*2.9. Others*

Twelve compounds with other structures were obtained by co-culture of marine fungi–bacteria (4 compounds, 33%) and fungi–fungi (8 compounds, 67%).

#### 2.9.1. Other Compounds Derived from the Co-Cultures of Di fferent Marine Fungi

A novel polysubstituted benzaldehyde derivative, ethyl-5-ethoxy-2-formyl-3-hydroxy-4- methylbenzoate (**143**) (Figure 29) was identified from the mixed fermentation of the two mangrove fungi, *Phomopsis* sp. K38 and *Alternaria* sp. E33 that were collected from the South China Sea [92]. Another two novel furanone derivatives were identified as sclerotiorumins A and B (**144**, **145**) (Figure 29) from the co-culture of the two marine fungi, *sclerotiorum* SCSGAF 0053 and *P. citrinum* SCSGAF 0052 isolated from gorgonian *Muricella flexuosa* collected from the South China Sea, Sanya (18◦11 N, 109◦25 E), Hainan Province, China [23]. Five diorcinols, including one novel diorcinol J (**146**) and four known diorcinols B-E (**147**–**150**) (Figure 29), were characterized during the co-culturing of two marine-derived fungi, *A. sulphureus* KMM 4640 and *I. felina* KMM 4639 [93].

**Figure 29.** Chemical structures of **143**–**150**.

Compound **143** showed in vitro inhibitory activity against *G. musae*, *F. graminearum*, *P. sojae* (*Kaufmann* and *Gerdemann*) and *Rhizoctonia solani* Kuhn at 0.25 mM with inhibition zone diameters of 11.57, 12.06, 8.5 and 10.21 mm, respectively. This suggested that **143** had broad inhibitory activity against these microbes [92]. **144** and **145** exhibited weak toxicity against brine shrimp (LC50 > 100 μM) and none of them displayed cytotoxicity against the liver hepatocellular carcinoma Huh7 and HepG2 (LC50 > 100 μM) and obvious inhibitory activities towards three marine-derived bacteria, *Bacillus stearothermophilus*, *Pseudoalteromonas nigrifaciens* and *Bacillus amyloliquefaciens*, and two common pathogens, *P. aeruginosa* and *S. aureus* [23].

Among the five diorcinols, only **146** showed apparent cytotoxicity against murine Ehrlich carcinoma cells and hemolytic activity against mouse erythrocytes. The significant hemolytic activity of **146** suggested that its cytotoxic activity against murine Ehrlich carcinoma cells was due to a membranolytic mechanism. It is well known that the heat shock protein 70 (HSP70) was frequently overexpressed in tumor cell lines as an ATP-dependent molecular chaperone and played a significant role in refolding misfolded proteins and promoting cell survival under stress [94]. Thus, compounds that could inhibit HSP70 had grea<sup>t</sup> potential in tumor therapy. **147** could decrease the expression of HSP70 in the Ehrlich carcinoma cells, which made it possible to develop as a new antitumor drug/lead. Diorcinol D (**149**) was studied for its combined therapy against planktonic *Candida albicans* with a broad-spectrum antifungal agen<sup>t</sup> fluconazole [95]. The combined therapy exhibited considerable antifungal activity against ten clinical isolates of *C. albicans* containing five fluconazole-resistant isolates

and five fluconazole-sensitive isolates, whereas fluconazole alone did not display antifungal activity. This suggested that diorcinol D (**149**) restored the susceptibility of fluconazole to *C. albicans*.

Moreover, the e fficiencies of fluconazole inhibiting mature biofilms were also drastically boosted by the addition of **149** [95]. The fractional inhibitory concentration index (FICI) model and Δ*E* model unclosed that the synergistic actions indeed existed in combination of diorcinol D (**149**) and fluconazole [95]. Two resistance mechanisms of azoles were overexpression of e fflux pumps genes and alterations of genes (point mutations). **149** mainly suppressed the activity of e fflux pump in cells partly by decreasing the expression of Cdr1 (one mediator of azole e fflux pumps) in *Candida albicans* CASA1. On the other hand, **149** also inhibited ergosterol synthesis and CYP51 (the target of fluconazole) expression [95]. Thus, the significant synergistic interaction and drug-resistant reversion of fluconazole combined with diorcinol D (**149**) were caused by the two latent mechanisms, the block of e fflux pump and ergosterol biosynthesis. Notably, **149** was still needed to further in vivo study in the combination therapy field to settle rock-ribbed clinical fungal infection in response to the azole resistance.

2.9.2. Other Compounds Derived from the Co-Cultures of Marine Fungi and Bacteria

Five known metabolites, diorcinol D (**149**), penicillanone (**151**), diorcinol G (**152**), diorcinol I (**153**) and radiclonic acid (**154**) (Figure 30) were obtained from the co-culture of the sponge-derived fungi *A. versicolor* and *B. subtilis* [38].

**Figure 30.** Chemical structures of **151**–**154**.

Compounds **149**, **152** and **153** displayed antibacterial activities against five Gram-positive microbes, including one *S. aureus*, two *E. faecalis* and two *E. faecium* with the MIC values of 12.5–50 μM. In addition, **152** displayed potent inhibitory activities against all tested bacteria with an MIC value of 12.5 μM. **149** displayed inhibitory activity against *E. coli* with an MIC value of 8 μg/mL; and **153** showed significant antibacterial activity against *S. aureus* with an MIC value of 6.25 μg/mL [96,97]. In contrast, **149**, **152** and **153** did not display any obvious activity against L5178Y cell lines, which suggested that the antimicrobial activities of these products were not associated with their respective general toxicities [38].
