Chemical aromatization of 19-hydroxyandrosta-1,4-diene-3,17-dione with acid or alkaline: Elimination of the 19-hydroxymethyl group as formaldehyde

Article abstract of DOI:10.1016/j.steroids.2008.10.011

In order to determine whether or not a 19-hydroxymethyl group of 19-hydroxyandrosta-1,4-diene-3,17-dione (2, 19-hydroxy ADD), an intermediate of aromatase-catalyzed estrone formation from ADD, a suicide substrate of aromatase, is eliminated as formaldehyde, we examine chemical nature of removal of the 19-hydroxymethyl group. 19-Acetate 3 and 19-tert-butyldimethylsiloxy compound 4 are known to convert rapidly to estrone with treatment of NaOH or n-Bu₄NF. Since compound 2 was unstable and unobtainable under these conditions, compounds 3 and 4 as equivalents to compound 2 were used in this study. The acetate 3 with 5 mol/l HCl in acetone and 10% KOH in MeOH along with the silyl ether 4 with 5 mol/l HCl in acetone and 1 mol/l n-Bu₄NF in THF gave formaldehyde and estrone in which a ratio of the aldehyde to estrone was near 1. This result indicates that the 19-hydroxymethyl groups of compound 3 and 4 are eliminated as formaldehyde along with estrone derived from the steroid skeleton under the acid or base treatment. The findings suggest that a single hydroxylation at the 19 carbon of ADD (1) would be, chemically, all that was required for estrone formation.

Full text of DOI:10.1016/j.steroids.2008.10.011

Steroids 74 (2009) 208–211
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Steroids
journal homepage: www.elsevier.com/locate/steroids
Chemical aromatization of 19-hydroxyandrosta-1,4-diene-3,17-dione with acid
or alkaline: Elimination of the 19-hydroxymethyl group as formaldehyde
∗
Mitsuteru Numazawa , Kouwa Yamashita, Nao Kimura, Madoka Takahashi
Tohoku Pharmaceutical University, 1-4 Komatsushima-4-chome, Sendai 981-8558, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 July 2008
Received in revised form 8 October 2008
Accepted 19 October 2008
Available online 30 October 2008
In order to determine whether or not a 19-hydroxymethyl group of 19-hydroxyandrosta-1,4-diene-3,17-
dione (2, 19-hydroxy ADD), an intermediate of aromatase-catalyzed estrone formation from ADD, a suicide
substrate of aromatase, is eliminated as formaldehyde, we examine chemical nature of removal of the 19-
hydroxymethyl group. 19-Acetate 3 and 19-tert-butyldimethylsiloxy compound 4 are known to convert
rapidly to estrone with treatment of NaOH or n-Bu4NF. Since compound 2 was unstable and unobtainable
under these conditions, compounds 3 and 4 as equivalents to compound 2 were used in this study. The
acetate 3 with 5 mol/l HCl in acetone and 10% KOH in MeOH along with the silyl ether 4 with 5 mol/l HCl
in acetone and 1 mol/l n-Bu4NF in THF gave formaldehyde and estrone in which a ratio of the aldehyde
to estrone was near 1. This result indicates that the 19-hydroxymethyl groups of compound 3 and 4
are eliminated as formaldehyde along with estrone derived from the steroid skeleton under the acid or
base treatment. The findings suggest that a single hydroxylation at the 19 carbon of ADD (1) would be,
chemically, all that was required for estrone formation.
Keywords:
19-Hydroxyandrosta-1,4-diene-3,17-dione
Chemical aromatization
Formaldehyde formation
Acid-catalyzed reaction
Base-catalyzed reaction
©
2008 Elsevier Inc. All rights reserved.
1. Introduction
Fig. 2) [21]. Deprotonation of the hydroxyl group of 19-hydroxyADD
2) formed conceivably could lead the release of formaldehyde and
(
The conversion of androgens to estrogens is catalyzed by aro-
matase, whose activity results in aromatization of the A-ring of
androgens to form the phenolic A-ring characteristic of estrogens,
with concomitant loss of the 19-methyl group [1–3]. Three oxy-
genative steps are involved, each of which requires 1 mol of O2 and
the phenolic steroid. However, the oxidation state of the C-19 car-
bon released during estrone formation has not been established.
Previously, it has been reported that removal of the acetyl ester
or the tert-butyldimethylsilyl ether of 19-hydroxy ADD (2), com-
pound 3 and 4, with NaOH or n-Bu NF, respectively, to obtain the
4
1
mol of NADPH [4–11]. The first two oxygenations are sequential
dien-19-ol 2 led to rapid A-ring aromatization to give estrone [22].
We then examined whether or not the 19-hydroxymethyl group is
chemically eliminated as formaldehyde during the hydrolysis of the
19-substituted ADDs, compounds 3 and 4.
hydroxylations at the C-19 methyl group and following the third
oxygenation, C-19 and 1␤,2␤-hydrogens are eliminated as formic
acid and water, respectively, to produce estrogens. Inhibitors of
aromatase are of interest in the treatment of established estrogen-
dependent breast cancer [12–15].
Androsta-1,4-diene-3,17-dione (1, ADD) (Fig. 1) is a prototypi-
cal mechanism-based inhibitor of aromatase; the 1,4-diene-3-one
is responsible for inactivation, although the inactivation mecha-
nism is currently unknown [16,17]. ADD can serve as a substrate
for aromatase with higher apparent Km compared to that for
androstenedione [18]. Estrone known to be formed from ADD is
postulated to arise by an alternate pathway involving hydroxylation
at C-19 methyl group [19–21]. Possibly a single hydroxylation at C-
2. Experimental
2.1. Materials and reagents
19-Acetoxyandrosta-1,4-diene-3,17-dione (3) [22], 19-tert-
butyldimethlsiloxy-androsta-1,4-diene-3,17-dione (4) [22], and
estradiol 3-methyl ether [23] were synthesized according to known
methods. Estradiol-d5 was prepared previously in our laboratory
[
18].
Formaldehyde, acetylacetone, ammonium acetate (NH OAc)
19 methyl group is all that is required for estrone formation (path A,
4
were obtained from Nakalai Tesque (Kyoto, Japan). High perfor-
mance liquid chromatography (HPLC) grade acetonitrile (MeCN),
methanol (MeOH), and ultra-pure water were purchased from
Wako Pure Chemical Industries (Osaka, Japan).
∗ Corresponding author. Tel.: +81 022 727 0138; fax: +81 022 727 0137.
E-mail address: numazawa@tohoku-pharm.ac.jp (M. Numazawa).
0
039-128X/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2008.10.011

M. Numazawa et al. / Steroids 74 (2009) 208–211
209
Fig. 3. Derivatization of formaldehyde with acetylacetone and ammonium acetate.
Lutidine derivative was determined by fluorometry (wavelength: excitation 412 nm,
+
emission 510 nm) or LC-MS (ESI, positive, m/z 194.2 [M + H ]).
◦
flow rate of 1 ml/min; injector temperature, 280 C; transfer line
◦
temperature, 200 C. MS condition: ionization energy, 70 eV; ion
◦
source temperature, 200 C; emission current 300 ␮A.
2.5. Determination of estrone by HPLC
Fig. 1. Structures of 19-hydroxy ADD substrates.
Estrone produced was determined by HPLC using estradiol-3-
methyl ether as an internal standard. To a solution of estrone (0,
.1, 0.2, 0.5 and 1.0 ␮g/0.1 ml MeOH) was added the internal stan-
0
Fluorescence intensity was measured with a Shimadzu RF-1500
spectro-fluorometer (Kyoto, Japan). Thin-layer chromatography
TLC) was carried out with pre-coated TLC plates (Kiesel Gel 60
F254, layer thickness 0.25 mm, Merck KGaA, Darmstadt, Germany).
dard (2.5 ␮g/0.1 ml MeOH) and H O (1 ml), and then extracted
2
with EtOAc (2 ml). The organic layer was washed with saturated
(
NaCl (1 ml), dried over anhydrous Na SO4 and then evaporated
2
to dryness under nitrogen stream. The residue was dissolved in
mobile phase (MeCN-H O = 60:40, 100 ␮l) and then injected to
2
2
.2. HPLC
HPLC. Calibration curve for estrone was obtained by assigning the
concentration of estrone to x and the peak area ratio of estrone to
the internal standard to y, monitoring the UV absorbance at 280 nm.
Subsequently, a linear regression was performed for constructing
calibration curve.
HPLC was carried out using a Waters 600 pump equipped with
a Waters 2487 Dual UV detector (Milford, MA, USA). Column was a
Puresil C18 (4.6 mm i.d. × 150 mm, 5 ␮m, Waters) and mobile phase
was MeCN-H O (60:40, v/v) at a flow rate of 1 ml/min. Wavelength
2
of detector was set at 280 nm.
2.6. Determination of formaldehyde by fluorometry
2
.3. Liquid chromatography-electrospray ionization-mass
Formaldehyde produced was determined by fluorometry after
conversion of formaldehyde to the corresponding lutidine deriva-
spectrometry (LC-ESI-MS)
tive using acetylacetone and NH OAc [24] (Fig. 3). To a solution of
4
LC-ESI-MS was run on a Finnigan TSQ Quantum quadrupole
mass spectrometer (Thermo Electrone, San Jose, CA, USA). LC
condition; Capcell Pak UG 120 5 ␮m, C18, 2 mm × 100 mm,
formaldehyde (0, 0.1, 0.3, 1.0 and 3.0 ␮g/0.1 ml H O) were added
2
0
.3 ml (acetone-H O = 1:1, v/v) and the reagent mixture (1 ml)
2
(
NH OAc 3 g/AcOH 0.06 ml/acetylacetone 0.04 ml in 20 ml H O).
4
2
acetonitrile-0.25% CH COOH = 40/60 v/v, 250 ␮l/min. MS condi-
3
tion; spray voltage 4500 V, sheath gas nitrogen, 35 arbitrary units
Table 1
(
gas pressure), auxiliary gas nitrogen, 20 arbitrary units (gas pres-
◦
Formaldehyde and estrone formation from the acetate 3 and the silyl ether 4 by acid
and basic conditions and 120 min reaction time.
sure), ion transfer capillary temperature 350 C, collision gas argon,
1.5 mTorr (gas pressure), ion polarity positive.
Substrate
Conditions
Product yields (%)
HCHO/estrone
2.4. Gas chromatography (GC)-MS
HCHO
Estrone
Acetate 3
Acetate 3
Silyl ether 4
Silyl ether 4
5 mol/l HCl/acetone
10% KOH/MeOH
34.0
14.6
99.5
73.3
41.3
50.1
101.3
85.9
0.82
0.29
0.98
0.85
A
Finnigan Trace DSQ GC-MS instrument (Thermo Elec-
trone) was used for the GC-MS analysis. GC conditions: column,
0 m × 0.25 mm i.d. fused silica DB5 (J&W Scientific, CA, USA); col-
5 mol/l HCl/acetone
+
−
n-Bu4N F /THF
3
◦
◦
umn temperature, from 50 to 280 C at 25 C/min; carrier gas, He
Each value represents the mean of duplicate experiments.
Fig. 2. Possible formation process of HCHO and HCOOH from 19-hydroxy ADD.

210
M. Numazawa et al. / Steroids 74 (2009) 208–211
Table 2
Intensity of the reaction mixture was determined by spectroflu-
orometer with excitation and emission wavelengths of 412 nm
and 510 nm, respectively. Calibration curve for formaldehyde was
obtained by assigning the amount of formaldehyde to x and the
fluorescence intensity to y. Subsequently, a linear regression was
performed for constructing calibration curve.
Detection of formaldehyde and estrone released from the acetate 3 and the silyl
ether 4 with 5 mol/l HCl in acetone by LC and GC-MS.
Substrate
nmol
Product (nmol)
HCHO
Estrone
Acetate 3
Acetate 3
Silyl ether 4
Silyl ether 4
2.4
24.0
2.4
1.50 ± 0.12
5.50 ± 0.04
1.25 ± 0.13
5.10 ± 0.14
1.87 ± 0.02
8.17 ± 0.05
1.35 ± 0.003
5.41 ± 0.19
2.7. Hydrolysis of the 19-acetate 3 and 19-tert-butyldimethylsilyl
24.0
ether 4 by acid or base
The substrate 3 or 4 in acetone (0.1 ml) was subjected to the reaction with 5 mol/l
HCl in acetone (50 ␮l) for 120 min. Each value represents the mean ± SD of four
experiments.
2
.7.1. Condition 1
Solution of compounds 3 (0.29 ␮mol) and 4 (0.24 ␮mol) in ace-
tone (0.1 and 0.2 ml) were treated with 5 mol/l HCl in acetone
35% HCl 1 ml in acetone 1.27 ml) at room temperature for 2 h,
(
determined by measuring the intensity of the M+ ion of the prod-
respectively. After this time, 0.1 ml portion of the reaction mix-
ture was added to 5 mol/l HCl in acetone (0.2 ml) to minimize
the fluorescence intensity of the reaction matrix. Estrone formed
from compounds 3 and 4 was determined as follows; estrone in
the HCl solution was extracted with EtOAc (2 ml) and subjected
to HPLC using estradiol 3-methyl ether as an internal standard, as
described in Section 2.5. Formaldehyde in the HCl solution, formed
from compounds 3 and 4, was diluted with 3 ml of distilled water
uct to that of the corresponding ion in the internal standard (tR:
estradiol 14.48 min, estradiol-d5 14.44 min).
2.7.4. Condition 4
The silyl ether 4 (10 mg, 24 ␮mol) was dissolved in CH Cl2
2
(
0.55 ml) and CH CN (0.35 ml). LiBF (7 mg, 75 ␮mol) was added to
3 4
this solution and the reaction mixture was stirred for 1 h at room
temperature.
and determined by adding the acetylacetone-NH OAc reagent as
4
described Section 2.6.
3. Results and discussion
2.7.2. Condition 2
A solution of compound 3 in MeOH (0.2 ml) or compound 4 in
Initially, in our hand, we attempted to obtain 19-hydroxyADD
THF (0.2 ml) was treated with 10% KOH methanolic solution (0.1 ml)
(2) by removal of the 19-acetate 3 and the 19-silyl ether 4 accord-
ing to the previous report [22]. Treatment of the acetate 3 with
0.5 mol equivalent of NaOH in MeOH for a brief time (10 min) and
that of the silyl ether 4 with 0.5 mol equivalent of n-Bu4NF in THF
for 20 min gave A-ring aromatized estrone (about 5–10%) along
with the respective unreacted substrate on TLC analysis. It was
previously reported that primary and secondary alcohols are regen-
erated from their corresponding tert-butyldimethylsilyl ethers by
treatment with LiBF4 [25]. Then, treatment of the silyl ether 4
with LiBF4 in CH2Cl2 and CH3CN for 1 h gave estrone (about 40%)
along with compound 4 (20%) and unidentified compounds (no UV
absorption) on TLC analysis. Thus, compound 2 was found to be not
isolated from the hydrolysis of the ester 3 or the ether 4 because of
its rapid conversion to estrone as previously reported [22].
Covey has suggested that a single hydroxylation at C-19 of ADD,
catalyzed aromatase, is all that is required for estrone formation
[21]. We then examined whether or not a 19-hydroxymethyl moiety
of the acetate 3 and the silyl ether 4 is removed as formalde-
hyde during their conversion to estrone using conditions 1 and 2.
Formaldehyde was determined by fluorescence fluorometry after
conversion to the lutidine derivative with the reaction of acety-
lacetone and NH4OAc (Fig. 3), excitation 412 nm and emission
or 1 mol/l n-Bu NF in THF (0.1 ml), respectively, at room tem-
4
perature for 2 h. The estrone and formaldehyde formations were
determined similarly as described above.
2.7.3. Condition 3
A mixture of the acetate 3 or the silyl ether 4 (1 ␮g, 2.4 nmol and
10 ␮g, 24 nmol), 5 mol/l HCl in acetone (50 ␮l), and acetone (100 ␮l)
was allowed to stand at room temperature for 2 h. After this time,
H O (2 ml) was added to the reaction mixture and the mixture was
2
carefully distilled to give the water phase and the residue. The water
phase was reacted with 20 ␮l of the acetylacetone-NH OAc reagent
4
◦
(
60 C, 10 min) and then subjected to LC-ESI-MS. The residue was
extracted with EtOAc (5 ml × 2) and the combined organic phase
was evaporated to dryness and dissolved in 100 ␮l of MeOH. NaBH
4
◦
(
500 ␮g) was added to this solution and allowed to stand at 0 C for
0 min. The internal standard estradiol-d5 (5 ng) and H O (5 ml)
3
2
were added and subjected to Oasis cartridge. After the elution,
and the trimethylsilylation, gas chromatography (GC)-MS was con-
ducted according to previous report [18]. The quantitative analysis
of the aromatized product was performed with a selected ion mon-
itoring method (EI mode). The amount of estradiol produced was
510 nm. Effect of matrix on fluorescence intensity in the assay of
formaldehyde in 5 mol/l HCl solution was very low and then the
HCl solution was used as a solvent of the fluorescence. Estrone
was determined by revere-phase HPLC. The acetate 3 was treated
with 5 mol/l HCl in acetone and 10% KOH solution in MeOH and the
silyl ether 4 was done with 5 mol/l HCl in acetone and n-Bu NF
4
in THF (Table 1). Treatment of compound 3 with 5 mol/l HCl in
acetone for 120 min gave formaldehyde and estrone at a ratio of
formaldehyde to estrone of 0.82, in contrast, treatment of 10% KOH
in MeOH produced them at the ratio of 0.29. The yield of estrone
was about 50% in each. The formaldehyde formation in the case
of the KOH-MeOH system was extremely low. It is supposed that
formaldehyde produced by alkaline condition may produce the fur-
ther oxidized product formic acid, by air oxidation, although there is
Fig. 4. Time course of the formation of formaldehyde and estrone in the silyl ether
4
with 5 mmol/l HCl–acetone solution. The formation rates (%) of formaldehyde and
no evidence. Treatment of the silyl ether 4 with the HCl or n-Bu NF
estrone were expressed as relative values to the corresponding values of complete
hydrolysis (5 mol/l HCl–acetone, 100%).
4
described above for 120 min yielded formaldehyde and estrone at

M. Numazawa et al. / Steroids 74 (2009) 208–211
211
the ratio of about 1 where yields of estrone were more than 86%
Table 1).
The time course of the formation of the aldehyde and estrone in
Ministry of Education, Culture, Sports, Science, and Technology of
Japan.
(
the reaction of compound 4 with 5 mmol/l HCl–acetone was deter-
mined (Fig. 4). The formations of the two products were gradually
increased to about 60% of the substrate and the relative amounts of
the two products were about the ratios of 0.90 during the reaction.
These results show that the 19-hydroxymethyl group of compound
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This work was supported, in part, by a Grant-in-Aid for Scien-
tific Research and High Technology Research Center Project from