Supplementary information to support article findings.

Abstract Antibody–drug conjugates (ADC) delivering pyrrolobenzodiazepine (PBD) DNA cross-linkers are currently being evaluated in clinical trials, with encouraging results in Hodgkin and non–Hodgkin lymphomas. The first example of an ADC delivering a PBD DNA cross-linker (loncastuximab tesirine) has been recently approved by the FDA for the treatment of relapsed and refractory diffuse large B-cell lymphoma. There has also been considerable interest in mono-alkylating PBD analogs. We conducted a head-to-head comparison of a conventional PBD bis-imine and a novel PBD mono-imine. Key Mitsunobu chemistry allowed clean and convenient access to the mono-imine class. Extensive DNA-binding studies revealed that the mono-imine mediated a type of DNA interaction that is described as “pseudo cross-linking,” as well as alkylation. The PBD mono-imine ADC demonstrated robust antitumor activity in mice bearing human tumor xenografts at doses 3-fold higher than those that were efficacious for the PBD bis-imine ADC. A single-dose toxicology study in rats demonstrated that the MTD of the PBD mono-alkylator ADC was approximately 3-fold higher than that of the ADC bearing a bis-imine payload, suggesting a comparable therapeutic index for this molecule. However, although both ADCs caused myelosuppression, renal toxicity was observed only for the bis-imine, indicating possible differences in toxicologic profiles that could influence tolerability and therapeutic index. These data show that mono-amine PBDs have physicochemical and pharmacotoxicologic properties distinct from their cross-linking analogs and support their potential utility as a novel class of ADC payload.

Synthetic pyrrolobenzodiazepine (PBD) dimers, where two PBD monomers are linked through their aromatic A-ring phenolic C8-positions via a flexible propyldioxy tether, are highly efficient DNA minor groove cross-linking agents with potent cytotoxicity. PBD dimer SG3199 is the released warhead component of the antibody-drug conjugate (ADC) payload tesirine (SG3249), currently being evaluated in several ADC clinical trials. SG3199 was potently cytotoxic against a panel of human solid tumour and haematological cancer cell lines with a mean GI(50) of 151.5 pM. Cells defective in DNA repair protein ERCC1 or homologous recombination repair showed increased sensitivity to SG3199 and the drug was only moderately susceptible to multidrug resistance mechanisms. SG3199 was highly efficient at producing DNA interstrand cross-links in naked linear plasmid DNA and dose-dependent cross-linking was observed in cells. Cross-links formed rapidly in cells and persisted over 36 hours. Following intravenous (iv) administration to rats SG3199 showed a very rapid clearance with a half life as short as 8 minutes. These combined properties of cytotoxic potency, rapid formation and persistence of DNA interstrand cross-links and very short half-life contribute to the emerging success of SG3199 as a warhead in clinical stage ADCs.

BACKGROUND:In order to investigate the mechanisms of acquired resistance to trabectedin, trabectedin-resistant human myxoid liposarcoma (402-91/T) and ovarian carcinoma (A2780/T) cell lines were derived and characterised in vitro and in vivo. METHODS:Resistant cell lines were obtained by repeated exposures to trabectedin. Characterisation was performed by evaluating drug sensitivity, cell cycle perturbations, DNA damage and DNA repair protein expression. In vivo experiments were performed on A2780 and A2780/T xenografts. RESULTS:402-91/T and A2780/T cells were six-fold resistant to trabectedin compared with parental cells. Resistant cells were found to be hypersensitive to UV light and did not express specific proteins involved in the nucleotide excision repair (NER) pathway: XPF and ERCC1 in 402-91/T and XPG in A2780/T. NER deficiency in trabectedin-resistant cells was associated with the absence of a G2/M arrest induced by trabectedin and with enhanced sensitivity (two-fold) to platinum drugs. In A2780/T, this collateral sensitivity, confirmed in vivo, was associated with an increased formation of DNA interstrand crosslinks. CONCLUSIONS:Our finding that resistance to trabectedin is associated with the loss of NER function, with a consequent increased sensitivity to platinum drugs, provides the rational for sequential use of these drugs in patients who have acquired resistance to trabectedin.

The design, synthesis, and DNA binding properties of azaHx-PI or p-anisyl-4-aza-benzimidazole-pyrrole-imidazole (5) are described. AzaHx, 2-(p-anisyl)-4-aza-benzimidazole-5-carboxamide, is a novel, fluorescent DNA recognition element, derived from Hoechst 33258 to recognize G·C base pairs. Supported by theoretical data, the results from DNase I footprinting, CD, ΔTM, and SPR studies provided evidence that an azaHx/IP pairing, formed from antiparallel stacking of two azaHx-PI molecules in a side-by-side manner in the minor groove, selectively recognized a C–G doublet. AzaHx-PI was found to target 5′-ACGCGT-3′, the Mlu1 Cell Cycle Box (MCB) promoter sequence with specificity and significant affinity (Keq 4.0±0.2×107M−1).

Hx-amides are fluorescent hybrids of imidazole (I)- and pyrrole (P)-containing polyamides and Hoechst 33258, and they bind in the minor groove of specific DNA sequences. Synthesis and DNA binding studies of HxII (5) complete our studies on the first set of Hx-amides: Hx-I/P-I/P. HxPP (2), HxIP (3) and HxPI (4) were reported earlier. Results from DNase I footprinting, biosensor-SPR, CD and ΔTM studies showed that Hx-amides interacted with DNA via the anti-parallel and stacked, side-by-side motif. Hx was found to mimic the DNA recognition properties of two consecutive pyrrole units (PP) in polyamides. Accordingly, the stacked Hx/PP pairing binds preferentially to two consecutive AT base pairs, A/T-A/T; Hx/IP prefers C-A/T; Hx/PI prefers A/T-C; and Hx/II prefers C-C. The results also showed that Hx-amides bound their cognate sequence at a higher affinity than their formamido-triamide counterparts.

A series of ferrocene analogues based on a 6,7-dimethoxy-N-phenylquinazolin-4-amine template has been synthesized, and two compounds were characterized in the solid state by X-ray crystallography. The compounds have been tested for in vitro anticancer activity, against epidermal growth receptor (EGFR), and submicromolar IC50 values have been determined.

Alchemix is an exemplar of a class of anthraquinone with efficacy against multidrug resistant tumours. We have explored further the mechanism of action of alchemix and investigated the effect of extending its side arm bearing the alkylating functionality with regard to DNA binding and activity against multidrug resistant cancer cells. Increasing the distance between the intercalating chromophore and the alkylating functionality of ICT2901 (propyl), ICT2902 (butyl) and ICT2903 (pentyl), led to a higher number of DNA alkylation sites, more potent topoisomerase II inhibition and generated more apoptotic and necrotic cells when analysed in p53-proficient HCT116 cells. Intriguingly, alchemix, the compound with the shortest distance between its intercalative chromophore and alkylating functionality (ethyl), did not conform to this SAR. A different toxicity pattern against DNA repair defective CHO cell lines as well as arrest of cells in G1 supports a somewhat distinct mode of action by alchemix compared with its analogues. Importantly, both alchemix and ICT2901 demonstrated greater cytotoxic activity against anthraquinone-resistant MCF-7/adr cells than wild-type MCF-7 cells. Subtle synthetic modification in this anthraquinone series has led to significant changes to the stability of DNA-compound complexes and cellular activity. Given that the failure of chemotherapy in the clinic is often associated with MDR, the results of both alchemix and ICT2901 represent important advances towards improved therapies.

Abstract Bendamustine has demonstrated substantial clinical efficacy in the treatment of hematologic malignancies and continues to distinguish itself from other alkylating agents. The mechanistic and clinical differences associated with bendamustine may be directly related to its unique structural features, although the precise mechanism of action is still poorly understood. We have undertaken a detailed study of the drug-DNA interactions of bendamustine. It alkylates DNA primarily at guanine-N7 positions with a sequence selectivity similar to other nitrogen mustards. It produces DNA interstrand cross-links (ICLs) in both solid tumor and hematological tumor cells at doses that cause growth inhibition. ICLs peak at 8h following a 1h treatment and persist over 48h in tumor cells including those that are proficient at repairing (unhooking) ICLs formed by melphalan or cisplatin. This persistence of ICLs is also observed in multiple myeloma cells from patients who have relapsed on melphalan therapy and which efficiently repair melphalan-induced ICLs compared to cells from treatment naive patients. The peak γ-H2AX response follows the ICL peak and also persists beyond 48h. Cells defective in ICL unhooking proteins ERCC1 and XPF, or defective in homologous recombination repair, show increased sensitivity to bendamustine, but at a level less than observed for other nitrogen mustards or cisplatin. Real time PCR profiling showed that at equivalent peak levels of DNA ICLs bendamustine induced fewer changes in DNA damage signaling and DNA repair gene expression compared to either cisplatin or melphalan. These data suggest that the critical DNA damage produced by bendamustine induces a different signaling and repair response to conventional cross-linking agents, including other nitrogen mustards, which may be an important contribution to its clinical efficacy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1766. doi:1538-7445.AM2012-1766

Background DNA interstrand cross-links (ICLs) are critical lesions produced by several cancer chemotherapy agents including platinum drugs and nitrogen mustards. We have previously shown in haematological (multiple myeloma) and solid tumours (ovarian cancer) that clinical sensitivity to such agents can result from a defect in DNA ICL processing leading to their persistence. Conversely, enhanced repair can result in clinical acquired resistance following chemotherapy. The repair of ICLs is complex but it is assumed that the ‘unhooking’ step is common to all ICLs. Methods Using a modification of the single cell gel electrophoresis (Comet) assay we measured the formation and unhooking of melphalan and cisplatin-induced ICLs in cell lines and clinical samples. DNA damage response in the form of γ-H2AX foci formation and the formation of RAD51 foci as a marker of homologous recombination were also determined. Real-time PCR of 84 genes involved in DNA damage signalling pathways was also examined pre- and post-treatment. Results Plasma cells from multiple myeloma patients known to be clinically resistant to melphalan showed significant unhooking of melphalan-induced ICLs at 48 hours, but did not unhook cisplatin-induced ICLs. In ovarian cancer cells obtained from patients following platinum-based chemotherapy, unhooking of cisplatin-induced ICLs was observed at 48 hours, but no unhooking of melphalan-induced ICLs. In vitro , A549 cells were proficient at unhooking both melphalan and cisplatin-induced ICLs. γ-H2AX foci formation closely followed the formation of ICLs for both drugs, and rapidly declined following the peak of formation. RPMI8226 cells unhooked melphalan, but not cisplatin-induced ICLs. In these cells, although cross-links form with cisplatin, the γ-H2AX response is weak. In A549 cells, addition of 3nM gemcitabine resulted in complete inhibition of cisplatin-induced ICL unhooking but no effect on repair of melphalan ICLs. The RAD51 foci response was both drug and cell line specific. Real time PCR studies highlighted differences in the damage response to melphalan and cisplatin following equi-ICL forming doses. Conclusions These data suggest that the mechanisms by which melphalan and cisplatin-induced ICLs are ‘unhooked’ in vitro are distinct, and the mechanisms of clinical acquired resistance involving repair of ICLs, are drug specific.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

1,3-Dihydro-2H-indol-2-one (1) undergoes microwave-mediated Knoevenagel condensations with 1,2,3,4,5-pentaphenylferrocene carboxaldehyde (2b) or (eta(4)-tetraphenyl-cyclobutadiene)(eta(5)-cyclopentadienylcarboxaldehyde)cobalt (2c) to afford metallocene-substituted 3-methylidene-1,3-dihydro-2H-indol-2-one 4 or 5, respectively, in excellent yields as separable E,Z-sfereoisomeric mixtures. Both 4 and 5 exhibited poor kinase inhibition and no appreciable cytotoxicity toward a leukemia cell line, attributed to the steric bulk of the metallocene substituents.

The synthesis of a series of azinomycin bisepoxides containing rigid linkers is achieved through two different strategies. Double copper-mediated C–N bond formation under Buchwald-type conditions can be realised but yields are poor with fully intact epoxide substrates. The bisepoxides are made in improved yields through the simultaneous formation of two amide bonds. Bioassays reveal that 5, containing a 1,3-diaminobenzene linker, is a potent in vitro DNA interstrand cross-linking agent with significant cytotoxicity in the NCI 60-human cancer cell panel (GI50=0.15μM).

We are seeking to develop more effective alkylating agents as antitumour agents. In previous work conformationally restricted nitrogen mustards were synthesised containing piperidine or pyrrolidine rings. The free bases were designed to be bifunctional alkylating agents via aziridinium ion formation and the effects of varying the distances between the two alkylating sites were studied. Some efficient cross-linkers of naked DNA were prepared but few of these compounds exhibited significant cytotoxicity in human tumour cells in vitro. We have extended this work by making tri- and tetra-azamacrocyclic compounds containing two to four potential alkylating sites. Most of these compounds were powerful DNA alkylating agents and showed cytotoxicity (IC(50) values 6-100microM) comparable with chlorambucil (45microM) and melphalan (8.5microM). In particular the cyclen derivative 2a was more than 10(4) times more effective at cross-linking DNA (2a XL(50)<<10nM) than chlorambucil (XL(50) 100microM), and showed significant cytotoxicity in human tumour cells in vitro.

The design, synthesis and in vitro activities of a series of cinnamoyl nitrogen mustard pyrazole analogues of tallimustine 8-13, in which the amidino moiety has been replaced by moieties of different physico-chemical features are described, and the structure-activity relationships are discussed. In spite of the relevance of these modifications on the amidino moiety, these derivatives showed significant growth inhibitory activity against mouse leukemia L1210 cells. A selected series of compounds have been evaluated for their sequence selective alkylating properties and cytotoxicity against human K562 leukemia cells. Therefore, the presence of the amidino moiety, and in general of a basic moiety, is not an absolute requirement for biological activity. Our preliminary results indicated that the compounds of this series have a pattern of alkylation similar to that of tallimustine, but they seem to be less reactive overall in alkylating naked DNA. (C) 2004 Elsevier Ltd. All rights reserved.

Neocarzinostatin (NCS) is an antitumor antibiotic comprising a 1:1 protein-chromophore complex and exhibits cytotoxic action through DNA cleavage via H-abstraction. Cytotoxic activity resides with the chromophore 1 alone, while the protein (apoNCS) protects and transports labile 1. The naphthoate portion (2) of NCS chromophore (1) is important for binding to apoNCS and DNA intercalation. In this paper we describe our attempts to use apoNCS to improve the hydrolytic stability of novel bifunctional DNA alkylating agents. The nitrogen mustards, melphalan and chlorambucil, were both conjugated to 2, and the biological activities of these conjugates were assessed. Chlorambucil did not benefit from conjugation. The melphalan conjugate (6) formed covalent DNA adducts at guanine bases and exhibited greater in vitro cytotoxic activity than unmodified melphalan. Fluorescence and NMR spectroscopy showed that 6 binds to apoNCS. Binding to apoNCS-protected 6 reduced the extent of hydrolysis of the conjugate. This novel approach demonstrates for the first time that an enediyne apo-protein can be used to improve the stability of substances that are of potential interest in cancer chemotherapy.

Compounds containing the naphthoate moiety of Neocarzinostatin chromophore or 2-hydroxynaphthoate have been synthesized and evaluated for cytotoxic activity against a leukemia cell line and a small panel of human-tumor cell lines. Those compounds containing a cyclopentenone moiety were active, with the carbonyl group being essential for biological activity.

A series of benzoyl and cinnamoyl nitrogen mustards tethered to different benzoheterocycles and to oligopyrroles structurally related to netropsin consisting of two pyrrole-amide units and terminating with an amidine moiety have been synthesised and a structure--activity relationship determined. Derivatives 3--10 have been evaluated for their sequence selective alkylating properties and cytotoxicity against human K562 leukaemia cells. They are 2- to 50-fold less cytotoxic than tallimustine, with compound 8 being the most potent member of this series. Among tallimustine isosters, the compounds with an indole 3 or benzothiophene 6 are 4-fold less cytotoxic than tallimustine, while the compounds with an N-methyl indole or benzofuran showed a 7- and 14-fold reduced cytotoxic potency, respectively. Our preliminary results indicate that these derivatives preferentially bind to AT-rich sequence with a sequence selectivity similar to tallimustine.

The design, synthesis, characterization, DNA binding properties, and cytotoxic activity of a novel series of hybrids, namely, a molecular combination of the natural antibiotic distamycin A and the antineoplastic agent uramustine, are reported, and the structure-activity relationships are discussed. This homologous series 29-34 consisted of the minor groove binder distamycin A joined to uramustine (uracil mustard) by suitable aliphatic carboxylic acid moieties containing a flexible polymethylene chain that is variable in length [(CH(2))(n)(), where n = 1-6). All the hybrid compounds in this series exhibit enhanced activity compared to both distamycin A and uramustine derivatives 22-27 used for conjugation, giving IC(50) values in the range 7.26-0.07 microM following a 1 h exposure of human leukemic K562 cells, with maximal activity shown when n = 6. The distance between the uramustine and distamycin frame is crucial for the cytotoxicity, with compounds having linker lengths of four to six being at least 20-fold more cytotoxic than linker lengths one to three. Taq polymerase stop experiments demonstrated selective covalent binding of uramustine-distamycin hybrids to A/T rich DNA sequences, which was again more efficient with compounds 32-34 with a longer linker length. Two consequences can be derived from our study: (a) the distamycin moiety directs binding to the minor groove of A/T rich DNA sequences and, consequently, is responsible for the alkylation regioselectivity found in footprinting studies; (b) the higher flexibility due to a longer linker between the distamycin and uracil moieties allows the formation of complexes with the mustard moiety situated more deeply in the minor groove and, hence, with better alkylating properties.