Design, synthesis and evaluation of progesterone-adenine hybrids as bivalent inhibitors of P-glycoprotein-mediated multidrug efflux

Article abstract of DOI:10.1016/j.bmcl.2010.03.085

Steroidal bivalent ligands were designed on the basis of the described closer proximity of the ATP-site and the putative steroid-binding site of P-glycoprotein (ABCB1). The syntheses of seven progesterone-adenine hybrids were described. Their abilities to inhibit P-glycoprotein-mediated daunorubicin efflux in K562/R7 human leukemic cells overexpressing P-glycoprotein were evaluated versus progesterone.

Full text of DOI:10.1016/j.bmcl.2010.03.085

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3165–3168
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and evaluation of progesterone–adenine hybrids as
bivalent inhibitors of P-glycoprotein-mediated multidrug efflux
a
b
Waël Zeinyeh a, Ghina Alameh a, Sylvie Radix a, , Catherine Grenot , Charles Dumontet ,
*
Nadia Walchshofer a,
*
a Université de Lyon, Université Lyon 1, ISPB-Faculté de Pharmacie, INSERM U863, 8 avenue Rockefeller, F-69373 Lyon cedex 08, France
b Université de Lyon, Université Lyon 1, Faculté de Médecine, Laboratoire de Cytologie analytique, INSERM U590, 8 avenue Rockefeller, 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:
Steroidal bivalent ligands were designed on the basis of the described closer proximity of the ATP-site
and the putative steroid-binding site of P-glycoprotein (ABCB1). The syntheses of seven progesterone–
adenine hybrids were described. Their abilities to inhibit P-glycoprotein-mediated daunorubicin efflux
in K562/R7 human leukemic cells overexpressing P-glycoprotein were evaluated versus progesterone.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 29 January 2010
Revised 23 March 2010
Accepted 25 March 2010
Available online 28 March 2010
Keywords:
Multidrug resistance
P-Glycoprotein
ABCB1
Bivalent ligand
Multivalent ligand
Progesterone
Adenine
Chemotherapeutics are the most effective treatment for meta-
static cancer, but their efficacy is limited by the multidrug resistance
(MDR) phenotype which results from multifactor mechanisms.1
Drug efflux is a significant contributor for this phenotype in cancer
cells, wherein drug-extrusion pumps belonging to the ABC super-
family of proteins are overexpressed.2 One of these human ATP-
dependent membrane transporters is P-glycoprotein (Pgp) whichal-
lows the efflux of a wide variety of structurally and functionally
unrelated compounds.3 Therefore, inhibition of Pgp may improve
chemotherapeutic treatments and numerous studies4 have focused
on the development of Pgp modulators. Whereas the more hydro-
philic steroids are transported by Pgp (e.g., cortisol), the more hydro-
phobic ones function as inhibitors (e.g., progesterone which is
always considered as the reference among steroidal inhibitors of
Pgp although it exhibits a low affinity to this protein).5 Until now,
some progesterone derivatives have been described as inhibitors
of Pgp-mediated antitumor drugs efflux.6–8 Moreover, it has been
postulated that the steroid-binding region would be adjacent to
the ATP-binding site of Pgp.9 If this could be confirmed, it would be
interesting to build on this feature to lead to more potent and selec-
tive Pgp inhibitors.
We decided to design and synthesize progesterone–adenine hy-
brids as potential bivalent ligands which may bind simultaneously
to these two sites in the Pgp molecule (Fig. 1). Indeed, bivalent li-
gands can be used as valuable tools to confirm the proximity of the
targeted binding sites,10 owing to the binding-affinity and selectiv-
ity improvements generally recorded with these compounds com-
pared to their monovalent counterparts.11 The synthesis of dimeric
compounds targeting substrate-binding sites of Pgp has already
been reported,12 but to our knowledge, heterobivalent compounds
Pgp
ATP-binding site
NH2
Pgp
steroid-
N
N
binding site
N
N
O
20
adenine
NH
Y
Y= linker
H
H
H
1
* Corresponding authors. Tel.: +33 478777253; fax: +33 478777158 (S.R.); tel.:
+33 478777006; fax: +33 478777158 (N.W.).
O
progesterone
E-mail addresses: sylvie.radix@univ-lyon1.fr (S. Radix), nadia.walchshofer@univ-
lyon1.fr (N. Walchshofer).
Figure 1. Structure of targeted bivalent compounds 1.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.03.085

3166
W. Zeinyeh et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3165–3168
a
b
a
R
NH
OH
BocHN
Cbz
OMs
HO
OH
MsO
OH
6
4, R=H
5, R=Boc
3
d
2
c
d
e
H2N
H2N
N
H
OH
OH
OH
COOH
7
8
9
Cbz
Cbz
e
H2N
H2N
N
H
OH
OH
COOH
12
11
13
10
e
OH
H2N
N
14
H
Scheme 1. Reagents and conditions: (a) MsCl, NEt3, THF, 0 °C to rt, overnight (45% for 3; 76% for 6); (b) NH4OH, rt, 1 h then Dowex (76%); (c) Boc2O, NEt3, THF, rt, overnight
(73%); (d) LiAlH4, THF, 0 °C to reflux, 3 h (70% for 8; 28% for 11); (e) Na2CO3, CbzCl, H2O, 0 °C to rt, overnight (70% for 9; 90% for 12; 79% for 14).
targeting ATP-binding site and/or steroid-binding region have
never been described. In order to evaluate our strategy, we chose,
in a first attempt, to synthesize the most readily accessible C20-
substituted progesterone derivatives (with a NH group instead of
the methyl group in the C21 position), and rather short-length
linkers with different conformational flexibilities. We report here
the synthesis of compounds 1 and their abilities to inhibit the
Pgp-mediated daunorubicin efflux in K562/R7 human leukemic
cells overexpressing P-glycoprotein.
compounds 16, 18, and 20. As Mitsunobu reaction failed with 5, we
used classical adenine N-alkylation conditions with mesylate 6 to
provide derivative 21. Alkyne 22, easily obtained by Boc deprotec-
tion of 21, was used as a common key precursor of amino com-
pounds 23, 2416 and 25. Reduction of 22 by LiAlH4 led to trans-
alkene 23. Catalytic hydrogenation of the same alkyne using Lind-
lar catalyst yielded cis-alkene 24, while the use of Pd on charcoal
provided 25.
At the same time, progesterone was converted to its 17b-car-
boxylic acid analog 26 by the haloform reaction using sodium
hypobromite (Scheme 3).17 The activated ester 27, obtained by
treatment of acid 26 with N-hydroxysuccinimide,18 was then al-
lowed to react with the adenine derivatives 16, 18, 20 or 22–25
to afford the corresponding bivalent compounds 1a–g in average
to good yields (cf. Supplementary data).
Syntheses of protected-amino linkers 6, 9, 12, and 14, starting
from commercially available compounds 2, 7, 10, and 13, are de-
scribed in Scheme 1. Monomesylation of diol 2 afforded mesylate
3. Treatment of 3 with NH4OH,13 followed by N-Boc-protection14
and methanesulfonylation of the remaining hydroxy function, pro-
vided the linker 6. Reduction of carboxylic acids 7 and 10 in pres-
ence of LiAlH4 gave alcohols
8
and 11, respectively.15 Cbz
The efficiency of progesterone–adenine hybrids to inhibit Pgp-
mediated cytotoxic drug efflux was evaluated by measuring the
intracellular accumulation of daunorubicin in the K562/R7 human
leukemic resistant cell line overexpressing Pgp (Table 1). Proges-
terone, used as a positive control, was found to improve daunoru-
bicin accumulation in K562/R7 cells by 25%. On the other hand,
protection of aminoalcohols 8, 11, and 13 led to linkers 9, 12,
and 14. Then, amino derivatives 14, 9, and 12 were allowed to react
with adenine using the Mitsunobu method, producing compounds
15, 17, and 19 (Scheme 2). Cbz deprotection of these products was
achieved by catalytic hydrogenation (H2, Pd/C) to afford free amino
NH2
N
NH2
N
N
N
N
a
N
N
b
N
N
N
N
15
16
(CH2)6NHCbz
(CH2)6NH2
NH2
NH2
N
N
N
b
a
NH2
N
N
N
N
N
17
N
18
NHCbz
NH2
NH2
N
H
NH2
N
N
b
a
c
N
N
N
N
adenine
N
N
19
20
NHCbz
NH2
NH2
NH2
N
N
N
N
N
d
N
N
22
21
NH2
NHBoc
g
e
f
NH2
NH2
NH2
N
N
N
N
N
N
N
N
N
N
N
N
(CH2)4NH2
23
24
25
NH2
NH2
Scheme 2. Reagents and conditions: (a) PPh3, DIAD, THF, 14 or 9 or 12 (41% for 15, 49% for 17, 18% for 19); (b) H2, Pd/C, MeOH or MeOH/AcOEt (100% for 16, 71% for 18, 77%
for 20); (c) K2CO3, DMF, 6 (75%); (d) (i) aq HCl; (ii) Dowex 1 Â 8 (ÀOH form) (80%); (e) LiAlH4, THF (29%); (f) H2, Lindlar Pd, quinoline, MeOH (67%); (g) H2, Pd/C, MeOH (90%).

W. Zeinyeh et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3165–3168
3167
O
O
O
O
20 CH3
OH
O
O
N
H
a
H
H
b
H
H
H
H
H
H
O
O
O
27
NH2
26
progesterone
c
N
N
N
N
H2N Y
16, 18, 20, 22-25
NH2
N
N
1d, Y =
1e, Y =
1f, Y =
CH2
CH2
(77%)
(79%)
(60%)
N
N
O
4
1a, Y =
1b, Y =
1c, Y =
(92%)
(55%)
(40%)
NH
Y
6
H
H
H
O
1a-g
1g, Y =
(61%)
Scheme 3. Reagents and conditions: (a) (i) NaOH, Br2, t-BuOH, 0 °C, 3 h; (ii) Na2SO3, rt, overnight (57%); (b) N-hydroxysuccinimide, THF, rt, overnight (55%); (c) (i-Pr)2NEt,
DMF, overnight, rt (1a–f) or 35 °C (1g).
Table 1
Acknowledgments
Capacity of the synthesized derivatives to improve daunorubicin accumulation in
K562/R7 human leukemic resistant cells19
We thank Dr. Luc Rocheblave (UCB Lyon 1) for his helpful mon-
itoring of cell culture and Dr. Ph. Lawton (UCB Lyon 1) for a careful
reading of the manuscript.
b
Compoundsa
Daunorubicin accumulation (% progesterone)
1a
1b
1c
1d
1e
1f
61.2 ( 8)
72.2( 6.7)
86.2 ( 2.6)
78.0 ( 8.7)
110. 9 ( 13.7)
98. 9 ( 6.2)
98.1 ( 0.5)
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.03.085.
1g
a
Compounds were tested at a 10
Accumulation of daunorubicin (1
l
l
M concentration.19
M) in the presence of progesterone (10 lM)
b
References and notes
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