*4.3. Antiparasitic Activity*

Chagas' disease, caused by *Trypanosoma cruzi*, is a widely spread endemic disease in American. Alvarez and Minini et al. selected phenazine **158** (Figure 19) from their own chemistry library and investigated its in-depth insight mechanism of inhibition; **158** could bind to a widespread enzyme, triosephosphate isomerase (TIM) from *T. cruzi*. It showed grea<sup>t</sup> inhibitory activity against TIM and could be further developed as inhibitors of TIM; **158** showed highly selective inhibition against *T. cruzi* enzyme (TcTIM) and weak inhibition against *T. brucei* (TbTIM), without affecting TIM from *H. sapiens* (HsTIM) and *Leishmania* sp. (LmTIM) [67,68].

**Figure 19.** The chemical structure of compound **158** and its inhibition assay data against Tc, Hs, Lm and TbTIMs.

## *4.4. Anticancer Activity*

#### 4.4.1. Phenazine 5,10-dioxide Derivatives

Phenazine-5,10-dioxide derivatives **159**–**166** are listed in Table 1. Hernández et al. investigated chemosensitizer effect of compounds **159** and **161** to cisplatin. They showed a significant increase of the antiproliferative activity compared with the control group treated with cisplatin alone, demonstrating sensitization to cisplatin therapy. In addition, **159** combined with cisplatin induced cell cycle arrest on bladder cancer cells, resensitizing the invasive and cisplatin resistant 253 J cell line. It also showed grea<sup>t</sup> inhibition activity against histone deacetylase (HDAC) and sensitized chemotherapeutic drugs to better access to DNA, which would cause DNA damage, leading to cell death [69].

**Table 1.** The chemical structures of phenazine-5,10-dioxide derivatives **159**–**166** and survival rate (%) in normoxia and hypoxia.


#### **Table 1.** *Cont.*

a SF norm = survival fraction in normoxia at 20 mM on V79 cells. b SF hypox = survival fraction in hypoxia at 20 mM on V79 cells. c Values are means of two different experiments. The assays were done by duplicate and using at least three repetitions. Nd = not determined.

Phenazine 5,10-dioxide derivatives have also been reported in development as bioreductive agents. This class of compounds all contain a bioreductive moiety, the N-oxide group and a planar heterocycle moiety, phenazine [70,71]. After the hypoxic selective bioreductive process, the phenazine moiety can interact with DNA causing cytotoxicity in the solid tumour cells [72]. Gonda et al. attempted to find selective hypoxic cytotoxins with additional ability to inhibit DNA topoisomerase II. Inhibitive values in normoxia and hypoxia condition of these compounds were shown in Table 1: **162**–**164** displayed some degree of selectivity; **165** showed non-selectivity towards both conditions, normoxia and hypoxia. Meanwhile, **159** and 1**63** showed the best selectivity; **164** exhibited inhibition of topoisomerase II in hypoxia; **166** showed no inhibition of topoisomerase II in hypoxia and normoxia. The DNA interaction abilities of phenazine 5,10-dioxide derivatives were related to cytotoxicity in normoxia or hypoxia. SAR implicated that the arylethenyl moieties were generally responsible for normoxic cytotoxicity. On the contrary, the group of sulfonamido did not produce selective cytotoxicity whether in normoxia or hypoxia [73].

## 4.4.2. Benzo[*a*]phenazine Derivatives

According to related reports, benzo[*a*]phenazine derivatives show significant activity of antiproliferation against HL-60 cell line. Topoisomerases, including topoisomerase I and II, have been proved to be effective anticancer targets in drug discovery due to highly over-expression in cancer cells [74,75].

Benzo[*a*]phenazine derivatives **167**–**174** are listed in Table 2. Zhuo et al. designed series of benzo[*a*]phenazine derivatives, which alkylated the phenolic hydroxyl group on ring B, introducing different substituted groups on ring D by condensation and followed up with amination. This class of compounds showed good antiproliferative activity. Compound **167** demonstrated good Topo I–DNA cleavage complex stabilizing ability in vivo. Compound **168** showed inhibition of ATPase. Compared with **167**, **168** and **169** were introduced a methoxy group, their inhibitory activity was significantly improved and there was a good correlation between the inhibitory activity and cytotoxic activity. Caspase-3/7 activation assay showed that this class of compounds could induce an apoptotic response in HL-60 cell line [76].


**Table 2.** The chemical structures of benzo[*a*]phenazine derivatives **167–174,** IC50 values of in vitro growth inhibitory, relative activity of Topo I-DNA cleavage and Topo II ATPase inhibition.

a IC50 = concentration required for 50% of the antiproliferative effect for a given cell population grown for 2 days in presence of the compound of interest.; b The relative Topo I cleavage complex stabilizing potencies of the compounds are presented as follows: −, no detectable activity; **+**, weak activity; **++**, weaker activity than that of CPT; **+++**, activity similar to that of CPT.; c The relative Topo II ATPase inhibitory potencies of the compounds are presented as follows: *−*, no detectable activity; **+**, weak activity; **++**, weaker activity than that of 1,4-naphthoquinone; **+++**, activity similar to that of 1,4-naphthoquinone, higher activity than that of 1,4-naphthoquinone; **++++**.; Nd = not determined.

Yao et al. synthesized a series of 7-alkylamino substituted benzo[*a*]phenazine derivatives. Most of these compounds showed better inhibitory activity in HL-60 cell line than the other tested cell lines. The structure–activity relationship studies revealed that the substitution of amino groups on terminal of side chain at N-7 position could improve the Topo I/II inhibitory activity and cytotoxicity: **170**–**172** with the dimethylamino terminal showed good Topo I and Topo II inhibitory activity; **170** could stabilize the Topo I-DNA cleavage complexes in vivo; **173** with methoxy group at position C-9 exhibited good Topo I and Topo II inhibitory activity at 25 mM concentration; **173** showed inhibition of ATPase [22].

## 4.4.3. Pyran[2,3-*c*]phenazine Derivatives

Phenazine derivatives and pyran derivatives are important heterocyclic compounds which possess good biological activity. The heterocyclic pyran structure usually is a functional framework which appears in amounts of important drugs and natural products. Molecular hybridization strategy shows grea<sup>t</sup> prospect in the present drug discovery to reduce the side effects and the occurrence of drug-resistance [77]. The aromatic interlayer coupling structure of phenazine, as well as the structural characteristics of fluted or specific enzyme binders, leads to selective high-affinity binders that target DNA and DNAenzyme complexes [78]. According to recent articles, pyran[2,3-*c*] phenazine derivatives mainly showed cytotoxicity against HepG2 cell line. In addition, the mechanism inducing apoptosis against cancer cells about this class of compounds is shown in Figure 3.

Lu et al. designed and synthesized a series of pyran[3,2-*a*] phenazine derivatives. Most pyran[3,2-*a*] phenazine derivatives demonstrated cytotoxicity against HCT116, MCF-7, HepG2 and A549 cancer cell lines in vitro. Especially, compounds **181**–**183** (Scheme 5) were found to show potent growth inhibitory activity against HepG2 cell line (IC50 of 2–6 μM). In addition, they also used experimental mouse models to test in vivo activity of these phenazine derivatives. Among these phenazine derivatives, **181** was selected to do tumor xenografts experiment to test the effect of inhibition. H22 cells was injected into ICR mice, inhibitions were 7.78% (5 mg/kg), 68.89% (10 mg/kg) and 77.78% (20 mg/kg), respectively. Further mechanism studies implicated **181** acted as topoisomerases I and II dual inhibitor, cell cycle arrester and apoptosis inducer against HepG2 cell line [23].

**Scheme 5.** Synthesis of compounds **180**–**183**. Reagents and conditions: (**a**) Conc. HCl, FeCl3, rt, overnight; (**b**) malononitrile or ethyl 2-cyanoacetate, DABCO, EtOH, reflux, 0.5–2.5 h.

Additionally, Liao et al. firstly found **181** as thioredoxin reductase I (TrxR1) inhibitor against HepG2 cell line. TrxR1 is a novel anticancer target different from topoisomerases I and II. Molecular docking was carried out to study the inhibitory possibility of compound **181** against TrxR1. Soon afterwards, they investigated the crucial downstream protein TrxR1 to confirm antiproliferation function of **181** against the HepG2 cell line. It supplied valuable information for further development of the TrxR1 inhibitors [24].

Lu et al. also designed phenazine derivatives containing phenazine, pyran, indole and 1,2,3-triazole pharmacophores. Among these derivatives, **187** (Scheme 6) demonstrated the best antiproliferative activity against A549 cancer cell line (IC50 of 5.4 μM) and no cytotoxicity against L02 and HUVEC non-cancer cell lines [18].

**Scheme 6.** Synthesis of compound **187**. Reagents and conditions: (**a**) Conc. HCl, FeCl3, rt, overnight; (**b**) malononitrile, DABCO, EtOH; (**c**) sodium ascorbate, CuSO4, THF/H2O.
