Role of hydrophilic interaction in binding of hydroxylated 3-deoxy C19 steroids to the active site of aromatase

Article abstract of DOI:10.1021/jm010282t

As part of our investigation into the structure - activity relationship of a novel class of aromatase inhibitors, C₁₉ steroids having no oxygen function at C-3, we tested aromatase inhibition activity of polar diol compounds 4,19-dihydroxyandrost-5-en-17-ones (25 and 27) and 6,19-dihydroxyandrost-4-en-17-ones (36 and 37). 4α,19-Diol 25 was synthesized from tert-butyldimethylsilyoxyandrost-4-ene steroid (9) through its OsO₄ oxidation, giving the 4α,5α-dihydroxy derivative 12, as a key reaction. Acetylation of 5β,6α-dihydroxy-19-acetate 30 and its 5α,6β-analogue 31 followed by dehydration with SOCl₂ and alkaline hydroxysis gave 6α,19-diol 36 and its 6βisomer 37, respectively. The stereochemistry of a hydroxy group at C-4 of compound 25 and that at C-6 of compounds 36 and 37 were determined on the basis of ¹H NMR spectroscopy in each case. 4β,19-Diol 27, previously synthesized, was identified as an extremely powerful competitive inhibitor of aromatase (K_i = 3.4 nM). In contrast, its 4α,19-dihydroxy isomer 25 and other series of diol compounds, 6,19-dihydroxy-4-en-17-one steroids, were moderate to poor competitive inhibitors (K_i = 110-800 nM). Through this series of analyses, it was concluded that hydrophilic interaction of a 4β,19-diol function with the active site of aromatase plays a critical role in the tight binding of 3-deoxy-5-ene steroids.

Full text of DOI:10.1021/jm010282t

J . Med. Chem. 2001, 44, 4277-4283
4277
Role of Hyd r op h ilic In ter a ction in Bin d in g of Hyd r oxyla ted 3-Deoxy C19
Ster oid s to th e Active Site of Ar om a ta se
Mitsuteru Numazawa,* Keiko Yamada, Syoko Nitta, Chika Sasaki, and Kanae Kidokoro
Tohoku Pharmaceutical University, 4-1 Komatsushima-4-chome, Aobaku, Sendai 981-8558, J apan
Received J une 21, 2001
As part of our investigation into the structure-activity relationship of a novel class of aromatase
inhibitors, C19 steroids having no oxygen function at C-3, we tested aromatase inhibition activity
of polar diol compounds 4,19-dihydroxyandrost-5-en-17-ones (25 and 27) and 6,19-dihydroxyan-
drost-4-en-17-ones (36 and 37). 4R,19-Diol 25 was synthesized from tert-butyldimethylsily-
oxyandrost-4-ene steroid (9) through its OsO4 oxidation, giving the 4R,5R-dihydroxy derivative
12, as a key reaction. Acetylation of 5â,6R-dihydroxy-19-acetate 30 and its 5R,6â-analogue 31
followed by dehydration with SOCl2 and alkaline hydroxysis gave 6R,19-diol 36 and its 6â-
isomer 37, respectively. The stereochemistry of a hydroxy group at C-4 of compound 25 and
that at C-6 of compounds 36 and 37 were determined on the basis of 1H NMR spectroscopy in
each case. 4â,19-Diol 27, previously synthesized, was identified as an extremely powerful
competitive inhibitor of aromatase (Ki ) 3.4 nM). In contrast, its 4R,19-dihydroxy isomer 25
and other series of diol compounds, 6,19-dihydroxy-4-en-17-one steroids, were moderate to poor
competitive inhibitors (Ki ) 110-800 nM). Through this series of analyses, it was concluded
that hydrophilic interaction of a 4â,19-diol function with the active site of aromatase plays a
critical role in the tight binding of 3-deoxy-5-ene steroids.
In tr od u ction
Aromatase, a unique cytochrome P-450-enzyme com-
plex, catalyzes the conversion of 4-en-3-one androgens,
androst-4-ene-3,17-dione (androstenedione, 1), and tes-
tosterone to the phenolic estrogens, estrone, and
estradiol.1-3 Inhibitors of this enzyme are useful in
treating estrogen-dependent diseases such as breast
cancer.4-9 For this reason, various steroids have been
tested in a number of laboratories as aromatase inhibi-
tors. Structure-activity studies on aromatase inhibi-
tors10-19 and a recent structural prediction of aromatase
by homology modeling20-25 predicted the existence of a
hydrophobic binding pocket extending roughly in the
plane of the substrate androstenedione (1) from the
position that would be occupied by its C4, C6, and C7
atoms. Thus, introducing a hydrophilic group, such as
a hydroxyl, at the C-626 and C-192 positions of steroid 1
decreases the affinity to the active site of aromatase.
We have previously reported that a 3-deoxy derivative
of compound 1, androst-4-en-17-one (2), is an excellent
competitive inhibitor of aromatase,19,27 although this is
F igu r e 1. Structures of androstenedione and 3-deoxy steroids.
lacking a carbonyl group at C-3 that is thought to be
essential for the proper binding of substrate 1 to the
active site of the enzyme.20-25 On the other hand,
androst-5-en-17-one (6), an isomer of 3-deoxy steroid 2,
is a fair competitive inhibitor28 (Figure 1). The structure-
activity relationships indicated that a 17-carbonyl func-
tion is necessary for an effective binding of 3-deoxy
steroids 2 and 6 to the active site and that binding
geometries of the two steroids are different in the region
of an A-B ring system of the steroid molecule.28,29 The
binding pocket also tolerates a polar hydroxy group at
the 6- or 19-position of 4-ene steroid 227,30 and at the
4â-position of 5-ene steroid 6.31 The 4-ene system plays
a critical role in the tight binding of a steroid without a
carbonyl function at C-3 to the active site.
In continuing the study of the 3-deoxy steroids as
conformational and catalytic probes for the active site
of aromatase, we were interested in steroids that have
a hydrophilic structure combining the 19-hydroxy group
with a hydroxy function at the allylic position, i.e., C-4
of the 5-ene 6 or C-6 of 4-ene steroid 2. This study
focused on the synthesis and biochemical evaluation of
4,19-dihydroxyandrost-5-en-17-ones and 6,19-dihydroxy-
androst-4-en-17-ones. 5-Ene-4â,19-diol 27 was found to
* To whom correspondence should be addressed. Phone: +81-22-
234-4181, extension 3303. Fax: +81-22-275-2013. E-mail: numazawa@
tohoku-pharm.ac.jp.
10.1021/jm010282t CCC: $20.00 © 2001 American Chemical Society
Published on Web 10/27/2001

4278 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24
Numazawa et al.
Sch em e 1a
Sch em e 2a
a
Reagents: CH3I, Ag2O.
by irradiation of the 4â-proton (δ ) 5.43 ppm). Further-
more, the stereochemistry was unambiguously proven
by a comparison of this compound with 4â-acetate 19
that was previously obtained.32 The structure of enol
acetate 22 was assigned on the basis of NMR spectros-
copy [1H, δ 2.12 ppm (4-OCOCH3); 13C, 130.2, 140.2,
169.1, and 221.1 ppm (four sp2 carbons)]. Alkaline
hydrolysis of compound 22 yielded 5R-4-one product 26,
also supporting the assigned structure. Treatment of
acetate 18 with K2CO3 afforded 4R-ol 24.
4R-Hydroxy derivative of 19-ol 7, compound 25, was
synthesized in approximate 10% yield, starting from 19-
tert-butyldimethylsilyloxy-4-ene-compound 9,28 in a se-
quence similar to that described above. This synthesis
involves, sequentially, OsO4 oxidation, acetylation of
4R,5R-diol 12 obtained after separation of the oxidation
products, dehydration, and deprotection of protecting
groups at C-4 with K2CO3 and at C-19 with dilute HCl
(Scheme 1). The stereochemistry of an acetoxy group
at C-4 of the intermediate 4R-acetoxy-5-ene steroid 20
1
was similarly assigned on the basis of H NMR spec-
troscopy. Irradiation of the 4â-proton (δ ) 5.05 ppm) of
4R-acetate 16 caused 11% NOE enhancement of the 19-
methylene proton (δ ) 3.71 ppm). In contrast, there was
no NOE enhancement of the 19-methylene proton
obtained through irradiation of the 4R-proton (δ ) 5.34
ppm) of 4â-acetate 17. In this sequence, the dehydration
of 4â-acetoxy-5â-ol 17 gave principally 4-acetoxy-4-ene
product 23 (56%), and the 5-en-4â-acetoxy compound
could not be isolated in a pure form. The dehydration
of another 4â-acetoxy-5â-ol 15 also gave 4-enol acetate
22 (47%) as a major product along with 5-en-4â-acetate
19 (24%), indicating that not only the conformations at
C-4 and C-5 but also the structure at C-19 of a
4-acetoxy-5-ol steroid affect the direction of dehydration
of the 5-hydroxy moiety with SOCl2.
a
Reagents: (a) (i) OsO4, pyridine, (ii) aqueous NaHSO3; (b)
(CH3CO)2O, pyridine; (c) SOCl2, pyridine; (d) dilute HCl, THF,
propan-2-ol; (e) K2CO3, MeOH, H2O.
be a very powerful competitive inhibitor, indicating that
a hydrophilic interaction between the active site and the
diol structure plays an important role in the tight
binding.
Resu lts
Treatment of 4â,19-diol 27, previously synthesized,33
with CH3I in the presence of Ag2O gave 4- and 19-
monomethyl ethers 28 and 29, respectively (Scheme 2).
Previously synthesized 19-acetoxy-5â,6R- and 5R,6â-
trans-diols 30 and 3134 were separately acetylated to
give 6R- and 6â-acetates 32 and 33, respectively, of
which treatment with SOCl2 followed by hydrolysis with
K2CO3 yielded 4-ene-6R,19-diol 36 (68% from 32) and
its 6â-isomer 37 (47% from 33), respectively (Scheme
3). The stereochemistry of the C-6 hydroxy moiety of
6,19-diols was determined on the basis of 1H NMR
spectroscopy [6â-H at 4.27 ppm (m) and 4-H at 6.07 ppm
(t, J ) 4.9 Hz) for the 6R-ol 36 and 6R-H at 4.29 ppm (t,
J ) 2.9 Hz) and 4-H at 5.86 (t, J ) 3.6 Hz) for the 6â-ol
37]. Irradiation of the 6â-proton of 6R-ol 36 produced
1.4% and 9.8% NOE enhancement of the 19-methylene
protons at 3.60 and 3.84 ppm, respectively, and 2.2%
Ch em istr y. 5-En-4R-ol 24, having the angular 19-
methyl group, was initially prepared to show the effect
of the 4R-hydroxy group of 5-ene steroid 6 on the affinity
to aromatase (Scheme 1). Treatment of 4-ene compound
2 with OsO4 followed by reductive cleavage of the
osmium adduct with NaHSO3 gave an approximate 3:1
mixture of 4R,5R-diol 10 to 4â,5â-diol 11. After separa-
tion of the mixture using silica gel column chromatog-
raphy, 5R-compound 10 was acetylated with Ac2O and
pyridine and subsequently dehydrated with SOCl2 to
give 5-en-4R-acetate 18 and 4-enol acetate 22 (75% and
18% from 4R-acetate 14, respectively). The stereochem-
istry of the 4R-acetoxy group was determined on the
1
basis of H NMR spectroscopy. Thus, the 41% nuclear
Overhauser effect (NOE) enhancement of the 19-methyl
protons (δ ) 1.07 ppm) of compound 18 was produced

Binding of Steroids to Aromatase
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24 4279
Sch em e 3a
Ta ble 1. Aromatase Inhibition by 4- or 6- Hydroxy and
5R-4-One Steroidsa
b
IC50
µM
,
apparent
compound
Ki,c nM
4-Hydroxy-5-ene Series
4R-ol 24
0.86
0.70
6.8
11.0
0.28
1.0
0.031
150
20% inhibition
at 10 µM
58
60
800
4R-acetate 18
4R,19-diol 25
4R-acetoxy-19-ol 21
4â-ol 8d
25
90
3.4
4â-acetate 19d
4â,19-diol 27
4â-methoxy-19-ol 28
4â-hydroxy-19-methoxide 29
6-Hydroxy-4-ene Series
6R-ol 4e
0.19
1.4
0.050
0.94
21
6R,19-diol 36
250
6.0
110
6â-ol 5e
6â,19-diol 37
For Comparison
4-ene steroid 2e
4-en-19-ol 3e
0.060
0.049
0.66
6.9
6.8
5.8
5-ene steroid 6f
5-en-19-ol 7f
120
1000
a
Reagents: (a) (CH3CO)2O, pyridine; (b) SOCl2, pyridine; (c)
K2CO3, MeOH, H2O.
a
Inhibition type was determined by Lineweaver-Burk plot. In
these experiments, the apparent Km for the substrate androstene-
dione was found to be about 33 nM. All of the compounds listed
NOE enhancement of the C-4 proton at 6.07 ppm. In
contrast, no significant NOE enhancement of the 19-
methylene protons was detected by irradiation of the
6R-proton of 6â-ol 37 whereas 11.5% NOE enhancement
of the 4-proton at 5.86 ppm was observed in the
irradiation.
b
showed a competitive type of inhibition. A concentration of 300
nM of [1â-3H] androstenedione and 20 µg of protein from human
placental microsomes were used. c Inhibition constant was ob-
tained by Dixon plot. Reference 31. e Reference 30. f Reference
d
29.
Bioch em ica l P r op er ties. Inhibition of aromatase
activity in human placental microsomes by 4,19-diols
25 and 27 and their derivatives, 6,19-diols 36 and 37,
and 4R-ol 24 and its acetate 18 was examined in vitro
by enzyme kinetics. The results are given in Table 1.
In addition to the above compounds, the parent 4- and
5-ene steroids 2 and 6 and their respective 19-ols 3 and
7 are listed for comparison. Aromatase activity in
placental microsomes was determined using a radio-
metric assay in which tritiated water released from [1â-
3H]androstenedione (1) into the incubation medium
during aromatization was measured.35 To characterize
the nature of the inhibitor binding to the active site of
aromatase, aromatization was measured at several
inhibitor and substrate concentrations. The results of
these studies were plotted on a typical Lineweaver-
Burk plot. In these studies, the apparent Km and Vmax
values for androstenedione (1) were approximately 33
nM and 128 (pmol/min)/mg of protein, respectively. All
the steroids studied exhibited clear competitive-type
inhibition. The apparent inhibition constants (Ki) were
obtained by Dixon plots. The Lineweaver-Burk plot of
aromatase inhibition by inhibitor 4â,19-diol 27 is shown
in Figure 2.
F igu r e 2. Lineweaver-Burk plot of inhibition of human
placental aromatase by 4â,19-diol 27 with androstenedione (1)
as the substrate. Each point represents the mean of two
determinations that varied by less than 5% of the mean. The
inhibition experiments with the other inhibitors examined in
this study gave essentially results similar to the results for
27 (data not shown).
5-ene-4â,19-diol 27 was an extremely potent competitive
inhibitor of aromatase in human placental microsomes,
and with an apparent Ki value of 3.4 nM, it is one of
the most powerful steroidal aromatase inhibitors re-
ported to date. In contrast, the 4R-isomer of inhibitor
27, compound 25, and 6,19-diols 36 and 37 with a 4-ene
structure were moderate to poor inhibitors of aromatase,
with Ki values ranging between 110 and 800 nM.
The introduction of a 4â-hydroxy group into 5-en-19-
ol 7 enhanced about 300-fold the affinity to aromatase.
This modification is equivalent to the introduction of a
19-hydroxy group into 4â-ol 8 (Ki ) 25 nM) where the
affinity was increased by 85-fold. The 4â-hydroxylated
19-ol 27 is the first powerful aromatase inhibitor having
Discu ssion
To clarify the role of hydrophilic interaction in the
binding of a series of 3-deoxy steroids to the active site
of aromatase, the effect of introducing a 6R- or 6â-
hydroxy group into 19-hydroxyandrost-4-en-17-one (3),
which has a high affinity to aromatase with a Ki of 5.8
nM,27 and that of a 4R- or 4â-hydroxy group into 19-
hydroxyandrost-5-en-17-one (7), of which the affinity is
very low,29 on aromatase inhibition were principally
tested. Among diol steroids examined in this study,

4280 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24
Numazawa et al.
such polar functions at the A,B-ring system. In contrast,
its 4R-isomer 25 was a poor inhibitor of aromatase with
almost the same affinity as the parent 19-ol 7 (Ki ) 800
vs 1000 nM for 25 vs 7) (Table 1); however, introducing
a 4R-hydroxy function into 19-methyl-5-ene compound
6 slightly increased the affinity to aromatase (Ki ) 58
vs 120 nM, 4R-ol 24 vs 6), as seen in the case of the
4â-hydroxylation.31 4R-Acetylation of 4R,19-diol 25 de-
creased the affinity (IC50 ) 6.8 and 11.0 µM for 25 and
4R-acetoxy-19-ol 21, respectively), although acetylation
of 4R-ol 24 does not change the affinity (Ki for 4R-acetate
18 is 60 nM). Methylation of either a 4â-hydroxy or a
19-hydroxy group of the powerful inhibitor 27 dramati-
cally decreased the affinity. Taken together, it is shown
that a combination of 4â- and 19-hydroxy groups of
4â,19-diol 27 is important in the formation of a ther-
modynamically stable inhibitor-aromatase complex.
On the other hand, the introduction of a hydroxyl
group at the C-6R or C-6â position of 4-en-19-ol 3, which
is equivalent to the introduction of a 19-hydroxy group
into either 6R-ol 4 or 6â-ol 5, lowered to a great extent
the affinity of each parent inhibitor with a high affinity
to aromatase. The results indicate that a 6R,19- or
6â,19-diol structure does not tolerate the access of 4-ene-
diols 36 and 37 to aromatase; either a 6â-hydroxy or a
19-hydroxy moiety existing in the â-side region of a
steroid nucleus is enough for production of the thermo-
dynamically stable complex with aromatase in the
3-deoxy-4-ene series, and the additional hydroxy group
interferes with the production of the stable complex.
Structure-activity relationships27-30 and the aro-
matase-catalyzed 19-oxygenation37 of a series of 3-deoxy
C19 steroids previously revealed that there is a marked
difference in the binding manner between the two
parent 3-deoxy-4-ene and 5-ene steroids, 2 and 6, in the
active site and that the binding aspect of the latter is
comparable to that of substrate 1. The mechanistic
proposals by Laughton’s group as well as Robinson’s and
Simpson’s groups incorporate critical information from
site-directed mutagenesis and molecular modeling stud-
ies that place glutamic acid (302Glu) as an essential
active-site amino acid.20,23,24,37 Substrate androstenedi-
one (1) is anchored by a hydrogen bond between an
active-site amino acid (128His) and the C-3 carbonyl
function.37 On the other hand, it is reported that 309Asp
and 310Thr are close to the catalytic site in the active
site,20,21,24 suggesting that a carboxyl group of 309Asp
and a hydroxy group of 310Thr may also play a role in
the catalytic mechanism.
Exp er im en ta l Section
Ma ter ia ls a n d Gen er a l Meth od s. Androst-4-en-17-one
(2)27 and its 19-tert-butyldimethylsiloxy derivative 9,28 4â-
hydroxyandrost-5-en-17-one (8),30 5â,6R-dihydroxy-19-ace-
toxyandrostan-17-one (30) and its 5R,6â-dihydroxy derivative
31,33 and 4â,19-diol 2733 were prepared according to known
methods. [1â-3H]Androstenedione (27.5 Ci/mmol) (3H distribu-
tion of 74-79% at 1â) was purchased from New England
Nuclear Corp. (Boston, MA) and NADPH from Kohjin Co. Ltd.
(Tokyo, J apan).
Melting points were measured on a Yanagimoto melting
point apparatus and were uncorrected. IR spectra were
recorded in KBr pellets, except for oily compounds of which
spectra were obtained in neat forms, on a Perkin-Elmer FT-
IR 1725X spectrophotometer. 1H and 13C NMR spectra were
obtained in CDCl3 solution with J EOL EX 270 (270 MHz for
1H and 67.5 MHz for 13C) and GSX 400 (400 MHz for 1H and
100 MHz for 13C) spectrometers, respectively (Tokyo, J apan),
using tetramethylsilane (δ ) 0.00) or CHCl3 (δ ) 7.26, for tert-
butyldimethylsilyl derivatives) as an internal standard, and
mass spectra (MS; electron impact) were obtained with a J EOL
J MS-DX 303 spectrometer. Thin-layer chromatography (TLC)
was performed on E. Merck precoated silica gel plates (Darm-
stadt, Germany). Column chromatography was conducted with
silica gel (E. Merck, 70-230 mesh).
Tr ea tm en t of th e 4-En e Ster oid 2 or 9 w ith OsO4. OsO4
(360 mg, 1.42 mmol) was added to a solution of 19-methyl
steroid 2 (354 mg, 1.3 mmol) in pyridine (14 mL), and the
mixture was stirred at room temperature for 3 h. After this
time, NaHSO3 (680 mg, 6.5 mmol) in H2O (10 mL) was added.
The mixture was stirred until the color changed to orange, and
then it was diluted with EtOAc, washed with NaCl solution,
and dried with Na2SO4. Evaporation of the solvent gave a
brown oil (380 mg) that was purified by column chromatog-
raphy (hexanes-EtOAc) followed by recrystallization from
hexanes-EtOAc to afford two products: 4R,5R-dihydroxyan-
drostan-17-one (10) and 4â,5â-dihydrosyandrostan-17-one (11).
Compound 10 (224 mg, 56%): mp 116-117 °C; 1H NMR (270
MHz) δ 0.86 (3H, s, 18-Me), 0.99 (3H, S, 19-Me), 4.02 (1H, dd,
J ) 5.3 and 11.2 Hz, 4â-H); MS m/z (rel intens) 306 (M+, 85),
288 (48), 270 (18), 234 (100), 219 (42); IR νmax 3471 (OH), 1740
(CdO) cm-1. Anal. (C19H30O3) C, H.
Compound 11 (75 mg, 17%): mp 172-173 °C; 1H NMR (270
MHz) δ 0.86 (3H, s, 18-Me), 0.96 (3H, s, 19-Me), 3.70 (1H, m,
4R-H); MS m/z (rel intens) 306 (M+, 68), 288 (35), 270 (20),
234 (90), 219 (38), 97 (100); IR νmax 3478 (OH), 1732 (CdO)
cm-1. Anal. (C19H30O3) C, H.
19-tert-butyldimethylsiloxy steroid 9 (1.91 g, 5 mmol) was
treated with OsO4 (1.33 g, 5.2 mmol) in a similar fashion
(pyridine, 52 mL; reaction period, 2.2 h; NaHSO3, 3.3 g, 31.7
mmol; H2O, 54 mL). The brown oily product was purified by
column chromatography (hexanes-EtOAc) to afford two prod-
ucts: 19-(tert-butyldimethylsilyloxy)-4R,5R-dihydroxyandrostan-
17-one (12) and 19-(tert-butyl-dimethylsiloxy)-4â,5â-dihy-
droxyandrostan-17-one (13).
Compound 12 (480 mg, 23%): mp 174-175 °C (from
acetone-hexane); 1H NMR δ 0.06 and 0.07 (3H each, s, 19-
OSiMe2), 0.87 (3H, s, 18-Me), 0.89 (9H, s, 19-OSiMe2CMe3),
3.63 and 3.91 (1H each, d, J ) 10.6 Hz, 19-CH2), 3.71 (1H, m,
4â-H); MS m/z (rel intens) 436 (M+, 7), 379 (31), 361 (43), 303
(26), 287 (100); IR νmax 3455 (OH), 1729 (CdO) cm-1. Anal.
(C25H44O4Si) C, H.
On the basis of these previous reports, it is likely that
4â,19-diol 27 would be anchored by hydrogen bonds
between two hydroxy groups of the inhibitor and two
polar amino acid residues in the active site such as
128His, 302Glu, 309Asp, and/or 310Thr. The remaining
carbonyl group at C-17 of compound 27 also becomes
very important in anchoring this inhibitor in the active
site, similar to the other 3-deoxy steroids.19,28-30 The
present findings regarding a hydrophilic interaction of
a series of hydroxylated 3-deoxy C19 steroids with
aromatase would be useful for understanding spatial
and electronic aspects of the binding (active) site of the
enzyme as well as for developing an effective aromatase
inhibitor.
Compound 13 (oil, 572 mg, 25%): 1H NMR δ 0.11 and 0.12
(3H each, s, 19-OSiMe2), 0.84 (3H, s, 18-Me), 0.93 (9H, s, 19-
OSiMe2CMe3), 3.59 and 4.34 (1H each, d, J ) 10.6 Hz, 19-
CH2), 3.89 (1H, m, 4R-H); MS m/z (rel intens) 436 (M+, 3), 379
(28), 361 (94), 287 (100), 269 (89); IR νmax 3448 (OH), 1740
(CdO) cm-1; HRMS (C25H44O4Si) found 436.3009, calcd
436.3030.
Acetyla tion of 4,5-Diols 10-13. 19-Methyl compounds 10
and 11 (200 mg, 0.68 mmol) and 19-siloxy compounds 12 and
13 (450 mg, 3 mmol) were separately acetylated with acetic
anhydride and pyridine (1 and 2 mL for 10 and 11, respec-
tively, and 2.2 and 4.5 mL for 12 and 13, respectively) (room

Binding of Steroids to Aromatase
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24 4281
temperature, overnight). After the usual treatment, the crude
product obtained was recrystallized from an appropriate
solvent.
2.12 (3H, s, 4-OCOMe), 3.71 and 3.83 (1H each, d, J ) 10.1
Hz, 19-CH2); MS m/z (rel intens) 403 (M+ - 57, 33), 273 (100);
IR νmax 1741 (CdO) cm-1. Anal. (C27H44O4Si) C, H.
4R-Acetoxy-5R-hydroxyandrostan-17-one (14) (198 mg,
4r-Acet oxy-19-h yd r oxya n d r ost -5-en -17-on e (21). 19-
Silyl ether 20 (120 mg, 0.26 mmol) was dissolved in propan-
2-ol (3.5 mL) and THF (1.7 mL), and 1 M hydrochloric acid
(1.8 mL, 1.8 mmol) was added to this solution. The mixture
was stirred at room temperature for 2 days, then diluted with
EtOAc (100 mL), washed with 5% NaHCO3 solution and water,
and dried with Na2SO4. Evaporation of the solvent gave an
oil that was purified by column chromatography (hexanes-
EtOAc) to yield 19-ol 21 (28 mg, 31%) along with the recovered
silyl ether 20 (73 mg, 61%).
1
87%): mp 178-179 °C (from EtOAc); H NMR (270 MHz) δ
0.85 (3H, s, 18-Me), 1.00 (3H, s, 19-Me), 2.09 (3H, s, 4-OCOMe),
5.38 (1H, m, 4â-H); MS m/z (rel intens) 348 (M+, 40), 288 (95),
270 (32), 234 (100), 219 (33); IR νmax 3538 (OH), 1729 (CdO)
cm-1. Anal. (C21H32O4) C, H.
4â-Acetoxy-5â-hydroxyandrostan-17-one (15) (193 mg, 85%):
mp 198-199 °C (from acetone); 1H NMR (270 MHz) δ 0.86 (3H,
s, 18-Me), 1.01 (3H, s, 19-Me), 2.07 (3H, s, 4-OCOMe), 4.99
(1H, t, J ) 8.2 Hz,4R-H); MS m/z (rel intens) 348 (M+, 60),
330 (10), 288 (100), 270 (36), 234 (91); IR νmax 3596 (OH), 1737
(CdO) cm-1. Anal. (C21H32O4) C, H.
Compound 21: mp 77-80 °C (from EtOAc-hexane); 1H
NMR (270 MHz) δ 0.94 (3H, s, 18-Me), 2.14 (3H, s. 4-OCOMe),
3.63 and 3.86 (1H each, d, J ) 11.9 Hz, 19-CH2), 5.33 (1H, m,
4-H), 5.79 (1H, m, 6-H); MS m/z (rel intens) 346 (M+, 3), 273
4R-Acet oxy-19-(ter t-bu t yldim et h ylsiloxy)-5R-h ydr oxy-
androstan-17-one (16) (330 mg, 67%): mp 220-221 °C (from
(61), 256 (100), 238 (25); IR νmax 3547 (OH), 1730 (CdO) cm-1
Anal. (C21H30O4) C, H.
.
1
acetone); H NMR (400 MHz) δ 0.082 and 0.085 (3H each, s,
19-OSiMe2), 0.86 (3H, s, 18-Me), 0.89 (9H, s, 19-OSiMe2CMe3),
2.07 (3H, s, 4-OCOMe), 3.71 and 3.92 (1H each, d, J ) 10.7
Hz, 19-CH2), 5.05 (1H, m, 4â-H); MS m/z (rel intens) 478 (M+,
1), 421 (25), 362 (100), 269 (22); IR νmax 3533 (OH), 1736 (Cd
O) cm-1. Anal. (C27H46O5Si) C, H.
Alk a lin e Hyd r olysis of 4r-Aceta tes 18 a n d 21. Com-
pounds 18 and 21 (0.75 mmol) were separately dissolved in
MeOH (70 mL). K2CO3 (120 mg, 0.87 mmol) in water (24 mL)
was added to this solution. The mixture was stirred at room
temperature for 15-19 h, diluted with EtOAc, washed with
water, and dried with Na2SO4. Evaporation of the solvent gave
a crude product, which was purified by column chromatogra-
phy and recrystallized from an appropriate solvent to afford
4R-ols 24 and 25, respectively.
4R-Hydroxyandrost-5-en-17-one (24) (75%): mp 152-153 °C
(from acetone-hexane); 1H NMR (270 MHz) δ 0.89 (3H, s, 18-
Me), 1.02 (3H s 19-Me), 4.25 (1H, d, J ) 10.1 Hz, 4â-H), 5.73
(1H, m, 6-H); 13C NMR (100 MHz) 13.6, 20.205, 20.218, 20.4,
21.9, 30.5, 31.1, 31.5, 35.9, 37.4, 38.6, 39.3, 47.4, 50.9, 51.9,
69.5, 114.8, 145.7, 221.1; MS m/z (rel intens) 288 (M+, 100),
273 (99), 255 (41), 199 (22); IR νmax 3452 (OH), 1727 (CdO)
cm-1. Anal. (C19H30O2) C, H.
4â-Acet oxy-19-(ter t-bu t yldim et h ylsiloxy)-5â-h ydr oxy-
androstan-17-one (17) (419 mg, 85%): mp 184-185 °C (from
acetone-hexane); 1H NMR (400 MHz) δ 0.10 and 0.11 (3H
each, s, 19-OSiMe2), 0.83 (3H, s, 18-Me), 0.92 (9H, s, 19-
OSiMe2CMe3), 2.10 (3H, s. 4-OCOMe), 3.62 and 4.30 (1H each,
d, J ) 10.6 Hz, 19-CH2), 5.34 (1H, m, 4R-H); MS m/z (rel
intens) 421 (M+ - 57, 3), 361 (100), 287 (46), 269 (77); IR νmax
3480 (OH), 1736 (CdO) cm-1. Anal. (C27H46O5Si) C, H.
Tr ea tm en t of th e 4-Acetoxy-5-ols 14-17 w ith SOCl2.
The 5-ols 14-17 (1 mmol) were separately dissolved in pyridine
(11 mL). SOCl2 (8.5 mmol) was added dropwise to this solution
at 0 °C, and the mixture was stirred at 0 °C for about 1 h.
Water was carefully added to the reaction mixture, and the
product was extracted with EtOAc, washed sequentially with
1% HCl, 5% NaHCO3 solution, and water, and dried with Na2-
SO4. Evaporation of the solvent gave a solid that was purified
by column chromatography (hexanes-EtOAc) and crystalliza-
tion from an appropriate solvent to give the 4-acetoxy product
along with 4-enol acetate in each case.
4R,19-Dihydroxyandrost-5-en-17-one (25) (88%): mp 167-
1
170 °C (from acetone); H NMR (270 MHz) δ 0.95 (3H, s, 18-
Me), 3.61 (1H, m, 19-CHa), 3.76 (1H, d, J ) 11.5 Hz, 19-CHb),
4.23 (1H, m, 4-OH), 6.13 (1H, m, 6-H); 13C NMR (100 MHz)
13.9, 20.7, 20.8, 21.7, 29.7, 31.7, 32.5, 35.7, 35.8, 37.2, 43.7,
47.7, 51.1, 52.6, 63.5, 69.2, 120.5, 140.4, 221.1; MS m/z (rel
intens) 304 (M+, 3), 273 (100), 255 (43), 237 (14); IR νmax 3480
and 3521 (OH), 1708 (CdO) cm-1. Anal. (C19H28O3) C, H.
Tr ea tm en t of En ol Aceta te 22 w ith K2CO3. Compound
22 (100 mg, 0.30 mmol) was treated with 1.2 mol equiv K2-
CO3 similarly as described for the hydrolysis of compounds
18 and 21. The crude product obtained was purified by
crystallization to yield androstane-4,17-dione (26) (75%): mp
4R-Acetoxyandrost-5-en-17-one (18) (75%): mp 149-151 °C
(from EtOAc); 1H NMR (270 MHz) δ 0.88 (3H, s, 18-Me), 1.07
(3H s 19-Me), 2.12 (3H, s, 4R-OCOMe), 5.43 (1H, m, 4â-H),
5.48 (1H, m, 6-H); 13C NMR (100 MHz) δ 13.5, 20.0, 20.1, 20.2,
21.2, 21.9, 30.4, 31.0, 31.5, 33.6, 35.8, 38.9, 39.0, 47.4, 50.8,
51.8, 71.6, 115.4, 141.0, 170.2, 221.0; MS m/z (rel intens) 330
(M+, 15), 288 (100), 273 (72), 255 (14); IR νmax 1742 (CdO) cm-1
Anal. (C21H30O3) C, H.
.
1
164-165 °C (from acetone) (lit.38 162-164 °C); H NMR (270
MHz) δ 0.77 (3H, s, 18-Me), 0.87 (3H, s, 19-Me); MS m/z (rel
4â-Acetoxyandrost-5-en-17-one (19) (24%): mp 129-130 °C
intens) 288 (M+, 100), 273 (14), 255 (20), 229 (10); IR νmax 1706
1
(from MeOH) (lit.32 120-122 °C); H NMR (270 MHz) δ 0.89
and 1737 (CdO) cm-1
.
(3H, s, 18-Me), 1.15 (3H s 19-Me), 2.02 (3H, s, 4â-OCOMe),
5.33 (1H, s, 4R-H), 5.77 (1H, t, J ) 2.7 Hz, 6-H).
Meth yla tion of 4r,19-Diol 27 w ith CH3I. A mixture of
the 4-ol (0.3 mmol), CH3I (3 mL), and Ag2O (100 mg, 0.43
mmol) was refluxed with stirring for 24-36 h. The solid
material was removed by filtration, and the filtrate was
evaporated to give an oil that was purified by preparative TLC
(hexanes-EtOAc) and recrystallization from an appropriate
solvent.
4â-Methoxy-19-hydroxyandrost-5-en-17-one (28) (15%): mp
159-163 °C (from acetone); 1H NMR (270 MHz) δ 0.93 (3H, s,
18-Me), 3.28 (3H, s, 4-OMe), 3.65 (2H, m, 19-CH2), 3.88 (1H,
dd, J ) 6.1 and 11.2 Hz, 4-H), 5.83 (1H, dd, J ) 2.3 and 4.8
Hz, 6-H); MS m/z (rel intens) 318 (M+, 3), 286 (60), 257 (100),
238 (43), 228 (23); IR νmax 3589 (OH), 1735 (CdO) cm-1. Anal.
(C20H30O3) C, H.
4â-Hydroxy-19-methoxyandrost-5-en-17-one (29) (16%) (oil):
1H NMR (270 MHz) δ 0.91 (3H, s, 18-Me), 3.34 (3H, s, 19-CH2-
OMe), 3.46 and 3.82 (1H each, d, J ) 9.1 Hz, 19-CH2), 4.22
(1H, s, 4-H), 5.83 (1H, dd, J ) 2.1 and 4.7 Hz, 6-H); MS m/z
(rel intens) 318 (M+, 4), 300 (12), 255 (100), 238 (30), 213 (15);
IR νmax 3449 (OH), 1737 (CdO) cm-1; HRMS (C20H30O3) found
318.2222, calcd 318.2195.
4-Acetoxyandrost-4-en-17-one (22) (18% from 14 or 47%
1
from 15): mp 108-109 °C (from acetone-hexane); H NMR
(270 MHz) δ 0.88 (3H, s, 18-Me), 1.08 (3H s 19-Me), 2.12 (3H,
s, 4-OCOMe); 13C NMR (100 MHz) δ 13.7, 18.8, 19.4, 20.9, 21.1,
21.8, 22.3, 27.7, 30.6, 31.5, 35.2, 35.8, 37.1, 37.5, 47.6, 51.0,
54.3, 130.2, 140.2, 169.1, 221.1; MS m/z (rel intens) 330 (M+,
21), 288 (100), 273 (62); IR νmax 1746 (CdO) cm-1. Anal.
(C21H30O3) C, H.
4R-Acet oxy-19-(ter t-bu t yldim et h ylsiloxy)a n dr ost -5-en -
17-one (20) (92%): mp 93-95 °C (from EtOAc); 1H NMR
(270 MHz) δ 0.039 and 0.054 (3H each, s, 19-OSiMe2), 0.86
(9H, s, 19-OSiMe2CMe3), 0.91 (3H, s, 18-Me), 2.12 (3H, s.
4-OCOMe), 3.61 and 3.76 (1H each, d, J ) 10.7 Hz, 19-CH2),
5.38 (1H, m, 4-H), 5.70 (1H, m, 6-H); MS m/z (rel intens) 403
(M+ - 57, 100), 343 (20), 269 (85), 255 (34); IR νmax 1742 (Cd
O) cm-1. Anal. (C27H44O4Si) C, H.
4-Acetoxy-19-(tert-butyldimethylsiloxy)androst-4-en-17-
one (23) (3% from 16 or 56% from 17): mp 127-130 °C (from
1
acetone); H NMR (270 MHz) δ 0.037 and 0.043 (3H each, s,
19-OSiMe2), 0.88 (9H, s, 19-OSiMe2CMe3), 0.89 (3H, s, 18-Me),

4282 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24
Numazawa et al.
Acetyla tion of 5,6-Diols 30 a n d 31. Compounds 30 and
31 (280 mg, 0.77 mmol) were separately dissolved in pyridine
(2.8 mL) and acetic anhydride (1.4 mL), and the mixture was
allowed to stand at room temperature overnight. After the
usual procedure, the crude product was purified by column
chromatography (hexanes-EtOAc) to afford 5â-hydroxy-6,19-
diacetoxyandrostan-17-one (32) and 5R-hydroxy-6,19-diace-
toxy-androstan-17-one (33), respectively.
son,35 in which tritiated water released from [1â-3H]AD into
the incubation medium during aromatization was used as an
index of the enzyme activity. All assays were carried out in
67 mM phosphate buffer, pH 7.5, in the presence of NADPH
(180 µM) at a final incubation volume of 0.5 mL under the
conditions similar to those used previously.16 Briefly, for the
screening assay, 20 µg of protein of the lyophilized microsomes,
a 20 min incubation time, 300 nM concentration of the 3H-
labeled substrate, and various concentrations of each of the
inhibitors were used, and for the kinetic study, 20 µg of protein,
various concentrations of each of the inhibitors, 12.4, 15.3,
Compound 32 (250 mg, 80%) (oil): 1H NMR (270 MHz) δ
0.86 (3H, s, 18-Me), 2.08 and 2.11 (3H each, s, 6R- and 19-
OCOMe), 4.22 and 4.37 (1H each, d, J ) 12.0 Hz, 19-CH2),
5.22 (1H, dd, J ) 4.8 and 12.0 Hz, 6-H); MS m/z (rel intens)
406 (M+, 24), 364 (51), 346 (23), 303 (37), 286 (100), 268(45);
IR νmax 1738 (CdO) cm-1; HRMS found 406.2325, calcd for
3
21.3, and 28.3 nM concentrations of the H-labeled substrate,
and a 5 min incubation time were used.
C
23H34O6 406.2355.
Ack n ow led gm en t. This work was supported in part
by a Grant-in-Aid for Scientific Research from the
Ministry of Education, Science, Sports and Culture of
J apan. We are grateful to Dr. Hideo Imaizumi of
Imaizumi Hospital, Sendai, for generously supplying
human term placenta.
Compound 33 (255 mg, 82%): mp 171-175 °C (from
1
acetone-hexane); H NMR (270 MHz) δ 0.88 (3H, s, 18-Me),
2.07 and 2.14 (3H each, s, 6â- and 19-OCOMe), 4.42 and 4.60
(1H each, d, J ) 12.7 Hz, 19-CH2), 4.67 (1H, s, 6-H); MS m/z
(rel intens) 406 (M+, 57), 346 (12), 328 (10), 286 (40), 268 (100);
IR νmax 3456 (OH), 1738 (CdO) cm-1. Anal. (C23H34O6) C, H.
Tr ea tm en t of 6-Acetoxy-5-ols 32 a n d 33 w ith SOCl2.
Compounds 32 and 33 (100 mg, 0.25 mmol) were separately
treated with SOCl2 (200 mg, 1.68 µmol) in pyridine (5.5 mL)
at 0 °C for 30 min in a similar fashion as described for the
synthesis of compounds 18-20. The crude product obtained
was purified by column chromatography to give 6R,19-diace-
toxyandrost-4-en-17-one (34) and 6â,19-diacetoxyandrost-4-en-
17-one (35), respectively.
Refer en ces
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Compound 34 (86 mg, 89%) (oil): 1H NMR (270 MHz) δ 0.89
(3H, s, 18-Me), 2.08 and 2.12 (3H each, s, 6R- and 19-OCOMe),
4.10 and 4.36 (1H each, d, J ) 11.4 Hz, 19-CH2), 5.37 (1H, m,
6â-H), 5.71 (1H, t, J ) 1.9 Hz, 4-H); MS m/z (rel intens) 388
(3) Yoshida, N.; Osawa, Y. Purification of Human Placental Aro-
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(15) Abul-Hajj, Y. J .; Liu, X.-P.; Hedge, M. Synthesis and Evaluation
of Substituted-4-androstene-3,17-dione Derivatives as Aro-
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(16) Numazawa, M.; Oshibe, M. 6-Alkyl- and 6-Arylandrost-4-ene-
3,17-dione as Aromatase Inhibitors. Synthesis and Structure-
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(M+, 3), 328(12), 273 (100), 255 (48); IR νmax 1740 (CdO) cm-1
HRMS found 388.2239, calcd for C23H32O5 388.2250.
;
Compound 35 (74 mg, 77%): mp 157-161 °C (from acetone);
1H NMR (270 MHz) δ 0.94 (3H, s, 18-Me), 2.00 and 2.03 (3H
each, s, 6â- and 19-OCOMe), 4.27 and 4.33 (1H each, d, J )
11.3 Hz, 19-CH2), 5.35 (1H, m, 6-H), 5.97 (1H, dd, J ) 1.8 and
4.5 Hz, 4-H); MS m/z (rel intens) 388 (M+, 11), 346 (24), 328
(20), 273 (100); IR νmax 1734 (CdO) cm-1. Anal. (C23H32O5) C,
H.
Alk a lin e Hyd r olysis of 6,19-Dia ceta tes 34 a n d 35.
Compounds 34 and 35 (86 mg, 0.22 mmol) were separately
treated with K2CO3 (86 mg, 0.62 mmol) in MeOH (7 mL) and
H2O (1 mL) at room temperature for 24 h as described above.
The crude product obtained was purified by column chroma-
tography and recrystallization to give 6R,19-dihydroxyandrost-
4-en-17-one (36) and 6â,19-dihydroxyandrost-4-en-17-one (37),
respectively.
Compound 36 (52 mg, 77%): mp 191-194 °C (from EtOAc);
1H NMR (270 MHz) δ 0.89 (3H, s, 18-Me), 3.59 (1H, dd, J )
7.9 and 10.6 Hz, 19-CHa), 3.84 (1H, d, J ) 8.9 Hz, 19-CHb),
4.27 (1H, m, 6â-H), 6.07 (1H, t, J ) 4.9 Hz, 4-H); 13C NMR
(100 MHz) δ 13.9, 19.5, 20.9, 21.7, 25.0, 31.7, 34.5, 34.5, 35.7,
41.0, 43.1, 47.9, 51.2, 53.4, 66.5, 69.0, 121.3, 141.4, 220.5; MS
m/z (rel intens) 286 (M+ - 18, 6), 273 (100), 255 (37), 237 (15);
IR νmax 3321 (OH), 1737 (CdO) cm-1. Anal. (C19H28O3) C, H.
Compound 37 (41 mg, 61%): mp 176-178 °C (from acetone);
1H NMR (270 MHz) δ 0.95 (3H, s, 18-Me), 3.65 and 3.86 (1H
each, d, J ) 10.7 Hz, 19-CH2), 4.29 (1H, t, J ) 2.9 Hz, 6-H),
5.86 (1H, t, J ) 3.6 Hz, 4-H); 13C NMR (100 MHz) δ 14.1, 19.2,
20.6, 21.7, 25.4, 30.9, 31.8, 35.8, 36.0, 38.1, 41.2, 48.1, 51.8,
53.9, 69.3, 74.0, 129.4, 140.9, 221.1; MS m/z (rel intens) 286
(M+ - 18, 6), 256 (100), 238 (15); IR νmax 3240 (OH), 1730 (Cd
O) cm-1. Anal. (C19H28O3) C, H.
P r ep a r a tion of P la cen ta l Micr osom es. Human term
placental microsomes (particles sedimenting at 105000g for
60 min) were obtained as described by Ryan.39 They were
washed once with 0.5 mM dithiothreitol solution, lyophilized,
and stored at -20 °C. No significant loss of aromatase activity
occurred over the period of this study (2 months).
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