Synthesis and antitumor activity of N-sulfonyl-3,7-dioxo-5β-cholan-24- amides, ursodeoxycholic acid derivatives

Article abstract of DOI:10.1016/j.steroids.2012.09.009

A series of N-sulfonyl-3,7-dioxo-5β-cholan-24-amides, ursodeoxycholic acid derivatives, have been designed and synthesized in nine steps starting from ursodeoxycholic acid. The in vitro antitumor activity of the target compounds has been evaluated against HCT-116, MCF-7, K562, and SGC-7901 cell lines. The pharmacological results showed that most of the prepared compounds display excellent selective cytotoxicity toward HCT-116, MCF-7, and K562 cell lines. Particularly, compounds 10c, 10f and 10g show high inhibitory activity on these human cancer cell lines (IC50: 2.39-9.34 μM). Conversely, all compounds are generally inactive against SGC-7901, with only 10b having IC₅₀ below 50 μM.

Full text of DOI:10.1016/j.steroids.2012.09.009

Steroids 78 (2013) 53–58
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis and antitumor activity of N-sulfonyl-3,7-dioxo-5b-cholan-24-amides,
ursodeoxycholic acid derivatives
a,
Jie Ren b, Yongchao Wang a, Junliang Wang a, Jun Lin a, Kun Wei a, , Rong Huang
⇑
⇑
a Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education, School of Chemical Science and Technology, Yunnan University, Kunming 650091, PR China
b School of Pharmaceutical Engineering & Life Science, Changzhou University, 1 Gehu Road, Changzhou, Jiangsu 213164, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 July 2012
Received in revised form 19 September 2012
Accepted 25 September 2012
Available online 2 November 2012
A series of N-sulfonyl-3,7-dioxo-5b-cholan-24-amides, ursodeoxycholic acid derivatives, have been
designed and synthesized in nine steps starting from ursodeoxycholic acid. The in vitro antitumor activity
of the target compounds has been evaluated against HCT-116, MCF-7, K562, and SGC-7901 cell lines. The
pharmacological results showed that most of the prepared compounds display excellent selective cyto-
toxicity toward HCT-116, MCF-7, and K562 cell lines. Particularly, compounds 10c, 10f and 10g show
high inhibitory activity on these human cancer cell lines (IC50: 2.39–9.34
pounds are generally inactive against SGC-7901, with only 10b having IC50 below 50
l
M). Conversely, all com-
M.
Keywords:
l
N-sulfonyl-3,7-dioxo-5b-cholan-24-amides
Anti-tumour
Crown Copyright Ó 2012 Published by Elsevier Inc. All rights reserved.
1. Introduction
2. Experimental section
Steroids are an important class of natural products which have
high ability to penetrate cells and bind to nucleus and membrane
receptors. They include great variations in structure and play a
very important role in life [1,2], such as cholesterol, bile acids,
sex hormones, vitamin D, corticoid hormones, cardiac aglycones,
antibiotics, and insect molting hormones. A variety of steroids with
unusual and interesting structures have been synthesized and
evaluated for their anti-tumor activity [3–8]. As a relatively inex-
pensive bile acid and a traditional medicine, ursodeoxycholic acid
has been used to synthesize some new derivatives with biological
activity [9–11].
Sulfonamides are also an important group of drugs. They exhibit
a broad spectrum of antibacterial activities. Recently, some sulfon-
amide derivatives have been reported to have potential antitumor
effects [12–19]. However there are few literature references avail-
able for steroid sulfonamide derivatives. As part of our current ef-
forts in understanding various steroid sulfonamide analogs as
antitumor agents, this paper presents a series of N-sulfonyl-3,7-di-
oxo-5b-cholan-24-amides, an ursodeoxycholic acid derivatives, de-
signed and synthesized by us. Their antitumor activity against
different cancer cell lines in vitro has been investigated in order
to screen potent and selective chemotherapy agents.
2.1. Chemistry
2.1.1. General methods
All compounds were fully characterized by spectroscopic tech-
niques. The NMR spectra were recorded on a Bruker DRX500 (1H:
500 MHz, 13C: 125 MHz), chemical shifts (d) are expressed in
ppm, and J values are given in Hz. Deuterated CDCl3-d6 was used
as a solvent. IR spectra were recorded on a FT-IR Thermo Nicolet
Avatar 360 using a KBr pellet. The reactions were monitored by
thin layer chromatography (TLC) using silica gel GF254. The melting
points were determined on an XT-4A melting point apparatus and
are uncorrected. HRMS was performed on an Agilent LC-MSD TOF
instrument.
All chemicals and solvents were used as received without fur-
ther purification unless otherwise stated. Column chromatography
was performed on silica gel (200–300 mesh).
Ursodeoxycholic acid and all of sulfonyl chlorides were pur-
chased from Adrich Corporation Limited.
2.1.2. 3a,7b-Dihydroxy-methylcholanat 2
Ursodeoxycholic acid (1) (5 g, 12.76 mmol) was dissolved in
cold methanol (50 mL) containing anhydrous HCl (generated
in situ by the dropwise addition of SOCl2 into methanol). After stir-
ring for 0.5 h at room temperature, the reagent was refluxed for
3 h. When the reaction was finished, the volatiles were removed
under reduced pressure. The crude product was column purified
on silica gel using 30% ethyl acetate in n-hexane to afford 2 in
95% yield as colorless crystals (4.92 g)1. mp 210–212 °C. 1H NMR
⇑
Corresponding authors. Tel./fax: +86 871 5033215 (K. Wei).
E-mail addresses: kunwei@ynu.edu.cn (K. Wei), huangrong@ynu.edu.cn (R.
Huang).
0039-128X/$ - see front matter Crown Copyright Ó 2012 Published by Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.steroids.2012.09.009

54
J. Ren et al. / Steroids 78 (2013) 53–58
25
(500 MHz, CDCl3-d) d 0.64 (3H, s, 18-CH3), 0.89 (3H, d, J = 6.5 Hz,
21-CH3), 0.91 (3H, s, 19-CH3), 1.04–1.97 (m, 24H), 2.18 (1H, m,
ane to afford 6 in quantitative yield as colorless oil (3.19 g). [
ꢀ15.5 °C (c 0.35, CHCl3); IR (KBr) mmax 3447, 3278, 2946, 2882,
1425, 1350, 1098, 699, 550 cmꢀ1 1H NMR (500 MHz, CDCl3-d) d
a]
D
23-H1), 2.32 (1H, m, 23-H2), 3.51(1H, m, 3b-H), 3.53(1H, m, 7
a
-
.
H), 3.62(3H, s, -OCH3). 13C NMR (125 MHz, CDCl3-d) d 12.5, 18.8,
21.6, 23.8, 27.3, 29.0, 30.6, 31.4, 31.5, 34.4, 35.4, 35.7, 37.5, 37.7,
39.7, 40.6, 42.9, 44.0, 44.1, 51.9, 55.4, 56.3, 71.5, 175.1.
0.70 (3H, s, 18-CH3), 0.94 (3H, d, J = 6.5 Hz, 21-CH3), 1.31 (3H, s,
19-CH3), 0.97–2.90 (m, 26H), 3.60 (2H, m, 24-H). 13C NMR
(125 MHz, CDCl3-d) d 12.5, 19.1, 22.5, 22.8, 25.2, 28.8, 29.8, 32.2,
35.8, 35.8, 35.8, 37.1, 39.3, 43.0, 43.2, 43.3, 45.4, 48.1, 49.2, 50.0,
55.4, 63.7, 210.8, 211.8. HRMS (TOF ES+): m/z calcd for C24H38NaO3
+
2.1.3. 3
a,7b,24-Trihydroxy-5b-cholan 3
A solution of ester 2 (4.92 g, 12.12 mmol) in dry ethanol
(100 mL) was stirred rapidly as sodium borohydride (1.2 eq.,
550 mg) was added carefully in small portions. The reaction was
continued overnight at room temperature before the suspension
was poured carefully onto cooled ethyl acetate (20 mL). The sus-
pension was filtered, and the filtrate was dried under reduced pres-
sure. The residual solids were extracted with water (300 mL) and
ethyl acetate (300 mL) for three times. The combined organic lay-
ers were dried under reduced pressure. The crude product was col-
umn purified on silica gel using 50% ethyl acetate in n-hexane to
afford 3 in 92% yield as colorless needles (4.21 g)2. mp 150–
152 °C. 1H NMR (500 MHz, CD3OD-d4) d 0.74 (3H, s, 18-CH3), 0.99
(3H, br.s., 21-CH3), 0.99 (3H, s, 19-CH3), 1.06–2.08 (m, 26H), 3.37
[(M+Na)+], 397.2713; found, 397.2706.
2.1.7. 3,7-Dioxo-24-cholan-methansulfonate 7
To a 8.52 mmol solution of 6 in 100 mL of CH2Cl2 at 0 °C was
added 17 mmol (2 eq., 2.37 mL) of Et3N followed by slow addition
of 10.22 mmol (1.2 eq., 816 lL) of methanesulfonyl chloride. The
mixture was stirred for 1 h at 0 °C and diluted with cold water.
The solution was extracted twice with 100 ml of CH2Cl2. The organ-
ic layer was washed successively with H2O to neutral pH and with
brine. The solution was dried over anhydrous MgSO4, filtered, and
concentrated to afford crude product which was chromatographed
on silica gel using 30% ethyl acetate in n-hexane to afford 7 in 93%
25
(2H, m, 24-H), 3.53 (2H, m, 3b-H and 7
a
-H). 13C NMR (125 MHz,
yield as amorphous solid (3.58 g). [
a]
ꢀ17.5 °C (c 0.25, CHCl3);
D
CD3OD-d4) d 13.11, 19.8, 22.8, 24.4, 28.4, 30.2, 30.8, 31.5, 33.7,
35.6, 36.6, 37.4, 38.5, 39.1, 41.2, 42.1, 44.5, 45.0, 45.2, 57.2, 58.0,
64.0, 72.4, 72.6.
IR (KBr) mmax 3445, 3272, 2947, 2872, 1422, 1321, 1085, 732,
521 cmꢀ1 1H NMR (500 MHz, CDCl3-d) d 0.63 (3H, s, 18-CH3),
.
0.88 (3H, d, J = 6.5 Hz, 21-CH3), 1.24 (3H, s, 19-CH3), 0.91–2.84
(m, 26H), 2.94 (3H, s, -OSO2CH3), 4.12 (2H, m, 24-H). 13C NMR
(125 MHz, CDCl3-d) d 12.5, 19.0, 22.5, 22.8, 25.1, 26.2, 28.7, 31.9,
35.5, 35.8, 35.8, 37.1, 37.7, 39.2 43.0, 43.2, 43.3, 45.4, 48.1, 49.2,
49.9, 55.2, 71.0, 210.6, 211.6. HRMS (TOF ES+): m/z calcd for C25H40-
NaO5S+ [(M+Na)+], 475.2489; found, 475.2482.
2.1.4. 3a,7b-Dihydroxy-24-triphenylmethoxy-5b-cholan 4
A solution of alcohol 3 (4.21 g, 11.14 mmol) in dry pyridine
(100 mL) was stirred rapidly under nitrogen as triphenylmethyl
chloride (1.5 eq., 4.64 g) was added in one portion. The reaction
was continued overnight at 90 °C before the solvent was removed
in vacuo. The resultant oily solid was purified by column chroma-
tography using 30% ethyl acetate in n-hexane to afford 4 in 85%
yield as colorless solid (5.87 g)2. mp 85–87 °C. 1H NMR
(500 MHz, CDCl3-d) d 0.65 (3H, s, 18-CH3), 0.91 (3H, d, J = 6.5 Hz,
21-CH3), 0.95 (3H, s, 19-CH3), 0.98–2.01 (m, 26H), 3.02 (2H, m,
2.1.8. 3,7-Dioxo-24-azido-5b-cholan 8
NaN3 (2 eq., 1.03 g) was added to a solution of mesylate 7
(3.58 g, 7.92 mmol) in DMF (30 mL) and stirred overnight at room
temperature. Water (50 mL) was added and the aqueous phase was
extracted with ethyl acetate (3 ꢁ 30 mL). The combined organic
extracts were washed with saturated aqueous NaCl solution
(2 ꢁ 100 mL) and dried over anhydrous MgSO4. The solvent was re-
moved under reduced pressure. Silica gel column chromatography
24-H), 3.59 (2H, m, 3b-H and 7a
-H), 7.20–7.45 (m, 15H, ArH). 13C
NMR (125 MHz, CDCl3-d) d 12.6, 19.2, 21.6, 23.8, 27.0, 27.3, 29.1,
30.1, 30.8, 32.8, 34.5, 35.4, 35.8, 37.2, 37.8, 39.6, 40.6, 42.9, 44.2,
44.2, 55.5, 56.2, 64.6, 71.9, 86.7, 127.2, 128.1, 129.1, 145.0.
using 10% ethyl acetate in n-hexane to afford 8 in 82% yield as col-
25
2.1.5. 3,7-Dioxo-24-triphenylmethoxy-5b-cholan 5
orless oil (2.59 g). [
a
]
D ꢀ9.5 °C (c 0.3, CHCl3); IR (KBr)
mmax 3455,
Chromium trioxide (5 eq., 4.74 g) was added to a dry dichloro-
methane (100 mL) solution containing pyridine (10 eq., 7.64 mL).
Under the condition of stirring, a solution of 4 (5.87 g, 9.47 mmol)
in dry dichloromethane (10 mL) was dropped in one portion. The
mixture was stirred for overnight at room temperature, and the
reaction progress was monitored by TLC. The mixture was then
poured into a silica gel column and chromatographied using
3280, 2945, 2870, 1325, 1095, 625, 533 cmꢀ1 1H NMR (500 MHz,
.
DMSO-d6) d 0.73 (3H, s, 18-CH3), 0.98 (3H, d, J = 6.5 Hz, 21-CH3),
1.28 (3H, s, 19-CH3), 1.01–3.00 (m, 26H), 3.48 (2H, m, 24-H). 13C
NMR (125 MHz, DMSO-d6) d 12.4, 19.1, 22.5, 22.8, 25.1, 25.9,
28.7, 33.3, 35.7, 35.8, 35.8, 37.1, 39.2, 43.0, 43.2, 43.3, 45.4, 48.1,
49.2, 49.9, 52.2, 55.2, 210.6, 211.5. HRMS (TOF ES+): m/z calcd for
+
C
24H37N3NaO2 [(M+Na)+], 422.2778; found, 422.2776.
dichloromethane to afford 5 in 90% yield as colorless oil (5.25 g).
25
[a
]
ꢀ25.0 °C (c 0.2, CHCl3); IR (KBr) mmax 3445, 3280, 2946,
D
2880, 1435, 1310, 1112, 702, 535 cmꢀ1
.
1H NMR (500 MHz, CDCl3-
2.1.9. 3,7-Dioxo-5b-cholan-24-amide 9
d) d 0.67 (3H, s, 18-CH3), 0.92 (3H, d, J = 6.5 Hz, 21-CH3), 1.25 (3H, s,
19-CH3), 0.95–2.87 (m, 26H), 3.02 (2H, m, 24-H), 7.19–7.45 (m,
15H, ArH). 13C NMR (125 MHz, CDCl3-d) d 12.5, 19.2, 22.6, 22.9,
25.2, 27.0, 28.7, 32.8, 35.8, 35.8, 35.8, 37.2, 39.3, 43.0, 43.3, 43.4,
45.4, 48.2, 49.3, 50.0, 55.4, 64.6, 86.7, 127.2, 128.1, 129.1, 145.0,
210.6, 211.6. HRMS (EI): m/z calcd for C43H52O3 [M+], 616.3916;
found, 616.3920.
A mixture of 8 (2.59 g, 6.49 mmol), 5% Pd-C (137 mg, 5% mol) in
100 mL methanol was hydrogenated under stirring overnight by
using hydrogen balloon. After hydrogenation, the reaction mixture
was filtered. The solvent was evaporated under reduced pressure.
The crude product was purified by column chromatography over
silica gel using 2% MeOH in CH2Cl2 to get the amine 9 in 80% yield
25
as amorphous solid (1.94 g). [
a
]
D ꢀ11.0 °C (c 0.2, CHCl3); IR (KBr)
mmax 3450, 3281, 2949, 2868, 1410, 1320, 1100, 661, 512 cmꢀ1
.
1H
2.1.6. 3,7-Dioxo-5b-cholan-24-ol 6
NMR (500 MHz, CDCl3-d) d 0.73 (3H, s, 18-CH3), 0.90 (3H, d,
J = 6.5 Hz, 21-CH3), 1.26 (3H, s, 19-CH3), 0.94–2.85 (m, 26H), 3.19
(2H, m, 24-H). 13C NMR (125 MHz, CDCl3-d) d 12.3, 18.9, 22.0,
22.2, 24.0, 24.7, 28.3, 32.7, 35.1, 35.2, 35.3, 36.7, 38.8, 42.0, 42.6,
42.9, 44.9, 47.2, 48.9, 49.1, 54.9, 63.2, 210.2, 211.5. HRMS (EI): m/
z calcd for C24H39NO2 [M+], 373.2981; found, 373.2975.
To a solution of 5 (5.25 g, 8.52 mol) in acetone (100 mL), 4-tol-
uenesulfonic acid (1.05 eq., 1.49 g) were added and stirred under
room temperature for 0.5 h (monitored by TLC). The solvent was
removed under reduced pressure. The residue obtained was puri-
fied by column chromatography using 25% ethyl acetate in n-hex-

J. Ren et al. / Steroids 78 (2013) 53–58
55
2.1.10. General procedure for the synthesis of N-sulfonyl-3,7-dioxo-5b-
cholan-24-amides 10a–o
0.92 (3H, d, J = 6.4 Hz, 21-CH3), 1.32 (3H, s, 19-CH3), 1.00–2.91
(m, 26H), 2.94 (2H, m, 24-H), 4.26 (2H, s, –NHSO2CH2C6H5), 4.41
(1H, br.s., –NHSO2–), 7.41 (5H, m). 13C NMR (125 MHz, CDCl3-d) d
12.5, 19.1, 22.5, 22.8, 25.2, 27.5, 28.8, 33.1, 35.7, 35.8, 35.8, 37.1,
39.3, 43.0, 43.3, 43.3, 44.6, 45.4, 48.1, 49.3, 50.0, 55.3, 59.1,
129.1, 129.2, 129.2, 130.0, 131.0, 131.0, 210.6, 211.6. HRMS (TOF
ES+): m/z calcd for C31H45NNaO4S+ [(M+Na)+], 550.2962; found,
550.2958.
A mixture of compounds 9 (100 mg, 0.268 mol) and a series of
sulfonyl chloride compounds in dry pyridine (2.0 mL) was stirred
overnight at room temperature (monitored by TLC). The solvent
was removed under reduced pressure. The residue obtained was
purified by column chromatography over silica gel using 2% ace-
tone in n-hexane to get the sulfonamides 10a–o in 78–94% yield
as colorless solid.
2.1.10.6. N-(40-methylbenzenesulfonyl)-3,7-dioxo-5b-cholan-24-amide
25
2.1.10.1. N-methanesulfonyl-3,7-dioxo-5b-cholan-24-amide (10a).
(10f). 120 mg (85% yield) as a colorless amorphous solid. [a]
D
25
111 mg (92% yield) as a colorless amorphous solid. [
a]
D ꢀ15.6 °C
ꢀ9.0 °C (c 0.2, CHCl3); IR (KBr) mmax 3478, 3281, 2944, 2872,
1708, 1646, 1325, 1157, 549 cmꢀ1 1H NMR (500 MHz, CDCl3-d) d
(c 0.25, CHCl3); IR (KBr) mmax 3456, 3283, 2946, 2872, 1707, 1616,
.
1442, 1325, 1140, 1096, 1050 cmꢀ1 1H NMR (500 MHz, CDCl3-d)
.
0.60 (3H, s, 18-CH3), 0.79 (3H, d, J = 6.5 Hz, 21-CH3), 1.24 (3H, s,
19-CH3), 0.89–2.45 (m, 26H), 2.37 (3H, s, 40-CH3), 2.83 (2H, m,
24-H), 5.17 (1H, t, J = 5.9 Hz, –NHSO2–), 7.25 (2H, d, J = 8.2 Hz, 30-
ArH and 50-ArH), 7.70 (2H, d, J = 8.2 Hz, 20-ArH and 60-ArH). 13C
NMR (125 MHz, CDCl3-d) d 12.2, 19.0, 21.8, 22.4, 22.7, 25.1, 26.5,
28.6, 33.1, 35.6, 35.7, 35.7, 37.1, 39.2, 43.0, 43.2, 43.2, 44.0, 45.4,
48.0, 49.2, 49.9, 55.2, 127.4, 127.4, 130.0, 130.0, 137.6, 143.5,
210.8, 211.7. HRMS (TOF ES+): m/z calcd for C31H45NNaO4S+
[(M+Na)+], 550.2962; found, 550.2958.
d 0.70 (3H, s, 18-CH3), 0.94 (3H, d, J = 6.5 Hz, 21-CH3), 1.31 (3H, s,
19-CH3), 0.98–2.91 (m, 26H), 2.95 (3H, s, –OSO2CH3), 3.09 (2H,
m, 24-H), 4.73 (1H, m, –NHSO2–). 13C NMR (125 MHz, CDCl3-d) d
12.5, 19.1, 22.5, 22.8, 25.1, 27.2, 28.8, 33.2, 35.7, 35.8, 35.8, 37.1,
39.2, 40.6, 43.0, 43.2, 43.3, 44.2, 45.4, 48.1, 49.2, 49.9, 55.3,
210.8, 211.8. HRMS (TOF ESꢀ): m/z calcd for C25H40NO4Sꢀ
[(MꢀH)ꢀ], 450.2684; found, 450.2687.
2.1.10.2. N-ethanesulfonyl-3,7-dioxo-5b-cholan-24-amide (10b). 112 mg
25
(90% yield) as a colorless amorphous solid. [
a
]
ꢀ7.5 °C (c 0.2,
2.1.10.7. N-(40-bromobenzenesulfonyl)-3,7-dioxo-5b-cholan-24-amide
D
25
CHCl3); IR (KBr) mmax 3456, 3287, 2944, 2868, 1706, 1633, 1450,
(10g). 140 mg (88% yield) as a colorless amorphous solid. [a]
D
1319, 1143, 1097, 1052, 725 cmꢀ1 1H NMR (500 MHz, CDCl3-d) d
.
ꢀ11.0 °C (c 0.2, CHCl3); IR (KBr) mmax 3450, 3281, 2949, 2868,
1707, 1606, 1432, 1322, 1156, 825, 529 cmꢀ1 1H NMR (500 MHz,
0.69 (3H, s, 18-CH3), 0.94 (3H, d, J = 6.5 Hz, 21-CH3), 1.31 (3H, s,
19-CH3), 1.36 (3H, t, J = 7.2 Hz, –OSO2CH2CH3), 0.94–2.91 (m,
26H), 3.03 (2H, q, J = 7.2 Hz, –OSO2CH2CH3), 3.07 (2H, m, 24-H),
4.63 (1H, t, J = 5.6 Hz, –NHSO2–). 13C NMR (125 MHz, CDCl3-d) d
8.7, 12.5, 19.1, 22.5, 22.8, 25.1, 27.5, 28.8, 33.2, 35.7, 35.8, 35.8,
37.1, 39.3, 43.0, 43.3, 43.3, 44.1, 45.4, 47.2, 48.1, 49.2, 49.9, 55.3,
210.7, 211.6. HRMS (TOF ESꢀ): m/z calcd for C26H42NO4Sꢀ
[(MꢀH)ꢀ], 464.2840; found, 464.2845.
.
CDCl3-d) d 0.63 (3H, s, 18-CH3), 0.82 (3H, d, J = 6.5 Hz, 21-CH3),
1.27 (3H, s, 19-CH3), 0.90–2.48 (m, 26H), 2.87 (2H, m, 24-H), 5.20
(1H, t, J = 5.9 Hz, –NHSO2–), 7.63 (2H, d, J = 8.4 Hz, 30-ArH and 50-
ArH), 7.71 (2H, d, J = 8.2 Hz, 20-ArH and 60-ArH). 13C NMR
(125 MHz, CDCl3-d) d 12.4, 19.0, 22.5, 22.8, 25.1, 26.6, 28.7, 33.1,
35.6, 35.8, 35.8, 37.1, 39.2, 43.0, 43.3, 43.3, 44.1, 45.4, 48.1, 49.2,
49.9, 55.2, 127.8, 129.0, 129.0, 132.7, 132.7, 139.8, 210.8, 211.7.
HRMS (TOF ES+): m/z calcd for C30H42BrNNaO4S+ [(M+Na)+],
614.1910; found, 614.1914.
2.1.10.3. N-n-butanesulfonyl-3,7-dioxo-5b-cholan-24-amide (10c).
25
112 mg (85% yield) as a colorless amorphous solid. [
a
]
ꢀ8.5 °C
D
(c 0.2, CHCl3); IR (KBr) mmax 3457, 3285, 2946, 2872, 1706, 1625,
2.1.10.8. N-(40-chlorobenzenesulfonyl)-3,7-dioxo-5b-cholan-24-amide
1452, 1319, 1143, 1097, 1053, 929, 894, 732 cmꢀ1 1H NMR
.
(10h). 115 mg (78% yield) as a colorless amorphous solid. [a 25
]
D
(500 MHz, CDCl3-d) d 0.69 (3H, s, 18-CH3), 0.93 (3H, d, J = 6.5 Hz,
21-CH3), 0.96 (3H, t, J = 7.4 Hz, –OSO2CH2CH2CH2CH3), 1.31 (3H,
s, 19-CH3), 1.00–3.02 (m, 30H), 3.00 (2H, t, J = 7.8 Hz, –OSO2CH2-
CH2CH2CH3), 3.07 (2H, m, 24-H), 4.63 (1H, t, J = 5.6 Hz, –NHSO2–
). 13C NMR (125 MHz, CDCl3-d) d 12.4, 14.0, 19.1, 21.9, 22.5, 22.8,
25.1, 26.1, 27.5, 28.8, 33.2, 35.7, 35.8, 35.8, 37.1, 39.3, 43.0, 43.3,
43.3, 44.1, 45.4, 48.1, 49.3, 49.9, 52.7, 55.4, 210.6, 211.6. HRMS
(TOF ESꢀ): m/z calcd for C28H46NO4Sꢀ [(MꢀH)ꢀ], 492.3153; found,
492.3155.
ꢀ18.0 °C (c 0.15, CHCl3); IR (KBr) mmax 3476, 3281, 2945, 2872,
1708, 1331, 1161, 1088, 752, 621 cmꢀ1 1H NMR (500 MHz, CDCl3-
.
d) d 0.66 (3H, s, 18-CH3), 0.85 (3H, d, J = 6.5 Hz, 21-CH3), 1.30 (3H,
s, 19-CH3), 0.93–2.87 (m, 26H), 2.90 (2H, m, 24-H), 5.16 (1H, t,
J = 5.8 Hz, –NHSO2–), 7.49 (2H, d, J = 8.4 Hz, 30-ArH and 50-ArH),
7.81 (2H, d, J = 8.4 Hz, 20-ArH and 60-ArH). 13C NMR (125 MHz, CDCl3-
d) d 12.4, 19.0, 22.5, 22.8, 25.1, 26.6, 28.7, 33.1, 35.6, 35.8, 35.8, 37.1,
39.2, 43.0, 43.3, 43.3, 44.1, 45.4, 48.1, 49.2, 49.9, 55.2, 128.9, 128.9,
129.7, 129.7, 139.2, 139.3, 210.8, 211.7. HRMS (TOF ES+): m/z calcd
for C30H42ClNNaO4S+ [(M+Na)+], 570.2415; found, 570.2416.
2.1.10.4. N-benzenesulfonyl-3,7-dioxo-5b-cholan-24-amide (10d).
25
126 mg (92% yield) as a colorless amorphous solid. [
a
]
D ꢀ13.5 °C
2.1.10.9. N-(40-fluorobenzenesulfonyl)-3,7-dioxo-5b-cholan-24-amide
25
(c 0.2, CHCl3); IR (KBr) mmax 3458, 3281, 2945, 2871, 1708, 1445,
(10i). 1271 mg (89% yield) as a colorless amorphous solid. [a]
D
1328, 1160, 1090, 735, 585 cmꢀ1 1H NMR (500 MHz, CDCl3-d) d
.
ꢀ14.0 °C (c 0.2, CHCl3); IR (KBr) mmax 3456, 3281, 2946, 2872,
1708, 1592, 1331, 1158, 1091, 837, 550 cmꢀ1 1H NMR (500 MHz,
0.65 (3H, s, 18-CH3), 0.84 (3H, d, J = 6.4 Hz, 21-CH3), 1.30 (3H, s,
19-CH3), 0.92–2.90 (m, 26H), 2.93 (2H, m, 24-H), 5.06 (1H, t,
J = 6.0 Hz, –NHSO2–), 7.52 (2H, m), 7.58 (1H, m), 7.87 (2H, m).
13C NMR (125 MHz, CDCl3-d) d 12.4, 19.0, 22.5, 22.8, 25.1, 26.7,
28.7, 33.1, 35.6, 35.8, 35.8, 37.1, 39.2, 43.0, 43.3, 43.3, 44.1, 45.4,
48.1, 49.2, 49.9, 55.3, 127.4, 127.4, 129.4, 129.4, 132.9, 140.6,
210.7, 211.6. HRMS (TOF ES+): m/z calcd for C30H43NNaO4S+
[(M+Na)+], 536.2805; found, 512.2800.
.
CDCl3-d) d 0.59 (3H, s, 18-CH3), 0.78 (3H, d, J = 6.5 Hz, 21-CH3),
1.24 (3H, s, 19-CH3), 0.86–2.45 (m, 26H), 2.83 (2H, m, 24-H), 5.35
(1H, t, J = 5.8 Hz, –NHSO2–), 7.13 (2H, t, J = 8.6 Hz, 30-ArH and 50-
ArH), 7.83 (2H, dd, J = 8.6, 5.0 Hz, 20-ArH and 60-ArH). 13C NMR
(125 MHz, CDCl3-d) d 12.4, 18.9, 22.4, 22.7, 25.1, 26.5, 28.6, 33.1,
35.5, 35.7, 35.7, 37.1, 39.2, 42.9, 43.2, 43.3, 44.0, 45.4, 48.1, 49.2,
49.9, 55.2, 116.5, 116.7, 130.1, 130.1, 136.7, 164.2, 166.3, 210.9,
211.8. HRMS (TOF ES+): m/z calcd for C30H42FNNaO4S+ [(M+Na)+],
554.2711; found, 554.2710.
2.1.10.5. N-(10-phenylmethanesulfonyl)-3,7-dioxo-5b-cholan-24-amide
(10e). 123 mg (87% yield) as a colorless amorphous solid. IR (KBr)
mmax 3460, 3287, 2943, 2871, 1708, 1450, 1323, 1146, 1078, 699,
2.1.10.10. N-(30-fluorobenzenesulfonyl)-3,7-dioxo-5b-cholan-24-amide
543 cmꢀ1
.
1H NMR (500 MHz, CDCl3-d) d 0.70 (3H, s, 18-CH3),
(10j). 128 mg (90% yield) as a colorless amorphous solid. [a]
D
25

56
J. Ren et al. / Steroids 78 (2013) 53–58
25
ꢀ16.5 °C (c 0.2, CHCl3); IR (KBr)
m
max 3452, 3283, 2946, 2872, 1708,
lid. [
a]
D ꢀ30.0 °C (c 0.35, CHCl3); IR (KBr)
m
max 3472, 3281, 2946,
1593, 1432, 1333, 1223, 1155, 1081, 794, 690, 587 cmꢀ1
.
1H NMR
2872, 1708, 1610, 1451, 1321, 1110, 862, 712 cmꢀ1 1H NMR
.
(500 MHz, CDCl3-d) d 0.66 (3H, s, 18-CH3), 0.86 (3H, d, J = 6.4 Hz,
21-CH3), 1.30 (3H, s, 19-CH3), 0.93–2.92 (m, 26H), 2.95 (2H, m,
24-H), 5.31 (1H, t, J = 5.7 Hz, –NHSO2–), 7.29 (1H, m), 7.52 (1H,
m), 7.57 (1H, d, J = 8.1 Hz), 7.67 (1H, d, J = 7.8 Hz). 13C NMR
(125 MHz, CDCl3-d) d 12.4, 19.0, 22.5, 22.8, 25.1, 26.6, 28.7, 33.1,
35.6 35.8, 35.8, 37.1, 39.2, 43.0, 43.2, 43.3, 44.2, 45.4, 48.1, 49.2,
49.9, 55.2, 114.7, 114.9, 120.0, 120.2, 123.2, 131.3, 142.7, 161.8,
163.8, 211.0, 211.9. HRMS (TOF ES+): m/z calcd for C30H42FNNaO4-
S+ [(M+Na)+], 554.2711; found, 554.2705.
(500 MHz, CDCl3-d) d 0.63 (3H, s, 18-CH3), 0.84 (3H, d, J = 6.2 Hz,
21-CH3), 1.24 (3H, s, 19-CH3), 2.10 (3H, s, –NHCOCH3), 1.00–2.96
(m, 26H), 3.40 (2H, m, 24-H), 7.41 (1H, t, J = 5.5 Hz, -NHSO2-),
7.71 (2H, d, J = 8.6 Hz, 20-ArH and 60-ArH), 7.77 (2H, d, J = 8.6 Hz,
30-ArH and 50-ArH), 10.32 (1H, s, –NHCOCH3). 13C NMR
(125 MHz, CDCl3-d) d 12.2, 18.9, 22.0, 22.2, 24.4, 24.7, 26.0, 28.2,
32.8, 35.0, 35.2, 35.3, 36.7, 38.7, 42.0, 42.6, 42.9, 43.4, 44.9, 47.2,
48.9, 49.0, 54.9, 119.0, 119.0, 128.0, 128.0, 134.8, 143.0, 169.3,
210.2, 211.5. HRMS (TOF ESꢀ): m/z calcd for C32H45N2O5Sꢀ
[(MꢀH)ꢀ], 569.3055; found, 569.3060.
2.1.10.11. N-(20-fluorobenzenesulfonyl)-3,7-dioxo-5b-cholan-24-amide
25
(10k). 124 mg (87% yield) as a colorless amorphous solid. [a]
D
ꢀ15.0 °C (c 0.2, CHCl3); IR (KBr) mmax 3290, 2946, 2872, 1708,
1595, 1466, 1336, 1162, 1079, 764, 591 cmꢀ1 1H NMR (500 MHz,
2.1.10.14. N-(40-(trifluoromethoxy)benzenesulfonyl)-3,7-dioxo-5b-
.
cholan-24-amide (10n). 131 mg (83% yield) as a colorless amor-
25
CDCl3-d) d 0.65 (3H, s, 18-CH3), 0.84 (3H, d, J = 6.4 Hz, 21-CH3),
1.29 (3H, s, 19-CH3), 0.92–2.89 (m, 26H), 2.96 (2H, m, 24-H), 5.15
(1H, br.s., –NHSO2–), 7.20 (1H, t, J = 9.4 Hz), 7.28 (1H, m), 7.57
(1H, m), 7.88 (1H, t, J = 7.4 Hz). 13C NMR (125 MHz, CDCl3-d) d
12.4, 19.0, 22.5, 22.8, 25.1, 26.6, 28.6, 33.1, 35.6 35.8, 35.8, 37.1,
39.2, 43.0, 43.3, 43.3, 44.2, 45.4, 48.1, 49.2, 49.9, 55.2, 117.2,
117.4, 124.8, 128.7, 130.7, 135.2, 158.2, 160.2, 210.8, 211.7. HRMS
(TOF ES+): m/z calcd for C30H42FNNaO4S+ [(M+Na)+], 554.2711;
found, 554.2704.
phous solid. [
a]
ꢀ11.2 °C (c 0.25, CHCl3); IR (KBr) mmax 3476,
D
3283, 2947, 2868, 1709, 1434, 1258, 1215, 1163, 1092, 819, 695,
603 cmꢀ1 1H NMR (500 MHz, CDCl3-d) d 0.66 (3H, s, 18-CH3),
.
0.84 (3H, d, J = 6.4 Hz, 21-CH3), 1.31 (3H, s, 19-CH3), 1.00–2.96
(m, 26H), 2.92 (2H, m, 24-H), 5.37 (1H, t, J = 5.8 Hz, -NHSO2-),
7.35 (2H, d, J = 8.4 Hz, 30-ArH and 50-ArH), 7.94 (2H, d, J = 8.4 Hz,
20-ArH and 60-ArH). 13C NMR (125 MHz, CDCl3-d) d 12.4, 18.9,
22.5, 22.7, 25.1, 26.6, 28.7, 33.1, 35.6 35.8, 35.8, 37.1, 39.2, 43.0,
43.3, 43.3, 44.1, 45.4, 48.1, 49.2, 49.9, 55.2, 121.4, 121.4, 129.6,
129.6, 139.1, 139.1, 152.3, 211.0, 211.8. HRMS (TOF ESꢀ): m/z calcd
for C31H41F3NO5Sꢀ [(MꢀH)ꢀ], 596.2663; found, 596.2660.
2.1.10.12. N-(40-nitrobenzenesulfonyl)-3,7-dioxo-5b-cholan-24-amide
25
(10l). 126 mg (84% yield) as a colorless amorphous solid. [
ꢀ15.5 °C (c 0.2, CHCl3); IR (KBr)
1623, 1334, 1011, 842, 551 cmꢀ1
a]
D
m
max 3456, 3285, 2945, 2868, 1706,
.
1H NMR (500 MHz, CDCl3-d) d
2.1.10.15. N-(thiophene-2-sulfonyl)-3,7-dioxo-5b-cholan-24-amide
25
0.63 (3H, s, 18-CH3), 0.83 (3H, d, J = 6.5 Hz, 21-CH3), 1.25 (3H, s, 19-
CH3), 0.88–2.89 (m, 26H), 2.96 (2H, m, 24-H), 5.37 (1H, t, J = 5.8 Hz,
–NHSO2–), 8.05 (2H, t, J = 8.7 Hz, 20-ArH and 60-ArH), 8.35 (2H, d,
J = 8.7 Hz, 30-ArH and 50-ArH). 13C NMR (125 MHz, CDCl3-d) d 12.4,
19.0, 22.5, 22.8, 25.1, 26.7, 28.7, 33.0, 35.5, 35.8, 35.8, 37.1, 39.2,
43.0, 43.3, 43.3, 44.3, 45.4, 48.1, 49.3, 49.9, 55.2, 124.8, 124.8,
128.7, 128.7, 146.7, 150.4, 210.9, 211.8. HRMS (TOF ESꢀ): m/z calcd
for C30H41N2O6Sꢀ [(MꢀH)ꢀ], 557.2691; found, 557.2686.
(10o). 111 mg (80% yield) as a colorless amorphous solid. [a]
D
ꢀ18.0 °C (c 0.2, CHCl3); IR (KBr) mmax 3278, 2945, 2871, 1707,
1430, 1333, 1156, 1081, 1014, 727, 589 cmꢀ1 1H NMR (500 MHz,
.
CDCl3-d) d 0.67 (3H, s, 18-CH3), 0.87 (3H, d, J = 6.4 Hz, 21-CH3),
1.30 (3H, s, 19-CH3), 0.94–2.90 (m, 26H), 3.01 (2H, m, 24-H), 5.09
(1H, br.s., –NHSO2–), 7.10 (1H, m), 7.60 (2H, m). 13C NMR
(125 MHz, CDCl3-d) d 12.4, 19.0, 22.5, 22.8, 25.1, 26.5, 28.7, 33.2,
35.6, 35.8, 35.8, 37.1, 39.2, 43.0, 43.3, 43.3, 44.4, 45.4, 48.1, 49.2,
50.0, 55.3, 127.8, 132.0, 132.3, 141.7, 210.8, 211.7. HRMS (TOF
ESꢀ): m/z calcd for C28H40NO4S2 [(MꢀH)ꢀ], 518.2404; found,
ꢀ
2.1.10.13. N-(40-(acetamide)benzenesulfonyl)-3,7-dioxo-5b-cholan-
24-amide (10m). 119 mg (78% yield) as a colorless amorphous so-
518.2408.
O
O
OH
O
OH
c
H
H
H
H
H
H
H
H
H
a
H
H 1
H
H
H
b
H
H3
H
HO
HO
O
OH
HO
O
OH
HO
O
OH
H
2
OTr
d
OTr
e
OH
f
H
H 4
H
H
H 5
H
O
H
H
O
OH
H
H
6
O
O S
O
N3
h
NH2
i
g
H
H
O
H
H
O
H
H
O
O
O
H
H
8
9
7
O
HN S R
O
H
H
H 10
H
O
O
Scheme 1. Synthesis of N-sulfonyl-3,7-dioxo-5b-cholan-24-amides. Reagents and conditions: Reagents and conditions: (a) SOCl2, CH3OH, reflux, 3 h, 95%; (b) LiAlH4, THF, rt,
1 h, 92%; (c) triphenylmethyl chloride (TrCl), pyridine, 90 °C, 24 h, 85%; (d) CrO3, pyridine, CH2Cl2, rt, 0.5 h, 95%; (e) TsOH, acetone, rt, 10 min, quantitative; (f) MsCl, CH2Cl2,
0 °C, 1 h, 92%; (g) NaN3, DMF, 70 °C, 3 h, 80%; (h) H2, Pd/C, CH3OH, rt, 5 h, 82%; (i) RSO2Cl, pyridine, rt, 12 h, 78–94%.

J. Ren et al. / Steroids 78 (2013) 53–58
57
Table 1
2.2. Biology
The tumor cell lines (HCT-116, MCF-7, K562, and SGC-7901)
Human cancer cell line growth inhibition values in vitroa (IC50
,
l
M/mLb) for the
synthesized compounds.
Compounds
R
IC50/(lM)
were obtained from Shanghai Institute of Pharmaceutical Industry.
The cytotoxic activity in vitro was measured using the MTT assay.
HCT-116 MCF-7 K562 SGC-7901
The MTT solution (10.0 lL/well) was added in RPMI-1640 media
(Sigma, St. Louis, MO) after cells were treated with the drug for
72 h, and cells were incubated for further 4 h at 37 °C. The purple
formazan crystals were dissolved in 100.0 lL DMSO. After
10 min, the plates were read on an automated microplate spectro-
photometer (Bio-Tek Instruments, Winooski, VT) at 570 and
630 nm. Assays were performed in triplicate on three independent
experiments. The concentration required for 50% inhibition of cell
viability (IC50) was calculated using the software ‘‘Dose–Effect
Analysis with Microcomputers’’. The tumor cell lines panel con-
sisted of HCT-116, MCF-7, K562, and SGC-7901. In all of these
experiments, three replicate wells were used to determine each
point.
6
7
8
9
10a
10b
10c
10d
–
–
–
–
70.23
31.61
88.5
24.11
13.74
3.39
76.38 39.44 73.26
42.4
65.2
38.5
>100 >100
24.34 >100
61.29 63.32
–CH3
–CH2CH3
–CH2CH2CH2CH3
24.47 16.58 53.04
15.89 12.75 26.79
9.07
15.46
8.99
6.54 59.2
15.29 6.76 72.75
10e
12.81
35.04 10.73 >100
H2
C
10f
10g
10h
10i
10j
3.71
8.86
9.05
8.49
16.4
8.62
9.34
4.94 >100
2.39 >100
CH3
Br
3. Results and discussion
10.36 4.51 >100
16.27 4.68 >100
18.28 6.98 >100
Cl
Cl
3.1. Chemistry
F
The synthetic route of target compounds is shown in Scheme 1.
Synthesis of amine 9 started from commercially available ursode-
oxycholic acid. A solution of ursodeoxycholic acid and thionyl chlo-
ride was refluxed for 2 h. After cooling to room temperature, the
solution was evaporated to dryness to form ester 2 [20]. Subse-
quently, the ester group of compound 2 was reduced by lithium
aluminum hydride in dry tetrahydrofuran to generate correspond-
ing compound 3. Then, 3 was selectively protected in the position
of primary alcohol by treatment with triphenylmethyl chloride
(TrCl) in the presence of catalytic amount of 4-dimethylaminopyr-
idine in pyridine to provide the key precursor 4 [21]. Next, Collins
oxidation furnished the ketone 5, followed by deprotection of the
Tr ether to afford 6 in quantitative yield. The mesylate 7 was pre-
pared from 6 by treatment with methanesulfonyl chloride, fol-
lowed by conversion of 7 in dimethylformamide with sodium
azide to give the azide 8. Thereafter, hydrogenation of 8 with Pd/
C in methanol provided amine 9. Finally, amine 9 was treated with
various sulfuryl chloride and refluxed in pyridine for 2–5 h to af-
ford various, N-sulfonyl-3,7-dioxo-5b-cholan-24-amides. All the
new compounds synthesized were characterized by 1H NMR, 13C
NMR, HRMS, and IR spectroscopy.
F
10k
12.06
9.3
5.19 >100
F
10l
11.97
6.16
50
16.4
7.37 72.36
NO2
10m
10n
10o
13.84 11.37 78.49
14.77 5.89 >100
31.31 9.48 68.97
NHCOCH3
OCF3
50
S
5-fu
1.93
14.18 45.4 2.19
a
Cytotoxicity as IC50 for each cell line, is the concentration of compound which
reduced by 50% the optical density of treated cells with respect to untreated cells
using the MTT assay.
b
Data represent the mean values of three independent determinations.
3.2. Biological activity
gated concentration range (>100
below 50 M. For the four cell lines, all sulfonamide compounds
displayed the higher cytotoxicity to K562 cell, which was superior
to 5-FU (IC50 = 45.4 M). Among 10a–o, 10g was found to have the
best activity on K562 cell line (IC50 = 2.39 M). Interestingly, the
result that all compounds showed potent activity for human colon
cancer cell line (HCT-16) was consistent to our design. In addition,
the comparisons of 7 and 10a revealed that the cytotoxicities of N-
sulfonyl-3,7-dioxo-5b-cholan-24-amides were more potent than
that of 3,7-dioxo-5b-cholan-24-sulfonate.
The results presented in Table 1 implied that the nature of the
substituent groups at the sulfonamide core play a critical role in
antitumor potency. Compounds 10a, 10b, and 10c with the ali-
phatic chain of methyl, ethyl, and n-butyl, respectively, exhibited
increasing cytotoxicity against MCF-7 and K562 cancer cells, sug-
gesting that the length of the aliphatic chain of the sulfonamide
groups is critical for the compound0s cytotoxic activity. Neverthe-
less, for HCT-116, ethyl substitution was superior to the others.
In addition, replacement of the aliphatic groups of the sulfonamide
lM), with only 10b having IC50
l
All the synthesized N-sulfonyl-3,7-dioxo-5b-cholan-24-amides
s were evaluated in vitro against a panel of four cancer cell lines
including HCT-116 (human colon cancer cell line), MCF-7 (human
breast adenocarcinoma cell line), K562 (human leukemia cell line),
and SGC-7901 (human gastric cancer cell line) by the standard
microculture tetrazolium (MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide) assay using 5-FU(5-Fluorouracil)
as a positive control. Antitumor potency of the compounds was
indicated by IC50 values that were calculated by linear regression
analysis of the concentration–response curves obtained for each
compound. The results of the cytotoxicity studies are summarized
in Table 1 (IC50 value, defined as the concentration corresponding
to 50% growth inhibition).
l
l
As shown in Table 1, all of N-sulfonyl-3,7-dioxo-5b-cholan-24-
amides exhibited potent inhibitory effects on HCT-116, MCF-7,
and K562 cell lines. Sulfonamides 10a–o, compared to amide 9,
displayed a higher cytotoxicity. However, these compounds dis-
played low cytotoxicity against SGC-7901 cell line in the investi-

58
J. Ren et al. / Steroids 78 (2013) 53–58
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In summary, a series of N-sulfonyl-3,7-dioxo-5b-cholan-24-
amides, ursodeoxycholic acid analogs, have been prepared and
evaluated against HCT-116, MCF-7, K562, and SGC-7901 cell lines.
The results showed that more of the prepared sulfonamides dis-
played the selective cytotoxicity toward HCT-116, MCF-7 and
K562 cell lines. From the structure–activity relationships we may
conclude that the introduction sulfonamide groups at 24-position
in 3,7-dioxo-5b-cholan is associated with enhanced cytotoxic
activity. This study may provide valuable information for further
designing and developing more potent anticancer agents.
Acknowledgements
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We sincerely thank the financial support from the National Nat-
ural Science Foundation of China (Grant No. 20902078).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.steroids.
2012.09.009.
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