Progesterone-adenine hybrids as bivalent inhibitors of P-glycoprotein- mediated multidrug efflux: Design, synthesis, characterization and biological evaluation

Article abstract of DOI:10.1016/j.steroids.2012.07.010

Bivalent ligands were designed on the basis of the described close proximity of the ATP-site and the putative steroid-binding site of P-glycoprotein (ABCB1). The syntheses of 19 progesterone-adenine hybrids are described. Their abilities to inhibit P-glycoprotein-mediated daunorubicin efflux in K562/R7 human leukemic cells overexpressing P-glycoprotein were evaluated versus progesterone. The hybrid with a hexamethylene linker chain showed the best inhibitory potency. The efficiency of these progesterone-adenine hybrids depends on two main factors: (i) the nature of the linker and (ii) its attachment point on the steroid skeleton.

Full text of DOI:10.1016/j.steroids.2012.07.010

Steroids 77 (2012) 1177–1191
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Progesterone–adenine hybrids as bivalent inhibitors of P-glycoprotein-mediated
multidrug efflux: Design, synthesis, characterization and biological evaluation
a
a
a
Waël Zeinyeh a,b, Zahia Mahiout a, Sylvie Radix a, , Thierry Lomberget , Axel Dumoulin , Roland Barret ,
⇑
Catherine Grenot c, Luc Rocheblave a, Eva-Laure Matera d, Charles Dumontet d, Nadia Walchshofer a,
⇑
a Université de Lyon, Université Lyon 1, EA 4446 Biomolécules Cancer Chimiorésistances (B2C), ISPB-Faculté de Pharmacie, UMS 3453 Santé Lyon-Est, 8 Avenue Rockefeller,
F-69373 Lyon Cedex 08, France
b Hospices Civils de Lyon, Laboratoire d’Hormonologie, Centre de Biologie Est, Groupement Hospitalier Est, Bron F-69677, France
c Université de Lyon, Université Lyon 1, INSERM U863, ISPB-Faculté de Pharmacie, 8 Avenue Rockefeller, F-69373 Lyon Cedex 08, France
d Université de Lyon, Université Lyon 1, INSERM U1052, CNRS UMR 5286, Centre de Recherche de Cancérologie de Lyon, HCL, 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:
Bivalent ligands were designed on the basis of the described close proximity of the ATP-site and the puta-
tive steroid-binding site of P-glycoprotein (ABCB1). The syntheses of 19 progesterone–adenine hybrids
are described. Their abilities to inhibit P-glycoprotein-mediated daunorubicin efflux in K562/R7 human
leukemic cells overexpressing P-glycoprotein were evaluated versus progesterone. The hybrid with a hex-
amethylene linker chain showed the best inhibitory potency. The efficiency of these progesterone-ade-
nine hybrids depends on two main factors: (i) the nature of the linker and (ii) its attachment point on
the steroid skeleton.
Received 23 March 2012
Received in revised form 5 July 2012
Accepted 15 July 2012
Available online 31 July 2012
Keywords:
Multidrug resistance
P-glycoprotein
Bivalent ligand
Adenine
Ó 2012 Elsevier Inc. All rights reserved.
Progesterone
PEG linker
1. Introduction
[6]. Among these molecules, the more hydrophilic ones are trans-
ported by Pgp (e.g. cortisol) while the more hydrophobic ones
Chemotherapeutics are the most effective treatment for meta-
static cancer, but their efficacy is limited by the multidrug resis-
tance (MDR) phenotype that remains a significant impediment to
successful cancer chemotherapy. MDR results from multifactor
mechanisms [1]. A reduced intracellular drug accumulation is
one of the prominent mechanisms of resistance in cancer cells,
wherein drug-efflux pumps belonging to ABC superfamily of pro-
teins are overexpressed [2]. One of these human ATP-dependant
membrane transporters is P-glycoprotein (Pgp) which allows the
efflux of a wide variety of structurally and functionally unrelated
compounds [3]. Inhibition of Pgp may therefore improve chemo-
therapeutic treatments and numerous works [4] have focused on
the development of Pgp modulators since the demonstration in
the early 1980s of the reversal of resistance in vitro by Verapamil
[5].
function as inhibitors (e.g. progesterone) [7]. Moreover, progester-
one and derivatives like progestagens (e.g. nomegestrone and
megestrol) or antiprogestin RU 486 inhibit in some extend Pgp-
mediated efflux of chemotherapeutic drugs such as vinblastine
[7a,8], vincristine [6b], doxorubicin [9] or adriamycin [10]. There-
fore, progesterone represents a lead compound for the design of
Pgp inhibitors.
Pgp consists of two homologous halves, each containing six
transmembrane domains (TMDs) and two nucleotide-binding do-
mains (NBD1 and NBD2). NBD1 and NBD2 are separated by
ꢀ30 Å, as revealed by the X-ray crystallographic study of murine
Pgp which has 87% sequence homology to human Pgp [11]. Pgp
contains at least two transport-active binding sites within the
TMDs. It remains unclear how steroidal compounds act at the
molecular level. Shapiro et al. [12] have shown that progesterone
could bind to a third allosteric binding site. Moreover, it has been
postulated that a steroid-binding region would be adjacent to the
ATP-binding site of Pgp [13]. Then, we decided to design and syn-
thesize progesterone–adenine hybrids as potential bivalent ligands
(Fig. 1) which may simultaneously bind to both sites of Pgp.
Bivalent ligands are known to often improve the binding affin-
ities and selectivities compared to the monovalent counterparts
Pgp is also present in various normal cells [2a]. In particular,
adrenal Pgp could play a role in secretion of steroid hormones
⇑
Corresponding author. Tel.: +33 478777253; fax: +33 478777158 (S. Radix), tel.:
+33 478777006; fax: +33 478777158 (N. Walchshofer).
E-mail addresses: sylvie.radix@univ-lyon1.fr (S. Radix), nadia.walchshofer
@univ-lyon1.fr (N. Walchshofer).
0039-128X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.steroids.2012.07.010

1178
W. Zeinyeh et al. / Steroids 77 (2012) 1177–1191
Pgp
O
Pgp
21
18
ATP-binding site
20 CH3
steroid-binding site
12
17
11
9
NH2
H13
8
19
10
N
N
7'
N
16
1
4
14
6'
5'
4'
N1'
spacer
2
15
8'
H
6
H
H
N
N
3
2'
N
N
7α
5
O
N
3'
9'
Y
O
2
Fig. 1. Design of steroid-purine hybrids.
= spacer
Y
[14]. They can be used as tools to confirm the proximity of targeted
binding sites [15]. This concept has been already applied to some
homodimeric compounds targeting substrate binding sites of Pgp
[16]. All these homodimers have been found to reverse the Pgp-
mediated multiple drug resistance phenotype more efficiently than
their respective monomers. For our part, we have recently de-
signed new hetero-bivalent MDR modulators combining a steroid
nucleus (progesterone) and a purine ring system (adenine) con-
nected by a spacer. Progesterone was a known steroidal Pgp mod-
ulator [17] and adenine was first used as a mimic analogue of ATP
because aromatic residues of the Pgp A-loop were known to inter-
act with the adenine ring of ATP [18]. In order to evaluate our strat-
egy, we have described the synthesis of C20-substituted
progesterone derivatives 1 using an amide link as a connecting
function and rather short-length spacers with different conforma-
tional flexibilities (Fig. 2) [19]. The Pgp-inhibition activity of these
progesterone–adenine hybrids in leukemic MDR cells was similar
to that of progesterone.
Fig. 3. Structures of C7-progesterone–adenine hybrids 2. Numbering system for
NMR data.
1H spectra were recorded with Bruker ALS300 and Bruker
DRX400 Fourier transform spectrometers, using an internal deute-
rium lock, operating at 300 MHz or 400 MHz, respectively. 13C
NMR spectra were recorded with a Bruker ALS300 and DRX400
Fourier transform spectrometers, using an internal deuterium lock,
operating at 75 and 100 MHz, respectively. All spectra were re-
corded at 25 °C. Chemical shifts are reported in parts per million
(ppm) relative to internal standard (tetramethylsilane, dH = 0.00;
CDCl3, dH = 7.26, dC = 77.36; DMSO, dH = 2.54, dC = 39.52; CD3OD,
dH = 3.34, dC = 49.86). Carbon multiplicity was determined by DEPT
experiments. Electron-Spray low-resolution mass spectra were re-
corded on a Thermo ALCQ Advantage spectrometer or Agilent 6120
spectrometer. High-resolution mass spectra were recorded on a
Bruker MicroTOF Q or ThermoQuest FINNIGAN MAT 95 XL appara-
tus operating at 70 eV. UV spectra were recorded on a Shimadzu
The structure of the linker and its attachment points on the two
moieties (steroid and adenine) are important to consider when
designing bivalent ligands. Therefore, we report here, on the one
hand, the synthesis of two new C20-hybrids with flexible PEG link-
ers as Y and, on the other hand, the synthesis of ten C7-hybrids 2
(Fig. 3) with their respective abilities to inhibit Pgp-mediated dau-
norubicin efflux in K562/R7 human leukemic cells overexpressing
P-glycoprotein. Our choice for the preparation of C7 derivatives
was driven by literature precedents describing some C-7 substi-
tuted progesterone derivatives: they are known to possess an
equivalent potency than cyclosporin A, a well-known Pgp modula-
tor, while the most potent of them do not bind to progesterone
receptors [9,20].
UV/Vis spectrometer UV-1700. Product purification was performed
either by flash column chromatography using Merck Silicagel 60 ÅA
0
(40–63
l
m) or by preparative TLC carried out using Merck com-
m layer thickness)
mercial glass plates (20 ꢁ 20 cm) coated (250
l
with Silicagel 60 F254 with visualization by ultraviolet. Analytical
thin layer chromatography (TLC) was carried out using Merck com-
mercial aluminum sheets coated (200 lm layer thickness) with Sil-
icagel 60 F254, with visualization by ultraviolet or by spraying
plates with diluted solution of H2SO4 or ninhydrine in ethanol fol-
lowed by drying with heat gun. Flow cytometry analyses were car-
ried out on a Beckton Dickinson FACS-Calibur.
2.2. Chemical syntheses
2. Experimental procedures
2.2.1. 6-(N-benzyloxycarbonylamino)hexan-1-ol (4)
To an ice-bath cooled solution of sodium carbonate (2.12 g,
20 mmol) and 6-aminohexan-1-ol 3 (1.17 g, 10 mmol) in water
(30 mL) was added dropwise benzyl chloroformate (2.41 mL,
17 mmol). The reaction mixture was stirred overnight at room
temperature then filtered. The precipitate was dissolved in dichlo-
romethane and the organic layer was washed with water, dried
over Na2SO4, filtered then concentrated in vacuo to give a solid
2.1. Material and methods
All commercially available chemicals and solvents were used as
received. All air- and moisture-sensitive reactions were carried out
under an argon atmosphere. All melting points were measured on a
Barnstead Electrothermal 9200 apparatus and were uncorrected.
NH2
7'
N
6'N1'
5'
CH2
CH2
8'
4
6
2'
4'
9'N
N
3'
O
18
20 NH
Y
Y
12
17
11
9
H13
8
19
10
16
1
4
14
2
15
H
6
H
1
3
7
5
O
Fig. 2. Structure of progesterone–adenine hybrids 1 (C20-hybrids). Numbering system for NMR data.

W. Zeinyeh et al. / Steroids 77 (2012) 1177–1191
1179
which was recristallised from cyclohexane to give compound 4 as a
white solid (1.98 g, 79% yield). Mp = 80–81 °C; 1H NMR (DMSO-d6)
d: 7.39–7.23 (m, 5H, 5 ꢁ H-phenyl), 7.21 (m, 1H, NH), 5.00 (s, 2H,
CH2Ph), 4.33 (t, 1H, J = 5.2 Hz, OH), 3.36 (q, 2H, J = 5.2 Hz, CH2O),
2.97 (q, 2H, J = 6.6 Hz, CH2N), 1.40–1.20 (m, 8H, 4 ꢁ CH2) ppm;
13C NMR (DMSO-d6) d: 156.9 (NHCOO), 138.18 (C-phenyl ipso),
129.19 (2 ꢁ C-phenyl meta, C-phenyl para), 128.56 (2 ꢁ C-phenyl
ortho), 65.91 (OCH2Ph), 61.50 (CH2OH), 41.11 (CH2), 33.34 (CH2),
30.33 (CH2), 27.02 (CH2), 26.08 (CH2) ppm; MS(ESI): m/z (%) 525
(70) [2M+Na]+, 274 (83) [M+Na]+, 252 (100) [M+H]+; HRMS (ESI):
m/z: calcd for C14H21NO3 + Na: 274.1419; found: 274.1423.
(425 mg, 90% yield). Mp = 112–113 °C; 1H NMR (CDCl3) d: 7.40–
7.20 (m, 9H, 9 ꢁ H-phenyl), 5.14 (s, 2H, CH2OCONH), 5.04 (m, 1H,
NH), 4.68 (d, 2H, J = 4.4 Hz, CH2OH), 4.38 (d, 2H, J = 5.8 Hz, CH2NH),
1.61 (m, 1H, OH) ppm; 13C NMR (CDCl3) d: 156.54 (COO), 140.36
(C-phenyl ipso), 137.99 (C-phenyl ipso), 136.58 (C-phenyl ipso),
128.69 (CH-phenyl), 128.32 (CH-phenyl), 128.30 (CH-phenyl),
127.89 (CH-phenyl), 127.47 (CH-phenyl), 67.05 (CH2), 65.17
(CH2), 45.02 (CH2) ppm; MS(ESI): m/z (%) 564 (18) [2M+Na]+, 294
(100) [M+Na]+; HRMS(ESI): m/z calcd for C16H17NO3 + Na:
294.1101; found: 294.1092.
2.2.5. 4-(Mesyloxy)but-2-yn-1-ol (12)
2.2.2. [Trans-4-(N-benzyloxycarbonylaminomethyl) cyclohexyl]
methanol (7)
To an ice-bath cooled solution of but-2-yne-1,4-diol 11 (2.58 g,
30 mmol) in anhydrous THF (35 mL) were added dropwise meth-
anesulfonyl chloride (2.30 mL, 30 mmol) and triethylamine
(4.20 mL, 30 mmol). After the addition, the reaction mixture was
allowed to warm up to room temperature. After 17 h stirring, the
reaction mixture was concentrated in vacuo. The residue was puri-
fied by flash chromatography on silica gel (CH2Cl2/methanol, 97:3)
to give compound 12 as a colorless oil (2.23 g, 45% yield). 1H NMR
(CDCl3) d: 4.89 (t, 2H, J = 1.8 Hz, CH2OMs), 4.34 (m, 2H, CH2OH),
3.12 (s, 3H, CH3), 1.92 (m, 1H, OH) ppm.
To an ice-bath cooled suspension of lithium aluminum hydride
(302 mg, 7.9 mmol) in anhydrous THF (20 mL) was added dropwise
a suspension of trans-4-(aminomethyl)cyclohexylcarboxylic acid 5
(500 mg, 3.18 mmol) in anhydrous THF (20 mL). The reaction mix-
ture was stirred at 0 °C for 1 h then it was heated under reflux for
3 h. The mixture was cooled to room temperature and quenched
with water. The aqueous phase was extracted by dichloromethane.
Addition of a few drops of aqueous solution of Rochelle salt avoids
emulsion formation. Combined organic phases were dried over
Na2SO4, filtered and concentrated in vacuo to give [trans-4-(amino-
methyl)cyclohexyl]methanol 6 as a yellow oil (320 mg, 70% yield)
which should be used without delay.
2.2.6. 4-(N-tert-butyloxycarbonylamino)but-2-yn-1-ol (14) [22]
Compound 12 (1.02 g, 6.02 mmol) was stirred in ammonium
hydroxide (15 mL) for 1 h then the solvent was evaporated. The
residue was treated with Dowex 1X8 R3N+Clꢂ prewashed with 4%
NaOH aq. solution. The filtrate was concentrated and dried under
To an ice-bath cooled suspension of compound 6 (320 mg,
2.23 mmol) and sodium carbonate (475 mg, 4.48 mmol) in water
(20 mL) was added dropwise benzyl chloroformate (540
lL,
vacuum to give 4-aminobut-2-yn-1-ol 13 as a yellow solid
3.79 mmol). The reaction mixture was stirred at room temperature
overnight then filtered. The precipitate was washed with water
then recristallised from cyclohexane to give compound 7 as a white
solid (432 mg, 70% yield). Mp = 95–96 °C; 1H NMR (CDCl3) d: 7.40–
7.30 (m, 5H, 5 ꢁ H-phenyl), 5.09 (s, 2H, CH2Ph), 4.80 (bs, 1H, NH),
3.45 (d, 2H, J = 6.2 Hz, CH2O), 3.05 (t, 2H, J = 6.3 Hz, CH2N), 1.9–0.9
(m, 11H, 10 ꢁ H-cyclohexyl, OH) ppm; 13C NMR (CDCl3) d: 156.7
(COO), 136.8 (C-phenyl ipso), 128.7 (2 ꢁ C-phenyl meta), 128.29
(2 ꢁ C-phenyl ortho), 128.26 (C-phenyl para), 68.6 (OCH2), 66.8
(OCH2), 47.4 (NCH2), 40.6 (CH), 38.5 (CH), 30.1 (2 ꢁ CH2 cyclo-
hexyl), 29.0 (2 ꢁ CH2 cyclohexyl) ppm; MS(ESI): m/z (%) 577
(100) [2M+Na]+, 300 (65) [M+Na]+; HRMS(ESI): m/z calcd for
(880 mg, 76% yield) which should be used without delay.
Mp = 58 °C (lit. 59–60 °C) [23]; 1H NMR (CDCl3) d: 4.22 (t, 2H,
J = 1.9 Hz, CH2O), 3.45 (t, 2H, J = 1.9 Hz, CH2N), 3.42 (s, 1H, OH),
2.50 (s, 2H, NH2) ppm.
To a solution of compound 13 (65 mg, 7.64 mmol) and di-tert-
butyl dicarbonate (1.67 g, 7.66 mmol) in anhydrous THF (30 mL),
was added dropwise triethylamine (1.08 mL, 7.69 mmol) at 0 °C.
The mixture was stirred overnight at room temperature then, the
solvent was evaporated and the residue was dissolved in dichloro-
methane. The organic phase was washed twice with water and the
aqueous phase was extracted by dichloromethane. The combined
organic extracts were dried over Na2SO4, filtered and concentrated.
The residue was purified by flash chromatography on silica gel
(CH2Cl2/MeOH, 95:5) to give compound 14 as a colorless oil
(1.03 g, 73% yield). 1H NMR (CDCl3) d: 5.18 (bs, 1H, NH), 4.18 (d,
2H, J = 5.2 Hz, CH2O), 3.88 (d, 2H, J = 5.2 Hz, CH2N), 3.71 (m, 1H,
OH), 1.38 (s, 9H, (CH3)3C) ppm.
C16H23NO3 + Na: 300.1570; found: 300.1577.
2.2.3. (4-Aminomethylphenyl)methanol (9) [21]
To a an ice-bath cooled suspension of lithium aluminum hy-
dride (629 mg, 16.55 mmol) in anhydrous THF (40 mL), 4-(amino-
methyl)benzoic acid 8 (1.0 g, 6.62 mmol) was added portion-wise.
The reaction mixture was stirred at 0 °C for 1 h then it was heated
under reflux for 3 h. The mixture was cooled to 0 °C and water was
added. The aqueous phase was extracted with a mixture of dichlo-
romethane/isopropanol (4:1). Addition of a few drops of aqueous
solution of Rochelle salt avoids emulsion formation. Combined or-
ganic phases were dried over sodium sulfate, filtered and concen-
trated in vacuo to give compound 9 as a yellow solid (250 mg, 28%).
1H NMR (CD3OD) d: 7.32 (m, 4H, H-phenyl), 4.58 (s, 2H, OCH2), 3.79
(s, 2H, NCH2) ppm.
2.2.7. 9-[(6-N-benzyloxycarbonylamino)hexyl]adenine (16)
To an ice-bath cooled solution of compound 14 (260 mg,
1.40 mmol) in anhydrous THF (5 mL), were added dropwise meth-
anesulfonyl chloride (130
lL, 1.68 mmol) and triethylamine
(240 L, 1.71 mmol). After the addition, the reaction mixture was
l
allowed to warm up to room temperature. After 17 h stirring, the
reaction mixture was filtered then concentrated in vacuo and the
residue was dissolved in dichloromethane. The organic layer was
washed with water, dried over Na2SO4, filtered then concentrated
in vacuo to give compound 15 as a yellow oil (380 mg, 76% yield)
which should be used without delay.
2.2.4. 4-[(N-benzyloxycarbonylamino)methylphenyl]methanol (10)
To a suspension of compound 9 (240 mg, 1.75 mmol) and so-
dium carbonate (371 mg, 3.5 mmol) in water (20 mL) was added
To a suspension of adenine (1.61 g, 11.9 mmol) and triphenyl-
phosphine (2.34 g, 8.9 mmol) in dry THF (10 mL) under argon
dropwise benzyl chloroformate (420
lL, 2.97 mmol). The reaction
atmosphere, was added a solution of compound 4 (1.74 g,
mixture was stirred at room temperature for 3 h then the aqueous
phase was extracted with dichloromethane. The combined organic
phases were washed with water, dried over Na2SO4, filtered, then
concentrated in vacuo to give a solid, which was recristallised from
ethyl acetate/cyclohexane to give compound 10 as a white solid
6.9 mmol) and diisopropylazadicarboxylate (1.77 mL, 8.9 mmol)
in dry THF (10 mL). The reaction mixture was stirred at room tem-
perature overnight then the solvent was evaporated. The precipi-
tate was washed with
a mixture dichloromethane/methanol
(90:10) then combined filtrates were concentrated in vacuo. The

1180
W. Zeinyeh et al. / Steroids 77 (2012) 1177–1191
residue was purified by flash chromatography on silica gel (CH2Cl2/
MeOH, 95:5) to give a crude material which was recristallised from
methanol to give compound 16 as a white solid (1.05 g, 41% yield).
Mp = 132–133 °C; UV (CHCl3) kmax 261 nm; 1H NMR (DMSO-d6) d:
8.15 (s, 1H, H-2), 8.13 (s, 1H, H-8), 7.40–7.10 (m, 8H, 5 ꢁ H-phenyl,
NH2, NHCOO), 4.99 (s, 2H, CH2Ph), 4.11 (t, 2H, J = 7 Hz, CH2N), 2.96
(q, 2H, J = 6.3 Hz, CH2NH), 1.77 (m, 2H, CH2), 1.45–1.15 (m, 6H,
3 ꢁ CH2) ppm; 13C NMR (DMSO-d6) d: 156.93 (NHCOO), 156.82
(C-6), 153.21 (C-2), 150.39 (C-4), 141.67 (C-8), 138.16 (C-phenyl
ipso), 129.17 (2 ꢁ C-phenyl meta, C-phenyl para), 128.56 (2 ꢁ C-
phenyl ortho), 119.61 (C-5), 65.93 (CH2Ph), 43.67 (CH2), 40.98
(CH2), 30.21 (CH2), 30.07 (CH2), 26.56 (CH2), 26.51 (CH2) ppm;
MS(ESI): m/z (%) 758 (13) [2M+Na]+, 369 (100) [M+H]+; HRMS
(ESI): m/z: calcd for C19H24N6O2 + H: 369.2039; found 369.2035.
2.59 (d, 2H, J = 7.1 Hz, CH2NH2), 1.89–1.68 (m, 3H, 3 ꢁ H-cyclo-
hexyl), 1.60–1.44 (m, 3H, 3 ꢁ H-cyclohexyl), 1.05–0.78 (m, 4H,
4 ꢁ H-cyclohexyl) ppm; 13C NMR (DMSO-d6) d: 155.93 (C-6),
152.36 (C-2), 149.70 (C-4), 141.24 (C-8), 118.64 (C-5), 48.61
(CH2), 44.17 (CH2), 29.14 (2 ꢁ CH2), 28.92 (2 ꢁ CH2), 37.29 (CH),
35.37 (CH) ppm; MS(CI): m/z (%) 261 (100) [M+H]+; HRMS (ESI):
m/z: calcd for C13H20N6 + H: 261.1828; found: 261.1826.
2.2.11. 9-[4-(N-benzyloxycarbonylaminomethyl)phenylmethyl]
adenine (20)
To an ice-bath cooled solution of triphenylphosphine (1.16 g,
4.42 mmol) in anhydrous THF (18 mL), was added dropwise diiso-
propylazadicarboxylate (880 lL, 4.44 mmol). The resulting mixture
was stirred for 30 min at room temperature. Adenine (598 mg,
4.43 mmol) and a solution of compound 10 (400 mg, 1.47 mmol)
in anhydrous THF (10 mL) were then added. The reaction mixture
was stirred at room temperature overnight then filtered. The pre-
cipitate was washed with a mixture dichloromethane/methanol
(90:10) then combined filtrates were concentrated in vacuo. The
residue was purified by preparative TLC (CH2Cl2/MeOH, 95:5) to
2.2.8. 9-(6-Aminohexyl)adenine (17)
To a solution of compound 16 (950 mg, 2.58 mmol) in methanol
(50 mL) was added palladium on carbon (400 mg, 10 wt.%). The
reaction mixture was maintained under H2 atmosphere (1 atm)
for 1 h. The resulting mixture was filtered over Celite and the fil-
trate was concentrated in vacuo to give compound 17 as a white
solid (600 mg, 100% yield). Mp = 160–161 °C; UV (CHCl3) kmax
261 nm; 1H NMR (DMSO-d6) d: 8.13 (s, 1H, H-2), 8.12 (s, 1H, H-
8), 7.17 (s, 2H, NH2), 4.11 (t, 2H, J = 7.1 Hz, CH2N), 2.93 (bs, 2H,
NH2), 2.47 (m, 2H, CH2NH2), 1.78 (qn, 2H, J = 7.2 Hz, CH2), 1.35–
1.15 (m, 6H, 3 ꢁ CH2) ppm; 13C NMR (DMSO-d6) d: 155.94 (C-6),
152.33 (C-2), 149.54 (C-4), 140.83 (C-8), 118.73 (C-5), 42.83
(CH2), 41.37 (CH2), 32.91 (CH2), 29.39 (CH2), 25.92 (CH2), 25.79
(CH2) ppm; MS(CI): m/z (%) 235 (100) [M+H]+; HRMS (CI): m/z:
calcd for C11H18N6 + H: 235.1671; found: 235.1671.
give compound 20 as
a white solid (100 mg, 18% yield).
Mp = 227–228 °C; 1H NMR (DMSO-d6) d: 8.26 (s, 1H, H-2), 8.16 (s,
1H, H-8), 7.79 (t, 1H, J = 6.1 Hz, NH), 7.48–7.18 (m, 11H, 9 ꢁ H-phe-
nyl + NH2), 5.33 (s, 2H, CH2N), 5.01 (s, 2H, CH2OCONH), 4.15 (d, 2H,
J = 6.1 Hz, CH2NH) ppm; 13C NMR (DMSO-d6) d: 156.31(NHCOO),
155.58 (C-6), 152.09 (C-2), 149.36 (C-4), 140.95 (C-8), 139.32
(C-phenyl ipso), 137.12 (C-phenyl ipso), 135.58 (C-phenyl ipso),
128.33 (CH-phenyl), 127.78 (CH-phenyl), 127.74 (CH-phenyl),
127.56 (CH-phenyl), 127.34 (CH-phenyl), 118.64 (C-5), 65.37
(CH2OCO), 45.96 (CH2), 43.50 (CH2) ppm; MS(ESI): m/z (%) 798
(72) [2M+Na]+, 776 (17) [2M+H]+, 411 (29) [M+Na]+, 389 (100)
[M+H]+; HRMS (ESI): m/z: calcd for C21H20N6O2 + H: 389.1721;
found: 389.1705.
2.2.9. 9-[Trans-4-(N-
benzyloxycarbonylaminomethyl)cyclohexylmethyl]adenine (18)
To an ice-cooled solution of triphenylphosphine (993 mg,
3.79 mmol) in anhydrous THF (15 mL), was added dropwise diiso-
2.2.12. 9-(4-aminomethylphenylmethyl)adenine (21)
propylazadicarboxylate (750
l
L, 3.79 mmol). The resulting mixture
To a solution of compound 20 (80 mg, 0.20 mmol) in methanol
(30 mL) and ethylacetate (30 mL) was added palladium on carbon
(80 mg, 10 wt.%). The reaction mixture was maintained under pres-
sure of H2 (13 atm) overnight. The resulting mixture was filtered
over Celite and the filtrate was concentrated in vacuo. The residue
was purified by column chromatography (CH2Cl2/MeOH 90/10
then CH2Cl2/MeOH/NH4OHaq., 90:10:2) to give compound 21 as a
white solid (40 mg, 77% yield). Mp (dec.); 1H NMR (CD3OD) d:
8.20 (s, 1H, H-2), 8.18 (s, 1H, H-8), 7.41–7.18 (m, 6H, H-phe-
nyl + NH2), 5.45 (s, 2H, CH2N), 3.98 (s, 2H, CH2NH2) ppm; 13C
NMR (CD3OD) d: 157.38 (C-6), 153.91 (C-2), 150.68 (C-4), 142.59
(C-8), 138.04 (C-phenyl ipso), 137.44 (C-phenyl ispo), 130.07
(CH-phenyl), 129.35 (CH-phenyl), 120.00 (C-5), 47.72 (CH2),
44.66 (CH2) ppm; MS(ESI): m/z (%) 255 (100) [M+H]+; HRMS
(ESI): m/z: calcd for C13H14N6 + H: 255.1353; found: 255.1342.
was stirred for 30 min at room temperature then it was added drop-
wise to a suspension in anhydrous THF (3 mL) of adenine (511 mg,
3.79 mmol) and compound 7 (350 mg, 1.26 mmol). The reaction
mixture was stirred at room temperature overnight then filtered.
The precipitate was washed with a mixture dichloromethane/
methanol (90:10) then combined filtrates were concentrated in va-
cuo. The residue was purified by flash chromatography on silica gel
(CH2Cl2/MeOH, 95:5) to give compound 18 as a white solid
(287 mg, 49 % yield). Mp = 229 °C; 1H NMR (DMSO-d6) d: 8.12 (s,
1H, H-2), 8.08 (s, 1H, H-8), 7.37–7.21 (m, 5H, 5 ꢁ H-phenyl), 7.16
(s, 2H, NH2), 4.98 (s, 2H, CH2-Ph), 3.97 (d, 2H, J = 7.0 Hz, CH2N),
2.82 (t, 2H, J = 6.1 Hz, CH2NHCbz), 1.80–0.70 (m, 10H, 10 ꢁ H-cyclo-
hexyl) ppm; 13C NMR (DMSO-d6) d: 156.23 (NHCOO), 155.94 (C-6),
152.36 (C-2), 149.72 (C-4), 141.20 (C-8), 137.30 (C-phenyl ipso),
128.32 (2 ꢁ C-phenyl meta, C-phenyl para), 127.70 (2 ꢁ C-phenyl
ortho), 118.64 (C-5), 65.09 (CH2Ph), 48.72 (CH2), 46.46 (CH2),
29.50 (2 ꢁ CH2), 29.40 (2 ꢁ CH2), 37.67 (2 ꢁ CH) ppm; MS(ESI):
m/z (%) 417 (36) [M+Na]+, 395 (6) [M+H]+; HRMS (ESI): m/z: calcd
for C21H26N6O2 + Na: 417.2015; found: 417.2012.
2.2.13. 9-[(4-N-tert-butyloxycarbonylamino)but-2-ynyl]adenine (22)
A suspension of adenine (380 mg, 2.8 mmol) and potassium car-
bonate (233 mg, 1.68 mmol) in DMF (40 mL) was heated to 75 °C
for 1 h. To the resulting mixture was added a solution of compound
15 (370 mg, 1.4 mmol) in DMF (10 mL) and dichloromethane
(1 mL). Stirring was carried on at 75 °C for 1h30 then solvents were
removed under vacuum. The residue was partitioned between
water and dichloromethane. Aqueous phase was extracted by
CH2Cl2 and the combined organic extracts were dried (Na2SO4), fil-
tered and concentrated. The residue was purified by flash chroma-
tography on silica gel (CH2Cl2/MeOH, 95:5) to give compound 22 as
a white solid (320 mg, 75% yield). Mp = 200 °C (dec.); 1H NMR
(DMSO-d6) d: 8.18 (s, 1H, H-2), 8.15 (s, 1H, H-8), 7.26 (s, 2H,
NH2), 5.00 (s, 2H, CH2N), 3.75 (d, 2H, J = 5.3 Hz, CH2NHCOO), 1.36
(m, 10H, NH, (CH3)3C) ppm; 13C NMR (DMSO-d6) d: 155.99 (C-6),
2.2.10. 9-(Trans-4-aminomethylcyclohexylmethyl)adenine (19)
To a solution of compound 18 (255 mg, 0.65 mmol) in methanol
(20 mL) was added palladium on carbon (100 mg, 10 wt.%). The
reaction mixture was maintained under pressure of H2 (5 atm) for
15 min. The resulting mixture was filtered over Celite and the fil-
trate was concentrated in vacuo. The residue was purified by pre-
parative TLC (CH2Cl2/MeOH/NH4OHaq., 80:20:1) to give compound
19 as a white solid (120 mg, 71% yield). Mp (dec.); UV (CHCl3) kmax
261 nm; 1H NMR (DMSO-d6) d: 8.12 (s, 1H, H-2), 8.10 (s, 1H, H-8),
7.61(bs, 2H, NH2), 7.18 (s, 2H, NH2), 3.98 (d, 2H, J = 7.1 Hz, CH2N),

W. Zeinyeh et al. / Steroids 77 (2012) 1177–1191
1181
155.23 (NHCOO), 152.64 (C-2), 149.06 (C-4), 140.03 (C-8), 118.54
(C-5), 82.61 (CBC), 78.27 (CBC), 75.79 (C(CH3)3), 34.47 (CH2),
29.50 (CH2), 28.15 (3 ꢁ CH3) ppm; MS(CI): m/z (%) 303 (100)
[M+H]+; HRMS (CI): m/z: calcd for C14H18N6O2 + H: 303.1569;
found: 303.1568.
(DMSO-d6) d: 155.93 (C-6), 152.33 (C-2), 149.54 (C-4), 140.83 (C-
8), 118.73 (C-5), 42.85 (CH2), 41.08 (CH2), 30.26 (CH2), 26.99
(CH2) ppm; MS(CI): m/z (%) 207 (100) [M+H]+; HRMS (ESI): m/z:
calcd for C9H14N6 + H: 207.1358; found: 207.1361.
2.2.18. N-[2-(2-hydroxyethoxy)ethyl]phtalimide (28a) [25]
2.2.14. 9-(4-Aminobut-2-ynyl)adenine (23)
To a solution of chloroethoxyethanol 27a (623 mg, 5 mmol) in
DMF (5 mL) was added potassium phtalimide (770 mg,
4.16 mmol). The reaction mixture was then stirred for 2 h at
130 °C. After addition of water (10 mL) and EtOAc (20 mL) and
decantation, the aqueous phase was extracted with EtOAc
(2 ꢁ 20 mL) and the organic phase was dried over Na2SO4. After fil-
tration and removal of the solvents under reduced pressure, the
residue was purified by flash chromatography (Cyclohexane/Ethyl
acetate, 30:70) to afford compound 28a (836 mg, 85%) as a yellow
oil. 1H NMR (CDCl3) d: 7.84 (m, 2H, 2 ꢁ H-phenyl), 7.71 (m, 2H,
2 ꢁ H-phenyl), 3.90 (t, 2H, J = 5.4 Hz, CH2), 3.73 (m, 2H, CH2),
3.67 (m, 2H, CH2), 3.60 (m, 2H, CH2), 2.32 (broad s, 1H, OH) ppm.
To a suspension of compound 22 (280 mg, 0.93 mmol) in water
(15 mL) was added dropwise concentrated HCl (3 mL) under vigor-
ous stirring. The reaction mixture was stirred for few minutes then
solvent was removed. The residue was dissolved in water and trea-
ted with Dowex 1X8 R3N+Clꢂ prewashed with 4% NaOH aq. Solu-
tion. The filtrate was concentrated and dried in vacuo to give
compound 23 as a white solid (150 mg, 80% yield) which was recri-
stallised from methanol. Mp = 135 °C (dec.); 1H NMR (DMSO-d6) d:
8.24 (s, 1H, H-2), 8.16 (s, 1H, H-8), 7.76 (bs, 2H, NH2), 7.29 (s, 2H,
NH2), 5.10 (m, 2H, CH2N), 3.71 (m, 2H, CH2NH2) ppm; 13C NMR
(CD3OD) d: 157.34 (C-6), 153.88 (C-2), 150.20 (C-4), 142.04 (C-8),
119.93 (C-5), 86.94 (CBC), 76.26 (CBC), 34.22 (CH2), 31.41 (CH2)
ppm; MS(CI): m/z (%) 203 (100) [M+H]+; HRMS (CI): m/z: calcd
for C9H10N6 + H: 203.1045; found: 203.1043.
2.2.19. N-{2-[2-(2-Hydroxyethoxy)ethoxy]ethyl}phtalimide (28b) [26]
To
a solution of chloroethoxy(ethoxy)ethanol 27b (1.09 g,
2.2.15. (E)-9-(4-aminobut-2-enyl)adenine (24) [24]
6.47 mmol) in DMF (7 mL) was added potassium phtalimide (1 g,
5.39 mmol). The reaction mixture was then stirred for 2 h at
130 °C. After addition of water (10 mL) and EtOAc (20 mL) and
decantation, the aqueous phase was extracted with EtOAc
(3 ꢁ 20 mL) and the organic phase was dried over Na2SO4. After fil-
tration and removal of the solvents under reduced pressure, the
residue was purified by flash chromatography (Cyclohexane/Ethyl
acetate, 30:70) to afford compound 28b (1.46 g, 97%) as a colorless
oil. 1H NMR (CDCl3) d: 7.85 (m, 2H, 2 ꢁ H-phenyl), 7.71 (m, 2H,
2 ꢁ H-phenyl), 3.91 (t, 2H, J = 5.7 Hz, CH2), 3.75 (t, 2H, J = 5.6 Hz,
CH2), 3.58–3.67 (m, 6H, 3 ꢁ CH2), 3.53 (m, 2H, CH2), 2.53 (broad
s, 1H, OH) ppm.
To a suspension of compound 23 (200 mg, 1 mmol) in anhy-
drous THF (10 mL) was added lithium aluminum hydride
(400 mg, 20 mmol). The mixture was stirred for two days at room
temperature then the reaction was quenched by adding an aque-
ous solution of sodium hydroxide (1N, 5 mL). The precipitate was
filtered, and washed with ethyl acetate. Combined filtrates were
concentrated in vacuo then purified by flash chromatography on
silica gel (CH2Cl2/MeOH/NH4OHaq., 20:20:1) to give compound 24
as a white solid (57 mg, 29% yield). UV (CHCl3) kmax 260 nm; 1H
NMR (DMSO-d6) d: 8.13 (s, 1H, H-2), 8.10 (s, 1H, H-8), 7.21 (s,
2H, NH2), 5.84–5.64 (m, 2H, HC = CH), 4.73 (d, 2H, J = 5.4 Hz,
CH2N), 3.40–3.10 (m, 4H, CH2NH2, NH2) ppm; 13C NMR (DMSO-
d6) d: 155.94 (C-6), 152.46 (C-2), 149.32 (C-4), 140.53 (C-8),
135.54 (CH = CH), 123.61 (CH = CH), 118.65 (C-5), 44.11 (CH2),
42.52 (CH2) ppm.
2.2.20. N-[2-(2-iodoethoxy)ethyl]phtalimide (29a) [25]
To a solution of compound 28a (1 g, 4.25 mmol) in Et2O/MeCN
(13.5 mL/4.5 mL) at 0 °C was added imidazole (868 mg,
12.7 mmol), PPh3 (1.67 g, 6.37 mmol) and I2 (1.62 g, 6.37 mmol).
The reaction mixture was stirred at 0 °C for 2 h and at room tem-
perature for 1 h. After addition of pentane, the resulting mixture
was filtered off on a sintered glass funnel filled with a pad of silica
gel 1 cm thick, and washed with EtOAc. After evaporation of the fil-
trate under reduced pressure, the residue was purified by flash
chromatography (Cyclohexane/Ethyl acetate, 70:30) to afford com-
pound 29a (1.06 g, 72%) as a yellow solid. Mp = 84–85 °C (lit. 79–
81 °C) [24]; 1H NMR (CDCl3) d: 7.85 (m, 2H, 2 ꢁ H-phenyl), 7.71
(m, 2H, 2 ꢁ H-phenyl), 3.90 (t, 2H, J = 5.1 Hz, CH2), 3.73 (m, 4H,
CH2), 3.18 (t, 2H, J = 6.6 Hz, CH2) ppm.
2.2.16. (Z)-9-(4-aminobut-2-enyl)adenine (25) [24]
To a solution of compound 23 (70 mg, 0.35 mmol) in methanol
(70 mL) was added Lindlar catalyst (7 mg, 5 wt.%) and two drops of
quinoline. The reaction mixture was maintained under H2 atmo-
sphere (1 atm) until the reaction was complete (TLC control). Then
the reaction mixture was filtered over Celite and the filtrate was
concentrated in vacuo. The residue was purified by preparative
TLC (CH2Cl2/MeOH/NH4OHaq., 30:20:1) to give compound 25 as a
white solid (47 mg, 67% yield). 1H NMR (CD3OD) d: 8.17 (s, 1H,
H-2), 8.09 (s, 1H, H-8), 5.85–5.60 (m, 2H, HC = CH), 5.00–4.70 (m,
6H, CH2N, 2 ꢁ NH2), 3.53 (d, 2H, J = 6.8 Hz, CH2NH2) ppm; 13C
NMR (CD3OD) d: 157.28 (C-6), 153.70 (C-2), 150.46 (C-4), 142.34
(C-8), 134.46 (CH = CH), 126.19 (CH = CH), 120.01 (C-5), 41.45
(CH2), 38.58 (CH2) ppm.
2.2.21. N-{2-[2-(2-iodoethoxy)ethoxy]ethyl}phtalimide (29b) [27]
To a solution of compound 28b (1.3 g, 4.65 mmol) in Et2O/
MeCN (13.5 mL/4.5 mL) at 0 °C was added imidazole (950 mg,
13.9 mmol), PPh3 (1.83 g, 6.98 mmol) and I2 (1.77 g, 6.98 mmol).
The reaction mixture was stirred at 0 °C for 2 h and at room tem-
perature for 1 h. After addition of pentane, the resulting mixture
was filtered off on a sintered glass funnel filled with a pad of silica
gel 1 cm thick, and washed with EtOAc. After evaporation of the fil-
trate under reduced pressure, the residue was purified by flash
chromatography (Cyclohexane/Ethyl acetate, 70:30) to afford com-
pound 29b (1.05 g, 58%) as a colorless viscous oil. 1H NMR (CDCl3)
d: 7.84 (m, 2H, 2 ꢁ H-phenyl), 7.71 (m, 2H, 2 ꢁ H-phenyl), 3.90 (t,
2H, J = 5.7 Hz, CH2), 3.75 (t, 2H, J = 5.7 Hz, CH2), 3.59–3.69 (m, 6H,
3 ꢁ CH2), 3.16 (td, 2H, J = 6.9 Hz, J = 1.0 Hz, CH2) ppm.
2.2.17. 9-(4-aminobutyl)adenine (26)
To a solution of compound 23 (250 mg, 1.24 mmol) in methanol
(70 mL) was added palladium on carbon (200 mg, 10 wt.%). The
reaction mixture was maintained under H2 atmosphere (1 atm)
until the reaction was complete (TLC control). Then the reaction
mixture was filtered over Celite and the filtrate was concentrated
in vacuo. The residue was purified by flash chromatography on sil-
ica gel (CH2Cl2/MeOH/NH4OHaq., 20:20:1) to give compound 26 as
a white solid (227 mg, 90% yield). Mp = 164–166 °C; 1H NMR
(DMSO-d6) d: 8.14 (s, 1H, H-2), 8.12 (s, 1H, H-8), 7.17 (s, 2H,
NH2), 4.12 (t, 2H, J = 7.3 Hz, CH2N), 1.81 (qn, J = 7.5 Hz, 2H,
CH2NH2), 1.28 (qn, J = 6.9 Hz, 4H, 2 ꢁ CH2) ppm; 13C NMR

1182
W. Zeinyeh et al. / Steroids 77 (2012) 1177–1191
2.2.22. N6,N6-Bis(tert-butoxycarbonyl)adenine (30) [28]
and then compound 29b (580 mg, 1.49 mmol) was added. The
resulting mixture was then stirred at 70 °C for 19 h. After addition
of water (20 mL) and EtOAc (30 mL) and decantation, the aqueous
phase was extracted with EtOAc (3 ꢁ 20 mL) and the organic phase
was dried over Na2SO4. After filtration and removal of the solvents
under reduced pressure, the residue was purified by flash chroma-
tography (cyclohexane/AcOEt, 10:90) to afford compound 31b
(412 mg, 46%) as a yellow oil. 1H NMR (CDCl3) d: 8.83 (s, 1H, s,
H-2), 8.31 (s, 1H, H-8), 7.85 (m, 2H, 2 ꢁ H-phenyl), 7.71 (m, 2H,
2 ꢁ H-phenyl), 4.41 (2H, t, J = 5.1 Hz, CH2), 3.90 (2H, t, J = 5.7 Hz,
CH2), 3.79 (2H, t, J = 4.8 Hz, CH2), 3.71 (2H, t, J = 5.7 Hz, CH2), 3.57
(m, 4H, 2 ꢁ CH2), 1.45 (s, 18H, C(CH3)3) ppm.
To a solution of adenine (1.35 g, 10 mmol) and DMAP (122 mg,
1 mmol) in THF (50 mL) under argon was added Boc2O (9.38 g,
43 mmol). The reaction was stirred 5 h at room temperature. After
removal of the solvent, EtOAc (400 mL) was added and the solution
was washed with HCl 1 N (30 mL) and then with NaCl
(3 ꢁ 100 mL). After drying over Na2SO4, filtration and removal of
the solvents under reduced pressure, the residue was dissolved
in methanol (100 mL) and NaHCO3 sat. (45 mL). The reaction was
then stirred for 1h30 at 50 °C and after removal of the solvent,
water (100 mL) was added. The aqueous phase was then extracted
with CHCl3 (2 ꢁ 300 mL) and, after drying over Na2SO4, the solvent
was removed under reduced pressure. The resulting crude product
was purified by flash chromatography (cyclohexane/AcOEt, 10:90)
to afford compound 30 (2.44 g, 73%) as a white solid. 1H NMR
(DMSO-d6) d: 13.69 (s, 1H, NH), 8.80 (s, 1H, H-2), 8.63 (s, 1H, H-
8), 1.36 (s, 18H, C(CH3)3) ppm.
2.2.26. 9-{2-[2-(2-Aminoethoxy)ethoxy]ethyl}adenine (32b)
Compound 32b according to the same procedure as for com-
pound 32a, scale: 31b (412 mg, 0.69 mmol), CH2Cl2 (7 mL), trifluo-
roacetic acid (1.92 mL, 25.8 mmol) reaction time 16 h. To a solution
of the obtained crude fully Boc deprotected compound (458 mg) in
absolute ethanol (9 mL) was slowly added methylhydrazine
2.2.23. N-{2-[2-(N6,N6-Bis(tert-butoxycarbonyl)adenin-9-yl)ethoxy]
ethyl}phtalimide (31a)
(572 lL, 10.77 mmol). The reaction mixture was then stirred at
To a solution of protected adenine 30 (295 mg, 0.88 mmol) in
DMF (10 mL) was added NaH (60% in mineral oil, 42 mg,
1.05 mmol). The reaction mixture was stirred at 70 °C for one hour
and then compound 29a (364 mg, 1.05 mmol) was added and the
reaction mixture was stirred for 4 h at 70 °C. After addition of
water (20 mL) and EtOAc (30 mL) and decantation, the aqueous
phase was extracted with EtOAc (2 ꢁ 30 mL) and the organic phase
was dried over Na2SO4. After filtration and removal of the solvents
under reduced pressure, the residue was purified by flash chroma-
tography (cyclohexane/AcOEt, 30:70) to afford compound 31a
(298 mg, 61%) as a colorless oil. 1H NMR (CDCl3) d: 8.83 (s, 1H,
H-2), 8.38 (s, 1H, H-8), 7.86 (m, 2H, 2 ꢁ H-phenyl), 7.72 (m, 2H,
2 ꢁ H-phenyl), 4.46 (2H, t, J = 5.0 Hz, CH2), 3.88 (2H, t, J = 5.4 Hz,
CH2), 3.82 (2H, t, J = 4.8 Hz, CH2), 3.68 (2H, t, J = 5.3 Hz, CH2), 1.46
(s, 18H, C(CH3)3) ppm; 13C NMR (CDCl3) d: 168.32 (C), 153.29 (C),
151.92 (CH), 150.59 (C), 150.05 (C), 146.11 (CH), 134.20 (CH),
132.04 (C), 128.24 (C), 123.51 (CH), 83.86 (C), 68.50 (CH2), 43.93
(CH2), 37.25 (CH2), 29.78 (CH2), 27.90 (CH3).
70 °C for 24 h. After removal of the solvents under reduced pres-
sure, the residue was purified by flash chromatography (CH2Cl2/
MeOH, 80:20 with 2% of Et3N) to afford compound 32b (178 mg,
97% from 31b) as a yellow oil. 1H NMR (DMSO-d6) d: 8.13 (s, 1H,
H-2), 8.09 (s, 1H, H-8), 7.81 (s, 2H, disappears after D2O addition,
NH2), 4.29 (t, 2H, J = 5.3 Hz, CH2), 3.78 (t, 2H, J = 5.4 Hz, CH2),
3.45 (m, 4H, 2 ꢁ CH2), 3.31 (t, 2H, J = 5.8 Hz, CH2), 2.64 (t, 2H,
J = 5.8 Hz, CH2) ppm; 13C NMR (DMSO-d6) d: 173.12 (C), 155.85
(C), 152.28 (CH), 149.44 (C), 141.13 (CH), 71.69 (CH2), 69.41
(CH2), 69.37 (CH2), 68.19 (CH2), 42.63 (CH2), 41.03 (CH2) ppm.
2.2.27. Androst-4-en-3-one-17b-carboxylic acid (33) [29]
A sodium hypobromite solution was prepared by slow addition
of bromine (730 lL, 14.2 mmol) to a solution of sodium hydroxide
(1.205 g, 30.1 mmol) in distilled water (5 mL) under argon atmo-
sphere. This first mixture was stirred for 1 h to provide an orange
solution. In a separate flask, a solution of sodium hydroxide
(625 mg, 15.6 mmol) in distilled water (5 mL) was added to a solu-
tion of progesterone (1.00 g, 3.18 mmol) in tert-butanol (10 mL) at
room temperature. This second mixture was stirred for 1 h at room
temperature then cooled in an ice bath before being treated with
the previously prepared sodium hypobromite solution. The result-
ing mixture was vigorously stirred at 0 °C for 3 h then the reaction
was quenched with an aqueous solution of sodium sulfite (2.1 M,
1.5 mL). Stirring was continued overnight at room temperature.
The mixture was concentrated in vacuo and extracted with toluene.
The combined organic phases were acidified with concentrated HCl
until pH 0. The resulting precipitate was filtered off and recristal-
lised from methanol/water (4:1) to give compound 33 as a white
solid (0.570 g, 57 % yield). Mp (dec.); 1H NMR (DMSO-d6) d: 5.62
(s, 1H, H-4), 2.40–0.80 (m, 17H), 1.14 (s, 3H, CH3), 0.67 (s, 3H,
CH3) ppm.
2.2.24. 9-[2-(2-Aminoethoxy)ethyl]adenine (32a)
To a solution of compound 31a (298 mg, 0.54 mmol) in CH2Cl2
(5 mL) was added trifluoroacetic acid (1.5 mL, 20.2 mmol). The
reaction mixture was then stirred at room temperature for 16 h.
After removal of the solvent under reduce pressure, EtOAc was
added and the precipitate was filtered to afford the fully Boc depro-
tected compound (197 mg, 78%, trifluoroacetate salt) as a white so-
lid. 1H NMR (DMSO-d6) d: 8.66 (s, 2H, NH2), 8.31 (s, 1H, H-2), 8.23
(s, 1H, H-8), 7.81 (m, 4H, H-phenyl), 4.32 (t, 2H, J = 4.8 Hz, CH2),
3.78 (t, 2H, J = 5.1 Hz, CH2), 3.68 (t, 2H, J = 4.8 Hz, CH2), 3.62 (t,
2H, J = 5.1 Hz, CH2) ppm.
To a solution of the fully Boc deprotected compound (197 mg,
0.42 mmol) in absolute ethanol (5 mL) was slowly added meth-
ylhydrazine (269 lL, 5.07 mmol). The reaction mixture was then
stirred at 70 °C for 17 h. After removal of the solvents under re-
duced pressure, the residue was purified by flash chromatography
(CH2Cl2/MeOH, 80:20 with 2% of Et3N) to afford compound 32a
(87 mg, 73% from 31a) as a yellow oil. 1H NMR (CDCl3) d: 8.13 (s,
1H, s, H-2), 8.11 (s, 1H, H-8), 7.17 (broad s, 2H, NH2), 4.30 (t, 2H,
J = 5.3 Hz, CH2), 3.75 (t, 2H, J = 5.3 Hz, CH2), 3.38 (m, 4H,
2 ꢁ CH2), 2.64 (broad s, 2H, NH2) ppm.
2.2.28. N-succinimidyl ester of androst-4-en-3-one-17b-carboxylic
acid (34) [30]
To a solution of N-hydroxysuccinimide (110 mg, 0.96 mmol)
and compound 33 (300 mg, 0.95 mmol) in anhydrous THF (6 mL)
was added a solution of N,N0-dicyclohexylcarbodiimide (DCC,
195 mg, 0.95 mmol) in anhydrous THF (3 mL). The reaction mix-
ture was stirred overnight at room temperature. The resulting mix-
ture was filtered and the filtrate was concentrated in vacuo. The
residue was purified by flash chromatography on silica gel
(CH2Cl2/MeOH, 99:1) followed by recristallisation from metha-
nol/water to give compound 34 as a white solid (216 mg, 55 %
yield). Mp = 233–236 °C; 1H NMR (DMSO-d6) d: 5.64 (s, 1H, H-4),
2.2.25. N-{2-[2-(2-(N6,N6-Bis(tert-butoxycarbonyl)adenin-9-yl)
ethoxy)ethoxy]ethyl}phtalimide (31b)
To a solution of protected adenine 30 (500 mg, 1.49 mmol) in
DMF (8 mL) was added NaH (60% in mineral oil, 65 mg,
1.64 mmol). The reaction mixture was stirred one hour at 70 °C

W. Zeinyeh et al. / Steroids 77 (2012) 1177–1191
1183
2.81 (m, 4H, 2 ꢁ CH2-succinimide), 2.50–0.90 (m, 20H), 1.16 (s, 3H,
NaOH aqueous solution (4 ꢁ 20 mL) then with water (4 ꢁ 20 mL).
The organic phase was dried over Na2SO4, filtered through What-
man #4 paper and concentrated in vacuo. The residue was purified
by flash chromatography on silica gel (ACOEt/cyclohexane, 1:2) to
give compound 35a as a white solid (1.23 g, 50% yield) and trace
amounts of epimer 35b (0.01 g, 4% yield).
CH3), 0.78 (s, 3H, CH3) ppm.
2.2.29. 17b-{[2-(2-(6-Aminopurin-9-yl)ethoxy)ethyl] carbamoyl}
androst-4-en-3-one (1h)
To a solution of compound 34 (47 mg, 0.11 mmol) and com-
pound 32a (25 mg, 0.11 mmol) in dry DMF (2 mL) was added
N,N-diisopropylethylamine (20
lL, 0.17 mmol). The resulting mix-
2.2.31.1. D
6-Progesterone (35a)[9,31]. Mp = 144–145 °C (Litt 144–
ture was stirred at 35 °C during 20 h. Water (20 mL) and ethyl ace-
tate (15 mL) were then added and, after extraction and
decantation, the aqueous phase was extracted with ethyl acetate
(2 ꢁ 15 mL). The organic phases were then dried over Na2SO4, fil-
tred and the solvents were removed in vacuo. The residue was puri-
fied by flash chromatography on silica gel (CH2Cl2/MeOH, 90:10) to
give compound 1h as a white solid (36 mg, 61% yield). Mp = 202–
145 °C) [9]; 1H NMR (CDCl3) d: 6.10 (m, 2H, H-6 et H-7), 5.67 (s,
1H, H-4), 2.64–2.51 (m, 2H), 2.42 (m, 1H), 2.29–1.10 (m, 13H),
2.12 (s, 3H, CH3-21), 1.10 (s, 3H, CH3-19), 0.70 (s, 3H, CH3-18)
ppm; 13C NMR (CDCl3) d: 209.15 (C-20), 199.58 (C-3), 163.53 (C-
5), 140.64 (CH-7), 128.26 (CH-6), 123.92 (CH-4), 63.37 (CH-17),
53.79 (CH), 50.68 (CH), 44.84 I, 38.69 (CH2), 37.72 (CH), 36.16 I,
34.03 (2 ꢁ CH2), 31.64 (CH3-21), 24.02(CH2), 23.02 (CH2), 20.76
(CH2), 16.42 (CH3-19), 13.39 (CH3-18) ppm.
1
203 °C; H NMR (DMSO-d6) d: 8.13 (s, 1H, H-20), 8.08 (s, 1H, H-
80), 7.33 (t, 1H, J = 5.7 Hz, NHCO), 7.17 (s, 2H, NH2), 5.62 (s, 1H,
H-4), 4.28 (t, 2H, J = 5.3 Hz, CH2), 3.76 (t, 2H, J = 5.3 Hz, CH2), 3.40
(m, 2H, CH2), 3.28 (m, 1H), 3.06 (m, 1H), 2.43–0.84 (m, 20H),
1.12 (s, 3H, CH3-19), 0.54 (s, 3H, CH3-18) ppm; 13C NMR (DMSO-
d6) d: 198.08 (C), 171.65 (C), 171.00 (C), 155.92 (C), 152.35 (CH),
149.52 (C), 141.11 (CH), 123.15 (CH), 118.57 (C), 69.04 (CH2),
68.10 (CH2), 55.42 (CH), 54.88 (CH), 53.24 (CH), 43.22 (C), 42.72
(CH2), 38.33 (CH2), 38.20 (C), 37.36 (CH2), 35.12 (CH2), 34.97
(CH), 33.62 (CH2), 32.00 (CH2), 31.69 (CH2), 24.20 (CH2), 23.12
(CH2), 20.31 (CH2), 16.87 (CH3), 13.21 (CH3) ppm; HRMS (ESI) m/
z: calc for C29H40N6O3 + H: 521.3235, found: 521.3220.
2.2.31.2. 17a-D
6-Progesterone (35b). Mp = 137–139 °C. 1H NMR
(CDCl3) d: 6.10 (dd, 1H, J1 = 9.8 Hz, J2 = 1.5 Hz, H-6), 6.05 (dd, 1H,
J1 = 9.8 Hz, J2 = 2.5 Hz, H-7), 5.62 (s, 1H, H-4), 2.82 (dd, 1H,
J1 = 8.3 Hz, J2 = 2.0 Hz, H-17), 2.60–2.42 (m, 1H), 2.60–2.42 (m,
1H), 2.39–2.29 (m, 1H), 2.15–1.10 (m, 13H), 2.10 (s, 3H, CH3-21),
1.06 (s, 3H, CH3-19), 0.96 (s, 3H, CH3-18) ppm; 13C NMR (CDCl3)
d: 212.45 (C-20), 199.48 (C-3), 163.59 (C-5), 141.28 (CH-7),
127.87 (CH-6), 123.69 (CH-4), 60.77 (CH-17), 50.05 (CH), 47.48
(CH), 46.33 I, 37.79 (CH), 36.03 I, 34.92 (CH2), 33.96 (CH2), 33.92
(CH2), 32.95 (CH3-21), 25.29 (CH2), 24.48 (CH2), 20.62 (CH3-
18 + CH2), 16.36 (CH3-19) ppm; MS (ESI): m/z (%) 647 (100)
[2M+Na]+, 335 (18) [M+Na]+, 313 (65) [M+H]+; HRMS (ESI): m/z:
calc for C21H28O2 + H: 313.2162, found: 313.2154.
2.2.30. 17b-{[2-(2-[2-(6-Aminopurin-9-yl)ethoxy]ethoxy)ethyl]
carbamoyl}androst-4-en-3-one (1i)
To a solution of compound 34 (104 mg, 0.25 mmol) and com-
pound 32b (67 mg, 0.25 mmol) in dry DMF (2 mL) was added diiso-
2.2.32. Progesterone-7
A solution of Et2AlCN (1 M in toluene, 16 mL, 16 mmol) was
added to a solution of 6-progesterone (35a) (1.23 g, 3.9 mmol)
a-carbonitrile (36)
propylethylamine (20
lL, 0.17 mmol). The resulting mixture was
stirred at 35 °C during 20 h. Water (20 mL) and ethyl acetate
(15 mL) were then added and, after extraction and decantation,
the aqueous phase was extracted with ethyl acetate (2 ꢁ 15 mL).
The organic phases were then dried over Na2SO4, filtred and the
solvents were removed in vacuo. The residue was purified by flash
chromatography on silica gel (CH2Cl2/MeOH, 90:10) to give 157 mg
of a yellow paste. This fraction was further purified (removal of
DMF traces) using TLC plates, eluting one time with CH2Cl2/MeOH,
90:10 and two times with CH2Cl2/MeOH, 95:5. The silica was
rinsed with AcOEt and then with anhydrous ethanol. Evaporation
of the ethanolic fraction gave compound 1i as a yellow paste
D
in anhydrous THF (20 mL) at room temperature. After 2 h stirring,
the mixture was cooled down in an ice bath and a 0.5 M NaOH
aqueous solution (80 mL) was added. Aqueous phase was extracted
by CH2Cl2 and the combined organic extracts were dried (MgSO4),
filtered and concentrated. The residue was purified by flash chro-
matography on silica gel (cyclohexane/AcOEt, 1:1) to give com-
pound 36 as a white solid (1.17 g, 88% yield). Mp = 164–165 °C.
1H NMR (CDCl3) d: 5.87 (d, 1H, H-4), 3.01 (ddd, J = 5.0 Hz,
J = 4.3 Hz, J = 2.2 Hz, 1H, H-7), 2.71–1.16 (m, 18H), 2.13 (s, 3H,
CH3-21), 1.19 (s, 3H, CH3-19), 0.68 (s, 3H, CH3-18) ppm; 13C (CDCl3)
d: 208.85 (C-20), 198.37 (C-3), 163.13 (C-5), 127.26 (CH-4), 118.96
(CN), 63.08 (CH-17), 52.82 (CH), 48.72 (CH), 43.97 (C), 38.34 (C),
37.88 (CH2), 36.77 (CH), 35.37 (CH2), 34.96 (CH2), 33.90 (CH2),
32.96 (CH), 31.63 (CH3-21), 23.90 (CH2), 22.94 (CH2), 20.90 (CH2),
17.49 (CH3-19), 13.39 (CH3-18) ppm; MS(ESI): m/z (%) 701 (42)
[2M+Na]+, 362 (100) [M+Na]+, 340 (65) [M+H]+; HRMS (ESI): m/z:
calcd for C22H29NO2 + H: 340.2271; found: 340.2281.
1
(39 mg, 27% yield). H NMR (DMSO-d6) d: 8.13 (s, 1H, H-20), 8.10
(s, 1H, H-80), 7.37 (broad t, 1H, J = 5.4 Hz, NHCO), 7.19 (s, 2H,
NH2), 5.62 (s, 1H, H-4), 4.29 (t, 2H, J = 5.4 Hz, CH2), 3.78 (t, 2H,
J = 5.3 Hz, CH2), 3.60 (m, 1H), 3.50 (m, 4H, 2 ꢁ CH2), 3.06 (m, 1H),
2.44–0.84 (m, 22H), 1.10 (s, 3H, CH3-19), 0.58 (s, 3H, CH3-18)
ppm; 13C NMR (DMSO-d6) d: 198.02 (C), 171.61 (C), 170.96 (C),
155.93 (C), 152.34 (CH), 149.49 (C), 141.12 (CH), 123.14 (CH),
118.56 (C), 69.43 (CH2), 69.37 (CH2), 69.25 (CH2), 68.30 (CH2),
55.36 (CH), 54.87 (CH), 53.23 (CH), 43.21 (C), 42.60 (CH2), 38.36
(CH2), 38.16 (C), 37.31 (CH2), 35.09 (CH2), 34.96 (CH), 33.61
(CH2), 31.99 (CH2), 31.67 (CH2), 24.19 (CH2), 23.09 (CH2), 20.36
(CH2), 16.84 (CH3), 13.24 (CH3) ppm; HRMS (ESI) m/z: calc for
2.2.33. Progesterone-7a-carboxylic acid (38)
A solution of DIBAL-H (1 M in toluene, 20.7 mL, 20.7 mmol) was
added dropwise to a solution of 36 (1.17 g, 3.4 mmol) in toluene
(80 mL) at ꢂ40 °C. After 2 h stirring at ꢂ40 °C, the reaction mixture
was quenched with methanol (15 mL) and a 1N HCl aqueous solu-
tion (30 mL). Aqueous phase was extracted with AcOEt. Combined
organic phases were washed with water, dried over Na2SO4, fil-
tered through Whatman #4 paper and concentrated in vacuo to
give compound 37 as a white solid (1.2 g) which was used in the
next step without further purification.
Jones’ reagent (8N, 4.32 mL, 34.6 mmol) was added dropwise to
a solution of 37 (1.2 g, 3.5 mmol) in acetone (60 mL) at ꢂ15 °C. The
reaction mixture was stirred for 30 min. then excess of Jones’ re-
agent was hydrolyzed with MeOH (40 mL). After concentration in
C31H44N6O4 + H: 565.3497, found: 565.3488.
2.2.31. Oxidation of progesterone by chloranil
To a solution of progesterone (2.44 g, 7.8 mmol) in tert-butanol
(170 mL) was added chloranil (4.6 g, 18.7 mmol) at room temper-
ature. The reaction mixture was heated to reflux for one hour then
cooled down to room temperature and filtered. The precipitate was
washed with chloroform. Combined filtrates were concentrated in
vacuo and the residue was diluted in chloroform (200 mL). Organic
phase was successively washed with water (3 ꢁ 20 mL), 1.2 M

1184
W. Zeinyeh et al. / Steroids 77 (2012) 1177–1191
vacuo, the residue was partitioned between AcOEt and water.
Aqueous phase was extracted with AcOEt. The combined organic
extracts were successively washed with brine and a saturated
NaHCO3 aqueous solution. The combined aqueous extracts were
cooled down by an ice bath then they were acidified by a concen-
trated HCl solution to pH 3. After extraction with AcOEt, the com-
bined organic extracts were dried over MgSO4, filtered and
concentrated in vacuo to give compound 38 (830 mg, 67% overall
yield from 36) as a white solid. Mp = 224–226 °C; 1H NMR
(DMSO-d6) d: 12.00 (bs, 1H, COOH), 5.74 (s, 1H, H-4), 2.84 (bs,
1H), 2.74–2.31 (m, 5H), 2.28–1.13 (m, 13H), 2.12 (s, 3H, CH3-21),
1.20 (s, 3H, CH3-19), 0.66 (s, 3H, CH3-18) ppm; 13C NMR (DMSO-
d6) d: 208.53 (C-20), 197.74 (C-3), 174.46 (COOH), 169.35 (C-5),
124.39 (CH-4), 62.40 (CH-17), 51.93 (CH), 45.71 (CH), 43.57 I,
42.96 (CH), 37.92 I, 37.64 (CH2), 36.23 (CH), 35.17 (CH2), 34.79
(CH2), 33.67 (CH2), 31.18 (CH3-21), 23.65 (CH2), 22.21 (CH2),
20.74 (CH2), 17.33 (CH3-19), 12.76 (CH3-18) ppm; MS (ESI): m/z
(%) 381 (100) [M+Na]+, 359 (67) [M+H]+; HRMS (ESI): m/z: calcd
for C22H30NO4 + H: 359.2222; found: 359.2217.
(CH3-19), 13.01 (CH3-18) ppm; MS (ESI) m/z (%): 690 (100)
[M+H]+, 410 (36); HRMS (ESI) m/z: calcd for C37H52N7O6 + H:
690.3974; found: 690.3974.
2.2.34.2. Coupling reaction with adenine derivative (23). Flash chro-
matography on silica gel (CH2Cl2/MeOH, 93:7) gave amide 2b (Rf
0.35, 13 mg, 9% yield) as a white solid and succinamide 39b (Rf
0.3, 7 mg, 5% yield) as a white solid.
2.2.34.2.1.
7a-[4-(6-Aminopurin-9-yl)but-2-ynylcarbamoyl]pregn-4-
en-3,20-dione (2b). Mp = 215–216 °C; 1H NMR (CDCl3) d: 8.32 (s,
1H, H-20), 8.00 (s, 1H, H-80), 6.24 (t, 1H, J = 4.8 Hz, NH), 6.19 (s,
2H, NH2), 5.68 (s, 1H, H-4), 4.95 (bs, 2H, CH2-40), 4.02 (bs, 2H,
NHCH2), 2.79–1.05 (m, 19H), 2.08 (s, 3H, CH3-21), 1.17 (s, 3H,
CH3-19), 0.60 (s, 3H, CH3-18) ppm; 13C NMR (CDCl3) d: 209.38
(C-20), 199.01 (C-3), 172.74 (CONH), 168.86 (C-5), 155.56 (C-60),
153.03 (CH-20), 149.57 (C-40), 140.05 (CH-80), 125.55 (CH-4),
119.47 (C-50), 82.59 (CB), 75.65 (BC), 63.16 (CH-17), 52.06 (CH),
46.16 (CH), 44.53 (CH), 44.40 (C), 38.28 (C), 38.15 (CH2), 37.81
(CH), 36.22 (CH2), 35.50 (CH2), 34.03 (CH2), 33.49 (CH2), 31.65
(CH3-21), 29.19 (CH2), 24.65 (CH2), 22.88 (CH2), 21.39 (CH2),
17.96 (CH3-19), 13.19 (CH3-18) ppm; MS (ESI) m/z (%): 543
(100%) [M+H]+; HRMS (ESI): m/z: calcd for C31H38N6O3 + H:
543.3078; found: 543.3086.
2.2.34. General procedure for coupling reactions between acid 38 and
adenine derivatives 17, 23–26
DCC (58 mg, 0.28 mmol) and N-hydroxysuccinimide (32 mg,
0.28 mmol) were successively added to a solution of acid 38
(100 mg, 0.28 mmol) in anhydrous DMF (4 mL). The mixture was
stirred overnight at room temperature. Then, adenine derivative
2.2.34.2.2. N-(3,20-dioxopregn-4-en-7-carbonyloxy)-N0-[4-(6-amin-
opurin-9-yl)but-2-ynyl] succinamide (39b). Mp = 110–111 °C; 1H
NMR (CDCl3) d: 8.29 (s, 1H, H-20), 8.09 (s, 1H, H-80), 6.93 (bs, 1H,
CONH), 6.51 (s, 2H, NH2), 5.70 (s, 1H, H-4), 4.93 (bs, 2H, CH2N),
4.04 (bs, 2H, NHCH2), 2.97 (bs, 1H, H-7), 2.86–1.10 (m, 18H), 2.58
(m, 4H, CO-CH2CH2-CO), 2.07 (s, 3H, CH3-21), 1.18 (s, 3H, CH3-
19), 0.62 (s, 3H, CH3-18) ppm; 13C NMR (CDCl3) d: 209.51 (C-20),
199.38 (C-3), 172.20 (CO), 171.28 (C-5), 170.50 (CO), 155.55 (C-
60), 152.88 (CH-20), 149.45 (C-40), 140.44 (CH-80), 126.00 (CH-4),
119.31 (C-5’), 83.02 (CB), 75.46 (BC), 63.16 (CH-17), 51.91 (CH),
46.39 (CH), 44.27 (C), 41.64 (CH), 38.34 (C), 38.06 (CH2), 37.40
(CH), 35.54 (CH2), 35.16 (CH2), 34.07 (CH2), 33.73 (CH2), 31.61
(CH3-21), 29.83 (CH2), 29.64 (CH2), 24.17 (CH2), 22.94 (CH2),
21.22 (CH2), 17.77 (CH3-19), 13.15 (CH3-18) ppm; MS (ESI) m/z
(%): 680 (7) [M+Na]+, 658 (100) [M+H]+; HRMS (ESI): m/z: calcd
for C35H43N7O6 + H: 658.3348; found: 658.3348.
(1.2 eq.)andN,N-diisopropylethylamine(DIEA) (720 lL, 0.41 mmol)
wereadded. Stirringwascontinuedfor4 h. Afterconcentrationinva-
cuo, theresiduewas partitionedbetween CH2Cl2 and water. Aqueous
phase was thoroughly extracted with CH2Cl2. The combined organic
extracts were concentrated and the residue was purified by flash
chromatography on silica gel.
2.2.34.1. Coupling reaction with adenine derivative (17). Flash chro-
matography on silica gel (CH2Cl2/MeOH, 92:8) gave amide 2a (Rf
0.35, 58 mg, 37% yield) as a white solid and succinamide 39a (Rf
0.3, 47 mg, 31% yield) as a white solid.
2.2.34.1.1.
7a-[4-(6-Aminopurin-9-yl)hexylcarbamoyl]pregn-4-en-
3,20-dione (2a). Mp = 132–133 °C; 1H NMR (CDCl3) d: 8.32 (s,
1H, H-20), 7.81 (s, 1H, H-80), 5.89 (s, 2H, NH2), 5.72 (t, 1H,
J = 6.0 Hz, NH), 5.68 (s, 1H, H-4), 4.17 (t, 2H, J = 7.0 Hz, CH2-600),
3.16 (q, 2H, J = 6.0 Hz, NHCH2), 2.73–0.99 (m, 27H), 2.09 (s, 3H,
CH3-21), 1.18 (s, 3H, CH3-19), 0.62 (s, 3H, CH3-18) ppm; 13C NMR
(CDCl3) d: 209.81 (C-20), 199.42 (C-3), 172.97 (CONH), 170.02 (C-
5), 155.54 (C-60), 152.81 (CH-20), 149.77 (C-40), 140.48 (CH-80),
125.15 (CH-4), 119.20 (C-50), 63.18 (CH-17), 52.15 (CH), 46.02
(CH), 44.77 (CH), 44.42 (C), 43.88 (CH2), 38.80 (CH2), 38.23 (C),
38.14 (CH2), 37.72 (CH), 36.52 (CH2), 35.39 (CH2), 33.91 (CH2),
31.59 (CH3-21), 30.05 (CH2), 26.27 (CH2), 26.16 (CH2), 24.98
(CH2), 24.61 (CH2), 22.81 (CH2), 21.35 (CH2), 17.92 (CH3-19),
13.17 (CH3-18) ppm; MS (ESI) m/z (%): 575 (100) [M+H]+; HRMS
(ESI) m/z: calcd for C33H47N6O3 + H: 575.3704; found: 575.3710.
2.2.34.1.2. N-(3,20-dioxopregn-4-en-7-carbonyloxy)-N0-[4-(6-amin-
opurin-9-yl)hexyl] succinamide (39a). Mp = 148–149 °C; 1H NMR
(CDCl3) d: 8.32 (s, 1H, H-20), 7.86 (s, 1H, H-80), 6.13 (m, 3H, NH2,
NHCO), 5.70 (s, 1H, H-4), 4.19 (t, 2H, J = 6.9 Hz, CH2N), 3.19 (q,
2H, J = 6.4 Hz NHCH2), 2.95 (bs, 1H, H-7), 2.74–1.10 (m, 26H),
2.54 (m, 4H, COCH2CH2CO), 2.07 (s, 3H, CH3-21), 1.19 (s, 3H,
CH3-19), 0.63 (s, 3H, CH3-18) ppm; 13C NMR (CDCl3) d: 210.09
(C-20), 199.82 (C-3), 172.36 (CO), 170.83 (C-5), 168.12 (CO),
155.46 (C-60), 152.60 (CH-20), 149.62 (C-40), 140.58 (CH-80),
125.72 (CH-4), 119.05 (C-50), 63.15 (CH-17), 51.77 (CH), 46.25
(CH), 44.26 (C), 43.94 (CH2), 41.50 (CH), 39.28 (CH2), 38.28 (C),
37.99 (CH2), 37.26 (CH), 35.35 (CH2), 35.09 (CH2), 33.84 (CH2),
31.48 (CH3-21), 31.10 (CH2), 28.96 (CH2), 28.52 (CH2), 26.13
(CH2), 26.11 (CH2), 24.02 (CH2), 22.84 (CH2), 21.14 (CH2), 17.64
2.2.34.3. Coupling reaction with adenine derivative (24). Flash chro-
matography on silica gel (CH2Cl2/MeOH, 90:10) to give amide
(2c) (Rf 0.35, 33 mg, 22% yield) as a white solid and succinamide
(39c) (Rf 0.3, 31 mg, 21% yield) as a white solid.
2.2.34.3.1. (E)-7a-[4-(6-Aminopurin-9-yl)but-2-enylcarbamoyl]pregn
-4-en-3,20-dione (2c). Mp = 130–131 °C; 1H NMR (CDCl3) d: 8.32
(s, 1H, H-20), 7.81 (s, 1H, H-80), 5.84 (t, 1H, J = 5.6 Hz, NH), 6.01 (s,
2H, NH2), 5.80 (dt, 1H, J = 15.3 Hz, J = 5.7 Hz, CH=), 5.62 (s, 1H, H-
4), 5.58 (dt, 1H, J = 15.3 Hz, J = 5.5 Hz, HC@), 4.78 (d, 2H,
J = 5.7 Hz, CH2-400), 3.92–3.75 (m, 2H, NHCH2), 2.67 (m, 1H, H-7),
2.64–1.10 (m, 19H), 2.08 (s, 3H, CH3-21), 1.17 (s, 3H, CH3-19),
0.61 (s, 3H, CH3-18) ppm; 13C NMR (CDCl3) d: 209.30 (C-20),
198.90 (C-3), 172.79 (CONH), 168.91 (C-5), 155.55 (C-60), 152.96
(CH-20), 149.95 (C-40), 140.50 (CH-80), 130.86 (HC@), 125.93
(@CH), 125.51 (CH-4), 119.63 (C-50), 63.14 (CH-17), 52.12 (CH),
46.05 (CH), 44.87 (CH), 44.83 (CH2), 44.38 (C), 40.37 (CH2), 38.26
(C), 38.15 (CH2), 37.80 (CH), 36.47 (CH2), 35.49 (CH2), 34.03
(CH2), 31.65 (CH3-21), 24.74 (CH2), 22.81 (CH2), 21.38 (CH2),
17.99 (CH3-19), 13.22 (CH3-18) ppm; MS (ESI) m/z (%): 567 (14)
[M+Na]+, 545 (100) [M+H]+; HRMS (ESI) m/z: calcd for
C
31H40N6O3 + H: 545.3235; found: 545.3239.
2.2.34.3.2.
N-(3,20-dioxopregn-4-en-7-carbonyloxy)-N0-[(E)-4-(6-
aminopurin-9-yl)but-2-enyl] succinamide (39c). Mp = 125–126 °C;
1H NMR (CDCl3) d: 11.19 (bs, 1H, NHOCO), 8.28 (s, 1H, H-20), 7.87
(s, 1H, H-80), 6.77 (bs, 1H, CONH), 6.47 (s, 2H, NH2), 5.82 (dt, 1H,
J = 15.3 Hz, J = 5.8 Hz, HC@), 5.68 (s, 1H, H-4), 5.62 (dt, 1H,

W. Zeinyeh et al. / Steroids 77 (2012) 1177–1191
1185
J = 15.3 Hz, J = 5.1 Hz, @CH), 4.75 (d, 2H, J = 5.1 Hz, CH2-400), 3.83
(bs, 2H, NHCH2), 2.94 (bs, 1H, H-7), 2.78–1.00 (m, 18H), 2.55 (m,
4H, COCH2CH2CO), 2.06 (s, 3H, CH3-21), 1.17 (s, 3H, CH3-19), 0.61
(s, 3H, CH3-18) ppm; 13C NMR (CDCl3) d: 209.64 (C-20), 199.44
(C-3), 172.38 (C), 171.35 (C), 170.58 (C), 167.65 (C), 155.68 (C-60),
152.89 (CH-20), 149.86 (C-40), 140.89 (CH-80), 131.35 (CH=),
126.05 (CH=), 125.55 (CH-4), 119.45 (C-50), 63.25 (CH-17), 51.97
(CH), 46.47 (CH), 45.06 (CH2), 44.35 (C), 41.70 (CH), 40.92 (CH2),
38.42 (C), 38.15 (CH2), 37.47 (CH), 35.63 (CH2), 35.24 (CH2),
34.15 (CH2), 31.70 (CH3-21), 24.23 (CH2), 23.01 (CH2), 21.30
(CH2), 17.84 (CH3-19), 13.24 (CH3-18) ppm; MS (ESI) m/z (%): 660
(100) [M+H]+; HRMS (ESI) m/z: calcd for C35H45N7O6 + H:
660.3504; found: 660.3513.
(C), 38.20 (CH2), 37.80 (CH), 36.54 (CH2), 35.53 (CH2), 34.04
(CH2), 31.67 (CH3-21), 27.63 (CH2), 26.60 (CH2), 24.75 (CH2),
22.85 (CH2), 21.41 (CH2), 17.99 (CH3-19), 13.25 (CH3-18) ppm;
MS (ESI) m/z (%): 569 (5) [M+Na]+, 547 (100) [M+H]+; HRMS (ESI)
m/z: calcd for C31H42N6O3 + H: 547.3391; found: 547.3392.
2.2.34.5.2. N-(3,20-dioxopregn-4-en-7-carbonyloxy)-N0-[4-(6-amin-
opurin-9-yl)butyl] succinamide (39e). Mp = 144–145 °C; 1H NMR
(CDCl3) d: 8.31 (s, 1H, H-20), 7.92 (s, 1H, H-80), 6.60 (t, 1H,
J = 5.5 Hz, CONH), 6.40 (s, 2H, NH2), 5.69 (s, 1H, H-4), 4.21 (t, 2H,
J = 7.0 Hz, CH2N), 3.28 (m, 2H, NHCH2), 2.97 (bs, 1H, H-7), 2.73–
1.10 (m, 22H), 2.56 (m, 4H, COCH2CH2CO), 2.07 (s, 3H, CH3-21),
1.18 (s, 3H, CH3-19), 0.63 (s, 3H, CH3-18) ppm; 13C NMR (CDCl3)
d: 209.44 (C-20), 199.20 (C-3), 172.46 (CO), 171.25 (C-5), 167.46
(CO), 155.52 (C-60), 152.45 (CH-20), 149.94 (C-40), 141.17 (CH-80),
125.99 (CH-4), 119.56 (C-50), 63.16 (CH-17), 51.87 (CH), 46.32
(CH), 44.26 (C), 43.48 (CH2), 41.60 (CH), 38.82 (CH2), 38.33 (C),
38.05 (CH2), 37.42 (CH), 35.56 (CH2), 35.14 (CH2), 34.08 (CH2),
31.61 (CH3-21), 27.42 (CH2), 26.22 (CH2), 24.16 (CH2), 22.95
(CH2), 21.20 (CH2), 17.76 (CH3-19), 13.15 (CH3-18) ppm; MS (ESI)
m/z (%): 662 (100) [M+H]+, 382 (15); HRMS (ESI) m/z: calcd for
2.2.34.4. Coupling reaction with adenine derivative (25). Flash chro-
matography on silica gel (CH2Cl2/MeOH, 90:10) gave amide (2d)
(Rf 0.35, 41 mg, 28% yield) as a white solid and succinamide
(39d) (Rf 0.3, 40 mg, 26% yield) as a white solid.
2.2.34.4.1. (Z)-7a-[4-(6-Aminopurin-9-yl)but-2-enylcarbamoyl]pregn
-4-en-3,20-dione (2d). Mp = 103–104 °C; 1H NMR (CDCl3) d: 8.29
(s, 1H, H-20), 7.87 (s, 1H, H-80), 7.41 (t, 1H, J = 5.2 Hz, NH), 5.94 (s,
2H, NH2), 5.79 (dd, 1H, J = 10.4 Hz, J = 7.8 Hz, CH=), 5.66 (m, 1H,
@H), 5.67 (s, 1H, H-4), 4.89 (d, 2H, J = 7.3 Hz, CH2-400), 4.15–3.95
(m, 2H, NHCH2), 2.68 (m, 1H), 2.61–1.13 (m, 18H), 2.09 (s, 3H,
CH3-21), 1.19 (s, 3H, CH3-19), 0.63 (s, 3H, CH3-18) ppm; 13C NMR
(CDCl3) d: 209.33 (C-20), 198.95 (C-3), 172.92 (CONH), 169.09 (C-
5), 155.65 (C-60), 152.39 (CH-20), 149.76 (C-40), 140.86 (CH-80),
130.31 (HC@), 126.63 (@H), 125.42 (CH-4), 120.11 (C-50), 63.21
(CH-17), 52.20 (CH), 46.11 (CH), 44.90 (CH), 44.46 (C), 41.07
(CH2), 38.23 (CH2), 37.85 (CH), 36.48 (CH2), 35.51 (CH2), 35.03
(CH2), 34.06 (CH2), 31.67 (CH3-21), 24.60 (CH2), 22.89 (CH2),
21.46 (CH2), 18.02 (CH3-19), 13.25 (CH3-18) ppm; MS (ESI) m/z
(%): 567 (12) [M+Na]+, 545 (100) [M+H]+; HRMS (ESI) m/z: calcd
for C31H40N6O3 + H: 545.3235; found: 545.3225.
C35H47N7O6 + H: 662.3661; found: 662.3675.
2.2.35. Progesterone-7
To a stirred solution of acid 38 (54 mg, 150
(2 mL) at 0 °C, MeOH (20 L, 490 mol), DMAP (2 mg, 16
and N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydrochlo-
ride (EDCI, 59 mg, 310 mol) were added. After stirring overnight
a
-carboxylic acid methyl ester (40)
mol) in CHCl3
mol)
l
l
l
l
l
at room temperature, water was added and the mixture was ex-
tracted by CH2Cl2. The combined organic extracts were washed
with water and brine, dried over Na2SO4, filtered and evaporated
to dryness. The residue was purified by preparative TLC (cyclohex-
ane/AcOEt, 60:40) to give the ester 40 (Rf 0.3, 15 mg, 27% yield) as
a white solid. 1H NMR (CDCl3) d: 5.64 (s, 1H, H-4), 3.57 (s, 3H,
COOCH3), 2.77 (bs, 1H), 2.61–2.32 (m, 5H), 2.21–1.78 (m, 13H),
2.13 (s, 3H, CH3-21), 1.14 (s, 3H, CH3-19), 0.59 (s, 3H, CH3-18)
ppm; MS (ESI): m/z (%) 767 (32) [2M+Na]+, 411 (68) [M+K]+, 395
(100) [M+Na]+, 373 (21) [M+H]+.
2.2.34.4.2.
N-(3,20-dioxopregn-4-en-7-carbonyloxy)-N0-[(Z)-4-(6-
aminopurin-9-yl)but-2-enyl] succinamide (39d). Mp = 92–93 °C;
1H NMR (CDCl3) d: 11.00 (bs, 1H, NHOCO), 8.32 (s, 1H, H-20), 7.96
(s, 1H, H-80), 7.59 (bs, 1H, CONH), 6.34 (s, 2H, NH2), 5.82–5.60
(m, 2H, HC = CH), 5.71 (s, 1H, H-4), 4.90 (d, 2H, J = 7.3 Hz, CH2-
400), 4.05 (t, 2H, J = 6.3 Hz, NHCH2), 2.98 (bs, 1H, H-7), 2.70–1.10
(m, 18H), 2.62 (m, 4H, COCH2CH2CO), 2.06 (s, 3H, CH3-21), 1.18
(s, 3H, CH3-19), 0.62 (s, 3H, CH3-18) ppm; 13C NMR (CDCl3) d:
209.62 (C-20), 199.35 (C-3), 172.36 (C), 171.33 (C), 170.77 (C),
167.58 (C), 155.69 (C-6), 152.59 (CH-20), 149.80 (C-40), 141.18
(CH-80), 130.49 (HC@), 126.60 (@H), 126.10 (CH-4), 119.79 (C-50),
63.26 (CH-17), 51.92 (CH), 48.58 (CH), 46.35 (CH), 44.36 (C),
41.72 (CH), 41.02 (CH2), 38.43 (C), 38.12 (CH2), 37.55 (CH), 36.22
(CH2), 35.16 (CH2), 35.24 (CH2), 34.17 (CH2), 31.71 (CH3-21),
24.29 (CH2), 23.07 (CH2), 21.29 (CH2), 17.85 (CH3-19), 13.25
(CH3-18) ppm; MS (ESI) m/z (%): 660 (100) [M+H]+; HRMS (ESI)
m/z: calcd for C35H45N7O6 + H: 660.3504; found: 660.3510.
2.3. Biological evaluation
The inhibition of Pgp activity by the studied compounds was
evaluated by their ability to prevent the efflux of daunorubicin in
the K562/R7 drug-selected human cell line [30]. One million
K562/R7 human leukemic cells expressing high levels of P-glyco-
protein were incubated for 1 h at 37 °C in 1 mL of RPMI 1640 med-
ium containing a final concentration of 10
lM daunorubicin, in the
presence or absence of inhibitor (at 10 or 50
l
M). After incubation,
the cells were then washed twice with ice-cold phosphate buffered
saline (PBS) and kept on ice until analysis by flow cytometry on a
FACS-Calibur (Becton–Dickinson Corp., Mountain View, CA). Pro-
gesterone was used as a positive control (mean inhibitory activity
of 100%). Assays were performed in duplicate, with at least two
separate experiments.
2.2.34.5. Coupling reaction with adenine derivative (26). Flash chro-
matography on silica gel (CH2Cl2/MeOH, 93:7) gave amide (2e)
(Rf 0.35, 48 mg, 32% yield) as a white solid and succinamide
(39e) (Rf 0.3, 35 mg, 23% yield) as a white solid.
3. Results and discussion
2.2.34.5.1.
7a-[4-(6-Aminopurin-9-yl)butylcarbamoyl]pregn-4-en-
3,20-dione (2e). Mp = 123–124 °C; 1H NMR (CDCl3) d: 8.32 (s, 1H,
H-20), 7.82 (s, 1H, H-80), 6.03 (t, 1H, J = 5.8 Hz, NH), 5.95 (s, 2H,
NH2), 5.66 (d, 1H, J = 1.2 Hz, H-4), 4.25 (t, 2H, J = 7.2 Hz, CH2N),
3.27 (m, 2H, NHCH2), 2.66 (m, 1H), 2.54–1.14 (m, 22H), 2.09 (s,
3H, CH3-21), 1.18 (s, 3H, CH3-19), 0.62 (s, 3H, CH3-18) ppm; 13C
NMR (CDCl3) d: 209.30 (C-20), 198.88 (C-3), 173.13 (CONH),
169.06 (C-5), 155.47 (C-60), 152.61 (CH-20), 150.13 (C-40), 140.83
(CH-80), 125.46 (CH-4), 119.79 (C-50), 63.16 (CH-17), 52.20 (CH),
46.10 (CH), 44.95 (CH), 44.40 (C), 43.40 (CH2), 38.32 (CH2), 38.26
3.1. Chemistry
As their syntheses have not been previously described in detail
[19], we report here the preparation of adenine-spacer-NH2 deriv-
atives 17, 19, 21 and 23–26, obtained from the building blocks 4, 7,
10 and 15, following the reaction pathways described in Schemes 1
and 2.
Of note, whatever the N-alkylating conditions of adenine we
used, this reaction produced the N-9 alkylated isomer. As shown

1186
W. Zeinyeh et al. / Steroids 77 (2012) 1177–1191
a
a
OH
Cbz
Cbz
OH
H2N
N
H
3
4
b
H2N
H2N
N
H
OH
OH
7
6
5
COOH
Cbz
a
b
H2N
H2N
N
H
OH
OH
COOH
9
10
8
c
d
c
R NH
13, R=H
14, R=Boc
OH
BocHN
OMs
HO
OH
MsO
OH
15
12
11
e
Scheme 1. Reagents and conditions: (a) Na2CO3, CbzCl, H2O, 0 °C to RT, overnight (79% for 4; 70% for 7; 90% for 10); (b) LiAlH4, THF, 0 °C to reflux, 3 h (70% for 6; 28% for 9); c)
MsCl, Et3N, THF, 0 °C to RT, overnight (45% for 12; 76% for 15); (d) NH4OH, RT, 1 h then Dowex (76%); (e) Boc2O, Et3N, THF, RT, overnight (73%).
NH2
NH2
N
N
N
N
N
N
a
N
N
b
N
16
N
17
(CH2)6NHCbz
(CH2)6NH2
NH2
NH2
N
N
N
a
b
N
N
18
N
NH2
N
19
NHCbz
NH2
N
N
NH2
NH2
N
H
N
b
N
N
a
c
N
N
N
N
adenine
N
N
20
N
21
NHCbz
NH2
NH2
NH2
N
N
N
N
d
N
N
22
23
NH2
NHBoc
g
e
f
NH2
NH2
NH2
N
N
N
N
N
N
N
N
N
N
N
N
(CH2)4NH2
26
24
25
NH2
NH2
Scheme 2. Reagents and conditions: (a) PPh3, DIAD, THF, 4 or 7 or 10 (41% for 16, 49% for 18, 18% for 20); (b) H2, Pd/C, MeOH or MeOH/AcOEt (100% for 17, 71% for 19, 77% for
21); (c) K2CO3, DMF, 15 (75%); (d) (i) aq. HCl, (ii) Dowex 550A (hydroxide form) (80%); (e) LiAlH4, THF (29%); (f) H2, Lindlar catalyst, quinoline, MeOH (67%); (g) H2, Pd/C,
MeOH (90%).
in Fig. 4, regioselectivity was confirmed by NMR analysis including
{1H; 13C} HMBC correlations recorded for compounds 16 and 24,
respectively obtained by Mistsunobu coupling reaction or nucleo-
philic substitution. In each case, long-distance coupling constants
3J were measured on one hand, between the adenine amino proton
(7.17 or 7.21 ppm) and the quaternary carbon C-5 (118.73 or
118.65 ppm) and on the other hand, between N-methylene proton
H-10 (4.11 or 4.73 ppm) and the other quaternary carbon C-4

W. Zeinyeh et al. / Steroids 77 (2012) 1177–1191
1187
The methodology previously described to synthesize com-
pounds 1a–g [19] was applied to prepare PEG hybrids 1h,i. The
activated N-hydroxysuccinimidyl ester 34 [30] was allowed to re-
act with the adenine derivatives 32a,b in the presence of DIEA in
DMF to afford the corresponding C20-progesterone-PEG-adenine
hybrids 1h–i in low to moderate yields.
H
N
H
H
H
N
N
N
N
5 N
5 N
N
8 H
8 H
4 N1'
H
4 N1'
H
At the same time, we have synthesized a new series of hybrids
whose linker arms were attached at the C7-position of the steroid
nucleus. 7-Substituted progesterone derivatives are generally
16
24
NH2
D
6-progesterone 35a
NHCbz
prepared by 1,6-addition of a nucleophile to
[34]. The latter was obtained by oxidation of progesterone by
tetrachloro-1,4-benzoquinone (chloranil) in refluxing tert-butanol
according to a literature procedure [35] (Scheme 5). This reaction
was monitored by UV spectroscopy after dilution of a reaction ali-
quot in absolute EtOH and carried out until the complete shift of
the absorption maximum from 240 nm (characteristic of proges-
terone) to 280 nm (characteristic of 3-oxo-4,6-diene steroids). In
our case, it is noteworthy that this oxidation step also led to traces
of the 17a-epimer 35b which made the reaction mixture more dif-
ficult to purify. The structure of 35b was unambiguously estab-
lished by comparison of its proton NMR spectrum in CDCl3 to
that of 35a. Indeed, CH3-18 and H17 proton signals of 17a-isomer
(d[CH3-18] = 0.96 ppm, d[H17] = 2.82 ppm) were significantly
deshielded compared with the corresponding protons in 17b-iso-
mer (d[CH3-18] = 0.70 ppm, d[H17] = 2.55 ppm) [31].
With the aim of using an amide link as a connection function
between the steroid moiety and the spacer, a carboxylic acid func-
tion had to be introduced in position 7 of progesterone. As de-
scribed in Scheme 6, the first step was the stereoselective
Fig. 4. Establishment of the regioselectivity of the N-alkylation of adenine by HMBC
NMR sequence. 1H NMR spectra were recorded in DMSO-d6.
(149.54 or 149.32 ppm). Furthermore, the structure of alkylated
isomers of adenine was also assigned by comparison with UV liter-
ature data [32]. The UV spectrum of N-9 substituted isomers in
CHCl3 showed one absorption maximum at 260 nm while a maxi-
mum at 270 nm is characteristic of N-7 substituted isomers.
Secondly, poly(ethyleneglycol)-modified adenine derivatives
32a,b were prepared as described in Scheme 3. As preliminary
studies have shown that the formation of the terminal amino
group after various reduction conditions of the corresponding ade-
nine-PEG azides (H2/Pd-C, PPh3, NaI/CeCl3) [33] have failed to pro-
duce pure adenine–PEG–NH2 derivative 32a, we turned our
attention to another method. This strategy involved an alkylation
step of potassium phtalimide with commercially available chloro-
alcohols 27a,b, followed by conversion of the alcohols 28a,b into
alkyl iodides 29a,b using Corey’s method (combination of PPh3
and iodine, in the presence of imidazole). (See Scheme 4).
In a first attempt, the building blocks 29a,b were coupled with
adenine under nucleophilic substitution conditions affording the
corresponding N-9-alkylated purine in low yields. This reaction
was then applied to N-6 diBoc adenine 30 to get diprotected com-
pounds 31a,b in more satisfactory yields. Subsequent N-Boc depro-
tections were carried out in acidic conditions and the primary
amino group was then recovered after hydrazinolysis of these
phtalimide functionalized PEGs to get the expected products
32a,b in good overall yields.
introduction of a cyano group at the 7a-position of the steroid
framework in a high 88% yield, by 1,6-nucleophilic addition to
the dienone 35a in presence of an excess of diethyl aluminum cya-
nide [36]. Under these very mild conditions, no epimerization was
detected. Stereochemistry was confirmed by proton NMR: the
width of the H7-proton NMR peak (d = 3.01 ppm) at half height
was 8.30 Hz, which matched with an equatorial proton. Further-
more, it was shown in literature that conformationally rigid sub-
strates favored axial attack of the cyanide anion [37].
Treatment of nitrile 36 with diisobutyl aluminum hydride in
anhydrous toluene at ꢂ40 °C induced the simultaneous reductions
O
O
O
a
b
O
O
O
+
N
I
N
OH
NK
Cl
N
OH
n
n
n
O
O
O
29a n = 1
29b n = 2
27a n = 1
27b n = 2
28a n = 1
28b n = 2
Boc
N
Boc
N
NH2
N
N
c
Boc
N
Boc
NH2
N
N
N
N
H
N
H
N
N
N
+
N
N
N
30
d
e
N
O
O
O
O
O
N
I
31a n = 1
31b n = 2
32a n = 1
NH2
n
N
O
n
n
32b n = 2
O
29a n = 1
29b n = 2
Scheme 3. Reagents and conditions: (a) DMF, 130 °C, 2 h (85% for 28a; 97% for 28b); (b) PPh3, I2, imidazole, Et2O, CH3CN, 0 °C for 2 h then RT for 1 h (72% for 29a; 58% for
29b); (c) (i) Boc2O, DMAP, THF, RT, 5 h (ii) NaHCO3, MeOH, 50 °C, 1h30 (73% over the two steps) (d) NaH 60%, DMF, 70 °C, 29a (5 h) or 29b (20 h) (61% for 31a; 46% for 31b); (e)
(i) TFA, CH2Cl2, RT, 16 h (ii) MeNHNH2, abs. EtOH, 70 °C, (17 h, 73% for 32a; 24 h, 97% for 32b).

1188
W. Zeinyeh et al. / Steroids 77 (2012) 1177–1191
O
O
O
O
20 CH3
OH
O
O
N
H
a
H
H
b
H
H
H
H
H
H
O
O
O
34
33
progesterone
NH2
N
N
N
c
N
H2N Y
NH2
N
17, 19, 21, 23-26, 32a-b
N
N
N
O
NH
Y
H
1a-i
H
=
H
O
=
CH2
Y
Y
=
Y
6
1b (60%)
1c (61%)
1a (79%)
CH2
=
=
Y
Y
=
Y
Y
=
4
1g (77%)
1f (40%)
1d (92%)
1e (55%)
1h n = 1 (61%)
1i n = 2 (27%)
O
Y
=
n
Scheme 4. Reagents and conditions: (a) (i) NaOH, Br2, t-BuOH, 0 °C, 3 h (ii) Na2SO3, RT, overnight (57%); (b) N,N0-dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide
(HOSu), THF, RT, overnight (55%) and (c) DIEA, DMF, overnight, RT (1a–b, 1d–g) or 35 °C (1c, 1h,i).
O
O
O
CH3
CH3
CH3
chloranil
t-BuOH, reflux, 1h
H
H
H
+
H
H
H
H
H
H
O
O
O
progesterone
35a (17β, 50%)
35b (17α, 4%)
Scheme 5. Synthesis of
D
6-progesterone.
of cyano group and both C3- and C20-ketones to afford the dihy-
droxy aldehyde 37 as a mixture of diastereoisomers. This mixture
was easily oxidized without further purification, by an excess of
Jones reagent (CrO3, H2SO4) at low temperature to obtain carbox-
ylic acid function at the position 7 and regenerate C3- and C20-ke-
tones. Compound 38 was isolated as the sole isomer in good overall
yield (29% from progesterone).
to 1:1. The formation of compounds 39a–e might arise from nucle-
ophilic attack of the amine at one of the carbonyl groups of the suc-
cinimide intermediate, thus leading to its ring-opening [38]. This
side reaction might be favored by a sterically more hindered N-
hydroxysuccinimidyl ester at C7 position of progesterone than at
C20 position. Amides 2a–e and succinamides 39a–e were obtained
in low to moderate total yields.
Finally, coupling reactions in C7 position were carried out in a
slightly different manner as previously described for C20 hybrids.
Because of the degradation of the activated N-hydroxysuccinimidyl
ester during its purification, coupling reactions with five adenine-
spacer-NH2 derivatives (17 and 23–26) were performed in a one-
pot sequential DCC/HOSu activation-amidation process. Somewhat
surprisingly, the reaction of acid 38 with each of the amines led not
only to amides 2a–e but also to succinamides 39a–e in a ratio close
The esterification of acid 38 with methanol was also carried out
(Scheme 7) in the presence of a catalytic amount of 4-(dimethyl-
amino)pyridine (DMAP) and the water soluble coupling reagent,
N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride
(EDCI).
This simpler 7a-carboxymethyl derivative 40 was used as a sec-
ondary control: its inhibitory activity was compared to those ob-
tained for C7-hybrids 2 and 39.

W. Zeinyeh et al. / Steroids 77 (2012) 1177–1191
1189
HO
O
O
CH3
CH3
CH3
a
b
H
H
H
H
H
H
H
CN
H
H
CHO
O
7
O
HO
35a
36
37
(mixture of diastereoisomers)
O
c
CH3
O
NH2
N
CH3
H
O
N
N
H
H
H
N
1) HOSu, DCC, DMF, RT, 15h
NH2
H
N
O
Y
H
H
2) DIEA,
, RT, 4h
O
+
2a-e
N
O
COOH
N
38
N
N
CH3
H2N Y
17, 23-26
NH2
N
H
N
N
H
H
H
O
O
O
N
N
O
NH
Y
O
39a-e
=
CH2
CH2
=
=
Y
Y
=
Y
=
Y
Y
4
6
2a / 39a
(37% / 31%)
2b / 39b
(9% / 5%)
2c / 39c
2d / 39d
(9% / 5%)
2e / 39e
(32% / 23%)
(22% / 21%)
Scheme 6. Reagents and conditions: (a) Et2AlCN (1 M in toluene), THF-toluene, RT, 2 h (88%); (b) DIBAL-H (1 M in toluene), toluene, ꢂ40 °C, 2 h. (c) CrO3, H2SO4, H2O, acetone,
ꢂ15 °C, 30 min (67% overall yield for 2 steps).
O
O
CH3
CH3
DMAP, EDCI, CH3OH
H
H
CH2Cl2, 0°C to RT, overnight
H
H
H
H
O
COOCH3
O
COOH
38
40 (27%)
Scheme 7. Esterification of carboxylic acid 38.
3.2. Biological evaluation
activity similar to that of progesterone. At a concentration of
50 M, the result for ester 40 confirmed that the substitution
at the position 7 of the progesterone did not prevent the bind-
ing of the steroid nucleus to P-glycoprotein, as already described
in literature [9,20]. On the other hand, at 50 M, all the bivalent
compounds are less efficient than progesterone, except hybrid 1a
(entry 1) which significantly enhanced the daunorubicin accu-
mulation by 30 % after one-hour incubation at 37 °C.
l
The efficiency of our hybrids progesterone-spacer-adenine 1, 2
and 39 to inhibit Pgp-mediated cytotoxic drug efflux was
evaluated by measuring the intracellular accumulation of dauno-
rubicin by flow cytometry in K562/R7 human leukemic cells
overexpressing Pgp (Table 1). Progesterone was used as positive
control.
a
l
All new synthesized compounds (1h,i, 2a–e, 39a–e and 40) and
the hybrid 1a with a hexamethylene linker chain, which has previ-
ously shown the best inhibitory potency [19], were evaluated at
As expected, both attachment point and nature of the linker
play a fairly crucial role in the ability of these bivalent com-
pounds to inhibit Pgp transport activity. Indeed, the connection
10
lM and at 50
lM as well, the latter concentration for which
of the hexamethylene linker at the position 7a of the progester-
progesterone was known to reverse significantly the drug resis-
tance in Pgp-overexpressing cells [17].
At a concentration of 10 lM, C20-PEG-hybrids (1h,i, entries 2
and 3), C7-hybrids (2a–e, 39a–e, entries 4–8 and 9–13) and ester
one was not favorable (entry 4). This could be explained by an
incorrect orientation of the steroid in its binding pocket. More-
over, tether flexibility induced by the use of a PEG linker (1i)
also led to less potent Pgp modulators compared to more rigid
hexamethylene one (entries 1 and 3).
40, used as a secondary control (entry 14), showed an inhibitory

1190
W. Zeinyeh et al. / Steroids 77 (2012) 1177–1191
Table 1
Capacity of the synthesized derivatives to improve daunorubicin accumulation in K562/R7 human leukemic resistant cells [39].
a,c
b,d
Entry
Compound
Daunorubicin accumulation (% progesterone)
Daunorubicin accumulation (% progesterone)
1
2
3
4
5
6
7
8
1a
1h
1i
2a
2b
2c
2d
2e
39a
39b
39c
39d
39e
40
110.6 ( 13.4)
94.0 ( 6.9)
90.3 ( 1.7)
102.1 ( 5.7)
101.1 ( 6.3)
90.1 ( 3.6)
98.2 ( 2.7)
96.0 ( 5.6)
98.2 ( 3.7)
97.6 ( 7.8)
98.8 ( 4.2)
95.4 ( 6.4)
91.2 ( 4.8)
101.4 ( 6.3)
133.2 ( 18.1)
62.0 ( 8.2)
65.9 ( 9.4)
52.7 ( 5.0)
54.4 ( 5.4)
52.2 ( 3.3)
51.8 ( 1.2)
50.6 ( 5.5)
54.8 ( 5.3)
55.7 ( 7.4)
55.5 ( 5.9)
51.6 ( 6.8)
54.1 ( 7.1)
81.9 ( 10.3)
9
10
11
12
13
14
a
b
c
Compounds were tested at a 10
Compounds were tested at a 50
l
l
M concentration.
M concentration.
Accumulation of daunorubicin (10
M) was expressed as percent of the accumulation in the presence of progesterone. Standard deviation is given in brackets.
Accumulation of daunorubicin (10 M) in the presence of progesterone (50 M) was considered as 100%. Daunorubicin accumulation in the presence of the tested
compounds (50 M) was expressed as percent of the accumulation in the presence of progesterone. Standard deviation is given in brackets.
lM) in the presence of progesterone (10 lM) was considered as 100%. Daunorubicin accumulation in the presence of the tested
compounds (10
l
d
l
l
l
[5] Tsuruo T, Iida H, Tsukagoshi S, Sakurai J. Overcoming of vincristine resistance
4. Conclusion
in P388 leukemia in vivo and in vitro through enhanced cytotoxicity of
vincristine and vinblastine by verapamil. Cancer Res 1981;41:1967–72.
[6] (a) Sugawara I, Nakahama M, Hamada H, Tsuruo T, Mori S. Apparent stronger
expression in the human adrenal cortex than in the human adrenal medulla of
Mr 170,000–180,000 P-glycoprotein. Cancer Res 1988;48:4611–4;
(b) Naito M, Yusa K, Tsuruo T. Steroid hormones inhibit binding of Vinca
alkaloid to multidrug resistance related P-glycoprotein. Biochem Biophys Res
Commun 1989;158:1066–71.
We have described herein the synthesis and the biological eval-
uation of 13 new progesterone–adenine hybrids. An efficient four-
step procedure was developed from progesterone to prepare C7
derivatives in a stereocontrolled manner.
a
The efficiency of progesterone–adenine hybrids might greatly
depend on two main factors: (i) the nature of the linker and (ii)
its attachment point on the steroid skeleton. Further studies are
necessary to determine if the use of longer poly-methylene or -eth-
yleneglycol chains as spacers connected at C20 or C7 position of
progesterone improve the inhibitory efficiency of these bivalent
compounds. Some analogs like flavonoid derivatives could mimic
the adenine moiety of ATP and bind to Pgp with high affinity at
NBD site. The use of such compounds instead of adenine should
therefore also be considered [40].
[7] (a) Barnes KM, Dickstein B, Cutler GB, Fojo T, Bates SE. Steroid transport,
accumulation, and antagonism of P-glycoprotein in multidrug-resistant cells.
Biochemistry 1996;35:4820–7;
(b) Li Y, Wang Y-H, Yang L, Zhang S-W, Liu C-H, Yang S-L. Comparison of
steroid substrates and inhibitors of P-glycoprotein by 3D-QSAR analysis. J Mol
Struct 2005;733:111–8.
[8] (a) Yang CP, Cohen D, Greenberger LM, Hsu SI, Horwitz SB. Differential
transport properties of two mdr gene products are distinguished by
progesterone. J Biol Chem 1990;265:10282–8;
(b) Perez-Victoria FJ, Conseil G, Munoz-Martinez F, Perez-Victoria JM, Dayan G,
Marsaud V, Castanys S, Gamarro F, Renoir JM, Di Pietro A. RU49953: a non-
hormonal steroid derivative that potently inhibits P-glycoprotein and reverts
cellular multidrug resistance. Cell Mol Life Sci 2003;60:526–35.
[9] Leonessa F, Kim J-YK, Ghiorghis A, Kulawiec RJ, Hammer C, Talebian A, Clarke
R. C-7 analogues of progesterone as potent inhibitors of the P-glycoprotein
efflux pump. J Med Chem 2002;45:390–8.
Acknowledgements
[10] Li J, Xu L-Z, He K-L, Guo W-J, Zheng Y-H, Xia P, Chen Y. Reversal effects of
nomegestrol acetate on multidrug resistance in adriamycin-resistant MCF7
breast cancer cell line. Breast Cancer Res 2001;3:253–63.
[11] Aller SG, Yu J, Ward A, Weng Y, Chittaboina S, Zhuo R, Harrell PM, Trinh YT,
Zhang Q, Urbatsch IL, Chang G. Structure of P-glycoprotein reveals a molecular
basis for poly-specific drug binding. Science 2009;323:1718–22.
[12] Shapiro AB, Fox K, Lam P, Ling V. Stimulation of P-glycoprotein mediated drug
transport by prazosin and progesterone. Evidence for a third drug-binding site.
Eur J Biochem 1999;259:841–50.
[13] (a) Dayan G, Jault JM, Baubichon-Cortay H, Baggetto LG, Renoir JM, Baulieu EE
et al. Binding of steroid modulators to recombinant cytosolic domain from
mouse P-glycoprotein in close proximity to the ATP site. Biochemistry
1997;36:15208–15; (b) Mares-Samano S, Badhan R, Penny J. Identification of
putative steroid-binding sites in human ABCB1 and ABCG2. Eur J Med Chem
2009;44:3601–11.
[14] (a) Halazy S, Perez M, Fourrier C, Pallard I, Pauwells PJ, Palmier C, et al.
Serotonin dimers: application of the bivalent ligand approach to the design of
new potent and selective 5-HT1B/1D agonists. J Med Chem 1996;39:4920–7;
(b) Mammen M, Choi S-K, Whitesides GM. Polyvalent interactions in biological
systems: implications for design and use of multivalent ligands and inhibitors.
Angew Chem Int Ed 1998;37:2754–94;
ZM thanks the Ministère de l’Enseignement Supérieur et de la
Recherche for a PhD fellowship.
References
[1] (a) Fojo T, Bates S. Strategies for reversing drug resistance. Oncogene
2003;22:7512–23;
(b) Lage H. An overview of cancer multidrug resistance: a still unsolved
problem. Cell Mol Life Sci 2008;65:3145–67.
[2] (a) Eckford PDW, Sharom FJ. ABC efflux pump-based resistance to
chemotherapy drugs. Chem Rev 2009;109:2989–3011;
(b) Szakacs G, Paterson JK, Ludwig JA, Booth-Genthe C, Gottesman MM.
Targeting multidrug resistance in cancer. Nat Rev Drug Discov 2006;5:219–34.
[3] Matsson P, Pedersen JM, Norinder U, Bergström CAS, Artusson P. Identification
of novel specific and general inhibitors of the three major human ATP-binding
cassette transporters Pgp, BCRP and MRP2 among registered drugs. Pharm Res
2009;26:1816–31.
[4] (a) Baumert C, Hilgeroth A. Recent advances in the development of Pgp
inhibitors. Anti-Cancer Agents Med Chem 2009;9:415-36 [and references
therein]; (b) Ramahete C, Molnar J, Mulhovo S, Rosario VE, Ferreira M-JU. New
potent P-glycoprotein modulators with the cucurbitane scaffold and their
synergistic interaction with doxorubicin on resistant cancer cells. Bioorg Med
Chem 2009;7:6942–51; (c) Viale M, Cordazzo C, Corimelli B, De Torero D,
Castagnola P, Aiello C, et al. Inhibition of MDR1 activity in vitro by a novel class
of diltiazem analogues: toward new candidates. J Med Chem 2009;52:259–66;
(d) Duan Z, Li X, Huang H, Yuan W, Zheng S-L, Liu X, et al. Synthesis and
evaluation of 2-(4-methoxyphenyl)-4-quinolinyl)-2-piperidinyl-methanol
(NSC23925) isomers to reverse multidrug resistance in cancer. J Med Chem
2012. doi: 10.1021/jm300117u.
(c) Meyer SC, Shomin CD, Gaj T, Ghosh I. Tethering small molecules to a phage
display library: discovery of a selective bivalent inhibitor of protein kinase A. J
Am Chem Soc 2007;129:13812–3;
(d) Bérubé M, Poirier D. Design, chemical synthesis, and in vitro biological
evaluation of simplified estradiol-adenosine hybrids as inhibitors of 17b-
hydroxysteroid dehydrogenase type 1. Can J Chem 2009;87:1180–99;
(e) Bérubé G. Natural and synthetic biologically active dimeric molecules:
anticancer agents, anti-HIV agents, steroid derivatives and opioid antagonists.
Curr Med Chem 2006;13:131–54.
[15] (a) Yeager AR, Finney NS. The first direct evaluation of the two-active site
mechanism for chitin synthase. J Org Chem 2004;69:613–8;

W. Zeinyeh et al. / Steroids 77 (2012) 1177–1191
1191
(b) Meltzer PC, Kryatova O, Pham-Huu D-P, Donovan P, Janowsky A. The
synthesis of bivalent 2b-carbomethoxy-3b-(3,4-dichlorophenyl)-8-
heterobicyclo[3.2.1]octanes as probes for proximal binding sites on the
dopamine and serotonin transporters. Bioorg Med Chem 2008;16:1832–41.
[16] (a) Sauna ZE, Andrus MB, Turner TM, Ambudkar SV. Biochemical basis of
[26] Sato H, Hayashi E, Yamada N, Yatagai M, Takahara Y. Further studies on the
site-specific protein modification by microbial transglutaminase. Bioconjugate
Chem 2001;12:701–10.
[27] Lukyanenko NG, Kirichenko TI, Scherbakov SV. Novel cryptands containing
thiourea units as a part of the macrocyclic framework. J Chem Soc Perkin Trans
1 2002;21:2347–51.
polyvalency as
a strategy for enhancing the efficacy of P-glycoprotein
(ABCB1) modulators: stipiamide homodimers separated with defined-length
spacers reverse drug efflux with greater efficacy. Biochemistry
2004;43:2262–71;
[28] Dey S, Garner P. Synthesis of tert-butoxycarbonyl (Boc)-protected purines. J
Org Chem 2000;65:7697–9.
[29] Ashford SW, Grega KC. Oxidative cleavage of 1,3-dicarbonyls to carboxylic
acids with oxone. J Org Chem 2001;66:1523–4.
(b) Chan K-F, Zhao Y, Burkett BA, Wong ILK, Chow LMC, Chan TH.
Flavonoid
dimers
as
bivalent
modulators
for
P-glycoprotein-
[30] Hoyte RM, Rosner W, Johnson IS, Zielinski J, Hochberg RB. Synthesis and
evaluation of potential radioligands for the progesterone receptor. J Med Chem
1985;28:1695–9.
based multidrug resistance: synthetic apigenin homodimers linked with
defined-length poly(ethylene glycol) spacers increase drug retention and
enhance chemosensitivity in resistant cancer cells.
2006;49:6742–59;
J
Med Chem
[31] Stoelwinder J, van Zoest WJ, van Leusen AM. Chemistry of N, P-acetals:
application to the synthesis of 20-ketosteroids. J Org Chem 1992;57:2252–7.
[32] Kim J-H, Kim HO, Lee KM, Chun MW, Moon HR, Jeong LS. Asymmetric
(c) Wong ILK, Chan K-F, Tsang KH, Lam CY, Zhao Y, Chan TH, et al.
Modulation of multidrug resistance protein (MRP1/ABCC1)-mediated
1
synthesis of homo-apioneplanocin
A
from D-ribose. Tetrahedron
multidrug resistance by bivalent apigenin homodimers and their
derivatives. J Med Chem 2009;52:5311–22;
2006;62:6339–42 [and references therein].
[33] For a recent method of reduction of azide by NaI/CeCl3 system, see: Bartoli G,
Di Antonio G, Giovannini R, Giuli S, Lanari S, Paoletti M. Efficient
transformation of azides to primary amines using the mild and easily
accessible CeCl3.7H2O/NaI system. J Org Chem 2008;73:1919–24.
(d) Namanja HA, Emmert D, Pires MM, Hrycyna CA, Chmielewski J.
Inhibition of human P-glycoprotein transport and substrate binding using
a galantamine dimer. Biochem Biophys Res Commun 2009;388:672–6;
(e) Pires MM, Emmert D, Hrycyna CA, Chmielewski J. Inhibition of P-
glycoprotein-mediated paclitaxel resistance by reversibly linked quinine
homodimers. Mol Pharmacol 2009;75:92–100;
[34] For 1,6-addition on
D
a
4,6-3-keto steroids, see: (a) Campbell JA, Babcock JC. The
- and 7/3-methyl steroid hormones. J Am Chem Soc
synthesis of some 7
1959;81:4069–74; (b) Modi SP, Gardner JO, Milowsky A, Wierzba M, Forgione
L, Mazur P, Solo AJ. Conjugate addition of Grignard reagents to enones and
dienones. J Org Chem 1989;54:2317–21; (c) Li G, Zhang Y. Stereoselective
(f) Pires MM, Hrycyna CA, Chmielewski J. Bivalent probes of the human
multidrug transporter P-glycoprotein. Biochemistry 2006;45:11695–702.
[17] Yang C-PH, DePinho SG, Greenberger LM, Arceci RJ, Horwitz SB. Progesterone
interacts with P-glycoprotein in multidrug-resistant cells and in the
endometrium of gravid uterus. J Biol Chem 1989;264:782–8.
synthesis of the two major metabolites of spironolactone,
hydroxy-7 -methylthio-17 -pregn-4-ene-21,17-carbolactone.
3
a
-
and 3b-
Steroids
a
a
2007;72:569–72; (d) Wynne JH, Lloyd CT, Mushrush GW. Synthesis of novel
7a-thiol-bridged progesterone derivatives. Synthetic Commun 2003;33:885–
[18] Ambudkar SV, Kim IW, Xia D, Sauna ZE. The A-loop,
a
novel conserved
aromatic acid subdomain upstream of the Walker A motif in ABC transporters,
is critical for ATP binding. FEBS Lett 2006;580:1049–55.
[19] Zeinyeh W, Alameh G, Radix S, Grenot C, Dumontet C, Walchshofer N. Design,
synthesis and evaluation of progesterone–adenine hybrids as bivalent
inhibitors of P-glycoprotein-mediated multidrug efflux. Bioorg Med Chem
Lett 2010;3165–68.
93; (e) Brueggemeier RW, Floyd EE, Counsell RE. Synthesis and biochemical
evaluation of inhibitors of estrogen biosynthesis. J Med Chem 1978;21:1007–
11.
[35] Agnello EJ, Laubach GD. A new method for dehydrogenation of corticosteroids.
J Am Chem Soc 1957;79:1257–8.
[36] Nickisch K, Bittler D, Laurent H, Losert W, Nishino Y, Schillinger E, Wiechert R.
[20] Grenot C, Cuilleron C. Inventors; Inserm, assignee. New steroid inhibitors of
Pgp for inhibiting multidrug resistance. International Patent WO 2011073419,
June 23, 2011.
Aldosterone antagonists. 3. Synthesis and activities of steroidal 7a-
(alkoxycarbonyl)-15,16-methylene
1990;33:509–13.
spirolactones.
J
Med
Chem
[21] Haddenham D, Pasumansky L, DeSoto J, Eagon S, Singaram B. Reductions of
[37] Nagata W, Yoshioka M. Hydrocyanation of conjugated carbonyl compounds.
Org React 1977;25:255–476.
[38] Degrand C, Limoges B, Blankespoor RL. Synthesis of nitroxides for use as
aliphatic
diisopropylaminoborane. J Org Chem 2009;74:1964–70.
[22] Spiegel DA, Schroeder FC, Duvall JR, Schreiber SL. An oligomer-based approach
and
aromatic
nitriles
to
primary
amines
with
procationic labels and their incorporation into nafion films.
1993;58:2573–7.
J Org Chem
to skeletal diversity in small-molecule synthesis.
2006;128:14766–7.
J Am Chem Soc
[39] Comte G, Daskiewicz J-P, Bayet C, Conseil G, Viorney-Vanier A, Dumontet C, Di
Pietro A, Barron D. C-Isoprenylation of flavonoids enhances binding affinity
toward P-glycoprotein and modulation of cancer cell chemoresistance. J Med
Chem 2001;44:763–8.
[23] Juenge EC, Spangler P. Study of 4-amino-2-butyn-1-ol and preparation of the
reverse carbamate of the selective herbicide, Barban.
1964;29:226–8.
J Org Chem
´
´
´
[24] Krištafor V, Raic-Malic S, Cetina M, Kralj M, Šuman L, Pavelic K, et al. Synthesis,
X-ray crystal structural study, antiviral and cytostatic evaluations of the novel
unsaturated acyclic and epoxide nucleoside analogues. Bioorg Med Chem
2006;14:8126–38.
[40] a Colabufo NA, Berardi F, Cantore M, Contino M, Inglese C, Niso M, Perrone R.
Perspectives of P-glycoprotein in modulating agents in oncomogy and
neurodegenerative diseases: pharmaceutical, biological, and diagnostic
potentials. J Med Chem 2010;53:1883–97;
[25] Rouge P, Becuwe M, Dassonville-Klimpt A, Da Nascimento S, Raimbert J-F,
Cailleu D, et al. Efficient synthesis of amino-protected calix[4]arenes
selectively functionalized with iron chelator ICL670 designed as platform for
iron recognition. Tetrahedron 2011;67:2916–24.
b Colabufo NA, Berardi F, Contino M, Inglese C, Perrone R. Flavonoids as MDR
modulating agents: SAR studies. In: Colabufo NA, editor. Multidrug resistance.
biological
and
pharmaceutical
advance
in
the
antitumour
treatment. Kerala: Research Signpost; 2008. p. 171–201.