Rearranged Terpenoids from the Marine Sponge Darwinella cf. oxeata

Feb 13, 2017 - ... R. Gubiani†, Lizbeth L. L. Parra†, Mario F. C. Santos†, Daiane D. Ferreira§, Juliana T. Mesquita§, Andre G. Tempone§, Antonio G. Fe...
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Rearranged Terpenoids from the Marine Sponge Darwinella cf. oxeata and Its Predator, the Nudibranch Felimida grahami Maria Camila A. Ramirez,† David E. Williams,‡ Juliana R. Gubiani,† Lizbeth L. L. Parra,† Mario F. C. Santos,† Daiane D. Ferreira,§ Juliana T. Mesquita,§ Andre G. Tempone,§ Antonio G. Ferreira,⊥ Vinícius Padula,∥ Eduardo Hajdu,# Raymond J. Andersen,‡ and Roberto G. S. Berlinck*,† †

Instituto de Química de São Carlos, Universidade de São Paulo, CP 780, CEP 13560-970, São Carlos, SP, Brazil Departments of Chemistry and Earth, Ocean & Atmospheric Sciences, University of British Columbia, Vancouver, BC V6T 1Z1, Canada § Centre for Parasitology and Mycology, Instituto Adolfo Lutz, Avenida Dr. Arnaldo, 351, 8° andar, 01246-000 São Paulo, SP, Brazil ⊥ Departamento de Química, Universidade Federal de São Carlos, Rodovia Washington Luis s/n, km 235, CEP 13565-905 São Carlos, SP, Brazil ∥ Departamento de Biotecnologia R. Kioto, Instituto de Estudos do Mar Almirante Paulo Moreira (IEAPM), 253 - Praia dos Anjos, 28930-000 Arraial do Cabo, RJ, Brazil # Museu Nacional, Universidade Federal do Rio de Janeiro, Quinta da Boa Vista, s/n, CEP 20940-040 Rio de Janeiro, RJ, Brazil ‡

S Supporting Information *

ABSTRACT: Marine sponges are a rich source of terpenoids with rearranged spongian carbon skeletons. Investigation of extracts from the sponge Darwinella cf. oxeata yielded four new rearranged diterpenoids, oxeatine (2) and oxeatamides H−J (35), as well as the known metabolites oxeatamide A (6), oxeatamide A methyl ester (7), and membranolide (1). Oxeatine (2) has a new heterocyclic skeleton, while oxeatamide J (5) has an N-methyl urea group included in a γ-lactam moiety. UPLC-QTOF analysis of the extract obtained from the mantle of the nudibranch Felimida grahami indicated the presence of 1 and 4.

S

often connected to the terpenoid skeletons of these metabolites via a pyrrole or an α,β-unsaturated γ-lactam, suggesting a biogenesis in which the amino acid amines are condensed with bis-aldehydes, leading to pyrroles, or to a carboxylic acid/ aldehyde to give the corresponding lactams.1 Sponge terpenoidpyrroles and terpenoid-lactams represent less reactive forms of their corresponding dialdehydes or aldehyde/carboxylic acids, which are often still bioactive, but their actual functions or ecological roles remain unknown.

ponge terpenoids condensed with intact or decarboxylated amino acids are a rare group of marine natural products. Molliorins A, B, and C isolated from the Mediterranean sponge Cacospongia mollior contain pyrrole groups that incorporate the nitrogen atoms of decarboxylated amino acids.1 In the case of dimeric molliorin B, pyrroles are formed at both the α and δ amino groups of the putative biogenetic precursor ornithine.1b A number of nitrogenous sponge terpenoids, including haumanamide, the spongolactams, the pyrodysinoic acids, and the fasciospongins, contain α,β-unsaturated γ-lactams or maleimides instead of a pyrrole.2−6 Coscinolactams A to F isolated from Coscinoderma sp. have intact glycine, alanine, and valine moieties, representing an important variation in this select class of terpenoids.7 The oxeatamides isolated from Darwinella oxeata,8 the hamigerans from Hamigera tarangaensis,9 and the ceylonamides from Spongia ceylonensis10 represent additional sponge diterpenoids condensed with decarboxylated amino acids. Intact or decarboxylated amino acids are most © 2017 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION

A collection of Darwinella cf. oxeata from the Cabo Frio archipelago along the Rio de Janeiro state coastline in Brazil Special Issue: Special Issue in Honor of Phil Crews Received: December 18, 2016 Published: February 13, 2017 720

DOI: 10.1021/acs.jnatprod.6b01160 J. Nat. Prod. 2017, 80, 720−725

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tetrasubstituted benzene ring and the three carbonyl functionalities identified in the NMR data. Therefore, oxeatine (2) contained two additional rings for a total of three rings, the same as oxeatamide A (6). As illustrated in Figure 1, detailed analysis of the gCOSY, gHSQC, and gHMBC spectra identified the same (1,3,3trimethylcyclohexyl)benzene moiety with substituents at C-8, C-13, and C-14 as seen in oxeatamide A (6).8 The substituent at C-13 was deduced to be a methyl ester, as gHMBC correlations were observed between both H-12 and a methoxy methyl [δH 3.88/δC 52.2 (Me-24)] and the carbonyl assigned to C-16 at δC 167.2. As in oxeatamide A (6), a 2-propionyl moiety was identified as the C-8 substituent. Similarly, at C-14 the substituent could be assigned as the C-15 methylene. In the gHMBC spectrum both the Me-17 doublet [(δH 1.50 (d, 6.8 Hz)/δC 16.9 (C-17)] and H-7 quartet [δH 4.47 (q, 6.8)/δC 41.5], along with the H-15 methylene protons (δH 5.05/4.98), correlated to the C-6 carbonyl at δC 174.3. Although no gHMBC correlations were observed for the resonances assigned to the C-22 methylene protons (H-22 at δH 4.70 and 3.80), the molecular formula of 2 required that the remaining unassigned C2H3NO2 fragment had to account for an additional ring and the third and final carbonyl group. A glycine residue in which the amino nitrogen belongs to a sixmembered lactam ring satisfies these requirements and connects C-15 to the C-6 carbonyl via N-21 to complete the planar structure of 2. In regard to the relative configuration of oxeatine (2), the 1H and 13 C chemical shift assignments for the (1,3,3trimethylcyclohexyl)benzene moiety of 2 were almost identical to those reported for oxeatamide A (6), for which the relative configuration was previously assigned by analysis of NMR data.8 Therefore, the configuration at C-10 of oxeatine (2) was assigned as 10(S*), the same as that in 6.8 With both the substituted benzene ring and Me-18 in axial orientations at C-4 and C-10, respectively, anisotropic shielding provides an explanation for the shielded δH 0.52 resonance assigned to the Me-18 protons.11−15 The NOEs observed between H-11 (δH 7.47) and both H-1a (δH 2.20) and H-2a (δH 1.81) and between H-7 [δH 4.47 (q, 6.8)] and both Me-20 (δH 1.33) and H-5a (δH 2.06) are consistent with the 7(R*) relative configuration previously established for 6. Oxeatine (2) is the first spongiane terpenoid with the new heterocyclic skeleton featuring a δ-lactam fused with the aromatic moiety at C-8 and C-14. The 1H and 13C NMR spectra of oxeatamide H (3) were remarkably similar to those of oxeatamide A (6).8 The HRESIMS spectrum of 3 gave a [M + H]+ ion at m/z 344.2220 and a [M + Na]+ ion at m/z 366.2036, which were appropriate for the molecular formula C21H29NO3, differing from that of oxeatamide A (6) by the loss of CO2 and requiring one less degree of unsaturation for a total of eight. The noticeable differences in NMR spectra of 3 and 6 were that the C-22 methylene and the carbonyl of the C-23 carboxylic acid in 6 were missing and instead a deshielded methyl singlet at δH 3.11 (Me-22) that correlated to a carbon at δC 29.4 (C-22) in the gHSQC was observed. In the HMBC spectrum this methyl singlet (Me-22, δH 3.11) correlated to both the resonance at δC 51.8 assigned to the C-15 methylene carbon and the resonance at δC 168.6 assigned to the C-16 carbonyl. An N-methyl at the lactam nitrogen satisfied all the spectroscopic requirements for the structure of 3. The relative configuration of 3 was shown to be the same as 6 by comparison of their 1H and 13C NMR data.

provided an extract with antiparasitic activity against Trypanosoma cruzi, the causative agent of Chagas disease (American trypanosomiasis). Bioassay-guided isolation yielded membranolide (1) as the active compound, along with the five new oxeatamide-related derivatives oxeatine (2) and oxeatamides H−J (3−5), as well as the known metabolites oxeatamide A (6)8 and oxeatamide A methyl ester (7).8 Analysis by UPLC-HRMS of the MeOH extract obtained from the mantle of the nudibranch Felimida grahami, which preys upon D. oxeata, showed the presence of membranolide (1) and oxeatamide I (4). The isolation and structure elucidation of compounds 2−5 are herein presented as well as the occurrence of 1 and 4 in F. grahami.

The 1H and 13C NMR spectra of oxeatine (2) exhibited similarities to the spectra obtained for oxeatamide A (6).8 The HRESIMS analysis of oxeatine (2) gave a [M + H]+ ion at m/z 402.2258 and a [M + Na]+ ion at 424.2074 that corresponded to the molecular formula C23H31NO5, which differs from that of 6 by the addition of CH2 but requires the same nine degrees of unsaturation. Resonances at δC 174.3 (C-6), 167.2 (C-16), and 178.8 (C-23) in the 13C NMR spectrum of 2 were assigned to three carbonyl carbons. Six 13C resonances at δC 138.9 (C-8), 151.7 (C-9), 125.5 (C-13), 136.2 (C-14), 125.9 (C-11), and 128.5 (C-12) were assigned to the carbons of an aromatic ring. A coupling constant of 8.7 Hz between two aromatic proton doublets resonating at δH 7.47 (H-11) and 7.80 (H-12), which correlated in the gHSQC spectrum to the carbons at δC 125.9 (C-11) and 128.5 (C-12), respectively, indicated the presence of a 1,2,3,4-tetrasubstituted benzene ring as in oxeatamide A (6). Additionally, four methyl singlets [δH 0.52/δC 27.8 (Me18), δH 0.94/δC 32.5 (Me-19), δH 1.33/δC 32.7 (Me-20), δH 3.88/δC 52.2 (Me-24)], one methyl doublet [δH 1.50/δC 16.9 (Me-17)], six methylenes, one methine, and two quaternary carbons were observed in the 1D and 2D NMR spectra (Table 1). Seven of the nine sites of unsaturation required by the molecular formula of 2 were accounted for by the 1,2,3,4721

DOI: 10.1021/acs.jnatprod.6b01160 J. Nat. Prod. 2017, 80, 720−725

a

722

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 22 23 24 NH

δH (J in Hz)

d (6.8) s s s d (18.6); 3.80, d (19.0)

3.88, s

1.50, 0.52, 0.94, 1.33, 4.70,

5.05, d (16.7); 4.98, d (16.7)

7.47, d (8.7) 7.80, d (8.7)

4.47, q (6.8)

2.06, d (14.3); 1.55, d (14.2)

2.20, bd (15.4); 1.45, d (10.1) 1.81, m; 1.68, m 1.28, m

CH2 CH2 CH2 C CH2 C CH C C C CH CH C C CH2 C CH3 CH3 CH3 CH3 CH3

δC, type 40.9, 19.9, 39.6, 31.8, 50.8, 176.7, 40.6, 134.7, 150.5, 39.7, 127.5, 122.1, 131.3, 141.2, 51.8, 168.6, 15.4, 27.1, 32.9, 32.8, 29.4,

CDCl3. bDMSO-d6. cAcetone-d6. dBroad singlet of low intensity.

δC, type

40.0, CH2 20.0, CH2 39.8, CH2 31.7, C 50.2, CH2 174.3, C 41.5, CH 138.9, C 151.7, C 39.7, C 125.9, CH 128.5, CH 125.5, C 136.2, C 49.9, CH2 167.2, C 16.9, CH3 27.8, CH3 32.5, CH3 32.7, CH3 48.8d, CH2 178.8, C 52.2, CH3

position

oxeatine (2)a

1.62, 0.38, 0.92, 1.46, 3.11,

d (7.3) s s s s

4.38, d (16.9); 4.22, d (16.9)

7.58, d (8.1) 7.63, d (8.1)

4.73, q (7.2)

2.06, d (14.2); 1.50, d (14.2)

2.35, bd (15.2); 1.44, m 1.81, m; 1.67, m 1.33, m; 1.26, m

δH (J in Hz)

oxeatamide H (3)a CH2 CH2 CH2 C CH2 C CH C C C CH CH C C CH2 C CH3 CH3 CH3 CH3

δC, type 40.3, 19.5, 39.1, 31.2, 49.8, 174.8, 40.4, 136.9, 149.5, 41.5, 126.5, 120.5, 130.8, 144.0, 44.3, 169.5, 15.5, 26.6, 32.7, 32.6, d (7.0) s s s

8.42 (bs)

1.48, 0.35, 0.89, 1.40,

4.37, d (17.0); 4.08, d (17.0)

7.55, d (8.2) 7.48, d (8.2)

4.50, m

2.10, d (14.1); 1.47, m

2.30, bd (13.7); 1.43, m 1.76, m; 1.63, m 1.30, m; 1.27, m

δH (J in Hz)

oxeatamide I (4)b

Table 1. NMR Spectroscopic Data (1H 600 MHz, 13C 150 MHz) for Oxeatine (2) and Oxeatamides H−J (3−5) δC, type 41.5, CH2 20.7, CH2 40.3, CH2 n.o., C 51.1, CH2 175.0, C 41.3, CH 137.8, C 154.0, C 40.7, C 129.2, CH 123.3, CH 129.7, C 141.4, C 48.4, CH2 170.0, C 18.0, CH3 27.4, CH3 32.3, CH3 32.3, CH3 147.0, C 33.2, CH3 d (7.0) s s s

8.15 (bs)

4.0

1.71, 0.44, 0.94, 1.51,

4.93, d (17.5); 4.73, d (17.5)

7.82, d (8.2) 7.78, d (8.2)

4.86, q (6.5)

2.23, d (14.0); 1.61, d (14.0)

2.43, bd (13.4); 1.50, m 1.87, m; 1.69, m 1.33, m

δH (J in Hz)

oxeatamide J (5)c

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DOI: 10.1021/acs.jnatprod.6b01160 J. Nat. Prod. 2017, 80, 720−725

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N−H (δH 8.42) of 4. Instead, in the gCOSY spectrum of 5 a deshielded methyl doublet at δH 4.0 (d, J = 4.6 Hz)/δC 33.2 (Me-23) was observed to couple to a N−H resonance at δH 8.15. The Me-23 doublet (δH 4.0) correlated in the gHMBC to a shielded carbonyl with a chemical shift at δC 147.0 (C-22) that was appropriate for a urea carbonyl. Because the remaining NMR data of oxeatamide J were essentially identical to the NMR data for oxeatamides H (3) and I (4), the CO-NHCH3 fragment had to be attached to the lactam nitrogen as an Nmethyl urea group. The remaining structure of 5, including the relative configuration, was identical to oxeatamides H (3) and I (4). The absolute configurations of 2−5 have not been assigned because these compounds failed to provide suitable crystals for X-ray diffraction analysis. A recent molecular analysis suggests that the nudibranch Felimida grahami (Thompson, 1980), originally described from the Caribbean Sea, and a similar species, F. paulomarcioi, described from Brazil,16 are the same species (V. Padula, unpublished data). In Brazil, F. grahami preys upon two sponge species: D. oxeata (as Darwinella sp.17) and Amphimedon viridis.17 A. viridis is a cosmopolitan sponge species that is the source of extremely polar and strongly bioactive poly alkylpyridinium salts in the halitoxin and amphitoxin families.18 As F. grahami is a small and uncommon nudibranch, we opted to analyze the mantle of F. grahami exclusively for rearranged diterpenoids. Using authentic standards of membranolide (1), oxeatine (2), oxeatamides I (4) and J (5), and oxeatamide A methyl ester (7), analysis of the MeOH extract of the mantle of

Figure 1. Bold bonds indicate COSY correlations, single arrows indicate key HMBC correlations, and double arrows indicate NOEs observed for oxeatine (2).

The 1H NMR spectrum of oxeatamide I (4) was identical to that of oxeatamide H (3), except for the absence of the Nmethyl resonance (Me-22, δH 3.11) present in 3 and the presence of an exchangeable proton singlet at δH 8.42 in 4 (Table 1), which indicated that 4 differed from 3 simply by the loss of the N-methyl group in 3. The HRESIMS spectrum of 4 gave a [M + Na]+ ion at m/z 352.1871 that corresponded to the molecular formula C20H27NO3, which is 14 mass units less than oxeatamide H (3). The HRMS and NMR data confirmed the structure of oxeatamide I as 4. The HRESIMS of oxeatamide J (5) gave a [M + Na]+ ion at m/z 409.2102 appropriate for the molecular formula C22H30N2O4, which differed from that of oxeatamide H (3) and oxeatamide I (4) by the addition of CHON and C2H3ON, respectively, and required an additional degree of unsaturation. The 1H NMR spectrum of 5 lacked the resonances assigned to either the N-Me singlet (Me-22, δH 3.11) observed for 3 or the

Figure 2. UPLC-QTOF comparison of the extract of the mantle of F. grahami with rearranged diterpenes isolated from Darwinella cf. oxeata. Line A: mantle extract of F. grahami. Line B: Membranolide (1). Line C: oxeatamide A methyl ester (7). Line D: oxeatamide J (5). Line E: oxeatamide I (4). Line F: oxeatine (2). Conditions: Acquity UPLC BEH C18 (1.7 μm, 2.1 × 100 mm) column; eluent: gradient of MeCN (0.01% HCO2H) in H2O (0.01% HCO2H), starting at 10% to 98% MeCN (0.01% HCO2H) in 8 min. Detection: MSE continuum during 2 min, m/z 185−1.000 molecular weight range; detection mode: ESI(+); scan time: 0.2 s; ramp collision energy: 20−30 V. 723

DOI: 10.1021/acs.jnatprod.6b01160 J. Nat. Prod. 2017, 80, 720−725

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Animal Material. Specimens of the marine sponge Darwinella cf. oxeata (identified by E.H.) were collected at a depth of 8 m, at Ilha Comprida, and Enseada 3, Ilha dos Papagaios, Cabo Frio, Rio de Janeiro state, in 2011 and 2015. Animals were immersed in 95% EtOH immediately after collection. A voucher specimen has been deposited at the Porifera collection, Museu Nacional, Universidade Federal do Rio de Janeiro (MNRJ 18412). Felimida grahami (identified by V.P.) nudibranchs were collected at a number of locations in Cabo Frio, Rio de Janeiro state, between 2008 and 2015. Specimens were immersed in 95% EtOH immediately after collection. A voucher specimen has been deposited at the Malacological collection, Zoological Museum of Universidade de São Paulo (MZSP 97648). Extraction of Darwinella cf. oxeata and Isolation of Rearranged Terpenes. Specimens of D. oxeata were removed from the EtOH (300 mL) and extracted with MeOH (3 × 300 mL, 30 min in an utrasound bath). The extracts were pooled and evaporated. The resulting organic extract was suspended in 95% MeOH and partitioned with hexane (3 × 200 mL). After evaporation, the MeOH extract was suspended in H2O and extracted with EtOAc (3 × 500 mL). The EtOAc extract was evaporated to give 1.79 g of crude material. The EtOAc fraction (1.79 g) was fractionated by Sephadex LH20 (MeOH) column chromatography, to give six fractions: E1A-1 (385.6 mg), E1A-2 (178.1 mg), E1A-3 (257.5 mg), E1A-4 (511.5 mg), E1A-5 (153.2 mg), and E1A-6 (72.9 mg). These fractions were analyzed by HPLC-UV-MS, using an analytical C18 reversed-phase column (Waters X-Terra MS C18, 3.5 μm, 2.1 × 50 mm) with a linear gradient of 1:1 MeOH/MeCN in H2O (with 0.1% HCO2H) as eluent, starting at 80% to 0% H2O over 22 min, at a flow rate of 1.0 mL/min. Detection was monitored by UV between λmax 200 and 400 nm and by positive ion ESIMS with a cone voltage of 25 V monitoring ions between m/z 180 and 700. Fractions E1A-3 and E1A-4 were pooled (769.0 mg) and subjected to a solid-phase extraction on a C18 reversed-phase silica-gel cartridge (10 g) eluted with 40:60, 50:50, 60:40, 70:30, and 90:10 MeOH/H2O and 100% MeOH. Six fractions were obtained: E1A-34-1 (153.4 mg), E1A-34-2 (165.4 mg), E1A-34-3 (190.9 mg), E1A-34-4 (132.9 mg), E1A-34-5 (71.2 mg), and E1A-34-6 (15.0 mg). Fractions E1A-34-1, E1A-34-2, E1A-34-3, and E1A-34-4 were subjected to a series of reversed-phase HPLC separations, to give membranolide (1, 10.8 mg), oxeatine (2, 1.7 mg), oxeatamide H (3, 1.4 mg), oxeatamide A (6, 8.4 mg), and oxeatamide A methyl ester (7, 2.1 mg). Fractions E1A-5 and E1A-6 were pooled (225.0 mg) and fractionated by a series of reversed-phase HPLC separations, to give oxeatamide I (4, 1.2 mg) and oxeatamide J (5, 2.3 mg). Details of the separation procedure are included in the Supporting Information. Oxeatine (2): colorless gum; [α]26D +1.6 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 210 (3.70), 246 (3.60); IR νmax 2976, 1723, 1676, 1632, 1606, 1453 cm−1; 1H NMR (CDCl3, 600 MHz), Table 1; 13 C NMR (CDCl3, 150 MHz), Table 1; HRESIMS m/z 402.2258 [M + H]+ (calcd for C23H31NO5, 402.2282), m/z 424.2074 [M + Na]+ (calcd for C23H31NO5Na, 424.2101). Oxeatamide H (3): colorless gum; [α]26D −39 (c 0.7, MeOH); UV (MeOH) λmax (log ε) 206 (3.80); IR νmax 2944, 1726, 1682, 1608, 1456 cm−1; 1H NMR (CDCl3, 600 MHz), Table 1; 13C NMR (CDCl3, 150 MHz), Table 1; HRESIMS m/z 344.2220 [M + H]+ (calcd for C21H29NO3, 344.2233), m/z 366.2036 [M + Na]+ (calcd for C21H29NO3, 366.2053). Oxeatamide I (4): colorless gum; [α]26D −2.2 (c 0.5; MeOH); UV (MeOH) λmax (log ε) 212 (3.70), 240 (3.50); IR νmax 2947, 1692, 1678, 1609, 1454 cm−1; 1H NMR (DMSO-d6, 600 MHz), Table 1; 13C NMR (DMSO-d6, 150 MHz), Table 1; HRESIMS m/z 352.1871 [M + Na]+ (calcd for C20H27NO3, 352.1890). Oxeatamide J (5): colorless gum; [α]26D +0.03 (c 0.1; MeOH); UV (MeOH) λmax (log ε) 204 (3.78), 245 (3.58); IR νmax 2944, 1726, 1680, 1608, 1456 cm−1; 1H NMR (CDCl3, 600 MHz), Table 1; 13C NMR (CDCl3, 150 MHz), Table 1; HRESIMS m/z 409.2102 [M + Na]+ (calcd for C22H30N2O4Na, 409.2103). Oxeatamide A (6): colorless gum [α]26D −40 (c 2.2; MeOH); lit.8 [α]D −60.3 (c 1.0; in MeOH).

F. grahami by UPLC-QTOF (Figure 2) revealed the presence of membranolide (1) and oxeatamide I (4). Membranolide (1) displayed weak antitrypanosomal activity against T. cruzi, with an IC50 of 29 μM against trypomastigotes and 20 μM against intracellular amastigotes. Compound 1 displayed cytotoxic activity against mammalian NCTC cells with a CC50 of 54 μM, being essentially inactive. Oxeatamide H (3) and the methyl ester of oxeatamide A (6) were not active against T. cruzi parasites. Membranolide (1) was first isolated from the sponge Dendrilla membranosa15 and subsequently from the Australian nudibranchs Goniobranchus reticulatus and G. splendidus.19 Spongian diterpenoids are commonly found in tissues of sponge-feeding nudibranchs.20 Nevertheless, this is the first time that a nitrogenous spongian-derived diterpenoid has been isolated from the mantle of a nudibranch. Oxeatine (2) and oxeatamide J (5) both have structural features without precedent in spongian-derived terpenoid alkaloids. The 4,8-dimethyl-5-(1,3,3-trimethylcyclohexyl)octahydro-1H-2λ2-isoquinoline heterocylic skeleton found in 2, which features a δ-lactam with the nitrogen atom bridging C6 and C-15 of a rearranged spongian carbon framework, is unknown in nature or from synthesis. Oxeatamide J (5) has a rare N-methylcarboxamide substituent on the γ-lactam nitrogen atom.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured using a Jasco P-2000 polarimeter with sodium light (λmax 589 nm). UV spectra were recorded with a UV/vis-NIR JASCO V-670 instrument. IR spectra were recorded with a Shimadzu IRAffinity 1 instrument. 1H and 13C NMR spectra were recorded on a Bruker AVANCE III (14.1 T) or a Bruker AV-600 (14.1 T) spectrometer. 1H chemical shifts were referenced to either the residual DMSO-d6 signal (δ H 2.49 ppm), the CDCl3 signal (δH 7.26 ppm), or the tetramethylsilane (TMS) internal standard when recorded in acetone-d6. 13C chemical shifts were referenced either to the solvent peak of DMSO-d6 (δC 39.5 ppm) or CDCl3 (δC 77.2 ppm) or to the TMS internal standard when recorded in acetone-d6. HRESIMS and direct insertion MS/MS analyses were performed on a Thermo Scientific Velos LTQ Orbitrap, with an API Zspray ion source and a high-flow electrospray ionization probe, operating in positive mode. Solvents used for extraction and column chromatography were glass distilled prior to use. HPLC-grade solvents were utilized without further purification in the HPLC purifications. HPLC semipreparative separations were performed with either a Waters quaternary pump 600, a double beam UV detector 2487, and data module 746 or with a Waters 600E system controller liquid chromatograph attached to a Waters 996 photodiode array detector, on which the UV spectra were recorded. HPLC-LRESIMS analyses were performed using a Waters Alliance 2695 coupled online with a Waters 2996 photodiode array detector, followed by a Micromass ZQ2000 mass spectrometry detector with an electrospray interface. The photodiode array scanned samples between 205 and 254 nm. The mass spectrometer detector was optimized to the following conditions: capillary voltage, 3.00 kV; source block temperature, 100 °C; desolvation temperature, 350 °C, operating in electrospray positive mode; detection range, 200−800 Da with total ion count extracting acquisition. The cone and desolvation gas flow were 50 and 350 L/h, respectively, and were obtained from a Nitrogen Peak Scientific N110DR nitrogen source. Data acquisition and processing were performed using Empower 2.0. UPLC-qTOFHRMS analysis was performed using an Acquity UPLC BEH C18 column (2.1 × 100.0 mm, 1.7 μm), with a 0.8 mL/min flow rate, using a gradient of MeCN (0.01% HCO2H) in H2O (0.01% HCO2H), starting at 10% to 98% MeCN in 8 min. Detection: MSE continuum during 2 min, m/z 185−1.000 molecular weight range; detection mode, ESI(+); scan time, 0.2 s; ramp collision energy, 20−30 V. 724

DOI: 10.1021/acs.jnatprod.6b01160 J. Nat. Prod. 2017, 80, 720−725

Journal of Natural Products

Article

UPLC-QTOF Analysis of Metabolites from the Mantle of the Nudibranch Felimida grahami. Eight specimens of the nudibranch F. grahami were removed from the EtOH (200 mL) used for preservation and subsequently extracted with MeOH (3 × 300 mL, 30 min in an ultrasound bath). The extracts were pooled and evaporated. The resulting organic extract was suspended in MeOH 95% and partitioned with hexane (3 × 200 mL). After partition, the MeOH extract was evaporated, suspended in H2O, and partitioned with EtOAc (3 × 500 mL). The EtOAc extract was evaporated to give 0.355 g of an organic extract. The EtOAc extract was diluted in MeOH and analyzed by UPLC-qTOF. UPLC-qTOF analysis conditions: Acquity UPLC BEH C18 column (2.1 × 100.0 mm, 1.7 μm), with a 0.8 mL/ min flow rate, using a gradient of MeCN (0.01% HCO2H) in H2O (0.01% HCO2H), starting at 10% to 98% MeCN in 8 min. Detection: MSE continuum during 2 min, from m/z 185 to m/z 1000 molecular weight range; detection mode: ESI(+); scan time: 0.2 s; ramp collision energy: 20−30 V.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b01160. Detailed isolation procedure, schemes of isolation, as well as 1H and 13C NMR spectra of compounds 2−5 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +55-16-33739954. Fax: +55-16-33739952. E-mail: [email protected]. ORCID

Raymond J. Andersen: 0000-0002-7607-8213 Roberto G. S. Berlinck: 0000-0003-0118-2523 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support was provided by BIOTA/BIOprospecTA FAPESP grants (2013/50228-8 and 2015/01017-0) to R.G.S.B. and by a NSERC grant to R.J.A. The authors also thank the Brazilian funding agencies CAPES and CNPq for scholarships awarded to M.C.A.R., J.R.G., L.L.L.P., M.F.C.S., and V.P.

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DEDICATION Dedicated to Professor Phil Crews, of the University of California, Santa Cruz, for his pioneering work on bioactive natural products.

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DOI: 10.1021/acs.jnatprod.6b01160 J. Nat. Prod. 2017, 80, 720−725