Synthesis and steroid sulfatase inhibitory activities of: N -phosphorylated 3-(4-aminophenyl)-coumarin-7-O-sulfamates

Article abstract of DOI:10.1039/c6md00113k

In the present work, we report convenient methods for the synthesis and biological evaluation of N-phosphorylated derivatives of 3-(4-aminophenyl)-coumarin-7-O-sulfamate as potential steroid sulfatase (STS) inhibitors. Their binding modes were modeled using docking techniques. The inhibitory effects of the synthesized compounds were tested on STS isolated from human placenta. All of the newly synthesised coumarin derivatives were powerful inhibitors of STS with IC₅₀ values ranging between 0.19 and 0.78 μM. In particular, we found that 3-[4-(diphenoxy-phosphorylamino)-phenyl]-coumarin-7-O-sulfamate 10e and 3-[4-(dibenzyloxy-phosphorylamino)-phenyl]-coumarin-7-O-sulfamate 10f produced the highest inhibitory effects, with IC₅₀ values of 0.19 and 0.24 μM, respectively (IC₅₀ values of 1.38 μM for coumarin-7-O-sulfamate 2 and 1.03 μM for coumate 3 used as reference). The structure-activity relationships of the synthesized coumarin derivatives toward the STS enzyme were discussed.

Full text of DOI:10.1039/c6md00113k

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Synthesis and steroid sulfatase inhibitory activities of N-
phosphorylated 3-(4-aminophenyl)-coumarin-7-O-sulfamates†
Mateusz Daśko,a Maciej Masłyk,b Konrad Kubiński,b Justyna Aszyk,c Janusz Rachona and Sebastian
Demkowicz*,a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
In the present work, we report convenient methods for the synthesis and biological evaluation of N-phosphorylated
derivatives of 3-(4-aminophenyl)-coumarin-7-O-sulfamate as potential steroid sulfatase (STS) inhibitors. Their binding
modes have been modeled using docking techniques. The inhibitory effects of the synthesized compounds were tested on
STS isolated from human placenta. All of the newly synthesised coumarin derivatives were powerful inhibitors of STS with
IC50 values ranging between 0.19 and 0.78 µM. In particular, we found that the 3-[4-(diphenoxy-phosphorylamino)-
phenyl]-coumarin-7-O-sulfamate 10e and 3-[4-(dibenzyloxy-phosphorylamino)-phenyl]-coumarin-7-O-sulfamate 10f
produced the largest inhibitory effects, with IC50 values of 0.19 and 0.24 µM, respectively (IC50 values of 1.38 µM for the
coumarin-7-O-sulfamate 2 and 1.03 µM for the coumate 3 used as references). The structure-activity relationships of the
synthesized coumarin derivatives toward the STS enzyme have been discussed.
www.rsc.org/
modulate several cancer specific enzymes such as aromatase
or steroid sulfatase.6 The first potent inhibitors based on the
Introduction
coumarin scaffold were coumarin-7-O-sulfamate
2 or 4-
Steroid sulfatase (STS) is
a target enzyme of growing
methylcoumarin-7-O-sulfamate (coumate) , which exhibited
3
therapeutic importance. STS is responsible for the hydrolysis of
steroid sulfates to their active forms (e.g., estrone sulfate to
estrone), therefore, inhibition of this enzyme decreases the
biosynthesis of active hormones- responsible for breast,
endometrial or prostate cancer.1 STS is widely distributed
throughout the body, and its action is involved in physiological
and pathological conditions.2 Approaches to development of
effective and potent STS inhibitors include three different
categories of compounds: alternative substrates (including
competitive reversible inhibitors), reversible inhibitors, and
irreversible inhibitors.3 Most of the STS inhibitors discovered
good activity when evaluated against placental microsomes.7
Further modifications of their structures led to a wide range of
tricyclic coumarin derivatives, which showed more potent
inhibitory activities than coumate
coumate (the first of steroid sulfatase inhibitors entered into
clinical trials for patients with hormone-dependent breast
cancer) and 6610- coumate have demonstrated potent
3. For example, 667-
4
5
activity toward STS with IC50 values of 8 nM and 1 nM,
respectively.8
O
R
to date act in an irreversible way. EMATE 1, one of the first
O
O
S
irreversible inhibitors, exhibited a very potent activity in MCF-7
cells with IC50 value of 65 pM.4 Despite the exceptional
O
O
O
O
O
H2N
S
O
H2N
R= H, coumarin-7-O-sulfamate 2
R= CH3, coumate 3
EMATE 1
potency of the EMATE 1, clinical trials for this compound have
been discontinued due to its estrogenic properties.5 The
attempts to synthesize nonsteroidal agents (devoid of
undesirable adverse endocrine effects in vivo) have promoted
the generation of the coumarin sulfamates. These compounds
are also able to mimic the AB rings of the natural substrate and
n
S
O
O
O
P
O
O
O
O
O
HO
S
O
O
O
H2N
n= 3, 667-coumate 4
n= 6, 6610-coumate 5
6
Fig.1 Chemical structures of the STS inhibitors 1-6
.
Encouraged by our previous research,9 we decided to
design and synthesize a series of N-phosphorylated derivatives
of 3-(4-aminophenyl)-coumarin-7-O-sulfamate as compounds
with potential STS inhibitory activity. We found that
introduction of different phosphate or thiophosphate moieties
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into the structures of coumarin scaffolds can significantly steroid sulfatase was performed using the Autodock Vina 1.1.2
increase the inhibitory properties of tested compounds software (The Molecular Graphic Laboratory, The Scripps
(analog
6, Fig.1.). Furthermore, as is widely recognized, Research Institute, La Jolla, CA, USA). Figure 2 shows a putative
phosphate or thiophosphate groups may undergo
a
enzyme-ligand complex before the presumed inactivation of
nucleophilic substitution reactions on the phosphorus atom or the STS and the superimposed best conformations of the three
create many electrostatic interactions (e.g., hydrogen bonds) N-phosphorylated derivatives of 3-(4-aminophenyl)-coumarin-
with a variety of amino acid residues found within the active 7-O-sulfamates 10d (yellow), 10e (CPK colored) and 10f (red).
site of the STS. Generally, the predicted hydrogen bonds or the
All candidates expressed satisfactory predicted free
ability to create an electrostatic interactions could favor the docking energies (in the range of -6.2 to -8.3 kcal/mol) and
binding and may have a significant impact on the enzyme- exhibited significantly lower values of the AutoDock Vina score
ligand complex stability.
compared to a reference compounds (for example, the value
of predicted free docking energy for coumate was -4.1
kcal/mol). The best docking result was obtained for the 3-[4-
(dibenzyloxy-phosphorylamino)-phenyl]-coumarin-7-O-
Results and discussion
sulfamate 10f, which led to value within the lowest energy
of -8.3 kcal/mol. Furthermore, we found that introduction of
N-phosphoryl moieties in para positions of the coumarin’s
phenyl rings was the most preferred, leading to the lowest free
docking energies of the enzyme-inhibitor complexes. Analysis
of the docking studies for the newly designed STS inhibitor
candidates showed that these compounds could adopt
substrate-like poses in active site of STS in a similar manner to
the mode of the reported STS inhibitor (coumate). We found
that sulfamate functional groups are directed to catalytic
amino acid FGly75 coordinated to Ca2+, and they are
surrounded by the proposed catalytic residues of Asp35,
Asp36, Arg79, Lys134, His136 and Lys368. Furthermore, the
coumarin scaffolds are well accommodated to the cavity
delimited by lipophilic amino acids in the enzyme pocket
(Arg98, Leu103, Phe104, Leu167, Val177, Phe178, Phe182,
Leu185, Phe230, Phe233, Phe237, Thr484, Val486, Phe488,
Trp550 and Phe553). Molecular modelling studies suggest that
increasing the hydrophobic properties of coumarin
frameworks (by introducing more hydrophobic esters of
phosphorus acid) could favor binding by the establishment of
hydrophobic interactions with lipophilic amino acids in the
enzyme pocket. Furthermore, as shown in Figure 2, and
Chemistry
The synthesis of all newly designed STS inhibitors based on N-
phosphorylated derivatives of 3-(4-aminophenyl)-coumarin
scaffolds were achieved using the pathway shown in Scheme
1. In the first step, we synthesized 7-hydroxy-3-(4-
nitrophenyl)-coumarin
7 starting from 4-nitrophenylacetyl
chloride obtained in situ by the treatment of 4-
nitrophenylacetic acid with thionyl chloride in dry
dichloromethane. For the synthesis of 7-hydroxy-3-(4-
nitrophenyl)-coumarin 7, raw 4-nitrophenylacetyl chloride was
refluxed with 2,4-dihydroxybenzaldehyde in the presence of
potassium carbonate. Next, reduction of 7-hydroxy-3-(4-
nitrophenyl)-coumarin
7 to 7-hydroxy-3-(4-aminophenyl)-
coumarin 8 using sodium hydrosulfite (Na2S2O4) was
performed. The progress of reaction was monitored by TLC
analysis, and after full conversion of starting material, the
product
satisfactory yield (58%). In the next step, 7-hydroxy-3-(4-
aminophenyl)-coumarin was N-phosphorylated with
8 was isolated from the reaction mixture with
8
corresponding chlorophosphates in dry pyridine. Upon work-
up and fractionation of the crude products by flash column
chromatography, the desired coumarin derivatives 9a-h were
obtained in a good yields (60 - 78%). Finally, OH groups of the
compounds 9a-h were sulfamoylated. In these cases, the
solutions of stable N-phosphorylated derivatives of 7-hydroxy-
3-(4-aminophenyl)-coumarin 9a-h in N,N-dimethylacetamide
(DMA) were treated with H2NSO2Cl (previously generated by
the reaction of chlorosulfonyl isocyanate and formic acid in the
presence of a catalytic amount of N,N-dimethylacetamide).
The yields of these reactions were high and reached 92%. After
standard isolation procedure we obtained desired compounds
exemplified by compounds 10d, 10e and 10f, the phosphoryl
groups of these compounds are within hydrogen bonding
distance from the backbone NH group of Arg98. This additional
interaction may be a contributing factor that further assists the
binding of these molecules to the enzyme active site.
STS enzyme assay
The ability of the compounds synthesized (10a-h) to inhibit
steroid sulfatase activity was tested using an in vitro STS assay,
according to the methods reported previously.10,11 The
screening tests were performed using STS enzyme extracted
from human placenta and purified by 3-step chromatography.
After the purification, the fractions were used directly as an
enzyme source. Table 1 shows a summary of the results. The
enzyme assay results showed that the highest efficiency was
exhibited by compounds containing a hydrophobic diphenoxy-
phosphorylamino and dibenzyloxy-phosphorylamino groups in
the structure of the coumarin scaffold (compounds 10e and
10f) which is in agreement with the data of molecular
modelling studies.
10a-h
.
Molecular modelling
To examine the possible interactions of N-phosphorylated
derivatives of 3-(4-aminophenyl)-coumarin-7-O-sulfamate with
amino acid residues within the active site of STS, these
molecules were docked into the crystal structure of the human
steroid sulfatase (STS). The X-ray structure of STS was
retrieved from the Protein Databank (Protein Data Bank
accession code 1P49) and prepared for docking using required
procedure (see experimental section). The docking of the
optimized inhibitors into the prepared structure of the human
2 | J. Name., 2012, 00, 1-3
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Fig. 2 Docked binding modes and distances to Arg98 for compounds 10d (yellow), 10e (CPK colored), 10f (red) and coumate (green).
CHO
OH
COCl
NO2
Na2S2O4
COOH
NH2
SOCl2
DCM
OH
H2O
acetone
K2CO3
HO
O
O
HO
O
O
acetone
7
8
NO2
NO2
NH2
O
O
Cl
O
P
O
HO
O
O
Cl
P
8
R1O
OR1
R1 R1
py
py
H
N
H
N
O
O
P
P
O
O
O
OR1
O
R1O
S
Cl
N
C
O
R1
R1
R1 = H
HO
O
O
HO
O
O
HCOOH
DMA
DCM
9a-f
9 g-h
R1 = Me
R1 = Et
R1 = i-Pr
9a
9b
9c
9 g
R1 = Me 9h
O
O
R1 = n-Bu 9d
DMA
S
DMA
H2N
Cl
R1 = Ph
R1 = Bn
9e
9f
H
O
N
H
O
P
N
O
P
O
OR1
R1O
O
O
O
R1
R1
S
O
O
O
O
S
H2N
O
O
O
10a-f
10 g-h
H2N
R1 = H
10 g
R1 = Me 10h
R1 = Me
R1 = Et
R1 = i-Pr
10a R1 = n-Bu
10b R1 = Ph
10c R1 = Bn
10d
10e
10f
Scheme 1 Synthesis of N-phosphorylated derivatives of 3-(4-aminophenyl)-coumarin-7-O-sulfamate 10a-h
.
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These results may suggest that the frameworks based on 3-[4- the gem
–
diol form, addition hydrogen atoms and
(diphenoxy-phosphorylamino)-phenyl]-7-hydroxy-coumarin
optimization of prepared structure of human STS) docking
and 3-[4-(dibenzyloxy-phosphorylamino)-phenyl]-7-hydroxy- analysis was carried out. A docking studies was carried out
coumarin are more suitable for anchoring the enzyme active using Autodock Vina 1.1.2 software (The Molecular Graphic
site and provided the best potency. In the course of Laboratory, The Scripps Research Institute, La Jolla, CA, USA).12
investigations, the highest inhibition was observed for 3-[4- For all the docking studies, a grid box size of 30 Å x 30 Å x 30 Å,
(diphenoxy-phosphorylamino)-phenyl]-coumarin-7-O-
centered on Cβ atom of FGly75, was used. Graphic
sulfamate 10e with IC50 value of 0.19 μM (IC50 values of 1.38 visualizations of the 3D model were generated using VMD 1.9
and 1.03 μM for the coumarin-7-O-sulfamate and coumate (University of Illinois at Urbana – Champaign, Urbana, IL, USA).
used as references). Furthermore, we found
a
strong
LogP calculations. LogP parameters were calculated using
correlation between the IC50 and calculated LogP values of ChemDraw Ultra 7.0 (CambridgeSoft Corporation, Cambridge,
tested STS inhibitors, which suggests that increase the MA, USA).
hydrophobic properties of N-phosphoryl moieties favor
binding by the establishment of stronger hydrophobic Biological assays
interactions with lipophilic amino acids lining the cavity of STS.
Enzyme purification. STS was extracted from human
However, our preliminary molecular docking studies suggest
placenta and purified to homogeneity following a multi-step
chromatography protocol as previously described.13
that P=O∙∙∙∙∙∙H interactions between the phosphoryl groups
and hydrogen bond donor residue Arg98 might also contribute
In vitro activity assay. The reaction mixture, at a final
volume of 100 mL, contained 20 mM Tris–HCl pH 7.4, 3 mM p-
to the high potency observed for tested compounds.
nitrophenyl sulfate (NPS), varied concentrations of an inhibitor
Table 1 Activities and calculated LogP parameters of the synthesized compounds
10a-h and reference inhibitors (coumate and coumarin-7-O-sulfamate) in the STS
enzyme assays
(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 ᴼC). The
reaction was performed at 37 ᴼC 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
Microplate Reader Biotek ELx800 (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.
No.
IC50 [μM]
LogP [-]
2.01
2.70
3.52
4.43
5.37
5.56
1.89
2.99
0.94
0.76
10a
0.78 ± 0.07
0.51 ± 0.02
0.47 ± 0.03
0.31 ± 0.03
0.19 ± 0.02
0.24 ± 0.05
0.70 ± 0.05
0.45 ± 0.03
1.03 ± 0.23
1.38 ± 0.13
10b
10c
10d
10e
10f
10 g
10h
Conclusions
coumate
coumarin-7-O-sulfamate
A
series of N-phosphorylated derivatives of 3-(4-
aminophenyl)-coumarin-7-O-sulfamate have been synthesized
and biologically evaluated using in vitro STS assay. This study
has determined that 3-[4-(diphenoxy-phosphorylamino)-
phenyl]-coumarin-7-O-sulfamate 10e exhibits good affinity for
STS and the highest inhibition potency, with IC50 value of 0.19
μM (IC50 values of 1.38 and 1.03 μM for the coumarin-7-O-
sulfamate and coumate used as references). In the course of
our research we found a strong correlation between the IC50
and calculated LogP values of tested STS inhibitors, which
suggest the substantial participation of hydrophobic
interactions for the enzyme-inhibitor complex stability.
Furthermore, occurrence of hydrogen bonding between the
phosphoryl residue of tested compounds and NH group of
Arg98 may influence for the increase of their inhibitory
potency. Further structural modifications for these compounds
Experimental
Computational studies
Molecular modelling. All the molecular structures of the
ligands were built with the program Portable HyperChem 8.0.7
Release (Hypercube, Inc., Gainesville, FL, USA) and were
energy minimized 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/Å. The X-ray structure of human steroid sulfatase
used for molecular modelling study were taken from the
Protein Databank (Protein Data Bank accession code 1P49).
After standard preparation procedures (including removal of
water molecules, conversion of catalytic amino acid FGly75 to
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will be conducted in order to increase the activities of the new
STS inhibitors.
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