Cytotoxic activity of some natural and synthetic guaianolides

Article abstract of DOI:10.1021/np0500575

Several natural guaianolides and synthetic derivatives of repin (1) were tested and found to be active against tumor cell replication. Repin (1) and both mono- and di-halohydrin analogues (2, 7-9, 11, 12) showed significant antitumor potency. A more effec

Full text of DOI:10.1021/np0500575

1042
J. Nat. Prod. 2005, 68, 1042-1046
Cytotoxic Activity of Some Natural and Synthetic Guaianolides
Maurizio Bruno,*,† Sergio Rosselli,† Antonella Maggio,† Rosa Angela Raccuglia,† Kenneth F. Bastow,‡ and
Kuo-Hsiung Lee‡
Dipartimento di Chimica Organica, Universita` di Palermo, Viale delle Scienze, 90128 Palermo, Italy, and Natural Products
Laboratory, School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
Received February 21, 2005
Several natural guaianolides and synthetic derivatives of repin (1) were tested and found to be active
against tumor cell replication. Repin (1) and both mono- and di-halohydrin analogues (2, 7-9, 11, 12)
showed significant antitumor potency. A more effective compound (17) was obtained by esterificating
repin with the paclitaxel side chain.
Sesquiterpene lactones have drawn significant interest
due to their interesting biological properties, particularly
antitumor and cytotoxic activities. Prior studies of natural
sesquiterpenes1-4 and synthetic analogues5 showed that
the main determining factor responsible for cytotoxicity of
the compounds studied is the presence of a OdC-CHd
CH2 system.1-4 This R,â-unsaturated system likely serves
as an alkylating center and can be part of an ester, ketone,
or lactone moiety. In helenalin analogues, an endocyclic
R,â-unsaturated ketone moiety (cyclopentenone) was found
to be more important than an exocyclic R-methylene-γ-
lactone system,1-4 and addition of lipophilic character,
particularly by incorporation of conjugated ester groups,
enhanced cytotoxicity.3
for compound 8, and δ 4.06 and 3.71 (J ) 10.8 Hz, H-15a
and H-15b) and δ 3.59 and 3.45 (J ) 10.3 Hz, H-3′a and
H-3′b) for compound 9]. The 13C NMR spectra of compounds
7-9 also confirmed the presence of the halogens on C-15
and C-3′. When only 1 equiv of lithium halide was used,
different products were obtained, depending on the reagent
used (see Experimental Section). With LiCl, the two
monochlorohydrins chlorohyssopifolin C (2), found for the
first time in Centaurea hyssopifolia,7 and solstitiolide (10),
a natural product isolated from Centaurea solstitialis,8 were
obtained. However, reaction with LiI produced the di-
iodohydrin 9 and one monoiodohydrin, 11, while LiBr gave
three derivatives: the dibromohydrin 8 and both possible
monobromohydrins 12 and 13.
Because compound 14, with the iodohydrin on the
guaiane skeleton, was not obtained with the former method,
diiodohydrin 9 was treated with t-BuOK to effect a dif-
ferential ring closure with formation of the oxirane ring
only on the ester side chain, most likely due to steric
hindrance of the other hydroxyl group.
Furthermore, with the aim of enhancing the cytotoxic
activity, we esterified the free C-3 hydroxy group of repin
with the side chain of paclitaxel. According to a procedure
previously reported,9 the commercially available (2R,3S)-
3-phenylisoserine hydrochloride was converted to com-
pound 15, which was then esterified with repin (1). Acidic
hydrolysis of the resulting ester 16 gave compound 17, with
the same side chain as paclitaxel (Scheme 1).
Compounds 10 and 13 were not stable and decomposed
completely within 1 week, even with storage at 5 °C. The
cytotoxic activities of the remaining synthetic derivatives
and natural guaianolides were evaluated, and the data are
reported in Table 1.
Repin (1) was tested and found to be significantly potent
against seven cell lines [A549 (lung cancer), MCF-7 (breast
cancer), 1A9 (ovarian cancer), KB (nasopharyngeal cancer),
KB-VIN (KB drug-resistant variant), HCT-8 (ileocecal
cancer), and SK-MEL-2 (melanoma)]. This compound and
the subsequent derivatives were generally least potent
against A549 cell replication.
Results and Discussion
Recently, we described the isolation of four known [repin
(1), chlorohyssopifolin C (2), janerin (3), and cebellin J (4)]
and two unreported [babylin A (5) and babylin B (6)]
sesquiterpene 8,12-lactones with a guaiane skeleton6 from
Centaurea babylonica (Asteraceae), a species used in
Lebanese folk medicine. These compounds were first tested
against replication of A549 and MCF-7 tumor cell lines
(Table 1). While compounds 4-6, which contain a diol
rather than a chlorohydrin or epoxide on the skeleton or
the ester side chain, were not active against either cell line,
compounds 2 and 1 showed good activity against MCF-7
and both cell lines, respectively. In light of these results,
we carried out the following chemical modifications on
repin (1), which was readily available, to study the struc-
ture-activity relationships (SAR) of this compound type.
The presence of two oxirane rings in 1 suggested the
preparation of various halohydrins containing chlorine,
bromine, and iodine. Accordingly, treatment of 1 with 6
equiv of the appropriate lithium halide gave excellent yields
of the dichlorohydrin chlorohyssopifolin A (7), a natural
product previously isolated from Centaurea hyssopifolia,7
and its two analogues dibromohydrin 8 and diiodohydrin
9. The structures of these compounds were clearly indicated
by the absence of 1H NMR signals for the two epoxide
methylenes of repin and by the presence of two pairs of
AB systems [δ 4.33 and 3.95 (J ) 11.8 Hz, H-15a and
H-15b) and δ 3.87 and 3.63 (J ) 11.1 Hz, H-3′a and H-3′b)
for compound 7, δ 4.26 and 3.88 (J ) 11.1 Hz, H-15a and
H-15b) and δ 3.76 and 3.56 (J ) 10.4 Hz, H-3′a and H-3′b)
Monoiodohydrin 14, with the halohydrin on the cyclic
skeleton and the epoxide in the side chain, displayed weak
activity (IC50 4.1 µM) against the KB cell line and was
inactive in the other tested cell lines. In contrast, signifi-
cant activity (IC50 0.8-2.0 µM) was seen against 1A9, KB,
and HCT-8 cell lines when the halohydrin was on the side
chain and the epoxide on the skeleton (2, 11, and 12).
However, these three monohalohydrins were generally
slightly less active than the parent diepoxide repin (1) (IC50
* Corresponding author. Phone: +39091596905. Fax: +39091596825.
E-mail: bruno@dicpm.unipa.it.
† Universita´ di Palermo.
‡ University of North Carolina at Chapel Hill.
10.1021/np0500575 CCC: $30.25
© 2005 American Chemical Society and American Society of Pharmacognosy
Published on Web 06/14/2005

Cytotoxic Activity of Guaianolides
Journal of Natural Products, 2005, Vol. 68, No. 7 1043
Table 1. Effect of Compounds 1-9, 11, 12, 14, 16, and 17 against Tumor Cell Lines
IC50 (µM)/Cell Linea
compound
A549
MCF-7
1A9
KB
KB-V
HCT-8
SK-MEL-2
2.5
1
2
3
4
5
6
7
8
9
2.5
9.3
1.1
1.5
0.3
0.8
5.0
1.4
2.0
3.0
0.8
1.5
25.9
0.8
1.6
5.5
19.9
>24.0
>26.3
>26.3
10.3
4.2
8.0
>24.0
>26.3
>26.3
0.7
0.4
1.3
0.8
1.1
5.6
0.8
0.2
4.1
1.1
2.8
1.3
5.5
1.8
6.3
18.6
2.6
7.5
3.6
1.0
1.3
2.6
11
7.3
2.0
12
4.3
1.1
1.3
4.5
0.5
0.5
3.6
9.4
1.5
1.0
5.8c
5.8c
14
13.7
3.4
4.1
16
2.6
17
0.8
0.3
cynaropicrin
chlorojanerin
aguerin B
paclitaxel
mitomycin C
16.5b
3.2b
3.3b
1.5
15.9c
15.1c
26.4b
0.002
0.001
0.6
0.013
0.6
0.3
a Cell line: A549 ) lung; MCF-7 ) breast; 1A9 ) ovarian; KB ) nasopharynx; KB-V ) nasopharynx MDR; HCT-8 ) ileocecal; SK-
MEL-2 ) melanoma. b Taken from ref 10. c Taken from ref 11.
Scheme 1
0.3-1.4 µM). The three dihalohydrins (7, 8, and 9) were
also quite active (IC50 0.4-4.1 µM) against most tested cell
lines; however, the potency range was broader. The effect
of the halogen (chlorine, bromine, iodine) was not consistent
among the cell lines.
It is known that the C-13 side chain of paclitaxel is
crucial for its strong antitumor activity.10,11 Esterification
of repin (1) with the side chain of paclitaxel to give
compound 17 led to increased potency (IC50 0.2-1.0 µM)
in all tested cell lines, with the exception of KB-VIN. The
dihydro-oxazole-protected intermediate (16) was generally
less potent, except against KB-VIN and HCT-8 cells.
In summary, the following structure/activity trends were
found with this series of repin analogues. The presence of
a diol (4, 6) rather than an epoxide on the cyclic skeleton
of repin (1) abolished activity. Likewise, compounds with
an allylhydroxy (3) or diol (5) in the side chain also lost
activity. However, the side chain epoxide or both epoxide
moieties of repin (1) could be modified to halohydrins (2,
11, 12 and 7, 8, 9, respectively) and still result in active
compounds.
of three structurally related guaianolides, whose activity
has been previously reported,12,13 and two positive controls
as paclitaxel and mitomycin C in Table 1.
Experimental Section
General Experimental Procedures. Optical rotations
1
To compare the cytotoxic effects of the compounds
presented in this report, we included the IC50 (µM) values
were determined on a JASCO P-1010 digital polarimeter. H
and 13C NMR spectra were recorded on a Bruker AC 250 E

1044 Journal of Natural Products, 2005, Vol. 68, No. 7
Bruno et al.
MHz NMR spectrometer, using the residual solvent signal (δ
amorphous solid; [R]25D +15.0° (c 1.0, CHCl3); 1H NMR (CDCl3,
250 MHz) δ 6.24 (1H, d, J ) 3.4 Hz, H-13a), 5.60 (1H, d, J )
3.0 Hz, H-13b), 5.24 (1H, br dd, J ) 9.4, 5.1 Hz, H-8), 5.20
(1H, br s, H-14a), 5.12 (1H, br s, H-14b), 4.75 (1H, dd, J )
11.2, 9.1 Hz, H-6), 4.11 (1H, br d, J ) 6.5 Hz, H-3), 4.06 (1H,
d, J ) 10.8 Hz, H-15a), 3.71 (1H, d, J ) 10.8 Hz, H-15b), 3.68
(1H, ddd, J ) 11.1, 8.5, 7.0 Hz, H-1), 3.59 (1H, d, J ) 10.3 Hz,
H-3′a), 3.45 (1H, d, J ) 10.3 Hz, H-3′b), 3.16 (1H, dddd, J )
9.4, 9.1, 3.4, 3.0 Hz, H-7), 2.69 (1H, dd, J ) 15.0, 5.1 Hz, H-9a),
2.58 (1H, br d, J ) 15.0 Hz, H-9b), 2.54 (1H, ddd, J ) 14.8,
11.1, 6.5 Hz, H-2a), 2.37 (1H, dd, J ) 11.1, 8.5 Hz, H-5), 1.69
(3H, s, Me-4′), 1.60 (1H, br dd, J ) 14.8, 7.0 Hz, H-2b); 13C
NMR (CDCl3, 62.7 MHz) δ 174.58 (C, C-1′), 168.39 (C, C-12),
142.37 (C, C-10), 136.87 (C, C-11), 122.41 (CH2, C-13), 118.51
(CH2, C-14), 83.47 (C, C-4), 79.81 (CH, C-6), 76.32 (CH, C-3),
75.86 (CH, C-8), 73.26 (C, C-2′), 56.52 (CH, C-5), 48.55 (CH,
C-7), 46.45 (CH, C-1), 37.82 (CH2, C-9), 34.59 (CH2, C-2), 22.68
(CH3, C-4′), 19.11 (CH2, C-15), 15.13 (CH2, C-3′); ESIMS m/z
(positive mode) 641 [M + Na]+ (48), 411 [M + Na - C4H7O3I]+
(62); anal. C 36.94%, H 3.90%, I 41.03%, calcd for C19H24I2O7,
C 36.91%, H 3.91%, I 41.06%.
1
) 7.27 in H and δ ) 77.00 in 13C for CDCl3 as 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% H2O, was used for column chromatog-
raphy. The optically pure (2R,3S)-3-phenylisoserine hydro-
chloride was purchased from Industrial Chemistry Research
(Poland). CH2Cl2 was dried by distillation over calcium hy-
dride. Tetrahydrofuran was dried by distillation from sodium
metal under a nitrogen atmosphere using benzophenone ketyl
as indicator.
General Halohydrin Synthesis Procedure. Repin (1, 50
mg, 0.138 mmol) was solubilized in 4 mL of dry THF and added
to either 1 or 6 equiv of the appropriate lithium halide and 1
(0.01 mL) or 2 (0.02 mL) equiv of acetic acid at room
temperature under an argon atmosphere. After stirring over-
night, the reaction was subjected to the usual workup by
adding brine and extracting with EtOAc. The organic layer
was dried over Na2SO4 and evaporated under reduced pres-
sure. Generally, the residue was purified by column chroma-
tography. This procedure gave the following derivatives.
Chlorohyssopifolin A (7). Treatment of 50 mg of repin
(1) with 6 equiv of lithium chloride (36 mg) gave, after column
chromatography (Si gel, 47:3 CH2Cl2-MeOH), 48 mg of chlo-
rohyssopifolin A (7), whose spectroscopic data were in perfect
agreement with literature values:7 1H NMR (CDCl3, 250 MHz)
δ 6.21 (1H, d, J ) 3.5 Hz, H-13a), 5.57 (1H, d, J ) 3.1 Hz,
H-13b), 5.20 (1H, ddd, J ) 9.6, 5.2, 1.6 Hz, H-8), 5.16 (1H, br
s, H-14a), 5.00 (1H, br s, H-14b), 4.73 (1H, dd, J ) 11.3, 9.0
Hz, H-6), 4.33 (1H, d, J ) 11.8 Hz, H-15a), 4.16 (1H, dd, J )
6.6, 1.5 Hz, H-3), 3.95 (1H, d, J ) 11.8 Hz, H-15b), 3.87 (1H,
d, J ) 11.1 Hz, H-3′a), 3.63 (1H, d, J ) 11.1 Hz, H-3′b), 3.62
(1H, ddd, J ) 11.1, 8.5, 8.0 Hz, H-1), 3.13 (1H, dddd, J ) 9.6,
9.0, 3.5, 3.1 Hz, H-7), 2.65 (1H, dd, J ) 15.3, 5.2 Hz, H-9a),
2.54 (1H, ddd, J ) 15.0, 11.1, 6.6 Hz, H-2a), 2.49 (1H, dd, J )
15.3, 1.6 Hz, H-9b), 2.31 (1H, dd, J ) 11.3, 8.5 Hz, H-5), 1.56
Compounds 2 and 10. Treatment of 50 mg of repin (1)
with 1 equiv of lithium chloride (6 mg) gave a mixture of two
compounds, which were separated by column chromatography
(Si gel, 24:1 CH2Cl2-MeOH as eluent), giving 10 mg of
chlorohyssopifolin C6 (2) and 12 mg of solstitiolide (10),
recovering 30 mg of repin (1).
Compound 10: amorphous solid; [R]25 +52.0° (c 0.7,
D
MeOH); 1H NMR (CDCl3, 250 MHz) δ 6.23 (1H, d, J ) 3.5 Hz,
H-13a), 5.59 (1H, d, J ) 3.1 Hz, H-13b), 5.16 (1H, br s, H-14a),
5.13 (1H, ddd, J ) 9.5, 5.0, 1.9 Hz, H-8), 4.86 (1H, br s, H-14b),
4.73 (1H, dd, J ) 11.2, 9.1 Hz, H-6), 4.33 (1H, d, J ) 11.8 Hz,
H-15a), 4.17 (1H, br d, J ) 6.6 Hz, H-3), 3.96 (1H, d, J ) 11.8
Hz, H-15b), 3.62 (1H, ddd, J ) 11.2, 8.5, 8.0 Hz, H-1), 3.18
(1H, d, J ) 5.8 Hz, H-3′a), 3.12 (1H, dddd, J ) 9.5, 9.1, 3.5,
3.1 Hz, H-7), 2.83 (1H, d, J ) 5.8 Hz, H-3′b), 2.63 (1H, dd, J
) 15.4, 5.0 Hz, H-9a), 2.52 (1H, ddd, J ) 15.0, 11.2, 6.6 Hz,
H-2a), 2.38 (1H, dd, J ) 15.4, 1.6 Hz, H-9b), 2.31 (1H, dd, J )
11.2, 8.5 Hz, H-5), 1.60 (3H, s, Me-4′), 1.58 (1H, br dd, J )
15.0, 8.0 Hz, H-2b); 13C NMR (CDCl3, 62.7 MHz) δ 169.67 (C,
C-1′), 168.51 (C, C-12), 141.64 (C, C-10), 136.85 (C, C-11),
122.48 (CH2, C-13), 118.28 (CH2, C-14), 84.51 (C, C-4), 77.21
(CH, C-6), 76.17 (CH, C-3), 74.99 (CH, C-8), 57.50 (CH, C-5),
53.83 (C, C-2′), 52.78 (CH2, C-3′), 49.86 (CH2, C-15), 47.07 (CH,
C-7), 46.43 (CH, C-1), 37.77 (CH2, C-9), 34.88 (CH2, C-2), 17.36
(CH3, C-4′); ESIMS m/z (positive mode) 423 [(M + 2) + Na]+
(12), 421 [M + Na]+ (40), 321 [(M + 2) + Na - C4H6O3]+ (21),
319 [M + Na - C4H6O3]+ (70); anal. C 57.26%, H 5.83%, Cl
8.90%, calcd for C19H23ClO7, C 57.22%, H 5.81%, Cl 8.89%.
Compounds 12 and 13. Treatment of 50 mg of repin (1)
with 1 equiv of lithium bromide (12 mg) gave a mixture of three
compounds, which were separated by column chromatography
(Si gel, 24:1 CH2Cl2-MeOH as eluent), giving 18 mg of 12, 12
mg of 13, and 12 mg of 8, as well as 16 mg of unreacted repin
(1).
(1H, ddd, J ) 15.0, 8.0, 1.5 Hz, H-2b), 1.55 (3H, s, Me-4′); 13
C
NMR (CDCl3, 62.7 MHz) δ 173.05 (C, C-1′), 168.32 (C, C-12),
141.80 (C, C-10), 136.87 (C, C-11), 122.39 (CH2, C-13), 118.51
(CH2, C-14), 84.62 (C, C-4), 77.15 (CH, C-6), 76.03 (CH, C-3),
75.85 (CH, C-8), 74.69 (C, C-2′), 57.54 (CH, C-5), 51.19 (CH2,
C-3′), 49.99 (CH2, C-15), 47.26 (CH, C-7), 46.39 (CH, C-1), 37.89
(CH2, C-9), 34.56 (CH2, C-2), 23.40 (CH3, C-4′).
Compound 8. Treatment of 50 mg of repin (1) with 6 equiv
of lithium bromide (73 mg) gave, after column chromatography
(Si gel, 97:3 CH2Cl2-MeOH), 66 mg of dibromo derivative 8:
1
amorphous solid; [R]25 +60.5° (c 0.4, CH3COCH3); H NMR
D
(CDCl3, 250 MHz) δ 6.24 (1H, d, J ) 3.6 Hz, H-13a), 5.59 (1H,
d, J ) 3.2 Hz, H-13b), 5.25 (1H, br dd, J ) 9.6, 5.2 Hz, H-8),
5.19 (1H, br s, H-14a), 5.08 (1H, br s, H-14b), 4.74 (1H, dd, J
) 11.2, 9.0 Hz, H-6), 4.26 (1H, d, J ) 11.1 Hz, H-15a), 4.18
(1H, br d, J ) 6.6 Hz, H-3), 3.88 (1H, d, J ) 11.1 Hz, H-15b),
3.76 (1H, d, J ) 10.4 Hz, H-3′a), 3.68 (1H, ddd, J ) 11.1, 8.5,
7.6 Hz, H-1), 3.56 (1H, d, J ) 10.4 Hz, H-3′b), 3.17 (1H, dddd,
J ) 9.6, 9.0, 3.6, 3.2 Hz, H-7), 2.68 (1H, dd, J ) 15.3, 5.2 Hz,
H-9a), 2.54 (1H, ddd, J ) 15.0, 11.1, 6.6 Hz, H-2a), 2.54 (1H,
br d, J ) 15.3 Hz, H-9b), 2.35 (1H, dd, J ) 11.2, 8.5 Hz, H-5),
1.62 (3H, s, Me-4′), 1.56 (1H, br dd, J ) 15.0, 7.6 Hz, H-2b);
13C NMR (CDCl3-CD3OD, 9:1, 62.7 MHz) δ 173.37 (C, C-1′),
170.25 (C, C-12), 143.75 (C, C-10), 138.17 (C, C-11), 122.75
(CH2, C-13), 118.08 (CH2, C-14), 84.61 (C, C-4), 77.95 (CH, C-6),
77.16 (CH, C-3), 76.06 (CH, C-8), 74.76 (C, C-2′), 58.81 (CH,
C-5), 48.65 (CH, C-7), 46.96 (CH, C-1), 40.38 (CH2, C-3′), 40.00
(CH2, C-9), 39.38 (CH2, C-15), 35.08 (CH2, C-2), 24.67 (CH3,
C-4′); ESIMS m/z (positive mode) 549 [(M + 4) + Na]+ (23),
547 [(M + 2) + Na]+ (48), 545 [M + Na]+ (25), 365 [(M + 2) +
Na - C4H7O3Br]+ (59), 363 [M + Na - C4H7O3Br]+ (61); anal.
C 43.58%, H 4.64%, Br 30.52%, calcd for C19H24Br2O7, C
43.53%, H 4.61%, Br 30.49%.
Compound 12: amorphous solid; [R]25 +40.6° (c 0.8,
D
CHCl3); 1H NMR (CDCl3, 250 MHz) δ 6.23 (1H, d, J ) 3.5 Hz,
H-13a), 5.60 (1H, d, J ) 3.1 Hz, H-13b), 5.27 (1H, ddd, J )
9.0, 5.2, 2.4 Hz, H-8), 5.22 (1H, br s, H-14a), 5.17 (1H, br s,
H-14b), 4.68 (1H, dd, J ) 11.3, 9.1 Hz, H-6), 4.01 (1H, dd, J )
7.2, 5.3 Hz, H-3), 3.76 (1H, d, J ) 10.4 Hz, H-3′a), 3.56 (1H, d,
J ) 10.4 Hz, H-3′b), 3.38 (1H, ddd, J ) 10.7, 8.6, 8.5 Hz, H-1),
3.36 (1H, d, J ) 4.8 Hz, H-15a), 3.10 (1H, dddd, J ) 9.1, 9.0,
3.5, 3.1 Hz, H-7), 3.09 (1H, d, J ) 4.8 Hz, H-15b), 2.77 (1H,
dd, J ) 14.7, 5.2 Hz, H-9a), 2.52 (1H, dd, J ) 14.7, 2.4 Hz,
H-9b), 2.49 (1H, ddd, J ) 14.0, 8.6, 7.2 Hz, H-2a), 2.05 (1H,
dd, J ) 11.3, 8.5 Hz, H-5), 1.83 (1H, ddd, J ) 14.0, 10.7, 5.3
Hz, H-2b), 1.62 (3H, s, Me-4′); 13C NMR (CDCl3, 62.7 MHz) δ
173.07 (C, C-1′), 169.35 (C, C-12), 141.04 (C, C-10), 137.16 (C,
C-11), 122.33 (CH2, C-13), 119.21 (CH2, C-14), 76.63 (CH, C-6),
76.30 (CH, C-3), 75.80 (CH, C-8), 74.00 (C, C-2′), 68.15 (C, C-4),
53.62 (CH, C-5), 48.48 (CH2, C-15), 47.75 (CH, C-7), 46.09 (CH,
C-1), 40.07 (CH2, C-3′), 37.84 (CH2, C-9), 35.53 (CH2, C-2),
24.03 (CH3, C-4′); ESIMS m/z (positive mode) 467 [(M + 2) +
Na]+ (44), 465 [M + Na]+ (45), 283 [M + Na - C4H7O3Br]+
Compound 9. Treatment of 50 mg of repin (1) with 6 equiv
of lithium iodide (112 mg) gave, after column chromatography
(Si gel, 97:3 CH2Cl2-MeOH), 82 mg of diiodo derivative 9:

Cytotoxic Activity of Guaianolides
Journal of Natural Products, 2005, Vol. 68, No. 7 1045
(58); anal. C 51.52%, H 5.18%, Br 18.08%, calcd for C19H23-
122.44 (CH2, C-13), 118.14 (CH2, C-14), 83.77 (C, C-4), 79.84
(CH, C-6), 76.80 (CH, C-3), 74.99 (CH, C-8), 56.53 (CH, C-5),
53.82 (C, C-2′), 52.71 (CH2, C-3′), 48.38 (CH, C-7), 46.29 (CH,
C-1), 37.70 (CH2, C-9), 34.67 (CH2, C-2), 19.07 (CH2, C-15),
17.38 (CH3, C-4′); ESIMS m/z (positive mode) 513 [M + Na]+
(50), 411 [M + Na - C4H6O3]+ (78); anal. C 46.50%, H 4.70%,
I 25.85%, calcd for C19H23IO7, C 46.54%, H 4.73%, I 25.88%.
Compound 16. The (4S,5R)-2,4-diphenyl-4,5-dihydro-ox-
azol-5-carboxylic acid 15 (44 mg, 0.165 mmol), synthesized as
previously reported,9 was dissolved in 5 mL of dry CH2Cl2 and
added to DMAP (5 mg, 0.025 mmol) and DCC (38 mg, 0.184
mmol). After stirring at room temperature for 15 min, repin
(1) (55 mg, 0.149 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, 49:1 CH2Cl2-MeOH as eluent) to give
BrO7, C 51.48%, H 5.23%, Br 18.03%.
Compound 13: amorphous solid; [R]25 +24.0° (c 0.5,
D
CHCl3); 1H NMR (CDCl3, 250 MHz) δ 6.25 (1H, d, J ) 3.4 Hz,
H-13a), 5.60 (1H, d, J ) 3.1 Hz, H-13b), 5.17 (1H, br s, H-14a),
5.13 (1H, br dd, J ) 9.3, 5.0 Hz, H-8), 4.87 (1H, br s, H-14b),
4.74 (1H, dd, J ) 11.0, 9.2 Hz, H-6), 4.25 (1H, d, J ) 11.0 Hz,
H-15a), 4.16 (1H, br d, J ) 6.3 Hz, H-3), 3.88 (1H, d, J ) 11.0
Hz, H-15b), 3.65 (1H, ddd, J ) 11.2, 8.5, 7.3 Hz, H-1), 3.19
(1H, d, J ) 5.7 Hz, H-3′a), 3.15 (1H, dddd, J ) 9.3, 9.2, 3.4,
3.1 Hz, H-7), 2.84 (1H, d, J ) 5.7 Hz, H-3′b), 2.62 (1H, dd, J
) 15.4, 5.0 Hz, H-9a), 2.52 (1H, ddd, J ) 15.1, 11.2, 6.3 Hz,
H-2a), 2.38 (1H, br d, J ) 15.4 Hz, H-9b), 2.30 (1H, dd, J )
11.0, 8.5 Hz, H-5), 1.63 (3H, s, Me-4′), 1.57 (1H, br dd, J )
15.1, 7.3 Hz, H-2b); 13C NMR (CDCl3, 62.7 MHz) δ 169.87 (C,
C-1′), 168.48 (C, C-12), 141.73 (C, C-10), 136.85 (C, C-11),
122.46 (CH2, C-13), 118.29 (CH2, C-14), 83.93 (C, C-4), 78.19
(CH, C-6), 76.12 (CH, C-3), 74.96 (CH, C-8), 57.48 (CH, C-5),
53.83 (C, C-2′), 52.78 (CH2, C-3′), 47.65 (CH, C-7), 46.35 (CH,
C-1), 41.40 (CH2, C-15), 37.72 (CH2, C-9), 34.66 (CH2, C-2),
17.37 (CH3, C-4′); ESIMS m/z (positive mode) 467 [(M + 2) +
Na]+ (50), 465 [M + Na]+ (52), 365 [(M + 2) + Na - C4H6O3]+
(72), 363 [M + Na - C4H6O3]+ (74); anal. C 51.50%, H 5.20%,
Br 18.00%, calcd for C19H23BrO7, C 51.48%, H 5.23%, Br
18.03%.
66 mg (yield 72%) of the ester 16: amorphous solid; [R]25
D
1
+1.0° (c 2.5, CHCl3); H NMR (CDCl3, 250 MHz) δ 8.05 (1H,
m, arom), 8.02 (1H, m, arom), 7.55-7.29 (8H, m, arom), 6.12
(1H, d, J ) 3.4 Hz, H-13a), 5.41 (1H, d, J ) 3.0 Hz, H-13b),
5.40 (1H, d, J ) 6.0 Hz, H-3′′), 5.16 (1H, br s, H-14a), 5.06
(1H, dd, J ) 7.1, 1.6 Hz, H-3), 4.95 (1H, d, J ) 6.0 Hz, H-2′′),
4.90 (1H, br s, H-14b), 4.71 (1H, ddd, J ) 9.6, 4.7, 3.9 Hz, H-8),
4.12 (1H, dd, J ) 11.4, 9.4 Hz, H-6), 3.40 (1H, ddd, J ) 11.0,
8.8, 8.2 Hz, H-1), 3.27 (1H, d, J ) 4.4 Hz, H-15a), 3.18 (1H, d,
J ) 4.4 Hz, H-15b), 3.14 (1H, d, J ) 5.7 Hz, H-3′a), 2.90 (1H,
dddd, J ) 9.6, 9.4, 3.4, 3.0 Hz, H-7), 2.82 (1H, d, J ) 5.7 Hz,
H-3′b), 2.67 (1H, ddd, J ) 15.5, 11.0, 7.1 Hz, H-2a), 2.39 (1H,
dd, J ) 14.7, 4.7 Hz, H-9a), 2.23 (1H, dd, J ) 14.7, 3.9 Hz,
H-9b), 1.97 (1H, dd, J ) 11.4, 8.2 Hz, H-5), 1.87 (1H, ddd, J )
15.5, 8.8, 1.6 Hz, H-2b), 1.61 (3H, s, Me-4′); 13C NMR (CDCl3,
62.7 MHz) δ 169.60 (C, C-1′′), 169.07 (C, C-1′), 167.92 (C, C-12),
163.42 (C, C-5′′), 140.43 (C, C-10), 139.85 (C, arom), 136.60
(C, C-11), 132.27 (CH, arom), 128.89 (2CH, arom), 128.72
(2CH, arom), 128.40 (2CH, arom), 128.23 (CH, arom), 126.90
(C, arom), 126.41 (2CH, arom), 121.87 (CH2, C-13), 119.28
(CH2, C-14), 82.82 (CH, C-2′′), 79.64 (CH, C-3), 75.40 (CH, C-6),
74.94 (CH, C-8), 74.55 (CH, C-3′′), 66.16 (C, C-4), 53.74 (CH,
C-5), 53.62 (C, C-2′), 52.73 (CH2, C-3′), 49.38 (CH, C-7), 48.06
(CH2, C-15), 45.71 (CH, C-1), 35.93 (CH2, C-9), 35.19 (CH2,
C-2), 17.25 (CH3, C-4′); ESIMS m/z (positive mode) 634 [M +
Na]+ (20), 266 (52); anal. C 68.70%, H 5.47%, N 2.27%, calcd
for C35H33NO9, C 68.73%, H 5.44%, N 2.29%.
Compound 11. Treatment of 50 mg of repin (1) with 1 equiv
of lithium iodide (19 mg) gave a mixture of two compounds,
which were separated by column chromatography (Si gel, 24:1
CH2Cl2-MeOH as eluent), giving 54 mg of 11, 8 mg of 9, and
5 mg of recovered repin (1).
Compound 11: amorphous solid; [R]25 +51.7° (c 0.4,
D
CHCl3); 1H NMR (CDCl3, 250 MHz) δ 6.24 (1H, d, J ) 3.5 Hz,
H-13a), 5.59 (1H, d, J ) 2.9 Hz, H-13b), 5.27 (1H, ddd, J )
9.1, 5.2, 2.9 Hz, H-8), 5.24 (1H, br s, H-14a), 5.21 (1H, br s,
H-14b), 4.68 (1H, dd, J ) 11.3, 9.2 Hz, H-6), 4.00 (1H, dd, J )
7.2, 5.9 Hz, H-3), 3.58 (1H, d, J ) 10.3 Hz, H-3′a), 3.44 (1H, d,
J ) 10.3 Hz, H-3′b), 3.39 (1H, ddd, J ) 10.0, 9.6, 8.3 Hz, H-1),
3.35 (1H, d, J ) 4.2 Hz, H-15a), 3.10 (1H, dddd, J ) 9.2, 9.1,
3.5, 2.9 Hz, H-7), 3.08 (1H, d, J ) 4.2 Hz, H-15b), 2.80 (1H,
dd, J ) 15.1, 5.2 Hz, H-9a), 2.56 (1H, dd, J ) 15.1, 2.9 Hz,
H-9b), 2.48 (1H, ddd, J ) 14.5, 9.6, 7.2 Hz, H-2a), 2.04 (1H,
dd, J ) 11.3, 8.3 Hz, H-5), 1.84 (1H, ddd, J ) 14.5, 10.0, 5.9
Hz, H-2b), 1.68 (3H, s, Me-4′); 13C NMR (CDCl3, 62.7 MHz) δ
175.35 (C, C-1′), 168.80 (C, C-12), 141.06 (C, C-10), 137.10 (C,
C-11), 122.29 (CH2, C-13), 119.32 (CH2, C-14), 76.48 (CH, C-6),
76.48 (CH, C-3), 75.81 (CH, C-8), 73.29 (C, C-2′), 68.16 (C, C-4),
53.54 (CH, C-5), 48.54 (CH2, C-15), 47.87 (CH, C-7), 46.09 (CH,
C-1), 37.78 (CH2, C-9), 35.71 (CH2, C-2), 21.16 (CH3, C-4′),
14.98 (CH2, C-3′); ESIMS m/z (positive mode) 513 [M + Na]+
(45), 283 [M + Na - C4H7O3I]+ (58); anal. C 46.51%, H 4.77%,
I 25.83%, calcd for C19H23IO7, C 46.54%, H 4.73%, I 25.88%.
Synthesis of Compound 14. Compound 9 (20 mg, 0.032
mmol) was solubilized in 2 mL of dry THF and then 1 equiv
of potassium tert-butoxide (4 mg) was added, and the reaction
mixture was stirred at room temperature for 24 h. The reaction
mixture was quenched by adding 2 mL of saturated aqueous
NH4Cl and extracted three times with EtOAc. The organic
layer was dried over Na2SO4 and evaporated under reduced
pressure. The residue was purified by column chromatography
(Si gel, 24:1 CH2Cl2-MeOH as eluent), giving 7 mg of 14, 1.5
mg of 13, and 4 mg of repin (1).
Compound 17. Compound 16 (36 mg, 0.059 mmol), dis-
solved in CH2Cl2 (6 mL), was stirred at room temperature with
p-toluenesulfonic acid (3 mg, 0.017 mmol). After completion
of the reaction 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 chromatography (Si gel, 49:1
CH2Cl2-MeOH as eluent) to give 28 mg (yield 75%) of
compound 17: amorphous solid; [R]25D +81.8° (c 0.33, CHCl3);
1H NMR (CDCl3, 250 MHz) δ 7.82 (1H, m, arom), 7.79 (1H, m,
arom), 7.58-7.30 (8H, m, arom), 7.03 (1H, d, J ) 9.7 Hz, NH),
6.28 (1H, d, J ) 3.4 Hz, H-13a), 5.88 (1H, dd, J ) 9.7, 1.9 Hz,
H-3′′), 5.62 (1H, d, J ) 3.0 Hz, H-13b), 5.40 (1H, ddd, J ) 9.1,
4.9, 2.2 Hz, H-8), 5.11 (1H, br s, H-14a), 4.85 (1H, br s, H-14b),
4.82 (1H, dd, J ) 8.3, 4.7 Hz, H-3), 4.77 (1H, dd, J ) 11.6, 9.3
Hz, H-6), 4.73 (1H, d, J ) 1.9 Hz, H-2′′), 3.40 (1H, d, J ) 4.2
Hz, H-15a), 3.31 (1H, ddd, J ) 12.0, 7.3, 7.3 Hz, H-1), 3.17
(1H, d, J ) 5.9 Hz, H-3′a), 3.08 (1H, d, J ) 4.2 Hz, H-15b),
3.03 (1H, dddd, J ) 9.3, 9.1, 3.4, 3.0 Hz, H-7), 2.82 (1H, d, J
) 5.9 Hz, H-3′b), 2.63 (1H, ddd, J ) 15.3, 8.3, 7.3 Hz, H-2a),
2.63 (1H, dd, J ) 15.5, 4.9 Hz, H-9a), 2.10 (1H, dd, J ) 15.5,
2.2 Hz, H-9b), 1.93 (1H, dd, J ) 11.6, 7.3 Hz, H-5), 1.73 (1H,
ddd, J ) 15.3, 12.0, 4.7 Hz, H-2b), 1.63 (3H, s, Me-4′); 13C NMR
(CDCl3, 62.7 MHz) δ 172.17 (C, C-1′′), 169.40 (C, C-1′), 168.55
(C, C-12), 167.05 (C, C-5′′), 140.27 (C, C-10), 138.21 (C, arom),
137.31 (C, C-11), 133.18 (C, arom), 132.21 (CH, arom), 128.92
(2CH, arom), 128.83 (2CH, arom), 128.06 (CH, arom), 127.17
(2CH, arom), 126.99 (2CH, arom), 122.24 (CH2, C-13), 119.61
(CH2, C-14), 81.90 (CH, C-3), 75.60 (CH, C-8), 75.06 (CH, C-6),
73.07 (CH, C-2′′), 66.04 (C, C-4), 55.14 (CH, C-3′′), 53.92 (CH,
C-5), 53.92 (C, C-2′), 52.72 (CH2, C-3′), 49.00 (CH, C-7), 48.35
Compound 14: amorphous solid; [R]25 +32.5° (c 0.6,
D
1
3
CHCl ); H NMR (CDCl3, 250 MHz) δ 6.24 (1H, d, J ) 3.5 Hz,
H-13a), 5.59 (1H, d, J ) 3.0 Hz, H-13b), 5.16 (1H, br s, H-14a),
5.14 (1H, br dd, J ) 9.3, 5.0 Hz, H-8), 4.86 (1H, br s, H-14b),
4.73 (1H, dd, J ) 11.0, 9.0 Hz, H-6), 4.12 (1H, br d, J ) 6.3
Hz, H-3), 4.05 (1H, d, J ) 10.8 Hz, H-15a), 3.70 (1H, d, J )
10.8 Hz, H-15b), 3.67 (1H, ddd, J ) 11.2, 8.5, 7.4 Hz, H-1),
3.19 (1H, d, J ) 5.8 Hz, H-3′a), 3.18 (1H, dddd, J ) 9.3, 9.0,
3.5, 3.0 Hz, H-7), 2.83 (1H, d, J ) 5.8 Hz, H-3′b), 2.62 (1H, dd,
J ) 15.4, 5.0 Hz, H-9a), 2.52 (1H, ddd, J ) 15.1, 11.2, 6.3 Hz,
H-2a), 2.38 (1H, br d, J ) 15.4 Hz, H-9b), 2.37 (1H, dd, J )
11.0, 8.5 Hz, H-5), 1.85 (1H, br dd, J ) 15.1, 7.4 Hz, H-2b),
1.64 (3H, s, Me-4′); 13C NMR (CDCl3, 62.7 MHz) δ 169.90 (C,
C-1′), 168.55 (C, C-12), 142.03 (C, C-10), 137.00 (C, C-11),

1046 Journal of Natural Products, 2005, Vol. 68, No. 7
Bruno et al.
(CH2, C-15), 46.73 (CH, C-1), 36.33 (CH2, C-9), 34.57 (CH2,
C-2), 17.44 (CH3, C-4′); ESIMS m/z (positive mode) 652 [M +
Na]+ (11), 268 (78); anal. C 66.72%, H 5.58%, N 2.20%, calcd
for C35H35NO10, C 66.76%, H 5.60%, N 2.22%.
References and Notes
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In Vitro Anticancer Assay.14 All stock cultures were
grown in T-25 flasks. Freshly trypsinized cell suspensions were
seeded in 96-well microtiter plates at densities of 1500-7500
cells per well with compounds added from DMSO-diluted stock.
After 3 days in culture, attached cells were fixed with cold 50%
trichloroacetic acid and then stained with 0.4% sulforhodamine
B (SRB). The absorbency at 562 nm was measured using a
microplate reader after solubilizing the bound dye. The mean
IC50 is the concentration of agent that reduces cell growth by
50% under the experimental conditions and is the average
from three determinations that were reproducible and statisti-
cally significant. The following human tumor cell lines were
used in the assay: A549 (non small cell lung cancer), MCF-7
(estrogen receptor positive breast cancer), HCT-8 (colon can-
cer), SK-Mel-2 (melanoma), 1A9 (ovarian cancer), KB (na-
sopharyngeal carcinoma), and KB-VIN (vincristine-resistant
KB subline). All cell lines were obtained from the Lineberger
Comprehensive Cancer Center (UNC-CH) or from ATCC
(Rockville, MD) and were cultured in RPMI-1640 medium
supplemented with 25 mM HEPES, 0.25% sodium bicarbonate,
10% fetal bovine serum, and 100 µg/mL kanamycin.
(12) Ha, T. J.; Jang, D. S.; Lee, J. R.; Lee, K. D.; Lee, J.; Hwang, S. W.;
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Acknowledgment. This work was supported by Italian
Government project MIUR “Sostanze Naturali ed Analoghi
Sintetici con Attivita` Antitumorale” awarded to M.B. and by
NIH grant CA-17625 awarded to K.H.L.
NP0500575