The synthesis and antitumor activity of lithocholic acid and its derivatives

Article abstract of DOI:10.1016/j.steroids.2017.06.009

In this paper, a new and concise synthetic route of lithocholic acid (LCA) using commercially available steroid source deoxycholic acid is reported. A series of amide derivatives of LCA were also synthesized and investigated for their activity against the growth of MCF-7 and MCF-7/ADR cells using the sulforhodamine B assay. For MCF-7, the most potent compound 20 showed a 20-fold higher antitumor activity than LCA. For MCF-7/ADR, the most potent compound 24 showed a 22-fold higher antitumor activity than LCA. The transwell migration assay of 20 was evaluated on MDA-MB-231 cells. The colony formation and apoptosis assays of 20 were performed on MCF-7 and MCF-7/ADR cell lines.

Full text of DOI:10.1016/j.steroids.2017.06.009

Accepted Manuscript
The synthesis and antitumor activity of lithocholic acid and its derivatives
Xiao-Long He, Yajing Xing, Xiang-Zhong Gu, Jie-Xin Xiao, Ying-Ying Wang,
Zhengfang Yi, Wen-Wei Qiu
PII:
S0039-128X(17)30102-2
http://dx.doi.org/10.1016/j.steroids.2017.06.009
STE 8120
DOI:
Reference:
Steroids
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Revised Date:
Accepted Date:
13 April 2017
14 May 2017
19 June 2017
Please cite this article as: He, X-L., Xing, Y., Gu, X-Z., Xiao, J-X., Wang, Y-Y., Yi, Z., Qiu, W-W., The synthesis
and antitumor activity of lithocholic acid and its derivatives, Steroids (2017), doi: http://dx.doi.org/10.1016/
j.steroids.2017.06.009
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The synthesis and antitumor activity of lithocholic acid and its derivatives
a
Xiao-Long He , Yajing Xing b, Xiang-Zhong Gu c, Jie-Xin Xiao a, Ying-Ying Wang a, Zhengfang
Yi b, Wen-Wei Qiu a,*
a Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development,
School of Chemistry and Chemical Engineering, East China Normal University, Shanghai 200241,
China
b Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of
Life Sciences, East China Normal University, Shanghai 200241, China
c Department of Research and Development, Jiangsu Jiaerke Pharmaceuticals Group Co., Ltd.,
Zhenglu Town, Changzhou 213111, China
Keywords: Lithocholic acid; Antitumor; Apoptosis; Migration; Breast cancer
* Corresponding author.
E-mail address: wwqiu@chem.ecnu.edu.cn (W.-W. Qiu).
ABSTRACT
In this paper, a new and concise synthetic route of lithocholic acid (LCA) using commercially
available steroid source deoxycholic acid is reported. A series of amide derivatives of LCA were
also synthesized and investigated for their activity against the growth of MCF-7 and MCF-7/ADR
cells using the sulforhodamine B assay. For MCF-7, the most potent compound 20 showed a
20-fold higher antitumor activity than LCA. For MCF-7/ADR, the most potent compound 24
showed a 22-fold higher antitumor activity than LCA. The transwell migration assay of 20 was
evaluated on MDA-MB-231 cells. The colony formation and apoptosis assays of 20 were
performed on MCF-7 and MCF-7/ADR cell lines.
1. Introduction
Lithocholic acid (LCA, Fig.1) is a hydrophobic secondary bile acid, formed by bacterial
7-dehydroxylation of chenodeoxycholic acid and ursodeoxycholic acid [1,2]. Recently, LCA and
its derivatives attracted the focus of research interests due to their variety of biological activities,

such as α-2,3-sialyltransferase inhibition [3], protein tyrosine phosphatase 1B inhibition [4],
vitamin D receptor modulation [5,6], TGR5 receptor activation [7], antibacterial and antifungal [8],
anti-aging [9] and especially antitumor activity [10, 11]. For instance, LCA not only has the ability
to selectively kill the neuroblastoma cell lines while sparing normal neuronal cells [12], but also
induces apoptosis selectively in androgen-dependent (LNCaP) and -independent (PC-3) prostate
cancer cells [13]; LCA amphiphiles possesses potent anticancer activity against colon cancer cells
[14]; amino acid conjugates of LCA can serve as antagonists of the EphA2 receptor as novel
antiangiogenetic agents [15].
Although, LCA and its derivatives play an increasingly important role in biological activity
studies of drug discovery, the practical approach for the preparation of LCA is rarely reported.
Willard et al. (1940) [16] provided LCA (50% overall yield) in 4 steps from methyl deoxycholate,
and the key reactions are oxidation of the C12-hydroxy with CrO3 and reduction of
C12-semicarbazone with Na at 200 oC. Fujinori et al. (1963) [17] also used the methyl
deoxycholate as the starting material for preparation of LCA (7.6% overall yield) in 4 steps, and
the key step is producing the C11-C12 alkene by dehydration of the C12-hydroxy with POCl3.
Mckenzie et al. (1946) [18] reported the synthesis of LCA (23.5% overall yield) in 7 steps from
commercially available deoxycholic acid, and the crucial steps are formation of C11-C12 alkene at
o
300 C and hydrogenation of the double bond with PtO2 and H2. There are deficiencies in above
three procedures, such as high temperature, non-environmental friendly or expensive reagents, and
very low overall yields. Accordingly, exploitation of an economical and efficient synthetic route of
LCA will promote the further development of LCA and its derivatives. Herein, we report an
efficient and concise synthetic route of LCA (Scheme 1) and its derivatives (Scheme 2) using
inexpensive reagents and commercially available steroid source deoxycholic acid. These synthetic
compounds were evaluated for their in vitro activity against breast cancer cell lines (MCF-7 and
MCF-7/ADR) using the sulforhodamine B (SRB) assay. The most potent derivative was screened
to determine its inhibitory effects on tumor cell migration and colony formation ability, and its
apoptotic behavior was also tested.
2. Experimental
2.1. General procedures

Starting steroid, deoxycholic acid was purchased from Aladdin Company, China. All the other
reagents and chemicals were purchased from commercial suppliers and used without further
purification unless otherwise stated. When needed, the reactions were carried out in oven-dried
glassware under a positive pressure of dry N2. Column chromatography was performed on silica
gel (QinDao, 200-300 mesh) using the indicated eluents. Thin-layer chromatography was carried
out on silica gel plates (QinDao) with a layer thickness of 0.25mm. Melting points were
1
determined using the MEL-TEMP 3.0 apparatus and uncorrected. H (400 MHz) and 13C (100
MHz) NMR spectra were recorded on Bruker AM-400 spectrometer with CDCl3 or DMSO-d6 as
solvent and tetramethylsilane (TMS) as the internal standard. All chemical shift values were
reported in units of δ (ppm). The following abbreviations were used to indicate the peak
multiplicity: s = singlet; d = doublet; t = triplet; m = multiplet; br = broad. High-resolution mass
data were obtained on a BrukermicroOTOF-Q II spectrometer.
2.2. Chemical synthesis
2.2.1. Methyl 3α,12α-dihydroxy-5β-cholan-24-oate (1)
To a solution of deoxycholic acid (20 g, 50.94 mmol) in CH3OH (300 mL) was added H2SO4 (5
mL). The reaction mixture was refluxed for 2 h and then concentrated. The residue was dissolved
in DCM (300 mL). Organic layer was washed with 5% aqueous NaOH and brine, dried over
anhydrous Na2SO4 and concentrated to give compound 1 (20.6 g, 99%) as a white solid. 1H NMR
(400 MHz, CDCl3) δ: 3.97 (s, 1H), 3.66 (s, 3H), 3.64–3.57 (m, 1H), 0.97 (d, J = 6.0 Hz, 3H), 0.91
(s, 3H), 0.67 (s, 3H). 13C NMR (100 MHz, CDCl3) δ: 174.8, 73.1, 71.7, 51.5, 48.2, 47.2, 46.5, 42.1,
36.4, 36.0, 35.3, 35.2, 34.1, 33.6, 31.1, 30.9, 30.3, 28.6, 27.5, 27.1, 26.1, 23.7, 23.1, 17.2, 12.7.
2.2.2. Methyl 3α-acetoxy-12α-hydroxy-5β-cholan-24-oate (2)
To a solution of 1 (20.6 g, 50.66 mmol) in DCM (300 mL) was added acetic anhydride (6 mL,
63.33 mmol), pyridine (8.2 mL, 101.32 mmol) and DMAP (309 mg, 2.53 mmol). The reaction
mixture was stirred for 2 h at room temperature and then poured into 2M HCl (150 mL). The
organic layer was washed with brine, dried over anhydrous Na2SO4 and concentrated. The residue
was purified by silica gel chromatography (petroleum ether/AcOEt, 5/1 v/v) to give 2 (20.9 g,
1
92%) as a white solid. H NMR (400 MHz, CDCl3) δ: 4.72–4.64 (m, 1H), 3.97 (s, 1H), 3.64 (s,
3H), 2.00 (s, 3H), 0.95 (d, J = 6.4 Hz, 3H), 0.90 (s, 3H), 0.66 (s, 3H). 13C NMR (100 MHz, CDCl3)
δ: 174.8, 170.8, 74.4, 73.2, 51.6, 48.4, 47.5, 46.6, 42.0, 36.1, 35.2, 35.0, 34.2, 33.7, 32.3, 31.2,

31.0, 28.8, 27.5, 27.1, 26.6, 26.1, 23.7, 23.2, 21.6, 17.4, 12.8.
2.2.3. Methyl 3α-acetoxy-12α- methanesulfonate-5β-cholan-24-oate (3)
To a solution of 2 (20.9 g, 46.6 mmol) in DCM (300 mL) was added MsCl (36 mL, 466 mmol)
and pyridine (37.5 mL, 466 mmol) under N2 at room temperature. The reaction mixture was
stirred for 5 h and then poured into 2M HCl (250 mL). The organic layer was washed with brine,
dried over anhydrous Na2SO4 and concentrated. The residue was purified by silica gel
1
chromatography (petroleum ether/AcOEt, 2/1 v/v) to give 3 (24.3 g, 99%) as a white solid. H
NMR (400 MHz, CDCl3) δ: 5.12 (s, 1H), 4.74–4.66 (m, 1H), 3.66 (s, 3H), 3.06 (s, 3H), 2.02 (s,
3H), 0.98 (d, J = 6.4 Hz, 3H), 0.92 (s, 3H), 0.76 (s, 3H). 13C NMR (100 MHz, CDCl3) δ: 174.6,
170.8, 85.2, 74.2, 51.6, 48.8, 47.3, 46.1, 41.9, 39.8, 35.9, 35.3, 34.9, 34.3, 34.1, 32.5, 31.1, 30.9,
27.6, 27.2, 27.1, 26.6, 25.9, 23.6, 23.3, 21.6, 17.8, 12.5.
2.2.4. Methyl 3α-acetoxy-5β-chol-11-enoate (4)
To a solution of 3 (24.3 g, 46 mmol) in DMPU (150 mL) was added potassium acetate (45.3 g,
460 mmol) under N2. The reaction mixture was stirred for 8 h at 130 oC and then poured into water
(500 mL) and extracted with AcOEt (50 mL × 3). The organic layer was washed with brine, dried
over anhydrous Na2SO4 and concentrated. The residue was purified by silica gel chromatography
1
(petroleum ether/AcOEt, 10/1 v/v) to give 4 (18.4 g, 93%) as a white solid. H NMR (400 MHz,
CDCl3) δ: 6.08 (d, J = 10 Hz, 1H), 5.38 (d, J = 10.4Hz, 1H), 4.75–4.68 (m, 1H), 3.64 (s, 3H), 2.00
(s, 3H), 0.98 (d, J = 6.4Hz, 3H), 0.87 (s, 3H), 0.71 (s, 3H). 13C NMR (100 MHz, CDCl3) δ: 174.7,
170.7, 138.8, 125.3, 74.2, 53.6, 51.9, 51.5, 45.0, 43.1, 40.9, 35.9, 35.0, 34.8, 34.3, 32.9, 31.0, 30.9,
28.4, 27.9, 26.5, 25.4, 23.6, 23.0, 21.4, 18.3, 16.6.
2.2.5. Methyl 3α-acetoxy-5β-cholan-24-oate (5)
To a solution of 4 (18.4 g, 42.78 mmol) in a mixed solvent (150 mL) of CH3OH-THF (4:1, v/v)
was hydrogenated in the presence of 5% Pd/C catalyst (500 mg) under H2 (5.0 × 105 Pa). After
stirring at 70 oC for 8 h, the catalyst was removed by filtration through Celite, and the solvent of
the mother liquor was evaporated under reduced. The residue was purified by silica gel
1
chromatography (petroleum ether/AcOEt, 2/1 v/v) to give 5 (18.3 g, 99%) as a white solid. H
NMR (400 MHz, CDCl3) δ: 4.73–4.67 (m, 1H), 3.65 (s, 3H), 2.01 (s, 3H), 0.90 (d, J = 8.8 Hz, 6H),
0.63 (s, 3H). 13C NMR (100 MHz, CDCl3) δ: 174.8, 170.7, 74.5, 56.6, 56.1, 51.6, 42.8, 42.0, 40.5,
40.3, 35.9, 35.5, 35.2, 34.7, 32.4, 31.2, 31.1, 28.3, 27.1, 26.7, 26.4, 24.3, 23.4, 21.6, 20.9, 18.4,

12.1.
2.2.6. Lithocholic acid (LCA)
To a solution of 5 (18.4 g, 42.5 mmol) in a mixed solvent (150 mL) of CH3OH-THF (4:1, v/v)
was added anhydrous potassium carbonate (58.7 g, 425 mmol) under N2. The reaction mixture
was stirred for 24 h at 60 oC, and then poured into 2M HCl (250 mL). The mixture was extracted
with DCM (60 mL × 3). The organic layer was washed with brine, dried over anhydrous Na2SO4
and concentrated. The residue was purified by silica gel chromatography (DCM/CH3OH, 40/1 v/v)
to give LCA (14.7g, 92%) as a white solid; mp: 185–187 oC [lit. 183-185 oC] [17]. 1H NMR (400
MHz, MeOD) δ: 3.58–3.50 (m, 1H), 0.96–0.94 (m, 6H), 0.70 (s, 3H). 13C NMR (100 MHz, MeOD)
δ: 178.2, 72.4, 57.9, 57.5, 43.9, 43.6, 41.9, 41.5, 37.3, 37.2, 36.7, 36.5, 35.7, 32.3, 32.0, 31.2, 29.2,
28.4, 27.7, 25.3, 23.9, 22.0, 18.8, 12.5. HRMS (ESI) m/z: calcd for C24H40NaO3 [M+Na]+,
399.2875, found 399.2881.
2.2.7. Synthesis of 3α-hydroxy-5β-chol-11-en-oic acid (6)
By a similar procedure described for LCA, compound 6 was obtained as a white solid; yield:
87%. 1H NMR (400 MHz, DMSO-d6) δ: 11.94 (s, 1H), 6.05 (dd, J = 10.4, 2.8 Hz, 1H), 5.40 (d, J
= 10.0 Hz, 1H), 4.43 (s, 1H), 3.41–3.36 (m, 1H), 0.95 (d, J = 6.4 Hz, 3H), 0.82 (s, 3H), 0.69 (s,
3H).13C NMR (100 MHz, DMSO-d6) δ: 174.8, 138.2, 125.6, 69.7, 53.2, 51.4, 44.5, 42.7, 40.6, 37.0,
35.4, 34.9, 34.6, 33.9, 30.7, 30.5, 30.3, 27.9, 27.7, 25.2, 23.6, 22.6, 18.1, 16.5. HRMS (ESI) m/z: calcd
for C24H38NaO3 [M+Na]+, 397.2719, found 397.2717.
2.2.8. Synthesis of 3α-tert-butyldimethylsilyloxy-5β-cholan-24- oic acid (7)
To a solution of LCA (10 g, 26.6 mmol) in DMF (120 mL) was added TBSCl (5.4 mg, 80 mmol)
and imidazole (12 mg, 80 mmol) under N2. The reaction mixture was stirred for 4 h at 80oC and
then poured into water and extracted with AcOEt (50 mL × 3). The organic layer was washed with
saturated Na2CO3 solution and brine, then dried over anhydrous Na2SO4 and concentrated. The
residue was purified by silica gel chromatography (petroleum ether/AcOEt, 2/1 v/v) to give 7 (11g,
1
84.4%) as a white solid. H NMR (400 MHz, CDCl3) δ: 3.62–3.54 (m, 1H), 0.92 (d, J = 6.4 Hz,
3H), 0.90 (s, 3H), 0.89 (s, 9H), 0.64 (s, 3H), 0.06 (s, 6H).
2.2.9. Synthesis of 3α-tert-butyldimethylsilyloxy-5β-chol-11-en-oic acid (8)
By a similar procedure described for 7, compound 8 was obtained as a white solid; yield: 80%.
1H NMR (400 MHz, CDCl3) δ: 6.07 (dd, J = 10.4, 3.2 Hz, 1H), 5.41 (dd, J = 10.4, 1.2 Hz, 1H),

3.63–3.55 (m, 1H), 1.01 (d, J = 6.4 Hz, 3H), 0.88 (s, 9H), 0.86 (s, 3H), 0.72 (s, 3H), 0.05 (s, 6H).
2.2.10. General procedure for the synthesis of compounds 9-16.
To a solution of 7 or 8 (0.61 mmol) in DCM (10 mL) was added EDCI (234 mg, 1.2 mmol),
HOBt (162 mg, 1.2 mmol), DMAP (293 mg, 2.4 mmol) and amine (120 mg, 1.2 mmol) under N2
at room temperature. The reaction mixture was stirred for 12 h and then poured into water (30 mL)
and extracted with DCM (20 mL × 3). The organic layer was washed with brine, dried over
anhydrous Na2SO4 and concentrated. The residue was purified through column chromatography
(petroleum ether/AcOEt, 5/1 v/v) to give the desired product.
2.2.11 N-(thiazol-2-yl)-3α-tert-butyldimethylsilyloxy-5β-cholan-24-amide (9)
White solid; yield: 79%. 1H NMR (400 MHz, CDCl3) δ: 11.03 (s, 1H), 7.44 (d, J = 3.6 Hz, 1H),
7.00 (d, J = 3.6 Hz, 1H), 3.63–3.54 (m, 1H), 0.96 (d, J = 6.0 Hz, 3H), 0.90 (s, 3H), 0.89 (s, 9H),
0.64 (s, 3H), 0.06 (s, 6H).
2.2.11. N-(thiazol-2-yl)-3α-tert-butyldimethylsilyloxy-5β-chol-11-en-24-amide (10)
1
White solid; yield: 83%. H NMR (400 MHz, CDCl3) δ: 7.43 (d, J = 3.6 Hz, 1H), 6.99 (d, J =
3.6 Hz, 1H), 6.07 (dd, J = 10.0, 2.8 Hz, 1H), 5.42 (dd, J = 10.4, 1.2 Hz, 1H), 3.63–3.56 (m, 1H),
1.05 (d, J = 6.0 Hz, 3H), 0.88 (s, 9H), 0.86 (s, 3H), 0.72 (s, 3H), 0.05 (s, 6H).
2.2.12. N-(5-methylthiazol-2-yl)-3α-tert-butyldimethylsilyloxy-5β-cholan-24-amide (11)
White solid; yield: 77%. 1H NMR (400 MHz, CDCl3) δ: 10.98 (s, 1H), 7.06 (d, J = 1.2 Hz, 1H),
3.61–3.54 (m, 1H), 2.41 (d, J = 1.2 Hz, 3H), 0.95 (d, J = 6.4Hz, 3H), 0.90 (s, 3H), 0.89 (s, 9H),
0.64 (s, 3H), 0.06 (s, 6H).
2.2.13. N-(5-methylthiazol-2-yl)-3α-tert-butyldimethylsilyloxy-5β-chol-11-en-24-amide (12)
White solid; yield: 77%. 1H NMR (400 MHz, CDCl3) δ: 10.95 (s, 1H), 7.05 (d, J = 1.2 Hz, 1H),
6.08 (dd, J = 10.4, 3.2 Hz, 1H), 5.42 (dd, J = 10.4, 1.2 Hz, 1H), 3.63–3.55 (m, 1H), 2.40 (d, J =
1.2 Hz, 3H), 1.04 (d, J = 6.0 Hz, 3H), 0.88 (s, 9H), 0.86 (s, 3H), 0.72 (s, 3H), 0.05 (s, 6H).
2.2.14. N-(benzo[d]thiazol-2-yl)-3α-tert-butyldimethylsilyloxy-5β-cholan-24-amide (13)
White solid; yield: 77%. 1H NMR (400 MHz, CDCl3) δ: 10.33 (s, 1H), 7.86 (d, J = 8.0 Hz, 1H),
7.78 (d, J = 8.0 Hz, 1H), 7.48–7.44 (m, 1H), 7.36–7.33 (m, 1H), 3.63–3.55 (m, 1H), 0.90 (s, 9H),
0.89 (s, 3H), 0.87 (d, J = 3.2 Hz, 3H), 0.58 (s, 3H) , 0.07 (s, 6H).
2.2.15. N-(benzo[d]thiazol-2-yl)-3α-tert-butyldimethylsilyloxy-5β-chol-11-en-24-amide (14)
1
White solid; yield: 81%. H NMR (400 MHz, CDCl3) δ: 7.84 (d, J = 8.4 Hz, 1H), 7.77 (d, J =

8.0 Hz, 1H), 7.47–7.43 (m, 1H), 7.35–7.31 (m, 1H), 6.03–6.00 (m, 1H), 5.40 (d, J = 10.4 Hz, 1H),
3.64–3.56 (m, 1H), 0.95 (d, J = 4.0 Hz, 3H), 0.89 (s, 9H), 0.85 (s, 3H), 0.67 (s, 3H) , 0.05 (s, 6H).
2.2.16. N-(1H-indol-6-yl)-3α-tert-butyldimethylsilyloxy-5β-cholan-24-amide (15)
White solid; yield: 63%. 1H NMR (400 MHz, CDCl3) δ: 8.27 (s, 1H), 8.13 (s, 1H), 7.53 (d, J =
8.4 Hz, 1H), 7.21 (s, 1H), 7.17 (t, J = 2.8, 1H), 6.81 (dd, J = 8.4, 1.2 Hz, 1H), 6.49 (s, 1H), 3.62–
3.54 (m, 1H), 0.97 (d, J = 6.4 Hz, 3H), 0.89 (s, 12H), 0.65 (s, 3H), 0.06 (s, 6H).
2.2.17. N-(1H-indol-6-yl)-3α-tert-butyldimethylsilyloxy-5β-chol-11-en-24-amide (16)
White solid; yield: 80%. 1H NMR (400 MHz, CDCl3) δ: 8.19 (s, 1H), 8.13 (s, 1H), 7.53 (d, J =
8.0 Hz, 1H), 7.18–7.17 (m, 2H), 6.81 (d, J = 8.4 Hz, 1H), 6.50 (s, 1H), 6.09 (dd, J = 10.4, 2.4 Hz,
1H), 5.41 (d, J = 10.4 Hz, 1H), 3.63–3.56 (m, 1H), 1.06 (d, J = 6.4 Hz, 3H), 0.88 (s, 9H), 0.86 (s,
3H), 0.73 (s, 3H), 0.05 (s, 6H).
2.2.18. General procedure for the synthesis of compounds 17–24
One of compounds 9–16 (0.26 mmol) was dissolved in THF (10 mL) and TBAF (204 mg, 0.78
mmol) was added under N2. The reaction mixture was stirred for 5 h at 60oC and then the reaction
mixture was poured into water (30 mL) and extracted with DCM (10 mL × 3). The organic layer
was washed with brine, dried over anhydrous Na2SO4 and concentrated. The residue was purified
through column chromatography (DCM/CH3OH, 40/1 v/v) to give the desired product.
2.2.19. N-(thiazol-2-yl)-3α-hydroxy-5β-cholan-24-amide (17)
White solid; yield: 90%; mp: 137–145 oC. 1H NMR (400 MHz, CDCl3) δ: 10.62 (s, 1H), 7.43 (d,
J = 3.6 Hz, 1H), 6.98 (d, J = 3.6 Hz, 1H), 3.66–3.58 (m, 1H), 0.95 (d, J = 6.0 Hz, 3H), 0.91 (s, 3H),
0.64 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ: 171.6, 158.0, 137.4, 113.1, 69.8, 56.0, 55.5, 42.2,
41.5, 39.9, 39.7, 36.2, 35.3, 35.1, 34.9, 34.2, 31.9, 31.0, 30.3, 27.7, 26.9, 26.1, 23.8, 23.2, 20.4,
18.2, 11.8. HRMS (ESI) m/z: calcd for C27H42N2NaO2S [M+Na]+, 481.2865, found 481.2870.
2.2.20. N-(thiazol-2-yl)-3α-hydroxy-5β-chol-11-en-24-amide (18)
1
White solid; yield: 91%; mp: 156–164 oC. H NMR (400 MHz, CDCl3) δ: 7.42 (d, J = 3.6 Hz,
1H), 6.97 (d, J = 3.6 Hz, 1H), 6.09 (dd, J = 10.4, 2.8 Hz, 1H), 5.43 (d, J = 11.6 Hz, 1H), 3.67–3.62
(m, 1H), 1.05 (d, J = 6.0 Hz, 3H), 0.88 (s, 3H), 0.73 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ:
171.5, 158.0, 138.2, 137.5, 125.6, 113.1, 69.7, 53.3, 51.3, 44.5, 42.7, 40.6, 37.0, 35.5, 34.9, 34.6,
33.9, 31.8, 30.8, 30.3, 27.9, 27.7, 25.2, 23.6, 22.6, 18.2, 16.5. HRMS (ESI) m/z: calcd for
C27H40N2NaO2S [M+Na]+, 479.2708, found 479.2724.

2.2.21. N-(5-methylthiazol-2-yl)-3α-hydroxy-5β-cholan-24-amide (19)
1
White solid; yield: 90%; mp: 202–207 oC. H NMR (400 MHz, CDCl3) δ: 7.06 (d, J = 1.2 Hz,
1H), 3.65–3.57 (m, 1H), 2.41 (s, 3H), 0.94 (d, J = 6.0 Hz, 3H), 0.91 (s, 3H), 0.63 (s, 3H). 13C
NMR (100 MHz, DMSO-d6) δ: 171.2, 156.2, 134.5, 125.7, 69.8, 56.0, 55.5, 42.2, 41.5, 39.9, 39.7,
36.3, 35.3, 35.1, 34.9, 34.2, 31.9, 31.0, 30.3, 27.7, 26.9, 26.1, 23.8, 23.2, 20.4, 18.2, 11.8, 11.0.
HRMS (ESI) m/z: calcd for C28H44N2NaO2S [M+Na]+, 495.3021, found 495.3022.
2.2.22. N-(5-methylthiazol-2-yl)-3α-hydroxy-5β-chol-11-en-24-amide (20)
White solid; yield: 90%; mp: 225–229 oC. 1H NMR (400 MHz, CDCl3) δ: 11.67 (s, 1H), 7.05 (d,
J = 1.2 Hz, 1H), 6.09 (dd, J = 10.4, 2.8 Hz, 1H), 5.42 (d, J = 9.6 Hz, 1H), 3.67–3.60 (m, 1H), 2.40
(d, J = 0.8 Hz, 3H), 1.04 (d, J = 6.4 Hz, 3H), 0.87 (s, 3H), 0.72 (s, 3H). 13C NMR (100 MHz,
DMSO-d6) δ: 171.2, 156.2, 138.2, 134.5, 125.7, 125.6, 69.7, 53.2, 51.3, 44.5, 42.7, 40.6, 36.9,
35.5, 34.9, 34.6, 33.9, 31.8, 30.9, 30.3, 27.9, 27.7, 25.2, 23.6, 22.6, 18.2, 16.5, 11.0. HRMS (ESI)
m/z: calcd for C28H42N2NaO2S [M+Na]+, 493.2865, found 493.2878.
2.2.23. N-(benzo[d]thiazol-2-yl)-3α-hydroxy-5β-cholan-24-amide (21)
o
1
White solid; yield: 90%; mp: 251–254 C. H NMR (400 MHz, DMSO-d6) δ: 12.30 (s, 1H),
7.96 (d, J = 8.0 Hz, 1H), 7.73 (d, J = 8.4 Hz, 1H), 7.45–7.41 (m, 1H), 7.31–7.27 (m, 1H), 4.43 (d,
J = 4.8 Hz, 1H), 3.41–3.35 (m, 1H), 0.92 (d, J = 6.4 Hz, 3H), 0.87 (s, 3H), 0.62 (s, 3H). 13C NMR
(100 MHz, DMSO-d6) δ: 172.6, 157.9, 148.5, 131.4, 125.9, 123.3, 121.5, 120.4, 69.8, 56.0, 55.4,
42.2, 41.5, 39.9, 39.6, 36.3, 35.3, 35.1, 34.9, 34.1, 32.2, 30.8, 30.3, 27.7, 26.9, 26.1, 23.8, 23.2,
20.4, 18.2, 11.8. HRMS (ESI) m/z: calcd for C31H44N2NaO2S [M+Na]+, 531.3021, found
531.3046.
2.2.24. N-(benzo[d]thiazol-2-yl)-3α-hydroxy-5β-chol-11-en-24-amide (22)
o
1
White solid; yield: 91%; mp: 233–238 C. H NMR (400 MHz, DMSO-d6) δ: 12.31 (s, 1H),
7.95 (d, J = 8.0 Hz, 1H), 7.72 (d, J = 8.0 Hz, 1H), 7.42 (t, J = 7.2 Hz, 1H), 7.29 (t, J = 7.2 Hz, 1H),
6.06 (dd, J = 10.0, 2.4 Hz, 1H), 5.40 (d, J = 10.4 Hz, 1H), 4.45 (d, J = 4.8 Hz, 1H), 3.44–3.37 (m,
1H), 1.00 (d, J = 6.4 Hz, 3H), 0.82 (s, 3H), 0.69 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ: 172.6,
157.9, 148.5, 138.1, 131.4, 125.9, 125.5, 123.3, 121.5, 120.4, 69.7, 53.2, 51.3, 44.5, 42.7, 40.5,
36.9, 35.5, 34.9, 34.6, 33.9, 32.1, 30.6, 30.3, 27.9, 27.7, 25.2, 23.6, 22.6, 18.2, 16.5. HRMS (ESI)
m/z: calcd for C31H42N2NaO2S [M+Na]+, 529.2865, found 529.2902.
2.2.25. N-(1H-indol-6-yl)-3α-hydroxy-5β-cholan-24-amide (23)

o
1
White solid; yield: 89%; mp: 252–257 C. H NMR (400 MHz, DMSO-d6) δ: 10.95 (s, 1H),
9.72 (s, 1H), 7.96 (s, 1H), 7.39 (d, J = 8.4 Hz, 1H), 7.23 (t, J = 2.4 Hz, 1H), 7.00 (dd, J = 8.4, 1.2
Hz, 1H), 6.32 (s, 1H), 4.43 (d, J = 4.4 Hz, 1H), 3.40–3.35 (m, 1H), 0.94 (d, J = 6.4 Hz, 3H), 0.87
(s, 3H), 0.62 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ: 171.1, 135.9, 133.5, 124.7, 123.6, 119.6,
112.2, 102.0, 100.8, 69.8, 56.1, 55.6, 42.3, 41.5, 39.9, 39.7, 36.3, 35.4, 35.1, 35.0, 34.2, 33.4, 31.5,
30.4, 27.7, 26.9, 26.1, 23.8, 23.2, 20.4, 18.4, 11.9. HRMS (ESI) m/z: calcd for C32H46N2NaO2
[M+Na]+, 513.3457, found 513.3486.
2.2.26. N-(1H-indol-6-yl)-3α-hydroxy-5β-chol-11-en-24-amide (24)
White solid; yield: 93%; mp: 246–250 oC. 1H NMR (400 MHz, CDCl3) δ: 10.95 (s, 1H), 9.73 (s,
1H), 7.96 (s, 1H), 7.39 (d, J = 8.4 Hz, 1H), 7.23 (t, J = 2.8 Hz, 1H), 6.99 (dd, J = 8.4, 1.6 Hz, 1H),
6.32 (s, 1H), 6.07 (dd, J = 10.4, 2.4 Hz, 1H), 5.40 (d, J = 10.0 Hz, 1H), 4.45 (d, J = 4.4 Hz, 1H),
3.40–3.36 (m, 1H), 1.01 (d, J = 6.4 Hz, 3H), 0.82 (s, 3H), 0.69 (s, 3H). 13C NMR (100 MHz,
DMSO-d6) δ: 171.1, 138.3, 135.9, 133.6, 125.6, 124.7, 123.7, 119.6, 112.2, 102.1, 100.8, 69.8,
53.3, 51.5, 44.6, 42.7, 40.6, 37.0, 35.6, 34.9, 34.7, 33.9, 33.4, 31.4, 30.4, 28.0, 27.8, 25.3, 23.7,
22.7, 18.4, 16.6. HRMS (ESI) m/z: calcd for C32H44N2NaO2 [M+Na]+, 511.3300, found 511.3322.
2.3 Biological assay
2.3.1 Cell lines and culture conditions
Human breast cancer cell lines MCF-7 and MDA-MB-231 were purchased from American Type
Culture Collection (ATCC). Human adriamycin-resistant breast cancer cell line MCF-7/ADR was
kindly provided from Tianjin Medical University. MCF-7 cell line was cultured in RPMI 1640
medium. MDA-MB-231 cell line was cultured in MEM with nonessential amino acids.
MCF-7/ADR cell line was cultured in the final concentration of 1 µg/mL of adriamycin RPMI
1640. Medium was supplemented with 10% FBS, and all tumor cells were incubated at 37 oC and
5% CO2 incubator.
2.3.2 Sulforhodamine B (SRB) assay
Cell viability was assessed by SRB assay according to previous methods [19]. Cells were
seeded in 96-well plate (3×103 cells/well). After cultured for 24 h, cells were treated with various
concentrations of compound for 96 h. Then cells were fixed with 25 µL cold 50% trichloroacetic
acid (TCA) and incubated for at least 1 h at 4 oC. Next, washing plates by deionized water 5 times
and plates were air dried. Adding 50 µL 0.4% (W/V) SRB to each well of the plates and incubated

at room temperature for 10-20 min. The unbound SRB was removed quickly by washing the wells
5 times with 1% acetic acid and 100 µL tris buffer was added. The absorption at 515 nm was
measured by a microplate spectrophotometer. The values are averaged from at least 3 independent
experiments.
2.3.3 Transwell migration assay
Transwell migration assay was performed as previously reported [20]. The inhibition of tumor
cell migration was determined using the transwell chamber (Corning Falcon) migration assay in
24-well cell culture plate with 8.0 µm pore. Briefly, the top chambers were seeded with 6×104
MDA-MB-231 cells in 200 µL of serum-free medium containing different dose of compound. The
bottom chambers were filled with 500 µL of complete medium supplemented with different dose
of compound. After 12 h incubation, non-migrated cells were removed with cotton swabs, and
migrated cells were fixed with cold 4% paraformaldehyde for 20 min and stained with 0.2%
crystal violet for 3 min. Then the chambers were washed using water, and the membrane was left
to dry. Images were taken with an inverted microscope (Olympus) and cells from three random
areas per filter were counted. The percentage of migrated cells was normalized to untreated
control cells. Cells from three random areas per filter were counted.
2.3.4 Colony formation assay
To mimic individual cell development into macroscopic cell colonies, the colony formation
assay was performed as previously described [21]. MCF-7 and MCF-7/ADR cells were seeded
2000 per well in 6-well plate and allowed to grow for 24 h. Then cells were treated with different
concentrations of compound for 7 days. Colonies were fixed with 4% paraformaldehyde, stained
with 0.2% crystal violet and counted manually. Three independent experiments were performed.
2.3.5 Cell apoptosis assay
Apoptosis was measured using the apoptosis detection kit (BD Biosciences) with flow
cytometry [22]. MCF-7 cells and MCF-7/ADR cells were cultured in 6-well plate with different
concentrations of compound for 72 h. Cells were collected and washed with PBS, then
resuspended in binding buffer and incubated with Annexin V-FITC and propidium iodide. After
20-min incubation, cells were analyzed immediately with flow cytometry (FACS Calibur, BD
Biosciences). Three independent experiments were performed.

3. Results and discussion
3.1. Chemistry
We report here synthesis of LCA, the target compound, from deoxycholic acid in 6 steps,
according to the pathway described in Scheme 1. The intermediate 2 was prepared according to
previously reported procedure [23,24], which was obtained by methyl esterification of LCA with
MeOH and H2SO4 with 99% yield firstly, and then selective esterification of the 3-hydroxy group
with Ac2O in the presence of pyridine and DMAP in 92% yield. Treatment of 2 with MsCl under
pyridine gave compound 3 with 99% yield. The key intermediate 4 was afforded by reaction of 3
under potassium acetate in DMPU with 93% yield. Reduction of the C11-C12 double bond of 4
with H2 and Pd in a mixed solvent of MeOH-THF obtained compound 5 with 95% yield. LCA was
synthesized by hydrolysis of 5 with potassium acetate in MeOH with 92% yield. Previously, three
methodologies were reported by Willard, Fujinori and Mckenzie et al., respectively. However the
expensive reagents or drastic reaction conditions and low overall yields (5.7-50%) were the
hindrance of the further studies of LCA and its derivatives. In our straightforward methodology,
most of the conversions are very efficient with an average yield of 96% in 6 steps and overall
yield up to 76%. The route is based on mild reaction conditions and inexpensive commercially
available reagents. All the steps can be up-scaled to industrial process potentially.
Introduction of the heterocyclic rings can significantly improve the biological activities of
steroids [25]. In order to obtain promising antitumor agents, we synthesized heterocyclic amide
derivatives of LCA according to the pathway described in Scheme 2. Hydrolysis of compound 4
with K2CO3 in THF and MeOH afforded compound 6. Protection of the 3-hydroxyl group of LCA
and 6 with TBSCl in the presence of imidazole in DMF gave compounds 7 and 8. Treatment of 7
and 8 with various amines under EDCI, HOBt and DMAP in CH2Cl2 produced compounds 9-16
with 61-83% yields. LCA derivatives 17-24 were obtained by deprotection of the TBS group of
9-16 with TBAF with 88-93% yields.

O
O
O
O
OH
OMs
OH
OH
OMe
OMe
OH
OMe
b
a
c
92%
99%
99%
AcO
AcO
HO
HO
1
3
Deoxycholic acid
2
O
O
O
OH
OMe
OMe
d
f
e
93%
AcO
92%
99%
HO
AcO
4
Lithocholic acid (LCA)
Total yield: 76.4%
5
Scheme 1. Reagents and conditions: (a) CH3OH, H2SO4, reflux, 99%; (b) Ac2O, Py, DMAP,
CH2Cl2, rt, 92%; (c) MsCl, Py, CH2Cl2, rt, 99%; (d) CH3COOK, DMPU, 130 oC, 93%; (e) H2
(5MPa), Pd, THF, CH3OH, 70 oC, 99%; (f) K2CO3, THF, CH3OH, 70 oC, 92%.
O
O
O
O
NHR
OH
NHR
OH
X
X
X
X
b
d
c
TBSO
TBSO
HO
HO
a
S
S
S
S
LCA X = single bond
X = double bond
7
8
X = single bond
17
18
9
R =
R =
R =
R =
N
N
N
4
6
X = double bond
10
N
S
S
S
S
19
20
21
22
11
12
13
14
R =
R =
R =
R =
R =
R =
R =
R =
N
N
N
N
S
S
N
S
N
S
N
N
H
N
H
N
23
24
15
16
R =
R =
R =
R =
H
N
H
N
9, 11, 13, 15 X = single bond
10, 12, 14, 16 X = double bond
17, 19, 21, 23 X = single bond
18, 20, 22, 24 X = double bond
Scheme 2. Reagents and conditions: (a) K2CO3, THF, CH3OH, 70 oC, 87%. (b) TBSCl, imidazole,
DMF, rt, 95%; (c) amines, EDCI, HOBt, DMAP, CH2Cl2, rt, 61–83%; (d) TBAF, THF, 60 oC,
88–93%.
3.2. Biological evaluation
3.2.1. In vitro evaluation of cytotoxic activity
The synthetic LCA and its derivatives were evaluated for their antitumor activity in vitro against
human breast cancer cell line (MCF-7) and multidrug-resistant human breast cancer cell line
(MCF-7/ADR) (Table 1). The results showed that LCA and its 11-ene isomer (compound 6)

possessed almost no antiproliferative activity (IC50 = 150.3–165.1 µM), while if their C-24
carboxylic acid groups were modified by heterocyclic amines, the inhibition of tumor cell growth
(IC50 = 6.78–37.82 µM) increased significantly. The 11-ene amide derivatives had much more
activity than corresponding LCA amide derivatives (compounds 18, 20 and 24 vs 17, 19 and 23),
except 22 and 21. For MCF-7, the most potent compound 20 showed an IC50 of 7.39 µM, which
was 20 folds higher activity than LCA. For MCF-7/ADR, the most potent compound 24 showed
an IC50 of 6.78 µM, which was 22 folds higher activity than LCA. It is amazing that for most
amide derivatives the multidrug-resistant MCF-7/ADR displayed almost the same antiproliferative
activity as MCF-7. Compounds 20 and 24 exhibited almost the same degree of antitumor activity
as the positive control tamoxifen and we chose 20 for further study.
Table 1
IC50 values of LCAand its derivatives against the growth of breast cancer cell lines MCF-7 and MCF-7/ADR.
IC50
IC50
Compound
Compound
MCF-7
150.3
165.1
32.60
10.79
15.02
7.39
MCF-7/ADR
MCF-7
7.83
MCF-7/ADR
LCA
6
150.6
159.7
37.82
15.96
16.74
6.95
21
22
10.81
37.84
14.02
6.78
11.19
13.66
8.80
17
18
19
20
23
24
Tamoxifen
7.05
4.28
From SRB assay after 96 h of treatment; the values are averaged from at least 3 independent experiments;
variation 10%.
3.2.2. Migration
Migration is an important step during the metastasis of cancer. MCF-7 cell line is malignant but
relatively noninvasive. While MDA-MB-231 is a highly invasive and metastatic breast cancer cell
line [26]. To determine anti-migration effect of compound 20, transwell migration assay was
performed on MDA-MB-231 cell line. Results showed that 20 decreased cells migration in a
dose-dependent manner after treated 12 h, with about 50% cell-migration inhibition at the
concentration of 10 µM compared to the control group (Fig. 1). The antiproliferative activity of 20

on MDA-MB-231 cells was also tested, which IC50 was 7.36 0.39 µM.
3.2.3. Colony formation
Colony formation assay was used to determine the cell proliferation ability. To further
determine the antiproliferation effect of compound 20, colony formation assay was conducted.
The results (Fig. 2) indicated that 20 inhibited colony formation of breast cancer cells MCF-7 and
MCF-7/ADR significantly including the number and size of colonies in a dose-dependent manner
and we did not observe any colonies at concentration of 10 µM.
3.2.4. Apoptosis
We evaluated whether the antiproliferative activity of compound 20 was resulted by cell
apoptosis using flow cytometry assay (Fig. 3). Results displayed that 20 caused a dose-dependent
induction of apoptosis in MCF-7 and MCF-7/ADR breast cancer cell lines and the apoptosis rates
increased to 60.47% (MCF-7) and 52.4% (MCF-7/ADR) at concentration of 20 µM.
4. Conclusion
Herein we report a new straightforward methodology for preparation of LCA with an average
yield of 96% in 6 steps and overall yield up to 76%. A series of derivatives of LCA and its 11-ene
isomer were also synthesized by modification of C-24 carboxylic acid with heterocyclic amines
and evaluated for their antiproliferative activity on MCF-7 and MCF-7/ADR cell lines using the
sulforhodamine B assay. Although their corresponding amide derivatives exhibited obvious
antiproliferative activity, LCA and its 11-ene isomer showed almost no activity. The 11-ene amide
derivatives showed more activity than corresponding LCA amide derivatives. The most potent
compound 20 showed an IC50 of 7.39 µM on MCF-7 cells that was 20 folds better than LCA and
compound 24 showed an IC50 of 6.78 µM on MCF-7/ADR cells that was 22 folds better than LCA.
Moreover, the transwell migration, colony formation and apoptosis assays were performed on
MCF-7 and MCF-7/ADR cell lines. The results revealed that 20 displayed potent effects in
transwell migration, colony formation and apoptosis assays.
Acknowledgment
This work was supported by Jiangsu Jiaerke Pharmaceuticals Group Co., Ltd.

Appendix A. Supplementary data
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120
Control
5 µM
10 µM
20 µM
100
80
60
40
20
0
***
***
***
¦ÌM
Fig. 1. Compound 20 inhibited breast cancer cells MDA-MB-231 migration in transwell chamber
compared with the control group. **p ＜ 0.01, ***p ＜ 0.001, compared with control group (0
µM).

120
100
80
60
40
20
0
2.5µM
Control
5µM
**
***
***
***
µM
120
100
80
60
40
20
0
***
***
***
***
µM
Fig. 2. Compound 20 inhibited the colony formation of MCF-7 and MCF-7/ADR cell lines. After
treatment by various doses of compound 20 in 6-well plates for 7 days, cells were fixed and
stained with crystal violet and the numbers of cell colonies were counted. **p ＜ 0.01, ***p ＜
0.001, compared with control group (0 µM).
Control
5µM
10µM
20µM
0
5.01%
4.48%
9.64%
6.20%
2.76%
11.47%
16.93%
50.83%
5.28%
9.23%
2.58%
5.66%
25.78%
26.62%
21.69%
21.75%
Annexin V-FITC
Fig. 3. Compound 20 induced MCF-7 and MCF-7/ADR cells apoptosis in dose-dependent manner

compared with control groups. Cells in the lower right quadrant indicate
Annexin-positive/Annexin-negative early apoptotic cells. Cells in the upper right quadrant
indicate Annexin-positive/PI-positive late apoptotic cells. The right quadrants of each diagram
(Annexin+/IP¯ and Annexin+/IP+) represent apoptotic cells for each compound treatment.

A concise synthesis of lithocholic acid and its amide derivatives was reported.
The antiproliferative activities of these derivatives were evaluated in vitro.
Transwell migration, colony formation and apoptosis assays were performed.

The synthesis and antitumor activity of lithocholic acid and its derivatives
Xiao-Long He, Yajing Xing, Xiang-Zhong Gu, Jie-Xin Xiao, Ying-Ying Wang, Zhengfang Yi,
Wen-Wei Qiu
O
NHR
O
O
X
OH
OH
OH
6 steps
HO
LCA Amides (17-24) R = Heterocycles
Total yield: 76%
HO
HO
S
20 R =
X = double bond
Lithocholic acid (LCA)
Deoxycholic acid
N
Control
20 (5 µM)
20 (20 µM)
MDA-MB-231
120
100
80
60
40
20
0
***
***
***
µM
A concise synthesis of lithocholic acid and its amide derivatives and evaluation of their activity
against the growth of breast cancer cell lines.