*3.2. Biological Activities of Azaphilones*

Azaphilones, besides being good compounds to replace synthetic pigments, aggregate valuable pharmacological properties. The wide broad range of biological activities that has been reported for azaphilones such as cytotoxic, anti-inflammatory, antimicrobial, antitumoral, antiviral and antioxidant is exemplified in Table 1.

Concerning the activities regarded to the new 101 azaphilones reported, the cytotoxic and antitumor potential are the most evaluated. Remarkably, compounds (**29**), (**30**), and (**33**) showed the most effective anti-gastric cancer activities (MGC803 and AGS cell lines) with IC50 values less than 1 μM, being more active than the positive control paclitaxel (3.8 μM) [41]. Additionally, (**29**) and (**30**) induced apoptosis in a concentration-dependent manner and (**30**) inhibited cell cycle progression. The authors also claim that 3,7-dimethyl-2,6-octadienyl group attached to N-2 contributed to the potent cytotoxic activities against MGC803 and AGS gastric cancer cell lines what can induce new investigations with semisynthetic azaphilone derivatives possessing this group [11]. The azaphilones (**39**) and (**40**) showed moderate activity against leukemia HL-60 and human breast cancer. However, (**39**) exhibited potent apoptosis induction activity by mediating caspase-3 activation and PARP degradation at 3 μM in leukemic cells HL-60 [44]. Another interesting result was the potent cytotoxic activity showed by the dimeric azaphilones (**89**) and (**90**) against five

different human cell lines. (**89**) showed more potent cytotoxicity against MGC-803 than cisplatin and possessed a unique 6/4/6 ring system suggesting the new ring may play an important role in cytotoxicity [57].

A great number of azaphilones present anti-inflammatory activity [35,37,38,42,49,50, 52,53,58,59]. The compounds (**21**), (**51**), (**52**) and (**100**) exhibited anti-inflammatory activities due to potent anti-NO production activity, with IC50 values of 11.9, 2.6, 12.5, and 10.0 μM, respectively, compared to the known iNOS inhibitor quercetin (34.6 ± 1.4 μM) on lipopolysaccharide (LPS) -induced nitric oxide (NO) production [35,46,57]. The antimicrobial activity of azaphilones also must be highlighted. Two dimeric azaphilones, penicitrinol Q (**24**) and penctrimertone (**80**), showed both excellent inhibitory activities against *B. subtilis* with MIC of 6.2 and 4.0 μg/mL, respectively. Moreover, (**24**) also presented inhibitory activity against bacteria *Staphylococcus aureus* (4.3 μg/mL) and *Pseudomonas aeruginosa* (11.2 μg/mL), and the yeast *C. albicans* (4.0 μg/mL) [36].

In vitro antiviral activity against HIV-1 was detected for phomopsones B and C (**82–83**) (7.6 and 0.5 μM, respectively [52]). Research in antiviral potential of azaphilones may be strengthened as they have been focused as possible drug leads for the development of effective antiviral agents against SARS-CoV-2 [60,61]. This worldwide impact-generated virus draws attention to the difficulty in developing new non-toxic antiviral drugs, as viruses use cell host metabolism for replication. This is corroborated by previous reports of antiviral activity of azaphilone metabolites, such as chermisinone B, isolated from the endophytic fungus *Nigrospora* sp. YE3033, and active against A/Puerto Rico/8/34 (H1N1) in CPE assay (IC50 0.80 μg/mL) with low cellular toxicity on MDCK cells (CC50 184.75 μg/mL) [62]. In vitro HIV-1 replication inhibitory effects in C8166 cells were demonstrated for Helotialins A and B (EC50 8.01 and 27.9 nM, respectively) [63]. In 2019, comazaphilone D was reported as a non-competitive inhibitor of neuraminidase from recombinant rvH1N1 (IC50 30.9 μM) while rubiginosin A was active against H5N1 (IC50 29.9 μM) [64]. The previous knowledge of the antiviral potential of azaphilone derivatives is an advantageous background for the development of new drugs to inhibit SARS-CoV-2.


**Table 1.** Azaphilones fungal sources and reported biological activities.




**Table 1.** *Cont.*

\* isolated as endophytic of *Polygonatum sibiricum*; \*\* closest relative being *Muyocopron atromaculans* (MUCL 34983); SF-268 (human glioblastoma carcinoma), MCF-7 (breast cancer), HepG-2 (liver cancer), HeLa (human cervix carcinoma), and A549 (lung cancer), BST = Brine Shrimp test.
