Rare norisodinosterol derivatives from Xenia umbellata: Isolation and anti-proliferative activity

Two new rare 30-norisodinosterol derivatives, 23,24-dimethylcholest-16-ene-3β,5α,6β,11α,20(R)-pentol 3-monoacetate (1) and 23,24-dimethylcholest-16-ene3β,5α,6β,20(R)-tertrol 3-monoacetate (2), along with a known steroid, 3β,5α,6β,11α,20β-pentahydroxygorgosterol (3), were identified from Xenia umbellata. The structures of the isolated compounds were determined by analyses of the measured spectra (1D and 2D nuclear magnetic resonance, mass spectrometry, and infrared). The biosynthetic pathway of the new norisodinosterols was proposed. Compound 1 exhibited potent cytotoxicity against HepG2, PC-3, and HT-29 with IC50 values of 4.70 ± 0.2, 5.60 ± 0.6, and 4.00 ± 0.4 μg/mL, respectively. On the contrary, compound 3 showed less potent cytotoxicity against HepG2 with IC50 value of 22.20 ± 1.0 μg/mL. Two DNA-binding dyes have been used for the morphological detection of viable, apoptotic, and necrotic cells. The early apoptotic cell death was observed in all types of treated tumour cells. The late apoptotic cells are highly present in HepG2 cells with compound 3 compared with other cancer cells except for compound 1. The anti-proliferative activity of compounds 1 and 3warranted further investigation.


Introduction
Alcyonacea (Phylum: Cnidaria; Class: Anthozoa) survives worldwide in tropical and subtropical seawaters and does not have the hard calcium carbonate skeleton. Members of Alcyonacea inhabit the inner reefs below the stony corals [1]. They are known for their productivity of secondary metabolites such as terpenoids and steroids. The soft corals are important member of marine fauna which have cells in the form of toxic stinging nematocysts with the absence of the rigid protective skeleton of scleractinians. They also have the ability to produce toxic substances [2][3][4].
Family Xeniidae (Alcyonacea) consists of 20 genera and 162 species. They live in tropical waters as the Red Sea. They present as yellow cylindrical clavate colonies [3]. They have many varieties of long feather-like tentacles and their polyps pump water into the colony, creating a rhythmic pulsing motion. They are named as pulsing Xenia and pom-pom Xenia. This genus is known for its productivity of terpenoids and steroids [5,6].

Determination of anti-proliferative effect of compounds 1 and 3
The cytotoxicity of the isolated compounds was evaluated against (HepG2, PC-3, and HT-29) human cancer cells using sulphorhodamine B assay (SRB), according to the previously published [8,9].

AO/EtBr staining for detection of apoptosis
The DNA-binding dyes AO and EtBr have been used for the morphological detection of viable, apoptotic, and necrotic cells. The procedures have been done as previously reported [11,12].

Statistical analysis
Data are presented as mean and SD. Statistical significance was acceptable to a level of p < 0.05. All statistical analyses were performed using GraphPad Prism software, version 6.00 (GraphPad Software, La Jolla, CA, USA).
Ethical approval: The conducted research is not related to either human or animal use.

Results and discussion
A Red Sea soft coral specimen, identified as X. umbellata, was extracted with a mixture of organic solvents at room temperature, yielding a viscous oily material (21.4 g).
The total extract was evaluated for its cytotoxic effect against HepG2 and displayed cytotoxicity with IC 50 (19.74 ± 1.98 μg/mL). The aforementioned promising anti-proliferative results directed the further chemical investigation of the X. umbellata extract. It was subjected to normal-phase silica gel column chromatography and PTLC to give two new steroidal derivatives, compounds 1 and 2 together with a previously identified steroid compound 3 ( Figure 1).   Figure S1s-v). The later deduction furnished the gross structure of 1 as 23,24-dimethylcholest-16en-pentahydroxy monoacetate. The position of the five hydroxyl groups was deduced from 13 C NMR, DEPT, and HSQC spectra. The methine proton resonating at δ H 5.13 (dddd, J = 11.1, 11.1, 5.1, and 5.1 Hz) implies acetylated hydroxyl located at C-3, since this is the sole available location flanked by two methylene groups. 1 H-1 H COSY and HMBC spectra recognized the positions of the other hydroxyl groups; H-3 and H-19 are both correlated with the quaternary carbon at δ C 76.4 (C-5) ppm as well as this carbon is also correlated with the proton resonating at δ H 3.55 (dd, J = 3.4 and 1.7 Hz, H-6), which implies that positions 5 and 6 are both hydroxylated. The fourth hydroxyl group was decided by observing the signal at δ H 3.99, which appeared as dt with J values 9.4 and 5.1 Hz. This proton could be positioned on several locations within the carboskeleton of 1; however, the HMBC correlations were observed between this proton and the two quaternary carbons at C-10 and C-13. The OH group is positioned on C-11. The fifth hydroxyl group was deduced from the HMBC to be depicted at C-20, based on the correlations between the methyl group resonated at (δ H 1.37, s) and the olefinic proton resonated at δ H 5.48 (dd, J = 3.4 and 1.7 Hz) with the C-20 resonated at δ C 75.9. The relative stereochemistry of 1 was elucidated on the basis of nuclear overhauser effect spectroscopy (NOESY) correlations ( Figure S1w-y) and analyses of J values. The multiplicity of the methine assignment at 5.13 ppm had the normal complexity for the 3α-carbinol proton of an A/B trans-steroid. This unusually downshifted signal is typical of 3β-hydroxysterols bearing a 5α-hydroxyl group, which esterified by acetyl moiety [13]. The downshift of the Me-19 signal at 1.35 was indicative of the β-orientation of the C-6 hydroxyl group. The large J-value of H-11 implies its axial orientation and hence α-OH. The strong NOESY correlations between H-3 and H-6 and between Me-19 and H-11 supported the proposed orientations.
Since the spectral data of 1 coincided with the reported data and the R configuration was recognized at C-20 [14]. From these data, compound 1 was concluded to be 23,24dimethylcholest-16-ene-3β,5α,6β,11α,20(R)-pentol 3monoacetate. Compound 2, [α] D 22 -81.1 (c 0.01, CHCl 3 ), was obtained as a gummy substance and had the molecular formula C 31 H 52 O 5 , as determined by HRESIMS, requiring six degrees of unsaturation. The IR spectrum of 1 showed absorptions due to hydroxyl and acetyl groups (λ max 3,387 and 1,730/cm, respectively). The 13 C NMR spectroscopical data of 2 ( Table 2) revealed the presence of 31 carbon signals, which were identified by assistance of the DEPT spectrum. They were categorized into six quaternary carbons, eight sp 3 methines, one sp 2 methine, eight methylenes, and eight methyls. Analysis of the spectral data of 2 and comparison with those of 1 revealed the great similarity between both structures. However, the lack of 16 mass unit in mass spectrum and the absence of signals due to CH-11 in the NMR spectra ( 1 H and 13 C NMR, DEPT, HSQC, COSY, HMBC, and NOESY) in case of compound 2 allowed the determination of its structure as 23,24-dimethylcholest-16-ene-3β,5α,6β,20(R)-tetrol 3-monoacetate. 3β,5α,6β,11α,20β-Pentahydroxygorgosterol (3) was identified by comparing the measured spectral data with the reported [15].

Biosynthesis of 30-norisodinosterols carbon skeleton
Dinosterol (4), a C-30 sterol isolated from free swimming dinoflagellates, is the biosynthetic precursor to the cyclopropyl-containing sterol gorgosterol (5) (Figure 1) [16]. Despite the reported information, gorgosterol was originally isolated from several gorgonian species and, moreover, was proved to be a symbiont product and was not isolated from the free swimming dinoflagellates [16].
The unsaturation (Δ 24 ) was common among the initial tetracyclic precursor, lanosterol and cycloartenol, in all sterol-building organisms. The presence of such unsaturation site renders the molecules prone to decorations by reduction (e.g. formation of cholesterol) and methylation, which started from simple methylation at C-24, may extend to multiple alkylations. This situation was common among marine organisms [17]. A hypothetical biosynthetic pathway of the current isolated norisodinosterol derivatives (1 and 2) could be started with brassicasterol (6) which undergoes methylation by a methyalting agent (mainly S-adenosylmethionine). Then reduction of Δ 5 and formation of Δ 16 could be performed. A more evidenced route to the biosynthesis of these C-29 sterols (Scheme 1) started from compound 4, which lead to the construction of 30-norisodinosterol skeleton through the reduction of Δ 22 , demethylation at C-4 and then formation of Δ 16 . It is worthy to mention that the isolation of 23,24dimethylchlesta-5,22-dien-3β-ol from the soft coral Sarcophyton elegans supports the speculation that dinosterol is the precursor of compounds 1 and 2 along with the symbiont production nature of these compounds [18].
The compounds isolated in the current manuscript are steroidal derivatives. They are characterized by the presence of four rings arranged in a unambiguous molecular configuration. Multi-functionality gave them unique molecular structures. Consequently, the steroids have potential diversity of bioactivity. Generally, steroids have two common biological functions: as vital components of cell membranes that alter membrane fluidity and as signalling molecules. The isolated steroids showed different chemical functionality and potent anti-proliferative activities. After further pharmacological investigation and mechanistically studies, they may be a lead of anti-cancer drug.

Conclusion
Xenia umbellata, a soft coral, was collected from the Red Sea and found to produce two new steroidal derivatives, compounds 1 and 2, together with a known steroid, compound 3. The chemical structures of the isolated compounds were determined by analyses of the measured spectroscopic data. The anti-proliferative activities of compounds 1 and 3 have been evaluated against hepatocellular carcinoma (HepG2), prostate adenocarcinoma (PC-3), and colorectal adenocarcinoma (HT-29) human cell lines. Compound 1 exhibited potent cytotoxic effect against tumour cell lines, HepG2, PC-3, and HT-29. Compounds 1 and 3 displayed late apoptotic effect in HepG2 cells. The anti-proliferative activity of compounds 1-3 warranted further investigation. Extra work should be carried out to establish the chemoecological functions of nordinosterols (1 and 2) and to assess their impact on the host soft coral organism.