6β,19-Bridged androstenedione analogs as aromatase inhibitors

Article abstract of DOI:10.1016/j.steroids.2009.06.001

Inhibition of aromatase is an efficient approach for the prevention and treatment of breast cancer. New 6β,19-bridged steroid analogs of androstenedione, 6β,19-epithio- and 6β,19-methano compounds 11 and 17, were synthesized starting from 19-hydroxyandros

Full text of DOI:10.1016/j.steroids.2009.06.001

Steroids 74 (2009) 884–889
Contents lists available at ScienceDirect
Steroids
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6␤,19-Bridged androstenedione analogs as aromatase inhibitors
a
a
a
b
a,∗
Sachiko Komatsu , Ayaka Yaguchi , Kouwa Yamashita , Masao Nagaoka , Mitsuteru Numazawa
a
Tohoku Pharmaceutical University, 4-1 Komatsushima-4-chome, Aoba-ku, Sendai 981-8558, Japan
Nihon Pharmaceutical University, 10281 Komuro, Ina-machi, Kita-adachi-gun, Saitama 362-0806, Japan
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Inhibition of aromatase is an efficient approach for the prevention and treatment of breast cancer. New
␤,19-bridged steroid analogs of androstenedione, 6␤,19-epithio- and 6␤,19-methano compounds 11
and 17, were synthesized starting from 19-hydroxyandrostenedione (6) and 19-formylandrost-5-ene-
␤,17␤-yl diacetate (12), respectively, as aromatase inhibitors. All of the compounds including known
steroids 6␤,19-epoxyandrostenedione (4) and 6␤,19-cycloandrostenedione (5) tested were weak to poor
competitive inhibitors of aromatase and, among them, 6␤,19-epoxy steroid 4 provided only moderate
inhibition (Ki: 2.2 ␮M). These results show that the 6␤,19-bridged groups of the inhibitors interfere with
binding in active site of aromatase.
Received 15 April 2009
Received in revised form 1 June 2009
Accepted 3 June 2009
6
3
Available online 12 June 2009
Keywords:
Aromatase inhibitor
6␤,19-Epithioandrostenedione
6␤,19-Methanoandrostenedione
6␤,19-Epoxyandrostenedione
6␤,19-Cycloandrostenedione
©
2009 Elsevier Inc. All rights reserved.
Human placental microsomes
1. Introduction
[28,29], 6␤,19-epithio- [30], 6␤,19-methano- [31], and 6␤,19-cyclo-
32] progesterones and its 21-hydroxy analogs, have been shown
[
Aromatase is the cytochrome P-450–enzyme complex respon-
sible for estrogen biosynthesis [1–3]. The aromatase reaction is
thought to proceed through three sequential oxygenations at C-19
of the androgen androst-4-ene-3,17-dione (androstenedione) and
testosterone [4–11] (Fig. 1). The first two oxygenations occur at C-19
positiontoproduce19-hydroxyand19,19-dihydroxyintermediates.
In the third step, C-19 and the 1␤,2␤-hydrogens are eliminated as
formic acid and water, respectively. There appear to be a major
clinical role for methods of controlling estrogen-dependent breast
cancer. The inhibitors of aromatase represent one route to such
control [12–15].
to possess new biological activities of steroid hormones (Fig. 2).
However, effect of 6␤,19-bridged structures on the aromatase activ-
ity have not been explored so far. In this paper, we describe the
synthesis of 6␤,19-bridged steroids, epoxy-, cyclo-, epithio-, and
methano-androstenediones (4, 5, 11, and 17), and their inhibitory
activity of human placental aromatase.
2. Experimental
2.1. Materials and general methods
A number of potent aromatase inhibitors, analogs of the natural
substrate androstenedione, have been the subjects of clinical trials.
Previously, we have reported a series of 6␤-alkylandrostenediones
as aromatase inhibitors of which 6␤-ethyl compound is extremely
powerful inhibitor with Ki of 1.4 nM [16–19]. The 6␤-hydroxy
derivative, a polar steroid, has also high activity to aromatase
6␤,19-Epoxyandrost-4-ene-3,17-dione (4) [33] and 6␤,19-
cycloandrost-4-ene-3,17-dione (5) [34] were synthesized by the
3
previous reported methods. [1␤- H] androstenedione (specific
activity, 27.5 Ci/mmol; 3H-distribution 74–79%) was obtained from
New England Nuclear (Boston, MA, USA). NADPH was purchased
from Sigma–Aldrich (St. Louise, MO, USA).
[
20,21]. On the other hand, l9-substituted (hydroxy, methyl and
Melting points were measured on a Yanagimoto melting point
apparatus (Kyoto, Japan) and are uncorrected. Infra red (IR) spectra
were recorded on a PerkinElmer FT-IR 1725X spectrophotometer
in a KBr pellet or Nujor form, and ultra-violet (UV) spectra were
determined in 95% ethanol on a Hitachi 150-20 spectrophotometer
(Tokyo, Japan). Nuclear magnetic resonance (NMR) spectra were
sulfur) androstenedione analogs have been reported as good to
powerful competitive inhibitors of the enzyme [22–27]. Consider-
ing the facts obtained by the 6␤- and 19-substituted steroids, we
aimed at compounds with 6␤,19-bridged androstenediones as aro-
matase inhibitors. Previously, 6␤,19-bridged steroids, 6␤,19-epoxy-
1
obtained in CDCl3 solution with JEOL LA 400 (400 MHz for H) and
JEOL LA 600 (600 MHz for 1H and 150 MHz for C) spectrome-
ter (Tokyo, Japan) using tetramethylsilane as an internal standard,
and mass (MS) spectra (electron impact mode) and high resolution
13
∗ Corresponding author. Tel.: +81 22 727 0138; fax: +81 22 727 0137.
E-mail address: numazawa@tohoku-pharm.ac.jp (M. Numazawa).
0
039-128X/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2009.06.001

S. Komatsu et al. / Steroids 74 (2009) 884–889
885
Fig. 1. Aromatase reaction of androstenedione by human placental aromatase.
(
HR)-MS with a JEOL JMS-700 spectrometer. Thin-layer chromatog-
2.3. 19-Acetylthioandrost-4-ene-3,17-dione (8)
raphy (TLC) was performed on E. Merck precoated plates (silica
gel 60F-254, Darmstadt, Germany). Column chromatography was
conducted with silica gel 60, 70–230 mesh (E. Merck).
A solution of 19-hydroxyandrostenedione (6) (1.08 g, 3.57 mmol)
in cold pyridine (10.8 mL) was added dropwise to a stirred solution
of trifluoromethanesulfonic anhydride (1.34 mL, 7.96 mmol) in cold
pyridine (7.4 mL) under nitrogen [30]. The mixture was allowed to
warm to room temperature for 1.5 h and diluted with EtOAc. After
acidification with 5% HCl, the organic layer was washed with sat.
NaHCO3 solution and water and dried with Na SO . A yellow solid,
a crude 19-trifilate (1.42 g), obtained by evaporation of the solvent,
was used without further purification in the following step.
A solution of the 19-triflate 7 (1.42 g) and potassium thioacetate
2.2. 6ˇ,19-Cycloandrost-4-ene-3,17-dione (5)
p-Toluenesulfonyl chloride (1.13 g, 5.93 mmol) was added to a
2
4
solution of 19-hydroxyandrostenedione (6) (556 mg, 1.84 mmol) in
◦
pyridine (27.8 mL) at 0 C [34]. The reaction mixture was stand for
3
0 min and allowed to warm to room temperature. After 116 h, the
mixture was poured to ice water, acidified by 10% HCl, extracted
with EtOAc. The organic layer was washed with 5% NaHCO3 solu-
(
1.42 g, 12.45 mmol) in acetone (73 mL) was stirred at room temper-
ature for 24 h under nitrogen. The reaction mixture was then diluted
tion and water and dried with Na SO . The evaporation of the
2
4
with EtOAc, washed with sat. NaHCO solution and water, and dried
3
organic solvent afforded 19-tosylate (802 mg, 96%). 28% NaOCH3
in CH OH solution (0.48 mL) was added to the 19-tosylate (402 mg,
with Na SO . Evaporation of the solvent yielded an oil which was
2
4
3
purified by column chromatography (hexane–EtOAc, 3:1, v/v) to
0
.88 mmol) in CH OH (12.4 mL) under nitrogen. The mixture was
afford 19-thioacetate 8 as pale yellow oil (558 mg, 43%). 1H NMR ı:
3
heated under reflux for 5.5 h, diluted with EtOAc, washed with sat.
NaHCO3 solution and water, and dried with Na SO . Evaporation
0
.95 (3H, s, 18-CH ), 2.34 (3H, s, SCOCH ), 3.20 and 3.52 (1H each,
3
3
2
4
−1
d, J = 14.0 Hz, 19-H ), 5.90 (1H, s, 4-H). IR (Nujor) cm : 3586 (OH),
2
of the solvent gave an oil which was purified by column chro-
matography (hexane–EtOAc, 5:1, v/v) followed by preparative TLC
1
3
733, 1683, and 1652 (C O). UV ꢀmax nm (ε): 238 (16,600). MS m/z:
60 (M , 31%), 317 (58), 284 (18), 272 (100). HR-MS calculated for
+
(hexane–EtOAc, 1:1, v/v). A solid material was recrystallized from
C21H28O S: 360.1759. Found: 360.1766.
3
acetone to give 6␤,19-cycloandrostenedione (5) (110 mg, 44%), mp
◦
◦
1
1
3
28–132 C (reported, 132 C [34]). H NMR ı: 0.97 (3H, s, 18-CH ),
3
2.4. 3,17-Diethylenedioxy-19-acetylthioandrost-5-ene (9)
.24 (1H, t, J = 5.6 Hz, 6-H), 5.61 (1H, s, 4-H). 13C NMR ı: 14.43 (C-
1
8), 21.28 (C-11), 24.39 (C-15), 30.67 (C-12), 31.69 (C-2), 32.35 (C-1),
A mixture of the 19-thioacetate 8 (502 mg, 1.39 mmol) and ethy-
3
4
3.72 (C-8), 33.95 (C-19), 35.73 (C-16), 43.24 (C-6), 49.02 (C-13),
9.48 (C-9), 49.79 (C-14), 49.99 (C-10), 111.06 (C-4), 180.19 (C-5),
lene glycol (3.5 mL) in benzene (16 mL) was distilled for 15 min to
remove traces water. p-Toluenesulfonic acid monohydrate (15 mg)
was added to the reaction mixture and the mixture was heated
under reflux for 4 h, diluted with EtOAc, and added sat. NaHCO3
solution. The organic layer was washed with water, dried with
−1
198.88 (C-3), 219.46 (C-17). IR (KBr) cm : 1732 and 1655 (C O).
+
UV ꢀmax nm (ε): 242 (13,900). MS m/z: 284 (M , 100%). Analy-
sis calculated for C19H24O : C, 80.24; H, 8.51. Found: C, 80.15; H,
2
8
.56.
Na SO , and evaporated to give a semi-solid which was purified by
2
4
column chromatography (hexane–EtOAc, 20:1, v/v) to afford ketal 9
(
432 mg, 69%) as an oil. 1H NMR ı: 0.87 (3H, s, 18-CH ), 2.31 (3H, s,
3
SCOCH ), 3.05 and 3.43 (1H each, d, J = 14.2 Hz, 19-H ), 3.85 (8H, m,
3
2
−
,17-OCH CH O–), 5.46 (1H, m, 6-H). IR (Nujor) cm : 1689 (C O).
2 2
1
3
+
UV ꢀmax nm (ε): 235 (5200). MS m/z: 448 (M , 21%), 405 (17), 359
32), 297 (69), 99 (100). HR-MS calculated for C25H36O5S: 448.2284.
Found: 448.2292.
(
2.5. 6ˇ,19-Epithioandrost-4-ene-3,17-dione (11)
A solution of the ketal 9 (287 mg, 0.64 mmol) in dry CH OH
3
(8 mL) was deoxygenated by bubbling dry nitrogen for 15 min. Then
a solution of potassium hydroxide (58 mg, 1.04 mmol) in CH OH
3
(
0.4 mL) was added and the mixture was stirred at room tempera-
ture for 30 min. The reaction mixture was neutralized with 5% HCl
and diluted EtOAc. The organic layer was washed with sat. NaHCO3
solution and water, and dried with Na SO , and evaporated to afford
2
4
the crude 19-thiol 10 (176 mg). Iodine (234 mg, 0.93 mmol) and tri-
ethylamine (61 mL, 0.43 mmol) in dry CH Cl2 (115 mL) were added
2
to a solution of the crude 19-thiol 10 (176 mg) in CH Cl (72 mL)
2
2
◦
at 0 C, then the mixture was stirred and allowed to warm to room
temperature. After 2 h, the reaction mixture was diluted with EtOAc
and washed with sat. Na2S2O3 solution and water, and dried with
Fig. 2. Structures of 6,19-bridged progesterone analogs and androstenediones.

8
86
S. Komatsu et al. / Steroids 74 (2009) 884–889
◦
Na SO . Evaporation of the solvent gave a solid which was puri-
in CH OH (1.5 mL) under nitrogen and warmed at 50 C. After
2
4
3
fied by column chromatography (hexane–EtOAc, 3:1, v/v) to yield a
crude 6,19-epithioandrostenedione 11 (33 mg, 16%). Recrystalliza-
tion of the crude compound from acetone afforded compound 11,
1.5 h, the reaction mixture was diluted with EtOAc and neu-
tralized with 5% HCl. The organic layer was washed with sat.
NaHCO3 solution and water, and dried with Na SO . Evaporation
of the solvent gave a crude 5␣-chloro-6␤,19-methanoandrostane-
3␤,17␤-diol (15) (43 mg, 99%).
2
4
◦
1
mp 251–256 C. H NMR ı: 0.99 (3H, s, 18-CH ), 2.61 and 3.07 (1H
3
each, d, J = 10.3 Hz, 19-H ), 3.95 (1H, m, 6-H), 5.81 (1H, s, 4-H). IR
2
−1
(
KBr) cm : 1738 and 1667 (C O). UV ꢀmax nm (ε): 241 (14,000).
MS m/z: 316 (M , 100%), 288 (10), 260 (18), 242 (15), 152 (34). Anal-
Pyridinium chlorochromate (113 mg, 0.53 mmol) and BaCO3
(66 mg, 0.34 mmol) were added to a solution of the crude diol 15
+
ysis calculated for C19H24O S: C, 72.11; H, 7.64; S, 10.13. Found: C,
(43 mg, 0.13 mmol) in CH Cl2 (1.5 mL) under nitrogen atmosphere.
2
2
72.35; H, 7.30; S, 10.20.
The reaction mixture was stirred for 1.5 h and passed through sil-
ica gel column (5 g) to remove a dark brawn solid. Elution with
2
3
.6. 5˛-Chloro-19a-hydroxy-6ˇ,19-methanoandrostane-
ˇ,17ˇ-yl diacetate (13)
CH Cl and evaporation of the solvent gave a crude 5␣-chloro-
6␤,19-methanoandrostane-3,17-dione (16) (31 mg, 72%).
2 2
A solution of the crude 5␣-androstane steroid 16 (29 mg,
A solution of 19-formylandrost-5-ene-3␤,17␤-yl diacetate (12)
0.09 mmol) in CH Cl2 (1 mL) was stirred for 6 h in the presence of
2
(
608 mg, 1.51 mmol) in dry CH Cl (6 mL) was added to a solution of
Al O (192 mg, 1.88 mmol). Al O was removed by filtration and the
solvent was evaporated to yield a solid. The solid was recrystallized
2
2
2 3 2 3
◦
titanium chloride (3 mL) in CH Cl2 (10 mL) at −70 C under a nitro-
2
◦
gen atmosphere [31]. The reaction mixture was stirred at −30 C
from acetone to afford 6␤,19-methanoandrost-4-ene-3,17-dione
◦
1
for 2 h, and C H5OH–water (1:3, 12 mL) was added. The resulting
(17) (10 mg, 42%), mp 196–199 C. H NMR ı: 0.95 (3H, s, 18-CH ),
2
3
◦
−1
solution was further stirred at 0 C for 15 min. The mixture was
diluted with EtOAc, washed with sat. NaHCO3 solution and water,
and dried with Na SO . The solvent was evaporated to give a solid
2.85 (1H, br. s, 6-H), 5.77 (1H, s, 4-H). IR (KBr) cm : 2927 (SR), 1738
and 1670 (C O). UV ꢀmax nm (ε): 243 (14,400). MS m/z: 298 (M ,
+
100%), 256 (84), 148 (44), 135 (82), 91 (39). Analysis calculated for
2
4
which was purified by column chromatography (hexane–EtOAc,
:1, v/v) and recrystallization from acetone to afford 5␣-chloro-
9a-hydroxy-6␤,19-methanoandrostane-3␤,17␤-yl diacetate (13)
C20H26O : C, 80.50; H, 8.78. Found: C, 80.28; H, 8.90.
2
8
1
2.9. Enzyme preparation
◦
266 mg, 40%), mp 158–162 C. H NMR ı: 0.79 (3H, s, 18-CH ),
3
1
(
2
(
.72 (1H, t, J = 12.8 Hz, 6␣-H), 4.27 (1H, d, J = 7.7 Hz, 19a-CH), 4.60
Human placental microsomes (sedimented after 60 min at
1H, t, J = 8.4 Hz, 17-H), 5.15 (1H, m, 3-H). 13C NMR ı: 12.49 (C-18),
1
05,000 × g) were obtained as described by Ryan [36]. They were
21.14 and 21.36 (3␤- or 17␤-OCOCH ), 21.67 (C-11), 22.96 (C-15),
3
washed once with 0.05 mM dithiothreitol, lyophilized and stored at
2
5.89 (C-1), 26.71 (C-2), 27.58 (C-16), 29.86 (C-7), 33.87 (C-8), 36.54
◦
−
80 C. No significant loss of activity occurred during the period (6
(
(
C-19), 36.70 (C-12), 41.45 (C-4), 43.31 (C-10), 47.50 (C-14), 48.03
C-13), 49.15 (C-9), 54.35 (C-6), 70.30 (C-3), 73.31 (C-19a), 82.08 (C-
months) of this study. The preparation of human placental micro-
somes was conducted under the approval of the ethical review
committeeofTohokuPharmaceuticalUniversityinaccordancewith
the standard of the Helsinki Declaration.
5
), 82.41 (C-17), 170.45 and 171.10 (3␤- or 17␤-OCOCH ). IR (KBr)
3
−
1
+
cm : 3465 (OH) and 1723 (C O). MS m/z: 438 (M , 0.5%), 378 (11),
34 (100), 298 (85). Analysis calculated for C24H35ClO5: C, 65.66;
H, 8.04. Found: C, 65.87; H, 8.25.
3
2
.10. Aromatase assay procedure
2
.7. 5˛-Chloro-6ˇ,19-methanoandrostane-3ˇ,17ˇ-yl diacetate
Aromatase activity was measured essentially according to the
(
14)
original procedure of Siiteri and Thompson [37]. The screening
assay for determination of IC50 value and the kinetic assay were
carried out essentially according to the assay methods described in
ourpreviouswork[21]. Briefly, 20 ␮gofproteinfromthelyophilized
microsomes and 20-min incubation period were used for the
screening assay, and 20 ␮g of protein from the microsomes and a
A mixture of DBU (110 ␮L) and CS2 (348 ␮L) was added to
a solution of the 19a-hydroxy-6␤,19-methano steroid 13 (91 mg,
.21 mmol) in dimethylformamide (1.25 mL) under nitrogen atmo-
0
sphere. The reaction mixture was stirred for 1 h, then CH I (2.5 mL)
was added [35]. After 2 h, the mixture was diluted with EtOAc which
was washed successively with sat. KHSO4 solution, sat. NaHCO3
solution, and water. The organic layer was dried with Na SO .
Evaporation of the solvent gave the residue which was dissolved
3
5
-min period were used for the kinetic assay. The assays were car-
◦
ried out at 37 C in 67 mM phosphate buffer, pH 7.4, in the presence
of NADPH under air.
2
4
Apparent K values were calculated using non-linear regression
i
in dry toluene (2 mL). Bu SnH (73 ␮L) was added to the solution
under nitrogen and the mixture was heated under reflux for 17 h
3
analysis with GraFit software [38].
and another Bu SnH (73 ␮L) was added to the mixture which was
3
heated under reflux for 6.5 h. Then the reaction mixture was diluted
with EtOAc and insoluble materials were filtered off. Evaporation
of the solvent yielded a solid which was purified by column chro-
matography (hexane–EtOAc, 10:1, v/v) followed by recrystallization
3. Results and discussion
3.1. Chemistry
from CH OH to give 5␣-chloro-6␤,19-methanoandrostane-3␤,17␤-
yl diacetate 14 (47 mg, 54%), mp 110–114 C. H NMR ı: 0.82 (3H,
6␤,19-Epoxyandrostenedione (4) [33] and 6␤,19-cycloandro-
stenedione (5) [34] were prepared according to the reported meth-
ods. The structure of the 6␤,19-cyclo compound 5, obtained by
reaction of 19-tosylate of 19-hydroxyandrostenedione (6) with
sodium methoxide, was reconfirmed by two-dimensional NMR
spectroscopy.
3
◦
1
s, 18-CH ), 4.60 (1H, t, J = 8.3 Hz, 17-H), 5.15 (1H, m, 3-H). IR (KBr)
3
−
1
+
cm : 1733 (C O). MS m/z: 422 (M , 6%), 362 (45), 326 (76), 119
100). Analysis calculated for C24H35ClO : C, 68.15; H, 8.34. Found:
(
4
C, 68.33; H, 8.51.
Burton et al. [30,31] have reported synthesis of series of
6␤,19-epithio- and 6␤,19-methano-progesterone analogs to expect
new biological activities as steroid hormones. In the course of
our studies, we were of interest in 6␤,19-bridged androstene-
dione analogs as the aromatase inhibitors. We initially tried
2
.8. 6ˇ,19-Methanoandrost-4-ene-3,17-dione (17)
Potassium hydroxide (23.5 mg, 0.42 mmol) in CH OH (0.71 mL)
3
was added to a solution of the diacetate 14 (50 mg, 0.12 mmol)

S. Komatsu et al. / Steroids 74 (2009) 884–889
887
Fig. 3. Synthesis of 6,19-epithioandrostenedione (11).
synthesis of 6␤,19-epithioandrostenedione (11) starting from 19-
hydroxyandrostenedione (6) (Fig. 3). The 19-hydroxy group of
compound 6 was converted to triflate 7 by treatment with tri-
fluoromethanesulfonic anhydride in pyridine and displaced with
procedure [40]. The 19a-alcohol 13 was converted into the 19a-
xanthate derivative by reaction with CS and methyl iodide and this
2
was treated with tributyltin hydride in toluene to give the deoxy-
genated compound 14. The 1H NMR spectrum of this compound
showed an absence of a hydroxymethine resonance at 3.5–4.5 ppm.
The 3␤,17␤-acetoxy moieties of compound 14 was eliminated by
the alkaline hydrolysis to yield 3␤,17␤-dihydroxy compound 15.
Pyridinium chlorochromate oxidation of compound 15 followed by
treatment with Al O afforded the other desired compound, 6␤,19-
potassium thioacetate in acetone to give 19-thioacetyl derivative
1
8
. The H NMR spectrum of this compound showed a singlet peak
of an acetylthio group (3H, 2.34 ppm) and two doublet peaks of
9-CH2 (3.20 and 3.52 ppm, J = 14.0 Hz). Compound 8 was con-
1
verted to ethyleneketal 9 in which the double bond migrated to
2
3
1
the 5,6-position ( H NMR: multiplet signal at 5.46 ppm of C-6) as
methano compound 17.
required for the 6␤,19-cyclization [30], and the 19-thioacetate was
hydrolyzed with potassium hydroxide in methanol to give the free
thiol 10. Treatment of the thiol 10 by triethylamine and iodine
resulted in the one-pot conversion of compound 10 into 6␤,19-
epithio compound 11 based upon the 1H NMR spectrum which
showed 2.61 and 3.07 ppm (1H each, d, J = 10.3 Hz) for 19-H, and
The structures of the compounds synthesized were confirmed
by the spectrometric analysis, HR-MS, and elemental analysis.
3.2. Biochemical properties
The inhibitory activities of human placental aromatase by the
6␤,19-bridged steroids 4, 5, 11 and 17 were tested. Aromatiza-
tion activity in placental microsomes was determined using a
3.95 ppm (1H, m) for 6-H.
Next, 6␤,19-methanoandrostenedione (17) was synthesized
3
from 19-formylandrost-5-ene-3␤,17␤-yl diacetate (12) through six
steps (Fig. 4). The key step in the synthesis was the cyclization of
an additional carbon atom at C-19a [31]. Titanium tetrachloride
has been the Lewis acid of choice for the Prins reaction to give
radiometric assay in which tritiated water released from [1␤- H]
androstenedione into the incubation medium during aromatiza-
tion was measured [37]. To characterize the nature of inhibitor
binding to the active site of aromatase, aromatization was mea-
sured at several concentrations and substrate concentrations. The
results of these studies were plotted on a typical Lineweaver–Burk
plot in all cases. The results are given in Table 1. In these studies,
apparent Km and Vmax values for androstenedione were 35 nM and
110 pmol/min/mg protein, respectively. All of the steroids showed
clear-cut competitive inhibition (Fig. 5). The apparent Ki values
were obtained by Dixon plots. The bridged steroids 4, 5, 11 and 17
were weak to poor competitive inhibitors to aromatase in human
placental microsomes in which the 6␤,19-epoxy steroid 4 was the
bridged chloroalcohol [39]. Treatment of compound 12 with the
◦
titanium salt in CH Cl2 at −50 C gave the Prins reaction product,
2
5
␣-chloro-6␤,19-methano steroid 13, with a hydroxyl group at the
C-19a position. The H NMR spectrum showed a methine proton at
1
4
.27 ppm (J = 7.7 Hz) for the 19a-position and 2.72 ppm (J = 12.8 Hz)
for the 6␣-position. Thus the formation of the 6␤,19-methano ring
proceeded through a stereo-controlled way; the stereochemistry of
the 19a-hydroxy group was not established by the NMR spectrome-
tries. This hydroxyl group was eliminated by Barton deoxygenation
Fig. 4. Synthesis of 6,19-methanoandrostenedione (17).

8
88
S. Komatsu et al. / Steroids 74 (2009) 884–889
Table 1
In vitro aromatase inhibition by 6␤,19-bridged ADs (4, 5, 11 and 17).
tively. The aromatase inhibitory activities of the 6␤,19-brigded
compounds were disappointingly low, however, their new biolog-
ical properties such as androgenic/antiandrogenic activities are of
interest and studies of them will be carried in our laboratory.
Compound
IC50 (␮Ma)
Apparent Ki (␮Mb)
6
6
6
6
,19-epoxy, 4
,19-cyclo, 5
,19-epithio, 11
,19-methano, 17
44 ± 2.0
>100
>100
2.2 ± 0.17
18 ± 1.0
18 ± 1.4
16 ± 1.0
Acknowledgments
>100
a
3
3
00 nM of [1␤- H]AD 20 ␮g protein from human placental microsomes and 20-
This work was supported in part by a High Technology Research
Center Project from the Ministry of Education, Culture, Sports, and
Technology of Japan. Human term placenta was kindly donated by
Dr. Michihiro Yuki of Yuki Lady’s Clinic.
min incubation time were used.
b
All of the inhibitors examined showed a competitive type inhibition on the basis
of the Lineweaver–Burk plot and apparent inhibition constant (Ki) was obtained by
Dixon plot. Twenty micrograms protein from the placental microsomes and 5-min
incubation time were used.
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