Hemisynthesis and antiproliferative properties of mono-[O-(14- benzoylaconine-8-yl)]esters and bis-[O-(14-benzoylaconine-8-yl)]esters

Article abstract of DOI:10.1016/j.ejmech.2012.05.015

A series of mono- and bifunctional acyl compounds, build from the 8-O-azeloyl-14-benzoylaconine scaffold and differing by the length of the alkyl linker chain, were synthesised and evaluated against a panel of human tumour cell lines, A-549 (lung cancer), MCF-7 (breast cancer) and HCT-15 (colon cancer). None of the mono-[O-(14-benzoylaconine-8-yl)]esters displayed in vitro activity against tumour cells (IC₅₀ > 60 μM). However, three bis-[O-(14-benzoylaconine-8-yl)]esters presented a noticeable in vitro cytotoxic activity, those bearing 7, 8 and 9 carbon atoms between the two aconitine moieties, with IC₅₀s ranging between 4 and 28 μM. The most active, bis[O-(14-benzoylaconine-8-yl)]suberate, was then evaluated in vivo in immunodeficient mice bearing human tumour xenografts originating from MCF-7 and HCT-15 cells. For MCF-7 cells, administration of five doses every 4 days, and weekly administration of 4 doses resulted in T/C percent values of 36% (p = 0.001) and 56% (p = 0.02) on day 45, respectively. For HCT-15 cells, administration of five doses every 3 days resulted in 49% tumour regression on the 25th day (p = 0.00001).

Full text of DOI:10.1016/j.ejmech.2012.05.015

European Journal of Medicinal Chemistry 54 (2012) 343e351
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Hemisynthesis and antiproliferative properties of mono-[O-(14-benzoylaconine-
8-yl)]esters and bis-[O-(14-benzoylaconine-8-yl)]esters
Ainura Chodoeva a, Jean-Jacques Bosc b, Jean Guillon b, Pierre Costet c, Alain Decendit d,
,
Jean-Michel Mérillon d, Jean-Michel Léger b, Christian Jarry b, Jacques Robert a
*
a Université Bordeaux Segalen, Institut Bergonié, 229 cours de l’Argonne, 33076 Bordeaux, France
b FRE CNRS 3396 e Pharmacochimie, Université Bordeaux Segalen, 146 rue Léo-Saignat, 33076 Bordeaux, France
c Animalerie spécialisée, Université Bordeaux Segalen, 146 rue Léo-Saignat, 33076 Bordeaux, France
d Groupe d’Étude des Substances Végétales à Activité Biologique, EA 3675, Université Bordeaux Segalen, 146 rue Léo-Saignat, 33076 Bordeaux cedex, France
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
< We synthesised
a
series of
compounds derived from Aconitum
alkaloids.
< Three of them displayed anti-
proliferative activity against human
tumour cell lines.
< Antitumour activity was detected in
human tumour xenografts in
immunodeficient mice.
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of mono- and bifunctional acyl compounds, build from the 8-O-azeloyl-14-benzoylaconine
scaffold and differing by the length of the alkyl linker chain, were synthesised and evaluated against
a panel of human tumour cell lines, A-549 (lung cancer), MCF-7 (breast cancer) and HCT-15 (colon
cancer). None of the mono-[O-(14-benzoylaconine-8-yl)]esters displayed in vitro activity against tumour
Received 8 March 2012
Received in revised form
4 May 2012
Accepted 10 May 2012
Available online 17 May 2012
cells (IC50 > 60
in vitro cytotoxic activity, those bearing 7, 8 and 9 carbon atoms between the two aconitine moieties,
with IC50s ranging between 4 and 28 M. The most active, bis[O-(14-benzoylaconine-8-yl)]suberate, was
mM). However, three bis-[O-(14-benzoylaconine-8-yl)]esters presented a noticeable
m
Keywords:
then evaluated in vivo in immunodeficient mice bearing human tumour xenografts originating from
MCF-7 and HCT-15 cells. For MCF-7 cells, administration of five doses every 4 days, and weekly
administration of 4 doses resulted in T/C percent values of 36% (p ¼ 0.001) and 56% (p ¼ 0.02) on day 45,
respectively. For HCT-15 cells, administration of five doses every 3 days resulted in 49% tumour
regression on the 25th day (p ¼ 0.00001).
Aconitum alkaloids
Antiproliferative activity
Tumour models
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
The plant kingdom harbours the largest reservoir of pharma-
cologically active natural compounds. About 40% of all drugs derive
directly or indirectly from plant alkaloids, and only a small fraction
of the biosphere has been yet analysed. Several potent anticancer
* Corresponding author. Tel.: þ33 556 33 33 27; fax: þ33 556 33 04 38.
E-mail address: robert@bergonie.org (J. Robert).
drugs of widespread use were isolated from plants, among which
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.05.015

344
A. Chodoeva et al. / European Journal of Medicinal Chemistry 54 (2012) 343e351
vinblastine, paclitaxel (TaxolÒ) and camptothecin are the most
renowned. In an attempt to identify new potential anticancer
drugs, we recently discovered that 8-O-azeloyl-14-benzoylaconine,
an aconitine derivative, extracted from the roots of the Asian plant
Aconitum karakolicum Rapcs displayed cytotoxic properties against
human tumour cells in culture [1].
In the present work, the previously described 8-O-azeloyl-
14-benzoylaconine [1], was used as a basis for the design and
hemisynthesis of new structural analogues. A series of mono-
and bifunctional acyl compounds, build from the 8-O-azeloyl-
14-benzoylaconine scaffold and differing by the length of the alkyl
linker, were synthesised and evaluated against a panel of human
tumour cell lines. The most active, bis[O-(14-benzoylaconine-8-yl)]
suberate was then evaluated in vivo in appropriate models of
immunodeficient mice bearing human tumour xenografts. We
report here the hemisynthesis method and preliminary anticancer
activity data of new mono-[O-(14-benzoylaconine-8-yl)]esters and
bis-[O-(14-benzoylaconine-8-yl)]esters.
Plants of the Aconitum genus have been used in traditional
medicine in many Asian and former USSR countries. In spite of their
high toxicity, Aconitum plants display analgesic, anti-inflammatory
and anti-arrhythmic activities, and their use has been proposed in
several diseases and pathological conditions [2]. The principal
chemical constituents of the Aconitum genus are diterpenoid
alkaloids such as aconitine, mesaconitine, lappaconitine and son-
gorine. Other new other new alkaloids have been recently isolated
and studied [3e6]. Toxicological and pharmacological properties of
purified compounds from Aconitum have been studied in vitro and
in vivo: aconitine, 3-acetylaconitine and hypaconitine were found
to be the most toxic alkaloids, inhibiting the respiratory and
cardiovascular systems [7]. The proposed mechanism of the toxic
effect of the principal alkaloid, aconitine, is the block of the voltage-
gated Naþ channels, inducing an influx of Naþ ions into cells, which
finally results in permanent neuron depolarisation and inhibition of
neuronal conductivity [8].
2. Results
2.1. Chemistry
Commercially available reagents were used as received without
additional purification. The aliphatic acid mono- and di-esters of
aconitine 1aeh and 2aef were prepared from commercially avail-
able aconitine using a modified procedure adapted from Pelletier
[13]. The reaction of aconitine with various aliphatic diacids in DMF
at 80 ꢀC led to the formation of the mono-[O-(14-benzoylaconine-8-
yl)]esters 1aeh and bis-[O-(14-benzoylaconine-8-yl)]esters 2aef
through a transesterification carried out on the 8-acetyl function-
ality of the aconitine moiety (Scheme 1). The reaction mixtures were
heated during 7 h in an oil bath for which the temperature was
regulated at 80 ꢀC, due to the heat-sensitivity of reaction products
[14]. Purification by HPLC of the reaction mixture led to the isolation
of 1 and 2 with yields ranging from 37 to 44% and from 18 to
25%, respectively. Structural identification was performed using
high resolution MSeMS mass spectrometry and 1H, 13C, 2D
NOESY NMR spectroscopy analysis. The isolation of a suitable tri-
fluoroacetate salt of 1d allowed to establish unambiguously its 3D
In spite of various and detailed studies of Aconitum alkaloids,
very little information is available on their antitumour activity. De
Ines et al. [9] showed the cytotoxic effect of various C19-nordi-
terpenoid alkaloids against several tumour cell lines. Hazawa et al.
[10e12] reported that semi-synthetic derivatives of C20-nordi-
terpenoid alkaloids, e.g. 11-veratroylpseudokobustine, 11-(m-tri-
fluoromethylbenzoyl)kobustine,
11-(m-trifluoromethylbenzoyl)
pseudokobustine and 11,15-dianisoylpseudokobusine, exhibited
cytotoxic activity against A172, A-549, HeLa and Raji cell lines. Yet,
the mechanism of the cytotoxic activity of Aconitum derivatives
remained unclear.
HO
OCH3
O
OCH3
CH3
HO
O
OH
N
O
(
)
n
OH
OCH3
O
CH3O
O
1a-h
HO
OCH3
OCH3
O
+
CH3
HO
O
HO
HOOC-(CH2)n-COOH
DMF, 80°C
OH
N
OCH3
O
O
CH3
OCH3
CH3O
OH
OCH3
O
CH3
O
CH3O
CH3O
OH
N
aconitine
CH3
N
O
HO
(
)
n
a : n = 2
b : n = 3
c : n = 4
d : n = 5
e : n = 6
f : n = 7
g : n = 8
h : n = 9
OCH3
O
OCH3
O
CH3O
O
HO
O
2a-f
OH
O
OCH3
Scheme 1. Synthesis of new mono-[O-(14-benzoylaconine-8-yl)]esters 1aeh and bis-[O-(14-benzoylaconine-8-yl)]esters 2aef.

A. Chodoeva et al. / European Journal of Medicinal Chemistry 54 (2012) 343e351
345
spatial structure by single crystal X-ray crystallography, with one
alkyl diacid moietyat C-8 of aconitine scaffold (Fig.1). Moreover, this
established the R-configuration of carbon C-8 of 1d. With reference
to aconitine [14], no epimerisation occurred during the trans-
esterification procedure.
Compound 2e was well tolerated and neither mortality nor toxic
effects were observed during animal experiments (Table 2).
Moreover, concerning weight variation of the treated mice, only
one group of treated mice presented a moderate weight loss of
9.34% in reference to the control group.
For MCF-7 cells, administration of five doses every 4 days, and
weekly administration of four doses resulted in T/C percent values
of 36% (p ¼ 0.001) and 56% (p ¼ 0.02) on day 45, respectively
(Fig. 2). For HCT-15 cells, administration of five doses every 3 days
resulted in 49% tumour regression on the 25th day (Fig. 2,
p ¼ 0.00001). These preliminary in vivo results are encouraging and
warrant further investigation, which is presently ongoing.
2.2. Biological activity
2.2.1. In vitro antiproliferative activity
The two series of compounds 1aeh and 2aef were tested
against three human tumour cell lines, A-549 (lung cancer), MCF-
7 (breast cancer), and HCT-15 (colon cancer). None of the mono-
[O-(14-benzoylaconine-8-yl)]esters displayed in vitro activity
against tumour cells (IC50 > 60
m
M) (Fig. 2). Concerning the bis-
3. Discussion
[O-(14-benzoylaconine-8-yl)]esters 2def, there was a discrep-
ancy between compounds with a C-4, C-5 or C-6 linker chain and
those with a C-7, C-8 or C-9 linker chain. As depicted in Table 1,
only the latter group presented a noticeable in vitro cytotoxic
In a previous study, we reported the preliminary screening
cytotoxic activity of the naturally occurring 8-O-azeloyl-14-
benzoylaconine, isolated from A. karakolicum, against three types
of in vitro human tumour cell lines [1]. We undertook further
progress of this work by the design and hemisynthesis of new
structural analogues bearing various alkyl chains on the heterocy-
clic moiety, and the evaluation of their antiproliferative activity.
From the results depicted in Table 1, it appears that the
replacement of the azeloyl moiety by C-4 to C-11 analogues
(compounds 1aeh) led to compounds devoid of cytotoxic activity
activity (IC50 ranging from 4 to 28 mM) (Figs. 2 and 3, Table 1).
2.2.2. In vivo testing of bis[O-(14-benzoylaconine-8-yl)]suberate
(2e)
An in vivo study was undertaken for the bis[O-(14-
benzoylaconine-8-yl)]suberate 2e. It was evaluated for anticancer
activity in immunodeficient mice xenografted subcutaneously with
human tumour cells, at the dose of 10 mg/kg. The same tumours as
those used for the in vitro evaluation were chosen for the in vivo
studies. However, the A-549 cell line grew very slowly in vivo and
was not considered as suitable for antitumour evaluation.
(IC50 > 60
mM). On the other hand, some related di-esterified
compounds
2
exhibited notable cytotoxic activities against
several tumour cell lines. These findings show that potency is
affected by structural change and also indicate that the binding site
Fig. 1. The ORTEP drawing of mono-[O-(14-benzoylaconine-8-yl)]succinate 1d as trifluoroacetate salt with thermal ellipsoids at 20% level.

346
A. Chodoeva et al. / European Journal of Medicinal Chemistry 54 (2012) 343e351
Fig. 2. In vitro cytotoxicity of compounds 1d, 1e, 2d and 2e against three human tumour cell lines: A-549; MCF-7; HCT-15. A representative experiment made in triplicate is shown.
of the target cancer cell may have a strict structural requirement.
Finally, this difference of in vitro cytotoxic behaviour between
mono- and bis-[O-(14-benzoylaconine-8-yl)]esters prompted us to
select the latter for further in vivo studies performed with breast
cancer (MCF-7) and colon cancer (HCT-15) cells.
Among compounds 2aef, the bis-[O-(14-benzoylaconine-8-yl)]
esters 2def linked by a C-7 to C-9 moiety, exhibited the best anti-
proliferative activity on the human tumour cell lines (IC50 from 4 to
The in vitro activity of the diester compounds 2def was
encouraging enough to test the activity of 2e on the same human
tumour models growing in vivo in immunodeficient mice. Anti-
tumour activity was clearly detected at the dose of 10 mg/kg, a dose
largely below the maximum tolerated dose (15 mg/kg). Thus,
obtaining an antitumour activity before any optimisation of the
dose to be injected, or of the tumour models to be tested, appears
very promising and prompted us to patent new hemisynthetic bis
[O-(14-benzoylaconine-8-yl)]esters [15].
28
m
M) in comparison with their C-4 to C-6 linked analogues 2aec
M). Hence, 2e was found as the most active compound
M). The replacement of the
(IC50 > 60
m
on the A-549 cell line (IC50 ¼ 7.53
m
4. Conclusion
suberate (2e) linker by a pimelate (2d) or an azelate one (2f) led to
a slight decrease in the antiproliferative activity (IC50 ¼ 9.50 and 19.5
In the present report, we described the synthesis of new series
of mono-[O-(14-benzoylaconine-8-yl)]esters 1aeh and bis-[O-(14-
mM, respectively). In the MCF-7 cell line, the diester 2e, bearing
a suberate linker, was found to have almost the same activity as its
pimelate analogue 2d with a 6.9 versus 7.56 M IC50. In the HCT-15
colon cancer cell line, the two diesters 2d and 2e bearing a suberate
or pimelate linker, were found equally active with 4.01e4.64
IC50. In addition, the bis-[O-(14-benzoylaconine-8-yl)]ester 2f was
benzoylaconine-8-yl)]esters 2aef through
a transesterification
m
carried out on the 8-acetyl function of the aconitine moiety. Their
antiproliferative activity on the human cancer cell lines A-549,
MCF-7 and HCT-15 shows that the bis-[O-(14-benzoylaconine-8-
yl)]esters 2aef are more active than their mono-[O-(14-
benzoylaconine-8-yl)]esters counterparts 1aeh, indicating that
mM
found less potent (IC50 ¼ 28
mM) against this colon cancer line.
1600
600
500
400
300
200
100
HCT-15
MCF-7
1400
1200
1000
800
600
*
*
400
200
0
*
*
*
*
*
*
0
0
0
5
10
15
20
25
10
20
30
40
50
Days
Days
Fig. 3. In vivo antitumour activity of bis[O-(14-benzoylaconine-8-yl)]suberate 2e on MCF-7 and HCT-15 tumour models grown in immunodeficient mice. Two schedules were used
for MCF-7 tumours (see Table 2). Tumour weights are means ꢁ s.e.m. of 8e10 individual values. Each asterisk indicates a significant difference between treated and control animals
at the p < 0.01 level.

A. Chodoeva et al. / European Journal of Medicinal Chemistry 54 (2012) 343e351
347
Table 1
In vitro cytotoxicity of bis[O-(14-benzoylaconine-8-yl)]esters 2aef against human tumour cell lines.
Compound
IC50 (mM)
A-549
MCF-7
HCT-15
bis-[O-(14-benzoylaconine-8-yl)]-succinate
bis-[O-(14-benzoylaconine-8-yl)]-glutarate
bis-[O-(14-benzoylaconine-8-yl)]-adipate
bis-[O-(14-benzoylaconine-8-yl)]-pimelate
bis-[O-(14-benzoylaconine-8-yl)]-suberate
bis-[O-(14-benzoylaconine-8-yl)]-azelate
2a
2b
2c
2d
2e
2f
>60
>60
>60
9.50 ꢁ 3.21
7.53 ꢁ 3.08
19.5
>60
>60
>60
7.56 ꢁ 0.84
6.90 ꢁ 1.62
16.9
>60
>60
>60
4.64 ꢁ 1.53
4.01 ꢁ 0.51
28.0
IC50 values are means ꢁ s.e.m. of 2e4 experiments.
the binding site of the target cancer cell may have a strict structural
requirement. Furthermore, compounds 2def linked by a pimelate,
a suberate or an azelate moiety are the most active with IC50
pressure liquid chromatography to give mono-[O-(14-
benzoylaconine-8-yl)]esters 1aeg and bis[O-(14-benzoylaconine-
8-yl)]esters 2aef. Flash chromatography on silica gel was used to
isolate the mono-[O-(14-benzoylaconine-8-yl)]ester 1h.
ranging from 4 to 28
the alkyl linker.
mM, enlightening the influence of the length of
Based on these preliminary results, further studies to enlarge
the biological evaluation of the most active bis-[O-(14-
benzoylaconine-8-yl)]esters are in progress in order to address its
mechanism of action. This series can be considered as a promising
lead for future rational design of novel antiproliferative agents.
5.1.1.1. 8-O-(14-Benzoylaconine)succinate
(1a). White powder
(39%); tR ¼ 7.8 min; 1H NMR spectrum (400.13 MHz, CDCl3)
d: 8.09
(m, 1H, NHþ), 8.03 (d, 2H, J ¼ 7.7 Hz, H-20/H-60), 7.64 (t, 1H,
J ¼ 7.3 Hz, H-40), 7.51 (t, 2H, J ¼ 7.6 Hz, H-30/H-50), 4.97 (d, 1H,
J ¼ 4.4 Hz, H-14), 4.55 (d, 1H, J ¼ 4.8 Hz, H-15), 4.32 (br s, 1H, H-3),
4.18 (d, 1H, J ¼ 6.0 Hz, H-6), 3.81 (s, 3H, 16eOCH3), 3.69 (m, 1H, H-
19b), 3.54 (br s, 1H, H-1), 3.47 (m, 1H, 18b), 3.41 (s, 3H, 1eOCH3),
5. Experimental procedures
3.39 (s, 1H, H-17), 3.31 (s, 3H, 18eOCH3), 3.29 (m, 4H,
NeCH2eCH3, H-16, H-18 ), 3.27 (s, 3H), 6(eOCH3), 3.21 (m, 1H, H-
19
), 3.02 (br s,1H, H-5), 2.97 (m,1H, H-9), 2.52 (d,1H, J ¼ 5.9 Hz, H-
7), 2.42 (m, 1H, H-2 ), 2.40 (br d, 1H, H-10), 2.34 (br d, 3H, H-12
8eCO2e(CH2)200), 1.88 (br d, 1H, H-12
), 1.55 (m, 3H, 8eCO2e(CH2)
100, H-2 ), 1.45 (br s, 3H, NeCH2eCH3). 13C NMR (CDCl3)
: 175.0
a
5.1. Chemistry
a
b
b,
Commercially available reagents were used as received without
additional purification. Melting points were determined with an
SM-LUX-POL Leitz hot-stage microscope and are uncorrected. IR
spectra were recorded on a BRUKER IFS-25 spectrophotometer.
NMR spectra were recorded with tetramethylsilane as an internal
standard using a BRUKER AVANCE III/600 spectrometer (2D-COSY,
HSQC and HMBC). Splitting patterns have been designated as
follows: s ¼ singlet; bs ¼ broad singlet; d ¼ doublet; t ¼ triplet;
q ¼ quartet; qt ¼ quintuplet; m ¼ multiplet. Analytical TLC was
carried out on 0.25 precoated silica gel plates (POLYGRAM SIL G/
UV254) with visualisation by irradiation with a UV lamp. The
conditions used for HPLC purification were the following: Column:
a
a
d
(COOH), 173.90 (8-COO), 166.0 (C-70), 134.16 (C-40), 129.86 (C-20/C-
60), 129.50 (C-10), 129.16 (C30/C-50), 90.48 (C-8), 89.92 (C-16), 81.99
(C-6), 79.87 (C-1), 78.72 (C-15), 78.58 (C-14), 76.23 (C-18), 74.06 (C-
13), 69.98 (C-3), 63.72 (C-17), 61.73 (16-OCH3), 59.22 (18-OCH3),
58.06 (6-OCH3), 55.40 (1-OCH3), 50.42 (C-19), 50.41 (C-11), 50.40
(NeCH2eCH3), 44.95 (C-4), 43.48 (C-4), 43.27 (C-9), 42.78 (C-7),
39.84 (C-10), 35.09 (C-12), 29.42 (C-2), 29.19 (C-100, C-200), 10.81
(NeCH2eCH3). HRMS [M]þ C36H49NO13 requires 703.78, found
704.3.
Ultrasep ES 10, RP 18 6.0
mm reversed phase, C18 column,
5.1.1.2. 8-O-(14-Benzoylaconine)glutarate
(1b). White powder
250 ꢂ 10 mm (Bischoff, Germany). Solvents: A: trifluoroacetic acid/
water 1/1000; B: trifluoroacetic acid/acetonitrile 1/1000. Elution
conditions: injection of a volume of 0.5 mL; gradient from 25% of B
to 50% in 50 min, followed by 100% of B during 10 min. Absorbance
was monitored at 234 nm. High-resolution ESI mass measurements
were performed on an Applied Biosystems QStar mass spectrom-
eter equipped with an electrospray source in positive mode.
(37%); tR ¼ 15.3 min; 1H NMR spectrum (400.13 MHz, CDCl3)
d: 8.18
(m,1H, NHþ), 8.02 (d, 2H, J ¼ 5.12 Hz, H-20/H-60), 7.64 (t,1H, J ¼ 5.0 Hz,
H-40), 7.51 (t, 2H, J ¼ 5.06 Hz, H-30/H-50), 4.93 (d,1H, J ¼ 3.0 Hz, H-14),
4.53 (d, 1H, J ¼ 3.5 Hz, H-15), 4.30 (br s, 1H, H-3), 4.16 (d, 1H,
J ¼ 4.08Hz, H-6), 3.82(s, 3H,16-OCH3), 3.70(m,1H, H-19
b), 3.52(br s,
1H, H-1), 3.50 (m,1H, H-18
3.30 (s, 3H, 18-OCH3), 3.29 (m, 2H, NeCH2eCH3), 3.28 (s, 1H, H-16),
3.27 (s,1H, H-18 ), 3.25 (s, 3H, 6-OCH3), 3.19 (m,1H, H-19 ), 2.97 (m,
2H, H-5, H-9), 2.59 (d,1H, J ¼ 3.8 Hz, H-7), 2.42 (br d,1H, H-2 ), 2.39
(br d, 1H, H-10), 2.36 (br d, 1H, H-12 ), 2.07 (m, 1H, 8eCO2e(CH2)
300 ), 2.0 (m, 2H, 8eCO2e(CH2)100 , 8eCO2e(CH2)300
),1.92 (d, 1H,
), 1.54 (m, 2H, 8eCO2e(CH2)100
, 8eCO2e(CH2)
, 8eCO2e(CH2)
: 175.0 (COOH), 174.42 (8-COO), 165.68 (C-
b), 3.40 (s, 3H,1-OCH3), 3.36 (s,1H, H-17),
a
a
5.1.1. General procedure for synthesis of mono-[O-(14-
benzoylaconine-8-yl)]esters (1) and bis[O-(14-benzoylaconine-8-
yl)]esters (2)
Aconitine (200 mg, 0.30 mmol) and aliphatic diacid (0.15 mmol)
in DMF (5 mL) were heated at 80 ꢀC under stirring during 7 h, then
stirred at room temperature overnight. The solvent was then
removed under reduced pressure at a temperature lower than
70 ꢀC. The oily residue was purified by semi-preparative high
b
b
b
b
a
J 0¼0 6.64 Hz, H-12
a
a
a
2
b
), 1.46 (br s, 3H, NeCH2eCH3), 1.44 (m, 2H, H-2
200
a
). 13C NMR (CDCl3)
d
70), 133.91 (C-40), 129.63 (C-20/C-60), 129.07 (C-10), 129.95 (C30/C-50),
Table 2
In vivo evaluation of antitumour efficacy of bis[O-(14-benzoylaconine-8-yl)]suberate (2e).
Tumour
Treatment schedule
Weight loss %
Tumour weight (mg)
Control
p value
%T/C
Treated
HCT-15
MCF-7
Q3D ꢂ 5, st. Day 7
Q7D ꢂ 4, st. Day 13
Q4D ꢂ 5, st. Day 9
9.34
0
0
1425 ꢁ 166
491 ꢁ 137
776 ꢁ 358
297 ꢁ 142
205 ꢁ 99
0.00001
0.0394
0.0022
49
56
36

348
A. Chodoeva et al. / European Journal of Medicinal Chemistry 54 (2012) 343e351
89.88 (C-8), 89.60 (C-16), 82.11 (C-6), 79.64 (C-1), 78.56 (C-15), 78.07
(C-14), 75.63 (C-18), 73.82 (C-13), 63.61 (C-3), 63.09 (C-17), 61.66
(16-OCH3), 59.03 (18-OCH3), 58.90 (6-OCH3), 55.15 (1-OCH3), 50.33
(C-19), 50.19 (C-11), 50.11 (NeCH2eCH3), 46.76 (C-4), 44.75 (C-5),
43.17 (C-9), 41.87 (C-7), 39.75 (C-10), 34.85 (C-12), 33.22 (C-100),
32.30 (C-300), 29.27 (C-2), 19.08 (C-200), 10.70 (NeCH2eCH3). HRMS
[M]þ C37H51NO13 requires 717.81, found 718.3.
16-OCH3), 3.52 (s, 1H, H-18
3.33 (s, 1H, H-17), 3.29 (s, 3H, 18-OCH3), 3.26 (s, 1H, 18
H-16), 3.23 (s, 3H, 6-OCH3), 3.20 (m, 2H, NeCH2eCH3), 3.15 (m, 1H,
H-19
), 2.96 (br s,1H, H-9), 2.93 (s,1H, H-5), 2.58 (d, 1H, J ¼ 6.05 Hz,
b
), 3.49 (s, 1H, H-1), 3.38 (s, 3H, 1-OCH3),
a
), 3.24 (s, 1H,
a
H-7), 2.44 (br s, 1H, H-2b), 2.37 (br s, 1H, H-12b), 2.34 (d, 1H,
J ¼ 6.23 Hz, H-10), 1.99 (q, 2H, J ¼ 7.2 Hz, 8eCO2e(CH2)500), 1.91 (m,
1H, H-12a b), 1.55 (m, 2H,
), 1.82 (m, 1H, 8eCO2e(CH2)100
8eCO2e(CH2)200), 1.50 (m, 1H, 8eCO2e(CH2)100
a
), 1.45 (t, 3H,
5.1.1.3. 8-O-(14-Benzoylaconine)adipate (1c). White powder (42%);
J ¼ 7.02 Hz, NeCH2eCH3), 1.40 (br s, 1H, H-2
a), 1.09 (q, 2H,
tR ¼ 5.65 min; 1H NMR spectrum (400.13 MHz, CDCl3)
d: 8.23 (m,1H,
J ¼ 7.5 Hz, 8eCO2e(CH2)400), 0.92 (q, 2H, J ¼ 7.35 Hz, 8eCO2e(CH2)
NHþ), 8.0 (d, 2H, J ¼ 7.2 Hz, H-20/H-60), 7.63 (t, 1H, J ¼ 7.4 Hz, H-40),
7.49 (t, 2H, J ¼ 7.6 Hz, H-30/H-50), 4.94 (d, 1H, J ¼ 4.8 Hz, H-14), 4.55
(d,1H, J ¼ 5.2 Hz, H-15), 4.30 (br s,1H, H-3), 4.15 (d,1H, J ¼ 6.0 Hz, H-
300). 13C NMR (CDCl3)
d: 177.66 (COOH), 175.11 (8-COO), 165.66 (C-
70), 133.79 (C-40), 129.62 (C-20/C-60), 129.09 (C-10), 128.84 (C30/C-50),
89.68 (C-16), 89.54 (C-8), 82.18 (C-6), 79.60 (C-1), 78.54 (C-15),
78.09 (C-14), 75.51 (C-18), 73.77 (C-13), 69.57 (C-3), 62.87 (C-17),
61.63 (16-OCH3), 59.0 (18-OCH3), 58.84 (6-OCH3), 55.10 (1-OCH3),
50.33 (C-19), 50.22 (C-11), 50.04 (NeCH2eCH3), 44.72 (C-5), 43.19
(C-9), 43.13 (C-4), 41.61 (C-7), 39.79 (C-10), 34.76 (C-12), 34.25 (C-
100), 33.58 (C-600), 29.27 (C-2), 28.29 (C-400), 28.23 (C-300), 24.21 (C-
200), 23.78 (C-500), 10.69 (NeCH2eCH3). HRMS [M]þ C40H57NO13
requires 759.89, found 760.4.
6), 3.80 (s, 3H, 16-OCH3), 3.75 (m, 1H, H-19b), 3.53 (m, 1H, H-18b),
3.5 (br s,1H, H-1), 3.39 (s, 3H,1-OCH3), 3.34 (s,1H, H-17), 3.29 (s, 3H,
18-OCH3), 3.28 (m,1H,18 ), 3.26 (br s, 2H, NeCH2eCH3), 3.25 (s,1H,
H-16), 3.24 (s, 3H, 6-OCH3), 3.19 (m, 1H, H-19 ), 2.96 (m, 1H, H-9),
2.94 (s, 1H, H-5), 2.59 (d, 1H, J ¼ 6.4 Hz, H-7), 2.43 (br d, 1H, H-2 ),
), 2.13 (t, 2H,
), 1.87 (m,
), 1.52 (m, 1H,
a
a
b
2.38 (d, 1H, J ¼ 4.4 Hz, H-10), 2.34 (br d, 1H, H-12
b
J ¼ 6.4 Hz, 8eCO2e(CH2)400), 1.92 (d, 1H, J ¼ 9.6 Hz, H-12
a
1H, 8eCO2e(CH2)100 ), 1.56 (m, 1H, 8eCO2e(CH2)100
b a
H-2
8eCO2e(CH2)200
(CDCl3)
: 177.70 (COOH),174.97 (8-COO),165.89 (C-70),134.2 (C-40),
a
), 1.46 (t, 3H,
J
¼
6.8 Hz, NeCH2eCH3), 1.28 (m, 3H,
5.1.1.6. 8-O-(14-Benzoylaconine)azelate (1f). White powder (44%);
b
, 300), 1.16 (m, 1H, 8eCO2e(CH2)200
a
). 13C NMR
tR ¼ 15.99min;1HNMRspectrum(400.13MHz,CDCl3)
d:8.16(brs,1H,
d
NHþ), 7.98 (d, 2H, J ¼ 7.2Hz, H-20/H-60), 7.6(t,1H, J ¼ 7.5 Hz, H-40), 7.46
(t, 2H, J ¼ 7.6 Hz, H-30/H-50), 4.89 (d, 1H, J ¼ 4.8 Hz, H-14), 4.49 (d,1H,
J ¼ 5.3 Hz, H-15), 4.26 (br s,1H, H-3), 4.13 (d,1H, J ¼ 5.85 Hz, H-6), 3.78
130.2 (C-20/C-60), 129.87 (C-10), 129.15 (C30/C-50), 92.18 (C-16), 90.07
(C-8) 83.5 (C-6), 82.52 (C-1), 79.92 (C-15), 78.85 (C-14), 75.83 (C-18),
74.09 (C-13), 69.86 (C-3), 63.19 (C-17), 61.88 (16-OCH3), 59.26 (18-
OCH3), 59.15 (6-OCH3), 55.35 (1-OCH3), 50.64 (C-19), 50.48 (C-11),
50.35 (NeCH2eCH3), 46.01 (C-4), 45.07 (C-5), 43.55 (C-9), 42.0 (C-
7), 40.12 (C-10), 35.12 (C-12), 34.29 (C-100), 33.0 (C-400þ), 29.79 (C-2),
24.04 (C-300, C-200), 8.83 (NeCH2eCH3). HRMS [M] C38H53NO13
requires 731.84, found 732.4.
(s, 3H,16-OCH3), 3.73e4.02 (m,1H, H-19b), 3.50 (s,1H, H-18b), 3.47 (s,
1H, H-1), 3.36 (s, 3H,1-OCH3), 3.32 (s,1H, H-17), 3.27 (s, 3H,18-OCH3),
3.25 (s, 1H, 18 ), 3.23 (s, 1H, H-16), 3.21 (s, 3H, 6-OCH3), 3.20 (m, 2H,
NeCH2eCH3), 3.14 (m, 1H, H-19 ), 2.95 (br s, 1H, H-9), 2.92 (s, 1H, H-
5), 2.56 (d,1H, J ¼ 6.25 Hz, H-7), 2.41 (br s,1H, H-2 ), 2.36 (br s,1H, H-
a
a
b
12
b
), 2.34 (d, 1H, J ¼ 6.73, H-10), 2.29 (t, 2H, J ¼ 7.5 Hz, 8eCO2e(CH2)
700),1.90 (m,1H, H-12
a
),1.78 (t,1H, J ¼ 7.6 Hz, 8eCO2e(CH2)100
b), 1.54
5.1.1.4. 8-O-(14-Benzoylaconine)pimelate
(1d). White
powder
(q, 2H, J ¼ 7.4 Hz, 8eCO2e(CH2)200), 1.47 (m, 1H, 8eCO2e(CH2)100
a),
(43%); tR ¼ 13.34 min; mp 107 ꢀC. 1H NMR spectrum (400.13 MHz,
1.44 (t, 3H, J ¼ 7.1 Hz, NeCH2eCH3), 1.20 (m, 4H, 8eCO2e(CH2)
CDCl3)
d
: 8.15 (br s, 1H, NHþ), 7.99 (d, 2H, J ¼ 7.6 Hz, H-20/H-60), 7.62
500,600), 1.05 (q, 2H, J ¼ 7.1 Hz, 8eCO2e(CH2)400), 0.92 (q, 2H,
(t, 1H, J ¼ 7.4 Hz, H-40), 7.48 (t, 2H, J ¼ 7.4 Hz, H-30/H-50), 4.92 (d, 1H,
J ¼ 4.92 Hz, H-14), 4.50 (d, 1H, J ¼ 5.2 Hz, H-15), 4.28 (br s, 1H, H-3),
J ¼ 6.9 Hz, 8eCO2e(CH2)300). 13C NMR (CDCl3)
d: 177.91 (8-COOH),
175.22 (8-COO00), 165.47 (C-70), 133.87 (C-40), 129.60 (C-20/C-60),
129.07 (C-10), 128.82 (C30/C-50), 89.66 (C-16), 89.50 (C-8), 82.15 (C-6),
79.59 (C-1), 78.53 (C-15), 78.07 (C-14), 75.53 (C-18), 73.77 (C-13), 69/
59 (C-3), 62.97 (C-17), 61.58 (16-OCH3), 58.99 (18-OCH3), 58.84 (6-
OCH3), 55.08 (1-OCH3), 50.33 (C-19), 50.14 (C-11), 50.07
(NeCH2eCH3), 44.72 (C-5), 43.19 (C-9), 43.10 (C-4), 41.60 (C-7), 39.75
(C-10), 34.77 (C-12), 34.27 (C-100), 33.81 (C-700), 29.94 (C-300), 29.23 (C-
2), 28.44 (C-500), 28.24 (C-400), 24.38 (C-200), 23.88 (C-600), 10.67
(NeCH2eCH3). HRMS [M]þ C41H59NO13 requires 773.92, found 774.5.
4.14 (d, 1H, J ¼ 5.6 Hz, H-6), 3.74 (m, 1H, H-19
b), 3.80 (s, 3H, 16-
OCH3), 3.51 (m, 1H, H-18
3.34 (s, 1H, H-17), 3.29 (s, 3H, 18-OCH3), 3.26 (s, 1H, 18
H-16), 3.22 (s, 3H, 6-OCH3), 3.18 (m, 2H, NeCH2eCH3), 3.13 (m, 1H,
H-19
), 2.96 (m,1H, H-9), 2.94 (s,1H, H-5), 2.56 (d,1H, J ¼ 5.2 Hz, H-
7), 2.41 (m,1H, H-2
b), 3.5 (s, 1H, H-1), 3.38 (s, 3H, 1-OCH3),
a), 3.25 (s, 1H,
a
b
), 2.38 (br s,1H, H-12b), 2.35 (m,1H, H-10), 2.23
(br s, 1H, H-12
1H, H-2
a
), 1.85 (q, 2H, J ¼ 8.4 Hz, 8eCO2e(CH2)500), 1.53 (br d,
a
), 1.44 (br s, 3H, NeCH2eCH3), 1.38 (m, 2H, 8eCO2e(CH2)
100), 1.31 (m, 2H, 8eCO2e(CH2)200), 1.23 (q, 1H, J ¼ 7.0 Hz,
8eCO2e(CH2)400
b
), 1.06 (q, 1H, J ¼ 6.6 Hz, 8eCO2e(CH2)400
a
), 0.96 (t,
5.1.1.7. 8-O-(14-Benzoylaconine)sebacate
(1g). White
powder
: 8.14
2H, J ¼ 6.0 Hz, 8eCO2e(CH2)300). 13C NMR (CDCl3)
d: 177.76 (COOH),
(33%); tR ¼ 11.12 min; 1H NMR spectrum (400.13 MHz, CDCl3)
d
175.21 (8-COO), 165.91 (C-70), 134.19 (C-40), 129.85 (C-20/C-60),
129.29 (C-10), 129.13 (C30/C-50), 89.93 (C-16), 89.76 (C-8), 82.27 (C-
6), 79.81 (C-1), 78.77 (C-15), 78.29 (C-14), 75.95 (C-18), 74.06 (C-
13), 69.78 (C-3), 63.46 (C-17), 61.80 (16-OCH3), 59.21 (18-OCH3),
59.03 (6-OCH3), 55.32 (1-OCH3), 50.50 (C-19), 50.40 (C-11), 50.36
(NeCH2eCH3), 44.95 (C-5), 43.36 (C-9), 43.10 (C-4), 42.11 (C-7),
39.84 (C-10), 35.04 (C-12), 34.37 (C-100), 33.94 (C-500), 29.46 (C-2),
28.29 (C-300), 24.34 (C-200), 23.89 (C-400), 10.86 (NeCH2eCH3). HRMS
[M]þ C39H55NO13 requires 745.86, found 746.4.
(m, 1H, NHþ), 7.99 (d, 2H, J ¼ 7.2 Hz, H-20/H-60), 7.61 (t, 1H,
J ¼ 7.4 Hz, H-40), 7.47 (t, 2H, J ¼ 7.6 Hz, H-30/H-50), 4.91 (d, 1H,
J ¼ 5.1 Hz, H-14), 4.51 (d, 1H, J ¼ 5.1 Hz, H-15), 4.26e4.32 (m, 1H, H-
3), 4.15 (d, 1H, J ¼ 5.7 Hz, H-6), 3.79 (s, 3H, 16-OCH3), 3.88e4.02 (m,
1H, H-19b), 3.51e3.54 (m, 1H, H-18b), 3.46e3.50 (m, 1H, H-1), 3.38
(s, 3H, 1-OCH3), 3.32 (s, 1H, H-17), 3.29 (s, 3H, 18-OCH3), 3.25e3.28
(m, 1H, 18 ), 3.22e3.25 (m, 1H, H-16), 3.23 (s, 3H, 6-OCH3),
3.22e3.25 (m, 2H, NeCH2eCH3), 3.11e3.20 (m, 1H, H-19 ), 2.95 (t,
1H, J ¼ 5.5 Hz, H-9), 2.93 (s, 1H, H-5), 2.60 (d, 1H, J ¼ 5.7 Hz, H-7),
a
a
2.38e2.46 (m, 1H, H-2b), 2.33e2.38 (m, 2H, H-10,12b), 2.30 (t, 2H,
5.1.1.5. 8-O-(14-Benzoylaconine)suberate
(1e). White
powder
J ¼ 7.6 Hz, 8eCO2e(CH2)800), 1.94 (br d, 1H, J ¼ 9.6 Hz, H-12
a), 1.82
(40%); tR ¼ 10.03 min; 1H NMR spectrum (400.13 MHz, CDCl3)
d:
(q, 1H,
J
¼
8.25 Hz, 8eCO2e(CH2)100
b), 1.58e1.66 (m, 2H,
8.20 (br s, 1H, NHþ), 7.99 (d, 2H, J ¼ 7.14 Hz, H-20/H-60), 7.62 (t, 1H,
J ¼ 7.41 Hz, H-40), 7.48 (t, 2H, J ¼ 7.62 Hz, H-30/H-50), 4.90 (d, 1H,
J ¼ 4.9 Hz, H-14), 4.50 (d, 1H, J ¼ 5.39 Hz, H-15), 4.29 (br s, 1H, H-3),
8eCO2e(CH2)200), 1.50e1.58 (m, 1H, 8eCO2e(CH2)100
a), 1.44 (t, 3H,
J ¼ 6.92 Hz, NeCH2eCH3), 1.13e1.34 (m, 7H, 8eCO2e(CH2)300, 400, 600,
700 ), 0.86e1.12 (m, 3H, 8eCO2e(CH2)500, 700 ). 13C NMR (CDCl3)
b a d:
4.14 (d, 1H, J ¼ 5.99 Hz, H-6), 3.75 (m, 1H, H-19
b
), 3.80 (s, 3H,
176.65 (8-COOH), 175.28 (8-COO00), 165.72 (C-70), 133.56 (C-40),

A. Chodoeva et al. / European Journal of Medicinal Chemistry 54 (2012) 343e351
349
129.47 (C-20/C-60), 129.03 (C-10), 128.66 (C30/C-50), 89.60 (C-16),
89.41 (C-8), 82.18 (C-6), 79.51 (C-1), 78.41 (C-15), 78.0 (C-14), 75.4
(C-18), 77.06 (C-13), 73.68 (C-3), 62.73 (C-17), 61.25 (16-OCH3),
58.87 (18-OCH3), 58.76 (6-OCH3), 54.89 (1-OCH3), 50.31 (C-19),
49.87 (C-11), 49.93 (NeCH2eCH3), 44.71 (C-5), 43.21 (C-4), 43.03
(C-9), 41.35 (C-7), 39.79 (C-10), 34.63 (C-12), 34.07 (C-100), 33.85 (C-
800), 28.83 (C-2), 28.82 (C-600), 28.76 (C-300), 28.61 (C-400), 28.4 (C-500),
24.50 (C-200), 23.68 (C-700), 10.61 (NeCH2eCH3). HRMS [M]þ
C42H61NO13 requires 787.95, found 787.8.
16-OCH3), 3.53 (d, 2H, J ¼ 5.16 Hz, H-18
b
), 3.51 (br s, 2H, H-1), 3.41
(s, 6H, 1-OCH3), 3.33 (m, 2H, H-17), 3.32 (s, 6H, 18-OCH3), 3.30 (m,
2H, 18 ), 3.27 (br s, 4H, NeCH2eCH3), 3.26 (s, 2H, H-16), 3.21 (s, 6H,
6-OCH3), 3.15 (m, 2H, H-19 ), 2.93 (m, 2H, H-9), 2.88 (br s, 2H, H-5),
2.62 (d, 2H, J ¼ 3.76, H-7), 2.44 (d, 2H, J ¼ 11.0 Hz, H-2 ), 2.38 (br s,
2H, H-10), 2.35 (d, 2H, J ¼ 8.32 Hz, H-12 ), 1.95 (d, 2H, J ¼ 7.24 Hz,
H-12 ), 1.48 (m,
), 1.56 (m, 4H, 8eCO2e(CH2)100 , 8eCO2e(CH2)300
6H, NeCH2eCH3), 1.45 (m, 2H, H-2 ), 1.25 (m, 4H, 8eCO2e(CH2)
100 , 8eCO2e(CH2)300 ), 1.05 (m, 4H, 8eCO2e(CH2)200). 13C NMR
(CDCl3)
: 173.68 (8-COO, 8-COO00), 165.50 (C-70), 133.62 (C-40),
a
a
b
b
a
b
b
a
a
a
d
5.1.1.8. 8-O-(14-benzoylaconine)undecanoate (1h). White powder
129.63 (C-20/C-60), 129.29 (C-10), 128.77 (C30/C-50), 90.20 (C-8),
89.85 (C-16), 82.34 (C-6), 79.61 (C-1), 78.53 (C-15), 78.20 (C-14),
75.55 (C-18), 73.80 (C-13), 69.63 (C-3), 62.80 (C-17), 61.78 (16-
OCH3), 59.06 (18-OCH3), 58.75 (6-OCH3), 55.06 (1-OCH3), 50.46
(C-19), 50.37 (C-11), 50.07 (NeCH2eCH3), 44.78 (C-5), 43.33 (C-9),
43.22 (C-4), 41.67 (C-7), 39.90 (C-10), 34.76 (C-12), 33.96 (C-100, Cþ-
300), 29.32 (C-2), 18.63 (C-200), 10.74 (NeCH2eCH3). HRMS [M]
C69H94N2O22 requires 1303.51, found 1303.3.
(34%); 1H NMR spectrum (400.13 MHz, CDCl3) : 8.16 (m, 1H, NHþ),
d
7.99 (d, 2H, J ¼ 7.2 Hz, H-20/H-60), 7.61 (t,1H, J ¼ 7.32 Hz, H-40), 7.47 (t,
2H, J ¼ 7.6 Hz, H-30/H-50), 4.90 (d, 1H, J ¼ 4.79 Hz, H-14), 4.51 (d, 1H,
J ¼ 4.50 Hz, H-15), 4.28 (br s, 1H, H-3), 4.14 (d, 1H, J ¼ 6.20 Hz, H-6),
3.79 (s, 3H, 16-OCH3), 3.71 (m, 1H, H-19b), 3.52 (s,1H, H-18b), 3.49 (s,
1H, H-1), 3.38 (s, 3H,1-OCH3), 3.33 (s,1H, H-17), 3.27 (s, 3H,18-OCH3),
3.26 (s, 1H, 18 ), 3.24 (s, 1H, H-16), 3.23 (s, 3H, 6-OCH3), 3.20 (m, 2H,
NeCH2eCH3), 3.17 (m, 1H, H-19 ), 2.96 (br s, 1H, H-9), 2.93 (s, 1H, H-
a
a
5), 2.57 (d, 1H, J ¼ 6.0 Hz, H-7), 2.43 (br s, 1H, H-2
b
), 2.37 (m, 2H, H-
5.1.1.11. Bis[O-(14-benzoylaconin-8-yl)]adipate (2c). White powder
10,12
b
), 2.34 (t, 2H, J ¼ 6.93 Hz, 8eCO2e(CH2)900),1.89 (m,1H, H-12
a),
(24%); tR ¼ 16.53 min; 1H NMR spectrum (400.13 MHz, CDCl3)
d:
1.80 (t, 1H, J ¼ 8.0 Hz, 8eCO2e(CH2)100
b
), 1.61 (q, 2H, J ¼ 7.4 Hz,
8.27 (m, 1H, NHþ), 8.01 (d, 4H, J ¼ 5.0 Hz, H-20/H-60), 7.58 (t, 2H,
J ¼ 5.0 Hz, H-40), 7.47 (t, 4H, J ¼ 5.1 Hz, H-30/H-50), 4.91 (d, 2H,
J ¼ 3.12 Hz, H-14), 4.49 (d, 2H, J ¼ 3.52 Hz, H-15), 4.33 (br s, 2H, H-
8eCO2e(CH2)200), 1.53 (m, 1H, 8eCO2e(CH2)100
a), 1.45 (m, 3H,
NeCH2eCH3), 1.20 (m, 8H, 8eCO2e(CH2)500, 600, 700, 800), 1.02 (q, 2H,
J ¼ 6.95 Hz, 8eCO2e(CH2)400), 0.90 (q, 2H, J ¼ 7.18 Hz, 8eCO2e(CH2)
3), 4.15 (d, 2H, J ¼ 3.96 Hz, H-6), 3.86 (m, 2H, H-19
b), 3.82 (s, 6H, 16-
), 3.52 (br s, 2H, H-1), 3.41 (s, 6H,1-OCH3),
300). 13C NMR (CDCl3)
d
: 177.05 (8-COOH), 175.25 (8-COO00),165.66 (C-
OCH3), 3.54 (m, 2H, H-18
3.35 (s, 2H, H-17), 3.33 (s, 6H,18-OCH3), 3.30 (m, 2H,18
4H, NeCH2eCH3), 3.27 (s, 2H, H-16), 3.23 (s, 6H, 6-OCH3), 3.21 (m,
2H, H-19 ), 2.95 (m, 2H, H-9), 2.93 (br s, 2H, H-5), 2.62 (d, 2H,
J ¼ 4.0 Hz, H-7), 2.44 (d, 2H, J ¼ 10.5 Hz, H-2 ), 2.39 (br s, 2H, H-10),
2.36 (d, 2H, J ¼ 8.24 Hz, H-12 ), 1.94 (d, 2H, J ¼ 7.4 Hz, H-12 ), 1.68
(m, 4H, 8eCO2e(CH2)100 8eCO2e(CH2)400
), 1.48 (m, 6H,
NeCH2eCH3), 1.47 (m, 2H, H-2
), 1.35 (m, 4H, 8eCO2e(CH2)100
8eCO2e(CH2)400 ), 0.91 (m, 4H, 8eCO2e(CH2)200
, 8eCO2e(CH2)
300 ), 0.75 (m, 4H, 8eCO2e(CH2)200 , 8eCO2e(CH2)300 ). 13C NMR
(CDCl3)
: 174.2 (8-COO, 8-COO00), 165.52 (C-70), 133.59 (C-40),
b
70), 133.66 (C-40), 129.62 (C-20/C-60), 129.15 (C-10), 128.8 (C30/C-50),
89.82 (C-16), 89.58 (C-8), 82.31 (C-6), 79.74 (C-1), 78.67 (C-15), 78.20
(C-14), 75.64 (C-18), 73.89 (C-13), 69.47 (C-3), 63.07 (C-17), 61.66 (16-
OCH3), 59.08 (18-OCH3), 58.92 (6-OCH3), 55.17 (1-OCH3), 50.45
(C-19), 50.36 (C-11), 50.20 (NeCH2eCH3), 44.88 (C-5), 43.32 (C-4),
43.31 (C-9), 41.79 (C-7), 39.93 (C-10), 34.99 (C-12), 34.85 (C-100), 34.45
(C-900), 29.50 (C-2), 28.99 (C-400), 28.92 (C-500), 28.86 (C-600), 28.84
(C-700), 28.64 (C-300), 24.09 (C-200), 23.74 (C-800), 10.73 (NeCH2eCH3).
HRMS [M]þ C43H63NO13 requires 801.97, found 802.1.
a
), 3.28 (br s,
a
b
b
a
b
,
b
a
a,
a
b
b
a
a
d
5.1.1.9. Bis[O-(14-benzoylaconin-8-yl)]succinate
(2a). White
129.67 (C-20/C-60), 129.28 (C-10), 128.82 (C30/C-50), 89.91 (C-8),
89.84 (C-16), 82.29 (C-6), 79.62 (C-1), 78.56 (C-15), 78.21 (C-14),
75.59 (C-18), 73.82 (C-13), 69.71 (C-3), 62.91 (C-17), 61.78 (16-
OCH3), 59.06 (18-OCH3), 58.79 (6-OCH3), 55.10 (1-OCH3), 50.42
(C-19), 50.30 (C-11), 50.11 (NeCH2eCH3), 44.77 (C-5), 43.28 (C-9),
43.22 (C-4), 41.73 (C-7), 39.87 (C-10), 34.79 (C-12), 33.80 (C-100, C-
400), 29.31 (C-2), 23.11 (C-200, C-300), 10; 73 (NeCH2eCH3). HRMS
[M]þ C70H96N2O22 requires 1317.53, found 1317.3.
powder (18%); tR ¼ 21.3 min; 1H NMR spectrum (400.13 MHz,
CDCl3)
d
: 8.11 (m,1H, NHþ), 7.98 (d, 4H, J ¼ 7.4 Hz, H-20/H-60), 7.61 (t,
2H, J ¼ 7.4 Hz, H-40), 7.45 (t, 4H, J ¼ 7.7 Hz, H-30/H-50), 4.92 (d, 2H,
J ¼ 4.7 Hz, H-14), 4.42 (d, 2H, J ¼ 5.3 Hz, H-15), 4.31 (br s, 2H, H-3),
4.11 (d, 2H, J ¼ 6.1 Hz, H-6), 3.81 (s, 6H, 16-OCH3), 3.66 (m, 2H, H-
19b
), 3.53 (br s, 2H, H-1), 3.48 (m, 2H, H-18
b), 3.41 (s, 6H, 1-OCH3),
3.35 (br s, 2H, H-17), 3.32 (s, 6H, 18-OCH3), 3.30 (m, 4H, 18
a
, 19 ),
a
3.26 (br s, 4H, NeCH2eCH3), 3.25 (s, 2H, H-16), 3.21 (s, 6H, 6-OCH3),
2.91 (br s, 2H, H-5), 2.89 (s, 2H, H-9), 2.54 (d, 2H, J ¼ 6.4 Hz, H-7),
5.1.1.12. Bis[O-(14-benzoylaconine-8-yl)]pimelate
(2d). White
2.44 (br s, 2H, H-2
1.89 (s, 2H, H-12
200
), 1.51 (m, 2H, H-2
8eCO2e(CH2)100 , 8eCO2e(CH2)200
b
), 2.39 (br s, 2H, H-10), 2.38 (br d, 2H, H-12
b
),
, 8eCO2e(CH2)
), 1.45 (m, 6H, NeCH2eCH3), 1.10 (m, 4H,
). 13C NMR (CDCl3)
: 172.99 (8-
powder (25%); tR ¼ 27.21 min; mp 151 ꢀC. 1H NMR spectrum
a
), 1.85 (br d, 4H, 8eCO2e(CH2)100
b
(400.13 MHz, CDCl3)
d
: 8.1 (d, 4H, J ¼ 7.6 Hz, H-20/H-60), 7.71 (t, 2H,
b
a
J ¼ 7.4 Hz, H-40), 7.58 (t, 4H, J ¼ 7.6, H-30/H-50), 5.04 (d, 2H,
J ¼ 4.4 Hz, H-14), 4.63 (d, 2H, J ¼ 5.2 Hz, H-15), 4.44 (br s, 2H, H-3),
a
a
d
COO, 8-COO00),165.66 (C-70),133.88 (C-40),129.84 (C-20/C-60),129.34
(C-10), 128.99 (C30/C-50), 90.67 (C-8), 89.98 (C-16), 81.99 (C-6), 79.83
(C-1), 78.48 (C-15), 78.33 (C-14), 76.18 (C-18), 73.98 (C-13), 70.0 (C-
3), 63.49 (C-17), 61.94 (16-OCH3), 59.25 (18-OCH3), 58.89 (6-OCH3),
55.36 (1-OCH3), 50.38 (C-19), 50.37 (C-11), 50.36 (NeCH2eCH3),
44.84 (C-9), 43.63 (C-4), 43.45 (C-5), 42.67 (C-7), 39.85 (C-10),
35.08 (C-12), 29.35 (C-2), 28.19 (C-100, C-200), 10.86 (NeCH2eCH3).
HRMS [M]þ C68H92N2O22 requires 1289.48, found 1289.5.
4.27 (br d, 2H, H-6), 3.92 (m, 2H, H-19b), 3.95 (s, 6H, 16-OCH3), 3.67
(s, 2H, H-18
17), 3.47 (s, 6H, 18-OCH3), 3.41 (s, 2H, 18
3.36 (s, 6H, 6-OCH3), 3.31 (m, 2H, H-19
NeCH2eCH3), 3.08 (m, 2H, H-9), 3.06 (s, 2H, H-5), 2.74 (br d, 2H, H-
7), 2.58 (br s, 2H, H-2 ), 2.51 (br d, 2H, H-12 ), 2.49 (m, 2H, H-10),
2.07 (d, 2H, J ¼ 10.0 Hz, H-12
), 1.87 (m, 4H, 8eCO2e(CH2)100b,
8eCO2e(CH2)600b), 1.60 (m, 12H, 8eCO2e(CH2)100
, 8eCO2e(CH2)
NeCH2eCH3, H-2
500 ), 1.12 (m, 4H, 8eCO2e(CH2)200
8eCO2e(CH2)400 ), 0.95 (m, 4H, 8eCO2e(CH2)200
b a, 8eCO2e(CH2)
b
), 3.64 (m, 2H, H-1), 3.53 (s, 6H,1-OCH3), 3.48 (s, 2H, H-
), 3.39 (br d, 2H, H-16),
), 3.33 (m, 4H,
a
a
b
b
a
a
a
,
a
b,
5.1.1.10. Bis[O-(14-benzoylaconin-8-yl)]glutarate
(2b). White
powder (22%); tR ¼ 25.13 min; 1H NMR spectrum (400.13 MHz,
400
a
), 0.72 (quint, 2H, J ¼ 7.2 Hz, 8eCO2e(CH2)300). 13C NMR
CDCl3)
d
: 8.27 (m, 1H, NHþ), 7.98 (d, 4H, J ¼ 5.04 Hz, H-20/H-60), 7.59
(CDCl3) d
: 174.9 (8-COO, 8-COO00), 165.76 (C-70), 133.8 (C-40), 129.9
(t, 2H, J ¼ 4.88 Hz, H-40), 7.45 (t, 4H, J ¼ 5.06 Hz, H-30/H-50), 4.92 (d,
2H, J ¼ 2.96 Hz, H-14), 4.47 (d, 2H, J ¼ 3.32 Hz, H-15), 4.32 (br s, 2H,
(C-20/C-60), 129.5 (C-10), 129 (C30/C-50), 90 (C-16), 89.98 (C-8), 82.48
(C-6), 79.83 (C-1), 78.12 (C-15), 78.45 (C-14), 75.79 (C-18), 74.02 (C-
13), 69.9 (C-3), 63.12 (C-17), 62.01 (16-OCH3), 59.3 (18-OCH3), 59.05
H-3), 4.12 (d, 2H, J ¼ 3.68 Hz, H-6), 3.92 (m, 2H, H-19
b), 3.82 (s, 6H,

350
A. Chodoeva et al. / European Journal of Medicinal Chemistry 54 (2012) 343e351
(6-OCH3), 55.36 (1-OCH3), 50.63 (C-19), 50.42 (C-11), 50.38
(NeCH2eCH3), 45.0 (C-5), 43.42 (C-4), 43.47 (C-9), 41.91 (C-7),
40.09 (C-10), 35.01 (C-12), 34.27 (C-100, C-500), 29.49 (C-200, C-400),
28.02 (C-2), 23.86 (C-300), 10; 99 (NeCH2eCH3). HRMS [M]þ
C71H98N2O22 requires 1331.56, found 1331.2.
methanol/chloroform/trifluoroacetic acid (20/80/0.5) solution:
monoclinic, space group P21, a ¼ 10.3646 (9) Å, b ¼ 14.7961 (10) Å,
c ¼ 13.6373 (9) Å,
a
¼ 90ꢀ,
b
¼ 110.022 (5)ꢀ,
g
¼ 90ꢀ, V ¼ 1965.0 (3) Å3,
Z ¼ 2,
d
(calcd) ¼ 1.436 Mg mꢃ3, FW ¼ 849.83 for C37H53NO14.CF3
-
COOH, F(000) ¼ 900. The data were collected on a Rigaku R-axis rapid
diffractometer equipped with micro-focus rotating anode CueK
5.1.1.13. 3Bis[O-(14-benzoylaconine-8-yl)]suberate
(2e). White
radiation (
l
¼ 1.5418 Å) mode at 133 (2) K. In the range of
powder (24%); tR ¼ 28.91 min; mp 153 ꢀC. 1H NMR spectrum
6.58ꢀ < < 72.03ꢀ, a totalof27,953reflections were collected, of which
7064 were independent (Rint ¼ 0.0834). The structure was solved by
direct methods with SHELXS-97 [16]. Non-hydrogen atoms were
refined by full-matrix least-squares techniques on F2 with anisotropic
thermal parameters, using SHELXL-97 [17]. Full crystallographic
results have been deposited at the Cambridge Crystallographic Data
Centre (CCDC-857072), UK, as supplementary Material [18].
(400.13 MHz, CDCl3)
d
: 8.00 (d, 4H, J ¼ 7.8 Hz, H-20/H-60), 7.58 (t, 2H,
J ¼ 7.32 Hz, H-40), 7.46 (t, 4H, J ¼ 7.66 Hz, H-30/H-50), 5.04 (d, 2H,
J ¼ 4.9 Hz, H-14), 4.64 (d, 2H, J ¼ 5.36 Hz, H-15), 4.43 (br s, 2H, H-3),
4.28 (d, 2H, J ¼ 6.1 Hz, H-6), 3.97 (d, 2H, J ¼ 12.96 Hz, H-19b), 3.94 (s,
6H, 16-OCH3), 3.67 (s, 2H, H-18b), 3.64 (m, 2H, H-1), 3.53 (s, 6H, 1-
OCH3), 3.47 (s, 2H, H-17), 3.43 (s, 6H, 18-OCH3), 3.41 (s, 2H, 18a),
3.39 (d, 2H, J ¼ 5.4 Hz, H-16), 3.36 (s, 6H, 6-OCH3), 3.31 (d, 2H,
J ¼ 12.96 Hz, H-19a), 3.30 (m, 4H, NeCH2eCH3), 3.10 (m, 2H, H-9),
3.07 (s, 2H, H-5), 2.74 (d, 2H, J ¼ 6.1 Hz, H-7), 2.58 (br s, 2H, H-2b),
2.51 (br d, 2H, H-12b), 2.49 (m, 2H, H-10), 2.07 (d, 2H, J ¼ 9.9 Hz, H-
12a), 1.94 (m, 2H, 8eCO2e(CH2)100b, 8eCO2e(CH2)600b), 1.63 (m, 2H,
8eCO2e(CH2)100a, 8eCO2e(CH2)600a), 1.60 (t, 6H, J ¼ 6.98 Hz,
NeCH2eCH3), 1.56 (br d, 2H, H-2a), 1.24 (m, 2H, 8eCO2e(CH2)200b,
8eCO2e(CH2)500b), 1.09 (m, 2H, 8eCO2e(CH2)200a, 8eCO2e(CH2)
500a), 0.83 (m, 4H, 8eCO2e(CH2)300, 8eCO2e(CH2)400). 13C NMR
5.3. In vitro testing
5.3.1. Tumour cell lines
The human tumour cell lines A-549 (lung cancer), MCF-7 (breast
cancer) and HCT-15 (colon cancers), were obtained from the
Developmental Therapeutics Program (DTP) of the National Cancer
Institute (NCI) (Rockville, MD, USA). Cells were routinely grown
with RPMI 1640 medium supplemented with 10% foetal calf serum,
both obtained from Biochrom AG (Berlin, Germany). They were
grown on Petri dishes (Nunc, Denmark) at 37 ꢀC in a humidified
atmosphere containing 5% CO2. Cells were replicated every 4e5
days and the medium changed once in-between.
(CDCl3) d
: 175.1 (8-COO, 8-COO00),165.8 (C-70),133.8 (C-40),129.9 (C-
20/C-60), 129.5 (C-10), 129 (C30/C-50), 90 (C-16), 89.97 (C-8), 82.51 (C-
6), 79.82 (C-1), 78.82 (C-15), 78.40 (C-14), 75.75 (C-18), 74.01 (C-
13), 70 (C-3), 63.13 (C-17), 61.95 (16-OCH3), 59.26 (18-OCH3), 59.01
(6-OCH3), 55.29 (1-OCH3), 50.64 (C-19), 50.51 (C-11), 50.28
(NeCH2eCH3), 45.0 (C-5), 43.43 (C-4), 43.40 (C-9), 41.87 (C-7),
40.06 (C-10), 35.0 (C-12), 34.49 (C-100, C-600), 29.50 (C-2), 28.54 (Cþ-
300, C-400), 24.05 (C-200, C-500), 10; 94 (NeCH2eCH3). HRMS [M]
C72H100N2O22 requires 1345.59, found 1345.68.
5.3.2. Cytotoxicity
Cytotoxicity was evaluated on exponentially growing cells
grown for 24 h in the presence of the compounds to be tested.
These were dissolved in distilled water, the same solvent being
used as a control. All samples were first sterilised using poly-
5.1.1.14. Bis[O-(14-benzoylaconine-8-yl)]azelate (2f). White powder
carbonate 0.22
m
m membrane filters (Millipore, Molsheim, France).
(24%); tR ¼ 32.24 min; 1H NMR spectrum (400.13 MHz, CDCl3)
d:
Briefly, 1000 cells were seeded in 96-well plates with 200 m
L of
8.03 (d, 4H, J ¼ 7.2 Hz, H-20/H-60), 7.56 (t, 2H, J ¼ 7.4 Hz, H-40), 7.45 (t,
4H, J ¼ 7.2 Hz, H-30/H-50), 5.09 (d, 2H, J ¼ 5.0 Hz, H-14), 4.51 (d, 2H,
J ¼ 5.2 Hz, H-15), 4.30 (br s, 2H, H-3), 4.12 (d, 2H, J ¼ 6.1 Hz, H-6),
4.06 (d, 2H, J ¼ 11.91 Hz, H-19b), 3.79 (s, 6H, 16-OCH3), 3.52 (s, 2H,
H-18b), 3.47 (m, 2H, H-1), 3.36 (s, 6H, 1-OCH3), 3.29 (s, 2H, H-17),
3.27 (s, 6H, 18-OCH3), 3.24 (s, 2H, 18a), 3.22 (br s, 2H, H-16), 3.19 (s,
6H, 6-OCH3), 3.11 (d, 2H, J ¼ 11.9 Hz, H-19a), 3.16 (m, 4H,
NeCH2eCH3), 2.90 (m, 2H, H-9), 2.86 (s, 2H, H-5), 2.55 (d, 2H,
J ¼ 6.1 Hz, H-7), 2.34 (br s, 2H, H-2b), 2.28 (br d, 2H, H-12b), 2.24 (m,
2H, H-10), 2.02 (br d, 2H, H-12a), 1.82 (m, 2H, 8eCO2e(CH2)100b,
8eCO2e(CH2)700b), 1.50 (m, 2H, 8eCO2e(CH2)100a, 8eCO2e(CH2)
700a), 1.42 (t, 6H, J ¼ 7.01 Hz, NeCH2eCH3), 1.35 (br s, 2H, H-2a),
1.13 (m, 4H, 8eCO2e(CH2)200b, 8eCO2e(CH2)300b, 8eCO2e(CH2)500b,
8eCO2e(CH2)600b), 1.04 (m, 4H, 8eCO2e(CH2)200a, 8eCO2e(CH2)
300a, 8eCO2e(CH2)500a, 8eCO2e(CH2)600a), 0.87 (m, 2H,
complete medium; 24 h later, the medium was supplemented with
a series of concentrations of the compounds to be tested and the
contact maintained for 24 h. The cells were then allowed to re-grow
for 48 h; viable and metabolically active cells were quantitatively
estimated by coloration with MTT (1-(4,5-dimethylthiazol-2-yl)-
3,5-diphenylformazan, SigmaeAldrich Chimie, Saint-Quentin-
Fallavier, France) [19]. The amount of substance allowing 50%
decrease in cell numbers is indicative of the cytotoxicity and
represents the IC50 of the tested compound.
5.4. In vivo testing
5.4.1. Animals
Experiments were performed in accordance with the European
Community Standards on the Care and Use of Laboratory Animals
and approved by the Animal Care and Use Committee of University
Bordeaux Segalen. All animals were bred and housed under specific
pathogen-free conditions and provided food and water ad libitum.
Eight-week old immunodeficient mice (NSG nomenclature: NOD.Cg-
Prkdcscid Il2rgtmw/SzJ jax mice 005557), males for the HCT-15 tumour
model and females for the MCF-7 tumour model, weighing 19e25 g,
were used for the experiments. Each experimental group contained
8e10 animals. For the MCF-7 tumour model, mice received
8eCO2e(CH2)400). 13C NMR (CDCl3) : 175.1 (8-COO, 8-COO00), 166
d
(C-70), 134 (C-40), 130 (C-20/C-60), 129.7 (C-10), 129.3 (C30/C-50), 90.3
(C-16), 90.1 (C-8), 82.9 (C-6), 80.1 (C-1), 79.0 (C-15), 78.7 (C-14),
76.0 (C-18), 74.3 (C-13), 70.3 (C-3), 63.4 (C-17), 60.9 (16-OCH3), 59.5
(18-OCH3), 59.06 (6-OCH3), 55.6 (1-OCH3), 50.7 (C-19), 50.51 (C-11),
50.50 (NeCH2eCH3), 45.4 (C-5), 43.7 (C-4), 43.40 (C-9), 41.9 (C-7),
40.5 (C-10), 35.3 (C-12), 34.49 (C-100,C-700), 28.8 (C-200, C-600), 26.54
(C-300, C-500), 24.05 (C-400), 11 (NeCH2eCH3). HRMS [M]þ
C73H102N2O22 requires 1359.61, found 1359.5.
17b-oestradiol (SigmaeAldrich Chimie, Saint-Quentin-Fallavier,
France) previously dissolved in ethanol (4.25 mg/mL), which was
5.2. X-ray data
added to the drinking water at a final concentration of 8.5 mg/mL.
The structure of compound 1d has been established by X-
5.4.2. Drug testing
ray
crystallography
(Fig.
1).
Colorless
single
crystal
For drug testing, 1.5 ꢂ 106 human tumour cells in culture
medium were inoculated subcutaneously into the right flank of the
(0.12 ꢂ 0.10 ꢂ 0.10 mm3) of 1dwasobtainedbyslowevaporation from

A. Chodoeva et al. / European Journal of Medicinal Chemistry 54 (2012) 343e351
351
roots of Aconitum karacolicum Rapcs, and its antiproliferative activities, Bioorg.
Med. Chem. 13 (2005) 6493e6501.
[2] F.P. Wang, Q.H. Chen, X.Y. Liu, Diterpenoid alkaloids, Nat. Prod. Rep. 27 (2010)
529e570.
[3] B.S. Huang, H.C. Wang, A. Lao, Y. Fujimoto, M. Kirisawa, Studies on the alka-
loids from Aconitum karakolicum Rap. Part I, Heterocycles 32 (1991)
2429e2432.
[4] A. Lao, Y. Wang, H.C. Wang, Y. Fujimoto, Studies on the alkaloids from Aco-
nitum karakolicum Rap. Part II, Heterocycles 43 (1996) 1267e1270.
[5] F. Gao, D.L. Chen, F.P. Wang, New aconitine-type C19-diterpenoid alkaloids
from Aconitum hemsleyanium var. circinacum, Arch. Pharm. Res. 30 (2007)
1497e1500.
[6] J.H. Yang, Z.Y. Li, L. Li, Y.S. Wang, Diterpenoid alkaloids from Aconitum epis-
copale, Phytochemistry 50 (1999) 345e348.
[7] U.T. Gutser, J. Friese, J.F. Heubach, T. Matthiesen, N. Selve, B. Wilffert, J. Gleitz,
Mode of antinociceptive and toxic action of alkaloids of Aconitum spec, Nau-
nyn-Schmiedeberg’s Arch. Pharmacol. 357 (1998) 39e48.
mice (day 0). When the tumour reached 4 ꢂ 4 mm in average,
animals were randomly divided into control and test groups, con-
sisting of at least 8 mice each, and the treatment was started.
Compound 2e (bis[O-(14-benzoylaconine-8-yl)]suberate) in 0.9%
solution of NaCl was injected i.v. at the dose of 10 mg/kg. This dose
was chosen after independent evaluation of the maximum toler-
ated dose of the compound in mice (15 mg/kg) by Phycher (Cestas,
France), a contract research organisation dedicated to toxicology
and agreed for regulatory evaluations. Two treatment schedules
were chosen for MCF-7 tumour cells: administration of five doses
every 4 days, and weekly administration of four doses. The mice
weighed three times a week, and only one for HCT-15 cells:
administration of five doses every 3 days, starting one week after
tumour cell graft.
[8] A. Ameri, The effect of Aconitum alkaloids on the central nervous system, Prog.
Neurobiol. 56 (1998) 211e235.
The length (L) and width (W) of the tumour were measured by
calipers three times a week and the tumour volume was calculated
as follows: T (mg) ¼ (L ꢂ W2)/2. Tumour regression was determined
[9] C. De Ines, M. Reina, J. Gavin, A. Gonzales-Coloma, In vitro cytotoxicity of
norditerpenoid alkaloids, Z. Naturforsch. 61 (2006) 11e18.
[10] M. Hazawa, K. Wada, K. Takahashi, T. Mori, N. Kawahara, I. Kashiwakura,
Suppressive effects of novel derivatives prepared from Aconitum alkaloids on
tumor growth, Invest. New Drugs 27 (2009) 111e119.
[11] K. Wada, M. Hazawa, K. Takahashi, T. Mori, N. Kawahara, I. Kashiwakura,
Inhibitory effects of diterpenoid alkaloids on the growth of A172 human
malignant cells, J. Nat. Prod. 70 (2007) 1854e1858.
[12] M. Hazawa, K. Takahashi, K. Wada, T. Mori, N. Kawahara, I. Kashiwakura,
Structure-activity relationships between the Aconitum C(20)-diterpenoid
alkaloid derivatives and the growth suppressive activities of Non-Hodgkin’s
lymphoma Raji cells and human hematopoietic stem/progenitor cells, Invest.
New Drugs 29 (2011) 1e8.
as % T/C ¼
D
T/
D
C ꢂ 100 where
DT is the difference between the
mean tumour volumes of the treated group at the end (Tfinal) and at
the start (Tstart) of the treatment, and C is the difference between
D
the mean tumour volumes of the control group at the end (Cfinal) at
the start (Cstart) of the treatment. According to the National Cancer
Institute standards, a T/C < 42% is considered as the minimum level
for activity [20]. At the end of experiments, animals were sacrificed
by cervical dislocation. Tumours were extracted and frozen in liquid
nitrogen for further immuno-histological investigations. The
tumour volumes of treated and control groups were compared
using bilateral Student’s test.
[13] Y. Bai, H.K. Desai, S.W. Pelletier, Long-chain fatty acid esters of some nordi-
terpenoid alkaloids, J. Nat. Prod. 57 (1994) 963e970.
[14] B. Borcsa, U. Widowitz, D. Csupor, P. Forgo, R. Bauer, J. Hohmann, Semisyn-
thesis and pharmacological investigation of lipo-alkaloids prepared from
aconitine, Fitoterapia 82 (2011) 365e368.
[15] J.J. Bosc, C. Jarry, A. Chodoeva, J. Guillon, I. Forfar, J. Robert, Preparation of bis-
[O-(14-benzoylaconine-8-yl)] esters for use in antitumor pharmaceutical
compositions WO 2010063638 A1, Chem. Abstr. 153 (2010) 11885.
[16] G.M. Sheldrick, SHELXS 97, Program for Crystal Structure Solution, University
of Göttingen, Germany, 1997.
[17] G.M. Sheldrick, SHELXL 97, Program for Crystal Structure Refinement,
University of Göttingen, Germany, 1997.
[18] Supplementary X-ray Crystallographic Data: Cambridge Crystallographic Data
Centre, University Chemical Lab, Lensfield Road, Cambridge, CB2 1EW, UK;
E-mail: deposit@chemcrys.cam.ac.uk.
Acknowledgements
This work was supported by funds from Conseil Régional
d’Aquitaine. We thank Dr Jean-Luc Chagnaud and Dr Jacques Sus-
perregui, from Aquitaine Valo, for their support.
References
[19] J. Carmichael, W.G. DeGraff, A.F. Gazdar, J.D. Minna, J.B. Mitchell, Cancer Res.
47 (1987) 936e942.
[20] M.G. Hollinshead, Antitumor efficacy testing in rodents, J. Natl. Cancer Inst.
100 (2008) 1500e1510.
[1] A. Chodoeva, J.J. Bosc, J. Guillon, A. Decendit, M. Petraud, C. Absalon, I.C. Vitry,
C. Jarry, J. Robert, 8-O-azeloyl-14-benzoylaconine: a new alkaloid from the