Unusual Anti-allergic Diterpenoids from the Marine Sponge Hippospongia lachne

Hipposponlachnins A (1) and B (2), possessing an unprecedented tetracyclo [9.3.0.02,8.03,7] tetradecane ring system, and the probable biogenetic precursor [3, (1R*,2E,4R*,7E,10S*,11S*,12R*)-10, 18-diacetoxydolabella-2,7-dien-6-one] of 1‒2 were isolated from the South China Sea marine sponge Hippospongia lachne. The structures of the novel compounds were determined using integrated spectroscopic methods in combination with single-crystal X-ray diffraction analysis. Compounds 1‒2 showed potent inhibitory activity on the release of β-hexosaminidase, a biomarker for degranulation, as well as the production of pro-inflammatory cytokine IL-4 and lipid mediator LTB4 in DNP-IgE-stimulated RBL-2H3 cells.

The planar structure of 1 was elucidated via a detailed analysis of 2D NMR data (Fig. 2). The COSY spectrum readily revealed the presence of four isolated spin systems: (a) C-2-C-3-C-7, (b) C-5-C-4-C-16, (c) C-9-C-10-C-11, and (d) C-12-C-13-C-14. The observed HMBC correlations from H 3 -16 to C-3, C-4, and C-5 and from H-12 to C-11 revealed the connectivities of C-3 and C-4 and of C11 and C-12, respectively. The HMBC correlations from H-3, H-4, H-5, and H-7 to C-6 indicated that C-5 and C-7 were connected via the carbonyl carbon C-6. The additional HMBC correlations of H 3 -17/C-2, C-7, C-8, and C-9, H 3 -15/C-1, C-2, C-11, and C-14, and H-12/C-10, tethered the remaining three fragments a, c, and d by inserting the "loose ends" of the quaternary   carbons C-1 and C-8, and located the three methyl groups at C-1, C-4, and C-8, respectively, demonstrating a tetracyclo [9.3.0.0 2,8 .0 3,7 ] tetradecane core in 1. Moreover, the presence of a hydroxy isopropyl group at C-12 was supported by the HMBC correlations from the two remaining methyl groups, H 3 -19 and H 3 -20, to C-12 and the oxygenated quaternary carbon at δ C 72.7 (C-18). The unassigned hydroxyl group was attached at the downfield-shifted carbon at (δ C 67.2, C-10). Thus, the planar structure of hipposponlachnin A (1) was established as shown in Fig. 2. The relative configuration of 1 was determined by NOESY data (Fig. 2) and H-7/H-9α suggested that they were oriented in opposite directions. Therefore, the configuration of 1 was determined and confirmed by single-crystal X-ray diffraction analysis with Cu Kα irradiation as shown in Fig. 3.
Hipposponlachnin B (2) was also obtained as a colorless crystal. Its molecular formula of C 20 H 32 O 3 was deduced from its HRESIMS data, consistent with that of 1. The overall appearance of the NMR spectrum of 2 revealed close structural similarity between 1 and 2, indicating the presence of the same tetracyclo [9.3.0.0 2,8 .0 3,7 ] tetradecane skeleton in both compounds. Further analysis of 1D and 2D NMR spectra of 2 established the planar structure of 2, which was identical to that of 1 (Fig. 2). Moreover, the almost mirror-image CD spectra of 1 and 2 (see Supplementary Fig. S21) indicated that these two compounds must be a pair of diastereoisomers with  Compound 3 was identified as a known dolabellane diterpene, named (1R*,2E,4R*,7E,10S*,11S*,12R*)-10,18-diacetoxydolabella-2,7-dien-6-one, by comparing its physical and spectroscopic features with the data reported in the literature 15,16 .
In addition, the biosynthetic pathway for 1-3 was proposed as shown in Fig. 4. 3 could be produced from geranylgeranyl diphosphate (GGPP) following a series of biosynthetic cyclization, migration, oxidation, and acetylation processes [17][18][19] . The four-membered ring in 1 and 2 might be formed from 3 via an intramolecular [2 + 2] cycloaddition reaction between two double bonds (Δ 2,3 and Δ 7,8 ) 7,20 . The deacetylation could be carried out by an esterase 21 . What's more, compound 3 with 7, 8-E geometry rather than 7,8-Z isomer is the predicted precursor of 1 and 2, because the high rotational barrier around the C 7 -C 8  Anti-allergic activity evaluation. Subsequently, RBL-2H3 cells were used as a model system to evaluate the anti-allergic activity of 1-2 22 . As indicated in Fig. 5A, no significant cytotoxicity was observed in RBL-2H3 cells after 24 h of treatment with 1 or 2. 1 and 2 exhibited higher activity (IC 50 49.37 and 23.91 μM, respectively) in the release of β-hexosaminidase inhibition (Fig. 5B), compared with the market-available anti-asthmatic drug, ketotifen fumarate (IC 50 = 63.88 μM). In addition, 1 and 2 suppressed IL-4 production in a dose-dependent manner (Fig. 5C) and significantly inhibited LTB4 release in activated RBL-2H3 cells compared with untreated control (Fig. 5D). The results indicated that 1 and 2 are promising new anti-allergic lead compounds.
In summary, two novel tetracyclic diterpenes (1-2) were isolated from the marine sponge H. lachne, together with their probable biogenetic precursor (3). The structures of new compounds were elucidated by spectroscopic and single-crystal X-ray diffraction analysis. To our knowledge, only one diterpenoid with a similar skeleton, namely vulgarisin A, derived from the Chinese Medicinal Plant Prunella vulgaris, has been reported to date 19 . These two novel compounds represented an unprecedented tetracyclo [9.3.0.0 2,8 .0 3,7 ] tetradecane ring system from marine source for the first time. Moreover, the potent inhibitory activity on the release of β-hexosaminidase for 1-2 suggested that these two bioactive diterpenoids can be the potential therapeutic agents for the treatment of allergy.

General Experimental Procedures.
Optical rotation data were recorded on a PerkinElmer model 341 polarimeter with a 10 cm length cell at room temperature. UV and IR (KBr) spectra were obtained on a Hitachi U-3010 spectrophotometer and Jasco FTIR-400 spectrometer, respectively. CD spectra were obtained on a Jasco J-715 spectropolarimeter in MeCN. NMR spectra including 1D and 2D spectra were acquired at room temperature on Bruker AMX-500 instrument. HRESIMS data were obtained on a Waters Q-Tof micro YA019 mass spectrometer. Reversed-phase HPLC was performed on YMC-Pack Pro C18 RS  Extraction and Isolation. The sponge H. lachne (1.2 kg, dry weight) was cut into small pieces and exhaustively extracted by percolation with 95% EtOH at room temperature to give 56.0 g extract, which was suspended in H 2 O and extracted sequentially with EtOAc to afford EtOAc-soluble extract (27.5 g). The EtOAc-soluble extract was dissolved in 90% aqueous MeOH, and extracted with petroleum ether to yield petroleum ether-soluble extract (15.2 g). The 90% aqueous MeOH phase was diluted to 60% MeOH with H 2 O, and extracted with CH 2 Cl 2 to yield CH 2 Cl 2 -soluble extract (10.0 g). The CH 2 Cl 2 -soluble extract was subjected to column chromatography on silica gel with a gradient elution of MeOH in CH 2 Cl 2 to give six fractions (A-F). Fraction C was subjected to column chromatography (CC) on Sephadex LH-20 eluting with CH 2 Cl 2 -MeOH (1:1) to afford three subfractions (C1-C3). Subfraction C2 was separated by CC on ODS to give nine subfractions (C2A-C2I) with 80% MeOH-H 2 O as elution. Fr. C2C was purified by reversed-phase semipreparative HPLC (65% MeCN/H 2 O, 2.0 mL/ min, 250 nm) to obtain 3 (3.0 mg, t R = 51.0 min). Fr. C2D was further purified by reversed-phase semipreparative HPLC (50% MeCN/H 2 O, 2.0 mL/min, 210 nm) to obtain 1 (2.4 mg, t R = 15.2 min) and 2 (2.8 mg, t R = 18.0 min).
Chemical structure data. All compounds were ≥ 98% pure, which were supported by the NMR spectra of the compounds provided in the Supporting Information.  Anti-allergic assay. Cell viability were determined using the MTT method. RBL-2H3 cells were plated into a 96-well plate at 5 × 10 5 cells per well (100 μL/well) for 24 h. Subsequently, cells were incubated with hipposponlachnins A (1) and B (2) for 24 hours and medium was replaced with MTT solution (250 μg/mL) and incubated at 37 °C for 4 h. The medium was carefully discarded and formazan was resuspended in 150 μL of dimethyl sulfoxide (DMSO). The absorbance was measured at 490 nm using a microplate reader. Values measured from untreated cells were considered to represent 100% viability. RBL-2H3 cells were seeded in a 24-well plate at 5 × 10 5 cells per well, and sensitized with dinitrophenyl (DNP)-specific IgE (DNP-IgE) (1 μg/mL) at 37 °C overnight. DNP-IgE-sensitized cells were preincubated with hipposponlachnins A (1) and B (2) for 30 min, and then stimulated with DNP-BSA for 1.5 h. To measure the activity of β-hexosaminidase released from the cells, cultured media were centrifuged (17,000 g, 10 min) at 4 °C. The supernatant (50 μL) was mixed with 50 μL of 0.1 M sodium citrate buffer (pH 4.5) containing 10 mM 4-nitrophenyl N-acetyl-β-D-glucosaminide in a 96-well plate, and then incubated for 90 min at 37 °C. The absorbance was measured at 405 nm after terminating the reaction by 0.2 M glycine (pH 10.0). To measure Interleukin-4 (IL-4) and Leukotriene B 4 (LTB 4 ) level in cultured media, all cultured media were centrifuged at 4 °C, and hipposponlachnins A (1) and B (2) were stored at − 80 °C until assay. IL-4 and LTB4 were quantified using an ELISA kit according to the manufacturer's instructions. The data were analyzed using a one-way ANOVA followed by Dunnett's Multiple Comparison Test with GraphPad Prism software (GraphPad Prism version 5.01 for Windows, San Diego, CA, USA). The values are expressed as the means ± SD. The differences with p < 0.05 were considered significant.