Unusual Anti-allergic Diterpenoids from the Marine Sponge Hippospongia lachne

Abstract

Hipposponlachnins A (1) and B (2), possessing an unprecedented tetracyclo [9.3.0.0^2,8.0^3,7] 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.

Introduction

Allergy is an abnormal adaptive immune response against allergens, such as pollen, food, dust mites, cosmetics, mold spores, and animal hairs^1,2. β-Hexosaminidase, typically released in addition to histamine by mast cells after stimulation with antigen, has been used as a biomarker for antigen-induced degranulation in rat basophilic leukemia (RBL-2H3) cells³. Thus, the release of β-hexosaminidase in RBL-2H3 cells has commonly been used as a reliable indicator of inhibitory activity against allergic reactions induced by natural products⁴. Natural products have been proved to be an important source for drug discovery and development. More than three hundreds natural compounds were reported to exhibit diverse biological properties, such as antitumor, antibacterial, antifungal, and a range of other bioactivities⁵. However, only a few natural compounds with anti-allergic activity have been isolated to date⁵. Therefore, there is a great opportunity in search for leads to treat allergy effectively.

Marine invertebrates such as sponges are well-known sources of bioactive natural products⁶. Sponges of the genus Hippospongia are reported to be rich sources of polyketides⁷, sesquiterpenes⁸, sesterterpenes and sulfates⁹, furanoterpenes¹⁰, triterpenoic acids¹¹, fatty acids¹², and sterols¹³. These metabolites exhibited a broad spectrum of biological activities, including cytotoxic⁹, antifungal⁷, anti-inflammatory⁸, coronary vasodilating¹⁴, and RCE-protease inhibitory activities¹¹. A literature survey revealed that no anti-allergic metabolites have been isolated from sponges belonging to this genus. As part of our searching for anti-allergic secondary metabolites from marine sponges, we investigated the sponge Hippospongia lachne, collected from the Xisha Islands of China in March 2013. The 95% ethanol extract of H. lachne showed promising β-hexosaminidase inhibitory activity with IC₅₀ value of 7.8 μg/mL. Further bioactivity-guided fractionation of this extract led to the isolation of compounds 1‒3 (Fig. 1). Of these three metabolites, hipposponlachnins A (1) and B (2) possessed a rare tetracyclo [9.3.0.0^2,8.0^3,7] tetradecane carbon skeleton, exemplifying the first diterpene scaffold obtained from marine source, and showed inhibitory activity on the release of β-hexosaminidase in anti-murine DNP-IgE-stimulated RBL-2H3 cells. Herein, we report the isolation, structure elucidation and anti-allergic activity of the two unprecedented diterpenoids. In addition, biogenetic relationship between these compounds was proposed, suggesting 1‒2 may be obtained from 3 via an intramolecular [2 + 2] cycloaddition reaction.

Figure 1

Structures of compounds 1‒3.

Results and Discussion

Isolation and structure elucidation

Air dried specimen of H. lachne were extracted with 95% ethanol and after evaporation under reduced pressure, the aqueous residue was extracted with EtOAc. The EtOAc layer was repeatedly chromatographed on Sephadex LH-20, silica gel, ODS column and reversed phase high performance liquid chromatography (HPLC) to yield hipposponlachnin A (1), hipposponlachnin B (2), and (1R*,2E,4R*,7E,10S*,11S*,12R*)-10,18-diacetoxydolabella-2,7-dien-6-one (3) (Fig. 1).

Hipposponlachnin A (1) was obtained as a colorless crystal. Its molecular formula was determined to be C₂₀H₃₂O₃ by HRESIMS (m/z 343.2251 [M + Na]⁺), indicating five degrees of unsaturation. The IR spectrum featured characteristic absorptions of hydroxyl (3280 cm⁻¹) and carbonyl (1732 cm⁻¹) groups. The ¹H NMR spectrum revealed the presence of four methyl singlets (δ_H 0.85, 1.11, 1.24, and 1.28), one methyl doublet (δ_H 0.97, d, J = 7.0 Hz), and one oxymethine proton (δ_H 3.72, td, J = 10.0, 4.5 Hz). The ¹³C and DEPT135 NMR spectra displayed 20 carbon resonances (Table 1), including five methyls, four methylenes, seven methines (one oxygenated), and four quaternary carbons (one oxygenated and one carbonyl). The aforementioned data accounted for one degree of unsaturation. The remaining four degrees of unsaturation suggested a tetracyclic core in 1.

Table 1 ¹H and ¹³C NMR Data for 1 and 2 (500, 125 MHz, CDCl₃, TMS, δ ppm).

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 H₃-16 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 H₃-17/C-2, C-7, C-8, and C-9, H₃-15/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, H₃-19 and H₃-20, 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.

Figure 2

Key HMBC and COSY and selected NOE correlations of 1 and 2.

The relative configuration of 1 was determined by NOESY data (Fig. 2). The NOESY correlations of H₃-15/H-2, H₃-15/H₃-17, H₃-15/H-10, H₃-15/H-12, H-2/H-4, and H-2/H₃-17 indicated that these protons and methyl groups were cofacially oriented, whereas the NOESY correlations of H-11/H-3, H-3/H₃-16, H₃-16/H-7, 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.

Figure 3

X-ray crystallographic structures of 1 and 2.

Hipposponlachnin B (2) was also obtained as a colorless crystal. Its molecular formula of C₂₀H₃₂O₃ 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 a variation of configurations at the chiral centers around the carbonyl chromophore. Detailed analysis of the single-crystal X-ray diffraction patterns of 2 confirmed this hypothesis and established the absolute configuration of 2 as 1R,2R,3S,4R,7S,8S,10S,11S,12R (Fig. 3).

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 esterase²¹. 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 C₇-C₈ bond in the intermediate of the 7, 8-Z isomer in the [2 + 2] cycloaddition process makes it difficult to form stable trans-cyclobutane products (1 and 2) (see Supplementary Fig. S26).

Figure 4

Proposed Biosynthetic Pathway of 1–3.

Anti-allergic activity evaluation

Subsequently, RBL-2H3 cells were used as a model system to evaluate the anti-allergic activity of 1‒2²². 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 (IC₅₀ 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 (IC₅₀ = 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.

Figure 5

(A) Cytotoxic effects of 1 and 2 on RBL-2H3 cells. (B) Inhibitory effects of 1 and 2 on the release of β-hexosaminidase in DNP-IgE-activated RBL-2H3 cells. (C) Inhibition of IL-4 production of 1 and 2 in RBL-2H3 cells. (D) Inhibitory activity on lipid mediator LTB₄ secretion of 1 and 2 in RBL-2H3 cells. Data are presented as the mean ± SD values of triplicate determinations. *P < 0.05, **P < 0.01, ***P < 0.001 versus DNP-BSA-treated group (n = 3). Data are analyzed by one-way ANOVA followed by Tukey’s Multiple Comparison Post-Test (GraphPad Prism 5.0).

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 date¹⁹. 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.

Methods

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 (5 μm) columns with a Waters 1525 separation module equipped with a Waters 2998 photodiode array detector. Purifications by column chromatography were performed on silica gel 60 (200–300 mesh; Yantai, China), Sephadex LH-20 (18–110 μm, Pharmacia Co.), and ODS (50 μm, YMC Co.). Analytical thin-layer chromatography was carried out using HSGF 254 plates and visualized by spraying with anisaldehyde-H₂SO₄ reagent.

Animal Material

The marine sponge H. lachne was collected from the Xisha Islands in the South China Sea in March 2013, and identified by Prof. Jin-He Li (Institute of Oceanology, Chinese Academy of Sciences, China). The voucher specimens of H. lachne (RM-2013) is deposited at Research Center for Marine Drugs, State Key Laboratory of Oncogenes and Related Genes, Department of Pharmacy, Renji Hospital, School of Medicine, Shanghai Jiao Tong University.

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 H₂O 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 H₂O, and extracted with CH₂Cl₂ to yield CH₂Cl₂-soluble extract (10.0 g). The CH₂Cl₂-soluble extract was subjected to column chromatography on silica gel with a gradient elution of MeOH in CH₂Cl₂ to give six fractions (A–F). Fraction C was subjected to column chromatography (CC) on Sephadex LH-20 eluting with CH₂Cl₂–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-H₂O as elution. Fr. C2C was purified by reversed-phase semipreparative HPLC (65% MeCN/H₂O, 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/H₂O, 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.

Hipposponlachnin A (1)

Colorless crystal;  + 49 (c 0.11, MeOH); UV (MeOH) λ_max (log ε) 203 (3.37) nm; CD (MeCN) λ_max (Δε) 200 (+0.28), 308 (+0.16) nm; IR (KBr) ν_max 3280, 3167, 2943, 2922, 2861, 1732, 1453, 1410, 1377, 1339, 1306, 1252, 1147, 1122, 1076, 1045, 1025, 962, 942, 905, 858, 806, 672 cm⁻¹; ¹H and ¹³C NMR data see Table 1; HRESIMS m/z 343.2251 [M + Na]⁺ (calcd for C₂₀H₃₂O₃Na, 343.2249).

Hipposponlachnin B (2)

Colorless crystal;  − 44 (c 0.24, MeOH); UV (MeOH) λ_max (log ε) 204 (3.47) nm; CD (MeCN) λ_max (Δε) 208 (−0.13), 308 (−0.25) nm; IR (KBr) ν_max 3375, 3331, 2953, 2926, 2900, 1726, 1704, 1456, 1412, 1377, 1360, 1329, 1263, 1240, 1171, 1130, 1088, 1032, 1009, 982, 957, 906, 874, 839, 804 cm⁻¹; ¹H and ¹³C NMR data see Table 1; HRESIMS m/z 343.2247 [M + Na]⁺ (calcd for C₂₀H₃₂O₃Na, 343.2249).

(1R*,2E,4R*,7E,10S*,11S*,12R*)-10,18-diacetoxydolabella-2,7-dien-6-one (3)

Colorless oil;  − 23 (c 0.15, MeOH); ¹H NMR (CDCl₃, 600 MHz) δ 6.05 (1H, s, H-7), 5.53 (1H, dd, J = 16.2 and 6.0 Hz, H-3), 4.99 (1H, d, J = 16.2 Hz, H-2), 4.81 (1H, dd, J = 10.2 and 3.0 Hz, H-10), 3.13 (1H, m, H-12), 2.69 (1H, dd, J = 11.4 and 3.6 Hz, H-5a), 2.55 (1H, m, H-4), 2.33 (3H, m, H-5b and H-9), 2.06 (3H, s, H-10-OAc), 2.01 (3H, br s, H-17), 1.95 (1H, m, H-13a), 1.92 (3H, s, H-18-OAc), 1.64 (1H, d, J = 10.2 Hz, H-11), 1.61 (3H, s, H-19), 1.49 (2H, m, H-13b and H-14a), 1.44 (1H, m, H-14b), 1.39 (3H, s, H-20), 1.02 (3H, d, J = 6.6 Hz, H-16), 0.86 (3H, s, H-15); ¹³C NMR (CDCl₃, 150 MHz) δ 201.5 (C, C-6), 170.2 (C, C-10-OAc), 170.0 (C, C-18-OAc), 146.3 (C, C-8), 138.2 (CH, C-2), 132.1 (CH, C-3), 131.3 (CH, C-7), 84.7 (C, C-18), 71.9 (CH, C-10), 53.8 (CH, C-11), 51.1 (CH₂, C-5), 47.8 (C, C-1), 44.9 (CH, C-12), 43.8 (CH₂, C-9), 39.1 (CH₂, C-14), 34.1 (CH, C-4), 26.7 (CH₃, C-19), 26.2 (CH₂, C-13), 23.3 (CH₃, C-20), 23.0 (CH₃, C-18-OAc), 21.5 (CH₃, C-17), 21.0 (CH₃, C-10-OAc), 18.3 (CH₃, C-16), 17.5 (CH₃, C-15); HRESIMS m/z 427.2451 [M + Na]⁺ (calcd for C₂₄H₃₆O₅Na, 427.2460).

X-ray Crystallographic Analysis of 1 and 2

Hipposponlachnins A (1) and B (2) were crystallized from MeOH at room temperature. The X-ray crystallographic data of them were measured on a Bruker Apex-II CCD diffractometer employing graphite monochromated Cu-Kα radiation (λ = 1.54178 Å) at 140(2) K (operated in the φ-ω scan mode). The structures were solved by direct method using SHELXS-97 program and refined with full-matrix least-squares calculations on F² using SHELXL-97. Crystallographic data for 1 and 2 have been deposited at the Cambridge Crystallographic Data Centre. Copies of these data can be obtained free of charge via the internet at www.ccdc.cam.ac.uk/conts/retrieving.html or on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [Tel: (+44) 1223-336-408; Fax: (+44) 1223-336-033; E-mail: deposit@ccdc.cam.ac.uk].

Crystallographic data of 1

Colorless crystal, C₂₀H₃₂O₃, M_r = 320.45, orthorhombic, space group P2₁2₁2₁, a = 6.62210(10) Å, b = 15.2395(3) Å, c = 18.4069(3) Å, α = β = γ = 90°, V = 1857.58(6) ų, T = 140 K, Z = 4, D_calcd = 1.146 g/cm³, crystal size 0.20 × 0.12 × 0.05 mm³, μ = 0.588 mm⁻¹, F(000) = 704, 8888 reflections measured (7.53° < 2θ < 139.318°), 3177 unique (R_int = 0.0311, R_sigma = 0.0335). The final R₁ value is 0.0354 [I > 2σ(I)] and wR₂ = 0.0953 (all data). The goodness of fit on F² was 1.060. Flack parameter = 0.13 (13). CCDC deposition number: 1501503.

Crystallographic data of 2

Colorless crystal, C₂₀H₃₂O₃, M_r = 320.45, monoclinic, space group P2₁, a = 8.17720(10) Å, b = 9.71010(10) Å, c = 11.2700(2) Å, α = γ = 90°, β = 92.8110(10)°, V = 893.78(2) ų, T = 140 K, Z = 2, D_calcd = 1.191 g/cm³, crystal size 0.20 × 0.10 × 0.02 mm³, μ = 0.611 mm⁻¹, F(000) = 352, 6940 reflections measured (7.854° < 2θ < 139.132°), 2953 unique (R_int = 0.0248, R_sigma = 0.0310). The final R₁ value is 0.0307 [I > 2σ(I)] and wR₂ = 0.0818 (all data). The goodness of fit on F² was 1.066. Flack parameter = −0.03 (9). CCDC deposition number: 1501504.

Anti-allergic assay

Cell viability were determined using the MTT method. RBL-2H3 cells were plated into a 96-well plate at 5 × 10⁵ 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 × 10⁵ 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 B₄ (LTB₄) 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.