Synthesis and in vitro evaluation of piperazinyl-ureido sulfamates as steroid sulfatase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2019.111614

Two new piperazinyl-ureido single ring aryl sulfamate-based inhibitor series were designed against the emerging oncology drug target steroid sulfatase (STS), for which there are existing potent steroidal and non-steroidal agents in clinical trials. 4-(Pip

Full text of DOI:10.1016/j.ejmech.2019.111614

European Journal of Medicinal Chemistry 182 (2019) 111614
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis and in vitro evaluation of piperazinyl-ureido sulfamates as
steroid sulfatase inhibitors
,
, c,
Davide Moi a 1, Paul A. Foster b 1, Lucy G. Rimmer b, Alisha Jaffri b,
Alessandro Deplano a 2, Gianfranco Balboni a, Valentina Onnis a *, Barry V.L. Potter d
,
,
, **
a Department of Life and Environmental Sciences, Unit of Pharmaceutical, Pharmacological and Nutraceutical Sciences, University of Cagliari, via Ospedale
72, Cagliari I, 09124, Italy
b Institute of Metabolism and Systems Research, University of Birmingham, 2nd Floor IBR Tower, Birmingham, B15 2TT, UK
c Centre for Endocrinology, Diabetes and Metabolism, Birmingham Health Partners, Birmingham, B15 2TH, UK
d Medicinal Chemistry & Drug Discovery, Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, OX1 3QT, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Two new piperazinyl-ureido single ring aryl sulfamate-based inhibitor series were designed against the
emerging oncology drug target steroid sulfatase (STS), for which there are existing potent steroidal and
non-steroidal agents in clinical trials. 4-(Piperazinocarbonyl)aminosulfamates (5e31) were obtained by
reacting 4-hydroxyarylamines with phenylchloroformate, subsequent sulfamoylation of the resulting
hydroxyarylcarbamates and coupling of the product with 1-substituted piperazines. Pyrimidinyl-
piperazinourea sulfamates (35e42) were synthesized by pyrimidine ring closure of 4-Boc-piperazine-
1-carboxamidine with 3-(dimethylamino)propenones, deprotection and coupling with the sulfamoylated
building block. Target ureidosulfamates 5e31 and 35e42 were evaluated both as STS inhibitors in vitro
using a lysate of JEG-3 human placenta choriocarcinoma cell line and in a whole cell assay. SAR con-
clusions were drawn from both series. In series 35e42 the best inhibitory activity is related to the
presence of a benzofuryl on the pyrimidine ring. In series 5e31 the best inhibitory activity was shown by
the ureas bearing 4-chlorophenyl, 3,4-dichlorophenyl groups or aliphatic chains at the piperazino 4-
nitrogen displaying IC50 in the 33e94 nM concentration range. Final optimization to the low nano-
molar level was achieved through substitution of the arylsulfamate ring with halogens. Four halogenated
arylsulfamates of high potency were achieved and two of these 19 and 20 had IC50 values of 5.1 and
8.8 nM respectively and are attractive for potential in vivo evaluation and further development. We
demonstrate the optimization of this new series to low nanomolar potency, employing fluorine sub-
stitution, providing potent membrane permeant inhibitors with further development potential indi-
cating piperazinyl-ureido aryl sulfamate derivatives as an attractive new class of STS inhibitors.
© 2019 Elsevier Masson SAS. All rights reserved.
Received 5 July 2019
Received in revised form
26 July 2019
Accepted 9 August 2019
Available online 12 August 2019
Keywords:
Piperazines
Ureido compounds
Sulfamates
Pyrimidines
Steroid sulfatase
Enzyme inhibition
1. Introduction
biosynthetic conversion of androgens to estrogens, the main stan-
dards of clinical care. More recently, a new drug target steroid
Estrogen signalling is a well-established target for breast cancer
drug discovery [1] with clinical drugs such as Tamoxifen, acting at
the estradiol receptor, and aromatase inhibitors blocking
sulfatase (STS) has become an emerging new therapeutic modality
with its inhibition showing clinical benefit and the first clinical trial
data published in 2006 [2]. STS is widely distributed throughout the
body and is involved in numerous physiological and pathological
conditions [3] including hormone-dependent cancers [4], lyso-
somal storage disorders, developmental abnormalities, and bacte-
rial pathogenesis [5]. Primarily, STS catalyzes the desulfation of
biologically inactive steroid sulfates into their bioactive forms. It
can work in an intracrine fashion, playing a crucial role in the in situ
formation of biologically active steroids such as estradiol or
androstenediol in tumor cells in hormone-dependent disease [6].
* Corresponding author.
** Corresponding author.
E-mail addresses: vonnis@unica.it (V. Onnis), barry.potter@pharm.ac.uk
(B.V.L. Potter).
1
These authors contributed equally to the present paper.
Present address: Pharmacelera, Placa Pau Vila, 1, Sector 1, Edificio Palau de Mar,
2
Barcelona 08039, Spain.
https://doi.org/10.1016/j.ejmech.2019.111614
0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.

2
D. Moi et al. / European Journal of Medicinal Chemistry 182 (2019) 111614
Numerous STS inhibitors have already been described in the
Recently, some of us explored further development of non-fused
aryl sulfamate esters as potential STS inhibitors. Thus, simple ary-
lamide derivatives A possessing especially terminal sulfamate
moieties (Fig. 2) were synthesized. One compound with n ¼ 2 had
an IC50 of 421 nM and was an effective STS inhibitor in whole cells,
showing potential for further optimization [34].
literature and these have mainly focused on the sulfamate ester
class of compound, first discovered by some of us [7]. Initial potent
STS inhibitors were steroid-based: estrone and estradiol-based aryl
sulfamate esters are known to be time and concentration depen-
dent irreversible active-site directed inhibitors [8]. However,
because of their very potent estrogenic activity in rodents they
were not deemed suitable for progression as anti-tumor drug
candidates, although one of them, estradiol 3-O-sulfamate (Fig. 1,
E2MATE), did reach phase II human clinical trials as a prodrug of
estradiol in hormone replacement therapy [9,10]. E2MATE is still
being pursued as a potential drug against the hormone-dependent
disease endometriosis [11,12], that was shown to have an important
STS component [13].
In the present study, we explored a series of broadly related
piperazinylureas bearing the arylsulfamate moiety to further opti-
mize this lead. In an effort to define the critical requirements for
activity and understand the molecular determinants of such novel
STS inhibitors, our approaches involved making structural modifi-
cations in the putative key pharmacophoric portions of the mole-
cule. Our starting approach was focused upon synthesizing a series
of sulfamate derivatives bearing on their scaffold a large number of
aliphatic/aromatic/heterocyclic moieties (substituting the second
nitrogen atom from the piperazine ring) in order to generate
chemical diversity and to incorporate a piperazine group to
improve physicochemical properties. Starting from the compound
A, the amide moiety was replaced with an ureido group to allow
strong hydrogen bonding which might lead to cooperative effects
[35,36]. Furthermore, the ureido linker was connected to a more
rigid heterocyclic system, the N-substituted piperazine ring.
Piperazine ring is a well-known heterocyclic structure present in
many biologically active molecules. Constraining the polar nitrogen
atoms into the piperazine ring can confer more drug-like properties
to molecules and enhance favourable interactions with macro-
molecules [37,38]. Herein we report the synthesis of a series of
these ligands and in vitro results of an STS inhibition study using the
lysate of JEG-3, a human placenta choriocarcinoma cell line known
to have high STS activity. Additionally, membrane permeability was
explored using STS inhibition in intact JEG-3 cells.
While many further steroidal based sulfamate-based inhibitors
have been designed [9,14,15], because of estrogenicity issues the
search for non-estrogenic STS inhibitors was prioritised and many
structurally diverse inhibitors with different scaffolds combined
with the sulfamate moiety have been reported [1,9]. Initially, those
possessing
a coumarin-based template were highly effective
[16e18]. Coumarin-7-O-sulfamate and derivatives such as COU-
MATE showed high STS inhibitory activity and no significant
estrogenicity [19]. Further development in this area led to the
clinical STS inhibitor Irosustat/STX64 (Fig. 1) [2,17]. Such aryl sul-
famate derivatives in particular have proved to be the most potent
STS inhibitors and have reached clinical trials [9,10]. The most
recent clinical reports relate to two studies, one with Irosustat in
combination with an aromatase inhibitor [20] and another
demonstrating the first effects of Irosustat in early breast cancer in
treatment-naïve patients [21]. Aryl sulfamateebased drugs are
thought to work by sulfamoyl group transfer to the active site hy-
drated formylglycine residue important for catalysis by STS, leading
to irreversible inactivation of the enzyme [10].
2. Results and discussion
Further work on STS inhibitor development included synthe-
sizing reversible inhibitors without a sulfamate moiety, although
these have not reached clinical evaluation [22,23]. Other types of
irreversible inhibitors that can produce reactive quinone methides
upon activation by STS [24] and other alternatives to the sulfamate
ester approach [25e27] have been investigated. Dual inhibitors of
both STS and aromatase have also been reported, some with dual
picomolar potency against both enzymes [28]. Sulfamate analogs of
N-acylated tyramine containing CeF bonds demonstrate that
introduction of a fluorine atom into tyramine-based STS inhibitors
remote from the aryl sulfamate moiety can enhance inhibitory ac-
tivity, albeit in the micromolar range [29]. Recent work by some of
us has shown that the introduction of halogens, particularly fluo-
rine into the aryl sulfamate ring can appreciably improve the ac-
tivity of STS inhibitors by lowering the pKa of the leaving phenol
after transfer of the sulfamoyl moiety, thus increasing the “sulfa-
moyl-transfer potential” of the inhibitor [9,10,30].
2.1. Chemistry
The synthesis of the new compounds is reported in Schemes 1
and 2. Hydroxyarylcarbamates 3 were obtained with good yields
by reacting 4-hydroxyarylamines 1 with phenylchloroformate 2 in
the presence of N,N-diisopropylethylamine (DIPEA). Sulfamoylation
of aryl carbamates 3 upon treatment with sulfamoyl chloride [39]
in N,N-dimethylacetamide (DMA) solution furnished the key in-
termediates 4. Coupling of 4 with 1-substituted piperazines in
DMSO solution gave the piperazinyl urea derivatives 5e31 (Scheme
1).
A pyrimidinyl-piperazinourea series 35e42 was synthesized by
heterocyclization of 4-Boc-piperazine-1-carboxamidine (32) with
3-(dimethylamino)propenones 33a-h in boiling 1-propanol, fol-
lowed by trifluoroacetic acid (TFA)-mediated deprotection in
dichloromethane (DCM) solution and then coupling with 4-((phe-
noxycarbonyl)amino)phenyl sulfamate (4a) (Scheme 2).
Non-steroidal STS inhibitors have often included a sulfamoy-
lated fused AB phenolic ring steroid surrogate motif, although early
examples with employing single ring also showed good potency
[31,32] as did that using a single ring of a biphenyl template [33].
2.2. STS inhibition
The in vitro STS inhibition of activity of the sulfamates 5e31 and
Fig. 2. Structures of arylamide sulfamates (A) and design of the new piperazino ureido
Fig. 1. Structures of the STS inhibitors E2MATE and Irosustat.
sulfamates of this paper.

D. Moi et al. / European Journal of Medicinal Chemistry 182 (2019) 111614
3
Scheme 1. Reagents and conditions: (i) DIPEA, THF, r.t. 24 h; (ii) ClSO2NH2, DMA, r.t. 12 h; (iii) Substituted piperazine, DMSO, r.t. 24 h.
Scheme 2. Reagents and conditions: (i) 1-propanol, reflux 12 h; (ii) TFA, DCM, r.t. 24 h; (iii) 4a, DIPEA, DMSO, r.t. 24 h.
35e42 was measured in an assay using a JEG-3 cell lysate. The
in vitro inhibition results are reported as % of residual STS at 10
of a second chlorine atom to give compound 13 produced reduc-
mM
tion in activity on the isolated enzyme and 3.6% 2.3 on JEC-
3 cells (Fig. 3). The replacement of the benzyl group of compound
5 with a benzofurylmethyl (urea 15) did not afforded significant
change in inhibitory activity. However, the replacement of the
benzyl group with aliphatic chains to give compounds 16e18
produced good inhibitory activity being the potency of the ureas
as much strong as the chain carbon atom number is increased. In
summary, the best inhibitory activity was showed by the ureas
bearing 4-chlorophenyl, 2,3-dimethylphenyl groups or aliphatic
chains.
inhibitor concentration (Tables 1e3), and IC50 values were deter-
mined in the relevant cases (Figs. 4e6). Compounds showing strong
STS inhibition were selected for whole cell experiments to assess
the ability to cross a lipid bilayer using intact monolayers of JEG-
3 cells (Figs. 3 and 6).
The following structure activity relationship (SAR) may be
noted regarding the inhibition data of compounds 5e18 shown in
Table 1 and Fig. 3. The benzylpiperazine urea 5 showed good STS
residual activity (18.7% 1.6) in isolated enzyme assay as well as in
intact JEG-3 cells (19.5% 1.3). The removal of the methylene
linker to give sulfamate 6 did not produce significant changes in
inhibitory activity against the isolated enzyme, while the activity
The compounds showing the best activity were evaluated for
their IC50 values in STS inhibition (Table 4). Compounds 13 and 16
showed IC50 values of 1.23 mM and 1.69 mM respectively. While
on JEG-3 was about
a
half as compared to compound
5
relatively modest, this was nevertheless encouraging, and further
substitutions produced compounds in the nM potency range, ie 17,
18, 8 and 14 for which IC50 values were determined as 43.7 nM,
33.2 nM, 94.0 nM and 66.0 nM respectively (Fig. 4).
It has been reported that introduction of fluorine atoms into of
biologically active compounds is a strategy to enhance their activity
and to modify their absorption, distribution, metabolism, and
excretion. In general, H/F exchange produces a more lipophilic
molecule. Moreover, in the case of STS inhibitors possessing the aryl
sulfamate pharmacophore generally substitution of the aromatic
ring with electron-withdrawing groups including halogenation,
even when not directly on the ring, lowers the pKa of the departing
phenolic group in the irreversible inhibition process and invariably
(32.8% 6.2). The introduction of a methyl group into 3-position of
aromatic ring (compound 7) produced an increase in activity
against isolated enzyme and cells (23.4% 1.1). The shift of the
methyl group to the 2-position to give urea 9 highly reduced the
activity. On the contrary, the introduction of a second methyl
group as in compound 14 produced high activity on the isolated
enzyme. The replacement of the 3-methyl group of compound 7
with a 3-methoxy group to give the urea 12 provided reduction of
inhibitory activity as well as the shift of the methoxy into 4-
position (urea 11). The 4-fluorine substituted urea 10 showed
poor activity, while the replacement of the fluorine with a 4-
chlorine atom afforded the high active urea 8. The introduction

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D. Moi et al. / European Journal of Medicinal Chemistry 182 (2019) 111614
Table 1
Table 2
STS inhibitory activity of sulfamates of the hydroxyarylcarbamate series 5e18.
STS inhibitory activity of sulfamates of the hydroxyarylcarbamate series 19e31.
Compound
X
R
Residual STS % activity SDa
7.1 0.7
Compound
R
Residual STS % activity SDa
18.7 1.6
19
2-F
5
20
21
22
2-F
2-F
2-Cl
n-octyl
n-decyl
11.4 0.8
15.2 3.3
110 10.8
6
7
16.9 3.3
10.4 1.9
23
2-Cl
15.1 0.2
8
9
14.9 1.6
47.9 6.3
24
25
26
2-Cl
2-Cl
3-F
n-octyl
n-decyl
13.9 1.5
25.4 0.4
107.9 6.2
27
3-F
108.8 3.2
10
11
12
13
93.5 6.5
37.0 2.4
26.3 1.9
23.0 3.4
28
29
30
31
3-F
3-F
3-Cl
3-Cl
n-octyl
n-decyl
n-octyl
n-decyl
29.6 1.2
7.8 1.2
71.6 4.8
102.1 3.5
a
Residual activity after JEG-3 cell lysate treatment with 10 mM inhibitor is shown.
(IC50 66 nM) and urea 23 (IC50 17 nM) activity indicated a positive
effect of the chlorine atom on the inhibitory activity. The compar-
ison of compound 20 and compound 24 (IC50 18.9 nM) indicated a
slight reduction in inhibitory activity, while the activity drop in
compound 25 (25.4% 0.4) confirming the trend showed by the
comparison of compounds 18 and 21. The shift on fluorine or
chlorine atoms into 3-position led reduction in activity as
compared with the unsubstituted analogs and the corresponding 2-
fluorine or 2-chlorine isomers except for compound 29 (7.8% 1.2)
that showed better than the isomer 21 (15.2% 3.3). Thus, final
optimization yielded a set of low nM potent halogenated inhibitors,
two of which 19 and 20 with IC50 values of 5.1 and 8.8 nM
respectively seem ideally suited for further development.
14
15
5.9 0.8
20.2 0.7
16
17
18
n-heptyl
n-octyl
n-decyl
27.7 1.6
12.4 2.7
3.5 0.7
a
Residual activity after JEG-3 cell lysate treatment with 10 mM inhibitor is shown.
The third series of ureidosulfamates was designed by intro-
duction on the piperazine nitrogen of 6-arylpyrimidine moieties
and led to ureas 35e41 showing STS residual activity ranging from
7.9 to 60.7% (Table 3).
increases overall potency considerably. Thus, we designed a second
series of ureas bearing substituted moieties unchanged but intro-
ducing fluorine and chlorine atoms on the sulfamoylated aromatic
ring (Tables 2 and 4 and Fig. 5).
The introduction of a 2-fluorine atom indeed produced an ex-
pected improvement in activity as shown by the paired comparison
of the activity of compound 8 (IC50 94 nM) with compound 19 (IC50
5.1 nM) activity and compound 17 (IC50 43.7 nM) with compound
20 (IC50 8.8 nM). On the contrary, compound 18 shows high activity
as compared with the fluorine analog 21. The replacement of the 2-
fluorine with a 2-chlorine produced variable results. Compound 22
(110% 10.8) is about ten times less active as compared to the
unsubstituted analog 8 (14.9% 1.6). The comparison of urea 14
The best activity in the case of a single aryl substitution was
shown by the 4-methoxyphenyl urea 36 (7.9% 1.3, HEC-3
13.7% 2.6, Fig. 6) both on isolated enzyme and whole cells. The
introduction of further methoxy groups led to a slight reduction in
activity as in compound 41 (19.0% 3.1) or a deep reduction of
activity as in compound 40 (60.7% 9.4). The removal of sub-
stituents as in compound 35 or the introduction of substituents into
the 3-position to afford ureas 37e39 produced reduction of activity.
The replacement of the aryl ring on the pyrimidine with a benzo-
furan ring to give compound 42 (4.7% 0.7) produced a high in-
crease in activity as compared to compound 36 especially in JEG-
3 cells (4.1% 0.9). With this encouraging potency sulfamate 42
was thus chosen for IC50 evaluation and it exhibited a value of

D. Moi et al. / European Journal of Medicinal Chemistry 182 (2019) 111614
5
Table 3
development and potential in vivo evaluation. Thus, the replace-
ment of arylamide moiety with a piperazinyl-ureido group high
increases STS inhibitory activity and piperazinyl-ureido aryl sulfa-
mate derivatives represent an attractive new class of STS inhibitors.
STS inhibitory activity of sulfamates 35e42.
4. Experimental section
4.1. General methods
Compound
Ar
Residual STS % activity SDa
65.9 5.4
35
All commercially available solvents and reagents were used
without further purification. NMR spectra were recorded on an
Inova 500 spectrometer (Varian, Palo Alto, CA, USA). The chemical
shifts are reported in part per million downfield from tetrame-
thylsilane (TMS) and the spectra were recorded in hexadeuter-
iodimethylsulphoxide (DMSO‑d6). Infrared spectra were recorded
on a Vector 22 spectrometer (Bruker, Bremen, Germany) in Nujol
mulls. The main bands are given in cmꢀ1. Positive-ion electrospray
ionization (ESI) mass spectra were recorded on a double-focusing
MAT 95 instrument (Finnigan, Waltham, MA, USA) with BE geom-
etry. Melting points (mp) were determined with a SMP1 Melting
Point apparatus (Stuart Scientific, Stone, UK) and are uncorrected.
All products reported showed spectral data in agreement with the
assigned structures. The purity of the tested compounds was
determined by combustion elemental analyses conducted by the
Microanalytical Laboratory of the Chemistry Department of the
University of Ferrara with a MT-5 CHN recorder elemental analyser
(Yanagimoto, Kyoto, Japan) and the values found were within 0.4%
of theoretical values. 4-(Phenoxycarbonyl)aminophenylsulfamate
4a [ 39], 4-Boc-piperazine-1-carboxamidine 32 [40] and sulfamates
5e8, 10e12, 14, 35e39, 41, 42 [41] were synthesized as previously
described.
36
37
7.9 1.3
26.7 8.5
38
39
39.4 9.9
44.3 1.0
40
41
60.7 9.4
19.0 3.1
42
4.7 0.7
4.2. Chemistry
a
Residual activity after JEG-3 cell lysate treatment with 10 mM inhibitor is shown.
4.2.1. 1-(Benzofuran-2-ylmethyl)piperazine
To a solution of N-Boc-piperazine (0.13 g, 0.7 mmol) in CH2Cl2
(10 mL) benzofuran-2-carboxaldehyde (0.13 mL, 1.1 mmol), sodium
NaHCO3 (0.07 g, 0.84 mmol) and sodium triacetoxyborohydride
(0.21 g, 1 mmol) were added; the mixture was then stirred at r.t. for
48 h. Then the reaction mixture was basified to pH 10 with a so-
lution of NaOH 0.1 N and extracted with CH2Cl2 (3 ꢁ 20 mL). The
organic phases were collected, dried over sodium Na2SO4, filtrated
and the solvent removed to obtain the desired compound. The
obtained residue was dissolved in dichloromethane (10 mL)
without further purification, added trifluoroacetic acid (5 mL) and
stirred at r.t. for 24 h. Then the solvent was removed under vacuum
and to the residue obtained diethyl ether (20 mL) was added,
leading to formation of a solid that was filtered to give the title
139 nM (Fig. 6) which already surpassed that of compound class A
[34]. IC50 values for the most optimized compounds are shown
collected in Table 4.
3. Conclusions
Following on from an initial arylamide series of aryl sulfamate-
based inhibitors of steroid sulfatase of moderate potency two new
series of piperazinyl-ureido aryl sulfamate-based STS inhibitors
were designed and synthesized. Hydroxyarylcarbamates 3 were
obtained by reacting 4-hydroxyarylamines with phenyl-
chloroformate and subsequent sulfamoylation and coupling of the
product with 1-substituted piperazines to give piperazinyl urea
derivatives 5e31. A pyrimidinyl-piperazinourea series 35e42 was
also synthesized by heterocyclization of 4-Boc-piperazine-1-
carboxamidine with 3-(dimethylamino)propenones, followed by
deprotection and subsequent coupling with the sulfamoylated
building block. The ability of the target compounds both to inhibit
STS in vitro using a JEG-3 human placenta choriocarcinoma cell line
lysate and to inhibit STS in a whole cell evaluation of membrane
permeability was explored using STS inhibition in intact JEG-3 cells.
SAR conclusions were drawn from both series and a progression
from the micromolar through to nanomolar inhibition level was
achieved by varying substitution patterns. Selected compounds
were chosen for final optimization through arylsulfamate ring
substitution with halogens. Ultimately, four compounds of low
nanomolar potency were achieved and two of these 19 and 20 with
IC50 values of 5.1 and 8.8 nM respectively are attractive for further
compound. Yield 74%. M.p. 123e124 ꢂC. 1H NMR (DMSO‑d6)
d 2.76
(s, 4H, CH2), 3.14 (s, 4H, CH2, 3.87 (s, 2H, CH2), 6.88 (s, 1H, Ar), 7.23
(d, J ¼ 7.5 Hz, 1H, Ar), 7.29 (d, J ¼ 8.0 Hz, 1H, Ar), 7.55 (d, J ¼ 7.5 Hz,
1H, Ar), 7.62 (d, J ¼ 8.0 Hz, 1H, Ar), 8.66 (s, 1H, NH). IR (Nujol)
1667 cmꢀ1 m/z 217 (M þ H)þ. Anal. Calcd. for C13H16N2O (216.28) C,
72.19; H, 7.46; N, 12.95. Found % C, 72.27; H, 7.48; N, 12.91.
4.2.2. General procedure for the preparation of phenyl(4-
hydroxylaryl)-carbamates (3b-e)
To an ice-cooled stirred solution of the appropriate 4-
hydroxyarylamine (5 mmol) and DIPEA (0.69 mL, 4 mmol) in
anhydrous THF (10 ml) phenylchloroformiate (0.5 mL, 4 mmol) was
added dropwise. The reaction mixture was stirred at room tem-
perature for 24 h, then water (100 mL) was added; the mixture was
stirred for additional 2 h, the formed solid filtered off, and vacuum
dried to give carbamates.

6
D. Moi et al. / European Journal of Medicinal Chemistry 182 (2019) 111614
Fig. 3. Evaluation of sulfamates 5e7 (A), 13 and 16 (B), 8, 17 and 18 (C) in whole cell JEG-3. All compounds were tested at 10
control. All data represents mean S.D., n ¼ 3.
mM, the reference inhibitor STX64 was used as positive
Fig. 4. IC50 of STS inhibition determined for sulfamates 8, 13, 16, 17, and 18, using JEG-3 protein. All data represents mean S.D., n ¼ 3.
4.2.2.1. Phenyl
N-(3-fluoro-4-hydroxyphenyl)carbamate
(3b).
7.21 (m, 3H, Ar), 7.35 (m, 1H, Ar), 7.43 (m, 2H, Ar), 9.54 (s, 1H, NH),
10.09 (s, 1H, OH). IR (Nujol) 3321, 1718, 1615 cmꢀ1 m/z 248
(M þ H)þ. Anal. Calcd. for C13H10FNO3 (247.22) C, 63.16; H, 4.08; N,
5.67. Found C, 63.23; H, 4.06; N, 5.70.
Following the general procedure, the title compound was prepared
starting from 2-fluoro-4-hydroxyphenyl amine. Yield 88%. M.p.
129e130 ꢂC. 1H NMR (DMSO‑d6)
d 6.91 (m, 1H, Ar), 7.07 (m, 1H, Ar),

D. Moi et al. / European Journal of Medicinal Chemistry 182 (2019) 111614
7
Fig. 5. The IC50 of the most potent haloarylcarbamate sulfamates (19, 20, 23 and 24) determined using JEG-3 protein. All data represents mean S.D., n ¼ 3.
Fig. 6. A) Evaluation of the STS inhibitory activity of sulfamate compounds 36 and 42 in whole cell JEG-3 tested at 10
mM, the reference inhibitor STX64 was used as positive control.
B) STS inhibition IC50 value of compound 42 determined using JEG-3 protein. All data represents mean S.D., n ¼ 3.
4.2.2.2. Phenyl
N-(3-chloro-4-hydroxyphenyl)carbamate
(3c).
Calcd. for C13H10FNO3 (247.22) C, 63.16; H, 4.08; N, 5.67. Found C,
63.09; H, 4.10; N, 5.64.
Following the general procedure, the title compound was prepared
starting from 3-chloro-4-hydroxyphenyl amine. Yield 79%. M.p.
132e133 ꢂC. 1H NMR (DMSO‑d6)
d 6.93 (m, 1H, Ar), 7.07 (m, 1H, Ar),
4.2.2.4. Phenyl
N-(2-chloro-4-hydroxyphenyl)carbamate
(3e).
7.22 (m, 2H, Ar), 7.36 (m, 1H, Ar), 7.43 (m, 2H, Ar), 7.52 (m, 1H, Ar),
9.89 (s, 1H, NH), 10.07 (s, 1H, OH). IR (Nujol) 3385, 1776, 1716,
1597 cmꢀ1 m/z 264 (M þ H)þ. Anal. Calcd. for C13H10ClNO3 (263.68)
C, 59.22; H, 3.82; N, 5.31. Found C, 59.16; H, 3.84; N, 5.28.
Following the general procedure, the title compound was prepared
starting from 2-chloro-4-hydroxyphenyl amine. Yield 90%. M.p.
128e129 ꢂC. 1H NMR (DMSO- d6)
d 6.76 (m, 1H, Ar), 6.90e7.41 (m,
7H, Ar), 9.30 (s,1H, NH), 9.89 (s,1H, OH). IR (Nujol) 3379,1736,1698,
1615 cmꢀ1 m/z 264 (M þ H)þ. Anal. Calcd. for C13H10ClNO3 (263.68)
C, 59.22; H, 3.82; N, 5.31. Found C, 59.28; H, 3.84; N, 5.35.
4.2.2.3. Phenyl
N-(2-fluoro-4-hydroxyphenyl)carbamate
(3d).
Following the general procedure, the title compound was prepared
starting from 3-fluoro-4-hydroxyphenyl amine. Yield 90%. M.p.
4.2.3. General procedure for the preparation of 4-
(phenoxycarbonyl)aminoarylsulfamate (4b-e)
126e127 ꢂC. 1H NMR (DMSO‑d6)
d
6.62 (m, 2H, Ar), 7.19 (m, 2H, Ar),
7.26 (m, 2H, Ar), 7.41 (m, 2H, Ar), 9.52 (s,1H, NH), 9.82 (s,1H, OH). IR
To
a stirred solution of phenyl(4-hydroxylaryl)-carbamate
(Nujol) 3425, 3403, 1730, 1639, 1610 cmꢀ1 m/z 248 (M þ H)þ. Anal.
(5 mmol) in anhydrous DMA (10 mL, 114 mmol), freshly prepared

8
D. Moi et al. / European Journal of Medicinal Chemistry 182 (2019) 111614
62%. M.p. 117e118 ꢂC. 1H NMR (DMSO‑d6)
d 6.44 (m, 1H, Ar), 6.76
Table 4
Summary IC50 values for STS inhibition.
(m, 1H, Ar), 7.31e7.36 (m, 6H, Ar), 8.20 (s, 2H, NH2), 9.78 (s, 1H, NH).
IR (Nujol) 3315, 3222, 1743, 1579 cmꢀ1 m/z 327 (M þ H)þ. Anal.
Calcd. for C13H11FN2O5S (326.30) C, 47.85; H, 3.40; N, 8.59. Found C,
47.92; H, 3.38; N 8.55.
4.2.3.4. 2-Fluoro-4-((phenoxycarbonyl)amino)phenyl sulfamate (4e).
Following the general procedure, the title compound was prepared
starting from phenyl (2-fluoro-4-hydroxyphenyl)carbamate. Yield
Compound
8
X
H
R
STS inhibition IC50
94.0 nM
56%. M.p.112e113 ꢂC. 1H NMR (DMSO‑d6)
d 7.27 (m, 4H, Ar), 7.42 (m,
3H, Ar), 7.68 (d, J ¼ 8.5 Hz, 1H, Ar), 8.13 (s, 2H, NH2), 9.81 (s, 1H, NH).
IR (Nujol) 3322, 3238, 1744, 1587 cmꢀ1 m/z 327 (M þ H)þ. Anal.
Calcd. for C13H11FN2O5S (326,30) C, 47.85; H, 3.40; N, 8.59. Found C,
47.79; H, 3.39; N, 8.63.
13
14
H
H
1.23 mM
66.0 nM
4.2.4. General procedure for the synthesis of 4-(piperazinocarbonyl)
aminosulfamates (5ꢀ31)
A mixture of 4-(phenoxycarbonyl)aminoarylsulfamate (1 mmol)
and substituted 1-substituted piperazine (1 mmol) and DIPEA
(0.5 mmol), in anhydrous DMSO (3 mL) was stirred at room tem-
perature for 24 h. Then, water (10 mL) was added and the mixture
was stirred at room temperature until a solid is formed. The solid
formed was filtered off, washed with water and air dried to give the
title ureas.
16
17
18
19
H
H
H
2-F
n-heptyl
n-octyl
n-decyl
1.69 mM
43.7 nM
33.2 nM
5.1 nM
20
23
2-F
2-Cl
n-octyl
n-octyl
8.8 nM
17.0 nM
4.2.4.1. 4-(4-(o-Tolyl)piperazine-1-carboxamido)phenyl
sulfamate
(9). Following the general procedure, the title compound was
prepared starting from o-tolylpiperazine. Yield 72%. M.p.
170e171 ꢂC. 1H NMR (DMSO‑d6)
d 2.29 (s, 3H, CH3), 2.85 (s, 4H, CH2),
24
42
2-Cl
H
18.9 nM
139 nM
3.60 (s, 4H, CH2), 6.98 (d, J ¼ 7.0 Hz, 1H, Ar), 7.04 (d, J ¼ 8.5 Hz, 1H,
Ar), 7.15 (d, J ¼ 8.0 Hz, 2H, Ar), 7.18 (d, J ¼ 7.0 Hz, 2H, Ar), 7.52 (d,
J ¼ 8.0 Hz, 2H, Ar), 7.86 (s, 2H, NH2), 8.70 (s, 1H, NH). 13C NMR
(DMSO‑d6)
d 17.8, 53.4 (2C), 55.8 (2C), 117.8, 120.8 (2C), 125.2 (2C),
126.6, 127.1, 130.2, 133.6, 139.8, 146.2, 152.3, 157.9. IR (Nujol) 3329,
1647, 1535 cmꢀ1 m/z 391 (M þ H)þ. Anal. Calcd. for C18H22N4O4S
(390,46) C, 55.37; H, 5.68 N, 14.35. Found C, 55.30; H, 5.66; N, 14.37.
sulfamoyl chloride (0.81 g, 7 mmol) in DMA (5 mL, mmol) was
added dropwise in 30 min. The mixture was stirred at room tem-
perature overnight, then water (20 mL) was added. The mixture
was stirred for an additional 2 h, then the white solid formed was
filtered off and dried to give sulfamates in good purity to be used in
the next step without further purification.
4.2.4.2. 4-(4-(3,4-Dichlorophenyl)piperazine-1-carboxamido)phenyl
sulfamate (13). Following the general procedure, the title com-
pound was prepared starting from 3,4-dichlorophenylpiperazine.
Yield 98%. M.p. 144e145 ꢂC. 1H NMR (DMSO‑d6)
d 3.23 (m, 4H, CH2),
3.58 (m, 4H, CH2), 6,97 (s,1H, Ar), 7.14 (d, J ¼ 8.5 Hz, 2H, Ar), 7.20 (m,
2H, Ar), 7.52 (d, J ¼ 8.5 Hz, 2H, Ar), 7.87 (s, 2H, NH2), 8.72 (s, 1H, NH).
13C NMR (DMSO‑d6)
d 52.3 (2C), 53.2 (2C), 111.2, 114.2, 122.4, 122.9,
4.2.3.1. 3-Chloro-4-((phenoxycarbonyl)amino)phenyl sulfamate (4b).
Following the general procedure, the title compound was prepared
starting from phenyl N-(3-chloro-4-hydroxyphenyl)carbamate.
123.6 (2C), 127.5, 133.0 (2C), 144.2, 144.8 (2C), 157.4. IR (Nujol) 3341,
1649 cmꢀ1 m/z 446 (M þ H)þ. Anal. Calcd. for C17H18Cl2N4O4S
(445,32) C, 45.85; H, 4.07; N, 12.58. Found C, 45.92; H, 4.05; N,
12.62.
Yield 40%. M.p. 109e110 ꢂC. 1H NMR (DMSO‑d6)
d 6.75 (m, 1H, Ar),
6.89 (m, 1H, Ar), 7.23e7.41 (m, 6H, Ar), 8.15 (s, 2H, NH2), 9.89 (s, 1H,
NH). IR (Nujol) 3319, 3224,1738,1588 cmꢀ1 m/z 343 (M þ H)þ. Anal.
Calcd. for C13H11ClN2O5S (342.75) C, 45.55; H, 3.23; N, 8.17. Found C,
45.61; H, 3.25; N, 8.20.
4.2.4.3. 4-(4-(Benzofuran-2-ylmethyl)piperazine-1-carboxamido)
phenyl sulfamate (15). Following the general procedure, the title
compound was prepared starting from 1-(benzofuran-2-ylmethyl)
piperazine. Yield 40%. M.p. 140e141 ꢂC. 1H NMR (DMSO‑d6)
d 2.54
4.2.3.2. 2-Chloro-4-((phenoxycarbonyl)amino)phenyl sulfamate (4c).
Following the general procedure, the title compound was prepared
starting from phenyl N-(2-chloro-4-hydroxyphenyl)carbamate.
(s, 2H, CH2), 3.30 (m, 4H, CH2), 3.51 (m, 4H, CH2), 6.81 (s, 1H, Ar),
7.13 (d, J ¼ 9.0 Hz, 1H, Ar), 7.16 (d, J ¼ 9.0 Hz, 1H, Ar), 7.26 (d,
J ¼ 9.5 Hz, 1H, Ar), 7.43 (d, J ¼ 7.0 Hz, 1H, Ar), 7.46 (d, J ¼ 7.0 Hz, 1H,
Ar), 7.49 (d, J ¼ 9.5 Hz, 1H, Ar), 7.47 (m, 2H, Ar), 7.88 (s, 2H, NH2),
Yield 36%. M.p. 120e121 ꢂC. 1H NMR (DMSO‑d6)
d 7.23 (m, 3H, Ar),
8.61 (s, 1H, NH). 13C NMR (DMSO‑d6)
d 50.1 (2C), 52.2 (2C), 58.3,
7.43 (m, 4H, Ar), 7.71 (s, 1H, Ar), 8.17 (s, 2H, NH2), 10.48 (s, 1H, NH).
IR (Nujol) 3302, 3216, 1752, 1591 cmꢀ1 m/z 343 (M þ H)þ. Anal.
Calcd. for C13H11ClN2O5S (342.75) C, 45.55; H, 3.23; N, 8.17. Found C,
45.49; H 3.24; N, 8.14.
103.0, 111.2, 120.9 (2C), 121.6, 123.4 (2C), 123.9, 125.2, 127.1, 131.8,
146.3, 155.1, 155.2, 157.4. IR (Nujol) 3386, 1645 cmꢀ1 m/z 431
(M þ H)þ. Anal. Calcd. for C20H22N4O5S (430,13) C, 55.80; H, 5.15; N,
13.02. Found C, 55.73; H, 5.13; N, 12.98.
4.2.3.3. 3-Fluoro-4-((phenoxycarbonyl)amino)phenyl sulfamate (4d).
Following the general procedure, the title compound was prepared
starting from phenyl (3-fluoro-4-hydroxyphenyl)carbamate. Yield
4.2.4.4. 4-(4-Heptylpiperazine-1-carboxamido)phenyl
(16). Following the general procedure, the title compound was
sulfamate

D. Moi et al. / European Journal of Medicinal Chemistry 182 (2019) 111614
9
2
prepared starting from n-heptylpiperazine. Yield 42%. M.p.
114e115 ꢂC. 1H NMR (DMSO‑d6)
0.87 (m, 3H, CH3), 1.28 (m, 12H,
47.1 (2C), 57.3 (2C), 62.2, 113.4 (d, JCF ¼ 23.7 Hz), 118.4 (d,
4 3
d
3JCF ¼ 5.4 Hz), 120.9 (d, JCF ¼ 4.4 Hz), 133.8 (d, JCF ¼ 9.5 Hz), 143.2
2
CH2),1.48 (m, 2H, CH2), 3.29 (m, 4H, CH2), 3.48 (m, 2H, CH2), 7.14 (s,
(d, JCF ¼ 16.2 Hz), 155.4 (d, 1JCF ¼ 232.0 Hz), 156.1. IR (Nujol) 3366,
2H, Ar), 7.48 (s, 2H, Ar), 7.86 (s, 2H, NH2), 8.63 (s, 1H, NH). 13C NMR
1644, 1519 cmꢀ1 m/z 459 (M þ H)þ. Anal. Calcd. for C21H35FN4O4S
(DMSO‑d6)
d
14.4, 23.2, 26.8, 28.4, 29.6, 32.2, 51.8 (2C), 56.8 (2C),
(458.59) C, 55.00; H, 7.69; N,12.22. Found C, 54.93; H, 7.71; N,12.26.
57.3, 121.1 (2C), 123.6 (2C), 132.5, 148.1, 156.2. IR (Nujol) 3348, 1642,
1538 cmꢀ1 m/z 399 (M þ H)þ. Anal. Calcd. for C18H30N4O4S (398.52)
C, 54.25; H, 7.59; N, 14.06. Found C, 54.31; H, 7.57; N, 14.09.
4.2.4.10. 2-Chloro-4-(4-(4-chlorophenyl)piperazine-1-carboxamido)
phenyl sulfamate (22). Following the general procedure, the title
compound was prepared starting from 4-chlorophenylpiperazine.
4.2.4.5. 4-(4-Octylpiperazine-1-carboxamido)phenyl sulfamate (17).
Following the general procedure, the title compound was prepared
starting from n-octylpiperazine. Yield 38%. M.p. 154e155 ꢂC. 1H
NMR (DMSO‑d6) 0.87 (m, 3H, CH3), 1.27 (m, 14H, CH2), 2.36 (s, 4H,
CH2), 3.43 (s, 4H, CH2), 7.14 (d, J ¼ 7.0 Hz, 2H, Ar), 7.49 (d, J ¼ 7.5 Hz,
Yield 82%. M.p. 84e85 ꢂC. 1H NMR (DMSO‑d6)
d
3.16 (s, 4H, CH2),
3.57 (s, 4H, CH2), 6.86 (d, J ¼ 8.0 Hz, 2H, Ar), 7.19 (m, 3H, Ar), 7.25 (m,
2H, Ar), 7.51 (s, 2H, NH2), 8.47 (s,1H, NH). 13C NMR (DMSO‑d6)
52.4
d
(2C), 54.2 (2C), 116.2 (2C), 117.2, 120.9,123.0, 124.7, 128.8,130.2 (2C),
133.5, 148.0, 152.6, 158.0 IR (Nujol) 3279, 1638, 1594 cmꢀ1 m/z 446
(M þ H)þ. Anal. Calcd. for C17H18Cl2N4O4S (445.32) C, 45.85; H, 4.07;
N, 12.58. Found C, 45.89; H, 4.09; N 12.61.
2H, Ar), 7.86 (s, 2H, NH2), 8.59 (s,1H, NH). 13C NMR (DMSO‑d6)
d 17.1,
25.2, 30.1, 31.8, 32.0, 34.4 (2C), 42.3 (2C), 55.8 (2C), 60.9, 123.5 (2C),
125.2 (2C), 142.1, 147.6, 158.0. IR (Nujol) 3373, 1642 cmꢀ1 m/z 413
(M þ H)þ. Anal. Calcd. for C19H32N4O4S (412.55) C, 55.32; H, 7.82; N,
13.58. Found C, 55.27; H, 7.98; N, 13.62.
4.2.4.11. 2-Chloro-4-(4-(2,3-dimethylphenyl)piperazine-1-
carboxamido)phenyl sulfamate (23). Following the general proced-
ure, the title compound was prepared starting from 2,3-
dimethylphenylpiperazine. Yield 60%. M.p. 89e90 ꢂC. 1H NMR
4.2.4.6. 4-(4-Decylpiperazine-1-carboxamido)phenyl sulfamate (18).
Following the general procedure, the title compound was prepared
starting from n-decylpiperazine. Yield 51%. M.p. 159e160 ꢂC. 1H
NMR (DMSO‑d6) 0.86 (m, 3H, CH3), 1.26 (m, 18H, CH2), 2.38 (s, 4H,
CH2), 3.44 (s, 4H, CH2), 7.13 (d, J ¼ 9.0 Hz, 2H, Ar), 7.50 (d, J ¼ 9.0 Hz,
(DMSO‑d6)
3.58 (s, 4H, CH2), 6.88 (m, 2H, Ar), 7.06 (m, 2H, Ar), 7.21 (m, 2H, Ar),
7.52 (s, 2H, NH2), 8.44 (s, 1H, NH). 13C NMR (DMSO‑d6)
16.8, 23.4,
d 2.17 (s, 3H, CH3), 2.20 (s, 3H, CH3), 2.80 (s, 4H, CH2),
d
47.4, (2C), 55.0 (2C), 119.8, 123.6 (2C), 125.3 (2C), 128.0, 128.9, 130.7,
140.5, 142.1, 147.7, 154.2, 158.2. IR (Nujol) 3321, 1638, 1592 cmꢀ1 m/z
439 (M þ H)þ. Anal. Calcd. for C19H23ClN4O4S (438.93) C, 51.99; H,
5.28; N, 12.76. Found C, 52.05; H, 5.26; N, 12.79.
2H, Ar), 7.86 (s, 2H, NH2), 8.60 (s,1H, NH). 13C NMR (DMSO‑d6)
d 17.1,
25.3, 31.8 (2C), 32.1 (2C), 32.2, 34.5 (2C), 42.5 (2C), 46.7 (2C), 60.9,
123.5 (2C), 125.2 (2C), 142.1, 147.6, 157.9.IR (Nujol) 3388,
1642 cmꢀ1 m/z 441 (M þ H)þ. Anal. Calcd. for C21H36N4O4S (440.60)
C, 57.25; H, 8.24; N, 12.72. Found C, 55.19; H, 8.21; N,12.76.
4.2.4.12. 2-Chloro-4-(4-octylpiperazine-1-carboxamido)phenyl sul-
famate (24). Following the general procedure, the title compound
was prepared starting from n-octylpiperazine. Yield 18%. M.p.
4.2.4.7. 4-(4-(4-Chlorophenyl)piperazine-1-carboxamido)-2-
fluorophenyl sulfamate (19). Following the general procedure, the
title compound was prepared starting from 4-(chlorophenyl)
108e110 ꢂC. 1H NMR (DMSO‑d6)
d 0.88 (m, 3H, CH3), 1.29 (m, 14H,
CH2), 2.34 (s, 4H, CH2), 3.46 (s, 4H, CH2), 7.17 (d, J ¼ 7 Hz, 1H, Ar),
piperazine. Yield 90%. M.p.102e103 ꢂC. 1H NMR (DMSO‑d6)
d 3.17 (s,
7.44 (s, 1H, Ar), 7.47 (d, J ¼ 7.5 Hz, 1H, Ar), 7.75 (s, 2H, NH2), 8.44 (s,
4H, CH2), 3.60 (s, 4H, CH2), 6.84 (m, 1H, Ar), 7.00 (d, J ¼ 8.0 Hz, 2H,
1H, NH). 13C NMR (DMSO‑d6)
d 15.0, 22.7, 26.2, 28.5, 29.0 (2C), 32.7,
Ar), 7.26 (d, J ¼ 8.0 Hz, 2H, Ar), 7.29 (m, 1H, Ar), 7.41 (m, 1H, Ar), 8.09
53.1 (2C), 58.3 (2C), 61.8, 119.4, 122.2, 123.6, 125.8, 130.7, 154.2,
155.5. IR (Nujol) 3346, 1641, 1595 cmꢀ1 m/z 447 (M þ H)þ. Anal.
Calcd. for C19H31ClN4O4S (446.99) C, 51.05; H, 6.99; N, 12.53. Found
C, 51.12; H, 7.02; N, 12.57.
(s, 2H, NH2), 8.99 (s, 1H, NH). 13C NMR (DMSO‑d6)
d 42.6 (2C), 51.2
2
3
(2C), 111.6 (d, JCF ¼ 19.1 Hz), 117.8 (d, JCF ¼ 8.3 Hz), 119.1 (d,
4JCF ¼ 3.5 Hz), 120.3 (2C), 125.8, 131.8 (2C), 134.4 (d, JCF ¼ 13.4 Hz),
2
3
1
135.6 (d, JCF ¼ 9.5 Hz), 142.7, 157.9 (d, JCF ¼ 242.1 Hz), 158.2. IR
(Nujol) 3325, 1645, 1605 cmꢀ1 m/z 429 (M þ H)þ. Anal. Calcd. for
4.2.4.13. 2-Chloro-4-(4-decylpiperazine-1-carboxamido)phenyl sul-
famate (25). Following the general procedure, the title compound
was prepared starting from n-decylpiperazine. Yield 70%. M.p.
C
17H18ClFN4O4S (428.87) C, 47.61; H, 4.23; N, 13.06, Found C, 47.66;
H, 4.21; N, 13.10.
92e93 ꢂC. 1H NMR (DMSO‑d6)
d 0.87 (m, 3H, CH3), 1.27 (m, 18H,
4.2.4.8. 2-Fluoro-4-(4-octylpiperazine-1-carboxamido)phenyl sulfa-
mate (20). Following the general procedure, the title compound
was prepared starting from 4-octylpiperazine. Yield 15%. M.p.
CH2), 2.36 (s, 4H, CH2), 3.46 (s, 4H, CH2), 7.11 (d, J ¼ 9.0 Hz, 1H, Ar),
7.46 (d, J ¼ 9.0 Hz, 1H, Ar), 7.53 (m, 1H, Ar), 7.61 (s, 2H, NH2), 9.36 (s,
1H, NH). 13C NMR (DMSO‑d6)
d 16.8, 25.2, 31.7 (2C), 31.9 (2C), 32.0,
86e87 ꢂC. 1H NMR (DMSO‑d6)
d
0.87 (t, J ¼ 6.5 Hz, 3H, CH3), 1.27 (m,
14H, CH2), 2.38 (s, 4H, CH2), 3.43 (s, 4H, CH2), 6.81 (m, 1H, Ar), 7.00
(m, 1H, Ar), 7.32 (m, 1H, Ar), 7.41 (s, 2H, NH2), 8.40 (s, 1H, NH). 13
33.1 (2C), 53.1 (2C), 57.6 (2C), 60.2, 119.2, 120.8, 122.4, 124.8, 131.3,
155.0, 157.4. IR (Nujol) 3348, 1633, 1596 cmꢀ1 m/z 475 (M þ H)þ.
Anal. Calcd. for C21H35ClN4O4S (474.04) C, 53.09; H, 7.43; N 11.79.
Found C, 53.16; H, 7.45; N, 11.76.
C
NMR (DMSO‑d6)
d 17.0, 25.2, 28.9, 30.0, 31.8, 34.4 (2C), 46.4 (2C),
55.6 (2C), 60.7, 111.6 (d, 2JCF ¼ 22.9 Hz), 119.0 (d, 3JCF ¼ 6.1 Hz), 120.2
4
3
2
(d, JCF ¼ 3.9 Hz), 135.6 (d, JCF ¼ 8.6 Hz), 142.5 (d, JCF ¼ 12.4 Hz),
154.3 (d, 1JCF ¼ 229.8 Hz), 158.1. IR (Nujol) 3323, 1643, 1605 cmꢀ1 m/
z 431 (M þ H)þ. Anal. Calcd. for C19H31FN4O4S (430.54) C, 53.00; H,
7.26; N, 13.01. Found C, 53.05; H, 7.24; N, 12.97.
4.2.4.14. 4-(4-(4-Chlorophenyl)piperazine-1-carboxamido)-3-
fluorophenyl sulfamate (26). Following the general procedure, the
title
chlorophenylpiperazine. Yield 20%. M.p. 94e95 ꢂC. 1H NMR
(DMSO‑d6) 3.16 (s, 4H, CH2), 3.55 (s, 4H, CH2), 6.55 (m,1H, Ar), 6.91
compound
was
prepared
starting
from
4-
d
4.2.4.9. 4-(4-Decylpiperazine-1-carboxamido)-2-fluorophenyl sulfa-
mate (21). Following the general procedure, the title compound
was prepared starting from 4-decylpiperazine. Yield 22%. M.p.
(m, 1H, Ar), 7.00 (d, J ¼ 8.0 Hz, 2H, Ar), 7.11 (m, 1H, Ar), 7.25 (d,
J ¼ 8.0 Hz, 2H, Ar), 8.10 (s, 2H, NH2), 8.87 (s, 1H, NH). 13C NMR
2
(DMSO‑d6)
d
45.3 (2C), 49.4 (2C), 109.4 (d, JCF ¼ 13.6 Hz), 115.8 (d,
4
48e49 ꢂC. 1H NMR (DMSO‑d6)
d
0.91 (t, J ¼ 7.0 Hz, 3H, CH3), 1.31 (m,
18H, CH2), 2.39 (s, 4H, CH2), 3.39 (s, 4H, CH2), 6.86 (m, 1H, Ar), 7.11
(m, 1H, Ar), 7.27 (m, 1H, Ar), 7.43 (s, 2H, NH2), 8.40 (s, 1H, NH). 13
NMR (DMSO‑d6) 17.0, 22.7, 25.22, 27.5, 28.2, 30.4, 32.2, 34.7 (2C),
3JCF ¼ 7.1 Hz), 117.2 (d, JCF ¼ 3.6 Hz), 121.3 (2C), 125.0 (d,
3JCF ¼ 8.6 Hz), 127.6, 130.8 (2C), 142.8 (d, JCF ¼ 9.5 Hz), 145.8, 153.9
3
1
C
(d, JCF ¼ 244.5 Hz), 163.0. IR (Nujol) 3431, 3165, 1645, 1627,
d
1597 cmꢀ1 m/z 429 (M þ H)þ. Anal. Calcd. for C17H18ClFN4O4S

10
D. Moi et al. / European Journal of Medicinal Chemistry 182 (2019) 111614
(428.87) C, 47.61; H, 4.23; N, 13.06. Found C, 47.66; H, 4.21; N, 13.10.
4.2.5. (E)-3-(Dimethylamino)-1-(3,4-dimethoxyphenyl)prop-2-en-
1-one (33f)
A mixture of 3,4-dimethoxyacetophenone (0.9 g, 5 mmol) and
DMF-DMA (1.79 g, 15 mmol) in anhydrous toluene (10 mL) was
refluxed for 1 h, then was allowed to reach the room temperature
and stirred for additional 24 h. The mixture was carefully concen-
trated in vacuum to the title compound in 77% yield. M.p.
4.2.4.15. 4-(4-(2,3-Dimethylphenyl)piperazine-1-carboxamido)-3-
fluorophenyl sulfamate (27). Following the general procedure, the
title
dimethylphenylpiperazine. Yield 58%. M.p. 87e88 ꢂC. 1H NMR
(DMSO‑d6) 2.21 (s, 3H, CH3), 2.23 (s, 3H, CH3), 2.70 (s, 2H, CH2),
compound
was
prepared
starting
from
2,3-
d
113e114 ꢂC (n-hexane). 1H NMR (DMSO‑d6)
d 2.91 (s, 3H, CH3), 3.12
2.87 (s, 2H, CH2), 3.57 (s, 4H, CH2), 6.57 (m, 2H, Ar), 6.91 (m, 3H, Ar),
7.11 (m, 1H, Ar), 8.07 (s, 2H, NH2), 9.04 (s, 1H, NH). 13C NMR
(s, 3H, CH3), 3.81 (s, 6H, OCH3), 5.81 (d, J ¼ 12.0 Hz, 1H, CH), 6.97 (d,
J ¼ 8.0 Hz, 1H, Ar), 7.45 (s, 1H, Ar), 7.53 (d, J ¼ 8.5 Hz, 1H, Ar), 7.65 (d,
J ¼ 12.0 Hz, 1H, CH). IR (Nujol) 3583, 1636 cmꢀ1 m/z 254 (M þ H)þ.
Anal. Calcd. for C13H17NO3 (253.28) C, 66.36; H, 7.28; N, 5.95. Found
C, 66.27; H, 7.31; N, 5.93.
(DMSO‑d6)
d
16.8, 23.4, 44.2 (2C), 53.8 (2C), 109.6 (d, 2JCF ¼ 15.2 Hz),
3
4
112.1, 115.7 (d, JCF ¼ 8.1 Hz), 117.0 (d, JCF ¼ 3.5 Hz), 121.2, 122.1,
3
3
124.9 (d, JCF ¼ 8.4 Hz), 129.3, 140.2, 142.7 (d, JCF ¼ 9.0 Hz), 149.4,
154.3 (d, 1JCF ¼ 243.6 Hz), 159.9. IR (Nujol) 3280, 1625,
1590 cmꢀ1 m/z 423 (M þ H)þ. Anal. Calcd. for C19H23FN4O4S
(422.47) C, 54.02; H, 5.49; N,13.26. Found C, 54.08; H, 5.47; N,13.22.
4.2.6. 4-(3,4-Dimethoxyphenyl)-2-(piperazin-1-yl)pyrimidine (34f)
A solution of (E)-3-(dimethylamino)-1-(3,4-dimethoxyphenyl)
prop-2-en-1-one (0.51 g, 2 mmol), 4-(tert-butoxycarbonyl)pipera-
zine-1-carboxamidine (1.02 g, 2.2 mmol) and sodium methylate
30% MeOH solution (0.8 ml, 4 mmol) in anhydrous EtOH (5 mL) was
refluxed 8 h. After cooling to room temperature the solvent was
removed under reduced pressure. The residue was treated with
ethyl acetate (20 mL) and washed with water (3 ꢁ 10 mL) and brine
(10 mL). After drying over sodium sulphate the solvent was
removed under reduced pressure. Then the residue was solubilised
in anhydrous dichloromethane (10 mL) and trifluoroacetic acid
(5 mL) was added. The mixture was stirred at room temperature
overnight and after evaporation of the solvent, the residue was
treated with a diethyl ether (20 mL) to obtain a solid that was
filtered off and dried. The formed solid was used in the next step
without further purification. Yield 48%. M.p. >240 ꢂC. 1H NMR
4.2.4.16. 3-Fluoro-4-(4-octylpiperazine-1-carboxamido)phenyl sul-
famate (28). Following the general procedure, the title compound
was prepared starting from n-octylpiperazine. Yield 11%. M.p.
78e79 ꢂC. 1H NMR (DMSO‑d6)
d 0.87 (m, 3H, CH3), 1.28 (m, 14H,
CH2), 2.45 (s, 4H, CH2), 3.38 (s, 4H, CH2), 7.22 (d, J ¼ 7.0 Hz, 1H, Ar),
7.51 (s, 1H, Ar), 7.59 (d, J ¼ 7.5 Hz, 1H, Ar), 7.73 (s, 2H, NH2), 8.51 (s,
1H, NH). 13C NMR (DMSO‑d6)
d 17.1, 25.1, 28.7, 29.9, 31.8, 34.0 (2C),
2
46.8 (2C), 55.5 (2C), 60.7, 109.2 (d, JCF ¼ 12.8 Hz), 116.0 (d,
4
3
3JCF ¼ 8.4 Hz), 117.3 (d, JCF ¼ 3.6 Hz), 125.0 (d, JCF ¼ 8.6 Hz), 142.4
3
(d, JCF ¼ 8.4 Hz), 155.0 (d, 1JCF ¼ 247.0 Hz), 161.2. IR (Nujol) 3430,
1644, 1596 cmꢀ1 m/z 431 (M þ H)þ. Anal. Calcd. for C19H31FN4O4S
(430.54) C, 53.00; H, 7.26; N 13.01. Found C, 52.94; H, 7.29; N 13.05.
4.2.4.17. 4-(4-Decylpiperazine-1-carboxamido)-3-fluorophenyl sul-
famate (29). Following the general procedure, the title compound
was prepared starting from n-decylpiperazine. Yield 12%. M.p.
(DMSO‑d6) d 1.91 (s,1H, NH), 2.79 (s, 4H, CH2), 3.33 (s, 4H, CH2), 4.31
(s, 6H, OCH3), 6.97 (d, J ¼ 7.0 Hz, 1H, Ar), 7.25 (s, 1H, Ar), 7.54 (d,
J ¼ 7.5 Hz, 1H, Ar), 7.85 (m, 2H, Ar). IR (Nujol) 3583, 1636 cmꢀ1 m/z
301 (M þ H)þ. Anal. Calcd. for C16H20N4O2 (300.36) C, 63.98; H,
6.71; N, 18.65. Found C, 63.87; H, 6.74; N, 18.70.
81e82 ꢂC. 1H NMR (DMSO‑d6)
d 0.92 (m, 3H, CH3), 1.95 (m, 18H,
CH2), 2.17 (s, 4H, CH2), 3.39 (s, 4H, CH2), 7.15 (m,1H, Ar), 7.44 (m,1H,
Ar), 7.77 (m, 1H, Ar), 7.80 (s, 2H, NH2), 8.49 (s, 1H, NH). 13C NMR
(DMSO‑d6) d 16.8, 25.2, 31.7 (2C), 31.9 (2C), 32.0, 33.1 (2C), 53.1 (2C),
4.2.7. General procedure for the synthesis of 4-(4-(4-aryl)
pyrimidin-2-yl)piperazinocarbonyl)aminophenyl sulfamates
(35e42)
57.6 (2C), 60.2, 109.3 (d, 2JCF ¼ 12.7 Hz), 115.9 (d, 3JCF ¼ 8.5 Hz), 117.5
4
3
3
(d, JCF ¼ 3.5 Hz), 124.8 (d, JCF ¼ 8.7 Hz), 142.2 (d, JCF ¼ 8.5 Hz),
155.6 (d, 1JCF ¼ 242.3 Hz), 159.1 IR (Nujol) 3346, 1644, 1614 cmꢀ1 m/
z 459 (M þ H)þ. Anal. Calcd. for C21H35FN4O4S (458.59) C, 55.00; H,
7.69; N 12.22. Found C, 55.06; H, 7.66; N, 12.26.
A
mixture of 4-(phenoxycarbonyl)aminophenylsulfamate
(0,31 g, 1 mmol) and pyrimidines 34a-h (1 mmol), in anhydrous
DMSO (3 mL) was stirred at room temperature for 24 h. Then, water
(10 mL) was added and the mixture was stirred at room tempera-
ture until a solid is formed. The formed solid was filtered off,
washed with water, air dried and recrystallized from EtOH to give
ureas 35e42.
4.2.4.18. 3-Chloro-4-(4-octylpiperazine-1-carboxamido)phenyl sul-
famate (30). Following the general procedure, the title compound
was prepared starting from n-octylpiperazine. Yield 38%. M.p.
106e107 ꢂC. 1H NMR (DMSO‑d6)
d 0.93 (m, 3H, CH3), 1.04 (m, 14H,
CH2), 2.18 (s, 4H, CH2), 3.65 (s, 4H, CH2), 7.24 (m, 1H, Ar), 7.60 (m,
4.2.7.1. 4-(4-(4-(3,4-Dimethoxyphenyl)pyrimidin-2-yl)piperazine-1-
carboxamido)phenyl sulfamate (40). Following the general proced-
ure, the title compound was obtained in 27% yield. Oil. 1H NMR
2H, Ar), 7.90 (s, 2H, NH2), 8.78 (s, 1H, NH). 13C NMR (DMSO‑d6)
d
17.0, 25.2, 29.7, 31.8, 31.9, 34.4 (2C), 45.9 (2C), 55.2 (2C), 60.3,
119.3, 121.9, 123.1, 124.6, 135.8, 151.2, 157.9. IR (Nujol) 3324, 1636,
1603 cmꢀ1 m/z 447 (M þ H)þ. Anal. Calcd. for C19H31ClN4O4S
(446.99) C, 51.05; H, 6.99; N, 12.53. Found C, 51.11; H, 6.96; N, 12.57.
(DMSO‑d6)
d 3.56 (s, 4H, CH2), 3.83 (s, 4H, CH2), 4.42 (s, 6H, CH2),
7.07 (m, 2H, Ar), 7.19 (s, 1H, Ar), 7.45 (d, J ¼ 7.5 Hz, 1H, Ar), 7.56 (d,
J ¼ 8.0 Hz, 2H, Ar), 7.77 (d, J ¼ 7.0 Hz,1H, Ar), 7.84 (s,1H, NH), 8.33 (s,
2H, NH2), 8.61 (d, J ¼ 8.5 Hz, 2H, Ar). 13C NMR (DMSO‑d6)
d 51.1 (2C),
4.2.4.19. 3-Chloro-4-(4-decylpiperazine-1-carboxamido)phenyl sul-
famate (31). Following the general procedure, the title compound
was prepared starting from n-decylpiperazine. Yield 35%. M.p.
52.0 (2C), 57.2 (2C), 105.3, 108.6, 110.8, 121.3 (2C), 122.0, 123.3 (2C),
130.3, 133.6, 148.1, 150.3 (2C), 154.2, 155.5, 163.5, 166.7. IR (Nujol)
3328, 1634, 1516 cmꢀ1 m/z 515 (M þ H)þ. Anal. Calcd. for
57e58 ꢂC. 1H NMR (DMSO‑d6)
d
0.95 (m, 3H, CH3), 1.95 (m, 16H,
C23H26N6O6S (514.55) C, 53.69; H, 5.09; N, 16.33. Found C, 53.62; H,
CH2), 2.11 (s, 2H, CH2), 3.46 (s, 4H, CH2), 3.88 (s, 2H, CH2), 4.10 (s, 2H,
CH2), 7.26 (m, 1H, Ar), 7.40 (m, 1H, Ar), 7.70 (m, 1H, Ar), 7.89 (s, 2H,
5.11; N, 16.37.
NH2), 8.88 (s, 1H, NH). 13C NMR (DMSO‑d6)
d
16.8, 25.2, 31.7 (2C),
4.3. STS assay
31.9 (2C), 32.0, 33.1 (2C), 53.1 (2C), 57.6 (2C), 60.2, 119.7, 120.9,
122.2, 124.2, 131.6, 155.4, 157.0. IR (Nujol) 3307, 1639, 1608 cmꢀ1 m/
z 476 (M þ H)þ. Anal. Calcd. for C21H35ClN4O4S (475.04) C, 53.09; H,
7.43; N, 11.79. Found C, 53.14; H, 7.45; N, 11.75.
STS inhibitory assays were performed as described previously
[19]. Briefly, the ability of a compound to inhibit STS activity was
determined using the lysate of JEG-3,
a human placenta

D. Moi et al. / European Journal of Medicinal Chemistry 182 (2019) 111614
11
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choriocarcinoma cell line known to have high STS activity. To
determine STS inhibition, activity was measured in the absence and
presence of the inhibitor (0.001e10
dpm, PerkinElmer) adjusted to 20
m
M) using [3H]E1S (4 ꢁ 105
m
M with unlabelled E1S sub-
strate. After incubation of the substrate-inhibitor with JEG-3 lysate
(125 g of protein/mL) for 1 h, the product formed estrone (E1) was
m
separated from the mixture by extraction with toluene (4 mL).
[4e14C]E1 (American Radiolabelled Chemicals) was also used
throughout the assay to monitor procedural losses.
To ascertain whether compounds were able to pass through the
cell lipid bilayer, intact monolayers of JEG-3 cells were incubated
for 20 h at 37 ꢂC with [3H]E1S (5 pmol, 7 ꢁ 105 dpm, 60 Ci/mmol) in
serum-free Eagle's Minimal Essential Medium (1.0 mL) with or
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4 substituted bicyclic coumarin sulfamates are potent steroid sulfatase in-
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without inhibitors (10 mM). After incubation, medium (0.5 mL) was
removed and product E1 separated from E1S by solvent partition
using toluene (4 mL). [14C] Estrone (7 ꢁ 103 dpm, 52 mCi/mmol)
was used to correct for procedural losses. An organic phase aliquot
was added to scintillation fluid and the 3H and 14C content
measured by scintillation spectrometry. The mass of E1S hydrolyzed
was calculated from the 3H counts detected (corrected for the
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volume of medium and organic solvent used and for recovery of 14
C
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Acknowledgment
This work was supported partially by the University of Cagliari,
Italy FIR funds to GB and VO.
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of steroid sulfatase with quinone methide-generating suicide inhibitors,
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Appendix A. Supplementary data
Supplementary data to this article can be found online at
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