Synthesis and antiprotozoal activity of 4-arylcoumarins

Article abstract of DOI:10.1016/j.ejmech.2009.10.022

A large series of 4-arylcoumarins was synthesized by Suzuki-Miyaura cross-coupling reaction and evaluated for antiprotozoal activity against Plasmodium falciparum and Leishmania donovani. Several compounds were found to strongly inhibit the proliferation

Full text of DOI:10.1016/j.ejmech.2009.10.022

European Journal of Medicinal Chemistry 45 (2010) 864–869
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antiprotozoal activity of 4-arylcoumarins
,
b
b
b
b
b
Jean-Thomas Pierson a , Aurelien Dumetre , Sebastien Hutter , Florence Delmas , Michele Laget ,
´
`
´
`
Jean-Pierre Finet a, Nadine Azas b , Sebastien Combes
,
a,
**
*
´
a UMR-CNRS 6264, Laboratoire Chimie Provence, Universite´s Aix-Marseille I, II and III, Faculte´ des Sciences de Saint-Je´roˆme, Avenue Escadrille-Normandie-Niemen,
13397 Marseille Cedex 20, France
b UMR-MD 3, Relations Hoˆte-Parasites, Pharmacologie et The´rapeutique, Universite´ Aix-Marseille II, Faculte´ de Pharmacie, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 5, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A large series of 4-arylcoumarins was synthesized by Suzuki-Miyaura cross-coupling reaction and
evaluated for antiprotozoal activity against Plasmodium falciparum and Leishmania donovani. Several
compounds were found to strongly inhibit the proliferation of human cell line and/or parasites. The
4-(3,4-dimethoxyphenyl)-6,7-dimethoxycoumarin exhibit a potent activity on L. donovani amastigotes
with a selectivity index (SI ¼ 265) twice than amphotericin B (SI ¼ 140).
Received 11 May 2009
Received in revised form
9 October 2009
Accepted 15 October 2009
Available online 23 October 2009
Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords:
Coumarins
Palladium-mediated cross-coupling
Neoflavonoids
Cytotoxicity
Antiprotozoal activity
1. Introduction
capacity to escape the host immune responses [6]. These similari-
ties allow to consider the potential antiparasitic activity of anti-
Malaria and leishmaniasis are the most important parasitic
diseases in the world. Malaria is the most threatening, with an
annual incidence of 250 million clinical cases and an annual
mortality of 1 million deaths, mainly in children in Sub-Saharan
countries [1]. Its therapeutic management has become problematic
since the emergence of multidrug-resistant strains of Plasmodium
falciparum [2]. The challenge is to find new active compounds
structurally different to current antiplasmodial molecules for which
resistance arises [3].
Leishmaniasis is endemic in 88 countries, leading to 500 000
new cases and 50 000 deaths each year for its visceral form caused
by Leishmania donovani [4]. The therapeutic is based on antimonial
derivatives as first-line drugs and amphotericin B and pentamidine
as second-line drugs. However, resistant parasites have emerged in
several endemic areas in India and Sudan, underlying the urgent
need of new antileishmanial drugs [5].
cancer compounds. For instance, miltefosine, an anticancer
alkyllysophospholipid has a potent activity on Leishmania amasti-
gotes and other trypanosomatid parasites [7].
We previously reported the synthesis and evaluation of 4-aryl-
coumarins as potent anticancer agents [8–10]. Two poly-
methoxylated and hydroxylated derivatives have shown very
interesting antiproliferative activity on human cancer cell lines. It is
therefore relevant to consider their potential activity towards
protozoan parasites. Moreover among their various biological
properties 4-arylcoumarins exhibited interesting pharmacological
activity in-vitro against chloroquine resistant strains of P. falcipa-
rum[11] and in-vivo against P. berghei [12]. Antileishmanial activity
of natural 4-arylcoumarins was also reported on promastigote and
amastigote forms of Leishmania amazonensis[13] and L. braziliensis
[14]. Overall, the mechanisms of action of such molecules remain
unclear in particular on the amastigote form of Leishmania parasites.
In this purpose, we prepared a large series of 4-arylcoumarins
variouslysubstitutedonphenyl ringsandinvestigatedtheirpotential
antiproliferative properties towards P. falciparum and L. donovani.
Protozoan parasites share several important features with
cancer cells regarding their proliferative properties and their
2. Chemistry
* Corresponding author. Tel./fax: þ33 491 835 564.
** Corresponding author. Tel.: þ33 491 288 291; fax: þ33 491 288 758.
E-mail addresses: nadine.azas@pharmacie.univ-mrs.fr (N. Azas), sebastien.
combes@univ-provence.fr (S. Combes).
The 4-Arylcoumarins were synthesized by ligand coupling
reaction of two aromatic units, the preformed coumarin ring and
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.10.022

J.-T. Pierson et al. / European Journal of Medicinal Chemistry 45 (2010) 864–869
865
R1
R2
O
O
R1
R2
O
O
R1
R2
O
C
O
a
b
R1- R3 = H, OMe
R4- R6 = H, OH, OMe
1 - 13, 15 - 18, 20 - 22, 24, 25
A
R3 OH
R3 OSO2CF3
R3
B
33-40
R4
R6
O
R5
c, d
e
R4
H
H
H
H
H
R5
OH
OH OMe
OMe OH
OMe OMe
OBn OBn
R6
H
MeO
R2
O
26
27
28
29
30
31
32
MeO
MeO
O
O
B(OH)2
R2 = H
R2 = OMe 23
14
19
MeO
MeO
R4
R6
R5
OMe OH OMe
OMe OMe OMe
OAc
OH
OMe
OH
Scheme 1. Synthesis of 4-arylcoumarins 1–25 by palladium-catalysed coupling between triflates 33–40 and boronic acids 26–32. Reagents and conditions: (a) (CF3SO2)2O, Et3N,
CH2Cl2; (b) ArB(OH)2, Pd(PPh3)4, CuI, Na2CO3, toluene, 110 ꢀC; (c) 30, Pd(PPh3)4, CuI, Na2CO3, toluene, 110 ꢀC; (d) aqueous HBr, 50 ꢀC, then H2O; (e) (CH3CO)2O, CH3CO2Na.
a conveniently substituted phenyl derivative. The 4-trifluoro-
methylsulfonyloxycoumarins were easily prepared in 73–97% yield
by treatment of the appropriate 4-hydroxycoumarins with triflic
anhydride [15]. In the modified Suzuki conditions, the C-4 arylation
of the 4-activated coumarins was performed with arylboronic acids
in presence of a palladium catalyst and copper (I) iodide as a co-
catalyst (Scheme 1) [8,15–17]. The resulting neoflavones were
isolated in good to excellent yields (59–99%, Table 1). Use of the
benzyl-protected arylboronic acid 30 in the palladium-copper
catalyzed Suzuki reaction led to the corresponding protected
hydroxycoumarin. Deprotection of the benzyloxy groups was
performed by treatment with hydrobromic acid [8], leading to the
expected dihydroxylated 4-arylcoumarin 19 in 77% yield. Acetyla-
tion of 12 and 21 was performed with acetic anhydride in presence
of sodium acetate [10], and led to esterified compounds 14 and 23 in
97% and 88% yield respectively (Table 1).
3. Pharmacology
The cytotoxicity of 4-arylcoumarins 1–25 was evaluated against
human monocytes THP1. Antiplasmodial activity was assessed
against the reference strain of P. falciparum type W2 resistant to
Table 1
Structure, yield, cytotoxic and antiprotozoal activity of 4-arylcoumarins 1–25 and reference drugs.
Product
R1-3
R4
R5
R6
Yield
(%)
Cytotoxicity (
m
M)a
Anti-Ld activity (
m
M)a
Anti-Pf activity (m
M)a
IC50
THP1
IC50
Pro
IC50
Ama
SI
IC50 W2
SI W2b
Amab
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
H
OMe
OMe
OMe
OMe
H
OMe
OMe
OMe
OMe
H
OMe
OMe
OMe
OMe
OH
OH
OMe
OH
OMe
OH
OH
OMe
OMe
OMe
OH
OMe
OMe
OH
H
H
H
H
H
H
H
OMe
OMe
H
H
H
H
H
OMe
OMe
H
H
H
H
H
H
H
OMe
OMe
82
93
99
88
85
74
81
72
68
86
82
74
75
81e
91
75
59
82
58e
83
84
78
88e
88
65
>176
64
>160
54
>176
25.6
>160
>54
>67
94.2
>292
99.3
32
>25.5
>152
>15
>146
>135
>139
>134
>88
>152
>45
>262
>14
>78
>12.5
>257
>248
–
26.4
> 25
5.4
12.8
>67
30.4
1.1
>6.7
NCc
>29.6
4.2
12
64
>14,7
1
NC
NC
NC
> 6,1
NC
> 6.2
NC
1.7
NC
TOX
>2.4
NC
>2.2
NC
1.4
>5.3
1.6
>9.7
TOX
3.0
TOX
NC
>3.4
–
–
7-methoxy
6-methoxy
5-methoxy
6,7-dimethoxy
>160
>54
>67
22.5
>117
22.4
>268
15.4
>122
>15
61.2
>135
64
>134
63.7
28.9
27.9
27.1
>14
25.8
>12.5
>103
72.3
–
>67
>137
>292
>139
>268
25.5
>152
<15
>146
>135
>139
>134
88
>152
45
>262
<14
78
<12.5
>257
>248
0.05
14
NC
>4.5
>265
>13.9
>4.3
1.1
>6.3
TOXd
>15.7
>51.9
>7.2
NC
1.4
>2.0
1.8
>109
TOX
18.1
TOX
NC
10
61.8
23.3
24.1
>15
9.3
5,7-dimethoxy
OMe
OH
OMe
OAc
OMe
OMe
OMe
H
OH
OMe
OH
OMe
OAc
OMe
OMe
2.6
19.1
> 25
64.5
74.2
25.3
2.4
13.9
4.3
3.5
> 50
>25
–
0.10
–
5,6-dimethoxy
5,6,7-trimethoxy
OH
OH
OMe
OMe
OMe
OH
25
OMe
NC
–
140
–
Doxorubicin
Amphotericin B
Chloroquine
0.38
–
–
0.70
40
57.1
a
Mean of three independent experiments.
b
c
Selectivity index was calculated according to the following formula: SI ¼ IC50 THP1/IC50 Parasite.
NC: not calculated due to insufficient solubility of compound.
TOX: toxic compound (SI < 1).
d
e
Overall yield including Suzuki coupling and acetylation or debenzylation.

866
J.-T. Pierson et al. / European Journal of Medicinal Chemistry 45 (2010) 864–869
chloroquine, pyrimethamine and proguanil. Antileishmanial
activity was evaluated against extracellular promastigotes and
intracellular amastigotes of the reference strain L. donovani (MHOM/
IN/80/DD8).
compound 7 is the most selective toward L. donovani amastigote
forms with an index twice than the reference amphotericin B. These
encouraging results justify a further prodrug derivatives develop-
ment to increase antileishmanial potency of 4-arylcoumarins.
4. Results and discussion
6. Experimental
Structure, cytotoxic and antiprotozoal activity of 4-arylcou-
marins are reported in Table 1. Compound 23 with a 3-acetoxy-4-
methoxyphenyl moiety was highly cytotoxic towards THP1 cells
(IC50THP1 <15 mM) as well as compounds 12 and 21 described
previously [9]. We reported the importance of the 3-hydroxy-4-
6.1. Chemistry
Melting points were taken on a Bu¨chi capillary apparatus and
are uncorrected. 1H and 13C NMR spectra were recorded at room
temperature on a Bruker AC 300 (300 MHz) spectrometer using the
methoxyphenyl ring leading to the cytotoxicity of compounds 12
deuterated solvent as an internal reference. Chemical shifts (
d) are
and 21 on human leukemia CEM cells (IC50 ¼ 0.083
m
M and 0.52
HBL100 cells
M respectively) [8,9]. These two mole-
mM
reported in ppm and J values are given in hertz. Combustion
´
respectively)
and
epithelial
mammary
analyses were performed at the Service d’Analyse Elementaire of
(IC50 ¼ 0.087
mM and 0.42
m
the Saint Jerome Faculty (Marseille, France). Separation by column
chromatography was performed using Merck Kieselgel 60. All
solvents were purified by standard techniques. Arylboronic acids
26–32 were synthesized according to the reported procedures
involving the reaction of the appropriate aryllithium with triiso-
propylborate [16,18]. 4-Trifluoromethylsulfonyloxy-coumarins 33–
40 were prepared as previously reported [16,19].
cules disturbed cell survival by depolymerizing the microtubule
network leading to a mitototic block and apoptosis induction.
Mechanisms of action on cell survival of acetylated derivative 23
were recently studied [10]. The three arylcoumarin analogues were
found to be able to promote apoptosis of CLL cells ex-vivo through
the caspase-dependent mitochondrial pathway. Despite their
promising use as antitumoral molecules, these three compounds
did not have the antiplasmodial or antileishmanial required selec-
tivity (Table 1).
6.1.1. 6-Methoxy-4-trifluoromethylsulfonyloxycoumarin (35)
Light yellow needles, 93%, mp 76–78 ꢀC. 1H NMR (CDCl3):
d 7.36
Among other 4-arylcoumarins tested, only compounds 3, 7, 14
and 20 displayed a significant antiprotozoal activity (Table 1,
column 9) associated to a weak cytotoxicity towards THP1 cells.
These four molecules had only an interesting activity against
(d, J ¼ 9.1 Hz, 1H, 8-H), 7.25 (dd, J ¼ 9.1, 2.8 Hz, 1H, 7-H), 7.08 (d,
J ¼ 2.8 Hz, 1H, 5-H), 6.51 (s, 1H, 3-H), 3.88 (s, 3H, OMe).
6.1.2. 5,6-Dimethoxy-4-trifluoromethylsulfonyloxycoumarin (40)
L. donovani amastigotes with an IC50Ama of 5.4, 1.1, 2.6 and 2.4
mM
Light yellow crystals, 97%, mp 95–98 ꢀC. 1H NMR (CDCl3):
d
7.24
(d, J ¼ 8.6 Hz, 1H, 7-H), 7.12 (d, J ¼ 8.6 Hz, 1H, 8-H), 6.31 (s, 1H, 3-H),
3.97 (s, 3H, OMe), 3.91 (s, 3H, OMe). 13C NMR (CDCl3):
159.5, 157.1,
respectively, and a selectivity index of at least 29.6, 265, 51.9 and
109 respectively. The selectivity index of compound 7 (SI ¼ 265) is
nearly twice than the antileishmanial reference molecule ampho-
tericin B (SI ¼ 140, Table 1).
d
149.6, 147.5, 144.7, 118.5, 118.3, 112.2, 109.2, 108.4, 61.6, 56.6.
Despite an important structural similarity between the reported
ranges of compounds 1–25, the difference of activity is quite
noteworthy. Considering to un- and monosubstituted coumarin
core derivatives 1–4, 6-methoxylated compound 3 exhibits the
highest activity on amastigote form. Importance of presence of 6-
OMe group for activity is observed for dimethoxylated analogues
6.1.3. Synthesis of 4-arylcoumarins by Suzuki coupling reaction –
General procedure
A mixture of 4-trifluoromethylsulfonyloxycoumarin (1 mmol),
arylboronic acid (1.5 eq.), tetrakis (triphenylphosphine) palladium
(0) (46 mg, 4 mol%), copper(I) iodide (210 mg, 1.1 eq.) and sodium
carbonate (742 mg, 7 eq.) in dry toluene (10 ml) and absolute
methanol (3 ml) was refluxed overnight. Then the reaction mixture
was diluted with chloroform (40 ml) and filtered through a short
pad of Celite. The filtrate was washed with a saturated aqueous
solution of sodium bicarbonate (3 ꢁ 20 ml) and the combined
aqueous layers were extracted with chloroform (3 ꢁ 20 ml). The
combined organic phases were washed with brine, dried over
Na2SO4 and the solvent was distilled under reduced pressure. The
residue was purified by column chromatography or directly by
crystallization to afford the 4-arylcoumarin derivative.
also. 6,7-diOMe compound
compared to 5,7-diOMe derivative 13, with an IC50Ama of 1.1 and
9.3 M and a selectivity index more than 265 and 15.7 respectively.
7 has a very significant activity
m
Finally, when the 6-OMe group is introduced on ring A to form the
5,6,7-trimethoxylated analogue 22, the activity is partially restored
(IC50Ama amounting to 4 mM). Further structure- activity rela-
tionship show that the best substitution pattern on ring B is either
a methoxy or acetoxy hydrophobic group on the 30- and 40-
position.
It is interesting to note major differences between activities of
compounds 3, 7, 14 and 20 against promastigote and amastigote
forms of L. donovani. As a consequence of the obligate intracellular
development of amastigotes inside the mammalian host macro-
phages, metabolism is distinct from promastigotes. Such differ-
ences will lead us to further investigate the mechanisms of action of
these four compounds. Preliminary results suggest that these
compounds may modulate the parasite phagocytosis by THP1 cells
and there is no evidence to indicate that the active compounds
exert their toxic effects through a tubulin mechanism.
6.1.3.1. 4-(30,40-dimethoxyphenyl)coumarin (1). Crystallised from
ether as fine white needles, 82%, mp 145 ꢀC (lit. [15], 145–146 ꢀC).
1H NMR (CDCl3,):
d
7.60 (d, J ¼ 7.9 Hz, 1H, 5-H), 7.56 (dd, J ¼ 8.1,
7.2 Hz, 1H, 7-H), 7.42 (d, J ¼ 8.1 Hz, 1H, 8-H), 7.25 (dd, J ¼ 7.9, 7.2 Hz,
1H, 6-H), 7.06 (dd, J ¼ 8.3,1.3 Hz,1H, 60-H), 7.01 (d, J ¼ 8.3 Hz,1H, 50-
H), 6.95 (d, J ¼ 1.3 Hz, 1H, 20-H), 6.38 (s, 1H, 3-H), 3.97 (s, 3H, OMe),
3.92 (s, 3H, OMe).
6.1.3.2. 4-(30,40-dimethoxyphenyl)-7-methoxycoumarin
(2). Crystallised from ether as white crystals, 93%, mp 158 ꢀC (lit.
5. Conclusion
[20], 151–153 ꢀC). 1H NMR (CDCl3):
d
7.47 (d, J ¼ 8.9 Hz, 1H, 5-H),
7.03 (dd, J ¼ 8.3, 1.8 Hz, 1H, 60-H), 6.98 (d, J ¼ 8.3 Hz, 1H, 50-H), 6.95
(d, J ¼ 1.8 Hz, 1H, 20-H), 6.88 (d, J ¼ 2.5 Hz, 1H, 8-H), 6.80 (dd, J ¼ 8.9,
2.5 Hz, 1H, 6-H), 6.20 (s, 1H, 3-H), 3.95(s, 3H, OMe), 3.91 (s, 3H,
OMe), 3.87 (s, 3H, OMe).
Among the various 4-arylcoumarins prepared, four compounds
exhibit potent antileishmanial activity. In contrast, no anti-
plasmodial activity was observed. The 30,40,6,7-tetramethoxylated

J.-T. Pierson et al. / European Journal of Medicinal Chemistry 45 (2010) 864–869
867
6.1.3.3. 4-(30,40-dimethoxyphenyl)-6-methoxycoumarin
(3). Crystallised from ether as white crystals, 99%, mp 178 ꢀC (lit.
210–213 ꢀC). 1H NMR (CDCl3):
d
7.16 (d, J ¼ 8.5 Hz, 2H, 20- and 60-H),
6.86 (d, J ¼ 8.5 Hz, 2H, 30- and 50-H), 6.52 (d, J ¼ 2.4 Hz, 1H, 8H), 6.25
(d, J ¼ 2.4 Hz, 1H, 6H), 5.99 (s, 1H, 3-H), 5.51 (s, 1H, OH), 3.87 (s, 3H,
OMe), 3.49 (s, 3H, OMe).
[15], 148–150 ꢀC). 1H NMR (CDCl3):
d
7.35 (d, J ¼ 9.0 Hz, 1H, 8-H),
7.13 (dd, J ¼ 9.0, 2.3 Hz,1H, 7-H), 7.00–7.06 (m, 3H, 20-, 50- and 60-H),
6.98 (d, J ¼ 2.9, 1H, 5-H), 6.38 (s, 1H, 3-H,), 3.97 (s, 3H, OMe), 3.92
(s, 3H, OMe), 3.75 (s, 3H, OMe). 13C NMR (CDCl3):
d
161.1, 155.9,
6.1.3.11. 4-(40-hydroxy-30-methoxyphenyl)-5,7-dimethoxycoumarin
(11). Purified by column chromatography, eluent CH2Cl2-EtOH
(49:1), and crystallized from ether as white needles, 82%, mp 172 ꢀC
155.1, 150.3, 149.3, 148.7, 127.8, 121.3, 119.6, 119.1, 118.3, 115.2, 111.6,
111.4, 109.9, 56.1, 56.0, 55.8.
(lit. [16], 172–173 ꢀC). 1H NMR (CDCl3):
d
6.92 (d, J ¼ 8.7 Hz, 1H, 50-
6.1.3.4. 4-(30,40-dimethoxyphenyl)-5-methoxycoumarin (4). Cry-
H), 6.81 (dd, J ¼ 8.7, 1.9 Hz, 1H, 60-H), 6.79 (d, J ¼ 1.9 Hz, 1H, 20-H),
6.52 (d, J ¼ 2.5 Hz, 1H, 8H), 6.26 (d, J ¼ 2.5 Hz, 1H, 6H), 6.02 (s, 1H, 3-
H), 5.54 (s, 1H, OH), 3.89 (s, 3H, OMe), 3.87 (s, 3H, OMe), 3.50 (s, 3H,
OMe).
stallised from ether as white crystals, 88%, mp 187 ꢀC. 1H NMR
(CDCl3):
d
7.47 (t, J ¼ 8.1 Hz, 1H, 7-H), 7.03 (d, J ¼ 8.1 Hz, 1H, 6-H),
6.89 (d, J ¼ 8.4 Hz,1H, 60-H), 6.87 (d, J ¼ 8.4 Hz,1H, 50-H), 6.82 (s,1H,
20-H), 6.70 (d, J ¼ 8.1 Hz, 1H, 8-H), 6.19 (s, 1H, 3-H), 3.94 (s, 3H,
OMe), 3.87(s, 3H, OMe), 3.53 (s, 3H, OMe). 13C NMR (CDCl3):
d
160.5,
6.1.3.12. 4-(3 -Hydroxy-4 -methoxyphenyl)-5,7-dimethoxycoumarine
(12). Purified by column chromatography, eluent CH2Cl2-EtOH
(49:1), and crystallized from ether as white needles, 74%, mp 152 ꢀC
0
0
´
´
´
157.3, 155.5, 155.0, 149.1, 148.0, 132.4, 132.3, 119.7, 116.0, 111.1, 110.2,
110.0, 109.3, 106.7, 56.0, 55.9, 55.6. Anal. Calcd for C18H16O5
(312.10): C, 69.22; H, 5.16. Found: C, 69.11; H, 5.15%.
(lit. [8], 152 ꢀC). 1H NMR (CDCl3):
d
6.84–6.87 (m, 2H, 20- et 50-H),
6.79 (dd, J ¼ 8.1, 1.9 Hz, 1H, 60-H), 6.51 (d, J ¼ 2.3 Hz, 1H, 8H), 6.24 (d,
J ¼ 2.3 Hz, 1H, 6H), 6.00 (s, 1H, 3-H), 5.64 (s, 1H, OH), 3.95 (s, 3H,
OMe), 3.87 (s, 3H, OMe), 3.50 (s, 3H, OMe).
6.1.3.5. 4-(40-hydroxyphenyl)-6,7-dimethoxycoumarin (5). Purified
by column chromatography, eluent CH2Cl2–EtOH (49:1), as light
yellow crystals, 85%, mp 240 ꢀC (lit. [21], 259–260 ꢀC). 1H NMR
(CDCl3):
d
7.38 (d, J ¼ 8.5 Hz, 2H, 20- and 60-H), 7.00 (d, J ¼ 8.5 Hz,
6.1.3.13. 4-(30,40-dimethoxyphenyl)-5,7-dimethoxycoumarin (13). Cry-
2H, 30- and 50-H), 6.93 (s, 1H, 5-H), 6.92 (s, 1H, 8-H), 6.22 (s, 1H,
stallised from ether as yellow crystals, 75%, mp 170 ꢀC (lit. [23],170–
3-H), 5.43 (s, 1H, OH), 3.97 (s, 3H, OMe), 3.78 (s, 3H, OMe). 13C NMR
171 ꢀC). 1H NMR (CDCl3):
d
6.89 (dd, J ¼ 8.3,1.3 Hz,1H, 60-H), 6.86 (d,
(CDCl3):
d
160.4, 158.0, 154.9, 152.2, 149.4, 145.3, 129.1, 126.1, 115.1,
J ¼ 8.3 Hz, 1H, 50-H), 6.81 (d, J ¼ 1.3 Hz, 1H, 20-H), 6.53 (d, J ¼ 2.5 Hz,
1H, 8H), 6.25 (d, J ¼ 2.5 Hz, 1H, 6H), 6.02 (s, 1H, 3-H), 3.94 (s, 3H,
OMe), 3.88 (s, 3H, OMe), 3.87 (s, 3H, OMe), 3.49 (s, 3H, OMe).
110.7, 110.5, 107.1, 100.2, 55.4, 55.3.
6.1.3.6. 4-(40-hydroxy-30-methoxyphenyl)-6,7-dimethoxycoumarin
(6). Purified by column chromatography, eluent EtOAc-petroleum
spirit (1:1), as white crystals, 74%, mp 164 ꢀC. 1H NMR (CDCl3):
6.1.3.14. 4-(30-acetoxy-40-methoxyphenyl)-5,7-dimethoxycoumarin
(14). Crystallised from ether as white crystals, 97%, mp 166 ꢀC
d
6.96–7.06 (m, 4H, 5-, 20-, 5’- and 60-H), 6.88 (s,1H, 8-H), 6.21 (s,1H,
(CHCl3–Et2O) (lit. [24], 196–198 ꢀC). 1H NMR (CDCl3):
d 7.15 (dd,
3-H), 6.11 (s, 1H, OH), 3.94 (s, 3H, OMe), 3.92 (s, 3H, OMe), 3.76
J ¼ 8.3, 2.2 Hz, 1H, 60-H), 6.98 (d, J ¼ 2.2 Hz, 1H, 20-H), 6.96 (d,
J ¼ 8.3 Hz, 1H, 50-H), 6.51 (d, J ¼ 2.4 Hz, 1H, 8H), 6.23 (d, J ¼ 2.4 Hz,
1H, 6H), 6.01 (s, 1H, 3-H), 3.89 (s, 3H, OMe), 3.87 (s, 3H, OMe), 3.51
(s, 3H, OMe). 13C NMR (CDCl3):
d
161.6, 155.5, 152.8, 150.1, 147.1,
146.9, 146.0, 127.5, 121.7, 114.8, 111.7, 111.4, 110.9, 107.4, 100.2, 56.3,
56.2, 56.1. Anal. Calcd for C18H16O6 (328.09): C, 65.85; H, 4.91.
Found: C, 65.82; H, 4.97%.
(s, 3H, OMe), 2.32 (s, 3H, Me). 13C NMR (CDCl3):
d 168.9,163.4, 160.8,
158.2, 157.2, 154.4, 151.1, 138.7, 132.2, 125.5, 122.8, 112.7, 111.2, 103.3,
95.6, 93.5, 56.0, 55.8, 55.5, 20.7.
6.1.3.7. 4-(30,40-dimethoxyphenyl)-6,7-dimethoxycoumarin (7). Cry-
stallised from ether as yellow crystals, 81%, mp 222–223 ꢀC (lit.
6.1.3.15. 4-(40-hydroxy-30,5’-dimethoxyphenyl)-5,7-dimethoxycoumarin
[22], 220–221 ꢀC). 1H NMR (CDCl3):
d
7.07 (dd, J ¼ 8.3, 1.9 Hz, 1H, 60-
(15). Crystallised from ether as fine white needles, 91%, mp 215 ꢀC
H), 7.02 (d, J ¼ 8.3 Hz, 1H, 50-H), 6.98 (d, 1H, J ¼ 1.9 Hz, 20-H), 6.97
(s, 1H, 5-H), 6.92 (s, 1H, 8-H), 6.25 (s, 1H, 3-H), 3.98 (s, 6H, 2ꢁ OMe),
3.92 (s, 3H, OMe), 3.79 (s, 3H, OMe).
(lit. [8], 215 ꢀC). 1H NMR (CDCl3):
d
6.54 (d, J ¼ 2.5 Hz, 1H, 8-H), 6.52
(s, 2H, 20- and 60-H), 6.26 (d, J ¼ 2.5 Hz, 1H, 6-H), 6.04 (s, 1H, 3-H),
5.62 (s, 1H, OH), 3.89 (s, 6H, 30- and 50-OMe), 3.88 (s, 3H, OMe), 3.51
(s, 3H, OMe).
6.1.3.8. 4-(40-hydroxy-30,50-dimethoxyphenyl)-6,7-dimethoxycoumarin
(8). Purified bycolumn chromatography, eluent CH2Cl2, as light pink
6.1.3.16. 4-(30,40,50-trimethoxyphenyl)-5,7-dimethoxycoumarin
crystals, 72%, mp 154 ꢀC. 1H NMR (CDCl3):
d
6.98 (s, 1H, 5-H), 6.89 (s,
(16). Crystallised from ether as yellow plates, 75%, mp 207 ꢀC (lit.
1H, 8-H), 6.69 (s, 2H, 20- and 60-H), 6.23 (s,1H, 3-H), 5.83 (s, 1H, OH),
[25], 204–206 ꢀC). 1H NMR (CDCl3):
d
6.54 (d, J ¼ 2.5 Hz, 1H, 8H),
3.95 (s, 3H, OMe), 3.92 (s, 6H, 30- and 50-OMe), 3.77 (s, 3H, OMe). 13C
6.49 (s, 2H, 20- and 60-H), 6.26 (d, J ¼ 2.5 Hz, 1H, 6H), 6.04 (s, 1H, 3-
H), 3.91(s, 3H, OMe), 3.88 (s, 3H, OMe), 3.85 (s, 6H, 30- and 50-OMe),
3.50 (s, 3H, OMe).
NMR (CDCl3):
d 160.5, 155.6, 152.8, 150.1, 147.3, 146.0, 136.1, 126.7,
111.8, 111.4, 107.3, 105.3, 100.3, 56.5, 56.4, 56.3. Anal. Calcd for
C19H18O7 (358.11): C, 63.68; H, 5.06. Found: C, 63.64; H, 5.10%.
6.1.3.17. 4-(30,40-dimethoxyphenyl)-5,6-dimethoxycoumarin (17). Puri-
6.1.3.9. 4-(30,40,50-trimethoxyphenyl)-6,7-dimethoxycoumarin
fied by column chromatography, eluent CH2Cl2–EtOH (49:1), as
(9). Crystallised from ether as white needles, 68%, mp 202 ꢀC. 1H
yellow crystals, 59%, mp 145 ꢀC. 1H NMR (CDCl3):
d 7.16 (d,
NMR (CDCl3):
and 60-H), 6.25 (s,1H, 3-H), 3.97 (s, 3H, OMe), 3.94 (s, 3H, OMe), 3.89
(s, 6H, 30- and 50-OMe), 3.78 (s, 3H, OMe). 13C NMR (CDCl3):
161.4,
d
6.97 (s, 1H, 5-H), 6.91 (s, 1H, 8-H), 6.67 (s, 2H, 20-
J ¼ 9.3 Hz, 1H, 7-H), 7.13 (d, J ¼ 9.3 Hz, 1H, 8-H), 6.92–6.87 (m, 3H,
20-, 5’- and 60-H), 6.24 (s, 3H, 3-H), 3.94 (s, 3H, OMe), 3.89 (s, 3H,
d
OMe), 3.87 (s, 3H, OMe), 3.21 (s, 3H, OMe). 13C NMR (CDCl3):
d 160.4,
155.4, 153.6, 153.0, 150.2, 146.1, 139.2, 131.1, 112.1, 111.3, 107.4, 105.6,
100.4, 61.0, 56.4, 56.4, 56.3. Anal. Calcd for C20H20O7 (372.12): C,
64.51; H, 5.41. Found: C, 64.28; H, 5.41%.
154.5, 149.7, 149.2, 148.4, 148.0, 146.4, 131.3, 120.1, 117.6, 116.3, 114.2,
112.2, 111.5, 110.2, 61.0, 56.6, 56.0, 55.9. Anal. Calcd for C19H18O6
(342.11): C, 66.66; H, 5.30. Found: C, 66.51; H, 5.41%.
6.1.3.10. 4-(40-hydroxyphenyl)-5,7-dimethoxycoumarin (10). Cry-
stallised from ether as fine white needles, 86%, mp 204 ꢀC (lit. [16],
6.1.3.18. 4-(40-hydroxyphenyl)-5,6,7-trimethoxy-coumarin (18). Cry-
stallised from ether as light pink plates, 82%, mp 232 ꢀC (lit. [8],

868
J.-T. Pierson et al. / European Journal of Medicinal Chemistry 45 (2010) 864–869
232 ꢀC). 1H NMR (CDCl3):
d
7.24 (d, J ¼ 8.6 Hz, 2H, 20- and 60-H), 6.88
6.2. Biology
(d, J ¼ 8.6 Hz, 2H, 30- and 50-H), 6.73 (s, 1H, 8-H), 6.06 (s, 1H, 3-H),
5.11 (s, 1H, OH), 3.94 (s, 3H, OMe), 3.81 (s, 3H, OMe), 3.29 (s, 3H,
OMe).
6.2.1. Reagents
Cell culture medium (RPMI 1640), foetal calf serum (FCS), L-
glutamine, non essential amino acids and others medium additives
were from Eurobio (Paris, France). All other chemicals were of
highest chemical purity and were purchased from Sigma unless
mention otherwise. Stock solutions of 4-arylcoumarin derivatives
and amphotericin B were prepared in DMSO. Stock solutions of
reference drugs (doxorubicin and chloroquine) were prepared in
ultrapure H2O.
6.1.3.19. 4-(30,40-dihydroxyphenyl)-5,6,7-trimethoxycoumarin
(19). Purified by column chromatography, eluent Et2O, as white
crystals, 77%, mp 199 ꢀC. 1H NMR (CDCl3):
d
6.92 (d, J ¼ 8.1 Hz, 1H,
50-H), 6.87 (d, J ¼ 1.9 Hz, 1H, 20-H), 6.77 (dd, J ¼ 8.1, 1.9 Hz, 1H, 60-H),
6.71 (s, 1H, 8-H), 6.25 (s, 1H, OH), 6.04 (s, 1H, 3-H), 5.75 (s, 1H, OH),
3.93 (s, 3H, OMe), 3.78 (s, 3H, OMe), 3.35 (s, 3H, OMe). 13C NMR
(acetone-d6):
d 160.5, 157.9, 156.2, 152.6, 152.2, 146.1, 144.9, 140.4,
131.9, 120.2, 115.9, 115.2, 114.2, 107.9, 97.1, 61.5, 61.1, 56.7. Anal.
Calcd for C18H16O7 (344.09): C, 62.79; H, 4.68. Found: C, 62.89; H,
4.70%.
6.2.2. Cytotoxic assays on human monocytes THP1
Cytotoxicity of 4-arylcoumarins was assessed on human mono-
cytes THP1 (ATCC, Manassas VA, USA) by colorimetric determination
of cell viability using the redox indicator Alamar BlueÒ [26]. Late log-
phase THP1 cells were incubated in RPMI 1640 (without phenol red)
6.1.3.20. 4-(40-hydroxy-30-methoxyphenyl)-5,6,7-trimethoxycoumarin
(20). Purified by column chromatography, eluent CH2Cl2–EtOH
(49:1), as white fine needles, 83%, mp 154 ꢀC (lit. [8], 154 ꢀC). 1H
supplemented with 10% FSC and 1% L-glutamine/penicillin–strepto-
mycin mix (complete RPMI medium), containing 10% Alamar BlueÒ
and a range of compound concentrations incorporated in duplicate
(final DMSO concentration less than 0.5%). Appropriate controls
treated with or without solvent (DMSO), and various concentrations
of doxorubicin, chloroquine and amphotericin B were added to each
set of experiments. After a 72 h incubation period at 37 ꢀC with 6%
CO2, reduction of Alamar BlueÒ from blue to red was measured by
absorbance monitoring at 570 and 630 nm. IC50THP1 was defined as
the concentration of compound required to induce a 50% decrease of
cell viability, corresponding to a 50% reduction of Alamar BlueÒ as
compared to the control culture. IC50 was calculated by non-linear
regression analysis processed on dose–response curves, using the
Table Curve software 2D v.5.0. IC50 values represent the mean value
calculated from three independent experiments.
NMR (CDCl3):
d
6.96 (d, J ¼ 8.5 Hz, 1H, 50-H), 6.85–6.89 (m, 2H, 20-
and 60-H), 6.72 (s, 1H, 8-H), 6.08 (s, 1H, 3-H), 5.72 (s, 1H, OH), 3.94
(s, 3H, OMe), 3.90 (s, 3H, OMe), 3.81 (s, 3H, OMe), 3.32 (s, 3H, OMe).
6.1.3.21. 4-(30-Hydroxy-40-methoxyphenyl)-5,6,7-trimethoxycoumarin
(21). Purified by column chromatography, eluent CH2Cl2–EtOH
(49:1), and crystallized from ether as fine white needles, 84%, mp
157 ꢀC (lit. [8], 157 ꢀC). 1H NMR (CDCl3):
d
6.92 (d, J ¼ 2.1 Hz, 1H, 20-
H), 6.89 (d, J ¼ 8.2 Hz, 1H, 50-H), 6.83 (dd, J ¼ 8.2, 2.1 Hz, 1H, 60-H),
6.71 (s, 1H, 8-H), 6.06 (s, 1H, 3-H), 3.95 (s, 3H, OMe), 3.94 (s, 3H,
OMe), 3.80 (s, 3H, OMe), 3.36 (s, 3H, OMe).
6.1.3.22. 4-(30,40-dimethoxyphenyl)-5,6,7-trimethoxycoumarin
(22). Crystallised from ether as fine white needles, 78%, mp 151 ꢀC.
1H NMR (CDCl3):
d
6.87–6.92 (m, 3H, 20-, 5’- and 60-H), 6.72 (s,1H, 8-
6.2.3. Antiplasmodial activity
H), 6.09 (s, 1H, 3-H), 3.94(s, 6H, 2ꢁ OMe), 3.89 (s, 3H, OMe), 3.81 (s,
Assays used culture-adapted P. falciparum reference strain W2.
Maintenance in continuous culture was done as described previously
[27]. Parasites were cultivated in 75 cm2-flasks containing RPMI
1640 (20 ml) supplemented with 25 mM HEPES, 25 mM NaHCO3,
10% of Aþ human serum and 1 ml of washed Aþ erythrocytes (final
haematocrit 2.5%). Parasitaemia was maintained daily between 1 and
6%. Dilutions used non-infected Aþ erythrocytes. Cultures were
incubated at 37 ꢀC, 10% O2, 6% CO2, 84% N2, with 90% humidity.
Cultures were monitored daily by microscopic examination of blood
smears fixed with methanol and stained with 10% Giemsa stain.
Parasite growth was assessed by flow cytometry using hydro-
ethidine (HE, Interchim, Montluçon, France) that is converted by
metabolically active parasites into ethidium [28]. After incubation
with hydroethidine, parasitized and uninfected erythrocytes were
all identified on the basis of fluorescence intensity and size.
Triplicate assays were performed in 96-well plates (Nunc Brand
3H, OMe), 3.31 (s, 3H, OMe). 13C NMR (CDCl3):
d
160.6, 156.7, 155.1,
151.7, 151.1, 149.0, 147.9, 139.5, 131.5, 119.8, 113.9, 111.3, 110.1, 107.2,
96.2, 61.2, 61.0, 56.2, 55.9, 55.8. Anal. Calcd for C20H20O7 (372.12): C,
64.51; H, 5.41. Found: C, 64.32; H, 5.42%.
6.1.3.23. 4-(30-acetoxy-40-methoxyphenyl)-5,6,7-trimethoxycoumarin
(23). Crystallised from ether as fine white needles, 88%, mp 122 ꢀC
(CHCl3–Et2O). 1H NMR (CDCl3):
d
7.19 (1H, dd, J 8.3 and 2.2, 60-H),
7.06 (1H, d, J 2.2, 20-H), 7.00 (1H, d, J 8.3, 50-H), 6.71 (1H, s, 8-H), 6.07
(1H, s, 3-H), 3.94 (3H, s, OMe), 3.89 (3H, s, OMe), 3.80 (3H, s, OMe),
3.34 (3H, s, OMe), 2.32 (3H, s, Me). 13C NMR (CDCl3):
d 168.7, 160.5,
156.9, 154.1, 151.7, 151.1, 139.4, 138.8, 131.3, 126.0, 122.5, 114.2, 111.3,
107.1, 96.2, 61.1, 56.2, 56.0, 20.6. Anal. Calcd for C21H20O8 (400.12):
C, 63.00; H, 5.03. Found: C, 62.98; H, 5.05%.
6.1.3.24. 4-(40-hydroxy-30,50-dimethoxyphenyl)-5,6,7-trimethox-
products, Fisher, Paris, France) containing 200
ml of asynchronous
ycoumarin (24). Crystallised from ether as fine white needles, 88%,
parasite cultures at 2% of parasitaemia and 2% haematocrit, and 5 ml
mp 205 ꢀC (lit. [8], 205 ꢀC). 1H NMR (CDCl3):
d
6.72 (s, 1H, 8-H), 6.58
of the appropriate extract dissolved in DMSO or ultrapure H2O.
Negative control treated by solvents (DMSO or H2O) and positive
controls (chloroquine) were added to each set of experiments. After
48 h incubation without medium change, plates were centrifuged
(s, 2H, 20- and 60-H), 6.10 (s, 1H, 3-H), 5.62 (s, 1H, OH), 3.94 (s, 3H,
OMe), 3.90 (s, 6H, 30- and 50-OMe), 3.81 (s, 3H, OMe), 3.36 (s, 3H,
OMe).
and the upper liquids were replaced with 200 ml hydroethidine
6.1.3.25. 4-(30,40,50-trimethoxyphenyl)-5,6,7-trimethoxycoumarin
solution (0.05 mg/ml in phosphate buffered saline (PBS)). Plates
were incubated 20 min in the dark at 37 ꢀC and washed three times
with PBS. Finally, cells were suspended in 1 ml of PBS to allow
determination of the number of cell events (around 300 per s) and
parasitaemia by flow cytometry using a FACSort flow cytometer
(Beckton Dickinson, Paris, France), equipped with an argon laser
(power of 15 mW, and wavelength of 488 nm). Settings were:
Forward Scatter (FSC-H), size: Voltage E-1, gain 1, mode Log; Side
(25). Crystallised from ether as white needles, 65%, mp 146 ꢀC. 1H
NMR (CDCl3):
d
6.70 (s, 1H, 8-H), 6.53 (s, 2H, 20- and 60-H), 6.08
(s, 1H, 3-H), 3.92 (s, 3H, OMe), 3.89 (s, 3H, OMe), 3.85 (s, 6H, 30- and
50-OMe), 3.78 (s, 3H, OMe), 3.36 (s, 3H, OMe). 13C NMR (CDCl3):
d
160.5, 156.9, 155.1, 152.5, 151.6, 151.0, 139.4, 137.8, 134.5, 113.8,
107.2,104.8, 96.2, 61.2, 61.0, 60.9, 56.2, 56.1. Anal. Calcd for C21H22O8
(402.13): C, 62.68; H, 5.51. Found: C, 62.84; H, 5.53%.

J.-T. Pierson et al. / European Journal of Medicinal Chemistry 45 (2010) 864–869
869
Scatter (SSC-H), granulosity: Voltage 250, gain 1, mode Log; Fluo-
rescence 2 (FL2), red fluorescence: Voltage 459, gain 1, mode Log.
The concentration of compounds required to induce a 50%
decrease of infected erythrocytes (IC50W2) was calculated from
three independent experiments as describe above. A specificity
index (SI) corresponding to the ratio between toxicity on THP1 and
antiplasmodial activity was expressed as follows: SI W2 ¼ IC50THP1/
IC50W2.
was calculated from three independent experiments as described
above. A specificity index (SI Ama) corresponding to the ratio
between cytotoxicity and activity against L. donovani amastigotes
was expressed as follows: SI Ama ¼ IC50THP1/IC50Ama.
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mM phor-
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(6% CO2). Appropriate controls treated with or without solvent
(DMSO) or a range of amphotericin B concentrations were added to
each set of experiments. After incubation, cells were fixed with
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David, Parasitol. Res. 88 (2002) 165–171.
IC50Ama was defined as the concentration of compound
required to induce a 50% decrease of infected macrophages, and