Selected redox chemotherapeutics in clinical development for oncological indications. The therapeutic performance of redox chemotherapeutics in advanced phases of clinical testing suggests that they represent novel pharmacological agents that target cancer depicted here as an invasive tumor in the process of metastatic cell dissemination (center insert). Selected investigational redox chemotherapeutics are depicted (clockwise from upper right): the orally active prooxidants elesclomol and artemisinin, the infusional redox drug ascorbate, the infusional SOD mimetic mangafodipir, the infusional redox cycler motexafin gadolinium, the orally active SOD1 inhibitor ATN-224, the infusional thioredoxin inhibitor PX-12, and the infusional arsenical darinaparsin. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at www.liebertonline.com/ars).

Targeting cancer cell redox homeostasis by prooxidant therapeutic intervention. Constitutive upregulation of oxidative stress contributes to genotypic and phenotypic changes characteristic of cancer cells and represents a redox vulnerabilty that can be targeted by prooxidant therapeutic intervention (POX). Differential redox set points in normal and malignant cells (as represented in a virtual ‘redox tachometer’) suggest that prooxidant-induced upregulation of cellular ROS specifically targets cancer cells. This process can be referred to as ‘redlining’ in analogy to the red bar (redline) displayed on car tachometers denoting the maximum speed at which an internal combustion engine is designed to operate without causing damage. In malignant cells already at a high setpot of constitutive oxidative stress, prooxidant deviation induces a redox shift that ‘redlines’ the cancer cell proliferative engine, leading to functional impairment, cell cycle arrest, and cell death. In contrast, the same prooxidant deviation from redox homeostasis is tolerated by nonmalignant cells. The width of the therapeutic window will be determined by the redox differential between normal and tumor cells and may limit efficacy of redox intervention. Exemplary redox chemotherapeutics for POX are depicted in the lower box including ( from left to right) the semisynthetic endoperoxide artemether, the metabolic modulator 2-deoxy-D-glucose, and the synthetic glutathione depleting agent imexon. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at www.liebertonline.com/ars).

Artemisinins: A novel class of endoperoxide-based anticancer redox chemotherapeutics. (A) The sesquiterpene endoperoxide artemisinin (1) and 1,2,4-trioxane-based, semisynthetic artemisinin-derivatives including dihydroartemisinin (3), artemether (4), arteether (5), artesunate (6), and artemisone (9) are established prooxidant antimalarial drugs and investigational chemotherapeutics for oncological indications. Deoxyartemisinin (2) is a redox-inactive artemisinin derivative. OZ277 (7) and the steroidal tetraoxane 5β-cholan-7α,12α,24-triol-3-spiro-6′-(1′,2′,4′,5′-tetraoxacyclohexane)-3′-spiro-cyclo-hexane (8), are fully synthetic drug-like 1,2,4-trioxolane-derivatives. (B) Artemisinin–glycoprotein bioconjugates for targeted prooxidant intervention. Artemisinin bioconjugation of transferrin and other glycoproteins (curved line: protein; squares and circle: oligosaccharide chain) occurs via hydrazone formation using artelinic acid hydrazide (10) after periodate-induced oxidation of vicinal sugar diol-groups. (C) The free radical mechanism of artemisinin drug action. The pharmacophoric moiety of artemisinins is an endoperoxide bridge that can be fragmented by intracellular iron [Fe(II)] species, leading to the formation of carbon-centered electrophilic radical species including a sterically unhindered primary radical (middle panel, upper left) and a sterically more congested secondary radical (middle panel, upper right). These organic free racicals together with ROS formed through Fenton chemistry are the suggested key mediators of artemisinin-induced cancer cell inactivation.

A selection of experimental and developmental redox chemotherapeutics and related derivatives. Agents discussed throughout the text and not shown in other specialized figures: Darinaparsin (11), motexafin gadolinium (12), menadione (13), toluidine blue O (14), geldanamycin (15), 17-allylamino-17-demethoxygeldanamycin (16), radicicol (17), 2-(phenyltelluryl)-3-methyl-[1,4]naphthoquinone (18), ebselen (19), 4,4′-dihydroxydiphenyl telluride (20), disulfiram (21), Dp44mT (22), triapine (23), lissoclinotoxin A (24), leinamycin (25), L-ascorbic acid (26), ATN-224 (27), 2-methoxyestradiol (28), M40403 (29), mangafodipir (30), TEMPO (31), imexon (32), PX-916 (33), auranofin (34), chaetocin (35), ES936 (36), DIBA (37), and elesclomol (38).

N-Acetyl-para-aminophenol and O-acetylsalicylic acid as tyrosinase-activated prooxidants. In melanoma cells, phenolic metabolites of the nonsteroidal anti-inflammatory agents N-acetyl-para-aminophenol (39) and O-acetylsalicylic acid (40) form hydroquinone (HQ), semiquinone free radical (SQ), and quinone (Q) metabolites through tyrosinase-dependent hydroxylation. These prooxidant metabolites are thought to induce therapeutically relevant glutathione depletion and other cytotoxic effects directed against tyrosinase expressing cells. Acetaminophen is now an investigational drug in clinical trials targeting metastatic melanoma.

Polysulfide drugs as prooxidant redox chemotherapeutics. Polysulfide-based redox reactivity is thought to underly the prooxidant cytotoxic activity of experimental anticancer agents including varacin (41) and diallyltrisulfide (DATS) (42). Bioreductive activation of polysulfide pharmacophores, through reaction with cellular thiols, leads to formation of strongly reducing polysulfide anion species upstream of ROS and reactive sulfur species (RSS) formation. RSS comprise a wide range of redox reactive molecules that also include neutral sulfur species such as S₃. Polysulfide-dependent formation of ROS and RSS occurs in a quasi-catalytic redox cycle driven by cellular reducing thiols.

Free radical mechanism of DNA cleavage by the polysulfide derivative calicheamicin γ₁^I. (A) Calicheamicin γ₁^I (43) with enediyne warhead and trisulfide bioreductive trigger. (B) After intracellular bioreductive activation of the trisulfide trigger by formation of a thiolate intermediate (a), an intramolecular Michael adduction enables subsequent Bergman cycloaromatization of the enediyne warhead (b), leading to the generation of a highly reactive diradical intermediate (c) in close proximity to target DNA. Oxygen-dependent DNA cleavage is initiated by hydrogen abstraction, leading to the formation of an unreactive reaction product (d).

Michael acceptors as experimental redox chemotherapeutics. Selective prooxidant redox intervention through ROS upregulation and covalent modification of redox sensitive target proteins can be achieved using Michael acceptor pharmacophores contained in small molecule natural products and synthetic derivatives. (A) The sesquiterpene lactone parthenolide (45) represents a lead compound containing a Michael acceptor pharmacophore that imparts anticancer activity through covalent modulation of redox targets. Helenalin (44) represents a structural analogue with unfavorable toxicity profile associated with its dual Michael acceptor pharmacophore. The Michael-inactive prodrug dimethylamino-parthenolide (46) represents a clinical candidate with improved solubility, oral bioavailability, and pharmacokinetic profile. (B) The pleiotropic anticancer Michael acceptor curcumin (47) has served as a lead compound for the design of improved drug-like derivatives, including the bisbenzylidenecyclopentanone-derivative (48) and the fluorinated drug-like analogue EF24 (49). (C) Michael acceptor-induced upregulation of oxidative stress response gene expression in A375 melanoma cells. The dietary factor trans-cinnamic aldehyde (CA, 50) contains a Michael pharmacophore. CA, but not closely related derivatives devoid of Michael acceptor activity including DHCA (51), COH (52), and CAC (53), displays antimelanoma activity in vivo accompanied by induction of a complex oxidative stress response, as revealed by gene array analysis performed on cultured A375 melanoma cells (25 μM CA, 24 h; RT² Human Oxidative Stress Profiler^TM PCR Expression Array technology) with upregulation of heme oxygenase-1 (HMOX1; 129-fold), glutathione peroxidase 2 (GPX2; sevenfold), sulfiredoxin 1 homolog (SRXN1; sevenfold), thioredoxin reductase 1 (TXNRD1; fourfold); early growth response gene 1 (EGR1; fivefold), DNA-damage-inducible transcript 3 (DDIT3; 13-fold), and cyclin-dependent kinase inhibitor 1A (CDKN1A; 11-fold) (modified with permission from (47)).

Neratinib and peltinib: ligand-guided Michael acceptors targeting a specific cysteine residue in oncogenic tyrosine receptor kinases. (A) ATP-competitive inhibitors of EGFR kinases such as the established anilinoquinazoline drugs gefitinib (54) and erlotinib (55) are ligands that inactivate receptor tyrosine kinases through reversible binding. (B) The 4-anilino-3-cyano quinoline derivatives neratinib (56) and peltinib (57) are investigational receptor tyrosine kinase inhibitors that act as irreversible suicide ligands of ErbB-1 and ErbB-2, combining ligand-based selectivity (ATP-competitive ligand moiety) with irreversible target inactivation through covalent adduction by an electrophilic pharmacophore (Michael acceptor moiety). (C) A conserved cysteine residue (Cys-805 in HER-2) contained in the ATP active site attacks the 4-(dimethylamino)crotonamide Michael acceptor pharmacophore (blue) of the suicide ligand neratinib facilitated by intramolecular amine catalysis. (D) Model of neratinib (Michael acceptor moiety in blue, covalently bound to Cys-805 (thiol in yellow) in the ATP active site of the HER-2 kinase catalytic domain (ATP-competitive ligand moiety in red). (Panel D adapted from (376) with permission from WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at www.liebertonline.com/ars).

Thioredoxin: A redox target for anticancer intervention. The small redox protein thioredoxin (Trx-1) is an important cancer drug target for redox chemotherapeutic intervention. Akyl-2-imidazolyl disulfides including the investigational drug PX-12 (58) inactivate thioredoxin by disulfide exchange leading to thioalkylation of a critical cysteine residue (Cys73) or oxidative formation of a disulfide bridge between Cys32 and Cys35. PMX464 (59) is an electrophilic double-Michael pharmacophore containing 4-hydroxycyclohexa-2,5-dien-1-one that targets cysteine residues 32 and 35 of the thioredoxin active site, potentially through dual Michael adduction.

Gliotoxin: A prooxidant peroxiredoxin mimetic and experimental redox chemotherapeutic. The disulfide-based mycotoxin gliotoxin (60) causes catalytic oxidation of thioredoxin [reaction cycle (1)] and glutathione depletion with ROS formation [redox cycle (2)]. In addition, gliotoxin inactivates target proteins by either covalent modification or free radical damage mediated by redox cycling.

Apurinic/apyrimidinic endonuclease/redox effector factor-1 (APE/Ref-1): A redox target for anticancer intervention. Reductive activation of DNA binding of redox-sensitive transcription factors that are involved in tumor initiation and progression depends on APE/Ref-1 enzymatic activity. (A) DNA repair activity of APE/Ref-1 is specifically inhibited by lucanthone (61) and CRT0044876 (62), investigational drugs that may confer tumor chemo- and radiosensensitization. E3330 (63) and resveratrol (64) are ligands that suppress redox-dependent transcription factor activation by APE/Ref-1, but do not interfer with DNA repair function of the enzyme. (B) PNRI-299 (65) is a small molecule inhibitor of APE/Ref-1 redox function that contains a redox active enedione pharmacophore that covalently traps (step a) and potentially oxidizes (step b) cysteine nucleophiles contained in the target protein APE/Ref-1, leading to functional inhibition of inflammatory signaling through the APE/Ref-1 client protein AP-1.

Cdc25 phosphatases: Redox targets for anticancer intervention. Hyperproliferative dysregulation and cell cycle progression in cancer cells can be controlled by oncogenic cell division cycle 25 (Cdc25) phosphatases that are redox-sensitive activators of cyclin-dependent kinases. The active site cysteine thiolate renders Cdc25 phosphatases susceptible to redox intervention by (a) ROS from redox cycler drugs including 2-(2-mercaptoethanol)-3-methyl-1,4-napthoquinone (NSC 67121, 66) and (b) organic electrophiles including F-NSC 67121 (67), leading to target modulation by covalent adduction. (c) Target inactivation may be achieved by inhibitory ligands including 7-[(2-methylbenzyl)-1H-indol-3-yl]-2,5-dihydroxy-1,4-benzoquinone (68).

Prooxidant anticancer intervention targeting glucose metabolism. Metabolic targeting can occur through administration of alternative energy substrates or small molecule modulators. These anatagonize or redirect glucose flux through the glycolytic and pentose phosphate pathways with induction of energy crisis and oxidative stress in cancer cells. Redox chemotherapeutics for metabolic targeting include 2-deoxy-D-glucose (69) and its improved derivative 2-fluoro-2-deoxy-D-glucose (70), 3-bromopyruvate (71), dichloroacetic acid (72), and oxythiamine (73).

Prooxidant anticancer intervention targeting mitochondria. Drugs that induce oxidative stress by modulation of mitochondrial targets include redox silent α-tocopherol derivatives, including α-tocopherylsuccinate ester (74) and its orally available esterase-resistant ether-derivative (75), 3,3′-diindolylmethane (76) and its potentiated derivative 5,5′-dibromo-3,3′-diindolylmethane (77), the 1,4-benzodiazepine-derivative Bz-423 (78), the quinazoline-derivative erastin (79), and RSL5 (80).

Redox chemotherapeutics targeting tumor hypoxia. Tumor tissue hypoxia (illustrated by the shaded triangular pO₂ gradient) can be targeted by (I) hypoxia-selective redox drugs, an important class of developmental redox chemotherapeutics, including the bioreductively activated hypoxia-selective prodrug tirapazamine (81) and its improved derivative 6-morpholinopropyloxy-1,2,4-benzotriazine 1,4-dioxide (82), the hypoxia-activated topoisomerase II-inhibitor prodrug AQ4N (83), the nitrogen mustard prodrug PR-104 (84), and (II) the HIF antagonist PX-478 (85). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at www.liebertonline.com/ars).