Methylene Blue Analogues Research Articles

Outcomes of folic acid esterification upon the properties of hydrophilic phenothiazinium dyes: New photosensitizers for antimicrobial photodynamic therapy

The utilization of three methylene blue analogues containing hydroxyl functionalized auxochrome units (MBA) and their corresponding novel folic acid esters (FAE) as singlet-oxygen photosensitizers for antimicrobial photodynamic therapy (aPDT) was investigated. A new environmentally friendly ultrasoundassisted procedure for the synthesis of MBA and an optimized protocol for folic acid esterification are reported. Binding of folic acid to MBA did not alter the optical absorption/emission properties of the dyes, the folates exhibiting higher or at least equal fluorescence quantum yields when compared to those of the parent dyes. The photodynamic effect of MBA and FAE was evaluated based on their singlet oxygen generation capacity. The 1O2 quantum yields, determined by the indirect detection technique using 1,3-diphenylisobenzofuran (DPBF) as a sensor, appeared to be ∼ 50 % for MBA and decreased to ∼ 20 % in the case of FAE. The antimicrobial efficacy of the new FAE on four bacterial strains (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Deinococcus radiodurans) showed remarkable results especially when combined with photodynamic therapy.

In vitro Antibacterial Activity of Dye Compounds

Background: Methylene blue and some of its analogues have known antibacterial activity, however their exact mechanism of action is unknown Objective: In this study, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of several methylene blue analogues were determined against five bacterial strains, whereafter the data were used to create and validate a pharmacophore model. Methods: The agar dilution method was used to screen the analogues for antibacterial activity, while the broth microdilution method was used to determine their MIC and MBC. A pharmacophore model was constructed and validated using the rank score, fit value, enrichment factor (EF10%), hit rate (HR10%) and receiver operating characteristic area under the curve (ROC-AUC) as metrics. Results: Against Staphylococcus aureus, pyronin B (0.125 µg/ml) was more active than tetracycline (1 µg/ml) and pyronin Y (0.5 µg/ml), 1,9-dimethylmethylene blue (2 µg/ml), basic blue 3 (2 µg/ml), new methylene blue (2 µg/ml) and Nile blue (2 µg/ml) had similar activity compared to tetracycline. Pyronin B, 1,9-dimethylmethylene blue and new methylene blue were bactericidal. A pharmacophore model was created (rank score: 36.55, max. fit value: 3), which was able to identify active analogues out of the test set (EF10%: 2.83, HR10%: 28.57%, ROC-AUC: 0.84 ± 0.04). The pharmacophore model highlighted that a positive ionisable, aromatic ring as well as a hydrophobic moiety are important for antibacterial activity. Conclusion: Methylene blue analogues were found to have potent antibacterial activity and a pharmacophore model was created to understand the structural requirements for activity.

New methylene blue analogues with N-piperidinyl-carbinol units: Synthesis, optical properties and in vitro internalization in human ovarian cancer cells

In this work, two new phenothiazinium dyes containing N-piperidinyl-carbinol auxochrome units have been designed, synthesized and investigated in vitro for ovarian cancer cell fluorescence imaging and cytotoxicity. A solvent free mechanochemical procedure is described as a greener alternative to the previously reported synthetic methodology applied in the preparation of methylene blue analogues by the substitution of the phenothiazinium tetraiodide substrate with nucleophilic secondary amines in solution. The optical properties of the new dyes were evaluated by UV–vis absorption/emission spectroscopy which revealed maxima situated in the red region (λmaxabsorption = 668-664 nm and λmaxemission = 715-698 nm) with large molar extinction coefficients (log ε = 4.5–5.1) and bathochromic shifts of 314–383 cm−1 occurring upon switching from less polar to more polar solvents. The theoretical DFT computational study performed in order to clarify the obtained data indicated that the vertical TD-DFT calculations (typically applied for simulating UV–vis spectra) were within ∼150 nm of the experimental data, but a better agreement with experiment was obtained by using the vibronic corrections which revealed a dominant vibronic component not only for the main absorption/emission maxima, but also for the displayed shoulders which are rather the effects of the vibronic coupling between S0 and S1 electronic states. The new dyes presented a hydrophilic character (logPn-octanol/water = −1.15 and −1.29 respectively) comparable to the parent methylene blue and no aggregation tendency was observed in aqueous solution at concentration range 75–150 μM used in this study. Cellular imaging experiments displayed the new dyes' ability to selectively stain the cytoplasm of tumoral OVCAR-3 and A2780 fixed cells. The evaluation of their biological activity encompassed cell grow inhibitory potential (dose-response activity and half maximal inhibitory concentration IC50), as well as their capacity to influence the tumoral OVCAR-3, A2780 and healthy BJ ovarian cells’ metabolic activity. Dye 2 exhibited moderate cytotoxicity against human ovarian cancer cell lines, being the most active against the A2780 ovarian adenocarcinoma cells where its cellular uptake was noteworthy and displayed a deeper influence on the chemoresistant, highly malignant OVCAR-3 cells function by modulating FOLR1, ICAM-1 and VCAM-1 proteins with role in the growth and migration of cancer cells.

Towards Asymmetrical Methylene Blue Analogues: Synthesis and Reactivity of 3-N'-Arylaminophenothiazines.

The search for new ways to obtain analogues of the well-known Methylene Blue dye is an important synthetic task. Herein, we proposed and developed an approach to the synthesis of 3-N′-arylaminophenothiazines and asymmetrical 3,7-di(N′-arylamino)phenothiazines. This approach included the optimization of synthetic strategy by quantification analysis of the positive charge distribution in the cation of 3-N′-arylaminophenothiazine derivative. The obtained experimental data are confirmed by DFT studies. Two synthetic routes for asymmetrical phenothiazine diarylamino derivatives were suggested and verified. The developed convenient and versatile synthetic approach makes it easy to obtain aromatic Methylene Blue isostructural analogues with various substituents. As a result, a series of novel 3-N′-arylaminophenothiazines and asymmetrical 3,7-di(N′-arylamino)phenothiazines containing ester, tert-butoxycarbonyl, sulfonic acid, hydroxyl and amine groups were obtained in high yields.

Methylene blue analogues: In vitro antimicrobial minimum inhibitory concentrations and in silico pharmacophore modelling

It has been shown that methylene blue has antimicrobial properties although few studies have determined its minimum inhibitory concentration (MIC), which is the gold standard used to measure antimicrobial activity. The exact antimicrobial mode of action of methylene blue is still unclear and to our knowledge no pharmacophore model has yet been created to investigate methylene blue's mode of action. The aim of this study was to determine the MIC of methylene blue and a number of its analogous against Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae, Salmonella enterica and Candida albicans, and to use these data to develop and validate a common feature pharmacophore model. Three statistical metrics, i.e. the enrichment factor (EF), hit rate (HR) and the area under the curve of a receiver operator characteristic (ROC-AUC), were used to determine the pharmacophore model's ability to differentiate between active/decoy compounds. The validated pharmacophore model was used to map similar antibacterial compounds in an attempt to elucidate methylene blue's antimicrobial mode of action. Most test compounds only had activity against S. aureus, S. epidermidis and K. pneumoniae. Dimethyl methylene blue, new methylene blue and acriflavine proved to be the most active compounds against S. aureus [1 µg/ml (dimethyl methylene blue); 4 µg/ml (new methylene blue); 8 µg/ml (acriflavine)], S. epidermidis [1 µg/ml (dimethyl methylene blue); 4 µg/ml (new methylene blue); 2 µg/ml (acriflavine)] and K. pneumoniae [8 µg/ml (dimethyl methylene blue); 0.5 µg/ml (new methylene blue); 2 µg/ml (acriflavine)]. A common feature pharmacophore model was created (rank score: 26.664, max. fit value: 4), which was able to accurately identify active methylene blue analogous out of the test set (EF2%: 51, HR2%: 100%, ROC-AUC: 1.00 ± 0.00). Six phenothiazine derivatives with known antibacterial activity had high fit values when mapped with the validated pharmacophore model, i.e. >3.4. Dimethyl methylene blue, new methylene blue and acriflavine had potent antibacterial activity against S. aureus, S. epidermidis and K. pneumoniae. The MIC data will allow other researchers to compare the activity across different laboratories. The common feature pharmacophore model was able to identify methylene blue analogous with known in vitro antibacterial activity out of a database containing active/decoy compounds and highlighted the importance of two hydrophobic features, an aromatic feature and a hydrogen bond acceptor. The validated pharmacophore model also correctly mapped six phenothiazine derivatives with known antibacterial activity suggesting that they may share a common antibacterial mechanism of action.

Methylene Blue Analogues with Marginal Monoamine Oxidase Inhibition Retain Antidepressant-like Activity.

Methylene blue (MB) possesses diverse medical applications. Among these, MB presents with antidepressant-like effects in animals and has shown promise in clinical trials for the treatment of mood disorders. As an antidepressant, MB may act via various mechanisms which include modulation of the nitric oxide cyclic guanosine monophosphate (NO-cGMP) cascade, enhancement of mitochondrial respiration and antioxidant effects. MB is also, however, a high potency inhibitor of monoamine oxidase (MAO) A, which most likely contributes to its antidepressant effect, but also to its adverse effects profile (e.g., serotonin toxicity). The latter has raised the question whether it is possible to design out the MAO inhibition properties of MB yet retaining its clinically useful attributes. This study explores this idea further by characterizing five newly synthesized low MAO-A active MB analogues and examining their antidepressant-like properties in the acute forced swim test (FST) in rats, with comparison to imipramine and MB. The results show that all five analogues exhibit antidepressant-like properties in the FST without confounding effects on locomotor activity. The magnitude of these effects is comparable to those of imipramine and MB. Moreover, these newly synthesized MB analogues are markedly less potent MAO-A inhibitors (IC50 = 0.518-4.73 μM) than MB (IC50 = 0.07 μM). We postulate that such lower potency MAO-A inhibitors may present with a reduced risk of adverse effects associated with MAO-A inhibition. While low level MAO-A inhibition still may produce an antidepressant effect, we posit that other MB-related mechanisms may underlie their antidepressant effects, thereby representing a novel group of antidepressant compounds.

Lipophilic methylene blue analogues enhance mitochondrial function and increase frataxin levels in a cellular model of Friedreich’s ataxia

Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disorder resulting from reduced expression of the protein frataxin (FXN). Although its function is not fully understood, frataxin appears to help assemble iron sulfur clusters; these are critical for the function of many proteins, including those needed for mitochondrial energy production. Finding ways to increase FXN levels has been a major therapeutic strategy for this disease. Previously, we described a novel series of methylene violet analogues and their structural optimization as potential therapeutic agents for neurodegenerative and mitochondrial disorders. Presently, a series of methylene blue analogues has been synthesized and characterized for their in vitro biochemical and biological properties in cultured Friedreich’s ataxia lymphocytes. Favorable methylene blue analogues were shown to increase frataxin levels and mitochondrial biogenesis, and to improve aconitase activity. The analogues were found to be good ROS scavengers, and able to protect cultured FRDA lymphocytes from oxidative stress resulting from inhibition of complex I and from glutathione depletion. The analogues also preserved mitochondrial membrane potential and augmented ATP production. Our results suggest that analogue 5, emerging from the initial structure of the parent compound methylene blue (MB), represents a promising lead structure and lacks the cytotoxicity associated with the parent compound MB.

Phenothiazine antioxidants increase mitochondrial biogenesis and frataxin levels in Friedreich's ataxia cells.

Friedreich's ataxia (FRDA) is a progressive neurodegenerative disease that is linked to transcriptional repression of the nuclear FXN gene encoding the essential mitochondrial protein frataxin (FXN). Compounds that increase frataxin levels may enable effective therapeutic intervention for blunting disease progression. Recently, we showed that lipophilic methylene violet (MV) and methylene blue (MB) analogues both conferred benefit to cultured FRDA cells in several regards, including ROS suppression, maintenance of mitochondrial membrane potential and increased ATP production. Some of the MB analogues were also shown to promote increased frataxin levels and mitochondrial biogenesis. Presently, we report that two of the MV analogues studied previously (1 and 2) also increased frataxin levels and mitochondrial biogenesis significantly. Because the substitution pattern in the two series of compounds was not the same, we also prepared new MV derivatives having the same substitution pattern as the original MB derivatives studied to enable a more direct comparison. Two of the new MV compounds, 4b and 6b, exhibited enhanced antioxidant capability, increased frataxin levels and mitochondrial biogenesis, and improved aconitase activity. These encouraging findings demonstrated that the MV analogues had better overall activity with less cytotoxicity.

Lipophilic methylene violet analogues as modulators of mitochondrial function and dysfunction

In an effort to identify methylene blue analogues having improved antioxidant activity, a series of new methylene violet analogues have been designed and synthesized. The analogues were prepared following a synthetic route that is more efficient than the previously reported methods, both in terms of yield and purity of the final products. The route involves the Smiles rearrangement as one of the crucial steps. Smiles rearrangement of suitably substituted diphenyl sulfide intermediates afforded the corresponding phenothiazine analogues in high yields, which were subsequently converted to the final products. The methylene violet analogues were evaluated for their ability to preserve mitochondrial function in Friedreich’s ataxia (FRDA) lymphocytes. The analogues were shown to be efficient ROS scavengers, and able to protect cultured FRDA lymphocytes from oxidative stress resulting from inhibition of complex I. The analogues also preserved mitochondrial membrane potential and augmented ATP production. The analogues were found to be better antioxidants than the parent compounds methylene blue and methylene violet.

The monoamine oxidase inhibition properties of selected structural analogues of methylene blue

The thionine dye, methylene blue (MB), is a potent inhibitor of monoamine oxidase (MAO) A, a property that may, at least in part, mediate its antidepressant effects in humans and animals. The central inhibition of MAO-A by MB has also been linked to serotonin toxicity (ST) which may arise when MB is used in combination with serotonergic drugs. Structural analogues and the principal metabolite of MB, azure B, have also been reported to inhibit the MAO enzymes, with all compounds exhibiting specificity for the MAO-A isoform. To expand on the structure-activity relationships (SARs) of MAO inhibition by MB analogues, the present study investigates the human MAO inhibition properties of five MB analogues: neutral red, Nile blue, new methylene blue, cresyl violet and 1,9-dimethyl methylene blue. Similar to MB, these analogues also are specific MAO-A inhibitors with cresyl violet (IC50=0.0037μM), Nile blue (IC50=0.0077μM) and 1,9-dimethyl methylene blue (IC50=0.018μM) exhibiting higher potency inhibition compared to MB (IC50=0.07μM). Nile blue also represents a potent MAO-B inhibitor with an IC50 value of 0.012μM. From the results it may be concluded that non-thionine MB analogues (e.g. cresyl violet and Nile blue) also may exhibit potent MAO inhibition, a property which should be considered when using these compounds in pharmacological studies. Benzophenoxazines such as cresyl violet and Nile blue are, similar to phenothiazines (e.g. MB), representative of high potency MAO-A inhibitors with a potential risk of ST.

In Vitro Photodynamic Activity of a Series of Methylene Blue Analogues¶

In Vitro Photodynamic Activity of a Series of Methylene Blue Analogues¶

Systematic Change in Stacking Structure Induced by Alkyl Chains and Spectral Change Due to Hetero-Aggregation in [Ni(dmit)2]− Salts of Methylene Blue Analogues

Abstract A series of [Ni(dmit)2]− (dmit = 2-thioxo-1,3-dithiole-4,5-dithiolato) salts of 3,7-bis(dialkylamino)phenothiazin-5-ium (alkyl = methyl (C1S), ethyl (C2S), propyl (C3S), and butyl (C4S)) together with those of 3,7-bis(diallylamino)phenothiazin-5-ium (AS) and 3,7-bis(diethylamino)phenoxazin-5-ium (C2O) have been prepared and analyzed by X-ray crystal structural analysis and diffuse reflectance spectra. All complex salts formed stacking columns containing cations and anions. In the crystal structure of (C1S)[Ni(dmit)2], both C1S cations and [Ni(dmit)2]− anions were found to form stacked dimers, and these dimers piled up alternately in columns. In the cases of (C2S)[Ni(dmit)2], (AS)[Ni(dmit)2], and (C2O)[Ni(dmit)2], cations and anions piled up alternately in the form of monomers. In the cases of (C3S)[Ni(dmit)2] and (C4S)[Ni(dmit)2], one anion was sandwiched between two cation dimers to form columns, and another anion layed between these columns. An absorption band appeared at 516–566 nm in the diffuse reflectance spectra, which originated from the absorption of blue cationic dyes, exhibited a large shift by the intermolecular interaction of cation–cation and cation–anion π-stacking. Compounds of (C1S)[Ni(dmit)2] and (C2O)[Ni(dmit)2] were found to behave as semiconductors, exhibiting room temperature conductivities of 3.8 × 10−6 and 4.5 × 10−7 S cm−1, respectively.

In vitro photodynamic activity of a series of methylene blue analogues.

We have synthesized a series of symmetrical phenothiazines in which the methyl groups of methylene blue have been substituted by longer alkyl chains. Intrinsic photosensitizing ability was not altered by increasing the chain length. However, in vitro phototoxicity after 2 h incubation of RIF-1 murine fibrosarcoma cells followed the order n-propyl > n-pentyl > n-butyl > n-hexyl > ethyl > methyl, with ethyl and n-propyl analogues being 14- and 130-fold more phototoxic than methylene blue, respectively. All analogues also had an improved ratio of phototoxicity: dark toxicity (4:1 to 27:1) compared with methylene blue (3:1). Phototoxicity did not correlate with cellular phenothiazine levels, suggesting that the site of subcellular localization may be more important. After 2 h incubation of RIF-1 cells with the phototoxicity LD50 concentration, methylene blue and all analogues were observed to be localized in the lysosomes by fluorescence microscopy. On exposure to light, methylene blue relocalized to the nucleus, the ethyl analogue did not relocalize, whereas the more phototoxic n-propyl - n-hexyl analogues relocalized to the mitochondria. Relocalization to the mitochondria was associated with an octanol: buffer partition coefficient > or = 1. Therefore, the longer-chain analogues of methylene blue show significantly improved phototoxicity in vitro and, in addition, are expected to avoid the problems of mutagenicity associated with the nuclear localization of methylene blue.

Synthesis of Substituted Phenothiazines Analogous to Methylene Blue by Electrophilic and Nucleophilic Aromatic Substitutions in Tandem. A Mechanistic Perspective.

3,7-Disubstituted phenothiazines analogous to methylene blue (1) were synthesized from phenothiazine (6) reacting first with an excess of bromine in acetic acid to give 3,7-dibromophenothiazin-5-ium bromide (8), according to a reaction sequence that involves two electrophilic aromatic substitutions and one oxidation. Subsequently, 8 reacting with diallylamine or allylmethylamine via two nucleophilic aromatic substitution steps gave 3,7-bis[di(2-propenyl)amino]phenothiazin-5-ium bromide (3) or 3,7-bis[methyl, (2-propenyl)amino]phenothiazin-5-ium bromide (4), compounds analogous to methylene blue. The choice of solvent for the second step is critically important as 3,7-dibromophenothiazin-5-ium bromide is very reactive and prone to irreversible oxidation, reduction, and ipso attack at the 3- and 7-positions. The best yields (∼63%) for the methylene blue analogues 3 and 4 were obtained when CHCl3 or CH2Cl2 were employed as solvents for the nucleophilic aromatic substitution step. These solvents dissolve the methylene blue analogue products, but not 3,7-dibromophenothiazin-5-ium bromide. © 1997 Elsevier Science Ltd.

Mode of antimalarial effect of methylene blue and some of its analogues on Plasmodium falciparum in culture and their inhibition of P. vinckei petteri and P. yoelii nigeriensis in vivo.

The antimalarial action of methylene blue (MB) was first noted by Paul Ehrlich in the late 19th century. Although it has only sporadically been adopted as a serviceable drug, the resolution of its antimalarial action seems warranted, as it is currently used for the treatment of various methemoglobinemias. In this work we have used MB, and its analogues Azures A (AZA), B (AZB), C (AZC), and thionin (TH), as well as the oxazine Celestine blue (CB) and azine Phenosaphranin (PS). All MB analogues inhibit the growth of various strains of Plasmodium falciparum in culture with IC50s in the 2 x 10(-9)-1 x 10(-7) M range, with the rank order MB approximately AZA > AZB > AZC > TH > PS > CB. The IC50s for a mammalian cell line were in the 3 x 10(-6)-4 x 10(-5) M range, and the rank order was TH approximately AZB > AZA approximately PS > AZC approximately CB > MB. As MB could affect cell growth through the oxidation of NADPH, we tested the action of the various compounds on the hexose-monophosphate shunt activity. Appreciable activation of the shunt was observed at 1 x 10(-5) M in both cell types, thus accounting for inhibition of growth of mammalian cells but not of parasites. All compounds were found to complex with heme in a rank order similar to their antimalarial effect. It is therefore suggested that MB and its congeners act by preventing the polymerization of heme, which is produced during the digestion of host cell cytosol in the parasite food vacuole, into hemozoin. In this respect, these compounds seem to act similarly to the 4-aminoquinoline antimalarials. All compounds effectively suppressed the growth of P. vinckei petteri in vivo with IC50 in the 1.2-5.2 mg/kg range, and MB and AZB suppressed P. yoelii nigeriensis in the 9-11 mg/kg range (i.e. at doses similar to those of chloroquine). The potential toxicity of these compounds may restrict their clinical use, but their impressive antimalarial activities suggest that the phenothiazine structure could serve as a lead compound for further drug development.