Deoxycholic acid derivatives as inhibitors of P-glycoprotein-mediated multidrug efflux

Article abstract of DOI:10.1016/j.steroids.2016.09.017

Deoxycholic acid derivatives were designed as P-glycoprotein (Pgp, ABCB1) inhibitors. Thus the synthesis and the biological activity of methyl deoxycholate derivatives 5–10 and their ether analogs 15–20 have been reported. The potency of these compounds to modulate Pgp-mediated MDR was evaluated through daunorubicin accumulation and potentiation of doxorubicin cytotoxicity in K562/R7 multidrug resistant cells overexpressing Pgp. In parallel, their intrinsic toxicity was appreciated on K562 sensitive cells. Methyl 12α-[(2R or 2S) tetrahydro-2H-pyran-2-yloxy]-3-oxo-5β-cholan-24-oate 9b has shown a good efficiency as a Pgp inhibitor and a low intrinsic toxicity. Therefore, this derivative constitutes a new lead compound which can be used as a starting point to improve the design of non-toxic Pgp modulators.

Full text of DOI:10.1016/j.steroids.2016.09.017

Steroids 116 (2016) 5–12
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Deoxycholic acid derivatives as inhibitors of P-glycoprotein-mediated
multidrug efflux
b
a
a
a
Luc Rocheblave a, , Marc Rolland de Ravel , Elodie Monniot , Jeremy Tavenard , Claude-Yves Cuilleron ,
⇑
Catherine Grenot a, Sylvie Radix a, Eva-Laure Matera b, Charles Dumontet b, Nadia Walchshofer a,
⇑
a Univ Lyon, Université Claude Bernard Lyon 1, ISPB-Faculté de Pharmacie, EA 4446, 8 avenue Rockefeller, F-69373 Lyon Cedex 08, France
b Univ Lyon, Université Claude Bernard Lyon 1, INSERM 1052, CNRS UMR5286, Centre Léon Bérard, Centre de Recherche en Cancérologie de Lyon, Cheney D, 28 rue Laënnec,
F-69373 Lyon Cedex 08, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Deoxycholic acid derivatives were designed as P-glycoprotein (Pgp, ABCB1) inhibitors. Thus the synthesis
and the biological activity of methyl deoxycholate derivatives 5–10 and their ether analogs 15–20 have
been reported. The potency of these compounds to modulate Pgp-mediated MDR was evaluated through
daunorubicin accumulation and potentiation of doxorubicin cytotoxicity in K562/R7 multidrug resistant
cells overexpressing Pgp. In parallel, their intrinsic toxicity was appreciated on K562 sensitive cells.
Received 30 August 2016
Accepted 27 September 2016
Available online 30 September 2016
Keywords:
Methyl 12a-[(2R or 2S) tetrahydro-2H-pyran-2-yloxy]-3-oxo-5b-cholan-24-oate 9b has shown a good
Multidrug resistance
P-glycoprotein inhibitors
Deoxycholic acid derivatives
efficiency as a Pgp inhibitor and a low intrinsic toxicity. Therefore, this derivative constitutes a new lead
compound which can be used as a starting point to improve the design of non-toxic Pgp modulators.
Ó 2016 Elsevier Inc. All rights reserved.
1. Introduction
active than compound 1 but also more toxic, like most cholic acid
derivatives compared to their 5 b-dihydroprogesterone analogs.
P-glycoprotein Pgp (ABCB1) [1] is an efflux pump which belongs
to the ATP-Binding Cassette (ABC) family. This transporter is lar-
gely involved in the multidrug resistance MDR, which is a major
impediment for cancer chemotherapy [2], decreasing intracellular
concentration of a wide variety of chemotherapeutic agents includ-
ing recent tyrosine kinase inhibitors [3].
Numerous Pgp inhibitors were currently developed in order to
suppress Pgp-mediated MDR, but none of them has been approved
as a drug, mainly due to toxicity effects [4].
Therefore, our study was carried out to design new bile acids
derivatives with retained potency as Pgp modulators but less toxic
than 2. Hence we reported here the synthesis of the methyl deoxy-
cholate derivatives 5–10 and their ether analogs 15–20. The
potency of these compounds to modulate Pgp-mediated MDR
was evaluated through daunorubicin accumulation and potentia-
tion of doxorubicin cytotoxicity in K562/R7 multidrug resistant
cells overexpressing Pgp. In parallel, their intrinsic toxicity was
appreciated on K562 sensitive cells.
The report of the weak activity of progesterone as a Pgp inhibi-
tor [5] led to further studies on steroid compounds. Yet only a few
reports have mentioned that bile acids or their salts could modu-
late Pgp activity [6] although these derivatives are mainly trans-
ported by a member of ABC transporters, the so-called Sister of
P-glycoprotein (ABCB11) [7]. As part of our work about the design
of steroidal modulators of Pgp [8], we described potent inhibitors
as 5b-dihydroprogesterone derivative 1 and cholic acid derivative
2 (Fig. 1) [9]. Compounds 1 and 2 have shown a better efficiency
than conventional modulators such as mifepristone (RU486), vera-
pamil and cyclosporine A (CsA). Compound 2 was found more
2. Materials and methods
2.1. General experimental procedures
All air- and moisture-sensitive reactions were carried out under
argon atmosphere. All commercially available chemicals were used
as received except where noted. Melting points were determined
with an Electrothermal 9300 capillary apparatus. 1H spectra were
recorded with Bruker DRX400 Fourier transform spectrometer,
using an internal deuterium lock, operating at 400 MHz. 13C NMR
spectra were recorded with Bruker DRX400 Fourier transform
spectrometer, using an internal deuterium lock, operating at
100 MHz. Chemical shifts are reported in part per million (ppm)
relative to residual protons of CDCl3 (d = 7.26 ppm for 1H NMR
⇑
Corresponding authors.
E-mail addresses: luc.rocheblave@univ-lyon1.fr (L. Rocheblave), nadia.walch-
shofer@univ-lyon1.fr (N. Walchshofer).
http://dx.doi.org/10.1016/j.steroids.2016.09.017
0039-128X/Ó 2016 Elsevier Inc. All rights reserved.

6
L. Rocheblave et al. / Steroids 116 (2016) 5–12
O
O
C
CH3
O
(R)
O
O
COOCH3
O
O
C
H
O
O
O
H
O
1
2
OR
OR
COOCH3
CH2OCH3
1'
O
6'
4'
O
O
H
H
2'
5'
1'
5
6
7
8
THP =
DHP =
R = COCH3
R = COC6H5
R = CH2C6H5
R = CH2OCH3
R = THP (S/R)
R = DHP
15
16
17
18
19a,b
20
*
3'
R = COCH3
R = COC6H5
R = CH2C6H5
R = CH2OCH3
R = THP (S/R)
R = DHP
3'
2'
9a,b
10
O
4'
5'
6'
Fig. 1. Compounds 1 and 2, identified as Pgp inhibitors in a previous study [9] and compounds 5–10/15–20 described in this study. DHP and THP: numbering system for NMR
data.
and d = 77.16 ppm for 13C NMR) and coupling constants J are
reported in hertz (Hz). Carbon multiplicity was determined by
DEPT experiments. The following abbreviations are used: s: sin-
glet; br s: broad signal; d: doublet; t: triplet; m: multiplet. Chro-
matographic separations were performed on silica gel columns
(Kieselgel 300–400 mesh). Preparative thin layer chromatography
plates were prepared using Kieselgel Silica Gel 60 F254 for thin-
layer chromatography. All reactions were monitored by TLC on
GF254 plates and spots were detected under a UV lamp (254 nm)
or by spraying plates with 10% v/v aqueous H2SO4 followed by dry-
ing with heat gun. Low- and high-resolution mass spectra were
performed on a ThermoFinnigan MAT 95 XL apparatus operating
at 70 eV.
2.2.3. Methyl 12a-(acetyloxy)-3-oxo-5b-cholan-24-oate (5)
To a stirred solution of compound 4 (150 mg, 0.37 mmole) and
N,N-dimethylaminopyridine (DMAP) (cat.) in pyridine (1 mL) at
room temperature under argon atmosphere was added dropwise
acetic anhydride (1 mL, 10.58 mmol). After stirring for 12 h, the
reaction mixture was cooled down to 0 °C and methanol (1 mL)
was added. After evaporation, the residue was dissolved in ethyl
acetate. The organic layer was washed with 1 M HCl (3 times),
10% w/v aqueous NaHCO3 (3 times) and brine. After drying by fil-
tration through a phase separation paper, the evaporation of all
volatiles afforded the crude compound. This dry residue was puri-
fied by flash chromatography (petroleum ether/ethyl acetate 4:1)
to give the title compound 5 (139 mg, 84%) as a colorless amor-
phous solid. Mp = 121–122 °C; 1H NMR (400 MHz, CDCl3) d 0.75
(s, 3H, 18-CH3), 0.80 (d, 3H, J = 6.36 Hz, 21-CH3), 0.98 (s, 3H, 19-
CH3), 2.06 (s, 3H, 12-OCOCH3), 2.68 (m, 1H), 3.65 (s, 3H, 24-
COOCH3), 5.11 (br s, 1H, 12-CH); 13C NMR (100 MHz, CDCl3) d
213.11, 174.72, 170.57, 75.86, 51.67, 49.51, 47.71, 45.18, 44.13,
42.35, 37.05, 36.69, 35.45, 34.78, 34.80, 34.38, 31.08, 30.91,
27.43, 26.53, 26.06, 25.46, 23.53, 22.48, 21.43, 17.64, 12.57; HRMS:
calcd for C27H42O5[M+Na]+, m/z 469.2924; found, 469.2926.
2.2. Synthetic methods
2.2.1. Methyl 3
a,12a-dihydroxy-5b-cholan-24-oate [10] (methyl
deoxycholate) (3)
A solution of deoxycholic acid (4.0 g, 10.2 mmol) in methanol
(30 mL) was stirred for 3 h at room temperature in presence of
1 mL of conc. HCl. The reaction was quenched with addition of sat-
urated aqueous NaHCO3. After evaporation of all volatiles, the
aqueous phase was extracted by ethyl acetate (3 times). The com-
bined organic phases were washed with water (3 times), dried over
Na2SO4 and concentrated under reduced pressure. The colorless
residue corresponding to the compound 3 (3.82 g, 94%) was
employed without further purification in the following step.
2.2.4. Methyl 12a-(benzoyloxy)-3-oxo-5b-cholan-24-oate (6)
A solution of compound 4 (100 mg, 0.25 mmol) and benzoyl
chloride (0.17 mL, 1.465 mmol) in pyridine (2.5 mL) was stirred
at room temperature for 72 h. The progress of the reaction was
monitored by TLC. The reaction mixture was cooled down to 4 °C
in an ice-bath and a 1:1 mixture of ethyl acetate and saturated
aqueous NaHCO3 (10 mL) was added. After stirring for 30 min,
the aqueous phase was extracted with ethyl acetate (3 times).
The combined organic phases were washed with water, dried by
filtration through a phase separation paper and concentrated
under reduced pressure. The dry residue was purified by prepara-
tive TLC (petroleum ether/ethyl acetate 3:1, RF 0.43) to give the
title compound 6 (96 mg, yield 76%) as an amorphous solid.
Mp = 40 °C; 1H NMR (400 MHz, CDCl3) d 0.81 (d, 3H, J = 6.6 Hz,
21-CH3), 0.84 (s, 3H, 18-CH3), 1.01 (s, 3H, 19-CH3), 2.63 (m, 1H),
3.61 (s, 3H, 24-COOCH3), 5.37 (br s, 1H, 12-CH), 7.46 (m, 2H,
2.2.2. Methyl 12a-hydroxy-3-oxo-5b-cholan-24-oate [11] (4)
A solution of ester 3 (7.1 g, 17.46 mmol) in toluene (250 mL)
was stirred for 3 h under reflux in a Dean-Stark water trap in pres-
ence of Ag2CO3/Celite reagent (50% w/w, 49.7 g, 90 mmol). The
reaction mixture was cooled down to room temperature, filtered
on a Celite pad and washed with toluene. The combined filtrates
were evaporated under reduced pressure. The title compound 4
(6.49 g, 92%) was recovered as an amorphous solid employed with-
out further purification in the following step. Mp = 101–102 °C.

L. Rocheblave et al. / Steroids 116 (2016) 5–12
7
m-ArH), 7.58 (m, 1H, p-ArH), 8.02 (m, 2H, o-ArH); 13C NMR
(100 MHz, CDCl3) 213.08, 174.68, 165.93, 133.28, 130.55,
129.44 (2C), 128.78 (2C), 76.40, 51.61, 50.09, 48.10, 45.70, 44.18,
42.33, 36.93, 36.73, 35.59, 35.05, 34.88, 34.43, 31.08, 30.89,
27.48, 26.55, 26.16, 25.60, 23.63, 22.53, 17.57, 12.70; HRMS: calcd
for C32H44O5[M+Na]+, m/z 531.3081; found, 531.3066.
44 mg, 18%) as an amorphous solid and the more polar
d
diastereoisomer 9b (RF 0.46, 26 mg, yield 11%) as a colorless oil.
Presumed 12a
-(S)OTHP isomer 9a: Mp 65–66 °C; 1H NMR
(400 MHz, CDCl3) d 0.69 (s, 3H, 18-CH3), 0.88 (d, 3H, J = 6.23 Hz,
21-CH3), 0.98 (s, 3H, 19-CH3), 2.75 (m, 1H), 3.40 (m, 1H, 60-CHH),
3.65 (s, 3H, 24-COOCH3), 3.78 (br s, 1H, 12-CH), 3.86 (m, 1H, 60-
CHH), 4.47 (m, 1H, 20-CH); 13C NMR (100 MHz, CDCl3) d 214.48,
174.88, 101.67, 82.85, 64.22, 51.64, 48.64, 46.65, 46.37, 44.60,
42.63, 37.23, 36.96, 35.81, 35.46, 34.86, 34.24, 31.89, 31.16,
31.04, 27.71, 26.90, 26.68, 25.67, 25.44, 23.88, 22.69, 21.24,
17.90, 12.66; HRMS: calcd for C30H48O5[M+Na]+, m/z 511.3394;
found, 511.3381.
2.2.5. Methyl 12a-(benzyloxy)-3-oxo-5b-cholan-24-oate (7)
To a vigorously stirred solution of compound 4 (100 mg,
0.25 mmol), silver triflate (137 mg, 0.53 mmol) and 2,6-di-tert-
butylpyridine (0.125 mL, 0.54 mmol) in dry CH2Cl2 (1.5 mL), was
added dropwise
a solution of benzyl bromide (0.122 mL,
Presumed 12a
-(R)OTHP isomer 9b: 1H NMR (400 MHz, CDCl3) d
0.57 mmol) in dry CH2Cl2 (0.5 mL). The mixture was refluxed for
3 h. After cooling down to room temperature, the precipitate was
filtered off and rinsed with CH2Cl2. The combined filtrates were
washed with 5 M HCl (3 times) and water (3 times), dried over
anhydrous Na2SO4 and concentrated under reduced pressure. The
residue was purified by flash chromatography on silica gel (petro-
leum ether/ethyl acetate 5:1) to give the title compound 7 (54 mg,
44%) as a pale yellow oil. 1H NMR (CDCl3, 400 MHz) d 0.74 (s, 3H,
18-CH3), 0.92 (d, 3H, J = 6.3 Hz, 21-CH3), 1.01 (s, 3H, 19-CH3),
2.37–1.10 (massif, 28H), 2.67 (m, 1H), 3.66 (s, 3H, COOCH3), 3.74
(br s, 1H, 12-CH), 4.38 (d, 1H, J = 11.5 Hz, O-CH2Ar), 4.54 (d, 1H,
J = 11.5 Hz, O-CH2Ar), 7.33–7.26 (massif, 5H, ArH); 13C NMR (CDCl3,
100 MHz) d 213.39, 174.91, 139.25, 128.54 (2C), 127.63, 127.43
(2C), 81.26, 70.75, 51.64, 48.85, 46.76, 46.51, 44.53, 42.50, 37.19,
37.11, 35.82, 35.40, 34.61, 34.19, 31.13, 27.69, 27.11, 26.75,
25.55, 23.80, 23.62, 22.68, 17.74, 12.90; HRMS: calcd for
0.73 (s, 3H, 18-CH3), 0.99 (d, 3H, J = 7.3 Hz, 21-CH3), 1.00 (s, 3H,
19-CH3), 2.69 (m, 1H), 3.45 (m, 1H, 60-CHH), 3.66 (s, 3H, 24-
COOCH3), 3.94 (m, 1H, 60-CHH), 3.98 (br s, 1H, 12-CH), 4.66 (m,
1H, 20-CH); 13C NMR (100 MHz, CDCl3) d 213.38, 175.00, 96.54,
77.47, 63.58, 51.60, 48.32, 46.60, 45.93, 44.47, 42.55, 37.26,
37.04, 36.20, 35.83, 34.66, 34.11, 32.35, 31.34, 31.25, 28.16,
26.84, 25.67, 25.65, 24.26, 24.08, 22.69, 20.40, 17.30,13.00; HRMS:
calcd for C30H48O5 [M+Na]+, m/z 511.3394; found, 511.3379.
2.2.8. Methyl 12a-(3,6-dihydro-2H-pyran-4-yloxy)-3-oxo-5b-cholan-
24-oate (10)
A solution of compound 4 (150 mg, 0.38 mmol), 4-methoxy-3,6-
dihydro-2H-pyrane (0.300 mL, 2.69 mmol) and p-toluenesulfonic
acid (1.5 mg) in anhydrous THF (1.5 mL) was stirred at room tem-
perature under argon atmosphere. The progress of the reaction was
monitored by TLC (petroleum ether/ethyl acetate 2:1, RF 0.49).
After 48 h, the reaction was quenched with saturated aqueous
NaHCO3. The aqueous phase was extracted with ethyl acetate (3
times). The combined organic phases were washed with water,
dried by filtration through a phase separation paper and concen-
trated under reduced pressure. The residue (228 mg) was purified
by flash chromatography on silica gel (petroleum ether/ethyl acet-
ate 3:1) to give the title compound 10 (120 mg, 67%) as a colorless
oil. 1H NMR (400 MHz, CDCl3) d 0.74 (s, 3H, 18-CH3), 0.84 (d, 3H,
J = 6.24 Hz, 21-CH3), 0.99 (s, 3H, 19-CH3), 2.68 (m, 1H), 3.66 (s,
3H, 24-COOCH3), 3.74–3.85 (massif, 2H, 20-CH2), 4.10–4.21 (massif,
2H, 60-CH2), 4.29 (br s, 1H, 12-CH), 4.55 (s, 1H, 50-CH); 13C NMR
(100 MHz, CDCl3) d 213.38, 174.84, 149.79, 93.81, 75.95, 64.82,
64.74, 51.64, 49.08, 47.01, 45.88, 44.26, 42.40, 36.95, 36.93,
35.50, 34.93, 34.62, 34.42, 31.01 (2C), 28.67, 27.48, 26.57, 25.51,
23.63, 23.48, 22.55, 17.70, 12.73; HRMS: calcd for C30H46O5[M
+Na]+, m/z 509.3237 found, 509.3235.
C
32H46O4[M+Na]+, m/z 517.3288; found, 517.3265.
2.2.6. Methyl 12 -(methoxymethoxy)-3-oxo-5b-cholan-24-oate (8)
a
A solution of compound 4 (100 mg, 0.25 mmol), N,N-diiso-
propylethylamine (0.11 mL, 0.63 mmol) and chloromethyl methyl
ether (0.05 mL, 0.66 mmol) in CH2Cl2 (1.3 mL) was stirred at room
temperature. The progress of the reaction was monitored by TLC
(petroleum ether/ethyl acetate 3:1, RF 0.40). After 72 h, the reac-
tion was quenched with saturated aqueous NaHCO3. The aqueous
phase was extracted with ethyl acetate (3 times). The combined
organic phases were washed with water, dried by filtration
through a phase separation paper and concentrated under reduced
pressure. The residue (82 mg) was purified by preparative TLC
(petroleum ether/ethyl acetate 4:1, RF 0.17) to give the title com-
pound 8 (81 mg, 73%) as an amorphous solid. Mp = 54–55 °C; 1H
NMR (400 MHz, CDCl3) d 0.71 (s, 3H, 18-CH3), 0. 92 (d, 3H,
J = 6.36 Hz, 21-CH3), 0.99 (s, 3H, 19-CH3), 2.71 (m, 1H), 3.36 (s,
3H, OCH2OCH3), 3.65 (s, COOCH3), 3.80 (br s, 12-CH), 4.63 (d, 1H,
J = 6.6 Hz, OCH2OCH3), 4.70 (d, 1H, J = 6.6 Hz, OCH2OCH3); 13C
NMR (100 MHz, CDCl3) d 213.54, 174.79, 96.78, 81.24, 56.21,
51.61, 48.52, 46.58, 46.46, 44.40, 42.50, 37.12, 37.02, 35.76,
35.66, 34.72, 34.36, 31.22, 31.11, 27.84, 26.81, 25.93, 25.70,
23.93, 22.64, 17.65, 12.76; HRMS: calcd for C27H44O5[M+Na]+, m/
z 471.3081; found, 471.3063.
2.2.9. Methyl 3,3-(ethylenedioxy)-12a-hydroxy-5b-cholan-24-oate
(11)
A solution of compound 4 (5 g, 12.35 mmol), ethylene glycol
(80 mL) and p-toluenesulfonic acid (360 mg) in toluene (280 mL)
was stirred for 1.5 h under reflux using a Dean-Stark water trap.
The reaction mixture was cooled at 4 °C, neutralized with solid
NaHCO3 then diluted with saturated aqueous NaHCO3. The aque-
ous phase was extracted with ethyl acetate (3 times). The com-
bined organic phases were washed with water, dried by filtration
through a phase separation paper and concentrated under reduced
pressure to give the title compound 11 (5.4 g, 97%) as an amor-
phous solid which was employed without further purification in
the following step. RF 0.48 (CH2Cl2/ethyl acetate 2:1). Mp = 99–
100 °C; 1H NMR (400 MHz, CDCl3) d 0.66 (s, 18-CH3), 0.92 (s, 19-
CH3), 0.95 (d, 3H, J = 6.24 Hz, 21-CH3), 3.64 (s, 3H, COOCH3), 3,92
(s, 4H, 3-OCH2CH2O), 3.96 (m, 1H, 12-CH); 13C NMR (100 MHz,
CDCl3) d 174.85, 110.14, 73.22, 64.31, 64.17, 51.63, 48.35, 47.40,
46.61, 40.99, 35.94, 35.74, 35.20, 34.24, 34.13, 33.10, 31.17,
30.99, 30.08, 29.05, 27.54, 26.79, 26.00, 23.72, 23.04, 17.40, 12.88.
2.2.7. Methyl 12a-[(2R or 2S) tetrahydro-2H-pyran-2-yloxy]-3-oxo-
5b-cholan-24-oate (9a) and (9b)
A solution of compound 4 (200 mg, 0.49 mmol), freshly distilled
3,4-dihydro-2H-pyrane (1 mL, 10.94 mmol) and p-toluenesulfonic
acid (5 mg) in anhydrous THF (4 mL) was stirred for 1 h at room
temperature. The reaction was quenched with saturated aqueous
NaHCO3. The aqueous phase was extracted with ethyl acetate (3
times). The combined organic phases were washed with water,
dried by filtration through a phase separation paper and concen-
trated under reduced pressure. The residue was purified by prepar-
ative TLC (petroleum ether/ methyl tert-butyl ether 6:1, 10
developments) to give the less polar diastereoisomer 9a (RF 0.54,

8
L. Rocheblave et al. / Steroids 116 (2016) 5–12
2.2.10. 3,3-(ethylenedioxy)-5b-cholane-12
a
,24-diol (12)
reaction mixture was cooled down to 0 °C and methanol (1 mL)
was added. After evaporation, the residue was dissolved in ethyl
acetate. The organic layer was washed with 1 M HCl (3 times),
10% w/v aqueous NaHCO3 (3 times) and brine. After drying by fil-
tration through a phase separation paper, the evaporation of all
volatiles afforded the crude compound. This dry residue was puri-
fied by flash chromatography on silica gel (petroleum ether/ethyl
acetate 4:1) to give the title compound 15 (102 mg, 92%) as an
amorphous solid; Mp = 110–111 °C;1H NMR (400 MHz, CDCl3) d
0.75 (s, 3H, 18-CH3), 0.81 (d, 3H, J = 6.6 Hz, 21-CH3), 0.99 (s, 3H,
19-CH3), 2.05 (s, 3H, 12-OCOCH3), 2.69 (m, 1H), 3.29–3.35 (massif,
5H, CH2OCH3, 24-OCH3), 5.12 (br s, 1H, 12-CH); 13C NMR (100 MHz,
CDCl3) d 213.19, 170.62, 75.97, 73.50, 58.71, 49.53, 47.87, 45.15,
44.17, 42.37, 37.07, 36.71, 35.48, 35.05, 34.81, 34.40, 32.19,
27.51, 26.55, 26.27, 26.06, 25.48, 23.55, 22.49, 21.45, 17.98,
12.59; HRMS: calcd for C27H44O4[M+Na]+, m/z 455.3132; found,
455.3123.
To a stirred suspension of lithium aluminum hydride (420 mg,
11 mmol) in dry THF at 0° C under argon was slowly added a solu-
tion of the compound 11 (4.2 g, 9.36 mmol) in dry THF (126 mL).
After stirring for 3 h at room temperature, the reaction mixture
was diluted with diethyl ether (50 mL), cooled down to 4 °C and
carefully hydrolysed by dropwise addition of water. The aqueous
phase was extracted with ethyl acetate (3 times). The combined
organic phases were washed with water, dried by filtration
through a phase separation paper and concentrated under reduced
pressure to give the title compound 12 (2.9 g, 74%) as a white solid
which was employed without further purification in the following
step. RF 0.32 (petroleum ether/ethyl acetate 1:3). 1H NMR
(400 MHz, CDCl3) d 0.68 (s, 3H, 18-CH3), 0.92 (s, 3H, 19-CH3),
0.97 (d, 3H, J = 6.48 Hz, 21-CH3), 3,92 (s, 4H, 3-OCH2CH2O), 3.98
(m, 1H, 12-CH); 13C NMR (100 MHz, CDCl3) d 110.17, 73.32,
64.31, 64.18, 63.65, 48.37, 47.70, 46.61, 41.01, 35.96, 35.75,
35.45, 34.26, 34.14, 33.12, 31.86, 30.08, 29.58, 28.96, 27.70,
26.81, 26.03, 23.75, 23.04, 17.79, 12.90.
2.2.14. 24-methoxy-3-oxo-5b-cholan-12a-yl benzoate (16)
A solution of compound 14 (150 mg, 0.384 mmol) and benzoyl
chloride (0.26 mL, 2.20 mmol) in pyridine (3.8 mL) was stirred at
room temperature for 72 h. The progress of the reaction was mon-
itored by TLC. The reaction mixture was cooled down to 4 °C in an
ice-bath and a 1:1 mixture of ethyl acetate and saturated aqueous
NaHCO3 (13 mL) was added. After stirring for 30 min, the aqueous
phase was extracted with ethyl acetate (3 times). The combined
organic phases were washed with water, dried by filtration
through a phase separation paper and concentrated under reduced
pressure. The dry residue was purified by preparative TLC (petro-
leum ether/ethyl acetate 3:1, RF 0.46) to give the title compound
16 (105 mg, 55%) as a colorless oil. 1H NMR (400 MHz, CDCl3) d
0.82 (d, 3H, J = 5.63 Hz, 21-CH3), 0.84 (s, 3H, 18-CH3), 1.02 (s, 3H,
19-CH3), 2.63 (m, 1H), 3.24–3.30 (massif, 5H, CH2OCH3, 24-
OCH3), 5.38 (br s, 1H, 12-CH), 7.46 (m, 2H, m-ArH), 7.58 (m, 1H,
p-ArH), 8.03 (m, 2H, o-ArH); 13C NMR (100 MHz, CDCl3) d 213.16,
165.97, 133.22, 130.64, 129.46 (2C), 128.75 (2C), 76.51, 73.42,
58.62, 50.10, 48.30, 45.67, 44.22, 42.35, 36.95, 36.75, 35.61,
35.20, 35.07, 34.44, 32.19, 27.59, 26.58, 26.35, 26.16, 25.63,
23.66, 22.54, 17.90, 12.71; HRMS: calcd for C32H47O4[M+Na]+, m/
z 517.3288; found, 517.3292; calcd for C32H47O4[M+H]+, m/z
495.3469; found, 495.3467.
2.2.11. 3,3-(ethylenedioxy)-24-methoxy-5b-cholan-12a-ol (13)
To a stirred solution of compound 12 (1 g, 2.38 mmol) in freshly
distilled dry THF (15 mL) under nitrogen atmosphere, was added
portion wise sodium hydride (60% in mineral oil, 172 mg,
4.28 mmol). The reaction mixture was stirred for 1 h at room tem-
perature. Then, methyl iodide (0.168 mL, 2.62 mmol) was added.
The reaction mixture was stirred successively for 3 h at room tem-
perature then for 3 h at 70 °C. After cooling down to room temper-
ature, water (15 mL) was added and the aqueous phase was
extracted with ethyl acetate (3 times). The combined organic
phases were washed with water, dried by filtration through a
phase separation paper and concentrated under reduced pressure.
The residue was purified by flash chromatography on silica gel
(petroleum ether/ethyl acetate 3:2, RF 0.30) to give the title com-
pound 13 (0.76 g, yield 74%) as a pale yellow paste. 1H NMR
(400 MHz, CDCl3) d 0.67 (s, 3H, 18-CH3), 0.92 (s, 3H, 19-CH3),
0.95 (d, 3H, J = 6.48 Hz, 21-CH3), 3.29–3.37 (massif, 5H, CH2OCH3,
24-OCH3), 3,92 (s, 4H, 3-OCH2CH2O), 3.97 (m, 1H, 12-CH); 13C
NMR (100 MHz, CDCl3) d 110.17, 73.53, 73.32, 64.31, 64.18,
58.65, 48.37, 47.76, 46.61, 41.02, 35.97, 35.76, 35.50, 34.26,
34.15, 33.12, 32.21, 30.08, 28.94, 27.67, 26.83, 26.43, 26.03,
23.74, 23.04, 17.77, 12.89.
2.2.12. 12
a-hydroxy -24-methoxy-5b-cholan-3-one (14)
2.2.15. 12a-(benzyloxy)-24-methoxy-5b-cholan-3-one (17)
A solution of compound 13 (200 mg, 0,46 mmol), p-toluenesul-
fonic acid (40 mg) and water (1.2 mL) in acetone (24 mL) was stir-
red for 2 days at room temperature. The reaction was quenched
with a cold saturated aqueous NaHCO3. The aqueous phase was
extracted with ethyl acetate (3 times). The combined organic
phases were washed with water, dried by filtration through a
phase separation paper and concentrated under reduced pressure
to give the title compound 14 (179 mg, 99%) as an amorphous
solid. RF 0.36 (petroleum ether/ethyl acetate 1:1); Mp = 107–
108 °C; 1H NMR (400 MHz, CDCl3) d 0.71 (s, 3H, 18-CH3), 0.98 (d,
3H, J = 4.4 Hz, 21-CH3), 0.99 (s, 3H, 19-CH3), 2.71 (m, 1H), 3.29–
3.35 (massif, 5H, CH2OCH3, 24-OCH3), 4.04 (s, 1H, 12-CH); 13C
NMR (100 MHz, CDCl3) d 213.54, 73.49, 73.19, 58.68, 48.28,
47.86, 46.68, 44.43, 42.47, 37.26, 37.01, 35.84, 35.43, 34.53,
33.98, 32.22, 28.93, 27.65, 26.68, 26.42, 25.60, 23.69, 22.53,
17.87, 12.89.
To a vigorously stirred solution of compound 14 (195 mg,
0.5 mmol), silver triflate (295 mg, 1.15 mmol) and 2,6-di-tert-
butylpyridine (0.277 mL, 1.2 mmol) in dry CH2Cl2 (3 mL) was
added dropwise
a solution of benzyl bromide (0.152 mL,
1.25 mmol) in dry CH2Cl2 (1 mL). Then, the mixture was refluxed
for 3 h. After cooling down to room temperature, the precipitate
was filtered off and rinsed with CH2Cl2 (10 mL). The combined fil-
trates were washed with 5 M HCl (3 times) and water (3 times),
dried over anhydrous Na2SO4 and concentrated under reduced
pressure. The residue was purified by flash chromatography on sil-
ica gel (ethyl acetate/petroleum ether 2:3) to give the title com-
pound 17 (56 mg, 23%) as
a
colorless oil. 1H NMR (CDCl3,
400 MHz) d 0.74 (s, 3H, 18-CH3), 0.94 (d, 3H, J = 6.6 Hz, 21-CH3),
1.01 (s, 3H, 19-CH3), 2.19–0.98 (massif, 28H), 2.67 (m, 1H), 3.31
(s, 3H, OCH3), 3.34 (m, 2H, CH2-OCH3), 3.75 (br s, 1H, 12-CH),
4.40 (d, 1H, J = 11.5 Hz, O-CH2Ar), 4.53 (d, 1H, J = 11.5 Hz, O-CH2Ar),
7.30–7.25 (massif, 5H, Ar-H); 13C NMR (CDCl3, 100 MHz) d 213.49,
139.34, 128.50 (2C), 127.58, 127.46 (2C), 81.36, 73.62, 70.81, 58.66,
48.85, 46.79, 46.74, 44.56, 42.51, 37.20, 37.13, 35.83, 35.78, 34.62,
34.19, 32.36, 27.83, 26.77, 26.43, 25.56, 23.83, 23.67, 22.68, 18.13,
12.91; HRMS: calcd for C32H48O3[M+Na]+, m/z 503.3496; found,
503.3496.
2.2.13. 24-methoxy-3-oxo-5b-cholan-12a-yl acetate (15)
To a stirred solution of compound 14 (100 mg, 0.26 mmole) and
N,N-dimethylaminopyridine (DMAP) (cat.) in pyridine (0.67 mL) at
room temperature under argon atmosphere was added dropwise
acetic anhydride (0.67 mL, 7.1 mmol). After stirring for 12 h, the

L. Rocheblave et al. / Steroids 116 (2016) 5–12
9
2.2.16. 24-methoxy-12
a
-(methoxymethoxy)-5b-cholan-3-one (18)
After 48 h, the reaction was quenched with saturated aqueous
NaHCO3. The aqueous phase was extracted with ethyl acetate (3
times). The combined organic phases were washed with water,
dried by filtration through a phase separation paper and concen-
trated under reduced pressure. The residue (189 mg) was purified
by flash chromatography on silica gel (petroleum ether/ ethyl acet-
ate, 3:1) to give the title compound 20 (134 mg, 74%) as a colorless
oil. 1H NMR (400 MHz, CDCl3) d 0.74 (s, 3H, 18-CH3), 0.84 (d, 3H,
J = 6.6 Hz, 21-CH3), 0.99 (s, 3H, 19-CH3), 2.68 (m, 1H), 3.29–3.34
(massif, 5H, CH2OCH3, 24-OCH3), 3.76–3.84 (massif, 2H, 20-CH2),
4.13–4.17 (massif, 2H, 60-CH2), 4.30 (br s, 1H, 12-CH), 4.55 (s, H,
50-CH); 13C NMR (100 MHz, CDCl3) 213.43, 149.82, 93.73, 76.02,
73.59, 64.84, 64.75, 58.67, 49.08, 47.24, 45.85, 44.29, 42.42, 36.96
(2C), 35.52, 35.28, 34.63, 34.43, 32.25, 28.67, 27.61 26.59, 26.27,
25.53, 23.66, 23.50, 22.56, 18.03, 12.73; HRMS: calcd for
A solution of compound 14 (100 mg, 0.26 mmol), N,N-diiso-
propylethylamine (0.11 mL, 0.63 mmol) and chloromethyl methyl
ether (0.05 mL, 0.66 mmol) in CH2Cl2 (1.3 mL) was stirred at room
temperature. The progress of the reaction was monitored by TLC
(petroleum ether/ethyl acetate, 3:1, RF 0.44). After 72 h, the reac-
tion was quenched with saturated aqueous NaHCO3. The aqueous
phase was extracted with ethyl acetate (3 times). The combined
organic phases were washed with water, dried by filtration
through a phase separation paper and concentrated under reduced
pressure. The residue (104 mg) was purified by preparative TLC
(petroleum ether/ethyl acetate, 3:1) to give the title compound
18 (86 mg, 77%) as a colorless oil. 1H NMR (400 MHz, CDCl3) d
0.72 (s, 18-CH3), 0. 94 (d, J = 6.6 Hz, 21-CH3), 1.00 (s, 19-CH3),
2.72 (m, 1H), 3.21–3.42 (massif, 8H, CH2OCH3, 24-OCH3), 3.82 (br
s, 1H, 12-CH), 4.65 (d, J = 6.6 Hz, 1H, OCH2OCH3), 4.71 (d,
J = 6.6 Hz, 1H, OCH2OCH3); 13C NMR (100 MHz, CDCl3) d 213.69,
96.88, 81.34, 73.58, 58.68, 56.23, 48.54, 46.68, 46.57, 44.45,
42.54, 37.16, 37.06, 35.99, 35.79, 34.75, 34.39, 32.35, 27.98,
26.85, 26.47, 26.03, 25.73, 23.98, 22.66, 18.06, 12.79; HRMS: calcd
for C27H46O4[M+Na]+, m/z 457.3288; found, 457.3272.
C
30H48O4[M+Na]+, m/z 495.3445 found, 495.3429.
2.3. Intracellular accumulation of daunorubicin measured by flow
cytometry
The accumulation of daunorubicin in the presence of steroid
modulators in resistant K562/R7 cells was measured by flow
cytometry using a reported procedure [12]. Cells (1 ꢀ 106 cells/-
tube) were incubated for 1 h at 37 °C in 1 mL of RPMI 1640 med-
2.2.17. 24-methoxy-12a-[(2R or 2S)-tetrahydro-2H-pyran-2-yloxy]-
5b-cholan-3-one (19a) and (19b)
A solution of compound 14 (200 mg, 0.51 mmol), freshly dis-
tilled 3,4-dihydro-2H-pyrane (0.8 mL, 3.88 mmol) and p-toluene-
sulfonic acid (4 mg) in anhydrous THF (4 mL) was stirred for 1 h
at room temperature. The reaction was quenched with saturated
aqueous NaHCO3. The aqueous phase was extracted with ethyl
acetate (3 times). The combined organic phases were washed with
water, dried by filtration through a phase separation paper and
concentrated under reduced pressure. The residue was purified
by preparative TLC (petroleum ether/methyl tert-butyl ether 5:1,
8 developments) to give the less polar diastereoisomer 19a (RF
0.62, 56 mg, 23%) as a colorless oil and the more polar diastereoiso-
mer 19b (RF 0.57, 56 mg, 23%) as an amorphous solid.
ium containing daunorubicin at 10
lM in the absence or
presence of steroid at 10 M. The cells were then washed twice
l
with ice-cold PBS and kept on ice until analysis by flow cytometry
on a FACS-II (Becton-Dickinson Corp., Mountain View, CA). Assays
were performed in duplicate in at least three separate experiments.
2.4. Potentiation of doxorubicin cytotoxicity using MTT reagent
Cell viability was determined on exponentially growing resis-
tant K562/R7 cells using the MTT assay. This assay is based on
the conversion by metabolically active cells of the MTT reagent
into formazan crystals. K562/R7 cells (8000 cells/well) were incu-
Presumed 12a
-(S)OTHP isomer 19a: 1H NMR (400 MHz, CDCl3) d
0.69 (s, 3H, 18-CH3), 0.90 (d, 3H, J = 6.6 Hz, 21-CH3), 0.98 (s, 3H, 19-
CH3), 2.68–2.75 (m, 2H), 3.32 (s, 3H, 24-OCH3), 3.32–3.33 (massif,
2H, CH2OCH3), 3.37 (m, 1H, 60-CHH), 3.79–3.87 (massif, 2H, 12-
CH and 60-CHH), 4.48 (m, 1H, 20-CH); 13C NMR (100 MHz, CDCl3)
d 214.57, 101.72, 82.91, 73.64, 64.20, 58.69, 48.63, 46.61, 46.59,
44.63, 42.64, 37.23, 36.98, 35.81, 35.79, 34.86, 34.23, 32.37,
31.86, 27.83, 26.91, 26.70, 26.27, 25.68, 25.45, 23.91, 22.69,
bated for 72 h at 37 °C in 100
bicin (at eight concentrations ranging from 10ꢁ7 to 10ꢁ4 M) and
M of cyclosporine A or steroid derivative in a final volume of
200 L of medium. After incubation, 20 L of MTT reagent (5 mg/
lL RPMI medium containing doxoru-
1
l
l
l
mL in PBS buffer) were added to each well, and the plate was fur-
ther incubated for 4 h, allowing viable cells to change the yellow
MTT into dark-blue formazan crystals. Supernatants were carefully
21.24, 18.26, 12.67; HRMS: calcd for
497.3601; found, 497.3587.
C
30H50O4[M+Na]+, m/z
discarded, and 100 lL of isopropanol containing 1 M HCl were
added to dissolve the formazan crystals. Absorbance in each well
was determined at 570 nm using a microplate reader (MultisKan
EX, Thermo Electron). Assays and controls, using support medium
only, were performed in triplicate in at least three separate exper-
iments. Results were expressed as the percentage of surviving cells
in each well compared with untreated cells. IC50 values, defined as
the concentration of doxorubicin inhibiting cell growth by 50%,
were estimated using the Sigma Plot 11.0 software.
Presumed 12a
-(R)OTHP isomer 19b: Mp = 88–89 °C; 1H NMR
(400 MHz, CDCl3) d 0.73 (s, 3H, 18-CH3), 0.99 (d, 3H, J = 5.99 Hz,
21-CH3), 1.00 (s, 3H, 19-CH3), 2.68 (m, 1H), 3.32 (s, 3H, 24-
OCH3), 3.32–3.36 (massif, 2H, CH2OCH3), 3.47 (m, 1H, 60-CHH),
3.95 (m, 1H, 60-CHH), 3.99 (br s, 1H, 12-CH), 4.68 (m, 1H, 20-CH);
13C NMR (100 MHz, CDCl3) d 213.48, 96.23, 77.33, 73.66, 63.41,
58.65, 48.29, 46.59, 46.22, 44.50, 42.56, 37.28, 37.04, 36.52,
35.84, 34.67, 34.10, 32.46, 32.28, 28.26, 27.11, 26.86, 26.65,
25.67, 24.15, 24.11, 22.69, 20.27, 17.71, 13.03; HRMS: calcd for
C
30H48O5 [M+Na]+, m/z 497.3601; found, 497.3585.
2.5. Intrinsic toxicity using MTT reagent
2.2.18. 12a-(3,6-dihydro-2H-pyran-4-yloxy)-24-methoxy-5b-cholan-
K562 cells (8000 cells/well) were incubated for 72 h at 37 °C in
3-one (20)
100
lL RPMI medium containing 10
lM of cyclosporine A or ster-
A solution of compound 14 (150 mg, 0.38 mmol), 4-methoxy-
3,6-dihydro-2H-pyrane (0.300 mL, 2.69 mmol) and p-toluenesul-
fonic acid (1.5 mg) in anhydrous THF (1.5 mL) was stirred at room
temperature under argon atmosphere. The progress of the reaction
was monitored by TLC (petroleum ether/ethyl acetate 2:1, RF 0.55).
oid derivative in a final volume of 200
l
L of medium. After incuba-
tion, cells were treated with MTT as described above. Each assay
was performed in triplicate, and results were expressed as the per-
centage of surviving cells in each well compared with untreated
cells.

10
L. Rocheblave et al. / Steroids 116 (2016) 5–12
In the same manner as mentioned above for 9a and 9b, pure
3. Results and discussion
tetrahydropyranyl derivatives 19a and 19b were obtained in low
yields from a mixture of two diastereoisomers, which were hardly
separated by preparative thin-layer chromatography.
3.1. Chemistry
At this point, assumption can be made to assign the putative
Methyl deoxycholate derivatives 5–10 were prepared following
the reaction pathway described in Scheme 1.
absolute configuration of carbon C-20 of 12
a-OTHP ether rings
by comparison with reported 13C NMR data on previously
described homologous structures [9]. Actually, in both series, a
strong characteristic difference was observed for the dioxygenated
C-20 carbon atom resonance which was found at ca. 101 ppm and
96 ppm for 9a/19a and 9b/19b respectively. Moreover, 9a and
19a isomers could be readily differentiated from their opposite iso-
mers, 9b and 19b, by a much stronger deshielding (ca. 5.5 ppm) of
the steroidal carbon atom bearing the OTHP substituent in the for-
mer stereoisomers (ca. 83 ppm and 77.5 ppm for 9a/19a and
9b/19b respectively). These data would suggest that 9a/19a and
9b/19b isomers should be (20S) and (20R) isomers respectively.
Finally, etherification of compounds 4 and 14 with 4-methoxy-
3,6-dihydro-2H-pyrane did not lead to the expected tetrahedral
adducts [16] (scheme 3). Indeed, the dihydropyranyl ethers 10
and 20 were respectively obtained by elimination of methanol
from the corresponding ketals, as confirmed by the 1H/13C NMR
analyses and mass spectrometry data.
After esterification of the deoxycholic acid in classical condi-
tions [13], methyl deoxycholate 3 was selectively oxidized with sil-
ver carbonate on celite [14] to lead to 3-keto compound 4. Then the
hydroxyl group in position 12 was O-substituted to afford com-
pounds 5–10. In each case, good yield was generally obtained using
classical conditions. Nevertheless, unusual conditions (AgOTf, 2,6-
di-tert-butylpyridine) [15] must have been applied to prepare the
benzyl derivative 7 in average yield. Moreover, tetrahydropyranyl
derivatives 9a and 9b were synthesized as a mixture of two
diastereoisomers. Pure samples of each isomer were obtained by
preparative thin-layer chromatography. However, these purifica-
tions provided low yields because they have required multiple
developments of the TLC plates.
12
a -O-substituted-24-methoxy-5 b -cholan-3-one derivatives
15–20 were prepared following the reaction pathway described in
scheme 2. After a mild protection of ketone 4 as 1,3-dioxolane in
presence of paratoluenesulfonic acid, the ester group in position
24 was converted into an ether group in two steps. First the reduc-
tion using LiAlH4 afforded the intermediary primary alcohol 12,
which was then selectively etherified with MeI in presence of
sodium hydride as a base in THF. Deprotection of the ketone group
following by O-substitution in position 12 under the same condi-
tions as above, led to compounds 15–20.
3.2. Biological evaluation
The efficiency of the 5 b -steroid derivatives as modulators of
Pgp (Table 1) was evaluated in K562/R7 human leukemic mul-
tidrug resistant cells through intracellular accumulation of
OR
OH
OH
OH
COOCH3
COOCH3
COOH
COOCH3
a
c
b
O
O
HO
HO
H
R = COCH3
H
H
H
Deoxycholic acid
3
4
5
6
7
8
R = CH2OCH3
R = COC6H5 9a,b R = THP (S/R)
10
R = CH2C6H5
R = DHP
Scheme 1. Reagents and conditions: a) MeOH, HCl, 94%. b) Ag2CO3/Celite, toluene, 92%. c) 5: Ac2O, pyridine, DMAP, 84%; 6: C6H5COCl, pyridine, 76%; 7: C6H5CH2Br, AgOTf,
2,6-di-tert-butylpyridine, 44%; 8: CH3OCH2Cl, (iPr)2NEt, CH2Cl2, 73%; 9a,b: DHP, TsOH, THF (9a: 18%; 9b: 11%); 10: 4-methoxy-3,6-dihydro-2H-pyrane, TsOH, THF, 67%.
OH
OH
OH
COOCH3
COOCH3
b
CH2OR
a
O
O
O
O
O
H
O
11
12
13
H
H
R = H
R = CH3
4
c
OR
OH
CH2OCH3
CH2OCH3
e
d
13
15
16
17
18
R = COCH3
R = COC6H5
R = CH2C6H5
R = CH2OCH3
O
H
H
14
19a,b R = THP (S/R)
20
R = DHP
Scheme 2. Reagents and conditions: a) HO(CH2)2OH, TsOH, toluene, 97%. b) LiAlH4, Et2O, 74%. c) CH3I, NaH-THF, 74%. d) TsOH, acetone, 99%. e) 15: Ac2O, pyridine, DMAP, 92%;
16: C6H5COCl, pyridine, 55%; 17: C6H5CH2Br, AgOTf, 2,6-di-tert-butylpyridine, 23%; 18: CH3OCH2Cl, (iPr)2NEt, CH2Cl2, 77%; 19a/b: DHP, TsOH, THF (19a: 23%; 19b: 23%); 20:
4-methoxy-3,6-dihydro-2H-pyrane, TsOH, THF, 74%.

L. Rocheblave et al. / Steroids 116 (2016) 5–12
11
O
OCH3
O
OH
O
R
O
R
R
-CH3OH
O
H
O
O
H
H
4
R = COOCH3
10 R = COOCH3
20 R = CH2OCH3
tetrahedral adducts
14 R = CH2OCH3
Scheme 3. Presumed mechanism leading to DHP ether compounds 10 and 20.
the OTHP group seemed to have a modest impact on the activity.
Indeed 9a was slightly less active than 9b, and 19a was somewhat
more active than 19b.
Table 1
Intrinsic toxicities on K562 sensitive cells and capacity of compounds 5–10 and 15–20
to improve daunorubicin accumulation and potentiate the cytotoxicity of doxorubicin
in K562/R7 human leukemic resistant cells vs compounds 1, 2 and cyclosporine A
(CsA).
The intrinsic toxicity of the steroids was expressed as a percent-
age of surviving cells. All the percentages were found above 50% for
the tested deoxycholic acid derivatives 5–10 and 15–20. Further-
more, all these compounds had a lower toxicity than cyclosporine
A and cholate derivative 2, especially 9b which showed one of the
highest percentage of surviving K562 cells. Moreover compound
9b was as active as 1 and cyclosporine A concerning the potentia-
tion of doxorubicin, and slightly more active with regard to
daunorubicin accumulation.
Compd Daunorubicin
accumulationa,b
doxorubicin IC50 ratio with/
Intrinsic
without steroid (1
lM)
toxicityb,c
CsA
1
2
5
6
3.47 0.15
3.70 0.19
3.88 0.10
3.65 0.85
3,54 0.06
3.27 0.02
3.70 0.04
4.29 0.26
4.41 0.06
2.86 0.19
3.92 0.05
3.11 0.47
2.70 0.11
4.35 0.09
3.83 0.17
4.09 0.32
3.02 0.09
0.15 0.10
0.09 0.02
0.01 0.005
0.23 0.12
0.12 0.11
0.44 0.09
0.35 0.05
0.21 0.14
0.09 0.01
0.43 0.11
0.30 0.13
0.36 0.23
0.81 0.38
0.36 0.31
0.29 0.12
0.44 0.19
0.50 0.31
18.62 0.03
82.86 0.05
43.50 0.80
59.75 0.01
54.63 0.01
63.92 0.08
74.59 0.04
69.46 0.05
78.59 0.10
70.10 0.06
66.21 0.10
67.97 0.09
57.41 0.10
75.01 0.10
82.54 0.08
86.54 0.09
69.46 0.07
7
8
9a
9b
10
15
16
17
18
19a
19b
20
4. Conclusion
The synthesis and biological evaluation of 14 deoxycholic acid
derivatives were described. They were obtained in moderate to
good overall yields from deoxycholic acid which is a readily acces-
sible compound. Biological results confirmed that bile acid deriva-
tives could be as potent as progesterone derivatives to modulate
Pgp-mediated MDR. Moreover, the 12
a-OTHP deoxycholate
All values are expressed as the mean SE. afluorescence ratio with/without tested
derivative 9b showed a better biological activity than 5 b-dihy-
droprogesterone derivative 1 and cyclosporine A, and a similar
one compared to cholate 2. Furthermore, 9b possesses a lower
intrinsic toxicity than cyclosporine A and compound 2. Therefore
9b represents a new lead compound in order to improve the design
of non-toxic potent Pgp modulators.
compound. bCompounds were tested at a 10
cells.
l
M concentration. c% of surviving K562
daunorubicin (10
bicin accumulation was expressed as the daunorubicin fluores-
cence ratio with/without the steroid (10 M), and the
potentiation of doxorubicin was expressed as the doxorubicin
IC50 ratio with/without the steroid (1 M). Intrinsic toxicity of
the compounds (10 M) was estimated on parental K562 non-
lM) and potentiation of doxorubicin. Daunoru-
Acknowledgement
l
The authors wish to thank the Ministère de l’Éducation natio-
nale, de l’Enseignement supérieur et de la Recherche (France) for
financial support.
l
l
resistant cells. The results are presented in Table 1.
Appendix A. Supplementary data
The analogs 15–20, where the 24-COOCH3 ester group has been
replaced by a 24-CH2OCH3 ether moiety, were synthesized owing
to the potentially higher resistance of an ether group to in vivo
hydrolysis. Although replacement of the ester group by an ether
function generally had little effect on steroid derivative activities
(5/15, 8/18), it can be noted that it led to a more marked decrease
of efficiency in the case of the most active OTHP series (9b/19b,
9a/19a). All compounds showing an equal or a better efficiency
than cyclosporine A through the potentiation of doxorubicin, pos-
sess an ester function in position 24.
Supplementary data (1H and 13C spectra) associated with this
article can be found, in the online version, at http://dx.doi.org/10.
1016/j.steroids.2016.09.017.
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