Cytotoxic styryl-lactones from the leaves and twigs of Polyalthia crassa

Article abstract of DOI:10.1021/np060323u

Four new styryl-lactones, crassalactones A-D (1-4), were isolated from a cytotoxic ethyl acetate-soluble extract of the leaves and twigs of Polyalthia crassa, together with seven known compounds, (+)-3-acetylaltholactone, (+)-altholactone, aristolactam All, cinnamic acid, (+)-goniofufurone, (+)-goniopypyrone, and (+)-howiinol A. Their structures were determined on the basis of spectroscopic methods. The absolute configuration of 1-3 was established by chemical conversions. Single-crystal X-ray analysis and the Mosher ester method were used to confirm the absolute stereochemistry of 4. Cytotoxic evaluation against several mammalian cancer cell lines was performed on all new isolates, aristolactam AII, and the modified (+)-tricinnamate derivative 11 obtained from 1.

Full text of DOI:10.1021/np060323u

1728
J. Nat. Prod. 2006, 69, 1728-1733
Cytotoxic Styryl-Lactones from the Leaves and Twigs of Polyalthia crassa
Patoomratana Tuchinda,*,† Bamroong Munyoo,† Manat Pohmakotr,† Pongchan Thinapong,† Samaisukh Sophasan,‡
Thawatchai Santisuk,§ and Vichai Reutrakul†
Department of Chemistry and Department of Physiology, Faculty of Science, Mahidol UniVersity, Rama VI Road, Bangkok 10400, Thailand,
and The Forest Herbarium, Royal Forest Department, Bangkok 10900, Thailand
ReceiVed July 6, 2006
Four new styryl-lactones, crassalactones A-D (1-4), were isolated from a cytotoxic ethyl acetate-soluble extract of the
leaves and twigs of Polyalthia crassa, together with seven known compounds, (+)-3-acetylaltholactone, (+)-altholactone,
aristolactam AII, cinnamic acid, (+)-goniofufurone, (+)-goniopypyrone, and (+)-howiinol A. Their structures were
determined on the basis of spectroscopic methods. The absolute configuration of 1-3 was established by chemical
conversions. Single-crystal X-ray analysis and the Mosher ester method were used to confirm the absolute stereochemistry
of 4. Cytotoxic evaluation against several mammalian cancer cell lines was performed on all new isolates, aristolactam
AII, and the modified (+)-tricinnamate derivative 11 obtained from 1.
Several members of the genus Polyalthia have been widely used
in traditional medicine in Asia. In Thailand, water decoctions from
the roots of P. eVecta and P. debilis are used as a galactagogue1a,b
and for the treatment of abdominal pain,1a respectively. Polyalthia
bullata is used in Malaysia as an aphrodisiac.2 From various
Polyalthia species, many constituents showing cytotoxic,3 antimi-
crobial,4 antimalarial,5 and anti-HIV6 activities have been reported
previously. Earlier chemical examinations of plants in the genus
Polyalthia yielded diterpenes,3,7 triterpenes,6a,8 benzopyrans,9 2-sub-
stituted furans,1b,6b,10 and various types of alkaloids.3d,5,11 Our
previous work on P. suberosa has resulted in the isolation of an
azaanthrazene alkaloid, kalasinamide,12 2-substituted furans,6b and
carboxamides.12a
As part of our ongoing project on the discovery of new cytotoxic
agents from plants, we describe herein the chromatographic
separation of a cytotoxic ethyl acetate extract of the leaves and
twigs of P. crassa, leading to the isolation of four new styryl-
lactones, crassalactones A-D (1-4), along with the known (+)-
howiinol A (5),13 (+)-goniofufurone (6),14 (+)-altholactone (7),15
(+)-3-acetylaltholactone (8),16 (+)-goniopypyrone (9),14a aristolac-
tam AII (10),17 and cinnamic acid. For further structural and
stereochemical studies of compound 1, (+)-tricinnamate 11 was
prepared from both (+)-1 and (+)-5, whereas the known (+)-6
could be converted to (+)-2 and (+)-(3). The modified compounds
were characterized on the basis of spectroscopic methods. The four
styryl-lactones 1-4, aristolactam AII (10), and the semisynthetic
(+)-tricinnamate 11 were evaluated for their cytotoxic activities
against a panel of five mammalian cancer cell lines.
Results and Discussion
spectroscopic data of 1 (Table 1) were closely comparable to those
observed for (+)-howiinol A (5) obtained from the same extract
(for spectroscopic data of 5, see Supporting Information) and
previously reported from Goniothalamus howii.13 The olefinic
signals at δ 6.21 (d, J ) 9.7 Hz, H-3) and 6.99 (dd, J ) 9.7 and
5.9 Hz, H-4) were typical of an R,â-unsaturated δ-lactone, while a
broad singlet at δ 2.75 (2H) confirmed the presence of two hydroxyl
protons in 1. The cinnamate group in 1 was characterized by a
pair of doublets (J ) 16 Hz each) at δ 6.32 (H-2′) and 7.63 (H-3′),
together with the aromatic signals at δ 7.50 (2H, H-5′ and H-9′)
and 7.37-7.41 (3H, H-6′, H-7′, H-8′). By analyses of its COSY
spectrum, the four oxygen-linked methine signals at δ 5.30 (dd, J
) 5.9 and 2.7 Hz), 4.79 (dd, J ) 5.6 and 2.7 Hz), 4.29 (t, J ) 5.6
Hz), and 4.91 (d, J ) 5.6 Hz) were assigned to H-5, H-6, H-7, and
H-8, respectively. The 13C NMR spectrum of 1 (Table 1) displayed
18 signals for 22 carbons, of which the carbon types were
determined by DEPT experiments. The connectivity of the cin-
(+)-Crassalactone A (1) displayed a molecular ion peak at m/z
380 in the EIMS, corresponding to the molecular formula C22H20O6
and confirmed by elemental analysis. The presence of two free
hydroxyls and a cinnamate group in 1 were indicated by the ion
peaks at m/z 362 (M+ - H2O) and 344 (M+ - 2H2O), 148
[(PhCHdCHCO2H)+, base peak], and 131 (PhCHdCHCO)+. The
IR spectrum of 1 showed an OH stretch at 3456 cm-1 and two
CdO absorptions at 1721 and 1711 cm-1. The R,â-unsaturated
δ-lactone character of 1 was confirmed by the presence of a small
peak at δC 162.3 in its 13C NMR spectrum. The 1H NMR
* Corresponding author. Tel: 662-2015159. Fax: 662-3547151. E-
mail: scptc@mahidol.ac.th.
† Department of Chemistry, Mahidol University.
‡ Department of Physiology, Mahidol University.
§ The Forest Herbarium, Royal Forest Department.
10.1021/np060323u CCC: $33.50
© 2006 American Chemical Society and American Society of Pharmacognosy
Published on Web 11/16/2006

Cytotoxic Styryl-Lactones from Polyalthia crassa
Journal of Natural Products, 2006, Vol. 69, No. 12 1729
Table 1. 1H and 13C NMR Spectroscopic Data of 1-4 (in CDCl3)
1
2
3
4
C/H
δ
H mult. (J)a
δC mult.b
δH mult. (J)a
δ
C mult.b
δ
H mult. (J)a
δC mult.b
δ
H mult. (J)a
δC mult.b
2
3
162.3 s
124.6 d
174.7 s
35.6 t
175.3 s
35.8 t
169.0 s
124.9 d
6.21 d (9.7)
2.72 dd (19.0, 5.8)
2.60 d (19.0)
5.02-5.05 m
5.02-5.05 m
5.75 d (2.6)
2.67 dd (18.8, 5.6)
2.56 d (18.8)
4.99-5.03 m
4.99-5.03 m
4.42 br s
6.28 d (5.6)
7.29 d (5.6)
4
5
6
6.99 dd (9.7, 5.9)
5.30 dd (5.9, 2.7)
4.79 dd (5.6, 2.7)
140.8 d
62.8 d
77.6 d
77.1 d
85.3 d
75.5 d
77.1 d
87.0 d
73.2 d
151.0 d
114.3 s
42.4 t
2.57 dd (14.3, 6.4)
2.31 dd (14.3, 1.9)
4.42 ddd (6.4, 1.9, 1.9)
5.39 d (1.9)
7
8
4.29 t (5.6)
4.91 d (5.6)
73.4 d
73.5 d
4.27 dd (8.6, 2.6)
4.68 d (8.6)
83.1 d
70.9 d
4.25 dd (9.2, 2.2)
6.00 d (9.2)
82.5 d
72.9 d
78.2 d
91.4 d
9
139.9 s
126.6 d
128.7 d
128.3 d
128.7 d
126.6 d
165.5 s
116.3 d
146.6 d
133.8 s
128.2 d
128.9 d
130.8 d
128.9 d
128.2 d
140.4 s
126.8 d
128.6 d
128.4 d
128.6 d
126.8 d
166.4 s
116.1 d
147.6 d
133.8 s
128.4 d
129.1 d
131.1 d
129.1 d
128.4 d
136.8 s
127.7 d
128.7 d
128.9 d
128.7 d
127.7 d
167.5 s
116.7 d
147.3 d
133.8 s
128.4 d
129.0 d
131.0 d
129.0 d
128.4 d
138.4 s
125.0 d
128.7 d
128.2 d
128.7 d
125.0 d
10
11
12
13
14
1′
2′
3′
4′
5′
6′
7′
8′
9′
OH-6
OH-7
OH-8
7.43 d (7.4)
7.34 t (7.4)
7.28 t (7.4)
7.34 t (7.4)
7.43 d (7.4)
7.31-7.46 m
7.31-7.46 m
7.31-7.46 m
7.31-7.46 m
7.31-7.46 m
7.48 dd (8.1, 1.5)
7.36-7.44 m
7.29-7.34 m
7.37-7.41 m
7.29-7.34 m
7.37-7.41 m
7.29-7.34 m
7.36-7.44 m
7.36-7.44 m
7.48 dd (8.1, 1.5)
6.32 d (16.0)
7.63 d (16.0)
6.55 d (16.0)
7.84 d (16.0)
6.46 d (15.9)
7.76 d (15.9)
7.50 dd (7.4, 2.0)
7.37-7.41 m
7.59 dd (7.4, 2.2)
7.31-7.46 m
7.53 dd (7.5, 2.1)
7.36-7.44 m
7.36-7.44 m
7.36-7.44 m
7.53 dd (7.5, 2.1)
4.18 br s
7.37-7.41 m
7.31-7.46 m
7.37-7.41 m
7.31-7.46 m
7.50 dd (7.4, 2.0)
7.59 dd (7.4, 2.2)
2.75 br s
2.75 br s
2.43 (br s)
2.91 br s
a Spectra recorded at 500 MHz in CDCl3, using TMS as an internal reference; J values (in Hz) in parentheses. b Spectra recorded at 125 MHz
in CDCl3, using CDCl3 signal at δC 77.0 as reference; multiplicites determined by DEPT experiments.
Scheme 1. Chemical Conversions of (+)-Crassalactone A (1) and (+)-Howiinol A (5) to the (+)-Tricinnamate 11
(a) Cinnamic acid (2.2 equiv), DCC (2.3 equiv), DMAP (4 equiv), Dry CH2Cl2, rt, 3 h; (b) cinnamic acid (2.2 equiv), DCC (2.3 equiv), DMAP (4 equiv), dry
CH2Cl2, rt, 14 h.
The molecular formula of (+)-crassalactone B (2) was deter-
mined as C22H20O6 by HRTOFMS (ESI positive) measurement for
C22H20O6Na at m/z 403.1158 (calcd 403.1158). The weak molecular
ion [M]+ at m/z 380, together with the fragment ions at m/z 362
(M+ - H2O), 148 [(PhCHdCHCOOH)+, base peak], and 131
(PhCHdCHCO)+ in the EIMS, suggested the presence of a hydroxy
and a cinnamate group in 2, which was confirmed by the IR
absorption bands at 3518 (OH) and 1719 cm-1 (CdO of an R,â-
unsaturated ester). Another carbonyl band at 1763 cm-1 indicated
1
that 2 is also a saturated γ-lactone. Similar to 1, the H NMR
spectrum of 2 (Table 1) exhibited a set of signals corresponding to
a cinnamate group at δ 6.55 (H-2′), 7.84 (H-3′), 7.31-7.46 (H-6′,
H-7′, and H-8′), and 7.59 (H-5′ and H-9′). The protons of another
Figure 1. X-ray ORTEP diagram of (+)-howiinol A (5).
phenyl group (H-10 through H-14) were observed in the same range
as H-6′, H-7′, and H-8′. Due to the proximity to the C-2 carbonyl
in the γ-lactone ring, the downfield shifts of the H-3a and H-3b
signals were observed at δ 2.72 (dd, J ) 19.0 and 5.8 Hz) and
2.60 (d, J ) 19.0 Hz), respectively. The COSY spectrum supported
the assignments of the overlapping signals at δ 5.02-5.05 (2H) as
H-4 and H-5, including the separated signals at δ 5.75 (d, J ) 2.6
Hz), 4.27 (dd, J ) 8.6 and 2.6 Hz), and 4.68 (d, J ) 8.6 Hz) as
H-6, H-7, and H-8, respectively. The COSY correlation between
the H-8 signal and a broad singlet at δ 2.91 confirmed the location
of the hydroxy group at position C-8. Eighteen carbon signals for
22 carbons were found present in the 13C NMR spectrum of 2 (Table
1). The carbon types and assignments were analyzed by DEPT and
2D-NMR data. The presence of the cinnamate group at C-6 was
established through the long-range HMBC correlations of H-6 to
namate group to C-5 was indicated by the HMBC correlations of
the H-5 signal to the C-2, C-3, C-4, C-6, C-7, C-1′, and C-2′ signals
(Table S1, Supporting Information). As both (+)-1 and (+)-5 could
be converted into the (+)-tricinnamate 11 (Scheme 1) and the
products obtained from both reactions (41 and 18% yields,
1
respectively) were found to be identical (mp, optical rotation, H
and 13C NMR data), the relative configuration of both compounds
was proved to be the same. The isolated (+)-howiinol A (5) and
the compound reported previously by Chen et al.13b were confirmed
by single-crystal X-ray diffraction analysis (see Figure 1). Since
the absolute stereochemistry of natural (+)-howiinol A (5) has been
proposed previously through synthesis,13a the absolute configuration
of (+)-crassalactone A (1) was determined therefore as 5S, 6R,
7R, and 8R.

1730 Journal of Natural Products, 2006, Vol. 69, No. 12
Tuchinda et al.
Scheme 2. Preparation of (+)-Crassalactone B (2) and (+)-Crassalactone C (3) from (+)-Goniofufurone (6)a
a (a) Cinnamoyl chloride (1 equiv), Et3N (2.5 equiv), and DMAP (4 equiv), rt, 4 h.
C-4, C-5, C-7, and C-1′ (Table S1, Supporting Information).
Compound 2 was further proved to possess the same stereochem-
istry as (+)-goniofufurone (6) isolated from the same extract (for
spectroscopic data of 6, see the Supporting Information) and
reported earlier from the stem bark of Goniothalamus giganteus.14a
By the reaction of (+)-6 with cinnamic acid (1 equiv) under the
conditions shown in Scheme 2, (+)-2 and (+)-3 were obtained in
13 and 12% yields, respectively. The physical and spectroscopic
data of the natural (+)-2 were identical (mp, optical rotation, IR,
1
UV, MS, H and 13C NMR) to the semisynthetic product. Hence,
(+)-crassalactone (2) was suggested to possess the same relative
configuration as its precursor (+)-goniofufurone (6). Since the
absolute configuration of (+)-goniofufurone (6) has been deter-
mined previously through synthetic work,14b-d the absolute con-
figuration of (+)-crassalactone B (2) was established as 4R, 5S,
6R, 7R, and 8R.
Figure 2. X-ray ORTEP diagram of crassalactone D (4).
(+)-Crassalactone C (3) was found to possess the molecular
formula C22H20O6 from the [M]+ peak at m/z 380 in the EIMS and
confirmed by elemental analysis. The fragment ions at m/z 362 (M+
- H2O), 148 (PhCHdCHCOOH)+, and 131 [(PhCHdCHCO)+,
base peak], as well as the IR absorptions at 3460 (OH stretch) and
1716 cm-1 (CdO stretch of cinnamate), indicated the presence of
hydroxy and cinnamate groups in 3. The absorption band at 1775
cm-1 was assigned to the CdO stretch of a saturated γ-lactone
1
moiety. By comparison of its H NMR data (Table 1) with those
Figure 3. Distribution of ∆δS-R values of the (S)- and (R)-MTPA
esters of (+)-crassalactone D (4).
of 2, the structure of 3 was closely related to 2. The locations of
the hydroxyl group at C-6 and cinnamate group at C-8 in 3 were
suggested by an upfield shift of the broad singlet at δ 4.42 (H-6)
and a downfield shift of doublets at δ 6.00 (H-8, J7,8 ) 9.2 Hz).
Further confirmation was obtained through the observed HMBC
correlations of OH-6 to C-5 and C-6, and H-8 to C-6, C-7, C-9,
C-10, C-14, and C-1′ (Table S1, Supporting Information). Other
proton signals were found in accordance with those of 2, whereas
the 13C NMR signals (Table 1) showed 18 signals for 22 carbons,
of which the carbon types were analyzed by DEPT experiments.
The two downfield peaks at δC 175.3 and 167.5 were attributable
to the carbonyls of saturated γ-lactone and cinnamate groups,
respectively. As mentioned earlier, compound 3 was also obtained
from the reaction of the isolated (+)-goniofufurone (6) with
cinnamoyl chloride under the conditions shown in Scheme 2. The
absolute configuration of (+)-crassalactone C (3) was thus proved
to be 4R, 5S, 6R, 7R, and 8R.
the signal of H-6a at δ 2.57 (dd, J ) 14.3 and 6.4 Hz) and the
signal of H-8 at δ 5.39 (d, J ) 1.9 Hz), but very weak cross-peaks
with the signal of H-6b at δ 2.31 (dd, J ) 14.3 and 1.9 Hz). The
hydroxyl group in 4 was suggested to be located at C-7 because a
rather broad signal was observed for H-7. The 13C NMR and DEPT
spectroscopic data of 4 (Table 1) indicated 11 signals for 13 carbons,
consisting of one carbonyl, two quarternary carbons, nine methines,
and one methylene. The signal of a quaternary carbon in the low-
field region (δC 114.3) was assigned to the spiroketal carbon (C-
5), since HMBC correlations of this signal to the H-3, H-4, H-6a,
H-6b, H-7, and H-8 signals were observed (Table S1, Supporting
Information). The complete structure and relative configuration of
4 were established by X-ray diffraction analysis. A view of the
structure is provided in Figure 2.
The absolute stereochemistry of 4 was determined by the Mosher
ester method.18 Crassalactone D (4) was reacted with (S)-(+)- and
(R)-(-)-R-methoxy-R-(trifluoromethyl)phenylacetyl chloride at room
temperature to give (R)- and (S)-MTPA esters [(R)- and (S)-R-
methoxy-R-(trifluoromethyl)phenylacetic acid esters] of 4 in 21 and
19% yields, respectively. The observed chemical shift differences
(∆δS-R), shown in Figure 3, unambiguously established the absolute
configuration at C-7 as S. Thus, the configuration in 4 was assigned
as 5S, 7S, and 8R.
Elemental analysis of (+)-crassalactone D (4) established its
molecular formula as C13H12O4. In its EIMS, the prominent peaks
at m/z 214 [M+ - H2O] suggested that 4 was a monohydroxy
alcohol. The OH stretch band at 3469 cm-1 in the IR spectrum
confirmed the alcoholic character of 4. The presence of an R,â-
unsaturated γ-lactone unit in 4 was indicated by the two carbonyl
absorbances at 1758 and 1750 cm-1 and confirmed by the peak at
δC 169.0 (C-2) in its 13C NMR spectrum (Table 1). The H NMR
1
spectrum of 4 (Table 1) showed signals for a phenyl group,
consisting of two sets of multiplets at δ 7.37-7.41 (2H) and 7.29-
7.34 (3H). The olefinic protons (H-3 and H-4) in the unsaturated
γ-lactone ring resonated as a pair of doublets (J ) 5.6 Hz each) at
δ 6.28 and 7.29, respectively. The assignments of the other protons
were made from its COSY spectrum. The signal of H-7 at δ 4.42
(ddd, J ) 6.4, 1.9, and 1.9 Hz) showed strong cross-peaks with
Pure isolated compounds (+)-1-(+)-4, the alkaloid aristolactam
AII (10), and the (+)-tricinnamate 11 were evaluated for cytotoxic
effects against a panel of cultured mammalian cell lines. The results
are shown in Table 2. (+)-Crassalactone A (1) and (+)-crassalac-
tone D (4) showed broad cytotoxic activity for all cell lines tested,
while (+)-crassalactone B (2), 10, and 11 exhibited moderate
cytotoxic effects only for the P-388 cell line.

Cytotoxic Styryl-Lactones from Polyalthia crassa
Journal of Natural Products, 2006, Vol. 69, No. 12 1731
Table 2. Cytotoxicity of Compounds 1-4, 10, and 11 against
recrystallization with EtOH. Fraction E4 (882.5 mg), after preparative
TLC (5% MeOH-CH2Cl2 as eluent) and recrystallization with EtOH,
gave (+)-crassalactone C (3) (147.5 mg). Fraction E5 was further
purified by column chromatography (40% EtOAc-hexane as eluent)
to provide fractions F1-F4. Fraction F3 (252.9 mg) yielded (+)-
crassalactone A (1) (67.7 mg) upon recrystallization with EtOAc-
hexane. Fraction F4 gave (+)-goniopypyrone (9) (62.9 mg) after column
chromatography (10% EtOAc-hexane as eluent) and recrystallization
from EtOAc-hexane. Fraction E6 (2.38 g) afforded (+)-howiinol A
(5) (390.9 mg) upon recrystallization with EtOH-CH2Cl2. An additional
quantitiy of (+)-5 (304.9 mg) was obtained after column chromatog-
raphy (30% acetone-hexane as eluent) and recrystallization. Fraction
D4 (941.6 mg) was further separated by passage on a silica gel column
(10% EtOAc-CH2Cl2 as eluent) to yield fractions G1-G6. Fraction
G1 yielded (+)-crassalactone D (4) (30.2 mg) after preparative TLC
(5% EtOAc-CH2Cl2) and recrystallization with EtOAc-hexane. Frac-
tion G3 (299.7 mg) provided (+)-goniofufurone (6) (87.8 mg) after
column chromatography (50% EtOAc-hexane as eluent) and recrys-
tallization with EtOAc-hexane. Fraction A10 (27.9 g, eluted with 50-
80% EtOAc-hexane) was rechromatographed on a silica gel column
(MeOH-CH2Cl2-hexane, 1:12:7, as eluent) to give fractions H1-H5.
Fraction H3 (1.74 g) yielded an additional amount of (+)-6 (128.3 mg)
after separation on two consecutive silica gel columns (10% acetone-
hexane and 2% MeOH-CH2Cl2 as eluents, respectively), followed by
recrystallization with EtOAc-hexane.
Human and Rat Cancer Cell Lines
cell line (ED50 µg/mL)a
compound
P-388
KB
1.7
>5
>5
3.3
>5
>5
0.65
Col-2
BCA-1
Lu-1
ASK
1
2
3
4
0.18
3.8
>5
1.1
2.7
3.1
0.52
1.9
>5
0.92
>5
>5
3.2
>5
>5
0.65
1.9
>5
1.6
>5
>5
>5
>5
4.0
>5
3.1
10
11
ellipticine
>5
>5
0.53
>5
>5
>5
0.60
>5
0.56
a Cytotoxic assay: ED50 e 5 µg/mL is considered active; P-388:
murine lymphocytic leukemia, KB: human nasopharyngeal carcinoma,
Col-2: human colon cancer, BCA-1: human breast cancer, Lu-1:
human lung cancer, ASK: rat glioma.
Although the presence of styryl-lactones in the genus Polyalthia
is unusual, altholactone has been reported previously from an
unnamed species of this genus.15 Our work represents the second
report on the discovery of styryl-lactones from the genus Polyalthia.
Experimental Section
General Experimental Procedures. Melting points (uncorrected)
were recorded on a digital Electrothermal melting apparatus. Optical
rotations were determined on a JASCO DIP 370 digital polarimeter
using a 50 mm microcell (1 mL). UV spectra were measured in ethanol
on a JASCO 530 spectrometer, and IR spectra were recorded on a
28
(+)-Crassalactone A (1): white powder, mp 133-134 °C; [R]D
+326.0 (c 0.1, CHCl3); [R]D30 +329.6 (c 0.5, EtOH); UV (EtOH) λmax
(log ꢀ) 224 (sh) (4.88), 277 (4.58) nm; IR (KBr) Vmax 3456, 1721, 1711,
1636, 1450, 1338, 1253, 1164, 1099, 1069, 985 cm-1; 1H and 13C NMR,
Table 1; EIMS m/z 380 [M]+ (0.7), 362 [M+ - H2O] (0.6), 344 [M+
- 2H2O] (0.4), 273 (10), 178 (15), 148 (89), 131 (100); anal. C 69.44%,
H 5.78%, calcd for C22H20O6, C 69.47%, H 5.30%.
1
Perkin-Elmer 2000 FT-IR spectrophotometer. The H and 13C NMR
spectra were recorded on a Bruker AV 500 spectrometer in CDCl3,
using TMS as internal standard. EIMS were recorded on a Thermo
Finnigan Polaris Q mass spectrometer at 70 eV (probe). The HRMS
were recorded on a Micromass model VQ-TOF spectrometer. Solvents
for extraction, chromatography, and recrystallization were distilled prior
to use. Silica gel 60 (Merck, 70-230 mesh) and silica gel plates (Merck,
Kieselgel 60F254, 0.5 mm) were used for column chromatography and
preparative TLC, respectively.
Plant Material. The leaves and twigs of P. crassa Parker (Annon-
aceae) were collected from Kangkrachan District, Petchburi Province,
in October 2001 and identified by one of us (T.S.). A voucher specimen
(BKF no. 109931) of P. crassa has been deposited at the Forest
Herbarium, Royal Forest Department, Bangkok, Thailand.
Extraction and Isolation. The air-dried and finely powdered mixed
leaves and twigs of P. crassa (2.6 kg) were sequentially percolated
with hexane (5 × 4 L), EtOAc (5 × 3.5 L), and MeOH (5 × 4 L), at
room temperature. Removal of solvents yielded the hexane, EtOAc,
and MeOH extracts in 91, 184, and 203 g quantities, respectively.
The cytotoxic EtOAc extract (183 g) was subjected to silica gel
column chromatography (1.6 kg), eluting with EtOAc-hexane (0-
100%), followed by MeOH-EtOAc (0-100%) to give fractions A1-
A11 after combination and removal of solvents. Fraction A5 (4.92 g,
eluted with 8-10% EtOAc-hexane) was further separated by column
chromatography (silica gel, 20% acetone-hexane as eluent), followed
by recrystallization with EtOAc-hexane to give cinnamic acid (185.5
mg). Fraction A7 (7.57 g, eluted with 20-30% EtOAc-hexane) was
purified by column chromatography (silica gel, 4% EtOAc-CH2Cl2
as eluent) to yield fractions B1-B6. Fraction B4 (100.6 mg) afforded
(+)-3-acetylaltholactone (8) (31.4 mg) after separation by preparative
TLC (20% acetone-hexane as eluent), followed by recrystallization
from EtOH-CH2Cl2. Fraction B5 (3.01 g) yielded (+)-altholactone (7)
(2.53 g) as a pure yellow oil after column chromatography (60%
EtOAc-hexane as eluent). Fraction A8 (8.09 g, eluted with 30-35%
EtOAc-hexane) was further separated using a silica gel column
(MeOH-EtOAc-CH2Cl2, 1:1:48, as eluent) to provide an additional
amount of pure 7 (4.56 g). Fraction A9 (16.6 g, eluted with 40-50%
EtOAc-hexane) was rechromatographed on a Sephadex LH-20 column
(MeOH as eluent) to give fractions C1-C4. Further separation of
fraction C4 (7.77 g) on a silica gel column (MeOH-CH2Cl2-hexane,
1:12:7, as eluent) yielded fractions D1-D5. Fraction D3 (882.5 mg)
was further separated by column chromatography (2% acetone-CH2-
Cl2 as eluent) to give fractions E1-E7. Fraction E1 (50.2 mg) afforded
aristolactam AII (10) (33.6 mg) upon recrystallization with MeOH-
C6H6. Fraction E2 yielded (+)-crassalactone B (2) (22.7 mg) after
Preparation of (+)-Tricinnamate 11 from (+)-Crassalactone A
(1). A mixture of (+)-crassalactone A (1) (38 mg, 0.1 mmol), cinnamic
acid (32.6 mg, 0.22 mmol), DCC (47.4 mg, 0.23 mmol), and DMAP
(48.9 mg, 0.40 mmol) in dry CH2Cl2 (6 mL) was stirred at room
temperature for 3 h. The solution was quenched with water (10 mL)
and extracted with CH2Cl2 (3 × 20 mL). The combined organic layers
were washed successively with brine and water and dried over
anhydrous MgSO4. After solvent removal, the crude product (122.8
mg) was further purified by preparative TLC (silica gel, 40% EtOAc-
hexane) to yield the (+)-tricinnamate 11 (26.4 mg, 41% yield).
Preparation of (+)-Tricinnamate 11 from (+)-Howiinol A (5).
A mixture of (+)-howiinol A (5) (38 mg, 0.1 mmol), cinnamic acid
(32.6 mg, 0.22 mmol), DCC (47.4 mg, 0.23 mmol), and DMAP (48.9
mg, 0.40 mmol) in dry CH2Cl2 (6 mL) was stirred at room temperature
overnight (14 h). The solution was quenched with water (10 mL) and
extracted with CH2Cl2 (3 × 20 mL). The combined organic layers were
washed successively with brine and water and dried over anhydrous
MgSO4. After solvent removal, the crude product (125.9 mg) was
purified by preparative TLC (silica gel, 2% MeOH-CH2Cl2) to afford
the (+)-tricinnamate 11 (11.3 mg, 18% yield). Pure (+)-11 was obtained
as a white powder by recrystallization from EtOH-CH2Cl2, mp 195-
29
197 °C; [R]D +175.8 (c 0.2, CHCl3); UV (EtOH) λmax (log ꢀ) 221
(sh) (2.79), 274 (2.89) nm; IR (KBr) νmax 1731, 1721, 1710, 1636,
1577, 1496, 1450, 1308, 1164, 1039, 979 cm-1; 1H NMR (CDCl3, 500
MHz) δ 7.76 (1H, d, J ) 16.0 Hz, H-3′′′), 7.62 (1H, d, J ) 16.0 Hz,
H-3′), 7.57 (1H, d, J ) 16.0 Hz, H-3′′), 7.56-7.53 (2H, m, ArH), 7.49
(2H, dd, J ) 7.9, 1.2 Hz, H-10, H-14), 7.43-7.30 (13H, m, Ar-H),
7.27-7.19 (3H, m, Ar-H), 7.05 (1H, dd, J ) 9.7, 5.5 Hz, H-4), 6.49
(1H, d, J ) 16.0 Hz, H-2′′′), 6.37 (1H, d, J ) 16.0 Hz, H-2′′), 6.28
(1H, d, J ) 6.7 Hz, H-8), 6.27 (1H, d, J ) 16.0 Hz, H-2′), 6.23 (1H,
d, J ) 9.7 Hz, H-3), 5.94 (1H, dd, J ) 6.7, 4.1 Hz, H-7), 5.52 (1H, dd,
J ) 5.5, 3.1 Hz, H-5), 4.93 (1H, dd, J ) 4.1, 3.1 Hz, H-6); 13C NMR
(CDCl3, 125 Hz) δ 165.5 (s, C-1′), 165.2 (s, C-1′′), 165.1 (s, C-1′′′),
161.8 (s, C-2), 146.7 (d, C-3′), 146.33 (d, C-3′′′), 146.27 (d, C-3′′),
140.5 (d, C-4), 135.7 (s, C-9), 134.1 (s, C-4′′′), 134.0 (s, C-4′′), 133.7
(s, C-4′), 130.64, 130.60, 130.57, 128.91, 128.85, 128.82, 128.78, 128.6,
128.3 (d each, ArCH), 127.3 (d each, C-10 and C-14), 124.6 (d, C-3),
117.1 (d, C-2′′′), 116.9 (d, C-2′′), 116.1 (d, C-2′), 75.5 (d, C-6), 73.4
(d, C-8), 71.4 (d, C-7), 63.0 (d, C-5); EIMS m/z 640 [M]+ (0.06), 147
(7), 131 (100), 103 (31), 77 (9).
(+)-Crassalactone B (2): white powder, mp 171-173 °C; [R]28
D
+8.0 (c 0.5, EtOH); UV (EtOH) λmax (log ꢀ) 224 (sh) (4.88), 279 (4.74)
nm; IR (KBr) νmax 3518, 1763, 1719, 1635, 1578, 1497, 1449, 1398,

1732 Journal of Natural Products, 2006, Vol. 69, No. 12
Tuchinda et al.
1
1333, 1315, 1163, 1045, 901 cm-1; H and 13C NMR, Table 1; EIMS
Acknowledgment. We thank the Thailand Research Fund for
financial support (DBG4880014) to P.T., a Ph.D. scholarship to B.M.
through the Royal Golden Jubilee Ph.D. Program, and an award of
Senior Research Scholar to V.R. Thanks are also due to the Higher
Education Development Project, Postgraduate Education and Research
Program in Chemistry (PERCH), for support.
m/z 380 [M]+ (0.6), 362 [M - H2O]+ (2), 232 (20), 173 (20), 160
(78), 148 (100), 131 (99), 126 (26); HRTOFMS (ESI positive) m/z
403.1158 (calcd for C22H20O6Na, 403.1158).
(+)-Crassalactone C (3): white powder, mp 147-150 °C; [R]30
D
+98.4 (c 0.5, EtOH); UV (EtOH) λmax (log ꢀ) 223 (sh) (4.65), 277
(4.46) nm; IR (KBr) νmax 3460, 1775, 1716, 1682, 1638, 1578, 1498,
1450, 1333, 1167, 1068, 1047, 1004, 981 cm-1; H and 13C NMR,
1
Table 1; EIMS m/z 380 [M]+ (1), 362 [M - H2O]+ (2), 335 (6), 275
(14), 232 (23), 173 (22), 148 (32), 131 (100); anal. C 69.19%, H 5.36%,
calcd for C22H20O6, C 69.47%, H 5.30%.
Supporting Information Available: HMBC correlations observed
for (+)-crassalactones A-D (1-4) and physical and spectroscopic
properties of (+)-howiinol A (5) and (+)-goniofufurone (6). This
material is available free of charge via the Internet at http://
pubs.acs.org.
(+)-Crassalactone D (4): colorless needles, mp 138-139 °C; [R]30
D
+7.0 (c 0.2, EtOH); UV (EtOH) λmax (log ꢀ) 252 (2.89), 258 (2.90),
264 (2.80), 268 (sh) (2.64) nm; IR (KBr) νmax 3469, 1758, 1750, 1608,
1500, 1455, 1413, 1347, 1312, 1191, 1129, 1097, 1056, 982, 925 cm-1
;
-
References and Notes
1H and 13C NMR, Table 1; EIMS m/z 233 [M + H]+ (1), 214 [M+
H2O] (3), 126 (22), 107 (100), 97 (27), 79 (42); anal. C 67.53%, H,
5.70%, calcd for C13H12O4, C 67.24%, H 5.21%.
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Preparation of the (R)-MTPA Ester of (+)-4.18 (S)-(+)-MTPA
Cl (25.3 mg, 0.100 mmol) was added to a CH2Cl2 (1 mL) solution of
crassalactone D (4) (11.9 mg, 0.051 mmol), DMAP (25.0 mg, 0.205
mmol), and Et3N (0.32 mL, 0.24 M solution in CH2Cl2) at 0 °C. The
reaction mixture was stirred at room temperature for 6 h. The solution
was quenched with water (10 mL) and extracted with CH2Cl2 (3 × 30
mL). The combined organic layers were washed successively with brine
and water and dried over anhydrous MgSO4. After solvent removal,
the crude product (18.6 mg) was purified by preparative TLC (silica
gel, 5% MeOH-CH2Cl2) to give the (R)-MTPA ester of 4 (4.9 mg,
21% yield).
Preparation of the (S)-MTPA Ester of (+)-4.18 (R)-(-)-MTPA
Cl (22.9 mg, 0.091 mmol) was added to a CH2Cl2 (1 mL) solution of
crassalactone D (4) (10.5 mg, 0.045 mmol), DMAP (22.1 mg, 0.181
mmol), and Et3N (0.28 mL, 0.24 M solution in CH2Cl2) at 0 °C. The
reaction mixture was stirred at room temperature for 4 h. The solution
was quenched with water (10 mL) and extracted with CH2Cl2 (3 × 30
mL). The combined organic layers were washed successively with brine
and water and dried over anhydrous MgSO4. After solvent removal,
the crude product (20.9 mg) was purified by preparative TLC (silica
gel, 5% MeOH-CH2Cl2) to give the (S)-MTPA ester of 4 (4.4 mg,
19% yield).
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X-ray Structure Determination of (+)-Crassalactone D (4).
C13H12O4, MW 232.24, monoclinic, P21, a ) 9.7649(9) Å, b ) 4.8852-
(3) Å, c ) 11.8369(11) Å, â ) 99.310 (3)°, V ) 557.22 (8) Å3, Dx )
1.384 g/cm3, Z ) 2, F(000) ) 244. A total of 6287 reflections, of
which 1994 were unique reflections (1784 observed, |Fo| > 4σ |Fo|),
were measured at room temperature from a 0.25 × 0.15 × 0.10 mm3
colorless crystal using graphite-monochromated Mo KR radiation (λ
) 0.71073 Å) on a Bruker-Nonius kappaCCD diffractometer. The
crystal structure was solved by direct methods using SIR-97, and then
all atoms except hydrogen atoms were refined anisotropically by full-
matrix least-squares methods on F2 using SHELXL-97 to give a final
R-factor of 0.0375 (Rw ) 0.0986 for all data).
X-ray Structure Determination of (+)-Howiinol A (5). C22H20O6,
MW 380.396, orthorhombic, P212121, a ) 6.0723(4) Å, b ) 12.0285-
(6) Å, c ) 25.741(2) Å, V ) 1880.1(2) Å3, Dx ) 1.344 g/cm3, Z ) 4,
F(000) ) 800. A total of 6605 reflections, of which 2632 were unique
reflections (1996 observed, |Fo| > 4σ|Fo|), were measured at room
temperature from a 0.20 × 0.05 × 0.05 colorless crystal using graphite-
monochromated Mo KR radiation (λ ) 0.71073 Å) on a Bruker-Nonius
kappaCCD diffractometer. The crystal structure was solved by direct
methods using SIR-97, and then all atoms except hydrogen atoms were
refined anisotropically by full-matrix least-squares methods on F2 using
SHELXL-97 to give a final R-factor of 0.0505 (Rw ) 0.1175 for all
data).
Crystallographic data for structures reported in this paper have been
deposited with the Cambridge Crystallographic Data Centre and
allocated the deposition numbers CCDC 612409 for compound 4 and
CCDC 612408 for compound 5. Copies of the data can be obtained,
free of charge, on application to the Director, CCDC, 12 Union Road,
Cambridge CB2 1EZ, UK (fax: +44-(0) 1223-336033 or e-mail:
deposit@ccds.cam.ac.uk).
Cytotoxicity Testing. Cytotoxicity assays of compounds 1-4, 10,
and 11 were performed employing the colorimetric method as described
by Skehan et al.,19 and ellipticine was used as a positive control.
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Cytotoxic Styryl-Lactones from Polyalthia crassa
Journal of Natural Products, 2006, Vol. 69, No. 12 1733
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NP060323U