Isolation of Sesquiterpenoids and Steroids from the Soft Coral Sinularia brassica and Determination of Their Absolute Configuration

Two undescribed rearranged cadinane-type sesquiterpenoids (1–2), named sinulaketol A-B, together with one new chlorinated steroid (3), one new gorgosterol (4), one known sesquiterpene (5), one known dibromoditerpene (6) and two known polyhydroxylated steroids (7–8) were isolated from the soft coral Sinularia brassica. The structures of these compounds were established by extensive spectroscopic analysis, including HRESIMS, 1D, and 2D NMR spectroscopy. Their absolute configurations were also determined by the ECD calculations and DP4+ probability analysis. Antileishmanial activity of compounds 1–8 was evaluated in vitro against the amastigote forms of Leishmania donovani, in which compounds 3, 6, and 7 inhibited the growth of L. donovani by 58.7, 74.3, 54.7%, respectively, at a concentration of 50 μM. Antimicrobial effect of the isolated compounds were also evaluated against Candida albicans, Staphylococcus aureus, and Escherichia coli. Compound 6, a brominated diterpene, exhibited antimicrobial effect against S. aureus.


Introduction
Soft corals of the genus Sinularia, which belong to the order Alcyonacea, are important sources of bioactive natural products and have been the target of study since the middle of twentieth century. Numerous secondary metabolites have been isolated from various Sinularia species, particularly sesquiterpenoids, steroids, diterpenoids, and others [1][2][3][4][5]. There is a hypothesis that constituents from soft corals may possess remarkable bioactivities, because their derivatives may act as chemical defense compounds against their predators in various marine ecological environment, to ensure their protection and survival [6]. The object of our study S. brassica is an invertebrate which is widely distributed in the Red Sea and Indo-Pacific. To date, only limited steroids, which belong to the withanolide, ergostane, and pregnene type, have been reported in earlier studies on this species [7,8]. Therefore, a further chemical investigation may likely discover more interesting compounds from the soft coral S. brassica. Our investigation on the chemical compositions of S. brassica had led to the discovery of two undescribed rearranged cadinane-type sesquiterpenoids (1-2), one new chlorinated steroid (3), one new gorgosterol (4) (Figure 1), one known sesquiterpene (5), one known dibromoditerpene (6) and two known polyhydroxylated steroids (7)(8). The structures of these compounds were established by spectrometric and spectroscopic approaches, quantum mechanics-based chemical shifts calculation with support of the DP4+ probability analysis, and comparison with previous literature. The antileishmanial activity of compounds 1-8 was evaluated in vitro against L. donovani amastigote forms. In these compounds were established by spectrometric and spectroscopic approaches, quantum mechanics-based chemical shifts calculation with support of the DP4+ probability analysis, and comparison with previous literature. The antileishmanial activity of compounds 1-8 was evaluated in vitro against L. donovani amastigote forms. In addition, the antimicrobial activities of all the isolates were tested against Candida albicans, Staphylococcus aureus, and Escherichia coli.
All the isolates 1-8 were tested in vitro against the amastigote forms of L. donovani. Among the compounds 1-8, the new chlorinated steroid (3), dibromoditerpene (6) and the polyhydroxylated steroid (7) inhibited the growth of L. donovani by 58.7, 74.3, and 54.7%, respectively, at a concentration of 50 µM, while compounds 3, 6, and 7 did not exhibit cytotoxicity against the THP-1 cells (Table 5). In addition, all the isolated compounds were evaluated for their antimicrobial activity against Candida albicans, Staphylococcus aureus, and Escherichia coli. Among the tested metabolites, compound 6 showed antimicrobial activity against S. aureus (Table 5, Figure S51).

Discussion
As discussed above, we isolated two rare rearranged cadinene-type sesquiterpenoids (1-2) and two new steroids (3)(4). Their structures were elucidated by 1D ( 1 H, 13 C) and 2D NMR experiments (HSQC, HMBC, COSY, and NOESY) and confirmed by HRESIMS. Their absolute configurations were comprehensively established by the ECD calculations and NMR chemical shifts calculations supported by DP4+ analysis.
Sesquiterpenoid is one of the significant metabolites of the genus Sinularia. During the period of 2013-2021, 35 new sesquiterpenes, including four new carbon skeletons, were isolated from this genus [5]. Although new sesquiterpenoids 1 and 2 belong to cadinane-type skeleton, it is the first report on the cadinane sesquiterpenoids with unprecedented carbon backbone at C-15. They might be derived from the cleavage of C1-C6 bond of ylangene-type sesquiterpenoids, rarely found in the soft corals belonging to the genus Dendronephthya and Lemnalia [17][18][19].
A new chlorinated steroid (3) suppressed the growth of L. donovani by 58.7% without cytotoxicity (at 50 µM). Pinnaterpene C (6), a dibrominated diterpene, displayed both antileishmanial and antimicrobial activities. This study is the first antileishmanial and antimicrobial investigation for known Pinnaterpene C.

Materials and Methods
The soft coral Sinularia brassica May 1898 was collected in Van Phong bay, Khanh Hoa province, Vietnam in May 2014 and identified by experts at Institute of Oceanography, Nha Trang, Vietnam. A voucher specimen (E54582) was deposited with the Oceanography Museum, Institute of Oceanography in Nha Trang, Vietnam.

Computational Details
The conformational searches of each possible isomer were performed by applying 10,000 steps of the Monte Carlo multiple minimum method with PRCG energy minimization using the Merck Molecular Force Field (MMFF) in gas phase to obtain five conformers for each isomer of 1 and 2, 18 and 16 conformers for 24S and 24R isomers of 3 respectively, with a 10 kJ/mol energy window limit. Those occurring conformers were then subjected to geometrical optimization and vibrational frequencies calculation using DFT/B3LYP/6-31G(d,p) level with Gaussian 16 package (Gaussian Inc., Wallingford, CT, USA). All optimized structures have no imaginary frequency, and those of compounds 1 and 2 were then proceeded to ECD calculations at TD-DFT/CAM-B3LYP/6-31+G(d,p) (CPCM, acetonitrile) level. ECD curves were Boltzmann averaged and extracted by SpecDis v.1.7 software with half-band of 0.3 eV.
The optimized conformers of two possible diastereomers of compound 3 (18 and 16 conformers for 24S and 24R isomers, respectively) were calculated for NMR shielding constants using the gauge-independent atomic orbitals (GIAO) method at the DFT/ rmPW1PW91/6-311+G(d,p) (CPCM, chloroform) level. Chemical shift values were calculated by an equation below where δ x calc is the calculated NMR shift for nucleus x, and σ 0 is the shielding tensor for the proton or carbon nuclei in tetramethylsilane calculated at the same condition.
The calculated NMR properties were averaged based on the Boltzmann populations of all conformers, and the DP4+ probability analysis was conducted using the Excel sheet provided by Grimblat et al [20,21].

Biological Assays
Antileishmanial activity was evaluated according to the protocol of Institut Pasteur Korea [22]. Antimicrobial activity was determined by dropping compounds on agar plate covered with S. aureus KCTC 3881 (bacterium), E. coli DH5α (bacterium) and C. albicans KCTC 27242 (fungus) (Korean Collection for Type Cultures, Daejeon, Korea). The cell culture was spread on each agar plate up to 10 8 cells/plate, and then 10 µL of each compound (0.3 and 3 mM) dissolved in 50% DMSO in water were dropped on the plate followed by incubating at 37 • C for 16 h. Antimicrobial activity was determined by the mark of the cell inhibition. Kanamycin and nystatin were used as positive controls against the bacterium and fungus, respectively. The IC 50 values of the compounds were determined using S. aureus KCTC 3881, E. coli DH5α and C. albicans KCTC 27242 in a 96-well-plate. The cell culture was diluted up to 0.5 McFarland Standard with sterilized media. For C. albicans, the culture broth was 100 times more diluted before use. Each well was filled with 95 µL of culture broth. The compounds dissolved in DMSO were added until the final concentrations (1, 2, 5, 10, 20, 50, 100, 200, and 500 µg/mL), and the final volume of each well was 100 µL [23]. The plate was incubated at 37 • C for 16 h. Cell inhibition was measured at 600 nm (for S. aureus and E. coli) and 530 nm (for C. albicans) using Multiskan™ GO Microplate Spectrophotometer (Thermo Scientific, Waltham, MA, USA). The IC 50 value was calculated using an exponential trend line calculated in Excel (Microsoft, Redmond, WA, USA). Kanamycin and nystatin were used as positive controls against the bacterium and fungus, respectively.