5-(6-Hydroxy-2,5,7,8-tetramethylchroman-2-yl)-2-methyl-pentanoic Acid Methyl Ester

Abstract

A natural diastereomeric mixture of 5-(6-hydroxy-2,5,7,8-tetramethyl-chroman-2-yl)-2-methyl-pentanoic acid methyl ester (1), was isolated from the soft coral Sinularia arborea. The structure of 1 was elucidated by spectroscopic methods and 1 displayed a significantly inhibitory effect on the generation of superoxide anion by human neutrophils.

Introduction

Octocorals belonging to the genus Sinularia have been proved to be rich sources of interesting secondary metabolites. The octocoral Sinularia arborea (phylum Cnidaria, class Anthozoa, subclass Octocorallia, order Alcyonacea, family Alcyoniidae) was studied, as their organic extract was found to show meaning signals in NMR studies. In previous studies on the chemical constituents of the soft coral Sinularia arborea [1], six new diterpenoids, arbolides A–C, (+)-sarcophytol T, sinularbols A and B, along with a trihydroxysteroid, crassarosterol A, were isolated [2,3,4]. In the continuing studies on this interesting species, a mixture of diastereoisomers, 5-(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)-2-methyl-pentanoic acid methyl ester (1), was isolated (Scheme 1). In this paper, we describe the isolation, structure determination and bioactivity of compound 1.

Results and Discussion

5-(6-Hydroxy-2,5,7,8-tetramethylchroman-2-yl)-2-methyl-pentanoic acid methyl ester (1) was isolated as a colorless oil. Its molecular formula was established as C₂₀H₃₀O₄ from a sodium salt ion at m/z 357 in the ESIMS and further supported by the HRESIMS at m/z 357.20365 (calcd. for C₂₀H₃₀O₄Na, 357.20363), thus implying six degrees of unsaturation. The presence of hydroxy and ester groups in 1 were evidenced by IR absorptions at υ_max 3,450 and 1,727 cm⁻¹. From the ¹³C and DEPT spectral data, six tetrasubstituted olefins were deduced from the signals at δ_C 145.40, 144.59, 122.58, 121.04, 118.48 and 117.25. A methyl esterified carboxyl group was supported by the ¹³C-NMR signals at δ_C 177.28 (C), 51.45 (CH₃) and the ¹H-NMR signal (

Table 1. ¹H (400 MHz, CDCl₃) and ¹³C (100 MHz, CDCl₃) NMR data, ¹H–¹H COSY and HMBC correlations for 1.

Position	δH (J in Hz)	δC, Multiple			1H–1H COSY	HMBC (H→C)
2		74.25		C
2a	1.22 s	23.67	23.65	CH3		C-2, -3, -1',
3	1.78 m	31.55		CH2	H2-4	C-4
4	2.60 t (6.8)	20.69		CH2	H2-3	C-2, -3, -4a, -5, -8b
4a		118.48		C
5		117.25		C
5a	2.11 s	11.26		CH3		C-4a, -5, -6
6		144.59		C
7		121.04		C
7a	2.10 s	11.75		CH3		C-7, -8
8		122.58		C
8a	2.16 s	12.19		CH3		C-7, -8
8b		145.40		C
1'	1.59 m, 1.51 m	39.35	39.31	CH2	H2-2'	n.o. a
2'	1.44 m	21.30	21.26	CH2	H2-1', H2-3'	n.o.
3'	1.66 m, 1.42 m	34.20		CH2	H2-2', H-4'	n.o.
4'	2.46 sext (5.6)	39.40	39.36	CH	H2-3', H3-4a'	n.o.
4a'	1.15 d (6.8)	17.06	17.02	CH3	H-4'	C-3', -4', -5'
5'		177.28	177.24	C
5'-OCH3	3.67 s	51.45	51.44	CH3		C-5'
6-OH	4.20 br s

^a n.o. = not observed.

) at δ_H 3.67 (3H, s, -OMe). Comparison of the ¹³C-NMR and DEPT spectra with the molecular formula indicated that there must be an exchangeable proton, requiring the presence of a hydroxy group. From the above data, four degrees of unsaturation were accounted for and 1 must be a compound with two rings.

From the ¹H–¹H COSY spectrum of 1 (Table 1 and Figure 1), the separate spin systems of H₂-3/H₂-4 and H₂-1'/H₂-2'/H₂-3'/H-4'/H₃-4a' were differentiated. These data, together with the key HMBC correlations between protons and quaternary carbons (Table 1 and Figure 1), such as H₃-2a, H₂-4/C-2; H₂-4, H₃-5a/C-4a; H₂-4, H₃-5a/C-5; H₃-5a/C-6; H₃-7a, H₃-8a/C-7; H₃-7a, H₃-8a/C-8; H₂-4/C-8b; and H₃-4a'/C-5', established the main carbon skeleton of 1. The vinyl methyls at C-5, C-7 and C-8 were confirmed by the HMBC correlations noted, thus, the remaining hydroxy group is positioned at C-6, an oxygen-bearing olefin carbon at δ_C 144.59. Based on the above findings, the structure of 1 is similar in structure to α-tocopherol. A set of additional carbon signals observed in the ¹³C-NMR spectrum of 1 (Table 1) supported the presence diastereoisomers. The stereochemistry of chiral carbons C-2 and C-4' was not determined at this stage.

It was found that the ¹H and ¹³C-NMR data of 1 are almost identical to those of a known synthetic compound, 5-(6-hydroxy-2,5,7,8-tetramethyl-chroman-2-yl)-2-methyl-pentanoic acid methyl ester, that was shown to exist as a set of diastereoisomers [5]. However, compound 1 has not been isolated previously from any natural sources.

In a previous study, the cembranoid, arbolide C, which was also isolated from S. arborea, showed an inhibitory effect on the release of elastase (IC₅₀ = 16.99 μM) by human neutrophils [4]. The in vitro anti-inflammatory effects of 1 was examined; 1 displayed an inhibitory effect on the generation of superoxide anion (IC₅₀ = 7.42 μM), but not active in inhibition of elastase release by human neutrophils, respectively.

Experimental

General

Infrared spectra were recorded on a Varian Diglab FTS 1000 FT-IR spectrometer (Varian Inc., Palo Alto, CA, USA); peaks are reported in cm⁻¹. NMR spectra were recorded on a Varian Mercury Plus 400 NMR spectrometer (Varian Inc.) using the residual CHCl₃ signal (δ_H 7.26 ppm) as the internal standard for ¹H-NMR and CDCl₃ (δ_C 77.1 ppm) for ¹³C-NMR. Coupling constants (J) are given in Hz. ESIMS and HRESIMS were recorded using a Bruker 7 Tesla solariX FTMS system (Bruker, Bremen, Germany). Column chromatography was performed on silica gel (230–400 mesh, Merck, Darmstadt, Germany). TLC was carried out on precoated Kieselgel 60 F₂₅₄ (0.25 mm, Merck); spots were visualized by spraying with 10% H₂SO₄ solution followed by heating. The normal phase HPLC (NP-HPLC) was performed using a system comprised of a Hitachi L-7110 pump (Hitachi Ltd. Tokyo, Japan), a Hitachi L-2455 photodiode array detector (Hitachi Ltd.) and a Rheodyne 7725 injection port (Rheodyne LLC. Rohnert Park, CA, USA). A normal phase column (Supelco Ascentis^® Si Cat #:581515-U, 25 cm × 21.2 mm, 5 µm, Sigma-Aldrich, St. Louis, MO, USA) was used for NP-HPLC. The reverse phase HPLC (RP-HPLC) was performed using a system comprised of a Hitachi L-7100 pump (Hitachi Ltd.), a Hitachi L-2455 photodiode array detector (Hitachi Ltd.), a Rheodyne 7725 injection port (Rheodyne LLC.) and a Varian Polaris 5 C-18-A column (25 cm × 10 mm, 5 μm).

Animal Material

Specimens of the octocoral Sinularia arborea [1] were collected by hand using SCUBA equipment off the coast of southern Taiwan on October, 2012, and stored at −20 °C until extraction. A voucher specimen (NMMBA-TWSC-1200X) was deposited in the National Museum of Marine Biology and Aquarium, Taiwan.

Extraction and Isolation

The freeze-dried and minced material of Sinularia arborea (wet weight 1.6 kg, dry weight 576 g) were minced and extracted with ethyl acetate (EtOAc). The extract (12.5 g) was separated by silica gel and eluted using a mixture of n-hexane/EtOAc in a stepwise fashion from 100:1 to pure EtOAc to yield 11 fractions A–K. Fraction E was purified by NP-HPLC, using a mixture of dichloromethane and methanol (90:1, flow rate: 2.0 mL/min) to yield 26 subfractions E1–E26. Fraction E20 was repurified by NP-HPLC, using a mixture of n-hexane and acetone (10:1, flow rate: 2.0 mL/min) to yield 9 subfractions E20A–E20I. Fraction E20G was separated by RP-HPLC, using a mixture of methanol and water (83:17, flow rate: 1.0 mL/min) to yield 5-(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)-2-methyl-pentanoic acid methyl ester (1, 6.2 mg, t_R = 27 min) as a colorless oil; IR (neat) ν_max 3450, 1727 cm⁻¹; ¹H (400 MHz, CDCl₃) and ¹³C (100 MHz, CDCl₃) NMR data, see Table 1; ESIMS: m/z 357 (M+Na)⁺; HRESIMS: m/z 357.20365 (calcd for C₂₀H₃₀O₄Na, 357.20363).

Generation of Superoxide Anions by Human Neutrophils

Human neutrophils were obtained by means of dextran sedimentation and Ficoll centrifugation. Measurements of superoxide anion generation were carried to previously described procedures [6,7]. Briefly, superoxide anion production was assayed by monitoring the superoxide dismutase-inhibitable reduction of ferricytochrome c. LY294002 (2-morpholin-4-yl-8-phenylchromen-4-one), a phosphatidylinositol-3-kinase inhibitor, was used as a positive control (IC₅₀ = 4.00 μM) for superoxide anion generation.