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.

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.

Total Synthesis of Ritterazine B.

The first total synthesis of the cytotoxic alkaloid ritterazine B is reported. The synthesis features a unified approach to both steroid subunits, employing a titanium-mediated propargylation reaction to achieve divergence from a common precursor. Other key steps include gold-catalyzed cycloisomerizations that install both spiroketals and late stage C-H oxidation to incorporate the C7' alcohol.

Synthesis and biological evaluation of novel benzo[a]pyridazino[3,4-c]phenazine derivatives.

A convenient microwave-assisted one-pot four-component synthetic approach was developed en route to novel functionalized benzo[a]pyridazino[3,4-c]phenazine derivatives starting from 2-hydroxy-1,4-naphthoquinone, aromatic aldehydes, methyl hydrazine and o-phenylenediamine. Nine new derivatives were successfully synthesized and subsequently evaluated in terms of their biological profiles. The results revealed good cytotoxic activities of compounds 6a, 6h against KB, HepG2, Lu1 and MCF7 human cancer cell lines. Besides that, compound 6d exhibited promising antimicrobial activities toward Staphylococcuc aureus and Bacillus subtilis bacterial strains with IC50 < 6 µM.

Design, synthesis and biological evaluations of diverse Michael acceptor-based phenazine hybrid molecules as TrxR1 inhibitors.

A series of novel phenazine derivatives (1~27) containing the Michael acceptor scaffolds were designed and synthesized in this study. Some compounds exhibited selective cytotoxicity against Bel-7402 cancer cell line in vitro, in which compound 26 were found to have the best antiproliferative activity. Meanwhile, compound 26 showed no obvious cell toxicity against human normal liver epithelial L02 cells, which means this compound possessed a better safety potential. In the following research, compound 26 was verified to inhibit TrxR1 enzyme activity, ultimately resulting in cellular molecular mechanism events of apoptosis including growth of intracellular ROS level, depletion of reduced Trx1, liberation of ASK1 and up-regulation of p38, respectively. Together, all these evidences implicated that compound 26 acted as the TrxR1 inhibitor against Bel-7402 cells, and could activate apoptosis through the ROS-Trx-ASK1-p38 pathway.

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.

Hierarchically Porous Biocatalytic MOF Microreactor as a Versatile Platform towards Enhanced Multienzyme and Cofactor-Dependent Biocatalysis.

Metal-organic frameworks (MOFs) have recently emerged as excellent hosting matrices for enzyme immobilization, offering superior physical and chemical protection for biocatalytic reactions. However, for multienzyme and cofactor-dependent biocatalysis, the subtle orchestration of enzymes and cofactors is largely disrupted upon immobilizing in the rigid crystalline MOF network, which leads to a much reduced biocatalytic efficiency. Herein, we constructed hierarchically porous MOFs by controlled structural etching to enhance multienzyme and cofactor-dependent enzyme biocatalysis. The expanded size of the pores can provide sufficient space for accommodated enzymes to reorientate and spread within MOFs in their lower surface energy state as well as to decrease the inherent barriers to accelerate the diffusion rate of reactants and intermediates. Moreover, the developed hierarchically porous MOFs demonstrated outstanding tolerance to inhospitable surroundings and recyclability.

Tailoring an HSO4- anion hybrid receptor based on a phenazine derivative.

A catechol-functionalized phenazine imidazole (PD) was tailored with 2,3-diaminophenazine and 3,4-dihydroxy benzaldehyde, and it served as a hybrid acceptor for capturing HSO4- anions. The selectivity and sensitivity of the PD receptor for anion sensing were studied. It was found that the PD receptor could not only display a preferable sensitivity to HSO4- ions with a "turn-off" fluorescence response, but also have a strong anti-interference ability toward other common anions, especially basic anions such as CH3COO-, HPO42-, and H2PO4-. The anion recognition mechanism of PD towards HSO4- is based on multiple hydrogen bond interactions. Finally, the strips for anion detection were prepared, which were verified to be a convenient and high-efficiency test kit for detecting HSO4- ions with the naked eye.

A NIR fluorescent probe based on phenazine with a large Stokes shift for the detection and imaging of endogenous H2O2 in RAW 264.7 cells.

Hydrogen peroxide (H2O2), one of the reactive oxygen species (ROS), plays vital roles in diverse physiological processes. Thus, herein, to improve the signal-to-noise ratio, a new near-infrared region (NIR) fluorophore (PCN) based on reduced phenazine was developed. PCN was further designed as a "turn on" fluorescent probe (PCN-BP) for the detection of H2O2 by introducing p-boratebenzyl. After H2O2 was added, the p-boratebenzyl group in PCN-BP was oxidized to p-hydroxy benzyl; it then self-departed, forming PCN, which displayed 24-fold NIR emission at 680 nm with a large Stokes shift (more than 200 nm). This probe presented an excellent linear relation with the concentration of H2O2 and good selectivity to various ions, ROS and biothiols; thus, it can be utilized as a colorimetric and fluorescence turn-on probe. More importantly, the probe was also employed for the exogenous and endogenous imaging of H2O2 in RAW 264.7 cells.

A Phenazine based colorimetric and fluorescent chemosensor for sequential detection of Ag+ and I- in aqueous media.

A new colorimetric and fluorescent probe MNTPZ based on 1H-imidazo[4,5-b]phenazine derivative has been designed and synthesized for successive detection of Ag+ and I- . The probe MNTPZ shows selective colorimetric response by a change in color from yellow to orange and "turn-off" fluorometric response upon binding with Ag+ in DMSO: Water (pH = 7, 1:1, v/v) over other cations. The binding mode of probe MNTPZ to Ag+ was studied by Job's plot, 1 H NMR studies, FT-IR spectroscopy and DFT calculations. Moreover, the situ generated probe MNTPZ+ Ag+ complex acted as an efficient fluorometric "turn-on" probe for I- via Ag+ displacement approach. The detection limit of probe MNTPZ for Ag+ and the resultant complex probe MNTPZ+ Ag+ for I- were determined to be 1.36 µmol/L and 1.03 µmol/L respectively. Notably, the developed probe was successfully used for quantitative determination of I- in real samples with satisfactory results.

Dual emission carbon dots as enzyme mimics and fluorescent probes for the determination of o-phenylenediamine and hydrogen peroxide.

A selective and sensitive fluorescent technique is proposed to determine o-phenylenediamine (OPD) and hydrogen peroxide (H2O2). This is carried out by utilizing enzyme mimics carbon dots (N/Cl-CDs) and fluorescent probe carbon dots (N/Zn-CDs). The synthesized N/Cl-CDs and N/Zn-CDs were characterized by transmission electron microscopy (TEM), X-ray powder diffraction (XRD), Fourier-transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), and Ultraviolet-visible spectrophotometry techniques. The particle size of synthesized carbon dots was found to be 4.1 ± 1.09 nm and 3.3 ± 1.82 nm for N/Cl-CDs and N/Zn-CDs, respectively. The N/Cl-CDs showed a noticeable intrinsic peroxidase-like activity, which could catalyze the oxidization of OPD by H2O2 to form the yellow colored product 2,3-diaminophenazine (DAP). The N/Zn-CD fluorescence was quenched directly by DAP through the inner filter effect (IFE). As a result, a novel double carbon dot system as enzyme mimics and fluorescent investigations to recognize OPD and H2O2 was obtained. The N/Cl-CDs were synthesized by deep eutectic solvent (DES) source and chamber electric furnace. N/Zn-CDs were also synthesized with the quantum yield of 27.52% through the tubular furnace technique using ethylenediamine tetra acetic acid disodium zinc salt and ascorbic acid as precursors. The detection limits were found to be 0.58 µM and 0.27 µM for OPD and H2O2, respectively. The nanoprobe was applied to real sample analysis, which showed excellent recovery in the range of 95.8-103.5% and 98.6-107.3% for OPD and H2O2, respectively. The dual emission carbon dots as enzyme mimic and fluorescent probe sensing system opens a new platform for determination of H2O2 and OPD in real samples. Graphical abstract Schematic illustration of the preparation of double carbon dots and determination process of o-phenylenediamine (OPD) and hydrogen peroxide (H2O2).

Phenazine Antibiotic-Inspired Discovery of Bacterial Biofilm-Eradicating Agents.

Bacterial biofilms are surface-attached communities of slow-growing and non-replicating persister cells that demonstrate high levels of antibiotic tolerance. Biofilms occur in nearly 80 % of infections and present unique challenges to our current arsenal of antibiotic therapies, all of which were initially discovered for their abilities to target rapidly dividing, free-floating planktonic bacteria. Bacterial biofilms are credited as the underlying cause of chronic and recurring bacterial infections. Innovative approaches are required to identify new small molecules that operate through bacterial growth-independent mechanisms to effectively eradicate biofilms. One source of inspiration comes from within the lungs of young cystic fibrosis (CF) patients, who often endure persistent Staphylococcus aureus infections. As these CF patients age, Pseudomonas aeruginosa co-infects the lungs and utilizes phenazine antibiotics to eradicate the established S. aureus infection. Our group has taken a special interest in this microbial competition strategy and we are investigating the potential of phenazine antibiotic-inspired compounds and synthetic analogues thereof to eradicate persistent bacterial biofilms. To discover new biofilm-eradicating agents, we have established an interdisciplinary research program involving synthetic medicinal chemistry, microbiology and molecular biology. From these efforts, we have identified a series of halogenated phenazines (HPs) that potently eradicate bacterial biofilms, and future work aims to translate these preliminary findings into ground-breaking clinical advances for the treatment of persistent biofilm infections.

Incorporating Morpholine and Oxetane into Benzimidazolequinone Antitumor Agents: The Discovery of 1,4,6,9-Tetramethoxyphenazine from Hydrogen Peroxide and Hydroiodic Acid-Mediated Oxidative Cyclizations.

The reactivity of hydrogen peroxide and catalytic hydroiodic acid toward 3,6-dimethoxy-2-(cycloamino)anilines is tunable to give ring-fused benzimidazoles or 1,4,6,9-tetramethoxyphenazine in high yield. Mechanisms via a detected nitroso-intermediate are proposed for oxidative cyclization and the unexpected intermolecular displacement of the oxazine. An aqueous solution of molecular iodine is capable of the same transformations. Oxidative demethylation gave targeted benzimidazolequinones, including without cleavage of the incorporated oxetane.

Identification of phenazine analogue as a novel scaffold for thioredoxin reductase I inhibitors against Hep G2 cancer cell lines.

Even though phenazines have been extensively reported as anticancer molecules, the molecular target of these compounds is severely lagging behind. Our study consequently focuses on the anticancer target of a phenazine analogue (CPUL1) for its potently antitumor activities in initial stage. Along with redox status courses of Hep G2 cells, thioredoxin reductase I (TrxR1) was speculated as anticancer target of CPUL1. By virtue of zymologic, immunological and molecular biological experiments, we demonstrated that TrxR1 could be the anticancer target of CPUL1. The knowledge on phenazine targeting to TrxR1 have not been reported previously. Thus, it can provide valuable information for further development of the TrxR1 inhibitors.

Synthesis and antifungal evaluation of PCA amide analogues.

To improve the physical and chemical properties of phenazine-1-carboxylic acid (PCA) and find higher antifungal compounds, a series of PCA amide analogues were designed and synthesized and their structures were confirmed by 1H NMR, HRMS, and X-ray. Most compounds showed some antifungal activities in vitro. Particularly, compound 3d exhibited inhibition effect against Pyriculariaoryzac Cavgra with EC50 value of 28.7 µM and compound 3q exhibited effect against Rhizoctonia solani with EC50 value of 24.5 µM, more potently active than that of the positive control PCA with its EC50 values of 37.3 µM (Pyriculariaoryzac Cavgra) and 33.2 µM (Rhizoctonia solani), respectively.

Vibration-Induced-Emission (VIE) for imaging amyloid ß fibrils.

This paper discusses the use of N,N'-disubstituted-dihydrodibenzo[a,c]phenazines with typical Vibration-Induced-Emission (VIE) properties for imaging amyloid ß (Aß) fibrils, which are a signature of neurological disorders such as Alzheimer's disease. A water-soluble VIEgen with a red fluorescence emission shows a pronounced, blue-shifted emission with Aß peptide monomers and fibrils. The enhancement in blue fluorescence can be ascribed to the restriction of the molecular vibration by selectively binding to Aß. We determine an increasing blue-to-red emission ratio of the VIEgen with both the concentration and fibrogenesis time of Aß, thereby enabling a ratiometric detection of Aß in its different morphological forms. Importantly, the VIEgen was proven to be suitable for the fluorescence imaging of small Aß plaques in the hippocampus of a transgenic mouse brain (five months old), with the blue and red emissions well overlapped on the Aß. This research offers a new rationale to design molecular VIE probes for biological applications.

Synthesis and bioactivities of Phenazine-1-carboxylic acid derivatives based on the modification of PCA carboxyl group.

Phenazine-1-carboxylic acid (PCA) as a natural product widely exists in microbial metabolites of Pseudomonads and Streptomycetes and has been registered for the fungicide against rice sheath blight in China. To find higher fungicidal activities compounds and study the effects on fungicidal activities after changing the carboxyl group of PCA, we synthesized a series of PCA derivatives by modifying the carboxyl group of PCA and their structures were confirmed by 1H NMR and HRMS. Most compounds exhibited significant fungicidal activities in vitro. In particular, compound 6 exhibited inhibition effect against Rhizoctonia solani with EC50 values of 4.35mg/L and compound 3b exhibited effect against Fusarium graminearum with EC50 values of 8.30mg/L, compared to the positive control PCA with its EC50 values of 7.88mg/L (Rhizoctonia solani) and 127.28mg/L (Fusarium graminearum), respectively. The results indicated that the carboxyl group of PCA could be modified to be amide group, acylhydrazine group, ester group, methyl, hydroxymethyl, chloromethyl and ether group etc. And appropriate modifications on carboxyl group of PCA were useful to extend the fungicidal scope.

Total synthesis and antileukemic evaluations of the phenazine 5,10-dioxide natural products iodinin, myxin and their derivatives.

A new efficient total synthesis of the phenazine 5,10-dioxide natural products iodinin and myxin and new compounds derived from them was achieved in few steps, a key-step being 1,6-dihydroxyphenazine di-N-oxidation. Analogues prepared from iodinin, including myxin and 2-ethoxy-2-oxoethoxy derivatives, had fully retained cytotoxic effect against human cancer cells (MOLM-13 leukemia) at atmospheric and low oxygen level. Moreover, iodinin was for the first time shown to be hypoxia selective. The structure-activity relationship for leukemia cell death induction revealed that the level of N-oxide functionality was essential for cytotoxicity. It also revealed that only one of the two phenolic functions is required for activity, allowing the other one to be modified without loss of potency.

Synthesis and bioactivities of amino acid ester conjugates of phenazine-1-carboxylic acid.

Phenazine-1-carboxylic acid (PCA) is a natural product that has been characterized by special chemical structures, interesting bioactivities and has been registered for fungicide against rice sheath blight in China. Phloem mobility is of great significance to long-distance transport of systemic pesticides in plants. In order to improve the phloem mobility and bioactivities of PCA, seventeen PCA-amino acid ester conjugates were designed and synthesized by conjugating PCA with different amino-acid esters. The conjugates were evaluated for their fungicidal activities against Rhizoctonia solani Kuhn and their phloem mobility was determined by HPLC. Results showed that conjugates a, b, c, d, e, l, m and p (EC50 values between 5.35 and 18.85µg/mL) were more active than PCA (25.66µg/mL). In particular, conjugates l and m exhibited the best fungicidal activities against Rhizoctonia solani Kuhn (EC50 values of them were 6.47µg/mL and 5.35µg/mL respectively). All these conjugates didn't have phloem mobility.

Metal catalyst free cyclization of 3-alkynyl substituted 2-(indol-3-yl)quinoxalines in TFA alone: a new synthesis of indolophenazines.

TFA alone was found to be remarkably effective for the intramolecular hydroarylation (IMHA) of alkynes when employed as a solvent in the cyclization of 3-alkynyl substituted 2-(indol-3-yl)quinoxalines. This simple and metal free cyclization method afforded a range of indolophenazines as new and potential cytotoxic agents. The use of excess TFA was found to be crucial for the success of this reaction.