Synthesis of bile acid derivatives and in vitro cytotoxic activity with pro-apoptotic process on multiple myeloma (KMS-11), glioblastoma multiforme (GBM), and colonic carcinoma (HCT-116) human cell lines

Article abstract of DOI:10.1016/j.ejmech.2010.03.016

The novelty of this work derives from the use of nitrogenous heterocycles as building block in the synthesis of conjugate bile acid derivatives. New piperazinyl bile acid derivatives were synthesized and tested in vitro against various human cancer cells

Full text of DOI:10.1016/j.ejmech.2010.03.016

European Journal of Medicinal Chemistry 45 (2010) 2912e2918
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of bile acid derivatives and in vitro cytotoxic activity with pro-apoptotic
process on multiple myeloma (KMS-11), glioblastoma multiforme (GBM), and
colonic carcinoma (HCT-116) human cell lines
,
b
a
c
Dominique Brossard a, Laïla El Kihel a , Monique Clément , Walae Sebbahi , Mohamed Khalid ,
*
Christos Roussakis d, Sylvain Rault a
a Centre d’Etudes et de Recherche sur le Médicament de Normandie, UPRES EA-4258, FR CNRS INC3M, Université de Caen Basse-Normandie, U.F.R. des Sciences Pharmaceutiques,
Boulevard Becquerel, 14032 Caen Cedex, France
b INSERM U892, 8 quai Moncousu, BP 70721 44007 Nantes Cedex, France
c Université Hassan Premier, Faculté des Sciences et Techniques, Km 3, Route de Casablanca, BP 577, 26000 Settat, Maroc
d Laboratoire de Pharmacologie, Institut ISOMer, 2 Rue de la Houssinière, 44322 Nantes, France
a r t i c l e i n f o
a b s t r a c t
Article history:
The novelty of this work derives from the use of nitrogenous heterocycles as building block in the synthesis
of conjugate bile acid derivatives. New piperazinyl bile acid derivatives were synthesized and tested in vitro
against various human cancer cells (GBM, KMS-11, HCT-116). The best pro-apoptotic activity was obtained
Received 1 July 2009
Received in revised form
11 March 2010
Accepted 15 March 2010
Available online 19 March 2010
with N-[4N-cinnamylpiperazin-1-yl)-3
perazin-1-yl)- ,7 -dihydroxy-5 -cholan-24-amide (7c) on these human cancer cell lines (IC50
8.5e31.4 M). This activity was associated with nuclear and DNA fragmentation, demonstrating that 7b
a,7b-dihydroxy-5b-cholan-24-amide (7b) and N-[4N-cinnamyllpi-
3a
a
b
:
m
induces cell death by an apoptotic process as 7c. This study shows the possibility of hydrid heterocycle-
steroids as new anticancer agents with improved bioactivity and easy to synthesize.
Ó 2010 Published by Elsevier Masson SAS.
Keywords:
Bile acid derivatives
Piperazinylsteroids
Pro-apoptotic compounds
Multiple myeloma
Glioblastoma multiforme
Colonic carcinoma
1. Introduction
derivatives, which are conjugates of bile acids with amino acids.
These compounds have shown antitumor effects in human cancer
Bile acids, polar derivatives of cholesterol, are normal constitu-
ents of the colon and play key roles in the absorption of dietary
lipids. A representative secondary bile acid, ursodeoxycholic acid
(UDCA), is known to prevent gastrointestinal disorders of patients
with various cancers (stomach, colon, lung, breast, and liver) [1e3]. It
was also evaluated in clinical phase III trials to prevent colorectal
adenoma recurrence [4]. Furthermore, recent clinical trials demon-
strate that bile acid sequestrants can significantly reduce glucose
levels in patients with type 2 diabetes mellitus [5,6]. The primary bile
cells such as stomach cancer cells [7], glioblastoma multiforme [8],
colon cancer [9], breast carcinoma [10], leukemic Tcells [11], prostate
cancer cells [12], cervical carcinoma [13], hepatocellular carcinoma
[14,15] and breast cancer cells [16]. Only a few conjugate bile acids
with amino acids are synthesized but their biological activities have
been widely studied. Due to their biological applicability, our
approach consisted to introduce a nitrogenous heterocycle on the
bile acids side chain. For this, we chose piperazine derivatives which
are a pharmacologically interesting class of heterocycles [17e19].
This paper is the continuation of a previous study [20].
acids (cholic acid (CA), (3a,7a,12a-trihydroxy-5b-cholan-24-oic acid)
and chenodeoxycholic acid (CDCA), 3
a
,7 -dihydroxy-5
a
b
-cholan-24-
We report here the synthesis of new piperazinyl bile acid
derivatives and discuss their cytotoxic activities on human cancer
cell lines (Fig. 1).
oic acid)) are conjugated with glycine or taurine in hepatocytes to
form the corresponding N-acyl conjugates. By analogy to these
natural compounds, some authors have synthesized several bile acid
2. Chemistry
Starting compounds were cholic acid (3
a
-,7
a-,12a-trihydrox-
* Corresponding author. Tel.: þ33 (0) 23 31 56 68 12; fax: þ33 (0) 2 31 56 68 03.
E-mail address: laila.elkihel@unicaen.fr (L. El Kihel).
ycholanic acid, 1a), ursodeoxycholic acid (3 -,7
a
b-dihydroxycholanic
0223-5234/$ e see front matter Ó 2010 Published by Elsevier Masson SAS.
doi:10.1016/j.ejmech.2010.03.016

D. Brossard et al. / European Journal of Medicinal Chemistry 45 (2010) 2912e2918
2913
H
N
R2
R2
H
N
N
N
N
H
O
O
H
H
H
H
HO
R1
HO
R1
H
H
7a; R1 = α OH, R2 = OH
7b; R1 = β OH, R2 = H
7c; R1 = α OH, R2 = H
5a; R1 = α OH, R2 = OH
5b; R1 = β OH, R2 = H
5c; R1 = α OH, R2 = H
Fig. 1. Piperazinyl bile acid derivatives.
acid, 1b) and chenodeoxycholic acid (3a-,7a-dihydroxycholanic acid,
IC50 8.5
mM, but 7b is quite similar in these three human cancer cell
1c) which were acetylated with acetic anhydride in pyridine to give
the acetates 2aec respectively. The latter acids 2aec were converted
into the corresponding mixed anhydrides by treatment with ethyl
chloroformate in the presence of triethylamine giving 3aec
respectively. The mixed anhydrides 3aec were reacted with
substituted piperazines (a or b), the products were identified as the
carboxamides (4aec and 6aec). Alcohol moieties were deprotected
by ethanolic potassium hydroxide to give the corresponding deace-
tylated amides 5aec and 7aec which were purified on alumina
deactivated with 6% water. The structures of the compounds
prepared were confirmed by analytical and spectral analyses
(Scheme 1).
lines with IC50 24.7e31.4
m
M.
The marked difference in activity among the piperazinyl deriv-
atives shows the importance of the cinnamyl system. The cinna-
mylpiperazine derivatives 7b and 7c appear to be the most active
compounds on these human cancer lines. It can be noticed that no
activity was observed with 7a which is a cinnamylpiperazinyl
building block with cholic acid.
On the other hand, the lipophilicity (LogP) of bile acid deriva-
tives 5aec and 7aec was predicted from structures using ALogP
program (Table 1). These data show that compounds 7b and 7c
have the highest lipophilicity suggesting that the lipophilicity plays
a role in the activity of these molecules.
The hydrolytic stability of compounds 5aec and 7aec was
studied under physiological conditions (phosphate buffer, pH 7.4 at
37 ꢀC). No degradation of these bile acid derivatives was observed.
The prediction of the lipophilicity of bile acid derivatives 5aec
and 7aec has been determined by ALogP program [21].
Following the results obtained with the 7b, and because the
apoptotic effect of 7c was already described [20], we decided to
investigate how 7b induces cell death.
As shown by annexin V staining which reveals the early stages of
apoptosis [22], the percentage of dead cells strongly increased after
a 24 h treatment with 20 mM of 7b (Fig. 2). Percentage of specific
apoptosis was of 96% (KMS-11), 78% (GBM-12, data not shown), 45%
(HCT-116). These data are in accordance with IC50 values and
suggest that induction of apoptosis is delayed in HCT-116 compared
to the two other cell lines.
3. Biology
3.1. Cytotoxicity
Characteristic images of apoptotic nuclei are shown in Fig. 3. Cell
density of KMS-11, evaluated after 24 h of treatment by counting
the number of nuclei using metamorph software, decreased with
The compounds synthesized were tested for their initial in vitro
cytotoxicity against a panel of tumor cells as described in the
experimental section.
increased 7b concentrations (84% for 10
mM, 59% for 20 mM, 35% for
40 M) relative to the control. Final DNA fragmentation which is
a consequence of apoptotic death [23], was observed after 48 hours
of treatment (Fig. 4).
Taken together, these results (exposure of phosphatidylserines
on the external surface of cells, nuclear and DNA fragmentation)
demonstrate that 7b induces cell death by an apoptotic process as
7c [20].
m
3.2. Pro-apoptotic activity
The pro-apoptotic activity of 7b was explored using various
approaches as described in the experimental section.
4. Results and discussion
Comparing 7a with 7b and 7c, it can be concluded that the
hydroxyl functionality at C-7 position of steroid nucleus in cinna-
mylpiperazinylsteroid is crucial for the cytotoxic activity. In addition,
this activity was enhanced with 7-hydroxyl group in the stereo-
chemistry alpha. Indeed, no activity was observed with N-[4N-cin-
Compounds 5aec and 7aec were synthesized to explore the
relationship between structure and cytotoxic activity. We have
investigated the role of the substituted lipid moiety in the cytotoxic
activity. Cholic acid (1a), ursodeoxycholic acid (1b) and cheno-
deoxycholic acid (1c) were chosen because they allow controlled
changes in the regiochemical and the stereochemical substitution
of the hydroxyl groups on the cholane skeleton.
The cytotoxicity experiments with the synthesized compounds
were performed in vitro in various human cancer cells: glioblas-
toma multiforme (GBM-12, brain tumor cell line), multiple
myeloma (KMS-11) and colonic carcinoma (HCT-116) cell lines. The
IC50 cytotoxicity values obtained are summarized in Table 1. These
compounds displayed micromolar cytotoxic concentrations. The
highest activity was obtained with bile acid derivatives 7b and 7c.
The best activity was observed for 7c on multiple myeloma with
namylpiperazin-1-yl)-3a-hydroxy-5b-cholan-24-amide without
hydroxyl group at the C-7position [20]. Moreover, the activity was
suppressed when the steroid possess the hydroxyl group on the C-
12 position (7a) even in the presence of the hydroxyl at C-7 position.
5. Conclusion
In conclusion, some new piperazinyl bile acid derivatives were
synthesized and their cytotoxic activities on human cancer cell
lines were evaluated. Some of them exhibited good activity in all

2914
D. Brossard et al. / European Journal of Medicinal Chemistry 45 (2010) 2912e2918
Scheme 1. Synthesis of piperazinyl bile acid derivatives. Reagents and conditions: (i) Ac2O, pyridine, r.t., 15 h; (ii) EtOCOCl, NEt3, CHCl3, r.t., 50 min; (iii) piperazine (a or b), NEt3,
CHCl3, r.t., 16 h; (iv) KOH, EtOH, reflux, 24 h.
cancer cells tested. The best activity shown by 7b and 7c may be
due to the presence of the cinnamylpiperazinyl group in the side
chain of the ursodeoxycholic and chenodeoxycholic acids bearing
a hydroxyl group at C-7 position. These derivatives constitute
a starting point towards a new platform development of this type of
steroid-heterocyclic hybrid, which could easily be prepared in large
amounts for biological evaluation. Further research in this area is in
progress in our laboratory.
6. Experimental
6.1. Chemistry
6.1.1. 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 silica gel
60 (0.063e0.2 mm) (Merck). Melting points were determined on
a Kofler block. IR spectra were recorded on a PerkineElmer 1600 FT-
IR spectrometer. EI mass spectra were recorded on a Jeol-GCmate
(GC-MS system) spectrometer with ionisation energy from 30 to
40 eV. ESI mass spectra were recorded on a LC/MS Waters alliance.
1H NMR and 13C NMR spectra were recorded using CDCl3 and
methanol-d4 respectively at 400 MHz (Jeol Lambda 400 spectrom-
eter) and at 100 MHz. Chemical shifts are reported relative to TMS; J
values are given in Hz. 13C NMR spectra are 1H-decoupled. Elemental
analyses were performed at the “Institut de Recherche en Chimie
Organique Fine” (Rouen, France).
Table 1
Cytotoxic activity of piperazinyl bile acid derivatives on three human cancerous cell
lines.a
Compounds
Log Pb
KMS-11
GBM IC50
(m
M)
HCT-116
IC50
(m
M)
IC50 (mM)
5a
5b
5c
7a
7b
7c
CA
CDCA
UDCA
2.162
3.264
3.264
4.705
5.807
5.807
2.912
4.014
4.014
>50
>50
ꢁ50
>50
28.1 ꢂ 5.2
8.5 ꢂ 0.5
>50
>50
>50
>50
>50
>50
>50
24.7 ꢂ 1.9
16.5 ꢂ 2
>50
ꢁ50
>50
31.4 ꢂ 6.3
18.5 ꢂ 1
>50
>50
>50
>50
>50
>50
>50
a
IC50 values were determined from the dose-response curves after 24 h of
incubation using the neutral red uptake assay. Results are expressed as the mean of
three experiments ꢂ standard error.
6.1.2. Chemical synthesis
6.1.2.1. General procedure for 2a, 2b and 2c. Acetic anhydride
(78.8 mmol, 6.7 mL) was added dropwise to a solution of 1a, 1b or
b
LogP was determined by ALogP program.

D. Brossard et al. / European Journal of Medicinal Chemistry 45 (2010) 2912e2918
2915
Fig. 2. Percentage of apoptotic KMS-11 and HCT-116 cells in the absence or presence of 7b (20 mM) determined after 24 h, by annexin V staining.
1c (8.8 mmol) in pyridine (25 mL). The reaction mixture was stirred
at room temperature under argon atmosphere for 15 h and then ice
water was added. The white precipitate formed was dissolved in
CH2Cl2 (100 mL), washed with 1 M HCl (2 ꢃ 20 mL), 5% NaHCO3
(2 ꢃ 20 mL), brine (2 ꢃ 20 mL) and then with water (20 mL). The
organic layer was dried over anhydrous magnesium sulfate and
evaporated under reduced pressure. The crude product was puri-
fied by column chromatography (silica gel, cyclohexane/ethyl
acetate, 8:2) to afford 2a, 2b or 2c.
give 2b (3.86 g, 92 %) as a white powder. IR (KBr): (
3050 (OeH acid), 2939e2870 (CeH alkane), 1729 (C]O ester and
n
cmꢄ1): 3493-
acid). 1H NMR (400 MHz, CDCl3, 25 ꢀC):
d
¼ 0.68 (s, 3H, Me-18), 0.90
(s, 3H, Me-19), 0.95 (d, 3J ¼ 8.0 Hz, 3H, Me-21), 2.02 (s, 6H,
2 ꢃ eOCOCH3), 4.65e4.67 (m, 1H, H-3), 4.73e4.79 (m, 1H, H-7)
ppm. 13C NMR (100 MHz, CDCl3, 25 ꢀC):
d
¼ 12.0 (C-18), 18.3 (C-21),
21.2, 21.4 (2 ꢃ eOCOCH3), 21.8 (C-19), 23.2, 25.6, 26.4, 28.4, 29.7,
30.8, 30.9, 32.9, 34.0, 34.5, 35.2, 39.4, 39.9, 40.0, 42.0, 43.6, 55.0,
55.2, 73.6 (C-3), 170.6 (eOCOCH3), 170.7 (eOCOCH3), 178.7 (C-24)
ppm. MS (30 eV, EI): m/z (%) ¼ 476.3 (1) [Mþ$], 434.3 (3), 416.3
[Mþ$e CH3CO2H], 374.3 (7), 356.3 (100) [Mþ$e 2 ꢃ CH3CO2H], 341.2
(33), 302.2 (10), 255.2 (22), 228.2 (38), 213.2 (28).
6.1.2.2. 3a,7a-,12a-triacetyloxy-5b-cholan-24-oic acid (2a). The crude
product was recrystallized from acetone to give 2a (4.15 g, 88 %) as
a white powder. IR (KBr): v (cmꢄ1): 3404e3010 (OeH acid),
2950e2869 (CeH alkane), 1736 (C]O ester), 1709 (C]O carboxylic
6.1.2.4. 3a,7a-diacetyloxy-5b-cholan-24-oic acid (2c). Compound
2c have been prepared according to a reported procedure [20].
acid). 1H NMR (400 MHz, CDCl3, 25 ꢀC):
d
¼ 0.73 (s, 3H, Me-18), 0.82
(d, 3J ¼ 6.8 Hz, 3H, Me-21), 0.91 (s, 3H, Me-19), 2.05 (s, 3H, eOCOCH3),
2.09 (s, 3H, eOCOCH3), 2.14 (s, 3H, eOCOCH3), 4.52e4.62 (m, 1H, H-
3), 4.86e4.95 (m, 1H, H-7), 5.08e5.12 (m, 1H, H-12) ppm. 13C NMR
6.1.3. General procedure for compounds 4a, 4b, 4c, 6a, 6b and 6c
Ethyl chloroformate (1.13 mL, 11.8 mmol) was added to a stirred
solution of acid 2a, 2b or 2c (8.9 mmol) in chloroform (30 mL) under
argon atmosphere at room temperature. Triethylamine (2.50 mL,
17.7 mmol) was added dropwise and the solution was kept under
stirring for 45 min. The reaction mixture was diluted with methylene
chloride (40 mL), washed with water (2 ꢃ 20 mL), dried over anhy-
drous magnesium sulfate and evaporated. The crude product of
anhydride (3a or 3b) was used to next step without purification.
Under nitrogen atmosphere, piperazine derivative (a or b)
(17.8 mmol) and triethylamine (1.25 mL, 8.9 mmol) were added to
a solution of anhydride 3a, 3b or 3c (8.9 mmol) in chloroform
(100 MHz, CDCl3, 25 ꢀC):
d
¼ 12.2 (C-18), 17.4 (C-21), 21.4 (eOCOCH3),
21.5(eOCOCH3), 21.6(eOCOCH3), 22.5(C-19), 22.7, 25.5, 26.8, 27.1,
28.8, 30.5, 30.8, 31.2, 34.3, 34.5 (2C), 34.6, 37.7, 40.8, 43.3, 45.0, 47.3,
70.7, 74.1, 75.3, 170.4(eOCOCH3), 170.5(eOCOCH3), 170.6(eOCOCH3),
179.6(C-24) ppm. MS (30 eV, EI): m/z (%) ¼ 418.5 (3) [Mþ$], 358.4
(100) [Mþ$e CH3CO2H], 343.4 (12), 304.3 (6), 257.3 (9), 230.3 (21),
215.3 (33).
6.1.2.3. 3
a,7b-diacetyloxy-5b-cholan-24-oic acid (2b). The crude
product was recrystallized from acetonitrile/diethyl ether (2/8) to
Fig. 3. Nuclear fragmentation of KMS-11 and HCT-116 cells induced by 7b treatment (20 mMe24 h) visualized by DNA staining with Hoëchst.

2916
D. Brossard et al. / European Journal of Medicinal Chemistry 45 (2010) 2912e2918
34.4, 35.2, 35.3, 39.3, 39.8, 41.9, 43.5, 45.6, 54.1, 54.5, 54.9, 55.0, 55.1,
55.4, 56.4, 66.7 (C-7), 73.4 (C-3), 170.4 (OeC]O), 170.5(OeC]O),
176.9 (O]CeN) ppm. MS (30 eV, EI): m/z (%) ¼ 573.4 (25) [Mþ$],
558.4 (19) [Mþ$e CH3], 198.1 (4) [Mþ$e StC23], 170.1 (100) 198.1 (4)
e
[Mþ$ StC21]. HRMS (EI) m/z [Mþ] calcd. for C33H55N3O5:
573.41413, found: 573.41556.
6.1.3.3. N-(4N-methylpiperazin-1-yl)-3
24-amide (4c). 3.49 g (69 % yield) as a yellow oil. IR (KBr): (
a
,7
a
-di-acetoxy-5
b
-cholan-
cmꢄ1):
n
3450e3220 (NeH amide), 2942e2871 (CeH alkane), 1735 (C]O
ester), 1665 (C]O amide), 1247 (CeN amine). 1H NMR (400 MHz,
CDCl3, 25 ꢀC):
d
¼ 0.65 (s, 3H, Me-18), 0.93 (s, 3H, Me-19), 0.96
(d, 3J ¼ 5.8 Hz, 3H, Me-21), 2.00 (s, 3 H, C3 eOCOCH3), 2.05 (s, 3 H, C7
eOCOCH3), 2.30 (s, 3H, NeCH3), 2.52e2.66 (m, 4H, 2 ꢃ eCH2-
piperazinyl), 2.72e2.96 (m, 4H, 2 ꢃ eCH2- piperazinyl), 4.50e4.62
(m, 1H, H-3), 4.82e4.92 (m, 1H, H-7), 6.66 (br s, 1H, NH, D2O
13
exchange) ppm. C NMR (100 MHz, CDCl3, 25 ꢀC):
d
¼ 11.5, 18.1,
20.4, 21.2(1 ꢃ eOCOCH3), 21.3(1 ꢃ eOCOCH3), 22.4, 23.3, 26.5, 27.8,
29.1, 31.0, 33.8, 34.3, 34.5, 34.6, 35.2, 35.4, 37.6, 39.2, 40.6, 42.4, 45.4,
50.1, 53.9, 54.3, 54.9, 55.6, 56.1, 70.1 (C-7), 73.9 (C-3), 170.3 (OeC]
O), 170.7 (OeC]O), 176.9 (O]CeN) ppm. MS (ESI): [M þ H]þ
574.10. HRMS (EI) m/z [Mþ] calcd. for C33H35N3O5: 573.41413,
found: 573.41588.
Fig. 4. Supernatants of control and 7b treated (20 mMe48 h) KMS-11 cultures analyzed
by agarose gel electrophoresis (MW, 100 bp DNA ladder).
6.1.3.4. N-(4N-cinnamylpiperazin-1-yl)-3a,7a,12a-tri-acetoxy-5b-
cholan-24-amide (6a). 3.78 g (59 % yield) as a yellow oil. IR (KBr): (
n
cmꢄ1): 2942e2808 (CeH alkane), 1732 (C]O ester), 1645 (C]O
1
(30 mL). The reaction mixture was stirred at room temperature for
12 h. The solution was diluted with methylene chloride (40 mL),
washed with water (2 ꢃ 20 mL), dried over anhydrous magnesium
sulfate and evaporated. The crude product was purified by column
chromatography on deactivated neutral alumina (6% water) eluting
by cyclohexane/ethyl acetate, 7:3.
amide), 1248 (CeN amine). H NMR (400 MHz, CDCl3, 25 ꢀC):
d
¼ 0.72 (s, 3H, Me-18), 0.82 (s, 3H, Me-19), 0.91 (d, 3H, J ¼ 6.6 Hz,
Me-21), 2.07 (s, 3H, eC3eOCOCH3), 2.08 (s, 3H, eC7eOCOCH3), 2.13
(s, 3H, eC12eOCOCH3), 2.46e2.50 (m, 4H, 2 ꢃ eCH2- piperazinyl),
3.17 (d, J ¼ 6.8 Hz, 2H, eCH2eCH]CH-Ph), 3.46e3.48 (m, 2H, 2 ꢃ eC
HAXHEQ- piperazinyl), 3.60e3.65 (m, 2H, 2 ꢃ eC HAXHEQ- piper-
azinyl), 4.53e4.65 (m, 1H, H-3), 4.87e4.95 (m, 1H, H-7), 5.05e5.12
(m, 1H, H-12), 6.25 (dt, dJ ¼ 15.6 Hz and tJ ¼ 6.8 Hz, 1H, eCH2eCH]
CH-Ph), 6.52 (d, J ¼ 15.6 Hz, 1H, eCH2eCH]CH-Ph), 7.22e7.39 (m,
6.1.3.1. N-[4N-methylpiperazin-1-yl)-3
cholan-24-amide (4a). 3.24 g (58 % yields) as a yellow oil. IR (KBr):
cmꢄ1): 3448e3150 (NeH amide), 2944e2872 (CeH alkane),1735
a,7a,12a-tri-acetoxy-5b-
(n
5H, Har) ppm. 13C NMR (100 MHz, CDCl3, 25 ꢀC):
d
¼ 12.1(C-18), 17.6
(C]O ester), 1666 (C]O amide). 1H NMR (400 MHz, CDCl3, 25 ꢀC):
(C-21), 21.3 (2 ꢃ eOCOCH3), 21.5 (1 ꢃ eOCOCH3), 22.4, 22.7, 25.4,
26.7, 27.1, 28.7, 30.2, 31.1, 34.1, 34.4, 34.5, 34.8, 37.5, 40.7, 41.3, 43.2,
44.9, 45.4, 47.5, 52.7, 53.1 (2C), 60.7 (eCH2eCH]CH-Ph), 70.5 (C-7),
73.9 (C-3), 75.2 (C-12), 125.7, 126.2 (2C), 127.5, 128.4 (2C), 133.4,
136.5, 170.2(C]O), 170.4 (2 C]O), 171.6 (O]CeN) ppm. MS (30 eV,
EI): m/z (%) ¼ 718.3 (100) [Mþ$], 658.3 (27) [Mþ$e CH3CO2H], 600.3
(36) [Mþ$e Ph-CH]CHeCH2], 542.3 (34), 358.2 (84), 313.1 (43),
257.1 (48).
d
¼ 0.74 (s, 3H, Me-18), 0.85 (d, 3J ¼ 6.8 Hz, 3H, Me-21), 0.92 (s, 3H,
Me-19), 2.05 (s, 3H, eC3eOCOCH3), 2.09 (s, 3H, eC7eOCOCH3), 2.15
(s, 3H, eC12eOCOCH3), 2.30 (s, 3H, NeCH3), 2.51e2.60 (m, 4H,
2 ꢃ eCH2- piperazinyl), 2.75e2.95 (m, 4H, 2 ꢃ eCH2e piperazinyl),
4.52e4.62 (m, 1H, H-3), 4.86e4.95 (m, 1H, H-7), 5.08e5.12 (m, 1H,
H-12), 6.65 (br s, 1H, NH, D2O exchange) ppm. 13C NMR (100 MHz,
CDCl3, 25 ꢀC):
d
¼ 12.0 (Me-18), 17.4 (Me-21), 21.2 (3 ꢃ eOCOCH3),
22.3 (Me-19), 22.5, 25.3, 26.6, 26.9, 28.6, 29.0, 30.8, 31.3, 34.0, 34.3,
34.5, 34.7, 37.4, 40.6, 43.1, 44.8, 45.4, 47.3, 54.0, 54.3, 55.0, 56.1, 70.4
(C-7), 73.8 (C-3), 75.1 (C-12), 170.1(eOCOCH3), 170.3(eOCOCH3),
170.5(eOCOCH3), 176.6 (O]CeN) ppm. MS (30 eV, EI): m/z (%) ¼
631.4 (82) [Mþ$], 616.5 [Mþ$e CH3], 572.4 (100) [Mþ$e CH3CO2],
556.4 (13) [Mþ$e CH3CO2H e CH3], 512.4 (16) [Mþ$e CH3CO2H e
CH3CO2], 413.3 (18), 354.3 (27), 253.2 (37.9), 170.1 (74). HRMS (EI)
m/z [Mþ] calcd. for C35H57N3O7: 631.41963, found: 631.41943.
6.1.3.5. N-(4N-cinnamylpiperazin-1-yl)-3
24-amide (6b). 4.16 g (71 % yield) as a yellow oil. IR (KBr): (
2948e2871 (CeH alkane), 1735 (C]O ester), 1647 (C]O amide). 1H
NMR (400 MHz, CDCl3, 25 ꢀC):
a
,7
b
-di-acetoxy-5
b
-cholan-
n
cmꢄ1):
d
¼ 0.68 (s, 3H, Me-18), 0.94
(d, J ¼ 6.8 Hz, 3H, Me-21), 0.97 (s, 3H, Me-19), 2.00 (s, 3H,
1 ꢃ eOCOCH3), 2.02 (s, 3H, 1 ꢃ eOCOCH3), 2.44e2.52 (m, 4H,
2 ꢃ eCH2- piperazinyl), 3.17 (d, J ¼ 5.8 Hz, 2H, eCH2eCH]CH-Ph),
3.46e3.48 (m, 2H, 2 ꢃeC HAXHEQ- piperazinyl), 3.60e3.64 (m, 2H,
2 ꢃ -C HAXHEQ- piperazinyl), 4.62e4.67 (m, 1H, H-3), 4.72e4.79
6.1.3.2. N-[4N-methylpiperazin-1-yl)-3
24-amide (4b). 3.14 g (62 % yield) as a yellow oil. IR (KBr): (
a
,7
b
-di-acetoxy-5
b
-cholan-
cmꢄ1):
t
n
(m, 1H, H-7), 6.25 (dt, dJ ¼ 15.6 Hz and J ¼ 6.8 Hz, 1H, eCH2eCH]
3445-3140 (NeH amide), 2948e2873 (CeH alkane), 1736 (C]O
CH-Ph), 6.53 (d, J ¼ 15.6 Hz, 1H, eCH2eCH]CH-Ph), 7.22e7.39
1
ester), 1667 (C]O amide). H NMR (400 MHz, CDCl3, 25 ꢀC):
(m, 5H, Har) ppm. 13C NMR (100 MHz, CDCl3, 25 ꢀC):
d
¼ 11.9, 18.5,
d
¼ 0.68 (s, 3H, Me-18), 0.90 (d, 3J ¼ 6.8 Hz, 3H, Me-21), 0.97 (s, 3H,
21.2 (eOCOCH3), 21.7(eOCOCH3), 23.0, 25.4, 26.1, 28.3, 30.0, 31.2,
32.7, 33.8, 34.3, 35.3, 39.2, 39.7, 41.3, 41.8, 43.4, 45.4, 52.7, 54.8, 55.0,
60.7 (eCH2eCH]CH-Ph), 73.3 (C-3 and C-7), 125.7, 126.1 (2C),
127.5, 128.4 (2C), 133.3, 136.5, 170.3 (C]O), 170.4 (C]O), 171.8 (O]
CeN) ppm. MS (30 eV, EI): m/z (%) ¼ 660.3 (30) [Mþ$], 600.3
Me-19), 2.00 (s, 3H, eC3eOCOCH3), 2.03 (s, 3H, eC7eOCOCH3), 2.31
(s, 3H, NeCH3), 2.52e2.61 (m, 4H, 2 ꢃ eCH2- piperazinyl),
2.70e3.70 (m, 4H, 2 ꢃ eCH2- piperazinyl), 4.62e4.70 (m, 1H, H-3),
4.72e4.80 (m, 1H, H-7), 6.27 (br s, 1H, NH, D2O exchange) ppm. 13
C
NMR (100 MHz, CDCl3, 25 ꢀC):
21.3(1 ꢃ eOCOCH3), 23.1, 25.5, 26.3, 28.4, 29.0, 31.2, 31.6, 32.7, 33.9,
d
¼ 12.0, 18.5, 21.1(1 ꢃ eOCOCH3),
(9) [Mþ$e Ph-CH]CH-CH2], 569.1 (10), 449.3 (9), 257.0 (43) [Mþ$
e
StC21], 172.0 (100).

D. Brossard et al. / European Journal of Medicinal Chemistry 45 (2010) 2912e2918
2917
6.1.3.6. N-(4N-cinnamylpiperazin-1-yl)-3
cholan-24-amide (6c). Compound 6c have been prepared according
to a reported procedure [20].
a
,7
a
-di-acetoxy-5
b
-
[Mþ] calcd. for C29H51N3O3: 489.39301, found: 489.39303. Anal.
calcd. for C29H51N3O3: C, 71.12; H, 10.50; N, 8.58; O, 9.80 Found: C,
71.31; H, 10.72; N, 8.60; O, 9.83.
6.1.4. Typical procedure of compounds 5a, 5b, 5c, 7a and 7b
An aqueous solution (1 mL) of potassium hydroxide (0.84 g,
15.04 mmol) was added to a stirred solution of 4a (1.18 g,1.88 mmol)
in ethanol (15 mL). 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 anhydrous sodium sulfate. The crude product was
purified by column chromatography on deactived neutral alumina
(6% water) (eluent: cyclohexane/ethyl acetate, 7:3) to afford 5a
(0.53 g, 56 %) as a white powder.
6.1.4.4. N-[4N-cinnamylpiperazin-1-yl)-3a,7a,12a-trihydroxy-5b-
cholan-24-amide (7a). 0.54 g (48 % yield) as a white solid. IR (KBr):
(
n
cmꢄ1): 3419 (br, OeH alcohol), 2934e2813 (CeH alkane), 1627
1
(C]O amide). H NMR (400 MHz, CDCl3, 25 ꢀC):
d
¼ 0.64 (s, 3H,
Me-18), 0.84 (s, 3H, Me-19), 1.00 (d, J ¼ 5.8 Hz, 3H, Me-21),
2.98e3.08 (m, 4H, 2 ꢃ eCH2- piperazinyl), 3.15 (d, J ¼ 5.8 Hz, 2H,
eCH2eCH]CH-Ph), 3.38-3.43 (m, 1H, 3-H), 3.39 (m, 2H, 2 ꢃ eC
HAXHEQ- piperazinyl), 3.61 (m, 2H, 2 ꢃ eCHAXHEQ- piperazinyl),
3.80 (m, 1H, 7-H), 3.94 (m, 1H, 12-H), 4.55 (s, 3H, 3 ꢃ OH, D2O
exchange), 6.18e6.30 (m, 1H, eCH2eCH]CH-Ph), 6.52
(d, J ¼ 15.6 Hz, 1H, eCH2eCH]CH-Ph), 7.21e7.38 (m, 5H, Har)
13
6.1.4.1. N-(4N-methylpiperazin-1-yl)-3
24-amide (5a). IR (KBr): (
cmꢄ1): 3417e3231 (OeH alcohol and
N-H amide), 2936e2862 (CeH alkane), 1629 (C]O amide). 1H NMR
(400 MHz, CDCl3, 25 ꢀC):
¼ 0.67 (s, 3H, Me-18), 0.88 (s, 3H, Me-19),
0.98 (d, J ¼ 6.8 Hz, 3H, Me-21), 2.06e2.26 (m, 4H, 2 ꢃ eCH2- piper-
azinyl), 2.29 (s, 3H, N-CH3), 2.51e2.68 (m, 2H, 2 ꢃ eC HAXHEQ
piperazinyl),
a
,7
a
,12
a
-trihydroxy-5
b
-cholan-
ppm. C NMR (100 MHz, CDCl3, 25 ꢀC):
d
¼ 12.3, 17.3, 22.3, 23.1,
n
26.1, 27.5, 27.9, 29.8, 30.0, 31.2, 34.5, 34.6, 35.2, 35.4, 39.3, 41.3,
41.4, 44.6 (2C), 45.4, 46.2, 46.5, 51.2, 51.9, 60.9, 68.1 (C-7), 71.4 (C-
3), 72.8 (C-12), 125.5, 126.2 (2C), 127.5, 128.4 (2C), 133.5, 136.5,
172.3 (C-24) ppm. MS (ESI): [M þ H]þ 593.2. Anal. calcd. for
C37H56N2O4: C, 74.96; H, 9.52; N, 4.73; O, 10.79 Found: C, 74.91; H,
9.57; N, 4.73; O, 10.80.
d
-
piperazinyl), 2.75e2.98 (m, 2H, 2 ꢃ eC HAXHEQ
-
3.35e3.46 (m,1H, H-3), 3.80e3.85 (m,1H, H-7), 3.92e3.96 (m,1H, H-
12), 5.30 (s, 3H, 3 x OH, D2O exchange), 6.61 (br s, 1H, eNH, D2O
6.1.4.5. N-[4N-cinnamylpiperazin-1-yl)-3
24-amide (7b). 0.68 g (63 % yield) as a white solid. IR (KBr):
cmꢄ1): 3410 (br, OeH alcohol), 2931e2813 (CeH alkane), 1628
(C]O amide). 1H NMR (400 MHz, CDCl3, 25 ꢀC):
a,7a-dihydroxy-5b-cholan-
13
exchange) ppm. C NMR (100 MHz, CDCl3, 25 ꢀC):
d
¼ 12.37, 17.5,
22.4, 23.3, 27.6, 27.7, 28.0, 30.3, 30.4, 31.2, 31.3, 34.8, 35.3, 35.8, 39.5,
41.5, 41.6, 45.5, 46.3, 47.0 (2C), 54.2 (2C piperazinyl), 55.9
(2C piperazinyl), 68.4 (C-7), 71.7 (C-3), 73.0 (C-12), 177.4 (C]O) ppm.
MS (30 eV, EI): m/z (%) ¼ 505.4 (28) [Mþ$], 490.4 (25) [Mþ$e CH3],
472.3 (8) [Mþ$e (H20 þ CH3)], 271.2 (18), 170.1 (100) [Mþ$e StC21].
HRMS (EI) m/z [Mþ] calcd. for C29H51N3O4: 505.38792, found:
505.38805. Anal. calcd. for C29H51N3O4: C, 68.87; H, 10.16; N, 8.31; O,
12.65 Found: C, 68.90; H, 10.20; N, 8.31; O, 12.66.
(n
d
¼ 0.68 (s, 3H, Me-
18), 0.94 (s, 3H, Me-19), 0.95 (d, J ¼ 5.8 Hz, 3H, Me-21), 2.43e2.52
(m, 4H, 2 ꢃ eCH2- piperazinyl), 3.17 (d, J ¼ 6.8 Hz, 2H, eCH2eCH]
CH-Ph), 3.42e3.51 (m, 4H, 2 ꢃ eCH2- piperazinyl), 3.52-3.61 (m,1H,
3-H), 3.62e3.68 (m, 1H, 7-C), 5.30 (s, 2H, 2 ꢃ OH, D2O exchange),
t
6.27 (dt, dJ ¼ 15.6 Hz and J ¼ 6.7 Hz, 1H, eCH2eCH]CH-Ph), 6.52
(d, J ¼ 15.6 Hz, 1H, eCH2eCH]CH-Ph), 7.23e7.39 (m, 5H, Har) ppm.
13C NMR (100 MHz, CDCl3, 25 ꢀC):
d
¼ 12.1 (C-18), 18.5 (C-21), 21.1
6.1.4.2. N-(4N-methylpiperazin-1-yl)-3
24-amide (5b). 0.61 g (66 % yield) as a yellow amorphous solid. IR
(KBr): (
cmꢄ1): 3424e3000 (OeH alcohol and NH amide),
2926e2852 (CeH alkane), 1654 (C]O amide). 1H NMR (400 MHz,
CDCl3, 25 ꢀC):
¼ 0.68 (s, 3H, Me-18), 0.92 (s, 3H, Me-19), 0.97
(d, J ¼ 5.9 Hz, 3H, Me-21), 2.31 (s, 3H, N-CH3), 2.58e2.80 (m, 2H,
a
,7
b
-dihydroxy-5
b
-cholan-
(C-19), 23.3, 26.8, 28.6, 30.2, 31.4, 33.0, 34.0, 34.9, 35.5, 36.8, 37.2,
39.1, 40.0, 41.4, 42.4, 43.6, 43.7, 45.5, 52.8, 53.2, 54.9, 55.6, 60.8, 71.2
(C-3 and C-7), 125.7, 126.3 (2C), 127.6, 128.5 (2C), 133.5, 136.6, 172.1
(C-24) ppm. MS (30 eV, EI): m/z (%) ¼ 576.3 (27) [Mþ$], 561.3 (3)
[Mþ e CH3], 485.3 (9), 257.1 (19), 201.1 (24), 172.1 (100). HRMS (EI)
m/z [Mþ] calcd. for C37H56N2O3: 576.42907, found: 576.42697. Anal.
calcd. for C37H56N2O3: C, 77.04; H, 9.78; N, 4.86; O, 8.82 Found: C,
77.08; H, 9.75; N, 4.85; O, 8.84.
n
d
2 ꢃ eC HAXHEQ- piperazinyl), 2.85-2.96 (m, 2H, 2 ꢃ eC HAXHEQ
-
piperazinyl), 3.55e3.79 (m, 2H, H-3 and H-7), 5.30 (s, 2H, 2 ꢃ OH,
D2O exchange), 6.18 (br s, 1H, -NH, D2O exchange) ppm. 13C NMR
(100 MHz, CD3OD, 25 ꢀC):
d
¼ 12.7, 19.1, 22.4, 24.0, 24.3, 28.0, 29.7,
6.1.4.6. N-[4N-cinnamyllpiperazin-1-yl)-3a,7a-dihydroxy-5b-
cholan-24-amide (7c). Compound 7c have been prepared according
to a reported procedure [20].
31.0, 34.1, 35.2, 36.1, 36.3, 37.3, 38.0, 38.7, 40.8, 41.6, 44.1, 44.5, 44.8,
56.8, 57.6, 72.0 (C-7), 72.2 (C-3), 180.4 (C]O) ppm. (Carbon signals
were probably included in Methanol-d4 signal). MS (30 eV, EI): m/z
(%) ¼ 489.4 (83) [Mþ$], 474.3 (48) [Mþ$e CH3], 447 (18), 170 (100)
[Mþ$e StC21]. HRMS (EI) m/z [Mþ] calcd. for C29H51N3O3:
489.39301, found: 489.39199. Anal. calcd. for C29H51N3O3: C, 71.12;
H, 10.50; N, 8.58; O, 9.80 Found: C, 71.34; H, 10.69; N, 8.58; O, 9.84.
6.1.5. Hydrolytic stability of the bile acid derivatives
Bile acid derivative (5aec and 7aec, 50 mg) was dissolved in
ethanol (0.5 mL) and added to aqueous solution of phosphate 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 monitored by TLC on Kieselgel-G
(Merck Si 254 F) layers (0.25 mm).
6.1.4.3. N-(4N-methylpiperazin-1-yl)-3
24-amide (5c). 0.59 g (64 % yield) as a yellow amorphous solid. IR
(KBr): (
cmꢄ1): 3419 (br, OeH alcohol and NH amide), 2935e2865
(CeH alkane), 1662 (C]O amide). 1H NMR (400 MHz, CD3OD,
25 ꢀC):
a,7a-dihydroxy-5b-cholan-
n
d
¼ 0.68 (s, 3H, Me-18), 0.97 (s, 3H, Me-19), 1.01
6.2. Bioassays
(d, J ¼ 5.84 Hz, 3H, Me-21), 2.30 (s, 3H, NeCH3), 2.15e2.48 (m, 4H,
2 ꢃ eCH2- piperazinyl), 2.15e2.48 (m, 4H, 2 ꢃ -CH2- piperazinyl),
3.21e3.40 (m, 2H, H-3 and H-7), 3.78 (br s, 1H, eNH) ppm. 13C NMR
Evaluation of cytotoxic activities against glioblastoma multi-
forme (GBM), multiple myeloma (KMS-11) and colonic carcinoma
(HCT-116) cell lines, were realized according to the method detailed
before in our previous work [20].
(100 MHz, CD3OD, 25 ꢀC):
d
¼ 12.2 (Me-18), 18.9 (Me-21), 21.8, 23.4
(Me-19), 24.6, 29.3, 31.4, 32.3, 33.2, 34.0, 35.3, 35.9, 36.2, 36.6, 36.9,
40.5, 40.8, 41.1, 43.2, 43.7, 45.6, 51.5, 55.1, 55.3, 55.8, 56.3, 69.0 (C-7),
72.8 (C-3), 174.3 (C]O) ppm. MS (30 eV, EI): m/z (%) ¼ 489.4 (76)
[Mþ$], 474.3 (56), 447 (12), 170 (100) [Mþ$e StC21]. HRMS (EI) m/z
For DNA fragmentation analysis, KMS-11 controls and 7b treated
(20
mM e 48 h) cultures were treated according to Gentra-Puregen
Kit of Qiagen recommandations.

2918
D. Brossard et al. / European Journal of Medicinal Chemistry 45 (2010) 2912e2918
[7] J.H. Jeong, J.S. Park, B. Moon, M.C. Kim, J.K. Kim, S. Lee, H. Suh, N.D. Kim, J.
Percentage of apoptotic cells was determined after FITC-conju-
gated annexin V labeling (Beckman Coulter) on a FACSCalibur flow
cytometer (BD Biosciences).
Nuclei were visualized after Hoëchst (SigmaeAldrich, St.
Louis, MO) staining using a LEICA DMI 6000B microscope and
data were quantified using Metamorph software of ROPER
(PICell, Nantes).
M. Kim, Y.C. Park, Y.H. Yoo, Ann. N.Y. Acad. Sci. 1010 (2003) 171e177.
[8] S.B. Yee, W.J. Yeo, B.S. Park, J.Y. Kim, S.J. Baek, Y.C. Kim, S.Y. Seo, S.H. Lee, J.
H. Kim, H. Suh, N.D. Kim, Y.J. Lim, Y.H. Yoo, Int. J. Oncol. 27 (2005) 653e659.
[9] S.E. Park, H.J. Choi, S.B. Yee, H.Y. Chung, H. Suh, Y.H. Choi, Y.H. Yoo, N.D. Kim,
Int. J. Oncol. 25 (2004) 231e236.
[10] E.O. Im, Y.H. Choi, K.J. Paik, H. Suh, Y. Jin, K.W. Kim, Y.H. Yoo, N.D. Kim, Cancer
Lett. 163 (2001) 83e93.
[11] Y.H. Choi, E.O. Im, H. Suh, Y. Jin, W.H. Lee, Y.H. Yoo, K.W. Kim, N.D. Kim, Int. J.
Oncol. 18 (2001) 979e984.
[12] Y.H. Choi, E.O. Im, H. Suh, Y. Jin, Y.H. Yoo, N.D. Kim, Cancer Lett. 199 (2003)
157e167.
Acknowledgments
[13] E. Im, S.H. Choi, H. Suh, Y.H. Choi, Y.H. Yoo, N.D. Kim, Cancer Lett. 229 (2005)
49e57.
[14] Y.H. Park, J.A. Kim, J.H. Baek, E.J. Jung, T.H. Kim, H. Suh, M.H. Park, K.W. Kim,
Arch. Pharm. Res. 20 (1997) 29e33.
[15] S.E. Park, S.W. Lee, M.A. Hossain, M.Y. Kim, M.N. Kim, E.Y. Ahn, Y.C. Park,
H. Suh, G.Y. Kim, Y.H. Choi, N.D. Kim, Cancer Lett. 270 (2008) 77e86.
[16] S.B. Yee, Y.S. Song, S.H. Jeong, H.S. Lee, S.Y. Seo, J.H. Kim, H. Suh, N.D. Kim, Y.
H. Yoo, Oncol. Rep. 17 (2007) 919e923.
We gratefully acknowledge the financial support from the
« Conseil Régional de Basse-Normandie ». We also thank Sophie
Maïga, Catherine David, for their technical helps.
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