Sulfamoyloxy-substituted 2-phenylindoles: Antiestrogen-based inhibitors of the steroid sulfatase in human breast cancer cells

Article abstract of DOI:10.1016/S0968-0896(02)00306-1

Estrone sulfate (E1S) is an endogenous prodrug that delivers estrone and, subsequently, estradiol to the target cells following the hydrolysis by the enzyme estrone sulfatase which is active in various tissues including hormone dependent breast cancer cells. Blockade of this enzyme should reduce the estrogen level in breast cancer cells and prevent hormonal growth stimulation. Sulfamates of a variety of phenolic compounds have been shown to be inhibitors of estrone sulfatase. Our rational is based on findings that these inhibitors can undergo hydrolysis and the pharmacological effects of the free hydroxy compounds contribute to the bioactivity of the sulfamates. A desirable action of the metabolites would be an estrogen antagonism to block stimulatory effects of residual amounts of estrogens. Thus, we synthesized a number of sulfamoyloxy-substituted 2-phenylindoles with side chains at the indole nitrogen that guarantee antiestrogenic activity. All of the new sulfamates were studied for their inhibitory effects on the enzyme estrone sulfatase from human breast cancer cells and their (anti)hormonal activities in stably transfected human MCF-7/2a mammary carcinoma cells. The hormonal profile of the sulfamates was partly reflected by the properties of the corresponding hydroxy precursors. Some of the sulfamoylated antiestrogens strongly inhibited estrone sulfatase activity with IC₅₀ values in the submicromolar range. They were devoid of agonist activity and suppressed estrone sulfate-stimulated gene expression mainly by blocking the enzyme. Examples are the disulfamates of the indoles ZK 119, 010 and ZK 164, 015. Their IC₅₀s for sulfatase inhibition were 0.3 and 0.2 μM, respectively, and 50 and 80 nM, respectively, for the inhibition of E1S-stimulated luciferase expression in transfected MCF-7 cells. With some of the new sulfamates an additional direct antiestrogenic effect was noticed which might be due to a partial hydrolysis during incubation and would improve the growth inhibitory effect on estrogen-sensitive breast cancer cells.

Full text of DOI:10.1016/S0968-0896(02)00306-1

Bioorganic & Medicinal Chemistry 10 (2002) 3941–3953
Sulfamoyloxy-Substituted 2-Phenylindoles:
Antiestrogen-Based Inhibitors of the Steroid Sulfatase
in Human Breast Cancer Cells
Thomas Golob, Renate Liebl and Erwin von Angerer*
Institut fur Pharmazie, Universitat Regensburg, D-93040 Regensburg, Germany
¨
¨
Received 25 February 2002;accepted 10 July 2002
Abstract—Estrone sulfate (E1S) is an endogenous prodrug that delivers estrone and, subsequently, estradiol to the target cells fol-
lowing the hydrolysis by the enzyme estrone sulfatase which is active in various tissues including hormone dependent breast cancer
cells. Blockade of this enzyme should reduce the estrogen level in breast cancer cells and prevent hormonal growth stimulation.
Sulfamates of a variety of phenolic compounds have been shown to be inhibitors of estrone sulfatase. Our rational is based on
findings that these inhibitors can undergo hydrolysis and the pharmacological effects of the free hydroxy compounds contribute to
the bioactivity of the sulfamates. A desirable action of the metabolites would be an estrogen antagonism to block stimulatory effects
of residual amounts of estrogens. Thus, we synthesized a number of sulfamoyloxy-substituted 2-phenylindoles with side chains at
the indole nitrogen that guarantee antiestrogenic activity. All of the new sulfamates were studied for their inhibitory effects on the
enzyme estrone sulfatase from human breast cancer cells and their (anti)hormonal activities in stably transfected human MCF-7/2a
mammary carcinoma cells. The hormonal profile of the sulfamates was partly reflected by the properties of the corresponding
hydroxy precursors. Some of the sulfamoylated antiestrogens strongly inhibited estrone sulfatase activity with IC50 values in the
submicromolar range. They were devoid of agonist activity and suppressed estrone sulfate-stimulated gene expression mainly by
blocking the enzyme. Examples are the disulfamates of the indoles ZK 119, 010 and ZK 164, 015. Their IC50s for sulfatase inhibi-
tion were 0.3 and 0.2 mM, respectively, and 50 and 80 nM, respectively, for the inhibition of E1S-stimulated luciferase expression in
transfected MCF-7 cells. With some of the new sulfamates an additional direct antiestrogenic effect was noticed which might be due
to a partial hydrolysis during incubation and would improve the growth inhibitory effect on estrogen-sensitive breast cancer cells.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
has been demonstrated that the steroid sulfatase present
in breast cancer cells plays a more important role than the
enzyme aromatase in the formation of free estrogens.4 In
mammary tumors the levels of free and conjugated
estrogens as well as the sulfatase activity is significantly
higher than in normal tissue.5 These findings have sti-
mulated the search for inhibitors of steroid sulfatase.
The majority of breast cancers are hormone-dependent,
at least in their early stages, and require estrogens for
growth. Blockade of estrogen receptors by antiestrogens
and inhibition of estrogen biosynthesis by aromatase
inhibitors are therapeutic options which both have
proved effective in the treatment of postmenopausal
patients although number and duration of remissions
are far from being acceptable. Studies on estrogen
metabolism in man have identified estrone sulfate as the
predominant form of circulating estrogens and has also
been detected in breast cancer tissue.1 Mammary tumor
tissue has been shown to be capable of cleaving this
conjugate to liberate free estrone which can subse-
quently be converted by 17b-HSD to 17b-estradiol.2, 3 It
The starting point was the natural substrate estrone
sulfate which was first modified chemically in the
3-position.6ꢀ8 The most favourable substituent in
respect to enzyme inhibition was the sulfamoyloxy
group but the inherent estrogenic potency9 made this
derivative (EMATE, Fig. 1) unsuitable for further
development as sulfatase inhibitor. Meanwhile a variety
of steroidal10,11 and nonsteroidal sulfamates12,13 have
been synthesized and evaluated as enzyme inhibitors.14
Some of these agents have shown estrogenic activity10,15
while others were devoid of a hormonal action.13 Since
these sulfamates can act as both inhibitor and substrate of
*Corresponding author. Tel.: +49-941-9434821;fax: +49-941-
9434820;e-mail: erwin.von-angerer@chemie.uni-regensburg.de
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00306-1

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T. Golob et al. / Bioorg. Med. Chem. 10 (2002) 3941–3953
steroid sulfatase the bioactivity of the free phenols is of
great importance and has to be considered in the design
of sulfatase inhibitors for therapeutic application. For
clinical use as drug for the treatment of estrogen-
dependent breast cancer it would be desirable to have
an inhibitor of estrone sulfatase which can be hydro-
lyzed to a potent antiestrogen to offer the possibility of
a dual mode of action (Fig. 2).
system as basic structure because of its inherent anti-
estrogenic properties, and converted the phenolic
hydroxy groups into sulfooxy functions with sodium as
the cation.16 The most active compound in this series
was the fluorinated derivative 15 (Fig. 1). The following
studies were aimed to increase the antiestrogenic
potency of the 2-phenylindole system by introduction of
functional side chains.17ꢀ19 Some of these new com-
pounds proved to be pure estrogen antagonists because
they lack residual estrogenic activity. Parallel to these
studies, other groups optimized the functional group
necessary for the interaction with steroid sulfatase, and
identified the sulfamoyloxy group as the most potent
function.20 Based on these findings, we also used this
element and converted 2-phenyl-3-methylindoles with
different side chains at the nitrogen and a variable
substitution pattern in the aromatic rings into the sulfa-
mates 8–14 (Fig. 1). All sulfamoyl derivatives were tested
in whole cell assays for inhibitory activity against steroid
sulfatase from human MCF-7 breast cancer cells, for
estrogenic and antiestrogenic activity in stably trans-
fected MCF-7/2a mammary carcinoma cells and for
their effects on estrone sulfate-stimulated MCF-7/2a
cells which mimic the in vivo situation. Both, the sulfa-
mates and the corresponding free hydroxy compounds
as possible products of a chemical or enzymatic hydro-
lysis were also tested for their ability of binding to the
estrogen receptor to estimate hormonal effects.
In our first study on non-steroidal inhibitors of the
enzyme steroid sulfatase we used the 2-phenylindole
Chemistry
Figure. 1. Chemical structures of estrone sulfate, estrone-3-sulfamate
(EMATE) and 2-phenylindole-based inhibitors of steroid sulfatase.
The 2-phenylindole-based sulfamates were prepared as
outlined in Schemes 1–3. The starting material for the
syntheses was 2-(4-methoxyphenyl)-3-methylindole and
2-phenyl-3-methylindole, respectively, with methoxy or
other groups in the 5- or 6-position (1).21 Three different
side chains which give rise to different degrees of estrogen
antagonism were introduced by a nucleophilic substitution
reaction. The ethyl group was used because it is also
found in the zindoxifene structure.22 The side chains
with a pyrrolidine ring or sulfone group were used in
analogy to the potent antiestrogens ZK 119.01023 and
ZK 164.01519 Ether cleavage of the derivatives 2–4 with
boron tribromide gave the compounds 5–7 (Scheme 1).
Generally, the side chains were introduced without fur-
ther modification except for the tertiary amines 11a and
11b which were obtained by LiAlH4 reduction of the
corresponding amides 6a and 6b after ether cleavage
(Scheme 2). All of the phenolic compounds were con-
verted to the corresponding sulfamates by reacting them
with sulfamoyl chloride. When hydroxy groups were
present in both aromatic rings often the mono-
sulfamates could be isolated as byproducts. In all these
cases, the phenolic group in the indole part reacted first
and it was not possible to obtain monosulfamoylated
products with the sulfamate function in the phenyl ring.
For the conversion of the two pyrrolidinohexyl deriva-
tives (11a, b) into the sulfamates the procedure had to
be modified due to the reduced reactivity of the hydroxy
functions. Prior to the reaction with sulfamoyl chloride
the phenolic groups had to be converted to the anions
by sodium hydride (Scheme 2).
Figure 2. Stimulation of breast cancer growth by estrone sulfate:
points of intervention by antiestrogen-based steroid sulfatase inhibi-
tors.

T. Golob et al. / Bioorg. Med. Chem. 10 (2002) 3941–3953
3943
Scheme 1.
Results
Since the compounds were evaluated for biological
activity in estrogen receptor (ER) positive breast cancer
cells their affinity for this particular receptor was deter-
mined as the first step of biological characterization. As
in previous studies18 calf uterine cytosol was used as the
receptor source and no attempt was made to distinguish
between ERa and ERb. The binding affinities decreased
considerably by the conversion of the phenols into the
sulfamates. Generally, the RBA values dropped by
one order of magnitude after one hydroxy group had
been sulfamoylated and two orders of magnitude
when both hydroxy functions had been modified
(Table 1).
The target enzyme of these investigations is the steroid
sulfatase present in various human tissues including
breast cancer cells. An appropriate source for this
enzyme are humam MCF-7 breast cancer cells which
express this enzyme in sufficient quantities24 and might
be more relevant than placental microsomes used by
others. The enzyme inhibitory activities were deter-
mined in a whole cell assay25 because intact cells were
Scheme 2.
Scheme 3.

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T. Golob et al. / Bioorg. Med. Chem. 10 (2002) 3941–3953
Table 1. Relative binding affinities of 2-phenylindole sulfamates and their parent hydroxy derivatives for the estrogen receptor
Compd
R1
R2
R3
R4
RBAa
5a
8a
9a
5b
8b
9b
5c
8c
5d
8d
5e
8e
6a
10a
6b
10b
11a
12a
11b
12b
7a
13a
14a
7b
13b
14b
7c
13c
7f
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
OH
OH
OSO2NH2
OH
OH
OSO2NH2
OH
OSO2NH2
H
OH
OSO2NH2
OSO2NH2
H
H
H
14
0.85
0.04
21
0.57
0.05
3.4
0.37
0.37
<0.001
0.47
0.01
18.9
0.18
12.2
0.03
33
0.64
8.4
0.25
4.9
0.45
0.07
2.4
H
OH
OSO2NH2
OSO2NH2
H
H
H
H
H
H
OH
H
H
OH
C2H5
C2H5
C2H5
H
H
OSO2NH2
H
H
OH
OSO2NH2
OH
OSO2NH2
OH
OSO2NH2
OH
OSO2NH2
OH
OH
OSO2NH2
OH
OH
OSO2NH2
OH
OSO2NH2
OH
OSO2NH2
OH
OSO2NH2
H
H
OH
OSO2NH2
H
H
OH
OSO2NH2
OSO2NH2
H
H
H
(CH2)5CON(CH2)4
(CH2)5CON(CH2)4
(CH2)5CON(CH2)4
(CH2)5CON(CH2)4
(CH2)6N(CH2)4
(CH2)6N(CH2)4
(CH2)6N(CH2)4
(CH2)6N(CH2)4
(CH2)10SO2C5H11
(CH2)10SO2C5H11
(CH2)10SO2C5H11
(CH2)10SO2C5H11
(CH2)10SO2C5H11
(CH2)10SO2C5H11
(CH2)10SO2C5H11
(CH2)10SO2C5H11
(CH2)10SO2C5H11
(CH2)10SO2C5H11
(CH2)10SO2C5H11
(CH2)10SO2C5H11
H
OH
OSO2NH2
H
H
OH
OSO2NH2
H
H
H
OH
H
H
H
H
OSO2NH2
OSO2NH2
0.12
0.01
0.19
0.06
0.93
0.03
0.24
n.d.
H
H
H
H
H
H
CONH2
CN
COOCH3
COOCH3
13f
7g
13g
OSO2NH2
OH
OSO2NH2
aRelative binding affinities for the calf uterine estrogen receptor, determined by incubation at 4 ꢁC for 20 h. RBA value for 17b-estradiol=100.
also used for the following assays. Cells were incubated
with [3H]estrone sulfate in the presence of an inhibitor
for 20 h. Enzyme activity was estimated by the amount
of [3H]estrone that was formed during incubation and
extracted with toluene. The extraction yield was quan-
tified by the addition of [14C]-labeled estrone and
measurement of both radioactive nuclei. The inhibitory
activity of the new sulfamates varied over a wide range
(Table 2). Some of the indole-based monosulfamates
were completely devoid of activity (8e, 13a, 13b). The
strongest inhibition was observed for the disulfamate 9b
(IC50: 5 nM) with an ethyl side chain and a sulfamoyl-
oxy group in position 6. Its effect was only one order of
magnitude weaker than that of the steroidal reference
drug estrone-3-sulfamate (EMATE) but higher than
other nonsteroidal sulfamates such as the coumarin-
based inhibitor COUMATE with an IC50 value of 380
nM.12 High inhibitory activity was also found for 9a,
for the two pyrrolidinocarbonyl derivatives (10a, 10b),
and the disulfamate 14b with a 10-(pentylsulfonyl)decyl
side chain with IC50 values in the range of other non-
steroidal structures tested in MCF-7 cells.12 The data
for the three monosulfamates 8c, 8d, and 8e without an
additional functional group in the other ring revealed
that the sulfamoyloxy group in the phenyl ring provides
the most favorable conditions for inhibition. The values
for 14a, 13f, and 13g indicate the importance of a polar
function in the indole moiety (Table 2).
The aim of this study was the identification of non-
steroidal compounds that inhibit the enzyme steroid
sulfatase without estrogenic side effects but possibly
with antiestrogenic activity. Both actions were deter-
mined in stably transfected MCF-7/2a breast cancer
cells. These cells express the estrogen receptor like the
wild-type cells and carry the luciferase gene under control
of an estrogen responsive element (ERE) as the reporter
for estrogen-driven gene expression. All of the 2-phenyl-
indole-based sulfamates with an ethyl group at the
nitrogen (8, 9) showed moderate to strong estrogenic
activity at a concentration of 1 mM (Table 2). The effect
of estradiol at 10ꢀ8 M, however, was not reached. All
sulfamates with side chains other than ethyl were devoid
of agonist activity in this assay (Table 2). When the
results were compared with data for the derivatives
lacking one or both the sulfamoyl groups no marked
difference was noticed (Fig. 3a and b). As expected from
the literature, EMATE proved to be a potent agonist and
exceeded the value for estradiol considerably when tested
at 1 mM (Table 2).

T. Golob et al. / Bioorg. Med. Chem. 10 (2002) 3941–3953
3945
Table 2. Sulfatase inhibition, estrogenic and antiestrogenic activities, and combined hormonal effects of 2-phenylindole sulfamates in wild-type or
transfected MCF-7 breast cancer cells
Compd
Inhibition of
estrone sulfatasea
Estrogenic activityb
(% of E2)
Antiestrogenic activityc
(% inhibition)
Inhibition of
E1 sulfate actiond
[IC50 (mM) or % at 1 mM]
IC50 (mM) or % at 1 mM
8a
9a
8b
9b
8c
8d
8e
10a
10b
12a
12b
13a
14a
13b
14b
13c
13f
13g
EMATE
0.5
0.01
0.2
0.005
0.2
73
73
82
79
72
41
15
0
18
0
0
0
1
0
0
0
0
0
128
ꢀ8
ꢀ7
ꢀ2
11
ꢀ24
1
ꢀ19%
ꢀ7%
ꢀ7%
ꢀ10%
ꢀ10%
5%
26%
0.20
0.40
20%
0%
ꢀ2
0.07
0.03
0.3
0.3
0%
4
8
8
4
22
37
24
16
7
0.05
0.07
0.14
0.08
0.46
0.44
0.67
0.10
0.2
0%
0.07
5
0.15
0.27
0.0003
5
6
6
0.20
ꢀ50%
aInhibitory effect on the conversion of [3H]estrone sulfate (2 nM) to [3H]estrone by MCF-7 cells.
bStimulation of luciferase activity by test compounds (1 mM) in stably transfected MCF-7/2a cells;given in% of the value for estradiol (10 nM).
Basal activity (15% of E2) was subtracted from all values.
cInhibition of estradiol stimulated luciferase activiy by test compounds (1 mM) in stably transfected MCF-7/2a cells;given in % of the value for
estradiol (10 nM). Negative values indicate an additional estrogenic effect.
dInhibition of estrone sulfate stimulated luciferase activity in stably transfected MCF-7/2a cells;given in% of the value for estrone sulfate (100 n M).
Negative values indicate an additional estrogenic effect.
Antiestrogenic properties were determined in a similar
way except that the MCF-7/2a cells were treated with
the 2-phenylindole derivatives (1 mM) in the presence of
10ꢀ8 M estradiol. All sulfamates with an ethyl group
were devoid of antiestrogenic activity as expected from
the test on agonist action (Table 2). The disulfamates
with pyrrolidinocarbonyl-pentyl or pyrrolidinohexyl as
side chains showed no significant antagonism, but three
out of the four phenols (6a, 11a, 11b) acted as anti-
estrogens (Fig. 4a). This activity has been previously
reported for 11a (ZK 119.010) in mice and rats.23 The
only sulfamates with antiestrogenic properties in this
assay are characterized by a (pentylsulfonyl)decyl side
chain which is typical for pure antiestrogens (Table 2).
The strongest effect was observed for the disulfamate
14a (37% inhibition at 1 mM). The value for the corre-
sponding monosulfamate 13a with a free hydroxy group
at the phenyl ring was 22% whereas the dihydroxy
derivative 7a completely antagonized the effect of estra-
diol as described previously (Fig. 4b).19
than inhibited it (Table 2). All of the compounds with
other side chains strongly inhibited the stimulation by
estrone sulfate as measured by the luciferase expression.
The IC50 values were in the submicromolar range (Table
2). The most potent derivatives were the two pyrrolidi-
nohexyl derivatives 12a and 12b (IC50: 50 and 70 nM,
respectively) and the indole 14a with a (pentylsulfo-
nyl)decyl side chain (IC50: 80 nM). The 2-phenylindoles
with functionalities other than sulfamoyloxy in the
indole part (13f, 13g) displayed similar activities. In
those cases (12a, 12b, 14a) in which the inhibitory effect on
estrone sulfate-stimulated luciferase expression in trans-
fected cells exceeded the inhibition of steroid sulfatase
measured in wild-type MCF-7 cells a contribution of
estrogen antagonism to the overall effect is likely
(Table 2).
Discussion
For the interpretation of these results both, enzyme
inhibition and hormonal effects on the cellular level
have to be considered. Possibly, the enzyme-catalyzed
hydrolysis of the sulfamates to the free hydroxy deriva-
tives can contribute to overall activity if the sulfamates
function as substrates. All sulfamoyloxy derivatives
with inherent estrogenic activity were incapable of
interfering with the estrone sulfate mediated stimulation
of gene expression although they displayed the strongest
inhibitory effects on the enzyme. Obviously, the ability
of binding to the active site of the enzyme coincides with
the capability of acting as agonist. The inhibitory activities
The majority of these compounds was shown to act as
steroid sulfatase inhibitors of variable potencies. The
overall effect of these agents on gene expression may
arise from both the inhibition of this particular enzym
and their (anti)hormonal action. After these three dif-
ferent activities had been quantified separately, the
combined effect of the sulfamates on the transcriptional
activity was estimated in MCF-7/2a cells by using
estrone sulfate (0.1 mM) as the agonist instead of estra-
diol or estrone. All ethyl derivatives with estrogenic
activity rather enhanced the effect of estrone sulfate

3946
T. Golob et al. / Bioorg. Med. Chem. 10 (2002) 3941–3953
Figure 3. Comparison of the estrogenic activities of 2-phenylindole
sulfamates (1 mM) and their corresponding hydroxy derivatives (1 mM)
in stably transfected MCF-7/2a breast cancer cells. Panel a: Deriva-
tives with pyrrolidine-containing side chains. Panel b: Derivatives with
10-(pentylsulfonyl)decyl side chains. E1=estrone (10 nM). Values are
means ꢂSD of three independent experiments.
Figure 4. Comparison of direct antiestrogenic effects of 2-phenyl-
indole sulfamates (1 mM) and their corresponding hydroxy derivatives
(1 mM) in stably transfected MCF-7/2a breast cancer cells. Panel a:
Derivatives with pyrrolidine-containing side chains. Panel b: Deriva-
tives with 10-(pentylsulfonyl)decyl side chains. E2=17b-estradiol (10
nM);ICI=fulvestrant (ICI 182, 780;1 mM);ESA=estrone sulfamate
(EMATE;1 mM). Values are means ꢂSD of three independent
experiments.
of the pyrrolidinocarbonyl derivatives 10a and 10b with
submicromolar IC50 values can be rationalized by the
enzyme inhibition. A contribution of the free hydroxy
compounds is unlikely for 10b but cannot be ruled out
for 10b because its hydroxy derivative (6a) displayed
significant antiestrogenic effects. The activity profile
changed when the carbonyl group had been reduced to
a methylene group to give 12a and 12b. These amino
derivatives proved to be the most active compounds in
this series in respect to the blockade of estrone sulfate
action in transfected MCF-7/2a cells with IC50 values of
50 and 70 nM, respectively. The observed increase in
overall activity was not paralleled by an enhanced
enzym inhibition but appears to be an additive effect of
estrogen antagonism due to the formation of the free
hydroxy derivatives 11a and 11b. This result supports
the assumption that these agents can act by a dual mode
of action.
estrone sulfate-stimulated MCF-7/2a cells was found for
the two monosulfamate 13f and 13g though their anti-
estrogenic effects were hardly significant. Their potency
can only be rationalized by a strong inhibition of the
steroid sulfatase. The comparison of the IC50 values of
compounds 14a and 14b revealed that the antiestrogenic
effect in this particular series has a greater share than
the enzyme inhibition because the stronger antagonist
14a inhibited the action of estrone sulfate better than
the isomer 14b despite its lower inhibitory effect on the
enzym (Table 2). The most favorable conditions are
provided by strong enzyme inhibition combined with
antiestrogenic activity of the free phenols as demon-
strated for the bis(sulfamoyloxy)-substituted 2-phenyl-
indole 14a with an IC50 value of 80 nM.
In the series of 2-phenylindoles with a (pentylsulfo-
nyl)decyl side chain which is responsible for the strong
antiestrogenic character of this type of molecule both,
mono- and disulfamoylated products were studied. It
was interesting to note that the monosulfamates 13a
and 13b though devoid of enzyme inhibition strongly
decreased the stimulatory effect of estrone sulfate.
Obviously, hydrolysis of estrone sulfate to estrone took
place but the monosulfamates blocked the action of
thus formed estrone and/or estradiol by virtue of their
antiestrogenic properties. A much higher activity in
Conclusion
The results of this study cleary showed that the 2-phe-
nylindole system is appropriate for the synthesis of new
steroid sulfatase inhibitors after one or two phenolic
hydroxy groups have been converted to sulfamates.
Sufficient potency can be reached when the indole moiety
carries a polar substituent besides a sulfamoyloxy group
in the phenyl ring. Though the 1-ethyl derivatives possess
the highest inhibitory activity they are inappropriate for
further development due to their inherent estrogenic

T. Golob et al. / Bioorg. Med. Chem. 10 (2002) 3941–3953
3947
activity. Obviously, sulfamates derived from agonists fit
much better into the active site of the enzyme than
antiestrogen based sulfamates because they lack the
functional side chain. Systematic variation of this side
chain, however, made it possible to keep the decrease in
enzyme inhibition to a minimum without changing the
hormonal profile of the parent compounds. All anti-
estrogen-derived sulfamates strongly inhibited estrone
sulfate-stimulated gene expression in stably transfected
MCF-7/2a breast cancer cells mainly by virtue of their
enzyme inhibiting activity. One particular compound
(14a), however, seems to block the estrogen receptor as
an antiestrogen after sulfatase catalyzed hydrolysis. A
careful analysis of the data for the two most active
compounds 12a and 12b revealed that they abrogate the
effect of estrone sulfate on gene activation by a dual
mode of action: enzyme inhibition and receptor blockade.
These findings can be taken as proof of principle and
provide the basis for further investigations.
5-Methoxycarbonyl-2-(4-methoxyphenyl)-3-methylindole
(1g). Prepared from methyl 4-aminobenzoate by a
method similar to that described for 1f. The crude pro-
duct was purified by chromatography and recrystallized
(EtOH) to give colorless crystals (20% yield), mp 161–
162 ꢁC. 1H NMR (DMSO-d6) d 2.41 (s, 3H, –CH3), 3.83
(s, 3H, –OCH3);3.86 (s, 3H, –OCH 3);7.10, 7.61
(AA0BB0, J=8.8 Hz, 4H, ArH);7.40 (dd, 3J=8.5 Hz,
3
5J=0.5 Hz, 1H, indole-H7);7.74 (dd, 3J=8.5 Hz,
4J=1.6 Hz, 1H, indole-H6);8.20 (dd, 4J=1.6 Hz,
5J=0.5 Hz, 1H, indole-H4);11.51 (s, 1H, –N–H). Anal.
(C18H17NO3): C, 73.19;H, 5.81;N, 4.74. Found: C,
72.63;H, 5.74;N, 4.61.
1-Ethyl-5-methoxy-3-methyl-2-phenylindole (2c). Under
N2, a solution of 5-methoxy-3-methyl-2-phenylindole
(1c, 1.19 g, 4.47 mmol) in dry DMF (25 mL) was added
slowly with stirring to an ice-cold suspension of NaH
(158 mg, 6.6 mmol) in dry DMF. Stirring was continued
until the gas evolution ceased. Then, ethyl bromide
(0.49 g, 4.47 mmol) in dry DMF (20 mL) was added
dropwise with cooling in an ice bath. After the addition,
the ice bath was removed and stirring continued for 2 h
at rt. The excess of NaH was destroyed carefully by
dropwise addition of water, followed by the addition of
water (50 mL) and EtOAc (50 mL). The organic layer
was separated, and the aqueous layer was extracted
three times with EtOAc. The combined organic layers
were washed with water and dried (MgSO4). The solvent
was removed in vacuo and residue purified by chroma-
tography (SiO2) with CH2Cl2/EtOAc mixtures as the
eluent. Recrystallization with EtOH yielded colorless
Experimental
Melting points were determined on a Buchi 510 apparatus
and are uncorrected. NMR spectra were obtained on a
Bruker AC-250 spectrometer with TMS as internal stan-
dard and were in accord with the assigned structures. Purity
of all compounds was checked by tlc. Elemental analyses
were performed by the Mikroanalytisches Laboratorium,
University of Regensburg. High-resolution mass spectra
were recorded on a MAT 95 (Varian). The syntheses of
1-ethyl-5-hydroxy-2-(4-hydroxyphenyl)-3-methylindole
(5a),21 1-ethyl-6-hydroxy-2-(4-hydroxyphenyl)-3-methyl-
indole (5b),21 5-hydroxy-2-(4-hydroxyphenyl)-3-methyl-
1-[5-(pyrrolidinocarbonyl)pentyl]indole (6a),18 5-hydroxy-
2-(4-hydroxyphenyl)-3-methyl-1-[10-(pentylsulfonyl)decyl]
indole (7a),19 and 5-hydroxy-2-(4-hydroxyphenyl)-3-
methyl-1-[6-(pyrrolidino)hexyl]indole (ZK119010, 11a)17
have been described previously.
crystals (59% yield), mp 96–97 ꢁC. H NMR (CDCl3) d
1
1.19 (t, 3J=7.2 Hz, 3H, CH3–CH2–);2.21 (s, 3H, –CH 3);
3
3.90 (s, 3H, –OCH3);4.02 (q, J=7.2 Hz, 2H, CH3–CH2–
4
);6.90 (dd, 3J=8.8 Hz, J=2.4 Hz, 1H, indole-H6);7.05
4
5
(dd, J=2.4 Hz, J=0.5 Hz, 1H, indole-H4);7.26 (dd,
3J=8.8 Hz, 5J=0.5 Hz, 1H, indole-H7), 7.38–7.53 (m, 5H,
phenyl-H). Anal. (C18H19NO): C, 81.46;H, 7.23;N, 5.28.
Found: C, 81.14;H, 6.90;N, 5.00.
1-Ethyl-6-methoxy-3-methyl-2-phenylindole (2d). Pre-
pared from 6-methoxy-3-methyl-2-phenylindole (1d) by
a method similar to that described for 2c. Colorless
5-Carbamoyl-2-(4-methoxyphenyl)-3-methylindole (1f).
A solution of 4-aminobenzamide (51.54 g, 0.38 mol) in
N,N-dimethylaniline (75 mL) was heated to 170 ꢁC fol-
lowed by the slow addition of 0.18 mol of a-bromo-4-
methoxypropiophenone (43.74 g, 0.18 mol) in xylene
(240 mL). After heating for another 3 h under reflux,
the mixture was cooled to rt and poored into 2 N HCl
(600 mL). The organic layer was separated and the
aqueous extracted three times with EtOAc. The com-
bined organic layers were washed with 2 N HCl and
dried (MgSO4). After the evaporation of the solvent, the
residue was crystallized with a small volume of warm
EtOH to give a greenish solid (22% yield), mp 245–
247 ꢁC. 1H NMR (DMSO-d6) d 2.42 (s, 3H, –CH3);3.82
crystals (75% yield), mp 62–63 ꢁC (EtOH). H NMR
1
3
(CDCl3) d 1.19 (t, J=7.2 Hz, 3H, CH3–CH2–);2.21 (s,
3H, –CH3);3.90 (s, 3H, –OCH );4.02 (q, 3J=7.2 Hz,
3
4
2H, CH3–CH2–);6.81 (dd, 3J=7.8 Hz, J=2.2 Hz, 1H,
indole-H5);6.83 (dd, 4J=2.2 Hz, 5J=0.5 Hz, 1H,
indole-H7), 7.36–7.51 (m, 6H, phenyl-H, indole-H4).
Anal. (C18H19NO): C, 81.46;H, 7.23;N, 5.28. Found:
C, 81.42;H, 7.33;N, 5.21.
1-Ethyl-2-(4-methoxyphenyl)-3-methylindole (2e). Pre-
pared from 2-(4-methoxyphenyl)-3-methylindole (1e) by
a method similar to that described for 2c. Colorless
needles (89% yield), mp 133 ꢁC (EtOH). 1H NMR
0
3
(s, 3H, –OCH3);7.09, 7.61 (AA BB0, J=8.8 Hz, 4H,
ArH);7.10 (s, br, 1H, –CO–NH);7.33 (dd, 3J=8.4 Hz,
5J=0.5 Hz, 1H, indole-H7);7.67 (dd, 3J=8.4 Hz,
4J=1.6 Hz, 1H, indole-H6);7.86 (s, br, 1H, –CO–NH);
3
(CDCl3) d 1.20 (t, J=7.1 Hz, 3H, CH3–CH2–);2.22 (s,
3H, –CH3);3.88 (s, 3H, –OCH );4.05 (q, 3J=7.1 Hz,
3
0
3
2H, CH3–CH2–);7.01, 7.31 (AA BB0, J=8.8 Hz, 4H,
phenyl-H);7.04–7.60 (m, 4H, indole-H). Anal.
(C18H19NO): C, 81.46;H, 7.23;N, 5.28. Found: C,
81.17;H, 7.50;N, 5.12.
4
5
8.15 (dd, J=1.6 Hz, J=0.5 Hz, 1H, indole-H4);11.30
(s, 1H, –N–H). Anal. (C17H16N2O2): C, 72.83;H, 5.76;
N, 9.99. Found: C, 72.50;H, 5.71;N, 9.56.

3948
T. Golob et al. / Bioorg. Med. Chem. 10 (2002) 3941–3953
6-Methoxy-2-(4-methoxyphenyl)-3-methyl-1-(5-pyrrolidi-
nocarbonylpentyl)indole (3b). Prepared from 6-methoxy-
2-(4-methoxyphenyl)-3-methylindole (1b)21 and 6-bromo-
hexanoyl-pyrrolidine by a method similar to that
described for 2c. Yellow oil (85% yield). 1H NMR
(CDCl3) d 1.13–1.20 (m, 2H, –(CH2)2–CH2–);1.49–1.62
(m, 4H, –CH2–CH2–CH2–, –CH2–CH2–), 1.85–1.94 (m,
to that described for 2c. Light yellow resin (76% yield).
1H NMR (CDCl3) d 0.92 (t, 3J=7.0 Hz, 3H, CH3–CH2–);
1.13–1.49 (m, 16H, –CH2);1.57–1.89 (m, 6H, N–CH –
CH2–, SO2(CH2–CH2–)2);2.24 (s, 3H, –CH 3);2.89–2.96
2
(m, 4H, SO2(CH2–)2);3.89 (s, 3H, –OCH );3.95 (s, 3H,
3
–COOCH3);4.01 (t, 3J=7.5 Hz, 2H, N–CH2–);7.02,
7.30 (AA0BB0, 3J=8.7 Hz, 4H, phenyl-H);7.31 (dd,
3J=8.7 Hz, 5J=0.5 Hz, 1H, indole-H7);7.92 (dd,
3J=8.7 Hz, 4J=1.6 Hz, 1H, indole-H6);8.34 (dd,
3
4H, –N(CH2–CH2–)2), 2.12 (t, J=7.5 Hz, 2H, –CO-
CH2–), 2.18 (s, 3H, –CH3), 3.30–3.42 (m, 4H, –N–
(CH2–)2), 3.87 (s, 3H, –OCH3), 3.89 (s, 3H, –OCH3),
3.95 (t, 3J=7.5 Hz, 2H, indole-N–CH2–), 6.80 (dd,
3J=9.1 Hz, 4J=2.2 Hz, 1H, indole-H5), 6.81 (dd,
4J=2.2 Hz, 5J=0.5 Hz, 1H, indole-H7), 6.99, 7.27
5
4J=1.6 Hz, J=0.5 Hz, 1H, indole-H4).
1-Ethyl-2-(4-hydroxyphenyl)-3-methylindole (5c). Under
N2 a solution of 2c (143 mg, 0.54 mmol) in dry CH2Cl2
(10 mL) was added dropwise with stirring to a solution
of BBr3 (0.54 g, 2.16 mmol) in dry CH2Cl2 (10 mL) that
had been cooled to ꢀ20 ꢁC. After the addition the cool-
ing bath was removed and stirring continued for 3 h.
With cooling satd NaHCO3 solution (60 mL) was added
slowly with stirring followed by the addition of EtOAc
(100 mL). The aqueous phase was separated and
extracted twice with EtOAc. The combined organic
layers were washed with satd NaHCO3 solution and
water, and dried (MgSO4). After the evaporation of the
solvent the residue was purified by chromatography
(SiO2) with CH2Cl2/EtOAc (10:1) as the eluent. Crys-
tallization from CH2Cl2 gave colorless crystals (52%
yield), mp 122–124 ꢁC (CH2Cl2/n-hexane 2:1). 1H NMR
(DMSO-d6) d 1.08 (t, 3J=7.1 Hz, 3H, CH3–CH2–);2.13
(s, 3H, –CH3);4.04 (q, 3J=7.1 Hz, 2H, CH3–CH2–);
(AA0BB0, J=8.7 Hz, 4H, phenyl-H), 7.44 (dd, J=9.1
3
3
Hz, J=0.5 Hz, 1H, indole-H4).
5
6-Methoxy-2-(4-methoxyphenyl)-3-methyl-1-[10-(pentylsul-
fonyl)decyl]indole (4b). Prepared from (1b) and 1-bromo-
10-(pentylsulfonyl)decane19 by a method similar to that
described for 2c. Colorless crystals (53% yield), mp 61–
63 ꢁC (Et2O). H NMR (CDCl3) d 0.92 (t, J=7.0 Hz,
1
3
3H, CH3–CH2–);1.13–1.45 (m, 16H, –CH –);1.54–1.90
2
(m, 6H, –N–CH2–CH2–, SO2(CH2–CH2–)2);2.19 (s,
3H, –CH3);2.89–2.96 (m, 4H, SO 2(CH2–)2);3.88 (s, 3H,
–OCH3);3.90 (s, 3H, –OCH 3);3.93 (t, 3J=7.5 Hz, 2H, –
N–CH2–);6.78–6.83 (m, 2H, indole-H 5+7);7.00, 7.28
(AA0BB0, J=8.8 Hz, 4H, phenyl-H);7.45 (dd, 3J=8.9
3
5
Hz, J=0.5 Hz, 1H, indole-H4). Anal. (C32H47NO4S):
C, 70, 92;H, 8.76;N, 2.59. Found: C, 70.78;H, 8.67;N,
2.63.
6.93, 7.23 (AA0BB0, J=8.6 Hz, 4H, phenyl-H);7.01–
3
7.51 (m, 4H, indole-H), 9.70 (s, br, 1H, –OH). Anal.
(C17H17NO): C, 81.23;H, 6.83;N, 5.57. Found: C,
81.07;H, 6.82;N, 5.58.
2-(4-Methoxyphenyl)-3-methyl-1-[10-(pentylsulfonyl)decyl]
indole (4c). Prepared from 1e and 1-bromo-10-(pentyl-
sulfonyl)decane19 by a method similar to that described
for 2c. Colorless solid (64% yield), mp 65 ꢁC. H NMR
1-Ethyl-6-hydroxy-3-methyl-2-phenylindole (5d). Pre-
pared from 2d by the method described for 5c. Colorless
crystals (61% yield), mp 149–150 ꢁC (CH2Cl2). 1H
NMR (DMSO-d6) d 1.05 (t, 3J=7.1 Hz, 3H, CH3–CH2–);
1
3
(CDCl3) d 0.92 (t, J=7.0 Hz, 3H, CH3–CH2–), 1.13–
1.49 (m, 16H, –CH2–);1.56–1.90 (m, 6H, N–CH 2–CH2–,
SO2(CH2–CH2–)2);2.22 (s, 3H, –CH 3);2.89–2.96 (m,
4H, SO2(CH2–)2), 3.89 (s, 3H, –OCH3);3.99 (t, 3J=7.5
3
2.11 (s, 3H, –CH3);3.94 (q, J=7.1 Hz, 2H, CH3–CH2–);
0
3
3
4
Hz, 2H, N–CH2–);7.02, 7.30 (AA BB0, J=8.7 Hz, 4H,
phenyl-H);7.09–7.34 (m, 3H, indole-H 5,6+7);7.56–7.59
(m, 1H, indole-H4). Anal. (C31H45NO3S): C, 72.74;H,
8.88;N, 2.74. Found: C, 72.46;H, 8.86;N, 2.73.
6.60 (dd, J=8.4 Hz, J=2.1 Hz, 1H, indole-H5);6.76
(dd, J=2.1 Hz, J=0.5 Hz, 1H, indole-H7);7.30 (dd,
3J=8.4 Hz, J=0.5 Hz, 1H, indole-H4);7.36–7.57 (m,
4
5
5
5H, phenyl-H);9.03 (s, 1H, –OH). Anal. (C 17H17NO):
C, 81.23;H, 6.83;N, 5.57. Found: C, 81.57;H, 6.79;N,
5.68.
5-Carbamoyl-2-(4-methoxyphenyl)-3-methyl-1-[10-(pentyl-
sulfonyl)decyl]indole (4f). Prepared from 1f and 1-
bromo-10-(pentylsulfonyl)decane19 by a method similar
to that described for 2c. Colorless crystals (80% yield),
mp 107–109 ꢁC (EtOH). 1H NMR (DMSO-d6) d 0.87 (t,
3J=7.1 Hz, 3H, CH3–CH2–);1.03–1.44 (m, 18H, –CH 2);
1.57–1.72 (m, 4H, SO2(CH2–CH2–)2);2.18 (s, 3H, –CH 3);
1-Ethyl-5-hydroxy-3-methyl-2-phenylindole (5e). Pre-
pared from 2e by the method described for 5c. Colorless
crystals (65% yield), mp 146–147 ꢁC. 1H NMR (DMSO-
3
d6) d1.04 (t, J=7.4 Hz, 3H, CH3–CH2–);2.08 (s, 3H,
–CH3);3.99 (q, 3J=7.4 Hz, 2H, CH3–CH2–);6.69 (dd,
3J=8.6 Hz, 4J=2.3 Hz, 1H, indole-H6);6.82 (dd,
4J=2.3 Hz, 5J=0.5 Hz, 1H, indole-H4);7.26 (dd,
3.00–3.07 (m, 4H, SO2(CH2–)2);3.84 (s, 3H, –OCH );
3
0
4.06 (t, 3J=7.1 Hz, 2H, NꢀCH2ꢀ);7.10, 7.36 (AA BB0,
5
3J=8.7 Hz, 4H, phenyl-H);7.10 (s, br, 1H, –CO–NH–);
3J=8.6 Hz, J=0.5 Hz, 1H, indole-H7);7.36–7.56 (m,
3
5
7.47 (dd, J=8.6 Hz, J=0.5 Hz, 1H, indole-H7);7.73
5H, phenyl-H);8.72 (s, 1H, –OH). Anal. (C 17H17NO): C,
81.23;H, 6.83;N, 5.57. Found: C, 81.19;H, 6.89;N, 5.56.
(dd, 3J=8.6 Hz, 4J=1.5 Hz, 1H, indole-H6);7.88 (s, br,
1H, –CO–NH–);8.16 (dd, 4J=1.5 Hz, J=0.5 Hz, 1H,
5
indole-H4). Anal. (C32H46N2O4S): C, 69.26;H, 8.37;N,
5.05. Found: C, 69.06;H, 8.30;N, 4.96.
6-Hydroxy-2-(4-hydroxyphenyl)-3-methyl-1-(5-pyrrolidi-
nocarbonylpentyl)indole (6b). Prepared from 3b by the
method described for 5c. Grey powder (64% yield), mp
1
5-Methoxycarbonyl-2-(4-methoxyphenyl)-3-methyl-1-[10-
(pentylsulfonyl)decyl]indole (4g). Prepared from 1g and
1-bromo-10-(pentylsulfonyl)decane19 by a method similar
182–183 ꢁC (EtOH). H NMR (DMSO-d6) d 1.03–1.09
(m, 2H, N–(CH2)2–CH2–);1.31–1.50 (m, 4H, N–CH –
–
2
CH2–, –CO–CH2–CH2–);1.70–1.86 (m, 4H, –N(CH
2

T. Golob et al. / Bioorg. Med. Chem. 10 (2002) 3941–3953
3949
CH2–)2);2.03–2.08 (m, 2H, –CO–CH –);2.07 (s, 3H,
2
1.05–1.45 (m, 16H, –CH2–);1.57–1.72 (m, 6H, N–CH –
2
3
–CH3);3.20–3.32 (m, 4H, –N–(CH –)2), 3.87 (t, J=7.2
2
CH2–, SO2(CH2–CH2–)2);2.17 (s, 3H, –CH 3);2.99–3.07
(m, 4H, SO2(CH2–)2);3.86 (s, 3H, –COOCH );3.99–
4
Hz, 2H, indole-N–CH2–);6.56 (dd, 3J=8.4 Hz, J=2.0
3
Hz, 1H, indole-H5);6.71 (d, 4J=2.0 Hz, 1H, indole-H7);
6.87, 7.17 (AA0BB0, 3J=8.5 Hz, 4H, phenyl-H);7.25 (d,
3J=8.4 Hz, 1H, indole-H4);8.93 (s, 1H, indole-OH);
9.60 (s, 1H, phenyl-OH). Anal. (C25H30N2O3): C,
73.85H, 7.45;N, 6.89. Found: C, 72.45;H, 7.49;N,
6.57.
4.09 (m, 2H, N–CH2–): 6.92, 7.23 (AA0BB0, 3J=8.5 Hz,
5
4H, phenyl-H);7.53 (dd, 3J=8.7 Hz, J=0.5 Hz, 1H,
indole-H7);7.77 (dd, 3J=8.7 Hz, 4J=1.6 Hz, 1H,
indole-H6);8.18 (dd, 4J=1.6 Hz, 5J=0.5 Hz, 1H,
indole-H4);9.70 (s, br, 1H, –OH);MS (EI, 70 eV) m/z
556.3 (MH+);HRMS calcd for C 32H45NO5S (M+)
555.30181, found 555.30179.
6-Hydroxy-2-(4-hydroxyphenyl)-3-methyl-1-[10-(pentyl-
sulfonyl)decyl]indole (7b). Prepared from 4b by the
method described for 5c. Colorless resin (91% yield). 1H
NMR (CDCl3) d 0.93 (t, 3J=7.0 Hz, 3H, CH3–CH2-ꢀ);
6-Hydroxy-2-(4-hydroxyphenyl)-3-methyl-1-(6-pyrrolidi-
nohexyl)indole (11b). A solution of 6b (700 mg, 1.72
mmol) in dry THF (60 mL) was added slowly to a
refluxing suspension of LiAlH4 (176 mg, 4.64 mmol) in
dry THF (50 mL). Heating was continued for 1 h fol-
lowed by stirring overnight at rt. For hydrolysis water
(75 mL) and satd NaHCO3 solution (25 mL) were
added. The mixture was extracted three times with
EtOAc. The combined organic layers were washed with
saline and water, and dried (MgSO4). After evaporation
of the solvents the crude product was purified by chro-
matography (SiO2) with EtOAc/MeOH (1:10) as eluent.
Brownish resin (81% yield). 1H NMR (DMSO-d6) d
1.06–1.08 (m, 4H, –CH2–(CH2–)2–CH2–);1.21–1.32 (m,
1.06–1.57 (m, 16H, –CH2–);1.68–1.97 (m, 6H, N–CH –
2
CH2–, SO2(CH2–CH2–)2);2.17 (s, 3H, –CH 3);2.95–3.02
(m, 4H, SO2(CH2–)2);3.90 (t, 3J=7.4 Hz, 2H, –N–
CH2–), 5.01 (s, br, 1H, indole-OH);6.13 (s, br, 1H,
phenyl-OH);6.69 (dd, 3J=8.5 Hz, 4J=2.2 Hz, 1H,
indole-H5);6.78 (dd, 4J=2.2 Hz, 5J=0.5 Hz, 1H,
indole-H7);6.95, 7.21 (AA 0BB0, 3J=8.6 Hz, 4H, phenyl-
5
H);7.39 (dd, 3J=8.5 Hz, J=0.5 Hz, 1H, indole-H4).
2-(4-Hydroxyphenyl)-3-methyl-1-[10-(pentylsulfonyl)decyl]
indole (7c). Prepared from 4e by the method described
for 5c. Colorless solid (80% yield), mp 68–69 ꢁC
(CH2Cl2/n-hexane 1:4). 1H NMR (CDCl3) d 0.93 (t,
3J=7.0 Hz, 3H, CH3–CH2–);1.07–1.48 (m, 16H, –CH 2–);
1.56–1.91 (m, 6H, N–CH2–CH2–, SO2(CH2–CH2–)2);
2.22 (s, 3H, –CH3);2.94–3.02 (m, 4H, SO 2(CH2–)2);
2H, N–CH2–CH2–);1.42–1.47 (m, 2H, indole-N–CH –
2
CH2–);1.60–1.68 (m, 4H, –N(CH 2–CH2–)2);2.07 (s,
3H, –CH3);2.22 (t, 3J=7.2 Hz, 2H, –N–CH2–);2.29–
2.34 (m, 4H, –N(CH2–)2);3.86 (t, 3J=7.2 Hz, 2H,
4
indole-N–CH2–);6.56 (dd, 3J=8.4 Hz, J=1.9 Hz, 1H,
3
4.01 (t, J=7.4 Hz, 2H, N–CH2–);6.12 (s, 1H, –OH);
6.96, 7.24 (AA0BB0, J=8.6 Hz, 4H, phenyl-H);7.09–
indole-H5);6.70 (dd, 4J=1.9 Hz, 5J=0.5 Hz, 1H,
indole-H7);6.86, 7.13 (AA 0BB0, 3J=8.5 Hz, 4H, phenyl-
3
5
7.25 (m, 2H, indole-H), 7.31–7.34 (m, 1H, indole-H);
7.56–7.59 (m, 1H, indole-H). Anal. (C30H43NO3S): C,
72.38;H, 8.72;N, 2.81. Found: C, 71.98;H, 8.89;N,
2.82.
H);7.24 (dd, 3J=8.4 Hz, J=0.5 Hz, 1H, indole-H4);
8.50–10.50 (s, br, 2H;–OH). Anal. (C25H32N2O2): C,
76.48;H, 8.22;N, 7.13. Found: C, 74.34;H, 8.32;N,
6.73.
5-Carbamoyl-2-(4-hydroxyphenyl)-3-methyl-1-[10-(pentyl-
sulfonyl)decyl]indole (7f). Prepared from 4f by the
method described for 5c. Light yellow resin (86% yield).
General procedure for the preparation of the sulfamates
A solution of the hydroxy-2-phenylindole (2.77 mmol)
in dry DMF (15 mL) was cooled to 10–15 ꢁC. Sulfamoyl
3
1H NMR (DMSO-d6) d 0.87 (t, J=7.0 Hz, 3H, CH3–
26
CH2–);1.05–1.44 (m, 18H, –CH –);1.54–1.72 (m, 4H,
SO2(CH2–CH2–)2);2.17 (s, 3H, –CH 3);3.00–3.07 (m,
choride, 13.9 mmol per hydroxy group, was added in
2
portions. After the addition, the mixture was stirred for
12 h under N2. It was hydrolyzed with water (40 mL)
followed by extraction with EtOAc. The combined
organic layers were washed with water and dried
(MgSO4). After evaporation of the solvent the residue
was chromatographed over SiO2 with CH2Cl2/EtOAc
mixtures as eluent. In the case of the dihydroxy deriva-
tives it was sometimes possible to isolate one of the
monosulfamates from the first fraction as a by-product.
4H, SO2(CH2–)2);4.05 (t, 3J=7.2 Hz, 2H, N–CH2–);
6.92, 7.23 (AA0BB0, J=8.5 Hz, 4H, phenyl-H);7.10 (s,
3
5
br, 1H, –CO–NH–);7.45 (dd, 3J=8.6 Hz, J=0.5 Hz,
4
1H, indole-H7);7.72 (dd, 3J=8.6 Hz, J=1.4 Hz, 1H,
indole-H6);7.87 (s, br, 1H, –CO–NH–);8.14 (dd,
5
4J=1.4 Hz, J=0.5 Hz, 1H, indole-H4);9.72 (s, br, 1H,
–OH).
2-(4-Hydroxyphenyl)-5-methoxycarbonyl-3-methyl-1-[10-
(pentylsulfonyl)decyl]indole (7g). Due to the sensitivity
of the ester function the cleavage reaction had to be
modified: To a solution of 4g (285 mg, 0.5 mmol) in dry
CH2Cl2 (10 mL) AlCl3 (200 mg, 1.5 mmol) was added
with stirring. After the addition stirring was continued
for 5 min followed by the addition of ethanethiol (155
mg, 2.5 mmol). After stirring for 3 h at rt under N2, the
mixture was pored into ice-water. The conditions for
working-up and purification were similar to those
described for 5c. Slightly red resin (46% yield). 1H
NMR (DMSO-d6) d 0.87 (t, 3J=7.0 Hz, 3H, CH3–CH2–);
1-Ethyl-2-(4-hydroxyphenyl)-3-methyl-5-sulfamoyloxyin-
dole (8a). Beige resin (13% yield). 1H NMR (DMSO-d6)
d 1.07 (t, 3J=7.1 Hz, 3H, CH3–CH2–);2.12 (s, 3H, –CH 3);
3
4.04 (q, J=7.1 Hz, 2H, CH3–CH2–);7.06 (dd, 3J=8.8
0
Hz, 4J=2.3 Hz, 1H, indole-H6);6.92, 7.24 (AA BB0,
3J=8.5 Hz, 4H, phenyl-H);7.40 (dd, 4J=2.3 Hz,
5J=0.5 Hz, 1H, indole-H4);7.48 (dd, 3J=8.8 Hz,
5J=0.5 Hz, 1H, indole-H7);7.78 (s, br, 2H, –SO NH2);
9.74 (s, br, 1H, phenyl-OH). MS (EI, 70 eV) m/z 347.2
2
(MH+);HRMS calcd for
347.10652, found 347.10650.
C
17H19N2O4S (MH+)

3950
T. Golob et al. / Bioorg. Med. Chem. 10 (2002) 3941–3953
1-Ethyl-3-methyl-5-sulfamoyloxy-2-(4-sulfamoyloxyphe-
nyl)indole (9a). Grey solid (21% yield), mp 193–195 ꢁC
3-Methyl-1-(5-pyrrolidinocarbonylpentyl)-5-sulfamoyl-
oxy-2-(4-sulfamoyloxyphenyl)indole (10a). Colorless
resin (66% yield). 1H NMR (DMSO-d6) d 1.01–1.10 (m,
1
3
(dec.). H NMR (DMSO-d6) d 1.10 (t, J=6.8 Hz, 3H,
CH3–CH2–);2.16 (s, 3H, –CH );4.10 (q, 3J=6.8 Hz,
2H, N–(CH2)2–CH2–);1.24–1.36 (m, 2H, –CO–CH –
3
2
4
2H, CH3–CH2–);7.11 (dd, 3J=8.9 Hz, J=2.3 Hz, 1H,
CH2–);1.41–1.50 (m, 2H, indole-N–CH –CH2–);1.70–
2
indole-H6);7.45 (dd, 4J=2.3 Hz, 5J=0.5 Hz, 1H,
indole-H4);7.46, 7.53 (AA 0BB0, 3J=8.7 Hz, 4H, phenyl-
H);7.55 (dd, 3J=8.9 Hz, J=0.5 Hz, 1H, indole-H7);
1.88 (m, 4H, –N(CH2–CH2–)2);2.04 (t, 3J=7.3 Hz, 2H,
–CO–CH2–);2.16 (s, 3H, –CH 3);3.19–3.31 (m, 4H, –N–
(CH2–)2);4.08 (t, 3J=7.0 Hz, 2H, indole–N–CH2–);
5
3
4
7.79 (s, br, 2H, indole-OSO2NH2);8.14 (s, br, 2H,
phenyl-OSO2NH2). Anal. (C17H19N3O6S2): C, 47.98;H,
4.51;N, 9.88. Found: C, 48.09;H, 4.83;N, 9.58.
7.10 (dd, J=8.8 Hz, J=2.2 Hz, 1H, indole-H6);7.44
(dd, J=2.4 Hz, J=0.5 Hz, 1H, indole-H4);7.44, 7.53
4
5
(AA0BB0, J=8.7 Hz, 4H, phenyl-H);7.54 (dd, 3J=8.8
3
Hz, 5J=0.5 Hz, 1H, indole-H7);7.80 (s, 2H, indole-
OSO2NH2);8.14 (s, 2H, phenyl-OSO NH2);MS (EI, 70
1-Ethyl-2-(4-hydroxyphenyl)-3-methyl-6-sulfamoyloxyin-
1
2
dole (8b). Brown resin (15% yield). H NMR (DMSO-
3
eV) m/z 565.2 (MH+);HRMS calcd for C 25H33N4O7S2
d6) d 1.09 (t, J=7.1 Hz, 3H, CH3–CH2–);2.13 (s, 3H,
(MH+) 565.17901, found 565.18031.
–CH3);4.02 (q, 3J=7.1 Hz, 2H, CH3–CH2–);6.98 (dd,
3J=8.6 Hz, 4J=2.0 Hz, 1H, indole-H5);6.92, 7.22
3-Methyl-1-(5-pyrrolidinocarbonylpentyl)-6-sulfamoyl-
oxy-2-(4-sulfamoyloxyphenyl)indole (10b). Colorless
resin (74% yield). 1H NMR (DMSO-d6) d 1.03–1.08 (m,
(AA0BB0, J=8.5 Hz, 4H, phenyl-H);7.36 (dd, 4J=2.0
3
5
Hz, J=0.5 Hz, 1H, indole-H7);7.52 (dd, 3J=8.6 Hz,
5J=0.5 Hz, 1H, indole-H4);7.81 (s, 2H, –SO 2NH2);
9.73 (s, 1H, phenyl-OH).
2H, –N–(CH2)2–CH2–);1.24–1.37 (m, 2H, –CO–CH –
2
CH2–);1.44–1.52 (m, 2H, indole-N–CH –CH2–);1.70–
2
1.88 (m, 4H, –N(CH2–CH2–)2);2.05 (t, 3J=7.3 Hz, 2H,
–CO–CH2–);2.18 (s, 3H, –CH 3);3.19–3.31 (m, 4H, –N–
(CH2–)2);4.04 (t, 3J=7.1 Hz, 2H, indole-N–CH2–);7.02
1-Ethyl-3-methyl-6-sulfamoyloxy-2(4-sulfamoyloxyphenyl)
indole (9b). Colorless solid (37% yield), mp 186–187 ꢁC
3
3
4
(dec.;MeOH). 1H NMR (DMSO-d6) d 1.12 (t, J=7.0
(dd, J=8.6 Hz, J=2.0 Hz, 1H, indole-H5);7.40 (dd,
Hz, 3H, CH3–CH2–);2.18 (s, 3H, –CH 3);4.05 (q,
3J=7.0 Hz, 2H, CH3–CH2–);7.02 (dd, 3J=8.5 Hz,
4J=1.7 Hz, 1H, indole-H5);7.40 (dd, 4J=1.7 Hz,
4J=2.0 Hz, 5J=0.5 Hz, 1H, indole-H7);7.44, 7.52
(AA0BB0, J=8.7 Hz, 4H, phenyl-H);7.57 (dd, 3J=8.6
3
5
Hz, J=0.5 Hz, 1H, indole-H4);7.84 (s, 2H, indole–
OSO2NH2);8.12 (s, 2H, phenyl-OSO NH2).
0
5J=0.5 Hz, 1H, indole-H7);7.45, 7.53 (AA BB0, 3J=8.7
2
5
Hz, 4H, phenyl-H);7.58 (dd, 3J=8.5 Hz, J=0.5 Hz,
1H, indole-H4);7.87 (s, br, 2H, indole-OSO 2NH2);8.10 (s,
br, 2H, phenyl-OSO2NH2). Anal. (C17H19N3O6S2): C,
47.98;H, 4.51;N, 9.88. Found: C, 48.39;H, 4.70;N, 9.75.
2-(4-Hydroxyphenyl)-3-methyl-1-[10-(pentylsulfonyl)decyl]-
5-sulfamoyloxyindole (13a). Colorless resin (27% yield).
1H NMR (CDCl3) d 0.93 (t, 3J=7.0 Hz, 3H, CH3–CH2–);
1.05–1.48 (m, 16H, –CH2–);1.50–1.93 (m, 6H, N–CH –
2
1-Ethyl-3-methyl-2-(4-sulfamoyloxyphenyl)indole
(8c).
CH2–, SO2(CH2–CH2–)2);2.18 (s, 3H, –CH 3);2.95–3.03
(m, 4H, SO2(CH2–)2): 4.00 (t, 3J=7.3 Hz, 2H, N–CH2–);
4.97 (s, br, 2H, –SO2NH2);6.26 (s, 1H, phenyl-OH);
6.94, 7.19 (AA0BB0, 3J=8.6 Hz, 4H, phenyl-H);7.17
Colorless crystals (58% yield), mp 173–175 ꢁC
1
3
(MeOH). H NMR (DMSO-d6) d 1.10 (t, J=7.1 Hz,
3H, CH3–CH2–);2.18 (s, 3H, –CH );4.09 (q, 3J=7.1
3
3
4
Hz, 2H, CH3–CH2–);7.05–7.22 (m, 2H, indole-H);
7.42–7.56 (m, 2H, indole-H);7.44, 7.54 (AA 0BB0,
3J=8.7 Hz, 4H, phenyl-H);8.13 (s, 2H, –SO 2NH2).
Anal. (C17H18N2O3S): C, 61.79;H, 5.50;N, 8.48.
Found: C, 61.65;H, 5.67;N, 8.28.
(dd, J=8.8 Hz, J=2.4 Hz, 1H, indole-H6);7.30 (dd,
3J=8.8 Hz, 5J=0.5 Hz, 1H, indole-H7);7.51 (dd,
5
4J=2.4 Hz, J=0.5 Hz, 1H, indole-H4).
3-Methyl-1-[10-(pentylsulfonyl)decyl]-5-sulfamoyloxy-2-
(4-sulfamoyloxyphenyl)indole (14a). Colorless resin
1
3
1-Ethyl-3-methyl-2-phenyl-6-sulfamoyloxyindole
Colorless crystals (47% yield), mp 163–165 ꢁC (dec.;
(8d).
(71% yield). H NMR (CDCl3) d (t, J=7.0 Hz, 3H,
CH3–CH2–);1.04–1.47 (m, 16H, –CH –);1.49–1.90 (m,
6H, N–CH2–CH2–, SO2(CH2–CH2–)2);2.19 (s, 3H,
2
1
3
MeOH). H NMR (DMSO-d6) d 1.09 (t, J=7.1 Hz,
3H, CH3–CH2–);2.16 (s, 3H, –CH );4.05 (q, 3J=7.1
–CH3);2.96–3.03 (m, 4H, SO (CH2–)2);3.98 (t, 3J=7.4
3
2
Hz, 2H, CH3–CH2–);7.02 (dd, 3J=8.5 Hz, J=2.0 Hz,
Hz, 2H, N–CH2–);5.08 (s, br, 2H, indole-OSO NH2);
4
2
1H, indole-H5);7.41 (dd, 4J=2.0 Hz, J=0.5 Hz, 1H,
5.51 (s, 2H, phenyl-OSO2NH2);7.20 (dd, 3J=8.8 Hz,
4J=2.3 Hz, 1H, indole-H6);7.31 (dd, 3J=8.8 Hz,
5J=0.5 Hz, 1H, indole-H7);7.36, 7.44 (AA 0BB0, 3J=8.8
5
indole-H7);7.57 (dd, 3J=8.5 Hz, 5J=0.5 Hz, 1H,
indole-H4);7.38–7.60 (m, 5H, phenyl-H);7.84 (s, 1H,
–SO2NH2). Anal. (C17H18N2O3S): C, 61.79;H, 5.50;N,
8.48. Found: C, 62.33;H, 5.67;N, 8.78.
5
Hz, 4H, phenyl-H);7.52 (dd, 4J=2.3 Hz, J=0.5 Hz,
1H, indole-H4);MS (EI, 70 eV) m/z 672.3 (MH+;
100%).
1-Ethyl-3-methyl-2-phenyl-5-sulfamoyloxyindole
(8e).
Colorless resin (81% yield). 1H NMR (DMSO-d6) d
1.07 (t, 3J=7.1 Hz, 3H, CH3–CH2–);2.15 (s, 3H, –CH 3);
4.08 (q, 3J=7.1 Hz, 2H, CH3—CH2–);7.10 (dd, 3J=8.8
2-(4-Hydroxyphenyl)-3-methyl-1-[10-(pentylsulfonyl)dec-
yl]-6-sulfamoyloxyindole (13b). Colorless resin (18%
yield). 1H NMR (CDCl3) d 0.93 (t, 3J=7.0 Hz, 3H,
4
Hz, J=2.3 Hz, 1H, indole-H6);7.44 (dd, 4J=2.3 Hz,
CH3–CH2–);1.05–1.51 (m, 16H, –CH –);1.54–1.92 (m,
2
5J=0.5 Hz, 1H, indole-H4);7.54 (dd, 3J=8.8 Hz,
5J=0.5 Hz, 1H, indole-H7);7.39–7.60 (m, 5H, phenyl-
H);7.78 (s, 2H, –SO NH2).
6H, N–CH2–CH2–, SO2(CH2–CH2–)2);2.19 (s, 3H,
–CH3);2.96–3.03 (m, 4H, SO (CH2–)2);3.97 (t, 3J=7.4
Hz, 2H, N–CH2–);5.03 (s, 2H, –SO NH2);7.08 (dd,
2
2
2

T. Golob et al. / Bioorg. Med. Chem. 10 (2002) 3941–3953
3951
3J=8.5 Hz, 4J=2.1 Hz, 1H, indole-H5), 6.94, 7.18
After evaporation of the solvent, the crude product was
purified by chromatography over SiO2 with EtOAc/
MeOH (1:10) as eluent to afford a colorless resin (64%
yield). 1H NMR (DMSO-d6) d 1.04–1.06 (m, 4H, –CH2–);
1.22–1.24 (m, 2H, –N–CH2–CH2–);1.44–1.46 (m, 2H,
(AA0BB0, J=8.6 Hz, 4H, phenyl-H);7.32 (dd, 4J=2.1
3
5
Hz, J=0.5 Hz, 1H, indole-H7);7.54 (dd, 3J=8.6 Hz,
5J=0.5 Hz, 1H, indole-H4);MS (EI, 70 eV) m/z 593.3
(MH+);HRMS calcd for C 30H45N2O6S2 (MH+)
593.27186, found 593.27105.
indole-N–CH2–CH2–);1.57–1.69 (m, 4H, –N(CH
CH2–)2);2.16 (s, 3H, –CH 3);2.21–2.24 (m, 2H, –N–
–
2
3-Methyl-1-[10-(pentylsulfonyl)decyl]-6-sulfamoyloxy-2-
(4-sulfamoyloxyphenyl)indole (14b). Colorless resin
CH2–);2.31–2.33 (m, 4H, –N(CH –)2);4.07 (t, 3J=7.0
2
4
Hz, 2H, indole-N–CH2–);7.09 (dd, 3J=8.8 Hz, J=2.3
1
3
5
(62% yield). H NMR (CDCl3) d 0.91 (t, J=7.0 Hz,
Hz, 1H, indole-H6);7.49 (dd, 4J=2.3 Hz, J=0.5 Hz,
1H, indole-H4);7.41, 7.45 (AA BB0, 3J=8.8 Hz, 4H,
0
3H, CH3–CH2–);1.03–1.88 (m, 22H, –CH –);2.18 (s,
3H, –CH3);2.95–3.01 (m, 4H, SO 2(CH2–)2);3.94 (t,
2
phenyl-H);7.52 (dd, 3J=8.8 Hz, 5J=0.5 Hz, 1H,
indole-H7);MS (FAB) m/z 551.3 (MH+);HRMS calcd
for C25H35N4O6S2 (MH+) 551.19975, found 551.20073.
3J=7.3 Hz, 2H, N–CH2–);5.28 (s, 2H, indole-
OSO2NH2);5.59 (s, 2H, phenyl-OSO NH2);7.09 (dd,
2
3J=8.6 Hz, 4J=2.0 Hz, 1H, indole-H5);7.32 (dd,
4J=2.0 Hz, 5J=0.5 Hz, 1H, indole-H7);7.34, 7.42
Methyl-1-(6-pyrrolidinohexyl)-6-sulfamoyloxy-2-(4-sulfa-
moyloxyphenyl)indole (12b). Prepared from 11b by the
method described for 12a. Colorless resin (56% yield).
1H NMR (DMSO-d6) d 1.05–1.06 (m, 4H, –CH2–);
1.23–1.49 (m, 2H, –N–CH2–CH2–);1.50–1.63 (m, 2H,
(AA0BB0, J=8.8 Hz, 4H, phenyl-H);7.53 (dd, 3J=8.6
3
5
Hz, J=0.5 Hz, 1H, indole-H4).
3-Methyl-1-[10-(pentylsulfonyl)decyl]-2-(4-sulfamoyloxy-
1
phenyl)indole (13c). Yellow resin (77% yield). H NMR
3
indole-N–CH2–CH2–);1.76–1.82 (m, 4H, –N(CH
–
2
(CDCl3) d 0.93 (t, J=7.0 Hz, 3H, CH3–CH2–);1.06–
1.47 (m, 16H, –CH2–);1.54–1.91 (m, 6H, N–CH 2–CH2–,
CH2–)2);2.18 (s, 3H, –CH 3);2.13–2.22 (m, 2H, –N–
CH2–);2.32–2.41 (m, 4H, –N(CH –)2);4.04 (t, 3J=6.9
2
4
SO2(CH2–CH2–)2);2.24 (s, 3H, –CH 3);2.96–3.02 (m,
3
Hz, 2H, indole-N–CH2–);7.01 (dd, 3J=8.5 Hz, J=1.9
5
4H, SO2(CH2–)2), 4.01 (t, J=7.4 Hz, 2H, N–CH2–);
5.48 (s, 2H, –SO2NH2);7.11–7.61 (m, 8H, indole-H,
phenyl-H).
Hz, 1H, indole-H5);7.38 (dd, 4J=1.9 Hz, J=0.5 Hz,
1H, indole-H7);7.42, 7.48 (AA BB0, 3J=8.9 Hz, 4H,
0
phenyl-H);7.56 (dd, 3J=8.5 Hz, 5J=0.5 Hz, 1H,
indole-H4). Anal. (C25H34N4O6S2): C, 51.17;H, 6.52;N,
9.54. Found: C, 51.28;H, 6.02;N, 9.00.
5-Cyano-3-methyl-1-[10-(pentylsulfonyl)decyl]-2-(4-sulfa-
moyloxyphenyl)indole (13f). Yellow resin (67% yield).
3
1H NMR (DMSO-d6) d 0.87 (t, J=7.0 Hz, 3H, CH3–
Materials and reagents for bioassays
CH2–);1.04–1.44 (m, 18H, –CH –);1.54–1.72 (m, 4H,
2
SO2(CH2–CH2–)2);2.20 (s, 3H, –CH 3);2.99–3.06 (m,
4H, SO2(CH2–)2);4.13 (t, 3J=7.0 Hz, 2H, N–CH2–);
7.46, 7.56 (AA0BB0, 3J=8.7 Hz, 4H, phenyl-H);7.53
[3H]17b-Estradiol, [3H]estrone sulfate (ammonium salt),
and [14C]estrone were purchased from New England
Nuclear (Dreieich, Germany);all other biochemicals
were obtained from Sigma (Munich, Germany). ICI
182, 780 was generously provided by Dr. M.R. Schnei-
der, Berlin (Germany). Hormone-sensitive human
MCF-7 breast cancer cells and hormone-independent
human MDA-MB 231 breast cancer cells were obtained
from the American Type Culture Collection (ATCC).
MCF-7/2a cells with the reporter construct integrated in
the genome had been cloned in authors’ laboratory.27
3
4
(dd, J=8.6 Hz, J=1.5 Hz, 1H, indole-H6);7.71 (dd,
3J=8.6 Hz, 5J=0.5 Hz, 1H, indole-H7);8.12 (dd,
5
4J=1.5 Hz, J=0.5 Hz, 1H, indole-H4);8.16 (s, br, 2H,
–SO2NH2).
5-Methoxycarbonyl-3-methyl-1-[10-(pentylsulfonyl)decyl]-
2-(4-sulfamoyloxyphenyl)indole (13g). Colorless resin
(64% yield). 1H NMR (DMSO-d6) d 0.87 (t, 3J=7.0 Hz,
3H, CH3–CH2–), 1.05–1.46 (m, 16H, –CH2–);1.56–1.72
(m, 6H, N–CH2–CH2–, SO2(CH2–CH2–)2);2.22 (s, 3H,
–CH3), 2.99–3.06 (m, 4H, SO2(CH2–)2);3.87 (s, 3H,
–OCH3);4.11 (t, 3J=7.0 Hz, 2H, N–CH2–);7.45, 7.55
Estrogen receptor binding assay
For the determination of relative binding affinities
(RBA), the previously described procedure was applied
with modifications.21 The 500 mL-incubation mixture
comprised 5 nM [3H]17b-estradiol [added in 100 mL
Tris–buffer (0.01 M, pH 7.5), supplemented with EDTA
(0.01 M) and NaN3 (0.003 M)], 10ꢀ9–10ꢀ5 M competing
ligand (in 100 mL buffer), 100 mL of calf uterine cytosol,
and buffer. The mixture was incubated for 18 h at 4 ꢁC,
after which 0.5 mL of dextran-coated charcoal (DCC)
slurry (0.8% charcoal Norit A and 0.008% dextran in
buffer) was added to the tubes, and the contents were
mixed. The tubes were incubated for 90 min at 4 ꢁC and
then centrifuged at 700g for 10 min to pellet the charcoal.
An aliquot (100 mL) of the supernatant was removed and
radioactivity was determined by liquid scintillation
spectrometry after addition of 3 mL of Quickszint 212
(Zinsser). Nonspecific binding was calculated using 5
(AA0BB0, J=8.7 Hz, 4H, phenyl-H);7.59 (dd, 3J=8.7
3
5
Hz, J=0.5 Hz, 1H, indole-H7);7.81 (dd, 3J=8.7 Hz,
4J=1.7 Hz, 1H, indole-H6);8.15 (s, 2H, –SO 2NH2);
4
5
8.23 (dd, J=1.7 Hz, J=0.5 Hz, 1H, indole-H4).
3-Methyl-1-(6-pyrrolidinohexyl)-5-sulfamoyloxy-2-(4-sul-
famoyloxyphenyl)indole (12a). Under N2 a solution of
11a (196 mg, 0.5 mmol) in dry DMF (5 mL) was added
slowly at 0 ꢁC to a stirred suspension of NaH (50 mg, 2
mmol) in dry DMF (5 mL). After stirring for 1.5 h,
sulfamoyl chloride (575 mg, 5 mmol) was added in por-
tions at 0 ꢁC. After removal of the cooling bath, the
mixture was stirred overnight and then poured into a
sat. NaHCO3 solution (40 mL). The aqueous mixture
was extracted twice with EtOAc. The combined organic
layers were washed with water and dried (MgSO4).

3952
T. Golob et al. / Bioorg. Med. Chem. 10 (2002) 3941–3953
mM 17b-estradiol as competing ligand. Radioactivity
was plotted as a function of the log concentration of
competing ligand in the assay. RBA was calculated as
the ratio of the molar concentrations of estradiol and
test compound required to decrease the amount of
bound radioactivity by 50%, multiplied by 100.
tetraacetate, 10% glycerol, and 1% Triton X-100 was
added to each well. After 20 min at room temperature
cells were collected with a rubber policeman, cleared by
centrifugation and stored at ꢀ20 ꢁC.
Luciferase activity was assayed using the Promega kit
according to manufactor’s protocol. The luminescence
of 30 mL-samples and Promega assay solution (100 mL)
was measured in a luminometer Lumat LB 9501 (Berthold,
Wildbad, Germany). Luminescence (in relative light
units, RLU) was integrated over 10 s. The background
was approximately 250 RLU/10s. With samples from
Photinus pyralis (Boehringer, Mannheim, Germany) a
linear correlation between luminescence (RLU/10s) and
the amount of luciferase was established in the range
from 0.0001 pg to 30 pg enzyme with 3000 RLU referring
to 10 fg luciferase. Measurements were corrected for the
protein content of the samples quantified according to
Bradford29 using bovine serum albumin as standard.
Unless stated otherwise, average values and the devia-
tions of three independent measurements are shown.
IC50 values were calculated from the dose-response
curves. Estrone (E1;10 nM) and 17 b-estradiol (E2;10
nM) were used as reference estrogens, estrone sulfate
(100 nM) for estimation of combined sulfatase inhibiting
and antiestrogenic effects.
Steroid sulfatase assay28
MCF-7 human breast cancer cells were grown in 75 mL-
flasks in MEM Eagles’s medium supplemented with
NaHCO3 (2.2 g/L), sodium pyruvate (110 mg/L), gen-
tamycin (50 mg/L), and 10% FCS at 37 ꢁC in a humi-
dified atmosphere containing 5% CO2. Shortly before
confluence they were harvested by addition of trypsin/
EDTA solution and suspended in 11 mL of fresh medium.
One mL-aliquots of the cell suspension were transfered
to 25 mL-flasks followed by the addition of 9 mL of
medium containing 10% FCS. After 3–4 days of incu-
bation the cells have reached 80% confluence and can
be used for this assay. After removal of the medium,
cells were washed with PBS (2.5 mL) followed by the
addition of medium without FCS (2.5 mL), 20 mL of a
0.25 mM [3H] estrone sulfate solution (520, 000 dpm),
leading to a final concentration of 2 nM, and 25 mL of
inhibitor dissolved in DMF. After an incubation period
of 20 h at standard conditions cells were cooled to 4 ꢁC
for 15 min before 1 mL of the supernatant was trans-
ferred into a test tube. After the addition of [14C]
estrone (7500 dpm) the mixture was vortexed with toluene
(5 mL). For an efficient separation of the two phases,
the mixture was centrifuged at 1000g for 10 min. Two
mL of the toluene layer and 3 mL of scintillation liquid
were used for the separate determination of both nuclei.
Values for maximum conversion to [3H] estrone (controls)
were obtained with 25 mL DMF without inhibitor.
Background radioactivity was determined in the
absence of cells.
Acknowledgements
The authors wish to thank the Deutsche Forschungs-
gemeinschaft for financial support.
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