Chemical Constituents from Soft Coral Clavularia spp. Demonstrate Antiproliferative Effects on Oral Cancer Cells

Five new eudensamane-type sesquiterpene lactones, clasamanes A–E (1–5), three new dolabellane-type diterpenes, clabellanes A–C (6–8), and fifteen known compounds (9–23) were isolated from the ethanolic extract of Taiwanese soft coral Clavularia spp. The structures of all undescribed components (1–8) were determined by analysis of IR, mass, NMR, and UV spectroscopic data. The absolute configuration of new compounds was determined by using circular dichroism and DP4+ calculations. The cytotoxic activities of all isolated marine natural products were evaluated. Compound 7 showed a significant cytotoxic effect against oral cancer cell line (Ca9-22) with an IC50 value of 7.26 ± 0.17 μg/mL.


Introduction
In south-central Asia, people are susceptible to oral cancers because of the usage of areca nuts as chewing gum.Taiwan has one of the world's highest incidences of oral cancer, which ranks fourth in the cause of cancer death among male Taiwanese [1].In Taiwan, about 3000 deaths yearly are due to oral cancers.The treatments for oral cancer are usually combined surgery and chemotherapy; however, chemotherapy drugs sometimes produce adverse effects [2].It is necessary to discover new anti-oral cancer drugs.
Marine sessile animals like sponges, soft corals, tunicates, and zoanthids are known to produce diverse secondary metabolites.Octacoral is one of the most abundant sources of bioactive marine natural products (MNPs) with unique backbones.Since 1977, the soft corals of the genus Clavularia have been found to have different kinds of MNPs, such as diterpenoids [3][4][5], sesquiterpenoids [6], prostanoids [7,8], and steroids [9].Those MNPs usually demonstrate considerable cytotoxic effects against several cancer cell lines.For example, dolabellane-type diterpenes could significantly inhibit P-388 leukemia cells with an ED 50 value of 0.052 µg/mL [4].Eudensamane-type sesquiterpene lactones were found to inhibit the growth of cancer cell lines.In our previous study on Taiwanese marine invertebrates, the methanol extract of Clavularia inflata exerts an apoptotic effect and DNA damage to oral cancer cells [10].These findings propel us to conduct the natural product investigation of this coral extract.
damage to oral cancer cells [10].These findings propel us to conduct the natural product investigation of this coral extract.
Clasamane C (3) possessed a molecular formula of C 18 H 22 O 5 , which is consistent with its positive sodiated HRESIMS ion at m/z 341.1362.The 1 H and 13 C NMR spectroscopic data of 3 were similar to those of 10, suggesting they are congeners.Comparison of the NMR spectra between 3 and 10 showed that 3 has an additional methoxy group (δ H 3.07 (s) (H 3 -1 ); δ C 50. 3 (C-1 )).This methoxy group situated at C-8 was evidenced by the HMBC correlation from H 3 -1 to C-8 (δ 105.7).The NOESY correlation between H-5 (δ 1.86) and H 3 -14 (δ 1.56) revealed they were on the same face (α-orientation) of the molecule.The absolute configuration of 3 was determined by ECD data analysis.Due to the consistency of ECD curves between 3 and 11 (Figure S3), the absolute configuration of 3 was defined as 5R,8S,10R.
Clasamane D (4) was isolated as a colorless oil and had the molecular formula of C 19 H 24 O 5 inferred from the sodiated HRESIMS ion at m/z 355.1518, which is 14 amu more than 3.The UV, IR and NMR data of 4 were quite similar to those of 3, except the methoxy group in 3 was replaced by an ethoxy group (δ H 3.38 (m), 3.09 (m) (H 2 -1 ); δ C 58.7 (C-1 ); δ H 1.14 (t) (H 3 -2 ); δ C 15.1 (C-2 )) in 4.This speculation is consistent with the difference in mass spectrometry between 4 and 3, and it was confirmed by the HMBC correlation from H 2 -1 to C-8 (δ 105.7).The similar ECD trends of 4 and 3 suggested these two compounds share the same absolute configuration.Thus, the structure of 4 was determined as shown.
Clasamane E (5) was a colorless oil, and its HRESIMS data showed a [M + Na] + ion at m/z 387.1414, suggesting the molecular formula of C 19 H 24 O 7 with seven indices of hydrogen deficiency.The 1 H and 13 C data (Tables 1 and 2   spectrometry between 4 and 3, and it was confirmed by the HMBC correlation from H2-1′ to C-8 (δ 105.7).The similar ECD trends of 4 and 3 suggested these two compounds share the same absolute configuration.Thus, the structure of 4 was determined as shown.Clasamane E (5) was a colorless oil, and its HRESIMS data showed a [M + Na] + ion at m/z 387.1414, suggesting the molecular formula of C19H24O7 with seven indices of hydrogen deficiency.The 1 H and 13 C data (Tables 1 and 2 Considering the molecular formula, two oxygen atoms were not assigned yet, and the carbon chemical shift of C-1 and C-4 suggested that these two carbons are oxygen bearing.Thus, a peroxide bridge between C-1 and C-4 was allocated.This assignment was also confirmed by the down-field shifted signals of H-2 and H-3 [17].The cis-decalin moiety of 5 was assured by the NOESY correlations between H3-14 and H-5, which were assigned on the α-face.In addition, the NOESY correlations of H2-15/H-5 and H3-14/H-1 revealed the peroxide bridge was on the β-face of the molecule.The NOESY correlation of H-9β (δ 2.26)/H2-1″ indicated the β-orientation of the ethoxy group.Therefore, the stereocenters of 5 could be temporarily assigned as 1S*,4R*,5S*,8S*,10R* or 1R*,4S*,5R*,8S*,10S* (Figure S45).The 1 H and 13 C data of those two isomers were calculated by Gaussian 16, and the data were applied to DP4+ probability analysis.The analytic result indicated that the 1S*,4R*,5S*,8S*,10R* isomer has 100% possibility, so the configuration of 5 was determined.and δ 24.1 (C-15)) by using 13 C NMR (Table 2) data together with DEPT-135 and HSQC spectra.Considering the above data and the reported compounds isolated from the genus Clavularia, 6 can be deduced as a dollabellane-type diterpenoid.The planar structure of 6 was established by COSY and HMBC correlations (Figure 4).Three proton sequences of H 2 -2 (δ 1.96 and δ 1.25)/H 2 -3 (δ 2.11 and δ 1.63), H 2 -5 (δ 2.42 and δ 2.28)/H 2 -6 (δ 2.11 and δ 1.90)/H-7 (δ 4.04), and H 2 -9 (δ 2.25)/H-10 (δ 2.86) were observed from the COSY spectrum.Those findings and the HMBC correlation from H 2 -16 to C-3, C-4, and C-5; from H 2 -17 to C-7, C-8, and C-9; from H 2 -9 to C-8 and C-11; and from H 3 -15 to C-1, C-2, and C-11 could build the cycloundecane moiety (C-1 to C-11) of 6, and it could confirm the presence of exomethylene (H 2 -16) connecting at C-4, an oxymethylene attaching at C-8, and a methyl (H 3 -15) connecting at C-1.In addition, the COSY correlations of H-12 (δ 2.21)/H 2 -13 (δ 1.90 and δ 1.63)/H 2 -14 (δ 1.76) together with the HMBC correlations from H-12 to C-11 and from H 3 -15 to C-1, C-11, and C-14 could establish the cyclopentane ring of 6.The HMBC correlations from H-12 and the geminal methyls (H 3 -19 and H 3 -20) to C-3 constructed the isopropyl alcohol group.The planar structure of 6 was found to be similar to that of clavinflol B (13) except that the chlorine atom in the molecular formula (C 20 H 33 BrO 4 ) of 13 was replaced by a bromine atom in 6.The halogen atom of 6 was allocated at C-7, the same as that of 13, due to the downfield shifted carbon chemical shift at this position (δ 66.2 in 13 and δ 63.0 in 6).The relative configuration of 6 was determined through the NOESY spectrum (Figure 4).The bromine atom was assigned on the α-orientation to avoid steric interaction, while the NOESY correlations of H-7/H-10/H 3 -15/H 3 -19 indicated those protons were on the β-face of the molecule.On the other hand, the NOESY correlation between H 2 -17 and H 2 -9 implied that the hydroxy group should be β-oriented.Therefore, the configuration of 6 could be defined as 1R*,7R*,10S*,12R*.The configuration of C-8 was deduced to be 8R by the DP4+ probability measurement.The 1R*,7R*,8R*,10S*,12R* isomer showed an overwhelming possibility (100%) compared with the 1R*,7R*,8S*,10S*,12R* isomer (0%), which confirmed the configuration of 6.
10/H3-15/H3-19 indicated those protons were on the β-face of the molecule.On the other hand, the NOESY correlation between H2-17 and H2-9 implied that the hydroxy group should be β-oriented.Therefore, the configuration of 6 could be defined as 1R*,7R*,10S*,12R*.The configuration of C-8 was deduced to be 8R by the DP4+ probability measurement.The 1R*,7R*,8R*,10S*,12R* isomer showed an overwhelming possibility (100%) compared with the 1R*,7R*,8S*,10S*,12R* isomer (0%), which confirmed the configuration of 6.   Clabellane B ( 7) was found to possess the molecular formula C 20 H 33 IO 4 according to the sodium adduct ion at m/z 487.1318 (calcd for 487.1316) in the HRESIMS.The UV, IR, and NMR data (Figures 2 and 3) were quite similar to that of 6, indicating they are close analogs.The major difference between 7 and 6 was found in the molecular formula; the bromine atom in 6 was replaced by an iodine atom in 7. The heavy atom effect of C-7 (δ 46.0 in 7 and δ 63.0 in 6) revealed that the bromine atom at C-7 in 6 was replaced by an iodine atom in 7. Since the specific rotation, ECD, and NOESY data of 7 also resembled those of 6, and the stereochemistry of 7 was thus assigned identically.
Clabellane C (8) has the molecular formula C 20 H 32 O 4 (IHD = 5), as deduced from HRESIMS and NMR spectrometric data.The IR spectrum of 8 indicated the presence of hydroxy (3378 cm −1 ) and exomethylene (1643 cm −1 ) functionalities.The 1 H and 13 C NMR data of 8 were analogous to those of 6, suggesting it is also a dolabellane-type diterpenoid.Clabellane B (7) was found to possess the molecular formula C20H33IO4 according to the sodium adduct ion at m/z 487.1318 (calcd for 487.1316) in the HRESIMS.The UV, IR, and NMR data (Figures 2 and 3) were quite similar to that of 6, indicating they are close analogs.The major difference between 7 and 6 was found in the molecular formula; the bromine atom in 6 was replaced by an iodine atom in 7. The heavy atom effect of C-7 (δ 46.0 in 7 and δ 63.0 in 6) revealed that the bromine atom at C-7 in 6 was replaced by an iodine atom in 7. Since the specific rotation, ECD, and NOESY data of 7 also resembled those of 6, and the stereochemistry of 7 was thus assigned identically.
Clabellane C (8) has the molecular formula C20H32O4 (IHD = 5), as deduced from HRESIMS and NMR spectrometric data.The IR spectrum of 8 indicated the presence of hydroxy (3378 cm −1 ) and exomethylene (1643 cm −1 ) functionalities.The 1 H and 13 C NMR data of 8 were analogous to those of 6, suggesting it is also a dolabellane-type diterpenoid.In our earlier investigation, we found that the methanol extract of Clavularia spp.had an apoptotic effect on oral cancer cells [10].Hence, most of the isolated compounds were evaluated in vitro for their antiproliferative effect against oral cancer cells (Ca9-22) using  S22, new iodinated dolabellane 7 exhibited strong cytotoxic effects with an IC 50 value of 15.7 µM, while the eudensamane-type sesquiterpenes were less active.It is noted that the cytotoxic effect of compound 15 (IC 50 = 24.9µM) was seven times higher than that of 16 (IC 50 = 166.7 µM), which implied the position of the C=C bond might change the bioactivity dramatically.For the halogenated dolabellanes 6, 7, and 13, the iodinated one showed the best cytotoxic activity and the chlorinated one was the weakest.Moreover, clasamane E (5) having a peroxide bridge showed a relatively good cytotoxic activity against the Ca9-22 cell among all isolated eudensamanetype sesquiterpene lactones.

General
Merck KGaA (Darmstadt, Germany) cellite 545 (0.02-0.1 mm) and silica gel 60 (0.015-0.040 mm) were used for dry sample and flash column chromatography, respectively.Phenomenex (Torrance, CA, USA) C 18 , phenyl-hexyl, and biphenyl columns were used for high-performance liquid chromatography (HPLC).The Shimadzu (Kyoto, Japan) HPLC instrument consisted of an LC-40D solvent delivery module, DGU-405 degassing unit, CBM-40 system controller, CTO-40S column oven, SPD-M40 photo diode array detector, and FRC-10A fraction collector.A Jasco (Tokyo, Japan) V-650 spectrophotometer was used for measuring UV data.A Jasco FT/IR-4X spectrophotometer was chosen for measuring IR data.A Jasco J-815 CD spectrometer was used for recording the circular dichroism data.Specific optical rotation was measured by a Jasco P-2000 polarimeter.NMR spectra were obtained from Varian (Palo Alto, CA, USA) Mercury Plus 400 MHz and VNMRS 600 MHz FT-NMR spectrometers.A Bruker (Bremen, Germany) APEX II spectrometer was used for detecting HRSIMS.

Animal Material
The coral materials were collected in May 2021 off the coast of Green Island, Taiwan.Coral specimens were identified as Clavularia spp.by Dr. Yuan-Bin Cheng.A voucher specimen (code: CI2021) was given, and the specimens were deposited at the Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung, Taiwan.It is noted that the coral materials were previously identified as Clavularia inflata [10].However, the materials contained more than one species and can only be recognized as Clavularia spp.
a Measured at 150 MHz in CDCl 3 .b Measured at 100 MHz in CDCl 3 .
a Measured in CDCl 3b Measured at 400 MHz in CDCl 3 .