New lithocholic and chenodeoxycholic piperazinylcarboxamides with antiproliferative and pro-apoptotic effects on human cancer cell lines

Article abstract of DOI:10.1016/j.bmc.2008.07.046

Six new synthetic bile acid derivatives were synthesized and tested in vitro against various human cancer cells (glioblastoma multiforme (GBM), multiple myeloma (KMS-11), and colonic carcinoma (HCT-116) cell lines. The best activity was obtained with comp

Full text of DOI:10.1016/j.bmc.2008.07.046

Bioorganic & Medicinal Chemistry 16 (2008) 8737–8744
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New lithocholic and chenodeoxycholic piperazinylcarboxamides
with antiproliferative and pro-apoptotic effects on human cancer cell lines
b
a
b
Laïla El Kihel a, , Monique Clément , Marc-Antoine Bazin , Géraldine Descamps ,
*
Mohamed Khalid c, Sylvain Rault a
a Centre d’Etudes et de Recherche sur le Médicament de Normandie, UPRES EA-3915, FR CNRS INC3M, Université de Caen Basse-Normandie, U.F.R. des Sciences Pharmaceutiques,
5 rue Vaubénard, 14032 Caen Cedex, France
b INSERM U601, Institut de Biologie, CHU Nantes, 9 Quai Moncousu, 44093 Nantes, France
c Université Hassan Premier, Faculté des Sciences et Techniques, Km 3, Route de Casablanca, BP 577, 26000 Settat (Maroc), France
a r t i c l e i n f o
a b s t r a c t
Article history:
Six new synthetic bile acid derivatives were synthesized and tested in vitro against various human cancer
cells (glioblastoma multiforme (GBM), multiple myeloma (KMS-11), and colonic carcinoma (HCT-116)
Received 3 April 2008
Revised 11 July 2008
Accepted 18 July 2008
Available online 23 July 2008
cell lines. The best activity was obtained with compound IIIb on multiple myeloma cells (LD50
8.5 0.5 M). This activity was associated with Mcl-1 and PARP-1 cleavage, inhibition of NF B signaling,
and DNA fragmentation, demonstrating an apoptotic cell death signaling pathway.
:
l
j
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Steroid-heterocycle
Bile acid derivatives
Apoptosis
Anticancer
Multiple myeloma
1. Introduction
In a previous study, it has been reported that several unconju-
gated bile acids may play some role in the development of intesti-
nal tumors.6 Meanwhile, these acids possess pharmacological
activities such as a potent and selective farnesoid X receptor
(FXR) agonist,7,8 and they also showed anticancer activities.9,10 It
is worth mentioning that the UDCA is used for the prevention of
gastrointestinal disorders in patients having various cancers
(stomach, colon, lung, breast, and liver).11 It is also evaluated in
clinical phase III trial therapy to prevent colorectal adenoma recur-
rence.12 Another way for cancer treatment consists of coupling bile
acids with anticancer molecules; cisplatin13 or heparin14 through
covalent bonds. On the other hand, a series of bile acid–polyamine
amides conjugated with 30-azido-30-deoxythymidine (AZT) were
synthesized and their cytotoxic activities were screened against
human cancer cells in vitro.15
Bile acids, the products of cholesterol catabolism, are synthe-
sized in the liver and excreted into the bile canaliculus and the
digestive tract. The primary bile acids (cholic acid, CA
(3a,7a
,12a-trihydroxy-5b-cholan-24-oic acid) and chenodeoxy-
cholic acid, CDCA (3
a
,7 -dihydroxy-5b-cholan-24-oic acid) are
a
conjugated with glycine or taurine in hepatocytes to form the cor-
responding N-acyl conjugates. As conjugates, they are secreted
from the hepatocyte into the bile that is evacuated in the intestine.
These conjugates are metabolized by enteric bacteria in the distal
small intestine and in the colon to produce secondary bile acids
by deconjugation followed by 7-dehydroxylation: deoxycholic acid
(3a,12a-dihydroxy-5b-cholan-24-oic acid, DCA) and lithocholic
acid (3
a
-hydroxy-5b-cholan-24-oic acid, LCA) are obtained from
cholic and chenodeoxycholic acids, respectively.1–4 In addition,
the bile contains 3b-, 7b- and 12b-epimers which mainly would
come from the bacterial action in the distal ileum or colon. The
Furthermore, the conjugation of bile acids with glycine and tau-
rine is one of the mechanisms by which an organism could de-
crease its cytotoxicity at physiological concentrations.16–18 For
this reason, several synthetic bile acid derivatives have been devel-
oped. Among these synthesized ones, a conjugate of UDCA or CDCA
urso family with 7b-epimer includes ursocholic acid (3
trihydroxy-5b-cholan-24-oic acid, UCA) and ursodeoxycholic acid
(3
,7b-dihydroxy-5b-cholan-24-oic acid, UDCA).5
a,7a,12a-
a
with L-phenylalanine benzyl ester led HS-1183 or HS-1199, respec-
tively. On the other hand, the conjugate of CDCA with b-alanine
benzyl ester gave HS-1200, which were intensively studied.19–21
These derivatives exerted antitumor effects against human cancer
cells such as prostate cancer cells,22 cervical carcinoma cells,23
* Corresponding author. Tel.: +33 (0) 2 31 93 41 69; fax: +33 (0) 2 31 93 11 88.
E-mail address: laila.elkihel@unicaen.fr (L. El Kihel).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.07.046

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L. El Kihel et al. / Bioorg. Med. Chem. 16 (2008) 8737–8744
colon cancer cells,24 hepatocellular carcinoma cells,20 stomach can-
cer cells,25 leukemic T cells,26 breast carcinoma cells,27 and glio-
blastoma multiforme cells.28 However, glycine or taurine
conjugated bile acids are cleaved by intestinal and hepatic en-
zymes.29 For this reason, another strategy was reported by intro-
duction of a heterocycle amino acid (proline) in the bile acids
side chain. The resulting compounds were found to be more resis-
tant to bacterial deamidation than the corresponding natural con-
jugated molecules, but no anticancer activity was described.30
We wish to describe herein the synthesis of some new bile acids
containing nitrogen heterocycles side chain. In vitro studies were
then accomplished on glioblastoma multiforme (GBM, brain tumor
cell line), multiple myeloma (KMS-11) and colonic carcinoma
(HCT-116) cell lines.
Three piperazine derivatives, substituted in the 1-position by
pyridine, pyrimidine or cinnamyl groups, were introduced in the
bile acid side chain in place of the proline moiety. Therefore, a ser-
ies of novel bile acid-substituted piperazine conjugates, derived
from chenodeoxycholic and lithocholic acids, have been prepared
(Scheme 1).
No degradation of these carboxamides was observed after 24 and
48 h.
2.2. Biology
The anticancer activity of compounds (Ia,b–IIIa,b) was evalu-
ated against three human cancer cells: glioblastoma multiforme
(GBM),31 colon (HCT-116),32 and multiple myeloma (KMS-11)33
using the neutral red uptake assay as described in Section 3. Multi-
ple myeloma is a plasma-cell neoplasm characterized by the clonal
expansion of malignant plasma cells within the hematopoietic
bone marrow, glioblastoma multiforme is the most frequent tumor
in the central nervous system; the prognosis of these cancers is ex-
tremely unfavorable. Both tumors are characterized by a potent
evasion of apoptosis. Inhibition of this programmed cell death
has been shown to be a major obstacle to chemo-resistance in
many cancer cells.34 Therefore, it would be important to identify
new active agents that trigger apoptosis in these cells. HCT-116 cell
line was chosen as bile acids may play an important role in intes-
tinal tumorigenesis; the discovery and development of new prod-
ucts for both prevention and therapy remain an important field of
research.
2. Results and discussion
2.1. Chemistry
The bile acids (Ia,b–IIIa,b), LCA, CDCA, and amine building
blocks (a, b, and c) were first tested at 50 lM. The percentage of
viability cell was determined after 24 and 48 h of incubation. A
representative experiment out of three is given in Figure 1(1 and
2). No cytotoxicity was observed for LCA, CDCA, amines (a, b,
and c), Ib and IIIa. Compound IIIb induced a complete death as
soon as 24 h, while compounds Ia and IIb showed various cytotox-
icities at this concentration. No significant difference of activity
was observed between 24 and 48 h of treatment as determined
by the student test.
In order to calculate the LD50 after a 24 h treatment, dose–re-
sponse experiments were carried out. An example is given inFigure
2A showing the dose–response curve of IIIb effect toward the three
cell lines. The bile acid derivative IIIb caused a concentration
dependent increase in cytotoxicity toward all the cells with KMS-
11 cell line being the more sensitive. Morphological modifications
Lithocholic and chenodeoxycholic acid derivatives were pre-
pared as depicted in Scheme 2. Hydroxyl groups of cholic acids
(3a-hydroxy-5b-cholan-24-oic acid (1a) and 3a,7a-dihydroxy-
5b-cholan-24-oic acid (1b)) were acetylated with acetic anhydride
in pyridine to give the acetates 2a and 2b, respectively. The latter
acids 2a and 2b were converted into the corresponding mixed
anhydride by treatment with triethylamine followed by ethyl chlo-
roformate giving 3a and 3b, respectively. When the mixed anhy-
drides 3a and 3b were reacted with substituted piperazines (a, b,
or c), the products were identified as the carboxamides (4a,b–
6a,b). The latter amides were saponified by ethanolic potassium
hydroxide to give the corresponding deacetylated amides Ia,b–
IIIa,b. However, during purification of the resulting amides on sil-
ica gel, they were strongly adsorbed on the carrier material, thus
they could be successfully purified on alumina deactivated with
6% water. The structures of the compounds prepared were con-
firmed by analytical and spectral analyses.
induced by IIIb (12 lM–24 h) are illustrated in Figure 2B.
The mean LD50 (lethal dose 50% standard error, calculated
from the results of at least three independent dose responsiveness
experiments, after 24 h treatment) for all products is summarized
in Table 1.
The hydrolytic stability of bile acids prepared was studied un-
der physiological conditions (phosphate buffer, pH 7.4, at 37 °C).
Taken together, these results showed that LCA, CDCA, Ib, and
IIIa had no cytotoxic activity. Compound IIIb was the most active
O
N
O
N
N
N
N
N
H
H
N
H
H
R
H
H
R
HO
HO
N
Ia; R = H
H
H
IIa; R = H
Ib; R = OH
IIb; R = OH
O
N
H
H
H
R
HO
IIIa; R = H
H
IIIb; R = OH
Scheme 1. Amide derivatives of lithocholic and chenodeoxycholic acids.

L. El Kihel et al. / Bioorg. Med. Chem. 16 (2008) 8737–8744
8739
O
O
O
O
OH
OH
O
OEt
H
H
H
i
ii
H
H
H
H
R
H
R
H
R
HO
AcO
AcO
3a; R = H
3b; R = OAc
1a; R = H
2a; R = H
H
H
N
H
1b; R = OH
2b; R = OAc
O
O
N
N
N
N
N
1-(2-pyridyl)
H
H
piperazine (a)
iv
H
H
H
R
H
R
iii
4a; R = H
Ia; R = H
AcO
HO
H
H
4b; R = OAc
Ib; R = OH
O
O
N
N
N
N
N
N
1-(2-pyrimidyl)
piperazine (b)
H
H
N
N
iv
H
H
H
R
H
R
iii
IIa; R = H
AcO
5a; R = H
HO
H
H
IIb; R = OH
5b; R = OAc
O
O
N
N
N
N
trans-1-cinnamyl
piperazine (c)
H
H
iv
H
H
R
H
H
R
iii
HO
IIIa; R = H
AcO
6a; R = H
H
H
IIIb; R = OH
6b; R = OAc
Scheme 2. Synthesis of lithocholic and chenodeoxycholic acid derivatives. Reagents and conditions: (i) Ac2O, pyridine, rt, 15 h; (ii) EtOCOCl, NEt3, CHCl3, rt, 50 min; (iii)
piperazine (a, b or c), NEt3, CHCl3, rt, 16 h; (iv) KOH, EtOH, reflux, 24 h.
24 h of treatment (50 µM)
48 h of treatment(50 µM)
110
100
90
80
70
60
50
40
30
20
10
0
110
100
90
80
70
60
50
40
30
20
10
0
KMS-11
GBM
HCT-116
Figure 1. Cell viability after 24 h (A) and 48h (B) exposure with bile acid derivatives (50 lM) evaluated using the neutral red assay. Results are expressed as the percentage of
untreated cells (one representative experiment).

8740
L. El Kihel et al. / Bioorg. Med. Chem. 16 (2008) 8737–8744
pletely inhibits cell proliferation (Fig. 3 illustrates one representa-
tive experiment; results are given as the percentage of untreated
cells).
Next, we analyzed the mechanisms of KMS-11 cell death with
emphasis to apoptotic processes. For this purpose, we investigated
whether 7A6 antigen (detected with the specific antibody APO2.7,
specifically expressed on the mitochondrial membrane during the
early stages of apoptosis)35 could be detected after cells exposure
to IIIb: a 24 h treatment leads to a significant dose dependent in-
crease in APO2.7-positive cells (p < 0.001) (Fig. 4), therefore sug-
gesting an apoptotic signal.
To confirm this KMS-11 cells apoptotic death, some intracellu-
lar signaling pathways were analyzed by immunoblotting. Western
blots revealed the cleavage of Mcl-1 (Myeloid Cell Leukemia-1) and
PARP-1 (Poly(ADP-Ribose)Polymerase-1) as well as the hyper-
phosphorylation of IkBa (Fig. 5A), an inhibitor of NFkB (nuclear fac-
tor-
activation of some critical apoptotic genes, while inactivated NF
is maintained in the cytoplasm compartment because of its bind-
ing to non-phosphorylated I
B.36 Modifications of these key signal-
jB). Activated NFjB is involved in the transcriptional
jB
j
ing proteins have clearly been associated with induction of
apoptosis in multiple myeloma cell lines.37–39 Moreover, nuclear
staining clearly proved DNA fragmentation, which is the final stage
of apoptosis (Fig. 5B).
These preliminary results showed that IIIb induced cell death in
three cancer cell lines. Further experiments are needed to better
understand the balance between cell cycle inhibition and apoptotic
process in IIIb cytotoxic activity. Difference of sensitivity between
cell lines could result from the implication of various survival fac-
tors and signaling pathways.
100
80
CA
CDCA
60
Figure 2. Dose–response effect of IIIb on KMS-11, GBM, and HCT-116 cell lines. (A)
Cell survival was determined using the neutral red uptake assay (one representative
experiment). Each concentration was done in triplicate, results are expressed in the
mean OD, error bars represent the standard deviation. (B) Morphological aspect of
IIIb
40
three cell lines treated with IIIb at 12 lM for 24 h.
20
Table 1
Conjugated bile acids cytotoxicity towards three human cancerous cell lines
0
Compound
KMS-11 LD50
(lM)
GBM LD50
(
lM)
HCT-116 LD50 (lM)
neutral red 3H thymidine
uptake
incorporation
Ia
Ib
IIa
IIb
IIIa
IIIb
LCA
CDCA
9
>50
50 15
17
>50
8.5 0.5
P50
3
50 10
>50
50 15
30 10
>50
40 10
>50
40 10
35 10
>50
Figure 3. Effects of IIIb, LCA, and CDCA (20
lM for 24 h) on cell viability (evaluated
by neutral red uptake) and cell proliferation (evaluated by tritiated thymidine
uptake) on KMS-11 multiple myeloma cell line. Results are expressed as the
percentage of untreated cells (one representative experiment).
8
18.5
>50
>50
1
16.5
>50
>50
2
P50
IIIb (10 μM)
IIIb (40 μM)
control
LD50 values were determined from the dose–response curves after 24 h of incuba-
tion using the neutral red uptake assay. Results are expressed as the mean of three
experiments standard error.
60
60
80
37%
86%
1%
0
toward the three cell lines, complete toxicity being obtained at
100 101 102 103 104
100 101 102 103 104 100 101 102 103 104
25 lM (data not shown); therefore, we focused our study on this
compound. The effect of IIIb on KMS-11 cell proliferation was eval-
Figure 4. Apoptotic index with Apo2.7 staining analyzed by flow cytometry of
KMS-11 cells after a 24 h treatment with IIIb at the indicated concentrations.
Results are presented as the percentage of APO2.7-positive cells.
uated by using the [3H]thymidine incorporation assay. In contrast
to LCA and CDCA, an exposure with IIIb at 20 lM for 24 h com-

L. El Kihel et al. / Bioorg. Med. Chem. 16 (2008) 8737–8744
8741
40 eV. ESI mass spectra were recorded on a LC/MS Waters alliance.
1H NMR and 13C NMR spectra were recorded using CDCl3 and
DMSO-d6, respectively, at 400 MHz (Jeol Lambda 400 spectrometer)
and at 100 MHz. Chemical shifts are reported relative to TMS; J val-
ues are given in Hz. 13C NMR spectra are 1H-decoupled.
0
10
20
IIIb (24 h - μM)
Mcl-1
(cleaved form)
3.1.2. General procedure for 2a and 2b
IκBα
p-IκBα
p116
Acetic anhydride (106.2 mmol, 10 mL) was added dropwise to a
solution of 1a or 1b (13.3 mmol) in pyridine (40 mL). The reaction
mixture was stirred at room temperature (rt) under argon atmo-
sphere for 15 h and then ice water was added. The white precipi-
tate formed was dissolved in CH2Cl2 (150 mL), washed with 1M
HCl (2ꢀ 20 mL), 5% NaHCO3 (2ꢀ 25 mL), brine (2ꢀ 25 mL), and
then with water (25 mL). The organic layer was dried over anhy-
drous sodium sulfate and evaporated under reduced pressure.
The crude product was purified by column chromatography (silica
gel, cyclohexane/ethyl acetate, 8:2) to afford 2a or 2b.
PARP
p85
actin
fragmented
nuclei (%)
1 1
5 1
61 4
3.1.2.1. 3a-Acetyloxy-5b-cholan-24-oic acid (2a). The crude
product was recrystallized from acetone to give 2a (4.73 g, 83%) as
a white powder. Mp 167 °C (lit.40 168–169 °C). IR (KBr): v (cmꢁ1):
3404 (br, O–H acid), 2950–2869 (C–H alkane), 1736 (C@O ester),
1709 (C@O carboxylic acid). 1H NMR (400 MHz, CDCl3, 25 °C):
d = 0.65 (s, 3H, Me-18), 0.92 (d, 3J = 5.4 Hz, 3H, Me-21), 0.93 (s, 3H,
Me-19), 2.03 (s, 3H, –OCOCH3), 4.68–4.76 (m, 1H, H-3), 10.86 (br s,
1H, OH) ppm. 13C NMR (100 MHz, CDCl3, 25 °C): d = 12.0 (C-18),
18.2 (C-21), 20.8, 21.4 (–OCOCH3), 23.3 (C-19), 24.1, 26.3, 26.5,
26.9, 28.1, 30.8, 31.2, 32.2, 34.5, 35.0, 35.3, 35.7, 40.1, 40.3, 41.8,
42.7, 55.9, 56.4, 74.4 (C-3), 170.7 (–OCOCH3), 180.2 (C-24) ppm.
MS (30 eV, EI): m/z (%) = 418.5 (3) [M+Å], 358.4 (100) [M+ÅꢁCH3CO2H],
343.4 (12), 304.3 (6), 257.3 (9), 230.3 (21), 215.3 (33).
5 µm
Figure 5. Pro-apoptotic effects of IIIb (24 h of treatment) on KMS-11 cells. (A)
Western blotting showing the cleavage of Mcl-1 and PARP-1 as well as the
3.1.2.2. 3a,7a-Bis(acetyloxy)-5b-cholan-24-oic acid (2b). The
crude product was recrystallized from acetonitrile/diethyl ether to
hyperphosphorylation of IjB. (B) DNA fragmentation is illustrated after nuclear
give 2b (6.25 g, 95%) as a white powder. Mp 99 °C (dec). IR (KBr):
staining with Hoëchst.
(
m
cmꢁ1): 3493 (br, O–H acid), 2939–2870 (C–H alkane), 1729
(C@O ester and acid). 1H NMR (400 MHz, CDCl3, 25 °C): d = 0.64 (s,
3H, Me-18), 0.89 (s, 3H, Me-19), 0.91 (d, 3J = 8.0 Hz, 3H, Me-21),
1.98 (s, 6H, 2ꢀ –OCOCH3), 3.81–3.84 (m, 1H, H-7), 4.50–4.59 (m,
1H, H-3) ppm. 13C NMR (100 MHz, CDCl3, 25 °C): d = 11.7 (C-18),
18.2 (C-21), 20.5, 21.5 (2ꢀ –OCOCH3), 22.7 (C-19), 23.6, 26.6, 28.1,
30.7, 31.0, 32.7, 34.3, 34.9, 35.0, 35.2, 35.3, 39.3, 39.5, 41.1, 42.7,
50.3, 55.7, 68.5 (C-7), 74.4 (C-3), 170.9 (2ꢀ –OCOCH3), 179.9 (C-
24) ppm. MS (30 eV, EI): m/z (%) = 476.3 (1) [M+Å], 434.3 (3), 416.3
[M+Åꢁ CH3CO2H], 374.3 (7), 356.3 (100) [M+ÅÅꢁ2ꢀ CH3CO2H], 341.2
(33), 302.2 (10), 255.2 (22), 228.2 (38), 213.2 (28).
In conclusion, we have developed a new series of cholic acids
combined with nitrogen heterocycles, and demonstrated that some
of these synthetic bile acid derivatives possess cytotoxic functions
against glioblastoma multiforme (GBM), colorectal adenocarci-
noma (HCT-116), and multiple myeloma (KMS-11) cell lines,
whereas LCA and CDCA have no effect. Concerning the structure–
activity relationship, the best activity was obtained with com-
pound IIIb having cinnamylpiperazinyl group in the side chain
and hydroxyl group at C-7 of steroid skeleton (compound IIIb in
the range of 8–20 lM). These results prompt us in a future study
to incorporate other amino substituted cinnamylpiperazinyl
groups in the side chain of chenodeoxycholic acid.
3.1.3. General procedure for 4a, 4b, 5a, 5b, 6a, and 6b
Ethyl chloroformate (1.13 mL, 11.8 mmol) was added to a stir-
red solution of acid 2a or 2b (5.9 mmol) in chloroform (20 mL) un-
der argon atmosphere at room temperature. Triethylamine
(1.66 mL, 11.8 mmol) was added dropwise and the solution was
kept under stirring for 50 min. The reaction mixture was diluted
with methylene chloride (30 mL), washed with water (2ꢀ
15 mL), dried over anhydrous magnesium sulfate, and evaporated.
The crude product of anhydride (3a or 3b) was used in next step
without purification.
Under nitrogen atmosphere, piperazine derivative (a, b, or c)
(59.0 mmol) and triethylamine (0.83 mL, 5.9 mmol) were added
to a solution of anhydride 3a or 3b (5.9 mmol) in chloroform
(20 mL). The reaction mixture was stirred at room temperature
for 16 h. The solution was diluted with methylene chloride
(20 mL), washed with water (2ꢀ 15 mL), dried over anhydrous
magnesium sulfate and evaporated. The crude product was puri-
fied by column chromatography on deactivated neutral alumina
(6% water) eluting by cyclohexane/ethyl acetate, 7:3.
3. Experimental
3.1. Chemistry
3.1.1. General remarks
All solvents were distilled and dried prior to use. Reagents and
materials were obtained from commercial suppliers and were used
without further purification. All the reactions were monitored by
TLC on Kieselgel-G (Merck Si 254 F) layers (0.25 mm thick). The spots
were detected using a UV lamp (254 nm) and by spraying sulfuric
acid/ethanol (2:8) on TLC and heating. Column chromatography
was carried out using deactived neutral alumina (6% water) and sil-
ica gel 60 (0.063–0.2 mm) (Merck). Melting points were determined
on a Kofler block. IR spectra were recorded on a Perkin-Elmer 1600
FT-IR spectrometer. EI mass spectra were recorded on a Jeol-GCmate
(GC–MS system) spectrometer with ionization energy from 30 to

8742
L. El Kihel et al. / Bioorg. Med. Chem. 16 (2008) 8737–8744
3.1.3.1. 3
a
-Acetoxy-17b-[1-methyl-3-(1N-(4N-(pyridin-2-yl))pip-
3.1.3.5. 3
zin-1-yl)carboxy-propyl]etiocholane (6a). 2.22 g, 61%. Yellow
oil. IR (KBr): (
cmꢁ1): 2938–2866 (C–H alkane), 1734 (C@O es-
a-Acetoxy-17b-[1-methyl-3-(1N-(4N-cinnamyl)pipera-
erazin-1-yl)carboxy-propyl]etiocholane (4a). 1.41 g, 43%. Yel-
low oil. IR (KBr): (
m
cmꢁ1): 2927–2860 (C–H alkane), 1733 (C@O
m
ester), 1657 (C@O amide), 1241 (C–N amine). 1H NMR (400 MHz,
CDCl3, 25 °C): d = 0.65 (s, 3H, Me-18), 0.92 (s, 3H, Me-19), 0.95
(d, J = 6.4 Hz, 3H, Me-21), 2.02 (s, 3H, –OCOCH3), 3.47–3.62 (m,
8H, 4 –CH2– piperazinyl), 4.68–4.74 (m, 1H, H-3), 6.64–6.68 (m,
2H, H-3py and H-5py), 7.49–7.53 (m, 1H, H-4py), 8.20 (d,
J = 4.7 Hz, 1H, H-6py) ppm. 13C NMR (100 MHz, CDCl3, 25 °C):
d = 12.1 (C-18), 18.5 (C-21), 20.8, 21.5 (–OCOCH3), 23.3 (C-19),
24.2, 26.3, 26.6, 27.0, 28.3, 30.4, 31.5, 32.2, 34.6, 35.0, 35.7, 35.8,
40.2, 40.4, 41.1, 41.9, 42.8, 45.2, 45.4 (2C), 56.1, 56.5, 74.4 (C-3),
107.3 (C-3py), 113.9 (C-5py), 137.7 (C-4py), 147.9 (C-6py), 158.9
(C-2py), 170.7 (–OCOCH3), 172.4 (C-24) ppm. MS (30 eV, EI): m/z
ter), 1646 (C@O amide), 1243 (C–N amine). 1H NMR (400 MHz,
CDCl3, 25 °C): d = 0.63 (s, 3H, Me-18), 0.91 (s, 3H, Me-19), 0.95
(d, 3H, J = 6.6 Hz, Me-21), 2.00 (s, 3H, –OCOCH3), 2.40–2.62 (m,
4H, 2 –CH2– piperazinyl), 2.97–3.06 (m, 2H, –CH2– piperazinyl),
3.15 (d, J = 5.9 Hz, 2H, –CH2–CH@CH–Ph), 3.44–3.66 (m, 2H,
–CH2– piperazinyl), 4.65–4.75 (m, 1H, H-3), 6.17–6.27 (m, 1H,
–CH2–CH@CH–Ph), 6.50 (d, J = 15.9 Hz, 1H, –CH2–CH@CH–Ph),
7.19–7.36 (m, 5H, Har), 7.66 (br s, 1H, NH) ppm. 13C NMR
(100 MHz, CDCl3, 25 °C): d = 12.0 (C-18), 18.5 (C-21), 20.8, 21.5
(–OCOCH3), 23.3 (C-19), 24.2, 26.3, 26.6, 27.0, 28.2, 30.3, 31.4,
32.2, 34.5, 35.0, 35.6, 35.7, 40.1, 40.4, 41.8, 42.7, 44.3, 45.5,
51.8, 52.8, 56.0, 56.5, 61.0, 74.4 (C-3), 125.7, 126.3 (2C), 127.7,
128.6 (2C), 133.6, 136.6, 170.7 (–OCOCH3), 172.2 (C-24) ppm.
MS (30 eV, EI): m/z (%) = 602.4 (71) [M+ꢂ], 542.3 (5)
[M+ÅꢁCH3CO2H], 511.3 (12), 451.3 (14), 257.1 (34), 201.1 (41),
172.1 (100).
(%) = 563.4 (56) [M+Å], 548.4 (19) [M+ÅꢁCH3 ], 218.1 (80), 205.1
Å
(55), 120.0 (100).
3.1.3.2.
yl))piperazin-1-yl) carboxypropyl]etiocholane (4b). 0.88 g,
24%. White powder. Mp 200 °C. IR (KBr): (
cmꢁ1): 2973–2849
3a,7a-Diacetoxy-17b-[1-methyl-3-(1N-(4N-(pyridin-2-
m
(C–H alkane), 1732 (C@O ester), 1619 (C@O amide), 1248 (C–N
amine). 1H NMR (400 MHz, CDCl3, 25 °C): d = 0.67 (s, 3H, Me-18),
0.92 (s, 3H, Me-19), 0.96 (d, J = 6.4 Hz, 3H, Me-21), 2.01 (s, 6H,
2ꢀ –OCOCH3), 3.47–3.76 (m, 8H, 4 –CH2– piperazinyl), 3.83–3.87
(m, 1H, H-7), 4.54–4.62 (m, 1H, H-3), 6.65–6.70 (m, 2H, H-3py
and H-5py), 7.47–7.53 (m, 1H, H-4py), 8.20 (d, J = 4.9 Hz, 1H, H-
3.1.3.6. 3
piperazin-1-yl)carboxy-propyl]etiocholane (6b). 2.07 g, 53%.
Yellow oil. IR (KBr): (
cmꢁ1): 2939–2868 (C–H alkane), 1732
a,7a-Diacetoxy-17b-[1-methyl-3-(1N-(4N-cinnamyl)-
m
(C@O ester), 1632 (C@O amide), 1247 (C–N amine). 1H NMR
(400 MHz, CDCl3, 25 °C): d = 0.66 (s, 3H, Me-18), 0.91 (s, 3H, Me-
19), 0.95 (d, J = 6.6 Hz, 3H, Me-21), 2.00 (s, 6H, 2ꢀ –OCOCH3),
2.46–2.50 (m, 4H, 2 –CH2– piperazinyl), 3.17 (d, J = 6.6 Hz, 2H, –
CH2–CH@CH–Ph), 3.47–3.67 (m, 4H, 2 –CH2– piperazinyl), 3.83–
3.87 (m, 1H, H-7), 4.53–4.61 (m, 1H, H-3), 6.25 (dt, dJ = 15.8 Hz
6
py) ppm. 13C NMR (100 MHz, CDCl3, 25 °C): d = 11.8 (C-18), 18.5
(C-21), 20.6, 21.5 (2ꢀ –OCOCH3), 22.7 (C-19), 23.8, 26.8, 28.3,
30.3, 31.4, 32.8, 34.4, 35.1 (2C), 35.3, 35.6, 39.4, 39.6, 41.2 (2C),
42.7, 45.3, 45.4 (2C), 50.4, 55.8, 68.5 (C-7), 74.4 (C-3), 107.2 (C-
t
and J = 6.6 Hz, 1H, –CH2–CH@CH–Ph), 6.53 (d, J = 15.8 Hz, 1H, –
3
py), 113.9 (C-5py), 137.7 (C-4py), 148.0 (C-6py), 159.0 (C-2py),
CH2–CH@CH–Ph), 7.22–7.39 (m, 5H, Har) ppm. 13C NMR
(100 MHz, CDCl3, 25 °C): d = 11.9 (C-18), 18.7 (C-21), 20.7, 21.7
(2ꢀ –OCOCH3), 22.9 (C-19), 23.9, 26.9, 28.4, 30.3, 31.6, 32.9, 34.6,
35.1, 35.2, 35.4, 35.8, 39.5, 39.7, 41.4, 41.6, 42.8, 45.8, 50.6, 53.1,
53.4, 56.0, 61.1, 68.6 (C-7), 74.5 (C-3), 126.0, 126.5 (2C), 127.8,
128.8 (2C), 133.7, 136.8, 170.9 (2ꢀ –OCOCH3), 172.3 (C-24) ppm.
MS (30 eV, EI): m/z (%) = 660.3 (18) [M+Å], 618.1 (96), 600.3 (17),
257.1 (41), 201.1 (39), 172.1 (100).
170.7 (2ꢀ –OCOCH3), 172.2 (C-24) ppm. MS (ESI): [M+H]+ 622.
3.1.3.3. 3
a-Acetoxy-17b-[1-methyl-3-(1N-(4N-(pyrimidyl-2-yl))pip-
erazin-1-yl)carboxypropyl]etiocholane (5a). 1.33 g, 40%. White
powder. Mp 190 °C. IR (KBr): (
m
cmꢁ1): 2928–2869 (C–H alkane),
1734 (C@O ester), 1652 (C@O amide), 1244 (C–N amine). 1H
NMR (400 MHz, CDCl3, 25 °C): d = 0.65 (s, 3H, Me-18), 0.92 (s, 3H,
Me-19), 0.95 (d, J = 6.6 Hz, 3H, Me-21), 2.03 (s, 3H, –OCOCH3),
3.52–3.87 (m, 8H, 4 –CH2– piperazinyl), 4.68–4.74 (m, 1H, H-3),
6.54 (t, J = 4.7 Hz, 1H, H-5py), 8.33 (d, J = 4.7 Hz, 2H, H-4py and H-
3.1.4. Typical procedure of compounds Ia, Ib, IIa, IIb, IIIa, and
IIIb
6
py) ppm. 13C NMR (100 MHz, CDCl3, 25 °C): d = 12.2 (C-18), 18.7
Compound 4a (1.06 g, 1.88 mmol) was added to a solution of
potassium hydroxide (0.84 g, 15.04 mmol) in ethanol. The mixture
was refluxed for 24 h. Aqueous 0.1 N HCl was added to neutral pH
and the solution was evaporated. The residue was dissolved in
chloroform, washed with 5% NaHCO3, water and dried over anhy-
drous sodium sulfate. The crude product was purified by column
chromatography on deactived neutral alumina (6% water) (elu-
ent/cyclohexane/ethyl acetate, 7:3) to afford Ia (0.29 g, 30%) as a
white powder.
(C-21), 21.0, 21.7 (–OCOCH3), 23.5 (C-19), 24.4, 26.5, 26.8, 27.2,
28.5, 30.7, 31.6, 32.4, 34.8, 35.2, 35.9, 36.0, 40.3, 40.6, 41.5, 42.1,
42.9, 43.8, 44.0, 45.6, 56.3, 56.7, 74.6 (C-3), 110.6 (C-5py), 158.0
(C-4py and C-6py), 161.7 (C-2py), 170.9 (–OCOCH3), 172.6 (C-24)
ppm. MS (30 eV, EI): m/z (%) = 564.2 (9) [M+Å], 549.2 (4) [M+ÅꢁCH3 ],
Å
489.2 (2) [M+Åꢁ(CH3Å + CH3CO2H)], 219.0 (30), 206.0 (100), 121.0
(74).
3.1.3.4.
2-yl))piperazin-1-yl)carboxy-propyl]etiocholane (5b). 2.50 g,
68%. White powder. Mp 80 °C. IR (KBr): (
cmꢁ1): 2937–2866 (C–
3a,7a-Diacetoxy-17b-[1-methyl-3-(1N-(4N-(pyrimidyl-
3.1.4.1. 17b-[1-Methyl-3-(1N-(4N-(pyridin-2-yl))piperazin-1-yl)-
m
carboxypropyl] etiocholan-3
a-ol (Ia). 0.29 g, 30%. White pow-
H alkane), 1732 (C@O ester), 1644 (C@O amide), 1248 (C–N
amine). 1H NMR (400 MHz, CDCl3, 25 °C): d = 0.67 (s, 3H, Me-18),
0.92 (s, 3H, Me-19), 0.97 (d, J = 6.4 Hz, 3H, Me-21), 2.01 (s, 6H,
2ꢀ –OCOCH3), 3.52–3.82 (m, 8H, 4 –CH2– piperazinyl), 3.83–3.87
(m, 1H, H-7), 4.53–4.63 (m, 1H, H-3), 6.54 (t, J = 4.7 Hz, 1H, H-
der. Mp 168 °C. IR (KBr): (
m
cmꢁ1): 3414 (br, O–H alcohol), 2928–
2861 (C–H alkane), 1638 (C@O amide), 1241 (C–N amine). 1H
NMR (400 MHz, CDCl3, 25 °C): d = 0.65 (s, 3H, Me-18), 0.92 (s, 3H,
Me-19), 0.96 (d, J = 6.6 Hz, 3H, Me-21), 3.48–3.76 (m, 9H, 4 –
CH2– piperazinyl, and H-3), 6.66–6.69 (m, 2H, H-3py, and H-5py),
7.49–7.54 (m, 1H, H-4py), 8.20 (d, J = 4.9 Hz, 1H, H-6py) ppm. 13C
NMR (100 MHz, CDCl3, 25 °C): d = 12.2 (C-18), 18.6 (C-19), 20.9
(C-21), 23.5, 24.4, 26.5, 27.3, 28.4, 30.5, 30.7, 31.6, 34.7, 35.5,
35.8, 36.0, 36.6, 40.3, 40.5, 41.2, 42.2, 42.9, 45.3, 45.5 (2C), 56.1,
56.6, 71.9 (C-3), 107.4 (C-3py), 114.0 (C-5py), 137.8 (C-4py), 148.1
(C-6py), 159.2 (C-2py), 172.5 (C-24) ppm. MS (ESI): [M+H]+ 522.1,
[M+H+MeCN]+ 564.1. HRMS (EI) m/z [M+] calcd. for C33H51N3O2:
521.3981, found: 521.4005.
5
py), 8.33 (d, J = 4.7 Hz, 2H, H-4py and H-6py) ppm. 13C NMR
(100 MHz, CDCl3, 25 °C): d = 11.8 (C-18), 18.5 (C-21), 20.6, 21.5
(2ꢀ –OCOCH3), 22.7 (C-19), 23.8, 26.7, 28.3, 30.3, 31.4, 32.8, 34.4,
35.0, 35.1, 35.3, 35.6, 39.4, 39.6, 41.2, 41.3, 42.7, 43.6, 43.8, 45.5,
50.5, 55.9, 68.5 (C-7), 74.4 (C-3), 110.5 (C-5py), 157.8 (C-4py and
C-6py), 161.8 (C-2py), 170.6 (2ꢀ –OCOCH3), 172.2 (C-24) ppm. MS
(30 eV, EI): m/z (%) = 622.6 (6) [M+Å], 580.5 (10), 563.4 (7), 269.0
(20), 206.1 (72), 121.0 (100).

L. El Kihel et al. / Bioorg. Med. Chem. 16 (2008) 8737–8744
8743
t
3.1.4.2. 17b-[1-Methyl-3-(1N-(4N-(pyridin-2-yl))piperazin-1-yl)-
3.57–3.68 (m, 1H, H-3), 6.27 (dt, dJ = 15.6 Hz and J = 6.8 Hz, 1H,
–CH2–CH@CH–Ph), 6.53 (d, J = 15.6 Hz, 1H, –CH2–CH@CH–Ph),
7.17–7.39 (m, 5H, Har) ppm. MS (ESI): [M+H]+ 561.5. MS (30 eV,
EI): m/z (%) = 560.4 (15) [M+Å], 469.3 (3), 358.2 (90), 343.2 (18),
230.2 (26), 215.1 (100), 202.1 (38). HRMS (EI) m/z [M+] calcd. for
carboxypropyl]etiocholan-3
powder. Mp 218 °C. IR (KBr): (
a
,7
m
a
-diol (Ib). Yield: 86%. White
cmꢁ1): 3402 (br, O–H alcohol),
2931–2845 (C–H alkane), 1606 (C@O amide), 1238 (C–N amine).
1H NMR (400 MHz, CDCl3, 25 °C): d = 0.66 (s, 3H, Me-18), 0.90 (s,
3H, Me-19), 0.96 (d, J = 6.6 Hz, 3H, Me-21), 3.45–3.50 (m, 3H, 1
–CH2– piperazinyl, and H-3), 3.56–3.63 (m, 4H, 2 –CH2– piperazi-
nyl), 3.72–3.75 (m, 2H, 1 –CH2– piperazinyl), 3.82–3.86 (m, 1H,
H-7), 6.64–6.68 (m, 2H, H-3py and H-5py), 7.50 (ddd, 3J = 8.4 Hz,
3J = 7.7 Hz and 4J = 2.2 Hz, 1H, H-4py), 8.19 (d, J = 4.9 Hz, 1H, H-
C37H56N2O2: 560.4342; found: 560.4354.
3.1.4.6. 17b-[1-Methyl-3-(1N-(4N-cinnamyl)piperazin-1-yl)carb-
oxypropyl]etiocholan-3 ,7 -diol (IIIb). Yield: 60%. Yellow
powder. Mp 126 °C. IR (KBr): (
cmꢁ1): 3412 (br, O–H alcohol),
a
a
m
6
py) ppm. 13C NMR (100 MHz, CDCl3, 25 °C): d = 11.9 (C-18), 18.7
2929–2864 (C–H alkane), 1629 (C@O amide). 1H NMR (400 MHz,
CDCl3, 25 °C): d = 0.65 (s, 3H, Me-18), 0.90 (s, 3H, Me-19), 0.94 (d,
J = 6.6 Hz, 3H, Me-21), 2.45–2.50 (m, 4H, 2 –CH2– piperazinyl),
3.17 (d, J = 6.9 Hz, 2H, –CH2–CH@CH–Ph), 3.47–3.50 (m, 3H, 1
–CH2– piperazinyl, and H-3), 3.62–3.67 (m, 2H, 1 –CH2– piperazi-
(C-19), 20.7 (C-21), 22.9, 23.9, 28.4, 30.3, 30.8, 31.5, 33.0, 34.7,
35.2, 35.4, 35.7, 39.5, 39.8, 40.0, 41.3, 41.6, 42.8, 45.3, 45.5 (2C),
50.6, 55.9, 68.6 (C-7), 72.1 (C-3), 107.4 (C-3py), 114.0 (C-5py),
137.8 (C-4py), 148.1 (C-6py), 159.2 (C-2py), 172.5 (C-24) ppm. MS
(30 eV, EI): m/z (%) = 537.2 (3) [M+Å], 519.3 (2) [M+ÅꢁH2O], 501.3
(1) [M+Åꢁ2 H2O], 218.1 (12), 206.1 (10), 120.1 (100), 107.0 (19).
HRMS (EI) m/z [M+] calcd. for C33H51N3O3: 537.3930, found:
537.3967.
nyl), 3.83–3.86 (m, 1H, H-7), 6.25 (dt, dJ = 15.8 Hz and J = 6.9 Hz,
t
1H, –CH2–CH@CH–Ph), 6.53 (d, J = 15.8 Hz, 1H, –CH2–CH@CH-Ph),
7.23–7.39 (m, 5H, Har) ppm. 13C NMR (100 MHz, CDCl3, 25 °C):
d = 11.9 (C-18), 18.6 (C-21), 20.7, 22.9 (C-19), 23.9, 28.3, 30.2,
30.8, 31.5, 33.0, 34.7, 35.2, 35.4, 35.7, 39.5, 39.7, 40.0, 41.6 (2C),
42.8, 45.7, 50.6, 53.0, 53.4, 55.9, 61.1, 68.7 (C-7), 72.1 (C-3),
126.0, 126.5 (2C), 127.7, 128.7 (2C), 133.7, 136.8, 172.1 (C-24)
ppm. MS (ESI): [M+H]+ 577.5. HRMS (EI) m/z [M+] calcd. for
3.1.4.3. 17b-[1-Methyl-3-(1N-(4N-(pyrimidyl-2-yl))piperazin-1-
yl)carboxypropyl] etiocholan-3
powder. Mp 144 °C. IR (KBr): (
a
-ol (IIa). Yield: 75%. White
m
cmꢁ1): 3422 (br, O–H alcohol),
2934–2862 (C–H alkane), 1652 (C@O amide), 1244 (C–N amine).
1H NMR (400 MHz, DMSO-d6, 25 °C): d = 0.61 (s, 3H, Me-18), 0.87
(s, 3H, Me-19), 0.90 (d, J = 6.4 Hz, 3H, Me-21), 3.49–3.53 (m, 4H,
2 –CH2– piperazinyl), 3.49–3.53 (m, 4H, 2 –CH2– piperazinyl),
3.67–3.76 (m, 4H, 2 –CH2– piperazinyl), 6.66 (t, J = 4.6 Hz, 1H,
H-5py), 8.38 (d, J = 4.6 Hz, 2H, H-4py and H-6py) ppm (H-3 signal
was probably included in H2O signal). 13C NMR (100 MHz,
DMSO-d6, 25 °C): d = 12.1 (C-18), 18.6 (C-19), 20.6 (C-21), 23.5,
24.1, 26.4, 27.1, 28.0, 29.6, 30.6, 30.9, 31.1, 34.4, 35.2, 35.3,
35.6, 36.5, 40.9, 41.7, 42.5, 43.3, 43.7, 44.8, 55.7, 56.3, 70.1 (C-
3), 110.6 (C-5py), 158.2 (C-4py and C-6py), 161.2 (C-2py), 171.4
(C-24) ppm (one carbon signal was probably included in
DMSO-d6 signal). MS (30 eV, EI): m/z (%) = 522.3 (8) [M+Å], 507.4
(4) [M+ÅꢁMeÅ], 219.1 (35), 206.1 (100), 164.1 (9), 121.1 (45).
HRMS (EI) m/z [M+] calcd. for C32H50N4O2: 522.3934; found:
522.3953.
C37H56N2O3: 576.4291; found: 576.4313.
3.1.5. Hydrolytic stability of the bile acid derivatives
Bile acid derivative (Ia, Ib, IIa, IIb, IIIa, or IIIb, 50 mg) was dis-
solved in ethanol (0.5 mL) and added to aqueous solution of phos-
phate buffer at pH 7.4 (0.1 M, 10 mL). The mixture solution was
stirred at 37 °C for 24 and 48 h. The solution was extracted with
methylene chloride, dried over anhydrous magnesium sulfate,
and evaporated. The crude product was found unchanged as mon-
itored by TLC on Kieselgel-G (Merck Si 254 F) layers (0.25 mm).
3.2. Biology
3.2.1. Cell lines and culture
Three tumor cell lines were tested in this study: KMS-11 (an
adherent plasma-cell line obtained from a patient with multiple
myeloma),31 GBM (astrocytoma cells obtained after tumor resec-
tion of patients with glioblastoma multiforme (GBM primary cul-
ture),32 HCT-116 (colorectal adenocarcinoma cells derived from a
patient with Lynch’s syndrome).33
3.1.4.4. 17b-[1-Methyl-3-(1N-(4N-(pyrimidyl-2-yl))piperazin-1-
yl)carboxypropyl] etiocholan-3
White powder. Mp 198 °C. IR (KBr): (
a
,7
m
a
-diol (IIb). Yield: 79%.
cmꢁ1): 3400 (br, O–H alco-
hol), 2962–2845 (C–H alkane), 1615 (C@O amide), 1250 (C–N
amine). 1H NMR (400 MHz, CDCl3, 25 °C): d = 0.65 (s, 3H, Me-18),
0.91 (s, 3H, Me-19), 0.95 (d, J = 6.6 Hz, 3H, Me-21), 3.53–3.56 (m,
2H, 1 –CH2– piperazinyl), 3.57–3.67 (m, 1H, H-3), 3.68–3.71 (m,
2H, 1 –CH2– piperazinyl), 3.81–3.88 (m, 5H, 2 –CH2– piperazinyl,
and H-7), 6.55 (t, J = 4.7 Hz, 1H, H-5py), 8.34 (d, J = 4.7 Hz, 2H, H-
4py and H-6py) ppm. 13C NMR (100 MHz, CDCl3, 25 °C): d = 11.9
(C-18), 18.7 (C-19), 20.7 (C-21), 22.9, 23.9, 28.4, 30.4, 30.8, 31.5,
33.0, 34.7, 35.2, 35.5, 35.7, 39.6, 39.8, 40.0, 41.5, 41.6, 42.9, 43.7,
44.0, 45.6, 50.6, 55.9, 68.7 (C-7), 72.2 (C-3), 110.6 (C-5py), 157.9
(C-4py and C-6py), 161.7 (C-2py), 172.6 (C-24) ppm. MS (30 eV,
EI): m/z (%) = 538.3 (11) [M+Å], 523.3 (5) [M+ÅꢁMeÅ], 487.4 (3)
[M+Åꢁ(2 H2O+MeÅ)], 335.1 (4), 267.1 (7), 219.2 (37), 206.1 (100),
164.1 (16), 121.1 (83). HRMS (EI) m/z [M+] calcd. for C32H50N4O3:
538.3883; found: 538.3866.
Cells were cultured in RPMI 1640 medium supplemented with
5% (KMS-1l) or 10% (GBM and HCT-116) fetal calf serum, 2 mM
glutamine, antibiotics (100 IU/mL penicillin and 100 mg/mL strep-
tomycin) (Life Technologies). Cells were subcultured at confluency
after dispersal with 0.025% trypsin in 0.02% EDTA. For experiments
cells were used at 70–80% confluency.
3.2.2. Evaluation of cytotoxicity (neutral red uptake assay)
Twenty thousand cells (GBM and HCT-116) and 50,000 cells
(KMS-11) (200
(Falcon) and incubated at 37 °C in 5% CO2. After 24 h, drugs were
added in 50 L fresh medium and after 45 h cells were loaded for
lL) were plated in 96-well culture microtiter plates
l
3 h with neutral red (3-amino-7,7-dimethylamino-2-methylphen-
azine hydrochloride) (Sigma–Aldrich, St Quentin, France) at a final
concentration of 50 lg/mL in culture medium. This weakly cationic
dye penetrates cell membranes by non-ionic diffusion, and binds
intracellularly to anionic carboxylic and/or phosphate groups of
the lysosomal matrix. Thereafter, the medium was removed, cells
were fixed for 5 min with a mixture of 1% formaldehyde–1% CaCl2
and the dye extracted with 0.2 mL of 1% acetic acid in 50% ethanol.
Plates were left overnight at 4 °C and absorbance (OD, optical den-
sity) was recorded at 570 nm (Multiskan EX—Thermo-Electron
Corporation). Control experiments were carried out using appro-
3.1.4.5. 17b-[1-Methyl-3-(1N-(4N-cinnamyl)piperazin-1-yl)car-
boxypropyl]etiocholan-3
a
-ol (IIIa). Yield: 73%. Beige powder.
Mp 159 °C (dec). IR (KBr): (
m
cmꢁ1): 3401 (br, O–H alcohol),
2960–2863 (C–H alkane), 1638 (C@O amide). 1H NMR (400 MHz,
CDCl3, 25 °C): d = 0.64 (s, 3H, Me-18), 0.91 (s, 6H, Me-19 and Me-
21), 2.45–2.57 (m, 4H, 2 –CH2– piperazinyl), 2.94–2.97 (m, 4H, 2
–CH2– piperazinyl), 3.17 (d, J = 6.8 Hz, 2H, –CH2–CH@CH–Ph),

8744
L. El Kihel et al. / Bioorg. Med. Chem. 16 (2008) 8737–8744
priate DMSO dilutions. Experiments were performed at least in
triplicate, four wells per cell line being used. LD50 (lethal dose
50%) values were calculated from the dose–response curves as
illustrated in Figure 2A. Values are expressed as the mean of at
least three independent experiments standard error. Statistical
analyses were performed by Student’s T test.
Acknowledgments
The authors thank A. Roger and H. Tauvel for their technical
assistance. Dr. Rémi Legay is gratefully acknowledged for his assis-
tance in measurement of mass spectra. We are grateful to L. Oliver
(CHU, Nantes, France) for providing GBM cells.
3.2.3. Evaluation of cell proliferation ([3H]thymidine
incorporation assay)
References and notes
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loading control, was purchased from Chemicon International
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