Chemical synthesis of the 17-propanamide derivatives of stereoisomeric Δ14-17α- and 17β-estradiols: Potential 17β-hydroxysteroid dehydrogenase inhibitors

Article abstract of DOI:10.1016/j.chemphyslip.2010.11.005

The 17-propanamide derivatives of diastereomeric Δ¹⁴- 17α- and 17β-estradiols, the potential candidates of a 17β-hydroxysteroid dehydrogenase (17β-HSD) inhibitor, were synthesized in 11 steps from estrone. The principal reactions employed involved in (1) conversion of estrone to the corresponding Δ¹⁴-estrone, (2) Grignard reaction of Δ¹⁴-estrone with allylmagnesium bromide followed by regioselective hydroboration of the resulting stereoisomeric 17ξ-allyl-Δ¹⁴-17ξ-ols with 9-borabicyclo[3.3.1]nonane (9-BBN), and (3) direct amidation of the 17ξ-O-/17ξ-C-spiro-γ- lactones with NH₃ under positive pressure of H₂.

Full text of DOI:10.1016/j.chemphyslip.2010.11.005

Chemistry and Physics of Lipids 164 (2011) 106–112
Contents lists available at ScienceDirect
Chemistry and Physics of Lipids
journal homepage: www.elsevier.com/locate/chemphyslip
Chemical synthesis of the 17-propanamide derivatives of stereoisomeric
ꢀ14-17␣- and 17␤-estradiols: potential 17␤-hydroxysteroid dehydrogenase
inhibitors
Takashi Iidaa,∗ , Shoujiro Ogawaa , Hideyuki Tamegaia , Yuuki Adachia , Hiroaki Saitob , Shigeo Ikegawac ,
Hiroaki Konishid, Akimitsu Takagid, Takeshi Matsuzakid
a Department of Chemistry, College of Humanities & Sciences, Nihon University, Setagaya, Sakurajousui, Tokyo 329-1151, Japan
b College of Pharmacy, Nihon University, Narashinodai, Funabashi, Chiba 274-8555, Japan
c Faculty of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashi-osaka, 577-8502, Japan
d Yakult Central Institute for Microbiological Research, Yakult Honsha Co., Ltd., Yaho, Kunitachi, Tokyo 186-8650, Japan
a r t i c l e i n f o
a b s t r a c t
The 17-propanamide derivatives of diastereomeric ꢀ14-17␣- and 17␤-estradiols, the potential candi-
dates of a 17␤-hydroxysteroid dehydrogenase (17␤-HSD) inhibitor, were synthesized in 11 steps from
Article history:
Received 20 September 2010
Received in revised form
22 November 2010
Accepted 25 November 2010
Available online 1 December 2010
estrone. The principal reactions employed involved in (1) conversion of estrone to the corresponding ꢀ14
-
estrone, (2) Grignard reaction of ꢀ14-estrone with allylmagnesium bromide followed by regioselective
hydroboration of the resulting stereoisomeric 17␰-allyl-ꢀ14-17␰-ols with 9-borabicyclo[3.3.1]nonane (9-
BBN), and (3) direct amidation of the 17␰-O-/17␰-C-spiro-␥-lactones with NH3 under positive pressure
of H2.
Keywords:
Steroid
© 2010 Elsevier Ireland Ltd. All rights reserved.
Estrogen
Estradiol
Estrone
Chemical synthesis
Spiro-␥-lactone
Propanamide
NMR
17␤-HSD
Inhibitor
1. Introduction
synthetic inhibitors of this key enzyme and such inhibitors would
have useful antitumor activity.
Estrogens are known to be an important factor involved in
the growth of estrogen-dependent breast cancers. The principal
human estrogen, 17␤-estradiol (estra-1,3,5(10)-trien-3,17␤-diol),
is a potent stimulator of the growth of estrogen receptor positive
neoplasms (Brodie and Njar, 1998; Vihko et al., 2003). In humans
17␤-hydroxysteroid dehydrogenase type 1 (17␤-HSD1) enzyme
involve in the last step in the biosynthesis of the most potent
mitogenic estrogen, 17␤-estradiol, from the less potent estro-
gen, estrone (3-hydroxyestra-1,3,5(10)-trien-17-one). The reverse
reaction, conversion of 17␤-estradiol to estrone, is catalyzed by
17␤-hydroxysteroid dehydrogenase type 2 (17␤-HSD2). Thus, the
17␤-hydroxysteroid dehydrogenases are attractive targets for the
Based on the three-dimensional structure of the active center
of 17␤-HSD1 (Ghosh et al., 1995; Ghosh and Vihko, 2001), a wide
variety of structurally different anti-estrogenic inhibitor candidates
have been proposed and their chemical syntheses as well as bio-
logical activities have been evaluated by Poirier (2003) and others
(Sam et al., 1998; Bellavance et al., 2009). Nonetheless, the thera-
peutic use of these synthetic inhibitors is still limited, because of
their estrogenic activity and cytotoxicity.
Considering the above, we judged it to be of interest to synthe-
size modified stereoisomeric derivatives at C-17 of 17␤-estradiol
as possible 17␤-HSD inhibitors. These compounds featured the
incorporation of an olefinic functionality in the steroid d-ring
as an electrophilic group that should mediate 17␤-HSD inac-
tivation and an amide moiety in the side chain that would
provide an antiestrogenic property (Sam et al., 1998; Poirier,
2003; Bellavance et al., 2009). The present paper deals with
∗
Corresponding author. Tel.: +81 3 3329 1151; fax: +81 3 3303 9899.
E-mail address: takaiida@chs.nihon-u.ac.jp (T. Iida).
0009-3084/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.chemphyslip.2010.11.005

T. Iida et al. / Chemistry and Physics of Lipids 164 (2011) 106–112
107
dine hydrochloride (0.5 g). The mixture was concentrated by slow
distillation using a Vigreux type fractionating column until the vol-
ume was reduced to 1/3–1/4 of the initial volume; this required
12 h. After cooling the mixture to room temperature, the reac-
tion product was extracted with EtOAc. The combined organic
layer was washed with saturated brine and water, dried with
Drierite® (CaSO4), and evaporated under reduced pressure to give
a pale yellow residue. The crude product 2b dissolved in dry pyri-
dine (15 mL) was then treated with acetic anhydride (23 mL) and
4-dimethylaminopyridine (60 mg) at room temperature for 2 h. Ice-
chips were added to the mixture, and the reaction product was
extracted with EtOAc. The combined extract was washed with
saturated brine and water, dried with Drierite, and evaporated
to dryness. The yellow product was chromatographed on a col-
umn of silica gel (250 g) eluting with hexane–EtOAc (9:1, v/v).
Recrystallization of the major product from EtOAc–hexane gave
the corresponding 3-acetoxy-17-ketal derivative (3) as colorless
amorphous crystals: yield, 6.0 g (91%); mp, 104–105 ◦C (lit., mp,
105–105.5 ◦C (Nambara et al., 1976)); IR (KBr) cm−1: 1772 (C O);
1H NMR (CDCl3) ı: 0.88 (3H, s, 18-H3), 2.28 (3H, s, OCOCH3), 2.84
(2H, m, 6-H2), 3.88–3.96 (4H, m, 19- and 20-H2), 6.78 (1H, d,
J = 2.4 Hz, 4-H), 6.83 (1H, dd, J1 = 2.7 Hz, J2 = 8.4 Hz, 2-H), 7.28 (1H, d,
J = 8.6 Hz, 1-H); LR-EI-MS m/z 356 (M+, 18%), 314 (9), 294 (25), 270
(M−OCH2CH2O−part of ring D, 9), 252 (38), 99 (100).
R1
R2
H
H
HO
R1
R2
CH2CH2CONH 2
OH
1a
CH2CH2CONH 2
1b
OH
Fig. 1. Structure of the propanamide derivative of 3,17␰-dihydroxy-ꢀ14-estradiols
prepared.
the chemical synthesis of diastereoisomeric 3-(3ꢀ,17␰ꢀ-dihydroxy-
1ꢀ,3ꢀ,5ꢀ(10ꢀ),14ꢀ-estratetraene-17ꢀ␰-yl)propanamide (1a and 1b)
starting from estrone (2a) (Fig. 1).
2.3.2.
16˛-Bromo-17,17ꢀ-ethylenedioxyestra-1,3,5(10)-triene-3-yl
2. Experimental
acetate (4)
2.1. Compounds and reagents
To
a magnetically stirred solution of the 3-acetoxy-17-
ketal 3 (6.0 g, 17 mmol) in dry tetrahydrofuran (THF; 90 mL),
phenyltrimethylammonium tribromide (9.0 g, 4 mmol) was added
with ice-bath cooling, and the mixture was allowed to stand for
12 h. After evaporation of the solvent under reduced pressure,
the yellow oily residue was extracted with EtOAc. The combined
extract was washed with 5% (w/v) NaHCO3 and water, dried over
Drierite, and evaporated to dryness; the crude product showed
essentially a single spot on TLC. Recrystallization from Et2O gave
the corresponding 3-acetoxy-16␣-bromo-17-ketal 4 as pale yellow
crystals: yield, 6.0 g (82%); mp, 151–153 ◦C (lit., mp, 154.5–156 ◦C
(Nambara et al., 1976)); IR (KBr) cm−1: 1764 (C O); 1H NMR (CDCl3)
ı: 0.91 (3H, s, 18-H3), 2.27 (3H, s, OCOCH3), 2.85 (2H, m, 6-H2),
3.96–4.26 (4H, m, 19- and 20-H2), 4.47 (1H, m, 16␤-H), 6.78 (1H,
d, J = 2.4 Hz, 4-H), 6.83 (1H, dd, J1 = 2.7, J2 = 8.1, 2-H), 7.26 (1H, d,
J = 8.4 Hz, 1-H); LR-EI-MS m/z 406 (M−CH2CH2, 12%), 99 (100).
Estrone (2a) was purchased from Wako Pure Chemical Indus-
tries Ltd. (Osaka, Japan). Sep-Pak Vac tC18 cartridges (adsorbent
weight, 5 g) for reversed-phase solid extraction was available from
Waters Co. (Milford, MA, USA) and successively conditioned by
washings with methanol and then water prior to use. Other chem-
ical reagents and solvents were of analytical reagent grade. All
solvents were dried by azeotropic distillation before use in reac-
tions.
2.2. Instruments
Melting points (mp) were determined on a micro hot-stage
apparatus and are uncorrected. Optical rotations were measured
by a JASCO P-1020 digital polarimeter (Tokyo, Japan) in methanol
or dimethyl sulfoxide (DMSO) at 28 ◦C. IR spectra were measured
on a JASCO FT-IR 4100 spectrometer (Tokyo, Japan) for samples
in the KBr tablets. 1H and 13C NMR spectra were obtained on
a JEOL JNM-EX 270 FT NMR instrument (Tokyo, Japan) at 270
and 68.80 MHz, respectively, with CDCl3, CD3OD or DMSO-d6 con-
taining Me4Si as the solvent; chemical shifts are expressed as
ı ppm relative to Me4Si. Electron ionization (EI) low-resolution
mass spectra (LR-EI-MS) were recorded on a JEOL GCmate gas
chromatograph–mass spectrometer at 70 eV. High-resolution mass
spectra (HR-MS) were obtained on a JEOL GCmate equipped with
an electron ionization and fast atom bombardment (FAB) under the
positive ion mode (PIM). TLC was performed on pre-coated silica gel
(0.25 mm layer thickness; E. Merck, Germany) using hexane–EtOAc
mixtures (4:1–3:7, v/v) as the developing solvent. Reversed phase
TLC was carried out on precoated RP-18F254s plates (Merck) using
methanol/water (7:3, v/v) as developing solvent.
2.3.3. 17-Ethylenedioxyestra-1,3,5(10),15-tetraen-3-ol (5)
To a solution of the 3-acetoxy-16␣-bromo-17-ketal 4 (6.0 g,
14 mmol) in dimethyl sulfoxide (DMSO; 70 mL) was added potas-
sium tert-butoxide (33 g, 0.3 mol), and the mixture was stirred
at 60 ◦C for 6 h. After cooling the mixture at room temperature,
the reaction product was extracted with EtOAc, and the com-
bined extract was washed and neutralized with 1% (v/v) acetic
acid and water. The organic phase was dried with Drierite, and
evaporated to dryness under reduced pressure. The resulting pale
green oil was purified by passing through a column of silica gel
(200 g) eluting with hexane–EtOAc (9:1, v/v). Recrystallization of
a single component effluent from acetone–hexane gave the ꢀ15
-
3-hydroxy-17-ketal 5 as colorless prisms: yield, 3.88 g (90%); mp,
208–210 ◦C (lit., mp, 210–214 ◦C (Nambara et al., 1976)); mp,
218–220 ◦C (Cantrall et al., 1964); IR (KBr) cm−1: 3349 (OH), 1619,
1498 (C C); 1H NMR (CDCl3) ı: 0.96 (3H, s, 18-H3), 2.84 (2H, m,
6-H2), 3.88–3.99 (4H, m, 19- and 20-H2), 5.75 (1H, dd, J1 = 3.2 Hz,
J2 = 5.9 Hz, 15-H), 6.26 (1H, dd, J1 = 1.4 Hz, J2 = 5.9 Hz, 16-H), 6.57
(1H, d, J = 2.7 Hz, 4-H), 6.62 (1H, dd, J1 = 2.7 Hz, J2 = 8.4 Hz, 2-H), 7.14
(1H, d, J = 8.1 Hz, 1-H); LR-EI-MS m/z 312 (M+, 100%), 297 (M−CH3,
7), 284 (M−CH2CH2, 2), 268 (M−O−CH2CH2, 15), 240 (60), 211 (40).
2.3. Chemical synthesis
2.3.1. 17,17ꢀ-Ethylenedioxyestra-1,3,5(10)-triene-3-yl acetate (3)
To
a solution of estrone 2a (5 g, 18 mmol) in toluene
(100 mL) was added ethylene glycol (10 mL, 0.18 mol) and pyri-

108
T. Iida et al. / Chemistry and Physics of Lipids 164 (2011) 106–112
2.3.4. 3-Hydroxyestra-1,3,5(10),15-tetraen-17-one (6)
2.3.7. 3,17ˇ-Dihydroxy-17˛-(1ꢀ-hydroxyprop-3ꢀ-yl)-
A solution of the ꢀ15-3-hydroxy-17-ketal 5 (2.6 g, 8.3 mmol)
and p-toluenesulfonic acid monohydrate (p-TSA; 100 mg) in
methanol (50 mL) was allowed to stand at room temperature for
1 h. After evaporating the solvent under reduced pressure, the pale
yellow oily residue was chromatographed on a column of silica
gel (200 g). Elution with hexane–EtOAc (8:2, v/v) gave conjugated
1,3,5(10),14-estratetraene (9a) and
3,17˛-dihydroxy-17ˇ-(1ꢀ-hydroxyprop-3ꢀ-yl)-1,3,5(10),14-
estratetraene
(9b)
To a mixture of the ꢀ14-17␰-allyl-17␰-hydroxy compounds 8
(1.2 g, 3.9 mmol) dissolved in dry THF (12 mL) was added slowly
0.5 M 9-borabicyclo[3.3.1]nonane (9-BBN) in a THF solution (28 mL)
with ice-bath cooling under a stream of N2. The mixture was stirred
at room temperature for 3 h; the reaction was monitored by TLC.
A solution of 3 M NaOH (8 mL) and then 30% H2O2 (10 mL) were
added gradually to the mixture with ice-bath cooling, and the
resulting suspension was let stand at room temperature for 4 h.
The reaction product was extracted with EtOAc, and the combined
extract was washed with saturated brine and water, dried with
Drierite, and evaporated to dryness under reduced pressure. The
resulting pale yellow oil, which consisted of a mixture of two com-
ponents by TLC, was passed through a column of silica gel (80 g).
Elution with hexane–EtOAc (9:1, v/v) provided two well resolved
fractions.
ꢀ
15-estrone (6), which was recrystallized from EtOAc–hexane as
colorless prisms: yield, 1.64 g (74%); mp, 246–248 ◦C (lit., mp,
250–252 ◦C (Cantrall et al., 1964)); IR (KBr) cm−1: 3232 (OH), 1686
(C O), 1619, 1503 (C C); 1H NMR (CDCl3) ı: 1.17 (3H, s, 18-CH3),
2.85 (2H, m, 6-H2), 6.22 (1H, dd, J1 = 2.7 Hz, J2 = 5.9 Hz, 15-H), 6.55
(1H, d, J = 2.4 Hz, 4-H), 6.64 (1H, dd, J1 = 2.7 Hz, J2 = 8.6 Hz, 2-H), 7.03
(1H, d, J = 8.6 Hz, 1-H), 7.62 (1H, dd, J1 = 2.4 Hz, J2 = 5.9 Hz, 16-H);
LR-EI-MS m/z 268 (M+, 93%), 253 (M−CH3, 7), 240 (M−CO, 7), 225
(M−CH3−CO, 6), 172 (100).
2.3.5. 3-Hydroxyestra-1,3,5(10),14-tetraen-17-one (7)
A solution of ꢀ15-estrone (6; 2.2 g, 8.2 mmol) and p-TSA (1.32 g)
in benzene (130 mL) was refluxed for 1 h; reaction was monitored
by TLC. After cooling at room temperature, the reaction mixture
was washed with 5% (w/v) NaHCO3 and water, dried with Drierite,
and evaporated to dryness to give a pale yellow oily residue. Chro-
matography of the oily product on a column of silica gel (200 g)
and elution with hexane–EtOAc (9:1, v/v) afforded unconjugated
A less polar, minor effluent, recrystallized from EtOAc–hexane
as colorless amorphous solids, was identified as the ꢀ14-17␤-
propyl-3,17␣,21-triol 9b: yield, 208 mg (16%); mp, 191–193 ◦C.
[˛]D = +87.2◦ (C = 1.00, methanol); IR (KBr) cm−1: 3365 (OH), 1612,
1502 (C C); 1H NMR (CD3OD) ı: 0.99 (3H, s, 18-CH3), 2.80 (2H,
m, 6-H2), 3.59 (2H, m, 21-H2), 5.28 (1H, brs, 15-H), 6.48 (1H, d,
J = 2.7 Hz, 4-H), 6.56 (1H, dd, J1 = 2.7 Hz, J2 = 8.6 Hz, 2-H), 7.12 (1H, d,
J = 8.1 Hz, 1-H); LR-EI-MS m/z 328 (M+, 15%), 310 (M−H2O, 85), 298
(M−CH3−H2O, 7), 277 (M−CH3−2H2O, 3), 251 (M−H2O−side chain
(S.C.), 26), 226 (M−S.C.−part of ring D, 100), 213 (M−S.C.−ring D,
94), 198 (M−CH3−S.C.−ring D, 25); HR-EI-MS (PIM) m/z Calc. for
ꢀ
14-estrone (7), which was recrystallized from EtOAc–hexane as
colorless needles: yield, 1.76 g (80%); mp, 183–184 ◦C (lit., mp,
185–189 ◦C (Cantrall et al., 1964)); IR (KBr) cm−1: 3430 (OH), 1731
(C O), 1609, 1502 (C C); 1H NMR (CDCl3) ı: 1.17 (3H, s, 18-
H3), 2.92 (2H, m, 6-H2), 5.61 (1H, d, J = 1.6 Hz, 15-H), 6.61 (1H,
d, J = 2.7 Hz, 4-H), 6.66 (1H, dd, J1 = 2.7 Hz, J2 = 8.4 Hz, 2-H), 7.16
(d, J = 8.1 Hz, 1-H); LR-EI-MS m/z 268 (M+, 100%), 253 (M−CH3,
1), 240 (M−CO, 43), 225 (M−CH3–CO, 18), 198 (M−CH3−ring D,
44).
C
21H28O3 [M]+ 328.2038, Found m/z 328.2040.
A more polar, major effluent was characterized as the cor-
responding 17-epimer 9a (ꢀ14-17␣-propyl-3,17␤,21-triol) of 9b,
which was recrystallized from methanol–water as colorless nee-
dles: yield, 833 mg (64%); mp, 173–174 ◦C. [˛]D = +182.5◦ (C = 1.00,
methanol); IR (KBr) cm−1: 3322 (OH), 1609, 1502 (C C); 1H NMR
(CD3OD) ı: 1.08 (3H, s, 18-CH3), 2.80 (2H, m, 6-H2), 3.59 (2H, m,
21-H2), 5.18 (1H, brs, 15-H), 6.50 (1H, d, J = 2.7 Hz„ 4-H), 6.54 (1H,
dd, J1 = 2.7 Hz, J2 = 9.2 Hz, 2-H), 7.06 (1H, d, J = 8.1 Hz, 1-H); LR-EI-
MS m/z 328 (M+, 18%), 310 (M−H2O, 76), 298 (M−CH3−H2O, 9),
277 (M−CH3−2H2O, 3), 251 (M−H2O−S.C., 29), 226 (M−S.C.−part
of ring D, 100), 213 (M−S.C.−ring D, 96), 198 (M−CH3−S.C.−ring D,
26); HR-EI-MS (PIM) m/z Calc. for C21H28O3 [M]+ 328.2038, Found
m/z 328.2038.
2.3.6. 3,17␰-Dihydroxy-17␰-allyl-1,3,5(10),14-estratetraene (8)
To a vigorously stirring mixture of metallic magnesium rib-
bon (2.64 g, 0.1 mol) and iodine (60 mg) in dry Et2O (180 mL) a
solution of allylbromide (0.2 g, 1.65 mmol) dissolved in dry Et2O
(25 mL) was added slowly at room temperature under an atmo-
sphere of N2, until the brown iodide color had disappeared. To the
white turbid suspension was added allylbromide (19.2 g, 0.16 mol)
and then ꢀ14-estrone 7 (2.0 g, 7.4 mmol) in dry Et2O (15 mL) over
a period of 30 min with ice-bath cooling. Stirring was further
continued at room temperature for 12 h. A saturated solution of
ammonium hydrogencarbonate was then added to the reaction
mixture, and the reaction product was extracted with Et2O. The
combined extract was washed with saturated brine and water,
dried with Drierite, and evaporated to dryness under reduced
pressure. The yellow oily residue was subjected to chromatog-
raphy on silica gel (200 g). Elution with EtOAc–hexane (95:5,
2.3.8. 3,17ˇ-Dihydroxy-19-nor-17˛-pregna-1,3,5(10),14-
tetraene-21-carboxylic acid-ꢁ-lactone
(10a)
A solution of the ꢀ14-17␣-propyl-3,17␤,21-triol 9a (100 mg,
0.30 mmol) and pyridinium dichromate (PDC; 300 mg, 0.80 mmol)
in dry N,N-dimethylformamide (DMF; 1.5 mL) was stirred at room
temperature for 6 h. The mixture was diluted with EtOAc and the
insoluble material was filtered off on Celite. The filtrate was washed
with saturated brine and water, dried with Drierite, and evap-
orated to dryness under reduced pressure. The dark brown oily
residue was passed through a column of silica gel (30 g) eluting with
hexane–EtOAc (2:8, v/v). After evaporation of the solvent, recrys-
tallization of a homogeneous effluent from EtOAc–hexane afforded
the ꢀ14-C-17␤-O-/C-21-spiro-␥-lactone 10a as pale yellow crys-
tals: yield, 52 mg (51%); mp, 230–231 ◦C; [˛]D = +151.7◦ (C = 1.01,
DMSO); IR (KBr) cm−1: 3359 (OH), 1735 (C O); 1H NMR (CD3OD)
ı: 1.11 (3H, s, 18-CH3), 2.85 (2H, m, 6-H2), 5.25 (1H, brs, 15-H),
6.50 (1H, d, J = 2.7 Hz, 4-H), 6.55 (1H, dd, J1 = 2.7 Hz, J2 = 8.1 Hz, 2-
H), 7.07 (1H, d, J = 10.8 Hz, 1-H); LR-EI-MS m/z 324 (M+, 100%), 309
(M−CH3, 7), 237 (M−CH3−S.C., 7), 213 (M−S.C.−ring D, 22), 198
v/v) provided a mixture of two ꢀ14-17␣-allyl-3,17␤- and ꢀ14
-
17␤-allyl-3,17␣-dihydroxy isomers 8 in an approximate ratio of
4:1 (estimated by 1H NMR): yield, 2.12 g (70%); mp, 122–123 ◦C.
The mixture of the two stereoisomers was used for the subse-
quent reaction. IR (KBr) cm−1: 3295 (OH), 1618, 1498 (C C);
1H NMR (CDCl3) ı: 1.10 (3H, s, 18-CH3), 2.86 (2H, m, 6-H2),
5.14 (1H, brs, 15-H), 5.20 (2H, m, 21-H2), 6.00 (1H, brm, 20-
H), 6.59 (1H, brs, 4-H), 6.64 (1H, d, J = 8.1 Hz, 2-H), 7.10 (1H, d,
J = 8.1 Hz, 1-H); LR-EI-MS (PIM) m/z 310 (M+, 70), 292 (M−H2O,
13), 251 (M−H2O−S.C., 24), 226 (M−S.C.−part of ring D, 97),
213 (M−S.C.−ring D, 100), 198 (M−CH3−S.C.−ring D, 19); HR-
EI-MS (PIM) m/z Calc. for C21H26O2 [M]+ 310.1933, Found m/z
310.1934.

T. Iida et al. / Chemistry and Physics of Lipids 164 (2011) 106–112
109
(M−CH3−S.C.−ring D, 20); HR-EI-MS (PIM) m/z Calc. for C21H24O3
3. Results and discussion
[M]+ 324.1725, Found m/z 324.1724.
Previous works on synthetic 17␤-HSD inhibitors have identi-
fied some of the essential factors governing the biological activities
(Sam et al., 1998; Poirier, 2003). These factors were deduced on
the basis of results obtained with a variety of 7-, 16- and/or
17-substituted derivatives of 3,17␰-estradiols. Such compounds
possess polar and/or electron-enriched functional groups and were
considered to be prototype enzyme inhibitors of 17␤-HSD. Accord-
ing to previous findings by Poirier (2003) (Sam et al., 1995;
Tremblay and Poirier, 1998; Bydal et al., 2004), the biological activ-
ities were significantly enhanced by polar or electron-enriched
functionality of the d-ring in the estradiol nucleus, because they
interacts strongly with a cofactor (NAD(P)H) binding domain. Oth-
erwise, introduction of a certain type of alkyl side chain at the
position C-7, C-16 or C-17 with a carbon length of C3–C4 bearing
an electron-enriched site (e.g., alkylamide, olefinic and acetylenic
linkages, alkyl alcohol or alkyl halide) as a spacer was shown to
enhance the inhibitory potency. In particular, estradiol deriva-
tives possessing lactone and/or alkylamide moieties in the steroidal
d-ring are now well recognized as significant pharmacophores
for the enzyme inhibition of 17␤-HSD. However, potent cyto-
toxicity of these functionalities has also been reported recently
(Bellavance et al., 2009; Bydal et al., 2004). These findings moti-
vated us to introduce an unsaturated ꢀ14-bond in the steroid d-ring
and propanamide linkage in the side chain onto the diastereoiso-
meric ꢀ14-17␣- and ꢀ14-17␤-estradiols as modified 17␤-HSD
inhibitor candidates (1a and 1b). These target compounds having
both electron-enriched and antiestrogenic sites would be expected
to inactivate the binding affinity of 17␤-HSD and to increase the
antiestrogenic activity.
Chemical synthesis of the target compounds (1a and 1b) was
achieved in 11 steps starting from estrone (2a). As outlined in
Fig. 2, a key intermediate in our synthesis was ꢀ14-estrone (7)
which was prepared from 2a in 6 steps. Thus, ketalization of the
17-oxo group in 2a with ethylene glycol and pyridine hydrochlo-
ride in boiling toluene under azeotropic removal of water yielded
the 17-ethylenedioxy ketal derivative 2b (Nambara et al., 1976).
Then, the crude product of 2b was subjected to the usual acetylation
with acetic anhydride-pyridine to yield the 17-ethylenedioxy ketal
derivatives 3. Treatment of 3 with phenyltrimethylammonium
tribromide in THF at 0 ◦C for 12 h gave the corresponding 16␣-
bromo-17-ketal 4. The configuration of a less sterically hindered
16␣-bromo group with the axially oriented C-18 methyl group
was determined by the appearance of a1H signal at 4.47 ppm as
a multiplet, indicating the presence of 16␤-H (Reeder and Joannou,
1996).
2.3.9. 3,17˛-Dihydroxy-19-nor-17ˇ-pregna-1,3,5(10),14-
tetraene-21-carboxylic acid-ꢁ-lactone
(10b)
The ꢀ14-17␤-propyl-3,17␣,21-triol 9b (40 mg, 0.12 mmol) was
subjected to the oxidation reaction with PDC (140 mg, 0.37 mmol)
and processed as described for the preparation of 10a; the yield was
a dark brown oil. The oily residue was chromatographed on a col-
umn of silica gel (30 mg) and eluted with hexane–EtOAc (2:8, v/v).
Recrystallization of a homogeneous effluent from EtOAc–hexane
gave the ꢀ14-C-17␣-O-/C-21-spiro-␥-lactone 10b as pale yel-
low amorphous crystals: yield, 20 mg (50%); mp, 208–210 ◦C;
[˛]D = +19.9◦ (C = 0.51, DMSO); IR (KBr) cm−1: 3374 (OH), 1746
(C O); 1H NMR (CD3OD) ı: 1.06 (3H, s, 18-CH3), 2.85 (2H, m, 6-
H2), 5.33 (1H, brs, 15-H), 6.51 (1H, d, J = 2.7 Hz, 4-H), 6.56 (1H,
dd, J1 = 2.7 Hz, J2 = 8.1 Hz, 2-H), 7.13 (1H, d, J = 10.8 Hz, 1-H); LR-EI-
MS m/z 324 (M+, 100%), 309 (M−CH3, 10), 237 (M−CH3−S.C., 7),
213 (M−S.C.−ring D, 16), 198 (M−CH3−S.C.−ring D, 13); HR-EI-MS
(PIM) m/z Calc. for C21H24O3 [M]+ 324.1725, Found m/z 324.1728.
2.3.10.
(3ꢀ,17ˇꢀ-Dihydroxy-1ꢀ,3ꢀ,5ꢀ(10ꢀ),14ꢀ-estratetraene-17ꢀ˛-yl)
propanamide (1a)
To a solution of the ꢀ14-C-17␤-O-/C-21-spiro-␥-lactone 10a
(50 mg, 0.15 mmol) dissolved in a methanolic saturated solution
with NH3 gas (5 mL) in an autoclave was added sodium cyanide
(10 mg) as a catalyst. The mixture was magnetically stirred at 60 ◦C
for 12 h under H2 with a positive pressure of 50 kPa. After evapora-
tion of the solvent under reduced pressure, the dark brown residue
was dissolved in a small amount of methanol and loaded onto a Sep-
Pak Vac tC18 cartridge. After being washed with water, elution with
methanol–water (1:9, v/v) gave the desired ꢀ14-3,17␤-estradiol
17␣-propanamide 1a. This compound was recrystallized from
acetone–water as colorless amorphous crystals: yield, 21 mg (40%);
mp, 172–174 ◦C; [˛]D = +128.3◦ (C = 1.00, methanol); IR (KBr) cm−1
:
3354 (OH), 1648 (C O); 1H NMR (DMSO) ı: 0.98 (3H, s, 18-CH3),
2.76 (2H, m, 6-H2), 5.13 (1H, brs, 15-H), 6.46 (1H, d, J = 2.4 Hz, 4-H),
6.60 (1H, dd, J1 = 2.7 Hz, J2 = 8.1 Hz, 2-H), 7.06 (1H, d, J = 8.1 Hz, 1-H);
LR-EI-MS m/z 341 (M+, 3%), 324 (M−NH3, 100), 309 (M−CH3−NH3,
9), 251 (M−H2O−S.C., 28), 198 (M−CH3−S.C.−ring D, 29); HR-FAB-
MS (PIM) m/z Calc. for C21H28NO3 [M + H]+ 342.2069, Found m/z
342.2067.
Table 1 compiles the 13C chemical shifts for the compounds
prepared in this study, except for 8. The 13C NMR data, together
with the 1H data, provided confirmatory evidence for the struc-
tures of these compounds. The 13C NMR signal assignments were
exclusively based on the data for the DEPT experiments.
2.3.11.
3-(3ꢀ,17˛ꢀ-Dihydroxy-1ꢀ,3ꢀ,5ꢀ(10ꢀ),14ꢀ-estratetraene-17ꢀˇ-yl)
propanamide (1b)
The ꢀ14-C-17␣-O-/C-21-spiro-␥-lactone 10b (20 mg, 58 ␮mol),
subjected to the amidation reaction with NH3 and processed as
described for the preparation of 1a, gave a dark brown oily residue.
Reversed-phase solid extraction of the oil on a Sep-Pak Vac tC18
cartridge and elution with water–methanol (95:5, v/v) afforded
the desired ꢀ14-3,17␣-estradiol 17␤-propanamide 1b, which was
recrystallized from acetone–water as colorless amorphous crys-
tals: yield, 6.3 mg (40%); mp, 188–190 ◦C; [˛]D = +42.4◦ (C = 0.51,
methanol); IR (KBr) cm−1: 3219 (OH), 1682 (C O); 1H NMR (DMSO)
ı: 0.90 (3H, s, 18-CH3), 2.75 (2H, m, 6-H2), 5.20 (1H, brs, 15-H),
6.46 (1H, d, J = 2.7 Hz, 4-H), 6.54 (1H, dd, J1 = 2.7 Hz, J2 = 8.1 Hz, 2-
H), 7.12 (1H, d, J = 8.1 Hz, 1-H); LR-EI-MS m/z 341 (M+, 3%), 324
(M−NH3, 100), 309 (M−CH3−NH3, 8), 251 (M−H2O−S.C., 37); HR-
FAB-MS (PIM) m/z Calc. for C21H28NO3 [M + H]+ 342.2069, Found
m/z 342.2069.
When the 16␣-bromo-17-ketal 4 was treated with potas-
sium tert-butoxide in DMSO and benzene at 60 ◦C for 6 h,
dehydrobromination reaction took place smoothly to produce
the 3-hydroxy-ꢀ15-17-ethylenedioxy ketal 5. Mild acid-catalyzed
cleavage of the 17-ketal linkage in 5 with p-TSA in aqueous ace-
tone at room temperature for 1 h afforded the conjugated enone,
ꢀ
15-estrone (6) (Cantrall et al., 1964). Subsequent acid-catalyzed
isomerization of the resulting 6 with p-TSA under vigorous con-
ditions in refluxing benzene for 2 h resulted in the formation of
the unconjugated enone, ꢀ14-estrone (7). Differentiation between
the two positional isomers of 6 and 7 was readily made on the
basis of the characteristic 1H signals arising from the olefinic pro-
tons attached at C-15 and/or C-16. In 6, the 15- and 16-H signals
appear at 6.22 and 7.62 ppm, both as a doublet of doublets, respec-

110
T. Iida et al. / Chemistry and Physics of Lipids 164 (2011) 106–112
O
O
O
O
O
H
H
Br
H
(iii)
(i)
H
H
H
H
H
H
HO
AcO
RO
4
2a
2b: R=H
3 : R=Ac
(ii)
Ac = COCH 3
(iv)
O
O
O
O
H
H
(vi)
(v)
H
H
H
H
H
H
HO
HO
HO
7
6
5
Fig. 2. Synthetic route to intermediary ꢀ14-estrone (7) from estrone (2a).
tively, while the 15-H signal in 7 is shifted up-field by 0.61 ppm and
resonates at 5.61 ppm as a doublet.
ing evidence that 8 is an epimeric mixture at C-17 was confirmed
by the occurrence of the 13C signals at 118.5 and 118.1 ppm (C-21),
134.7 and 134.2 ppm (C-20), and 41.8 and 41.6 ppm (C-19).
As shown in Fig. 3, ꢀ14-estrone (7) served as a key intermediate
for the preparation of our targeted molecules. Grignard reaction
of 7 with allylmagnesium bromide (Bydal et al., 2004; Garrouj
et al., 1993) at room temperature for 12 h afforded a mixture of the
two stereoisomeric ꢀ14-17␣-allyl-17␤- and ꢀ14-17␤-allyl-17␣-
dihydroxyestradiol derivatives 8 in an approximate ratio of 4:1
(estimated by the peak height of the 13C NMR spectrum; see below).
Because our attempt to separate the two diastereoisomers by chro-
matography failed, the mixture 8 was directly subjected to the
subsequent reaction. The 1H NMR spectrum of the mixture showed
the characteristic 1H signals at 5.20 ppm (21-H2) as a multiplet and
at 6.00 ppm (20-H) as a broad multiplet arising from the allyl group
at C-17. On the other hand, the 13C signal due to C-17 resonated at
83.0 ppm in the 13C NMR spectrum. Furthermore, strong support-
Attempted regioselective hydroxylation of the allyl group in
8 initially was unsuccessful. When less sterically hindered dib-
orane (BH3) (Hosoda et al., 1975; Pelletier, 1994; Sam et al.,
1994) followed by alkaline hydrogen peroxide were employed as
oxidative-hydroboration agents, the main reaction product was the
15␰,21␰-dihydroxylated derivative of 8. However, by changing the
hydroborating agent to the more sterically bulky 9-BBN, highly
regioselective hydroxylation at the C-21 position in 8 was attained
without simultaneous formation of either 15-hydroxylated or
15,21-dihydroxylated analogs of 8. The reaction product with 9-
BBN at room temperature for 3 h was found to be a mixture of
the diastereoisomeric ꢀ14-3,17␤,21-triol 9a and ꢀ14-3,17␣,21-
triol 9b in an approximate ratio of 4:1. The two epimers were
Table 1
13C NMR chemical shifts for compounds prepared.a
Carbon
1a
1b
3
4
5
6
7
9a
9b
10a
10b
1
2
3
4
5
6
126.7
113.2
155.3
115.3
137.4
31.0
127.2
113.3
155.3
115.2
137.6
30.8
126.4
118.5
148.4
121.4
138.3
30.6
126.2
118.6
148.4
121.5
138.0
30.5
126.2
112.6
153.4
115.2
138.1
29.6
128.1
113.4
153.4
115.0
137.6
30.4
126.8
113.2
153.7
115.4
138.0
29.5
127.4
113.9
156.0
116.1
138.7
30.8
128.0
113.9
156.0
116.2
138.9
30.9
127.0
113.5
155.4
115.9
138.3
30.0
128.0
114.0
156.1
116.1
138.8
30.3
7
27.5
27.6
26.8
26.5
27.2
27.7
26.5
28.7
28.6
27.6
28.3
8
39.1
42.0
38.6
38.0
36.0
35.6
38.9
40.5
40.5
39.5
40.5
9
5.1
43.9
43.8
47.4
44.3
38.0
44.6
47.2
44.0
46.5
44.3
10
11
12
13
14
15
16
17
18
19
20
21
130.4
25.6
32.1
130.2
25.5
29.6
138.1
25.9
29.5
46.1
49.4
22.3
34.2
119.4
14.3
137.5
25.6
29.3
45.6
55.2
35.1
43.6
116.8
14.6
132.6
25.7
29.3
49.9
55.5
132.2
136.2
119.6
16.2
132.6
27.4
31.4
47.8
54.7
132.8
162.2
214.8
22.0
131.5
24.8
33.2
132.2
27.0
34.8
132.1
26.7
30.9
131.3
26.3
34.6
131.6
26.7
30.8
51.6
51.5
51.0
53.2
53.0
52.0
51.8
152.2
114.6
42.6
82.1
18.0
29.5
33.2
175.9
151.5
115.4
42.5
82.2
20.8
30.9
31.9
175.8
152.4
113.0
41.6
222.8
20.0
154.0
115.3
43.6
84.6
17.8
33.4
28.7
63.8
153.2
116.1
44.8
85.0
21.4
33.2
28.8
63.8
153.1
114.3
44.4
98.2
17.4
30.2
29.8
179.0
153.0
116.0
44.0
99.8
20.3
30.4
28.5
179.6
OCH2
OCH2
64.6
65.2
66.2
66.5
64.2
65.3
Ac(CH3)
Ac(CO)
21.1
169.9
21.1
169.8
a
In ppm downfield from TMS.

T. Iida et al. / Chemistry and Physics of Lipids 164 (2011) 106–112
111
Fig. 3. Synthetic route to the target compounds (1a, 1b, 10a, and 10b) from ꢀ14-estrone.
efficiently separated by silica gel column chromatography with
hexane–EtOAc as the eluent.
signals at 98.2 and 99.8 ppm are assigned to C-17 and at 179.0
and 179.6 ppm to C-21, respectively, supporting the formation of
a spiro-␥-lactone ring by intramolecular esterification of 9a and
9b. The C-18 signal appearing at 17.4 ppm in the 17␤-O-lactone
10a is deshielded by 2.9 ppm and resonates at 20.3 ppm in the
corresponding 17␣-O-isomer 10b. Similar trends have also been
reported for the analogous lactone derivatives of 3,17␰-estradiols
(Bydal et al., 2004; Dionne and Poirier, 1995; Dionne et al., 1997).
Furthermore, the 13C signal at C-12 occurring at 34.6 ppm in 10a is
shielded by 3.8 ppm and resonates at 30.8 ppm in 10b, indicating
the validity of their stereochemical assignments.
For obtaining the targeted final compounds 1a and 1b, our
exploratory experiment revealed that direct amidation of the
carboxyl group at C-21 in intermediary 3-(3ꢀ,17ꢀ␤-dihydroxy-
1ꢀ,3ꢀ,5ꢀ(10),14ꢀ-estratetraene-17ꢀ␣-yl)propanoic acid (11a) with
NH3 in the presence of a coupling reagent such as diethyl
The presence of a ꢀ14-bond in 9a and 9b was confirmed by the
1H signal of 15-H occurring at 5.18 ppm in 9a and 5.28 ppm in 9b as
a broad singlet. On the other hand, the 20-H and 21-H2 signals (at
6.00 and 5.20 ppm as a multiplet, respectively) observed in 8 disap-
pears in the spectra of 9a and 9b. The stereochemical configuration
at C-17 in 9a and 9b was evidenced by the 1H signal of 18-H3: this
signal resonates at 0.99 ppm as a singlet in 9b, but it shifts to down-
field by 0.09 ppm and appears at 1.08 ppm in 9a, probably owing
to the steric hindrance of a bulky 17␤-hydroxypropyl group. Simi-
larly, the 13C signal arising from C-18 in 9a resonates at 17.8 ppm,
whereas the corresponding signal in 9b appears at 21.4 ppm (ꢂ-
value, 3.6 ppm). A comparison of the 13C signal at C-12 also revealed
that the resonance position is at 34.8 ppm in 9a and at 30.9 ppm in
9b (ꢂ-value, 3.9 ppm), which are in accord with those reported for
the analogous derivatives of 3,17␰-estradiols reported previously
(Dionne and Poirier, 1995; Dionne et al., 1997). These observed dif-
ferences in the 13C NMR spectra of the two epimers permit them
to be distinguished from each other.
phosphorocyanide,
4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-
methylmorpholinium chloride or water soluble carbodiimide
(WSCD) in peptide synthesis was unsuccessful (Iida et al., 1995,
2006; Momose et al., 1997); 11a was prepared by alkaline hydrol-
ysis of 10a (unpublished data), but subsequent amidation of the
resulting 11a did not proceed at all. However, when the ꢀ14-17␤-
O-/17␣-C-spiro-␥-lactone 10a was subjected to direct amidation
with a saturated methanolic NH3 solution containing sodium
cyanide as a catalyst at 60 ◦C for 24 h under positive pressure
(50 kPa) of H2, the reaction proceeded smoothly to give the desired
As has been previously reported for steroid derivatives bear-
ing both primary and tertiary hydroxy groups at C-16/C-17 or
C-17/C-17 position in the same molecule, the carboxyl group
resulting from oxidation of the primary hydroxy group, via the alde-
hyde intermediate, undergoes intramolecular lactonization with
the tertiary hydroxy group to produce the corresponding lactone
derivatives (Bydal et al., 2004). In fact, when 9a and 9b were
subjected to oxidation with PDC in DMF at room temperature
for 6 h, facile intramolecular lactonization occurred between the
17- and 21-hydroxy groups to yield directly the corresponding
ꢀ
14-17␤-estradiol 17␣-propanamide 1a in isolated yield of 40%.
An essentially identical result was also obtained by transforma-
tion of the stereoisomeric
14-17␣-O-/17␤-C-spiro-␥-lactone
ꢀ
10b into the corresponding 17␣-hydroxy-17␤-propanamide 1b.
Total yields of 1a and 1b from estrone (2a) were 4.3 and 1.1%,
respectively.
The IR spectra of 1a and 1b showed the characteristic absorption
peak at 1648–1682 cm−1 arising from a carbonyl group of the amide
linkage. A characteristic feature serving to differentiate between
the two stereoisomeric pairs was observed in the 1H and 13C NMR
spectra. The 1H NMR spectra of the two isomeric pairs are very
ꢀ
14-17␤-O-/17␣-C-spiro-␥-lactone 10a and its ꢀ14-17␣-O-/17␤-
C-isomer 10b, respectively. The presence of the spiro-␥-lactone
ring in 10a and 10b was characterized by the IR absorption peak
at 1735–1746 cm−1 due to the carbonyl group of the lactone moi-
ety. Although no significant difference in the 1H NMR spectra was
observed between the two C-17 epimeric 10a and 10b, they were
distinctly differentiated by the 13C NMR. In 10a and 10b, the 13C

112
T. Iida et al. / Chemistry and Physics of Lipids 164 (2011) 106–112
similar to each other, except for the 18-H3. The angular 18-H3 signal
appearing at 0.98 ppm in 1a (measured in DMSO-d6) is shifted to
up-field by 0.08 ppm and resonates at 0.90 ppm in 1b, because of
the ␣-configuration of a sterically crowded propanamide group.
Both the epimers exhibited the 13C signal at 114.6 (1a) and 115.4
(1b) ppm (ꢂ-value, 0.8 ppm) owing to C-15. In addition, while 1a
showed the 13C signals at 18.0 ppm (C-18) and 32.1 ppm (C-12),
the corresponding signals in 1b occurred at 20.8 ppm (C-18) and
29.6 ppm (C-12). These differences between the two epimeric pairs
are in good agreement with those observed for analogous pairs at
C-17 in stereoisomeric17␰-estradiol derivatives (Bydal et al., 2004;
Dionne and Poirier, 1995; Dionne et al., 1997).
Thus, we described herein the chemical synthesis of stereoiso-
meric 3-(3ꢀ,17ꢀ␰-dihydroxy-1ꢀ,3ꢀ,5ꢀ(10ꢀ),14ꢀ-estratetraene-17ꢀ␰-yl)
propanamide (1a and 1b). The biological evaluation of the syn-
thesized estrogen derivatives, which includes enzyme-inhibitory
effect, cytotoxicity, and antitumor activity against 17␤-HSD and
cancer cell lines is now progressing in our laboratory, and the result
will be reported elsewhere.
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Kakiyama contributed able technical assistance. This work was
supported in part by a Grant-in-Aid for Scientific Research from
the Ministry of Education, Culture, Sports, Science and Technol-
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of Natural Sciences, Nihon University, Joint Research Grant for
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