Sporiolides A and B, New Cytotoxic Twelve-Membered Macrolides from a Marine-Derived Fungus Cladosporium Species

Abstract

Two new cytotoxic twelve-membered macrolides, sporiolides A (1) and B (2), were isolated from the cultured broth of a fungus Cladosporium sp., which was separated from an Okinawan marine brown alga Actinotrichia fragilis, and the structures were elucidated by spectroscopic data. Sporiolides A (1) and B (2) exhibited cytotoxicity against murine lymphoma L1210 cells. Spoliolide A (1) showed antifungal activity against Cryptococcus neoformans and Neurospora crassa.

Introduction

Marine microorganisms such as bacteria, fungi, and microalgae have proven to be a rich source of structurally novel and biologically active secondary metabolites [1]. In our search for new substances from marine microorganisms [2], two new cytotoxic twelve-membered macrolides, sporiolides A (1) and B (2), were isolated from the cultured broth of a fungus Cladosporium sp., which was separated from an Okinawan marine brown alga Actinotrichia fragilis. In this paper we describe the isolation and structure elucidation of 1 and 2.

Results and Discussion

The fungus Cladosporium sp. (L037) was separated from the brown alga collected off Seragaki Beach, Okinawa Island, and grown in SC broth [starch (1%) and casein (0.1%) in 50% sea water, pH 7.4] at 28°C for 14 days. The filtrate of the cultured broth (10 L) was extracted with EtOAc (1 L × 2). The EtOAc-soluble portions (58 mg) were subjected to a silica gel column (hexane/EtOAc, 70:30) followed by C₁₈ reversed-phase HPLC (Develosil ODS-5, Nomura Chemical, 1.0 × 25 cm: flow rate 2.5 mL/min; UV detection at 254 nm; eluent: MeOH/H₂O, 70:30) to give sporiolides A (1, 2.7 mg) and B (2, 11.5 mg) together with a known related macrolide, cladospolide D [3] (7.0 mg). On the other hand, other known compounds, cladospolide A [4–6] (5.0 mg), iso-cladospolide B [7] (2.0 mg), and seco-patulolide C [7] (3.0 mg), were isolated from the EtOAc extract of the mycelium.

Sporiolide A (1) {[α]_D²⁵ −14° (c 0.2, MeOH)} was obtained as colorless amorphous solid. The molecular weight of 1 was elucidated to be 348 Dalton on the basis of FABMS data that showed the pseudomolecular ion at m/z 371 (M+Na)⁺. The molecular formula, C₁₉H₂₄O₆, of 1 was established by HRFABMS data [m/z 371.1483, (M+Na)⁺, Δ +1.2 mmu]. The IR spectrum suggested the presence of hydroxy (3426 cm⁻¹), unsaturated ester and/or ketone carbonyl (1724 cm⁻¹) groups. The UV absorptions at 237 (9200) and 209 (11700) nm indicated the presence of benzoyl chromophore. The ¹H NMR (

Table 1. ¹H and ¹³C NMR Data of Sporiolides A (1) and B (2) in CDCl₃.

	1					2
position	δHa			δCb		δHa			δCb
1				168.8	s				171.5	s
2	3.52	dd	18.0, 9.8	40.5	t	3.30	m		42.2	t
	2.95	d	18.0			2.66	m
3	5.90	d	9.8	67.4	d	4.46	dd	9.0,2.0	74.1	d
4				207.1	s				207.8	s
5	4.40	m		76.0	d	4.36	dd	6.1,1.8	75.8	d
6	2.02	m		30.5	t	1.99	m		30.5	t
	1.77	m				1.69	m
7	1.34	m		19.0	t	1.47	m		22.8	t
	1.17	m				1.05	m
8	1.50	m		26.6	t	1.37a	m		26.6	t
	1.12	m
9	1.27	m		22.6	t	1.50	m		23.5	t
	1.21	m				1.42	m
10	1.67	m		33.4	t	1.59	m		33.6	t
	1.40	m				1.32	m
11	4.89	m		74.4	d	4.89	m		73.6	d
12	1.46b	d	5.3	20.8	q	1.22b	d	6.5	21.0	q
13				165.5	s	3.45b	s		58.2	q
14				129.2	s
15, 19	8.05	m		129.9	d
16,18	7.43	m		128.4	d
17	7.56	m		133.5	d

^a2H

^b3H.

) spectrum of 1 showed proton signals due to a benzoyl group [δ_H 8.05 (2H, m), 7.56 (1H, m), and 7.43 (2H, m)].

Analysis of the ¹H-¹H COSY spectrum (Figure 1) revealed connectivities of C-2 to C-3 and C-5 to C-12. HMBC correlations of H-3 (δ_H 5.90) to C-1 (δ_C 168.8), C-4 (δ_C 207.1), and C-5 (δ_C 76.0) and H-11 (δ_H 4.89) to C-1 indicated that 1 possessed a twelve-membered macrocyclic lactone with a ketone group at C-4 and a hydroxy at C-5. An HMBC correlation between H-3 to C-13 (δ_C 165.5) revealed that the benzoyl group was attached to C-3. Thus, the structure of sporiolide A was assigned as 1, which corresponded to be a 3-O-benzoyl form of pandangolide 1 [7].

Sporiolide B (2) {[α]_D²⁵ −33° (c 0.3, MeOH)} was obtained as colorless amorphous solid. The molecular weight of 2 was elucidated by m/z 281(M+Na)⁺ in the positive mode FABMS. The molecular formula, C₁₃H₂₂O₅, of 2 was established by HRFABMS data (m/z 281.1367 [M+Na]⁺, Δ+ 0.2mmu). The IR spectrum suggested the presence of hydroxy (3429 cm⁻¹), unsaturated ester and/or ketone carbonyl (1710 cm⁻¹) groups. Detailed analysis of ¹H, ¹³C, and 2D NMR data revealed that the structure of 2 was similar to that of 1, except for functional group at C-3. An HMBC correlation of H-3 (δ_H 4.46) to C-13 (δ_C 58.2, MeO) indicated the presence of a methoxy group at C-3. Thus, the structure of sporiolide B (2) was elucidated to be a 3-O-methoxy form of pandangolide 1 [7].

Sporiolides A (1) and B (2) were new twelve-membered macrocyclic lactones from the cultured broth of a marine-derived fungus Cladosporium sp. [8], although similar twelve-membered macrocyclic lactone such as cladospolide A has been obtained from a terrestrial fungus Cladosporium sp. and more recently, cladospolide D [3], iso-cladospolide B, seco-patulolide C, and pandangolides 1 and 2 have been isolated from an unidentified marine fungus [6,7], while pandagolides 2 and 3 were isolated from a marine-derived fungus Cladosporium herbarum [9]. Sporiolides A (1) and B (2) exhibited cytotoxicity against murine lymphoma L1210 cells with IC₅₀ values of 0.13 and 0.81 μg/mL, respectively. Sporiolide A (1) showed antifungal activity against Candida albicans, Cryptococcus neoformans, Aspergillus niger, and Neurospora crassa and antibacterial activity against Micrococcus luteus, while sporiolide B (2) had antibacterial activity against Micrococcus luteus (

Table 2. Antimicrobial Activity of Sporiolides A (1) and B (2).

Test organisms	MIC (μg/ml)
	1	2
Micrococcus luteus	16.7	16.7
Bacillus subtilis	>33.3	>33.3
Escherichia coli	>33.3	>33.3
Candida albicans	16.7	>33.3
Cryptococcus neoformans	8.4	>33.3
Paecilomyces variotii	>33.3	>33.3
Aspergillus niger	16.7	>33.3
Neurospora crassa	8.4	>33.3

Mueller Hinton broth and Sabouraud dextrose broth were used for bacteria and fungi, respectively.

).

Experimental

General

Optical rotations were measured on a JASCO DIP-1000 polarimeter. The IR and UV spectra were recorded on a JASCO FT/IR-5300 and a Shimadzu UV-1600PC spectrophotometer, respectively. CD spectra were measured on a JASCO J-720 spectropolarimeter. NMR spectra were recorded on a Bruker AMX-600 spectrometer. FAB mass spectrum was obtained on a JEOL HX-110 spectrometer using nitrobenzyl alcohol as a matrix.

Fungal Material and Fermentation

The fungus Cladosporium sp. (L037) was separated from the brown alga Actinotrichia fragilis, which was collected off Seragaki Beach at Okinawa Island. Subcultures of the organism are deposited at Graduate School of Pharmaceutical Sciences, Hokkaido University. The fungus was grown in SC broth [starch (1%) and casein (0.1%) in 50% sea water, pH 7.4] at 28°C for 14 days. The cultured broth (10 L) was filtered.

Extraction and Separation

The filtrate of the cultured broth (10 L) was extracted with EtOAc (1 L × 2). The EtOAc-soluble portions (58 mg) were subjected to a silica gel column (hexane/EtOAc, 70:30) followed by C₁₈ reversed-phase HPLC [Develosil ODS-5, Nomura Chemical, 1.0 × 25 cm: flow rate 2.5 mL/min; UV detection at 254 nm; eluent: MeOH/H₂O, 70:30] to give sporiolides A (1, 2.7 mg) and B (2, 11.5 mg) together with cladospolide D (7.0 mg). On the other hand, cladospolide A, isocladospolide B, and seco-patulolide C were isolated from the EtOAc extract of the mycelium.

Spectral Data

Sporiolide A (1): colorless amorphous solid; [_α]_D²⁵ −14° (c 0.2, MeOH)}; UV (MeOH) _λ^max 237 (_ɛ9200) and 209 (11700) nm; IR (KBr) _ν^max 3426, 1724, and 1633 cm^{−1; 1}H and ¹³C NMR (Table 1); FABMS m/z 371 [M+Na]⁺; HRFABMS m/z 371.1483 [M+Na]⁺ (calcd for C₁₉H₂₄O₆Na, 371.1471).

Sporiolide B (2): colorless amorphous solid; [α]_D²⁵ −33° (c 0.3, MeOH); IR (KBr) _ν^max 3429, 1710, and 1646 cm^{−1; 1}H and ¹³C NMR (Table 1); FABMS m/z 281 [M+Na]⁺; HRFABMS m/z 281.1367 [M+Na]⁺ (calcd for C₁₃H₂₂O₅Na, 281.1365).