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Mar. Drugs 2014, 12(7), 4096-4109; https://doi.org/10.3390/md12074096

Article
New Hippolide Derivatives with Protein Tyrosine Phosphatase 1B Inhibitory Activity from the Marine Sponge Hippospongia lachne
1
Laboratory of Marine Drugs, Department of Pharmacy, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China
2
Key Laboratory for Marine Drugs, Department of Pharmacy, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China
3
Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, China
*
Authors to whom correspondence should be addressed.
Received: 23 May 2014; in revised form: 15 June 2014 / Accepted: 19 June 2014 / Published: 8 July 2014

Abstract

:
Five new sesterterpenoids, compounds 15, have been isolated from the sponge Hippospongia lachne off Yongxing Island in the South China Sea. The structures of compounds 15 were elucidated through extensive spectroscopic analysis, including HRMS, 1D, and 2D NMR experiments. The stereochemistry, including absolute configurations of these compounds, was determined by spectroscopic, chemical, and computational methods. Compounds 1 and 5 showed moderate protein tyrosine phosphatase 1B (PTP1B) inhibitory activities with IC50 values of 5.2 μM and 8.7 μM, respectively, more potent than previously reported hippolides.
Keywords:
marine sponge; Hippospongia lachne; sesterterpenoids; absolute configuration; PTP1B

1. Introduction

Protein tyrosine phosphatase 1B (PTP1B), as a therapeutic target for the treatment of Type-II diabetes and obesity, has been the subject of intense study over the past decade [1,2,3,4,5]. Approximately 300 new or known natural products with PTP1B inhibitory activity, have been isolated and identified from various natural resources, many of which are of marine origin [2]. Since the discovery of sulfircin, a sesterterpene sulfate as the first reported marine natural product with PTP1B inhibitory activity, isolated from a deep-water sponge Ircinia (unknown species), marine sponges have proven to be a valuable source of structurally diverse molecules with PTP1B inhibitory activity [4], such as polybromodiphenyl ether from Lamellodysidea herbacea [6], and sesquiterpenoids and sesquiterpene quinones from sponge Dysidea villosa [7].
Marine sponges of the genus Hippospongia (family Spongiidae, order Dictyoceratida) have attracted a great deal of attention as they contain bioactive sesquiterpenes [8,9,10,11], sesterterpenes and sulfates [12,13,14], furanoterpenes [15], triterpenoic acids [16], and polyketides [17]. In our screening program to search for bioactive marine natural products from the South China Sea sponge Hippospongia lachne, we have previously obtained eight new cytotoxic acyclic manoalide-related sesterterpenes, hippolides A–H, with potent cytotoxicity against A549, HeLa, and HCT-116 cell lines [15]. Interestingly, further investigation of crude fractions with PTP1B inhibitory activity from the same sample led to the isolation of a series of new hippolides-related sesterterpenes, compounds 15 (Figure 1), which exhibited different levels of PTP1B inhibitory activity with an IC50 values of 5.2, >50, 33, 14, and 8.7 μM, respectively. Herein, we report the isolation, structure elucidation, and bioactivity of these compounds.
Figure 1. Structures of compounds 15.
Figure 1. Structures of compounds 15.
Marinedrugs 12 04096 g001

2. Results and Discussion

The sponge H. lachne was collected off Yongxing Island and seven connected islets in the South China Sea. The organic extract (110 g) was subjected to chromatography on a silica gel column, followed by consecutive Sephadex LH-20 and RP-HPLC chromatography to yield five new sesterterpenoids, compounds 15 (Figure 1).
Table 1. 13C NMR Data of Compounds 15 (CDCl3).
Table 1. 13C NMR Data of Compounds 15 (CDCl3).
Carbon1 a2 a3 b4 b5 b
1186.5 CH174.5 qC178.5 qC181.9 qC190.2 CH
2117.3 qC33.9 CH230.4 CH242.0 CH2108.0 CH
3146.5 CH53.1 qC58.6 qC123.3 qC151.8 qC
465.6 CH67.8 CH67.4 CH153.5 qC139.7 CH
531.0 CH229.8 CH231.5 CH2117.0 CH114.2 CH
6120.1 CH122.2 CH119.7 CH128.2 CH150.9 CH
7134.8 qC135.1 qC138.8 qC134.5 qC138.2 qC
833.0 CH233.2 CH231.9 CH235.4 CH224.5 CH2
926.0 CH225.7 CH226.8 CH230.5 CH227.4 CH2
10124.0 CH123.1 CH123.6 CH123.9 CH123.5 CH
11135.7 qC136.2 qC136.0 qC135.8 qC136.1 qC
1239.7 CH239.7 CH239.7 CH239.9 CH239.7 CH2
1326.6 CH226.6 CH226.7 CH226.8 CH226.7 CH2
14123.2 CH124.0 CH124.1 CH124.4 CH124.2 CH
15126.2 qC135.0 qC135.1 qC135.1 qC135.0 qC
1639.7 CH239.7 CH239.7 CH239.9 CH239.7 CH2
1726.8 CH226.7 CH226.7 CH226.9 CH226.8 CH2
18124.4 CH124.3 CH124.4 CH124.6 CH124.4 CH
19131.4 qC131.4 qC131.3 qC131.4 qC131.3 qC
2025.7 CH325.7 CH325.7 CH325.7 CH325.7 CH3
2117.7 CH317.7 CH317.7 CH317.7 CH317.7 CH3
2216.0 CH316.0 CH316.0 CH316.0 CH316.0 CH3
2316.2 CH316.1 CH316.1 CH316.0 CH316.1 CH3
2476.9 CH79.7 CH70.8 CH130.9 CH194.2 CH
25 175.2 qC182.9 qC
a Recorded at 100 MHz; b Recorded at 125 MHz.
Compound 1 was obtained as colorless oil. The HR-ESI-MS (high resolution electrospray ionization mass spectrometry) data for 1 gave an adduct ion [M + Na]+ at 392.2567, consistent with a molecular formula of C24H35NO2, implying eight degrees of unsaturation. The 1D and 2D NMR spectra (Supplementary Information) clearly indicated the presence of one aldehyde group (δC 186.5; δH 8.97 1H, s); ten olefinic carbons (δC 117.3, 146.5, 120.1, 134.8, 124.0, 135.7, 123.2, 126.2, 124.4, and 131.4), correlated with five olefinic protons at δH 7.13 (1H, d, J = 5.6 Hz), 5.66 (1H, d, J = 5.0 Hz), 5.11 (1H, m), 5.10 (1H, m), and 5.08 (1H, m); two oxygen-bearing sp3 methines (δC 65.6, δH 5.06; δC 76.9, δH 5.03); seven methylenes, and four methyl carbons (Table 1 and Table 2). The 1H NMR spectrum also displayed a NH proton resonance at δH 5.78 (1H, br s) with no correlation in HSQC spectrum. The COSY correlations for H-8/H-9/H-10, H-12/H-13/H-14, and H-16/H-17/H-18, together with the HMBC correlations of four methyl groups, H3-20/C-18, C-19, and C-21, H3-21/C-18, C-19, H3-22/C-14, C-15, and C-16, and H3-23/C-10, C-11, and C-12, delineated the presence of a farnesyl moiety (Figure 2). In addition, the COSY correlations of H-4/H-5/H-6, and H-3/NH/H-24 as well as the HMBC correlations from H-6 to C-4 and C-24, H-4 to C-2, C-3 and C-24, and H-3 to C-24, revealed a 9-oxa-2-azabicyclo-[3,3,1]-nona-3,7-diene moiety. Furthermore, the HMBC correlations of H-1/C-2, C-3, and C-4 indicated that the aldehyde group was attached to the bicyclo-moiety at C-2. The aforementioned two moieties were connected at C-7, with the evidence of 2JCH and 3JCH correlations from H-8 to C-7 and C-6, C-24, respectively. Therefore, the eight degrees of unsaturation were accounted for by five double bonds, one aldehyde moiety, and two rings.
Table 2. 1H NMR Data of Compounds 15 (CDCl3, J in Hz).
Table 2. 1H NMR Data of Compounds 15 (CDCl3, J in Hz).
Position1 a2 a3 b4 b5 b
18.97, s 9.75, d (3.5)
2 2.93, d (18.0); 2.77, d (18.0)2.69, s3.53, s
37.13, d (5.6) 7.70, dt (3.5, 11.5)
45.03, d (5.0)4.21, dd (7.0, 1.8)4.22, dd (9.5, 3.6) 6.64, d (14.7)
52.64, dd (5.0, 20.0) 2.12, m2.46, m; 2.29, m2.39, m; 2.06, m6.78, d (8.0)6.66, m
65.66, d (5.0)5.74, t (3.9)5.35, t (3.9)6.93, d (8.0)6.86, d (11.0)
81.99, m2.19, m2.06, m2.51, t (8.0)2.39, t (7.5)
92.13, m2.17, m2.06, m2.22, m2.12, m
105.11, m5.09, m5.10, m5.17, t (6.8)5.16, t (7.5)
121.97, m1.97, m2.69, m1.98, m1.96, m
132.05, m2.07, m2.06, m2.06, m2.05, m
145.10, m5.09, m5.10, m5.11, t (6.8)5.11, t (6.8)
161.97, m1.97, m1.98, m1.98, m1.95, m
172.05, m2.07, m2.06, m2.06, m2.05, m
185.08, m5.09, m5.10, m5.10, t (6.3)5.09, t (6.5)
201.68, s1.68, s1.68, s1.68, s1.68, s
211.60, s1.60, s1.59, s1.60, s 1.60, s
221.60, s1.60, s1.59, s1.60, s1.58, s
231.60, s1.60, s1.59, s1.57, s1.60, s
245.06, d (4.1)5.14, s4.58, s6.89, br s9.37, s
25
OH c 4.99, br s11.04, s; 4.02, br s
NH5.78, br s (in CDCl3)7.56 c, s; 7.26, s5.42 c, d (7.5) 8.20 c, m
a Recorded at 400 MHz; b Recorded at 500 MHz; c Recorded at 500 MHz in DMSO.
Figure 2. Selected 1H–1H COSY and 1H–13C HMBC correlations of 15.
Figure 2. Selected 1H–1H COSY and 1H–13C HMBC correlations of 15.
Marinedrugs 12 04096 g002
The relative configuration of 1 was determined by the analysis of NOESY correlations (Figure 3) and computational method (Supplementary Information). The NOESY correlations of H-10/H-12, H-9/H3-23, H-14/H-16, and H-13/H3-22 suggested a 10E, 14E double bond geometry in the farnesyl moiety. However, the remaining stereochemistry of 1 could not be ambiguously assigned by the observed NOESY correlations as shown in Figure 3. Consequently, the computational approach was used to determine the relative structure of 9-oxa-2-azabicyclo-[3,3,1]-nona-3,7-diene moiety. To reduce computational cost, the aliphatic chain of 1 was shortened, as the long aliphatic chain may generate various conformations but has little effect on systematic analysis of all the possible stereoisomers [18,19,20]. On the basis of the relative geometry of H-4 and H-24, as well as the NOSEY correlations indicated in 1a (Figure 3), only four configurations were modeled for theoretical calculations to identify the most energetically reasonable configuration of 1 (Supplementary Information). Conformational analysis using MMFF94, followed by energy optimization at HF/6-31G(d) levels in GAUSSIAN 03 (for details see Supplementary Information), suggested that the cis orientation of H-4/H-24, with syn relationships to the oxo bridge as shown in 1A (Supplementary Information, Energy minimization and ECD calculations) retained the most favorable configuration with respect to the energy minimization. The trans relationships for H-4 and H-24 were eliminated due to the severe distortions of the sp3 atoms with highly unfavorable energy involved in the associated rings (Supplementary Information). To establish the absolute configuration of compound 1, its electronic circular dichroism (ECD) spectrum was experimentally recorded, which showed positive Cotton effect at 219 and 297 nm. The theoretical ECD of 1a and its enantiomer 1b were then calculated with a time-dependent density function theory (TD-DFT) method at the b3lyp/aug-cc-pvdz level, and the polarizable continuum model (PCM) was adopted to consider solvent effects using the dielectric constant of methanol. The calculated ECD spectra were produced by SpecDis software in Figure 4. The overall pattern of calculated spectrum of 1b was in good agreement with the experimental one. Thus, the absolute configurations at C-4, and C-24 of 1 were determined as R and S, respectively.
Figure 3. Selected NOE correlations of 15.
Figure 3. Selected NOE correlations of 15.
Marinedrugs 12 04096 g003
Figure 4. (a) Simplified structures (1a,b) of two possible enantiomers of 1 and computational modeling of 1b (4R, 24S); (b) Calculated ECD spectra of (4S, 24R)- and (4R, 24S)- two enantiomers and experimental ECD spectrum of 1.
Figure 4. (a) Simplified structures (1a,b) of two possible enantiomers of 1 and computational modeling of 1b (4R, 24S); (b) Calculated ECD spectra of (4S, 24R)- and (4R, 24S)- two enantiomers and experimental ECD spectrum of 1.
Marinedrugs 12 04096 g004
Compound 2, was assigned a molecular formula of C25H37NO4 with 8 degrees of unsaturation, based on HR-ESI-MS data for [M + Na]+ m/z 438.2619. The 13C NMR and DEPT spectra indicated 25 resonances for four methyls, eight methylenes, six methines (two oxygen-bearing sp3 methines), and seven quaternary carbons (two carbonyl carbons) (Table 1). The 1H NMR spectrum displayed resonances for four olefinic protons at δH 5.74 (1H, t, J = 3.9 Hz ), and 5.09 (3H, m, overlapped), two oxygenated methine protons at δH 5.14 (1H, s), and 4.21 (1H, dd, J = 7.0, 1.8 Hz), four methyl groups at δH 1.68 (3H, s), and 1.60 (9H, s, overlapped), in addition to two NH2 protons at δH 7.56 (1H, s, in DMSO-d6) and 7.26 (1H, s, in DMSO-d6), and one OH proton at δH 4.99 (1H, s, in DMSO-d6). Inspection of the 1H and 13C NMR, HMBC and COSY spectra of 2 suggested that it was structurally related to compound 1, possessing a farnesyl moiety. Six of the eight degrees of unsaturation of 2 were accounted for by four double bonds and two carbonyl carbons, implying that the structure retained two rings as well. Interpretation of the COSY correlations of H-4/H-5a, 5b/H-6, and H-4 with an exchangeable proton at δH 4.99 (4-OH in DMSO-d6, not shown), together with the HMBC correlations of H-24/C-6 and C-3, H-5a/C-3, and C-4, indicated the presence of a 4-hydroxycyclohexenyl moiety. The connection of the farnesyl group to the 4-hydroxycyclohexenyl moiety at C-7 was supported by the HMBC correlations of H-8/C-6 and C-7. The remaining ring structure was assigned to a butanolide moiety attached to the 4-hydroxycyclohexenyl moiety via an ester linkage at C-24 and a quaternary carbon at C-3, which was delineated by the HMBC correlations of H-24/C-1 and C-3, H-2a, H-2b/C-1 and C-3 and C-24. The last unassigned substructure CONH2 was determined as an acetamide motif based on the chemical shift of the carbonyl carbon (δC 175.2), and two NH2 protons at δH 7.56, 7.26 in DMSO-d6, as well as the IR absorption bands (νmax 3427, 1671 cm−1). Furthermore, the 3JCH correlations from H-2, H-4, and H-24 to C-25 indicated that the acetamide group was attached to the bicyclo-moiety at C-3. The NOESY correlations for H-10/H-12, H-9/H3-23, H-14/H-16, and H-13/H3-22 (Figure 2) suggested the 10E, 14E double bond geometry in the farnesyl moiety. The syn-relationship of H-4, H-24 and NH2 was determined on the basis of NOESY correlations of H-4/H-24/NH2 (Figure 3). The absolute configuration of C-4 was assigned by application of the modified Mosher method [14]. The (S)- and (R)-MTPA esters of 2 were prepared by treatment with (R)- and (S)-MTPA chloride, respectively. The ∆δS–R values of the Mosher ester observed for the protons near the secondary C-4 hydroxy group indicated the R configuration for the carbinol stereogenic center in 2 (Figure 5). On the basis of its relative configuration (Figure 3), the absolute configuration of 2 was, thus, determined as 3S, 4R, 24S.
Compound 3 exhibited the same molecular formula of C25H37NO4 ([M + Na]+ m/z 438.2622) as that of 2. A detailed analysis of the 1D and 2D NMR spectroscopic data confirmed that 3 possessed the farnesyl and 4-hydroxycyclohexenyl moieties as well. However, careful comparison of 1H NMR spectra of compounds 2 and 3 revealed that they are isomers, in which the δH 5.14 of H-24 in 2 (1H, s, in CDCl3) changed to δH 4.58 (1H, s, in CDCl3) in 3, and displayed clear correlation with NH proton at δH 5.42 (1H, d, J = 7.5 Hz, in DMSO-d6) in the COSY spectrum of 3. In addition, the resonance of 1H NMR for 3 indicated the presence of one carboxyl proton at δH 11.04 (1H, s, in DMSO-d6). The HMBC of H-2/C-1, C-3, C-4, C-24, and C-25, and COSY correlations of NH/H-24, H-6/H-5a/H-4 (Figure 2) suggested that 3 possessed a pyrrolidin-2-one moiety and a carboxy group (C-25) at C-3 instead of butenolide moiety and carbamoyl group in 2. The NOESY correlations for H-10/H-12, H-9/H3-23, H-14/H-16, and H-13/H3-22 (Figure 3) confirmed that the farnesyl moiety in 3 remained the same configuration as the 10E, 14E in 2. The syn-relationship of H-4 and H-24 was indicated by the NOESY correlations of H-4/H-24 (Figure 3). The absolute configuration of C-4 was assigned by application of the modified Mosher method as well. The ∆δS–R values observed for the protons near the secondary C-4 hydroxy group for the esters indicated the R configuration for the carbinol stereogenic center in 3 (Figure 5). On the basis of its relative configuration, as well as the analysis of CD spectra of compounds 2 and 3 showing similar Cotton effects near 195 nm (Figure 6), the absolute configuration of compound 3 was suggested as 3S, 4R, 24S.
Figure 5. ∆δS–R values (ppm) for the MTPA derivatives of 2 and 3 in CDCl3.
Figure 5. ∆δS–R values (ppm) for the MTPA derivatives of 2 and 3 in CDCl3.
Marinedrugs 12 04096 g005
Figure 6. CD curves of compounds 2 and 3.
Figure 6. CD curves of compounds 2 and 3.
Marinedrugs 12 04096 g006
The adduct ion of compound 4 at m/z 393.2404 [M + Na]+ in HR-ESI-MS determined the molecular formula of 4 as C24H34O3, which is supported by 13C NMR data (Table 1). The 1H, 13C NMR spectra of 4 were indicative of the hippolide-like metabolite, containing a farnesyl group with the proton signals at δH 5.17 (1H, t, J = 6.8 Hz), 5.11 (1H, t, J = 6.8 Hz), and 5.10 (1H, t, J = 6.3 Hz). In addition, the analysis of HSQC and DEPT data delineated the presence of a substituted benzene ring with three signals at δC 117.0/δH 6.78, at δC 128.2/δH 6.93, and at δC 130.9/δH 6.89. The HMBC correlations of H-5/C-3, C-4, and C-7, H-6/C-4, C-5, and C-24, H-8/C-6, C-7, and C-24, as well as the COSY correlations of H-5/H-6 revealed that this benzene ring is substituted at three positions, C-3, C-4 and C-7, with the connection of the farnesyl group at C-7. The 13C NMR and DEPT data indicated C-4 is an oxygenated quaternary carbon, which characterized the phenolic identity of compound 4. Furthermore, an acetic acid moiety was elucidated to be attached to the phenolic ring at C-3 using the assemblage of 1D and 2D experiments, in which methylene protons of H-2 (δH 3.53, 2H, s) have displayed HMBC correlations with C-1 (δC 181.9, a carboxylic carbon), C-3, C-4, and C-24. The NOESY correlations for H-10/H-12, H-9/H3-23, H-14/H-16, and H-13/H3-22 (Figure 3) suggested the 10E, 14E-configuration of the farnesyl group.
The molecular formula of compound 5 was established as C24H35NO2 on the basis of HR-ESI-MS (m/z 392.2564, [M + Na]+) and NMR spectral data (Table 1 and Table 2), indicative of eight degrees of unsaturation. The 1H NMR spectrum displayed resonances for two aldehyde protons at 9.75 (1H, d, J = 3.5 Hz) and 9.37 (1H, s), seven olefinic protons at δH 7.70 (1H, dt, J = 3.5, 11.5 Hz), 6.86 (1H, d, J = 11.0 Hz), 6.66 (1H, m), 6.64 (1H, d, J = 14.7 Hz), 5.16 (1H, t, J = 7.5 Hz), 5.11 (1H, t, J = 6.8 Hz), and 5.09 (1H, t, J = 6.5 Hz), and four methyl groups at δH 1.68 (3H, s), 1.60 (6H, s, overlapped), and 1.58 (3H, s). The HMBC and COSY correlations (Figure 1) suggested that 5 possessed a farnesyl group as well. A -NH2 motif was clarified by the presence of two exchangeable NH2 proton resonances at δH 8.20 (2H, m, in DMSO-d6), as well as the presence of IR band at 3379 cm−1. In addition, the olefinic proton at δH 7.70 (H-2) displayed the 4JH-1/H-2 and 3JH-3/NH as doublets of triplets (1H, dt, J = 3.5, 11.5 Hz), revealing the NH2 motif to be attached at C-3 position. Furthermore, the HMBC correlations of H-1/C-2, H-4/C-1, C-2, C-3, and H-24/C-6, C-7, and C-8, together with the COSY correlations of H-1/H-3 (W effect), and H-4/H-5/H-6 confirmed the presence of an 2-(aminomethylene) hepta-3,5-dienedial moiety connected with the farnesyl group at C-7. Therefore, the eight degrees of unsaturation were accounted for by six double bonds, two aldehyde moieties. The NOESY correlations for H-3/H-4, H-5/H-8, H-6/H-24, H-10/H-12, H-9/H3-23, H-14/H-16, and H-13/H3-22 (Figure 3) suggested the geometry of double bonds Δ2,3, Δ4,5, Δ6,7, Δ10,11, Δ14,15, as 3Z, 4E, 6E, 10E, 14E, respectively.
To further confirm the original PTP1B activity from the crude fraction of the title sponge, compounds 15 were evaluated in vitro for PTP1B inhibitory activity. Compound 1 and 5 exhibited moderate PTP1B inhibitory activities with IC50 values of 5.2 and 8.7 μM, but compounds 3, and 4 exhibited weak PTP1B inhibitory activities with an IC50 values of 33, 14 μM, respectively. The oleanolic acid was used as a positive control for the PTP1B experiment with an IC50 value of 2.0 µM. We have also evaluated the cytotoxicity of compounds 15 against A549, HeLa, and HCT-116 cancer cell lines, however, only compound 1 exhibited weak activity against HCT-116 cell line with an IC50 value of 11.6 µM and no activity was observed for other compounds.

3. Experimental Section

3.1. General Experimental Procedures

IR spectra were recorded on a Bruker vector 22 spectrometer (Bruker Optics, Inc., Billerica, MA, USA) with KBr pellets. Optical rotation data were recorded on a Perkin-Elmer model 341 polarimeter (Perkin-Elmer, Inc., Waltham, MA, USA) with 1 dm cell. The CD spectra were obtained with a JASCO J-715 spectropolarimeter (Jasco Inc., Tokyo, Japan). The NMR experiments were measured on Bruker AMX-400 MHz and AMX-500 MHz instruments (Bruker Biospin Corp., Billerica, MA, USA) in CDCl3 with TMS as an internal standard. ESIMS and HRESIMS spectra were recorded on a Waters Q-Tof micro YA019 mass spectrometer (Waters Corp., Milford, MA, USA). Reversed-Phase HPLC was performed on a YMC-Pack Pro C18 RS column (250 × 10 mm, 5 µm) using a Waters 600 HPLC instrument (Waters Corp., Milford, MA, USA) with Waters 996 UV detector. Column chromatography (CC) was performed on Sephadex LH-20 (Pharmacia Fine Chemicals, Piscataway, NJ, USA) and YMC ODS-A (50 μm) (250 × 10 mm, YMC Co., Ltd., Kyoto, Japan). Vacuum liquid chromatography (VLC) was performed on silica gel (200–300 mesh, Qingdao Ocean Chemical Co., Jinan, China); the fractions were monitored by TLC (HSGF 254, Yantai Huiyou Co., Yantai, China) and spots were visualized by heating silica gel plates sprayed with 10% H2SO4 in H2O.

3.2. Animal Material

A specimen of H. lachne was collected off Yongxing Island and seven connected islets in the South China Sea in June 2007, and was identified by Jin-He Li (Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China). A voucher sample (No. B-2) was deposited in Laboratory of Marine Drugs, Department of Pharmacy, Changzheng Hospital, Second Military Medical University, Shanghai, China.

3.3. Extraction and Isolation

The sponge (3.6 kg, wet weight) was extracted with 95% aq. EtOH and combined extracts were concentrated under reduced pressure to yield the crude extract (671 g). This extract was suspended in H2O and extracted with EtOAc and n-BuOH to afford the EtOAc- and n-BuOH-soluble extracts (232 g and 78 g, respectively). The EtOAc-soluble extract was partitioned between MeOH–H2O (9:1) and petroleum ether to yield a brownish red oil (84 g). The MeOH–H2O (9:1) phase was diluted 3:2 with H2O and extracted with CH2Cl2 to afford the CH2Cl2-soluble extract (110 g). This CH2Cl2-soluble extract was subjected to VLC on silica gel using CH2Cl2/MeOH (100:1, 50:1, 30:1, 20:1, 10:1, 5:1, and 1:1) as eluent to give nine sub-fractions (A-G). Sub-fraction C with PTP1B inhibitory activity was subjected to CC on Sephadex LH-20 (CH2Cl2:MeOH = 1:1) and silica gel using Petroleum ether/Acetone (30:1, 20:1, 10:1, 5:1, 3:1, and 1:1) to give eight sub-fractions (a–i). Sub-fractions (b–d) were further purified by HPLC (YMC-Pack Pro C18 RS, YMC Co., Ltd., Kyoto, Japan, 5 µm, 10 × 250 mm, 2.0 mL/min, UV detection at 210 and 254 nm) eluting with MeOH/H2O (80:20) to yield pure compounds 1 (6.4 mg), 4 (8.9 mg), and 5 (12.0 mg) at 56, 85, and 93 min, respectively. Similarly, compounds 2 (3.9 mg), and 3 (5.1 mg) were purified from sub-fractions h and e using HPLC (YMC-Pack Pro C18 RS, 5 µm, 10 × 250 mm, 2.0 mL/min, UV detection at 210 and 254 nm) in MeOH/H2O (83:17) at 28 and 23 min, respectively.
Compound 1: colorless oil; Marinedrugs 12 04096 i001 +67° (c 0.120, MeOH); CD (MeOH) λmax (∆ε) 219 (3.40), 266 (−0.80), 297 (2.77) nm; UV (MeOH) λmax (log ε) 210 (1.68), 233 (1.64), 282 (1.56) nm; IR (KBr) νmax 3369, 2926, 1596, 1448, 1212, 1067, 840, 673 cm−1; 1H and 13C NMR data, see Table 1 and Table 2; HRESIMS m/z 392.2567 [M + Na]+ (calcd for C24H35NO2Na+, 392.2560).
Compound 2: colorless oil; Marinedrugs 12 04096 i001 −15° (c 0.10, MeOH); CD (MeOH) λmax (∆ε) 189 (−3.48), 196 (5.71) nm; UV (MeOH) λmax (log ε) 190 (3.30) nm; IR (KBr) νmax 3427, 2924, 1773, 1671, 1377, 1189, 1097, 981 cm−1; 1H and 13C NMR data, see Table 1 and Table 2; 1H NMR data (in DMSO): δH 7.56 (1H, s, NH), 7.26 (1H, s, NH), 5.54 (1H, br s, H-24), 4.99 (1H, br s, OH), 5.14 (1H, m, H-6), 4.10 (1H, m, H-4); HRESIMS m/z 438.2619 [M + Na]+ (calcd for C25H37NO4Na+, 438.2615).
Compound 3: colorless oil; Marinedrugs 12 04096 i001 −10° (c 0.12, MeOH); CD (MeOH) λmax (∆ε) 184 (−3.30), 198 (4.39) nm; UV (MeOH) λmax (log ε) 195 (3.84), 223 (1.48) nm; IR (KBr) νmax 3434, 2923, 1712, 1374, 1199, 1038, 742 cm−1; 1H and 13C NMR data, see Table 1 and Table 2; 1H NMR data (in DMSO): δH 11.04 (1H, s, OH), 5.42 (1H, d, J = 7.5 Hz, NH), 5.22 (1H, m, H-6), 4.30 (1H, br s, H-24), 4.02 (1H, br s, OH), 3.86 (1H, m, H-4); HRESIMS m/z 438.2622 [M + Na]+ (calcd for C25H37NO4Na+, 438.2615).
Compound 4: yellow oil; UV (MeOH) λmax (log ε) 228 (3.20), 282 (3.16) nm; IR (KBr) νmax 3415, 2923, 1566, 1439, 1377, 1248, 1015, 823, 651 cm−1; 1H and 13C NMR data, see Table 1 and Table 2; HRESIMS m/z 393.2404 [M + Na]+ (calcd for C24H34O3Na+, 393.2400).
Compound 5: yellow oil; UV (MeOH) λmax (log ε) 365 (2.56) nm; IR (KBr) νmax 3379, 2925, 1582, 1441, 1380, 1234, 1188, 1126 cm−1; 1H and 13C NMR data, see Table 1 and Table 2; 1H NMR data (in DMSO): δH 9.33 (1H, s, H-1), 9.04 (1H, s, H-24), 8.20 (2H, m, NH2), 7.70 (1H, m, H-5), 7.29 (1H, m, H-3), 6.97 (1H, d, J = 14.5 Hz, H-4), 6.92 (1H, d, J = 12.0 Hz, H-6); HRESIMS m/z 392.2564 [M + Na]+ (calcd for C24H35NO2Na+, 392.2560).

3.4. PTP1B Inhibitory Assay

PTP1B inhibitory activity was determined using a PTP1B inhibitory assay as described in a previous report [21]. The enzymatic activities of the PTP1B catalytic domain were determined at 30 °C by monitoring the hydrolysis of pNPP. Dephosphorylation of pNPP generates product pNP, which was monitored at an absorbance of 405 nm. In a typical 100 μL assay mixture containing 50 mmol/L 3-[N-morpholino] propanesulfonic acid (MOPs), pH 6.5, 2 mmol/L pNPP, and 30 nmol/L recombinant PTP1B, activities were continuously monitored and the initial rate of the hydrolysis was determined using the early linear region of the enzymatic reaction kinetic curve.

3.5. Computational Details of Calculated ECD

All quantum-chemical calculations were performed by the Gaussian 03 program. The TD calculations were calculated by b3lyp/aug-cc-pvdz method under Self-Consistent Reaction Field model of solvent (MeOH). Details of the DFT calculation see Supplementary Information.

3.6. Preparation of MTPA Esters

A previously described modified Mosher’s method was used [15]. The (S)- and (R)-MTPA esters of 2 (3S, 3R) and 3 (4S, 4R) were obtained by treatment of 2 (0.5 mg and 0.6 mg, respectively) and 3 (0.7 mg and 0.7 mg, respectively) with (R)- and (S)-MTPA chlorides (10 μL) in dry pyridine (0.5 mL), and stirred at room temperature overnight. The MTPA esters were purified by mini-column chromatography on silica gel (200 mesh, CH2Cl2/MeOH, 13:1 of 2 and 3).

4. Conclusions

Protein tyrosine phosphatase 1B (PTP1B), one of the protein tyrosine phosphatases (PTPases), is known to be a negative regulator of insulin signal transduction by dephosphorylating the insulin receptor as well as its substrate, insulin receptor substrates [2]. The PTP1B inhibitors are recognized as potential therapeutic agents for the treatment of type ΙΙ diabetes and obesity [3]. Interestingly, in comparison of our previously discovered hippolides A–H [14], in which only hippolides A and B displayed weak PTP1B inhibitory activity (23.8 and 39.7 μM), Compound 1 and 5, with IC50 values of 5.2 μM and 8.7 μM, are the most potent compounds isolated from marine sponges of the genus Hippospongia relevant to PTP1B inhibitory activity. Biogenetically, all the hippolides discovered so far are sesterterpenoid derivatives. The sesterterpenoids are a group of pentaprenyl terpenoids whose structures are derivable from geranylfarnesyl diphosphate [22]. Hypothetically, compounds 15 could be biosynthetically formed via multiple reactions involving oxidations, decarboxylations, aminations, dehydrations and double bond formations and shifts, etc. with the same precursor, acyclic carboxylic sesterterpenoid.

Supplementary Files

  • Supplementary File 1:

    Supplementary Information (PDF, 3204 KB)

  • Acknowledgments

    The authors thank Jin-He Li (Institute of Oceanology, Chinese Academy of Sciences) for identifying the marine sponge and the authors are grateful for the helpful discussions of biosynthetic pathways provided by Harald Gross (Department of Pharmaceutical Biology, University of Tübingen). This research was supported by the National Natural Science Fund for Distinguished Young Scholars of China (81225023), the National Natural Science Fund of China (Nos. 41106127, 81072573, 81172978, 81302691, 81373321, 81001394 and 81202441), Shanghai Subject Chief Scientist (12XD1400200), the Innovation Program of Shanghai Municipal Education Commission (No. 14YZ037), and National High Technology Research and Development Program of China (863 Projects, Nos. 2011AA09070107 and 2013AA092902). The calculation part was supported by the HPC Center of Kunming Institute of Botany, CAS (Chinese Academy of Sciences).

    Author Contributions

    Shu-Juan Piao and Bing-Nan Han conducted natural products isolation and structural determination and drafted the manuscript; Bing-Nan Han, Wei-Hua Jiao, Fan Yang and Yang-Hua Yi gave constructive comments for the results and discussion parts of the manuscript; Ying-Tong Di performed the chemical computation of compound 1; Hou-Wen Lin conceived and designed the research.

    Conflicts of Interest

    The authors declare no conflict of interest.

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