Antivascular and anti-parasite activities of natural and hemisynthetic flavonoids from New Caledonian Gardenia species (Rubiaceae)

Article abstract of DOI:10.1016/j.ejmech.2015.01.012

A series of 16 flavonoids were isolated and prepared from bud exudate of Gardenia urvillei and Gardenia oudiepe, endemic to New Caledonia. Most of them are rare polymethoxylated flavones. Some of these compounds showed noticeable activity against Leishmania (Leishmania) amazonensis, Plasmodium falciparum and Trypanosoma brucei gambiense, in addition to tubulin polymerization inhibition at low micromolar concentration. We also provide a full set of NMR data as some of the flavones were incompletely described.

Full text of DOI:10.1016/j.ejmech.2015.01.012

European Journal of Medicinal Chemistry 93 (2015) 93e100
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Antivascular and anti-parasite activities of natural and hemisynthetic
flavonoids from New Caledonian Gardenia species (Rubiaceae)
Linh H. Mai a, Guy G. Chabot b, Philippe Grellier c, Lionel Quentin b, Vincent Dumontet d,
Cyril Poulain d, Laila S. Espindola e, Sylvie Michel a, Hue T.B. Vo f, Brigitte Deguin a,
a, *
€
Raphael Grougnet
a
ꢀ
ꢀ
ꢀ
Laboratoire de Pharmacognosie, UMR/CNRS 8638, Faculte des Sciences Pharmaceutiques et Biologiques, Universite Paris Descartes, Sorbonne Paris Cite, 4,
Avenue de l'Observatoire, 75006 Paris, France
b
ꢀ ꢀ
ꢀ
Laboratoire de Pharmacologie Chimique, Genetique et Imagerie U1022 Inserm-UMR8151 CNRS, Faculte des Sciences Pharmaceutiques et Biologiques,
ꢀ
ꢀ
Universite Paris Descartes, Sorbonne Paris Cite, 4, Avenue de l'Observatoire, 75006 Paris, France
c
ꢀ
ꢀ
Museum National d'Histoire Naturelle, UMR 7245 CNRS, Molecules de Communication et Adaptation des Micro-organismes, 61 rue Buffon, F-75231 Paris
Cedex 05, France
d Centre de recherche de Gif, Institut de Chimie des Substances Naturelles, UPR2301 CNRS, Avenue de la Terrasse, Gif-sur-Yvette Cedex, France
e Laboratorio de Farmacognosia, Universidade de Brasilia, Campus Universitario Darcy Ribeiro, Asa Norte, 70910-900 Brasilia, DF, Brazil
f Faculty of Pharmacy, Ho Chi Minh University of Medicine and Pharmacy, 41 Dinh Tien Hoang St., District 1, Ho Chi Minh City, Viet Nam
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 16 flavonoids were isolated and prepared from bud exudate of Gardenia urvillei and Gardenia
oudiepe, endemic to New Caledonia. Most of them are rare polymethoxylated flavones. Some of these
compounds showed noticeable activity against Leishmania (Leishmania) amazonensis, Plasmodium fal-
ciparum and Trypanosoma brucei gambiense, in addition to tubulin polymerization inhibition at low
micromolar concentration. We also provide a full set of NMR data as some of the flavones were
incompletely described.
Received 15 September 2014
Received in revised form
6 January 2015
Accepted 7 January 2015
Available online 8 January 2015
Dedicated to the memory of Prof. François
Tillequin.
© 2015 Elsevier Masson SAS. All rights reserved.
Keywords:
Flavonoids
Antivascular agents
Endothelial cells
Antiplasmodial
Trypanocidal
Antileishmanial
1. Introduction
combretastatin A4 and analogs, able to compromise selectively
established tumor vasculature [2]. In this case, exploitation of the
Microtubules are composed of
a
- and
b
-tubulin heterodimers
differences between normal and immature tumor blood vessels
leads to hemorrhagic necrosis within the tumors.
which are in constant dynamic interaction. They are key compo-
nents of the cytoskeleton and are deeply involved in cell division
and mitosis. Over the last two decades, several anticancer agents
were developed targeting the tubulin polymerization of endothelial
cells of neovessels formed during the tumoral process [1]. They can
act as antiangiogenics by preventing the formation of tumor
vasculature, or as vascular disrupting agents, such as
The protozoal cytoskeleton is also deeply involved in the suc-
cessive steps of the life cycle of parasite species [3e5]. The high
mortality and morbidity rate caused by malaria, sleeping sickness
and leishmaniasis in developing countries makes the fight against
these parasitic infections a priority in public health policy.
Some natural flavonoids have previously been shown to possess
antiangiogenic, antivascular, anti-proliferative, anti-plasmodial,
trypanocidal and leishmanicidal properties [6e16]. In this context,
the aim of this study was to further explore some of these biological
activities of flavone derivatives exhibiting rare substitution
* Corresponding author.
E-mail address: raphael.grougnet@parisdescartes.fr (R. Grougnet).
http://dx.doi.org/10.1016/j.ejmech.2015.01.012
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

94
L.H. Mai et al. / European Journal of Medicinal Chemistry 93 (2015) 93e100
patterns on A ring, isolated from the exudate of Gardenia urvillei
and Gardenia oudiepe (Rubiaceae), both endemic from New Cale-
donia and considered as non-endangered species. Flavonoids were
extracted from concentrated plant exudate using a minimum of
organic solvent. This extraction methodology is efficient and
respectful to both the phytoresource and the environment. Meth-
ylated and acetylated derivatives were then semi-synthetized in
order to establish structureeactivity relationships within this series
of compounds.
parasitic activities, as described hereafter.
2.2. Biological evaluation
2.2.1. Cytotoxic activity on L6 and B16 cell lines
The flavonoids were initially tested for their cytotoxicity against
the L6 rat skeletal myoblast cell line and the B16 murine melanoma
cell line. The results presented in Table 1 show that most com-
pounds were relatively non-cytotoxic to L6 cells with 13 out of the
16 compounds showing less than 50% of inhibition of cell prolif-
eration (IC50) at a concentration of 10 mM.
2. Results and discussion
For the B16 murine melanoma cell line, most compounds also
demonstrated relatively high IC50 values with the exception of 4
2.1. Preparation of the flavonoids library
(IC50 ¼ 11.5
mM). There was no apparent correlation between the
two cell lines in terms of cytotoxicity.
The exudate covering the flowering buds and leaf bases of G.
urvillei or G. oudiepe (Rubiaceae) was dissolved in a minimum
volume of dichloromethane. The solution obtained was filtered to
remove all small plant pieces and evaporated to dryness under
reduced pressure. The use of exudate is highly appreciated for
studies working with endemic species because the material
collection does not cause damage to the tree. Moreover, vegetal
extraction is not required because the exudate, sometimes referred
to as ‘gum’ or ‘resin’, is a concentrate of secondary metabolites
[17e19] that allows us to save extraction time and to avoid the use
of large volumes of organic solvents.
2.2.2. Morphological activity on EA.hy926 endothelial cells
The structural stilbene similarity of methoxylated flavonoids
with the antivascular agent combretastatin A4 (Fig. 1) makes them
promising candidates as potential inhibitors of tumor blood vessels.
We therefore tested our library of compounds on endothelial cells
using immortalized HUVEC (EA.hy926), and evaluated their
rounding-up effect, which is considered predictive of a potential
in vivo antivascular activity [20,21].
Four compounds (1, 2, 3 and 8) presented low micromolar
rounding up activity on EA.hy926 endothelial cells (Table 1),
ranging from 1.56 to 25 mM. Fig. 2 depicts the typical rounding up
Chromatographic separations on silica gel permitted the isola-
tion of the following flavonoids: 5,7-dihydroxy-3,30,40,6-
tetramethoxyflavone
(1)
and
30,5,7-trihydroxy-3,40,50,6-
40,5,7-trihydroxy-3,6,8-
activity of compound 2 on EA.hy926 endothelial cells after a 2 h
exposure time. As stated above, this alteration in endothelial cell
morphology is an indicative feature of potential antivascular ac-
tivity. Despite several publications concerning the antiangiogenic
and antivascular properties of flavonoids and their modified ana-
logs [7,8,22e25], this study is, to the best of our knowledge, the first
to explore the 3-methoxy-flavones bearing quite rare substitution
patterns on A ring, i.e. 5-OH, 6-OMe, 7-OH, 8-H, along with their
methylated derivatives in the last 15 years [26e28].
tetramethoxyflavone (2) from G. oudiepe; 5,7-dihydroxy-
3,30,40,50,6-pentamethoxyflavone
(3),
trimethoxyflavone (6), 40,5,7-trihydroxy-3,6-dimethoxyflavone
(7), santin (5,7-dihydroxy-3,40,6-trimethoxyflavone) (8), and 3-
methoxy-kaempferol
(kaempferol-3-monomethylether)
(12),
from G. urvillei (Scheme 1). Some of these natural products were
rarely mentioned in the literature, sometimes without or with
incomplete NMR data (See Supporting Information). Moreover,
noticeable anti-tubulin and anti-parasitic activities were reported
for closely related flavones [16].
2.2.3. Inhibition of tubulin polymerization (ITP)
This led us to proceed, in a second step, to methylation and
acetylation in order to perform a screening of bioactivities on a li-
brary of 3-methoxykaempferol derivatives which were never
evaluated together on such targets. Thus, compounds 3, 8 and 12
were subsequently methylated (Scheme 2). 4 and 5 were generated
from 3, 9 and 10 from 8, and 13 and 14 from 12. Two acetylated
derivatives 15 and 16 were also semi-synthetized from commercial
kaempferol (11) (Scheme 3).
Some flavones were reported to inhibit tubulin polymerization
[26e28]. This prompted us to test our library of compounds for this
activity. We initially performed a screening test at the concentra-
tion of 30 mM to compounds 1, 2, 3 and 8, previously selected for
their morphological effects on EA.hy926 cells. All samples exhibited
potential inhibitory activity at this concentration (Table 1). The IC50
values of these compounds were then determined in the low
micromolar concentration range and therefore presented signifi-
cant inhibitory activity on the tubulin polymerization. Santin (8)
We then submitted this library of polymethoxylated and poly-
acetylated flavonoids (cf. Schemes 1e3) to various in vitro assays
aimed at detecting cytotoxic, antivascular, anti-tubulin and anti-
showed the most promising profile (5.7
mM), while 1, 2 and 3
exhibited a similar activity with IC50 of 19.3, 17.8 and 21.4
mM,
Scheme 1. Flavonoids from G. urvillei and G. oudiepe and modified analogues.

L.H. Mai et al. / European Journal of Medicinal Chemistry 93 (2015) 93e100
95
Scheme 2. Methylation of natural compounds 3, 8 and 12. (a) Reagents and conditions: Me2SO4 4 equiv., DBU 2 equiv. in dried acetone, room temperature, 1 h. Neutralization with
HCl 10% to pH7, precipitation with ice water.
or 30-amino-40-methoxy substituted B ring [29,35]. A too bulky
substitution on the B ring seems to lead to a slight decrease of ITP as
observed in the case of flavones isolated from G. urvillei and
G. oudiepe, exemplified by a 30-hydroxy-40,50-dimethoxyl 2 or
Scheme 3. Acetylation of kaempferol (11). (b) Reagents and conditions: Ac2O 4 equiv.
in dried pyridine, room temperature, 15 min or 4 h, precipitation with ice water.
30,40,50-trimethoxyl substitution 3 [28].
Interestingly, the role of the methoxyl group on 40 in this flavone
series seems similar of the one played by the 40 methoxyl group of
the B ring in combretastatin series and like in this series the ITP
activity is well correlated with the morphologic effect on EA.hy926
cells [20,21].
respectively.
2.2.4. Structure e activity ITP and morphological effect
relationships
Furthermore, the role of a methoxyl group on position 3 is also
predominant as illustrated by the lack of activity of compounds
exactly corresponding to 1 and 8 but bearing a single proton on 3.
These compounds are inactive (compounds 45 and 73 of ref. [30]).
Generally, the methylated semi-synthetic derivatives on posi-
tion 7 (compounds 4, 9 and 13) and on position 7 and 5 (com-
pounds 5, 10 and 14) were all less active on EA.hy926 cells than the
natural products used as starting material, and were therefore not
evaluated for ITP.
Following the determination of inhibition of tubulin polymeri-
zation (ITP) and morphological effects induced by several com-
pounds, structural requirements could be established. The good
activity of 5,7-dihydroxy-3,30,40,6-tetramethoxyflavone (1) and
santin (8) was also presented by centaureidin (30,5,7-trihydroxy-
3,40,6-trimethoxy-flavone) [26e28] the only 3-methoxy-flavone
bearing this kind of substitution on the A ring evaluated for ITP. The
structural differences are in 30 on the B ring: the lack of a hydroxyl
group for 1 and a methoxyl group instead of a hydroxyl one for 8.
This underlines the importance of a 40-methoxyl substitution on
the B ring and is supported by the fact that 7, which bears a phenol
on B ring at the 40 position, did not show any rounding-up activity.
Similarly, product with phenol on 40 and methoxyl group on 30 was
shown a weak inhibitor of tubulin polymerization [28]. The sub-
stitution pattern presented by 8, with only one methoxyl group on
40 was shown to express the strongest biological effect in our study.
Further substitution by additional methoxyl and/or hydroxyl group
as in the case of 1, 2 and 3 led to a 3-fold less active metabolite in
the ITP assay. These structureeactivity relationships are illustrated
in Fig. 3.
2.2.5. Anti-parasitic activity
Noticeable anti-parasitic activity has been reported for flavones
and related secondary metabolites such as flavanones, chalcones or
catechins [22,24] and for semisynthetic analogs [28]. Nevertheless,
with the exception of santin (8) [12], 5,7-dihydroxy-3,6-
dimethoxyflavones have never been screened against this type of
target.
This led us to perform an evaluation of natural and semi-
synthetic flavones against the promastigote form of Leishmania (L.)
amazonensis, the FcB1-Colombia strain of Plasmodium falciparum
and Trypanosoma brucei gambiense.
The ITP observed for 8 and 1 may also be considered in com-
parison with the results published for calycopterin-40-O-methyl
ether [29] and for 5-hydroxy-3,30,40,6,7,8-hexamethoxyflavone
[28], respectively. Surprisingly, these last two compounds were
not active, despite bearing the same substitution pattern on the A
ring, similarly to the very strong inhibitors of tubulin polymeriza-
tion, e.g., 5,30-dihydroxy-3,6,7,8,40-pentamethoxy-flavone [28,30]
and 30-amino-5-hydroxy-3,6,7,8,40-pentamethoxyflavone [29]. In
this case, for a same B ring, active compounds were those with a
santin-type A ring. This statement attracted our attention and let us
hypothesize that the mode and/or site of action is not the same as
for 3-methoxy-flavones with 5-hydroxy-6,7,8-trimethoxyl substi-
tution. Indeed, even if flavonoids seem to bind closely to the
colchicine binding site [31], the exact position remains to be
determined, data furnished by the literature are sometimes
controversial for related compounds [32e34].
2.2.5.1. Leishmania (L.) amazonensis. The leishmanicidal activity
results in Table 2 report inhibitory activity for some of the flavones:
natural products 1, 2, 3 and 7 were active with an IC50 value lower
than 10 mM, while 9, 12 and 15 presented an IC50 lower than 15 mM.
Moreover, some structureeactivity relationships emerge from this
screening. The flavonoids isolated from both exudates all exhibit
inhibitory activity, with the exception of 6 and 8. Products 6, 7, 12
and 13 bear a single phenol on the B ring, a methoxyle group at
position 3, and a phenol on position 5. Compound 7, which also
possesses an additional phenol at position 7 and a methoxyle at
position 6 is the most potent within this series. The lack of the
methoxyle group at position 6, together with an additional
methoxyle on position 8, leads to a 3e4 fold decrease in the activity,
as observed for flavones 12 and 6 respectively. These data may also
be compared to those published for hispidulin [12], which differs
from 7 by the presence of a proton at position 3, and was found to
be very active against Leishmania mexicana. Santin (8) demon-
strated a moderate activity against Leishmania (L.) amazonensis, as
against L. mexicana [12], confirming that a single methoxyle group
at position 40 is detrimental to leishmanicidal activity. However,
natural compounds 1, 2 and 3, with a bulkier B ring, were found to
possess the same anti-leishmanial properties as 7.
Natural product 6 did not show any rounding up activity.
However, this could arise from the presence on the B ring of a single
phenol only on 40. This kind of substitution appears to be smaller
than the minimum hindrance required, at least a methoxyl group
on 40, as in the case of 7 compared to compound 8, as described
above.
As a kind of illustration, the analogs of 6 with a 30,40-dihydroxy
or 40-hydroxy-30-methoxy [28] showed no ITP activity or cytotox-
icity. Nevertheless, this ability to inhibit tubulin polymerization has
already been observed in the case of flavones with a 5,7-dihydroxy-
6,8-dimethoxyl pattern on the A ring and a 30-hydroxy-40-methoxy
In the case of flavones trimethoxylated at 30,40,50, further
methoxylation at position 7 and 5 led to less active derivatives,
while methoxylation of santine at position 7 increased the anti-
leishmanial effect.

96
L.H. Mai et al. / European Journal of Medicinal Chemistry 93 (2015) 93e100
Table 1
Flavonoids 1-16: cytotoxicity against L6 and B16 cell lines, rounding-up activity on endothelial cells (EA.hy926), and inhibition of tubulin polymerization (ITP).
Compound
Cytotoxicity
L6 cellsa
EA.hy926c cells rounding-up (
m
M)
ITP 30
m
Md
ITP IC50emM)
B16 cellsb IC50 m
M
10
mM % viable cells
1 mM % viable cells
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
94.7
44.2
78.2
53.5
81.2
80.4
73.5
40.8
96.5
93.6
100.0
61.0
38.7
100.0
94.8
99.7
e
99.7
97.6
96.1
99.2
93.8
88.4
86.2
90.9
85.3
88.3
93.8
85.0
81.3
99.1
90.2
91.3
e
28.9
33.9
91.2
11.5
>100
>100
74.1
45.7
>100
>100
53.7
75.8
57.5
>100
39.8
40.7
e
25.0
1.6
12.5
75.1
99.0
69.9
e
19.3
17.8
21.4
e
>100
>100
>100
>100
12.5
>100
50e100
>100
>100
50.0
e
e
e
e
e
e
100.0
e
5.7
e
e
e
e
e
e
e
e
e
100.0
>100
>100
0.27
e
e
e
e
e
e
Combrestatin A4
Colchicine
e
e
e
e
e
e
e
0.36
a
Percentage of viable L6 cells at 10
m
M (rat skeletal myoblast cell line).
b
c
Concentration required to cause 50% cytotoxicity to murine B16 melanoma cells (IC50 mM).
Concentration ( M) required for the endothelial cells to adopt rounding shape within 2 h.
Percentage of inhibition of tubulin polymerization (ITP) at 30 M.
Concentration required to cause 50% inhibition (IC50 M) of tubulin polymerization.
m
d
e
m
m
For kaempferol (11), the moderate activity is in agreement with
2.2.5.2. P. falciparum and T. b. gambiense. Some of the flavones
demonstrated noticeable activity against Plasmodium and Trypa-
nosoma. The screening for anti-plasmodial activity against the
FcB1-Colombia strain of P. falciparum resulted in detectable inhi-
previous reports on Leishmania donovani [36], Leishmania peruviana
and Leishmania braziliensis [37]. Regarding its methylated and
acetylated derivatives, 3,7-kaempferoldimethylether (12) and tri-
bition at 10 m
M for compounds 1, 3, 4 and 5. They possess a 30,40-
dimethoxy or a 30,40,50-trimethoxy substitution on the B ring. For
these latter, on the A ring, the activity increased with the degree of
methoxylation.
After this first step of selection, the most active derivative 5,
semi-synthesized from 3, was found to be active against P. falcip-
arum with an IC50 of 5.8
mM and an IC90 of 10.8 mM by using the
[3H]-hypoxanthine assay. These data firmly support those pub-
Fig. 1. Combretastatin A4.
Fig. 2. A) Control endothelial cells EA.hy926 incubated with 1% DMSO (solvent), B) Endothelial cells exposed to compound 2 at 6.25 mM for 2 h.
acetylkaempferol (15) are the most promising compounds, while
the high IC50 of 16 agrees with former reports whereby the latter
did not show any strong effect against L. peruviana and L. braziliensis
[37].
lished for 30,40,50,5,6,7-hexamethoxyflavone isolated from Ager-
atum conyzoides [11], which bears an hydrogen on position 3.
However, this natural product exhibited cytotoxicity against L6 rat
skeletal myoblast cell line, while 5 was non-toxic when tested

L.H. Mai et al. / European Journal of Medicinal Chemistry 93 (2015) 93e100
97
Fig. 3. Comparison of the substitutions needed for activity in the inhibition of tubulin polymerization assay on the B ring for the 5,7-dihydroxy-3,6-methoxy-flavone derivatives.
against the L6 and B16 melanoma cell lines. This leads to the
conclusion that the 3-methoxyl substitution does not change the
antiplasmodial activity, but affords improved target selectivity
which could permit to avoid detrimental side effects.
When screened against T. b. gambiense, good inhibition levels
were observed at 50 mM for 1, 2, 7, 8, 11 and 12. All of the afore-
4.2. Chemistry
4.2.1. Isolation of flavones 1-3, 6-8, 12
30.0 g of the exudate covering the flowering buds and leaf bases
of G. urvillei were dissolved in 200 mL of dichloromethane. The
solution was filtered under suction through a Büchner funnel and
evaporated to dryness under reduced pressure to furnish 27.0 g of
exudate free from buds and leaf pieces.
400.0 g of flowering buds and leaf bases of G. oudiepe covered
with exudate was dissolved in 1L of dichloromethane. The solution
was filtered under suction through a Buchner funnel and evapo-
rated to dryness under reduced pressure to furnish 102.0 g of
exudate free from buds and leaf pieces.
mentioned compounds are naturally occurring, have relatively low
degree of methylation and, with the exception of 1 and 2, share a B
ring substituted only at 40. At 10
mM, only 1 continued to demon-
strate a strong effect against parasite growth. The IC50 of 1 was
determined as 3.7 mM.
Repeated chromatographic separations on silica gel column
using cyclohexane-dichloromethane and dichloromethane-
methanol mixtures of increasing polarity as eluent permitted the
isolation of 5,7-dihydroxy-3,30,40,6-tetramethoxyflavone (1)
(210 mg) and 30,5,7-trihydroxy-3,40,50,6-tetramethoxyflavone (2)
(12 mg) from G. oudiepe exudate; 5,7-dihydroxy-3,30,40,50,6-
pentamethoxyflavone (3) (170 mg), 40,5,7-trihydroxy-3,6,8-
3. Conclusion
Our work once again demonstrates the importance of natural
products as a source of leader compounds for antiangiogenesis or
vascular disruption [28,29,34,35]. It is noteworthy that this series of
naturally occurring compounds should be regarded as a model for
future development and as valuable starting materials for phar-
macomodulation. These promising data are also enhanced by the
fact that the most active flavonoids were found to be specific for
different targets, without any acute cytotoxicity on mammalian
cells (B16 and L6 cells). Moreover, santin (8), the strongest inhibitor
of tubulin polymerization from our library, was also found nontoxic
at high concentrations on human peripheral mononuclear blood
cells [38] and on lymphoid mice cells [12] and exhibited strong
cytotoxicity against several cancer cell lines [38], while 5,7-
trimethoxyflavone
(6)
(10
mg),
40,5,7-trihydroxy-3,6-
dimethoxyflavone (7) (150 mg), santin (5,7-dihydroxy-3,40,6-
trimethoxyflavone) (8) (320 mg), and 3-methoxy-kaempferol
(isokaempferide) (12) (155 mg), from G. urvillei exudate.
4.2.2. Semi-synthesis of flavones 4, 5, 9, 10, 13, 14
The methylation reactions were performed in one step by stir-
ring the substrate with dimethyl sulfate (Me2SO4, 4 equiv.) and 1,8-
diazabicyclo[5.4.0]undec-7-en (DBU, 2 equiv.) in dried acetone at
room temperature for 1 h. The crudes were precipitated and
washed with iced water. The resulting residue were solubilized
with ethyl acetate (15 mL) and treated with a solution of 1N HCl
(3 mL). The final products were extracted with ethyl acetate
(3 ꢀ 10 mL); the organic phases were washed with a saturated
solution of NaCl and dried over Na2SO4. After filtration and solvent
evaporation, methylated compounds were purified by chromatog-
raphy in a silica gel column, using a mixture of CH2Cl2/CH3OH (95/5,
v/v) as eluent.
dihydroxy-3,30,40,50,6-pentamethoxyflavone (3) showed
selectivity index when comparing its high rounding-up activity
with its very low cytotoxicity on B16 and L6 cells.
Regarding the anti-parasite evaluation, the antileishmanial ac-
tivity of some derivatives is noticeable and may also constitute
promising alternatives for a highly incapacitating disease currently
treated by drugs with serious side effects.
As some compounds were incompletely described, we also
provide a complete set of 1H and 13C NMR data (supporting
information).
a high
Methylation of 125 mg (0.39 mmol) of 5,7-dihydroxy-
3,30,40,50,6-pentamethoxyflavone (3) gave 4 and 5 with a 3/1 ratio
and an overall yield of 86%.
Methylation of 150 mg (0.44 mmol) of santin (8) gave 9 and 10
with a 3/1 ratio and an overall yield of 92%.
4. Experimental section
Methylation of 120 mg (0.40 mmol) of 3-methoxykaempferol
(12) gave 13 and 14 with a 3/1 ratio and an overall yield of 95%.
4.1. Plant material
Bud exudates of G. urvillei were collected by two of us (V.D. and
C.P.) in August 2007 in the dry forest at the surroundings Noumea,
South Province of New Caledonia, and bud exudates of G. oudiepe
4.2.3. Semi-synthesis of flavone 15
80 mg (0.28 mmol) of commercial kaempferol (11) were acet-
ylated by stirring with acetic anhydride in dry pyridine at room
temperature for 15 min. The crudes were precipitated and washed
in iced water. The resulting was recrystallized in CH2Cl2/CH3OH (9/
1, v/v) to furnish 108 mg (0.26 mmol) of triacetyl derivative 15
(yield: 95%).
^
were collected in October 2008 in Foret Plate, North Province of
New Caledonia. Voucher specimens (POU-0143 and POU-0290,
respectively) were deposited at the Herbarium of the Botanical
and Tropical Ecology Department of the IRD Center, Noumea, New
Caledonia.

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L.H. Mai et al. / European Journal of Medicinal Chemistry 93 (2015) 93e100
Table 2
Activity of compounds 1-16 against L. (L.) amazonensis, P. falciparum, and T. brucei gambiense.a
Compound
L. (L.) amazonensis
IC50 g/mL)
L. (L.) amazonensis
IC50 M)
P. falciparum FcB1
T. b. gambiense
(m
(
m
At 10
m
Mb
At 50
m
Mb
At 10 m
Mb
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
3.05
3.99
3.9
8.15
10.23
9.65
21.77
31.61
27.33
8.07
63.15
14.80
37.17
27.56
14.29
22.95
33.00
12.27
48.52
21.3
0.0
13.2
21.7
35.0
0.6
0.0
0.0
0.0
0.0
0.0
2.9
7.2
3.9
7.6
3.6
e
99.8
82.6
38.1
40.5
72.4
50.7
88.8
83.1
8.0
24.9
86.5
93.4
26.4
19.9
5.6
76.2
37.8
42.9
17.6
25.9
14.6
40.7
33.2
18.3
29.4
55.1
36.6
25.2
4.6
9.1
13.66
9.84
2.67
21.73
5.3
13.83
8.27
4.49
7.53
13.6
5.57
14.56
0.067
e
0.0
0.0
13.7
e
Amphotericin B
Chloroquine (IC50 nM)
0.0725
e
e
0.110
e
e
Pentamidine (IC50
m
M)
e
e
e
e
0.007
a
Each sample was tested in three assays.
Percentage of parasite growth inhibition.
b
4.2.4. Semi-synthesis of flavone 16
controls with DMEM only and MTT were run to subtract back-
ground absorbance. The compound concentration that inhibited
cell viability by 50% (inhibitory concentration for 50% of cells, or
IC50) was determined using the GraphPad Prism software.
80 mg (0.28 mmol) of commercial kaempferol (11) were acet-
ylated by stirring with acetic anhydride in anhydrous pyridine at
room temperature for 4 h. The crudes were precipitated and
washed in ice water. The resulting was recrystallized in CH2Cl2/
CH3OH (9/1, v/v) to furnish 120 mg (0.26 mmol) of tetraacetyl de-
rivative 16 (yield: 95%).
4.5. Endothelial cell morphology assay
To assess the effects of the compounds on the morphology of
endothelial cells, we used the EA.hy926 endothelial cell line, which
is derived from the fusion of human umbilical vein endothelial cells
(HUVEC) with the permanent human cell line A549 [39]. The
EA.hy926 cell line is considered as one of the best immortalized
HUVEC cell lines because these cells express most of the
biochemical markers of parental HUVEC cells [40]. EA.hy926 cells
4.3. Cytotoxicity evaluation on L6 cells
The cytotoxicity analysis in the rat skeletal myoblast cell line (L6
cells) was conducted in 96-well microtiter plates, each well
receiving 100
m
L of culture medium with approximately 4 ꢀ 104
cells. After 24 h, the medium was removed from all wells, and serial
drug dilutions prepared at 10
concentration was tested in triplicate. After 72 h of incubation,
plates were inspected under an inverted microscope to ensure
growth of the controls and sterile conditions, and then 10
added to each well for 2 h. After formazan crystal formation, the
culture medium supernatant was removed from the wells by
m
M and 1
m
M. Each compound at each
were grown in DMEM containing 2 mM
bovine serum, 100 U/mL penicillin and 100
humidified atmosphere (37 ꢁC, 5% CO2). Exponentially growing
EA.hy926 cells were plated onto 96 well plates at 5000 cells/100 L/
well. 24 h after plating, the medium was aspirated, and 100 L of
L
-glutamine, 10% fetal
mg/mL streptomycin in a
mL of MTT
m
m
medium containing the test compound added to the well con-
aspiration without disturbing the formazan precipitate. 100 mL of
taining the cells (in triplicate) and incubated for 2 h (37 ꢁC, 5% CO2).
detergent reagents were then added to each well, and plates left at
room temperature in the dark for 2 h. Absorbance was measured at
570 nm in order to determine the cell proliferation rate.
The highest concentration used was 100 mM followed by serial two-
fold dilutions down to low nanomolar concentrations, if needed for
the most morphologically active compounds. After the 2 h-incu-
bation period, digital photographs were taken of representative
center areas of each well at a magnification of 100X and 200X. The
results are presented as the minimum concentration that could
elicit the rounding up of more than 15% of cells in a field in order to
exclude normal mitotic cells which is less that 15% in control 1%
DMSO treated cells. Triplicate concentrations on the same plate
were routinely performed with combretastatin A4 included in the
experiments as a positive internal standard.
4.4. Cytotoxicity evaluation on B16 cells
Murine B16 melanoma cells were grown in DMEM medium
containing 2 mM
penicillin and 100
L
-glutamine, 10% fetal bovine serum, 100 U/mL
m
g/mL streptomycin (37 ꢁC, 5% CO2). All com-
pounds were initially dissolved in DMSO at a stock concentration of
2.5 mg/mL and were further diluted in cell culture medium.
Exponentially growing cells were plated onto 96-well plates at
5000 cells per well in 100
100 L of medium containing the compound at final concentrations
ranging from 0.01 to 100 M were added to the wells containing the
m
L of culture medium. 24 h after plating,
4.6. Inhibition of tubulin polymerization assay (ITP assay)
m
m
Tubulin assembly in microtubules was carried out using the
fluorescent dye DAPI (40,6-diamidino-2-phenylindole) [41] in a 96-
well plate format as described previously [42,43]. The standard
assay was performed as follows: wells were charged with purified
tubulin (Cytoskeleton, 97% pure, final concentration 1 mg/mL) in
cells (in triplicate), and incubated for 48 h at 37 ꢁC with 5% CO2.
After the 48 h exposure period to the test compounds, cell viability
was assayed using the MTT test and absorbance read at 562 nm in a
microplate reader (BioKinetics Reader, EL340). Appropriate

L.H. Mai et al. / European Journal of Medicinal Chemistry 93 (2015) 93e100
99
PME buffer (100 mM PIPES (1,4-piperazinebis(ethanesulfonic acid);
1 mM MgSO4; 2 mM EGTA) with 10 M DAPI and varying con-
centrations of the test compounds using colchicine at 30 M as a
positive internal standard control. The final concentrations used for
the test compounds were started at 30 M and diluted in 3-fold
decrements until no inhibition was observed. Triplicate wells
were run for each concentration. After pre-incubation at room
temperature for 45 min, 1 mM GTP (5 mL) was added to each well to
initiate tubulin polymerization, and the plate was then transferred
to a thermostatically-controlled Victor plate reader at 37 ꢁC for an
additional 2 h. Fluorescence was then read at the excitation
wavelength of 360 nm and emission wavelength of 450 nm. The
4.9. Leishmania (Leishmania) amazonensis in vitro assay
m
m
The compounds were tested against promastigotes of Leish-
mania (Leishmania) amazonensis (L(L)a)-MHOM/BR/PH8) main-
tained in Schneider's culture medium (Sigma) with 20% heat-
inactivated fetal calf serum, at 22e25 ꢁC. Compounds were dis-
solved in DMSO (Sigma) or DMSO/Tween 80 (Sigma) (50/50)
mixture, and diluted in Schneider's medium (Sigma). The final
DMSO concentration for each experiment did not exceed 0.1% and
growth controls with/without DMSO were performed. Experiments
were performed in 96-well plates with compounds at concentra-
m
tions ranging from 100.00 to 1.56 mg/mL. The inoculum consisted of
percentage of inhibition was determined as follows: 1 e (
ple/ F(control) X 100, where
F control ¼ F(no inhibition) e
F(complete inhibition), and
F sample ¼ F(sample) e F(complete
D
F(sam-
105 parasites per well in logarithmic growth phase. The cells were
incubated at 22e25 ꢁC for 48 h. Amphotericin B (Sigma) was used
as the reference drug. Negative controls were performed with 105
parasites in Schneider's and culture medium only. Promastigote
viability was based on cellular conversion of the soluble tetrazo-
D
D
D
inhibition with colchicine). The IC50 for compound-induced inhi-
bition of tubulin polymerization is the concentration at which the
extent of inhibition of polymerization is 50% of the maximum value,
as determined from the semi-logarithmic plot of percentage inhi-
bition as a function of the logarithm of compound concentration
fitted to a sigmoidal model with variable slope using the nonlinear
regression program SigmaPlot (Jandel Scientific). Under these
conditions the IC50 value for colchicine inhibition of tubulin poly-
lium
salt
MTT
(3-[4.5-dimethylthiazol-2-yl]-2.5-
diphenyltetrazolium bromide, Sigma) into insoluble formazan by
mitochondrial enzymes. MTT (5 mg/mL) was dissolved in
Schneider's medium, 33
dissolved in DMSO (50
m
L/well, for 4 h at 37 ꢁC. Formazan was
mL/well). The number of viable promasti-
gotes was determined spectrophotometrically at 570 nm. All assays
were performed in triplicate.
merization was 0.36 mM.
Conflict of interest
4.7. P. falciparum in vitro assay
The authors have no conflicts of interest to disclose.
Acknowledgments
Antiplasmodial activity was determined on the FCB1 strain from
Colombia P. falciparum. A modified [3H]-hypoxanthine incorpora-
tion assay was used. Briefly, infected human red blood cells in
culture were exposed to serial drug dilutions (50 and 10 mM) in 96-
This work was financially supported by the Agence Universitaire
de la Francophonie (AUF, grant Hoang Linh Mai), by the Centre
National de la Recherche Scientifique (CNRS), the Institut National
well microtiter plates. Each compound at each concentration was
tested in triplicate. After 48 h of incubation at 37 ꢁC in a reduced
oxygen atmosphere, 25 m
L of [3H]-hypoxanthine was added to each
ꢀ
ꢀ
de la Sante et de la Recherche Medicale (INSERM) and by a grant
from the Institut National du Cancer (INCa, Boulogne-Billancourt,
France). We thank Dr. Alexandre Maciuk (Paris Sud University) for
providing some NMR facilities. The authors are very grateful to the
South Province and the North Province of New Caledonia who
facilitated our field investigations.
well. Cultures were incubated for a further 24 h prior to harvesting
on glass-fiber filters and washing with distilled water. Radioactivity
was counted using a 1450 Microbeta trilux (Wallac) (Liquid scin-
tillation and luminescence counter). The results were recorded as
counts per minute per well at each drug concentration and
expressed as a percentage of the untreated controls. Some com-
pounds that revealed moderate activity were then selected to
determine IC50 values. Repeated assays with decreasing concen-
Appendix A. Supplementary data
trations (from 100 to 0.195 mM) were conducted. IC50 values were
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.01.012.
calculated using the sigmoidal inhibition curves. All assays were
performed in triplicate. The antimalarial drug chloroquine was
used as positive control.
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