Cytotoxic lignans from fruits of Cleistanthus indochinensis: Synthesis of cleistantoxin derivatives

Article abstract of DOI:10.1021/np3003832

Two new aryl-tetralin lignans, 1 and 2, were isolated from the fruits of Cleistanthus indochinensis by bioassay-guided purification. Their structures were determined by spectroscopic analysis including MS and 2D NMR. The absolute configurations of 1 and 2 were established from examination of their CD spectra. Compound 1 was cytotoxic against KB cells with an IC₅₀ value of 0.022 μM, while compound 2 had weaker cytotoxicity, with an IC₅₀ value of 1.4 μM. When tested against other cancer cell lines (MCF-7, MCF-7R, and HT29), 1 showed an IC₅₀ of 0.014 against MCF-7R cells and an IC₅₀ of 0.036 μM against MCF-7 cells. A series of amide derivatives of a new lactone, homoderivatives of 1, were prepared. Of these derivatives, only compound 3 had weak cytotoxicity against KB cells.

Full text of DOI:10.1021/np3003832

Article
pubs.acs.org/jnp
Cytotoxic Lignans from Fruits of Cleistanthus indochinensis: Synthesis
of Cleistantoxin Derivatives
Van Trinh Thi Thanh,† Van Cuong Pham,†,* Huong Doan Thi Mai,† Marc Litaudon,‡
Franco̧
ise Gueritte,‡ Pascal Retailleau,‡ Van Hung Nguyen,† and Van Minh Chau†,*
́
†Institute of Marine Biochemistry of the Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet Road, Cau
Giay, Hanoi, Vietnam
‡Institut de Chimie des Substances Naturelles, Gif-sur Yvette, France
S
* Supporting Information
ABSTRACT: Two new aryl-tetralin lignans, 1 and 2, were
isolated from the fruits of Cleistanthus indochinensis by
bioassay-guided purification. Their structures were determined
by spectroscopic analysis including MS and 2D NMR. The
absolute configurations of 1 and 2 were established from
examination of their CD spectra. Compound 1 was cytotoxic
against KB cells with an IC50 value of 0.022 μM, while
compound 2 had weaker cytotoxicity, with an IC50 value of 1.4
μM. When tested against other cancer cell lines (MCF-7, MCF-7R, and HT29), 1 showed an IC50 of 0.014 against MCF-7R cells
and an IC50 of 0.036 μM against MCF-7 cells. A series of amide derivatives of a new lactone, homoderivatives of 1, were
prepared. Of these derivatives, only compound 3 had weak cytotoxicity against KB cells.
he genus Cleistanthus comprises about 140 species and is
T
known for its lignan and terpenoid content.1−8 Lignan
lactones from several species of this genus have structural
similarity to podophyllotoxin and its semisynthetic anticancer
derivatives such as etopoide and teniposide.1,9,10 In a previous
study, we reported the isolation of a new triterpenoid skeleton
from the leaves of Cleistanthus indochinensis.11 In our screening
program, a fruit extract of C. indochinensis Merr. ex Croiz
(Euphorbiaceae) showed strong cytotoxicity against KB cells
(>90% inhibition at 1 μg/mL). In this paper, we report the
isolation and structural elucidation of two new cytotoxic
lignans, 1 and 2, from the fruits of C. indochinensis. Compound
1 was a principle active component of the fruits against KB,
MCF-7, MCF-7R, and HT9 cell lines. The structure of 1 is
similar to that of podophyllotoxin, which has been found to
epimerize easily at C-7 in acidic medium and at C-8′ in basic
solution. The epimerization of podophyllotoxins at C-7 affords
epipodophyllotonxins that change their anticancer properties
(inhibition of microtubule polymerization vs topoisomerase II
inhibition),12,13 while the epimerization at C-8′ in many cases
reduces their cytotoxicity.14,15 A number of stable podophyllo-
toxin derivatives with lactone ring-opening have been reported,
and some of them could be promising new antitumor drug
candidates.16−18 In order to obtain more stable derivatives of 1,
a series of a new type of related lignans were prepared for
evaluation of their biological activities.
This crude extract showed strong inhibition against KB cells
and then was chromatographed by open column chromatog-
raphy (CC). The resulting fractions were retested against KB
cells, and the two active fractions were further purified by CC
to give compounds 1 (2.1 g) and 2 (0.25 g).
Compound 1 was isolated as a white, microcrystalline solid.
Its HRESI mass spectrum showed the pseudomolecular ion [M
+ Na]+ consistent with the molecular formula C21H18O8. The
1H NMR spectrum of 1 presented a singlet aromatic proton at
δH 6.22 (H-3) and an ABX ring system, characterized from
three aromatic protons at δH 6.72 (H-2′), 6.66 (H-5′), and 6.64
(H-6′). The presence of a methoxy and two methylendioxy
RESULTS AND DISCUSSION
■
Dried and ground fruits of C. indochinensis (0.5 kg) were
extracted with CH2Cl2 at room temperature. The solvent was
removed under reduced pressure to give a residue of 28.5 g.
Received: May 31, 2012
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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1
Table 1. NMR Data for 1 and 2 (CDCl3, H: 500 MHz, 13C: 125 MHz)
1
2
position
δC, type
δH
mult. (J in Hz)
position
δC
δH
mult. (J in Hz)
1
2
3
4
5
6
7
8
9
124.7, C
132.9, C
104.1, CH
149.4, C
134.9, C
141.5, C
70.4, CH
38.7, CH
71.8, CH2
1
2
3
4
5
6
7
8
9
119.7
131.6
104.4
150.1
135.2
140.5
72.1
6.22
s
6.20
s
4.99
d (9.0)
5.81
3.13
3.62
3.71
5.92
5.93
br d (5.0)
m
2.84
dddd (9.0, 9.0, 10.5, 15.0)
dd (9.0, 10.5)
dd (9.0, 9.0)
d (1.5)
46.6
4.02, Hβ
4.59, Hα
5.88
59.4
br dd (8.0, 10.5)
br dd (7.5, 10.5)
d (1.0)
10
101.2, CH2
10
101.3
5.91
d (1.5)
d (1.0)
1′
2′
3′
4′
5′
6′
7′
8′
9′
10′
133.0, C
1′
2′
3′
4′
5′
6′
7′
8′
9′
10′
135.2
109.8
147.7
147.0
108.2
122.7
43.2
111.1, CH
147.2, C
6.72
d (1.5)
6.49
br s
146.5, C
107.6, CH
124.1, CH
43.9, CH
44.6, CH
174.2, C
6.66
6.64
4.47
2.71
d (8.0)
6.73
6.56
4.24
2.94
d (9.0)
dd (1.5, 8.0)
d (4.5)
br d (9.0)
d (4.5)
dd (4.5, 15.0)
47.2
ddd (1.0, 4.5, 4.5)
175.7
101.1
100.9, CH2
5.86
5.87
4.12
d (1.3)
d (1.3)
s
5.93
4.04
br s
s
OMe
59.8, CH3
OMe
60.3
groups was also noted, together with six protons in the aliphatic
region. The 13C NMR and DEPT spectra showed signals of 12
aromatic carbons, a carboxylic group, four sp3 methines, a sp3
methylene, a methoxy, and two dioxymethylenes. The chemical
shifts of C-4, C-5, C-6, C-3′, and C-4′ (Table 1) were
characteristic of oxygenated aromatic carbons. Similarly, the 1H
and 13C chemical shifts of CH-7 and CH2-9 suggested their
1
linkage to oxygen. Analysis of the H−1H COSY spectrum
revealed two spin−spin coupling systems: (a) correlations of
the ABX system; (b) cross-peaks of H-8 (δH 2.84) to H-7 (δH
4.99), CH2-9 (δH 4.02 and 4.59), and H-8′ (δH 2.71), which
was further correlated to H-7′ (δH 4.47). These analyses
suggested that 1 was an aryl-tetralin lignan, which was
supported by HMBC analysis. Cross-peaks of C-7′ (δC 43.9)
with H-3, H-2′, and H-6′ indicated linkage of C-7′ to both the
A- and B-rings. Correlation of C-1 (δC 124.7) with H-8 and
that of C-6 (δC 141.5) to H-7 assigned the C-1−C-7
connection. The lactone ring was revealed from HMBC
cross-peaks of the carboxylate carbon C-9′ (δC 174.2) to
CH2-9 protons. The dioxymethylene protons at δH 5.88 and
5.91 were correlated to C-4 (δC 149.4) and C-5 (δC 134.9),
indicating the position of this dioxymethylene group at C-4 and
C-5 of the B-ring. Similarly, bonding of the dioxymethylene at
δH 5.86 and 5.87 to C-3′ and C-4′ was established from their
HMBC correlations. Finally, C-6 was correlated to the methoxy
signal at δH 4.12 that defined the presence of an OCH3 group at
C-6.
Figure 1. Selected HMBC correlations for 1.
observation was confirmed by a NOESY experiment in which
interaction of H-7 and H-8′ was noted.
The absolute configuration (7′R) was established by
examination of the circular dichroism (CD) spectrum of 1,
which gave a positive Cotton effect at 295 nm (Δε +2.16).
Since the sign of the first couplet is determined by the
configuration of the aryl substituent at C-7′, namely, negative
for 7′S and positive for 7′R, C-7′ was assigned the R-
configuration.19−21 On the basis of the relative configuration
determined above, the R-configuration was assigned for the
remaining chiral centers, C-7, C-8, and C-8′. Compound 1 was
thus identified as (7R,8R,7′R,8′R)-7-hydroxy-6-methoxy-
3′,4′:4,5-bis(methylenedioxy)-2,7′-cyclolignan-9′,9-olide. This
is the first report of a compound with this structure, and
compound 1 has been named cleistantoxin. The structure of 1
is similar to that of podophyllotoxin, which is well known for its
antiviral and antitumor properties.22
1
The relative configuration of 1 was deduced from H−1H
coupling constant analysis. H-7 presented an anti coupling
constant (J = 9.0 Hz), assigning its pseudodiaxial relationship
with H-8. H-8′ was observed as a doublet of doublets in the 1H
NMR spectrum and had both gauche (J = 4.5 Hz) and anti (J =
15.0 Hz) coupling constants. This indicated that H-8′ had a
pseudoaxial orientation, while H-7′ was pseudoequatorial. This
The 13C NMR spectrum of compound 2 presented signals
1
similar to those of 1. However, their H NMR spectra were
remarkably different, especially in the aliphatic region. The
1H−1H COSY spectrum analysis indicated the same spin−spin
coupling systems as 1, and analysis of the HMBC spectrum of 2
B
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indicated the same A-, B-, and C-ring systems as in the structure
of 1. However, cross-peaks of carboxylate C-9′ (δC 175.7) with
H-7 (δH 5.81) in the HMBC spectrum of 2 indicated that the
lactone ring was formed from the carbonyl C-9′ and the OH
group at C-7. Further HMBC analysis defined the planar
structure of 2 (Figure 2).
Cleistantoxin (1) is the major active component from the
fruits of C. indochinensis. It is easily epimerized at C-7 in acidic
medium and at C-8′ in basic solution, as observed for
podophyllotoxin. In order to obtain a small library of stable
derivatives of 1 for evaluation of their biological properties, a
series of amide derivatives with a C−C bond at C-7 instead of a
C−O linkage, and with lactone ring modification, were
prepared as shown in Scheme 1. Accordingly, compound 1
was treated with Me3SiCN in the presence of InCl3 and
Me3SiBr in CH2Cl2 at reflux,23 providing compound 3 in 64%
yield. Configuration inversion at C-7 was observed for 3, as
indicated by the small coupling constant of H-7 (J = 5.5 Hz).
Exposure of 3 to a dioxane/aqueous 2 N HCl mixture at 70 °C
for 6 h afforded 4. The HMBC experiment indicated the
presence of a new lactone ring by correlations of the
carboxylate carbon C-7a (δ 175.2) to H-7 (δ 4.39) and CH2-
9 (δ 4.15 and 4.53). The structure of 4 was then confirmed by
X-ray diffraction (Figure 3), in which the configuration
inversion at C-7 was clearly observed. Due to the highest
anomalous contribution from oxygen atoms,24 the absolute
configuration of 7S,8S,7′R,8′R was assigned for 4, which was in
agreement with the CD spectrum analysis of 1. The formation
of the new lactone ring in 4 could be explained by the Pinner
reaction followed by hydrolysis.25 Compound 4 was converted
into the acid chloride with oxalyl chloride and then treated with
different amines in CH2Cl2 at room temperature to afford
amides 5a−h. These compounds were found to be more stable
in alkaline solutions, such as pyridine, than cleistantoxin (1). It
is important to note that cleistantoxin (1) is easily epimerized
at C-8′ in pyridine or DBU at room temperature.
Figure 2. Key HMBC correlations for 2.
The relative configuration of 2 was also established from
proton coupling constant analysis and a NOESY experiment.
H-7′ (δ 4.24) was strongly correlated with H-8′ (δ 2.94) and
CH2-9 (δ 3.62 and 3.71) in the NOESY spectrum, which
indicated that H-7′, H-8′, and CH2-9 were on the same face of
the C-ring. H-7′ and CH2-9 were assigned as being pseudoaxial.
The positive Cotton effects at 293 nm (Δε +6.2) in the CD
spectrum of 2 indicated the R-configuration for C-7′.19−21 The
absolute configuration of 2 was thus identical to that of 1.
Compound 2 was identified as (7R,8R,7′R,8′R)-9-hydroxy-6-
methoxy-3′,4′:4,5-bis(methylenedioxy)-2,7′-cyclolignan-9′,7-
olide and was named neocleistantoxin.
Lignans 1 and 2 were evaluated for their cytotoxicity.
Cleistantoxin (1) had strong activity against KB cells with an
IC50 value of 0.022 μM. Compound 2 had moderate
cytotoxicity (IC50: 1.4 μM). Cleistantoxin (1) also had
significant activity against MCF-7 (IC50 0.036 μM), MCF-7R
(IC50 0.014 μM), and HT29 (IC50 0.035 μM) cancer cell lines.
Compounds 4 and 5a−h were inactive against KB cells.
All of the derivatives (5a−h) were tested against KB cells.
Loss of cytotoxicity was observed for all of these synthetic
derivatives in comparison with cleistantoxin (1). The most
active derivative was 3 (IC50 1.2 μg/mL), followed by 5d (IC50
26 μg/mL) and 5h (IC50 42 μg/mL). The other compounds
were noncytotoxic at a concentration of 100 μg/mL. However,
this is the first synthesis of aryl-tetralin lignan derivatives with a
lactone ring between C-9 and the carboxylate carbon attached
a
Scheme 1
a
(I) Me3SiCN, InCl3, Me3SiBr, CH2Cl2, reflux, 5 h, 64%; (II) 2 N HCl, dioxane, 70 °C, 6 h, 70%; (III) 1. (COCl)2, CHCl3, reflux, 3 h; 2. amines,
Et3N, CH2Cl2, 25 °C, 40 −75% for two steps.
C
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Neocleistantoxin (2): white powder; mp 225−226 °C; [α]30
D
−44.4 (c 0.225, CHCl3); IR (KBr): IR νmax 3447, 2923, 1753, 1621,
1483, 1377, 1242, 1084, 1040 cm−1; UV (CHCl3) λmax nm (log ε)
212.3 (3.40), 240.0 (4.23), 286.7 (3.82); NMR data see Table 1;
HRESIMS (positive mode) m/z 421.0897 [M + Na]+ (calcd 421.0899
for C21H18NaO8).
Compound 3. To a solution of 1 (1.0 g, 2.51 mmol) in dry CH2Cl2
(15 mL) was added InCl3 (157 mg, 0.71 mmol), Me3SiCN (0.2 mL,
2.51 mmol), and Me3SiBr (0.38 g, 2.51 mmol). The mixture was
heated at reflux for 5 h and then cooled to room temperature. Water
(40 mL) was added, and the resulting mixture was extracted with
CH2Cl2 (3 × 40 mL). The CH2Cl2 extracts were combined and dried
over MgSO4, and the solvent was removed under reduced pressure.
The residue was purified by CC on silica gel, eluted with a mixture of
CH2Cl2/MeOH (99.5/0.5) to afford 3 (650 mg, 64%): white powder;
mp 173−174 °C; [α]30D −7.5 (c 0.5, CHCl3); 1H NMR (500.13 MHz,
CDCl3) δ 6.67 (1H, d, J = 8.5 Hz, H-5′), 6.53 (1H, d, J = 1.5 Hz, H-
2′), 6.52 (1H, dd, J = 1.5 and 8.5 Hz, H-6′), 6.25 (1H, s, H-3), 5.95
(2H, br s, CH2-10), 5.90 (1H, d, J = 1.5, Hb-10′), 5.89 (1H, d, J = 1.5
Hz, Ha-10′), 4.60 (1H, d, J = 5.2 Hz, H-7′), 4.47 (1H, dd, J = 7.5 and
9.0 Hz, Hb-9), 4.37 (1H, d, J = 5.5 Hz, H-7), 4.34 (1H, dd, J = 9.0 and
10.5 Hz, Ha-9), 4.17 (3H, s, OMe-6), 3.03 (1H, dd, J = 5.2 and 14.0
Hz, H-8′), 2.89 (1H, m, H-8); 13C NMR (125.76 MHz, CDCl3) δ
172.8 (C, C-9′), 150.5 (C, C-4), 147.5 (C, C-3′), 146.9 (C, C-4′),
140.5 (C, C-6), 135.1 (C, C-5), 133.0 (C, C-1′), 132.7 (C, C-2), 124.1
(CH, C-6′), 117.6 (C, C-1), 115.3 (C, C-7a), 110.9 (CH, C-2′), 107.8
(CH, C-5′), 104.7 (CH, C-3), 101.6 (CH2, C-10), 101.1 (CH2, C-
10′), 68.8 (CH2, C-9), 59.7 (CH3, OMe-6), 43.4 (CH, C-8′), 42.9
(CH, C-7′), 32.8 (CH, C-8), 28.7 (CH, C-7); HRESIMS (positive
mode) m/z 408.1085 [M + H]+ (calcd 408.1083 for C22H18NO7).
Compound 4. To a solution of 3 (600 mg, 1.47 mmol) in dioxane
(10 mL) was added 2 N HCl (10 mL). The mixture was heated at 70
°C for 6 h and cooled to room temperature. EtOAc (100 mL) was
added, and the aqueous layer was removed. The EtOAc solution was
washed with 5% NaHCO3 (2 × 20 mL), then with water (50 mL), and
dried over MgSO4. The solvent was removed under reduced pressure,
and the residue was chromatographed on a silica gel column (5−20%
MeOH in CH2Cl2) to give 4 (440 mg, 70%): white powder; mp 234−
235 °C; [α]30D −10.6 (c 1.8, CHCl3); IR νmax 3468, 2930, 1778, 1629,
1481, 1388, 1242, 1115, 1048 cm−1; UV (CHCl3) λmax nm (log ε)
207.5 (3.21), 229.7 (4.04), 287.7 (3.61); 1H NMR (500.13 MHz,
CD3COCD3) δ 6.67 (1H, d, J = 8.0 Hz, H-5′), 6.53 (1H, d, J = 2.0 and
8.0 Hz, H-6′), 6.46 (1H, d, J = 2.0 Hz, H-2′), 6.26 (1H, s, H-3), 5.98
(1H, d, J = 1.0 Hz, Hb-10), 5.97 (1H, d, J = 1.0 Hz, Ha-10), 5.92 (1H,
d, J = 1.0 Hz, Hb-10′), 5.90 (1H, d, J = 1.0 Hz, Ha-10′), 4.53 (1H, dd, J
= 5.5 and 9.5 Hz, Hb-9), 4.52 (1H, d, J = 5.5 Hz, H-7′), 4.39 (1H, d, J
= 7.0 Hz, H-7), 4.15 (1H, d, J = 9.5 Hz, Ha-9), 4.06 (3H, s, OMe-6),
3.08 (1H, ddd, J = 5.5, 7.0, and 13.5 Hz, H-8), 2.95 (1H, dd, J = 5.5
and 13.5 Hz, H-8′); 13C NMR (125.76 MHz, CDCl3): δ 175.2 (C, C-
7a), 172.9 (C, C-9′), 149.9 (C, C-4), 148.3 (C, C-3′), 147.4 (C, C-4′),
142.7 (C, C-6), 136.6 (C, C-5), 135.9 (C, C-1′), 132.7 (C, C-2), 123.3
(CH, C-6′), 116.7 (C, C-1), 110.2 (CH, C-2′), 108.4 (CH, C-5′),
104.1 (CH, C-3), 102.1 (CH2, C-10), 101.9 (CH2, C-10′), 70.5 (CH2,
C-9), 59.9 (CH3, OMe-6), 46.9 (CH, C-7′), 45.8 (CH, C-8′), 39.9
(CH, C-7), 33.0 (CH, C-8); HRESIMS (positive mode) m/z
427.1025 [M + H]+ (calcd 427.1029 for C22H19O9).
Figure 3. X-ray crystal structure with 30% probability ellipsoids for 4.
to C-7. This new class of lignan-like compounds should be
subjected to further biological screening.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Melting points were
recorded on a Buchi B-545 instrument and are uncorrected. Optical
rotations were recorded on a Polax-2 L polarimeter in CHCl3. UV
spectra were recorded on an UV-1601 spectrometer. CD spectra were
measured on a JASCO J-810 spectrophotometer. IR spectra were
measured on a Nicolet Impact-410 FT-IR spectrometer. NMR spectra
were recorded on a Bruker AM500 FT-NMR spectrometer operating
at 500.13 and 125.76 MHz for 1H NMR and 13C NMR spectra,
respectively. 1H chemical shifts were referenced to CDCl3 at 7.27 ppm,
and 13C chemical shifts were referenced to the central peak of CDCl3
at 77.0 ppm. The HMBC measurements were optimized to 7.0 Hz
long-range couplings, and NOESY experiments were run with a 150
ms mixing time. High-resolution ESIMS were measured on a Varian
910 spectrometer.
Plant Material. Fruit of C. indochinensis was collected in Quy
Chau, Nghe An, Vietnam, on May 6, 2003. The plant material was
identified by Dr. Nguyen Quoc Binh, and a voucher specimen (VN-
1086) has been deposited at Herbarium of Institute of Ecology and
Biological Resources of the Vietnam Academy of Science and
Technology, Hanoi, Vietnam.
Extraction and Isolation. Dried and ground fruits (0.5 kg) of C.
indochinensis were extracted with CH2Cl2 at room temperature (3 L ×
5 times) for one day each time. The CH2Cl2 solution was concentrated
under reduced pressure to dryness, and the residue (28.5 g) was
chromatographed on a silica gel column, eluted with mixtures of
CH2Cl2/MeOH (0 to 100% CH2Cl2 in MeOH), to give 15 fractions,
which were tested against KB cells. Two active fractions (7 and 8)
were combined (25.1 g) and subjected to CC on silica gel, using a
stepwise gradient of acetone in CH2Cl2, to give 1 (2.1 g) and 2 (250
mg).
General Procedure for Synthesis of 5a−h. Compound 4 (30
mg, 0.07 mmol) was dissolved in CHCl3 (1 mL), and oxalyl chloride
(0.5 mL) was added dropwise. The resulting mixture was heated at 60
°C for 4 h and then concentrated under vacuum to dryness. The
residue was dissolved in 1 mL of CH2Cl2. To this solution were added
amines (1.5 equiv) and Et3N (3 equiv). The mixture was stirred at
room temperature for 12 h and then concentrated under reduced
pressure. The residues were purified by CC on silica gel using a
stepwise gradient of MeOH in CH2Cl2, to give amides 5a−h.
Cleistantoxin (1): white, microcrystalline solid; mp 195−196 °C
Compound 5a: 40% yield; white powder; mp 198−199 °C; [α]30
(acetone); [α]30 −148.0 (c 0.5, CHCl3); IR (KBr) νmax 3565, 3466,
D
D
2931, 1773, 1617, 1483, 1297, 1230, 1142, 1047 cm−1; UV (CHCl3)
λmax nm (log ε) 207.3 (3.39), 240.0 (4.18), 286.7 (3.83); NMR data
see Table 1; HRESIMS (positive mode) m/z 421.0888 [M + Na]+
(calcd 421.0899 for C21H18NaO8).
−147.3 (c 1.2, CHCl3); IR νmax 3461, 2930, 1773, 1633, 1479, 1248,
1120 cm−1; UV (CHCl3) λmax nm (log ε) 210.5 (2.52), 233.5 (4.15),
1
288.1 (3.78); H NMR (500.13 MHz, CDCl3) δ 7.43 (1H, br d, J =
2.0 Hz, H-5″), 6.57 (1H, d, J = 8.0 Hz, H-5′), 6.37 (1H, dd, J = 2.0 and
D
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3.5 Hz, H-4″), 6.25 (1H, br d, J = 3.5 Hz, H-3″), 6.23 (1H, dd, J = 1.5
and 8.0 Hz, H-6′), 6.20 (1H, d, J = 1.5 Hz, H-2′), 6.12 (1H, s, H-3),
5.90 (1H, d, J = 1.5 Hz, Hb-10), 5.89 (1H, d, J = 1.5 Hz, Ha-10), 5.88
(1H, d, J = 1.5 Hz, Hb-10′), 5.87 (1H, d, J = 1.5 Hz, Ha-10′), 4.50,
(1H, dd, J = 4.5 and 15.0 Hz, Hb-6″), 4.41 (1H, dd, J = 4.7 and 10.0
Hz, Hb-9), 4.27 (1H, dd, J = 4.5 and 15.0 Hz, Ha-6″), 4.22 (1H, d, J =
7.0 Hz, H-7), 4.19 (1H, d, J = 5.2 Hz, H-7′), 4.11 (3H, s, OMe-6),
4.08 (1H, d, J = 10.0 Hz, Ha-9), 3.10 (1H, ddd, J = 4.7, 7.0, and 12.5
Hz, H-8), 2.78 (1H, dd, J = 5.2 and 12.5 Hz, H-8′); 13C NMR (125
MHz, CDCl3) δ 175.3 (C, C-7a), 170.5 (C, C-9′), 151.0 (C, C-2″),
149.2 (C, C-4), 147.4 (C, C-3′), 146.5 (C, C-4′), 142.3 (CH, C-5″),
141.5 (C, C-6), 135.6 (C, C-5), 134.1 (C, C-1′), 131.4 (C, C-2), 122.1
(CH, C-6′), 114.9 (C, C-1), 110.6 (CH, C-4″), 109.2 (CH, C-2′),
108.0 (CH, C-3″), 107.9 (CH, C-5′), 103.4 (CH, C-3), 101.1 (CH2,
C-10′), 101.0 (CH2, C-10), 70.3 (CH2, C-9), 59.8 (CH3, OMe-6),
47.6 (CH, C-7′), 46.3 (CH, C-8′), 39.6 (CH, C-7), 36.3 (CH2, C-6″),
32.3 (CH, C-8); HRESIMS (positive mode) m/z 506.1490 [M + H]+
(calcd 506.1451 for C27H24NO9).
1.63 (2H, m, Hb-3″ and Hb-6″), 1.62 (2H, m, Hb-4″ and Hb-5″), 1.50
(2H, m, Ha-4″ and Ha-5″), 1.49 (2H, m, Ha-3″ and Ha-6″), 1.47 (1H,
m, Ha-2″), 1.40 (1H, m, Ha-7″); 13C NMR (125 MHz, CDCl3) δ
175.4 (C, C-7a), 169.3 (C, C-9′), 149.2 (C, C-4), 147.4 (C, C-3′),
146.6 (C, C-4′), 141.5 (C, C-6), 135.5 (C, C-5), 134.4 (C, C-1′),
131.7 (C, C-2), 122.2 (CH, C-6′), 114.9 (C, C-1), 109.3 (CH, C-2′),
107.8 (CH, C-5′), 103.4 (CH, C-3), 101.1 (CH2, C-10), 101.0 (CH2,
C-10′), 70.4 (CH2, C-9), 59.8 (CH3, OMe-6), 50.6 (CH, C-1″), 47.7
(CH, C-7′), 46.4 (CH, C-8′), 39.6 (CH, C-7), 32.4 (CH, C-8), 35.1
(CH2, C-7″), 35.0 (CH2, C-2″), 24.1 (CH2, C-6″), 24.0 (CH2, C-3″),
28.2 (CH2, C-5″), 28.1 (CH2, C-4″); HRESIMS (positive mode) m/z
522.2125 [M + H]+ (calcd 522.2128 for C29H32NO8).
Compound 5e: 40% yield; yellow powder; mp 91−92 °C; [α]30
D
−214.9 (c 1.5, CHCl3); IR νmax 3434, 2897, 1780, 1640, 1479, 1385,
1233, 1113, 1043 cm−1; UV (CHCl3) λmax nm (log ε) 207.5 (3.06),
1
235.2 (4.15), 288.1 (3.78); H NMR (500.13 MHz, CDCl3) δ 7.39
(4H, m, H-3″, H-5″, H-3‴ and H-5‴), 7.36 (1H, m, H-4‴), 7.35 (1H,
m, H-4″), 7.27 (2H, br d, J = 7.5 Hz, H-2″ and H-6″), 7.16 (2H, br d, J
= 7.5 Hz, H-2‴ and H-6‴), 6.57 (1H, d, J = 8.0 Hz, H-5′), 6.20 (1H,
d, J = 1.5 Hz, H-2′), 6.15 (1H, dd, J = 1.5 and 8.0 Hz, H-6′), 6.05 (1H,
s, H-3), 5.93 (1H, d, J = 1.5 Hz, Hb-10), 5.92 (1H, d, J = 1.5 Hz, Ha-
10), 5.88 (1H, d, J = 1.5 Hz, Hb-10′), 5.86 (1H, d, J = 1.5 Hz, Ha-10′),
5.20 (1H, d, J = 14.0 Hz, Hb-7″), 4.87 (1H, d, J = 18.5 Hz, Hb-7‴),
4.47 (1H, dd, J = 4.7 and 10.0 Hz, Hb-9), 4.43 (1H, d, J = 18.5 Hz, Ha-
7‴), 4.24 (1H, d, J = 7.0 Hz, H-7), 4.11 (1H, d, J = 10.0 Hz, Ha-9),
4.09 (3H, s, OMe-6), 4.07 (1H, d, J = 4.7 Hz, H-7′), 3.84 (1H, d, J =
14.0 Hz, Ha-7″), 3.31 (1H, ddd, J = 4.7, 7.0, and 12.0 Hz, H-8), 3.21
(1H, dd, J = 4.7 and 12.0 Hz, H-8′); 13C NMR (125.76 MHz, CDCl3)
δ 174.9 (C, C-7a), 170.9 (C, C-9′), 149.2 (C, C-4), 147.5 (C, C-3′),
146.6 (C, C-4′), 141.7 (C, C-6), 136.9 (C, C-1″), 135.7 (C, C-5 and
C-1‴), 133.9 (C, C-1′), 131.4 (C, C-2), 129.7 (CH, C-2″ and C-6″),
129.3 (CH, C-3″ and C-5″), 128.7 (CH, C-3‴ and C-5‴), 128.2 (CH,
C-4‴), 127.8 (CH, C-4″), 126.6 (CH, C-2‴ and C-6‴), 122.6 (CH,
C-6′), 114.9 (C, C-1), 109.6 (CH, C-2′), 107.8 (CH, C-5′), 103.4
(CH, C-3), 101.1 (CH2, C-10 and C-10′), 70.4 (CH2, C-9), 59.9
(CH3, OMe-6), 49.7 (CH2, C-7‴), 48.3 (CH2, C-7″), 46.6 (CH, C-
7′), 43.0 (CH, C-8′), 39.8 (CH, C-7), 33.7 (CH, C-8); HRESIMS
(positive mode) m/z 606.2129 [M + H]+ (calcd 606.2128 for
C36H32NO8).
Compound 5b: 60% yield; yellow powder, mp 244−245 °C; [α]30
D
−139.0 (c 1.1, CHCl3); IR νmax 3480, 2926, 1778, 1736, 1628, 1472,
1238, 1102, 1049 cm−1; UV (CHCl3) λmax nm (log ε) 239.0 (4.19),
278.3 (3.81); 1H NMR (500.13 MHz, CDCl3) δ 8.24 (1H, br s, H-2″),
7.50 (1H, br s, H-4″), 7.23 (1H, br s, H-5″), 6.57 (1H, d, J = 8.0 Hz,
H-5′), 6.13 (1H, s, H-3), 6.01 (1H, d, J = 1.5 Hz, H-2′), 5.93 (1H, dd,
J = 1.5 and 8.0 Hz, H-6′), 5.92 (2H, br s, CH2-10), 5.89 (2H, br s,
CH2-10′), 4.51 (1H, dd, J = 5.0 and 10.5 Hz, Hb-9), 4.41 (1H, d, J =
5.0 Hz, H-7′), 4.34 (1H, d, J = 7.0 Hz, H-7), 4.14 (3H, s, OMe-6),
4.05 (1H, d, J = 10.5 Hz, Ha-9), 3.59 (1H, dd, J = 5.0 and 12.0 Hz, H-
8′), 3.32 (1H, ddd, J = 5.0, 7.0, and 12.0 Hz, H-8); 13C NMR (125.76
MHz, CDCl3) δ 174.2 (C, C-7a), 168.7 (C, C-9′), 149.6 (C, C-4),
147.9 (C, C-3′), 147.2 (C, C-4′), 141.6 (C, C-6), 136.2 (CH, C-2″),
135.9 (C, C-5), 132.7 (C, C-1′), 132.0 (CH, C-5″), 129.9 (C, C-2),
121.6 (CH, C-6′), 115.9 (CH, C-4″), 114.4 (C, C-1), 108.7 (CH, C-
2′), 108.2 (CH, C-5′), 103.3 (CH, C-3), 101.3 (CH2, C-10′), 101.2
(CH2, C-10), 69.8 (CH2, C-9), 59.9 (CH3, OMe-6), 47.4 (CH, C-7′),
46.2 (CH, C-8′), 39.3 (CH, C-7), 32.2 (CH, C-8); HRESIMS
(positive mode) m/z 477.1297 [M + H]+ (calcd 477.1298 for
C25H21N2O8).
Compound 5c: 47% yield; yellow powder; mp 186−187 °C; [α]30
D
−97.9 (c 0.62, CHCl3); IR νmax 3454, 2965, 1774, 1643, 1480, 1249,
1156, 1048 cm−1; UV (CHCl3) λmax nm (log ε) 207.9 (3.01), 231.8
(4.17), 288.3 (3.75); 1H NMR (500 MHz, CDCl3) δ 6.64 (1H, d, J =
8.0 Hz, H-5′), 6.35 (1H, br d, J = 8.0 Hz, H-6′), 6.26 (1H, br s, H-2′),
6.11 (1H, s, H-3), 5.90 (2H, br s, CH2-10), 5.88 (2H, br s, CH2-10′),
4.41 (1H, dd, J = 5.2 and 10.0 Hz, Hb-9), 4.21 (1H, d, J = 7.0 Hz, H-
7), 4.16 (1H, d, J = 5.2 Hz, H-7′), 4.12 (1H, m, H-1″), 4.11 (3H, s,
OMe-6), 4.08 (1H, d, J = 10.0 Hz, Ha-9), 3.08 (1H, ddd, J = 5.2, 7.0,
and 12.5 Hz, H-8), 2.68 (1H, dd, J = 5.2 and 12.5 Hz, H-8′), 1.98 (2H,
m, Hb-2″ and Hb-5″), 1.71 (2H, m, Hb-3″ and Hb-4″), 1.62 (2H, m,
Ha-3″ and Ha-4″), 1.45 (1H, m, Ha-2″) 1.34 (1H, m, Ha-5″); 13C
NMR (125 MHz, CDCl3) δ 175.4 (C, C-7a), 170.0 (C, C-9′), 149.2
(C, C-4), 147.5 (C, C-3′), 146.6 (C, C-4′), 141.5 (C, C-6), 135.6 (C,
C-5), 134.3 (C, C-1′), 131.5 (C, C-2), 122.1 (CH, C-6′), 114.9 (C, C-
1), 109.3 (CH, C-2′), 107.8 (CH, C-5′), 103.3 (CH, C-3), 101.1
(CH2, C-10), 101.0 (CH2, C-10′), 70.4 (CH2, C-9), 59.8 (CH3, OMe-
6), 51.3 (CH, C-1″), 47.7 (CH, C-7′), 46.4 (CH, C-8′), 39.6 (CH, C-
7), 32.4 (CH, C-8), 33.0 (CH2, C-2″ and C-5″), 23.7 (CH2, C-3″ and
C-4″); HRESIMS (positive mode) m/z 494.1817 [M + H]+ (calcd
494.1815 for C27H28NO8).
Compound 5f: 55% yield; yellow powder; mp 150−151 °C; [α]30
D
−126.6 (c 0.96, CHCl3); IR νmax 3434, 2904, 1779, 1638, 1479, 1384,
1231, 1156, 1045 cm−1; UV (CHCl3) λmax nm (log ε) 232.6 (4.18),
288.1 (3.90); 1H NMR (500.13 MHz, CDCl3) δ 7.24 (2H, dd, J = 6.0
and 8.0 Hz, H-2″ and H-6″), 7.04 (2H, t, J = 8.5 Hz, H-3″ and H-5″),
6.55 (1H, d, J = 8.0 Hz, H-5′), 6.19 (1H, br d, J = 8.0 Hz, H-6′), 6.15
(1H, s, H-2′), 6.04 (1H, s, H-3), 5.88 (2H, m, CH2-10′), 5.87 (2H, m,
CH2-10), 4.39 (1H, m, Hb-9), 4.38 (1H, m, Hb-7″), 4.25 (1H, dd, J =
5.5 and 14.5 Hz, Ha-7′), 4.18 (1H, d, J = 7.0 Hz, H-7), 4.11 (1H, d, J =
5.5 Hz, H-7′), 4.08 (3H, s, OMe-6), 4.05 (1H, d, J = 10.0 Hz, Ha-9),
3.07 (1H, ddd, J = 5.5, 7.0, and 12.5 Hz, H-8), 2.70 (1H, dd, J = 5.5
and 12.5 Hz, H-8′); 13C NMR (125.76 MHz, CDCl3) δ 175.5 (C, C-
7a), 170.6 (C, C-9′), 161.3−163.2 (C, C-4″), 149.2 (C, C-4), 147.5
(C, C-3′), 146.6 (C, C-4′), 141.7 (C, C-6), 135.5 (C, C-5), 134.1 (C,
C-1′), 133.8 (C, C-1″), 131.4 (C, C-2), 130.1 (CH, C-2″ and C-6″),
122.1 (CH, C-6′), 115.5−115.6 (CH, C-3″ and C-5″), 114.7 (C, C-1),
109.2 (CH, C-2′), 107.9 (CH, C-5′), 103.3 (CH, C-3), 101.1 (CH2,
C-10), 101.0 (CH2, C-10′), 70.4 (CH2, C-9), 59.8 (CH3, OMe-6),
47.5 (CH, C-7′), 46.3 (CH, C-8′), 43.0 (CH2, C-7″), 39.6 (CH, C-7),
32.3 (CH, C-8); HRESIMS (positive mode) m/z 534.1564 [M + H]+
(calcd 534.1564 for C29H25FNO8).
Compound 5d: 75% yield; yellow powder; mp 147−148 °C; [α]30
D
−128.5 (c 1.6, CHCl3); IR νmax 3426, 2930, 1777, 1647, 1481, 1390,
1252, 1049 cm−1; UV (CHCl3) λmax nm (log ε) 245.9 (4.31), 288.5
(4.37); 1H NMR (500 MHz, CDCl3) δ 6.63 (1H, d, J = 8.0 Hz, H-5′),
6.35 (1H, dd, J = 1.5 and 8.0 Hz, H-6′), 6.26 (1H, d, J = 1.5 Hz, H-2′),
6.09 (1H, s, H-3), 5.90 (1H, d, J = 1.5 Hz, Hb-10), 5.89 (1H, d, J = 1.5
Hz, Ha-10), 5.87 (2H, brs, CH2-10′), 4.38 (1H, dd, J = 5.0 and 9.5 Hz,
Hb-9), 4.19 1H, (d, J = 7.0 Hz, H-7), 4.14 (1H, d, J = 5.2 Hz, H-7′),
4.11 (3H, s, OMe-6), 4.06 (1H, d, J = 9.5 Hz, Ha-9), 3.86 (1H, m, H-
1″), 3.08 (1H, ddd, J = 5.0, 7.0, and 12.5 Hz, H-8), 2.65 (1H, dd, J =
5.2 and 12.5 Hz, H-8′), 1.92 (1H, m, Hb-2″), 1.84 (1H, m, Hb-7″),
Compound 5g: 75% yield; yellow powder; mp 194−195 °C; [α]30
D
−140.1 (c 1.7, CHCl3); IR νmax 3427, 2932, 1774, 1642, 1477, 1388,
1248, 1123, 1046 cm−1; UV (CHCl3) λmax nm (log ε) 207.9 (2.86),
1
233.1 (4.39), 286.8 (3.90); H NMR (500.13 MHz, CDCl3) δ 7.40
(1H, d, J = 8.5 Hz, H-5″), 7.38 (1H, d, J = 1.5 Hz, H-2″), 7.10 (1H,
dd, J = 1.5 and 8.5 Hz, H-6″), 6.55 (1H, d, J = 8.0 Hz, H-5′), 6.17 (1H,
br d, J = 8.0 Hz, H-6′), 5.99 (1H, s, H-3), 6.10 (1H, br s, H-2′), 5.88
(2H, m, CH2-10), 5.87 (2H, m, CH2-10′), 4.36 (1H, dd, J = 5.0 and
10.0 Hz, Hb-9), 4.30 (1H, dd, J = 5.5 and 14.5 Hz, Hb-7″), 4.20 (1H,
dd, J = 6.0 and 14.5 Hz, Ha-7″), 4.19 (1H, d, J = 7.0 Hz, H-7), 4.08
E
dx.doi.org/10.1021/np3003832 | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(1H, d, J = 5.7 Hz, H-7′), 4.07 (3H, s, OMe-6), 4.02 (1H, d, J = 10.0
Hz, Ha-9), 3.07 (1H, ddd, J = 5.0, 7.0, and 13.0 Hz, H-8), 2.63 (1H,
dd, J = 5.7 and 13.0 Hz, H-8′); 13C NMR (125.76 MHz, CDCl3) δ
176.0 (C, C-7a), 170.9 (C, C-9′), 149.2 (C, C-4), 147.4 (C, C-3′),
146.6 (C, C-4′), 141.4 (C, C-6), 138.4 (C, C-1″), 135.4 (C, C-5),
134.2 (C, C-1′), 132.5 (C, C-3″), 131.5 (C, C-2 and C-4″), 130.5
(CH, C-5″), 130.3 (CH, C-2″), 127.8 (CH, C-6″), 122.0 (CH, C-6′),
114.5 (C, C-1), 109.1 (CH, C-2′), 107.9 (CH, C-5′), 103.3 (CH, C-
3), 101.2 (CH2, C-10), 101.1 (CH2, C-10′), 70.6 (CH2, C-9), 59.8
(CH3, OMe-6), 47.3 (CH, C-7′), 46.1 (CH, C-8′), 39.6 (CH, C-7),
32.3 (CH, C-8), 27.3 (CH2, C-7″); HRESIMS (positive mode) m/z
584.0879 [M + H]+ (calcd 584.0879 for C29H24Cl2NO8).
AUTHOR INFORMATION
Corresponding Author
*Tel: 84 (0)4 37564995. Fax: 84 (0)4 37917054. E-mail:
phamvc@imbc.vast.vn (V.C.P.); cvminh@vast.ac.vn (V.M.C.).
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank Mr. D. D. Cuong and Mr. N. Q. Binh
(VAST, Vietnam) for plant collection and botanical determi-
nation. The Centre National de la Recherche Scientifique
(CNRS, France) is gratefully acknowledged for the Franco-
Vietnamese Cooperation Program and The Vietnam National
Foundation for Science and Technology Development
(NAFOSTED) for financial support (grant no. 104.01.75.09).
Compound 5h: 55% yield; yellow powder; mp 233−234 °C; [α]30
D
−147.4 (c 1.4, CHCl3); IR νmax 3444, 2946, 1778, 1640, 1477, 1387,
1245, 1043 cm−1; UV (CHCl3) λmax nm (log ε) 207.9 (2.64), 238.6
(4.18), 288.1 (3.90); 1H NMR (500.13 MHz, CDCl3) δ 6.63 (1H, d, J
= 8.0 Hz, H-5′), 6.28 (1H, dd, J = 1.5 and 8.0 Hz, H-6′), 6.22 (1H, d, J
= 1.5 Hz, H-2′), 6.16 (1H, s, H-3), 5.91 1H, (d, J = 1.0 Hz, Ha-10),
5.90 (1H, d, J = 1.0 Hz, Hb-10), 5.89 1H, (d, J = 1.5 Hz, Ha-10′), 5.88
(1H, d, J = 1.5 Hz, Hb-10′), 4.43 (1H, dd, J = 5.0 and 10.0 Hz, Hb-9),
4.23 (1H, d, J = 7.0 Hz, H-7), 4.12 (3H, s, OMe-6), 4.11 (1H, d, J =
5.0 Hz, H-7′), 4.02 (1H, d, J = 10.0 Hz, Ha-9), 3.61 (1H, m, Hb-2″),
3.59 (2H, m, CH2-6′), 3.44 (1H, ddd, J = 4.0, 7.5, and 13.5 Hz, Ha-2′),
3.20 (1H, ddd, J = 5.0, 7.0, and 12.0 Hz, H-8), 3.12 (1H, dd, J = 5.0
and 12.0 Hz, H-8′), 1.72 (1H, m, Hb-5″), 1.71 (2H, m, CH2-4″), 1.64
(1H, m, Ha-5″), 1.61 (2H, m, CH2-3″); 13C NMR (125.76 MHz,
CDCl3) δ 175.3 (C, C-7a), 168.7 (C, C-9′), 149.2 (C, C-4), 147.4 (C,
C-3′), 146.5 (C, C-4′), 141.6 (C, C-6), 135.6 (C, C-5), 134.2 (C, C-
1′), 131.3 (C, C-2), 122.0 (CH, C-6′), 115.2 (C, C-1), 109.2 (CH, C-
2′), 107.8 (CH, C-5′), 103.4 (CH, C-3), 101.1 (CH2, C-10), 101.0
(CH2, C-10′), 70.8 (CH2, C-9), 59.8 (CH3, OMe-6), 46.7 (CH2, C-
6″), 46.1 (CH, C-7′), 42.9 (CH2, C-2″), 42.4 (CH, C-8′), 39.7 (CH,
C-7), 33.0 (CH, C-8), 26.9 (CH2, C-5″), 25.6 (CH2, C-3″), 24.5
(CH2, C-4″); HRESIMS (positive mode) m/z 494.1814 [M + H]+
(calcd 494.1815 for C27H28NO8).
X-ray Crystallographic Analysis of Compound 4. X-ray
crystallographic data were collected at room temperature (293(2)
K) on a Rigaku diffractometer (for additional details see Supporting
Information). Crystallographic data have been deposited with the
Cambridge Crystallographic Data Centre (deposit no. CCDC
883776). 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)223-336033 or e-mail: deposit@ccdc.cam.ac.
uk).
Cytotoxic Activity Assay. The human KB tumor (oral
epidermoid carcinoma) cell line was obtained originally from ATCC
(Manassas, VA, USA). KB cells were maintained in Dulbecco’s D-
MEM medium, supplemented with 10% fetal calf serum, L-glutamine
(2 mM), penicillin G (100 UI/mL), streptomycin (100 μg/mL), and
gentamicin (10 μg/mL). Stock solutions of compounds were prepared
in DMSO/H2O (1:9), and the cytotoxicity assays were carried out in
96-well microtiter plates against human nasopharynx carcinoma KB
cells (3 × 103 cells/mL) using a modification of the published
method.26 After 72 h incubation at 37 °C in air/CO2 (95:5) with or
without test compounds, cell growth was estimated by colorimetric
measurement of stained living cells by neutral red. Optical density was
determined at 540 nm with a Titertek Multiscan photometer. The IC50
value was defined as the concentration of sample necessary to inhibit
the cell growth to 50% of the control. Taxotere was used as a reference
compound.
REFERENCES
■
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S
* Supporting Information
NMR spectra of 1−4 and 5a−h; crystallographic information
file (CIF) for 4. This material is available free of charge via the
Internet at http://pubs.acs.org.
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