H. D. Sarath Liyanage

Nitro-chloromethylbenzindolines: hypoxia-activated prodrugs of potent adenine N3 DNA minor groove alkylators

Hypoxia represents an important therapeutic target in tumors because of the resistance of hypoxic cells to radiotherapy and chemotherapy and because it is more severe in many tumors than in normal tissues. Here, we describe a class of prodrugs, nitro-chloromethylindolines, which undergo hypoxia-selective activation by endogenous nitroreductases in tumor cells to form the corresponding amino compounds. The latter are chemically related to the cyclopropylindoline antitumor antibiotics and they share the same properties of sequence-selective DNA minor groove alkylation and high cytotoxic potency. Of three alkylating subunits investigated, the chloromethylbenzindoline (CBI) structure provided the most favorable prodrug properties: aerobic cytotoxic potency of the amines was approximately 90- to 3,000-fold higher than the corresponding nitro compounds, and the nitro compounds showed air/anoxia potency differentials of up to 300-fold. Selective alkylation of adenine N3 in calf thymus DNA by an amino-CBI was shown by characterization of the thermal depurination product; the same adduct was shown in hypoxic RIF-1 cells exposed to the corresponding nitro-CBI prodrug under hypoxic (but not oxic) conditions. The amino metabolite generated from a nitro-CBI by cells expressing Escherichia coli nfsB nitroreductase in multicellular layer cultures was shown to elicit bystander killing of surrounding cells. Nitro-CBI prodrugs were >500-fold less toxic to mice than amino-CBIs by i.p. administration and provided selective killing of hypoxic cells in RIF-1 tumors (although only at maximally tolerated doses). Nitro-CBIs are novel lead hypoxia-activated prodrugs that represent the first examples of hypoxia-selective generation of potent DNA minor groove alkylating agents. [Mol Cancer Ther 2009;8(10):2903–13]

The flavoprotein FOXRED2 reductively activates nitro-chloromethylbenzindolines and other hypoxia-targeting prodrugs

The nitro-chloromethylbenzindoline prodrug SN29428 has been rationally designed to target tumour hypoxia. SN29428 is metabolised to a DNA minor groove alkylator via oxygen-sensitive reductive activation initiated by unknown one-electron reductases. The present study sought to identify reductases capable of activating SN29428 in tumours. Expression of candidate reductases in cell lines was modulated using forced expression and, for P450 (cytochrome) oxidoreductase (POR), by zinc finger nuclease-mediated gene knockout. Affymetrix microarray mRNA expression of flavoreductases was correlated with SN29428 activation in a panel of 23 cancer cell lines. Reductive activation and cytotoxicity of prodrugs were measured using mass spectrometry and antiproliferative assays, respectively. SN29428 activation under hypoxia was strongly attenuated by the pan-flavoprotein inhibitor diphenyliodonium, but less so by knockout of POR suggesting other flavoreductases contribute. Forced expression of 5-methyltetrahydrofolate-homocysteine methyltransferase reductase (MTRR), as well as POR, increased activation of SN29428 in hypoxic HCT 116 cells. SN29428 activation strongly correlated with expression of POR and also FAD-dependent oxidoreductase domain containing 2 (FOXRED2), in cancer cell lines. This association persisted after removing the effect of POR enzyme activity using first-order partial correlation. Forced expression of FOXRED2 increased SN29428 activation and cytotoxicity in hypoxic HEK293 cells and also increased activation of hypoxia-targeted prodrugs PR-104A, tirapazamine and SN30000, and increased cytotoxicity of the clinical-stage prodrug TH-302. Thus this study has identified three flavoreductases capable of enzymatically activating SN29428, one of which (FOXRED2) has not previously been implicated in xenobiotic metabolism. These results will inform future development of biomarkers predictive of SN29428 sensitivity.

The Cytotoxicity of Duocarmycin Analogues is Mediated through Alkylation of DNA, not Aldehyde Dehydrogenase 1: A Comment

Duocarmycin SA (1) is one member of a small group of natural products that are notable for their extreme cytotoxicity, which is generally believed to be a consequence of DNA alkylation. Much evidence has been advanced to support a mechanism of action involving sequence-selective reaction at N3 of adenine at the base of the minor groove; this evidence includes, for example, the isolation of adducts such as 2 following thermal depurination of alkylated DNA (Scheme 1). It was recently reported that duocarmycin analogues (S,S)-3 and (S)-4, bearing synthetic seco variants of the alkylating subunit (with the same enantiomeric form as the natural products), are able to alkylate aldehyde dehydrogenase 1 (ALDH1A1), and indeed these compounds were described as selective and potent inhibitors of ALDH1A1 in A549 lung cancer cells. The evidence for ALDH1A1 alkylation included activity-based protein profiling (ABPP) of the “clickable” analogue (S)-4, MS/MS sequencing of recombinant ALDH1A1 alkylated with (S)-4, and competition between (S,S)-3 and (S)-4 in the ABPP assay. ALDH1A1 inhibition was investigated by using recombinant enzyme and A549 cell lysates rather than living cells, thereby making it difficult to relate the observed inhibitory potency to cytotoxicity. Nevertheless, the authors concluded that inhibition of ALDH1A1 contributes to the cytotoxicity of the duocarmycin family of compounds, and that in particular for (S,S)-3 this inhibition is likely to be the preferred mechanism of action. This inference was based on the claim that (S,S)-3 does not bind to or alkylate DNA, citing previous work from the same group that failed to detect an interaction between (S,S)-3 and oligonucleotides by circular dichroism, or evidence for significant interstrand crosslinks by mass spectrometry. These observations stand in marked contrast to earlier studies in which dimers of a similar structure to (S,S)-3 were shown to crosslink DNA and a correlation between crosslinking ability and cytotoxicity was demonstrated. Prompted by these observations we have investigated (S,S)-3 and (S)-4 and find that while these compounds are indeed capable of alkylating ALDH1A1 (and other proteins), their cytotoxicity, including the marked picomolar toxicity of (S,S)-3, can be attributed to the alkylation of DNA. (S,S)-3 and (S)-4 and their previously unreported enantiomers (R,R)-3 and (R)-4 were prepared using slight modifications of the published procedure. To investigate if (S,S)-3 can alkylate DNA a solution of this compound (0.1 mm) was incubated with calf thymus DNA (ctDNA, 250 mm in base pairs) at 37 8C in aqueous buffer containing 10% dimethylacetamide (DMA; Figure 1). Under these conditions and in the absence of DNA (S,S)-3 is completely converted via the monocyclopropyl intermediate [(S,S)-S3 in the Supporting Information] to the biscyclopropyl compound (S,S)-5 over about 3 h; the products were characterized by LC–MS and UVabsorbance spectra. Incubation with DNA Scheme 1. Structures of duocarmycin SA, its N3 adenine adduct, and the seco duocarmycin analogues 3 and 4.

Preparation and Antitumour Properties of the Enantiomers of a Hypoxia‐Selective Nitro Analogue of the Duocarmycins

Racemic 2‐{[1‐(chloromethyl)‐5‐nitro‐3‐{5‐[2‐(dimethylamino)ethoxy]indol‐2‐carbonyl}‐1,2‐dihydro‐3H‐benzo[e]indol‐7‐yl]sulfonyl}aminoethyl dihydrogen phosphate, a synthetic nitro derivative of the duocarmycins, is a hypoxia‐selective prodrug active against radiation‐resistant tumour cells at nontoxic doses in mice. An intermediate in the synthesis of this prodrug was resolved by chiral HPLC and the absolute configuration assigned by X‐ray crystallography. The intermediate was used to prepare the prodrug′s enantiomers, and also the enantiomers of the active nitro and amino metabolites. In vitro analysis in the human cervical carcinoma cell line SiHa showed that both nitro enantiomers are hypoxia‐selective cytotoxins, but the “natural” S enantiomer is at least 20‐fold more potent. Examination of extracellular amino metabolite concentrations demonstrated no enantioselectivity in the hypoxia‐selective reduction of nitro to amino. Low levels of amino derivative were also found in aerobic cell suspensions, sufficient to account for the observed oxic toxicity of the nitro form. At an equimolar dose in SiHa‐tumour bearing animals, the (−)‐R enantiomer of the prodrug was inactive, while the (+)‐S enantiomer caused significantly more hypoxic tumour cell kill than the racemate. At this dose, the combination of (+)‐S‐prodrug and radiation eliminated detectable colony‐forming cells in four out of five treated tumour‐bearing animals.

Reductive Metabolism Influences the Toxicity and Pharmacokinetics of the Hypoxia-Targeted Benzotriazine Di-Oxide Anticancer Agent SN30000 in Mice

3-(3-Morpholinopropyl)-7,8-dihydro-6H-indeno[5,6-e][1,2,4]triazine 1,4-dioxide (SN30- 000), an analog of the well-studied bioreductive prodrug tirapazamine (TPZ), has improved activity against hypoxic cells in tumor xenografts. However, little is known about its biotransformation in normal tissues. Here, we evaluate implications of biotransformation of SN30000 for its toxicokinetics in NIH-III mice. The metabolite profile demonstrated reduction to the 1-N-oxide (M14), oxidation of the morpholine side-chain (predominantly to the alkanoic acid M18) and chromophore, and subsequent glucuronidation. Plasma pharmacokinetics of SN30000 and its reduced metabolites was unaffected by the presence of HT29 tumor xenografts, indicating extensive reduction in normal tissues. This bioreductive metabolism, as modeled by hepatic S9 preparations, was strongly inhibited by oxygen indicating that it proceeds via the one-electron (radical) intermediate previously implicated in induction of DNA double strand breaks and cytotoxicity by SN30000. Plasma pharmacokinetics of SN30000 and M14 (but not M18) corresponded closely to the timing of reversible acute clinical signs (reduced mobility) and marked hypothermia (rectal temperature drop of ∼8°C at nadir following the maximum tolerated dose). Similar acute toxicity was elicited by dosing with TPZ or M14, although M14 did not induce the kidney and lung histopathology caused by SN30000. M14 also lacked antiproliferative potency in hypoxic cell cultures. In addition M14 showed much slower redox cycling than SN30000 in oxic cultures. Thus a non-bioreductive mechanism, mediated through M14, appears to be responsible for the acute toxicity of SN30000 while late toxicities are consistent with DNA damage resulting from its one-electron reduction. A two-compartment pharmacokinetic model, in which clearance of SN30000 is determined by temperature-dependent bioreductive metabolism to M14, was shown to describe the non-linear PK of SN30000 in mice. This study demonstrates the importance of non-tumor bioreductive metabolism in the toxicology and pharmacokinetics of benzotriazine di-oxides designed to target tumor hypoxia.

Preparation and properties of clickable amino analogues of the duocarmycins: factors that affect the efficiency of their fluorescent labelling of DNA.

Herein we report the synthesis of three DNA‐alkylating amino analogues of the duocarmycins that carry an alkyne functional group suitable for copper‐catalysed click chemistry. The alkyne‐containing substituents are connected via a side chain position which projects away from the minor groove, and have only a small effect on DNA alkylation and cytotoxicity. The efficiency of click reactions with fluorophore azides was studied using alkylated ctDNA by analysing the adenine adducts produced after thermal depurination. Click reactions “on DNA” were sensitive to steric effects (tether length to the alkyne) and, surprisingly, to the nature of the fluorophore azide. With the best combination of click partners and reagents, adducts could be detected in the nuclei of treated cells by microscopy or flow cytometry, provided that an appropriate detergent (Triton X‐100 and not Tween 20) was used for permeabilisation. The method is sensitive enough to detect adducts at physiologically relevant concentrations, and could have application in the development of nitro analogues of the duocarmycins as hypoxia‐activated anticancer prodrugs.

Mechanism of Action of AminoCBIs: Highly Reactive but Highly Cytotoxic Analogues of the Duocarmycins

Duocarmycins are highly cytotoxic natural products that have potential for development into anticancer agents. Herein we describe proposed but previously unidentified NH analogues of the DNA‐alkylating subunit and characterise these by solvolysis studies, NMR and computational modelling. These compounds are shown to be the exclusive intermediates in the solvolysis of their seco precursors and to possess very similar structural features to the widely studied O‐based analogues, apart from an unusually high basicity. The measured pKa of 10.5 implies that the NH compounds are fully protonated under physiological conditions. Remarkably, their extremely high reactivity (calculated hydrolysis rate 108 times higher for protonated NH compared to the neutral O analogue) is still compatible with potent cytotoxicity, provided the active species is formed in the presence of cells. These surprising findings are of relevance to the design of duocarmycin‐based tumour‐selective therapies.

Influence of a Basic Side Chain on the Properties of Hypoxia-Selective Nitro Analogues of the Duocarmycins: Demonstration of Substantial Anticancer Activity in Combination with Irradiation or Chemotherapy

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 were completely tumor free at day 100.

Drug-DNA adducts as biomarkers for metabolic activation of the nitro-aromatic nitrogen mustard prodrug PR-104A

Graphical abstract Figure. No Caption available. ABSTRACT PR‐104A is a clinical‐stage nitrogen mustard prodrug that is activated for DNA alkylation by reduction of a nitro group to the corresponding hydroxylamine (PR‐104H) or amine (PR‐104M). Metabolic reduction is catalysed by flavoreductases such as cytochrome P450 oxidoreductase (POR) under hypoxia, or by aldo‐ketoreductase 1C3 (AKR1C3) independently of hypoxia. The unstable reduced metabolites are challenging to measure in biological samples, and biomarkers of the metabolic activation of PR‐104A have not been used in the clinical evaluation of PR‐104 to date. Here, we employ a selected reaction monitoring mass spectrometry assay for DNA crosslinks to assess the capacity of human cancer cells to bioactivate PR‐104A. We also test whether the more abundant DNA monoadducts could be used for the same purpose. DNA monoadducts and crosslinks from PR‐104A itself, and from its reduced metabolites, accumulated over 4 h in AKR1C3‐expressing TF1 erythroleukaemia cells under hypoxia, whereas intracellular concentrations of unstable PR‐104H and PR‐104M reached steady state within 1 h. We then varied rates of PR‐104A reduction by manipulating hypoxia or reductase expression in a panel of cell lines, in which AKR1C3 and POR were quantified by targeted proteomics. Hypoxia or reductase overexpression induced large increases in PR‐104A sensitivity (inhibition of proliferation), DNA damage response (&ggr;H2AX formation), steady‐state concentrations of PR‐104H/M and formation of reduced drug‐DNA adducts but not DNA adducts retaining the dinitro groups of PR‐104A. The fold‐change in the sum of PR‐104H and PR‐104M correlated with the fold‐change in reduced crosslinks or monoadducts (R2 = 0.87 for both), demonstrating their potential for assessing the capacity of cancer cells to bioactivate PR‐104A.