Cytotoxic Terpenoids from the Formosan Soft Coral Nephthea brassica
Chang-Yih Duh,*,†Shang-Kwei Wang,‡Ying-Ling Weng,†Michael Y. Chiang,§and Chang-Feng Dai⊥Department of Marine Resources, National Sun Yat-sen University, Department of Microbiology, Kaohsiung Medical College, Department of Chemistry, and National Sun Yat-sen University, Kaohsiung, Taiwan, Republic of China, and Institute of Oceanography, National Taiwan University, Taipei, Taiwan, Republic of China
Received May 5, 1999
Two new cytotoxic cembranoid diterpenes, brassicolide (1) and brassicolide acetate (2); a new cytotoxic sesquiterpene, (-)-4R-O-acetyl-selin-11-en (3); and six cytotoxic terpenoids, (-)-selin-11-en-4R-ol (4), 2-hydroxynephthenol (5), nephthenol (6), cembrene A (7), epoxycembrene A (8), and (-)-β-elemene (9), have been isolated from the Formosan soft coral Nephthea brassica. The structures of compounds 1-9 were determined by spectral, chemical, and X-ray crystallographic analysis.
As part of our search for bioactive substances from marine organisms, the soft coral Nephthea brassica
Ku¨ kenthal (Nephtheidae) was studied based on the CH2
-Cl2extracts showing significant cytotoxicity in A549
(hu-man lung adenocarcinoma), HT-29 (hu(hu-man colon adeno-carcinoma), KB (human epidermoid adeno-carcinoma), and P-388 (mouse lymphocytic leukemia) cell cultures as determined
by standard procedures.1,2Bioassay-guided fractionations
resulted in the isolation of two new cytotoxic cembranoid diterpenes, brassicolide (1) and brassicolide acetate (2); a new cytotoxic sesquiterpene, (-)-4R-O-acetyl-selin-11-en (3); six cytotoxic terpenoids, (-)-selin-11-en-4R-ol (4), 2-hy-droxynephthnol (5), nephthenol (6), cembrene A (7), ep-oxycembrene A (8), and (-)-β-elemene (9).
Results and Discussion
Methylene chloride extraction of the freeze-dried animals followed by Si gel chromatography and recrystallization from hexane-acetone yielded 1 as a colorless crystalline solid, mp 115-116°. HRFABMS and the DEPT (135)
established the molecular formula of 1 as C20H30O3. Thus,
6 degrees of unsaturation were determined for 1. The IR spectrum of 1 exhibited the presence of a carbonyl group
of an R,β-unsaturated γ-lactone (νmax 1750 cm-1) and a
hydroxyl group (νmax3502 cm-1). A strong UV absorption
at λmax236 nm suggested the presence of a R,β-unsaturated
γ-lactone.1H and13C NMR spectral data (Table 1) showed
the structure of 1 contained an isopropyl carbinol (δC26.4,
28.5, q, C-16, 17; 73.0, s, C-15; δH 1.30, 1.32, 3H each, s
each, H-16, 17), an R,γ-disubstituted R,β-unsaturated
γ-lac-tone (δC172.9, s, C-18; 133.1, s, C-4; 149.7, d, C-3; 83.1, d,
C-2; δH7.08, s, H-3; 4.89, d, J ) 7.8 Hz, H-2), two isolated
methyl-bearing trisubstituted double bonds (δH4.96, 1H,
m, H-7; 4.98, 1H, m, H-11; 1.54, 3H, br s, H-19; 1.61, 3H,
br s, H-20; δC123.3, d, C-7; 133.2, s, C-8; 126.9, d, C-11;
135.9, s, C-12), one methine carbon (δC50.8, d, C-1), and
six methylene carbons (δC24.8, t, C-6; 25.0, t, C-5; 25.2, t,
C-10; 25.5, t, C-14; 38.4, t, C-9; 39.4, t, C-13). These data suggested that 1 possessed a cembrane skeleton with functionalities of an isopropyl carbinol, an R,γ-disubstituted R,β-unsaturated γ-lactone, and two isolated methyl-bearing trisubstituted double bonds. In the HMBC experiment (Table 1) of 1, the isopropyl carbinol group attached to C-1
was confirmed by long-range correlations between H-1 to C-2, C-3, C-13, C-14, C-15, C-16, and C-17; H-16 to C-1, C-15, and C-17; and H-17 to C-1, C-15, and C-16. The positioning of the R,β-unsaturated γ-lactone at C-4 (R), C-3 (β), C-2 (γ), and C-18 (carbonyl carbon) was deduced from HMBC correlations between H-2 and C-1, C-3, C-4, C-14, and C-15; H-3 and C-2, C-4, C-5, C-18; and H-5 and C-3, C-4, C-6, C-7, and C-18. The vinyl methyl group attached at C-8 was confirmed by HMBC correlations between H-19 and C-7, C-8, and C-9; H-7 and C-6 and C-19; and H-9 and C-7, C-8, C-10, and C-19. The other vinyl methyl group attached at C-12 was revealed by the HMBC correlations * To whom correspondence should be addressed. Tel.: 886-7-525-2000,
ext. 5036. Fax: 886-7-525-5020.
†Department of Marine Resources, National Sun Yat-sen University. ‡Kaohsiung Medical College.
§Department of Chemistry, National Sun Yat-sen University. ⊥National Taiwan University.
1518 J. Nat. Prod. 1999, 62, 1518-1521
10.1021/np990212d CCC: $18.00 © 1999 American Chemical Society and American Society of Pharmacognosy Published on Web 09/25/1999
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between H-20 and C-11, C-12, and C-13; H-11 and C-9, C-10, C-13, and C-20; and H-13 and C-11, C-14, and C-20. The relative configuration of 1 was finally established by
its X-ray diffraction analysis (Figure 1).3
Compound 2 was isolated as a colorless oil, whose
molecular formula, C22H32O4, was revealed by HRFABMS.
The IR spectrum of 2 exhibited the presence of a carbonyl
group of an R,β-unsaturated γ-lactone (νmax1755 cm-1) and
an ester carbonyl (νmax1725 cm-1). A strong UV absorption
at λmax234 nm suggested the presence of a R,β-unsaturated
γ-lactone.1H and13C NMR spectral data (Table 2) showed
the structure of 2 contained an acetoxyisopropyl side chain
(δC24.7, 24.6, q, C-16, 17; 83.5, s, C-15, 22.5, q; 170.0, s;
δH 1.59, 1.56, 3H each, s each, H-16, 17; 2.06, 3H, s), an
R,γ-disubstituted R,β-unsaturated γ-lactone (δC 173.3, s,
C-18; 134.6, s, C-4; 148.6, d, C-3; 81.9, d, C-2; δH7.05, s,
H-3; 5.01, d, H-2), two isolated methyl-bearing
trisubsti-tuted double bonds (δH4.98, 1H, m, H-7; 4.95, 1H, m, H-11;
1.50, 3H, br s, H-19; 1.55, 3H, br s, H-20; δC124.0, d, C-7;
134.6, s, C-8; 125.8, d, C-11; 133.9, s, C-12), one methine
carbon (δC49.4, d, C-1), and six methylene carbons (δC25.6,
t, C-6; 25.0, t, C-5; 24.1, t, C-10; 25.7, t, C-14; 38.8, t, C-9;
39.4, t, C-13). These data suggested that 2 possessed a cembrane skeleton with functionalities of an acetoxyiso-propyl, an R,γ-disubstituted R,β-unsaturated γ-lactone, and two isolated methyl-bearing trisubstituted double bonds. In the HMBC spectrum (Table 2) of 2, the acetoxyisopropyl attached to C-1 was confirmed by correlations between H-1 to C-13, C-14, C-16, and C-17; H-16 to C-1, C-15, and C-17; H-17 to C-1, C-15, and C-16. HMBC correlations between H-2 and C-1, C-3, C-4, and C-14; H-3 and C-2, C-4, C-5, and C-18; H-5 and C-6, C-7, and C-18 revealed the position of R,β-unsaturated γ-lactone. The vinyl methyl group attached at C-8 was confirmed by the HMBC correlations between H-19 and C-7, C-8, and C-9; H-7 and C-9 and C-19; H-9 and C-7, C-10, and C-19. The other vinyl methyl group attached at C-12 was confirmed by the HMBC correlations between H-20 and C-11, C-12, and C-13; H-13 and C-11, C-12, C-14, and C-20. NMR data of 2 looked similar to those
of 1 except for an additional methyl and a carbonyl at δH
2.06, 3H, s; δC 22.5, q and δC 170.0, s, respectively. In
addition, downfield shift of H-16, 17 from δ 1.30, 1.32 in 1 to δ 1.56, 1.58 in 2, as well as 10.5-ppm downfield shift of C-15 from δ 73.0 in 1 to δ 83.5 in 2 indicated that 2 may be a 15-acetate of 1. The relative configuration of 2 was
deduced from similarity of NOESY spectra and 1H-1H
coupling patterns between 2 and 1. Thus, 2 was confirmed as 15-acetate of 1.
Compound 4 was isolated as colorless needles, mp
92-94°, [R]20
D-16° (c 0.5, CHCl3). IR absortion at 3305 and
1641 cm-1indicated the presence of a hydroxyl group and
a terminal double bond. In the1H and13C NMR spectra,
the presence of the following moieties were deduced: an
isopropenyl group [δH4.65, 2H, d, J ) 6 Hz; 1.68, 3H, s; δC
108.1, t; 150.5, s; 22.5, q], two aliphatic tertiary methyl
groups [δH0.83, 3H, s; 1.06, 3H, s; δC18.6, q; 21.0, q], an
aliphatic quaternary carbon [δC 34.5], an oxygenated
qua-ternary carbon [δC 72.1], two sp3methine carbons [δC46.2,
d; 54.8, d], and six sp3methylene carbons [δ
C20.0, 25.9,
26.8, 41.0, 43.3, 44.6]. The identity of 4 as
(-)-selin-11-en-4R-ol was established by direct comparison of the [R]20
D,
IR, EIMS,1H NMR, and13C NMR data with those reported
in the literature.4-6
Table 1. NMR Data of 1a
position δH, mult. (Hz) δC; mult. HMBC NOESY
1 1.62-1.63 m 50.8; dc 2, 3, 13, 14, 15, 16, 17 13 2 4.89 d (7.8)b 83.1; d 1, 3, 4, 14, 15 1, 3, 14β 3 7.08 s 149.7; d 2, 4, 5, 18 2, 14R, 14β 4 133.1; s 5R 2.31-2.33 m 25.0; t 3, 4, 6, 7, 18 6R β 2.51-2.53 m 3, 4, 6, 7, 18 6β 6R 2.07-2.09 m 24.8; t 4, 5, 7 5R β 2.46-2.49 m 4, 5, 7 5β, 7, 19 7 4.96 m 123.3; d 6, 19 6R, 9 8 133.2; s 9 2.10-2.13 m 38.4; t 7, 8, 10, 19 7 10R 2.18-2.20 m 25.2; t 9, 11 20 β 2.42-2.44 m 9, 11 11 4.98 m 126.9; d 9, 10, 13, 20 12 135.9; s 13 2.03-2.06 m 39.4; t 11, 14, 20 1, 20 14R 1.11-1.15 m 25.5; t 1, 2, 13 1, 3 β 1.61-1.63 m 1, 2, 13 2, 16, 17 15 73.0; s 3 16 1.32 s 28.5; q 1, 15, 17 14β 17 1.30 s 26.4; q 1, 15, 16 14β 18 172.9; s 19 1.54 s 16.1; q 7, 8, 9 6β 20 1.61 s 16.0; q 11, 12, 13 10R, 13 aSpectra recorded in CDCl
3.bJ values (in Hz) in parentheses.
cMultiplicity deduced by DEPT and indicated by usual symbols.
Figure 1. Molecular structure (relative configuration) of compound 1.
Table 2. NMR Data of 2a
position δH, mult. (Hz) δC; mult. HMBC NOESY
1 2.18-2.21 m 49.4; dc 13, 14, 16, 17 2, 13 2 5.01 d (6.3)b 81.9; d 1, 3, 4, 14 1, 3, 14β 3 7.05 s 148.6; d 2, 4, 5, 18 2, 14R, 14β 4 134.6; s 5R 2.39-2.41 m 25.0; t 6, 7, 18 6R β 2.49-2.52 m 6, 7, 18 6β 6R 2.17-2.19 m 25.6; t 7, 8 5R, 7 β 2.52-2.55 m 7, 8 5β, 19 7 4.98 m 124.0; d 9, 19 6R 8 134.6; s 9 2.06-2.09 m 38.8; t 7, 10, 19 7 10R 2.06-2.09 m 24.1; t 9, 11 20 β 2.15-2.18 m 9, 11 11 4.95 m 125.8; d 12 133.9; s 13 2.03-2.06 m 39.4; t 11, 12, 14, 20 1, 20 14R 1.12-1.16 m 25.7; t 2 1, 3, 11 β 2.16-2.20 m 1, 13 2, 16, 17 15 83.5; s 3 16 1.59 s 24.7; q 1, 15, 17 14β 17 1.56 s 24.6; q 1, 15, 16 14β 18 173.3; s 19 1.50 s 15.5; q 7, 8, 9 6β, 10, 14 20 1.55 s 15.9; q 11, 12, 13 10R, 13 OAc 2.06 s 22.5; q 170.0; s aSpectra recorded in CDCl
3.bJ values (in Hz) in parentheses.
cMultiplicity deduced by DEPT and indicated by usual symbols. Cytotoxic Terpenoids from Coral Journal of Natural Products, 1999, Vol. 62, No. 11 1519
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Compound 3 was isolated as a colorless oil, whose
molecular formula, C17H28O2, was established by HREIMS.
IR absortion at 1730 and 1643 cm-1indicated the presence
of an ester carbonyl group and a terminal double bond. The
1H and13C NMR data (Table 3) showed the structure of 3
contained an isopropenyl group [δH4.73, 1H, s; 4.71, 1H,
s; 1.76, 3H, s; δC 108.2, t; 150.8, s; 20.9, q], two aliphatic
tertiary methyl groups [δH0.92, 3H, s; 1.41, 3H, s; δC18.8,
q; 29.1, q], an aliphatic quaternary carbon [δC 34.7], an
oxygenated quaternary carbon [δC85.8], two sp3methine
carbons [δC46.2, d; 51.9, d], and six sp3methylene carbons
[δC19.6, 26.3, 26.9, 37.8, 40.6, 44.9]. NMR data of 3 were
similar to those of 4 except for an additional methyl and a
carbonyl at δH 1.95, 3H, s; δC 22.8, q and δC 170.5, s,
respectively. The 0.29 ppm downfield shift of H-14 from δ 1.12 in 4 to δ 1.41 in 3 and 14.0 ppm downfield shift of C-4 from δ 71.8 in 4 to δ 85.8 in 3 indicated 3 to be a 4-acetyl derivative of 4. The C-4-acetoxy substitution in 3 was further confirmed by HMBC correlations (Table 3) between H-14 and C-3, C-4, and C-5; H-5 and C-3, C-4, C-6, and
C-14. Hydrolysis of 3 with methanolic K2CO3 at 40 °C
afforded 3. The structure of 4 was thus established as (-)-4R-O-acetyl-selin-11-en. The identity of compounds 5-9
was established by direct comparison of the [R]20
D, IR,
EIMS, 1H NMR, and 13C NMR data with literature
data.7-11
Cytotoxicity of compounds 1-9 was shown in Table 4. Compounds 2 and 5-8 exhibited cytotoxicity against A549, HT-29, KB, and P-388 cell lines. Compounds 1 and 4 were not active against A549 cells; however, their acetates (2 and 3) exhibited cytotoxicity toward A549 cells. Compound 3 was not cytotoxic against HT-29 cells.
Experimental Section
General Experimental Procedures. Melting points were determined using a Yanagimoto micromelting point apparatus and are reported uncorrected. Optical rotations were deter-mined on a JASCO DIP-181 polarimeter. UV spectra were obtained on a Shimadzu UV-160A spectrophotometer, and IR spectra were recorded on a Hitachi 26-30 spectrophotometer.
1H and13C NMR spectra were recorded with a Brucker AMX
400 NMR spectrometer at 400 and 100.6 MHz, respectively, in CDCl3using TMS as internal standard. EIMS were obtained with a JEOL JMS-SX/SX 102A mass spectrometer at 70 ev. Si gel 60 (Merck, 230-400 mesh) was used for column chromatography; precoated Si gel plates (Merck, Kieselgel 60 F254, 0.25 mm) were used for TLC analysis.
Animal Material. The soft coral N. brassica (Nephtheidae) was collected at Liouchou Island of Taiwan in September 1996, at a depth of 10 m and was stored for 2 days in a freezer until extraction. A voucher specimen, NSUSL-1001, was deposited in the Department of Marine Resources, National Sun Yat-sen University, Taiwan.
Extraction and Isolation. The bodies of the soft coral N.
brassica were freeze-dried to give 610 g of a solid, which was
extracted with CH2Cl2(1.5 L× 3). After removal of solvent in vacuo, the residue (52 g) was chromatographed over Si gel 60 using CHCl3and CHCl3-MeOH mixtures of increasing polar-ity. Elution by CHCl3afforded fractions containing compounds 3, 4, 6, and 7. Elution by CHCl3-MeOH (99:1) afforded fractions containing cembranoids 8 and 9. Elution by CHCl3 -MeOH (19:1) afforded fractions containing compounds 1, 2, and 5. Compounds 7, 3, 4, and 6 were obtained by Si gel column chromatography, by eluting with n-hexanes-EtOAc (100:1),
n-hexanes-EtOAc (50:1), n-hexanes-EtOAc (30:1), and
n-hex-anes-EtOAc (10:1), respectively. Compounds 9 and 8 were obtained by Si gel column chromatography, by eluting with
n-hexanes-EtOAc (100:1) and n-hexanes-EtOAc (30:1),
re-spectively. Compounds 2, 1, and 5 were obtained by Si gel column chromatography, by eluting with n-hexane-acetone (6:1), n-hexane-acetone (3:1), and n-hexane-acetone (2:1), respectively.
Brassicolide (1): colorless prism (193 mg); mp 115-116 °C; [R]25
D+50.3° (c 3.7, CHCl3); UV (MeOH) λmax(log ) 236 (4.1) nm; IR (KBr) νmax 3502, 2972, 1750 cm-1;1H and 13C NMR, see Table 1; FABMS m/z 319 [M]+(4), 301 (33), 255 (12), 215 (8), 165 (16), 133 (100), 121 (27), 91 (60, 77 (44); HR-FABMS m/z 319.2253 (calcd for C20H31O3319.2265).
Brassicolide acetate (2): colorless oil (60 mg); [R]25 D +101.0° (c 0.25, MeOH); UV (MeOH) λmax(log ) 234 (4.4) nm; IR (KBr) νmax 2924, 1755, 1725 cm-1;1H and13C NMR, see Table 2; FABMS m/z 361 [MH]+(1), 301 (27), 255 (7), 137 (45), 81 (86), 69 (100); HRFABMS m/z 361.2380 (calcd for C22H33O4 361.2370).
(-)-4r-O-Acetyl-selin-11-en (3): colorless oil (56 mg); [R]25
D-41.6° (c 0.45, CHCl3); IR (KBr) νmax1730, 1643 cm-1;
1H and13C NMR, see Table 3; EIMS m/z 264 [M]+
(1), 204 (59), 189 (100), 161 (37), 147 (46), 133 (67), 108 (52), 43 (60); HREIMS [M]+m/z 264.2162 (calcd for C17H28O2264.2171).
Hydrolysis of (-)-4r-O-Acetyl-selin-11-en (3).
Com-pound 3 (7 mg) was treated with 0.5% K2CO3-MeOH at 40
°C for 48 h. The resulting mixture was purified by column chromatography over Si gel using n-hexanes-EtOAc (10:1) as eluting solvent to afford 4 (4 mg).
Single-crystal X-ray Analysis of Brassicolide (1). Crys-tal data: C20H30O3, space group P21, a ) 8.95 (6) Å, b ) 24.3 (2) Å, c ) 8.79 (1) Å, V ) 1913 (19) Å3, Z ) 4, Dcalc ) 1.106 g/cm3, λ (Mo KR) ) 0.71069 Å. Intensity data were measured on a Rigaku AFC6S diffractometer up to 2θ of 50.1°. A total of 1966 reflections were observed [I >3σ(I)]. The structure was solved by the direct methods (SIR92),12and the final structure parameters were obtained by a full-matrix least-squares process. In view of the absence of heavy atom in the structure, Friedel pairs were not collected, and the absolute configuration of brassicolide (1) was not determined via diffraction method. Calculated hydrogen positions were put in the final cycle of
Table 3. NMR Data of 3a
position δH, mult. (Hz) δC; mult. HMBC NOESY
1 1.14 m 40.6; tc 2 1.37 m 2 1.53 m 19.6; t 1 3R 2.63 m 37.8; t 1, 4, 5 5 β 1.50 m 4 85.8; s 5 1.62 dd (10.1, 3.1)b 51.9; d 3, 4, 6 3R 6 1.76 m 26.3; t 5 7 1.98 m 46.2; d 8 1.52 m 26.9; t 6 9 1.27 m 44.9; t 5, 8, 15 1.41 m 10 34.7; s 11 150.8; s 12 4.72 d (6.0) 108.2; t 7, 13 13 1.76 s 20.9; q 7, 11, 12 14 1.41 s 19.1; q 4, 5 15 0.92 s 18.8; q 1, 5, 9, 10 OAc 1.95 s 22.8; q 170.5; q aSpectra recorded in CDCl
3.bJ values (in Hz) in parentheses.
cMultiplicity deduced by DEPT and indicated by usual symbols.
Table 4. Cytotoxicitiyaof 1-9 (n ) 8)
ED50(µg /mL) in indicated cell line
compound A549 HT-29 KB P-388 1 >50 5.81 2.40 2.44 2 3.03 0.81 0.72 1.20 3 0.12 >50 >50 2.44 4 >50 >50 >50 3.86 5 0.71 1.02 0.23 1.80 6 2.72 3.01 1.81 0.42 7 1.11 3.42 3.73 1.10 8 3.61 2.72 3.41 0.40 9 >50 >50 >50 0.92
aFor significant activity of pure compounds, an ED
50of e 4.0
µg/mL is required.1
1520 Journal of Natural Products, 1999, Vol. 62, No. 11 Duh et al.
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structure factor calculation but not refined. The agreement indices were R(F) ) 0.079, Rw(F) ) 0.050 with anisotropic refinement done on all nonhydrogen atoms. Experimental details are shown in Table 5. Final atomic coordinates are listed in Table 6.
Cytotoxicity Testing. KB and P-388 cells were supplied by Prof. J. M. Pezzuto, University of Illinois at Chicago; A549 and HT-29 were purchased from the American Type Culture Collection.
The P-388 cells were cultured in Fisher’s medium supple-mented with 10% heat-inactivated fetal calf serum (FCS). The KB cells were maintained in Basal Medium Eagle (BME) containing 10% heat-inactivated FCS. The A549 cell line was cultured in Eagle Minimum Essential Medium (EMEM) containing Earle’s salts and supplemented with 0.1 mM of nonessential amino acids and 10% heat-inactivated FCS. The HT-29 cell lines were maintained in Rosewell Park Memorial Institute (RPMI) 1640 medium containing 10% heat-inacti-vated FCS. All the cell lines were maintained in an incubator at 37 °C in humidified air containing 5% CO2. For routine cytotoxicity assay, all four cell lines were adapted to one single medium, RPMI 1640 supplemented with 10% FCS and 1 mM glutamate.
The cytotoxic activities of tested compounds or fractions against P-388, KB, A549, and HT-29 were assayed with modification of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphen-yltetrazolium bromide] colorimetric method. For P-388 cells, 200 µL of culture was established at 1500 cells/well in 96-well tissue culture plates (Falcon). Tested compounds were subse-quently dispensed to the established culture plate at eight concentrations, each with three replicates. After 3 days of incubation, P-388 cells were enumerated with MTT.
To measure the cytotoxic activities of pure compounds or crude fractions against A549, HT-29, KB, and P-388, each cell line was initiated at 750, 750, 2000, and 1500 cells/well, respectively, in 96-well microtiter plates. Three to eight concentrations encompassing an 8- to 128-fold range were evaluated on each cell line. A549, HT-29, KB, and P-388 cells were enumerated using MTT after the exposure to test samples for 6, 6, 3, and 3 days, respectively. Of 1 mg/mL MTT, 50 mL was added to each well, and plates were incubated at
37 °C for a further 4 h. The supernatant was aspirated with a Dynatech automatic washer. Formazan crystals were redis-solved in DMSO (Merck) for 10 min with shaking, and the plate was read immediately on a microtiter plate reader
(Dynatech) at a wavelength of 540 nm. The ED50value was
defined as the concentration of test compound resulting in a 50% reduction of absorbance compared to untreated cell in the MTT assay.13
Acknowledgment. We thank Prof. J. M. Pezzuto, Program for Collaborative Research in Pharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago, for the provision of P-388 and KB cell lines. This work was supported by a grant from the National Science Council of Taiwan (NSC-89-2314-B-110-002) awarded to C.-Y. Duh.
Supporting Information Available: X-ray crystallograpphic data
for brassicolide (1), including an ORTEP drawing, experimental and calculation details, and tables of atomic coordinates, anisotropic displacement parameters, bond lengths and angles, and nonbonded contacts. This material is available free of charge via the Internet at http://pubs.acs.org.
References and Notes
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(2) Hou, R.-S.; Duh, C.-Y.; Chiang, M. Y.; Lin, C.-N. J. Nat. Prod. 1995,
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(3) Crystallographic data for 1 have been deposited with the Cambridge Crystallographic Data Centre. Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (Fax: +44-(0)1223-336033 or E-mail: [email protected]).
(4) Kesselmans, R. P. W.; Wijnberg, J. B. P. A.; Minnaard, A. J.; Walinga, R. E.; de Groot, A. J. Org. Chem. 1991, 56, 7237-7244.
(5) Chetty, G. L.; Zalkow, V. B.; Zalkow, L. H. Tetrahedron Lett. 1968, 3223-3225.
(6) Corbett, R. E.; Smith, R. A. J. Tetrahedron Lett. 1967, 1009-1012. (7) Suzuki, M.; Shimada, A.; Kato, T. Chem. Lett. 1978, 759-762. (8) Schwabe, R.; Farkas, I.; Pfander, H. Helv. Chim. Acta 1988, 71,
292-297.
(9) Vanderah, D. J.; Rutledge, N.; Schimitz, F. J.; Ciereszko, L. S. J. Org.
Chem. 1978, 43, 1614-1616.
(10) Bowden, B. F.; Coll, J. C.; Mitchell, S. J.; Kazlauskas, R. Aust. J.
Chem. 1981, 34, 1551-1556.
(11) Irie, T.; Yamato, K.; Masamune, T. Bull. Chem. Soc. Jpn. 1964, 37, 1053-1055.
(12) Attomare, A.; Cascarano, M.; Giacovazzo, C.; Guagliardi, A. J. Appl.
Cryst. 1993, 26, 343-348.
(13) Mosmann, T. J. Immunol. Method 1983, 65, 55-63.
NP990212D
Table 5. Crystal Data and Intensity Collection Data for
Brassicolide (1)
empirical formula C20H30O3
fw 318.46
color, habit colorless, prism diffractormeter used Rigaku AFC6S space group P212121(#19) a, Å 8.95 (6) b, Å 24.3 (2) c, Å 8.79 (1) V, Å3 1913 (19) Z 4 Dcalcd, g cm-3 1.106 g/cm3 λ(Mo KR), Å 0.71069 F(000) 696.00
unit cell detn (no. 2θ range, deg) 21, 2θ (17.1-25.3°)
scan type ω-2θ
µ(Mo KR), cm-1 0.72 cm-1
crystal size, mm 0.33× 0.66 × 0.72 mm transm factor 0.6273-1.000
temp, °C 23.0
no. of measd reflns 1966 no. of obsd reflns (No) 1119
R,aR
wa 0.079, 0.050
GOFa 4.53
no. of ref params (Np) 208
max peak in final diff. map 0.33 e-/Å3
min peak in final diff. map -0.31 e-/Å3
aR ) [∑||F
o| - |Fc||/∑|Fo|. Rw) [∑w(|Fo| - |Fc|)2/∑w|Fo|2]1/2.
GOF ) [∑w(|Fo| - |Fc|)2/(No- Np)]1/2.
Table 6. Atomic Parameters for Brassicolide (1)
atom x y z Beq O(1) 0.7896(8) 0.3674(3) 0.5101(6) 4.3(2) O(2) 0.8212(9) 0.4021(3) 0.7445(7) 6.7(2) O(3) 0.9032(7) 0.4048(2) 0.0538(6) 3.7(2) C(1) 0.908(1) 0.3424(3) 0.2717(10) 2.9(2) C(2) 0.812(1) 0.3869(4) 0.3541(9) 3.2(2) C(3) 0.872(1) 0.4446(4) 0.3755(9) 3.6(3) C(4) 0.888(1) 0.4568(4) 0.524(1) 3.7(3) C(5) 0.952(1) 0.5084(4) 0.599(1) 4.5(3) C(6) 1.123(1) 0.5080(4) 0.604(1) 3.9(3) C(7) 1.189(1) 0.4593(3) 0.697(1) 3.3(2) C(8) 1.321(1) 0.4378(4) 0.675(1) 4.2(3) C(9) 1.378(1) 0.3913(4) 0.781(1) 4.8(3) C(10) 1.418(1) 0.3368(4) 0.702(1) 4.3(3) C(11) 1.293(1) 0.3158(3) 0.604(1) 3.8(3) C(12) 1.289(1) 0.3050(3) 0.457(1) 3.2(2) C(13) 1.141(1) 0.2939(3) 0.380(1) 3.6(3) C(14) 1.0725(10) 0.3491(4) 0.326(1) 3.5(2) C(15) 0.884(1) 0.3474(4) 0.0955(10) 3.4(2) C(16) 0.726(1) 0.3281(3) 0.053(1) 4.7(3) C(17) 0.993(1) 0.3105(3) 0.010(1) 5.6(3) C(18) 0.833(1) 0.4080(4) 0.609(1) 4.4(3) C(19) 1.428(1) 0.4547(4) 0.554(1) 5.9(3) C(20) 1.421(1) 0.3089(4) 0.353(1) 5.6(3)
Cytotoxic Terpenoids from Coral Journal of Natural Products, 1999, Vol. 62, No. 11 1521
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