Synthesis and biological evaluation of bile carboxamide derivatives with pro-apoptotic effect on human colon adenocarcinoma cell lines

Article abstract of DOI:10.1016/j.ejmech.2014.07.080

We previously reported that the cinnamylpiperazinyl group in the side chain of the chenodeoxycholic acid showed apoptosis-inducing activity on multiple myeloma cancer cell line KMS-11. In the present study, we synthesized and tested the pro-apoptotic pote

Full text of DOI:10.1016/j.ejmech.2014.07.080

Accepted Manuscript
Synthesis and biological evaluation of bile carboxamide derivatives with pro-apoptotic
effect on human colon adenocarcinoma cell lines
Dominique Brossard, Mathilde Lechevrel, Laïla El Kihel, Céline Quesnelle, Mohamed
Khalid, Safa Moslemi, Jean-Marie Reimund
PII:
S0223-5234(14)00695-3
10.1016/j.ejmech.2014.07.080
EJMECH 7201
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 March 2014
Revised Date: 9 July 2014
Accepted Date: 23 July 2014
Please cite this article as: D. Brossard, M. Lechevrel, L. El Kihel, C. Quesnelle, M. Khalid, S. Moslemi,
J.-M. Reimund, Synthesis and biological evaluation of bile carboxamide derivatives with pro-apoptotic
effect on human colon adenocarcinoma cell lines, European Journal of Medicinal Chemistry (2014), doi:
10.1016/j.ejmech.2014.07.080.
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ACCEPTED MANUSCRIPT
Graphical abstract
Synthesis and biological evaluation of bile carboxamide derivatives with pro-
apoptotic effect on human colon adenocarcinoma cell lines.
Dominique Brossard, Mathilde Lechevrel, Laïla El Kihel, Céline Quesnelle, Mohamed
Khalid, Safa Moslemi, and Jean-Marie Reimund.
O
O
N
N
R2
H
OH
R2
H
aryl group
R1 = αOH, R2 = OH
H
H
R1
H
H
R1
R1 = βOH, R2 = H
R1 = αOH, R2 = H
HO
HO
H
H
R1 = H, R2 = OH
R1 = H, R2 = H

ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of bile carboxamide derivatives with pro-apoptotic
effect on human colon adenocarcinoma cell lines.
Dominique Brossard,a,b Mathilde Lechevrel,a,c Laïla El Kihel,a,b * Céline Quesnelle,a,c
Mohamed Khalid,d Safa Moslemi,a,c and Jean-Marie Reimund.a,c
aUniversité de Caen/Basse-Normandie, Esplanade de la Paix, 14032 Caen cedex, France.
bUFR des Sciences Pharmaceutiques, Centre d’Etudes et de Recherche sur le Médicament de
Normandie (CERMN), CNRS INC3M – SFR ICORE 146, Bd Becquerel, F-14032 Caen
cedex, France
cUFR de Médecine, Laboratoire Microenvironnement Cellulaire et Pathologies (MILPAT,
EA 4652), SFR ICORE 146, Avenue de la Côte de Nacre, 14032 Caen cedex, France.
dUniversité Hassan Premier, Faculté des Sciences et Techniques, Km 3, Route de Casablanca,
BP 577, 26000 Settat, Maroc.
Key words: bile carboxamide derivatives, colorectal cancer, cytotoxicity, apoptosis,
piperazinyl steroids.
Abstract.
We previously reported that the cinnamylpiperazinyl group in the side chain of the
chenodeoxycholic acid showed apoptosis-inducing activity on multiple myeloma cancer cell
line KMS-11. In the present study, we synthesized and tested the pro-apoptotic potency of
fifteen new piperazinyl bile carboxamide derived from cholic, ursodeoxycholic,
chenodeoxycholic, deoxycholic and lithocholic acids on human colon adenocarcinoma cell
lines DLD-1, HCT-116, and HT-29. Cell viability was first measured using XTT assay. The
most of the synthetic bile carboxamide derivatives decreased significantly cell viability in a
dose-dependent manner. HCT-116 and DLD-1 cell lines were more sensitive than HT-29 to
tested compounds. 9c, 9d showed the best in vitro results in term of solubility and dose-
response effect on the three colon adenocarcinoma cell lines. Additionally, flow cytometric
* Corresponding Author Tel: 00 33 (0) 2 31 56 68 12; Fax: 00 33 (0) 2 31 56 68 03, E-mail
address: laila.elkihel@unicaen.fr.
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and Western-blotting analysis showed that 9c induced pro-apoptosis in DLD-1 and HCT-116
whereas 9d did not. We conclude that the benzyl group improved anti-proliferative activity
and that the α-hydroxyl group was found to be more beneficial at the 7-position in steroid
skeleton.
1. Introduction
In Western countries, colorectal cancer (CRC) is a major health care problem. In France,
despite nationwide screening campaigns and therapeutic advances, the 5-year overall survival
rate remains limited to 40-60% [1].
Currently, colon cancer treatment strategy is well defined and primarily depends on colon
cancer extension (i.e. Stage). Usually, if possible, the treatment remains primarily surgical
through standard open surgery, but the use of minimally invasive techniques in accordance
with the principles recommended oncological surgery of the colon is increasingly sought [2].
Following surgery, additional chemotherapy may be necessary; in patients with lymph node
metastasis and/or with distant metastasis affecting other organs (the most frequently
concerned being the liver). This chemotherapy consists, in the first line, of combinations of 5-
flurouracil (5-FU), leucovorin, and oxaliplatin (the most used in patients only presenting
lymph node as adjuvant chemotherapy named FOLFOX),or 5-FU, leucovorin and irinotecan
(FOLFIRI), all of which have similar effects but exhibit different side effects [3]. In some
cases, resection of liver metastasis could be performed. Radiotherapy is only considered in
rectal (but not colon) cancer. In patients failing to have a progression-free (usually due to
resistance to one or several of the used drugs) [4], or presenting adverse events not
authorizing to continue the used chemotherapy regimen, the treatment is modified [3]. In
order to have an idea of the efficacy of these regimens, the best results obtained in metastatic
colon cancer have been reported using FOLFOX with a time to progression of 8.7 months,
response rate of 45% and median survival of 19.5 months [5-7].
Biotherapy using monoclonal antibodies directed against Epidermal Growth Factor Receptor
(EGFR) (cetuximab) or Vascular Endothelial Growth Factor (VEGF) (bevacizumab) remains
currently a second or third line treatment. For example, the combination of bevacizumab and
FOLFIRI prolonged survival from 15 months to 20 months [8-11]. Despite the recent targeted
therapies the improved overall survival in some patients, is still insufficient; so, improving
CRC treatment remains a key challenge for the future.
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Taurine (and glycine)-conjugated bile acids and bile alcohol sulfates, the functional end
products of cholesterol metabolism, continue to be of interest because of newly discovered
physiological functions in the small [12-13] and large [14] intestine, as well as recently
discovered regulatory properties [15].
Indeed, it has been demonstrated that bile salts have various biologic effects under
physiologic conditions besides their essential role in the digestion of lipids. For example,
Taurodeoxycholate (TDCA) has been shown to increase esophageal mucosal growth in an
explant model [16]. Another study has been demonstrated that the bile salt deoxycholate
(DCA) was able to modulate p53 gene expression [17]. Furthermore, TDCA has also been
shown to increase intestinal epithelial cell migration by increasing TGF-beta expression, and
that the transcription factor NF-қB regulates TDCA induced cell migration after injury [18].
Recent studies showed that a co-treatment of celecoxib and tauro-ursodeoxycholic acid
(TUDCA) could inhibit cell growth in familial adenomatous polyposis derived LT97colon
adenoma cells [19]. TUDCA is well characterized as a potent inhibitor of apoptosis in
different cell types and its effects has expanded to several experimental models of
neurological disorders, including Alzheimer disease [20]. More recent studies showed that
tauro-chenodeoxycholic acid has an effect in preventing pulmonary fibrosis in mice [21], and
to induce apoptosis of fibroblast-like synoviocytes [22].
By analogy of these natural conjugated bile acids, a few synthetic bile acid analogues have
been described as antitumoral agents against several cancers including stomach [23],
glioblastoma multiforme [24], colon [25-26], breast [27], leukemic T [28], prostate [29],
cervical [30], and hepatocellular cancer cells [31].
In the same way, our group had recently reported the synthesis of bile acid derivatives and
their in vitro evaluation against various human cancer cells such as glioblastoma multiforme
(GBM), multiple myeloma (KMS-11), and colon carcinoma (HCT-116) cell lines. The best
activity was obtained with trans-cinnamylchenodeoxycholic derivative (N-[4N-trans-
cinnamyl(piperazin-1-yl)]-3α,7α-dihydroxy-5β-cholan-24-amide, compound I (Figure 1)) on
KMS-11. This activity was associated with apoptotic mechanism characterized by Mcl-1 and
poly (ADP-ribose) polymerase (PARP-1) cleavage, inhibition of NFқB signaling and DNA
fragmentation [32-33]. In the present investigation, we continue the effort to develop novel
piperazine bile acids endowed with better pharmacological profiles and elucidate the
antitumor structure-activity relationships. We report here the synthesis, in vitro evaluation of
the pro-apoptotic mechanism and structure-activity relationship study for the piperazine with
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various arylated alkyl substituents using as a building block with bile carboxamide
derivatives. Their cell death-inducing activity was analysed in three colon cell lines including
DLD-1, HCT-116, and HT-29. As expected with compound I (used here as positive control),
some synthetic derivatives induced cell death with more efficiency. A structure-cell death
relationship among this new series of synthetic bile carboxamide derivatives was established.
This work is the continuation of our previous study [32,33].
Figure 1.
2. Results and discussion
2.1 Chemistry
This methodology have already been reported by as [32,33] and briefly modified : Treatment
of bile acids such as cholic (1a), ursodeoxycholic (1b), chenodeoxycholic (1c), deoxycholic
(1d) and lithocholic (1e) acids with acetic anhydride in the presence of 4,4-
dimethylaminopyridine (DMAP) in anhydrous dichloromethane afforded the corresponding
ester (2a-e). The latter acids were converted into the corresponding mixed anhydride by
treatment with triethylamine followed by ethyl chloroformate giving 3a-e, respectively. When
the mixed anhydrides 3a-e were reacted with substituted piperazines (4-
chlorobenzhydrylpiperazine (a), 4,4'-Difluorobenzhydrylpiperazine (b), or 1-benzylpiperazine
(c), the products were identified as the carboxamides (4a-e – 6a-e). The latter amides were
saponified by an aqueous solution of potassium hydroxide in ethanol to give the
corresponding deacetylated amides 7a-e – 9a-e. However, during purification of the resulting
amides on silica gel, they were strongly adsorbed on the carrier material, thus they could be
successfully purified on alumina deactivated with 6% water. Compounds 7a-e– 9a-e were
characterized by IR, 1HNMR, I3CNMR and HRMS, respectively.
1
The H-NMR and 13C-NMR spectra of 7a-e – 9a-e series were the basis of their structure
1
elucidation. For 7a-e series, we choose 7a compound for the descriptive example. The H-
NMR spectrum of compound 7a showed the expected signals for steroid moiety signals at m
3.5 (C3-H), m 3.83 (C7-H), and m 4.13 (C12-H). The presence of signals at 2.20-2.37 (CH2-
piperazinyl), m 7.25-7.28 (Har) and m 7.30-7.37 (Har), and signals at 172.2 (O=C24-NH),
142.0 (C1, Ph), 141 (C1, (PhCl), and 75.2 (-CHPhPhCl) in the 13CNMR confirmed the
1
formation of piperazinylsteroid carboxamide. The H-NMR spectrum of 8a showed the
expected signals for the steroid moiety signals at m 3.59 (C3-H), m 3.81 (C7-H) and m 3.93
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(C12-H). The presence of signals at 2.31-2.35 and 3.43-3.47 (CH2-piperazinyl), t 7.34 (Har)
and t 6.98 (Har), and signals at 172.3 (O=C24-NH), 161.9 (2 x C4-F, Ar), 137.6 (2 x C1, Ar)
1
and 74.2 (CH-(PhF)2) in the 13CNMR confirmed the formation of compound 8a. The H-
NMR spectrum of 9a showed the expected signals for the steroid moiety signals at m 3.61
(C3-H), m 3.82 (C7-H) and m 3.95 (C12-H). The presence of signals at 2.40-2.44 and 3.43-
3.48 (CH2-piperazinyl), s 3.52 (CH2-benzyl), m 7.30-7.33 (Har), and signals at 172.4
(O=C24-NH), 137.5 (C1- Ar), 129.1 (C3 and C5-Ar), 128.3 (C2 and C6-Ar), 127.2 (C4-Ar),
and 62.8 (CH2-Ph) in the 13CNMR confirmed the formation of compound 9a.
Scheme 1.
2.2 Biology
The effect of bile carboxamide derivatives 7a-e – 9a-e against a panel of colon cell lines
(HCT-116, DLD-1 and HT-29) was investigated and compared with the reference compound
I described by us in the previous studies [32-33]. The colon cell line panel consisted of colon
carcinoma DLD-1, HCT-116, and HT-29.
Compounds 7e, 8b, 8d and 9b were converted into their corresponding hydrochloride salts
because they have low solubility in the dimethyl sulfoxide (DMSO). However, precipitation
of these compounds in culture medium was observed. So, the investigations could not be
achieved.
2.2.1. Anti-proliferative evaluation of the synthetic bile carboxamide derivatives on
DLD-1, HCT-116 and HT-29
The first approach of the structure-activity relationships concerned a general comparison of
the anti-proliferative activities among the synthesized three classes of bile acid piperazine
derivatives. The effects of bile acid derivatives on tumour cell viability were determined by
XTT assays after 24 and 48 h incubation with or without each tested compound at four
concentrations (20, 30, 40 and 50 ꢀM). No significant difference of activity was observed
between 24 and 48 h of treatment as determined by the student test.
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The inhibitory effects of these compounds on cell viability were evaluated after 24 h
incubation and compared to that of compound I, and correlation tests were used to identify the
best anticancer compounds. A representative experiment out of three is given in Figure 2.
Figure 2-3
The most of the synthetic bile carboxamide derivatives, except for 7b, 7d, 8c and 9e,
decreased significantly cell viability in a dose-dependent manner as well as compound I used
as positive control (Figure 2). However, some of them, such as 7a, 7c, 8a, have displayed
brutal cytotoxic responses namely on DLD-1 and HCT-116 even at low doses making
difficult the determination of their graphical IC50 values. This kind of compounds may not be
good candidates for a further evaluation (for instance; in vivo evaluation) because of the
difficulty to determine the optimal dose to avoid any non specific or side effects. In contrast,
the responses obtained with 9c and 9d were promising as illustrated by dose-response curves
and IC50 determination, even with HT-29 which generally was more resistant (Figure 3). The
IC50 values of compound I, 9c and 9d, determined graphically from dose-response curves of
Figure 3, were respectively 36, 33 and 26 µM for DLD-1, 34, 23 and 22 µM for HCT-116,
and 35, 35 and 32 µM for HT-29 (Table 1). Moreover, research cell response remains useful
to study an induction of apoptotic pathways.
Table 1
On the basis of these results it can be noted that N-substitution by diphenylmethyl group on
piperazine in the series 7a-e and 8a-e did not give any further advantages, neither for
cytotoxic effects nor for solubility (as mentioned above). On the other hand, introduction of
benzyl group to piperazinyl ring improved the cytotoxicity of 9c and 9d as compared to
compound I (Figure 2) especially in HCT-116; for instance, IC50 of 23 and 22 µM for each 9c
and 9d against 35 µM for compound I (Table 1).
These results showed that 9c and 9d were more active towards DLD-1 and HCT-116 cell lines
whereas HT-29 appeared more resistant to these compounds. Since 9c and 9d have exhibited
the best in vitro results in term of solubility and dose-response toxicities, we therefore focused,
for the further experiments, only on 9c, 9d and I as positive control.
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2.2.2. Cell cycle modulation and induction of apoptosis by the synthetic bile
carboxamide derivatives
Compounds 9c and 9d, altering cell growth, led us to examine the mechanism behind this
activity. In our previous study on cancer myeloma cell line [32-33], we demonstrated that
compound I induced cell death by apoptosis. So, we firstly investigated whether 9c, 9d and I
compounds had anti-proliferative and pro-apoptotic effects on the three cell lines studied in
this work by flow cytometry after 24h treatment with 30 ꢀM of each compound and staining
with propidium iodide (PI) which is a popular red-fluorescent nuclear and chromosome
counter stain. Since PI is impermeant to live cells, it is also commonly used to evaluate cell
viability and DNA content in cell cycle analysis.
Figure 4
As shown in Figure 4 and table 2, an arrest of cell cycle progression at the G0-G1 phase was
observed for 9c, 9d and I compounds on the three cell lines. This effect was observed even
with lower dose of 20 ꢀM (Data not shown). These cells have also displayed a marked
accumulation of G1 subfraction and a decrease of the percentage of cells blocked in their
G2/M phase. This profile was particularly observed for compound I (respectively for sub-G1
and G2-M populations; 49.77% and 6.22% in DLD-1, 31.23% and 11.34% in HCT-116,
48.38% and 4.77% in HT-29) and was less pronounced for 9c (respectively for sub-G1 and
G2-M populations; 10.67% and 32.1% in DLD-1, 12.98% and 26.64% in HCT-116, 18.13%
and 13.63% in HT-29) and 9d (respectively for sub-G1 and G2-M populations; 14.28% and
38.97% in DLD-1, 14.85% and 28.89% in HCT-116, 34.81% and 11.14% in HT-29) as
compared with DMSO treated cells (Table 2).
Table 2
To confirm the sub-G1 peak, which represents the apoptotic cell population we further
analysed APO2.7 positive cells treated with 30 ꢀM of each compound 9c, 9d and I.
APO 2.7 reacts with a 38-kDa protein that is exposed on the outer mitochondrial membranes
of cells undergoing apoptosis and used a marker for detection of early apoptotic events. As
shown in Figure 5, the percentages of apoptotic cells in DLD-1 and HCT-116 were higher
after 24 h treatment with compound I (34.87 in DLD-1, 37.73% in HCT-116) and 9c (22.54,
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20.33%) than with 9d (2.49, 4.59%). As expected for HT-29, the rates of apoptotic cells were
very low: 8.79 % with compound I, 8.57 % with 9c and 1.26% with 9d, close to the DMSO-
treatment (1.11%, Table 3).
Figure 5
Table 3
These results, showing an early stage of apoptosis, were in accordance with those previously
published. Indeed, it has been reported that synthetic or natural bile acids interfered with the
cell cycle and that the most of them induced cell death by apoptotic mechanism [22, 34-35].
We then further explored apoptosis induction by 9c, 9d and I in DLD-1, HCT-116 and HT-29
cell lines by Western-blotting of Poly (ADP-ribose) polymerase (PARP) cleavage. PARP is a
nuclear enzyme of 116 kD involved in DNA repair and its cleavage by caspase-3 is admitted
to be a hallmark of late stage of cell death. As shown in Figure 6, an inactivation of PARP
was observed by the appearance of 89 kD fragment with all cell lines incubated 24h in the
presence of 30 ꢀM of each compound 9c, 9d and I. However, when the data obtained by flow
cytometry of APO2.7 staining (Figure 5 and Table3) are compared with those of Western-
blotting (Figure 6), we can consider that I and 9c act as apoptosis inducers in DLD-1 and
HCT-116 whereas 9d does not. Furthermore, programmed cell death or apoptosis seems to
not be involved in HT-29. The different responses to 9d treatment could be related to the
position of a hydroxyl group. Indeed the only molecular difference between 9c and 9d is the
position of hydroxyl group at C7 and C12 steroid skeleton respectively (Figure 1).
Figure 6
In this study, HT-29 has always responded differently compared to the other cell lines
whatever the treatment used. Several hypotheses could explain this difference of sensitivity:
(i) The involvement of various genetic background may explain this difference. For examples:
it is known that tumour suppressor gene TP53 is mutated in HT-29 but not in DLD-1 and
HCT-116 cells [36] making the first one more resistant to programmed cell death.
Additionally, the expression profiling of xenobiotic-metabolizing enzymes and drug efflux
transporters in HT-29 might be different from that in DLD-1 and HCT-116 [37].
(ii) Time- and dose-treatments used in this work were insufficient to HT-29 to elicit a
response; we kept the dose of 30 ꢀM to study apoptosis and cycle cell because it was close to
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the IC50 values defined for 9c, 9d and I in the three human colon cancer cell lines. However,
Park et al. showed an apoptosis induction of other synthetic bile acid derivates in HT-29 at
doses higher than 40 ꢀM [24]. Our results are consistent with those of Martinez et al. [35]
who reported that DLD-1 and HCT-116 cells responded to drug treatments more rapidly than
HT-29 cells which displays generally a slower cell cycle time.
(iii) Finally, survival and/or cell death signalling pathways could be different between colon
cancer cell lines [31,36]; another cell death pathway than apoptosis could be induced in HT-
29 following the cleavage and inactivation of PARP (Figure 6). PARP cleavage and
inactivation, known as late stage of cell death following DNA damage, appears to be involved,
besides apoptosis, in several cell death pathways such as autophagy in response to exogenous
stress [38].
3. Conclusion
In conclusion, new fifteen piperazinyl bile acid derived from cholic, ursodeoxycholic,
chenodeoxycholic, deoxycholic and lithocholic acids were synthesized and tested on human
colon adenocarcinoma cell lines HT-29, HCT-116, and DLD-1.
The most of the synthetic bile acid derivatives decreased significantly cell viability
significantly in a concentration dependent manner. HCT-116 and DLD-1 cell lines were more
sensitive than HT-29 to different treatments. Although it has been observed a low different
response for these cell lines, it can be noted that 9c and 9d provided the best in vitro results in
term of the solubility and dose-response effect on DLD-1 and HCT-116 cell lines, and are
more promising than compound I. Additionally, flow cytometric and Western-blotting
analysis showed that 9c induced apoptosis in DLD-1 and HCT-116 whereas 9d did not.
Benzyl group improve the apoptotic activity and the α-hydroxyl group was found to be more
beneficial at the 7-position in steroid skeleton. These results suggested that this group of
compounds can be good candidates for a further in vivo evaluation and explored for
developing potent anticancer drugs.
4. Experimental section
4.1. Chemistry
All solvents were distilled prior to use. Reagents and materials were obtained from
commercial suppliers and were used without further purification. The reactions were
monitored by TLC on Kieselgel-G (Merck Si 254 F) layers (0.25 mm thick). The spots were
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detected using a UV lamp (254 nm) and by spraying with sulfuric acid/ethanol (2:8) on TLC
and subsequent heating. Column chromatography was carried out using silica gel 60 (0.063–
0.2 mm) (Merck). Melting points were determined on a Kofler block. IR spectra were
recorded on a Perkin-Elmer BX FT-IR spectrometer. HRMS were recorded on a Jeol-GCmate
(GC-MS system) spectrometer with ionisation energy from 30 to 40 eV. MS detection was
performed by positive or negative ESI.1H NMR and 13C NMR spectra were recorded
respectively, at 400 MHz and at 100 MHz (Jeol Lambda 400 spectrometer) using CDCl3 as
solvent. Chemical shifts are reported relative to TMS; J values are given in Hz. 13C NMR
spectra are 1H-decoupled.
4.1.1. General procedure for 2a-2e: Acetic anhydride (26.43 mmol, 2.5 mL) was added
dropwise to a solution of 1a (7.34 mmol) and DMAP (1.47 mmol, 180 mg) in CH2Cl2 (30
mL). The reaction mixture was stirred at room temperature under nitrogen atmosphere for 1 h.
The mixture was then washed with HCl 1N aqueous solution (3×30 mL), 5% NaHCO3
solution (3×30 mL), saturated NaCl solution (3×20 mL) and water (1×20 mL). The organic
layer was dried over MgSO4 and concentrated. The crude product was purified by column
chromatography (silica gel, cyclohexane/ethyl acetate, 8:2) to afford 2a.
4.1.1.1. 3α-,7α-,12α-triacetoxy-5β-cholan-24-oic acid (2a)
White powder (3.27, 83 %) was obtained. C30H46O8. IR (KBr) υ: 3404 (C-OH acid), 2950-
2869 (CH alkane), 1736 (C=O ester), 1709 (C=O carboxylic acid) cm-1. 1H NMR (400 MHz,
CDCl3) δ (ppm): 4.89 (m, 1H, C12-H), 4.58 (m, 1H, C7-H), 3.86 (m, 1H, C3-H), 2.22 (s, 3H,
-OCOCH3), 2.07 (s, 3H, -OCOCH3), 2.03 (s, 3H, -OCOCH3), 0.99 (d, 3H, J = 5.8 Hz, 21-Me),
0.93 (s, 3H, 19-Me), 0.69 (s, 3H, 18-Me). 13C NMR (100 MHz, CDCl3) δ (ppm): 179.6, 170.6,
170.5, 170.4, 75.3, 74.1, 70.7, 56.4, 55.9, 42.7, 41.8, 40.3, 35.7, 35.3, 35.0, 34.5, 32.2, 30.8,
28.1, 26.9, 26.5, 26.3, 24.1, 23.3, 21.6, 21.5, 21.4, 20.8, 18.2, 12.0. MS (30eV, IE): m/z
(r.i. %) = 534.7 (3) [M+.], 358.4 (100) [M+.-CH3CO2H], 343.4 (12), 304.3 (6), 257.3 (9), 230.3
(21), 215.3 (33).
4.1.1.2. 3α-,7β-diacetoxy-5β-cholan-24-oic acid (2b)
White powder (3.32 g, 95 %) was obtained. C28H44O6. IR (KBr) υ: 3404 (C-OH acid), 2950-
2869 (CH alkane), 1736 (C=O ester), 1709 (C=O carboxylic acid) cm-1. 1H NMR (400 MHz,
CDCl3) δ (ppm): 4.71 (m, 1H, C7-H), 3.99 (m, 1H, C3-H), 2.02 (s, 6H, 2×-OCOCH3), 0.98 (d,
3H, J = 5.8 Hz, 21-Me), 0.92 (s, 3H, 19-Me), 0.69 (s, 3H, 18-Me). 13C NMR (100 MHz,
CDCl3) δ (ppm): 179.6, 170.6, 170.4, 75.3, 70.7, 56.4, 55.9, 42.7, 41.8, 40.7, 40.3, 35.7, 35.3,
35.0, 34.5, 32.2, 30.8, 28.1, 26.9, 26.5, 26.3, 24.1, 23.3, 21.6, 21.4, 20.8, 18.2, 12.0. MS
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(30eV, IE): m/z (r.i. %) = 476.3 (1) [M+.], 434.3 (3), 416.3 (54.2) [M+.-CH3CO2H], 374.3 (7),
356.3 (100) [M+.-2×CH3CO2H], 341.2 (33), 302.2 (10), 255.2 (22), 228.2 (38), 213.2 (28).
4.1.1.3. 3α-,7α-diacetoxy-5β-cholan-24-oic acid (2c)
White powder (3.30 g, 94 %). C28H44O6. IR (KBr) υ: 3404 (C-OH acid), 2950-2869 (CH
1
alkane), 1736 (C=O ester), 1709 (C=O carboxylic acid) cm-1. H NMR (400 MHz, CDCl3) δ
(ppm): 4.57 (m, 1H, C7-H), 3.85 (m, 1H, C3-H), 2.22 (s, 3H, -OCOCH3), 2.02 (s, 3H, -
OCOCH3), 0.94 (d, 3H, J = 6.8 Hz, 21-Me), 0.92 (s, 3H, 19-Me), 0.66 (s, 3H, 18-Me). 13C
NMR (100 MHz, CDCl3) δ (ppm): 179.6, 170.6, 170.4, 75.3, 70.7, 56.4, 55.9, 42.7, 41.8,
40.7, 40.3, 35.7, 35.3, 35.0, 34.5, 32.2, 30.8, 28.1, 26.9, 26.5, 26.3, 24.1, 23.3, 21.6, 21.4,
20.8, 18.2, 12.0. MS (30eV, IE): m/z (r. i. %) = 476.3 (1) [M+.], 434.3 (3), 416.3 (62.8) [M+.-
CH3CO2H], 374.3 (7), 356.3 (100) [M+.-2×CH3CO2H], 341.2 (33), 302.2 (10), 255.2 (22),
228.2 (38), 213.2 (28).
4.1.1.4. 3α-,12α-diacetoxy-5β-cholan-24-oic (2d)
Amorphous white solid (3.30 g, 94 %) was obtained. C28H44O6. IR (KBr) υ: 3404 (C-OH
1
acid), 2950-2869 (CH alkane), 1736 (C=O ester), 1709 (C=O carboxylic acid) cm-1. H NMR
(400 MHz, CDCl3) δ (ppm): 4.71 (m, 1H, C12-H), 3.99 (m, 1H, C3-H), 2.02 (s, 6H, 2×-
OCOCH3), 0.98 (d, 3H, J = 5.8 Hz, 21-Me), 0.92 (s, 3H, 19-Me), 0.69 (s, 3H, 18-Me). 13C
NMR (100 MHz, CDCl3) δ (ppm): 179.6, 170.6, 170.4, 75.3, 70.7, 56.4, 55.9, 42.7, 41.8,
40.3, 35.7, 35.3, 35.0, 34.5, 32.6, 32.2, 30.8, 28.1, 26.9, 26.5, 26.3, 24.1, 23.3, 21.6, 21.4,
20.8, 18.2, 12.0. MS (30eV, IE) m/z (r.i. %) = 476.3 (1) [M+.], 434.3 (3), 416.3 (61.1) [M+.-
CH3CO2H], 374.3 (7), 356.3 (100) [M+.-2×CH3CO2H], 341.2 (33), 302.2 (10), 255.2 (22),
228.2 (38), 213.2 (28).
4.1.1.5. 3α-acetoxy-5β-cholan-24-oic (2e)
White powder (3.00 g, 98 %) was obtained. C26H42O4. IR (KBr) υ: 3404 (C-OH acid), 2950-
2869 (CH alkane), 1736 (C=O ester), 1709 (C=O carboxylic acid) cm-1. 1H NMR (400 MHz,
CDCl3) δ (ppm): 4.72 (m, 1H, C3H), 2.03 (s, 3H, -OCOCH3), 0.93 (s, 3H, 19-Me), 0.92 (d, 3H,
J = 5.4 Hz, 21-Me), 0.65 (s, 3H, 18-Me). 13C NMR (100 MHz, CDCl3) δ (ppm): 180.2, 170.7,
74.4, 56.4, 55.9, 42.7, 41.8, 40.3, 40.1, 35.7, 35.3, 35.0, 34.5, 32.2, 31.2, 30.8, 28.1, 26.9, 26.5,
26.3, 24.1, 23.3, 21.4, 20.8, 18.2, 12.0. MS (30eV, IE) m/z (r.i. %) = 418.5 (3) [M+.], 358.4
(100) [M+.-CH3CO2H], 343.4 (12), 304.3 (6), 257.3 (9), 230.3 (21), 215.3 (33).
4.1.2. General procedure for compounds 7a-7e, 8a-8e and 9a-9e: Ethyl chloroformate (2
equivalents) was added to a stirred solution of acid 2a-2e (1 equivalent) in anhydrous CH2Cl2
11

ACCEPTED MANUSCRIPT
(5 mmol in 20 mL) under nitrogen atmosphere at room temperature. Triethylamine (2
equivalents) was added dropwise and the solution was stirred for 1 h. The reaction mixture
was diluted with CH2Cl2, washed with water (3 × 20 mL), dried over MgSO4 and
concentrated. The crude product of anhydride (3a-3e) was used to next step without
purification.
Under nitrogen atmosphere, piperazine derivative (a, b or c) (1.5 equivalents) and
triethylamine (2 equivalents) were added to a solution of mixed anhydride 3a-3e (1
equivalent) in anhydrous CH2Cl2. The reaction mixture was stirred at room temperature for 16
h. The solution was diluted with CH2Cl2, washed with water (3 × 20 mL), dried over MgSO4
and evaporated. The crude product (4a-e, 5a-e and 6a-e) was used to the next step without
purification.
An aqueous solution of potassium hydroxide (8 equivalents) was added to a stirred solution of
intermediate compound (4a-e, 5a-e and 6a-e, 1 equivalent) in EtOH (8 mmol in 20 mL). The
mixture was refluxing for 16 h. Aqueous 1 N HCl was added to neutral pH and the solution
was evaporated. The residue was dissolved in CH2Cl2, washed with 5% NaHCO3 (3 × 50 mL),
water (3 × 20 mL), dried over MgSO4 and concentrated. The crude product was purified by
column chromatography on deactivated basic alumina (6% water).
4.1.2.1. N-(4N-[(4-chlorophenyl)phenylmethyl]piperazin-1-yl)-3α,7α,12α-tri-hydroxy-5β-
cholan-24-amide (7a)
1.2 g (24% yield) as a beige amorphous solid. IR (KBr) υ: 3400 (br, C-OH alcohol), 2932-
1
2810 (CH alkane), 1626 (C=O amide), 1488 (C=C aromatic) cm-1. H NMR (400MHz,
CDCl3) δ (ppm): 7.37-7.30 (m, 4H, Har), 7.28-7.25 (m, 5H, Har), 4.19 (s, 1H, -CHPhPhCl),
4.13 (m, 1H, C12-H), 3.83 (m, 1H, C7-H), 3.50 (m, 1H, C3-H), 2.37-2.30 (m, 4H, 2×-CH2-
piperazinyl), 2.25-2.20 (m, 4H, 2×-CH2-piperazinyl), 0.97 (d, 3H, J = 4.8 Hz, 21-Me), 0.88 (s,
3H, 19-Me), 0.66 (s, 3H, 18-Me). 13C NMR(100MHz, CDCl3) δ (ppm): 172.2 (O=C-NH),
142.0 (C1Ar, Ph), 141 (C1Ar, PhCl), 132.5 (C4-Cl, PhCl)), 129.2 (C3Ar and C5Ar, PhCl),
128.7 (C2Ar and C6Ar, PhCl), 128.6 (C3Ar and C5Ar, Ph), 127.9 (C4Ar, Ph), 127.2 (C2Ar and
C6Ar, Ph), 75.9 (C12), 75.2 (CPhPhCl), 71.9 (C3), 68.8 (C7), 53.0, 52.1, 51.6, 46.8, 46.4,
45.8, 41.7, 39.5, 35.5, 35.3, 34.7, 31.4, 30.5, 30.1, 28.2, 27.6, 26.4, 23.2, 22.5, 21.1 (Me-19),
17.5 (Me-21), 12.5 (Me-18). HRMS (ESI) m/z [M+H]+ : calcd for C41H58N2O4Cl: 677.4085,
found: 677.4106.
12

ACCEPTED MANUSCRIPT
4.1.2.2.
N-(4N-[(4-chlorophenyl)phenylmethyl]piperazin-1-yl)-3α,7β-di-hydroxy-5β-
cholan-24-amide (7b)
Beige amorphous solid (1.4 g, 28%) was obtained. IR (KBr) υ: 3415 (br, C-OH alcohol),
1
2930-2862 (CH alkane), 1629 (C=O amide), 1489 (C=C aromatic) cm-1. H NMR (400MHz,
CDCl3) δ (ppm): 7.37-7.35 (m, 4H, Har), 7.30-7.19 (m, 5H, Har), 4.21 (s, 1H, -CHPhPhCl),
3.59-3.55 (m, 4H, 1×-CH2-piperazinyl, C7-H and C3-H), 3.46-3.44 (m, 2H, 1×-CH2-
piperazinyl), 2.36-2.31 (m, 4H, 2×-CH2-piperazinyl), 0.93 (d, 3H, J = 5.8 Hz, 21-Me), 0.91 (s,
3H, 19-Me), 0.66 (s, 3H, 18-Me). 13C NMR (100MHz, CDCl3) δ (ppm): 172.0 (O=C-NH),
141.6 (C1Ar, Ph), 140.7 (C1Ar, PhCl), 132.7 (C4-Cl, PhCl)), 129.0 (C3Ar and C5Ar, PhCl),
128.7 (C2Ar and C6Ar, PhCl), 128.6 (C3Ar and C5Ar, Ph), 127.7 (C4Ar, Ph), 127.3 (C2Ar and
C6Ar, Ph), 75.1 (CPhPhCl), 71.2 (C3), 68.7 (C7), 55.6, 54.8,51.9, 51.5, 45.7, 43.6, 42.3, 41.5,
40.0, 39.1, 37.2, 36.8, 35.5, 34.8, 34.0, 31.4, 30.2, 28.6, 26.8, 23.3 (Me-19), 21.1, 18.5 (Me-
21), 12.1 (Me-18). HRMS (ESI) m/z [M+H]+: calcd for C41H58N2O3Cl: 661.4136, found:
661.4144.
4.1.2.3.
N-(4N-[(4-chlorophenyl)phenylmethyl]piperazin-1-yl)-3α,7α-di-hydroxy-5β-
cholan-24-amide (7c)
0.70 g (14% yield) as an orange amorphous solid. IR (KBr) υ: 3426 (br, C-OH alcohol), 2929-
1
2865 (CH alkane), 1627 (C=O amide), 1488 (C=C aromatic) cm-1. H NMR (400MHz,
CDCl3) δ (ppm): 7.37-7.35 (m, 4H, Har), 7.30-7.18 (m, 5H, Har), 4.19 (s, 1H, -CHPhPhCl),
3.83 (m, 1H, C7-H), 3.60 (m, 1H, C3-H), 3.45-3.42 (m, 4H, 2×-CH2-piperazinyl), 2.35-2.32
(m, 4H, 2×-CH2-piperazinyl), 0.91 (d, 3H, J = 6.8 Hz, 21-Me), 0.90 (s, 3H, 19-Me), 0.64 (s,
3H, 18-Me). 13C NMR (100MHz, CDCl3) δ (ppm): 172.0 (O=C-NH), 142.0 (C1Ar, Ph), 141.2
(C1Ar, PhCl), 132.7 (C4-Cl, PhCl)), 129.2 (C3Ar and C5Ar, PhCl), 128.7 (C2Ar and C6Ar,
PhCl), 128.5 (C3Ar and C5Ar, Ph), 127.8 (C4Ar, Ph), 127.1 (C2Ar and C6Ar, Ph), 75.2
(CPhPhCl), 71.3 (C3), 68.9 (C7), 55.7, 53.0, 52.0, 50.4, 46.1, 45.7, 42.6, 41.4, 39.8, 39.6,
39.3, 35.5, 35.0, 32.8, 31.4, 30.6, 30.1, 28.2, 23.7, 22.7 (Me-19), 20.5, 18.4 (Me-21), 11.7
(Me-18). HRMS (ESI) m/z [M+H]+: calcd for C41H58N2O3Cl: 661.4136, found: 661.4160.
4.1.2.4.
N-(4N-[(4-chlorophenyl)phenylmethyl]piperazin-1-yl)-3α,12α-di-hydroxy-5β-
cholan-24-amide (7d)
1.1 g (23% yield) as a beige amorphous solid. IR (KBr) υ: 3430 (br, C-OH alcohol), 2934-
1
2861 (CH alkane), 1630 (C=O amide), 1489 (C=C aromatic) cm-1. H NMR (400MHz,
CDCl3) δ (ppm): 7.38-7.35 (m, 4H, Har), 7.31-7.19 (m, 5H, Har), 4.21 (s, 1H, -CHPhPhCl),
3.96 (m, 1H, C12-H), 3.63-3.58 (m, 3H, 1×-CH2-piperazinyl and C3-H), 3.46-3.42 (m, 2H,
13

ACCEPTED MANUSCRIPT
1×-CH2-piperazinyl), 2.36-2.32 (m, 4H, 2×-CH2-piperazinyl), 0.96 (d, 3H, J = 6.8 Hz, 21-Me),
0.90 (s, 3H, 19-Me), 0.66 (s, 3H, 18-Me). 13C NMR (100MHz, CDCl3) δ (ppm): 172.0 (O=C-
NH), 141.6 (C1Ar, Ph), 140.7 (C1Ar, PhCl), 132.7 (C4-Cl, PhCl)), 129.1 (C3Ar and C5Ar,
PhCl), 128.8 (C2Ar and C6Ar, PhCl), 128.7 (C3Ar and C5Ar, Ph), 127.7 (C4Ar, Ph), 127.4
(C2Ar and C6Ar, Ph), 75.2 (C12), 73.3 (C-PhPhCl), 71.7 (C3), 52.0, 51.5, 48.2, 47.1, 46.4,
45.7, 42.0, 41.6, 36.3, 35.9, 35.3, 35.1, 34.1, 33.6, 31.3, 30.4, 30.1, 28.6, 27.5, 27.1, 26.1, 23.6,
23.1 (Me-19), 17.4 (Me-21), 12.7 (Me-18). HRMS (ESI) m/z [M+H]+: calcd for
C41H58N2O3Cl: 661.4136, found: 661.4140.
4.1.2.5. N-(4N-[(4-chlorophenyl)phenylmethyl]piperazin-1-yl)-3α-hydroxy-5β-cholan-24-
amide (7e)
2.5 g (50% yield) as an orange amorphous solid. IR (KBr) υ: 3410 (br, C-OH alcohol), 2969-
1
2806 (CH alkane), 1599 (C=O amide), 1487 (C=C aromatic) cm-1. H NMR (400MHz,
CDCl3) δ (ppm): 7.36-7.34 (m, 4H, Har), 7.30-7.18 (m, 5H, Har), 4.19 (s, 1H, -CHPhPhCl),
3.63-3.55 (m, 3H, 1×-CH2-piperazinyl and C3H), 3.48-3.42 (m, 2H, 1×-CH2-piperazinyl),
2.88-2.85 (m, 4H, 2×-CH2-piperazinyl), 0.91 (s, 3H, 19-Me), 0.89 (d, 3H, J = 6.6 Hz, 21-Me),
13
0.62 (s, 3H, 18-Me). C NMR (100MHz, CDCl3) δ (ppm): 172.2 (O=C-NH), 142.0 (C1Ar,
Ph), 141.3 (C1Ar, PhCl), 132.5 (C4-Cl, PhCl)), 129.1 (C3Ar and C5Ar, PhCl), 128.7 (C2Ar and
C6Ar, PhCl), 128.6 (C3Ar and C5Ar, Ph), 127.8 (C4Ar, Ph), 127.1 (C2Ar and C6Ar, Ph), 71.7
(C-PhPhCl), 71.2 (C3), 60.4, 56.5, 55.9, 52.0, 51.5, 46.1, 42.7, 40.3, 40.1, 36.4, 35.8, 35.6,
35.3, 34.5, 31.5, 30.5, 30.3, 28.3, 27.2, 26.4, 24.2, 23.4, 20.8 (Me-19), 18.4 (Me-21), 12.0
(Me-18). HRMS (ESI) m/z [M+H]+: calcd for C41H58N2O2Cl: 645.4146, found: 645.4151.
4.1.2.6.
N-(4N-[bis(4-fluorophenyl)methyl]piperazin-1-yl)-3α,7α,12α-tri-hydroxy-5β-
cholan-24-amide (8a)
3.5 g (70% yield) as a beige solid, mp = 265°C. IR (KBr) υ: 3490 (br, C-OH alcohol), 2944-
2804 (CH alkane), 1619 (C=O amide), 1504 (C=C aromatic) cm-1. 1H NMR(400MHz, CDCl3)
δ (ppm): 7.34 (t, 4H, J = 8.7 Hz, Har), 6.98 (t, 4H, J = 8.7 Hz, Har), 4.22 (s, 1H, -CH(PhF)2),
3.93 (m, 1H, C12-H), 3.81 (m, 1H, C7-H), 3.59 (m, 1H, C3-H), 3.47-3.43 (m, 4H, 2×-CH2-
piperazinyl), 2.35-2.31 (m, 4H, 2×-CH2-piperazinyl), 0.97 (d, 3H, J = 5.8 Hz, 21-Me), 0.86 (s,
3H, 19-Me), 0.65 (s, 3H, 18-Me). 13C NMR (100MHz, CDCl3) δ (ppm): 172.3 (O=C-NH),
161.9 (2 x C4-F Ar), 137.6 (2 x C1 Ar), 129.2 (2 x (C2 and C6Ar)), 115.5 (2 x (C3 and C5 Ar)),
74.8 (C12), 74.2 (-C(PhF)2), 73.0 (C3), 68.4 (C7), 52.0, 51.5, 46.8, 46.4, 45.8, 41.6, 41.4,
39.5, 39.4, 35.5, 35.3, 34.7, 34.6, 31.3, 30.4, 30.3, 30.1, 28.1, 27.6, 26.3, 23.2, 22.4 (Me-19),
14

ACCEPTED MANUSCRIPT
17.5 (Me-21), 12.4 (Me-18). HRMS (ESI) m/z [M+H]+: calcd for C41H57N2O4F2: 679.4286,
found: 679.4297.
4.1.2.7. N-(4N-[bis(4-fluorophenyl)methyl]piperazin-1-yl)-3α,7β-di-hydroxy-5β-cholan-
24-amide (8b)
1.8 g (37% yield) as an orange amorphous solid. IR (KBr) υ: 3408 (br, C-OH alcohol), 2935-
-1
1
2803 (CH alkane), 1630 (C=O amide), 1506 (C=C aromatic) cm . H NMR (400MHz,
CDCl3) δ(ppm): 7.34 (t, 4H, J = 6.8 Hz, Har), 6.98 (t, 4H, J = 6.8 Hz, Har), 4.20 (s, 1H, -
CH(PhF)2), 3.60 (m, 1H, C7-H), 3.46 (m, 1H, C3-H), 2.89-2.87 (m, 4H, 2×-CH2-piperazinyl),
2.34-2.31 (m, 4H, 2×-CH2-piperazinyl), 0.94 (s, 3H, 19-Me), 0.92 (d, 3H, J = 6.8 Hz, 21-Me),
0.66 (s, 3H, 18-Me). 13C NMR (100MHz, CDCl3) δ (ppm): 172.0 (O=C-NH), 161.7 (2 x C4-F
Ar), 138.1 (C1 PhF), 129.2 (2x (C2 and C6 PhF)), 115.3 (2 x (C3 and C5 PhF)), 74.9 (C12),
74.3 (C-(PhF)2), 71.2 (C3), 55.7, 54.9, 52.9, 51.5, 46.0, 43.7, 42.4, 41.6, 40.1, 39.1, 37.3, 36.8,
35.5, 34.9, 34.0, 31.4, 30.2, 28.7, 26.9, 23.3 (Me-19), 21.1, 18.5 (Me-21), 12.1 (Me-18).
HRMS (ESI) m/z [M+H]+: calcd for C41H57N2O3F2: 663.4337, found: 663.4348.
4.1.2.8. N-(4N-[bis(4-fluorophenyl)methyl]piperazin-1-yl)-3α,7α-di-hydroxy-5β-cholan-
24-amide (8c)
2.1 g (41% yield) as a beige amorphous solid. IR (KBr) υ: 3506 (br, C-OH alcohol), 2933-
1
2805 (CH alkane), 1635 (C=O amide), 1504 (C=C aromatic) cm-1. H NMR (400MHz,
CDCl3) δ (ppm): 7.34 (t, 4H, J = 8.7 Hz, Har), 6.98 (t, 4H, J = 8.7 Hz, Har), 4.23 (s, 1H, -
CH(PhF)2), 3.85 (m, 1H, C7-H), 3.62-3.58 (m, 2H, 1×-CH2-piperazinyl), 3.47-3.44 (m, 3H,
1×-CH2-piperazinyl and C3-H), 2.36-2.32 (m, 4H, 2×-CH2-piperazinyl), 0.92 (d, 3H, J = 5.8
13
Hz, 21-Me), 0.90 (s, 3H, 19-Me), 0.65 (s, 3H, 18-Me). C NMR (100MHz, CDCl3) δ (ppm):
172.1 (O=C-NH), 161.9 (2 x C4-F Ar), 137.7 (2 x C1 Ar), 129.2 (2 x (C2 and C6Ar)), 115.6 (2
x (C3 and C5 Ar)), 74.3 (-C(PhF)2), 72.0 (C3), 68.5 (C7), 55.7, 52.0, 51.5, 50.5, 45.8, 42.7,
41.6, 41.4, 39.9, 39.6, 39.4, 35.6, 35.3, 35.0, 34.6, 32.8, 31.4, 30.6, 30.1, 28.2, 23.7, 22.8 (Me-
19), 20.6, 18.5 (Me-21), 11.8 (Me-18). HRMS (ESI) m/z [M+H]+: calcd for C41H57N2O3F2:
663.4337, found: 663.4356.
4.1.2.9. N-(4N-[bis(4-fluorophenyl)methyl]piperazin-1-yl)-3α,12α-di-acetoxy-5β-cholan-
24-amide (8d)
1.5 g (31% yield) as a yellow amorphous solid. IR (KBr) υ: 3412 (br, C-OH alcohol), 2936-
1
2802 (CH alkane), 1603 (C=O amide), 1506 (C=C aromatic) cm-1. H NMR (400MHz,
15

ACCEPTED MANUSCRIPT
CDCl3) δ (ppm): 7.36-7.32 (m, 4H, Har), 6.98 (t, 4H, J = 8.7 Hz, Har), 4.20 (s, 1H, -
CH(PhF)2), 3.59 (m, 1H, C12-H), 3.45 (m, 1H, C3-H), 2.88-2.86 (m, 4H, 2×-CH2-
piperazinyl), 2.34-2.29 (m, 4H, 2×-CH2-piperazinyl), 0.96 (d, 3H, J = 5.8 Hz, 21-Me), 0.90 (s,
3H, 19-Me), 0.66 (s, 3H, 18-Me). 13C NMR (100MHz, CDCl3) δ (ppm): 172.0 (O=C-NH),
161.7 (2 x C4-F Ar), 138.0 (2 x C1 Ar), 129.2 (2 x (C2 and C6Ar)), 115.3 (2 x (C3 and C5 Ar)),
74.8 (C12), 72.9 (-C(PhF)2), 71.4 (C3), 52.7, 51.9, 51.4, 48.2, 46.0, 42.0, 41.5, 36.4, 35.9,
35.2, 34.0, 33.5, 31.3, 30.4, 30.1, 28.7, 27.4, 27.1, 26.1, 23.6, 23.1 (Me-19), 17.4 (Me-21),
12.7 (Me-18). HRMS (ESI) m/z [M+H]+: calcd for C41H57N2O3F2: 663.4337, found:
663.4357.
4.1.2.10.
N-(4N-[bis(4-fluorophenyl)methyl]piperazin-1-yl)-3α-acetoxy-5β-cholan-24-
amide (8e)
2.9 g (58% yield) as a white amorphous solid. IR (KBr) υ: 3329 (br, C-OH alcohol), 2944-
1
2801 (CH alkane), 1616 (C=O amide), 1504 (C=C aromatic) cm-1. H NMR (400MHz,
CDCl3) δ (ppm): 7.36-7.33 (m, 4H, Har), 7.01-696 (m, 4H, Har), 4.22 (s, 1H, -CH(PhF)2),
3.62-3.58 (m, 3H, 1×-CH2-piperazinyl and C3-H), 3.47-3.43 (m, 2H, 1×-CH2-piperazinyl),
2.34-2.31 (m, 4H, 2×-CH2-piperazinyl), 0.91 (s, 3H, 19-Me), 0.89 (d, 3H, J = 6.6 Hz, 21-Me),
0.63 (s, 3H, 18-Me). 13C NMR (100MHz, CDCl3) δ (ppm): 172.1 (O=C-NH), 161.9 (2 x C4-F
Ar), 137.7 (2 x C1 Ar), 129.2 (2 x (C2 and C6Ar)), 115.5 (2 x (C3 and C5 Ar)), 72.3 (-C(PhF)2),
71.8 (C3), 56.4, 55.9, 52.0,51.5, 45.7, 42.7, 40.4, 40.1, 36.4, 35.8, 35.6, 35.3, 34.5, 31.4, 30.5,
30.2, 28.2, 27.1, 26.4, 24.2, 23.3, 20.8 (Me-19), 18.4 (Me-21), 12.0 (Me-18). HRMS (ESI)
m/z [M+H]+: calcd for C41H57F2N2O2: 647.4388, found: 647.4391.
4.1.2.11. N-((4N-benzyl)piperazin-1-yl)-3α,7α,12α-tri-acetoxy-5β-cholan-24-amide (9a)
0.87 g (21% yield) as a beige amorphous solid. IR (KBr) υ: 3403 (br, C-OH alcohol), 2937-
1
2852 (CH alkane), 1601 (C=O amide), 1472 (C=C aromatic) cm-1. H NMR (400MHz,
CDCl3) δ (ppm): 7.33-7.30 (m, 5H, Har), 3.95 (m, 1H, C12-H), 3.82 (m, 1H, C7-H), 3.61 (m,
1H, C3-H), 3.52 (s, 2H, -CH2-benzyl), 3.48-3.43 (m, 4H, 2×-CH2-piperazinyl), 2.44-2.40 (m,
4H, 2×-CH2-piperazinyl), 0.99 (d, 3H, J = 4.8 Hz, 21-Me), 0.87 (s, 3H, 19-Me), 0.66 (s, 3H,
18-Me). 13C NMR (100MHz, CDCl3) δ (ppm): 172.4 (O=C-NH), 137.5 (C1 Ar), 129.1 (C3
and C5Ar), 128.3 (C2 and C6 Ar), 127.2 (C4 Ar), 73.0 (C12), 71.5 (C3), 68.4 (C7), 62.8 (C-Ph),
53.1, 52.7, 50.6, 46.7, 46.3, 45.6, 41.4, 39.4, 35.6, 35.3, 34.7, 34.6, 31.4, 30.3, 30.2, 28.0, 27.6,
26.2, 23.2, 22.4 (Me-19), 17.5 (Me-21), 12.4 (Me-18). HRMS (ESI) m/z [M+H]+: calcd for
C35H55N2O4: 567.4162, founded: 567.4179.
16

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4.1.2.12. N-((4N-benzyl)piperazin-1-yl)-3α,7β-di-acetoxy-5β-cholan-24-amide (9b)
0.70 g (17% yield) as an orange amorphous solid. IR (KBr) υ: 3401 (br, C-OH alcohol), 2929-
1
2862 (CH alkane), 1627 (C=O amide), 1453 (C=C aromatic) cm-1. H NMR (400MHz,
CDCl3) δ (ppm): 7.32-7.31 (m, 5H, Har), 3.62-3.54 (m, 4H, C7-H, C3-H and 1×-CH2-
piperazinyl), 3.52 (s, 2H, -CH2-benzyl), 3.47-3.43 (m, 2H, 1×-CH2-piperazinyl), 2.43-2.39
(m, 4H, 2×-CH2-piperazinyl), 0.95 (s, 3H, 21-Me), 0.93 (s, 3H, 19-Me), 0.68 (s, 3H, 18-Me).
13C NMR (100MHz, CDCl3) δ (ppm): 172.1 (O=C-NH), 137.7 (C1 Ar), 129.1 (C3 and C5Ar),
128.3 (C2 and C6 Ar), 127.3 (C4 Ar), 71.5 (C3), 68.4 (C7), 62.9 (C-Ph), 55.7, 54.7, 53.2, 52.8,
45.7, 43.8, 42.4, 41.5, 40.1, 39.1, 37.3, 36.8, 35.6, 34.2, 34.1, 31.5, 30.3, 28.7, 26.9, 23.4 (Me-
19), 21.2, 18.6 (Me-21), 12.1 (Me-18). HRMS (ESI) m/z [M+H]+: calcd for C35H55N2O3:
551.4213, found: 551.4210.
4.1.2.13. N-((4N-benzyl)piperazin-1-yl)-3α,7α-di-acetoxy-5β-cholan-24-amide (9c)
0.79 g (19% yield) as an orange amorphous solid. IR (KBr) υ: 3413 (br, C-OH alcohol), 2928-
1
2811 (CH alkane), 1630 (C=O amide), 1454 (C=C aromatic) cm-1. H NMR (400MHz,
CDCl3) δ (ppm): 7.33-7.32 (m, 5H, Har), 3.85 (m, 1H, C7-H), 3.62 (m, 1H, C3-H), 3.52 (s,
2H, -CH2-benzyl), 3.47-3.44 (m, 4H, 2×-CH2-piperazinyl), 2.43-2.41 (m, 4H, 2×-CH2-
13
piperazinyl), 0.94 (d, 3H, J = 5.8 Hz, 21-Me), 0.91 (s, 3H, 19-Me),0.66 (s, 3H, 18-Me). C
NMR (100MHz, CDCl3) δ (ppm): 172.1 (O=C-NH), 137.5 (C1 Ar), 129.1 (C3 and C5Ar),
128.3 (C2 and C6 Ar), 127.3 (C4 Ar), 72.0 (C3), 68.5 (C7), 62.9 (C-Ph), 55.7, 53.2, 52.8, 50.4,
45.6, 42.7, 41.4, 39.6, 39.4, 35.6, 35.3, 35.0, 34.6, 32.8, 31.4, 30.6, 30.1, 28.2, 23.7, 22.8 (Me-
19), 20.6, 18.5 (Me-21), 11.8 (Me-18). HRMS (ESI) m/z [M+H]+: calcd for C35H55N2O3:
551.4213, found: 551.4199.
4.1.2.14. N-((4N-benzyl)piperazin-1-yl)-3α,12α-di-acetoxy-5β-cholan-24-amide (9d)
0.87 g (21% yield) as a yellow amorphous solid. IR (KBr) υ: 3420 (br, C-OH alcohol), 2934-
1
2809 (CH alkane), 1628 (C=O amide), 1450 (C=C aromatic) cm-1. H NMR (400MHz,
CDCl3) δ (ppm): 7.35-7.28 (m, 5H, Har), 3.96 (m, 1H, C12-H), 3.62-3.54 (m, 3H, 1×-CH2-
piperazinyl and C3-H), 3.51 (s, 2H, -CH2-benzyl), 3.46-3.43 (m, 2H, 1×-CH2-piperazinyl),
2.42-2.47 (m, 4H, 2×-CH2-piperazinyl), 0.99 (d, 3H, J = 5.8 Hz, 21-Me), 0.90 (s, 3H, 19-Me),
13
0.67 (s, 3H, 18-Me). C NMR (100MHz, CDCl3) δ (ppm): 172.0 (O=C-NH), 137.5 (C1 Ar),
129.0 (C3 and C5Ar), 128.2 (C2 and C6 Ar), 127.1 (C4 Ar), 72.9 (C12), 71.5 (C3), 62.8 (C-Ph),
53.1, 52.7, 48.1, 47.0, 46.4, 45.5, 41.9, 41.4, 36.3, 35.9, 35.3, 35.1, 34.0, 33.5, 31.2, 30.3, 30.1,
17

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28.6, 27.4, 27.0, 26.0, 23.6, 23.1 (Me-19), 17.4 (Me-21), 12.7 (Me-18). HRMS (ESI) m/z
[M+H]+: calcd for C35H55N2O3: 551.4213, found: 551.4197.
4.1.2.15. N-((4N-benzyl)piperazin-1-yl)-3α-acetoxy-5β-cholan-24-amide (9e)
0.72 g (17% yield) as an orange oil. IR (KBr) υ: 3424 (br, C-OH alcohol), 2932-2808 (CH
alkane), 1643 (C=O amide), 1448 (C=C aromatic) cm-1. 1H NMR (400MHz, CDCl3) δ (ppm):
7.32-7.30 (m, 5H, Har), 3.62-3.59 (m, 3H, 1×-CH2-piperazinyl and C3H), 3.52 (s, 2H, -CH2-
benzyl), 3.50-3.44 (m, 2H, 1×-CH2-piperazinyl), 2.43-2.40 (m, 4H, 2×-CH2-piperazinyl), 0.92
13
(s, 3H, 19-Me), 0.91 (d, 3H, J = 6.6 Hz, 21-Me), 0.64 (s, 3H, 18-Me). C NMR (100MHz,
CDCl3) δ (ppm): 172.1 (O=C-NH), 137.6 (C1 Ar), 129.1 (C3 and C5Ar), 128.3 (C2 and C6 Ar),
127.2 (C4 Ar), 71.7 (C3), 62.8 (C-Ph), 56.4, 55.9, 53.1, 52.8, 45.6, 42.7, 40.3, 40.1, 36.4, 35.8,
35.6, 35.3, 34.5, 31.4, 30.5, 30.3, 28.2, 27.1, 26.4, 24.1, 23.3, 20.8 (Me-19), 18.4 (Me-21),
12.0 (Me-18). HRMS (ESI) m/z [M+H]+: calcd for C35H55N2O2: 535.4264, found: 535.4261.
4.2. Biology
4.2.1. Cell culture and treatment
Cell lines HCT-116, DLD-1 and HT-29 were purchased from ECACC (European Collection
of Cell Culture) in Health Protection Agency, UK. Cultures medium consisted in Dubelcco’s
Modified Eagle Medium (Lonza, Verviers, France) supplemented with 10% fetal bovine
serum (PAA Laboratories, les Mureaux, France), 100 U/mL and 100 µg/mL streptomycin and
2 mM L-Glutamine (Lonza). Cells were maintained at 37°C with 5% CO2 / 95% air and
routinely passaged using trypsin-EDTA 0.25% (Invitrogen, Carlsbad, USA).
Stock solutions of the synthetic chenodeoxycholic acid were prepared in dimethyl sulfoxide
(DMSO) (Sigma, St Louis, MO) at 10−2 M, and dissolved in culture medium to obtain the
appropriate final concentrations ranging from 20 to 50 ꢀM. In that treatment range the final
concentration of DMSO in the medium was < 0.5% (vol/vol) and showed no influence on cell
growth.
4.2.2. Cell viability assessment
18

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Measurement of cell viability was determined using the XTT assay (Cell Titer 96 Aqueous
One Solution Cell Proliferation Assay, Promega, Lyon, France). Cells were incubated in 96-
well microtiter plates for 24 hours at 37oC in a 5% CO2 / 95% air atmosphere in the presence
of different concentrations of chemical derivatives. XTT Assay was performed according to
the manufacturer’s instructions. The data were expressed as percentage of the vehicle
(DMSO) treated control. Additionally, XTT enabled us to determine a half maximal inhibition
of cell viability induced by products, noted IC50.
4.2.3. Western-blotting
The cell extract-associated proteins (20 µg) were resolved on 8% polyacrylamide gel, using
1% SDS/Tris glycine buffer. The proteins were then electrotransferred on a polyvinylidene
difluoride (PVDF) membrane (Millipore, Molsheim, France). Free protein-binding sites of the
PVDF membranes were blocked for 1 h 30 min in Tris buffer saline (TBST, 20 mM Tris, 137
mM NaCl, 0.1% Tween 20) containing 10% non-fat dry milk and the membranes were
incubated with an anti-PARP (1:1000 dilution), or anti-GAPDH (1:2000 dilution) antibodies
(Santa Cruz Biotechnology Inc., Heidelberg, Germany). After overnight incubation at 4°C,
the membranes were washed and incubated for 1 h with a secondary antibody (HRP-
conjugated goat anti-rabbit antibody) at 1:10000 dilution for PARP and at 1:5000 dilution for
GAPDH. Protein signals were revealed using a Western-blotting Chemiluminescence
Luminol Reagent (Tebu-Bio, Le Parray-en-Yvelines, France) and exposing of membranes on
X-ray films (Kodak, X-Omat, Perkins Elmers SAS, Courtaboeuf, France).
4.2.4. Cell cycle analysis
Cells were trypsined, washed with PBS, and fixed in 80% of ethanol at 4°C for 30 min. Prior
to analyses, cells were again washed with PBS, and then suspended in a cold propidium
iodide solution (PI, Sigma) supplemented with 100 ꢀg/mL RNase (Roche Molecular
Biochemicals, Meylan, France) and incubated at 37°C for 30 min. Flow cytometry analyses
were performed using Gallios flow cytometer (Beckman Coulter, France).
4.2.5. APO2.7 staining
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Cell death was assessed using the APO2.7 kit (Beckman Coulter, France) according to
manufacturer’s instruction (phycoerythrin-labeled APO2.7 antibody binds the 7A6 antigen
exposed on mitochondria of cells undergoing apoptosis). Flow cytometry analyses were
performed using Gallios flow cytometer (Beckman Coulter, France).
Acknowledgment
This work was financially supported by “Conseil Régional Basse-Normandie”.
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Captions
Scheme 1. Synthesis of piperazinyl bile acid derivatives
Reagents and conditions: (i) Ac2O, DMAP, CH2Cl2, r.t., 1 h; (ii) EtOCOCl, NEt3, CH2Cl2, r.t.,
1 h; (iii) piperazine (a, b or c), NEt3, CH2Cl2, r.t., 16 h; (iv) KOH, EtOH, reflux, 16 h.
Figure 1. Piperazinyl bile carboxamide derivatives
Figure 2. Effects of synthetic bile caeboxamide derivatives on viability of human colon
carcinoma cell lines. DLD-1, HCT-116 and HT-29 cell lines were treated with increasing
concentrations of different synthetic bile carboxamide derivatives (0-50 ꢀM) for 24 h and cell
survival was determined using the XTT assay. Data are expressed as percentage of the vehicle
treated with DMSO and represent the mean ± SD of triplicate determinations. Error bars
represent the standard deviation.
Figure 3. Dose-response effects of I, 9c and 9d on the proliferation of DLD-1, HCT-116 and
HT-29 cells. Cells were treated with different concentrations of bile acid derivatives for 24h.
Results are expressed as the mean of three independent experiments. Each concentration was
done in triplicate, results are expressed in the mean ± SD, and Error bars represent the
standard deviation. Standard deviations were added in table 1.
Figure 4. Representative results of cell cycle analysis of DLD-1 cell line. Exponentially
growing cells at 50-60% confluence were treated for 24h with 30 ꢀM of each compound.
Cells were harvested and analyzed by flow cytometry. The green and red colours represent
untreated and treated cells with (a) I, (b) 9c and (c) 9d respectively. Arrows indicates
accumulation in Sub-G1.
24

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Figure 5. Apoptotic index with Apo2.7 staining analyzed by flow cytometry after 24 h
treatment with 30 ꢀM of each compound (a) I, (b) 9c and (c) 9d.
Figure 6. Effects of I, 9c and 9d on pro-apoptotic protein expression in DLD-1, HCT-116 and
HT-29 cell lines. The cell lines were treated with compounds (30 ꢀM each) for 24 h and pro-
apoptotic protein expression was assayed using Western-blotting analysis of whole cell
extracts. GAPDH was used as a loading control. Results are representative of two independent
experiments showing the same profile.
Table 1. IC50 values of I, 9c and 9d were determined from the dose–response curves after 24
h of incubation using the XTT assay. Results are expressed as the mean of three experiments
± standard error (Figure 3).
Table 2. Percentage of cells in sub G1, G0-G1, S or G2-M phases of the cell cycle of the
three cell lines are indicated for each compound (Completed data of figure 4).
Table 3. APO2.7 staining and analyzes by flow cytometry of the three cell lines DLD-1,
HCT-116, and HT-29 in the presence or no of compounds I, 9c, and 9d. Results are presented
as the percentage of APO2.7-positive cells.
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Table 1
IC50 (µM)
I
9c
9d
DLD-1
HCT-116
HT-29
36±1.6
34 ±1,2
35 ±3
33 ±0.3
23 ±1.2
36 ± 1.3
26 ±0.3
22 ±3.7
32 ±0.3

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Table 2
Cell line
DLD-1
HCT-116
HT-29
Compound
DMSO
I
9c
9d
DMSO
I
9c
9d
DMSO
I
9c
9d
Sub-G1
G0-G1
S
1.73
49.77
23.96
18.82
10.67
40.01
14.13
14.28
28.54
15.41
1.78
31.23
32.99
22.47
12.98
36.85
17.83
14.85
33.57
18.39
3.18
48.38
28.49
18.65
18.13
49.08
17.11
34.81
36.89
15.67
35.37
21.08
42.61
18.80
43.78
28.28
G2-M
39.85
6.22
32.19
38.97
33.69
11.34
26.64
28.89
21.66
4.77
13.63
11.14

ACCEPTED MANUSCRIPT
Table 3
Cell line
DLD-1
HCT-116
HT-29
Compound
DMSO
I
9c
9d
DMSO
I
9c
9d
DMSO
I
9c
9d
APO2.7
(%)
1.01
34.87
22.54
2.49
1.19
37.73
20.33
4.59
1.11
8.79
8.57
1.26