chain –(OCH2)1-12-, a polyethylene glycol chain -(OCH2CH2)1-8-, which chains may be interrupted by one or more P, O, S and/or NH DB1/ 159693888.5 33

133186-5030-WO g_{roups and/or C5-9 heteroarylene and/or phenylene, wherein each C5-9 heteroarylene group} and/or each phenylene group is optionally substituted; and RS is a univalent saccharide substituent, preferably glycosyl or O-glycosyl. 5 In embodiments, D is a moiety of formula (IIa). In embodiments, the moiety of formula (IIa) is a moiety of any one of formula (G1) to (G8): R₈ DB1/ 159693888.5 34

133186-5030-WO In embodiments, D comprises a moiety of formula (IIb): R₅ R₇ R₁ H N Q T' B 5 wherein: the dotted line indicates the optional presence of a double bond between one or more of C1 and C2, C2 and C3, and C3 and C4; 10 the wavy line indicates the point of attachment to L; m is 0 or 1; R1, R3 and R4 are independently selected from H and R29; R2 is selected from H, L2-R58, R29, and –LS-RS, or one of R1 and R2, R2 and R3, or R3 and R4, together with the carbon atoms to which they are attached form a 6-membered aryl, or a 5- or 6-membered cyclic, 15 heterocyclic, or heteroaryl ring optionally substituted with 1, 2 or 3 independently selected optional R₂₀ groups; R5 is selected from H, OH and OC1-6 alkyl; R_{7 and R9 are independently selected from H and R20;} R8 is selected from H, SR24, SCH2Ph, R20, L2-R58, and –LS-RS; 20 R_A is selected from (CH₂)_j-OH, (CH₂)_j-CO₂R₂₆, C(=O)-O-(CH₂)_k-NR₂₆R₂₇, (CH₂)_jNR₂₆R₂₇, C(=O)-NH-( L₂ is a bond or a lin P, O, S or halogen, and optionally incorporates ether, oxo, carboxamidyl, urethanyl, branched, cyclic, unsaturated, heterocyclyl, aryl or heteroaryl moieties; and 25 R58 is RA, O-(CH2)k-NR26R26, or NHNH2; each R29 is independently selected from R20, R21, =CH2, =CH-(CH2)s-CH3, =CH-(CH2)s-R21, =O, (CH₂)_s-OR₂₁, (CH₂)_s-CO₂R₂₁, (CH₂)_s-NR₂₁R₂₄, O-(CH₂)_t-NR₂₁R₂₄, NH-C(O)-R₂₁, O- (CH₂)_t-NH-C(O)-R₂₁, O-(CH₂)_t-C(O)-NH-R₂₁, (CH₂)_s-SO₂R₂₁, O-SO₂R₂₁, (CH₂)_s-C(O)R₂₁ and (CH2)s-C(O)NR21R24; DB1/ 159693888.5 35

133186-5030-WO each R₂₀ is independently selected from F, Cl, Br, (CH₂)_j-OH, C_1-6 alkyl, OC_1-6 alkyl, OR_S, OCH2Ph, (CH2)j-CO2R26, O-(CH2)k-NR26R27, C(=O)-O-(CH2)k-NR26R27, C(=O)-NR26R27, (CH2)j-NR26R27, NR26NH2, C(=O)-NH-(CH2)j-NR26R27, C(=O)-NH-C6H4-(CH2)j-R26, C(=O)-NH-(CH2)k-C(=NH)NR26R27, –L2-R58, S(O)2-(C1-6 alkyl), O-(CH2)k-O-(C1-6 alkyl), 5 (CH2)j-S(O)2-NR26R27, C(=NH)-O-(C1-6 alkyl), (CH2)k-O-(C1-6 alkyl), CN, NCO, Cy, C(O)-NH-(CH₂)_j-Cy, C(O)-Cy, NH-C(O)-NR₂₆R₂₇ and r 6; each k and t is independently selected from 1, 2, 3, 4, 5 or 6; 10 each R21 is independently selected from H, C1-12 alkyl, C5-6 heterocyclyl, C5-9 heteroaryl, C6-15 heteroarylalkyl, phenyl and C_7-12 aralkyl groups; wherein the heterocyclyl, heteroaryl, heteroarylalkyl, phenyl and aralkyl groups are optionally substituted with 1, 2 or 3 independently selected optional R₂₀ groups; each R24, R26 and R27 is independently selected from H and C1-12 alkyl; 15 each Cy is independently selected from a C5-6 heterocyclyl or C5-6 heteroaryl group, wherein the heterocyclyl or heteroaryl groups are optionally substituted with 1 or 2 R20 groups; LS is a bond, an amino acid, a peptide chain having from 2 to 6 amino acids, an alkylene chain containing from 1 to 12 carbon atoms which may contain one or more carbon-carbon double or triple bonds, a paraformaldehyde chain –(OCH₂)_1-12-, a polyethylene glycol 20 chain -(OCH2CH2)1-6-, which chains may be interrupted by or optionally incorporates one o_{r more of P, O, S, NH, C5-9 heteroarylene, phenylene, heterocyclyl, cycloalkyl, ether,} oxo, carboxamidyl, and/or urethanyl moieties wherein the C5-9 heteroarylene, phenylene, heterocyclyl, and/or cycloalkyl moieties are optionally substituted, optionally LS is: ; 25 charide substituent, preferably glycosyl or O-glycosyl; Q is a linker; B is an DNA binding amide-containing chain; and T’ is an end group. DB1/ 159693888.5 36

133186-5030-WO In embodiments, D is a moiety of formula (IIb). In embodiments, the moiety of formula (IIb) is a moiety of any one of formula (H1) to (H8): In embodiments, R1 is H. In embodiments, R3 is H. In embodiments, R7 is H. In embodiments, R₉ is H. In embodiments, one or more of R₁, R_3, R₇, and R₉ are each H. In embodiments, each of R₁, R_3, R₇, and R₉ are H. DB1/ 159693888.5 37

133186-5030-WO RS In some embodiments, the carbohydrate substituent of the compounds of the disclosure (e.g. a compound of formula (I)) is a univalent saccharide substituent, represented by RS. In some 5 embodiments, R_S is glycosyl or O-glycosyl. “Saccharide”, refers to a sugar that may be a monosaccharide or disaccharide, and “RS” refers to monosaccharide and/or disaccharide univalent substituents derived from a monosaccharide and/or a disaccharide. The saccharide may suitably be a pentose or hexose or a disaccharides containing a pentose and/or a hexose. Examples of monosaccharides include glucose, fructose, 10 galactose, ribose, ribulose and stereoisomers of these sugars. In this specification, saccharide refers to the sugar in the furanose, acyclic, and/or pyranose forms, and formulae showing sugars in one form are also meant to include the sugar in the other forms unless the context otherwise requires. Saccharides that are univalent substituents may comprise glycosyl groups of the corresponding mono (or di-) saccharide obtainable by removing a hydroxyl group from 15 monosaccharide or disaccharide or by removing a hydrogen from a hydroxyl group of the mono- or di-saccharide. Non-limiting examples of furanoses include arabinofuranose, lyxofuranose, ribofuranose, xylofuranse, etc. Non-limiting examples of pyranoses include aliopyranose, altropyranose, glucopyranose, mannopyranose, gulopyranose, idopyranose, galactopyranose, talopyranose, etc. 20 In embodiments, RS is selected from any one of formula (S11), (S12), or (S13): wherein R49 is independently at each occurrence selected from H and C1-6 alkyl, optionally methyl. 25 In embodiments, R_S is selected from any one of formula (S101), (S102), or (S103): . DB1/ 159693888.5 38

133186-5030-WO In embodiments, wherein R_S is selected from any one of formula (S21), (S22), (S23), or (S24): wh 5 me y . In embodiments, RS is selected from any one of formula (S201), (S202), (S203), or (S204): 10 In embodiment, D comprises at least one RS. In embodiments, L comprises at least one RS. in embodiments, L and D each independently comprise at least one R_S. X1 In embodiments, X1 is selected from O, S, NH, CH2, CH2O, C(=O), C(=O)NR13, NR13C(=O), O- 15 C(O) and C(O)-O; In embodiments, X1 is selected from O, C(=O), C(=O)NR13 and NR13C(=O). In embodiments, X₁ is selected from O, C(=O)NH and NHC(=O). In embodiments, X1 is O. X2 20 In embodiments, X2 is selected from O, S, NH, CH2, CH2O, C(=O), C(=O)NR15, NR15C(=O), O- C(O) and C(O)O or is absent. In embodiments, X₂ is C(=O)NR₁₅ or NR₁₅C(=O). In embodiments, R₁₅ is H. In embodiments, X₂ is selected from O, C(=O)NH and NHC(=O). In embodiments, X2 is the same as X1. DB1/ 159693888.5 39

133186-5030-WO In embodiments, X₂ is O. L In embodiments, L is a linker group. Suitably, any of the peptide chain, alkylene chain, paraformaldehyde chain or polyethylene glycol chain may be interrupted by, or substituted with, 5 one or more hetero-atoms (e.g., P, N or NH, O, and S) and/or one or more C5-9 heteroarylene groups (e.g., pyrrolylene, pyrazolylene, pyrazolylene, 1,2,3-triazolylene, pyridinylene) and/or one or more phenylene groups, wherein each C5-9 heteroarylene group (e.g., pyrrolylene, pyrazolylene, pyrazolylene, 1,2,3-triazolylene, pyridinylene) and/or each phenylene group is optionally substituted. In embodiments, the chains may be interrupted by from one to three 10 hetero-atoms (e.g., P, O, S, NH) and/or from one to three C_5-9 heteroarylene groups and/or from one to three phenylene groups. In embodiments, L is an amino acid, a peptide chain having from 2 to 6 amino acids, an alkylene chain containing from 1 to 12 carbon atoms which may contain one or more carbon- carbon double or triple bonds, a paraformaldehyde chain –(OCH2)1-12-, a polyethylene glycol 15 chain -(OCH2CH2)1-6-, which chains may be interrupted by or optionally incorporates one or m_{ore of O, S, NH, C5-9 heteroarylene, phenylene, heterocyclyl, cycloalkyl, ether, oxo,} carboxamidyl, and/or urethanyl moieties wherein the C5-9 heteroarylene, phenylene, heterocyclyl, and/or cycloalkyl moieties are optionally substituted. In embodiments, the peptide, alkylene, paraformaldehyde, and polyethylene glycol chains and/or the C_5-9 20 heteroarylene, phenylene, heterocyclyl, and cycloalkyl moieties are optionally substituted with 1, 2 or 3 independently selected optional R20 groups and/or Ls-Rs groups. In embodiments, L may comprise L₂-R₅₈ and/or L_s-R_s. In some embodiments, L may comprise L₂-R₂₈. In some embodiments, L may comprise L_s-R_s. In embodiments, linker L may comprise one or more groups selected from sulfamino, sulfamyl, 25 sulfate, sulfhydryl, sulfinamino, sulfinate, sulfino, sulfinyl, sulfinyloxy, sulfo, sulfonamido, sulfonamino, sulfonate, sulfonyl, sulfonyloxy, phosphate ester, phosphoramidate, thiophosphate ester, phosphonate, and thiophosphonate. In embodiments, linker L and/or L₂ comprises a moiety that permits branching. In some embodiments, linker L and/or L₂ comprises the formula: 30 ; DB1/ 159693888.5 40

133186-5030-WO wherein R_S is a univalent saccharide substituent, preferably glycosyl or O-glycosyl; and L_C is selected from an amino acid, an amino acid derivative, a peptide chain having from 2 to 6 amino acids or amino acid derivatives, an alkylene chain containing from 1 to 12 carbon atoms which may contain one or more carbon-carbon double or triple bonds, a paraformaldehyde chain – 5 (OCH₂)_1-12-, a polyethylene glycol chain –(OCH₂CH₂)_1-8-, which chains may be interrupted by o_{ne or more P, O, S and/or NH groups and/or C5-9 heteroarylene and/or phenylene, wherein each} C_5-9 heteroarylene group and/or each phenylene group is optionally substituted. In embodiments, L is or comprises selected from a peptide chain having from 2 to 5 amino acids, from 2 to 4 amino acids, from 2 to 3 amino acids; an alkylene chain containing from 1 to 10 11 carbon atoms, from 1 to 10 carbon atoms, from 1 to 9 carbon atoms, from 1 to 8 carbon atoms, from 1 to 7 carbon atoms, from 1 to 6 carbon atoms, from 1 to 5 carbon atoms, from 1 to 4 carbon atoms, from 1 to 3 carbon atoms, which may contain one or more carbon-carbon double or triple bonds; a paraformaldehyde chain –(OCH2)1-12-, –(OCH2)1-11-, –(OCH2)1-10-, – (OCH2)1-9-, –(OCH2)1-8-, –(OCH2)1-7-, –(OCH2)1-6-, –(OCH2)1-5-, –(OCH2)1-4-, –(OCH2)1-3- a 15 polyethylene glycol chain –(OCH₂CH₂)_1-5-, chain –(OCH₂CH₂)_1-4-, chain –(OCH₂CH₂)_1-3-; which chain may be interrupted by one or more hetero-atoms and/or from one to three C_5-9 heteroarylene groups and/or from one to three phenylene groups, wherein the C_5-9 heteroarylene and/or phenylene groups are optionally substituted with 1, 2 or 3 independently selected optional R20 groups and/or Ls-Rs groups. In embodiments, L comprises one or more C5-9 20 heteroarylene and/or phenylene groups optionally substituted with L2-R28 and/or Ls-Rs. In embodiments, L comprises one or more C_5-9 heteroarylene and/or phenylene groups optionally substituted with L₂-R₂₈. In some embodiments, L comprises one or more C_5-9 heteroarylene and/or phenylene groups optionally substituted with Ls-Rs. In embodiments, L is or comprises an alkylene chain containing from 1 to 12 carbon atoms 25 which may contain one or more carbon-carbon double or triple bonds, optionally wherein L is an alkylene chain containing 3 carbon atoms. In embodiments, L is or comprises an alkylene chain containing from 1 to 12 carbon atoms which may contain one or more carbon-carbon double or triple bonds. In embodiments, L is or comprises CH=CH, CH2, CH2CH2, CH2CH2CH2, CH2CH2CH2CH2 and 30 CH2CH2CH2CH2CH2. DB1/ 159693888.5 41

133186-5030-WO In embodiments, –L–D has the formula (IVa): wherein: 5 L has the formula –Q–B–T–; Q is a linker; B is an DNA binding amide-containing chain; and T is an end group. 10 In embodiments, –L–D has the formula (IVb): In embodiments, L comprises –L_S2–L_C2–Z^*–, wherein: 15 LS2 is a bond, an amino acid, a peptide chain having from 2 to 6 amino acids, an alkylene chain containing from 1 to 12 carbon atoms which may contain one or more carbon-carbon double or triple bonds, a paraformaldehyde chain –(OCH2)1-12-, a polyethylene glycol chain -(OCH2CH2)1-6-, which chains may be interrupted by or o_{ptionally incorporates one or more of P, O, S, NH, C5-9 heteroarylene, phenylene,} 20 heterocyclyl, cycloalkyl, ether, oxo, carboxamidyl, and/or urethanyl moieties wherein the C_5-9 heteroarylene, phenylene, heterocyclyl, and/or cycloalkyl moieties are optionally substituted, optionally LS2 is: d LC2 oups selected from an amino acid, an amino acid 25 derivative, a peptide chain having from 2 to 6 amino acids or amino acid derivatives, an alkylene chain containing from 1 to 12 carbon atoms which may contain one or more carbon-carbon double or triple bonds, a paraformaldehyde DB1/ 159693888.5 42

133186-5030-WO chain –(OCH₂)_1-12-, a polyethylene glycol chain -(OCH₂CH₂)_1-8-, which chains m_{ay be interrupted by one or more P, O, S and/or NH groups and/or C5-9} heteroarylene and/or phenylene, wherein each C5-9 heteroarylene group and/or each phenylene group is optionally substituted; and 5 Z^* is a reactive moiety that has reacted with a functional group. In embodiments, Q comprises X₁-L-X₂, wherein: X1 is selected from O, S, NR13, CR13R14, CR13R14O, C(=O), C(=O)NR13, NR13C(=O), O-C(O)10 and C(O)-O, or is absent; L is selected from an amino acid, a peptide chain having from 2 to 6 amino acids, an alkylene chain containing from 1 to 12 carbon atoms which may contain one or more carbon- carbon double or triple bonds, a paraformaldehyde chain –(OCH₂)_1-12-, a polyethylene glycol chain -(OCH2CH2)1-6-, which chains may be interrupted by one or more P, O, S _{15 and/or NH groups and/or C5-9 heteroarylene and/or phenylene, wherein each C5-9} heteroarylene group and/or each phenylene group is optionally substituted; X2 is selected from O, S, NR15, CR15R16, CR15R16O, C(=O), C(=O)NR15, NR15C(=O), O-C(O) and C(O)-O or is absent; and R₁₃, R_14, R₁₅ and R₁₆ are independently selected from H and C_1-6 alkyl. 20 L2 In embodiments, linker L2 is a bond or is a moiety having 1-200 nonhydrogen atoms selected from C, N, P, O, S, or halogen, and optionally incorporates ether, oxo, carboxyl, carboxamide, carboxamidyl, urethanyl, branched, cyclic, unsaturated, amino acid, heterocyclyl, aryl or 25 heteroaryl moieties. In embodiments, linker L₂ is a bond or comprises one or more groups selected from an amino acid, a peptide chain having from 2 to 100 amino acids, an alkylene chain containing from 1 to 50 carbon atoms which may contain one or more carbon-carbon double or triple bonds, a paraformaldehyde chain –(OCH2)1-50-, and a polyethylene glycol chain -(OCH2CH2)1-50-, which chains may be interrupted by or optionally incorporate one or more of _{30 P, O, S, NH, C5-9 heteroarylene, phenylene, heterocyclyl, cycloalkyl, ether, oxo, carboxamidyl,} and/or urethanyl moieties wherein the C_5-9 heteroarylene, phenylene, heterocyclyl, and/or cycloalkyl moieties are optionally substituted. In some embodiments, the peptide, alkylene, paraformaldehyde, and polyethylene glycol chains and/or the C5-9 heteroarylene, phenylene, heterocyclyl, and cycloalkyl moieties are optionally substituted with 1, 2 or 3 independently 35 selected optional R20 groups and/or Ls-Rs groups. In embodiments, L2 is unbranched or branched, flexible or rigid, short or long and may incorporate any combination of moieties as deemed useful. In embodiments, at least a portion DB1/ 159693888.5 43

133186-5030-WO of L₂ has a polyalkylene oxide polymeric region, which may enhance solubility of the antibody- drug conjugate of formula (I). In embodiments, L2 has a repeating unit of ethylene glycol, and optionally has a number of repeating ethylene glycol units of about 1 to about 25, or any number therebetween. In embodiments, L2 includes about 3 to about 20, about 4 to about 15, 5 about 5 to about 12 or about 6 to about 10 ethylene glycol units. In embodiments, at least a portion of L₂ includes one or more amino acid moieties which provide enhanced solubility for the antibody-drug conjugate of formula (I) and/or may provide amino acid sequences to enhance target binding, enhance compatibility with a targeting agent, and/or enhance target binding recognition. In embodiments, L2 includes one or more amino acid moieties that provide a 10 suitable substrate motif for a protease. In embodiments, when a set of amino acid moieties are incorporated into the L2 that provide a substrate motif specific for a selected protease, the cytotoxic drug compound of the ADC (e.g. the ADC of formula (I)) may be released from a target bound conjugate to provide localized cytotoxic effects. Such substrate motifs are known in the art and may be incorporated into L2 as desired to provide selective release from the target 15 bound conjugate. This selectivity can be based on known presence of a desired protease within the localized delivery region of the conjugate drug. In embodiments, other polymeric types of moieties are incorporated in L2, such as polyacids, polysaccharides, or polyamines. In embodiments, other moieties such as substituted aromatic or heteroaromatic moieties are used to enhance rigidity or provide synthetically accessible sites on substituents therein for linking to 20 reactive moieties or to form the antibody-drug conjugate of formula (I). In embodiments, linker L₂ comprises one or more groups selected from sulfamino, sulfamyl, sulfate, sulfhydryl, sulfinamino, sulfinate, sulfino, sulfinyl, sulfinyloxy, sulfo, sulfonamido, sulfonamino, sulfonate, sulfonyl, sulfonyloxy, phosphate ester, phosphoramidate, thiophosphate ester, phosphonate, and thiophosphonate. 25 In embodiments, L₂ includes a variety of other connecting groups that connect the ethylene glycol portion to the amino acid sequence and/or to a drug moiety of the ADC. For example, the amino acid sequence can be connected to a drug moiety of the ADC via a 4- amino benzyl carboxylate group. In embodiments, the ethylene glycol portion can be directly linked to R28. In some embodiments, the linker L2 has the formula: 30 . In embodiments, L₂ may be selected from: DB1/ 159693888.5 44

133186-5030-WO (i) (ii) (iii) (iv) ; 5 In embodiments, X_AA may be L-valyl-L-alanine. For example, the linker L2 can include ethylene glycol repeating units, and/or an amino acid sequence. In some embodiments, linker L₂ includes the formula: -[CH₂CH₂O]_0-50-X_AA- 10 wherein X_AA is an amino acid sequence. In some embodiments, the linker L₂ comprises or consists of the formula: HN L_C L_{C O ;} t, preferably glycosyl or O-glycosyl. 15 In some embodiments, the linker L2 is or comprises: HN L O . In embodiments, LC comprises one or more groups selected from an amino acid, an amino acid 20 derivative, a peptide chain having from 2 to 6 amino acids or amino acid derivatives, an alkylene chain containing from 1 to 12 carbon atoms which may contain one or more carbon- carbon double or triple bonds, a paraformaldehyde chain –(OCH₂)_1-12-, a polyethylene glycol DB1/ 159693888.5 45

133186-5030-WO chain -(OCH₂CH₂)_1-8-, which chains may be interrupted by one or more P, O, S and/or NH g_{roups and/or C5-9 heteroarylene and/or phenylene, wherein each C5-9 heteroarylene group and/or} each phenylene group is optionally substituted. In some embodiments, LC has the formula , optionally or . 5 In some embodiments, L_C comprises a po y y gy - , p nally - (OCH₂CH₂)₈-. In some embodiments, LC is selected from: , nd 10 In embodiments, L_S includes ethylene glycol repeating units, and/or an amino acid sequence. In embodiments, LS2 is or comprises the formula: -[CH2CH2O]0-50-XAA- wherein XAA is an amino acid sequence. 15 O)- DB1/ 159693888.5 46

133186-5030-WO X20 is a single bond, , or . In some embodiments, LC has the formula . In em o e s, C as e o mula . In some embodiments, p is an m 5 to 10, e.g.5, 6, 7, 8, 9, or 10. In s iments, p is 8. In some embodiments, X₁₀ is -HN-[CH₂-CH₂-O]_p-(CH₂)₂ -C(O)-. In embodiments, X₁₀ is a single bond. 5 In some embodiments, X₁₀ is -HN-[CH₂-CH₂-O]_p-(CH₂)₂-C(O)-N-. In embodiments, X₁₀ is . In embodiments, Alk is -CH3. In embodiments, X20 is a single bond. In embodiments, X20 is . In embodiments, X20 is . L S2 In embodiments, LS2 is a bond or a linker moiety. In embodiments, LS2 is or comprises a bond, 10 an amino acid, a peptide chain having from 2 to 6 amino acids, an alkylene chain containing from 1 to 12 carbon atoms which may contain one or more carbon-carbon double or triple bonds, a paraformaldehyde chain –(OCH₂)_1-12-, a polyethylene glycol chain -(OCH₂CH₂)_1-6-, w_{hich chains may be interrupted by or optionally incorporates one or more of P, O, S, NH, C5-9} heteroarylene, phenylene, heterocyclyl, cycloalkyl, ether, oxo, carboxamidyl, and/or urethanyl 15 moieties wherein the C5-9 heteroarylene, phenylene, heterocyclyl, and/or cycloalkyl moieties are optionally substituted. In embodiments, the C5-9 heteroarylene, phenylene, heterocyclyl, and/or cycloalkyl moieties are optionally substituted with 1, 2 or 3 independently selected optional R20 groups. 2 is 20 In embodiments, LS2 is a peptide chain having from 2 to 6 amino acids, optionally 2 amino acids, optionally -valine-alanine-. 25 In embodiments, L_S2 is selected from: DB1/ 159693888.5 47

133186-5030-WO (i) (ii) (iii) (iv) ; 5 In embodiments, LS2 is unbranched or branched, flexible or rigid, short or long and may incorporate any combination of moieties as deemed useful. In embodiments, at least a portion of L_S2 has a polyalkylene oxide polymeric region, which may enhance solubility of the 10 antibody-drug conjugate of formula (I). In embodiments, L_S2 has a repeating unit of ethylene glycol, and optionally has a number of repeating ethylene glycol units of about 1 to about 25, or any number therebetween. In embodiments, LS2 includes about 3 to about 20, about 4 to about 15, about 5 to about 12 or about 6 to about 10 ethylene glycol units. In embodiments, at least a portion of L_S2 includes one or more amino acid moieties which provide enhanced solubility for 15 the antibody-drug conjugate of formula (I) and/or may provide amino acid sequences to enhance target binding, enhance compatibility with a targeting agent, and/or enhance target binding recognition. In embodiments, LS2 includes one or more amino acid moieties that provide a suitable substrate motif for a protease. In embodiments, when a set of amino acid moieties are incorporated into the LS2 that provide a substrate motif specific for a selected protease, the 20 cytotoxic drug compound of the ADC (e.g. the ADC of formula (I)) may be released from a target bound conjugate to provide localized cytotoxic effects. Such substrate motifs are known in the art and may be incorporated into LS2 as desired to provide selective release from the target bound conjugate. This selectivity can be based on known presence of a desired protease within the localized delivery region of the conjugate drug. In embodiments, other polymeric types of 25 moieties are incorporated in LS2, such as polyacids, polysaccharides, or polyamines. In embodiments, other moieties such as substituted aromatic or heteroaromatic moieties are used to enhance rigidity or provide synthetically accessible sites on substituents therein for linking to reactive moieties or to form the antibody-drug conjugate of formula (I). In some embodiments, L_S2 comprises one or more groups selected from sulfamino, sulfamyl, 30 sulfate, sulfhydryl, sulfinamino, sulfinate, sulfino, sulfinyl, sulfinyloxy, sulfo, sulfonamido, DB1/ 159693888.5 48

133186-5030-WO sulfonamino, sulfonate, sulfonyl, sulfonyloxy, phosphate ester, phosphoramidate, thiophosphate ester, phosphonate, and thiophosphonate. I_{n embodiments, –LS2– is selected from:} 5 . , C2 p g ps selected from an amino acid, an amino acid derivative, a peptide chain having from 2 to 6 amino acids or amino acid derivatives, an10 alkylene chain containing from 1 to 12 carbon atoms which may contain one or more carbon- carbon double or triple bonds, a paraformaldehyde chain –(OCH₂)_1-12-, a polyethylene glycol chain -(OCH2CH2)1-8-, which chains may be interrupted by one or more P, O, S and/or NH g_{roups and/or C5-9 heteroarylene and/or phenylene, wherein each C5-9 heteroarylene group and/or} each phenylene group is optionally substituted. 15 In some embodiments, LC2 has the formula , optionall . In some embodiments, L_C2 comprises a polyethylene glycol chain -(OCH₂CH₂)_1-10-, optionally - (OCH2CH2)8-. In some embodiments, L_C2 is selected from: 20 nd DB1/ 159693888.5 49

133186-5030-WO . In embodiments, –L_C2–L_S2–s include ethylene glycol repeating units, and/or an amino acid s_{equence. In some embodiments, –LC2–LS2– includes the formula:} 5 -[CH2CH2O]0-50-XAA- wherein XAA is an amino acid sequence. In embodiments, LC2 has or comprises the formula , wherein X10 is a single bond, -HN-[CH2-CH2-O]p-(CH2)1-5 HN-[CH2-CH2-O]p-(CH2)1-5-C(O)- NH-, or , wherein p is independently at each 10 occurre 1-50, and Alk is a C₁-C₅alkyl; and X20 is a single bond . In embodiments, LC2 has or comprises the formula . In some embodiments, LC2 has or comprises the formula . In some e , p is an integer from 5 to 10, e.g.5, 6, 7, 8, 9, or 10. In e , p is 8. In embodiments, X₁₀ is -HN-[CH₂-CH₂-O]_p-(CH₂)₂-C(O)-. In embodiments, X₁₀ is a single 15 bond. In embodiments, X₁₀ is -HN-[CH₂-CH₂-O]_p-(CH₂)₂-C(O)-N-. In embodiments, X₁₀ is . In some embodiments, Alk is -CH3. In embodiments, X₂₀ is a single bond. In some embodiments, X₂₀ . In embodiments, . –_{LC2–LS2– is selected from:} DB1/ 159693888.5 50

133186-5030-WO . Any suitable number of ethylene glycol units can be used in L₂, L_C2, and/or L_S2 of the present 5 disclosure. For example, L₂, L_C2, and/or L_S2 can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 16, 19, 20, 23, 24, 35, 36, 37, 48, 49, or more ethylene glycol units. In embodiments, L2, LC2, and/or LS2 includes 8 ethylene glycol units. Several commercially available ethylene glycol groups (polyethylene glycol, PEG) are suitable in L2, LC2, and/or LS2, such as H2N-PEG8- C(O)OH, having a discrete (“d”) polyethylene glycol having 8 ethylene glycol repeating units. 10 Other discrete PEG units are commercially available and known to one of skill in the art, such as by Advanced ChemTech. In embodiments, L₂, L_C2, and/or L_S2 includes the formula: -HN-PEG-C(O)-XAA- wherein PEG has 1-50 ethylene glycol units, and XAA is an amino acid sequence. In embodiments, the L₂, L_C2, and/or L_S2 includes the formula: 15 -HN-[CH2-CH2-O]p-(CH2)1-5-C(O)-XAA- wherein p is an integer from 1-50, and XAA is an amino acid sequence. In embodiments, p is an integer from 5 to 10, e.g.5, 6, 7, 8, 9, or 10. The amino acid portion of L2, LC2, and/or LS2 can include any suitable number of amino acid moieties, as described above. For example, the amino acid sequence XAA can include from 1 to 20 100 amino acid moieties, or from 1 to 10 amino acid moieties, or from 1 to 5 amino acid moieties. In embodiments, XAA is an amino acid sequence comprising 1 to 30 amino acids. In some embodiments, XAA is an amino acid sequence comprising 1 to 25 amino acids, 1 to 20 amino acids, 1 to 15 amino acids, 2 to 15 amino acids, 1 to 10 amino acids, 2 to 10 amino acids, 1 to 9 25 amino acids, 2 to 9 amino acids, 1 to 8 amino acids, 2 to 8 amino acids, 1 to 7 amino acids, 2 to 7 amino acids, 1 to 6 amino acids, 2 to 6 amino acids, 1 to 5 amino acids, 2 to 5 amino acids, 1 to 4 amino acids, 2 to 4 amino acids, 1 to 3 amino acids, or 2 to 3 amino acids. For example, DB1/ 159693888.5 51

133186-5030-WO X_AA can be an amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids. In embodiments, XAA can include 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid moieties. In embodiments, XAA is an amino acid sequence comprising 1 to 10 amino acids. In embodiments, XAA is an amino acid 5 sequence comprising 2 to 8 amino acids. In embodiments, XAA is an amino acid sequence comprising 2 to 6 amino acids. In embodiments, X_AA is an amino acid sequence comprising 2 to 4 amino acids. In embodiments, X_AA is an amino acid sequence comprising 4 amino acids. In embodiments, X_AA is an amino acid sequence comprising 3 amino acids. In embodiments, X_AA includes 2 amino acid moieties. 10 In embodiments, X_AA is Gly-Gly-Phe-Gly. In embodiments, X_AA is Val-Cit. In some embodiments, X_AA is Ala-Ala. In embodiments, X_AA is Val-Ala. In embodiments, X_AA is Ala- Ala-Ala. In embodiments, XAA is Val-Ala-Ala. In embodiments, the linker L₂ includes the formula: -HN-PEG₈-C(O)-Val-Ala- 15 wherein PEG8 has 8 ethylene glycol units. Q In embodiments, Q is X₁-L-X₂. H N O In embodiments, Q has the formu . B 20 In embodiments, B comprises (A)_q, wherein: q is selected from 0, 1, 2, 3, 4, 5 and 6; A is selected from: ; ₂₅ from N-R30, S and O; and the other of Y₃ and Y₄ is CH; and Y₅ is independently selected from CR₃₀, N, S and COH; DB1/ 159693888.5 52

133186-5030-WO for each A2 group one of Y₆ and Y₇ is independently selected from N and CH; and the other of Y6 and Y7 is CR30; and each R30 is independently selected from H, C1-6 alkyl, L2-R58 and RS. In embodiments, B is (A)_q, 5 In embodiments, q is 1 or 2. In embodiments, q is 1. In embodiments, q is 2. In embodiments, A is A1: ; wherein : 10 Y3 is N-R30; Y4 is CH; Y5 is CR30; optionally wherein each R30 is H, methyl, or RS. H ^N O H N N N , or 15 In embodiments, T comprises a group of formula: Y₂ wherein: 20 p is 0 or 1; DB1/ 159693888.5 53

133186-5030-WO R_T is selected from –L₂–, phenylene, and C_5-9 heteroarylene, wherein the phenylene and C_5-9 heteroarylene groups are optionally substituted with up to three optional substituent groups selected from OH, C1-6 alkyl, OC1-6 alkyl, (CH2)j-CO2R11, O-(CH2)k-NR11R12, (CH2)j-NR11R12, C(=O)-NH-(CH2)k-NR11R12, C(=O)-NH-R4, and C(=O)-NH-(CH2)k- 5 C(=NH)NR₁₁R₁₂, optionally with the proviso that the optionally substituted C_5-9 heteroarylene is not disubstituted indolyl. R₁₉ is selected from H, C_1-6 alkyl, L₂-R₅₈, R_S, and (CH₂)_t-NR₂₀R₂₁; Y₁ and Y₂ are independently N or CR₃₁, wherein at least one of Y₁ and Y₂ is CR₃₁; each R31 is independently selected from H, C1-6 alkyl, L2-R58 and RS; 10 R11, R12, and R24 are independently selected from H, –L2-R58, C1-6 alkyl, or a bond connecting the atom to which it is bound to –LS2–, with the proviso that both R11 and R12 cannot be a bond connecting the atom to which it is bound to –L_S2–. In embodiments, T has the formula: . 15 In embodiments, p is 0. In In embodiments, Y₁ and Y₂ are CR₃₁, R₃₁ are each H, and R₁₉ is H. In embodiments, RT is selected from –L2–, phenylene, and C5-9 heteroarylene, wherein the phenylene and C_5-9 heteroarylene groups are optionally substituted with up to three optional substituent groups. Hence, any of the phenylene or C_5-9 heteroarylene groups selected for R_T 20 may be optionally substituted with up to three optional substituent groups. In embodiments, RT is selected from divalent phenyl, divalent pyrrolyl, divalent N- methylpyrrolyl, divalent furanyl, divalent thiophenyl, divalent imidazolyl, divalent N- methylimidazolyl, divalent oxazolyl, divalent thiazolyl, divalent pyridyl, divalent benzofuranyl, divalent benzothiophenyl, divalent benzimidazolyl, divalent N-methylbenzoimidazolyl, divalent 25 benzooxazolyl and divalent benzothiazolyl, wherein the divalent phenyl, divalent pyrrolyl, divalent N-methylpyrrolyl, divalent furanyl, divalent thiophenyl, divalent imidazolyl, divalent N-methylimidazolyl, divalent oxazolyl, divalent thiazolyl, divalent pyridyl, divalent benzofuranyl, divalent benzothiophenyl, divalent benzimidazolyl, divalent N- methylbenzoimidazolyl, divalent benzooxazolyl and divalent benzothiazolyl groups are 30 optionally substituted with up to three optional substituent groups selected from OH, C1-6 alkyl, DB1/ 159693888.5 54

133186-5030-WO OC_1-6 alkyl, L₂-R₅₈, (CH₂)_j-CO₂R₁₁, O-(CH₂)_k-NR₁₁R₁₂, (CH₂)_j-NR₁₁R₁₂, C(=O)-NH-(CH₂)_k- NR11R12; C(=O)-NH-R24 and C(=O)-NH-(CH2)k-C(=NH)NR11R12. In embodiments, R_T is selected from divalent phenyl, divalent pyrrolyl, divalent N- methylpyrrolyl, divalent furanyl, divalent thiophenyl, divalent imidazolyl, divalent N- 5 methylimidazolyl, divalent oxazolyl, divalent thiazolyl, divalent benzofuranyl, divalent benzothiophenyl, divalent benzimidazolyl, divalent N-methylbenzoimidazolyl, divalent benzooxazolyl and divalent benzothiazolyl, wherein the divalent phenyl, divalent pyrrolyl, divalent N-methylpyrrolyl, divalent furanyl, divalent thiophenyl, divalent imidazolyl, divalent N-methylimidazolyl, divalent oxazolyl, divalent thiazolyl, divalent benzofuranyl, divalent 10 benzothiophenyl, divalent benzimidazolyl, divalent N-methylbenzoimidazolyl, divalent benzooxazolyl and divalent benzothiazolyl, groups are optionally substituted with one or two optional substituent groups selected from OH, C1-6 alkyl, OC1-6 alkyl, L2-R58, (CH2)j-CO2R11, O- (CH2)k-NR11R12, (CH2)j-NR11R12, C(=O)-NH-(CH2)k-NR11R12; C(=O)-NH-R24 and C(=O)-NH- (CH2)k-C(=NH)NR11R12. 15 In embodiments, RT is selected from divalent phenyl, divalent N-methylpyrrolyl, divalent thiophenyl, divalent N-methylimidazolyl, divalent oxazolyl, divalent thiazolyl, divalent benzothiophenyl, divalent N-methylbenzoimidazolyl and divalent benzothiazolyl, wherein the divalent phenyl, divalent N-methylpyrrolyl, divalent thiophenyl, divalent N-methylimidazolyl, divalent oxazolyl, divalent thiazolyl, divalent benzothiophenyl, divalent N- 20 methylbenzoimidazolyl and divalent benzothiazolyl, are optionally substituted with one or two optional substituent groups selected from OH, C1-6 alkyl, OC1-6 alkyl, L2-R58, (CH2)j-CO2R11 O- (CH2)k-NR11R12, (CH2)j-NR11R12, C(=O)-NH-(CH2)k-NR11R12; C(=O)-NH-R24 and C(=O)-NH- (CH2)k-C(=NH)NR11R12. In embodiments, RT is optionally substituted with up to three optional substituent groups 25 selected from OH, C1-6 alkyl, OC1-6 alkyl, L2-R58, (CH2)j-CO2R11, O-(CH2)k-NH2, (CH2)j-NH2, C(=O)-NH-(CH2)k-NH2; C(=O)-NH-R24 and C(=O)-NH-(CH2)k-C(=NH)NH2. In embodiments, R_T is substituted with up to three optional substituent groups selected from OH, C_1-6 alkyl, OC_1-6 alkyl, L₂-R₅₈, (CH₂)_j-CO₂R₁₁, O-(CH₂)_k-NH₂, (CH₂)_j-NH₂, C(=O)-NH-(CH₂)_k-NH₂; C(=O)-NH- R₂₄ and C(=O)-NH-(CH₂)_k-C(=NH)NH₂. 30 In embodiments, RT is an optionally substituted C(=O)-NH-R24, wherein R24 is -C6H4-(CH2)j- R₁₈, and the phenylene group –C₆H₄- is para substituted. In embodiments, R_T is substituted C(=O)-NH-R₂₄, wherein R₂₄ is -C₆H₄-(CH₂)_j-R₁₈, and the phenylene group –C₆H₄- is para substituted. DB1/ 159693888.5 55

133186-5030-WO In embodiments, R_T is optionally substituted with up to three substituent groups selected from OH, methyl, ethyl, OCH3, OCH2CH3, L2-R58, CO2H, CO2CH3, CO2CH2CH3, O-(CH2)k-NH2 and (CH2)j-NH2. In embodiments, RT is substituted with up to three substituent groups selected from OH, methyl, ethyl, OCH3, OCH2CH3, L2-R58, CO2H, CO2CH3, CO2CH2CH3, O-(CH2)k-NH2 and 5 (CH2)j-NH2. In embodiments, RT is optionally substituted with one or two substituent groups. In embodiments, RT is substituted with one or two substituent groups. In embodiments, R_T is optionally substituted with one substituent group. In embodiments, R_T is substituted with one substituent group. H N 10 In embodiments, T has the formul . T’ In embodiments, T’ comprises a group of formula: wherein: 15 p is 0 or 1; RT is selected from –L2-R58, phenyl, and C5-9 heteroaryl, wherein the phenyl and C5-9 heteroaryl groups are optionally substituted with up to three optional substituent groups selected from OH, C1-6 alkyl, OC1-6 alkyl, –L2-R58, (CH2)j-CO2R11, O-(CH2)k-NR11R12, (CH2)j- NR₁₁R₁₂, C(=O)-NH-(CH₂)_k-NR₁₁R₁₂, C(=O)-NH-R₂₄, and C(=O)-NH-(CH₂)_k-20 C(=NH)NR₁₁R₁₂, optionally with the proviso that the optionally substituted C_5-9 heteroaryl is not indolyl; R19 is selected from H, C1-6 alkyl, L2-R58, RS, and (CH2)t-NR20R21; Y1 and Y2 are independently N or CR31, wherein at least one of Y1 and Y2 is CR31; each R31 is independently selected from H, C1-6 alkyl, L2-R58 and RS; and 25 R₁₁, R₁₂, and R₂₄ are independently selected from H, –L₂-R₅₈, C_1-6 alkyl, or substituted aryl. DB1/ 159693888.5 56

133186-5030-WO In embodiments, T’ has the formula: . , . mbodiments, p is 1. In embodiments, Y1 and Y2 are CR31, R31 are each H, and R19 is H. 5 In embodiments, RT’ is selected from L2-R58, phenyl, and C5-9 heteroaryl, wherein the phenyl and C5-9 heteroaryl groups are optionally substituted with up to three optional substituent groups. Hence, any of the phenyl group or the C5-9 heteroaryl groups selected for RT’ may be optionally substituted with up to three optional substituent groups. In embodiments, RT’ is selected from L2-R58, phenyl, pyrrolyl, N-methylpyrrolyl, furanyl, 10 thiophenyl, imidazolyl, N-methylimidazolyl, oxazolyl, thiazolyl, pyridyl, benzofuranyl, benzothiophenyl, benzimidazolyl, N-methylbenzoimidazolyl, benzooxazolyl and benzothiazolyl, wherein the phenyl, pyrrolyl, N-methylpyrrolyl, furanyl, thiophenyl, imidazolyl, N-methylimidazolyl, oxazolyl, thiazolyl, pyridyl, benzofuranyl, benzothiophenyl, benzimidazolyl, N-methylbenzoimidazolyl, benzooxazolyl and benzothiazolyl groups are 15 optionally substituted with up to three optional substituent groups selected from OH, C1-6 alkyl, OC1-6 alkyl, L2-R58, (CH2)j-CO2R11, O-(CH2)k-NR11R12, (CH2)j-NR11R12, C(=O)-NH-(CH2)k- NR₁₁R₁₂; C O H R d C O H CH C H R R In embodiments, RT’ is selected from L2-R58, phenyl, pyrrolyl, N-methylpyrrolyl, furanyl, thiophenyl, imidazolyl, N-methylimidazolyl, oxazolyl, thiazolyl, benzofuranyl, 20 benzothiophenyl, benzimidazolyl, N-methylbenzoimidazolyl, benzooxazolyl and benzothiazolyl, wherein the phenyl, pyrrolyl, N-methylpyrrolyl, furanyl, thiophenyl, imidazolyl, N-methylimidazolyl, oxazolyl, thiazolyl, benzofuranyl, benzothiophenyl, benzimidazolyl, N- methylbenzoimidazolyl, benzooxazolyl and benzothiazolyl groups are optionally substituted with one or two optional substituent groups selected from OH, C1-6 alkyl, OC1-6 alkyl, L2-R58,25 (CH2)j-CO2R11, O-(CH2)k-NR11R12, (CH2)j-NR11R12, C(=O)-NH-(CH2)k-NR11R12; C(=O)-NH- DB1/ 159693888.5 57

133186-5030-WO In embodiments, R_T’ is selected from L₂-R₅₈, phenyl, N-methylpyrrolyl, thiophenyl, N- methylimidazolyl, oxazolyl, thiazolyl, benzothiophenyl, N-methylbenzoimidazolyl and benzothiazolyl, wherein the phenyl, N-methylpyrrolyl, thiophenyl, N-methylimidazolyl, oxazolyl, thiazolyl, benzothiophenyl, N-methylbenzoimidazolyl and benzothiazolyl groups are 5 optionally substituted with one or two optional substituent groups selected from OH, C1-6 alkyl, OC_1-6 alkyl, L₂-R₅₈, (CH₂)_j-CO₂R₁₁ O-(CH₂)_k-NR₁₁R₁₂, (CH₂)_j-NR₁₁R₁₂, C(=O)-NH-(CH₂)_k- NR₁₁R₁₂; C(=O)-NH-R₂₄ and C(=O)-NH-(CH₂)_k-C(=NH)NR₁₁R₁₂. In embodi CH2)j- R₁₈, and the phenylene group –C₆H₄- is para substituted. In embodiments, R_T’ is substituted 10 C(=O)-NH-R₂₄, wherein R₂₄ is -C₆H₄-(CH₂)_j-R₁₈, and the phenylene group –C₆H₄- is para substituted. In embodiments, RT’ is C5-9 heteroaryl. In embodiments, the C5-9 heteroaryl is substituted with C_1-6 alkyl. In embodiments, the C_5-9 heteroaryl is optionally substituted pyrrole. In embodiments, the C_5-9 heteroaryl is pyrrole substituted with C_1-6 alkyl. In embodiments, the C_5-9 15 heteroaryl is pyrrole substituted with methyl. In embodiments, RT’ is C5-9 heteroaryl substituted with methyl and/or C(=O)-NH-R24. In embodiments, RT’ is pyrrole substituted with methyl and/or C(=O)-NH-R24. In embodiments, R24 is substituted aryl, optionally wherein R24 is H₂N 20 In embodiments, RT’ is phenylene. In embodiments, the phenylene is substituted with (CH2)j- NR11R12. In embodiments, R11 and R12 are each H. In embodiments, R_T’ is optionally substituted with up to three substituent groups selected from OH, methyl, ethyl, OCH₃, OCH₂CH₃, L₂-R₅₈, CO₂H, CO₂CH₃, CO₂CH₂CH₃, O-(CH₂)_k-NH₂ and 25 (CH2)j-NH2. In embodiments, RT’ is substituted with up to three substituent groups selected from OH, methyl, ethyl, OCH3, OCH2CH3, L2-R58, CO2H, CO2CH3, CO2CH2CH3, O-(CH2)k- NH2 and (CH2)j-NH2. In embodiments, RT’ is optionally substituted with one or two substituent groups. In embodiments, RT’ is substituted with one or two substituent groups. 30 In embodiments, R_T’ is optionally substituted with one substituent group. In embodiments, R_T’ is substituted with one substituent group. In embodiments, R_T’ is selected from: R_S DB1/ 159693888.5 58

133186-5030-WO ; wh Z₂ Z₃ is selected from S and O; 5 Z4 is selected from CH, CRS and N; R22 is selected from –L2-R58, (CH2)jCO2R11, (CH2)jNR11R12 and C(=O)-NH-C6H4-(CH2)j-R18; R18 is selected from –L2-R58, CO2R11 and NR11R12; j is selected from an integer from 0 to 6; R₁₁ and R₁₂ are independently selected from H, –L₂-R₅₈, and C_1-6 alkyl; and 10 R23 is selected from H, RS, –L2-R58, and C1-6 alkyl. The wavy line indicates the point of attachment of the above R_T’ group to the rest of the compound (e.g. a compound of formula (I)). In embodiments, RT’ is selected from: nd 15 wherein Z1 is selected from NH, N-CH3, N-RS, S and O; Z₂ is selected from CH and N; and Z₃ is selected from S and O; 20 Z4 is selected from CH, RS and N; R11 is selected from H, L2-R58, and C1-6 alkyl; and R23 is selected from H, RS, L2-R58, and C1-6 alkyl. DB1/ 159693888.5 59

133186-5030-WO In embodiments, T’ has the formula . In embodiments, T’ has the formula . Z^* 5 In embodiments, Z* is a reactive moiety that has reacted with a functional group such as aldehydes, amines, disulfides, ketones thiols in an antibody or binding fragment thereof, or in Staudinger reactions, Pictet-Spengler reactions and/or Click-type chemistry of an antibody or fragment thereof (e.g. a reactive moiety Z*). In embodiments, the reactive moiety (e.g. Z*) is selected from succinimide, a heterocycle (e.g. a triazole), an amide, a thioether, an oxime, an 10 imine (e.g. chiral sulfinyl imine), an alkenyl phosphorous group, and an alkyl phosphorous group. In embodiments, Z* is selected from succinimide, an oxime, an imine (e.g. chiral sulfinyl imine), a heterocycle, an amide, and a thioether, an alkenyl phosphorous group, and an alkyl 15 phosphorous group. In embodiments, the heterocycle is formed by a cycloaddition (e.g. click chemistry) between two or more unsaturated moieties. Non-limiting examples of heterocycles include a triazole (including but not limited to triazoles formed from cycloaddition (e.g. click chemistry) of an azide and an alkyne (such as a cyclooctenyl ring (e.g. dibenzocyclooctenyl (DBCO) ring)), and an isoxazolidine (including but not limited to isoxazolidine rings formed by 20 a cycloaddition of a nitrone and an alkyne (such as a cyclooctenyl ring (e.g. dibenzocyclooctenyl (DBCO) ring)). In embodiments, Z* is succinimide (i.e., a succinimidyl moiety, “2,5-dioxo-3λ³-pyrrolidin-1- yl”): 25 . In embodiments, Z* is a heterocycle. In embodiments, the heterocycle is formed by a cycloaddition (e.g. click chemistry) between two or more unsaturated moieties. In embodiments, the heterocycle is fused to one or more additional rings, including but not limited DB1/ 159693888.5 60

133186-5030-WO to a cyclooctynyl ring. In embodiments, Z* is a triazole (triazolyl). In a non-limiting example, the triazole (triazolyl) is formed by a cycloaddition reaction (e.g. click chemistry) between an azide and an alkyne. In embodiments, Z* is an isoxazolidine (isoxazolidinyl). In a non-limiting example the (isoxazolidine (isoxazolidinyl) is formed by a cycloaddition of a nitrone and an 5 alkyne. In some embodiment, the alkyne is a cyclooctynyl group. Non-limiting examples of cyclooctynyl groups include dibenzocyclooctynye (DBCO) and derivatives thereof. In embodiments, the cyclooctynyl ring is fused to one or more additional rings, including but not limited to cycloalkyl rings and/or heterocycloalkyl rings. Non-limiting examples of heterocycles 10 , , US 20160107999, US 20170008858, US 20170298145, and 20230099074 for additional examples 15 of cyclooctynyl groups, all of which are incorporated by reference herein in their entireties. In embodiments, Z* is or comprises an oxime group. In embodiments, the oxime is or comprises the formula , wherein L is a linker, including but not limited to any DB1/ 159693888.5 61

133186-5030-WO linker L groups disclosed herein. In embodiments, L comprises or consists of a substituted or unsubstituted alkyl group. In embodiments, the formula is or comprises the formula , and n is an integer from 1 to 10. In embodiments, n is an integer from 1 to odiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In embodiments, L comprises or 5 consists of one or more polyethyleneglycol (PEG) groups. In embodiments, the formula is or comprises the formula , and n is an integer from 1 to 10. In embodiments, n is an integer f mbodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In embodiments, n is 4. See, for example, US 20230295320, US 11773150, US 20160052966, all of which are incorporated by reference herein in their entireties. 10 In embodiments, Z* is or comprises an imine. Non-limiting examples of imines include chiral sulfinyl imines. In embodiments, the imine is or comprises the formula , wherein L is a linker, including but not limited to any linker L groups disclosed In embodiments, Z* is or comprises an alkenyl phosphorous group. In embodiments, the alkenyl 15 phosphorous group is or comprises the formu , wherein R is an alkyl or aromatic group. In embodiments, R is a PEG ing about 1 to about 10 PEG moieties. In embodiments, Z* is or comprises an alkyl phosphorous group. In embodiments, the alkyl phosphorous group is or comprises the formu , wherein R is an alkyl or aromatic group. In embodiments, R is a PEG group about 1 to about 10 PEG 20 moieties. See, for example US 20230330258 and US 20230158154, both of which are incorporated by reference herein in their entireties. In embodiments, Z^* is succinimide . 25 In embodiments, –Z^*–LC2– is selected from: , DB1/ 159693888.5 62

133186-5030-WO nd I_{n embodiments, –Z*–LC2–LS2– is selected from:} ; DB1/ 159693888.5 63

133186-5030-WO DB1/ 159693888.5 64

133186-5030-WO . . In embodiments, L_S is a bond or a linker moiety that is attached or connected to R_S. In some embodiments, LS is a bond, an amino acid, a peptide chain having from 2 to 6 amino acids, an DB1/ 159693888.5 65

133186-5030-WO alkylene chain containing from 1 to 12 carbon atoms which may contain one or more carbon- carbon double or triple bonds, a paraformaldehyde chain –(OCH2)1-12-, a polyethylene glycol chain -(OCH2CH2)1-6-, which chains may be interrupted by or optionally incorporates one or m_{ore of P, O, S, NH, C5-9 heteroarylene, phenylene, heterocyclyl, cycloalkyl, ether, oxo,} 5 carboxamidyl, and/or urethanyl moieties wherein the C5-9 heteroarylene, phenylene, heterocyclyl, and/or cycloalkyl moieties are optionally substituted. In some embodiments, the C_5-9 heteroarylene, phenylene, heterocyclyl, and/or cycloalkyl moieties are optionally substituted with 1, 2 or 3 independently selected optional R₂₀ groups. In some embodiments, L_S comprises L₂, R₅₈, or L₂-R₅₈. 10 In some embodiments, linker Ls comprises one or more groups selected from sulfamino, sulfamyl, sulfate, sulfhydryl, sulfinamino, sulfinate, sulfino, sulfinyl, sulfinyloxy, sulfo, sulfonamido, sulfonamino, sulfonate, sulfonyl, sulfonyloxy, phosphate ester, phosphoramidate, thiophosphate ester, phosphonate, and thiophosphonate. In some embodiments, LS comprises a benzyl carboxylate group. In some embodiments, LS 15 comprises: each R40 is independently selected from -NO2, -SO3R26, -C(=O)-R26, -C(=O)-Cl, -C1-6 fluoroalkyl, -C1-6 fluoroalkoxy, R20, R58, -[CH2CH2O]1-50-R58, and -NH-XAA-R58, wherein XAA is an amino acid sequence having from 1 to 20 amino acid20 moieties. In some embodiments, v is 1, 2, or 3; and each R40 is independently selected from - NO2, -SO3R26, -C(=O)-R26, -C(=O)-Cl, -C1-6 fluoroalkyl, -C_1-6 fluoroalkoxy, -F, -OH, -NH₂, -CN, -NCO, -(CH₂)_j-CO₂R₂₆, -C(=O)-NR₂₆R₂₇, L₂-R₅₈, id 25 In some embodiments, LS is: . DB1/ 159693888.5 66

133186-5030-WO In embodiments, –L–D is selected from: , DB1/ 159693888.5 67

133186-5030-WO weren _S s a unvaen sacc ar e sus uen, preera y gycosy or -gycosy. DB1/ 159693888.5 69

133186-5030-WO In embodiments, –L–D is: O H N N O O O O O O O _O ^O O _{HO H} O O _{HN O N} O . O . 5 . In an aspect, antibody-drug conjugates of the disclosure (e.g. antibody-drug conjugates of formula (I)), comprising a pyrridinobenzodiazepine (PDD) and at least one carbohydrate substituent RS have enhanced or increased hydrophilicity compared to antibody-drug conjugates 10 comprising a PDD and not comprising at least one R_S. In embodiments, conjugates of the disclosure (e.g. antibody-drug conjugates of formula (I)) comprising a PDD and at least one carbohydrate substituent RS have enhanced or increased hydrophilicity compared to antibody- drug conjugates comprising a PDD and not comprising at least one RS. In some embodiments, conjugates of the disclosure (e.g. antibody-drug conjugates of formula (I)) comprising a PDD 15 and at least one carbohydrate substituent R_S have enhanced or increased hydrophilicity compared to antibody-drug conjugates comprising a PDD and not comprising at least one R_S. In some embodiments, compounds of formula (I), formula (IV), and/or conjugates thereof, DB1/ 159693888.5 70

133186-5030-WO comprising at least one R_S have about 1-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, or about 100-fold enhanced or increased hydrophilicity compared to antibody-drug conjugates comprising a PDD and not comprising at least one RS. In a non- limiting example increased or enhanced hydrophilicity allows for efficient conjugation of the 5 compounds of formula (I) and/or formula (IV) to a targeting agent and/or at a higher DAR (e.g., DAR 8). In some embodiments, compounds of formula (I) with a G- alkylating DNA group of formula (II) or one of G1-G8 as the D moiety, compounds of formula (IV), and/or conjugates thereof, comprising at least one carbohydrate substituent have enhanced or increased hydrophilicity 10 compared to antibody-drug conjugates comprising a PDD and not comprising at least one R_S. In some embodiments, compounds of formula (I) with a G- alkylating DNA group of formula (II) or one of G1-G8 as the D moiety, compounds of formula (IV), and/or conjugates thereof, comprising at least one RS have enhanced or increased hydrophilicity compared to antibody- drug conjugates comprising a PDD and not comprising at least one RS. In some embodiments, 15 compounds of formula (I) with a G- alkylating DNA group of formula (II) or one of G1-G8 as the D moiety, compounds of formula (IV), and/or conjugates thereof, comprising at least one R_S have about 1-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, or about 100-fold enhanced or increased hydrophilicity compared to antibody-drug conjugates comprising a PDD and not comprising at least one RS. In a non-limiting example increased or 20 enhanced hydrophilicity allows for efficient conjugation of the compounds of formula (I) with a G- alkylating DNA group of formula (II) or one of G1-G8 as the D moiety and/or compounds of formula (IV) to a targeting agent and/or at a higher DAR (e.g., DAR 8). In an aspect, antibody-drug conjugates of the disclosure (e.g. antibody-drug conjugates of formula (I)), comprising a pyrridinobenzodiazepine (PDD) and at least one carbohydrate 25 substituent R_S have enhanced or increased tolerability compared to antibody-drug conjugates comprising a PDD and not comprising at least one R_S. In some embodiments, antibody-drug conjugates of the disclosure (e.g. antibody-drug conjugates of formula (I)), comprising a pyrridinobenzodiazepine (PDD) and at least one RS have enhanced or increased tolerability compared to antibody-drug conjugates comprising a PDD and not comprising at least one RS. In 30 some embodiments, antibody-drug conjugates of the disclosure (e.g. antibody-drug conjugates of formula (I)), comprising a pyrridinobenzodiazepine (PDD) and at least one at least one R_S have enhanced or increased tolerability compared to antibody-drug conjugates comprising a PDD and not comprising at least one RS. In some embodiments, antibody-drug conjugates of the disclosure (e.g. antibody-drug conjugates of formula (I)), comprising a 35 pyrridinobenzodiazepine (PDD) and at least one RS have a 2-fold to 10-fold or more increase in DB1/ 159693888.5 71

133186-5030-WO tolerability compared to antibody-drug conjugates comprising a PDD and not comprising at least one RS. In some embodiments, antibody-drug conjugates of formula (I) comprising a moiety of formula (IIa), (IIb), or one of G1-G8 or H1-H8 as the D moiety and at least one R_S have enhanced or 5 increased tolerability compared to antibody-drug conjugates of formula (I) comprising a moiety of formula (IIa), (IIb), or one of G1-G8 or H1-H8 as the D moiety and RS not comprising at least one RS. In embodiments, antibody-drug conjugates of formula (I) comprising a moiety of formula (IIa), (IIb), or one of G1-G8 or H1-H8 as the D moiety and at least one RS have enhanced or increased tolerability compared to antibody-drug conjugates of formula (I) 10 comprising a moiety of formula (IIa), (IIb), or one of G1-G8 or H1-H8 as the D moiety and at least one RS. In some embodiments, compounds of formula (I) with a G- alkylating DNA group of formula (II) or one of G1-G8 as the D moiety and at least one RS have a 2-fold to 10-fold or more increase in tolerability compared to antibody-drug conjugates of formula (I) comprising a moiety of formula (IIa), (IIb), or one of G1-G8 or H1-H8 as the D moiety and not comprising at 15 least one R_S. In some embodiments, antibody-drug conjugates of formula (I) comprising a moiety of formula (IIa), (IIb), or one of G1-G8 or H1-H8 as the D moiety comprising RS at the R6 position and/or R₈ position have enhanced or increased hydrophilicity compared to antibody-drug conjugates of formula (I) comprising a moiety of formula (IIa), (IIb), or one of G1-G8 or H1-H8 as the D 20 moiety not comprising RS at the R6 position and/or R8 position. In embodiments, antibody-drug conjugates of formula (I) comprising a moiety of formula (IIa), (IIb), or one of G1-G8 or H1-H8 as the D moiety comprising R_S at the R₈ position have a 2-fold to 10-fold or more increase in tolerability compared to antibody-drug conjugates of formula (I) with a moiety of formula (IIa), (IIb), or one of G1-G8 or H1-G8 as the D moiety not comprising 25 RS at the R8 position. In embodiments, antibody-drug conjugates of formula (I) a moiety of formula (IIa), (IIb), or one of G1-G8 or H1-H8 as the D moiety comprising RS at the R6 position have the same or substantially the same tolerability compared to antibody-drug conjugates of formula (I) comprising a moiety of formula (IIa), (IIb), or one of G1-G8 or H1-H8 as the D moiety thereof 30 not comprising RS at the R6 position. COMPOUNDS In one aspect, the disclosure provides a compound having the following formula: DB1/ 159693888.5 72

133186-5030-WO . , . In one aspect, the disclosure provides an antibody-drug conjugate having formula (I): 5 Ab-[L–D]n formula (I) wherein in formula (I): -L-D- has the formula: . 10 In one aspect, the disclosure provides an antibody-drug conjugate having formula (I): Ab-[L–D]n formula (I) wherein in formula (I): -L-D- has the formula: DB1/ 159693888.5 73

133186-5030-WO . In one aspect, the disclosure provides an antibody or binding fragment thereof useful within the antibody-drug conjugates (ADCs), linkers, and other compounds and/or conjugates described 5 herein. In some embodiments, the antibody or binding fragment thereof binds CDCP1. In some embodiments the antibody and/or binding fragment thereof is an antibody or antigen-binding portion thereof that is specific for CDCP1. CUB domain-containing protein 1 (CDCP1) CDCP1 (HGNC: 24357; NCBI Entrez Gene: 64866; Ensembl: ENSG00000163814; 10 UniProtKB/Swiss-Prot: Q9H5V8) has a large extracellular domain (665 amino acids in size) containing three CUB domains that mediate protein-protein interactions and are likely involved in cell adhesion and interaction with the extracellular matrix. The CDCP1 gene has been found to be strongly expressed in cancer, and has been previously disclosed as a therapeutic target in at least WO 2020/097336 and WO 2018/112334, which are herein incorporated by reference in 15 their entireties. Transmembrane protein CDCP1 associates with Src and PKCδ and all three proteins display increases in tyrosine phosphorylation when CDCP1 is activated. Src phosphorylates and binds to CDCP1, followed by the binding of CDCP1 to the C2 domain which is part of the regulatory domain of PKCδ. Tyr-734 was identified as the site that is phosphorylated by Src and Src 20 Family Kinases, and as such, P-Tyr-734 is a biomarker of CDCP1 activation. The full length CDCP1 protein is 135 kDa, but in some cells, the extracellular domain is proteolytically cleaved to a ~75 kDa transmembrane protein. Table 1. Exemplary CDCP1 sequences SEQ Sequence name Amino acid sequence D / 59693888.5 74

133186-5030-WO 9 CUB domain-containing MAGLNCGVSIALLGVLLLGAARLPRGAEAFEIALPRESNITVLIKLGTPT protein 1 isoform 1 LLAKPCYIVISKRHITMLSIKSGERIVFTFSCQSPENHFVIEIQKNIDCM recrsor [Homo saiens] SGPCPFGEVQLQPSTSLLPTLNRTFIWDVKAHKSIGLELQFSIPRLRQIG PW LM DK LS FL KL CP VT CQ KQ NK DS PE In some aspects, the CDCP1 is human CDCP1. In some aspects, the CDCP1 is cynomologus monkey (cyno) CDCP1. In some aspects, the CDCP1 is mouse CDCP1. In some aspects, the CDCP1 is primate CDCP1. An exemplary CDCP1 sequence is provided in Table 1. 5 The immunoglobulin disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. The immunoglobulins can be derived from any species. In one aspect, however, the immunoglobulin is of human, murine, or rabbit origin. In embodiments, the antibody or binding fragment thereof comprises a variable heavy chain 10 sequence that comprises an amino acid sequence with at least about 95%, about 96%, about 97%, about 98%, or about 99%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In embodiments, the antibody or binding fragment thereof retains the binding and/or functional activity of an antibody or binding fragment thereof that comprises the variable heavy chain sequence of SEQ ID NO 1. In embodiments, the antibody or binding fragment thereof 15 comprises the variable heavy chain sequence of SEQ ID NO: 1 and has one or more conservative amino acid substitutions, e.g., 1, 2, 3, 4, 5, 1-2, 1-3, 1-4 or 1-5 conservative amino acid substitutions in the heavy chain variable sequence. In embodiments, the one or more conservative amino acid substitutions fall within one or more framework regions in SEQ ID NO: 1 (based on the numbering system of Kabat). 20 In embodiments, the antibody or binding fragment thereof comprises a variable heavy chain sequence with at least about 95%, about 96%, about 97%, about 98%, or about 99% sequence DB1/ 159693888.5 75

133186-5030-WO identity to the binding protein heavy chain variable region sequence set forth in SEQ ID NO: 1 comprises one or more conservative amino acid substitutions in a framework region (based on the numbering system of Kabat), and retains the binding and/or functional activity of a binding protein that comprises a variable heavy chain sequence as set forth in SEQ ID NO: 1 and a 5 variable light chain sequence as set forth in SEQ ID NO: 5. In embodiments, the antibody or binding fragment thereof comprises a variable light chain sequence that comprises an amino acid sequence with at least about 95%, about 96%, about 97%, about 98%, or about 99%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 5. In embodiments, antibody or binding fragment thereof retains the binding and/or 10 functional activity of a targeting agent, antibody, or binding fragment thereof that comprises the variable light chain sequence of SEQ ID NO: 5. In embodiments, the antibody or binding fragment thereof comprises the variable light chain sequence of SEQ ID NO: 5 and has one or more conservative amino acid substitutions, e.g., 1, 2, 3, 4, 5, 1-2, 1-3, 1-4 or 1-5 conservative amino acid substitutions in the light chain variable sequence. In embodiments, the one or more 15 conservative amino acid substitutions fall within one or more framework regions in SEQ ID NO: 5 (based on the numbering system of Kabat). In embodiments, the antibody or binding fragment thereof comprises a variable light chain sequence with at least about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the binding protein light chain variable region sequence set forth in SEQ ID NO: 5 20 comprises one or more conservative amino acid substitutions in a framework region (based on the numbering system of Kabat), and retains the binding and/or functional activity of a binding protein that comprises a variable heavy light chain sequence as set forth in SEQ ID NO: 5 and a variable heavy chain sequence as set forth in SEQ ID NO: 1. In embodiments, the antibody or binding fragment thereof comprises a variable heavy chain 25 sequence that comprises an amino acid sequence with at least about 95%, about 96%, about 97%, about 98%, or about 99%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 15. In embodiments, the antibody or binding fragment thereof retains the binding and/or functional activity of an antibody or binding fragment thereof that comprises the variable heavy chain sequence of SEQ ID NO: 15. In embodiments, the antibody or binding 30 fragment thereof comprises the variable heavy chain sequence of SEQ ID NO: 15 and has one or more conservative amino acid substitutions, e.g., 1, 2, 3, 4, 5, 1-2, 1-3, 1-4 or 1-5 conservative amino acid substitutions in the heavy chain variable sequence. In embodiments, the one or more conservative amino acid substitutions fall within one or more framework regions in SEQ ID NO: 15 (based on the numbering system of Kabat). DB1/ 159693888.5 76

133186-5030-WO In embodiments, the antibody or binding fragment thereof comprises a variable heavy chain sequence with at least about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the binding protein heavy chain variable region sequence set forth in SEQ ID NO: 15 comprises one or more conservative amino acid substitutions in a framework region (based on 5 the numbering system of Kabat), and retains the binding and/or functional activity of a binding protein that comprises a variable heavy chain sequence as set forth in SEQ ID NO: 15 and a variable light chain sequence as set forth in SEQ ID NO: 19. In embodiments, the antibody or binding fragment thereof comprises a variable light chain sequence that comprises an amino acid sequence with at least about 95%, about 96%, about 10 97%, about 98%, or about 99%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 19. In embodiments, antibody or binding fragment thereof retains the binding and/or functional activity of a targeting agent, antibody, or binding fragment thereof that comprises the variable light chain sequence of SEQ ID NO: 19. In embodiments, the antibody or binding fragment thereof comprises the variable light chain sequence of SEQ ID NO: 19 and has one or 15 more conservative amino acid substitutions, e.g., 1, 2, 3, 4, 5, 1-2, 1-3, 1-4 or 1-5 conservative amino acid substitutions in the light chain variable sequence. In embodiments, the one or more conservative amino acid substitutions fall within one or more framework regions in SEQ ID NO: 19 (based on the numbering system of Kabat). In embodiments, the antibody or binding fragment thereof comprises a variable light chain 20 sequence with at least about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the binding protein light chain variable region sequence set forth in SEQ ID NO: 19, 6, 7, or 8, comprises one or more conservative amino acid substitutions in a framework region (based on the numbering system of Kabat), and retains the binding and/or functional activity of a binding protein that comprises a variable heavy chain sequence as set forth in SEQ ID NO: 15 25 and a variable light chain sequence as set forth in SEQ ID NO: 19. In embodiments, the antibody or binding fragment thereof specifically bind CDCP1. Sequences of exemplary antibodies are shown in WO 2018/112334, which is incorporated by reference herein in its entirety. In some embodiments, the ADC is used to treat cancer. Table 2A. Anti-CDCP1 sequences SEQ ID Sequence Amino acid sequence DB1/ 159693888.5 77

133186-5030-WO TVTVSS 2 CDRH1 GFTFSNYAMN Table 2B. Anti-CDCP1 sequences SEQ ID Sequence Amino acid sequence DB1/ 159693888.5 78

133186-5030-WO 15 Heavy Chain QVQLVQSGAE VKKPGSSVKV SCKASGGTFS SYAMSWVRQA variable region PGQGLEWMGG IIPILGTTNY AQKFQGRVTI TADKSTSTAY MELSSLRSED TAVYYCAREG LYAFDIWGQG TMVTVSS (VH) In embodiments, the antibody or binding fragment thereof comprises a VH that comprises and/or consists of an amino acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least 5 about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence of SEQ ID NO: 1, and comprises a VL that comprises and/or consists of an amino acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence of SEQ ID NO: 5. 10 In embodiments, the antibody or binding fragment thereof comprises a VH that comprises and/or consists of an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 1, and comprises a VL that comprises and/or consists of an amino acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 15 99%, or about 100% identical to the amino acid sequence of SEQ ID NO: 5. In embodiments, the antibody or binding fragment thereof comprises and/or consists of a VH that comprises and/or consists of an amino acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the 20 amino acid sequence of SEQ ID NO: 1, and comprises a VL that comprises and/or consists of an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 5. DB1/ 159693888.5 79

133186-5030-WO In embodiments, the antibody or binding fragment thereof comprises and/or consists of a VH that comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 1, and comprises a VL that comprises and/or consists of an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 5. 5 In embodiments, the antibody or binding fragment thereof comprises a VH that comprises and/or consists of an amino acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence of SEQ ID NO: 15, and comprises a VL that comprises and/or consists of an amino 10 acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence of SEQ ID NO: 19. In embodiments, the antibody or binding fragment thereof comprises a VH that comprises and/or consists of an amino acid sequence that is 100% identical to the amino acid sequence of 15 SEQ ID NO: 15, and comprises a VL that comprises and/or consists of an amino acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence of SEQ ID NO: 19. In embodiments, the antibody or binding fragment thereof comprises and/or consists of a VH 20 that comprises and/or consists of an amino acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence of SEQ ID NO: 15, and comprises a VL that comprises and/or consists of an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 19. 25 In embodiments, the antibody or binding fragment thereof comprises and/or consists of a VH that comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 15, and comprises a VL that comprises and/or consists of an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 19. Any combination of a VH sequence and a VL sequence shown in Table A is also encompassed 30 by the present disclosure. Ab SEQ _{VH SEQ VL Source} name: ID NO ID NO DB1/ 159693888.5 80

133186-5030-WO h12A04 23 QVTLRESGPALVKPTQTLTLT 177 DIVMTQSPLSLPVTPGEPA US20230050380A1 1- CTFSGFSLSTSGMGVSWIRQP SISCRSSQSIVHSSGNTYL VH1/VL PGKALEWLAHIYWDDDKRYNP EWYLQKPGQSPQLLIYKVS 1 1 1 1 1 1 1 1 DB1/ 159693888.5 81

133186-5030-WO h14A06 26 EVQLVESGGGLVQPGGSLRLS 186 EIVLTQSPATLSLSPGERA US20230050380A1 3VH4/V CAASGFTFSSYTMSWVRQAPE TLSCRASESVDSYGNSFMH L1 KRLEWVAYISSGGGSTYYPDT WYQQKPGQAPRLLIFLASN 1 1 1 1 1 DB1/ 159693888.5 82

133186-5030-WO CL03 34 EVQLVESGGGLVQPGGSLRLS 188 DIQMTQSPSSLSASVGDRV WO2022212876 Fab CAASGFDFSSYSIHWVRQAPG TITCRASQSVSSAVAWYQQ KGLEWVASIYPYSGSTYYADS KPGKAPKLLIYSASSLYSG DB1/ 159693888.5 83

133186-5030-WO IgG3H7 40 EVQLVESGGGLVQPGGSLRLS 188 DIQMTQSPSSLSASVGDRV WO2022212876 L CAASGFDFSSYSIHWVRQAPG TITCRASQSVSSAVAWYQQ KGLEWVASIYPYSGSTYYADS KPGKAPKLLIYSASSLYSG DB1/ 159693888.5 84

133186-5030-WO IgGA10 45 EVQLVESGGGLVQPGGSLRLS 193 DIQMTQSPSSLSASVGDRV WO2022212876 CAASGFDFSSYSIHWVRQAPG TITCRASQSVSSAVAWYQQ KGLEWVASIYPYSGSTYYADS KPGKAPKLLIYSASSLYSG DB1/ 159693888.5 85

133186-5030-WO IgGC03 45 EVQLVESGGGLVQPGGSLRLS 201 DIQMTQSPSSLSASVGDRV WO2022212876 CAASGFDFSSYSIHWVRQAPG TITCRASQSVSSAVAWYQQ KGLEWVASIYPYSGSTYYADS KPGKAPKLLIYSASSLYSG DB1/ 159693888.5 86

133186-5030-WO IgGD07 33 EVQLVESGGGLVQPGGSLRLS 209 DIQMTQSPSSLSASVGDRV WO2022212876 CAASGFDFSSYSIHWVRQAPG TITCRASQSVSSAVAWYQQ KGLEWVASIYPYSGSTYYADS KPGKAPKLLIYSASSLYSG DB1/ 159693888.5 87

133186-5030-WO CL03 55 EISEVQLVESGGGLVQPGGSL 210 MASDIQMTQSPSSLSASVG WO2022212876 Fab RLSCAASGFNFSSYSIHWVRQ DRVTITCRASQSVSSAVAW APGKGLEWVASIYPYSGSTYY YQQKPGKAPKLLIYSASSL DB1/ 159693888.5 88

133186-5030-WO CL03 58 EVQLVESGGGLVQPGGSLRLS 213 DIQMTQSPSSLSASVGDRV WO2022212876 IgG CAASGFDFSSYSIHWVRQAPG TITCRASQSVSSAVAWYQQ H1- KGLEWVASIYPYSGSTYYADS KPGKAPKLLIYSASSLYSG DB1/ 159693888.5 89

133186-5030-WO CL03 60 EVQLVESGGGLVQPGGSLRLS 215 DIQMTQSPSSLSASVGDRV WO2022212876 IgG CAASGFDFSSYSIHWVRQAPG TITCRASQSVSSAVAWYQQ H1- KGLEWVASIYPYSGSTYYADS KPGKAPKLLIYSASSLYSG DB1/ 159693888.5 90

133186-5030-WO CL07 63 EVQLVESGGGLVQPGGSLRLS 218 DIQMTQSPSSLSASVGDRV WO2022212876 IgG CAASGFNFSSSSIHWVRQAPG TITCRASQSVSSAVAWYQQ KGLEWVASIYPYSGSTSYADS KPGKAPKLLIYSASSLYSG DB1/ 159693888.5 91

133186-5030-WO IgG3H7 65 EVQLVESGGGLVQPGGSLRLS 220 MTQSPSSLSASVGDRVTIT WO2022212876 L CAASGFDFSSYSIHWVRQAPG CRASQSVSSAVAWYQQKPG KGLEWVASIYPYSGSTYYADS KAPKLLIYSASSLYSGVPS DB1/ 159693888.5 92

133186-5030-WO IgGA03 67 EVQLVESGGGLVQPGGSLRLS 222 MTQSPSSLSASVGDRVTIT WO2022212876 CAASGFDFSSYSIHWVRQAPG CRASQSVSSAVAWYQQKPG KGLEWVASIYPYSGSTYYADS KAPKLLIYSASSLYSGVPS DB1/ 159693888.5 93

133186-5030-WO IgGA05 69 EVQLVESGGGLVQPGGSLRLS 224 MTQSPSSLSASVGDRVTIT WO2022212876 CAASGFDFSSYSIHWVRQAPG CRASQSVSSAVAWYQQKPG KGLEWVASIYPYSGSTYYADS KAPKLLIYSASSLYSGVPS DB1/ 159693888.5 94

133186-5030-WO IgGA08 71 EVQLVESGGGLVQPGGSLRLS 226 MTQSPSSLSASVGDRVTIT WO2022212876 CAASGFDFSSYSIHWVRQAPG CRASQSVSSAVAWYQQKPG KGLEWVASIYPYSGSTYYADS KAPKLLIYSASSLYSGVPS DB1/ 159693888.5 95

133186-5030-WO IgGA10 73 EVQLVESGGGLVQPGGSLRLS 228 MTQSPSSLSASVGDRVTIT WO2022212876 CAASGFDFSSYSIHWVRQAPG CRASQSVSSAVAWYQQKPG KGLEWVASIYPYSGSTYYADS KAPKLLIYSASSLYSGVPS DB1/ 159693888.5 96

133186-5030-WO IgGB01 75 EVQLVESGGGLVQPGGSLRLS 230 MTQSPSSLSASVGDRVTIT WO2022212876 CAASGFDFSSYSIHWVRQAPG CRASQSVSSAVAWYQQKPG KGLEWVASIYPYSGSTYYADS KAPKLLIYSASSLYSGVPS DB1/ 159693888.5 97

133186-5030-WO IgGB04 77 EVQLVESGGGLVQPGGSLRLS 232 MTQSPSSLSASVGDRVTIT WO2022212876 CAASGFDFSSYSIHWVRQAPG CRASQSVSSAVAWYQQKPG KGLEWVASIYPYSGSTYYADS KAPKLLIYSASSLYSGVPS DB1/ 159693888.5 98

133186-5030-WO IgGB08 79 EVQLVESGGGLVQPGGSLRLS 234 MTQSPSSLSASVGDRVTIT WO2022212876 CAASGFDFSSYSIHWVRQAPG CRASQSVSSAVAWYQQKPG KGLEWVASIYPYSGSTYYADS KAPKLLIYSASSLYSGVPS DB1/ 159693888.5 99

133186-5030-WO IgGC03 81 EVQLVESGGGLVQPGGSLRLS 236 MTQSPSSLSASVGDRVTIT WO2022212876 CAASGFDFSSYSIHWVRQAPG CRASQSVSSAVAWYQQKPG KGLEWVASIYPYSGSTYYADS KAPKLLIYSASSLYSGVPS DB1/ 159693888.5 100

133186-5030-WO IgGC05 83 EVQLVESGGGLVQPGGSLRLS 238 MTQSPSSLSASVGDRVTIT WO2022212876 CAASGFDFSSYSIHWVRQAPG CRASQSVSSAVAWYQQKPG KGLEWVASIYPYSGSTYYADS KAPKLLIYSASSLYSGVPS DB1/ 159693888.5 101

133186-5030-WO IgGC09 85 EVQLVESGGGLVQPGGSLRLS 240 MTQSPSSLSASVGDRVTIT WO2022212876 CAASGFDFSSYSIHWVRQAPG CRASQSVSSAVAWYQQKPG KGLEWVASIYPYSGSTYYADS KAPKLLIYSASSLYSGVPS DB1/ 159693888.5 102

133186-5030-WO IgGD03 87 EVQLVESGGGLVQPGGSLRLS 242 MTQSPSSLSASVGDRVTIT WO2022212876 CAASGFDFSSYSIHWVRQAPG CRASQSVSSAVAWYQQKPG KGLEWVASIYPYSGSTYYADS KAPKLLIYSASSLYSGVPS DB1/ 159693888.5 103

133186-5030-WO IgGD07 89 EVQLVESGGGLVQPGGSLRLS 244 MTQSPSSLSASVGDRVTIT WO2022212876 CAASGFDFSSYSIHWVRQAPG CRASQSVSSAVAWYQQKPG KGLEWVASIYPYSGSTYYADS KAPKLLIYSASSLYSGVPS DB1/ 159693888.5 104

133186-5030-WO IgGD09 91 EVQLVESGGGLVQPGGSLRLS 246 MTQSPSSLSASVGDRVTIT WO2022212876 CAASGFNFSSSSIHWVRQAPG CRASQSVSSAVAWYQQKPG KGLEWVASIYPYSGSTSYADS KAPKLLIYSASSLYSGVPS 1 2 1 DB1/ 159693888.5 105

133186-5030-WO Mab 95 QVTLKESGPGILQSSQTLSLT 250 DVVMTQTPLTLSVTIGQPA US20110070246A1 CDCP1- CSFSGFSLSTAGMGVSWIRQP SISCKSSQSLLYTNGKTYL (LAROCHE) 012 SGKGLEWLAHIYWDDDKRYNP IWLLQRPGQSPKRLIYLVS 1 DB1/ 159693888.5 106

133186-5030-WO Antibo 104 QVQLVQSGAEVKKPGSSVKVS 259 SYELTQPPSVSVSPGQTAR US2022011954A1 dy 76 CKASGGTFSSYAISWVRQAPG ITCSGDALPKKYAFWYQQK QGLEWMGGIIPIFGTANYAQK SGQAPVLVIYEDSKRPSGI DB1/ 159693888.5 107

133186-5030-WO 18C06 113 QVQLQESGPGQVKPSETLSLT 268 EIVMTQSPATLSVSPGERA US2022011954A1 CTVSGGSISSSFWSWIRQPPG TLSCRASQSVSSNLAWYQQ KGLEWIGYIYYSESTNYNPSL KPGQAPRLLIYGASTRATG DB1/ 159693888.5 108

133186-5030-WO 12C2 122 QEQLVESGGGVVQPGRSLRLS 277 DILMTQSPSSLSASVGDRV US2022011954A1 CVTSGFTFSSYGMHWVRQAPG TITCRASQGINYYLAWYQQ KGLEWVAVIWYDGTNKYYADT KPGKVPKLLIYTASTLQSG DB1/ 159693888.5 109

133186-5030-WO 39D7 131 EVQLLESGGGLVQPGGSLRLS 286 DIVMTQSPLSLPVTPGEPA US2022011954A1 CAASGFTFSSYAMSWVRQAPG SISCRSSQSLLHSNGYNYL KGLEWVSTISGSGGSTYYADS DWYLQKSGQSPQLLIYLGS DB1/ 159693888.5 110

133186-5030-WO 21F2 140 QVQLVESGGGVVQPGRSLRLS 295 DIQMTQSPSTLSASVGDRV US2022011954A1 CAASGFTFSSYGMFWVRQTPG TITCRASQSISSWLAWYQQ KGLEWVANIWYDGSNKYYTDS KPGKAPKLLIYKASSLESG 1 1 DB1/ 159693888.5 111

133186-5030-WO hHC4- 147 EVQLVESGGGLVQPGGSLRLS 302 EVQLVESGGGLVQPGGSLR US 9346886 B2 H- CAASGFTFNSYGMSWVRQAPG LSCAASGFTFNSYGMSWVR humani KGLEWVATISSGGSYKYYVDS QAPGKGLEWVATISSGGSY DB1/ 159693888.5 112

133186-5030-WO hHC4- 155 EVQLLESGGGLVQPGGSLRLS 310 EVQLLESGGGLVQPGGSLR US 9346886 B2 07- CAASGFTFNSYAMSWVRQAPG LSCAASGFTFNSYAMSWVR humani KGLEWVSTISSGGSYTYYPDS QAPGKGLEWVSTISSGGSY DB1/ 159693888.5 113

133186-5030-WO M103L/ 164 EVQLLESGGGLVQPGGSLRLS 315 DIQLTQSPSFLSASVGDRV WO2024/127366 M108L CAASGFTFSNYAMNWVRQAPG TITCRASQGISSYLAWYQQ A1 KGLEWVSAISGGGGSTYYADS KPGKAPKLLIYAASTLQGG 5 5 5 5 5 5 5 DB1/ 159693888.5 114

133186-5030-WO CDCP1- 172 EVQLVESGGGLVQPGGSLRLS 323 DIQMTQSPSSLSASVGDRV US 2024/0366815 009 CAASGFNLYSYYIHWVRQAPG TITCRASQSVSSAVAWYQQ A1 KGLEWVASIYPYYSSTSYADS KPGKAPKLLIYSASSLYSG 5 5 5 Several human, humanized, and chimeric anti-CDCP1 antibodies are disclosed in the patent literature (see, e.g., Table A above depicting the heavy chain, heavy chain variable region, light chain, and/or light chain variable regions of such antibodies. Such sequences can be found, for 5 example, in Japanese Application Publication No. JP 2007/112734, PCT Application Publication No. WO 2022/212876, as well as U.S. Application Publication Nos.2023/0050380 A1, 2022/0389113 A1, 2022/011954 A1, 2008/0008719 A1, and U.S. Patent No.9,346,886, which are herein incorporated by reference. In embodiments, the antibody or binding fragment thereof comprises a heavy chain (or heavy 10 chain variable region) comprising an amino acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence of any one of SEQ ID NOs: 23-176 and a light chain (or a light chain variable region) comprising an amino acid sequence at least about 90%, at least about 15 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence of SEQ ID NOs: 177-326, respectively. In embodiments, the antibody or binding fragment thereof comprises a heavy chain comprising an amino acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 20 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least DB1/ 159693888.5 115

133186-5030-WO about 98%, at least about 99%, or about 100% identical to the amino acid sequence of SEQ ID NO: 10, and comprises a light chain comprising an amino acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% 5 identical to the amino acid sequence of SEQ ID NO: 11. In embodiments, the antibody or binding fragment thereof comprises: (i) a heavy chain variable region (VH) that comprises: (a) a CDRH1 comprising and/or consisting of the amino acid sequence of SEQ ID NO: 2, 10 (b) a CDRH2 comprising and/or consisting of the amino acid sequence of SEQ ID NO: 3, and (c) a CDRH3 comprising and/or consisting of the amino acid sequence of SEQ ID NO: 4, and (ii) a light chain variable region (VL) that comprises: 15 (a) a CDRL1 comprising and/or consisting of the amino acid sequence of SEQ ID NO: 6, (b) a CDRL2 comprising and/or consisting of the amino acid sequence of SEQ ID NO: 7, and (c) a CDRL3 comprising and/or consisting of the amino acid sequence of SEQ ID 20 NO:8. In embodiments, the antibody or binding fragment thereof comprises: (i) a heavy chain variable region (VH) that comprises: (a) a CDRH1 comprising and/or consisting of the amino acid sequence of SEQ ID NO: 12, 25 (b) a CDRH2 comprising and/or consisting of the amino acid sequence of SEQ ID NO: 13, and DB1/ 159693888.5 116

133186-5030-WO (c) a CDRH3 comprising and/or consisting of the amino acid sequence of SEQ ID NO: 14, and (ii) a light chain variable region (VL) that comprises: (a) a CDRL1 comprising and/or consisting of the amino acid sequence of SEQ ID NO: 5 6, (b) a CDRL2 comprising and/or consisting of the amino acid sequence of SEQ ID NO: 7, and (c) a CDRL3 comprising and/or consisting of the amino acid sequence of SEQ ID NO:8. 10 In embodiments, the antibody or binding fragment thereof comprises: (i) a heavy chain variable region (VH) that comprises: (a) a CDRH1 comprising and/or consisting of the amino acid sequence of SEQ ID NO: 16, (b) a CDRH2 comprising and/or consisting of the amino acid sequence of SEQ ID NO: 15 17, and (c) a CDRH3 comprising and/or consisting of the amino acid sequence of SEQ ID NO: 18, and (ii) a light chain variable region (VL) that comprises: (a) a CDRL1 comprising and/or consisting of the amino acid sequence of SEQ ID NO: 20 20, (b) a CDRL2 comprising and/or consisting of the amino acid sequence of SEQ ID NO: 21, and (c) a CDRL3 comprising and/or consisting of the amino acid sequence of SEQ ID NO: 22. 25 In embodiments, the antibody or binding fragment thereof described herein comprises an Fc domain. The Fc domain can be derived from IgA (e.g., IgA1 or IgA2), IgG, IgE, or IgG (e.g., IgG₁, IgG₂, IgG₃, or IgG₄). In some embodiments, the Fc domain comprises wild type sequence DB1/ 159693888.5 117

133186-5030-WO of an Fc domain. In some embodiments, the Fc domain comprises one or more mutations resulting in altered biological activity. For example, mutations may be introduced into the Fc domain to increase the homogeneity during the production of the recombinant protein. In some embodiments, the Fc domain is the Fc domain of human IgG. In some embodiments, the lysine 5 located in the C-terminal position of the Fc domain is deleted to increase the homogeneity during the production of the recombinant protein. In some embodiments, the lysine located in the C-terminal position of the Fc domain is present. In embodiments, the polypeptide comprising the targeting agent, antibody, or binding fragment thereof, described herein is encoded by a cDNA polynucleotide sequence. As is well- 10 understood in the art, introduction of the cDNA into a competent mammalian cell will result in the production of the polypeptide comprising the targeting agent, antibody, or binding fragment thereof. Exemplary methods of antibody production by these means are disclosed in at least US Pat. Nos.8,008,449, 10,934,571 and 11,339,215, which are herein incorporated by reference. In embodiments, the cDNA comprises a polynucleotide encoding a polypeptide comprising an 15 immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs H1, H2, and H3 with the amino acid sequences set forth in SEQ ID NOs: 2, 3, and 4, respectively, and a VL comprising CDRs L1, L2, and L3 with the amino acid sequences set forth in SEQ ID NOs: 6, 7, and 8, respectively. In embodiments, the cDNA comprises a polynucleotide encoding a polypeptide comprising an 20 immunoglobulin heavy chain or a fragment thereof comprising a VH with the amino acid sequence set forth in SEQ ID NO: 1, and comprising a VL with the amino acid sequence set forth in SEQ ID NO: 5. In embodiments, the cDNA comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs H1, H2, 25 and H3 with the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively, and a VL comprising CDRs L1, L2, and L3 with the amino acid sequences set forth in SEQ ID NOs: 20, 21, and 22, respectively. In embodiments, the cDNA comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH with the amino acid 30 sequence set forth in SEQ ID NO: 15, and comprising a VL with the amino acid sequence set forth in SEQ ID NO: 19. Also provided by the present disclosure is an antibody or binding fragment thereof that binds to the same epitope (e.g. CDCP1) as any of the antibodies, or binding fragment thereofs thereof, DB1/ 159693888.5 118

133186-5030-WO described herein. For example, antibody competition assay (and overlapping epitope analysis) can be assessed by surface plasmon resonance (SPR) or bio-layer interferometry (BLI), as described in detail herein. The antibodies and binding fragment thereof provided by the invention include monoclonal 5 antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab’, F(ab’)2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, heteroconjugate antibodies (e.g. antibody-drug conjugates), single chain (ScFv), mutants thereof, fusion proteins comprising an antibody portion, domain antibodies (dAbs), humanized antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required 10 specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. The antibodies and binding fragment thereof may be murine, rat, human, or any other origin (including chimeric or humanized antibodies). In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a chimeric, humanized or human antibody. In certain embodiments, the antibody is an 15 antibody-drug conjugate. In some embodiments, the antibody or binding fragment thereof comprises one or more conservative amino acid substitutions. A person of skill in the art will recognize that a conservative amino acid substitution is a substitution of one amino acid with another amino acid that has similar structural or chemical properties, such as, for example, a similar side chain. 20 Exemplary conservative substitutions are described in the art, for example, in Watson et al., Molecular Biology of the Gene, The Benjamin/Cummings Publication Company, 4th Ed. (1987). “Conservative modifications” refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequences. 25 Conservative modifications include amino acid substitutions, additions and deletions. Conservative substitutions are those in which the amino acid is replaced with an amino acid residue having a similar side chain. The families of amino acid residues having similar side chains are well defined and include amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine), nonpolar side chains (e.g., 30 alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), uncharged polar side chains (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine, tryptophan), aromatic side chains (e.g., phenylalanine, tryptophan, histidine, tyrosine), aliphatic side chains (e.g., glycine, alanine, valine, leucine, isoleucine, serine, threonine), amide (e.g., asparagine, glutamine), beta- branched side chains (e.g., threonine, valine, isoleucine) and sulfur-containing 35 side chains (cysteine, methionine). Furthermore, any native residue in the polypeptide may also DB1/ 159693888.5 119

133186-5030-WO be substituted with alanine, as has been previously described for alanine scanning mutagenesis (MacLennan et al. (1998) Acta Physiol Scand Suppl 643: 55-67; Sasaki et al. (1998) Adv Biophys 35: 1-24). Amino acid substitutions to the antibodies of the invention may be made by known methods for example by PCR mutagenesis (U.S. Patent No.4,683,195). 5 In embodiments, the antibody or binding fragment thereof comprises a variable heavy chain sequence that comprises an amino acid sequence with at least about 95%, about 96%, about 97%, about 98%, or about 99%, sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1; 15; 23-176. In embodiments, the antibody or binding fragment thereof retains the binding and/or functional activity of an antibody or fragment thereof that comprises the variable 10 heavy chain sequence of SEQ ID NOs: 1; 15; 23-176. In embodiments, the antibody or binding fragment thereof comprises the variable heavy chain sequence of SEQ ID NOs: 1; 15; 23-176 and have one or more conservative amino acid substitutions, e.g., 1, 2, 3, 4, 5, 1-2, 1-3, 1-4 or 1- 5 conservative amino acid substitutions in the heavy chain variable sequence. In embodiments, the antibody or binding fragment thereof comprises a variable heavy chain 15 sequence with at least about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the antibody or fragment thereof heavy chain variable region sequence set forth in SEQ ID NOs: 1; 15; 23-176, comprises one or more conservative amino acid substitutions in a framework region (based on the numbering system of Kabat), and retains the binding and/or functional activity of an antibody or fragment thereof that comprises a variable heavy chain 20 sequence as set forth in SEQ ID NOs: 1; 15; 23-176 and a variable light chain sequence as set forth in SEQ ID NOs: 5; 19; 177-326. In embodiments, the antibody or binding fragment thereof comprises a variable light chain sequence that comprises an amino acid sequence with at least about 95%, about 96%, about 97%, about 98%, or about 99%, sequence identity to the amino acid sequence set forth in SEQ 25 ID NOs: 5; 19; 177-326. In other embodiments, the antibody or fragment thereof retains the binding and/or functional activity of an antibody or fragment thereof that comprises the variable light chain sequence of SEQ ID NOs: 5; 19; 177-326. In embodiments, the antibody or fragment thereof comprises the variable light chain sequence of SEQ ID NOs: 5; 19; 177-326 and have one or more conservative amino acid substitutions, e.g., 1, 2, 3, 4, 5, 1-2, 1-3, 1-4 or 1- 30 5 conservative amino acid substitutions in the light chain variable sequence. In embodiments, the antibody or binding fragment thereof comprises a variable light chain sequence with at least about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the antibody or fragment thereof light chain variable region sequence set forth in SEQ ID NOs: 5; 19; 177-326, comprises one or more conservative amino acid substitutions in a DB1/ 159693888.5 120

133186-5030-WO framework region (based on the numbering system of Kabat), and retains the binding and/or functional activity of an antibody or fragment thereof that comprises a variable heavy chain sequence as set forth in SEQ ID NOs: 1; 15; 23-176 and a variable light chain sequence as set forth in SEQ ID NOs: 5; 19; 177-326. 5 The binding affinity of a targeting agent or antibody can be expressed as an equilibrium dissociation constant (KD) value, which refers to the dissociation rate of a particular antigen- antibody interaction. KD is the ratio of the rate of dissociation, also called the “off-rate (koff)”, to the association rate, or “on-rate (kon)”. Thus, KD equals koff/kon (dissociation/association) and is expressed as a molar concentration (M), and the smaller the K_D, the stronger the affinity of 10 binding. K_D values for antibodies can be determined using methods well established in the art. Unless otherwise specified, “binding affinity” refers to monovalent interactions (intrinsic activity; e.g., binding of an antibody to an antigen through a monovalent interaction). In embodiments, the antibody or binding fragment thereof has an affinity (K_D) value of or less than about 350 nM, about 325 nM, about 323.10 nM, about 300 nM, about 286.44 nM, about 15 275 nM, about 250 nM, about 232.13 nM, about 225 nM, about 219.13 nM, about 200 nM, about 195.54 nM, about 175 nM, about 158 nM, about 150 nM, about 125 nM, or about 100 nM. In embodiments, the antibody or binding fragment thereof binds an epitope (e.g. CDCP1) with a KD value of or less than about 95 nM, about 90 nM, about 80 nM, about 79.89 nM, about 75 20 nM, about 70 nM, about 69.50 nM, about 65 nM, about 63.44 nM, about 60 nM, about 55 nM, about 52.88 nM, about 50 nM, about 45 nM, about 44.50 nM, about 41.99 nM, about 40 nM, about 35 nM, about 30 nM, about 25 nM, about 20 nM, about 10 nM, about 5 nM, or about 1 nM. In embodiments, the antibody or binding fragment thereof binds an epitope (e.g. CDCP1) with a 25 K_D value of or less than about 5 nM, about 4.5 nM, about 4 nM, about 3.5 nM, about 3.12 nM, about 3 nM, about 2.90 nM, about 2.5 nM, about 2 nM, about 1.5 nM, about 1 nM, about 900pM, about 800pM, about 700pM, about 600pM, about 500pM, about 400pM, about 300pM, about 250pM, about 200pM, about 150pM, about 100pM, about 50pM, about 40pM, about 30pM, about 25pM, about 20pM, about 15pM, about 10pM, about 5pM, or about 1pM. 30 The value of KD can be determined directly by well-known methods, and can be computed even for complex mixtures by methods such as those, for example, set forth in Caceci et al., (1984, Byte 9: 340-362). For example, the KD may be established using a double-filter nitrocellulose filter binding assay such as that disclosed by Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 5428-5432). Other standard assays to evaluate the binding ability of ligands such as DB1/ 159693888.5 121

133186-5030-WO antibodies towards target antigens are known in the art, including for example, ELISAs, Western blots, RIAs, and flow cytometry analysis, and other assays exemplified elsewhere herein. One exemplary method for measuring binding affinity (K_D) value is surface plasmon resonance 5 (SPR), typically using a biosensor system such as a BIACORE® system. SPR refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIACORE® system. BIAcore kinetic analysis comprises analyzing the binding and dissociation of an antigen from a chip with an immobilized molecule (e.g., a molecule 10 comprising an antigen-binding domain), on their surface; or the dissociation of an antibody, or binding fragment thereof, from a chip with an immobilized antigen. In embodiments, the SPR measurement is conducted using a BIACORE® T100 or T200 instrument. For example, a standard assay condition for surface plasmon resonance can be based on antibody immobilization of approximately 100-500 Response Units (RU) of IgG on the SPR 15 chip. Purified target proteins are diluted in buffer to a range of final concentrations and injected at a requisite flow rate (e.g., 10-100 µl/min) to allow the calculation of Ka. Dissociation is allowed to proceed to establish off-rate, followed by 3 M MgCl2 (or 20 mM NaOH) for regeneration of the chip surface. Sensorgrams are then analyzed using a kinetics evaluation software package. In an exemplary embodiment, the SPR assay is according to the conditions as 20 set forth in the Examples. In embodiments, the binding affinity (K_D) value is measured using solution-based kinetic exclusion assay (KinExA™). In a particular embodiment, the KinExA measurement is conducted using a KinExA™ 3200 instrument (Sapidyne). The Kinetic Exclusion Assay (KinExA™) is a general purpose immunoassay platform (basically a flow spectrofluorimeter) 25 that is capable of measuring equilibrium dissociation constants, and association and dissociation rate constants for antigen/antibody interactions. Since KinExA™ is performed after equilibrium has been obtained it is an advantageous technique to use for measuring the K_D of high affinity interactions where the off-rate of the interaction may be very slow. The KinExA™ methodology can be conducted generally as described in Drake et al., (2004) Analytical Biochem.328, 35-43. 30 Another method for determining the KD of an antibody is by using Bio-Layer Interferometry (BLI), typically using OCTET® technology (e.g., Octet QKe system) from ForteBio. In certain embodiments, the BLI measurement is conducted according to the following: sensor tips coated with a proprietary anti-human antibody (ForteBio) undergo BLI signal stabilization by dipping in running buffer (such as 10mM Hepes Buffered Saline (HBS) containing 0.05% tween-20) for DB1/ 159693888.5 122

133186-5030-WO 120s. The antibody is then captured by dipping the sensors into a running buffer solution (buffer may contain 1-10ug/mL of the antibody) for 300s. The signal is then stabilized by dipping the sensor tips back into running buffer for 120s. The tips are then transferred into solution containing the cognate antigen. The binding of antibody-antigen is measured over 180s prior to 5 the sensor tips being transferred to running buffer in order to monitor receptor dissociation over 180s. In the case of CDCP1, typically a 7-point dose response of the antigen (may range from 1-2nM in doubling dilutions) is measured. Additionally, sensor tips with no antibody captured are exposed to the antigen in order to monitor non-specific binding of the receptors to the sensor 10 tips. A 2^nd reference type also includes a tip with antibody captured upon on it but with subsequent exposure to running buffer only with no antigen. This allows for double-referencing to eliminate both non-specific binding as well as system noise and the underlying baseline drift attributed to the antibody dissociating from the anti-human Fc sensor tip. The raw under goes double reference subtraction and is then fit to a 1:1 Langmuir type binding model to determine 15 affinity and kinetic parameters. In embodiments, the CDCP1 is a human CDCP1, cyno CDCP1 or mouse CDCP1. In general, an anti-CDCP1 antibody should bind to CDCP1 with high affinity. It is desirable that the anti- CDCP1 antibody have binding affinities (K_D) to human CDCP1 in low nanomolar range, such as about 40 nM or lower. In some embodiments, the CDCP1 is a human CDCP1 and the K_D 20 value is about 40 nM, about 45 nM or about 50 nM. In some embodiments, the CDCP1 is a cyno CDCP1 and the KD value is about 62 nM, about 64 nM, about 66 nm, about 68 nM, or about 70 nM. Pharmaceutical Compositions In embodiments, an active pharmaceutical ingredient or combination of active pharmaceutical 25 ingredients, such as any of the conjugates, drug moieties, linkers, compounds, and/or compositions of the disclosure, is provided as a pharmaceutically acceptable composition. In embodiment, the disclosure relates to a pharmaceutical composition including a therapeutically effective amount of one or more antibody-drug conjugates, drug moieties, linkers, compounds, and /or compositions of the disclosure (e.g. antibody-drug conjugates of 30 formula (I)); and a physiologically compatible carrier medium, wherein the disease is cancer. In one embodiment, the diseases is a cancer such as pancreatic cancer, breast cancer, prostate cancer, lymphoma, skin cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain cancer, primary brain carcinoma, head-neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, head or neck carcinoma, breast carcinoma, ovarian DB1/ 159693888.5 123

133186-5030-WO carcinoma, lung carcinoma, small-cell lung carcinoma, Wilms’ tumor, cervical carcinoma, testicular carcinoma, bladder carcinoma, pancreatic carcinoma, stomach carcinoma, colon carcinoma, prostatic carcinoma, genitourinary carcinoma, thyroid carcinoma, esophageal carcinoma, myeloma, multiple myeloma, adrenal carcinoma, renal cell carcinoma, endometrial 5 carcinoma, adrenal cortex carcinoma, malignant pancreatic insulinoma, malignant carcinoid carcinoma, choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervical hyperplasia, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, chronic granulocytic leukemia, acute granulocytic leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi’s 10 sarcoma, polycythemia vera, essential thrombocytosis, Hodgkin’s disease, non-Hodgkin’s lymphoma, soft-tissue sarcoma, osteogenic sarcoma, primary macroglobulinemia, or retinoblastoma, and the like. In other embodiments, the cancer is acoustic neuroma, adenocarcinoma, angiosarcoma, astrocytoma, basal cell carcinoma, bile duct carcinoma, bladder carcinoma, brain cancer, breast cancer, triple-negative breast cancer (TNBC), bronchogenic 15 carcinoma, cervical cancer, chordoma, choriocarcinoma, colon cancer, colorectal cancer, craniopharyngioma, cystadenocarcinoma, embryonal carcinoma, endotheliocarcinoma, ependymoma, epithelial carcinoma, esophageal cancer, Ewing’s tumor, fibrosarcoma, gastric cancer, glioblastoma multiforme, glioma, head and neck cancer, hemangioblastoma, hepatoma, kidney cancer, leiomyosarcoma, liposarcoma, lung cancer, lymphangioendotheliosarcoma, 20 lymphangiosarcoma, medullary carcinoma, medulloblastoma, melanoma, meningioma, mesothelioma, myxosarcoma, nasal cancer, neuroblastoma, oligodendroglioma, oral cancer, osteogenic sarcoma, ovarian cancer, pancreatic cancer, papillary adenocarcinoma, papillary carcinoma, pinealoma, prostate cancer, rabdomyosarcoma, rectal cancer, renal cell carcinoma, retinoblastoma, sarcoma, sebaceous gland carcinoma, seminoma, skin cancer, squamous cell 25 carcinoma, stomach cancer, sweat gland carcinoma, synovioma, testicular cancer, small cell lung carcinoma, throat cancer, uterine cancer, Wilm’s tumor, blood cancer, acute erythroleukemic leukemia, acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, acute lymphoblastic leukemia, acute megakaryoblastic leukemia, acute monoblastic leukemia, acute myeloblastic leukemia, acute myelomonocytic leukemia, acute nonlymphocytic 30 leukemia, acute promyelocytic leukemia, acute undifferentiated leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, hairy cell leukemia, multiple myeloma, heavy chain disease, Hodgkin’s disease, multiple myeloma, non-Hodgkin’s lymphoma, polycythemia vera, or Waldenstrom’s macroglobulinemia. In some embodiments, the disease is triple-negative breast cancer (TNBC). 35 In embodiments, the disease is selected from kidney cancer, bladder cancer, testicular cancer, prostate cancer, rhabdomyosarcoma, ovarian cancer, Diffuse Large B-Cell Lymphoma, gastric cancer, breast cancer, squamous cell carcinoma (SCC), pancreatic cancer, neuroblastoma, DB1/ 159693888.5 124

133186-5030-WO leukemia (e.g. megakaryoblastic leukemia), lymphoma (e.g. Burkitt's lymphoma), cervical cancer, thyroid cancer, adenocarcinoma, hepatoma, mantle cell lymphoma, colorectal cancer, multiple myeloma, lung cancer (e.g. small cell lung cancer), endometrial carcinoma, neuroblastoma, uterine cancer, gastric cancer, and glioblastoma. 5 In embodiments, the concentration of each of the active pharmaceutical ingredients provided in the pharmaceutical compositions of the disclosure, such as any of the conjugates, drug moieties, linkers, compounds, and /or compositions of the disclosure (e.g. antibody-drug conjugates of formula (I)), is less than, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 10 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v, or v/v of the pharmaceutical composition. In embodiments, the concentration of each of the active pharmaceutical ingredients provided in 15 the pharmaceutical compositions of the disclosure, such as any of the conjugates, drug moieties, linkers, compounds, and /or compositions of the disclosure (e.g. antibody-drug conjugates of formula (I)), is greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25% 19%, 18.75%, 18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25% 17%, 16.75%, 16.50%, 16.25% 16%, 15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%, 14.25% 14%, 13.75%, 13.50%, 20 13.25% 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25% 11%, 10.75%, 10.50%, 10.25% 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%, 8.50%, 8.25% 8%, 7.75%, 7.50%, 7.25% 7%, 6.75%, 6.50%, 6.25% 6%, 5.75%, 5.50%, 5.25% 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 25 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v, or v/v of the pharmaceutical composition. In embodiments, the concentration of each of the active pharmaceutical ingredients provided in the pharmaceutical compositions of the disclosure, such as any of the conjugates, drug moieties, 30 linkers, compounds, and /or compositions of the disclosure (e.g. antibody-drug conjugates of formula (I)), is in the range from about 0.0001% to about 50%, about 0.001% to about 40%, about 0.01% to about 30%, about 0.02% to about 29%, about 0.03% to about 28%, about 0.04% to about 27%, about 0.05% to about 26%, about 0.06% to about 25%, about 0.07% to about 24%, about 0.08% to about 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 35 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about DB1/ 159693888.5 125

133186-5030-WO 17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12% or about 1% to about 10% w/w, w/v, or v/v of the pharmaceutical composition. In embodiments, the concentration of each of the active pharmaceutical ingredients provided in 5 the pharmaceutical compositions of the disclosure, such as any of the conjugates, drug moieties, linkers, compounds, and /or compositions of the disclosure (e.g. antibody-drug conjugates of formula (I)), is in the range from about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, 10 about 0.09% to about 1%, about 0.1% to about 0.9% w/w, w/v, or v/v of the pharmaceutical composition. In embodiments, the amount of each of the active pharmaceutical ingredients provided in the pharmaceutical compositions of the disclosure, such as any of the foregoing conjugates, drug moieties, linkers, compounds, and /or compositions of the disclosure (e.g. antibody-drug 15 conjugates of formula (I)), is equal to or less than 10 g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g, 4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g, 0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g, 0.007 g, 0.006 g, 0.005 g, 0.004 g, 0.003 g, 0.002 g, 0.001 g, 0.0009 g, 0.0008 g, 0.0007 g, 0.0006 g, 20 0.0005 g, 0.0004 g, 0.0003 g, 0.0002 g, or 0.0001 g. In embodiments, the amount of each of the active pharmaceutical ingredients provided in the pharmaceutical compositions of the disclosure, such as any of the conjugates, drug moieties, linkers, compounds, and /or compositions of the disclosure (e.g. antibody-drug conjugates of formula (I)), is more than 0.0001 g, 0.0002 g, 0.0003 g, 0.0004 g, 0.0005 g, 0.0006 g, 0.0007 g, 25 0.0008 g, 0.0009 g, 0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035 g, 0.004 g, 0.0045 g, 0.005 g, 0.0055 g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g, 0.008 g, 0.0085 g, 0.009 g, 0.0095 g, 0.01 g, 0.015 g, 0.02 g, 0.025 g, 0.03 g, 0.035 g, 0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g, 0.065 g, 0.07 g, 0.075 g, 0.08 g, 0.085 g, 0.09 g, 0.095 g, 0.1 g, 0.15 g, 0.2 g, 0.25 g, 0.3 g, 0.35 g, 0.4 g, 0.45 g, 0.5 g, 0.55 g, 0.6 g, 0.65 g, 0.7 g, 0.75 g, 0.8 g, 0.85 g, 0.9 g, 0.95 g, 1 g, 1.5 g, 2 g, 30 2.5, 3 g, 3.5, 4 g, 4.5 g, 5 g, 5.5 g, 6 g, 6.5 g, 7 g, 7.5 g, 8 g, 8.5 g, 9 g, 9.5 g, or 10 g. Each of the active pharmaceutical ingredients according to the disclosure is effective over a wide dosage range. For example, in the treatment of adult humans, dosages independently range from 0.01 to 1000 mg, from 0.5 to 100 mg, from 1 to 50 mg per day, and from 5 to 40 mg per day are examples of dosages that may be used. The exact dosage will depend upon the route of DB1/ 159693888.5 126

133186-5030-WO administration, the form in which the compound is administered, the gender and age of the subject to be treated, the body weight of the subject to be treated, and the preference and experience of the attending physician. The clinically-established dosages of the conjugates, drug moieties, linkers, compounds, and /or compositions of the disclosure (e.g. formula (I) formula 5 (II), and/or formula (III)), of the disclosure may also be used if appropriate. In embodiments, the molar ratio of two active pharmaceutical ingredients in the pharmaceutical compositions is in the range from 10:1 to 1:10, preferably from 2.5:1 to 1:2.5, and more preferably about 1:1. In an embodiment, the weight ratio of the molar ratio of two active pharmaceutical ingredients in the pharmaceutical compositions is selected from the group 10 consisting of 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, and 1:20. In an embodiment, the weight ratio of the molar ratio of two active pharmaceutical ingredients in the pharmaceutical compositions is selected from the group consisting of 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 15 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, and 1:20. Described below are non-limiting pharmaceutical compositions and methods for preparing the same. Dosages and Dosing Regimens 20 The amounts of the pharmaceutical compositions administered using the methods herein, such as the dosages of conjugates, drug moieties, linkers, compounds, and /or compositions of the disclosure (e.g. antibody-drug conjugates of formula (I)), will be dependent on the human or mammal being treated, the severity of the disorder or condition, the rate of administration, the disposition of the active pharmaceutical ingredients and the discretion of the prescribing 25 physician. However, an effective dosage is in the range of about 0.001 to about 100 mg per kg body weight per day, such as about 1 to about 35 mg/kg/day, in single or divided doses. For a 70 kg human, this would amount to about 0.05 to 7 g/day, such as about 0.05 to about 2.5 g/day. An effective amount of the combination of the active pharmaceutical ingredient may be administered in either single or multiple doses by any of the accepted modes of administration 30 of agents having similar utilities, including rectal, buccal, intranasal and transdermal routes, by DB1/ 159693888.5 127

133186-5030-WO intra-arterial injection, intravenously, intraperitoneally, parenterally, intramuscularly, subcutaneously, orally, topically, or as an inhalant. In some embodiments, the compositions described herein further include controlled-release, sustained release, or extended-release therapeutic dosage forms for administration of the 5 compounds described herein, which involves incorporation of the compounds into a suitable delivery system in the formation of certain compositions. This dosage form controls release of the compound(s) in such a manner that an effective concentration of the compound(s) in the bloodstream may be maintained over an extended period of time, with the concentration in the blood remaining relatively constant, to improve therapeutic results and/or minimize side effects. 10 Additionally, a controlled-release system would provide minimum peak to trough fluctuations in blood plasma levels of the compound. E_{mbodiment 1. An antibody-drug conjugate having formula (I):} Ab-[L–D]_n formula (I) 15 wherein in formula (I): Ab is an antibody or binding fragment thereof that binds CUB Domain-Containing Protein-1 (CDCP1); D is a drug moiety comprising a pyrridinobenzodiazepine (PDD); L is a linker; 20 wherein D and/or L comprise at least one carbohydrate substituent R_S. E_{mbodiment 2. The antibody-drug conjugate of embodiment 1, wherein RS is a} univalent saccharide substituent, preferably RS is glycosyl or O-glycosyl. E_{mbodiment 3. The antibody-drug conjugate of embodiment 1 or embodiment 2,}25 wherein D comprises a moiety of formula (IIa): wherein: DB1/ 159693888.5 128

133186-5030-WO the dotted line indicates the optional presence of a double bond between one or more of C1 and C2, C2 and C3, and C3 and C4; the wavy line indicates the point of attachment to L; m is 0 or 1; 5 R1, R3 and R4 are independently selected from H and R29; R2 is selected from H, RS, L2-R58, R29, and –L_S-R_S, or one of R₁ and R₂, R₂ and R₃, or R₃ and R₄, together with the carbon atoms to which they are attached form a 6-membered aryl, or a 5- or 6-membered cyclic, heterocyclic, or heteroaryl ring optionally substituted with 1, 2 or 3 independently selected optional R20 groups; 10 R5 and R6 are selected such that either (i) R5 is selected from H, OH and OC1-6 alkyl; and R6 is selected from H, SO3H, –LS-RS, nitrogen protecting groups, –L2-R58 and RA; (ii) R5 is oxo or H, and R₆ is H or C_1-6 alkyl; or (iii) R₅ and R₆ together form a double bond; R_{7 and R9 are independently selected from H and R20;} R8 is selected from H, SR24, SCH2Ph, R20, L2-R58, and –LS-RS; 15 RA is selected from (CH2)j-OH, (CH2)j-CO2R26, C(=O)-O-(CH2)k-NR26R27, (CH2)jNR26R27, C(=O)-NH-(CH2)j-NR26R27 and C(=O)-NH-(CH2)k-C(=NH)NR26R27; L2 is a bond or a lin P, O, S or halogen, and optionally incorporates ether, oxo, carboxamidyl, urethanyl, branched, cyclic, unsaturated, heterocyclyl, aryl or heteroaryl moieties; and 20 R₅₈ is R_A, O-(CH₂)_k-NR₂₆R₂₆, or NHNH₂; each R29 is independently selected from R20, R21, =CH2, =CH-(CH2)s-CH3, =CH-(CH2)s-R21, =O, (CH2)s-OR21, (CH2)s-CO2R21, (CH2)s-NR21R24, O-(CH2)t-NR21R24, NH-C(O)-R21, O- (CH2)t-NH-C(O)-R21, O-(CH2)t-C(O)-NH-R21, (CH2)s-SO2R21, O-SO2R21, (CH2)s-C(O)R21 and (CH₂)_s-C(O)NR₂₁R₂₄; 25 each R₂₀ is independently selected from F, Cl, Br, (CH₂)_j-OH, C_1-6 alkyl, OC_1-6 alkyl, R_S, OCH₂Ph, (CH₂)_j-CO₂R₂₆, O-(CH₂)_k-NR₂₆R₂₇, C(=O)-O-(CH₂)_k-NR₂₆R₂₇, C(=O)-NR₂₆R₂₇, (CH2)j-NR26R27, NR26NH2, C(=O)-NH-(CH2)j-NR26R27, C(=O)-NH-C6H4-(CH2)j-R26, C(=O)-NH-(CH2)k-C(=NH)NR26R27, –L2-R58, S(O)2-(C1-6 alkyl), O-(CH2)k-O-(C1-6 alkyl), (CH2)j-S(O)2-NR26R27, C(=NH)-O-(C1-6 alkyl), (CH2)k-O-(C1-6 alkyl), CN, NCO, Cy, 30 nd , , , , , or 6; each k and t is independently selected from 1, 2, 3, 4, 5 or 6; each R21 is independently selected from H, C1-12 alkyl, C5-6 heterocyclyl, C5-9 heteroaryl, C6-15 35 heteroarylalkyl, phenyl and C7-12 aralkyl groups; wherein the heterocyclyl, heteroaryl, DB1/ 159693888.5 129

133186-5030-WO heteroarylalkyl, phenyl and aralkyl groups are optionally substituted with 1, 2 or 3 independently selected optional R20 groups; each R24, R26 and R27 is independently selected from H and C1-12 alkyl; each Cy is independently selected from a C5-6 heterocyclyl or C5-6 heteroaryl group, wherein the 5 heterocyclyl or heteroaryl groups are optionally substituted with 1 or 2 R20 groups; L_S is a bond, an amino acid, a peptide chain having from 2 to 6 amino acids, an alkylene chain containing from 1 to 12 carbon atoms which may contain one or more carbon-carbon double or triple bonds, a paraformaldehyde chain –(OCH₂)_1-12-, a polyethylene glycol chain -(OCH2CH2)1-6-, which chains may be interrupted by or optionally incorporates one _{10 or more of P, O, S, NH, C5-9 heteroarylene, phenylene, heterocyclyl, cycloalkyl, ether,} oxo, carboxamidyl, and/or urethanyl moieties wherein the C5-9 heteroarylene, phenylene, heterocyclyl, and/or cycloalkyl moieties are optionally substituted, optionally L_S is: L_{C O} HN _O ; m an amino acid, an amino acid derivative, a 15 peptide chain having from 2 to 6 amino acids or amino acid derivatives, an alkylene chain containing from 1 to 12 carbon atoms which may contain one or more carbon-carbon double or triple bonds, a paraformaldehyde chain –(OCH2)1-12-, a polyethylene glycol chain -(OCH2CH2)1-8-, which chains may be interrupted by one or more P, O, S and/or NH g_{roups and/or C5-9 heteroarylene and/or phenylene, wherein each C5-9 heteroarylene group}20 and/or each phenylene group is optionally substituted; and R_S is a univalent saccharide substituent, preferably glycosyl or O-glycosyl. E_{mbodiment 4. The antibody-drug conjugate of embodiment 3, wherein the moiety of} formula (IIa) is a moiety of any one of formula (G1) to (G8): 25 DB1/ 159693888.5 130

133186-5030-WO R₅ ^R6 R₇ R_{1 H} N R_{8 5 10 Embodiment 5. The antibody-drug conjugate of embodiment 1 or embodiment 2,} wherein D comprises a moiety of formula (IIb): R₅ R₇ R H N Q T' wherein: 15 the dotted line indicates the optional presence of a double bond between one or more of C1 and C2, C2 and C3, and C3 and C4; the wavy line indicates the point of attachment to L; m is 0 or 1; DB1/ 159693888.5 131

133186-5030-WO R₁, R₃ and R₄ are independently selected from H and R₂₉; R₂ is selected from H, L₂-R_58, R₂₉, and –LS-RS, or one of R1 and R2, R2 and R3, or R3 and R4, together with the carbon atoms to which they are attached form a 6-membered aryl, or a 5- or 6-membered cyclic, heterocyclic, or heteroaryl ring optionally substituted with 1, 2 or 3 independently 5 selected optional R20 groups; R₅ is selected from H, OH and OC_1-6 alkyl; R_{7 and R9 are independently selected from H and R20;} R₈ is selected from H, SR₂₄, SCH₂Ph, R₂₀, L₂-R₅₈, and –L_S-R_S; RA is selected from (CH2)j-OH, (CH2)j-CO2R26, C(=O)-O-(CH2)k-NR26R27, (CH2)jNR26R27,10 C(=O)-NH-(CH2)j-NR26R27 and C(=O)-NH-(CH2)k-C(=NH)NR26R27; L2 is a bond or a linker moiety having 1-200 non-hydrogen atoms selected from C, N, P, O, S or halogen, and optionally incorporates ether, oxo, carboxamidyl, urethanyl, branched, cyclic, unsaturated, heterocyclyl, aryl or heteroaryl moieties; and R58 is RA, O-(CH2)k-NR26R26, or NHNH2; 15 each R29 is independently selected from R20, R21, =CH2, =CH-(CH2)s-CH3, =CH-(CH2)s-R21, =O, (CH2)s-OR21, (CH2)s-CO2R21, (CH2)s-NR21R24, O-(CH2)t-NR21R24, NH-C(O)-R21, O- (CH2)t-NH-C(O)-R21, O-(CH2)t-C(O)-NH-R21, (CH2)s-SO2R21, O-SO2R21, (CH2)s-C(O)R21 and (CH₂)_s-C(O)NR₂₁R₂₄; each R₂₀ is independently selected from F, Cl, Br, (CH₂)_j-OH, C_1-6 alkyl, OC_1-6 alkyl, OR_S, 20 OCH₂Ph, (CH₂)_j-CO₂R₂₆, O-(CH₂)_k-NR₂₆R₂₇, C(=O)-O-(CH₂)_k-NR₂₆R₂₇, C(=O)-NR₂₆R₂₇, (CH2)j-NR26R27, NR26NH2, C(=O)-NH-(CH2)j-NR26R27, C(=O)-NH-C6H4-(CH2)j-R26, C(=O)-NH-(CH2)k-C(=NH)NR26R27, –L2-R58, S(O)2-(C1-6 alkyl), O-(CH2)k-O-(C1-6 alkyl), (CH2)j-S(O)2-NR26R27, C(=NH)-O-(C1-6 alkyl), (CH2)k-O-(C1-6 alkyl), CN, NCO, Cy, C(O)-NH-(CH )-C C(O)-C NH-C(O)-NR R nd 25 or 6; each k and t is independently selected from 1, 2, 3, 4, 5 or 6; each R21 is independently selected from H, C1-12 alkyl, C5-6 heterocyclyl, C5-9 heteroaryl, C6-15 heteroarylalkyl, phenyl and C_7-12 aralkyl groups; wherein the heterocyclyl, heteroaryl, 30 heteroarylalkyl, phenyl and aralkyl groups are optionally substituted with 1, 2 or 3 independently selected optional R₂₀ groups; each R24, R26 and R27 is independently selected from H and C1-12 alkyl; each Cy is independently selected from a C5-6 heterocyclyl or C5-6 heteroaryl group, wherein the heterocyclyl or heteroaryl groups are optionally substituted with 1 or 2 R20 groups; DB1/ 159693888.5 132

133186-5030-WO L_S is a bond, an amino acid, a peptide chain having from 2 to 6 amino acids, an alkylene chain containing from 1 to 12 carbon atoms which may contain one or more carbon-carbon double or triple bonds, a paraformaldehyde chain –(OCH2)1-12-, a polyethylene glycol chain -(OCH2CH2)1-6-, which chains may be interrupted by or optionally incorporates one 5 _{or more of P, O, S, NH, C5-9 heteroarylene, phenylene, heterocyclyl, cycloalkyl, ether,} oxo, carboxamidyl, and/or urethanyl moieties wherein the C_5-9 heteroarylene, phenylene, heterocyclyl, and/or cycloalkyl moieties are optionally substituted, optionally L_S is: ; charide substituent, preferably glycosyl or O-glycosyl; 10 Q is a linker; B is an DNA binding amide-containing chain; and T’ is an end group. E_{mbodiment 6. The antibody-drug conjugate of embodiment 5, wherein the moiety of} 15 formula (IIb) is a moiety of any one of formula (H1) to (H8): R₅ R₇ R₇ R₁ H N Q _T' T' T' 20 DB1/ 159693888.5 133

133186-5030-WO E_{mbodiment 7. The antibody-drug conjugate of any one of embodiments 3-6, wherein} 5 each of R1, R3, R7, and R9 are H. E_{mbodiment 8. The antibody-drug conjugate of embodiment 7, wherein –L–D has the} formula (IVa): 10 wherein: L has the formula –Q–B–T–; Q is a linker; B is an DNA binding amide-containing chain; and 15 T is an end group. Embodiment 9. The antibody-drug conjugate of embodiment 7, wherein –L–D has the formula (IVb): 20 E_{mbodiment 10. The antibody-drug conjugate of any one of embodiments 1-9, wherein} L comprises –LS2–LC2–Z^*–, wherein: LS2 is a bond, an amino acid, a peptide chain having from 2 to 6 amino acids, an alkylene 25 chain containing from 1 to 12 carbon atoms which may contain one or more carbon-carbon double or triple bonds, a paraformaldehyde chain –(OCH₂)_1-12-, a DB1/ 159693888.5 134

133186-5030-WO polyethylene glycol chain -(OCH₂CH₂)_1-6-, which chains may be interrupted by or o_{ptionally incorporates one or more of P, O, S, NH, C5-9 heteroarylene, phenylene,} heterocyclyl, cycloalkyl, ether, oxo, carboxamidyl, and/or urethanyl moieties wherein the C5-9 heteroarylene, phenylene, heterocyclyl, and/or cycloalkyl moieties 5 are optionally substituted, optionally L_S2 is: d LC2 oups selected from an amino acid, an amino acid derivative, a peptide chain having from 2 to 6 amino acids or amino acid derivatives, an alkylene chain containing from 1 to 12 carbon atoms which may 10 contain one or more carbon-carbon double or triple bonds, a paraformaldehyde chain –(OCH2)1-12-, a polyethylene glycol chain -(OCH2CH2)1-8-, which chains m_{ay be interrupted by one or more P, O, S and/or NH groups and/or C5-9} heteroarylene and/or phenylene, wherein each C5-9 heteroarylene group and/or each phenylene group is optionally substituted; and 15 Z^* is a reactive moiety that has reacted with a functional group. E_{mbodiment 11. The antibody-drug conjugate of embodiment 10, wherein LS2 is:} . _{20 Embodiment 12. The antibody-drug conjugate of embodiment 10, wherein LS2 is a} peptide chain having from 2 to 6 amino acids, optionally 2 amino acids, optionally -valine- alanine-. E_{mbodiment 13. The antibody-drug conjugate of any one of embodiments 5-12, wherein} 25 Q comprises X1-L-X2, wherein: X1 is selected from O, S, NR13, CR13R14, CR13R14O, C(=O), C(=O)NR13, NR13C(=O), O-C(O) and C(O)-O, or is absent; L is selected from an amino acid, a peptide chain having from 2 to 6 amino acids, an alkylene30 chain containing from 1 to 12 carbon atoms which may contain one or more carbon- carbon double or triple bonds, a paraformaldehyde chain –(OCH2)1-12-, a polyethylene glycol chain -(OCH2CH2)1-6-, which chains may be interrupted by one or more P, O, S DB1/ 159693888.5 135

133186-5030-WO a_{nd/or NH groups and/or C5-9 heteroarylene and/or phenylene, wherein each C5-9} heteroarylene group and/or each phenylene group is optionally substituted; X2 is selected from O, S, NR15, CR15R16, CR15R16O, C(=O), C(=O)NR15, NR15C(=O), O-C(O) and C(O)-O or is absent; and 5 R13, R14, R15 and R16 are independently selected from H and C1-6 alkyl. E_{mbodiment 14. The antibody-drug conjugate of embodiment 13, wherein X1 is O. Embodiment 15. The antibody-drug conjugate of embodiment 13 or 14, wherein L is an} alkylene chain containing from 1 to 12 carbon atoms which may contain one or more carbon- carbon double or triple bonds, optionally wherein L is an alkylene chain containing 3 carbon10 atoms. E_{mbodiment 16. The antibody-drug conjugate of any one of embodiments 13-15,} wherein X2 is C(=O)NR15 or NR15C(=O), optionally wherein R15 is H. E_{mbodiment 17. The antibody-drug conjugate of any one of embodiments 13-16,} H N O wherein Q has the formu . _{15 Embodiment 18. The antibody-drug conjugate of any one of embodiments 5-17, wherein} B comprises (A)q, wherein: q is selected from 0, 1, 2, 3, 4, 5 and 6; A is selected from: _{20 ;} from N-R₃₀, S and O; and the other of Y3 and Y4 is CH; and Y5 is independently selected from CR30, N, S and COH; 25 for each A2 group one of Y6 and Y7 is independently selected from N and CH; and the other of Y6 and Y7 is CR30; and each R₃₀ is independently selected from H, C_1-6 alkyl, L₂-R₅₈ and R_S. E_{mbodiment 19. The antibody-drug conjugate of embodiment 18, wherein q is 1 or 2.} DB1/ 159693888.5 136

133186-5030-WO E_{mbodiment 20. The antibody-drug conjugate of embodiment 18 or 19, wherein} A is A1: ; 5 wherein : Y₃ is N-R₃₀; Y4 is CH; Y5 is CR30; optionally wherein each R30 is H, methyl, or RS. _{10 Embodiment 21. The antibody-drug conjugate of any one of embodiments 5-20, wherein} B has the formula H_{O OH} HO OH . _{Embodiment 22. The antibody-drug conjugate of any one of embodiments 8 or 10-21,} wherein T comprises a group of formula: Y₂ * ^L R 15 wherein: p is 0 or 1; R_T is selected from –L₂–, phenylene, and C_5-9 heteroarylene, wherein the phenylene and C_5-9 heteroarylene groups are optionally substituted with up to three optional substituent 20 groups selected from OH, C_1-6 alkyl, OC_1-6 alkyl, (CH₂)_j-CO₂R₁₁, O-(CH₂)_k-NR₁₁R₁₂, (CH2)j-NR11R12, C(=O)-NH-(CH2)k-NR11R12, C(=O)-NH-R4, and C(=O)-NH-(CH2)k- C(=NH)NR11R12, optionally with the proviso that the optionally substituted C5-9 heteroarylene is not disubstituted indolyl. R₁₉ is selected from H, C_1-6 alkyl, L₂-R₅₈, R_S, and (CH₂)_t-NR₂₀R₂₁; 25 Y₁ and Y₂ are independently N or CR₃₁, wherein at least one of Y₁ and Y₂ is CR₃₁; DB1/ 159693888.5 137

133186-5030-WO each R₃₁ is independently selected from H, C_1-6 alkyl, L₂-R₅₈ and R_S; R11, R12, and R24 are independently selected from H, –L2-R58, C1-6 alkyl, or a bond connecting the atom to which it is bound to –LS2–, with the proviso that both R11 and R12 cannot be a bond connecting the atom to which it is bound to –LS2–. 5 _{Embodiment 23. The antibody-drug conjugate of embodiment 22, wherein p is 0. Embodiment 24. The antibody-drug conjugate of embodiment 22 or 23, wherein RT is} phenylene, optionally wherein the phenylene is substituted with (CH2)j-NR11R12, optionally wherein R₁₁ is H and R₁₂ is a bond connecting the atom to which it is bound to –L_S2–. 10 E_{mbodiment 25. The antibody-drug conjugate of any one of embodiments 8 or 10-24,} H N wherein T has the formul . E_{mbodiment 26. The antibody-drug conjugate of any one of embodiments 5-7 or 9-14,}15 wherein T’ comprises a group of formula: wherein: p is 0 or 1; RT is selected from –L2-R58, phenyl, and C5-9 heteroaryl, wherein the phenyl and C5-9 heteroaryl 20 groups are optionally substituted with up to three optional substituent groups selected from OH, C_1-6 alkyl, OC_1-6 alkyl, –L₂-R₅₈, (CH₂)_j-CO₂R₁₁, O-(CH₂)_k-NR₁₁R₁₂, (CH₂)_j- NR₁₁R₁₂, C(=O)-NH-(CH₂)_k-NR₁₁R₁₂, C(=O)-NH-R₂₄, and C(=O)-NH-(CH₂)_k- C(=NH)NR11R12, optionally with the proviso that the optionally substituted C5-9 heteroaryl is not indolyl; 25 R19 is selected from H, C1-6 alkyl, L2-R58, RS, and (CH2)t-NR20R21; Y1 and Y2 are independently N or CR31, wherein at least one of Y1 and Y2 is CR31; each R₃₁ is independently selected from H, C_1-6 alkyl, L₂-R₅₈ and R_S; and R₁₁, R₁₂, and R₂₄ are independently selected from H, –L₂-R₅₈, C_1-6 alkyl, or substituted aryl. DB1/ 159693888.5 138

133186-5030-WO E_{mbodiment 27. The antibody-drug conjugate of embodiment 26, wherein p is 1. Embodiment 28. The antibody-drug conjugate of embodiment 26 or 27, wherein Y1 and} Y2 are CR31, R31 are each H, and R19 is H. 5 E_{mbodiment 29. The antibody-drug conjugate of any one of embodiments 26-28,} wherein R_T is C_5-9 heteroaryl, optionally pyrrole, optionally substituted with C_1-6 alkyl, optionally methyl, and/or C(=O)-NH-R₂₄, optionally wherein R₂₄ is substituted aryl, optionally H₂N h r in R i . 10 E_{mbodiment 30. The antibody-drug conjugate of any one of embodiments 26-29,} wherein RT is phenylene, optionally wherein the phenylene is substituted with (CH2)j-NR11R12, optionally wherein R11 and R12 are each H. _{15 Embodiment 31. The antibody-drug conjugate of any one of embodiments 5-7, 9-14, or} H₂N 26-30, wherein T’ has the formul . E_{mbodiment 32. The antibody-drug conjugate of any one of embodiments 10-25,} wherein Z^* is selected from a succinimide, a heterocycle (e.g. a triazole), an amide, a thioether, 20 an oxime, an imine (e.g. chiral sulfinyl imine), an alkenyl phosphorous group, and an alkyl phosphorous group. E_{mbodiment 33. The antibody-drug conjugate of any one of embodiments 10-25 or 32,} wherein L_S2 is selected from: 25 , , DB1/ 159693888.5 139

133186-5030-WO ; E_{mbodiment 34. The antibody-drug conjugate of any one of embodiments 10-25, 32, or} O O 5 33, wherein L_C2 comprises ^1-7 , optionally ^O or . E_{mbodiment 35. Th} e _{antibody-drug co njugate of any one of embodiments 10-25 or 32-} 34, wherein LC2 comprises a polyethylene glycol chain -(OCH2CH2)1-8-, optionally - (OCH₂CH₂)₈-. 10 E_{mbodiment 36. The antibody-drug conjugate of any one of embodiments 10-25 or 32-} 35, wherein –Z^*–LC2– is selected from: O O N nd 15 E_{mbodiment 37. The antibody-drug conjugate of any one of embodiments 32-36,} wherein XAA is L-valyl-L-alanine. _{20 Embodiment 38. The antibody-drug conjugate of any one of embodiments 10-25 or 32-} 37, wherein Z^* is succinimide: DB1/ 159693888.5 140

133186-5030-WO . _{Embodiment 39. The antibody-drug conjugate of any one of embodiments 10-25 or 32- 38, wherein –LS2– is selected from:} 5 . E_{mbodiment 40. The antibody-drug conjugate of any one of embodiments 10-25 or 32- 39, wherein –LC2–LS2– is selected from:} 10 . E_{mbodiment 41. The antibody-drug conjugate of any one of embodiments 1-40, wherein} D comprises at least one RS. 15 E_{mbodiment 42. The antibody-drug conjugate of any one of embodiments 1-41, wherein} L comprises at least one R_S. E_{mbodiment 43. The antibody-drug conjugate of any one of embodiments 1-42, wherein} 20 L and D each independently comprise at least one RS. DB1/ 159693888.5 141

133186-5030-WO Embodiment 44. The antibody-drug conjugate of any one of embodiments 10-25 or 32-43, w_{herein –Z*–LC2–LS2– is selected from:} , DB1/ 159693888.5 142

133186-5030-WO . DB1/ 159693888.5 143

133186-5030-WO E_{mbodiment 45. The antibody-drug conjugate of any one of embodiments 1-44, wherein} D is selected from , , DB1/ 159693888.5 144

133186-5030-WO nd O-glycosyl. 5 E_{mbodiment 46. The antibody-drug conjugate of any one of embodiments 1-45, wherein} –L–D is selected from: , DB1/ 159693888.5 146

133186-5030-WO DB1/ 159693888.5 147

133186-5030-WO , 5 glycosyl. E_{mbodiment 47. The antibody-drug conjugate of any one of embodiments 1-46, wherein} RS is selected from any one of formula (S11), (S12), or (S13): 10 wherein R49 is independently at each occurrence selected from H and C1-6 alkyl, optionally methyl. DB1/ 159693888.5 149

133186-5030-WO E_{mbodiment 48. The antibody-drug conjugate of any one of embodiments 1-47, wherein} RS is selected from any one of formula (S101), (S102), or (S103): . 5 E_{mbo d ment . e ant o y- rug conjugate o any one o em o ments - 8, wherein} RS is selected from any one of formula (S21), (S22), (S23), or (S24): 10 wh methyl. E_{mbodiment 50. The antibody-drug conjugate of any one of embodiments 1-49, wherein} 15 R_S is selected from any one of formula (S201), (S202), (S203), or (S204): E_{mbodiment 51. The antibody-drug conjugate of any one of embodiments 1-50, wherein} 20 the antibody or binding fragment thereof comprises: (i) a heavy chain variable region (VH) that comprises: (a) a VH complementarity determining region one (CDRH1) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 2, (b) a VH complementarity determining region two (CDRH2) comprising and/or 25 consisting of the amino acid sequence of SEQ ID NO: 3, and (c) a VH complementarity determining region three (CDRH3) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 4, DB1/ 159693888.5 150

133186-5030-WO and (ii) a light chain variable region (VL) that comprises: (a) a VL complementarity determining region one (CDRL1) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 6, (b) a VL complementarity determining region two (CDRL2) comprising and/or 5 consisting of the amino acid sequence of SEQ ID NO: 7, and (c) a VL complementarity determining region three (CDRL3) comprising and/or consisting of the amino acid sequence of SEQ ID NO:8. E_{mbodiment 52. The antibody-drug conjugate of any one of embodiments 1-51, wherein} 10 the antibody or binding fragment thereof comprises: (i) a heavy chain variable region (VH) that comprises: (a) a VH complementarity determining region one (CDRH1) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 12, (b) a VH complementarity determining region two (CDRH2) comprising and/or 15 consisting of the amino acid sequence of SEQ ID NO: 13, and (c) a VH complementarity determining region three (CDRH3) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 14, and (ii) a light chain variable region (VL) that comprises: (a) a VL complementarity determining region one (CDRL1) comprising and/or 20 consisting of the amino acid sequence of SEQ ID NO: 6, (b) a VL complementarity determining region two (CDRL2) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 7, and (c) a VL complementarity determining region three (CDRL3) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 8. 25 E_{mbodiment 53. The antibody-drug conjugate of any one of embodiments 1-52, wherein} the antibody or binding fragment thereof comprises a VH that comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid 30 sequence of SEQ ID NO: 1, and/or a VL that comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 5. DB1/ 159693888.5 151

133186-5030-WO E_{mbodiment 54. The antibody-drug conjugate of embodiment 53, wherein the antibody} or binding fragment thereof comprises a VH that comprises the amino acid sequence of SEQ ID NO: 1 and/or a VL that comprises the amino acid sequence of SEQ ID NO: 5. 5 _{Embodiment 55. The antibody-drug conjugate of any one of embodiments 1-54, wherein} the antibody or binding fragment thereof comprises: (i) a heavy chain variable region (VH) that comprises: (a) a VH complementarity determining region one (CDRH1) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 16, 10 (b) a VH complementarity determining region two (CDRH2) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 17, and (c) a VH complementarity determining region three (CDRH3) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 18, and (ii) a light chain variable region (VL) that comprises: 15 (a) a VL complementarity determining region one (CDRL1) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 20, (b) a VL complementarity determining region two (CDRL2) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 21, and (c) a VL complementarity determining region three (CDRL3) comprising and/or 20 consisting of the amino acid sequence of SEQ ID NO: 22. E_{mbodiment 56. The antibody-drug conjugate of any one of embodiments 1-52 or 55,} wherein the antibody or binding fragment thereof comprises a VH that comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at 25 least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 15, and/or a VL that comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 19. _{30 Embodiment 57. The antibody-drug conjugate of embodiment 56, wherein the antibody} or binding fragment thereof comprises a VH that comprises the amino acid sequence of SEQ ID NO: 15 and/or a VL that comprises the amino acid sequence of SEQ ID NO: 19. E_{mbodiment 58. The antibody-drug conjugate of any one of embodiments 1-57, wherein} 35 the antibody or binding fragment thereof comprises a heavy chain comprising an amino acid DB1/ 159693888.5 152

133186-5030-WO sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 10 and/or a light chain comprising an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 5 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 11. E_{mbodiment 59. The antibody-drug conjugate of any one of embodiments 1-50, wherein} the antibody or binding fragment thereof comprises a VH that comprises an amino acid 10 sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from SEQ ID NOs: 23-158 and/or a VL that comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from 15 SEQ ID NOs: 159-295. E_{mbodiment 60. The antibody-drug conjugate of embodiment 59, wherein the antibody} or binding fragment thereof comprises a VH that comprises an amino acid sequence selected from SEQ ID NOs: 23-158 and/or a VL that comprises an amino acid sequence selected from 20 SEQ ID NOs: 159-295. Embodiment 61. The antibody-drug conjugate of any one of embodiments 1-50, 59, or 60, wherein the antibody or binding fragment thereof comprises a heavy chain comprising an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, 25 at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from SEQ ID NOs: 23-158 and/or a light chain comprising an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from SEQ ID NOs: 159-295. 30 E_{mbodiment 62. The antibody-drug conjugate of embodiment 61, wherein the antibody} or binding fragment thereof comprises a heavy chain that comprises an amino acid sequence selected from SEQ ID NOs: 23-158 and/or a light chain that comprises an amino acid sequence selected from SEQ ID NOs: 159-295. 35 E_{mbodiment 63. The antibody-drug conjugate of any one of embodiments 1-62, wherein} the antibody-drug conjugate has a drug-to-antibody ratio (DAR) ranging from about 1 to about DB1/ 159693888.5 153

133186-5030-WO 10, optionally wherein the DAR is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10, optionally DAR is about 4, optionally DAR is about 8. E_{mbodiment 64. A pharmaceutical composition comprising the antibody drug conjugate} 5 of any one of embodiments 1-65; and a pharmaceutically acceptable carrier. E_{mbodiment 65. A method of treating a cancer comprising administering to a subject in} need thereof a therapeutically effective amount of the antibody drug conjugate of any one of embodiments 1-63, or the pharmaceutical composition of embodiment 64. 10 E_{mbodiment 66. The method of embodiment 65, wherein the cancer is selected from} pancreatic cancer, breast cancer, prostate cancer, lymphoma, skin cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain cancer, primary brain carcinoma, head- neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, 15 head or neck carcinoma, breast carcinoma, ovarian carcinoma, lung carcinoma, small-cell lung carcinoma, Wilms’ tumor, cervical carcinoma, testicular carcinoma, bladder carcinoma, pancreatic carcinoma, stomach carcinoma, colon carcinoma, prostatic carcinoma, genitourinary carcinoma, thyroid carcinoma, esophageal carcinoma, myeloma, multiple myeloma, adrenal carcinoma, renal cell carcinoma, endometrial carcinoma, adrenal cortex carcinoma, malignant 20 pancreatic insulinoma, malignant carcinoid carcinoma, choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervical hyperplasia, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, chronic granulocytic leukemia, acute granulocytic leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi’s sarcoma, polycythemia vera, essential thrombocytosis, Hodgkin’s 25 disease, non-Hodgkin’s lymphoma, soft-tissue sarcoma, osteogenic sarcoma, primary macroglobulinemia, or retinoblastoma, and the like. In other embodiments, the cancer is acoustic neuroma, adenocarcinoma, angiosarcoma, astrocytoma, basal cell carcinoma, bile duct carcinoma, bladder carcinoma, brain cancer, breast cancer, triple-negative breast cancer (TNBC), bronchogenic carcinoma, cervical cancer, chordoma, choriocarcinoma, colon cancer, 30 colorectal cancer, craniopharyngioma, cystadenocarcinoma, embryonal carcinoma, endotheliocarcinoma, ependymoma, epithelial carcinoma, esophageal cancer, Ewing’s tumor, fibrosarcoma, gastric cancer, glioblastoma multiforme, glioma, head and neck cancer, hemangioblastoma, hepatoma, kidney cancer, leiomyosarcoma, liposarcoma, lung cancer, lymphangioendotheliosarcoma, lymphangiosarcoma, medullary carcinoma, medulloblastoma, 35 melanoma, meningioma, mesothelioma, myxosarcoma, nasal cancer, neuroblastoma, oligodendroglioma, oral cancer, osteogenic sarcoma, ovarian cancer, pancreatic cancer, papillary adenocarcinoma, papillary carcinoma, pinealoma, prostate cancer, rabdomyosarcoma, DB1/ 159693888.5 154

133186-5030-WO rectal cancer, renal cell carcinoma, retinoblastoma, sarcoma, sebaceous gland carcinoma, seminoma, skin cancer, squamous cell carcinoma, stomach cancer, sweat gland carcinoma, synovioma, testicular cancer, small cell lung carcinoma, throat cancer, uterine cancer, Wilm’s tumor, blood cancer, acute erythroleukemic leukemia, acute lymphoblastic B-cell leukemia, 5 acute lymphoblastic T-cell leukemia, acute lymphoblastic leukemia, acute megakaryoblastic leukemia, acute monoblastic leukemia, acute myeloblastic leukemia, acute myelomonocytic leukemia, acute nonlymphocytic leukemia, acute promyelocytic leukemia, acute undifferentiated leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, hairy cell leukemia, multiple myeloma, heavy chain disease, Hodgkin’s disease, multiple myeloma, 10 non-Hodgkin’s lymphoma, polycythemia vera, and Waldenstrom’s macroglobulinemia. E_{mbodiment 67. The method of embodiment 65 or 66, wherein the cancer in triple-} negative breast cancer (TNBC). _{15 Embodiment 68. An antibody-drug conjugate having formula (I):} Ab-[L-D]n formula (I) wherein in formula (I): Ab is an antibody or binding fragment thereof that binds CUB Domain-Containing 20 Protein-1 (CDCP1); n is an integer from 1 to 20; and L-D has the formula: . _{25 Embodiment 69. An antibody-drug conjugate having formula (I):} Ab-[L-D]n formula (I) wherein in formula (I): Ab is an antibody or binding fragment thereof that binds CUB Domain-Containing 30 Protein-1 (CDCP1); n is an integer from 1 to 20; and L-D has the formula: DB1/ 159693888.5 155

133186-5030-WO . _{o e . a o y- ug co uga e av g o mula (I):} Ab-[L-D]n formula (I) 5 wherein in formula (I): Ab is an antibody or binding fragment thereof that binds CUB Domain-Containing Protein-1 (CDCP1); n is an integer from 1 to 20; and L-D has the formula: 10 . E_{mbodiment 71. The antibody-drug conjugate of any one of embodiments 68-70,} wherein the antibody or binding fragment thereof comprises: (i) a heavy chain variable region (VH) that comprises: (a) a VH complementarity determining region one (CDRH1) comprising and/or 15 consisting of the amino acid sequence of SEQ ID NO: 2, (b) a VH complementarity determining region two (CDRH2) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 3, and (c) a VH complementarity determining region three (CDRH3) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 4, 20 and (ii) a light chain variable region (VL) that comprises: DB1/ 159693888.5 156

133186-5030-WO (a) a VL complementarity determining region one (CDRL1) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 6, (b) a VL complementarity determining region two (CDRL2) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 7, and 5 (c) a VL complementarity determining region three (CDRL3) comprising and/or consisting of the amino acid sequence of SEQ ID NO:8. E_{mbodiment 72. The antibody-drug conjugate of any one of embodiments 68-70,} wherein the antibody or binding fragment thereof comprises: 10 (i) a heavy chain variable region (VH) that comprises: (a) a VH complementarity determining region one (CDRH1) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 12, (b) a VH complementarity determining region two (CDRH2) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 13, and 15 (c) a VH complementarity determining region three (CDRH3) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 14, and (ii) a light chain variable region (VL) that comprises: (a) a VL complementarity determining region one (CDRL1) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 6, 20 (b) a VL complementarity determining region two (CDRL2) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 7, and (c) a VL complementarity determining region three (CDRL3) comprising and/or consisting of the amino acid sequence of SEQ ID NO:8. _{25 Embodiment 73. The antibody-drug conjugate of any one of embodiments 68-70,} wherein the antibody or binding fragment thereof comprises a VH that comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1, and/or a VL that comprises an amino acid sequence at least 90%, at 30 least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 5. DB1/ 159693888.5 157

133186-5030-WO E_{mbodiment 74. A method of treating a cancer comprising administering to a subject in} need thereof a therapeutically effective amount of the antibody-drug conjugate of any one of embodiments 68-70. E_{mbodiment 75. A compound having the following formula:} 5 . _{m o men . compoun av ng e o ow ng ormu a:} . E_{mbodiment 77. An antibody-drug conjugate having formula (I):} Ab-[L–D]n 10 formula (I) wherein in formula (I): -L-D- has the formula: . _{15 Embodiment 78. An antibody-drug conjugate having formula (I):} Ab-[L–D]n formula (I) wherein in formula (I): DB1/ 159693888.5 158

133186-5030-WO -L-D- has the formula: . _{m o men . me o o rea ng a cancer compr s ng administering to a subject in} need thereof a therapeutically effective amount of the antibody-drug conjugate of any one of 5 embodiments 77 or 78. The following examples describe the disclosure in further detail. These examples are provided for illustrative purposes only, and should in no way be considered as limiting the disclosure. SYNTHETIC STRATEGIES The antibody-drug conjugates of formula (I), and any linkers/drug moieties therein, may be 10 prepared using the techniques described below. Some of the schemes and examples may omit details of common reactions, including oxidations, reductions, and so on, separation techniques (extraction, evaporation, precipitation, chromatography, filtration, trituration, crystallization, and the like), and analytical procedures, which are known to persons of ordinary skill in the art of organic chemistry. The details of such reactions and techniques can be found in a number of 15 treatises, including Richard Larock, Comprehensive Organic Transformations, A Guide to Functional Group Preparations, ²ⁿd Ed (2010), and the multi-volume series edited by Michael B. Smith and others, Compendium of Organic Synthetic Methods (1974 et seq.). Starting materials and reagents may be obtained from commercial sources or may be prepared using literature methods. Some of the reaction schemes may omit minor products resulting from 20 chemical transformations (e.g., an alcohol from the hydrolysis of an ester, CO2 from the decarboxylation of a diacid, etc.). In addition, in some instances, reaction intermediates may be used in subsequent steps without isolation or purification (i.e., in situ). In some of the reaction schemes and examples below, certain compounds can be prepared using protecting groups, which prevent undesirable chemical reaction at otherwise reactive sites. 25 Protecting groups may also be used to enhance solubility or otherwise modify physical properties of a compound. For a discussion of protecting group strategies, a description of DB1/ 159693888.5 159

133186-5030-WO materials and methods for installing and removing protecting groups, and a compilation of useful protecting groups for common functional groups, including amines, carboxylic acids, alcohols, ketones, aldehydes, and so on, see T. W. Greene and P. G. Wuts, Protecting Groups in Organic Chemistry, ^4th Edition, (2006) and P. Kocienski, Protective Groups, ^3rd Edition (2005). 5 Generally, the chemical transformations described throughout the specification may be carried out using substantially stoichiometric amounts of reactants, though certain reactions may benefit from using an excess of one or more of the reactants. Additionally, many of the reactions disclosed throughout the specification may be carried out at about room temperature (RT) and ambient pressure, but depending on reaction kinetics, yields, and so on, some reactions may be 10 run at elevated pressures or employ higher temperatures (e.g., reflux conditions) or lower temperatures (e.g., -78°C. to 0°C.). Any reference in the disclosure to a stoichiometric range, a temperature range, a pH range, etc., whether or not expressly using the word "range," also includes the indicated endpoints. Many of the chemical transformations may also employ one or more compatible solvents, which 15 may influence the reaction rate and yield. Depending on the nature of the reactants, the one or more solvents may be polar protic solvents (including water), polar aprotic solvents, non-polar solvents, or some combination. Representative solvents include saturated aliphatic hydrocarbons (e.g., n-pentane, n-hexane, n-heptane, n-octane); aromatic hydrocarbons (e.g., benzene, toluene, xylenes); halogenated hydrocarbons (e.g., methylene chloride, chloroform, 20 carbon tetrachloride); aliphatic alcohols (e.g., methanol, ethanol, propan-1-ol, propan-2-ol, butan-1-ol, 2-methyl-propan-1-ol, butan-2-ol, 2-methyl-propan-2-ol, pentan-1-ol, 3-methyl- butan-1-ol, hexan-1-ol, 2-methoxy-ethanol, 2-ethoxy-ethanol, 2-butoxy-ethanol, 2-(2-methoxy- ethoxy)-ethanol, 2-(2-ethoxy-ethoxy)-ethanol, 2-(2-butoxy-ethoxy)-ethanol); ethers (e.g., diethyl ether, di-isopropyl ether, dibutyl ether, 1,2-dimethoxy-ethane, 1,2-diethoxy-ethane, 1- 25 methoxy-2-(2-methoxy-ethoxy)-ethane, 1-ethoxy-2-(2-ethoxy-ethoxy)-ethane, tetrahydrofuran, 1,4-dioxane); ketones (e.g., acetone, methyl ethyl ketone); esters (methyl acetate, ethyl acetate); nitrogen-containing solvents (e.g., formamide, N,N-dimethylformamide, acetonitrile, N-methyl- pyrrolidone, pyridine, quinoline, nitrobenzene); sulfur-containing solvents (e.g., carbon disulfide, dimethyl sulfoxide, tetrahydro-thiophene-1,1,-dioxide); and phosphorus-containing 30 solvents (e.g.,HMPA, hexamethylphosphoramide). One synthetic strategy to add a monosaccharide unit to PDD compounds (including PDD compounds useful within the antibody-drug conjugates of the disclosure (e.g. antibody-drug conjugates of formula (I)) may involve (stereochemistry not shown): Glucose-based N11-carbamate DB1/ 159693888.5 160

133186-5030-WO ht) or secondary alcohol (left: any of the four groups); or 5 Glucuronide-based mide (right). In some aspects, the present disclosure relates to a compound of formula (I) and salts and 10 solvates thereof, for use in preparing a drug in an antibody-drug conjugate. Suitably, PDD compounds and salts and solvates thereof, may be used directly to prepare an antibody-drug conjugate of the disclosure (e.g. antibody-drug conjugate of formula (I)) when a linker and/or drug moiety (e.g. a PDD) and salts and solvates thereof, contains one or more functional groups (such as amine, hydroxyl or carboxylic acid groups) for attaching the drug to the antibody either 15 directly or via a linker group. Suitably, PDD compounds and salts and solvates thereof, may be used in preparing antibody-drug conjugates of the disclosure (e.g. antibody-drug conjugates of formula (I)) by being modified to contain one or more functional groups (such as amine, hydroxyl or carboxylic acid groups) for attaching the drug to the antibody either directly or via a linker group. Suitably, a PDD compound, such as those PDD compounds and moieties 20 described herein, and salts and solvates thereof, may be used in preparing an antibody-drug conjugate by being modified to contain one or more antibody linker groups, wherein the antibody is attached to the drug through the one or more antibody linker groups. Therefore, the present disclosure provides for PDD compound,s such as those PDD compounds and moieties DB1/ 159693888.5 161

133186-5030-WO described herein, further comprising one or more antibody linker group. Suitably, a PDD compound, such as those PDD compounds and moieties described herein, may contain 1, 2, or 3 antibody linker groups. Suitably, a PDD compound, such as those PDD compounds and moieties described herein, may contain 1 or 2 antibody linker groups. Suitably, a PDD 5 compound, such as those PDD compounds and moieties described herein, may contain 1 antibody linker group. In some aspects, one or more atoms or groups of the PDD compound, such as those PDD compounds and moieties described herein, may be eliminated during the attachment of the drug to the antibody or the attachment of the antibody linker to the drug or the modification of the drug to contain one or more functional groups (such as amine, hydroxyl or 10 carboxylic acid groups) for attaching the drug to the antibody either directly or via an antibody linker group. A variety of suitable antibody linker groups are known in the art and may be used as described herein. For example, the maleimide methodology is routinely used as a method to attach antibodies to drug compounds by providing an antibody linker attached to the drug with a 15 terminal succinimide group (forming a succinimide-antibody complex). In addition, methodologies using diarylcyclooctyne moeities (such as, but not limited to, DBCO, dibenzylcyclooctyne) are also alternatives used in the art. Diarylcyclooctynes react with azides to provide attachment via the formation of stable triazoles. Diarylcyclooctynes are thermostable with very narrow and specific reactivity toward azides, resulting in almost quantitative yields of 20 stable triazoles. Furthermore, the reaction does not require a cytotoxic Cu(I) catalyst (that is toxic to most organisms) and thus, prevents its use in many biological systems. Still further, alkoxyamine methodologies are also alternatives used in the art. For site-specific conjugation of the drug to an antibody, the antibodies may comprise a “tag” (which may be proprietary) that will react with a dairylcyclooctyne (for example, DBCO), an alkyloxyamine and/or maleimide 25 group to attach the antibody to the drug. The tag in some instances may be a mutated amino acid. Suitable antibody linker groups incorporating the various groups described above are available in the art. EXAMPLES Example 1: Synthesis of PDD-C8 subsituted compounds, PDD-N11 substituted 30 m nds, and control compounds General synthetic methods. All reagents and solvents were purchased from standard commercial suppliers and used as purchased. Anhydrous reactions were carried out under an inert atmosphere of argon using anhydrous solvents which were used as purchased, without further drying. Thin Layer Chromatography (TLC) was performed on silica gel aluminium 35 plates (Merck 60, F₂₅₄), and flash column chromatography was carried out using a Biotage DB1/ 159693888.5 162

133186-5030-WO Isolera One (automated flash chromatography system), whilst monitoring by TLC (UV, 254 nm). All Nuclear Magnetic Resonance (NMR) spectra were obtained at room temperature using a Bruker 600 MHz Ultrashield with Cryoprobe (Bruker Avance NEO console with Cryoplatform) 5 or a Varian Mercury Vx Agilent 400 MHz spectrometer, for which chemical shifts are expressed in ppm relative to the solvent and coupling constants are expressed in Hz. Microwave reactions were carried out on a Biotage Initiator+ microwave synthesizer. High Resolution Mass Spectrometry (HRMS) was performed on a Thermo Scientific-Exactive HCD Orbitrap Mass Spectrometer. Yields refer to isolated material (homogeneous by TLC and NMR) unless 10 otherwise stated and names are assigned according to IUPAC nomenclature. Liquid Chromatography Mass Spectrometry (LCMS) analysis Methods A-C were performed on a Waters Alliance 2695 with water (A) and acetonitrile (B) comprising the mobile phases. Formic acid (0.1%) was added to both acetonitrile and water to ensure acidic conditions throughout the analysis. Function type: Diode array (535 scans). Column type: Monolithic C18 15 50 X 4.60 mm. Mass spectrometry data were collected using a Waters Micromass ZQ instrument coupled to the HPLC with a Waters 2996 PDA. Waters Micromass ZQ parameters used were: Capillary (kV), 3.38; Cone (V), 35; Extractor (V), 3.0; Source temperature (°C), 100; De-solvation Temperature (°C), 200; Cone flow rate (L/h), 50; De-solvation flow rate (L/h), 250. Gradient conditions are described as follows. 20 Method A (10 min): from 95% A/5% B to 50% B over 3 min. Then from 50% B to 80% B over 2 min. Then from 80% B to 95% B over 1.5 min and held constant for 1.5 min. This was then reduced to 5% B over 0.2 min and maintained to 5% B for 1.8 min. The flow rate was 0.5 mL/min, 200 μL was split via a zero dead volume T piece which passed into the mass spectrometer. The wavelength range of the UV detector was 220-400 nm. 25 Method B (5 min): from 95% A/5% B to 90% B over 3 min. Then from 90% B to 95% B over 0.5 min and held constant for 1 min. This was then reduced to 5% B over 0.5 min. The flow rate was 1.0 mL/min, 100 μL was split via a zero dead volume T piece which passed into the mass spectrometer. The wavelength range of the UV detector was 220-500 nm. Method C (5 min): from 95% A/5% B, which was increased to 90% B over 3 min and to 95% 30 B over a further 0.5 min. The gradient was then held at 95% B for 1 min and then returned to 5% B over 0.5 min. The total duration of the run was 5 minutes and the solvent flow rate was 1 mL/min, 100 μL was split via a zero dead volume T piece which passed into the mass spectrometer. The wavelength range of the UV detector was 220-500 nm. DB1/ 159693888.5 163

133186-5030-WO Liquid Chromatography Mass Spectrometry (LCMS) analysis Methods D-G were performed on a Shimadzu LC-20AD series, Binary Pump, Diode Array Detector. Column type: Agilent Poroshell 120 EC-C18, 2.7 μm, 4.6×50 mm. Mobile phase: A: 0.05% formic acid in water (v/v); B: 0.05% formic acid in acetonitrile (v/v). Flow Rate: 1 mL/min at 25 °C. Detector: 214 nm, 5 254 nm. Gradient stop time: 5 min. MS: 2020, Quadrupole LC/MS, Ion Source: API-ESI, TIC: 100-1300 m/z, Drying gas flow: 15 L/min, Nebulizer pressure: 1.5 L/min, Drying gas temperature: 250 °C, Vcap: 4500V. Sample preparation: samples were dissolved in methanol at 1-10 μg/mL, then filtered through a 0.22 μm filter membrane. Injection volume: 1-10 μL. Gradient conditions are described as follows. 10 Method D (5 min): 20% A/80% B for 0.5 min, which was increased to 100% B over 3.5 min, then held at 100% B for 0.5 min. This was then returned to 20% A/80% B for 0.5 min. Method E (5 min): 50% A/50% B for 0.5 min, which was increased to 100% B over 3.5 min, then held at 100% B for 0.5 min. This was then returned to 50% A/50% B for 0.5 min. Method F (5 min): 85% A/15% B for 0.5 min, which was increased to 100% B over 3.5 min, 15 then held at 100% B for 0.5 min. This was then returned to 85% A/15% B for 0.5 min. Method G (5 min): 97% A/3% B for 0.5 min, which was increased to 30% A/70% B over 3.5 min, then to 100% B over 0.5 min. This was then returned to 97% A/3% B for 0.5 min. Optical rotations were measured on an SGWzz-1 automatic Polarimeter (Shanghai Shen Guang Instrument Co., Ltd.) or Bellingham-Stanley ADP 440+ Polarimeter. 20 Reverse phase Preparative HPLC was carried out on a Shimadzu LC with CTC IFC, using a Phenomenex Gemini NX 5m, C18, 110 Å, 150 x 50 mm column, eluting with mobile phase: A) water (0.1% TFA), B) acetonitrile, at a flow of 50 mL/min. PDD C8-substituted compounds DB1/ 159693888.5 164

133186-5030-WO 194 PDD-N11-substituted compounds DB1/ 159693888.5 166

133186-5030-WO S_{tructure Compound} Number DB1/ 159693888.5 167

133186-5030-WO 180 Control compounds DB1/ 159693888.5 168

133186-5030-WO Example 1A: Synthesis of compound (72). Scheme 1. Synthesis of compound (72). y y y yp y 5 A mixture of 3-(benzyloxy)-4-hydroxybenzaldehyde (61) (10.0 g, 44.0 mmol), methyl 4- bromobutanoate (8.3 mL, 65.0 mmol) and potassium carbonate (12.6 g, 88.0 mmol) in acetone (100 mL) was heated to 40 ˚C for 18 h. The reaction mixture was diluted with water (500 mL) and extracted with ethyl acetate (2 x 300 mL). The organic layer was concentrated in vacuo. The 10 resulting residue was purified by column chromatography (silica), eluting with acetone/dichloromethane (from 0% to 30%) to give the title compound (13.8 g, 96%) as a yellow oil. ¹H NMR (400 MHz, CDCl3) δ 9.82 (s, 1H), 7.49 – 7.42 (m, 4H), 7.38 (t, J = 7.3 Hz, 2H), 7.35 – 7.29 (m, 1H), 7.00 (d, J = 8.0 Hz, 1H), 5.17 (s, 2H), 4.16 (t, J = 6.2 Hz, 2H), 3_{.68 (s, 3H), 2.57 (t, J = 7.2 Hz, 2H), 2.24 – 2.15 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 190.9,} 15 173.5, 154.6, 149.0, 136.7, 130.2, 128.7, 128.1, 127.3, 126.9, 112.2, 71.0, 67.9, 51.8, 30.3, 24.5; MS (ES+): m/z = 329 (M+H)⁺; LCMS (Method B): t_R = 4.00 min. DB1/ 159693888.5 169

133186-5030-WO Methyl 4-(2-(benzyloxy)-4-formyl-5-nitrophenoxy)butanoate (63) _{l) in} trifluoroacetic acid (30 mL) at 0 °C was added to a stirring solution of potassium nitrate (2.7 g, 5 26.3 mmol) in trifluoroacetic acid (30 mL), dropwise. The resulting mixture was stirred at room temperature for 30 min and then quenched with a saturated aqueous solution of sodium hydrogen carbonate (100 mL) and extracted with ethyl acetate (2 x 80 mL). The organic layer was concentrated in vacuo. The resulting residue was purified by column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60 °C (from 0% to 50%) to give the title 10 compound (5.60 g, 68%) as a yellow solid. ¹H NMR (400 MHz, CDCl3) δ 10.41 (s, 1H), 7.62 (s, 1H), 7.48 (s, 1H), 7.44 – 7.35 (m, 5H), 5.26 (s, 2H), 4.22 (t, J = 6.2 Hz, 2H), 3.69 (s, 3H), 2_{.57 (t, J = 7.1 Hz, 2H), 2.26 – 2.19 (m, 2H);13C NMR (100 MHz, CDCl3) δ 187.6, 173.2, 152.5,} 152.1, 143.9, 135.3, 128.7, 128.4, 127.3, 125.3, 111.6, 108.5, 71.1, 68.6, 51.7, 30.1, 24.1; MS (ES+): m/z = 374 (M+H)⁺; LCMS (Method B): tR = 4.15 min. 15 Methyl 4-(4-formyl-2-hydroxy-5-nitrophenoxy)butanoate (64) so ut on o met y -( -(benzy oxy)- - ormy-5-ntrop enoxy)butanoate (63) (5.50 g, .7 mmol) in trifluoroacetic acid (30 mL) was heated to 80 °C for 30 min. It was then cooled to room temperature and quenched with a saturated aqueous solution of sodium hydrogen 20 carbonate (30 mL). The aqueous mixture was extracted with ethyl acetate and the organic layer was concentrated in vacuo. The resulting residue was purified by column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60 °C (from 0% to 50%), to give the title c_{ompound (2.10 g, 50%) as a brown solid. 1H NMR (400 MHz, CDCl3) δ 10.41 (s, 1H), 7.62 (s,} 1H), 7.45 (s, 1H), 6.75 (br. s, 1H), 4.27 (t, J = 6.0 Hz, 2H), 3.73 (s, 3H), 2.60 – 2.55 (m, 2H), 25 2.29 – 2.22 (m, 2H); ¹³C NMR (100 MHz, CD₃OD) δ 189.5, 175.4, 152.5, 115.1, 112.3, 110.9, 109.9, 101.4, 69.5, 52.1, 31.2, 25.4; MS (ES-): m/z = 282 (M-H)-; LCMS (Method B): tR = 3.47 min. DB1/ 159693888.5 170

133186-5030-WO Methyl 4-(4-formyl-5-nitro-2-((2-(trimethylsilyl)ethoxy)methoxy)phenoxy)butanoate (65) m_{mol), 2-(trimethylsilyl)ethoxymethyl chloride (1.50 g, 8.90 mmol) and potassium carbonate} 5 (2.60 g, 18.5 mmol) in acetone (30 mL) was stirred at room temperature for 2 h. The reaction mixture was diluted with water (150 mL) and extracted with ethyl acetate (2 x 60 mL). The organic layer was concentrated in vacuo. The resulting residue was purified by column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60 °C (from 0% to 50%) t_{o give the title compound (2.50 g, 82%) as a brown oil. 1H NMR (400 MHz, CDCl3) δ 10.36} 10 (s, 1H), 7.65 (s, 1H), 7.64 (s, 1H), 5.41 (s, 2H), 4.26 (t, J = 6.2 Hz, 2H), 3.85 – 3.80 (m, 2H), 3.71 (s, 3H), 2.60 (t, J = 7.1 Hz, 2H), 2.24 (p, J = 6.6 Hz, 2H), 1.00 – 0.96 (m, 2H), 0.02 (s, 9H) ; ¹³C NMR (100 MHz, CDCl₃) δ 187.2, 172.9, 152.1, 150.9, 144.2, 125.1, 114.0, 108.4, 93.4, 68.4, 67.1, 51.5, 30.0, 24.0, 17.8, -1.6; MS (ES-): m/z = 412 (M-H)-; LCMS (Method B): tR = 4.50 min. 15 4-(4-Methoxy-4-oxobutoxy)-2-nitro-5-((2-(trimethylsilyl)ethoxy)methoxy)benzoic acid (66) so ut on o sod um c or te ( .30 g, .9 mmo) and sodum monop osp ate ( .00 g, 8. 0 mmol) in water (30 mL) was added to a mixture of methyl 4-(4-formyl-5-nitro-2-((2- (trimethylsilyl)ethoxy)methoxy)phenoxy)butanoate (65) (2.50 g, 6.00 mmol). Hydrogen 20 peroxide (50% in water, 2.8 mL) was then added dropwise, and the resulting solution was stirred at 40 °C for 1 h. The reaction mixture was quenched with a saturated aqueous solution of sodium metabisulfite (60 mL) and acidified with acetic acid (1.5 mL). The aqueous mixture was extracted with ethyl acetate (2 x 60 mL) and the organic layer was concentrated in vacuo. The resulting residue was purified by column chromatography (silica), eluting with ethyl 25 acetate/petroleum spirit, 40-60 °C (from 0% to 50%) to give the title compound (2.40 g, 93%) a_{s a brown oil. 1H NMR (400 MHz, CD3OD) δ 7.42 (s, 1H), 7.41 (s, 1H), 5.30 (s, 2H), 4.08 (t, J} = 6.6 Hz, 2H), 3.78 – 3.74 (m, 2H), 3.61 (s, 3H), 2.49 (t, J = 8.1 Hz, 2H), 2.09 – 2.03 (m, 2H), 0.91 – 0.86 (m, 2H), -0.07 (s, 9H); ¹³C NMR (100 MHz, CD3OD) δ 175.0, 167.8, 151.9, 150.7, DB1/ 159693888.5 171

133186-5030-WO 144.2, 121.8, 117.0, 109.6, 94.8, 69.6, 67.9, 52.2, 31.1, 25.3, 18.7, -1.3; MS (ES-): m/z = 428 (M-H)-; LCMS (Method B): tR = 4.08 min. Methyl (S)-4-(4-(2-(hydroxymethyl)piperidine-1-carbonyl)-5-nitro-2-((2- (trimethylsilyl)ethoxy)methoxy)phenoxy)butanoate (67) 5 a_{cid (66) (2.40 g, 5.60 mmol) in anhydrous dichloromethane (30 mL) was charged with HATU} (2.60 g, 6.70 mmol) and anhydrous triethylamine (1.5 mL, 11.2 mmol). The reaction mixture was stirred at room temperature for 5 min. (S)-Piperidin-2-ylmethanol (711 mg, 6.20 mmol) was 10 then added and the resulting mixture was stirred at room temperature for 17 h. The reaction mixture was diluted with ethyl acetate (90 mL) and washed with brine (90 mL). The organic layer was concentrated in vacuo. The resulting residue was purified by column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60 °C (from 0% to 100%) to give the title c_{ompound (2.60 g, 88%) as a brown oil. 1H NMR (400 MHz, DMSO-d6) δ 7.71 (s, 1H), 7.15 (s,} 15 1H), 5.40 (s, 2H), 4.80 (t, J = 5.6 Hz, 1H), 4.18 – 4.09 (m, 2H), 3.76 – 3.70 (m, 2H), 3.60 (s, 3H), 3.57 – 3.39 (m, 2H), 3.34 – 2.70 (m, 3H), 2.50 – 2.46 (m, 2H), 2.07 – 1.97 (m, 2H), 1.85 – 1_{.28 (m, 6H), 0.92 – 0.84 (m, 2H), -0.04 (d, J = 3.6 Hz, 9H); 13C NMR (100 MHz, CDCl3) δ} 173.2, 168.0, 152.3, 148.6, 138.2, 127.2, 112.6, 108.7, 93.5, 68.2, 67.1, 60.3, 53.5, 51.6, 51.1, 43.4, 37.3, 30.1, 24.1, 18.0, -1.5; MS (ES-): m/z = 527 (M+H)⁺; LCMS (Method B): t_R = 4.10 20 min. Methyl (S)-4-(5-amino-4-(2-(hydroxymethyl)piperidine-1-carbonyl)-2-((2- (trimethylsilyl)ethoxy)methoxy)phenoxy)butanoate (68) A solution of ammonium chloride (15.8 g, 297 mmol) in water (25 mL) was added to a mixture25 of methyl (S)-4-(4-(2-(hydroxymethyl)piperidine-1-carbonyl)-5-nitro-2-((2- (trimethylsilyl)ethoxy)methoxy)phenoxy)butanoate (67) (2.60 g, 4.90 mmol) in acetone (30 mL) and tetrahydrofuran (30 mL), and charged with zinc powder (65.4 g, 148 mmol). The resulting mixture was stirred at room temperature for 30 min and then filtered through celite. DB1/ 159693888.5 172

133186-5030-WO The cake was washed with ethyl acetate. The filtrate was concentrated in vacuo and the resulting residue was purified by column chromatography (silica), eluting with m_{ethanol/dichloromethane (from 0% to 10%) to give the title compound (2.40 g, 98%) as an} orange viscous oil. ¹H NMR (400 MHz, CDCl3) 6.99 - 6.68 (m, 2 H), 5.20 (s, 2 H), 5.05 (br. s., 5 1 H), 3.97 (br. s., 3 H), 3.63 (dd, J = 5.9, 7.8 Hz, 3 H), 3.57 (br. s., 3 H)– 3.52 - 3.15 (m, 3 H), 2.42 (br. s., 2 H), 2.03 (br. s., 2 H)– 1.82 - 1.33 (m, 6 H)– 0.89 - 0.76 (m, 2 H), -0.09 (br. s., 9 H); ¹³C NMR (100 MHz, CDCl₃) δ 173.8, 173.6, 171.8, 152.9, 141.1, 125.5, 112.1, 108.2, 94.2, 77.2, 68.0, 66.5, 57.8, 53.6, 51.8, 51.3, 30.6, 30.3, 29.2, 25.6, 24.3, 18.1, -1.4; MS (ES-): m/z = 497 (M+H)⁺; LCMS (Method B): tR = 3.85 min. 10 Methyl (S)-4-(5-(((allyloxy)carbonyl)amino)-4-(2-(hydroxymethyl)piperidine-1-carbonyl)- 2-((2-(trimethylsilyl)ethoxy)methoxy)phenoxy)butanoate (69) (trimethylsilyl)ethoxy)methoxy)phenoxy)butanoate (68) (2.40 g, 4.80 mmol) and pyridine (973 15 µL, 12.1 mmol) in anhydrous dichloromethane (20 mL) was cooled to -10 °C and charged dropwise with a solution of allylchloroformate (468 µL, 4.30 mmol) in anhydrous dichloromethane (10 mL). The reaction mixture was stirred at room temperature for 40 min. The reaction mixture was sequentially washed with a saturated aqueous solution of copper (II) sulfate (20 mL), water (20 mL) and a saturated aqueous solution of sodium hydrogen carbonate 20 (20 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated. The resulting residue was purified by column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60 °C (from 0% to 100%) to give the title compound (2.10 g, 75%) a_{s a brown viscous oil. 1H NMR (400 MHz, CDCl3) δ 8.44 (s, 1H), 7.70 (s, 1H), 7.03 (s, 1H),} 5.90 (ddt, J = 15.9, 10.7, 5.5 Hz, 1H), 5.31 (d, J = 17.2 Hz, 1H), 5.21 – 5.11 (m, 3H), 4.58 (d, J 25 = 5.5 Hz, 2H), 4.05 (t, J = 6.8 Hz, 3H), 3.86 (s, 1H), 3.74 (t, J = 8.5 Hz, 2H), 3.66 – 3.51 (m, 5H), 3.40 – 3.21 (m, 1H), 3.11 – 2.82 (m, 1H), 2.49 (t, J = 7.4 Hz, 2H), 2.15 – 2.08 (m, 2H), 1_{.66 – 1.44 (m, 6H), 0.93 – 0.86 (m, 2H), -0.03 (s, 9H); 13C NMR (100 MHz, CDCl3) δ 173.5,} 171.2, 170.7, 153.8, 150.7, 132.6, 117.8, 116.3, 106.0, 94.1, 67.7, 66.4, 65.7, 60.4, 57.8, 51.7, 30.6, 25.6, 24.4, 21.1, 19.8, 18.1, 14.2, -1.4; MS (ES-): m/z = 581 (M+H)⁺; LCMS (Method B): 30 tR = 4.27 min. DB1/ 159693888.5 173

133186-5030-WO Allyl 6-hydroxy-3-(4-methoxy-4-oxobutoxy)-12-oxo-2-((2-(trimethylsilyl)ethoxy)methoxy)- 6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (70) 5 1-carbonyl)-2-((2-(trimethylsilyl)ethoxy)methoxy)phenoxy)butanoate (69) (2.10 g, 3.60 mmol) in dichloromethane (30 mL) was added TEMPO (226 mg, 1.40 mmol) and (diacetoxyiodo)benzene (2.90 mg, 9.00 mmol). The reaction mixture was heated to 50 °C for 6 h, and was then sequentially washed with a saturated aqueous solution of sodium metabisulfite (15 mL), a saturated aqueous solution of sodium hydrogen carbonate (15 mL) and brine (15 10 mL). The organic layer was concentrated in vacuo. The resulting residue was purified by column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60 °C (from 0% t_{o 100%) to give the title compound (900 mg, 43%) as a brown oil. 1H NMR (400 MHz,} CDCl3) δ 7.40 (s, 1H), 6.66 (s, 1H), 5.95 – 5.73 (m, 2H), 5.34 – 5.11 (m, 5H), 4.74 – 4.61 (m, 1H), 4.55 – 4.30 (m, 2H), 4.08 – 3.98 (m, 2H), 3.84 – 3.75 (m, 2H), 3.68 (s, 3H), 3.53 – 3.45 15 (m, 1H), 3.08 – 2.99 (m, 1H), 2.52 (t, J = 7.2 Hz, 2H), 2.17 – 2.09 (m, 2H), 1.93 – 1.64 (m, 6H), 1.01 – 0.91 (m, 2H), 0.00 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 173.4, 168.7, 156.1, 150.8, 146.4, 131.9, 128.4, 125.4, 118.0, 115.9, 113.8, 93.8, 82.2, 77.2, 67.8, 66.7, 66.6, 55.2, 51.7, 38.6, 30.3, 24.2, 23.2, 23.0, 18.3, 17.9, -1.4; MS (ES-): m/z = 579 (M+H)⁺; LCMS (Method A): tR = 8.00 min. 20 Allyl 3-(4-methoxy-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-2-((2- (trimethylsilyl)ethoxy)methoxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2- a][1,4]diazepine-5(12H)-carboxylate (71) A mixture of allyl 6-hydroxy-3-(4-methoxy-4-oxobutoxy)-12-oxo-2-((2-25 (trimethylsilyl)ethoxy)methoxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine- 5_{(12H)-carboxylate (70) (900 mg, 1.90 mmol), 3,4-dihydro-2H-pyran (1.71 mL, 18.8 mmol) and p-toluenesulfonic acid monohydrate (47.0 mg, 0.180 mmol) in dichloromethane (15 mL)} was stirred at 45 °C for 3 h. The reaction mixture was diluted with ethyl acetate (50 mL) and washed with a saturated aqueous solution of sodium hydrogen carbonate (20 mL) and brine (30 DB1/ 159693888.5 174

133186-5030-WO mL). The organic layer was concentrated in vacuo. The resulting residue was purified by c_{olumn chromatography (silica), eluting with methanol/dichloromethane (from 0% to 5%) to give the title compound (770 mg, 61%) as a cream solid. 1H NMR (400 MHz, CDCl3) δ 7.41 (s,} 1H), 6.57 (s, 1H), 6.23 – 5.97 (m, 1H), 5.91 – 5.65 (m, 1H), 5.34 – 5.00 (m, 5H), 4.71 – 4.55 5 (m, 1H), 4.48 – 4.20 (m, 2H), 4.08 – 3.98 (m, 2H), 3.91 – 3.75 (m, 2H), 3.73 – 3.52 (m, 5H), 3.52 – 3.40 (m, 1H), 3.11 – 2.96 (m, 1H), 2.53 (t, J = 7.1 Hz, 2H), 2.17 – 2.09 (m, 2H), 1.83 – 1.66 (m, 6H), 1.64 – 1.41 (m, 6H), 1.03 – 0.89 (m, 2H), 0.00 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 173.5, 169.2, 168.9, 150.7, 147.0, 132.3, 132.1, 128.9, 126.2, 117.1, 116.1, 115.7, 114.9, 114.4, 112.1, 100.3, 98.8, 93.9, 87.9, 83.9, 67.7, 66.7, 57.8, 55.1, 53.5, 51.7, 38.9, 30.7, 10 30.3, 25.3, 24.2, 23.1, 20.1, 19.7, 18.3, 18.0, -1.3; MS (ES-): m/z = 663 (M+H)⁺; LCMS (Method B): tR = 4.83 min. 4-((5-((Allyloxy)carbonyl)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-2-((2- (trimethylsilyl)ethoxy)methoxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2- a][1,4]diazepin-3-yl)oxy)butanoic acid (72) 15 , , 3-(4-methoxy-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-2-((2- (trimethylsilyl)ethoxy)methoxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine- 5_{(12H)-carboxylate (71) (300 mg, 0.450 mmol) in 1,4-dioxane (5 mL) and the resulting mixture} 20 was stirred at room temperature for 16 h. The mixture was then diluted into brine (30 mL) and acidified to pH = 3 with an aqueous solution of acetic acid (1 M, 15 mL). The aqueous layer was extracted with ethyl acetate (2 x 50 mL) and the combined organic extracts were dried over s_{odium sulfate, filtered, and concentrated in vacuo to give the title compound (200 mg, 69%) as} a brown oil. The product was carried through to the next step without any further purification. _{25 1H NMR (400 MHz, CDCl3) δ 7.39 (d, J = 11.8 Hz, 1H), 6.84-6.53 (m, 1H), 6.20-5.95 (m, 1H),} 5.87 – 5.64 (m, 1H), 5.31 (d, J = 6.9 Hz, 1H), 5.28 (s, 2H), 5.20 (d, J = 6.6 Hz, 1H), 5.11 – 5.02 (m, 2H), 4.63 – 4.25 (m, 3H), 4.05 (t, J = 7.2 Hz, 2H), 3.86 (dd, J = 16.1, 8.3 Hz, 1H), 3.77 (t, J = 8.4 Hz, 2H), 3.56 (dd, J = 25.6, 14.5 Hz, 1H), 3.08 – 2.98 (m, 1H), 2.54 (q, J = 6.9 Hz, 2H), 2.15 – 2.08 (m, 2H), 1.76 – 1.50 (m, 12H), 0.99 – 0.92 (m, 2H), -0.02 (s, 9H); ¹³C NMR (100 _{30 MHz, CDCl3) δ 176.5, 169.3, 169.1, 150.7, 147.0, 132.1, 126.6, 126.1, 117.2, 115.7, 114.9,} 114.5, 100.4, 99.3, 93.9, 88.0, 84.0, 67.1, 66.7, 63.3, 55.2, 53.5, 38.9, 31.1, 30.7, 25.3, 24.1, 23.3, 23.1, 20.9, 20.2, 18.2, 18.0, -1.3; MS (ES+/-): m/z = 649 (M+H)⁺, 648 (M-H)-; LCMS (Method B): tR = 4.42 min. DB1/ 159693888.5 175

133186-5030-WO Example 1B: Synthesis of compound (79). Scheme 2. Synthesis of compound (79). , , 5 yl)carbamate (75) -( - m nop eny)- , , - r uoroace am e ( ) ( mg, . mmo) was a e o a mx ure of 4-((tert-butoxycarbonyl)amino)-1-methyl-1H-pyrrol-2-carboxylic acid (74) (647 mg, 2.69 mmol), N,N-dimethylpyridin-4-amine (898 mg, 7.35 mmol) and N-(3-dimethylaminopropyl)-N′- 10 ethylcarbodiimide hydrochloride (1.2 g, 6.25 mmol) in N,N-dimethylformamide (8 mL), which was previously stirred for 30 min. The resulting solution was stirred at room temperature for 18 h. The reaction mixture was quenched with a saturated aqueous solution of sodium hydrogen carbonate (15 mL) and brine (80 mL). The aqueous phase was extracted with ethyl acetate (2 x 50 mL). The combined organic extracts were concentrated in vacuo. The resulting residue was 15 purified by column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60 °_{C (from 0% to 100%), to give the title compound (839 mg, 80%) as a cream solid. 1H NMR} (600 MHz, DMSO-d6) δ 11.17 (s, 1H), 9.85 (s, 1H), 9.11 (s, 1H), 7.74 (d, J = 9.0 Hz, 2H), 7.58 (d, J = 9.0 Hz, 2H), 6.94 (s, 1H), 3.81 (s, 3H), 1.46 (s, 9H); ¹³C NMR (150 MHz, DMSO-d6) δ 160.2, 153.3, 1375, 131.6, 123.1, 122.9, 121.8, 120.8, 118.4, 117.3, 115.4, 105.2, 78.8, 36.6, 20 28.7; MS (ES+): m/z = 427 (M+H)⁺; LCMS (Method B): t_R = 3.83 min. DB1/ 159693888.5 176

133186-5030-WO Allyl (6aS)-3-(4-((1-methyl-5-((4-(2,2,2-trifluoroacetamido)phenyl)carbamoyl)-1H-pyrrol- 3-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-2-((2- (trimethylsilyl)ethoxy)methoxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2- a][1,4]diazepine-5(12H)-carboxylate (76) 5 p_{yrrol-3-yl)carbamate (75) (100 mg, 0.23 mmol) in 1,4-dioxane (2 mL) and methanol (2 mL),} hydrogen chloride (4 M in 1,4-dioxane) (4 mL) was added dropwise. The reaction mixture was s_{tirred for 4 h and then concentrated in vacuo. The resulting residue was added to a mixture of}10 4-((5-((allyloxy)carbonyl)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-2-((2- (trimethylsilyl)ethoxy)methoxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2- a][1,4]diazepin-3-yl)oxy)butanoic acid (72) (150 g, 0.23 mmol), N,N-dimethylpyridin-4-amine (85 mg, 0.70 mmol) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (111 mg, 0.58 mmol) in N,N-dimethylformamide (5 mL) which was previously stirred for 30 min. 15 The reaction mixture was stirred at room temperature for 18 h, then diluted with ethyl acetate (60 mL) and washed with brine (2 x 40 mL). The organic layer was concentrated in vacuo. The resulting residue was purified by column chromatography (silica), eluting with m_{ethanol/dichloromethane (from 0% to 10%) to give the title compound (160 g, 72%) as a} cream viscous oil. MS (ES+): m/z = 957.5 (M+H)⁺, (ES-): m/z = 955.6 (M+H)-; LCMS 20 (Method B): tR = 4.42 min. Allyl(6aS)-2-hydroxy-3-(4-((1-methyl-5-((4-(2,2,2-trifluoroacetamido)phenyl)carbamoyl)- 1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)- 6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (77) 25 Tetrabutylammonium fluoride (1 M in tetrahydrofuran, 4 mL, 4.00 mmol) was added to a solution of allyl (6aS)-3-(4-((1-methyl-5-((4-(2,2,2-trifluoroacetamido)phenyl)carbamoyl)-1H- pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-2-((2- (trimethylsilyl)ethoxy)methoxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine- 5_{(12H)-carboxylate (76) (192 mg, 0.201 mmol) in tetrahydrofuran (2 mL). The reaction mixture} 30 was heated to 80 ˚C for 8.5 h and then stirred at room temperature for further 16 h. The reaction mixture was diluted into brine (30 mL) and then acidified with acetic acid (0.2 mL), before DB1/ 159693888.5 177

133186-5030-WO extracting with ethyl acetate (2 x 20 mL). The combined organic extracts were concentrated in vacuo. The resulting residue was purified by column chromatography (silica), eluting with m_{ethanol/dichloromethane (from 0% to 8%). This was then solubilised in dichloromethane (2} mL) and stirred with tert-butyl(methoxy)diphenylsilane (270 mg, 0.998 mmol) for 1 h and the 5 mixture was then re-concentrated and further purified by column chromatography (silica), e_{luting with methanol/dichloromethane (from 0% to 8%) to give the title compound (146 mg, 88%) as a brown viscous oil. MS (ES+): m/z = 827.3 (M+H)+, (ES-): m/z = 825.5 (M+H)-;} LCMS (Method B): t_R = 3.70 min. Allyl(6aS)-3-(4-((5-((4-aminophenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-10 oxobutoxy)-2-hydroxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10- hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (78) (6aS)-2-hydroxy-3-(4-((1-methyl-5-((4-(2,2,2-trifluoroacetamido)phenyl)carbamoyl)-1H-pyrrol-15 3-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10- hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (77) (63 mg, 0.076mmol) in 1,4-dioxane (2 mL) and methanol (2 mL) and the resulting mixture was stirred at room temperature for 18 h. This was then diluted into brine (60 mL) and extracted with ethyl acetate (2 x 50 mL). The combined organic extracts were concentrated to give the title compound (52 _{20 mg, 93%) as a yellow viscous oil. MS (ES+): m/z = 731.4 (M+H)+, (ES-): m/z = 729.4 (M+H)-;} LCMS (Method B): tR = 3.08 min. (S)-N-(4-Aminophenyl)-4-(4-((2-hydroxy-12-oxo-6a,7,8,9,10,12- hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole- 2-carboxamide (79) 25 T_{o a solution of allyl (6aS)-3-(4-((5-((4-aminophenyl)carbamoyl)-1-methyl-1H-pyrrol-3-} yl)amino)-4-oxobutoxy)-2-hydroxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10- h_{exahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (78) (52 mg, 0.071 mmol),} in dichloromethane (4 mL) was sequentially added tetrakis(triphenylphosphine)palladium(0) (4 30 mg, 5 mol%), and pyrrolidine (8 µL, 0.106 mmol). The reaction mixture was stirred at room DB1/ 159693888.5 178

133186-5030-WO temperature for 30 min, then concentrated in vacuo and subjected to high vacuum for 1h. The resulting residue was purified by column chromatography (silica), eluting with methanol/dichloromethane (from 0% to 20%), to give the title compound (24 mg, 62%) as a tan solid. ¹H NMR (600 MHz, DMSO) δ 9.77 (s, 1H), 9.44 – 9.35 (m, 2H), 7.88 (d, J = 5.7 Hz, 5 1H), 7.32 – 7.28 (m, 3H), 7.11 – 7.09 (m, 1H), 6.78 (d, J = 1.8 Hz, 1H), 6.68 (s, 1H), 6.55 (d, J = 8.9 Hz, 2H), 3.99 – 3.85 (m, 3H), 3.73 (s, 3H), 3.65 – 3.57 (m, 1H), 3.02 (ddd, J = 15.2, 9.4, 4.0 Hz, 1H), 2.39 (t, J = 7.4 Hz, 2H), 2.00 – 1.93 (m, 3H), 1.80 – 1.73 (m, 2H)– 1.51 - 144 (m, 3H); MS (ES+): m/z = 545.2(M+H)⁺, (ES-): m/z = 543.2 (M+H)-; LCMS (Method B): t_R = 2.58 min. 10 Example 1C: Synthesis of compound (82). Scheme 3. Synthesis of compound (82). ( , , ,5 , )- -( cetoxymet y)- -((( a )-5-((a yoxy)carbony )- -( -(( -met y-5-(( - (2,2,2-trifluoroacetamido)phenyl)carbamoyl)-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-15 6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2- a][1,4]diazepin-2-yl)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (80) To a solution of allyl (6aS)-2-hydroxy-3-(4-((1-methyl-5-((4-(2,2,2- trifluoroacetamido)phenyl)carbamoyl)-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6-20 ((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine- 5(12H)-carboxylate (77) (102 mg, 0.145 mmol) in acetone (6 mL) was added potassium carbonate (50 mg, 0.363mmol) and (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-bromotetrahydro- DB1/ 159693888.5 179

133186-5030-WO 2H-pyran-3,4,5-triyl triacetate (72 mg, 0.174 mmol). The mixture was stirred at 40 °C under reflux for 4 h. The reaction mixture was then diluted with brine (50 mL) and extracted with ethyl acetate (2 x 30mL). The combined organic fractions were concentrated under reduced pressure. The resulting residue was purified by flash column chromatography (silica) eluting 5 with methanol/dichloromethane (from 0% to 10%), to give the title compound (98 mg, 58%) as a cream solid. MS (ES+): m/z = 1157.7 (M+H)⁺, (ES-): m/z = 1155.7 (M+H)-; LCMS (Method B): t_R = 4.08 min. Allyl (6aS)-3-(4-((5-((4-aminophenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4- oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-2-(((2S,3R,4S,5S,6R)-3,4,5-10 trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10- hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (81) methyl-5-((4-(2,2,2-trifluoroacetamido)phenyl)carbamoyl)-1H-pyrrol-3-yl)amino)-4-15 oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12- octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-2-yl)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (80) (98 mg, 0.085 mmol) in 1,4-dioxane (3 mL) and methanol (0.5 mL) was added an aqueous solution of sodium hydroxide (1 M, 3 mL, 3 mmol). The reaction mixture was stirred at room temperature for 2 h, and then at 40 °C for an additional 2 h. The mixture was concentrated 20 in vacuo and filtered through a pad of silica, then washed with dichloromethane and methanol. The elutant was then concentrated and purified by flash column chromatography (silica), eluting with methanol/dichloromethane (from 0% to 30%) to give the title compound (50 mg, 66%) as a white solid. MS (ES+): m/z = 893.5 (M+H)⁺, (ES-): m/z = 891.4 (M+H)-; LCMS (Method B): tR = 2.95 min. 25 N-(4-Aminophenyl)-1-methyl-4-(4-(((S)-12-oxo-2-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-6a,7,8,9,10,12- hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1H-pyrrole-2- carboxamide (82) . 180

133186-5030-WO To a solution of (2R,3R,4S,5R,6S)-2-(acetoxymethyl)-6-(((6aS)-5-((allyloxy)carbonyl)-3-(4-((1- methyl-5-((4-(2,2,2-trifluoroacetamido)phenyl)carbamoyl)-1H-pyrrol-3-yl)amino)-4- oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12- octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-2-yl)oxy)tetrahydro-2H-pyran-3,4,5-triyl 5 triacetate (80) (86 mg, 0.074 mmol) in 1,4-dioxane (3 mL) and methanol (0.5 mL) was added an aqueous solution of sodium hydroxide (1 M, 3 mL, 3 mmol). The reaction mixture was stirred at room temperature for 18 h. The mixture was then diluted with methanol (30 mL) and concentrated in vacuo. The resulting residue was re-suspended in dichloromethane (3 mL) and methanol (1 mL). To the resulting mixture was sequentially added 10 tetrakis(triphenylphosphine)palladium(0) (8.6 mg, 10 mol%), and pyrrolidine (7 µL, 0.089 mmol). The reaction mixture was stirred at room temperature for 30 min and then concentrated in vacuo and subjected to high vacuum for 1 h. The resulting residue was purified by flash column chromatography (silica) eluting with methanol/dichloromethane (from 0% to 35%), to give the title compound (13 mg, 25%) as a white solid. ¹H NMR (600 MHz, DMSO-d6) δ 9.8415 (s, 1H), 9.42 (s, 1H), 8.02 (d, J = 5.7 Hz, 1H), 7.45 (s, 1H), 7.28 (d, J = 8.7 Hz, 2H), 7.16 (dd, J = 4.0, 1.8 Hz, 1H), 6.85 – 6.80 (m, 2H), 6.50 (d, J = 8.7 Hz, 2H), 5.04 – 4.83 (m, 6H)– 4.02 - 3.95 (m, 3H), 3.79 (s, 3H), 3.71 – 3.62 (m, 5H), 3.60 – 3.54 (m, 3H), 3.13 – 3.10 (m, 1H), 2.47 – 2.44 (m, 2H), 2.04 – 2.01 (m, 3H), 1.68 – 1.64 (m, 2H), 1.52 – 1.48 (m, 3H); MS (ES+): m/z = 7_{07.5 (M+H)+; LCMS (Method A): tR = 4.50 min.} 20 Example 1D: Synthesis of compound (85). Scheme 4. Synthesis of compound (85). (2R,3S,4S,5R,6S)-2-(Acetoxymethyl)-6-(((6aS)-5-((allyloxy)carbonyl)-3-(4-((1-methyl-5-((4- (2,2,2-trifluoroacetamido)phenyl)carbamoyl)-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo- DB1/ 159693888.5 181

133186-5030-WO 6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2- a][1,4]diazepin-2-yl)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (83) 5 trifluoroacetamido)phenyl)carbamoyl)-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6- ((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine- 5(12H)-carboxylate (77) (198 mg, 0.24 mmol) in dichloromethane (3 mL) was added potassium carbonate (116 mg, 0.84 mmol), tetrabutylammonium bromide (155 mg, 0.48 mmol) and (2R,3S,4S,5R,6R)-2-(acetoxymethyl)-6-bromotetrahydro-2H-pyran-3,4,5-triyl triacetate (296 10 mg, 0.72 mmol). The mixture was stirred at room temperature for 18 h. The reaction mixture was then diluted into ethyl acetate (50 mL) and washed with brine (40 mL). The organic layer was concentrated in vacuo. The resulting residue was purified by flash column chromatography (silica), eluting with methanol/dichloromethane (from 0% to 10%) to give the title compound (141 mg, 51%) as a cream solid. MS (ES+): m/z = 1157.7 (M+H)⁺, (ES-): m/z = 1155.9 (M+H)- 15 ; LCMS (Method B): t_R = 3.97 min. Allyl (6aS)-3-(4-((5-((4-aminophenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4- oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-2-(((2S,3R,4S,5R,6R)-3,4,5- trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10- hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (84) 20 To a solution of (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(((6aS)-5-((allyloxy)carbonyl)-3-(4-((1- methyl-5-((4-(2,2,2-trifluoroacetamido)phenyl)carbamoyl)-1H-pyrrol-3-yl)amino)-4- oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12- octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-2-yl)oxy)tetrahydro-2H-pyran-3,4,5-triyl 25 triacetate (83) (141 mg, 0.122 mmol) in 1,4-dioxane (3 mL) and methanol (0.5 mL) was added an aqueous solution of sodium hydroxide (1 M, 3 mL, 3 mmol). The reaction mixture was stirred at room temperature for 18 h. The mixture was then quenched with an aqueous solution of acetic acid (1 M, 2 mL, 2 mmol) and concentrated in vacuo. The resulting residue was DB1/ 159693888.5 182

133186-5030-WO purified by reverse-phase chromatography (C18), eluting with acetonitrile-water (from 5% to 100%) to give the title compound (76 mg, 70%) as a cream solid. MS (ES+): m/z = 893.6 (M+H)⁺; LCMS (Method B): tR = 2.93 min. N-(4-Aminophenyl)-1-methyl-4-(4-(((S)-12-oxo-2-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6- 5 (hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-6a,7,8,9,10,12- hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1H-pyrrole-2- carboxamide (85) 10 yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-2-(((2S,3R,4S,5R,6R)-3,4,5- trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10- h_{exahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (84) (19 mg, 0.021 mmol),} in dichloromethane (3 mL) and methanol (1.5 mL) was sequentially added tetrakis(triphenylphosphine)palladium(0) (12 mg, 50 mol%), and pyrrolidine (4 µL, 0.053 15 mmol). The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was then concentrated in vacuo. The resulting residue was purified by reverse-phase column chromatography eluting with acetonitrile-water (from 5% to 100%), to give the title compound (10 mg, 66%) as a white solid. ¹H NMR (600 MHz, DMSO-d6) δ 9.82 -9.77 (m, 1H), 9.43 – 9.40 (m, 1H), 8.02 (d, J = 5.7 Hz, 1H), 7.35 – 7.24 (m, 3H), 7.17 (dd, J = 3.8, 1.8 Hz, 1H), 6.90 20 – 6.79 (m, 2H), 6.52 – 6.49 (m, 2H), 4.88 – 4.81 (m, 3H), 4.68 – 4.52 (m, 3H), 4.08 – 3.91 (m, 3H), 3.79 (s, 3H), 3.76 – 3.65 (m, 2H), 3.63 – 3.53 (m, 3H), 3.50 – 3.52 (m, 3H), 3.10 (ddd, J = 18.0, 11.1, 5.3 Hz, 1H), 2.47 – 2.43 (m, 2H), 2.04 (dd, J = 23.8, 17.1 Hz, 3H), 1.87 – 1.75 (m, 2H), 1.61 – 1.48 (m, 3H); MS (ES+): m/z = 707.3 (M+H)⁺, (ES-): m/z = 706.4 (M+H)- ; LCMS (Method B): tR = 2.38 min. DB1/ 159693888.5 183

133186-5030-WO Example 1E: Synthesis of compound (88). Scheme 5. Synthesis of compound (88). 5 trifluoroacetamido)phenyl)carbamoyl)-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6- ((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2- a][1,4]diazepin-2-yl)oxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (86) 10 o a so u o o a y a - - y o y- - - - e y - - - , , - trifluoroacetamido)phenyl)carbamoyl)-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6- ((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine- 5(12H)-carboxylate (77) (200 mg, 0.242 mmol) in dichloromethane (3 mL) was added an aqueous solution (1 mL) of potassium carbonate (117 mg, 0.847 mmol), tetrabutylammonium15 bromide (156 mg, 0.484 mmol) and (2R,3R,4S,5S,6S)-2-bromo-6-(methoxycarbonyl)tetrahydro- 2H-pyran-3,4,5-triyl triacetate (289 mg, 0.726 mmol). The mixture was stirred at room temperature for 18 h. The reaction mixture was then diluted into ethyl acetate (50 mL) and washed with brine (40 mL). The organic layer was concentrated in vacuo. The resulting residue was purified by flash column chromatography (silica) eluting with methanol/dichloromethane 20 (from 0% to 10%), to give the title compound (104 mg, 38%) as a cream solid. MS (ES+): m/z = 1143.8 (M+H)⁺, (ES-): m/z = 1141.6 (M+H)-; LCMS (Method A): tR = 7.58 min. DB1/ 159693888.5 184

133186-5030-WO (2S,3S,4S,5R,6S)-6-(((6aS)-5-((Allyloxy)carbonyl)-3-(4-((5-((4-aminophenyl)carbamoyl)-1- methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)- 5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-2-yl)oxy)-3,4,5- trihydroxytetrahydro-2H-pyran-2-carboxylic acid (87) 5 - trifluoroacetamido)phenyl)carbamoyl)-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6- ((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2- a][1,4]diazepin-2-yl)oxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (86) 10 (104 mg, 0.091 mmol) in 1,4-dioxane (3 mL) and methanol (0.5 mL) was added an aqueous solution of sodium hydroxide (1 M, 3 mL, 3 mmol). The reaction mixture was stirred at room temperature for 18 h. The mixture was then quenched with an aqueous solution of acetic acid (1 M, 2 mL, 2 mmol) and concentrated in vacuo. The resulting residue was purified by reverse- phase chromatography, eluting with acetonitrile-water (from 5% to 100%) to give the title 15 compound (57 mg, 69%) as a cream solid. MS (ES+): m/z = 907.0 (M+H)⁺, (ES-): m/z = 905.0 (M+H)- ; LCMS (Method B): t_R = 2.98 min. (2S,3S,4S,5R,6S)-6-(((S)-3-(4-((5-((4-Aminophenyl)carbamoyl)-1-methyl-1H-pyrrol-3- yl)amino)-4-oxobutoxy)-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2- a][1,4]diazepin-2-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (88) 20 _{To a solution of (2S,3S,4S,5R,6S)-6-(((6aS)-5-((allyloxy)carbonyl)-3-(4-((5-((4-} aminophenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6- ((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2- a_{][1,4]diazepin-2-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (87) (27 mg,} 25 0.03 mmol), in dichloromethane (3 mL) and methanol (1.5 mL) was sequentially added tetrakis(triphenylphosphine)palladium(0) (17 mg, 50 mol%), and pyrrolidine (6 µL, 0.07 mmol). The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was then concentrated in vacuo. The resulting residue was purified by reverse-phase column DB1/ 159693888.5 185

133186-5030-WO chromatography, eluting with acetonitrile-water (from 5% to 100%) to give the title compound (11 mg, 51%) as a cream solid. ¹H NMR (600 MHz, DMSO-d6) δ 10.17 – 10.07 (m, 1H), 9.43 (t, J = 8.0 Hz, 1H), 8.02 (d, J = 5.7 Hz, 1H), 7.46 (s, 1H), 7.29 (d, J = 8.8 Hz, 2H), 7.17 – 7.14 (m, 1H), 6.85 (d, J = 1.8 Hz, 1H), 6.81 (s, 1H), 6.52 – 6.48 (m, 2H), 4.87 (d, J = 7.4 Hz, 1H), 5 4.69 – 4.55 (m, 1H), 4.15 – 3.86 (m, 4H), 3.78 (s, 3H), 3.76 – 3.63 (m, 2H), 3.19 – 3.08 (m, 5H), 2.55 – 2.51 (m, 2H), 2.08 – 1.99 (m, 3H), 1.74 – 1.68 (m, 2H), 1.58 – 1.51 (m, 3H); MS (_{ES+): m/z = 721.4 (M+H)+, (ES-): m/z = 719.3 (M+H)- ; LCMS (Method B): tR = 2.38 min.} Example 1F: Synthesis of compound (91). Scheme 6. Synthesis of compound (91). 10 Allyl (6aS)-3-(4-((5-((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3- methylbutanamido)propanamido)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4- oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-2-(((2S,3R,4S,5S,6R)-3,4,5- trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10- 15 hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (89) Allyl (6aS)-3-(4-((5-((4-aminophenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4- oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-2-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy- 6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2- _{20 a][1,4]diazepine-5(12H)-carboxylate (81) (50 mg, 0.056 mmol) was added to a mixture of} ((allyloxy)carbonyl)-L-valyl-L-alanine (17 mg, 0.062 mmol), N,N-dimethylpyridin-4-amine (21 mg, 0.168 mmol) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (28 mg, 0.14 mmol) in N,N-dimethylformamide (2 mL), which was previously stirred for 30 min. The DB1/ 159693888.5 186

133186-5030-WO resulting solution was stirred at room temperature for 18 h. The reaction mixture was then diluted with toluene (30 mL) and concentrated in vacuo. The resulting residue was purified by f_{lash column chromatography (silica), eluting with methanol/dichloromethane (from 0% to 30%), to give the title compound (35 mg, 54%) as a cream solid. MS (ES+): m/z = 1147.8} 5 (M+H)⁺, (ES-): m/z = 1146.6 (M+H)-; LCMS (Method B): t_R = 3.37 min. N-(4-((S)-2-((S)-2-Amino-3-methylbutanamido)propanamido)phenyl)-1-methyl-4-(4-(((S)- 12-oxo-2-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2- yl)oxy)-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)- 1H-pyrrole-2-carboxamide (90) 10 methylbutanamido)propanamido)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4- oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-2-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy- 6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2- _{15 a][1,4]diazepine-5(12H)-carboxylate (89) (35 mg, 0.03 mmol), in dichloromethane (2 mL) and} methanol (0.5 mL) was sequentially added tetrakis(triphenylphosphine)palladium(0) (3.5 mg, 10 mol%), and pyrrolidine (3 µL, 0.04 mmol). The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was then concentrated in vacuo. The resulting residue was purified by reverse-phase column chromatography, eluting with acetonitrile-water (from 20 5% to 100%), to give the title compound (21 mg, 79%) as a cream solid. MS (ES+): m/z = 877.5 (M+H)⁺, (ES-): m/z = 875.4 (M+H)-; LCMS (Method A): t_R = 4.75 min. N-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3- methylbutanamido)propanamido)phenyl)-1-methyl-4-(4-(((6aS)-12-oxo-2-(((3R,4S,5S,6R)- 3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-6a,7,8,9,10,12-25 hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1H-pyrrole-2- carboxamide (91) DB1/ 159693888.5 187

133186-5030-WO N-(4-((S)-2-((S)-2-Amino-3-methylbutanamido)propanamido)phenyl)-1-methyl-4-(4-(((S)-12- oxo-2-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)- 6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1H- p_{yrrole-2-carboxamide (21 mg, 0.024 mmol) was added to a mixture of 6-(2,5-dioxo-2,5-} 5 dihydro-1H-pyrrol-1-yl)hexanoic acid (90) (5.6 mg, 0.026 mmol) and 2-ethoxy-1- e_{thoxycarbonyl-1,2-dihydroquinoline (7.1 mg, 0.029 mmol) in dichloromethane (2 mL) and} methanol (0.2 mL) which was previously stirred at room temperature for 30 min. The resulting solution was stirred at room temperature for 18 h. The reaction mixture was concentrated in vacuo and the resulting residue was purified by flash column chromatography (silica), eluting _{10 with methanol/dichloromethane (from 0% to 25%), to give the title compound (11 mg, 43%) as} a cream solid. ¹H NMR (600 MHz, DMSO-d6) δ 9.82 (s, 2H), 9.74 (s, 1H), 8.39 (d, J = 7.5 Hz, 1H), 8.11 (d, J = 7.0 Hz, 1H), 7.95 (d, J = 7.7 Hz, 1H), 7.80 (d, J = 8.6 Hz, 1H), 7.61 (d, J = 6.9 Hz, 2H), 7.51 (d, J = 9.0 Hz, 2H), 7.23 – 7.18 (m, 2H), 7.00 – 6.98 (m, 2H), 6.94 (s, 1H), 4.73 – 4.62 (m, 2H), 4.59 – 4.49 (m, 2H), 4.06 – 3.96 (m, 5H), 3.81 (s, 3H), 3.70 – 3.54 (m, 6H), 3.50 15 – 3.43 (m, 4H), 3.13 – 3.08 (m, 1H), 2.48 – 2.45 (m, 2H), 2.26 (dd, J = 15.2, 7.6 Hz, 1H), 2.18 – 2.12 (m, 2H), 2.04 (dd, J = 13.5, 7.1 Hz, 3H), 1.98 – 1.89 (m, 2H), 1.68 – 1.58 (m, 5H), 1.30 (dd, J = 7.1, 2.1 Hz, 3H), 1.22 – 1.14 (m, 4H), 0.86 – 0.82 (m, 6H); MS (ES+): m/z = 1070.8 (M+H)⁺, (ES-): m/z = 1068.7 (M+H)-; LCMS (Method A): t_R = 5.70 min. Example 1G: Synthesis of compound (96). 20 Scheme 7. Synthesis of compound (96). Allyl (6aS)-3-(4-((5-((4-(5-(methoxycarbonyl)-1-methyl-1H-pyrrol-3-yl)phenyl)carbamoyl)- 1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)- DB1/ 159693888.5 188

133186-5030-WO 2-((2-(trimethylsilyl)ethoxy)methoxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2- a][1,4]diazepin-5(12H)-carboxylate (92) 5 carboxamido)phenyl)-1-methyl-1H-pyrrol-2-carboxylate (15) (700 mg, 1.55 mmol) in 1,4- dioxane (3 mL) and methanol (3 mL), hydrogen chloride (4 M in 1,4-dioxane) (6 mL) was a_{dded dropwise. The reaction mixture was stirred for 2 h and then concentrated in vacuo. The} residue was added to a mixture of 4-((5-((allyloxy)carbonyl)-12-oxo-6-((tetrahydro-2H-pyran-2- yl)oxy)-2-((2-(trimethylsilyl)ethoxy)methoxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2- 10 a][1,4]diazepin-3-yl)oxy)butanoic acid (72) (1.10 g, 1.70 mmol), N,N-dimethylpyridin-4-amine (622 mg, 5.10 mmol) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (813 mg, 4.24 mmol) in N,N-dimethylformamide (8 mL) which was previously stirred for 30 min. The reaction mixture was stirred at room temperature for 19 h and then quenched with a saturated aqueous solution of sodium hydrogen carbonate (20 mL) and brine (100 mL). The 15 aqueous phase was extracted with ethyl acetate (2 x 70 mL). The combined organic extracts were concentrated in vacuo. The resulting residue was purified by column chromatography (_{silica), eluting with methanol/dichloromethane (from 0% to 10%) to give the title compound (1.20 g, 72%) as a brown oil. 1H NMR (600 MHz, DMSO-d6) δ 9.88 (s, 1H), 9.77 (s, 1H), 7.68} (d, J = 8.7 Hz, 2H), 7.55 (d, J = 1.9 Hz, 1H), 7.51 (d, J = 8.4 Hz, 2H)– 7.24 - 717 (m, 3H), 6.97 20 – 6.82 (m, 2H), 6.05 – 5.91 (m, 1H), 5.87 – 5.75 (m, 1H), 5.29 – 5.23 (m, 2H), 5.11 – 5.01 (m, 3H), 4.63 – 4.39 (m, 2H), 4.11 – 3.97 (m, 4H), 3.89 (s, 3H), 3.86 – 3.79 (m, 4H), 3.77 (s, 3H), 3.75 – 3.71 (m, 2H), 3.49 – 3.44 (m, 1H), 2.88 (dd, J = 15.9, 8.3 Hz, 1H), 2.43 (t, J = 6.7 Hz, 2H), 2.09 – 1.99 (m, 2H), 1.74 – 1.40 (m, 12H), 0.90 (ddd, J = 8.6, 7.1, 3.1 Hz, 2H), -0.03 (s, 9H); ¹³C NMR (150 MHz, DMSO-d₆) δ 168.8, 167.9, 167.8, 160.8, 159.6, 146.1, 137.3, 132.7, 25 128.9, 127.0, 124.6, 122.8, 122.7, 122.2, 122.1, 120.4, 118.7, 113.8, 104.7, 99.2, 93.4, 87.5, 83.4, 68.2, 65.8, 59.7, 56.0, 54.9, 51.0, 38.1, 36.5, 36.4, 31.7, 30.5, 30.1, 24.8, 22.6, 20.7, 18.5, 17.8, 17.5, 14.1, -1.4; MS (ES+/-): m/z = 984 (M+H)⁺, 982 (M-H)-; LCMS (Method A): tR = 9.35 min. 4-(4-(4-(4-(((6aS)-5-((Allyloxy)carbonyl)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-2-((2-30 (trimethylsilyl)ethoxy)methoxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2- DB1/ 159693888.5 189

133186-5030-WO a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrol-2-carboxamido)phenyl)-1- methyl-1H-pyrrol-2-carboxylic acid (93) 5 yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro- 2H-pyran-2-yl)oxy)-2-((2-(trimethylsilyl)ethoxy)methoxy)-6,6a,7,8,9,10- hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-5(12H)-carboxylate (92) (1.10 g, 1.12 mmol) in 1,4-dioxane (20 mL) was added an aqueous solution of sodium hydroxide (1 M, 20 mL, 20 mmol). The reaction mixture was stirred at room temperature for 18 h and was then 10 concentrated in vacuo, after which water (200 mL) was added and the aqueous solution was acidified to pH = 4 with an aqueous solution of acetic acid (5 M, 20 mL). This was then extracted with ethyl acetate (2 x 120 mL). The combined organic extracts were concentrated, and the resulting residue was purified by column chromatography (silica), eluting with m_{ethanol/dichloromethane (from 0% to 10%), to give the title compound (292 mg, 27%) as a} 15 yellow oil. ¹H NMR (600 MHz, DMSO-d₆) δ 12.16 (s, 1H), 9.88 (s, 1H), 9.77 (s, 1H), 7.67 (d, J = 7.9 Hz, 2H), 7.52 – 7.45 (m, 3H), 7.26 – 7.18 (m, 3H), 7.12 (d, J = 1.9 Hz, 1H), 6.95 – 6.83 (m, 2H), 6.02 (d, J = 9.9 Hz, 1H), 5.83 – 5.71 (m, 1H), 5.27 – 5.24 (m, 2H), 5.10 – 5.00 (m, 3H), 4.60 – 4.41 (m, 2H), 4.07 – 3.98 (m, 4H), 3.87 (s, 3H), 3.83 (s, 3H), 3.78 – 3.74 (m, 3H), 3.52 (dd, J = 10.9, 5.3 Hz, 1H), 2.88 (dd, J = 15.0, 8.6 Hz, 1H), 2.43 (t, J = 6.8 Hz, 2H), 2.08 – 20 2.01 (m, 2H), 1.64 – 1.43 (m, 12H), 0.91 – 0.88 (m, 2H), -0.03 (s, 9H); ¹³C NMR (150 MHz, DMSO-d₆) δ 172.0, 170.3, 168.8, 167.8, 162.0, 159.6, 146.1, 137.2, 132.7, 129.2, 126.3, 124.5, 122.7, 122.4, 122.0, 120.4, 118.7, 116.5, 114.7, 113.6, 104.7, 99.2, 94.2, 93.4, 87.5, 83.4, 68.2, 68.0, 65.8, 62.8, 62.0, 59.7, 54.8, 38.1, 36.5, 36.1, 31.8, 30.5, 30.1, 24.9, 22.6, 21.1, 20.7, 18.9, 17.8, 17.5, 14.1, -1.4; δ; MS (ES+/-): m/z = 970 (M+H)⁺, 968 (M-H)-; LCMS (Method B): tR = 25 4.43 min. Allyl (6aS)-3-(4-((1-methyl-5-((4-(1-methyl-5-(phenylcarbamoyl)-1H-pyrrol-3- yl)phenyl)carbamoyl)-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H- pyran-2-yl)oxy)-2-((2-(trimethylsilyl)ethoxy)methoxy)-6,6a,7,8,9,10- hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-5(12H)-carboxylate (94) 30 To a solution of 4-(4-(4-(4-(((6aS)-5-((Allyloxy)carbonyl)-12-oxo-6-((tetrahydro-2H-pyran-2- yl)oxy)-2-((2-(trimethylsilyl)ethoxy)methoxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2- DB1/ 159693888.5 190

133186-5030-WO a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrol-2-carboxamido)phenyl)-1-methyl- 1_{H-pyrrol-2-carboxylic acid (93) (200 mg, 0.210 mmol) in N,N-dimethylformamide (3 mL)} were added N,N-dimethylpyridin-4-amine (76 mg, 0.62 mmol) and N-(3-dimethylaminopropyl)- N′-ethylcarbodiimide hydrochloride (100 mg, 0.520 mmol). The reaction mixture was stirred for 5 30 min at room temperature. Aniline (29 µL, 0.31 mmol) was added and the solution was stirred for a further 16 h. The reaction mixture was quenched with a saturated aqueous solution of sodium hydrogen carbonate (8 mL) and diluted into brine (70 mL). The aqueous phase was extracted with ethyl acetate (2 x 50 mL). The combined organic extracts were concentrated in vacuo. The resulting residue was purified by column chromatography (silica), eluting with ethyl _{10 acetate/dichloromethane (from 0% to 100%) to give the title compound (100 mg, 46%) as a} yellow oil. ¹H NMR (400 MHz, CDCl3) – 8.56 - 8.41 (m, 2 H)– 8.26 - 8.11 (m, 1 H)– 7.72 - 7.62 (m, 4 H)– 7.44 - 7.31 (m, 6 H)– 7.22 - 7.01 (m, 5 H)– 6.51 - 6.23 (m, 1 H)– 6.04 - 5.72 (m, 1 H)– 5.26 - 5.07 (m, 4 H), 4.69 (br. s., 1 H)– 4.45 - 4.34 (m, 1 H), 4.12 (br. s., 2 H), 3.99 (br. s., 4 H), 3.88 (br. s., 4 H), 3.75 (br. s., 2 H), 3.63 (br. s., 3 H)– 3.10 - 3.04 (m, 1 H), 2.51 (br. s., 2 15 H), 2.20 (br. s., 2 H)– 1.80 - 1.50 (m, 12 H)– 1.30 - 0.82 (m, 2 H), -0.02 (br. s., 9 H); ¹³C NMR (100 MHz, CDCl3) δ 173.8, 169.7, 169.0, 160.1, 155.9, 155.7, 138.4, 132.1, 131.9, 130.1, 129.9, 128.9, 126.6, 125.2, 125.1, 123.9, 123.3, 122.9, 121.6, 120.8, 120.2, 119.8, 116.1, 109.8, 106.4, 104.5, 104.0, 100.4, 93.9, 88.0, 81.6, 68.8, 66.7, 66.6, 64.1, 55.2, 39.0, 37.0, 36.8, 33.0, 31.0, 30.6, 25.2, 23.1, 20.9, 19.5, 18.4, 18.2, 17.9, -1.4; MS (ES+/-): m/z = 1045 (M+H)⁺, 1043 (M- 20 H)- LCMS (Method B): tR = 4.82 min. Allyl (6aS)-2-hydroxy-3-(4-((1-methyl-5-((4-(1-methyl-5-(phenylcarbamoyl)-1H-pyrrol-3- yl)phenyl)carbamoyl)-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H- pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-5(12H)- carboxylate (95) 25 solution of allyl (6aS)-3-(4-((1-methyl-5-((4-(1-methyl-5-(phenylcarbamoyl)-1H-pyrrol-3- yl)phenyl)carbamoyl)-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2- yl)oxy)-2-((2-(trimethylsilyl)ethoxy)methoxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2- 30 a][1,4]diazepin-5(12H)-carboxylate (94) ( 100 mg, 0.100 mmol) in tetrahydrofuran (1 mL). The reaction mixture was heated to 80 ˚C for 9 h. The reaction mixture was diluted with a 0.1 M solution of acetic acid in water (30 mL) and washed with ethyl acetate (2 x 20 mL). The combined organic extracts were concentrated in vacuo. The resulting residue was purified by c_{olumn chromatography (silica), eluting with methanol/dichloromethane (from 0% to 10%) to} DB1/ 159693888.5 191

133186-5030-WO g_{ive the title compound (66.0 mg, 75%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 8.92 –} 8.42 (m, 3H), 7.63 (d, J = 7.9 Hz, 2H), 7.44 (d, J = 6.6 Hz, 3H), 7.31 – 7.20 (m, 5H), 7.17 – 6.97 (m, 4H), 6.91 (s, 1H), 6.35 – 6.14 (m, 1H–,6.02 - 5.85 (m, 1H), 5.77 – 5.61 (m, 1H), 5.38 – 4.93 (m, 3H), 4.69 – 4.16 (m, 2H), 4.06 – 3.95 (m, 1H), 3.90 (s, 3H), 3.86 – 3.77 (m, 1H), 3.74 5 (s, 3H), 3.68 – 3.42 (m, 4H), 3.07 – 2.94 (m, 1H), 2.53 – 2.30 (m, 2H), 2.16 – 2.03 (m, 2H), 1.74 – 1.44 (m, 12H); ¹³C NMR (100 MHz, CDCl₃) δ 175.3, 171.1, 171.0, 169.6, 163.0, 160.3, 155.9, 155.5, 138.4, 136.3, 132.2, 130.3, 128.9, 126.8, 126.6, 125.2, 123.9, 123.2, 123.0, 121.5, 121.0, 120.5, 120.0, 118.8, 117.3, 114.3, 113.8, 110.0, 104.6, 100.9, 100.4, 98.4, 88.1, 73.7, 70.2, 66.5, 63.2, 39.1, 37.0, 36.7, 33.0, 32.0, 31.0, 30.7, 29.7, 25.3, 23.0, 20.9, 18.3, 18.2; MS 10 (ES+/-): m/z = 915 (M+H)⁺, 913 (M-H)-; LCMS (Method A): tR = 7.98 min. (S)-4-(4-((2-Hydroxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin- 3-yl)oxy)butanamido)-1-methyl-N-(4-(1-methyl-5-(phenylcarbamoyl)-1H-pyrrol-3- yl)phenyl)-1H-pyrrole-2-carboxamide (96) ₁₅ - 1H-pyrrol-3-yl)phenyl)carbamoyl)-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro- 2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-5(12H)- carboxylate (95) (110 mg, 0.120 mmol), in dichloromethane (6 mL) was sequentially added tetrakis(triphenylphosphine)palladium(0) (14 mg, 10 mol%), and pyrrolidine (20 µL, 0.240 20 mmol). The reaction mixture was stirred at room temperature for 10 min. The reaction mixture was then concentrated in vacuo and subjected to high vacuum for 1h. The resulting residue was purified by flash column chromatography (silica), eluting with methanol/dichloromethane (from 0% to 10%), to give the title compound (14 mg, 16%) as a cream solid. [α]_D ²⁰ = 33° (c 0.071, (CH3)2SO); ¹H NMR (600 MHz, DMSO-d6) δ 9.89 (s, 1H), 9.83 (s, 1H), 9.80 (s, 1H), 9.47 (s, 25 1H), 7.96 (d, J = 5.7 Hz, 1H), 7.74 (d, J = 7.6 Hz, 2H), 7.72 (d, J = 8.7 Hz, 2H), 7.51 (d, J = 8.7 Hz, 2H), 7.47 (d, J = 1.8 Hz, 1H), 7.41 (d, J = 1.9 Hz, 1H), 7.36 – 7.31 (m, 2H), 7.22 (d, J = 1.7 Hz, 1H), 7.20 (s, 1H), 7.06 (dd, J = 11.6, 4.2 Hz, 1H), 6.97 (d, J = 1.8 Hz, 1H), 6.76 (s, 1H), 4.15-4.12 (m, 1H), 4.07 – 3.96 (m, 2H), 3.91 (d, J = 14.5 Hz, 3H), 3.84 (s, 3H), 3.69 (dd, J = 9.8, 5.7 Hz, 1H), 3.09 (ddd, J = 13.8, 11.3, 4.1 Hz, 1H), 2.48 (t, J = 7.4 Hz, 2H), 2.09 – 2.00 (m, 30 3H), 1.89 – 1.82 (m, 1H), 1.79 – 1.68 (m, 3H), 1.61 – 1.54 (m, 1H); ¹³C NMR (150 MHz, DMSO-d6) δ 169.1, 166.4, 164.0, 159.7, 159.6, 149.3, 145.2, 139.3, 138.7, 137.1, 129.5, 128.5, 125.4, 124.4, 123.0, 122.7, 122.1, 122.0, 121.2, 120.5, 119.9, 118.8, 115.2, 110.5, 110.0, 104.8, 67.7, 49.2, 36.5, 36.2, 31.9, 29.0, 24.8, 23.8, 22.6, 17.8; MS (ES+): m/z = 728 (M+H)⁺; LCMS DB1/ 159693888.5 192

133186-5030-WO (Method A): t_R = 7.02 min; MS (ES+): m/z = 855 (M+H)⁺; LCMS (Method A): t_R = 6.07 min; HRMS (ESI, m/z): calc. for C41H42N7O6⁺ ([M]+H)⁺ 728.3191 found 728.3175. Example 1H: Synthesis of compound (98). Scheme 8. Synthesis of compound (98). 5 (2R,3R,4S,5R)-2-(Acetoxymethyl)-6-(((6aS)-5-((allyloxy)carbonyl)-3-(4-((1-methyl-5-((4-(1- methyl-5-(phenylcarbamoyl)-1H-pyrrol-3-yl)phenyl)carbamoyl)-1H-pyrrol-3-yl)amino)-4- oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12- octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-2-yl)oxy)tetrahydro-2H-pyran-3,4,5-triyl 10 triacetate (97) m xture o a y ( a )- - y roxy- -( -(( -met y -5-(( -( -met y -5-(p eny car amoy)- - pyrrol-3-yl)phenyl)carbamoyl)-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H- pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-5(12H)- 15 carboxylate (95) (66 mg, 0.072 mmol), acetobromo-α-D-glucose (60 mg, 0.144 mmol) and potassium carbonate (22.0 mg, 0.180 mmol) in acetone (3 mL) was stirred at 60 ˚C for 6 h. The reaction mixture was diluted into brine (30 mL) and extracted with ethyl acetate (2 x 20 mL). The organic layer was concentrated in vacuo. The resulting residue was purified by column chromatography (silica), eluting with acetone/dichloromethane (from 0% to 40%), to give the 20 title compound (53.0 mg, 61%) as a colourless oil. ¹H NMR (400 MHz, CDCl3) δ 8.34 (s, 1H), 8.19 – 8.02 (m, 2H), 7.62 (d, J = 7.8 Hz, 3H), 7.55 (d, J = 7.4 Hz, 2H), 7.39 (d, J = 8.4 Hz, 2H), 7.36 – 7.28 (m, 3H), 7.22 (s, 1H), 7.09 (t, J = 7.4 Hz, 1H), 7.02 (s, 2H), 6.61 (s, 1H), 6.27-5.95 (m, 1H), 5.91 – 5.65 (m, 1H), 5.49 – 5.19 (m, 3H), 5.16 – 4.91 (m, 4H), 4.71 – 4.14 (m, 5H), DB1/ 159693888.5 193

133186-5030-WO 4.08 – 3.97 (m, 6H), 3.89 (s, 3H), 3.85 – 3.75 (m, 1H), 3.65 – 3.42 (m, 2H), 3.12 – 2.96 (m, 1H), 2.53 – 2.43 (m, 2H), 2.26 – 2.16 (m, 2H), 2.05 (d, J = 10.0 Hz, 12H), 1.81 – 1.43 (m, 12H); 1³C NMR (100 MHz, CDCl3) δ 171.0, 170.3, 169.6, 168.8,168.5, 160.0, 155.6, 139.3, 138.3, 136.4, 132.1, 131.9, 131.6, 130.5, 130.4, 129.0, 126.6, 125.5, 125.3, 124.0, 123.3, 120.8, 120.7, 5 120.2, 120.0, 117.5, 115.4, 114.8, 114.3, 109.6, 103.9, 100.9, 98.8, 83.9, 73.8, 72.5, 72.2, 71.3, 68.2, 67.8, 66.5, 64.7, 63.6, 61.8, 39.1, 37.1, 36.8, 31.2, 31.0, 30.8, 29.4, 25.3, 25.2, 23.4, 23.1, 21.0, 20.8, 20.7, 18.2; MS (ES+): m/z = 1245 (M+H)⁺; LCMS (Method A): t_R = 8.52 min. 1-Methyl-4-(4-(1-methyl-4-(4-(((S)-12-oxo-2-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-6a,7,8,9,10,12-10 hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1H-pyrrole-2- carboxamido)phenyl)-N-phenyl-1H-pyrrole-2-carboxamide (98) methyl-5-((4-(1-methyl-5-(phenylcarbamoyl)-1H-pyrrol-3-yl)phenyl)carbamoyl)-1H-pyrrol-3-15 yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12- octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-2-yl)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (97) (53 g, 0.043 mmol) in 1,4-dioxane (3 mL) was added an aqueous solution of sodium hydroxide (1 M, 3 mL, 3 mmol). The reaction mixture was stirred at room temperature for 18 h and then quenched with an aqueous solution of acetic acid (1 M, 3 mL, 3 mmol). The 20 resulting mixture was then concentrated in vacuo. The resulting residue was then suspended in dichloromethane (4 mL) and tetrakis(triphenylphosphine)palladium(0) (5 mg, 10 mol%), and pyrrolidine (8 µL, 0.08 mmol) were sequentially added. The reaction mixture was stirred at room temperature for 10 min, then concentrated in vacuo and subjected to high vacuum for 1 h. The resulting residue was purified by reverse-phase column chromatography (C18), eluting with 25 acetonitrile/water containing 0.1% formic acid (from 5% to 95%), to give the title compound (22 mg, 57%) as a cream solid. [α]_D ²⁴ = 93° (c 0.074, (CH₃)₂CO); ¹H NMR (400 MHz, DMSO- d₆) δ 9.86 – 9.77 (m, 3H), 8.01 (d, J = 5.7 Hz, 1H), 7.71 (t, J = 8.7 Hz, 4H), 7.52 – 7.42 (m, 4H), 7.39 (s, 1H), 7.31 (t, J = 7.4 Hz, 2H), 7.20 (s, 1H), 7.04 (t, J = 7.6 Hz, 1H), 6.95 (s, 1H), 6.82 (s, 1H), 4.89 (d, J = 6.2 Hz, 1H), 4.70 – 4.40 (m, 1H), 4.22 – 3.92 (m, 3H), 3.89 (s, 3H), 3.82 (s, 30 3H), 3.70 – 3.48 (m, 4H), 3.40 – 3.25 (m, 8H), 2.44 (dd, J = 11.6, 4.0 Hz, 2H), 2.07 – 1.97 (m, 3H), 1.84 – 1.54 (m, 5H); ¹³C NMR (100 MHz, DMSO-d6) δ 169.1, 166.2, 165.3, 159.7, 159.6, 150.7, 145.1, 140.9, 139.3, 137.1, 131.5, 131.4, 129.4, 128.7, 128.5, 126.1, 125.4, 124.4, 123.0, 122.7, 122.0, 121.9, 120.4, 119.9, 118.8, 110.4, 104.8, 76.9, 76.6, 73.3, 69.2, 64.9, 54.9, 49.2, DB1/ 159693888.5 194

133186-5030-WO 48.6, 36.5, 36.2, 31.8, 24.7, 23.7, 22.6, 17.7, 15.2; MS (ES+): m/z = 891 (M+H)⁺; LCMS (Method B): tR = 3.32 min; HRMS (ESI, m/z): calc. for C47H52N7O11⁺ ([M]+H)⁺ 890.3719 found 890.3753. Example 1I: Synthesis of compound (102). 5 Scheme 9. Synthesis of compound (102). , , trifluoroacetamido)phenyl)carbamoyl)-1H-pyrrol-3-yl)phenyl)carbamoyl)-1H-pyrrol-3- yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-2-((2-10 (trimethylsilyl)ethoxy)methoxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2- a][1,4]diazepine-5(12H)-carboxylate (99) o a so uton o -( -( -( -((( a )-5-((a yoxy)car ony )- -oxo- -((tetra y ro- -pyran- - yl)oxy)-2-((2-(trimethylsilyl)ethoxy)methoxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-15 a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrol-2-carboxamido)phenyl)-1-methyl- 1_{H-pyrrol-2-carboxylic acid (93) (144 mg, 0.15 mmol) in N,N-dimethylformamide (6 mL) were} added N,N-dimethylpyridin-4-amine (54 mg, 0.45 mmol) and N-(3-dimethylaminopropyl)-N′- ethylcarbodiimide hydrochloride (74 mg, 0.37 mmol). The reaction mixture was stirred for 30 min at room temperature. N-(4-Aminophenyl)-2,2,2-trifluoroacetamide (73) (33 mg, 0.16 20 mmol) was added and the solution was stirred for further 16 h. The reaction mixture was diluted into brine (60 mL). The aqueous phase was extracted with ethyl acetate (2 x 20 mL). The combined organic extracts were concentrated in vacuo. The resulting residue was purified by c_{olumn chromatography (silica), eluting with methanol/dichloromethane (from 0% to 10%) to} DB1/ 159693888.5 195

133186-5030-WO g_{ive the title compound (141 mg, 82%) as a brown oil. MS (ES+/-): m/z = 1156.0 (M+H)+,} 1153.5 (M-H)-; LCMS (Method B): tR = 4.53 min. Allyl (6aS)-2-hydroxy-3-(4-((1-methyl-5-((4-(1-methyl-5-((4-(2,2,2- trifluoroacetamido)phenyl)carbamoyl)-1H-pyrrol-3-yl)phenyl)carbamoyl)-1H-pyrrol-3- 5 yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10- hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (100) solution of allyl (6aS)-3-(4-((1-methyl-5-((4-(1-methyl-5-((4-(2,2,2-10 trifluoroacetamido)phenyl)carbamoyl)-1H-pyrrol-3-yl)phenyl)carbamoyl)-1H-pyrrol-3- yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-2-((2- (trimethylsilyl)ethoxy)methoxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine- 5(12H)-carboxylate (99) (141 mg, 0.12 mmol) in tetrahydrofuran (1 mL). The reaction mixture was heated to 80 ˚C for 7 h and then stirred at room temperature for 18 h. The reaction mixture 15 was diluted into brine (60 mL), acidified with acetic acid (0.2 mL), and extracted with ethyl acetate (2 x 40 mL). The combined organic extracts were concentrated in vacuo. The resulting residue was purified by column chromatography (silica), eluting with m_{ethanol/dichloromethane (from 0% to 10%) to give the title compound (72 mg, 58%) as a} brown oil. MS (ES+/-): m/z = 1025.7 (M+H)⁺, 1023.8 (M-H)-, LCMS (Method B): tR = 4.00 20 min. (2S,3R,4S,5S,6S)-2-(((6aS)-5-((Allyloxy)carbonyl)-3-(4-((1-methyl-5-((4-(1-methyl-5-((4- (2,2,2-trifluoroacetamido)phenyl)carbamoyl)-1H-pyrrol-3-yl)phenyl)carbamoyl)-1H- pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)- 5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-2-yl)oxy)-6- 25 (methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (101) A mixture of allyl (6aS)-2-hydroxy-3-(4-((1-methyl-5-((4-(1-methyl-5-((4-(2,2,2- trifluoroacetamido)phenyl)carbamoyl)-1H-pyrrol-3-yl)phenyl)carbamoyl)-1H-pyrrol-3- yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10- _{30 hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (100) (72 mg, 0.07 mmol),} DB1/ 159693888.5 196

133186-5030-WO (2R,3R,4S,5S,6S)-2-bromo-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (42 mg, 0.105 mmol) and potassium carbonate (29 mg, 0.21 mmol) in acetone (6 mL) was stirred at room temperature for 18 h. The reaction mixture was diluted with a saturated aqueous solution of sodium chloride (50 mL) and extracted with ethyl acetate (2 x 30 mL). The organic layer was 5 concentrated in vacuo. The resulting residue was purified by column chromatography (silica), eluting with methanol-dichloromethane (from 0% to 10%) to give the title compound (40 mg, 43%) as a cream oil. MS (ES+): m/z = 1341.9 (M+H)⁺, LCMS (Method B): t_R = 4.15 min. (2S,3S,4S,5R,6S)-6-(((S)-3-(4-((5-((4-(5-((4-aminophenyl)carbamoyl)-1-methyl-1H-pyrrol- 3-yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-10 6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-2-yl)oxy)-3,4,5- trihydroxytetrahydro-2H-pyran-2-carboxylic acid (102) To a solution of (2S,3R,4S,5S,6S)-2-(((6aS)-5-((allyloxy)carbonyl)-3-(4-((1-methyl-5-((4-(1- methyl-5-((4-(2,2,2-trifluoroacetamido)phenyl)carbamoyl)-1H-pyrrol-3-yl)phenyl)carbamoyl)-15 1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)- 5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-2-yl)oxy)-6- (methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (101) (40 g, 0.03 mmol) in 1,4- dioxane (3 mL) was added an aqueous solution of sodium hydroxide (1 M, 3 mL, 3 mmol). The reaction mixture was stirred at room temperature for 18 h and was then quenched with an 20 aqueous solution of acetic acid (2 M, 1.5 mL, 3 mmol). The resulting mixture was concentrated in vacuo and then suspended in dichloromethane (3 mL), to which tetrakis(triphenylphosphine)palladium(0) (3.5 mg, 10 mol%) and pyrrolidine (4 µL, 0.04 mmol) were added. The reaction mixture was stirred at room temperature for 3 h and then concentrated in vacuo. Purification by reverse-phase column chromatography (C18), eluting with 25 acetonitrile-water containing 0.1% formic acid (from 5% to 95%), gave the title compound (15 mg, 55%) as a cream solid. MS (ES+/-): m/z = 919.6 (M+H)⁺, 917.5 (M-H)-, LCMS (Method A): t_R = 5.47 min. DB1/ 159693888.5 197

133186-5030-WO Example 1J: Synthesis of compound (112). Scheme 10. Synthesis of compound (112). ( , , , , n- 5 3,4,5-triyl triacetate (105) sou on o ( , , , , )- -romo- -(me oxycar ony)era yro- -pyran- , ,-ry triacetate (103) (12.0 g, 30.2 mmol) and 4-hydroxy-3-nitrobenzaldehyde (104) (5.0 g, 30.0 mmol) in anhydrous acetonitrile (200 mL) was charged with silver(I) oxide (13.9 g, 60 mmol) 10 and the resulting suspension was stirred at room temperature under argon for 16 h. The solid was then filtered off, and washed with 50 mL of acetonitrile. The resulting combined acetonitrile solution was employed in the subsequent step without further intermediate purification. DB1/ 159693888.5 198

133186-5030-WO (2S,3R,4S,5S,6S)-2-(4-(Hydroxymethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro- 2H-pyran-3,4,5-triyl triacetate (106) 5 nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (105), was charged with isopropyl alcohol (20 mL) and sodium borohydride (1.14 g, 30 mmol) and the resulting mixture was stirred at room temperature for 30 min, whereupon it was quenched by the cautious addition of water (200 mL). After extraction with ethyl acetate (2 x 200 mL), the combined organic layers were dried over anhydrous sodium sulfate, then filtered and 10 concentrated in vacuo, to give the title compound as a tan solid, which was employed in the subsequent step without any further intermediate purification. (2S,3R,4S,5S,6S)-2-(4-(((tert-butyldimethylsilyl)oxy)methyl)-2-nitrophenoxy)-6- (methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (107) 15 so ut on o cru e ( , , ,5 , )- -( -( y roxymet y)- -n trop enoxy)- - (methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (106) in anhydrous dichloromethane (200 mL) was charged with imidazole (5.1 g, 75 mmol) and tert- butyldimethylsilyl chloride (6.8 g, 45 mmol). The resulting mixture was stirred at room temperature for 1 h, then quenched with water (200 mL). This was then extracted with 20 dichloromethane (2 x 200 mL) and the combined organic extracts were then dried over magnesium sulfate, filtered, and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with ethyl acetate/hexanes (from 10% to 30%), gave the title compound (13.7 g, 76% over three steps) as a white solid. DB1/ 159693888.5 199

133186-5030-WO (2S,3S,4S,5R,6S)-6-(4-(((tert-Butyldimethylsilyl)oxy)methyl)-2-nitrophenoxy)-3,4,5- trihydroxytetrahydro-2H-pyran-2-carboxylic acid (108) 5 (methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (107) (12.0 g, 20 mmol) in acetonitrile (200 mL) was charged with an aqueous solution of sodium hydroxide (1 N, 120 mL) and the mixture was then stirred at room temperature for 1 h. Hydrochloric acid (1 N, 120 mL) was then added and the mixture was concentrated in vacuo to give the crude title compound, that was used in the next step without further purification. 10 Allyl (2S,3S,4S,5R,6S)-6-(4-(((tert-butyldimethylsilyl)oxy)methyl)-2-nitrophenoxy)-3,4,5- trihydroxytetrahydro-2H-pyran-2-carboxylate (109) , , , , y y y y y nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (108) in N,N- 15 dimethylformamide (150 mL) was charged with caesium carbonate (6.5 g, 20 mmol) and allyl bromide (3.5 mL, 40 mmol) and the resulting mixture was stirred at room temperature for 16 h. Water (300 mL) was then added and the mixture extracted with ethyl acetate (3 x 200 mL). The combined organic extracts were then washed with brine (200 mL), dried over sodium sulfate, and concentrated in vacuo to give the crude title compound as a tan solid, which was employed 20 in the next step without any further purification. Allyl (2S,3S,4S,5R,6S)-3,4,5-tris(((allyloxy)carbonyl)oxy)-6-(4-(((tert- butyldimethylsilyl)oxy)methyl)-2-nitrophenoxy)tetrahydro-2H-pyran-2-carboxylate (110) A solution of crude allyl (2S,3S,4S,5R,6S)-6-(4-(((tert-butyldimethylsilyl)oxy)methyl)-2- 25 nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylate (109) in anhydrous pyridine DB1/ 159693888.5 200

133186-5030-WO (150 mL) was cooled to 0 °C and charged with allyl chloroformate (45 mL), dropwise. The resulting mixture was then stirred at room temperature for 18 h, then concentrated in vacuo. The residue was then re-solubilised in dichloromethane (500 mL) and washed with hydrochloric acid (1 N, 200 mL), and water (200 mL). The organic layer was then dried over magnesium 5 sulfate, filtered, and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with ethyl acetate/hexanes (from 0% to 20%), gave the title compound (8.9 g, 59% over three steps) as a colourless syrup. Allyl (2S,3S,4S,5R,6S)-3,4,5-tris(((allyloxy)carbonyl)oxy)-6-(4-(hydroxymethyl)-2- nitrophenoxy)tetrahydro-2H-pyran-2-carboxylate (111) 10 butyldimethylsilyl)oxy)methyl)-2-nitrophenoxy)tetrahydro-2H-pyran-2-carboxylate (110) (8.0 g, 10.6 mmol) in tetrahydrofuran (100 mL) was charged with triethylamine trihydrofluoride (4.8 mL, 30 mmol) and the resulting mixture was stirred at room temperature for 16 h. After diluting 15 into dichloromethane (300 mL), the mixture was then washed with a saturated aqueous solution of sodium hydrogen carbonate (150 mL) and water (200 mL). The organic layer was then dried over magnesium sulfate, filtered, and concentrated in vacuo to give the crude title compound, which was employed in the next step without further purification. Allyl (2S,3S,4S,5R,6S)-3,4,5-tris(((allyloxy)carbonyl)oxy)-6-(2-nitro-4- 20 ((((perfluorophenoxy)carbonyl)oxy)methyl)phenoxy)tetrahydro-2H-pyran-2-carboxylate (112) A solution of crude allyl (2S,3S,4S,5R,6S)-3,4,5-tris(((allyloxy)carbonyl)oxy)-6-(4- (hydroxymethyl)-2-nitrophenoxy)tetrahydro-2H-pyran-2-carboxylate (111) in anhydrous N,N- 25 dimethylformamide (50 mL) was charged with bis(pentafluorophenyl) carbonate (8.4 g, 21.1 mmol) and N,N-diisopropylethylamine (0.35 mL, 2.0 mmol). The resulting mixture was stirred at room temperature for 1 h, then diluted into ethyl acetate (300 mL). This was then washed with hydrochloric acid (0.5 N, 200 mL) and water (200 mL), dried over sodium sulfate, filtered, DB1/ 159693888.5 201

133186-5030-WO and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with ethyl acetate/hexanes (10% to 30%) gave the title compound (7.7 g, 86% over two steps) as a colourless syrup. Example 1K: Synthesis of compound (127). 5 Scheme 11. Synthesis of compound (127). 4-(Benzyloxy)-3-methoxybenzaldehyde (114) A _{mixture of compound vanillin (113) (200 g, 1.31 mol), benzyl bromide (236 g, 1.38 mol) and} 10 potassium carbonate (545 g, 3.94 mol) in methanol (1.20 L) was refluxed for 5 h. The reaction DB1/ 159693888.5 202

133186-5030-WO mixture was filtered, and the filtrate evaporated under reduced pressure to afford the title compound (271 g, 85%) as a pale yellow solid. ¹H NMR (400 MHz, CDCl3) δ 9.83 (s, 1H), 7.47-7.35 (m, 6H), 7.33 (d, J=7.2 Hz, 1H), 6.98 (d, J=8.2 Hz, 1H), 5.24 (s, 2H), 3.94 (s, 3H); 1³C NMR (100 MHz, CDCl3) δ 191.0, 153.6, 150.1, 136.0, 130.3, 128.7, 128.2, 127.2, 126.6, 5 112.3, 109.3, 70.9, 56.1; MS (ES+): m/z = 243 (M+H)⁺; LCMS (Method A): t_R = 7.53 min. 4-(Benzyloxy)-5-methoxy-2-nitrobenzaldehyde (115) cetic acid (600 mL) was charged with a solution of potassium nitrate (65 g, 644 mmol), in 10 trifluoroacetic acid (600 mL) dropwise at 0°C. The reaction mixture was stirred for 1 h and then diluted into water (2.40 L). The resulting precipitate was filtered and washed with cold water (500 mL × 2) to afford the title compound (125 g, 81%) as a yellow solid. ¹H NMR (400 MHz, CDCl3) δ 10.43 (s, 1H), 7.67 (s, 1H), 7.46-7.30 (m, 6H), 5.27 (s, 2H), 4.02 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 187.8, 153.7, 151.4, 134.85, 129.0, 128.9, 128.7, 127.6, 125.7, 110.0, 15 108.9, 71.6, 56.7; MS (ES-): m/z = 286 (M-H) -; LCMS (Method A): t_R = 7.87 min. 4-Hydroxy-5-methoxy-2-nitrobenzaldehyde (116) A solution of 4-(benzyloxy)-5-methoxy-2-nitrobenzaldehyde (115) (100 g, 348 mmol) in glacial acetic acid (800 mL) was charged with an aqueous solution of hydrobromic acid (48% v/v, 88.0 20 mL, 522 mmol) and heated to 85 °C, with stirring for 1 h, after which the reaction was judged to be complete by TLC. After allowing the resulting mixture to cool to room temperature, it was then diluted in water (1.6 L), and the resulting precipitate filtered, and washed with cold water (100 mL x 3) to give the title compound (50.0 g, 73%) as a yellow solid, which was used immediately in the subsequent step without further purification. ¹H NMR (400 MHz, DMSO- 25 d6) δ 11.11 (br s, 1 H), 10.15 (br s, 1 H), 7.50 (s, 1 H), 7.35 (s, 1 H), 3.94 (s, 3 H); MS (ES-): m/z = 196 (M-H)-; LCMS (Method B): tR = 2.55 min. DB1/ 159693888.5 203

133186-5030-WO 5-Methoxy-2-nitro-4-((triisopropylsilyl)oxy)benzaldehyde (117) triisopropylsilyl chloride (59.7 mL, 279 mmol) and imidazole (51.8 g, 761 mmol) was heated 5 and stirred at 100 °C for 30 min. The reaction mixture was poured onto ice-water and extracted with ethyl acetate (500 mL × 3). The combined organic extracts were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60 °C (5%) to give the title compound (57.5 g, 64%) as a yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 10.42 10 (s, 1 H), 7.59 (s, 1 H), 7.40 (s, 1 H), 3.95 (s, 3 H), 1.33-1.24 (m, 3 H), 1.07 (s, 18 H). 5-Methoxy-2-nitro-4-((triisopropylsilyl)oxy)benzoic acid (118) so u o o so u c o e , . g, o a so u p osp a e o o as c dihydrate (35.5 g, 228 mmol) in water (200 mL) was added to a solution of 5-methoxy-2-nitro- _{15 4-((triisopropylsilyl)oxy)benzaldehyde (117) (57.5 g, 163 mmol) in tetrahydrofuran (800 mL) at} room temperature. Hydrogen peroxide (30% w/w, 235 mL, 2.28 mol) was added to the vigorously stirred biphasic mixture. The starting material dissolved, and the temperature of the reaction mixture rose to 45 °C. After 30 min, the reaction was judged to be complete by TLC. The mixture was subsequently acidified to pH = 3-4 with citric acid and extracted with ethyl 20 acetate (500 mL × 3). The combined organic extracts were washed with water (150 mL) and brine (150 mL), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was then purified by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60 °C (10%) then methanol/dichloromethane (10%) to afford the title compound (38.0 g, 63%) as a yellow oil. ¹H NMR (400 MHz, CDCl3) δ 9.81 (s, 1H), 7.35 25 (s, 1H), 7.25 (s, 1H), 3.91 (s, 3H), 1.26 (q, J=7.4 Hz, 3H), 1.09 (d, J=7.4 Hz, 18H); MS (ES–): m/z = 368 (M-H) -; LCMS (Method D): tR = 4.75 min. DB1/ 159693888.5 204

133186-5030-WO (S)-(2-(Hydroxymethyl)piperidin-1-yl)(5-methoxy-2-nitro-4 ((triisopropylsilyl)oxy)phenyl)methanone (119) 5 mmol), HATU (31.7 g, 83.4 mmol) and dry triethylamine (44 mL) in dry dichloromethane (300 mL) was stirred at room temperature for 30 min. (S)-Piperidin-2-ylmethanol (11.4 g, 98.5 mmol) was added and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was then partitioned between dichloromethane (500 mL × 2) and water (100 mL). The combined organic extracts were then dried over sodium sulfate, filtered, and concentrated in 10 vacuo. The resulting residue was purified by column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60 °C (from 50% to 75%), to give the title compound (20.0 g, 57%) as a yellow solid. ¹H NMR (400 MHz, CDCl3) mixture of rotamers, δ 7.68-7.65 (m, 1H), 7.03- 6.65 (m, 1H), 5.04-4.69 (m, 1H), 4.12-4.05 (m, 0.4H), 4.01-3.95 (m, 0.5H), 3.92-3.89 (m, 2.6H), 3.83-3.74 (m, 1.5H), 3.64-3.59 (m, 0.4H), 3.45-3.40 (m, 0.3H), 3.21-3.01 (m, 1.4H), 15 2.87-2.79 (m, 0.4H), 1.97-1.94 (m, 0.6H), 1.88-1.77 (m, 0.6H), 1.73-1.62 (m, 3H), 1.56-1.44 (m, 2H), 1.29-1.24 (m, 3H), 1.09 (d, J=7.3 Hz, 18H); MS (ES+): m/z = 467 (M+H)⁺; LCMS (Method B): tR = 4.75 min. (S)-(2-Amino-5-methoxy-4-((triisopropylsilyl)oxy)phenyl)(2-(hydroxymethyl)piperidin-1- yl)methanone (120) 20 A solution of (S)-(2-(hydroxymethyl)piperidin-1-yl)(5-methoxy-2-nitro-4 ((triisopropylsilyl)oxy)phenyl)methanone (119) (1.00 g, 2.14 mmol) in tetrahydrofuran (5 mL) was charged with palladium on activated charcoal (10 wt. % basis, 100 mg), ammonium formate (1.10 g, 17.1 mmol) and water (1 mL), and stirred at room temperature, under argon, 25 for 2 h. The resulting mixture was filtered through celite, the filter cake was washed with ethyl acetate (50 mL) and water (50 mL), and the filtrate separated. The organic phase was then extracted with brine (50 mL x 2), and dried over magnesium sulfate, filtered, and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with ethyl DB1/ 159693888.5 205

133186-5030-WO acetate/petroleum spirit, 40-60 °C (from 50% to 67%), gave the title compound (892 mg, 95%) as a yellow solid. ¹H NMR (400 MHz, CDCl3) δ 6.67 (s, 1H), 6.30 (s, 1H), 4.00-3.81 (m, 4H), 3.72 (s, 3H), 3.57 (s, 1H), 3.08 (s, 1H), 1.68-1.64 (m, 4H), 1.57-1.43 (m, 2H), 1.28-1.17 (m, 3H), 1.08 (d, J=7.4 Hz, 18H); ¹³C NMR (100 MHz, CDCl3) δ 171.8, 147.9, 143.7, 133.2, 112.5, 5 110.0, 109.5, 68.7, 61.0, 56.4, 30.9, 25.8, 19.9, 17.9, 12.9; MS (ES+): m/z = 437 (M+H)⁺; LCMS (Method C): t_R = 4.07 min. tert-Butyl (S)-(2-(2-(hydroxymethyl)piperidine-1-carbonyl)-4-methoxy-5- ((triisopropylsilyl)oxy)phenyl)carbamate (121) 10 (_{hydroxymethyl)piperidin-1-yl)methanone (120) (5.0 g, 11.5 mmol) in anhydrous} dichloromethane (100 mL) was charged with di-tert-butyl dicarbonate (3.7 g, 17.0 mmol) and triethylamine (0.5 mL, 3.56 mmol) and the resulting mixture was stirred at room temperature for 3 days. This resulting mixture was employed in the subsequent step without any intermediate 15 purification. tert-Butyl (S)-(5-hydroxy-2-(2-(hydroxymethyl)piperidine-1-carbonyl)-4- methoxyphenyl)carbamate (122) The dichloromethane solution from the previous step, containing tert-Butyl (S)-(2-(2- 20 (hydroxymethyl)piperidine-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamate (121), was charged with a solution of triethylamine trihydrofluoride (1.85 g, 11.5 mmol) in dichloromethane (20 mL) and stirred for 70 min. The resulting mixture was then washed with hydrochloric acid (0.5 M, 40 mL), then water (40 mL), and brine (40 mL). The organic phase was then dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The resulting 25 oil was then re-dissolved in diethyl ether (30 mL) and precipitated by addition to hexanes (120 mL). The precipitate was filtered and dried under strong vacuum, to give the crude title DB1/ 159693888.5 206

133186-5030-WO compound (4.4 g) as a tan solid, that was employed in the subsequent step without further purification. 2-(Trimethylsilyl)ethyl (S)-4-(5-((tert-butoxycarbonyl)amino)-4-(2- (hydroxymethyl)piperidine-1-carbonyl)-2-methoxyphenoxy)butanoate (123) 5 methoxyphenyl)carbamate (122) (2.3 g, 6.1 mmol) in anhydrous N,N-dimethylformamide (20 mL) was charged with 2-(trimethylsilyl)ethyl 4-bromobutanoate (2.3 g, 8.5 mmol) and caesium carbonate (5.9 g, 18.2 mmol) and the resulting mixture was stirred at 50 °C for 3 h. Purification 10 by reverse phase preparative HPLC (gradient: 25-95% acetonitrile over 20 min; compound eluted at 20 min) then gave the title compound (2.0 g, 58%) as a tan solid. 2-(Trimethylsilyl)ethyl (S)-4-(5-amino-4-(2-(hydroxymethyl)piperidine-1-carbonyl)-2- methoxyphenoxy)butanoate trifluoroacetate (124) 15 m xture o -(tr met y s y )et y (S)- -(5-((tert-butoxycarbony)am no)- -( - (hydroxymethyl)piperidine-1-carbonyl)-2-methoxyphenoxy)butanoate (123) (2.0 g, 3.5 mmol) in acetonitrile (6 mL) and trifluoroacetic acid (6 mL) was stirred at room temperature for 20 min. Purification by reverse phase preparative HPLC (gradient: 10-75% acetonitrile over 20 min; compound eluted at 18 min), gave the title compound (0.97 g, 47%) as the TFA salt.20 Allyl (2S,3S,4S,5R,6S)-3,4,5-tris(((allyloxy)carbonyl)oxy)-6-(4-((((2-((S)-2- (hydroxymethyl)piperidine-1-carbonyl)-4-methoxy-5-(4-oxo-4-(2- DB1/ 159693888.5 207

133186-5030-WO (trimethylsilyl)ethoxy)butoxy)phenyl)carbamoyl)oxy)methyl)-2-nitrophenoxy)tetrahydro- 2H-pyran-2-carboxylate (125) 5 carbonyl)-2-methoxyphenoxy)butanoate trifluoroacetate (124) (0.97 g, 1.7 mmol) in anhydrous N,N-dimethylformamide (10 mL) was charged with allyl (2S,3S,4S,5R,6S)-3,4,5- tris(((allyloxy)carbonyl)oxy)-6-(2-nitro-4- ((((perfluorophenoxy)carbonyl)oxy)methyl)phenoxy)tetrahydro-2H-pyran-2-carboxylate (112) (1.63 g, 1.9 mmol), 1-hydroxy-7-azabenzotriazole (46 mg, 0.34 mmol) and N,N- 10 diisopropylethylamine (0.67 mL, 3.8 mmol). The resulting mixture was stirred at room temperature for 2 h. Purification by reverse phase preparative HPLC (gradient: 35-95% acetonitrile over 20 min; compound eluted at 21 min), gave the title compound (1.44 g, 61%) as a tan solid. 4-(((2S,3R,4S,5S,6S)-6-((Allyloxy)carbonyl)-3,4,5-tris(((allyloxy)carbonyl)oxy)tetrahydro-15 2H-pyran-2-yl)oxy)-3-nitrobenzyl (6aS)-6-hydroxy-2-methoxy-12-oxo-3-(4-oxo-4-(2- (trimethylsilyl)ethoxy)butoxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2- a][1,4]diazepine-5(12H)-carboxylate (126) A solution of allyl (2S,3S,4S,5R,6S)-3,4,5-tris(((allyloxy)carbonyl)oxy)-6-(4-((((2-((S)-2-20 (hydroxymethyl)piperidine-1-carbonyl)-4-methoxy-5-(4-oxo-4-(2- (trimethylsilyl)ethoxy)butoxy)phenyl)carbamoyl)oxy)methyl)-2-nitrophenoxy)tetrahydro-2H- pyran-2-carboxylate (125) (386 mg, 0.34 mmol) in dichloromethane (22 mL) was charged with (diacetoxyiodo)benzene (275 mg, 0.85 mmol) and TEMPO (5.3 mg, 34 µmol) and the resulting DB1/ 159693888.5 208

133186-5030-WO mixture was stirred at room temperature for 6 days. After concentrating in vacuo, the resulting residue was re-solubilised in acetonitrile (4 mL) and water (0.5 mL) and then purified by reverse phase preparative HPLC (gradient: 25-95% acetonitrile over 20 min; compound eluted at 22 min), to give the title compound (239 mg, 62%) as a tan solid. 5 4-(((6aS)-5-(((4-(((2S,3R,4S,5S,6S)-6-((Allyloxy)carbonyl)-3,4,5- tris(((allyloxy)carbonyl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-3-nitrobenzyl)oxy)carbonyl)- 6-hydroxy-2-methoxy-12-oxo-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2- a][1,4]diazepin-3-yl)oxy)butanoic acid (127) 10 tris(((allyloxy)carbonyl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-3-nitrobenzyl (6aS)-6-hydroxy-2- methoxy-12-oxo-3-(4-oxo-4-(2-(trimethylsilyl)ethoxy)butoxy)-6,6a,7,8,9,10- hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (126) (239 mg, 0.21 mmol) in dichloromethane (3 mL) was charged with trifluoroacetic acid (2 mL) and the resulting 15 mixture was stirred at room temperature for 40 min. Acetonitrile (2 mL) was then added, and the mixture concentrated to a volume of approximately 3 mL, then purified by reverse phase preparative HPLC (gradient: 20-80% acetonitrile over 20 min; compound eluted at 21 min), to give the title compound (132 mg, 61%) as a white solid. Example 1L: Synthesis of compound (131). 20 Scheme 12. Synthesis of compound (131). DB1/ 159693888.5 209

133186-5030-WO tert-Butyl (5-((4-aminophenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)carbamate (128) g, 4.16 mmol) in dichloromethane (8 mL) was charged with triethylamine (2.4 mL, 17.5 mmol) 5 and HATU (1.66 g, 4.37 mmol) and then stirred at room temperature for 5 min before adding 1,4-diaminobenzene (450 mg, 4.16 mmol) and then stirring for 16 h. The resulting mixture was diluted into dichloromethane (100 mL), then washed with a saturated aqueous solution of sodium hydrogen carbonate (50 mL) and brine (50 mL) and then dried over magnesium sulfate, before filtering and concentrating in vacuo to give the title compound (1.38 g, crude) as a beige 10 solid, which was used in the subsequent step without further purification. MS (ES+): m/z = 331 (M+H)⁺; LCMS (Method A): t_R = 5.28 min. tert-Butyl (S)-(5-((4-(2-aminopropanamido)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3- yl)carbamate (129) 15 so u o o e - u y - -a op e y ca a oy - - e y - -py o- -y ca a a e (128) (31 mg, 0.093 mmol) in anhydrous N,N-dimethylformamide (2 mL) was charged with (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine (29 mg, 0.093 mmol), N,N- diisopropylethylamine (16 µL, 93 mmol) and PyAOP (49 mg, 0.093 mmol). The resulting mixture was stirred at room temperature for 2 h, upon which DBU (70 mg, 0.46 mmol) was 20 added and stirred for another 10 min to afford the title compound, which was used in the next step without further purification. Allyl ((S)-1-(((S)-1-((4-(4-((tert-butoxycarbonyl)amino)-1-methyl-1H-pyrrole-2- carboxamido)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2- yl)carbamate (130) 25 DB1/ 159693888.5 210

133186-5030-WO The solution from the previous step containing tert-butyl (S)-(5-((4-(2- aminopropanamido)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)carbamate (129) was charged with trifluoroacetic acid (43 µL), ((allyloxy)carbonyl)-L-valine (19 mg, 0.093 mmol), N,N- diisopropylethylamine (16 µL, 0.093 mmol) and PyAOP (49 mg, 0.093 mmol), and the resulting 5 mixture was stirred at room temperature for 10 min. Ethyl acetate (60 mL) was added to this mixture and the resulting solution was washed with water (40 mL), then hydrochloric acid (1 N, 40 mL), and finally with brine (40 mL). The organic phase was dried over sodium sulfate, filtered, and concentrated in vacuo, to give the crude title compound, which was employed in the subsequent step without further purification. 10 Allyl ((S)-1-(((S)-1-((4-(4-amino-1-methyl-1H-pyrrole-2-carboxamido)phenyl)amino)-1- oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate trifluoroacetate (131) carboxamido)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate 15 (130, residue from previous step) in dichloromethane (3 mL) and trifluoroacetic acid (2 mL) was stirred at room temperature for 10 min. The mixture was then concentrated to 2 mL and reduced pressure, and then purified directly by reverse phase preparative HPLC (gradient: 5- 60% acetonitrile over 20 min; compound eluted at 17 min), to give the title compound (31 mg, 55% over three steps) as a white solid. DB1/ 159693888.5 211

133186-5030-WO Example 1M: Synthesis of compound (134). Scheme 13. Synthesis of compound (134). 4-(((2S,3R,4S,5S,6S)-6-((Allyloxy)carbonyl)-3,4,5-tris(((allyloxy)carbonyl)oxy)tetrahydro- 5 2H-pyran-2-yl)oxy)-3-nitrobenzyl (6aS)-3-(4-((5-((4-((S)-2-((S)-2- (((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)phenyl)carbamoyl)-1- methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-6-hydroxy-2-methoxy-12-oxo-6,6a,7,8,9,10- hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (132) 10 A solution of 4-(((6aS)-5-(((4-(((2S,3R,4S,5S,6S)-6-((allyloxy)carbonyl)-3,4,5- tris(((allyloxy)carbonyl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-3-nitrobenzyl)oxy)carbonyl)-6- hydroxy-2-methoxy-12-oxo-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3- yl)oxy)butanoic acid (127) (23 mg, 22.4 µmol) in anhydrous N,N-dimethylformamide (1 mL) was charged with allyl ((S)-1-(((S)-1-((4-(4-amino-1-methyl-1H-pyrrole-2- 15 carboxamido)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate trifluoroacetate (131) (13.4 mg, 22.4 µmol), N,N-diisopropylethylamine (15.6 µL, 90 µmol) and PyAOP (11.7 mg, 22.4 µmol) and the resulting mixture was stirred at room temperature for 10 min. This was then directly purified by reverse phase preparative HPLC (gradient: 20-80% acetonitrile over 20 min; compound eluted at 22 min), to give the title compound (30 mg, 91%) 20 as a tan solid. DB1/ 159693888.5 212

133186-5030-WO (2S,3S,4S,5R,6S)-6-(4-((((6aS)-3-(4-((5-((4-((S)-2-((S)-2-Amino-3- methylbutanamido)propanamido)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4- oxobutoxy)-6-hydroxy-2-methoxy-12-oxo-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2- a][1,4]diazepine-5-carbonyl)oxy)methyl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H- 5 pyran-2-carboxylic acid trifluoroacetate (133) tris(((allyloxy)carbonyl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-3-nitrobenzyl (6aS)-3-(4-((5-((4- ((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-10 methylbutanamido)propanamido)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4- oxobutoxy)-6-hydroxy-2-methoxy-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2- a][1,4]diazepine-5(12H)-carboxylate (132) (23 mg, 16 µmol) in dichloromethane (1 mL) and N,N-dimethylformamide (1 mL) was charged with formic acid (9 µL, 239 µmol), pyrrolidine (19 µL, 239 µmol) and tetrakis(triphenylphosphine)palladium(0) (3.6 mg, 3.1 µmol) and the 15 resulting mixture was stirred at room temperature for 10 min, before removing the dichloromethane under reduced pressure. After re-solubilising in acetonitrile (1 mL) and water (0.5 mL), the residue was purified by reverse phase preparative HPLC (gradient: 5-45% acetonitrile over 20 min; compound eluted at 18 min), to give the title compound (18 mg, 90%) as the TFA salt. 20 (2S,3S,4S,5R,6S)-6-(4-((((6aS)-3-(4-((5-((4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)phenyl)carbamoyl)-1- methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-6-hydroxy-2-methoxy-12-oxo- 5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5-carbonyl)oxy)methyl)-2- nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (134) 25 A solution of (2S,3S,4S,5R,6S)-6-(4-((((6aS)-3-(4-((5-((4-((S)-2-((S)-2-Amino-3- methylbutanamido)propanamido)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4- oxobutoxy)-6-hydroxy-2-methoxy-12-oxo-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2- a][1,4]diazepine-5-carbonyl)oxy)methyl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H- 30 pyran-2-carboxylic acid trifluoroacetate (133) (17.6 mg, 14.3 µmol) in N,N-dimethylformamide DB1/ 159693888.5 213

133186-5030-WO (2 mL) was charged with perfluorophenyl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (6.5 mg, 17.2 µmol) and N,N-diisopropylethylamine (12 µL, 69 µmol) and the resulting mixture was stirred at room temperature for 1 h. Purification by reverse phase preparative HPLC (gradient: 10-70% acetonitrile over 20 min; compound eluted at 17 min), gave the title 5 compound (17.6 mg, 94%) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 12.84 (s, 1H), 9.89 (s, 1H), 9.83 (s, 1H), 9.75 (s, 1H), 8.12 (d, J=8.0 Hz, 1H), 7.81 (d, J=8.0 Hz, 1H), 7.74 (s, 1H), 7.61 (d, J=8.0 Hz, 2H), 7.51 (d, J=8.0 Hz, 3H), 7.40 (d, J=8.0 Hz, 1H), 7.22 (s, 1H), 7.03 (s, 1H), 7.00 (s, 2H), 6.93 (s, 1H), 6.78 (s, 1H), 6.52 (br s, 1H), 5.78 (d, J=8.0 Hz, 1H), 5.29 (d, J=4.0 Hz, 2H), 5.15 (d, J=8.0 Hz, 1H), 4.94 (d, J=8.0 Hz, 1H), 4.38 (t, J=6.0 Hz, 1H), 4.18-4.10 10 (m, 2H), 3.98 (d, J=8.0 Hz, 4H), 3.81 (d, J=4.0 Hz, 7H), 3.38-3.25 (m, 5H), 2.88 (br, 1H), 2.45 (br, 2H), 2.20-2.13 (m, 2H), 2.04-2.03 (m, 2H), 1.96 (q, J=4.0 Hz, 1H), 1.86 (br, 1H), 1.67-1.63 (m, 2H), 1.55-1.45 (m, 8H), 1.30 (d, J=8 Hz, 3H), 1.20 (t, J=6.0 Hz, 2H), 0.87 (d, J=4.0 Hz, 3H), 0.83 (d, J=4.0 Hz, 3H); MS (ES+): m/z = 1312.8 (M+H)⁺; LCMS (5 min): t_R = 2.13 min; HPLC (15 min): 6.05 min (100% purity, 220 nm). 15 Example 1N: Synthesis of compound (138). Scheme 14. Synthesis of compound (138). Allyl (4-(4-((tert-butoxycarbonyl)amino)-1-methyl-1H-pyrrole-2- carboxamido)phenyl)carbamate (135) 20 A solution of tert-butyl (5-((4-aminophenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)carbamate (128) (1.38 g, 4.16 mmol) in dichloromethane (8 mL) was cooled to 0 °C and charged with pyridine (777 µL, 9.60 mmol) and allyl chloroformate (488 µL, 4.59 mmol), and the resulting DB1/ 159693888.5 214

133186-5030-WO mixture was stirred at room temperature for 2 h. After diluting into dichloromethane (100 mL), the reaction mixture was then washed with a saturated aqueous solution of copper sulfate (2 x 50 mL), a saturated aqueous solution of sodium hydrogen carbonate (2 x 50 mL), and brine (50 mL), before drying over magnesium sulfate, filtering, and concentrating in vacuo. Purification 5 by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60 °C (50%), gave the title compound (469 mg, 27% over two steps) as a yellow solid. MS (ES+): m/z = 415 (M+H)⁺; LCMS (Method A): t_R = 7.07 min. Allyl (4-(4-amino-1-methyl-1H-pyrrole-2-carboxamido)phenyl)carbamate hydrochloride (136) 10 pyrrol-3-yl)carbamate (135) (469 mg, 1.13 mmol) in HCl (4 M in 1,4-dioxane) (5 mL) was stirred at room temperature for 2 h and then precipitated in diethyl ether. The resulting solid was filtered under reduced pressure and washed with diethyl ether. Strong vacuum was then applied 15 for 1 h, to give the title compound (250 mg, 63%) as a green solid, which was employed in the subsequent step without further purification. MS (ES+): m/z = 315 (M+H)⁺; LCMS (Method A): t_R = 5.05 min. 4-(((2S,3R,4S,5S,6S)-6-((Allyloxy)carbonyl)-3,4,5-tris(((allyloxy)carbonyl)oxy)tetrahydro- 2H-pyran-2-yl)oxy)-3-nitrobenzyl (6aS)-3-(4-((5-((4-20 (((allyloxy)carbonyl)amino)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4- oxobutoxy)-6-hydroxy-2-methoxy-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2- a][1,4]diazepine-5(12H)-carboxylate (137) A solution of 4-(((6aS)-5-(((4-(((2S,3R,4S,5S,6S)-6-((allyloxy)carbonyl)-3,4,5-25 tris(((allyloxy)carbonyl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-3-nitrobenzyl)oxy)carbonyl)-6- hydroxy-2-methoxy-12-oxo-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3- yl)oxy)butanoic acid (127) (10 mg, 9.7 µmol) in anhydrous N,N-dimethylformamide (1 mL) was charged with allyl (4-(4-amino-1-methyl-1H-pyrrole-2-carboxamido)phenyl)carbamate hydrochloride (136) (3.4 mg, 9.7 µmol), N,N-diisopropylethylamine (6.8 µL, 39 µmol) and DB1/ 159693888.5 215

133186-5030-WO PyAOP (5.1 mg, 9.7 µmol), and the resulting mixture was stirred at room temperature for 10 min. Purification by reverse phase preparative HPLC (gradient: 30-90% acetonitrile over 20 min; compound eluted at 19.5 min), gave the title compound (11 mg, 86%) as a tan solid. (2S,3S,4S,5R,6S)-6-(4-((((6aS)-3-(4-((5-((4-Aminophenyl)carbamoyl)-1-methyl-1H-pyrrol- 5 3-yl)amino)-4-oxobutoxy)-6-hydroxy-2-methoxy-12-oxo-5,6,6a,7,8,9,10,12- octahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5-carbonyl)oxy)methyl)-2-nitrophenoxy)- 3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (138) 10 tris(((allyloxy)carbonyl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-3-nitrobenzyl (6aS)-3-(4-((5-((4- (((allyloxy)carbonyl)amino)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)- 6-hydroxy-2-methoxy-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine- 5(12H)-carboxylate (137) (11 mg, 8.2 µmol) in dichloromethane (1 mL) and N,N- dimethylformamide (1 mL) was charged with formic acid (4.3 µL, 114 µmol), pyrrolidine (10.2 15 µL, 123 µmol) and tetrakis(triphenylphosphine)palladium(0) (3.6 mg, 3.1 µmol). The resulting mixture was stirred at room temperature for 10 min before removing the dichloromethane in vacuo. Acetonitrile (1 mL) and water (0.5 mL) were then added, and purification by reverse phase preparative HPLC gave the title compound (6.1 mg, 70%) as the TFA salt. MS (ES+): m/z = 948.5 (M+H)⁺; LCMS (5 min): tR = 1.45 min; HPLC (15 min): 7.54 min (100% purity, 20 220 nm). Example 1O: Synthesis of compound (141). DB1/ 159693888.5 216

133186-5030-WO Scheme 15. Synthesis of compound (141). tert-Butyl (S)-(5-((4-(5-((4-(2-aminopropanamido)phenyl)carbamoyl)-1-methyl-1H-pyrrol- 3-yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)carbamate (139) 5 yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)carbamate (31) (49.2 mg, 93 µmol) in anhydrous N,N-dimethylformamide (2 mL) was charged with (((9H-fluoren-9- yl)methoxy)carbonyl)-L-alanine (29 mg, 93 µmol), N,N-diisopropylethylamine (65 µL, 370 10 µmol) and HATU (35 mg, 93 µmol) and the resulting mixture was stirred at room temperature for 2 h. DBU (70 mg, 460 µmol) was then added to this solution, which was then stirred for 10 min to give the title compound, which was carried through to the next step without further purification. tert-Butyl (5-((4-(5-((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-15 methylbutanamido)propanamido)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3- yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)carbamate (140) To the solution from the previous step containing tert-butyl (S)-(5-((4-(5-((4-(2- aminopropanamido)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)phenyl)carbamoyl)-1-methyl-20 1H-pyrrol-3-yl)carbamate (139), was added trifluoroacetic acid (43 µL), ((allyloxy)carbonyl)-L- valine (19 mg, 93 µmol), N,N-diisopropylethylamine (16 µL, 93 µmol) and PyAOP (49 mg, 93 DB1/ 159693888.5 217

133186-5030-WO µmol). The resulting mixture was then stirred at room temperature for 10 min. Ethyl acetate (60 mL) was then added and the resulting solution was washed with water (40 mL), followed by hydrochloric acid (1 M, 40 mL), and then brine (40 mL). The organic phase was dried over sodium sulfate, filtered, and concentrated in vacuo, to give the crude title compound, which was 5 carried through to the next step without any further purification. Allyl ((S)-1-(((S)-1-((4-(4-(4-(4-amino-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1- methyl-1H-pyrrole-2-carboxamido)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1- oxobutan-2-yl)carbamate trifluoroacetate (141) 10 methylbutanamido)propanamido)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3- yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)carbamate (140, residue from previous step) in dichloromethane (3 mL) was charged with trifluoroacetic acid (2 mL) and the resulting solution was stirred at room temperature for 10 min. After concentrating under reduced pressure to a 15 volume of 2 mL, the mixture was purified by reverse phase preparative HPLC (gradient: 5-60% acetonitrile over 20 min; compound eluted at 17.5 min), to give the title compound (30 mg, 41% over three steps) as a white solid. DB1/ 159693888.5 218

133186-5030-WO Example 1P: Synthesis of compound (144). Scheme 16. Synthesis of compound (144). 4-(((2S,3R,4S,5S,6S)-6-((Allyloxy)carbonyl)-3,4,5-tris(((allyloxy)carbonyl)oxy)tetrahydro- 5 2H-pyran-2-yl)oxy)-3-nitrobenzyl (6aS)-3-(4-((5-((4-(5-((4-((S)-2-((S)-2- (((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)phenyl)carbamoyl)-1- methyl-1H-pyrrol-3-yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)- 6-hydroxy-2-methoxy-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine- 5(12H)-carboxylate (142) 10 A solution of 4-(((6aS)-5-(((4-(((2S,3R,4S,5S,6S)-6-((allyloxy)carbonyl)-3,4,5- tris(((allyloxy)carbonyl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-3-nitrobenzyl)oxy)carbonyl)-6- hydroxy-2-methoxy-12-oxo-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3- yl)oxy)butanoic acid (127) (30 mg, 29.2 µmol) in anhydrous N,N-dimethylformamide (1 mL)15 was charged with allyl ((S)-1-(((S)-1-((4-(4-(4-(4-amino-1-methyl-1H-pyrrole-2- carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)amino)-1-oxopropan-2- yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate trifluoroacetate (141) (23.3 mg, 29.2 µmol), DB1/ 159693888.5 219

133186-5030-WO N,N-diisopropylethylamine (20.4 µL, 117 µmol) and PyAOP (15.2 mg, 29.2 µmol), and the resulting mixture was stirred at room temperature for 10 min, before directly purifying by reverse phase preparative HPLC (gradient: 30-90% acetonitrile over 20 min; compound eluted at 20 min), to give the title compound (37 mg, 76%) as a tan solid. 5 (2S,3S,4S,5R,6S)-6-(4-((((6aS)-3-(4-((5-((4-(5-((4-((S)-2-((S)-2-Amino-3- methylbutanamido)propanamido)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3- yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-6-hydroxy-2- methoxy-12-oxo-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5- carbonyl)oxy)methyl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic 10 acid (143) tris(((allyloxy)carbonyl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-3-nitrobenzyl (6aS)-3-(4-((5-((4-(5- ((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-15 methylbutanamido)propanamido)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3- yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-6-hydroxy-2-methoxy- 12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (142) (37.2 mg, 22 µmol) in dichloromethane (1 mL) and N,N-dimethylformamide (1 mL) was charged with formic acid (9 µL, 239 µmol), pyrrolidine (19 µL, 239 µmol) and 20 tetrakis(triphenylphosphine)palladium(0) (3.6 mg, 3.1 µmol) and the resulting mixture was stirred at room temperature for 10 min. The dichloromethane was removed under reduced pressure and purification by reverse phase preparative HPLC (gradient: 5-55% acetonitrile over 20 min; compound eluted at 18.5 min), to give the title compound (18 mg, 58%) as the TFA salt. 25 (2S,3S,4S,5R,6S)-6-(4-((((6aS)-3-(4-((5-((4-(5-((4-((S)-2-((S)-2-(6-(2,5-Dioxo-2,5-dihydro- 1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)phenyl)carbamoyl)-1- methyl-1H-pyrrol-3-yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)- 6-hydroxy-2-methoxy-12-oxo-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2- a][1,4]diazepine-5-carbonyl)oxy)methyl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H- 30 pyran-2-carboxylic acid (144) DB1/ 159693888.5 220

133186-5030-WO A solution of (2S,3S,4S,5R,6S)-6-(4-((((6aS)-3-(4-((5-((4-(5-((4-((S)-2-((S)-2-Amino-3- methylbutanamido)propanamido)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3- yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-6-hydroxy-2-methoxy- 12-oxo-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5- 5 carbonyl)oxy)methyl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid trifluoroacetate (143) (21.4 mg, 15 µmol) in N,N-dimethylformamide (2 mL) was charged with perfluorophenyl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (6.5 mg, 17.2 µmol) and N,N-diisopropylethylamine (12 µL, 69 µmol) and the resulting mixture was stirred at room temperature for 1 h. Purification by reverse phase preparative HPLC (gradient: 10-70% 10 acetonitrile over 20 min; compound eluted at 16 min), gave the title compound (18.6 mg, 82%) as a white solid. ¹H NMR (400 MHz, DMSO-d6) δ 12.84 (br s, 1H), 9.91 (s, 1H), 9.85 (s, 1H), 9.80 (s, 2H), 8.13 (d, J=8.0 Hz, 1H), 7.82 (d, J=4.0 Hz, 1H), 7.74-7.70 (m, 3H), 7.65 (d, J=8.0 Hz, 2H), 7.54 (d, J=8.0 Hz, 2H), 7.50 (d, J=4.0 Hz, 3H), 7.45 (s, 1H), 7.41-7.38 (m, 2H), 7.23 (s, 1H), 7.03 (s, 1H), 7.00 (s, 2H), 6.97 (s, 1H), 6.79 (s, 1H), 6.53 (s, 1H), 5.78-5.76 (m, 1H), 15 5.29 (d, J=4.0 Hz, 1H), 5.16 (d, J=12.0 Hz, 2H), 4.95 (d, J=9.0 Hz, 1H), 4.40-4.37 (m, 1H), 4.19-4.16 (m, 1H), 4.13-4.10 (m, 1H), 3.98 (d, J=8.0 Hz, 3H), 3.91 (s, 3H), 3.83-3.81 (m, 6H), 3.27-3.25 (m, 6H), 2.88 (br s, 1H), 2.48-2.44 (m, 4H), 2.20-2.15 (m, 2H), 2.10-2.07 (m, 2H), 1.99-1.96 (m, 1H), 1.88-1.86 (m, 1H), 1.67-1.54 (m, 2H), 1.54-1.47 (m, 7H), 1.31 (d, J=8.0 Hz, 3H), 1.23-1.17 (m, 2H), 0.88 (d, J=4.0 Hz, 3H), 0.84 (d, J=4.0 Hz, 3H); MS (ES+): m/z = 20 1510.6 (M+H)⁺; LCMS (5 min): tR = 2.42 min; HPLC (15 min): 8.12 min (100% purity, 220 nm). Example 1Q: Synthesis of compound (146). Scheme 17. Synthesis of compound (146). 25 4-(((2S,3R,4S,5S,6S)-6-((Allyloxy)carbonyl)-3,4,5-tris(((allyloxy)carbonyl)oxy)tetrahydro- 2H-pyran-2-yl)oxy)-3-nitrobenzyl (6aS)-3-(4-((5-((4-(5-((4- (((allyloxy)carbonyl)amino)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3- yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-6-hydroxy-2- DB1/ 159693888.5 221

133186-5030-WO methoxy-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)- carboxylate (145) 5 tris(((allyloxy)carbonyl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-3-nitrobenzyl)oxy)carbonyl)-6- hydroxy-2-methoxy-12-oxo-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3- yl)oxy)butanoic acid (127) (10 mg, 9.7 µmol) in anhydrous N,N-dimethylformamide (1 mL) was charged with 5-((4-(5-((4-(((allyloxy)carbonyl)amino)phenyl)carbamoyl)-1-methyl-1H- pyrrol-3-yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-aminium 2,2,2-trifluoroacetate (33) (6.1 10 mg, 9.7 µmol), N,N-diisopropylethylamine (6.8 µL, 39 µmol) and PyAOP (5.1 mg, 9.7 µmol) and the resulting mixture was stirred at room temperature for 10 min. Purification by reverse phase preparative HPLC (gradient: 30-95% acetonitrile over 20 min; compound eluted at 19.5 min), gave the title compound (10.6 mg, 72%) as a white solid. (2S,3S,4S,5R,6S)-6-(4-((((6aS)-3-(4-((5-((4-(5-((4-Aminophenyl)carbamoyl)-1-methyl-1H-15 pyrrol-3-yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-6-hydroxy- 2-methoxy-12-oxo-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5- carbonyl)oxy)methyl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (146) 20 A solution of 4-(((2S,3R,4S,5S,6S)-6-((allyloxy)carbonyl)-3,4,5- tris(((allyloxy)carbonyl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-3-nitrobenzyl (6aS)-3-(4-((5-((4-(5- ((4-(((allyloxy)carbonyl)amino)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3- yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-6-hydroxy-2-methoxy- 12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (145) 25 (10.6 mg, 7.0 µmol) in dichloromethane (1 mL) and N,N-dimethylformamide (1 mL) was charged with formic acid (4.3 µL, 114 µmol), pyrrolidine (10.2 µL, 123 µmol) and tetrakis(triphenylphosphine)palladium(0) (3.6 mg, 3.1 µmol) and the resulting mixture was DB1/ 159693888.5 222

133186-5030-WO stirred for 10 min. The dichloromethane was then under reduced pressure and to the remaining solution was added acetonitrile (1 mL) and water (0.5 mL). Purification by reverse phase preparative HPLC, gave the title compound (7.4 mg, 84%) as a white solid. MS (ES+): m/z = 1146.7 (M+H)⁺; LCMS (5 min): tR = 1.68 min; HPLC (15 min): 9.90 min (99.8% purity, 220 5 nm). Example 1R: Synthesis of compound (150). Scheme 18. Synthesis of compound (150). Allyl (2S,3S,4S,5R,6S)-3,4,5-tris(((allyloxy)carbonyl)oxy)-6-(4-((((2-((S)-2-10 formylpiperidine-1-carbonyl)-4-methoxy-5-(4-oxo-4-(2- (trimethylsilyl)ethoxy)butoxy)phenyl)carbamoyl)oxy)methyl)-2-nitrophenoxy)tetrahydro- 2H-pyran-2-carboxylate (147) A solution of allyl (2S,3S,4S,5R,6S)-3,4,5-tris(((allyloxy)carbonyl)oxy)-6-(4-((((2-((S)-2-15 (hydroxymethyl)piperidine-1-carbonyl)-4-methoxy-5-(4-oxo-4-(2- (trimethylsilyl)ethoxy)butoxy)phenyl)carbamoyl)oxy)methyl)-2-nitrophenoxy)tetrahydro-2H- pyran-2-carboxylate (125) (38 mg, 34 µmol) in dichloromethane (2 mL) was charged with (diacetoxyiodo)benzene (27 mg, 85 µmol) and TEMPO (0.5 mg, 3.4 µmol) and the resulting mixture was stirred at room temperature for 2 h. The resulting solution was employed directly in 20 the subsequent step without further purification. DB1/ 159693888.5 223

133186-5030-WO 4-(5-((((4-(((2S,3R,4S,5S,6S)-6-((Allyloxy)carbonyl)-3,4,5- tris(((allyloxy)carbonyl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-3- nitrobenzyl)oxy)carbonyl)amino)-4-((S)-2-formylpiperidine-1-carbonyl)-2- methoxyphenoxy)butanoic acid (148) 5 containing allyl (2S,3S,4S,5R,6S)-3,4,5-tris(((allyloxy)carbonyl)oxy)-6-(4-((((2-((S)-2- formylpiperidine-1-carbonyl)-4-methoxy-5-(4-oxo-4-(2- (trimethylsilyl)ethoxy)butoxy)phenyl)carbamoyl)oxy)methyl)-2-nitrophenoxy)tetrahydro-2H- 10 pyran-2-carboxylate (147) and the resulting mixture was stirred at room temperature for 40 min. Acetonitrile (2 mL) was then added and the mixture was concentrated to a volume of 3 mL. Purification by reverse phase preparative HPLC (gradient: 20-80% acetonitrile over 20 min; compound eluted at 22 min) then gave the title compound (21 mg, 60% over two steps) as a tan solid. 15 4-(5-((((4-(((2S,3R,4S,5S,6S)-6-((Allyloxy)carbonyl)-3,4,5- tris(((allyloxy)carbonyl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-3- nitrobenzyl)oxy)carbonyl)amino)-2-methoxy-4-((S)-2-((methoxyimino)methyl)piperidine- 1-carbonyl)phenoxy)butanoic acid (149) 20 A solution of 4-(5-((((4-(((2S,3R,4S,5S,6S)-6-((allyloxy)carbonyl)-3,4,5- tris(((allyloxy)carbonyl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-3- nitrobenzyl)oxy)carbonyl)amino)-4-((S)-2-formylpiperidine-1-carbonyl)-2- methoxyphenoxy)butanoic acid (148) (21 mg, 20 µmol) in anhydrous N,N-dimethylformamide (1 mL) was charged with O-methylhydroxylamine hydrochloride (1.7 mg, 20 µmol) and the 25 resulting mixture was stirred at room temperature for 1.5 h and the resulting mixture was employed in the subsequent step without further purification. DB1/ 159693888.5 224

133186-5030-WO (2S,3S,4S,5R,6S)-6-(4-((((5-(3-Carboxypropoxy)-4-methoxy-2-((S)-2- ((methoxyimino)methyl)piperidine-1-carbonyl)phenyl)carbamoyl)oxy)methyl)-2- nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (150) 5 step, containing 4-(5-((((4-(((2S,3R,4S,5S,6S)-6-((Allyloxy)carbonyl)-3,4,5- tris(((allyloxy)carbonyl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-3- nitrobenzyl)oxy)carbonyl)amino)-2-methoxy-4-((S)-2-((methoxyimino)methyl)piperidine-1- carbonyl)phenoxy)butanoic acid (149), followed by pyrrolidine (19 µL, 239 µmol), and 10 tetrakis(triphenylphosphine)palladium(0) (3.6 mg, 3.1 µmol). The resulting mixture was stirred at room temperature for 10 min, upon which acetonitrile (1 mL) and water (0.5 mL) were added. Purification by reverse phase preparative HPLC (gradient: 10-70% acetonitrile over 20 min; compound eluted at 16 min) then gave the title compound (4.1 mg, 27% over two steps) as a white solid. MS (ES+): m/z = 765.3 (M+H)⁺; LCMS (5 min): t_R = 2.08 min; HPLC (15 min): 15 7.69 min (99.4% purity, 220 nm). DB1/ 159693888.5 225

133186-5030-WO Example 1S: Synthesis of compound (163). Scheme 19. Synthesis of compound (163). e y -( - ormy- -me oxyp enoxy) u anoa e ( ) 5 A mixture of vanillin (113) (20.0 g, 131 mmol), methyl 4-bromobutanoate (17.5 mL, 139 mmol) and potassium carbonate (27.2 g, 197 mmol) in N,N-dimethylformamide (100 mL) was stirred at room temperature for 18 h. The reaction mixture was diluted with water (500 mL) and the title compound (30.2 g, 91%) was obtained by filtration as a white solid. The product was 10 employed in the next step without any further purification. ¹H NMR (400 MHz, CDCl3) ^ 9.84 (s, 1H), 7.46-7.37 (m, 2H), 6.98 (d, J=8.2 Hz, 1H), 4.16 (t, J=6.3 Hz, 2H), 3.91 (s, 3H), 3.69 (s, 3H), 2.56 (t, J=7.2 Hz, 2H), 2.20 (quin, J=6.7 Hz, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 190.9, DB1/ 159693888.5 226

133186-5030-WO 173.4, 153.8, 149.9, 130.1, 126.8, 111.6, 109.2, 67.8, 56.0, 51.7, 30.3, 24.2; MS (ES+): m/z = 253 (M+H)⁺; LCMS (Method A): tR = 6.48 min. Methyl 4-(4-formyl-2-methoxy-5-nitrophenoxy)butanoate (152) 5 d_{ropwise with a solution of methyl 4-(4-formyl-2-methoxyphenoxy)butanoate (151) (20.0 g,} 79.2 mmol) in TFA (50 mL). The reaction mixture was stirred at room temperature for 1 h. It was then concentrated in vacuo and then re-solubilised in ethyl acetate (400 mL). The organic layer was washed with brine (3 x 100 mL) and a saturated aqueous solution of sodium hydrogen 10 carbonate (2 x 80 mL). The organic phase was dried over sodium sulfate, filtered, and c_{oncentrated in vacuo, to give the title compound (23.5 g, 100%) as a yellow solid. The product} was employed in the next step without any further purification. ¹H NMR (400 MHz, CDCl3) δ 10.42 (s, 1H), 7.60 (s, 1H), 7.39 (s, 1H), 4.21 (t, J=6.3 Hz, 2H), 3.98 (s, 3H), 3.70 (s, 3H), 2.61- 2.53 (m, 2H), 2.22 (quin, J=6.6 Hz, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 187.8, 173.2, 153.5, 15 151.7, 143.8, 125.5, 109.9, 108.1, 68.6, 56.6, 51.8, 30.2, 24.1; MS (ES+): m/z = 298 (M+H)⁺; LCMS (Method A): tR = 6.97 min. 5-Methoxy-4-(4-methoxy-4-oxobutoxy)-2-nitrobenzoic acid (153) To a solution of methyl 4-(4-formyl-2-methoxy-5-nitrophenoxy)butanoate (152) (23.0 g, 77.4 20 mmol) in acetone (600 mL) was added a hot (70 °C) solution of potassium permanganate (46.0 g, 291 mmol) in water (400 mL). The reaction mixture was stirred at 70 °C for 3 h. The reaction mixture was then cooled to room temperature and passed through celite. The celite cake was washed with hot water (200 mL). A saturated aqueous solution of sodium bisulfite (100 mL) and hydrochloric acid (1 M, 200 mL) was added to the filtrate which was extracted with 25 dichloromethane (2 x 400 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated. The resulting residue was purified by column chromatography (silica), eluting w_{ith methanol/dichloromethane (from 0% to 50%) to give the title compound (17.0 g, 70%) as a} DB1/ 159693888.5 227

133186-5030-WO pale-yellow solid. ¹H NMR (400 MHz, MeOD) δ 7.47 (s, 1H), 7.25 (s, 1H), 4.13 (t, J=6.2 Hz, 2H), 3.94 (s, 3H), 3.68 (s, 3H), 2.54 (t, J=7.2 Hz, 2H), 2.17-2.06 (m, 2H); ¹³C NMR (100 MHz, MeOD) ^ 175.3, 168.6, 153.8, 151.3, 143.1, 122.8, 112.4, 109.2, 69.6, 57.0, 52.2, 31.2, 25.5; MS (ES+): m/z = 314 (M+H)⁺; LCMS (Method A): tR = 6.22 min. 5 Methyl (S)-4-(4-(2-(hydroxymethyl)piperidine-1-carbonyl)-2-methoxy-5- nitrophenoxy)butanoate (154) mmol), oxalyl chloride (6.60 mL, 77.0 mmol) and anhydrous N,N-dimethylformamide (2 drops) 10 in anhydrous dichloromethane (100 mL) was stirred at room temperature for 1 h. Anhydrous toluene (20 mL) was added to the reaction mixture which was then concentrated in vacuo. A solution of the resulting residue in anhydrous dichloromethane (10 mL) was added dropwise to a solution of (S)-piperidin-2-ylmethanol (3.80 g, 33.4 mmol) and triethylamine (10.7 mL, 77.0 mmol) in anhydrous dichloromethane (90 mL) at -10 °C. The reaction mixture was stirred at 15 room temperature for 2 h and then washed with hydrochloric acid (1 M, 50 mL) and brine (50 mL), dried over sodium sulfate, filtered, and concentrated. The resulting residue was purified by c_{olumn chromatography (silica), eluting with methanol/dichloromethane (from 0% to 5%) to give the title compound (9.20 g, 73%) as a yellow oil. 1H NMR (400 MHz, CDCl3) ^ 7.68-} 7.64 (m, 1H), 6.77-6.70 (m, 1H), 4.16-4.07 (m, 3H), 3.93-3.89 (m, 3H), 3.83 (s, 1H), 3.67 (s, 20 3H), 3.15 (d, J=1.4 Hz, 1H), 3.11 (s, 1H), 2.78 (s, 1H), 2.56-2.50 (m, 3H), 2.21-2.12 (m, 4H), 1.74-1.55 (m, 4H); ¹³C NMR (100 MHz, CDCl₃) δ 173.3, 168.1, 154.6, 148.2, 137.4, 127.6, 111.4, 108.3, 68.3, 60.6, 56.7, 53.5, 51.7, 43.3, 38.0, 34.9, 30.3, 24.1, 19.7; MS (ES+): m/z = 411 (M+H)⁺; LCMS (Method A): tR = 6.28 min. Methyl (S)-4-(5-amino-4-(2-(hydroxymethyl)piperidine-1-carbonyl)-2- 25 methoxyphenoxy)butanoate (155) DB1/ 159693888.5 228

133186-5030-WO To a solution of methyl (S)-4-(4-(2-(hydroxymethyl)piperidine-1-carbonyl)-2-methoxy-5- nitrophenoxy)butanoate (9.20 g, 22.4 mmol) in ethanol (40 mL) and ethyl acetate (10 mL) was added palladium on activated charcoal (10% wt. basis) (920 mg). The reaction mixture was hydrogenated at 35 psi for 3 h in a Parr apparatus. The reaction mixture was filtered through 5 celite, and the resulting cake was washed with ethyl acetate. The filtrate was concentrated in v_{acuo to give the title compound (9.00 g, 90%) as a pink solid. The product was carried through} to the next step without any further purification. ¹H NMR (400 MHz, CDCl₃) δ 6.69 (s, 1H), 6.27-6.18 (m, 1H), 4.03-3.94 (m, 3H), 3.94-3.82 (m, 3H), 3.81-3.76 (m, 1H), 3.74 (s, 3H), 3.73- 3.68 (m, 1H), 3.67-3.65 (m, 3H), 3.56 (d, J=4.8 Hz, 1H), 3.03 (s, 1H), 2.51 (t, J=7.2 Hz, 2H), 10 2.11 (quin, J=6.7 Hz, 2H), 1.68-1.59 (m, 4H), 1.55-1.40 (m, 2H); ¹³C NMR (100 MHz, CDCl3) ^ 173.6, 171.2, 150.3, 141.8, 141.1, 113.2, 112.3, 102.4, 67.5, 60.8, 60.4, 56.8, 51.6, 30.4, 25.8, 24.3, 21.0, 19.9, 14.2; MS (ES+): m/z = 381 (M+H)⁺; LCMS (Method A): t_R = 5.52 min. Methyl (S)-4-(5-(((allyloxy)carbonyl)amino)-4-(2-(hydroxymethyl)piperidine-1-carbonyl)- 2-methoxyphenoxy)butanoate (156) 15 y y y y p p y methoxyphenoxy)butanoate (155) (9.00 g, 23.7 mmol) and pyridine (4.40 mL, 54.4 mmol) in anhydrous dichloromethane (100 mL) at -10 °C was added dropwise a solution of allylchloroformate (2.60 mL, 24.8 mmol) in anhydrous dichloromethane (20 mL). The reaction 20 mixture was stirred at room temperature for 30 min. The reaction mixture was sequentially washed with a saturated aqueous solution of copper (II) sulfate (80 mL), water (80 mL) and a saturated aqueous solution of sodium hydrogen carbonate (80 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting residue (2.00 g of the 11.0 g unpurified reaction mixture) was purified by column chromatography (silica), eluting _{25 with methanol/dichloromethane (from 0% to 1%) to give the title compound (930 mg, 47% based on the amount purified) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 8.30 (br s, 1H),} 7.63 (br s, 1H), 6.76 (br s, 1H), 5.92 (ddt, J=17.2, 10.6, 5.4, 5.4 Hz, 1H), 5.37-5.28 (m, 1H), 5.20 (dq, J=10.4, 1.3 Hz, 1H), 4.65-4.56 (m, 2H), 4.06 (t, J=6.2 Hz, 2H), 3.94-3.82 (m, 1H), 3.79 (s, 3H), 3.66 (s, 3H), 3.62-3.54 (m, 1H), 3.40 (br s, 1H), 3.10-2.88 (m, 1H), 2.52 (t, J=7.430 Hz, 2H), 2.22-2.04 (m, 3H), 1.64 (br s, 4H), 1.56-1.31 (m, 2H); ¹³C NMR (100 MHz, CDCl3) δ 173.5, 170.6, 153.9, 149.7, 144.8, 132.6, 130.1, 117.6, 116.9, 110.8, 107.1, 106.0, 67.7, 65.6, DB1/ 159693888.5 229

133186-5030-WO 60.7, 56.3, 53.5, 51.6, 43.1, 30.5, 25.7, 24.4, 19.7; MS (ES+): m/z = 465 (M+H)⁺; LCMS (Method A): tR = 6.47 min. Allyl (6aS)-6-hydroxy-2-methoxy-3-(4-methoxy-4-oxobutoxy)-12-oxo-6,6a,7,8,9,10- hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (157) 5 1-carbonyl)-2-methoxyphenoxy)butanoate (156) (930 mg, 2.00 mmol) in dichloromethane (45 mL) was added TEMPO (32.0 mg, 0.200 mmol) and PIDA (773 mg, 2.40 mmol). The reaction mixture was stirred at room temperature for 16 h, and was then sequentially washed with a 10 saturated aqueous solution of sodium metabisulfite (20 mL), a saturated aqueous solution of sodium hydrogen carbonate (20 mL), water (20 mL) and brine (20 mL). The organic layer was then dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting residue was p_{urified by column chromatography (silica), eluting with methanol/dichloromethane (from 0% to 5%) to give the title compound (825 mg, 89%) as a cream solid. MS (ES+): m/z = 463} 15 (M+H)⁺; LCMS (Method A): t_R = 6.30 min. Allyl (6aS)-2-methoxy-3-(4-methoxy-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2- yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (158) 20 A mixture of allyl (6aS)-6-hydroxy-2-methoxy-3-(4-methoxy-4-oxobutoxy)-12-oxo- 6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (157) (825 mg, 1.80 mmol), 3,4-dihydro-2H-pyran (1.70 mL, 18.2 mmol) and p-toluenesulfonic acid m_{onohydrate (8.50 mg, 1% w/w) in ethyl acetate (12 mL) was stirred at room temperature for} 16 h. The reaction mixture was then diluted with ethyl acetate (50 mL) and washed with a 25 saturated aqueous solution of sodium hydrogen carbonate (20 mL) and brine (30 mL). The DB1/ 159693888.5 230

133186-5030-WO organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting residue was purified by column chromatography (silica), eluting with m_{ethanol/dichloromethane (from 0% to 2%) to give the title compound (820 mg, 84%) as a} cream solid. MS (ES+): m/z = 547 (M+H)⁺; LCMS (Method A): tR = 7.70 min. 5 4-(((6aS)-5-((Allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)- 5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoic acid (159) 10 pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)- carboxylate (158) (770 mg, 1.40 mmol) in 1,4-dioxane (10 mL) was added an aqueous solution of sodium hydroxide (0.5 M, 10.0 mL, 5.00 mmol). The reaction mixture was stirred at room temperature for 2 h and was then concentrated in vacuo, after which water (20 mL) was added and the aqueous layer was with an aqueous solution of acetic acid (1 M, 5 mL). The aqueous 15 layer was then extracted with ethyl acetate (2 x 50 mL). The combined organic extracts were then washed with brine (50 mL), dried over sodium sulfate, filtered, and concentrated to give t_{he title compound (700 mg, 93%) as a yellow oil. The product was carried through to the next} step without any further purification. MS (ES+): m/z = 533 (M+H)⁺; LCMS (Method A): t_R = 6.98 min. 20 Allyl (6aS)-2-methoxy-3-(4-((5-(methoxycarbonyl)-1-methyl-1H-pyrrol-3-yl)amino)-4- oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10- hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (160) A solution of 4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2- 25 yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoic acid (159) (17.0 g, 31.9 mmol) in N,N-dimethylformamide (300 mL) was charged with N-(3- dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (12.2 g, 63.8 mmol), 4- DB1/ 159693888.5 231

133186-5030-WO (dimethylamino)pyridine (1.17 g, 9.58 mmol), N,N-diisopropylethylamine (8.25 g, 63.8 mmol) and methyl 4-amino-1-methyl-1H-pyrrole-2-carboxylate hydrochloride (44) (6.08 g, 31.9 mmol) and the resulting mixture was stirred at room temperature for 4 h. This was then diluted into water (1 L) and extracted with ethyl acetate (2 L). The organic phase was washed with brine (1 5 L), dried over sodium sulfate, and concentrated in vacuo. Purification by flash column chromatography (silica), eluting with ethyl acetate/petroleum spirit, 40-60 °C (50%) gave the title compound (19.1 g, 90%) as an off-white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 9.92 (s, 1H), 7.34 (d, J=1.9 Hz, 1H), 7.05 (d, J=2.2 Hz, 1H), 6.92 (s, 1H), 6.72 (d, J=1.9 Hz, 1H), 6.04 (6.04 (d, J=10.0 Hz, 1H), 5.77-5.76 (m, 1H), 5.40-4.92 (m, 2H), 4.70-4.35 (m, 1H), 4.12-3.90 10 (m, 5H), 3.82-3.81 (m, 6H), 3.75-3.71 (m, 3H), 3.57-3.43 (m, 1H), 3.37-3.29 (m, 4H), 2.45-2.37 (m, 2H), 2.08-1.86 (m, 3H), 1.77-1.39 (m, 10H); MS (ES-): m/z = 667 (M-H)^–; LCMS (Method F): t_R = 2.25 min. 4-(4-(((6aS)-5-((Allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)- 5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1- 15 methyl-1H-pyrrole-2-carboxylic acid (161) yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10- hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (160) (19.1 g, 28.6 mmol) 20 in tetrahydrofuran (250 mL) was charged with an aqueous solution of sodium hydroxide (0.5 M, 230 mL, 114.2 mmol) and the resulting mixture stirred rapidly at room temperature for 48 h. After diluting into water (1 L), the mixture was neutralised to pH 6-7 with a saturated aqueous solution of citric acid (500 mL) and extracted with ethyl acetate (1 L). The organic phase was washed with brine (1 L), dried over sodium sulfate, filtered, and concentrated in vacuo, to give 25 the title compound (18.2 g, 97%) as a white solid. ¹H NMR (400 MHz, DMSO-d6) δ 12.08 (s, 1H), 9.83 (s, 1H), 7.27 (d, J=2.0 Hz, 1H), 7.04 (d, J=2.4 Hz, 1H), 6.83 (br, 1H), 6.64 (d, J=2.0 Hz, 1H), 6.03-5.89 (m, 1H), 5.40-4.95 (m, 3H), 4.65-4.35 (m, 2H), 4.15-3.90 (m, 3H), 3.81-3.74 (m, 7H), 3.55-3.42 (m, 1H), 2.89 (s, 1H), 2.40-2.37 (m, 2H) , 1.90 (s, 2H), 1.85-1.40 (m, 12H); MS (ES-): m/z = 653 (M-H)^–; LCMS (Method F): t_R = 1.95 min. DB1/ 159693888.5 232

133186-5030-WO Allyl (6aS)-3-(4-((5-((4-aminophenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4- oxobutoxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10- hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (162) 5 pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3- yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxylic acid (161) (13.0 g, 19.9 mmol), N,N- dimethylpyridin-4-amine (728 mg, 5.96 mmol), N-(3-dimethylaminopropyl)-N′- ethylcarbodiimide hydrochloride (7.61 g, 39.7 mmol) and N,N-diisopropylethylamine (5.13 g, 10 39.7 mmol) in N,N-dimethylformamide (300 mL) which was previously stirred for 30 min, was added benzene-1,4-diamine (2.25 g, 20.8 mmol). The reaction mixture was stirred at room temperature for 4 h. The reaction mixture was partitioned between water (1 L) and ethyl acetate (2 x 1 L). The organic layer was then dried over sodium sulfate, filtered and concentrated in vacuo. The resulting residue was purified by column chromatography (silica), eluting with _{15 methanol/dichloromethane (from 2.5% to 5%) to give the title compound (19.2 g, 62%) as a} white solid. MS (ES+): m/z = 745 (M+H)⁺; LCMS (Method B): tR = 2.83 min. (S)-N-(4-Aminophenyl)-4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12- hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole- 2-carboxamide (163) (Control 1) 20 To a solution of allyl (6aS)-3-(4-((5-((4-aminophenyl)carbamoyl)-1-methyl-1H-pyrrol-3- yl)amino)-4-oxobutoxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10- hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (6.50 g, 8.73 mmol) in dichloromethane (65 mL) was added tetrakis(triphenylphosphine)palladium(0) (504 mg, 5 25 mol%) and pyrrolidine (860 ^L, 10.5 mmol). The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was subjected to high vacuum until excess pyrrolidine was thoroughly removed. The resulting residue was purified by column chromatography (_{silica), eluting with methanol/dichloromethane (from 0% to 10%) to give the title compound (4.80 g, 98%) as a cream solid. [α]D20 = 93.0° (c 1.36, DMSO); 1H NMR (600 MHz, DMSO-d6)} 30 δ 9.85 (s, 1H), 9.43 (s, 1H), 8.00 (d, J = 5.7 Hz, 1H), 7.31-7.26 (m, 3H), 7.17 (d, J = 1.8 Hz, 1H), 6.83 (d, J = 1.8 Hz, 1H), 6.80 (s, 1H), 6.51 (d, J = 8.8 Hz, 2H), 4.84 (s, 2H), 4.14-4.05 (m, DB1/ 159693888.5 233

133186-5030-WO 2H), 3.82 (s, 3H), 3.79 (s, 3H), 3.74-3.64 (m, 3H), 2.43 (t, J = 7.4 Hz, 2H), 2.04 (dt, J = 13.9, 6.8 Hz, 2H), 1.90-1.83 (m, 1H), 1.76-1.55 (m, 5H); ¹³C NMR (150 MHz, DMSO-d6) δ 168.8, 166.3, 164.7, 159.3, 150.2, 147.1, 144.7, 139.9, 128.2, 123.2, 122.1, 121.9, 120.7, 118.1, 113.7, 111.4, 109.5, 104.0, 67.8, 55.6, 54.9, 48.6, 39.5, 36.01, 31.9, 24.7, 23.7, 22.6, 17.7; MS (ES+): 5 m/z = 559 (M+H)⁺; LCMS (Method B): t_R = 2.53 min; HRMS (ESI, m/z): calc. for C₃₀H₃₅N₆O₅ ⁺ ([M]+H)⁺ 559.2663 found 559.2651. Example 1T: Synthesis of compound (167). Scheme 20. Synthesis of compound (167). 10 Allyl (6aS)-2-methoxy-3-(4-((5-((4-(5-(methoxycarbonyl)-1-methyl-1H-pyrrol-3- yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6- ((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2- a][1,4]diazepine-5(12H)-carboxylate (164) 15 To a solution of methyl 4-(4-(4-((tert-butoxycarbonyl)amino)-1-methyl-1H-pyrrole-2- carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylate (15) (440 mg, 0.970 mmol) in 1,4- dioxane (2 mL) and methanol (2 mL), hydrochloric acid (4 M in 1,4-dioxane) (4 mL) was added d_{ropwise. The reaction mixture was stirred for 4 h and then concentrated in vacuo. The residue} was added to a mixture of 4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-20 2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3- yl)oxy)butanoic acid (159) (470 mg, 0.880 mmol), N,N-dimethylpyridin-4-amine (322 mg, 2.64 mmol) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (422 mg, 2.20 DB1/ 159693888.5 234

133186-5030-WO mmol) in N,N-dimethylformamide (7 mL) which was previously stirred for 30 min. The resulting solution was stirred at room temperature for 18 h. The reaction mixture was quenched with a saturated aqueous solution of sodium hydrogen carbonate (10 mL) and washed with a saturated aqueous solution of sodium chloride (90 mL). The aqueous phase was extracted with 5 ethyl acetate (2 x 50 mL). The combined organic extracts were concentrated in vacuo. The resulting residue was purified by column chromatography (silica), eluting with a_{cetone/dichloromethane (from 0% to 30%) to give the title compound (600 mg, 78%) as an} orange solid. ¹H NMR (400 MHz, CDCl₃) δ 7.42 (d, J=8.3 Hz, 2H), 7.22-7.10 (m, 4H), 7.04 (s, 2H), 6.76 (br s, 1H), 6.02-5.87 (m, 1H), 5.74-5.68 (m, 1H), 5.38-5.25 (m, 1H), 5.11-5.05 (m, 10 1H), 4.38-4.26 (m, 1H), 4.11 (br s, 2H), 3.93 (s, 3H), 3.88 (br s, 5H), 3.82 (s, 6H), 3.78 (br s, 2H), 3.62 (br s,3H), 2.48-2.39 (m, 2H), 2.12-2.03 (m, 2H), 1.75-1.50 (m, 12H); MS (ES+): m/z = 867 (M+H)⁺; LCMS (Method B): t_R = 4.17 min. 4-(4-(4-(4-(((6aS)-5-((Allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2- yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-15 yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2- carboxylic acid (165) 3-yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-20 2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)- carboxylate (164) (600 mg, 0.690 mmol) in 1,4-dioxane (10 mL) was added an aqueous solution of sodium hydroxide (1 M, 10 mL, 10.0 mmol). The reaction mixture was stirred at room temperature for 18 h and was then concentrated in vacuo, after which water (100 mL) was added and the aqueous layer was acidified to pH = 4 with an aqueous solution of acetic acid (5 25 M, 20 mL). The aqueous layer was then extracted with ethyl acetate (2 x 100 mL). The combined organic extracts were dried over sodium sulfate, filtered and concentrated in vacuo to g_{ive the title compound (558 mg, 97%) as a cream solid. The product was carried through to the} next step without any further purification. ¹H NMR (400 MHz, CD3OD) δ 7.58-7.54 (m, 2H), 7.46 (d, J=8.3 Hz, 2H), 7.24 (s, 1H), 7.18 (s, 2H), 7.13 (s, 1H), 6.88 (br s, 2H), 6.17 (d, J=9.8 30 Hz, 1H), 5.78-5.74 (m, 1H), 4.66-4.38 (m, 3H), 4.26-4.12 (m, 1H), 4.06 (m, 3H), 3.91 (s, 3H), 3.87 (s, 3H), 3.84 (br s, 4H), 3.67-3.49 (m, 2H), 3.44 (br s, 1H), 3.11-2.96 (m, 1H), 2.51 (t, J=7.3 Hz, 2H), 2.15-2.12 (m, 2H), 1.72-1.48 (m, 12H); ¹³C NMR (100 MHz, CD3OD) δ 175.6, 172.2, 171.4, 164.6, 162.2, 152.1, 150.9, 137.8, 133.5, 132.1, 129.2, 127.6, 126.1, 125.0, 124.7, 124.6, 123.4, 122.4, 117.6, 115.8, 115.6, 106.4, 85.5, 69.5, 67.7, 56.6, 40.2, 37.3, 37.0, 31.8, DB1/ 159693888.5 235

133186-5030-WO 26.5, 26.4, 24.0, 21.0, 20.6, 19.1; MS (ES+): m/z = 853 (M+H)⁺; LCMS (Method B): t_R = 3.83 min. Allyl (6aS)-3-(4-((5-((4-(5-((4-aminophenyl)carbamoyl)-1-methyl-1H-pyrrol-3- yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-2-methoxy-12-oxo-6- 5 ((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2- a][1,4]diazepin-5(12H)-carboxylate (166) pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-10 yl)oxy)butanamido)-1-methyl-1H-pyrrol-2-carboxamido)phenyl)-1-methyl-1H-pyrrol-2- c_{arboxylic acid (165) (210 mg, 0.246 mmol) in N,N-dimethylformamide (8 mL) was charged} with 4-(dimethylamino)pyridine (90 mg, 0.74 mmol) and N-(3-dimethylaminopropyl)-N′- ethylcarbodiimide hydrochloride (118 mg, 0.615 mmol), and stirred for 30 min at room temperature. To the resulting mixture, 1,4-benzenediamine (40 mg, 0.370 mmol) was added and 15 stirred for a further 18 h. The reaction mixture was then diluted into a saturated aqueous solution of sodium hydrogen carbonate (100 mL) and extracted with ethyl acetate (2 x 40 mL). The combined organic extracts were concentrated in vacuo. Purification by flash column c_{hromatography (silica), eluting with methanol/dichloromethane (from 0% to 20%), gave the} title compound (110 mg, 47%) as a cream solid. ¹H NMR (600 MHz, DMSO-d6) δ 9.89 (s, 1H), 20 9.78 (s, 1H), 9.48 (s, 1H), 7.70 (d, J=8.6 Hz, 2H), 7.48 (d, J=8.5 Hz, 2H), 7.39 (d, J=1.7 Hz, 1H), 7.36-7.32 (m, 2H), 7.30 (d, J=1.8 Hz, 1H), 7.23-7.20 (m, 1H), 7.06 (d, J=4.1 Hz, 1H), 6.97-6.94 (m, 1H), 6.80 (s, 1H), 6.57-6.52 (m, 2H), 6.03 (d, J=10.0 Hz, 1H), 5.81-5.72 (m, 1H), 5.26-4.95 (m, 3H), 4.92 (s, 2H), 4.64-4.36 (m, 2H), 4.14-3.95 (m, 3H), 3.89 (s, 3H), 3.83 (dd, J=6.0, 2.4 Hz, 6H), 3.81-3.70 (m, 1H), 3.55-3.34 (m, 2H), 3.00-2.82 (m, 1H), 2.44 (t, J=7.0 Hz, 25 2H), 2.09-1.99 (m, 2H), 1.94-1.35 (m, 12H); ¹³C NMR (150 MHz, DMSO-d6) δ 169.4, 168.6, 160.1, 159.7, 149.2, 145.1, 137.5, 133.2, 128.8, 127.1, 125.1, 124.8, 123.2, 122.6, 122.4, 122.3, 121.0, 119.2, 117.0, 114.3, 110.9, 110.8, 110.1, 105.2, 99.7, 94.7, 88.0, 83.9, 68.5, 68.3, 66.0, 63.2, 56.2, 55.4, 38.7, 38.6, 36.8, 36.6, 32.2, 32.1, 30.9, 30.6, 25.4, 25.2, 24.9, 23.1, 23.0, 19.6, 19.4, 18.3; MS (ES+): m/z = 943 (M+H)⁺; LCMS (Method B): t_R = 3.45 min. DB1/ 159693888.5 236

133186-5030-WO (S)-N-(4-aminophenyl)-4-(4-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12- hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrol- 2-carboxamido)phenyl)-1-methyl-1H-pyrrol-2-carboxamide (167) (Control 2) 5 yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-2-methoxy-12-oxo-6- ((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin- 5(12H)-carboxylate (166) (250 mg, 0.265 mmol) in dichloromethane (3 mL) was charged with tetrakis(triphenylphosphine)palladium(0) (15 mg, 5mol%), and pyrrolidine (26 µL, 0.32 mmol) 10 and the resulting mixture was stirred at room temperature for 20 min, before being subjected to strong vacuum for 30 min. The resulting residue was then purified by flash column chromatography (silica), eluting with methanol/dichloromethane (from 0% to 10%) to give the title compound (118 mg, 59%) as a cream solid. [α]_D ²³ = 85° (c 0.143, DMSO); ¹H NMR (600 MHz, DMSO-d₆) δ 9.90 (s, 1H), 9.78 (s, 1H), 9.48 (s, 1H), 8.00 (d, J=5.6 Hz, 1H), 7.70 (d, 15 J=8.6 Hz, 2H), 7.48 (d, J=8.6 Hz, 2H), 7.39 (d, J=1.5 Hz, 1H), 7.33 (d, J=8.7 Hz, 2H), 7.30 (d, J=1.5 Hz, 1H), 7.27 (s, 1H), 7.21 (d, J=1.4 Hz, 1H), 6.96 (d, J=1.5 Hz, 1H), 6.80 (s, 1H), 6.53 (d, J=8.7 Hz, 2H), 4.86 (s, 2H), 4.13 (dt, J=9.6, 6.3 Hz, 1H), 4.07- 3.97 (m, 2H), 3.88 (s, 3H), 3.83 (d, J=5.1 Hz, 6H), 3.72 (dt, J=8.4, 4.1 Hz, 1H), 3.16-3.08 (m, 1H), 2.44 (t, J=7.4 Hz, 2H), 2.07- 2.02 (m, 3H), 1.89-1.59 (m, 5H); ¹³C NMR (150 MHz, DMSO-d6) δ 168.9, 166.3, 164.7, 20 159.6, 159.2, 150.2, 147.2, 144.8, 139.9, 137.0, 129.7, 128.2, 126.7, 124.7, 124.4, 122.7, 122.1, 121.9, 121.8, 120.7, 120.5, 118.8, 113.7, 111.4, 109.6, 109.5, 104.8, 67.8, 55.6, 49.3, 36.3, 36.2, 31.9, 24.7, 23.7, 22.6, 17.7; MS (ES+): m/z = 757 (M+H)⁺; LCMS (Method A): tR = 5.80 min; HRMS (EI, m/z): calculated for C42H45N8O6⁺ (M+H)⁺ 757.3457 found 757.3457. DB1/ 159693888.5 237

O ^W-03 0 .⁾ ₀ 8₁ ⁽ dn u_o p m o_cf o ^s _i s_e h_t n_y S_: U ⁵ _. 1 88 e_l 83 p 96 m 9 a 51 x _/ E 1B D

133186-5030-WO Allyl (2S,3S,4S,5R,6S)-3,4,5-tris(((allyloxy)carbonyl)oxy)-6-(2-amino-4-(((tert- butyldimethylsilyl)oxy)methyl)phenoxy)tetrahydro-2H-pyran-2-carboxylate (169) 5 , , , , , , -(4-(((tert- butyldimethylsilyl)oxy)methyl)-2-nitrophenoxy)tetrahydro-2H-pyran-2-carboxylate (168, 780 mg, 1.04 mmol) in methanol (10 mL) was charged with formic acid (0.6 mL) and zinc powder (1.0 g) and the resulting mixture was stirred at room temperature for 30 min. The mixture was diluted into ethyl acetate (100 mL), filtered, and washed with water (100 mL), then with brine 10 (100 mL), dried over anhydrous sodium sulfate, and concentrated in vacuo to give the title compound (755 mg) as a tan solid. Allyl (2S,3S,4S,5R,6S)-6-(2-(1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22,25,28-nonaoxa-4- azahentriacontan-31-amido)-4-(hydroxymethyl)phenoxy)-3,4,5- 15 tris(((allyloxy)carbonyl)oxy)tetrahydro-2H-pyran-2-carboxylate (170) A solution of allyl (2S,3S,4S,5R,6S)-3,4,5-tris(((allyloxy)carbonyl)oxy)-6-(2-amino-4-(((tert- butyldimethylsilyl)oxy)methyl)phenoxy)tetrahydro-2H-pyran-2-carboxylate (169, 200 mg, 0.277 mmol) in anhydrous N,N-dimethylformamide (2 mL) was charged with 1-(9H-fluoren-9- 20 yl)-3-oxo-2,7,10,13,16,19,22,25,28-nonaoxa-4-azahentriacontan-31-oic acid (184 mg, 0.277 mmol), N,N-diisopropylethylamine (144 mL, 0.831 mmol), and PyAOP (145 mg, 0.277 mmol). The resulting mixture was stirred at room temperature for 24 h. Triethylamine trihydrofluoride (135 mg, 0.831 mmol) was then added to this mixture and stirring continued for an additional 3 h. The mixture was purified by reverse phase preparative HPLC (gradient: 20-90% acetonitrile 25 over 20 min; compound eluted at 20 min), to give the title compound (247 mg, 71%) as a white solid. DB1/ 159693888.5 239

133186-5030-WO Allyl (2S,3S,4S,5R,6S)-6-(2-(1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22,25,28-nonaoxa-4- azahentriacontan-31-amido)-4-((((perfluorophenoxy)carbonyl)oxy)methyl)phenoxy)-3,4,5- tris(((allyloxy)carbonyl)oxy)tetrahydro-2H-pyran-2-carboxylate (171) 5 _8- nonaoxa-4-azahentriacontan-31-amido)-4-(hydroxymethyl)phenoxy)-3,4,5- tris(((allyloxy)carbonyl)oxy)tetrahydro-2H-pyran-2-carboxylate (170, 247 mg, 0.197 mmol) in N,N-dimethylformamide (3 mL) was charged with bis(pentafluorophenyl) carbonate (156 mg, 0.394 mmol) and stirred at room temperature for 4 h. Purification by reverse phase preparative 10 HPLC (gradient: 30-95% acetonitrile over 20 min; compound eluted at 20 min), gave the title compound (252 mg, 88%) as a white solid. Allyl (2S,3S,4S,5R,6S)-6-(2-(1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22,25,28-nonaoxa-4- azahentriacontan-31-amido)-4-((((2-((S)-2-(hydroxymethyl)piperidine-1-carbonyl)-4- methoxy-5-(4-oxo-4-(2-15 (trimethylsilyl)ethoxy)butoxy)phenyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5- tris(((allyloxy)carbonyl)oxy)tetrahydro-2H-pyran-2-carboxylate (173) A solution of allyl (2S,3S,4S,5R,6S)-6-(2-(1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22,25,28- nonaoxa-4-azahentriacontan-31-amido)-4-20 ((((perfluorophenoxy)carbonyl)oxy)methyl)phenoxy)-3,4,5- tris(((allyloxy)carbonyl)oxy)tetrahydro-2H-pyran-2-carboxylate (171, 218 mg, 0.149 mmol) in N,N-dimethylformamide (3 mL) was charged with 2-(trimethylsilyl)ethyl (S)-4-(5-amino-4-(2- (hydroxymethyl)piperidine-1-carbonyl)-2-methoxyphenoxy)butanoate (172, 86 mg, 0.149 mmol), HOAt (13.7 mg, 0.10 mmol) and N,N-diisopropylethylamine (78 mL, 0.44 mmol), and 25 the resulting mixture was stirred at room temperature for 24 h. Purification by reverse phase preparative HPLC (gradient: 30-95% acetonitrile over 20 min; compound eluted at 20 min), gave the title compound (252 mg, 88%) as a white solid. 3-(1-(9H-Fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22,25,28-nonaoxa-4-azahentriacontan-31- amido)-4-(((2S,3R,4S,5S,6S)-6-((allyloxy)carbonyl)-3,4,5-30 tris(((allyloxy)carbonyl)oxy)tetrahydro-2H-pyran-2-yl)oxy)benzyl (6aS)-6-hydroxy-2- methoxy-12-oxo-3-(4-oxo-4-(2-(trimethylsilyl)ethoxy)butoxy)-6,6a,7,8,9,10- hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (174) DB1/ 159693888.5 240

133186-5030-WO y , , , , y , , , , , , , , nonaoxa-4-azahentriacontan-31-amido)-4-((((2-((S)-2-(hydroxymethyl)piperidine-1-carbonyl)- 4-methoxy-5-(4-oxo-4-(2- 5 (trimethylsilyl)ethoxy)butoxy)phenyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5- tris(((allyloxy)carbonyl)oxy)tetrahydro-2H-pyran-2-carboxylate (173, 228 mg, 0.13 mmol) in dichloromethane (10 mL) was charged with (diacetoxyiodo)benzene (85 mg, 0.26 mmol) and TEMPO (2 mg, 13 mmol). The mixture was stirred at room temperature for 5 days, before removing the solvent in vacuo. Purification by reverse phase preparative HPLC (gradient: 30- 10 95% acetonitrile over 20 min; compound eluted at 22 min), gave the title compound (146 mg, 64%) as a tan solid. 4-(((6aS)-5-(((3-(1-(9H-Fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22,25,28-nonaoxa-4- azahentriacontan-31-amido)-4-(((2S,3R,4S,5S,6S)-6-((allyloxy)carbonyl)-3,4,5- tris(((allyloxy)carbonyl)oxy)tetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)-6-15 hydroxy-2-methoxy-12-oxo-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2- a][1,4]diazepin-3-yl)oxy)butanoic acid (175) A solution of 3-(1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22,25,28-nonaoxa-4- azahentriacontan-31-amido)-4-(((2S,3R,4S,5S,6S)-6-((allyloxy)carbonyl)-3,4,5-20 tris(((allyloxy)carbonyl)oxy)tetrahydro-2H-pyran-2-yl)oxy)benzyl (6aS)-6-hydroxy-2-methoxy- 12-oxo-3-(4-oxo-4-(2-(trimethylsilyl)ethoxy)butoxy)-6,6a,7,8,9,10- hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (174, 146 mg, 84 mmol) in dichloromethane (3 mL) was charged with trifluoroacetic acid (1 mL) and the mixture was stirred at room temperature for 30 min, , before removing the solvent in vacuo. Purification by 25 reverse phase preparative HPLC (gradient: 20-85% acetonitrile over 20 min; compound eluted at 22 min), gave the title compound (69 mg, 50%) as a white solid. 3-(1-(9H-Fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22,25,28-nonaoxa-4-azahentriacontan-31- amido)-4-(((2S,3R,4S,5S,6S)-6-((allyloxy)carbonyl)-3,4,5- tris(((allyloxy)carbonyl)oxy)tetrahydro-2H-pyran-2-yl)oxy)benzyl (6aS)-3-(4-((5-((4-((tert-30 butoxycarbonyl)amino)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)- DB1/ 159693888.5 241

133186-5030-WO 6-hydroxy-2-methoxy-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine- 5(12H)-carboxylate (177) 5 trifluoroacetate salt (176, 19 mg, 42 mmol) in anhydrous N,N-dimethylformamide (2 mL) was charged with 4-(((6aS)-5-(((3-(1-(9H-Fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22,25,28-nonaoxa-4- azahentriacontan-31-amido)-4-(((2S,3R,4S,5S,6S)-6-((allyloxy)carbonyl)-3,4,5- tris(((allyloxy)carbonyl)oxy)tetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)-6-hydroxy-2- methoxy-12-oxo-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-10 yl)oxy)butanoic acid (175, 69 mg, 42 mmol), PyAOP (22 mg, 42 mmol) and N,N- diisopropylethylamine (30 mL, 168 mmol) and the resulting mixture was stirred at room temperature for 20 min, then purified directly. Purification by reverse phase preparative HPLC (gradient: 35-95% acetonitrile over 20 min; compound eluted at 21 min), gave the title compound (26 mg, 30%) as a white solid. 15 (2S,3S,4S,5R,6S)-6-(2-(1-(9H-Fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22,25,28-nonaoxa-4- azahentriacontan-31-amido)-4-((((6aS)-3-(4-((5-((4-((tert- butoxycarbonyl)amino)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)- 6-hydroxy-2-methoxy-12-oxo-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2- a][1,4]diazepine-5-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran- 20 2-carboxylic acid (178) A solution of 3-(1-(9H-Fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22,25,28-nonaoxa-4- azahentriacontan-31-amido)-4-(((2S,3R,4S,5S,6S)-6-((allyloxy)carbonyl)-3,4,5- tris(((allyloxy)carbonyl)oxy)tetrahydro-2H-pyran-2-yl)oxy)benzyl (6aS)-3-(4-((5-((4-((tert-25 butoxycarbonyl)amino)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-6- hydroxy-2-methoxy-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine- 5(12H)-carboxylate (177, 26 mg, 12.6 mmol) in dichloromethane (2 mL) and N,N- dimethylformamide (2 mL) was charged with formic acid (7 mL), pyrrolidine (16 mL), and DB1/ 159693888.5 242

133186-5030-WO tetrakis(triphenylphosphine)palladium(0) (3.6 mg, 3.1 mmol), and the resulting mixture was stirred at room temperature for 20 min. Then dichloromethane was evaporated under vacuum. The remaining DMF solution was purified directly. Purification by reverse phase preparative HPLC (gradient: 35-95% acetonitrile over 20 min; compound eluted at 15 min), gave the title 5 compound (18 mg, 86%) as a white solid. (2S,3S,4S,5R,6S)-6-(2-(1-Amino-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-amido)-4- ((((6aS)-3-(4-((5-((4-((tert-butoxycarbonyl)amino)phenyl)carbamoyl)-1-methyl-1H-pyrrol- 3-yl)amino)-4-oxobutoxy)-6-hydroxy-2-methoxy-12-oxo-5,6,6a,7,8,9,10,12- octahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5-carbonyl)oxy)methyl)phenoxy)-3,4,5- 10 trihydroxytetrahydro-2H-pyran-2-carboxylic acid (179) so ut on o ( S,3S, S,5R,6S)-6-( -( -(9H- uoren-9-y)-3-oxo- ,7, 0, 3, 6, 9, , 5, 8- nonaoxa-4-azahentriacontan-31-amido)-4-((((6aS)-3-(4-((5-((4-((tert- butoxycarbonyl)amino)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-6-15 hydroxy-2-methoxy-12-oxo-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepine- 5-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (178, 18 mg, 10.8 mmol) in N,N-dimethylformamide (1 mL) was charged with piperidine (0.1 mL) and the resulting mixture stirred at room temperature for 20 min, before purifying directly. Purification by reverse phase preparative HPLC (gradient: 5-60% acetonitrile over 20 min; 20 compound eluted at 18 min), gave the title compound (9 mg, 54%) as a TFA-salt. (2S,3S,4S,5R,6S)-6-(4-((((6aS)-3-(4-((5-((4-Aminophenyl)carbamoyl)-1-methyl-1H-pyrrol- 3-yl)amino)-4-oxobutoxy)-6-hydroxy-2-methoxy-12-oxo-5,6,6a,7,8,9,10,12- octahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5-carbonyl)oxy)methyl)-2-(1-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)-3-oxo-7,10,13,16,19,22,25,28-octaoxa-4-azahentriacontan-31- 25 amido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid trifluoroacetate salt (181) DB1/ 159693888.5 243

133186-5030-WO A solution of (2S,3S,4S,5R,6S)-6-(2-(1-amino-3,6,9,12,15,18,21,24-octaoxaheptacosan-27- amido)-4-((((6aS)-3-(4-((5-((4-((tert-butoxycarbonyl)amino)phenyl)carbamoyl)-1-methyl-1H- pyrrol-3-yl)amino)-4-oxobutoxy)-6-hydroxy-2-methoxy-12-oxo-5,6,6a,7,8,9,10,12- octahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5-carbonyl)oxy)methyl)phenoxy)-3,4,5- 5 trihydroxytetrahydro-2H-pyran-2-carboxylic acid (179, 9 mg, 5.8 mmol) in anhydrous N,N- dimethylformamide (1 mL) was charged with 3-maleimidopropionic acid N- hydroxysuccinimide ester (1.7 mg, 6.4 mmol) and N,N-diisopropylethylamine (6 mL, 34 mmol) and the resulting mixture was stirred at room temperature for 2 h. after concentrating in vacuo, the residue was redissolved in dichloromethane (1.5 mL) and trifluoroacetic acid (0.5 mL) and 10 stirred for 10 min, whereupon the solvent was evaporated under vacuum and the residue was purified by reverse phase preparative HPLC (gradient: 2-45% acetonitrile over 20 min; compound eluted at 18 min), to give a trifluoroacetate salt of compound 180 (compound 181) (7.7 mg, 83%) as a colourless solid. 1H NMR (400 MHz, DMSO-d₆) δ 9.88 (s, 1H), 9.78 (s, 1H), 9.10 (s, 1H), 8.40-8.13 (m, 1H),15 8.00 (t, J=8 Hz, 1H), 7.63 (br, 2H), 7.22 (s, 1H), 7.02-6.91 (m, 8H), 6.70-6.53 (m, 1H), 5.86- 5.76 (m, 2H), 5.12-4.83 (m, 2H), 4.13-4.10 (m, 1H), 3.93-3.89 (m, 2H), 3.81-3.74 (m, 6H), 3.70 (t, J=6 Hz, 2H), 3.59 (t, J=6 Hz, 2H), 3.52 (br, 32H), 3.20 (s, 2H), 3.14 (q, J=6 Hz, 3H), 2.90- 2.85 (m, 1H), 2.68-2.64 (m, 2H), 2.44 (br, 2H), 2.36-2.31 (m, 3H), 2.08 (br, 2H), 1.86 (br, 1H), 1.69-1.55 (m, 4H); MS (ES+): m/z = 1492.9 (M+H)⁺; LCMS (5 min): t_R = 1.55 min; HPLC (15 20 min): 8.31 min (96.6% purity, 220 nm). Example 1V: Synthesis of compound (186) Scheme 22. Synthesis of compound (186). (S)-32-(((Allyloxy)carbonyl)amino)-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27- 25 azatritriacontan-33-oic acid (184) DB1/ 159693888.5 244

133186-5030-WO 179 mg, 0.396 mmol) in anhydrous N,N-dimethylformamide (2 mL) was charged with 1,8- diazabicyclo[5.4.0]undec-7-ene (0.12 mL), and the resulting mixture was stirred at room 5 temperature for 10 min. Perfluorophenyl 2,5,8,11,14,17,20,23-octaoxahexacosan-26-oate (183, 229 mg, 0.396 mmol) was then added to the mixture and stirring was continued for a further 10 min, before purifying directly. Purification by reverse phase preparative HPLC (gradient: 10- 65% acetonitrile over 20 min; compound eluted at 15 min), gave the title compound (206 mg, 83%) as a colourless oil. 10 Perfluorophenyl (S)-32-(((allyloxy)carbonyl)amino)-26-oxo-2,5,8,11,14,17,20,23-octaoxa- 27-azatritriacontan-33-oate (185) A solution of (S)-32-(((Allyloxy)carbonyl)amino)-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27- azatritriacontan-33-oic acid (184, 91 mg, 0.145 mmol) in anhydrous dichloromethane (2 mL) 15 was charged with pentafluorophenol (54 mg, 0.290 mmol) ’nd N,N'-diisopropylcarbodiimide (23 µL, 0.145 mmol) and the resulting mixture was stirred at room temperature for 40 min, before concentrating in vacuo. Purification by reverse phase preparative HPLC (gradient: 15- 75% acetonitrile over 20 min; compound eluted at 19 min), gave the title compound (67 mg, 58%) as a colourless oil. 20 (2S,3R,4S,5S,6S)-2-(2-(3-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4- ((((perfluorophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H- pyran-3,4,5-triyl triacetate (189) A solution of (2S,3R,4S,5S,6S)-2-(2-(3-((((9H-fluoren-9-25 yl)methoxy)carbonyl)amino)propanamido)-4-(hydroxymethyl)phenoxy)-6- (methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (188, 300 mg, 0.400 mmol) in N,N- DB1/ 159693888.5 245

133186-5030-WO dimethylformamide (3 mL) was charged with bis(pentafluorophenyl) carbonate (240 mg, 0.600 mmol) and N,N-diisopropylethylamine (18 µL, 0.100 mmol). The mixture was stirred at room temperature for 16 h, before diluting into ethyl acetate (60 mL) and washing successively with water (60 mL) and brine (50 mL). The organic layer was dried over anhydrous sodium sulfate 5 and concentrated to dryness under reduced pressure. Purification by flash column chromatography (silica), eluting with ethyl acetate/hexanes (17%), gave the title compound (280 mg, 73%) as a white solid. DB1/ 159693888.5 246

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133186-5030-WO (2S,3R,4S,5S,6S)-2-(2-(3-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4- ((((4-(4-(4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2- yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3- yl)oxy)butanamido)-1-methyl-1H-pyrrole-2- 5 carboxamido)phenyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro- 2H-pyran-3,4,5-triyl triacetate (191) y p y y y py yl)amino)-4-oxobutoxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10- 10 hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (190, 117 mg, 0.156 mmol) and (2S,3R,4S,5S,6S)-2-(2-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4- ((((perfluorophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H- pyran-3,4,5-triyl triacetate (189, 150 mg, 0.156 mmol) in anhydrous N,N-dimethylformamide (2 mL) was charged with 1-hydroxy-7-azabenzotriazole (14 mg, 0.10 mmol) and N,N- 15 diisopropylethylamine (54 µL, 0.31 mmol). The resulting mixture was stirred at room temperature for 16 h. Ethyl acetate (100 mL) was then added, and the solution was washed with water (100 mL), then brine (50 mL), then dried over anhydrous sodium sulfate, filtered, and concentrate in vacuo to give the title compound (240 mg) as a gum, that was used in the subsequent step without further purification. 20 (2S,3S,4S,5R,6S)-6-(4-((((4-(4-(4-(((6aS)-5-((Allyloxy)carbonyl)-6-hydroxy-2-methoxy-12- oxo-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)- 1-methyl-1H-pyrrole-2-carboxamido)phenyl)carbamoyl)oxy)methyl)-2-(3- aminopropanamido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (192) 25 A solution of (2S,3R,4S,5S,6S)-2-(2-(3-((((9H-Fluoren-9- yl)methoxy)carbonyl)amino)propanamido)-4-((((4-(4-(4-(((6aS)-5-((allyloxy)carbonyl)-2- methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12- octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2- DB1/ 159693888.5 248

133186-5030-WO carboxamido)phenyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H- pyran-3,4,5-triyl triacetate (191, 240 mg, 0.156 mmol) in acetonitrile (2 mL) and water (2 mL) was charged with sodium hydroxide (1 N in water, 1 mL) and the resulting mixture was stirred at room temperature for 1 h. Hydrochloric acid (37% in water, 0.5 mL) was then added and 5 stirring continued for an additional 30 min. Purification by reverse phase preparative HPLC (gradient: 2-50% acetonitrile over 20 min; compound eluted at 19 min), gave the title compound (40 mg, 24%) as a colourless oil. (2S,3S,4S,5R,6S)-6-(4-((((4-(4-(4-(((6aS)-5-((Allyloxy)carbonyl)-6-hydroxy-2-methoxy-12- oxo-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-10 1-methyl-1H-pyrrole-2-carboxamido)phenyl)carbamoyl)oxy)methyl)-2-((S)-32- (((allyloxy)carbonyl)amino)-26,33-dioxo-2,5,8,11,14,17,20,23-octaoxa-27,34- diazaheptatriacontan-37-amido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2- carboxylic acid (193) 15 A solution of (2S,3S,4S,5R,6S)-6-(4-((((4-(4-(4-(((6aS)-5-((allyloxy)carbonyl)-6-hydroxy-2- methoxy-12-oxo-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3- yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)carbamoyl)oxy)methyl)-2-(3- aminopropanamido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (192, 36 mg, 30 µmol) in anhydrous N,N-dimethylformamide (2 mL) was charged with perfluorophenyl20 (S)-32-(((allyloxy)carbonyl)amino)-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azatritriacontan- 33-oate (185, 26 mg, 33 µmol) and N,N-diisopropylethylamine (31 µL, 0.18 mmol) and the resulting mixture was stirred at room temperature for 20 min. Purification by reverse phase preparative HPLC (gradient: 2-65% acetonitrile over 20 min; compound eluted at 18 min), gave the title compound (48 mg, 76%) as a colourless oil. 25 (2S,3S,4S,5R,6S)-6-(2-((S)-32-Amino-26,33-dioxo-2,5,8,11,14,17,20,23-octaoxa-27,34- diazaheptatriacontan-37-amido)-4-((((4-(4-(4-(((S)-2-methoxy-12-oxo-6a,7,8,9,10,12- hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole- 2-carboxamido)phenyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H- pyran-2-carboxylic acid (194) DB1/ 159693888.5 249

133186-5030-WO , , , , onyl)-6-hydroxy-2- methoxy-12-oxo-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3- yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)carbamoyl)oxy)methyl)-2- 5 ((S)-32-(((allyloxy)carbonyl)amino)-26,33-dioxo-2,5,8,11,14,17,20,23-octaoxa-27,34- diazaheptatriacontan-37-amido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (193, 48 mg, 28 µmol) in anhydrous N,N-dimethylformamide (2 mL) was charged with tetrakis(triphenylphosphine)palladium(0) (6.6 mg, 5.7 µmol) and pyrrolidine (5 µL, 60 µmol) and the resulting mixture was purged with argon, then stirred at room temperature for 1 h. Ethyl 10 acetate (50 mL) was then added and the resulting solution was washed with water (50 mL) and brine (20 mL), and then dried over anhydrous sodium sulfate, before concentrating in vacuo to give the title compound (45 mg) as a gum, which was used immediately in the next step without further purification. (2S,3S,4S,5R,6S)-6-(2-((S)-32-(6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-15 26,33-dioxo-2,5,8,11,14,17,20,23-octaoxa-27,34-diazaheptatriacontan-37-amido)-4-((((4-(4- (4-(((S)-2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3- yl)oxy)butanamido)-1-methyl-1H-pyrrole-2- carboxamido)phenyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H- pyran-2-carboxylic acid triethylammonium salt (196) 20 DB1/ 159693888.5 250

133186-5030-WO A solution of (2S,3S,4S,5R,6S)-6-(2-((S)-32-Amino-26,33-dioxo-2,5,8,11,14,17,20,23-octaoxa- 27,34-diazaheptatriacontan-37-amido)-4-((((4-(4-(4-(((S)-2-methoxy-12-oxo-6a,7,8,9,10,12- hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2- carboxamido)phenyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2- 5 carboxylic acid (194, 45 mg, 28 µmol) in anhydrous N,N-dimethylformamide (2mL) was charged with perfluorophenyl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (195, 11 mg, 28 µmol) and N,N-diisopropylethylamine (10 µL, 60 µmol) and the resulting mixture was stirred at room temperature for 20 min. Purification by reverse phase preparative HPLC (gradient: 5-50% acetonitrile over 20 min; compound eluted at 20 min), gave the title compound 10 (20 mg, 37%) as a colourless oil. 1H NMR (400 MHz, DMSO-d6) δ 9.89 (s, 1H), 9.72 (s, 1H), 9.66 (s, 1H), 9.27 (s, 1H), 8.19 (s, 1H), 8.06 (s, 1H), 8.00 (d, J=8 Hz, 1H), 7.91 (d, J=8 Hz, 1H), 7.83 (br, 1H), 7.58 (d, J=8 Hz, 2H), 7.38 (d, J=8 Hz, 2H), 7.27 (s, 1H), 7.20 (s, 1H), 7.12 (dd, J=16, 4 Hz, 2H), 7.01 (s, 2H), 6.91 (s, 1H), 6.80 (s, 1H), 5.76-5.66 (m, 1H), 5.12-5.02 (m, 3H), 4.69 (br, 1H), 4.13-4.11 (m,15 2H), 4.04-4.02 (m, 2H), 3.82 (s, 3H), 3.81 (s, 3H), 3.71-3.67 (m, 2H), 3.58-3.55 (m, 3H), 3.51- 3.48 (m, 28H), 3.46-3.44 (m, 4H), 3.23 (s, 4H), 3.15-3.10 (m, 1H), 2.95-2.92 (m, 4H), 2.59-2.55 (m, 2H), 2.45-2.42 (m, 2H), 2.28 (t, J=4 Hz, 2H), 2.07-2.02 (m, 5H), 1.90-1.80 (m, 1H), 1.79- 1.65 (m, 3H), 1.64-1.50 (m, 2H), 1.49-1.35 (m, 5H), 1.34-1.20 (m, 4H), 1.15-1.00 (m, 10H); MS (ES+): m/z = 1688.2 (M+H)⁺; LCMS (5 min): tR = 2.06 min; HPLC (15 min): 10.19 min 20 (96.5% purity, 220 nm). DB1/ 159693888.5 251

O ^W-03 0 ⁵-6 _{1 1} S )₇ 0₂ ⁽ dn u_o p m o_cf o ^s _i s_e h_t n_y S_: X ⁵ _. 1 88 e_l 83 p 96 m 9 a 51 x _/ E 1B D

133186-5030-WO 4-(((6aS)-5-((Allyloxy)carbonyl)-2-hydroxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)- 5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoic acid (198) 5 -2-yl)oxy)-2-((2- (trimethylsilyl)ethoxy)methoxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine- 5(12H)-carboxylate (197, 2.50 g, 3.80 mmo253olubilizedbilised into a solution of tetra-n- butylammonium fluoride (1 M in THF, 30 mL), then charged with freshly activated molecular sieves (4 Å, 2.0 g), and the resulting suspension was stirred at 45 °C for 16 h. Ethyl acetate (200 10 mL) was then added, and the molecular sieves were removed by filtration. The filtrate was washed with water (100 mL) and brine (100 mL), and the organic layer was dried over anhydrous sodium sulfate, then concentrated in vacuo. The resulting residue was redissolved in methanol (40 mL), to which sodium hydroxide (1 M in water, 8 mL) was added. After stirring at room temperature for 3 h, hydrochloric acid (1 M, 4 mL) was added dropwise and the mixture 15 was concentrated in vacuo, to give the title compound, which was employed in the subsequent step without further purification. Allyl (6aS)-2-hydroxy-3-(4-((4-methoxybenzyl)oxy)-4-oxobutoxy)-12-oxo-6-((tetrahydro- 2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)- carboxylate (199) 20 4-(((6aS)-5-((Allyloxy)carbonyl)-2-hydroxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)- 5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoic acid (198, residue from previous into N,N-dimethylformamide (20 mL) and charged with sodium hydrogen carbonate (1.68 g), followed by 4-methoxybenzyl chloride (1.1 mL). The resulting mixture was 25 stirred at room temperature for 24 h, then partitioned between ethyl acetate (200 mL) and water (200 mL). The organic layer was dried over anhydrous sodium sulfate, then concentrated in vacuo. Purification by flash column chromatography (silica), eluting with ethyl acetate/hexanes (30-60%), gave the title compound (1.2 g, 50% yield over two steps) as an off-white solid. (2S,3R,4S,5S,6S)-2-(((6aS)-5-((Allyloxy)carbonyl)-3-(4-((4-methoxybenzyl)oxy)-4-30 oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12- DB1/ 159693888.5 253

133186-5030-WO octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-2-yl)oxy)-6-(methoxycarbonyl)tetrahydro- 2H-pyran-3,4,5-triyl triacetate (201) 2-oxo-6- 5 ((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine- 5(12H)-carboxylate (189, 640 mg, 1.00 mmol) in dichloromethane (15 mL) was charged with a solution of potassium carbonate (0.69 g, 5.00 mmol) in water (15 mL), tetrabutylammonium bromide (966 mg, 3.00 mmol) and acetobromo-α-D-glucuronic acid methyl ester (200, 2.00 g, 5.00 mmol). The resulting mixture was stirred at 40 °C for 24 h, before diluting into 10 dichloromethane (150 mL) and washing with water (50 mL x 2). The organic layer was concentrated in vacuo. Purification by flash column chromatography (silica), eluting with ethyl acetate/hexanes (from 30% to 60%) gave the title compound (430 mg, 45%) as a white solid. 4-(((6aS)-5-((Allyloxy)carbonyl)-6-hydroxy-12-oxo-2-(((2S,3R,4S,5S,6S)-3,4,5-triacetoxy-6- (methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12- 15 octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoic acid (202) (2S,3R,4S,5S,6S)-2-(((6aS)-5-((Allyloxy)carbonyl)-3-(4-((4-methoxybenzyl)oxy)-4-oxobutoxy)- 12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2- a][1,4]diazepin-2-yl)oxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (201, 20 240 mg, 0.251 mmol) was added to a mixture of trifluoroacetic acid/dichloromethane (1:9, 3 mL) and stirred at room temperature for 30 min. Acetonitrile/water (1:1, 4 mL) was then added and the solution concentrated under reduced pressure, to remove the dichloromethane. Purification by reverse phase preparative HPLC (gradient: 20-70% acetonitrile over 20 min; compound eluted at 19 min), gave the title compound (125 mg, 66%) as a colourless oil. 25 (2S,3R,4S,5S,6S)-2-(((6aS)-5-((Allyloxy)carbonyl)-3-(4-((5-((4-((S)-2-((S)-2- (((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)phenyl)carbamoyl)-1- methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-6-hydroxy-12-oxo-5,6,6a,7,8,9,10,12- DB1/ 159693888.5 254

133186-5030-WO octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-2-yl)oxy)-6-(methoxycarbonyl)tetrahydro- 2H-pyran-3,4,5-triyl triacetate (204) 5 triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12- octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoic acid (202, 108 mg, 0.144 mmol) and allyl ((S)-1-(((S)-1-((4-(4-amino-1-methyl-1H-pyrrole-2- carboxamido)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate trifluoroacetate salt (203, 85 mg, 0.142 mmol) in N,N-dimethylformamide (2 mL) was charged 10 with (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (80 mg, 0.153 mmol) and N,N-diisopropylethylamine (0.11 mL) and the resulting mixture was stirred at room temperature for 30 min. Purification by reverse phase preparative HPLC (gradient: 20-85% acetonitrile over 20 min; compound eluted at 20 min), gave the title compound (128 mg, 73%) as a colourless oil. 15 (2S,3S,4S,5R,6S)-6-(((6aS)-5-((Allyloxy)carbonyl)-3-(4-((5-((4-((S)-2-((S)-2- (((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)phenyl)carbamoyl)-1- methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-6-hydroxy-12-oxo-5,6,6a,7,8,9,10,12- octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-2-yl)oxy)-3,4,5-trihydroxytetrahydro-2H- pyran-2-carboxylic acid (205) 20 A suspension of (2S,3R,4S,5S,6S)-2-(((6aS)-5-((Allyloxy)carbonyl)-3-(4-((5-((4-((S)-2-((S)-2- (((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)phenyl)carbamoyl)-1-methyl- 1H-pyrrol-3-yl)amino)-4-oxobutoxy)-6-hydroxy-12-oxo-5,6,6a,7,8,9,10,12- octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-2-yl)oxy)-6-(methoxycarbonyl)tetrahydro-2H- 25 pyran-3,4,5-triyl triacetate (204, 128 mg, 0.105 mmol) in acetonitrile/water (4:1, 5 mL) was charged with an aqueous solution of sodium hydroxide (1 N, 0.7 mL) and the resulting mixture was stirred at room temperature for 2 h. Hydrochloric acid (1 N, 0.4 mL) was then added and the resulting mixture was directly purified. Purification by reverse phase preparative HPLC (gradient: 10-70% acetonitrile over 20 min; compound eluted at 18 min), gave the title 30 compound (108 mg, 96%) as a white powder. DB1/ 159693888.5 255

133186-5030-WO (2S,3S,4S,5R,6S)-6-(((S)-3-(4-((5-((4-((S)-2-((S)-2-Amino-3- methylbutanamido)propanamido)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4- oxobutoxy)-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-2-yl)oxy)- 3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (206) 5 , , , , (S)-2-((S)-2- (((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)phenyl)carbamoyl)-1-methyl- 1H-pyrrol-3-yl)amino)-4-oxobutoxy)-6-hydroxy-12-oxo-5,6,6a,7,8,9,10,12- octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-2-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2- 10 carboxylic acid (205, 30 mg, 0.025 mmol) in N,N-dimethylformamide (1.5 mL) was charged with tetrakis(triphenylphosphine)palladium(0) (7 mg) and pyrrolidine (14 µL), and the resulting mixture was stirred under argon for 20 min, then diluted with N,N-dimethylformamide (2 mL) and N,N-diisopropylethylamine (14 µL), concentrated under reduced pressure to approximately 2 mL, and then employed in the subsequent step with no further intermediate purification. 15 (2S,3S,4S,5R,6S)-6-(((S)-3-(4-((5-((4-((S)-2-((S)-2-(6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1- yl)hexanamido)-3-methylbutanamido)propanamido)phenyl)carbamoyl)-1-methyl-1H- pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2- a][1,4]diazepin-2-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (207) 20 The unpurified reaction mixture from the previous step, containing (2S,3S,4S,5R,6S)-6-(((S)-3- (4-((5-((4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)phenyl)carbamoyl)-1-methyl- 1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2- a][1,4]diazepin-2-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (206) was charged with perfluorophenyl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (195, 44 mg, 25 0.117 mmol), followed by N,N-diisopropylethylamine (12 µL) and the resulting mixture was stirred at room temperature for 30 min, before purifying directly. Purification by reverse phase preparative HPLC (gradient: 5-60% acetonitrile over 20 min; compound eluted at 19 min), gave the title compound (16 mg, 53%) as a white solid. DB1/ 159693888.5 256

133186-5030-WO 1H NMR (400 MHz, DMSO-d₆) δ 10.40-10.08 (m, 1H), 9.82 (s, 1H), 9.77 (s, 1H), 8.12 (d, J=8 Hz, 1H), 8.03-8.01 (m, 1H), 7.81 (d, J=8 Hz, 1H), 7.62 (d, J=8 Hz, 2H), 7.50 (d, J=8 Hz, 2H), 7.45 (s, 1H), 7.21-7.19 (m, 1H), 7.01-6.81 (m, 3H), 5.80-5.71 (m, 5H), 5.50-5.15 (m, 1H), 5.03- 4.85 (m, 1H), 4.39-4.36 (m, 1H), 4.19-4.16 (m, 1H), 4.08-3.99 (m, 2H), 3.81-3.80 (m, 3H), 5 3.79-3.65 (m, 1H), 2.74 (br, 7H), 2.16-2.13 (m, 2H), 2.10-1.97 (m, 4H), 1.91 (s, 2H), 1.80-1.60 (m, 3H), 1.51-1.46 (m, 6H), 1.30 (d, J=8 Hz, 3H), 1.20-1.17 (m, 2H), 0.87 (d, J=4 Hz, 3H), 0.83 (d, J=4 Hz, 3H); MS (ES+): m/z = 1084.4 (M+H)⁺; LCMS (5 min): t_R = 2.00 min; HPLC (15 min): 9.35 min (99.5% purity, 220 nm). Example 1Y: Synthesis of compound (213) 10 Scheme 25. Synthesis of compound (213). (2R,3R,4S,5R,6S)-2-(Acetoxymethyl)-6-(4-(((tert-butyldimethylsilyl)oxy)methyl)-2- nitrophenoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (209) 15 A solution of (2R,3R,4S,5R,6S)-2-(acetoxymethyl)-6-(4-(hydroxymethyl)-2- nitrophenoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (208, 2.00 g, 4.10 mmol) in anhydrous dichloromethane (25 mL) was charged with tert-butyldimethylsilyl chloride (0.93 g, 6.20 mmol) and imidazole (0.70 g, 10.3 mmol) and the resulting mixture was stirred at room temperature for 1 h. After diluting into dichloromethane (60 mL), the mixture was washed with water (50 mL), 20 then brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo, to give the title compound (2.5 g, quant.) as a tan solid. (2S,3R,4S,5S,6R)-2-(4-(((tert-Butyldimethylsilyl)oxy)methyl)-2-nitrophenoxy)-6- (hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol (210) DB1/ 159693888.5 257

133186-5030-WO butyldimethylsilyl)oxy)methyl)-2-nitrophenoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (209, 2.5 g, 4.10 mmol) in methanol (50 mL), was charged with sodium methoxide (25% solution in 5 methanol, 0.2 mL) and the resulting mixture was stirred at room temperature for 1 h, after which evaporation under reduced pressure gave the title compound as a tan solid, that was employed in the subsequent step without further purification. Triallyl ((2R,3R,4S,5R,6S)-2-((((allyloxy)carbonyl)oxy)methyl)-6-(4-(((tert- butyldimethylsilyl)oxy)methyl)-2-nitrophenoxy)tetrahydro-2H-pyran-3,4,5-triyl) 10 tricarbonate (211) A solution of (2S,3R,4S,5S,6R)-2-(4-(((tert-butyldimethylsilyl)oxy)methyl)-2-nitrophenoxy)-6- (hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol (210, 4.10 mmol, residue from previous step) in pyridine (30 mL) was cooled to 0 °C and charged with allyl chloroformate (10 mL, 94 15 mmol), dropwise with stirring, for 16 h. The solvent was then removed under reduced pressure and the residue redissolved into dichloromethane (200 mL), then washed with water (200 mL x 2), brine (200 mL), dried over anhydrous sodium sulfate, and concentrated in vacuo, to give the title compound (3.2 g) as a tan solid. Triallyl ((2R,3R,4S,5R,6S)-2-((((allyloxy)carbonyl)oxy)methyl)-6-(4-(hydroxymethyl)-2- 20 nitrophenoxy)tetrahydro-2H-pyran-3,4,5-triyl) tricarbonate (212) A solution of triallyl ((2R,3R,4S,5R,6S)-2-((((allyloxy)carbonyl)oxy)methyl)-6-(4-(((tert- butyldimethylsilyl)oxy)methyl)-2-nitrophenoxy)tetrahydro-2H-pyran-3,4,5-triyl) tricarbonate (211, 3.2 g, 4.10 mmol) in acetonitrile (10 mL) was charged with triethylamine trihydrofluoride 25 (2.0 g, 12.3 mmol) and the resulting mixture was stirred at room temperature for 1 h. The solvent was then removed under reduced pressure and the residue redissolved into dichloromethane (200 mL), then washed with water (200 mL x 2), brine (200 mL), dried over anhydrous sodium sulfate, and concentrated in vacuo. Purification by flash column DB1/ 159693888.5 258

133186-5030-WO chromatography (silica), eluting with ethyl acetate/hexanes (40%), gave the title compound (2.1 g, 79% yield over 4 steps), as a white solid. Triallyl ((2R,3R,4S,5R,6S)-2-((((allyloxy)carbonyl)oxy)methyl)-6-(2-nitro-4- ((((perfluorophenoxy)carbonyl)oxy)methyl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyl) 5 tricarbonate (213) , , , , -(4- (hydroxymethyl)-2-nitrophenoxy)tetrahydro-2H-pyran-3,4,5-triyl) tricarbonate (212, 1.50 g, 2.20 mmol) in N,N-dimethylformamide (10 mL) was charged with bis(pentafluorophenyl) 10 carbonate (0.86 g, 2.3 mmol), and the resulting mixture was stirred at room temperature for 4 h. The solvent was then evaporated under reduced pressure, then co-evaporated with acetonitrile, t_{o give the title compound (2.5 g) as a white solid.} DB1/ 159693888.5 259

O ^W-03 0 ⁵-6 S _H ^H O H O _, N O N _{H N} 02 C H 2 H O 2 e O N O O M N O _, O ⁾ _. O _{N H} q c o c O a l^{l o} _, A _l ^l O A H _{N H} ^M c l^o O O 8 O 1 l _O O H N 12 ⁽ H A _O 51 O O O N2 2 H _N O _{H N} a N O O O _)i ⁱ _i; )₀ Oc O _{N H} ^. _t ^. _r O _, 2 M 2 ^o _l ^l O A ( _{N H} C D O _, d O n P u_{o H N} M E O T p _i ⁾ O _, m N A D ^o _{I c N H f} c O ^P _i ^{) o o} l H H _l ^i; _. A ^t _. ^r . s_i O ^{, s} _F ^t _. ^r e 2 ^, H N M_F D h M t N O _, AD n_y E_, P_I AE S ) _i ^v _i ^{) D,} _t ^P _{I :} v D Z _{O O} 41 A_, ⁵ _. 1 2 O^p _f H^P- 88 e_l 3_, C 8 p 1 3 2 ^M i⁾ _i ⁾ 96 m v 9 a O 51 x O _/ E 1B D

133186-5030-WO Methyl 4-(4-((S)-2-(hydroxymethyl)piperidine-1-carbonyl)-2-methoxy-5-((((3-nitro-4- (((2S,3R,4S,5R,6R)-3,4,5-tris(((allyloxy)carbonyl)oxy)-6- ((((allyloxy)carbonyl)oxy)methyl)tetrahydro-2H-pyran-2- yl)oxy)benzyl)oxy)carbonyl)amino)phenoxy)butanoate (215) 5 hyl)piperidine-1-carbonyl)-2- methoxyphenoxy)butanoate trifluoroacetate salt (214, 377 mg, 0.76 mmol) in anhydrous N,N- dimethylformamide (2 mL) was charged with triallyl ((2R,3R,4S,5R,6S)-2- ((((allyloxy)carbonyl)oxy)methyl)-6-(2-nitro-4- 10 ((((perfluorophenoxy)carbonyl)oxy)methyl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyl) tricarbonate (213, 836 mg, 0.95 mmol), 1-hydroxy-7-azabenzotriazole (46 mg, 0.34 mmol) and N,N-diisopropylethylamine (0.27 mL, 1.5 mmol) and the resulting mixture was stirred at room temperature for 5 h. Purification by reverse phase preparative HPLC (gradient: 35-95% acetonitrile over 20 min; compound eluted at 18 min), gave the title compound (600 mg, 73%) 15 as a tan solid. 3-Nitro-4-(((2S,3R,4S,5R,6R)-3,4,5-tris(((allyloxy)carbonyl)oxy)-6- ((((allyloxy)carbonyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl 6-hydroxy-2- methoxy-3-(4-methoxy-4-oxobutoxy)-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2- a][1,4]diazepine-5(12H)-carboxylate (216) 20 A solution of methyl 4-(4-((S)-2-(hydroxymethyl)piperidine-1-carbonyl)-2-methoxy-5-((((3- nitro-4-(((2S,3R,4S,5R,6R)-3,4,5-tris(((allyloxy)carbonyl)oxy)-6- ((((allyloxy)carbonyl)oxy)methyl)tetrahydro-2H-pyran-2- yl)oxy)benzyl)oxy)carbonyl)amino)phenoxy)butanoate (215, 550 mg, 0.51 mmol) in 25 dichloromethane (10 mL) was charged with (diacetoxyiodo)benzene (412 mg, 1.28 mmol) and DB1/ 159693888.5 261

133186-5030-WO TEMPO (8 mg, 51 µmol) and the resulting mixture was stirred at room temperature for 5 days. The solvent was then removed under reduced pressure and the resulting residue redissolved in acetonitrile (7 mL) and water (1 mL). Purification by reverse phase preparative HPLC (gradient: 25-95% acetonitrile over 20 min; compound eluted at 19 min), gave the title compound (268 5 mg, 49%) as a tan solid. 4-((6-Hydroxy-2-methoxy-5-(((3-nitro-4-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)-12-oxo- 5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoic acid (217) 10 so u on o -n ro- -((( , , , , )- , , - r s(((a yoxy)carbonyl)oxy)-6- ((((allyloxy)carbonyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl 6-hydroxy-2-methoxy- 3-(4-methoxy-4-oxobutoxy)-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2- a][1,4]diazepine-5(12H)-carboxylate (216, 348 mg, 0.33 mmol) in acetonitrile (5 mL) was 15 charged with an aqueous solution of sodium hydroxide (1 N, 3 mL), and the resulting mixture was stirred at room temperature for 4 h, before concentrating in vacuo to a volume of 5 mL, which was purified directly. Purification by reverse phase preparative HPLC (gradient: 5-50% acetonitrile over 20 min; compound eluted at 17 min), gave the title compound (127 mg, 54%) as a white solid. 20 3-Nitro-4-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2- yl)oxy)benzyl (6aS)-3-(4-((5-((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3- methylbutanamido)propanamido)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4- oxobutoxy)-6-hydroxy-2-methoxy-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2- a][1,4]diazepine-5(12H)-carboxylate (218) 25 A solution of 4-((6-hydroxy-2-methoxy-5-(((3-nitro-4-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)-12-oxo- 5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoic acid (217, DB1/ 159693888.5 262

133186-5030-WO 50 mg, 69.3 µmol) in anhydrous N,N-dimethylformamide (1 mL) was charged with allyl ((S)-1- (((S)-1-((4-(4-amino-1-methyl-1H-pyrrole-2-carboxamido)phenyl)amino)-1-oxopropan-2- yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate trifluoroacetate salt (193, 42 mg, 70.2 µmol), N,N-diisopropylethylamine (60 µL, 345 µmol) and (7-azabenzotriazol-1- 5 yloxy)tripyrrolidinophosphonium hexafluorophosphate (36.2 mg, 69.3 µmol) and the resulting mixture was stirred at room temperature for 30 min, before purifying directly. Purification by reverse phase preparative HPLC (gradient: 10-60% acetonitrile over 20 min; compound eluted at 19 min), gave the title compound (80 mg, 98%) as a tan solid. 3-Nitro-4-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-10 yl)oxy)benzyl (6aS)-3-(4-((5-((4-((S)-2-((S)-2-amino-3- methylbutanamido)propanamido)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4- oxobutoxy)-6-hydroxy-2-methoxy-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2- a][1,4]diazepine-5(12H)-carboxylate (219) 15 A solution of 3-nitro-4-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H- pyran-2-yl)oxy)benzyl (6aS)-3-(4-((5-((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3- methylbutanamido)propanamido)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4- oxobutoxy)-6-hydroxy-2-methoxy-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2- a][1,4]diazepine-5(12H)-carboxylate (218, 80 mg, 67 µmol) in dichloromethane (2 mL) and 20 N,N-dimethylformamide (2 mL) was charged with formic acid (38 µL) and pyrrolidine (83 µL), followed by tetrakis(triphenylphosphine)palladium(0) (3.6 mg, 3.1 µmol), and the resulting mixture was stirred at room temperature for 20 min. The dichloromethane was then evaporated under reduced pressure and the remaining solution was purified directly. Purification by reverse phase preparative HPLC (gradient: 5-45% acetonitrile over 20 min; compound eluted at 1825 min), gave the title compound (63 mg, 77%) as the TFA salt. 3-Nitro-4-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2- yl)oxy)benzyl (6aS)-3-(4-((5-((4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)hexanamido)-3-methylbutanamido)propanamido)phenyl)carbamoyl)-1-methyl-1H- pyrrol-3-yl)amino)-4-oxobutoxy)-6-hydroxy-2-methoxy-12-oxo-6,6a,7,8,9,10- 30 hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (220) DB1/ 159693888.5 263

133186-5030-WO pyran-2-yl)oxy)benzyl (6aS)-3-(4-((5-((4-((S)-2-((S)-2-amino-3- methylbutanamido)propanamido)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4- 5 oxobutoxy)-6-hydroxy-2-methoxy-12-oxo-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2- a][1,4]diazepine-5(12H)-carboxylate (219, 63 mg, 52 µmol) in N,N-dimethylformamide (2 mL) was charged with perfluorophenyl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (195, 24 m_{g, 63 µmol) and N,N-diisopropylethylamine (27 µL, 190 µmol) and the resulting mixture was} stirred at room temperature for 1 h, before purifying directly. Purification by reverse phase 10 preparative HPLC (gradient: 10-65% acetonitrile over 20 min; compound eluted at 18 min), gave the title compound (51.6 mg, 77%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.89 (s, 1H), 9.83 (s, 1H), 9.75 (s, 1H), 8.12 (d, J=8 Hz, 1H), 7.81 (d, J=8 Hz, 1H), 7.73 (s, 1H), 7.61 (d, J=8 Hz, 2H), 7.51 (d, J=8 Hz, 2H), 7.37 (d, J=8 Hz, 1H), 7.21 (s, 1H), 7.02 (s, 1H), 7.00 (s, 2H), 6.93 (s, 1H), 6.79 (d, J=8 Hz, 1H), 6.53 (br, 1H), 15 5.78 (d, J=8 Hz, 1H), 5.16-5.12 (m, 3H), 4.97-4.91 (m, 1H), 4.39-4.36 (m, 1H), 4.18-4.15 (m, 1H), 4.12-4.10 (m, 1H), 3.97 (br, 2H), 3.81 (s, 3H), 3.80 (s, 3H), 3.66 (br, 3H), 3.19-3.16 (m, 5H), 2.89-2.87 (m, 1H), 2.45-2.43 (m, 2H), 2.20-2.15 (m, 2H), 2.03-1.87 (m, 4H), 1.70-1.61 (m, 2H), 1.52-1.45 (m, 7H), 1.29 (d, J=8 Hz, 3H), 1.21-1.17 (m, 2H), 0.87 (d, J=4 Hz, 3H), 0.83 (d, J=4 Hz, 3H); MS (ES+): m/z = 1298.5 (M+H)⁺; LCMS (5 min): tR = 1.89 min; HPLC (15 min): 20 5.93 min (99.8% purity, 220 nm). Example 1AA: Synthesis of compound (222) DB1/ 159693888.5 264

133186-5030-WO Scheme 27. Synthesis of compound (222). 3-Nitro-4-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2- yl)oxy)benzyl (6aS)-3-(4-((5-((4-((tert-butoxycarbonyl)amino)phenyl)carbamoyl)-1-methyl- 5 1H-pyrrol-3-yl)amino)-4-oxobutoxy)-6-hydroxy-2-methoxy-12-oxo-6,6a,7,8,9,10- hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (221) A solution 4-((6-hydroxy-2-methoxy-5-(((3-nitro-4-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)-12-oxo- 10 5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoic acid (217, 6.8 mg, 9.4 µmol) in anhydrous N,N-dimethylformamide (1 mL) was charged with tert-butyl (4- (4-amino-1-methyl-1H-pyrrole-2-carboxamido)phenyl)carbamate trifluoroacetate salt (176, 4.2 mg, 9.4 µmol), N,N-diisopropylethylamine (6.8 µL, 39 µmol), and (7-azabenzotriazol-1- yloxy)tripyrrolidinophosphonium hexafluorophosphate (5.1 mg, 9.7 µmol) and the resulting 15 mixture was stirred at room temperature for 10 min, before purifying directly. Purification by reverse phase preparative HPLC (gradient: 10-65% acetonitrile over 20 min; compound eluted at 19.5 min), gave the title compound (8 mg, 82%) as a white solid. 3-Nitro-4-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2- yl)oxy)benzyl (6aS)-3-(4-((5-((4-aminophenyl)carbamoyl)-1-methyl-1H-pyrrol-3-20 yl)amino)-4-oxobutoxy)-6-hydroxy-2-methoxy-12-oxo-6,6a,7,8,9,10- hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate trifluoroacetate salt (222) DB1/ 159693888.5 265

133186-5030-WO )tetrahydro-2H- pyran-2-yl)oxy)benzyl (6aS)-3-(4-((5-((4-((tert-butoxycarbonyl)amino)phenyl)carbamoyl)-1- methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-6-hydroxy-2-methoxy-12-oxo-6,6a,7,8,9,10- 5 hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (221, 8 mg, 7.7 µmol) in dichloromethane (2 mL) was charged with trifluoroacetic acid (1 mL), and the resulting mixture was stirred at room temperature for 10 min, before concentrating in vacuo. The residue was then redissolved into acetonitrile (1 mL) and water (0.5 mL) and purified directly. Purification by reverse phase preparative HPLC (gradient: 2-40% acetonitrile over 20 min; compound eluted at 10 19 min), gave the title compound (6.5 mg, 80%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.89 (s, 1H), 9.71 (s, 1H), 7.85 (s, 1H), 7.73 (s, 1H), 7.56 (d, J=8 Hz, 2H), 7.49 (br, 1H), 7.37 (d, J=8 Hz, 1H), 7.20 (s, 1H), 7.02 (s, 1H), 6.92-6.91 (m, 3H), 6.79-6.78 (m, 1H), 6.53 (br, 1H), 5.77 (br, 1H), 5.40-5.25 (m, 1H), 5.15-5.12 (m, 4H), 5.30-4.95 (m, 1H), 4.11 (d, J=8 Hz, 1H), 4.30-3.91 (m, 2H), 3.81 (s, 3H), 3.80 (s, 3H), 3.67 (d, J=8 Hz, 15 1H), 3.23-3.16 (m, 6H), 2.89 (br, 1H), 2.44-2.36 (m, 2H), 2.07-2.02 (m, 2H), 1.87 (br, 1H), 1.68-1.63 (m, 2H), 1.61-1.41 (m, 3H); MS (ES+): m/z = 934.2 (M+H)⁺; LCMS (5 min): t_R = 1.43 min; HPLC (15 min): 7.40 min (99.5% purity, 220 nm). DB1/ 159693888.5 266

O ^W-03 0 ⁵-6 ⁸ ₁3³ ₁ ) 1₉ ⁽ dnuop m o_cf o ^s _i ^s _e h_tnyS_: B B ⁵ _. 1 88 e_l 83 p 96 m 9 a 51 x _/ E 1B D

133186-5030-WO (2R,3S,4S,5R,6S)-2-(Acetoxymethyl)-6-(((6aS)-5-((allyloxy)carbonyl)-3-(4-methoxy-4- oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12- octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-2-yl)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (224) 5 o-2H-pyran-2- yl)oxy)-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoic acid (198, 200 mg, 0.37 mmol) in dichloromethane (6 mL) was charged with a solution of potassium carbonate (0.25 g, 1.9 mmol) in water (3 mL), tetrabutylammonium bromide (362 mg, 1.10 10 mmol) and acetobromo-α-D-galactose (223, 0.83 g, 1.90 mmol), and the resulting mixture was stirred at room temperature for 4 h. The mixture was then diluted into ethyl acetate (60 mL) and washed with water (40 mL x 2). The organic layer was then concentrated in vacuo and the residue was employed in the next step without further purification. 4-(((6aS)-5-((Allyloxy)carbonyl)-6-hydroxy-12-oxo-2-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-15 6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12- octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoic acid (225) The residue from the previous step, containing (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(((6aS)-5- ((allyloxy)carbonyl)-3-(4-methoxy-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-20 5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-2-yl)oxy)tetrahydro-2H-pyran- 3,4,5-triyl triacetate (224) was suspended into acetonitrile/water (4:1, 8 mL), and charged with an aqueous solution of sodium hydroxide (1 N, 3 mL), then stirred at room temperature for 2 h. Hydrochloric acid (11 M, 0.6 mL) was then added and the resulting mixture was stirred for a further 30 min, before purifying directly. Purification by reverse phase preparative HPLC 25 (gradient: 5-45% acetonitrile over 20 min; compound eluted at 19 min), gave the title compound (140 mg, 63% yield over two steps) as a white powder. Allyl (6aS)-3-(4-((5-((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3- methylbutanamido)propanamido)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4- oxobutoxy)-6-hydroxy-12-oxo-2-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6- DB1/ 159693888.5 268

133186-5030-WO (hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10- hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (226) 5 trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-5,6,6a,7,8,9,10,12- octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoic acid (225, 75 mg, 0.126 mmol) and allyl ((S)-1-(((S)-1-((4-(4-amino-1-methyl-1H-pyrrole-2-carboxamido)phenyl)amino)-1- oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate trifluoroacetate salt (203, 75 mg, 0.125 mmol) in N,N-dimethylformamide (3 mL) was charged with (7-azabenzotriazol-1-10 yloxy)tripyrrolidinophosphonium hexafluorophosphate (72 mg, 0.138 mmol), followed by N,N- diisopropylethylamine (0.10 mL) and the resulting mixture was stirred at room temperature for 15 min, whereupon it was purified directly. Purification by reverse phase preparative HPLC (gradient: 10-60% acetonitrile over 15 min; compound eluted at 13 min), gave the title compound (110 mg, 82%) as a white solid. 15 N-(4-((S)-2-((S)-2-Amino-3-methylbutanamido)propanamido)phenyl)-1-methyl-4-(4-(((S)- 12-oxo-2-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2- yl)oxy)-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)- 1H-pyrrole-2-carboxamide (90) 20 A solution of allyl (6aS)-3-(4-((5-((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3- methylbutanamido)propanamido)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4- oxobutoxy)-6-hydroxy-12-oxo-2-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2- a][1,4]diazepine-5(12H)-carboxylate (89, 30 mg, 0.028 mmol) in N,N-dimethylformamide (1.5 25 mL) was charged with tetrakis(triphenylphosphine)palladium(0) (7 mg) and pyrrolidine (14 µL) and the resulting mixture was stirred under argon at room temperature for 20 min. The unpurified reaction mixture was used in the subsequent step with no further intermediate purification. N-(4-((S)-2-((S)-2-(6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-30 methylbutanamido)propanamido)phenyl)-1-methyl-4-(4-(((S)-12-oxo-2- DB1/ 159693888.5 269

133186-5030-WO (((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)- 6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1H- pyrrole-2-carboxamide (91) 5 methylbutanamido)propanamido)phenyl)-1-methyl-4-(4-(((S)-12-oxo-2-(((2S,3R,4S,5R,6R)- 3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-6a,7,8,9,10,12- hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1H-pyrrole-2- carboxamide (90) was diluted with N,N-dimethylformamide (2 mL) and charged with N,N-10 diisopropylethylamine (14 µL) and then concentrated to 2 mL total volume. Perfluorophenyl 6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (195, 24 mg, 0.064 mmol) was added, followed by N,N-diisopropylethylamine (12 µL). The resulting mixture was stirred at room temperature for 30 min and then purified directly. Purification by reverse phase preparative HPLC (gradient: 5-50% acetonitrile over 30 min; compound eluted at 26 min), gave the title 15 compound (26 mg, 87%) as a white solid. 1H NMR (400 MHz, DMSO-d₆) δ 9.85-9.83 (m, 2H), 9.75 (s, 1H), 8.13-8.01 (m, 2H), 7.82 (d, J=4 Hz, 1H), 7.61 (d, J=8 Hz, 2H), 7.51 (d, J=8 Hz, 2H), 7.21 (s, 1H), 7.00 (s, 2H), 6.94 (s, 1H), 6.83 (s, 1H), 5.76-5.40 (m, 1H), 5.09-4.96 (m, 1H), 4.87 (d, J=8 Hz, 2H), 4.71-4.50 (m, 2H), 4.39-4.36 (m, 1H), 4.18-4.15 (m, 2H), 4.10-3.90 (m, 3H), 3.81 (s, 3H), 3.71-3.70 (m, 2H), 20 3.65-3.55 (m, 3H), 3.15-3.00 (m, 1H), 2.47-2.46 (m, 6H), 2.20-2.11 (m, 2H), 2.05-2.02 (m, 2H), 1.97-1.96 (m, 1H), 1.90-1.60 (m, 3H), 1.52-1.45 (m, 6H), 1.30 (d, J=8 Hz, 3H), 1.20-1.17 (m, 2H), 0.87 (d, J=4 Hz, 3H), 0.83 (d, J=4 Hz, 3H); MS (ES+): m/z = 1070.5 (M+H)⁺; LCMS (5 min): tR = 1.78 min; HPLC (15 min): 8.85 min (100% purity, 220 nm). Example 1CC: Alternative synthesis of compound (180). 25 This Example describes an alternative synthetic procedure for compound 180 (compound 180 provides CDCP1-180 upon conjugation to CDCP1 antibody). DB1/ 159693888.5 270

133186-5030-WO Part 1. The prepar ation of compound 1.13 The overview of an exemplary complete synthesis scheme to produce intermediate compound 1.13 is depicted in Figure 24A. 5 _{Step 1.1.1. The preparation of compound 1.2} A solution of 1.1 (methyl 1-methyl-4-nitropyrrole-2-carboxylic acid; 156 g, 847.8 mmol, 1 eq) in THF (1600 mL) was charged with NaOH (67.8 g, 1695.6 mmol, 2 eq), add water(1600 ml) to it and the resulting mixture was stirred at 60^oC for 2 hrs. After completion, the reaction was quenched by addition of an aqueous solution of HCl (1 M) until the pH was adjusted to 3-4. 10 Then extracted with ethyl acetate (3 x 1.5 L). The combined organic extracts were then washed with water (2 x 1 L) and brine (1 L), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. Dried in vacuum to give the title compound 1.2 (130.41 g.90% yield) as a yellow solid (Figure 24B). LCMS: r.t = 0.714 mins, MS m/z (ESI): 171.0 [M+H]⁺. ¹H NMR (400 MHz, DMSO) δ 13.14 15 (s, 1H), 8.23 (d, J = 2.0 Hz, 1H), 7.26 (d, J = 2.0 Hz, 1H), 3.92 (s, 3H). Step 1.1.2. The preparation of compound 1.4 A solution of 1.2 (130.41 g, 767.1 mmol, 1 eq), HATU (320.6 g, 843.8 mmol, 1.1 eq) and DIEA (247.4 g, 1917.8 mmol, 2.5 eq) in DMF (1400 mL) was stirred at room temperature for 30 min. Compound 1.3 (2-methylpropan-2-yl [(4-aminophenyl)amino] methanoate; 159.6 g, 767.1 20 mmol, 1 eq) was added and the reaction mixture was stirred at room temperature for 2 hrs. After completion, H2O (3 L) was added slowly to the reaction mixture under stirring at room temperature for 10 mins, solid appeared. Filtered and collected the solid and dried in vacuum to g_{ive the title compound 1.4 (167.2 g, 60% yield) as a yellow solid (Figure 24C).} DB1/ 159693888.5 271

133186-5030-WO LCMS: r.t = 1.143 mins, MS m/z (ESI): 383.1 [M+Na]⁺. ¹H NMR (400 MHz, DMSO) δ 10.04 (s, 1H), 9.30 (s, 1H), 8.20 (d, J = 1.6 Hz, 1H), 7.66 (d, J = 2.0 Hz, 1H), 7.57 (d, J = 9.2 Hz, 2H), 7.40 (d, J = 8.8 Hz, 2H), 3.95 (s, 3H), 1.47 (s, 9H). Step 1.1.3. The preparation of compound 1.5 5 A solution of 1.4 (167.2 g, 464.4 mmol, 1 eq) in DMF (1700 mL) was charged with B2(OH)4 (125.4 g, 1393.3 mmol, 3 eq) and 4,4'-Bipyridine (3.6 g, 23.2 mmol, 0.05 eq) at 0^oC and the resulting mixture was stirred at room temperature for 30 mins. After completion, the reaction mixture was then partitioned between water (10 L), stirred at room temperature for 10 mins. Filtered and collected the solid and purified by column chromatography (MeOH=10/1) to get 10 compound 1.5 (66 g, 43% yield) as yellow solid (Figure 24D). LCMS: r.t = 0.762 mins, MS m/z (ESI): 331.2 [M+H]⁺. ¹H NMR (400 MHz, DMSO) δ 9.41 (s, 1H), 9.21 (s, 1H), 7.54 (d, J = 9.2 Hz, 2H), 7.34 (d, J = 8.8 Hz, 2H), 6.42 (d, J = 2.0 Hz, 1H), 6.29 (d, J = 2.0 Hz, 1H), 3.84 (s, 2H), 3.72 (s, 3H), 1.47 (s, 9H). Step 1.1.4. The preparation of compound 1.7 15 A solution of 1.6 (5-methoxy-2-nitro-4-{[tri(prop-2-yl)silyl]oxy}benzoic acid; 300 g, 811.93 mmol), HATU (340 g, 893.12 mmol) and dry triethylamine (205 g, 2023.83 mmol) in dry dichloromethane (3 L) was stirred at room temperature for 90 min. Then 1.6a ([(2S)- hexahydropyridin-2-yl]methanol; 93.52 g, 811.93 mmol) was added and the reaction mixture was stirred at room temperature for 12 hrs. After completion, the reaction mixture was then 20 partitioned between dichloromethane (1500 mL × 3). The combined organic extracts were then dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting residue was purified by column chromatography (silica), eluting with ethyl acetate/petroleum ether (from 5_{0% to 75%) to give the title compound 1.7 (300 g, crude) as yellow oil. The crude product was} added EA (500 mL) and PE (1500mL) and stirred at rt. for 30min, filtered and the product was 25 dried in vacuo to give pure 1.7 (200 g, 52.8% yield) as a white solid (Figure 24E). LCMS: r.t = 1.477 mins, MS m/z (ESI): 467 (M+H)⁺. Step 1.1.5. The preparation of compound 1.8 A mixture of 1.7 (143 g, 306.9 mmol, 1 eq) in THF (1500 mL) and TBAF (460.3 mL, 460.3 mmol, 1.5 eq) was stirred at room temperature for 2 hrs. After completion, the 30 solvent was removed under reduced pressure and dried in vacuum to get crude 1.8 (121 g) as yellow oil and used directly for the next step without further purification (Figure 24F). LCMS: r.t = 0.757 mins, MS m/z (ESI): 311.1 [M+H]⁺. DB1/ 159693888.5 272

133186-5030-WO Step 1.1.6. The preparation of compound 1.10 A solution of the above crude 1.8 (121 g, 390.3 mmol, 1 eq), 1.9 (methyl 4-bromobutanoate; 70.2 g, 390.3 mmol, 1 eq), and K2CO3 (107.7 g, 780.6 mmol, 2 eq) in DMF (1300 mL) was stirred at 50℃ for 16 hrs. The reaction mixture was then partitioned between EtOAc (1 L × 3) 5 and water (2 L). The combined organic extracts were then dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to afford crude 1.10 (84 g, 66% yield 2 steps) as yellow oil. Trace TBAF remained (Figure 24G). LCMS: r.t = 1.200 mins, MS m/z (ESI): 411.2 [M+H]⁺. Step 1.1.7. The preparation of compound 1.11 10 A solution of above crude 1.10 (84 g, 204.4 mmol, 1 eq) in THF (800 mL) was charged with an NaOH (16.3 g, 408.7 mmol), water (160 ml) was added to it and the resulting mixture was stirred at 60℃ for 2 hrs. After completion, it was quenched by addition of an aqueous solution of HCl (1 M) until the pH to 3-4. Then extracted with ethyl acetate (3 x 1 L). The combined organic extracts were then washed with water (1 L) and brine (1 L), dried over anhydrous 15 sodium sulfate. Filtered and concentrated in vacuo to give the title compound 1.11 (63 g.77% yield) as a white solid (Figure 24H). LCMS: r.t = 0.763 mins, MS m/z (ESI): 397.1 [M+H]⁺. Step 1.1.8. The preparation of compound 1.12 A solution of 1.11 (63 g, 159 mmol, 1 eq) in anhydrous DMF (800 mL) was charged with 1.5 20 (52.5 g, 159 mmol, 1 eq), DIEA (51.3 g, 398 mmol, 2.5 eq) and PyBOP (91 g, 175 mmol, 1.1 eq). The resulting mixture was stirred at room temperature for 2 hrs. After completion, the solution was purified by RP-HPLC (gradient: 20-80% acetonitrile) to give the title compound 1.12 (45 g, 40% yield) as a brown solid (Figure 24I). LCMS: r.t = 1.059 mins, MS m/z (ESI): 709.3 [M+H]⁺. 25 Step 1.1.9. The preparation of compound 1.13 A solution of 1.12 (45 g, 63.5 mmol) in MeOH (800 mL) was charged with palladium on activated charcoal (10 wt. % basis, 4.5 g). Then it was stirred at room temperature under H₂ balloon for 24 hrs. After completion, the resulting mixture was filtered through celite, the filter cake was washed with MeOH (200 mL). Concentrated and dried in vacuum to afford the title 30 compound 1.13 (36.2 g, 84% yield) as a yellow solid (Figure 24H). LCMS: r.t = 0.970 mins, MS m/z (ESI): 679.4 [M+H]⁺. DB1/ 159693888.5 273

133186-5030-WO Part 2. The preparation of compound (180) The overview of an exemplary complete synthesis scheme to produce the subject compound 2005 is depicted in Figure 25A. Step 1.2.1. The preparation of compound 1.14 5 To a solution of 1.14a (42.5 g, 93.92 mmol) in DCM (500 mL) were added Fmoc-NH-PEG8- CH₂CH₂COOH (65.04 g, 97.99 mmol) and EEDQ (27.69 g, 111.99 mmol). Then the reaction was stirred at room temperature for 24 hrs. After completion, the residue was purified by silica gel column chromatography eluting with DCM/MeOH (30:1) to afford 1.14 (81 g, 78.82% yield) as yellowish oil (Figure 25B). _{10 LCMS: r.t = 1.170 min, MS m/z (ESI): 1101.4 (M+H)+.} Step 1.2.2. The preparation of compound 1.15 To a solution of 1.14 (81 g, 73.56 mmol) in DMF (800 mL) were added DPFPC (57.98 g, 147.12 mmol), DIEA (28.52 g, 220.68 mmol), and the reaction was stirred at room temperature for 2 hrs. After completion, the solution was directly purified by reverse HPLC under acid 15 conditions ((0.1% TFA/H2O) /ACN) to afford the title compound 1.15 (69 g, 71.54% yield) as yellowish oil (Figure 25C). LCMS: r.t =1.392 mins, MS m/z (ESI): 1311.4 (M+H)⁺. Step 1.2.3. The preparation of compound 1.16 To a solution of 1.15 (69 g, 52.62 mmol) in DMF (700 mL) were added 1.13 (32.15 g, 47.36 20 mmol), DIEA (18.36 g, 142.08 mmol), HOAt (8.6 g, 63.15 mmol), and the reaction was stirred at room temperature for 24 hrs. After completion, the solution was directly purified by reverse HPLC under neutral conditions (H2O /ACN) to afford 1.16 (42.8 g, 45.04% yield) as yellowish oil (Figure 25D). LCMS: r.t = 1.296 mins, MS m/z (ESI): 1805.7 (M+H)⁺. 25 Step 1.2.4. The preparation of compound 1.17 To a solution of 1.16 (42.8 g, 23.7 mmol) in anhydrous DMSO (400 mL) were added IBX (13.27 g, 47.4 mmol), and the reaction was stirred at room temperature for 18 hrs. After completion, the solution was directly purified by reverse HPLC under acid conditions ((0.1% TFA/H2O) /ACN) to afford 1.17 (36.1 g, 84.44% yield) as a white solid (Figure 25E). 30 LCMS: r.t = 1.306 mins, MS m/z (ESI): 1803.7 (M+H)⁺. DB1/ 159693888.5 274

133186-5030-WO Step 1.2.5. The preparation of compound 1.19 A solution of 1.17 (36.1 g, 0.599 mmol) in MeOH (500mL)/water (125mL)/DIEA (94 mL) was stirred at room temperature for 2 hrs. After completion (and formation of intermediate 1.18, which was not isolated), DEA (360 mL) was added to the aforementioned reaction mixture, and 5 stirred at room temperature for 2 hrs. After completion, the solution was directly purified by reverse HPLC under acid conditions ((0.1% TFA/H₂O) /ACN) to afford 1.19 (19.4 g, 67.25% yield for 2 steps) as a white solid (Figure 25F). LCMS: r.t = 0.898 mins, MS m/z (ESI): 1441.6 (M+H)⁺. Step 1.2.6. The preparation of compound 1.20 10 To a solution of 1.19 (19.4 g, 13.46 mmol) in DMF (200 mL) were added 1.20a (4.3 g, 16.15 mmol), DIEA (3.48 g, 26.92 mmol), and the reaction was stirred at 0℃ temperature for 1 hr. The solution was directly purified by Prep-HPLC under acid conditions ((0.1% TFA/H2O) /ACN) to afford 1.20 (10 g, 46.66% yield) as a white solid (Figure 25G). LCMS: r.t = 0.924 mins, MS m/z (ESI): 1592.6 (M+H)⁺. 15 Step 1.2.7. The preparation of compound 2005 To a solution of 1.20 (5 g, 3.14 mmol) in DCM (50 mL) were added TFA (10 mL), and the reaction was stirred at 0℃ temperature for 1 hr. After completion, concentrated the reaction solution under reduced pressure to get crude residue which was purified by Prep-HPLC under acid conditions ((0.1% TFA/H2O)/ACN) to afford the title compound (180) (1.84 g, 39.27% 20 yield) as a yellowish solid (Figure 25H). LCMS: r.t = 0.745 mins, MS m/z (ESI): 1492.6 (M+H)⁺, purity: 99.36% (HPLC).¹H NMR (400 MHz, DMSO) δ 9.88 (s, 1H), 9.84 (s, 1H), 9.10 (s, 1H), 8.17 – 8.07 (m, 1H), 8.00 (t, J = 5.5 Hz, 1H), 7.69 (d, J = 8.7 Hz, 2H), 7.22 (s, 1H), 7.13 – 6.96 (m, 8H), 6.70 (d, J = 0.6 Hz, 1H), 6.52 (s, 1H), 5.97 – 5.69 (m, 2H), 5.18 (s, 1H), 5.10 (s, 1H), 4.86 (d, J = 7.3 Hz, 2H), 4.11 (d, J = 25 13.7 Hz, 1H), 3.90 (d, J = 9.6 Hz, 4H), 3.80 (d, J = 7.9 Hz, 6H), 3.70 (t, J = 6.3 Hz, 2H), 3.60 (d, J = 7.2 Hz, 3H), 3.54 – 3.47 (m, 32H), 3.14 (q, J = 5.8 Hz, 3H), 2.89 (s, 1H), 2.64 (dd, J = 11.8, 6.1 Hz, 2H), 2.44 (s, 2H), 2.32 (t, J = 7.3 Hz, 3H), 2.01 (s, 2H), 1.85 (s, 1H), 1.54 (s, 6H). Example 2. Biological Characterisation In vitro cytotoxicity. The in vitro cytotoxicity of a selection of compounds was determined in 30 a panel of cell lines using the MTT assay for a 72-hour incubation period (Table 1 and Table 2). DB1/ 159693888.5 275

133186-5030-WO Table 1: In vitro cytotoxicity of a selection of compounds Compound Cell line (cancer type); IC50 (nM) Number Table 2: In vitro cytotoxicity of a selection of compounds Compound Cell line (cancer type); IC50 (nM) Number 0 DB1/ 159693888.5 276

133186-5030-WO 167 0.218 0.147 0.207 Pro-druggng/ mas ng o PDDs d m ns ed t e ab ty o pay oad to a y ate DN un ess sugar group is cleaved (e.g., payload reaches lysosome). See, Figure 22 and Table 3 below. Table 3: Comparative cytotoxicity data between PDD-pro-drug compounds before and after 5 cleavage of the sugar group Cytotoxicity (nM) SKBR3 MCF-7 As shown pared to the payload without the pro-drug (i.e., compound 138 versus compound 163), which indicated that the pro-drug sugar on the imine prevented the payload from alkylating DNA. Surprisingly, despite historic Sibiromycin data, the data shown for compound 88 in Table 3 suggests that 10 inclusion of a sugar on the C8 position of the PDD (analogous position to Sibiromycin on a PBD) reduces potency of the first generation PDD, resulting in IC₅₀ > 5µM. Free payloads (compound 163 and compound 79) also had substantially different cytotoxicity profiles, although only differing by a methyl group. In vivo, it was hypothesized that all sugars would be cleaved by enzymes at the tumour site (forming compound 163 and compound 79). 15 Example 2a. In vitro Efficacy of compound (180) The in vitro potency of free payload, compound (180), was tested across a panel of cell lines according to the following protocol: Day -1: Cell seeding 1. The cells were harvested during the logarithmic growth period and counted using Vi-cell 20 cell counter (The cell viability were measured by trypan blue exclusion assay). DB1/ 159693888.5 277

133186-5030-WO 2. The seeding density per well was determined and adjusted to 180 µL of cell suspensions per well in a 96-well plate according to the plate map in appendix 2. 3. An additional plate was set up for day 0 reading (T0) and the other plates were read at the end point of the assay. 5 4. The plates were incubated overnight in a humidified incubator at 37 °C with 5% CO2. Day 0: T0 plate reading and compound treatment 5. 50 μL of CellTiter-Glo® Reagent was added to each well. 6. The plate was shaken for 10 minutes on an orbital shaker to facilitate cell lysis. 10 7. The plate was incubated at room temperature for 10 minutes to stabilize luminescent signal. 8. Luminescence was recorded using EnVision Multi Label Plate Reader. 9. The test articles were prepared and the plates were placed back into the incubator until endpoint CTG reading. 15 Day 6: Endpoint CTG reading 10. 50 μL of CellTiter-Glo® Reagent was added to each well. 11. The plate was shaken for 10 minutes on an orbital shaker to facilitate cell lysis. 12. The plate was incubated at room temperature for 10 minutes to stabilize luminescent 20 signal. 13. Luminescence was recorded using EnVision Multi Label Plate Reader. 14. The formula for calculating survival rate is shown below and the absolute IC50 was calculated according to the dose-response curve generated. 25 The survival rate (%) = (LumTest article-Lumblank control)/ (LumNone treated-Lumblank control)×100%. Inhibition rate (%) = 1 - surviving rate (%) Results of the in vitro potency assay are set forth in Table B (● represents IC50 ≤ 60 nM; ●● 30 represents 60 nM < IC50 ≤ 120 nM; and ●●● represents 120 nM < IC50). Table B: In vitro IC50 values of compound 180 across various cell lines. Cell-Line IC50 (nM) DB1/ 159693888.5 278

133186-5030-WO 22Rv1 ● A172 ●● DB1/ 159693888.5 279

133186-5030-WO NCIH2172 ●●● NCIH441 ●● Example 3. Enzymatic Cleavage A number of LC-MS-based studies were undertaken to show that the sugar moieties (i.e., glucuronide, glucose, galactoside) were all cleaved through addition of relevant enzyme. In all 5 cases, the sugar moiety was observed to be cleaved from the payload structure, releasing the active payload. The rate of cleavage of each sugar moiety varied depending on the structural location and type of moiety cleaved. β-Glucosidase assay 10 1-Methyl-4-(4-(1-methyl-4-(4-(((S)-12-oxo-2-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2- a][1,4]diazepin-3-yl)oxy)butanamido)-1H-pyrrole-2-carboxamido)phenyl)-N-phenyl-1H- pyrrole-2-carboxamide (98) (0.89 mg, 0.001 mmol) was dissolved in dimethyl sulfoxide (100 µL). A 0.1 M citrated buffer solution (pH 5, 30% PEG 600) of β-glucosidase from almonds 15 (Sigma-Aldrich, 100 U/mL) was then added and the resulting aqueous solution was shaken at 37 °C for 180 h. Aliquots of 100 µL were taken and analysed by LC-MS at 5 min, 30 min, 1 h, 2 h, 18 h, 36 h, 60 h, 84 h and 132 h. Figures 1-9 illustrate results of the β-Glucosidase assay. DB1/ 159693888.5 280

133186-5030-WO β-Galactosidase assay (hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2- 5 a][1,4]diazepin-3-yl)oxy)butanamido)-1H-pyrrole-2-carboxamide (85) (0.71 mg, 0.001 mmol) was dissolved in dimethyl sulfoxide (100 µL). The solution was diluted with 1X PBS buffer (pH 7, 834 µL) and β-galactosidase suspension (Sigma-Aldrich, 100 U, 66 µL) was added. The resulting aqueous solution was shaken at 37 °C for 20 h and aliquots of 100 µL were taken and analysed by LC-MS at 5 min, 30 min, 90 min, 4.5 h, 7.5 h and 20 h. 10 Figures 10A-10F illustrate results of the β-Galactosidase assay. β-Glucuronidase assay ( , , ,5 , )- -((( )- -( -((5-(( -am nop eny )car amoy)- -met y- -pyrro - - yl)amino)-4-oxobutoxy)-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin- 15 2-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (88) (0.72 mg, 0.001 mmol) was dissolved in dimethyl sulfoxide (100 µL). The solution was diluted with a solution of β- glucuronidase from E. coli (Sigma-Aldrich, 100-500 U) in 1X PBS buffer (pH 6.8, 900 µL). The resulting aqueous mixture was shaken at 37 °C for 4.5 h and aliquots of 100 µL were taken and analysed by LC-MS at 5 min, 30 min and 90 min. LC-MS based analysis in Figures 11A- 20 11D indicated that the sugar-masked payload peak (compound 88) was fully converted to the free payload (compound 79) after 90 minutes. DB1/ 159693888.5 281

133186-5030-WO yl)amino)-4-oxobutoxy)-6-hydroxy-2-methoxy-12-oxo-5,6,6a,7,8,9,10,12- octahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5-carbonyl)oxy)methyl)-2-nitrophenoxy)-3,4,5- 5 trihydroxytetrahydro-2H-pyran-2-carboxylic acid (138) (0.95 mg, 0.001 mmol) was dissolved in dimethyl sulfoxide (100 µL). The solution was diluted with a solution of β-glucuronidase from e. coli (Sigma-Aldrich, 100-500 U) in 1X PBS buffer (pH 6.8, 900 µL). The resulting aqueous mixture was shaken at 37 °C for 4.5 h and aliquots of 100 µL were taken and analysed by LC- MS at 5 min, 30 min, 90 min and 150 min. LC-MS based analysis shown in Figures 12A-12E 10 indicated that conversion from glucuronide-containing payload (compound 138) to the glucuronide-free payload (compound 163) was extremely quick. Fig.12B shows that the glucuronide peak (2.03, 947.9) of the imine-protected construct 138 completely disappeared after 5 mins in the presence of small amounts of β-glucuronidase enzyme and was fully converted to active form 163. 15 The conversion of compound 138 to compound 163 was much faster than the conversion of compound 88 to compound 79, suggesting that the sugar group at the C8 position (as in compound 88) is not as enzyme-accessible as the sugar group at the N11 position (as in compound 138). The slow conversion of C8-sugar substituted payloads to free payloads has potential benefits relating to TI (therapeutic index), as higher enzyme concentrations and longer 20 exposure times (typically present in tumours) will be needed to cleave pro-drugs. (2S,3S,4S,5R,6S)-6-(4-((((6aS)-3-(4-((5-((4-(5-((4-aminophenyl)carbamoyl)-1-methyl-1H- pyrrol-3-yl)phenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-6-hydroxy-2- methoxy-12-oxo-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5- 25 carbonyl)oxy)methyl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (146) (1.15 mg, 0.001 mmol) was dissolved in dimethyl sulfoxide (100 µL). The solution was DB1/ 159693888.5 282

133186-5030-WO diluted with a solution of β-glucuronidase from e. coli (Sigma-Aldrich, 100-500 U) in 1X PBS buffer (pH 6.8, 900 µL). The resulting aqueous mixture was shaken at 37 °C for 4.5 h and aliquots of 100 µL were taken and analysed by LC-MS at 5 min, 30 min, 90 min and 150 min. 5 ((methoxyimino)methyl)piperidine-1-carbonyl)phenyl)carbamoyl)oxy)methyl)-2- nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (150) (0.77 mg, 0.001 mmol) was dissolved in dimethyl sulfoxide (100 µL). The solution was diluted with a solution of β-glucuronidase from e. coli (Sigma-Aldrich, 100-500 U) in 1X PBS buffer (pH 6.8, 900 µL). 10 The resulting aqueous mixture was shaken at 37 °C for 4.5 h and aliquots of 100 µL were taken and analysed by LC-MS at 5 min, 30 min, 90 min and 150 min. Example 4. Methodology MTT Cytotoxicity 1_{. Methodology} 15 Tumor cell lines were maintained in RPMI1640 medium supplemented with 10% heat- inactivated fetal bovine serum, 2mM L-glutamine and 1mM sodium pyruvate.1800 cells per well were seeded in a volume of 180μl in a 96-well flat bottom polystyrene plate. The cells were allowed to adhere overnight at 37°C in a CO2 incubator. Ligands were initially formulated in DMSO, and stocks stored at -80°C. They were then further formulated at 10x concentration in 20 RPMI1640 medium.20ul of diluted samples were added into each treatment well. On each plate, blank wells with no cells, and untreated wells containing cells, were included. Plates were then cultured at 37°C in a CO2 incubator for 72hrs. Cytotoxicity was evaluated using a tetrazolium salt-based assay, the MTT assay. After 72hours, the supernatant was removed from each well and 200μl of a sterile filtered 500μg/ml MTT solution in water added to each well. 25 The plates were then incubated at 37°C in a CO2 incubator for 4hrs. The supernatant was then removed and the formazan crystals formed solubilized by adding 150μl of DMSO to each well. The plate was then read on a plate reader at 540nm, and percentage cell survival calculated as DB1/ 159693888.5 283

133186-5030-WO follows: ((mean absorbance treated wells at concentration x – mean absorbance blank wells) ÷ (mean absorbance untreated wells at concentration x – mean absorbance blank wells)) x 100. Data were plotted as concentration in nM vs. % cell survival in Microsoft Excel, and IC50 values (concentration where cell survival is reduced by a half) were determined from the graph. 5 Example 5. ADC Conjugation Conjugation of Payload to Antibody. An example of the ADC conjugation methodology is provided below.21.5 mg IgG1 antibody (8.0 mg/ml in PBS) were charged with EDTA to a final concentration of 2 mM. Reduction was attained by adding 1.27 molar equivalents TCEP (10 mM in water) and incubating for 2 hours at 20°C. After 1.5 hours, a reduction in-process test 10 conjugation with Mal-vcMMAE was performed, and analyzed by HIC to test for the reduction level. As the target reduction level had not been reached, another 0.1 molar equivalents TCEP were added and the reduction time extended by 1 hour. After 0.5 hours, a second in-process test was run. After confirmation of the desired reduction level, 20% (v/v) Propylene glycol was added to the reduced antibody followed by 6.4 molar equivalents 31 (10 mM stock in DMSO). 15 The solution was incubated for 1 hour at rt. The reaction was quenched by adding 6.4 molar equivalents N-Acetylcysteine (10 mM in water). The ADC was buffer exchanged via G25 into PBS and washed by dead-end filtration (Vivaspin-20, 30 kDa MWCO, 0.0006 m2) for 10 DVs. Samples were taken for analysis by HIC, SEC, PLRP, free toxin linker, Endosafe, and the concentration was determined using a SEC calibration curve. Aliquotting was carried out under 20 laminar flow, and the product was stored at -80°C. Only disposable, sterile and pyrogen/DNA/RNA-free plasticware was used. Stochastic Conjugation 1_{.1 Conjugation of 91 to IgG1 antibody (forming ADC1). 91 was conjugated to an IgG1} antibody (Trastuzumab) targeted to HER2 in a stochastic manner. _{25 1.2 Antibody QC. The antibody was of good quality with high monomer content and} expected reduced profile in HIC studies (Figure 15). PLRP showed the expected pattern for reduced Light and Heavy chains (Figure 16). 1_{.3 Conjugation of 91 to Trastuzumab. DAR (Drug-Antibody Ratio) assignment was} possible through HIC analysis (Figure 17), with an overlay of DAR 4 ADC, and SEC 30 analysis (Figure 18). DB1/ 159693888.5 284 133186-5030-WO Example 6. In vivo studies In vivo efficacy and tolerability studies were performed using adaptations of the same basic protocol. An example of this is provided below: Antitumour activity of the ADC was assessed in tumour xenograft models obtained by 5 inoculation of the relevant cell-line (i.e., SK-BR-3) in nude mice (CD-1 or appropriate). Maximum tolerated dose (MTD) of the relevant ADC was established on 6 CD1 mice (or equivalent) at a number of concentrations through IV administration on day 1. For efficacy studies, tumours were implanted onto the flank of the mice using a 23-gauge needle, and were randomly assigned to groups (e.g., control or ADC). After implantation, 10 tumours were measured 3 times per week using digital calipers. The length and width of the tumour was measured and volume calculated using the following formula: volume = (length x width²)/2. The bodyweight of all mice on the study was measured and recorded 3 times per week. Mice were observed daily and any signs of distress or changes to general condition (e.g., starred fur, lack of movement, difficulty breathing). Specific criteria were set for early 15 termination, and this only occurred if tumour volume exceeded 1500mm³, weight loss of ≥15% occurred or animals became compromised (e.g., inability to eat/drink). Mice were housed in IVC cages (5 mice per cage) with individual mice identified by ear punch. Cages, bedding and water were sanitized before use. Animals were provided with Corn-o-cobs enrichment bedding to provide environment enrichment and nesting material. All animals had 20 free access to a standard certified commercial diet and water. The animal holding room was maintained as follows - room temperature at 20-24°C, humidity at 30-70% and a 12h light/dark cycle used. Cages were changed once a week with food and water replaced when necessary. All procedures were carried out under the guidelines of the Animal (Scientific Procedures) Act 1986. 25 In vivo Tolerability of Trastuzumab-134. The comparative data set forth in Table 4 below indicated that protecting the imine with a glucuronide-PABC unit of compound 134 had no effect on the MTD of the Trasbusumab-134 ADC when compared to the MTD of the Trastuzumab-control 3 ADC. Table 4: Comparative MTD data between control 3 and compound 134 ADC MTD ) 285

133186-5030-WO control 3 established, even at doses as high as 100 mg/kg (Figure 19). In this instance, loss of 15% of body weight was considered a toxic dose. Although not wishing to be bound by any particular theory, this trastuzumab-based study suggests that compound 91 with a sugar on the C8 position 5 can be efficiently conjugated at DAR 8 with tolerability exceeding 100 mg/kg. The MTD of the corresponding control 3-DAR 8 construct (which does not contain a sugar group at the C8 position) is 10 mg/kg. Although not wishing to be bound by any particular theory, this result suggests that “masking” the C8 position with a sugar group can increase tolerability by more than 10x compared to corresponding unmasked species. 10 In vivo Efficacy of ADC1. The efficacy data for a 5 mg/kg single dose of Tmab-91 (DAR 8) (i.e., Trastuzumab-91 (DAR 8)) shown in Figure 20 and in Table 5 below suggests that compound 91 is equally efficacious as control 3 at DAR 4. HER2 ADCs comprising compound 180 and control 3 were prepared (DAR 8). Mice were administered vehicle, the HER2 ADCs, or Enhertu. The HER2 ADC comprising compound 180 15 was found to show good activity versus Enhertu in a HER2-low xenograft model (Figure 23). The maximum tolerated dose of the HER2 ADC comprising compound 180 was found to be ≥ 50 mg/kg in mice (single dose). DB1/ 159693888.5 286

133186-5030-WO Table 5: Comparative MTD, Efficacy, and TI data for ADCs of control 3 and compound 91 MTD ED^a PDD DAR TI^b Compound E of structure: (E) 5 After cleavage of the sugar moiety in a tumour environment (in vitro), the product E’ is: (E’) 10 Previously it had been considered that PBD compounds containing a sugar moiety would have increased potency compared to the unsubstituted parent. However, surprisingly, the present inventors have discovered that sugar-containing molecules according to the disclosure are approximately 50-100x less potent than the parent molecule in vitro. The sugar group may be cleavable in the microenvironment of a tumour, to form the 15 parent molecule. Thus, in the present case compounds according to the disclosure have a DB1/ 159693888.5 287

133186-5030-WO substantially increased tolerability profile over the parent compound as a payload, and increased solubility. Examples of compounds according to the present disclosure are as set out in the following table: Structure No. A B C E DB1/ 159693888.5 288

133186-5030-WO F w e e S s a u va e sacc a e su s ue , p e e a y gycosy o -g ycosy. Summary Sugar-substituted A- and G-alkylating constructs were initially prepared as an attempt to increase hydrophilicity and enable more efficient conjugation. Differences in tolerability 5 profiles were not expected. The studies confirmed that addition of a sugar group enhanced hydrophilicity, which allowed for efficient antibody conjugation at higher DARs (i.e., DAR 8 is easily achieved). Results also indicated that traditional pro-drugging of the alkylating imine PDD N11 position, as in compound 134, had no impact on tolerability, which was surprising given the cytotoxicity data of naked payloads presented here and in the prior art. A ‘masking’ 10 approach, in which sugars were substituted at the PDD C8 position (e.g., compound 91), resulted in enhanced tolerability by more than 10x in ADC form (i.e., MTD of >100 mg/kg for compound 91-DAR8 compared to MTD of 10 mg/kg for control 3-DAR8). This was completely unexpected. The compound 91-DAR8 construct appeared to have similar efficacy to that of the control 3- 15 DAR4 construct, while the tolerability of compound 91-DAR8 was 2x greater than that of control 3-DAR4, thereby substantially widening the therapeutic window. Changing the C8 group from methoxy (C8-OMe of compound 163) to hydroxy (C8-OH of compound 79) had a dramatic effect on payload cytotoxicity, and exhibited reduced potency compared to the C8- OMe compound (compound 163). While not being bound by any particular theory, this result is 20 hypothesized to be due to membrane penetration issues of the C8-OH compounds (e.g. the inability of the compounds to cross cell membranes). In embodiments, and while not wishing to be bound by any particular theory, the C8-OH compounds exhibit reduced toxicity (e.g. off- target toxicity) compared to C8-OMe (or other C8-Oalkyl compounds) because once the payload is internalized into the cell by the ADC, the C8-OH compounds remain in the cell (e.g. 25 they cannot exit the cell because they cannot cross the cell membrane). It was observed that the type of sugar group and its substitution position on the payload scaffold is critical to tolerability profile. For example, N11-sugar (compound 134) had no impact on tolerability compared to unsubstituted agent, while the C8-sugar (compound 91) showed dramatic increase in tolerability compared to unsubstituted agent. DB1/ 159693888.5 289

133186-5030-WO Example 7. Conjugation to CDCP1 and in vivo efficacy 71 CDCP1 Antibody QC The antibody was of good quality with 99% monomer content via Size Exclusion Chromatography (SEC) (Figure 26A) and HIC (Figure 26B). PLRP showed the expected pattern 5 for reduced Light and Heavy chain. The minor peaks eluting after the main L0 and H0 are likely the result of intrachain disulfide reduction. 7.2. Conjugation to CDCP1 mAb All ADC conjugations were completed using a similar methodology, an example of which is provided below.21.5 mg anti-CDCP1 antibody (8.0 mg/ml in PBS) were charged with EDTA to 10 a final concentration of 2 mM. Reduction was attained by adding 1.27 molar equivalents TCEP (10 mM in water) and incubating for 2 hours at 20°C. After 1.5 hours, a reduction in-process test conjugation with Mal-vcMMAE was performed, and analyzed by HIC to test for the reduction level. As the target reduction level had not been reached, another 0.1 molar equivalents TCEP were added and the reduction time extended by 1 hour. After 0.5 hours, a 15 second in-process test was run. After confirmation of the desired reduction level, 20% (v/v) Propylene glycol was added to the reduced antibody followed by 6.4 molar equivalents of linker-payload (10 mM stock in DMSO). The solution was incubated for 1 hour at rt. The reaction was quenched by adding 6.4 molar equivalents N-Acetylcysteine (10 mM in water). The ADC was buffer exchanged via G25 into PBS and washed by dead-end filtration (Vivaspin- 20 20, 30 kDa MWCO, 0.0006 m²) for 10 DVs. Samples were taken for analysis by HIC, SEC, PLRP, free toxin linker, Endosafe, and the concentration was determined using a SEC calibration curve. Aliquotting was carried out under laminar flow, and the product was stored at -80°C. Only disposable, sterile and pyrogen/DNA/RNA-free plasticware was used. The anti- CDCP1 antibody includes the heavy chain variable region (VH) as set forth in SEQ ID NO: 1 25 and the light chain variable region (VL) as set forth in SEQ ID NO: 5. Example conjugation of a linker-payload to the antibody. DAR was assigned using HIC (Figure 27A). The conjugation process caused no significant aggregation compared to the starting antibody (Figure 27B). 30 Limited free toxin linker could be detected in the ADC sample. 7.3. In vivo Efficacy Antitumour activity of the selected ADCs was assessed in tumour xenograft models (both cancer-derived and patient-derived) obtained by inoculation of the relevant cell-line in mice DB1/ 159693888.5 290

133186-5030-WO Briefly, tumours were implanted onto the flank of the mice using a 23-gauge needle, and were randomly assigned to groups (e.g., control or ADC). After implantation, tumours were measured 3 times per week using digital calipers. The length and width of the tumour was measured and volume calculated using the following formula: volume = (length x width2)/2. The bodyweight 5 of all mice on the study was measured and recorded 3 times per week. Mice were observed daily and any signs of distress or changes to general condition (e.g., starred fur, lack of movement, difficulty breathing). Specific criteria were set for early termination, and this only occurred if tumour volume exceeded 1500mm3, weight loss of ≥15% occurred or animals became compromised (e.g., inability to eat/drink). 10 Mice were housed in IVC cages (5 mice per cage) with individual mice identified by ear punch. Cages, bedding and water were sanitized before use. Animals were provided with Corn-o-cobs enrichment bedding to provide environment enrichment and nesting material. All animals had free access to a standard certified commercial diet and water. The animal holding room was maintained as follows - room temperature at 20-24°C, humidity at 30-70% and a 12h light/dark 15 cycle used. Cages were changed once a week with food and water replaced when necessary. All procedures were carried out under the guidelines of the Animal (Scientific Procedures) Act 1986. F_{igure 28 depicts a graph illustrating mean tumour volume versus time after three doses of the} ADC (day 1, day 8, day 15) against an antigen positive prostate cancer CDX. Dose dependent 20 regression was observed. The ADC included anti-CDCP1 antibody including the heavy chain variable region (VH) as set forth in SEQ ID NO: 1 and the light chain variable region (VL) as set forth in SEQ ID NO: 5, and the antibody was conjugated to compound 180. References [1] a.) D. Antonow, D. E. Thurston, Chem Rev 2011, 111, 2815-2864; b.) L. Cipolla, A. C. 25 Araujo, C. Airoldi, D. Bini, Anticancer Agents Med Chem 2009, 9, 1-31; c.) B. Gerratana, Med Res Rev 2012, 32, 254-293; d.) J. A. Hartley, Expert Opin Investig Drugs 2011, 20, 733-744; e.) A. Kamal, K. L. Reddy, V. Devaiah, N. Shankaraiah, D. R. Reddy, Mini Rev Med Chem 2006, 6, 53-69. [2] D. S. Bose, G. B. Jones, D. E. Thurston, Tetrahedron 1992, 48, 751-758. 30 [3] L. H. Hurley, T. Reck, D. E. Thurston, D. R. Langley, K. G. Holden, R. P. Hertzberg, J. R. Hoover, G. Gallagher, Jr., L. F. Faucette, S. M. Mong, et al., Chem Res Toxicol 1988, 1, 258-268. DB1/ 159693888.5 291

133186-5030-WO [4] G. Wells, C. R. Martin, P. W. Howard, Z. A. Sands, C. A. Laughton, A. Tiberghien, C. K. Woo, L. A. Masterson, M. J. Stephenson, J. A. Hartley, T. C. Jenkins, S. D. Shnyder, P. M. Loadman, M. J. Waring, D. E. Thurston, Journal of medicinal chemistry 2006, 49, 5442-5461. 5 [5] a.) F. Brucoli, R. M. Hawkins, C. H. James, P. J. Jackson, G. Wells, T. C. Jenkins, T. Ellis, M. Kotecha, D. Hochhauser, J. A. Hartley, P. W. Howard, D. E. Thurston, Journal of medicinal chemistry 2013, 56, 6339-6351; b.) M. Kotecha, J. Kluza, G. Well’, C. C. O'Hare, C. Forni, R. Mantovani, P. W. Howard, P. Morris, D. E. Thurston, J. A. Hartley, D. Hochhauser, Mol Cancer Ther 2008, 7, 1319-1328. 10 [6] a.) M. S. Puvvada, J. A. Hartley, T. C. Jenkins, D. E. Thurston, Nucleic Acids Res 1993, 21, 3671-3675; b.) P. H. Clingen, I. U. De Silva, P. J. McHugh, F. J. Ghadessy, M. J. Tilby, D. E. Thurston, J. A. Hartley, Nucleic Acids Res 2005, 33, 3283-3291. [7] M. S. Puvvada, S. A. Forrow, J. A. Hartley, P. Stephenson, I. Gibson, T. C. Jenkins, D. E. Thurston, Biochemistry 1997, 36, 2478-2484. 15 [8] a.) M. D. Barkley, S. Cheatham, D. E. Thurston, L. H. Hurley, Biochemistry 1986, 25, 3021-3031; b.) J. Seifert, S. Pezeshki, A. Kamal, K. Weisz, Organic & Biomolecular Chemistry 2012, 10, 6850-6860. [9] M. Smellie, D. S. Bose, A. S. Thompson, T. C. Jenkins, J. A. Hartley, D. E. Thurston, Biochemistry 2003, 42, 8232-8239. 20 [10] M. L. Kopka, D. S. Goodsell, I. Baikalov, K. Grzeskowiak, D. Cascio, R. E. Dickerson, Biochemistry 1994, 33, 13593-13610. [11] R. Kizu, P. H. Draves, L. H. Hurley, Biochemistry 1993, 32, 8712-8722. [12] S. J. Gregson, P. W. Howard, J. A. Hartley, N. A. Brooks, L. J. Adams, T. C. Jenkins, L. R. Kelland, D. E. Thurston, Journal of medicinal chemistry 2001, 44, 737-748. 25 [13] I. Puzanov, W. Lee, A. P. Chen, M. W. Calcutt, D. L. Hachey, W. L. Vermeulen, V. J. Spanswick, C. Y. Liao, J. A. Hartley, J. D. Berlin, M. L. Rothenberg, Clinical Cancer Research 2011, 17, 3794-3802. [14] M. L. Miller, N. E. Fishkin, W. Li, K. R. Whiteman, Y. Kovtun, E. E. Reid, K. E. Archer, E. K. Maloney, C. A. Audette, M. F. Mayo, A. Wilhelm, H. A. Modafferi, R. DB1/ 159693888.5 292

133186-5030-WO Singh, J. Pinkas, V. Goldmacher, J. M. Lambert, R. V. Chari, Mol Cancer Ther 2016, 15, 1870-1878. [15] a.) M. G. Brazhnikova, N. V. Konstantinova, A. S. Mesentsev, J Antibiot (Tokyo) 1972, 25, 668-673; b.) L. H. Hurley, C. Gairola, M. Zmijewski, Biochim Biophys Acta 1977, 5 475, 521-535. [16] P. J. Jackson, C. H. James, T. C. Jenkins, K. M. Rahman, D. E. Thurston, ACS Chem Biol 2014, 9, 2432-2440. [17] B. S. Reddy, Y. Damayanthi, J. W. Lown, Anticancer Drug Des 2000, 15, 225-238. [18] a.) S. C. Jeffrey, J. De Brabander, J. Miyamoto, P. D. Senter, ACS medicinal chemistry 10 letters 2010, 1, 277-280; b.) S. J. Gregson, A. M. Barrett, N. V. Patel, G. D. Kang, D. Schiavone, E. Sult, C. S. Barry, B. Vijayakrishnan, L. R. Adams, L. A. Maste’son, F. D'Hooge, M. Snaith, J. Harper, J. A. Hartley, P. W. Howard, Eur J Med Chem 2019, 179, 591-607. [19] a.) T. Satomaa, H. Pynnonen, A. Vilkman, T. Kotiranta, V. Pitkanen, A. Heiskanen, B. 15 Herpers, L. S. Price, J. Helin, J. Saarinen, Antibodies (Basel) 2018, 7; b.) T. Legigan, J. Clarhaut, B. Renoux, I. Tranoy-Opalinski, A. Monvoisin, C. Jayle, J. Alsarraf, M. Thomas, S. Papot, Eur J Med Chem 2013, 67, 75-80. [20] J. Mantaj, P. J. Jackson, K. M. Rahman, D. E. Thurston, Angew Chem Int Ed Engl 2017, 56, 462-488. 20 [21] Hurley L, Thurston D. Pyrrolo(l,4)benzodiazepine antitumor antibiotics: chemistry, interaction with DNA, and biological implications. Pharm. Res.1984, 1, 52-59. All publications mentioned in the above specification are herein incorporated by reference. Although illustrative embodiments of the disclosure have been disclosed in detail herein, with reference to the accompanying drawings, it is understood that the disclosure is not limited to the 25 precise embodiment and that various changes and modifications can be effected therein by one skilled in the art without departing from the scope of the disclosure as defined by the appended claims and their equivalents. DB1/ 159693888.5 293

Claims

133186-5030-WO CLAIMS 1_{. An antibody-drug conjugate having formula (I):} Ab-[L–D]_n formula (I) wherein in formula (I): Ab is an antibody or binding fragment thereof that binds CUB Domain-Containing Protein-1 (CDCP1); D is a drug moiety comprising a pyrridinobenzodiazepine (PDD); L is a linker; wherein D and/or L comprise at least one carbohydrate substituent R_S. 2_{. The antibody-drug conjugate of claim 1, wherein RS is a univalent saccharide} substituent, preferably RS is glycosyl or O-glycosyl. 3_{. The antibody-drug conjugate of claim 1 or claim 2, wherein D comprises a moiety of} formula (IIa): wherein: the dotted line indicates the optional presence of a double bond between one or more of C1 and C2, C2 and C3, and C3 and C4; the wavy line indicates the point of attachment to L; m is 0 or 1; R₁, R₃ and R₄ are independently selected from H and R₂₉; R₂ is selected from H, R_S, L₂-R_58, R₂₉, and –LS-RS, or one of R1 and R2, R2 and R3, or R3 and R4, together with the carbon atoms to which they are attached form a 6-membered aryl, or a 5- or 6-membered cyclic, heterocyclic, or heteroaryl ring optionally substituted with 1, 2 or 3 independently selected optional R₂₀ groups; DB1/ 159693888.5 294

133186-5030-WO R₅ and R₆ are selected such that either (i) R₅ is selected from H, OH and OC_1-6 alkyl; and R₆ is selected from H, SO3H, –LS-RS, nitrogen protecting groups, –L2-R58 and RA; (ii) R5 is oxo or H, and R6 is H or C1-6 alkyl; or (iii) R5 and R6 together form a double bond; R_{7 and R9 are independently selected from H and R20;} R8 is selected from H, SR24, SCH2Ph, R20, L2-R58, and –LS-RS; R_A is selected from (CH₂)_j-OH, (CH₂)_j-CO₂R₂₆, C(=O)-O-(CH₂)_k-NR₂₆R₂₇, (CH₂)_jNR₂₆R₂₇, C(=O)-NH-(CH₂)_j-NR₂₆R₂₇ and C(=O)-NH-(CH₂)_k-C(=NH)NR₂₆R₂₇; L₂ is a bond or a linker moiety having 1-200 non-hydrogen atoms selected from C, N, P, O, S or halogen, and optionally incorporates ether, oxo, carboxamidyl, urethanyl, branched, cyclic, unsaturated, heterocyclyl, aryl or heteroaryl moieties; and R58 is RA, O-(CH2)k-NR26R26, or NHNH2; each R₂₉ is independently selected from R₂₀, R₂₁, =CH₂, =CH-(CH₂)_s-CH₃, =CH-(CH₂)_s-R₂₁, =O, (CH₂)_s-OR₂₁, (CH₂)_s-CO₂R₂₁, (CH₂)_s-NR₂₁R₂₄, O-(CH₂)_t-NR₂₁R₂₄, NH-C(O)-R₂₁, O- (CH2)t-NH-C(O)-R21, O-(CH2)t-C(O)-NH-R21, (CH2)s-SO2R21, O-SO2R21, (CH2)s-C(O)R21 and (CH2)s-C(O)NR21R24; each R20 is independently selected from F, Cl, Br, (CH2)j-OH, C1-6 alkyl, OC1-6 alkyl, RS, OCH2Ph, (CH2)j-CO2R26, O-(CH2)k-NR26R27, C(=O)-O-(CH2)k-NR26R27, C(=O)-NR26R27, (CH₂)_j-NR₂₆R₂₇, NR₂₆NH₂, C(=O)-NH-(CH₂)_j-NR₂₆R₂₇, C(=O)-NH-C₆H₄-(CH₂)_j-R₂₆, C(=O)-NH-(CH₂)_k-C(=NH)NR₂₆R₂₇, –L₂-R₅₈, S(O)₂-(C_1-6 alkyl), O-(CH₂)_k-O-(C_1-6 alkyl), (CH₂)_j-S(O)₂-NR₂₆R₂₇, C(=NH)-O-(C_1-6 alkyl), (CH₂)_k-O-(C_1-6 alkyl), CN, NCO, Cy, C(O)-NH-(CH2)-Cy C(O)-Cy NH-C(O)-NR26R27 and or 6; each k and t is independently selected from 1, 2, 3, 4, 5 or 6; each R₂₁ is independently selected from H, C_1-12 alkyl, C_5-6 heterocyclyl, C_5-9 heteroaryl, C_6-15 heteroarylalkyl, phenyl and C7-12 aralkyl groups; wherein the heterocyclyl, heteroaryl, heteroarylalkyl, phenyl and aralkyl groups are optionally substituted with 1, 2 or 3 independently selected optional R20 groups; each R₂₄, R₂₆ and R₂₇ is independently selected from H and C_1-12 alkyl; each Cy is independently selected from a C_5-6 heterocyclyl or C_5-6 heteroaryl group, wherein the heterocyclyl or heteroaryl groups are optionally substituted with 1 or 2 R₂₀ groups; LS is a bond, an amino acid, a peptide chain having from 2 to 6 amino acids, an alkylene chain containing from 1 to 12 carbon atoms which may contain one or more carbon-carbon double or triple bonds, a paraformaldehyde chain –(OCH2)1-12-, a polyethylene glycol chain -(OCH2CH2)1-6-, which chains may be interrupted by or optionally incorporates one DB1/ 159693888.5 295

133186-5030-WO o_{r more of P, O, S, NH, C5-9 heteroarylene, phenylene, heterocyclyl, cycloalkyl, ether,} oxo, carboxamidyl, and/or urethanyl moieties wherein the C5-9 heteroarylene, phenylene, heterocyclyl, and/or cycloalkyl moieties are optionally substituted, optionally LS is: L_{C O} HN _O ; m an amino acid, an amino acid derivative, a pept e c a n avng rom to am no acids or amino acid derivatives, an alkylene chain containing from 1 to 12 carbon atoms which may contain one or more carbon-carbon double or triple bonds, a paraformaldehyde chain –(OCH₂)_1-12-, a polyethylene glycol chain -(OCH₂CH₂)_1-8-, which chains may be interrupted by one or more P, O, S and/or NH g_{roups and/or C5-9 heteroarylene and/or phenylene, wherein each C5-9 heteroarylene group} and/or each phenylene group is optionally substituted; and RS is a univalent saccharide substituent, preferably glycosyl or O-glycosyl. 4_{. The antibody-drug conjugate of claim 3, wherein the moiety of formula (IIa) is a moiety} of any one of formula (G1) to (G8): R₈ DB1/ 159693888.5 296

133186-5030-WO 5_{. The antibody-drug conjugate of claim 1 or claim 2, wherein D comprises a moiety of} formula (IIb): R₅ R₇ R₁ H N Q T' wherein: the dotted line indicates the optional presence of a double bond between one or more of C1 and C2, C2 and C3, and C3 and C4; the wavy line indicates the point of attachment to L; m is 0 or 1; R₁, R₃ and R₄ are independently selected from H and R₂₉; R₂ is selected from H, L₂-R_58, R₂₉, and –LS-RS, or one of R1 and R2, R2 and R3, or R3 and R4, together with the carbon atoms to which they are attached form a 6-membered aryl, or a 5- or 6-membered cyclic, heterocyclic, or heteroaryl ring optionally substituted with 1, 2 or 3 independently selected optional R₂₀ groups; R₅ is selected from H, OH and OC_1-6 alkyl; R_{7 and R9 are independently selected from H and R20;} R8 is selected from H, SR24, SCH2Ph, R20, L2-R58, and –LS-RS; DB1/ 159693888.5 297

133186-5030-WO R_A is selected from (CH₂)_j-OH, (CH₂)_j-CO₂R₂₆, C(=O)-O-(CH₂)_k-NR₂₆R₂₇, (CH₂)_jNR₂₆R₂₇, C(=O)-NH-(CH2)j-NR26R27 and C(=O)-NH-(CH2)k-C(=NH)NR26R27; L2 is a bond or a linker moiety having 1-200 non-hydrogen atoms selected from C, N, P, O, S or halogen, and optionally incorporates ether, oxo, carboxamidyl, urethanyl, branched, cyclic, unsaturated, heterocyclyl, aryl or heteroaryl moieties; and R₅₈ is R_A, O-(CH₂)_k-NR₂₆R₂₆, or NHNH₂; each R₂₉ is independently selected from R₂₀, R₂₁, =CH₂, =CH-(CH₂)_s-CH₃, =CH-(CH₂)_s-R₂₁, =O, (CH₂)_s-OR₂₁, (CH₂)_s-CO₂R₂₁, (CH₂)_s-NR₂₁R₂₄, O-(CH₂)_t-NR₂₁R₂₄, NH-C(O)-R₂₁, O- (CH2)t-NH-C(O)-R21, O-(CH2)t-C(O)-NH-R21, (CH2)s-SO2R21, O-SO2R21, (CH2)s-C(O)R21 and (CH2)s-C(O)NR21R24; each R20 is independently selected from F, Cl, Br, (CH2)j-OH, C1-6 alkyl, OC1-6 alkyl, ORS, OCH₂Ph, (CH₂)_j-CO₂R₂₆, O-(CH₂)_k-NR₂₆R₂₇, C(=O)-O-(CH₂)_k-NR₂₆R₂₇, C(=O)-NR₂₆R₂₇, (CH₂)_j-NR₂₆R₂₇, NR₂₆NH₂, C(=O)-NH-(CH₂)_j-NR₂₆R₂₇, C(=O)-NH-C₆H₄-(CH₂)_j-R₂₆, C(=O)-NH-(CH2)k-C(=NH)NR26R27, –L2-R58, S(O)2-(C1-6 alkyl), O-(CH2)k-O-(C1-6 alkyl), (CH2)j-S(O)2-NR26R27, C(=NH)-O-(C1-6 alkyl), (CH2)k-O-(C1-6 alkyl), CN, NCO, Cy, C(O)-NH-(CH2)j-Cy, C(O)-Cy, NH-C(O)-NR26R27 and or 6; each k and t is independently selected from 1, 2, 3, 4, 5 or 6; each R21 is independently selected from H, C1-12 alkyl, C5-6 heterocyclyl, C5-9 heteroaryl, C6-15 heteroarylalkyl, phenyl and C7-12 aralkyl groups; wherein the heterocyclyl, heteroaryl, heteroarylalkyl, phenyl and aralkyl groups are optionally substituted with 1, 2 or 3 independently selected optional R20 groups; each R₂₄, R₂₆ and R₂₇ is independently selected from H and C_1-12 alkyl; each Cy is independently selected from a C_5-6 heterocyclyl or C_5-6 heteroaryl group, wherein the heterocyclyl or heteroaryl groups are optionally substituted with 1 or 2 R20 groups; LS is a bond, an amino acid, a peptide chain having from 2 to 6 amino acids, an alkylene chain containing from 1 to 12 carbon atoms which may contain one or more carbon-carbon double or triple bonds, a paraformaldehyde chain –(OCH₂)_1-12-, a polyethylene glycol chain -(OCH₂CH₂)_1-6-, which chains may be interrupted by or optionally incorporates one o_{r more of P, O, S, NH, C5-9 heteroarylene, phenylene, heterocyclyl, cycloalkyl, ether,} oxo, carboxamidyl, and/or urethanyl moieties wherein the C5-9 heteroarylene, phenylene, heterocyclyl, and/or cycloalkyl moieties are optionally substituted, optionally LS is: DB1/ 159693888.5 298

133186-5030-WO ; charide substituent, preferably glycosyl or O-glycosyl; B is an DNA binding amide-containing chain; and T’ is an end group. 6_{. The antibody-drug conjugate of claim 5, wherein the moiety of formula (IIb) is a moiety} of any one of formula (H1) to (H8): R₅ R₇ R R₇ R₁ H N ₅ Q _{T' N} Q T' B H B T' . DB1/ 159693888.5 299

133186-5030-WO 7. The antibody-drug conjugate of any one of claims 3-6, wherein each of R₁, R_3, R₇, and R9 are H. 8. The antibody-drug conjugate of claim 7, wherein –L–D has the formula (IVa): wherein: L has the formula –Q–B–T–; Q is a linker; B is an DNA binding amide-containing chain; and T is an end group. 9. The antibody-drug conjugate of claim 7, wherein –L–D has the formula (IVb): 10. The antibody-drug conjugate of any one of claims 1-9, wherein L comprises –LS2–LC2– Z^*–, wherein: L_S2 is a bond, an amino acid, a peptide chain having from 2 to 6 amino acids, an alkylene chain containing from 1 to 12 carbon atoms which may contain one or more carbon-carbon double or triple bonds, a paraformaldehyde chain –(OCH₂)_1-12-, a polyethylene glycol chain -(OCH2CH2)1-6-, which chains may be interrupted by or o_{ptionally incorporates one or more of P, O, S, NH, C5-9 heteroarylene, phenylene,} heterocyclyl, cycloalkyl, ether, oxo, carboxamidyl, and/or urethanyl moieties wherein the C_5-9 heteroarylene, phenylene, heterocyclyl, and/or cycloalkyl moieties are optionally substituted, optionally L_S2 is: d DB1/ 159693888.5 300

133186-5030-WO L_C2 comprises one or more groups selected from an amino acid, an amino acid derivative, a peptide chain having from 2 to 6 amino acids or amino acid derivatives, an alkylene chain containing from 1 to 12 carbon atoms which may contain one or more carbon-carbon double or triple bonds, a paraformaldehyde chain –(OCH₂)_1-12-, a polyethylene glycol chain -(OCH₂CH₂)_1-8-, which chains m_{ay be interrupted by one or more P, O, S and/or NH groups and/or C5-9} heteroarylene and/or phenylene, wherein each C_5-9 heteroarylene group and/or each phenylene group is optionally substituted; and Z^* is a reactive moiety that has reacted with a functional group. 11. The antibody-drug conjugate of claim 10, wherein LS2 is: . 12. The antibody-drug conjugate of claim 10, wherein L_S2 is a peptide chain having from 2 to 6 amino acids, optionally 2 amino acids, optionally -valine-alanine-. 13. The antibody-drug conjugate of any one of claims 5-12, wherein Q comprises X1-L-X2, wherein: X1 is selected from O, S, NR13, CR13R14, CR13R14O, C(=O), C(=O)NR13, NR13C(=O), O-C(O) and C(O)-O, or is absent; L is selected from an amino acid, a peptide chain having from 2 to 6 amino acids, an alkylene chain containing from 1 to 12 carbon atoms which may contain one or more carbon- carbon double or triple bonds, a paraformaldehyde chain –(OCH2)1-12-, a polyethylene glycol chain -(OCH2CH2)1-6-, which chains may be interrupted by one or more P, O, S a_{nd/or NH groups and/or C5-9 heteroarylene and/or phenylene, wherein each C5-9} heteroarylene group and/or each phenylene group is optionally substituted; X₂ is selected from O, S, NR₁₅, CR₁₅R₁₆, CR₁₅R₁₆O, C(=O), C(=O)NR₁₅, NR₁₅C(=O), O-C(O) and C(O)-O or is absent; and R13, R14, R15 and R16 are independently selected from H and C1-6 alkyl. 14. The antibody-drug conjugate of claim 13, wherein X₁ is O. 15. The antibody-drug conjugate of claim 13 or 14, wherein L is an alkylene chain containing from 1 to 12 carbon atoms which may contain one or more carbon-carbon double or triple bonds, optionally wherein L is an alkylene chain containing 3 carbon atoms. DB1/ 159693888.5 301

133186-5030-WO 1_{6. The antibody-drug conjugate of any one of claims 13-15, wherein X2 is C(=O)NR15 or} NR15C(=O), optionally wherein R15 is H. 1_{7. The antibody-drug conjugate of any one of claims 13-16, wherein Q has the formula} H N O . _{. e a o y-drug conjugate of any one of claims 5-17, wherein B comprises (A)q,} wherein: q is selected from 0, 1, 2, 3, 4, 5 and 6; A is selected from: ; for each A1 group one of Y₃ and Y₄ is independently selected from N-R₃₀, S and O; and the other of Y₃ and Y₄ is CH; and Y₅ is independently selected from CR₃₀, N, S and COH; for each A2 group one of Y6 and Y7 is independently selected from N and CH; and the other of Y6 and Y7 is CR30; and each R30 is independently selected from H, C1-6 alkyl, L2-R58 and RS. 1_{9. The antibody-drug conjugate of claim 18, wherein q is 1 or 2. 20. The antibody-drug conjugate of claim 18 or 19, wherein} A is A1: ; wherein: Y3 is N-R30; Y₄ is CH; Y₅ is CR₃₀; optionally wherein each R₃₀ is H, methyl, or R_S. 2_{1. The antibody-drug conjugate of any one of claims 5-20, wherein B has the formula} DB1/ 159693888.5 302

133186-5030-WO . T comprises a group of formula: wherein: p is 0 or 1; R_T is selected from –L₂–, phenylene, and C_5-9 heteroarylene, wherein the phenylene and C_5-9 heteroarylene groups are optionally substituted with up to three optional substituent groups selected from OH, C_1-6 alkyl, OC_1-6 alkyl, (CH₂)_j-CO₂R₁₁, O-(CH₂)_k-NR₁₁R₁₂, (CH2)j-NR11R12, C(=O)-NH-(CH2)k-NR11R12, C(=O)-NH-R4, and C(=O)-NH-(CH2)k- C(=NH)NR11R12, optionally with the proviso that the optionally substituted C5-9 heteroarylene is not disubstituted indolyl. R₁₉ is selected from H, C_1-6 alkyl, L₂-R₅₈, R_S, and (CH₂)_t-NR₂₀R₂₁; Y₁ and Y₂ are independently N or CR₃₁, wherein at least one of Y₁ and Y₂ is CR₃₁; each R₃₁ is independently selected from H, C_1-6 alkyl, L₂-R₅₈ and R_S; R11, R12, and R24 are independently selected from H, –L2-R58, C1-6 alkyl, or a bond connecting the atom to which it is bound to –LS2–, with the proviso that both R11 and R12 cannot be a bond connecting the atom to which it is bound to –LS2–. DB1/ 159693888.5 303

133186-5030-WO 2_{3. The antibody-drug conjugate of claim 22, wherein p is 0. 24. The antibody-drug conjugate of claim 22 or 23, wherein RT is phenylene, optionally} wherein the phenylene is substituted with (CH2)j-NR11R12, optionally wherein R11 is H and R12 is a bond connecting the atom to which it is bound to –LS2–. 2_{5. The antibody-drug conjugate of any one of claims 8 or 10-24, wherein T has the} H N formul . 2_{6. The antibody-drug conjugate of any one of claims 5-7 or 9-14, wherein T’ comprises a} group of formula: wherein: p is 0 or 1; RT is selected from –L2-R58, phenyl, and C5-9 heteroaryl, wherein the phenyl and C5-9 heteroaryl groups are optionally substituted with up to three optional substituent groups selected from OH, C1-6 alkyl, OC1-6 alkyl, –L2-R58, (CH2)j-CO2R11, O-(CH2)k-NR11R12, (CH2)j- NR₁₁R₁₂, C(=O)-NH-(CH₂)_k-NR₁₁R₁₂, C(=O)-NH-R₂₄, and C(=O)-NH-(CH₂)_k- C(=NH)NR₁₁R₁₂, optionally with the proviso that the optionally substituted C_5-9 heteroaryl is not indolyl; R19 is selected from H, C1-6 alkyl, L2-R58, RS, and (CH2)t-NR20R21; Y1 and Y2 are independently N or CR31, wherein at least one of Y1 and Y2 is CR31; each R31 is independently selected from H, C1-6 alkyl, L2-R58 and RS; and R₁₁, R₁₂, and R₂₄ are independently selected from H, –L₂-R₅₈, C_1-6 alkyl, or substituted aryl. DB1/ 159693888.5 304

133186-5030-WO 2_{7. The antibody-drug conjugate of claim 26, wherein p is 1. 28. The antibody-drug conjugate of claim 26 or 27, wherein Y1 and Y2 are CR31, R31 are} each H, and R19 is H. 2_{9. The antibody-drug conjugate of any one of claims 26-28, wherein RT is C5-9 heteroaryl,} optionally pyrrole, optionally substituted with C_1-6 alkyl, optionally methyl, and/or C(=O)-NH- H₂N R24, optionally wherein R24 is substituted aryl, optionally wherein R24 is . 3_{0. The antibody-drug conjugate of any one of claims 26-29, wherein RT is phenylene,} optionally wherein the phenylene is substituted with (CH2)j-NR11R12, optionally wherein R11 and R12 are each H. 3_{1. The antibody-drug conjugate of any one of claims 5-7, 9-14, or 26-30, wherein T’ has} H₂N . 3_{2. The antibody-drug conjugate of any one of claims 10-25, wherein Z* is selected from a} succinimide, a heterocycle (e.g. a triazole), an amide, a thioether, an oxime, an imine (e.g. chiral sulfinyl imine), an alkenyl phosphorous group, and an alkyl phosphorous group. 3_{3. The antibody-drug conjugate of any one of claims 10-25 or 32, wherein LS2 is selected} from: , , DB1/ 159693888.5 305

133186-5030-WO ; 3_{4. The antibody-drug conjugate of any one of claims 10-25, 32, or 33, wherein LC2} O O comprises ^1-7 , optionally ^O or . 3_{5. T} h_{e antibody-drug co njugate of any one of claims 10-25 or 32-34, wherein LC2} comprises a polyethylene glycol chain -(OCH2CH2)1-8-, optionally -(OCH2CH2)8-. 3_{6. The antibody-drug conjugate of any one of claims 10-25 or 32-35, wherein –Z*–LC2– is} selected from: O O N nd 3_{7. The antibody-drug conjugate of any one of claims 32-36, wherein XAA is L-valyl-L-} alanine. 3_{8. The antibody-drug conjugate of any one of claims 10-25 or 32-37, wherein Z* is} succinimide: DB1/ 159693888.5 306

133186-5030-WO . _{39. The antibody-drug conjugate of any one of claims 10-25 or 32-38, wherein –LS2– is} selected from: . 4_{0. The antibody-drug conjugate of any one of claims 10-25 or 32-39, wherein –LC2–LS2– is} selected from: . 41. The antibody-drug conjugate of any one of claims 1-40, wherein D comprises at least one RS. 42. The antibody-drug conjugate of any one of claims 1-41, wherein L comprises at least one R_S. 43. The antibody-drug conjugate of any one of claims 1-42, wherein L and D each independently comprise at least one RS. DB1/ 159693888.5 307

133186-5030-WO 44. The antibody-drug conjugate of any one of claims 10-25 or 32-43, wherein –Z*–L_C2– L_{S2– is selected from:} , DB1/ 159693888.5 308

133186-5030-WO . 45. The antibody-drug conjugate of any one of claims 1-44, wherein D is selected from DB1/ 159693888.5 309

133186-5030-WO nd O-glycosyl. 4_{6. The antibody-drug conjugate of any one of claims 1-45, wherein –L–D is selected from:} , DB1/ 159693888.5 312

133186-5030-WO DB1/ 159693888.5 313

133186-5030-WO , glycosyl. 47. The antibody-drug conjugate of any one of claims 1-46, wherein R_S is selected from any one of formula (S11), (S12), or (S13): wherein R49 is independently at each occurrence selected from H and C1-6 alkyl, optionally methyl. DB1/ 159693888.5 315

133186-5030-WO 48. The antibody-drug conjugate of any one of claims 1-47, wherein RS is selected from any one of formula (S101), (S102), or (S103): . 49. T e ant body-drug conjugate o any one o c a ms - 8, w ere n RS s se ected from any one of formula (S21), (S22), (S23), or (S24): wh methyl. 50. The antibody-drug conjugate of any one of claims 1-49, wherein RS is selected from any one of formula (S201), (S202), (S203), or (S204): 51. The antibody-drug conjugate of any one of claims 1-50, wherein the antibody or binding fragment thereof comprises: (i) a heavy chain variable region (VH) that comprises: (a) a VH complementarity determining region one (CDRH1) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 2, (b) a VH complementarity determining region two (CDRH2) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 3, and (c) a VH complementarity determining region three (CDRH3) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 4, DB1/ 159693888.5 316

133186-5030-WO and (ii) a light chain variable region (VL) that comprises: (a) a VL complementarity determining region one (CDRL1) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 6, (b) a VL complementarity determining region two (CDRL2) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 7, and (c) a VL complementarity determining region three (CDRL3) comprising and/or consisting of the amino acid sequence of SEQ ID NO:8. 52. The antibody-drug conjugate of any one of claims 1-51, wherein the antibody or binding fragment thereof comprises a VH that comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1, and/or a VL that comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 5. 53. The antibody-drug conjugate of claim 52, wherein the antibody or binding fragment thereof comprises a VH that comprises the amino acid sequence of SEQ ID NO: 1 and/or a VL that comprises the amino acid sequence of SEQ ID NO: 5. 54. The antibody-drug conjugate of any one of claims 1-53, wherein the antibody or binding fragment thereof comprises: (i) a heavy chain variable region (VH) that comprises: (a) a VH complementarity determining region one (CDRH1) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 16, (b) a VH complementarity determining region two (CDRH2) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 17, and (c) a VH complementarity determining region three (CDRH3) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 18, and (ii) a light chain variable region (VL) that comprises: (a) a VL complementarity determining region one (CDRL1) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 20, (b) a VL complementarity determining region two (CDRL2) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 21, and DB1/ 159693888.5 317

133186-5030-WO (c) a VL complementarity determining region three (CDRL3) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 22. 55. The antibody-drug conjugate of any one of claims 1-52 or 54, wherein the antibody or binding fragment thereof comprises a VH that comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 15, and/or a VL that comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 19. 56. The antibody-drug conjugate of claim 55, wherein the antibody or binding fragment thereof comprises a VH that comprises the amino acid sequence of SEQ ID NO: 15 and/or a VL that comprises the amino acid sequence of SEQ ID NO: 19. 57. The antibody-drug conjugate of any one of claims 1-56, wherein the antibody or binding fragment thereof comprises a heavy chain comprising an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 10 and/or a light chain comprising an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 11. 58. The antibody-drug conjugate of any one of claims 1-50, wherein the antibody or binding fragment thereof comprises a VH that comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from SEQ ID NOs: 23-176 and/or a VL that comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from SEQ ID NOs: 177-326. 59. The antibody-drug conjugate of claim 58, wherein the antibody or binding fragment thereof comprises a VH that comprises an amino acid sequence selected from SEQ ID NOs: 23- 176 and/or a VL that comprises an amino acid sequence selected from SEQ ID NOs: 177-326. 60. The antibody-drug conjugate of any one of claims 1-50, 58, or 59, wherein the antibody or binding fragment thereof comprises a heavy chain comprising an amino acid sequence at DB1/ 159693888.5 318

133186-5030-WO least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from SEQ ID NOs: 23-176 and/or a light chain comprising an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from SEQ ID NOs: 177-326. 61. The antibody-drug conjugate of claim 60, wherein the antibody or binding fragment thereof comprises a heavy chain that comprises an amino acid sequence selected from SEQ ID NOs: 23-176 and/or a light chain that comprises an amino acid sequence selected from SEQ ID NOs: 177-326. 62. The antibody-drug conjugate of any one of claims 1-61, wherein the antibody-drug conjugate has a drug-to-antibody ratio (DAR) ranging from about 1 to about 10, optionally wherein the DAR is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10, optionally DAR is about 4, optionally DAR is about 8. 63. A pharmaceutical composition comprising the antibody drug conjugate of any one of claims 1-64; and a pharmaceutically acceptable carrier. 64. A method of treating a cancer comprising administering to a subject in need thereof a therapeutically effective amount of the antibody drug conjugate of any one of claims 1-62, or the pharmaceutical composition of claim 63. 65. The method of claim 64, wherein the cancer is selected from pancreatic cancer, breast cancer, prostate cancer, lymphoma, skin cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain cancer, primary brain carcinoma, head-neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, head or neck carcinoma, breast carcinoma, ovarian carcinoma, lung carcinoma, small-cell lung carcinoma, Wilms’ tumor, cervical carcinoma, testicular carcinoma, bladder carcinoma, pancreatic carcinoma, stomach carcinoma, colon carcinoma, prostatic carcinoma, genitourinary carcinoma, thyroid carcinoma, esophageal carcinoma, myeloma, multiple myeloma, adrenal carcinoma, renal cell carcinoma, endometrial carcinoma, adrenal cortex carcinoma, malignant pancreatic insulinoma, malignant carcinoid carcinoma, choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervical hyperplasia, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, chronic granulocytic leukemia, acute granulocytic leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi’s DB1/ 159693888.5 319

133186-5030-WO sarcoma, polycythemia vera, essential thrombocytosis, Hodgkin’s disease, non-Hodgkin’s lymphoma, soft-tissue sarcoma, osteogenic sarcoma, primary macroglobulinemia, or retinoblastoma, acoustic neuroma, adenocarcinoma, angiosarcoma, astrocytoma, basal cell carcinoma, bile duct carcinoma, bladder carcinoma, brain cancer, breast cancer, triple-negative breast cancer (TNBC), bronchogenic carcinoma, cervical cancer, chordoma, choriocarcinoma, colon cancer, colorectal cancer, craniopharyngioma, cystadenocarcinoma, embryonal carcinoma, endotheliocarcinoma, ependymoma, epithelial carcinoma, esophageal cancer, Ewing’s tumor, fibrosarcoma, gastric cancer, glioblastoma multiforme, glioma, head and neck cancer, hemangioblastoma, hepatoma, kidney cancer, leiomyosarcoma, liposarcoma, lung cancer, lymphangioendotheliosarcoma, lymphangiosarcoma, medullary carcinoma, medulloblastoma, melanoma, meningioma, mesothelioma, myxosarcoma, nasal cancer, neuroblastoma, oligodendroglioma, oral cancer, osteogenic sarcoma, ovarian cancer, pancreatic cancer, papillary adenocarcinoma, papillary carcinoma, pinealoma, prostate cancer, rabdomyosarcoma, rectal cancer, renal cell carcinoma, retinoblastoma, sarcoma, sebaceous gland carcinoma, seminoma, skin cancer, squamous cell carcinoma, stomach cancer, sweat gland carcinoma, synovioma, testicular cancer, small cell lung carcinoma, throat cancer, uterine cancer, Wilm’s tumor, blood cancer, acute erythroleukemic leukemia, acute lymphoblastic B- cell leukemia, acute lymphoblastic T-cell leukemia, acute lymphoblastic leukemia, acute megakaryoblastic leukemia, acute monoblastic leukemia, acute myeloblastic leukemia, acute myelomonocytic leukemia, acute nonlymphocytic leukemia, acute promyelocytic leukemia, acute undifferentiated leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, hairy cell leukemia, multiple myeloma, heavy chain disease, Hodgkin’s disease, multiple myeloma, non-Hodgkin’s lymphoma, polycythemia vera, and Waldenstrom’s macroglobulinemia. 66. The method of claim 64 or 65, wherein the cancer in triple-negative breast cancer (TNBC). 67. An antibody-drug conjugate having formula (I): Ab-[L-D]_n formula (I) wherein in formula (I): Ab is an antibody or binding fragment thereof that binds CUB Domain-Containing Protein-1 (CDCP1); n is an integer from 1 to 20; and L-D has the formula: DB1/ 159693888.5 320

133186-5030-WO . 68. An antibody-drug conjugate having formula (I): Ab-[L-D]_n formula (I) wherein in formula (I): Ab is an antibody or binding fragment thereof that binds CUB Domain-Containing Protein-1 (CDCP1); n is an integer from 1 to 20; and L-D has the formula: . 69. An antibody-drug conjugate having formula (I): Ab-[L-D]n formula (I) wherein in formula (I): Ab is an antibody or binding fragment thereof that binds CUB Domain-Containing Protein-1 (CDCP1); n is an integer from 1 to 20; and L-D has the formula: DB1/ 159693888.5 321

133186-5030-WO . 7 . e ant o y- rug conjugate o any one o c a ms 7- , wherein the antibody or binding fragment thereof comprises: (i) a heavy chain variable region (VH) that comprises: (a) a VH complementarity determining region one (CDRH1) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 2, (b) a VH complementarity determining region two (CDRH2) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 3, and (c) a VH complementarity determining region three (CDRH3) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 4, and (ii) a light chain variable region (VL) that comprises: (a) a VL complementarity determining region one (CDRL1) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 6, (b) a VL complementarity determining region two (CDRL2) comprising and/or consisting of the amino acid sequence of SEQ ID NO: 7, and (c) a VL complementarity determining region three (CDRL3) comprising and/or consisting of the amino acid sequence of SEQ ID NO:8. 71. The antibody-drug conjugate of any one of claims 67-69, wherein the antibody or binding fragment thereof comprises a VH that comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1, and/or a VL that comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 5. DB1/ 159693888.5 322

133186-5030-WO 72. A method of treating a cancer comprising administering to a subject in need thereof a therapeutically effective amount of the antibody-drug conjugate of any one of claims 67-69. 73. A compound having the following formula: . 7 . compoun avng t e o ow ng ormua: . 75. An antibody-drug conjugate having formula (I): Ab-[L–D]n formula (I) wherein in formula (I): -L-D- has the formula: . 76. An antibody-drug conjugate having formula (I): Ab-[L–D]_n formula (I) wherein in formula (I): -L-D- has the formula: DB1/ 159693888.5 323

133186-5030-WO . . a oy- ug co ugae av g o ua : Ab-[L-D]_n formula (I) wherein in formula (I): L-D has the formula: . 78. An antibody-drug conjugate having formula (I): Ab-[L-D]_n formula (I) wherein in formula (I): Ab is an antibody or binding fragment thereof that binds CUB Domain-Containing Protein-1 (CDCP1); n is an integer from 1 to 20; and L-D has the formula: DB1/ 159693888.5 324

133186-5030-WO , n amino acid sequence as set forth in SEQ ID NO: 1, and a VL that comprises an amino acid sequence as set forth in SEQ ID NO: 5. 79. An antibody-drug conjugate having formula (I): Ab-[L-D]n formula (I) wherein in formula (I): Ab is an antibody or binding fragment thereof that binds CUB Domain-Containing Protein-1 (CDCP1); n is an integer from 1 to 20; and L-D has the formula: , wherein the antibody or binding fragment thereof comprises a VH that comprises an amino acid sequence as set forth in SEQ ID NO: 1, and a VL that comprises an amino acid sequence as set forth in SEQ ID NO: 5. 80. An antibody-drug conjugate having formula (I): Ab-[L-D]_n formula (I) wherein in formula (I): Ab is an antibody or binding fragment thereof that binds CUB Domain-Containing Protein-1 (CDCP1); n is an integer from 1 to 20; and L-D has the formula: DB1/ 159693888.5 325

133186-5030-WO , wee e a oy o g ag e eeo co pises a VH that comprises an amino acid sequence as set forth in SEQ ID NO: 1, and a VL that comprises an amino acid sequence as set forth in SEQ ID NO: 5. 81. A method of treating a cancer comprising administering to a subject in need thereof a therapeutically effective amount of the antibody-drug conjugate of any one of claims 75-80. DB1/ 159693888.5 326