Papuamides E and F, Cytotoxic Depsipeptides from the Marine Sponge Melophlus sp

Abstract

Two known papuamides C (1) and D (2) together with two new depsipeptides, papuamides E (3) and F (4), were isolated from an undescribed sponge of the genus Melophlus collected in the Solomon Islands. The planar structures of the compounds were elucidated on the basis of spectroscopic studies. Papuamides C–F (1–4) showed cytotoxicity against brine shrimp with LD₅₀ values between 92 and 106 μg/mL.

1 Introduction

Marine sponges that are active against human immunodeficiency virus (HIV) have served as a rich source of cyclic depsipeptides incorporating rare amino acid residues. The marine sponges Theonella swinhoei and Theonella mirabilis (family Theonellidae) from Papua New Guinea have yielded papuamides A–C, cyclic depsipeptides with structurally unique features including unprecedented acid moieties.¹ Callipeltin A, isolated from the New Caledonian marine sponge Callipelta sp.², neamphamide A, obtained from Neamphius huxlei³ and mirabamides A–D from Siliquariaspongia mirabilis⁴ are also well recognized for their potent HIV-inhibitory activity. Mirabamide C and novel congeners mirabamides E–H with anti-HIV activity were also recently reported from Stelletta clavosa (family Ancorinidae).⁵

As part of our continuing search for bioactive metabolites from marine invertebrates, in the present study, we isolated two new depsipeptides (3–4) along with the previously reported papuamides C and D¹ from the butanol extract of a marine sponge Melophlus sp. Marine sponges of the genus Melophlus (family Ancorinidae) have been reported to yield compounds belonging to tetramic acids and saponins.^6–9 This is the first report of depsipeptides from Melophlus sp. In this paper, we describe the isolation, structure elucidation and bioactivity of these new depsipeptides.

2 Results and discussion

The sponge was extracted three times at room temperature using MeOH followed by CH₂Cl₂. The extracts were combined, evaporated in vacuo and partitioned between H₂O and CH₂Cl₂. The aqueous layer was further partitioned with n-BuOH and the active n-BuOH extract was subjected to bioassay guided fractionation using C18 bonded silica. Further purification by reverse-phase C18 HPLC afforded compounds (1–4).

The major metabolites, compounds 1 and 2, showed protonated molecular ions [M+H]⁺ at m/z 1399.73678 and 1385.7203 in the HRMS (ESI), respectively. A marinlit (2010) search for the corresponding masses revealed a match to the known papuamides C and D.¹⁰ The NMR spectral data of 1 and 2 were identical to the literature values of papuamides C and D, respectively.

Compounds 3 and 4 were isolated in smaller quantities compared with 1 and 2. Comparison of the ¹H NMR spectra of 3 and papuamide C (1) indicated strong similarities over most of the molecule. Detailed 2D NMR data analysis of 3 together with comparison to 1 enabled us to identify 11 amino acid residues present in 3 to be identical to the corresponding residues in 1: two glycine (Gly) residues, threonine (Thr), aminobutenoic acid (Aba), 3,4-dimethylglutamine (3,4-DiMeGln), 3-hydroxyleucine (3-OHLeu), 3-methoxyalanine (3-OMeAla), alanine (Ala), N-methylthreonine (NMeThr), β-methoxytyrosine (β-OMeTyr) and homoproline (Hpr) (Table 1).

Table 1.

NMR Spectroscopic Data (400 MHz, CD₃OD), for Papuamides E (3) and F (4)

	Papuamide E (3)		Papuamide F (4)
	δC	δHa	δC	δHa
Homoproline (Hpr)
1	n.o.	–	n.o.	–
2	52.7	5.32 m	52.7	5.18 d (9.6)
3	27.3	2.20 m, 1.77 m	27.5	2.25 m
4	21.1	1.26 m	21.7	1.75 s
5	26.9	1.76 m	25.8	1.72 ovl
6	44.3	4.15 m	44.9	4.06 m
β-Methoxytyrosine (β-OMeTyr)
1	n.o.	–	n.o.	–
2	54.0	5.15 d (5.2)	54.0	5.22 m
3	85.5	4.25 d (9.6)	84.9	4.33 m
3-OMe	56.1	3.10 s	56.9	3.15 s
1′	129.3	–	129.1	–
2′, 6′	130.6	7.21 d (8.4)	130.3	7.20 d (8.4)
3′, 5′	116.0	6.77 d (8.4)	116.0	6.76 d (8.4)
4′	159.2	–	158.7
N-Methylthreonine (NMeThr/Thr-2)
1	n.o.	–	n.o.	–
2	59.3	3.37 m	60.1	3.93 m
3	64.1	3.89 m	66.8	3.94 m
4	20.0	0.52 d (6.4)	19.7	0.76 d (6.0)
N-Me	31.1	3.15 s
Alanine (Ala)
1	n.o.	–	175.0	–
2	51.1	4.68 m	51.1	4.32 m
3	15.8	1.46 d (7.2)	17.4	1.47 d (7.6)
Glycine (Gly-1)
1	n.o.	–	n.o.	–
2	43.8	3.85 s	43.9	4.13 m, 3.85 m
3-Methoxyalanine (3-OMeAla)
1	n.o.	–	n.o.	–
2	56.0	4.18 m	56.0	4.35 m
3	70.9	3.44 m, 3.46 m	71.9	3.83 m, 3.66 m
3-OMe	59.5	3.44 s	59.2	3.38 s
3-Hydroxyleucine (3-OHLeu)
1	n.o.	–	n.o.	–
2	54.0	4.75 m	54.9	4.87 m
3	77.4	5.40 m	77.6	5.40 m
4	29.6	2.04 m	29.4	2.03 m
5	17.6	0.92 m	16.8	0.91 ovl
5′	19.8	0.90 m	19.6	0.90 ovl
3,4 - Dimethylglutamine (3,4-DiMeGln)
1	n.o.	–	n.o.	–
2	58.7	4.21 m	58.5	4.32 m
3	39.7	2.15 m	38.0	2.20 m
4	42.9	2.64 m	42.4	2.59 m
5	180.5	–	n.o.	–
3-Me	13.6	0.95 ovl	14.0	0.98 d (6.8)
4-Me	15.5	1.19 d (7.2)	15.5	1.16 d (7.2)
Aminobutenoic acid (Aba)
1	n.o.	–	n.o.	–
2	n.o.	–	n.o.	–
3	132.0	6.55 m	131.1	6.57 q (7.2)
4	13.0	1.77 d (4.3)	12.9	1.78 d (4.0)
Threonine (Thr-1)
1	n.o.	–	n.o.	–
2	59.6	4.60 d (3.2)	59.7	4.60 d (3.2)
3	68.9	4.40 m	68.9	4.40 dd (6.4, 3.6)
4	19.8	1.24 d (6.4)	19.8	1.24 d (6.4)
Glycine (Gly-2)
1	n.o.	–	n.o.	–
2	44.0	4.14 m	45.0	3.39 m, 3.38 m
2,3-Dihydroxy-2,6,8-trimethyl-4,6-decadienoic acid (Dhtda)
1	178.5	–	178.4	–
2	48.7	2.41 m	48.7	2.42 m
3	71.5	4.72 m	71.3	4.69 t (9.6)
4	129.8	5.29 ovl	129.8	5.29 m
5	137.9	6.09 m	137.8	6.08 dd (11.6, 2.8)
6	132.0	–	132.0	–
7	139.6	5.26 m	139.7	5.26 m
8	35.4	2.36 m	35.4	2.37 m
9	31.3	1.35 m, 1.30 m	31.1	1.39 m, 1.29 m
10	12.3	0.89 ovl	12.3	0.88 ovl
2-Me	14.2	1.05 d (6.4)	14.1	1.05 d (6.8)
6-Me	16.8	1.80 s	16.8	1.80 s
8-Me	20.8	0.98 d (6.8)	20.8	0.98 ovl

Ovl: overlapped; n.o.: not observed.

^aCoupling constants are in parentheses and given in hertz. Although an HMBC was recorded, more assignments thus appear not possible.

A molecular formula of C₆₆H₁₀₂N₁₂O₂₀ for 3 was suggested by a pseudomolecular ion at 1369.7250 by HRMS (ESI) ([M+H]⁺; calcd. for C₆₆H₁₀₃N₁₂O₂₀ 1369.7250; = 0.0 ppm) which differs from papuamide C (1) by loss of an oxygen atom. The quaternary oxygenated carbon signal at δ_C 78.8 ppm in 1 has been replaced in 3 by a methine at δ_H 2.41 ppm, δ_C 48.7 ppm as suggested by the HMBC correlation of the proton Me-2_Htda (δ_H 1.05 ppm) to the carbon C-2_Htda (δ_C 48.7 ppm) and the absence of any HMBC correlations in 3 to the quaternary carbon (C-2_Dhtda in 1). The upfield shift of H-3_Dhtda (from δ_H 4.88 ppm to δ_H 4.72 ppm) and Me-2_Dhtda (from δ_C 22.4 ppm to δ_C 14.2 ppm) are further consistent with the replacement of the 2,3-dihydroxy-2,6,8-trimethyl-4,6-decadienoic acid moiety (Dhtda) of papuamide C (1) with a 3-hydroxy-2,6,8-trimethyl-4,6-decadienoic acid moiety (Htda) in 3, accounting for the difference in molecular formula between the two compounds. The chemical shifts for the Htda moiety in 3 were consistent with the proton and carbon chemical shifts assigned for the Htda moiety in mirabamide H⁵. However, a large (~15 Hz) coupling was not evident even if the actual coupling could not be resolved for H4 and H5 of Htda of compound 3. Furthermore, the agreement of the chemical shifts of all the NMR signals of Htda moiety in compound 3 to the Htda moiety in mirabamide H suggests a Z geometry to the C4-C5 olefin and E geometry to the C6-C7 double bond.

The HRESI(+)MS of papuamide F (4) indicated a molecular formula of C₆₅H₁₀₀N₁₂O₂₀ which differed from that of papuamide E (3) by loss of CH₂. The N-methyl (δ_C 31.1 ppm, δ_H 3.15 ppm) signal in 3 was absent in the HSQC spectrum of 4. Compound 3 differed from compound 4 in that the threonine residue of 3 was N-methylated while 4 was not. This relationship was similar to that of papuamides C and D in that papuamide C was N-methylated while papuamide D was not. Thus, 4 and 3 are analogues of 1 and 2, respectively, in which the Dhtda moiety has been replaced with Htda. Moreover, the C4–C5 olefin of the Htda was assigned a Z geometry on the basis of coupling of approximately 11 Hz between H4 and H5 as in the papuamides¹ and mirabamides⁵. The agreement of the chemical shifts of all NMR signals in Htda near the C6–C7 double bond suggests the same stereochemistry (E geometry) as in 1 and 2.

The optical rotation signs reported for all papuamides were same to that of the obtained for the compounds 3 and 4 indicating that all the amino acid residues in compounds 3 and 4 possessed identical configurations to papuamides A–D¹. The compounds, 3 and 4, were obtained as optically active white powders that are concluded as new analogues of papuamides A–D and proposed as papuamides E and F, respectively.

Brine shrimp assay¹¹ revealed these analogues (1–4) to be cytotoxic with LD₅₀ values of 92, 92, 104 and 106 μg/mL, respectively. However, papuamides C–F (1–4) were found to be inactive against methicillin resistant Staphylococcus aureus (ATCC 10537), vancomycin resistant Enterococcus faecium (ATCC 12952), wild type Candida albicans (ATCC 32354) and amphotericin resistant Candida albicans (ATCC 90873).

3 Experimental section

3.1 General experimental procedures

Optical rotations were recorded on a Bellingham Stanley ADP220 polarimeter. NMR spectra were recorded on a Varian spectrometer operating at 400 MHz. Chemical shifts are referenced to residual MeOH (δ_C 49.0; δ_H 3.31) in CD₃OD. High resolution ESI-MS analyses were obtained using Thermo Scientific LTQ Orbitarp Discovery LC-MS in positive electrospray ionisation mode. Reverse phase flash chromatography was performed on Bakerbond C18 40μm prep LC packing. Semi-preparative HPLC was performed using a Waters 515 HPLC system with a Alltech 10 μm C18 (250 x 10 mm) column.

3.2 Animal material (collection and taxonomy)

The marine sponge Melophlus sp. (family Ancorinidae) was collected by hand using scuba at a depth of 10 m from Karumolum Pt, Russell Island in the Solomon Islands (S8° 85.76′ and E159° 6.98′) on 21^st June 2006. The marine sponge was identified by Prof. John Hooper of Queensland Museum, Australia. Voucher specimens of the sponge, SOL06-1-018, are preserved at University of Utah and The University of the South Pacific.

3.3 Extraction, isolation and purification

The frozen sponge was cut into small pieces, extracted using MeOH (3 x 1000 mL) and followed by CH₂Cl₂ (3 x 1000 mL). The extracts were combined and evaporated to dryness under vacuum. The crude (4.5 g) was partitioned with CH₂Cl₂–H₂O (3:1). The aqueous layer was further partitioned with n-BuOH. The resulting biologically active n-BuOH extract was subjected to RP-silica chromatography pre-equilibrated with aqueous MeOH (20% H₂O). The column was eluted with a stepwise gradient of 20–100% MeOH_(aq). The active (100% MeOH) elute was rechromatographed on RP-silica using stepwise gradient 20–100% MeOH_(aq) to yield 12 fractions. The eighth fraction 80% MeOH_(aq) was subjected on isocratic RP-HPLC using 83% MeOH_(aq) at a flow rate of 4.0 mL/min and monitoring at a wavelength of 254 nm to yield 5 fractions. The fourth fraction was further purified on RP-HPLC using isocratic elution with 42% MeCN_(aq) to yield pure compounds 1 (4.2 mg, t_R = 25.3 min), 2 (4.1 mg, t_R = 18.5 min), 3 (1.8 mg, t_R = 31.0 min) and 4 (2.0 mg, t_R = 22.1 min).

3.3.1 Papuamide E (3)

white powder; [α]²⁵_D +68.5 (c 0.07, MeOH); ¹H NMR (400 MHz, CD₃OD), see Table 1; HRMS (ESI): m/z 1383.7407 [M + H]⁺ (calcd for C₆₆H₁₀₃N₁₂O₂₀, 1383.7406), HRMS (ESI): m/z 1405.7230 [M + Na]⁺ (calcd for C₆₆H₁₀₂N₁₂O₂₀Na, 1405.7226).

3.3.2 Papuamide F (4)

white powder; [α]²⁵_D +107.1 (c 0.03, MeOH); ¹H NMR (400 MHz, CD₃OD), see Table 1; HRMS (ESI): m/z 1369.7250 [M + H]⁺ (calcd for C₆₅H₁₀₁N₁₂O₂₀, 1369.7250), HRMS (ESI): m/z 1391.7067 [M + Na]⁺ (calcd for C₆₅H₁₀₀ N₁₂O₂₀Na, 1391.7069).