Synthesis and biological evaluation of N-acylated tyramine sulfamates containing C–F bonds as steroid sulfatase inhibitors

Article abstract of DOI:10.1111/cbdd.12931

Steroid sulfatase (STS) is responsible for the hydrolysis of biologically inactive sulfated steroids into their active un-sulfated forms and promotes the growth of various hormone-dependent cancers (e.g., breast cancer). Therefore, the STS enzyme is a promising therapeutic target for the treatment of steroid-sensitive cancers. Herein, we report the synthesis and biological evaluation of sulfamate analogs as potential STS inhibitors based on N-acylated tyramines that contain C–F bonds. The inhibitory effects of the analogs were tested using STS isolated from human placenta. Of the analogs tested, 4-(2-perfluoroundecanoylaminoethyl)-phenyl sulfamate, 5r, demonstrated the greatest inhibitory effect, with an IC₅₀ value of 2.18?μm (IC₅₀ value of 2.13?μm for coumarin-7-O-sulfamate was used as a reference). These findings were supported by the results our computational analyses performed using molecular docking techniques.

Full text of DOI:10.1111/cbdd.12931

DR. SEBASTIAN DEMKOWICZ (Orcid ID : 0000-0002-4252-4297)
Received Date : 28-Oct-2016
Revised Date : 30-Nov-2016
Accepted Date : 13-Dec-2016
Article type : Research Letter
Synthesis and biological evaluation of N-acylated tyramine sulfamates containing C-F
bonds as steroid sulfatase inhibitors
running title: Steroid sulfatase inhibition
keywords: tyramine, steroid sulfatase, breast cancer, STS inhibitors, sulfamates
Mateusz Daśko,1,* Janusz Rachon,1 Maciej Masłyk,2 Konrad Kubiński2 and Sebastian
Demkowicz1,*
1
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.
2
Department of Molecular Biology, Faculty of Biotechnology and Environmental Sciences,
The John Paul II Catholic University of Lublin, Konstantynów 1i, 20-708 Lublin, Poland.
Abstract
Steroid sulfatase (STS) is responsible for the hydrolysis of biologically inactive sulfated
steroids into their active un-sulfated forms and promote the growth of various hormone-
dependent cancers (e.g., breast cancer). Therefore, the STS enzyme is a promising therapeutic
target for the treatment of steroid-sensitive cancers. Herein, we report the synthesis and
biological evaluation of sulfamate analogs as potential STS inhibitors based on N-acylated
tyramines that contain C-F bonds. The inhibitory effects of the analogs were tested using STS
isolated from human placenta. Of the analogs tested, 4-(2-perfluoroundecanoylaminoethyl)-
phenyl sulfamate, 5r, demonstrated the greatest inhibitory effect, with an IC50 value of 2.18
μM (IC50 value of 2.13 μM for coumarin-7-O-sulfamate was used as a reference). These
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doi: 10.1111/cbdd.12931
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findings were supported by the results our computational analyses performed using molecular
docking techniques.
Different enzymes are involved in the conversion of androgens and estrogens during
steroidogenesis. One such enzyme that regulates the level of steroids is steroid sulfatase
(STS), one of the fifteen known human sulfatases. STS catalyzes the desulfation of sulfated
steroids, exemplified by the conversion of estrone sulfate (E1S) into estrone (E1) that
regulates the function of many physiological molecules (1). The solubility and half-life of
E1S is several fold higher than that of its un-sulfated form E1. This allows E1S to act as a
steroid reservoir, with its local concentrations reaching up to ten times that of E1 in tissues,
efficiently stimulating tumor growth (2,3). Sulfatases have been implicated in the
development of numerous pathophysiological conditions, including hormone-dependent
cancers, lysosomal storage disorders, developmental abnormalities, and bacterial
pathogenesis (4). In patients with hormone-dependent cancers, the reduction of estrogen and
androgen concentrations is crucial. Therefore, because STS is involved in biosynthesis of
active steroids, it is a promising therapeutic target for the treatment of steroid-sensitive
cancers. In addition to their utility in breast cancer treatment, STS inhibitors may be effective
in the treatment of endometrial and prostate cancers as well as other hormone-dependent
cancers (5).
Various approaches have been used in the design of effective STS inhibitors. Extensive
research efforts in the recent years have focused on finding novel steroidal and non-steroidal
inhibitors of STS containing different functional groups, e.g., the sulfamate moiety (6). One
of the most promising compounds was steroidal EMATE (Figure 1), which exhibited a very
high activity in MCF-7 cells, with an IC50 value of 65 pM. Despite its high inhibitory activity,
EMATE was withdrawn from clinical trials due to its estrogenicity (7). To avoid such
adverse side effects, several research groups attempted the synthesis of new compounds
based on non-steroidal structures. Coumarin derivatives are another important class of potent
STS inhibitors. An earlier study reported that coumarin-7-O-sulfamate and its derivatives,
such as COUMATE (IC50 value of 380 nM in an assay with placental microsomes), showed
no significant estrogenicity (8).
The objective of the present study was to design new STS inhibitors based on N-acylated
tyramine scaffolds containing C-F bonds. Fluorination is one of the most common strategies
in drug design. The introduction of fluorine atoms into the structure of biologically active
compounds alters nearly all physical properties of the compounds and modifies its
absorption, distribution, metabolism, and excretion. In general, H/F exchange produces a
more lipophilic molecule. Increased lipophilicity can lead to higher free energy of binding
due to a more preferential partitioning of the drug between polar aqueous solutions and the
hydrophobic nature of the receptor site (9). Although the C-F unit is a poor H-bond acceptor,
studies on the C-F···H-X (where X = O, N, S) and C-F···H-Cα contact points (where Cα
corresponds to the α-carbon of the amino acids) have shown that multipolar interactions of
the C-F dipole inside the active site of various enzymes is favorable (10). In addition, the C-
F···H-N (backbone amide) interactions are abundantly found in the PDB (9). Our previous
work with fluorinated 3-phenylcoumarin-7-O-sulfamate derivatives showed that the
introduction of a fluorinated phenyl ring into coumarin scaffolds results in increased
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inhibitory activity. Two of these fluorinated compounds, 1 and 2, demonstrated the highest
inhibitory activity in the STS enzyme assay, with identical IC50 values of 270 nM (11). In
most cases, compounds containing C-F bonds showed slightly higher STS inhibitory activity
than the corresponding analogs without the fluorine atom. Furthermore, the use of the
tyramine-based scaffolds in the design of STS inhibitors is scientifically justified, as some
tyramine derivatives have already been used as potent STS inhibitors with very promising
activities. Recently, we synthesized a series of phosphate and thiophosphate analogs based on
the frameworks of N-alkanoyl tyramine and studied their biological activities (12). We found
that 4-(2-dodecanoylamino-ethyl)-phenyl dimethyl phosphate, 3, potently inhibited STS
isolated from human placenta, with an IC50 value of 390 nM. Although, different functional
groups including: sulfonates, sodium methylenesulfonates, sulfonamides, sulfonyl halides,
methylenesulfonyl derivatives, phosphates and phosphonates have been used in designing
new steroidal and non-steroidal inhibitors of STS, the pharmacophore based on sulfamate
moiety seems to be the most promising. Therefore we decided to choose the above-mentioned
sulfamate group for the synthesis of compounds described in the manuscript.
Biogenic amines (including tyramine) play key roles in important physiological processes,
including neurotransmission, immune response, cellular growth, and differentiation (13). Due
to their potent biological activity and use in the design of new drugs, their synthesis has been
a key area of interest to many investigators. Many synthetic and enzymatic methods exist for
the preparation of tyramine and its derivatives. These methods include the Curtius
rearrangement of β-(p-hydroxyphenyl)propionyl azide, the hydrogenation of (α-chloro-α-
oximino) acetophenone by transition metal catalyst, the decarboxylation of tyrosine,
enzymatic decarboxylation using tyrosine decarboxylase, and the reduction of β-nitrostyrenes
(12).
Encouraged by the results of our previous work and the high biological activities of biogenic
amines reported in the literature, we decided to synthesize a series of sulfamate analogs of N-
acylated tyramines containing C-F bonds as potential STS inhibitors. Synthesis of all newly
designed STS inhibitors were accomplished using the pathway shown in Scheme 1. In the
first step, the N-acylated tyramine frameworks 4a-r were obtained from perfluorodecanoyl
and perfluoroundecanoyl chlorides as well as fluorinated derivatives of benzoyl and
phenylacetoyl chlorides (obtained in situ from the corresponding carboxylic acids by reaction
with thionyl chloride) in the presence of potassium carbonate. Next, we performed the direct
functionalization of the hydroxyl group of analogs 4a-r. In this case, solutions of the stable
compounds 4a-r in N,N-dimethylacetamide (DMA) were treated with H2NSO2Cl (previously
generated by the reaction of chlorosulfonyl isocyanate and formic acid in the presence of
catalytic amounts of N,N-dimethylacetamide). The yields of these reactions were high and
typically reached 90%. Standard isolation procedures were then followed to obtain the
desired derivatives 5a-r.
To verify the putative binding modes of N-acylated derivatives of tyramine sulfamates
containing C-F bonds, we conducted molecular docking studies using properly prepared
crystal structure of human steroid sulfatase (PDB accession code: 1P49). The results of our
docking experiments clearly revealed that the proposed structures of the candidate inhibitors
were able to efficiently bind to the active site of STS. The predicted free energies of docking
are presented in Table 1. (calculated using AutoDock Vina software) for all candidates 5a-r
were satisfactory (in the range of –4.4 to -6.7 kcal/mol) and were significantly lower than that
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of the reference inhibitor (the value of predicted free energy of docking for coumarin-7-O-
sulfamate was –2.9 kcal/mol). The 5r analog produced the best docking result (the predicted
free energy of docking was -6.7 kcal/mol).
The putative complexes, with the structures of the selected analogs (5d - blue, 5m - red, 5r -
CPK colored) docked into the active site of STS, are shown in Figure 2. As expected, the new
STS inhibitor candidates docked in a manner similar to that shown by the reported STS
inhibitor coumarin-7-O-sulfamate (green). We found that the sulfamate functional groups
were directed toward the catalytic amino acid FGly75 coordinated to Ca2+ ion and that they
were 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 one of the hydroxyl groups of FGly75 was short (approximately 3Å).
Furthermore, the cores of the candidate inhibitors were well accommodated by the cavity
delimited by the lipophilic amino acid residues located in the active site of the enzyme. The
results of our molecular modeling studies suggested that the hydrophobicity of the inhibitor
cores favored binding via 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 2 and exemplified by compound 5r, the fluorine atoms of the designed
compounds were within a short distance from the heteroatoms of several amino acid residues
(e.g., NH groups of Arg98 and OH group of Thr484), suggesting the presence of additional
interactions. These additional interactions may stabilize the inhibitor-enzyme complexes.
After their efficient synthesis, we evaluated the new candidate inhibitors for their ability to
inhibit STS using reported methods (14,15). Screening assays were performed with STS
enzymes extracted from human placenta and purified by 3-step chromatography. The results
of the screening are presented in Table 1.
The results showed that the newly synthesized inhibitors moderately inhibited STS. The
highest inhibitory activity was determined to be the 5r analog, with an IC50 value of 2.18 μM,
which was comparable to that of the reference inhibitor (the IC50 value for coumarin-7-O-
sulfamate was 2.13 μM). These results are consistent with the data obtained from molecular
modeling studies, which showed that 5r had the lowest free binding energy (-6.7 kcal/mol).
Furthermore, most of the fluorinated compounds inhibited the enzyme more effectively (the
IC50 values for nine out of thirteen fluorinated derivatives were below 10 µM) than their
nonfluorinated analogs (the IC50 values for compounds 5a, 5g, 5h and 5o were 54.02, 17.55,
18.82 and 28.70 µM, respectively). Only compound 5i was significantly less active (the IC50
value was 83.45 µM). These results clearly showed that the introduction of fluorine atom into
the structure of tyramine-based STS inhibitors increased the inhibitory activity of the
compounds.
Conclusions
In this manuscript, we described the synthesis and evaluation of a series of novel STS
inhibitors based on sulfamate analogs of N-acylated tyramine containing C-F bonds. Docking
studies showed that the new STS inhibitor candidates were well accommodated by the active
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site of STS. In addition, the fluorine atoms of the newly synthesized compounds were within
a short distance from the heteroatoms of several amino acid residues (e.g., NH groups of
Arg98 and OH group of Thr484), suggesting the presence of additional interactions that may
stabilize the inhibitor-enzyme complexes formed in the active site. The results of the
molecular modeling studies were confirmed with the help of in vitro assays using isolated
STS. Analog 5r showed the highest inhibitory activity, with an IC50 value of 2.18 μM (the
free energy of binding was -6.7 kcal/mol, which was the lowest among the entire series of
new compounds). Most of the fluorinated compounds inhibited STS more effectively (the
IC50 values for nine of the thirteen fluorinated derivatives were below 10 µM) than their
nonfluorinated analogs (the IC50 values for compounds 5a, 5g, 5h and 5o were 54.02, 17.55,
18.82 and 28.70 µM, respectively). These results clearly showed that the introduction of a
fluorine atom into the structure of tyramine-based STS inhibitors increased the inhibitory
activity of the compounds. Considering the influence of the introduced fluorine atoms on the
lipophilicity of the compounds, we expect that these new STS inhibitors containing C-F
bonds will show promising biological activities both in in vitro and in vivo compared to
nonfluorinated analogs in assays that involve interactions of the compounds with cell
membranes.
Acknowledgements
We gratefully acknowledge the National Science Centre (Poland) for financial support (grant
no. 2015/19/N/NZ7/00938).
Conflict of Interest
The authors declare no conflict of interest.
For more detailed information please read the Methods S1 file.
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Figure 1: Chemical structures of STS inhibitors.
Scheme 1: Synthesis of N-acylated tyramine sulfamates 5a-r (R1, R2, R3, R4, R5 = H, F, CF3,
OCF3, NO2; n = 0, 1; m = 8, 9).
Figure 2: Docked binding modes and distances to Arg98 and Thr484 for compounds 5d
(blue), 5m (red), 5r (CPK colored) and coumarin-7-O-sulfamate (green).
Table 1: Activities of the newly synthesized compounds 5a-r and that of the reference
inhibitor (coumarin-7-O-sulfamate) assessed using the STS enzyme assay.
COCl
COOH
n
n
R1
R2
R5
R1
R2
R5
SOCl2
DCM
R4
R4
R3
R3
R5
n
R5
H
N
n
H
N
NH2
R4
R3
DMA
R4
R3
O
O
O
K2CO3
S
O
HO
HO
R1
4a-o
H2N
O
R1
5a-o
acetone
R2
R2
HCOOH
DMA
O
O
O
O
S
S
Cl
N
C
O
DCM
Cl
NH2
m
m
F2
C
F2
C
H
N
H
N
NH2
acetone
CF3
CF3
O
O
K2CO3
S
O
O
DMA
HO
H2N
O
HO
4p-r
5p-r
m
m
F2
C
F2
C
SOCl2
Toluene
OH
Cl
F3C
F3C
O
O
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Free energy of binding
[kcal mol-1]
IC50
[µM]
No.
n
0
m
R1
R2
R3
R4
R5
5a
5b
5c
5d
5e
5f
H
H
H
F
H
H
F
H
F
H
H
H
F
H
H
H
H
F
-5.5
-5.3
-5.6
-6.3
-6.0
-5.7
-6.1
-5.1
-5.4
-5.3
-5.9
-6.0
-6.3
-5.5
-4.4
-5.9
-6.7
54.02 1.02
17.74 1.03
5.39 0.43
8.62 0.49
2.91 0.13
8.59 0.55
17.55 1.09
18.82 1.01
83.45 1.45
16.43 0.89
4.72 0.35
9.78 0.43
3.88 0.22
3.92 0.26
28.70 1.00
13.10 0.77
2.18 0.22
F
H
F
F
F
F
F
H
H
H
H
H
F
H
H
H
F
CF3
NO2
H
H
H
H
H
F
H
H
H
H
H
F
5 g
5h
5i
-
F
5j
F
F
5k
5l
F
F
F
1
H
CF3
H
H
-
CF3
H
H
H
-
F
H
CF3
H
H
-
H
H
H
H
-
5m
5n
5o
5p
5r
H
OCF3
NO2
-
-
-
8
9
-
-
-
-
-
coumarin-7-O-
sulfamate
-
-
-
-
-
-
-2.9
2.13 0.13
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