N-butyl-N-methyl-11-(3'-hydroxy-21',17'-carbolactone-19'-nor-17'α- pregna-1',3',5'(10')-trien-7'α-yl)-undecanamide: An inhibitor of type 2 17β-hydroxysteroid dehydrogenase that does not have oestrogenic or androgenic activity

Article abstract of DOI:10.1016/S0223-5234(00)00124-0

It is well known that 17β-hydroxysteroid dehydrogenases (17β-HSDs) play a key role in the formation and inactivation, from circulating precursors, of several active androgens and oestrogens. These enzymes can thus regulate tumoural cell proliferation in androgen- and oestrogen- dependent cancers. Recently, we discovered that adding a spiro-γ-lactone to the oestradiol nucleus results in a novel inhibitor of type 2 17β-HSD, an enzyme that catalyses the interconversions between 4-androstene-3,17-dione and testosterone, and between oestrone and oestradiol. This finding motivated our introducing the spiro-γ-lactone moiety onto an anti-oestrogenic nucleus. The N-butyl-N-methyll-1-(3'-hydroxy-21',7'-carbolactone-19'-nor-17'α-pregna- 1',3',5'(10')-trien-7'α-yl)-undecanamide (4) was then efficiently synthesized and its biological activity was assessed in vitro. Despite the presence of a bulky alkylamide side chain, the spiro-γ-lactone function conserved its ability to inhibit type 2 17β-HSD (IC₅₀ = 0.35 and 0.25 μM, with and without side chain, respectively). Furthermore, the selective inhibition by lactone 4 toward type 2 17β-HSD (microsomal fraction of human placenta) was demonstrated by the absence of inhibitory activity toward type 1 17β-HSD (cytosolic fraction of human placenta). Cell proliferation assays indicated that compound 4 had no oestrogenic activity but did show anti- oestrogenic activity on ER⁺ cell line ZR-75-1. No androgenic activity could be detected when assayed on the AR⁺ cell line Shionogi either. Based on these facts, we report the synthesis of a new steroidal derivative, one that inhibits type 2 17β-HSD while possessing anti-oestrogenic activity. (C) 2000 Editions scientifiques et medicales Elsevier SAS.

Full text of DOI:10.1016/S0223-5234(00)00124-0

Eur. J. Med. Chem. 35 (2000) 217−225
© 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved
S0223523400001240/FLA
217
Original article
N-Butyl-N-methyl-11-(3'-hydroxy-21',17'-carbolactone-19'-nor-17'α-pregna-
1',3',5'(10')-trien-7'α-yl)-undecanamide: an inhibitor of type 2
17â-hydroxysteroid dehydrogenase that does not have oestrogenic
or androgenic activity
Kay-Mane Sam, Fernand Labrie, Donald Poirier*
Medicinal Chemistry Division, Laboratory of Molecular Endocrinology, Laval University Medical Research Center, Centre hospitalier
universitaire de Québec and Laval University, 2705 Laurier Boulevard, Québec G1V 4G2, Canada
Received 30 May 1999; revised 28 July 1999; accepted 5 August 1999
Abstract – It is well known that 17â-hydroxysteroid dehydrogenases (17â-HSDs) play a key role in the formation and inactivation, from
circulating precursors, of several active androgens and oestrogens. These enzymes can thus regulate tumoural cell proliferation in androgen-
and oestrogen-dependent cancers. Recently, we discovered that adding a spiro-γ-lactone to the oestradiol nucleus results in a novel inhibitor
of type 2 17â-HSD, an enzyme that catalyses the interconversions between 4-androstene-3,17-dione and testosterone, and between oestrone
and oestradiol. This finding motivated our introducing the spiro-γ-lactone moiety onto an anti-oestrogenic nucleus. The N-butyl-N-methyl-
11-(3'-hydroxy-21',17'-carbolactone-19'-nor-17'α-pregna-1',3',5'(10')-trien-7'α-yl)-undecanamide (4) was then efficiently synthesized and its
biological activity was assessed in vitro. Despite the presence of a bulky alkylamide side chain, the spiro-γ-lactone function conserved its
ability to inhibit type 2 17â-HSD (IC50 = 0.35 and 0.25 µM, with and without side chain, respectively). Furthermore, the selective inhibition
by lactone 4 toward type 2 17â-HSD (microsomal fraction of human placenta) was demonstrated by the absence of inhibitory activity toward
type 1 17â-HSD (cytosolic fraction of human placenta). Cell proliferation assays indicated that compound 4 had no oestrogenic activity but
did show anti-oestrogenic activity on ER+ cell line ZR-75-1. No androgenic activity could be detected when assayed on the AR+ cell line
Shionogi either. Based on these facts, we report the synthesis of a new steroidal derivative, one that inhibits type 2 17â-HSD while possessing
anti-oestrogenic activity. © 2000 Éditions scientifiques et médicales Elsevier SAS
hydroxysteroid dehydrogenase / enzyme / inhibitor / anti-oestrogen / steroid / lactone
1. Introduction
size inhibitors of 17â-HSD that do not have agonistic
activity [7, 16]. In this report, we focus on type 2 17â-
HSD, which is the only one of five known human
17â-HSD isoforms [1, 17] that catalyses the transforma-
tion of both androgenic (C19-steroids) and oestrogenic
(C18-steroids) substrates (figure 1) [1–3, 18, 19]. In fact,
type 2 17â-HSD catalyses the interconversion of DHEA,
∆4-dione and E1 into the ∆5-diol, T and E2, respectively.
In intact cells, however, it was reported that type 2 prefers
the oxidative process, contrary to types 1, 3 and 5, which
prefers the reductive one [20–22]. Despite the growing
interest in type 2 17â-HSD [16–33], the role of this
isoform remains to be clarified and the development of its
specific inhibitors should prove useful.
The 17â-hydroxysteroid dehydrogenases, or 17â-
HSDs, play a pivotal role in the formation and inactiva-
tion of active androgens and oestrogens from circulating
steroid precursors [1–4]. During the past few years we
have been developing inhibitors of human placenta cyto-
solic 17â-HSD (type 1) [5–7] and inhibitors of human
placenta microsomal 17â-HSD (type 2) [8, 9], but the
C18-steroid nucleus limits the therapeutic use of these
inhibitors because of its oestrogenic activity. Our group
has concentrated much effort toward the development of
new weapons to fight oestrogen- and androgen-dependent
diseases [10–15], and one of our challenges is to synthe-
Based on our previous observation that an oestradiol
nucleus bearing a spiro-γ-lactone at position 17 (com-
*Correspondence and reprints: donald.poirier@crchul.ulaval.ca

218
erative and antiproliferative activities on oestrogen-
sensitive (ER+) ZR-75-1 cells, and the proliferative
activity on androgen-sensitive (AR+) Shionogi cells.
2. Chemistry
The six-step synthesis of spiro-γ-lactone 4 is shown in
figure 3. The starting compound 5, prepared according to
known methodology [36], was oxidized with 2.7 M Jones
reagent to give the corresponding carboxylic acid which,
upon treatment with tributyl amine, isobutylchlorofor-
mate, and N-methyl, N-butyl amine, yielded 73% of
amide 6 (two steps). In the process, the 17â-hydroxyl
1
group was oxidized to a ketone. Results of H-NMR
spectroscopy demonstrated that the methyl of amide
group (CONCH3) appeared as two singlets at 2.90 and
2.95 ppm, while the methylene (CONCH2) exhibits two
triplets at 3.24 and 3.35 ppm. Moreover, the CH3 of the
N-butyl group (two triplets at 0.92 and 0.94 ppm) as well
as other protons surrounding the amide (but masked by
steroidal protons) were also duplicated. The duplication
of these NMR signals can be explained by the two
conformations of the amide bond and is typical of such
alkylamide compounds [37]. Further evidence of the
formation of amide 6 was observed in 13C-NMR spec-
troscopy [38].
According to the Salman procedure [39], compound 6
was alkylated at position 17 with tetrahydro-2-
(propynyloxy)-2H-pyran and n-BuLi to give compound 7
in 75% yield. Under these conditions the benzoate (Bz)
group in position 3 was cleaved to generate a phenolic
group. In addition to the attack on the ester group the
lithium acetylenide derivative attacked the C17 carbonyl
by the more accessible steroidal α-face. In this case, the
angular CH3-18 (on steroidal â-face) accounted for the
high stereoselectivity of the alkylation that limited syn-
thesis to that of the 17α-side chain derivative. The triple
bond of compound 7 was then subjected to catalytic
hydrogenation with a 1:1 mixture of 10% Pd/C and 5%
Figure 1. Enzymatic transformations performed by different
isoforms (types 1–5) of human 17â-hydroxysteroid dehydroge-
nase (17â-HSD). DHEAS: dehydroepiandrosterone sulfate;
DHEA: dehydroepiandrosterone; ∆5-diol: 5-andosten-3,17â-
diol; ∆4-dione: 4-androstene-3,17-dione; T: testosterone; E1:
oestrone; E2: oestradiol.
pound 1, figure 2) inhibits type 2 17â-HSD [8, 9] and that
this isoenzyme can accommodate certain types of bulky
7α-side chain [16], we decided to add the same spiro-γ-
lactone (the inhibiting group) to an anti-oestrogenic
nucleus such as 2. Indeed, it is well known that the long
methyl butyl alkanamide side chain at position 7α of
oestradiol plays an essential role in the anti-oestrogenic
activity of compounds 2 [34–36] and 3 [12]. Thus, the
target compound 4 was suspected to inhibit type 2 17â-
HSD, but without residual oestrogenic activity. The
spiro-γ-lactone
4
(N-butyl-N-methyl-11-(3'-hydroxy-
21',17'-carbolactone-19'-nor-17'α-pregna-1',3',5'(10')-trien-
7'α-yl)-undecanamide) was then efficiently synthesized
and evaluated using different in vitro assays; namely the
inhibition of human placental 17â-HSD type 2 (microso-
mal fraction) and type 1 (cytosolic fraction), the prolif-
Figure 2. Chemical structures of type 2 17â-HSD inhibitors (1 and 4) and pure anti-oestrogens (2 and 3).

219
Figure 3. Chemical synthesis of lactone 4 from starting alcohol 5.
Pd/CaCO3 in ethyl acetate to produce the saturated
compound 8 in 92% yield. The amide 8 was then treated
with amberlyst 15 ion-exchange acid resin in methanol to
cleave the THP group. The crude alcohol was oxidized
with 2.7 M Jones reagent to yield the corresponding
carboxylic acid, which underwent cyclization with the
17â-OH group, resulting in the more stable γ-lactone
function (compound 4). A 48% yield was obtained for this
of lactone 4, lactone 1 or ∆4-dione (non-tritiated sub-
strate) were used to obtain the inhibition curves. As
indicated in figure 4 a total inhibition of type 2 17â-HSD
(microsomal fraction) was obtained at a concentration of
10 µM with an IC50 value of 0.35 µM for lactone 4. This
IC50 value was better than the IC50 value of the non-
tritiated substrate itself (1.27 µM for ∆4-dione). Interest-
ingly, the presence of a bulky alkylamide side chain
slightly decreased the ability of the spiro-γ-lactone group
to inhibit type 2 17â-HSD. Indeed, IC50 values of 0.35
and 0.25 µM were obtained, respectively, for lactones 4
and 1 (with and without an alkylamide side chain). The
17â-HSD activity in the cytosolic fraction of human
placenta (type 1 17â-HSD) was also used to evaluate the
ability of lactone 4 to inhibit the transformation of
oestrone to oestradiol. No inhibition of type 1 17â-HSD
was observed with lactone 4 at either of the two concen-
trations used (0.1 and 1 µM). At these same concentra-
tions the previously reported inhibitor of type 1 17â-
HSD, 16α-(bromopropyl)-estradiol [7, 40], inhibited 9%
and 66% of the enzyme activity, respectively.
1
last sequence of reactions. The IR, H-NMR, 13C-NMR,
HRMS, and CHN analyses were in agreement with the
chemical structure of spiro-γ-lactone 4.
3. Biological results
3.1. Inhibition of 17â-HSD
The 17â-HSD activity found in the microsomal frac-
tion of human placenta (type 2 17â-HSD) was used to
evaluate the ability of lactone 4 to inhibit the transforma-
tion of 4-androstenedione (∆4-dione) into testosterone
(T) [9]. In our enzymatic assay, increasing concentrations

220
tion of the E2-induced stimulation of ZR-75-1 cells
enabled us to assess the anti-oestrogenic activity of
lactone 4. As depicted in figure 5B, at a low concentration
of lactone 4 (0.03 µM), no significant anti-oestrogenic
effect was observed. However, at a higher concentration
(1 µM) an important anti-oestrogenic effect was obtained.
In fact, at the latter concentration lactone 4 reversed the
oestrogenic effect induced by 0.1 nM of oestradiol by
87%. In comparison, the pure anti-oestrogen EM-139
(compound 3, figure 2) [12] fully inhibited the E2 oestro-
genic effect induced at 1 µM.
3.3. Androgenic activity
An androgen-sensitive (AR+) human cancer cell line
(Shionogi) was used to evaluate the ability of lactone 4 to
stimulate the proliferation of cells (androgenic activity).
As can be seen in figure 6, no androgenic or proliferative
effect was observed for lactone 4 at either concentration
used (0.01 and 1 µM). Similar results were also obtained
for hydroxy-flutamide (OH-FLU), the active metabolite
of the widely used pure anti-androgen flutamide [42].
Conversely, a 3-fold proliferative effect was obtained
when Shionogi cells were stimulated by 0.1 nM of a
known androgen, dihydrotestosterone (DHT).
Figure 4. Effect of increasing concentrations of androst-4-en-
3,17-dione (∆4-dione), lactone 1 and lactone 4 on enzymatic
activity of type 2 17â-HSD (human placental microsomes). The
enzyme preparation was incubated for 60 min at 37 °C in a pH
7.4 phosphate buffer containing 3.2 nM of [3H]∆4-dione,
NADH in excess and the indicated concentration of inhibitor. In
this assay, the interfering aromatase activity was selectively
blocked by an inhibitor as previously described [9]. The results
are expressed as a percent of transformation of [3H]∆4-dione to
[3H]testosterone. The IC50 values are 1.27, 0.25 and 0.35 µM,
respectively, for ∆4-dione, lactone 1, and lactone 4. Data points
represent the mean values ± SEM from triplicate incubations.
4. Discussion
The successful and efficient synthesis of an oestradiol
derivative with a 17-spiro-γ-lactone and a 7α-alkylamide
side chain (lactone 4) was reported in figure 3. Starting
from alcohol 5, a six step sequence (Jones oxidation,
amidation, alkylation at 17α-position, catalytic hydroge-
nation of the triple bond, cleavage of THP group and
oxidation) provided a good overall yield (24%) of lactone
4. All synthetic intermediates, in addition to the final
lactone 4, were fully characterized by conventional meth-
1
ods (IR, H-NMR, 13C-NMR, and HRMS). This novel
3.2. Oestrogenic and anti-oestrogenic activities
spiro-γ-lactone derivative was found to selectively inhibit
type 2 17â-HSD (no inhibition was observed toward type
1 17â-HSD) due mostly to the presence of the spiro-γ-
lactone group. Furthermore, the bulky alkylamide side
chain at position 7α of the steroidal nucleus blocked the
oestrogenic activity associated with lactone 1, thus con-
ferring anti-oestrogenic activity. Inhibition of type 2 17â-
HSD by lactone 4 was demonstrated by its IC50 value of
0.35 µM. In the absence of the 7α-alkylamide chain, the
corresponding lactone 1 had an IC50 value of 0.25 µM.
Both lactones (1 and 4) fit the enzymatic pocket better
than did the substrate itself, as indicated by the IC50 value
of 1.27 µM obtained when non-tritiated ∆4-dione was
used to compete with tritiated ∆4-dione. We have already
The ability of lactone 4 to stimulate the proliferation
(oestrogenic effect) and/or to inhibit the 0.1 nM E2-
induced proliferation (anti-oestrogenic effect) of
oestrogen-sensitive (ER+) human breast cancer ZR-75-1
cells was evaluated as previously reported [41]. As shown
in figure 5A, lactone 4 was tested at two concentrations
(0.03 and 1 µM) and no proliferative effect was observed
at either concentration when lactone 4 was used alone.
We can see that a 0.1 nM concentration of oestradiol
provided a 2.4-fold stimulation of basal cell proliferation,
while lactone 4 showed no proliferative effect at concen-
trations 300-fold and even 10 000-fold higher than the
oestradiol reference concentration (0.1 nM). The inhibi-

221
Figure 5. Effects of lactone 4 and pure anti-oestrogen EM-139 [12] on the proliferation (oestrogenic activity) and the inhibition of
oestradiol (E2)-induced proliferation (anti-oestrogenic activity) of oestrogen-sensitive human breast cancer ZR-75-1 cells. Three days
after plating, cells were incubated for 9 days with the incubated concentrations of test compound in the absence (A) or presence (B)
of E2 (0.1 nM). Media were changed every second day. Results were expressed as the mean of DNA content (µg) ± SEM, in triplicate
analysis.
reported that lactone 1 acts as a reversible inhibitor [9]
and we expect that lactone 4 with its spiro-γ-lactone will
share this same mechanism. In addition to its capacity to
inhibit the formation of testosterone from ∆4-dione,
lactone 4 did not exhibit androgenic activity.
The data presented here demonstrated that lactone 4 is
an inhibitor of type 2 17â-HSD that exerts neither oestro-
genic nor androgenic activities and can be considered as
an anti-oestrogen. The further development of an inhibi-
tor of human placenta microsomal 17â-HSD (type 2),
that is at the same time an anti-oestrogen, will certainly
advance the elucidation of the physiological role and
action of this 17â-HSD isoform.
Gel 60 F254 plates (E. Merck, Darmstadt, Germany) while
a 230–400 mesh ASTM Silica Gel 60 (E. Merck) was
used for flash column chromatography. Infrared spectra
(IR) obtained using Perkin Elmer 1600 (FTIR Series)
spectrophotometer were reported in cm–1. Nuclear mag-
netic resonance spectra (NMR) were obtained at 300
MHz for 1H and 75 MHz for 13C with a Bruker AC/F300
spectrometer. The chemical shifts (δ in ppm) were
1
referenced to CDCl3 (7.26 ppm for H or 77.00 ppm for
13C). For 13C-NMR spectra, only the important carbons
were assigned (between []) while duplication of signals
attributed to the amide group [37] were reported between
parentheses. High resolution fast atom bombardment
mass spectra (HR-FABMS) were provided by the centre
régional de spectrométrie de masse (Université de Mon-
tréal, Montréal, Canada). The C, H, N analysis of
compound 4 was performed by Galbraith Laboratories
(Knoxville, TN).
5. Experimental
5.1. Chemical synthesis
General methods: unless otherwise noted, chemical
solvents and reagents were obtained from commercial
suppliers and used without further purification. Thin layer
chromatography (TLC) was performed on 0.20 mm Silica
5.1.1. Synthesis of N-butyl-N-methyl-11-(3'-benzyloxy-
17'-oxo-1',3',5'(10')-oestratrien-7'α-yl)-undecanamide 6
To a solution of 10.57 g (19.3 mmol) of alcohol 5 [36]
in 30 mL of acetone was added, dropwise, 16.9 mL of a

222
(hexane/EtOAc, 85:15) to give 8.83 g (73% yield) of
amide 6. Colourless oil; IR ν (film): 1 738 (C=O, ketone
1
and ester), 1 643 (C=O, amide); H-NMR δ (CDCl3):
0.92 (s, CH3-18'), 0.92 and 0.94 (2t, J = 7.4 Hz, CH3 of
butyl), 2.89 (ABX system, J6,7 = 5.0 Hz and
J
6,6 = 16.9 Hz, ∆ = 20.5 Hz, CH2-6'), 2.90 and 2.95 (2s,
CONCH3), 3.24 and 3.35 (2t, J = 7.4 Hz, CONCH2), 6.94
(d, J = 2.2 Hz, CH-4'), 6.99 (dd, J1 = 2.3 Hz and
J2 = 8.5 Hz, CH-2'), 7.33 (d, J = 8.5 Hz, CH-1'), 7.50 (m,
2H, meta protons of OBz), 7.63 (m, 1H, para proton of
OBz), 8.19 (d, J = 8.9 Hz, 2H, ortho protons of OBz);
13C-NMR δ (CDCl3): 13.74 [CH3 of butyl], 14.10
[C-18'], 19.88 (20.00) [CH2CH3 of butyl], 20.92, 25.03
(25.41)
[CH2CH2CON],
25.72,
26.68,
28.11,
29.39–29.88 (6×), 29.39 (30.57) [CONCH2CH2], 31.71,
32.61, 32.90 (33.56) [CH2CON], 33.21 (35.16)
[CONCH3], 34.37 [C-6'], 35.69, 38.27, 41.28, 46.98,
47.91, 47.30 (49.65) [CONCH2], 118.84 [C-2'], 122.48
[C-4'], 126.95 [C-1'], 128.45 (2×) [OBz], 129.63 [C-10'],
130.02 (2×) [OBz], 133.40 [OBz], 136.65 [OBz], 136.89
[C-5'], 148.83 [C-3'], 165.27 [CO of OBz], 172.73
(172.85) [CON], 220.47 [C-17']; HR-FABMS: calculated
for C41H58O4N (M+ + H) 628.4366, found 628.4378.
Figure 6. Effect of lactone 4 and pure anti-androgen hydroxy-
flutamide (OH-FLU) on the proliferation (androgenic activity)
of androgen-sensitive human breast cancer Shionogi cells. One
day after plating cells were incubated for 10 days with the
indicated concentration of test compound. Media were changed
every 3–4 days. Results were expressed as the mean of DNA
content (µg) ± SEM, in triplicate analysis.
5.1.2. Synthesis of N-butyl-N-methyl-11-{3',17'â-
dihydroxy-17'α-[3'-(tetrahydro-2'-H-pyran-2'-yloxy)-
propynyl]-1',3',5'(10')-oestratrien-7'α-yl}-
undecanamide 7
To a solution of 2.15 mL (15.3 mmol) of tetrahydro-2-
(2-propynyloxy)-2H-pyran (Aldrich, Milwaukee, USA)
in 50 mL of dry THF, was added at 0 °C, 9.28 mL of
n-BuLi 1.6 M in hexane and the mixture was stirred for
40 min. The reaction mixture was then cooled to –78 °C
and a solution of ketone 6 (1.75 g, 2.8 mmol) in 30 mL of
dry THF was added dropwise. After 3 h of stirring the
mixture was poured into a saturated solution of NaHCO3
and the aqueous phase was extracted with EtOAc
(3 × 100 mL). The organic layers were combined, washed
with brine, dried over MgSO4, filtered and evaporated
under reduced pressure. The brownish oil was purified by
flash chromatography (hexane/EtOAc, 57:43) to yield
1.59 g (75% yield) of compound 7. Yellow oil; IR ν
(film): 3 374 (OH, alcohol and phenol), 2 242 (C≡C),
1 626 (C=O, amide); 1H-NMR δ (CDCl3): 0.87 (s,
CH3-18'), 0.91 and 0.94 (2t, J = 7.3 Hz, CH3 of butyl),
2.76 (ABX system, J6,7 = 4.8 Hz and J6,6 =t16.8 Hz,
∆ = 20.2 Hz, CH2-6'), 2.92 and 2.97 (2s, CONCH3), 3.25
and 3.36 (2t, J = 7.5 Hz, CONCH2), 3.53 and 3.86 (2m,
OCH2 of THP), 4.36 (s, CH2OTHP), 4.87 (t, J = 3.1 Hz,
CH of THP), 6.59 (d, J = 2.5 Hz, CH-4'), 6.66 (dd,
J1 = 8.4 Hz and J2 = 2.5 Hz, CH-2'), 7.10 (d, J = 8.5 Hz,
CH-1'); 13C-NMR δ (CDCl3): 12.75 [C-18'], 13.78 [CH3
2.7 M Jones reagent at 0 °C and the orange mixture was
stirred for 2 h. Then, 1 mL of isopropanol was added and
the organic solvent was evaporated under reduced pres-
sure to yield a green solid which was then dissolved in
water (50 mL) and EtOAc (30 mL). The aqueous phase
was extracted with EtOAc (4 × 100 mL). The organic
layers were combined, washed with brine, dried over
MgSO4, filtered, and evaporated under reduced pressure
to yield a yellow viscous oil which was used, unpurified,
for the next step.
The crude carboxylic acid obtained above was dis-
solved in 100 mL of dry CH2Cl2 and 5.84 mL of tributyl-
amine. After the mixture was cooled to –10 °C, isobutyl
chloroformate (3.54 mL) was added dropwise and al-
lowed to react for 80 min. At this time excess
N-methylbutylamine (26.8 mL) was added and the cool-
ing bath removed. After 2.5 h, CH2Cl2 was added and the
organic phase washed with 10% aqueous HCl (v/v), brine
and dried over MgSO4. The solvent was removed and the
crude amide was purified by flash chromatography

223
of butyl], 19.12 [THP], 19.88 (20.00) [CH2CH3 of butyl],
22.31, 25.29 [THP], 25.09 (25.47) [CH2CH2CON],
27.37, 27.76, 29.17–29.49 (6×), 29.49 (30.55)
[CONCH2CH2], 30.29 [THP], 32.96 (33.64) [CH2CON],
33.07, 33.24, 33.53 (35.42) [CONCH3], 34.63 [C-6'],
37.88, 38.76, 42.66, 46.01, 47.44, 47.58 (49.87)
[CONCH2], 54.39 [C≡CCH2OTHP], 62.11 [CH2O of
THP], 79.92 and 81.59 [C≡C], 89.66 [C-17'], 96.59
(96.64) [CH of THP], 113.03 [C-2'], 116.21 [C-4'],
126.78 [C-1'], 130.73 [C-10'], 136.66 [C-5'], 154.36
[C-3'], 173.40 (173.51) [CON]; HR-FABMS: calculated
for C42H64O5NNa (M+ + Na) 685.4682, found 685.4662.
ion-exchange resin (Aldrich, Milwaukee, USA) and the
mixture was stirred at room temperature for 12 h. The
solution was then filtered and evaporated to dryness. The
crude alcohol, not having the THP group, was treated
with Jones reagent 2.7 M (0.26 mL) in acetone at 0 °C for
25 min. Then, isopropanol (2 mL) was added and the
green solution stirred 5 min before the acetone evapo-
rated. The residue was dissolved in water (20 mL) and
EtOAc (30 mL). The aqueous phase was extracted with
EtOAc (3 × 50 mL). The organic layers were combined,
washed with brine, dried over MgSO4, filtered and
evaporated under reduced pressure. Purification by flash
chromatography (hexane/EtOAc/MeOH, 70:29:1) of the
residue yielded 122 mg (48% yield) of lactone 4. Colour-
less oil; IR ν (film): 3 272 (OH, phenol), 1 770 (C=O,
5.1.3. Synthesis of N-butyl-N-methyl-11-{3',17'â-dihydro-
xy-17'α-[3'-(tetrahydro-2'-H-pyran-2'-yloxy)-propynyl]-
1',3',5'(10')-oestratrien-7'α-yl}-undecanamide 8
1
γ-lactone), 1 620 (C=O, amide); H-NMR δ (CDCl3):
0.91 and 0.94 (2t, J = 7.4 Hz, CH3 of butyl), 0.96 (s,
CH3-18'), 2.77 (ABX system, J6,7 = 4.7 Hz and
J6,6 = 16.6 Hz, ∆ = 20.4 Hz, CH2-6'), 2.93 and 2.97 (2s,
CONCH3), 3.26 and 3.37 (2m, CONCH2), 6.60 (d,
J = 2.4 Hz, CH-4'), 6.67 (dd, J1 = 2.5 Hz and J2 = 8.4 Hz,
CH-2'), 7.09 (d, J = 8.5 Hz, CH-1'); 13C-NMR δ (CDCl3):
13.79 [CH3 of butyl], 14.46 (CH3-18'), 19.88 (19.99)
[CH2CH3 of butyl], 22.12, 25.24, 25.05 (25.40)
[CH2CH2CON], 26.98, 27.67, 29.16–29.37 (7×), 29.61
(30.54) [CONCH2CH2], 31.41, 32.05, 32.94 (33.62)
[CH2CON], 33.08, 33.54 (35.42) [CONCH3], 34.59
[C-6'], 35.58, 37.85, 42.25, 45.14, 46.15, 47.59 (49.87)
[CONCH2], 96.23 [C-17'], 113.15 [C-2'], 116.24 [C-4'],
126.75 [C-1'], 130.11 [C-10'], 136.51 [C-5'], 154.57
[C-3'], 173.41 [CON], 177.04 (CO of γ-lactone); HR-
FABMS: calculated for C37H58O4N (M+ + H) 580.4366,
found 580.4375; Anal. (C37H57O4N): calc. C, 76.64; H,
9.91; N, 2.42, found C, 76.23; H, 9.70; N, 2.40.
A solution of alkyne 7 (751 mg, 1.13 mmol) in 100 mL
of EtOAc containing 113 mg of a 1:1 mixture of Pd/C
(10%) and Pd/CaCO3 (5%) was stirred at room tempera-
ture under H2 atmosphere for 64 h. The reaction mixture
was then filtered through celite, washed with EtOAc and
the solvent was evaporated to dryness. Purification by
flash chromatography (hexane/EtOAc, 5:5) afforded
690 mg (92% yield) of saturated compound 8. Yellow
foam; IR ν (film): 3 320 (OH, alcohol and phenol); 1 625
1
(C=O, amide); H-NMR δ (CDCl3): 0.91 (s, CH3-18'),
0.91 and 0.94 (2t, J = 7.3 Hz, CH3 of butyl), 2.77 (ABX
system, J6,7 = 4.8 Hz, J6,6 = 16.8 Hz, ∆ = Hz, CH2-6'),
2.92 and 2.97 (2s, CONCH3), 3.25 and 3.36 (2t,
J = 7.5 Hz, CONCH2), 3.50 and 3.85 (2m, 4H, OCH2 of
17α-side chain and OCH2 of THP), 4.62 (br, CH of THP,
6.59 (d, J = 2.5 Hz, CH-4'), 6.65 (dd, J1 = 2.5 Hz and
J2 = 8.4 Hz, CH-2'), 7.11 (d, J = 8.4 Hz, CH-1');
13C-NMR δ (CDCl3): 13.71 [CH3 of butyl], 14.28
[C-18'], 19.46 [THP], 19.79 (19.89) [CH2CH3 of butyl],
5.2. Biological evaluation
22.79,
23.90,
25.29
[THP],
25.01
(25.29)
[CH2CH2CON], 27.20, 27.91, 29.27–29.63 (6×), 29.27
(30.52) [CONCH2CH2], 30.47 [THP], 31.72, 32.85
(33.40) [CH2CON], 33.15, 33.18, 33.53 (35.31)
[CONCH3], 34.33, 34.60 [C-6'], 37.83, 42.71, 45.61,
46.75, 47.47 (49.76) [CONCH2], 62.25 [CH2O of THP],
68.21 [CH2OTHP], 83.09 [C-17'], 98.69 (98.78) [CH of
THP], 112.91 [C-2'], 116.14 [C-4'], 126.56 [C-1'], 130.57
[C-10'], 136.55 [C-5'], 154.47 [C-3'], 173.26 (173.37)
[CON]; HR-FABMS: calculated for C42H69O5N (M+)
667.5176, found 667.5211.
5.2.1. Inhibition of human placenta microsomal 17â-
hydroxysteroid dehydrogenase (type 2 17â-HSD)
The partially purified 17â-HSD activity obtained from
the microsomal fraction of human placenta (type 2 17â-
HSD) was used to assess the ability of newly synthesized
lactone 4 to inhibit the transformation of [3H]4-
androstene-3,17-dione ([3H]∆4-dione) to [3H]testoster-
one. Briefly, this enzymatic assay was performed at
pH 7.4 with a low concentration of tritiated ∆4-dione
(3.2 nM) to approximate physiological conditions.
NADH was used as cofactor. The time of incubation and
temperature were designated as 60 min and 37 °C. Sev-
eral concentrations of the tested compound (1 nM to 10
µM) were used to obtain the inhibition curve which
enabled us to determine the IC50 value, or the concentra-
5.1.4. Synthesis of N-butyl-N-methyl-11-(3'-hydroxy-
21',17'-carbolactone-19'-nor-17'α-pregna-1',3',5'(10')-
trien-7'α-yl)-undecanamide 4
To a solution of compound 8 (290 mg, 0.43 mmol) in
25 mL of methanol, was added 2.5 g of amberlyst 15

224
tion of inhibitor that inhibits 50% of target enzyme
activity. The procedure of the enzymatic assay has
previously been reported [9] and was used without
modification.
ethanol and their concentration adjusted to give a final
concentration of 0.01% ethanol in the culture medium.
Cells were incubated at 37 °C in a humidified atmosphere
of 5% CO2 and 95% air for 10 days with medium changes
every 3–4 days. At the end of the incubation period cell
growth was assessed by quantifying DNA content using a
modified Fiszer-Szafarz method [43], as previously de-
scribed [44]. The medium was then carefully removed
and 150 µL of methanol was added. Plates were then left
to dry at room temperature and were either frozen until
assayed or processed immediately. Salmon testis DNA
was used as a standard (0.5–20 µg/tube). Charcoal-
treated 3,5-diaminobenzoic acid (DABA) reagent
(200 mg/mL) was added (150 µL) to standards and dishes
containing dried, fixed cells. The reaction was carried out
for 60 min at 60 °C before cooling on ice and diluting
with 1.5 mL of HCl 1.0 N. Fluorescence was measured
with an LS2B Perkin-Elmer fluorimeter.
5.2.2. Inhibition of human placenta cytosolic 17â-
hydroxysteroid dehydrogenase (type 1 17â-HSD)
The activity of partially purified 17â-HSD obtained
from the cytosolic fraction of human placenta (type
1 17â-HSD) was used to assess the ability of lactone 4 to
inhibit the transformation of [3H]oestrone to [3H]oestra-
diol. Briefly, a low concentration of tritiated oestrone
(5 nM) and a pH of 7.4 was used to approximate
physiological conditions. The cofactor was NADH and
incubation time and temperature were 30 min and 37 °C,
respectively. Two concentrations of tested compound (0.1
and 1 µM) were used. A previously reported proce-
dure [40] for the enzymatic assay was implemented
without modification.
5.2.3. Oestrogenic and anti-oestrogenic activities
A proliferative assay on oestrogen-sensitive human
breast cancer cells (ZR-75-1) was used to assess in vitro
oestrogenic and anti-oestrogenic activity. The ability of
lactone 4 to stimulate proliferation of ZR-75-1 cells
demonstrated oestrogenic activity, while the ability of
lactone 4 to inhibit 0.1 nM oestradiol-induced prolifera-
tion of ZR-75-1 cells demonstrated anti-oestrogenic ac-
tivity. The tested compounds were evaluated at two
concentrations (0.03 and 1 µM). The procedure of this
assay has been previously reported [41] and was used
without modification.
Acknowledgements
We are grateful for the financial support provided
through grants from The Medical Research Council of
Canada (MRC) and Le Fonds de la Recherche en Santé
du Québec (FRSQ). We would like to extend this grati-
tude to our collaborators: Jacques Simard, who provided
the cell proliferation assays, Diane Michaud, who per-
formed the cell culture assays, and Serge Auger, who
performed the enzymatic assays.
5.2.4. Androgenic activity
References
The androgenic activity was evaluated by testing the
ability of lactone 4 to stimulate the proliferation of
androgen-sensitive human breast cancer Shionogi cells.
Two concentrations of tested compound (0.01 and 1 µM)
were used in the assay.
[1] Labrie F., Luu-The V., Lin S.X., Labrie C., Simard J., Breton R.,
Bélanger A., Steroids 62 (1996) 148–158.
[2] Moghrabi N., Andersson S., Trends Endocrinol. Metab. 9 (1998)
265–270.
[3] Penning T.M., Endocrine-Related Cancer 3 (1996) 41–56.
[4] Labrie F., Mol. Cell Endocrinol. 78 (1991) C113–C118.
Assay procedure: one clone of SC115 Shionogi cells
was selected for its elevated sensitivity to DHT. Cells
were harvested at the exponential growth phase with
0.1% pancreatine in HEPES buffer containing 3 mM
EDTA, then resuspended in MEM supplemented with
non-essential amino acids (1%), penicillin (10 IU/mL),
streptomycin sulfate (50 µg/mL) and 2% dextran-coated
charcoal-treated foetal calf serum. Cells were plated in
Falcon 24-well tissue culture plates (2 cm2/well) at a
density of 20 000 cells/dish. Twenty-four hours after
plating, the medium was changed and dihydrotestoster-
one (DHT) (0.3 nM), lactone 4 (0.01 or 1 µM) or
hydroxy-flutamide (0.01 or 1 µM) were added. Stock
solutions of DHT and tested compounds were prepared in
[5] Poirier D., Dionne P., Auger S., J. Steroid Biochem. Mol. Biol. 64
(1998) 83–90.
[6] Sam K.M., Boivin R.P., Tremblay M.R., Auger S., Poirier D., Drug
Design Discovery 15 (1998) 157–180.
[7] Tremblay M.R., Poirier D., J. Steroid Biochem. Mol. Biol. 66 (1998)
179–191.
[8] Auger S., Luu-The V., Sam K.M., Poirier D., Bioorg. Med. Chem.
Lett. 4 (1994) 2045–2048.
[9] Sam K.M., Auger S., Luu-The V., Poirier D., J. Med. Chem. 38
(1995) 4518–4528.
[10] Gauthier S., Caron B., Cloutier J., Dory Y.L., Favre A., Larouche D.
et al., J. Med. Chem. 40 (1997) 2117–2122.
[11] Auger S., Mérand Y., Pelletier J.D., Poirier D., Labrie F., J. Steroid
Biochem. Mol. Biol. 52 (1995) 547–565.

225
[12] Lévesque C., Mérand Y., Dufour J.M., Labrie C., Labrie F., J. Med.
Chem. 34 (1991) 1624–1630.
[27] Miettinen M.M., Mustonen M.V.J., Poutanen M.H., Isomaa V.V.,
Vihko R.K., Biochem. J. 314 (1996) 839–845.
[13] Poirier D., Mérand Y., Labrie C., Labrie F., Bioorg. Med. Chem.
Lett. 6 (1996) 2537–2542.
[28] Carsol J.L., Adamski J., Guirou O., Gérard H., Martin P.M., de
Launoit Y., Neuroendocrinology 64 (1996) 70–78.
[14] Poirier D., Boivin R.P., Bioorg. Med. Chem. Lett. 8 (1998)
1891–1896.
[29] Akinola L.A., Poutanen M., Vihko R., Endocrinology 137 (1996)
1572–1579.
[15] Lourdusamy M., Côté J., Laplante S., Labrie F., Singh S.M., Bioorg.
Med. Chem. 5 (1997) 305–310.
[30] Labrie Y., Durocher F., Lachance Y., Turgeon C., Simard J., Labrie
C., Labrie F., DNA Cell Biol. 14 (1995) 849–861.
[16] Tremblay M.R., Luu-The V., Leblanc G., Noël P., Breton E., Labrie
F., Poirier D., Bioorg. Med. Chem. 7 (1999) 1013–1023.
[31] Beaudoin C., Blomquist C.H., Tremblay Y., Endocrinology 136
(1995) 3807–3814.
[17] Carruba G., Adamski J., Calabro M., Dora Miceli M., Cataliotti A.,
Bellavia V. et al., Mol. Cell Endocrinol. 131 (1997) 51–57.
[32] Blomquist C.H., J. Steroid Biochem. Mol. Biol. 55 (1995) 515–524.
[33] Andersson S., J. Steroid Biochem. Mol. Biol. 55 (1995) 533–534.
[34] Wakeling A.E., Bowler J., J. Endocrinol. 112 (1987) R7–R10.
[35] Wakeling A.E., Bowler J., J. Steroid Biochem. 40 (1988) 141–147.
[18] Blomquist C.H., Lindemann N.J., Hakanson E.Y., Arch. Biochem.
Biophys. 239 (1985) 206–215.
[19] Wu L., Einstein M., Geissler W.M., Chan H.K., Elliston K.O.,
Andersson S., J. Biol. Chem. 268 (1993) 12964–12969.
[36] Bowler J., Lilley T.J., Pittam J.D., Wakeling A.E., Steroids 54 (1989)
71–99.
[20] Blomquist C.H., Leung B.S., Beaudoin C., Poirier D., Tremblay Y.,
J. Endocrinol. 153 (1997) 453–464.
[37] Dionne P., Singh S.M., Labrie F., Steroids 59 (1994) 493–497.
[38] Dionne P., Poirier D., Steroids 60 (1995) 830–836.
[21] Moghrabi N., Head J.R., Andersson S., J. Clin. Endocrinol. Metab.
82 (1997) 3872–3878.
[39] Salman M., Ruiz A.A., Stotter P.L., Chamness G.C., J. Steroid
Biochem. 26 (1987) 383–391.
[22] Luu-The V., Zhang Y., Poirier D., Labrie F., J. Steroid Biochem.
Mol. Biol. 55 (1995) 581–587.
[40] Tremblay M.R., Auger S., Poirier D., Bioorg. Med. Chem. 3 (1995)
505–523.
[23] Mustonen M.V.J., Poutanen M.H., Isomaa V.V., Vihko P.T., Vihko
R.K., Biochem. J. 325 (1997) 199–205.
[41] Poirier D., Auger S., Mérand Y., Simard J., Labrie F., J. Med. Chem.
37 (1994) 1115–1125.
[24] Castagnetta L.A.M., Carruba G., Traina A., Granata O.M., Markus
M., Pavone-Macaluso M., Blomquist C.H., Adamski J.,
Endocrinology 138 (1997) 4876–4882.
[42] Labrie F., Cancer 72 (1993) 3816–3827.
[43] Fiszer-Szafarz B., Skafarz D., Guevara de Murillo A., Anal. Bio-
chem. 110 (1981) 165–170.
[25] Akinola L., Poutanen M., Vihko R., Vihko P., Endocrinology 138
(1997) 2886–2892.
[44] Simard J., Dauvois S., Haagensen D.E., Lévesque C., Mérand Y.,
Labrie F., Endocrinology 126 (1990) 3223–3231.
[26] Zhang Y., Word R.A., Fesmire S., Carr B.R., Rainey W.E., J. Clin.
Endocrinol. Metab. 81 (1996) 3594–3598.