Phorbasins G-I: Three new diterpenoids from the sponge Phorbas gukulensis

Article abstract of DOI:10.1248/cpb.56.1198

Phorbasins G-I (1-3) were isolated from the sponge Phorbas gukulensis collected from Gagu-Do, Korea. The complete structure of the compounds was determined by NMR and MS spectroscopy, along with chemical reaction. Phorbasin G (1) was found to be a diterpene possessing a taurine residue.

Full text of DOI:10.1248/cpb.56.1198

1198
Notes
Chem. Pharm. Bull. 56(8) 1198—1200 (2008)
Vol. 56, No. 8
Phorbasins G—I: Three New Diterpenoids from the Sponge Phorbas
gukulensis
Hyi-Seung LEE,a Su Young PARK,b Chung Ja SIM,c and Jung-Rae RHO*,b
a Marine Natural Products Laboratory, Korea Ocean Research & Development Institute; Ansan P.O. Box 29, Seoul
425–600, Korea: b Department of Oceanography, Kunsan National University; Jeonbuk 673–701, Korea: and c Department
of Biological Sciences, College of Life Sciences and Nano Technology, Hannam University; Daejeon 305–811, Korea.
Received April 7, 2008; accepted May 19, 2008; published online May 26, 2008
Phorbasins G—I (1—3) were isolated from the sponge Phorbas gukulensis collected from Gagu-Do, Korea.
The complete structure of the compounds was determined by NMR and MS spectroscopy, along with chemical
reaction. Phorbasin G (1) was found to be a diterpene possessing a taurine residue.
Key words phorbasin G; phorbasin H; phorbasin I; Phorbas gukulensis; taurine residue; 2 dimensional NMR
Biologically active compounds with unusual functional H-2, -3 and -4 to the carbonyl carbon at C-20 (d 178.8) pro-
groups have been isolated from marine sponges over the last vided the attachment of the carbonyl group at the C-3 posi-
four decades.1) In our search for bioactive natural products tion of the cyclohexane. Next, the remaining molecular for-
from marine sponges, three new diterpenoids were isolated mula, C2H5NO3SNa, was determined to be constituted by
from the extract of the sponge Phorbas gukulensis with the two methylenes, an amine and three heteroatoms (Na, S, O)
cytotoxic gagunin A—D compounds reported previously.2) In based on the 13C-NMR chemical shift values and the COSY
the literature survey, metabolites from the genus Phorbas spectrum. An ethylene unit was readily assigned by two
were reported to feature heterocyclic phorboxazoles,3) phor- triplet proton signals at d 3.57 and 2.94 which were not ac-
bazoles,4) phobasterones,5) and diterpene phorbasins.6,7) commodated in diterpene, and linked with the given diter-
Though we could not isolate these compounds from our penoid through an amide bond on the basis of the HMBC
specimen, our three compounds are structural analogs with correlation of the methylene protons at d 3.57 (2H, t, Jꢂ
skeletons similar to diterpene phorbasins and are character- 6.8 Hz) with the carbonyl carbon at C-20 (d 178.8). An
ized by modified functional groups. Here we describe the amide moiety was supported by the typical absorption band
isolation and structure determination of the three diterpene at 1637 and 1453 cmꢁ1 in the IR spectrum and the chemical
phorbasins G (1), H (2) and I (3).
shift value of C-2ꢃ (d 51.4) suggested a sulfur-bearing car-
Phorbasin G (1) was isolated in a yellowish gum and de- bon, not an oxygen-bearing one. Thus, the molecular for-
termined to have a molecular formula of C22H36NO4SNa on mula, C2H5NO3SNa, was elucidated to be a taurine residue.8)
the basis of high resolution FAB-MS ([MꢀNa]ꢀ peak at m/z
The geometry of the double bonds for 1 was determined
456.2159). An FAB tandem MS spectrum of 1 also showed by the large coupling constants, Jꢂ15.1 Hz for the olefinic
intense fragment ion peaks at m/z 372, 319, 286, 224, 169, protons at C-9, -10 and the NOE correlations on H-9/H-18,
140, and 126. The IR spectrum exhibited strong characteris- H-9/H-19, H-8/H-10, and H14/H-16, indicating that all of the
tic absorption bands at 1210 cmꢁ1 (SꢂO stretching), 1039 double bonds had the (E) configurations. And 1,4-symmetri-
cmꢁ1 (asymmetric OꢂSꢂO stretching), as well as a peak at cal cyclohexane was formed to be the chair form from the
1637 cmꢁ1 (CꢂO stretching). This suggested the presence of NOE correlations on H-3/H1b, H-3/H5b and H-6/H2a,
a sulfonic acid salt, together with a fragment ion at m/z 126 H-6/H4a. In addition, to determine the configuration at C-
[SO3Na2]ꢀ in the positive FAB tandem MS. The 13C-NMR 11, a chemical degradation was performed.9) Treatment of
spectrum displayed 20 distinct signals: four methyls, six 1 with NaIO4 in the presence of RuCl3 as a catalyst gave
methylenes, seven methines, and three quaternary carbons by (S)-2-methylglutaric acid, which was confirmed by both
1
an analysis of both the edited HSQC spectrum and the inte- comparison of the H-NMR spectrum with an authentic
gration of the H-NMR spectrum. Two of these were as- sample and measurement of optical rotation {[a]D24ꢀ23°
1
signed to degenerate methylenes at d 32.0 and 30.7, which (cꢂ0.02, MeOH)}.10) Therefore, the molecular structure of 1
positions were readily recognized by the doubled integration was determined to be sodium 1ꢃ-[{(6-[(7E, 9E)-7,11,15-
ratio of their corresponding protons. And 1 had three double trimethyldeca-7,9,14-trien-7-yl]cyclohexyl)carbonyl}amino]-
bonds (d 141.2, 139.4, 132.1, 126.5, 125.8, and 124.6) and a
carbonyl group (d 178.8).
1
From a detailed analysis of the H–1H COSY and edited
HSQC spectra, in conjunction with a gradient HMBC spec-
trum, 1 was found to contain a diterpene moiety composed
of three substructures: 2,6-dimethylundeca-2,7,9-triene, 1,4-
disubstituted cyclohexane, and a carbonyl group. The linkage
between the linear chain and a symmetrical cyclohexane was
revealed by the HMBC correlations from the methyl protons
at d 1.69 (3H, s) to the neighboring carbons at C-6, -7, and
-8(d 47.9, 141.2, 124.6). And also HMBC correlations from
Fig. 1. Chemical Structures of Phorbasins G—I (1—3) Isolated from the
Sponge Phorbas gukulensis
∗ To whom correspondence should be addressed. e-mail: jrrho@kunsan.ac.kr
© 2008 Pharmaceutical Society of Japan

August 2008
1199
Table 1. NMR Spectral Data of Phorbasins G (1) and I (3) in CD3OD
Phorbasin G (1)
Phorbasin I (3)
Position
dC, mult.
32.0, CH2
30.7, CH2
d
H (J in Hz)
HMBC
dC, mult.
31.9, CH2
30.5, CH2
dH (J in Hz)
1, 5
2, 4
1.76, br dd (12.5, 2.8),
1.31, m
1.89, br d (12.5)
1.48, dd (12.5, 3.4)
2.12, tt (12.5, 3.4)
1.89, m
1.77, br dd (12.9, 3.2)
1.32, qd (12.9, 3.2)
2.03, br dd (12.9, 3.2)
1.45, qd (12.9, 3.2)
2.22, tt, (12.2, 3.7)
1.92, tt, (11.9, 10.8)
2, 4, 6
1, 3, 5
1, 3, 5, 20
2, 4, 20
1, 5
3
6
7
8
9
46.3, CH
47.9, CH
141.2, qC
124.6, CH
126.5, CH
139.4, CH
38.0, CH
38.5, CH2
26.9, CH2
125.8, CH
132.1, qC
25.9, CH3
17.8, CH3
21.4, CH3
15.0, CH3
178.8, qC
36.5, CH2
51.4, CH2
44.5, CH
48.2, CH
142.6, qC
125.3, CH
123.8, CH
137.2, CH
135.3, qC
133.3, CH
27.2, CH2
40.0, CH2
28.9, CH
22.9, CH3
22.9, CH3
12.5, CH3
15.1, CH3
180.2, qC
5.78, d (10.8)
6, 9, 10, 19
7, 8, 11
9,11,18
9, 10, 12, 18
11, 14
12, 14, 15
13, 17
5.88, d (10.8)
6.34, dd (15.1, 10.8)
6.13, d (15.1)
6.20, dd (15.1, 10.8)
5.39, dd (15.1, 8.3)
2.14, m
1.31, m
1.94, dd (14.9, 7.1)
5.08, tq (7.1, 1.2)
10
11
12
13
14
15
16
17
18
19
20
1ꢃ
2ꢃ
5.43, t (7.3)
2.15, dt (7.8, 7.3)
1.26, dt (8.8, 6.8)
1.55, quint (6.6)
0.88, d (6.6)
0.88, d (6.6)
1.76, s
1.66, br s
1.57, br s
0.98, d (6.8)
1.69, s
14, 15, 17
14, 15, 16
10, 11, 12
6, 7, 8
1.75, d
3.57, t (6.8)
2.94, t (6.8)
20, 2ꢃ
1ꢃ
ethanesulfonate.
Unlike the previous phorbasins A—F, phorbasins G—I have
Phorbasin H (2) was isolated in a yellowish gum and es- a symmetrical cyclohexane moiety and a carbonyl group at
tablished as a molecular formula C20H32O2 on the basis of the C-20 position. In particular, phorbasin G (1) was deter-
high resolution FAB-MS and 13C-NMR data. This molecule mined as a diterpenoid containing a taurine residue. Very re-
contained five double bond equivalents and was made up of cently, three other diterpenyl-taurines, containing phorbasins
four methyls, eight methylenes, seven methines, and three D similar to phorbasin G, were reported from an Australian
1
quaternary carbons as analyzed by a combination of the H- marine sponge, Phorbas sp.13)
1
NMR and the edited HSQC spectra. The H- and 13C-NMR
Unfortunately, phorbasins G—I did not exhibit a signifi-
spectra of 2 were highly similar to those of 1, except for the cant activation effect on AMPK, though phorbasins H and I
absence of two methylene signals and a difference in the car- showed a weak effect to a concentration of 10 mM in a West-
bon chemical shift in the carbonyl group. The intense ab- ern blot analysis. We are under collaboration for investiga-
sorption band at 1704 cmꢁ1 in the IR spectrum and the mo- tion into other biological activities.
lecular formula suggested the presence of the carboxyl
Experimental
group. Thus, the structure of 2 produced from the loss of a
taurine moiety could be deduced to be the basic skeleton of
General Optical rotations were measured on a JASCO P-1010 po-
larimeter with a 5 cm cell. UV spectra were obtained in MeOH using a Shi-
1. Accordingly, 2 was determined to be 6-[(7E,9E)-7,11,15-
trimethyldeca-7,9,15-trien-7-yl]cyclohexanecarboxylic acid.
Phorbasin I (3) was obtained as a yellow gum and its mo-
lecular formula was given to be the same as that of 2 by the
high resolution FAB-MS. However, the UV and NMR spec-
madzu UV-1700 and IR spectra were measured on a JASCO FT/IR 4100
spectrometer. All NMR spectra were recorded in a CD3OD solution on a
Varian VNMRS 500 spectrometer. Chemical shifts of the proton and carbon
spectra were reported in reference to residual solvent peaks at 3.30 ppm and
49.0 ppm, respectively. High resolution mass spectra were obtained on a
JEOL JMS-700 spectrometer and mass spectrometric analysis was per-
formed using a four-sector tandem mass spectrometer (JMS-HX110/110A,
JEOL) with the mass resolution set at 1000 (10% valley).
Animal Material The specimens of Phorbas gukulensis (Sample No.
07G-6) were collected by hand using SCUBA at a depth of 30 m in 2007 off
the shore of Gagu-Do at the West Sea, Korea. The sponge had a thick mass;
it measured 110ꢄ95 mm and was 20 mm thick. Oscules were very rare and
the texture was very soft. The color in life was red. In the skeleton, the
megascleres were tornotes (340—420ꢄ6—10 mm), small acanthostyles
(160—195ꢄ5—10 mm), and the large acanthostyles (390—470ꢄ8—
11 mm), and microscleres were isochelas (25—30 mm). A voucher specimen
is deposited at the Natural History Museum, Hannam University, Korea.
Extraction and Isolation The frozen organism (1.2 kg) was cut into
small pieces and extracted twice with MeOH at room temperature. The
methanolic extract (ca. 140 g) was partitioned between the n-BuOH and H2O
layers. The butanolic layer was evaporated under reduced pressure and
repartitioned between n-hexane and 15% aqueous MeOH for defatting. Then
the aqueous MeOH fraction was subjected to reversed-phase silica gel flash
column chromatography eluting with solvents of decreasing polarity
(MeOH/H2Oꢂ50/50, 60/40, 70/30, 80/20, 90/10; 100% MeOH; acetone) to
1
tra of 3 were different from those of 1 and 2. The H-NMR
spectrum of 3 showed a characteristic large methyl peak
corresponding to a dimethyl group, and two broad singlet
methyl signals. The sequential couplings from the dimethyl
protons at d 0.88 to the olefinic proton at d 5.43 were ob-
served in the COSY and TOCSY spectra. From the interpre-
tation of the 2D NMR spectra, the only structural difference
from 2 was the position of one double bond placed between
C-11 and -12. Stereochemical assignment of the D11,12) dou-
ble bond was established as an (E) configuration based on the
NOE correlation on H-10/H-12. Therefore, 3 was determined
to be 6-[(7E,9E,11E)-7,11,15-trimethyldeca-7,9,11-trien-7-
yl]cyclohexanecarboxylic acid.
In the course of finding an activator for the adenosine
monophosphate kinase (AMPK) protein in cells from marine
specimens,11,12) we isolated three new phorbasin compounds.

1200
Vol. 56, No. 8
give seven fractions. Compound 1 was isolated from a 30% aqueous MeOH
fraction by reversed phase HPLC (YMC ODS-A column, 250 mmꢄ10 mm
ture, the solvents were removed under vacuum. The residue was redissolved
in MeOH and filtered. The filtrate was dried and separated by reversed-phase
ID) eluting with 35% aqueous MeOH to yield 5.5mg. Compounds 2 and 3 HPLC (YMC ODS column, 5% aqueous MeOH) to give 0.8 mg of 2-
were obtained from a 100% MeOH fraction after purification on reversed- methylglutaric acid: [a]D ꢀ23° (cꢂ0.02, MeOH); 1H-NMR (CDCl3) d: 2.45
phase HPLC using 10% aqueous MeOH to obtain 5.0 mg and 2.6 mg, re-
spectively.
(1H, m), 2.32 (2H, m), 1.89 (1H, m), 1.72 (1H, m), 1.16 (3H, d, Jꢂ6.8 Hz).
Phorbasin G (1) was obtained as a pale yellowish gum. 1H- and 13C-NMR
data are given in Table 1. [a]D24 ꢀ30.1° (cꢂ0.10, MeOH). UV lmax (MeOH)
Acknowledgments This research was supported by a grant from Marine
Biotechnology Project funded by Ministry of Land, Transport and Maritime
nm (log e): 242 (4.12). IR (film) cmꢁ1: 2930, 2865, 1637, 1453, 1210, 1039. Affairs, and the Brain Korea 21 (BK21) project, Ministry of Education &
HR-FAB-MS m/z: 456.2159 (Calcd for C22H36NO4SNa2 : 456.2157). LR-
FAB-MS/MS m/z: 456.1, 372, 386, 224, 140, 126.
Human Resources Development, Republic of Korea.
Phorbasin H (2) was obtained as a pale yellowish gum. 1H-NMR References
(CD3OD), d: 6.21 (1H, dd, Jꢂ15.1, 11.4 Hz, H-9), 5.80 (1H, br d, Jꢂ
11.4 Hz, H-8), 5.40 (1H, dd, Jꢂ15.1, 8.3 Hz, H-10), 5.09 (1H, tq, Jꢂ7.3,
1.4 Hz, H-14), 2.22 (1H, tt, Jꢂ12.4, 3.7 Hz, H-3), 2.14 (1H, m, H-11), 2.03
(2H, dd, Jꢂ12.4, 3.2 Hz, H-2b, 4b), 1.95 (2H, quint, Jꢂ7.3 Hz, H-13), 1.89
(1H, tt, Jꢂ12.4, 2.8 Hz, H-6), 1.78 (2H, dd, Jꢂ12.4, 2.8 Hz, H-1a, 5a), 1.71
(3H, d, Jꢂ0.9 Hz, H-19), 1.67 (3H, s, H-16), 1.59 (3H, s, H-17), 1.46 (2H,
qd, Jꢂ12.4, 3.7 Hz, H-2a, 4a), 1.34 (2H, qd, Jꢂ12.4, 3.2 Hz, H-1b, 5b),
1.31 (2H, m, H-12), 0.98 (3H, d, Jꢂ6.9 Hz, H-18); 13C-NMR (CD3OD) d:
180.2 (C, C-20), 141.2 (C, C-7), 139.4 (CH, C-10), 132.1 (C, C-15), 126.5
(CH, C-9), 125.8 (CH, C-14), 124.7 (CH, C-8), 48.0 (CH, C-6), 44.5 (CH,
C-3), 38.5 (CH2, C-12), 38.0 (CH, C-11), 31.9 (CH2, C-1, -5), 30.5 (CH2, C-
2, -4), 26.9 (CH2, C-13), 25.9 (CH3, C-16), 21.4 (CH3, C-18), 17.8 (CH3, C-
17), 15.0 (CH3, C-19). [a]D24 ꢀ51.2° (cꢂ0.10, MeOH). UV lmax (MeOH)
nm (log e): 242 (4.31). IR (film) cmꢁ1: 2922, 2858, 1704, 1450. HR-FAB-
MS m/z: 327.2304 (Calcd for C20H32O2Na: 327.2300).
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Phorbasin I (3) was obtained as a pale yellowish gum. H- and 13C-NMR
data are given in Table 1. UV lmax (MeOH) nm (log e): 244 (3.35), 278
(3.71). IR (film) cmꢁ1: 3348, 2940, 1708, 1028. HR-FAB-MS m/z: 305.2481
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(0.012 mmol) of phorbasin G and 21.7 mg (0.101 mmol) of NaIO4 in a mix-
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