You are on page 1of 9

Phytochemistry xxx (2011) xxxxxx

Contents lists available at ScienceDirect

Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem

Phytochemical studies of the southern Australian marine alga, Laurencia elata


Daniel Anthony Dias, Sylvia Urban
School of Applied Sciences, Health Innovations Research Institute (HIRi) RMIT University, G.P.O. Box 2476V, Melbourne, Victoria 3001, Australia

a r t i c l e

i n f o

a b s t r a c t
Chemical proling of the southern Australian marine alga Laurencia elata (Rhodomelaceae) employing on-ow and stop-ow HPLCNMR methodology followed by off-line chemical investigations resulted in the isolation of two C16 chamigrenes, cycloelatanene A and B together with three previously reported sesquiterpenes, (3Z)-chlorofucin, pacifenol and elatenyne. The chemical structures were elucidated via detailed spectroscopic analyses. 2011 Elsevier Ltd. All rights reserved.

Article history: Received 3 February 2011 Received in revised form 24 June 2011 Available online xxxx Keywords: Rhodomelaceae Laurencia elata Antitumour Antiviral and antimicrobial activities C16 terpenes (3Z)-chlorofucin Elatenyne Pacifenol Cycloelatanene A Cycloelatanene B

1. Introduction Red algae belonging to the genus Laurencia (Ceramiales, Rhodomelaceae) are a prolic source of secondary metabolites, predominantly producing sesquiterpenes, diterpenes, triterpenes, C15 acetogenins, acetylenes and chamigrenes (Erickson, 1983). Laurencia is a common genus occurring along southern Australian coasts, with numerous species being found in this region (Knig and Wright, 1997). The search for bioactive natural products from this genus of red algae has been an active area of research since the early 1970s (Sims and Fenical, 1972) with the discovery of many interesting structural classes including C15 acetogenins, such as laurendecumenyne A (1) (Nai-Yun et al., 2007) and pannosallene (2) (Suzuki et al., 1996). Many of the chamigrenes reported from the genus are halogenated and contain a spiro centre, along with cyclohexane moieties such as mailione (3) (Francisco and Erickson, 2001) and the sesquiterpene bromo diether (4) (Kikuchi et al., 1985). As part of the activities of the Marine And Terrestrial NAtural Product (MATNAP) research group at RMIT University, which studies the chemistry and biological activity of southern Australian marine organisms, we examined a specimen of the red alga, Laurencia elata collected from St. Pauls Beach, Sorrento, Victoria,
Corresponding author. Tel.: +61 3 9925 3376; fax: +61 3 9925 3747.
E-mail address: sylvia.urban@rmit.edu.au (S. Urban). 0031-9422/$ - see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2011.06.012

Australia. The crude extract of the alga displayed slight antitumour activity as well as antifungal and antiviral activities. We describe herein the chemical proling strategy employed (on-ow and stop-ow HPLCNMR) to preliminary examine the crude extract of the specimen, together with the subsequent chemical investigation that led to the isolation and structure determination of two C16 chamigrenes, cycloelatanene A (23) and cycloelatanene B (28). This investigation also resulted in the isolation of several previously described compounds including (3Z)-chlorofucin (5), pacifenol (8) and elatenyne (10). 1D NOE NMR spectroscopy was utilised to assign the relative conguration of cycloelatanene A (23) and cycloelatanene B (28). In addition, an attempt to address the relative conguration for the recently revised structure of elatenyne (10) was also undertaken. 2. Results and discussion 2.1. Extraction of L. elata for on-ow HPLCNMR proling An initial small-scale DCM extraction of the alga was undertaken according to the procedure outlined in Section 4.1.4. The intention was to employ HPLCNMR to quickly prole the class of secondary metabolites present in the crude extract as well as to attempt to identify the presence of possible previously unrecognised constituents. Initial chemical proling of the L. elata crude extract in CH3CN employing on-ow HPLCNMR analysis resulted in a WET1D 1H

Please cite this article in press as: Dias, D.A., Urban, S. Phytochemical studies of the southern Australian marine alga, Laurencia elata. Phytochemistry (2011), doi:10.1016/j.phytochem.2011.06.012

D.A. Dias, S. Urban / Phytochemistry xxx (2011) xxxxxx

HOO O H O Br O laurendecumenyne A (1) Br O H Br O (3Z)-chlorofucin (4) Cl Br Br


3

H Br C H

HO Br ma'ilione (3) Cl H H O

O Br

pannosallene (2)

H H Cl O H O H
3

O H Br H O H (3E)-chlorofucin (6) H
12 10 9 14 7 3

(5)

5 3

O HO
1

O Cl Br pacifenol
1

Br

(3Z)-venustinene (7)

elatenyne (incorrect structure) (9)

(8) H

Br O
15 13 10 9 7

Br O

O Br Cl

O O lauroxolane (12) Br

Br

elatenyne (revised structure) (10)

(E)-dactomelyne (11)

Br H H3C O H (13) O Br H3C

Br

H O H (14)

O Br

NMR spectrum that suggested the presence of halogenated terpenes, which represent a class of compound typical of this genus (Fig. 1). Subsequent stop-ow HPLCNMR allowed for the individual WET1D 1H NMR spectra of the major metabolites to be obtained (Fig. 1). This enabled a partial identication of three of the secondary metabolites (5, 8 and 10). In all cases signicant signal suppression was observed and it was concluded that an off-line isolation and complete structure determination of these compounds would be necessary to permit their unequivocal identication. A combined chemical proling approach was adopted that involved both on-line (HPLCNMR analysis) and off-line (HPLC isolation and subsequent NMR and MS) studies to conrm and establish the nature of the principle components in the crude extract. 2.2. Extraction of L. elata for larger scale off-line chemical investigation A large-scale extraction of the marine alga, L. elata was undertaken according to the procedure outlined in Section 4.1.3. (3Z)-chlorofucin (5) was isolated as an unstable yellow oil. The positive mode ESIMS displayed a m/z value of 369 [M+Na]+, which supported a molecular formula of C15H20BrClO2. The structure of 5 was conrmed by a direct comparison of the proton and 13C NMR data with those in the literature (Suzuki et al., 2001; Suzuki, 1980). The corresponding isomer (3E)-chlorofucin (6) had been previously

isolated from the marine algae, Laurencia snyderae Dawson (Howard et al., 1980), Laurencia pannosa (Suzuki et al., 1996) and also from a Malaysian species of Laurencia (Vairappan et al., 2008). A comparison of the NMR data of (5) and (6), together with a comparison of the proton 3J coupling constants for the D3,4 double bond [(dH 5.55, dt, J = 2.5, 11.0 Hz) and (dH 6.03, ddd, J = 7.5, 8.0, 10.5 Hz)] in (5) to those of (3Z)-venustinene (7) [(dH 5.55, brd, J = 11.0 Hz) and (dH 6.00, ddd, J = 7.0, 11.0, 11.0 Hz)] (Suzuki et al., 1983) conrmed 5 to be the (3Z)-isomer of chlorofucin. Pacifenol (8) was isolated as stable colourless needles. The ESIMS showed the presence of a peak at m/z 464 [M+Cl], consistent with a molecular formula of C15H21Br2ClO2. The 1D and 2D spectroscopic NMR data and the mass spectrum were found to be identical to those previously reported (Argandoa et al., 1993; Kaiser et al., 2001). Pacifenol (8) is a sesquiterpene which contains a spiro[5.5]undecane skeleton and was rst isolated from the red alga, L. pacica and previously characterised by spectroscopic methods as well as by single-crystal X-ray analysis (Sims et al., 1971). Pacifenol (8) has been reported from a number of marine organisms including: Laurencia nidica (Kimura et al., 1999; Waraszkiewicz and Erickson, 1974), Laurencia nipponica Yamada (Suzuki, 1980), Laurencia claviformis (Argandoa et al., 1993), Laurencia majuscula (Caccamese et al., 1986) and from the Brazilian mollusc Aplysia dactilomela (Kaiser et al., 2001).

Please cite this article in press as: Dias, D.A., Urban, S. Phytochemical studies of the southern Australian marine alga, Laurencia elata. Phytochemistry (2011), doi:10.1016/j.phytochem.2011.06.012

D.A. Dias, S. Urban / Phytochemistry xxx (2011) xxxxxx

Br H H3C O H (15) O H3C Br

Br H O H (16) O Br

Br H H3C O H (17) O Br H Cl HO O O Cl

Br O

O Cl H3C

Br H O H (19) O Br

notoryne

(Z)-dactomelyne (18) H O Cl (21) Br CH3 CH3


7

HO H3C O

O Cl (22)

O H laurendecumenyne B (20) Br
3 14

H
13

1 5

CH3 CH3
6 8 10

Br
9

11

Br Br H O H3C
12

CH3 CH3
5

CH3 CH3 Cl CH3

H3C O H3C
12

Cl
16

CH3 HO Cl O H

H cycloelatanene A (23) CHO CH3 CH3


5 1 3 15

CH3

Br deoxyprepacifenol (24) Br
3 14

laureacetal-B (25) Br

Br halgenated epoxide chamigrene (26) Br H Cl HO H3C O (30) H Cl CH3

CH3 CH3 H
6 8 10

H
13

1 5

CH3 CH3

CH3 CH3

O laurencial (27)

CH3 Cl

H3C O H3C
12

Cl H
16

CH3

HO H3C O (29) H

CH3

cycloelatanene B (28)

Elatenyne (9), the structure of which has been only recently been revised to (10) (Sheldrake et al., 2006; Sheldrake et al., 2009; Smith et al., 2008; Usami, 2009), was rst reported in 1986 when it was isolated from L. elata and its structure deduced on the basis of 1H and 13C NMR spectroscopic analyses (Hall and Reiss, 1986). It has also been reported from the marine red algae, L. majuscula (Wright et al., 1993) and Laurencia decumbens (NaiYun et al., 2007). Synthesis of several derivatives together with the use of on and off resonance decoupling, double resonance and lanthanide shift NMR experiments have been previously employed in an attempt to establish the relative conguration of elatenyne (Hall, 1984; Hall and Reiss, 1986). In our current study of L. elata, elatenyne (10) was isolated as an unstable colourless oil. EIMS identied the presence of fragments at m/z 329, 327, 325 (1:2:1), representing the loss of [MC5H5]+ and indicated an isotopic ratio that supported the presence of two bromine atoms. Although the molecular ion was not observed, EIMS fragmentation was found to be almost identical to that previously reported (Hall and Reiss, 1986). On the basis of the 1H and 13 C NMR spectroscopic data together with HSQCAD and DEPT NMR experiments, the presence of one methyl, four methylenes, nine methines and one quaternary carbon was conrmed. However, the alkyne methine at position 1 (dC 82.8) was not observed in the DEPT 90 NMR spectrum until the coupling constant parameter was varied from the default setting (J = 120 Hz) to that typical

for sp hybridised carbons (J = 210 Hz). Extensive 1D and 2D NMR data was acquired (as shown in Tables 1 and 2) and it was quickly ascertained that the NMR data was in close agreement with those published (Hall and Reiss, 1986). Curiously differences were apparent in the proton chemical shifts in CDCl3 but the carbon NMR assignments were in accordance with the literature data published by Hall and Reiss. The complete NMR assignment of the spectra of elatenyne (10) in C6D6 had been previously been carried out (Hall and Reiss, 1986). Owing to the differences noted in CDCl3, the NMR data were re-acquired in C6D6 for a denitive comparison as well as to improve the resolution of the signals between dH 3.964.22 in an attempt to obtain further coupling constant information. Both the 1H and 13C NMR chemical shifts of 10 recorded in C6D6 were found to be in very close agreement with those assigned by Hall and Reiss (1986). However, we were able to conclude that the carbon chemical shifts previously assigned to positions 7 (pent-en-yne-substituted ring) and 12 (ethyl-substituted ring) by Hall and Reiss at 49.2 and 48.9 ppm, respectively, should be reversed. This was concluded on the basis of the direct proton to carbon correlations observed in the HSQCAD NMR spectrum (see Table 2). Comparison of the NMR spectroscopic data of Sheldrake et al. (2006, 2009) with those of our re-isolated elatenyne clearly identied several anomalies. Major differences were observed for the 13C NMR chemical shifts with differences of dDH $1.4 ppm and dDC

Please cite this article in press as: Dias, D.A., Urban, S. Phytochemical studies of the southern Australian marine alga, Laurencia elata. Phytochemistry (2011), doi:10.1016/j.phytochem.2011.06.012

D.A. Dias, S. Urban / Phytochemistry xxx (2011) xxxxxx

[A]

(23)

[B]

(28)

(5)

(10)

(8)

Fig. 1. [A] 2D HPLCNMR contour plot (on-ow HPLCNMR) illustrating the presence of compounds 5, 8, 10, 23 and 28; [B] Stop-ow HPLCNMR WET 1D NMR spectra for all compounds.

$8 ppm were observed for the positions 6, 9 10 and 13 stereocentres. In the case of elatenyne that was isolated in our study, the 13C NMR spectroscopic data was compared with those of (E)-dactome mus lyne (11), (Aydog et al., 2004; Gopichand et al., 1981; Lee et al., 1995) and to those reported for the 2,20 -bifuranyl, lauroxolane (12) (In Kyu et al., 1989). It was evident that our elatenyne and (E)-dactomelyne (11) showed large discrepancies in their 13C NMR data whereas elatenyne and lauroxolane (12) did not. We conclude that the correct skeletal structure of the elatenyne isolated in this work is as shown in 10.

In an attempt to address the relative conguration of elatenyne (10) 1D NOE NMR studies were carried out in CDCl3 (Table 1) and the spectroscopic data evaluated for compliance with the four diastereoisomers proposed by Smith and co-workers (1316). These four most likely diastereoisomers were concluded on the basis of GIAO (Gauche-Including Atomic Orbitals) 13C NMR calculations (Smith et al., 2008). However, owing to overlapping ring junction protons, no key NOE NMR enhancements were observed that could rule out any of the four possible stereoisomers proposed (Smith et al., 2008). While single irradiation studies were not conducted in C6D6 (owing to sample degradation) the overlapping signals

Table 1 1 H (500 MHz) and Position 1 2 3 4 5a 5b 6 7 8 9 10 11 12 13 14a 14b 15


a b c

13

C (125 MHz) NMR of elatenyne (10) in CDCl3. dH (J in Hz) 3.14 dd (2.5, 0.5) 5.60 ddt (11.0, 2.5, 1.0) 6.05 ddt (11.0, 7.5, 1.0) 2.57 m 2.66 ddd (8.5, 5.5, 1.0) 4.22 dt (6.0, 5.5) 4.05 dt (7.0, 5.5) b 2.33 m b 4.15 ddd (12.0, 7.0, 5.5) b 4.15 ddd (12.0, 7.0, 5.5) b 2.33 m 3.96 dt (5.5, 5.5) 4.00 dt (7.5, 5.5) 1.50 ddq (14.0, 7.5, 7.5) 1.66 ddq (14.0, 5.0, 7.5) 0.98 t (7.5) dCa, mult 82.8c d 80.1 s 111.6 d 140.1 d 34.9 t 86.8 48.9 39.1 80.3 79.5 39.2 49.3 89.1 27.1 d d tb db db tb d d t gCOSY 3 1, 4 3, 5a, 5b 4, 5b, 6 4, 5a, 6 5a, 5b, 7 6, 8 7, 9 8 11 10, 12 11 14a, 14b 13, 14b, 15 13, 14a, 15 14a, 14b gHMBC 3, 4 1, 2, 4, 5 2, 3, 5, 6 3, 4, 6, 7 3, 4, 6, 7 4, 7, 8, 9 5, 9 6, 7, 9, 10 8, 10, 11 8, 9, 11, 13 9, 10, 12, 13 10, 11, 14 12, 15 12, 13, 15 12, 13, 15 13, 14 1D NOE 3 1, 4 3, 5a, 5b, 6, 7 4, 6, 7 3, 4, 6, 7 4, 5a, 5b, 7, 8 4, 5a, 5b, 6, 8 7 8 11 12, 13 11, 14a, 15 11, 14a, 14b, 15 12, 13, 14b, 15 12, 13, 14a, 15 12, 13, 14a, 14b

10.5 q

Carbon assignments based on HSQCAD and DEPT NMR experiments. Signals may be interchanged. Observed in the DEPT90 NMR experiment (J = 210 Hz).

Please cite this article in press as: Dias, D.A., Urban, S. Phytochemical studies of the southern Australian marine alga, Laurencia elata. Phytochemistry (2011), doi:10.1016/j.phytochem.2011.06.012

D.A. Dias, S. Urban / Phytochemistry xxx (2011) xxxxxx Table 2 1 H (500 MHz) and Position 1 2 3 4 5a 5b 6 7 8 9 10 11a 11b 12 13 14a 14b 15
a b c

13

C (125 MHz) NMR of elatenyne (10) in C6D6. dCa, mult 82.9 d 80.2 s 111.2 d 140.1 d 34.7 t 86.5 d 48.9 d 39.1b t 80.1b d 79.5b d 39.3b t 49.2 d 88.6 d 26.8 t 10.1 q
c

dH (J in Hz) 2.77 5.36 5.72 2.41 2.49 4.06 3.66 1.91 3.86 3.86 2.03 1.98 3.53 3.89 1.23 1.37 0.81 d (1.8) dd (11.0, 1.8) dt (11.0, 7.5) ddd (14.5, 7.5, ddd (14.5, 6.5, dt (5.5, 5.5) dt (7.5, 5.5) ddd (14.0, 7.5, m m m m dt (7.0, 5.5) dt (7.5, 5.0) ddq (14.0, 7.5, ddq (14.0, 5.0, t (7.5)

gCOSY

gHMBC 3, 4

7.5) 6.5)

7.5)

7.5) 7.5)

4 3, 5a, 5b 4, 6 4, 6 5a, 5b, 7 6, 8 7, 9 8 11a, 11b 10, 12 12 11b, 13 12, 14a 13, 15 13, 15 14a, 14b,

1, 2, 3, 3, 4, 5, 6, 7, 6, 9,

4, 5 3, 5, 6 4, 6, 7 4, 6, 7 7, 8 9 9, 10 8 11 8, 11, 12 12, 13 11, 12, 13, 13, 14 14 15 15 15

10, 11, 12, 12, 13,

Carbon assignments based on HSQCAD and DEPT NMR experiments. Signals may be interchanged. Observed in the DEPT90 NMR experiment (J = 210 Hz).

for the protons at positions 9 and 10 would again preclude a denitive assignment although the position 8 and 11 proton resonances were resolved in this solvent. Notoryne (17) is the chlorinated relative of elatenyne (10) and a constitutional isomer of (Z)-dactylomelyne (18). Importantly, the absolute conguration of 17 had previously been established utilising chemical degradation methods (Kikuchi et al., 1991). A comparison of the NMR spectroscopic data (in particular the coupling constants) of compounds 10 and 17 was carried out by comparing the data recorded in the same NMR solvent. It was evident that the proton coupling constants for the ring junction protons in notoryne (17) (dH 3.98, ddd, J = 8.3, 6.8, 5.5 Hz and dH 4.26, ddd, J = 7.3, 7.3, 5.5 Hz) differed from those in elatenyne (10) (dH 4.15, ddd, J = 12.0, 7.0, 5.5 Hz and dH 4.15, ddd, J = 12.0, 7.0, 5.0 Hz) principally in that the latter compound showed a larger coupling constant. In addition a comparison of the proton chemical shifts in 17 with those in 10 indicated that the greatest difference was only at the ring junction protons. On these grounds we would tentatively propose a further possible diastereoisomer as a likely candidate for the structure of elatenyne, namely compound (19). We would suggest that either compounds (16) or (19) to be the most likely candidates for the possible diastereoisomer of elatenyne. In either case one of the two possible COCO Gauche conformations about C9C10 gives an anti HCCH arrangement. It would appear that the absolute conguration of elatenyne (10) may only be unequivocally conrmed either by total synthesis and/or by single-crystal X-ray crystallography (Sheldrake et al., 2006). To date, attempts to secure the absolute conguration by either of these methods has been unsuccessful (Hall and Reiss, 1986). Our efforts to secure crystals of elatenyne (10) were in vain with the compound being unstable and degradation observed with each crystallisation attempt. As recently as 20062007, the structure of elatenyne was still being reported incorrectly (Dez et al., 2006; Nai-Yun et al., 2007). On the basis of the structural revision of elatenyne (10), it became apparent that a number of other secondary metabolites assigned by comparison to (9) have required revisions. A case in point is the structure of laurenedecumenyne B (20), only recently revised from 20 to that of 17 which the authors conclude to be the same as notoryne (17) (Nai-Yun et al., 2007, 2010b). However, we have noted the existence of signicant differences in the 1H NMR resonances for laurendecumenyne B (20) and notoryne (17), suggesting that a possible stereoisomer may have been iso-

lated. Burton and co-workers also synthesised (21) a natural product assigned the same structure skeleton originally reported for elatenyne (9) and (E)-dactomelyne (11) on the basis of extensive NMR spectroscopic analysis (Wright et al., 1993). On the basis of the position 9 and 10 carbon resonances being >76 ppm, together with the fact that the synthetic pyrano[3,2-b]pyranyl did not match the natural product data, the structure of 20 was revised to the 2,20 -bifuranyl structure (22). Cycloelatanene A (23) was isolated as a colourless oil for which high resolution ESIMS established the molecular formula as C16H24BrClO2 (m/z 385.0541 [M+Na]+, calc for C16H2479Br35ClNaO2, 385.0546 and m/z 363.0721 [M+H]+; calc for C16H2579Br35ClO2, 363.0648) indicating four degrees of unsaturation. Analysis of the 1 H, 13C and HSQCAD NMR spectroscopic data indicated the presence of one methoxy moiety (dH 3.63, dC 60.3), four singlet methyls (dH 1.29, dC 30.1), (dH 1.13, dC 29.2), (dH 1.14, dC 22.7), and (dH 1.62, dC 28.1); three methylenes [(dH 2.21, ddd, J = 4.0, 7.5, 12.0 Hz), (dH 1.68, d, J = 12.5 Hz, dC 33.4); (dH 1.98, m), (dH 1.54, ddd, J = 7.0, 12.5, 13.5 Hz, dC 31.6); (dH 1.83, dd, J = 7.0, 13.5 Hz), (dH 2.94, ddd, J = 8.0, 13.0, 13.5 Hz, dC 37.5), one olenic methine [(dH 6.21, d, J = 6.0 Hz, dC 128.2); two deshielded methines (dH 3.67, dd, J = 6.0, 0.5 Hz, dC 82.4), (dH 4.30, dd, J = 7.5, 1.0 Hz, dC 84.3) and ve quaternary carbons (dC 45.4, 139.1, 85.7, 48.8, 74.7) (Table 3 and Fig. 2). COSY NMR correlations were observed from the olenic methine at position 3 (dH 6.21, d, J = 6.0 Hz) to a deshielded oxymethine (dH 3.67, dd, J = 6.0, 0.5 Hz, H4). In turn this oxymethine at position 4 showed key HMBC NMR correlations to the methyl group at (dC 30.1, C12), to the methoxy moiety at (dC 60.3, C13), to an olenic carbon (dC 139.1, C2) and to a carbon at 48.8 (C6). This allowed placement of the methoxy substituent to position 4. The olenic methine at position 3 (dH 6.21) displayed HMBC NMR correlations to 45.4 (C1) and to the deshielded quaternary carbon 85.7 (C5). Two geminal methyls (dH 1.14) and (dH 1.13) showing HMBC NMR correlations to each other and to an olenic moiety (dC 139.1, C2) were also identied. Further interpretation of the carbon resonances for this rst structural subunit of cycloelatanene A (23) indicated that the carbon at position 5 was deshielded by its direct attachment to an oxygen and that the olenic carbon resonance at position 2 was substituted with a halogen. The halogen was concluded to be bromine rather than chlorine on the basis of the chemical shift of the position 2 carbon and was further conrmed by comparison to related literature compounds such as deoxyprepacifenol (24) (Fronczek, 1989; Watanabe et al., 1989). On the basis of these combined COSY and HMBC NMR correlations structure fragment A could be assembled (Fig. 2). Upon further inspection of the literature it was evident that this structural fragment resembled part of the structure of laureacetal-B (25) (Suzuki and Kurosawa, 1979). Chemical shifts comparisons supported this structure fragment. In particular the coupling constant for the deshielded methine at position 4 (dH 3.67, dd, J = 6.0, 0.5 Hz) was consistent with that reported for laureacetal-B (25) (dH 3.98, d, J = 7.0 Hz) (Suzuki and Kurosawa, 1979). In addition the coupling constant for the olenic methine at position 3 (dH 6.21, d, J = 6.0 Hz) was also consistent with that reported for laureacetal-B (25) (dH 6.60, d, J = 7.0 Hz), which again supported the relative conguration assignment at position 4 (Suzuki and Kurosawa, 1979). The absolute conguration of laureacetal-B (25) had been previously established by a chemical degradation approach (Suzuki and Kurosawa, 1979). Both the 1H and 13C NMR assignments for structure fragment A were found to be similar to those for the bromotrimethylcyclohexene moiety present in 25 as well as to those reported for other related chamigrenes (Kimura et al., 1999; Kurata et al., 1983a; Suzuki et al., 1985). A second proposed structure fragment B (Fig. 2) was formulated on the basis of the following COSY and HMBC NMR correlations. The deshielded oxymethine at position 8 (dH 4.30) showed key HMBC NMR correlations to (dC

Please cite this article in press as: Dias, D.A., Urban, S. Phytochemical studies of the southern Australian marine alga, Laurencia elata. Phytochemistry (2011), doi:10.1016/j.phytochem.2011.06.012

6 Table 3 1 H (500 MHz) and Position 1 2 3 4 5 6 7a 7b 8 9 10a 10b 11a 11b 12 13 14 15 16

D.A. Dias, S. Urban / Phytochemistry xxx (2011) xxxxxx

13

C (125 MHz) NMR of cycloelatanene A (23) in CDCl3. dH (J in Hz) dCa, mult 45.4 s 139.1 s 128.2 d 82.4 d 85.7 s 48.8 s 33.4 t 84.3 d 74.7 s 37.5 t 31.6 t 30.1 60.3 29.2 22.7 28.1 q q q q q gCOSY gHMBC 1D NOE

6.21 d (6.0) 3.67 dd (6.0, 0.5)

4 3

1, 2, 5 2, 3, 5, 6, 12, 13

4, 14

2.21 ddd (12.0, 7.5, 4.0) 1.68 d (12.5) 4.30 dd (7.5, 1.0) 2.94 1.83 1.98 1.54 1.29 3.63 1.13 1.14 1.62 ddd (13.5, 13.0, 8.0) dd (13.5, 7.0) m ddd (13.5, 12.5, 6.5) s s s s s

7b, 8, 11a(w) 7a 7a 10a, 10b, 11a, 11b 10a, 11b 7a, 10a, 11b 10a, 10b, 11a, 16(w)

6, 8, 9, 11 8, 9, 11 5, 6, 9, 10 9, 6, 6, 5, 4, 4 2, 2, 8, 11, 16 8, 9, 11, 16 7, 9, 10 6, 7, 10 5, 6 15 14 9, 10

7b, 8, 12, 15 7a, 8, 14 7a, 7b, 12, 13, 15, 16 10b, 11a, 13 10a, 11b 11b, 15 11a, 14 3, 4, 10a, 12 12, 13(w) 8, 10b

11b(w)

w = Weak correlation or enhancement. a Carbon assignments based on HSQCAD and DEPT NMR experiments.

48.8, C6), (dC 74.7, C9) and to the methylene (dC 37.5, C10). The carbon chemical shift of the position 8 methine, once again, suggested that it was directly attached to oxygen. The quaternary carbon at position 6 (dC 48.8) was common to both fragments A and B and concluded to be a spiro centre on the basis of comparisons to other related spiro terpenoids isolated from the genus (Jongaramruong et al., 2002; Kimura et al., 1999; San-Martn et al., 1997). The methyl group at (dH 1.62) also showed a HMBC NMR correlation to the quaternary carbon at (dC 74.7, C9). The carbon chemical shift at position 9 is consistent with the presence of a methyl and a chlorine substituent and is a common structural feature in previously isolated chamigrenes such as the halogenated chamigrene epoxide (26) rst isolated from the red alga Laurencia okamurai (Ojika et al., 1982). Furthermore, the coupling constant for the oxymethine proton at position 8 (dH 4.30, dd, J = 7.5, 1.0 Hz) supported the presence of the chloromethylcyclohexane moiety in fragment B as in the case of laurencial (27) (dH 4.11, d, J = 6.0 Hz) (Kurata et al., 1983b) (Fig. 2). Additional HMBC NMR correlations from the methylene at position 11 (dH 1.98, m) and (dH 1.54, ddd, J = 6.5, 12.5, 13.5 Hz) to the position 5 carbon (dC 85.7) and to the spiro centre at position 6 (dC 48.8) and also from the adjacent methylenes at positions C10 (dC 37.5) and C7 (dC 33.4), respectively (Fig. 2) also supported this second structure fragment. The key HMBC NMR correlations (Fig. 3) that allowed for the connection of structure fragments A and B were those observed from the oxymethine at position 8 (dH 4.30) and the position 11 methylene (dH 1.54) to the carbon (dC 85.7) at position 5. These correlations concluded that a third ve membered ring was also present in the structure of 23 and accounted for the nal double bond equivalent. The relative conguration of cycloelatanene A (23) was established on the basis of selective 1D NOE NMR experiments and coupling constants. Key ID NOE NMR enhancements were observed from one of the geminal methyls at position 15 (dH 1.14) to the 12-CH3 (dH 1.29) and a weak correlation to the methoxy protons at position 13 (dH 3.63). The deshielded methine at position 8 (dH 4.30) showed key NOE enhancements to the methyl at 1.14, the deshielded methyls at 1.29 and to the methoxy protons 3.63. In turn, the position 13 methoxy moiety protons displayed an NOE to one of the position 10 methylene protons (dH 2.94, H10a). Also observed were key NOE enhancements between one of the position 7 methylene protons (dH 2.21, H7a) to both 12-CH3 (dH 1.29) and 15-CH3 (dH 1.14). Another important NOE enhancement was

A
H H H3 CO H3C
3

Br C H3
1 5

B
6 8 10

C H3

Cl

C H3
16

Fig. 2. Structural fragments of cycloelatanene A (23).

Br

CH3 CH3
1 6 8

H H3 C O

3 5

10

H3C O

Cl CH3

Fig. 3. Key HMBC NMR correlations of cycloelatanene A (23).

observed from the other position 7 proton (dH 1.68, H7b) to the position 14 methyl (dH 1.13). These NOE enhancements allowed the complete relative conguration assignment of 23 (Fig. 4), with H7a, H8, H10a, H11a, 12-CH3, 13-CH3, and 15-CH3 on the same face of the molecule. Cycloelatanene B (28) was isolated as a colourless oil and high resolution EIMS conrmed the molecular formula C16H24BrClO2 (364.0633 [M]+, calculated for C16H2480Br35ClO2, 364.0628) indicating that it possessed four degrees of unsaturation and was isobaric with 23. That 28 was a stereoisomer of 23 was concluded on the basis of the similarity of the IR, UV, 1H and 13C NMR spectra. Analysis of the 1H NMR, 13C NMR and HSQCAD NMR spectroscopic data once again indicated the presence of one methoxy moiety, (dH 3.53, dC 58.2) four methyl singlets [(dH 1.27, 22.7), (dH 1.18, dC 22.6), (dH 1.09, dC 28.6) and (dH 1.60, dC 26.9)], three methylenes [(dH 2.22, ddd, J = 4.0, 7.5, 12.0 Hz, dH 1.68, d, J = 12.0 Hz, dC 32.5); (dH 1.70,

Please cite this article in press as: Dias, D.A., Urban, S. Phytochemical studies of the southern Australian marine alga, Laurencia elata. Phytochemistry (2011), doi:10.1016/j.phytochem.2011.06.012

D.A. Dias, S. Urban / Phytochemistry xxx (2011) xxxxxx

Br
3 13

H
5

CH 3 CH3
6 10 8

14

H 3C O H3 C

12

H O

Cl CH 3
16

Fig. 4. Key 1D NOE NMR enhancements of cycloelatanene A (23).

m, dH 1.56, m, dC 30.4); (dH 2.58, ddd, J = 7.0, 14.0, 20.0 Hz, dH 1.95, dd, J = 6.0, 11.0 Hz, dC 38.2)]; one olenic methine [(dH 5.98, d, J = 1.6 Hz, dC 131.0); two deshielded methines (dH 4.17, d, J = 1.6 Hz, dC 80.3); (dH 4.29, brd, J = 7.5 Hz, dC 84.5) and ve quaternary carbons (dC 45.1, 135.2, 86.8, 50.1 and 72.7) (Table 4). Analysis of the 1H NMR spectrum of cycloelatanene B (28) identied some slight changes in both chemical shifts and coupling constants when compared with the NMR spectroscopic data of cycloelatanene A (23). In particular comparison of the chemical shift and coupling constant of the position 4 methine in 28 (dH 4.17, d, J = 1.6 Hz) with that present in 8-epi-laureacetal B (25) (dH 3.91, d, J = 1.5 Hz) supported a stereochemical change at this centre (Suzuki and Kurosawa, 1979). Analysis of the 1H NMR spectra of both compounds concluded that 23 and 28 were diastereoisomers. The most signicant difference observed between the 1H NMR spectra of 23 and 28 was in the chemical shifts associated with the oxymethine at position 4, (dH 4.17 in 23) compared to (dH 3.67 in 28). The chemical shift difference for the position 3 olenic methine 5.98 in 28 compared to 6.21 in 23, was inuenced by the change in conguration at position 4 and was supported by literature data (Kurata et al., 1983a,b; Nai-Yun et al., 2010a). The relative conguration of cycloelatanene B (28) was proposed on the basis of selective 1D NOE NMR experiments and coupling constants. Key 1D NOE NMR enhancements included those from the deshielded methine at position 8 (dH 4.29) to the 12-CH3 (dH 1.27) which in turn showed NOE enhancements to the 15-CH3 (dH 1.18) and one of the position 7 methylene protons (dH 2.22,

H7a). This methylene proton showed an important NOE to the 15-CH3. Other key NOEs were observed from the position 4 methine (dH 4.17) to one of the position 10 methylene protons (dH 2.58, H10a) as well as one of the methylene protons (dH 1.70, H11a). Finally the 14-CH3 (dH 1.09) displayed an NOE to one of the position 11 methylene protons (dH 1.56, H11b) and the other position 7 proton (dH 1.68, H7b). This spectral evidence established the relative conguration and concluded the structure of 28 to be epimeric at position 4 in relation to cycloelatanene A (23) with H4, H7a, H8, H10a, H11a, 12-CH3 and 15-CH3, on one face of the molecule whilst H11b, 13-CH3 and 14-CH3 were on the another. During the course of this investigation, the two compounds 10bromo-3-chloro-2,7-epoxychamigr-9-en-8b-ol (29) and 10-bromo-3-chloro-2,7-epoxychamigr-9-en-8a-ol (30) were isolated from two separate marine red algae, namely Laurencia composita and Laurencia saitoi, respectively (Nai-Yun et al., 2009, 2010a). These sesquiterpenes (29) and (30) are very closely related to cycloelatanene A (23) and cycloelatanene B (28) with both the 1H and 13C spectroscopic data found to be in close agreement with those reported for 28 and 29 (Nai-Yun et al., 2009). However, the relative conguration assignments at C5 of cycloelatanene A (23) and cycloelatanene B (28) were concluded to differ from those of compounds (29) and (30). The relative congurations of 29 and 30 had been assigned on the basis of ROESY and NOESY NMR enhancements, respectively (Nai-Yun et al., 2009). Some of the key NOE NMR enhancements reported by these authors differed to those found in cycloelatanene A (23) and cycloelatanene B (28). In addition the relative conguration assignments reported for 29 and 30 were also made on the basis of NOEs not observed. Our relative conguration assignments for cycloelatanene A (23) and cycloelatanene B (28) are solely based on NOE NMR enhancements actually observed. The epoxychamigrane class of sesquiterpenes are classied as a recent addition to the array of sesquiterpenes occurring in the genus Laurencia, with only four compounds (4), (27), (29) and (30) of this class reported to date (Kikuchi et al., 1985; Kurata et al., 1983b; Nai-Yun et al., 2009, 2010a). The isolation of the two undescribed C16-chamigrene derivatives, cycloelatanenes A (23) and B (28) represent further variations in this class, and unlike compounds (29) and (30), were found to co-occur in the one marine alga specimen.

Table 4 1 H (500 MHz) and Position 1 2 3 4 5 6 7a 7b 8 9 10a 10b 11a 11b 12 13 14 15 16

13

C (125 MHz) NMR of cycloelatanene B (28) in CDCl3. dH (J in Hz) dCa, mult 45.1 s 135.2 s 131.0 d 80.3 d 86.8 s 50.1 s 32.5 84.5 72.7 38.2 t d s t gCOSY gHMBC 1D NOE

5.98 d (1.6) 4.17 d (1.6)

4 3

1, 2, 5 2, 3, 5, 13

4, 12, 13 3, 10a, 11a, 12, 13

2.22 ddd (12.0, 7.5, 4.0) 1.68 d (12.0) 4.29 brd (7.5) 2.58 1.95 1.70 1.56 1.27 3.53 1.09 1.18 1.60 ddd (14.0, 13.5, 7.0) dd (13.0, 6.0) m ddd (14.5, 14.5, 6.0) s s s s s

7b, 8, 7a 7a 10b, 11a, 11b, 16 10a, 11b 10a 10a, 10b

5, 6, 9, 11 5, 6, 9, 11 5, 9, 10 9, 11, 16 6, 8, 9, 11,16 5, 4, 4 1, 1, 8, 6, 7, 10 5, 6 2, 6, 15 2, 6, 14 9, 10

7b, 8, 11a, 12, 15 7a, 14 7a, 7b, 12, 16 4, 10b, 11a, 11b 10a, 11a, 11b 4, 7a, 8, 15 10a, 10b, 11a, 13, 14 3, 4, 7a, 8, 15 3, 4 7b, 11b, 15 7a, 11a, 12, 14 8, 10b

30.4 t 22.7q 58.2 q 28.6 q 22.6 q 26.9 q

10a(w)

w = Weak correlation or enhancement. a Carbon assignments based on HSQCAD and DEPT NMR experiments.

Please cite this article in press as: Dias, D.A., Urban, S. Phytochemical studies of the southern Australian marine alga, Laurencia elata. Phytochemistry (2011), doi:10.1016/j.phytochem.2011.06.012

D.A. Dias, S. Urban / Phytochemistry xxx (2011) xxxxxx

3. Conclusions The present work reports on a chemical proling strategy (HPLC NMR) which was used to analyse the crude extract of the marine alga L. elata and ultimately assisted in the detection and then off-line isolation of two C16 chamigrene natural products (23 and 28), together with several previously reported compounds (5, 8 and 10). The relative congurations of cycloelatanene A (23) and cycloelatanene B (28) were determined using 1D NOE NMR spectroscopy. An attempt to address the relative conguration of the recently revised structure of elatenyne (10) was also undertaken and suggested two most likely stereoisomers (15 or 19). None of the compounds 5, 8, 10, 23 and 28 displayed any appreciable antitumour activity. 4. Experimental 4.1. General experimental procedures Details of the general experimental procedures please see (Dias et al., 2009). Analytical HPLC analyses were run using either a gradient method 02 min 10% CH3CN/H2O; 1424 min 75% CH3CN/ H2O; 2630 min 100% CH3CN and 32 40 min 10% CH3CN/H2O or an isocratic method (75% CH3CN/H2O) on a Phenomenex Prodigy ODS (3) C18 100 250 4.6 (5 l) and on a Phenomenex Luna ODS (3) C18 100 250 4.6 (5 l) column at a ow rate of 1.0 mL/min. Reversed phased semi-preparative HPLC was carried out on a Varian Prostar 210 (Solvent Delivery Module) equipped with a Varian Prostar 335 PDA detector using STAR LC WS Version 6.0 software using a an isocratic method (75% CH3CN/H2O) on a Phenomenex Prodigy ODS (3) 100 C18 250 10 (5 lm) column at a ow rate of 3.5 mL/min. HPLC-NMR was carried out under isocratic HPLC conditions (75% CH3CN/D2O) on a Varian Polaris C18 150 4.6 (5 lm) or a Varian Pursuit C18 250 4.6 (5 lm) column at 1.0 mL/min. For both the on-ow and stop-ow HPLCNMR experiments a 50 lL injection (1333 lg) of the DCM partition was used, monitoring at 215 and 254 nm. 4.1.1. Biological evaluation An extract of the alga (2 g extracted in 40 mL of 3:1 MeOH/ DCM) was evaluated (tested at 50 mg/mL) in a number of biological assays including against a P388 Murine Leukaemia cell line (antitumour assay), against Herpes simplex and Polio viruses (antiviral assays) as well as against a number of bacteria and fungi (antimicrobial assays) at the University of Canterbury, Christchurch, New Zealand. For further details on the biological assays please see (Dias et al., 2009). Only slight antitumour activity was observed for the crude extract of the alga (IC50 of 168,632 ng/ mL). The extract was found to be cytotoxic activity against Herpes simplex and Polio viruses as well as displaying modest antimicrobial activity against Trichophyton mentagrophytes. The crude extract displayed no activity against Eschericha coli, Pseudomonas aeruginosa, Candida albicans, Bacillus subtilis or Cladosporium resinae. All of the compounds isolated (5, 8, 10, 23 and 28) displayed no appreciable antitumour activity (IC50 > 12,500 ng/mL). 4.1.2. Marine alga material The marine red alga L. elata was collected on the 3rd January 2003 from St. Pauls Beach, Sorrento, Victoria, Australia at low tide. The alga was identied by Dr. Gerald Kraft (Honorary Principal Fellow), School of Botany, University of Melbourne, Australia. A voucher specimen designated, the code 200305 is deposited at the School of Applied Sciences, RMIT University. 4.1.3. Extraction and isolation L. elata (30 g) (8 g dr. wt.) was extracted with MeOH/DCM (3:1 v/v 800 mL). The crude extract was decanted, concentrated under

reduced pressure and triturated into DCM, MeOH and water soluble extracts, respectively. The DCM extract was fractionated using ash silica column chromatography (20% stepwise elution from nhexane to DCM to EtOAc and nally to MeOH). Several of the resultant silica column fractions were combined on the basis of their similar 1H NMR spectra and subjected to reversed phased HPLC (75% CH3CN/H2O) to yield (3Z)-chlorofucin (5), (12.3 mg, 0.15%), pacifenol (8) (7.8 mg, 0.09%), elatenyne (10) (14.2 mg, 0.18%), cycloelatanene A (23) (3.4 mg, 0.04%) and cycloelatanene B (28) (2.3 mg, 0.03%) (% based on the mass of the dried weight of the marine alga). 4.1.4. On-ow and stop-ow HPLCNMR Proling For the HPLCNMR chemical proling of the alga, a frozen sample of L. elata (2 g) was diced and extracted with 100% DCM (10 mL) overnight. The extract was decanted and concentrated under reduced pressure then re-solubilised using HPLCNMR grade CH3CN (100%) and nally ltered through a 0.45 lm PTFE membrane (HP045 Advantec, Japan). For both the on-ow and stop-ow HPLC-NMR experiments 50 lL (1333 lg of the extract) was injected and monitored at kmax 215 and 254 nm, under isocratic conditions (75% CH3CN/D2O). 4-bromo-6-chloro-3-ethyl-9-((Z)-pent-2-en-4-ynyl)-2,8-dioxabicyclo[5.2.1]decane (3Z-chlorofucin) (5), isolated as a yellow oil; 1 H and 13C NMR data were found to be identical to those previously reported (Suzuki et al., 2001; Suzuki, 1980); ESIMS (positive mode) m/z 369 [M+Na]+. 3,8-Dibromo-4-chloro-4,11,12,12-tetramethyl-7-oxa-tricyclo[6.3.1.01,6]dodec-9-en-11-ol (pacifenol) (8), isolated as a colourless viscous oil; 1H and 13C NMR data were found to be identical to that previously reported (Argandoa et al., 1993; Kaiser et al., 2001); ESIMS (negative mode) m/z 464 [M+Cl]. 4,4-dibromo-5-ethyl-5-((Z)-pent-2-en-4-ynyl)octahydro[2,2]-bifuran (elatenyne) (10); isolated as a yellow oil; 1H and 13C NMR spectroscopic data see Tables 1 (CDCl3) and 2 (C6D6); GC/ MS(EI) m/z (rel. int.) 329 (13), 327 (21), 325 (10) [C10H15Br2O2]+; 247 (100), 245 (95) [C10H15BrO2]+; 179 (72), 177 (70) [C5H6BrO2]+; 151 (25), 149 (28); 105 (24); 97 (12); 69 (5); 55 (5); 43 (13); 41 (53). 1-Bromo-4-(2-chloro-1-ethyl-2-methyl-pentyloxy)-3-methoxy-4,6,6-trimethyl-cyclohexene (cycloelatanene A) (23); isolated EtOH as a colourless viscous oil; a25 D 79.8 (c 0.1, CHCl3); UV kmax (log e) CHCl3 1 256 nm (3.8); IR mmax cm 2927, 2850, 2091, 1735, 1619, 1148, 1371, 1115, 1039; 1H and 13C NMR spectroscopic data see Table 3; HRESIMS m/z 385.0541 [M+Na]+; calc for C16H2479Br35ClNaO2, 385.0546 and m/z 363.0721 [M+H]+; calc for C16H2579Br35ClO2, 363.0648. 1-Bromo-4-(2-chloro-1-ethyl-2-methyl-pentyloxy)-3-epi-methoxy-4,6,6-trimethyl-cyclohexene (cycloelatanene B) (28); isolated EtOH as a colourless viscous oil; a25 D 62.2 (c 0.1, CHCl3); kmax (log e) CHCl3 1 256 nm (3.4); IR mmax cm 2980, 2062, 1733, 1618, 1149, 1381, 1112, 1100; 1H and 13C NMR spectroscopic data see Table 4; HRGC/MS(EI) m/z (rel. int.) 364.0633 [M]+ (calc for C16H2480Br35ClO2, 364.0628). Acknowledgments The Marine And Terrestrial NAtural Product (MATNAP) research group thanks Dr. G. Kraft (University of Melbourne, Victoria, Australia) for the alga identication; Dr. R. Tinker for assistance in the collection of the marine alga; Ms G. Ellis (University of Canterbury, Christchurch, New Zealand) for the biological testing; Ms. S. Duck (School of Chemistry, Faculty of Science, Monash University) for high resolution mass spectrometry analyses; Dr. Julie Niere (RMIT University, Victoria, Australia) for useful NMR discussions;

Please cite this article in press as: Dias, D.A., Urban, S. Phytochemical studies of the southern Australian marine alga, Laurencia elata. Phytochemistry (2011), doi:10.1016/j.phytochem.2011.06.012

D.A. Dias, S. Urban / Phytochemistry xxx (2011) xxxxxx

Ms. S. Khan for assistance with preliminary fractionation of the extract; Dr. J. Burton (Department of Chemistry, University of Oxford, formerly at the University of Cambridge, United Kingdom) for providing invaluable discussions and information with regards to the revised structure of elatenyne and nally we acknowledge the support and award of an Australian Postgraduate Award (APA) scholarship.

References
Argandoa, V.H., San-Martn, A., Rovirosa, J., 1993. Halogenated sesquiterpenes Pacifenol and Pacifenol derivatives on the asphid Schizaphis graminum. Phytochemistry 32, 11591161. mus Aydog , Z., Imre, S., Ersoy, L., Wray, V., 2004. Halogenated secondary metabolites from Laurencia obtusa. Nat. Prod. Res. 18, 4349. Caccamese, S., Compagnini, A., Maria Toscano, R., 1986. Pacifenol from the Mediterranean red alga Laurencia majuscula. J. Nat. Prod. 49, 173174. Dias, D.A., White, J.M., Urban, S., 2009. Laurencia liformis: phytochemical proling by conventional and HPLCNMR approaches. Nat. Prod. Commun. 4, 157172. Dez, D., Nnez, M.G., Moro, R.F., Lumeras, W., Marcos, I.S., Basabe, P., Urones, J.G., 2006. Enantioselective synthesis of cis-3-oxy-2,2,6,6,-tetrasubstituted tetrahydropyrans. Synlett 939, 941. Erickson, K.L., Scheuer, P.J., 1983. Marine Natural Products: Chemical and Biological Perspectives. Academic Press, New York, pp. 131257. Francisco, M.E.Y., Erickson, K.L., 2001. Mailiohydrin, a cytotoxic chamigrene dibromhydrin from a Philippine Laurencia species. J. Nat. Prod. 64, 790791. Fronczek, F., 1989. Redetermination of the absolute conguration of deoxyprepacifenol, from the Mediterranean red alga, Laurencia majuscula. Acta Crystallogr., Sect. C 45, 11021104. Gopichand, Y., Schmitz, F.J., Shelley, J., Rahman, A., Van der Helm, D., 1981. Marine natural products: halogenated acetylenic ethers from the sea hare Aplysia dactylomela. J. Org. Chem. 46, 51925197. Hall, J.G., 1984. Ph.D. Thesis. Latrobe University. Hall, J.G., Reiss, J.A., 1986. Elatenyne-a Pyrano[3,2-b]pyranyl vinyl acetylene from the red alga Laurencia elata. Aust. J. Chem. 39, 14011409. Howard, B.M., Schulte, G.R., Fenical, W., 1980. Three new vinyl acetylenes from the marine red alga Laurencia. Tetrahedron 36, 17471751. In Kyu, K., Brennan, M.R., Erickson, K.L., 1989. Lauroxolanes from the marine alga Laurencia majuscula. Tetrahedron 30, 17571760. Jongaramruong, J., Blackman, A.J., Skelton, B.W., White, A.H., 2002. Chemical relationships between the sea hare Aplysia parvula and the red seaweed Laurencia liformis from Tasmania. Aust. J. Chem. 55, 275280. Kaiser, C.R., Pitombo, L.F., Pinto, A.C., 2001. Complete 1H and 13C NMR assignments of chamigrenes from Aplysia dactilomela. Magn. Reson. Chem. 39, 147149. Kikuchi, H., Suzuki, T., Suzuki, M., Kurosawa, E., 1985. A new chamigrene-type bromo diether from the red alga Laurencia nipponica Yamada. Bull. Chem. Soc. Jpn. 58, 24372438. Kikuchi, H., Suzuki, T., Kurosawa, E., Suzuki, M., 1991. The structure of notoryne, a halogenated C15 terpenoid with a novel carbon skeleton from the red alga Laurencia nipponica Yamada. Bull. Chem. Soc. Jpn. 64, 17631775. Kimura, J., Kamada, N., Tsujimoto, Y., 1999. Fourteen chamigrene derivatives from a red alga, Laurencia nidica. Bull. Chem. Soc. Jpn. 72, 289292. Knig, G.M., Wright, A.D., 1997. Laurencia rigida: chemical investigations of its antifouling dichloromethane extract. J. Nat. Prod. 60, 967970. Kurata, K., Suzuki, T., Suzuki, M., Kurosawa, E., Furusaki, A., Matsumoto, T., 1983a. Laureacetal-D and -E, two new secochamigrene derivatives from the red alga Laurencia nipponica Yamada. Chem. Lett. 557560. Kurata, K., Suzuki, T., Suzuki, M., Kurosawa, E., Furusaki, A., Matsumoto, T., 1983b. Laurenical, a novel sesquiterpene a,b-unsaturated aldehyde from the red alga Laurencia nipponica Yamada. Chem. Lett. 299300.

Lee, U., Park, C.M., Yun, J.S., 1995. Total synthesis of dactomelynes. J. Am. Chem. Soc. 117, 80178018. Nai-Yun, J., Xiao-Ming, L., Ke, L., Bin-Gui, W., 2007. Laurendecumallenes A-B and Laurendecumenynes A-B, halogenated nonterpenoid C15-acetogenins from the marine red alga Laurencia decumbens. J. Nat. Prod. 70, 14991502. Nai-Yun, J., Xiao-Ming, L., Ke, L., Bin-Gui, W., 2009. Halogenated sesquiterpenes from the marine red alga Laurencia saitoi (Rhodomelaceae). Helv. Chim. Acta 92, 18731879. Nai-Yun, J., Xiao-Ming, L., Bin-Gui, W., 2010a. Sesquiterpenes from other metabolites from the marine red alga Laurencia composita (Rhodomelaceae). Helv. Chim. Acta 93, 22812286. Nai-Yun, J., Xiao-Ming, L., Li, K., Bin-Gui, W., 2010b. Laurendecumallenes A-B and Laurendecumenynes A-B, halogenated nonterpenoid C15-acetogenins from the marine red alga Laurencia decumens. J. Nat. Prod. 73, 1192. Ojika, M., Shizuri, Y., Yamada, K., 1982. A halogenated chamigrane epoxide and six related halogen-containing sesquiterpenes from the red alga Laurencia okamurai. Phytochemistry 21, 24102411. San-Martn, A., Darias, J., Soto, H., Contreras, C., Herrera, J.S., Rovirosa, J., 1997. A new C15 acetogenin from the marine alga Laurencia claviformis. J. Nat. Prod. 10, 303 311. Sheldrake, H.M., Jamieson, C., Burton, J.W., 2006. The changing faces of halogenated marine natural products: total synthesis of the reported structures of Elatenyne and an enyne from Laurencia majuscula. Angew. Chem., Int. Ed. 45, 71997202. Sheldrake, H.M., Jamieson, C., Pascu, S.I., Burton, J.W., 2009. Synthesis of the originally proposed structures of elatenyne and an enyne from Laurencia majuscula. Org. Biomol. Chem. 7, 238252. Sims, J.J., Fenical, W., 1972. Marine natural products III. Johnstonol, an unusual halogenated epoxide from the red alga Laurencia johnstonii. Tetrahedron Lett. 3, 195198. Sims, J.J., Fenical, W., Wing, R.M., Radlick, P., 1971. Marine natural products. I: Pacifenol, a rare sesquiterpene containing bromine and chlorine from the red alga, Laurencia pacica. J. Am. Chem. Soc. 33743375. Smith, S.G., Paton, R.S., Burton, J.W., Goodman, J.M., 2008. Stereostructure assignment of exible ve-membered rings by GIAO 13C NMR calculations: prediction of the stereochemistry of elatenyne. J. Org. Chem. 73, 40534062. Suzuki, T., 1980. Two new sesquiterpene alcohols containing bromine from the marine alga, Laurencia nipponica Yamada. Chem. Lett. 541542. Suzuki, T., Kurosawa, E., 1979. New bromo acetal from the marine alga, Laurencia nipponica Yamada. Chem. Lett. 301304. Suzuki, M., Kurosawa, E., Furusaki, A., Matsumoto, T., 1983. The structures of (3Z)epoxyvenustin, (3Z)-venustin, and (3Z)-venustinene, new halogenated C15nonterpenoids from the red alga Laurencia venusta Yamada. Chem. Lett. 12, 779782. Suzuki, M., Segawa, M., Suzuki, T., Kurosawa, E., 1985. Structures of two new halochamigrene derivatives from the red alga Laurencia nipponica Yamada. Bull. Chem. Soc. Jpn. 58, 24352436. Suzuki, M., Takahashi, Y., Matsuo, Y., Masuda, M., 1996. Pannosallene, a brominated C15 nonterpenoid from Laurencia pannosa. Phytochemistry 41, 11011103. Suzuki, M., Daitoh, M., Vairappan, C.S., Abe, T., Masuda, M., 2001. Novel halogenated metabolites from the Malaysian Laurencia pannosa. J. Nat. Prod. 64, 597602. Usami, Y., 2009. Recent synthetic studies leading to structural revisions of marine natural products. Mar. Drugs 7, 314330. Vairappan, C.S., Suzuki, M., Ishii, T., Okino, T., Abe, T., Masuda, M., 2008. Antibacterial activity of halogenated sesquiterpenes from Malaysian Laurencia spp.. Phytochemistry 69, 24902494. Waraszkiewicz, S.M., Erickson, K.L., 1974. Halogenated sesquiterpenes from the Hawaiian marine alga Laurencia nidica: Nidicene and Nididiene. Tetrahedron Lett. 23, 20032006. Watanabe, K., Umeda, K., Miyakado, M., 1989. Isolation and identication of three insecticidal principles from the red alga Laurencia nipponica Yamada. Agric. Biol. Chem. 53, 25132515. Wright, A.D., Knig, G.M., De Nys, R., Sticher, O., 1993. Seven new metabolites from the marine red alga Laurencia majuscula. J. Nat. Prod. 56, 394401.

Please cite this article in press as: Dias, D.A., Urban, S. Phytochemical studies of the southern Australian marine alga, Laurencia elata. Phytochemistry (2011), doi:10.1016/j.phytochem.2011.06.012

You might also like