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Background: Aberrant expression of the MET receptor tyrosine kinase is an oncogenic determinant and a drug target for cancer therapy. Currently, antibody-based biotherapeutics targeting MET are under clinical trials. Objective: Here, we report the preclinical and therapeutic evaluation of a novel anti-MET antibody- drug conjugate PCMC1D3-duocarmycin SA (PCMC1D3-DCM) for targeted cancer therapy. Methods: The monoclonal antibody PCMC1D3 (IgG1a/κ), generated by a hybridoma technique and specific to one of the MET extracellular domains, was selected based on its high specificity to human MET with a binding affinity of 1.60 nM. PCMC1D3 was conjugated to DCM via a cleavable valine-citrulline dipeptide linker to form an antibody-drug conjugate with a drug-to-antibody ratio of 3.6:1. PCMC1D3-DCM in vitro rapidly induced MET internalization with an internalization efficacy ranging from 6.5 to 17.2h dependent on individual cell lines. Results: Studies using different types of cancer cell lines showed that PCMC1D3-DCM disrupted the cell cycle, reduced cell viability, and caused massive cell death within 96h after treatment initiation. The calculated IC50 values for cell viability reduction were 1.5 to 15.3 nM. Results from mouse xenograft tumor models demonstrated that PCMC1D3-DCM in a single dose injection at 10 mg/kg body weight effectively delayed xenograft tumor growth up to two weeks without signs of tumor regrowth. The calculated tumoristatic concentration, a minimal dose required to balance tumor growth and inhibition, was around 2 mg/kg body weight. Taken together, PCMC1D3-DCM was effective in targeting the inhibition of tumor growth in xenograft models. Conclusion: This work provides the basis for the development of humanized PCMC1D3-DCM for MET-targeted cancer therapy in the future.

Among my recent work on the syntheses of complex natural products based on the development of a novel synthetic method for the heteroaromatic skeleton, this article primarily deals with the total syntheses of (+)-CC-1065, isobatzeline A/B, and batzeline A. These syntheses were accomplished via a novel indole synthesis utilizing a ring expansion reaction of benzocyclobutenone oxime sulfonate as the key step. The 1,2-dihydro-3H-pyrrolo[3,2-e]indole segments of (+)-CC-1065 were rapidly constructed via a two-directional double-ring expansion strategy. Highly substituted pyrrolidine-fused common 5-chloro-2-methylthioindoles of isobatzeline A/B and batzeline A were constructed using a ring expansion reaction of benzocyclobutenone oxime sulfonate with NaSMe and a benzyne-mediated cyclization/functionalization reaction.

A wide range of diseases have been shown to be influenced by the accumulation of senescent cells, from fibrosis to diabetes, cancer, Alzheimer’s and other age-related pathologies. Consistent with this, clearance of senescent cells can prolong healthspan and lifespan in in vivo models. This provided a rationale for developing a new class of drugs, called senolytics, designed to selectively eliminate senescent cells in human tissues. The senolytics tested so far lack specificity and have significant off-target effects, suggesting that a targeted approach could be more clinically relevant. Here, we propose to use an extracellular epitope of B2M, a recently identified membrane marker of senescence, as a target for the specific delivery of toxic drugs into senescent cells. We show that an antibody–drug conjugate (ADC) against B2M clears senescent cells by releasing duocarmycin into them, while an isotype control ADC was not toxic for these cells. This effect was dependent on p53 expression and therefore more evident in stress-induced senescence. Non-senescent cells were not affected by either antibody, confirming the specificity of the treatment. Our results provide a proof-of-principle assessment of a novel approach for the specific elimination of senescent cells using a second generation targeted senolytic against proteins of their surfaceome, which could have clinical applications in pathological ageing and associated diseases.

Duocarmycins are a class of DNA minor-groove-binding alkylating molecules. For the past decade, various duocarmycin analogues have been used as payloads in the development of antibody-drug conjugates (ADCs). Currently, more than 15 duocarmycin-based ADCs have been studied preclinically, and some of them such as SYD985 have been granted Fast-Track Designation status. Nevertheless, progress in duocarmycin-based ADCs also faces challenges, with setbacks including the termination of BMS-936561/MDX-1203. In this review, we discuss issues associated with the efficacy, pharmacokinetic profile, and toxicological activity of these biotherapeutics. Furthermore, we summarize the latest advances in duocarmycin-based ADCs that have different target specificities and linker chemistries. Evidence from preclinical and clinical studies has indicated that duocarmycin-based ADCs are promising biotherapeutics for oncological application in the future.

The duocarmycins belong to a class of agent that has fascinated scientists for over four decades. Their exquisite potency, unique mechanism of action, and efficacy in multidrug-resistant tumour models makes them attractive to medicinal chemists and drug hunters. However, despite great advances in fine-tuning biological activity through structure-activity relationship studies (SARS), no duocarmycin-based therapeutic has reached clinical approval. In this review, we provide an overview of the most promising strategies currently used and include both tumour-targeted prodrug approaches and antibody-directed technologies.

Solid phase synthesis allowed the rapid generation of a peptide-drug conjugate. A peptide targeting the Thomsen-Friedenreich antigen (TFα) was conjugated to the alkylating subunit of the potent cytotoxin duocarmycin SA. The compound, containing a cathepsin B cleavable linker, was shown to be active and selective against TFα expressing tumour cell lines.

Microbes produce a broad spectrum of antibiotic natural products, including many DNA-damaging genotoxins. Among the most potent of these are DNA alkylating agents in the spirocyclopropylcyclohexadienone (SCPCHD) family, which includes the duocarmycins, CC-1065, gilvusmycin, and yatakemycin. The yatakemycin biosynthesis cluster in Streptomyces sp. TP-A0356 contains an AlkD-related DNA glycosylase, YtkR2, that serves as a self-resistance mechanism against yatakemycin toxicity. We previously reported that AlkD, which is not present in an SCPCHD producer, provides only limited resistance against yatakemycin. We now show that YtkR2 and C10R5, a previously uncharacterized homolog found in the CC-1065 biosynthetic gene cluster of Streptomyces zelensis, confer far greater resistance against their respective SCPCHD natural products. We identify a structural basis for substrate specificity across gene clusters and show a correlation between in vivo resistance and in vitro enzymatic activity indicating that reduced product affinity—not enhanced substrate recognition—is the evolutionary outcome of selective pressure to provide self-resistance against yatakemycin and CC-1065., Microbial DNA glycosylases associated with the biosynthesis of DNA-damaging antibiotics have evolved self-resistance for their cognate natural products. Here, the authors provide evidence that cellular self-resistance is enabled by reduced affinity of the glycosylases for the excision products of the corresponding DNA lesions.

Duocarmycin natural products are promising anticancer cytotoxins but too potent for systemic use. Re-engineering of the duocarmycin scaffold has enabled the discovery of prodrugs designed for bioactivation by tissue-specific cytochrome P450 (P450) enzymes. Lead prodrugs bioactivated by both P450 isoforms CYP1A1 and CYP2W1 have shown promising results in xenograft studies; however, to fully understand the potential of these agents it is desirable to compare dual-targeting compounds with isoform-selective analogs. Such redesign requires insight into the molecular interactions with these P450 enzymes. Herein binding and metabolism of the individual stereoisomers of the indole-based duocarmycin prodrug ICT2700 and a nontoxic benzofuran analog ICT2726 were evaluated with CYP1A1 and CYP2W1, revealing differences exploitable for drug design. Although enantiomers of both compounds bound to and were metabolized by CYP1A1, the stereochemistry of the chloromethyl fragment was critical for CYP2W1 interactions. CYP2W1 differentially binds the S enantiomer of ICT2726, and its metabolite profile could potentially be used as a biomarker to identify CYP2W1 functional activity. In contrast to benzofuran-based ICT2726, CYP2W1 differentially binds the R isomer of the indole-based ICT2700 over the S stereoisomer. Thus the ICT2700 R configuration warrants further investigation as a scaffold to favor CYP2W1-selective bioactivation. Furthermore, structures of both duocarmycin S enantiomers with CYP1A1 reveal orientations correlating with nontoxic metabolites, and further drug design optimization could lead to a decrease of CYP1A1 bioactivation. Overall, distinctive structural features present in the two P450 active sites can be useful for improving P450—and thus tissue-selective—bioactivation. SIGNIFICANCE STATEMENT: Prodrug versions of the natural product duocarmycin can be metabolized by human tissue-specific cytochrome P450 (P450) enzymes 1A1 and 2W1 to form an ultrapotent cytotoxin and/or high affinity 2W1 substrates to potentially probe functional activity in situ. The current work defines the binding and metabolism by both P450 enzymes to support the design of duocarmycins selectively activated by only one human P450 enzyme.

Summary We identify a selective nanomolar inhibitor of blood-stage malarial proliferation from a screen of microbial natural product extracts. The responsible compound, PDE-I2, is a precursor of the anticancer duocarmycin family that preserves the class's sequence-specific DNA binding but lacks its signature DNA alkylating cyclopropyl warhead. While less active than duocarmycin, PDE-I2 retains comparable antimalarial potency to chloroquine. Importantly, PDE-I2 is >1,000-fold less toxic to human cell lines than duocarmycin, with mitigated impacts on eukaryotic chromosome stability. PDE-I2 treatment induces severe defects in parasite nuclear segregation leading to impaired daughter cell formation during schizogony. Time-of-addition studies implicate parasite DNA metabolism as the target of PDE-I2, with defects observed in DNA replication and chromosome integrity. We find the effect of duocarmycin and PDE-I2 on parasites is phenotypically indistinguishable, indicating that the DNA binding specificity of duocarmycins is sufficient and the genotoxic cyclopropyl warhead is dispensable for the parasite-specific selectivity of this compound class.

Duocarmycins [including cyclopropyl pyrroloindole (CPI) or cyclopropyl benzoindole (CBI)] are a class of DNA minor-groove alkylators and seco-CPI/CBIs are synthetic pro-forms that can spirocyclize to CPI/CBI. Bis-CPI/CBIs are potential drug candidates because of their enhanced cytotoxicity from DNA crosslinking, but it is difficult to analyze them for structure-activity correlation because of their DNA reactivity. To study their DNA alkylation, neutral thermal hydrolysis has been frequently applied to process depurination. However, unwanted side reactions under this condition have been reported, which could lead to poor correlation of DNA alkylation data with efficacy results, especially for bis-CPI/CBIs. In this study, an acidic depurination method was developed and applied for analysis of DNA alkylation and shown to be an easier and milder method than the traditional neutral thermal hydrolysis. DNA alkylation and stability of three bis-seco-CBIs were characterized in comparison with two mono-seco-CPIs. The results suggested that: 1) The acidic depurination method was capable of capturing a more representative population, sometimes a different population, of DNA adducts as they existed on DNA compared with the heat depurination method. 2) Di-adenine adducts were captured as expected for the CBI dimers, although the major type of adduct was still mono-adenine adducts. 3) The rate of DNA alkylation, DNA adduct profile, and relative amounts of di-adduct versus mono-adduct were significantly affected by the size, and possibly lipophilicity, of the nonalkylating part of the molecules. 4) Spirocyclization and amide hydrolysis represented two major pathways of degradation. Overall, by applying acidic depurination analyses, this study has illustrated DNA adduct characteristics of novel bis-seco-CBIs with dominating mono-alkylation and provides an alternative method for evaluating DNA minor-groove alkylators. These findings provide an effective analytical tool to evaluate DNA alkylators and to study the DNA alkylation that is a disposition mechanism of these compounds.

This study describes the characterization of conjugation sites for a random, lysine conjugated 2-iminothiolane (2-IT) based antibody-drug-conjugate synthesized from an IgG1 antibody and a duocarmycin analog-based payload-linker. Of the 80 putative lysine sites, 78 were found to be conjugated via tryptic peptide mapping and LC-HRMS. Surprisingly, seven cysteine-linked conjugated peptides were also detected resulting from the conjugation of cysteine residues derived from the four inter-chain disulfide bonds during the reaction. This unexpected finding could be attributed to the free thiols of the 2-IT thiolated antibody intermediates and/or the 4-mercaptobutanamide by-product resulting from the hydrolysis of 2-IT. These free thiols could cause the four inter-chain disulfide bonds of the antibody to scramble via intra- or inter-molecular attack. The presence of only pair of non-reactive (unconjugated) lysine residues, along with the four intact intra-chain disulfide bonds, is attributed to their poor accessibility, which is consistent with solvent accessibility modeling analysis. We also discovered a major by-product derived from the hydrolysis of the amidine moiety of the N-terminus conjugate. In contrast, the amidine moiety in lysine-linked conjugates appeared stable. Based on our results, we propose plausible formation mechanisms of cysteine-linked conjugates and the hydrolysis of the N-terminus conjugate, which provide scientific insights that are beneficial to process development and drug quality control.

Engineering cysteines at specific sites in antibodies to create well-defined ADCs for the treatment of cancer is a promising approach to increase the therapeutic index and helps to streamline the manufacturing process. Here, we report the development of an

Senescence is a stable growth arrest that impairs the replication of damaged, old or preneoplastic cells, therefore contributing to tissue homeostasis. Senescent cells accumulate during ageing and are associated with cancer, fibrosis and many age‐related pathologies. Recent evidence suggests that the selective elimination of senescent cells can be effective on the treatment of many of these senescence‐associated diseases. A universal characteristic of senescent cells is that they display elevated activity of the lysosomal β‐galactosidase, and this has been exploited as a marker for senescence (senescence‐associated β‐galactosidase activity). Consequently, we hypothesized that galactose‐modified cytotoxic prodrugs will be preferentially processed by senescent cells, resulting in their selective killing. Here, we show that different galactose‐modified duocarmycin (GMD) derivatives preferentially kill senescent cells. GMD prodrugs induce selective apoptosis of senescent cells in a lysosomal β‐galactosidase (GLB1)‐dependent manner. GMD prodrugs can eliminate a broad range of senescent cells in culture, and treatment with a GMD prodrug enhances the elimination of bystander senescent cells that accumulate upon whole‐body irradiation treatment of mice. Moreover, taking advantage of a mouse model of adamantinomatous craniopharyngioma (ACP), we show that treatment with a GMD prodrug selectively reduced the number of β‐catenin‐positive preneoplastic senescent cells. In summary, the above results make a case for testing the potential of galactose‐modified duocarmycin prodrugs to treat senescence‐related pathologies., Galactose‐modified duocarmycin prodrugs get preferentially processed in senescent cells and behave as senolytics.

Background Antibody-drug conjugates (ADCs) targeting the RON receptor, a tumorigenic factor contributing to cancer malignancy, has been considered as a novel strategy for cancer therapy. Here we describe a humanized antibody recognizing the RON plexin-semaphorin-integrin (PSI) domain with increased drug delivery capability for potential clinical application. Method Monoclonal antibody PCM5B14 specific to the human and monkey RON PSI domain was generated and characterized by various immunological methods. Humanized antibody H5B14 was created by grafting PCM5B14 complementarity-determining regions into human IgG1/κ acceptor frameworks and conjugated with monomethyl auristatin E and duocarmycin to form two H5B14-based ADCs. Stability of H5B14-based ADCs in human plasma was measured using hydrophobic interaction chromatography. Various biochemical and biological assays were used to determine ADC- regulated RON internalization, cell viability, spheroid formation, and death of cancer stem-like cells. Efficacies of H5B14-based ADCs in vivo were validated using tumor xenograft models. Maximal tolerated doses of H5B14-based ADCs were established in mice. Results H5B14 was highly specific to the human RON PSI domain and superior over other anti-RON ADCs in induction of RON internalization in various cancer cell lines tested. H5B14-based ADCS had a drug to antibody ratio of ~ 3.70:1 and were stable in human plasma with a minimal dissociation within a 10-day period. Functionally, H5B14-mediated drug delivery decreased cell viability at early stages with an average IC50 at ~ 20 nM in multiple cancer cell lines examined. H5B14-based ADCs also inhibited spheroid formation and caused death of cancer stem-like cells with RON+/CD44+/ESA+ phenotypes. In vivo, H5B14-based ADCs in a single injection inhibited tumor xenograft growth mediated by multiple cancer cell lines. Tumoristatic concentrations calculated from xenograft tumor models were in the range of 0.63 to 2.0 mg/kg bodyweight. Significantly, H5B14-based ADCs were capable of eradicating tumors at variable levels across multiple xenograft models regardless their malignant statuses. Toxicologically, H5B14-based ADCs were well tolerated in mice up to 60 mg/kg. Conclusion H5B14-based ADCs targeting the RON PSI domain are superior in inducing RON internalization, leading to robust drug delivery and overall inhibition and eradication of tumors in multiple xenograft models. These findings warrant H5B14-based ADCs for clinical trials in the future.

Small cell lung cancer (SCLC) is of a highly invasive and metastatic lung cancer subtype and there had not been effective targeted therapies. CD56, a cell surface marker highly expressed on most SCLC, is a promising therapeutic target for treatment of this aggressive cancer. In this study, we generated a novel anti-CD56 antibody named promiximab, characterized by high affinity, internalization and tumor specificity. Then, the promiximab was conjugated with a potent DNA alkylating agent duocarmycin via reduced interchain disulfides to yield the promiximab-Duocarmycin (promiximab-DUBA) conjugates. Mass spectrometry analysis showed promiximab-DUBA had an average DAR (Drug-to-Antibody Ratio) of about 2.04. In vitro, promiximab-DUBA exerted strong inhibitory effects on SCLC cell lines NCI-H526, NCI-H524 and NCI-H69, with IC50 values of 0.07 nmol/L, 0.18 nmol/L and 0.29 nmol/L, respectively. In vivo antitumor activity, promiximab-DUBA at the dose of 5 mg/kg and 10 mg/kg every three days with a total of three times were sufficient to induce sustained regression of NCI-H526 tumors over control treatment with promiximab. Mostly, no recurrence was observed until 65 days post treatment with promiximab-DUBA. In the NCI-H69 subcutaneous xenograft model, significant inhibition of tumor growth was also observed following administration of promiximab-DUBA at the dose of 5 mg/kg or 10 mg/kg. Moreover, body weight and histopathology of major organs (liver, spleen, heart, lung and kidney) showed no significant changes after treatment of promiximab-DUBA. In conclusion, promiximab-DUBA is highly efficacious in small cell lung cancer xenograft models, and provides a new immunotherapy approach for SCLC.

The duocarmycins belong to a class of agent which has great potential for use in cancer therapy. Their exquisite potency means they are too toxic for systemic use, and targeted approaches are required to unlock their clinical potential. In this study, we have explored seco-OH-chloromethylindoline (CI) duocarmycin-based bioprecursors for their potential for cytochrome P450 (CYP)-mediated cancer cell kill. We report on synthetic and biological explorations of racemic seco-CI-MI, where MI is a 5-methoxy indole motif, and dehydroxylated analogues. We show up to a 10-fold bioactivation of de-OH CI-MI and a fluoro bioprecursor analogue in CYP1A1-transfected cells. Using CYP bactosomes, we also demonstrate that CYP1A2 but not CYP1B1 or CYP3A4 has propensity for potentiating these compounds, indicating preference for CYP1A bioactivation.

Epithelial ovarian cancer (EOC) is an aggressive and heterogeneous disease.10% of EOC demonstrate HER2/neu 3+ receptor over-expression. However, moderate to low (i.e., 2+ and 1+) HER2/neu expression is reported in up to 50% of EOC. The objective of this study was to compare the anti-tumor activity of SYD985, a novel HER2-targeting antibody-drug conjugate (ADC), to trastuzumab emtansine (T-DM1) in EOC models with differential HER2/neu expression.The cytotoxicity of SYD985 and T-DM1 was evaluated using ten primary EOC cell lines with 0/1+, 2+, and 3+ HER2/neu expression in antibody-dependent cellular cytotoxicity (ADCC), proliferation, viability and bystander killing experiments. Finally, the in vivo activity of SYD985 and T-DM1 was also studied in ovarian cancer xenografts.SYD985 and T-DM1 induced similar ADCC in the presence of peripheral blood lymphocytes (PBL) against EOC cell lines with differential HER2/neu expression. In contrast, SYD985 was 3 to 42 fold more cytotoxic in the absence of PBL when compared to T-DM1 (p0.0001). Unlike T-DM1, SYD985 induced efficient bystander killing of HER2/neu 0/1+ tumor cells when admixed with HER2/neu 3+ EOC cells. In vivo studies confirmed that SYD985 is significantly more active than T-DM1 against HER2/neu 3+ EOC xenografts.SYD985 is a novel ADC with remarkable activity against EOC with strong (3+) as well as moderate to low (i.e., 2+ and 1+) HER2/neu expression. SYD985 is more potent than T-DM1 in comparative experiments and unlike T-DM1, it is active against EOC demonstrating moderate/low or heterogeneous HER2/neu expression.

Yatakemycin (YTM) is an extraordinarily toxic DNA alkylating agent with potent antimicrobial and antitumor properties and is the most recent addition to the CC-1065 and duocarmycin family of natural products. Though bulky DNA lesions the size of those produced by YTM are normally removed from the genome by the nucleotide-excision repair (NER) pathway, YTM adducts are also a substrate for the bacterial DNA glycosylases AlkD and YtkR2, unexpectedly implicating base-excision repair (BER) in their elimination. The reason for the extreme toxicity of these lesions and the molecular basis for the way they are eliminated by BER have been unclear. Here, we describe the structural and biochemical properties of YTM adducts that are responsible for their toxicity, and define the mechanism by which they are excised by AlkD. These findings delineate an alternative strategy for repair of bulky DNA damage and establish the cellular utility of this pathway relative to that of NER.

A new series of nitro analogues of the duocarmycins was prepared and evaluated for hypoxia-selective anticancer activity. The compounds incorporate 13 different amine-containing side chains designed to bind in the minor groove of DNA while spanning a wide range of base strength from pKa 9.64 to 5.24. The most favorable in vitro properties were associated with strongly basic side chains, but the greatest in vivo antitumor activity was found for compounds containing a weakly basic morpholine. This applies to single-agent activity and for activity in combination with irradiation or chemotherapy (gemcitabine or docetaxel). In combination with a single dose of γ irradiation 50 at 42 μmol/kg eliminated detectable clonogens in some SiHa cervical carcinoma xenografts, and in combination with gemcitabine using a well-tolerated multidose schedule, the same compound caused regression of all treated A2780 ovarian tumor xenografts. In the latter experiment, three of seven animals receiving the combination treatment we...

CC-1065 is the first characterized member of a family of naturally occurring antibiotics including yatakemycin and duocarmycins with exceptionally potent antitumor activity. CC-1065 contains three benzodipyrroles (1a-, 1b-, and 1c-) of which the 1a-subunit is remarkable by being composed of a cyclopropane ring, and the mechanism for the biological formation of benzodipyrrole rings remains elusive. Previously, biosynthetic studies of CC-1065 were limited to radioactively labeled precursor feeding experiments, which showed that tyrosine (Tyr) and serine (Ser) were incorporated into the two benzodipyrrole (1b- and 1c-) subunits via the same mode but that this was different from the key cyclopropabenzodipyrrole (1a-) subunit with N1-C2-C3 derived from Ser. Herein, the biosynthetic gene cluster of CC-1065 has been cloned, analyzed, and characterized by a series of gene inactivations. Significantly, a key intermediate bearing a C7-OH group derived from a Δc10C mutant exhibited improved cytotoxicity. Moreover, this data inspired us to suspect that the 1a-subunit might employ the same precursor incorporation mode as the 1b- and 1c-subunits. Subsequently

The currently marketed antibody-drug conjugates (ADC) destabilize microtubule assembly in cancer cells and initiate apoptosis in patients. However, few tumor antigens (TA) are expressed at high densities on cancer lesions, potentially minimizing the therapeutic index of current ADC regimens. The peptide/human leukocyte antigen (HLA) complex can be specifically targeted by therapeutic antibodies (designated T cell receptor [TCR]-like antibodies) and adequately distinguish malignant cells, but has not been the focus of ADC development. We analyzed the killing potential of TCR-like ADCs when cross-linked to the DNA alkylating compound duocarmycin. Our data comprise proof-of-principle results that TCR-like ADCs mediate potent tumor cytotoxicity, particularly under common scenarios of low TA/HLA density, and support their continued development alongside agents that disrupt DNA replication. Additionally, TCR-like antibody ligand binding appears to play an important role in ADC functionality and should be addressed during therapy development to avoid binding patterns that negate ADC killing efficacy.

Here we describe a new approach for tumor targeting in which augmented concentrations of Fe(II) in cancer cells and/or the tumor microenvironment triggers drug release from an Fe(II)-reactive prodrug conjugate. The 1,2,4-trioxolane scaffold developed to enable this approach can in principle be applied to a broad range of cancer therapeutics and is illustrated here with Fe(II)-targeted forms of a microtubule toxin and a duocarmycin-class DNA-alkylating agent. We show that the intrinsic reactivity/toxicity of the duocarmycin analog is masked in the conjugated form and this greatly reduced toxicity in mice. This in turn permitted elevated dosing levels, leading to higher systemic exposure and a significantly improved response in tumor xenograft models. Overall our results suggest that Fe(II)-dependent drug delivery via trioxolane conjugates could have significant utility in expanding the therapeutic index of a range of clinical and preclinical stage cancer chemotherapeutics.

A Pd-catalysed amination method is used to convert seco-CBI, a synthetic analogue of the alkylating subunit of the duocarmycin natural products, from the phenol to amino form. This allows efficient enantioselective access to the more potent S enantiomer of aminoCBI and its incorporation into analogues of DNA minor groove cross-linking agents. Evaluation in a panel of nine human tumour cell lines shows that the bifunctional agents containing aminoCBI are generally less cytotoxic than their phenolCBI analogues and more susceptible to P-glycoprotein-mediated resistance. However, all bifunctional agents are potent cytotoxins, some in the sub-pM IC50 range, with in vitro properties that compare favourably with established microtubule-targeted ADC payloads.