Isopetrosynol, a New Protein Tyrosine Phosphatase 1B Inhibitor, from the Marine Sponge Halichondria cf. panicea Collected at Iriomote Island

Abstract

A new polyacetylene compound, isopetrosynol (1), was isolated from the Okinawan marine sponge Halichondria cf. panicea together with petrosynol (2), adociacetylene D (3), (5R)-3,15,27-triacontatriene-1,29-diyn-5-ol (4), and petrosterol (5). The structure of 1 was assigned on the basis of spectroscopic data for 1 and 2. Compound 1 inhibited protein tyrosine phosphatase 1B (PTP1B) activity with an IC₅₀ value of 8.2±0.3 µM, while compound 2, a diastereomer of 1, showed only 28.9±4.5% inhibition at 21.6 µM. The IC₅₀ values of compounds 3 and 4 were 7.8±0.5 and 12.2±0.5 µM, respectively. Oleanolic acid, a positive control, inhibited PTP1B activity at 0.7±0.1 µM (IC₅₀) in the same experiment. The inhibitory activity of 1 was stronger than that of its diastereomer (2). This is the first study to show the inhibitory effects of polyacetylene compounds on PTP1B.

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of the insulin and leptin signaling pathways.^1–3) Therefore, the overexpression of PTP1B has been implicated in type 2 diabetes mellitus and obesity.^1–3) Despite the identification of a large number of PTP1B inhibitors from natural and synthetic compounds, the clinical application of PTP1B inhibitors has not yet been successful.⁴⁾ Thus, the search for new inhibitors of PTP1B with novel structural features is ongoing.

During our search for PTP1B inhibitors from marine invertebrates and microorganisms, we have reported the isolation and PTP1B inhibitory activities of new compounds, such as hyattellactones,⁵⁾ trichoketides,⁶⁾ verruculides,⁷⁾ and 26-O-ethylstrongylophorine-14.⁸⁾ With continuous efforts on the extracts of marine organisms, we found that the EtOH extract of the marine sponge Halichondria cf. panicea collected at Iriomote Island exhibited inhibitory activity against PTP1B. Bioassay-guided separation led to the isolation of a new polyacetylene, named isopetrosynol (1), together with four known compounds^9–14): petrosynol (2), adociacetylene D (3), (5R)-3,15,27-triacontatriene-1,29-diyn-5-ol (4), and petrosterol (5) (Fig. 1). We describe herein the isolation, structures, and biological activities of compounds 1–5.

Fig. 1. Structures of Compounds 1–5 Isolated from the Marine Sponge Halichondria cf. panicea Collected at Iriomote Island

Results and Discussion

Isolation

The ethanol extract of the marine sponge inhibited PTP1B activity (approximately 75% at 50 µg/mL) and was separated into seven fractions using an octadecyl silica (ODS) column. The bioactive fractions were then purified by repeated HPLC using an ODS column to yield compounds 1 (3.3 mg), 2 (16.4 mg), 3 (7.6 mg), 4 (2.2 mg), and 5 (6.5 mg).

Compounds 2–5 were identified as petrosynol (2),^9,10) adociacetylene D (3),^11,12) (5R)-3,15,27-triacontatriene-1,29-diyn-5-ol (4),¹²⁾ and petrosterol (5),^13,14) respectively, by comparing their spectroscopic data with those in the literature.

Structure Elucidation

The molecular formula of isopetrosynol (1) was deduced as C₃₀H₄₀O₄ from high resolution (HR)-FAB-MS (m/z 487.2838 [M+Na]⁺, Δ+1.4 mmu), which was the same as that of petrosynol (2). ¹H- and ¹³C-NMR data (Table 1) and the UV spectrum of 1 were also similar to those of 2.^9,10) Therefore, the planar structure of 1 including the geometry of C-4, C-15, and C-26 was presumed to be the same as that of 2 and was confirmed by an analysis of the ¹H–¹H correlation spectroscopy (COSY) and heteronuclear multiple bond connectivity (HMBC) spectra of 1 (Fig. 2).

Table 1. ¹H- and ¹³C-NMR Data for Isopetrosynol (1) and Petrosynol (2) in CDCl₃

No.	Isopetrosynol (1)		Petrosynol (2)
	δC	δH Mult. (J in Hz)	δC	δH Mult. (J in Hz)
1, 30	74.0	2.55, d (2.4)	73.9	2.54, d (2.4)
2, 29	83.3		83.3
3, 28	62.8	4.82, br d (5.8)	62.6	4.80, br d (5.6)
4, 27	128.6	5.59, dd (15.2, 6.0)	128.5	5.57, dd (15.5, 6.0)
5, 26	134.3	5.89, dt (15.2, 6.8)	134.1	5.86, dt (15.5, 6.7)
6, 25	31.7	2.05, m	31.6	2.03, m
7, 24	28.4	1.23–1.41, m	28.4	1.24–1.39, m
8, 23	28.5	1.23–1.41, m	28.4	1.24–1.39, m
9, 22	28.5	1.23–1.41, m	28.4	1.24–1.39, m
10, 21	28.3	1.48, m	28.2	1.46, m
11, 20	18.7	2.22, m	18.6	2.17, m
12, 19	87.4		86.4
13, 18	78.9		79.7
14, 17	62.2	4.89, br s	58.4	5.23, dd (4.3, 3.9)
15, 16	131.4	5.98, br d (2.4)	131.9	5.65, dd (4.8, 1.9)

Fig. 2. ¹H–¹H COSY and Key HMBC Correlations for 1

The symmetric structure of 1 was revealed from the ¹H- and ¹³C-NMR spectra similar to compounds 2 and 3. The ¹H and ¹³C chemical shifts at the C-3/28 positions of 1–3 were very similar to each other. The significant difference in the chemical shifts between 1 and 2 was detected on the signals due to the C-14/17 positions (Table 1). These ¹H- and ¹³C-NMR data suggested that the relative configuration of C-14/17 of 1 and 2 were the opposite each other. The absolute configurations of 2 and 3 were determined as (3S, 14S, 17S, 28S)¹⁰⁾ and (3S, 28S),¹¹⁾ respectively, by the analysis of electronic circular dichroism (ECD) spectra of the benzoyl derivatives of 2¹⁰ and by the modified Mosher’s method for 3.¹¹⁾

To elucidate the absolute configuration of 1, the ECD spectrum of (3S, 14R, 17R, 28S)-isomer was calculated and compared with the experimental ECD spectrum of 1. However, compound 1 did not show a marked ECD curve (201 nm, Δε+1.0), while 2 displayed a significant positive cotton effect (200 nm, Δε+26.8). The specific rotation of 1 ([α]_D+11.0, c 0.30, CHCl₃) was quite smaller than that of 2 ([α]_D+120.4, c 0.30, CHCl₃). Therefore, compound 1 will be a mixture of (3S, 14R, 17R, 28S) and (3R, 14S, 17S, 28R)-isomers.

Biological Activity

The inhibitory effects of compounds 1–5 on the activity of PTP1B were examined, and the IC₅₀ values of 1–5 and a positive control (oleanolic acid¹⁵⁾) are listed in Table 2. The inhibitory activity of compound 1 was stronger than that of 2. Therefore, the configurations of OH groups at C-14 and C-17 markedly affect this activity. Compounds 3 and 4 inhibited PTP1B activity with IC₅₀ values of 7.8±0.5 and 12.2±0.5 µM, respectively, whereas compound 5 was not active at 24.3 µM.

Table 2. PTP1B Inhibitory Activities of Compounds 1–5

Compound	IC50 (µM)
1	8.2±0.3
2	28.9±4.5% inhibition at 21.6 µM
3	7.8±0.5
4	12.2±0.5
5	0% inhibition at 24.3 µM
Oleanolic acida)	0.7±0.1

a) Positive control.¹⁵⁾

Compound 2 was previously reported to exhibit inhibitory activity against mitotic cell division in sea urchin eggs,⁹⁾ antimicrobial activity,^9,10) anti-human immunodeficiency virus (HIV) activity,¹⁶⁾ and cytotoxicity.¹⁷⁾ Compound 3 inhibited neutrophil leukocyte adhesion,¹¹⁾ and compounds 3 and 4 exerted inhibitory effects on the cell division of fertilized ascidian eggs and toxicity against brine shrimp.¹²⁾ Therefore, this is the first study to show that polyacetylenes inhibit PTP1B activity.

Experimental

General Experimental Procedures

Specific rotations were determined with a JASCO P-2300 digital polarimeter. UV spectra were measured on a U-3310 UV-Visible spectrophotometer (Hitachi) and IR spectra on a PerkinElmer, Inc. Spectrum One Fourier transform infrared spectrometer. ECD spectra were measured with a JASCO J-720 spectrometer. NMR spectra were recorded on a JEOL JNM-AL-400 NMR spectrometer (400 MHz for ¹H and 100 MHz for ¹³C) in CDCl₃ (δ_H 7.24, δ_C 77.0). FAB-MS and HR-FAB-MS were performed using a JMS-MS 700 mass spectrometer (JEOL). Preparative HPLC was carried out with a Hitachi L-6200 system.

Materials

PTP1B was purchased from Enzo Life Sciences (Farmingdale, NY, U.S.A.). p-Nitrophenyl phosphate (pNPP) was purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Oleanolic acid was purchased from Tokyo Chemical Industry (Tokyo, Japan). Plastic plates (96-well) were purchased from Corning Inc. (Corning, NY, U.S.A.). All other chemicals including organic solvents were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

Marine Sponge and Isolation of Compounds 1–5

The marine sponge was collected by scuba diving in the coral reef at Iriomote Island, Okinawa, Japan, in September 2013 and subsequently identified as Halichondria sp. (cf. H. panicea). A voucher specimen was deposited at the Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University as 13–9–7=2–3.

The sponge (595.8 g, wet weight) was cut into small pieces, and extracted with ethanol. The EtOH extract was evaporated to dryness (14.3 g), and applied to an ODS column (100 g). The column was eluted stepwise with 500 mL each of 0, 30, 50, 70, 85, and 100% CH₃OH in H₂O and then with CH₃OH containing 0.05% trifluoroacetic acid (TFA) into seven fractions (Frs. 1–7). Fraction 5 (460.9 mg, eluted with 85% CH₃OH) was subjected to preparative HPLC [column, PEGASIL ODS (10 mm i.d.×250 mm: Senshu Sci. Co., Ltd., Tokyo, Japan); solvent, 65% CH₃OH; flow rate, 2.0 mL/min; detection, UV 210 nm] to give isopetrosynol (1) (3.3 mg, t_R=46 min), petrosynol (2) (16.4 mg, t_R=35 min), and adociacetylene D (3) (7.6 mg, t_R=62 min). (5R)-3,15,27-Triacontatriene-1,29-diyn-5-ol (4) (2.2 mg, t_R=57 min) was isolated from Fr. 6 (1075.3 mg, eluted with 100% CH₃OH) by preparative HPLC [column, PEGASIL ODS (10 mm i.d.×250 mm); solvent, 90% CH₃OH; flow rate, 2.0 mL/min; detection, UV 210 nm]. Fraction 7 (1197.3 mg, eluted with CH₃OH containing 0.05% TFA) was separated by preparative HPLC [column, PEGASIL ODS (10 mm i.d.×250 mm); solvent, 96% CH₃OH; flow rate, 2.0 mL/min; detection, UV 210 nm] to afford petrosterol (5) (6.5 mg, t_R=50 min).

Isopetrosynol (1)

Yellow oil; [α]_D²⁸+11.0 (c 0.30, CHCl₃); IR (KBr) ν_max 3420, 2931, 2863, 2347, 2213, 1709, 1668, 1453, 1385, 1164, 1032 cm⁻¹; UV (CH₃OH) λ_max nm (log ε) 202 (4.0); ECD (c 0.010, CH₃CN) λ_max nm (Δε) 201 (+1.0); FAB-MS m/z 487 [M+Na]⁺; HR-FAB-MS m/z 487.2838 (Calcd for C₃₀H₄₀O₄Na [M+Na]⁺, 487.2824); ¹H- and ¹³C-NMR (CDCl₃), see Table 1.

Petrosynol (2)

Yellow oil; [α]_D²⁸+120.4 (c 0.30, CHCl₃); lit. [α]_D²³+107 (c 0.37, CHCl₃)^9,10); UV (CH₃OH) λ_max nm (log ε) 202 (3.8); ECD (c 0.010, CH₃CN) λ_max nm (Δε) 200 (+26.8); FAB-MS m/z 487 [M+Na]⁺; ¹H- and ¹³C-NMR (CDCl₃), see Table 1.

Conformational Analysis and Calculation of ECD Spectra

The most stable conformer of isopetrosynol (1) was predicted using Spartan ’14 (Wavefunction, Inc., Irvine, CA, U.S.A.) by a preliminary conformational analysis with the MMFF94 force field followed by geometry optimization using the density functional theory (DFT) with the B3LYP functional and the 6-31G(d) basis set.

The ECD spectrum in CH₃CN was calculated for the predicted most stable conformer using Gaussian 09 (Gaussian, Inc., Wallingford, CT, U.S.A.) by the time-dependent DFT (TDDFT) with the B3LYP functional and the 6-31+G(d,p) basis set. No Boltzmann averaging was performed since the relative energies of the other conformers were >1 kcal mol⁻¹ with respect to the most stable one. The solvent effect was introduced by the polarizable continuum model (PCM). Fifty low-lying excited states were calculated corresponding to the wavelength region down to about 181 nm, and the calculated spectrum was displayed using GaussView 5.0.9 (Semichem, Inc., Shawnee Mission, KS, U.S.A.) with the peak half-width at half height being 0.333 eV.

PTP1B Inhibitory Assay

PTP1B inhibitory activity was determined by measuring the rate of hydrolysis of the substrate, pNPP, according to the reported method with a slight modification.^18,19) Briefly, PTP1B (100 µL of 0.5 µg/mL stock solution) in 50 mM citrate buffer (pH 6.0) containing 0.1 M NaCI, 1 mM dithiothreitol (DTT), and 1 mM N,N,N′,N′-ethylenediaminetetraacetic acid (EDTA) was added to each well of a 96-well plastic plate. Each sample (2.0 µL in CH₃OH) was added to each well to make the final concentration and incubated for 10 min at 37°C. The reaction was initiated by the addition of pNPP (100 µL of 4.0 mM stock solution) to the citrate buffer, incubated at 37°C for 30 min, and terminated with the addition of 10 µL of a stop solution (10 M NaOH). The optical density of each well was measured at 405 nm using an MTP-500 microplate reader (Corona Electric Co., Ltd.). PTP1B inhibitory activity (%) was defined as [1−(ABS_sample−ABS_blank)/(ABS_control−ABS_blank)]×100, in which ABS_blank was the absorbance of wells containing only the buffer and pNPP, ABS_control was the absorbance of p-nitrophenol liberated by the enzyme in the assay system without a test sample, and ABS_sample was that with a test sample. Oleanolic acid, a known phosphatase inhibitor,¹⁵⁾ was used as a positive control. Data are expressed as the mean±standard error (S.E.) (n=4).

Acknowledgments

This work was supported in part by the Sasakawa Grants for Science Fellows from the Japan Science Society to H.Y. and the Foundation for Japanese Chemical Research to H.Y. The calculations by Gaussian 09 were performed using supercomputing resources at the Cyberscience Center, Tohoku University. We express our thanks to Dr. K. Ogawa of Z. Nakai Laboratory for identifying the marine sponge and to Mr. T. Matsuki and S. Sato of Tohoku Medical and Pharmaceutical University for measurements of mass spectra.

Conflict of Interest

The authors declare no conflict of interest.

References

• 1) Barr A. J., Future Med. Chem., 2, 1563–1576 (2010).
• 2) Zhang S., Zhang Z. Y., Drug Discov. Today, 12, 373–381 (2007).
• 3) Lee S., Wang Q., Med. Res. Rev., 27, 553–573 (2007).
• 4) Jiang C. S., Liang L. F., Guo Y. W., Acta Pharmacol. Sin., 33, 1217–1245 (2012).
• 5) Abdjul D. B., Yamazaki H., Takahashi O., Kirikoshi R., Mangindaan R. E. P., Namikoshi M., Bioorg. Med. Chem. Lett., 25, 904–907 (2015).
• 6) Yamazaki H., Saito R., Takahashi O., Kirikoshi R., Toraiwa K., Iwasaki K., Izumikawa Y., Nakayama W., Namikoshi M., J. Antibiot., 68, 628–632 (2015).
• 7) Yamazaki H., Nakayama W., Takahashi O., Kirikoshi R., Izumikawa Y., Iwasaki K., Toraiwa K., Ukai K., Rotinsulu H., Wewengkang D. S., Sumilat D. A., Mangindaan R. E. P., Namikoshi M., Bioorg. Med. Chem. Lett., 25, 3087–3090 (2015).
• 8) Lee J.-S., Abdjul D. B., Yamazaki H., Takahashi O., Kirikoshi R., Ukai K., Namikoshi M., Bioorg. Med. Chem. Lett., 25, 3900–3902 (2015).
• 9) Fusetani N., Kato Y., Matsunaga S., Hashimoto K., Tetrahedron Lett., 24, 2771–2774 (1983).
• 10) Fusetani N., Shiragaki T., Matsunaga S., Hashimoto K., Tetrahedron Lett., 28, 4313–4314 (1987).
• 11) Kobayashi M., Mahmud T., Tajima H., Wang W., Aoki S., Nakagawa S., Mayumi T., Kitagawa I., Chem. Pharm. Bull., 44, 720–724 (1996).
• 12) Ochi M., Ariki S., Tatsukawa A., Kotsuki H., Fukuyama Y., Shibata K., Chem. Lett., 1994, 89–92 (1994).
• 13) Sica D., Zollo F., Tetrahedron Lett., 19, 837–838 (1978).
• 14) Mattia C. A., Mazzarella L., Puliti R., Sica D., Zollo F., Tetrahedron Lett., 19, 3953–3954 (1978).
• 15) Zhang Y. N., Zhang W., Hong D., Shi L., Shen Q., Li J.-Y., Li J., Hu L.-H., Bioorg. Med. Chem., 16, 8697–8705 (2008).
• 16) Isaacs S., Kashman Y., Loya S., Hizi A., Loya Y., Tetrahedron, 49, 10435–10438 (1993).
• 17) Juan Y.-S., Lee C.-C., Tsao C.-W., Lu M.-C., El-Shazly M., Shih H.-C., Chen Y.-C., Wu Y.-C., Su J.-H., Int. J. Mol. Sci., 15, 16511–16521 (2014).
• 18) Cui L., Na M. K., Oh H., Bae E. Y., Jeong D. G., Ryu S. E., Kim S., Kim B. Y., Oh W. K., Ahn J. S., Bioorg. Med. Chem. Lett., 16, 1426–1429 (2006).
• 19) Yamazaki H., Sumilat D. A., Kanno S., Ukai K., Rotinsulu H., Wewengkang D. S., Ishikawa M., Mangindaan R. E. P., Namikoshi M., J. Nat. Med., 67, 730–735 (2013).