Cytotoxic activity of some natural and synthetic ent-kauranes

Article abstract of DOI:10.1021/np060504w

Atractyligenin (1) and several synthetic derivatives were tested and found to be active against tumor cell replication. Compound 1 was readily converted to the 2,15-diketo (3) or 15-keto (4) derivatives, which contain an α,β-unsaturated ketone. Compounds

Full text of DOI:10.1021/np060504w

J. Nat. Prod. 2007, 70, 347-352
347
Cytotoxic Activity of Some Natural and Synthetic ent-Kauranes
Sergio Rosselli,† Maurizio Bruno,*,† Antonella Maggio,† Gabriella Bellone,† Tzu-Hsuan Chen,‡ Kenneth F. Bastow,§ and
Kuo-Hsiung Lee*,‡
Dipartimento di Chimica Organica, UniVersita` di Palermo, Viale delle Scienze, 90128 Palermo, Italy, Natural Products Research Laboratories,
School of Pharmacy, UniVersity of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, and DiVision of Medicinal Chemistry
and Natural Products, School of Pharmacy, UniVersity of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
ReceiVed October 13, 2006
Atractyligenin (1) and several synthetic derivatives were tested and found to be active against tumor cell replication.
Compound 1 was readily converted to the 2,15-diketo (3) or 15-keto (4) derivatives, which contain an R,â-unsaturated
ketone. Compounds 3 and 4 showed significant cytotoxic activity against all six tested cancer cell lines and were most
potent against 1A9 ovarian cancer cells with EC50 values of 0.2 and 0.3 µM, respectively. These two 1-analogues are
promising lead compounds for further investigation.
ent-Kauranes are naturally occurring diterpenoids isolated from
several plant families, such as the Asteraceae and Lamiaceae. These
compounds have attracted interest because of their structures and
biological activities, including antitumor, anti-HIV, and antibacterial
effects.1 Extensive chemical work2,3 has been carried out on the
structure of atractyligenin, the nor-diterpene aglycon of the gluco-
side atractyloside, which occurs, together with its homologous
diterpene, carboxyatractyloside, in the roots of Atractylis gummifera
L. (Asteraceae). Interest in these compounds was stimulated by the
high toxicity4 of both glucosides, which have caused many deadly
poisonings, and by the antimicrobial and cytotoxic activities of some
derivatives.5
Figure 1.
Scheme 1a
In this paper, we report the conversion of the 15-hydroxy group
of atractyligenin (1, Figure 1) to a ketone in order to incorporate
an R,â-unsaturated ketone into the ent-kaurane skeleton. It is well
known that a main structural determinant for cytotoxicity is the
presence of an R,â-unsaturated system, which likely serves as an
alkylating center and can be part of an ester, ketone, or lactone
moiety.6 We also report the derivatization of the 2-hydroxy group.
a (i) MnO2, dry THF, rt.
Results and Discussion
The presence of a free hydroxy group allowed us to prepare ester
derivatives of 4 in order to evaluate the influence of an ester side
chain on the biological properties of atractyligenin (Scheme 2). First,
we synthesized the more lipophilic acetyl derivative 5 by treating
4 with Ac2O-pyridine. Because it was observed previously that
the introduction of piperonyl esters enhanced the biological
properties of some ent-kaurane derivatives,7 we prepared the
piperonyl ester 6 by treating 4 with piperonylic acid, DCC, and
DMPA. Finally, as previously reported by us for some guaiane
derivatives,8 in order to enhance the cytotoxic activity, the free C-2
hydroxyl group of compound 4 was esterified with the side chain
of paclitaxel. Treatment of alcohol 4 with compound 7, prepared
according to a previously reported procedure9 from commercially
available (2R,3S)-3-phenylisoserine hydrochloride, gave ester 8.
Acidic hydrolysis yielded 9, with the same side chain as paclitaxel
(Scheme 2).
To evaluate whether an oxygenated function at C-2 was needed
for biological activity, we planned to remove the C-2 keto group
of 3. Our synthetic design included the selective formation of a
C-2 thioketal followed by a Mozingo reduction. However, because
the R,â-unsaturated ketone in 3 could undergo an unwanted Michael
reaction, we decided to remove this system temporarily. As shown
in Scheme 3, compound 3 was treated with ethylene glycol and
PTSA in benzene to give 10. The 1H and 13C NMR spectra showed
signals for the protecting group in the range δH 4.05-3.76 (m, 4H),
the presence of a ketal carbon (δC 108.1 s), and the absence of the
carbonyl group at δC 207.7 (C-2). Reduction of compound 10 with
Atractyligenin methyl ester (2, Figure 1), prepared by treating 1
with CH2N2, was reacted with MnO2 for 15 min until the starting
material disappeared completely on TLC. A single product (3) was
obtained. Its 1H and 13C NMR spectra showed a downfield shift of
the exocyclic protons (δH 5.96, H-17a; δH 5.28, H-17b), the presence
of two ketones at δC 207.7 (C-2) and δC 209.6 (C-15), and the
absence of hydroxy groups. Consequently, compound 3 is consistent
with the structure of 2,15-diketoatractyligenin methyl ester. How-
ever, when the same reaction was stopped after 3 min, TLC showed
the presence of 3 as a minor product and a more polar compound
(4) as the main product (Scheme 1). The 1H and 13C NMR spectra
of 4 also indicated a downfield shift of the exocyclic protons (δH
5.95, H-17a; δH 5.27, H-17b), but showed only one ketone at δC
210.3 (C-15) with a hydroxy group (δH 4.25, H-2), rather than a
ketone, at C-2. Compound 4 was assigned the structure of
15-ketoatractyligenin methyl ester.
Dedicated to the late Dr. Kenneth L. Rinehart of the University of
Illinois at Urbana-Champaign for his pioneering work on bioactive natural
products.
* Corresponding authors. (K.H.L.) Tel: 919-962-0066. Fax: 919-966-
3893. E-mail: khlee@unc.edu. (M.B.) Tel: +39091596905. Fax:
+39091596825. E-mail: bruno@dicpm.unipa.it.
† Universita` di Palermo.
‡ Natural Products Research Laboratories, University of North Carolina
at Chapel Hill.
§ Division of Medicinal Chemistry and Natural Products, University of
North Carolina at Chapel Hill.
10.1021/np060504w CCC: $37.00
© 2007 American Chemical Society and American Society of Pharmacognosy
Published on Web 01/05/2007

348 Journal of Natural Products, 2007, Vol. 70, No. 3
Rosselli et al.
Scheme 2a
a (i) Ac2O, Py, rt; (ii) piperonylic acid, DCC, DMAP, CH2Cl2, rt; (iii) (4S,5R)-2,4-diphenyl-4,5-dihydro-oxazol-5-carboxylic acid (7), DCC, DMAP, CH2Cl2, rt; (iv)
p-TsOH, CH2Cl2, rt; (v) TsCl, Py, rt;
Scheme 3a
a (i) HOCH2CH2OH, p-TsOH, C6H6, reflux; (ii) NaBH4, MeOH, rt; (iii) p-TsOH, MeOH/H2O, rt; (iv) HSCH2CH2SH, p-TsOH, C6H6 reflux; (v) H2, Pd/C, MeOH,
rt; (vi) PCC, CH2Cl2, rt.
Scheme 4
NaBH4 in MeOH gave alcohol 11 with a 15â-hydroxy group (δH
3.76, H-15R), the opposite stereochemistry of that of atractyligenin
(1). Removal of the C-2 protecting group gave ketone 12, as clearly
indicated by a carbonyl signal at δC 208.8 (C-2). At this point, we
attempted the thioketalization of 12 by reflux with ethanedithiol
and PTSA. However, the 1H and 13C NMR spectra of the resulting
product showed unexpected signals. In fact, there were no signals
for the thioketal group, the exocyclic double bond, or the C-15
hydroxy group, whereas resonances for a methyl group (δH 1.09,
CH3-17; δC 9.9, C-17) and for two ketones (δC 207.8, C-2; δC 223.7,
C-15) were observed. Consequently, this product was assigned the
structure 13. Hypothetically, compound 13 could be produced
1
through formation of a C-16 carbocation in the strongly acidic
medium, followed by proton transposition and formation of a
saturated ketone (Scheme 4). This proposed mechanism would
require the C-17 methyl group to be â. To verify this stereochem-
istry, atractyligenin methyl ester (2) was catalytically reduced with
H2 and Pd/C to give two products in a 4:1 ratio (Scheme 3). The
δH 3.62, d, 1H, J ) 7.6 Hz, H-15) were observed in the H NMR
spectrum of the minor product. The values of the H-15/H-16
coupling constant allowed us to assign the major product as 14
with a â-oriented C-17 methyl group and the anomeric 15 to the
minor product with R-orientation. Oxidation of 14 with PDC gave
13, confirming the proposed stereochemistry. Since our first
reduction strategy failed, we next planned to remove the C-2
oxygenated moiety by forming the C-2 tosylate, followed by LiAlH4
reduction and reoxidation of the C-15 hydroxyl group. Treatment
1
major product showed H NMR signals for a secondary methyl
group (δH 1.14, d, 3H, CH3-17) and a secondary alcohol (δH 3.26
d, 1H, J ) 4.2 Hz, H-15). Similar signals (δH 0.96, d, 3H, CH3-17;

Cytotoxic ActiVity of ent-Kauranes
Journal of Natural Products, 2007, Vol. 70, No. 3 349
Scheme 5a
a (i) p-TsNHNH2, EtOH, 70 °C; (ii) NaBH3CN, p-TsOH, DMF/sulfolane, 120 °C; (iii) PCC, CH2Cl2, rt.
Table 1. Effect of 1-6, 8, 9, 16, 17, and 19 against Tumor
H2O, was used for column chromatography. Preparative TLC was
performed using Merck glass plates (product code 1.13895.0001). The
optically pure (2R,3S)-3-phenylisoserine hydrochloride was purchased
from Industrial Chemistry Research (Warsaw, Poland). CH2Cl2 was
dried by distillation over calcium hydride.
Cell Line Replication
EC50 (µM)
compound
A549
PC-3
1A9
MCF-7
KB
KB-VIN
1
2
3
4
5
6
8
9
NA
NA
3.4
1.0
3.3
19.0
2.2
1.7
2.3
24.5
4.4
NA
NA
1.1
4.0
1.4
9.2
2.2
0.6
2.6
15.8
3.1
2.0
NA
NA
0.2
0.3
0.5
3.0
1.8
0.4
0.5
7.5
0.7
0.1
NA
NA
1.0
2.0
0.9
21.4
11.8
1.5
4.3
18.0
5.0
NA
NA
1.6
1.5
3.6
1.1
3.0
2.6
1.4
18.1
1.3
0.4
NA
NA
1.6
2.6
4.0
3.0
4.8
3.5
3.5
20.4
1.3
1.7
Synthesis of Compound 2. Compound 2 was prepared from
atractyligenin (1) and CH2N2 as previously reported,11 and its physical
and spectroscopic data agreed with those reported in the literature.12
Oxidation of Atractyligenin Methyl Ester (2). A solution of 2 (3
g, 9 mmol) in 300 mL of dry THF was added to 3.6 g (42 mmol) of
MnO2 and left to stir for 3 min at room temperature. After filtration
through a Millipore filter (45 µm) and column chromatography (Si gel,
2:1 petroleum ether-EtOAc as eluent), compounds 3 (600 mg, 20%
yield) and 4 (2.1 g, 70% yield) were obtained.
Compound 3: amorphous solid; [R]D25-154.6 (c 0.05, CHCl3); IR
(film) νmax 2985, 1724, 1647, 1431, 1377, 1247, 1193, 1173, 1138,
959 cm-1; 1H NMR (CDCl3, 250 MHz) δ 5.96 (1H, brs, H-17a), 5.28
(1H, brs, H-17b), 3.65 (3H, s, OCH3), 3.07 (1H, m, H-13), 3.05 (1H,
m, H-4), 2.87 (1H, ddd, J ) 14.8, 2.3, 2.3 Hz, H-3R), 2.58 (1H, dd, J
) 14.1, 2.3 Hz, H-1R), 2.42 (1H, dd, J ) 14.8, 7.5 Hz, H-3â), 2.28
(1H, d, J ) 12.1 Hz, H-14a), 1.84 (1H, d, J ) 14.1 Hz, H-1â), 0.94
(3H, s, Me-20); 13C NMR (CDCl3, 62.7 MHz) δ 209.6 (C, C-15), 207.7
(C, C-2), 173.7 (C, C-19), 148.7 (C, C-16), 115.0 (CH2, C-17), 55.2
(CH2, C-1), 51.6 (CH3, OMe), 51.6 (C, C-8), 50.0 (CH, C-9), 47.4
(CH, C-5), 45.1 (CH, C-4), 42.9 (C, C-10), 42.8 (CH2, C-3), 37.7 (CH,
C-13), 35.8 (CH2, C-14), 32.7 (CH2, C-7), 31.6 (CH2, C-12), 24.0 (CH2,
C-6), 18.0 (CH2, C-11), 16.6 (CH3, C-20); EIMS m/z 330 [M]+ (4),
296 (100), 268 (21), 189 (16), 143 (11), 107 (12), 91 (62); anal. C
72.72%, H 7.90%, calcd for C20H26O4, C 72.70%, H 7.93%.
Compound 4: amorphous solid; [R]D25 -170.4 (c 0.11, CHCl3); IR
(film) νmax 3433, 3055, 2933, 1718, 1672, 1643, 1448, 1265, 1198,
1047, 931 cm-1; 1H NMR (CDCl3, 250 MHz) δ 5.95 (1H, brs, H-17a),
5.27 (1H, brs, H-17b), 4.25 (1H, dddd, J ) 11.8, 11.8, 4.3, 4.3 Hz,
H-2), 3.67 (3H, s, OCH3), 3.06 (1H, m, H-13), 2.70 (1H, m, H-4),
2.42 (1H, m, H-3R), 2.35 (1H, d, J ) 12.0 Hz, H-14a), 2.20 (1H, dd,
J ) 11.8, 4.3 Hz, H-1R), 0.96 (3H, s, Me-20), 0.74 (1H, dd, J ) 11.8,
11.8 Hz, H-1â); 13C NMR (CDCl3, 62.7 MHz) δ 210.3 (C, C-15), 175.3
(C, C-19), 149.1 (C, C-16), 114.9 (CH2, C-17), 64.0 (CH, C-2), 52.2
(C, C-8), 51.4 (CH3, OMe), 50.8 (CH, C-9), 48.3 (CH2, C-1), 48.3
(CH, C-5), 43.7 (CH, C-4), 40.8 (C, C-10), 38.0 (CH, C-13), 37.4 (CH2,
C-3), 36.5 (CH2, C-14), 33.2 (CH2, C-7), 32.0 (CH2, C-12), 24.4 (CH2,
C-6), 18.2 (CH2, C-11), 16.2 (CH3, C-20); EIMS m/z 332 [M]+ (12),
314 (71), 282 (100), 267 (37), 255 (63), 198 (23), 131 (23), 119 (26),
105 (63), 91 (67); anal. C 72.23%, H 8.52%, calcd for C20H28O4, C
72.26%, H 8.49%.
Synthesis of Acetyl 15-Ketoatractyligenin Methyl Ester (5).
Compound 4 (50 mg, 0.15 mmol) was dissolved in a mixture of Ac2O-
pyridine 1:1 (5 mL) and allowed to stir for 24 h at room temperature.
The solution was diluted with 30 mL of CH2Cl2 and 20 mL of aqueous
HCl. The organic layer was separated, washed with H2O, and dried
over Na2SO4. After evaporation of the solvent, 45 mg (80% yield) of
acetyl 15-ketoatractyligenin methyl ester (5) was obtained.
16
17
19
doxorubicin
0.9
0.2
a Cell line: A549 ) lung; PC-3 ) prostate; 1A9 ) ovarian; MCF-7
) breast; KB ) nasopharynx; KB-VIN ) nasopharynx MDR.
of 4 with p-toluenesulfonyl chloride cleanly gave tosylate 16
(Scheme 2), but reduction with LiAlH4 gave a complex mixture of
unidentifiable products. Since this synthetic strategy also failed,
we decided to prepare the tosylhydrazone of 12. Reduction of
intermediate 17 with NaBH3CN afforded alcohol 18, which was
oxidized with PCC to give the desired ketone 19 (Scheme 5).
Compounds 1-6, 8, 9, 16, 17, and 19 were screened against a
panel of human tumor cell lines including A549 (lung), PC-3
(prostate), 1A9 (ovarian), MCF-7 (breast), KB (nasopharyngeal),
and KB-VIN (multidrug-resistant KB subline) in order to explore
their anticancer spectra and critical drug-resistance profile.10 The
results are shown in Table 1. Compounds 1, 2, and 17, which do
not contain an R,â-unsaturated ketone, were inactive, while the
remaining compounds, which do contain this moiety, were active
against all or some cell lines. Thus, as proposed in the literature,6
the R,â-unsaturated ketone is likely the active center, possibly acting
as an alkylation site. Compounds 3-5 and 9 showed significant
activity against all six tested cell lines, while 16 and 19 were slightly
less active against the MCF-7 or A549 cell lines. Accordingly, a
wide range of substituents (ketone, hydroxyl, acetate, paclitaxel side
chain, tosylate, and hydrogen, respectively) could be present at the
C-2 position, without losing significant potency. However, com-
pounds 6 (2-piperonyl ester) and 8 (2-oxazole ester) lost activity
against certain cell lines. The 1A9 cell line was highly susceptible
to all active tested compounds, particularly to compounds 3 and 4
with EC50 values of 0.2 and 0.3 µM.
In conclusion, the ready conversion of 1 to the 2,15-diketo (3)
or 15-keto (4) derivatives with an R,â-unsaturated ketone provided
new lead compounds for further investigation, including in vivo
evaluation as new anticancer drug candidates.
Acetyl 15-Ketoatractyligenin Methyl Ester (5): amorphous solid;
25
[R]D -61.6 (c 0.25, CHCl3); IR (film) νmax 2950, 1726, 1645, 1446,
Experimental Section
1
1365, 1247, 1173, 1028, 930 cm-1; H NMR (CDCl3, 250 MHz) δ
General Experimental Procedures. Optical rotations were deter-
mined on a JASCO P-1010 digital polarimeter. IR spectra were obtained
on a Shimadzu FTIR-8300 spectrophotometer. 1H and 13C NMR spectra
were recorded on a Bruker AC-250 spectrometer, using the residual
5.96 (1H, brs, H-17a), 5.34 (1H, dddd, J ) 11.8, 11.8, 4.3, 4.3 Hz,
H-2), 5.28 (1H, brs, H-17b), 3.70 (3H, s, OCH3), 3.06 (1H, m, H-13),
2.74 (1H, m, H-4), 2.43 (1H, m, H-3R), 2.36 (1H, d, J ) 12.1 Hz,
H-14a), 2.29 (1H, dd, J ) 11.8, 4.3 Hz, H-1R), 2.03 (3H, s, Ac), 1.02
(3H, s, Me-20), 0.78 (1H, dd, J ) 11.8, 11.8 Hz, H-1â); 13C NMR
(CDCl3, 62.7 MHz) δ 210.0 (C, C-15), 174.5 (C, C-19), 149.2 (C, C-16),
114.8 (CH2, C-17), 67.9 (CH, C-2), 52.1 (C, C-8), 51.5 (CH3, OMe),
50.8 (CH, C-9), 48.3 (CH, C-5), 44.6 (CH2, C-1), 43.5 (CH, C-4), 40.8
(C, C-10), 38.0 (CH, C-13), 36.5 (CH2, C-14), 33.6 (CH2, C-3), 33.2
solvent signal (δ ) 7.27 in H and δ ) 77.00 in 13C for CDCl3) as
1
reference. 13C NMR assignments were determined by DEPT spectra.
ESIMS was obtained with an Applied Biosystem API-2000 mass
spectrometer. Elemental analysis was carried out with a Perkin-Elmer
240 apparatus. Merck Si gel (70-230 mesh), deactivated with 15%

350 Journal of Natural Products, 2007, Vol. 70, No. 3
Rosselli et al.
(CH2, C-7), 31.9 (CH2, C-12), 24.4 (CH2, C-6), 18.2 (CH2, C-11), 16.1
(CH3, C-20); ESIMS (positive mode) m/z 413 [M + K]+ (100), 397
[M + Na]+ (95), 375 [M + H]+ (4), 315 [M + H - AcOH]+ (8);
anal. C 70.60%, H 8.03%, calcd for C22H30O5, C 70.56%, H 8.07%.
Synthesis of Ester 6. Compound 4 (50 mg, 0.15 mmol) was
dissolved in dry CH2Cl2 (5 mL), and this was added to 25 mg of
piperonylic acid, 1 equiv of DMAP, and 1 equiv of DCC, under argon,
followed by 1 equiv of 1-hydroxybenzotriazole hydrate. The reaction
mixture was allowed to stir for 10 h at room temperature. The reaction
was stopped by evaporation in vacuo of the solvent, and the residue
was purified by preparative TLC (4:1 petroleum ether-EtOAc as eluent)
2.08 (1H, dd, J ) 11.8, 4.3 Hz, H-1R), 0.93 (3H, s, Me-20), 0.80 (1H,
dd, J ) 11.8, 11.8 Hz, H-1â); 13C NMR (CDCl3, 62.7 MHz) δ 209.5
(C, C-15), 174.6 (C, C-19), 172.4 (C, C-1′), 166.3 (C, C-5′), 149.1 (C,
C-16), 138.6 (C, arom), 133.2 (C, arom), 131.7 (CH, arom), 128.7 (4CH,
arom), 127.8 (CH, arom), 127.1 (2CH, arom), 126.9 (2CH, arom), 114.7
(CH2, C-17), 73.4 (CH, C-2′), 71.3 (CH, C-2), 54.6 (CH, C-3′), 51.9
(C, C-8), 51.5 (CH3, OMe), 50.4 (CH, C-9), 48.0 (CH, C-5), 43.8 (CH2,
C-1), 43.4 (CH, C-4), 40.8 (C, C-10), 37.9 (CH, C-13), 36.5 (CH2,
C-14), 33.3 (CH2, C-3), 33.1 (CH2, C-7), 31.9 (CH2, C-12), 24.3 (CH2,
C-6), 17.8 (CH2, C-11), 16.0 (CH3, C-20); ESIMS (positive mode) m/z
638 [M + K]+ (17), 622 [M + Na]+ (100), 600 [M + H]+ (25); anal.
C 72.03%, H 6.90%, N 2.32%, calcd for C36H41NO7, C 72.10%, H
6.89%, N 2.34%.
25
to give 27 mg (20% yield) of compound 6: amorphous solid; [R]D
-16.6 (c 0.13, CHCl3); IR (film) νmax 2923, 1762, 1645, 1443, 1279,
1257, 1159, 968, 933 cm-1; 1H NMR (CDCl3, 250 MHz) δ 7.63 (1H,
dd, J ) 8.1, 1.3 Hz, H-7′), 7.45 (1H, d, J ) 1.3 Hz, H-3′), 6.81 (1H,
d, J ) 8.1 Hz, H-6), 6.03 (2H, s, H-8′), 5.96 (1H, brs, H-17a), 5.55
(1H, dddd, J ) 11.8, 11.8, 4.3, 4.3 Hz, H-2), 5.27 (1H, brs, H-17b),
3.72 (3H, s, OCH3), 3.07 (1H, m, H-13), 2.79 (1H, m, H-4), 2.55 (1H,
m, H-3R), 2.40 (1H, d, J ) 12.0 Hz, H-14a), 2.40 (1H, dd, J ) 11.8,
4.3 Hz, H-1R), 1.07 (3H, s, Me-20), 0.91 (1H, dd, J ) 11.8, 11.8 Hz,
H-1â); 13C NMR (CDCl3, 62.7 MHz) δ 210.2 (C, C-15), 174.5 (C,
C-19), 165.2 (C, C-1′), 151.4 (C, C-5′), 149.1 (C, C-16), 147.6 (C,
C-4′), 125.2 (CH, C-7′), 124.7 (C, C-2′), 114.9 (CH2, C-17), 109.5
(CH, C-3′), 107.9 (CH, C-6′), 101.7 (CH2, C-8′), 68.6 (CH, C-2), 52.1
(C, C-8), 51.6 (CH3, OMe), 50.8 (CH, C-9), 48.3 (CH, C-5), 44.6 (CH2,
C-1), 43.5 (CH, C-4), 40.8 (C, C-10), 37.9 (CH, C-13), 36.5 (CH2,
C-14), 33.6 (CH2, C-3), 33.2 (CH2, C-7), 31.9 (CH2, C-12), 24.3 (CH2,
C-6), 18.2 (CH2, C-11), 16.1 (CH3, C-20); ESIMS (positive mode) m/z
519 [M + K]+ (36), 503 [M + Na]+ (100), 481 [M + H]+ (11); anal.
C 69.95%, H 6.75%, calcd for C28H32O7, C 69.98%, H 6.71%.
Synthesis of Ester 8. (4S,5R)-2,4-Diphenyl-4,5-dihydro-oxazol-5-
carboxylic acid 7 (71.5 mg, 0.3 mmol), synthesized as previously
reported,9 was dissolved in 10 mL of dry CH2Cl2, and this was added
to DMAP (5.51 mg, 0.04 mmol) and DCC (62.58 mg, 0.3 mmol). After
stirring at room temperature for 15 min, compound 4 (50 mg, 0.15
mmol) was added, and the mixture was stirred for an additional 3 h.
The reaction mixture was filtered, the solution evaporated in vacuo,
and the residue purified by chromatography (Si gel, 9:1 petroleum
ether-EtOAc as eluent) to give ester 8 (64 mg, 73% yield): amorphous
Synthesis of Compound 10. Compound 3 (550 mg, 1.6 mmol),
dissolved in benzene (40 mL), was refluxed in a Dean-Stark apparatus
with ethylene glycol (3.5 mL) and p-toluenesulfonic acid (454 mg, 2.4
mmol) for 5 h. The reaction was stopped by adding saturated aqueous
NaHCO3 and a small amount of Na2CO3 and extracted three times with
CHCl3 (25 mL). The organic layer was dried over Na2SO4, filtered,
and evaporated to dryness, leaving a residue, which was purified by
chromatography (Si gel, 4:1 petroleum ether-EtOAc as eluent) to give
561 mg (93% yield) of compound 10: amorphous solid; [R]D25 -37.3
(c 0.39, CHCl3); IR (film) νmax 2929, 2869, 1724, 1645, 1448, 1265,
1
1163, 1074, 931, 738, 704 cm-1; H NMR (CDCl3, 250 MHz) δ 5.95
(1H, brs, H-17a), 5.27 (1H, brs, H-17b), 4.05-3.76 (4H, m, CH2CH2),
3.67 (3H, s, OCH3), 3.06 (1H, m, H-13), 2.70 (1H, m, H-4), 2.58 (1H,
ddd, J ) 14.0, 2.0, 2.0 Hz, H-3R), 2.42 (1H, d, J ) 12.2 Hz, H-14a),
1.94 (1H, dd, J ) 13.6, 2.0 Hz, H-1R), 1.26 (3H, s, Me-20); 13C NMR
(CDCl3, 62.7 MHz) δ 210.0 (C, C-15), 173.7 (C, C-19), 149.1 (C, C-16),
114.4 (CH2, C-17), 108.1 (C, C-2), 64.1 (CH2, CH2O), 62.9 (CH2,
CH2O), 52.1 (C, C-8), 51.1 (CH3, OMe), 50.9 (CH, C-9), 48.1 (CH2,
C-1), 47.1 (CH, C-5), 43.1 (CH, C-4), 40.3 (C, C-10), 37.7 (CH, C-13),
36.2 (CH2, C-3), 34.7 (CH2, C-14), 33.2 (CH2, C-7), 31.6 (CH2, C-12),
24.2 (CH2, C-6), 18.1 (CH2, C-11), 16.9 (CH3, C-20); ESIMS (positive
mode) m/z 397 [M + Na]+ (100), 375 [M + H]+ (38); anal. C 70.50%,
H 8.05%, calcd for C22H30O5, C 70.56%, H 8.07%.
Synthesis of Compound 11. Compound 10 (486 mg, 1.3 mmol),
dissolved in MeOH (60 mL), was stirred at room temperature with
NaBH4 (77.3 mg, 2 mmol). After 10 min, the reaction was stopped by
adding water (50 mL) and extracted three times with CH2Cl2 (25 mL).
The organic layer was dried over Na2SO4, filtered, and evaporated in
vacuo to dryness, leaving a residue, which was purified by chroma-
tography (Si gel, 7:3 petroleum ether-EtOAc as eluent) to give 440
25
solid; [R]D -48.0 (c 0.23, CHCl3); IR (film) νmax 2931, 2858, 1751,
1724, 1655, 1450, 1267, 1230, 1064, 1026, 960, 931, 737, 698 cm-1
;
1H NMR (CDCl3, 250 MHz) δ 8.10-8.04 (2H, m, arom), 7.60-7.20
(8H, m, arom), 5.97 (1H, brs, H-17a), 5.56 (1H, dddd, J ) 11.8, 11.8,
4.3, 4.3 Hz, H-2), 5.40 (1H, d, J ) 6.3 Hz, H-3′), 5.28 (1H, brs, H-17b),
4.87 (1H, d, J ) 6.3 Hz, H-2′), 3.70 (3H, s, OCH3), 3.07 (1H, m, H-13),
2.78 (1H, m, H-4), 2.48 (1H, m, H-3R), 2.45 (1H, d, J ) 12.0 Hz,
H-14a), 2.35 (1H, dd, J ) 11.8, 4.3 Hz, H-1R), 1.04 (3H, s, Me-20),
0.90 (1H, dd, J ) 11.8, 11.8 Hz, H-1â); 13C NMR (CDCl3, 62.7 MHz)
δ 209.8 (C, C-15), 174.3 (C, C-19), 169.4 (C, C-1′), 164.1 (C, C-5′),
149.0 (C, C-16), 141.2 (C, arom), 131.9 (C, arom), 128.8 (2CH, arom),
128.7 (2CH, arom), 128.4 (2CH, arom), 128.0 (CH, arom), 126.8 (CH,
arom), 126.4 (2CH, arom), 114.9 (CH2, C-17), 83.1 (CH, C-2′), 74.6
(CH, C-3′), 69.9 (CH, C-2), 52.0 (C, C-8), 51.5 (CH3, OMe), 50.7 (CH,
C-9), 48.2 (CH, C-5), 44.2 (CH2, C-1), 43.4 (CH, C-4), 40.6 (C, C-10),
37.9 (CH, C-13), 36.5 (CH2, C-14), 33.4 (CH2, C-3), 33.1 (CH2, C-7),
31.6 (CH2, C-12), 24.3 (CH2, C-6), 18.2 (CH2, C-11), 16.0 (CH3, C-20);
ESIMS (positive mode) m/z 582 [M + H]+ (28), 474 (100); anal. C
74.30%, H 6.73%, N 2.39%, calcd for C36H39NO6, C 74.33%, H 6.76%,
N 2.41%.
25
mg (90% yield) of compound 11: amorphous solid; [R]D -50.0 (c
1.12, CHCl3); IR (film) νmax 3444, 2922, 1718, 1661, 1465, 1377, 1263,
1
1193, 1070, 972, 948, 827, 709 cm-1; H NMR (CDCl3, 250 MHz) δ
5.09 (1H, brs, H-17a), 4.96 (1H, brs, H-17b), 4.04-3.76 (4H, m, CH2-
CH2), 3.76 (1H, m, H-15), 3.65 (3H, s, OCH3), 2.65 (1H, m, H-13),
2.65 (1H, m, H-4), 2.58 (1H, ddd, J ) 14.0, 2.0, 2.0 Hz, H-3R), 2.05
(1H, d, J ) 12.0 Hz, H-14a), 2.00 (1H, dd, J ) 13.6, 2.0 Hz, H-1R),
1.27 (1H, d, J ) 13.6 Hz, H-1â), 1.12 (3H, s, Me-20); 13C NMR
(CDCl3, 62.7 MHz) δ 174.2 (C, C-19), 158.2 (C, C-16), 108.5 (C, C-2),
104.9 (CH2, C-17), 84.2 (CH, C-15), 64.3 (CH2, CH2O), 62.9 (CH2,
CH2O), 51.1 (CH3, OMe), 48.9 (CH2, C-1), 47.4 (CH, C-5), 45.6 (CH,
C-9), 45.5 (C, C-8), 43.3 (CH, C-4), 39.9 (CH, C-13), 39.6 (C, C-10),
38.5 (CH2, C-7), 36.1 (CH2, C-3), 34.7 (CH2, C-14), 32.6 (CH2, C-12),
25.7 (CH2, C-6), 18.3 (CH2, C-11), 17.2 (CH3, C-20); ESIMS m/z 377
[M + H]+ (100); anal. C 70.15%, H 8.60%, calcd for C22H32O5, C
70.18%, H 8.57%.
Synthesis of Ester 9. Compound 8 (50 mg, 0.09 mmol), dissolved
in CH2Cl2 (5 mL), was stirred at room temperature with p-toluene-
sulfonic acid (3 mg, 0.017 mmol). After completion of the reaction (4
days) the solution was neutralized with saturated aqueous NaHCO3,
diluted with water (10 mL), and extracted three times with CHCl3 (15
mL). The organic layer was dried over Na2SO4, filtered, and evaporated
to dryness, leaving a residue, which was purified by preparative TLC
(4:1 petroleum ether-EtOAc as eluent) to give 38 mg (75% yield) of
compound 9: amorphous solid; [R]D25 -21.4 (c 0.29, CHCl3); IR (film)
νmax 3431, 2928, 1724, 1664, 1647, 1514, 1485, 1448, 1265, 1211,
Synthesis of Compound 12. Compound 11 (390 mg, 1.0 mmol),
dissolved in a 1:1 mixture of MeOH-H2O (40 mL), was stirred at
room temperature with p-toluenesulfonic acid (107 mg, 0.5 mmol) for
2 h. The reaction was stopped by adding saturated aqueous NaHCO3
and extracted with CH2Cl2 (25 mL × 3). The organic layer was dried
over Na2SO4, filtered, and evaporated in vacuo to dryness, leaving a
residue, which was crystallized to give 289 mg (87% yield) of
25
compound 12: amorphous solid; C20H28O4; [R]D -96.0 (c 0.92,
CHCl3); IR (film) νmax 3555, 2933, 1713, 1664, 1433, 1257, 1197, 1074,
954 cm-1; 1H NMR (CDCl3, 250 MHz) δ 5.10 (1H, brs, H-17a), 4.99
(1H, brs, H-17b), 3.82 (1H, m, H-15), 3.65 (3H, s, OCH3), 3.02 (1H,
m, H-4), 2.87 (1H, ddd, J ) 14.4, 2.0, 2.0 Hz, H-3R), 2.68 (1H, m,
H-13), 2.65 (1H, dd, J ) 14.4, 2.0 Hz, H-1R), 2.39 (1H, dd, J ) 14.4,
7.6 Hz, H-3â), 1.92 (1H, d, J ) 14.4 Hz, H-1â), 1.87 (1H, d, J ) 12.0
Hz, H-14a), 0.89 (3H, s, Me-20); 13C NMR (CDCl3, 62.7 MHz) δ 208.8
(C, C-2), 173.9 (C, C-19), 157.7 (C, C-16), 105.3 (CH2, C-17), 82.1
1
1117, 739, 704 cm-1; H NMR (CDCl3, 250 MHz) δ 7.77-7.73 (2H,
m, arom), 7.48-7.28 (8H, m, arom), 7.03 (1H, d, J ) 9.3 Hz, NH),
5.95 (1H, brs, H-17a), 5.76 (1H, dd, J ) 9.3, 2.1 Hz, H-3′), 5.47 (1H,
dddd, J ) 11.8, 11.8, 4.3, 4.3 Hz, H-2), 5.25 (1H, brs, H-17b), 4.63
(1H, d, J ) 2.1 Hz, H-2′), 3.69 (3H, s, OCH3), 3.00 (1H, m, H-13),
2.75 (1H, m, H-4), 2.43 (1H, m, H-3), 2.30 (1H, d, J ) 12.0 Hz, H-14a),

Cytotoxic ActiVity of ent-Kauranes
Journal of Natural Products, 2007, Vol. 70, No. 3 351
(CH, C-15), 56.0 (CH2, C-1), 51.7 (CH3, OMe), 47.8 (CH, C-5), 45.3
(CH, C-4), 45.2 (C, C-8), 44.5 (CH, C-9), 42.9 (CH2, C-3), 42.5 (C,
C-10), 39.8 (CH, C-13), 37.9 (CH2, C-7), 35.6 (CH2, C-14), 32.6 (CH2,
C-12), 25.4 (CH2, C-6), 18.0 (CH2, C-11), 16.7 (CH3, C-20); ESIMS
(positive mode) m/z 687 [2M + Na]+ (100), 371 [M + K]+ (4), 355
[M + Na]+ (60), 333 [M + H]+ (4); anal. C 72.24%, H 8.52%, calcd
for C20H28O4, C 72.26%, H 8.49%.
H-14a), 0.92 (3H, s, Me-20); 13C NMR (CDCl3, 62.7 MHz) δ 209.7
(C, C-15), 174.2 (C, C-19), 148.9 (C, C-16), 144.4 (C, C-4′), 134.7
(C, C-1′), 129.7 (2CH, C-3′ and C-5′), 127.8 (2CH, C-2′ and C-6′),
115.0 (CH2, C-17), 77.5 (CH, C-2), 51.9 (C, C-8), 51.5 (CH3, OMe),
50.6 (CH, C-9), 47.8 (CH, C-5), 45.6 (CH2, C-1), 43.5 (CH, C-4), 41.0
(C, C-10), 37.9 (CH, C-13), 36.5 (CH2, C-14), 34.2 (CH2, C-3), 33.0
(CH2, C-7), 31.8 (CH2, C-12), 24.1 (CH2, C-6), 21.6 (CH3, C-7′), 18.1
(CH2, C-11), 15.9 (CH3, C-20); ESIMS (positive mode) m/z 525 [M +
K]+ (15), 509 [M + Na]+ (100), 487 [M + H]+ (4); anal. C 66.60%,
H 7.00%, S 6.55% calcd for C27H34O6S, C 66.64%, H 7.04%, S 6.59%.
Reduction of Compound 16. Compound 16 (40 mg. 0.08 mmol)
was dissolved in dry THF (5 mL), added to 10 mg of LiAlH4 (0.24
mmol), and allowed to stir for 2 h at room temperature. The reaction
was stopped by adding saturated NH4Cl solution (5 mL). The residue
was filtered off, and the solution was extracted three times with EtOAc
(10 mL). The organic layer was dried over Na2SO4, filtered, and
evaporated in vacuo to dryness. TLC analysis of the residue showed
an unresolvable complex mixture of products.
Synthesis of Compound 17. Compound 12 (210 mg, 0.63 mmol),
dissolved in EtOH (3 mL), was heated at 70 °C with p-toluenesulfonyl
hydrazine (140 mg, 0.75 mmol) for 5 h. The reaction was stopped by
removal of the solvent in vacuo, and the residue was purified by
preparative TLC (7:3 petroleum ether-EtOAc as eluent) to give 305
mg (97% yield) of compound 17: amorphous solid; [R]D25 54.1 (c 0.39,
CHCl3); IR (film) νmax 3435, 3217, 2930, 1703, 1643, 1599, 1450, 1337,
1267, 1167, 1091, 925 cm-1; 1H NMR (CDCl3, 250 MHz) δ 8.62 (1H,
brs, NH), 7.89 (2H, d, J ) 8.2 Hz, H-2′ and H-6′), 7.26 (2H, d, J )
8.2 Hz, H-3′ and H-5′), 5.10 (1H, brs, H-17a), 4.98 (1H, brs, H-17b),
3.77 (1H, brs, H-15), 3.55 (3H, s, OCH3), 3.09 (1H, ddd, 13.7, 1.6, 1.6
Hz, H-3R), 2.79 (1H, m, H-4), 2.65 (1H, m, H-13), 2.47 (1H, dd, J )
12.1, 1.6 Hz, H-1R), 2.41 (3H, s, Me-Ar), 0.34 (3H, s, Me-20); 13C
NMR (CDCl3, 62.7 MHz) δ 175.7 (C, C-19), 158.1 (C, C-2), 157.4
(C, C-16), 143.1 (C, C-1′), 135.3 (C, C-4′), 128.7 (CH, C-3′ and C-5′),
128.1 (CH, C-2′ and C-6′), 104.9 (CH2, C-17), 81.8 (CH, C-15), 51.6
(CH3, OMe), 49.5 (CH2, C-1), 49.0 (CH, C-5), 45.3 (CH, C-4), 45.2
(C, C-8), 44.3 (CH, C-9), 42.3 (C, C-10), 39.7 (CH, C-13), 37.8 (CH2,
C-7), 35.8 (CH2, C-14), 32.6 (CH2, C-12), 29.8 (CH2, C-3), 25.5 (CH2,
C-6), 21.3 (CH3, Me-Ar), 16.1 (CH2, C-11), 14.2 (CH3, C-20); ESIMS
m/z 539 [M + K]+ (11), 523 [M + Na]+ (57), 501 [M + H]+ (100);
anal. C 64.72%, H 7.22%, N 5.58%, S 6.42%, calcd for C27H36N2O5S,
C 64.77%, H 7.25%, N 5.60%, S 6.40%.
Reduction of Compound 17. Compound 17 (160 mg, 0.31 mmol),
dissolved in a 1:1 mixture of DMF/sulfolane (14 mL), was heated at
120 °C with p-toluenesulfonic acid (6 mg) and NaBH3CN (80 mg, 1.3
mmol) for 24 h. The reaction was stopped by adding saturated aqueous
NaCl and extracted three times with Et2O (25 mL). The organic layer
was dried over Na2SO4, filtered, and evaporated to dryness, leaving a
residue, which was purified by chromatography (Si gel, 4:1 petroleum
ether-EtOAc as eluent) to give 48 mg (48% yield) of compound 18:
amorphous solid; [R]D25 -116.3 (c 0.55, CHCl3); IR (film) νmax 3480,
3054, 2930, 1722, 1447, 1265, 1192, 1002, 896 cm-1; 1H NMR (CDCl3,
250 MHz) δ 5.10 (1H, brs, H-17a), 4.97 (1H, brs, H-17b), 3.76 (1H,
brs, H-15), 3.66 (3H, s, OCH3), 2.67 (1H, m, H-13), 2.47 (1H, m, H-4),
2.12 (1H, brd, 12.2 Hz, H-3R), 1.98 (1H, d, 12.0 Hz, H-14), 0.89 (3H,
s, Me-20); 13C NMR (CDCl3, 62.7 MHz) δ 176.0 (C, C-19), 158.4 (C,
C-16), 104.9 (CH2, C-17), 82.6 (CH, C-15), 51.4 (CH3, OMe), 49.1
(CH, C-5), 46.0 (C, C-8), 45.2 (CH, C-9), 43.2 (CH, C-4), 40.3 (CH2,
C-1), 40.1 (CH, C-13), 38.7 (C, C-10), 37.5 (CH2, C-7), 36.3 (CH2,
C-14), 33.0 (CH2, C-12), 28.5 (CH2, C-3), 26.7 (CH2, C-6), 18.7 (CH2,
C-2), 18.1 (CH2, C-11), 15.2 (CH3, C-20); ESIMS m/z 659 [2M +
Na]+ (89), 357 [M + K]+ (32), 341 [M + Na]+ (100), 319 [M + H]+
(32); anal. C 75.45%, H 9.47%, calcd for C20H30O3, C 75.43%, H
9.50%.
Oxidation of Compound 18. Compound 18 (30 mg, 0.09 mmol),
dissolved CH2Cl2 (6 mL), was stirred at room temperature with PCC
(54 mg, 0.25 mmol). The reaction was stopped after 1 h by filtering
over Florisil with EtOAc as eluent. The solvent was evaporated to
dryness, leaving a residue, which was purified by chromatography (Si
gel, 4:1 petroleum ether-EtOAc as eluent) to give 20 mg (67% yield)
of compound 19: amorphous solid; [R]D25 -157.8 (c 0.22, CHCl3); IR
(film) νmax 2927, 2855, 1727, 1644, 1447, 1255, 1197, 1173, 1038,
945 cm-1; 1H NMR (CDCl3, 250 MHz) δ 5.94 (1H, brs, H-17a), 5.24
(1H, brs, H-17b), 3.66 (3H, s, OCH3), 3.04 (1H, m, H-13), 2.49 (1H,
m, H-4), 2.39 (1H, d, 12.0 Hz, H-14), 2.15 (1H, brd, 12.2 Hz, H-3R),
0.94 (3H, s, Me-20); 13C NMR (CDCl3, 62.7 MHz) δ 210.5 (CH, C-15),
Synthesis of Compound 13. Compound 12 [50 mg, 0.15 mmol,
dissolved in benzene (25 mL)] was refluxed in a Dean-Stark apparatus
with ethanedithiol (25 µL) and p-toluenesulfonic acid (34 mg, 0.17
mmol) for 2 h. The reaction was stopped by adding saturated aqueous
NaHCO3 and extracted with EtOAc (25 mL × 3). The organic layer
was dried over Na2SO4, filtered, and evaporated to dryness, leaving a
residue, which was purified by chromatography (Si gel, 4:1 petroleum
ether-EtOAc as eluent) to give 23 mg (47% yield) of compound 13:
amorphous solid; [R]D25 -115.2 (c 0.55, CHCl3); IR (film) νmax 2931,
1728, 1438, 1251, 1190, 1167, 1070, 953 cm-1; 1H NMR (CDCl3, 250
MHz) δ 3.65 (3H, s, OCH3), 3.03 (1H, m, H-4), 2.85 (1H, ddd, J )
14.7, 1.8, 1.8 Hz, H-3R), 2.54 (1H, dd, J ) 13.9, 1.8 Hz, H-1R), 2.39
(1H, dd, J ) 14.7, 7.6 Hz, H-3â), 2.29 (1H, d, J ) 12.4 Hz, H-14a),
2.22 (1H, quint., J ) 6.9 Hz, H-16), 1.09 (3H, d, J ) 6.9 Hz, Me-17),
0.91 (3H, s, Me-20); 13C NMR (CDCl3, 62.7 MHz) δ 223.7 (C, C-15),
207.8 (C, C-2), 173.7 (C, C-19), 55.2 (CH2, C-1), 51.8 (C, C-8), 51.6
(CH3, OMe), 50.1 (CH, C-9), 47.6 (CH, C-5), 47.5 (CH, C-16), 45.2
(CH, C-4), 43.0 (CH2, C-3), 42.6 (C, C-10), 36.6 (CH2, C-7), 34.7 (CH,
C-13), 33.3 (CH2, C-12), 24.3 (CH2, C-6), 24.2 (CH2, C-14), 17.9 (CH2,
C-11), 16.6 (CH3, C-20), 9.9 (CH3, C-17); ESIMS m/z 687 [2M +
Na]+ (100), 371 [M + K]+ (18), 355 [M + Na]+ (64), 333 [M + H]+
(4); anal. C 72.29%, H 8.45%, calcd for C20H28O4, C 72.26%, H 8.49%.
Catalytic Reduction of Atractyligenin Methyl Ester (3). Com-
pound 3 (300 mg, 0.9 mmol), dissolved in MeOH (150 mL), was
reduced in a Parr apparatus with Pd/C (410 mg) and H2 (1 atm) for 1
h. The reaction was stopped and the solvent evaporated, leaving a
residue, which was purified by chromatography (Si gel, 3:7 petroleum
ether-EtOAc as eluent) to give, in order of increasing polarity, 50 mg
(17% yield) of compound 15 and 230 mg (77% yield) of compound
14.
Compound 14: amorphous solid; [R]D25 -66.2 (c 0.23, CHCl3); IR
(film) νmax 3380, 2925, 2870, 1722, 1450, 1263, 1192, 1178, 1030 cm-1
;
1H NMR (CDCl3, 250 MHz) δ 4.27 (1H, dddd, J ) 11.8, 11.8, 4.3,
4.3 Hz, H-2), 3.67 (3H, s, OCH3), 3.26 (1H, d, J ) 4.2 Hz, H-15),
2.67 (1H, m, H-4), 2.45 (1H, m, H-3R), 2.23 (1H, dd, J ) 11.8, 4.3
Hz, H-1R), 1.89 (1H, d, J ) 12.0 Hz, H-14a), 1.14 (3H, d, J ) 7.1 Hz,
Me-17), 0.90 (3H, s, Me-20), 0.70 (1H, dd, J ) 11.8, 11.8 Hz, H-1â);
ESIMS m/z 695 [2M + Na]+ (60), 375 [M + K]+ (7), 359 [M + Na]+
(100), 337 [M + H]+ (2); anal. C 71.38%, H 9.61%, calcd for C20H32O4,
C 71.39%, H 9.59%.
Compound 15: amorphous solid; [R]D25 -82.1 (c 0.52, CHCl3); IR
(film) νmax 3374, 2932, 2862, 1718, 1448, 1264, 1194, 1176, 1025 cm-1
;
1H NMR (CDCl3, 250 MHz) δ 4.26 (1H, dddd, J ) 11.8, 11.8, 4.3,
4.3 Hz, H-2), 3.67 (3H, s, OCH3), 3.62 (1H, d, J ) 7.6 Hz, H-15),
2.67 (1H, m, H-4), 2.45 (1H, m, H-3R), 2.22 (1H, dd, J ) 11.8, 4.3
Hz, H-1R), 0.96 (3H, d, J ) 7.6 Hz, Me-17), 0.89 (3H, s, Me-20),
0.72 (1H, dd, J ) 11.8, 11.8 Hz, H-1â); ESIMS m/z 695 [2M + Na]+
(58), 375 [M + K]+ (8), 359 [M + Na]+ (100), 337 [M + H]+ (2);
anal. C 71.35%, H 9.56%, calcd for C20H32O4, C 71.39%, H 9.59%.
Oxidation of Compound 14. Compound 14 (60 mg, 0.18 mmol),
dissolved in CH2Cl2 (12 mL), was stirred at room temperature with
PCC (108 mg) for 1 h. The reaction was stopped by filtering over
Florisil (CH2Cl2 and EtOAc as eluents), and the residue was purified
by chromatography (Si gel, 3:2 petroleum ether-EtOAc as eluent) to
give 40 mg (67% yield) of compound 13.
Synthesis of Compound 16. Compound 4 (50 mg. 0.15 mmol) was
dissolved in dry pyridine (5 mL), added to 43 mg of p-toluenesulfonyl
chloride (0.22 mmol), and allowed to stand for 7 days at room
temperature. The reaction was stopped by evaporation in vacuo of the
solvent with toluene, and the residue was purified by preparative TLC
(4:1 petroleum ether-EtOAc as eluent) to give 49 mg (66% yield) of
25
compound 16: amorphous solid; [R]D -43.3 (c 0.68, CHCl3); IR
(film) νmax 2927, 2861, 1725, 1644, 1598, 1448, 1360, 1257, 1188,
1
1188, 1012, 933 cm-1; H NMR (CDCl3, 250 MHz) δ 7.84 (2H, d, J
) 9.9 Hz, H-2′ and H-6′), 7.35 (2H, d, J ) 9.9 Hz, H-3 and H-5′),
5.96 (1H, brs, H-17a), 5.28 (1H, brs, H-17b), 5.10 (1H, dddd, J )
11.8, 11.8, 4.3, 4.3 Hz, H-2), 3.64 (3H, s, OCH3), 3.06 (1H, m, H-13),
2.65 (1H, m, H-4), 2.45 (3H, s, Me-7′), 2.30 (1H, d, J ) 13.1 Hz,

352 Journal of Natural Products, 2007, Vol. 70, No. 3
Rosselli et al.
(2) Piozzi, F.; Quilico, A.; Mondelli, R.; Ajello, T.; Sprio, V.; Melera,
A. Tetrahedron 1966, 515-529.
(3) Piozzi, F.; Quilico, A.; Ajello, T.; Sprio, V.; Melera, A. Tetrahedron
Lett. 1965, 1829-1836.
(4) Piozzi, F. In Atractyloside: Chemistry, Biochemistry and Toxicology;
Santi, R., Lucani, S., Eds.; Piccin Medical Books: Padova, Italy,
1978; pp 13-32.
(5) Camarda, L.; Di Stefano, V.; Schillaci, D. Pharmazie 2002, 57, 374-
376.
(6) (a) Lee, K. H.; Meck, R.; Piantadosi, C.; Huang, E. S. J. Med. Chem.
1973, 16, 299-301. (b) Lee, K. H.; Hall, I. H.; Mar, E. C.; Starnes,
C. O.; ElGebaly, S. A.; Waddell, T. G.; Hadgraft, R. I.; Ruffner, C.
G.; Weidner, I. Science 1977, 196, 533-536.
(7) Bruno, M.; Rosselli, S.; Pibiri, I.; Kilgore, N.; Lee, K. -H. J. Nat.
Prod. 2002, 65, 1594-1597.
(8) Bruno, M.; Rosselli, S.; Maggio, A.; Raccuglia, R. A.; Bastow, K.
F.; Lee, K. H. J. Nat. Prod. 2005, 68, 1042-1046.
(9) Kopczacki, P.; Gumulka, M.; Masnyk, M.; Grabarczyk, H.; Nowak,
G.; Daniewski, W. M. Phytochemistry 2001, 58, 775-787.
(10) Rubinstein, L. V.; Shoemaker, R. H.; Paull, K. D.; Simon, R. M.;
Tosini, S.; Skehan, P.; Scudiero, D. A.; Monks, M. R. J. Natl. Cancer
Inst. 1990, 82, 1113-1118.
175.8 (C, C-19), 149.5 (C, C-16), 114.5 (CH2, C-17), 52.6 (C, C-8),
51.2 (CH, C-9), 51.1 (CH3, OMe), 48.8 (CH, C-5), 43.0 (CH, C-4),
39.7 (C, C-10), 39.6 (CH2, C-1), 38.1 (CH, C-13), 36.5 (CH2, C-14),
33.3 (CH2, C-7), 32.1 (CH2, C-12), 28.3 (CH2, C-3), 25.3 (CH2, C-6),
18.5 (CH2, C-2), 18.1 (CH2, C-11), 15.1 (CH3, C-20); ESIMS m/z 355
[M + K]+ (32), 339 [M + Na]+ (100), 317 [M + H]+ (28); anal. C
75.96%, H 8.90%, calcd for C20H28O3, C 75.91%, H 8.92%.
In Vitro Cytotoxicity Assay. The sulforhodamine B assay was used
according to the procedures developed and validated at NCI.10 Doxo-
rubicin was used as the positive control antitumor drug. The in vitro
anticancer activities are expressed as EC50 values, which is the test
compound concentration (µM) that reduced the cell number by 50%
after 72 h of continuous treatment. The values were interpolated from
dose-response data. Each test was performed in triplicate with variation
less than 5%. The EC50 values determined in each of independent tests
varied less than 10%. Compound stock solutions were prepared in
DMSO with the final solvent concentration e1% DMSO (v/v), a
concentration without effect on cell replication. The cells were cultured
at 37 °C in RPMI-1640 supplemented with 25 mM N-2-hydroxyeth-
ylpiperazine-N′-2-ethanesulfonic acid (HEPES), 2% (w/v) sodium
bicarbonate, 10% (v/v) fetal bovine serum, and 100 µg/mL kanamycin
in a humidified atmosphere containing 5% CO2.
(11) Ajello, T.; Piozzi, F.; Quilico, A.; Sprio, V. Gazz. Chim. Ital. 1963,
93, 867-915.
(12) Hanson, J. R.; Siverns, M.; Piozzi, F.; Savona, G. J. Chem. Soc.,
Perkin Trans. 1 1976, 114-117.
Acknowledgment. This work was supported by Italian Government
project MIUR (M.B.) and by NIH grant CA17625 (K.H.L.).
References and Notes
(1) Hanson, J. R. Nat. Prod. Rep. 2005, 22, 594-602, and previous
reviews in this series.
NP060504W