Synthesis and anti-Trypanosoma cruzi activity of naphthoquinone-containing triazoles: Electrochemical studies on the effects of the quinoidal moiety

In our continued search for novel trypanocidal compounds, twenty-six derivatives of para- and ortho-naphthoquinones coupled to 1,2,3-triazoles were synthesized. These compounds were evaluated against the infective bloodstream form of Trypanosoma cruzi, the etiological agent of Chagas disease. Compounds 17–24, 28–30 and 36–38 are described herein for the first time. Three of these novel compounds (28–30) were found to be more potent than the standard drug benznidazole, with IC₅₀/24 h values between 6.8 and 80.8 μM. Analysis of the toxicity to heart muscle cells led to LC₅₀/24 h of <125, 63.1 and 281.6 μM for 28, 29 and 30, respectively. Displaying a selectivity index of 34.3, compound 30 will be further evaluated in vivo. The electrochemical properties of selected compounds were evaluated in an attempt to find correlations with trypanocidal activity, and it was observed that more electrophilic quinones were generally more potent.     

Trypanosoma cruzi: Insights into naphthoquinone effects on growth and proteinase activity

In this study we compared the effects of naphthoquinones (α-lapachone, β-lapachone, nor-β-lapachone and Epoxy-α-lap) on growth of Trypanosoma cruzi epimastigotes forms, and on viability of VERO cells. In addition we also experimentally analyzed the most active compounds inhibitory profile against T. cruzi serine- and cysteine-proteinases activity and theoretically evaluated them against cruzain, the major T. cruzi cysteine proteinase by using a molecular docking approach. Our results confirmed β-lapachone and Epoxy-α-lap with a high trypanocidal activity in contrast to α-lapachone and nor-β-lapachone whereas Epoxy-α-lap presented the safest toxicity profile against VERO cells. Interestingly the evaluation of the active compounds effects against T. cruzi cysteine- and serine-proteinases activities revealed different targets for these molecules. β-Lapachone is able to inhibit the cysteine-proteinase activity of T. cruzi proteic whole extract and of cruzain, similar to E-64, a classical cysteine-proteinase inhibitor. Differently, Epoxy-α-lap inhibited the T. cruzi serine-proteinase activity, similar to PMSF, a classical serine-proteinase inhibitor. In agreement to these biological profiles in the enzymatic assays, our theoretical analysis showed that E-64 and β-lapachone interact with the cruzain specific S2 pocket and active site whereas Epoxy-α-lap showed no important interactions. Overall, our results infer that β-lapachone and Epoxy-α-lap compounds may inhibit T. cruzi epimastigotes growth by affecting T. cruzi different proteinases. Thus the present data shows the potential of these compounds as prototype of protease inhibitors on drug design studies for developing new antichagasic compounds.     

Protection of NAD(P)H:quinone oxidoreductase 1 against renal ischemia/reperfusion injury in mice

Ischemia/reperfusion (I/R) is the most common cause of acute renal injury. I/R-induced reactive oxygen species (ROS) are thought to be a major factor in the development of acute renal injury by promoting the initial tubular damage. NAD(P)H:quinone oxidoreductase 1 (NQO1) is a well-known antioxidant protein that regulates ROS generation. The purpose of this study was to investigate whether NQO1 modulates the renal I/R injury (IRI) associated with NADPH oxidase (NOX)-derived ROS production in an animal model. We analyzed renal function, oxidative stress, and tubular apoptosis after IRI. NQO1^−/− mice showed increased blood urea nitrogen and creatinine levels, tubular damage, oxidative stress, and apoptosis. In the kidneys of NQO1^−/− mice, the cellular NADPH/NADP⁺ ratio was significantly higher and NOX activity was markedly higher than in those of NQO1^+/+ mice. The activation of NQO1 by β-lapachone (βL) significantly improved renal dysfunction and reduced tubular cell damage, oxidative stress, and apoptosis by renal I/R. Moreover, the βL treatment significantly lowered the cellular NADPH/NADP⁺ ratio and dramatically reduced NOX activity in the kidneys after IRI. From these results, it was concluded that NQO1 has a protective role against renal injury induced by I/R and that this effect appears to be mediated by decreased NOX activity via cellular NADPH/NADP⁺ modulation. These results provide convincing evidence that NQO1 activation might be beneficial for ameliorating renal injury induced by I/R.     

β-Lapachone induces programmed necrosis through the RIP1-PARP-AIF-dependent pathway in human hepatocellular carcinoma SK-Hep1 cells

β-Lapachone activates multiple cell death mechanisms including apoptosis, autophagy and necrotic cell death in cancer cells. In this study, we investigated β-lapachone-induced cell death and the underlying mechanisms in human hepatocellular carcinoma SK-Hep1 cells. β-Lapachone markedly induced cell death without caspase activation. β-Lapachone increased PI uptake and HMGB-1 release to extracellular space, which are markers of necrotic cell death. Necrostatin-1 (a RIP1 kinase inhibitor) markedly inhibited β-lapachone-induced cell death and HMGB-1 release. In addition, β-lapachone activated poly (ADP-ribosyl) polymerase-1(PARP-1) and promoted AIF release, and DPQ (a PARP-1 specific inhibitor) or AIF siRNA blocked β-lapachone-induced cell death. Furthermore, necrostatin-1 blocked PARP-1 activation and cytosolic AIF translocation. We also found that β-lapachone-induced reactive oxygen species (ROS) production has an important role in the activation of the RIP1-PARP1-AIF pathway. Finally, β-lapachone-induced cell death was inhibited by dicoumarol (a NQO-1 inhibitor), and NQO1 expression was correlated with sensitivity to β-lapachone. Taken together, our results demonstrate that β-lapachone induces programmed necrosis through the NQO1-dependent ROS-mediated RIP1-PARP1-AIF pathway.     

Novel naphtho[2,1-d]oxazole-4,5-diones as NQO1 substrates with improved aqueous solubility: Design,synthesis, and in vivo antitumor evaluation

A new series of ortho-naphthoquinone analogs of β-lapachone were designed, synthesized and evaluated. The biological results indicated that most of our compounds were efficient substrates for NQO1. The new scaffold with water-soluble side chain resulted in greater solubility under acidic condition compared to β-lapachone. Thus avoiding the use of hydroxylpropyl β-cyclodextrin which would finally cause the rapid drug clearance from the blood and dose-limiting toxicity in the form of hemolytic anemia. The most soluble and promising compound in this series was 2-((4-benzylpiperazin-1-yl)methyl)naphtho[2,1-d]oxazole-4,5-dione (3k), which inhibited cancer cell (NQO1-rich A549 cell line) growth at IC₅₀ values of 4.6 ± 1.0 μmol·L⁻¹. Furthermore, compound 3k had in vivo antitumor activity in an A549 tumor xenografts mouse model comparable to the activity obtained with β-lapachone. The results indicated that these ortho-naphthoquinones could serve as promising leads for further optimization as novel substrates for NQO1.     

β-Lapachone: A naphthoquinone with promising antischistosomal properties in mice

The activity of β-lapachone (3,4-dihydro-2,2-dimethyl-2H-naphthol[1,2-b]pyran-5,6-dione, β-lap) against different stages of Schistosoma mansoni was investigated in mice. Mice infected with 50 cercariae (BH strain) were intraperitoneally treated at a dose of 50 mg/kg for 5 consecutive days, starting on the 1st, 14th, 28th and 45th days after infection, to evaluate the effect of β-lap on skin schistosomula, lung schistosomula, young worms (before oviposition) and adult worms (after oviposition), respectively. All animals were euthanized 60 days after infection. β-Lap significantly reduced (p < 0.001) the number of worms in 29.78%, 37.2%, 24.2% and 40.22% when administered during the phases of skin schistosomula, lung schistosomula, young worms and adult worms, respectively. Significant reduction was also achieved in terms of female burden. In all groups, there was significant reduction in the number of eggs and granulomas in the hepatic tissue. When the intervention was performed during the phase of adult worms, β-lap reduced the size of hepatic granulomas and changed the oogram pattern, lowering the percentage of immature eggs and increasing the percentage of mature and dead eggs. Our data indicate that β-lap has moderate antischistosomal properties. Its molecule may also be used as a prototype for synthesis of new naphthoquinone derivatives with potential schistosomicidal properties. Further studies with different formulations containing β-lap are needed to clearly establish the best dose and route of administration and its mechanism of action against schistosomes.     

2-Phenyl-beta-lapachone can affect mitochondrial function by redox cycling mediated oxidation

2-Phenyl-beta-lapachone (3,4-dihydro-2-methyl-2-phenyl-2H-naphtho[1,2b]pyran-5,6-dione) (2PBL) is a o-naphthoquinone synthesized as a possible antitumoral agent. The addition of micromolar concentrations of 2PBL to rat liver mitochondria (in the presence of malate-glutamate or succinate, as respiratory substrates): (1) stimulated O(2) consumption in state 4 and inhibited O(2) consumption in state 3, thus decreasing respiratory control index (RCI); and (2) collapsed the mitochondrial membrane potential. The addition of 2PBL to rat liver submitochondrial particles: (1) stimulated NADH oxidation in the presence of rotenone, antimycin, myxothiazol or cyanide; (2) stimulated (.-)O(2)(-) production in the presence of NADH and antimycin; and (3) led to 2PBL semiquinone radical production. Control studies carried out with two p-naphthoquinones, menadione and atovaquone, did not produced equivalent effects. These findings support the hypothesis that 2PBL, undergoes redox cycling and affects mitochondrial function. The 2PBL effect is complex, involving inhibition of electron transfer, uncoupling of oxidative phosphorylation, collapse of mitochondrial membrane potential and (.-)O(2)(-) production by redox cycling. The mitochondrion could be a target organelle for 2PBL cytotoxicity.     

β-Lapachone activity in synergy with conventional antimicrobials against methicillin resistant Staphylococcus aureus strains

The aim of this study was to evaluate the antimicrobial activity of lapachol, α-lapachone, β-lapachone and six antimicrobials (ampicillin, amoxicillin/clavulanic acid, cefoxitin, gentamicin, ciprofloxacin and meropenem) against twelve strains of Staphylococcus aureus from which resistance phenotypes were previously determined by the disk diffusion method. Five S. aureus strains (LFBM 01, LFBM 26, LFBM 28, LFBM 31 and LFBM 33) showed resistance to all antimicrobial agents tested and were selected for the study of the interaction between β-lapachone and antimicrobial agents, busing checkerboard method. The criteria used to evaluate the synergistic activity were defined by the Fractional Inhibitory Concentration Index (FICI). Among the naphthoquinones, β-lapachone was the most effective against S. aureus strains. FICI values ranged from 0.07 to 0.5, suggesting a synergistic interaction against multidrug resistant S. aureus (MRSA) strains. An additive effect was observed with the combination β-lapachone/ciprofloxacin against the LFBM 33 strain. The combination of β-lapachone with cefoxitin showed no added benefit against LFBM 31 and LFBM 33 strains. This study demonstrated that, in general, β-lapachone combined with beta lactams antimicrobials, fluoroquinolones and carbapenems acts synergistically inhibiting MRSA strains.     

New tricks for old drugs: the anticarcinogenic potential of DNA repair inhibitors

Defective or abortive repair of DNA lesions has been associated with carcinogenesis. Therefore it is imperative for a cell to accurately repair its DNA after damage if it is to return to a normal cellular phenotype. In certain circumstances, if DNA damage cannot be repaired completely and with high fidelity, it is more advantageous for an organism to have some of its more severely damaged cells die rather than survive as neoplastic transformants. A number of DNA repair inhibitors have the potential to act as anticarcinogenic compounds. These drugs are capable of modulating DNA repair, thus promoting cell death rather than repair of potentially carcinogenic DNA damage mediated by error-prone DNA repair processes. In theory, exposure to a DNA repair inhibitor during, or immediately after, carcinogenic exposure should decrease or prevent tumorigenesis. However, the ability of DNA repair inhibitors to prevent cancer development is difficult to interpret depending upon the system used and the type of genotoxic stress. Inhibitors may act on multiple aspects of DNA repair as well as the cellular signaling pathways activated in response to the initial damage. In this review, we summarize basic DNA repair mechanisms and explore the effects of a number of DNA repair inhibitors that not only potentiate DNA-damaging agents but also decrease carcinogenicity. In particular, we focus on a novel anti-tumor agent, β-lapachone, and its potential to block transformation by modulating poly(ADP-ribose) polymerase-1.     

Microbial transformation of β-lapachone to its glycosides by Cunninghamella elegans ATCC 10028b

β-lapachone (1) has entered phases I and II clinical trials for the treatment of solid tumors and the therapeutic efficacy of β-lapachone is closely related to its metabolic process. In order to contribute to a better understanding of human metabolism of β-lapachone, Cunninghamella elegans ATCC 10028b was used as a microbial model of mammalian metabolism to biotransform β-lapachone and two new glycosylated derivatives were produced. The chemical structures were elucidated as 6-hydroxy-2,2-dimethyl-3,4-dihydro-2H-naphtho[1,2-b]pyran-5-O-β-d-glucopyranoside (2) and 5-hydroxy-2,2-dimethyl-3,4-dihydro-2H-naphtho[1,2-b]pyran-6-O-β-d-glucopyranoside (3) by ¹H NMR, ¹³C NMR, HMBC, HMQC, COSY and HRMS analyses. The major derivative (3) displayed a lower activity against breast cancer cell line SKBR-3 (IC₅₀ = 312.5 μM) than β-lapachone (IC₅₀ = 5.6 μM), but did not show cytotoxicity against normal fibroblasts cell line GM07492-A, whereas β-lapachone was highly toxic (IC₅₀ = 7.25 μM). These metabolites were reported here for the first time and are similar to those that occur in phase II of human metabolism