Structural and stereochemical studies of α-methylene-γ-lactone- bearing cembrane diterpenoids from a South China Sea soft coral Lobophytum crassum

Article abstract of DOI:10.1021/np800081p

Four new α-methylene-γ-lactone-bearing cembranoids, 20-acetylsinularolide B (6), presinularolide B (7), 3-dehydroxylpresinularolide B (8), and 3-dehydroxyl-20-acetylpresinularolide B (9), together with five known analogues, sinularolides B - E (1-4) and 20-acetylsinularolide C (5), were isolated from a South China Sea soft coral Lobophytum crassum. Their structures and relative stereochemistry were established by a combination of detailed spectroscopic data analysis and chemical correlations. The structures of 1-9 were further confirmed by an X-ray diffraction study on a single crystal of sinularolide B (1). The absolute configurations of sinularolide B (1) and presinularolide B (7) were determined by a novel solid-state CD/TDDFT approach and by a modified Mosher's method, respectively. This study also revealed that the coupling constant between the lactonic methine protons ³J _1,2) varies considerably with different functional groups on the cembrane ring and that the determination of the stereochemistry of lactone ring fusion based on this coupling constant is risky. In a bioassay, sinularolides B and C (1 and 2) and new cembranoids 7 and 8 showed in vitro cytotoxicity against the tumor cell lines A-549 and P-388.

Full text of DOI:10.1021/np800081p

J. Nat. Prod. 2008, 71, 961–966
961
Structural and Stereochemical Studies of r-Methylene-γ-lactone-Bearing Cembrane
Diterpenoids from a South China Sea Soft Coral Lobophytum crassum
Wen Zhang,†,‡ Karsten Krohn,‡ Jian Ding,† Ze-Hong Miao,† Xiu-Hong Zhou,† Si-Han Chen,† Gennaro Pescitelli,§
Piero Salvadori,§ Tibor Kurtan, and Yue-Wei Guo*,†
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Zhang
Jiang Hi-Tech Park, Shanghai, 201203, People’s Republic of China, Department of Chemistry, UniVersity of Paderborn, Warburger Strasse
100, 33098 Paderborn, Germany, Dipartimento di Chimica e Chimica Industriale, UniVersita` di Pisa, Via Risorgimento 35, 56126 Pisa, Italy,
and Department of Organic Chemistry, UniVersity of Debrecen, P.O.B. 20, H-4010 Debrecen, Hungary
ReceiVed February 4, 2008
Four new R-methylene-γ-lactone-bearing cembranoids, 20-acetylsinularolide B (6), presinularolide B (7), 3-dehydroxy-
lpresinularolide B (8), and 3-dehydroxyl-20-acetylpresinularolide B (9), together with five known analogues, sinularolides
B-E (1-4) and 20-acetylsinularolide C (5), were isolated from a South China Sea soft coral Lobophytum crassum.
Their structures and relative stereochemistry were established by a combination of detailed spectroscopic data analysis
and chemical correlations. The structures of 1-9 were further confirmed by an X-ray diffraction study on a single
crystal of sinularolide B (1). The absolute configurations of sinularolide B (1) and presinularolide B (7) were determined
by a novel solid-state CD/TDDFT approach and by a modified Mosher’s method, respectively. This study also revealed
that the coupling constant between the lactonic methine protons (3J1,2) varies considerably with different functional
groups on the cembrane ring and that the determination of the stereochemistry of lactone ring fusion based on this
coupling constant is risky. In a bioassay, sinularolides B and C (1 and 2) and new cembranoids 7 and 8 showed in Vitro
cytotoxicity against the tumor cell lines A-549 and P-388.
Cembrane diterpenoids are a large family of natural products
having many structural variations, including further cyclizations
and a multitude of functional groups, e.g., lactone, epoxide, furan,
ester, ether, hydroxyl, aldehyde, and carboxylic moieties.1 In
particular, R-methylene-γ-lactone-bearing cembranoids represent
an interesting group within this family, exhibiting mainly antitumor
activity,2 as well as other pharmacological effects such as anti-
HIV3 and antituberculosis.2d The occurrence of such lactonic
cembranoids in gorgonians of the genus Eunicea and alcyonarians
(soft corals) of the genera Lobophytum, Sinularia, Sarcophyton,
and ClaVularia was recently reported in an increasing number of
examples.2–4
In the course of our ongoing project aimed at searching for
biologically active secondary metabolites from South China Sea
octocorals,5 we had an opportunity to reinvestigate the soft coral
Lobophytum crassum collected from the coast of Sanya, Hainan
Island, China, which led to the isolation and structural elucidation
of four new trans-fused R-methylene-γ-lactone cembranoids,
namely, 20-acetylsinularolide B (6), presinularolide B (7), 3-de-
hydroxylpresinularolide B (8), and 3-dehydroxyl-20-acetylpresinu-
larolide B (9), together with five analogues, sinularolides B-E
(1-4) and 20-acetyl sinularolide C (5). We herein report the
isolation, structural elucidation, and bioactivities of these compounds.
careful comparison with reported data and further confirmed by an
X-ray diffraction study on a single crystal of sinularolide B (1)
(Figure 1). Sinularolides B-E (1-4) were recently reported to be
isolated from the Hainan soft coral Sinularia gibberosa.2a In fact,
sinularolide B (1) was first isolated from a Red Sea soft coral L.
crassum,6 which also produced the 20-acetyl analogue (5).7A
complete and rigorous 1H and 13C NMR assignment of 5 was then
performed, since its relative stereochemistry had not been reported
7
1
in the literature. A preliminary H NMR analysis of the newly
isolated molecules revealed a structural similarity for all of them
and indicated the presence of the common R-methylene-γ-lactone
cembranoid frameworks in compounds 6-9, according to previous
chemical studies on L. crassum.7 The chemical analysis of 6-9
was conducted starting from the main metabolite sinularolides B
(1), followed by the remaining cembranoids 6-9. Accordingly, the
structure elucidation details of these new molecules are described
in this order.
On the basis of the knowledge of the relative relationships of
the chiral centers around the 14-membered cembrane ring of
sinularolide B (1), advanced stereochemical studies were performed.
A novel solid-state CD/TDDFT (time-dependent density functional
theory) approach8 was applied to determine the absolute stereo-
chemistry of 1. The CD spectra of (-)-1 were recorded in both
acetonitrile solution and the solid state as a KCl disk and showed
Results and Discussion
Freshly collected specimens of L. crassum were immediately
frozen to -20 °C and stored at that temperature before extraction.
The Et2O-soluble portion of the acetone extract was repeatedly
chromatographed over silica gel, Sephadex LH-20 columns, and
RP-HPLC to afford nine diterpenoids (1-9). The structures and
relative stereochemistry of the known compounds 1-5 were
determined by extensive spectroscopic analysis combined with
* Corresponding author. Tel: 86-21-50805813. Fax: 86-21-50805813.
E-mail: ywguo@mail.shcnc.ac.cn.
† Shanghai Institute of Materia Medica-CAS.
‡ University of Paderborn.
§ Universita` di Pisa.
University of Debrecen.
10.1021/np800081p CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 05/31/2008

962 Journal of Natural Products, 2008, Vol. 71, No. 6
Zhang et al.
Presinularolide B (7) was obtained as an optically active colorless
oil. HRESIMS, 13C NMR, and DEPT spectra established the
molecular formula as C20H28O4, indicating seven double-bond
equivalents. IR bands at 1736, 1728, and 1660 cm-1 and the strong
UV absorption at 210 nm suggested the presence of an R-methylene-
γ-lactone moiety. This conclusion was supported by the observation
of the signals for a lactone group (δH 4.23; δC 169.8, qC and 83.7,
CH) and an exocyclic double bond (δH 6.30 and 5.71; δC 138.5,
qC and 123.4, CH2) in the 1H and 13C NMR spectra, which
accounted for three degrees of unsaturation. The remaining degrees
of unsaturation were assigned to three double bonds and an
additional ring by 1D and 2D NMR (HSQC and HMBC) experi-
ments (Tables 1 and 2).
A comparison of the 1H and 13C NMR data of 7 with those of 1
revealed a close similarity with the exception that the epoxy group
(δH 2.88; δC 63.4, CH and 62.8, qC) in 1 was replaced by a
trisubstituted double bond (δH 5.20; δC 140.3, qC and 124.6, CH2)
in 7. This fact suggested the gross structure of 7 to be a 12,13-
double bond precursor of 1, which was strongly supported by the
1H-1H COSY and HMBC experiments, as shown in Figure 3a.
The trans ring fusion and R-orientation of H-3 were deduced from
the significant NOE effect between H-1 and H-3 and the lack of
NOE effect between H-2 and both H-1 and H-3 in NOE difference
spectra (Figure 3b).
Figure 1. Molecular structure of sinularolide B (1) from the X-ray
diffraction experiment.
similar profiles in the two cases (Figure 2a), which is in agreement
with a pronounced rigidity of the macrocycle due to its many
unsaturated bonds. The CD spectrum calculated with the TDDFT
method9 at the BH&HLYP/TZVP level, using as input geometry
the X-ray coordinates for 1 with (1R,2R,3R,12S,13R) configurations,
is shown in Figure 2b. It clearly reproduces the shapes of the main
experimental bands very well, except for a 20 nm wavelength shift
and some intensity overestimation. The smaller positive band at
224 nm (experimental spectrum) and the stronger negative one at
197 nm are assigned to the π-π* transitions of the enone and the
isolated alkenes, respectively. The absolute configurations of the
five stereogenic centers in 1 are thus determined to be 1R, 2R, 3R,
12S, 13R.
Compound 6, 20-acetylsinularolide B, was isolated as an optically
active {[R]20D -85.0 (c 0.25, CHCl3)} colorless oil. The molecular
formula C22H30O6 was established by HRESIMS, 13C NMR, and
DEPT spectra. The IR spectrum of 6 indicated the presence of a
hydroxyl group (3446 cm-1), an ester group (1743 cm-1), and an
R-methylene-γ-lactone moiety (1766, 1660 cm-1) in the molecule.
The presence of an R-methylene-γ-lactone moiety was also sup-
ported by the observation of a strong UV absorption at 207 nm
and a pair of characteristic exocyclic olefinic protons (δ 6.32, 6.03)
in the 1H NMR spectrum. The 1H NMR data of 6 were very similar
to those of 1, except for the presence of an additional signal at δ
2.13 (s) (Table 1), which suggested the presence of an acetyl moiety
in 6, consistent with two carbon resonances at δ 170.7 (qC) and
20.7 (qC) in its 13C NMR spectrum (Table 2). The acetylation was
determined to occurr at C-20, since the AB-type proton signals of
H2-20 were significantly downfield shifted from δ 3.82 and 3.58
to δ 4.38 and 3.87 compared to 1, respectively. Acetylation of 1
and 6 yielded the same diacetate 10, giving further support to this
conclusion. Thus, compound 6 was established as the 20-acetyl
derivative of 1.
The presence of a secondary alcohol at C-3 of 7 allowed us to
determine its absolute stereochemistry by utilizing the modified
Mosher’s method.10 (S)- and (R)-MTPA esters of presinularolide
B (7) were prepared by treatment with (R)- and (S)-R-methoxy-R-
trifluoromethylphenylacetyl (MTPA) chloride in dry pyridine at
room temperature, respectively. Compound 7 was converted to the
corresponding MTPA esters 7S and 7R, respectively. Significant
∆δ values (∆δ ) δS-MTPA-ester - δR-MTPA-ester) for the protons near
the chiral center C-3 were observed as shown in Figure 4. According
to the Mosher’s rule, the absolute configuration at C-3 was
determined as R. As a consequence, the absolute configurations of
the other two stereogenic centers in 7 were determined to be 1R,
2R, on the basis of the established relative stereochemistry, which
is in agreement with the configuration of the related compound 1
determined above.
Although compound 1 was the 12,13-epoxide of 7, the marked
difference between the coupling constants of the protons attached
to the bridgehead carbons in 1 (3J1,2 ) 6.6 Hz) and 7 (3J1,2 ) 2.3
Hz) was puzzling. To further confirm the established structure of
7, a Sharpless epoxidation11 was performed to produce 1 from 7
by using (R,R)-diethyl tartrate. The structure and stereochemistry
of 7 were thus unambiguously determined.
Compound 8, 3-dehydroxylpresinularolide B, was an optically
active colorless oil. It had the molecular formula C20H28O3 as
deduced from HRESIMS, 13C NMR, and DEPT spectra and thus
one oxygen atom less than 7. The IR and UV spectra of 8 closely
1
resembled those of 7. The H and 13C NMR data of 8 (Tables 1
and 2) were also closely related to those of 7, with only a difference
at C-3 [the former possessed a methylene (δH 2.44, 2.02; δC 44.9,
CH2) and the latter had a hydroxyl-bearing methine (δH 3.75; δC
78.9, CH)]. Due to the loss of the 3-OH, 13C NMR chemical shifts
of C-2-C-5 were all upfield shifted while that of C-1 was downfield
shifted with respect to those of 7. This evidence suggested that 8
was a 3-deoxy derivative of 7. A series of obvious proton
A search of the literature revealed that compound 6 is identical
in all aspects, except for optical rotation sign and magnitude, to a
“known” cembranoid (namely, 13-hydroxylobolide7), previously
isolated from a Red Sea soft coral of L. crassum. Unfortunately,
the authors of the paper gave only a planar structure of 13-
hydroxylobolide without stereochemical comment. Since the [R]D
1
connectivities of H-1/H-2/H2-3 as established by a H-1H COSY
sign of compound 6 is opposite that of 13-hydroxylobolide {[R]24
D
experiment and the diagnostic long-range correlations of H2-3 with
C-1, C-2, C-4, C-5, and C-18 and of H3-18 with C-3, C-4, and C-5
observed in the HMBC spectrum confirmed this conclusion about
the structure 8.
+16.0 (c 0.9, CHCl3)}, it is obvious that 6 should be an enantiomer
of 13-hydroxylobolide. However, because the 13-epimer (3-epimer
in our atom-numbering system) of 6 and that of 13-hydroxylobolide
were also isolated from both Chinese and Israeli samples and they
are identical including the [R]D values and signs, it is necessary to
remeasure the optical rotation value of 13-hydroxylobolide. In fact,
we twice recorded the [R]D value of compound 6 in order to confirm
the correctness of our measurement.
Compound 9, 3-dehydroxyl-20-acetylpresinularolide B, had
a molecular formula of C22H30O4 as determined by HRESIMS
{m/z 381.2016 [M + Na]+, corresponding to C22H30O4Na
1
(calculated for 381.2042)}. Its UV, IR, and H and 13C NMR

Cembrane Diterpenoids from Lobophytum crassum
Journal of Natural Products, 2008, Vol. 71, No. 6 963
Figure 2. (a) Experimental CD spectra of (-)-1 in acetonitrile and in the solid state as a KCl disk. (b) TDBH&HLYP/TZVP-calculated CD
spectrum of (1R,2R,3R,12S,13R)-1 using X-ray coordinates; vertical bars represent rotational strengths.
a b
,
Table 1. 1H NMR Data for Compounds 6-9
6
7
8
9
position
δ mult. (J in Hz)
2.74, m
4.07, dd (9.1, 6.6)
4.02, d (9.1)
δ mult. (J in Hz)
δ mult. (J in Hz)
δ mult. (J in Hz)
1
2
3
2.73, dd (7.8, 2.3)
4.23, dd (7.5, 2.3)
3.75, d (7.5)
2.73, ddd (8.2, 3.6, 2.8)
4.33, dt (9.6, 2.8)
2.44, br d (14.1)
2.02,dd (14.1, 9.6)
5.03, m
2.72, ddd (8.3, 3.7, 2.7)
4.31, dt (9.4, 2.7)
2.44, br d (14.1)
2.03, dd (14.1, 9.4)
5.04, m
5
6
5.46, m
2.53, ddd (22.7, 9.1, 4.1)
2.20, m
5.34, dd (8.5, 3.5)
2.33, m
2.14, m
2.30, m
2.09, m
2.29, m
2.09, m
7
2.35, m
2.13, m
2.08, m
2.06, m
2.11, m
5.02, t, 7.1
2.26, m
2.08, dd (18.0, 9.8)
4.89, br d (7.2)
2.35, m
2.06, m
4.93, m
2.30, m
2.04, m
4.88, m
2.28, m
9
10
2.14,
m
2.18, m
2.21, m
2.22, m
11
2.33, m
2.32, m
2.35, m
2.28, m
1.24, dt (11.9, 1.5)
2.85, dd (7.3, 3.0)
1.87, ddd (14.5, 3.4, 3.0)
1.50, ddd (14.5, 9.7, 7.3)
6.32, d (2.3)
6.03, d (2.3)
1.72, s
1.63, s
2.21, m
5.20, dd (8.2, 7.3)
2.36, m
2.23, m
5.22, t (7.7)
2.33, m
2.21, m
5.29, t (7.7)
2.32, m
13
14
2.29, m
2.29, m
2.28, m
17
6.30, d (2.1)
5.71, d (2.1)
1.72, s
1.62, s
4.10, s
6.27, d (1.6)
5.68, d (1.6)
1.66, s
1.61, s
4.12, s
6.28, d (2.0)
5.69, d (2.0)
1.66, s
1.60, s
4.56, s
18
19
20
4.38, d (12.2)
3.87, d (12.2)
2.13, s
4.10, s
4.12, s
4.56, s
2.06, s
OAc
a Bruker DPX-300 spectrometer, chemical shifts referenced to CHCl3 (δ 7.26 ppm). b Assignments made by HSQC, 1H-1H COSY, and HMBC
experiments.
data (Tables 1 and 2) were very similar to those of 8, suggesting
it shares the same framework as 8. In fact, the presence of an
acetyl group (δH 2.13, s; δC 170.9, qC and 20.9, CH3) as
compared to 8 was the only difference recognized in the
spectroscopic data of 9. Furthermore, the apparent downfield
shifted 1H NMR resonance of H2-20 (from δH 4.12 to 4.56)
indicated that the 20-hydroxyl group was acetylated. Compound
9 was therefore the 20-acetyl derivative of 8.
interpretation of its NMR spectra, we suspect that the above-
mentioned differences between the 13C NMR data may be caused
by the different geometry of the double bonds in “20-deacetyl-
lobolide”. This observation creates a need to reanalyze the NMR
spectra of “20-deacetyllobolide” so as to verify its correct structure.
“3,4-Deepoxylobolide” and compound 9 shared almost identical
NMR data, indicating their structures might be the same if the
former had the same optical rotation sign as 9.
It is worthy to point out that both compounds 8 and 9 are formally
identical to “20-deacetyllobolide” and “3,4-deepoxylobolide”, both
of which were previously reported from the same species of Red
Sea origin by Kashman et al.6 However, neither relative stereo-
chemistry nor [R]D values of “20-deacetyllobolide” and “3,4-
deepoxy lobolide” were disclosed.6 In particular, to our surprise,
the 13C NMR data of “20-deacetyllobolide”6 were found to be quite
different from those of compound 8 (Table 2). Moreover, careful
comparison of their 13C NMR data with ours revealed that the
significant differences between them were observed for three
olefinic carbons (C-5, C-8, and C-12) and the methylene carbons
(C-7, C-7, C-10, and C-14) that linked to these double bonds. Since
the NMR data assignments of 8 were correct even after careful
The close biosynthetic relationship between metabolites 1-9 is
apparent. They differ from each other only in the oxidative pattern
of the 14-membered ring. Compounds 7-9 could be the formal
precursors of epoxides 1-6, as suggested by the chemical conver-
sion of 7 to 1. Moreover, since the absolute configuration of 1 and
7 was determined, it is reasonable to assign the same configurations
for the corresponding chiral centers within molecules 2-6, 8, and
9 based on biogenetic consideration. In addition, it is interesting to
note that the coupling constant between the lactone methine protons
(3J1,2) was significantly changed during the transformation of 7 to
1 (e.g., 3J1,2 ) 2.3 Hz in 7 and 6.6 Hz in 1) in spite of the fact that
the same ring junction is present in the two groups of metabolites.
This fact supports Coll’s suggestion that the coupling constant

964 Journal of Natural Products, 2008, Vol. 71, No. 6
Zhang et al.
a b
,
Table 2. 13C NMR Data for Compounds 6-9
6
7
8
9
8c
1
2
3
4
5
6
7
8
42.2, CH
82.1, CH
81.0, CH
131.9, qC
132.1, CH
24.8, CH2
38.5, CH2
135.1, qC
124.2, CH
23.6, CH2
32.6, CH2
60.4, qC
62.4, CH
31.5, CH2
138.5, qC
168.8, qC
124.2, CH2
12.4, CH3
15.8, CH3
64.0, CH2
20.7, CH3
170.7, qC
42.2, CH
83.7, CH
78.9, CH
131.9, qC
131.2, CH
24.2, CH2
37.6, CH2
134.0, qC
123.6, CH
24.4, CH2
34.7, CH2
140.3, qC
124.6, CH
33.8, CH2
138.5, qC
169.8, qC
123.4, CH2
13.2, CH3
17.0, CH3
60.0, CH2
44.7, CH
81.1, CH
44.5, CH
81.1, CH
45.0, CH2
129.3, qC
128.5, CH
24.6, CH2
37.9, CH2
134.0, qC
123.8, CH
24.4, CH2
34.6, CH2
135.5, qC
126.9, CH
33.8, CH2
139.2, qC
170.0, qC
122.8, CH2
17.4, CH3
16.7, CH3
61.7, CH2
20.9, CH3
170.9, qC
44.8, CH
81.2, CH
38.0, CH2
128.5, qC
124.7, CH
24.5, CH2
34.6, CH2
129.5, qC
124.2, CH
29.6, CH2
33.7, CH2
134.1, qC
125.6, CH
30.9, CH2
142.0, qC
169.2, qC
122.8, CH2
17.5, CH3
16.8, CH3
60.0, CH2
44.9, CH2
129.4, qC
128.4, CH
24.5, CH2
37.9, CH2
134.0, qC
124.0, CH
24.4, CH2
34.6, CH2
140.1, qC
124.7, CH
33.5, CH2
139.3, qC
170.2, qC
122.8, CH2
17.5, CH3
16.8, CH3
60.1, CH2
9
10
11
12
13
14
15
16
17
18
19
20
OAc
a Varian INOVA-600 NMR spectrometer; chemical shifts referenced to CDCl3 (δC 77.0). b Assignments made by DEPT, HSQC, and HMBC
experiments. c Data reported for 20-deacetyllobolide in ref 6.
much greater caution. Our observation of the dependence of the
3J1,2 coupling constant on the transformation of a ∆12(13) double
bond (7-9) into an epoxy group (1-6) demonstrates the sensitivity
of such parameters to the nature of different functional groups
attached to the cembrane ring scaffold. Similar phenomena were
also observed upon oxidation of 20-acetylsinularolide C (5, 3J1,2
)
6.5) to its 3-ketone analogue (3J1,2 ) 3.3) and conversion of ent-
lobophilide A (3J1,2 ) 6.8) into the corresponding 4,5-epoxy-3-
ketone derivative (3J1,2 ) 3.3).7 These observations indicate that
the conformational flexibility of the macrocycle renders somewhat
risky the stereochemical determination of the ring fusion based on
the proton coupling constants. To obtain an unambiguous stereo-
chemical assignment, other techniques, for example, more advanced
NMR methods and X-ray diffraction analysis, are necessary.
All the compounds were examined for growth-inhibition activities
in Vitro toward human lung adenocarcinoma A-549 cells and murine
leukemia P-388 cells. 3-Dehydroxylpresinularolide B (8) showed
significant cytotoxicity against both A-549 and P-388 cell lines with
95.7% and 100% inhibition at 10-5 mol/L, while sinularolide B
(1) demonstrated cytotoxic activity against P-388 with an inhibition
of 100% at 10-5 mol/L. Moderate antitumor activities were observed
for presinularolide B (7) against A-549 cells and for sinularolide
C (2) against P-388 cells with 64.0% and 63.8% inhibition at a
concentration of 10-5 mol/L, respectively.
1
Figure 3. (a) H-1H COSY (bold bonds) and selected HMBC
correlations (curved arrows) for 7. (b) Key NOESY correlations
for 7.
Experimental Section
Figure 4. ∆δ (δS - δR) values (in ppm) for the MTPA ester of 7.
General Experimental Procedures. Melting points were measured
on an X-4 digital micromelting point apparatus and are uncorrected.
Optical rotations were measured on a Perkin-Elmer polarimeter 341 at
the sodium D-line, cell length 100 mm. CD spectra were measured on
a Jasco J-810 spectropolarimeter (for the solid-state CD protocol, see
refs 8b,e). UV spectra were recorded on a 756 CRT spectrophotometer.
IR spectra were recorded on a Nicolet Magna FT-IR 750 spectropho-
tometer; peaks are reported in cm-1. The NMR spectra were recorded
at 293 K on Bruker DPX-300, Bruker Avance-500, and Varian INOVA-
600 NMR spectrometers, using the residual CHCl3 signal (δH 7.26 ppm)
around the five-membered lactone ring does not permit unequivocal
stereochemical assignments due to the flexibility of the cembrane
ring.12 Unfortunately, Coll’s suggestion has not been widely
appreciated and the 3J1,2 value is still widely used to deduce relative
stereochemistry (e.g., a small coupling constant indicates a transoid
ring junction, whereas a large coupling constant suggests a cisoid
ring linkage). This general trend was proposed mainly on the basis
of several early X-ray studies6,13 and played a crucial role in the
assignment of the bridgehead fusion pattern of lactonic cembranoids.
A literature survey revealed that the coupling constants of protons
(3J1,2) for the trans ring junction ranged from 2.9 to 7.5 Hz, while
those for the corresponding cis linkage extended over the range
6.5 to 9.3 Hz.2–4,6,7,12,13 It seems to be appropriate to employ
coupling constants in stereochemical assignments of the bicyclic
fusion pattern when these values lie at either of the two extremes,
while those lying in the middle (i.e., 6-8 Hz) should be used with
as an internal standard for 1H NMR and CDCl3 (δC 77.0 ppm) for 13
C
NMR; coupling constants (J) are given in Hz. 1H and 13C NMR
assignments were supported by 1H-1H COSY, HSQC, HMBC,
ROESY, and NOEDIFF experiments. EI mass spectra were obtained
on a MAT 8200 mass spectrometer, and ESI mass spectra were
performed on a Q-TOF Micro LC-MS-MS mass spectrometer. The
X-ray diffraction study was carried out on a Bruker SMART APEX
CCD diffractometer with Mo KR radiation (λ ) 0.71073 Å). Com-
mercial silica gel (Qing Dao Hai Yang Chemical Group Co., 200-300

Cembrane Diterpenoids from Lobophytum crassum
Journal of Natural Products, 2008, Vol. 71, No. 6 965
and 400-600 mesh) was used for column chromatography. Precoated
silica gel plates (Yan Tai Zi Fu Chemical Group Co., G60 F-254) were
used for analytical thin-layer chromatography (TLC).
Animal Material. The soft coral L. crassum was collected off the
coast of Sanya, Hainan Province, China, in December 2004, at a depth
of 20 m and identified by Professor R.-L. Zhou of South China Sea
Institute of Oceanology, Chinese Academy of Sciences. A voucher
specimen (LS-167) is available for inspection at Shanghai Institute of
Materia Medica, CAS.
3-Dehydroxyl 20-acetylpresinularolide B (9): colorless oil: [R]20
D
+81.0 (c 0.26, CHCl3); UV (MeOH) λmax (log ε) 208 (3.97) nm; IR
(KBr) νmax 2920, 2852, 1766, 1738, 1664, 1232, 1118, 1026 cm-1; 1H
and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS m/z
381.2016 (calcd for C22H30O4Na, 381.2042).
Acetylation of 1 and 6. To 1 (2.0 mg) and 6 (2.0 mg) in dry pyridine
(0.5 mL) was added 1 drop of Ac2O, respectively. Both mixtures were
stirred at room temperature overnight, and the reactions were stopped
by adding 1 drop of H2O. The crude acetylated products, after
evaporating the solvent in Vacuo, were purified on silica gel column
chromatography (petroleum ether/EtOAc in gradient), respectively, to
Extraction and Isolation. The frozen animals (630 g, dry weight)
were cut into pieces and exhaustively extracted with acetone at room
temperature (1.5 L × 3). The organic extract was evaporated to give a
residue, which was partitioned between ether and H2O. The ether
solution was concentrated under reduced pressure to give a dark green
residue (3.8 g), which was fractionated by gradient silica gel column
chromatography (0-100% acetone in petroleum ether), yielding 16
fractions. Fractions 6-11 showed interesting red TLC spots after
spraying with H2SO4. Fraction 6 gave compound 9 (9.0 mg) after a
column chromatography (CC) on Sephadex LH-20 (CHCl3). Fraction
8 was subjected to silica gel CC (400-600 mesh, petroleum ether/
acetone, 85:15), followed by CC on Sephadex LH-20 (CHCl3) to yield
8 (136.2 mg) and crude 5 (9.4 mg), which afforded pure 5 (6.0 mg)
after purification by RP-HPLC (semipreparative ZORBAX ODS (5 µm,
250 × 9.4 mm), MeO/H2O (7:3), 2.5 mL/min, 8.4 min). Fraction 9
was first split by Sephadex LH-20 CC (CHCl3/MeOH/petroleum ether,
2:1:1) and then chromatographed on a silica gel column (400-600
mesh, petroleum ether/acetone, 85:15) to afford 3 (39.0 mg) and 6 (18.3
mg), respectively. Compounds 1 (35.0 mg) and 2 (77.6 mg) were
isolated from fraction 10 by a CC sequence on Sephadex LH-20
(CHCl3/MeOH/petroleum ether, 2:1:1) and silica gel (400-600 mesh,
petroleum ether/acetone, 4:1) as well. Last, CC of fraction 11 on
Sephadex LH-20 CC (CHCl3/MeOH/petroleum ether, 2:1:1) yielded 4
(50.0 mg) and 7 (227.0 mg).
1
afford the expected diacetate 10: H NMR (500 MHz, CDCl3) δ 6.32
(1H, d, J ) 2.9 Hz, H-16a), 6.00 (1H, d, J ) 2.9 Hz, H-16b), 5.53
(1H, dd, J ) 8.2, 5.2 Hz, H-5), 5.21 (1H, d, J ) 8.8 Hz, H-3), 5.04
(1H, t, J ) 7.0 Hz, H-9), 4.36 (1H, d, J ) 12.2 Hz, H-20a), 4.19 (1H,
dd, J ) 8.8, 6.7 Hz, H-2), 3.89 (1H, d, J ) 12.2 Hz, H-20b), 2.88 (1H,
dd, J ) 6.8, 3.6 Hz, H-13), 2.86 (1H, m, H-1), 2.44 (1H, m, H-6a),
2.34 (2H, m, H-7a, H-11a), 2.24 (1H, m, H-10a), 2.17 (1H, m, H-6b),
2.14 (1H, m, H-10b), 2.14 (3H, s, H3-OAc-20), 2.10 (3H, s, H3-OAc-
3), 2.10 (1H, m, H-7b), 1.85 (1H, dt, J ) 14.6, 3.7 Hz, H-14a), 1.70
(3H, s, H3-18), 1.63 (s, 3H, H3-19), 1.58 (1H, ddd, J ) 14.6, 9.3, 7.0
Hz, H-14b), 1.28 (1H, dt, J ) 11.2, 2.9 Hz, H-11b) ppm; 13C NMR
(125 MHz, CDCl3) δ 170.7 (qC, CdO-OAc-20), 169.6 (qC, CdO-
OAc-3), 168.9 (qC, C-17), 138.2 (qC, C-15), 135.1 (qC, C-8),
133.5(CH, C-5), 129.2 (qC, C-4), 124.3 (CH, C-9), 124.0 (CH2, C-16),
80.1 (CH, C-3), 79.7 (CH, C-2), 64.1 (CH2, C-20), 62.0 (CH, C-13),
60.4 (qC, C-12), 42.1 (CH, C-1), 38.3 (CH2, C-7), 32.4 (CH2, C-11),
31.7 (CH2, C-14), 24.8 (CH2, C-6), 23.5 (CH2, C-10), 21.0 (CH3, Me-
OAc-3), 20.8 (CH3, Me-OAc-20), 15.9 (CH3, C-19), 13.3 (CH3, C-18)
ppm; HREIMS m/z 432.21508 (calcd for C24H32O7, 432.21481) {[R]20
-27 (c 0.10 CHCl3)}.
D
Esterification of 7 with MTPA Chlorides. Both (S)- and (R)-MTPA
esters of 7 (7S, 7R) were obtained by treatment of 7 (3.0 mg,
respectively) with (R)- and (S)-MTPA chlorides (10 µL) in dry pyridine
(0.5 mL) catalyzed with dimethylaminopyridine and stirred at room
temperature overnight. The MTPA esters (6.0 mg, 87% yield) were
purified by minicolumn chromatography on silica gel (300 mesh,
petroleum ether/EtOAc, 7:1).
Sinularolide B (1): colorless crystals, mp 128-130 °C; [R]20
D
-123.6 (c 0.47, CHCl3); CD (MeCN, λ [nm] (∆ꢀ), c ) 7.4 × 10-4
)
225 (2.09), 198 (-31.58), CD (KCl, λ [nm] φ [mdeg], 47 µg of 1 and
250 mg of KCl, 224 (3.18), 197 (- 21.28); UV (MeOH) λmax (log ε)
208 (3.83) nm; IR (KBr) νmax 3610, 3460, 2949, 2866, 1751, 1659,
1261, 1149, 1043 cm-1
C20H28O5Na, 371.1834).
;
HRESIMS m/z 371.1833 (calcd for
1H NMR data of 7S (500 MHz, CDCl3): δ 6.29 (1H, s, H-16a), 5.67
(1H, d, J ) 1.6 Hz, H-16b), 5.46 (1H, t, J ) 6.9 Hz, H-5), 5.29 (1H,
t, J ) 7.3 Hz, H-13), 5.13 (1H, d, J ) 7.6 Hz, H-3), 4.82 (1H, d, J )
12.1 Hz, H-20a), 4.81 (1H, m, H-9), 4.70 (1H, d, J ) 12.1 Hz, H-20b),
4.21 (1H, d, J ) 7.6 Hz, H-2), 2.69 (1H, br d, J ) 10.9 Hz, H-1), 2.34
(1H, m, H-14a), 2.33 (1H, m, H-10a), 2.28 (1H, m, H-6a), 2.15 (2H,
m, H2-11), 2.14 (1H, m, H-10b), 2.13 (1H, m, H-14b), 2.10 (3H, m,
H-6b, H2-7), 1.69 (3H, s, H3-18), 1.59 (3H, s, H3-19) ppm.
1H NMR data of 7R (300 MHz, CDCl3): δ 6.31 (1H, s, H-16a),
5.71 (1H, s, H-16b), 5.23 (1H, t, J ) 6.8 Hz, H-13), 5.19 (1H, t, J )
7.0 Hz, H-5), 4.84 (1H, d, J ) 8.6 Hz, H-3), 4.74 (2H, s, H2-20), 4.72
(1H, m, H-9), 4.21 (1H, dd, J ) 8.6, 1.6 Hz, H-2), 2.69 (1H, br d, J )
10.0 Hz, H-1), 2.36 (1H, m, H-14a), 2.18 (2H, m, H-10a, H-14b), 2.14
(2H, m, H-6a, H-11a), 2.13 (1H, m, H-10b), 2.11 (1H, m, H-11b), 2.10
(1H, m, H-6b), 1.97 (2H, m, H2-7), 1.61 (3H, s, H3-18), 1.54 (3H, s,
H3-19) ppm.
20-Acetylsinularolide C (5): colorless oil; [R]20 -89.0 (c 0.18,
D
CHCl3); UV (MeOH) λmax (log ε) 207 (3.84) nm; IR (KBr) νmax 3466,
2924, 2870, 1765, 1743, 1660, 1267, 1145, 1037 cm-1; 1H NMR (300
MHz, CDCl3) δ 6.27 (1H, d, J ) 3.1 Hz, H-16a), 5.98 (1H, d, J ) 3.1
Hz, H-16b), 5.62 (1H, m, H-5), 5.03 (1H, t, J ) 7.8 Hz, H-9), 4.46
(1H, br s, H-3), 4.35 (1H, d, J ) 12.1 Hz, H-20a), 4.22 (1H, dd, J )
6.8, 1.7 Hz, H-2), 3.87 (1H, d, J ) 12.1 Hz, H-20b), 3.29 (1H, m,
H-1), 2.90 (1H, dd, J ) 7.7, 2.8 Hz, H-13), 2.54 (1H, m, H-6a), 2.35
(1H, m, H-7a), 2.30 (1H, m, H-11a), 2.22 (1H, m, H-10a), 2.19 (1H,
m, H-7b), 2.13 (1H, m, H-6b), 2.13 (3H, s, H3-OAc), 2.11 (1H, m,
H-10b), 1.79 (1H, ddd, J ) 14.5, 3.6, 2.8 Hz, H-14a), 1.71 (3H, s,
H3-18), 1.61 (3H, s, H3-19), 1.49 (1H, ddd, J ) 14.5, 8.7, 7.7 Hz,
H-14b), 1.25 (1H, dt, J ) 12.4, 2.9 Hz, H-11b) ppm; 13C NMR (150
MHz, CDCl3) δ 170.9 (qC, C ) O-OAc), 170.1 (qC, C-17), 139.8 (qC,
C-15), 135.2 (qC, C-8), 131.5 (qC, C-4), 127.8 (CH, C-5), 124.3 (CH,
C-9), 123.6 (CH2, C-16), 81.9 (CH, C-2), 73.7 (CH, C-3), 64.3
(CH2, C-20), 62.6 (CH, C-13), 60.5 (qC, C-12), 38.8 (CH2, C-7), 37.4
(CH, C-1), 33.1 (CH2, C-14), 32.8 (CH2, C-11), 24.8 (CH2, C-6), 23.7
(CH2, C-10), 21.0 (CH3, Me-OAc), 16.1 (CH3, C-19), 15.7 (CH3, C-18)
ppm; HRESIMS m/z 413.1970 (calcd for C22H30O6Na, 413.1940).
Sharpless Epoxidation of 7. To a mixture of Ti(OiPr)4 (0.0041 mL,
0.01 mmol) and 4 Å molecular sieves (2 mg) in CH2Cl2 (0.5 mL) were
added L-(+)-DET (0.0034 mL, 0.02 mmol) and t-BuOOH (0.018 mmol,
0.0057 mL, 3.16 M in toluene) sequentially under an Ar atmosphere
at -20 °C. The reaction mixture was stirred for an additional 30 min,
and 7 (3 mg, 0.009 mmol) in CH2Cl2 (0.5 mL) was added dropwise.
The mixture was stirred for an additional 2 h at -20 to -15 °C until
TLC showed disappearance of 7. Then 10% aqueous tartaric acid
solution (0.5 mL) was added and the mixture stirred for 1 h. The mixture
was evaporated to remove the solvent and was then extracted with Et2O
(3 mL × 3). The ethereal solution was washed with saturated NaHCO3
solution (1.5 mL × 2) and brine (1.5 mL × 2), evaporated, and then
subjected to a minicolumn chromatography (petroleum ether/EtOAc,
20-acetylsinularolide B (6): colorless oil; [R]20 -85.0 (c 0.25,
D
CHCl3); UV (MeOH) λmax (log ε) 207 (3.80) nm; IR (KBr) νmax 3446,
1
2923, 2850, 1766, 1743, 1660, 1263, 1140, 1040 cm-1; H and 13C
NMR spectroscopic data, see Tables 1 and 2; HRESIMS m/z 413.1939
(calcd for C22H30O6Na, 413.1940).
Presinularolide B (7): colorless oil; [R]20D +33.8 (c 0.54, CHCl3);
UV (MeOH) λmax (log ε) 210 (4.06) nm; IR (KBr) νmax 3411, 3323,
1
2910, 2850, 1736, 1728, 1660, 1271, 1138, 1020 cm-1; H and 13C
1:1) to afford the pure product 1 (36%) { [R]20 -125.7 (c 0.11,
D
NMR spectroscopic data, see Tables 1 and 2; HRESIMS m/z 355.1883
(calcd for C20H28O4Na, 355.1885).
CHCl3)}.
X-ray Crystallographic Studies of Sinularolide B (1). Colorless
block crystals of 1 were obtained by recrystallization in CH3OH/H2O
(100:1). The crystal (0.475 × 0.466 × 0.345 mm) belongs to the
orthorhombic system, C20H28O5 ·H2O (Mr ) 366.44), space group
P2(1)2(1)2(1) with a ) 9.2900(14) Å, b ) 9.3200(15) Å, c ) 21.944(4)
Å, R ) ꢀ ) γ ) 90.0°, V ) 1899.9(5) Å3, Z ) 4, Dcalcd ) 1.281
3-Dehydroxylpresinularolide B (8): colorless oil; [R]20D +66.1 (c
0.32, CHCl3); UV (MeOH) λmax (log ε) 209 (3.96) nm; IR (KBr) νmax
1
3479, 2908, 2846, 1732, 1664, 1271, 1140, 1028 cm-1; H and 13C
NMR spectroscopic data, see Tables 1 and 2; HRESIMS m/z 339.1955
(calcd for C20H28O3Na, 339.1936).

966 Journal of Natural Products, 2008, Vol. 71, No. 6
Zhang et al.
mg/m3, λ ) 0.71073 Å. Intensity data were measured on a Bruker
SMART APEX CCD diffractometer. A total of 11 090 reflections were
collected to a maximum 2θ value of 54.60° by using the ω/ω scan
technique at 293(2) K. The structure was solved by the direct method
and was refined by the full matrix least-squares procedure. The
collection data were reduced using the Saint program, and the empirical
absorption correction was performed using the SADABS program. All
non-hydrogen atoms were given anisotropic thermal parameters,
whereas the hydrogen atom positions were geometrically idealized and
allowed to ride on their parent atoms. The refinement converged to the
final R ) 0.0434, Rw ) 0.0949 for 2473 observed reflections (I > 2σ(I),
2θ e 51.28°) and 277 variable parameters. Crystallographic data for
the structure of 1 have been deposited in the Cambridge Crystal-
lographic Data Center with the deposition no. CCDC 653250, which
contains the supplementary crystallographic data for this paper. These
data can be obtained free of charge from The Cambridge Crystal-
lographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
Computational Section. TDDFT calculations were run with the
Gaussian’03 program (Gaussian, Inc., Pittsburgh, PA, 2003). The input
geometry was obtained starting from the solid-state structure of 1, where
only the C-H bond distances were reoptimized with the DFT method
at the B3LYP/6-31G(d) level.14 The native hybrid functional BH&HLYP
and Ahlrich’s TZVP basis set were employed for the TDDFT
calculations. CD spectra were generated using dipole length-computed
rotational strengths to which a Gaussian band-shape was applied with
5800 cm-1 half-height width (corresponding to 23 at 200 nm). Dipole
velocity-computed rotational strengths differed from dipole length
values by less than 5% for the main bands.
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Acknowledgment. This research work was financially supported by
National Marine “863” Project (Nos. 2006AA09Z412 and 2006-
AA09Z447), Natural Science Foundation of China (Nos. 20572116,
30730108, 20721003), CAS Key Project (grant KSCX2-YW-R-18),
and STCSM Projects (Nos. 07XD14036, 06DZ22028). T.K. thanks the
Hungarian Scientific Research Fund (OTKA, F-043536). We are
grateful to E. Mollo for collection of the animal. We are greatly indebted
to R. Riguera (Universidad de Santiago de Compostela, Santiago de
Compostela, Spain) for his kind advice concerning the Mosher
experiment.
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References and Notes
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NP800081P