Cytotoxic activity of (S)-goniothalamin and analogues against human cancer cells

Article abstract of DOI:10.1016/j.bmc.2005.08.036

(R)- and (S)-Goniothalamin (1) and analogues 2-9 were efficiently prepared in high overall yield and enantiomeric purity, and their cytotoxic activities were evaluated against eight human cancer cell lines. A structure-activity relationship study (SAR) al

Full text of DOI:10.1016/j.bmc.2005.08.036

Bioorganic & Medicinal Chemistry 14 (2006) 622–631
Cytotoxic activity of (S)-goniothalamin and analogues
against human cancer cells
a
Angelo de Fatima, Luciana K. Kohn, Joa˜o Ernesto de Carvalho and Ronaldo A. Pilli
b
b
a,
*
ˆ
´
a
´
Instituto de Quımica, Unicamp, CP 6154, 13084-971, Campinas, Sa˜o Paulo, Brazil
´
b
´ ´ ´
Centro Pluridisciplinar de Pesquisas Quımica, Biologicas e Agrıcolas, Unicamp, CP 6171, 13083-970, Paulınia, Sa˜o Paulo, Brazil
Received 14 July 2005; revised 22 August 2005; accepted 22 August 2005
Available online 3 October 2005
Abstract—(R)- and (S)-Goniothalamin (1) and analogues 2–9 were efficiently prepared in high overall yield and enantiomeric purity,
and their cytotoxic activities were evaluated against eight human cancer cell lines. A structure–activity relationship study (SAR)
allowed us to establish the relevant structural features for the cytotoxic activity of goniothalamin analogues. In addition, we have
identified non-natural form of goniothalamin (S)-1 and analogue 5 as the highest and more selective cytotoxic compounds against
kidney cancer cell growth (786-0) with IC50 = 4 and 5 nM, respectively, and compound 8 (IC50 = 4 nM) as the more potent against
breast cancer cells with resistance phenotype for adryamycin (NCI.ADR).
ꢀ 2005 Elsevier Ltd. All rights reserved.
O
1. Introduction
O
Small exogenous molecules can exert powerful effects on
cell functions making them useful for understanding life
processes and treating life-threatening diseases. In the
(R)-Goniothalamin [(R)-1]
center of such scientific endeavor is the molecule which
Figure 1. Structure of (R)-goniothalamin [(R)-1].
needs to be made. A lead structure may be inspired
either from nature (target-oriented approach) or from
diversity-oriented synthesis. In both cases, an additional
dimension is provided by the chirality of the compounds
which may significantly influence the biological activity
of such chemical entities.
minimal on non-malignant cells. In vivo studies have
shown that (R)-1 displays tumoricidal and tumoristatic
effects on Sprague–Dawley rats with 7,12-dimethylbenz-
anthracene (DMBA)-induced mammary tumors.13 We
have previously reported on the cytotoxic activity of
(R)-goniothalamin (1) against eight different human can-
The styryl-lactones are a group of secondary metabolites
cer cell lines.14 Previous studies about the mechanism of
action of (R)-1 showed that this compound induces apop-
tosis in Jurkat T-cells and MCF-7 cells by activation of
caspases-3 and -7.8,11,12 Subsequent studies with HL-60
cells demonstrated that (R)-1-induced apoptosis occurs
via the loss of mitochondrial membrane potential
(Dwm) and activation of initiator caspase-9.9 Chien11
and Azimahtol12 showed that (R)-1 was able to modulate
the Bax expression increasing pro-apoptotic protein
(Bax) levels without affecting the anti-apoptotic Bcl-2
expression. It was also demonstrated in vivo that p53 tu-
mor suppressor protein accumulation was more pro-
nounced in rat tumor treated with (R)-1.13 In spite of
biological activities exhibited by (R)-1, no studies on the
cytotoxic activity of its enantiomer, (S)-goniothalamin
[(S)-1] or derivatives have been reported yet.
commonly isolated from the genus Goniothalamus,1 and
recent studies have demonstrated that these compounds
display cytotoxic and antitumor properties.2–7 Among
them, (R)-goniothalamin (1) (Fig. 1) has shown in vitro
cytotoxic effects especially by inducing apoptosis on dif-
ferent cancer cell lines [cervical carcinoma (Hela), gastric
carcinoma (HGC-27), breast carcinoma (MCF-7, T47D,
and MDA-MB-231), leukemia carcinoma (HL-60), and
ovarian carcinoma (Caov-3)].2,8–12 This cytotoxic activity
was observed on cancer cell lines but was shown to be
Keywords: (R)- and (S)-Goniothalamin; Styryl lactones; Cytotoxic
activity; Cancer cell lines.
*
Corresponding author. Tel.: +55 19 37883083; fax: +55 19
3788311; e-mail: pilli@iqm.unicamp.br
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.08.036

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A. de Fatima et al. / Bioorg. Med. Chem. 14 (2006) 622–631
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623
Figure 2. The l-oxo bis (binaphthoxy)(isopropoxy)titanium complexes
(S,S)-A developed by Maruoka and co-workers.25,26
Several asymmetric syntheses of (R)-1 have been pub-
lished,15–21 including the non-natural enantiomer, (S)-
1.22–24 Recently, we have reported a highly enantioselec-
tive synthesis of (R)-1 in three steps, 73% overall yield
and 96% enantiomeric excess from trans-cinnamalde-
hyde (2).14,17 This methodology was employed to pro-
vide (S)-1 and analogues (S)-2 to (S)-9 to evaluate
their cytotoxic activity on various cancer cell lines and
to carry out a structure–activity relationship (SAR)
study aiming to identify the pharmacophoric groups.
Bis(binaphthoxy)(isopropoxy)titanium oxide catalysts
(R,R)- or (S,S)-A (Fig. 2), described by Maruoka and
co-workers25,26 for the enantioselective allylation of cin-
namaldehyde, were employed for the total syntheses of
(R)- and (S)-1, respectively.16,24
Figure 3. Percentage growth of cancer cells for 48 h with different
concentrations (0.25, 2.5, 25, and 250 lg/mL) of (R)- or (S)-gonio-
thalamin [(R)- or (S)-1]. A, (S)-goniothalamin [(S)-1]; B, (R)-gonio-
thalamin [(R)-1]. Positive values in relation to y axis correspond to
cytostatic activity, while the others refer to cytotoxic activity of
compounds analyzed. The experiments were done in triplicate as
described in Section 4 using a panel of NCI human tumoral cells.
2. Results and discussion
Since it is well known that different cell lines display dif-
ferent sensitivities toward a cytotoxic compound, in the
present study we have used cell lines of various histolog-
ical origin {MCF-7 (breast), NCI.ADR (breast express-
ing the resistance phenotype for adryamycin), NCI.460
(lung, non-small cells), UACC.62 (melanoma), 786-0
(kidney), OVCAR03 (ovarian), PCO.3 (prostate), and
HT-29 (colon), which were grown in vitro [cell lines were
kindly provided by Frederick MA, National Cancer
Institute/USA]} for the initial evaluation of the cytotox-
icity of (R)- and (S)-goniothalamin [(R)- and (S)-1]. Cell
proliferation was determined by spectrophotometric as-
say using sulforhodamine B as protein-binding dye.27–29
(R)- and (S)-1 were used at 0.25–250 lg/mL and doxoru-
bicin (DOX, at the same concentration range), as posi-
tive control (see Section 4 for details). Concentration
that elicits 50% inhibition of cell growth (IC50) was
determined after 48 h of cell treatment.
(IC50 = 6.4 lM). Additionally, the IC50 values for (S)-1
against ovarian (OVCAR03) and prostate (PCO.3) were
lower than the ones found for natural goniothalamin
[(R)-1]. Additionally, both enantiomers presented simi-
lar IC50 values for breast cancer cell line (MCF-7) with
natural goniothalamin [(R)-1] being two times more po-
tent than (S)-1 against lung (NCI.460) and colon (HT-
29) cancer cell lines and slightly more active for melano-
ma (UACC.62).
The extremely high antiproliferative activity (Table 1)
and selectivity (Fig. 3A) presented by (S)-1 for 786-0 cell
line (IC50 4 nM) prompted us to synthesize analogues 2–
9 (Fig. 4) to identify the pharmacophoric groups respon-
sible for this activity.
Both enantiomers of goniothalamin (1) displayed anti-
proliferative activity in a concentration-dependent way
against the cancer cell lines tested (Fig. 3). IC50 (lM) for
(R)- and (S)-1 as well as DOX are summarized in Table 1.
The synthesis of 2 was carried out from (S)-1 by selective
reduction of the conjugated double bond of the pyra-
30–33
none ring using StrykerÕs reagent [(Ph3P)CuH]6
in
It is noteworthy that goniothalamin (1) enantiomers dis-
played higher potency against 786-0 cell line (kidney tu-
mor) and NCI.ADR (breast expressing the resistance
phenotype for adryamycin) than DOX. While for breast
resistant cancer cell line (NCI.ADR), (R)-1 showed to be
10 and 20 times more powerful than (S)-1 and DOX,
respectively, for kidney cells (786-0), we found (S)-1
(IC50 = 4 nM) to be 1, 600-fold more potent than (R)-1
benzene (63% yield, Scheme 1), while catalytic hydroge-
nation (10% Pd/C, 1 atm of H2) provided 3 in 79% yield
(Scheme 1).
MaruokaÕs catalyst [(S,S)-A] also performed well in the
asymmetric allylation of substituted aromatic and ali-
phatic aldehydes 11–16 thus extending the scope of this
methodology (Table 2). As observed in Table 2,

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624
Table 1. IC50 values, given in lM, for (R)- and (S)-goniothalamin [(R)- and (S)-1] and doxorubicin (DOX) necessary for inhibiting tumor cell
proliferationa
Compound
MCF-7
NCI.ADR
NCI.460
UACC.62
786-0
6.4
0.004
>100
OVCAR03
PCO.3
HT-29
(R)-(1)
(S)-(1)
DOXb
10.5
9.4
2.3
23.5
48.7
6.4
14.6
1.8
17.4
27.6
9.8
39.0
17.0
11.7
>100
24.3
18.6
11.2
22.5
5.3
3.3
a Concentration that elicits inhibition by 50% of cell growth (IC50) was determined from non-linear regression analysis using the GraphPad Prism
software (r2 > 0.9).
b Doxorubicin (DOX) was the positive control.
excellent enantiomeric excesses were observed in all
cases. While the presence of electron-withdrawing
groups in the aromatic ring did not affect either the yield
or the enantiomeric excess in the allylation reaction (en-
tries 3 and 4, Table 2), electron-donating groups as in
aldehydes 14 and 15 slowed down the reaction rate
probably due to additional complexation sites for the
catalyst and only provided good yields (61–79%) when
20 mol% of (S,S)-A was employed. However, similar
enantioselectivity in the allylation reaction was observed
when 10 or 20 mol% (entries 5 and 6, Table 2) of (S,S)-A
was used. Aldehyde 16 furnished the corresponding
homoallylic alcohol in excellent enantiomeric excess
and good yield (entry 7, Table 2).
Figure 4. Structures of analogues 2–9 of (S)-goniothalamin [(S)-1].
The next step involved acylation of homoallylic alcohols
17–23 with acryloyl chloride and ring closing metathesis
reaction catalyzed by GrubbsÕ ruthenium complex
Scheme 1. Syntheses of analogues 2 and 3 from (S)-goniothalamin
[(S)-1].
Table 2. Total syntheses of (S)-goniothalamin [(S)-1] and analogues 4–9
O
O
OH
O
a
O
b
c
RCHO
R
R
R
(17-23)
(24-30)
(10-16)
[ (S)-1 and 4-9]
Entry
R
Product (yield, enantiomeric excess)
Step b
Step a
Step c
1
2
3
4
5
6
7
17 (94%, 96% ee)b
18 (98%, 99% ee)c
19 (94%, 95% ee)c
20 (82%, 96% ee)c
21 (61%a, 94% ee)c
22 (79%a, 92% ee)c
23 (64%, 99% ee)b
24 (80%)
25 (86%)
26 (89%)
27 (85%)
28 (84%)
29 (79%)
30 (85%)
(S)-1 (98%)
(10)
(11)
4 (99%)
6 (77%)
7 (88%)
8 (78%)
9 (80%)
5 (94%)
(12)
O N
2
(13)
F
(14)
MeO
O
(15)
O
(16)
Reagents and conditions: (a) (S,S)-A [(S)-BINOL (10 mol%), Ti(OiPr)4 (15 mol%), TiCl4 (5 mol%), Ag2O (10 mol%)], allyltributyltin (1.1 equiv),
CH2Cl2, ꢀ20 ꢁC, 24 h; (b) acryloyl chloride (1.8 equiv), Et3N (3.6 equiv), CH2Cl2, 0 ꢁC; (c) GrubbsÕ catalyst [(PCy3)2Cl2Ru@CHPh] (10 mol%),
CH2Cl2.
a 20 mol% of (S,S)-A was used.
b The enantiomeric excess was determined by chiral GC analysis (CP-Chirasil-Dex-CB) or c1H NMR or 19F NMR analysis of the correspondent
Mosher esters derived from alcohols 17–23 (see Supplementary Material for details).

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Table 3. IC50 values, given in lM, for analogues 2–9 and doxorubicin (DOX) necessary for inhibiting tumor cell proliferationa
Compound
MCF-7
NCI.ADR
NCI.460
UACC.62
786-0
19.0
>100
20.8
0.005
OVCAR03
PCO.3
HT-29
2
12.5
>100
20.0
4.8
0.1
>100
6.0
11.0
>100
18.3
16.5
27.3
53.2
16.9
38.1
1.8
5.5
>100
23.7
2.4
3.5
>100
16.3
1.5
>100
>100
20.8
15.5
26.5
24.3
14.8
21.7
18.6
8.5
1.9
3
4
28.7
5
19.4
11.2
18.7
0.004
23.7
48.7
>100
6
16.5
14.4
6.5
13.0
23.8
5.9
2.2
9.6
9.4
9.4
34.8
28.2
23.7
5.3
7
26.1
20.8
24.6
11.7
8
7.4
31.9
9
40.1
3.3
26.8
9.8
DOXb
>100
a Concentration that elicits inhibition of 50% of cell growth (IC50) was determined from non-linear regression analysis using the GraphPad Prism
software (r2 > 0.9).
b DOX was employed as positive control.
Critical for activity
34
PhCH@RuCl2(PCy3)2 (Table 2). This approach fur-
nished analogues 4–9 in 3 steps, good overall yields
(40–83%) and enantiomeric excesses (92–99% ee): (R)-
O
O
Michael acceptor:
essential for activity
Hydrophobic tail
or (S)-1 (73%, 96% ee), 4 (83%, 99% ee), 5 (51%, 99%
ee), 6 (64%, 95% ee), 7 (61%, 96% ee), 8 (40%, 94%
ee), and 9 (50%, 92% ee). At this point, our efforts were
directed to evaluate the antiproliferative activity of 2–9
against the cancer cell panel described above (see Sec-
tion 4 for details). IC50 values (lM) for analogues 2–9
and DOX are summarized in Table 3.
binding to substrate
Absolute S configuration
is essential
Goniothalamin (1)
Figure 5. Pharmacophoric groups of goniothalamin (1) identified by
their cytotoxic activity against kidney cancer cell line (786-0).
and (S)-5, respectively, likely interacts with a hydropho-
bic domain of a biomolecule present in the cancer cell
since these compounds were shown to be the most active
in inhibiting cell proliferation at nanomolar concentra-
tions. From all these results, we have identified the phar-
macophoric groups of goniothalamin (1) as presented in
Figure 5.
(S)-1 was 4, 750- and 5200-fold more potent in inhibit-
ing proliferation of kidney cancer cells (786-0) than
analogues 2 and 4, respectively (Tables 1 and 3).
Remarkably, (S)-5 with a cyclohexyl substituent con-
served the same cytotoxic activity as (S)-goniothalamin
(1) against kidney cancer cells (786-0) being active at
the nanomolar level (IC50 = 5 nM). No antiproliferative
activity was found for analogue 3 under our experi-
mental conditions. These results demonstrated that
the endo and exo double bonds in the pyranone ring
are essential for the activity of (S)-1 against kidney
cancer cell line (786-0). It is noteworthy that analogues
(S)-2, (S)-3, and (S)-4 lacking either one or both dou-
ble bonds had much lower activity than (S)-1. Recent-
ly, the importance of the E-configuration of the styryl
moiet in (R)-1 for its antiproliferative activity was
shown14 and the a,b-unsaturated lactone group has al-
ready been implicated in the biological activity of other
natural lactones such as cytostatin35 and fostriecin.36
This behavior is most probably due to its possible role
as a Michael acceptor for nucleophilic amino acid res-
idues (cysteine, lysine, serine or threonine) present in
the natural receptors which interact with these
compounds.
Even though the most expressive activity of (S)-gonio-
thalamin [(S)-1] was against kidney cancer cells, very
promising results were also obtained for other cancer
cell cultures. All analogues evaluated in this study were
more potent than DOX against breast expressing the
resistance phenotype for adryamycin (NCI.ADR), par-
ticularly compound (S)-8 which displayed IC50 = 4 nM.
For melanoma cell line (UACC.62), (R)-1 was more po-
tent than (S)-1, although analogues (S)-2, (S)-5, and (S)-
8 were 3-, 7- and 3-fold more potent, respectively, than
(R)-1 (Tables 1 and 3). For breast cancer cells (MCF-
7), the substitution of the aromatic group for a cyclohex-
yl group (compound 5) or the addition of a 4-OMe
group in the aromatic ring [compound (S)-8] enhanced
the potency originally displayed by (S)-1. Similarly,
compounds (S)-5 and (S)-8 were more potent than (S)-
1 and significantly more than (R)-1 in inhibiting prostate
cancer cell (PCO.3) proliferation (Tables 1 and 3). In the
case of ovarian cancer cells (OVCAR03), the analogues
2 and 5 were 5- and 7-fold more potent than (S)-1,
respectively, while colon cancer cells (HT-29) were more
sensitive to analogue (S)-3 [IC50 = 190 nM]. As to the
cytotoxic activity against lung cancer cell line
(NCI.460), none of the compounds evaluated displayed
better activity than (R)-1 (Tables 1 and 3). In our stud-
ies, doxorubicin (DOX, an anticancer drug used as a po-
sitive control with IC50 = 9.8 lM) presented lower
activity for melanoma cancer cells (UACC.62) than ana-
logues (S)-2 [IC50 = 5.5 lM], (S)-5 [IC50 = 2.4 lM], and
Interestingly, (R)-2 and (R)-3 were shown to be the main
metabolites detected and identified in urine and blood
samples of (R)-1-treated Sprague–Dawley rats thus sig-
naling the double bond reduction in (R)-1 as a potential
detoxification route by this species.37 Moreover, the re-
sults obtained from the treatment of kidney cancer cells
with pyranones (S)-5 to (S)-9 demonstrated that elec-
tron-donating or electron-withdrawing groups in the
aromatic ring decreased their potency when compared
to (S)-1. Overall, the findings for kidney cancer cell line
(786-0) suggest that aromatic ring or cyclohexyl in (S)-1

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A. de Fatima et al. / Bioorg. Med. Chem. 14 (2006) 622–631
626
(S)-8 [IC50 = 5.9 lM]. On ovarian cancer cells (OV-
CAR03), the analogues (S)-2 [IC50 = 3.5 lM], (S)-5
[IC50 = 1.5 lM], and (S)-6 [IC50 = 9.4 lM] were more
potent than DOX [IC50 = 11.7 lM], while roughly the
same values were observed for compounds (S)-5 and
(S)-8 for prostate cancer cells (Table 3).
as from their neighbors. Moreover, at 250 lg/mL, the
analogue (S)-2 only inhibited the growth of kidney can-
cer cells (786-0) by 9% (Fig. 6D) and similar morpholog-
ical features are observed between untreated and treated
cells (Figs. 6A and D). Overall, our results show that
a,b-unsaturated lactone group is essential for antiprolif-
erative activity observed for (S)-1.
Analogues (S)-2 and (S)-3 presented very low antiprolif-
erative activity, independently of the cancer cell line
when compared with (R)- or (S)-1. Since the difference
in the kidney cancer cell (786-0) viability was greatest
for (R)- or (S)-1 and analogue (S)-2, we have taken
phase-contrast microscopic pictures from 786-0 cells
treated with (R)- or (S)-1 or analogue (S)-2 for 48 h
(Fig. 6).
3. Conclusion
In conclusion, we have applied our previously described
approach to (R)-goniothalamin (1) to the preparation of
(S)-goniothalamin (1) and analogues (S)-2 to (S)-9 in
high overall yields and enantiomeric purities. Our stud-
ies demonstrated that MaruokaÕs catalyst (S,S)-A was
efficient for the stereoselective addition of allyltributyl-
tin not only to aromatic aldehydes 11–15 related to
trans-cinnamaldehyde but also to aliphatic aldehyde
16. In addition, we have identified (S)-1 and analogue
(S)-5 as the highest and more selective cytotoxic com-
pounds against kidney cancer cell growth (786-0) with
IC50 = 4 and 5 nM, respectively, and compound (S)-8
(IC50 = 4 nM) as the more potent against breast
cancer cells with resistance phenotype for adryamycin
(NCI.ADR). A structure–activity relationship study
(SAR) allowed us to establish the relevant structural
features for cytotoxic activity of goniothalamin ana-
logues. Studies are underway to prepare and evaluate
As shown in Figure 6A, untreated kidney cancer cells
(786-0) exhibited typical growth patterns. After 48 h,
significant morphological modifications were observed
for 786-0 cancer cells treated with (R)- or (S)-1 at
2.5 lg/mL (Figs. 6B and C). Under these conditions,
(R)-1 caused decrease of cancer cell growth by 43%,
while (S)-1 triggered 100% of death (Fig. 6B, arrows
ÔaÕ). At 2.5 lg/mL of (R)-1, cancer cells came about to
present irregular shape with lamellipodia elongation
and many detached cells (Fig. 6C, arrows ÔbÕ). The
apparent lamellipodia elongation of the (S)-1-treated
cells may have led to the disruption of cell adhesion
and consequent cell detachment from the substratum
Figure 6. Morphological observation of kidney cancer cell line (786-0) after administration of (R)-, (S)-goniothalamin [(R)- or (S)-1] or analogue 2.
A, Confluent untreated 786-0 cells at 48 h showed normal morphology. Typical phase-contrast microscopic images of 786-0 cells following treatment
with: B, (S)-goniothalamin [(S)-1] (2.5 lg/mL; a, cells died); C, (R)-goniothalamin [(R)-1] (2.5 lg/mL, b, cells became irregularly shaped with
elongated lamellipodia), and D, analogue 2 (250 lg/mL). Magnification = original · 200.

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A. de Fatima et al. / Bioorg. Med. Chem. 14 (2006) 622–631
´
627
the cytotoxic activity of other goniothalamin derivatives
as well as to probe its mode of action.
isomer. HRMS (EI) m/z calculated for M+. = 174.10447;
found: 174.10496. The enantiomeric purity of 17 was
determined to be 96% by chiral GC analysis (CP-Chira-
sil-Dex-CB) and comparing with racemic standard (see
page S5 of Supporting Information for details).
4. Experimental
4.1. Chemistry
4.1.4. Homoallylic alcohol 18. White solid. Mp: 37.8–
38.6 ꢁC IR (neat tablet): 3559, 3360, 3063, 3026, 2930,
2860, 1648, 1595, 1495, 1453, 1048, 992, 915,
4.1.1. General procedures. Reagents and solvents are
commercial grade and were used as supplied, except
dichloromethane and triethylamine which were distilled
from calcium hydride. Chromatography separations
were performed using 70–230 mesh silica gel. Thin-layer
chromatography was carried out on Merck silica plates
(0.25 mm layer thickness). IR spectra were obtained on
Nicolet Impact 410 FT (film) and ThermoNicolet
IR200 spectrometer (neat tablet). 1H NMR and 13C
NMR data were recorded on a Varian Gemini 2000
(7.0 T) or Varian Inova 500 (11.7 T) spectrometer.
Chemical shifts are reported in d [ppm relative to
701 cmꢀ1 1H NMR (300 MHz, CDCl3): d 7.30–7.15
.
(m, 5H), 5.88–5.74 (m, 1H), 5.16–5.11 (m, 1H), 3.72–
3.62 (m, 1H), 2.85–2.63 (m, 2H), 2.36–2.13 (m, 2H),
1.85–1.61 (m, 2H). 13C NMR (75 MHz, CDCl3): d
141.8, 134.4, 128.3 (2C), 128.2 (2C), 125.7, 118.1, 69.9,
25
D
(EI) m/z calculated for
42.1, 38.5, 32.1. ½aꢁ = +23.5 (c 2.2, CHCl3). HRMS
M
+. = 176.12012; found:
176.11543. The enantiomeric purity of 18 was deter-
mined to be 99% by chiral GC analysis (CP-Chirasil-
Dex-CB) and comparing with racemic standard (see
page S6 of Supporting Information for details).
1
(CH3)4Si] for H NMR and CDCl3 for 13C NMR. For
1H NMR, the chemical shifts were followed by multi-
plicity (s, single; sl, large single; d, doublet; dd, double
doublet; ddd, double double doublet; t, triplet; dt,
double triplet; q, quartet; dq, double quartet; m, multi-
plet) and coupling constant J reported in Hertz
(Hz). High-solution mass spectra (HRMS) were
measured on a VG Autospec-Micromass spectrometer.
Chiral GC analyses were performed with a capillary
column CP-Chirasil-DEX CB fused silica WCOT
(25 m · 0.25 mm · 0.25 lm) on Agilent 6890 series GC
system. Optical rotations were measured at 25 ꢁC with
Perkin–Elmer 241 or Perkin–Elmer 341 instruments.
4.1.5. Homoallylic alcohol 19. Viscous yellow liquid. IR
(film): 3320, 3206, 3073, 2976, 2933, 2906, 1641, 1594,
1
1513, 1338, 1109, 1086, 973, 916, 871, 746 cmꢀ1
.
H
NMR (300 MHz, CDCl3): d 8.19 (d, 2H, J = 8.9 Hz),
7.52 (d, 2H, J = 8.9 Hz), 6.72 (d, 1H, J = 16.1 Hz),
6.44 (dd, 1H, J = 16.1 and 5.5 Hz), 5.93–5.79 (m, 1H),
5.25–5.20 (m, 2H), 4.45–4.41 (m, 1H), 2.54–2.34 (m,
3H). 13C NMR (75 MHz, CDCl3): d 146.9, 143.2,
136.4, 133.4 (2C), 127.9, 126.9, 123.9 (2C), 119.1, 70.9,
25
D
41.8. ½aꢁ = ꢀ45.0 (c 1.0, CHCl3). HRMS (EI) m/z cal-
culated for M+. = 219.08954; found: 219.08669. The
enantiomeric purity of 19 was determined to be 95%
by H NMR analysis after their conversion to Mosher
1
4.1.2. General procedure for catalytic asymmetric allyla-
tion. To a stirred solution of TiCl4 (0.12 mmol) in CH2Cl2
(1.0 mL for each 1.0 mmol of the aldehyde) was added Ti
(OiPr)4 (0.36 mmol) at 0 ꢁC under argon. The solution
was allowed to warm to room temperature. After 1 h,
recently prepared Ag2O (0.24 mmol) was added at room
temperature, and the whole mixture was stirred for 5 h un-
der exclusion of direct light. The mixture was diluted with
CH2Cl2 (2.0 mL for each 1.0 mmol of the aldehyde) and
treated with (S)-BINOL (0.48 mmol) at room tempera-
ture for 2 h. After cooling this mixture to ꢀ15 ꢁC, it was
treated sequentially with aldehyde (2.4 mmol) and allyl-
tributyltin (2.7 mmol). After 24 h, the whole mixture
was quenched with satd aqueous NaHCO3 and extracted
with ether. The organic extracts were dried over MgSO4.
Evaporation of solvents and purification of this residue by
column chromatography on silica gel furnished the corre-
sponding homoallylic alcohol.
ester and comparing with racemic standard (see page
S7 of Supporting Information for details).
4.1.6. Homoallylic alcohol 20. Viscous colorless liquid.
IR (film): 3363, 3076, 2978, 2927, 2860, 1641, 1601,
1
1508, 1230, 1157, 968, 918, 850, 816 cmꢀ1. H NMR
(300 MHz, CDCl3): d 7.35–7.28 (m, 2H), 7.01–6.95 (m,
2H), 6.55 (d, 1H, J = 15.9 Hz), 6.15 (dd, 1H, J = 15.9
and 6.4 Hz), 5.91–5.77 (m, 1H), 5.20–5.14 (m, 2H),
4.36–4.29 (m, 1H), 2.47–2.31 (m, 2H), 2.05 (sl, 1H).
13C NMR (75 MHz, CDCl3):
d
J = 246.6 Hz), 133.8, 132.7, 131.1, 129.1, 127.8 (d, 2C,
162.2 (d,
J = 7.9 Hz), 118.5, 115.4 (d, 2C, J = 21.9 Hz), 71.6,
25
D
42.1. ½aꢁ = ꢀ29.5 (c 1.2, CHCl3). HRMS (EI) m/z cal-
culated for M+. = 192.09504; found: 192.09303. The
enantiomeric purity of 20 was determined to be 96%
by H NMR analysis after their conversion to Mosher
1
ester and comparing with racemic standard (see page
S8 of Supporting Information for details).
4.1.3. Homoallylic alcohol 17. Viscous colorless liquid.
IR (film): 3381, 3078, 3026, 2978, 2927, 2870, 1641,
4.1.7. Homoallylic alcohol 21. Viscous colorless liquid.
IR (film): 3400, 3074, 2929, 2910, 2835, 1639, 1606,
1493, 1446, 1030, 966, 916, 692 cmꢀ1
.
1H NMR
1
(500 MHz, CDCl3): d 7.41–7.24 (m, 5H), 6.63 (d, 1H,
J = 16.0 Hz), 6.27 (dd, 1H, J = 16.0 and 6.2 Hz), 5.92–
5.84 (m, 1H), 5.23–5.17 (m, 2H), 4.40–4.36 (m, 1H),
2.49–2.37 (m, 2H), 2.00 (sl, 1H). 13C NMR (125 MHz,
1510, 1300, 1248, 1174, 1034, 968, 814 cmꢀ1. H NMR
(300 MHz, CDCl3): d 7.39–7.25 (m, 2H), 6.87–6.82 (m,
2H), 6.53 (d, 1H, J = 15.9 Hz), 6.10 (dd, 1H, J = 15.9
and 6.6 Hz), 5.92–5.78 (m, 1H), 5.20–5.15 (m, 2H),
4.28–4.38 (m, 1H), 3.80 (s, 3H), 2.48–2.31 (m, 2H),
1.78 (d, 1H, J = 3.7 Hz). 13C NMR (125 MHz, CDCl3):
d 159.3, 134.1, 130.0, 129.4 (2C), 129.3, 127.6, 118.4,
CDCl3): d 136.6, 134.0, 131.5, 130.3 (2C), 128.5, 127.6,
25
D
126.4 (2C), 118.4, 71.6, 41.9. ½aꢁ = +22.2 (c 2.0, CHCl3)
25
for R isomer and ½aꢁ = ꢀ22.4 (c 2.0, CHCl3) for S
D

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A. de Fatima et al. / Bioorg. Med. Chem. 14 (2006) 622–631
´
628
25
D
25
39.0. ½aꢁ = +82.8 (it c 1.2, CHCl3) for R isomer and
m/z calculated for M+. = 228.11503; found: 228.11981.
114.0 (2C), 71.9, 55.3, 42.1. ½aꢁ = ꢀ29.8 (c 1.6, CHCl3).
D
25
D
HRMS (EI) m/z calculated for M+. = 204.11503; found:
204.11298. The enantiomeric purity of 21 was deter-
mined to be 94% by 19F NMR analysis after their con-
version to Mosher ester and comparing with racemic
standard (see page S9 of Supporting Information for
details).
½aꢁ = ꢀ80.7 (c 1.2, CHCl3) for S isomer. HRMS (EI)
4.1.12. Acrylic ester 25. Viscous colorless liquid. IR (film):
3078, 3026, 2951, 2860, 1940, 1722, 1651, 1469, 1406,
1269, 1196, 1047, 985, 808, 700 cmꢀ1
.
1H NMR
(300 MHz, CDCl3): d 7.29–7.14 (m, 5H), 6.40 (dd, 1H,
J = 17.2 and 1.5 Hz), 6.13 (dd, 1H, J = 17.2 and
10.2 Hz), 5.82 (dd, 1H, J = 10.2 and 1.5 Hz), 5.78–5.69
(m, 1H), 5.11–5.00 (m, 3H), 2.73–2.55 (m, 2H), 2.38 (t,
2H, J = 7.0 Hz), 1.99–1.87 (m, 2H). 13C NMR (75 MHz,
CDCl3): d 165.8, 141.4, 133.3, 130.5, 128.7, 128.4 (2C),
4.1.8. Homoallylic alcohol 22. Viscous colorless liquid. IR
(film): 3556, 3384, 3074, 2978, 2777, 1847, 1639, 1606,
1
1504, 1489, 1444, 1245, 1190, 1039, 928, 800 cmꢀ1. H
NMR (300 MHz, CDCl3): d 7.12–6.93 (m, 3H), 6.71 (d,
1H, J = 15.7 Hz), 6.25 (dd, 1H, J = 15.7 and 6.2 Hz),
6.15 (s, 2H), 6.12–5.98 (m, 1H), 5.41–5.34 (m, 2H), 4.52
(q, 1H, J = 6.2 Hz), 2.66–2.56 (m, 2H), 2.05 (s, 1H). 13C
NMR (75 MHz, CDCl3): d 147.9, 147.1, 134.0, 131.0,
128.3 (2C), 125.9, 117.9, 73.0, 38.6, 35.3, 31.6.
25
D
½aꢁ = +10 (c 2.2, CHCl3). HRESI-MS = 231.13851 m/z
calculated for [M+H]+. = 231.1385; found: 231.1385.
130.0, 129.7, 121.1, 118.2, 108.2, 105.7, 101.0, 71.7, 42.0.
25
½aꢁ = ꢀ33.1 (c 1.11, CHCl3). HRMS (EI) m/z calculated
4.1.13. Acrylic ester 26. Yellow solid. Mp: 53.3–54.0 ꢁC.
IR (neat tablet): 3079, 2919, 2850, 1721, 1597, 1516,
1405, 1342, 1265, 1186, 1110, 1044, 970, 919, 863, 808,
D
for M+. = 218.09427; found: 218.09430. The enantiomer-
ic purity of 22 was determined to be 92% by 19F NMR
analysis after their conversion to Mosher ester and com-
paring with racemic standard (see page S10 of Supporting
Information for details).
1
746 cmꢀ1. H NMR (300 MHz, CDCl3): d. 8.18 (d, 2H,
J = 8.8 Hz), 7.51 (d, 2H, J = 8.8 Hz), 6.69 (d, 1H,
J = 15.9 Hz), 6.46 (dd, 1H, J = 17.4 and 1.5 Hz), 6.36
(dd, 1H, J = 15.9 and 6.6 Hz), 6.17 (dd, 1H, J = 17.4
and 10.4 Hz), 5.89 (dd, 1H, J = 10.4 and 1.5 Hz), 5.85–
5.73 (m, 1H), 5.59 (ddd, 1H, J = 12.8, 6.6, and 0.9 Hz),
5.20–5.13 (m, 2H), 2.56 (td, 2H, J = 6.6 and 0.9 Hz).13C
NMR (75 MHz, CDCl3): d 165.0, 147.0, 142.5, 132.3,
4.1.9. Homoallylic alcohol 23. Viscous colorless liquid.
IR (film): 3348, 3075, 2923, 2850, 1641, 1447, 1029,
967, 911 cmꢀ1. H NMR (300 MHz, CDCl3): d 5.87–
1
5.73 (m, 1H), 5.62 (dd, 1H, J = 15.4 and 6.6 Hz), 5.43
(ddd, 1H, J = 15.4, 6.6, and 1.1 Hz); 5.17–5.10 (m,
2H), 4.14–4.08 (m, 1H), 2.32–2.22 (m, 2H), 1.97–1.91
(m, 2H), 1.73–1.63 (m, 5H), 1.34–0.87 (m, 5H). 13C
NMR (75 MHz, CDCl3): d 137.9, 134.4, 129.5, 117.9,
131.8, 131.1, 130. 1 (2C), 128.2, 127.0, 123.8 (2C), 118.5,
25
D
73.2, 38.9. ½aꢁ = ꢀ70.6 (c 0.92, CHCl3). HRMS (EI) m/
z calculated for M+. = 273.10011; found: 273.09830.
71.9, 42.0, 40.2, 38.8 (2C), 26.1, 26.0 (2C).
4.1.14. Acrylic ester 27. Viscous colorless liquid. IR
(film): 3078, 3039, 2981, 2941, 1722, 1601, 1510, 1404,
25
D
½aꢁ = ꢀ12.0 (c 1.5, CHCl3). HRMS (EI) m/z calculated
for M+. = 180.15141; found: 180.15515. The enantio-
meric purity of 23 was determined to be 99% by H
1267, 1230, 1190, 1043, 966, 810 cmꢀ1 1H NMR
.
1
(500 MHz, CDCl3): d. 7.36–7.32 (m, 2H), 7.02–6.97
(m, 2H), 6.60 (d, 1H, J = 16.6 Hz), 6.43 (dd, 1H,
J = 16.6 and 1.4 Hz), 6.17–6.07 (m, 2H), 5.85–5.75 (m,
2H), 5.56–5.52 (m, 1H), 5.16–5.09 (m, 2H), 2.57–2.48
(m, 2H).13C NMR (125 MHz, CDCl3): d 165.3, 162.5
(d, J = 247.1 Hz), 132.9, 132.6 (d, J = 3.1 Hz), 131.6,
130.8, 128.6, 128.1 (d, 2C, J = 8.3 Hz), 126.6 (d,
NMR analysis after their conversion to Mosher ester
and comparing with racemic standard (see page S11 of
Supporting Information for details).
4.1.10. General procedure for acylation. To a solution of
homoallylic alcohol (2.0 mmol), obtained as described
above, in CH2Cl2 (1.0 mL/1.0 mmol of the homoallylic
alcohol) and cooled to 0 ꢁC were added acryloyl chloride
(3.5 mmol) and Et3N (7.0 mmol). The mixture was
warmed to room temperature and stirred for 2–4 h.
The resulting mixture was filtered through a short pad
of Celite, poured into water, and then the product was
extracted with CH2Cl2. Solvent evaporation under re-
duced pressure and purification of this residue by col-
umn chromatography on silica gel furnished the
corresponding acrylic esters.
J = 2.1 Hz), 118.2, 115.4 (d, 2C, J = 21.8 Hz), 73.8,
25
D
39.0. ½aꢁ = ꢀ66.0 (c 2.03, CHCl3). HRMS (EI) m/z cal-
culated for M+. = 246.10561; found: 246.10285.
4.1.15. Acrylic ester 28. Viscous colorless liquid. IR
(film): 3076, 3035, 3003, 2955, 2910, 2837, 1720, 1606,
1512, 1404, 1252, 1190, 1036, 966, 808 cmꢀ1 1H
.
NMR (300 MHz, CDCl3): d 7.32–7.29 (m, 2H), 7.25–
6.81 (m, 2H), 6.58 (d, 1H, J = 15.7 Hz), 6.42 (dd, 1H,
J = 17.2 e 1.5 Hz), 6.15 (dd, 1H, J = 17.2 and 10.2 Hz),
6.04 (dd, 1H, J = 15.7 and 7.3 Hz), 5.86–5.72 (m, 2H),
5.53 (q, 1H, J = 6.6 Hz), 5.16–5.06 (m, 2H), 3.79 (s,
3H), 2.54–2.49 (m, 2H). 13C NMR (75 MHz, CDCl3):
d 165.2, 159.3, 133.0, 132.3, 130.5, 128.9 (2C), 128.6,
4.1.11. Acrylic ester 24. Viscous yellow liquid. IR (film):
3080, 3060, 3026, 2979, 2939, 2848, 1722, 1637, 1495,
1404, 1265, 1188, 1043, 964, 918, 750, 692 cmꢀ1 1H
.
NMR (500 MHz, CDCl3): d 7.41–7.25 (m, 5H), 6.66 (d,
1H, J = 16.6 Hz), 6.46 (dd, 1H, J = 16.6 and 1.5 Hz),
6.19 (ddd, 2H, J = 17.5, 11.9, and 8.8 Hz), 5.86 (dd, 1H,
J = 10.4 and 1.5 Hz), 5.85–5.78 (m, 1H), 5.61–5.56 (m,
1H), 5.19–5.11 (m, 2H), 2.61–2.51 (m, 2H). 13C NMR
(125 MHz, CDCl3): d 165.3, 136.2, 132.9, 132.7, 130.7,
128.6, 128.5 (2C), 127.9, 126.8 (2C), 126.5, 118.1, 73.9,
127.7, 124.6, 117.9, 113.9 (2C), 74.2, 55.3, 39.2.
25
D
½aꢁ = ꢀ88.5 (c 1.15, CHCl3). HRMS (EI) m/z calculat-
ed for M+. = 258.12559; found: 258.12450.
4.1.16. Acrylic ester 29. Viscous colorless liquid. IR
(film): 3076, 2979, 2900, 2779, 1720, 1653, 1504, 1489,
1
1446, 1404, 1252, 1188, 1039, 964, 930, 808 cmꢀ1. H

ˆ
A. de Fatima et al. / Bioorg. Med. Chem. 14 (2006) 622–631
´
629
NMR (300 MHz, CDCl3): d 6.92–6.74 (m, 3H), 6.56 (d,
1H, J = 16.1 Hz), 6.46–6.39 (m, 1H), 6.19–6.09 (m, 1H),
6.02 (dd, 1H, J = 16.1 and 6.6 Hz), 5.95 (s, 2H), 5.87–
5.65 (m, 2H), 5.53 (q, 1H, J = 6.6 Hz), 5.17–5.04 (m,
2H), 2.73–2.47 (m, 2H). 13C NMR (75 MHz, CDCl3):
d 165.4, 147.9, 147.5, 133.0, 132.5, 130.7, 130.6, 128.6,
CDCl3): d. 6.88 (dt, 1H, J = 9.7 and 4.4 Hz), 6.04 (dt,
1H, J = 9.7 and 1.8 Hz), 5.79 (dd, 1H, J = 15.4 and
6.8 Hz), 5.53 (ddd, 1H, J = 15.4, 6.8, and 1.5 Hz), 4.86
(q, 1H, J = 7.3 Hz), 2.45–2.40 (m, 2H), 2.00–1.95 (m,
13
1H), 1.74–1.62 (m, 5H), 1.35–1.01 (m, 5H). C NMR
(75 MHz, DCl3): d 164.0, 144.6, 141.1, 124.1, 121.5,
25
78.5, 40.3, 32.5, 32.4, 30.0, 26.1, 25.9 (2C). ½aꢁ = ꢀ53
125.1, 121.5, 118.1, 108.2, 105.7, 101.0, 74.0, 39.1.
D
(c 1.24, CHCl3). HRMS (EI) m/z calculated for
25
D
½aꢁ = ꢀ74.8 (c 0.65, CHCl3). HRMS (EI) m/z calculat-
ed for M+. = 272.10486; found: 272.10490.
M
+. = 206.13068; found: 206.13208.
4.1.17. Acrylic ester 30. Viscous colorless liquid. IR
(film): 2925, 2852, 1724, 1637, 1448, 1403, 1294, 1265,
4.1.22. Analogue 6. Yellow solid. Mp: 125.8–127.6 ꢁC.
IR (neat tablet): 3062, 3034, 1719, 1593, 1510, 1383,
1
1
1187, 1043, 968, 916, 808 cmꢀ1. H NMR (300 MHz,
1336, 1254, 1106, 1085, 971, 823, 742 cmꢀ1. H NMR
CDCl3): d 6.39 (dd, 1H, J = 17.4 and 1.6 Hz), 6.11
(dd, 1H, J = 17.4 and 10.3 Hz), 5.80 (dd, 1H, J = 10.3
and 1.5 Hz), 5.77–5.65 (m, 2H), 5.43–5.30 (m, 2H),
5.12–5.04 (m, 2H), 2.49–2.34 (m, 2H), 2.00–1.91 (m,
1H), 1.73–1.63 (m, 5H), 1.44–1.00 (m, 5H). 13C NMR
(75 MHz, CDCl3): d 165.2, 140.1, 133.3, 130.2, 128.8,
(300 MHz, CDCl3): d. 8.21 (d, 2H, J = 8.8 Hz), 7.54
(d, 2H, J = 8.8 Hz), 6.95 (ddd, 1H, J = 8.4, 5.3, and
3.2 Hz), 6.84 (d, 1H, J = 16.1 Hz), 6.44 (dd, 1H,
J = 16.1 and 5.4 Hz), 6.13 (ddd, 1H, J = 3.2, 1.8, and
1.4 Hz), 5.20–5.13 (m, 1H), 2.66–2.55 (m, 2H). 13C
NMR (125 MHz, CDCl3): d 163.3, 147.4, 144.3, 142.1,
124.9, 117. 6, 74.2, 40.3, 39.2, 32.6 (2C), 26.2, 26.0
130.5 (2C), 130.2 (2C), 127.2, 124.0, 121.7, 76.9, 29.6.
25
D
25
D
ed for M+. = 245.06881; found: 245.06891.
(2C). ½aꢁ = ꢀ36.0 (c 1.6, CHCl3). HRMS (EI) m/z cal-
½aꢁ = ꢀ205.0 (c 1.0, CHCl3). HRMS (EI) m/z calculat-
culated for M+. = 234.16198; found: 234.15814.
4.1.18. General procedure for ring closing metathesis. To a
stirred solution of GrubbsÕ catalyst ([P(C6H11)3]2RuCl2
CHPh, 10 mol%) in dichloromethane (1.0 mL/1.0 mmol
of the acrylic ester) at 55–60 ꢁC was added the acrylic ester
(0.7 mmol), obtained as described above, dissolved in
dichloromethane (65 mL). The resulting mixture was
heated for 12–18 h. After this period, the mixture was
cooled at room temperature and evaporated under re-
duced pressure. The residue was purified by column chro-
matography on silica gel to give the analogues of
goniothalamin.
4.1.23. Analogue 7. White solid. Mp: 125.4–126.5 ꢁC. IR
(neat tablet): 3066, 2958, 2925, 1702, 1598, 1508, 1428,
1
1384, 1249, 1228, 1056, 1010, 966, 860, 815 cmꢀ1. H
NMR (300 MHz, CDCl3): d. 7.39–7.35 (m, 2H), 7.07–
6.99 (m, 2H), 6.96–6.90 (m, 1H), 6.70 (d, 1H,
J = 15.9 Hz), 6.20 (dd, 1H, J = 15.9 and 6.2 Hz), 6.10
(dt, 1H, J = 9.9 and 1.8 Hz), 5.13–5.06 (m, 1H), 2.57–
2.52 (m, 2H). 13C NMR (75 MHz, CDCl3): d 163.6,
162.6 (d, J = 247.8 Hz); 144.3, 131.8, 128.2 (d, 2C,
J = 8.5 Hz), 128.1, 125.3, 121.6, 115.6 (d, 2C,
25
D
J = 20.7 Hz), 77.8, 29.9. ½aꢁ = ꢀ158.0 (c 1.0, CHCl3).
HRMS (EI) m/z calculated for M+. = 218.07431; found:
218.07497.
4.1.19. (S)-goniothalamin [(S)-1]. White solid. Mp: 81–
82 ꢁC, (Ref. 38: 85 ꢁC). IR (neat tablet): 3024, 2924,
2854, 1720, 1381, 1246, 1018, 968, 814, 748, 694 cmꢀ1
.
4.1.24. Analogue 8. White solid. Mp: 102.5–102.8 ꢁC. IR
(neat tablet): 2962, 2933, 2838, 1708, 1604, 1511, 1245,
1H NMR (300 MHz, CDCl3): d 7.41–7.25 (m, 5H),
6.92 (dt, 1H, J = 9.5 and 4.0 Hz), 6.72 (d, 1H,
J = 15.9 Hz), 6.27 (dd, 1H, J = 15.9 and 6.2 Hz), 6.08
(d, 1H, J = 9.5 Hz), 5.10 (q, 1H, J = 6.9 Hz), 2.56–2.52
(m, 2H). 13C NMR (75 MHz, CDCl3): d 163.5, 144.5,
1
1178, 1024, 968, 848, 809 cmꢀ1. H NMR (300 MHz,
CDCl3): d 7.35–7.30 (m, 2H), 6.94–6.84 (m, 3H), 6.65
(d, 1H, J = 15.9 Hz), 6.14 (dd, 1H, J = 15.9 and
6.6 Hz), 6.08 (dt, 1H, J = 9.5 and 1.8 Hz), 5.11–5.03
(m, 1H), 3.81 (s, 3H), 2.55–2.51 (m, 2H). 13C NMR
(75 MHz, CDCl3): d 163.8, 159.6, 144.4, 132.7, 128.4
135.5, 132.8, 128.5 (2C), 128.1 (2C), 126.4, 125.5,
25
D
121.4, 77.8, 29. 8 ½aꢁ = ꢀ170 (c 1.7, CHCl3); Ref. 21:
25
½aꢁ = ꢀ178.5, (c 0.2, CHCl3). HRMS (EI) m/z calculat-
(2C), 127.8, 123.3, 121.6, 114.0 (2C), 78.2, 55.3, 30.0.
D
25
D
ed for M+. = 200.08373; found: 200.07891.
½aꢁ = ꢀ133.7 (c 1.02, CHCl3). HRMS (EI) m/z calculat-
ed for M+. = 230.09430; found: 230.09428.
4.1.20. Analogue 4. White solid. Mp: 45.2–45.8 ꢁC. IR
(neat tablet): 3060, 3026, 2937, 2866, 1716, 1495, 1454,
4.1.25. Analogue 9. White solid. Mp: 86.2–87.5 ꢁC. IR
(neat tablet): 3072, 2962, 2915, 1704, 1650, 1504, 1444,
1
1387, 1248, 1146, 1036, 958, 816, 752, 702 cmꢀ1. H
1
NMR (300 MHz, CDCl3): d 7.31–7.16 (m, 5H), 6.88–
6.26 (m, 1H), 6.01 (dt, 1H, J = 9.9 and 1.8 Hz), 4.45–
4.35 (m, 1H), 2.93–2.71 (m, 2H), 2.35–2.30 (m, 2H),
2.19–2.06 (m, 1H), 1.99–1.87 (m, 1H). 13C NMR
1376, 1243, 1097, 1037, 1008, 968, 933, 809 cmꢀ1. H
NMR (300 MHz, CDCl3): d 7.18–6.80 (m, 5H); 6.33–
6.26 (m, 2H); 6.16 (s, 2H); 5.26 (dq, 1H, 13 J = 7.3
and 1.1 Hz); 2.90–2.65 (m, 2H). 13C NMR (75 MHz,
CDCl3): d 163.8, 148.1, 147.8, 144.5, 132.9, 130.1,
(75 MHz, CDCl3): d 164.3, 144.9, 140.7, 128.4 (2C),
25
D
128.3 (2C), 126.0, 121.3, 76.5, 36.4, 30.8, 29.3. ½aꢁ
=
123.8, 121.7, 121.6, 108.3, 105.8, 101.2, 78.0, 29.9.
25
D
ꢀ50 (c 1.5, CHCl3). HRMS (EI) m/z calculated for
+. = 202.09938; found: 202.09229.
½aꢁ = ꢀ141.7 (c 0.93, CHCl3). HRMS (EI) m/z calculat-
M
ed for M+. = 244.07356; found: 244.07358.
4.1.21. Analogue 5. White solid. Mp: 45.0–46.1 ꢁC. IR
(neat tablet): 2925, 2852, 1708, 1675, 1448, 1384, 1247,
4.1.26. Synthesis of analogue (S)-2 from (S)-1. To a solu-
tion of (S)-goniothalamin [(S)-1] (0.060 g 0.30 mmol) in
degassed benzene (17 mL) under argon atmosphere was
1
1160, 1054, 1022, 977, 829 cmꢀ1. H NMR (300 MHz,

ˆ
´
A. de Fatima et al. / Bioorg. Med. Chem. 14 (2006) 622–631
630
added StrykerÕs reagent, {[(Ph3)CuH]6, 0.178 g,
0.090 mmol}. After 20 min, the mixture was exposed
to air for 2 h, quenched with satd aqueous NaHCO3,
and extracted with ether. The organic extracts were
dried over MgSO4. Evaporation of solvents and purifi-
cation of this residue by column chromatography on sil-
ica gel (hexanes/ethyl acetate, 7:3) furnished (S)-2
(0.039 g, 0.19 mmol) in 63% yield. White solid. Mp:
74.6–76.0 ꢁC. IV (neat tablet): 3024, 2951, 2879, 1736,
786-0, HT-29), 4 · 104 (NCI.460, PCO.3), 3 · 104
(UACC.62), and 7 · 104 (OVCAR03) cells/mL. This ini-
tial cell concentration was determined from individual
growth curves and ensured that the cells would be in
the logarithmic growth throughout the experiment.
The microtiter plates containing cells were pre-incubat-
ed for 24 h at 37 ꢁC to allow stabilizations prior to addi-
tion (100 lL) of goniothalamin (1) and analogues (2–9)
as well as doxorubicin (DOX). The plates were incubat-
ed with the test substance for 48 h at 37 ꢁC and 5%
CO2.28,29
1493, 1331, 1236, 1186, 1034, 968, 928, 748, 694 cmꢀ1
.
1H NMR (300 MHz, CDCl3): d 7.40–7.23 (m, 5H),
6.67 (d, 1H, J = 15.8 Hz), 6.20 (dd, 1H, J = 15.8 and
6.0 Hz), 5.02–4.96 (m, 1H), 2.69–2.47 (m, 2H), 2.11–
1.69 (m, 4H). 13C NMR (75 MHz, CDCl3): d 171.9,
4.2.4. Solubilization and dilution of test substance.
Goniothalamin (1) and analogues (2–9) were tested at
four concentrations (0.25, 2.5, 25, and 250 lg/mL), each
in triplicate wells. The substances tested were initially
solubilized in dimethylsufoxide (DMSO) (Sigma) at
400· the final maximum test concentration. The stock
solution was diluted with complete medium containing
50 lg gentamicin/mL (Schering-Plough).
135.9, 132.1, 128.6 (2C), 128.1, 127.0, 126.6 (2C), 80.3,
25
29.6, 28.5, 18.3. ½aꢁ = ꢀ5.5 (c 1.45, CHCl3). HRMS
D
(EI) m/z calculated for
202.09840.
M
+. = 202.09938; found:
4.1.27. Synthesis (S)-3 from (S)-1. To a solution of (S)-
goniothalamin [(S)-1] (0.050 g, 0.25 mmol) in 2.0 mL of
ethanol under H2 atmosphere (1 atm) was added 10%
Pd/C (5 mg). After 2 h, the solvent was removed under re-
duced pressure. The residue was purified by column chro-
matography on silica gel (hexanes/ethyl acetate, 7:3) to
give (S)-3 in 87% yield (0.036 g, 0.18 mmol). Viscous col-
orless liquid. IR (film): 3024, 2930, 2858, 1728, 1495, 1452,
4.2.5. Sulforhodamine B (SRB) assay. Sulforhodamine
B (SRB) is an aminoxanthine with a bright pink color
that has two sulfonic groups. Since it is an anionic dye
in weak acid solution, it is capable of bonding to basic
groups of proteins in cells fixed with trichloroacetic
acid. Therefore, this non-clonogenic methodology per-
mits a highly sensitive protein with a straight relation-
ship to cell culture. The SRB assay was done as
described by Skehan et al.29 Briefly, the cells were fixed
with 50% TCA (sigma) at 4 ꢁC (50 lL/well, final con-
centration 10%) for 1 h. The supernatant was then dis-
carded and the plates were washed five times with tap
water. The cells were stained for 30 min with 0.4% SRB
in 1% acetic acid (50 lL/well) (Sigma) and subsequent-
ly washed 4· with 1% acetic acid to remove unbound
dye. The plates were air-dried and protein-bound dye
was solubilized with 150 lL (100 mM) of Trizma buffer
(Sigma). The resulting optical density was read in a
multiwell plate reader at 540 nm. For cells grown in
suspension (e.g., leukemia), the same method was used,
but the TCA concentration was 80% to fix the cells to
the plate bottom.
1388, 1240, 1178, 1047, 930, 748 cmꢀ1
.
1H NMR
(300 MHz, CDCl3): d 7.31–7.18 (m, 5H), 4.30–4.22 (m,
1H), 2.91–2.69 (m, 2H), 2.64–2.40 (m, 2H), 2.09–1.76
(m, 5H), 1.64–1.52 (m, 1H). 13C NMR (75 MHz, CDCl3):
d 171.8, 141.0, 128.4 (4C), 126.0, 79.3, 37.4, 31.0, 29.4,
25
D
27.8, 18.4. ½aꢁ = +49 (c 1.0, CHCl3). HRMS (EI) m/z cal-
culated for M+. = 204.11503; found: 204.11466.
4.2. Biological assay
4.2.1. Cell lines. The human tumoral MCF-7 (breast),
NCI.ADR (breast expressing the resistance phenotype
for adryamycin), NCI.460 (lung, non-small cells),
UACC.62 (melanoma), 786-0 (kidney), OVCAR03
(ovarian), PCO.3 (prostate), and HT-29 (colon) were
obtained from the Frederick MA, National Cancer
Institute/USA.
4.2.6. Data calculations. Absorbances were calculated
using the Excellꢂ program (Microsoft Office package)
and the mean SE of the mean were calculated for 4
wells in triplicate samplings. The background absor-
bances were subtracted from the appropriate control
and drug-blank measurements. To assess the effect of
goniothalamin (1) and analogues (2–9) on cell growth,
three measurements were obtained at time zero (T0) val-
ues for all cells at the beginning of incubation, and con-
trol (C) and test (T) values at the end of incubation
without and with the test substance, respectively. For
T value 6T0 (cytostatic effect), the calculation was
100 · [(T ꢀ T0)/C ꢀ T0]. While for T < T0 (cytocidal ef-
fect), the calculation was 100 · [(T ꢀ T0)/T0]. The IC50
values (test substance concentration eliciting 50% inhibi-
tion) were determined by non-linear regression analysis.
These results presented here refer to a representative
experiment since all assays were run in triplicate and
the average standard error was always <5%.
4.2.2. Cell culture. The cells were maintained in RPMI-
1640 medium (Sigma Chemical Co., St. Louis, MO,
USA), supplemented with 10% fetal calf serum (FCS:
Nutricell Nutrientes Celulares, Campinas, SP, Brazil)
at 37 ꢁC with 5% CO2. The medium was changed every
2 days until the cells reached confluence, at which point
they were subcultured.
4.2.3. Antiproliferative assay. The adherent cell lines
were detached from the culture flasks by adding
0.5 mL trypsin solution 0.025% (Nutricell). The trypsin
was inactivated by adding 5 mL of 5% FCS in RPMI-
1640 medium. Single-cell suspensions were obtained by
a gentle pipetting acton. After counting, the cells were
diluted to appropriate seeding densities and transferred
to 96-well microtiter plates (Nunc Brand Products) in
a fixed volume of 100 lM per well. The seeding densities
were as follows: 6.5 · 104 (MCF-7), 5 · 104 (NCI.ADR,

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A. de Fatima et al. / Bioorg. Med. Chem. 14 (2006) 622–631
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631
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14. de Fatima, A.; Kohn, L. K.; Antonio, M. A.; de Carvalho,
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Tetrahedron Lett. 2000, 41, 583–586.
Supplementary Material
Determination of enantiomeric purity and spectra of
alcohols homoallylic 17–23, and spectra of goniothalam-
in (1) and analogues 2–9 cited in this article are available
free via internet at http://www.sciencedirect.com.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.
2005.08.036.
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Acknowledgments
`
The authors thank Fundac¸a˜o de Amparo a Pesquisa do
Estado de Sa˜o Paulo (FAPESP) for financial support
and FAPESP studentship (AF, Grants 02/00522-2),
´
Conselho Nacional para o Desenvolvimento Cientıfico
e Tecnologico (CNPq) for fellowship (RAP and JEC).
The authors thank Professor Dr. Armin de Meijere,
Georg-August Universita¨t, Go¨ttingen, Germany, for
critical reading and suggestions.
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