1H NMR-Based Isolation of Anti-Inflammatory 9,11-Secosteroids from the Octocoral Sinularia leptoclados

Octocoral Sinularia leptoclados has been identified as a source of bioactive 9,11-secosteroids. This study adopted a targeted isolation approach to the discovery and analysis of five 9,11-secosteroids, including two novel compounds named sinleptosterols A (1) and B (2) as well as five known analogues (8αH-3β,11-dihydroxy-24-methylene-9,11-secocholest-5-en-9-one (3), 8βH-3β,11-dihydroxy-24-methylene-9,11-secocholest-5-en-9-one (4), leptosterol A (5), (24S)-3β,11-dihydroxy-24-methyl-9,11-secocholest-5-en-9-one (6), and 3β,11-dihydroxy-9,11-secogorgost-5-en-9-one (7)) in terms of 1H-NMR patterns and potency against neutrophilic inflammation. The structure of secosteroids 1 and 2 was deduced from general spectroscopic analysis and an examination of NMR spectra. Among the above-mentioned isolates, compound 4 had the most pronounced effect in inhibiting elastase release and superoxide anion generation, with the IC50 values of 2.96 and 1.63 μM, respectively.


Introduction
Inflammation is one aspect of the regular host reaction to injury or infection caused by toxic chemicals, dead cells, pathogens, irritants, or allergens. The important role of neutrophils in a variety of infectious and inflammatory diseases makes them an attractive target for therapeutic interventions [1]. Numerous herbs and plant-derived compounds have been found to alleviate inflammation [2]; however, very few sources have been identified in marine environments.
Soft corals are reported to produce a variety of secondary metabolites with diverse pharmacological activities. Most of the metabolites from soft corals are sesquiterpenes, diterpenes, and steroids [3]. The octocoral Sinularia leptoclados belongs to the order Alcyonacea, which has been shown to produce a remarkable diversity of steroids in large quantities [4][5][6]. The 9,11-secosteroids found in marine invertebrates such as sponges, corals, ascidian, and mollusk can be structurally characterized by the C-9/11 oxidative cleavage of the C-ring [7,8]. The potent inhibitory effects of 9,11-secosteroids toward neutrophilic inflammation [9][10][11] motivated the current study of chemical compositions with this structural feature.
Mar. Drugs 2020, 18, x 2 of 10 of infectious and inflammatory diseases makes them an attractive target for therapeutic interventions 46 [1]. Numerous herbs and plant-derived compounds have been found to alleviate inflammation [2]; however, very few sources have been identified in marine environments.

48
Soft corals are reported to produce a variety of secondary metabolites with diverse 49 pharmacological activities. Most of the metabolites from soft corals are sesquiterpenes, diterpenes, 50 and steroids [3]. The octocoral Sinularia leptoclados belongs to the order Alcyonacea, which has been 51 shown to produce a remarkable diversity of steroids in large quantities [4][5][6]. The 9,11-secosteroids 52 found in marine invertebrates such as sponges, corals, ascidian, and mollusk can be structurally 53 characterized by the C-9/11 oxidative cleavage of the C-ring [7,8]. The potent inhibitory effects of 9,11-54 secosteroids toward neutrophilic inflammation [9][10][11] motivated the current study of chemical 55 compositions with this structural feature.

Chemical Identification of 9,11-Secosterols
Compound 1 was obtained as a colorless oil. Positive mode high resolution electrospray ionization mass spectrum ((+)-HRESIMS)) of 1 revealed a sodiated adduct ion peak at m/z 451.31811, which established the molecular formula C 28 H 44 O 3 (calcd. for C 28 H 44 O 3 + Na, 451.31827), indicating seven degrees of unsaturation. IR absorption observed at 3369, 2958, and 1708 cm −1 suggested the presence of hydroxy, alkene, and ketonic groups (see Supplementary Materials Figure S2). The 13 C and distortionless enhancement by polarization transfer (DEPT) spectroscopic data revealed 28 carbon signals in this compound, (Table 1), including five methyls, eight sp 3 methylenes (including an oxymethylene), one sp 2 methylene, six sp 3 methines (including one oxymethine), two sp 3 quaternary carbons, three sp 2 methines, and three sp 2 quaternary carbons (including two olefin carbons and one ketonic carbonyl) (see Supplementary Materials Figure S5). It was found that the quaternary carbon signal at δ C 217.5 (C-9) and the proton shifts at δ H 1.40 (s, H 3 -19)   The relative stereochemistry of 1 was explained in terms of correlations observed in a nuclear Overhauser effect spectroscopy (NOESY) experiment, and through a comparison of NMR data between 1 and known secosterol 5. The results suggest that these two compounds possess the same 9,11-secosterol skeleton as well as the same core A-, B-, and D-rings [5]. The configurations at C-3, C-8, C-10, C-13, C-14, and C-17 in 1 were found to be identical to those of 5. Key NOESY correlations for 1 displayed interactions between H-8/H 3 -18 and H-8/H 3 -19. Thus, H-8 should be located on the β-face (Figure 3). A large coupling constant (J = 16.0 Hz) indicated a trans relationship between H-22 and H-23. All these data allowed to identify compound (1) as depicted in Figure 1 and it was named sinleptosterol A. 9,11-secosterol skeleton as well as the same core A-, B-, and D-rings [5]. The configurations at C-3, C-119 8, C-10, C-13, C-14, and C-17 in 1 were found to be identical to those of 5. Key NOESY correlations  The molecular formula of compound (2) was the same as that of 1 (C 28 H 44 O 3 ), as determined by (+)-HRESIMS at m/z 451.3239 (calcd. for C 28 H 44 O 3 + Na, 451.3188), with seven degrees of unsaturation. Its IR bands revealed the presence of hydroxy (3406 cm −1 ) and ketone (1708 cm −1 ) groups (see Supplementary Materials Figure S11). On the basis of the above analysis and a comparison of one-dimensional and two-dimensional NMR experiments on 2 (Table 2), the core structural systems of secosterol 2 were established. It was found that the 1 H and 13 C NMR chemical shifts of 2 (including coupling patterns and coupling constants) resembled those of 1; however, the signals corresponding to the disubstituted alkene between C-22/23 in 1 were replaced by aliphatic methylenes in 2, and one of the methyl groups at C-25 in 1 (Me-26) was replaced by an exocyclic carbon-carbon bond in 2. The observed HMBC correlations fully supported the locations of the functional groups. An olefinic bond was located at C-25/26 from H 2 -26, H 3 -27, H 3 -28 to C-25, respectively; therefore, sinleptosterol B (2) was designated as structure 2. The relative configurations at C-3, C-8, C-10, C-13, C-14, and C-17 of 2 were found to be the same as those of 1 in the core rings A-C. Note that the stereogenic carbons were identical to those of 1, which were in agreement with the observed 1 H and 13 C NMR chemical shifts and proton coupling constants. Therefore, compound (2) was unambiguously identified as presented in Figure 1 and it was named sinleptosterol B.
It is worth noting that the configurations of H-8 for compounds 3 and 4 were elucidated by comparing their 1 H NMR data [12]. A large downfielding of the H-8 as well an upfielding of C-8 were identified to resonate from δ H 2.69/δ C 48.6 (compound 3) to δ H 3.02/δ C 43.8 (compound 4). Moreover, the 1 H-and 13 C-NMR spectroscopic features for positions 7 and 11 were also found to be different between these two compounds  Figures S19-S22).

Anti-Inflammatory Assessment of Isolated 9,11-Secosterols
The anti-inflammatory properties of metabolites 1-7 were characterized by assessing the inhibition of superoxide anion generation and elastase release by human neutrophils in response to fMLP/CB (Table 3). Compounds 1-5 were shown to inhibit superoxide anion generation and elastase release, at concentrations ranging from 1.63 to 8.07 µM. The IC 50 values of compounds 3 and 4 were lower than the other isolates. Secosteroid 7 presented activity at a concentration of 10 µM, indicating that the unique gorgosterol side chain nullified the anti-inflammatory activities.  N-Formyl peptide receptors (FPRs) are a family of G-protein coupled receptors involved in the switching on of leucocyte responses during inflammation. Human FPR1 is expressed primarily in neutrophils, monocytes, and macrophages. It also initiates immune reactions in response to several formyl peptide ligands derived from bacteria or mitochondria [13]. Researchers have previously proven that compounds acting as FPR1 antagonists exhibit anti-inflammatory activity in vitro and in vivo [14][15][16].
Flow cytometry was used to determine whether compounds 1-7 possess binding affinity to FPR1, the receptor-binding assay of N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein (fNLFNYK), an FPR1-specific fluorescent analog, on the surface of neutrophils. The results revealed that fMLF (10 µM) entirely inhibited the binding of fNLFNYK (2 nM), whereas only compound 2 presented a low affinity toward the FPR1 receptor at a concentration of 10 µM (Figure 4).

Coral Material
In November 2018, samples of the soft coral Sinularia leptoclados were obtained by hand using self-contained underwater breathing apparatus (SCUBA) off the coast of Pingtung, Taiwan. The samples were stored in a freezer at −20 • C until extraction. A specimen voucher was deposited with the Research Center for Chinese Herbal Medicine, Chang Gung University of Science and Technology, Taiwan (specimen No.: CGUST-C004-2018-NOV).

Human Neutrophil Superoxide Anion Generation and Elastase Release
Blood samples were collected from healthy adult donors (20-32 years) via venipuncture in accordance with the standard protocol approved by the local institutional review board. Human neutrophils were isolated from peripheral blood through dextran sedimentation, centrifugation in a Ficoll-Hypaque gradient, and hypotonic lysis of red blood cells. In accordance with the above mentioned protocol, assays were performed to measure superoxide anion generation based on the superoxide dismutase-inhibitable reduction of ferricytochrome C. Note that methoxy-succinylalanyl-alanyl-prolyl-valine-p-nitroanilide (MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide) was used as the enzyme substrate for the detection of elastase release [17,18].

Receptor Binding Assay
Receptor binding assays were performed via FACScan analysis of fNLFNYK binding, a fluorescent analog of fMLF, as described in [16]. Neutrophils, differentiated THP-1, or FPR1-expressed HEK-293 were preincubated with test compounds at 4 • C for 5 min and then labelled with fNLFNYK for 30 min. Cells were immediately analyzed via flow cytometry.

Statistical Analysis
All experiments were conducted at least three times and the results are presented as the mean ± SEM. Statistical analysis was performed using the Student's t-test, and a p value < 0.05 was considered statistically significant. Sigma Plot software (version 8.0, Systat Software, San Jose, CA, USA) was used for all statistical analysis [17].

Conclusions
Marine 9,11-secosteroid was first isolated from the gorgonian Pseudopterogorgia americana in 1972 [19]. Research since that time has revealed a variety of compounds in this group from a variety of invertebrates, including sponges, gorgonians, octocorals, ascidian, mollusk, and sea hare [7,20,21]. In recent years, 9,11-secosteroids have attracted considerable attention for their anti-inflammatory and antihepafibrosis properties [9,11,[22][23][24][25]. This study strategically isolated two novel metabolites, sinleptosterols A and B, and five known compounds from the soft coral Sinularia leptoclados. The effectiveness of compounds 1-7 in inhibiting fMLP/CB-induced neutrophilic inflammation was also assessed. Compound 3 and 4 presented the most potent anti-inflammatory activities in terms of superoxide anion production and elastase emancipation. Cell free binding assays also demonstrated that the anti-inflammatory properties of these 9,11-secosteroids did not follow the pathway of conventional FPR1 antagonists.