The Oxeatamides: Nitrogenous Spongian Diterpenes from the New

Article pubs.acs.org/jnp

The Oxeatamides: Nitrogenous Spongian Diterpenes from the New Zealand Marine Sponge Darwinella oxeata Joanna M. Wojnar, Katie O. Dowle, and Peter T. Northcote* School of Chemical and Physical Sciences, Victoria University of Wellington, P.O. Box 600, Wellington, New Zealand S Supporting Information *

ABSTRACT: As part of our ongoing search for novel and bioactive compounds from New Zealand marine organisms, we investigated the extracts of the sponge Darwinella oxeata. NMR-guided fractionation led to the isolation of nine new nitrogenous spongian diterpenes, oxeatamide A (1), iso-oxeatamide A (2), oxeatamides B−G (3−8), and oxeatamide A 23-methyl ester (9), as well as two known compounds, membranolides C and D (10, 11).

I

n order to isolate new natural products from marine organisms, we have developed a chromatographic and NMR spectroscopy-based approach to detect chemical entities unique to a particular organism. Preparative-scale reversed-phase solidphase extraction is used to partition amphiphilic (of medium polarity) secondary metabolites from the polar and nonpolar primary metabolites. Extensive 2D NMR spectroscopy is used to search for correlations unique to the extracted organism. A bright yellow sponge collected in northern New Zealand waters attracted our attention due to a series of diastereotopic methylenes that were detected in an HSQC spectrum of the medium-polarity fraction. We now report the isolation of a series of nitrogenous rearranged spongane diterpenes of the aplysulphurane skeleton1 from the New Zealand dendroceratid sponge Darwinella oxeata Berquist 1961 (Darwinellidae). This usually thin incrusting yellow sponge that occurs throughout New Zealand waters has often been mis-identified as Aplysilla sulfurea. Sponges of the nonspiculiferous orders Dictyoceradidae and Dendroceratidae are well known to produce diterpenoids, particularly those of the spongane class.2

■

RESULTS AND DISCUSSION Initial fractionation was carried out on a methanolic extract of the sponge using a polystyrene/divinylbenzene support and mixtures of acetone and H2O. Fractions containing the diterpenes as indicated by 1H NMR were further separated on a Diol bonded phase column with CH2Cl2 and MeOH. The poor chromatographic behavior of the major component (streaking) and initial NMR data suggested the presence of carboxylic acids, and therefore final purification was achieved by semipreparative HPLC using C18 and mixtures of methanol and 0.1 M formic acid to afford compounds 1−11. The major compound isolated (1) displayed a protonated molecule and an adduct ion [M + Na]+ by HREIMS corresponding to a molecular formula of C22H29NO5 and requiring nine degrees of unsaturation. All 22 carbons and 27 of the 29 protons were accountable in the 13C, 1H, and HSQC spectra, suggesting the presence of two exchangeable protons. Salient features of the molecule indicated by the NMR spectra © XXXX American Chemical Society and American Society of Pharmacognosy

included three methyl singlets, six aromatic carbons, and three carbonyls. Six methylenes, most of which displayed proton resonances with significantly disparate chemical shifts, indicated the presence of neighboring chirality and/or restricted rotation. The three methyl singlets, four of the methylenes, and two quaternary carbons were assembled as a 1,1,3,3-substituted cyclohexane ring from a series of COSY and HMBC correlations as depicted in Figure 1A. Two of the methyl singlets (CH3-18: δC 27.4, δH 0.40, and CH3-19: δC 33.1, δH 0.91) displayed a diagnostic pattern of HMBC correlations unique to a gem-dimethyl arrangement on the quaternary carbon C-4 (δC 31.9) and adjacent to a spin-isolated methylene (CH2-5, δC 50.9, δH 2.04, 1.49) and a methylene (CH2-3, δC Received: July 7, 2014

A

dx.doi.org/10.1021/np500549g | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

provide further evidence of this connectivity. The remaining isolated methylene, CH2-22 (δC 43.8, δH 4.77, 3.97) also revealed a large 1JCH (142 Hz) consistent with attachment to nitrogen, and these protons showed HMBC correlations to the amide carbonyl C-16, the lactam methylene C-15, and the remaining carbonyl C-23 (δC 173.9), clearly demonstrating a CH2-COO attachment to the amide nitrogen (Figure 1D). With the nine degrees of unsaturation and all of the atoms apart from two exchangeable protons accounted for, the functionalized carbonyls C-6 and C-23 were assigned as free carboxylic acids. The dicarboxylic acid nature of 1 manifested itself when a NOESY experiment was performed (in CDCl3) in an effort to establish the relative configuration. Unusually for such a relatively small molecule, the observed NOEs were all negative, indicating a slow tumbling rate in solution more typical of a large molecule. Moreover, NOEs were observed between protons (H-22 and H3-18, H3-19) at opposite ends of the molecule. Carboxylic acids are well known to form strongly bound hydrogen-bonded dimers in nonpolar solvents, and a dicarboxylic acid would have the potential to oligomerize, forming high molecular weight complexes. This would explain the negative NOEs; complexation could also lead to intermolecular NOEs between disparate ends of the molecule. In order to overcome this behavior and to confirm the presence of free carboxylic acids, 1 was subjected to TMS-diazomethane to produce the dimethyl ester 12. The expected two methyls were observed in the 1H NMR spectrum (δH 3.65 and 3.75). Satisfyingly, the NOESY experiment performed on 12 displayed positive NOEs as expected for a small molecule, and anomalous correlations attributed to intermolecular NOEs in 1 were not present. A series of NOE correlations and longrange W-couplings observed in the COSY experiment established the conformation of the cyclohexane ring as illustrated in Figure 2. The placement of the large aromatic substituent axial in the predominant conformation has been observed previously1,4,9 and explains the atypically low chemical shift of CH3-18 at 0.42 ppm, as it lies in the anisotropic shielding zone of the aromatic ring. NOEs observed between H3-18 and H3-17, between H-15a and H3-17, and between H-7 and H3-20 established the relative stereochemistry as 7R*, 10S* in accordance with previously reported metabolites of the same carbon skeleton.1,3−6,10−12 The structure and relative configuration of compound 1, named oxeatamide A, is therefore proposed as depicted. A protonated molecule and an adduct ion [M + Na]+ observed by HRESIMS for compound 2 indicated a molecular formula of C22H29NO5, suggesting it was a structural isomer of oxeatamide A (1). The NMR features of 2 were almost identical to those of 1, indicating they are structural analogues. As compared to 1, the only significant differences were chemical shift changes of the two aromatic protons (H-11 and H-12) at δH 7.61 and 7.23 (compared to δH 7.58 and 7.63 for 1), the methyl doublet (H3-17) at δH 1.77 (compared to δH 1.58), and the methine quartet (H-7) at δH 4.53 (compared to δH 4.71). The majority of the carbon skeleton of 2 could be constructed in a similar fashion to that of 1. However, instead of H-11 and H-12 showing correlations to a carbonyl resonance (the amide carbonyl carbon of oxeatamide A), they correlated to a methylene carbon, which was therefore assigned as CH2-16 (δC 49.8, δHa 4.37, δHb 4.28). Both proton resonances of CH216 showed weak HMBC correlations to C-12 (whereas before

Figure 1. Key COSY and HMBC correlations establishing the substructures of oxeatamide A (1).

39.8, δH 1.32, 1.27), which was part of a linear chain of three methylenes (CH2-1: δC 41.0, δH 2.31, 1.44; CH2-2: δC 20.1, δH 1.80, 1.65; CH2-3) as determined by COSY correlations. The remaining methyl singlet, CH3-20 (δC 32.9, δH 1.40), showed HMBC correlations to a quaternary carbon, C-10 (δC 39.8), and methylenes C-1 and C-5, establishing the cyclohexane ring. The 13C resonances of C-10 and CH2-3 overlap, but the established position of CH2-3 precludes an HMBC correlation from CH3-20. Two deshielded 1H resonances attached to olefinic carbons (CH-11: δC 127.8, δH 7.58 and CH-12: δC 122.7, δH 7.63) showed couplings to each other of 8.1 Hz, typical of ortho protons on a 1,2,3,4-tetrasubstituted aromatic ring. The relative positions of the substituted aromatic carbons were determined by the typical pattern of weak two-bond, strong three-bond, and absent four-bond HMBC correlations from H-11 and H-12. The attachment of the cyclohexane ring from C-10 to C-9 (δC 151.5) of the aromatic ring adjacent to CH-11 was established via HMBC correlations from CH3-20 to C-9 and H-11 to C-10 (see Figure 1B). The remaining methyl (doublet) CH3-17 (δC 15.5, δH 1.58) and its associated methine CH-7 (δC 40.9, δH 4.71) proton resonances both showed HMBC correlations to the next substituted position of the aromatic ring (C-8, δC 135.2) and to a functionalized carbonyl C-6 at δC 179.2. This combination of a cyclohexane ring attached to an aromatic ring ortho to a functionalized isopropyl moiety has precedence in the membranolides3,4 and aplysulphurins,1 where the C-6 carbonyl is part of a lactone ring. The more recently described pourewic acid and 15-methoxypourewic acid B5,6 match more closely in chemical shift of C-6, suggesting a free carboxylic acid at C-6 in 1. None of the previously reported compounds contain nitrogen, however, indicating that the oxeatamides are new members of the class. HMBC correlations from both aromatic protons (H-11 and H-12) to a shielded carbonyl δC 169.4 (C-16) established its connection to C-13 of the aromatic ring. Similarly HMBC correlations from an isolated methylene (CH2-15) to C-8 and C-9 placed this isolated system on C-14, completing the substitution of the aromatic ring (Figure 1C). The large 1JCH of CH2-15 (142 Hz) and the chemical shift of C-16 (δC 169.4), which was indicative of an amide carbonyl, suggested placement of a nitrogen between these two carbons, and this was confirmed by an 15N HMBC correlation from both CH2-15 protons to a nitrogen (δN −264.5) consistent with an amide.7,8 HMBC correlations from both protons of CH2-15 to C-16 B



Article

cyclohexane ring was again in the same chair conformation with the aromatic ring axial. Although the NOEs observed for 2 were once again negative, no intermolecular enhancements were apparent (perhaps due to a dilution effect). The NOE enhancements were consistent with the relative configuration of 7R*, 10S*. Therefore, the structure of 2 was determined as indicated and named iso-oxeatamide A. Compound 3 eluted quite early off the reversed-phase HPLC column, streaking over several minutes, indicating a significant difference in its structure from 1 and 2. A protonated molecule was observed by positive ion mode HRESIMS indicating a molecular formula of C25H34N3O3 for 3, requiring 11 doublebond equivalents. Analysis of the NMR spectra (in DMSO-d6, as 3 was only sparingly soluble in CDCl3) and comparison with compound 1 revealed that most signals were consistent with the diterpene portion of oxeatamide A (1). The diterpene lactam accounted for 20 of the carbons, 26 of the protons, the three oxygens, one of the nitrogens, and eight degrees of unsaturation. The remaining carbons and protons consisted of two methylenes, two aromatic methines (whose carbon signals were broadened), and a nonprotonated aromatic carbon. The two methylenes showed COSY and HMBC correlations to each other; a correlation from H2-22 in the 1 H−15N HMBC to N-21, as well as HMBC correlations to C15 and C-16, and a large 1JCH coupling constant (138 Hz) indicated CH2-22 was attached to the nitrogen of the lactam. The three aromatic carbons as well as the two nitrogens and three degrees of unsaturation that remained unaccounted for suggested an imidazole moiety linked to the methylenes; this was supported by COSY and HMBC correlations. In addition, the chemical shifts were consistent with average shifts reported for histidine residues in proteins,13 and 1JCH coupling constants (188 and 210 Hz) were consistent with calculated and observed coupling constants of imidazole rings.14,15 Chemical shift considerations, proton coupling constants, and W-coupling observed in the COSY as well as NOE enhancements were once again consistent with a 7R*, 10S* configuration, and the structure of 3 is assigned as depicted and named oxeatamide B.

Figure 2. Key NOE enhancements of oxeatamide A dimethyl ester (12) establishing the relative configuration. W-Coupling is shown in shades of green.

they had correlated to C-8), supporting this assignment. Compound 2 therefore had the amide functionality in the opposite orientation of that of oxeatamide A (Figure 3). Weak but clearly perceptible NOE enhancements from H-12 to H16a and H-16b confirmed this assignment. As with oxeatamide A, coupling constants and long-range Wcouplings observed in the COSY spectrum indicated the

Figure 3. Key correlations establishing the orientation of the lactam, and the attachment and connectivity of the nitrogen substituent of isooxeatamide A (2), oxeatamides B−G (3−8), and oxeatamide A 23-methyl ester (9). C



Article

isolated spin system of two methylenes was constructed through observation of correlations between H2-22 and H2-23 in the COSY spectrum. These two substructures were connected to each other through observation of HMBC correlations between H2-23 and C-25 and H-25 and C-23. Finally, the whole ethyl phenyl moiety was connected to the oxeatamide backbone through observation of HMBC correlations from H2-22 to C-15 and C-16, placing it as the lactam substituent. A large 1JCH coupling constant observed for CH222 gave further evidence for this placement. Due to the similary in chemical shifts and coupling constants, the relative configuration of compound 6 (named oxeatamide E) was assumed to be analogous to that of the other oxeatamides, and therefore its structure is given as depicted. The positive ion HRESIMS analysis of compound 7 showed an [M + Na]+ adduct ion indicating the molecular formula of C25H37NO3, which required eight double-bond equivalents. Analysis of the spectroscopic data once again suggested that 7 shares the same carbon framework as oxeatamide A, accounting for C20H26NO3 of the molecular formula. This left the fragment C5H11 as the N-21 substituent, comprising of two methylenes, one methine, and two methyls. Correlations observed in the COSY spectrum established a 2-methylbutyl moiety (see Figure 3). Methylene CH2-22 had a large 1JCH value of 137 Hz, suggesting it was attached to N-21; HMBC correlations from H2-22 to C-15 and C-16 confirmed this placement. The structure for compound 7 is therefore given as oxeatamide F, with the 7R*, 10S* relative configuration assumed based on similarity with the previously assigned oxeatamides. Compound 8 displayed a [M + Na]+ adduct ion indicating the molecular formula of C25H37NO3, identical to oxetamide F (7), establishing them as structural isomers. A notable difference was the methyl signals observed in the 13C NMR for C-25 (δC 22.5) and C-26 (δC 22.5), which appeared to be shifted downfield in 8, as compared to 7 (δC 11.2 and δC 16.9). The majority of the oxeatamide skeleton could be constructed in a similar fashion to that of 1, once again leaving C5H11 as the side chain of N-21. The remaining two methylenes, one methine, and two almost identical methyls were constructed into a 3-methyl butyl moiety based on COSY correlations (Figure 3). Similarly to the other oxeatamides, the downfield chemical shift and large 1JCH of methylene CH2-22, together with observed HMBC correlations from H2-22 to C-15 and C16, established it as the N-21 substituent. The configuration of the C-23 carbon was not determined. With the relative configuration of the aplisulphurane skeleton once again assumed as analogous to the other oxeatamides, the structure of oxeatamide G is assigned as 8. Positive ion mode HRESIMS analysis of compound 9 gave rise to a protonated molecule and an adduct ion ([M + Na]+) indicative of a molecular formula of C23H31NO5. Once again, the NMR spectra of 9 were very similar to those of oxeatamide A (1); the immediately obvious difference was the presence of a new OMe resonance at δC 53.5, δH 3.73. The proposed molecular formula differs by 14 Da from that of 1 and can be accounted for by the additional methoxy. Interestingly, the 1H chemical shifts of the H2-22 methylene pair had converged, from being 0.80 ppm apart in oxeatamide A to being separated by only 0.03 ppm. This sort of behavior was seen previously in oxeatamide A dimethyl ester (12), where H22a is only 0.02 ppm away from H-22b. The large chemical shift difference observed between H-22a and H-22b of 1 is highly unusual and is most likely due to restricted rotation

The molecular formula C22H32N2O3 for compound 4 was determined from an HRESIMS of the [M + Na]+ adduct ion, requiring eight double-bond equivalents. Like oxeatamide B (3), 4 was only sparingly soluble in CDCl3. The use of DMSOd6 revealed three exchangeable protons as a broad (3H) signal around 4 ppm. The 1H NMR spectrum accounted for all 32 protons; only 13 resonances were observed in the 13C NMR spectrum, in part due to overlap with the residual solvent signal. The other nine resonances were observed indirectly in the HSQC and/or HMBC spectra and were consistent with the formula. In a similar fashion to the other oxeatamides, COSY and HMBC correlations were used to construct the oxeatamide backbone, leaving a C2H6N fragment as the lactam substituent. The remaining signals visible in the 1H and 13C NMR spectra were two methylenes, which were placed adjacent to each other based on a correlation observed in the COSY spectrum. Both also displayed large 1JCH values (137 and 140 Hz), indicating both were adjacent to a nitrogen. An HMBC correlation observed from H2-22 to C-16 indicated that CH2-22 was adjacent to the lactam nitrogen N-21. With all the carbons thus assigned, the remaining NH2 unit was assigned as a primary amine on CH2-23, as depicted in Figure 3. Similarity of the chemical shifts and coupling constants of compound 4 to the other oxeatamides suggested the same relative configuration around C-7 and C-10. Therefore, the structure of this compound (named oxeatamide C) is given as 4. The molecular formula C24H35NO3 for compound 5 was determined by HRESIMS from the [M + Na]+ adduct ion. The 13 C NMR spectrum accounted for only 19 resonances; an additional five resonances were observed indirectly in the HSQC and/or HMBC spectra. Only 34 protons were apparent in the 1H NMR spectrum, suggesting one exchangeable proton. Once again, the majority of the resonances were consistent with the oxeatamide backbone, with one methylene CH2-22, one methine CH-23, and two methyls 2 × CH3-24 in identical environments left as the lactam subsituent. COSY correlations from the methyls CH3-24 to CH-23 and from the methine to CH2-22 established an isopropyl moiety. Observation of HMBC correlations from H2-22 to C-16 and C15 (Figure 3), as well as a large 1JCH coupling of 137 Hz, placed CH2-22 as the N-21 substituent. Once again, compound 5 displayed negative NOE enhancements in CDCl3. In order to prevent the formation of dimers, a NOESY experiment was run in the protic solvent CD3OD, resulting in positive NOE enhancements. Similarly to 12, the NOE correlations observed were also consistent with a 7R*, 10S* configuration. The structure of oxeatamide D is therefore given as 5. The molecular formula C28H35NO3 for compound 6 was determined from an HRESIMS adduct ion ([M + Na]+) and required 12 double-bond equivalents. Only 26 unique carbon resonances were observed in the 13C NMR spectrum, indicating spectral overlap or symmetry. Ten aromatic resonances were apparent, six making up the 1,2,3,4-substituted aromatic ring of the oxeatamide backbone. The remaining four aromatic resonances, C-24 (δC 138.7), CH-25 (δC 128.8, δH 7.20), CH-26 (δC 128.6, δH 7.25), and CH-27 (δC 126.5, δH 7.19), suggested a second, monosubstituted benzene ring. The majority of the signals of the molecule were assigned to the oxeatamide backbone through COSY and HMBC correlations, as well as by comparison with NMR data of the other oxeatamides. The remaining C8H9 fragment consisted of the monosubstituted benzene unit, which accounted for the symmetry observed in the NMR spectra. In addition, an D



Article

Table 1. 1H NMR Data for Oxeatamide A (1), Iso-oxeatamide A (2), Oxeatamides B−G (3−8), Oxeatamide A 23-Methyl Ester (9), and Dimethyl Oxeatamide A (12) (600 MHz, CDCl3 Unless Otherwise Noted) pos. 1a 1b 2a 2b 3a 3b 5a 5b 6-OH 7 11 12 15a 15b 16a 16b 17 18 19 20 22a 22b 23a 23b 24 25 26 28 pos. 1a 1b 2a 2b 3a 3b 5a 5b 7 11 12 15a 15b 17 18 19 20 22a 22b 23a 24a 24b 25 26 27 6-OMe 23-OMe

a

1 2.31, 1.44, 1.80, 1.65, 1.32, 1.27, 2.04, 1.49,

d (13.9) dd (14.2, 2.7) m m dt (12.8, 3.6) td (12.6, 3.9) d (13.9) d (14.2)

4.71, q (7.2) 7.58, d (8.1) 7.63, d (8.1) 4.40 d (16.5) 4.31 d (16.5)

1.58, 0.40, 0.91, 1.40, 4.77, 3.97,

d (7.1) s s s d (18.1) d (18.1)

2.27, 1.42, 1.80, 1.66, 1.31, 1.26, 2.10, 1.49,

br d (14.2) m m m m m d (14.0) d (14.2) q (7.3) d (8.2) d (8.2) d (16.9) d (16.9) d (7.2) s s s dt (14.1 7.1) dt (14.1 7.3) m

7.20, d (7.6) 7.25, t (7.1) 7.19, t (6.8)

d (14.8) m m dd (10.0, 7.3) m td (11.3, 3.8) d (13.9) d (14.2)

4.53, q (6.8) 7.61, d (8.3) 7.23, d (8.2)

4.37, 4.28, 1.77, 0.43, 0.91, 1.32, 4.78, 3.75,

6 2.34, 1.46, 1.81, 1.66, 1.31, 1.29, 2.03, 1.49, 4.69, 7.59, 7.67, 4.14, 4.01, 1.52, 0.38, 0.91, 1.44, 3.83, 3.74, 2.94,

3a

2

d d d s s s d d

(16.0) (16.0) (6.8)

(17.5) (18.3)

7 2.35, 1.51, 1.82, 1.67, 1.31, 1.27, 2.06, 1.52, 4.75, 7.61, 7.68, 4.38, 4.18, 1.62, 0.41, 0.92, 1.46, 3.42, 3.39, 1.78, 1.39, 1.16, 0.89, 0.88,

br d (13.0) m m dq (18.3, 4.8) m m d (13.9) d (14.0) q (7.5) d (8.2) d (8.2) d (17.0) d (17.0) d (7.2) s s s dd (13.9, 7.1) dd (13.7, 7.9) m m m t (7.6) d (7.1)

2.30, 1.38, 1.75, 1.61, 1.24,

4a

d (13.5) m m m m

2.08, d (14.0) 1.45, m 4.55, 7.56, 7.49, 4.47, 4.10,

q d d d d

(7.1) (8.3) (8.1) (17.1) (17.2)

1.48, 0.34, 0.89, 1.38, 3.73, 3.69, 2.82, 2.78,

d (7.2) s s s dt (13.7, dt (13.5, dt (14.7, dt (14.5,

7.8) 7.6) 7.7) 7.4)

br d (14.0) dd (14.0, 3.0) qt (11.7, 3.4) m m m br d (14.0) d (14.0) br s q (7.1) d (8.2) d (8.2) (d 17.3) (d 17.3)

2.46, 1.47, 1.82, 1.68, 1.33, 1.27, 2.06, 1.51,

br d (12.7) m m m dt (10.9, 4.0) td (13.0, 4.3) d (14.1) d (14.1)

4.75, 7.61, 7.68, 4.38, 4.21,

q d d d d

1.48, 0.50, 0.87, 1.37, 3.73, 3.66, 2.72, 2.62, 4.02,

d (7.3) s s s ddd (14.0, ddd (13.9, ddd (12.9, ddd (12.9, br s

1.63, 0.41, 0.92, 1.46, 3.38, 3.35, 2.02,

d (7.2) s s s dd (14.5, 7.5) dd (14.5 7.5) non (6.9)

7.56, br s 6.82, br s 8 2.34, 1.45, 1.82, 1.67, 1.31, 1.26, 2.06, 1.49, 4.72, 7.59, 7.65, 4.37, 4.19, 1.62, 0.39, 0.91, 1.43, 3.61, 3.52, 1.48, 1.59,

d (13.5) m qt (11.1, 3.2) dt (13.9, 4.2) m m d (13.8) m q (7.2) d (8.2) d (8.2) d (16.8) d (16.9) d (7.1) s s s dt (14.1, 7.8) dt (13.8, 7.7) m non (6.6)

5

2.29, 1.40, 1.74, 1.61, 1.25, 1.22, 2.07, 1.45, 4.02, 4.54, 7.54, 7.47, 4.67, 4.06,

9.7, 9.7, 9.9, 9.7,

5.5) 6.0) 6.1) 5.5)

0.92, d (6.9) 0.92, d (6.9)

9 2.36, 1.47, 1.83, 1.68, 1.34, 1.28, 2.07, 1.51, 4.74, 7.64, 7.71, 4.53, 4.33, 1.62, 0.41, 0.92, 1.46, 4.39, 4.35,

d (13.7) m m m dt (12.8, 4.1) td (12.3, 3.9) d (13.8) d (14.2) q (7.5) d (8.3) d (8.3) d (16.3) d (16.3) d (7.0) s s s d (17.7) d (17.6)

(7.3) (8.1) (8.1) (17.0) (17.0)

12 2.35, 1.46, 1.84, 1.68, 1.34, 1.27, 2.07, 1.51, 4.69, 7.64, 7.71, 4.50, 4.14, 1.61, 0.42, 0.92, 1.45, 4.38, 4.36,

d (14.2) ddd (14.3, 12.2, 2.9) qt (11.2, 3.6) dt (14.2, 4.6) dt (12.8, 3.6) ddd (12.8, 11.6, 4.2) d (13.8) d (14.0) q (7.1) d (8.2) d (8.2) d (16.5) d (16.5) d (7.1) s s s d (17.5) d (17.5)

0.93, d (6.6)

3.73 s

3.65 s 3.75 s

Data in DMSO-d6. E



Article

Table 2. 13C NMR Data (Assigned from 13C, HSQC and/or HMBC Spectra) for Oxeatamide A (1), Iso-oxeatamide A (2), Oxeatamides B−G (3−8), Oxeatamide A 23-Methyl Ester (9), and Dimethyl Oxeatamide A (12) (150 MHz, CDCl3 Unless Otherwise Noted.)

a

pos.

1

2

3a

4a

5

6

7

8

9

12

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 22 23 24 25 26 27 28 6-OMe 23-OMe

41.0 20.1 39.8 31.9 50.9 179.2 40.9 135.2 151.5 39.8 127.8 122.7 130.2 142.0 50.2 169.4 15.5 27.4 33.1 32.9 43.8 173.9

41.1 20.2 39.9 31.9 51.0 181.0 40.6 139.4 146.8 39.4 130.4 121.4 140.9 130.4 170.0 49.8 18.3 27.3 33.1 33.1 44.5 175.3

40.8 19.7 39.7 31.4 50.0 174.4 40.8 136.3 149.6 39.2 126.9 120.9 131.0 141.8 49.1 166.8 15.5 26.7 32.9 32.7 41.8 26.0 134.4

40.8 20.1 39.7 31.8 50.4 175.1 40.8 136.6 150.0 39.6 127.3 121.1 131.5 142.4 49.9 167.1 15.8 27.2 33.3 33.2 39.4 50.0

41.0 20.0 39.6 31.8 50.8 175.8 40.5 134.3 150.3 39.7 127.6 122.4 131.6 141.3 50.1 168.4 15.5 27.3 32.9 32.9 50.0 27.8 20.1 20.1

40.9 20.0 39.6 31.7 50.7 176.8 40.8 135.1 150.5 39.7 127.5 122.0 131.2 141.5 50.3 168.4 15.4 27.1 33.0 32.8 44.1 34.7 138.7 128.8 128.6 126.5

41.0 20.0 39.6 31.8 50.8 176.3 40.6 134.5 150.4 39.7 127.6 122.4 131.6 141.3 50.1 168.6 15.5 27.3 32.9 32.9 48.5 34.0 27.0 11.2 16.9

41.0 20.0 39.7 31.8 50.8 176.8 40.6 134.7 150.3 39.6 127.6 122.3 131.7 141.3 49.4 168.0 15.5 27.2 32.9 32.8 40.8 37.1 26.0 22.5 22.5

41.1 20.1 39.9 31.9 50.9 175.4 40.6 134.7 151.1 39.8 127.8 122.8 130.7 142.0 50.1 168.6 15.6 27.4 33.1 33.0 43.6 169.8

41.0 20.0 39.7 31.8 50.7 174.7 40.7 135.3 151.1 39.7 127.7 122.6 130.6 141.8 49.9 168.6 15.6 27.3 33.0 33.9 43.8 169.6

52.5

53.5 52.3

134.9 116.3

Data in DMSO-d6.

around the N-21/C-22 bond caused by the dimerization of the C-23 carboxylic acid. Methylation of the acid disrupts this effect, as was seen for 12. Furthermore, C-23 (δC 169.8) resonated further upfield than in oxeatamide A (δC 173.9), consistent with esterification. The new OMe resonance also showed a strong HMBC correlation to C-23. These data suggested that 9 is the 23-methyl ester of oxeatamide A. Compound 9 has nearly identical 1H and 13C chemical shifts to oxeatamide A (1) as well as similar (positive) NOE enhancements to oxeatamide A dimethyl ester (12), consistent once again with the relative configuration of 7R*, 10S*, as was established above. The final structure, therefore, is assigned as oxeatamide A 23-methyl ester (9). As MeOH was used for extraction of the sponge and subsequent chromatographic steps, this compound may be an artifact of isolation, although the equivalent 6-methyl ester or dimethyl ester was not detected. Compounds 10 and 11 displayed a different chromophore from that of the oxeatamides. Preliminary NMR analysis showed similarities to the diterpene skeleton of the oxeatamides, but the signals characteristic of the nitrogenous moiety were notably absent, and two acetal signals were apparent instead. A literature search and comparison of NMR data revealed the compounds to be membranolides C (10) and D (11), previously isolated by Ankisetty et al. from the Antarctic sponge Dendrilla membranosa.4

The oxeatamides described herein represent new members of the aplysulphurane class of rearranged spongian diterpenes. Their relative configuration was assigned based on observed NOE enhancements as 7R*, 10S*, consistent with various other aplysulphurane compounds whose relative configurations have been established through a combination of single-crystal X-ray diffraction analysis,1,3,10,11 NMR data,1,4−6,12 and chemical degradation experiments.12 The biogenetic mechanism proposed by Karuso et al.1 suggests the absolute configuration should be 7R and 10S, as has been established for various spongian and related compounds,2 but degradation reactions or total synthesis will be required for unequivocal proof. Compounds 1−9 were tested for cytotoxicity in a 48 h MTT assay against HL-60 cells and were found to be inactive (IC50 >10 μM). The unusual nitrogenous portion of the oxeatamides is most likely amino acid derived. While an abundance of nitrogenous terpenes has been isolated from marine invertebrates and their predators, the majority of them are isocyanides and the related isothiocyanides, thiocyanates, isocyanates, formamides, and the (uniquely marine) dichloroimines; inorganic cyanide has been established as the common precursor of their nitrogen.16 The oxeatamides, on the other hand, contain such nitrogenous moieties as 2-amino acetic acid (glycine), 2-(1H-imidazol-5yl)ethanamine (decarboxylated histidine or histamine), 2methylpropan-1-amine (decarboxylated valine), 2-phenylethylamine (decarboxylated phenylalanine), 2-methylbutan-1-amine F



Article

NMR (DMSO-d6) Table 1; 13C NMR (DMSO-d6) Table 2; HRESIMS m/z 424.2592 [M + H]+ (calcd for C25H34N3O3, 424.2600). Oxeatamide C (4): amorphous, white solid; [α]20 D −60.3 (c 0.15, MeOH); 1H NMR (CDCl3) Table 1; 13C NMR (CDCl3) Table 2; HRESIMS m/z 395.2318 [M + Na]+ (calcd for C22H32N2O3Na, 395.2316). Oxeatamide D (5): amorphous, white solid; [α]20 D −59.2 (c 0.20, MeOH); 1H NMR (CDCl3) Table 1; 13C NMR (CDCl3) Table 2; HRESIMS m/z 408.2520 [M + Na]+ (calcd for C24H35NO3Na, 408.2515). Oxeatamide E (6): amorphous, white solid; [α]20 D −60.1 (c 0.17, MeOH); 1H NMR (CDCl3) Table 1; 13C NMR (CDCl3) Table 2; HRESIMS m/z 456.2518 [M + Na]+ (calcd for C28H35NO3Na, 456.2515). Oxeatamide F (7): amorphous white solid; [α]20 D −59.9 (c 0.19, MeOH); 1H NMR (CDCl3) Table 1; 13C NMR (CDCl3) Table 2; HRESIMS m/z 422.2669 [M + Na]+ (calcd for C25H37NO3Na, 422.2671). Oxeatamide G (8): amorphous, white solid; [α]20 D −60.1 (c 0.20, MeOH); 1H NMR (CDCl3) Table 1; 13C NMR (CDCl3) Table 2; HRESIMS m/z 422.2670 [M + Na]+ (calcd for C25H37NO3Na, 422.2671). Oxeatamide A 23-methyl ester (9): amorphous, white solid; [α]20 D −62 (c 0.05, MeOH); UV (MeOH) λmax, nm (log ε) 203 (4.64) 239 1 13 (4.26); H NMR (CDCl3) see Table 1; C NMR (CDCl3) see Table 2; HRESIMS m/z 424.2112 [M + Na]+ (calcd for C23H31NO5Na, 424.2100). Oxeatamide A dimethyl ester (12): amorphous, white solid; [α]20 D −120 (c 0.1, MeOH); UV (MeOH) λmax, nm (log ε) 207 (4.62) 241 (4.25); 1H NMR (CDCl3) see Table 1; 13C NMR (CDCl3) see Table 2; HRESIMS m/z 416.2434 [M + H]+ (calcd for C24H34NO5, 416.2431) m/z 438.2254 [M + Na]+ (calcd for C24H33NO5Na 438.2251). Methylation of 1. TMSCHN2 (50 μL, 2 M in hexanes) was added to 5 mg of 1 dissolved in dry CH2Cl2 (1 mL) under argon. After 1 h, the reaction was opened and left overnight. To the dry residue was added 1 mL of MeOH, followed by 1 mL of 2% (v/v) aqueous acetic acid (1 mL). The sample was loaded onto a column of HP20SS (1 × 2 cm). The column was then washed with H2O (1 mL) and eluted with 6 mL fractions of (1) 20% Me2CO/H2O, (2) 40% Me2CO/H2O, (3) 60% Me2CO/H2O, (4) 80% Me2CO/H2O, and (5) Me2CO. Fraction 5 was concentrated under reduced pressure and further purified on a semipreparative C18 reversed-phase HPLC column that was eluted with 90% MeOH in H2O at a flow rate of 5 mL/min to yield oxeatamide A dimethyl ester (12) (1.1 mg).

(decarboxylated isoleucine), and 3-methylbutan-1-amine (decarboxylated leucine), which suggest amino acid biogenetic origins of the lactam substituent. The oxeatamides may therefore be one of the examples of compounds of mixed terpene/amino acid origins.17−31 Apart from haumanamide26 and the spongolactams A−C,27 the oxeatamides are the only nitrogenous spongian diterpenes reported.

■

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were performed on a PerkinElmer 241 polarimeter, and UV spectra were recorded on a Varian Carey 100 Scan UV−visible spectrophotometer. NMR spectra were obtained using a Varian DirectDrive 600 MHz spectrometer equipped with an inverse-detected triple resonance HCN probe operating at 25 K. 1H and 13C chemical shifts (δ) were internally referenced to the residual solvent peak,32 15N chemical shifts were externally referenced to nitromethane (CH3NO2) as 0 ppm. High-resolution mass spectra were obtained using a PE Biosystem Mariner 5158 TOF or a Micromass Q-TOF Premier mass spectrometer. HPLC purifications were performed on a Rainin Dynamax SD-200 HPLC system with 25 mL pump heads and a Varian ProStar 335 diode array detector, using Phenomenex Prodigy C18 analytical (4.6 × 250 mm, 5 μm) or semipreparative (10 × 250 mm, 10 μm) columns. Animal Material. The sponge specimens were collected by hand using scuba from Port Hardy, D’Urville Island (40°46.20 S, 173°59.70 E), in April 2000 and from Archway Bay, Cape Brett, Northland, New Zealand, in November 2006 and stored frozen. Voucher specimens (MNP1002 and PTN3-06A) are held at the School of Chemical and Physical Sciences, Victoria University of Wellington, New Zealand. Extraction and Isolation. The D’Urville Island sponge (505 g wet weight) was cut up and extracted twice overnight with MeOH. The extracts were loaded onto HP20 beads by successive dilution with H2O to 25% MeOH in H2O. The column was washed with H2O and eluted with (1) 20% Me2CO/H2O, (2) 40% Me2CO/H2O, (3) 60% Me2CO/H2O, (4) 80% Me2CO/H2O, and (5) Me2CO. A portion of fraction 1 and a portion of fraction 2 were partitioned on Diol eluted with CH2Cl2/MeOH. The 10% MeOH/CH2Cl2 fractions were further purified on a semipreparative C18 reversedphase HPLC column, which was eluted with 70% MeOH/0.1 M HCOOH, giving oxeatamide A (1) as the major compound (15.0 mg), as well as iso-oxeatamide A (2) (0.9 mg) and oxeatamide B (3) (6.2 mg). Fractions eluting from the HPLC after oxeatamide A (1) were combined and subjected to further RP-HPLC to yield oxeatamide A 23-methyl ester (9) (0.5 mg). A second sponge sample (535 g), from Archway Bay, was treated in a similar fashion. The 20% and 40% Me2CO/H2O fractions, after chromatography on Diol and final purification on RP-HPLC as above, afforded more of compounds 1 (34.2 mg), 2 (2.2 mg), and 3 (3.1 mg), as well as 0.5 mg of oxeatamide C (4). The 60% Me2CO/H2O fraction was also partitioned on Diol and eluted with CH2Cl2/MeOH. The CH2Cl2 and 1% MeOH/CH2Cl2 fractions were pooled based on TLC and subjected to RP-HPLC on a semipreparative C18 column, which was eluted using 80% MeOH/0.1 M HCOOH. This yielded 0.5 mg of oxeatamide D (5), 1.0 mg of oxeatamide E (6), 0.8 mg of oxeatamide F (7), and 0.6 mg of oxeatamide G (8). Oxeatamide A (1): amorphous, white solid; [α]20 D −60.3 (c 1.0, MeOH); UV (MeOH) λmax, nm (log ε) 206 (4.65) 250 (4.27); 1H NMR (CDCl3) Table 1; 13C NMR (CDCl3) Table 2; HRESIMS m/z 388.2115 [M + H]+ (calcd for C22H30O5, 388.2124), m/z 410.1940 [M + Na]+ (calcd for C22H29NO5Na, 410.1943). Iso-oxeatamide A (2): amorphous, white solid; [α]20 D −59.6 (c 0.22, MeOH); UV (MeOH) λmax, nm (log ε) 209 (4.57) 248 (4.05); 1H NMR (CDCl3) Table 1; 13C NMR (CDCl3) Table 2; HRESIMS m/z 388.2131 [M + H]+ (calcd for C22H30NO5, 388.2124), m/z 410.1949 [M + Na]+ (calcd for C22H29NO5Na 410.1943). Oxeatamide B (3): amorphous, white solid; [α]20 D −60.2 (c 0.50, MeOH); UV (MeOH) λmax, nm (log ε) 207 (4.39) 243 (3.96); 1H

■

ASSOCIATED CONTENT

S Supporting Information *

NMR spectra for all new compounds. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +64 4 463 5960. Fax: +64 4 463 5237. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by the New Zealand Cancer Society Training Scholarship and the Curtis-Gordon Scholarship in chemistry.

■

REFERENCES

(1) Karuso, P.; Skelton, B. W.; Taylor, W. C.; White, A. H. Aust. J. Chem. 1984, 37, 1081−1093. G



Article

(2) Keyzers, R. A.; Northcote, P. T.; Davies-Coleman, M. T. Nat. Prod. Rep. 2006, 23, 321−334. (3) Manríquez, V.; San-Martin, A.; Rovirosa, J.; Darias, J.; Peters, K. Acta Crystallogr. 1990, C46, 2486−2487. (4) Ankisetty, S.; Amsler, C. D.; McClintock, J. B.; Baker, B. J. J. Nat. Prod. 2004, 67, 1172−1174. (5) Díaz-Marrero, A. R.; Dorta, E.; Cueto, M.; San-Martín, A.; Darias, J. Tetrahedron 2004, 60, 1073−1078. (6) Keyzers, R. A.; Northcote, P. T.; Zubkov, O. A. Eur. J. Org. Chem. 2004, 419−425. (7) Williamson, K. L.; Roberts, J. D. J. Am. Chem. Soc. 1976, 98, 5082−5086. (8) Ruostesuo, P.; Häkkinen, A.-M.; Peltola, K. Spectrochim. Acta 1985, 41A, 739−742. (9) Shapiro, B. L.; Gattuso, M. J.; Hepfinger, N. F.; Shone, R. L.; White, W. L. Tetrahedron Lett. 1971, 12, 219−222. (10) Tischler, M.; Andersen, R. J.; Choudhary, M. I.; Clardy, J. J. Org. Chem. 1991, 56, 42−47. (11) Buckelton, J. S.; Bergquist, P. R.; Cambie, R. C.; Clark, G. R.; Karuso, P.; Rickard, C. E. F. Acta Crystallogr. 1987, C43, 2430−2432. (12) Molinski, T. F.; Faulkner, D. J. J. Org. Chem. 1987, 52, 296−298. (13) Biological Magnetic Resonance Data Bank. Statistics Calculated for Selected Chemical Shifts from Atoms in the 20 Common Amino Acids. http://bmrb.postgenomicnmr.net/ref_info/statsel.htm#9 (accessed Jan 11, 2008). (14) Bhattacharyya, A.; Nirmala, R.; Subramanian, S. THEOCHEM 1995, 339, 245−254. (15) Tori, K.; Nakagawa, T. J. Phys. Chem. 1964, 68, 3163−3169. (16) Garson, M. J.; Simpson, J. S. Nat. Prod. Rep. 2004, 21, 164−179. (17) Cafieri, F.; de Napoli, L.; Fattorusso, E.; Santacroce, C.; Sica, D. Tetrahedron Lett. 1977, 18, 477−480. (18) Cafieri, F.; de Napoli, L.; Fattorusso, E.; Santacroce, C. Experientia 1977, 33, 994−995. (19) Cafieri, F.; de Napoli, L.; Iengo, A.; Santacroce, C. Experientia 1978, 34, 300−301. (20) Cafieri, F.; de Napoli, L.; Iengo, A.; Santacroce, C. Experientia 1979, 35, 157−158. (21) Liu, Y.; Mansoor, T. A.; Hong, J.; Lee, C.-O.; Sim, C. J.; Im, K. S.; Kim, N. D.; Jung, J. H. J. Nat. Prod. 2003, 66, 1451−1456. (22) Liu, Y.; Hong, J.; Lee, C.-O.; Im, K. S.; Kim, N. D.; Choi, J. S.; Jung, J. H. J. Nat. Prod. 2002, 65, 1307−1314. (23) Shin, J.; Rho, J.-R.; Seo, Y.; Lee, H.-S.; Cho, K. W.; Sim, C. J. Tetrahedron Lett. 2001, 42, 3005−3007. (24) Goetz, G. H.; Harrigan, G. G.; Likos, J. J. Nat. Prod. 2001, 64, 1486−1488. (25) Williams, D. E.; Marques, S. O.; Hajdu, E.; Peixinho, S.; Andersen, R. J.; Berlinck, R. G. S. J. Nat. Prod. 2009, 72, 1691−1694 PMID: 19722506. (26) Pham, A. T.; Carney, J. R.; Yoshida, W. Y.; Scheuer, P. J. Tetrahedron Lett. 1992, 33, 1147−1148. (27) Mori, D.; Kimura, Y.; Kitamura, S.; Sakagami, Y.; Yoshioka, Y.; Shintani, T.; Okamoto, T.; Ojika, M. J. Org. Chem. 2007, 72, 7190− 7198. (28) Yao, G.; Chang, L. C. Org. Lett. 2007, 9, 3037−3040. (29) Yao, G.; Kondratyuk, T. P.; Tan, G. T.; Pezzuto, J. M.; Chang, L. C. J. Nat. Prod. 2009, 72, 319−323. (30) Marino, S. D.; Festa, C.; D’Auria, M. V.; Bourguet-Kondracki, M.-L.; Petek, S.; Debitus, C.; András, R. M.; Terencio, M. C.; Payá, M.; Zampella, A. Tetrahedron 2009, 65, 2905−2909. (31) Kim, C.-K.; Song, I.-H.; Park, H. Y.; Lee, Y.-J.; Lee, H.-S.; Sim, C. J.; Oh, D.-C.; Oh, K.-B.; Shin, J. J. Nat. Prod. 2014, 77, 1396−1403. (32) Gottlieb, H. E.; Kotlyar, V.; Nudelman, A. J. Org. Chem. 1997, 62, 7512−7515.

H


The Oxeatamides: Nitrogenous Spongian Diterpenes from the New

Recommend Documents