**2. Results**

### *2.1. Compound Purification and Structure Elucidation*

Chromatographic fractionation of ethyl acetate solubles from *C. flava* afforded four dolabellane diterpenes, **1**–**4**, as well as two secosteroids, **5** and **6**. The three known dolabellane diterpenes, **2**–**4** were identified by comparison of their spectral data with those of reported literatures [5,6]. The structures of new compounds, **1**, **5**, and **6** were elucidated by analysis of 1D and 2D spectral data (Figures S1–S24).

Clavinflol C (**1**) had a molecular formula of C20H33O4Cl as deduced from HR-ESI-MS (Figure S1) and NMR data. Its IR bands (Figure S2) indicated the presence of exo-methylene (1,639, 961 cm<sup>−</sup>1) and hydroxyl (3343 cm<sup>−</sup>1) groups. The 1H NMR data of **1** (Figure S3) showed a pair of exo-methylene singlets (δ 4.91, 5.00) and an AB quartet for hydroxy-methyl group (δ 3.39, 3.82, J = 11.6 Hz), three methyl singlets (δ 1.03, 1.32, 1.40), an oxygenated methine proton (δ 4.18), and a chlorinated methine proton (δ 4.00). To determine the proton sequence of **1**, a COSY spectrum (Figure S6) revealed the connectiveness of H-2/H-3, H-5/H-6/H-7, H-9/H-10, and H-13/H-14. The 13C NMR (Figure S4) and HSQC spectra (Figure S5) of **1** showed signals for three methyl carbons, eight methylene carbons including the exomethylene (δ 113.9, 147.7), two methine carbons, and five quaternary carbons. Detailed analyses of the 1H, 13C NMR, and HSQC spectra revealed that **1** is a dolabellane diterpene with a 5/11 membered ring and a tetrasubstituted olefin at the C-11/C-12 positions. This type of skeleton was further confirmed from the observation of long range correlations of H2-16/C-3, C-5; H2-2/C-4; H2-7/C-8, C-17; H-10/C-1, C-7, C-8, C-9, C-11, H3-15/C-1, C-2, C-11, C-13; H2-13/C-1, C-11, C-12; H2-14/ C-11, C-12; H3-19/C-12, C-18, C-20; H3-20/C-12, C-18, C-19 in the HMBC spectrum (Figure S7). The relative stereochemistry of **1** was determined from NOESY experiments as illustrated in Figure S8. Assuming that H3-15 is α-oriented, key NOESY correlations from H3-15 to H-10 and from H-7 to H-10 suggested that H-10 and H-7 were in the α-orientation. NOESY correlations between H-9a/H-6a and H-9b/H-17b suggested that OH-8 was in the β-orientation.

Compound **5** was isolated as a white amorphous powder, showing a pseudo-molecular ion peak at *m*/*z* 469.32880 [M + Na]+ in the HR-ESI-MS (Figure S9), consistent with the molecular formula C28H46 NaO4 (calculated for 469.32883), requiring six degrees of unsaturation. The presence of an oxymethylene and a keto carbonyl carbon was confirmed by the 1H NMR (Figure S11) (δH 3.88 (m, H-11a) and 3.74 (m, H-11b)) and 13C NMR (Figure S12) (δC 59.2 (CH2), 212.5 (qC), and 216.2 (qC)) data, as well as from the IR absorption (Figure S10) at 3396 and 1704 cm<sup>−</sup>1. The diagnostic NMR signals of a 9,11-secosterol were confirmed by the 1H–1H COSY correlation (Figure S14) from H2-11 to H2-12 as well as HMBC correlations (Figure S15) from H3-18 to C-12, C-13, C-14, and

C-17; from H3-19 to C-1, C-5, C-9, and C-10. The NMR features of **5** were analogous to those of 3,11-dihydroxy-24-methyl-9,11-secocholest-5-en-9-one [7], except for the presence of a ketone (δC 201.1 (qC)) at C-23. Based on NOESY correlations (Figure S16) of H3-19/H-1, H3-19/H-2, H3-19/H-4, H3-19/H-8, H-3/H-1, H-3/H-2, H-3/H-4, H-8/H3-18, H-8/H-7, H3-18/H-15, H3-18/H-16, H3-18/H-20, and H-14/H-7, the relative stereochemistry at C-3, C-8, C-10, C-13, C-14, C-17, and C-20 in **5** was found to be the same as those of 3β,11-dihydroxy-24-methyl-9,11-secocholest-5-en-9-one [7]. On the basis of the above-mentioned findings, the structure of **5** was consistent with the structure shown as 3β,11-dihydroxy-24-methyl-9,11-secocholest-5-en-9,23-dione.

Compound **6** appeared as a white amorphous powder like **5**. Careful inspection of the 2D NMR spectroscopic data (Figures S21–23) of **6** led to the establishment of the same nucleus as that of **5**. The NMR spectroscopic data (Figures S19 and S20) of **6** were analogous to those of **5**, except for NMR signals due to the conjugated enone in **6**. The location of the conjugated enone was identified by the HMBC correlations (Figure S23) from the methylene protons (H2-22) to the carbonyl carbon (C-23) and from H3-26, 27 to C-24, securing the structure of **6**, which was shown as 3β,11-dihydroxy-24-methylene-9,11-secocholest-5-en-9,23-dione.

### *2.2. Identification of Marine Compounds Showed High-Content Characteristics of Proteasome Inhibition*

The proteasome inhibition assay was performed by following the standard operation protocol of high-content screening (HCS) of EGFP-UL76 aggresome as described previously [4]. A stringent proteasome inhibition was considered as the HCS measurements of marine compounds with an increase greater than 0.2-fold relative to those of the control without treatment. Under this validity criterion, we demonstrated the identification of six compounds with proteasome inhibition and their e ffects were statistically significant. Four compounds with dolabellane-based structures designated clavinflol C (**1**), stolonidiol (**2**), stolonidiol-17-acetate (**3**), and clavinflol B (**4**) (Figures 2 and 3) [5,8]. Additionally, two unprecedent compounds with secosteroid-based structures designated compound **5** and **6** (Figures 4 and 5). Prior to HCS experiments, the in vitro cell-based MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cytotoxicity assays were performed against four cell lines: A549 (human lung adenocarcinoma), HT-29 (human colon adenocarcinoma), and P-388 (mouse lymphocytic leukemia). Plasmid pEGFP-UL76 transfected HEK293T (human embryonic kidney large-T antigen-transformed) cell expressing EGFP-UL76 for HCS assay was assessed the ED50 using both MTT and high-content nuclear count measurements (Table S1). The ED50 values for respective compounds were as follows: compound **1**, >50 μg/mL, >50 μg/mL, >50 μg/mL, >25 μg/mL, and 6.14 μg/mL; stolonidiol (**2**), 3.9 μg/mL, >50 μg/mL, 0.6 μg/mL, >25 μg/mL, and >25 μg/mL; stolonidiol-17-acetate (**3**), >50 μg/mL, >50 μg/mL, >50 μg/mL, >25 μg/mL, and 19.56 μg/mL; clavinflol B (**4**), >50 μg/mL, >50 μg/mL, >50 μg/mL, >25 μg/mL, and 21.33 μg/mL; compound **5**, >50 μg/mL, 3.2 μg/mL, 4.6 μg/mL, 12.28 μg/mL, and 12.13 μg/mL; compound **6**, 5.3 μg/mL, >50 μg/mL, 4.8 μg/mL, >25 μg/mL, and 10.92 μg/mL. Both the MTT assay and high-content nucleus counts were performed to assess the ED50 values of HEK293T cells expressing EGFP-UL76 for bortezomib which were 11.95 nM and 24.29 nM and for MG132 were 1.18 μM and 1.91 μM, respectively. Clavinflol B (**4**) showed moderate cytotoxicity in previous reports, which was consistent with our data (Table S1) [6].

Following the HCS assay, the high-content EGFP-UL76 aggresome measurements integrated intensity and average intensity per cell were analyzed and the relative ratios were obtained by normalization to the control. For the ratio of EGFP-UL76 aggresome integrated intensity per cell (Figures 2A and 3A, top panels), the highest ratios for compounds **1**, **2**, **3**, **4**, **5,** and **6** were 1.22 (*p* = 0.0390), 2.12 (*p* < 0.0010), 1.74 (*p* = 0.0020), 1.33 (*p* < 0.0010), 2.03 (*p* < 0.0010), and 1.72 (*p* < 0.0010), respectively. The highest ratios of average intensity per cell presented for compounds **1**, **2**, **3**, **4**, **5,** and **6** were 1.32 (*p* = 0.0371), 1.75 (*p* = 0.0021), 1.40 (*p* < 0.0010), 1.19 (*p* = 0.0117), 1.85 (*p* < 0.0010), and 1.34 (*p* = 0.0089), respectively (Figures 2A and 3A, bottom panels). Furthermore, all these increases in ratios achieved statistical significance.

Consequently after the assay procedure, we performed Western blotting analysis and q-PCR experiments to examine the levels of EGFP-UL76 protein and mRNA transcript under the same experimental conditions (Figure 2B,C and Figure 3B,C). In these two experiments, cells treated with bortezomib 25 nM and MG132 1 μM were used in parallel as proteasome inhibitory controls. We obtained similar results that the ratios of EGFP-UL76/tubulin under treatment with bortezomib and MG132 showed no difference from the control level, which was consistent with a previous report [4].

**Figure 2.** The assessment of proteasome inhibitory activity of marine dolabellanes-based compounds (**1**, **2**, **3**, and **4**) using a standard operation protocol of high-content EGFP-UL76 aggresomes screening assay. Pure compounds modulated high-content measurements of EGFP-UL76 aggresomes. (**A**) Assessment of the integrated and average intensities of EGFP-UL76 aggresomes (1 to 50 μm) per cell. The tested concentrations were 0.2, 1, 5, and 25 μg/mL for pure compounds **1**, **2**, **3**, and **4**, respectively. The integrated (top panel) and average (bottom panel) intensities per cell were measured, and the ratios were obtained by normalization to the control without proteasome inhibitor treatment, which is denoted by **C** throughout the text. (**B**) Validation of the EGFP-UL76 protein levels upon the addition of the tested marine compounds. Western blot imaging and densitometric analyses were performed to quantitate the EGFPUL76/tubulin protein ratio with the addition of tested compound treatment at 1, 5, and 25 μg/mL. The molecular mass markers are shown on the left in kDa. (**C**) Quantitative PCR was conducted to assess the transcript ratio of EGFP-UL76/GAPDH in HEK293T cells treated with pure compounds at 1, 5, and 25 μg/mL. The high-content measurements of EGFP-UL76 with the addition of the proteasome inhibitors, bortezomib (25 nM, denoted **B**) and MG132 (1 μM, denoted **M**), were used as positive controls. All data points are the averages of at least three repetitive experiments. The error bars indicate standard deviations. The following symbols are used to indicate statistical significance throughout the text: \* 0.01 < *p* < 0.05; \*\* 0.001 < *p* < 0.01; \*\*\* *p* < 0.001.

**Figure 3.** The assessment of proteasome inhibitory activity of marine secosteroid-based compounds (**5** and **6**) using a standard operation protocol of high-content EGFP-UL76 aggresomes screening assay as described in Figure 2 legend.

Compound **1** did not affect protein ratios of EGFP-UL76/tubulin proteins at 1, 5, and 25 μg/mL of any kind. However, for cells treated with compound **2** at 1, 5, and 25 μg/mL, the ratios were 1.00 (*p* = 0.9466), 0.91 (*p* = 0.0359), and 0.88 (*p* < 0.0010), respectively (Figures 2B and 3B). Nevertheless, the cytotoxic ED50 for compound **2** was greater than 25 μg/mL for HEK293T cell in both assays (Table S1). The protein ratios for compound **3** were 0.91 (*p* < 0.0010), 0.96 (*p* = 0.1484), and 0.86 (*p* < 0.0010), respectively. The protein ratios for compound **4** were 0.92 (*p* = 0.0045), 0.95 (*p* = 0.0420), and 0.86 (*p* = 0.0014), respectively. However, HEK293T cells treated with compound **3** and **4** at 25 μg/mL exhibited a lighter toxicity with ED50 values of 19.56 and 21.33 μg/mL, respectively. Compound **5** exhibited significant reduction at 5 and 25 μg/mL; the ratios were 0.78 (*p* < 0.0010) and 0.15 (*p* < 0.0010), respectively, which is consistent with cytotoxicity ED50 values (Table S1). Compound **6** did not affect protein ratios at any tested concentrations. The results from both the quantitative PCR and

Western blotting analyses revealed that in the tested compound treatment neither the protein nor the mRNA ratios for EGFP-UL76/GADPH were elevated (Figures 2C and 3C). Taking all these results in account, we suggested that the increase in EGFP-UL76 high-content measurement was likely due to the modulation of protein conformation.

After the results, we investigated the phenotypic size of aggresomes and analyzed the high-content data from Figures 2 and 3 by diameter with methods described previously [4]. As shown in the top panels of Figures 4 and 5, compounds **1**, **2**, **3**, **4**, **5**, and **6** exhibited the highest ratio increases for count, which were as follows: for pit aggresomes: 1.18 (*p* = 0.0331), 1.73 (*p* < 0.0010), 1.76 (*p* < 0.0010), 1.48 (*p* < 0.0010), 1.29 (*p* = 0.0236), and 1.32 (*p* = 0.0062), respectively; for vesicle aggresomes, 1.43 (*p* = 0.0028), 1.88 (*p* < 0.0010), 1.84 (*p* < 0.0010), 1.54 (*p* < 0.0010), 1.53 (*p* < 0.0010), and 1.37 (*p* = 0.0071), respectively. Similar profiles were observed for the ratios of integrated intensity per cell (Figures 4 and 5, middle panels). Compounds **1**, **2**, **3**, **4**, **5**, and **6** showed the highest ratio increases which were as follows: for pit aggresomes, 1.18 (*p* = 0.0130), 1.48 (*p* < 0.0010), 1.31 (*p* = 0.0055), 1.33 (*p* < 0.0010), 1.41 (*p* < 0.0010), and 1.21 (*p* = 0.0097), respectively; for vesicle aggresomes, 1.16 (*p* = 0.0220), 1.73 (*p* < 0.0010), 1.76 (*p* < 0.0010), 1.05 (*p* = 0.5352), 1.29 (*p* = 0.0236), and 1.32 (*p* =0.0063), respectively. The ratios of average intensity per cell were the same for pit and vesicle aggresomes (Figures 4 and 5, bottom panels). For compounds **1**, **2**, **3**, **4**, **5**, and **6** showed the highest increases in ratios observed which were as follows: 1.27 (*p* = 0.0099), 1.42 (*p* < 0.0010), 1.25 (*p* = 0.0034), 1.22 (*p* = 0.0628), 1.42 (*p* < 0.0010), and 1.19 (*p* = 0.0135), respectively.

**Figure 4.** Classification of the high-content measurements of EGFP-UL76 aggresomes by size with marine dolabellanes (**1**, **2**, **3**, and **4**) at 0.2, 1, 5, and 25 μg/mL treatment. Pit and vesicle denote aggresomes 1 to 5 μm and 5 to 20 μm in diameter, respectively. Measurements of pit and vesicle aggresomes per cell were as follows: count number, integrated intensity, and average intensity. The relative ratio was normalized to control values without pure compound treatment. All data points are the averages of at least three repetitive experiments. The error bars indicate standard deviations. The following symbols are used to indicate statistical significance throughout the text: \* 0.01 < *p* < 0.05; \*\* 0.001 < *p* < 0.01; \*\*\* *p* < 0.001.

**Figure 5.** Classification of the high-content measurements of EGFP-UL76 aggresomes by size with marine secosteroid-based compounds (**5** and **6**). Treatment as described in Figure 4 legend.
