**1. Introduction**

Natural products are considered to be especially valuable resources for drug discovery. With the rapid development of technologies for isolation, purification and detection, grea<sup>t</sup> interest has been shown in the underexplored natural products. Natural phenazines are mainly discovered from microorganisms of marine and terrestrial. More than 100 natural phenazine derivatives and over 6000 synthetic phenazine derivatives have been investigated so far [1–4]. Phenazine derivatives are a large group of planar nitrogencontaining heterocyclic compounds and the most important core structure is a pyrazine ring (1,4-diazabenzene) with two annulated benzenes [5–7]. Phenazine derivatives differ in their chemical and physical properties based on the type and position of present functional groups. Their oxidation–reduction (redox) and fluorescent properties have attracted increasing attention. Some of them are significant dyes applied in medical and biological industry, while others are developed as efficient fluorescent probes to study the change of biochemical profile in vivo [8,9]. Natural phenazines are produced directly from various microorganisms, including *Pseudomonas* spp., *Streptomyces* spp., *Actinomycete* spp. in terrestrial and marine environments. They, like most other important secondary metabolites, possessed various biological activities and have been extensively studied for a long period

**Citation:** Yan, J.; Liu, W.; Cai, J.; Wang, Y.; Li, D.; Hua, H.; Cao, H. Advances in Phenazines over the Past Decade: Review of Their Pharmacological Activities, Mechanisms of Action, Biosynthetic Pathways and Synthetic Strategies. *Mar. Drugs* **2021**, *19*, 610. https:// doi.org/10.3390/md19110610

Academic Editor: Bill J. Baker

Received: 4 October 2021 Accepted: 22 October 2021 Published: 27 October 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

of time [10,11]. Phenazines and their derivatives exhibit a broad range of biological activities, such as antimicrobial, antiparasitic, neuroprotective, insecticidal, anti-inflammatory, anticancer activity and so on [12–15]. Phenazine derivatives could be used as prodrugs due to biological activities, for which pharmacologists and chemists have committed themselves to make them into patent medicines. For example, clofazimine (Figure 1) is successfully applied in clinic as widely used antileprosy and antitubercular drug due to antimicrobial activity and immunosuppressive properties [16]. XR11576, XR5944, NC-182 and NC-190 (Figure 1) belong to fused aryl phenazine derivatives, also they show significant anticancer activity and are under clinical studies [7]. Phenazine derivatives display antibacterial activity mainly against methicillin-resistant due to redox properties [17]. According to reports in recent years, phenazine derivatives possessed antiproliferative activities against various cancer cell lines [18–21]. Additionally, phenazine derivatives were candidates to be developed as inhibitors of disease-related targets and reported to show activity of inhibition to multiple enzymes [22–25]. Although phenazine derivatives possessed a broad activity spectrum, the in-depth study was hindered due to the limited resource. Many research groups devoted themselves to carrying out the synthetic work to investigate biological activities of synthetic phenazine derivatives.

**Figure 1.** The chemical structures of clofazimine, XR11576, NC-182, NC-190 and XR5944.

Laursen et al. reviewed natural and synthetic phenazine derivatives with regard to biological activities in 2004 [6]. Phenazines and their derivatives had been associated with anticancer activity since 1959. On the basis of significant anticancer activity of phenazines and their derivatives, Cimmino et al. excellently reviewed natural and synthetic phenazines and derivatives about their anticancer activity and mechanisms of action in 2012, covering articles from 2000 to 2011 [26]. In recent years, researchers found various novel structures of natural phenazine derivatives and investigations of pharmacological activity were involved in many aspects. In this review, phenazines isolated from microorganisms, synthetic phenazine derivatives, their pharmacological activities and mechanisms of action were summarized, covering the articles from 2012 to 2021.

#### **2. Natural Phenazine Derivatives**

In the past decades, according to the published articles, many researchers investigated known phenazines deeply and further evaluated their potent biological activity. Other researchers tried to find novel phenazines from natural sources. Natural phenazines can be categorized according to the types of functional groups and their linking positions on the phenazine core.

#### *2.1. Biological Activity of Known Phenazines*

Compound **1** (phenazine-1-carboxylic acid, Figure 2) is also called tubermycin B due to its antibiotic activity against *Mycobacterium tuberculosis*. It is widely distributed in various microorganisms as a precursor of many natural phenazine derivatives. Gorantla et al. firstly reported its antifungal activity against major human pathogen, *Trichophyton rubrum*, which could be responsible for causing athlete's foot, jock itch, ringworm and fingernail fungus infections. The minimum inhibitory concentration (MIC) was 4 mg/mL [27]. Varsha et al. first isolated it from *Lactococcus* BSN307 and investigated its anticancer activity against HeLa cell line (IC50 = 20 μg/mL) and MCF-7 cells (IC50 = 24 μg/mL). It showed inhibitory activity towards leucine and proline aminopeptidases; thus, it would be used as a potential metalloenzyme inhibitor [28].

**Figure 2.** The chemical structures of classical phenazines **1**–**8**.

Compound **2** (Figure 2) is also a significant phenazine-type metabolite produced by various microorganisms. Cardozo et al. investigated its antibacterial activity against MRSA (Methicillin-resistant *Staphylococcus aureus*) strains and found its synergic effect when combined with silver nanoparticles produced by *Fusarium oxysporum* [29]. Thanabalasingam et al. first isolated it from the leaves of a medicinal plant *Coccinia grandis* [30]. Tupe et al. tested its activity against human pathogen *Candida albicans* (MIC = 32–64 μg/mL), demonstrating its mechanism of antibacterial and antifungal activities via reactive oxygen species (ROS)-mediated apoptotic death; **2** could lead to production of intracellular ROS. ROS caused hyperpolarization of mitochondrial membrane, following externalizing phosphatidylserine, chromatin condensation and DNA fragmentation, thus, inducing apoptosis and, finally, cell death [31]. Kennedy et al. and Ali et al. further investigated anticancer mechanism of **2**. The anticancer activity mechanism was also connected with ROS. p53, Bax and cytochrome C (Cyto-C) were overexpressed while caspase-3 was activated and oncogenic, anti-apoptotic proteins such as poly ADP-ribose polymerase (PARP) and B-cell lymphoma-2 (Bcl-2) family proteins (Bcl-2, Bcl-w and Bcl-xL) were inhibited (Figure 3) [32,33].

Pyocyanin (**<sup>7</sup>**, Figure 2) is a redox-active phenazine. It is a major virulent factor produced by *Pseudomonas aeruginosa*, which exerts damage effects on mammalian cells. Chai et al. explored pathogenesis of **7** on macrophages. Biological data showed it could promote IL-8 secretion and mRNA expression in a concentration-dependent manner. Signal pathways of the protein kinase C (PKC) and nuclear factor-κ-gene binding (NF-κB) were involved in phorbol 12-myristate 13-acetate (PMA)-differentiated U937 cells infected by **7** [34]. Forbes et al. aimed to investigate the pyocyanin role of redox-sensitive mitogenactivated protein kinase (MAPK) by inducing toxicity in A549 cell line. The results showed that pyocyanin-induced cytotoxicity was different from c-Jun N-terminal Kinase (JNK) and p38MAPK signaling pathways. Acute ROS production and subsequent oxidative stress strengthened its toxicity [35]. 1,6-Dihydroxyphenazine 5,10-dioxide (**8**, Figure 2) is also called iodinin. It was discovered to show anti-bacterial activity and weak activity against a mouse tumor model. Sletta et al. firstly isolated it from *Streptosporangium* sp. DSM 45942 from the fjord sediment; **8** showed grea<sup>t</sup> antibacterial and antifungal activities against *Candida glabrata* and *Enterococcus faecium*, MIC ranging from 0.35–0.71 μg/mL.

Compared with normal rat kidney (NRK) fibroblasts, **8** showed higher selectivity towards leukemia cell line. It was a promising compound to be developed as an anticancer drug, especially those targeting leukemia [36]. Myhren et al. further investigated its anticancer potential against acute myeloid leukemia (AML) and acute promyelocytic leukemia (APL) cells. The results demonstrated its anticancer potency against two selective cancer cell lines and weak toxicity to normal cells. Molecular modeling results suggested that it could intercalate between bases in the DNA, leading to DNA-strand break. The apoptosis progress was associated with Fms-like tyrosine kinase (FLT3) internal tandem duplications, mutated/deficient p53 and activation of caspase-3 [37].

**Figure 3.** The anticancer mechanisms of action of reported phenazines which induced apoptosis associated via mitochondria mediated apoptotic pathways.

Lee et al. isolated compounds **1**, **3** and **4** (saphenic acid, Figure 2) from a deep-sea sediment-derived yeast-like fungus *Cystobasidium larynigs* IV17-028. These compounds could decrease the production of NO, thus showing inhibitory activity against lipopolysaccharide (LPS)-induced murine macrophage RAW 264.7 cells with EC50 values of 17.06 mg/mL (76.1 μM), 14.67 mg/mL (54.7 μM) and 6.15 mg/mL (22.9 μM), respectively [38].

Hifnawy et al. isolated compounds **1**, **5** (phenazine-1,6-dicarboxylate) and **6** (phencomycin) from actinomycetes, *Micromonospora* sp. UR56 and *Actinokineospora* sp. EG49. These compounds demonstrated high to moderate antibacterial and antibiofilm activities against four bacterial strains (*Staphylococcus aureus*, *Bacillus subtilis*, *Escherichia coli* and *Pseudomonas aeruginosa*), with modest cytotoxicity against four cell lines (WI38, HCT116, HepG-2 and MCF-7). They took *Staphylococcus* DNA gyrase-B and pyruvate kinase as targets. Subsequently, in vitro data showed that **1**, **5** and **6** (Figure 2) exerted their bacterial inhibitory activities through inhibiting *Staphylococcus* DNA gyrase-B and pyruvate kinase [39].

## *2.2. Natural Phenazines*
