In Vitro and In Vivo Trypanocidal Efficacy of Synthesized Nitrofurantoin Analogs.

African trypanosomes cause diseases in humans and livestock. Human African trypanosomiasis is caused by Trypanosoma brucei rhodesiense and T. b. gambiense. Animal trypanosomoses have major effects on livestock production and the economy in developing countries, with disease management depending mainly on chemotherapy. Moreover, only few drugs are available and these have adverse effects on patients, are costly, show poor accessibility, and parasites develop drug resistance to them. Therefore, novel trypanocidal drugs are urgently needed. Here, the effects of synthesized nitrofurantoin analogs were evaluated against six species/strains of animal and human trypanosomes, and the treatment efficacy of the selected compounds was assessed in vivo. Analogs 11 and 12, containing 11- and 12-carbon aliphatic chains, respectively, showed the highest trypanocidal activity (IC50 < 0.34 µM) and the lowest cytotoxicity (IC50 > 246.02 µM) in vitro. Structure-activity relationship analysis suggested that the trypanocidal activity and cytotoxicity were related to the number of carbons in the aliphatic chain and electronegativity. In vivo experiments, involving oral treatment with nitrofurantoin, showed partial efficacy, whereas the selected analogs showed no treatment efficacy. These results indicate that nitrofurantoin analogs with high hydrophilicity are required for in vivo assessment to determine if they are promising leads for developing trypanocidal drugs.

Synthesis and in vitro antileishmanial efficacy of benzyl analogues of nifuroxazide.

Leishmaniasis is a vector-borne parasitic disease that mostly affects populations in tropical and subtropical countries. There is currently no vaccine to protect against and only a handful of drugs are available to treat this disease. Leishmaniasis is curable, but its eradication and elimination are hindered by the emergence of multidrug resistant strains of the causative pathogens, accentuating the need for new and effective antileishmanial drugs. In search for such agents, nifuroxazide, a clinical antibiotic, was evaluated through investigation of its benzyl analogues for in vitro antileishmanial efficacy against promastigotes of various Leishmania (L.) strains. The monobenzylated analogues 1 and 2 were the most potent of all, possessing nanomolar activities up to 10-fold higher than the parent drug nifuroxazide against all three tested Leishmania strains. Both analogues stand as antipromastigote hits for further lead investigation into their potential to act as new antileishmanial agents.

Analogs of nitrofuran antibiotics are potent GroEL/ES inhibitor pro-drugs.

In two previous studies, we identified compound 1 as a moderate GroEL/ES inhibitor with weak to moderate antibacterial activity against Gram-positive and Gram-negative bacteria including Bacillus subtilis, methicillin-resistant Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, and SM101 Escherichia coli (which has a compromised lipopolysaccharide biosynthetic pathway making bacteria more permeable to drugs). Extending from those studies, we developed two series of analogs with key substructures resembling those of known antibacterials, nitroxoline (hydroxyquinoline moiety) and nifuroxazide/nitrofurantoin (bis-cyclic-N-acylhydrazone scaffolds). Through biochemical and cell-based assays, we identified potent GroEL/ES inhibitors that selectively blocked E. faecium, S. aureus, and E. coli proliferation with low cytotoxicity to human colon and intestine cells in vitro. Initially, only the hydroxyquinoline-bearing analogs were found to be potent inhibitors in our GroEL/ES-mediated substrate refolding assays; however, subsequent testing in the presence of an E. coli nitroreductase (NfsB) in situ indicated that metabolites of the nitrofuran-bearing analogs were potent GroEL/ES inhibitor pro-drugs. Consequently, this study has identified a new target of nitrofuran-containing drugs, and is the first reported instance of such a unique class of GroEL/ES chaperonin inhibitors. The intriguing results presented herein provide impetus for expanded studies to validate inhibitor mechanisms and optimize this antibacterial class using the respective GroEL/ES chaperonin systems and nitroreductases from E. coli and the ESKAPE bacteria.

Anti-Diarrheal Drug Repositioning in Tumour Cell Cytotoxicity.

BACKGROUND: Drug repositioning is becoming an ideal strategy to select new anticancer drugs. In particular, drugs treating the side effects of chemotherapy are the best candidates. OBJECTIVE: In this present work, we undertook the evaluation of anti-tumour activity of two anti-diarrheal drugs (nifuroxazide and rifaximin). METHODS: Anti-proliferative effect against breast cancer cells (MDA-MB-231, MCF-7 and T47D) was assessed by MTT analysis, the Brdu incorporation, mitochondrial permeability and caspase-3 activity. RESULTS: Both the drugs displayed cytotoxic effects on MCF-7, T47D and MDA-MB-231 cells. The lowest IC50 values were obtained on MCF-7 cells after 24, 48 and 72 hours of treatment while T47D and MDA-MB-231 were more resistant. The IC50 values on T47D and MDA-MB-231 cells became significantly low after 72 hours of treatment showing a late cytotoxicity effect especially of nifuroxazide but still less important than that of MCF-7 cells. According to the IC50 values, the non-tumour cell line HEK293 seems to be less sensitive to cytotoxicity especially against rifaximin. Both the drugs have shown an accumulation of rhodamine 123 as a function of the rise of their concentrations while the Brdu incorporation decreased. Despite the absence of a significant difference in the cell cycle between the treated and non-treated MCF-7 cells, the caspase-3 activity increased with the drug concentrations rise suggesting an apoptotic effect. CONCLUSION: Nifuroxazide and rifaximin are used to overcome the diarrheal side effect of anticancer drugs. However, they have shown to be anti-tumour drugs which make them potential dual effective drugs against cancer and the side effects of chemotherapy.

Hypoxia-Activated, Small-Molecule-Induced Gene Expression.

Hypoxia, a condition of reduced oxygen, occurs in a wide variety of biological contexts, including solid tumors and bacterial biofilms, which are relevant to human health. Consequently, the development of chemical tools to study hypoxia is vital. Here we report a hypoxia-activated, small-molecule-mediated gene expression system using a bioreductive prodrug of the inducer isopropyl 1-thio-ß-d-galactopyranoside. As a proof-of-concept we have placed the production of a green fluorescent protein under the control of hypoxia. Our system has the potential to be extended to regulate the production of any given protein of choice.

Conjugation of a 5-nitrofuran-2-oyl moiety to aminoalkylimidazoles produces non-toxic nitrofurans that are efficacious in vitro and in vivo against multidrug-resistant Mycobacterium tuberculosis.

Within the general nitrofuran carboxamide chemotype, chimera derivatives incorporating diversely substituted imidazoles attached via an alkylamino linker were synthesized. Antimycobacterial evaluation against drug-sensitive M. tuberculosis H37Rv strain identified five active druglike compounds which were further profiled against patient-derived M. tuberculosis strains in vitro. One of the compounds displayed promising potent activity (MIC 0.8 µg/mL) against one of such strains otherwise resistant to such first- and second-line TB therapies as streptomycin, isoniazid, rifampicin, ethambutol, kanamycin, ethionamide, capreomycin and amikacin. The compound was shown to possess low toxicity for mice (LD50 = 900.0 ±â€¯83.96 mg/kg) and to be similarly efficacious to etambutol, in the mouse model of drug-sensitive tuberculosis, and to neurotoxic cycloserine in mice infected with MDR tuberculosis.

Synthesis and evaluation of nitrofuranyl methyl N-heterocycles derivatives as novel antitubercular agents.

AIM: Tuberculosis (TB) is one of the world's deadliest chronic infectious diseases caused mainly by Mycobacterium tuberculosis (MTB). Many nitrofuran derivatives were found to possess promising anti-TB potential and have been widely studied. In our previous study, we discovered diazaspiro-nitrofuran IMB1701-1702 as potent anti-TB agents. METHODOLOGY: We report herein a series of nitrofuranyl methyl N-heterocycles based on IMB1701-1702. Results reveal that most of them show potent activity (minimum inhibitory concentration: <0.016-0.062 µg/ml) against MTB H37Rv strain. Especially, compound 7h without cytotoxicity, has the same minimum inhibitory concentration value of ≤0.016 µg/ml as PBTZ169 against both MTB H37Rv strain and two clinically isolated multidrug-resistant MTB strains. CONCLUSION: The newly designed compound 7h might be a promising anti-TB candidate.

Computer-aided discovery of two novel chalcone-like compounds active and selective against Leishmania infantum.

Leishmaniasis are infectious diseases caused by parasites of genus Leishmania that affect affects 12 million people in 98 countries mainly in Africa, Asia, and Latin America. Effective treatments for this disease are urgently needed. In this study, we present a computer-aided approach to investigate a set of 32 recently synthesized chalcone and chalcone-like compounds to act as antileishmanial agents. As a result, nine most promising compounds and three potentially inactive compounds were experimentally evaluated against Leishmania infantum amastigotes and mammalian cells. Four compounds exhibited EC50 in the range of 6.2-10.98µM. In addition, two compounds, LabMol-65 and LabMol-73, exhibited cytotoxicity in macrophages >50µM that resulted in better selectivity compared to standard drug amphotericin B. These two compounds also demonstrated low cytotoxicity and high selectivity towards Vero cells. The results of target fishing followed by homology modeling and docking studies suggest that these chalcone compounds could act in Leishmania because of their interaction with cysteine proteases, such as procathepsin L. Finally, we have provided structural recommendations for designing new antileishmanial chalcones.

Pentacyclic nitrofurans that rapidly kill nifurtimox-resistant trypanosomes.

OBJECTIVES: In response to reports of Trypanosoma brucei resistance to the nitroaromatic drug nifurtimox, we evaluated the potential of antituberculosis nitrofuran isoxazolines as inhibitors of trypanosome growth. METHODS: The susceptibility of T. brucei brucei was assessed in vitro. The lowest effective concentration to inhibit growth (EC90) against drug-susceptible and -resistant parasites, time-kill kinetics, reversibility of inhibition and propensity for P-glycoprotein-mediated exclusion from the blood-brain barrier were determined. RESULTS: Nitrofuran isoxazolines were potent inhibitors of T. brucei brucei proliferation at nanomolar concentrations, with pentacyclic nitrofurans being 100-fold more potent than nifurtimox. Activity was sustained against nifurtimox-resistant parasites, suggesting the possibility of a unique mechanism of activation and potential for use in the treatment of drug-resistant infections. Exposure of parasites to the maximum concentrations of Compound 15 achieved in vivo with oral dosing yielded >2 logs of irreversible killing in <4 h, indicating rapid trypanocidal activity. CONCLUSIONS: Pentacyclic nitrofuran isoxazolines warrant further development for the treatment of drug-susceptible and nifurtimox-resistant trypanosome infections.

Exploring 5-nitrofuran derivatives against nosocomial pathogens: synthesis, antimicrobial activity and chemometric analysis.

The burden of nosocomial or health care-associated infection (HCAI) is increasing worldwide. According to the World Health Organization (WHO), it is several fold higher in low- and middle-income countries. Considering the multidrug-resistant infections, the development of new and more effective drugs is crucial. Herein, two series (I and II) of 5-nitrofuran derivatives were designed, synthesized and assayed against microorganisms, including Gram-positive and -negative bacteria, and fungi. The pathogens screened was directly related to either the most currently relevant HCAI, or to multidrug-resistant infection caused by MRSA/VRSA strains, for instance. The sets I and II were composed by substituted-[N'-(5-nitrofuran-2-yl)methylene]benzhydrazide and 3-acetyl-5-(substituted-phenyl)-2-(5-nitro-furan-2-yl)-2,3-dihydro-1,3,4-oxadiazole compounds, respectively. The selection of the substituent groups was based upon physicochemical properties, such as hydrophobicity and electronic effect. The compounds have showed better activity against Staphylococcus aureus, Escherichia coli, and Enterococcus faecalis. The findings from S. aureus strain, which was more susceptible, were used to investigate the intersamples and intervariables relationships by applying chemometric methods. It is noteworthy that the compound 4-butyl-[N'-(5-nitrofuran-2-yl)methylene]benzhydrazide has showed similar MIC value to vancomycin, which is the reference drug for multidrug-resistant S. aureus infections. Taken the findings together, the 5-nitrofuran derivatives might be indeed considered as promising hits to develop novel antimicrobial drugs to fight against nosocomial infection.

Pentacyclic nitrofurans with in vivo efficacy and activity against nonreplicating Mycobacterium tuberculosis.

The reductively activated nitroaromatic class of antimicrobials, which include nitroimidazole and the more metabolically labile nitrofuran antitubercular agents, have demonstrated some potential for development as therapeutics against dormant TB bacilli. In previous studies, the pharmacokinetic properties of nitrofuranyl isoxazolines were improved by incorporation of the outer ring elements of the antitubercular nitroimidazole OPC-67683. This successfully increased stability of the resulting pentacyclic nitrofuran lead compound Lee1106 (referred to herein as 9a). In the current study, we report the synthesis and antimicrobial properties of 9a and panel of 9a analogs, which were developed to increase oral bioavailability. These hybrid nitrofurans remained potent inhibitors of Mycobacterium tuberculosis with favorable selectivity indices (>150) and a narrow spectrum of activity. In vivo, the pentacyclic nitrofuran compounds showed long half-lives and high volumes of distribution. Based on pharmacokinetic testing and lack of toxicity in vivo, 9a remained the series lead. 9a exerted a lengthy post antibiotic effect and was highly active against nonreplicating M. tuberculosis grown under hypoxia. 9a showed a low potential for cross resistance to current antitubercular agents, and a mechanism of activation distinct from pre-clinical tuberculosis candidates PA-824 and OPC-67683. Together these studies show that 9a is a nanomolar inhibitor of actively growing as well as nonreplicating M. tuberculosis.

Nitroreductase detection and hypoxic tumor cell imaging by a designed sensitive and selective fluorescent probe, 7-[(5-nitrofuran-2-yl)methoxy]-3H-phenoxazin-3-one.

A highly selective and sensitive fluorescence probe, 7-[(5-nitrofuran-2-yl)methoxy]-3H-phenoxazin-3-one (1), is developed for imaging the hypoxic status of tumor cells via the indirect detection of nitroreductase. The detection mechanism is based on the fact that nitroreductase can selectively catalyze the reduction of the nitro group in 1 to a hydroxylamine or amino group in the presence of reduced nicotinamide adenine dinucleotide as an electron donor that is indispensable, followed by the 1,6-rearrangement-elimination and the release of resorufin. As a result, the reaction produces a distinct color and fluorescence change from almost colorless and nonfluorescent to pink and strong red fluorescence. The fluorescence increase of probe 1 at λ(550/585 nm) is directly proportional to the concentration of nitroreductase in the range of 15-300 ng/mL, with a detection limit of 0.27 ng/mL. The ready reduction of the nitro group in 1 under hypoxic conditions leads to the establishment of a sensitive and selective fluorescence method for imaging the hypoxic status of tumor cells, and with this method Hela and A549 cells under normoxic and hypoxic conditions (even for different extents of hypoxia) can be differentiated successfully. This method is simple and may be useful for the imaging of disease-relevant hypoxia.

Chemical, biochemical and microbiological properties of a brominated nitrovinylfuran with broad-spectrum antibacterial activity.

A di-bromo substituted nitrovinylfuran with reported broad-spectrum antibacterial activity was found to be a potent inhibitor of MurA, a key enzyme in peptidoglycan biosynthesis. Further characterization of the compound was carried out to assess its reactivity towards thiol nucleophiles, its stability and degradation under aqueous conditions, inhibitory potential at other enzymes, and antibacterial and cytotoxic activity. Our results indicate that the nitrovinylfuran derivative is reactive towards cysteine residues in proteins, as demonstrated by the irreversible inhibition of MurA and bacterial methionine aminopeptidase. Experiments with proteins and model thiols indicate that the compound forms covalent adducts with SH groups and induces intermolecular disulfide bonds, with the intermediate formation of a monobromide derivative. The parent molecule as well as most of its breakdown products are potent antibiotics with MIC values below 4 µg/mL and are active against multiresistant strains such as methicillin-resistant Staphylococcus aureus (MRSA). Further development of the bromonitrovinyl scaffold towards antibiotics with clinical relevance, however, requires optimization of the antibiotic-cytotoxic selectivity profile.

Antitubercular nitrofuran isoxazolines with improved pharmacokinetic properties.

A series of tetracyclic nitrofuran isoxazoline anti-tuberculosis agents was designed and synthesized to improve the pharmacokinetic properties of an initial lead compound, which had potent anti-tuberculosis activity but suffered from poor solubility, high protein binding and rapid metabolism. In this study, structural modifications were carried on the outer phenyl and piperidine rings to introduce solubilizing and metabolically blocking functional groups. The compounds generated were evaluated for their in vitro antitubercular activity, bacterial spectrum of activity, solubility, permeability, microsomal stability and protein binding. Pharmacokinetic profiles for the most promising candidates were then determined. Compounds with phenyl morpholine and pyridyl morpholine outer rings were found to be the most potent anti-tuberculosis agents in the series. These compounds retained a narrow antibacterial spectrum of activity, with weak anti-Gram positive and no Gram negative activity, as well as good activity against non-replicating Mycobacterium tuberculosis in a low oxygen model. Overall, the addition of solubilizing and metabolically blocked outer rings did improve solubility and decrease protein binding as designed. However, the metabolic stability for compounds in this series was generally lower than desired. The best three compounds selected for in vivo pharmacokinetic testing all showed high oral bioavailability, with one notable compound showing a significantly longer half-life and good tolerability supporting its further advancement.

5-Nitrofuranes and 5-nitrothiophenes with anti-Trypanosoma cruzi activity and ability to accumulate squalene.

Chagas disease represents a serious public health problem in South America. The first line of treatment is Nifurtimox and Benznidazole which generate toxic effects in treated patients. We have recently shown that a number of 5-nitrofuranes possess activity against Trypanosoma cruzi through oxidative stress and inhibition of parasite ergosterol biosynthesis, specifically at the level of squalene epoxidase. Here, we identify new 5-nitrofuranes and the thia-analogues with excellent effects on the viability of T. cruzi and adequate parasite/mammal selectivity indexes. Analysis of the free sterols from parasite incubated, during 120h, with the compounds showed that some of them accumulated squalene suggesting the squalene epoxidase activity inhibition of the parasite. Nifurtimox was able to accumulate squalene only at lower incubation times. Due to this fact some derivatives were also tested as antifungal agents. Quantitative structure-activity relationship studies were also performed showing relevant features for further new derivatives design. Taken together, the results obtained in the present work point to a more general effect of 5-nitrofuranes and 5-nitrothiophenes in trypanosomatids, opening potential therapeutic possibilities of them for these infectious diseases.

Synthesis of novel hybrid molecules from precursors with known antiparasitic activity.

Three novel new compounds derived from antiparasitic precursors have been synthesized and tested for their antiamoebic and antigiardial activities. The condensation of 2-(2-methyl-5-1H-nitroimidazolyl)ethylamine (6) with 5-nitro-2-furylacrylic acid (7) gave 3-(5-nitrofuran-2-yl)-N-[2-(5-nitroimidazol-1-yl)ethyl]acrylamide (8). Condensation of 7 with 7-chloro-4-(piperazin-1-yl)quinoline (9) afforded 1-[4-(7-chloroquinolin-4-yl)piperazin-1-yl)-3-(5-nitrofuran-2-yl)propenone as a mixture of two isomers; 10-a (the E-isomer) and 10-b (the Z-isomer). In addition, the reaction of 9 with 1-(2-bromoethyl)-2-methyl-5-nitroimidazole (11) in the presence of K(2)CO(3) and NaI yielded 7-chloro-4-(4-[2-(5-nitroimidazol-1-yl)ethyl]-piprazin-1-yl)quinoline (12). On the basis of preliminary screening data for these new compounds, compound 12 exhibited potent lethal activities against Entamoeba histolytica and Giardia intestinalis; its IC(50) (about 1 microM) was lower, at least by a factor of five, compared to the standard drug, metronidazole. In addition, the IC(50) of compound 12 against the tested parasites is 600 times below that against Hep-2 and Vero cells. Compounds 8 and 10-a also exhibited potent or moderate antiamoebic and antigiardial activities with IC(50 values) of about 5.5 microM, and 140 microM, respectively, against the tested parasites. These two hybrid molecules, 8, 10-a, were also non-cytotoxic at the lethal concentrations against the parasites.

Design, synthesis, antimicrobial activity and molecular modeling studies of novel benzofuroxan derivatives against Staphylococcus aureus.

Molecular modification is a quite promising strategy in the design and development of drug analogs with better bioavailability, higher intrinsic activity and less toxicity. In the search of new leads with potential antimicrobial activity, a new series of 14 4-substituted [N'-(benzofuroxan-5-yl)methylene]benzohydrazides, nifuroxazide derivatives, were synthesized and tested against standard and multidrug-resistant Staphylococcus aureus strains. The selection of the substituent groups was based on physicochemical properties, such as hydrophobicity and electronic effect. These properties were also evaluated through the lipophilic and electrostatic potential maps, respectively, considering the compounds with better biological profile. Twelve compounds exhibited similar bacteriostatic activity against standard and multidrug-resistant strains. The most active compound was the 4-CF(3) substituted derivative, which presented a minimum inhibitory concentration (MIC) value of 14.6-13.1 microg/mL, and a ClogP value of 1.87. The results highlight the benzofuroxan derivatives as potential leads for designing new future antimicrobial drug candidates.

Quantitative structure-activity relationship studies on nitrofuranyl anti-tubercular agents.

A series of nitrofuranylamide and related aromatic compounds displaying potent activity against Mycobacterium tuberculosis have been investigated utilizing 3-dimensional quantitative structure-activity relationship (3D-QSAR) techniques. Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) methods were used to produce 3D-QSAR models that correlated the minimum inhibitory concentration (MIC) values against M. tuberculosis with the molecular structures of the active compounds. A training set of 95 active compounds was used to develop the models, which were then evaluated by a series of internal and external cross-validation techniques. A test set of 15 compounds was used for the external validation. Different alignment and ionization rules were investigated as well as the effect of global molecular descriptors including lipophilicity (cLogP, LogD), polar surface area (PSA), and steric bulk (CMR), on model predictivity. Models with greater than 70% predictive ability, as determined by external validation, and high internal validity (cross-validated r(2)>.5) have been developed. Incorporation of lipophilicity descriptors into the models had negligible effects on model predictivity. The models developed will be used to predict the activity of proposed new structures and advance the development of next generation nitrofuranyl and related nitroaromatic anti-tuberculosis agents.

Heteroallyl-containing 5-nitrofuranes as new anti-Trypanosoma cruzi agents with a dual mechanism of action.

New heteroallyl-containing 5-nitrofuranes were synthesized as potential anti-Trypanosoma cruzi agents with a dual mechanism of action, oxidative stress and inhibition of membrane sterol biosynthesis. Some of the derivatives were found to have high and selective activity against the proliferative stages of the parasite, with IC(50) values against the clinically relevant intracellular amastigote forms in the low micromolar to sub-micromolar range. Oxidative stress was verified measuring cyanide dependent respiration. Inhibition of the de novo sterol biosynthesis at the level of squalene epoxidase was confirmed, using high-resolution gas-liquid chromatography coupled to mass spectrometry, by the disappearance of the parasite's mature sterols and the concomitant accumulation of squalene. The in vitro activities of these novel compounds were superior to that of nifurtimox, a nitrofuran currently used in the treatment of human Chagas' disease, and terbinafine, a commercially available allylamine-based squalene epoxidase inhibitor. The results support further in vivo studies of some of these nitrofuran derivatives.