Synthesis and Biological Evaluation of Fluorinated 3-Phenylcoumarin-7-O-Sulfamate Derivatives as Steroid Sulfatase Inhibitors

Article abstract of DOI:10.1111/cbdd.12652

In the present work, we report the initial results of our study on a series of 3-phenylcoumarin sulfamate-based compounds containing C-F bonds as novel inhibitors of steroid sulfatase. The new compounds are potent steroid sulfatase inhibitors, possessing more than 10 times higher inhibitory potency than coumarin-7-O-sulfamate. In the course of our investigation, compounds 2b and 2c demonstrated the highest inhibitory effect on the enzymatic steroid sulfatase assay; both had IC₅₀ values of 0.27 μm (the IC₅₀ value of coumarin-7-O-sulfamate is 3.5 μm, used as a reference).

Full text of DOI:10.1111/cbdd.12652

Chem Biol Drug Des 2016; 87: 233–238
Research Letter
Synthesis and Biological Evaluation of Fluorinated
3-Phenylcoumarin-7-O-Sulfamate Derivatives as
Steroid Sulfatase Inhibitors
1
,
1
Sebastian Demkowicz *, Mateusz Da sꢀ ko , Witold
In recent years, there has been intensive research toward
finding novel steroidal and non-steroidal inhibitors of STS
containing different functional groups, for example, phos-
phate, thiophosphate, or sulfamate moieties (3–8). The
most promising compound was estrone-3-O-sulfamate
1
1
1
Kozak , Katarzyna Krawczyk , Dariusz Witt ,
Maciej Masłyk , Konrad Kubi nꢀ ski and Janusz
2
2
1
Rachon
1
(
EMATE) (Figure 1)—a time- and concentration-dependent
Department of Organic Chemistry, Chemical Faculty,
Gdansk University of Technology, Narutowicza 11/12,
0-233 Gdansk, Poland
irreversible steroidal inhibitor, which exhibited very high
activity in MCF-7 cells, with an IC50 value of 65 pM (9).
Unfortunately, EMATE is not used for the treatment of
breast cancer therapy due to its estrogenic properties. An
important class of compounds that exhibits high activity
against STS includes coumarin derivatives. 4-methyl-
coumarin-7-O-sulfamate (COUMATE) (IC50 value of 380 nM
when evaluated against placental microsomes) and its tri-
cyclic derivatives, for example, 667-COUMATE (IC50 value
of 8 nM), have been shown to lack significant estrogenicity.
Coumarin analogs are classified as irreversible inhibitors
whose effects are time and concentration dependent
(10,11).
8
2
Department of Molecular Biology, Faculty of
Biotechnology and Environment Sciences, The John Paul
II Catholic University of Lublin, Konstantyn oꢀ w 1i, 20-708
Lublin, Poland
*Corresponding author: Sebastian Demkowicz,
sebdemko@pg.gda.pl
In the present work, we report the initial results of
our study on a series of 3-phenylcoumarin sulfamate-
based compounds containing C-F bonds as novel
inhibitors of steroid sulfatase. The new compounds
are potent steroid sulfatase inhibitors, possessing
more than 10 times higher inhibitory potency than
coumarin-7-O-sulfamate. In the course of our investi-
gation, compounds 2b and 2c demonstrated the high-
est inhibitory effect on the enzymatic steroid sulfatase
assay; both had IC50 values of 0.27 lM (the IC50 value
of coumarin-7-O-sulfamate is 3.5 lM, used as a refer-
ence).
With respect to the binding mode, our docking experi-
ments revealed that sulfamated 3-phenylcoumarin analogs
containing C-F bonds could, at least theoretically, bind
STS. The new STS inhibitor candidates dock in a similar
manner to the mode of the reported STS inhibitor (cou-
marin-7-O-sulfamate) (Figure 2). Sulfamate functional
groups are directed to catalytic amino acid FGly75 co-
Key words: breast cancer, coumarin, steroid sulfatase, ster-
oid sulfatase inhibitors, sulfamates
2+
ordinated to Ca , and they are in the proximity of the pro-
posed catalytic residues of Asp35, Asp36, Arg79, Lys134,
His136, His290, Asp342, and Lys368. Furthermore, the
fluorine-substituted phenyl rings of coumarin scaffolds are
in close proximity to several lipophilic amino acids (Leu74,
Arg98, Leu103, Leu167, Val177, Phe178, Thr484, His485,
Val486, and Phe488), which are implicated in substrate
recognition. Molecular modeling studies suggest that
increasing the hydrophobic properties of coumarin frame-
works (by introducing the substituted phenyl rings) could
favor binding by the establishment of hydrophobic interac-
tions, wherein the cavity is delimited by lipophilic amino
acids in the enzyme pocket. All candidates expressed sim-
ilar free docking energies (predicted free docking energies
in the range of À6.2 to À6.9 kcal/mol). In contrast, the
compounds lacking the hydrophobic substituents in the
Received 12 May 2015, revised 20 July 2015 and accepted
for publication 11 August 2015
Hormone-dependent breast cancer (HDBC) is a major
cause of mortality, and there is a pressing need to
develop novel treatment methods. One approach for
treating HDBC involves the use of inhibitors for enzymes
that are responsible for the biosynthesis of estrogens in
peripheral tissues, for example, steroid sulfatase (STS)
(1). STS is responsible for the hydrolysis of steroid sul-
fates to their active forms; therefore, it has a crucial role
in regulating the formation of biologically active steroids.
STS is widely distributed throughout the body, and its
action is involved in physiological and pathological condi-
tions (2).
3
rd position of the coumarin scaffolds exhibited a poor
ability to bind STS, resulting in an increase in the predicted
ª 2015 John Wiley & Sons A/S. doi: 10.1111/cbdd.12652
233

Demkowicz et al.
sized scaffolds was performed. Solutions of stable 7-hy-
droxy-3-phenylcoumarin derivatives in N,N-
dimethylacetamide (DMA) were treated with H NSO Cl
previously generated by the reaction of chlorosulfonyl iso-
2
2
(
cyanate and formic acid in the presence of a catalytic
amount of N,N-dimethylacetamide). The yields of these
reactions were high and reached 90%.
The inhibitory effects of the synthesized compounds 2a-g
were tested using the in vitro STS assay according to
reported methods (13,14). STS enzyme was extracted
from human placenta and purified to homogeneity follow-
ing a multistep chromatography protocol, as previously
described (15). The results of the biochemical evaluation
are presented in Table 1.
Figure 1: Chemical structures of steroid sulfatase inhibitors.
As observed in the data collected in the Table 1, com-
pounds 2b and 2c exhibited the highest inhibitory activi-
ties for STS (IC50 values of 0.27 lM in both cases). Most
of the synthesized inhibitors possessed significantly
greater inhibitory potency than reported for coumarin-7-
O-sulfamate (IC50 value of 3.50 lM). Only compound 2e
was less active. These results indicate that increasing
the hydrophobic properties of coumarin scaffolds by
introducing a substituted phenyl ring in the 3rd position
favors binding through the creation of hydrophobic inter-
actions, wherein the cavity is delimited by lipophilic
amino acids in the STS enzyme pocket. Furthermore, in
most cases, the compounds containing C-F bonds had
slightly higher STS inhibition than inhibitor 2a, which was
probably due to the stronger hydrophobic effect of the
aromatic substituent.
Figure 2: Docked binding modes and distances to Thr484 and
Arg98 for compounds 2a (yellow), 2c (blue), 2d (red), and
coumarin-7-O-sulfamate (green).
free binding energies (e.g., the value of the AutoDock Vina
score for the reference compound—coumarin-7-O-sulfa-
mate was—2.9 kcal/mol). Although the distances between
the fluorine atoms of the newly designed STS inhibitors
and amino acid residues (Thr484 and Arg98) suggest the
possibility for creating several weak hydrogen bonds,
recent research indicates that covalently bound fluorine (in
contrast to anionic fluoride) hardly ever acts as a hydro-
gen-bond acceptor (12). Therefore, we suggest that only
hydrophobic interactions between the fluorine-substituted
phenyl rings and lipophilic amino acids located in the STS
cavity may influence the enzyme–inhibitor complex stability
and they have a significant impact on the STS inhibitory
potency.
Experimental
Molecular modeling
All the molecular structures of the ligands were built with
the program Portable HyperChem 8.0.7 Release (Hyper-
cube, Inc., Gainesville, FL, USA) and were energy-mini-
mized using the MM+ force field and Polak–Ribiere
conjugate gradient algorithm. The iteration procedure was
continued until energy gradients became less than
ꢁ
0.05 kcal/mol/A. The X-ray structure of human steroid sul-
fatase used for molecular modeling study was taken from
the Protein Databank (Protein Data Bank accession code
1P49). After standard preparation procedures (including
the removal of water molecules, conversion of catalytic
amino acid FGly75 to the gem–diol form, addition of
hydrogen atoms, and optimization of prepared structure of
human STS), docking analysis was carried out. A docking
study was carried out using AUTODOCK VINA 1.1.2 software
(The Molecular Graphic Laboratory, The Scripps Research
Institute, La Jolla, CA, USA). For all the docking studies, a
All newly designed STS inhibitors were obtained using
the pathway shown in Scheme 1. In the synthesis of
7
-hydroxy-3-phenylcoumarin
derivatives,
phenylacetyl
chlorides (obtained in situ from the corresponding pheny-
lacetic acids by the reaction with thionyl chloride) were
refluxed with commercially available 2,4-dihydroxyben-
zaldehyde in the presence of potassium carbonate. The
reactions proceeded well and had good yields (62–80%).
Next, sulfamoylation of the hydroxyl group of the synthe-
ꢁ
ꢁ
ꢁ
grid box size of 25 A 9 25 A 9 25 A, centered on Cb
atom of FGly75, was used. Graphic visualizations of the
3D model were generated using VMD 1.9 (University of Illi-
nois at Urbana — Champaign, Urbana, IL, USA).
2
34
Chem Biol Drug Des 2016; 87: 233–238

1 2 3 4 5 3 3
Scheme 1: Synthesis of sulfamate 3-phenylcoumarin derivatives 2a-g (R , R , R , R , and R = H, F, CF , and OCF ).
Table 1: Activities of the synthesized compounds 2a-g and a
reference inhibitor (coumarin-7-O-sulfamate) in the STS enzyme
assays
Chemical shifts are reported in ppm relative to the resi-
1
dua solvent peak (DMSO-d
6
2.49 ppm for H, acetone-
13
d
6
29.92 ppm for C). Coupling constants are given in
Hertz. IR spectra were measured on Nicolet 8700. Ele-
mental analysis was performed using CHNS-Carlo Erba
EA-1108. Mass spectra were recorded on Agilent 6540
accurate mass Q-TOF LC/MS systems. Column chro-
matography was performed using silica gel 60 (230–400
mesch, Merck). Preparative thin-layer chromatography
was performed with Polygram SIL G/UV254 silica gel
Compound
R
1
R
2
R
3
R
4
R
5
IC50 [lM]
2
2
2
2
2
2
2
a
b
c
d
e
f
H
H
H
F
CF
H
H
–
H
F
F
H
F
F
F
H
F
H
H
F
H
H
H
F
H
H
H
–
0.36 Æ 0.022
0.27 Æ 0.020
0.27 Æ 0.021
0.28 Æ 0.019
4.18 Æ 0.24
0.30 Æ 0.019
0.44 Æ 0.020
3.50 Æ 0.320
F
F
3
H
CF
H
–
CF
H
H
–
3
3
(
Macherey- Nagel GmbH & Co. KG, D u€ ren, Germany).
g
OCF
–
3
Coumarin-7-O-
sulfamate
General method for the synthesis of 7-hydroxy-3-
phenylcoumarin derivatives 1a-g
Materials and methods
2
SOCl (1.65 mL) was added to a solution of pheny-
Phenylacetic acid, 3,4-difluorophenylacetic acid, 3,4,5-tri-
fluorophenylacetic acid, 2,3,4,5,6-pentafluorophenylacetic
acid, 2,5-Bis(trifluoromethyl)phenylacetic acid, [4-fluoro-3-
lacetic acid derivatives (2.0 mmol) in dry CH Cl2
2
(1.65 mL), and the suspension was refluxed for 2.5 h.
The resulting solution was evaporated, the residue was
dissolved in dry acetone (10 mL), and 2,4-dihydroxyben-
zaldehyde (2.0 mmol) was added. The mixture was
(
trifluoromethyl)phenyl]acetic
phenylacetic acid, thionyl chloride, potassium carbonate,
,4-dihydroxybenzaldehyde, chlorosulfonyl isocyanate, N,
acid,
4-(trifluoromethoxy)
2
2 3
refluxed with anhydrous K CO (1.104 g, 8.0 mmol) for
N-dimethylacetamide, and formic acid are commercially
available from Aldrich. Dichloromethane and acetone
were dried and distilled using standard procedures. Melt-
ing points (uncorrected) were determined with a Stuart
Scientific SMP30 apparatus. NMR spectra were
recorded on a Varian Gemini 500 MHz spectrometer.
4 h. Then, acetone was removed under reduced pres-
sure and cold water (20 mL) was added. The mixture
was extracted with EtOAc to give the crude solid. The
crude solid was crystallized from methanol, resulting in
the corresponding 7-hydroxy-3-phenylcoumarin deriva-
tives 1a-g.
Chem Biol Drug Des 2016; 87: 233–238
235

Demkowicz et al.
7
-hydroxy-3-phenylcoumarin 1a
NMR (500 MHz, DMSO), d (ppm): 10.75 (s, 1H, OH),
8.11–8.00 (m, 4H), 7.58 (d, 1H, J = 8.30 Hz), 6.84 (dd,
1H, J = 8.55 Hz, J = 2.20 Hz), 6.80 (d, 1H, J = 1.95 Hz);
À1
Yield 80%; mp: 210–212 °C; vmax (KBr)/cm 3203, 1671,
1
1
587, 1519, 1445, 1131, 858, 736, 632; H NMR
13
(500 MHz, DMSO), d (ppm): 10.20–11.00 (brs, 1H, OH),
C NMR (200 MHz, DMSO), d (ppm): 162.2, 160.2,
155.8, 143.6, 135.9, 133.9–131.3 (m), 130.6, 130.0 (m),
13
8
.16 (s, 1H), 7.30–7.80 (m, 6H), 6.70–6.90 (m, 2H);
C
1
NMR (500 MHz, acetone-d ), d (ppm): 161.2, 160.2,
128.1 (m), 126.4, 123.7 (q, J
= 274 Hz), 120.4, 114.0,
6
C-F
1
1
55.7, 140.7, 135.8, 130.1, 128.6, 128.4, 128.3, 123.7,
13.4, 113.0, 102.2; Anal. Calcd for: C15 : C, 75.62;
8 6 3
111.4, 102.5; Anal. Calcd for: C17H F O : C, 54.56; H,
+
H
10
O
3
2.15; found: C, 54.41; H, 2.04; HRMS (m/z) [M+H] calcd
375.0456, found 375.0536.
+
H, 4.23; found: C, 75.67; H, 4.19; HRMS (m/z) [M+H]
calcd 318.0436, found 318.0469.
7
-hydroxy-3-[4-fluoro-3-(trifluoromethyl)
phenyl]coumarin 1f
Yield 65%; mp: 241–244 °C; vmax (KBr)/cm 3204, 1674,
7
-hydroxy-3-(3,4-difluorophenyl)coumarin 1b
À1
À1
Yield 73%; mp: 288–289 °C; vmax (KBr)/cm 3255, 1692,
1
1
1
610, 1573, 1520, 1423, 1132, 1101, 862, 738, 629;
NMR (500 MHz, DMSO), d (ppm): 10.70 (s, 1H, OH), 8.24
s, 1H), 7.82–7.76 (m, 1H), 7.62–7.46 (m, 3H), 6.83 (dd,
H
1589, 1566, 1506, 1431, 1122, 1112, 846, 741, 630; H
NMR (500 MHz, DMSO), d (ppm): 10.71 (s, 1H, OH), 8.31
(s, 1H), 8.12–8.04 (m, 2H), 7.63–7.56 (m, 2H), 6.86–6.82
(
1
3
1
H, J = 8.55 Hz, J = 2.20 Hz), 6.75 (d, 1H, J = 1.95 Hz);
C NMR (200 MHz, DMSO), d (ppm): 162.0, 160.2,
(m, 1H), 6.76 (d, 1H, J = 1.47 Hz); C NMR (200 MHz,
1
3
1
DMSO), d (ppm): 162.1, 160.4, 158.7 (d, JC-F = 211 Hz),
155.5, 142.6, 135.4 (m), 132.5 (m), 130.7, 127.3 (m),
1
1
1
2
1
55.4, 149.6 (d, JC-F = 250 Hz), 148.8 (d, JC-F
50 Hz), 142.2, 132.9 (m), 130.6, 125.6 (m), 120.2, 117.9,
17.5, 114.0, 112.2, 102.2; Anal. Calcd for: C15
=
1
123.0 (q,
J
C-F = 272 Hz), 120.0, 117.7, 117.3, 114.0,
H
8
F
2
O
3
:
112.2, 102.2; Anal. Calcd for: C16
H
8
F
4
O
3
: C, 59.27; H,
+
C, 65.70; H, 2.94; found: C, 65.61; H, 2.86; HRMS (m/z)
2.49; found: C, 59.39; H, 2.41; HRMS (m/z) [M+H] calcd
+
[M+H] calcd 275.0520, found 275.0551.
325.0488, found 325.0525.
7
-hydroxy-3-(3,4,5-trifluorophenyl)coumarin 1c
7-hydroxy-3-[4-(trifluoromethoxy)phenyl]coumarin
1g
À1
Yield 68%; mp: 251–254 °C; vmax (KBr)/cm 3426, 1695,
1
À1
1
615, 1579, 1528, 1437, 1132, 1103, 858, 739, 628;
NMR (500 MHz, DMSO), d (ppm): 10.76 (s, 1H, OH), 8.32
s, 1H), 7.72 (dd, 2H, J = 9.28 Hz, J = 6.84 Hz), 7.58 (d,
H, J = 8.79 Hz), 6.84 (dd, 1H, J = 8.54 Hz,
J = 2.20 Hz), 6.76 (d, 1H, J = 2.44 Hz);
H
Yield 62%; mp: 219–222 °C; vmax (KBr)/cm 3310, 1708,
1
1606, 1583, 1514, 1421, 1129, 1109, 848, 743, 631;
H
(
1
NMR (500 MHz, DMSO), d (ppm): 10.67 (s, 1H, OH), 8.22
(s, 1H), 7.81 (d, 2H, J = 8.79 Hz), 7.59 (d, 1H,
J = 8.79 Hz), 7.43 (d, 2H, J = 8.79 Hz), 6.83 (dd, 1H,
13
C
NMR
13
(
(
1
200 MHz, DMSO), d (ppm): 162.4, 160.0, 155.5, 150.3
J = 8.55 Hz, J = 2.20 Hz), 6.76 (d, 1H, J = 1.95 Hz);
C
1
1
d,
32.2 (m), 130.8, 119.0, 114.2, 113.2 (m), 112.0, 102.2;
Anal. Calcd for: C H F O : C, 61.65; H, 2.41; found: C,
JC-F = 245 Hz), 142.9, 138.7 (d,
JC-F = 250 Hz),
NMR (200 MHz, DMSO), d (ppm): 161.9, 160.4, 155.5,
1
148.3, 142.1, 134.8, 130.6, 130.5, 121.1, 120.5 (q, J
C-F
=
C
256 Hz), 113.9, 112.3, 102.2; Anal. Calcd for:
16 9 3 4
H F O : C, 59.64; H, 2.82; found: C, 59.75; H, 2.91;
1
5 7 3 3
+
6
1.55; H, 2.47; HRMS (m/z) [M+H] calcd 293.0426,
+
found 293.0443.
HRMS (m/z) [M+H] calcd 323.0531, found 323.0557.
7
-hydroxy-3-pentafluorophenylcoumarin 1d
General method for the synthesis of 3-
À1
Yield 74%; mp: 225–227 °C; vmax (KBr)/cm 3377, 1699,
phenylcoumarin-7-O-sulfamate derivatives 2a-g
A mixture of formic acid (1.54 mmol) and N,N-dimethyl
acetamide (0.016 mmol) was added to a stirred solution of
chlorosulfonyl isocyanate (1.50 mmol) in dry dichloro-
methane (0.5 mL) at 40 °C within a period of 3.5 h. Then,
a stirred solution of 7-hydroxy-3-phenylcoumarin deriva-
tives 1a-g (0.238 g, 1.00 mmol) in N,N-dimethyl aceta-
mide (3.4 mL) was added to the mixture. The mixture was
stirred at ambient temperature overnight and then poured
onto water (10 mL). Eventually, a white precipitate was
formed. The suspension was stirred at ambient tempera-
ture for another 2 h and then filtered. The crude product
was purified by column chromatography using a mixture of
1
1
606, 1575, 1519, 1429, 1124, 1108, 851, 740, 630;
H
NMR (500 MHz, DMSO), d (ppm): 10.92 (s, 1H, OH), 8.28
1
3
(
(
s, 1H), 7.68–7.58 (m, 1H), 6.92–6.74 (m, 2H); C NMR
500 MHz, acetone-d ), d (ppm): 162.8, 158.4, 156.6,
6
1
1
1
2
1
5
46.8, 145.2 (d,
J
C-F = 247 Hz), 141.4 (d,
C-F
J =
1
52 Hz), 137.9 (d,
J
C-F = 250 Hz), 130.8, 114.0, 111.8,
: C,
4.90; H, 1.54; found: C, 54.98; H, 1.49; HRMS (m/z)
5 5 3
10.8 (m), 110.1, 102.7; Anal. Calcd for: C15H F O
+
[M+H] calcd 329.0237, found 329.0271.
7
-hydroxy-3-[2,5-Bis(trifluoromethyl)
phenyl]coumarin 1e
Yield 72%; mp: 116–120 °C; vmax (KBr)/cm 3276, 1699,
EtOAc: CH Cl (1:30) as eluent, resulting in the corre-
3
À1
sponding 3-phenylcoumarin-7-O-sulfamate derivatives
2a-g.
1
1
610, 1575, 1506, 1418, 1130, 1116, 843, 751, 625;
H
2
36
Chem Biol Drug Des 2016; 87: 233–238

3
-phenylcoumarin-7-O-sulfamate 2a
3-[2,5-Bis(trifluoromethyl)phenyl]coumarin-7-O-
À1
Yield 90%; mp: 198–201 °C; vmax (KBr)/cm 3267, 3205,
sulfamate 2e
Yield 86%; mp: 212–214 °C; vmax (KBr)/cm
1
À1
1672, 1608, 1494, 1363, 1186, 1137, 757; H NMR
3361,
(
500 MHz, DMSO), d (ppm): 8.40–8.20 (m, 3H), 7.88 (d,
3267, 1722, 1616, 1510, 1366, 1184, 1130, 1112, 752;
1
1
1
H, J = 8.30 Hz), 7.80–7.60 (m, 2H), 7.60–7.20 (m, 5H);
2
H NMR (500 MHz, DMSO), d (ppm): 8.30 (s, 2H, NH ),
3
C NMR (500 MHz, acetone-d ), d (ppm): 159.5, 154.2,
8.20 (s, 1H), 8.14–8.06 (m, 3H), 7.86 (d, 1H,
J = 8.79 Hz), 7.43 (d, 1H, J = 1.95 Hz), 7.33 (dd, 1H,
6
1
1
5
3
3
52.7, 139.7, 135.1, 129.9, 128.9, 128.8, 128.5, 127.7,
13
19.0, 118.6, 110.0; Anal. Calcd for: C15
H
11NO
5
S: C,
J = 8.55 Hz, J = 2.20 Hz);
C NMR (200 MHz, ace-
6.78; H, 3.49; N, 4.41; S, 10.11; found: C, 56.90; H,
tone-d ), d (ppm): 159.0, 154.5, 153.1, 142.1, 135.1,
6
+
.58; N, 4.32; S, 10.03; HRMS (m/z) [M+H] calcd
133.7–132.0 (m), 130.0, 129.2 (m), 127.7 (m), 126.2,
1
18.0436, found 318.0469.
125.3, 123.5 (q, JC-F = 271 Hz), 119.0, 117.3, 110.2;
Anal. Calcd for: C17
9 6 5
H F NO S: C, 45.04; H, 2.00; N,
3
.09; S, 7.07; found: C, 45.15; H, 1.93; N, 2.97; S,
+
3
-(3,4-difluorophenyl)coumarin-7-O-sulfamate 2b
6.99; HRMS (m/z) [M+H] calcd 454.0184, found
À1
Yield 87%; mp: 190–193 °C; vmax (KBr)/cm
3394,
454.0308.
3
290, 1707, 1616, 1520, 1369, 1180, 1113, 1098, 750;
1
H NMR (500 MHz, DMSO), d (ppm): 8.38 (s, 1H), 8.28
(
(
s, 2H, NH ), 7.87–7.80 (m, 2H), 7.64–7.52 (m, 2H), 7.37
3-[4-fluoro-3-(trifluoromethyl)phenyl]coumarin-7-
2
13
s, 1H), 7.28–7.34 (m, 1H); C NMR (200 MHz, acetone-
O-sulfamate 2f
Yield 79%; mp: 173–176 °C; vmax (KBr)/cm 3359, 3196,
1707, 1617, 1504, 1358, 1192, 1123, 1112, 755;
1
À1
d ), d (ppm): 159.0, 154.0, 152.8, 150.2 (d, JC-F
=
6
1
1
2
1
1
51 Hz), 149.6 (d,
J
C-F = 249 Hz), 140.1, 132.2 (m),
H
29.8, 125.4 (m), 125.0, 118.8, 117.9, 117.4 (m), 117.0,
09.8; Anal. Calcd for: C H F NO S: C, 50.99; H, 2.57;
NMR (500 MHz, DMSO), d (ppm): 8.44 (s, 1H), 8.29 (s,
2H, NH ), 8.16–8.08 (m, 2H), 7.87(d, 1H, J = 8.30 Hz),
15
9
2
5
2
N, 3.96; S, 9.08; found: C, 51.14; H, 2.51; N, 3.81; S,
7.65 (t, 1H, J = 9.77 Hz), 7.39 (d, 1H, J = 1.95 Hz), 7.32
+
13
9.21; HRMS (m/z) [M+H]
calcd 354.0248, found
(dd, 1H, J = 8.55 Hz, J = 2.20 Hz); C NMR (200 MHz,
1
354.0277.
acetone-d
6
), d (ppm): 159.4 (d,
JC-F = 256 Hz), 159.2,
1
54.2, 152.9, 140.6, 135.1 (m), 131.7 (m), 129.9, 127.5
1
(m), 124.9, 122.8 (q, JC-F = 271 Hz), 118.8, 118.0, 117.2,
3
2
-(3,4,5-trifluorophenyl)coumarin-7-O-sulfamate
116.8, 109.8; Anal. Calcd for: C16 NO S: C, 47.65; H,
H
9
F
4
5
c
2.25; N, 3.47; S, 7.95; found: C, 47.47; H, 2.18; N, 3.31;
À
+
Yield 85%; mp: 185–187 °C; vmax (KBr)/cm 3386, 3284,
S, 8.11; HRMS (m/z) [M+H] calcd 404.0216, found
1
1
711,1610, 1530, 1359, 1183, 1118, 1109, 765; H NMR
500 MHz, DMSO), d (ppm): 8.44 (s, 1H), 8.29 (s, 2H,
NH ), 7.84 (d, 1H, J = 8.79 Hz), 7.79–7.72 (m, 2H), 7.38
d, 1H, J = 1.95 Hz), 7.32 (dd, 1H, J = 8.30 Hz,
404.0254.
(
2
(
3-[4-(trifluoromethoxy)phenyl]coumarin-7-O-
1
3
J = 1.95 Hz); C NMR (200 MHz, acetone-d
6
), d (ppm):
sulfamate 2g
Yield 88%; mp: 190–193 °C; vmax (KBr)/cm 3349, 3199,
1710, 1615, 1510, 1363, 1183, 1143, 1111, 753;
1
À1
1
1
1
58.8, 154.1, 153.0, 150.5 (d, JC-F = 255 Hz), 140.9,
39.4 (d, JC-F = 252 Hz), 131.4 (m), 130.0, 123.9, 118.9,
17.8, 113.1 (m), 109.8; Anal. Calcd for: C H F NO S:
1
1
H
NMR (500 MHz, DMSO), d (ppm): 8.36 (s, 1H), 8.27 (s,
2H, NH ), 7.89–7.84 (m, 3H), 7.48 (d, 2H, J = 8.30 Hz),
7.37 (d, 1H, J = 1.95 Hz), 7.31 (dd, 1H, J = 8.55 Hz,
15
8
3
5
C, 48.52; H, 2.17; N, 3.77; S, 8.64; found: C, 48.39; H,
2
+
2
3
.09; N, 3.91; S, 8.72; HRMS (m/z) [M+H] calcd
72.0154, found 372.0184.
13
J = 2.20 Hz); C NMR (200 MHz, acetone-d
6
), d (ppm):
1
1
59.2, 154.1, 152.7, 149.1, 140.1, 134.0, 130.5, 129.7,
1
25,9, 120.7, 120.5 (q, JC-F = 256 Hz), 118.8, 118.1,
NO S: C, 47.89; H, 2.51;
3
-pentafluorophenylcoumarin-7-O-sulfamate 2d
109.8; Anal. Calcd for: C16
H
10
F
3
6
À1
Yield 82%; mp: 157–160 °C; vmax (KBr)/cm
3412,
N, 3.49; S, 7.99; found: C, 48.01; H, 2.40; N, 3.65; S,
+
3
301, 1732, 1620, 1527, 1365, 1175, 1122, 1105, 750;
7.88; HRMS (m/z) [M+H] calcd 402.0259, found
402.0310.
1
H NMR (500 MHz, DMSO), d (ppm): 8.48 (s, 1H), 8.30
s, 2H, NH ), 7.93 (d, 1H, J = 8.30 Hz), 7.50–7.25 (m,
H); C NMR (500 MHz, acetone-d ), d (ppm): 157.7,
55.1, 154.1, 146.2, 145.1 (d, JC-F = 248 Hz), 141.7
(
2
1
2
1
3
6
1
In vitro activity assay
1
1
(
1
d,
J
C-F = 253 Hz), 137.9 (d,
19.5, 117.2, 114.6, 110.5, 110.1 (t, JC-F = 18 Hz);
NO S: C, 44.24; H, 1.48; N,
J
C-F = 251 Hz), 130.7,
The reaction mixture, at a final volume of 100 lL, con-
tained 20 lM Tris-HCl pH 7.4, 3 mM NPS, various con-
centrations of inhibitor (0.01–100 lM), and
2
Anal. Calcd for: C15
H
6
F
5
5
5 U of
3
7
4
.44; S, 7.87; found: C, 44.11; H, 1.59; N, 3.29; S,
.99; HRMS (m/z) [M+H] calcd 407.9965, found
08.0019.
purified enzyme (1 is the amount of enzyme
that hydrolyzes 100 lM of NPS in 1 h at 37 °C). The
reaction was performed at 37 °C for 15 min. It was
U
+
Chem Biol Drug Des 2016; 87: 233–238
237

Demkowicz et al.
halted by the addition of 100 lL of 1 M NaOH. The
absorbance of the released p-nitrophenol was measured
at 405 nm using a Microplate Reader Biotek ELx800
phate tricyclic coumarin analogs as steroid sulfatase
inhibitors: synthesis and biological activity. RSC
Adv;4:44350.
(
BioTek Instruments, Inc. Winooski, VT, USA) (SERVA).
4. Demkowicz S., Kozak W., Da ꢀs ko M., Masłyk M.,
Kubi nꢀ ski K., Rachon J. (2015) Phopshate and thiopho-
sphate biphenyl analogs as steroid sulfatase inhibitors.
IC50 values were calculated using GRAPHPAD PRISM soft-
ware (GraphPad Software, Inc., La Jolla, CA, USA). All
measurements were performed in triplicate.
Drug Dev Res;76:94–104.
5
. Demkowicz S., Kozak W., Da ꢀs ko M., Masłyk M., Giel-
niewski B., Rachon J. (2015) Synthesis of bicoumarin
thiophosphate derivatives as steroid sulfatase inhibitors.
Eur J Med Chem;101:358.
Conclusions
6. Demkowicz S., Kozak W., Dasko M., Wołos A., Masłyk
ꢀ
We have efficiently designed, synthesized, and biologi-
cally evaluated a series of potent STS inhibitors. Two
compounds, 2b and 2c, demonstrated the highest
activity in the STS enzyme assay with IC50 values of
M., Kubi
nꢀ ski K., Składanowski A., Misiak M., Rachon
J. (2015) Synthesis and steroid sulfatase inhibitory
activities of N-alkanoyl tyramine phosphates and thio-
phosphates. RSC Adv;5:32594.
0
.27 lM in both cases. According to the inhibitory data,
supported by the molecular modeling study, the intro-
duction of substituted phenyl rings in the 3rd position
of the coumarin scaffolds significantly improved the bio-
logical activities of tested compounds. Furthermore, sta-
bilization of the enzyme–ligand complex by establishing
hydrophobic interactions may increase their inhibitory
potency. Furthermore, taking into account the influence
of fluorine atoms on the lipophilicity of the compounds,
we expect the new STS inhibitors containing C-F
bonds 2b-g will have promising biological activities
compared to 2a and coumarin-7-O-sulfamate in cell
line or in vivo assays that involve interactions with cell
membranes.
7. Purohit A., Williams G.J., Howarth N.M., Potter B.V.L.,
Reed M.J. (1995) Inactivation of steroid sulfatase by an
active site-directed inhibitor, estrone-3-O-sulfamate.
Biochemistry;34:11508.
8. Thomas M.P., Potter B.V.L. (2015) Discovery and
Development of the Aryl O-Sulfamate Pharmacophore
for Oncology and Women’s Health. J Med Chem
doi:10.1021/acs.jmedchem.5b00386.
9. Purohit A., Reed M.J., Morris N.C., Williams G.J., Pot-
ter B.V.L. (1996) Regulation and inhibition of steroid
sulfatase activity in breast cancer. Ann N Y Acad
Sci;784:40.
10. Woo L.W.L., Howarth N.M., Purohit A., Hatem A.M.,
Reed M.J., Potter B.V.L. (1998) Steroidal and nonster-
oidal sulfamates as potent inhibitors of steroid sulfa-
tase. J Med Chem;41:1068–1083.
Acknowledgments
1
1. Malini B., Purohit A., Ganeshapillai D., Woo L.W.L.,
Potter B.V.L., Reed M.J. (2000) Inhibition of steroid
sulphatase activity by tricyclic coumarin sulphamates.
J Steroid Biochem Mol Biol;75:253.
We gratefully acknowledge the National Science Centre
(Poland) for financial support (grant no. 2011/03/D/NZ7/
0
3985).
1
1
2. Dunitz J.D., Taylor R. (1997) Organic fluorine hardly
ever accepts hydrogen bonds. Chem Eur J;3:89.
3. Vaccaro A.M., Salvioli R., Muscillo M., Renola L.
Conflict of Interest
(1987) Purification and properties of arylsulfatase C
from human placenta. Enzyme;37:115.
The authors declare no conflict of interest.
1
1
4. Woo L.W.L., Jackson T., Putey A., Cozier G., Leonard
P., Acharya K.R., Chander S.K., Purohit A., Reed
M.J., Potter B.V.L. (2010) Highly potent first examples
of dual aromatase-steroid sulfatase inhibitors based on
a biphenyl template. J Med Biol;53:2155.
5. Hernandez-Guzman F.G., Higashiyama T., Osawa Y.,
Ghosh D. (2001) Purification, characterization and
crystallization of human placental estrone/dehydroe-
piandrosterone sulfatase, a membrane-bound enzyme
of the endoplasmic reticulum. J Steroid Biochem Mol
Biol;78:441.
References
1
2
3
. Purohit A., Foster P.A. (2012) Steroid sulfatase inhibi-
tors for estrogen- and androgen-dependent cancers. J
Endocrinol;212:99.
. Reed M.J., Purohit A., Woo L.W.L., Newman S.P.,
Potter B.V.L. (2005) Steroid sulfatase: molecular biol-
ogy, regulation, and inhibition. Endocr Rev;26:171.
. Kozak W., Da ꢀs ko M., Masłyk M., Pieczykolan J.S.,
Gielniewski B., Rachon J., Demkowicz S. (2014) Phos-
2
38
Chem Biol Drug Des 2016; 87: 233–238