Synthesis and structure–activity relationships of 2- and/or 4-halogenated 13β- and 13α-estrone derivatives as enzyme inhibitors of estrogen biosynthesis

Article abstract of DOI:10.1080/14756366.2018.1490731

Ring A halogenated 13α-, 13β-, and 17-deoxy-13α-estrone derivatives were synthesised with N-halosuccinimides as electrophile triggers. Substitutions occurred at positions C-2 and/or C-4. The potential inhibitory action of the halogenated estrones on human aromatase, steroid sulfatase, or 17β-hydroxysteroid dehydrogenase 1 activity was investigated via in vitro radiosubstrate incubation. Potent submicromolar or low micromolar inhibitors were identified with occasional dual or multiple inhibitory properties. Valuable structure–activity relationships were established from the comparison of the inhibitory data obtained. Kinetic experiments performed with selected compounds revealed competitive reversible inhibition mechanisms against 17β-hydroxysteroid dehydrogenase 1 and competitive irreversible manner in the inhibition of the steroid sulfatase enzyme.

Full text of DOI:10.1080/14756366.2018.1490731

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Synthesis and structure–activity relationships of
2- and/or 4-halogenated 13β- and 13α-estrone
derivatives as enzyme inhibitors of estrogen
biosynthesis
Ildikó Bacsa, Bianka Edina Herman, Rebeka Jójárt, Kevin Stefán Herman,
János Wölfling, Gyula Schneider, Mónika Varga, Csaba Tömböly, Tea Lanišnik
Rižner, Mihály Szécsi & Erzsébet Mernyák
To cite this article: Ildikó Bacsa, Bianka Edina Herman, Rebeka Jójárt, Kevin Stefán Herman,
János Wölfling, Gyula Schneider, Mónika Varga, Csaba Tömböly, Tea Lanišnik Rižner,
Mihály Szécsi & Erzsébet Mernyák (2018) Synthesis and structure–activity relationships of
2- and/or 4-halogenated 13β- and 13α-estrone derivatives as enzyme inhibitors of estrogen
biosynthesis, Journal of Enzyme Inhibition and Medicinal Chemistry, 33:1, 1271-1282, DOI:
10.1080/14756366.2018.1490731
To link to this article: https://doi.org/10.1080/14756366.2018.1490731
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2018, VOL. 33, NO. 1, 1271–1282
https://doi.org/10.1080/14756366.2018.1490731
RESEARCH PAPER
Synthesis and structure–activity relationships of 2- and/or 4-halogenated 13b- and
13a-estrone derivatives as enzyme inhibitors of estrogen biosynthesis
a
b
a
a
a
a
ꢀ
ꢀ
ꢀ
ꢀ ꢀ
ꢁ
ꢀ
ꢀ
€
Ildiko Bacsa , Bianka Edina Herman , Rebeka Jojart , Kevin Stefan Herman , Janos Wolfling , Gyula Schneider ,
c
d
e
b
a
ꢀ
€
€
ꢁ
ꢀ
ꢀ
ꢀ
ꢀ
Monika Varga , Csaba Tomboly , Tea Lanisnik Rizner , Mihaly Szecsi and Erzsebet Mernyak
b
aDepartment of Organic Chemistry, University of Szeged, Szeged, Hungary; 1st Department of Medicine, University of Szeged, Szeged,
c
d
Hungary; Department of Microbiology, University of Szeged, University of Szeged, Szeged, Hungary; Laboratory of Chemical Biology, Institute
of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, HungaryeInstitute of Biochemistry, Faculty of
Medicine, University of Ljubljana, Ljubljana, Slovenia
ABSTRACT
ARTICLE HISTORY
Received 22 March 2018
Revised 5 June 2018
Accepted 15 June 2018
Ring A halogenated 13a-, 13b-, and 17-deoxy-13a-estrone derivatives were synthesised with N-halosuccini-
mides as electrophile triggers. Substitutions occurred at positions C-2 and/or C-4. The potential inhibitory
action of the halogenated estrones on human aromatase, steroid sulfatase, or 17b-hydroxysteroid
dehydrogenase 1 activity was investigated via in vitro radiosubstrate incubation. Potent submicromolar or
low micromolar inhibitors were identified with occasional dual or multiple inhibitory properties. Valuable
structure–activity relationships were established from the comparison of the inhibitory data obtained.
Kinetic experiments performed with selected compounds revealed competitive reversible inhibition mech-
anisms against 17b-hydroxysteroid dehydrogenase 1 and competitive irreversible manner in the inhibition
of the steroid sulfatase enzyme.
KEYWORDS
Estrone; halogenations;
aromatase; STS; 17b-HSD1
Introduction
the sulfate ester by steroid sulfatase (STS). The next, hormone-acti-
vating process is the formation of active hormone E2 from E1,
which is mainly catalyzed by 17b-hydroxysteroid dehydrogenase
type 1 (17b-HSD1). Activities of STS and 17b-HSD1 are higher in
breast cancer tissue compared to other tissues, that is this may be
Estrogens play an important role in cell proliferation and their
overproduction stimulates the growth of hormone-sensitive cells,
leading to hormone-dependent diseases, such as breast and endo-
metrial cancer1. Inhibition of enzymes involved in the final steps
of estrogen biosynthesis is a powerful route to prevent the prolif-
erative action of estrogens. Cytochrome P450 aromatase is respon-
sible for the conversion of nonaromatic androgens 1 and 2 to
estrone (E1, 4, Scheme 1) or 17b-estradiol (E2, 5), respectively.
Estrogens are originated not only from nonaromatic steroids, but
also from estrone-3-sulfate (E1S) 3, which exists as a large circula-
tory reservoir. E1S is transported into cells by organic anion trans-
porters (OATPs) and several other members of the SoLute Carrier
the main route of local estrogen production in the tumors3,4
.
Inhibition of the above-mentioned enzymes may be achieved
by inhibitors designed on the estrane core. A-ring halogenation of
E1 results in 2- and 4-regioisomeric (6, 7) and/or 2,4-bis-substi-
tuted compounds (8, Figure 1)5–7. The substitution pattern of the
aromatic ring and the nature of the introduced halogen greatly
influence the inhibitory properties of estrone derivatives halogen-
ated at the A-ring. Compounds obtained by introduction of the
(SLC) protein family2. After entering the cells, E1 is released from same substituent to different positions of the aromatic ring may
ꢀ
ꢀ
ꢀ
1st Department of Medicine, University of Szeged, Koranyi fasor 8–10, H-6720, Szeged, Hungary;
bobe@chem.u-szeged.hu Department of Organic Chemistry, University of Szeged, Dom ter 8, H-6720, Szeged, Hungary
CONTACT Mihaly Szecsi
szecsi.mihaly@med.u-szeged.hu
ꢀ
ꢀ
ꢀ
ꢀ
Erzsebet Mernyak
Supplemental data for this article can be accessed here.
These authors contributed equally to this work.
ß 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
ꢀ
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

1272
I. BACSA ET AL.
O
OH
H
H
H
H
H
H
O
O
2
1
Androst-4-ene-3,17-dione
Aromatase
Testosterone
Aromatase
O
OH
O
H
H
H
17β-HSD1
STS
O
HO
O
H
H
H
H
H
H
S
HO
HO
O
5
3
4
17β-Estradiol
Estrone-3-sulfate
Estrone
Scheme 1. The role of aromatase, STS and 17b-HSD1 enzymes in the estrogen biosynthesis.
Figure 1. Ring A halogenated derivatives 6–8 of estrone.
possess high binding affinities to different enzymes. 2-Bromo- (6b) negligible estrogenic potential8,10,11. Certain other chemical modi-
or 2-chloroestrone (6c) are potent aromatase inhibitors with low fications of the estrane skeleton, such as the inversion of the con-
micromolar IC50 values5. Their 4-substituted counterparts (7b,c) figuration at C-13 or the opening of ring D, may result in the
display only moderate aromatase inhibitory potential. Moller et al. complete loss of hormonal activity12–15. We have promising pre-
€
described that 2-haloestrones (6b,c) as 17b-HSD1 inhibitors can liminary results concerning the design, synthesis and biochemical
suppress the E1–E2 conversion with IC50 values in the submicro- evaluation of 17b-HSD1 inhibitors based on hormonally inactive
molar range6. 4-Halogenated counterparts (7) have not been 13a-estrane core16. 13a-Estrone (9, Scheme 2) itself was proved to
tested against 17b-HSD1 by this research group. However, the be a potent inhibitor with an IC50 comparable to that of the
4-haloestrones (7b,d) are known to be efficient STS inhibitors7. natural substrate E1. Additionally, the previously synthesised
Thus, introduction of Br or F onto C-4 of E1 led to a significant 13a-estrone derivatives (10a,b–12a,b) brominated or iodinated in
increase in STS inhibitory potential. None of the mentioned refer- the A-ring exerted low micromolar or submicromolar inhibitory
ences discusses the affinity of 2-iodo- and 4-iodoestrones (6a, 7a) potential17. Chlorinations in the 13a-estrone series leading to
10c–12c have not been performed up to now. Concerning recent
promising results, that 13a-estrone derivatives might possess valu-
able 17b-HSD1 inhibitory potential, it seemed rational to evaluate
these compounds as potential aromatase or STS inhibitors, too.
Based on the above-mentioned literature results here we
aimed to perform aromatic halogenations on 13a-estrone (9) and
its 17-deoxy counterpart (13, Scheme 2, 3). Chlorination of
13a-estrone (9) and introduction of chlorine, bromine or iodine
or 2,4-bis-compounds (8) for these three enzymes.
Concerning the results obtained so far for certain A-ring halo-
genated estrones it seems that substitution of E1 at C-2 may
enhance aromatase and 17b-HSD1 inhibitory potential, but 4-halo-
genation may lead to efficient STS inhibitors.
The use of the estrone-based inhibitors of the mentioned ster-
oidogenic enzymes in the therapy is limited because of their
retained estrogenic activity. The availability of inhibitors acting
selectively without hormonal behavior would be of particular onto positions 2 and/or 4 of 17-deoxy-13a-estrone (13) were
interest. Literature data reveal that estrogenic effect of estrone is planned. Synthesis of the halogenated 13b-estrone derivatives
a 5–35% extent of that of 17b-estradiol8–10. Chloro substitution at (6a,b,c–8a,b,c) was also designed with the aim of preparing the
position C-4 of estrone or 17b-estradiol retains the estrogenicity, 13b-counterparts for comparative investigations (Figure 1). We
however, 4-bromo and 4-iodo, as well as 2-chloro, -bromo, and additionally intended to investigate the potential in vitro inhibitory
-iodo compounds exert suppressed effect compared to that of the effects of the basic and the halogenated compounds
parent compounds. Data vary according to the methods applied, (6a,b,c–8a,b,c; 9–16) towards aromatase, STS and 17b-HSD1
but it can be stated that estrogenic effect decreases with the enzymes. Comparison of the inhibitory results and evaluation of
increasing size of the introduced halogen. C-2 substituted estrone structure–activity relationships were also planned. Representatives
or 17b-estradiol compounds are usually less estrogenic than their from the most effective test compounds were selected for mech-
C-4 counterparts. The 2,4-disubstituted analogs, nevertheless, exert anistic and kinetic investigations.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1273
Scheme 2. Ring A substitutions in the 13a-estrone series.
Scheme 3. Introduction of different halogens onto the aromatic ring of 17-deoxy-13a-estrone
Table 1. Synthesis of halogenated compounds (6–8, 10c–12c, 14–16)
Materials and methods
Starting
Entry compound
Electrophilic reagent
(solvent)
Chemical syntheses and characterisation data of the reported
compounds, as well as experimental conditions of enzymatic
assays performed are described in the Supporting Information.
Product
10c þ 11c
Yield (%)
16 þ 63
1
2
3
4
5
6
7
8
9
9
13
13
13
13
4
4
4
4
4
NCS (1.0 equiv., TFA)
NCS (2.0 equiv., TFA)
NIS (1.5 equiv., TFA)
NBS (1.1 equiv., CH2Cl2)
NCS (1.5 equiv., CH3CN)
NCS (3.0 equiv., CH3CN)
NIS (1.0 equiv., TFA)
NIS (2.0 equiv., TFA)
NBS (1.0 equiv., CH2Cl2)
NBS (2.0 equiv., CH2Cl2)
11c þ 12c
57 þ 19
14a þ 15a þ 16a 48 þ 24 þ 24
14b þ 15b þ 16b 44 þ 33 þ 11
14c þ 15c
15c þ 16c
6a þ 7a
48 þ 24
26 þ 53
58 þ 30
59 þ 30
40 þ 41
12 þ 72
30 þ 45
41 þ 40
Results and discussion
Chemistry
7a þ 8a
We recently described the halogenations of the aromatic ring of 9
with different groups at position 3 (OH, OMe, OBn)17. Bromination
or iodination was carried out with N-halosuccinimides as electro-
phile triggers in different solvents. In the 3-OH series both 2- and
4-substituted regioisomers were formed and bis-substitutions also
occurred. The mixtures of the desired products (10a,b–12a,b)
could efficiently be separated by flash chromatography.
9
6b þ 7b
7b þ 8b
10
11
12
NCS (1.1 equiv., MeCN/TFA) 6c þ 7c
4
NCS (2.2 equiv., CH2Cl2)
7c þ 8c
halosuccinimides in different solvents (Scheme 3, Table 1).
Iodination of 13 with 1.5 equivalent of NIS in trifluoroacetic acid
yielded the mixture of the two regioisomers (14a and 15a) and
the bis-compound (16a) in a ratio of 2:1:1 (Table 1, Entry 3).
Bromination of 13 with 1.1 equivalent of reagent in dichlorome-
thane led to a 4:3:1 mixture of 14b, 15b and 16b (Table 1, Entry
4). Chlorination of 13 with 1.5 equivalent of NCS yielded the 2:1
mixture of the two regioisomers 14c and 15c (Table 1, Entry 5).
The bis-chloro compound (16c) could be synthesised by using 3.0
As a completion of our previous results, here we performed
the chlorination of the aromatic ring of 9 in order to get the
appropriate 2-, 4-, or 2,4-bis-chloro derivatives (10c–12c) for fur-
ther structure–activity examinations. Reactions with N-chlorosucci-
nimide led to a mixture of products (10c–12c, Scheme 2, Table 1,
Entries 1, 2), which were separated by flash chromatography.
Expecting to acquire valuable structure–activity relationship
data, it seemed reasonable to synthesise the appropriate halogen-
ated 13a compounds in the 17-deoxy series, too. The halogena-
tions of 17-deoxy-13a-estrone 13 were carried out using N- equivalent of NCS (Table 1, Entry 6).

1274
I. BACSA ET AL.
In order to have the appropriate 13b-estrone derivatives inhibitor binding4. Aside from the differences in the active sites
and catalytic mechanisms, these three enzymes may be inhibited
by similar, estrone-based inhibitors. Slight differences in the struc-
tures of the potential inhibitors, involving regioisomerism, may
influence not only the extent but also the nature of inhibition.
This seems to be true for the ring A halogenated derivatives in
the 13b-estrone series5–7. Literature data suggest that substitu-
tions at C-2 are advantageous concerning 17b-HSD1 and aroma-
tase, but 4-regioisomers are better STS inhibitors. Nevertheless,
there is no thorough comparative investigation in the literature
concerning the aromatase, STS and 17b-HSD1 inhibitory activities
of 2-, 4-, and 2,4-bis-chloro, -bromo, and -iodo estrones. The litera-
ture data are incomplete in this sense. The involvement of
17-keto- and 17-deoxy-13a-estrone compounds in the structur-
e–activity determinations seems also reasonable. To the best of
our knowledge, there has not been found any exact correlation
between the structural characteristics of the investigated estrone
derivative (regarding the conformation and the presence of the
17-keto function) and good aromatase, STS, or 17b-HSD1 inhibi-
tory potential.
As a part of our efforts to get valuable pieces of information,
which could help the development of more potent single or
multiple inhibitors of estrogen biosynthesis, we describe here
the evaluation of halogenated 17-keto-13b-, 17-keto-13a-, and
17-deoxy-13a-estrone derivatives (6a,b,c–8a,b,c, 9–16) as 17b-
HSD1, aromatase, and/or STS inhibitors.
Enzyme inhibition studies were performed by in vitro radiosub-
strate incubations using human term placenta cytosol and micro-
somas as enzyme sources. Aromatase inhibition was measured on
testosterone (2) to E2 (5) conversion, STS inhibition was investi-
gated via hydrolytic release of E1 (4) from E1S (3), whereas the
influence on 17b-HSD1 was tested by the transformation of (4) to
E2 (5). Relative conversions compared to non-inhibited controls
(100%) were measured in the presence of 10 mM concentration of
the test compound. For more efficient compounds, IC50 values
were determined and inhibitory potentials were assessed also in
comparison to IC50 data of the corresponding substrate. Reference
IC50 parameters measured for the substrates and the basic com-
(6a,b,c–8a,b,c) for the comparative enzyme inhibition studies, hal-
ogenations were carried out in this series, too. Our primary goal
was to get the two regioisomers and the bis-compounds for
the in vitro tests; therefore the regioselectivity was inessential.
Different conditions were needed for the convenient synthesis of
mono- and disubstituted 13b compounds. Monosubstitutions
occurred using nearly 1 equiv. of N-halosuccinimide (Table 1,
Entries 7, 9, 11), but the excess (nearly 2 equiv.) of the halogenat-
ing agent led to disubstitution (Table 1, Entries 8, 10, 12), too. The
aimed halogenations at C-2 and/or C-4 were achieved and the
flash chromatographic separations of the reaction mixtures fur-
nished the desired compounds (6–8).
Enzyme inhibition studies
Aromatase, STS and 17b-HSD1 belong to different enzyme families
with distinct catalytic mechanisms18–20. Specific inhibitors of these
enzymes may be developed because of the differences in their
active sites4. Dual or multiple inhibition might also be of value
since inhibition of only one of these three enzymes is not
adequate in treatment of hormone-dependent breast cancer.
Since aromatase is needed for the synthesis of E1, hormone-
dependent breast cancer may be more effectively treated by dual
inhibition of aromatase and STS. Among the inhibitors of the
mentioned three enzymes, aromatase inhibitors are clinically the
most effective for hormone-dependent breast cancer.
Beside their efficiency, the estrogen deprivation is accompa-
nied with resistance and side effects. It would be crucial to design
new drug candidates without such side effects. Innovative treat-
ment strategies combining inhibitors of STS or 17b-HSD1 with aro-
matase inhibitor could lower the dose of the latter and extend
the disease progression. The tumor-selective lowering of E2 levels
could be achieved by the use of inhibitors of these two enzymes
together with the aromatase inhibitor.
Literature data reveal that three-dimensional structures of
these enzymes have not met expectations in drug design, but
they are useful in understanding the catalytic mechanisms and pound E1 (4) are listed in Table 2. Mechanistic and kinetic
Table 2. Reference IC50 parameters of the substrate compounds. Relative conversions (Rel. conv., control incu-
bation with no inhibition is 100%) measured in the presence of 10 lM concentration of the compound tested.
Mean SD, n ¼ 3.
Comp.
Structure
Arom
STS
17b-HSD1
IC50 SD (mM)
IC50 SD (mM)
IC50 SD (mM)
OH
H
H
Testosterone (2)
0.52 0.14
H
O
E1S (3)
E1 (4)
5.2 1.2
>10 Rel. conv. 78 7%
24 10
0.63 0.11
IC50: the inhibitor concentration decreasing the enzyme activity to 50%. Ki: inhibitor constant determined from
Dixon plot; SD: standard deviation.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1275
investigations were performed and inhibitory constants (Ki) were STS
determined for selected compounds.
Numerous STS inhibitors have already been described in the litera-
ture7. Estrone aryl sulfamates are known as irreversible, suicide
inhibitors. EMATE is a highly potent STS inhibitor, but because of
its estrogenic activity it is not an adequate antitumor drug candi-
date. As literature data show the 17-deoxy analog of EMATE
(NOMATE) displays similar STS inhibitory potential as its 17-keto
counterpart26,27. This suggests that the presence of the 17-keto
function is not essential for the effective inhibition of 3-sulfa-
Aromatase
According to literature information, introduction of F, Cl, Br, Me,
and formyl groups to C-2 of E1 affords compounds with high
binding affinity to aromatase enzyme5. 2-Bromoestrone (6b)
proved to be an efficient inhibitor with an IC50 value of 2.4 mM,
whereas 2-chloroestrone (6c, Ki ¼ 0.13 mM) seemed to be more mates. E1 displays weak binding to STS, but its certain counter-
parts substituted in ring A exert substantial inhibition. This proves
that appropriately substituted 3-OH E1 derivatives may also be
good inhibitor candidates. It was established that substitution at
C-4 of E1 with relatively small electron withdrawing-groups, such
as F, Br, CN, formyl, or NO2, lead to improvement in inhibitory
potency, which may be attributed to H-bonding and/or steric or
other interactions. It is known that 4-formylestrone is a time- and
concentration-dependent irreversible inhibitor of STS7, and it inac-
tivates the enzyme by reacting with active site residues. Phan
et al. proposed that the 4-formyl function is involved in Schiff
base formation with amino groups in appropriate side-chains
including Lys-368, Lys-134, and Arg-797. As reported in the litera-
ture, the 3-OH function of the inhibitor may be involved in H-
bonding with certain amino acid residues, with His346 and/or
formylGly75 hydrate among others. Concerning hydrogen-bonding
abilities, these side-chains are bifunctional. His346 may accept
proton through its p-cloud and nitrogen, but may donate its NH
proton. FormylGly75 hydrate may establish hydrogen bonds with
its carbonyl group as a proton acceptor, whereas its OH group
may behave as a proton donor. On the part of the steroid, both
the OH function and its phenolate may form H-bonds or specific
interactions. The electron-withdrawing properties of the intro-
duced ring A substituents may greatly influence the polarisation
and the acidity of the 3-OH group. This substituent effect depends
on the position, number and nature of the introduced groups.
Certain substituents at the ortho positions may additionally be
involved in intramolecular H-bonding with the 3-OH group, which
may reduce the affinity of the inhibitor to the enzyme. Phan et al.
have not found direct correspondence between the estimated pKa
values (taken from the corresponding o-substituted phenols) and
the inhibitor potentials of their examined compounds7.
potent displaying about a 20-fold enhancement in its affinity com-
pared to E1 (Ki ¼ 2.50 mM). Concerning 6c as a powerful competi-
tive inhibitor, no evidence of enzymatic generation of a reactive
substance was observed. Exact correlations between inhibitory
activity and size and/or electronegativity of substituents at C-2
could not be established. Inhibitory activities of estrone analogs
were found to be higher than those of the corresponding estra-
diol derivatives. Consequently, a 17-carbonyl function plays a cru-
cial role in the binding of estrogens to the active site of
aromatase enzyme, as observed in the cases of the androgen
derivatives21–24. Halogenation at the C-4 position, except for fluor-
ination, markedly decreased affinities. Osawa et al. suggested that
substrates bind to the active site of aromatase through two con-
formations [b-side up (normal) and a-side up (upside down)], or
have the opportunity and space to rotate around the binding
site25. In the b-side up binding mode, C-2 is located close to the
heme. This binding allows the catalysis of 2-hydroxylation. The
estradiol molecule may rotate by 180ꢁ through the O(3)–O(17)
axis, resulting in the a-side up binding mode, which allows
4-hydroxylation but to a lesser extent. The higher inhibitory
potential of C-2-substituted E1 analogs compared with those of
their C-4 substituted counterparts may be related to the high
aromatase C-2 hydroxylation activities. These literature results
indicate that concerning estrone-based aromatase inhibitors, the
nature of the C-17 substituent, the substitution pattern of the
aromatic ring, and the conformation of the compound greatly
influence their inhibitory behavior.
In this contribution we also report in vitro aromatase inhibition
tests of the synthesised 13b- and 13a-estrone derivatives. Certain
2-halogenated 13b-estrone derivatives (6b and 6c) displayed low
micromolar inhibition (Table 3). 2-Chloroestrone (6c) was found
to be the most effective with its IC50 value of 6.0 mM.
2-Bromoestrone (6b) was slightly less potent (IC50 ¼ 8.7 mM). These
results are in a good agreement with those of Numazawa et al.5.
2-Iodoestrone (6a) displayed weaker inhibition with a relative con-
version of 66% at a 10 mM test concentration (IC50 > 10 mM).
Nevertheless, both derivatives have enhanced efficiency compared
to their parent compound E1. The results obtained for the 2-halo-
genated 13b-estrone derivatives reveal that the inhibitory poten-
tial decreases with the increasing size of the halogen substituent.
Other test compounds including 13a-estrone (9), its 17-deoxy
counterpart (13), and their halogenated derivatives (10–12,
14–16) exerted very weak inhibitory effect: their relative conver-
sion data are higher than 80% at a 10 mM test concentration. The
empirical rules previously established in the 13b-series have not
been observable in the 13a-estrone series, while the affinity for
aromatase enzyme of the two basic 13a-estrone derivatives (9 and
13) could not be improved by attaching halogens onto ring A.
This might be explained by the lack of ability of 13a-estrones
for binding to the active site, because of their core-modi-
fied structure.
Taking into account the above-mentioned literature results, it
can be stated that not only the presence of a 3-O-sulfamoyl group
but also the introduction of a relatively small electron-withdraw-
ing group to carbon 4 of E1 may be a general possibility to
enhance the potency of estrone-based STS inhibitors.
Here we start with the evaluation of the 2- and/or 4-chlori-
nated, brominated or iodinated 13b-estrone derivatives
(6a,b,c–8a,b,c). The 4-iodo compound (7a) exerted outstanding
submicromolar inhibition (Table 3). Its 0.23 mM IC50 value indicates
a 22-fold higher affinity compared to the E1S substrate and an
affinity increased by 100-fold compared to E1. 6a its 2-sbstituted
counterpart exerted 100-fold weaker inhibition according to its
IC50 value. 4-Bromo derivative 7b displayed submicromolar inhibi-
tory potential with an IC50 value nearly twice as high as that of its
2-counterpart (6b). Phan et al. recently reported that the inhib-
ition potential of 6c is modest, whereas its 4-counterpart 7b dis-
plays considerable inhibition7. Even so we found that both
isomers are potent inhibitors. This difference may be ascribed to
different substrates used in the two methods. Phan et al. used an
artificial substrate (4-methylumbelliferyl sulfate), whereas we
applied the natural substrate estrone-3-sulfate (3). The different
binding specificity of these substrates may result in different

1276
I. BACSA ET AL.
Table 3. In vitro inhibition of enzyme activities by the test compounds.
Arom
STS
17b-HSD1
Rel.conv. SD (%) IC50 SD (mM)
Comp.
Structure
Rel.conv. SD (%)
IC50 SD (mM)
Rel.conv. SD (%)
IC50 SD (mM)
62
88
1
2
64
3
0.064 0.034
0.36 0.25
6a
7a
0.23 0.09
Ki ¼ 0.36 0.05 lM
8a
86
6
80 13
55 7
6b
7b
8.7 2.8
2.0 0.4
0.095 0.031
0.30 0.20
91
81
6
5
0.89 0.3
8b
2.1 0.6
0.96 0.45
6c
7c
6.0 1.2
2.4 0.4
1.6 0.3
0.18 0.02
0.60 0.16
92
82
3
4
8c
3.0 0.9
0.59 0.16
9
85 13
82 10
96
83
1
3
1.2 0.2 [12]
Ki ¼ 1.9 0.2 lM
0.59 0.23 [13]
10a
11a
90
7
6.0 1.6
1.0 0.3 [13]
Ki ¼ 2.2 0.3 lM
(continued)

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1277
Table 3. Continued.
Arom
Rel.conv. SD (%)
STS
17b-HSD1
Rel.conv. SD (%) IC50 SD (mM)
Comp.
Structure
IC50 SD (mM)
Rel.conv. SD (%)
IC50 SD (mM)
12a
91
1
2.4 0.5
0.38 0.08 [13]
Ki¼ 0.94 0.15 lM
10b
11b
100
78
5
1
81
6
1.2 0.3 [13]
2.1 1.2 [13]
8.5 3.1
12b
108 11
71
4
2.7 0.1 [13]
10c
11c
106
98
6
3
57
80
1
7
0.33 0.10
2.6 1.0
12c
101
4
70
3
2.2 0.6
13
95 12
76
5
1.1 0.33
Ki ¼ 2.0 0.4lM
14a
15a
89
92
9
7
3.9 1.6
2.7 1.3
2.9 1.6
57
61
9
7
16a
94
2
59 13
14b
15b
97
90
5
8
4.1 1.3
3.7 1.2
1.3 0.8
49 12
11 4
16b
14c
82
6
7.5 2.0
7.0 1.9
4.1 2.5
2.6 1.3
88 10
(continued)

1278
I. BACSA ET AL.
Table 3. Continued.
Arom
Rel.conv. SD (%)
STS
17b-HSD1
Rel.conv. SD (%) IC50 SD (mM)
Comp.
Structure
IC50 SD (mM)
Rel.conv. SD (%)
IC50 SD (mM)
15c
16c
89
1
6.3 1.8
4.5 2.0
82 12
1.3 0.4 Ki ¼ 1.9 0.2 lM
53
2
inhibition results. Nevertheless, our results are in good agreement
with those obtained recently by Phan et al. concerning the effect
of the regioisomerism on the inhibitory potential7. The two chlori-
nated regioisomers (6c and 7c) displayed commensurate inhibi-
tory properties in the low micromolar range. Concerning all data
obtained for the monosubstituted compounds (6, 7), it can be
stated that introduction of a large halogen substituent onto C-4 is
particularly advantageous. The 2,4-bis-iodo compound (8a) had no
marked influence on the conversion of E1S–E1. The bis-bromo
compound (8b) displayed low micromolar inhibition, commensur-
able with that of its bis-chloro counterpart (8c).
7a
16c
100
80
60
40
20
With all compounds and their inhibitory data in hand we were
additionally interested in the investigation of the potential correl-
ation between their predicted pKa values and the measured
inhibitory potentials. pKa values were calculated using computer
software available online28. Data are not listed separately.
Predicted pKa values suggest that our monosubstituted derivatives
bear protonated 3-OH function (pKa > 7.6), whereas bis com-
pounds (pKa ¼ 6.8–7.4) are partially or completely deprotonated
under the physiological conditions (pH ¼7.3) applied in our
experiments. The obtained inhibition potentials do not have
apparent direct relationship with the number and electronegativ-
ity of the introduced halogens, neither reflect the predicted pKa
data. Differences observed between inhibitory potentials of the
two regioisomers indicate that electronic properties of the intro-
duced halogens are not the determining factors in binding inter-
actions of the 3-OH group7.
In the 13a-estrone series, the inhibitory potential of the com-
pounds tested greatly depended on both the nature and the pos-
ition of the halogen introduced. Introduction of iodine onto ring
A was advantageous over substitution with smaller halogens (Br
or Cl). 4-Bromo and 4-iodo compounds (11a and 11b) were effect-
ive inhibitors with IC50 values of 8.5 and 6.0 mM. These regioisom-
ers displayed better inhibitory potentials than their two
counterparts (10a and 10b). In the iodinated series, the best
inhibitor was bis-iodo derivative 12a with its low micromolar IC50
value. The chlorinated compounds (10c–12c) displayed weak
inhibition with relative conversions over 50%.
0
I
II
III IV
V
I
II
III IV
V
Figure 2. Investigation of STS inhibition reversibility of selected 13b-estrone com-
pounds 7a, 16c. Inhibitor compounds were preincubated with human placental
microsomes for 10 and 20min. Following a 50-fold dilution step and 20 min sec-
ondary incubation to allow dissociation, the usual enzyme activity measurement
was applied. Mean SD of three separate experiments. Experimental conditions: I
No preincubation, 7a 0.03 lM; 16c 0.2 lM, II No preincubation, 7a 1.5 lM; 16c
10 lM, III Preincubation, 2.5min, 7a 1.5lM; 16c 10 lM, IV Preincubation, 10 min,
7a 1.5lM; 16c 10 lM, V Preincubation, 20 min, 7a 1.5lM; 16c 10 lM.
inhibitory potential greatly depended on the nature of the halo-
gen introduced. Surprisingly, the most potent 16c compound in
the 17-deoxy series, displaying an IC50 value of 1.3 mM, bears the
smallest halogens. Compound 16c exerts inhibition comparable to
that of 4-bromoestrone 7b (IC50 ¼ 1.3 and 0.89 mM, respectively),
but lower to that of 4-iodoestrone 7a (IC50 ¼ 0.23 mM).
The most potent representative test compounds 7a and 16c
were selected for mechanistic and kinetic investigations. In the
reversibility tests, placental microsomas were preincubated with
high concentration of the inhibitor and enzyme activities were
measured after dilution of the samples. Relative conversions in
these preincubated samples (Figure 2, III and V) show suppressed
enzyme activities, similar to those obtained for incubations with
high inhibitor concentrations (Figure 2, II). Results indicate that 7a
and 16c molecules do not dissociate upon dilution and they are
bound to the enzyme in an irreversible manner. The time depend-
ence of the reversibility test reveals that irreversible binding is
nearly completed within a short 2.5 min time for both compounds
(Figure 2, III).
The data obtained for the halogenated 17-deoxy-13a-estrone
derivatives (14–16) reveal that all monohalogenated compounds
(14, 15), independently from the regioisomerism, are potent STS
inhibitors in the low micromolar range. In general, these com-
pounds displayed higher inhibitory potential than those of their
17-keto counterparts (10, 11). We found that the presence of the
17-keto function on the halogenated 13a-estrane core is not
essential for STS inhibitory activity. Additionally, empirical rules
established previously do not predominate in this series.
Furthermore, the IC50 values of the two regioisomers are compar-
able and the nature of introduced halogen is not crucial.
Kinetic analyses of selected compounds 7a and 16c were per-
formed by measurement of enzyme activities using different fixed
substrate concentrations and varied inhibitor concentrations. Lines
of the Dixon’s plots intersect in the second quadrant revealing
competitive inhibition mechanisms (Figure 3, B). To confirm the
competitive nature of the inhibition of 7a, replot of slopes vs.
1/substrate concentration was constructed (inset B/7a in Figure 3).
The resulting straight line passing through the origin supports the
Concerning the bis-substituted compounds (16a–16c), their kinetics obtained from the Dixon’s plot29,30. Phan and coworkers

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1279
7a
16c
(A)
Rel. conv. %
Rel. conv. %
100
100
IC50= 0.23 µM
IC50= 1.3 µM
80
60
40
20
80
60
40
20
0.01
0.1
1
10
100
0.01
0.1
1
10
100
[I] (µM)
[I] (µM)
1/V (1/µM)
15
1/V (1/µM)
10
(B)
Slope
Ki= 1.9 µM
Ki= 0.36 µM
20
15
Substrate conc.
1 µM
10
5
10
5
0
5
1.5 µM
2 µM
0.0
0.5
1.0
1/[S] (1/µM)
5
5 µM
−2
0
2
−0.4
−0.2
0.0
[I] (µM)
0.2
0.4
[I] (µM)
Figure 3. Concentration-dependent STS inhibition (A) and Dixon’s kinetic analysis (B) of selected 13b-estrone compounds 7a, 16c. Inset in B/7a shows the secondary
plot of slopes of the Dixon’s lines vs. 1/substrate concentration.
investigated 4-nitroestrone as a representative estrone derivatives parent compound. We previously extended their research to the
substituted at position C-4 with various electron-withdrawing 13a-estrone series by performing brominations or iodinations on
its aromatic ring17. All synthesised 3-OH derivatives (10a,b–12a,b)
functions. They have found the binding to be noncompetitive
indicating that 4-nitroestrone is bound to the enzyme outside of
its active site7. Our test compounds, 4-iodoestrone 7a and 2,4-bis-
chloro-17-deoxy-13a-estrone 16c selected for the mechanistic
investigations displayed a competitive mechanism, which alludes
to binding within the active site of the enzyme.
Ki parameters were determined from the intersections of the
Dixon’s plots, and these values were found to be 0.36 mM for 7a
and 1.9 mM for the 16c. These measured Ki values reflected inhibi-
tory potentials ranked according to the IC50 data.
displayed outstanding low or submicromolar 17b-HSD1 inhibitory
activities, but their IC50 values depended on the number, the
nature and the position of the A-ring substituents (Table 3). The
iodo derivatives (10a–12a) proved to be more potent than their
bromo counterparts (10b–12b). The finding, that the 2,4-bis-iodo
compound (12a) displays a submicromolar IC50 of 0.38 mM, was a
novel result17.
Concerning that the iodo derivatives in the 13a-estrone series
displayed high inhibitory potential, the iodo counterparts in the
13b-estrone series (6a–8a) have also been tested. Both iodo
regioisomers (6a, 7a) exerted outstanding inhibition, but the
2-iodo compound (6a) proved to be more potent with an IC50
value of 0.064 mM (Table 3). 2-Bromo- and 2-chloro-13b-estrone
(6b and 6c) exerted somewhat weaker potentials as their iodo
counterpart (6a), nevertheless, we observed their improved bind-
17b-hsd1
The crystal structure of 17b-HSD1 in its complex with E2 has been
recently reported31. On the basis of this structure, molecular
dynamic simulations and ligand–protein docking studies showed
that there is an unoccupied lipophilic tunnel to the exterior of the ing affinity compared to the parent compound E1.
protein located near the C-2 atom of E26. These results may serve
as the basis for the development of potent inhibitors, while the
Efficiency of the 4-substituted counterparts (7a, 7b) was found
to be slightly weaker. These results are in a good agreement with
empirical rules established previously concerning the two substitu-
tion to be up against the four substitution. New inhibition results
binding affinity of the potential inhibitor might be enhanced by
6
€
introducing a lipophilic moiety to position 2. Moller et al. have
found that 2-bromo and 2-chloro derivatives of E1 exerted potent of the iodo compounds (6a–7a) are particularly remarkable, and
inhibition and displayed similar binding affinity to that of the the inhibition potential of the 2-iodo regioisomer is outstanding.

1280
I. BACSA ET AL.
9
11a
12a
Additionally, all chlorinated derivatives (6c–8c) displayed outstand-
ing commensurate submicromolar inhibition.
100
80
60
40
20
0
The three newly synthesised chloro 13a-estrone compounds
(10c–12c) proved to be potent inhibitors. The 2-regioisomer (10c)
seemed to be the most prominent with its submicromolar IC50
value of 0.33 mM. This value is commensurate with that of com-
pound 6c. This result indicates a twofold better binding than that
of E1, and an affinity increased by fourfold in comparison to par-
ent compound 13a-estrone 9. The 4-chloro and the 2,4-bis-chloro
derivatives (11c and 12c) exerted somewhat weaker inhibitions
(IC50 values were found to be 2.6 and 2.2 mM, respectively).
Comparing recent results obtained for bromo and iodo com-
pounds in both the 13a- and 13b-series with those of the chloro-
13a derivatives, it can be stated that there is less difference in the
inhibitory potential of the two regioisomers in the 13a-series than
in the natural estrone series except for the chloro-13a compounds.
The inhibitory potential of 13a-estrone (9) increased four-fold by
adding chlorine, a relatively small electron-withdrawing group, to
C-2.
I
II III IV
I
II III IV
I
II III IV
Figure 4. Investigation of 17b-HSD1 inhibition reversibility of selected 13a-
estrone compounds 9, 11a, 12a. Inhibitor compounds were preincubated with
human placental cytosol. Following a 50-fold dilution step, the usual enzyme
activity measurement was applied. Mean SD of three separate experiments.
Experimental conditions: I No preincubation, 0.2lM, II No preincubation, 10 lM,
III Preincubation, 10 lM, 2.5min, IV Preincubation, 10 lM, 20 min.
We were interested in the comparison of the inhibitory results
concerning the 17-keto and the 17-deoxy-13a derivatives. The
basic deoxy compound 13 displayed a surprising but outstanding
inhibitory potential comparable with that of reference E1, the nat-
ural substrate of the enzyme. We expected that this promising
result might be improved by the introduction of halogens to the
aromatic ring of 13. Nevertheless, the 1.1 mM IC50 value of 13
could not be lowered significantly. All halogenated derivatives
(14–16) displayed comparable or higher values than that obtained
for compound 13. In this series the 2-halogenated compounds
(14a–c) were found to be the best inhibitors (IC50 values 2.6, 1.3,
and 2.9 mM, respectively). The 4-chloro (15c) and the 2,4-bis-bromo
derivative (16b) exerted modest inhibition (IC50¼4–5 mM), whereas
other 17-deoxy-13a derivatives displayed weaker inhibition: their
relative conversions exceeded 50% indicating IC50 > 10 mM.
It was established that the advantage of the 2-substitution
over the 4-halogenation shows up in the 17-deoxy series, too.
However, none of the new 17-deoxy inhibitor candidates dis-
played submicromolar IC50 values.
Potent 13a-estrone derivatives, basic compound 9, its 4-iodo
(11a) and 2,4-bis-iodo derivative 12a, were selected for mechanis-
tic and kinetic studies. To provide some information about the
mechanism of action, we performed reversibility tests by preincu-
bation of inhibitors with human placental cytosol. Figure 4 shows
relative conversions obtained for the selected inhibitors according
to preincubation conditions I–IV. The results indicate that the rela-
tive conversions in preincubated and diluted samples (Figure 4, III
and IV), were similar to that obtained for incubation with the
lower concentration of the inhibitor (Figure 4, I). This means that
inhibitors 9, 11a and 12a can be released from binding by dilu-
tion that is they bind to the enzyme in a reversible manner.
In order to characterise the inhibition type and determine the
Ki, inhibition experiments were performed for the selected inhibi-
tors 9, 11a and 12a at different fixed substrate concentrations in
the presence of cofactor excess. On the Dixon’s plot, data for each
substrate concentration fall on straight lines which intersect in the
second quadrant, alluding to competitive inhibition mechanism
(Figure 5, B)29,30. Inhibitors that bind to the steroid site of 17b-
HSD1 can bind to both the free enzyme and the binary enzyme–-
cofactor complex by random kinetic mechanism. This mixed-type
inhibition, nevertheless, is simplified if the enzyme is saturated
bound in the substrate-binding cavity of 17b-HSD1 with non-cova-
lent interactions.
Ki parameters were determined from the intersections, and
they were found to be 1.9, 2.2, and 0.94 mM for compounds 9,
11a, and 12a, respectively. Inhibitory potentials on the basis of
these Ki data were comparable to those obtained by the
IC50 values.
The 3-OH group of E1 and related inhibitors plays an important
role in the binding to 17b-HSD1 forming a hydrogen-bonding sys-
tem with the His221 and Glu282 residues at the recognition end
of the active site33,34. However, literature data show high affinity
of certain 3-OMe derivatives indicating that ligands may establish
effective binding even in the absence of proton on the C-3 sub-
stituent16,17,35–37. Halogen substituents at C-2 and/or C-4 position
modify binding abilities of the 3-OH substituent. The electron-
withdrawing effect increases polarisation of the O–H bond and
may induce deprotonation of this group under physiological pH
conditions. ortho Substituents might also be able to form intramo-
lecular hydrogen bond with the 3-OH group, which is
disadvantageous6.
Our inhibition results demonstrate that introduction of halogen
atoms to C-2 and/or to C-4 position increases affinity to 17b-
HSD1. However, there does not appear to be a direct relationship
between the number and electronegativity of the halogens and
the inhibitor potency.
Multiple or specific inhibition
Certain dual 17b-HSD1 and STS inhibitors were identified in both
the 13b- and 13a-estrone series. Two 4-halo-17-keto-13b com-
pounds (7a and 7b) elicited submicromolar inhibitory effect
towards both enzymes. Certain additional 17-keto compounds
(6b, 6c, 7c, 8c, 12a) possess dual inhibitory properties with IC50
values in submicromolar or low micromolar range. In the
17-deoxy-13a-estrone series, all two-halogenated compounds (14),
the 2,4-bis-bromo- (16b) and 4-chloro derivative (15c) exerted
potent low micromolar dual action. Two compounds, namely
2-bromo- and 2-chloro-13b-estrones 6b and 6c exerted consider-
able inhibitions towards the three investigated enzymes.
It is interesting to note that inhibitory potentials of iodo deriv-
with cofactor first and displays competitive patterns32. Reversible atives in the 13b-estrone series display outstanding variations.
and apparently competitive mechanism of the inhibition observed 2-Iodo compound 6a is a highly specific 17b-HSD1 inhibitor, but
in our experiments shows that inhibitors 9, 11a, and 12a are 7a its 4-counterpart has dual STS and 17b-HSD1 inhibitory

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1281
9
11a
12a
Rel. conv.%
Rel. conv.%
Rel. conv.%
100
100
100
IC50= 0.38 µM
IC50= 1.0 µM
IC50= 1.1 µM
80
60
40
20
80
60
40
20
80
60
40
20
0.01
0.1
1
10
100
0.01
0.1
1
10
100
0.01
0.1
1
10
100
[I] (µM)
[I] (µM)
[I] (µM)
1/V (1/µM)
40
1/V (1/µM)
80
1/V (1/µM)
200
Ki= 1.9 µM
Ki= 0.94 µM
Ki= 2.2 µM
Substrate conc.
0.1 µM
150
100
50
60
40
20
30
20
10
0.15 µM
0.3 µM
0.5 µM
0.75 µM
1 µM
3 µM
−3
−2
−1
0
1
2
3
−2
0
2
4
6
8
10
−2
0
2
4
6
8
10
[I] (µM)
[I] (µM)
[I] (µM)
Figure 5. Concentration-dependent 17b-HSD1 inhibition (A) and Dixon’s kinetic analysis (B) of selected 13a-estrone compounds 9, 11a, 12a.
potential. The disubstituted derivative (8a) exerts weak effects
towards the enzymes investigated.
Funding
ꢀ
ꢀ
The work of Erzsebet Mernyak in this project was supported by
Our results confirm that structurally different enzymes with dis-
tinct catalytic mechanisms might be inhibited by the same inhibi-
tor compounds. Newly detected multiple 17b-HSD1, STS and/or
aromatase inhibitors might be superior to compounds affecting
the action of only a single enzyme. These multiple inhibitors may
serve as good candidates for efficient suppression of local estro-
gen production in breast cancer tissues.
ꢀ
the Janos Bolyai Research Scholarship of the Hungarian Academy
ꢀ
of Sciences and by the UNKP-17–4 “New National Excellence
Program Of The Ministry Of Human Capacities”. This work was
supported by National Research, Development and Innovation
Office-NKFIH through project Orszagos Tudomanyos Kutatasi
Alapprogramok (OTKA) SNN 124329.
ꢀ
ꢀ
ꢀ
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