Nakijiquinone S and Nakijinol C, New Meroterpenoids from a Marine Sponge of the Family Spongiidae

Abstract

New meroterpenoids, nakijiquinone S (1) and nakijinol C (2), have been isolated from an Okinawan marine sponge of the family Spongiidae. The gross structures and relative stereochemistries of 1 and 2 were elucidated on the basis of their spectral data. Nakijiquinone S (1) and nakijinol C (2) were new meroterpenoids consisting of a clerodane-type decalin ring connected to a 2-butoxy-5-hydroxy-benzoquinone unit or methyl 2,3,4-trihydroxybenzoate unit through a methylene, respectively. Nakijiquinone S (1) and nakijinol C (2) showed antimicrobial activities against several bacteria and fungi.

Marine sponges of the family Spongiidae have been demonstrated to be a rich source of unique bioactive meroterpenoids and acetylenic fatty acid derivatives.^1–5) During our search for bioactive metabolites from marine organisms, we investigated the extract of a sponge family Spongiidae (SS-1202) and resulted in the isolation of two new meroterpenoids, nakijiquinone S (1) and nakijinol C (2). Here we describe the isolation and structure elucidation of 1 and 2 (Fig. 1).

Fig. 1. Structures of Nakijiquinone S (1) and Nakijinol C (2)

The sponge family Spongiidae (SS-1202) collected at Unten Port, Okinawa, was extracted with MeOH to obtain the extract, which was partitioned between EtOAc and H₂O. EtOAc-soluble materials were separated by gel filtration, SiO₂ column chromatography, and C18 HPLC to afford nakijiquinone S (1, 0.000042%, wet weight) and nakijinol C (2, 0.00011%). Seven known related meroterpenoids, arenarol,⁶⁾ dictyoceratins A and B,⁷⁾ 18-hydroxyhyrtiophenol,⁸⁾ isospongiaquinone,⁹⁾ and nakijiquinones E and Q,^2,10) have been isolated in the purification process of 1 and 2.

Nakijiquinone S (1) was obtained as optically active brown oil. High resolution-electrospray ionization (HR-ESI) MS analysis established the molecular formula of 1 to be C₂₅H₃₆O₄. IR absorptions (3330, 1640 cm⁻¹) indicated the presence of OH and quinone functionalities. UV absorptions (λ_max 292, 520 nm) suggested the presence of a quinone chromophore. Inspection of the heteronuclear multiple quantum coherence (HMQC) spectrum with ¹H- and ¹³C-NMR data of 1 (Table 1) disclosed the existence of twenty-five carbons consisting of two carbonyl carbons (δ_C 182.5, 182.1), four sp² quaternary carbons (δ_C 161.3, 153.1, 144.0, 117.6), two sp² methines (δ_C 120.9, 102.2), two sp³ quaternary carbons (δ_C 43.1, 38.5), two sp³ methines (δ_C 48.1, 37.9), eight sp³ methylenes (δ_C 70.0, 36.0, 32.4, 30.1, 27.9, 27.0, 19.8, 19.1), and five sp³ methyls (δ_C 20.1, 18.2, 17.7, 17.3, 13.6). Among them, two carbonyl carbons, three sp² quaternary carbons (δ_C 161.3, 153.1, 117.6), and one sp² methine (δ_C 102.2) were ascribed to a 2,5-dihydroxy-1,4-benzoquinone moiety (C-16–C-21).^2,9,10) Analysis of the ¹H–¹H correlated spectroscopy (COSY) spectrum of 1 revealed three partial structures, a (C-3 to C-10), b (C-6 to C-13), and c (C-22 to C-25) (Fig. 2). Heteronuclear multiple bond correlation (HMBC) correlations of H₃-11/C-3 and H₃-11/C-4 disclosed that 1,2-disubstituted propene moiety (C-3, C-4, C-11). HMBC correlations of H₃-11/C-5, H₃-12/C-4, H₃-12/C-5, H₃-12/C-6, and H₃-12/C-10 uncoverd that C-4, C-6, C-10, and C-12 were connected to C-5. Linkings of C-8, C-10, C-14, and C-15 through C-9 were inferred from HMBC correlations of H₃-13/C-9, H₃-14/C-9, H₂-15/C-8, H₂-15/C-9, and H₂-15/C-10. HMBC correlations of 17-OH/C-16, 17-OH/C-17, 17-OH/C-18, H-19/C-17, H-19/C-18, H-19/C-20, and H-19/C-21 supported the presence of a 2,5-dihydroxy-1,4-benzoquinone moiety instead of a 4,5-dihydroxy-1,2-benzoquinone moiety. Attachment of a 2,5-dihydroxy-1,4-benzoquinone unit to C-15 was deduced from HMBC correlations of H₂-15/C-16, H₂-15/C-17, and H₂-15/C-21. An HMBC correlation of H₂-22/C-20 revealed that one hydroxy group of 2,5-dihydroxy-1,4-benzoquinone unit was n-butylated. Thus, the gross structure of nakijiquinone S (1) was elucidated to be shown in Fig. 1.

Table 1. ¹H- and ¹³C-NMR Data of Nakijiquinone S (1) and Nakijinol C (2) in DMSO-d₆

Position	1			Position	2
	δHa)	Multi (J in Hz)	δCb)		δHa)	Multi (J in Hz)	δCb)
1	2.03	me)	19.8	1	2.03	me)	19.6
	1.46	me)			1.57	me)
2	2.00	me)	27.0	2	2.14	me)	26.0
	1.86	me)			2.08	me)
3	5.13	br s	120.9	3	5.14	br s	120.5
4	—		144.0	4	—		144.4
5	—		38.5	5	—		38.2
6	1.62	ddd (12.7, 3.3, 3.3)	36.0	6	1.57	me)	35.9
	1.04	me)			0.92	me)
7	1.34c)	me)	27.9	7	1.35c)	me)	27.7
8	1.28	me)	37.9	8	1.30	me)	35.9
9	—		43.1	9	—		41.4
10	1.04	me)	48.1	10	1.18	d (11.8)	45.4
11	1.53d)	br s	18.2	11	1.51d)	s	18.1
12	1.00d)	s	20.1	12	1.02d)	s	20.0
13	0.96d)	d (6.2)	17.7	13	1.01d)	d (6.2)	17.7
14	0.83d)	s	17.3	14	0.84d)	s	17.5
15	2.60	d (13.7)	32.4	15	2.68	d (14.3)	36.3
	2.48	d (13.7)			2.60	d (14.3)
16	—		117.6	16	—		117.2
17	—		153.1	17	—		149.1
18	—		182.1	18	—		130.3
19	5.81	s	102.2	19	—		147.1
20	—		161.3	20	—		104.4
21	—		182.5	21	7.18	s	124.8
22	3.95c)	t (6.6)	70.0	22	—		170.7
23	1.84c)	me)	30.1	23	3.90d)	s	52.0
24	1.46c)	me)	19.1	17-OH	10.74f)	br s	—
25	0.96d)	t (7.4)	13.6	18-OH	5.52f)	br s	—
17-OH	7.49	s	—	19-OH	5.98f)	br s	—

a) 600 MHz. b) 150 MHz. c) 2H. d) 3H. e) J-Values were not determined because of overlapping with other signals. f) These signals might be exchangeable.

Fig. 2. Selected 2D-NMR Correlations for Nakijiquinone S (1)

The relative stereochemistry of a decalin moiety in nakijiquinone S (1) was deduced from nuclear Overhauser enhancement and exchange spectroscopy (NOESY) correlations as shown in Fig. 3. The α-configuration of H-10 and β-configurations of C-12, C-13, and C-14 were inferred from NOESY correlations of H-8/H-10, H-10/H₂-15, and H₃-12/H₃-14, indicating a twist-boat conformation of ring A (C-1 to C-5 and C-10) and a chair conformation of ring B (C-5 to C-10).

Fig. 3. Selected NOESY Correlations and Relative Stereochemistry for a Decalin Moiety (C-1–C-15) of Nakijiquinone S (1) (Hydrogen Atoms of Methyl Groups Were Omitted)

Nakijinol C (2) was obtained as colorless amorphous solid and its molecular formula, C₂₃H₃₂O₅, was clarified by HR-ESI-MS analysis. IR absorptions (3411, 1671 cm⁻¹) implied the existence of OH and carbonyl functionalities. The UV absorption (λ_max 221 nm) suggested the presence of an aromatic chromophore. Analyses of one dimensional (1D)- and 2D-NMR spectra disclosed that 2 had a clerodane-type decalin moiety (C-1–C-15) identical with that of 1 (Fig. 4) and the remaining part of 2 was composed by one carbonyl carbon (δ_C 170.7), five sp² quaternary carbons (δ_C 149.1, 147.1, 130.3, 117.2, 104.4), one sp² methine (δ_C 124.8), one methoxy methyl (δ_C 52.0), and three hydroxy groups. Comparison of NMR data between 2 and known related meroterpenoids⁷⁾ as well as HMBC correlations of H-21/C-17, H-21/C-19, H-21/C-22, and H₃-23/C-22 exposed the existence of monosubstituted methyl 2,3,4-trihydroxybenzoate unit. An HMBC correlation of H₂-15/C-21 clarified that a methyl 2,3,4-trihydroxybenzoate unit was attached to C-15 via C-16. Thus, the gross structure of nakijinol C (2) was assigned as shown in Fig. 1.

Fig. 4. Selected 2D NMR Correlations for Nakijinol C (2)

Inspection of the NOESY spectrum of nakijinol C (2) suggested that its relative stereochemistry and conformation of a decalin moiety was the same as that of 1 (Fig. 5).

Fig. 5. Selected NOESY Correlations and Relative Stereochemistry for a Decalin Moiety (C-1–C-15) of Nakijinol C (2) (Hydrogen Atoms of Methyl Groups Were Omitted)

Nakijiquinone S (1) and nakijinol C (2) are new meroterpenoids consisting of a clerodane-type decalin ring connected to a 2-butoxy-5-hydroxy-benzoquinone unit or methyl 2,3,4-trihydroxybenzoate unit through a methylene, respectively. Antimicrobial assay¹¹⁾ of nakijiquinone S (1) and nakijinol C (2) revealed their antimicrobial activities against several bacteria and fungi as shown in Table 2, while 1 and 2 did not show cytotoxicity (IC₅₀>10 µg/mL) against murine lymphoma L1210 and human epidermoid carcinoma KB cells in vitro.

Table 2. Antimicrobial Activities of Nakijiquinone S (1) and Nakijinol C (2)

Strains	1	2
Escherichia coli	8.0	>32.0
Staphylococcus aureus	1.0	8.0
Bacillus subtilis	1.0	>32.0
Micrococcus luteus	1.0	4.0
Aspergillus niger	4.0	8.0
Trichophyton mentagrophytes	4.0	8.0
Candida albicans	16.0	32.0
Cryptococcus neoformans	>32.0	32.0

The units for values indicating activities against bacteria and fungi are MIC (µg/mL) and IC₅₀ (µg/mL), respectively.

Experimental

General Methods

Optical rotations were recorded on a JASCO P-1030 polarimeter. IR spectra were recorded on JASCO FT/IR-230 spectrometer. ¹H- and ¹³C-NMR spectra were recorded on a Bruker AMX-600 spectrometer using 2.5 mm micro cell. The 7.20 and 77.0 ppm resonances of residual CHCl₃ and CDCl₃ were used as internal references for ¹H- and ¹³C-NMR spectra, respectively. Electron ionization (EI)-MS spectra were recorded on a JEOL JMS-700TZ mass spectrometer. ESI-MS spectra were recorded on JEOL JMS-700TZ and Thermo Scientific Exactive mass spectrometers.

Sponge Description

The sponge (SS-1202, family Spongiidae) was collected at Unten Port, Okinawa, and kept frozen until used. The sponge was medium brown mound with a spikey, conulose surface, hispid with a fine adherent membrane, and alcohol brown stains. Dermis was unarmored. The sponge has a reticulate fibre skeleton with some pithing of fibres centrally. The reticulation is irregular and all fibres are uncored. Primary fibres are ca. 50 µm wide. Secondary fibres are ca. 40 µm wide with finer fibres between as a tertiary skeleton, 10 µm wide. The voucher specimen was deposited at Graduate School of Pharmaceutical Sciences, Hokkaido University.

Extraction and Separation

The sponge family Spongiidae (SS-1202, 1.25 kg, wet weight) collected at Okinawa, was extracted with MeOH (2 L×2) to obtain the extract (73.3 g), which was partitioned between EtOAc (500 mL×3) and H₂O (500 mL) to give EtOAc-soluble materials (11.8 g). A part of EtOAc soluble materials (1.0 g) was fractionated by gel filtration (Sephadex LH-20, GE Healthcare; eluent, MeOH). A fraction was purified by SiO₂ column chromatography (Wakosil C-300, Wako Pure Chemical Industries, Ltd.; eluent, CH₃Cl–MeOH, 95 : 5 to 0 : 100) and C18 HPLC (Wakosil-II 5C18 AR, 10×250 mm, Wako Pure Chemical Industries, Ltd.; eluent, MeOH–H₂O, 85 : 15; flow rate, 2.0 mL/min; UV detection at 300 nm) to afford nakijiquinone S (1, 0.52 mg, 0.000042%, wet weight). Another fraction eluted from the LH-20 column after the fraction contained 1 was purified by SiO₂ column chromatography (Wakosil C-300, Wako Pure Chemical Industries, Ltd.; eluent, CH₃Cl–MeOH, 95 : 5 to 0 : 100) and C18 HPLC (Wakosil-II 5C18 AR, 10×250 mm, Wako Pure Chemical Industries, Ltd.; eluent, MeOH–H₂O, 85 : 15; flow rate, 2.0 mL/min; UV detection at 300 nm) to afford nakijinol C (2, 1.32 mg, 0.00011%, wet weight).

Nakijiquinone S (1): Brown oil; [α]_D²³ +42 (c=0.10, CHCl₃); UV (MeOH) λ_max (ε) nm: 292 (11900), 520 (670); IR (neat) ν_max cm⁻¹: 3330, 1730, 1640, 1610, 1460, 1380, 1230, 1210; ¹H- and ¹³C-NMR, see Table 1; HR-EI-MS m/z 400.2600 [M⁺, Δ −1.4 mmu], Calcd for C₂₅H₃₆O₄.

Nakijinol C (2): Colorless amorphous solid; [α]_D²⁴ +46 (c=0.52, CHCl₃); UV (MeOH) λ_max (ε) nm: 202 (2127), 221 (1812); IR (film) ν_max cm⁻¹: 3411, 2937, 2857, 1671, 1560, 1438, 1169, 1083, 1023, 962, 788, 758; ¹H- and ¹³C-NMR, see Table 1; HR-ESI-MS m/z 387.21778 [(M−H)⁻, Δ+0.08 mmu], Calcd for C₂₃H₃₁O₅.

Acknowledgment

We thank Mr. Z. Nagahama and Mr. K. Uehara, Okinawa, for their help with sponge collection, and Ms. S. Oka, Instrumental Analysis Division, Equipment Management Center, Creative Research Institution, Hokkaido University, for measurements of ESI-MS. This work was supported by The Naito Foundation, Cooperative Research Program of Medical Mycology Research Center, Chiba University, and Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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