Diarylheptanoid glucosides from Pyrostria major and their antiprotozoal activities

Article abstract of DOI:10.1002/ejoc.201101414

Eight new diarylheptanoid glucosides 1-8 have been isolated from the leaves of Pyrostria major (A. Rich & DC.) Cavaco [syn. Canthium major (A. Rich & DC.)] Cavaco. Their structures were determined by 1D and 2D NMR spectroscopic and HRMS analyses. The abso

Full text of DOI:10.1002/ejoc.201101414

FULL PAPER
DOI: 10.1002/ejoc.201101414
Diarylheptanoid Glucosides from Pyrostria major and Their Antiprotozoal
Activities
Mehdi A. Beniddir,[a] Philippe Grellier,[b] Philippe Rasoanaivo,[c] Philippe M. Loiseau,[d]
Christian Bories,[d] Vincent Dumontet,[a] Françoise Guéritte,[a] and Marc Litaudon*[a]
Keywords: Natural products / Medicinal chemistry / Carbohydrates / Structure elucidation / Configuration determination
Eight new diarylheptanoid glucosides 1–8 have been isolated
from the leaves of Pyrostria major (A. Rich & DC.) Cavaco
[syn. Canthium major (A. Rich & DC.)] Cavaco. Their struc-
tures were determined by 1D and 2D NMR spectroscopic and
HRMS analyses. The absolute configurations of the diaryl-
heptanoids 1–4 were determined to be 3S and 5S by the ap-
plication of the CD exciton chirality method to the corre-
sponding 3,5-bis(p-bromobenzoyl) (1–3) and 3,5-bis(p-di-
methylaminobenzoyl) (4) derivatives. Their antiparasitic ac-
tivities against Plasmodium falciparum, Leishmania dono-
vani (amastigote forms), and Trypanosoma brucei (trypomas-
tigote bloodstream forms) as well as their cytotoxic activities
against the HL-60, KB, and MRC5 cell lines of naturally oc-
curring diarylheptanoid glucosides and their derivatives are
reported.
plant extracts prepared from various species of the Mada-
gascarian biodiversity for their inhibitory activity against
blood stages of the Plasmodium falciparum strain. The re-
sults of this screening led us to investigate chemically the
leaves of Pyrostria major (Rubiaceae). The ethyl acetate ex-
tract yielded, besides β-sitosterol as one of the major
components, eight new diarylheptanoid glucosides 1–8
(Scheme 1), closely related to the previously reported Tacca
chantrieri metabolites.[5] If β-sitosterol displayed moderate
antiplasmodial activity, the diarylheptanoid glucosides were
found to be inactive. However, we discovered by chance that
minor structural modifications of these glucosides led to
active compounds. Moreover, despite the fact that a large
number of Rubiaceae species have been chemically investi-
gated, so far no studies have revealed the presence of diaryl-
heptanoids in this family. Naturally occurring diarylhept-
anoids are common in Zingiberaceae and Betulaceae, and
are encountered in many small botanical families such as
Myricaceae and Casuarinaceae.[6] They possess various bio-
logical activities, such as anti-inflammatory, antioxidant,
and anti-tumoral properties.[7]
Introduction
Neglected tropical diseases are a group of chronic, disa-
bling, and disfiguring pathologies caused by parasitic, bac-
terial, and other infections. Among numerous parasitic dis-
eases, malaria is the most important one in the world. It
represents a major problem for public health services in
tropical and subtropical regions of the planet. About 300
million people contract malaria each year, with 2–3 million
dying annually,[1] mainly due to an increase in parasite resis-
tance to available drugs and the failure to apply existing
effective drugs in areas where they would be of greatest ben-
efit. The introduction of new drugs, notably the artemisi-
nin-based combination therapies or ACTs,[2] could replace
ineffective treatments (chloroquine and sulfadoxine-pyri-
methamine), but remain more expensive than single-agent
treatments.[3] In addition, lately, there have been signs that
the efficacy of ACTs and artesunate monotherapy have de-
creased in western Cambodia.[4] There is therefore an ur-
gent need for the discovery of new efficient and cheap anti-
malarial drugs to cure this disease and to prevent the emer-
gence of resistance. Recently, we screened a series of 40
In this paper we describe the isolation, structure elucida-
tion, and biological activities of the natural diarylheptanoid
glucosides and their derivatives.
[a] Centre de Recherche de Gif, Institut de Chimie des Substances
Naturelles, CNRS, Labex LERMIT,
1, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France
Fax: +33-1-69077247
Results and Discussion
E-mail: marc.litaudon@icsn.cnrs-gif.fr
[b] Muséum National d’Histoire Naturelle, UMR 7245 CNRS,
Team APE,
The leaves of Pyrostria major (375 g) were extracted with
EtOAc to give a dry crude extract (13 g) after removal of
the solvent. This extract exhibited a significant antiplasmo-
dial activity (100% at 10 μgmL–1) with no cytotoxicity
against the MRC-5 cell line. It was subjected to silica gel
chromatography using as eluent a gradient of CH2Cl2/
CP 52, 61, Rue Buffon, 75231 Paris Cedex 05, France
[c] Institut Malgache de Recherches Appliquées,
B. P. 3833, 102 Antananarivo, Madagascar
[d] Groupe Chimiothérapie Antiparasitaire UMR 8076 CNRS,
Labex LERMIT, Faculté de Pharmacie, Université Paris-Sud,
Rue Jean-Baptiste-Clément, 92296 Châtenay Malabry Cedex,
France
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M. Litaudon et al.
FULL PAPER
Compounds 1–8 are closely related to the diarylheptano-
ids isolated from the rhizomes of Tacca chantrieri.[5] Indeed,
they share several common spectral characteristics: The UV
spectra show absorption bands at 223 and 278 nm, consis-
tent with substituted aromatic rings, and the IR spectra ex-
hibit bands arising from hydroxy groups (3350 cm–1) and
aromatic rings (1600 cm–1). The 1H NMR spectra show sig-
nals for substituted aromatic rings, an aliphatic moiety, and
an anomeric proton belonging to a sugar moiety.
Compound 1 was obtained as an amorphous solid.
HRESIMS indicates a [M + Na]+ ion peak at m/z =
529.2406, which suggests a molecular formula of C27H38O9.
1
The H NMR spectrum shows signals for two 1,4-disubsti-
tuted aromatic ring protons [δH = 6.80 (d, J = 8.5 Hz, 2 H),
6.81 (d, J = 8.5 Hz, 2 H), 7.09 (d, J = 8.5 Hz, 2 H), and
7.14 (d, J = 8.5 Hz, 2 H) ppm]. In addition, a signal for two
methoxy groups [δH = 3.74 (s, 2 ϫ3 H, 4Ј-OMe and 4ЈЈ-
OMe) ppm] and a signal due to an anomeric proton [δH
=
4.39 (d, J = 7.8 Hz) ppm] are observed, which indicate the
β configuration. Hydrolysis of 1 gave d-glucose and 1a. d-
Glucose, including its absolute configuration, was identified
by direct supercritical fluid chromatography (SFC) analysis
of the hydrolysate lyophilized against d- and l-glucose as
references. The 13C and DEPT 135 13C NMR spectra reveal
signals for 12 sp2 aromatic carbon atoms and 15 sp3 carbon
atoms, among which there are five methylenes and two hy-
droxymethines for the aliphatic moiety, one hydroxymethyl-
ene and five methines for the sugar moiety, and two meth-
oxy groups, which suggests a diarylheptanoid glucoside
structure. Each structural fragment constituting the C7 unit
of 1 was connected by analysis of the 1H–1H COSY,
HMQC, and HMBC correlations. Finally, HMBC corre-
lations of 2ЈЈ-H and 6ЈЈ-H to C-7, and of 2Ј-H and 6Ј-H to
C-1 allowed the two aromatic rings to be located at the
terminal methylene groups. Furthermore, the linkage posi-
tion of d-glucose at C-3 can be determined by a long-range
correlation from the anomeric proton at δH = 4.29 ppm to
the C-3 carbon at δC = 78.3 ppm. All these connectivities
are similar to those reported by Yokosuka et al.[5]
The absolute configurations of the 3,5-dihydroxy groups
were determined by application of the CD exciton chirality
method[8] to acyclic 1,3-dibenzoates.[9] Compound 1a was
converted into the corresponding 3,5-bis(p-bromobenzoate)
derivative 1b. Its CD spectrum shows positive (252.7 nm,
Δε
= =
+9.3 m–1 cm–1) and negative (235.5 nm, Δε
–9.2 m–1 cm–1) Cotton effects, which is consistent with a
positive chirality. Thus, the absolute configurations at C-3
and C-5 were assigned as S. The structure of 1 was thus
shown to be (3S,5S)-3,5-dihydroxy-1,7-bis(4-methoxy-
Scheme 1. Chemical structures of natural diarylheptanoid gluco-
sides 1–8 and their derivatives 1a, 1b, 2a–2c, 3a, 4a–4c.
MeOH (1:0 to 8:2) of increasing polarity, which led to 16 phenyl)heptyl 3-O-β-d-glucopyranoside.
fractions. Only fraction 5, manly containing β-sitosterol,
Compound 2 was obtained as an amorphous solid. Its
showed a significant antiplasmodial activity (100% at molecular formula was determined to be C27H38O10 on the
1 μgmL–1). All the fractions were then subjected to HPLC basis of the ion peak [M + Na]+ at m/z = 545.2410 in the
1
analysis to assess qualitatively and quantitatively their HRESIMS spectrum. The H NMR spectrum shows aro-
chemical content. Owing to their highly complex composi- matic signals for 1,3,4-trisubstituted and 1,4-disubstituted
tions, we decided to investigate chemically fractions 15 and benzene rings, and signals for two methoxy groups. HMBC
16, and subsequent HPLC preparative purification afforded correlations were observed from the methoxy protons at δH
compounds 1–8 (Figure 1).
= 3.72 ppm to C-4ЈЈ, and at δH = 3.78 ppm to C-4Ј. Acidic
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Diarylheptanoid Glucosides and Their Antiprotozoal Activities
hydrolysis of 2 gave 2a and d-glucose. The location of the served from the anomeric proton signal (δH = 4.39 ppm) to
sugar at C-3 was confirmed by an HMBC correlation of C-3 (δC = 78.4 ppm) of the heptane moiety. Other HMBC
the anomeric proton signal at δH = 4.39 ppm to C-3 at δC correlations indicate that 5 is an isomer of 2. Unfortunately,
= 78.3 ppm. Compound 2a was methylated to form the tri- the small amount of 5 did not allow us to determine its
methylated derivative 2b, which was then converted into the absolute configuration. However, compound 5 is supposed
3,5-bis(p-bromobenzoate) derivative 2c. The CD spectrum to have the same absolute configuration as compounds 1–4
of 2c exhibits similar Cotton effects to 1b. All of these data on the basis of the same negative sign of their specific rota-
are consistent with the structure (3S,5S)-3,5-dihydroxy- tions. Thus, the structure was tentatively assigned as
1-(3-hydroxy-4-methoxyphenyl)-7-(4-methoxyphenyl)heptyl (3S,5S)-3,5-dihydroxy-7-(3-hydroxy-4-methoxyphenyl)-1-
3-O-β-d-glucopyranoside, which was assigned to compound (4-methyoxyphenyl)heptyl 3-O-β-d-glucopyranoside.
2.
The molecular formula C26H36O11 of compound 6 was
Compound 3 was obtained as an amorphous solid. The deduced from the [M + Na]+ ion peak at m/z = 547.2166
HRESIMS spectrum exhibits a [M + Na]+ ion peak at m/z in the HRESIMS spectrum. The 1H NMR spectrum exhib-
= 531.2285, which corresponds to the molecular formula its a signal for an anomeric proton at δH = 4.39 (d, J =
C26H36O10, lower by CH2 than that of 2. The H NMR 7.8 Hz, 1 H) ppm, signals for six aromatic protons, and a
1
spectrum of 3 shows signals for a 1,3,4-trisubstituted aro- signal for a methoxy group. In addition, the 13C NMR spec-
matic ring, a 1,4-disubstituted aromatic ring, and a signal trum shows signals for four oxygenated sp2 carbon atoms
for only one methoxy group. An HMBC correlation be- at δC = 144.2, 146.1, 147.2, and 147.5 ppm, which suggests
tween these protons and C-4ЈЈ confirmed the location of the presence of two 1,3,4-trisubstituted aromatic rings.
the methoxy group. Acidic hydrolysis of 3 gave 3a and d- HMBC correlations between the anomeric proton and C-3
glucose, and consecutive methylation of 3a afforded 2b. confirmed the location of the sugar moiety. The closeness
Consequently, 3 was identified as (3S,5S)-1-(3,4-dihydroxy- of the carbon resonances of C-3ЈЈ (δC = 147.5 ppm) and
phenyl)-3,5-dihydroxy-7-(4-methoxyphenyl)heptyl 3-O-β-d- C-4ЈЈ (δC = 147.2 ppm) makes it difficult to determine the
glucopyranoside.
attachment of the methoxy group. The NOESY correlation
Compound 4 was obtained as an amorphous solid. Its between the aromatic proton 5ЈЈ-H at δH = 6.80 ppm and
molecular formula C25H34O11 was deduced from the ion the methoxy protons at δH = 3.81 ppm confirmed the loca-
peak [M + Na]+ at m/z = 533.2015 in the HRESIMS spec- tion of this group at C-4ЈЈ. Thus, the planar structure of 6
1
trum. The H NMR spectrum of 4 exhibits a signal for an was elucidated as 1-(3,4-dihydroxyphenyl)-3,5-dihydroxy-7-
anomeric proton at δH = 4.39 (d, J = 7.8 Hz, 1 H) ppm and (3-hydroxy-4-methoxyphenyl)heptyl 3-O-β-d-glucopyranos-
signals for six aromatic protons, but it did not exhibit any ide. The negative sign of the specific rotation of 6, similar
signals for methoxy groups. Furthermore, the 13C NMR to compounds 1–4, allowed us to tentatively assign the ab-
spectrum shows signals for four oxygenated sp2 carbon solute configuration 3S,5S to compound 6.
atoms (δC = 144.2, 144.3, 146.1, and 146.2 ppm), which sug-
Compound 7 was obtained as an amorphous solid. Its
gests the presence of two 3,4-dihydroxyphenyl groups. molecular formula C27H36O11 was deduced from the quasi-
Acidic hydrolysis of 4 gave 4a and d-glucose. Thus, the molecular peak [M + Na]+ at m/z = 559.2241 in the HRES-
1
planar structure of 4 was identified as 1,7-bis(3,4-dihydroxy- IMS spectrum. Comparison of the H and DEPT 135 13C
phenyl)-3,5-dihydroxyheptyl 3-O-β-d-glucopyranoside by NMR spectra of 7 and 3 revealed that 7 is closely related
comparison of its NMR spectroscopic data with those re- to 3 but the spectra contain additional deshielded signals at
ported in the literature.[5] To assign the absolute configura- δH = 5.19 (m, 1 H) and 8.12 (s, 1 H) ppm, and at δC
=
tion of compound 4, 4a was methylated to give derivative 163.5 ppm. These data, along with the presence of a strong
4b, which was converted into the 3,5-bis[p-(dimethylamino)- IR absorption at 1710 cm–1, clearly indicate the presence
benzoate] derivative 4c. Its CD spectrum exhibits positive of a formyloxy group. The HMBC correlation between the
(319.8 nm, Δε = +24.5 m–1 cm–1) and negative (296 nm, Δε formyl proton at δH = 8.12 ppm and the C-5 carbon at δC
= –22.1 m–1 cm–1) Cotton effects, which is consistent with a = 73.1 ppm confirmed that the formyloxy group is attached
positive chirality by comparison with reported values.[10] to carbon C-5. Compound 7 is therefore the formyl ester of
The resulting exciton chirality interaction between the two 3 and the planar structure of 7 was assigned as 1-(3,4-
(dimethylamino)benzoyloxy chromophores, in a redshifted dihydroxyphenyl)-5-formyl-3-hydroxy-7-(4-methoxyphen-
region, provides an unambiguous configurational assign- yl)heptyl 3-O-β-d-glucopyranoside. The negative sign of the
ment.[11] Moreover, compounds 4a and 4b are enantiomers specific rotation of 7, similar to compounds 1–4, allowed
of the corresponding arylheptanoids described by Mimaki us to assign tentatively the absolute configuration 3S,5S to
and co-workers,[5] as illustrated by their similar values and compound 7.
opposite signs of specific rotation. Thus, compound 4 is
shown to be (3S,5S)-3,5-dihydroxy-1,7-bis(3,4-dihydroxy- anoid glucoside structurally related to 4. The H and 13C
phenyl)heptyl 3-O-β-d-glucopyranoside. NMR spectroscopic data for 8 are very similar to those of
Compound 8 (C26H34O12) was assigned as a diarylhept-
1
Compound 5 was shown to have a molecular formula of compound 4 but with signals for an additional formyl
C27H38O10 on the basis of the ion peak [M + Na]+ at m/z group, as was the case for 7. The location of this group at
= 545.2404 in the HRESIMS spectrum, which is the same C-5 was deduced from HMBC correlation between the for-
as that of 2. In the HMBC spectrum, correlation was ob- myl proton at δH = 8.12 ppm and the C-5 carbon at δC
=
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1041

M. Litaudon et al.
FULL PAPER
73.1 ppm. Other 1H–1H COSY and HMBC correlations are 5, KB, and HL-60 cell lines (at Ͼ100 μm for 4b and Ͼ25 μm
similar to those of compound 4 and confirm that com- for 4c), which makes 4b a good candidate for the prepara-
pound
8 is 1,7-bis(3,4-dihydroxyphenyl)-5-formyl-3-hy- tion of analogues for further study. Note that, in contrast
droxyheptyl 3-O-β-d-glucopyranoside. The negative sign of to 4b, its enantiomer was reported to be cytotoxic against
the specific rotation of 8, similar to compounds 1–4, al- the HL-60 cancer cell line[6] (IC50 = 12.1 μm). The lack of
lowed us to assign tentatively the absolute configuration cytotoxic activity of saturated arylheptanoids was con-
3S,5S to compound 8.
firmed by Ishida et al.,[12] who showed that the saturation
Naturally occurring diarylheptanoid glucosides and their of the olefinic bonds of curcumin analogues eliminated
derivatives were evaluated in vitro for their antiparasitic ac- cytotoxic activity. The antiplasmodial activity of the EtOAc
tivity against Plasmodium falciparum (erythrocytic stage), leaves extract is probably due to the presence of β-sitosterol
Leishmania donovani (amastigote forms), and Trypanosoma (IC50 = 2.1 μm) as a major constituent of the extract. This
brucei (trypomastigote bloodstream forms) as well as for activity could be due to a stomatocytogenic effect, as was
their cytotoxic activity against the HL-60, KB, and MRC- reported for lupeol.[13] Further investigation using trans-
5 cell lines (Table 1). The diarylheptanoid glucosides and mission electron microscopy would be required to confirm
their derivatives showed weak or no antiparasitic activity this hypothesis.
and no cytotoxic activity except for the derivatives 4b and
4c against P. falciparum, compound 2c against the intra-
Conclusions
macrophage amastigotes of L. donovani, and compound 6,
which exhibited significant antitrypanosomal activity. Al-
though the antiplasmodial activity is low, because com-
pounds are considered interesting in vitro against P. falcipa-
rum with activities in the nanomolar range, the antileish-
manial activity of compound 2c and the antitrypanosomal
activity of compound 6 against T. brucei in the micromolar
range, compared with pentamidine, could justify further in
vivo evaluation.
A chemical investigation of the ethyl acetate extract of
leaves of Pyrostria major led to the isolation of eight new
diarylheptanoid glucosides 1–8, described for the first time
in a plant of the Rubiaceae family. Their absolute configu-
rations were successfully determined by the CD exciton chi-
rality[4] method. Surprisingly, this structural study has al-
lowed us to obtain two selective antiplasmodial compounds
4b and 4c with no cytotoxicity, as well as a potential anti-
leishmanial-active compound 2c. Further structure–activity
relationship studies, as well as in vivo experimentation, are
expected to identify serious drug candidates for use against
malaria and leishmaniasis.
Table 1. Antiplasmodial, antileishmanial, and antitrypanosomal
activities of compounds 1–8 and their derivatives.[a]
Compound
IC50 [μm]
P. falciparum
L. donovani LV9 T. brucei
strain FcB1
intramacroph-
age amastigotes MEC
CMP strain,
Experimental Section
1
Ͼ20
39.7Ϯ3.2
53.0Ϯ4.2
35.0Ϯ2.8
31.2Ϯ2.5
37.4Ϯ2.9
66.8Ϯ5.3
10.5Ϯ0.8
33.2Ϯ2.6
58.5Ϯ4.7
33.3Ϯ2.6
67.5Ϯ5.4
Ͼ100
26.1Ϯ2.0
41.4Ϯ3.3
Ͼ100
33.0Ϯ2.6
34.6Ϯ2.7
n.d.
Ͼ100
Ͼ100
Ͼ100
Ͼ100
50
50
25
Ͼ100
25
25
50
50
Ͼ100
Ͼ100
12.5
25
25
n.d.
3.1
General Procedures: Optical rotations were measured at 25 °C with
a JASCP P1010 polarimeter. IR spectra were recorded with a Nico-
let FTIR 205 spectrophotometer. The UV spectra were recorded
with a Perkin–Elmer Lambda 5 spectrophotometer. CD spectra
were recorded at 25 °C with a JASCO J-810 spectropolarimeter
using the software SpecDis version 1.51.[14] The NMR spectra were
recorded with a Bruker Avance 500 instrument for all compounds
except 2–4 for which a Bruker 300 MHz instrument was employed.
CD3OD or CDCl3 was used as solvent. HRESIMS were recorded
with a Thermoquest TLM LCQ Deca ion-trap spectrometer. Kro-
masil analytical and preparative C18 columns (250ϫ4.6 mm,
250ϫ21.2 mm i.d. 5 μm Thermo) were used for preparative HPLC
separations using a “Waters autopurification system” equipped
with a sample manager (Waters 2767), a column fluidics organizer,
a binary pump (Waters 2525), a UV/Vis diode-array detector (190–
600 nm, Waters 2996), and a PL-ELS 1000 ELSD Polymer Labora-
tory detector. SFC analyses were performed with a Thar Waters
SFC Investigator II System using a photodiode array detector
(Waters 2998), an ELS detector (Waters 2424), and a chiralpack
analytical IA column (250ϫ4.6 mm, 5 μm, Daicel Chemical indus-
tries). Silica gel 60 (6–35 μm) and analytical TLC plates (silica gel
60 F254) were purchased from SDS (France). All other chemicals
and solvents were purchased from SDS (France).
1a
60.40Ϯ0.92
27.25Ϯ7.25
Ͼ20
40.78Ϯ1.03
32.71Ϯ3.47
16.45Ϯ0.79
Ͼ20
25.39Ϯ2.44
Ͼ20
45.44Ϯ2.31
4.1Ϯ0.8
4.5Ϯ0.2
Ͼ20
1b
2
2a
2b
2c
3
3a
4
4a
4b
4c
5
6
Ͼ20
Ͼ20
Ͼ20
0.05Ϯ0.02
n.d.
7
8
Chloroquine
Pentamidine
11.2Ϯ1.3
0.10Ϯ0.02
Amphotericin n.d.
B
n.d.
[a] Data are IC50 values in μm Ϯ standard errors and are the
average of three independent experiments unless indicated other-
wise. FcB1: Chloroquine-resistant strain of Plasmodium falciparum;
MEC: minimum effective concentration; n.d.: not done.
Although compounds 4b and 4c exhibited a moderate
antiplasmodial activity (IC50 = 4.1 and 4.5 μm, respec-
Plant Material: Leaves of Pyrostria major were collected in the Ma-
hanoro region of the Eastern coastal rainforest of Madagascar in
tively), they showed no cytotoxic activity against the MRC- September 2005. The plant was identified by Armand Rakotozafy
1042 Eur. J. Org. Chem. 2012, 1039–1046
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Diarylheptanoid Glucosides and Their Antiprotozoal Activities
Table 2. 13C NMR data for compounds 1, 1a, 2, 2a, 2b, and 3a.[a]
by comparison with an authentic specimen held in the Department
of Botany, Parc Botanique et Zoologique de Tsimbazaza, Antanan-
arivo. Botanical identification was confirmed by Dr. Sylvain Razaf-
imandimbison (The Bergius Foundation, Royal Swedish Academy
of Sciences, SE-10504, Stockholm, Sweden). A voucher specimen
(MAD-0086) was deposited at the Institut Malgache de Recherches
Appliqueés.
C atom
δC [ppm]
1
1a
2
2a
2b
3
3a
1
2
3
4
5
6
7
31.6
39.1
78.3
43.1
68.4
41.3
32.3
31.4
39.5
69.1
42.7
69.1
39.5
31.4
31.8
38.9
78.3
42.9
68.4
41.2
32.2
31.7
39.2
69.1
42.7
69.1
39.4
31.4
32.0
39.5
69.2
42.8
69.2
39.5
31.4
31.9
39.1
78.4
43.0
68.5
41.3
32.3
32.5
41.4
69.8
45.7
68.9
41.4
32.2
Extraction and Isolation: The plant material (dry wt., 375 g) was
extracted with EtOAc three times (each 2 L) at 40 °C. The EtOAc
extract was concentrated in vacuo at 40 °C to yield 13 g of residue.
The EtOAc extract (10 g) was subjected to silica gel column
chromatography by using a gradient of CH2Cl2/MeOH (1:0 to 8:2)
of increasing polarity, which gave 16 fractions on the basis of TLC.
Fraction 5 (63.2 mg) was identified as β-sitosterol. Fraction 15
(200 mg) was subjected to preparative C18 column chromatography
by using the mobile phase MeOH/H2O (60:40 to 100:0 + 0.1%
formic acid) over 20 min at 21 mLmin–1 to afford 1 (80 mg,
6.2ϫ10–6 %), 2 (50 mg, 2.7ϫ10–6 %), and 5 (4 mg, 7ϫ10–7 %).
Fraction 16 (400 mg) was subjected to preparative C18 chromatog-
raphy by using MeOH/H2O (40:60 to 70:30 + 0.1% formic acid)
over 20 min at 21 mLmin–1 to afford 3 (30 mg, 5.2ϫ10–6 %), 4
1Ј
2Ј
3Ј
4Ј
5Ј
6Ј
1ЈЈ
2ЈЈ
3ЈЈ
4ЈЈ
5ЈЈ
6ЈЈ
136.0[b] 134.0 136.9 135.3 134.7 135.6[b] 135.4
130.5 129.4 116.8 114.8 111.9 116.9 116.7
114.9 114.1 147.3 145.7 149.1 146.1 146.2
159.4 158.0 147.1 145.0 147.5 144.2 144.3
114.9 114.1 113.0 110.9 111.5 116.4 116.4
130.5 129.4 120.8 119.9 120.3 120.9 120.8
135.8[b] 134.0 135.8 134.1 134.0 135.8[b] 135.8
130.4 129.4 130.4 129.4 129.4 130.4 130.4
114.8 114.1 114.9 114.1 114.1 114.9 114.9
159.3 158.0 159.3 158.0 158.1 159.4 159.7
114.8 114.1 114.9 114.1 114.1 114.9 114.9
130.4 129.4 130.4 129.4 129.4 130.4 130.4
(20 mg, 3.87ϫ10–6 %),
6
(2 mg, 3.4ϫ10–7 %),
7
(2 mg,
Glc
1
2
3
4
5
104.4
75.6
78.3
71.8
78.0
62.9
104.2
75.5
78.1
71.7
78.0
62.9
104.3
75.6
78.4
71.8
78.0
62.5
3.4ϫ10–7 %), and 8 (2 mg, 3.4ϫ10–7 %).
Compound 1: Amorphous solid. [α]2D5 = –39 (c = 0.05, MeOH). UV
(MeOH): λmax [log(ε/m–1 cm–1)] = 224 [4.36], 278 nm [3.6]. IR: ν
˜
max
= 1634, 1512, 1244, 1078, 710, 2155, 3220 cm–1. 1H NMR
(CD3OD, 500 MHz): δH = 1.61–1.75 (m, 4 H, 4-H2, 6-H2), 1.79 (m,
1 H, 2a-H), 1.93 (m, 1 H, 2b-H), 2.56 (m, 1 H, 7a-H), 2.64 (t, J =
8.5 Hz, 2 H, 1-H2), 2.68 (m, 1 H, 7b-H), 3.19 (t, J = 8.5 Hz, 1 H,
Glc-2), 3.26 (m, 1 H, Glc-5), 3.33 (m, 1 H, Glc-4), 3.34 (m, 1 H,
Glc-3), 3.71 (d, J = 11.8, 5.5 Hz, 1 H, Glc-6a), 3.74 (s, 2 ϫ3 H, 2
ϫOMe), 3.87 (dd, J = 11.8, 2.7 Hz, 1 H, Glc-6b), 3.91 (m, 1 H, 5-
H), 3.96 (m, 1 H, 3-H), 4.39 (d, J = 7.8 Hz, 1 H, Glc-1), 6.80 (d, J
= 8.5 Hz, 2 H, 3Ј-H, 5Ј-H), 6.81 (d, J = 8.5 Hz, 2 H, 3ЈЈ-H, 5ЈЈ-H),
7.09 (d, J = 8.5 Hz, 2 H, 2ЈЈ-H, 6ЈЈ-H), 7.14 (d, J = 8.5 Hz, 2 H,
2Ј-H, 6Ј-H) ppm. 13C NMR: see Table 2. HRMS (ESI): calcd. for
C27H38O9Na [M + Na]+ 529.2414; found 529.2406.
6
3Ј-OMe
4Ј-OMe
3ЈЈ-OMe
4ЈЈ-OMe
56.0
56.1
55.7
55.7
55.4
55.4
56.6
55.7
56.2
55.4
55.5
55.8
55.7
[a] Spectra recorded in CD3OD (1–3, 3a) or CDCl3 (1a, 2a, 2b) at
75 (2) and 125 MHz (1–3, 1a, 2a, 2b, 3a). [b] May be interchanged.
(40 mg), and the mixture was stirred at 60 °C for 9 h. The reaction
mixture was diluted with H2O (20 mL) and extracted with EtOAc
(3 ϫ10 mL). After concentration of the organic phase, it was puri-
fied by chromatography on silica gel eluting with CH2Cl2/MeOH
(9.5:0.5) to yield 1b (12 mg).
Compound 1b: Amorphous solid. [α]2D5 = +10 (c = 0.1, CHCl3). UV
(CHCl3): λmax [log(ε/m–1 cm–1)] = 318 [3.6], 247 [4.3], 228 nm [4.1].
CD (EtOH): λmax (Δε) = 252.7 (+9.31), 235.6 nm (–9.22 m–1 cm–1).
Acid Hydrolysis of 1: Compound 1 (23 mg) in 1 n HCl (10 mL) was
heated at 100 °C for 10 h. The resulting hydrolysate was extracted
with EtOAc (3ϫ10 mL), dried (Na2SO4), and evaporated under
reduced pressure to afford pure 1a (14 mg). The aqueous phase was
freeze-dried. The residue containing the sugar was analyzed by
SFC under the following conditions: Co-solvent (%): MeOH
(12%), CO2 flow rate: 3.52 mLmin–1, co-solvent flow rate:
0.48 mLmin–1, total flow: 4 mLmin–1, detection: ELSD. The d-glu-
cose present in the sugar fraction was identified by comparison of
its retention time with those of authentic samples of d- and l-
glucose [tR = 5.20/6.20 (α/β form) and 4.50/5.50 min (α/β form),
respectively].
IR: ν
= 2930, 2864, 1716, 1589, 1513, 1032, 1011, 848, 809,
˜
max
682 cm–1. 1H NMR (CDCl3, 500 MHz): δH = 1.86–2.08 (m, 4 H,
2-H2, 6-H2), 2.10 (m, 2 H, 4-H2), 2.59 (m, 4 H, 1-H2, 7-H2), 3.73
(s, 2 ϫ3 H, 2 ϫOMe), 5.23 (m, 2 H, 3-H, 6-H), 6.75 (d, J = 8.5 Hz,
4 H, 3Ј-H, 5Ј-H, 3ЈЈ-H, 5ЈЈ-H), 7.02 (d, J = 8.5 Hz, 4 H, 2Ј-H, 6Ј-
H, 2ЈЈ-H, 6ЈЈ-H), 7.43 (d, J = 8.5 Hz, 4 H), 7.68 (d, J = 8.5 Hz, 4
H) ppm. 13C NMR (CDCl3, 125 MHz): δC = 30.9 (t, C-1, C-7),
36.4 (t, C-2, C-6), 38.2 (t, C-4), 55.4 (q, 4Ј-OMe, 4ЈЈ-OMe), 71.5
(d, C-3, C-5), 114.1 (d, C-3Ј, C-5Ј, C-3ЈЈ, C-5ЈЈ), 129.4 (d, C-2Ј, C-
6Ј, C-2ЈЈ, C-6ЈЈ), 133.2 (s, C-1Ј, C-1ЈЈ), 158.0 (s, C-4Ј, C-4ЈЈ); p-
bromobenzoate substituents: 128.1 (2 s), 129.3 (2 s), 131.2 (4 d),
131.7 (4 d), 165.4 (2 s) ppm. HRMS (ESI): calcd. for
C35H34Br2O6Na [M + Na]+ 731.0620; found 731.0616.
Compound 1a: Amorphous solid. [α]2D5 = –3.7 (c = 0.05, CHCl3).
UV (CHCl3): λmax [log(ε/m–1 cm–1)] = 241 [3.2], 278 nm [3.5]. IR:
ν
= 1513, 1246, 3373, 761, 768 cm–1. 1H NMR (CDCl3,
˜
max
500 MHz): δH = 1.64 (t, J = 5.7 Hz, 2 H, 4-H2), 1.67–1.84 (m, 4
H, 2-H2, 6-H2), 2.10 (br. s, 2 H, 3-OH, 5-OH), 2.56–2.72 (m, 4 H,
1-H2, 7-H2), 3.76 (s, 2 ϫ3 H, 2 ϫOMe), 3.95 (m, 2 H, 3-H, 5-H),
6.81 (d, J = 8.5 Hz, 4 H, 3Ј-H, 5Ј-H, 3ЈЈ-H, 5ЈЈ-H), 7.09 (d, J =
8.5 Hz, 4 H, 2Ј-H, 6Ј-H, 2ЈЈ-H, 6ЈЈ-H) ppm. 13C NMR: see Table 2.
HRMS (ESI): calcd. for C21H28O4Na [M + Na]+ 367.1885; found
367.1884.
Compound 2: Amorphous solid. [α]2D5 = –31 (c = 0.17, MeOH). UV
(MeOH): λmax [log(ε/m–1 cm–1)] = 224 [4.36], 278 nm [3.7]. IR: ν
˜
max
= 3291, 1512, 1432, 1364, 1271, 1232, 1078, 1033, 815 cm–1. 1H
NMR (CD3OD, 300 MHz): δH = 1.59–1.74 (m, 4 H, 4-H2, 6-H2),
1.75 (m, 1 H, 2a-H), 1.94 (m, 1 H, 2b-H), 2.51–2.73 (m, 4 H, 7-
H2, 1-H2), 3.19 (t, J = 8.5 Hz, 1 H, Glc-2), 3.26 (m, 1 H, Glc-5),
3.33 (m, 1 H, Glc-4), 3.35 (m, 1 H, Glc-3), 3.71 (dd, J = 11.8,
5.5 Hz, 1 H, Glc-6a), 3.72 (s, 3 H, 4ЈЈ-OMe), 3.78 (s, 3 H, 4Ј-OMe),
Bis(p-bromobenzoate) 1b of 1a: NEt3 (32 μL) was added dropwise
to a solution of 1a (13 mg) in anhydrous CH2Cl2 (0.6 mL) contain-
ing DMAP (15 mg) as catalyst and p-bromobenzoyl chloride
Eur. J. Org. Chem. 2012, 1039–1046
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1043

M. Litaudon et al.
FULL PAPER
3.87 (dd, J = 11.8, 2.7 Hz, 1 H, Glc-6b), 3.91 (m, 1 H, 5-H), 3.96 NMR (CD3OD, 300 MHz): δH = 1.59–1.72 (m, 4 H, 4-H2, 6-H2),
(m, 1 H, 3-H), 4.39 (d, J = 7.8 Hz, 1 H, Glc-1), 6.63 (dd, J = 8.5, 1.75 (m, 1 H, 2a-H), 1.94 (m, 1 H, 2b-H), 2.51–2.61 (m, 3 H, 7a-
2 Hz, 1 H, 6Ј-H), 6.73 (d, J = 2 Hz, 1 H, 2Ј-H), 6.78 (d, J = 8.5 Hz, H, 1-H2), 2.67 (m, 1 H, 7b-H), 3.20 (t, J = 8.5 Hz, 1 H, Glc-2),
1 H, 5Ј-H), 6.79 (d, J = 8.5 Hz, 2 H, 3ЈЈ-H, 5ЈЈ-H), 7.07 (d, J = 3.30 (m, 1 H, Glc-5), 3.33 (m, 1 H, Glc-4), 3.36 (m, 1 H, Glc-3),
8.5 Hz, 2 H, 2ЈЈ-H, 6ЈЈ-H) ppm. 13C NMR, see Table 2. HRMS
3.71 (dd, J = 11.8, 5.5 Hz, 1 H, Glc-6a), 3.74 (s, 3 H, 4ЈЈ-OMe),
(ESI): calcd. for C27H38O10Na [M + Na]+ 545.2363; found 3.89 (dd, J = 11.8, 2.7 Hz, 1 H, Glc-6b), 3.90 (m, 1 H, 5-H), 3.96
545.2410.
(m, 1 H, 3-H), 4.39 (d, J = 7.8 Hz, 1 H, Glc-1), 6.52 (dd, J = 8,
2 Hz, 1 H, 6Ј-H), 6.66 (d, J = 8 Hz, 1 H, 5Ј-H), 6.71 (d, J = 2 Hz,
1 H, 2Ј-H), 6.81 (d, J = 8.5 Hz, 2 H, 3ЈЈ-H, 5ЈЈ-H), 7.08 (d, J =
8.5 Hz, 2 H, 2ЈЈ-H, 6ЈЈ-H) ppm. 13C NMR, see Table 2. HRMS
(ESI): calcd. for C26H36O10Na [M + Na]+ 531.2206; found
531.2285.
Acid Hydrolysis of 2: Compound 2 (30 mg) was hydrolyzed by the
same procedure as described for 1 to yield 2a (22 mg). SFC analysis
of the sugar fraction under the same conditions as in the case of 1
showed the presence of d-glucose.
Compound 2a: Amorphous solid. [α]2D5 = –10 (c = 0.1, CHCl3). UV
Acid Hydrolysis of 3: Compound 3 (30 mg) was hydrolyzed by the
same procedure as described for 1 to yield 3a (22 mg). SFC analysis
of the sugar fraction under the same conditions as in the case of 1
showed the presence of d-glucose.
(CHCl3): λmax [log(ε/m–1 cm–1)] = 279 nm [3.6]. IR: ν
= 3407,
˜
max
1590, 1514, 812, 764 cm–1. 1H NMR (CDCl3, 500 MHz): δH = 1.62
(t, J = 5.7 Hz, 2 H, 4-H2), 1.66–1.83 (m, 4 H, 2-H2, 6-H2), 2.51–
2.71 (m, 4 H, 1-H2, 7-H2), 3.76 (s, 3 H, 4ЈЈ-OMe), 3.83 (s, 3 H, 4Ј-
OMe), 3.94 (m, 2 H, 3-H, 5-H), 6.64 (dd, J = 8.5, 2 Hz, 1 H, 6Ј-
H), 6.74 (d, J = 8.5 Hz, 1 H, 5Ј-H), 6.75 (d, J = 2 Hz, 1 H, 2Ј-H),
6.79 (d, J = 8.5 Hz, 2 H, 3ЈЈ-H, 5ЈЈ-H), 7.08 (d, J = 8.5 Hz, 2 H,
2ЈЈ-H, 6ЈЈ-H) ppm. 13C NMR, see Table 2. HRMS (ESI): calcd. for
C21H28O5Na [M + Na]+ 383.1834; found 383.1816.
Compound 3a: Amorphous solid. [α]2D5 = –10 (c = 0.1, MeOH). UV
(MeOH): λmax [log(ε/m–1 cm–1)] = 222 [4.2], 284 nm [3.6]. IR: ν
˜
max
= 3340, 1513, 1246, 824, 797 cm–1. H NMR (CD3OD, 500 MHz):
δH = 1.54 (t, J = 6.2 Hz, 2 H, 4-H2), 1.68 (m, 4 H, 2-H2, 6-H2),
2.46–2.71 (m, 4 H, 1-H2, 7-H2), 3.74 (s, 3 H, 4Ј-OMe), 3.80 (m, 2
H, 3-H, 5-H), 6.50 (dd, J = 8.5, 2 Hz, 1 H, 6Ј-H), 6.63 (d, J = 2 Hz,
1 H, 2Ј-H), 6.66 (d, J = 8.5 Hz, 1 H, 5Ј-H), 6.80 (d, J = 8.5 Hz, 2
H, 3Ј-H, 5Ј-H), 7.09 (d, J = 8.5 Hz, 2 H, 2ЈЈ-H, 6ЈЈ-H) ppm. 13C
NMR, see Table 2. HRMS (ESI): calcd. for C20H26O5Na
[M + Na]+ 369.1678; found 369.1678.
1
Methylation of 2a: Compound 2a (20 mg) and Cs2CO3 (13 mg)
were dissolved in THF (0.5 μL). A large excess of CH3I was added
to the reaction mixture, which was stirred at 50 °C for 9 h. The
reaction mixture was purified by chromatography on silica gel elut-
ing with CH2Cl2/MeOH (9.5:0.5) to yield 2b (12 mg).
Compound 2b: Amorphous solid. [α]2D5 = –2.5 (c = 0.07, MeOH).
UV (MeOH): λmax [log(ε/m–1 cm–1)] = 225 [4.8], 279 nm [4.3]. IR:
Methylation of 3a: Compound 3a (7 mg) was methylated by the
same procedure as described for 2 to yield 2b (4 mg).
ν
= 3407, 1611, 1590, 1155, 1030, 812, 764 cm–1. 1H NMR
Compound 4: Amorphous solid. [α]2D5 = –3.7 (c = 0.1, MeOH). UV
˜
max
(CDCl3, 500 MHz): δH = 1.65 (t, J = 5.7 Hz, 2 H, 4-H2), 1.68–1.84
(m, 4 H, 2-H2, 6-H2), 2.56–2.73 (m, 4 H, 1-H2, 7-H2), 3.76 (s, 3 H,
4ЈЈ-OMe), 3.83 (s, 3 H, 4Ј-OMe), 3.84 (s, 3 H, 3Ј-OMe), 3.96 (m, 2
H, 3-H, 5-H), 6.70 (d, J = 2 Hz, 1 H, 2Ј-H), 6.71 (dd, J = 8.5,
2.0 Hz, 1 H, 6Ј-H), 6.77 (d, J = 8.5 Hz, 1 H, 5Ј-H), 6.81 (d, J =
8.5 Hz, 2 H, 3ЈЈ-H, 5ЈЈ-H), 7.09 (d, J = 8.5 Hz, 2 H, 2ЈЈ-H, 6ЈЈ-
H) ppm. 13C NMR, see Table 2. HRMS (ESI): calcd. for
C21H28O5Na [M + Na]+ 383.1834; found 383.1816.
(MeOH): λmax [log(ε/m–1 cm–1)] = 227 [3.6], 281 nm [3.5]. IR: ν
=
˜
max
1634, 1512, 1244, 1078, 710, 2155, 3220 cm–1. H NMR (CD3OD,
300 MHz): δH = 1.57–1.81 (m, 5 H, 4-H2, 6-H2, 2a-H), 1.93 (m, 1
H, 2b-H), 2.46–2.67 (m, 4 H, 7-H2, 1-H2), 3.20 (t, J = 8.5 Hz, 1 H,
Glc-2), 3.28 (m, 1 H, Glc-5), 3.33 (m, 1 H, Glc-4), 3.37 (m, 1 H,
Glc-3), 3.71 (dd, J = 11.8, 5.5 Hz, 1 H, Glc-6a), 3.89 (dd, J = 11.8,
2.7 Hz, 1 H, Glc-6b), 3.93 (m, 1 H, 5-H), 3.96 (m, 1 H, 3-H), 4.39
(d, J = 7.8 Hz, 1 H, Glc-1), 6.50 (dd, J = 8, 2 Hz, 1 H, 6ЈЈ-H), 6.53
(dd, J = 8, 2 Hz, 1 H, 6Ј-H), 6.63 (d, J = 2 Hz, 1 H, 2ЈЈ-H), 6.66
(d, J = 8 Hz, 2 H, 5Ј-H, 5ЈЈ-H), 6.70 (d, J = 2 Hz, 1 H, 2Ј-H) ppm.
13C NMR, see Table 3. HRMS (ESI): [M + Na]+ calcd. for
C25H34O11Na 533.1999; found 533.2015.
1
Bis(p-bromobenzoate) 2c of 2b: Compound 2b (6 mg) was treated
by the same procedure as for 1a to yield 2c (6 mg).
Compound 2c: Amorphous solid. [α]2D5 = +30 (c = 0.1, CHCl3). UV
(CHCl3): λmax [log(ε/m–1 cm–1)] = 225 [4.3], 246 nm [4.6]. CD
(EtOH): λmax (Δε) = 253 nm (+15.5), 237 nm (–11.77 m–1 cm–1). IR:
Acid Hydrolysis of 4: Compound 4 (27 mg) was hydrolyzed by the
same procedure as described for 1 to yield 4a (15 mg). SFC analysis
of the sugar fraction under the same conditions as in the case of 1
showed the presence of d-glucose.
ν
= 2930, 1720, 1513, 1480, 1270, 1140, 1016, 809, 755,
˜
max
682 cm–1. 1H NMR (CDCl3, 500 MHz): δH = 1.86–2.08 (m, 4 H,
2-H2, 6-H2), 2.10 (m, 2 H, 4-H2), 2.59 (m, 4 H, 1-H2, 7-H2), 3.73
(s, 3 H, 4ЈЈ-OMe), 3.79 (s, 3 H, 4Ј-OMe), 3.80 (s, 3 H, 3Ј-OMe),
5.26 (m, 2 H, 3-H, 6-H), 6.63 (dd, J = 8.5, 2.0 Hz, 1 H, 6Ј-H), 6.64
(d, J = 2.0 Hz, 1 H, 2Ј-H), 6.70 (d, J = 8.5 Hz, 1 H, 5Ј-H), 6.75 (d,
J = 8.5 Hz, 2 H, 3ЈЈ-H, 5ЈЈ-H), 7.02 (d, J = 8.5 Hz, 2 H, 2ЈЈ-H, 6ЈЈ-
H), 7.43 (d, J = 8.5 Hz, 4 H), 7.68 (d, J = 8.5 Hz, 4 H) ppm. 13C
NMR (CDCl3, 125 MHz): δC = 30.9 (t, C-7), 31.4 (t, C-1), 36.4 (t,
C-2, C-6), 38.3 (t, C-4), 55.4 (q, 4ЈЈ-OMe), 56.0 (q, 4Ј-OMe), 56.1
(q, 3Ј-OMe), 71.4–71.5 (d, C-3, C-5), 111.4 (d, C-5Ј), 111.9 (d, C-
2Ј), 114.0 (d, C-3ЈЈ,C-5ЈЈ), 120.3 (d, C-6Ј), 129.4 (d, C-2ЈЈ, C-6ЈЈ),
133.2 (s, C-1ЈЈ), 133.7 (s, C-1Ј), 147.5 (s, C-3Ј), 149.1 (s, C-4Ј), 158.1
(s, C-4ЈЈ); p-bromobenzoate substituents: 128.2 (2 s), 129.3 (2 s),
131.2 (4 d), 131.7 (4 d), 165.4 (2 s) ppm. HRMS (ESI): calcd. for
C36H36Br2O7Na [M + Na]+ 761.0725; found 761.0761.
Compound 4a: Amorphous solid. [α]2D5 = –10 (c = 0.1, MeOH). UV
(MeOH): λmax [log(ε/m–1 cm–1)] = 221 [4.0], 283 nm [3.7]. IR: ν
˜
max
= 3440, 1519, 1370, 1058, 815, 782 cm–1. 1H NMR (CD3OD,
500 MHz): δH = 1.53 (t, J = 6.2 Hz, 2 H, 4-H2), 1.67 (m, 4 H, 2-
H2, 6-H2), 2.46–2.64 (m, 4 H, 1-H2, 7-H2), 3.80 (m, 2 H, 3-H, 5-
H), 6.51 (dd, J = 8.5, 2 Hz, 2 H, 6Ј-H, 6ЈЈ-H), 6.63 (d, J = 2 Hz, 2
H, 2Ј-H, 2ЈЈ-H), 6.66 (d, J = 8.5 Hz, 2 H, 5Ј-H, 5ЈЈ-H) ppm. 13C
NMR, see Table 3. HRMS (ESI): calcd. for C19H25O6 [M + H]+
349.1651; found 349.1653.
Methylation of 4a: Compound 4a (11 mg) was methylated by the
same procedure as described for 2 to yield 4b (5 mg).
Compound 4b: Amorphous solid. [α]2D5 = –20 (c = 0.1, MeOH). UV
Compound 3: Amorphous solid. [α]2D5 = –10 (c = 0.13, MeOH). UV
(MeOH): λmax [log(ε/m–1 cm–1)] = 228 [5.0], 279 nm [4.6]. IR: ν
˜
max
(MeOH): λmax [log(ε/m–1 cm–1)] = 222 [4.2], 284 nm [3.6]. IR: ν
= 3280, 1600, 1580, 1420, 1145, 1020, 803, 787, 765 cm–1. 1H NMR
˜
max
= 3291, 1512, 1432, 1364, 1271, 1232, 1078, 1033, 815 cm–1. 1H
(CDCl3, 500 MHz): δH = 1.65 (t, J = 5.7 Hz, 2 H, 4-H2), 1.70–1.86
1044
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© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2012, 1039–1046

Diarylheptanoid Glucosides and Their Antiprotozoal Activities
Table 3. 13C NMR data for compounds 4, 4a, 4b, and 5–8.[a]
1 H, Glc-6b), 3.90 (m, 1 H, 5-H), 3.95 (m, 1 H, 3-H), 4.39 (d, J =
7.8 Hz, 1 H, Glc-1), 6.62 (dd, J = 8.5, 2 Hz, 1 H, 6ЈЈ-H), 6.66 (d,
J = 2 Hz, 1 H, 2ЈЈ-H), 6.80 (d, J = 8.5 Hz, 1 H, 5ЈЈ-H), 6.81 (d, J
= 8.5 Hz, 2 H, 3Ј-H, 5Ј-H), 7.12 (d, J = 8.5 Hz, 2 H, 2Ј-H, 6Ј-
H) ppm. 13C NMR, see Table 3. HRMS (ESI): calcd. for
C27H38O10Na [M + Na]+ 545.2363; found 545.2404.
C atom
4
4a
4b
5
6
7
8
1
2
3
4
5
6
7
31.8
39.1
78.4
43.0
68.5
41.3
32.5
32.5
41.4
68.9
45.7
68.9
41.4
32.5
32.0
39.5
69.2
42.8
69.2
39.5
32.0
31.6 31.9
39.2 39.2
78.4 78.4
42.0 43.1
68.4 68.5
41.2 41.2
32.5 32.6
31.6
38.8
77.1
40.6
73.1
38.0
31.7
31.7
38.9
77.2
40.7
73.3
38.1
32.0
Compound 6: Amorphous solid. [α]2D5 = –16 (c = 0.016, MeOH).
UV (MeOH): λmax [log(ε/m–1 cm–1)] = 221 [4.1], 282 nm [3.7]. IR:
ν
= 3291, 1512, 1432, 1364, 1271, 1232, 1078, 1033, 815 cm–1.
˜
max
135.6[b] 135.4 134.6 136.0 135.6 135.4 135.4
116.9 116.7 112.0 130.5 116.9 117.0 116.9
146.1 146.2 149.1 114.8 146.1 146.2 146.3
144.3 144.2 147.5 159.3 144.2 144.3 144.3
116.5 116.4 111.6 114.8 116.4 116.4 116.5
120.9 120.8 120.4 130.5 120.9 121.0 120.8
135.5[b] 135.4 134.6 136.9 136.9 134.9 134.5
116.7 116.7 112.0 116.6 116.7 130.4 116.7
146.2 146.2 149.1 114.8 147.5 115.0 146.1
144.2 144.2 147.5 147.2 147.2 159.6 144.5
116.4 116.4 111.6 113.1 113.1 115.0 116.4
120.7 120.8 120.4 120.6 120.7 130.4 121.0
1H NMR (CD3OD, 500 MHz): δH = 1.60–1.72 (m, 4 H, 4,-H2, 6-
H2), 1.74 (m, 1 H, 2a-H), 1.93 (m, 1 H, 2b-H), 2.48–2.66 (m, 4 H,
1-H2, 7-H2), 3.19 (t, J = 8.5 Hz, 1 H, Glc-2), 3.28 (m, 1 H, Glc-5),
3.33 (m, 1 H, Glc-4), 3.37 (m, 1 H, Glc-3), 3.71 (dd, J = 11.8,
5.5 Hz, 1 H, Glc-6a), 3.81 (s, 3 H, 4ЈЈ-OMe), 3.89 (dd, J = 11.8,
2.7 Hz, 1 H, Glc-6b), 3.90 (m, 1 H, 5-H), 3.95 (m, 1 H, 3-H), 4.39
(d, J = 7.8 Hz, 1 H, Glc-1), 6.53 (dd, J = 8.5, 2.0 Hz, 1 H, 6Ј-H),
6.61 (dd, J = 8.5, 2.0 Hz, 1 H, 6ЈЈ-H), 6.65 (d, J = 8.5 Hz, 1 H, 5Ј-
H), 6.66 (d, J = 2.0 Hz, 1 H, 2ЈЈ-H), 6.69 (d, J = 2.0 Hz, 1 H, 2Ј-
H), 6.80 (d, J = 8.5 Hz, 1 H, 5ЈЈ-H) ppm. 13C NMR, see Table 3.
HRMS (ESI): calcd. for C26H36O11Na [M + Na]+ 547.2155; found
547.2166.
1Ј
2Ј
3Ј
4Ј
5Ј
6Ј
1ЈЈ
2ЈЈ
3ЈЈ
4ЈЈ
5ЈЈ
6ЈЈ
Glc
1
2
3
4
5
6
104.3
75.6
78.3
71.7
78.0
62.9
104.4 104.3 104.0 104.1
75.6 75.7
78.3 78.3
71.8 71.8
78.0 78.1
62.9 63.0
75.5
78.3
71.8
78.0
62.9
75.6
78.3
71.8
78.0
62.9
Compound 7: Amorphous solid. [α]2D5 = –10 (c = 0.032, MeOH).
UV (MeOH): λmax [log(ε/m–1 cm–1)] = 223 [4.1], 279 nm [3.6]. IR:
ν
= 3291, 1512, 1432, 1364, 1271, 1232, 1078, 1033, 815 cm–1.
˜
max
1H NMR (CD3OD, 500 MHz): δH = 1.75 (m, 1 H, 2a-H), 1.80–
1.95 (m, 5 H, 4-H2, 6-H2, 2b-H), 2.49–2.60 (m, 4 H, 1-H2, 7-H2),
3.20 (t, J = 8.5 Hz, 1 H, Glc-2), 3.23 (m, 1 H, Glc-5), 3.32 (m, 1
H, Glc-4), 3.34 (m, 1 H, Glc-3), 3.71 (dd, J = 11.8, 5.5 Hz, 1 H,
Glc-6a), 3.74 (m, 1 H, 3-H), 3.75 (s, 3 H, 4ЈЈ-OMe), 3.87 (dd, J =
11.8, 2.7 Hz, 1 H, Glc-6b), 4.39 (d, J = 7.8 Hz, 1 H, Glc-1), 5.19
(m, 1 H, 5-H), 6.52 (dd, J = 8.0, 2.0 Hz, 1 H, 6Ј-H), 6.66 (d, J =
8.0 Hz, 1 H, 5Ј-H), 6.70 (d, J = 2.0 Hz, 1 H, 2Ј-H), 6.82 (d, J =
8.5, 2.0 Hz, 2 H, 3ЈЈ-H, 5ЈЈ-H), 7.06 (d, J = 8.5, 2.0 Hz, 2 H, 2ЈЈ-
H, 6ЈЈ-H), 8.12 (s, 1 H, CHO-5) ppm. 13C NMR, see Table 3.
HRMS (ESI): calcd. for C27H36O11Na [M + Na]+ 559.2255; found
559.2241.
3Ј-OMe
4Ј-OMe
3ЈЈ-OMe
4ЈЈ-OMe
5-CHO
56.0
56.1
56.0
56.1
56.6 56.7
55.7
55.8
163.5 163.5
[a] Spectra recorded in CD3OD (4–8, 4a) or CDCl3 (4b) at 75 (4)
and 125 MHz (5–8, 4a, 4b). [b] Assignments may be interchanged.
(m, 4 H, 2-H2, 6-H2), 2.54–2.74 (m, 4 H, 1-H2, 7-H2), 3.83 (s, 2 ϫ3
H, 4Ј-OMe, 4ЈЈ-OMe), 3.85 (s, 2 ϫ3 H, 3Ј-OMe, 3ЈЈ-OMe), 3.98
(m, 2 H, 3-H, 5-H), 6.70 (d, J = 2 Hz, 2 H, 2Ј-H, 2ЈЈ-H), 6.71 (dd,
J = 8.5, 2 Hz, 2 H, 6Ј-H, 6ЈЈ-H), 6.77 (d, J = 8.5 Hz, 2 H, 5Ј-
H, 5ЈЈ-H) ppm. 13C NMR, see Table 3. HRMS (ESI): calcd. for
C23H32O6Na [M + Na]+ 427.2097; found 427.2084.
Compound 8: Amorphous solid. [α]2D5 = –61 (c = 0.024, MeOH).
UV (MeOH): λmax [log(ε/m–1 cm–1)] = 222 [3.9], 281 nm [3.6]. IR:
1
ν
= 3409, 1660, 1069 cm–1. H NMR (CD3OD, 500 MHz): δH
˜
max
Bis[p-(dimethylamino)benzoate] (4c) of 4b: NEt3 (10 μL) was added
dropwise to a solution of 4b (4 mg) in anhydrous CH2Cl2 (0.6 mL)
containing DMAP (15 mg) as catalyst and p-(dimethylamino)ben-
zoyl chloride (10 mg), and the mixture was stirred at 60 °C for 9 h.
The reaction mixture was diluted with H2O (20 mL) and extracted
with EtOAc (3ϫ10 mL). After concentration of the organic phase,
it was purified by chromatography on silica gel eluting with
CH2Cl2/MeOH (9.5:0.5) to yield 4c (2 mg).
= 1.75 (m, 1 H, 2a-H), 1.80–1.95 (m, 5 H, 4-H2, 6-H2, 2b-H), 2.49–
2.60 (m, 4 H, 1-H2, 7-H2), 3.18 (t, J = 8.5 Hz, 1 H, Glc-2), 3.23
(m, 1 H, Glc-5), 3.32 (m, 1 H, Glc-4), 3.34 (m, 1 H, Glc-3), 3.71
(dd, J = 11.8, 5.5 Hz, 1 H, Glc-6a), 3.72 (m, 1 H, 3-H), 3.87 (dd,
J = 11.8, 2.7 Hz, 1 H, Glc-6b), 4.39 (d, J = 7.8 Hz, 1 H, Glc-1),
5.19 (m, 1 H, 5-H), 6.47 (dd, J = 8.0, 2.0 Hz, 1 H, 6ЈЈ-H), 6.52 (dd,
J = 8.0, 2.0 Hz, 1 H, 6Ј-H), 6.60 (d, J = 2.0 Hz, 2ЈЈ-H), 6.65 (d, J
= 8.0 Hz, 1 H, 5Ј-H), 6.66 (d, J = 8.0 Hz, 1 H, 5ЈЈ-H), 6.70 (d, J =
2.0 Hz, 1 H, 2Ј-H), 8.12 (s, 1 H, CHO-5) ppm. 13C NMR, see
Table 3. HRMS (ESI): calcd. for C26H34O12Na [M + Na]+
561.1948; found 561.2037.
Compound 4c: Amorphous solid. [α]2D5 = +30 (c = 0.07, EtOH). UV
(EtOH): λmax [log(ε/m–1 cm–1)] = 229 [4.4], 311 nm [4.7]. CD
(EtOH): λmax (Δε) = 319.8 (+24.5), 296 nm (–22.1 m–1 cm–1). IR:
ν
= 2989, 1702, 1435, 1020, 803, 787, 765 cm–1. NMR spectro-
˜
max
Antiplasmodial Evaluation: The chloroquine-resistant strain FcB1/
Colombia of Plasmodium falciparum was maintained in vitro on
human erythrocytes in RPMI 1640 medium supplemented by 8%
(v/v) heat-inactivated human serum at 37 °C under an atmosphere
of 3% CO2, 6% O2, and 91% N2. In vitro drug susceptibility was
measured by [3H]hypoxanthine incorporation as described pre-
viously.[15] Stock solutions of drugs were prepared in DMSO. Com-
scopic data: see ref.[10]. HRMS (ESI): calcd. for C41H54N3O8 [M +
NH4]+ 716.3911; found 716.3916.
Compound 5: Amorphous solid. [α]2D5 = –6 (c = 0.05, MeOH). UV
(MeOH): λmax [log(ε/m–1 cm–1)] = 223 [4.7], 278 nm [3.25]. IR: ν
˜
max
= 3291, 1512, 1432, 1364, 1271, 1232, 1078, 1033, 815 cm–1. 1H
NMR (CD3OD, 500 MHz): δH = 1.60–1.77 (m, 4 H, 4-H2, 6-H2),
1.79 (m, 1 H, 2a-H), 1.94 (m, 1 H, 2b-H), 2.51 (m, 1 H, 7a-H), pounds were serially diluted two-fold with 100 μL culture medium
2.62 (m, 1 H, 7b-H), 2.64 (t, J = 7.7 Hz, 2 H, 1-H2), 3.19 (t, J = in 96-well plates. Asynchronous parasite cultures (100 μL, 1%
8.5 Hz, 1 H, Glc-2), 3.26 (m, 1 H, Glc-5), 3.33 (m, 1 H, Glc-4),
3.35 (m, 1 H, Glc-3), 3.71 (dd, J = 11.8, 5.5 Hz, 1 H, Glc-6a), 3.74
(s, 3 H, 4Ј-OMe), 3.80 (s, 3 H, 4ЈЈ-OMe), 3.87 (dd, J = 11.8, 2.7 Hz,
parasitemia and 1% final haematocrit) were then added to each
well, which were incubated for 24 h at 37 °C prior to the addition
of 0.5 μCi of [3H]hypoxanthine (GE Healthcare, 1–5 CimmolmL–1)
Eur. J. Org. Chem. 2012, 1039–1046
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1045

M. Litaudon et al.
FULL PAPER
per well. After further incubation for 24 h, the plates were frozen
and thawed. Cell lysates were then collected on glass-fiber filters
and counted in a liquid scintillation spectrometer. The growth inhi-
bition at each drug concentration was determined by comparison
of the radioactivity incorporated in the treated culture with that in
the control culture maintained on the same plate. The concentra-
tion causing 50% growth inhibition (IC50) was determined from the
drug concentration–response curve and the results are expressed
as the mean values Ϯ standard deviations determined from three
independent experiments. Chloroquine diphosphate, used as a posi-
tive control, was purchased from Sigma (purity Ͼ99%).
into a mouse to confirm that the non-motile parasites were really
killed and not able to divide.
Cytotoxicity Evaluation: The human KB tumor cell line (mouth
epidermoid carcinoma) was originally obtained from ATCC. The
human diploid embryonic lung cells MRC-5 were obtained from
ECACC. The human promyelocytic leukemia cells HL-60 were ob-
tained from ICSN. They were seeded into 96-well microplates at
2000 cells per well. The cytotoxicity assays were performed accord-
ing to a published procedure.[17] Taxotere (in-house product, purity
Ͼ99%) was used as a reference compound.
Acknowledgments
Antileishmanial Evaluation: Promastigote forms of the Leishmania
donovani (MHOM/ET/67/HU3) line, called LV9, were grown in M-
199 medium (Sigma, Saint-Quentin Fallavier, France) sup-
plemented with 10% inactivated foetal calf serum (Invitrogen, Er-
agnie, France), 40 mm HEPES (VWR, Paisley, Scotland), 100 μm
adenosine (Sigma, Saint-Quentin Fallavier, France), and 0.5 mgL–1
Hemin (Sigma, Saint-Quentin Fallavier, France) in the presence of
50 μgmL–1 gentamicin at 26 °C in 5% CO2. Peritoneal macro-
phages were harvested from female CD1 mice (Charles River,
Cléon, France) three days after an intraperitoneal injection of
1.5 mL of sodium thioglycolate (Biomérieux) and were dispensed
into eight-well chamber slides (LabTek Ltd.) at a density of 5ϫ104
per well (400 μL per well) in RPMI 1640 medium supplemented
with 10% hi-FCS, 25 mm HEPES, and 2 mm l-glutamine (Life
Technologies, Cergy-Pontoise, France). Four hours after the macro-
phages were plated, they were washed to eliminate fibroblasts. After
a 24 h incubation period, the macrophages were infected with pro-
mastigote forms of L. donovani LV9 in a stationary phase in a ratio
of 10 parasites per macrophage to obtain 87% of infected macro-
phages and 10Ϯ3 amastigotes per macrophage. After 18 h the free
promastigotes were eliminated and intramacrophagic amastigotes
were treated with various concentrations of the compounds. Penta-
midine and amphotericin B were used as reference compounds. The
culture medium was renewed 48 h later and a new culture medium
containing the drug was added. The experiment was stopped after
five days and the percentages of infected macrophages were evalu-
ated microscopically after Giemsa staining. The 50% inhibitory
concentrations (IC50) were determined by linear regression analysis
and expressed in μm Ϯ standard errors. Each experiment was per-
formed in triplicate.
We are grateful to Prof. D. Crich for the grant accorded to this
project (M. A. B.). We express our thanks to G. Aubert, who per-
formed the cytotoxic assays. We also thank Dr. Elisabeth Mouray
from the National Museum of Natural History for his help with
the antiplasmodial assays.
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Antitrypanosomal Evaluation: The method used has been described
previously by Loiseau et al.[16] Briefly, the bloodstream forms of T.
brucei brucei were maintained in vitro for 24 h in the dark at 37 °C
in a 5% CO2 atmosphere in a minimum essential medium (Gibco
BRL) that includes 25 mm HEPES and Earle’s salts and is sup-
plemented with 2 mm l-glutamine, 1 gL–1 of additional glucose,
10 mLL–1 of minimum essential medium nonessential amino acids
(100ϫ, Gibco BRL), 0.2 mm 2-mercaptoethanol, 2 mm sodium pyr-
uvate, 0.1 mm hypoxanthine, 0.016 mm thymidine, 15% heat-inacti-
vated horse serum (Gibco BRL), and 50 μgmL–1 of gentamycin.
The drug evaluation was carried out in 96-well tissue culture plates
in a final volume of 200 μL containing 2ϫ105 trypomastigotes pre-
viously purified by centrifugation from the blood of an infected
mouse, collected aseptically from the retro-orbital sinus and the
compounds to be tested. Pentamidine was used as reference com-
pound. The minimum effective concentration (MEC) was defined
as the minimum concentration at which no viable parasite was ob-
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peritoneally the culture from the well corresponding to the MEC
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[17] C. Tempête, G. H. Werner, F. Favre, A. Rojas, N. Langlois, Eur.
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Received: September 27, 2011
Published Online: December 13, 2011
1046
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© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2012, 1039–1046