Free radicals in quinone containing antitumor agents: The nature of the diaziquone (3,6-diaziridinyl-2,5-bis(carboethoxyamino)-1,4-benzoquinone) free radical

The enzymatically generated free radical of the antitumor agent diaziquone is analyzed with the help of two analogs where either the aziridine rings (RQ14) or the carboethoxyamino groups (RQ2) were substituted by chlorine atoms. The hyperfine couplings observed in the diaziquone free radical are due to the nitrogens in the aziridine group. Unresolved coupling and hindered rotation contribute to line broadening. We find that diaziquone free radicals are more stable than RQ14 but less stable than RQ2 free radicals. The reason for this is that the carboethoxyamino groups make the aromatic ring unstable, while the aziridines contribute to its stability. The free radical observed in diaziquone is in all probability that of the parent compound and not that of an intermediate metabolite.     

Free radicals in quinone containing antitumor agents. The nature of the diaziquone (3,6,-diaziridinyl-2,5-bis(carboethoxyamino)-1,4-benzoquinone) free radical

The enzymatically generated free radical of the antitumor agent diaziquone is analyzed with the help of two analogs where either the aziridine rings (RQ14) or the carboethoxyamino groups (RQ2) were substituted by chlorine atoms. The hyperfine couplings observed in the diaziquone free radical are due to the nitrogens in the aziridine group. Unresolved coupling and hindered rotation contribute to line broadening. We find that diaziquone free radicals are more stable than RQ14 but less stable than RQ2 free radicals. The reason for this is that the carboethoxyamino groups make the aromatic ring unstable, while the aziridines contribute to its stability. The free radical observed in diaziquone is in all probability that of the parent compound and not that of an intermediate metabolite.     

Mechanism(s) of bioreductive activation. The example of diaziquone (AZQ)

Bioreduction in the activation of diaziquone (2,5-diaziridinyl-3,6-bis (carboethoxyamino)-1,4-benzoquinone) has been investigated by exploring its reduction by whole cells, rat liver microsomes and purified enzymes. The mechanism of bioreduction was further investigated by exploring the chemical and electrochemical reduction of diaziquone as well as its photochemistry. Reduced diaziquone (by several means) was then tested for activity against parent compound. It appears that reduced diaziquone in most cases is more active than the oxidized form. Diaziquone redox cycles, but it is easily reduced to the hydroquinone which oxidizes to the semiquinone yielding free radicals under aerobiosis. The most probable mechanism of action is that of bioreductive alkylation where the alkylating aziridines are protonated after reduction facilitating the opening of the aziridine rings and thus alkylation.     

The chemical reduction of diaziquone: products and free radical intermediates

Sodium borohydride reduced diaziquone (AZQ) can cause cross-links between DNA molecules, between DNA and proteins and cause single- and double-strand DNA breaks. In order to understand these effects better, we investigated the reduction of diaziquone by borohydride, and looked at reaction products. We found that a major product was formed during the oxidation of the colorless 2-electron reduced AZQ, and that this product was a monoaziridinyl quinone. We interpret this result to mean that both the leaving aziridine as well as the remaining one can alkylate. This mode of alkylation does not explain cross-links which may occur by a different mechanism requiring simultaneous opening of the aziridine rings. Most of the antitumor activity of borohydride reduced AZQ is probably exerted during the oxidation of the 2-electron reduced AZQ (AZQH2).     

The reduction of anti-tumour diaziridinyl benzoquinones

The properties of the semiquinone radicals produced for 2,5-bis(carboethoxyamino)-3,6-diaziridinyl-1,4-benzoquinone (AZQ) and 2,5-bis(2-hydroxyethylamino)-3,6-diaziridinyl-1,4-benzoquinone (BZQ), have been investigated. AZQ semiquinone radicals can be produced from the reduction of AZQ by superoxide radicals, whereas BZQ semiquinone radicals are unstable in the presence of oxygen. The one-electron reduction potentials of the couples Q/Q-. at pH 7.0 were determined as -70 +/- 10 mV for AZQ and -376 +/- 15 mV for BZQ. The difference in these potentials is explained. As a consequence of ESR studies on the enzymatically produced radicals, we have considered the factors which determine the detection of ESR signals for reduced quinones produced in a biological system.     

Activation And Deactivation Of Quinones Catalyzed By Dt-Diaphorase. Evidence For Bioreductive Activation Of Diaziquone (Azq) In Human Tumor Cells And Detoxification Of Benzene Metabolites In Bone Marrow Stroma

Bioactivation of diaziquone (AZQ) in HT-29 human colon carcinoma cells and detoxification of benzene metabolites in bone marrow stromal cells were used as examples of the potential role of DT-diaphorase in both activation and deactivation processes. HT-29 cell cytosol contained high levels of DT-diaphorase activity and removed AZQ in the presence of either NADH or NADPH. Prior boiling of cytosol. omission of NADH or NADPH or inclusion of dicoumarol. an inhibitor of DT-diaphorase. inhibited removal of AZQ. AZQ-inouced cytotoxicity in HT-29 cells was also inhibited by dicoumarol. Chemical reduction of AZQ in a cell free system enhanced formation of a GSH conjugate of AZQ. Two of the major cell types in bone marrow stroma are macrophages and fibroblastoid stromal cells. A fibroblastoid cell line derived from stromal cells contained approximately fourfold higher levels of DT-diaphorase than macrophages. Inclusion of dicournarol in incubations containing ¹⁴C-hydroquinone and the respective stromal cell type, significantly increased covalent binding of radiolabel to macromolecules in stromal fibroblasts but not in macrophages     

Diaziquone as a potential agent for photoirradiation therapy: formation of the semiquinone and hydroxyl radicals by visible light

When diaziquone was irradiated with 500 nm visible light, hydroxyl free radicals as well as the diaziquone semiquinone were produced. The diaziquone semiquinone is a stable free radical that exhibits a characteristic 5-line electron spin resonance (ESR) spectrum. Since hydroxyl free radicals are short lived, and not observable by conventional ESR, the nitrone spin trap 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) was used to convert hydroxyl radicals into longer lived ESR detectable spin adducts. The formation of hydroxyl radicals was further confirmed by investigating reactions in which hydroxyl radical scavangers, sodium formate and dimethylsulfoxide, compete with the spin traps DMPO or POBN (alpha-(4-Pyridyl-1-oxide)-N- tert-butylnitrone) for hydroxyl free radicals. The products of these scavenging reactions were also trapped with DMPO or POBN. If drug free radicals and hydroxyl free radicals are important in the activity of quinone-containing antitumor agents, AZQ may have a potential in photoirradiation therapy or photodynamic therapy.     

Quantitative evaluation of the effects of human carcinogens and related chemicals on human foreskin fibroblasts

Ten compounds representative of diverse classes of chemicals were evaluated for their cytotoxicity and transforming ability to human skin fibroblasts in vitro. Only five of the ten compounds were highly cytotoxic in the 0-100 µg/ml range and their order of cytotoxicity was: 2,5-bis(1-aziridinyl)-3,6-bis(carboethoxyamino)-1,4-benzoquinone (AZQ) > cis platin > bis(chloromethyl)ether (BCME) > acrylonitrile > afatoxin BI (AFBI). The other five compounds, afatoxin B2 (AFB2), methylmethacrylate, 1-naphthylamine (1-NA), 2-naphthylamine (2-NA), and cyclophosphamide, exhibited less than 40% inhibition of colony formation even at 100 µg/ml of the compound (the maximum concentration of AFB2 used was 50 µg/ml due to its low solubility). Anchorage-independent growth of exposed cells in soft agar was used as a biological endpoint for the expression of chemical transformation. AFB₁ had strong transforming ability, whereas AFB₂ was a weak transforming agent. The transforming abilities of acrylonitrile, AZQ, BCME, cis-platin, methylmethacrylate and 2-NA ranged between those of AFBI and AFB2. 1-NA also induced the soft agar growth property in the treated cells even though this compound has not been shown to be carcinogenic. AFB₁, AZQ, cisplatin, cyclophosphamide and 1-NA exhibited a dose dependent increase in soft agar growth frequency for at least three consecutive concentrations. The data suggest that anchorage-independent colony forming ability of exposed cells is a reliable marker to measure the carcinogenic potential of various hazardous chemicals.Abbreviations AZQ 2,5-bis(1-aziridinyl)-3,6-bis(carboethoxyamino)-1,4-benzoquinone - AFB aflatoxin B₁ - AFB2 aflatoxin 132 - AI anchorage independent - B[a]P benzo[a]pyrene - BCME bis(chloromethyl)ether; cis-platin, cis-diammine-dichloroplatinum - CM complete medium - E.D.50 effective dose which produced 50% cytotoxicity - CP cyclophosphamide - HNF human neonatal foreskin - HPLC high pressure liquid chromatography - 1-NA 1-naphthylamine - 2-NA 2-naphthylamine - PDL population doubling     

Trace detection of hydroxyl radicals during the redox cycling of low concentrations of diaziquone: a new approach

Quantifying oxygen radicals that arise during the redox cycling of quinone-containing anticancer agents such as diaziquone (AZQ) has been difficult, as has been their detection at low drug concentrations. This is due to the fact that EPR spin trapping, the method most often used for *OH detection, requires the use of high drug concentrations. Using a new highly sensitive technique that employs a fluorescamine-derivatized nitroxide, we show that low levels of NADPH-cytochrome P450 reductase (4.25 microg/ml) catalyze the production of hydroxyl radicals at very low, clinically relevant AZQ concentrations. Thus, at this enzyme concentration, we were able to detect a rate of 0.10 nM s(-1) hydroxyl radical production by 5 microM AZQ, a clinically relevant concentration. The Michaelis-Menten constants for AZQ-mediated hydroxyl radical production are: K(M) = 10.7 +/- 1.4 microM, and V(max) = 5.2 +/- 0.9 x 10(-8) M s(-1) (mg protein)(-1). Experiments employing catalase, superoxide dismutase, and NADPH-cytochrome P450 reductase, confirm the previously deduced conclusions from high drug concentrations, that is, that at low concentrations, AZQ acts to shuttle reducing equivalents from the enzyme to oxygen, thus generating the redox cycle. The data presented here suggest that the levels and locations of redox active metal ions may be the principal controlling factor in the pathway of AZQ activity that involves oxidative stress.     

The lethal effect of bis-type azridinylnaphthoquinone derivative on oral cancer cells (OEC-M1) associated with anti-apoptotic protein bcl-2

Several drugs of aziridinylbenzoquinone analogs have undergone clinical trials as potential antitumor drugs. These bioreductive compounds are designed to kill tumor cells preferentially within the hypoxic microenvironment. From our previous reported data, it was found that the synthesized 2-aziridin-1-yl-3-[(2-[2-[(3-aziridin-1-yl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)thio]ethoxy]ethyl)thio]naphthoquinone (AZ-1) is a bioreductive compound with potent lethal effect on oral cancer cell, OEC-M1. It was found in this study that the lethal effect of the oral cancer cell lines OEC-M1 induced by AZ-1 was mediated through the cell cycle arrest and apoptosis pathway. The LC50 values of OEC-M1 and KB cells induced by AZ-1 compound were 0.72 and 1.02 microM, respectively, which were much lower than that of normal fibroblast cells (SF with LC50 = 5.6 microM) with more than 90% of normal fibroblasts surviving as compared to control at a concentration of AZ-1 as high as 2 microM. It was interesting to note that the LC50 of monotype diaziridinylbenzoquinone compound, diaziquone (AZQ), was 50 microM on OEC-M1 cells. Comparing the cytotoxicity of AZ-1 and AZQ on OEC-M1 cells, AZ-1 is approximately 70 times more potent than AZQ. By using Western blot, both G2/M phase cell cycle arresting protein, cyclin B, and anti-apoptotic protein, bcl-2, were expressed in OEC-M1 cell when the concentrations of AZ-1 were increased from 0.125 to 0.5 microM and then decreased from 1 to 2 microM of AZ-1 treatment as compared with control for 24 h. Both proteins were expressed most abundantly at 0.5 microM AZ-1. However, the expression of bcl-2 protein in OEC-M1 was significantly decreasing in a dose-dependent manner and was only about 50% protein level at 2 microM AZ-1 for 48h as compared with control. The cell survival check protein p53 increased from 1.72- to 2.8-fold and 1.36- to 2.16-fold at concentrations of AZ-1 from 0.125 to 2.0 microM in a dose-dependently increasing manner on OEC-M1 as compared with control for 24 and48 h treatments, respectively. The apoptotic-related phenomena were observed, which included apoptotic body formation and the enzyme activity change of caspase-3. The apoptotic bodies and caspase-3 activity of OEC-M1 were induced only at 2 microM AZ-1 for a 24h treatment, yet apoptotic body formation was observed at as low as 0.5 microM AZ-1 and in a dose-dependently increasing manner for a 48 h treatment. The caspase-3 activity was increased 20.6%, 26.8%, and 84.2%, respectively, at 0.5, 1, and 2muM concentrations of AZ-1 for a 48 h treatment as compared with control. These results indicate that AZ-1 induced the cell death of OEC-M1 through the G2/M phase arrest of cell cycle and anti-apoptosis first and then apoptosis following a 48 h treatment. All of the pathway might be associated with bcl-2 and p53 protein expression. We propose that the AZ-1 could be used as anti-oral cancer drug for future studies with animal models.     

Free radical formation and DNA strand breakage during metabolism of diaziquone by NAD(P)H quinone-acceptor oxidoreductase (DT-diaphorase) and NADPH cytochrome c reductase.

One-electron reduction of diaziquone (AZQ) by purified rat liver NADPH cytochrome c reductase was associated with formation of AZQ semiquinone, superoxide anions, hydrogen peroxide, and hydroxyl radicals as indicated by ESR spin-trapping studies. Reactive oxygen formation correlated with AZQ-dependent production of single and double PM2 plasmid DNA strand breaks mediated by this system as detected by gel electrophoresis. Direct two-electron reduction of AZQ by purified rat liver NAD(P)H (quinone acceptor) oxidoreductase (QAO) was also associated with formation of AZQ semiquinone, superoxide anions, hydrogen peroxide, and hydroxyl radicals as detected by ESR spin trapping. Furthermore, PM2 plasmid DNA strand breaks were detected in the presence of this system. Plasmid DNA strand breakage was inhibited by dicumarol (49 +/- 5%), catalase (57 +/- 2.3%), SOD (42.2 +/- 3.6%) and ethanol (41.1 +/- 3.9%) showing QAO and reactive oxygen formation was involved in the PM2 plasmid DNA strand breaks observed. These results show that both one- and two-electron enzymatic reduction of AZQ give rise to formation of reactive oxygen species and DNA strand breaks. Autoxidation of the AZQ semiquinone and hydroquinone in the presence of molecular oxygen appears to be responsible for these processes. QAO appears to be involved in the metabolic activation of AZQ to free radical species. The cellular levels and distribution of this enzyme may play an important role in the response of tumor and normal cells to this antitumor agent.     

Decreased skp2 expression is necessary but not sufficient for therapy-induced senescence in prostate cancer

Therapy-induced senescence (TIS), a cytostatic stress response in cancer cells, is induced inefficiently by current anticancer agents and radiation. The mechanisms that mediate TIS in cancer cells are not well defined. Herein, we characterize a robust senescence response both in vitro and in vivo to the quinone diaziquone (AZQ), previously identified in a high-throughput senescence-induction small-molecule screen. Using AZQ and several other agents that induce senescence, we screened a series of cyclin-dependent kinase inhibitors and found that p27(Kip1) was induced in all investigated prostate cancer cell lines. The ubiquitin-ligase Skp2 negatively regulates p27(Kip1) and, during TIS, is translocated to the cytoplasm before its expression is decreased in senescent cells. Overexpression of Skp2 blocks the effects of AZQ on senescence and p27(Kip1) induction. We also find that stable long-term short hairpin RNA knockdown of Skp2 decreases proliferation but does not generate the complete senescence phenotype. We conclude that Skp2 participates in regulating TIS but, alone, is insufficient to induce senescence in cancer cells.     

Covalent binding studies on the 14C-labeled antitumor compound 2,5-bis(1-aziridinyl)-1,4-benzoquinone. Involvement of semiquinone radical in binding to DNA, and binding to proteins and bacterial macromolecules in situ.

2,5-Bis(1-aziridinyl)-1,4-benzoquinone (BABQ) is a compound from which several antitumour drugs are derived, such as Trenimone, Carboquone and Diaziquone (AZQ). The mechanism of DNA binding of BABQ was studied using 14C-labeled BABQ and is in agreement with reduction of the quinone moiety and protonation of the aziridine ring, followed by ring opening and alkylation. The one-electron reduced (semiquinone) form of BABQ alkylates DNA more efficiently than two-electron reduced or non reduced BABQ. Covalent binding to polynucleotides did not unambiguously reveal preference for binding to specific DNA bases. Attempts to elucidate further the molecular structure of DNA adducts by isolation of modified nucleosides from enzymatic digests of reacted DNA failed because of instability of the DNA adducts. The mechanism of covalent binding to protein (bovine serum albumin, BSA) appeared to be completely different from that of covalent binding to DNA. Binding of BABQ to BSA was not enhanced by reduction of the compound and was pH dependent in a way that is opposite to that of DNA alkylation. Glutathione inhibits binding of BABQ to BSA and forms adducts with BABQ in a similar pH dependence as the protein binding. The aziridine group therefore does not seem to be involved in the alkylation of BSA. Incubation of intact E. coli cells, which endogenously reduce BABQ, resulted in binding to both DNA and RNA, but also appreciable protein binding was observed.     

Diaziquone-induced micronuclei in cytochalasin B-blocked mouse peripheral blood lymphocytes

A mouse peripheral blood lymphocyte (PBL) micronucleus (MN) test was developed using a modification of the technique for assessing MN in human PBLs described by Fenech and Morley (1985). Male C57Bl/6 mice (5/dose) were injected i.p. with either 0, 2.5, 5.0, 7.5, or 10.0 mg diaziquone (AZQ)/kg. After 24 h the mice were bled by cardiac puncture, PBLs were isolated on a Ficoll-density gradient and then cultured in RPMI 1640 medium using 8 micrograms phytohemagglutinin/ml. In some cultures cytochalasin B (CYB) was added at 21 h during the medium change to block cytokinesis. In other cultures, CYB was omitted to compare the sensitivity of analyzing MN in binucleate versus unblocked mononucleate cells. All doses of AZQ yielded significant increases in MN-containing binucleated PBLs. The use of CYB in the mouse PBL MN test increased the sensitivity approximately 3-fold. The MN test in mouse PBLs should be useful in comparative cytogenetic studies of mice and humans.     

Total synthesis and biological evaluation of ustiloxin natural products and two analogs

Synthetic investigations of ustiloxin natural products are described. The first total synthesis of ustiloxin F was completed in 15 steps via ethynyl aziridine ring-opening by a phenol derivative. The results of biological tests of synthetic ustiloxins D and F, and two analogs, O-Me-ustiloxin D and 6-Ile-ustiloxin, demonstrated that the free hydroxyl group ortho to the ether linkage is critical for activity and variations at the Val/Ala site produce changes in the biological activity suggesting the need for further perturbations at this site to more extensively study the tubulin binding.     

The reductive metabolism of diaziquone (AZQ) in the S9 fraction of MCF-7 cells: Free radical formation and NAD(P)H: Quinone-acceptor oxidoreductase (DT-diaphorase) activity

The S9 fraction of MCF-7 human breast carcinoma cells has NAD(P)H (quinone-acceptor) oxidoreductase activity as measured by the reduction of dichlorophenol-indophenol (DCPIP). This reduction is dependent on the activators Tween-20 and bovine serum albumin and it is inhibitable by dicumarol. The S9 fraction also has cytochrome c reductase activity which is approximately 29 times less than the two-electron reduction activity of NAD(P)H (quinone-acceptor) oxidoreductase. Diaziquone (AZQ) is a substrate for this NAD(P)H oxidoreductase active S9 fraction as judged by its enzymatic reduction detected spectrophotometrically and by electron spin resonance spectroscopy. Two-electron mediated enzymatic reduction of AZQ was evidenced by the formation of the colorless dihydroquinone (AZQH2) which could be followed at 340 nm. The production of the dihydroquinone was inhibitable by dicumarol implicating NAD(P)H oxidoreductase in its formation. Under aerobic conditions, electron spin resonance spectroscopy showed evidence for the production of AZQ semiquinone (AZQH) and oxygen radicals. Under anaerobic conditions no oxygen radicals were observed, but the semiquinone was stable for hours. These results are also inhibitable by dicumarol and suggest a two-step one-electron oxidation process of the dihydroquinone. The production of semiquinone and oxygen radicals as detected by electron spin resonance spectroscopy was more sensitive to dicumarol when NADPH was used as cofactor (68% inhibition of OH and 65% inhibition of AZQH) than when NADH was used (28% inhibition of OH and 5% inhibition of AZQH). This suggests that NADH flavin reductases play a more important role in the one-electron reduction pathway of AZQ in MCF-7 S9 fraction than NADPH reductases. The reduction of AZQ by NAD(P)H (quinone-acceptor) oxidoreductase may play an important role in the bioreductive alkylating properties of AZQ.     

Finding more active antioxidants and cancer chemoprevention agents by elongating the conjugated links of resveratrol

Resveratrol is the subject of intense research as a natural antioxidant and cancer chemopreventive agent. There has been a great deal of interest and excitement in understanding its action mechanism and developing analogs with antioxidant and cancer chemoprevention activities superior to that of the parent compound in the past decade. This work delineates that elongation of the conjugated links is an important strategy to improve the antioxidant activity of resveratrol analogs, including hydrogen atom- or electron-donating ability in homogeneous solutions and antihemolysis activity in heterogeneous media. More importantly, C3, a triene bearing 4,4'-dihydroxy groups, surfaced as an important lead compound displaying remarkably increased antioxidant, cytotoxic, and apoptosis-inducing activities compared with resveratrol.     

Structure-activity studies on a novel series of cholinergic channel activators based on a heteroaryl ether framework.

Analogs of compound 1 with a variety of azacycles and heteroaryl groups were synthesized. These analogs exhibited Ki values ranging from 0.15 to > 10,000 nM when tested in vitro for cholinergic channel receptor binding activity (displacement of [3H](-) cytisine from whole rat brain synaptic membranes).     

Discovery of benzo[d]imidazo[5,1-b]thiazole as a new class of phosphodiesterase 10A inhibitors

The design, synthesis and structure activity relationship studies of a series of compounds from benzo[d]imidazo[5,1-b]thiazole scaffold as phosphodiesterase 10A (PDE10A) inhibitors are discussed. Several potent analogs with heteroaromatic substitutions (9a–d) were identified. The anticipated binding mode of these analogs was confirmed by performing the in silico docking experiments. Later, the heteroaromatics were substituted with saturated heteroalkyl groups which provided a tool compound 9e with excellent PDE10A activity, PDE selectivity, CNS penetrability and with favorable pharmacokinetic profile in rats. Furthermore, the compound 9e was shown to be efficacious in the MK-801 induced psychosis model and in the CAR model of psychosis.     

Reduction of the toxicity and mutagenicity of aziridine in mammalian cells harboring the Escherichia coli fpg gene.

Aziridine (ethyleneimine) reacts with DNA in vitro, mainly at the N7 position of guanine and N3 of adenine, then imidazole ring opening of the modified guanine results in formation of formamidopyrimidine (FaPy) residues. The Escherichia coli fpg gene encodes a DNA glycosylase that removes FaPy residues from DNA. To determine whether aziridine produces FaPy lesions in mammalian cells we have expressed the E.coli fpg gene in CHO cells. The transfected cells, expressing high levels of the bacterial protein, are more resistant to the toxic and mutagenic effects of aziridine than the control population. Less DNA damage was measured by quantitative PCR analysis in transfected than in control cells treated with equimolar concentrations of aziridine. The results suggest that aziridine produces in vivo FaPy residues that could account for the deleterious effects of this compound.