Halichonic Acid B, a Rearranged Nitrogenous Bisabolene-Type Sesquiterpene from a Marine Sponge Axinyssa sp.

Abstract

A new rearranged nitrogenous bisabolone-type sesquiterpene, halichonic acid B (1), was isolated from a marine sponge Axinyssa sp. together with halichonic acid (2) and (6R,7S)-7-amino-7,8-dihydro-α-bisabolene (3). The structure of 1 was determined by extensive NMR and MS analyses, revealing an unprecedented carbon framework, and its absolute configuration was elucidated by time-dependent density-functional theory (TDDFT)-based electronic circular dichroism (ECD) spectrum calculation. We propose that 1 and 2 may be biosynthesized in the same pathway, involving the reaction between farnesyl pyrophosphate and glycine, followed by cyclization.

Introduction

Marine sponges belonging to the family Halichondriidae are rich sources of nitrogenous bisabolene-type sesquiterpenoids. Sesquiterpenes with amine and isocyanate substituents were isolated from marine sponges Halichondria sp.¹⁾ and Ciocalypta sp.,²⁾ and other nitrogenous sesquiterpenes with formamide,^3–5) isonitrile,⁶⁾ isothiocyanate,^5,7) and urea⁷⁾ have been isolated from marine sponges of the genus Axinyssa. Recently, we also isolated a new rearranged nitrogenous bisabolene-type sesquiterpene, halichonic acid (2), with a novel 3-azabicyclo[3.3.1]nonene skeleton from Halichondria sp.⁸⁾ These nitrogenous bisabolene-type sesquiterpenoids have been reported to exhibit various biological functions, including antimicrobial,^1–4) cytotoxic,^1,6,8) and protein tyrosine phosphatase 1B inhibitory activities.⁷⁾

In our current research on bioactive secondary metabolites from marine sponges, a new rearranged nitrogenous bisabolene-type sesquiterpene, halichonic acid B (1), together with two known biosynthetically related sesquiterpenes [2⁸⁾ and (6R,7S)-7-amino-7,8-dihydro-α-bisabolene¹⁾ (3)] were isolated from Axinyssa sp. collected in Indonesia (Fig. 1). Here, we describe the structural elucidation of 1 and the antimicrobial activities of 1–3.

Fig. 1. Chemical Structures of Compounds 1–3

Results and Discussion

Halichonic acid B (1) was obtained as a yellowish amorphous solid with a molecular formula of C₁₇H₂₉NO₃ as established by high resolution-electrospray ionization-time-of-flight (HR-ESI-TOF)-MS. ¹H- and ¹³C-NMR spectra (methanol-d₄) displayed characteristic signals reminiscent of bisabolene-type sesquiterpenes, including four methyl singlets [δ_H 1.24/δ_C 28.9 (C-12); δ_H 1.31/δ_C 25.9 (C-13); δ_H 1.26/δ_C 20.7 (C-14); δ_H 1.67/δ_C 23.3 (C-15)], a trisubstituted double bond [δ_H 5.43/δ_C 120.1 (C-2); δ_C 135.4 (C-3)], two quaternary carbons [δ_C 61.0 (C-7); δ_C 72.6 (C-11)], three methines [δ_H 2.02 (m)/δ_C 36.4 (C-6); δ_H 2.02 (m)/δ_C 47.8 (C-10); δ_H 3.59 (d, J = 10.1 Hz)/δ_C 58.8 (C-2′)], a carbonyl carbon [δ_C 174.2 (C-1′)], and five methylenes (Table 1). Correlation spectroscopy (COSY) correlations showed two spin systems: H-2/H₂-1/H-6/H₂-5/H₂-4 and H₂-8/H₂-9/H-10/H-2′ (Fig. 2). Heteronuclear multiple bond correlations (HMBCs) from H₃-15 to C-2, C-3, and C-4 (δ_C 31.6), from H-2 to C-4 and C-6, and from H₂-5 to C-1 (δ_C 25.5) and C-3 established substructure A (Fig. 2). HMBCs from H-2′ to C-7, C-9 (δ_C 22.2), C-11, and C-1′, from H₃-14 to C-7 and C-8, from H₃-12 and H₃-13 to C-10 and C-11, and from H-10 to C-1′ established substructure B. The molecular formula and chemical shifts of C-7 (δ_C 61.0) and C-2′ (δ_C 58.8) suggest that these carbons are linked via a nitrogen atom, and the chemical shift of C-11 (δ_C 72.6) indicates that this carbon is oxygenated. For the three remaining hydrogen atoms, HMBCs between H-1β and H-5β/C-7 and H₃-14 and H₂-8/C-6 showed the connection of C-6 and C-7 and the tetrasubstituted piperidine structure with a carboxylic acid at C-2′ and hydroxy group at C-11 for substructure B. Thus, the planar structure of 1 was determined.

Table 1. NMR Data for Compound 1 in Methanol-d₄ and Pyridine-d₅

Position	Methanol-d4			Pyridine-d5
	δC, mult.	δH, mult. (J in Hz)	HMBC	δC, mult.	δH, mult. (J in Hz)
1	25.5, CH2	α 1.91, m		25.4, CH2	α 2.10, m
		β 2.03, m	2, 3, 5, 6, 7		β 2.40, m
2	120.1, CH	5.43, br d (4.3)	1, 4, 15	121.2, CH	5.34, br d (4.3)
3	135.4, C			133.5, C
4	31.6, CH2	α 2.01, m	15	31.1, CH2	α 1.89, m
		β 2.10, m			β 2.02, m
5	24.8, CH2	α 1.29, m	1, 4, 6	24.3, CH2	α 1.17, m
		β 1.75, m	1, 3, 4, 6, 7		β 1.61, m
6	36.4, CH	2.02, m		35.1, CH	2.20, m
7	61.0, C			56.4, C
8	32.2, CH2	α 1.61, m	6, 7, 10, 14	33.7, CH2	α 1.65, m
		β 2.14, d (4.1)	6, 7, 10, 14		β 2.05, m
9	22.2, CH2	α 1.86, m	7, 8, 10, 11, 2′	22.7, CH2	α 1.83, m
		β 1.62, m			β 1.63, m
10	47.8, CH	2.02, m		48.9, CH	2.37, m
11	72.6, C			71.3, C
12	28.9, CH3	1.24, s	10, 11, 13	30.1, CH3	1.43, s
13	25.9, CH3	1.31, s	10, 11, 12	25.4, CH3	1.48, s
14	20.7, CH3	1.26, s	6, 7, 8	22.6, CH3	1.24, s
15	23.3, CH3	1.67, s	2, 3, 4	23.4, CH3	1.60, s
1′	174.2, C			175.2, C
2′	58.8, CH	3.59, d (10.1)	7, 9, 10, 11, 1′	57.8, CH	4.01, d (10.5)

Fig. 2. COSY (Bold Lines) Correlations and Key HMBCs (Arrows) of Compound 1

The relative configuration of 1 was determined by nuclear Overhauser effect (NOE) correlations in pyridine-d₅ (Table 1) owing to the overlap of H-1β, H-4β, H-6, and H-10 in methanol-d₄. NOE correlations for H-2′/H-1β, H-2′/H-6, H-6/H-9β, H-2′/H-9β, H-9β/H₃-12, and H-2′/H₃-13 suggest β-orientations of H-2′, H-9β, a 4-methylcyclohex-3-enyl group at C-7, and a 2-hydroxyisopropyl group at C-10 (Fig. 3). On the other hand, NOE correlations for H-8α/H₃-14 and H-8α/H-10 suggest that they are α-oriented.

Fig. 3. Key NOE Correlations of Compound 1

The absolute configuration of 1 was determined by the theoretical electronic circular dichroism (ECD) spectrum. As 3 has a 6R,7S-configuration,¹⁾ the ECD spectrum of the biosynthetically expected 6R,7S,10S,2′S-1 was calculated using a standard time-dependent density-functional theory (TDDFT) procedure (Fig. 4). The experimental and calculated spectra matched well, and thus the absolute configuration of 1 was assigned.

Fig. 4. Experimental ECD Spectrum of Compound 1 and Calculated Spectrum of 6R,7S,10S,2′S-1

As shown in Chart 1, the biosynthesis of 1 may occur from farnesyl pyrophosphate and glycine in the same pathway as 2.⁸⁾ First, the nitrogen atom of glycine attacks farnesyl pyrophosphate, followed by cyclization and elimination of pyrophosphate to afford 4. Subsequent oxidation and dehydration form the imine 5, followed by cyclization to afford 1. Alternatively, cyclization with the formation of a new bond between C-2 and C-2′may yield 2.

Chart 1. Plausible Biosynthetic Pathway of Compounds 1–3

The antimicrobial activities of 1–3 were tested against Chromobacterium violaceum. Among them, 3 inhibited bacterial growth at an IC₅₀ value of 36 µM, whereas 1 and 2 had no antimicrobial activity even at 400 µM.

Experimental

General

Optical rotation was measured on a JASCO DIP-1000 polarimeter in MeOH. UV spectrum was measured on a JASCO V-550 spectrophotometer in CH₃CN. ECD spectrum was measured on a JASCO J-820 spectropolarimeter in CH₃CN. IR spectrum was recorded on a PerkinElmer, Inc. Frontier FT-IR spectrophotometer. NMR spectra were recorded on a Bruker Avance III 600 NMR spectrometer (¹H-NMR: 600 MHz, ¹³C-NMR: 150 MHz). Chemical shifts were referenced to the residual solvent peaks (δ_H 3.31 and δ_C 49.0 for methanol-d₄; δ_H 7.58 and δ_C 135.5 for pyridine-d₅). Mass spectrum was measured on a Waters Xevo G2-XS Qtof mass spectrometer. The preparative medium-pressure liquid chromatography (MPLC) was performed on a Biotage Isolera I. The HPLC system comprised a Waters 515 HPLC pump, Waters 2489 UV/visible detector, and Pantos Unicorder U-228.

Animal Material

Marine sponge Axinyssa sp. was collected by scuba at a depth of 10 m in North Sulawesi, Indonesia, in September 2014, and immediately soaked in EtOH. The sponge was identified by one of the authors (Y.I.). A voucher specimen (14M051) has been deposited at the Department of Natural Medicines, Graduate School of Pharmaceutical Sciences, Kumamoto University, Japan.

Extraction and Isolation

The sponge specimen (116 g, wet weight) was extracted with EtOH. After evaporation, the residual aqueous solution was extracted with EtOAc and then n-BuOH. The n-BuOH fraction (1.23 g) was subjected to MPLC (Purif SI silica gel, 30 g; Biotage Japan Ltd., Tokyo, Japan) eluted with a gradient system (0–15% n-Hexane–EtOAc for 0–16 min; 0–30% CH₂Cl₂–MeOH for 16–47 min) to yield five fractions (Frs. A1–A5). Fraction A3 was subjected to MPLC (SNAP Ultra C18, 10 g; Biotage Japan Ltd.) with a gradient system (10–100% MeOH–H₂O for 0–12 min) to yield three fractions (Frs. B1–B3). Fraction B2 was subjected to HPLC (COSMOSIL 5C18-MS-II, 20 × 250 mm, Nacalai Tesque Inc., Kyoto, Japan) eluted with 20–30% CH₃CN–H₂O containing 1% formic acid for 0–30 min to afford 1 (2.52 mg). Fraction B3 was subjected to HPLC (COSMOSIL 5C18-MS-II, 20 × 250 mm; Nacalai Tesque Inc.) eluted with 30% CH₃CN–H₂O containing 1% formic acid to afford 2 (3.60 mg). Fraction A3 was subjected to MPLC (SNAP Ultra C18, 10 g) with a gradient system (10–100% MeOH–H₂O for 0–11 min) to yield four fractions (Frs. C1–C4). Fraction C3 was subjected to HPLC (Asahipack GS-310P column, 21.5 × 500 mm; Asahi Chemical Industry Co., Ltd., Tokyo, Japan) eluted with MeOH to afford 3 (9.68 mg).

Halichonic Acid B (1)

Yellowish amorphous solid, [α]²¹_D +47 (c 0.40, MeOH); UV (CH₃CN) no absorption maximum above 195 nm; ECD (CH₃CN) λ_max (Δε) 201 (+1.9) and 226 (−0.8) nm; IR (film) ν_max 3367, 2966, 2924, 2856, 1628, 1602, 1441, and 1386 cm⁻¹; ¹H- and ¹³C-NMR data, see Table 1; HR-ESI-TOF-MS m/z 294.2049 [M–H]⁻ (Calcd for C₁₇H₂₈NO₃, 294.2075).

Conformational Analysis and ECD Calculation for 1

Conformational analysis and ECD calculations were performed as previously described⁹⁾ using Spartan’18 instead of Spartan’16. ECD calculations were performed at the BHandHLYP/TZVP level.

Antimicrobial Test

The antimicrobial activities of 1–3 against C. violaceum were evaluated using a broth microdilution method. C. violaceum was cultured in a 96-well plate in Luria–Bertani (LB) (Lennox) broth containing 1% dimethyl sulfoxide (DMSO) and the tested compounds. After 1-d incubation at 27 °C, bacterial growth was assessed using the resazurin assay. To each well of bacterial suspension, 50 µL of resazurin sodium salt in distilled water (60 µM) was added, and the plate was shaken at 27 °C for 40 min. The fluorescence of resorufin, which was generated by microbial reduction of resazurin, was measured (EX: 520 nm/EM: 590 nm) to evaluate bacterial growth. The IC₅₀ value of chloramphenicol (positive control) was 7.8 µM.

Acknowledgments

This work was supported by JSPS KAKENHI Grant Numbers JP26305005 (S.T.), JP20H03396 (S.T.), JP20K16026 (Y.H.), and JP21K15282 (A.H.H.E).

Conflict of Interest

The authors declare no conflict of interest.

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