Semisynthesis and antiproliferative evaluation of a series of 3′-aminoflavones

Article abstract of DOI:10.1016/j.bmcl.2009.05.008

A series of 3′-aminoflavones 5,6,7,8-tetra- or 5,7-dioxygenated on the A-ring was synthesized from tangeretin or naringin, two natural Citrus flavonoids. These flavones were evaluated for antiproliferative activity, activation of apoptosis, and inhibition

Full text of DOI:10.1016/j.bmcl.2009.05.008

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3502–3506
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Bioorganic & Medicinal Chemistry Letters
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Semisynthesis and antiproliferative evaluation of a series of 30-aminoflavones
Jérôme Quintin a, Didier Buisson b, Sylviane Thoret c, Thierry Cresteil c, 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 Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601 CNRS, 45 rue des Sts Pères, 75006 Paris, France
c ICSN, CNRS - UPR 2301, 91190 Gif-sur-Yvette, France
a r t i c l e i n f o
a b s t r a c t
A series of 30-aminoflavones 5,6,7,8-tetra- or 5,7-dioxygenated on the A-ring was synthesized from tan-
geretin or naringin, two natural Citrus flavonoids. These flavones were evaluated for antiproliferative
activity, activation of apoptosis, and inhibition of tubulin assembly. The most antiproliferative flavones
exhibit a common 5-hydroxy-6,7,8-trimethoxy substitution pattern on the A-ring.
Ó 2009 Elsevier Ltd. All rights reserved.
Article history:
Received 30 March 2009
Revised 30 April 2009
Accepted 3 May 2009
Available online 7 May 2009
Keywords:
Tangeretin
Flavones
Aminoflavones
Antiproliferative activity
Tubulin
In 1998, Beutler et al. reported results of a comparative in vitro
antitumor screening and subsequent tubulin polymerization stud-
ies carried out with a series of natural and synthetic flavones.1
Most of the studied flavones contained only hydrogen, hydroxyl
and methoxyl substituents. Some structure–activity relationships
for cytotoxicity and associated inhibitory effects on tubulin poly-
merization were apparent from these results. Maximum potencies
for cytotoxicity and tubulin interaction were found only with com-
pounds bearing a hydroxyl group at C-5 on the A-ring, 30-hydroxy-
40-methoxy groups on the B-ring and a methoxyl at C-3 on the C-
ring. The substitution of the A-ring was apparently not critical
for activity (except hydroxylation at C-5), but it could be observed
that 1,2–4 the most potent of the studied flavones, was trimethoxy-
lated at C-6, C-7 and C-8. We noticed that the required substitution
pattern on the B-ring was the same as in combretastatin A4, 2, a
powerful inhibitor of tubulin assembly now under clinical investi-
gation as a phosphate prodrug. As SARs within the combretastatins
series reveal a slight increase in potency for the amino analog 3
compared to combretastatin A4,5 we decided to prepare a series
of 30-amino-40-methoxyflavones and to evaluate them for activities
related to cancer (antiproliferative and proapoptotic activities and,
inhibition of tubulin assembly). Owing to their substitution pat-
terns, two Citrus flavonoids, tangeretin, 4, a polymethoxyflavone
(PMF) that occurs in high concentrations in the peel of various Cit-
rus species such as sweet orange [Citrus sinensis (L.) Pers.] and man-
darin (Citrus reticulata Blanco), and naringin, 5, which are easily
available from grapefruit (Citrus x paradisi Macfad), were chosen
as starting materials for the semisynthesis.
Synthesis of 30-aminoflavones with a 5,6,7,8-tetrasubstituted A-
ring. Synthesis of these analogs was achieved from tangeretin (4)
via the intermediate 30-nitroflavones. First attempts of nitration
were undertaken on tangeretin itself. As tangeretin has been de-
scribed to yield by oxidation with excess nitric acid a red-coloured
5,8-flavoquinone structure,6 we carried out nitration of 4 under
various conditions: (a) with one equivalent of nitric acid in acetic
acid at 60 °C (to dissolve 4) or in trifluoroacetic acid at 0 °C; (b)
with other reagents (nitric acid in acetic anhydride, NO2BF4 in ace-
tonitrile, (NH4)2Ce(NO3)6 in trifluoroacetic acid, Bi(NO3)3 on Mont-
morillonite K10) already used with para-methoxybenzoic and
cinnamic acids, or with para-methoxy aromatic ketones.7–11 None
of these reactions allowed isolation of the expected 30-nitro deriv-
ative, but led either to red-coloured unstable products (at least two
according to TLC), or to recovery of 4. These results demonstrated
that oxidation of the tetramethoxylated A-ring is favored over
nitration of the B-ring at C-30. So we decided to enhance reactivity
of the B-ring by replacing the methoxyl group at C-40 by the more
strongly activating phenol group. Since the chemical regioselective
demethylation of 4 at C-40 seemed very unlikely to occur according
to the major studies of Horie et al.,12 semisynthesis of 40-O-dem-
ethyltangeretin 6 from 4 was achieved in 70% yield by biotransfor-
mation using an Aspergillus niger strain as previously described in
our laboratory.13 When 6 was subjected to conditions of nitration
used with tangeretin (1 equiv nitric acid, TFA, 0 °C), the expected
30-nitro compound 7 was isolated in 45% yield, which confirms
our hypothesis on the inversion of A and B-ring reactivities
* Corresponding author. Tel.: +33 146835593; fax: +33 146835399.
E-mail address: guy.lewin@u-psud.fr (G. Lewin).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.008

J. Quintin et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3502–3506
3503
scale from 4 (0.15 mmol starting amount) was a very neat and
effective method, we opted on a larger scale for the more classical
chemical pathway that begins by the basic degradation of tangere-
tin to acetophenone 9.14 Preparation of 8 from 9 was then per-
formed in two steps through the intermediate nitrochalcone 10
according to Scheme 1.
4'
OMe
OMe
8
B
7
6
MeO
MeO
O
C
3' R3
A
3
R2
5
R1
O
As pointed out on Scheme 2, 30-aminotangeretin, 11, was then
obtained from 8 by catalytic hydrogenation over palladium, while
preparation of 19 requires a regioselective 5-O-demethylation step
with aluminium bromide into nitroflavone 18 prior to the reduc-
tion of the nitro group.15 Access to C-3 oxygenated derivatives
from 8 was anticipated, but the two attempted methods proved
unsuccessful: Classical C-3 hydroxylation process with dimethyldi-
oxirane provided only a complex mixture,16,17 while hypervalent
iodine oxidation with iodobenzene diacetate according to Moriarty
et al. led to total recovery of 8.18,19 An alternative approach to C-3
oxygenated flavones from nitrochalcone 10 by means of the Algar–
Flynn–Oyamada method (AFO method) was also unfruitful, since
2-hydroxy-3,4,5,6-tetramethoxybenzoic acid and 4-methoxy-3-
nitrobenzoic acid were the major isolated compounds of the reac-
tion.20,21 As we could not access to C-3 oxygenated flavones, we
then turned to synthesis of 3-chloro and bromo analogs, since such
substituents effect on antiproliferative activity had previously been
unexplored. 3-Chlorination and bromination of 8 that led to 12 and
15, respectively, were carried out by N-chlorosuccinimide in a mix-
ture dichloromethane-pyridine as previously reported in our labo-
ratory,22 and by N-bromosuccinimide according to the two-steps
method of Bird et al. via a 2-methoxy-3-bromoflavanone interme-
diate.23 Regioselective 5-O-demethylation of the nitroflavones 12
and 15 with aluminium bromide provided 5-hydroxynitroflavones
13 and 16, then 14 and 17, respectively, by a final reduction step of
the nitro group by stannous chloride, dihydrate.
R1
R2
R3
OH
OMe
H
OH
H
1
OMe
OH
4
H
H
23
OMe
OH
H
H
OH
OH
24
25
MeO
MeO
OMe
OMe
R
R = OH
R = NH2
2
3
OH
RO
O
OH
O
Synthesis of 30-aminoflavones with a 5,7-disubstituted A-ring.
Nitroflavone 20 (=30-nitroacacetin) has been previously semisyn-
thesized in four steps in our laboratory from naringin 5.24 Starting
from 20, 5,7-dioxygenated aminoflavones 21 and 22 were prepared
by catalytic hydrogenation over palladium for 21 (82%), and a sub-
sequent regioselective methylation (iodomethane, KHCO3, dimeth-
ylformamide) for 22 (48% from 20).
R = neohesperidosyl
5
between 4 and 6. In a following step, methylation of 7 (iodometh-
ane, K2CO3, dimethylformamide) afforded 30-nitrotangeretin 8 in a
quantitative yield. Though this synthesis carried out on a small
Table 1
Antiproliferative, proapoptotic and antitubulin activities of synthesized flavones
Compd
Cytotoxicity on KB cellsa IC50
(
lM)
Activation of apoptosis in HL60b
ITA activityd
30-Aminoflavones
14
19
17
22
21
11
86% IC50 = 0.14
83% IC50 = 0.16
55%
30%
7%
1
1
1
l
l
l
M (ꢀ4.5)
M (ꢀ6.0)
M (ꢀ3.6)
15% inhibition
27% inhibition
No activation at 100
100
No activation at 100
l
M
M
lM (ꢀ4.0)
6%
l
30-Nitroflavones
16
8
18
23%
3%
2%
n.d.
n.d.c
n.d.
30-Unsubstituted flavones
23
4
28%
12%
100
lM (ꢀ3.8)
No activation at 100
lM
30-Hydroxyflavones
1
25
92% IC50 = 0.02
IC50 = 0.08
0.1
l
M (ꢀ5.7)
IC50 = 10
IC50 = 86
l
l
M 3e
1
l
M (ꢀ3.8) 10
lM (ꢀ5.2)
M 27e
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 10ꢁ6 M flavone
a
concentration, or as IC50, calculated only for the most active compounds.
b
Activation of caspases 3/7 activity: optimal concentration of compound and fold activation.
c
Not determined.
d
Results are expressed as the percentage of ITA at 6.6 ꢀ 10ꢁ5 M, or as IC50, calculated only for 1 and 25.
e
IC50 1 or 25/IC50 deoxypodophyllotoxin.

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J. Quintin et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3502–3506
Synthesis of 24 from tangeretin 4 gave 11% yield by the same clas-
OMe
R3
sical three-steps pathway (steps d–f) as described for 8 in the
Scheme 1, but with isovanillin instead of 3-nitro-4-methoxybenz-
aldehyde at the step e. The 5-O-demethylation step (4ꢂ23 and
24ꢂ25) was carried out as mentioned in the Scheme 2 (step d).
The antiproliferative effect of flavones was assayed on KB human
buccal carcinoma cells and the activation of apoptosis with DEVD-
AMC as substrate in HL60 human leukemia cells. Inhibition of tubu-
lin assembly (ITA) was determined according to Zavala and Gue-
nard’s method26 for the most antiproliferative compounds only.
Results were given as the percentage of ITA at 6.6 ꢀ 10ꢁ5 M or as
IC50, calculated and also expressed in relation to deoxypodophyllo-
toxin (DPPT) in terms of the IC50/IC50 DPPT ratio. As depicted in Table
1, the three most antiproliferative and proapoptotic 30-aminoflav-
ones, 14, 19 and 17, have in common a persubstituted A-ring with
a phenol function at C-5 and three methoxyl groups at C-6, C-7 and
C-8. When one of these structural requirements is lacking (5-meth-
oxylated flavone 11 vs 19; 5,7-dioxygenated flavones 21 and 22 vs
19), the biological response is very weak. Loss of antiproliferative
activity by methylation of the 5-phenol group confirms the
R2
O
O
R1
R1
R2
R3
OH
OH
OH
OH
NO2
NH2
NH2
20
21
22
OH
OMe
Lastly, in order to evaluate SARs within this series, three analogs
without nitrogen on the B-ring, 5-hydroxy-6,7,8,40-tetramethoxyf-
lavone (=5-O-demethyltangeretin = gardenin B) 23, 30-hydroxy-
5,6,7,8,40-pentamethoxyflavone 24 (=30-hydroxytangeretin), and
its 5-O-demethyl derivative 25 (=gardenin D) were also prepared.25
tangeretin 4
a
d
OH
OMe
OMe
MeO
MeO
O
MeO
MeO
OH
OMe O
OMe O
6
9
e
b
OMe
NO2
OH
NO2
OMe
OMe
O
MeO
MeO
OH
MeO
MeO
OMe O
OMe O
10
7
c
f
OMe
NO2
OMe
MeO
MeO
O
OMe O
8
Scheme 1. Reagents and conditions: (a) Aspergillus niger, 70%; (b) 1 equiv HNO3, TFA, 0 °C, 0.5 h, 45%; (c) iodomethane, K2CO3, DMF, rt, 1 h, quantitative yield; (d) EtOH–40%
aq KOH, reflux, 5 h, 56%; (e) MeOH–50% aq KOH 10:1, 3-nitro-4-methoxybenzaldehyde, rt, 15 h, 60%; (f) I2, pyridine, 120 °C, 10 h, 52%.

J. Quintin et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3502–3506
3505
OMe
NH2
OMe
NO2
OMe
OMe
MeO
MeO
O
MeO
O
MeO
X
OMe O
OMe O
12 X = Cl
15 X = Br
b or c
11
a
3'-nitrotangeretin 8
d
d
OMe
NO2
OMe
NO2
OMe
O
OMe
OH
MeO
MeO
MeO
MeO
O
X
OH
O
O
13 X = Cl
16 X = Br
18
e
a
OMe
NH2
OMe
NH2
OMe
OH
OMe
MeO
MeO
O
MeO
MeO
O
X
O
OH
O
14 X = Cl
17 X = Br
19
Scheme 2. Reagents and conditions: (a) H2, Pd–C 10%, DMF, rt, 3 h, 93%, (11), 95% (19) ; (b) NCS, CH2Cl2–pyridine 4:1, rt, 48 h, 95% ; (c) NBS, CH2Cl2–MeOH 2:1, rt, 5 h;
extraction then THF–NaOH N 1:1, rt, 0.25 h, 95%; (d) AlBr3, acetonitrile, 0 °C, then HCl 1 N, 50 °C, 0.33 h, 60% (13), 64% (16), 69% (18); (e) SnCl2, 2H2O, MeOH, 60 °C, 14 h, 53%
(14), 41% (17).
previously reported importance of the 5-hydroxyl group.1,27 Contri-
References and notes
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Though failing in its initial goal of access to the amino analog of
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to our knowledge. Preparation of this lacking amino analog is in
progress by an alternative synthetic process and its biological eval-
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Acknowledgments
We thank Jean-Christophe Jullian for NMR measurements, Dr.
Françoise Gueritte for a sample of 1, and Kevin Spelman for English
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