*3.9. Cytotoxic Activity*

Compounds **3** and **4**, which were new β-bergamotane sesquiterpenoids, were separated by SiO2/RP-18/HPLC from the marine-associated *Aspergillus fumigatus-*YK-7 EtOAc extract. Their antiproliferative effects on the U937 and PC-3 cell lines were measured in vitro in an MTT assay. Compound **4** revealed a weak growth inhibition capacity (IC50 of 84.9 μM) versus the U937 cell line, while **3** had no activity (IC50 > 100 μM) compared with doxorubicin hydrochloride (IC50 of 0.021 μM). On the other sides, both had no effect versus PC-3 cells [29]. Wu et al. reported the separation of two new derivatives, xylariterpenoids

A and B (**16** and **17**), from the EtOAc extract of *Xylariaceae* fungus by Sephadex LH-20/ODS CC and reversed-phase HPLC processing [33]. Their structures and stereo-configuration were proved utilizing NMR and CD methods. They are C-10 epimers having 2S/6S/7S/10R and 2S/6S/7S/10S configurations, respectively. Unfortunately, they (IC50 > 40 μM) exhibited no cytotoxic potential versus HL-60, MCF-7, SMMC-7721, A-549, and SW480 in an MTT assay [33].

From *Paraconiothynium brasiliense* Verkley, new bergamotane sesquiterpenoids brasilamides K-N (**49**–**52**), featuring 4-oxatricyclo-(3.3.1.02,7)-nonane (as in **49**) and 9-oxatricyclo- (4.3.0.04,7)-nonane (as in **50**–**52**) skeletons in addition to the formerly reported brasilamides A and C (**45** and **46**), were purified from the fungus scale-up fermentation cultures using SiO2/Sephadex LH-20/HPLC processing. They were elucidated via NMR analyses and compound **52**'s configuration was assured using modified Mosher's method. Compound **49** is a **45**-hydrogenated analog that has a tetrahydro-2H-pyrone unit linked at C-2 and C-5 to the bicyclo(3.1.1)heptane framework, forming a 4-oxatricyclo-(3.3.1.0 2,7)-nonane skeleton, whereas compounds **50**–**52** displayed unusual 9-oxatricyclo-(4.3.0.0 4,7)-nonane skeletons. Compounds **50** and **51** are hydrogenated and oxygenated derivatives of **46**, respectively, while **52** differed from **46** by having a C-8-carbonyl, C-1-methyl, and C-12 hydroxyl group instead of methylene, oxy-methylene, and ketone carbonyl, respectively. These metabolites (concentration of 50 μM) possessed no potential versus A549, A375, MCF-7, CNE1-LMP1, EC109, MGC, PANC-1, and Hep3B-2 in the MTS assay [44].

*Montagnula donacina* (edible mushroom) biosynthesized rare tetracyclic bergamotane sesquiterpenoids, donacinolides A (**82**) and B (**76**) and donacinoic acids A (**88**) and B (**6**), which were separated using SiO2 CC/Sephadex LH-20 CC/HPLC processing and were characterized using spectroscopic data, X-ray diffraction analysis, and computational methods. Compounds **76** and **82** are C9 epimers with a spiroketal moiety having 1S/5S/6S/9R and 1S/5S/6S/9S configurations, respectively, whereas **88** and **6** exhibited α,β-unsaturated carboxylic acid moiety and had 1R/2R/5S/6S/9S/14S and 1R/3S/5R/6R/9S configurations, respectively. These metabolites lacked a marked cytotoxic potential (IC50 > 40 μM) versus HL-60, SW480, A549, SMMC-7721, and MCF-7 [32].

In addition, purpurolides B (**83**) and C (**84**) had no cytotoxicity versus M14, HCT-116, U87, A2780, BGC-823, Bel-7402, and A549 [47], whereas compounds **85**–**87** (concentration of 50 μM) were inactive versus HCT-116, BGC-823, and Bel-7402 cell lines [48].

The chemical investigation of Arctic fungus *Eutypella* sp. D-1 s EtOAc extract yielded new derivatives, eutypellacytosporins A–D (**94**–**97**), which were established by spectroscopic analysis and modified Mosher's method. Structurally, these metabolites are related to decipienolides and cytosporins. They exhibited (IC50s ranging from 4.9 to 17.1 μM) weak-to-moderate cytotoxic influence versus DU145, SW1990, Huh7, and PANC-1 in the CCK-8 assay, whereas Huh7 and SW1990 cell lines had more sensitivity to **94**–**97** (IC50s ranging from 4.9 to 8.4 μM). On the other hand, compounds **95** and **97** possessed noticeable potential versus PANC-1 (IC50s of 7.9 and 7.5 μM, respectively) compared with cisplatin (IC50 4.5 μM). The results revealed that the decipienolide moiety was substantial for activity; however, the C-33 configuration did not affect the activity [52]. It was proposed that compounds **94**–**97** are created from gentisaldehyde precursor with subsequent isoprenyl unit addition, double bond epoxidation, keto group hydrogenation, and an aliphatic chain addition (Scheme 6). The other precursor, the 14-OH of decipienolide A **74** or B **75**, is produced from hydroxylation, allylic oxidation, and cyclization of farnesyl diphosphate to give **I** with a bicycle[3.1.1]heptane. Additionally, (14S)-14-OH-expansolide C, (14R)- 14-OH-expansolide C, (14S)-14-OH-expansolide D, and (14R)-14-OH-expansolide D are formed via two steps of reface- and si-face attacks of the OH groups on the ketone and aldehyde groups, respectively. After these steps, compounds **94**–**97** were produced from the two groups of 14-OH-expansolides C and D through condensation reactions with (S)-3-hydroxy-2,2-dimethylbutanoic acid and cytosporin D, respectively [52].

**Scheme 6.** Biosynthetic pathway of eutypellacytosporins A–D (**94**–**97**) [52].
