recent developments in the isolation biological function biosynthesis and synthesis of phenazine natural products

Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc Review article Recent developments in the isolation, biological function, biosynthesis, and synthesis of phenazine natural products Nikolaus Guttenberger a,b, Wulf Blankenfeldt c,d, Rolf Breinbauer a,⇑ a Institute of Organic Chemistry, Graz University of Technology, Stremayrgasse 9, 8010 Graz, Austria Institute of Chemistry-Analytical Chemistry, University of Graz, Universitaetsplatz 1, 8010 Graz, Austria c Structure and Function of Proteins, Helmholtz Centre for Infection Research, Inhoffenstr 7, 38124 Braunschweig, Germany d Institute for Biochemistry, Biotechnology and Bioinformatics, Technische Universität Braunschweig, Spielmannstr 7, 38106 Braunschweig, Germany b a r t i c l e i n f o Article history: Received 11 November 2016 Revised 29 December 2016 Accepted January 2017 Available online xxxx Keywords: Antibiotics Anticancer Biofilm Biosynthesis Natural product Phenazine a b s t r a c t Phenazines are natural products which are produced by bacteria or by archaeal Methanosarcina species The tricyclic ring system enables redox processes, which producing organisms use for oxidation of NADH or for the generation of reactive oxygen species (ROS), giving them advantages over other microorganisms In this review we summarize the progress in the field since 2005 regarding the isolation of new phenazine natural products, new insights in their biological function, and particularly the now almost completely understood biosynthesis The review is complemented by a description of new synthetic methods and total syntheses of phenazines Ó 2017 The Authors Published by Elsevier Ltd This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Contents Introduction Biological activity 2.1 Modes of action 2.2 Phenazines and glutathione (GSH) 2.3 Anticancer activity 2.4 Phenazines as antibiotics and their role to host defence 2.5 Phenazines and biofilms 2.6 Isolation of new phenazines 2.7 Synthetic phenazines with exceptional biological activity Biosynthesis 3.1 Early studies 3.2 Anthranilate synthase genes 3.3 Current understanding Synthesis 4.1 Classic methods 4.2 Cu- and Pd-catalyzed coupling reactions 4.3 Transition metal-catalyzed C-H functionalization 4.4 One-pot procedures and multicomponent reactions (MCRs) 4.5 Miscellaneous 4.6 Summary of modern strategies 4.7 Biomimetic synthesis of phenazine-1,6-dicarboxylic acid (PDC) 4.8 Total syntheses of streptophenazine A (51) ⇑ Corresponding author E-mail address: breinbauer@tugraz.at (R Breinbauer) http://dx.doi.org/10.1016/j.bmc.2017.01.002 0968-0896/Ó 2017 The Authors Published by Elsevier Ltd This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Please cite this article in press as: Guttenberger N., et al Bioorg Med Chem (2017), http://dx.doi.org/10.1016/j.bmc.2017.01.002 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 N Guttenberger et al / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx 4.9 Total syntheses Conclusion Acknowledgments References and notes of dermacozines A (61), B (64) and C (65) In this review we aim to describe the current state of phenazine natural product chemistry with a special focus on the literature since 2005, when the last seminal reviews have been published by Nielsen1 and Beifuss.18 Introduction Phenazines are a large class of redox-active secondary metabolites produced by many Gram-positive (e.g Streptomyces) and Gram-negative bacteria (e.g Pseudomonas), or by archaeal Methanosarcina species (Fig 1).1 The core structure of phenazines is a pyrazine ring (1,4-diazabenzene) exhibiting two annulated benzenes Their early discovery in the mid-19th century has been facilitated by the fact that phenazines are intensively colored compounds.2 In 1859 Fordos described the extraction of a blue pigment, which is responsible for the coloration of the ‘‘blue pus”, observed in patients suffering from severe purulent wounds, and named the blue pigment ‘‘pyocyanine” (nowadays more common as pyocyanin (PYO)) from the Greek words for ‘‘pus” and ”blue”.3,4 Since then >180 phenazine natural products have been described in the literature.5 It has been shown that some phenazines exhibit antibiotic, antifungal, insecticidal, antitumor, cancer chemopreventive, antiplasmodial, antimalarial, and antiparasitic activities Phenazines are produced at late growth stages at high cell densities and it has been demonstrated that phenazine-producing organisms exhibit a longer lifespan in the natural environment than their non phenazine-producing counterparts.1 In addition, a mouse model with PYO-deficient strains of P aeruginosa showed that PYO is important for their hosts in lung infections.6 Several modes of actions of phenazines have been identified, which include the reduction of molecular oxygen to reactive oxygen species (ROS),7 the facilitation of energy generation,8–10 involvement in iron homeostasis via Fe(III) reduction,11,12 participation as signal molecules via the activation of the Fe-containing transcription factor SoxR,13–15 DNA p–p interaction and intercalation,16 and biofilm morphogenesis17 through influencing the intracellular redox state O O N N N Biological activity This section highlights some newly isolated as well as synthetic phenazines, exhibiting exceptional structures or showing promising anticancer, antibiotic or biofilm-eradicating activities Some important modes of action are discussed and a closer look is taken at the connection between glutathione (GSH) and phenazines, as new findings indicate a possible mode of action in intracellular GSH level depletion 2.1 Modes of action Although a detailed description of the modes of action of phenazines would be far beyond this review, some aspects that have already reviewed elsewhere1,19–22 are indispensable to impart a better understanding and will be therefore recapitulated shortly Phenazines are able to both donate and to accept electrons, dependent on its relative redox potential to other electron transfer molecules.19 Reactive oxygen species (ROS) formation is a major mode of action of phenazines and can be beneficial to the host, e.g via an inhibition of pathogenic organisms or detrimental by an interference with normal cell functions.19 PYO production by P aeruginosa has shown to play a major role in lung infection via ROS generation.6,19,23–25 PYO is able to supply toxic superoxide (OÅÀ ) and hydrogen peroxide (H2O2) via cellular redox cycling of N endophenazine A (S cinnamomensis) CONH R N N N HO O H N H H OH CO2 H N N pyocyanin (PYO) (P aer uginosa) HO SCH N O N CO2 H OH O CH3 izuminoside B (St rept omy ces sp.) N N phenazinolin A (R=OH) phenazinolin B (R=H) (St rept omy ces sp.) N H CONH2 CO 2Me HO dermacozine A (Der macoccus aby ssi) N O N CO 2Me N methanophenazine (M sarcina Gö1) 00 00 00 00 streptophenazine A (St rept omy ces sp HB202) Figure Selected naturally-occurring phenazine derivatives Please cite this article in press as: Guttenberger N., et al Bioorg Med Chem (2017), http://dx.doi.org/10.1016/j.bmc.2017.01.002 N Guttenberger et al / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx molecular oxygen and various reducing agents such as NADH and NADPH.22,26 Muller could demonstrate that upon treatment of endothelial cells with PYO, hydrogen peroxide was formed accompanied with depletion of the glutathione (GSH) level.27 In addition, intracellular NADPH levels increased O’Malley et al found that PYO depletes GSH level in airway epithelial cells and overexpression of catalase could not fully prevent the decline in cellular GSH.28 Counterintuitively, GSH can have a detrimental effect on lung cells as it can reduce PYO with concomitant formation of 22 OÅÀ Hydroxyl radical formation from the interaction of pro2 tease-cleaved Fe-transferrin with redox cycling of pyocyanin has been associated with endothelial cell injury.29 Lactoperoxidase and related dual oxidases (Duox) produce mild oxidants harmful for several airway pathogens like Staphylococcus aureus, Burkholderia cepacia, and Pseudomonas aeruginosa.30 The expression of PYO leads to oxidative stress via a consumption of NADPH and superoxide formation leading to a competitive inhibition of epithelial Duox activity.30 Bacterial nitric oxide synthases (bNOS) catalyze the formation of NO from arginine and NO has shown to increase the resistance of bacteria to various antibiotics via chemical modification of toxic compounds and mitigation of oxidative stress.31 PYO was found to inhibit the growth of the B subtilis nos-deletion strain significantly more compared to the wild type.31 As a protection from oxidative stress, P aeruginosa contains cytoplasmic superoxide dismutases (SODs).22,32 Results obtained in the Newman lab9,10 have shown that the intracellular redox state in P aeruginosa can be influenced by PYO in the absence of other electron acceptors via reoxidation of NADH to NAD+ and phenazines enable survival in P aeruginosa under anaerobic conditions via electron-shuttling Another role performed by phenazines is the ability to reductively dissolve ferrihydrite and hematite in the pH range of 5–8 thus making iron more bioavailable as shown for electrochemically-reduced PYO, phenazine-1-carboxylic acid (PCA), phenazine-1-carboxamide and 1-hydroxyphenazine by Wang and Newman.12 Oxygen was shown to inhibit the reduction, as O2 can compete with Fe(III) as the final oxidant The authors speculated that the different phenazines may accomplish different functions dependent on oxygen and iron availability Further work in the group of Newman13,14 has indicated that phenazines can act as signalling molecules via the activation of the Fe-containing transcription factor SoxR PYO was found to trigger the upregulation of transport genes and downregulates genes involved in ferric iron acquisition PYO was lately linked to an aberrant entrapment and killing by neutrophil extracellular trap (NET) release leading to a damage of host tissues, found in cystic fibrosis (CF).33 The role of PYO in P aeruginosa infection has been reviewed,23,25 and a more general discussion of phenazines in Pseudomonas spp was recently given.34 2.2 Phenazines and glutathione (GSH) GSH, an important antioxidant ubiquitous in mammalian cells, is important for detoxification (e.g of carcinogens) and can protect against DNA damage that is caused by ROS.35 In addition, GSH is a regulator of the thiol-redox status and plays an important role in many disease states and in critical cell signalling pathways.35 GSH homeostasis is of utmost importance as for example a low intracellular GSH level decreases the antioxidant capacity, whereas higher GSH levels are observed in cancer cells and lead to increased chemo-resistance.35 PYO has been known to reduce the intracellular GSH level with accompanied formation of mixed disulfides.27,28 Exogenous GSH is protective against PYO toxicity,36 and it has been hypothesized that this is due to the formation of a cell-impermeant GSH-PYO conjugate.37 Ray et al could show that a chronic, low-level exposure to PCA, PYO and 1-hydroxyphenazine increased protein misfolding and neurotoxic phenotypes in the model organism C elegans.38 It was demonstrated that these phenotypes are not directly linked to ROS production, as the addition of the anti-oxidant N-acetyl cysteine did not prevent the formation of the phenotypes Recently, two new phenazines exhibiting a thiol ether linkage between PCA and pantetheine were identified by Heine et al.39 The new phenazines, namely panphenazine A and B, were discovered via metabolic profiling of concentrated culture extracts of the rare actinomycete Kitasatospora sp HKI 714.39 The biosynthesis gene cluster40 revealed no genes that could be responsible for CAS bond formation and upon irradiation of PCA in the presence of pantetheine, a mixture of panphenazine A and B was formed most likely via a radical mechanism (Scheme 1) These findings suggest a possible mode of action for the intracellular GSH level depletion, caused by PYO.27,28 In addition, the authors could demonstrate that phenazines readily form S-conjugates with different proteins exhibiting cysteine side-chains, which could explain phenazineinduced protein misfolding in C elegans.38 Further research has to be undertaken in order to clarify the role of phenazines in protein misfolding processes 2.3 Anticancer activity Anticancer activities of phenazines have been recently summarized and critically evaluated by Cimmino et al.41,42 It was found that an implementation of phenazines as anticancer agents is problematic because of nonselective DNA intercalation leading to general toxicity.41 However, selectivity can be enhanced using derivatives to overcome the ‘‘flatland structure” of phenazines.41 Potent structures have been found e.g amongst dimeric or those bearing a pendant protonatable group.41 The so called ‘‘prodrug approach” using phenazine-5,10-dioxides rather than phenazines CO2 H N CO2 H N HS H N H N O N PCA OH N H N S O OH OH H N OH O panphenazine A O hv (370 nm) or AIBN, Et N + OH HO O CO2 H N H N H N S N O panphenazine B Scheme Formation of panphenazines via a non-enzymatic reaction.39 Please cite this article in press as: Guttenberger N., et al Bioorg Med Chem (2017), http://dx.doi.org/10.1016/j.bmc.2017.01.002 N Guttenberger et al / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx Table Several newly discovered phenazines exhibiting extraordinary structures or activities Structure HO O R H N N H N HO H N H O H H N O H HO 2C N N N Ref Phenazinolin A-C (1–3) exhibited in vitro cytotoxicity against P388, GLC, H460, and XWLC human cancer cell lines with IC50 values between 14–40 lM and antibiotic activity against Bacillus subtilis, Staphylococcus aureus, Aspergillus niger, and Botrytis cinerea MIC values were between 12–27 lM Streptomyces sp 64 Neuronal cell protective effect on glutamate-induced mouse hippocampal HT-22 cell damage Streptomyces sp 82 Cytotoxic to murine P388 leukemia cells at 50 lM (only tested at this concentration)-proliferation inhibition by 78% Bacillus sp 57 IC50 = 489 nM (A549 lung cancer cell line) P putida 69 Streptomyces sp 58 phenazinolin C ( 3) N OH N H Species H H O phenazinolin A ( 1, R= OH) phenazinolin B (2, R= H) HO HO 2C HO N N Biological effect H N OH N N HO N OH H OH phenazinolin E ( 5) phenazinolin D ( 4) R1 OMe O N N N H O R2 pontemazine A R1 =R = NH2 , pontemazine B R 1= OH, R2 = NH2 , N N O O CO2 H N N CH3 5-methyl phenazine1-carboxylic acid betaine O O N N 10 IC50 = 459 nM (MDA MB-231 breast cancer cell line) Growth-free inhibition zones (diameter) ranged from 11 to 23 mm towards pathogenic bacteria tested at this concentration)- proliferation inhibition by 78% IC50 of TNF-a-induced NFjB activity (10: 4.1 lM, 11: 24.2 lM) Br N Br N 11 IC50 of LPS-induced nitric oxide production (10: >48.6 lM, 11: 15.1 lM) IC50 of PGE2 production (10: 7.5 lM, 11: 0.89 lM) Please cite this article in press as: Guttenberger N., et al Bioorg Med Chem (2017), http://dx.doi.org/10.1016/j.bmc.2017.01.002 N Guttenberger et al / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx Table (continued) Structure OH OH HO OH O O HO CH N SCH3 N N CO 2H N CO 2H OH O O Biological effect Species Ref Compound 13 (10 lM) displayed a 22% decrease in cell viability (AGS cell line) in the presence of TRAIL (100 ng mLÀ1), compared to the cell viability where no TRAIL is present Streptomyces sp 59– Kitasatospora sp 91 62 CH izuminoside B, 13 izuminoside A, 12 OH HO CH3 HO O HO N N O O H N CO2 H N N N CO 2H OH O H OH izumiphenazine A, 15 izuminoside C, 14 CO2H N O N CO2 H S H CO O O O H N O N H N CH3 aotaphenazine, 17 yorophenazine, 16 Compound 14 (60 lM): 19% decrease Compound 15 (40 lM): 19% decrease Compound 17 (12.5 lM): 35% decrease Izuminoside A (12) and yorophenazine (16) showed no decrease in cell viability in the presence of TRAIL OH CONH N HO OH OH O HO HO Inhibition zone (mm) OH O O O N O O O O N N O HO N HO O 18 N compound Bacillus subtilis 18 Escherichia coli 19 12 20 14 21 22 15 23 20 19 OH HO OH CO 2H N O HO HO O O HO N O O O CO2 H N N N 21 22 OH N 23 Please cite this article in press as: Guttenberger N., et al Bioorg Med Chem (2017), http://dx.doi.org/10.1016/j.bmc.2017.01.002 N Guttenberger et al / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx represents a more promising strategy against cancer.41,43–45 A review on fused aryl phenazine derivatives summarizes their anticancer activities.46 PCA was identified to exhibit both protective and anticancer activities against human skin melanoma cell line SK-MEL-2.47 Further efforts in testing both synthetic48–53 and naturally-occuring54–69 phenazines as anticancer agents have been made recently O HO O O OH NH2 NH O OH N N N N endophenazine E endophenazine A1 2.4 Phenazines as antibiotics and their role to host defence OH Research into new antibiotics is particularly urgent as antibiotic drug-resistance has emerged widely and most antibiotics rely only on structures identified during the 1940s to 1960s, known as the ‘‘golden era” of antibiotic discovery.70 Most antibiotics during this time were identified via the screening of soil-derived actinomycetes.70 Phenazines could present a promising scaffold for the development of a new class of antibiotics In a seminal publication, Moura-Alves et al could show that the ligand-dependent transcription factor aryl hydrocarbon receptor (AhR) plays a crucial role to host defence against both acute and chronical bacterial infections as AhR-deficient mice were more vulnerable to both P aeruginosa and M tuberculosis.71 Upon phenazine binding to AhR, several processes like the transcription of canonical detoxifying genes or a regulation of cytokine and chemokine production are initiated This work has now established the sought–after mechanistic connection for phenazines in host defence interaction O OH O OH N N O N N endophenazine F endophenazine G Figure Endophenazine E,86 A1,40 F40 and G.40 CH O O Br N OH Br N N 25 R Br ~2-fold increase in potency N 33 H 26: R= iPr 27: R= Ph N O O Br 4-fold decrease in potency N 2.5 Phenazines and biofilms Biofilm formation represents a major burden in antimicrobial therapy The term ‘‘biofilm” has been defined as an ‘‘aggregate of microorganisms in which cells that are frequently embedded within a self-produced matrix of extracellular polymeric substance (EPS) adhere to each other and/or to a surface”.72 Extracellular DNA (eDNA) stabilizes bacterial biofilms and protects against physical and chemical stress, thereby being a promising target against bacterial infections.73 A biofilm offers protection against antimicrobials by reducing their amount to a sublethal concentration, which in turn can lead to resistance Resistance can also emerge via an alleviated horizontal gene transfer.74 Slow growth states are also expected to account for the failure of antibiotic treatment.74 PYO was found to play a central role in P aeruginosa biofilm formation, as PYO promotes the release of eDNA.75 In addition, PYO influences the binding of eDNA to P aeruginosa PA14 cells via intercalation.76,77 Sakhtah et al recently discovered that 5-methylphenazine-1-carboxylate (5-Me-PCA) is transported by the efflux pump MexGHI-OpmD that controls gene expression and biofilm development in P aeruginosa.78 O’May et al showed that iron supply in P aeruginosa biofilms is important and that iron chelators can facilitate to prevent biofilm formation.79 The importance of phenazines on biofilms has been outlined in a review.34,80 HO HO O HO O O OH O N N N N Figure Unprecedented glyceride phenazines showed O-acyl isomerism.81 Br same activity cyclohexyl O O Br N N 32 OH N OH N Br N Br N Br 24: MIC= 1.56 µM inactive H H 28 inactive OMe OH N Br N H N 29 NH N 31 Br N Br Br inactive inactive N 30 Br inactive Figure SAR of phenazine 24 against S aureus.92 2.6 Isolation of new phenazines A plethora of novel phenazines have been discovered in the last couple of years and some noteworthy examples are outlined in this section Important examples of newly isolated phenazines exhibiting exceptional structures or biological activities are listed in Table Dimeric phenazines: Dimeric phenazines are rare and some isolates exhibit extraordinary structures Worthwhile mentioning are phenazinolins, dimeric phenazines exhibiting uncommon azabicyclo[3.3.1]nonadienol (1–3) and oxabicyclo[3.3.1]nonadienol (4 and 5) ring systems These phenazines have been isolated from Streptomyces sp and showed anticancer and antibiotic activity.64 Diastaphenazine is a further example of dimeric phenazines and was Please cite this article in press as: Guttenberger N., et al Bioorg Med Chem (2017), http://dx.doi.org/10.1016/j.bmc.2017.01.002 N Guttenberger et al / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx weakly cytotoxic against five human tumor cell lines and showed weak antimicrobial activity against S aureus (MIC = 64 lg mLÀ1).63 Uncommon phenazines: Wu et al isolated unprecedented glyceride O-acyl isomeric phenazines and HPLC analysis revealed its spontaneous interconversion (Fig 2).81 Another example of uncommon phenazines present pontemazines A (6) and B (7), exhibiting an unusual methyl amine linkage and showed protective effect to HT-22 neuronal cells.82 The uncommon phenazine exhibits anticancer activity.57 Uncommon 5-methyl phenazine-1carboxylic acid betaine (MPCAB, 9) isolated by Kennedy et al from P putida displayed antimicrobial as well as anticancer activities.69 Terpenoid phenazines: The terpenoid phenazines 10 and 11 have been isolated by Kondratyuk et al from Streptomyces sp and are potential cancer chemopreventive83 agents.58 Further examples of recently isolated terpenoid phenazines are phenaziterpenes A and B84 isolated from Streptomyces niveus A genome mining/isolation approach by Heine et al of Kitasatospora sp HKI 714 led to the discovery of formerly unknown terpenoid endophenazine derivatives, namely endophenazine A1, F and G (Fig 3) showing antibacterial activity.40 The total synthesis of endophenazine G has been disclosed recently.85 Via heterologous expression of the biosynthetic gene cluster for endophenazines from Streptomyces anulatus 9663 in engineered host strains, derived from Streptomyces coelicolor M145, C–isoprenylated endophenazine E was produced Endophenazine E is a conjugate between endophenazine A, and (Fig 3).86 Six new phenazines, named chromophenazines, exhibiting a prenyl group attached on one of the two nitrogens were tested for antimicrobial activity, but only one chromophenazine displayed moderate activity against B subtilis, E coli, and the fungus M miehei.87 Geranylphenazinediol, isolated from Streptomyces sp showed some activity against the enzyme acetylcholinesterase.88 Glycosylated phenazines: Much progress has been made towards the isolation of bioactive phenazines by the group of Ishibashi in their screening program89 for new natural products from actinomycetes (see compounds 12–17) Several phenazines displayed activity in overcoming tumor necrosis factor-related apoptosis inducing ligand (TRAIL) resistance in AGS cells.59–62,68 Some of the new phenazines, namely izuminosides A–C (12–14), belong to the rare class90 of glycosylated phenazines Further examples of glycosylated phenazines have been published lately by Wu et al.,81,91 who reported the isolation of several glycosylated endophenasides (see compounds 18–22) Six new glycosylated phenazines, named solphenazines AÀF were isolated by Rusman et al from Streptomyces sp.65 These phenazines exhibit one or two rhamnose moieties and three compounds showed some cytotoxicity against HCT-116 cancer cells No antibacterial activity was observed, which is uncommon for carbohydrate-containing phenazines.1 L-glutamine Cl N N N N H clofazimine MIC (µg/mL) = 0.12 IC50 (Vero) (µg/mL) = 68.6 log CFU/lung* = 3.54 Cl N N O MIC = 2.2 µM against M tuber culosis H 37 Rv and rifampicin-resistant strain ATCC 35338 Figure A phenazine exhibiting an allyl-pyran group was active against M tuberculosis H37Rv and rifampicin-resistant strain ATCC 35338 OH Cl N OH Br Cl N Cl N 37 36 MRSA MBEC= 12.5 µM MRSE MBEC= 1.56 µM VRE MBEC= 0.20 µM I MtB MIC= 3.13 µM Figure HPs with promising activity against persistent bacteria.102,103 MBEC: minimum biofilm eradication concentration, MRSA: methicillin-resistant S aureus, MRSE: methicillin-resistant S epidermidis, VRE:vancomycin-resistant Enterococcus 2.7 Synthetic phenazines with exceptional biological activity A diverse library of several phenazines was synthesized in the Huigens lab from which 24 was identified as a lead antibiotic displaying a MIC value of 1.56 lM against S aureus (Fig 4).92 Via systematic structural diversification, a twofold increase in potency could be realized for compound 25 Furthermore, a structureactivity relationship (SAR) was established, which will be of use for further focused libraries Clofazimine93 and derivatives94 have shown remarkable in vitro activity against multidrug-resistant tuberculosis (MDR-TB) and clofazimine has been in clinical trials for the treatment of MDR-TB.95 A library of clofazimine derivatives has been established containing compounds that exhibit lower logP values compared to clofazimine in order to reduce undesired side effects like skin discoloration which is caused by accumulation in skin and fat tissues.96–99 Some compounds displayed good in vitro activity against M tuberculosis and were further tested for their acute toxicity and pharmacokinetic properties Compounds, exhibiting a significantly reduced skin discoloration potential were selected for further evaluation in a mouse model of acute MDR-TB infection Clofazimine and two other promising candidates for the treatment O N N 34 N I Cl N Cl N H Cl O N N N N H N O MIC (µg/mL) = 0.016 IC50 (Vero) (µg/mL) = >64 log CFU/lung* = 4.04 N 35 MIC (µg/mL) = 0.016 IC50 (Vero) (µg/mL) = 51 log CFU/lung* = 3.25 Figure Active compounds against MDR-TB ⁄after 20 days of treatment in BALB/C mice infected with clinical isolated MDR-TB dosed orally at 20 mg/kg CFU = colony forming unit.98 Please cite this article in press as: Guttenberger N., et al Bioorg Med Chem (2017), http://dx.doi.org/10.1016/j.bmc.2017.01.002 N Guttenberger et al / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx of MDR-TB are depicted in Fig In this regard the results from Coelho et al should be mentioned, who tested several phenazine derivatives for their in vitro activity against M tuberculosis H37Rv (ATCC 27294) and the Rifampicin-resistant strain (ATCC 35338) containing a His-526-Tir mutation in the rpoB gene and the most active derivative showed a MIC value of 2.2 lM for both strains (Fig 6).100 A small focused library of halogenated phenazines (HPs) was developed in the Huigens lab,101–103 among which some show promising activity against persistent bacteria In addition, a SAR was established Most importantly, it was demonstrated that the HPs were selective for bacterial cells over mammalian cells Compound 36 exhibits the most potent biofilm-eradicating activities to date against several multi-resistant germs (which can be expressed as MBEC (minimum biofilm eradication concentration)) and compound 37 was identified as a potent inhibitor of the slow-growing M tuberculosis (Fig 7) Compounds containing an N-(methylsulfonyl)amide substituent have been recently synthesized by Conda-Sheridan et al exhibiting MICs against MRSA comparable to Vancomycin.85 In addition, two QSAR models were reported which could be of use in the future Synthetic phenazine derivatives have shown to exhibit antiplasmodial and antiprotozoal,104–107 as well as insecticidal activities.108 3.2 Anthranilate synthase genes Biosynthesis Research into phenazine biosynthesis has continued to attract interest since early studies in the second half of the 19th century Comprehensive reviews, dealing with phenazine biosynthesis have been published recently and important facts are iterated.2,109–113 The focus of this section is to give a short overview of seminal findings in the field of phenazine biosynthesis including the most recent mechanistic insights 3.1 Early studies Studies on early phenazine biosynthesis have been reviewed by Turner and Messenger and some important milestones will be iterated here.114 Initial research into phenazine biosynthesis was mainly focused on PYO and it was found that the amount of PYO formed is significantly affected by culture conditions and bacterial origin.112 Early studies on phenazine biosynthesis turned out to be troublesome as O H O P O OH OH HO OH erythrose 4-phosphate OH O P O O OH H 2O - Pi CH2 CO2 H HO CO2H O PhzC + HO rich media with different bacterial isolates of poorly defined composition had been used.109 Jordan115 was the first to take a more systematic approach with synthetic media and his achievements led to the development of a medium for the detection of P aeruginosa in the clinic (e.g King’s A medium116).109 P aeruginosa was found to produce a variety of additional colored compounds.112 Importantly, it was realized that trace amounts of iron and exclusion of air led to an increase in phenazine production in several strains of Pseudomonas117 but the physiological role of phenazines as ‘‘respiratory pigments” was demonstrated only recently by Price–Whelan et al.9 In the 1950s, Blackwood and Neish118 could show that glycine, alanine, leucine and isoleucine were the preferred amino acid substrates for phenazine biosynthesis and that 14 C-incorporated glycerol and dihydroxyacetone were incorporated into PYO Millican119 performed further studies with 14Clabeled shikimic acid and proved some incorporation into PYO, whereas anthranilate was found not to be incorporated These results were somewhat conflictive to previous findings120 where anthranilate was shown to stimulate PYO formation and phenazine biosynthesis had been proposed to originate in anthranilate.121 By means of feeding experiments with 14C-shikimic acid, Hollstein and McCamey122 proposed two identical C-6- or C-1-N-substituted chorismic acids as precursors in the biosynthesis of the phenazine moiety PiO OH HO OH OH OH DAHP shikimic acid phosphoenolpyruvate CO2H O CO2 H OH chorismic acid Scheme Chorismic acid is biosynthesized via the shikimate pathway, starting from erythrose 4-phosphate and phosphoenolpyruvate Research into the biosynthesis of strain-specific phenazines had been difficult because of unstable intermediates and the discovery of specific genes has leveraged our current knowledge of the biosynthetic pathway towards phenazines.112 Essar et al identified anthranilate synthase genes in PYO-producing strains of P aeruginosa,123 which upon inactivation caused a significant decrease in PYO levels such that they concluded that phenazine biosynthesis proceeds via anthranilate, which is in contrast to our current knowledge of phenazine biosynthesis It was later confirmed that anthranilate synthase genes are indeed responsible for the generation of anthranilate but rather for the generation of the Pseudomonas Quinolone Signal (PQS), than being a precursor for the synthesis of phenazines.124 PQS plays an important role in quorum sensing,125 a cell density-dependant regulatory mechanism to express specific genes, in P aeruginosa phenazine biosynthesis.126 3.3 Current understanding Seminal results were obtained by McDonald et al.,127 who found that 2-amino-2-deoxyisochorismic acid (ADIC) is completely converted into phenazine-1-carboxylic acid (PCA) in cell-free extracts of E coli containing phz gene products On the contrary, anthranilate was not converted to PCA, indicating that phenazine biosynthesis branches off from primary shikimate pathway at ADIC Pierson et al.128,129 were the first to report genes directly involved in phenazine biosynthesis and genes for phenazine biosynthesis have been discovered in several bacterial phenazine producers.9,130–132 The five enzymes responsible in phenazine synthesis, namely PhzB, PhzD, PhzE, PhzF, and PhzG are conserved among all phenazine-producing bacteria and it is assumed that all phenazines found in nature share a small number of common precursors as the gene cluster has most likely spread via horizontal gene transfer.109,133,134 Chorismic acid, an intermediate in the shikimate pathway (Scheme 2) is a common precursor for many primary and secondary metabolites such as vitamin K, aromatic amino acids, folate, ubiquinone or the siderophores Chorismic acid is also the first substrate in the core biosynthetic pathway for the synthesis of strain-specific phenazines (Scheme 2) Pseudomonas and some Please cite this article in press as: Guttenberger N., et al Bioorg Med Chem (2017), http://dx.doi.org/10.1016/j.bmc.2017.01.002 N Guttenberger et al / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx chorismic acid O OH PhzE CO2H CO 2H NH2 Gln Glu - H2 O CO2H pyruvate AOCHC H PhzD O CO 2H NH PhzB PhzF H CO 2H NH 3H OH H CO2 H NH OH O HHPDC HHPDCa CO 2H H N CO 2H H N - H2 O O AOCHC DHHA ADIC CO 2H NH2 CO 2H N N CO2 H O2 H2 O2 CO2 H O2 PhzG H2 O2 CO THPDC CO 2H H N CO2H H N N CO2H H N N CO2 H O2 PhzG N H THPCA O2 H2 O2 H2 O2 CO PhzG H2 O2 CO2H H N CO2H H N N DHPDC H N DHPCA H CO2 H H N strain-specific phenazines N H DHPHZ O2 O2 O2 H2 O2 H2 O2 H2 O2 CO2H CO2H N N CO2 H PDC THPCAa O2 N N N N PCA phenazine Scheme Current understanding of the biosynthesis towards strain-specific phenazines starting from chorismic acid.2,136 other bacteria possess the phzC gene, which encodes a type-II 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase, which catalyzes the first step of the shikimate pathway, the reaction of erythrose 4-phosphate, water, and phosphoenolpyruvate to give DAHP.112 It is believed that in the case of inhibition of other DAHP synthases, PhzC acts to ensure sufficient flow for the phenazine biosynthesis.112 The core biosynthetic pathway towards strain-specific phenazines (Scheme 3) commences with the conversion of chorismic acid to ADIC, catalyzed by Mg(II)-dependent PhzE,135 and the proof that PhzE is an effective ADIC synthase was given by Li et al.135 PhzE consists of two domains In the first domain chorismate is converted to ADIC, whereas in the second domain ammonia, needed for this reaction, is generated from glutamine.135 It was found that upon binding of chorismic acid, a channel of approximately 25 Å in length is induced in order to prevent the loss of ammonia to the solvent.135 Fascinatingly, stereochemistry in ADIC is induced due to the fact that the channel ends at the Si-face at C-2 of chorismate.135 An enzyme related to PhzE is anthranilate synthase (AS),137–139 exhibiting virtually an identical active site, but in contrast to AS, pyruvate elimination takes not place in PhzE and ADIC enters the consecutive biocatalytic cascade for the synthesis of strain-specific phenazines The vinyl ether functional group of ADIC is cleaved off in the next step of the biosynthetic cascade catalyzed by PhzD,140 to give pyruvate and trans-2,3-dihydro-3hydroxyanthranilic acid (DHHA), the last stable intermediate in the biosynthesis of phenazines PhzD is an isochorismatase with major structure similarities to other structures from a subfamily of a/b-hydrolase enzymes that includes pyrazinamidase and N-carbamoylsarcosine amidohydrolase.140 Different to related structures, PhzD does not contain a nucleophilic cysteine but rather uses aspartic acid to protonate the vinyl ether functionality of ADIC Furthermore, PhzD catalyzes a dissimilar reaction compared to the aforementioned related structures.140 The subsequent double bond isomerization is catalyzed by PhzF141,142 and the underlying mechanism is still under discussion.143,144 PhzF exhibits two active sites which are occupied by sulfate ions in the available crystal structure.141 The enzyme-substrate complex suggests that a conserved glutamate E45 abstracts a proton from C-3 of DHHA, which is then attached to C-1 after double-bond shift to yield an enol.142 Catalyzed by PhzF144 the obtained enol tautomerizes to 6-amino-5-oxocyclohex-2-ene-1-carboxylic acid (AOCHC) and it is suggested that a Please cite this article in press as: Guttenberger N., et al Bioorg Med Chem (2017), http://dx.doi.org/10.1016/j.bmc.2017.01.002 10 N Guttenberger et al / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx cavity in dimeric PhzF could be suited for the ensuing ketone condensation of two molecules AOCHC for the generation of hexahydrophenazine-1,6-dicarboxylic acid (HHPDC).142 The head-to tail condensation of two molecules AOCHC to give HHPDC can proceed spontaneously in vitro, but involves PhzB, a small dimeric protein of the D5-3-ketosteroid isomerase/nuclear transport factor family in vivo.145 It is thought that AOCHC is toxic because of possible side reactions with other amines, e.g on proteins, thus its accumulation has to be limited.2 By means of crystallization experiments with product and substrate analogues, it has been proposed that double-imine formation is catalyzed through orienting two substrate molecules and by protonation of the tetrahedral intermediate.145 Pseudomonas species contain an approximately 70% sequence identical copy of the phzB gene, namely phzA, and PhzA has shown to play a role127 in the biosynthesis of phenazines.2 HHPDC is a central intermediate towards the likely end products of the pathway, 5,10-dihydro-PDC (DHPDC) and 5,10-dihydro-PCA (DHPCA) that are central precursors for strain-specific phenazines.136 Earlier, PCA and PDC have been claimed as final products of the pathway.136 As HHPDC is not stable, it undergoes rapid oxidative decarboxylation to tetrahydrophenazine-1,6-carboxylic acid (THPCA) Starting from HHPDC, two oxidative decarboxylation reactions and a spontaneous oxidation lead to the unsubstituted phenazine.136 The final steps of the biosynthesis of DHPDC and DHPCA involve flavin-dependant PhzG-catalyzed oxidation reactions.136,146 PhzG was found to exhibit close similarities to PdxH that catalyzes the final step in pyridoxal-50 -phosphate (PLP) biosynthesis.146 PhzG is not perfectly specific, which explains the appearance of PCA, PDC and unsubstituted phenazine and that competition between PhzG-catalyzed oxidation reactions and spontaneous oxidative decarboxylations governs the ratio of these compounds.136 Further modifications of the phenazine core involve e.g hydroxylation, methylation or N-oxidation Zhao et al.147 recently found that the aromatic N-monooxogenase LaPhzNO1, which is homologous to BaeyerÀVilliger flavoproteins, catalyzes in a substrateselective fashion phenazine N-oxidation and its possible use in chemoenzymatic aromatic N-oxidation reactions is speculated Chin-A-Woeng et al showed that the introduction of the gene phzH of Pseudomonas chlororaphis can efficiently extend the range of the biocontrol ability of bacterial strains.148 Methanophenazine, isolated from Methanosarcina mazei Gö1 is suggested to play an important role in membrane-bound electron transport,149 and its synthesis might proceed via a biosynthetic pathway different to that in bacteria.2 Future research has to be directed towards phenazine-modifying enzymes as there are major gaps in understanding when it comes to the biochemistry of modifications or the generation of species-specific phenazine compounds.34,109 Synthesis Classic synthetic strategies towards phenazines have been reviewed1,18,150–152 and an annual update153 on diazines and benzo derivatives can also be found The following section gives an overview of highlights in the field of phenazine synthesis since 2004, as extensive progress both in methodology and natural product synthesis has been made Established synthetic strategies towards phenazines will be touched shortly in order to convey an integral overview 4.1 Classic methods An overview of classic methods for the synthesis of phenazines is given in Scheme These methods are primarily based on the construction of the central heterocyclic ring and suffer from major disadvantages like limited substrate scope, harsh reaction conditions, low yields or the requirement of several synthetic steps rather than a one-step synthesis from commercially available starting materials.1,18 This fact, combined with a constant interest in differently substituted phenazines has spurred the development of new methods for the preparation of phenazines An old but rarely applied method, discovered by Wohl and Aue,154 involves the fusion of anilines and nitrobenzenes at high temperatures under basic conditions The Bamberger-Ham155 procedure comprises the reaction of two para-substituted nitrosobenzenes under acidic conditions and suffers from various limitations The Beirut-reaction156–158 can be used for the synthesis of phenazines by reacting benzofurazan oxide with phenols to give 5,10-phenazine dioxides, which can be easily reduced to phenazines This approach offers some advantages compared to the two strategies mentioned earlier like a broader substrate scope and milder reaction conditions Broad application has been found for the condensation of substituted 1,2-benzoquinones (can be in-situ generated from catechols) with 1,2-diaminobenzenes.159 Many strategies compromise either a reductive cyclization of diphenylamines with an ortho-nitro160–163, ortho,ortho0 -dinitro,163 or ortho-nitro,ortho0 fluoro164 arrangement The oxidative cyclization of diphenylamines with an ortho,ortho0 -diamino arrangement (Tomlinson oxidation)165,166 is also described in the literature.165,167 A very promising approach involves a sequential aniline arylation followed by aniline arylative intramolecular cyclication via a Buchwald-Hartwig coupling reaction.167,168 4.2 Cu- and Pd-catalyzed coupling reactions Winkler et al succeeded in a homocoupling of substituted bromoanilines via two subsequent Pd-catalyzed Buchwald-Hartwig amination,169 followed by an in-situ oxidation to yield symmetrical phenazines in moderate to good yields (Scheme 8(a)) This concept was extended by Yu et al in the same year by using an environmentally friendly aqueous system to yield substituted phenazines in moderate to high yields under Cu-catalysis (Scheme 8(b)).170 Laha et al achieved the synthesis of various phenazines in good to high yields via a Pd-catalyzed coupling reaction between readily available 1,2-diaminobenzenes and 1,2-dibromobenzenes (Scheme 8(e)).171 Monoarylated products could be isolated when the reaction was stopped earlier, indicating a domino reaction pathway 1,2-Dichlorobenzenes were also tested as substrates but gave the corresponding phenazines in lower yields This approach gives access to unsymmetrical phenazines (no C2-axis) 4.3 Transition metal-catalyzed C-H functionalization Seth et al accomplished a synchronous twofold C-N bond formation via an oxidative ortho-aryl C-H activation in poor to very good yields (Scheme 8(c)).172 The reaction was catalyzed by a binary Pd-Ag nanocluster Azoarenes were identified as sideproducts and the lowest yields were observed for a substrate containing a thioether substituent The presented protocol suffers from the need of a stoichiometric amount of Ag2CO3 and only symmetrical phenazines have been synthesized Upon further assessing substrate scope and reaction conditions, this strategy would be perfectly suited for the establishment of a library for SAR assessment, as differently substituted anilines are commercially available In a seminal publication, Lian et al disclosed the Rh(III)-catalyzed, formal [3 + 3] annulation of aromatic azides with aromatic azobenzenes to yield phenazines (Scheme 8(d)).173 This strategy offers several advantages as unsymmetrical, disubstituted phenazines are easily accessible and the strategy is also applicable for the synthesis of acridines This strategy, when applied for Please cite this article in press as: Guttenberger N., et al Bioorg Med Chem (2017), http://dx.doi.org/10.1016/j.bmc.2017.01.002 11 N Guttenberger et al / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx which upon protonation recycles Cp⁄Rh(III), which re-enters the catalytic cycle and VI is formed Acid-catalyzed intramolecular electrophilic aromatic substitution yields VII, which aromatizes upon elimination of arylamine to give phenazine VIII Wang et al recently disclosed a Cu-catalyzed CAH amination/ Ullman coupling domino reaction of phenanthrene-9,10-diamines with aryl iodides to give 9,14-diaryl-9,14-dihydrodibenzo[a,c] phenazines (Scheme 4).174 This methodology suffers from the requisite of harsh reaction conditions (210 °C reaction temperature), a high catalyst loading of 25 mol% along with the fact that 1,2-diaminobenzenes are not suited as substrates 2,7-Disubstituted 5,10-diaryl-5,10-dihydrophenazines have been synthesized in poor yields via an Fe-catalyzed intramolecular CAH amination reaction by Aoki et al (Scheme 5).175 A very promising approach for the facile access to differently substituted benzophenazines was lately given by Kumar et al (Scheme 8(i)).176 The Pd-catalyzed intramolecular alkynehydroarylation under strong acidic conditions offers various advantages like easily accessible starting materials, high atom economy, good yields, and a broad substrate scope Ar Ar Ar 25 mol% Cu(OTf)2 equiv K2CO3 1,3,5-trichlorobenzene NH HN + Ar'-I Ar N N Ar' ref lux 1.5 equiv Scheme Cu-catalyzed CAH amination/Ullmann coupling domino reaction for the synthesis of 9,14-diaryl-9,14-dihydrodibenzo[a,c]phenazines.174 R R R BuMgBr FeBr 3, 1,2-dibromoethane R Bu O, 70-100 °C HN N N R N R R R 4.4 One-pot procedures and multicomponent reactions (MCRs) Scheme Fe-catalyzed intramolecular CAH amination reaction for the synthesis of 2,7-disubstituted 5,10-diaryl-5,10-dihydrophenazines.175 A non-convergent multistep synthesis that includes several isolation and purification procedures is far from being ‘‘ideal” An ‘‘ideal synthesis” would require several characteristics, some of which are: being environmentally friendly, simple and affording the product in a 100% yield in one operation.177,178 Several parameters can be used to assess a reaction being economic, one of which is ‘‘pot-economy”.179,180 Running a multistep reaction in one single pot provides several advantages like saving time, resources and avoiding purification steps between reaction steps.180 phenazine synthesis, suffers from the loss of one equivalent of arylamine throughout the catalytic cycle The proposed mechanism for the formal [3 + 3] annulation is depicted in Scheme Orthodirected C-H-bond activation of azobenzene I gives complex II, which upon coordination of an aromatic azide forms IV Rh-C bond insertion of the azide and concomitant N2 loss gives complex V, N R1 H 2N R2 R1 N + Wohl-Aue harsh conditions low yields limited substrate scope R2 NO2 R1/2 Bamberger-Ham harsh conditions low yields limited substrate scope deoxygenation necessary ON + R1/2 NO R 1/2 = EDG N R1 R2 R1 N O HO N O + N H N N R2 O H/NO R1 N R2 + NH2 R1 Condensation regioselectivity O NH2 R1 Beirut limited substrate scope deoxygenation necessary R2 R2 NO2 NH H N R1 H N R2 NO2 R1 NH2 R2 F Reductive/Oxidative Cyclization several steps required H N R1 Br R2 Buchwald-Hartwig several steps required NH Scheme Classic methods for the preparation of phenazines.1,18 Please cite this article in press as: Guttenberger N., et al Bioorg Med Chem (2017), http://dx.doi.org/10.1016/j.bmc.2017.01.002 12 N Guttenberger et al / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx Ar N N N R2 R1 N R2 R1 N VIII N H VI Ar NH N ArNH R H R + N H+ N R1 R2 N N Ar N R1 Rh(III)Cp* N Ar Rh(III) II Cp* N R1 V Ar I Cp*Rh(III) H+ N H VII R1 N Ar Rh(III)Cp* N- + N2 N2 R N3 R2 III IV Scheme Proposed reaction mechanism of the Rh(III)-catalyzed amination/cyclization/aromatization cascade reaction for the synthesis of phenazines.173 The development of a uniform SAR assessment for anticancer activities has turned out to be intricate as many different biochemical techniques have been used for phenazines.41 It would be therefore advantageous to have access to a large, systematic library of differently substituted phenazines The construction of such a library would require a straightforward protocol that ideally starts from commercially available or easily accessible starting materials Progress in this respect has been made in the last years using multicomponent reactions (MCRs, Scheme 9) MCRs involve the reaction of three or more starting materials in one single pot and the reactions could either proceed simultaneously (so-called ‘‘domino” or ‘‘cascade” reactions) or via a sequential addition of reagents without any solvent exchange and provide many benefits compared to divergent multistep syntheses.178,181 MCRs are practical, the atom economy is high and many products can be synthesized from a few starting materials.178,182 Raju et al developed a new one-pot protocol for the synthesis of benzophenazines in moderate to very high yields by the reaction of benzoxepine-4-carboxylates with benzene-1,2-diamines in the presence of mol% Bi(OTf)3 (Scheme 8(g)).183 Benzophenazines have shown dual inhibition of topoisomerase I and II,184 two key enzymes influencing DNA topology at different points in the cell cycle Mishra and Moorthy185 recently succeeded in the one-pot synthesis of substituted benzo[a]phenazine derivatives via the IBX-mediated formation of 1,2-naphthoquinones and subsequent cyclocondensation reaction Some differently substituted 5arylthio-and 5-aminobenzo[a]phenazines were synthesized in fair to very good yields in DMF at rt Wang et al used a microwave-assisted186 MCR of 2-hydroxynaphthalene-1,4-dione, diamines, aldehydes and malononitrile for the synthesis of a small library of benzo[a]pyrano[2,3-c] phenazine derivatives in very good to excellent yields.187 Worth mentioning is the operational simplicity and the avoidance of elaborate purification procedures Other approaches using microwave-assisted MCRs have also been used for the synthesis of differently decorated benzopyrano phenazines.188–190 Differently substituted benzo[a]phenazines have been synthesized using an environmentally benign p-TSA (10 mol%)/PEG-400 reaction medium via a one-pot four-component protocol by Khurana et al in very high to excellent yields.191 Benzo[a]pyrano[2,3c]phenazine derivatives have been synthesized using AcOH,187,192 caffeine,188 theophylline,193 DABCO,189,194 pyridine,195 PTSA,196 ionic-liquid,197 oxalic acid,198 bifunctional thiourea-based organocatalyst199 and nano copper(II) oxide200 catalyzed MCRs MCRs involving isocyanides are especially versatile regarding accessible scaffolds and are perfectly suited for the establishment of large libraries for biological testing.178,182 Isocyanides have been applied in the synthesis of biologically interesting benzo[a]pyrano [2,3-c]phenazines emerging in the synthesis of an array of differently composed products.201,202 It should be referred to further studies using MCRs for the synthesis of phenazine derivatives.203–205 4.5 Miscellaneous The oxidation of 2-fluoro-substituted anilines using K3Fe(CN)6/ KOH gave fluorophenazines in low to moderate yields.206,207 There are also electrochemical methods towards fluorophenazines, via an anodic oxidation of fluorinated anilines, described in the literature.208–210 Recently, Gulevskaya disclosed a promising ICl-promoted 6endo-dig cyclization of 3-alkynyl-2-arylquinoxalines for the synthesis of benzo[a]-, naphtho[1,2-a]- and naphtho[2,1-a]phenazines (Scheme 8(h)).211 Most importantly, starting materials are easily accessible and the iodinated phenazine product could be further modified However, regioselectivity issues have been observed when e.g 3-(ethynyl)-2-m-tolylquinoxalines were used as starting materials Furthermore, a spirocyclic compound was isolated when 2-(4-methoxyphenyl)-3-(p-tolylethynyl)quinoxaline was used as starting material Unfortunately, only a limited substrate scope has been investigated so far for this interesting access to benzophenazines Laha et al.212 reported that an oxidative removal of benzylic methylene group in 10,11-dihydro-5H-dibenzo[b,e][1,4]diazepines using either DDQ or K2S2O8 is accompanied with a C-N bond formation resulting in the formation of various phenazines This transformation involves radical formation (Scheme 8(f)) In an effort to synthesize 1,3-dimesitylbenzimidazolium tetrafluoroborate as intermediate for a promising N-heterocyclic carbene (NHC), Borguet et al observed an unexpected formation of dihydrophenazines.213 Several N,N’-diarylbenzene-1,2-diamines were treated with sodium periodate on wet silica to give dihydrophenazines (Scheme 10) It was proposed that this reaction proceeds via a thermal, disrotatory 6p-electrocyclic process In the case of 2,6-diisopropylphenyl substituents on the nitrogens, a migration of the isopropyl groups was observed to give a mixture of isomers, tentatively via a [1,5]- and [1,3]-sigmatropic rearrangement to release steric stress Please cite this article in press as: Guttenberger N., et al Bioorg Med Chem (2017), http://dx.doi.org/10.1016/j.bmc.2017.01.002 13 N Guttenberger et al / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx N R R N Pd(OAc)2 phosphine ligand Cs 2CO3 toluene, 120 °C NH (a) R Br (c) R Br/I H O, 120 °C NH2 [PdCl2(PPh 3) 2] Ag 2CO3 TBAB O 2, DEF/DMA, 100 °C R N R1 N R Yu et al 2012 R Seth et al 2016 R2 Lian et al 2013 R2 Laha et al 2013 R2 Laha et al 2014 N N N R N (Cp*RhCl2) AgBC24 F20 Me + Winkler et al 2012 R Me (d) R N sulf onato-Cu(salen) complex (n-Bu)4 NBr K2 CO NH (b) N R N R1 AcOH, 110 °C, 24 h N R2 N N3 R1 R2 N NH2 (e) Br R1 R2 NH2 Br Pd(OAc)2 S-Phos Cs 2CO3 N R1 toluene, 110 °C N N R1 R2 N N NH (f) R1 K2 S2O R2 N H R1 MeCN, 90 °C, 30 CO2 C2 H5 R N N R2 N R3 R1 CO 2C H5 (g) O R3 R2 H 2N CO2 C2 H5 R N H 2N O R2 Raju et al 2014 Bi(OTf) or AcOH, toluene ref lux N R3 R1 N N R1 N I R2 R1 N ICl (h) Gulevskaya 2016 CH 2Cl2, -20 °C, 48 h N N I R2 R2 N N N R1 R2 R1 N Pd(OAc)2 CF3 COOH, 65 °C, 24 h (i) N R1 Kumar et al 2016 N R2 R2 Scheme Modern strategies for the synthesis of phenazines.169–171,173,176,183,211,212 Please cite this article in press as: Guttenberger N., et al Bioorg Med Chem (2017), http://dx.doi.org/10.1016/j.bmc.2017.01.002 14 N Guttenberger et al / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx conventional synthesis B A C B E A F F CO2 Et 1) H2 , Pd/C NHBoc 2) IBX, DMSO 65 % (2 steps) OH CO2 Et NHBoc 38 39 B C A O TFA CH2 Cl2 3h or (b) C E MCR (a) E CO2 Et N F 1) TFA removal 2) air CO2 Et NH N Scheme Comparison between conventional synthesis and multicomponent reactions (MCRs) Conventional synthesis: starting materials A and B react to give E, which is isolated and reacts with C to give final product F MCR: three or more starting materials A–C react to a final product F in one pot without any solvent exchange (a): no consecutive addition of the starting materials, (b): consecutive addition of the starting materials 41 O CO2Et air Pd/C xylenes 200 °C 53 % from 39 CO2 Et N N Ar R NH NaIO 4, SiO NH Ar 42 40 CO2 Et N N CO2Et 43 N KOt -Bu Et2O/H O 83 % R2 N R1 Ar R1 CO2Et R1 R1 Ar= CO2 H N R1 N R R R 1= Me, i-Pr R 2= H, Cl, Me N R1 iPr migration* R2 N Ar R2 N Ar + R1 N examples 79-90% R2 *observed for R1 = iPr, R2 = H Scheme 10 New method for the preparation of dihydrophenazines.213 CO 2H N N CO2 H O CO 2H OH chorismic acid CO2 H NH O AOCHC PDC CO2H Scheme 12 Biomimetic synthesis of PDC.230 R1 N Ar N PDC CO2 H or CO2 H N N Pd-catalyzed domino double-arylations by Laha et al.171 will significantly facilitate access to differently decorated phenazines Importantly, unsymmetrical phenazines are attainable with both strategies and starting materials are comparably easily accessible Some benzophenazines have shown dual inhibition of topoisomerase I and II and progress towards their synthesis has been made, mainly via MCRs We anticipate that a more target-oriented approach for the synthesis of benzophenazines, as recently disclosed by Kumar et al.176 will attract increasing attention Their approach via a Pd-catalyzed intramolecular alkyne-hydroarylation of substituted 2-ethynyl-3-phenylquinoxalines gives facile access to differently decorated benzophenazines Most importantly, starting materials are easily accessible via a one-pot sequential Sonogashira/Suzuki coupling of 2,3-dichloroquinoxalines It can be expected that the modern strategies presented herein will promote the development of additional phenazine libraries for biological testing and serve as a stimulus for research into the expansion of modern methods for phenazine ring assembly PCA Scheme 11 Biosynthesis of PDC and PCA Further progress214–226 has been made in method development, but a detailed discussion would go beyond the scope of this review 4.6 Summary of modern strategies Tremendous progress has been made in the implementation of new strategies for phenazine ring assembly A summary of modern, very promising methods for the ring construction of phenazines is given in Scheme We expect that the Rh-catalyzed aminationcyclization-aromatization cascade by Lian et al.173 and the 4.7 Biomimetic synthesis of phenazine-1,6-dicarboxylic acid (PDC) PDC and PCA are the oxidized forms of DHPDC and DHPDA, precursors of other naturally occurring phenazines,112,136 and are biosynthesized from chorismic acid, enabled by various enzymes encoded in the phz operon (Scheme 11).112 Clark et al succeeded in a biomimetic synthesis of PDC starting from literature-known227–229 38 (Scheme 12).230 Ketone 39 was synthesized starting from 38 via a hydrogenation reaction followed by an IBX-mediated oxidation of the secondary alcohol to the corresponding ketone Compound 39 shows strong resemblance to an intermediate in the biosynthesis towards PDC and PCA, namely AOCHC Boc-deprotection was accomplished using TFA and it is assumed that two molecules of Please cite this article in press as: Guttenberger N., et al Bioorg Med Chem (2017), http://dx.doi.org/10.1016/j.bmc.2017.01.002 15 N Guttenberger et al / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx NO2 Cl CO2H HO K2 CO 1-pentanol 150 °C 62 % 44 + HO HO NaBH NaOEt EtOH, 65 °C 65 % NO CO2 H N H NO2 90 % N H2 N 55 54 N CO2H 47 46 CO2 Me 54, r ac-BINAP Pd(OAc)2 Br Cs2 CO Fe, NH4 Cl MeOH 80 °C, h CO2 Me Br CO2 Me NH NH toluene, 110 °C 12 h 66 % NO2 Br CO2 Me 56 dimethyl sulfate KOH, acetone reflux, h 82 % MeI, K2CO3 DMF, rt 82 % 45 CO2 Me CO 2Me + MnO CH 2Cl2 , rt 84 % N N N Ph 53 O O HO N O HN O 59 O Br 58 N NO2 Br CO2 Me CO2 Me 57 Pd2 (dba)3 , dppf, Cs 2CO3 toluene, 120 °C, 48 h 68 % O Ph Cl n-BuLi, THF 66 % N aq NaOH EtOH/THF (2:1) reflux, 10 h 62 % 52 O N Ph 1) LiOH, H 2O THF, H 2O, °C 2) TMSCHN2 MeOH, °C CO2 Me CONH CO 2H HO HATU, NH4 Cl DIPEA DMF, rt, 12 h 67 % N N 60 % (two steps) N CO2 Me N NH2 48 O O N H CO2 Me N CO2 Me N CO 2Me 49 MgCl2 TMSCl Et 3N EtOAc, rt 75 % N HO O CO2 Me Fe, NH4 Cl DMF 100 °C, h 59 % of 58 24 % of 59 N CO2 Me N H CO2 H N N H CONH2 dermacozine A (61) 60 streptophenazine A (51) Scheme 14 Total synthesis of dermacozine A (61).237 50 Scheme 13 Total synthesis of streptophenazine A (51).234 40 condensed upon TFA removal and subjection to air to give 42 Full oxidation of the tricycle was achieved using Pd/C at 200 °C to yield 43 Saponification of 43 using KOt-Bu followed by an acidification afforded PDC in an overall yield of 29% starting from 38 CO 2Me O AlCl3, benzoyl chloride CH 2Cl2, 40 °C, 16 h N 49 % N H CO2 Me CO2 Me N N H CO2 Me 59 4.8 Total syntheses of streptophenazine A (51) The previously unknown streptophenazines A-H have been isolated by Mitova et al.231 and the structures have been revised.232–234 Streptophenazines I-K have been isolated by Kunz et al.232 and streptophenazines I–L by Bunbamrung et al.235 Streptophenazines M-O have been isolated by Liang et al.236 Yang et al.234 succeeded in the synthesis of the originally proposed structure231 of streptophenazine A, but spectral data turned out to differ from the natural isolate An alternative structure for streptophenazine A (51) was proposed and the synthesized product showed identity to the natural isolate (compound 51, Scheme 13) Phenazine ring construction was achieved using a reductive cyclization of diphenylamine with an ortho-nitro160–163 arrangement The key step in the synthesis towards (–)–streptophenazine A (51) was an asymmetric aldol reaction of the chiral oxazolidinone 53 with aldehyde 49 The absolute configuration was determined to be 10 S,20 R Streptophenazines B and E, as well as streptophenazine G233 have also been synthesized by Yang et al using the same strategy 4.9 Total syntheses of dermacozines A (61), B (64) and C (65) Dermacozines are reduced phenazine derivatives and have been isolated from deep-sea Dermacoccus abyssi, MT1.1 and MT1.2.66,67 62 N aq NaOH EtOH/THF (2:1) 90 °C, 16 h O CONH2 N N H CONH O HATU, NH4 Cl DIPEA DMF, rt, 12 h 42 % (2 steps) dermacozine B (64) CO 2H N N H CO2 H 63 Scheme 15 Total synthesis of dermacozine B (64).237 Some derivatives were moderately cytotoxic against leukaemia cell line K562 and dermacozine C (65) displayed a DPPH radical scavenger activity with an IC50 value of 8.4 lM.67 Wagner et al recently disclosed the isolation of dermacozines HÀJ from Dermacoccus abyssi.66 Dermacozine H, exhibiting a rare thioether linkage, revealed the best radical scavenging activity amongst dermacozines HÀJ, but lower than that reported for dermacozine C (65) Ghanta et al succeeded in the first total syntheses of dermacozines A (61), B (64) and C (65) (Schemes 14–16).237 The key step in the three syntheses is the central ring formation via inter- and intramolecular Pd-catalyzed N-arylation reactions (Buchwald- Please cite this article in press as: Guttenberger N., et al Bioorg Med Chem (2017), http://dx.doi.org/10.1016/j.bmc.2017.01.002 16 N Guttenberger et al / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx CO 2Me Cl O NH CN N + N H CO 2Me CN Cl NO plethora of novel synthetic protocols for the phenazine ring assembly has been published in the last years Importantly, facile access to unsymmetrical phenazines starting from easily accessible starting materials is now possible A large, systematic library of differently substituted phenazines would be expedient for biological testing and much progress has been made in the synthesis of differently substituted benzophenazines, primarily via multicomponent reactions (MCRs) Acknowledgments O CONH N N H CO2 H dermacozine C (65) Scheme 16 Total synthesis of dermacozine C (65) 237 Hartwig amination).167,168 Reduction of 57 using Fe and NH4Cl afforded a mixture of 58 and the already N-arylated 59 An effective N-arylation of 58 was achieved using Pd2(dba)3, dppf and Cs2CO3 in toluene at 120 °C to give 59 in a yield of 68% Upon saponification using aqueous NaOH and a final amidation using HATU/DIPEA dermacozine A (61) was isolated in a 13 % overall yield (7 steps) A selective Friedel-Crafts benzoylation of 59 was envisaged for the synthesis of dermacozine B (64) and various conditions were tested Mono-benzoylated 62 could be obtained in a yield of 49 % (along with 12 % of the di-benzoylated side product) when optimized reaction conditions were applied Dermacozine B (64) could be isolated in an overall yield of 6% Dermacozine C (65) was synthesized by masking one of the carboxylic acid ester starting materials as nitrile that could be transformed to the corresponding amide in the end Dermacozine C (65) could be isolated starting from methyl 3-amino-2chlorobenzoate in an overall yield of 9% Conclusion The most recent results summarized in this review article document that over the last decade phenazine research has made tremendous progress The molecular enzymology of the biosynthesis of the core structures phenazine 5,10-dihydro-PCA (DHPCA) and 5,10-dihydro-PDC (DHPDC) has been clarified in molecular detail With the available crystal structures at hand inhibitors can be designed,238 which should enable chemical control about DHPCA and DHPDC biosynthesis Such inhibitors could serve as antibiotic agents, when they are able to prevent the synthesis of virulent phenazine natural products such as PYO However, there are still gaps in understanding, for example the biochemistry of phenazine modifications has to be clarified in the future Another goal will be to increase our understanding of the biological function of additional phenazine natural products, especially in the context of host-pathogen interactions and in the symbiotic relationships within the bacterial rhizosphere Many novel phenazines have been isolated in the last couple of years, some of which have shown promising antibacterial and anticancer activity Until recently, synthetic access to differently decorated phenazines was limited by methods which suffer from major disadvantages like narrow substrate scope, harsh reaction conditions, low yields, or the requirement of several synthetic steps rather than a onestep synthesis from commercially available starting materials A Financial support by the ERA-Chemistry network through funding by the Deutsche Forschungsgemeinschaft (DFG; Grant no BL587-3 to W.B.) and the Austrian Science Fund (FWF; project I668 to R.B.), NAWI Graz, and BioTechMed Graz is gratefully acknowledged References and notes 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 Laursen JB, Nielsen J Chem Rev 2004;104:1663–1686 Blankenfeldt W, Parsons JF Curr Opin Struct Biol 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