Synthesis and biological evaluation of fluorinated N-benzoyl and N-phenylacetoyl derivatives of 3-(4-aminophenyl)-coumarin-7-O-sulfamate as steroid sulfatase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.01.028

In the present work, we report convenient methods for the synthesis of 3-(4-aminophenyl)-coumarin-7-O-sulfamate derivatives N-acylated with fluorinated analogues of benzoic or phenylacetic acid as steroid sulfatase (STS) inhibitors. The design of these potential STS inhibitors was supported by molecular modeling techniques. Additionally, computational docking methods were used to determine the binding modes of the synthesized inhibitors and to identify potential interactions between inhibitors and amino acid residues located in the active site of STS. The inhibitory effects of the synthesized compounds were tested on STS isolated from human placenta and against estrogen receptor-(ER)-positive MCF-7 and T47D cells, as well as ER-negative MDA-MB-231 and SkBr3 cancer cell lines. In the course of our investigation, compounds 6c and 6j demonstrated the highest inhibitory effect in enzymatic STS assays, both with IC₅₀values of 0.18?μM (the IC₅₀value of coumarin-7-O-sulfamate is 1.38?μM, used as a reference). Compound 6j exhibited the highest potency against the MCF-7 and T47D cell lines (15.9?μM and 8.7?μM, respectively). The GI₅₀values of tamoxifen (used as a reference) were 6.8; 10.6; 15.1; 12.5?μM against MCF-7, T47D, MDA-MB-231 and SkBr3 cancer cell lines, respectively. Despite the slightly lower activity of compounds 1 and 2 (both in enzymatic and cell-based experiments) compared to 6g and 6j, analogues 1 and 2 proved to selectively inhibit the growth of ER- and PR-positive cell lines.

Full text of DOI:10.1016/j.ejmech.2017.01.028

Accepted Manuscript
Synthesis and biological evaluation of fluorinated N-benzoyl and N-phenylacetoyl
derivatives of 3-(4-aminophenyl)-coumarin-7-O-sulfamate as steroid sulfatase
inhibitors
Mateusz Daśko, Maja Przybyłowska, Janusz Rachon, Maciej Masłyk, Konrad
Kubiński, Majus Misiak, Andrzej Składanowski, Sebastian Demkowicz
PII:
S0223-5234(17)30038-7
10.1016/j.ejmech.2017.01.028
EJMECH 9179
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 November 2016
Revised Date: 30 December 2016
Accepted Date: 21 January 2017
Please cite this article as: M. Daśko, M. Przybyłowska, J. Rachon, M. Masłyk, K. Kubiński, M. Misiak,
A. Składanowski, S. Demkowicz, Synthesis and biological evaluation of fluorinated N-benzoyl and N-
phenylacetoyl derivatives of 3-(4-aminophenyl)-coumarin-7-O-sulfamate as steroid sulfatase inhibitors,
European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.01.028.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of fluorinated N-benzoyl and N-
phenylacetoyl derivatives of 3-(4-aminophenyl)-coumarin-7-O-sulfamate as
steroid sulfatase inhibitors
,
a
a
a
b
b
Mateusz Daśko,* Maja Przybyłowska, Janusz Rachon, Maciej Masłyk, Konrad Kubiński,
c
c
,a
Majus Misiak, Andrzej Składanowski, and Sebastian Demkowicz,*
a
Department of Organic Chemistry, Faculty of Chemistry, Gdansk University of Technology,
Narutowicza 11/12, 80-233 Gdansk, Poland, e-mail: matdasko@gmail.com,
sebdemko@pg.gda.pl.
b
Department of Molecular Biology, Faculty of Biotechnology and Environment Sciences,
The John Paul II Catholic University of Lublin, Konstantynów 1i, 20-708 Lublin, Poland
c
Department of Pharmaceutical Technology and Biochemistry, Faculty of Chemistry, Gdansk
University of Technology, Narutowicza 11/12, 80-233 Gdansk, Poland
Keywords: steroid sulfatase, STS inhibitors, breast cancer, coumarin, sulfamates
Abstract
In the present work, we report convenient methods for the synthesis of 3-(4-aminophenyl)-
coumarin-7-O-sulfamate derivatives N-acylated with fluorinated analogs of benzoic or
phenylacetic acid as steroid sulfatase (STS) inhibitors. The design of these potential STS
inhibitors was supported by molecular modeling techniques. Additionally, computational
docking methods were used to determine the binding modes of the synthesized inhibitors and
to identify potential interactions between inhibitors and amino acid residues located in the
active site of STS. The inhibitory effects of the synthesized compounds were tested on STS
isolated from human placenta and against estrogen receptor-(ER)-positive MCF-7 and T47D
cells, as well as ER-negative MDA-MB-231 and SkBr3 cancer cell lines. In the course of our
investigation, compounds 6c and 6j demonstrated the highest inhibitory effect in enzymatic
STS assays, both with IC values of 0.18 µM (the IC value of coumarin-7-O-sulfamate is
50
50
1
.38 µM, used as a reference). Compound 6j exhibited the highest potency against the MCF-7
and T47D cell lines (15.9 µM and 8.7 µM, respectively). The GI values of tamoxifen (used
5
0
as a reference) were 6.8; 10.6; 15.1; 12.5 µM against MCF-7, T47D, MDA-MB-231 and
SkBr3 cancer cell lines, respectively. Despite the slightly lower activity of compounds 1 and 2
(both in enzymatic and cell-based experiments) compared to 6g and 6j, analogues 1 and 2
proved to selectively inhibit the growth of ER- and PR-positive cell lines.
1
. Introduction
The World Health Organization (WHO) lists biologically active hormones, including
estrogens, as one of the most important factors inducing the development of breast cancer.
Novel treatment strategies for breast cancer involve inhibitors, which prevent the synthesis of
estrogens in peripheral tissues [1]. Steroid sulfatase (STS) is one enzyme responsible for the
formation of active estrogens in the breast tissue of postmenopausal women. STS hydrolyses
estrone sulfate (E1S) and dehydroepiandrosterone sulfate (DHEAS) into estrone (E1) and
dehydroepiandrosterone (DHEA), which can be converted into steroids that exhibit estrogenic
properties (estradiol and androstenediol) [2].
The design and synthesis of compounds that regulate hormone levels in tissues through
inhibition of STS activity is a major challenge for modern medicinal chemistry. Most known
STS inhibitors act irreversibly. To date, various research groups have synthesized steroidal

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and non-steroidal irreversible inhibitors of STS substituted with various functional groups
including phosphates, thiophosphates or sulfamates [3-10]. Because the sulfamate moiety of
STS inhibitors such as EMATE was designed to mimic sulfate moiety of natural substrates, it
is assumed that the inhibition mechanism of these compounds involves an FGly75 residue
located inside the active site of STS. The FGly75 residue coordinates to a calcium ion and
plays a crucial role in the enzymatic hydrolysis of sulfated substrates. One proposed
mechanism of inhibition assumes nucleophilic attack of the FGly75 hydroxyl group on the
sulfur atom of the inhibitor containing the sulfamate moiety (Figure 1), which results in
irreversible inactivation of the enzyme [11].
NH2
O
S
O
O
R
O
O
H
S
O
O
NH
2
H
H
-ROH
O
O
E
FGly75
E
I
Fig. 1. Proposed mechanism of STS inhibition by compounds containing sulfamate moiety (E
enzyme; R – core of inhibitors).
–
Many STS inhibitors had been discontinued from clinical trials due to their estrogenic
properties. For example, the steroid derivative EMATE (Figure 2) is an efficient inhibitor of
STS activity (IC value of 65 pM in MCF-7 cell line assay) but was withdrawn from clinical
5
0
trials due to its estrogenicity [12]. To avoid adversary side effects, several research groups
have designed new compounds based on non-steroidal structures. An important class of potent
inhibitors of STS includes coumarin derivatives. Coumarin-7-O-sulfamate and its derivatives,
e.g., COUMATE (IC value of 380 nM in an assay with placental microsomes) [13] have
5
0
been shown to lack significant estrogenicity.
O
H
2
NO
2
SO
H
2
NO
2
SO
O
O
EMATE
coumarin-7-O-sulfamate
R
1
R
R
2
3
H
2
NO
2
SO
COUMATE
O
O
H
2
NO
2
SO
O
O
1,
R
R
1
, R
, R
2
2
3
= F, R = H
, R , = F
3
2
,
1
Fig. 2. Chemical structures of STS inhibitors.
A key strategy in drug design is the introduction of fluorine atoms to their chemical structure.
Since 1957, more than 150 fluorinated drugs have come to the market and now make up
approximately 20% of all pharmaceuticals [14-16]. Organofluorine affects nearly all physical
properties of the drug, as well as its absorption, distribution, metabolism, and excretion. In
this paper we describe the synthesis of new fluorine-containing coumarins as potential
inhibitors of STS. In the course of previous investigations with fluorinated 3-phenylcoumarin-

ACCEPTED MANUSCRIPT
7
-O-sulfamate derivatives [17], we found that introduction of fluorinated phenyl rings to
coumarin scaffolds results in increased inhibitory activity. Two fluorinated compounds, 1 and
, demonstrated the highest activity in the STS enzyme assay, with IC values of 270 nM in
2
5
0
both cases. In most cases, compounds containing C-F bonds showed slightly higher STS
inhibition than analogues without fluorine atom in its structure.
2
. Results and discussion
2
.1. Chemistry
The synthesis of coumarin and their derivatives has been the subject of extensive research
over many decades. Many convenient synthetic methods have been described including
Pechmann [18], Perkin [19], Knoevenagel condensation [20], Reformatsky [21] and Wittig
reactions [22]. Synthesis of newly designed STS inhibitors based on fluorinated N-benzoyl
and N-phenylacetoyl derivatives of 3-(4-aminophenyl)-coumarin-7-O-sulfamate was
accomplished using the pathway shown in Scheme 1. In the first step, 4-nitrophenyl acetyl
chloride was obtained in situ by the treatment of 4-nitrophenyl acetic acids with thionyl
chloride in dry dichloromethane. For the synthesis of 7-hydroxy-3-(4-nitrophenyl)-coumarin
3
, raw 4-nitrophenyl acetyl chloride was refluxed with 2,4-dihydroxybenzaldehyde in the
presence of potassium carbonate. Next, 7-hydroxy-3-(4-nitrophenyl)-coumarin 3 was reduced
to 7-hydroxy-3-(4-aminophenyl)-coumarin 4 using sodium hydrosulfite (Na S O ). The
2
2
4
progress of the reaction was monitored by TLC and, after full conversion of the starting
material, product 4 was isolated from the reaction mixture in satisfactory yield (58%). In the
next step, 7-hydroxy-3-(4-aminophenyl)-coumarin 4 was N-acylated with corresponding
phenylacetoyl or benzoyl chlorides containing C-F bonds (obtained in situ from
corresponding phenylacetic or benzoic acids reacted with thionyl chloride) in the presence of
potassium carbonate. The reactions proceeded well and the desired products 5a-k were
obtained in good yield (62 - 75%). Finally, OH groups of the coumarin scaffolds 5a-k were
sulfamoylated. In brief, solutions of stable compounds 5a-k in N,N-dimethylacetamide
(
DMA) were treated with H NSO Cl (previously generated by the reaction of chlorosulfonyl
2 2
isocyanate and formic acid in the presence of a catalytic amount of N,N-dimethylacetamide).
The yields of these reactions were high, reaching 90%. After standard isolation, we obtained
the desired derivatives 6a-k. The detailed synthesis is shown in Scheme 1. Additionally, we
synthesized 3-(4-nitrophenyl)-coumarin-7-O-sulfamate 7 in the reaction of 7-hydroxy-3-(4-
nitrophenyl)-coumarin 3 with H NSO Cl.
2
2

ACCEPTED MANUSCRIPT
CHO
OH
COOH
COCl
NO
2
NH2
SOCl
2
Na S O
OH
2
2
4
DCM
K CO
H
2
O
2
3
acetone
HO
O
O
HO
O
O
acetone
3
4
NO
2
NO
2
NH
2
HO
COCl
O
O
4
COOH
n
n
R
R
1
2
R
5
4
SOCl
2
R
1
2
R
R
5
4
2 3
K CO
acetone
DCM
R
1
4
R
R
H
N
n
R
R
2
3
R
3
R
3
O
R
5
R
HO
O
O
O
5a-k
R
1
H
n
N
R
R
2
3
DMA
HCOOH
DMA
O
O
O
O
O
O
R
5
S
S
S
R4
Cl
N
C
O
DCM
H
N
Cl
6a-k
2
H
2
N
O
O
O
NO
2
NO
2
O
O
S
H
2
N
Cl
O
O
DMA
S
HO
O
O
H
2
N
O
O
O
3
7
Scheme 1. Synthesis of 3-(4-benzoylamino-phenyl)- coumarin-7-O-sulfamate and 3-(4-
phenylacetylamino-phenyl)-coumarin-7-O-sulfamate derivatives 6a-k (n = 0, 1; R , R , R ,
1
2
3
R , R = H, F, CF , OCF ) and 3-(4-nitrophenyl)-coumarin-7-O-sulfamate 7.
4
5
3
3
2
.2. Molecular modeling
To examine the possible interactions of N-benzoyl and N-phenylacetoyl derivatives of 3-(4-
aminophenyl)-coumarin-7-O-sulfamate with amino acid residues within the active site of
STS, potential inhibitors were docked into the crystal structure of the human steroid sulfatase
(Protein Data Bank accession code 1P49). The procedures for docking analyses as well as
protein and inhibitor preparations are described in detail in the experimental section.
Our docking experiments revealed that fluorinated N-benzoyl and N-phenylacetoyl derivatives
of 3-(4-aminophenyl)-coumarin-7-O-sulfamate could, at least theoretically, efficiently bind to
the active site of STS. All candidates 6a-k expressed satisfactory predicted free docking
energies (in the range of -7.6 to -9.5 kcal/mol) and exhibited significantly lower AutoDock
Vina scores compared to a reference inhibitor (the value of predicted free docking energy for
coumarin-7-O-sulfamate, 1 and 2 were -2.9, -6.7 and -6.8 kcal/mol, respectively). The best
docking result among the N-benzoyl derivatives was obtained for the 6c analogue (predicted
free docking energy was -8.9 kcal/mol), whereas the best docking result among the N-
phenylacetoyl derivatives was obtained for the 6h analogue (predicted free docking energy
was -9.5 kcal/mol). Furthermore, we found that the AutoDock Vina score for compound 7
was -7.2 kcal/mol, lower than reference inhibitors (Table 1).

ACCEPTED MANUSCRIPT
Figure 3 shows a putative enzyme-ligand complex before the presumed inactivation of STS
and the superimposed best conformations of the three selected potential inhibitors 6c (blue),
6
h (red), 6j (CPK colored). The STS inhibitor candidates docked in a similar manner to the
mode of the reported STS inhibitor coumarin-7-O-sulfamate (green). We found that sulfamate
2+
functional groups are directed to the catalytic amino acid FGly75 coordinated to the Ca ion
and are surrounded by several putative catalytic residues (Asp35, Asp36, Arg79, Lys134,
His136 and Lys368). In each case, the distance between the sulfur atom of the designed
compounds and a hydroxyl group of FGly75 are short (less than 3 Å) suggesting the
possibility of an electrostatic interactions (e.g., hydrogen bonds) between the sulfamate
moiety and an OH group of FGly75 in its gem-diol form. Furthermore, the cores of the
potential inhibitors are well accommodated by the cavity delimited by lipophilic amino acids
residues located near the active site of the STS. Molecular modeling studies suggest that the
hydrophobicity of coumarin cores could favor binding through the establishment of
hydrophobic interactions with lipophilic amino acids in the enzyme active site (Leu103,
Phe104, Leu167, Val177, Phe178, Phe182, Leu185, Phe230, Phe233, Phe237, Thr484,
Val486, Phe488, Trp550 and Phe553). In addition, as shown in Figure 3 and exemplified by
compound 6j, amide moieties and fluorine atoms (e.g., in CF groups) of designed compounds
3
are within a short distance of the backbone NH groups of Arg98, suggesting the presence of
additional stabilizing interactions (e.g., hydrogen bonds). These additional interactions may
influence the binding of potential drug molecules to the enzyme active site.
Fig. 3. Docked binding modes and distances to Arg98 for compounds 6c (blue), 6h (red), 6j
(CPK colored) and coumarin-7-O-sulfamate (green).
2
.3. STS enzyme assays
The inhibitory effects of the synthesized compounds 6a-k were tested using in vitro STS
assays according to reported methods [23,24]. The screening assays were performed with STS
enzyme extracted from human placenta and purified by three-step chromatography.
The results of the enzymatic assays are presented in Table 1. As observed in the data collected
in the Table 1, the IC50 values are all fairly similar suggesting that the nature of R -R5
1
substituents does not significantly affect the binding. Nevertheless, the compounds containing
fluorine atoms in their structures, in most cases showed a higher activity against STS which
was probably due to the stronger hydrophobic effect of the fluorine substituted aromatic ring.
In the course of our investigation, we found that all the newly synthesized inhibitors
possessed significantly greater inhibitory potency than reported for coumarin-7-O-sulfamate
(
IC value of 1.38 µM). The highest inhibitory activity among the N-benzoyl derivatives was
50
exhibited by the 6c analogue, with an IC50 value of 0.18 µM (consistent with molecular
modeling data) whereas the highest inhibition among the N-phenylacetoyl derivatives was

ACCEPTED MANUSCRIPT
observed for the 6j analogue, with an IC value of 0.18 µM (the value of the predicted free
5
0
docking energy calculated by AutoDock Vina software was -8.6 kcal/mol). Furthermore, the
IC value for compound 7 was 0.29 µM. The best newly synthesized compounds 6c and 6j
5
0
were slightly more potent than inhibitors 1 and 2, whose design and evaluation we previously
described [17].
Table 1.
Activities of the synthesized compounds 6a-k and 7, and a reference inhibitors (coumarin-7-
O-sulfamate, 1 and 2) in the STS enzyme assays.
Free binding
IC50
No.
n
0
R1
R2
R3
R4
R5
energy
-1
[µM]
[
kcal mol ]
-7.6
6a
H
H
H
F
H
H
F
H
F
H
H
H
F
H
H
H
F
0.49 ± 0.04
0.21 ± 0.01
0.18 ± 0.01
0.23 ± 0.01
0.26 ± 0.02
0.36 ± 0.03
0.30 ± 0.03
0.34 ± 0.03
0.38 ± 0.02
0.18 ± 0.01
0.44 ± 0.02
0.29 ± 0.03
0.28 ± 0.02
0.29 ± 0.02
6b
-8.6
-8.9
-8.8
-8.2
-8.7
-7.8
-9.5
-8.9
-8.6
-8.4
-7.2
-6.7
-6.8
-2.9
6c
F
6d
F
F
6e
H
H
H
F
H
F
H
F
H
H
F
H
H
H
F
6f
6
g
h
F
F
6
6
1
F
F
F
6
i
H
CF
H
H
-
3
F
H
H
H
H
-
j
CF
H
-
3
H
OCF
-
CF
H
-
3
6k
3
7
-
-
-
1
2
-
-
-
-
-
-
-
-
-
-
coumarin-7-
O-sulfamate
-
-
-
-
-
-
1.38 ± 0.13
2
.4. Cancer cell viability assay
According to the results of the enzymatic assay, we selected the six most active fluorinated
derivatives from three different families (6b and 6c, 6g and 6j, 1 and 2) for cytotoxicity
studies. In our panel of test cell lines, we included two estrogen- (ER-) and progesterone-
(PR-) positive (MCF-7, T47D) and two ER- and PR-negative (SkBr3, MDA-MB-231) breast
cancer cell lines. The results are presented in Table 2.
Despite the high activity of compounds 6b and 6c in in vitro experiments, their cytotoxicity
against the tested cell lines ranged from moderate (mid-micromolar) to none. Analogue 6j
demonstrated the most potent antiproliferative activity (GI values for MCF-7 and T47D cell
5
0
lines of 15.9 µM and 8.7 µM, respectively). However, it was not selective towards estrogen-
dependent cells and effectively inhibited the growth of ER- and PR-negative cell lines (GI50
values in assays with SkBr3 and MDA-MB-231 cell lines of 18.8 µM and 8.1 µM,
respectively). The activities of compounds 6g, 6j, 1 and 2 were found to be comparable or
approximately 2-3 times lower in comparison with tamoxifen used as a reference. The GI50
values of tamoxifen were 6.8; 10.6; 15.1; 12.5 µM against MCF-7, T47D, MDA-MB-231 and

ACCEPTED MANUSCRIPT
SkBr3 cancer cell lines, respectively. In comparison, coumarin-7-O-sulfamate, used as a
reference, was inactive against all of the aforementioned cells. No selectivity observed for
compounds 6g and 6j towards estrogen-dependent cells could be a consequence of their low
binding affinity to the human estrogen receptors ERα and ERβ (confirmed by independent
experiments of molecular docking). In addition, other processes including a membrane
transport and cellular metabolism could be crucial for the antiproliferative properties of these
compounds. Despite the slightly lower activity of compounds 1 and 2 (both in enzymatic and
cell-based experiments) in comparison with 6g and 6j, analogues 1 and 2 proved to selectively
inhibit the growth of ER- and PR-positive cell lines (e.g., for compound 2 the GI values
5
0
were approximately 30 µM with MCF-7 and T47D cell lines and above 55 µM for SkBr3 and
MDA-MB-231 cells). It is noteworthy that the compounds 1 and 2 showed selectivity for the
estrogen-dependent cells, although molecular docking studies to the ERα and ERβ showed no
affinity. These observations may suggest that the compounds 1 and 2 could be involved
mainly in the inhibition of estrogen biosynthesis by interacting with the STS.
Table 2.
Antiproliferative activities of selected compounds and references (coumarin-7-O-sulfamate
and tamoxifen).
GI [µM]
50
No.
MCF-7
T47D
SkBr3
MDA-MB-231
+
+
+
+
+
-
-
-
+
-
-
-
ER ; PR ; Her2
>100
ER ; PR ; Her2
>100
ER ; PR ; Her2
>100
ER ; PR ; Her2
>100
6
b
6
6
6
c
>100
>100
>100
>100
g
j
31.3 ± 6.9
15.9 ± 3.7
30.0 ± 2.3
32.6 ± 2.6
13.7 ± 1.0
8.7 ± 0.1
43.0 ± 1.7
30.7 ± 3.9
36.1 ± 0.3
18.8 ± 0.4
>100
11.7 ± 3.0
8.1 ± 1.7
61.5 ± 3.9
56.3 ± 0.6
1
2
61.3 ± 3.8
coumarin-7-
O-sulfamate
>
100
>100
>100
>100
tamoxifen
6.8 ± 2.2
10.6 ± 0.6
12.5 ± 0.6
15.1 ± 0.1
a
ER, estrogen receptor; PR, progesterone receptor; Her2 epithelial growth factor receptor 2.
3
. Conclusion
We have efficiently synthesized and biologically evaluated a series of new STS inhibitors.
Computational analyses supported the design of the structures of potential inhibitors. In the
course of our docking studies, we found that fluorinated N-benzoyl and N-phenylacetoyl
derivatives of 3-(4-aminophenyl)-coumarin-7-O-sulfamate are well accommodated to the
active site of STS. Furthermore, the amide moieties and fluorine atoms (e.g., in CF groups)
3
of the designed compounds are within a short distance of the backbone NH groups of Arg98,
suggesting the presence of additional stabilizing interactions (e.g., hydrogen bonds). The
results of our molecular modeling studies were mostly confirmed in enzymatic experiments.
An in vitro assay with isolated STS showed that all newly synthesized inhibitors possessed
significantly greater inhibitory potency than reported for coumarin-7-O-sulfamate (IC value
5
0
of 1.38 µM). The highest inhibitory activity among the N-benzoyl derivatives was exhibited
by the 6c analogue, whereas the highest inhibition among the N-phenylacetoyl derivatives was
observed for the 6j analogue, both with IC50 value of 0.18 µM. Six of the most active
fluorinated derivatives in the enzymatic assay were grouped into three different families (6b

ACCEPTED MANUSCRIPT
and 6c, 6g and 6j, 1 and 2) and were selected for cytotoxicity studies. In the course of our
investigation, we found that compound 6j exhibited the highest potency against the MCF-7
and T47D cell lines at 15.9 µM and 8.7 µM, respectively. However, it was not selective
towards estrogen-dependent cells and effectively inhibited the growth of ER- and PR-negative
cells. Despite the slightly lower activity of compounds 1 and 2 (both in enzymatic and cell-
based experiments) compared to 6g and 6j, analogues 1 and 2 proved to selectively inhibit the
growth of ER- and PR-positive cell lines.
4
. Experimental
4
.1. Materials and methods
Benzoic acid, 4-fluorobenzoic acid, 3,4-difluorobenzoic acid, pentafluorobenzoic acid,
phenylacetic acid, 4-nitrophenylacetic acid, 3,4-difluorophenylacetic acid, 3,4,5-
trifluorophenylacetic
Bis(trifluoromethyl)phenylacetic acid, [4-fluoro-3-(trifluoromethyl)phenyl]acetic acid, 4-
trifluoromethoxy)phenylacetic acid, thionyl chloride, potassium carbonate, 2,4-
dihydroxybenzaldehyde, sodium hydrosulfite, chlorosulfonyl isocyanate, N,N-
acid,
2,3,4,5,6-pentafluorophenylacetic
acid,
2,5-
(
®
dimethylacetamide, formic acid, tamoxifen (TraceCERT ) are commercially available from
Aldrich. Dichloromethane and acetone were dried and distilled using standard procedures.
Melting points (uncorrected) were determined with a Stuart Scientific SMP30 apparatus.
NMR spectra were recorded on a Varian Gemini 200 MHz and Varian Unity Plus 500
spectrometers. Chemical shifts are reported in ppm relative to the residual solvent peak
1
13
(
DMSO-d 2.49 ppm for H, acetone-d 29.92 ppm for C). Coupling constants are given in
6 6
Hertz. IR spectra were measured on a Nicolet 8700 spectrometer. Elemental analysis was
performed using CHNS-Carlo Erba EA-1108. Mass spectra were recorded on an Agilent 6540
Accurate Mass Q-TOF LC/MS System. Column chromatography was performed using silica
gel 60 (230-400 mesh, Merck). Preparative thin-layer chromatography was performed with
Polygram SIL G/UV254 silica gel (Macherey- Nagel GmbH & Co. KG, Düren, Germany).
4
.2. Preparation of 7-hydroxy-3-(4-nitrophenyl)-coumarin (3).
SOCl (33 mL) was added to a solution of 4-nitrophenylacetic acid (7.25 g, 40 mmol) in dry
2
CH Cl (33 mL), and the suspension was refluxed for 4 h. The resulting solution was
2
2
evaporated, the residue was dissolved in dry acetone (200 mL) and 2,4-
dihydroxybenzaldehyde (5.52 g, 40 mmol) was added. The mixture was refluxed with
anhydrous K CO (22.08 g, 160 mmol) for 4 h. Acetone was removed under reduced pressure
2
3
and cold water (400 mL) was added; 3 N HCl was then added until the solution became
acidic. The resulting precipitate was filtered, washed with water and recrystallized from
ethanol to give the desired product 3.
-
1
Yield 68%; mp 298-299 ºC (with decomposition); v (KBr)/cm 3201, 1685, 1588, 1508,
max
1
1
415, 1336, 1286, 1219, 1169, 1118, 993, 848; H NMR δ (500 MHz, DMSO) 10.81 (1H, s,
H
OH), 8.39 (1H, s, CH), 8.28 (2H, d, J 8.8, Ar-H), 8.00 (2H, d, J 8.3, Ar-H), 7.64 (1H, d, J 8.8,
1
3
Ar-H), 6.85 (1H, d, J 8.8, Ar-H), 6.77 (1H, s, Ar-H); C NMR (125 MHz, DMSO), δ (ppm):
62.9, 160.3, 156.1, 147.3, 143.9, 142.5, 131.3, 129.9, 124.0, 120.4, 114.4, 112.4, 102.5.
Anal. Calcd for: C H NO : C, 63.61; H, 3.20; N, 4.95. Found: C, 63.70; H, 3.27; N, 4.84%.
1
1
5
9
5
-
HRMS (m/z) [M-H] calcd 282.0402. Found 282.0444.
4
.3. Preparation of 3-(4-aminophenyl)-7-hydroxy-coumarin (4).

ACCEPTED MANUSCRIPT
First, 7-hydroxy-3-(4-nitrophenyl)-coumarin 3 (7.08 g, 25 mmol) was added to a mixture of
acetone (1300 mL) and water (650 mL). The resulting solution was heated to 50 ˚C (until the
solution became completely clear). Next, sodium hydrosulfite (43.53 g, 250 mmol) was added
in three portions, and the reaction mixture was heated to reflux. After 2 h, the acetone was
evaporated, and the resulting precipitate was filtered, washed with water and recrystallized
from acetone to give the desired product 4.
-1
Yield 58%; mp 292-295 ºC (with decomposition); vmax (KBr)/cm 3338, 3278, 1670, 1608,
1
1
565, 1507, 1467, 1333, 1284, 1211, 1162, 1132, 994, 846; H NMR δ (500 MHz, DMSO)
H
1
0.45 (1H, s, OH), 7.94 (1H, s, CH), 7.53 (1H, d, J 8.3, Ar-H), 7.41 (2H, d, J 8.8, Ar-H), 6.77
13
(1H, d, J 8.8, Ar-H), 6.71 (1H, s, Ar-H), 6.59 (2H, d, J 8.8, Ar-H), 5.43 (2H, br s, NH ); C
2
NMR (125 MHz, DMSO), δ (ppm): 137.6, 137.5, 131.4, 126.0, 114.8, 106.5, 106.2, 99.9,
9.4, 90.6, 90.4, 89.6, 78.8. Anal. Calcd for: C H NO : C, 71.14; H, 4.38; N, 5.53. Found:
9
1
5
11
3
-
C, 71.01; H, 4.41; N, 5.65%. HRMS (m/z) [M-H] calcd 252.0661. Found 252.0705.
4
.4. General method for the synthesis of N-benzoyl and N-phenylacetoyl derivatives of
3
-(4-aminophenyl)-7-hydroxy-coumarin (5a-k).
SOCl (1.65 mL) was added to a solution of benzoic or phenylacetic acid derivatives (2
2
mmol) in dry CH Cl (1.65 mL), and the suspension was refluxed for 4 h. The resulting
2
2
solution was evaporated, the residue was dissolved in dry acetone (10 mL) and 3-(4-
aminophenyl)-7-hydroxy-coumarin 4 (0.51 g, 2 mmol) was added. The mixture was refluxed
with anhydrous K CO (1.104 g, 8 mmol) for 4 h. Acetone was removed under reduced
2
3
pressure and cold water (20 mL) was added. 3 N HCl was added until the solution became
acidic. The resulting precipitate was filtered, washed with water and recrystallized from
methanol or ethanol to give the desired products 5a-k.
3
-(4-benzoylamino-phenyl)-7-hydroxy-coumarin (5a). Yield 72%; mp 281-284 ºC (with
-1
decomposition); vmax (KBr)/cm 3363, 3198, 1696, 1664, 1601, 1516, 1410. 1325, 1283,
1
1
202, 1158, 1129, 988, 837; H NMR δ (500 MHz, DMSO) 10.59 (1H, s, OH), 10.37 (1H, s,
H
NH), 8.16 (1H, s, CH), 7.97 (2H, d, J 7.1, Ar-H), 7.85 (2H, d, J = 8.8, Ar-H), 7.71 (2H, d, J
.8, Ar-H), 7.62-7.50 (4H, m, Ar-H), 6.82 (1H, dd, J 8.5, 2.3, Ar-H), 6.75 (1H, d, J 2.2, Ar-
8
13
H); C NMR (125 MHz, DMSO), δ (ppm): 166.3, 161.8, 160.8, 155.5, 141.0, 139.7, 135.5,
32.4, 130.9, 130.6, 129.2, 129.1, 128.4, 122.4, 120.5, 114.1, 112.8, 102.4. Anal. Calcd for:
C H NO : C, 73.94; H, 4.23; N, 3.92. Found: C, 73.99; H, 4.16; N, 3.83%. HRMS (m/z)
1
2
2
15
4
-
[
M-H] calcd 356.0923. Found 356.0960.
3
-[4-(4-fluoro-benzoylamino)-phenyl]-7-hydroxy-coumarin (5b). Yield 66%; mp 321-324 ºC
-1
(with decomposition); vmax (KBr)/cm 3544, 3337, 1705, 1665, 1599, 1501, 1412, 1321,
1
1
282, 1213, 1153, 1118, 990, 822; H NMR δ (500 MHz, DMSO) 10.59 (1H, s, OH), 10.38
H
(
(
1H, s, NH), 8.16 (1H, s, CH), 8.08-8.02 (2H, m, Ar-H), 7.83 (2H, d, J = 8.8, Ar-H), 7.71
2H, d, J 8.8, Ar-H), 7.59 (1H, d, J 8.3, Ar-H), 7.40-7.34 (2H, m, Ar-H), 6.82 (1H, dd, J 8.3,
.4, Ar-H), 6.75 (1H, d, J 2.4, Ar-H); C NMR (50 MHz, DMSO), δ (ppm): 164.4, 164.1 (d,
1
3
2
1
JC-F 249), 161.0, 160.1, 154.7, 140.2, 138.9, 131.2 (m). 130.5, 130.3, 129.8, 128.4, 121.7,
19.8, 115.3 (m), 113.3, 112.0, 101.7. Anal. Calcd for: C H FNO : C, 70.40; H, 3.76; N,
1
5
3
2
2
14
4
-
.06. Found: C, 70.49; H, 3.83; N, 4.98%. HRMS (m/z) [M-H] calcd 374.0829. Found
74.0883.

ACCEPTED MANUSCRIPT
3
-[4-(3,4-difluoro-benzoylamino)-phenyl]-7-hydroxy-coumarin (5c). Yield 65%; mp 334-337
-1
ºC (with decomposition); vmax (KBr)/cm 3577, 3337, 1703, 1666, 1600, 1503, 1411, 1322,
1
1
288, 1214, 1160, 1122, 994, 824; H NMR δ (200 MHz, DMSO) 10.61 (1H, s, OH), 10.44
H
(
1H, s, NH), 8.18 (1H, s, CH), 8.16-8.00 (2H, m, Ar-H), 7.90-7.56 (6H, m, Ar-H), 6.88-6.76
1
3
(2H, m, Ar-H); C NMR (50 MHz, DMSO), δ (ppm): 163.2, 161.1, 159.7, 154.7, 150.0 (m),
1
1
6
47.8 (m), 140.3, 138.6, 132.1 (m), 130.5, 129.9, 128.5, 125.3 (m), 121.6, 119.9, 117.4 (m),
13.4, 112.0, 101.7. Anal. Calcd for: C H F NO : C, 67.18; H, 3.33; N, 3.56. Found: C,
2
2
13
2
4
-
7.14; H, 3.40; N, 3.63%. HRMS (m/z) [M-H] calcd 392.0734. Found 392.0777.
7
-hydroxy-3-(4-pentafluorobenzoylamino-phenyl)-coumarin (5d). Yield 62%; mp 243-245 ºC
-1
(with decomposition); vmax (KBr)/cm 3307, 3201, 1695, 1666, 1608, 1502, 1412, 1335,
1
1
282, 1214, 1158, 1126, 992, 844; H NMR δ (500 MHz, DMSO) 11.11 (1H, s, OH), 10.61
H
(
1H, s, NH), 8.17 (1H, s, CH), 7.78-7.68 (4H, m, Ar-H), 7.60 (1H, d, J = 8.8, Ar-H), 6.81
1
3
(1H, d, J 8.5, Ar-H), 6.75 (1H, s, Ar-H); C NMR (50 MHz, DMSO), δ (ppm): 161.2, 160.0,
1
1
1
1
2
54.8, 143.1 (d, J 255), 140.6, 137.6, 137.0 (d, J 252), 131.4, 129.9, 129.1, 128.9,
C-F C-F
21.4, 119.2, 113.4, 112.7 (m), 112.0, 101.7. Anal. Calcd for: C H F NO : C, 59.07; H,
2
2
10
5
4
-
.25; N, 3.13. Found: C, 59.11; H, 2.14; N, 3.21%. HRMS (m/z) [M-H] calcd 446.0452.
Found 446.0502.
7
-hydroxy-3-(4-phenylacetylamino-phenyl)-coumarin (5e). Yield 75%; mp 237-239 ºC (with
-1
decomposition); vmax (KBr)/cm 3351, 3190, 1705, 1655, 1609, 1517, 1410, 1321, 1285,
1
1
211, 1165, 1129, 991, 840; H NMR δ (500 MHz, DMSO) 10.59 (1H, s, OH), 10.30 (1H, s,
H
NH), 8.12 (1H, s, CH), 7.68-7.54 (5H, m, Ar-H), 7.36-7.28 (4H, m, Ar-H), 7.26-7.22 (1H, m,
13
Ar-H), 6.80 (1H, d, J 8.3, Ar-H), 6.73 (1H, s, Ar-H), 3.65 (2H, s, CH ); C NMR (125 MHz,
2
DMSO), δ (ppm): 169.9, 161.7, 160.8, 155.4, 140.8, 139.7, 136.6, 130.6, 130.5, 129.8, 129.3,
1
7
3
29.0, 127.3, 122.4, 122.4, 119.3, 114.1, 112.8, 102.4, 44.0. Anal. Calcd for: C H NO : C,
4.38; H, 4.61; N, 3.77. Found: C, 74.52; H, 4.55; N, 3.86%. HRMS (m/z) [M-H] calcd
70.1079. Found 370.1092.
23 17 4
-
3
2
1
1
7
2
1
-{4-[2-(3,4-difluoro-phenyl)-acetylamino]-phenyl}-7-hydroxy-coumarin (5f). Yield 68%; mp
-
1
81-283 ºC (with decomposition); vmax (KBr)/cm 3350, 3215, 1702, 1658, 1614, 1514, 1412,
1
318, 1283, 1200, 1164, 1129, 994, 841; H NMR δ (500 MHz, DMSO) 10.58 (1H, s, OH),
H
0.30 (1H, s, NH), 8.12 (1H, s, CH), 7.68-7.62 (4H, m, Ar-H), 7.58 (1H, d, J 8.3, Ar-H), 7.42-
.34 (2H, m, Ar-H), 7.20-7.14 (1H, m, Ar-H), 6.80 (1H, dd, J 8.8, 2.1, Ar-H), 6.73 (1H, d, J
1
3
.1, Ar-H), 3.67 (2H, s, CH ); C NMR (50 MHz, DMSO), δ (ppm): 168.5, 161.0, 160.1,
2
1
1
54.7, 149.2 (d, J 245), 148.5 (d, J 244), 140.1, 138.9, 133.4 (m), 129.8, 128.6, 126.0
C-F
C-F
(m), 121.6, 118.7 (m), 118.1, 117.3, 117.0, 113.3, 112.0, 101.7, 42.0. Anal. Calcd for:
C H F NO : C, 67.81; H, 3.71; N, 3.44. Found: C, 67.77; H, 3.58; N, 3.61%. HRMS (m/z)
2
3
15
2
4
-
[
M-H] calcd 406.0891. Found 406.0929.
7
-hydroxy-3-{4-[2-(3,4,5-trifluoro-phenyl)-acetylamino]-phenyl}-coumarin (5 g). Yield 67%;
-1
mp 280-282 ºC (with decomposition); vmax (KBr)/cm 3342, 3243, 1691, 1662, 1614, 1525,
1
1
413, 1322, 1282, 1201, 1161, 1130, 993, 842; H NMR δ (500 MHz, DMSO) 10.58 (1H, s,
H
OH), 10.31 (1H, s, NH), 8.12 (1H, s, CH), 7.68-7.60 (4H, m, Ar-H), 7.58 (1H, d, J 8.3, Ar-H),
.32-7.26 (2H, m, Ar-H), 6.80 (1H, dd, J 8.8, 2.0, Ar-H), 6.74 (1H, d, J 2.0, Ar-H), 4.08 (2H,
7
1
3
1
s, CH ); C NMR (50 MHz, DMSO), δ (ppm): 168.0, 161.0, 160.1, 154.7, 150.0 (d, J
2
C-F
2
1
6
52), 140.2, 138.8, 135.2 (m), 133.0 (m), 129.8 (m), 128.6, 121.6, 118.7, 114.2, 113.8, 113.3,
12.0, 101.7, 41.8. Anal. Calcd for: C H F NO : C, 64.94; H, 3.32; N, 3.29. Found: C,
2
3
14
3
4
-
4.81; H, 3.35; N, 3.35%. HRMS (m/z) [M-H] calcd 424.0797. Found 424.0868.

ACCEPTED MANUSCRIPT
7
2
1
1
-hydroxy-3-[4-(2-pentafluorophenyl-acetylamino)-phenyl]-coumarin (5h). Yield 73%; mp
-
1
91-293 ºC (with decomposition); vmax (KBr)/cm 3322, 3245, 1685, 1655, 1606, 1503, 1416,
1
327, 1291, 1194, 1159, 1132, 1001, 842; H NMR δ (500 MHz, DMSO) 10.59 (1H, s, OH),
H
0.50 (1H, s, NH), 8.13 (1H, s, CH), 7.70-7.60 (4H, m, Ar-H), 7.58 (1H, d, J 8.8, Ar-H), 6.80
1
3
(1H, dd, J 8.8, 2.4, Ar-H), 6.74 (1H, d, J 2.4, Ar-H), 3.92 (2H, s, CH ); C NMR (50 MHz,
2
1
1
DMSO), δ (ppm): 165.7, 161.0, 160.0, 154.7, 144.9 (d, J 244), 140.2, 138.6 (d, J 234),
C-F
C-F
1
1
2
2
38.5, 136.8 (d, J 249), 130.1, 129.8, 128.7, 121.5, 118.7, 113.3, 112.0, 109.9 (m), 101.6,
9.8. Anal. Calcd for: C H F NO : C, 59.88; H, 2.62; N, 3.04. Found: C, 60.01; H, 2.53; N,
.99%. HRMS (m/z) [M-H] calcd 460.0608. Found 460.0670.
C-F
23
12
5
4
-
3
-{4-[2-(4-fluoro-3-trifluoromethyl-phenyl)-acetylamino]-phenyl}-7-hydroxy-coumarin (5i).
-1
Yield 62%; mp 267-271 ºC (with decomposition); vmax (KBr)/cm 3384, 3262, 1697, 1669,
1
1
601, 1505, 1410, 1326, 1287, 1209, 1159, 1122, 990, 833; H NMR δ (500 MHz, DMSO)
H
1
0.58 (1H, s, OH), 10.35 (1H, s, NH), 8.12 (1H, s, CH), 7.75 (1H, d, J 6.8, Ar-H), 7.74-7.62
(
5H, m, Ar-H), 7.58 (1H, d, J 8.3, Ar-H), 7.52-7.40 (1H, m, Ar-H), 6.81 (1H, dd, J 8.3, 2.0,
13
Ar-H), 6.74 (1H, d, J 2.0, Ar-H), 3.80 (2H, s, CH ); C NMR (50 MHz, DMSO), δ (ppm):
2
1
1
1
5
3
68.5, 161.0, 160.1, 157.7 (d, J 252), 154.7, 140.1, 138.8, 136.1 (m), 132.9 (m), 129.8,
28.6, 127.9 (m), 122.7 (q, J 272), 121.6, 120.0, 118.6, 117.1, 116.7, 113.3, 112.0, 101.6,
C-F
1
C-F
0.0. Anal. Calcd for: C H F NO : C, 63.02; H, 3.31; N, 3.06. Found: C, 62.97; H, 3.24; N,
24
15
4
4
-
.11%. HRMS (m/z) [M-H] calcd 456.0859. Found 456.0872.
3
-{4-[2-(2,5-bis-trifluoromethyl-phenyl)-acetylamino]-phenyl}-7-hydroxy-coumarin
(5j).
-1
Yield 68%; mp 271-272 ºC (with decomposition); v (KBr)/cm 3308, 3262, 1669, 1611,
1
max
1
516, 1412, 1318, 1290, 1187, 1168, 1114, 997, 838; H NMR δ (500 MHz, DMSO) 10.58
H
(1H, s, OH), 10.41 (1H, s, NH), 8.12 (1H, s, CH), 7.98-7.94 (2H, m, Ar-H), 7.89 (1H, d, J 8.3,
Ar-H), 7.70-7.54 (5H, m, Ar-H), 6.80 (1H, dd, J 8.3, 2.4, Ar-H), 6.74 (1H, d, J 2.4, Ar-H),
1
3
4
.09 (2H, s, CH ); C NMR (50 MHz, DMSO), δ (ppm): 167.5, 161.0, 160.1, 154.7, 140.2,
2
1
38.8, 135.6, 132.5, 131.8, 131.6, 130.6 (m), 129.8, 128.7, 127.0 (m), 124.4 (m), 123.7 (q,
1
1
JC-F 274), 123.5 (q, JC-F 274), 121.6, 118.6, 113.3, 112.0, 101.7. Anal. Calcd for:
C H F NO : C, 59.18; H, 2.98; N, 2.76. Found: C, 59.26; H, 2.91; N, 2.81%. HRMS (m/z)
2
5
15
6
4
-
[
M-H] calcd 506.0827. Found 506.0851.
7
7
1
-hydroxy-3-{4-[2-(4-trifluoromethoxy-phenyl)-acetylamino]-phenyl}-coumarin (5k). Yield
-1
0%; mp 261-262 ºC; vmax (KBr)/cm 3352, 3260, 1696, 1661, 1612, 1504, 1412, 1322, 1280,
1
197, 1156, 1129, 991, 841; H NMR δ (500 MHz, DMSO) 10.58 (1H, s, OH), 10.33 (1H, s,
H
NH), 8.12 (1H, s, CH), 7.68-7.62 (4H, m, Ar-H), 7.58 (1H, d, J 8.8, Ar-H), 7.45 (2H, d, J 8.3,
Ar-H), 7.32 (2H, d, J 8.3, Ar-H), 6.80 (1H, dd, J 8.3, 2.0, Ar-H), 6.74 (1H, d, J 2.0, Ar-H),
1
3
3
1
1
6
.71 (2H, s, CH ); C NMR (50 MHz, DMSO), δ (ppm): 168.8, 161.0, 160.1, 154.7, 147.1,
2
1
40.1, 138.9, 135.4, 131.0, 129.8, 128.6, 121.6, 120.9, 120.1 (q, J 256), 118.6, 113.3,
12.0, 101.6, 44.9. Anal. Calcd for: C H F NO : C, 63.30; H, 3.54; N, 3.08. Found: C,
3.19; H, 3.45; N, 3.15%. HRMS (m/z) [M-H] calcd 454.0902. Found 454.0960.
C-F
2
4
16
3
5
-
4
.5. General method for the synthesis of 3-(4-benzoylamino-phenyl)-coumarin-7-O-
sulfamate and 3-(4-phenylacetylamino-phenyl)-coumarin-7-O-sulfamate derivatives (6a-k).
A mixture of formic acid (70.9 mg, 1.54 mmol) and N,N-dimethyl acetamide (1.4 mg, 0.016
mmol) was added to a stirred solution of chlorosulfonyl isocyanate (212.3 mg, 1.50 mmol) in
dry dichloromethane (0.5 mL) at 40 ºC over 3.5 h. Then, a stirred solution of 3-(4-
benzoylamino-phenyl)-7-hydroxy-coumarin and 7-hydroxy-3-(4-phenylacetylamino-phenyl)-
coumarin derivatives 5a-k (1.00 mmol) in N,N-dimethyl acetamide (3.4 mL) was added to the

ACCEPTED MANUSCRIPT
mixture. The mixture was stirred at ambient temperature overnight and then poured into water
(10 mL). Eventually, a white precipitate formed. The suspension was stirred at ambient
temperature for an additional two hours. The resulting precipitate was filtered, washed with
water and recrystallized from acetonitrile or acetone to give the desired products 6a-k.
3
-(4-benzoylamino-phenyl)-coumarin-7-O-sulfamate (6a). Yield 79%; mp 267-270 ºC (with
-1
decomposition); vmax (KBr)/cm 3332, 3294, 1701, 1668, 1602, 1513, 1412, 1331, 1244,
1
1
8
190, 1118, 979, 843; H NMR δ (500 MHz, DMSO) 10.42 (1H, s, NH), 8.31 (1H, s, CH),
.26 (2H, s, NH ), 7.97 (2H, d, J 6.3, Ar-H), 7.92-7.84 (3H, m, Ar-H), 7.80-7.74 (2H, m, Ar-
H
2
1
3
H), 7.66-7.52 (3H, m, Ar-H), 7.36 (1H, d, J 2.0, Ar-H), 7.29 (1H, dd, J 8.3, 2.0, Ar-H); C
NMR (125 MHz, DMSO), δ (ppm): 166.4, 160.2, 153.9, 152.6, 140.4, 139.7, 135.5, 132.4,
1
30.4, 130.2, 129.5, 129.1, 128.4, 126.6, 120.5, 119.4, 118.7, 110.2. Anal. Calcd for:
C H N O S: C, 60.54; H, 3.70; N, 6.42; S, 7.35. Found: C, 60.61; H, 3.74; N, 6.53; S,
2
2
16
2
6
-
7
.31%. HRMS (m/z) [M-H] calcd 435.0651. Found 435.0655.
3
-[4-(4-fluoro-benzoylamino)-phenyl]-coumarin-7-O-sulfamate (6b). Yield 85%; mp 255-258
-1
ºC; v (KBr)/cm 3399, 3345, 1701, 1660, 1610, 1499, 1412, 1327, 1253, 1181, 1112, 993,
max
1
8
8
34; H NMR δ (200 MHz, DMSO) 10.44 (1H, s, NH), 8.33 (1H, s, CH), 8.27 (2H, s, NH ),
.14-8.02 (2H, m, Ar-H), 7.96-7.72 (5H, m, Ar-H), 7.48-7.26 (4H, m, Ar-H); C NMR (50
H
2
1
3
1
MHz, DMSO), δ (ppm): 164.4, 164.1 (d, J 249), 161.0, 160.1, 159.4, 154.7, 153.2, 151.9,
C-F
1
1
3
40.2, 139.5, 139.0, 131.2, 130.4 (m), 129.6 (m), 128.6 (m), 125.8, 121.7, 119.8, 118.6,
17.9, 115.3 (m), 113.3, 112.0, 109.4, 101.7. Anal. Calcd for: C H FN O S: C, 58.15; H,
.33; N, 6.16; S, 7.06. Found: C, 58.11; H, 3.41; N, 6.29; S, 6.99%. HRMS (m/z) [M-H]
2
2
15
2
6
-
calcd 453.0557. Found 453.0608.
3
2
1
-[4-(3,4-difluoro-benzoylamino)-phenyl]-coumarin-7-O-sulfamate (6c). Yield 88%; mp 258-
-1
60 ºC (with decomposition); vmax 1(KBr)/cm 3416, 3342, 1699, 1651, 1607, 1506, 1411,
327, 1246, 1184, 1113, 991, 832; H NMR δ (200 MHz, DMSO) 10.62 (1H, s, NH), 8.33
H
(
1H, s, CH), 8.27 (2H, s, NH ), 8.20-8.00 (1H, m, Ar-H), 7.92-7.56 (7H, m, Ar-H), 7.40-7.26
2
1
3
(2H, m, Ar-H); C NMR (125 MHz, DMSO), δ (ppm): 164.0, 160.1, 154.0, 152.6, 152.2 (d,
1
1
JC-F 252), 149.8 (d, J 247), 141.1, 139.8 (m), 132.8 (m), 130.5 (m), 129.6, 129.2, 126.5,
C-F
1
26.0, 120.6, 119.4, 118.3 (m), 114.1, 112.8, 110.1, 102.4. Anal. Calcd for: C H F N O S:
22 14 2 2 6
C, 55.93; H, 2.99; N, 5.93; S, 6.79. Found: C, 56.01; H, 3.04; N, 5.81; S, 6.71%. HRMS (m/z)
-
[
M-H] calcd 471.0462. Found 471.0486.
3
2
1
-(4-pentafluorobenzoylamino-phenyl)-coumarin-7-O-sulfamate (6d). Yield 81%; mp 259-
-1
65 ºC (with decomposition); vmax 1(KBr)/cm 3257, 3194, 1711, 1666, 1601, 1501, 1417,
336, 1253, 1191, 1118, 994, 846; H NMR δ (500 MHz, DMSO) 11.16 (1H, s, NH), 8.31
H
(
1H, s, CH), 8.25 (2H, s, NH ), 7.87 (1H, d, J 8.3, Ar-H), 7.82-7.72 (4H, m, Ar-H), 7.36 (1H,
2
1
3
d, J 2.4, Ar-H), 7.30 (1H, dd, J 8.3, 2.4, Ar-H); C NMR (50 MHz, DMSO), δ (ppm): 161.2,
1
2
1
1
593, 154.8 (m), 153.3, 152.0, 143.1 (d, J 252), 140.6, 139.4, 138.2, 137.6, 136.9 (d, J
C-F C-F
47), 131.4, 130.7, 129.9 (m), 129.2, 128.9, 125.6, 121.4, 119.2, 118.6, 117.8, 113.4, 112.0
(
m), 109.4, 101.7. Anal. Calcd for: C H F N O S: C, 50.20; H, 2.11; N, 5.32; S, 6.09.
22 11 5 2 6
-
Found: C, 50.32; H, 2.05; N, 5.29; S, 6.00%. HRMS (m/z) [M-H] calcd 525.0180. Found
5
25.0177.
3
-(4-phenylacetylamino-phenyl)-coumarin-7-O-sulfamate (6e). Yield 85%; mp 208-210 ºC;
-1
vmax (KBr)/cm 3289, 3185, 1712, 1665, 1601, 1499, 1415, 1347, 1248, 1182, 1112, 991,
1
8
43; H NMR δ (500 MHz, DMSO) 10.30 (1H, s, NH), 8.26 (1H, s, CH), 8.24 (2H, s, NH ),
H
2
7
.84 (1H, d, J 8.8, Ar-H), 7.72-7.66 (4H, m, Ar-H), 7.36-7.22 (7H, m, Ar-H), 3.66 (2H, s,

ACCEPTED MANUSCRIPT
13
CH ); C NMR (125 MHz, DMSO), δ (ppm): 170.0, 160.1, 153.9, 152.6, 140.4, 139.6, 136.6,
2
1
30.4, 129.9, 129.8, 129.7, 129.6, 129.0, 127.3, 126.5, 119.4, 119.3, 118.7, 110.1, 44.0. Anal.
Calcd for: C H N O S: C, 61.32; H, 4.03; N, 6.22; S, 7.12. Found: C, 61.26; H, 3.95; N,
2
3
18
2
6
-
6
.11; S, 7.25%. HRMS (m/z) [M-H] calcd 449.0807. Found 449.0813.
3
8
1
-{4-[2-(3,4-difluoro-phenyl)-acetylamino]-phenyl}-coumarin-7-O-sulfamate (6f). Yield
-1
7%; mp 212-214 ºC; v (KBr)/cm 3313, 3192, 1714, 1595, 1515, 1414, 1357, 1247, 1185,
max
1
115, 992, 840; H NMR δ (500 MHz, DMSO) 10.35 (1H, s, NH), 8.27 (1H, s, CH), 8.24
H
(
2H, s, NH ), 7.85 (1H, d, J 8.8, Ar-H), 7.74-7.64 (4H, m, Ar-H), 7.44-7.36 (2H, m, Ar-H),
2
7
.35 (1H, d, J 2.0, Ar-H), 7.28 (1H, dd, J 8.3, 2.0, Ar-H), 7.20-7.16 (1H, m, Ar-H), 3.69 (2H,
1
3
s, CH ); C NMR (50 MHz, DMSO), δ (ppm): 168.7, 161.0, 159.4, 153.2, 151.8, 149.2 (d,
2
1
1
JC-F 244), 148.4 (d, J 244), 140.1, 139.5, 138.9, 133.4 (m), 129.8, 129.6 (m), 126.1 (m),
C-F
1
18.7 (m), 117.0, 116.5, 113.3, 109.4, 101.7, 42.0. Anal. Calcd for: C H F N O S: C, 56.79;
23 16 2 2 6
-
H, 3.32; N, 5.76; S, 6.59. Found: C, 56.91; H, 3.25; N, 5.79; S, 6.50%. HRMS (m/z) [M-H]
calcd 485.0619. Found 485.0621.
3
9
1
8
-{4-[2-(3,4,5-trifluoro-phenyl)-acetylamino]-phenyl}-coumarin-7-O-sulfamate (6 g). Yield
-1
0%; mp 216-219 ºC; v (KBr)/cm 3304, 3188, 1704, 1677, 1603, 1504, 1415, 1354, 1248,
max
1
185, 1115, 991, 841; H NMR δ (500 MHz, DMSO) 10.35 (1H, s, NH), 8.27 (1H, s, CH),
H
.24 (2H, s, NH ), 7.84 (1H, d, J 8.3, Ar-H), 7.74-7.64 (4H, m, Ar-H), 7.36-7.24 (4H, m, Ar-
2
1
3
H), 3.72 (2H, s, CH ); C NMR (50 MHz, DMSO), δ (ppm): 168.1, 161.0, 159.4, 153.2,
2
1
1
1
3
51.8, 149.7 (d, J 250), 139.4, 138.9, 135.3 (m), 132.9 (m), 129.7 (m), 128.9 (m), 125.7,
18.7, 117.9, 114.0 (m), 109.4, 101.7, 41.8. Anal. Calcd for: C H F N O S: C, 54.76; H,
.00; N, 5.55; S, 6.36. Found: C, 54.71; H, 3.11; N, 5.48; S, 6.40%. HRMS (m/z) [M-H]
C-F
23
15
3
2
6
-
calcd 503.0525. Found 503.0586.
3
-[4-(2-pentafluorophenyl-acetylamino)-phenyl]-coumarin-7-O-sulfamate (6h). Yield 82%;
-1
mp 248-250 ºC; vmax (KBr)/cm 3304, 3188, 1705, 1678, 1603, 1505, 1415, 1354, 1250,
1
1
8
186, 1115, 991, 842; H NMR δ (500 MHz, DMSO) 10.55 (1H, s, NH), 8.28 (1H, s, CH),
.24 (2H, s, NH ), 7.85 (1H, d, J 8.8, Ar-H), 7.73 (2H, d, J 8.8, Ar-H), 7.66 (2H, d, J 8.8, Ar-
H
2
1
3
H), 7.35 (1H, d, J 2.0, Ar-H), 7.29 (1H, dd, J 8.3, 2.4, Ar-H), 3.93 (2H, s, CH ); C NMR (50
MHz, DMSO), δ (ppm): 165.8, 161.0, 159.4, 153.2, 151.9, 145.0 (d, J 245), 139.0, 138.3
2
1
C-F
1
1
(d, J 245), 132.2 (d, J 245), 129.4 (m), 129.0 (m), 125.7, 118.7, 117.9, 113.3, 110.4
C-F C-F
(
m), 109.4, 101.6, 29.9. Anal. Calcd for: C H F N O S: C, 51.12; H, 2.42; N, 5.18; S, 5.93.
2
3
13
5
2
6
-
Found: C, 51.23; H, 2.45; N, 5.21; S, 5.88%. HRMS (m/z) [M-H] calcd 539.0336. Found
5
39.0396.
3
-{4-[2-(4-fluoro-3-trifluoromethyl-phenyl)-acetylamino]-phenyl}-coumarin-7-O-sulfamate
-1
(6i). Yield 83%; mp 187-190 ºC; v (KBr)/cm 3299, 3188, 1711, 1667, 1611, 1508, 1413,
max
1
1
356, 1244, 1187, 1115, 991, 842; H NMR δ (500 MHz, DMSO) 10.39 (1H, s, NH), 8.26
H
(
(
(
1H, s, CH), 8.24 (2H, s, NH ), 7.84 (1H, d, J 8.8, Ar-H), 7.78-7.62 (6H, m, Ar-H), 7.52-7.44
2
13
1H, m, Ar-H), 7.34 (1H, s, Ar-H), 7.28 (1H, d, J 9.3, Ar-H), 3.80 (2H, s, CH ); C NMR
2
1
125 MHz, DMSO), δ (ppm): 169.4, 160.1, 156.2 (d, JC-F 245), 153.9, 152.6, 140.9, 140.2,
39.7, 136.9 (m), 133.6 (m), 130.4 (m), 129.7 (m), 128.7 (m), 126.5, 123.5 (q, J 274),
1
1
C-F
1
19.4, 118.0, 117.6 (m), 114.1, 110.1, 102.4, 42.3. Anal. Calcd for: C H F N O S: C, 53.73;
2
4
16
4
2
6
-
H, 3.01; N, 5.22; S, 5.98. Found: C, 53.69; H, 2.90; N, 5.14; S, 6.09%. HRMS (m/z) [M-H]
calcd 535.0587. Found 535.0593.
3
-{4-[2-(2,5-bis-trifluoromethyl-phenyl)-acetylamino]-phenyl}-coumarin-7-O-sulfamate (6j).
-1
Yield 89%; mp 208-212 ºC; vmax (KBr)/cm 3305, 3211, 1710, 1671, 1598, 1517, 1414, 1342,

ACCEPTED MANUSCRIPT
1
1
249, 1185, 1115, 991, 843; H NMR δ (500 MHz, DMSO) 10.46 (1H, s, NH), 8.27 (1H, s,
H
CH), 8.24 (2H, s, NH ), 8.00-7.94 (2H, m, Ar-H), 7.89 (1H, d, J 8.3, Ar-H), 7.85 (1H, d, J
2
8
.8, Ar-H), 7.71 (2H, d, J 8.8, Ar-H), 7.66 (2H, d, J 8.8, Ar-H), 7.35 (1H, d, J 2.0, Ar-H), 7.29
1
3
(1H, d, J 8.3, Ar-H), 4.11 (2H, s, CH ); C NMR (50 MHz, DMSO), δ (ppm): 167.6, 161.0,
2
1
1
1
59.4, 153.2, 151.8, 140.2, 139.4, 138.9, 135.5, 130.5 (m), 129.6 (m), 129.0 (m), 127.0 (m),
1
1
25.7, 124.4 (m), 123.7 (q, J 274), 123.5 (q, J 274), 118.6 (m), 117.9, 113.3, 109.4,
C-F
C-F
01.7. Anal. Calcd for: C H F N O S: C, 51.20; H, 2.75; N, 4.78; S, 5.47. Found: C, 51.09;
25
16
6
2
6
-
H, 2.68; N, 4.88; S, 5.52%. HRMS (m/z) [M-H] calcd 585.0555. Found 585.0555.
3
-{4-[2-(4-trifluoromethoxy-phenyl)-acetylamino]-phenyl}-coumarin-7-O-sulfamate
(6k).
-1
Yield 88%; mp 252-254 ºC; v (KBr)/cm 3304, 3217, 1711, 1670, 1611, 1509, 1413, 1354,
max
1
1
248, 1182, 1111, 991, 842; H NMR δ (500 MHz, DMSO) 10.38 (1H, s, NH), 8.26 (1H, s,
H
CH), 8.24 (2H, s, NH ), 7.84 (1H, d, J 8.8, Ar-H), 7.74-7.64 (4H, m, Ar-H), 7.45 (2H, d, J
2
1
3
8
.3, Ar-H), 7.36-7.30 (3H, m, Ar-H), 7.28 (1H, dd, J 8.3, 2.0, Ar-H), 3.72 (2H, s, CH ); C
2
NMR (50 MHz, DMSO), δ (ppm): 168.9, 161.0, 154.7, 153.2, 151.8, 147.1, 140.1, 139.5,
1
1
1
38.9, 135.3, 131.0, 129.6 (m), 129.1 (m), 125.8, 121.6, 120.9, 120.1 (q, J 255), 118.6,
C-F
17.5, 113.3, 112.0, 109.4, 101.6, 42.3. Anal. Calcd for: C H F N O S: C, 53.93; H, 3.21;
N, 5.24; S, 6.00. Found: C, 60.02; H, 3.16; N, 5.15; S, 6.11%. HRMS (m/z) [M-H] calcd
33.0630. Found 533.0681.
2
4
17
3
2
7
-
5
4
.6. Preparation of 3-(4-nitrophenyl)-coumarin-7-O-sulfamate (7).
A mixture of formic acid (70.9 mg, 1.54 mmol) and N,N-dimethyl acetamide (1.4 mg, 0.016
mmol) was added to a stirred solution of chlorosulfonyl isocyanate (212.3 mg, 1.50 mmol) in
dry dichloromethane (0.5 mL) at 40 ºC over 3.5 h. A stirred solution of 7-hydroxy-3-(4-
nitrophenyl)-coumarin 3 (283.2 mg, 1.00 mmol) in N,N-dimethyl acetamide (3.4 mL) was
added to the mixture. The mixture was stirred at ambient temperature overnight and then
poured into water (10 mL). Eventually, a white precipitate formed. The suspension was stirred
at ambient temperature for an additional two hours. The resulting precipitate was filtered,
washed with water and recrystallized from acetonitrile or acetone to give the desired product
7
.
-
1
Yield 91%; mp 200-203 ºC; v (KBr)/cm 3387, 3217, 1697, 1595, 1507, 1341, 1247, 1191,
max
1
1
111, 988, 853; H NMR δ (500 MHz, DMSO) 8.5 (1H, s, CH), 8.33 (2H, d, J 9.3, Ar-H),
H
8
.30 (2H, s, NH ), 8.02 (2H, d, J 8.8, Ar-H), 7.90 (1H, d, J 8.3, Ar-H), 7.40 (1H, d, J 2.0, Ar-
2
1
3
H), 7.33 (1H, d, J 8.8, 2.0, Ar-H); C NMR (125 MHz, DMSO), δ (ppm): 159.7, 154.5,
53.4, 147.9, 142.7, 141.8, 131.1, 130.5, 125.1, 124.1, 119.5, 118.2, 110.2. Anal. Calcd for:
C H N O S: C, 49.72; H, 2.78; N, 7.73; S, 8.85. Found: C, 49.65; H, 2.69; N, 7.80; S,
1
15
10
2
7
+
8
.98%. HRMS (m/z) [M+H] calcd 363.0287. Found 363.0277.
4
.7. Molecular modeling
Prior to docking procedures, the potential inhibitors were constructed using Portable
HyperChem 8.0.7 Release (Hypercube, Inc., Gainesville, FL, USA). Each ligand was
optimized using a MM + force field and the Polak–Ribiere conjugate gradient algorithm
-1
-1
(
terminating at a gradient of 0.05 kcal mol Å ). For docking the X-ray structure of human
STS (obtained from Protein Data Bank – accession code 1P49) was prepared for docking
using the standard procedure. First, the water molecules from crystallization were removed
from the structure and the catalytic amino acid FGly75 were converted to the gem-diol form
using the Protein Preparation Wizard module, delivered with Maestro (Schrödinger, LLC,

ACCEPTED MANUSCRIPT
New York, NY, USA). Next, hydrogen atoms were built onto the structure and prepared
model of enzyme will be optimized using the OPLS-AA force field. Docking of the optimized
ligands to the prepared structure of human STS was carried out with Autodock Vina 1.1.2
software (The Molecular Graphic Laboratory, The Scripps Research Institute, La Jolla, CA,
USA) [25]. For all of the docking studies, a grid box size of 30 Å x 30 Å x 30 Å, centered on
the Cβ atom of amino acid 75, was used. The best poses for a particular ligand were inspected
visually. Illustrations of the 3D model were generated using VMD 1.9 (University of Illinois
at Urbana – Champaign, Urbana, IL, USA).
4
4
.8. Biological assays
.8.1. Enzyme purification
STS was extracted from human placenta and purified to homogeneity following a multi-step
chromatography protocol as previously described [26].
4
.8.2. In vitro activity assay
The reaction mixture, at a final volume of 100 µL, contained 20 mM Tris–HCl pH 7.4, 3 mM
p-nitrophenyl sulfate (NPS), varied concentrations of an inhibitor (0.01–100 µM) and 5 U of
purified enzyme (1 U is the amount of enzyme that hydrolyzes 100 µM of NPS in 1 h at 37
˚C). The reaction was performed at 37 ˚C for 15 min and halted by the addition of 100 mL of
1
M NaOH. The absorbance of the released p-nitrophenol was measured at 405 nm using a
Biotek ELx800 Microplate Reader (BioTek Instruments, Inc. Winooski, VT, USA). IC50
values were calculated using GraphPad Prism software (GraphPad Software, Inc., La Jolla,
CA, USA). All measurements were performed in triplicate.
4
.8.3. Cell culture and viability assay
TM
MCF7 (ATCC® HTB-22™), T47D (ATCC ® HTB-133 ), MDA-MB-231 (ATCC® HTB-
6™) and SkBr3 (ATCC® HTB-30™) cells lines have been kindly provided by prof.
2
Wesierska-Gadek (Medical University of Vienna, Vienna, Austria) in 2016. Cells were
cultured in phenol red-free DMEM high glucose media supplemented with 10% FBS and
antibiotics: penicillin (62.6 µg/mL) and streptomycin (40 µg/mL). All cells were maintained
at 37 ˚C in a humidified atmosphere of 10% CO and 90% air and routinely screened for
2
Mycoplasma contamination. To determine cytotoxicity, exponentially grown cells were
exposed to the indicated concentrations of the studied compounds for 120 hours, and viability
was determined using the MTT assay. Dose–response curves were plotted using GraphPad
Prism software (GraphPad Software, Inc., La Jolla, CA, USA) and represent data from two to
three independent experiments performed in quadruplicate.
Acknowledgements
We gratefully acknowledge the National Science Centre (Poland) for financial support (grant
no. 2015/19/N/NZ7/00938).
References

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[
1] Ireson, C. R.; Chander, S. K.; Purohit, A.; Parish, D. C.; Woo, L. W. L.; Potter, B. V. L.;
Reed, M. J. Pharmacokinetics of the nonsteroidal steroid sulphatase inhibitor 667 COUMATE
and its sequestration into red blood cells in rats. Br. J. Cancer 2004, 91, 1399-1404.
[
[
2] Labrie, F. Intracrinology. Mol. Cell. Endocrinol. 1991, 78, 113-118.
3] Kozak, W.; Daśko, M.; Masłyk, M.; Pieczykolan, J. S.; Gielniewski, B.; Rachon J.;
Demkowicz S. Phosphate tricyclic coumarin analogs as steroid sulfatase inhibitors: synthesis
and biological activity. RSC Adv. 2014, 4, 44350-44358.
[
4] Demkowicz, S.; Kozak, W.; Daśko, M.; Masłyk, M.; Kubiński, K.; Rachon, J. Phosphate
and thiophosphate biphenyl analogs as steroid sulfatase inhibitors. Drug Dev. Res. 2015, 76,
4-104.
5] Demkowicz, S.; Kozak, W.; Daśko, M.; Masłyk, M.; Gielniewski, B.; Rachon, J.
9
[
Synthesis of bicoumarin thiophosphate derivatives as steroid sulfatase inhibitors. Eur. J. Med.
Chem. 2015, 101, 358-366.
[
6] Demkowicz, S.; Kozak, W.; Daśko, M.; Wołos, A.; Masłyk, M.; Kubiński, K.;
Składanowski, A.; Misiak, M.; Rachon, J. Synthesis and steroid sulfatase inhibitory activities
of N-alkanoyl tyramine phosphates and thiophosphates. RSC Adv. 2015, 5, 32594-32603.
[
7] Kozak, W.; Daśko, M.; Masłyk, M.; Gielniewski, B.; Rachon, J.; Demkowicz, S.
Synthesis and biological evaluation of thiophosphate tricyclic coumarin derivatives as steroid
sulfatase inhibitors. J. Asian Nat. Prod. Res. 2015, 17, 1091-1096.
[
8] Kozak, W.; Daśko, M.; Masłyk, M.; Kubiński, K.; Rachon, J.; Demkowicz, S. Steroid
sulfatase inhibitors based on phosphate and thiophosphate flavone analogs. Drug Dev. Res.
015, 76, 450-462.
9] Purohit, A.; Williams, G. J.; Howarth, N. M.; Potter, B. V. L.; Reed, M. J. Inactivation of
steroid sulfatase by an active site-directed inhibitor, estrone-3-O-sulfamate. Biochemistry
995, 34, 11508-11514.
10] Thomas, M. P.; Potter, B. V. L. Discovery and development of the aryl O-sulfamate
pharmacophore for oncology and women’s health. J. Med. Chem. 2015, 19, 7634-7658.
11] Reed, M. J.; Purohit, A.; Woo, L. W. L.; Newman, S. P.; Potter, B. V. L. Steroid
sulfatase: Molecular biology, regulation, and inhibition. Endocr. Rev. 2005, 26, 171-202.
12] Purohit, A.; Reed, M. J.; Morris, N. C.; Williams, G. J.; Potter, B. V. L. Regulation and
inhibition of steroid sulfatase activity in breast cancer. Ann. N. Y. Acad. Sci. 1996, 784, 40-49.
13] Woo, L. W. L.; Howarth, N. M.; Purohit, A.; Hatem, A. M.; Reed, M. J.; Potter, B. V. L.
Steroidal and nonsteroidal sulfamates as potent inhibitors of steroid sulfatase. J. Med. Chem.
998, 41, 1068-1083.
14] Bégué, J. P.; Bonnet-Delpon, D. Recent advances (1995-2005) in fluorinated
pharmaceuticals based on natural products. J. Fluorine Chem. 2006, 127, 992-1012.
15] Isanbor, C.; O’Hagan, D. Fluorine in medicinal chemistry: A review of anti-cancer
agents. J. Fluorine Chem. 2006, 127, 303-319.
16] Kirk, K.L. Fluorine in medicinal chemistry: Recent therapeutic applications of
fluorinated small molecules. J. Fluorine Chem. 2006, 127, 1013-1029.
17] Demkowicz, S.; Daśko, M.; Kozak, W.; Krawczyk, K.; Witt, D.; Masłyk, M.; Kubiński,
2
[
1
[
[
[
[
1
[
[
[
[
K.; Rachon, J. Synthesis and biological evaluation of fluorinated 3-phenylcoumarin-7-O-
sulfamate derivatives as steroid sulfatase inhibitors. Chem. Biol. Drug Des. 2016, 87, 233-
2
38.
[
18] Maheswara, M.; Siddaiah, V.; Damu, G. L. V.; Rao, Y. K.; Rao, C. V. A solvent-free
synthesis of coumarins via Pechmann condensation using heterogeneous catalyst. J. Mol.
Catal. A: Chem. 2006, 255, 49-52.
[
19] Perkin, W. H. VI. - On the artificial production of coumarin and formation of its
homologues. J. Chem. Soc. 1868, 21, 53-63.

ACCEPTED MANUSCRIPT
[
20] Valizadeh, H.; Gholipour, H. Imidazolium-based phosphinite ionic liquid (IL-OPPh ) as
2
reusable catalyst and solvent for the Knoevenagel condensation reaction. Synth. Commun.
010, 40, 1477-1485.
21] Dittmer, D. C.; Li, Q.; Avilov, D. V. Synthesis of coumarins, 4-hydroxycoumarins, and
2
[
4
4
-hydroxyquinolinones by Tellurium-Triggered cyclizations. J. Org. Chem. 2005, 70, 4682-
686.
[
22] Upadhyay, P. K.; Kumar, P. A novel synthesis of coumarins employing triphenyl(α-
carboxymethylene)phosphorane imidazolide as a C-2 synthon. Tetrahedron Lett. 2009, 50,
36-238.
23] Vaccaro, A. M.; Salvioli, R.; Muscillo, M.; Renola, L. Purification and properties of
arylsulfatase C from human placenta. Enzyme 1987, 37, 115-126.
24] Woo, L. W. L.; Jackson, T.; Putey, A.; Cozier, G.; Leonard, P.; Acharya, K. R.; Chander,
2
[
[
S. K.; Purohit, A.; Reed, M. J.; Potter, B. V. L. Highly potent first examples of dual
aromatase-steroid sulfatase inhibitors based on a biphenyl template. J. Med. Chem. 2010, 53,
2
[
155-2170.
25] Trott, O.; Olson, A. J. AutoDock Vina: Improving the speed and accuracy of docking
with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem.
010, 31, 455-461.
26] Hernandez-Guzman, F. G.; Higashiyama, T.; Osawa, Y. Ghosh, D.; Purification,
2
[
characterization and crystallization of human placental estrone/dehydroepiandrosterone
sulfatase, a membrane-bound enzyme of the endoplasmic reticulum. J. Steroid Biochem. Mol.
Biol. 2001, 78, 441-450.

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Highlights:
1
2
3
4
5
.
.
.
.
.
A series of 3-(4-aminophenyl)-coumarin-7-O-sulfamate derivatives N-acylated with
fluorinated analogs of benzoic or phenylacetic acid have been synthesized.
Computational docking methods were used to determine the binding modes of the
synthesized inhibitors.
Compounds 6c and 6j demonstrated the highest inhibitory effect in enzymatic STS
assays, both with IC values of 0.18 µM.
5
0
Compound 6j exhibited the highest potency against the MCF-7 and T47D cell lines
15.9 µM and 8.7 µM, respectively).
(
Analogues 1 and 2 proved to selectively inhibit the growth of ER- and PR-positive cell
lines.