Phenazine Cations as Anticancer Theranostics†.

The biological properties of two water-soluble organic cations based on polypyridyl structures commonly used as ligands for photoactive transition metal complexes designed to interact with biomolecules are investigated. A cytotoxicity screen employing a small panel of cell lines reveals that both cations show cytotoxicity toward cancer cells but show reduced cytotoxicity to noncancerous HEK293 cells with the more extended system being notably more active. Although it is not a singlet oxygen sensitizer, the more active cation also displayed enhanced potency on irradiation with visible light, making it active at nanomolar concentrations. Using the intrinsic luminescence of the cations, their cellular uptake was investigated in more detail, revealing that the active compound is more readily internalized than its less lipophilic analogue. Colocalization studies with established cell probes reveal that the active cation predominantly localizes within lysosomes and that irradiation leads to the disruption of mitochondrial structure and function. Stimulated emission depletion (STED) nanoscopy and transmission electron microscopy (TEM) imaging reveal that treatment results in distinct lysosomal swelling and extensive cellular vacuolization. Further imaging-based studies confirm that treatment with the active cation induces lysosomal membrane permeabilization, which triggers lysosome-dependent cell-death due to both necrosis and caspase-dependent apoptosis. A preliminary toxicity screen in the Galleria melonella animal model was carried out on both cations and revealed no detectable toxicity up to concentrations of 80 mg/kg. Taken together, these studies indicate that this class of synthetically easy-to-access photoactive compounds offers potential as novel therapeutic leads.

Study on the Design, Synthesis, Bioactivity and Translocation of the Conjugates of Phenazine-1-carboxylic Acid and N-Phenyl Alanine Ester.

The natural pesticide phenazine-1-carboxylic acid (PCA) is known to lack phloem mobility, whereas Metalaxyl is a representative phloem systemic fungicide. In order to endow PCA with phloem mobility and also enhance its antifungal activity, thirty-two phenazine-1-carboxylic acid-N-phenylalanine esters conjugates were designed and synthesized by conjugating PCA with the active structure N-acylalanine methyl ester of Metalaxyl. All target compounds were characterized by 1H NMR, 13C NMR and HRMS. The antifungal evaluation results revealed that several target compounds exhibited moderate to potent antifungal activities against Sclerotinia sclerotiorum, Bipolaris sorokiniana, Phytophthora parasitica, Phytophthora citrophthora. In particular, compound F7 displayed excellent antifungal activity against S. sclerotiorum with an EC50 value of 6.57 µg/mL, which was superior to that of Metalaxyl. Phloem mobility study in castor bean system indicated good phloem mobility for the target compounds F1-F16. Particularly, compound F2 exhibited excellent phloem mobility; the content of compound F2 in the phloem sap of castor bean was 19.12 µmol/L, which was six times higher than Metalaxyl (3.56 µmol/L). The phloem mobility tests under different pH culture solutions verified the phloem translocation of compounds related to the "ion trap" effect. The distribution of the compound F2 in tobacco plants further suggested its ambimobility in the phloem, exhibiting directional accumulation towards the apical growth point and the root. These results provide valuable insights for developing phloem mobility fungicides mediated by exogenous compounds.

Dihydrophenazine: a multifunctional new weapon that kills multidrug-resistant Acinetobacter baumannii and restores carbapenem and oxidative stress susceptibilities.

AIMS: The current work aims to fully characterize a new antimicrobial agent against Acinetobacter baumannii, which continues to represent a growing threat to healthcare settings worldwide. With minimal treatment options due to the extensive spread of resistance to almost all the available antimicrobials, the hunt for new antimicrobial agents is a high priority. METHODS AND RESULTS: An Egyptian soil-derived bacterium strain NHM-077B proved to be a promising source for a new antimicrobial agent. Bio-guided fractionation of the culture supernatants of NHM-077B followed by chemical structure elucidation identified the active antimicrobial agent as 1-hydroxy phenazine. Chemical synthesis yielded more derivatives, including dihydrophenazine (DHP), which proved to be the most potent against A. baumannii, yet it exhibited a marginally safe cytotoxicity profile against human skin fibroblasts. Proteomics analysis of the cells treated with DHP revealed multiple proteins with altered expression that could be correlated to the observed phenotypes and potential mechanism of the antimicrobial action of DHP. DHP is a multipronged agent that affects membrane integrity, increases susceptibility to oxidative stress, interferes with amino acids/protein synthesis, and modulates virulence-related proteins. Interestingly, DHP in subinhibitory concentrations re-sensitizes the highly virulent carbapenem-resistant A. baumannii strain AB5075 to carbapenems providing great hope in regaining some of the benefits of this important class of antibiotics. CONCLUSIONS: This work underscores the potential of DHP as a promising new agent with multifunctional roles as both a classical and nonconventional antimicrobial agent that is urgently needed.

Tepuazines A-E: Phenazine Glycosides from a Venezuelan Quartz-Rich (Tepui) Cave Soil-Derived Streptomyces virginiae CMB-CA091.

Investigation of the secondary metabolites of Streptomyces virginiae CMB-CA091 isolated from the quartz-rich (tepui) soil of a cave in Venezuela yielded two new dimeric phenazine glycosides, tepuazines A and B (1 and 2); three new monomeric phenazine glycosides, tepuazines C-E (3-5); and a series of known analogues, baraphenazine G (6), phenazinolin D (7), izumiphenazine C (8), 4-methylaminobenzoyl-l-rhamnopyranoside (9), and 2-acetamidophenol (10). Structures were assigned to 1-10 on the basis of detailed spectroscopic analysis and biosynthetic considerations, with 1 and 2 featuring a rare 2-oxabicyclo[3.3.1]nonane-like ring C/D bridge shared with only a handful of known Streptomyces natural products. We propose a plausible convergent biosynthetic relationship linking all known members of this structure class that provides a rationale for the observed ring C/D configuration.

Bacillus sp. G2112 Detoxifies Phenazine-1-carboxylic Acid by N5 Glucosylation.

Microbial symbionts of plants constitute promising sources of biocontrol organisms to fight plant pathogens. Bacillus sp. G2112 and Pseudomonas sp. G124 isolated from cucumber (Cucumis sativus) leaves inhibited the plant pathogens Erwinia and Fusarium. When Bacillus sp. G2112 and Pseudomonas sp. G124 were co-cultivated, a red halo appeared around Bacillus sp. G2112 colonies. Metabolite profiling using liquid chromatography coupled to UV and mass spectrometry revealed that the antibiotic phenazine-1-carboxylic acid (PCA) released by Pseudomonas sp. G124 was transformed by Bacillus sp. G2112 to red pigments. In the presence of PCA (>40 µg/mL), Bacillus sp. G2112 could not grow. However, already-grown Bacillus sp. G2112 (OD600 > 1.0) survived PCA treatment, converting it to red pigments. These pigments were purified by reverse-phase chromatography, and identified by high-resolution mass spectrometry, NMR, and chemical degradation as unprecedented 5N-glucosylated phenazine derivatives: 7-imino-5N-(1'ß-D-glucopyranosyl)-5,7-dihydrophenazine-1-carboxylic acid and 3-imino-5N-(1'ß-D-glucopyranosyl)-3,5-dihydrophenazine-1-carboxylic acid. 3-imino-5N-(1'ß-D-glucopyranosyl)-3,5-dihydrophenazine-1-carboxylic acid did not inhibit Bacillus sp. G2112, proving that the observed modification constitutes a resistance mechanism. The coexistence of microorganisms-especially under natural/field conditions-calls for such adaptations, such as PCA inactivation, but these can weaken the potential of the producing organism against pathogens and should be considered during the development of biocontrol strategies.

Luminescent 11-{Naphthalen-1-yl}dipyrido[3,2-a:2',3'-c]phenazine-Based Ru(II)/Ir(III)/Re(I) Complexes for HCT-116 Colorectal Cancer Stem Cell Therapy.

Due to a number of unpleasant considerations, marketed drugs have steadily lost their importance in the treatment of cancer. In order to find a viable cancer cell diagnostic agent, we therefore focused on metal complexes that displayed target adequacy, permeability to cancer cells, high standard water solubility, cytoselectivity, and luminescent behavior. In this aspect, luminescent 11-{naphthalen-1-yl} dipyrido [3,2-a:2',3'-c] phenazine based Ru(II)/Ir(III)/Re(I) complexes have been prepared for HCT-116 colorectal cancer stem cell therapy. Our study successfully established the possible cytotoxicity of IrL complex at different doses on HCT-116 colorectal cancer stem cells (CRCSCs). Additionally, an immunochemistry analysis of the complex IrL showed that the molecule was subcellularly localized in the nucleus and other regions of the cytoplasm, where it caused nuclear DNA damage and mitochondrial dysfunction. The level of BAX and Bcl-2 was further quantified by qRT-PCR. The expression of proapoptotic BAX showed increased expression in the complex IrL-treated cell compared to the control, indicating the potential of complex IrL for apoptotic induction. Upon further validation, complex IrL was developed as an inhibitor of autophagy for the eradication of cancer stem cells.

One-pot multicomponent synthesis of benzophenazine tethered tetrahydropyridopyrimidine derivatives.

A simple, facile, and efficient green methodology has been developed for the synthesis of benzophenazine tethered tetrahydropyridopyrimidine derivatives by the one-pot four-component reaction of cinnamaldehyde/crotonaldehyde, 2-hydroxy-1,4-naphthoquinone, 1,3-dimethyl-6-amino uracil, and o-phenylenediamine in ethanol medium under reflux conditions using p-TSA as a catalyst. In this environmentally benign methodology, three C-N and two C-C bonds are formed in one pot. The hybrid products have three bioactive moieties such as benzophenazine, tetrahydropyridine, and pyrimidine. Operational simplicity, metal-free conditions, wide substrate scope, readily available starting materials, moderate to good yields of the desired products, presence of pharmaceutically active moieties, and easy purification process are the notable features of this methodology.

Photophysical Properties and DNA Binding of Two Intercalating Osmium Polypyridyl Complexes Showing Light-Switch Effects.

The synthesis and photophysical characterization of two osmium(II) polypyridyl complexes, [Os(TAP)2dppz]2+ (1) and [Os(TAP)2dppp2]2+ (2) containing dppz (dipyrido[3,2-a:2',3'-c]phenazine) and dppp2 (pyrido[2',3':5,6]pyrazino[2,3-f][1,10]phenanthroline) intercalating ligands and TAP (1,4,5,8-tetraazaphenanthrene) ancillary ligands, are reported. The complexes exhibit complex electrochemistry with five distinct reductive redox couples, the first of which is assigned to a TAP-based process. The complexes emit in the near-IR (1 at 761 nm and 2 at 740 nm) with lifetimes of >35 ns with a low quantum yield of luminescence in aqueous solution (∼0.25%). The Δ and Λ enantiomers of 1 and 2 are found to bind to natural DNA and with AT and GC oligodeoxynucleotides with high affinities. In the presence of natural DNA, the visible absorption spectra are found to display significant hypochromic shifts, which is strongly evident for the ligand-centered π-π* dppp2 transition at 355 nm, which undergoes 46% hypochromism. The emission of both complexes increases upon DNA binding, which is observed to be sensitive to the Δ or Λ enantiomer and the DNA composition. A striking result is the sensitivity of Λ-2 to the presence of AT DNA, where a 6-fold enhancement of luminescence is observed and reflects the nature of the binding for the enantiomer and the protection from solution. Thermal denaturation studies show that both complexes are found to stabilize natural DNA. Finally, cellular studies show that the complexes are internalized by cultured mammalian cells and localize in the nucleus.

One pot three component synthesis of DNA targeting phototoxic Ru(II)-p-cymene dipyrido[3,2-a:2',3'-c]phenazine analogues.

We have developed a one pot three component synthetic protocol for half-sandwich Ru(II)-p-cymene dipyrido[3,2-a:2',3'-c]phenazine analogues for selective cancer therapy under light irradiation. On average, the cytotoxicity of all the complexes is indeed doubled upon light irradiation and also exhibited significant photo and dark selectivity against cancer cells with respect to normal cells. Out of five Ru(II) complexes (RuL1-RuL5), [(η6-p-cymene)RuIICl(K2-N,N-11-nitrodipyrido[3,2-a:2',3'-c]phenazine]PF6 (RuL4) exhibited the best phototoxicity (lowest IC50 under light irradiation). Intracellular ROS generation was studied by the 2',7'-dichlorofluorescein diacetate (DCFH-DA) assay. Moreover, these complexes exhibited a strong serum albumin and DNA binding capacity. These complexes also exhibited good stability in 10% DMSO-buffer and under 1 mM GSH conditions. Overall, the remarkable photocytotoxic efficacy of new Ru(II)-p-cymene dipyrido[3,2-a:2',3'-c]phenazine analogues (RuL1-RuL5) makes them potential photochemotherapeutics as an alternative of current PDT agents.

Ratiometric Flapping Force Probe That Works in Polymer Gels.

Polymer gels have recently attracted attention for their application in flexible devices, where mechanically robust gels are required. While there are many strategies to produce tough gels by suppressing nanoscale stress concentration on specific polymer chains, it is still challenging to directly verify the toughening mechanism at the molecular level. To solve this problem, the use of the flapping molecular force probe (FLAP) is promising because it can evaluate the nanoscale forces transmitted in the polymer chain network by ratiometric analysis of a stress-dependent dual fluorescence. A flexible conformational change of FLAP enables real-time and reversible responses to the nanoscale forces at the low force threshold, which is suitable for quantifying the percentage of the stressed polymer chains before structural damage. However, the previously reported FLAP only showed a negligible response in solvated environments because undesirable spontaneous planarization occurs in the excited state, even without mechanical force. Here, we have developed a new ratiometric force probe that functions in common organogels. Replacement of the anthraceneimide units in the flapping wings with pyreneimide units largely suppresses the excited-state planarization, leading to the force probe function under wet conditions. The FLAP-doped polyurethane organogel reversibly shows a dual-fluorescence response under sub-MPa compression. Moreover, the structurally modified FLAP is also advantageous in the wide dynamic range of its fluorescence response in solvent-free elastomers, enabling clearer ratiometric fluorescence imaging of the molecular-level stress concentration during crack growth in a stretched polyurethane film.

Pyrazine and Phenazine Heterocycles: Platforms for Total Synthesis and Drug Discovery.

There are numerous pyrazine and phenazine compounds that demonstrate biological activities relevant to the treatment of disease. In this review, we discuss pyrazine and phenazine agents that have shown potential therapeutic value, including several clinically used agents. In addition, we cover some basic science related to pyrazine and phenazine heterocycles, which possess interesting reactivity profiles that have been on display in numerous cases of innovative total synthesis approaches, synthetic methodologies, drug discovery efforts, and medicinal chemistry programs. The majority of this review is focused on presenting instructive total synthesis and medicinal chemistry efforts of select pyrazine and phenazine compounds, and we believe these incredible heterocycles offer promise in medicine.

A Second Near-Infrared Ru(II) Polypyridyl Complex for Synergistic Chemo-Photothermal Therapy.

The clinical success of cisplatin ushered in a new era of the application of metallodrugs. When it comes to practice, however, drug resistance, tumor recurrence, and drug systemic toxicity make it implausible to completely heal the patients. Herein, we successfully transform an electron acceptor [1, 2, 5]thiadiazolo[3,4-g]quinoxaline into a novel second near-infrared (NIR-II) fluorophore H7. After PEGylation and chelation, HL-PEG2k exhibits a wavelength bathochromic shift, enhanced photothermal conversion efficiency (41.77%), and an antineoplastic effect against glioma. Its potential for in vivo tumor tracking and image-guided chemo-photothermal therapy is explored. High levels of uptake and high-resolution NIR-II imaging results are thereafter obtained. The hyperthermia effect could disrupt the lysosomal membranes, which in turn aggravate the mitochondria dysfunction, arrest the cell cycle in the G2 phase, and finally lead to cancer cell apoptosis. HL-PEG2k displays a superior biocompatibility and thus can be a potential theranostic platform to combat the growth and recurrence of tumors.

Characterization of the diastaphenazine/izumiphenazine C biosynthetic gene cluster from plant endophyte Streptomyces diastaticus W2.

Two phenazine compounds, diastaphenazine and izumiphenazine C, with complex structures and promising antitumour activity have been isolated from the plant endophytic actinomycete Streptomyces diastaticus W2. Their putative biosynthetic gene cluster (dap) was identified by heterologous expression and gene knockout. There are twenty genes in the dap cluster. dap14-19 related to shikimic pathway were potentially involved in the precursor chorismic acid biosynthesis, and dapBCDEFG were confirmed to be responsible for the biosynthesis of the dibenzopyrazine ring, the nuclear structure of phenazines. Two transcriptional regulatory genes dapR and dap4 played the positive regulatory roles on the phenazine biosynthetic pathway. Most notably, the dimerization of the dibenzopyrazine ring in diastaphenazine and the loading of the complex side chain in izumiphenazine C could be catalysed by the cyclase homologous gene dap5, suggesting an unusual modification strategy tailoring complex phenazine biosynthesis. Moreover, metabolite analysis of the gene deletion mutant strain S. albus::23C5Δdap2 and substrate assay of the methyltransferase Dap2 clearly revealed the biosynthetic route of the complex side chain in izumiphenazine C.

Synthesis, Crystal Structure and Bioactivity of Phenazine-1-carboxylic Acylhydrazone Derivatives.

A phenazine-1-carboxylic acid intermediate was synthesized from the reaction of aniline and 2-bromo-3-nitro-benzoic acid. It was then esterified and reacted with hydrazine hydrate to afford phenazine-1-carboxylic hydrazine. Finally, 10 new hydrazone compounds 3a-3j were obtained by the condensation reaction of phenazine-1-carboxylic acid hydrazide and the respective aldehyde-containing compound. The structures were characterized by 1H and 13C NMR spectroscopy, MS and single crystal X-ray diffraction. The antitumor activity of the target compounds in vitro (HeLa and A549) was determined by thiazolyl blue tetrazolium bromide. The results showed that compound (E)-N'-(2-hydroxy-4-(2-(piperidine-1-yl) ethoxy) benzyl) phenazine-1-carbonyl hydrazide 3d exhibited good cytotoxic activity.

Zn2+-Dependent peptide nucleic acid-based artificial ribonucleases with unprecedented efficiency and specificity.

We present Zn2+-dependent dimethyl-dipyridophenazine PNA conjugates as efficient RNA cleaving artificial enzymes. These PNAzymes display site-specific RNA cleavage with 10 minute half-lives and cleave clinically relevant RNA models.

Adenine Radical Cation Formation by a Ligand-Centered Excited State of an Intercalated Chromium Polypyridyl Complex Leads to Enhanced DNA Photo-oxidation.

Assessment of the DNA photo-oxidation and synthetic photocatalytic activity of chromium polypyridyl complexes is dominated by consideration of their long-lived metal-centered excited states. Here we report the participation of the excited states of [Cr(TMP)2dppz]3+ (1) (TMP = 3,4,7,8-tetramethyl-1,10-phenanthroline; dppz = dipyrido[3,2-a:2',3'-c]phenazine) in DNA photoreactions. The interactions of enantiomers of 1 with natural DNA or with oligodeoxynucleotides with varying AT content (0-100%) have been studied by steady state UV/visible absorption and luminescence spectroscopic methods, and the emission of 1 is found to be quenched in all systems. The time-resolved infrared (TRIR) and visible absorption spectra (TA) of 1 following excitation in the region between 350 to 400 nm reveal the presence of relatively long-lived dppz-centered states which eventually yield the emissive metal-centered state. The dppz-localized states are fully quenched when bound by GC base pairs and partially so in the presence of an AT base-pair system to generate purine radical cations. The sensitized formation of the adenine radical cation species (A•+T) is identified by assigning the TRIR spectra with help of DFT calculations. In natural DNA and oligodeoxynucleotides containing a mixture of AT and GC of base pairs, the observed time-resolved spectra are consistent with eventual photo-oxidation occurring predominantly at guanine through hole migration between base pairs. The combined targeting of purines leads to enhanced photo-oxidation of guanine. These results show that DNA photo-oxidation by the intercalated 1, which locates the dppz in contact with the target purines, is dominated by the LC centered excited state. This work has implications for future phototherapeutics and photocatalysis.

Dynamic Polymer Free Volume Monitored by Single-Molecule Spectroscopy of a Dual Fluorescent Flapping Dopant.

Single-molecule spectroscopy (SMS) of a dual fluorescent flapping molecular probe (N-FLAP) enabled real-time nanoscale monitoring of local free volume dynamics in polystyrenes. The SMS study was realized by structural improvement of a previously reported flapping molecule by nitrogen substitution, leading to increased brightness (22 times) of the probe. In a polystyrene thin film at the temperature of 5 K above the glass transition, the spectra of a single N-FLAP molecule undergo frequent jumps between short- and long-wavelength forms, the latter one indicating planarization of the molecule in the excited state. The observed spectral jumps were statistically analyzed to reveal the dynamics of the molecular environment. The analysis together with MD and QM/MM calculations show that the excited-state planarization of the flapping probe occurs only when sufficiently large polymer free volume of more than, at least, 280 Å3 is available close to the molecule, and that such free volume lasts for an average of 1.2 s.

Dermacozine N, the First Natural Linear Pentacyclic Oxazinophenazine with UV-Vis Absorption Maxima in the Near Infrared Region, along with Dermacozines O and P Isolated from the Mariana Trench Sediment Strain Dermacoccus abyssi MT 1.1T.

Three dermacozines, dermacozines N-P (1-3), were isolated from the piezotolerant Actinomycete strain Dermacoccus abyssi MT 1.1T, which was isolated from a Mariana Trench sediment in 2006. Herein, we report the elucidation of their structures using a combination of 1D/2D NMR, LC-HRESI-MSn, UV-Visible, and IR spectroscopy. Further confirmation of the structures was achieved through the analysis of data from density functional theory (DFT)-UV-Visible spectral calculations and statistical analysis such as two tailed t-test, linear regression-, and multiple linear regression analysis applied to either solely experimental or to experimental and calculated 13C-NMR chemical shift data. Dermacozine N (1) bears a novel linear pentacyclic phenoxazine framework that has never been reported as a natural product. Dermacozine O (2) is a constitutional isomer of the known dermacozine F while dermacozine P (3) is 8-benzoyl-6-carbamoylphenazine-1-carboxylic acid. Dermacozine N (1) is unique among phenoxazines due to its near infrared (NIR) absorption maxima, which would make this compound an excellent candidate for research in biosensing chemistry, photodynamic therapy (PDT), opto-electronic applications, and metabolic mapping at the cellular level. Furthermore, dermacozine N (1) possesses weak cytotoxic activity against melanoma (A2058) and hepatocellular carcinoma cells (HepG2) with IC50 values of 51 and 38 µM, respectively.

Riminophenazine Derivatives as Potential Antituberculosis Agents: Synthesis, Biological, and Electrochemical Evaluations.

A series of novel riminophenazine derivatives, having ionizable alkyl substituents at N-5 and a variety of substituents on the C-3 imino nitrogen, at C-8 and on the pendant aryl group, have been designed and synthesized. Preliminary investigations into the relationship between lipophilicity, redox potential, and antimycobacterial activity were conducted, using the in vitro activity against Mycobacterium tuberculosis H37Rv, mammalian cytotoxicity, and the redox potential of the compounds determined by cyclic voltammetry as measures. Results revealed an activity "cliff" associated with C-8 substitution (10l and 10m) that, along with defined redox activity, point to a new class of riminophenazines as potential anti-tuberculosis agents having reasonable activity (MIC99 ~1 µM).

Optimization of the clofazimine structure leads to a highly water-soluble C3-aminopyridinyl riminophenazine endowed with improved anti-Wnt and anti-cancer activity in vitro and in vivo.

Triple-negative breast cancer (TNBC) is a cancer subtype critically dependent upon excessive activation of Wnt pathway. The anti-mycobacterial drug clofazimine is an efficient inhibitor of canonical Wnt signaling in TNBC, reducing tumor cell proliferation in vitro and in animal models. These properties make clofazimine a candidate to become first targeted therapy against TNBC. In this work, we optimized the clofazimine structure to enhance its water solubility and potency as a Wnt inhibitor. After extensive structure-activity relationships investigations, the riminophenazine 5-(4-(chlorophenyl)-3-((2-(piperazin-1-yl)ethyl)imino)-N-(pyridin-3-yl)-3,5-dihydrophenazin-2-amine (MU17) was identified as the new lead compound for the riminophenazine-based targeted therapy against TNBC and Wnt-dependent cancers. Compared to clofazimine, the water-soluble MU17 displayed a 7-fold improved potency against Wnt signaling in TNBC cells resulting in on-target suppression of tumor growth in a patient-derived mouse model of TNBC. Moreover, allowing the administration of reduced yet effective dosages, MU17 displayed no adverse effects, most notably no clofazimine-related skin coloration.