Isolation of Norsesterterpenes and Spongian Diterpenes from

Article pubs.acs.org/jnp

Isolation of Norsesterterpenes and Spongian Diterpenes from Dorisprismatica (= Glossodoris) atromarginata Ken W. L. Yong,† I. Wayan Mudianta,†,‡ Karen L. Cheney,§ Ernesto Mollo,⊥ Joanne T. Blanchfield,*,† and Mary J. Garson*,† †

School of Chemistry and Molecular Biosciences and §School of Biological Sciences, The University of Queensland, Brisbane QLD 4072, Australia ‡ Department of Analytical Chemistry, Faculty of Mathematics and Natural Sciences, Ganesha University of Education, Bali 81161, Indonesia ⊥ Consiglio Nazionale delle Ricerche, Istituto di Chimica Biomoleculare, Via Campi Flegrei 34, 80078 Pozzuoli (Na), Italy S Supporting Information *

ABSTRACT: Ten new norscalarane metabolites (1−10) with the mooloolabene skeleton in which the C-8 methyl substituent of a scalarane is replaced by a C-7/C8 double bond are described from the nudibranch Doriprismatica (= Glossodoris) atromarginata and characterized by extensive 1D and 2D NMR studies, together with MS data. Also isolated was the known scalarane 12-deacetoxy-12-oxodeoxoscalarin together with 26 furanoterpenes, nine of which (11−19) are reported for the first time. The high diversity of chemical compounds and variation between individuals and locations could reflect a varied sponge diet or an enzymatic detoxification mechanism.

P

C-8 methyl substituent of a scalarane framework. The norscalarane compounds are named as mooloolabenes F−O (1−10) after the collection site (Mooloolaba) of the sponge Hyatella intestinalis, from which the first example of mooloolabene structures (A−E) were identified by our group in 2006.8 The sponge taxonomy has since been revised to Spongia sp. #1997 on the advice of Dr. John N. A. Hooper from the Queensland Museum. In addition, 26 furanoditerpenes, nine (11−19) of which are new, and the known scalarane sesterterpene 12-deacetoxy-12-oxo-deoxoscalarin (20)5 were isolated from D. atromarginata.

opulations of the nudibranch species Doriprismatica atromarginata (syn Glossodoris1 family Chromodorididae) collected from Indo-Pacific locations are known to contain furanoditerpenes of the spongian series. Six new spongian furanoditerpenes were reported by de Silva et al. from eight specimens of D. atromarginata (formerly referred to as Casella atromarginata) collected in Sri Lanka, 2 together with spongiadiol diacetate and spongiatriol triacetate previously isolated from an Australian Spongia sp.3 On the basis of only this chemical evidence, the authors speculated that the sponge Spongia sp. was most likely the preferred diet of D. atromarginata. Fontana et al. isolated five new spongian diterpenes from an Egyptian population of D. atromarginata along with four known spongian diterpenes including epispongiadiol and epispongiatriol triacetate;4 this mollusk specimen was later reassigned as Glossodoris cincta.5 In contrast, five specimens of D. atromarginata collected from India provided cytotoxic scalarane sesterterpenes together with the known sponge metabolite heteronemin.5 We previously reported on the chemistry of a small population (n = 5) of D. atromarginata collected from Mooloolaba, South East Queensland, which yielded two new spongian metabolites along with five known derivatives, with one of the new diterpenes containing a C-3 keto group.6,7 We now report that investigation of a large collection of D. atromarginata made in the same location as the original collection has yielded norsesterterpene metabolites lacking the © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSIONS A collection of 23 specimens (crawling length 1−2 cm each) of D. atromarginata from Mudjimba Island and the Gneerings Reef (Mooloolaba) was extracted with acetone, then partitioned into diethyl ether. Silica flash chromatography (CH2Cl2/ EtOAc) followed by RP-HPLC (MeOH/H2O) and then repetitive NP-HPLC (hexanes/EtOAc) gave nine new norscalaranes, named mooloolabenes F−N (1−9). The structures and relative configurations of the new norsesterterpenes were elucidated based on 2D NMR spectroscopic Special Issue: Special Issue in Honor of William Fenical Received: October 12, 2014

A

DOI: 10.1021/np500797b J. Nat. Prod. XXXX, XXX, XXX−XXX

B

4.71, br ddd (11.8, 2.3, 2.3) 4.66, br ddd (11.8, 1.4, 1.4) 0.87, s 0.92, s

20a

a

1.00, ddd (13.1, 13.1, 3.7) 1.52, m 1.43, m 1.42, m 1.16, ddd (13.7, 13.7, 3.7) 1.10, dd (12.1, 4.3) 1.99, m 1.92, m 5.41, br d (6.4) 1.72, m 1.57, m 1.35, m

1.52, m

1.07, s 0.71, s

1.08, s 1.14, s

4.67, br d (11.6)

4.72, br ddd (11.6, 2.4, 2.4)

2.84, br m

m m m br m

m m m br m

0.84, s 0.63, s 2.65, d (5.2)

0.87, s 0.92, s

4.20, br d (11.4)

2.23, br m 5.24, dd (5.5, 5.2) 4.52, br d (11.4)

1.92, 2.17, 1.94, 5.54,

1.35, m

1.33, ddd (13.3, 13.3, 3.9)

2.01, 2.25, 2.01, 5.79,

1.91, m

2.22, br d (18.5) 2.03, m 5.47, br d (5.9) 1.80, m 1.67, m 1.44, dddd (13.3, 13.3, 13.3, 3.4) 2.62, ddd (13.3, 3.4, 3.4)

1.53, m

2.71, ddd (14.6, 14.6, 5.3) 2.27, ddd (14.6, 3.7, 3.7)

1.83, br d (13.1)

3b

2.16, br d (12.5)

2c

m m br d (7.9) m m m

m m m br m

0.98, 3.90, 3.51, 0.82, 0.63, 2.67,

s d (11.1) d (11.1) s s br

4.20, br d (11.8)

4.52, br d (11.8)

2.23, br m 5.25, d (5.1)

1.92, 2.16, 1.93, 5.54,

1.35, m

1.91, m

2.06, 1.93, 5.40, 1.76, 1.58, 1.35,

1.05, ddd (12.8, 12.8, 4.8) 1.49, m 1.46, m 1.88, m 0.95, ddd (13.2, 13.2, 3.9) 1.28, m

1.87, m

4b


m m m br m

s d (10.9) d (10.9) s s d (5.0) 2.06, s

0.97, 4.35, 3.98, 0.84, 0.63, 2.64,

4.21, br d (11.3)

4.52, br d (11.3)

2.23, br m 5.24, t (5.0)

1.92, 2.16, 1.92, 5.54,

1.34, m

1.91, m

2.09, 1.94, 5.40, 1.76, 1.58, 1.35,

1.78, m 1.00, ddd (12.7, 12.7, 4.1) 1.28, m

1.04, ddd (12.8, 12.8, 4.8) 1.46, m

1.88, m

5b

br md dd (12.5, 5.4) m m

1.10, 0.68, 2.64, 2.04,

s s m s

0.98, s 1.12, s

4.21, br d (11.3)

4.52, br d (11.3)

2.25, br m 5.25, d (3.7)

1.38, ddd (13.5, 13.5, 3.4) 1.95, m 2.19, br m 1.95, m 5.54, br md

1.95, m

5.58, 1.73, 1.63, 1.47,

5.55 md

1.09, ddd (13.0, 13.0, 3.2) 1.63, m 1.45, m 1.40, br d (13.2) 1.21, ddd (13.2, 13.2, 3.2) 1.29, m

1.87, br d (13.0)

6c

0.88, s 0.92, s

0.88, s 0.93, s

4.64, dd (16.9, 1.3) 4.54, d (16.9)

2.62, ddd (13.3, 2.3, 2.3) 1.25, ddd (13.3, 13.3, 4.3) 1.90, m 1.98, m 1.57, m 2.40, dd (19.0, 5.9) 2.31, ddd (19.0, 6.9, 4.2)

0.99, ddd (13.1, 13.1, 3.1) 1.51, m 1.43, m 1.43, m 1.16, ddd (13.2, 13.2, 3.4) 1.09, dd (12.1, 4.2) 1.99, m 1.92, m 5.40, br d (6.0) 1.74, br m 1.62, m 1.52, m

1.83, br d (13.1)

7c

Chemical shifts (ppm) referenced to CHCl3 (δC 7.26). bAt 500 MHz. cAt 750 MHz. dUnresolved chemical shifts due to overlapping signals.

23 24 19-OH 6-OCOCH3 22OCOCH3

21 22

20b

0.84, s 0.68, s

2.82, br m

18 19

16

1.99, 2.22, 2.00, 5.77,

m m m br m

2.58, ddd (12.7, 3.2, 3.2) 1.37, m

14 15

12

7 9 11


1.98, 1.92, 5.42, 1.73, 1.58, 1.37,

6

5

3

2

1.84, dddd (13.1, 2.7, 2.7, 2.7, 2.7) 0.99, ddd (13.1, 13.1, 3.7) 1.49, m 1.43, m 1.41, br d (13.3) 1.16, ddd (13.3, 13.3, 3.9) 1.09, dd (12.0, 4.4)

1b

1

position

Table 1. 1H NMR Assignments for Mooloolabenes 1−10a

m m br m m m m

s d (11.0) d (11.0 s s

2.06, s

0.97, 4.35, 4.00, 0.89, 0.97,

4.64, dd (16.9, 1.2) 4.58, d (16.9)

1.90, m 1.97, md 1.58, m 2.41, dd (19.0, 5.7) 2.39, ddd (19.0, 6.8, 4.4)

2.64, ddd (12.9, 3.0, 3.0) 1.26, m

2.10, 1.97, 5.40, 1.79, 1.63, 1.52,

1.79 1.00, ddd (13.7, 13.7, 3.8) 1.28, m

1.04, ddd (13.4, 13.4, 4.0) 1.45−1.52d

1.87, m

8c

br d (12.2) br dd (13.5, 7.0) m dd (19.0, 5.9)

2.04, s

1.15, s 0.95, s

0.98, s 1.13, s

4.59, d (17.0)

4.65, dd (17.0, 1.3)

1.93, 1.98, 1.61, 2.41,

1.30, m

s d, (10.9) d (10.9) s s

2.06, s

0.96, 4.34, 3.99, 0.84, 0.68,

4.65−4.70, m

2.82 br s


2.30, ddd (19.0, 6.8, 4.3)

1.99, 1.95, 5.41, 1.77, 1.56, 1.33,

2.59, ddd (12.7, 3.4, 3.4) 1.37, dd (12.7, 2.7) 1.99. m 2.23, m 2.00, m 5.78, br s

dt (5.9, 2.0) br m m m

m m m m 1.07, m

1.47, 1.44, 1.44, 1.25,

1.02, m

1.89, m

10b

2.67, ddd (13.2, 2.6, 2.6)

5.59, 1.75, 1.74, 1.67,

5.56, br m

1.08, ddd (13.2, 13.2, 3.8) 1.62, m 1.46, ddd (13.8, 3.5, 3.5) 1.41, br d (13.2) 1.21, ddd (13.2, 13.2, 2.7) 1.28, m

1.87. br d (13.2)

9c

Journal of Natural Products Article


Journal of Natural Products

Article

Table 2. 13C NMR Assignments for Mooloolabenes 1−10a position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 6-OCOCH3 6-OCOCH3 22-OCOCH3 22-OCOCH3 a

1b 40.0, 19.0, 42.3, 32.9, 49.8, 23.9, 120.7, 137.2, 53.3, 35.8, 20.3, 37.5, 35.0, 45.3, 25.9, 121.1, 129.8, 51.2, 175.5, 70.0, 33.7, 22.5, 15.2, 12.3,

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

2c 38.3, 34.8, 217.0, 47.5, 51.4, 25.8, 120.3, −d 52.5, 35.8, 20.3, 37.3, 35.2, 45.2, 24.2, 121.0, −d 51.1, −d 70.2, 25.7, 22.7, 14.9, 12.3,

3b CH2 CH2 C C CH CH2 CH CH C CH2 CH2 C CH CH2 CH CH CH2 CH3 CH3 CH3 CH3

40.0, 18.9, 42.2, 32.8, 49.7, 23.8, 120.3, 137.6, 53.2, 35.7, 20.3, 40.0, 33.9, 45.3, 26.1, 117.1, 136.0, 59.2, 99.9, 69.0, 33.6, 22.5, 15.1, 12.9,

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

4b 39.9, CH2 18.6, CH2 35.4, CH2 37.9, C 50.9, CH 23.3, CH2 120.2, CH 138.5.C 53.4, CH 35.7, C 20.6, CH2 39.0, CH2 34.0, C 45.3, CH 26.1, CH2 117.0, CH 136.0, C 59.2, CH 99.9, CH 69.0, CH2 26.9, CH3 65.4, CH2 16.3, CH3 12.9, CH3

5b 39.8, 18.6, 36.2, 35.5, 50.8, 23.4, 120.0, 137.5. 53.2, 35.5, 20.5, 38.9, 33.9, 45.2, 26.1, 116.9, 136.1, 59.2, 99.9, 69.1, 27.5, 67.0, 16.2, 13.0,

6c

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

171.4, C 21.0, CH3

42.0, CH2 19.0, CH2 44.8, CH2 33.7, C 52.4, CH 67.8, CH 119.0, CH 142.8.C 53.5, CH 35.7, C 20.5, CH2 38.7, CH2 34.2, C 45.2, CH 26.0, CH2 116.9, CH 136.0, C 59.0, CH 99.6, CH 69.0, CH2 33.0, CH3 24.5, CH3 17.2, CH3 13.2, CH3 170.7, C 21.9, CH3

7c 40.0, 18.7, 42.2, 32.8, 50.2, 23.4, 119.6, 137.5, 52.6, 35.8, 19.9, 33.5, 34.8, 48.3, 20.3, 24.4, 159.4, 134.4, 172.6, 70.8, 33.7, 22.3, 15.1, 17.8,

8c CH2 CH2 CH2 C CH CH2 CH C CH C CH2 CH2 C CH CH2 CH2 C C C CH2 CH3 CH3 CH3 CH3

39.8, 18.4, 36.3, 36.1, 51.2, 23.5, 119.3, −d 52.8, 35.7, 20.3, 33.6, 34.7, 48.3, 20.3, 24.5, 159.3, 134.4, 172.5, 70.9, 27.5, 67.0, 16.4, 17.8,

9c CH2 CH2 CH2 C CH CH2 CH CH C CH2 CH2 C CH CH2 CH2 C C C CH2 CH3 CH2 CH3 CH3

42.2, 19.0, 44.8, 33.6, 52.7, 67.7, 118.2, 142.8, 53.1, 36.0, 20.6, 33.7, 35.0, 48.3, 20.4, 24.5, 159.4, 134.1, 172.5, 71.0, 33.4, 24.6, 17.4, 17.9, 170.8, 22.2,

171.3, C 21.1, CH3

10b CH2 CH2 CH2 C CH CH CH C CH C CH2 CH2 C CH CH2 CH2 C C C CH2 CH3 CH3 CH3 CH3 C CH3

39.5, 19.0, 42.3, 36.1, 50.5, 23.9, 120.3, 137.2, 53.2, 35.6, 20.3, 37.3, 34.9, 45.0, 25.9, 120.9, 129.8, 51.1, 175.5, 70.0, 27.3, 66.9, 16.2, 12.3,

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

171.5, C 21.1, CH3

Chemical shifts (ppm) taken from 2D spectra referenced to CDCl3 (δC 77.16). bAt 500 MHz. cAt 750 MHz. dNot detected.

irradiation of H-16 at δH 5.77 showed enhancement of H2-15 at δH 2.22/2.00 and H-14 at δH 1.99, while HMBC correlations from H2-20 to C-16, C-17, C-18, and to C-19 at δC 175.5 confirmed the γ-lactone moiety. The relative configuration followed from NOESY correlations between H-5/H-9, H-9/H14, H-14/H-18, and H-18/H-20a on one face of the molecule, and between H3-22/H3-23 and H3-23/H3-24 on the other face. The NOE correlation between H3-23/H3-24 corresponded to an interproton distance of 2.8 Å (estimated by molecular modeling with ChemBio 3D Ultra software (Cambridge)) and was also noted in the NOESY data of the other mooloolabenes isolated in the current study. The [α]D value of 1 was −35.4, and by comparison with the value of −6.4 for the synthetic sesterterpenoid 21,5 the absolute configuration shown for 1 was suggested. Apart from the C-7/C-8 double bond, 1 is closely related to 21. Enantiospecific syntheses of other marine sesterterpenes with the scalarane skeleton have so far verified the same absolute configuration as for 1 and 21.9 The remaining mooloolabenes isolated in this study were assigned the same absolute configurations as 1. Mooloolabene G (2), isolated as a colorless oil, had a molecular formula of C24H32O3 by HRESIMS. The 1H and 13C NMR data displayed four methyl singlets (δH 0.71, 1.07, 1.08, and 1.14), two olefinic protons (δH 5.47 and 5.78), and an AB system (δH 4.67, br d, J = 11.6 Hz and 4.72, br d, J = 11.6 Hz; (δC 70.2) that were all closely similar to the signals in 1, but showed a keto group (δC 217.0) located at C-3 by HMBC correlations from H3-21 and H3-22. Owing to the small quantity of 2 available, full 13C data could not be obtained from the 2D NMR experiments, and assignments for the methine and methylene protons were made by selective 1D TOCSY

analysis and HRESIMS data. Subsequently, collections of D. atromarginata from various S.E. Queensland dive sites were extracted individually using the same protocol, and one such specimen collected at Mudjimba Island yielded an additional norsesterterpene named mooloolabene O (10). Also isolated from the pooled collection was the known sesterterpenoid 12deacetoxy-12-oxodeoxoscalarin (20)5 together with a diverse array of furanoditerpenes, including eight new examples with the spongian diterpene skeleton (11−18). One other new furanoditerpene, 19, characterized from an individual specimen of D. atromarginata, was also detected in the pooled extract. A summary of the known furanoditerpenes isolated is provided in the Supporting Information. Mooloolabenes. Mooloolabene F (1) was isolated as a colorless oil and had a molecular formula of C24H34O2 inferred from HRESIMS. The 1H NMR and HSQC-edited data (Table 1) showed the presence of four methyl singlets (δH 0.68, 0.84, 0.87, and 0.92) and two olefinic signals (δH 5.42 and 5.77; δC 120.7 and 121.1) that were suggestive of a norsesterterpene skeleton,8 as well as an AB system (δH 4.71, br d, J = 11.6 Hz and 4.66, br d, J = 11.6 Hz; δC 70.0) and other signals. The 13C NMR spectrum (Table 2) showed 24 carbons, including nonprotonated carbons at δC 137.2, 129.8, and 175.5. The planar structure was completed by 2D NMR experiments and by comparison with literature data,8 which enabled all resonances to be assigned. The C-7/C-8 alkene was assigned by HMBC correlations from H-7 at δH 5.42 to C-5, C-9, and C14 (δC 49.8, 53.3, and 45.3), from H-5 (δH 1.09) and H-14 (δH 1.99) to C-7, and from H2-11 (δH 1.58 and 1.37) to C-8 at δC 137.2. The upfield shifts of H3-23 and H3-24 were consistent with the loss of a γ-methyl substituent.8 Selective 1D TOCSY C



Article

ester carbonyl at δC 171.4, established that 5 is the 22-acetate derivative of 4. The 13C chemical shift for Me-21 at δC 27.5 indicated an equatorial orientation. NOESY correlations observed between H2-22/H3-23, H3-23/H3-24, H3-24/H-19, and H-14/H-18 were all consistent with the relative configuration shown, as was the 5.0 Hz coupling between H18 and H-19. Mooloolabene K (6), a white, amorphous solid, had the same molecular formula as 5 by HRESIMS. The 1H NMR data were comparable with those of 3 except for signals in the vicinity of C-6. An oxymethine proton at δH 5.54 showed HMBC correlations to C-5, C-7, C-8, and C-10 and to an ester carbonyl at δC 170.5; this, together with HMBC correlations from an acetyl group at δH 2.04 to the same ester carbonyl, confirmed a C-6 acetate, thus matching the ring B substitution pattern of mooloolabene D (23) previously reported by our group from Spongia sp.8 The 3J coupling of 3.7 Hz observed between H-18/H-19, although smaller than the 5 Hz coupling observed in 3−5, together with a NOESY correlation between H-19/H3-24 supported the trans arrangement of H-18 and H19. NOESY correlations from H-6 to both H-5 and the equatorial H3-21 (δC 33.0) together with correlations from both H3-22 and H3-23 to the C-6 acetate methyl indicated that H-6 was equatorial. Mooloolabene L (7) was isolated as a white, amorphous solid with a molecular formula of C24H34O2 and thus was isomeric with 1. Signals for H2-20 at δH 4.64 and 4.54 showed HMBC correlations to C-17, C-18, and a carbonyl at δC 172.6, while other signals closely resembled those of scalarolide from Spongia idia;12 together, these data identified an α,βunsaturated lactone in ring E. Although the 1H chemical shifts of two of the four methyl groups were identical (δH 0.88, 6H), they could be assigned to C-21 and C-23 based on the corresponding 13C chemical shifts observed at δC 33.7 (C-21) and at δC 15.1 (C-23) in the HSQC spectrum, respectively. NOESY correlations were consistent with the relative configuration shown. Mooloolabene M (8) was isolated as a colorless, amorphous solid with a molecular formula of C26H36O4 by HRESIMS, again suggesting an acetate substituent. In the 1H NMR, signals for an alkene proton (δH 5.40) and an AB system (δH 4.58 and 4.64) were similar to those of 7, suggesting the same lactone skeleton. There were also an acetyl methyl (δH 2.06) and three methyl signals (δH 0.97, 0.92, and 0.89) plus a second AB system (δH 4.35 and 4.00) that showed HMBC correlations to C-3, C-4, Me-21, and an ester carbonyl at δC 171.3 for a C-22 acetate substituent, as in 4. The axial C-22 methylene showed an NOE correlation with H3-23, while a chemical shift of δC 27.5 confirmed that Me-21 was equatorial. Mooloolabene N (9), isolated as a colorless, amorphous solid, was an isomer of 8, based on the identical molecular formula of C26H36O4 inferred from the HRESIMS. Diagnostic signals for an olefinic proton (δH 5.59), an AB system (δH 4.59 and 4.65), and a Δ7,8,17,18-norscalarane fused lactone skeleton were similar to those in 7 and 8. Signals for an acetyl methyl (δH 2.04) and an oxymethine proton (δH 5.56), showing HMBC correlations to C-5, C-7, C-8, C-10, and a carbonyl at δC 170.8, revealed that 9 had the same C-6 acetate substituent as 6. There were NOESY correlations from the equatorial H321 (δC 33.4) to both H-5 and H-6 and from the C-6 acetate signal at δH 2.04 to H3-22 and H3-23, as well as from H3-23 to H3-24.

experiments. In selective 1D NOE experiments, NOEs from H3-22 at δH 1.14 to H3-23 at δH 1.07, from H-9 to H-5, and from H-18 to H-14 were consistent with the proposed configuration, as were the chemical shifts for C-13, C-14, and C-24. Mooloolabene G is structurally related to 3-keto scalaranes isolated previously from the nudibranch Chromodoris inornata10 and from the sponge Hyrtios erecta.11 Mooloolabene H (3) was isolated as a white, amorphous solid with a molecular formula of C24H36O2 from HRESIMS. The presence of four methyl singlets (δH 0.63, 0.84, 0.87, and 0.92), two alkenes (δH 5.41and 5.54), and an AB system (δH 4.52 and 4.20; δC 69.0) in the 1H and 13C NMR spectra established the mooloolabene framework. Signals for a hemiacetal moiety (δH 5.24, dd, J = 5.5, 5.2 Hz; δC 99.9) adjacent to a hydroxy signal at δH 2.65 (d, J = 5.5 Hz) were similar to data for (−)-12-deacetoxy-12-oxo-deoxoscalarin (20).5 There were HMBC correlations from the hemiacetal methine proton to the signals assigned to C-13, C-18, and C20, from the hydroxy proton to both C-18 and C-19, and from H2-20 to C-16 and C-17. NOESY data confirmed that 3 had the same relative configuration as 1 and 2. NOESY correlations between H-14 and H-18 and between H-19 and H3-24 suggested a trans arrangement of H-18 and H-19. The 3 JH‑18/H‑19 of 5.2 Hz for 3 supports a trans configuration by analogy with the equivalent value of 5.0 Hz observed in the scalarane 22, for which the relative configuration has been secured by an X-ray study.11 Mooloolabene I (4) was obtained as a colorless, amorphous solid with a molecular formula of C24H36O3 by HRESIMS that indicated an additional oxygen atom compared to 3. The 1H NMR data showed two olefinic protons (δH 5.54 and 5.40), a hemiacetal (δH 5.25), an AB system (δH 4.52 and 4.20), and other signals that corresponded to those of 3. The presence of three methyl singlets (δH 0.63, 0.82, and 0.98) together with a second AB system (δH 3.90 and 3.51) suggested that 4 differed from 3 by the replacement of a methyl group by a −CH2OH moiety. HMBC correlations from these methylene signals to C4 and C-21 confirmed a hydroxy substituent at C-22. The NOESY correlation between H2-22/H3-23 identified an axial orientation of C-22, with the carbon chemical shift for C-21 (δC 26.9) consistent with its equatorial orientation.6,8 Mooloolabene J (5) was obtained as a white, amorphous solid, and the molecular formula of C26H38O4 by HRESIMS suggested acetate substitution. In its 1H NMR spectrum, 5 showed three methyl singlets (δH 0.63, 0.84, and 0.97), two AB systems (δH 3.98 and 4.35; δH 4.21 and 4.52), and an acetyl singlet at δH 2.06. HMBC correlations from the methylene signals at δH 4.35 and 3.98 (H2-22) and from the methyl group at δH 0.97 to C-3, C-4, and C-5, as well as from H2-22 to an D



Article

Table 3. 1H NMR Assignments for Furanoditerpenes 11−19a 11b

12b

2.75, d (12.7)

2.62, d (12.1)

2.49, d (15.0)

2.13, d (12.7)

2.20, d (12.1)

2.25, d (15.0)

3

3.94, s

4.93, d (0.9)

4.99, s

5

1.71, dd (11.9, 1.8) 1.86, m

1.67, md

1.53, md

1.79, md 1.68, md 2.19, md

1.64, md

1.68, md 1.48, md

1.64, md 1.41, dd, (11.0, 2.2) 1.64−1.68, md

position 1

13b

2

6 7

2.15, md

9

2.19, ddd (12.9, 3.2, 3.2) 1.63, md 1.50, md

11

1.67, md

1.65, md

12

2.82

15 16 17

2.48 7.11, d (1.3) 7.07, q (1.3) 1.22, s

2.80, ddd (15.6, 3.5, 3.5) 2.47, md 7.11, d (1.3) 7.07, q (1.3) 1.22, s

2.47, 7.11, 7.07, 1.24,

18 19

1.32, s 4.02, d (12.0)

1.11, s 0.89, s

0.99, s 1.01, s

2.80, ddd (16.3, 5.5, 5.5) md d (1.3) q (1.3) s

3.94, d (12.0) 20 2-OCOCH3 3-OCOCH3 19OCOCH3 2-OH 3-OH a

0.92, s

0.91, s

1.14, s

2.19, s

2.14, s

14b 2.33, dd (14.5, 3.4) 1.23, m 4.14, q (3.9)

15c

17b

1.35, br m 5.25, q (3.9)

2.37, dd (15.0, 3.3) 1.28, md 5.33, q (3.4)

3.39, dd (3.8, 1.1) 1.09, dd (11.8, 2.2) 1.74, md 1.55, md 2.13, md

3.42, br s

3.57, md

1.16, md

1.25, md

1.94, md

1.79, md 1.69, md 2.15, md

1.55, md 1.13, dd (11.5, 1.5) 1.82, md 1.66, m 2.79, br dd (16.3, 6.2)

1.55, md 1.17, md

1.82, md 1.52, md 2.54, ddd (13.2, 3.0, 3.0) 1.32, md 1.37, dd (12.2, 1.8) 1.77, md 1.67, md 2.79, br dd (16.6, 6.3)

2.44, 7.07, 7.04, 1.20,

2.44, 7.07, 7.04, 1.22,

m d (1.5) q (1.5) s

1.27, s 4.72, d (10.5) 3.54, dd (10.5, 1.1) 1.19, s

2.04, s

2.26, br m

16c

1.74, md 1.65, md 2.78, br dd (16.2, 6.2) md d (1.5) q (1.5) s

md br s br s t (10.8 d (10.8) s d (11.1)

1.20, s 4.73, d (11.5)

2.51, 7.17, 7.16, 3.77, 3.47, 1.26, 4.61,

4.25, d (11.5)

3.56, d (11.1)

1.13, s 2.11, s

1.13, s 2.08, s

6.60, s

18c

19b

2.37, t (12.0)

2.65, d (18.6)

1.62, md 4.58, ddd (12.0, 6.9, 4.0)

2.19, d (18.6)

4.69, d (4.9) 1.39, m

1.72, md

1.90, dd (11.8, 4.4) 1.62, md

2.19, md

2.12, md

2.49, m

1.65, md 1.50, md

1.61, md 1.39, dd (10.2, 3.3) 1.68, md

1.30, m 1.47−1.52, md

2.80, br dd (16.2, 6.0)

2.82, dd (16.4, 6.1)

1.92, md 1.65, md 2.83, br dd (16.1, 6.2) 2.51, 7.09, 7.07, 1.28,

m d (1.5) q (1.5) s

1.37, s 3.94, d (11.1) 3.61, dd (11.1, 1.1) 1.21, s

2.48, 7.11, 7.07, 1.19,

md d (1.3) q (1.3) s

1.16, s 1.17, s

1.70−1.80, md

1.64−1.78. md

2.52, 7.20, 7.18, 3.86, 3.50, 0.85, 4.16,

m br s br s t (10.9) d (10.9) s d (11.6)

4.07, d (11.6) 0.80, s

2.09, s

1.30, s

2.12 5.86, s

3.60, d (4.0) 3.39, d (4.9)

Chemical shifts (ppm) referenced to CHCl3 (δC 7.26). bAt 500 MHz.. cAt 750 MHz. dUnresolved chemical shifts due to overlapping signals.

with the presence of an acetate moiety. The 1H NMR spectrum was reminiscent of that of 1, except for the presence of only three methyl signals and an acetyl methyl group at δH 2.06. The individual protons of an AB methylene system (δH 4.33 and 3.99) each showed HMBC correlations to C-4 at δC 36.1 and to an acetoxy carbonyl at δC 171.5; hence 10 was a C-22 acetate derivative of mooloolabene F. NOESY correlations between H22a and H3-23 indicated that C-22 was axially oriented. The carbon chemical shift of C-21 (δC 27.3) verified its equatorial orientation. Traces of 10 were also identified in HPLC fractions from the pooled sample of D. atromarginata. Spongian Diterpenes. Compound 11, obtained as a colorless, amorphous solid after RP-HPLC, gave the molecular formula C22H30O5 by HRESIMS, corresponding to the monoacetate derivative of a spongian diol.2,4,6,13 The planar structure was deduced by 1H and 13C NMR data (Tables 3 and 4), including 2D experiments, and by comparison with literature data that enabled all resonances to be assigned. In the 1H NMR spectrum, in addition to the acetate methyl (δH 2.04) there were two α-furan signals (δH 7.11 and 7.07) and three methyl groups (δH 0.92, 1.22, and 1.32). The chemical shift of an oxymethine proton at δH 3.94 was diagnostic of H-3

In a parallel study, separate Et2O extracts were prepared from D. atromarginata specimens collected from two different diving sites (Mudjimba Island, Mooloolaba, and Shag Rock, North Stradbroke Island) in S.E. Queensland. The 1H NMR spectra of two individuals collected from Mudjimba Island and one individual from Shag Rock were consistent with the presence of furanoditerpenes; however the extract of a third specimen (#240) from Mudjimba Island lacked furan signals, instead containing alkene signals diagnostic for mooloolabenes. Chemical investigation of this specimen afforded the new norsesterterpene mooloolabene O (10) along with mooloolabenes D and F and mooloolaldehyde.8 Mooloolabene F (1) was found as the major component, whereas mooloolabenes D (23) and O (10) and mooloolaldehyde (24) were obtained as minor components. Individual specimens (n = 4) of D. atromarginata from a third location, Nelson Bay (New South Wales), were found to contain mooloolabene metabolites alone within the limits of detection provided by 1H NMR analysis of the extracts, but no detailed chemical studies were undertaken to identify individual metabolites owing to the small sample size. Mooloolabene O (10) was isolated as a colorless oil, and the molecular formula of C26H36O4 from HRESIMS was consistent E



Article

Table 4. 13C NMR Assignments for Furanoditerpenes 11−19a position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 2-OCOCH3 2−OCOCH3 3-OCOCH3 3-OCOCH3 19-OCOCH3 19-OCOCH3 a

11b 53.2, 209.6, 82.2, 47.8, 55.3, 19.5, 41.0, 34.4, 56.3, 43.1, 18.7, 20.6, 119.3, 136.5, 135.1, 136.9, 25.8, 24.2, 64.6, 16.8,

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

12b 54.1, 203.8, 84.1, 43.6, 55.8, 18.8, 40.6, 34.5, 55.9, 43.2, 18.5, 20.6, 119.3, 136.7, 135.1, 136.9, 26.0, 29.0, 17.4, 17.1,


170.5, C 20.6, CH3

13b 53.5, 206.7, 82.8, 41.0, 52.3, 19.6, 40.3, 34.4, 56.4, 41.5, 19.0, 20.8, 119.3, 136.5, 135.2, 137.0, 25.8, 26.3, 21.9, 18.2,


14b 43.1, 70.8, 80.2, 41.8, 55.9, 18.5, 41.3, 34.2, 56.9, 36.7, 18.6, 20.7, 119.7, 137.2, 135.1, 136.9, 26.1, 23.9, 66.9, 17.8,

15c

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

41.5, 72.8, 77.5, 41.9, 55.5, 18.7, 41.0, 34.1, 56.5, 37.0, 18.5, 20.4, 119.4, 137.1, 134.8, 136.7, 26.0, 23.2, 66.7, 17.3, 171.0, 21.2,

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

16c 40.5, 70.9, 77.5, 38.0, 55.1, 18.0, 35.1, 40.6, 57.2, 36.6, 17.9, 20.1, 119.6, 129.6, 137.3, 138.3, 62.2, 25.3, 66.7, 18.3, 170.0, 21.4,

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

17b 129.1, 144.4, 200.5, 49.3, 54.5, 19.1, 40.6, 34.7, 51.6, 38.7, 18.8, 20.5, 119.2, 136.7, 134.8, 136.8, 26.7, 21.9, 64.8, 21.1,

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

18c 51.0, 68.6, 219.2, 45.5, 51.9, 20.0, 39.5, 33.9, 55.1, 36.9, 19.3, 20.8, 119.5, 136.4, 136.6, 137.1, 25.1, 29.7, 19.6, 18.6,

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

19c 54.0, 212.1, 75.2, 44.9, 54.7, 19.6, 34.2, -d 56.8, 38.4, 18.7, 20.5, 119.4, 129.2, 137.4, 138.3, 62.0, 20.7, 66.8, 20.6,

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

170.2, C 20.9, CH3

170.7, C 21.0, CH3

171.2, C 20.9, CH3

170.7, C 21.2, CH3

Chemical shifts (ppm) taken from 2D spectra referenced to CDCl3 (δC 77.16). bAt 500 MHz. cAt 750 MHz. dNot detected

showed HMBC correlations to C-3 and to a carbonyl at δC 203.8 (C-2). HMBC correlations from H-3 to an ester carbonyl at δC 170.5 placed the acetate substituent at C-3. The substitution pattern of 12 was identical to epispongiadiol diacetate3 except for the lack of the 19-acetate substituent. The 1 H NMR spectrum of 13 was closely analogous to that of 12, except that the H-3 signal appeared at δH 4.99, suggestive of 3αacetoxy substitution.3 The conclusion that 12 and 13 were epimers was further reinforced by 13C NMR data (C-3: δC 84.1 for 12 vs δC 82.8 for 13). Literature trends reveal that the C-3 chemical shift in 3β-substituted spongian diterpenes is deshielded relative to the value for the 3α-substituted equivalent.3,13,14 In 12, NOESY cross-peaks observed between H-3/H-1a, H-3/H-5, H-1a/H-5, and H-1a/H-9 established both a chair conformation in the A ring and the equatorial C-3 acetate group. In contrast in 13, a boat conformation for the A ring was inferred from the NOE between H-3/H3-20. The C-3 acetate substituent was pseudoequatorial. Similar conformational differences (3β-hydroxy: chair vs 3α-hydroxy: boat) have been noted previously in NOE data for epimeric spongian diterpenes.15 In their original publication on spongian diterpenes, Kazlauskas et al. reported the triacetate derivative of spongiatriol as having a ring A boat conformation by X-ray analysis.3 However, in an X-ray study, we found that only 29% of the conformational population of spongiadiol (26) is in a distorted boat conformation, with the 3-OH in an equatorial orientation, whereas 71% of the conformational population adopts the chair conformation; in contrast, epispongiadiol (25) adopts a ring A chair conformation.16 Compound 14, isolated as a white, amorphous solid, had a molecular formula of C20H30O4 by HRESIMS; as expected for a diterpene triol, there were three methyl groups (δH 1.19, 1.20,

in 3β-hydroxy-substituted spongian diterpenes (H-3: δH 3.97 for epispongiadiol 25 vs δH 4.71 in spongiadiol 26).3 The chemical shifts of an AB system at δH 4.02 and 3.94 supported a 19-acetoxy substituent,3,4,13 as did HMBC correlations from Me-18 at δH 1.32 to C-3 at δC 82.2 and C-4, C-5, and C-19 at δC 64.6 and correlations from H-19a to C-5, C-6, C-18, and an ester carbonyl (δC 170.7). The diagnostic H-1 signals (δH 2.11 and 2.73) showed HMBC correlations to the C-2 keto (δC 209.4) as well as to C-3, C-5, and C-10. NOESY correlations were observed between H-1b/H-3 and H-3/H-5, consistent with a chair conformation of ring A, and between H2-19/H3-20 and H3-20/H3-17. These data confirmed 11 as a new epispongiadiol derivative, 3β-hydroxy-19-acetoxyspongia13(16),14-dien-2-one.

Compounds 12 and 13 both had the molecular formula C22H30O4 from HRESIMS. The 1H NMR spectrum of 12 showed an acetate methyl at δH 2.19 as well as four methyl signals at δH 0.89, 0.91, 1.11, and 1.22. The oxymethine H-3 at δH 4.93, diagnostic of a 3β-acetoxy substituent,3,4,13 was linked by HSQC to a carbon at δC 84.1, which showed HMBC correlations from H3-18 and H3-19. The AB methylene system (δH 2.62 and 2.20), assigned to H2-1 by comparison with 11, F



Article

and 1.27). In the 1H NMR spectrum, signals at δH 4.14 and at δH 3.39 were similar to signals at δH 4.02 (H-2) and δH 3.28 (H-3) in 2β,3β,17,19-tetrahydroxyspongia-13(16),14-diene (27).13 The presence of an oxygenated AB system (δH 4.72 and 3.54) showing HMBC correlations to C-3, C-4, and C-18, indicated a 19-OH substituent. There were also HMBC correlations from H3-17 at δH 1.20 to C-7, C-8, and C-14 and from H3-18 at δH 1.27 and H2-19 to C-3. NOESY correlations observed between H-3 and H-5 and between H219/Me-20 suggested a chair conformation for the A ring. The small coupling values (3JH‑2/H‑3 3.8 Hz) observed between H-2 and H-3,14,15 in contrast to the 9.7 Hz coupling reported for a synthetic analogue in which H-2 and H-3 were trans,3,17 placed H-2 in an equatorial orientation. Compound 15 was isolated as a colorless, amorphous solid of molecular formula C24H34O6 by HRESIMS. The majority of 1H NMR signals for the tetracyclic skeleton were similar to those in 14, apart from the changes expected for the presence of two acetate groups. HMBC correlations from H-2 at δH 5.25 to C-3 at δC 77.5, C-4, C-10, and an ester carbonyl at δC 171.0 confirmed an acetoxy substituent at C-2. HMBC correlations from H3-18 at δH 1.20 and from H2-19 to C-3, C-4, and a carbonyl at δC 171.2 confirmed C-19 was acetoxy-substituted. Selective 1D NOE irradiation of H-3 showed an enhancement of H-1b and H-5, consistent with a ring A chair conformation. The cis arrangement of substituents at C-2 and C-3 followed from the NOE between H-2/H-3 and the 3JH‑2/H‑3 of 3.8 Hz. Compound 16, isolated as a white, amorphous solid, had a molecular formula of C22H32O6 from HRESIMS and was therefore a monoacetate derivative of a tetraol. The 1H/13C NMR data revealed that there were two oxymethylene systems (C-17: δH 3.77 and 3.47, δC 62.2; C-19: δH 4.61 and 3.56, δC 66.7), two oxymethine centers (C-2: δH 5.33, δC 70.9 and C3:3.57, δC 77.5), and two methyl singlets (δH 1.26 and 1.13). These data were also reminiscent of signals for 2β,3β,17,19tetrahydroxyspongia-13(16),14-diene (27),14 except for the presence of an acetate signal at δH 2.08, which explained the downfield shift of H-2. HMBC correlations from H-2 to C-4, C-10, and an acetate carbonyl (δC 170.0) confirmed the acetoxy substituent at C-2.4 There were also correlations from H3-18 at δH 1.26 to C-3, C-4, C-5, and C-19, from H2-19 to C-3, C-4, and C-18, from the methylene protons (δH 2.37 and 1.28) assigned to C-1 to C-2, C-3, C-5, C-10, and C-20, and from H217 to C-7 and C-8. All other HMBC correlations were fully consistent with the furanoditerpene skeleton of 16. The small coupling value (∼3 Hz) between H-2 and H-3 confirmed their cis relationship. NOESY correlations between H-2/H-18, H-2/ H-3, H-3/H3-18, H2-17/H3-20, and H2-19/H3-20 were all consistent with the proposed relative configuration.4,14 Compound 17 was isolated as a white, amorphous solid with a molecular formula of C20H26O4 by HRESIMS. The 1H NMR data were closely related to those of 17,19-dihydroxyspongia13(16),14-diene-2,3-dione (28) previously reported from G. atromarginata,2 as well as from Spongia sp.,18 and showed three methyl singlets (δH 1.21, 1.28, and 1.37) and an oxygenated AB system (δH 3.94 and 3.61) positioned at C-19 by HMBC correlations to C-3 and C-18. An alkene proton at δH 6.60 was assigned to H-1 by HMBC correlations to C-2 at δC 144.4, to a carbonyl at δC 200.5 assigned to C-3, and to C-5, C-9, and C20. An exchangeable proton signal at δH 5.86 showed HMBC correlations to C-1 and to C-2 and so confirmed the enolized α-diketone moiety in the A ring.2 In the NOESY spectrum,

there were correlations between H-5/H-9, H2-19/Me-20, and H3-17/Me-20. Compound 18 was isolated as a colorless, amorphous solid, with a molecular formula of C20H28O3 by HRESIMS. The 1H NMR data were closely similar to those of isospongiadiol (29) from a Caribbean Spongia sp.17 and to the data for the epimeric pair of 2,17-dihydroxyspongia-13(16),14-dien-3-ones (30, 31) from a West Australian Spongia sp., 15 except for the replacement of their C-17 hydroxymethylene by a methyl group. In 18, a carbonyl at δC 219.2 was assigned to C-3 based on HMBC correlations from Me-18 and Me-19. A hydroxy group was identified at C-2 since H-2 at δH 4.58, linked to a carbon at δC 68.6, was coupled to an exchangeable signal at δH 3.60 (J = 4.0 Hz). Both the hydroxy proton and H-2 showed HMBC correlations to C-3, and there was a correlation from H-1 at δH 1.60 to C-2. Searle and Molinski15 reported 3JH‑1/H‑2 values close to 12 Hz in both 30 and 31, implying a ring A conformation for each epimer such that H-2 is axial. In 18, H-2 was also axial from the 3JH‑1a/H‑2 of 12.0 Hz. Selective 1D experiments revealed NOEs between H-2/H-5, H-2/H3-18, H5/H-9, H-5/H3-18, H3-20/H3-17, and H3-20/H3-19. These data indicated a twist-boat conformation for ring A, as noted before in the X-ray structure of the related spongia-13(16),14dien-3-one (32)16 and in contrast to the solution conformation of 29, for which a chair conformation was deduced from the NOE between H-2 and H3-20.17 In the twist-boat conformation indicated for 18, the internuclear distance between H-2/H-5 was estimated as 2.46 Å by modeling studies; the 2-hydroxy substituent was close to coplanar with the keto group. Compound 18, named 2β-hydroxyspongia-13(16),14-dien-3one, decomposed on storage. During our field collections, D. atromarginata was frequently encountered on sponge substrates. Analysis of samples collected in January 2005 yielded spongian diterpenes from both D. atromarginata and the small piece of sponge on which the mollusks were found.6 In November 2008, two specimens of D. atromarginata were found on Spongia sp. #1997. Mooloolabenes A, C, and D were isolated from the sponge tissue, while the furanoterpenes spongiadiol, epispongiadiol, and epispongiadiol acetate and traces of other spongian diterpenes were found in the mantle tissue of the mollusk, and mooloolabene F was found in the gut tissue of the mollusk. In June 2009, a single specimen of D. atromarginata was found feeding on an unidentified sponge. 1H NMR investigation of both sponge and mollusk tissue extracts revealed that each extract contained furanoditerpenes. The sponge tissue contained spongia-13(16),14-dien-3-one, epispongiadiol, and spongiadiol as the major metabolites, among other furan diterpenes. Analysis of the gut contents of the mollusk yielded the same major furanoditerpenes as in the sponge extract, while the mantle tissue provided spongiatriol, epispongiatriol, and the new metabolite 19. We had previously detected both spongiadiol and epispongiatriol in the mantle tissue of D. atromarginata, while a fully acetylated diterpenoid was also detected in the mantle glands.6 Spongian diterpene 19 was isolated as an amorphous solid with a molecular formula of C22H30O6 by HRESIMS. Apart from the characteristic NMR signals for a furan and for a keto group (δC 212.1) assigned to C-2 by HMBC correlations from H2-1, there were two methyl signals and two oxymethylene systems (C-17: δH 3.86 and 3.50; δC 62.0; C-19: δH 4.16 and 4.07; δC 66.8), suggesting that C-17 and C-19 were hydroxyand acetoxy-substituted, respectively. There were HMBC G



Article

solutions in CHCl3. IR spectra were recorded on a PerkinElmer 400 FT-IR spectrophotometer for solutions in CHCl3. NMR spectra were recorded at ambient probe temperature on a Bruker Avance 500 or a Bruker Avance 750 spectrometer. All NMR spectra were run in CDCl3 and referenced to solvent signals at δH 7.26 (1H) and δC 77.16 (13C). Positive ion electrospray mass spectra (LRESIMS) were determined using a Bruker Esquire HCT (or HRESIMS) using a MicrOTOF Q instrument each with a standard ESI source. Normal-phase flash column chromatography was performed using silica gel 60 (40−63 μm; Scharlau). Normal-phase HPLC was performed using a Waters 515 pump connected to a Gilson 132 series refractive index detector with a Waters μPorasil (10 μm, 7.8 × 300 mm) column. Separations were performed using isocratic solvent conditions using premixed, filtered, and degassed mobile phases. Reversed-phase HPLC was carried out on an Agilent 1100 series instrument fitted with a variablewavelength UV and refractive index detector, an Agilent D1311A quaternary pump, and a semipreparative Phenomenex C18 Gemini 5 μ 110 Å column (10 mm × 250 mm) or a Phenomenex Gemini 5 μ C18 column (4.6 × 250 mm). Biological Material. Specimens of Dorisprismatica atromarginata (23 animals) were collected from dive sites at Mudjimba Island and at the Inner Gneerings Reef, Mooloolaba, in South East Queensland while scuba diving at a depth of 10−15 m during December 2004 and combined into a single collection prior to analysis. Collections of mollusks and associated sponge material were made at the Inner Gneerings Reef (November 2008, two mollusks; June 2009, one mollusk). These samples were all transported to the University of Queensland on ice and stored at −20 °C until extraction. Mollusk collections made at Shag Rock (September 2012, one specimen) and Mudjimba Island (October 2012, three mollusks) were transported live to the University of Queensland, then stored at −20 °C until extraction (individual specimens). Four specimens of D. atromarginata from Nelson Bay, NSW, collected in November 2013 were transported on ice to the University of Queensland and stored at −20 °C prior to individual extraction. Extraction and Purification. The pooled collection of D. atromarginata (wet wt ∼25 g; crawling size 1−2 cm each) was extracted by crushing the whole animals, followed by sonication in acetone (20 mL) twice. The extract was removed, then evaporated under reduced pressure to give an aqueous residue, which was then partitioned with Et2O (20 mL). The organic layer was removed, dried with anhydrous Na2SO4, and then concentrated under reduced pressure to give 70 mg of a dark yellow oil. The extract was then chromatographed by RP-HPLC (Phenomenex C18 Gemini 5 μ 110 Å column (10 mm × 250 mm); flow rate 1.5 mL/min; UV detection at 230 nm) using 50−100% MeOH/H2O over 40 min and then 100% MeOH isocratic for another 20 min to afford 16, 17, diterpene mixture A, 11, diterpene mixture B, 18, 15, 12, 13, norsesterterpene mixture C, and norsesterterpene mixture D in order of elution. The residual fraction contained norsesterterpene mixture E. Diterpene mixture B was rechromatographed separately by RP-HPLC (flow rate 1.0 mL/ min; UV detection at 220 nm; Phenomenex Gemini 5 μ C18 4.6 × 250 mm) using 50−80% MeOH/H2O over 60 min to afford 14. The norsesterterpene mixture C was rechromatographed on NP-HPLC (Waters 10 μ μPorasil 7.8 × 300 mm column; flow rate 2.0 mL/min; RI detection) using EtOAc/hexanes (1:9) to afford compounds 8 and 9 in order of elution. The norsesterterpene mixture D was rechromatographed by NP-HPLC using isocratic EtOAc/hexanes (15:85) to afford compounds 1, 7, and 3 in order of elution. Finally the norsesterterpene mixture E from the residual fraction was rechromatographed by NP-HPLC using isocratic EtOAc/hexanes (1:4) to afford a fraction containing traces of 10 followed by 2, 5, 6, 20, and 4 in order of elution. A specimen of D. atromarginata collected in June 2009 was separated into gut and mantle tissue by dissection. Each tissue sample was extracted in a similar fashion to give crude extracts, 2.0 and 5.0 mg, respectively, and the mantle extract was purified by RP-HPLC (flow rate 1.0 mL/min; UV detection at 230 nm; Phenomenex Gemini 5 μ C18 4.6 × 250 mm using 50−100% MeOH/H2O over 60 min) to afford 19 (0.6 mg).

correlations from H-19 to C-3, C-4, C-18, and an ester carbonyl at δC 170.7 and from H-17 to C-7 and C-14. The chemical shift of C-14 at δC 129.2 is diagnostic of a 17-hydroxy-substituted furanoditerpene4,14,15 (cf. the 13C NMR data for 16). The chemical shift of H-3 at δH 4.69 established that the hydroxy group in 19 was α-oriented by comparison with data for spongiatriol and derivatives.2,3 The suite of nudibranch metabolites isolated in both this and our earlier study6 display considerable variation in their substitution patterns. α-Diketones, or their enolized equivalents, are likely to be involved in the interconversion of spongian diterpenes. Oxidation of either epispongiadiol (25) or spongiadiol (26) could give a diketo intermediate, which equilibrates to the enolized diterpenoid (17). Selective reduction would then yield the 3-keto diterpene (e.g., 18). 2,3-Dioxygenated metabolites (such as 14−16) may derive from reduction of either the diketones or their 3-keto equivalents. A noticeable feature of spongian diterpene chemistry is the oxidation of the axial methyl groups at C-17 and C-19, likely catalyzed by P450 enzymes. The diverse chemistry shown between individuals of D. atromarginata could reflect their individual sponge diets. Differences in chemistry between mollusks collected from different locations must reflect local environmental conditions and the availability of individual sponges at these collection sites. The presence of mooloolabenes F−O in D. atromarginata, compared to mooloolabenes A−E in the sponge source, is suggestive of an enzymatic detoxification mechanism similar to that proposed previously for related species from the Glossodoris genus. 19 Only mooloolabene D (23) and mooloolaladehyde (24) were isolated from both D. atromarginata and Spongia sp. #1997.

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CONCLUSIONS Nine new norsesterterpene scalaranes, mooloolabenes F−N (1−9), and a known sesterterpene, 12-deacetoxy-12-oxodeoxoscalarin (20), were isolated from a large pooled sample of D. atromarginata collected near Mooloolaba in S.E. Queensland, in addition to a large number of furanoditerpenes, eight of which (11−18) were previously unreported. One additional mooloolabene, O (10), and a further furanoditerpene metabolite (19) were isolated from individual specimens of D. atromarginata collected near Mooloolaba. Animals collected at Nelson Bay, New South Wales, contained mooloolabenes alone, within the detection limits of our analyses. There were differences in chemistry in individual specimens of D. atromarginata, as well as differences in chemistry between mollusks collected from different locations. Our study is a significant step toward mapping the diversity of chemical constituents present in D. atromarginata, as earlier studies2,4,6 had found only either spongian diterpenoids or sesterterpenes in this mollusk. Our observations clearly support the presumed dietary relationship between D. atromarginata and the local sponge fauna, as the mooloolabenes are a unique set of metabolites that have so far been reported from a single sponge source, Spongia sp. #1997 collected at Mooloolaba. A detailed ecological study of the chemical defenses encountered in D. atromarginata will be published separately.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured at 589 nm at ambient temperature using a 1 mL quartz cell (10 cm path length) with a JASCO P-2000 polarimeter for H



Article

Spongian diterpene 16: amorphous solid (0.6 mg); [α]D −27 (c 0.04, CHCl3); 1H NMR (CDCl3, 750 MHz), Table 3; 13C NMR (CDCl3, 750 MHz), Table 4; HRESIMS m/z 415.2090 (calcd for C22H32NaO6, 415.2091). Spongian diterpene 17: amorphous solid (0.5 mg); [α]D −20 (c 0.03, CHCl3); 1H NMR (CDCl3, 500 MHz), Table 3; 13C NMR (CDCl3, 500 MHz), Table 4; HRESIMS m/z 353.1714 (calcd for C20H36NaO4, 353.1723). Spongian diterpene 18: amorphous solid (0.4 mg); [α]D +27 (c 0.03, CHCl3); 1H NMR (CDCl3, 750 MHz), Table 3; 13C NMR (CDCl3, 750 MHz), Table 4; HRESIMS m/z 339.1920 (calcd for C20H28NaO3, 339.1931). Spongian diterpene 19: amorphous solid (0.6 mg); [α]D +27 (c 0.03, CHCl3); 1H NMR (CDCl3, 500 MHz), Table 3; 13C NMR (CDCl3, 500 MHz), Table 4; HRESIMS m/z 413.1980 (calcd for C22H30NaO6, 413.1935).

Specimens of D. atromarginata that were collected from Shag Rock (Gold Coast) and Mudjimba Island (Sunshine Coast) in 2012 were individually extracted in a similar fashion to the above pooled collection. Specimen #240 (length 15 mm) from Mudjimba Island gave a crude extract (4.4 mg), which was subjected to NPHPLC using 9:1 (hexanes/EtOAc) as eluent to provide mooloolabene F (1, 0.5 mg), mooloolabene O (10, 0.3 mg), mooloolaldehyde (24, 0.1 mg), and mooloolabene D (23, 0.2 mg). Mooloolabene F, 1: amorphous solid (0.6 mg); [α]D −35 (c 0.04, CDCl3); IR (CHCl3) νmax 2922, 1774, 1124, 1008 cm−1; 1H NMR (CDCl3, 500 MHz), Table 1; 13C NMR (CDCl3, 500 MHz), Table 2; HRESIMS m/z 377.2446 [M + Na]+ (calcd for C24H34NaO2, 377.2451). Mooloolabene G, 2: amorphous solid (0.2 mg); [α]D +17 (c 0.01, CHCl3); 1H NMR (CDCl3, 750 MHz), Table 1; 13C NMR (CDCl3, 750 MHz), Table 2; HRESIMS m/z 391.2239 [M + Na]+ (calcd for C24H32NaO3, 391.2244). Mooloolabene H, 3: amorphous solid (0.4 mg); [α]D −46 (c 0.03, CHCl3); 1H NMR (CDCl3, 500 MHz), Table 1; 13C NMR (CDCl3, 500 MHz), Table 2; HRESIMS m/z 379.2621 [M + Na]+ (calcd for C24H36NaO2, 379.2608). Mooloolabene I, 4: amorphous solid (0.9 mg); [α]D −5 (c 0.06, CHCl3); 1H NMR (CDCl3, 750 MHz), Table 1; 13C NMR (CDCl3, 750 MHz), Table 2; HRESIMS m/z 395.2566 [M + Na]+ (calcd for C24H36NaO3, 395.2557). Mooloolabene J, 5: amorphous solid (0.5 mg); [α]D −19 (c 0.04, CHCl3); 1H NMR (CDCl3, 500 MHz), Table 1; 13C NMR (CDCl3, 500 MHz), Table 2; HRESIMS m/z 437.2660 [M + Na]+ (calcd for C26H38NaO4, 437.2662). Mooloolabene K, 6: amorphous solid (0.2 mg); [α]D −140 (c 0.02, CHCl3); 1H NMR (CDCl3, 750 MHz), Table 1; 13C NMR (CDCl3, 750 MHz), Table 2; HRESIMS m/z 437.2664 [M + Na]+ (calcd for C26H38NaO4, 437.2662). Mooloolabene L, 7: amorphous solid (1.0 mg); [α]D −24 (c 0.06, CHCl3); 1H NMR (CDCl3, 750 MHz), Table 1; 13C NMR (CHCl3, 750 MHz), Table 2; HRESIMS m/z 377.2450 [M + Na]+ (calcd for C24H34NaO2, 377.2451). Mooloolabene M, 8: amorphous solid (0.5 mg); [α]D −130 (c 0.03, CHCl3); 1H NMR (CDCl3, 750 MHz), Table 1; 13C NMR (CHCl3, 750 MHz), Table 2; HRESIMS m/z 435.2506 [M + Na]+ (calcd for C26H36NaO4, 435.2506). Mooloolabene N, 9: amorphous solid (1.2 mg); [α]D −59 (c 0.08, CHCl3); 1H NMR (CDCl3, 750 MHz), Table 1; 13C NMR (CDCl3, 750 MHz), Table 2; HRESIMS m/z 435.2506 [M + Na]+ (calcd for C26H36NaO4, 435.2506). Mooloolabene O, 10: colorless oil (0.3 mg); [α]D −64 (c 0.02, CHCl3); IR (CHCl3) νmax 2927, 1772, 1737, 1371, 1239 cm−1; 1H NMR (CDCl3, 500 MHz), Table 1; 13C NMR (CDCl3, 500 MHz), Table 2; HRESIMS m/z 435.2497 [M + Na]+ (calcd for C26H36NaO4 435.2506). Spongian diterpene 11: amorphous solid (0.3 mg); [α]D +21 (c 0.02, CHCl3); 1H NMR (CDCl3, 500 MHz), Table 3; 13C NMR (CDCl3, 500 MHz), Table 4; HRESIMS m/z 397.1975 (calcd for C22H30NaO5, 397.1985). Spongian diterpene 12: amorphous solid (0.5 mg); [α]D −27 (c 0.04, CHCl3); 1H NMR (CDCl3, 500 MHz), Table 3; 13C NMR (CDCl3, 500 MHz), Table 4; HRESIMS m/z 381.2039 (calcd for C22H30NaO4, 381.2036). Spongian diterpene 13: amorphous solid (0.5 mg); [α]D −27 (c 0.03, CHCl3); 1H NMR (CDCl3, 750 MHz), Table 3; 13C NMR (CDCl3, 750 MHz), Table 4; HRESIMS m/z 381.2027 (calcd for C22H30NaO4, 381.2036). Spongian diterpene 14: amorphous solid (0.3 mg); [α]D −16 (c 0.02, CHCl3); 1H NMR (CDCl3, 500 MHz), Table 3; 13C NMR (CDCl3, 500 MHz), Table 4; HRESIMS m/z 357.2042 (calcd for C20H30NaO4, 357.2036). Spongian diterpene 15: amorphous solid (0.7 mg); [α]D −33 (c 0.05, CHCl3); 1H NMR (CDCl3, 750 MHz), Table 3; 13C NMR (CDCl3, 750 MHz), Table 4; HRESIMS m/z 441.2259 (calcd for C24H34NaO6, 441.2248).

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ASSOCIATED CONTENT

S Supporting Information *

Figures S1−S21. Structures of known furanoditerpenes isolated from D. atromarginata and NMR data for mooloolabenes 1−10 and spongian diterpenes 11−19. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*(M. J. Garson) Tel: +61-7-3365 3605. Fax: +61-7-3365 4273. E-mail: [email protected]. *(J. T. Blanchfield) Tel: +61 7 3365 3622. Fax: +61 7 3365 4299. E-mail: j.blanchfi[email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Australian Research Council, The University of Queensland, for financial support, including postgraduate scholarships to K.W.L.Y. and I.W.M. We thank Dr. M. Somerville, Dr. R. Swasono, and Ms. A. Winters for assistance with field collections and/or preliminary sample preparation. Dr. T. Le and Ms. L. Lambert assisted with NMR measurements, while Dr. A. Piggott and Mr. G. McFarlane undertook high-resolution MS measurements. We thank Prof. R. Capon for access to the JASCO P-2000 polarimeter. The mollusk collection was made under appropriate permit with the assistance of ScubaWorld and SunReef Diving, both of Mooloolaba. We thank G. Cobb for permission to use an image of D. prismatica from the website “Nudibranchs of the Sunshine Coast” (http://www.nudibranch.com.au/).

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DEDICATION Dedicated to Dr. William Fenical of Scripps Institution of Oceanography, University of California−San Diego, for his pioneering work on bioactive natural products.

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REFERENCES

(1) Bouchet, P.; Caballer, M. (2014). Glossodoris atromarginata (Cuvier, 1804). See the World Register of Marine Species at http:// www.marinespecies.org/aphia.php?p=taxdetails&id=214507. (2) Dilip de Silva, E.; Scheuer, P. J. Heterocycles 1982, 17, 167−170. (3) Kazlauskas, R.; Murphy, P. T.; Wells, R. J.; Noack, K.; Oberhänsli, W. E.; Schönholzer, P. Aust. J. Chem. 1979, 32, 867−880. (4) Fontana, A.; Mollo, E.; Ricciardi, D.; Fakhr, I.; Cimino, G. J. Nat. Prod. 1997, 60, 444−448. (5) Fontana, A.; Cavaliere, P.; Ungur, N.; D’Souza, L.; Parameswaram, P. S.; Cimino, G. J. Nat. Prod. 1999, 62, 1367−1370. I



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Isolation of Norsesterterpenes and Spongian Diterpenes from

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