Insights into idarubicin antimicrobial activity against methicillin-resistant Staphylococcus aureus.

BACKGROUND: MRSA is a major concern in community settings and in health care. The emergence of biofilms and persister cells substantially increases its antimicrobial resistance. It is very urgent to develop new antimicrobials to solve this problem. OBJECTIVE: Idarubicin was profiled to assess its antimicrobial effects in vitro and in vivo, and the underlying mechanisms. METHODS: We investigated the antimicrobial effects of idarubicin against MRSA by time-kill analysis. The antibiofilm efficacy of idarubicin was assessed by crystal violet and XTT staining, followed by laser confocal microscopy observation. The mechanisms underlying the antimicrobial effects were studied by transmission electron microscopy, all-atom molecular dynamic simulations, SYTOX staining, surface plasma resonance, and DNA gyrase inhibition assay. Further, we addressed the antimicrobial efficacy in wound and subcutaneous abscess infection in vivo. RESULTS: Idarubicin kills MRSA cells by disrupting the lipid bilayers and interrupting the DNA topoisomerase IIA subunits, and idarubicin shows synergistic antimicrobial effects with fosfomycin. Through synergy with a single dose treatment fosfomycin and the addition of the cell protector amifostine, the cytotoxicity and cardiotoxicity of idarubicin were significantly reduced without affecting its antimicrobial effects. Idarubicin alone or in combination with fosfomycin exhibited considerable efficacy in a subcutaneous abscess mouse model of MRSA infection. In addition, idarubicin also showed a low probability of causing resistance and good postantibiotic effects. CONCLUSIONS: Idarubicin and its analogs have the potential to become a new class of antimicrobials for the treatment of MRSA-related infections.

Synthesis of (±)-Idarubicinone via Global Functionalization of Tetracene.

Anthracyclines are archetypal representatives of the tetracyclic type II polyketide natural products that are widely used in cancer chemotherapy. Although the synthesis of this class of compounds has been a subject of several investigations, all known approaches are based on annulations, relying on the union of properly prefunctionalized building blocks. Herein, we describe a conceptually different approach using a polynuclear arene as a starting template, ideally requiring only functional decorations to reach the desired target molecule. Specifically, tetracene was converted to (±)-idarubicinone, the aglycone of the FDA approved anthracycline idarubicin, through the judicious orchestration of Co- and Ru-catalyzed arene oxidation and arenophile-mediated dearomative hydroboration. Such a global functionalization strategy, the combination of site-selective arene and dearomative functionalization, provided the key anthracycline framework in five operations and enabled rapid and controlled access to (±)-idarubicinone.

Idarubicin Stimulates Cell Cycle- and TET2-Dependent Oxidation of DNA 5-Methylcytosine in Cancer Cells.

The topoisomerase II inhibitor idarubicin (Ida) is an effective anticancer anthracycline drug and has been used for clinical therapies of multiple cancers. It is well-known that Ida and its analogues can induce DNA double strand breakage (DSB) by inhibiting topoisomer II and kill tumor cells. To date, it remains unknown whether they alter DNA epigenomes. Here, we show that Ida significantly stimulates the oxidation of a key epigenetic mark DNA 5-methyl-2'-deoxycytidine (5mC), which results in elevation of 5-hydroxymethyl-2'-deoxycytidine (5hmC) in four tested cell lines. Similarly, Ida analogues also display elevated 5hmC. DSB-causing topoisomer II inhibitor etopside fails to induce 5hmC change even at very high dose, which suggests the independence of the DSB. Moreover, the structure comparison supports that the histone eviction-associated amino sugar moiety is a characteristic of the anthracyclines required to promote the 5hmC elevation. Noteworthy, we also found that the 5mC oxidation is also cell-cycle dependent and mainly occurs during the S and G2/M phases. TET2 depletion diminishes the observed 5hmC elevation, which suggests that the Ida stimulation of 5hmC formation is mainly TET2-dependent. Deep-sequencing shows that 5hmC increases in all regions of the tested genome of T47D cells. The observation of a novel effect of Ida as well as other anthracycline compounds on epigenetic DNA modifications may help to further elucidate their biological and clinical effects.

5'-OH-5-nitro-Indirubin oxime (AGM130), an Indirubin derivative, induces apoptosis of Imatinib-resistant chronic myeloid leukemia cells.

Imatinib is a highly effective drug for the treatment of chronic myeloid leukemia (CML) that targets the BCR-ABL kinase. However, a number of patients have CML that is resistant to Imatinib treatment. In this report, we developed AGM130 as a potential therapeutic drug for Imatinib-resistant CML treatment. The AGM130 compound is derived from Indirubin, which is an ingredient of Danggui Longhui Wan and known as a cyclin-dependent kinase (CDK) inhibitor. The water solubility of AGM130 is more enhanced than that of the original form of Indirubin, which has very poor water solubility. Our data showed that the AGM130 compound efficiently decreased the viability of CML-derived K562 cells. Moreover, this compound also efficiently decreased the viability of Imatinib-resistant K562 cells in in vitro and in vivo systems. In addition, like Indirubin, AGM130 also inhibited phosphorylation of retinoblastoma protein (Rb), which is a major substrate of CDK. Conclusively, our data suggest that AGM130 is a strong candidate for treating Imatinib-resistant CML.

Anthracycline glycosides of 2,6-dideoxy-2-fluoro-alpha-L-talopyranose.

The methyl beta-glycoside of the title sugar, obtained from 2-deoxy-2-fluoro-beta-D-glucopyranose tetraacetate by a sequence with detailed characterization of all intermediates, was converted by acetolysis-bromination into 3,4-di-O-acetyl-2,6-dideoxy-2-fluoro-alpha-L-talopyranosyl bromide, coupling of which with (7S,9S)-4-demethoxydaunomycinone afforded the 3,4-diacetate of 4-demethoxy-9-O-(2,6-dideoxy-2-fluoro-alpha-L-talopyranosyl)daunomycinone (19). The antitumor-active 19 was converted by way of its 14-bromo derivative into the 14-hydroxy analogue, the antitumor-active 4-demethoxyadriamycinone glycoside 21.

Enantioselective synthesis of (+)-8-hydroxy-8-methylidarubicinone.

An asymmetric Diels-Alder reaction methodology was employed to construct the tetracyclic structure of the anthracyclinone. A five-step sequence was needed to furnish the target (+)-8-hydroxy-8-methylidarubicinone.

New chiral pool approach to anthracyclinones. The stereoselective synthesis of idarubicinone.

In the present work, a new chiral pool approach has been developed for the synthesis of anthracyclinones. Thus, enone 8, readily available from l-rhamnose, has been converted via addition of 2,5-dimethoxybenzyllithium to the carbonyl group and a series of six reactions into a suitably protected aldehyde 21. The SnCl(4)-promoted stereospecific cyclization of the latter afforded enantiopure key intermediate 22. Silylation of benzylic hydroxyl of 21 followed by anodic oxidation and selective hydrolysis gave ketoacetals 25 and 26 to which 3-cyano-1(3H)-isobenzofuranone 27 was annelated. Removal of the isopropylidene group in the resulting 28, subsequent oxidation of the C(13) hydroxyl and full deprotection led to idarubicinone (4).

Partial inhibition of the P-glycoprotein-mediated transport of anthracyclines in viable resistant K562 cells after irradiation in the presence of a verapamil analogue.

P-glycoprotein (P-gp) is a membranous ATPase responsible for the multidrug resistance phenotype. The effect on P-gp-mediated transport of anthracyclines of cell irradiation in the presence of 2,2-diphenyl-5-[N-1-(o-azidophenyl)ethylamino]valeronitrile (VP*), a photoactivable analogue of verapamil was studied in viable K562/ADR cells. The derivatives were daunorubicin (DNR), idarubicin (IDA), 8-(S)-fluoro-idarubicin (F-IDA), 2'-bromo-4'-epidaunorubicin (Br-DNR) and pirarubicin (PIRA). It was observed that the irradiation in the presence of the verapamil analogue was unable to completely inhibit the P-gp-mediated efflux of anthracyclines and we estimated that P-gp retained 10-20% of its ability to pump these toxins. The ability of verapamil, DNR, IDA, F-IDA, Br-DNR and PIRA to inhibit the effect of VP* was studied. For this purpose, cells were irradiated in the presence of VP* and various concentrations of either verapamil or of one of the anthracyclines and then the P-gp functionality was checked by its ability to pump pirarubicin. It was observed that (i) the effect observed, when cells were irradiated in the presence of VP*, was completely blocked by the presence of verapamil; (ii) that anthracyclines are able to partially inhibit the VP* effect. This inhibition occurs at low concentration of anthracycline and depends on the nature of the derivative used. With those used in that study, after the photoirradiation of K562 ADR cells in the presence of VP* and anthracycline, P-gp has retained 50 +/- 5% of its functionality. The anthracycline concentration required for this inhibition is rather low, the total drug concentration yielding 50% of the effect ranged from 0.5 (Br-DNR) to 4 microM (F-IDA). The corresponding cytosolic concentrations are highly correlated with the values of Km determined previously.

Topoisomerase poisoning activity of novel disaccharide anthracyclines.

Doxorubicin and idarubicin are very effective anticancer drugs in the treatment of human hematological malignancies and solid tumors. These agents are well known topoisomerase II poisons; however, some anthracycline analogs recently have been shown to poison topoisomerase I. In the present work, we assayed novel disaccharide analogs and the parent drug, idarubicin, for their poisoning effects of human topoisomerase I and topoisomerases IIalpha and IIbeta. Drugs were evaluated with a DNA cleavage assay in vitro and with a yeast system to test whether the agents were able to poison the enzymes in vivo. We have found that the test agents are potent poisons of both topoisomerases IIalpha and IIbeta. The axial orientation of the second sugar relative to the first one of the novel disaccharide analogs was shown to be required for poisoning activity and cytotoxicity. Interestingly, idarubicin and the new analogs stimulated topoisomerase I-mediated DNA cleavage at low levels in vitro. As expected, the cytotoxic level of the drug was highly affected by the content of topoisomerase II; nevertheless, the test agents had a yeast cell-killing activity that also was weakly dependent on cellular topoisomerase I content. The results are relevant for the full understanding of the molecular mechanism of topoisomerase poisoning by anticancer drugs, and they define structural determinants of anthracyclines that may help in the rational design of new compounds directed against topoisomerase I.

Relation among the resistance factor, kinetics of uptake, and kinetics of the P-glycoprotein-mediated efflux of doxorubicin, daunorubicin, 8-(S)-fluoroidarubicin, and idarubicin in multidrug-resistant K562 cells.

Multidrug resistance (MDR) is frequently associated with decreased cellular drug accumulation resulting from enhanced drug efflux. This is correlated with the presence of a membrane protein, the P-glycoprotein, which pumps a wide variety of drugs out of cells, reducing their intracellular concentration and thus their toxicity. The influx and efflux of drugs across the cell membrane are in large part responsible for their intracellular concentrations, and in the search for new compounds able to overcome MDR, it is of prime importance to determine the molecular parameters whose modification would lead to an increase in the kinetics of uptake and/or to a decrease in the P-glycoprotein-medicated efflux. Four anthracycline derivatives, doxorubicin, daunorubicin, 8-(S)-fluoroidarubicin, and idarubicin, which have the same amino sugar, were used to analyze the respective contribution of the kinetics of uptake and the P-glycoprotein-mediated efflux in their impaired accumulation in MDR cells. The kinetics of uptake of the four drugs vary over a very large range: the kinetics of uptake of daunorubicin, 8-(S)-fluoroidarubicin, and idarubicin are 16, 200, and 400 times higher than that of doxorubicin, respectively. However, the four drugs are extruded by P-glycoprotein at comparable rates. The apparent Km values for P-glycoprotein-mediated transport, the intracellular free cytosolic drug concentrations at half-maximal velocity for the cell lines used, were approximately 2.2 microM for daunorubicin and and approximately 1 microM for idarubicin and 8-(S)-fluoroidarubicin.

Comparison of DNA sequence selectivity of anthracycline antibiotics and their 3'-hydroxylated analogs.

The sequence selectivity of three anthracyclines and their 3' hydroxylated analogs (in which an OH replaces NH3+ in the daunosamine at neutral pH) was examined in DNase I footprinting experiments on a 158-bp DNA fragment. We found that chemical modification of the daunosamine at C3' has more drastic consequences for sequence selectivity than chemical modification at C4 and C14 of the aglycone moiety. All anthracyclines and hydroxylated derivatives selectively recognize the triplet PyAPy. The importance of NH3+ in stabilizing the interaction was evidenced. First of all, comparable protection patterns require 5 times more hydroxyanthracycline than regular anthracycline. Furthermore, it is only after the replacement of NH3+ by OH that an additional protection site - CGC--appears. GGC is the site of best selectivity of the hydroxyanthracyclines. Anthracyclines can be considered both intercalators (aglycone moiety) and minor groove binders (sugar moiety). Since intercalating drugs show a slight preference for GC base pairs, we suggest hydroxylated anthracyclines to have a sequence specificity closer that of pure intercalators. Chemical modifications at C4 and C14 only modify the hydrogen bonding stabilization of the DNA-aglycone moiety complex: the more the anthracycline or its analog is lipophilic, the less it will interact with the sugar-phosphate chain.

High-performance liquid chromatographic analysis of idarubicin and fluorescent metabolites in biological fluids.

A specific, sensitive, and reliable high-performance liquid chromatographic (HPLC) method for the determination of idarubicin (IDA) and its known fluorescent metabolites idarubicinol (IDAol) and 4-demethoxy-daunomycinone (AG1) in biological fluids (human plasma and urine) was developed and tested. Plasma samples were solid-phase-extracted (C18 bonded silica cartridges). Complete separation of unchanged drugs and metabolites was achieved on a Cyanopropyl chromatographic column (25 cm x 4.6 mm inside diameter; particle size, 5 microns) using fluorescence detection (excitation wavelength, 470 nm; emission wavelength, 580 nm). Sensitivity was better than 0.2 ng/ml for all analytes; rates of recovery of unchanged drug and metabolites were better than 84.5% (IDA), 80.3% (IDAol), and 83.9% (AG1). The interassay coefficient of variation was 6.5% for IDA, 5.8% for IDAol, and 9.8% for AG1. Mean intra-assay precision was 4.6% for IDA, 5.9% for IDAol, and 5.0% for AG1 at sample concentrations of above 1 ng/ml and 12.1% for IDA, 10.8% for IDAol, and 14.1% for AG1 at sample concentrations of below 1 ng/ml.

DNA damage and cytotoxicity induced by metabolites of anthracycline antibiotics, doxorubicin and idarubicin.

This study assessed the ability of major metabolites of two types of anthracycline antibiotics, doxorubicin and idarubicin (4-demethoxydaunorubicin) to damage DNA in mouse fibrosarcoma 935.1 cells. Since DNA lesions by anthracyclines may be mediated by topoisomerase II, we also characterized the ability of the drugs to inhibit this enzyme. The C-13 alcohol and aglycone metabolites of doxorubicin and idarubicin were compared to the parent drugs in terms of induction of DNA single strand breaks measured by filter elution. In whole cells, the maximal DNA strand breakage induced by the C-13 alcohol metabolites was similar to that of their respective parent drugs. In isolated nuclei, however, the alcohol metabolites were two times more potent than the parent drugs. The aglycone metabolites produced very little damage in either whole cells or nuclei. The doxorubicin compounds differed markedly from idarubicin drugs in the way their ability to induce DNA breaks was related to cytotoxic activity. Doxorubicin and doxorubicinol cytotoxic effects (50% cell growth inhibition at 0.2 and 4 microM, respectively) coincided (in terms of drug concentrations) with the induction of significant breakage of cellular DNA. In contrast, the concentrations of idarubicin and idarubicinol needed to produce 50% growth inhibition (0.005 and 0.006 microM, respectively) were about 20 times lower than drug levels that induced significant DNA damage. All six compounds inhibited the catalytic activity of isolated topoisomerase II. While the alcohol metabolites produced this inhibition at concentrations similar to those of their parent drugs (5-10 microM), the aglycones were again much less active.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis and antitumor activity of novel 4-demethoxyanthracyclines.

A versatile and efficient synthetic route to 4-demethoxyanthracyclinones has been utilized in the preparation of a number of aglycons having 9-alkyl, 9-(hydroxylalkyl), or 9-carbamoyl substituents. Silver trifluoromethanesulfonate catalyzed coupling of these aglycons with various daunosamine derivatives has yielded a series of novel anthracyclines which have been evaluated as antitumor agents. 9-Alkylanthracyclines 22, 23, 33, and 34 have higher efficacy vs L-1210 leukemia than the parent 4-demethoxydaunorubicin (21), or the natural anthracyclines daunorubicin (1) and doxorubicin (2). 9-(Hydroxyalkyl) derivatives have in most cases high efficacy but are slightly less potent than 21. 9-Methyl analogue 22 has higher efficacy vs P388 leukemia than other anthracyclines tested, while 9-(hydroxymethyl) derivative 37 retains similar efficacy to anthracyclines 1, 2, and 21 but is considerably more potent. The N-substituted 9-carbamoylanthracyclines are devoid of antitumor activity.

Total chemical synthesis and antitumor evaluation of 4-demethoxy-10,10-dimethyldaunomycin.

The novel anthracycline analogue 4-demethoxy-10,10-dimethyldaunomycin was prepared in nine chemical steps from 5,8-dimethoxy-2-tetralone. It proved to be inactive as an antitumor agent in the mouse P388 lymphocytic leukemia model.

Toxicological studies with a novel synthetic anthracycline, the bis-hydrazone bridged analog of 4-demethoxydaunorubicin (SC-33428).

The toxicological effects of SC-33428, a bis-hydrazone bridged analog of 4-demethoxydaunorubicin were evaluated in rats. The bone marrow depressant effects were more pronounced than those caused by equal doses of doxorubicin, but the cardiotoxic and the nephrotoxic effects were clearly less marked. The concentrations of SC-33428 in serum, heart and kidneys after single and repeated i.p. injections were considerably lower than those reached with doxorubicin. Since the drug is at least as active as doxorubicin in various tumor models, it is likely that it will distinguish itself in the clinic by a broader margin of safety.