4- and 6-(p-Sulphamoylphenyl)androstenediones: Studies of aromatase inhibitor-based oestrone sulphatase inhibition

Article abstract of DOI:10.1016/j.steroids.2010.05.011

4-(p-Sulphamoylphenyl)androstenedione (3) and 6α-p-sulphamoylphenyl analogues 12-14 were synthesised and tested as aromatase inhibitors as well as oestrone sulphatase inhibitors in human placental microsomes. All of the p-sulphamoylphenyl compounds synthesised were powerful inhibitors of aromatase with apparent K_i values ranging between 30 and 97 nM. In addition, the aromatase inhibitory activities of 6α-p-hydroxyphenyl compounds 9-11, which may be produced from their respective sulphamoylphenyl compounds by action of oestrone sulphatase, were also high in a range of 23 and 75 nM of the K _i values. On the other hand, all of the sulphamoylphenyl compounds were poor inhibitors of oestrone sulphatase with more than about 200 μM of IC₂₅ values. Although the present findings of the oestrone sulphatase inhibition are disappointing, such attempts may be valuable to develop a new class of drugs having a dual function, aromatase inhibitor and oestrone sulphatase inhibitor, for the treatment of oestrogen-dependent breast cancer.

Full text of DOI:10.1016/j.steroids.2010.05.011

Steroids 75 (2010) 891–896
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
4- and 6-(p-Sulphamoylphenyl)androstenediones: Studies of aromatase
inhibitor-based oestrone sulphatase inhibition
Yoko Wataria, Satoshi Yamaguchia, Madoka Takahashia, Masao Nagaokab,
Mitsuteru Numazawaa,∗
a Tohoku Pharmaceutical University, 4-1 Komatsushima-4-Chome, Aobaku, Sendai 981-8558, Japan
b Nihon Pharmaceutical University, 10281 Komuro, Ina-machi, Kitaadachi-gun, Saitama 362-0806, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
4-(p-Sulphamoylphenyl)androstenedione (3) and 6␣-p-sulphamoylphenyl analogues 12–14 were syn-
thesised and tested as aromatase inhibitors as well as oestrone sulphatase inhibitors in human placental
microsomes. All of the p-sulphamoylphenyl compounds synthesised were powerful inhibitors of aro-
matase with apparent Ki values ranging between 30 and 97 nM. In addition, the aromatase inhibitory
activities of 6␣-p-hydroxyphenyl compounds 9–11, which may be produced from their respective sul-
phamoylphenyl compounds by action of oestrone sulphatase, were also high in a range of 23 and 75 nM
of the Ki values. On the other hand, all of the sulphamoylphenyl compounds were poor inhibitors of
oestrone sulphatase with more than about 200 ␮M of IC25 values. Although the present findings of the
oestrone sulphatase inhibition are disappointing, such attempts may be valuable to develop a new class
of drugs having a dual function, aromatase inhibitor and oestrone sulphatase inhibitor, for the treatment
of oestrogen-dependent breast cancer.
Received 10 March 2010
Received in revised form 7 May 2010
Accepted 7 May 2010
Available online 19 June 2010
Keywords:
p-Sulphamoylphenyl-substituted
androstenedione
Aromatase inhibitor
Oestrone sulphatase inhibitor
Human placental microsomes
© 2010 Elsevier Inc. All rights reserved.
In post-menopausal women with oestrogen-dependent breast
cancer, the oestrogen concentration in tumour tissues is much
higher than that circulating in plasma [1–5]. Peripheral and intra-
tumoural synthesis of oestrogens has been initially considered
almost exclusively through the aromatase pathway. The enzyme,
aromatase, which converts androstenedione into oestrone, has
therefore been among the prime targets for depleting oestrogen
levels [5–7] (Fig. 1). The sole inhibition of this enzyme could
not afford an effective reduction of oestrogenic stimulation to
tumours, the reason being that other pathways are involved in
oestrogen bio-synthesis [8,9]. The sulphatase pathway, where the
inactive precursor oestrone sulphate is converted into oestrone
by the enzyme, oestrone sulphatase, is now considered to be the
main source for the local production of oestrogens in tumours,
providing 10-fold more oestrone than the aromatase pathway
[10–13]. Oestrone sulphate has been suggested to act as a reser-
voir for the synthesis of active steroids in post-menopausal
women [14].
reported that 6␣- and 6␤-alkyl- [17,18] or 6-aryl- [19] substituted
androstenediones and their 1,4-diene-, 4,6-diene- and 1,4,6-triene-
derivatives [20–22] are potent competitive inhibitors of aromatase.
On the other hand, 4-alkyl- and 4-aryl-substituted androstene-
dione derivatives have been reported to be moderate-to-powerful
inhibitors of aromatase [23].
The most notable and potent of steroid sulphatase inhibitors is
oestrone 3-sulphamate [24–26]. Oestrone 3-sulphamate has been
reported to act as an active site-directed irreversible inhibitor
of oestrone sulphatase [25]. Cleavage of a sulphamoyl moiety of
oestrone 3-sulphamate is the prerequisite for the irreversible inac-
tivation [27–29], and oestrone is released during the sulphatase
inactivation. Oestrone formation is unsuitable for use in the treat-
ment of oestrogen-dependent breast cancer.
We have then postulated that the aromatase inhibitor-based
sulphamates are useful for the treatment of the breast cancer.
Recently, 3-sulphamates of 2-, 4- or 6-substituted oestrogens,
which act as aromatase inhibitors [30], have been synthesised
and evaluated as aromatase inhibitor-based oestrogen sulphatase
inhibitors; some of them are effective inhibitors of both aro-
matase and oestrone sulphatase [31]. In this article, considering
that 4-phenyl- [23] and 6-phenyl- [19] androstenedione analogues
are good aromatase inhibitors, we synthesised 4- and 6␣-(p-
sulphamoylpheny) derivatives of androstenedione analogues and
examined them as aromatase inhibitors as well as oestrone sul-
phatase inhibitors.
Inhibitors of aromatase are of interest in the treatment of
established oestrogen-dependent breast cancer [7,15,16]. Various
analogues of androstenediones have been synthesised as aro-
matase inhibitors by various laboratories. We have previously
∗
Corresponding author. Tel.: +81 022 727 0138; fax: +81 022 727 0137.
E-mail address: numazawa@tohoku-pharm.ac.jp (M. Numazawa).
0039-128X/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2010.05.011

892
Y. Watari et al. / Steroids 75 (2010) 891–896
Fig. 1. Oestrogen formation by aromatase and oestrone sulphatase pathways in peripheral and breast tissue.
1. Experimental
at room temperature with stirring for 2 h. The reaction mix-
ture was then poured in chilled 5% NaHCO3 solution (200 ml),
extracted with EtOAc (200 ml), washed with saturated NaCl solu-
tion, and dried with Na2SO4. Evaporation of the solvent gave
an oily substance that was purified by column chromatogra-
phy (hexane–EtOAc) and preparative thin-layer chromoatography
(TLC) (hexane–EtOAc) followed by recrystallisation to give a
solid. The solid was recrystallised from acetone to produce 4-
(p-sulphamoy1phenyl)androst-4-ene-3,17-dione (3) in 26% yield
(23 mg, 51 ␮mol), mp 240–244 ◦C. Compound 3: IR (KBr): 1721
1.1. Material and general methods
Dextran-coated charcoal was obtained from Sigma–Aldrich
Corporation (St. Louis, MI, USA) and nicotinamide adenine
dinucleotide phosphate (NADPH) was from Kohjin Co., Ltd.
(Tokyo, Japan). [6,7-3H]Oestrone was purchased from Perkin-Elmer
Japan, Co. Ltd. (Yokohama, Japan) and [1␤-3H]androstenedione
(25.4 Ci mmol−1) (3H distribution: ␤/␣ = 74.2/25.8) was from New
England Nuclear Corporation (Boston, MA, USA). The compound
4-(p-methoxyphenyl)androstenedione (1) [23] and 3,3;17,17-
bis(ethylenedioxy)androstan-5␣,6␣-epoxide (4) [17] were synthe-
sised according to the previous methods. All other chemicals were
purchased from Sigma–Aldrich Corporation (St. Louis, MI, USA).
Melting points were measured on a Yanagimoto melting-point
apparatus (Kyoto, Japan) and are uncorrected. Infrared (IR) spec-
tra were recorded on a Perkin-Elmer Fourier transform-infrared
spectroscopy (FT-IR) 1725X spectrophotometer in a KBr pellet,
and ultraviolet (UV) spectra were determined in 95% EtOH on a
Hitachi 150-20 spectrophotometer (Tokyo, Japan). 1H nuclear mag-
netic resonance (NMR) spectra were obtained in CDCl3 solution
with a JEOL LA 400 (400 MHz) and JEOL LA 600 (600 MHz) spec-
trometers (Tokyo, Japan) using tetramethylsilane as an internal
standard and mass spectra (MS) (electron impact, (EI) mode) with a
JEOL JMS-DX 303 spectrometer. Thin-layer chromatography (TLC)
was performed on E. Merck pre-coated silica gel plates (silica gel
60F-254, Darmstadt, Germany). Column chromatography was con-
ducted with silica gel 60, 70–230 mesh (E. Merck).
and 1663 cm−1 (C O), 1387 and 1163 cm−1 (SO2NH2); UV ꢀmax
:
230 nm (ε = 12,000); 1H NMR ı: 0.97 (3H, s, 18-Me), 1.38 (3H, s, 19-
Me), 7.03–7.06 (2H, d, J = 8.4 Hz, aromatic protons), 7.31–7.34 (2H,
d, J = 8.7 Hz, aromatic protons); MS m/z: 457 (M+, 4), 376 (64), 279
(5), 200 (100). Anal. calculated for C25H31NO5S: C, 65.62; H, 6.83;
N, 3.06. Found C, 65.33; H, 6.75; N, 3.00.
1.3. Synthesis of 6-(p-hydroxyphenyl)androstenedione
derivatives 9–11
To a solution of the bis(ethylenedioxy)-5␣,6␣-epoxide (4)
(500 mg, 1.28 mmol) in tetrahydrofuran (THF) (20 ml) was added
20 molar equivalent of p-methoxyphenylmagnesium bromide
in THF (10 ml), and the mixture was heated under reflux
for 4 h in a N2 stream. After the solution was cooled, sat-
urated NH4Cl solution (100 ml) was added to this and the
product was extracted with EtOAc (200 ml × 2). The com-
bined organic layers were washed with water to neutrality,
dried (Na2SO4), and evaporated to dryness leaving the residue,
which was purified by column chromatography (hexane–EtOAc)
and recrystallised from EtOAc to give 6␤-(p-methoxyphenyl)-
3,3;17,17-bis(ethylenedioxy)androstan-5␣-ol (5) in 90% yield
(1.64 g, 3.24 mmol), mp 211–212 ◦C. Compound 5: IR (KBr):
3538 cm−1 (OH); 1H NMR ı: 0.68 (3H, s, 19-Me), 0.96 (3H, s, 18-Me),
2.91 (1H, d, J = 5.9 Hz, 6␣-H), 3.78 (3H, s, OCH3), 3.84–4.06 (8H, m,
3-O2(CH2)2 and 17-O2(CH2)2), 6.79 (2H, d, J = 8.9 Hz, aromatic pro-
tons), 7.26–7.34 (2H, m, aromatic protons); MS m/z: 498 (M+, 12),
480 (10), 121 (19), 99 (100). Anal. calculated for C30H42O6: C, 72.26;
H, 8.49. Found C, 72.37; H, 8.77.
3 M HClO4 (1.13 ml) was added to a solution of compound
5 (1.62 g, 3.21 mmol) in THF (24 ml), and the reaction mix-
ture was stirred at room temperature for 5 h. After this time,
the mixture was diluted with EtOAc (200 ml), washed with
5% NaHCO3 solution and water, dried (Na2SO4) and evapo-
rated to yield the residue. The residue was purified by column
chromatography followed by recrystallisation from acetone to pro-
duced 6␤-(p-methoxyphenyl)-5␣-hydroxyandrostane-3,17-dione
(6) (1.32 g, 3.22 mmol, 98% yield), mp 201–203 ◦C. Compound 6: IR
(KBr): 3452 cm−1 (OH), 1738 and 1708 cm−1 (C O); 1H NMR ı: 0.98
(3H, s, 19-Me), 1.02 (3H, s, 18-Me), 2.86 (1H, d, J = 6.1 Hz, 6␣-H), 3.81
(3H, s, OCH3), 6.80–6.86 (2H, m, aromatic protons), 7.26–7.33 (2H,
m, aromatic protons); MS m/z: 410 (M+, 89), 392 (55), 339 (100),
1.2. Synthesis of 4-(p-sulphamoylphenyl)androstenedione (3)
The compound 4-(p-methoxyphenyl)androst-4-ene-3,17-dione
(1) (75 mg, 89 ␮mol) in 47% HBr in acetic acid (8 ml) was heated
at 120 ◦C for 2 h, and the reaction mixture was poured into ice-
water (100 ml). The product was extracted with ethyl acetate
(EtOAc) (200 ml) and the organic layer was washed with 5% sodium
bicarbonate (NaHCO3) solution and water and dried (Na2SO4).
Evaporation of the solvent gave the crude solid, which was
recrystallised from acetone to yield 4-(p-hydroxyphenyl)androst-
4-ene-3,17-dione (2) (32 mg, 42 ␮mol) in 48% yield, mp 287–289 ◦C.
Compound 2: IR (KBr): 3307 cm−1 (OH), 1720 and 1666 cm−1
(C O); UV ꢀmax: 229 nm (ε = 13,000); 1H NMR ı: 0.93 (3H, s, 18-
Me), 1.31 (3H, s, 19-Me), 6.76 (2H, d, J = 8.7 Hz, aromatic protons),
6.85 (2H, d, J = 8.7 Hz, aromatic protons); MS m/z: 378 (M+, 64), 200
(100), 188 (20), 172 (12). Anal. calculated for C25H30O3: C, 79.33;
H, 7.99. Found C, 79.31; H, 8.01.
Sodium hydride (60% oil suspension, 73 mg, 1.8 mmol) was
separately added to stirred solutions of compound 2 (71 mg,
0.18 mmol) in anhydrous dimethylforamide (DMF) (6 ml) at 0 ◦C.
Subsequently, sulphamoyl chloride (0.25 ml, 2.35 mmol) was
added carefully, and the resulting mixture was allowed to stand

Y. Watari et al. / Steroids 75 (2010) 891–896
893
241 (63). Anal. calculated for C26H34O4: C, 76.06; H, 8.35. Found C,
76.07; H, 8.63.
gave the crude product, which was purified by a column chro-
matography (hexane–EtOAc) and recrystallised from acetone to
afford 6-(p-hydroxyphenyl)androsta-1,4,6-triene-3,17-dione (11)
(130 mg, 0.35 mmol, 61% yield), mp 254–255 ◦C. Compound 11:
IR (KBr): 3610 cm−1 (OH), 1742 and 1647 cm−1 (C O), 1598 cm−1
(C C); UV ꢀmax: 242 nm (ε = 19,000) and 290 nm (ε = 8,700); 1H
NMR ı: 1.02 (3H, s, 18-Me), 1.35 (3H, s, 19-Me), 5.98 (1H, s, 4-H),
6.06 (1H, s, 7-H), 6.31–6.35 (1H, m, 2-H), 6.76–6.80 (2H, m, aro-
matic protons), 7.02–7.06 (2H, m, aromatic protons), 7.15 (1H, d,
J = 10.2 Hz, 1-H); MS m/z: 374 (M+, 100), 359 (26), 211 (51), 107 (22).
Anal. calculated for C25H26O3: C, 80.18; H, 7.00. Found C, 79.99; H,
7.12.
Thionyl chloride (1.9 ml, 26 mmol) was added to a chilled solu-
tion of compound 6 (1.30 g, 3.20 mmol) in dry pyridine (19 ml),
and the mixture was stirred for 3 min at 0 ◦C, poured into
ice-water (100 ml), extracted with EtOAc (100 ml × 2). The com-
bined organic layers were washed with water, dried (Na2SO4)
and evaporated to afford the crude product, which was puri-
fied by column chromatography (hexane–EtOAc) and recrystallised
from acetone–hexane, giving 6␤-(p-methoxyphenyl)androst-4-
ene-3,17-dione (7) (1.01 g, 2.58 mmol, 80% yield), mp 182–183 ◦C.
Compound 7: IR (KBr): 1737 and 1678 cm−1 (C O); UV ꢀmax
:
231 nm (ε = 18,400) and 240 nm (ε = 13,600); 1H NMR ı: 0.70 (3H,
s, 19-Me), 0.91 (3H, s, 18-Me), 3.76 (1H, d, J = 4.9 Hz, 6␣-H), 3.81
(3H, s, OCH3), 6.04 (1H, s, 4-H), 6.85–6.89 (2H, m, aromatic pro-
tons), 7.22–7.26 (2H, m, aromatic protons); MS m/z: 392 (M+, 25),
377 (13). Anal. calculated for C26H32O3: C, 79.56; H, 8.22. Found C,
79.42; H, 8.50.
1.4. Synthesis of 6-(p-sulphamoylphenyl)androstenedione
derivatives 12–14
Compounds 9–11 (0.30 mmol) were sulphamoylated by the sim-
ilar procedure to the synthesis of the 4-sulphamoyloxy derivative 3
with sulphamoyl chloride (300 mg, 2.6 mmol) and NaH (3.0 mmol).
The crude products were purified by column chromatography
and recrystallised from MeOH to give the corresponding 6-
(sulphamoylphenyl)androstene derivatives 12–14 in 23–30% yield.
Compound 12: mp 199–203 ◦C. IR (KBr): 1736 and 1656 cm−1
Compound 7 (560 mg, 1.43 mmol) was dissolved in 95% EtOH
(18 ml) and 1 M HCl (1.8 ml) was added to the solution. The mix-
ture was heated under reflux for 12 h. After removing most of the
solvent, the mixture was diluted with EtOAc (200 ml), washed with
5% NaHCO3 solution and water, dried (Na2SO4) and evaporated.
The residue was subjected to column chromatography followed by
recrystallisation to afford 6␣-(p-methoxyphenyl)androst-4-ene-
3,17-dione (8) (432 mg, 1.10 mmol, 77% yield), mp 198–201 ◦C.
(C O), 1388 and 1157 cm−1 (SO2NH2); UV ꢀmax
: 216 nm
(ε = 14,000) and 240 nm (ε = 12,200); 1H NMR ı: 0.96 (3H, s, 18-Me),
1.36 (3H, s, 19-Me), 3.61–3.66 (1H, m, 6␤-H), 5.11 (1H, d, J = 1.8 Hz,
4-H), 7.15–7.19 (2H, m, aromatic protons), 7.26–7.32 (2H, m, aro-
matic protons); MS m/z: 378 (M+, 100), 363 (29), 322 (14), 107 (25).
Anal. calculated for C25H31NO5S: C, 65.62; H, 6.83; N, 3.06. Found
C, 65.24; H, 6.84; N, 3.06.
Compound 8: IR (KBr): 1737 and 1672 cm−1 (C O); UV ꢀmax
:
226 nm (ε = 18,800) and 240 nm (ε = 13,600); 1H NMR ı: 0.95 (3H,
s, 18-Me), 1.34 (3H, s, 19-Me), 3.52–3.58 (1H, m, 6␤-H), 3.81 (3H, s,
OCH3), 5.20 (1H, d, J = 1.5 Hz, 4-H), 6.86 (2H, d, J = 2.0 Hz, aromatic
protons), 6.89 (2H, d, J = 2.8 Hz, aromatic protons); MS m/z: 392 (M+,
100), 377 (46), 336 (26), 121 (33). Anal. calculated for C26H32O3: C,
79.56; H, 8.22. Found C, 79.66; H, 8.35.
Compound 13: mp 210–215 ◦C. IR (KBr): 1731 and 1641 cm−1
(C O), 1387 and 1156 cm−1 (SO2NH2); UV ꢀmax
: 290 nm
(ε = 16,300); 1H NMR ı: 1.01 (3H, s, 18-Me), 1.26 (3H, s, 19-Me), 5.53
(1H, s, 4-H), 6.12 (1H, d, J = 2.1 Hz, 7-H), 7.14–7.19 (2H, m, aromatic
protons), 7.28–7.33 (2H, m, aromatic protons); MS m/z: 376 (M+,
100), 361 (38), 319 (17), 228 (33). Anal. calculated for C25H29NO5S:
C, 65.91; H, 6.42; N, 3.07. Found C, 65.69; H, 6.23; N, 2.96.
Compound 8 (1.06 g, 2.70 mmol) in 48% HBr in AcOH (50 ml)
was heated at 40 ◦C for 24 h, and the reaction mixture was
similarly treated to the method described for the synthesis
of compound 2. The crude product was subjected to column
chromatography followed by recrystallisation from acetone to
give 6␣-(p-hydroxyphenyl)androst-4-ene-3,17-dione (9) (650 mg,
1.73 mmol, 64% yield), mp 250–253 ◦C. Compound 9: IR (KBr):
3423 cm−1 (OH), 1736 and 1656 cm−1 (C O); UV ꢀmax: 226 nm
(ε = 15,400) and 240 nm (ε = 12,500); 1H NMR ı: 0.95 (3H, s, 18-Me),
1.34 (3H, s, 19-Me), 3.50–3.56 (1H, m, 6␤-H), 5.24 (1H, d, J = 1.3 Hz,
4-H), 6.78 (2H, d, J = 8.6 Hz, aromatic protons), 6.95 (2H, d, J = 8.4 Hz,
aromatic protons); MS m/z: 378 (M+, 100), 363 (27), 322 (19), 107
(30). Anal. calculated for C25H30O3: C, 79.33; H, 7.99. Found C, 79.61;
H, 7.77.
Compound 14: mp 198–204 ◦C. IR (KBr): 1729 and 1648 cm−1
(C O), 1599 cm−1 (C C), 1386 and 1158 cm−1 (SO2NH2); UV ꢀmax
:
240 nm (ε = 16,000) and 308 nm (ε = 8,000); 1H NMR ı: 1.04 (3H, s,
18-Me), 1.36 (3H, s, 19-Me), 5.90 (1H, d, J = 1.8 Hz, 4-H), 6.05 (1H,
d, J = 2.1 Hz, 7-H), 6.31 (1H, dd, J = 1.8 and 10.2 Hz, 2-H), 7.17 (1H,
d, J = 10.2 Hz, 1-H), 7.21–7.25 (2H, m, aromatic protons), 7.30–7.36
(2H, m, aromatic protons); MS m/z: 374 (M+, 100), 289 (25), 276
(48), 250 (40). Anal. calculated for C25H27NO5S: C, 66.20; H, 6.00;
N, 3.09. Found C, 66.02; H, 5.70; N, 3.08.
A mixture of compound 9 (300 mg, 0.79 mmol), chloranil
(284 mg, 1.16 mmol), p-toluenesulphanic acid (p-TsOH) monohy-
drate (6.9 mg, 0.36 mmol), and xylene (23 ml) was heated under
reflux for 5 h and cooled to room temperature. The reaction mix-
ture was subjected directly to a column of silica gel (20 g). Elution
with hexane–EtOAc gave the crude product, which was subjected to
recrystallisation to give 6-(p-hydroxyphenyl)androsta-4,6-diene-
3,17-dione (10) (227 mg, 0.60 mmol, 76% yield), mp 217–218 ◦C.
Compound 10: IR (KBr): 3526 cm−1 (OH), 1737 and 1640 cm−1
(C O); UV ꢀmax: 232 nm (ε = 9,600) and 280 nm (ε = 14,800); 1H
NMR ı: 1.00 (3H, s, 18-Me), 1.24 (3H, s, 19-Me), 5.69 (1H, s, 4-H),
6.07 (1H, d, J = 2.1 Hz, 7-H), 6.74–6.79 (2H, m, aromatic protons),
6.95–6.99 (2H, m, aromatic protons); MS m/z: 376 (M+, 100), 361
(22), 343 (10), 228 (23). Anal. calculated for C25H28O3: C, 79.75; H,
7.50. Found C, 79.76; H, 7.54.
1.5. Enzyme preparation
Human placental microsomes (sedimenting at 105,000 × g for
60 min) were obtained as described by Ryan [32]. They were
washed once with 0.05 mM dithiothreitol solution, lyophilised, and
stored at −20 ◦C until use.
1.6. Aromatase assay
Aromatase activity was measured essentially according to the
original procedure of Siiteri and Thompson [33]. The screening
assay for determination of IC50 value and the kinetic assay were car-
ried out essentially according to the assay method described in our
previous work [34]. Briefly, 20 ␮g of protein from the lyophilised
microsomes, and 20-min-incubation period were used for the
screening assay, and 20 ␮g of protein from the microsomes and a
5-min period were used for the kinetic assay. The assays were car-
ried out at 37 ◦C in 67 mM phosphate buffer, pH 7.5, in the presence
of NADPH (180 ␮M) under air.
A solution of compound 10 (200 mg, 0.53 mmol) and 2,3-
dichloro-5,6-dicyano-p-benzoquinone (DDQ) (180 mg, 0.79 mol)
in benzene (31 ml) was refluxed for 10 h, and then the reaction
mixture was loaded onto a column of Al2O3, eluting with EtOAc

894
Y. Watari et al. / Steroids 75 (2010) 891–896
Fig. 2. Synthesis of 4-(p-sulphamoylphenyl)androstenedione (3).
Apparent Ki values were calculated using non-linear regression
analysis with GraFit software [35].
somes, various concentrations of inhibitors and 25 ␮M of MeOH.
Incubations were carried out at 37 ◦C for 5 min and terminated
by adding of 3 ml of toluene, followed by vortexing for 45 s. After
centrifugation at 2500 revolutions per minute (rpm) for 5 min
at 4 ◦C, an aliquot (1.0 ml) in duplicate was removed from the
organic layer and added to scintillation mixture for determination
of the (3H)oestrone production. Control samples with no inhibitor
were incubated simultaneously. Blank samples were obtained by
incubating with boiled microsomes (95 ◦C for 10 min). The basal
sulphatase activity (without the inhibitor) for the IC50 assay was
1.7. Oestrone sulphate assay
Oestrone sulphatase activity was determined essentially by the
standard literature method [25]. All enzymatic studies were carried
out in 67 mM phosphate buffer, pH 7.5, at a final incubation volume
of 0.5 ml. The incubation mixture contained 20 ␮M of (3H)oestrone
sulphate (2 × 105 dpm), 20 ␮g of protein of the lyophilised micro-
Fig. 3. Synthesis of 6-(p-sulphamoylphenyl)androstenediones (12–14).

Y. Watari et al. / Steroids 75 (2010) 891–896
895
found to be 3.6 nmol min−1 mg−1 protein (approximately 3.6% con-
version of the used substrate).
Table 1
In vitro aromatase inhibition by p-hydroxyphenyl- and p-sulphamoylyphenyl-
substituted steroids.
Compound
IC50, nMa
Apparent Ki, nMb
2. Results and discussion
p-Hydroxyphenyl-substituted steroid
4-Ene, 2
>2000
–
6␣-4-Ene, 9
6-4,6-Diene, 10
6-1,4,6-Triene, 11
300
240
890
27 1.1
23 1.3
75 2.4
We initially synthesised 4- and 6-(p-sulphamoylphenyl)
androstenediones (3 and 12–14) through the following pathways
(Fig. 2). The compound 4-(p-methoxyphenyl)androstenedione (1)
[23] was demethylated with 47% HBr in acetic acid to give
4-p-hydroxyphenyl compound 2. Treatment of phenol 2 with sul-
phamoyl chloride in the presence of NaH in anhydrous DMF
afforded the 4-p-sulphamoyl compound 3 in 13% yield from com-
pound 1.
p-Sulphamoylphenyl-substituted steroid
4-Ene, 3
6␣-4-Ene, 12
6-4,6-Diene, 13
6-1,4,6-Triene, 14
990
400
300
680
97 5.0
39 1.8
30 2.1
56 3.7
Substrate: 300 nM of [1␤-3H]androstenedione.
Apparent Ki values were obtained by Dixon plot in which Km for androstenedione
was 33 nM.
a
b
6␣-(p-Hydroxyphenyl)androstenedione (9) was synthesised
from 3,3;17,17-bis(ethylenedioxy)androstan-5␣,6␣-epoxide (4)
[17] in five steps in 34% yield (Fig. 3). Reaction of com-
pound
4 with p-methoxyphenylmagnesium bromide in THF
phamolyl group of a para position of the phenyl ring at the C-4 and
C-6 positions of androstenediones did not change the affinity to
aromatase. Aromatase would tolerate such hydrophilic groups as
phenol and sulphamate, at these positions.
giving 6␤-(p-methoxyphenyl)-5␣-ol 5, which was de-protected
with HClO4 in THF to yield 5␣-hydroxy-3,17-diketone 6. Dehy-
dration of compound
6 with SOCl2 in dry pyridine gave
6␤-p-methoxyphenyl-4-ene-3-one 7 of which isomerisation with
acid produced 6␣-p-methoxyphenyl isomer 8 and finally demethy-
lation of compound 8 with 48% HBr in acetic acid afforded
6␣-p-hydroxyphenyl compound 9, which has thermodynamically
more stablequasi-equatorial conformationthan the quasi-axial6␤-
p-hydroxypnenyl isomer. Compound 9 was then converted into the
6␣-p-sulphamoylphenyl compound 12 (23% yield), similarly to the
synthesis of the sulphamoyl compound 3. On the other hand, com-
pound 9 was treated, under reflux, with chloranil in the presence
of p-TsOH in xylene to yield 4,6-diene derivative 10 (76% yield) of
which dehydrogenation with DDQ under reflux in dry benzene gave
1,4,6-triene compound 11 (61% yield). Both compounds 10 and 11
were finally converted into the 6-sulphamoylphenyl derivatives 13
and 14 in about 30% yields.
The inhibitory activities of oestrone sulphatase in placental
microsomes by the 4- and 6-p-sulphamoylphenyl derivatives 3 and
12–14 were next examined, and the results are shown in Table 2. In
Spectral and analytical data of the new compounds were con-
sistent with their respectively assigned structures.
We initially studied inhibition of aromatase in human pla-
cental microsomes by the sulphamoylphenyl derivatives of
androstenedione and its 4,6-diene and 1,4,6-triene compounds, the
sulphamates 3 and 12–14. In addition to the above compounds, the
aromatase inhibitory activities of p-hydroxyphenyl derivatives 2
and 9–11, which may be produced by hydrolysis of the correspond-
ing sulphamoylphenyl derivatives by oestrone sulphatase [27–29],
were also examined. Aromatase activity in placental microsomes
was determined using a radiometric assay in which tritiated
water released from [1␤-3H]androstenedione in the incubation
medium during aromatisation was measured [33]. To characterise
the nature of inhibitor binding to the active site of aromatase, aro-
matisation was measured at several inhibitor concentrations and
substrate concentrations. The results of these studies were plotted
on a typical Lineweaver–Burk plot. The results are shown in Table 1.
In the studies, the apparent Km and Vmax values for the substrate
androstenedione were 33 nM and 110 pmol min−1 mg−1 protein,
respectively. All of the sulphamates and the phenols, except for
compound 2, showed clear-cut competitive inhibition (Fig. 4).
The apparent Ki values were obtained by Dixon plots. All of the
steroids 3 and 9–14 examined in this study, were potent compet-
itive inhibitors of human placental aromatase with the apparent
Kis ranging from 23 to 97 nM. The Ki values were almost compa-
rable with the Km value of the substrate, ∼33 nM. It has previously
been reported that the affinities to aromatase of the non-polar 4-
and 6-phenyl derivatives of androstenedione and its 4,6-diene and
1,4,6-triene analogues are very high and the Ki values are almost
similar to the Km value of the substrate [17,22,23]. This study
indicated that a hydrophilic substitution such as a hydroxyl or sul-
Fig. 4. Lineweaver–Burk plots of inhibition of human placental aromatase by
p-hydroxyphenyl- and p-sulphamoylphenyl-substituted steroids with AD as the
substrate. (A) Concentrations of p-hydroxyphenyl-substituted steroid 10: (ꢀ)
control (0 nM); (᭹) 20 nM; (ꢀ) 40 nM; (ꢁ) 80 nM. (B) Concentrations of p-
sulphamoylphenyl-substituted steroid 13: (ꢀ) control (0 nM); (᭹) 10 nM; (ꢀ)
30 nM; (ꢁ) 60 nM. The inhibition experiments with all the other steroids examined
gave essentially similar to (A) and (B) (data not shown).

896
Y. Watari et al. / Steroids 75 (2010) 891–896
Table 2
[5] Reed MJ, Owen AM, Lai LC, Coldham NG, Ghilchik MW, Shaik NA, et al. In
situ oestrone synthesis in normal breast and breast tumour tissues: effect of
treatment with 4-hydroxyandrostenedione. J Cancer 1989;44:233–7.
[6] James VHT, McNeill JM, Lai LC, Newton CJ, Ghilchik MW, Reed MJ. Aromatase
activity in normal breast and breast tumor tissues: in vivo and in vitro studies.
Steroids 1987;50:269–79.
[7] Miller WR. Aromatase inhibitors. Endocr Rel Cancer 1996;3:65–79.
[8] Castiglione-Gertsch M. New aromatase inhibitors: more selectivity, less toxic-
ity, unfortunately, the same activity. Eur J Cancer 1996;32A:393–5.
In vitro sulphatase inhibition by p-sulphamoylphenyl-substituted steroids.
Compound
IC25, ␮Ma
4-Ene, 3
190 12
390 23
6␣-4-Ene, 12
6-4,6-Diene, 13
6-1,4,6-Triene, 14
210
9
200 13
[9] Miller WR. Aromatase inhibitors—where are we now? Br
1996;73:415–7.
[10] Samojik E, Santen RJ, Worgul TJ. Plasma estrone sulfate assessment of reduced
estrogen production during treatment of metastatic breast carcinoma. Steroids
1982;39:497–507.
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For comparison
Oestrone 3-sulphamate
IC50 = 0.32 0.0062
a
Substrate: 20 ␮M of [3H]oestrone sulphate.
[11] James VHT, McNeill JK, Lai LC, Newton CJ, Ghilchik MW, Reed MJ. Aro-
matase activity in normal breast tissues: in vivo and in vitro studies. Steroids
1987;50:269–79.
[12] Pasqualini JR, Gelly C, Nguyen BL. Metabolism and biologic response of estrogen
sulfates in hormone-dependent and hormone-independent mammary cancer
cell lines. Ann NY Acad Sci 1990;595:106–16.
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fatase pathway in breast tumors: relative importance versus the aromatase
pathway. J Clin Endocrinol Metab 1984;59:29–33.
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sulfate in postmenopausal woman: lack of diurnal variation, and effect of
ovariectomy, age, and weight. J Steroid Biochem 1981;14:1101–5.
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overview and perspective. Breast Cancer Res Treat 1994;30:1–6.
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375–405.
[17] Numazawa M, Oshibe M. 6-Alkyl- and 6-aryl-androst-4-ene-3,17-diones as
aromatase inhibitors. Synthesis and structure–activity relationships. J Med
Chem 1994;37:1312–9.
[18] Numazawa M, Oshibe M. Further studies on 6-alkylandrost-4-ene-3,17-
diones as aromatase inhibitors: elongation of the 6-alkyl chain. Steroids
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[19] Numazawa M, Yamaguchi S. 6-Phenylaliphatic-substituted androst-4-ene-
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addition to the above compounds, oestrone 3-sulphamate, a typi-
cal oestrone sulphatase inhibitor previously reported [24–26], was
tested as a positive control. The oestrone sulphatase activity in the
placental microsomes was determined by the radiometric method
in which the amount of [3H]oestrone released from the substrate
[3H]oestrone sulphate during the incubation was used as an index
of the sulphatase activity [25]. In the studies, the apparent Km for
oestrone sulphate was found to be approximately 18 nM. Disap-
pointingly, the p-sulphamoylphenyl derivatives 3 and 12–14 were
very weak inhibitors of oestrone sulphatase. Then, 25% inhibition
values of the control, IC25 values, of the sulphamates 3 and 12–14
were obtained to be ranging from 190 to 400 ␮M. The oestrone sul-
phatase activity was not effectively but slightly blocked by these
sulphamates examined. The results reveal that the introduction of
a p-sulphamoylphenyl moiety to C-4 or C-6 position of androstene-
dione analogues fail to obtain the effective inhibitory activity of
oestrone sulphatase; the position is unsuitable for the inhibitor of
oestrone sulphatase.
[20] Numazawa M, Oshibe M, Yamaguchi S, Tachibana M. Time-dependent inactiva-
tion of aromatase by 6-alkylandrosta-1,4-diene-3,17-diones. Effects of length
and configuration of the 6-alkyl group. J Med Chem 1996;39:1033–8.
[21] Numazawa M, Oshibe M, Yamaguchi S. 6-Alkylandrosta-4,6-diene-3,17-
diones and their 1,4,6-triene analogs as aromatase inhibitors. Steroids
1997;62:595–602.
[22] Numazawa M, Yamaguchi S. Synthesis and structure–activity relationships of
6-phenylaliphatic-substituted C19 steroids having a 1,4-diene, 4,6-diene, or
1,4,6-triene structure as aromatase inhibitors. Steroids 1999;64:187–96.
[23] Yusuf JAH, Xing-Ping L, Matthew H. Synthesis and evaluation of 4-
substituted-4-androstene-3,17-dione derivatives as aromatase inhibitors. J
Steroid Biochem Mol Biol 1995;54:111–9.
3. Conclusion
Compounds 3 and 12–14 introduced by a p-sulphamolyphenyl
group at C-4 or C-6 position of androstenedione analogues were
the powerful aromatase inhibitors but not effective oestrone
sulphatase inhibitors. It seems that the position of the sul-
phamoylphenyl group other than the C-4 or C-6 position will be
useful for causing a dual function, the aromatase inhibitor and
the oestrone sulphatase inhibitor. Synthesis of other sulphamoyl-
phenyl derivatives of androstenedione analogues, which are pow-
erful for oestrone sulphatase, is now conducted in our laboratory.
[24] Howarth NM, Purohit A, Reed MJ, Potter BVL. Estrone sulfamates: potent
inhibitors of estrone sulfatase with therapeutic potential.
1994;37:219–21.
J Med Chem
[25] Purohit A, Williams GA, Howarth NM, Potter VL, Reed MJ. Inactivation of steroid
sulfatase by an active-site directed inhibitor, estrone-3-sulfamate. Biochem-
istry 1995;34:11508–14.
Acknowledgements
[26] Reed MJ, Potter BVL. Steroid sulphatase inhibitors. US Patent 5,616; 1997.
[27] Woo LWL, Lightowler M, Purohit A, Reed MJ, Potter BVL. Heteroatom-
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Biochem Mol Biol 1996;57:79–88.
[28] Williams GJ, Woo LWL, Mahon MF, Purohit A, Reed MJ, Potter BVL. X-ray crystal
structure and mechanism of action of oestrone 3-O-sulphamate, a synthetic
active site-directed inhibitor of oestrone sulfatase. Pharm Sci 1966;2:11–6.
[29] Lawrence Woo LW, Howarth NM, Purohit A, Hejaz HMA, Reed MJ, Potter BVL.
Steroidal and nonsteroidal sulfatase as potent inhibitors of steroid sulfatase. J
Med Chem 1998;41:1068–83.
[30] Numazawa M, Ando M, Watari Y, Tominaga T, Hayata Y, Yoshimura A.
Structure–activity relationships of 2-, 4-, or 6-substituted estrogens as aro-
matase inhibitors. J Steroid Biochem Mol Biol 2005;96:51–8.
[31] Numazawa M, Tominaga T, Watari Y, Tada Y. Inhibition of estrone sulfatase by
aromatase inhibitor-based estrogen 3-sulfamates. Steroids 2006;71:371–9.
[32] Ryan KJ. Biochemical aromatization of steroids. J Biol Chem 1959;234:268–
72.
This work was supported, in part, by a High Technology Research
Centre 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.
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