Sponge-Derived 24-Homoscalaranes as Potent Anti-Inflammatory Agents

Scalarane-type sesterterpenoids are known for their therapeutic potential in cancer treatments. However, the anti-inflammatory properties of this class of metabolites remain elusive. Our current work aimed to investigate the anti-inflammatory scalaranes from marine sponge Lendenfeldia sp., resulting in the isolation of six new 24-homoscalaranes, lendenfeldaranes E–J (1–6). The structures of the new metabolites were determined by extensive spectroscopic analyses, and the absolute configuration of 1 was established by electronic circular dichroism (ECD) calculations. Compounds 2 and 3 were discovered to individually reduce the generation of superoxide anions, and compound 1 displayed an inhibitor effect on the release of elastase. These three compounds were proven to be the first anti-neutrophilic scalaranes.


Introduction
The marine sponges of genus Lendenfeldia have been studied for decades since first being reported in 1982 [1]. Further investigation of this genus revealed more than 50 compounds categorized into scalarane-type sesterterpenoids [1][2][3][4][5], other types of sesterterpenoids [4], amino acids [6], steroids [7,8], iminosugars [9], naphthalenes [10], lipid [10,11], and diphenyl ethers [12]. Our group has extensively studied scalarane-type sesterterpenoids over the past few years and has found that they demonstrate a wide structural diversity [5,[13][14][15][16]. This class of compounds possesses a pentamethyl-D-homoandrostane skeleton. Alkylated scalaranes are usually known as homoscalarane, exhibiting methylation at C-20 or C-24. In the current report, we summarize a series of structural classification for scalaranes from the Lendenfeldia sp. sponge. In detail, a normal 25carbon scalarane represents the basic type of scalarane, while the 26-carbon ones can be further sorted into tetra-and pentacyclic homoscalarane groups. Nor-homoscalaranes were characterized with a missing methyl at position 18 and bishomoscalaranes are defined for the scalaranes with a pair of methylations at both C-20 and C-24. Additionally, it is noteworthy that most of them present a different oxidation in positions C-12, C-16, C-22, C-24, and C-25 [17]. The biological properties of scalarane-type sesterterpenoids were extensively studied with special emphasis on cytotoxic and anti-proliferative properties [5]. For instance, the scalaranes isolated from sponges of Hyrtios, Hippospongia, Lendenfeldia, Phyllospongia, and Psammocinia genus were examined to show potent cytotoxicity against A498, ACHN, MIA-paca, PANC-1, CV-1, molt-4, K562, DLD-1, HCT-116, and T-47D cancer cell lines at low concentrations (< 4 μM) [5,15,18]. The previous pharmacological studies on scalaranes have also revealed several possible anti-proliferative mechanisms, including the inhibition of Hsp90 and topoisomerase II [16], and the binding of DNA [19]. In addition, these sesterterpenoids were also reported to exhibit other pharmacological activities, such as antimicrobial, anti-fungal, anti-viral, and so on [16]. However, only few studies have explored the antiinflammatory activity of this class of metabolites. A sponge-derived scalarane, named deacetylphylloketa, was reported to exhibit anti-inflammatory activity by regulating the expression levels of pro-inflammatory factors (TNF-α, IL-6, and IL-1β) and anti-inflammatory factors (Nrf-2 and HO-1). It could downregulate the expressions of iNOS and COX-2, as well as attenuate nuclear translocation of NF-κB [20]. Recently, we focused our ongoing studies on a marine sponge identified as Lendenfeldia sp. From the result of our studies on this species, we report herein the isolation, structural determination, and bioactivity of six new 24-homoscalaranes, lendenfeldaranes E-J (1-6) ( Figure 1). Moreover, the extensive biological screening suggested the isolates significantly inhibited superoxide anion generation and elastase release in neutrophils responding to N-formyl-methionylleucyl-phenylalanine (fMLF).

Results and Discussion
Specimens of the marine sponge Lendenfeldia sp. were collected by hand by self-contained underwater breathing apparatus (scuba) diving off the coast of Southern Taiwan in 2012, and stored frozen at -20 °C until extraction. The frozen sponge was minced and extracted with ethyl acetate (EtOAc). The fractionation of the EtOAc-soluble extract led to the production of 11 fractions A-K. Fractions I and J were further purified by normal-phase and reversed-phase HPLC to afford scalaranes 1-6.
Compound 3 (lendenfeldarane G) was obtained as an amorphous powder. Its molecular formula was determined to be C29H48O6 by (+)-HRESIMS with six degrees of unsaturation. The IR spectra indicated the presence of OH (3462 cm -1 ) and ester carbonyl (1729 cm -1 ) functionalities. 1D and 2D NMR data disclosed a 6/6/6/6/5 pentacyclic skeleton, which was closely related to compound 2. The only difference between these two compounds was a reductive substitution at C-12 in 3. Comparing the 1 H and 13 C NMR data ( Table 2) Figures S17-S24).
Lendenfeldarane H (4) was also obtained as an amorphous powder. The (+)-HRESIMS (m/z 501.31879, calculated for C28H46O6 + Na, 501.31866) and NMR data of 4 indicated a molecular formula C28H46O6 with six degrees of unsaturation. The IR spectra revealed the presence of OH (3292 cm -1 ) and ester carbonyl (1740 cm -1 ) groups. Based on the analysis of the NMR spectra between 3 and 4 ( Table 2), a missing methoxy group signal was found, together with different assignments at position 25 (4: δH 5.35, 1H, dd, J = 6.8, 3.2 Hz/δC 96.5; 3: δH 4.85, 1H, d, J = 6.4 Hz/δC 103.9). The HMBC cross peak ( Figure 2) further revealed the replacement of an α-hydroxy group in 4. The configuration of 4 was confirmed to be identical to that of 3 by NOESY experiment (Figure 5). Compound 4 was finally assigned, as shown in Figure 1 (Supplementary Materials, Figures S25-S32).  Lendenfeldarane I (5) was isolated as a white powder. The (+)-HRESIMS at m/z 497.28757 (calculated for C28H42O6 + Na, 497.28736) indicated a molecular formula of C28H42O6. The IR spectra showed absorptions for OH (3460 cm -1 ), C=O (1701 cm -1 ), and ester carbonyl (1745 cm -1 ) functionalities. The 13 C NMR data (Table 3) revealed 28 carbons signals stored by HSQC and DEPT, including a ketone at δC 211.9 and two ester carbonyls at δC 170.7 and 172.3. Therefore, three degrees of unsaturation were built up, then the rest of the five unsaturated degrees were speculated to come from a pentacyclic homoscalarane. The 1D and 2D NMR data disclosed the existence of a compound 5-like 6/6/6/6/5 pentacyclic skeleton. The only found divergence was located at E-ring, the disappearance of the ketal carbon in 2 was replaced by an ester carbonyl in 5. Then the HMBC ( Figure  2) cross-peak from H-24 to C-17, C-18, and C-25 allowed the establishment of a γ-valerolactone. The stereochemical configuration was identical to that of other scalarane sesterterpenes [5] (Table 3)   Several lines of scientific and clinical evidences indicated that neutrophil oxidants and elastase secreted by inflammatory cells play critical roles in the pathogenesis of several inflammation-related disorders, such as psoriasis, arthritis, acute respiratory distress syndrome, and systemic lupus erythematos [22]. NADPH oxidase type 2 (NOX2) is an important enzyme that causes superoxide generation during respiratory burst, a predominant neutrophil function against foreign pathogens. An excessive amount of superoxide release can damage host tissues and lead to neutrophilic inflammation. Besides, another critical role, elastase, can contribute to neutrophil migration toward the inflammatory site, and activates neutrophil degranulation that causes the release of more elastolytic proteases to degrade the proteins from invading pathogens. Many recent studies have revealed that the pharmacological inhibition of NOX2 and elastase can restrict inflammatory responses, indicating the promising therapeutic potential of NOX2 and elastase inhibitors for treating neutrophil-dominant inflammatory disorders [23].
In the current study, the inhibition of fMLF-activated superoxide anion generation and elastase release were evaluated on metabolites 1-6 to characterize their property of anti-neutrophilic inflammation (Table 4). From these results, compound 1 showed the most potent inhibitory effect independently against elastase release, as well as a slight enhancing property in superoxide generation. With an additional acetyl functionality at C-22, compounds 2 and 3 both displayed activity of superoxide inhibition, but not elastase inhibition. These results suggest a crucial role of C-22-acetylation of homoscalarane on specifically affecting neutrophilic targets, such as NOX2 and elastase. Percentage of inhibition (Inh %) at 10 μM. Results are presented as mean ± S.E.M. (n = 3~5). * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the control (DMSO). a Concentration necessary for 50 % inhibition (IC50). b Inh = inhibition, Enh = Enhancement.

General Experimental Procedures
Optical rotations spectra were recorded on a JASCO P-1010 polarimeter (cell length 10 mm) (JASCO, Tokyo, Japan). IR spectra were obtained with a Thermo Scientific Nicolet iS5 FT-IR spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). ECD spectra were recorded on JASCO-815 CD spectrometer. The NMR spectra were obtained on a JEOL ECZ 400S or an ECZ 600R NMR (JEOL, Tokyo, Japan), using the residual CHCl3 signals (δH 7.26 ppm) and CDCl3 (δC 77.0 ppm) as the internal standards for 1 H and 13

Animal Material
Specimen of the marine sponges Lendenfeldia sp. was collected by hand using self-contained underwater breathing apparatus (scuba) diving off the coast of Southern Taiwan on September 5, 2012, and stored in a freezer until extraction. The specimen was identified by one of the authors (Y.M. Huang). A voucher specimen (NMMBA-TWSP-12006) was deposited in the National Museum of Marine Biology and Aquarium, Pingtung, Taiwan.

Superoxide Anion Generation and Elastase Release by Human Neutrophils
Human neutrophils were obtained from healthy human volunteers and were isolated by Ficoll centrifugation and dextran sedimentation. Purified neutrophils were re-suspended in calcium (Ca 2+ )free Hank's balanced salt solution (HBSS) buffer at pH 7.4 and were maintained at 4 °C before use. For the superoxide anion generation assay, neutrophils (6 × 10 5 cell/mL) were equilibrated in ferricytochrome c (0.6 mg/mL) and Ca 2+ (1 mM) at 37 °C for 5 min and incubated with DMSO (0.1%) or tested compounds for another 5 min [24]. Cells were activated with formyl-methionyl-leucylphenylalanine (fMLF, 0.1 μM) for 10 min after priming with cytochalasin B (CB, 1 μg/mL) for 3 min.
The change in absorbance was monitored continuously at 550 nm with a spectrophotometer (Hitachi U-3010). For the elastase release assay, neutrophils (6 × 10 5 cell/mL) were equilibrated in MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide (100 μM) and Ca 2+ (1 mM) at 37 °C for 5 min and incubated with dimethyl sulfoxide (DMSO) (0.1%) or test compounds for another 5 min. The cells were activated with fMLF (0.1 μM) for 10 min after the priming with CB (0.5 μg/mL) for 3 min. The change in absorbance was monitored continuously at 405 nm with a spectrophotometer [24]. The results are recorded as the mean ± SEM of three measurements. The inhibition % was measured at 10 μM concentration of each compound, and IC50 values were estimated from dose-response curves. Statistical analysis was carried out using Student's t-tests with SigmaPlot (Systat Software, San Jose, CA, USA).

Conclusions
The current work is the first to illustrate the anti-neutrophilic inflammatory properties of scalarane-type sesterterpenoids, and reported a series of metabolites with novel structures, lendenfeldaranes E-J (1-). These results also suggested a structural dependent specificity of C-22acetylation in neutrophilic targets, which motivates future research to illustrate structural dependent specificity as well as further clarify the corresponding molecular mechanisms of the active leads.