Hyrtinadines C and D, New Azepinoindole-Type Alkaloids from a Marine Sponge Hyrtios sp.

Abstract

New bisindole alkaloids, hyrtinadines C (1) and D (2), have been isolated from an Okinawan marine sponge Hyrtios sp. The structures of hyrtinadines C (1) and D (2) were elucidated based on analyses of the spectral data. Hyrtinadines C (1) and D (2) were the relatively rare alkaloids possessing a 3,4-fused azepinoindole skeleton. Hyrtinadines C (1) and D (2) showed antimicrobial activity.

Marine sponges of the genus Hyrtios have been recognized as a rich source of unique bioactive products.¹⁾ During our search for new bioactive metabolites from marine organisms, a series of indole alkaloids have been isolated from marine sponges of the genus Hyrtios.^2–8) Recently, we have isolated two new bisindole alkaloids, hyrtinadines C (1) and D (2) from the extract of a sponge Hyrtios sp. (SS-1171). Here we describe the isolation and structure elucidation of 1 and 2 (Fig. 1).

Fig. 1. Structures of Hyrtinadines C (1) and D (2)

The sponge Hyrtios sp. (SS-1171) collected at Okinawa was extracted with MeOH, and the extract was partitioned between organic solvents (EtOAc and BuOH sequentially) and H₂O. BuOH soluble materials were separated by Sephadex LH-20 column, C₁₈ column, and hydrophilic interaction liquid chromatography (HILIC) HPLC to obtain hyrtinadines C (1, 0.0019%, wet weight) and D (2, 0.0003%, wet weight). Two known indole alkaloids, dragmacidonamines A and B,⁹⁾ have been isolated in the purification process of 1 and 2.

Hyrtinadine C (1) was obtained as an optical active yellow amorphous solid. The molecular formula of 1 was revealed to be C₂₀H₁₅N₃O₅ by high resolution-electrospray ionization (HR-ESI) MS data [m/z 400.09269 (M+Na)⁺, Δ +1.75 mmu]. The UV absorption [λ_max 429 (ε 7400) nm] was attributed to heteroaromatic rings, while IR absorption (3429 cm⁻¹) indicated the presence of hydroxy and/or amino functionalities. Inspection of the heteronuclear single quantum coherence (HSQC) spectrum with ¹H- and ¹³C-NMR data of 1 (Table 1) disclosed the existence of twenty carbons consisting of one carbonyl carbon (δ_C 169.3), one imino carbon (δ_C 163.9), nine sp² quaternary carbons (δ_C 157.0, 153.9, 131.2, 130.2, 125.6, 125.2, 117.9, 109.8, 105.2), seven sp² methines (δ_C 136.8, 127.9, 120.8, 113.7, 113.1, 112.7, 103.8), and two sp³ methines (δ_C 68.4, 65.0). Among them, nine sp² quaternary carbons and seven sp² methines were ascribed to two 5-hydroxyindole moieties (N-1, C-2–C-7a and N-1′, C-2′–C-7a′), which was supported by ¹H–¹H correlated spectroscopy (COSY) and heteronuclear multiple bond correlation (HMBC) spectra of 1 (Fig. 2). HMBC correlations for H-6/C-8′ and H-2′/C-8′ suggested that two 5-hydroxyindole units were attached to an imino carbon (C-8′) by C-4 and C-3′, respectively. ¹H–¹H COSY correlations for H-8/8-OH and H-8/H-9 and an HMBC correlation for H-9/C-11 revealed that the existence of a 3-hydroxypropionic acid moiety (C-8, C-9, C-11). HMBC correlations for H-2/C-8 and H-9/C-3 disclosed the connection of C-3 and C-8. The connectivity of C-9 and imino carbon (C-8′) through N-10 was inferred from an HMBC correlation for H-9/C-8′. Thus, the gross structure of 1 was elucidated to be a new bisindole alkaloids possessing a 3,4-fused azepinoindole skeleton (N-1, C-2–C-9, N-10, and C-8′) as shown in Fig. 1, which correspond to 8-hydroxy derivative of hyrtiazepine.¹⁰⁾ The relative configuration of 1 was deduced by comparison of ³J_H-8/H-9 of 1 with that of the calculated stable conformers for H-8/H-9 erythro form (1a) and H-8/H-9 threo form (1b) of 1. The dihedral angles of H-8 and H-9 for the most stable conformers of 1a and 1b were 72 and 174 degrees, and the ³J_H-8/H-9 were 0.5 and 10.4 Hz, respectively (Fig. 3). The relative configuration of H-8/H-9 was assigned as erythro, since the ³J_H-8/H-9 (≈0 Hz) of 1 was close to that of 1a rather than that of 1b.

Table 1. ¹H- and ¹³C-NMR Data of Hyrtinadines C (1) and D (2) in DMSO-d₆

Position	1			2
	δHa)	Multi (J in Hz)	δCb)	δHa)	Multi (J in Hz)	δCb)
1	11.73	br s	—	11.56	br s	—
2	7.54	d (2.2)	127.9	7.57	br s	127.7
3	—	—	117.9	—	—	113.4
3a	—	—	125.6	—	—	125.4
4	—	—	105.2	—	—	105.2
5	—	—	157.0	—	—	156.4
6	6.98	br d (8.6)	112.7	6.73	br d (8.7)	112.9
7	7.69	d (8.7)	120.8	7.59	d (8.9)	121.3
7a	—	—	130.2	—	—	128.8
8	5.53	br s	68.4	5.17	s	78.1
9	3.86	br s	65.0	3.76	s	63.7
11	—	—	169.3	—	—	168.2
5-OH	9.42	br s	—	9.27	br s	—
8-OH	5.28	br s	—	—	—	—
8-OCH3	—	—	—	3.13	s	54.5
1′	12.49	br s	—	12.09	br s	—
2′	7.79	s	136.8	7.76	s	135.5
3′	—	—	109.8	—	—	110.5
3a′	—	—	125.2	—	—	125.2
4′	7.37	br s	103.8	7.31	br s	103.9
5′	—	—	153.9	—	—	153.5
6′	6.84	dd (8.6, 1.3)	113.1	6.77	dd (8.6, 1.7)	112.9
7′	7.38	d (8.7)	113.7	7.33	d (8.8)	113.3
7a′	—	—	131.2	—	—	131.2
8′	—	—	163.9	—	—	162.1
5′-OH	10.37	br s	—	10.16	br s	—

a) 600 MHz. b) 150 MHz.

Fig. 2. Selected 2D-NMR Correlations for Hyrtinadine C (1)

Fig. 3. The Most Stable Conformers of H-8/H-9 erythro Form (1a) and H-8/H-9 threo Form (1b) of Hyrtinadine C (1)

Hyrtinadine D (2) was obtained as an optical active red amorphous solid. The molecular formula of 2 was established as C₂₁H₁₇N₃O₅ by HR-ESI-MS data [m/z 414.10936 (M+Na)⁺, Δ +2.77 mmu]. The molecular formula and NMR data of 2 (Table 1) implied that 2 was an O-methyl derivative of 1, which was supported by two dimensional (2D)-NMR spectra of 2. An HMBC correlation between methoxy protons (δ_H 3.13) and C-8 (δ_C 78.1) uncovered that the methoxy group was attached to C-8. The relative configuration of H-8 and H-9 was assigned as erythro from ³J_H-8/H-9 (≈0 Hz) of H-8/H-9. Thus, the structure of hyrtinadine D (2) was elucidated as shown in Fig. 1. Since the circular dichroism (CD) spectra of 1 and 2 exhibited a similar pattern, the absolute configurations of 1 and 2 were expected to be the same.

Though the number of natural products possessing a 3,4-fused azepinoindole skeleton was limited,^6,8,10–18) those compounds were attracting great interest as synthetic targets.¹⁹⁾ Therefore, 1 and 2 were the new members of relatively rare class of natural products. Antimicrobial activities of 1 and 2 against bacteria (Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Micrococcus luteus) and fungi (Aspergillus niger, Trichophyton mentagrophytes, Candida albicans, Cryptococcus neoformans) were tested. Hyrtinadine C (1) showed antifungal activity against A. niger (IC₅₀ 32 µg/mL), while hyrtinadine D (2) showed antibacterial activity against E. coli (minimum inhibitory concentration (MIC) 16 µg/mL) and B. subtilis (MIC 16 µg/mL). They did not show antimicrobial activity against other bacteria (MIC >32 µg/mL) and fungi (IC₅₀ >32 µg/mL).

Experimental

General Methods

Optical rotations were recorded on a JASCO P-2200 polarimeter. UV spectra were recorded on a JASCO V-630BIO spectrophotometer. IR spectra were recorded on a HORIBA FT710 spectrophotometer. CD spectra were recorded on a JASCO J-600 spectropolarimeter. ¹H- and ¹³C-NMR spectra were recorded on a Bruker Avance II 600 MHz NMR spectrometer equipped with a cryoplatform using 3.0 mm micro cells (Shigemi Co., Ltd., Japan) for DMSO-d₆. The 2.49 ppm resonance of residual DMSO-d₅ and 39.5 ppm resonance of DMSO-d₆ were used as internal references for ¹H- and ¹³C-NMR spectra, respectively. MS spectra were recorded on a JMS-T100LP spectrometer.

Extraction and Separation

The sponge (SS-1171) Hyrtios sp. was collected off Unten Port, Okinawa, in 2006, and kept frozen until used. The sponge (0.2 kg, wet weight) was extracted with MeOH (1.5 L×3) to obtain the extract (8.55 g). The extract was partitioned stepwise between organic solvents [EtOAc (500 mL×3) and BuOH (500 mL×3)] and H₂O (500 mL) to give EtOAc materials (1.31 g) and BuOH-soluble materials (1.68 g). The BuOH soluble materials (1.68 g) were fractionated by gel filtration (Sephadex LH-20, GE Healthcare (U.S.A.); eluent, MeOH), and a fraction was separated by C₁₈ column chromatography (Silica gel 120 (spherical) RP-18, Kanto Chemical Co., Inc. (Japan); eluent, MeOH–H₂O–trifluoroacetic acid (TFA), 50 : 50 : 0 to 100 : 0 : 0.1) and HILIC HPLC (Cosmosil HILIC, 10×250 mm, Nacalai Tesque Inc. (Japan); eluent, MeCN–H₂O, 60 : 40; flow rate, 2.0 mL/min; UV detection at 220 nm) to yield hyrtinadine C (1, 3.7 mg, 0.0019%, wet weight) and a fraction including hyrtinadine D (2). Hyrtinadine D (2, 0.6 mg, 0.0003%, wet weight) was purified by HILIC HPLC (Cosmosil HILIC, 10×250 mm, Nacalai Tesque Inc.; eluent, MeCN–H₂O, 70 : 30; flow rate, 2.0 mL/min; UV detection at 220 nm).

Hyrtinadine C (1)

Yellow amorphous solid; [α]_D²⁶ −157.8 (c=0.25, MeOH); UV (MeOH) λ_max (ε) nm: 220 (23700), 429 (7400); IR (KBr plate) ν_max cm⁻¹: 3429, 2916, 1624, 1585, 1369, 1207, 1138; CD (MeOH) Δε₂₁₆ −0.21, Δε₂₃₆ 0.05, Δε₂₅₅ 0.00, Δε₂₆₇ 0.03, Δε₂₈₉ −0.03, Δε₃₃₇ 0.03, Δε₃₆₂ 0.00, Δε₄₀₄ 0.08; ¹H- and ¹³C-NMR see Table 1; ESI-MS m/z 400 (M+Na)⁺; HR-ESI-MS m/z 400.09269 [(M+H)⁺, Δ +1.75 mmu]. Calcd for C₂₀H₁₅N₃O₅Na.

Hyrtinadine D (2)

Red amorphous solid; [α]_D²⁵ −764.7 (c=0.25, MeOH); UV (MeOH) λ_max (ε) nm: 218 (21100), 400 (9800); IR (KBr plate) ν_max cm⁻¹: 3402, 2916, 1624, 1585, 1356, 1207, 1140, 1064; CD (MeOH) Δε₂₁₅ −0.30, Δε₂₄₁ 0.11, Δε₂₅₄ 0.04, Δε₂₆₄ 0.07, Δε₂₈₉ −0.05, Δε₃₃₄ 0.03, Δε₃₆₃ 0.01, Δε₄₃₁ 0.11; ¹H- and ¹³C-NMR see Table 1; ESI-MS m/z 414 (M+Na)⁺; HR-ESI-MS m/z 414.10936 [(M+Na)⁺, Δ +2.77 mmu]. Calcd for C₂₁H₁₇N₃O₅Na.

Calculations

Conformational searches were performed with Spartan 14 (Wavefunction, Inc. (U.S.A.)). Two possible structures (1a, 1b) of hyrtinadine C (1) were submitted to a conformational search, which was carried out with MMFF force-field. The top 20 stable conformers for each of 1a and 1b that were selected from 10000 conformers, were optimized by ab initio molecular orbital calculations at the HF/3-21G level. The stable conformers were further optimized by density functional theory (DFT) calculations at the EDF2/6-31G* level assuming solvent-less (vacuum) conditions.

Antimicrobial Activity Test

Antimicrobial activity tests of hyrtinadines C (1) and D (2) against Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Micrococcus luteus, Aspergillus niger, Candida albicans, Cryptococcus neoformans, and Trichophyton mentagrophytes were carried out as previously described.²⁰⁾

Acknowledgments

We thank late Mr. Z. Nagahama and Mr. K. Uehara for his help with sponge collection, and Mr. K. Chiba, the Instrument Analysis Equipment Research Center, Showa Pharmaceutical University, for measurements of MS. This work was partially supported by JSPS KAKENHI Grant Number 25460115.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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