Synthesis of antiproliferative flavones from calycopterin, major flavonoid of Calycopteris floribunda Lamk.

Article abstract of DOI:10.1016/j.bmc.2010.11.035

Eighteen new analogues of 5,3′-dihydroxy-3,6,7,8,4′- pentamethoxy-flavone, a potent natural cytotoxic and antimitotic flavone, were synthesized from calycopterin, the major flavonoid of Calycopteris floribunda Lamk., a traditional Asian medicinal plant. O

Full text of DOI:10.1016/j.bmc.2010.11.035

Bioorganic & Medicinal Chemistry 19 (2011) 186–196
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of antiproliferative flavones from calycopterin, major flavonoid
of Calycopteris floribunda Lamk.
Guy Lewin a, , Narayan Bhat Shridhar b, Geneviève Aubert c, Sylviane Thoret c, Joëlle Dubois c,
⇑
Thierry Cresteil c
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 Department of Pharmacology and Toxicology, KVAFSU Veterinary College, Hebbal, Bangalore 560 024, India
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
Eighteen new analogues of 5,30-dihydroxy-3,6,7,8,40-pentamethoxy-flavone, a potent natural cytotoxic and
antimitotic flavone, were synthesized from calycopterin, the major flavonoid of Calycopteris floribunda
Lamk., a traditional Asian medicinal plant. One of them, the 30-amino substituted analogue, displayed
almost the same activity as the reference compound. Pharmacomodulation at C-30 on the B-ring, and at
C-5,6,7 and 8 on the A-ring allowed to refine structure–activity relationships within the cytotoxic flavones
series.
Article history:
Received 28 September 2010
Revised 13 November 2010
Accepted 16 November 2010
Available online 20 November 2010
Keywords:
Calycopterin
Ó 2010 Elsevier Ltd. All rights reserved.
Calycopteris floribunda
Flavones
Antiproliferative activity
Antimitotic
Tubulin
Combretastatins
1. Introduction
OR1
4'
OMe
B
8
7
6
MeO
MeO
O
C
3' R2
In 1998, Beutler et al. reported results of a comparative cytotox-
icity screening and subsequent tubulin polymerization studies
carried out with a series of 79 natural and synthetic flavones.1
Most of the studied flavones exhibited only hydrogen, hydroxy
and methoxy substituents. Maximum potencies for cytotoxicity
and tubulin interaction were found only with compounds bearing
an OH group at C-5 on the A-ring, 30-hydroxy-40-methoxy groups
on the B-ring and an OCH3 at C-3 on the C-ring. The best activity
was found with 5,30-dihydroxy-3,6,7,8,40-pentamethoxy-flavone
1, a natural flavone first isolated by Mabry et al. in 1986 from
Gutierrezia microcephala.2 In 1994 then 1995, the Sévenet3 then
Lee4 groups reported, respectively the strong cytotoxic and IPT
(Inhibition of Polymerization of Tubulin) properties of 1, which
remains the most antimitotic natural flavone isolated to date.
Though the substitution pattern of the A-ring was apparently not
critical for activity (except hydroxylation at C-5), a trimethoxyla-
tion at C-6, C-7 and C-8 appeared the most favourable, as it has
been recently confirmed in the laboratory.5
A
3
OMe
5
OH
O
R1
R2
Me
H
OH
H
1
4
5
NH2
Me
MeO
MeO
OMe
OMe
R
R = OH
R = NH2
2
3
We noticed also that the substituents of 1 at C-6, C-7, C-8 on
⇑
Corresponding author. Tel.: +33 146835593; fax: +33 146835399.
E-mail address: guy.lewin@u-psud.fr (G. Lewin).
the A-ring and C-30, C-40 on the B-ring were the same as in
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.11.035

G. Lewin et al. / Bioorg. Med. Chem. 19 (2011) 186–196
187
combretastatin A4 2, a powerful inhibitor of tubulin assembly now
under clinical investigation as its phosphate prodrug. SARs within
the combretastatin series reveal that the hydroxy group, though
highly favourable to the activity, was not crucial. A slight increase
in potency compared to combretastatin A4 was even reported for
the amino analogue 3,6 an other combretastatin under clinical
investigation as its serine prodrug. Therefore, we decided to
prepare and to evaluate for activities related to cancer (antiprolif-
erative and proapoptotic activities, inhibition of tubulin assembly)
a series of original analogues of 1, which would differ from the ref-
erence compound only in the substituent at C-30. Access to these
analogues could be planned by total synthesis, but this approach
was turn down owing to multi-step processes, partly due to the
tetramethoxylation pattern of ring A. A semisynthetic method
was then envisaged from a natural flavone. The ideal starting
compound would be a 3,5,6,7,8,40-hexa-oxygenated flavone bear-
ing at C-40 an OH, as strong activating group of the 30 ortho position
towards electrophilic reagents. A thorough examination of the lit-
erature in the field of natural flavones pointed out calycopterin,
5,40-dihydroxy-3,6,7,8-tetramethoxy-flavone 4, as a choice raw
material to achieve our purpose. Calycopterin, the main flavonoid
of leaves of Digitalis thapsi L. (Plantaginaceae) and Calycopteris
floribunda Lamk. (Combretaceae) was isolated both by Karrer7
and Ratnagiriswaran et al.8 in 1934. Calycopteris floribunda is a tra-
ditional Asian medicinal plant which has been recently suspected
to cause morbidity and mortality in grazing cattle in India. In
2006, one of us (N.B. Shridhar) reported on the toxicity of this plant
in calf, rabbit and rat,9 and subsequently provided us with dried
leaves of C. floribunda for extraction and isolation of calycopterin.
carried out with the crystallized mixture 6a–6b and consisted suc-
cessively of: a reduction of the isomeric quinones (H2, Pd–C 10%, rt,
15 min in DMF) to the corresponding ortho and para-diphenols; a
methylation of the crude resulting mixture (iodomethane, K2CO3,
rt in DMF) leading after purification to 5-hydroxy-30-nitro-
3,6,7,8,40-pentamethoxyflavone 9 as main product (48% from
6a–6b), and 30-nitro-3,5,6,7,8,40-hexamethoxyflavone 10 (4.5%
from 6a–6b) as minor one; a reduction of 9 (H2, Pd–C 10%, rt,
5 h in DMF) to the expected 30-amino-5-hydroxy-3,6,7,8,40-penta-
methoxyflavone 5, analogue of 1 (78%). Lastly, with the view of
biological evaluation, the dimethylaminoflavone 11 was also pre-
pared from 5 (aq formaldehyde, NaBH3CN, rt, 24 h in acetic acid)
in 66% yield.
Since SAR’s of combretastatins indicate that analogues of com-
bretastatin A4 with an hydrogen, a fluoride or a boronic acid group
in place of the hydroxyl retain a good biological activity,6 in a second
time we undertook the synthesis of the three same corresponding
derivatives of 1. The 30-deoxy analogue of 1 is the known natural
calycopterin 40O-methyl ether 12,13,14 and it was prepared from 4
(iodomethane, KHCO3, rt in DMF) in 61% yield. Access to the 30-boro-
nic acid analogue was attempted via the synthesis of 30-iodoflavone
15, and its subsequent palladium-catalyzed cross-coupling
reaction with bis(pinacolato)diboron according to Miyaura et al.15
(Scheme 2).
Iodination of calycopterin 4 was carried out with N-iodosuccin-
imide (NIS), followed by 40-O-methylation (in the reverse order,
oxidation of the A-ring to the mixture of quinones was observed
in place of 30-iodination). Direct monoiodination of 4 at C-30
occurred in poor yield, for 1 equiv NIS leads to a mixture of remain-
ing 4, mono and diiodo derivatives. Therefore access to the ex-
pected 30-iodoflavone 15 was achieved according to the following
three-step sequence with a 51% overall yield: (a) diodination of 4
(2.2 equiv NIS, rt in CH2Cl2–MeOH) to 30,50-diodo-calycopterin 13
(71%); (b) 40-O-methylation of 13 (iodomethane, KHCO3, rt in
DMF) to 14 (86%); (c) monodeiodination at C-50 (Zn in acetic acid,
95 °C)16 to 15 (85%). Unfortunately, applying Miyaura’s condi-
tions15 [bis(pinacolato)diboron, PdCl2(dppf), potassium acetate,
80 °C in DMSO] to the iodoflavone 15 did not provide 30-boronic es-
ter, but two compounds resulting from a 30-30 dimerization (16,
22%) and a 30-deiodination (12, 8%), respectively. The dimeric and
symmetrical structure of 16 was unambiguously deduced from
mass and NMR spectra. Its isolation as main compound of the reac-
tion is unexpected according to literature data,17,18 but could be
explained by quenching of the boronic ester intermediate by the
starting iodoflavone 15. Synthesis of the 30-fluoro analogue from
calycopterin was no more fruitful: all attempts were carried out
with N-fluorobenzenesulfonimide (NFSI, Accufluor™) in various
solvents (CH2Cl2–MeOH, CH2Cl2–MeCN, DMF) and under solvent-
free fluorination conditions,19 but they led to complex mixtures
of coloured compounds.
2. Chemistry
2.1. Synthesis of analogues of flavone 1 differing in the
30-substituent
Owing to the strong activity of the aminocombretastatin 3, we
began the study by the synthesis of flavone 5, the 30-amino ana-
logue of the reference compound 1 (Scheme 1).
Access to 5 was attempted via nitration of 4 with HNO3 (1 equiv)
in TFA at 0 °C, as previously and successfully used in the laboratory
for 30-nitration of some other 40-hydroxyflavones.10,11 Under these
conditions, instead of the expected 30-nitrocalycopterin, a mixture
(6–4) of two orange red-coloured compounds 6a (major) and 6b
(minor) was obtained (yield 77% by crystallization in MeOH). Purifi-
cation of an aliquot part of these crystals led to pure 6a and 6b. EIMS
(pseudomolecular ion [M+Na]+ 426) and 1H NMR spectroscopy
(three remaining OCH3 singlets) indicated that 6a and 6b are iso-
meric 30-nitro-5,6-ortho and 5,8-para-flavoquinones. Structures of
these two quinones were established unambiguously after reduc-
tion (H2, Pd–C 10%, rt, 5 h in DMF) into the corresponding 30-aminof-
lavones 7a and 7b, and were inferred from NOESY experiments (for
7a, significant NOE correlations were observed between both OCH3
signals at C-3 and C-8 and H-20 and H-60; for 7b, significant NOE cor-
relations were observed between OCH3 signal at C-3 and H-20 and H-
60 on one hand, and signals of OCH3 at C-6 and OH at C-5 and on the
other hand). These experiments proved unambiguously 6a to be
30-nitro-5,6-ortho-flavoquinone, and 6b 30-nitro-5,8-para-flavoqui-
none. Changing nitration reagent of 1 for 1 equiv NO2BF4 in acetoni-
trile led to the mixture 45–55 of 8a and 8b, an other couple of ortho
and para-flavoquinones, non nitrated on the B-ring. These results
demonstrate that oxidation of the A-ring into ortho and para-qui-
nones occurs before nitration of the B-ring at C-30 (recovery of 6a
and 6b can then be explained by oxidation of the A-ring by HNO3 fol-
lowed by nitration at the B-ring by resulting nitrite ion in TFA, as
already reported in the literature12). Nextsteps of thesynthesis were
The last series of original 30-substituted analogues of 1 resulted
from formylation studies carried out on calycopterin 4. Indeed, the
formyl group appears to us as an excellent starting function to
introduce easily chemical diversity at C-30 (Scheme 3).
Formylation was achieved by a Duff reaction [(CH2)6N4 in
CF3COOH at reflux], which afforded 30-formyl-calycopterin 17 in
55% yield. Despite the presence of two chelated phenol groups at
C-5 and C-40, 17 underwent a surprisingly selective methylation
at 40 (iodomethane, KHCO3, rt in DMF), which led to 18, the 30-for-
myl analogue of 1, in 89% yield. Flavone 18 was the hub for the
access to the following 30-substituted analogues: 30-hydroxy-
methyl 19 (NaBH4, rt in CH2Cl2–MeOH, 94% yield), 30-aminomethyl
and 30-dimethylaminomethyl 20 and 21, respectively [20: NH4OAc,
NaBH3CN, rt in MeOH, 34% yield; 21: (CH3)2NH, HCl, NaBH3CN, rt
in MeOH, 23% yield] and 30-cyano 22 (NH2OH, HCl, reflux in
HCOOH, 56% yield).

188
G. Lewin et al. / Bioorg. Med. Chem. 19 (2011) 186–196
OH
OH
OMe
O
O
MeO
O
O
O
MeO
O
+
OMe
MeO
OMe
O
O
c
8a
8b
calycopterin 4
OH
OH
OMe
OH
OH
MeO
HO
O
O
MeO
MeO
O
NH2
NH2
OMe
OMe
OH
O
a
7a
7b
b
b
OH
OH
OMe
O
O
MeO
O
O
O
MeO
MeO
O
+
NO2
NO2
OMe
OMe
O
O
6a
6b
d, e
OMe
R
OMe
R
OMe
OH
OMe
MeO
MeO
O
O
MeO
MeO
O
+
OMe
OMe
OMe O
9 R = NO2
10 R = NO2
31 R = NH2
g
b
5 R = NH2
f
11 R = NMe2
Scheme 1. Reagents and conditions: (a) HNO3 1 equiv in TFA, 0 °C, 0.5 h, 77% [6a, 6b in mixture (6:4)]; (b) H2, Pd–C 10% in DMF, rt, 5 h; (c) NO2BF4 1.4 equiv in MeCN, 80 °C,
15 min, 45% [8a, 8b in mixture (45:55)]; (d) H2, Pd–C 10% in DMF, rt, 15 min; (e) iodomethane, K2CO3 in DMF, rt, 5.75 h, 48% 9 and 4.5% 10 (from 6a, 6b); (f) 37% aq HCHO,
NaBH3CN in AcOH, rt, 24 h, 66%; (g) HCOONH4, Pd–C 10% in MeOH, reflux, 2 h, 86%.
2.2. Synthesis of analogues of aminoflavone 5 differing in the
A-ring substitution
in common (Scheme 4): (a) 40-O-benzylation (benzyl bromide,
KHCO3 in DMF, 115 °C) to 23 (72% yield); (b) 5-O-triflation of 23
(PhN(Tf)2, NaHMDS, rt in THF)21 to 24 (75% yield); (c) transfer
hydrogenolysis (Pd–C 10%, HCOONH4, in MeOH at reflux) of 24 to
25 (80% yield); (d) 30-nitration of 25 (1 equiv HNO3 1 equiv in
TFA at 0 °C) to 26; (e) 40-O-methylation of 26 (iodomethane,
K2CO3, rt in DMF) to 30-nitro-3,6,7,8,40-pentamethoxy-flavone 27
(69% from 25). A new nitration step carried out with 27 led in a
very weak yield (16%) to 5,30-dinitroflavone 28, which provided
29, the expected 5-amino analogue of 1, by a last hydrogenation
step (85% yield). When 27 was directly submitted to hydrogena-
tion, the 5-deoxy analogue 30 was isolated in 86% yield. Lastly,
though a 5-methoxy group is known to be detrimental to the cyto-
toxic activity,22 31, the 5-O-methyl ether of 5, was also prepared
from the side compound 10 (cf. Scheme 1) by hydrogenation of
the 30-nitro group (86% yield).
Since the flavone 5 appeared the most potent of the synthesized
analogues (cf. biological results), we prepared a second series of
flavones which differs from 5 by the A-ring substitution pattern.
We successively focused on the 5-hydroxyl known to be critical
for the activity,1 then on the 6,7,8-trimethoxy substitution pattern.
2.2.1. Substituent at C-5
We first studied the influence of the 5-hydroxyl by its substitu-
tion for an amino group (such a substituent at C-5 is present in
some previously reported very cytotoxic flavones20). In the course
of the synthesis of this 5-amino analogue 29, the 5-deoxy analogue
30 was also isolated. Access to 29 and 30 was achieved from caly-
copterin 4 by multi-step sequences having the following five steps

G. Lewin et al. / Bioorg. Med. Chem. 19 (2011) 186–196
189
I
I
OH
OMe
I
OMe
OMe
OH
MeO
O
O
MeO
O
a
b
I
calycopterin 4
MeO
OMe
OMe
OMe
MeO
OMe
OH
O
14
13
O
O
OMe
OMe
MeO
OMe
OMe
c
MeO
MeO
O
OMe
MeO
OH
O
OMe
I
OMe
OH
16
MeO
MeO
O
d
+
OMe
OMe
OMe
OH
O
MeO
MeO
O
15
OMe
O
12
Scheme 2. Reagents and conditions: (a) NIS 2.2 equiv in CH2Cl2–MeOH 1–1, rt, 15 h, 71%; (b) iodomethane, KHCO3 in DMF, rt, 8 h, 86%; (c) Zn powder in AcOH, 95 °C, 50 min,
85%; (d) bis(pinacolato)diboron 1.3 equiv, KOAc 4 equiv, PdCl2 (dppf) 5% in anhydrous DMSO, 90 °C, 18 h, 22% 16 and 8% 12.
OR
OMe
MeO
MeO
O
O
19
a
CHO
4
R =CH2OH
OMe
c
OH
b
20
OMe
R
17 R = H
d
OMe
OH
R =CH2NH2
MeO
MeO
O
O
18 R = Me
OMe
e
f
21
R =CH2NMe2
22
R =CN
Scheme 3. Reagents and conditions: (a) hexamethylenetetramine 1.06 equiv in TFA, reflux, 5 h, 55%; (b) iodomethane, KHCO3 in DMF, rt, 2.5 h, 89%; (c) NaBH4 in
MeOH–CH2Cl2 5–1, rt, 30 min, 94%; (d) NH4OAc 15 equiv, NaBH3CN in MeOH, rt, 20 h, 34%; (e) Me2NH, HCl 6 equiv, NaBH3CN in MeOH, rt, 20 h, 23%; (f) NH2OH, HCl in
HCOOH, reflux, 2 h, 56%.
2.2.2. Substituent at C-6, C-7 and C-8
3. Biology
In a first attempt, the antiproliferative effect of flavones assayed
Role of the 6,7,8-trimethoxy substitution was investigated by
preparing each of the three mono O-demethyl derivatives
(Scheme 5). Access to the 6 and 8-O-demethyl analogues was
achieved from the 30-nitroflavone 9 by the following two-step
sequence: (a) oxidation (1 equiv HNO3 in TFA at 0 °C) to the cou-
ple of isomeric ortho and para-flavoquinones; (b) reduction of
both quinone and nitro groups (H2, Pd–C 10%, rt, 1.5 h in DMF)
to the corresponding 5,6 and 5,8-dihydroxy-30-aminoflavones 32
and 33. Structure of each isomer was deduced from NOESY exper-
iments, as mentioned for 7a and 7b (two observed NOE correla-
tions between H-20, 60 and 3 and 8 methoxy groups in 32; one
only with the 3-methoxyl in 33). Lastly, reaction of 5 with LiCl/
DMF, as demethylating reagent, provided the third analog 34 by
an expected selective 7-O-demethylation.23
on KB human buccal carcinoma cells was associated with the acti-
vation of caspases 3/7 in HL60 human leukemia cells and the
in vitro inhibition of tubulin polymerization for the most active
compounds. The biological evaluation was undertaken with four-
teen analogues of 1 having a various B-ring substitution pattern:
11 flavones (5, 9, 11, 12, 15, 16, 18–22) differ only in the substitu-
ent at C-30, the three others being calycopterin 4, and two synthetic
intermediates, the diiodoflavone 14 and 30-formyl-calycopterin 17
(Table 1). As expected, the 30-aminoflavone 5 possesses strong bio-
logical activities (antiproliferative, proapoptotic and IPT) very near
from 1, though a little less potent. No interesting responses were
noticed with other synthesized flavones, except calycopterin

190
G. Lewin et al. / Bioorg. Med. Chem. 19 (2011) 186–196
OBn
b
OBn
c
OH
OMe
OH
OMe
OMe
MeO
MeO
O
O
MeO
MeO
O
O
MeO
MeO
O
a
4
OMe
OMe
OMe
OTf
O
23
25
24
d
OMe
R
OMe
R
OH
OMe
OMe
OMe
MeO
MeO
O
O
MeO
MeO
O
O
MeO
MeO
O
e
f
NO2
OMe
OMe
OMe
R
O
26
R = NO2
R = NH2
R = NO2
R = NH2
28
29
27
30
g
g
Scheme 4. Reagents and conditions: (a) benzyl bromide 3 equiv, KHCO3 in DMF, 115 °C, 3 h, 72%; (b) NaHMDS 2.1 equiv, PhN(Tf)2 2.7 equiv in THF, rt, 22 h, 75%;
(c) HCOONH4, Pd–C 10% in MeOH, reflux, 1 h, 80%; (d) HNO3 1 equiv in TFA, 0 °C, 0.5 h, 90%; (e) iodomethane, K2CO3 in DMF, rt, 6 h, 69% from 25; (f) HNO3 1 equiv in TFA, 0 °C,
2 h, 16%; (g) HCOONH4, Pd–C 10% in MeOH, reflux, 2 h, 85% 29 and 86% 30.
OMe
NH2
Table 1
R2
Antiproliferative, proapoptotic and antitubulin activities of synthesized flavones
MeO
R1
O
O
a, b
Compd Cytotoxicity on KB cellsa Activation of caspases
IPT activityc
9
IC50 (nM)
3/7 in HL60b
OMe
1
96% IC50 = 8
100 nM (ꢀ5.7)
8.9 l
M (3)d
OH
Analogues of 1 differing in the B-ring substitution
4
5
9
11
12
R1= OH
R2 = OMe
R1 = OMe R2 = OH
32
0%
nd
nd
13
nd
nd
2%
96% IC50 = 25
13%
13%
100 nM (ꢀ3.2)
l
M (4.5)d
33
nd
nd
73% IC50 = 720
100
lM (ꢀ4.5)
Inhibitionc
nd
14–22
613%
nd
OMe
NH2
OMe
OH
Analogues of 5 differing in the A-ring substitution
HO
MeO
O
O
c
29
30
31
78% IC50 = 128
84% IC50 = 78
17%
100
100
l
l
M (ꢀ3.6)
24
12
5%
l
l
M (8.3)d
M (4)d
5
M (ꢀ3.1)
OMe
No activation at 100 lM
Inhibitionc
32
33
34
38% IC50 ꢁ 10,000
34% IC50 ꢂ10,000
IC50 = 6200
nd
nd
nd
nd
nd
85
34
l
M (29)d
Scheme 5. Reagents and conditions: (a) HNO3 1 equiv in TFA, 0 °C, 0.5 h; (b) H2,
Pd–C 10% in DMF, rt, 1.5 h, 6% 32 and 6% 33 from 9; (c) LiCl, 3.5 equiv in DMF,
180 °C, 17 h, 26%.
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 10ꢃ6 M flavone
concentration, or as IC50 (nM), calculated only for the most active compounds.
b
Activation of caspases 3/7 activity: optimal concentration of compound and fold
40O-methyl ether 12, which displays a fair cytotoxicity, but without
IPT property. So, it appears clearly that homologation of the 30-hy-
droxy or amino substituents (19, 20), such as replacement by some
electron donating (11) or withdrawing (18, 22) groups of small size
are highly unfavourable to the activity. From SAR’s point of view,
the strong activities observed only with flavones bearing 30-hydro-
xy or amino substituents confirmed the analogy with the combre-
tastatins family. Potent activities of 5 led us, in a second time, to
synthesize and evaluate analogues of this aminoflavone differing
in the substitution pattern on the A-ring (Table 1). Replacement
of the 5-hydroxyl by an amino group (29), or deoxygenation at
C-5 (30) resulted in a decrease of antiproliferative and proapoptot-
ic activities, which remain however strong enough. On the con-
trary, methylation of this phenol group (31) is very detrimental
to the activity, as previously reported.1 Lastly, the biological evalu-
ation of 32–34, the three monodemethylated analogues at C-6, C-8
and C-7, respectively, indicated a strong decrease of the antiprolif-
erative activity. This observation, as well as results of our recent
study,5 confirms that A-ring pattern substitution of 1 seems highly
favorable for strong antiproliferative and IPT activities within class
activation over controls.
c
Results are expressed as the percentage of IPT at ꢁ2 ꢀ 10ꢃ5 M, or as IC50
(lM).
d
IC50 compound/IC50 deoxypodophyllotoxin. nd: not determined.
of flavones. Compound 5 was the most potent newly synthesized
flavone, and we compared its biological activity with that of the
reference flavone 1 in several human cancer cell lines (Table 2).
The antiproliferative activity was comparable for both flavones, ex-
cept in PC3 which was resistant to the two compounds. One can
also observed a striking difference within HT29 cell line, which is
sensible to 5 but resistant to 1. To go further, apoptosis was ex-
plored by flow cytometry (Table 3): compounds 1 and 5 similarly
induced apoptosis as early as 24 h leading later to the activation
of caspases 3/7 reported in Table 1. The inhibition of tubulin poly-
merization was correlated with the blockade of cell cycle in phase
G2/M after 24 h of treatment, prior to a partial DNA degradation
(apparent S phase) and finally cell death (sub G1 phase). A similar
behavior was reported for combretastatin A4 2 and, to a lesser ex-
tent, compounds 1 and 5 (Table 4).

G. Lewin et al. / Bioorg. Med. Chem. 19 (2011) 186–196
191
Table 2
Table 4
Comparison of antiproliferative activities of 1 and 5
Cell cycle analysis of KB cells treated with 1 and 5a
a
Cell line
IC50 (nM)
Compd
% Cells in
1
5
Sub G1
G0/G1
S
G2/M
HCT-116b
HCT-15b
HT29b
23
14
3140
17
40
25
12
50
34
105
37
32
>1000
33
81
24 h
Control
0.7
13.8
14.9
7.3
72.9
9.5
21.3
39.3
6.9
19.6
50.1
45.3
34.5
C-A4b (20 nM)
1 (50 nM)
5 (50 nM)
26.7
18.5
19.2
MCF7Rc
OVCAR-8d
SK-OV-3d
A549e
22
58
48 h
Control
C-A4 (20 nM)
1 (50 nM)
5 (50 nM)
1.2
43.5
22.2
15.5
66.6
27.4
27.4
38.5
18.4
44
38.6
32
13.8
0.8
11.8
14
PC-3f
>1000
12
174
186
36
Mia PaCa-2g
HepG2h
SF-268i
HL-60j
400
36
14
a
Expressed as the percentage of KB cells in the various mitotic phases.
Combretastatin A4.
HL-60Rj
K-562j
5
7
b
9
a
As measured by the MTS assay after 72 h incuba-
5. Experimental section
tion of cells with drug.
b
Colon cancer.
Breast cancer.
Ovarian cancer.
Non-small cell lung cancer.
Prostate cancer.
Pancreas cancer.
Liver cancer.
CNS cancer.
c
5.1. General experimental procedures
d
e
Melting points were determined with a micro-Koffler apparatus
and are uncorrected. NMR spectra, including NOESY, 1H–13
f
g
C
h
(HMQC and HMBC) experiments, were recorded on Bruker AC-
300 (300 MHz) or Bruker AM-400 (400 MHz) spectrometers. ESIMS
were recorded on a Navigator Aqua thermoquest spectrometer or
an Agilent HP 1100 MSD spectrometer (ESI source) and APCIMS
on a Esquire-LC Bruker 00040 spectrometer. Flash chromatogra-
phies were performed with Silica Gel 60 (9385 Merck) or alumin-
ium oxide 90 (1097 Merck). Preparative tlc were performed with
60 F 254 silica gel (5715 Merck) or 60 F 254 aluminium oxide
(5713 Merck).
i
j
Leukemia.
Table 3
Early and late apoptosis in HL60 cells treated with 1 and 5a
Compd
Viableb
% Cells
In early apoptosisc
In late apoptosisd
24 h
Control
Doxorubicin (200 nM)
1 (50 nM)
5 (50 nM)
5.2. Plant material
97.2
52.8
72.4
77.1
2.6
45.9
26.7
20.7
0.2
1.2
0.8
2.1
The plant was collected in India from a place named ‘Talaguppa’
Shimoga District, Karnataka State and its identification confirmed
by Dr. Gopalakrishna Bhat, Professor and Head, Department of Bot-
any, Poornaprajna College, Udupi.
48 h
Control
Doxorubicin (200 nM)
1 (50 nM)
5 (50 nM)
97.4
5.2
30.8
60.1
2.5
89.2
61.8
37.6
0.1
5.6
7.2
2.2
5.3. Extraction and isolation of calycopterin 4
a
Expressed as the percentage of viable cells, cells in early and late apoptosis,
Dried and ground leaves of C. floribunda (2.5 kg) were extracted
with CH2Cl2 in a Soxhlet apparatus. Extract was filtered over a
Celite pad, then evaporated to dryness. The residue (98 g) was
purified by flash chromatography (silica gel, CH2Cl2 then CH2CL2–
MeOH 99–1). The most interesting fractions were combined and
evaporated to dryness. Crystallization of the dried residue (36 g)
with MeOH afforded pure calycopterin (8.6 g, 0.34%). Bright-yellow
crystals: mp: 225–226 °C (lit.14,25: 226 °C; 225–226 °C); 1H NMR
(DMSO-d6) d ppm 3.80, 3.82, 3.90 and 4.02 (4s, 12H, OMe-3, 6, 7
and 8), 6.98 (d, J = 8.7 Hz, 2H, H-30 and 50), 7.98 (d, J = 8.7 Hz, 2H,
H-20 and 60), 12.46 (br s, 1H, 5-OH).
measured by respective fluorescence intensities of 7-AAD (7-Aminoactinomycin D)
versus annexin V-PE.
b
Annexin negative/7-AAD negative.
Annexin positive/7-AAD negative.
Annexin positive/7-AAD.
c
d
4. Conclusion
In conclusion, this study reports synthesis and evaluation of the
antiproliferative activity of 20 3-methoxy-flavones including 18
new compounds. Nineteen of these flavones were prepared by
semisynthesis from calycopterin, a flavonoid isolated from leaves
of Calycopteris floribunda Lamk., a traditional Asian medicinal plant.
One of the prepared products, aminoflavone 5, displayed a strong
antiproliferative activity and proved to possess the same mecha-
nism and about the same potency (though slightly weaker) as
5,30-dihydroxy-3,6,7,8,40-pentamethoxy-flavone 1, a natural fla-
vone, which remains the most antimitotic natural flavone isolated
to date. From a pharmacomodulation point of view relating to anti-
proliferative activity within the flavone series, this study refines
the previously predicted structure–activity relationships.1,24 Lastly,
access to these original flavones by semisynthesis confirms the
interest of natural products as raw materials for medicinal
chemistry.
5.4. Synthesis of flavones from calycopterin
5.4.1. 30-Nitro-5,6-ortho-flavoquinone 6a and 30-nitro-5,8-para-
flavoquinone 6b
A solution of 1 (1.122 g, 3 mmol) in trifluoroacetic acid (30 mL)
at 0 °C was added with 11.19 N HNO3 (1 equiv) then stirred for
0.5 h. The reaction mixture was taken up in ice water, then ex-
tracted with CH2Cl2. Standard work-up of the organic layer affor-
ded
a red dried residue, which was crystallized in MeOH
(0.931 g, 77%). According to 1H NMR spectrum, the crystals consist
of a mixture (6–4) of 6a and 6b, which were separated from an ali-
quot part by preparative tlc (silica gel, CH2CH2–MeOH 97.5–2.5).

192
G. Lewin et al. / Bioorg. Med. Chem. 19 (2011) 186–196
Compound 6a orange red crystals: mp 195–200 °C (MeOH); 1H
NMR (DMSO-d6) d ppm 3.90, 3.92 and 4.18 (3s, 9H, OMe-3, 7, 8),
7.32 (d, J = 8.8 Hz, 1H, H-50), 8.10 (dd, J = 8.8 and 2.3 Hz, 1H, H-
60), 8.48 (d, J = 2.3 Hz, 1H, H-20). ESIMS (+) m/z 404 [M+H]+, 426
[M+Na]+, [M+K]+ 442. Compound 6b orange red crystals mp:
217–221 °C (MeOH); 1H NMR (DMSO-d6) d ppm 3.88, 3.92 and
4.00 (3s, 9H, OMe-3, 6, 7), 7.32 (d, J = 8.8 Hz, 1H, H-50), 8.10 (dd,
J = 8.8 and 2.3 Hz, 1H, H-60), 8.46 (d, J = 2.3 Hz, 1H, H-20). ESIMS
(+) m/z 404 [M+H]+, 426 [M+Na]+, [M+K]+ 442.
7.25 (d, J = 8.8 Hz, 1H, H-50), 8.37 (dd, J = 8.8 and 2.3 Hz, 1H, H-60),
8.70 (d, J = 2.3 Hz, 1H, H-20), 12.21 (s, 1H, 5-OH). APCIMS (+) m/z
448 [M+H]+.
5.4.5. 30-Amino-5-hydroxy-3,6,7,8,40-pentamethoxy-flavone 5
A solution of 9 (0.2 g, 0.46 mmol) in DMF (15 mL) was hydroge-
nated under 1 atm pressure hydrogen with 10% Pd–C (0.2 g) at
room temperature for 5 h. The catalyst was separated and the fil-
trate concentrated to dryness. Dried residue was purified by flash
chromatography (silica gel, CH2CH2–MeOH 98–2), and provided
pure expected flavone 5, which crystallized by evaporation to dry-
ness (0.146 g, 78%). Compound 5 bright-yellow crystals: mp 116–
118 °C; 1H NMR (CDCl3) d ppm 3.85 (s, 3H, OMe-3), 3.94 (s, 9H,
OMe-6, 8 and 40), 4.10 (s, 3H, OMe-7), 6.91 (d, J = 8.8 Hz, 1H, H-
50), 7.55 (d, J = 2.3 Hz, 1H, H-20), 7.63 (dd, J = 8.8 and 2.3 Hz, 1H,
5.4.2. 30-Amino-5,6-40-trihydroxy-3,7,8-trimethoxy-flavone 7a
and 30-amino-5,8-40-trihydroxy-3,6,7-trimethoxy-flavone 7b
Solutions of 6a (14 mg, 0.035 mmol) and 6b (12 mg, 0.03 mmol)
in DMF (3 mL) were hydrogenated under 1 atm pressure hydrogen
with 10% Pd–C (15 mg) at room temperature for 5 h. The catalyst
was separated and the filtrate concentrated to dryness. Dried res-
idues were purified by tlc (silica gel, CH2CH2–MeOH 95–5) and
afforded pure 7a (4.5 mg) and 7b (4 mg). Compound 7a Amorphe;
1H NMR (DMSO-d6) d ppm 3.75, 3.87 and 3.92 (3s, 9H, OMe-3, 7, 8),
6.81 (d, J = 8.8 Hz, 1H, H-50), 7.26 (dd, J = 8.8 and 2.3 Hz, 1H, H-60),
7.38 (d, J = 2.3 Hz, 1H, H-20), 12.2 (br s, 1H, 5-OH). ESIMS (+) m/z
376 [M+H]+, 398 [M+Na]+. Compound 7b Amorphe; 1H NMR
(DMSO-d6) d ppm 3.75, 3.80 and 3.90 (3s, 9H, OMe-3, 6, 7), 6.81
(d, J = 8.8 Hz, 1H, H-50), 7.29 (dd, J = 8.8 and 2.3 Hz, 1H, H-60),
7.42 (d, J = 2.3 Hz, 1H, H-20), 12.2 (s, 1H, 5-OH). ESIMS (+) m/z
376 [M+H]+, 398 [M+Na]+.
13
H-60), 12.45 (s, 1H, 5-OH). C NMR (CDCl3) d ppm 55.6 (OMe-40),
60.1 (OMe-3), 61.1, 61.7 and 62.1 (OMe-6, 7, 8), 107.5 (C-10),
110.1 (C-50), 114.4 (C-20), 120.1 (C-60), 123.1 (C-10), 132.8 (C-8),
136.0 (C-6 and C-30), 138.7 (C-3), 144.9 (C-9), 149.1 and 149.7
(C-5 and C-40), 152.8 (C-7), 156.5 (C-2), 179.3 (C-4). HRESIMS (+)
m/z [M+H]+ 404.1323 (calcd for C20H22NO8, 404.1339).
5.4.6. 30-Dimethylamino-5-hydroxy-3,6,7,8,40-pentamethoxy-
flavone 11
A solution of 5 (12 mg, 0.03 mmol) in acetic acid (4 mL) was
added withaqueous37%formaldehydeand NaBH3CN in excess, then
stirred for 24 h at rt. Standard work-up of the reaction, then purifica-
tionof theresidue bypreparativetlc (silica gel, CH2CH2–MeOH98.5–
1.5) led to pure 11 in 66% yield. Compound 11 Amorphous; 1H NMR
(CDCl3) d ppm 2.86 (s, 6H, N(CH3)2), 3.87, 3.93, 3.94, 3.97 (s, 12H,
OMe-3, 6, 8 and 40), 4.09 (s, 3H, OMe-7), 6.98 (d, J = 8.8 Hz, 1H, H-
50), 7.82 (d, J = 2.3 Hz, 1H, H-20), 7.86 (dd, J = 8.8 and 2.3 Hz, 1H, H-
60), 12.40 (s, 1H, 5-OH). ESIMS (+) m/z 432 [M+H]+, 454 [M+Na]+,
[M+K]+ 470.
5.4.3. 5,6-ortho-Flavoquinone 8a and 5,8-para-flavoquinone 8b
A mixture of 1 (0.187 g, 0.5 mmol) in acetonitrile (20 mL) was
heated at 80 °C till dissolution, left at rt for 15 min then added with
nitronium tetrafluoroborate (0.092 g, 0.7 mmol). The reaction was
stirred for 15 min at rt under nitrogen, then diluted with CH2Cl2.
Standard work-up of the organic layer afforded an orange reddish
dried residue, which was crystallized in MeOH (0.080 g, 45%).
According to 1H NMR spectrum, the crystals consist of a mixture
(45–55) of 8a and 8b. 1H NMR (DMSO-d6) signals of 8a at d ppm
3.82, 3.90 and 4.18 (3s, 9H, OMe-3, 7, 8), 6.95 (d, J = 8.7 Hz, 2H,
H-30 and 50), 7.86 (d, J = 8.7 Hz, 2H, H-20 and 60); signals of 8b at d
ppm 3.82, 3.92 and 4.00 (3s, 9H, OMe-3, 6, 7), 6.98 (d, J = 8.7 Hz,
2H, H-30 and 50), 7.88 (d, J = 8.7 Hz, 2H, H-20 and 60).
5.4.7. 5-Hydroxy-3,6,7,8,40-pentamethoxy-flavone (calycopterin
40O-methyl ether) 12
A solution of calycopterin 4 (0.094 g, 0.25 mmol) in DMF (5 mL)
was stirred for 24 h at rt, and successively added with KHCO3
(0.027 g, 0.27 mmol) and iodomethane (0.095 mL, 1.5 mmol) at
t = 0, 2, 4, 6 and 22 h. Standard work-up of the reaction, then puri-
fication of the dried residue by flash chromatography (silica gel,
CH2CH2–MeOH 99–1) and crystallization with MeOH gave pure
compound 12 (0.059 g, 61%). Bright-yellow crystals: mp
119–122 °C (lit.13,14: 122–123 °C; 125–127 °C); 1H NMR (CDCl3) d
ppm 3.87, 391 and 3.96 (3s, 12H, OMe-3, 6, 8, 40), 4.11 (s, 3H,
OMe-7), 7.05 (d, J = 8.7 Hz, 2H, H-30 and 50), 8.17 (d, J = 8.7 Hz,
2H, H-20 and 60), 12.45 (br s, 1H, 5-OH). 13C NMR (CDCl3) d ppm
55.5 (OMe-40), 60.1 (OMe-3), 61.2, 61.8, 62.2 (OMe-6, 7, 8), 107.5
(C-10), 114.3 (C-30 and 50), 122.9 (C-10), 130.3 (C-20 and 60), 132.9
(C-8), 136.2 (C-6), 138.6 (C-3), 144.9 (C-9), 149.2 (C-5), 152.9
(C-7), 156.1 (C-2), 161.9 (C-40), 179.3 (C-4).
5.4.4. 5-Hydroxy-30-nitro-3,6,7,8,40-pentamethoxy-flavone 9 and
30-nitro-3,5,6,7,8,40-hexamethoxy-flavone 10
A solution of the mixture (6–4) 6a–6b (0.806 g, 2 mmol) in DMF
(80 mL) was hydrogenated under 1 atm pressure hydrogen with
10% Pd–C (0.4 g) at room temperature for 15 min. The catalyst was
separated, the filtrate was concentrated to 40 mL, added with
K2CO3 (0.345 g, 2.5 mmol) and iodomethane (0.9 mL, 14 mmol),
then the reaction mixture was stirred for 5 h at room temperature.
After monitoring of the reaction by tlc, same amounts of K2CO3
and iodomethane were added and the medium stirred for further
45 min. The reaction mixture was diluted with iced water, and thor-
oughly extracted by CH2Cl2. Standard work-up of the organic layer
afforded a dried residue (0.622 g). Two successive purifications by
flash chromatography (silica gel, CH2CH2–MeOH 99–1) provided
the pure 5-hydroxylated flavone 9, which was crystallized in MeOH
(0.415 g, 48%). Fractions (0.1 g) containing mainly the 5-methoxy-
lated flavone 10 were purified by a third flash chromatography (alu-
mina, CH2CH2–cyclohexane 3–1), and led to pure compound 10,
which crystallized by evaporation to dryness (0.045 g, 4.5%). Com-
pound 9 bright-yellow crystals: mp 160–163 °C (MeOH); 1H NMR
(CDCl3) d ppm 3.97, 3.98, 4.10 and 4.15 (4s, 15H, OMe-3, 6, 7, 8, 40),
7.25 (d, J = 8.8 Hz, 1H, H-50), 8.39 (dd, J = 8.8 and 2.3 Hz, 1H, H-60),
8.70 (d, J = 2.3 Hz, 1H, H-20). APCIMS (+) m/z 434 [M+H]+. Compound
10 light-yellow crystals mp: 151–154 °C; 1H NMR (CDCl3) d ppm
3.97, 3.98, 4.00, 4.03, 4.10 and 4.15 (6s, 18H, OMe-3, 5, 6, 7, 8, 40),
5.4.8. 30,50-Diodo-calycopterin 13
A solution of calycopterin 4 (0.374 g, 1 mmol) in CH2Cl2–MeOH
1–1 (80 mL) was added with NIS (0.495 g, 2.2 mmol), and stirred
overnight at rt. Standard work-up of the reaction (with washing
of the organic phase with 1 N aqueous thiosulfate), then purifica-
tion of the dried residue by crystallization with MeOH then flash
chromatography (silica gel, CH2CH2–MeOH 99–1) led to pure dio-
doflavone 13 (0.444 g) in 71% yield. Bright-yellow crystals: mp
206–209 °C; 1H NMR (CDCl3) d ppm 3.88, 393 (2s, 9H, OMe-3, 6,
8), 4.09 (s, 3H, OMe-7), 6.10 (s, 1H, OH-40), 8.49 (s, 2H, H-20 and
60), 12.23 (s, 1H, 5-OH). 13C NMR (CDCl3) d ppm 60.4 (OMe-3),
61.2, 61.8, 62.2 (OMe-6, 7, 8), 80.3 (C-30and 50), 107.5 (C-10),
126.5 (C-10), 132.8 (C-8), 136.4 (C-6), 139.1 (C-3), 139.4 (C-20 and

G. Lewin et al. / Bioorg. Med. Chem. 19 (2011) 186–196
193
60), 144.7 (C-9), 149.2 (C-5), 152.4 and 153.3 (C-7 and C-40), 155.8
(C-2), 179.1 (C-4).
CH2Cl2. Standard work-up of the organic phase, then purification
by flash chromatography provide pure 17 which crystallized by
evaporation to dryness (0.292 g, 55%). Compound 17 bright-yellow
crystals: mp 125–127 °C; 1H NMR (CDCl3) d ppm 3.92 (s, 3H, OMe-
3), 3.94 and 3.95 (2s, 6H, OMe-6 and 8), 4.11 (s, 3H, OMe-7), 7.16
(d, J = 8.8 Hz, 1H, H-50), 8.34 (dd, J = 8.8 and 2.3 Hz, 1H, H-60),
8.47 (d, J = 2.3 Hz, 1H, H-20), 10.01 (s, 1H, CHO), 11.36 (s, 1H, 4-
OH0), 12.28 (s, 1H, 5-OH). 13C NMR (CDCl3) d ppm 60.3 (OMe-3),
61.2, 61.7 and 62.1 (OMe-6, 7, 8), 107.5 (C-10), 118.5 (C-50),
120.5 (C-30), 122.6 (C-10), 132.9 (C-8), 134.6 (C-20), 136.1 (C-6),
136.4 (C-60), 138.9 (C-3), 144.8 (C-9), 149.3 (C-5), 153.2 (C-7),
154.1 (C-2), 163.5 (C-40), 179.2 (C-4), 195.4 (CHO).
5.4.9. 30,50-Diodo-5-hydroxy-3,6,7,8,40-pentamethoxy-flavone 14
A solution of diodoflavone 13 (0.406 g, 0.65 mmol) in DMF
(35 mL) was added with 5 equiv KHCO3 (0.325 g, 3.25 mmol) and
iodomethane (0.3 mL, 4.8 mmol) and stirred for 5 h at rt. New
amounts of KHCO3 (0.16 g, 1.6 mmol) and iodomethane (0.2 mL,
3.2 mmol) were added, and the mixture stirred 3 h more. The reac-
tion was diluted with CH2Cl2, filtered, and concentrated to dryness.
The dried residue was taken up with CH2Cl2, and the organic phase
submitted to the standard work-up. Crystallization of the residue
with MeOH gave pure compound 12 (0.357 g, 86%). Bright-yellow
crystals: mp 170–172 °C; 1H NMR (CDCl3) d ppm 3.94, 395 and
3.96 (3s, 12H, OMe-3, 6, 8, 40), 4.12 (s, 3H, OMe-7), 8.54 (s, 2H,
H-20 and 60), 12.12 (s, 1H, 5-OH). 13C NMR (CDCl3) d ppm 60.5,
60.9, 61.2, 61.8, 62.2 (OMe-3, 6, 7, 8, 40), 90.6 (C-30 and 50), 107.5
(C-10), 130.0 (C-10), 132.9 (C-8), 136.4 (C-6), 139.6 (C-3), 139.8
(C-20 and 60), 144.9 (C-9), 149.2 (C-5), 152.1 and 153.4 (C-2 and
C-7), 161.0 (C-40), 179.2 (C-4). ESIMS (+) m/z 641 [M+H]+, 663
[M+Na]+, [M+K]+ 679.
5.4.13. 30-Formyl-5-hydroxy-3,6,7,8,40-pentamethoxy-flavone 18
A solution of compound 17 (0.241 g, 0.6 mmol) in DMF (25 mL)
was added with 5 equiv KHCO3 (0.3 g, 3 mmol) and iodomethane
(0.15 mL, 2.4 mmol) and stirred for 2.5 h at rt. The reaction was di-
luted with H2O, and extracted with CH2Cl2. Standard work-up of
the organic phase, then crystallization of the residue with MeOH
gave pure compound 18 (0.223 g, 89%). Compound 18 bright-yellow
crystals: mp 159–163 °C; 1H NMR (CDCl3) d ppm 3.92 (s, 3H, OMe-3),
3.95 (s, 3H, OMe-6), 3.97 (s, 3H, OMe-8), 4.05 (s, 3H, OMe-40), 4.11 (s,
3H, OMe-7), 7.16 (d, J = 8.8 Hz, 1H, H-50), 8.39 (dd, J = 8.8 and 2.3 Hz,
1H, H-60), 8.62 (d, J = 2.3 Hz, 1H, H-20), 10.52 (s, 1H, CHO), 12.30 (s,
5.4.10. 5-Hydroxy-30-iodo-3,6,7,8,40-pentamethoxy-flavone 15
A mixture of flavone 14 (0.34 g, 0.53 mmol) in acetic acid was
stirred at 95 °C till solubilization, then added with Zn powder
(0.34 g) and heated to the same temperature 50 min more. The
reaction mixture was diluted with H2O and thoroughly extracted
with CH2Cl2. Standard work-up of the reaction, then crystallization
of the dried residue with MeOH provided pure compound 15
(0.235 g) in 86% yield. Compound 15 bright-yellow crystals: mp
137–139 °C; 1H NMR (CDCl3) d ppm 3.89 (s, 3H, OMe-3), 3.96
and 3.99 (2s, 9H, OMe-6, 8 and 40), 4.11 (s, 3H, OMe-7), 6.95
(d, J = 8.8 Hz, 1H, H-50), 8.19 (dd, J = 8.8 and 2.3 Hz, 1H, H-60),
8.61 (d, J = 2.3 Hz, 1H, H-20), 12.25 (s, 1H, 5-OH). 13C NMR (CDCl3)
d ppm 56.6 (OMe-40), 60.2 (OMe-3), 61.2, 61.8 and 62.2 (OMe-6, 7,
8), 80.1 (C-30) 107.5 (C-10), 110.6 (C-50), 124.7 (C-10), 130.3 (C-20),
132.9 (C-8), 136.3 (C-6), 138.9 (C-3), 139.6 (C-60), 144.9 (C-9),
149.2 (C-5), 153.1 (C-7), 154.4 (C-2), 160.2 (C-40), 179.3 (C-4).
ESIMS (+) m/z 514 [M+H]+, 537 [M+Na]+, [M+K]+ 553.
13
1H, 5-OH). C NMR (CDCl3) d ppm 56.1 (OMe-40), 60.3 (OMe-3),
61.2 and 61.7 (OMe-6 and 8), 62.1 (OMe-7), 107.5 (C-10), 112.1 (C-
50), 123.2 (C-10), 124.9 (C-30), 129.2 (C-20), 132.8 (C-8), 135.8 (C-60),
136.2 (C-6), 139.0 (C-3), 144.9 (C-9), 149.1 (C-5), 153.1 (C-7), 154.7
(C-2), 163.2 (C-40), 179.3 (C-4), 188.7 (CHO). HRESIMS (+) m/z
[M+H]+ 417.1201 (calcd for C21H21O9, 417.1179), [M+Na]+
439.1015 (calcd for C21H20O9Na, 439.0999).
5.4.14. 30-Hydroxymethyl-5-hydroxy-3,6,7,8,40-pentamethoxy-
flavone 19
A solution of compound 18 (16 mg, 0.04 mmol) in CH2Cl2 (2 mL)
was diluted to 10 mL with MeOH, added with NaBH4 in excess,
then stirred for 30 min at rt. The mixture was taken up with water
at pH 4, then extracted with CH2Cl2. Standard work-up of the or-
ganic phase, then crystallization of the residue with MeOH gave
pure compound 19 (15 mg, 94%). Compound 19 bright-yellow
crystals: mp 171–174 °C; 1H NMR (CDCl3) d ppm 3.87 (s, 3H,
OMe-3), 3.95 and 3.96 (2s, 9H, OMe-6, 8 and 40), 4.10 (s, 3H,
OMe-7), 4.77 (s, 2H, CH2OH), 7.02 (d, J = 8.8 Hz, 1H, H-50), 8.14
(d, J = 2.3 Hz, 1H, H-20), 8.15 (dd, J = 8.8 and 2.3 Hz, 1H, H-60),
5.4.11. 30,30-Biflavone 16
A mixture of flavone 15 (0.103, 0.2 mmol), bis(pinacolato)dibo-
ron (0.066 g, 0.26 mmol), potassium acetate (0.078, 0.8 mmol) and
PdCl2 (dppf) (8 mg, 0.01 mmol) in anhydrous DMSO (5 mL) was stir-
red at 90 °C for 18 h. The reaction mixture was diluted with H2O, and
thoroughly extracted with Et2O. Standard work-up of the organic
phase gave a rather complex dried residue (0.05 g). Purification by
preparative tlc (silica gel, CH2CH2–MeOH 99.5–0.5) allowed isola-
tion of pure biflavone 16 (0.017 g, 22%) and calycopterin 40O-methyl
ether 12 (0.006 g, 8%). Compound 16 light-yellow crystals (MeOH):
mp 148–150 °C; 1H NMR (CDCl3) dppm 3.90, 3.93 and 3.96, (3s, 12H,
OMe-3, 6, 8 and 40), 4.10 (s, 3H, OMe-7), 7.19 (d, J = 8.8 Hz, 1H, H-50),
8.19 (d, J = 2.3 Hz, 1H, H-20), 8.24 (dd, J = 8.8 and 2.3 Hz, 1H, H-60),
13
12.38 (s, 1H, 5-OH). C NMR (CDCl3) d ppm 55.6 (OMe-40), 60.1
(OMe-3), 61.1 (OMe-6 and 8), 61.7 (CH2OH), 62.1 (OMe-7), 107.5
(C-10), 110.4 (C-50), 122.9 (C-10), 128.7 (C-20), 129.6 (C-30), 129.9
(C-60), 132.8 (C-8), 136.1 (C-6), 138.7 (C-3), 144.9 (C-9), 149.1 (C-
5), 152.9 (C-7), 155.9 (C-2), 159.6 (C-40), 179.3 (C-4). ESIMS (+)
m/z 419 [M+H]+, 441 [M+Na]+.
5.4.15. 30-Aminomethyl-5-hydroxy-3,6,7,8,40-pentamethoxy-
flavone 20
13
12.36 (s, 1H, 5-OH). C NMR (CDCl3) d ppm 55.9 (OMe-40), 60.2
A solution of compound 18 (21 mg, 0.05 mmol) in 10 mL MeOH
was added with 15 equiv NH4OAc (77 mg, 0.75 mmol) and
NaBH3CN in excess, then stirred for 20 h at rt. Standard work-up
of the reaction, then purification of the residue by tlc (silica gel,
CH2CH2–MeOH 96.5–3.5) led to pure aminoflavone 20 (7 mg) in
34% yield. Compound 20 Amorphous; 1H NMR (CDCl3) d ppm
3.86 (s, 3H, OMe-3), 3.90, 3.92 and 3.95 (3s, 9H, OMe-6, 8 and
40), 3.94 (s, 2H, CH2NH2), 4.08 (s, 3H, OMe-7), 7.00 (d, J = 8.8 Hz,
1H, H-50), 8.11 (dd, J = 8.8 and 2.3 Hz, 1H, H-60), 8.15 (d,
J = 2.3 Hz, 1H, H-20), 12.38 (br s, 1H, 5-OH). 13C NMR (CDCl3) d
ppm 48.4 (CH2NH2), 55.6 (OMe-40), 60.1 (OMe-3), 61.1, 61.7 and
62.0 (OMe-6, 7 and 8), 107.4 (C-10), 110.3 (C-50), 122.7 (C-10),
126.9 (C-30), 129.3 (C-60), 130.0 (C-20), 132.8 (C-8), 136.1 (C-6),
(OMe-3), 61.2, 61.8 and 62.2 (OMe-6, 7, 8), 107.6 (C-10) 111.2 (C-
50), 122.7 (C-10), 127.0 (C-30), 130.0 and 132.1 (C-20 and 60), 132.9
(C-8), 136.2 (C-6), 138.8 (C-3), 145.0 (C-9), 149.2 (C-5), 152.9 (C-
7), 156.1 (C-2), 159.3 (C-40), 179.3 (C-4). HRESIMS (+) m/z [M+H]+
775.2252 (calcd for C40H39O16, 775.2226), [M+Na]+ 797.2063 (calcd
for C40H38O16Na, 797.2046).
5.4.12. 30-Formyl-calycopterin 17
A solution of calycopterin 4 (0.497 g, 1.33 mmol) in trifluoro-
acetic acid (15 mL) was added with 1.06 equiv hexamethylenetet-
ramine (0.2 g, 1.41 mmol) and heated for 5 h at reflux. The mixture
was diluted with iced water, stirred for 5 min, then extracted with

194
G. Lewin et al. / Bioorg. Med. Chem. 19 (2011) 186–196
138.2 (C-3), 144.9 (C-9), 149.1 (C-5), 152.8 (C-7), 156.1 (C-2), 159.9
extracted with CH2Cl2. Standard work-up of the organic phase,
then purification of the dried residue by flash chromatography
(alumina, CH2Cl2–cyclohexane 1–1) gave pure triflate 24 which
was crystallized with MeOH (0.249 g, 75%). Pale-yellow crystals:
mp 120–121 °C.
(C-40), 179.2 (C-4). HRESIMS (+) m/z [M+Na]+ 440.1319 (calcd for
C21H23NO8Na, 440.1315).
5.4.16. 30-Dimethylaminomethyl-5-hydroxy-3,6,7,8,40-
pentamethoxy-flavone 21
From 18 (21 mg), same work-up than for compound 20, but
with 6 equiv (CH3)2NH, HCl in place of NH4OAc. Purification of
the dried residue of the reaction by tlc (silica gel, CH2CH2–MeOH
94–6), led to 21 (5 mg) in 23% yield. Compound 21 Amorphous;
1H NMR (CDCl3) d ppm 2.37 (s, 6H, N(CH3)2) 3.60 (s, 2H, C-30-
CH2–), 3.87 (s, 3H, OMe-3), 3.94 (s, 3H, OMe-40), 3.95 (s, 6H,
OMe-6 and 8), 4.10 (s, 3H, OMe-7), 7.02 (d, J = 8.8 Hz, 1H, H-50),
8.12 (dd, J = 8.8 and 2.3 Hz, 1H, H-60), 8.13 (d, J = 2.3 Hz, 1H, H-
20), 12.42 (s, 1H, 5-OH). 13C NMR (CDCl3) d ppm 45.2 (N(CH3)2),
55.5 (OMe-40), 58.1 (C-30–CH2–), 60.0 (OMe-3), 61.2, 61.8 and
62.0 (OMe-6, 7 and 8), 107.7 (C-10), 110.9 (C-50), 122.8 (C-10),
126.7 (C-30), 130.8 and 131.2 (C-20 and 60), 132.9 (C-8), 136.2 (C-
6), 138.7 (C-3), 149.2 (C-5), 153.1 (C-7), 160.7 (C-40); C-2, C-4
and C-9 not detected. ESIMS (+) m/z 446 [M+H]+, 468 [M+Na]+.
5.4.20. 40-Hydroxy-3,6,7,8-tetramethoxy-flavone (5-deoxy-
calycopterin) 25
A solution of 24 (0.23 g, 0.38 mmol) was heated at reflux in
MeOH (20 mL) till dissolution. Pd–C 10% (0.23 g) and HCOONH4
(0.29 g, 4.5 mmol) were added, and the mixture heated at reflux
for 1 h. The reaction was filtered, diluted with H2O, brought to
pH 6 with 2 N aqueous HCl, and extracted with CH2Cl2. Standard
work-up of the organic phase gave a dried residue, which provided
pure flavone 25 by crystallization in MeOH (0.11 g, 80%). Com-
pound 25 pale-yellow crystals: mp 243–245 °C; 1H NMR (DMSO-
d6) d ppm 3.78 (s, 3H, OMe-3), 3.90 and 3.92 (2s, 6H, OMe-6 and
8), 4.00 (s, 3H, OMe-7), 6.97 (d, J = 8.7 Hz, 2H, H-30 and 50), 7.26
(s, 1H, H-5), 7.94 (d, J = 8.7 Hz, 2H, H-20 and 60). APCIMS (+) m/z
359 [M+H]+.
5.4.17. 30-Cyano-5-hydroxy-3,6,7,8,40-pentamethoxy-flavone 22
A solution of 18 (25 mg, 0.06 mmol) in anhydrous formic acid
(5 mL) was added with 1.9 equiv NH2OH, HCl (8 mg), then heated
at reflux for 2 h. Standard work-up of the reaction gave a dried res-
idue, which was purified by crystallization with MeOH, then flash
chromatography (silica gel, CH2CH2–MeOH 99.5–0.5) to provide
pure 22 (14 mg) in 56% yield. Compound 22 bright-yellow crystals:
mp 148–149 °C; 1H NMR (CDCl3) d ppm 3.91 (s, 3H, OMe-3), 3.94
(s, 6H, OMe-6 and 8), 4.03 (s, 3H, OMe-40), 4.11 (s, 3H, OMe-7),
7.16 (d, J = 8.8 Hz, 1H, H-50), 8.39 (dd, J = 8.8 and 2.3 Hz, 1H, H-
60), 8.40 (d, J = 2.3 Hz, 1H, H-20), 12.17 (s, 1H, 5-OH). 13C NMR
(CDCl3) d ppm 56.5 (OMe-40), 60.4 (OMe-3), 61.2, 61.8 and 62.2
(OMe-6, 7 and 8), 102.7 (C-30), 107.5 (C-10), 111.7 (C-50), 115.7
(CN), 123.6 (C-10), 132.9 (C-8), 134.0 and 134.6 (C-20 and 60),
136.4 (C-6), 139.2 (C-3), 144.8 (C-9), 149.2 (C-5), 153.3 (C-7),
162.7 (C-40), 179.2 (C-4); C-2 not detected. HRESIMS (+) m/z
[M+H]+ 414.1210 (calcd for C21H20NO8, 414.1183), [M+Na]+
436.1032 (calcd for C21H19NO8Na, 436.1003).
5.4.21. 30-Nitro-3,6,7,8,40-pentamethoxy-flavone 27
A solution of 25 (0.093 g, 0.26 mmol) in trifluoroacetic acid
(5 mL) at 0 °C was added with 11.19 N HNO3 (1 equiv) then stirred
for 0.5 h. The reaction mixture was taken up in iced water, then ex-
tracted with CH2Cl2. Standard work-up of the organic layer affor-
ded a dried residue of crude crystallized nitroflavone 26 (0.094 g,
90%)., Without further purification, this residue was dissolved in
DMF (5 mL), added twice with K2CO3 (0.035 g, 0.25 mmol) and
iodomethane (0.1 mL, 1.6 mmol) at t = 0 and 2 h, and stirred for
6 h at rt. The mixture was diluted with H2O, brought to pH 6 with
1 N aqueous HCl, and extracted with CH2Cl2. Standard work-up of
the organic phase gave a dried residue, which led to pure com-
pound 27 (0.075 g, 69% from 25) by crystallization with MeOH
and tlc (alumina, CH2Cl2). Compound 27 pale-yellow crystals: mp
188–189 °C; 1H NMR (CDCl3) d ppm 3.96, 3.97, 4.05 and 4.08 (4s,
15H, OMe-3, 6,7,8 and 40), 7.25 (d, J = 8.8 Hz, 1H, H-50), 7.35 (s,
1H, H-5), 8.41 (dd, J = 8.8 and 2.3 Hz, 1H, H-60), 8.71 (d, J = 2.3 Hz,
1H, H-20). APCIMS (+) m/z 418 [M+H]+.
5.4.18. 40-Benzyloxy-5-hydroxy-3,6,7,8-tetramethoxy-flavone
(calycopterin 40O-benzyl ether) 23
5.4.22. 5,30-Dinitro-3,6,7,8,40-pentamethoxy-flavone 28
A solution of 27 (0.063 g, 0.15 mmol) in trifluoroacetic acid
(5 mL) at 0 °C was added with 11.19 N HNO3 (1.25 equiv) then stir-
red for 2 h. Same work-up as described for 27 led by crystallization
with MeOH to dinitroflavone 28 (0.011 g) in 16% yield. Compound
28 pale-yellow crystals: mp 174–176 °C; 1H NMR (CDCl3) d ppm
3.97, 3.99, 4.08 and 4.12 (4s, 15H, OMe-3, 6, 7, 8 and 40), 7.25 (d,
J = 8.8 Hz, 1H, H-50), 8.40 (dd, J = 8.8 and 2.3 Hz, 1H, H-60), 8.67
(d, J = 2.3 Hz, 1H, H-20). APCIMS (+) m/z 463 [M+H]+.
A solution of calycopterin 4 (0.3 g, 0.8 mmol) in DMF (10 mL)
was added with KHCO3 (0.1 g, 1 mmol) and benzyl bromide
(0.19 mL, 1.6 mmol) and stirred for 1.5 h at 115 °C. New amounts
of KHCO3 (0.05 g, 0.5 mmol) and benzyl bromide (0.095 mL,
8 mmol) were added and the reaction was carried on for 1.5 h
more. The reaction was diluted with CH2Cl2, filtered, and concen-
trated to dryness. The dried residue was taken up with CH2Cl2,
and the organic phase submitted to the standard work-up. Crystal-
lization of the residue with MeOH gave pure compound 23
(0.268 g, 72%). Compound 23 bright-yellow crystals: mp 117–
118 °C; 1H NMR (CDCl3) d ppm 3.89 (s, 3H, OMe-3), 3.98 (s, 6H,
OMe-6 and 8), 4.11 (s, 3H, OMe-7), 5.17 (s, 2H, C6H5–CH2) 7.11
(d, J = 8.7 Hz, 2H, H-30 and 50), 7.35–7.47 (m, 5H, C6H5–CH2), 8.16
(d, J = 8.7 Hz, 2H, H-20 and 60), 12.40 (s, 1H, 5-OH).
5.4.23. 5,30-Diamino-3,6,7,8,40-pentamethoxy-flavone 29
A solution of 28 (9.5 mg, 0.02 mmol) was heated at reflux in
MeOH (5 mL) till dissolution. Pd–C 10% (10 mg) and HCOONH4
(14 mg, 0.22 mmol) were added, and the mixture heated at reflux
for 2 h. The reaction was filtered, diluted with H2O, brought to
pH 6 with 2 N aqueous HCl, and extracted with CH2Cl2. Standard
work-up of the organic phase gave a dried residue, which was
purified by tlc (silica gel, CH2CH2–MeOH 98–2) to give pure diam-
inoflavone 29 (7 mg, 85%). Compound 29 Amorphous; 1H NMR
(CDCl3) d ppm 3.82 (s, 3H, OMe-3), 3.84 (s, 3H, OMe-6), 3.91 (s,
3H, OMe-8), 3.93 (s, 3H, OMe-40), 4.08 (s, 3H, OMe-7), 6.90 (d,
J = 8.8 Hz, 1H, H-50), 7.56 (d, J = 2.3 Hz, 1H, H-20), 7.62 (dd, J = 8.8
5.4.19. 40-Benzyloxy-3,6,7,8-tetramethoxy-5-trifluoromethane-
sulfonyloxy-flavone 24
A solution of 23 (0.26 g, 0.56 mmol) in dry THF (4 mL) was
added at rt under N2 with NaHMDS (2 M) in THF (0.4 mL,
0.8 mmol), and the mixture was stirred for 15 min. PhN(Tf)2
(0.4 g, 1.12 mmol) was added and the reaction mixture stirred for
17 h more. New amounts of NaHMDS (0.2 mL, 0.4 mmol) and
PhN(Tf)2 (0.133 g, 0.37 mmol) were added and the reaction stirred
for 5 h more. The reaction was diluted with H2O, and thoroughly
13
and 2.3 Hz, 1H, H-60). C NMR (CDCl3) d ppm 55.7 (OMe-40), 59.9
and 60.2 (OMe-3 and 6), 61.2 (OMe-7), 61.8 (OMe-8), 106.3
(C-10), 110.0 (C-50), 114.4 (C-20), 119.8 (C-60), 123.6 (C-10), 130.4
(C-8), 133.3 (C-6), 136.1 (C-30), 139.2 (C-3), 149.1 (C-40), 151.3

G. Lewin et al. / Bioorg. Med. Chem. 19 (2011) 186–196
195
(C-7); C-2, C-4, C-5 and C-9 non detected. HRESIMS (+) m/z [M+H]+
403.1471 (calcd for C20H23N2O7, 403.1499).
J = 2.3 Hz, 1H, H-20), 7.63 (dd, J = 8.8 and 2.3 Hz, 1H, H-60), 12.65
(s, 1H, 5-OH). APCIMS (+) m/z 390 [M+H]+.
5.4.24. 30-Amino-3,6,7,8,40-pentamethoxy-flavone 30
5.5. Biological evaluation
Hydrogenation of 27 (10 mg, 0.024 mmol) under same condi-
tions of transfer hydrogenation as described from 29, then purifica-
tion of the dried residue by tlc (silica gel, CH2CH2–MeOH 97.5–2.5)
led to pure crystallized aminoflavone 30 (8 mg, 86%). Compound
30 bright-yellow crystals: mp 193–195 °C; 1H NMR (CDCl3) d
ppm 3.87, 3.95, 3.97, 4.03 and 4.05 (5s, 15H, OMe-3, 6, 7, 8 and
40), 6.92 (d, J = 8.8 Hz, 1H, H-50), 7.40 (s, 1H, H-5), 7.56 (d,
J = 2.3 Hz, 1H, H-20), 7.62 (dd, J = 8.8 and 2.3 Hz, 1H, H-60). 13C
NMR (CDCl3) d ppm 55.6 (OMe-40), 59.9 (OMe-3), 56.3, 61.5 and
62.1 (OMe-6, 7, 8), 99.8 (C-5), 110.1 (C-50), 114.4 (C-20), 119.8 (C-
60), 120.1 (C-10), 123.8 (C-10), 136.2 (C-30), 140.5 (C-3), 142.0 (C-
8), 144.8 (C-6), 147.0 (C-9), 149.3 (C-40), 150.9 (C-7), 156.2 (C-2),
177.8 (C-4). HRESIMS (+) m/z [M+H]+ 388.1416 (calcd for
5.5.1. Cell culture
Human cell lines were purchased from ATCC or ECACC or
obtained from the NCI. The human cell lines KB, MiaPaca and
HepG2 were cultured in D-MEM medium supplemented with
10% fetal calf serum, in the presence of penicillin, streptomycin
and fungizone in 75 cm2 flasks under 5% CO2, whereas all other cell
lines were cultured in complete RPMI medium.
5.5.2. Cell proliferation assay
Cells (600 cells/well) were plated in 96-well tissue culture
microplates in 200
compounds dissolved in DMSO at concentrations that ranged
0.5 nM–10 with Biomek 3000 automation workstation
lL of medium and treated 24 h later with
C20H22NO7, 388.1396).
lM
a
(Beckman-Coulter). Control cells receivedthe same volumeof DMSO
(1% final volume). After 72 h exposure to the drug, MTS reagent (Pro-
mega) was added and incubated for 3 h at 37 °C. Experiments were
performed in triplicate: the absorbance was monitored at 490 nm
and results were expressed as the inhibition of cell proliferation cal-
culated as the ratio [(1 ꢃ (OD490 treated/OD490 control)) ꢀ 100]. For
IC50 determinations (50% inhibition of cell proliferation) experi-
ments were performed in duplicate.
5.4.25. 30-Amino-3,5,6,7,8,40-hexamethoxy-flavone 31
Hydrogenation of 10 (10 mg, 0.022 mmol) under same condi-
tions of transfer hydrogenation as described from 29, then purifica-
tion of the dried residue by tlc (silica gel, CH2CH2–MeOH 97.5–2.5)
led to pure crystallized aminoflavone 31 (8 mg, 86%). Compound 5
light-yellow crystals: mp 160–162 °C; 1H NMR (CDCl3) d ppm 3.86
(s, 3H, OMe-3), 3.94 (2s, 6H, OMe-6 and 40), 3.97 (s, 3H, OMe-5),
3.99 (s, 3H, OMe-8), 4.09 (s, 3H, OMe-7), 6.91 (d, J = 8.8 Hz, 1H,
H-50), 7.56 (d, J = 2.3 Hz, 1H, H-20), 7.62 (dd, J = 8.8 and 2.3 Hz,
5.5.3. Activation of caspases 3/7
13
1H, H-60). C NMR (CDCl3) d ppm 55.6 (OMe-40), 59.8 (OMe-3),
HL60 cells (20,000 cells/well) were plated in 96-well black
tissue culture microplates and treated for 24 and 48 h with com-
pounds dissolved in DMSO. Control cells received the vehicle only
and positive control cells were treated with 1 lM doxorubicine.
Cells were lysed with a buffer consisting in 25 mM HEPES, pH
61.3–61.8 (OMe-5, 6, 7, 8), 110.1 (C-50), 114.3 (C-20), 119.6 (C-60),
123.1 (C-10), 136.0 (C-30), 137.6 (C-8), 140.5 (C-3), 143.4 (C-6),
147.9 (C-5), 149.3 (C-40), 151.5 (C-7), 153.5 (C-2); C-4, C-9, C-10
not detected. APCIMS (+) m/z 418 [M+H]+.
7.5, 0.5 mM EDTA, 0.05% NP40, 0.001% SDS and 5 mM DTT contain-
ing 50 lM Ac-DEVD-AMC (Biomol). Fluorescence was monitored
(kex = 360 nm, kem = 465 nm) over a 3 h period.
5.4.26. 30-Amino-5,6-dihydroxy-3,7,8,40-tetramethoxy-flavone 32
and 30-amino-5,8-dihydroxy-3,6,7,40-tetramethoxy-flavone 33
A solution of 9 (33 mg, 0.075 mmol) in trifluoroacetic acid
(4 mL) at 0 °C was added with 11.19 N HNO3 (1 equiv) then stirred
for 0.5 h. The reaction mixture was taken up in iced water, then ex-
tracted with CH2Cl2. Standard work-up of the organic layer affor-
5.5.4. Inhibition of tubulin polymerization assay
Sheep brain microtubule proteins were purified by two cycles
of assembly/disassembly at 37 °C/0 °C in MES buffer: 100 mM
MES (2-[N-morpholino]-ethanesulfonic acid, pH 6.6), 1 mM EGTA
(ethyleneglycol-bis[b-aminoethyl ether]-N,N,N0,N0-tetraacetic acid),
0.5 mM MgCl2. All samples were dissolved in DMSO. The evaluated
ded
a red dried residue, which was submitted to catalytic
hydrogenolysis in DMF (4 mL) under 1 atm pressure hydrogen with
10% Pd–C (35 mg) at room temperature for 1.5 h. The catalyst was
separated, and the filtrate concentrated to dryness (19 mg). The
purification by tlc (silica gel, CH2CH2–MeOH 99–1) was very diffi-
cult owing to the very near Rf of two isomers, but it allowed isola-
tion of pure 32 and 33 in very weak yields (6% for each isomer).
Compound 32 Amorphous; 1H NMR (CDCl3) d ppm 3.85 (s, 3H,
OMe-3), 3.95 (s, 3H, OMe-40), 3.96 (s, 3H, OMe-8), 4.13 (s, 3H,
OMe-7), 6.91 (d, J = 8.8 Hz, 1H, H-50), 7.55 (d, J = 2.3 Hz, 1H, H-20),
7.63 (dd, J = 8.8 and 2.3 Hz, 1H, H-60), 12.25 (s, 1 h, 5-OH). APCIMS
(+) m/z 390 [M+H]+. Compound 33 Amorphous; 1H NMR (CDCl3) d
ppm 3.84 (s, 3H, OMe-3), 3.94 (s, 3H, OMe-40), 3.97 (s, 3H, OMe-6),
4.12 (s, 3H, OMe-7), 6.90 (d, J = 8.8 Hz, 1H, H-50), 7.54 (d, J = 2.3 Hz,
1H, H-20), 7.63 (dd, J = 8.8 and 2.3 Hz, 1H, H-60), 12.18 (s, 1H, 5-OH).
APCIMS (+) m/z 390 [M+H]+.
compound (1 lL) was added to microtubular solution (150 lL) that
was incubated at 37 °C for 10 min and at 0 °C for 5 min. The tubulin
polymerization rate was measured by turbidimetry at 350 nm
according to Zavala and Guénard’s protocol26 using deoxypodo-
phyllotoxin as reference compound. Compounds were tested at
ꢁ2 ꢀ 10ꢃ5 M, and results were given as the percentage of IPT or as
IC50, calculated for the most active compounds, and also expressed
in relation to deoxypodophyllotoxin (DPPT) in terms of the IC50/IC50
DPPT ratio.
5.5.5. Measurement of annexin-V-PE/7-AAD staining
HL60 cells (5000 cells/well in 96-well microplates) were
exposed for 24 and 48 h at 37 °C under 5% CO2 with chemicals in
100
of DMSO (1% final volume). 7-AAD (6.5
tion) and human recombinant annexinV-PE (6.5
added to 1 ml binding buffer consisting in 30 mM Hepes buffer,
pH7.4, 420 mM NaCl and 7.5 mM CaCl2 immediately prior to addi-
tion to cells. After a 20 min incubation at room temperature in the
dark, cells were analyzed by flow cytometry with a Guava EasyCyte
plus cytometer (Millipore). Cells were classified according to their
fluorescence and results expressed as the percentage of cells in
each group, calculated on 5000 events.
l
l complete RPMI medium. Controls received the same volume
l of 1 mg/ml ethanol solu-
l, Bender) were
5.4.27. 30-Amino-5,7-dihydroxy-3,6,8,40-tetramethoxy-flavone 34
A mixture of 5 (8 mg, 0.02 mmol) and 3.5 equiv LiCl (3 mg,
0.07 mmol) in DMF was stirred under N2 at 180 °C for 17 h. The
reaction was diluted with H2O and extracted by n-butanol. Concen-
tration to dryness of the organic phase and purification of the res-
idue by tlc (silica gel, CH2CH2–MeOH 98–2) provided pure
amorphous 34 in 26% yield. Amorphous; 1H NMR (CDCl3) d ppm
3.85 (s, 3H, OMe-3), 3.95 (s, 3H, OMe-40), 3.98 (s, 3H, OMe-6),
4.04 (s, 3H, OMe-8), 6.91 (d, J = 8.8 Hz, 1H, H-50), 7.55 (d,
l
l

196
G. Lewin et al. / Bioorg. Med. Chem. 19 (2011) 186–196
3. Lichius, J. J.; Thoison, O.; Montagnac, A.; Païs, M.; Guéritte-Voegelein, F.;
5.5.6. Cell cycle analysis
Sévenet, T. J. Nat. Prod. 1994, 57, 1012.
KB cells (25,000 cells/well in 96-well microplates) were
4. Shi, Q.; Li, L.; Chang, J.-J.; Autry, C.; Kozuka, M.; Konoshima, T.; Estes, J. R.; Lin, C.
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100 ll complete RPMI medium. Controls received the same volume
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and gently centrifuged to collect floating cells, adherent cells har-
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2 h at 4 °C, cells were spun down by centrifugation, washed with
2% FCS in PBS and stained with 50
tonic buffer in the presence of RNase A (50
room temperature shielding away from light, before be analyzed
by flow cytometry with a Guava Easycyte cytometer (Millipore).
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Acknowledgments
We thank J.-C. Jullian for NMR measurements, and Dr. F. Gueritte
for a sample of 1.
Supplementary data
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Supplementary data associated with this article can be found, in
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