Xenicane-type diterpenes with cytotoxicity from Xenia florida

Article abstract of DOI:10.1021/np058110c

Chromatographic investigation of an acetone extract of the octocoral Xenia florida afforded three new xenicane diterpenes, namely, florxenilide A (1), florxenilide B (2), and florxenilide C (3), in addition to seven known xenicane diterpenes and two known

Full text of DOI:10.1021/np058110c

J. Nat. Prod. 2006, 69, 675-678
675
Xenicane-Type Diterpenes with Cytotoxicity from Xenia florida
Yuan-Bin Cheng,† Jiun-Yang Jang,† Ashraf Taha Khalil,† Yao-Haur Kuo,‡ and Ya-Ching Shen*,†
Institute of Marine Resources, National Sun Yat-sen UniVersity, 70 Lien-Hai Road, Kaohsiung, Taiwan 80424, Republic of China, and
National Research Institute of Chinese Medicine, Taipei 112, Taiwan, Republic of China
ReceiVed October 10, 2005
Chromatographic investigation of an acetone extract of the octocoral Xenia florida afforded three new xenicane diterpenes,
namely, florxenilide A (1), florxenilide B (2), and florxenilide C (3), in addition to seven known xenicane diterpenes
and two known cadinene sesquiterpenes. Structures were elucidated through spectroscopic analysis, especially 2D NMR,
and chemical derivatization. The absolute configuration of 1 was determined by NOESY, CD, and Mosher’s methods.
Florxenilides A (1) and B (2) exhibited cytotoxicity against human colon cancer (WiDr) cells at 4.5 and 3.7 µM,
respectively.
Table 1. 1H NMR Data (CDCl3, 300 MHz) of Compounds
Octocorals have been widely studied, as they produce a huge
array of skeletal classes of terpenes with unique substitution patterns
and functionalities.1 Xenia (order Alcyonaceae, family Xeniidae)
1-3a
1
2
3
commonly occurs in clear water of the tropics in the form of small
yellowish cylindrical or clavate colonies.2 They are a rich source
of diterpenoids containing a nine-membered monocarbocyclic ring.
The structures of Xenia diterpenoids have been divided into three
groups: xenicins (containing a dihydropyran-cyclononane skeleton),
xeniolides (possessing a δ-lactone-cyclononane skeleton), and
xeniaphyllanes (with a bicyclo[7.2.0]undecane skeleton).3,4 In our
survey of bioactive marine natural products, we have investigated
the constituents of marine octocorals inhabiting Taiwan’s coral reefs.
Three new xenicane derivatives were isolated from Xenia florida
together with seven known xenicane diterpenes, xeniafaraunol A,5
florlide C,6 florlide D,6 9-deoxyxeniolide B,7 9-deoxyxeniolide A,8
xeniafaraunol B,5 and florlide A.6 Two cadinene sesquiterpenes,
xenitorin A9 and xenitorin B,9 were identified in the course of
chromatographic fractionation. The new metabolites were named
florxenilide A (1), florxenilide B (2), and florxenilide C (3). The
structure elucidation was based on spectroscopic analysis, especially
2D NMR and chemical derivatization. The isolated compounds were
evaluated against human KB, WiDr, and Hepa tumor cell lines for
their cytotoxic activity.
1
3
5.85 br s
6.40 br s
5.97 s
6.45 s
4.94 d (12.0)
4.35 d (12.0)
2.81 m
1.86 m
1.85 m
1.68 m
1.26 m
3.42 br s
2.05 m
1.77 m
4a
5
2.15 m
2.01 m
4.35 d (12.0)
2.30 m
2.12 m
2.16 m
2.14 m
1.70 m
2.35 m
1.26 m
6
8
9
5.19 d (8.7)
5.74 br d (8.7)
3.11 d (9.3)
4.79 d (9.3)
10
11a
12
13
14
16
17
18
19
4.37 br s
2.46 br s
5.67 d (3.6)
4.41 s
2.83 s
5.66 d (3.6)
2.58 m
2.74 s
6.00 d (11.0)
5.65 dd (9.2, 3.6) 5.62 dd (8.9, 3.6) 6.18 dd (15.0, 11.0)
5.28 d (9.2)
1.73 s
1.73 s
1.89 s
5.04 s
4.99 s
2.10 s
5.28 d (8.9)
1.74 s
1.77 s
1.55 s
5.18 s
5.08 s
2.12 s
2.03 s
2.11 s
5.91 d (15.0)
1.34 s
1.34 s
1.06 s
1.90 d (13.2)
1.50 d (13.2)
1-OAc
12-OAc 2.00 s
13-OAc 2.03 s
OBz
3′,7′
4′,6′
5′
The molecular formula C33H40O10 was established for 1 by
HRESIMS, which showed a quasi-molecular ion peak at m/z
619.2522 [M + Na]+. The IR spectrum displayed absorption bands
diagnostic of hydroxyl (3477 cm-1), esters (1744, 1728, 1711 cm-1),
double bond (1653 cm-1), and aromatic (1601 cm-1) functionalities.
8.01 d (7.5)
7.41 t (7.5)
7.52 t (7.5)
8.07 d (7.5)
7.47 t (7.5)
7.60 t (7.5)
OMe
3.23 s
1
The H NMR spectroscopic data (Tables 1 and 2) indicated the
a J values (in Hz) are cited in parentheses.
presence of three acetate methyls (δH 2.10, 2.03, and 2.00) and a
benzoyl ester (δH 8.01, 7.52, and 7.41). The oxymethine signal at
δH 5.85, the olefinic at δH 6.40 (both br s), and three methyls at δH
1.73 (6H) and 1.89 (3H) suggested the presence of a xenicane
skeleton.3,10,11 The proton at δH 5.85 (H-1) coupled with a proton
at δH 2.46 (H-11a, COSY spectrum) and 3J-correlated with an
olefinic CH at δC 137.2 (C-3) and an acetate carbonyl at δC 169.7
(HMBC spectrum). The proton at δH 6.40 (H-3, br s) coupled to a
8.7 Hz, H-9), which vicinally coupled with two protons at δH 5.19
(d, J ) 8.7 Hz, H-8) and 4.37 (br s, H-10), thus indicating that a
benzoyloxy group was attached to C-9 and a hydroxyl to C-10.
HMBC correlations between H-9 and carbons at δC 137.5 (s, C-7)
and 150.0 (s, C-11) as well as of exomethylene protons (δH 5.04,
4.99, H-19) with C-11a (CH, δC 41.9) and C-10 (CH, δC 81.5)
constructed the cyclononane ring with exomethylene at C-11. On
3
proton at δH 2.15 (H-4a) and J-correlated to a CH at δC 38.0 (C-
3
4a), confirming the presence of a 1-acetoxy dihydropyran moiety.
Furthermore, the COSY spectrum revealed vicinal coupling between
H-4a/H-5/H-6, while the methyl proton at δH 1.89 (H-18) 3J-
correlated with the methylene at δC 40.6 (C-6) and the olefinic CH
at δC 122.3 (C-8), indicating C-7/C-8 unsaturation. The benzoyl
carbonyl at δC 166.0 correlated with the proton at δH 5.74 (d, J )
the other hand, H-3 was J-correlated with an oxymethine at δC
70.6, which was attached to a proton at δH 5.67 (d, J ) 3.6 Hz,
H-12), which in turn was coupled to δH 5.65 (dd, J ) 9.2, 3.6 Hz,
H-13). The chemical shifts of H-12 and H-13 as well as their HMBC
correlation to two carbonyls at δC 169.6 and 170.2 located two
acetoxy moieties at C-12 and C-13. Obviously, the olefinic proton
at δC 5.28 (d, J ) 9.2 Hz, H-14) was coupled to H-13 and attached
to a carbon at δC 117.8 (d, C-14). The two equivalent methyl signals
* To whom correspondence should be addressed. Tel: 886-7-525-2000,
ext. 5058. Fax: 886-7-525-5020. E-mail: ycshen@mail.nsysu.edu.tw.
† National Sun Yat-Sen University.
2
at δH 1.73 (6H, s) were J-correlated to the quaternary olefinic at
‡ National Research Institute of Chinese Medicine.
δC 140.8 (C-15) and 3J-correlated to H-14 as well as to the methyl
10.1021/np058110c CCC: $33.50
© 2006 American Chemical Society and American Society of Pharmacognosy
Published on Web 03/28/2006

676 Journal of Natural Products, 2006, Vol. 69, No. 4
Notes
Table 2. 13C NMR Data (CDCl3, 300 MHz) of Compounds
1-3a
1
2
3
1
3
4
4a
5
6
7
8
9
10
11
11a
12
13
14
15
16
17
18
19
1-OAc
91.8 (CH)
137.2 (CH)
113.1 (qC)
38.0 (CH)
28.7 (CH2)
40.6 (CH2)
137.5 (qC)
122.3 (CH)
78.0 (CH)
81.5 (CH)
150.0 (qC)
41.9 (CH)
70.6 (CH)
71.5 (CH)
117.8 (CH)
140.8 (qC)
25.7 (CH3)
18.7 (CH3)
18.1 (CH3)
115.5 (CH2)
169.7 (qC)
20.8 (CH3)
169.6 (qC)
20.8 (CH3)
170.2 (qC)
20.9 (CH3)
91.2 (CH)
138.1 (CH)
112.8 (qC)
38.4 (CH)
26.0 (CH2)
40.5 (CH2)
60.1 (qC)
171.4 (qC)
70.9 (CH2)
136.9 (qC)
37.8 (CH)
34.0 (CH2)
37.9 (CH2)
36.9 (qC)
73.8 (CH)
27.8 (CH2)
21.2 (CH2)
75.7 (qC)
53.9 (CH)
127.7 (CH)
120.5 (CH)
145.0 (CH)
70.8 (qC)
29.8 (CH3)
29.7 (CH3)
30.5 (CH3)
38.4 (CH2)
61.6 (CH)
80.6 (CH)
80.6 (CH)
148.0 (qC)
40.6 (CH)
70.5 (CH)
71.2 (CH)
118.0 (CH)
141.2 (qC)
25.8 (CH3)
18.8 (CH3)
17.9 (CH3)
117.9 (CH2)
169.7 (qC)
21.1 (CH3)
169.6 (qC)
21.0 (CH3)
170.2 (qC)
20.9 (CH3)
Figure 1. Selected HMBC correlations of 1.
12-OAc
13-OAc
benzoyl
CO
2′
3′,7′
4′,6′
5′
Figure 2. Selected NOESY correlations of 1.
166.0 (qC)
129.9 (qC)
129.4 (CH)
128.3 (CH)
133.1 (CH)
166.7 (qC)
129.8 (qC)
129.6 (CH)
128.5 (CH)
133.6 (CH)
OMe
48.5 (CH3)
a Assignments were guided by DEPT, HMQC, and HMBC spectral
data.
Table 3. Cytotoxicity of Compounds 1-5 against Human
Cancer Cells (ED50, µg/mL)a
compound
KBb
>20
11.4
>20
WiDrc
Hepad
1
2
3
4
5
6
2.73
1.88
>20
8.03
>20
>20
>20
>20
0.04
>20
>20
>20
>20
0.35
>20
>20
>20
mitomycin
0.56
a The concentration that inhibits 50% of the growth of human tumor
cell lines after 72 h exposure according to the method described in the
Experimental Section. b Oral epidermoid carcinoma. c Human colon
adenocarcinoma. d Human liver carcinoma.
signals at δC 25.7 and 18.7, confirming the attachment of 2,3-
diacetoxy-5-methylhex-4-ene group at C-4.12
The coupling constants J12,13 and J13,14 were identical to the
reported values,12 indicating the same configuration at C-12 and
C-13. The NOE interaction between H-8/H-4a indicated the
E-geometry of the C-7/C-8 double bond. The NOESY correlations
between H-11a/H-1; H-18/,Hâ-6, H-9; H-10/H-9,H-19; and H-4a/
H-12 suggested the relative stereochemistry of 1 (Figure 2).
The absolute configuration of 1 was determined by application
of Mosher’s method.13 Compound 1 was converted to (R)-R-
methoxy-R-(trifluoromethyl)phenylacetyl (MTPA) ester and (S)-
MTPA ester. The 1H NMR spectrum for each ester was measured,
followed by calculation of chemical shift differences (∆δ ) δS -
δR). The obtained differences were ∆δH -0.18, +0.07, +0.12, and
+0.32 for H-1, H-8, H-9, and H-10, respectively, indicating the
R-configuration at C-9 and the S-configuration at C-10. This was
in accordance with the result of the circular dichroism (CD) exciton
chirality of the dibenzoate ester (5) of 1, which exhibited a positive
Cotton effect at 239 nm. On the basis of the dibenzoate rule, these
two benzoyl chromophores are right-handed and clockwise.14 It is
noteworthy that this is the first report of oxy-substitution at both
C-9 and C-10, although 9R-oxy-substitution was previously reported
in a few related xenicanes such as xenialactol.15 The structure of 1
was thus established as florxenilide A.
Compound 2 had the molecular formula C33H40O11 as established
by HRESIMS (m/z 635.2465, [M + Na]+). The spectroscopic data
of 2 were similar to those of 1 (Tables 1 and 2), implying the
presence of the xenicane skeleton with an exomethylene at C-11
and three acetoxy and one benzoyloxy groups. A significant
difference was found in the chemical shifts of C-7 and C-8 (δC

Notes
Journal of Natural Products, 2006, Vol. 69, No. 4 677
recorded on a Bruker FT-300 spectrometer or on a Varian Unity INOVA
Scheme 1. Pausible Biogenetic Pathway of 3
1
500 FT-NMR at 500 MHz for H and 125 MHz for 13C, respectively,
using TMS as internal standard. The chemical shifts are given in δ
values (ppm) and coupling constants in Hz. Low-resolution EIMS and
FABMS were recorded on a VG Quattro 5022 mass spectrometer, and
HREIMS were measured on a JEOL JMS-SX 102 spectrometer. Silica
gel 60 (Merck) was used for column chromatography (CC), and
precoated silica gel plates (Merck, Kieselgel 60 F-254, 1 mm) were
used for preparative TLC. Sephadex LH-20 (Amersham Pharmacia
Biotech AB, Uppsala, Sweden) was used. S-(+)-R-Methoxy-R-trifluo-
romethylphenyl acetyl chloride and R-(-)-R-methoxy-R-trifluoro-
methylphenyl acetyl chloride (MTPA esters) were obtained from
ACROS Organics (NJ). Chromium oxide was obtained from Showa
Chemicals Co. LTD (Japan).
60.1 and 61.6, respectively) that replaced the olefinic signals
assignable to C-7 and C-8 in 1. The oxymethine at δH 3.11 (d, J )
9.3 Hz, H-8), attached to a carbon at δC 61.6, revealed HMBC
correlations to a quaternary carbon at δC 60.1 (C-7) and a methylene
at δC 40.5 (C-6), whereas H-18 (δH 1.55, s) correlated with C-6,
C-7, and C-8, indicating the presence of a 7,8-epoxy ring. NOESY
correlations were detected between H-1/H-11a; H-8/H-4a; and H-18/
H-9. These correlations favored the R-orientation of H-9 and H-10
as well as the â-orientation of H-1.8 Compound 2 was therefore
identified as florxenilide B.
Animal Material. The soft coral Xenia florida was collected from
Green Island, off Taiwan, in December 2003, at a depth of 20 m, and
immediately stored in a freezer. A voucher specimen (GSCII-2) is
deposited at the Institute of Marine Resources, National Sun Yat-sen
University, Kaohsiung, Taiwan.
Extraction and Isolation. The wet organism (1400 g) was extracted
with acetone three times using a stirrer. The acetone extract was
evaporated under vacuum and then partitioned between EtOAc-H2O
(1:1) to give a crude extract (29 g). The extract was chromatographed
on a silica gel column using a gradient of n-hexane-EtOAc to give 18
fractions. Fraction F3 (207 mg) was chromatographed on a silica gel
column using a gradient of n-hexane-EtOAc to furnish xenitorin A
(70 mg), xenitorin B (37 mg), and xeniflorlide A (8 mg). A similar
separation of F4 (280 mg) afforded an additional amount of xeniflorlide
A (37 mg). Separation of F5 (900 mg) on Sephadex LH-20, eluting
with MeOH, followed by column chromatography on silica gel using
n-hexane-EtOAc (10:1 to 1:2) yielded 1 (166 mg) and a mixture that
gave 2 (2 mg) after NP-HPLC purification [n-hexane-EtOAc (2:1)].
Fraction F7 (354 mg) was subjected to NP-HPLC using n-hexane-
EtOAc (3:1) followed by RP-HPLC using MeOH-H2O (4:1) to give
florlide C (8 mg) and florlide D (9 mg). Fraction F10 (179 mg) was
chromatographed on NP-HPLC, eluting with n-hexane-EtOAc (2:1),
to give 9-deoxyxeniolide B (11 mg). Fraction F11 (982 mg) was
chromatographed on a silica gel column using a gradient of n-hexane-
acetone to give eight subfractions (F11-A to F11-H). Subfraction F11-C
(191 mg) was separated using NP-HPLC, eluting with n-hexane-EtOAc
(2:1), to yield 9-deoxyxeniolide A (16 mg) and xeniafarunol B (18.3
mg). Fraction F17 (371 mg) was separated on NP-HPLC, eluting with
n-hexane-EtOAc (2:1), to give florlide A (64 mg) and a mixture that
was further purified using RP-HPLC, eluting with MeOH-H2O-CH3-
CN (10:10:1), to yield 3 (13 mg). Fraction F18 (240 mg) was fractionated
on a silica gel column using CH2Cl2-MeOH (20:1 to 10:1) for elution
to furnish four subfractions (F18-A to F18-D). Subfraction F18-B (143
mg) was further separated using RP-HPLC, eluting with MeOH-H2O
(1:1), to yield additional amounts of florlide A (15 mg) and 3 (12 mg).
The HRESIMS of 3 revealed a quasi-molecular ion peak at m/z
387.2146 [M + Na]+, consistent with the molecular formula
C21H32O5 and six degrees of unsaturation. The IR spectrum
suggested the presence of hydroxyl, carbonyl, and double bonds,
whereas the UV spectrum suggested a conjugated diene system (λmax
244 nm). The 1H NMR displayed an E-diene system at δH 6.00 (d,
J ) 11.0 Hz, H-12), 6.18 (dd, J ) 15.0, 11.0 Hz, H-13), and 5.91
(d, J ) 15.0 Hz, H-14). The oxymethylene protons at δH 4.94 and
3
4.35 (each d, J ) 12.0 Hz) were J-correlated to a carbonyl at δC
171.4 (C-1) and an olefinic CH at δC 127.7 (C-12). Three methyl
singlets were observed at δH 1.34 (3H × 2) and 1.06 (3H) along
with isolated AB methylene proton signals at δH 1.90 and 1.50
(each d, J ) 13.2 Hz) with HMBC correlations to a quaternary
carbon at δC 36.9 (C-7) and 75.7 (C-11) and an oxymethine at δC
73.8 (C-8). This suggested the presence of a bicyclic [4.3.1] ring
system containing two hydroxyl groups at C-8 and C-11. The NMR
data of 3 matched those of florlide A except for the presence of a
methoxy group at δH 3.23 (δC 48.5) whose protons correlated to
an oxy-quaternary carbon at δC 75.7 (C-11). This proved that the
hydroxyl group of florlide A was substituted by an OCH3 in 3,
and this was augmented by MS data at m/z 349 [M - Me]+ (see
Experimental Section). Therefore, 3 was identified as florlide A-11-
O-methyl ether or florxenilide C.6,16 A possible biogenetic pathway
of 3 might be explained by incorporation of a molecule of MeOH
to floride C at C-11, as illustrated in Scheme 1.6
Florxenilide A (1): [R]25 +8 (c 0.1, CH2Cl2); UV λmax (log ꢀ)
D
228 (3.16) nm; IR (CH2Cl2) νmax 3477 (OH), 2936 (CH), 1744, 1728,
1711 (CdO esters), 1653 (double bond), 1601 and 1490 (Ar CH), 1260,
1
1231 (C-O), 1023, 947, 714 cm-1; H NMR (300 MHz, CDCl3), see
Chromium oxide oxidation of 1 was performed in anhydrous
medium (Collin’s reagent)17 to afford 10-dehydroflorxenilide A (4).
The spectroscopic data are cited in the Experimental Section. The
isolated natural xenicane diterpenes, including the new compounds
1-3 together with the new xenicane derivatives 4-6, were tested
for cytotoxic activities against three human tumor cell lines. The
results revealed that florxenilides A (1) and B (2) exhibited
significant cytotoxicity against human colon cancer cells with IC50
values of 2.7 and 1.9 µg/mL (4.5, 3.7 µM), respectively, while
florxenilide B (1) had a very weak activity against KB and Hepa
tumor cells at 11.4 and 8.0 µg/mL, respectively. The derivatives
4-6 were all inactive (>20 µg/mL) against the three tumor cell
lines.
Table 1; 13C NMR (75 MHz, CDCl3), see Table 2; ESIMS m/z 619 [M
+ Na]+; HRESIMS m/z 619.2522 (calcd for C33H40O10Na, 619.2519).
Preparation of (R)- and (S)-MTPA Esters of Florxenilide A (1).
S-(+)- or R-(-)-MTPA chloride (one drop) was added to a solution of
1 (5 mg in 0.5 mL of pyridine), and the solution was allowed to stand
at room temperature for 7 h. After purification using preparative thin-
layer chromatography, the ester (4.8 mg, 96% yield) was submitted to
1H NMR analysis, and ∆δ values (δS - δR) were calculated for H-1,
H-8, H-9, and H-10.
Benzoylation of Florxenilide A (1). Thirty milligrams of 1 was
treated with benzoyl chloride (0.1 mL) and pyridine (2 mL) at 50 °C
for 15 h. Usual workup and extraction with EtOAc gave a residue.
Purification of the product by LH-20 using MeOH as eluent gave 27
mg of florxenilide A-10-O-benzoate (5), which showed the following:
[R]25D +0.8 (c 2.5, CH2Cl2); UV (MeOH) λmax (log ꢀ) 231 (0.79), 215
(0.49) nm; CD λmax (Mol. CD) 224 (-4.241), 239 (+4.438) nm; IR
(CH2Cl2) νmax 3061, 2931 (CH), 1737, 1730, 1715 (CdO esters), 1673,
Experimental Section
General Experimental Procedures. Optical rotations were recorded
on a JASCO DIP-1000 polarimeter. IR and UV spectra were measured
on Hitachi T-2001 and Hitachi U-3210 spectrophotometers, respectively.
The CD spectrum was recorded on a Jasco J-715 CD polarimeter. The
1H and 13C NMR, COSY, HMQC, HMBC, and NOESY spectra were
1650 (CdC), 1601, 1484 (Ar CH), 1245 (C-O), 1025, 947, 711 cm-1
;
1H NMR (CDCl3, 300 MHz) δ 8.06 (2H, d, 3′,7′-Bz), 7.98 (2H, d,
3′′,7′′-Bz), 7.57 (2H, m, 5′, 5′′-Bz), 7.46 (2H, t, 4′,6′-Bz), 7.43 (2H, t,
4′′,6′′-Bz), 6.44 (s, H-3), 5.96 (d, J ) 8.6 Hz, H-9), 5.78 (s, H-1), 5.74

678 Journal of Natural Products, 2006, Vol. 69, No. 4
Notes
(s, H-10), 5.69 (d, J ) 3.6 Hz, H-12), 5.65 (dd, J ) 10.0, 3.6 Hz,
H-13), 5.41 (s, H-19a), 5.28 (d, J ) 10.0 Hz, H-14), 5.26 (d, J ) 8.6
Hz, H-8), 5.21 (s, H-19b), 2.49 (br s, H-11a), 2.38 (br d, J ) 12.2 Hz,
H2-6), 2.10 (m, H2-5), 2.10 (3H, s), 2.08 (m, H-4a), 2.04 (3H, s), 1.97
(s, H-18), 1.97 (3H, s), 1.79, 1.76 (s, H-16 and H-17); 13C NMR (CDCl3,
75 MHz) δ 170.2 (C, CH3CO), 169.8 (C, CH3CO), 168.9 (C, CH3CO),
165.2 (C, CO Bz), 164.6 (C, CO Bz), 146.3 (C, C-11), 141.1 (C, C-15),
137.9 (CH, C-3), 137.8 (C, C-7), 133.3 (CH, C-5 Bz), 133.1 (CH, C-5
Bz), 130.3 (C, C-2 Bz), 129.9 (CH, C-3 Bz), 129.6 (C, C-2 Bz), 128.4
(CH, C-4 Bz), 128.3 (CH, C-4 Bz), 122.8 (CH, C-8), 118.5 (CH2, C-19),
117.8 (CH, C-14), 113.1 (C, C-4), 91.3 (CH, C-1), 82.0 (CH, C-10),
74.8 (CH, C-9), 71.5 (CH, C-13), 70.6 (CH, C-12), 43.6 (CH, C-11a),
38.4 (CH, C-4a), 29.0 (CH2, C-5), 25.8 (CH3, C-17), 20.9 (CH3, CH3-
CO), 20.8 (CH3, CH3CO), 20.6 (CH3, CH3CO), 18.6 (CH3, C-16), 18.3
(CH3, C-18).
128.4 (CH, 4′,6-Bz), 125.5 (CH, C-8), 117.4 (CH2, C-19), 116.1 (CH,
C-14), 113.4 (C, C-4), 91.2 (CH, C-1), 77.2 (CH, C-9), 71.5 (CH, C-13),
71.1 (CH, C-12), 42.3 (CH, C-11a), 41.4 (CH2, C-6), 37.7 (CH, C-4a),
28.1 (CH2, C-5), 25.8 (CH3, C-17), 21.1, 21.1, 21.0 (each CH3, 3x CH3-
CO), 18.8 (CH3, C-18), 18.2 (CH3, C-16).
Cytotoxicity Assay. The cytotoxicity assay depends on the binding
of methylene blue to fixed monolayers of three human tumor cell lines
(KB, WiDr, and Hepa), respectively. Samples and control standard
drugs were prepared at a concentration of 1, 10, 40, and 100 g/mL.
After seeding 2880 cells/well in a 96-well microtiter plate for 3 h, 20
L of sample or standard agent was placed in each well and incubated
at 37 °C for 3 days. The absorbance was measured on a microtiter
plate reader (Dynatech, MR 7000) at 650 nm. The ED50 value was
defined, by comparison with the untreated cells, as the concentration
of a test sample resulting in 50% reduction of absorbance. Mitomycin
was used as a positive control.
Florxenilide B (2): [R]25D +114 (c 0.1, CH2Cl2); UV (MeOH) λmax
(log ꢀ) 228 (3.15) nm; IR (CH2Cl2) νmax 3523 (OH), 2927 (CH), 1746,
1
1640, 1731 (CdO esters), 1228, 1025, 713 cm-1; H NMR (CDCl3,
Acknowledgment. The authors are grateful to the National Science
Council, Taipei, Taiwan, for financial support (grant #NSC 93-2323-
B-110-001). We thank Y.-S. Ching, NSC Southern MS Instrument
Center in the National Sun Yat-sen University, for measurement of
MS spectra.
300 MHz), see Table 1; 13C NMR (CDCl3, 75 MHz), see Table 2;
EIMS (30 eV) m/z 546, 490, 475, 398, 295, 277, 127, 105, 85, 77;
ESIMS m/z 635 [M + Na]+; HRESIMS m/z 635.2465 (calcd for
C33H40O11Na, 635.2468).
Florxenilide C (3): [R]25 +204 (c 0.02, CH2Cl2); UV (MeOH)
D
λmax (log ꢀ) 244 (3.72) nm; IR (CH2Cl2) νmax 3440 (OH), 3053 (Cd
References and Notes
C-H), 2930, 2931 (C-H), 1742 (CdO), 1655 (CdC), 1026, 735 cm-1
;
1H NMR (CDCl3, 300 MHz), see Table 1; 13C NMR (CDCl3, 75 MHz),
see Table 2; EIMS m/z 364 [M]+, 349 [M - Me]+, 346 [M - H2O]+,
141, 123, 105, 91, 79; FABMS m/z 387 [M + Na]+, 365 [M + H]+;
HRESIMS m/z 387.2146 (calcd for C21H32O5Na, 387.2147).
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Florxenilide C monoacetate (6): [R]25 +102 (c 0.02, CH2Cl2);
D
IR (CH2Cl2) νmax 3481 (OH), 2968, 2931 (C-H), 1746 (CdO), 1731
1
(CdO ester), 1653 (CdC), 1250 (C-O acetate), 1026, 733 cm-1; H
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NMR (CDCl3, 300 MHz) δ 6.17 (dd, J ) 15.0, 11.0 Hz, H-13), 6.02
(d, J ) 11.0 Hz, H-12), 5.91 (d, J ) 15.0 Hz, H-14), 4.94 (d, J ) 12.0
Hz, H-3a), 4.67 (br s, H-8), 4.37 (d, J ) 12.0 Hz, H-3a), 3.24 (s, OMe),
2.83 (m, H-4a), 2.78 (br s, H-11a), 2.11 (s, OAc), 1.35 (s, H-16 and
H-17), 0.99 (s, H-18); ESIMS m/z 429 [M + Na]+.
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Oxidation of 1 with Collin’s Reagent [(C5H5N)2CrO3]. Two drops
of chromium trioxide (40 mg in 3 mL of pyridine) were added to a
solution of 1 (20 mg in 2 mL of CH2Cl2), and the reaction mixture
was stirred at RT for 30 min. After filtration to remove CrO3, the filtrate
was concentrated and purified by NP-HPLC using n-hexane-CH2Cl2-
MeOH (20:15:1) for elution to provide 10-dehydroflorxenilide A (4, 5
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mg): [R]25 +101.7 (c 0.12, CH2Cl2); UV (MeOH) λmax (log ꢀ) 230
D
(3.11) nm; IR (CH2Cl2) νmax 3060 (CdC-H), 2924 (CH), 1754, 1746,
1731, 1714 (CdO), 1681, 1601 (Ar C-H), 1271, 1226 (C-O), 1024,
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Soc. 1991, 113, 4092-4096.
1
948, 712 cm-1; H NMR (CDCl3, 300 MHz), δ 8.12 (2H, d, J ) 7.8
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Mill Valley, CA, 1983; pp 15-17 and 79-80.
Hz, 3′,7′-Bz), 7.58 (t, J ) 7.8 Hz, 5′-Bz), 7.46 (2H, t, J ) 7.8 Hz,
4′,6′-Bz), 6.40 (s, H-3), 6.24 (d, J ) 6.5 Hz, H-9), 6.12 (br s, H-1),
5.57, (s, H-19), 5.52 (dd, J ) 9.4, 3.5 Hz, H-13), 5.49 (d, J ) 3.5 Hz,
H-12), 5.44 (s, H-19), 5.42 (d, J ) 6.5 Hz, H-8), 5.17 (d, J ) 9.4 Hz,
H-14), 2.25 (m, H-4a), 2.11 (s, OAc), 2.01 (s, OAc), 1.75 (3H, s, H-18),
1.67 (s, OAc), 1.26 (3H, s, H-16 & H-17); 13C NMR (CDCl3, 75 MHz),
δ 196.5 (C, C-10), 170.3, 169.8, 169.0 (each C, 3 × CH3CO), 165.7
(C, CdO Bz), 147.4 (C, C-11), 141.4 (C, C-15), 138.2 (C, C-7), 138.1
(CH, C-3), 133.2 (CH, 5′-Bz), 131.7 (C, 2′-Bz), 129.9 (CH, 3′,7′-Bz),
(15) Hiyaoka, H.; Mitome, H.; Nakano, M.; Yamada, Y. Tetrahedron
2000, 56, 7737-7740.
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NP058110C