New Hippolide Derivatives with Protein Tyrosine Phosphatase 1B Inhibitory Activity from the Marine Sponge Hippospongia lachne

Five new sesterterpenoids, compounds 1–5, have been isolated from the sponge Hippospongia lachne off Yongxing Island in the South China Sea. The structures of compounds 1–5 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.


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].

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 1-5 ( Figure 1).  16, and H 3 -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 2 J CH and 3 J CH 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.   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 sp 3 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.     13 C NMR and DEPT spectra indicated 25 resonances for four methyls, eight methylenes, six methines (two oxygen-bearing sp 3 methines), and seven quaternary carbons (two carbonyl carbons) ( Table 1). The 1 H NMR spectrum displayed resonances for four olefinic protons at δ H 5.74 (1H, t, J = 3.9 Hz ), and 5.09 (3H, m, overlapped), two oxygenated methine protons at δ H 5.14 (1H, s), and 4.21 (1H, dd, J = 7.0, 1.8 Hz), four methyl groups at δ H 1.68 (3H, s), and 1.60 (9H, s, overlapped), in addition to two NH 2 protons at δ H 7.56 (1H, s, in DMSO-d 6 ) and 7.26 (1H, s, in DMSO-d 6 ), and one OH proton at δ H 4.99 (1H, s, in DMSO-d 6 ). Inspection of the 1 H and 13 C 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 δ H 4.99 (4-OH in DMSO-d 6 , 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 CONH 2 was determined as an acetamide motif based on the chemical shift of the carbonyl carbon (δ C 175.2), and two NH 2 protons at δ H 7.56, 7.26 in DMSO-d 6 , as well as the IR absorption bands (ν max 3427, 1671 cm −1 ). Furthermore, the 3 Figure 3). The absolute configuration of C-4 was assigned by application of the modified Mosher method as well. The ∆δ S-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.  The adduct ion of compound 4 at m/z 393.2404 [M + Na] + in HR-ESI-MS determined the molecular formula of 4 as C 24 H 34 O 3 , which is supported by 13 C NMR data ( Table 1). The 1 H, 13 C NMR spectra of 4 were indicative of the hippolide-like metabolite, containing a farnesyl group with the proton signals at δ H 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 δ C 117.0/δ H 6.78, at δ C 128.2/δ H 6.93, and at δ C 130.9/δ H 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 13 C 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 (δ H 3.53, 2H, s) have displayed HMBC correlations with C-1 (δ C 181.9, a carboxylic carbon), C-3, C-4, and C-24. The To further confirm the original PTP1B activity from the crude fraction of the title sponge, compounds 1-5 were evaluated in vitro for PTP1B inhibitory activity. Compound 1 and 5 exhibited moderate PTP1B inhibitory activities with IC 50 values of 5.2 and 8.7 μM, but compounds 3, and 4 exhibited weak PTP1B inhibitory activities with an IC 50 values of 33, 14 μM, respectively. The oleanolic acid was used as a positive control for the PTP1B experiment with an IC 50 value of 2.0 µM. We have also evaluated the cytotoxicity of compounds 1-5 against A549, HeLa, and HCT-116 cancer cell lines, however, only compound 1 exhibited weak activity against HCT-116 cell line with an IC 50 value of 11.6 µM and no activity was observed for other compounds.

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.

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.

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 IC 50 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 1-5 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.