Synthesis and biological evaluation of a series of tangeretin-derived chalcones

Article abstract of DOI:10.1016/j.bmcl.2008.10.126

A series of chalcones polyoxygenated on the ring A (with pentamethoxy or 2′-hydroxy-3′,4′,5′,6′-tetramethoxy substitution patterns) was synthesized from tangeretin, a natural Citrus flavonoid. These chalcones were evaluated for their antiproliferative, ac

Full text of DOI:10.1016/j.bmcl.2008.10.126

Bioorganic & Medicinal Chemistry Letters 19 (2009) 167–169
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of a series of tangeretin-derived chalcones
Jérôme Quintin a, Julie Desrivot a, Sylviane Thoret b, Patrick Le Menez c, Thierry Cresteil b, Guy Lewin a,
*
a Laboratoire de Pharmacognosie (Univ. Paris-Sud 11, BIOCIS, UMR-8076 CNRS), Faculté de Pharmacie, av. J.B. Clément, 92296 Châtenay-Malabry Cedex, France
b ICSN-CNRS-UPR 2301, 91190 Gif-sur-Yvette, France
c Laboratoire de Chimie Thérapeutique (Univ. Paris-Sud 11, BIOCIS, UMR-8076 CNRS), Faculté de Pharmacie, av. J.B. Clément, 92296 Châtenay-Malabry Cedex, France
a r t i c l e i n f o
a b s t r a c t
A series of chalcones polyoxygenated on the ring A (with pentamethoxy or 20-hydroxy-30,40,50,60-tetra-
methoxy substitution patterns) was synthesized from tangeretin, a natural Citrus flavonoid. These chal-
cones were evaluated for their antiproliferative, activation of apoptosis, inhibition of tubulin assembly
and antileishmanial activities. Comparison with the reference analogous 30,40,50-trimethoxylated chal-
cones showed that such peroxygenated substitution patterns on the ring A were less beneficial to these
activities.
Article history:
Received 23 September 2008
Revised 27 October 2008
Accepted 28 October 2008
Available online 5 November 2008
Keywords:
Chalcones
Ó 2008 Elsevier Ltd. All rights reserved.
Tangeretin
Antiproliferative activity
Tubulin
Antileishmanial drugs
Chalcones are natural or synthetic compounds bearing the 1,3-
diphenylprop-2-en-1-one 1 framework, that have displayed a wide
pharmacological spectrum including, among others, cytotoxic,
antitumour, antiviral, and antiprotozoal activities.1 Many studies
of chalcones related to cancer have demonstrated the positive
influence of a polymethoxylated ring A on cytotoxicity, though
the optimal substitution pattern remains to be defined.2,3 For in-
stance, a 30,40,50-trimethoxyphenyl ring A is present in the strongly
cytotoxic chalcones 2, 3, and 5, that interfere with the mitotic
phase of the cell cycle.4–7 The biological profile of 2, 3, and 5 can
be easily related to the structural analogy of these chalcones with
combretastatin A4 6 and its amino analog 7, two powerful inhibi-
tors of tubulin assembly now under clinical investigation as their
respective prodrugs 8 and 9.8
More recently, it was shown that dimethoxylation or trimeth-
oxylation at 20,40,60-carbons were highly beneficial to cell cycle ar-
rest at G2/M, while hydroxylation at 20 was generally detrimental9
(this last observation was in contrast with previous results show-
ing high cytotoxicity of 20-hydroxylated chalcones against Jurkat
and U 937 cancer cells10). So a clear correlation between the poly-
methoxylation pattern of chalcones on the ring A and their cyto-
toxicity seems difficult to establish. Starting from tangeretin 10
we used this natural polymethoxylated flavone isolated from Citrus
for a straightforward access to chalcones peroxygenated on the
ring A through the easily available acetophenones 11a and 11b.
Until now, only two of such chalcones, pedicin 12, and isodidymo-
carpin 13, have been studied with regard to cytotoxicity that
proved to be weak [(12: IC50 (KB cells) 21.2 M; IC50 (Inhibition
of tubulin assembly) 300
M.11 Compound 13: IC50 (P-388 cells)
11.1
M12]. As chalcones 12 and 13 are unsubstituted on the ring
l
l
l
B, we undertook synthesis and cytotoxicity evaluation of chalcones
14a and 14b, and 16a and 16b bearing, respectively, on the ring B
the substitution pattern of reference antimitotic chalcones 2 and 5.
Chemistry: Synthesis of these new chalcones was achieved from
tangeretin 10 in two, three, or four steps according to scheme 1: (a)
basic degradation of 10 into acetophenone 11a;13 (b) methy-
lation of 11a to the pentamethoxyacetophenone 11b; (c)
Claisen–Schmidt condensation of 11a or 11b with isovanillin in a
mixture MeOH–aqueous KOH giving 14a and 14b respectively;
(d) same Claisen–Schmidt condensation of 11a or 11b but with
3-nitro-4-methoxybenzaldehyde leading to 15a and 15b, respec-
tively;14 (e) reduction of nitrochalcones 15a or 15b with SnCl2
providing the aminochalcones 16a or 16b. Reference 30,40,50-tri-
methoxylated chalcones 2, 4, and 5 were prepared in the same
way from 30,40,50-trimethoxyacetophenone. Lastly,
a Claisen–
Schmidt reaction between 3-nitrobenzaldehyde and 30,40,50-tri-
methoxyacetophenone or acetophenone 11a led to compounds
173 or 18, the 4-demethoxylated analogs of 3-nitrochalcones 4
and 15a (vide infra Biology).
From a spectral point of view, a striking difference between ser-
ies a (20-OH) and b (20-OMe) could be noted in the 1H NMR spectra:
in the series a, signals of both olefinic protons were deshielded
with chemical shifts for
Ha and Hb very near [2 doublets
(J = 16 Hz) at d 7.75 and 7.85 ppm in 15a; 2 doublets (J = 16 Hz)
* Corresponding author. Tel.: +33 146835593; fax: +33 146835399.
at d 7.77 and 7.78 ppm in 16a], or identical in 14a (one singlet of
E-mail address: guy.lewin@u-psud.fr (G. Lewin).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.126

168
J. Quintin et al. / Bioorg. Med. Chem. Lett. 19 (2009) 167–169
5
2
OMe
4
3
6
OMe
5'
2'
β
MeO
MeO
O
6'
4'
3'
α
O
OMe O
1
10
OMe
R1
OMe
OMe
OR
MeO
MeO
MeO
MeO
OMe
O
R2
O
R1
R2
R
OH
OH
NO2
NH2
H
Me
H
2
H
11a
11b
3
4
5
Me
H
R2
R1
OMe
O
R1
OMe OH
OH OMe 13
R2
MeO
MeO
OMe
OMe
R
MeO
12
OH
R4
R3
OMe
MeO
MeO
R6'
R
OH
NH2
6
7
8
9
R2'
R6’
OMe OH
O
OPO3Na2
NHserine, HCl
R2’
R3
R4
OH
OMe 14a
OMe 14b
OMe 15a
OMe 15b
OMe 16a
OMe 16b
OMe OMe OH
OH OMe NO2
OMe OMe NO2
OH OMe NH2
OMe OMe NH2
two protons at d 7.79 ppm) ; in the series b, the difference between
and Hb chemical shifts was much more marked (about
0.4 ppm) as in compounds 2, 4, and 5 [two doublets (J = 16 Hz)
for H and Hb, respectively, at 6.93 and 7.30 ppm in 15b, 6.87
and 7.26 ppm in 16b and 6.85 and 7.26 ppm in 14b]. The coupling
constant of 16 Hz observed in all the spectra (except 14a) indicates
the formation of only the expected E isomers. The strong variation
of the olefinic signals between series a and b correlates certainly
with a marked difference of conformations, that is related to the
presence in the series a of an intramolecular hydrogen bonding be-
tween the 20-hydroxyl proton and carbonyl oxygen.
Biology: The antiproliferative effect of chalcones was asseyed on
KB human buccal carcinoma cells and the activation of apoptosis
with DEVD-AMC as substrate in HL60 human leukemia cells.
Inhibition of tubulin assembly (ITA) was determined according to
Zavala and Guenard’s method.15 Compounds were tested at
0.1 mg/ml (ꢀ2 ꢁ 10ꢂ4 M) and estimated inactive when they de-
creased by less than 30% the maximum assembly rate of tubulin
in the absence of drug. The IC50 was calculated only for the most
active compounds and expressed in relation to deoxypodophyllo-
toxin (DPPT) in terms of the IC50/IC50 DPPT ratio. As depicted in Ta-
ble 1, all the chalcones polyoxygenated on the ring A displayed a
weak capacity to inhibit cell proliferation (still weaker for com-
pounds 14b–16b of the series b). This was in contrast to the strong
activity observed with the 30,40,50-trimethoxylated chalcones 2 and
5. The measured ITA of the four most active chalcones 2, 5 and 14a,
16a was in good correlation with the cell growth inhibition and
matched the activation of apoptosis: 2 and 5 were potent activa-
Ha
H
H
NO2
H
H
17
18
a
OH
OMe NO2
intramolecular bond between the 20-hydroxyl and carbonyl func-
tion (14a and 16a) exhibit significantly higher ITA that those which
do not have this bond (14b and 16b).
In other respects, a recent article reporting some highly selec-
tive antileishmanial 3-nitrochalcones led us to evaluate antileish-
14a
c
e
e
d
a
10
11a
15a
15b
16a
16b
b
d
11b
c
14b
Scheme 1. Reagents and conditions: (a) EtOH–40% aq KOH, reflux, 5 h, 56%; (b)
iodomethane, K2CO3, DMF, rt, 1 h, quantitative yield; (c) MeOH–50% aq KOH 1:1,
isovanillin, rt, 15 h, 31% (14a), 64% (14b); (d) MeOH–50% aq KOH 10:1, 3-nitro-4-
methoxybenzaldehyde, rt, 15 h, 60% (15a), 50% (15b); (e) SnCl2, MeOH, 60 °C, 2 h,
74% (16a), 34% (16b).
tors (100 nM) compared to 14a and 16a (10
inactive. Lastly, it was noticed that compounds having the
lM) while 15a was

J. Quintin et al. / Bioorg. Med. Chem. Lett. 19 (2009) 167–169
169
Table 1
Antiproliferative, proapoptotic, antitubulin, and antileishmanial activities of synthesized chalcones
Compound Cytotoxicity on KB
cellsa IC50
M)
Activation of
ITA activity
IC50 M)
Antileishmanial activity on L. donovani
Antileishmanial activity on L. amazonensis
(
l
apoptosis in HL60b
(
l
promastigotesf IC50
(lM)
amastigotesg IC50
(lM)
2
4
5
14a
14b
15a
15b
16a
16b
17
100% IC50 = 0.017
44%
100% IC50 = 0.031
100 nM: 3.6ꢁ
IC50 = 4.3 1.3c
ndd
nd
IC50 = 2.8
nd
IC50 = 25
IC50 = 20
IC50 = 5.3
nd
IC50 = 44
nd
nd
Inactiveh
100 nM: 3.6ꢁ
IC50 = 3.7 1.0c
IC50 = 13 3.8c
Inactivee
Inactivee
Inactivee
IC50 = 14 4.1c
Inactivee
nd
nd
49%
22%
43%
19%
40%
29%
29%
31%
10
10
10
lM: 3.2ꢁ
lM: 1.2ꢁ
lM: 2.9ꢁ
Inactiveh
nd
Inactiveh
nd
Inactiveh
nd
IC50 = 0.4
IC50 = 4.4
IC50 = 26 ISi = 2.8
IC50 = 26 IS = 2.5
18
nd
a
As measured by the MTS assay after 72 h incubation of cells with drug; results are expressed as the percentage of inhibition of cell growth with 10 to 6 M chalcone
concentration; IC50 was calculated only for the two most active compounds.
b
Activation of caspases 3/7 activity: optimal concentration of compound and fold activation.
IC50 chalcone/IC50 deoxypodophyllotoxin.
Not determined.
Estimated inactive when decreasing by less than 30% the maximum assembly rate of tubulin without drug.
As measured by the MTT assay after 72 h incubation of parasite with the drug.
As measured after 30 h incubation of infected macrophages with the drug.
Estimated inactive when amastigotes are still lodged within parasitophore vacuole, inside macrophages, as compared to DMSO control.
c
d
e
f
g
h
i
Index of selectivity defined by the ratio IC50
macrophages/IC50
.
amastigotes
murine
manial activity of the intermediate nitrochalcones 4 and 15a.16 As
the same letter displayed also the detrimental influence of almost
all substitutions at C-4 on this activity, we decided to compare 4
and 15a to their 4-unsubstituted analogs 17 and 18. The antileish-
manial activity (Table 1) was determined in vitro against prom-
astigotes of Leishmania donovani strain LV9 (MHOM/ET/67/HU3)
clone which were grown as described previously,17 and against
intracellular amastigotes of Leishmania amazonensis strain LV9
(MPROB/BR/1972/M1841) which were isolated from lesions and
purified as described earlier.18 Cytotoxicity against murine macro-
phages allowed evaluation of the compound selectivity (Index of
selectivity IS = IC50 macrophages/IC50 amastigotes). The evaluation on
promastigotes of compounds 14a–16a confirmed the positive ef-
fect of a nitro group at C-3 since 15a is much more active than
14a and 16a, but comparison 15a vs 4 was slightly in favor of 4.
Removing the methoxyl group at C-4 increased the antipromastig-
otes activity within the 30,40,50-trimethoxylated series (17 vs 4), but
not within the series a (18 vs 15a). In compounds 17 and 18 the
lack of substitution at C-4 had a significant impact on efficacy
against amastigotes. Only these two compounds lacking substitu-
tion at C-4 showed any efficacy in this regard.
Sud 11 (Laboratoire de Pharmacognosie, groupe de Chimiothérapie
Antiparistaire). Phytosynthèse is greatly thanked for J. Desrivot
financial support.
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In conclusion, our study showed that the peroxygenated substi-
tution patterns of the ring A present in the series a and b were less
beneficial to antiproliferative, activation of apoptosis, antimitotic,
and antileishmanial activities than the more classical and available
30,40,50-trimethoxy substitution. However, this SAR was deduced
from only few examples of chalcones, and needs to be generalized
to a larger series bearing other substitution patterns on the ring B.
14. When the reaction with 3-nitro-4-methoxybenzaldehyde was performed in a
mixture EtOH–aqueous KOH (classical conditions), the reaction compounds
were a mixture of 15a and its 4-OEt analog resulting from a SNAr mechanism
at C-4.
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Acknowledgments
E. Prina from Pasteur Institute (Unité d’Immunologie et Parasit-
isme Intracellulaire) is aknowledged for its help in amastigotes
toxicity evaluation, as well as P.M. Loiseau from Université Paris-