Chemical synthesis of 16β-propylaminoacyl derivatives of estradiol and their inhibitory potency on type 1 17β-hydroxysteroid dehydrogenase and binding affinity on steroid receptors

Article abstract of DOI:10.1016/S0039-128X(01)00116-7

The 17β-hydroxysteroid dehydrogenases (17β-HSDs) are members of a family of enzymes that catalyze the interconversion of weakly active sexual hormones (ketosteroids) and potent hormones (17β-hydroxysteroids). Among the known isoforms of 17β-HSD, the type

Full text of DOI:10.1016/S0039-128X(01)00116-7

Steroids 66 (2001) 821–831
Chemical synthesis of 16␤-propylaminoacyl derivatives of estradiol and
their inhibitory potency on type 1 17␤-hydroxysteroid dehydrogenase
and binding affinity on steroid receptors
Martin R. Tremblay, Sheng-Xiang Lin, Donald Poirier*
Medicinal Chemistry Division, Oncology and Molecular Endocrinology Research Center, Laval University Medical Center (CHUL),
2705 Laurier Boulevard, Sainte-Foy, Quebec G1V 4G2, Canada
Received 25 August 2000; received in revised form 31 January 2001; accepted 7 February 2001
Abstract
The 17␤-hydroxysteroid dehydrogenases (17␤-HSDs) are members of a family of enzymes that catalyze the interconversion of weakly
active sexual hormones (ketosteroids) and potent hormones (17␤-hydroxysteroids). Among the known isoforms of 17␤-HSD, the type 1
catalyzes the NAD(P)H-mediated reduction of estrone (E1) to estradiol (E2), a predominant mitogen for the breast cancer cells. Therefore,
the inhibition of this particular enzyme is a logical approach to reduce the concentration of estradiol in breast tumors. To develop inhibitors
of type 1 17␤-HSD activity, we hypothesized that molecules containing both hydrophobic and hydrophilic components should be interesting
candidates for interacting with both the steroid binding domain and some amino acid residues of the cofactor binding domain of the enzyme.
Firstly, a conveniently protected 16␤-(3-aminopropyl)-E2 derivative was synthesized from commercially available E1. Then, a represen-
tative of all class of NHBoc-protected amino acids (basic, acid, aromatic, aliphatic, hydroxylated) were coupled using standard procedures
to the amino group of the precursor. Finally, cleavage of all protecting groups was performed in a single step to generate a series of
16␤-propylaminoacyl derivatives of E2. The enzymatic screening revealed that none of the novel compounds can inhibit the reductive
activity of type 1 17␤-HSD. On the other hand, all of these E2 derivatives did not show any significant binding affinity on four steroid
receptors including the estrogen receptor. Additional efforts aimed at improving the inhibitory potency of these steroidal derivatives on type
1 17␤-HSD without providing estrogenic activities is under investigation using a combinatorial chemistry approach. © 2001 Elsevier
Science Inc. All rights reserved.
Keywords: Steroid; Peptidosteroid; Estradiol; Hydroxysteroid dehydrogenase; Chemical synthesis; NMR data
1. Introduction
systems and are now used in clinical trials for the prevention
and the treatment of breast cancers. The history of steroidal
antiestrogens is shorter, but resulted in interesting activities
displayed by ICI 164384 [14], ICI 182780 [15], and RU
58810 [16]. These studies have clearly demonstrated the
importance of a long alkylamide side chain at position 7␣ or
11␤ of estradiol (E2) for their antiestrogenic activities. On
the other hand, our studies proved that similar side chains do
not provide potent antiestrogenic activity when they substi-
tute position 17␣, 16␣, 15␣ and 15␤ of E2 [17,18]. Several
other derivatives of E2 and their relative ER binding activ-
ities were reported resulting in very interesting structure-
activity relationships (SAR) summarized in a review article
[19]. All these data are very important because the 3D
structure of ER is only partially known [20,21].
Estrogens and peptide growth factors are known to pro-
mote the proliferation of an important proportion (46–77%)
of breast tumor cells [1,2]. Estrogens exert their action via
nuclear estrogen receptors (ER␣ and ER␤) giving transcrip-
tional activator complexes that bind to specific regulating
sequences of estrogen sensitive genes [3]. Based on this
principle, antiestrogens were designed to interfere with the
binding of estrogens to their receptors and/or the binding of
the complex to DNA. Nonsteroidal antiestrogen such as
tamoxifen [4,5], raloxifen [6,7], and EM-800 [8–13] have
shown very promising effects in both in vitro and in vivo
* Corresponding author. Tel.: ϩ1-418-654-2296; fax: ϩ1-418-654-
2761.
In a complementary approach to treat estrogen-sensitive
diseases such as breast cancer, we want to develop inhibi-
E-mail address: donald.poirier@crchul.ulaval.ca (D. Poirier).
0039-128X/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved.
PII: S0039-128x(01)00116-7

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M.R. Tremblay et al. / Steroids 66 (2001) 821–831
Fig. 1. Hypothesis for the design of prototype type 1 17␤-HSD inhibitors.
tors of type 1 17␤-hydroxysteroid dehydrogenase (17␤-
HSD), which is responsible for the last enzymatic step in the
biosynthesis of E2, the most potent estrogen. This enzyme
catalyzes preferentially the reduction of estrone (E1) to E2 in
intact cells [22–25] and is expressed in about 50% of the
breast tumor specimens [25–27]. For their effectiveness,
these inhibitors should be devoid of any estrogenic activity
and, if possible, they may display antiestrogenic activity
(dual-action blockers). Recently, the 3D structures of type 1
17␤-HSD, alone and complexed with substrates E2 and
cofactor, have been elucidated [28–31]. It has been clearly
indicated that the consensus sequence Tyr-X-X-X-Lys to-
gether with a serine are the most important amino acid
residues involved in the catalytic reaction. Particularly, Tyr-
155 and Ser-142 are involved in an interaction with either
the ketone or the hydroxyl at the position 17. Moreover,
residues that allows high specificity in binding of C18
steroid: His-221 and Glu-282 forms hydrogen bond with the
phenolic hydroxyl group at position 3. As expected, several
residues that interact with the substrate are hydrophobic
(Val, Met, Leu, Pro, Phe), while most of the residues that
bind to the cofactor are polar (Cys, Ser, Arg, Asp, Asn,
Lys). Therefore, we hypothesized that an inhibitor of type 1
17␤-HSD should possess both these structural elements,
namely hydrophobic and hydrophilic components. In an
exploratory study, we then decided to synthesize E2 deriv-
atives (compounds 12–20) bearing various polar chemical
groups (Fig. 1 and Scheme 1) as prototype inhibitors of type
1 17␤-HSD.
Fisher Scientific (Montreal, Canada) and were used as re-
ceived. Anhydrous solvents were obtained from Aldrich in
SureSeal bottles, which were conserved under positive ar-
gon pressure. Tetrahydrofuran (THF) was distilled from
sodium/benzophenone ketyl under argon. All anhydrous
reactions were performed in oven-dried glassware under
positive argon pressure. Flash chromatographies were per-
formed on E. Merck 60 230-400 mesh silica gel. Thin-layer
chromatographies were performed on 0.25 mm E. Merck
silica gel 60 F254 plates and visualized by UV (254 nm)
and/or cerium ammonium molybdate. Infrared (IR) spectra
were obtained from a KBr pellet with the solid compound or
from a thin film on NaCl pellet with the solubilized com-
pound (usually in CDCl3). They were recorded on a Pelkin-
Elmer series 1600 FT-IR spectrometer (Norwalk, CT, USA)
and only significant bands were reported in cmϪ1. Nuclear
magnetic resonance (NMR) spectra were reported in ppm
1
and recorded at 300 MHz for H and 75.5 MHz for 13C on
a Bruker AC/F300 spectrometer (Bruker, Billerica, MA,
1
USA). Only significant signals were reported for H NMR
while all signals were listed for 13C NMR. Low-resolution
mass spectra were recorded with a PE Sciex API-150ex
apparatus (Foster City, CA) equipped with a turbo ionspray
source.
2.2. Synthesis of 16␤-propylaminoacyl derivatives of E2
(Scheme 1)
Preparation of the intermediate amine 2: To a suspen-
sion of prereduced palladium 5% on activated carbon (300
mg) in EtOAc (20 ml) was added a solution of the azide 1
[32] (3.43 g; 6.20 mmol) in 30 ml of EtOAc. The resulting
mixture was stirred under hydrogen atmosphere at 25°C for
16 h. Then, celite was added to the reaction mixture and the
slurry was filtered through a celite pad eluting with EtOAc.
The organic solvent was evaporated under reduced pressure
2. Experimental
2.1. General methods
Reagents were obtained from Sigma-Aldrich Canada Co.
(Oakville, ON, Canada). Usual solvents were obtained from

M.R. Tremblay et al. / Steroids 66 (2001) 821–831
823
Scheme 1. Reagents and conditions: (a) H2, Pd/C, EtOAc, rt, 1 atm; (b) NHBoc-L-AA-COOH, DCC, HOBt, CH2Cl2, 0°C (30–66%, two steps); Method A:
3 N HCl, MeOH, rt (47–90%); Method B: HCl (anh.), CH2Cl2, 0°C (33–92%); Method C: i. 3 N HCl, MeOH, rt, ii. H2, Pd/C, EtOH, 60 psi (46%, two steps).
to give 3.4 g of the amine 2 in excellent purity. Therefore,
this compound was used for the coupling reaction without
further purification.
(sapp, 1H, 4-CH), 6.59 (dapp, J ϭ 8.4 Hz, 1H, 2-CH), 7.11
(2d, J ϭ 8.4 Hz, 1H, 1-CH); LRMS: calcd for
C39H65N2O6Si [MϩH]ϩ 685.5, found 685.6 m/z.
Typical procedure for amino acid coupling using DCC/
HOBt method: The N-t-Boc amino acid or peptide obtained
by a standard method [33] (1.0 eq.) was dissolved in dry
CH2Cl2 under argon and the solution was cooled at 0°C
before sequential addition of DCC and HOBt (1.0 eq.,
each). The resulting mixture was stirred for 1 h at 0°C.
Then, the amine 2 (1.0 eq.), dissolved in dry CH2Cl2, was
added dropwise and the reaction mixture was allowed to
warm very slowly to room temperature. After 3 h, the
reaction mixture was filtered and diluted in CH2Cl2. The
organic phase was washed successively with a saturated
aqueous solution of NaHCO3, water, a 1N HCl solution, and
brine. The organic layers were dried over MgSO4, and the
solvents were evaporated to dryness. The crude compound
was purified by flash chromatography to give pure amino-
acyl estradiol derivatives 3–11.
N-{3-[3-(t-butyldimethylsilyloxy)-17␤-(tetrahydro-2H-
pyran-2-yl-oxy)-estra-1, 3, 5 (10)-trien-16␤-yl]-propyl}-2-
(S)-isobutyl-2-(t-butoxycarbonylamino)-acetamide (4).
White amorphous solid (40% yield); IR (NaCl, film): 3300
1
(NH), 1700 (CϭO, carbamate), 1650 (CϭO, amide); H
NMR (CDCl3): 0.17 (s, 6H, Si(CH3)2), 0.77 and 0.82 (2s,
3H, 18-CH3), 0.93 (d, J ϭ 5.9 Hz, 6H, 2 ϫ CH3 of isobutyl),
0.97 (s, 9H, SiC(CH3)3), 1.43 (s, 9H, (CH3)3C of N-Boc),
2.78 (m, 2H, 6-CH2), 3.23 (m, 2H, NCH2 of 16␤-propyl),
3.49 and 3.92 (2m, 2H, OCH2 of THP), 3.71 and 3.78 (2d,
J ϭ 9.6 Hz, 1H, 17␣-CH), 4.10 (m, 1H, NCH of leucyl),
4.62 and 4.68 (2m, 1H, CH of THP), 5.00 (m, 1H, NH), 6.30
(m, 1H, NH), 6.54 (d, J ϭ 2.4 Hz, 1H, 4-CH), 6.58 (dd, J1 ϭ
8.5 Hz and J2 ϭ 2.4 Hz, 1H, 2-CH), 7.11 (d, J ϭ 8.5 Hz, 1H,
1-CH); LRMS: calcd for C43H71N2O6Si [M-H]Ϫ 739.5,
found 739.4 m/z.
N-{3-[3-(t-butyldimethylsilyloxy)-17␤-(tetrahydro-2H-
pyran-2-yl-oxy)-estra-1, 3, 5 (10)-trien-16␤-yl]-propyl}-2-
(t-butoxycarbonylamino)-acetamide (3). White amorphous
solid (54% yield); IR (NaCl, film): 3320 (NH), 1705 (CϭO,
N-{3-[3-(t-butyldimethylsilyloxy)-17␤-(tetrahydro-2H-
pyran-2-yl-oxy)-estra-1, 3, 5 (10)-trien-16␤-yl]-propyl}-2-
(S)-benzyl-2-(t-butoxycarbonylamino)-acetamide (5). White
amorphous solid (49% yield); IR (NaCl, film): 3300 (NH),
1
1
carbamate), 1665 (CϭO, amide); H NMR (CDCl3): 0.17
1700 (CϭO, carbamate), 1655 (CϭO, amide); H NMR
(s, 6H, Si(CH3)2), 0.77 and 0.82 (2s, 3H, 18-CH3), 0.96 (s,
9H, SiC(CH3)3), 1.45 (s, 9H, (CH3)3C of N-Boc), 2.78 (m,
2H, 6-CH2), 3.24 (m, 2H, NCH2 of 16␤-propyl), 3.47 and
3.87 (2m, 2H, OCH2 of THP), 3.71 (d, J ϭ 9.7 Hz, H,
17␣-CH), 3.76 (m, 2H, NCH2 of glycyl), 4.59 and 4.66 (2m,
1H, CH of THP), 5.20 (m, 1H, NH), 6.20 (m, 1H, NH), 6.54
(CDCl3): 0.18 (s, 6H, Si(CH3)2), 0.76 and 0.81 (2s, 3H,
18-CH3), 0.97 (s, 9H, SiC(CH3)3), 1.41 (s, 9H, (CH3)3C of
N-Boc), 2.79 (m, 2H, 6-CH2), 3.04 (m, 2H, CH2Ph), 3.20
(m, 2H, NCH2 of 16␤-propyl), 3.48 and 3.92 (2m, 2H,
OCH2 of THP), 3.69 and 3.76 (2d, J ϭ 9.8 Hz, 1H, 17␣-
CH), 4.26 (m, 1H, NCH of phenylalanyl), 4.57 and 4.67

824
M.R. Tremblay et al. / Steroids 66 (2001) 821–831
(2m, 1H, CH of THP), 5.10 (m, 1H, NH), 5.76 and 5.85
(2m, 1H, NH), 6.54 (d, J ϭ 2.3 Hz, 1H, 4-CH), 6.60 (dd,
J1 ϭ 8.4 Hz and J2 ϭ 2.3 Hz, 1H, 2-CH), 7.11 (2d, J ϭ 8.4
Hz, 1H, 1-CH), 7.25 (m, 5H, Ph of phenylalanyl); LRMS:
calcd for C46H71N2O6Si [MϩH]ϩ 775.5, found 776.0 m/z.
N-{3-[3-(t-butyldimethylsilyloxy)-17␤-(tetrahydro-2H-
pyran-2-yl-oxy)-estra-1, 3, 5 (10)-trien-16␤-yl]-propyl}-2-
(S)-(N-t-butoxycarbonylpyrrolidinyl)-acetamide (6). White
amorphous solid (44% yield); IR (NaCl, film): 3325 (NH),
1692 (CϭO, carbamate and amide); 1H NMR (CDCl3): 0.17
(s, 6H, Si(CH3)2), 0.77 and 0.82 (2s, 3H, 18-CH3), 0.96 (s,
9H, SiC(CH3)3), 1.45 (s, 9H, (CH3)3C of N-Boc), 2.78 (m,
2H, 6-CH2), 3.20 (m, 2H, NCH2 of 16␤-propyl), 3.50 (m,
3H, NCH2 of pyrrolidinyl and 0.5 ϫ OCH2 of THP), 3.90
(2m, 1H, 0.5 ϫ OCH2 of THP), 3.70 and 3.78 (2d, J ϭ 9.8
Hz, 1H, 17␣-CH), 4.20 (m, 1H, NCH of propyl), 4.58 and
4.69 (2m, 1H, CH of THP), 6.53 (d, J ϭ 2.4 Hz, 1H, 4-CH),
6.59 (dd, J1 ϭ 8.3 Hz and J2 ϭ 2.4 Hz, 1H, 2-CH), 7.10 (2d,
J ϭ 8.3 Hz, 1H, 1-CH); LRMS: calcd for C42H69N2O6Si
[MϩH]ϩ 725.5, found 725.6 m/z.
amino)-acetamide (9). White amorphous solid (50% yield);
IR (NaCl, film): 3330 (NH), 1730 (CϭO, ester), 1690
(CϭO, carbamate), 1658 (CϭO, amide); 1H NMR (CDCl3):
0.18 (s, 6H, Si(CH3)2), 0.78 and 0.82 (2s, 3H, 18-CH3), 0.97
(s, 9H, SiC(CH3)3), 1.43 and 1.45 (s, 18H, (CH3)3C of
N-Boc and t-butyl ester), 2.78 (m, 2H, 6-CH2), 3.25 (m, 2H,
NCH2 of 16␤-propyl), 3.49 and 3.93 (2m, 2H, OCH2 of
THP), 3.71 and 3.78 (2d, J ϭ 9.9 Hz, 1H, 17␣-CH), 4.08
(m, 1H, NCH of glutamyl), 4.59 and 4.69 (2m, 1H, CH of
THP), 5.30 (m, 1H, NH), 6.33 (m, 1H, NH), 6.54 (d, J ϭ 2.2
Hz, 1H, 4-CH), 6.59 (dapp, J ϭ 8.4 Hz, 1H, 2-CH), 7.09 and
7.11 (2d, J ϭ 8.4 Hz, 1H, 1-CH); LRMS: calcd for
C41H69N2O7Si [MϩH-THP]ϩ 729.5, found 729.0 m/z.
N-{3-[3-(t-butyldimethylsilyloxy)-17␤-(tetrahydro-2H-
pyran-2-yl-oxy)-estra-1, 3, 5 (10)-trien-16␤-yl]-propyl}-2-
(S)-(1-t-butoxycarbonylpropan-3-yl)-2-{N␣-[N␤-(t-butoxy-
carbonylamino)-L-alanyl]-L-alanyl}-acetamide (10). White
amorphous solid (35% yield); IR (NaCl, film): 3280 (NH),
1731 (CϭO, ester), 1700 (CϭO, carbamate), 1667 and 1633
1
(CϭO, amide); H NMR (CDCl3): 0.18 (s, 6H, Si(CH3)2),
N-{3-[3-(t-butyldimethylsilyloxy)-17␤-(tetrahydro-2H-
pyran-2-yl-oxy)-estra-1, 3, 5 (10)-trien-16␤-yl]-propyl}-2-
(S)-[4-(t-butoxycarbonylamino)-butyl]-2-(t-butoxycar-
bonyl-amino)-acetamide (7). White amorphous solid (66%
yield); IR (NaCl, film): 3320 (NH), 1696 (CϭO, carbam-
0.77 and 0.81 (2s, 3H, 18-CH3), 0.97 (s, 9H, SiC(CH3)3),
1.39 and 1.41 (2d, J ϭ 6.4 Hz, 6H, 2 ϫ CH3 of alanyl), 1.43
and 1.44 (2s, 18H, (CH3)3C of N-Boc and t-butyl ester),
2.78 (m, 2H, 6-CH2), 3.22 (m, 2H, NCH2 of 16␤-propyl),
3.50 and 3.90 (2m, 2H, OCH2 of THP), 3.71 and 3.78 (2d,
J ϭ 9.6 Hz, 1H, 17␣-CH), 4.12 (m, 1H, NCH of NHBoc-
Ala), 4.35 (m, 2H, NCH of alanyl and glutamyl), 4.59 and
4.67 (2m, 1H, CH of THP), 5.05 (m, 1H, NH), 6.54 (d, J ϭ
2.4 Hz, 1H, 4-CH), 6.59 (dd, J1 ϭ 8.5 Hz and J2 ϭ 2.4 Hz,
1H, 2-CH), 6.78 (m, 2H, NH), 7.05 and 7.09 (2d, J ϭ 8.5
Hz, 1H, 1-CH), 7.13 (m, 1H, NH); LRMS: calcd for
C52H87N4O10Si [MϩH]ϩ 955.6, found 955.8 m/z.
1
ate), 1660 (CϭO, amide); H NMR (CDCl3): 0.17 (s, 6H,
Si(CH3)2), 0.77 and 0.82 (2s, 3H, 18-CH3), 0.97 (s, 9H,
SiC(CH3)3), 1.44 (s, 18H, (CH3)3C of N-Boc), 2.78 (m, 2H,
6-CH2), 3.10 (m, 2H, NCH2 of lysyl), 3.25 (m, 2H, NCH2
of 16␤-propyl), 3.50 and 3.90 (2m, 2H, OCH2 of THP), 3.70
and 3.77 (2d, J ϭ 9.6 Hz, 1H, 17␣-CH), 4.00 (m, 1H, NCH
of lysyl), 4.58 and 4.69 (2m, 1H, CH of THP), 4.60 (m, 1H,
NH), 5.10 (m, 1H, NH), 6.20 (m, 1H, NH), 6.54 (d, J ϭ 2.3
Hz, 1H, 4-CH), 6.59 (dd, J1 ϭ 8.4 Hz and J2 ϭ 2.3 Hz, 1H,
2-CH), 7.10 (2d, J ϭ 8.4 Hz, 1H, 1-CH); LRMS: calcd for
C48H82N3O8Si [MϩH]ϩ 856.6, found 856.5 m/z.
N-{3-[3-(t-butyldimethylsilyloxy)-17␤-(tetrahydro-2H-
pyran-2-yl-oxy)-estra-1, 3, 5 (10)-trien-16␤-yl]-propyl}-2-
(S)-methyl-2-{N␣-[N␤-(t-butoxycarbonylamino)-L-alanyl]-
L-alanyl}-acetamide (8). White amorphous solid (26%
yield); IR (NaCl, film): 3275 (NH), 1698 (CϭO, carbam-
ate), 1670 and 1630 (CϭO, amide); 1H NMR (CDCl3): 0.17
(s, 6H, Si(CH3)2), 0.77 and 0.81 (2s, 3H, 18-CH3), 0.97 (s,
9H, SiC(CH3)3), 1.44 (s, 9H, (CH3)3C of N-Boc), 2.77 (m,
2H, 6-CH2), 3.23 (m, 2H, NCH2 of 16␤-propyl), 3.50 and
3.90 (2m, 2H, OCH2 of THP), 3.70 and 3.77 (2d, J ϭ 9.6
Hz, 1H, 17␣-CH), 4.20 (m, 1H, NCH of NHBoc-Ala), 4.42
(q, J ϭ 6.8 Hz, 1H, NCH of alanyl), 4.54 (q, J ϭ 7.5 Hz, 1H,
NCH of alanyl), 4.58 and 4.72 (2m, 1H, CH of THP), 5.35
(m, 1H, NH), 6.54 (d, J ϭ 2.4 Hz, 1H, 4-CH), 6.59 (dd, J1 ϭ
8.5 Hz and J2 ϭ 2.4 Hz, 1H, 2-CH), 6.90 (m, 1H, NH), 7.09
(m, 2H, 1-CH and NH), 7.22 (m, 1H, NH); LRMS: calcd for
C46H77N4O8Si [MϩH]ϩ 841.5, found 841.7 m/z.
N-{3-[3-(t-butyldimethylsilyloxy)-17␤-(tetrahydro-2H-
pyran-2-yl-oxy)-estra-1, 3, 5 (10)-trien-16␤-yl]-propyl}-2-
(S)-(1-benzyloxymethyl)-2-(t-butoxycarbonylamino)-
acetamide (11). White amorphous solid (33% yield); IR
(NaCl, film): 3325 (NH), 1715 (CϭO, carbamate), 1660
1
(CϭO, amide); H NMR (CDCl3): 0.19 (s, 6H, Si(CH3)2),
0.78 and 0.83 (2s, 3H, 18-CH3), 0.98 (s, 9H, SiC(CH3)3),
1.45 (s, 9H, (CH3)3C of N-Boc), 2.79 (m, 2H, 6-CH2), 3.30
(m, 2H, NCH2 of 16␤-propyl), 3.50 and 3.90 (2m, 4H,
OCH2 of THP and OCH2 of seryl), 3.70 and 3.78 (2d, J ϭ
9.8 Hz, 1H, 17␣-CH), 4.25 (m, 1H, NCH of seryl), 4.55 (m,
2H, OCH2Ph), 4.58 and 4.67 (2m, 1H, CH of THP), 5.40
(m, 1H, NH), 6.50 (m, 1H, NH), 6.55 (d, J ϭ 2.1 Hz, 1H,
4-CH), 6.60 (dd, J1 ϭ 8.5 Hz and J2 ϭ 2.1 Hz, 1H, 2-CH),
7.05 and 7.09 (2d, J ϭ 8.5 Hz, 1H, 1-CH), 7.31 (m, 5H, Ph);
LRMS: calcd for C47H73N2O7Si [MϩH]ϩ 805.5, found
805.4 m/z.
Typical procedure for the deprotection of acid labile
protecting groups. Method A (synthesis of compounds 12–
17): To cooled solutions of protected compounds 3–8 in
MeOH at 0°C was added concentrated hydrochloric acid to
give a final concentration of 3 N. The mixture was stirred at
0°C for 2 h and the solvent was evaporated to dryness at
room temperature under vacuum. Then, the crude com-
N-{3-[3-(t-butyldimethylsilyloxy)-17␤-(tetrahydro-2H-
pyran-2-yl-oxy)-estra-1, 3, 5 (10)-trien-16␤-yl]-propyl}-2-
(S)-(1-t-butoxycarbonylpropan-3-yl)-2-(t-butoxycarbonyl-

M.R. Tremblay et al. / Steroids 66 (2001) 821–831
825
pounds were triturated in diethyl ether, filtered, and dried
under vacuum. Method B (synthesis of compounds 18 and
19): To cooled solutions of the protected compounds 9 and
10 in dry CH2Cl2 at 0°C, anhydrous hydrogen chloride was
gently bubbled twice for 15 min. Then, solvent was evap-
orated, the crude compounds were triturated with diethyl
ether, washed with CH2Cl2, filtered, and dried under vac-
uum. Method C (synthesis of compound 20): To a cooled
solution of the protected compound 11 in MeOH at 0°C was
added concentrated hydrochloric acid to give a final con-
centration of 3 N. The mixture was stirred at 0°C for 2 h and
the solvent was evaporated to dryness at room temperature
under vacuum. The crude compound was then suspended in
EtOH containing 10% palladium on activated carbon, and
the resulting slurry was submitted to hydrogen pressure (60
psi) for 24 h at room temperature. Afterward, the suspension
was filtered through celite and the solvent was evaporated
under reduced pressure. The crude compound was triturated
in diethyl ether, filtered, and dried under vacuum.
(s, 3H, 18-CH3), 2.75 (m, 2H, 6-CH2), 2.95 (m, 2H, NCH2
of pyrolidinyl), 3.20 (m, 2H, NCH2 of 16␤-propyl), 3.61 (m,
1H, NCH of prolyl), 3.68 (d, J ϭ 9.8 Hz, 1H, 17␣-CH), 6.47
(d, J ϭ 2.6 Hz, 1H, 4-CH), 6.52 (dd, J1 ϭ 8.4 Hz and J2 ϭ
2.6 Hz, 1H, 2-CH), 7.05 (d, J ϭ 8.4 Hz, 1H, 1-CH); 13C
NMR (Table 1); LRMS: calcd for C26H39N2O3
[M-HClϩH]ϩ 427.3, found 427.5 m/z.
N-{3-[3, 17␤-dihydroxy-estra-1, 3, 5 (10)-trien-16␤-yl]-
propyl}-2-(S)-(1-ammonium
hydrochlroride-but-4-yl)-2-
(ammonium hydrochloride)-acetamide (16). White solid
(82% yield); IR (KBr): 3300 (br, OH and NH), 1667 (CϭO,
amide); 1H NMR (CD3OD): 0.76 (s, 3H, 18-CH3), 2.75 (m,
2H, 6-CH2), 2.96 (t, J ϭ 7.7 Hz, 2H, NCH2 of lysyl), 3.26
(t, J ϭ 6.4 Hz, 2H, NCH2 of 16␤-propyl), 3.70 (d, J ϭ 9.7
Hz, 1H, 17␣-CH), 3.89 (t, J ϭ 6.7 Hz, 1H, NCH of lysyl),
6.47 (d, J ϭ 2.6 Hz, 1H, 4-CH), 6.53 (dd, J1 ϭ 8.4 Hz and
J2 ϭ 2.6 Hz, 1H, 2-CH), 7.06 (d, J ϭ 8.4 Hz, 1H, 1-CH);
13C NMR (Table 1); LRMS: calcd for C27H44N3O3
[M-2HClϩH]ϩ 458.3, found 458.5 m/z.
N-{3-[3, 17␤-dihydroxy-estra-1, 3, 5 (10)-trien-16␤-yl]-
propyl}-2-(ammonium hydrochloride)-acetamide (12).
White solid (47% yield); IR (KBr): 3200 (br, OH and NH),
N-{3-[3, 17␤-dihydroxy-estra-1, 3, 5 (10)-trien-16␤-yl]-
propyl}-2-(S)-methyl-2-{N␣-[N␤-(ammonium
hydrochlo-
ride)-L-alanyl]-L-alanyl}-acetamide (17). White solid (81%
yield); IR (KBr): 3300 (br, OH and NH), 1634 (CϭO,
1
1693 (CϭO, amide); H NMR (CD3OD): 0.76 (s, 3H, 18-
1
CH3), 2.75 (m, 2H, 6-CH2), 3.25 (m, 2H, NCH2 of 16␤-
propyl), 3.65 (s, 2H, NCH2 of glycyl), 3.71 (d, J ϭ 9.8 Hz,
1H, 17␣-CH), 6.47 (d, J ϭ 2.6 Hz, 1H, 4-CH), 6.53 (dd,
J1 ϭ 8.5 Hz and J2 ϭ 2.6 Hz, 1H, 2-CH), 7.06 (d, J ϭ 8.5
Hz, 1H, 1-CH); 13C NMR (Table 1); LRMS: calcd for
C23H35N2O3 [M-HClϩH]ϩ 387.3, found 387.4 m/z.
amide); H NMR (CD3OD): 0.76 (s, 3H, 18-CH3), 1.34 (d,
J ϭ 7.2 Hz, 3H, CH3 of alanyl), 1.40 (d, J ϭ 7.1 Hz, 3H, CH3
of alanyl), 1.53 (d, J ϭ 7.0 Hz, 3H, CH3 of alanyl), 2.75 (m,
2H, 6-CH2), 3.19 (m, 2H, NCH2 of 16␤-propyl), 3.69 (d, J ϭ
9.8 Hz, 1H, 17␣-CH), 3.95 (q, J ϭ 7.0 Hz, 1H, NCH of alanyl),
4.29 (q, J ϭ 7.1 Hz, 1H, NCH of alanyl), 4.38 (q, J ϭ 7.1 Hz,
1H, NCH of alanyl), 6.46 (d, J ϭ 2.3 Hz, 1H, 4-CH), 6.52 (dd,
J1 ϭ 8.4 Hz and J2 ϭ 2.5 Hz, 1H, 2-CH), 7.06 (d, J ϭ 8.5 Hz,
1H, 1-CH); 13C NMR (Table 1); LRMS: calcd for
C30H47N4O5 [M-HClϩH]ϩ 543.4, found 543.6 m/z.
N-{3-[3, 17␤-dihydroxy-estra-1, 3, 5 (10)-trien-16␤-yl]-
propyl}-2-(S)-isopropyl-2-(ammonium hydrochloride)-acet-
amide (13). White solid (81% yield); IR (KBr): 3300 (br,
1
OH and NH), 1645 (CϭO, amide); H NMR (CD3OD):
0.76 (s, 3H, 18-CH3), 1.00 (AB system, J ϭ 4.0 Hz, 6H, 2 ϫ
CH3 of isopropyl), 2.75 (m, 2H, 6-CH2), 3.25 (m, 2H, NCH2
of 16␤-propyl), 3.70 (d, J ϭ 9.8 Hz, 1H, 17␣-CH), 3.83 (AB
system, J ϭ 5.6 Hz, 1H, NCH of leucyl), 6.47 (d, J ϭ 2.5
Hz, 1H, 4-CH), 6.53 (dd, J1 ϭ 8.5 Hz and J2 ϭ 2.5 Hz, 1H,
2-CH), 7.05 (d, J ϭ 8.5 Hz, 1H, 1-CH); 13C NMR (Table 1);
LRMS: calcd for C27H43N2O3 [M-HClϩH]ϩ 443.3, found
443.5 m/z.
N-{3-[3, 17␤-dihydroxy-estra-1, 3, 5 (10)-trien-16␤-yl]-
propyl}-2-(S)-(1-hydroxycarbonylpropan-3-yl)-2-(ammoni-
um hydrochloride)-acetamide (18). White solid (33%
yield); IR (KBr): 3300 (br, OH and NH), 1715 (CϭO, acid),
1
1670 (CϭO, amide); H NMR (CD3OD): 0.77 (s, 3H, 18-
CH3), 2.47 (t, J ϭ 7.9 Hz, 2H, CH2COOH), 2.76 (m, 2H,
6-CH2), 3.26 (m, 2H, NCH2 of 16␤-propyl), 3.71 (d, J ϭ 9.8
Hz, 1H, 17␣-CH), 3.87 (t, J ϭ 6.5 Hz, 1H, NCH of glu-
tamyl), 6.47 (d, J ϭ 2.5 Hz, 1H, 4-CH), 6.53 (dd, J1 ϭ 8.5
Hz and J2 ϭ 2.5 Hz, 1H, 2-CH), 7.06 (d, J ϭ 8.5 Hz, 1H,
1-CH); 13C NMR (Table 1); LRMS: calcd for C26H39N2O5
[M-HClϩH]ϩ 459.3, found 459.4 m/z.
N-{3-[3, 17␤-dihydroxy-estra-1, 3, 5 (10)-trien-16␤-yl]-
propyl}-2-(S)-benzyl-2-(ammonium hydrochloride)-acet-
amide (14). White solid (51% yield); IR (KBr): 3345 (br,
1
OH and NH), 1654 (CϭO, amide); H NMR (CD3OD):
0.75 (s, 3H, 18-CH3), 2.76 (m, 2H, 6-CH2), 3.08 (m, 4H,
NCH2 of 16␤-propyl and CH2Ph), 3.68 (d, J ϭ 9.8 Hz, 1H,
17␣-CH), 4.04 (t, J ϭ 7.4 Hz, 1H, NCH of phenylalanyl),
6.47 (d, J ϭ 2.6 Hz, 1H, 4-CH), 6.53 (dd, J1 ϭ 8.4 Hz and
J2 ϭ 2.6 Hz, 1H, 2-CH), 7.05 (d, J ϭ 8.4 Hz, 1H, 1-CH),
7.32 (m, 5H, Ph); 13C NMR (Table 1); LRMS: calcd for
C30H41N2O3 [M-HClϩH]ϩ 477.3, found 477.5 m/z.
N-{3-[3, 17␤-dihydroxy-estra-1, 3, 5 (10)-trien-16␤-yl]-
propyl}-2-(S)-(pyrrolinylammonium hydrochloride)-acet-
amide (15). White solid (65% yield); IR (KBr): 3290 (br,
OH and NH), 1654 (CϭO amide); 1H NMR (CD3OD): 0.75
N-{3-[3, 17␤-dihydroxy-estra-1, 3, 5 (10)-trien-16␤-yl]-
propyl}-2-(S)-(1-hydroxycarbonylpropan-3-yl)-2-{N␣-[N␤-
(ammonium hydrochloride)-L-alanyl]-L-alanyl}-acetamide
(19). White solid (90% yield); IR (KBr): 3320 (br, OH and
1
NH), 1710 (CϭO, acid), 1650 (CϭO, amide); H NMR
(CD3OD): 0.77 (s, 3H, 18-CH3), 1.38 (d, J ϭ 7.2 Hz, 3H,
CH3 of alanyl), 1.52 (d, J ϭ 7.2 Hz, 3H, CH3 of alanyl),
2.40 (qapp, J ϭ 7.6 Hz, 2H, CH2COOH), 2.75 (m, 2H,
6-CH2), 3.19 (m, 2H, NCH2 of 16␤-propyl), 3.70 (d, J ϭ
10.1 Hz, 1H, 17␣-CH), 3.93 (q, J ϭ 7.1 Hz, 1H, NCH of
alanyl), 4.37 (m, 2H, NCH of glutamyl and alanyl), 6.47 (d,

826
M.R. Tremblay et al. / Steroids 66 (2001) 821–831
Table 1
13C NMR chemical shifts (ppm) of peptidosteroids 12–20 and steroids 25 and 26*
Carbons 12 R ϭ 13 R ϭ
14 R ϭ 15 R ϭ
16 R ϭ
17 ءء R
18 R ϭ
19 ءء R ϭ
20 R ϭ 25 R ϭ 26 R ϭ
H
CH2CH(CH3)2 CH2Ph CH2CH2CH2 (CH2)4NH2 ϭ CH3
(CH2)2COOH (CH2)2COOH CH2OH NH2HCl
H
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
127.19
113.73
155.90
116.03
138.74
30.72
28.74
39.98
45.39
132.65
27.55
39.00
45.22
49.93
33.51
41.29
83.11
13.26
30.28
29.57
40.90
167.02
–
127.17
113.72
155.88
116.02
138.72
30.72
28.73
39.97
45.36
132.61
27.55
38.97
45.21
49.91
33.53
41.28
83.07
13.28
30.35
29.57
40.92
170.45
–
127.16 127.16
113.71 113.74
155.88 155.91
116.01 116.05
138.72 138.76
127.18
113.73
155.90
116.03
138.73
30.72
28.74
39.98
45.37
132.62
27.55
38.99
45.21
49.92
33.48
41.23
83.11
13.32
30.32
29.43
40.88
169.83
–
127.17
113.74
155.91
116.05
138.76
30.73
28.75
40.00
45.41
132.64
27.57
39.04
45.20
49.99
33.57
41.34
83.18
13.26
30.26
29.61
40.74
170.84
174.10
174.64
50.14
50.46
50.55
18.52
–
127.18
113.73
155.93
116.02
138.76
30.73
28.76
40.02
45.43
132.65
27.58
39.02
45.25
49.98
33.56
41.32
83.12
13.24
30.38
29.63
40.98
169.58
–
127.17
113.71
155.91
116.02
138.77
30.74
28.76
40.01
45.42
132.67
27.57
39.04
45.20
49.99
33.57
41.35
83.18
13.26
30.33
29.62
40.75
170.84
174.42
174.97
54.10
50.11
50.68
28.88
31.02
173.26
127.17
113.75
155.91
116.04
138.76
30.73
28.75
40.01
45.41
132.67
27.57
39.02
45.24
49.97
33.57
41.28
83.13
13.25
30.30
29.57
40.98
168.01
–
127.20
113.75
155.95
116.03
138.73
30.74
28.77
39.99
45.39
132.58
27.56
38.95
45.28
49.94
33.53
41.20
82.89
13.22
29.90
28.58
41.02
–
126.48
112.68
153.43
115.23
138.21
29.63
27.39
38.33
43.98
132.70
26.30
37.70
44.12
48.55
32.33
39.68
82.57
12.36
33.70
21.70
14.29
–
30.71
28.74
39.96
45.35
30.72
28.73
39.98
45.40
132.60 132.65
27.54
38.97
45.20
49.90
33.49
41.27
83.06
13.25
30.28
29.47
40.89
27.57
39.02
45.18
49.97
33.58
41.30
83.12
13.23
30.28
29.73
40.49
17
18
1Ј
2Ј
3Ј
NHCO
NHCOЈ
NHCOЉ
CHR
CHRЈ
CHRЉ
R
169.27 176.89
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
50.04
–
–
–
–
53.14
–
–
41.83
25.51
22.32
55.84
–
61.70
–
54.24
–
–
32.18
23.03
28.00
54.02
–
–
28.05
30.87
176.17
56.34
–
–
61.82
–
–
–
–
38.76
135.69
130.52
(2x)
130.03
(2x)
128.80
–
32.15
26.99
47.99
Љ
Љ
–
–
Љ
–
22.98
–
40.30
–
–
–
–
–
Љ
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
RЈ
RЉ
17.61
17.95
17.56
17.80
–
* NMR solvent: CD3OD for all compounds except 26 in CDCl3; (**) Val-Val (COCH(CH3)NHCOCH(CH3)NH2) added at the terminal NH2 group.
J ϭ 2.3 Hz, 1H, 4-CH), 6.53 (dd, J1 ϭ 8.4 Hz and J2 ϭ 2.3
Hz, 1H, 2-CH), 7.06 (d, J ϭ 8.5 Hz, 1H, 1-CH); 13C NMR
(Table 1); LRMS: calcd for C32H49N4O7 [M-HClϩH]ϩ
601.4, found 601.5 m/z.
of seryl and CH2OH), 6.47 (d, J ϭ 2.6 Hz, 1H, 4-CH), 6.54
(dd, J1 ϭ 8.4 Hz and J2 ϭ 2.6 Hz, 1H, 2-CH), 7.06 (d, J ϭ
8.4 Hz, 1H, 1-CH); 13C NMR (Table 1); LRMS: calcd for
C24H37N2O4 [M-HClϩH]ϩ 417.3, found 417.4 m/z.
N-{3-[3, 17␤-dihydroxy-estra-1, 3, 5 (10)-trien-16␤-yl]-
propyl}-2-(S)-(1-hydroxymethyl)-2-(ammonium hydrochlo-
ride)-acetamide (20). White solid (46%, two steps); IR
2.3. Synthesis of Mosher amides 21 and 24 (Scheme 2)
1
(KBr): 3320 (br, OH and NH), 1672 (CϭO, amide); H
The compound 22 was obtained using the same coupling
protocol as the compound 5, but NHBoc-D-Phe-COOH was
used instead of the corresponding L-isomer. At this step,
spectral data of 22 were identical to those of compound 5.
NMR (CD3OD): 0.77 (s, 3H, 18-CH3), 2.76 (m, 2H,
6-CH2), 3.25 (t, J ϭ 7.1 Hz, 2H, NCH2 of 16␤-propyl), 3.70
(d, J ϭ 10.4 Hz, 1H, 17␣-CH), 3.80 and 3.90 (2m, 3H, NCH

M.R. Tremblay et al. / Steroids 66 (2001) 821–831
827
Scheme 2. Reagents and conditions: (a) NHBoc-L-Phe-COOH, DCC, HOBt, CH2Cl2, 0°C; (b) 3 N HCl, MeOH, rt; (c) (S)-MTPA, DCC, HOBt, (i-Pr)2EtN,
DMF, 25°C; (d) NHBoc-D-Phe-COOH, DCC, HOBt, CH2Cl2, 0°C.
The deprotection was performed by the method A to give
compound 23 whose spectral data were again identical to
those of the compound 14. Consequently, the amino group
of each compound (14 and 23) was separately transformed
to its corresponding Mosher amide following this typical
procedure. To a cooled solution of (S)-(-)-␣-methoxy-␣-
(trifluoromethyl)-phenylacetic acid (MTPA) (3 mg; 0.015
mmol or 7 mg; 0.03 mmol) in dry DMF (300 ␮l or 500 ␮l)
at 0°C was added dicyclohexylcarbodiimide (3 mg; 0.015
mmol or 6 mg; 0.03 mmol) and 1-hydroxybenzotriazole (2
mg; 0.015 mmol or 4 mg; 0.03 mmol) and the mixture was
allowed to reach room temperature for 1 h. The reaction
mixture was cooled at 0°C before the addition of diisopro-
pylethylamine (2.5 ␮l; 0.015 mmol or 5 ␮l; 0.03 mmol)
and compound 14 or 23 (7 mg; 0.015 mmol or 16 mg;
0.03 mmol) and then the reaction was stirred at 25°C for
2 h. Afterward, water following by 1 N hydrochloric acid
solution were successively added and the crude com-
pounds were extracted three times with EtOAc. The com-
bined organic layers were washed with a saturated
NaHCO3 solution, water, brine, and dried over MgSO4.
After evaporation of the solvent, the crude compounds
were purified by flash chromatography (hexanes:EtOAc,
1:1) to give respectively the amides 21 (10 mg, 98%
yield) and 24 (12 mg; 55%). Only pertinent 1H NMR
(CDCl3) spectral data are reported. For 21: 4.66 (dd of
CH␣-amide), 3.05 (m of CH2Ph) and 3.29 (s of OCH3).
For 24: 4.57 (dd of CH␣-amide), 3.11 (m of CH2Ph) and
3.23 (s of OCH3). All signals were significantly different
when both compounds 21 and 24 were mixed together in
the same NMR tube.
2.4. Preparation of 16␤-(aminopropyl)-E2 (25) and 16␤-
(propyl)-E2 (26) (Table 2)
3-(3, 17␤-dihydroxy-estra-1, 3, 5 (10)-trien-16␤-yl)-pro-
pylamine hydrochloride (25). A solution of amine 2 (100
mg; 0.19 mmol) in 5 ml of MeOH containing 2% (v/v) of
hydrochloric acid was stirred for 3 h at room temperature.
Then, solvents were evaporated to dryness to give 68 mg
(98% yield) of compound 25. White solid; IR (KBr): 3250
1
(br, OH and NH2); H NMR (CD3OD): 0.78 (s, 3H, 18-
CH3), 2.75 (m, 2H, 6-CH2), 2.93 (m, 2H, NCH2 of 16␤-
propyl), 3.73 (d, J ϭ 9.8 Hz, 1H, 17␣-CH), 6.46 (d, J ϭ 2.3
Hz, 1H, 4-CH), 6.53 (dd, J1 ϭ 8.4 Hz and J2 ϭ 2.4 Hz, 1H,
2-CH), 7.06 (d, J ϭ 8.5 Hz, 1H, 1-CH); 13C NMR (Table 1);
LRMS: calcd for C21H32NO2 [M-HClϩH]ϩ 330.2, found
330.4 m/z.
16␤-propyl-estra-1, 3, 5 (10)-triene-3, 17␤-diol (26). A
solution of 3-t-butyldimethylsilyloxy-17␤-(tetrahydro-2H-
pyran-2-yl-oxy)-16␤-allyl-estra-1, 3, 5 (10)-triene [32] (5.3
g; 10.3 mmol) in 215 ml of MeOH/THF (93:7) containing
2% (v/v) of hydrochloric acid was stirred for 5 h at room
temperature. Then, water was added and organic solvents
were evaporated. The crude compound was extracted with
EtOAc (3X) and the combined organic was washed with
brine, dried over MgSO4, and concentrated to dryness. To a
solution of the crude alkene (1.2 g) in MeOH (100 ml) was

828
M.R. Tremblay et al. / Steroids 66 (2001) 821–831
Table 2
Biological activities of 16␤-propylaminoacyl derivatives of E2 (12–20) and compounds 25 and 26 as revealed by the inhibition of type 1 reductive 17␤-
HSD activity and the binding affinity on four steroid receptors
Compounds
Amino acyl
derivative
Inhibition (%)
Type 1 17␤-HSD
(E1 to E2)
Binding affinity (%)
AR
PR
GR
ER
0.1 ␮M
1.0 ␮M
10 nM
1.0 ␮M
10 nM
1.0 ␮M
10 nM
1.0 ␮M
10 nM
1.0 ␮M
12
13
14
15
16
17
18
19
Gly
Leu
Phe
Pro
Lys
Ala-Ala-Ala
Glu
Glu-Ala-Ala
0
0
16
0
0
4
3
0
0
1
7
24
-
-
-
0
0
6
0
0
16
5
6
3 Ϯ 2
3 Ϯ 2
7 Ϯ 1
6 Ϯ 2
2 Ϯ 2
8 Ϯ 1
5 Ϯ 1
6 Ϯ 2
6 Ϯ 1
0 Ϯ 2
5 Ϯ 1
-
6 Ϯ 2
6 Ϯ 2
8 Ϯ 3
5 Ϯ 1
0 Ϯ 1
7 Ϯ 3
9 Ϯ 2
6 Ϯ 2
3 Ϯ 4
1 Ϯ 2
4 Ϯ 2
-
0 Ϯ 2
0 Ϯ 2
1 Ϯ 2
0 Ϯ 3
0 Ϯ 3
0 Ϯ 2
0 Ϯ 4
0 Ϯ 2
0 Ϯ 2
0 Ϯ 2
0 Ϯ 1
-
0 Ϯ 2
0 Ϯ 2
0 Ϯ 2
1 Ϯ 2
0 Ϯ 2
0 Ϯ 3
1 Ϯ 2
0 Ϯ 2
0 Ϯ 2
0 Ϯ 1
6 Ϯ 2
-
0 Ϯ 4
0 Ϯ 3
0 Ϯ 4
0 Ϯ 3
0 Ϯ 3
0 Ϯ 2
0 Ϯ 4
1 Ϯ 4
3 Ϯ 3
0 Ϯ 3
0 Ϯ 3
-
0 Ϯ 3
1 Ϯ 4
1 Ϯ 3
0 Ϯ 3
0 Ϯ 3
0 Ϯ 5
5 Ϯ 3
0 Ϯ 4
0 Ϯ 3
0 Ϯ 3
0 Ϯ 3
-
0 Ϯ 2
0 Ϯ 1
0 Ϯ 2
0 Ϯ 2
1 Ϯ 1
0 Ϯ 2
0 Ϯ 1
1 Ϯ 4
0 Ϯ 2
0 Ϯ 2
2 Ϯ 1
-
5 Ϯ 1
6 Ϯ 3
6 Ϯ 1
5 Ϯ 1
5 Ϯ 1
6 Ϯ 2
12 Ϯ 1
5 Ϯ 4
5 Ϯ 1
12 Ϯ 1
69 Ϯ 1
-
20
Ser
0
25*
26**
E1
DHT
R5050
DEX
E2
-
-
-
-
-
-
-
22
48
55
-
-
-
70 Ϯ 1
1 Ϯ 4
0 Ϯ 1
0 Ϯ 2
100 Ϯ 1
28 Ϯ 2
2 Ϯ 1
34 Ϯ 1
3 Ϯ 2
65 Ϯ 2
0 Ϯ 3
6 Ϯ 3
40 Ϯ 2
99 Ϯ 2
1 Ϯ 2
25 Ϯ 2
2 Ϯ 2
9 Ϯ 2
66 Ϯ 2
5 Ϯ 2
6 Ϯ 2
85 Ϯ 2
99 Ϯ 1
12 Ϯ 2
2 Ϯ 2
5 Ϯ 2
0 Ϯ 3
75 Ϯ 1
4 Ϯ 1
5 Ϯ 2
0 Ϯ 1
100 Ϯ 1
-
-
* Compound 25: 16␤-(aminopropyl)-E2; **Compound 26: 16␤-(propyl)-E2; Abbreviations of steroids used as standard: DHT: dihydrotestosterone; R5050:
synthetic progestin; DEX: dexamethasone; E2: estradiol; E1: estrone.
added 5% palladium on activated carbon (200 mg). The
resulting mixture was stirred under hydrogen atmosphere at
25°C for 20 h. The slurry was filtered through a celite pad
eluting with MeOH and EtOAc. The organic solvent was
evaporated under reduced pressure. Purification by flash
chromatography (hexanes:EtOAc, 85:15) gave 1.0 g (31%
yield, two steps) of the compound 26. White solid; IR
(KBr): 3466 (br, OH); 1H NMR (CDCl3): 0.78 (s, 3H,
18-CH3), 0.93 (t, J ϭ 7.1 Hz, 3H, CH3 of propyl), 2.80 (m,
2H, 6-CH2), 3.75 (d, J ϭ 10 Hz, 1H, 17␣-CH), 6.56 (d, J ϭ
2.3 Hz, 1H, 4-CH), 6.62 (dd, J1 ϭ 8.4 Hz and J2 ϭ 2.5 Hz,
1H, 2-CH), 7.15 (d, J ϭ 8.4 Hz, 1H, 1-CH); 13C NMR
(Table 1); LRMS: calcd for C21H34NO2 [MϩNH4]ϩ 332.3,
found 332.4 m/z.
mixture was incubated for 1 h at 37°C, and the reaction
was stopped by adding an excess of unlabeled E1 and E2.
Steroids were extracted with diethyl ether and solvent
was removed under reduced pressure. The residue was
dissolved in CH2Cl2, spotted on a silica gel plate (TLC,
20 cm ϫ 20 cm ϫ 0.2 cm, Kieselgel 60 F254) and eluted
with CH2Cl2/EtOAc (9:1). Less polar E1 and more polar
E2 were identified on TLC as two rows of visible spots
under UV light. Radioactivity signals associated to [14C]-
E1 and [14C]-E2 were detected and quantified using a
Phosphor Imager (Sunny Vale, CA). The percentage of
transformation of [14C]-E1 into [14C]-E2 was calculated
as follows: % trans. ϭ 100 ϫ [14C]-E2 (cpm) / ([14C]-E1
(cpm) ϩ [14C]-E2 (cpm)). Subsequently, the percentage
of inhibition ϭ 100 ϫ [(% trans. of control Ϫ % trans. of
compound)/ (% trans. of control)].
2.5. Inhibition of type 1 17␤-HSD (Table 2)
This enzymatic assay on crude preparation of type 1
17␤-HSD was performed as previously described [22].
Briefly, Human Embryonic Kidney (HEK)-293 cells
transfected with cDNA encoding for type 1 17␤-HSD
were sonicated to liberate the crude enzyme that was used
as the enzymatic pool without further purification. The
enzymatic assay was performed as follows: a stock solu-
tion was first prepared containing the radiolabeled sub-
strate [14C]-E1 (0.1 ␮M), NADH (1 mM) in a phosphate
buffer (pH 7.4, 50 mM KH2PO4, EDTA 1 mM, 20%
glycerol). For the assay, 890 ␮l of the stock solution and
10 ␮l of a solution of inhibitor dissolved in EtOH were
added in a tube. The reaction was started by adding 100
␮l of a solution of crude enzyme prepared as above. The
2.6. Steroid receptor binding affinity screening (Table 2)
The affinity binding assays on estrogen and progestin
receptors from rat uterine were carried out under the stan-
dard procedure established in our laboratory [34]. Assays
for androgen receptor from rat ventral prostate were per-
formed according to the procedure described by Luo and
co-workers [35]. For binding assay on glucocorticoid recep-
tor from rat liver, a slightly modified version of the proce-
dure described by Asselin and co-workers was used [36]. In
this case, separation of bound and free steroids was
achieved with dextran-coated charcoal adsorption instead of
protamine sulfate precipitation.

M.R. Tremblay et al. / Steroids 66 (2001) 821–831
829
3. Results
Type 1 17␤-HSD has a strong preference towards C18-
cedure was not appropriate when the side-chain contained a
t-butyl ester (compounds 9 and 10). We then tried 3 N HCl
in EtOAc, but again deprotected compound was contami-
nated with about 15% of 17␤-OAc from transesterification
of solvent. Consequently, we used anhydrous HCl in
CH2Cl2 to produce glutamyl derivatives of estradiol (com-
pounds 18 and 19). Finally, we obtained the seryl derivative
20 from 11 by a two-step deprotection procedure using first
3 N HCl in MeOH, and then hydrogenolysis of the benzyl
ether group under pressure.
steroid substrates compare to C19-steroids like andro-
stenedione and dehydroepiandrosterone [22,37]. Steroidal
inhibitors should obviously contain a C18-steroid nucleus as
E2 to bind to the substrate binding domain. Moreover, we
wanted to attach the additional interacting polar group on
the D-ring because the reaction site and the cofactor binding
domain surround this part of E2 molecule. Based on our
previous studies [38–40], a 16␤-propyl side-chain was used
as a spacer group between the E2 moiety and the amino acyl
group (Fig. 1). The polar nature of NADH or NADPH
cofactor motivates us to choose polar chemical groups.
Because of their polarity, molecular diversity, chirality,
structural complementary with protein, and accessibility,
amino acids are excellent building blocks. Representatives
of all groups of amino acid residues (aliphatic [Gly, Leu],
aromatic [Phe], structural constrained [Pro], basic [Lys],
acid [Glu], alcohol [Ser] as well as two tripeptides [Glu-
Ala-Ala, Ala-Ala-Ala]) were thus added to the E2 derivative
(compounds 12–20) to probe different areas of the cofactor
binding domain. The natural L-series of amino acids were
arbitrarily chosen. Two additional control molecules, with-
out an amino acid residue, were used as control to evaluate
the influence of amino acyl residues: 16␤-aminopropyl-E2
(25) and 16␤-propyl-E2 (26).
All compounds were purified by trituration in diethyl
ether to yield pure solid characterized by IR, 1H NMR, 13
C
NMR (Table 1) and mass spectrometry. Furthermore, opti-
cal purity of the ␣-carbon center was proved by the synthe-
sis of one representative of D-series amino acid as well as
the Mosher amides of L-Phe and D-Phe derivatives (Scheme
1
2). Comparison of H NMR spectra established that, as
expected, no racemization occurred during the coupling
reaction and the subsequent deprotection. Finally, 16␤-
(aminopropyl)-E2 (25) was obtained from compound 2 after
hydrolysis of TBDMS and THP protecting groups while
16␤-(propyl)-E2 (26) was synthesized from an allyl inter-
mediate [32] after removal of TBDMS and THP protecting
groups and reduction of double bond. Both compounds
were used as references to evaluate the biological impor-
tance of the amino acid moiety.
3.2. Biological evaluation
3.1. Chemical synthesis
The 16␤-propylaminoacyl derivatives of E2 (12–20)
were tested on two systems to evaluate either their ability to
inhibit the formation of E2 from E1 by type 1 17␤-HSD or
their affinities on steroid receptors according to established
procedures (Table 2) [22,34–36]. An enzymatic screening
test was performed using homogenated HEK-293 cells
transfected with cDNA encoding for type 1 17␤-HSD. It
revealed that none of the added amino acids on the E2
nucleus provided a good inhibitory potency on the reductive
activity of type 1 17␤-HSD. In fact, the 16␤-propyl-E2 (26)
displayed a better inhibitory effect than the 16␤-aminopro-
pyl-E2 (25), which was also better than all the 16␤-pro-
pylaminoacyl derivatives of E2 (12–20). The ammonium
group seems to interact unfavorably with the enzyme and
none of the amino acid residue can overcome this effect.
Affinities toward steroid receptors of all 16␤-(propylamino-
acyl) derivatives of E2 (12–20) were also evaluated. This
biological data is very valuable to detect eventual com-
pounds lacking selectivity, which can interact with other
proteins that bind to steroids and provoke undesired effects
in more complex biological systems. All compounds did not
bind significantly to androgen, progestin, and glucocorticoid
receptors. No significant binding affinities were also ob-
served for all tested compounds with the estrogen receptor,
except for the 16␤-propyl-E2 (26), which displayed a better,
but unsuitable, affinity for this receptor. As an interesting
consequence, the addition of an amino acid residue to es-
A well-known synthetic sequence described by our lab-
oratory was used to generate the azide intermediate 1
(Scheme 1) [32,41]. Thereafter, the reduction of the azide
using palladium-catalyzed hydrogenation yielded the amine
precursor 2 that was used for N-Boc protected amino acid
coupling according to the Ko¨nig and Geiger procedure [33],
which gave the amides 3–11. For the synthesis of the pep-
tidosteroids 8 and 10, the tripeptide building blocks NH-
Boc-Ala-Ala-Ala-COOH and NHBoc-Glu-Ala-Ala-COOH
were obtained using standard synthetic methods [42,43].
Modest yields obtained in the coupling reaction could be
attributed to steric hindrance, partial deprotection of the
phenolic TBDMS group under the reaction conditions, and
difficulty during the purification step. The t-butoxy carbam-
ate (t-Boc)-protected amino acids were chosen because the
precursor 2 had already two other acid-labile protecting
groups (THP, TBDMS).
According to the side-chain amino acid residue, three
kinds of acidic conditions were used to yield the target
compounds 12–20. It is noteworthy that usual conditions for
t-Boc cleavage with trifluoroacetic acid led to several side-
products such as 17␤-O-trifluoroacetate derivatives. Thus,
protected compounds 3–8 bearing alkyl (Gly, Leu, Pro,
Ala-Ala-Ala), benzyl (Phe), or basic (Lys) were submitted
to 3 N HCl in MeOH to produce fully deprotected com-
pounds 12–17. Because of transesterification, the latter pro-

830
M.R. Tremblay et al. / Steroids 66 (2001) 821–831
tradiol drastically reduced its affinity for the estrogen re-
ceptor.
charged polar group. This approach is under current inves-
tigation in our laboratory. Secondly, the 16␤-propyl side-
chain could be further lengthened to prevent the postulated
negative interaction of functional groups with the Lys-159,
which seems to surround the extremity of the propyl side-
chain. The functional groups at the end of a longer side-
chain could hopefully interact with other areas of the co-
factor binding domain. However, this exploratory study has
revealed the complexity of maximizing interactions with the
cofactor binding domain. Consequently, we are currently
developing a combinatorial chemistry program that should
allow us to investigate the hybrid-molecule prototype with
much more power [32,41,44,45]. The present study has
revealed that, although amino acids are interesting building
blocks, they may not be ideally suited for our biological
system. Therefore, the present study help us to re-orientate
the future design of libraries of such hybrids as type 1
17␤-HSD inhibitors without estrogenic properties.
4. Discussion
We have described the preparation of a series of 16␤-
propylaminoacyl derivatives of E2 (12–20) containing several
kinds of functional groups such as alkyl, aryl, carboxylic acid,
alcohol, amine, and amides. All the newly synthesized com-
pounds showed very low inhibitory potencies on type 1 17␤-
HSD activity. This observation means that whatever the sub-
stitution made on the starting molecule, no specific binding on
the enzyme was created by this substitution. Moreover, all the
substitutions led to clearly unfavorable interactions with the
enzyme as witnessed by the decreasing inhibitory effect of the
16␤-propyl-E2 (26) Ͼ 16␤-(aminopropyl)-E2 (25) ϾϾ 16␤-
(propylaminoacyl)-E2 (12–20). Two major factors can proba-
bly explain these results. The ammonium group, which is
present in all inactive molecules, might surround that of the
Lys-159 of the enzyme. If so, none of the amino acid residues
introduced in ␣ of this ammonium succeeded in changing the
conformation of the molecule to provide any point of contact
into the enzyme. Particularly, the carboxylate groups of com-
pounds 18 and 19 were initially thought to interact with the
ammonium group of the Lys-159, but it seems that the con-
formation of the molecule into the enzyme did not allow this
salt bridge. This same situation was observed with the seryl
group whose hydroxy group could have been the mimic of the
hydroxy group of the cofactor ribose moiety, which is known
to form a hydrogen bond with the Lys-159 [28–31]. The other
factor that may contribute to the observed unfavorable binding
properties of these compounds is the amide bond(s).
Conformationally pseudo-rigid structures that can be
introduced by amide bond might expose the functional groups
of the molecule at the wrong place in the enzyme pocket. In
addition, the presence of one and more amide bonds can also
direct the ammonium group of the molecule toward that of the
Lys-159 in the enzyme. Therefore, the combination of these
two negative effects may be the explanation of why these
compounds were inactive in inhibiting type 1 17␤-HSD.
These 16␤-propylaminoacyl derivatives of E2 (12–20) did
not show any affinity toward androgen, progestin, and glu-
cocorticoid receptors. Interestingly, the addition of the amino
acyl group led to E2 derivatives possessing greatly reduced
binding properties to estrogen receptor. While the binding
affinities of 16␣-substituted E2 derivatives were extensively
studied [19], the properties of 16␤-substituted E2 derivatives
remained unknown. Our results demonstrated that polar and
moderate size substituents are not tolerated by the estrogen
receptor. These results are in agreement with the overall ob-
servation that E2 derivatives bearing polar substituents gener-
ally show low binding affinity on the estrogen receptor [19].
In the light of this study, the prototype chemical structure
can be modified to ultimately obtain a lead structure. Firstly,
the amino group should be changed for a non positively
Acknowledgments
We thank the Medical Research Council (MRC) of Can-
ada and Le Fonds de la recherche en sante´ du Que´bec
(FRSQ) for operating grants. M.R.T is holder of a NSERC
and FCAR scholarships. We are grateful to Mr. Gilles
Leblanc for providing biological evaluation on steroid re-
ceptors. We also thank Dr. Fernand Labrie and the Division
of Medicinal Chemistry for providing chemical facilities.
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