Scalarane-Type Sesterterpenoids from the Marine Sponge Lendenfeldia sp. Alleviate Inflammation in Human Neutrophils

Sponge-derived scalaranes are remarkable sesterterpenoids previously found to exhibit profound inhibitory effects against neutrophilic inflammation. In our current work, we constructed the metabolomic profile of marine sponge Lendenfeldia sp. for the first time using a tandem mass spectrometry (MS/MS) molecular networking approach. The results highlighted the rich chemical diversity of these scalaranes, motivating us to conduct further research to discover novel scalaranes targeting neutrophilic inflammation. MS- and NMR-assisted isolation and elucidation led to the discovery of seven new homoscalaranes, lendenfeldaranes K–Q (1–7), characterized by methylation at C-24, together with five known derivatives, lendenfeldarane B (8), 25-nor-24-methyl-12,24-dioxoscalar-16-en-22-oic acid (9), 24-methyl-12,24,25-trioxoscalar-16-en-22-oic acid (10), felixin B (11), and 23-hydroxy-20-methyldeoxoscalarin (12). Scalaranes 1–4 and 6–12 were assayed against superoxide anion generation and elastase release, which represented the neutrophilic inflammatory responses of respiratory burst and degranulation, respectively. The results indicated that 1–3 and 6–12 exhibited potential anti-inflammatory activities (IC50 for superoxide anion scavenging: 0.87~6.57 μM; IC50 for elastase release: 1.12~6.97 μM).

Lendenfeldarane L (2) had a molecular formula of C28H40O6 at m/z 495.27187 (calcd. for C28H40O6 + Na, 495.27171) according to (+)-HRESIMS, corresponding to nine degrees of unsaturation. 1 H and 13 C data (Table 1) analyses indicated that 2 was of the 24-homoscalarane class, which was similar to 1. The most striking difference between 1 and 2 was the presence of signals assigned to the hydroxymethyl group (δH 4.03,1H,d,J = 12.0 Hz;3.87,1H,d,J = 12.0,1.2 Hz/δC 62.8, at C-10 in 1 being replaced by a carboxylic acid (δC 178.8) in 2. Interpretation of the 2D NMR spectroscopic data of 2 confirmed the above elucidation, and thus established the planar structure ( Figure 5). The correlations from the NOESY experiment of 2 indicated configurations of the stereogenic centers in core rings AE of 2 that were identical to those of 1 ( Figure 5). Therefore, the configurations of the stereogenic carbons of 2 were elucidated as (5S,8S,9S,10R,12S,13S,14S,24S). Accordingly, the structure of lendenfeldarane L (2) was established. Compound 3 was isolated as an amorphous powder, and the molecular formula was determined by (+)-HRESIMS as C26H38O5 at m/z 453.26085 (calcd. for C26H38O5 + Na, 453.26115). Comparison of the 1 H and 13 C NMR data of 3 ( Table 2) with those of 2, the chemical shifts of CH-12 in 2 (δH 5.56, dd, J = 2.8, 2.8 Hz/δC 73.6) being shifted up-field in 3 (δH 4.65, br s/δC 69.7), along with missing acetyl signals, suggesting that the 12-acetoxy group in 2 was replaced by a hydroxy group in 3. Interpretation of the 2D NMR spectroscopic data of 3 confirmed the above elucidation, and thus established the planar structure ( Figure 6). The configurations of the stereogenic centers in 3 were assigned as (5S,8S,9S,10R,12S,13S,14S,24S), the same as those in 2, according to the NOESY spectrum ( Figure 6). Thus, the structure of 3 was elucidated, and the compound was named lendenfeldarane M.   In the NOESY experiment of 1 (Figure 4), the acetoxy group at C-12 was assigned on the α-face, according to a NOESY correlation between H-12 and H 3 -23. It was found that the NOESY correlations of 1 were similar to those of 13, suggesting close configurations of these two molecules. Therefore, lendenfeldarane K (1) was assigned as having a structure with the same relative stereochemistry as lendenfeldarane C (13) owing to the stereogenic carbons that 1 had in common with 13, and the configurations of the stereogenic centers of 1 were elucidated as (5S*,8R*,9S*,10R*,12S*,13S*,14S*,24S*). Accordingly, the structure of 1 was established. Additionally, scalaranes 1-12 were obtained from the same target organism, Lendenfeldia sp., and the absolute configuration of 10 was determined by single-crystal X-ray diffraction analysis. Therefore, it is biogenetically reasonable to conclude that 1-9, 11, and 12 have the same absolute configurations as 10, and the stereogenic carbons of 1 were elucidated as (5S,8R,9S,10R,12S,13S,14S,24S).
Lendenfeldarane L (2) had a molecular formula of C 28 H 40 O 6 at m/z 495.27187 (calcd. for C 28 H 40 O 6 + Na, 495.27171) according to (+)-HRESIMS, corresponding to nine degrees of unsaturation. 1 H and 13 C data (Table 1) analyses indicated that 2 was of the 24-homoscalarane class, which was similar to 1. The most striking difference between 1 and 2 was the presence of signals assigned to the hydroxymethyl group (δ H 4.03, 1H, d, J = 12.0 Hz; 3.87, 1H, d, J = 12.0, 1.2 Hz/δ C 62.8, CH 2 -22) at C-10 in 1 being replaced by a carboxylic acid (δ C 178.8) in 2. Interpretation of the 2D NMR spectroscopic data of 2 confirmed the above elucidation, and thus established the planar structure ( Figure 5). The correlations from the NOESY experiment of 2 indicated configurations of the stereogenic centers in core rings A-E of 2 that were identical to those of 1 ( Figure 5). Therefore, the configurations of the stereogenic carbons of 2 were elucidated as (5S,8S,9S,10R,12S,13S,14S,24S). Accordingly, the structure of lendenfeldarane L (2) was established.
Lendenfeldarane L (2) had a molecular formula of C28H40O6 at m/z 495.27187 (calcd. for C28H40O6 + Na, 495.27171) according to (+)-HRESIMS, corresponding to nine degrees of unsaturation. 1 H and 13 C data (Table 1) analyses indicated that 2 was of the 24-homoscalarane class, which was similar to 1. The most striking difference between 1 and 2 was the presence of signals assigned to the hydroxymethyl group (δH 4.03,1H,d,J = 12.0 Hz;3.87,1H,d,J = 12.0,1.2 Hz/δC 62.8, at C-10 in 1 being replaced by a carboxylic acid (δC 178.8) in 2. Interpretation of the 2D NMR spectroscopic data of 2 confirmed the above elucidation, and thus established the planar structure ( Figure 5). The correlations from the NOESY experiment of 2 indicated configurations of the stereogenic centers in core rings AE of 2 that were identical to those of 1 ( Figure 5). Therefore, the configurations of the stereogenic carbons of 2 were elucidated as (5S,8S,9S,10R,12S,13S,14S,24S). Accordingly, the structure of lendenfeldarane L (2) was established. Compound 3 was isolated as an amorphous powder, and the molecular formula was determined by (+)-HRESIMS as C26H38O5 at m/z 453.26085 (calcd. for C26H38O5 + Na, 453.26115). Comparison of the 1 H and 13 C NMR data of 3 ( Table 2) with those of 2, the chemical shifts of CH-12 in 2 (δH 5.56, dd, J = 2.8, 2.8 Hz/δC 73.6) being shifted up-field in 3 (δH 4.65, br s/δC 69.7), along with missing acetyl signals, suggesting that the 12-acetoxy group in 2 was replaced by a hydroxy group in 3. Interpretation of the 2D NMR spectroscopic data of 3 confirmed the above elucidation, and thus established the planar structure ( Figure 6). The configurations of the stereogenic centers in 3 were assigned as (5S,8S,9S,10R,12S,13S,14S,24S), the same as those in 2, according to the NOESY spectrum ( Figure 6). Thus, the structure of 3 was elucidated, and the compound was named lendenfeldarane M.   In the NOESY experiment of 1 (Figure 4), the acetoxy group at C-12 was assigned on the α-face, according to a NOESY correlation between H-12 and H3-23. It was found that the NOESY correlations of 1 were similar to those of 13, suggesting close configurations ), HMBC ( of these two molecules. Therefore, lendenfeldarane K (1) was assigned as having a structure with the same relative stereochemistry as lendenfeldarane C (13) owing to the stereogenic carbons that 1 had in common with 13, and the configurations of the stereogenic centers of 1 were elucidated as (5S*,8R*,9S*,10R*,12S*,13S*,14S*,24S*). Accordingly, the structure of 1 was established.
Lendenfeldarane L (2) had a molecular formula of C28H40O6 at m/z 495.27187 (calcd. for C28H40O6 + Na, 495.27171) according to (+)-HRESIMS, corresponding to nine degrees of unsaturation. 1 H and 13 C data (Table 1) analyses indicated that 2 was of the 24-homoscalarane class, which was similar to 1. The most striking difference between 1 and 2 was the presence of signals assigned to the hydroxymethyl group (δH 4.03, 1H, d, J = 12.0 Hz; 3.87, 1H, d, J = 12.0, 1.2 Hz/δC 62.8, CH2-22) at C-10 in 1 being replaced by a carboxylic acid (δC 178.8) in 2. Interpretation of the 2D NMR spectroscopic data of 2 confirmed the above elucidation, and thus established the planar structure ( Figure 5). The correlations from the NOESY experiment of 2 indicated configurations of the stereogenic centers in core rings AE of 2 that were identical to those of 1 ( Figure 5). Therefore, the configurations of the stereogenic carbons of 2 were elucidated as (5S,8S,9S,10R,12S,13S,14S,24S). Accordingly, the structure of lendenfeldarane L (2) was established. Compound 3 was isolated as an amorphous powder, and the molecular formula was determined by (+)-HRESIMS as C26H38O5 at m/z 453.26085 (calcd. for C26H38O5 + Na, 453.26115). Comparison of the 1 H and 13 C NMR data of 3 ( Table 2) with those of 2, the chemical shifts of CH-12 in 2 (δH 5.56, dd, J = 2.8, 2.8 Hz/δC 73.6) being shifted up-field in 3 (δH 4.65, br s/δC 69.7), along with missing acetyl signals, suggesting that the 12-acetoxy group in 2 was replaced by a hydroxy group in 3. Interpretation of the 2D NMR spectroscopic data of 3 confirmed the above elucidation, and thus established the planar structure ( Figure 6). The configurations of the stereogenic centers in 3 were assigned as (5S,8S,9S,10R,12S,13S,14S,24S), the same as those in 2, according to the NOESY spectrum ( Figure 6). Thus, the structure of 3 was elucidated, and the compound was named lendenfeldarane M.
), and protons with NOESY (  Compound 3 was isolated as an amorphous powder, and the molecular formula was determined by (+)-HRESIMS as C 26 H 38 O 5 at m/z 453.26085 (calcd. for C 26 H 38 O 5 + Na, 453.26115). Comparison of the 1 H and 13 C NMR data of 3 ( Table 2) with those of 2, the chemical shifts of CH-12 in 2 (δ H 5.56, dd, J = 2.8, 2.8 Hz/δ C 73.6) being shifted up-field in 3 (δ H 4.65, br s/δ C 69.7), along with missing acetyl signals, suggesting that the 12-acetoxy group in 2 was replaced by a hydroxy group in 3. Interpretation of the 2D NMR spectroscopic data of 3 confirmed the above elucidation, and thus established the planar structure ( Figure 6). The configurations of the stereogenic centers in 3 were assigned as (5S,8S,9S,10R,12S,13S,14S,24S), the same as those in 2, according to the NOESY spectrum ( Figure 6). Thus, the structure of 3 was elucidated, and the compound was named lendenfeldarane M.      In the NOESY experiment of 1 (Figure 4), the acetoxy group at C-12 was assigned on the α-face, according to a NOESY correlation between H-12 and H3-23. It was found that the NOESY correlations of 1 were similar to those of 13, suggesting close configurations of these two molecules. Therefore, lendenfeldarane K (1) was assigned as having a structure with the same relative stereochemistry as lendenfeldarane C (13) owing to the stereogenic carbons that 1 had in common with 13, and the configurations of the stereogenic centers of 1 were elucidated as (5S*,8R*,9S*,10R*,12S*,13S*,14S*,24S*). Accordingly, the structure of 1 was established. Additionally, scalaranes 1-12 were obtained from the same target organism, Lendenfeldia sp., and the absolute configuration of 10 was determined by single-crystal X-ray diffraction analysis. Therefore, it is biogenetically reasonable to conclude that 1-9, 11, and 12 have the same absolute configurations as 10, and the stereogenic carbons of 1 were elucidated as (5S,8R,9S,10R,12S,13S,14S,24S).
Lendenfeldarane L (2) had a molecular formula of C28H40O6 at m/z 495.27187 (calcd. for C28H40O6 + Na, 495.27171) according to (+)-HRESIMS, corresponding to nine degrees of unsaturation. 1 H and 13 C data (Table 1) analyses indicated that 2 was of the 24-homoscalarane class, which was similar to 1. The most striking difference between 1 and 2 was the presence of signals assigned to the hydroxymethyl group (δH 4.03, 1H, d, J = 12.0 Hz; 3.87, 1H, d, J = 12.0, 1.2 Hz/δC 62.8, CH2-22) at C-10 in 1 being replaced by a carboxylic acid (δC 178.8) in 2. Interpretation of the 2D NMR spectroscopic data of 2 confirmed the above elucidation, and thus established the planar structure ( Figure 5). The correlations from the NOESY experiment of 2 indicated configurations of the stereogenic centers in core rings AE of 2 that were identical to those of 1 ( Figure 5). Therefore, the configurations of the stereogenic carbons of 2 were elucidated as (5S,8S,9S,10R,12S,13S,14S,24S). Accordingly, the structure of lendenfeldarane L (2) was established. Compound 3 was isolated as an amorphous powder, and the molecular formula was determined by (+)-HRESIMS as C26H38O5 at m/z 453.26085 (calcd. for C26H38O5 + Na, 453.26115). Comparison of the 1 H and 13 C NMR data of 3 ( Table 2) with those of 2, the chemical shifts of CH-12 in 2 (δH 5.56, dd, J = 2.8, 2.8 Hz/δC 73.6) being shifted up-field in 3 (δH 4.65, br s/δC 69.7), along with missing acetyl signals, suggesting that the 12-acetoxy group in 2 was replaced by a hydroxy group in 3. Interpretation of the 2D NMR spectroscopic data of 3 confirmed the above elucidation, and thus established the planar structure ( Figure 6). The configurations of the stereogenic centers in 3 were assigned as (5S,8S,9S,10R,12S,13S,14S,24S), the same as those in 2, according to the NOESY spectrum ( Figure 6). Thus, the structure of 3 was elucidated, and the compound was named lend-), and protons with NOESY (   Compound 4 was isolated as an amorphous powder, which was determined to have a molecular formula of C 30 H 44 O 7 by (+)-HRESIMS at m/z 539.29763 (calcd. for C 30 H 44 O 7 + Na, 539.29792), requiring nine degrees of unsaturation. Analysis of the 1D NMR data (Table 2) indicated that compound 4 was an analogue of a known 24-homoscalarane, lendenfeldarane D (14) (Figure 1) [2]. The main difference was the presence of an additional hydroxy group at C-24 in 4, which was supported by MS data, less shielding of C-24 (from δ C 77.7 to 103.4), and a combination of HMBC cross-peaks from H 3 -26 (δ H 1.59, s) to C-24 (δ C 103.4) and C-17 (δ C 161.6) (Figure 7). The configuration of 4 was established by comparing the NOESY correlations ( Figure 7) to those of 14. The NOESY interactions of H 2 -22 with H 3 -20 and H 3 -21; and H 3 -23 with H-12 and H 3 -21, revealed the β-orientations of H 3 -20, H 3 -21, H 2 -22, H 3 -23, and H-12. H-5 showed an interaction with H-9, and H-9 was correlated with H-14, suggesting the α-orientations of H-5, H-9, and H-14. According to the above analyses, the structure of 4 was determined and the stereogenic centers were assigned as (5S,8R,9S,10R,12S,13S,14S). This compound was named lendenfeldarane N, although the stereochemistry of C-24 in 4 was not determined at this stage owing to the lack of a NOESY correlation between H 3 -24 and any protons.
Lendenfeldarane L (2) had a molecular formula of C28H40O6 at m/z 495.27187 (calcd. for C28H40O6 + Na, 495.27171) according to (+)-HRESIMS, corresponding to nine degrees of unsaturation. 1 H and 13 C data (Table 1) analyses indicated that 2 was of the 24-homoscalarane class, which was similar to 1. The most striking difference between 1 and 2 was the presence of signals assigned to the hydroxymethyl group (δH 4.03, 1H, d, J = 12.0 Hz; 3.87, 1H, d, J = 12.0, 1.2 Hz/δC 62.8, CH2-22) at C-10 in 1 being replaced by a carboxylic acid (δC 178.8) in 2. Interpretation of the 2D NMR spectroscopic data of 2 confirmed the above elucidation, and thus established the planar structure ( Figure 5). The correlations from the NOESY experiment of 2 indicated configurations of the stereogenic centers in core rings AE of 2 that were identical to those of 1 ( Figure 5). Therefore, the configurations of the stereogenic carbons of 2 were elucidated as (5S,8S,9S,10R,12S,13S,14S,24S). Accordingly, the structure of lendenfeldarane L (2) was established. Compound 3 was isolated as an amorphous powder, and the molecular formula was determined by (+)-HRESIMS as C26H38O5 at m/z 453.26085 (calcd. for C26H38O5 + Na, 453.26115). Comparison of the 1 H and 13 C NMR data of 3 ( Table 2) with those of 2, the chemical shifts of CH-12 in 2 (δH 5.56, dd, J = 2.8, 2.8 Hz/δC 73.6) being shifted up-field in 3 (δH 4.65, br s/δC 69.7), along with missing acetyl signals, suggesting that the 12-acetoxy group in 2 was replaced by a hydroxy group in 3. Interpretation of the 2D NMR spectroscopic data of 3 confirmed the above elucidation, and thus established the planar structure ( Figure 6). The configurations of the stereogenic centers in 3 were assigned as (5S,8S,9S,10R,12S,13S,14S,24S), the same as those in 2, according to the NOESY spectrum ( Figure 6). Thus, the structure of 3 was elucidated, and the compound was named lendenfeldarane M.
Lendenfeldarane L (2) had a molecular formula of C28H40O6 at m/z 495.27187 (calc for C28H40O6 + Na, 495.27171) according to (+)-HRESIMS, corresponding to nine degre of unsaturation. 1 H and 13 C data (Table 1) analyses indicated that 2 was of the 24-hom scalarane class, which was similar to 1. The most striking difference between 1 and 2 w the presence of signals assigned to the hydroxymethyl group (δH 4.03,1H,d,J = 12.0 H 3.87,1H,d,J = 12.0,1.2 Hz/δC 62.8, at C-10 in 1 being replaced by a carboxylic ac (δC 178.8) in 2. Interpretation of the 2D NMR spectroscopic data of 2 confirmed the abo elucidation, and thus established the planar structure ( Figure 5). The correlations from t NOESY experiment of 2 indicated configurations of the stereogenic centers in core rin AE of 2 that were identical to those of 1 ( Figure 5). Therefore, the configurations of t stereogenic carbons of 2 were elucidated as (5S,8S,9S,10R,12S,13S,14S,24S). According the structure of lendenfeldarane L (2) was established. Compound 3 was isolated as an amorphous powder, and the molecular formula w determined by (+)-HRESIMS as C26H38O5 at m/z 453.26085 (calcd. for C26H38O5 + N 453.26115). Comparison of the 1 H and 13 C NMR data of 3 ( Table 2) with those of 2, t chemical shifts of CH-12 in 2 (δH 5.56,dd,J = 2.8,2.8 Hz/δC 73.6) being shifted up-field 3 (δH 4.65, br s/δC 69.7), along with missing acetyl signals, suggesting that the 12-aceto group in 2 was replaced by a hydroxy group in 3. Interpretation of the 2D NMR spectr scopic data of 3 confirmed the above elucidation, and thus established the planar structu ( Figure 6). The configurations of the stereogenic centers in 3 were assigned (5S,8S,9S,10R,12S,13S,14S,24S), the same as those in 2, according to the NOESY spectru ( Figure 6). Thus, the structure of 3 was elucidated, and the compound was named len enfeldarane M.
), and protons with NOESY (  Compound 7 was found to have the molecular formula C 25 H 36 O 5 , as deduced from a (+)-HRESIMS peak at m/z 439.24547 (calcd. for C 25 H 36 O 5 + Na, 439.24550), revealing eight degrees of unsaturation. The 13 C and distortionless enhancement by polarization transfer (DEPT) spectra (Table 4) showed 25 carbon signals, which were classified as five methyls, seven sp 3 methylenes, four sp 3 methines, four sp 3 non-protonated carbons, one sp 2 methine, and four sp 2 non-protonated carbons. Based on the 1 H and 13 C spectra (Table 4), four degrees of unsaturation were accounted for, and the remaining four degrees were attributed to a tetracyclic ring. The consecutive COSY correlations ( Figure 10 -5, H-9, H-14, and H-16. According to the above analyses, the stereogenic carbons of this compound were assigned as (5S,8S,9S,10R,13R,14S,16S); the structure of 7 was determined, and the compound was named lendenfeldarane Q.

Assessments of O2 •− Generation and Elastase Release in fMLF-Activated Human Neutrophils
Neutrophils can be induced by N-formyl-methionyl-leucyl-phenylalanine (fMLF), and pathogen-associated molecular patterns (PAMPs) lead to a series of inflammatory responses such as respiratory burst (O2 •− generation) and degranulation (elastase release)   In the NOESY experiment of 1 (Figure 4), the acetoxy group at C-12 was assigned the α-face, according to a NOESY correlation between H-12 and H3-23. It was found the NOESY correlations of 1 were similar to those of 13, suggesting close configurat ), HMBC ( of these two molecules. Therefore, lendenfeldarane K (1) was assigned as having a str ture with the same relative stereochemistry as lendenfeldarane C (13) owing to the st ogenic carbons that 1 had in common with 13, and the configurations of the stereoge centers of 1 were elucidated as (5S*,8R*,9S*,10R*,12S*,13S*,14S*,24S*). Accordingly, structure of 1 was established. Additionally, scalaranes 1-12 were obtained from the same target organism, Lend feldia sp., and the absolute configuration of 10 was determined by single-crystal X-ray fraction analysis. Therefore, it is biogenetically reasonable to conclude that 1-9, 11, and have the same absolute configurations as 10, and the stereogenic carbons of 1 were el dated as (5S,8R,9S,10R,12S,13S,14S,24S).
Lendenfeldarane L (2) had a molecular formula of C28H40O6 at m/z 495.27187 (ca for C28H40O6 + Na, 495.27171) according to (+)-HRESIMS, corresponding to nine degr of unsaturation. 1 H and 13 C data (Table 1) analyses indicated that 2 was of the 24-hom scalarane class, which was similar to 1. The most striking difference between 1 and 2 w the presence of signals assigned to the hydroxymethyl group (δH 4.03, 1H, d, J = 12.0 3.87, 1H, d, J = 12.0, 1.2 Hz/δC 62.8, CH2-22) at C-10 in 1 being replaced by a carboxylic a (δC 178.8) in 2. Interpretation of the 2D NMR spectroscopic data of 2 confirmed the ab elucidation, and thus established the planar structure ( Figure 5). The correlations from NOESY experiment of 2 indicated configurations of the stereogenic centers in core ri AE of 2 that were identical to those of 1 ( Figure 5). Therefore, the configurations of stereogenic carbons of 2 were elucidated as (5S,8S,9S,10R,12S,13S,14S,24S). Accordin the structure of lendenfeldarane L (2) was established. Compound 3 was isolated as an amorphous powder, and the molecular formula w determined by (+)-HRESIMS as C26H38O5 at m/z 453.26085 (calcd. for C26H38O5 + 453.26115). Comparison of the 1 H and 13 C NMR data of 3 ( Table 2) with those of 2, chemical shifts of CH-12 in 2 (δH 5.56, dd, J = 2.8, 2.8 Hz/δC 73.6) being shifted up-field ), and protons with NOESY (   the top 3 peaks in the ±50 Da window throughout the spectrum. A network was then created, in which edges were filtered to have a cosine score above 0.70 and more than four matched peaks. The spectra in the network were annotated based on the experimental MS 2 fragmentations of isolated scalaranes. The library spectra were filtered in the same manner as the input data. The molecular network was visualized and presented using Cytoscape 3.8.2 (Cytoscape 3.8.2, NRNB, San Diego, La Jolla, CA, USA).

Preparation of Human Neutrophils
Blood was acquired from human donors (20~30 years old) by venipuncture under the approval and supervision of the Institutional Review Board (IRB) at Chang Gung Memorial Hospital. Neutrophils were purified utilizing a protocol of dextran sedimentation, hypotonic lysis, and Ficoll Hypaque gradient of erythrocytes according to previous reported methods [22]. Isolated human neutrophils were suspended in a calcium (Ca 2+ )-free HBSS buffer at pH 7.4 and examined by the trypan blue exclusion method (>98% viable cells). Then, neutrophil assessments were performed in HBSS containing 1 mM CaCl 2 at 37 • C.

Measurement of Superoxide Anion (O 2
•− ) Generation O 2 •− generation was assessed using superoxidase dismutase (SOD) inhibitable reduction of ferricytochrome c [22]. After supplementation with ferricytochrome c (0.6 mg/mL), neutrophils (6 × 10 5 cells/mL) were equilibrated at 37 • C and incubated for 5 min before treatment with pure compounds or DMSO (0.1%, control). Since cytochalasin B (CB) can convert neutrophils from phagocytic into secretory cells and facilitate respiratory burst and degranulation through disaggregation of intracellular actin network [24], it was then added (1 µg/mL) to magnify the reaction and the mixture was left for 3 min after activation with 0.1 µM fMLF. Absorbance changes with reduction of ferricytochrome c were monitored continuously at 550 nm using a spectrophotometer (U-3010, Hitachi, Tokyo, Japan).

Measurement of Elastase Release
Degranulation of azurophilic granules was evaluated using an elastase release assay and performed using MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide as the elastase substrate [1]. In brief, neutrophils (6 × 10 5 cells/mL) were equilibrated at 37 • C after supplementation with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide (100 µM), then incubated for 5 min before treatment with pure compounds. CB (0.5 g/mL) was added to magnify the reaction, followed by the addition of fMLF (0.1 µM) to induce cell activation. The variations in absorbance at 405 nm were monitored continuously to assess elastase release.

Statistics
The results were expressed as the mean ± standard deviation (SD). Comparison in each experiment was performed using an unpaired Student's t-test, and a p value of less than 0.05 was considered statistically significant.

Conclusions
The current study revealed the MN-based metabolomic profile of marine sponge Lendenfeldia sp. for the first time, which indicated a splendid diversity of scalarane-type sesterterpenoids. Subsequent isolation of compounds of this type led to the identification of seven new 24-homoscalaranes, lendenfeldaranes K-Q (1-7), together with five known derivatives. Anti-neutrophilic assessments of these isolates not only revealed their great anti-inflammatory potential towards activated neutrophils, but also highlighted the bioactivity-relevant structural importance of the functional group at C-12 in the 24-homoscalarane analogues. These results indicate a great potential of this class of compounds for further development as anti-neutrophilic agents, especially the accomplishment in the synthetic protocol of a scalarane-type sesterterpenoid (16-deacetoxy-12-episcalarafuranacetate) [25], which could give sustainable supply for future industrial development.