A new series of aryl sulfamate derivatives: Design, synthesis, and biological evaluation

Article abstract of DOI:10.1016/j.bmc.2020.115406

Steroid sulfatase (STS) has recently emerged as a drug target for management of hormone-dependent malignancies. In the present study, a new series of twenty-one aryl amido-linked sulfamate derivatives 1a-u was designed and synthesized, based upon a cycloh

Full text of DOI:10.1016/j.bmc.2020.115406

Journal Pre-proofs
A new series of aryl sulfamate derivatives: Design, synthesis, and biological
evaluation
Mohammed I. El-Gamal, Seyed-Omar Zaraei, Paul A. Foster, Hanan S.
Anbar, Randa El-Gamal, Raafat El-Awady, Barry V.L. Potter
PII:
S0968-0896(20)30211-X
https://doi.org/10.1016/j.bmc.2020.115406
BMC 115406
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
24 January 2020
27 February 2020
29 February 2020
Please cite this article as: M.I. El-Gamal, S-O. Zaraei, P.A. Foster, H.S. Anbar, R. El-Gamal, R. El-Awady,
B.V.L. Potter, A new series of aryl sulfamate derivatives: Design, synthesis, and biological evaluation,
Bioorganic & Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.bmc.2020.115406
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version
will undergo additional copyediting, typesetting and review before it is published in its final form, but we are
providing this version to give early visibility of the article. Please note that, during the production process, errors
may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Elsevier Ltd. All rights reserved.

A new series of aryl sulfamate derivatives: Design, synthesis, and biological
evaluation
Mohammed I. El-Gamal a,b,c,$,*, Seyed-Omar Zaraei b,$, Paul A. Foster d,e*, Hanan S. Anbar
f, Randa El-Gamal g, Raafat El-Awady b,h, and Barry V.L. Potter i
a Department of Medicinal Chemistry, College of Pharmacy, University of Sharjah, Sharjah
27272, United Arab Emirates.
b Sharjah Institute for Medical Research, University of Sharjah, Sharjah 27272, United Arab
Emirates.
c Department of Medicinal Chemistry, Faculty of Pharmacy, University of Mansoura, Mansoura
35516, Egypt.
d Institute of Metabolism and Systems Research, 2nd Floor IBR Tower, University of
Birmingham, Birmingham, B15 2TT, United Kingdom.
e Centre for Endocrinology, Diabetes and Metabolism, Birmingham Health Partners,
Birmingham, B15 2TH, United Kingdom.
f Department of Pharmacology and Pharmacy Practice, Dubai Pharmacy College, Dubai, United
Arab Emirates.
g Department of Medical Biochemistry, Faculty of Medicine, University of Mansoura, Mansoura
35516, Egypt.
h Department of Pharmacy Practice and Pharmacotherapeutics, College of Pharmacy, University
of Sharjah, Sharjah 27272, United Arab Emirates.
i
Medicinal Chemistry & Drug Discovery, Department of Pharmacology, University of Oxford,
Oxford, OX1 3QT, United Kingdom.
*
E-mail addresses of the corresponding authors: drmelgamal2002@gmail.com &
malgamal@sharjah.ac.ae (M.I. El-Gamal) & P.A.Foster@bham.ac.uk (P.A. Foster).
$ Both Mohammed I. El-Gamal and Seyed-Omar Zaraei are joint first authors.
Page 1 of 31

Abstract
Steroid sulfatase (STS) has recently emerged as a drug target for management of hormone-
dependent malignancies. In the present study, a new series of twenty-one aryl amido-linked
sulfamate derivatives 1a-u was designed and synthesized, based upon a cyclohexyl lead
compound. All members were evaluated as STS inhibitors in a cell-free assay. Adamantyl
derivatives 1h and 1p-r were the most active with more than 90% inhibition at 10 µM
concentration and, for those with the greatest inhibitory activity, IC50 values were determined.
These compounds exhibited STS inhibition within the range of ca 25-110 nM. Amongst them,
compound 1q possessing a o-chlorobenzene sulfamate moiety exhibited the most potent STS
inhibitory activity with an IC50 of 26 nM. Furthermore, to assure capability to pass through the cell
lipid bilayer, compounds with low IC50 values were tested against STS activity in JEG-3 whole-
cell assays. Consequently, 1h and 1q demonstrated IC50 values of ca 14 and 150 nM, respectively.
Thus, compound 1h is 31 times more potent than the corresponding cyclohexyl lead (IC50 value
= 421 nM in a JEG-3 whole-cell assay). Furthermore, the most potent STS inhibitors (1h and 1p-
r) were evaluated for their antiproliferative activity against the estrogen-dependent breast cancer
cell line T-47D. They showed promising activity with single digit micromolar IC50 values (ca 1-6
µM) and their potency against T-47D cells was comparable to that against STS enzyme. In
conclusion, this new class of adamantyl-containing aryl sulfamate inhibitor has potential for
further development against hormone-dependent tumours.
Keywords: Adamantyl; Antiproliferative activity; Breast cancer; JEG-3; Steroid sulfatase; T-47D.
1. Introduction
Steroid sulfatase (STS) catalyzes the desulfation reaction of the inactive metabolites estrone
sulfate (E1S), androstendiol sulfate, and dehydroepiandrosterone sulfate into their corresponding
active analogues. These active hormones (estrone, androstendiol, and DHEA) are mitogens for
proliferation of hormone-dependent malignancies such as breast, prostate, and endometrial
cancers. Thus, inhibition of STS represents an attractive avenue for treatment of those hormone-
Page 2 of 31

dependent cancers [1, 2]. Numerous molecules possessing the unsubstituted sulfamate moiety have
been reported and developed as STS inhibitors [3-16]. An aryl sulfamate moiety is essential for
potent inhibition [3,5]. Such agents irreversibly inhibit STS enzyme likely through covalent
binding of the sulfamoyl moiety with the hydrated N-formylglycine residue of the enzyme [5].
Amongst inhibitors reported, Irosustat (STX 64) (Figure 1) is the most successful [8] as it has been
investigated in clinical trials for treatment of estrogen-dependent breast and endometrial cancer.
Treatment resulted in stable disease in cancer patients and it significantly decreased the serum
levels of estrogenic steroids as a result of STS inhibition [17]. It was also investigated in men in
androgen-dependent prostate cancer [5].
In previous work, we published a series of sulfonate and sulfamate derivatives as inhibitors of
STS. As anticipated, the free (ie non-N-substituted) sulfamate lead compound illustrated in Figure
2 was the most active among the series. Its IC50 value against STS was 421 nM in whole-cell JEG-3
placental choriocarcinoma cells [12]. In the current work, this aryl sulfamate moiety was retained
to ensure covalent binding and inhibition of the enzyme, and the cyclohexyl ring was replaced
either with smaller (cyclopentyl) or larger (cycloheptyl or adamantyl) cycloalkyl rings, or replaced
with (substituted) aryl rings. The purpose of these substitutions was to explore enhancing the
strength of hydrophobic interactions with the enzyme and potentially to increase the ability of
agents to cross the cell membrane. The most active compounds were adamantyl derivatives. It is
assumed that cycloalkyl moieties (e.g. adamantyl) are more appropriate than the planar aryl rings
as they mimic the 5-membered ring D of estrone sulfate and the 7-membered ring of Irosustat
(Figure 1).
Page 3 of 31

H2N
O
S
R
N
H
N
O
O
O
O
O
O
S
S
O
O
H2N
O
X
X
C
D
O
-O
O
O
A
B
CH3
S
H
N
O
N
H
O
O
O
O
Estrone sulfate
S
H2N
O
H2N
O
Reversed amides
R = H. CH3
X = H, F, Cl, Br
O
O
S
H2N
O
O
O
H2N
S
O
O
Irosustat (STX 64)
O
O
N
N
H
H
Target adamantyl-containing sulfamate derivatives
Figure 1. Structures of estrone sulfate, Irosustat (STX 64), and the target adamantyl-containing
sulfamate compounds.
Further structural modifications were performed, such as insertion of o-halo substituents [16]
or modification of the amide linker as a reversed amide or urea in order to investigate their effects
on STS inhibition (Figure 2). Moreover, indole cyclic analogues (Figure 3) were also synthesized
and tested for STS inhibition to compare between the open chain amide and cyclic analogues as
STS inhibitors. The most potent compounds were evaluated for in vitro antiproliferative activity
against the T-47D estrogen-dependent breast cancer cell line. We now report here results,
structure-activity relationship, and experimental protocols.
Page 4 of 31

H
N
Cyclopentyl or cycloheptyl or aryl
O
O
S
O
H2N
O
Target cycloalkyl- or aryl-containing sulfamate derivatives
H
H2N
O
N
S
R
N
H
N
O
O
O
O
S
O
O
O
H2N
O
O
O
S
S
O
H2N
O
Lead compound
X
X
Inhibitory effects on STS enzyme:
72% at 20 µM concentration.
55.7% at 10 µM concentration.
IC50 against STS in JEG-3 whole-cell
assay = 421 nM.
O
CH3
H
N
N
H
O
O
O
O
S
H2N
O
H2N
O
Reversed amides
R = H. CH3
X = H, F, Cl, Br
H2N
O
S
O
O
O
N
N
H
H
Target adamantyl-containing sulfamate derivatives
Figure 2. Rational design of the target sulfamate derivatives.
O
R
H
N
N
O
O
S
O
O
H2N
O
S
O
H2N
O
Indole analogues
Lead compound
R = cyclohexyl or 1-adamantyl
Figure 3. Rational design of the target indole-containing sulfamate derivatives.
Page 5 of 31

2. Results and discussion
2.1. Chemistry
The pathways depicted in Schemes 1-6 have been utilized for synthesis of the target sulfamate
compounds 1a-u. The reaction of the appropriate acid chloride reagents 3a,b, 5a-d, or 8 with 4-
aminophenol (2) or other aminophenol derivatives 7a-d in presence of dry potassium carbonate
led to formation of the corresponding carboxamide-containing phenolic intermediates 4a,b, 6a-d,
or 9a-e (Schemes 1-3). The inverted amide phenolic intermediates 13a-d were synthesized via
reaction of the appropriate methoxybenzoic acid reactants 10a-d with amantadine (11) in the
presence of HOBt, EDCI.HCl, and triethylamine followed by demethylation of compounds 12a-d
using boron trifluoride methyl sulfide complex (Scheme 4). The phenolic intermediate 15
possessing a urea spacer was synthesized through reaction of 4-aminophenol (2) with 1-adamantyl
isocyanate (14) (Scheme 5). The cyclic indole phenolic intermediates 19a,b were synthesized
through reaction of 5-acetoxyindole (16) with cyclohexanecarbonyl chloride (17) or 1-
adamantylcarbonyl chloride (8) in the presence of sodium hydride, and subsequent ester hydrolysis
using lithium hydroxide in aqueous THF (Scheme 6). All the phenolic intermediates formed via
all the six schemes were converted into sulfamate target compounds 1a-u via reaction with
sulfamoyl chloride in presence of sodium hydride. Most of the target sulfamate products were
obtained in relatively low yield due to the possibility of side reaction between the sulfamate amino
group and sulfamoyl chloride in presence of sodium hydride in a polymerization fashion. We
therefore recommend performing the sulfamoylation reaction in absence of sodium hydride in the
future in order to obtain the product in higher yield. The target compound structures are presented
in Tables 1 and 2.
H
H
N
NH2
N
a
b
n
O
O
n
Cl
+
O
S
O
n
HO
HO
H2N
O
O
2
3a,b
4a,b
1a,b
n = 1, 3
Scheme 1. Reagents and conditions: (a) anhydrous K2CO3, acetone, 0 ºC, rt, 4 h; (b) sulfamoyl
chloride, NaH, anhydrous DMAc, 0 ºC, rt, overnight.
Page 6 of 31

H
H
N
Ar
NH2
N
Ar
a
b
O
O
Cl
Ar
+
O
S
O
HO
HO
H2N
O
O
2
5a-d
6a-d
1c-g
Scheme 2. Reagents and conditions: (a) anhydrous K2CO3, acetone, 0 ºC, rt, 4 h; (b) sulfamoyl
chloride, NaH, anhydrous DMAc, 0 ºC, rt, overnight.
X
X
HO
H2N
O
R1
N
S
R1
N
X
a
b
NH
R1
n
O
O
Cl
+
HO
R2
n
n
R2
O
R2
O
O
9a-e
2 or 7a-d
8
1h-n
R1, R2 = H, CH3
X = H, F, Cl, Br
n = 0, 1
Scheme 3. Reagents and conditions: (a) anhydrous K2CO3, acetone, 0 ºC, rt, 4 h; (b) sulfamoyl
chloride, NaH, anhydrous DMAc, 0 ºC, rt, overnight.
O
O
O
COOH
b
N
N
c
N
a
O
O
H
H
H
+
H2N
S
HO
O
H2N
O
O
X
X
X
X
13a-d
12a-d
1o-r
11
10a-d
X = H, F, Cl, Br
Scheme 4. Reagents and conditions: (a) HOBt, EDCI.HCl, triethylamine, DMF, rt, overnight; (b)
BF3.Me2S, rt, overnight; (c) sulfamoyl chloride, NaH, anhydrous DMAc, 0 ºC, rt, overnight.
O
H2N
S
O
O
HO
O
NH2
O
a
b
+
OCN
N
N
N
N
H
H
HO
H
H
1s
15
14
2
Scheme 5. Reagents and conditions: (a) anhydrous THF, rt, 12 h; (b) sulfamoyl chloride, NaH,
anhydrous DMAc, 0 ºC, rt, overnight.
O
O
R
R
O
H
N
N
a
b
N
O
O
Cl
R
+
HO
O
O
8,17
19a,b
18a,b
16
17: R = cyclohexyl
8: R = 1-adamantyl
c
O
R
N
O
O
S
H2N
O
1t,u
Page 7 of 31

Scheme 6. Reagents and conditions: (a) NaH, anhydrous THF, -20 oC, 30 min; (b) LiOH, aq. THF,
rt, 24 h; (c) sulfamoyl chloride, NaH, anhydrous DMAc, 0 ºC, rt, overnight.
2.2.
Biological screening
2.2.1. Cell-free assay against STS enzyme in JEG-3 cell lysate
The target compounds 1a-u were evaluated for STS inhibitory effect in an assay using JEG-3
placental choriocarcinoma cell lysate. In the first stage, compounds 1a-h were tested for STS
inhibition percentage at 10 µM concentration (Table 1). The results indicated that the adamantyl
derivative 1h was the most active among the eight tested compounds. It is more active than the
other derivatives possessing cyclopentyl, cycloheptyl, or aryl rings and is more potent than our
previously reported cyclohexyl lead compound [12]. It was thus concluded that the adamantyl
moiety is most optimal for STS inhibition by this series of compounds.
Table 1. Structures of the target sulfamate compounds 1a-h and their inhibitory effect against
STSat 10 µM concentration.
Compound No.
1a
Structure
% inhibition a
61.98 ± 9.47
H
N
O
O
S
O
H2N
O
1b
52.32 ± 3.26
H
N
O
O
S
O
H2N
O
1c
25.37 ± 1.74
24.56 ± 0.50
H
N
O
O
S
O
H2N
O
Cl
1d
H
N
O
O
S
O
H2N
O
Page 8 of 31

O
1e
72.68 ± 2.24
H
N
O
O
S
O
H2N
O
1f
1g
1h
12.89 ± 2.92
53.59 ± 4.78
93.90 ± 0.80
H
N
N
O
O
S
O
H2N
O
H
N
O
S
O
O
O
H2N
H
N
O
O
S
O
H2N
O
Irosustat
99.10 ± 0.46
a
Results are expressed as % STS inhibition compared to untreated controls. Data is mean ±
standard error of mean (S.E.M) (n=3).
In the next stage, adamantyl-containing sulfamate derivatives were designed and
synthesized as derivatives of compound 1h. The new derivatives include o-halo substituents, a
reversed amide and urea linker, or methylated analogues. The structures of adamantyl derivatives
1i-s and their inhibitory effect against STS at 10 µM concentration are shown in Table 2. The
methylated derivatives 1m,n are less active than compound 1h, so these inserted methyl groups
might affect the activity through potential steric effects or through inhibition of hydrogen bond
formation by the amide linker with the enzyme active site. Moreover, the extended linkers in
compounds 1l and 1s were detrimental for activity. In addition, the reversed amide analogue 1o is
somewhat less active than compound 1h. On the other hand, the ortho-halogenated analogues of
compound 1o, compounds 1p-r, exerted stronger inhibitory effect than both 1h and 1o. Indeed, it
has been previously reported that o-halo substituents increase potency [16]. Furthermore, the
halogenated reversed amide derivatives 1p-r (94.60~98.30% inhibition) are more active than the
corresponding halogenated amide analogues 1i-k (82.74~88.58% inhibition). This could be
rationalized since the reversed amide linker in compounds 1p-r (especially in concert with the o-
halo motif) is an electron-withdrawing moiety at para position of the free sulfamate group. It can
Page 9 of 31

deactivate the ortho and para positions by mesomeric effect. In addition, the ortho-halo group has
greater influence on the sulfamate group due to both resonance and inductive effects. Both reversed
amide and ortho-halo enhance the electrophilicity of the sulfamate sulfur atom and also lowers the
pKa of the phenolic side product after irreversible inhibition of the enzyme by transfer of the
sulfamoyl group to hydrated N-formylglycine amino acid residue. This is presumed to enhance the
formation of the active electrophilic sulfonylamine in the STS active site [5].
Table 2. Structures of the target sulfamate compounds 1i-s and their inhibitory effect against
steroid sulfatase (STS) enzyme at 10 µM concentration.
Compound No.
1i
Structure
% inhibition a
86.26 ± 6.02
H
N
O
O
S
S
S
O
H2N
O
F
1j
88.58 ± 3.16
82.74 ± 6.33
H
N
O
O
O
O
H2N
O
Cl
Br
1k
H
N
O
O
H2N
O
H2N
O
1l
-5.30 ± 8.80
60.97 ± 1.68
S
H
O
O
N
O
1m
CH3
N
O
O
S
S
S
O
H2N
O
1n
1o
58.48 ± 14.63
90.00 ± 0.64
CH3
H
N
O
O
O
H2N
O
O
N
H
O
O
H2N
O
Page 10 of 31

1p
1q
1r
1s
97.20 ± 0.00
98.30 ± 0.10
94.60 ± 0.90
5.32 ± 14.56
O
O
O
N
H
O
O
S
S
S
H2N
O
F
N
H
O
O
H2N
O
Cl
Br
N
H
O
O
H2N
O
O
H2N
S
O
O
O
N
N
H
H
a Results are expressed as % STS inhibition compared to untreated controls. Data is mean ± S.E.M
(n=3).
We also decided to synthesize the indole derivatives 1t,u (Table 3) as cyclic analogues of
the lead cyclohexyl compound (Fig. 2) and compound 1h, respectively. Upon comparing the
activities, we found that the cyclic analogues are much less active than the corresponding open
chain amide compounds. The 5-membered ring of indole might weaken the activity through
hindering hydrogen bond formation that could be done by NH in case of open chain amide
derivatives, or its increased bulkiness compared with the open chain analogues might be
detrimental for the activity. Further exploration of such indole derivatives was non-promising.
Table 3. Structures of the target sulfamate compounds 1t,u and their inhibitory effect against
steroid sulfatase (STS) enzyme at 10 µM concentration.
Compound No.
Structure
% inhibition a
Page 11 of 31

O
1t
14.34 ± 6.39
63.35 ± 2.12
N
O
O
S
H2N
O
1u
O
N
O
O
S
H2N
O
a Results are expressed as % STS inhibition compared to untreated controls. Data is mean ± S.E.M
(n=3).
Compounds 1h and 1p-r showing the highest inhibitory effect at 10 µM concentration were
evaluated for their IC50 values against STS enzyme in JEG-3 cell lysate. The results are illustrated
in Figure 4. The chloro derivative 1q is the most potent among them (IC50 = 25.8 nM) and is ca
4.2 times more potent than the non-halogenated compound 1h with a non-reversed amide. Notably,
1q was 2.6- and 2.9-fold more potent than the corresponding fluoro and bromo analogues,
respectively.
IC50 (nM)
25.8
1q
67.7
74.1
109.5
6.4
1p
1r
1h
Irosustat
150
100
50
0
1000
100
10
1
Concentration (nM)
Figure 4. IC50 Concentrations for the most potent compounds and Irosustat against STS enzyme
activity in cell-free JEG-3 lysate.
2.2.2. Whole-cell assay against STS enzyme in JEG-3 cells.
Page 12 of 31

Compounds 1h and 1q were also evaluated for STS inhibition in a whole-cell assay against
JEG-3 cells, to examine their ability to cross the cell membrane and inhibit STS enzyme inside
cells. Both compounds were active, but with different potency and IC50 values are shown in Figure
5. Compound 1h is ca 11 times more potent than compound 1q in this assay. In addition, compound
1h is 31-fold more potent than the corresponding cyclohexyl lead compound (Figure 2, IC50 = 421
nM).
IC50 (nM)
150
13.6
150.0
2.1
1h
1q
Irosustat
100
50
0
1000
100
10
1
0.1
Concentration (nM)
Figure 5. IC50 determination for compounds 1h, 1q, and Irosustat against STS enzyme activity in
JEG-3 placental choriocarcinoma cells.
2.2.3. Antiproliferative activity
The most potent STS inhibitors of this series, compounds 1h and 1p-r, were tested for
antiproliferative activity against T-47D estrogen-dependent breast cancer cells. Irosustat was
utilized as a reference standard molecule in this assay.
The cells were grown in charcoal-stripped fetal bovine serum containing no estrogen and
provided with estradiol sulfate (E2S). The cellular STS enzyme should convert E2S into the
estradiol needed for cellular proliferation. The inability to do that indicates STS inhibition by the
evaluated compound, and subsequently leads to inhibition of proliferation. The dose-response
curves are illustrated in Figure 6 and IC50 values of the tested compounds relative to Irosustat are
Page 13 of 31

shown in Table 6. The results are consistent and proportional to their potency against STS enzyme
in the cell lysate. All four tested compounds exhibited single-digit micromolar IC50 values.
Compound 1q possessing both o-chloro and reversed amide moieties, which is the most potent
STS inhibitor, also exhibited the strongest antiproliferative activity. Its potency is close to that of
Irosustat. The halogen substituents increase lipophilicity and can enhance the ability to penetrate
the cell membrane compared to the non-halogenated analogues. This could account for the stronger
potency of compounds 1p-r than that of the non-halogenated derivative 1h.
Figure 6. Dose-response curves of compounds 1h, 1p-r, and Irosustat against the T-47D breast
cancer cell line.
Table 6. IC50 values of compounds 1h, 1p-r, and Irosustat against T-47D breast cancer cell line
Compound No.
IC50 value (µM) a
Page 14 of 31

1h
1p
5.78 ± 0.18
3.40 ± 0.10
1.04 ± 0.08
4.67 ± 0.25
0.92 ± 0.03
1q
1r
Irosustat
a The results are expressed as means of triplicate assay ± standard error of mean (S.E.M.).
3. Conclusion
We report here a new series of aryl sulfamate derivatives as STS inhibitors. It is presumed that
they act irreversibly similar to all such inhibitors [3,5]. Structure-activity relationship (SAR)
correlations and lead optimization led to discovery of the adamantyl derivatives 1h and 1p-r as
the most potent STS inhibitors among this series. Compound 1h is the most potent in a whole-cell
STS assay (IC50 = 13.6 nM). It was found that the adamantyl moiety, reversed amide linker, and
o-halo substituents (especially chloro) are optimal for STS inhibitory effect of this series of
compounds. The o-chloro derivative 1q is the most potent STS inhibitor in JEG-3 placental
choriocarcinoma cell lysate (IC50 = 25.8 nM). It also exhibited the highest antiproliferative activity
against T-47D estrogen-dependent cancer (IC50 = 1.04 µM) with a potency very close to that of
Irosustat. These promising halogenated adamantyl sulfamate structures represent an attractive new
class of STS inhibitory agents for potential future optimization and application to treatment of
hormone-dependent cancers.
4. Experimental
4.1.
General
A Stuart melting point apparatus was used for measuring melting points and are uncorrected.
1H NMR and 13C NMR spectra were recorded on Bruker Avance (500 spectrometer). The identity
and purity of all the target compounds were confirmed by standard spectral and elemental analysis.
The samples were dried for 1h using a high vacuum oil pump prior to elemental analysis. All
Page 15 of 31

solvents and reagents were purchased from commercial vendors and used as supplied. Column
chromatography (silica gel with pore size of 0.040~0.063 mm, 230-400 mesh) with technical grade
solvents were used for purification of the final compounds.
4.2.
Synthesis of the phenolic intermediates 4a,b, 6a-d, and 9a-e
The appropriate aminophenol derivative (1.5 mmol) was dissolved in acetone (1.5 mL) under
N2 (g), cooled to 0 oC, charged with K2CO3 (622 mg, 4.5 mmol), and stirred for 15 minutes. After
that, the appropriate acyl chloride (1.55 mmol) dissolved in acetone (1 mL) was added dropwise
to the reaction mixture at 0 oC. The reaction was monitored by TLC and LC-MS spectrometry.
Once reaction completion was confirmed, the mixture was filtered, and the filtrate was
concentrated in vacuo. The solid was extracted using ethyl acetate (10 mL) and brine (10 mL). The
organic layer was collected and dried over anhydrous Na2SO4 and concentrated in vacuo. The
product was sulfamoylated without further purification.
4.3.
Synthesis of the phenolic intermediates 13a-d
Compound 10a-d (0.33 mmol) was dissolved in anhydrous DMF (1 mL), cooled to 0 oC under
N2 (g) while stirred. Triethylamine (120 μL, 0.86 mmol) was added and further stirred for 15
minutes before of the addition of HOBt (91 mg, 0.67 mmol) and EDCI.HCl (105 mg, 0.67 mmol)
to the mixture. After stirring for 15 minutes, the mixture was charged with amantadine (100 mg,
0.66 mmol) and kept at room temperature while being stirred. The reaction was quenched with ice,
the mixture extracted with ethyl acetate (10 mL) and brine (10 mL) and the organic layer collected,
dried over anhydrous Na2SO4, and evaporated to dryness. The resultant products 12a-d (0.60
mmol) were dissolved in anhydrous dichloromethane (1 mL), and BF3.MeS (630 μL, 10 mmol)
was added to the reaction that was kept under a nitrogen atmosphere at room temperature overnight
while stirring. The reaction was monitored with TLC. Upon completion, the reaction mixture was
basified with aqueous K2CO3 solution and extracted with ethyl acetate (10 mL). The organic layer
was dried over anhydrous Na2SO4 and concentrated in vacuo. The resultant product was used for
the sulfamoylation step.
Page 16 of 31

4.4.
Synthesis of the phenolic intermediate 15
4-Aminophenol (2, 280 mg, 2.57 mmol) was dissolved in anhydrous THF (1 mL) under
nitrogen gas while stirring. Then 1-adamantyl isocyanate (14, 910 mg, 5.13 mmol) dissolved in
anhydrous THF (1 mL) was added, and the reaction mixture was stirred overnight at room
temperature. The mixture was concentrated in vacuo and product purified using column
chromatography. The resultant solid was then used for the sulfamoylation step without further
purification.
4.5.
Synthesis of the 5-hydroxyindole intermediates 19a,b
5-Acetoxyindole (16, 343 mg, 1.96 mmol) was dissolved in anhydrous THF (1 mL), cooled to
-20 oC, and the mixture charged with NaH (60% dispersion in mineral oil, 94 mg, 2.35 mmol) and
stirred for 15 minutes. The appropriate carbonyl chloride 8 or 17 (2.15 mmol) was dissolved in
o
anhydrous THF (0.5 mL), and added to the mixture dropwise at -20 C. The mixture was then
extracted using ethyl acetate (10 mL) and brine (10 mL). The organic layer was collected and dried
on anhydrous Na2SO4, and concentrated in vacuo . The solid obtained was dissolved in THF (4
mL), and the mixture charged with LiOH (60 mg, 2.5 mmol) dissolved in H2O (1.5 mL). The
reaction was then checked after 3h and completion was confirmed via TLC and LC-MS. The
mixture was acidified with 1M HCl, extracted using ethyl acetate (10 mL), dried with brine (10
mL) and the organic extract was dried over anhydrous sodium sulfate and evaporated to dryness
in vacuo . The solid product was then used for sulfamoylation step.
4.6.
Synthesis of the target sulfamate derivative 1a-u
The appropriate phenolic intermediate 4a,b, 6a-d, 9a-e, 13a-d, 15, or 19a,b (0.72 mmol) was
dissolved in anhydrous DMAc (1.3 mL), the mixture charged with NaH (60% dispersion in mineral
oil, 50 mg, 1.25 mmol) and stirred at 0oC under N2 (g) for 15 minutes. Sulfamoyl chloride (411 mg,
3.6 mmol) dissolved in anhydrous DMAc (1 mL) was added dropwise at 0oC, and the mixture was
stirred under N2 (g) overnight. The reaction mixture was quenched with ice, extracted between ethyl
Page 17 of 31

acetate (10 mL) and distilled water (10 mL). The organic layer was collected and dried over
anhydrous Na2SO4, concentrated in vacuo, and product purified with flash column
chromatography.
1
4-(Cyclopentanecarboxamido)phenyl sulfamate (1a): Yield: 30%; mp: 166-9 °C; H NMR
(Acetone-d6, 500 MHz) δ 9.20 (brs, 1H), 7.70 (d, 2H, J = 9.0 Hz), 7.23 (d, 2H, J = 9.0 Hz), 7.03
13
(s, 2H), 2.84-2.78 (m, 1H), 1.90-1.80 (m, 4H), 1.75-1.68 (m, 2H), 1.61-1.55 (m, 2H); C NMR
(Acetone-d6, 125 MHz) δ 175.3 (carbonyl carbon), 146.7 (1 C), 139.2 (1 C), 123.5 (2 x CH), 120.9
(2 x CH) [aromatic carbons], 46.8 (cyclopentyl CH, 1C), 31.0 (cyclopentyl 2 x CH2), 26.7
(cyclopentyl 2 x CH2); LC-MS m/z: 285.1 (M + H)+; CHN analysis: calculated C:50.69%,
H:5.67%, N:9.85%; found: C:50.64%, H:5.72%, N:9.97%.
1
4-(Cycloheptanecarboxamido)phenyl sulfamate (1b): Yield: 9%; mp: 139-42 °C; H NMR
(Acetone-d6, 500 MHz) δ 9.00 (brs, 1H), 7.57 (d, 2H, J = 9.0 Hz), 7.10 (d, 2H, J = 9.0 Hz), 6.90
13
(s, 2H), 2.40-2.37 (m, 1H), 1.80-1.76 (m, 2H), 1.66-1.57 (m, 4H), 1.46-1.29 (m, 6H); C NMR
(Acetone-d6, 125 MHz) δ 176.2 (carbonyl carbon), 146.6 (1 C), 139.2 (1 C), 123.5 (2 x CH), 120.9
(2 x CH) [aromatic carbons], 48.2 (cycloheptyl CH, 1C), 32.2 (cycloheptyl 2 x CH2), 29.1
(cycloheptyl 2 x CH2), 27.2 (cycloheptyl 2 x CH2); LC-MS m/z: 313.08 (M + H)+; CHN analysis:
calculated C:53.83%, H:6.45%, N:8.97%; found: C:53.76%, H:6.30%, N:9.08%.
4-(Benzamido)phenyl sulfamate (1c): Yield: 11%; mp: 211-3 °C; 1H NMR (Acetone-d6, 500 MHz)
δ 9.66 (brs, 1H), 8.00 (d, 2H, J = 9.0 Hz), 7.89 (m, 2H, J = 9.0 Hz), 7.60-7.57 (m, 1H), 7.53-7.50
(m, 2H), 7.31 (d, 2H, J = 9.0 Hz), 7.09 (s, 2H); 13C NMR (Acetone, 125 MHz) δ 166.4 (carbonyl
carbon), 147.2 (1 C), 138.8 (1 C), 136.0 (1 C), 132.5 (1 C), 129.3 (2 x CH), 128.3 (2 x CH), 123.5
(2 x CH), 122.0 (2 x CH) [aromatic carbons]; LC-MS m/z: 292.97 (M + H)+; CHN analysis:
calculated C:53.42%, H:4.14%, N:9.58%; found: C:53.65%, H:4.08%, N:9.45%.
Page 18 of 31

4-(4-Chlorobenzamido)phenyl sulfamate (1d): Yield: 22%; mp: 223-6 °C; 1H NMR (Acetone-d6,
500 MHz) δ 9.73 (brs, 1H), 8.02 (d, 2H, J = 9.0 Hz), 7.88 (d, 2H, J = 9.0 Hz), 7.56 (d, 2H, J = 9.0
13
Hz), 7.32 (d, 2H, J = 9.0 Hz), 7.10 (brs, 2H); C NMR (Acetone, 125 MHz) δ 165.3 (carbonyl
carbon), 147.4 (1 C), 138.6 (1 C), 138.1 (1 C), 134.7 (1 C), 130.2 (2 x CH), 129.5 (2 x CH), 123.5
(2 x CH), 122.1 (2 x CH) [aromatic carbons]; LC-MS m/z: 327.07 (M + H)+; CHN analysis:
calculated C:47.78%, H:3.39%, N:8.57%; found: C:47.63%, H:3.30%, N:8.64%.
4-(4-Methoxybenzamido)phenyl sulfamate (1e): Yield: 11%; mp: 205-7 °C; 1H NMR (DMSO-d6,
500 MHz) δ 10.20 (brs, 1H), 7.98-7.95 (m, 4H), 7.83 (d, 2H, J = 9.0 Hz), 7.26 (d, 2H, J = 8.5 Hz),
7.08 (d, 2H, J = 8.5 Hz), 3.85 (s, 3H); 13C NMR (DMSO-d6, 125 MHz) 164.9 (carbonyl carbon),
162.0 (1 C), 145.6 (1 C), 137.7 (1 C), 129.6 (2 x CH), 126.7 (1 C), 122.4 (2 x CH), 121.3 (2 x CH),
113.6 (2 x CH) [aromatic carbons], 55.4 (methoxy carbon); LC-MS m/z: 323.03 (M + H)+; CHN
analysis: calculated C:52.17%, H:4.38%, N:8.69%; found: C:52.04%, H:4.30%, N:8.88%.
1
4-(Nicotinamido)phenyl sulfamate (1f): Yield: 10%; mp: 203-4 °C; H NMR (Acetone-d6, 500
MHz) δ 9.84 (brs, 1H), 9.17 (s, 1H), 8.76 (d, 1H, J = 4.0 Hz), 8.32 (d, 1H, J = 8.0 Hz), 7.90-7.87
(m, 2H), 7.55-7.52 (m, 1H), 7.34-7.31 (m, 2H), 7.11 (s, 2H); 13C NMR (Acetone-d6, 125 MHz) δ
165.0 (carbonyl carbon), 153.2 (1 C), 149.6 (1 C), 147.5 (1 C), 138.5 (1 C), 136.0 (1 C), 131.6 (1
C), 124.3 (1 C), 123.6 (2 C), 122.2 (2 C) [aromatic carbons]; LC-MS m/z: 293.72 (M + H)+; CHN
analysis: calculated C:49.14%, H:3.78%, N:14.33%; found: C:49.11%, H:3.83%, N:14.21%.
4-(2-Naphthamido)phenyl sulfamate (1g): Yield: 29%; mp: 215-8 °C; 1H NMR (Acetone-d6, 500
MHz) δ 8.59 (s,1H), 8.08-7.99 (m, 5H), 7.96-7.93 (m, 2H), 7.66-7.60 (m, 2H), 7.35-7.32 (m, 2H),
7.11 (s, 2H); 13C NMR (Acetone-d6, 125 MHz) 166.4 (carbonyl carbon), 147.3 (1 C), 138.9 (1 C),
135.8 (1 C) , 133.5 (1 C), 133.3 (1 C), 129.8 (1 C), 129.1 (1 C), 128.7 (1 C), 128.6 (2 C), 127.7 (1
C), 125.1 (1 C), 123.6 (2 C), 122.0 (2 C) [aromatic carbons]; LC-MS m/z: 342.77 (M + H)+; CHN
analysis: calculated C:59.64%, H:4.12%, N:8.18%; found: C:59.41%, H:4.03%, N:8.30%.
Page 19 of 31

1
4-(1-Adamantylcarboxamido)phenyl sulfamate (1h): Yield: 10%; mp: 182-3 °C; H NMR
(DMSO-d6, 500 MHz) δ 9.23 (s, 1H), 7.90 (s, 2H), 7.71 (d, 2H, J = 9.0 Hz), 7.19 (d, 2H, J = 9.0
Hz), 2.03 (s, 3H), 1.91 (d, 6H, J = 2.5 Hz), 1.71 (s, 6H); 13C NMR (Acetone-d6, 125 MHz) δ 176.7
(carbonyl carbon), 146.8 (1 C), 139.0 (1 C), 123.3 (2 x CH), 121.8 (2 x CH) [aromatic carbons],
42.3 (1 C, attached to carbonyl), 39.6 (3 C), 37.2 (3 C), 29.2 (3 C) [adamantyl carbons]; LC-MS
m/z: 351.14 (M + H)+; CHN analysis: calculated C:58.27%, H:6.33%, N:7.99%; found: C:58.11%,
H:6.25%, N:8.06%.
1
2-Fluoro-4-(1-adamantylcarboxamido)phenyl sulfamate (1i): Yield: 24%; mp: 159-62 °C; H
NMR (Acetone-d6, 500 MHz) δ 8.76 (brs, 1H), 7.87 (dd, 1H, J = 2.5, 13.0 Hz), 7.44-7.41 (m, 1H),
7.34 (t, 1H, J = 8.5 Hz), 7.25 (brs, 2H), 2.06-2.04 (m, 3H), 1.99 (d, 6H, J = 2.5 Hz), 1.79-1.73 (m,
6H); 13C NMR (Acetone-d6, 125 MHz) δ 177.0 (carbonyl carbon), 156.3 (1 C), 154.3 (1 C), 140.1
(1 C, d, JCF = 10.0 Hz), 133.6 (1 C, d, JCF = 12.6 Hz), 125.5 (1 C), 116.3 (1 C, d, JCF = 3.3 Hz),
109.2 (1 C, d, JCF = 24.0 Hz) [aromatic carbons], 42.4 (1 C, attached to carbonyl), 39.5 (3 C), 37.1
(3 C), 29.1 (3 C) [adamantyl carbons]; LC-MS m/z: 369.1 (M + H)+; CHN analysis: calculated
C:55.42%, H:5.75%, N:7.60%; found: C:55.23%, H:5.73%, N:7.66%.
1
2-Chloro-4-(1-adamantylcarboxamido)phenyl sulfamate (1j): Yield: 48%; mp: 158-61 °C; H
NMR (Acetone-d6, 500 MHz) δ 8.75 (brs, 1H), 8.05 (d, 1H, J = 2.5 Hz), 7.65 (dd, 1H, J = 2.5, 8.5
Hz), 7.44 (d, 1H, J = 9.0 Hz), 7.29 (brs, 2H), 2.08-2.05 (m, 3H), 2.01 (d, 6H, J = 2.5 Hz), 1.81-
1.74 (m, 6H); 13C NMR (Acetone-d6, 125 MHz) δ 177.0 (carbonyl carbon), 142.6 (1 C), 139.7 (1
C), 127.7 (1 C), 124.8 (1 C), 122.2 (1 C), 120.0 (1 C) [aromatic carbons], 42.4 (1 C, attached to
carbonyl), 39.5 (3 C), 37.1 (3 C), 29.1 (3 C) [adamantyl carbons]; LC-MS m/z: 385.04 (M + H)+;
CHN analysis: calculated C:53.05%, H:5.50%, N:7.28%; found: C:52.94%, H:5.34%, N:7.46%.
1
2-Bromo-4-(1-adamantylcarboxamido)phenyl sulfamate (1k): Yield: 49%; mp: 184-6 °C; H
NMR (Acetone-d6, 500 MHz) δ 8.71 (brs, 1H), 8.18 (d, 1H, J = 2.5 Hz), 7.69 (dd, 1H, J = 2.5, 9.0
Hz), 7.43 (d, 1H, J = 9.0 Hz), 7.27 (brs, 2H), 2.06-2.04 (m, 3H), 2.00 (d, 6H, J = 2.5 MHz), 1.79-
Page 20 of 31

1.73 (m, 6H); 13C NMR (Acetone-d6, 125 MHz) δ 177.0 (carbonyl carbon), 144.0 (1 C), 139.8 (1
C), 125.3 (1 C), 124.4 (1 C), 120.7 (1 C), 116.6 (1 C) [aromatic carbons], 42.4 (1 C, attached to
carbonyl), 39.5 (3 C), 37.1 (3 C), 29.1 (3 C) [adamantyl carbons]; LC-MS m/z: 429.09 (M + H)+;
CHN analysis: calculated C:47.56%, H:4.93%, N:6.52%; found: C:47.58%, H:4.89%, N:6.60%.
4-(1-Adamantylcarboxamidomethyl)phenyl sulfamate (1l): Yield: 44%; mp: 167-70 °C; 1H NMR
(Acetone-d6, 500 MHz) δ 7.32-7.30 (m, 3H), 7.24-7.23 (m, 2H), 7.09 (s, 2H), 4.39 (d, 2H, J = 6.0
Hz), 2.00 (s, 3H), 1.92 (d, 6H, J = 2.5 Hz), 1.77-1.70 (m, 6H); 13C NMR (Acetone-d6, 125 MHz)
δ 177.9 (carbonyl carbon), 150.3 (1 aromatic C, attached to sulfamate), 139.9 (2 x CH, aromatic
carbons next to sulfamate), 129.1 (2 x CH, aromatic carbons next to methylene) , 123.0 (1 aromatic
C, attached to methylene), 42.6 (1 adamantyl carbon attached to carbonyl), 41.3 (CH2 attached to
NH), 40.0 (3 adamantyl carbons), 37.3 (3 adamantyl carbons), 29.2 (3 adamantyl carbons); LC-
MS m/z: 364.81 (M + H)+; CHN analysis: calculated C:59.32%, H:6.64%, N:7.69%; found:
C:59.26%, H:6.78%, N:7.62%.
1
4-(N-Methyl-1-adamantylcarboxamido)phenyl sulfamate (1m): Yield: 20%; mp: 168-71 °C; H
NMR (Acetone-d6, 500 MHz) δ 7.39-7.38 (m, 4H), 7.21 (s, 2H), 3.12 (s, 3H), 1.82 (s, 3H), 1.76
13
(d, 6H, J = 2.5 Hz), 1.59 (d, 3H, J = 12.0 Hz), 1.52-1.50 (m, 3H); C NMR (Acetone-d6, 125
MHz) δ 177.5 (carbonyl carbon), 150.7 (1 aromatic C, attached to sulfamate), 144.9 (1 aromatic
C, attached to amide nitrogen), 130.9 (2 x CH, next to sulfamate), 123.8 (2 x CH, next to amide),
44.3 (1 adamantyl carbon attached to carbonyl), 41.7 (CH3), 41.0 (3 adamantyl carbons), 37.1 (3
adamantyl carbons), 29.3 (3 adamantyl carbons); LC-MS m/z: 364.81 (M + H)+; CHN analysis:
calculated C:59.32%, H:6.64%, N:7.69%; found: C:59.20%, H:6.54%, N:7.75%.
1
3-Methyl-4-(1-adamantylcarboxamido)phenyl sulfamate (1n): Yield: 48%; mp: 143-6 °C; H
NMR (Acetone-d6, 500 MHz) δ 8.10 (brs, 1H), 7.49 (d, 1H, J = 8.5 Hz), 7.16 (d, 1H, J = 2.5 Hz),
7.11-7.09 (m, 3H), 2.24 (s, 3H), 2.06-2.01 (m, 9H), 1.81-1.75 (m, 6H); 13C NMR (Acetone-d6, 125
MHz) δ 176.5 (carbonyl carbon), 148.4 (1 C), 136.2 (1 C), 134.8 (1 C), 127.1 (1 C), 124.7 (1 C),
Page 21 of 31

120.6 (1 C) [aromatic carbons], 42.1 (1 adamantyl carbon attached to carbonyl), 39.9 (3 adamantyl
carbons), 37.3 (3 adamantyl carbons), 29.2 (3 adamantyl carbons), 18.9 (CH3); LC-MS m/z:
365.26 (M + H)+; CHN analysis: calculated C:59.32%, H:6.64%, N:7.69%; found: C:59.24%,
H:6.43%, N:7.81%.
1
4-(1-Adamantylaminocarbonyl)phenyl sulfamate (1o): Yield: 12%; mp: 139-42 °C; H NMR
(Acetone-d6, 500 MHz) δ 7.87 (d, 2H, J = 9.0 Hz), 7.33 (d, 2H, J = 8.5 Hz), 7.18 (brs, 2H), 7.00
(brs, 1H), 2.18 (d, 6H, J = 2.5 Hz), 2.09 (s, 3H), 1.73 (s, 6H); 13C NMR (Acetone-d6, 125 MHz) δ
166.0 (carbonyl carbon), 153.2 (1 aromatic C, attached to sulfamate), 135.7 (1 aromatic C, attached
to carbonyl), 129.6 (2 x CH, next to sulfamate), 122.7 (2 x CH, next to carbonyl), 52.8 (1 adamantyl
carbon attached to amide), 42.1 (3 adamantyl carbons), 37.2(3 adamantyl carbons), 30.5 (3
adamantyl carbons); LC-MS m/z: 351.08 (M + H)+; CHN analysis: calculated C:58.27%, H:6.33%,
N:7.99%; found: C:58.34%, H:6.21%, N:8.05%.
1
2-Fluoro-4-(1-adamantylaminocarbonyl)phenyl sulfamate (1p): Yield: 11%; mp: 181-4 °C; H
NMR (Acetone-d6, 500 MHz) δ 8.11-8.06 (m, 2H), 7.78-7.75 (m, 2H), 7.60 (brs, 1H), 7.47 (t, 1H,
J = 9.0 Hz), 7.12 (brs, 1H), 2.20-2.17 (m, 6H), 2.10-2.09 (m, 3H), 1.74 (s, 6H); 13C NMR (Acetone-
d6, 125 MHz) δ 164.8 (1 aromatic C, d, JCF = 1.5 Hz), 156.5 (carbonyl carbon), 154.4 (1 aromatic
C), 140.8 (1 aromatic C, d, JCF = 13.4 Hz), 137.2 (1 aromatic C, d, JCF = 5.4 Hz), 128.8 (1 aromatic
C, d, JCF = 6.8 Hz), 116.9 (1 aromatic C), 53.0 (1 adamantyl carbon attached to amide), 42.0 (3
adamantyl carbons), 37.2 (3 adamantyl carbons), 30.5 (3 adamantyl carbons); LC-MS m/z: 369.1
(M + H)+; CHN analysis: calculated C:55.42%, H:5.75%, N:7.60%; found: C:55.30%, H:5.70%,
N:7.70%.
1
2-Chloro-4-(1-adamantylaminocarbonyl)phenyl sulfamate (1q): Yield: 52%; mp: 61-4 °C; H
NMR (Acetone-d6, 500 MHz) δ 7.93 (d, 1H, J = 2.0 Hz), 7.81 (dd, 1H, J = 2.0, 8.5 Hz), 7.56 (d,
1H, J = 8.5 Hz), 7.44 (brs, 2H), 7.15 (brs, 1H), 2.17 (d, 6H, J = 2.5 Hz), 2.09-2.04 (m, 3H), 1.73
(s, 6H); 13C NMR (Acetone-d6, 125 MHz) δ 164.8 (carbonyl carbon), 149.1 (1 aromatic C), 136.7
Page 22 of 31

(1 aromatic C), 130.3 (1 aromatic C), 128.0 (1 aromatic C), 127.7 (1 aromatic C), 124.3 (1 aromatic
C), 53.1 (1 adamantyl carbon attached to amide), 42.0 (4 adamantyl carbons), 37.2 (5 adamantyl
carbons); LC-MS m/z: 385.23 (M + H)+; CHN analysis: calculated C:53.05%, H:5.50%, N:7.28%;
found: C:53.25%, H:5.30%, N:7.41%.
1
2-Bromo-4-(1-adamantylaminocarbonyl)phenyl sulfamate (1r): Yield: 29%; mp: °C; H NMR
(Acetone-d6, 500 MHz) δ 8.71 (brs, 1H), 8.17 (d, 1H, J = 2.5 Hz), 7.69 (dd, 1H, J = 2.5, 9.0 Hz),
7.43 (d, 1H, J = 9.0 Hz), 7.27 (brs, 2H), 2.06-2.04 (m, 3H), 2.00 (d, 6H, J = 2.5 Hz), 1.79-1.73 (m,
6H); 13C NMR (Acetone-d6, 125 MHz) δ 177.0 (carbonyl carbon), 144.0 (1 aromatic C), 139.8 (1
aromatic C), 125.3 (1 aromatic C), 124.4 (1 aromatic C), 120.7 (1 aromatic C), 116.6 (1 aromatic
C), 42.4 (1 adamantyl carbon attached to amide), 39.5 (3 adamantyl carbons), 37.1 (3 adamantyl
carbons), 29.1 (3 adamantyl carbons); LC-MS m/z: 429.09 (M + H)+; CHN analysis: calculated
C:47.56%, H:4.93%, N:6.52%; found: C:47.55%, H:4.86%, N:6.66%.
4-[3-(1-Adamantyl)ureido]phenyl sulfamate (1s): Yield: 53%; mp: 204-5 °C; 1H NMR (Acetone-
d6, 500 MHz) δ 7.80 (brs, 1H), 7.46 (d, 2H, J = 9.0 Hz), 7.15 (d, 2H, J = 9.0 Hz), 6.96 (brs, 2H),
5.48 (brs, 1H), 2.06-2.04 (m, 8H), 1.69 (s, 7H); 13C NMR (Acetone, 125 MHz) δ 154.8 (carbonyl
carbon), 145.4 (1 aromatic C, attached to sulfamate), 140.5 (1 aromatic C, attached to urea), 123.4
(2 x CH, aromatic carbons next to sulfamate), 119.3 (2 x CH, aromatic carbons next to urea), 51.3
(1 adamantyl carbon), 42.8 (3 adamantyl carbons), 37.2 (3 adamantyl carbons), 29.4 (3 adamantyl
carbons); LC-MS m/z: 366.14 (M + H)+; CHN analysis: calculated C:55.87%, H:6.34%,
N:11.50%; found: C:55.90%, H:6.26%, N:11.62%.
1
1-(Cyclohexanecarbonyl)indole-5-ol sulfamate (1t): Yield: 11%; mp: 158-60 °C; H NMR
(Acetone-d6, 500 MHz) δ 8.47 (d, 1H, J = 9.0 Hz), 8.00 (d, 1H, J = 4.0 Hz), 7.55 (d, 1H, J = 2.0
Hz), 7.28 (dd, 1H, J = 2.0, 9.0 Hz), 7.04 (brs, 2H), 6.77 (d, 1H, J = 9.0 Hz), 3.34-3.30 (m, 1H),
2.02-1.99 (m, 2H), 1.85-1.82 (m, 2H), 1.76-1.73 (m, 1H), 1.67-1.59 (m, 2H), 1.55-1.47 (m, 2H),
1.33-1.29 (m, 1H); 13C NMR (Acetone, 125 MHz) δ 175.8 (carbonyl carbon), 147.6 (1 C), 134.7
Page 23 of 31

(1 C), 132.2 (1 C), 128.2 (1 C), 120.0 (1 C), 117.9 (1 C), 115.1 (1 C), 109.0 (1 C) [aromatic
carbons], 43.6 (cyclohexyl CH attached to carbonyl), 30.4 (2 x CH2, cyclohexyl), 26.5 (2 x CH2,
cyclohexyl), 26.0 (2 x CH2, cyclohexyl); LC-MS m/z: 323.16 (M + H)+; CHN analysis: calculated
C:55.88%, H:5.63%, N:8.69%; found: C:55.61%, H:5.80%, N:8.60%.
1
1-[(1-Adamantanecarbonyl)]indole-5-ol sulfamate (1u): Yield: 14%; mp: 198-201 °C; H NMR
(Acetone-d6, 500 MHz) δ 8.44 (d, 1H, J = 9.0 Hz), 8.31 (d, 1H, J = 4.0 Hz), 7.54 (d, 1H, J = 2.5
Hz), 7.26 (dd, 1H, J = 2.0, 9.0 Hz), 7.03 (s, 2H), 6.75 (d, 1H, J = 4.0 Hz), 2.27-2.26 (d, 6H, J =
2.5 Hz), 2.14 (s, 3H), 1.90 (d, 3H, J = 11.5 Hz), 1.83 (d, 3H, J = 12.5 Hz); 13C NMR (CDCl3, 125
MHz) δ 177.1 (carbonyl carbon), 147.5 (1 C), 135.8 (1 C), 131.0 (1 C), 128.8 (1 C), 119.9 (1 C),
118.5 (1 C), 114.8 (1 C), 108.4 (1 C) [aromatic carbons], 45.0 (1 adamantyl carbon attached to
carbonyl), 40.0 (3 adamantyl carbons), 37.0 (3 adamantyl carbons), 29.3 (3 adamantyl carbons);
LC-MS m/z: 375.09 (M + H)+; CHN analysis: calculated C:60.94%, H:5.92%, N:7.48%; found:
C:60.88%, H:6.02%, N:7.41%.
4.7.
Cell-free STS enzyme assay
STS inhibitory assays were performed as described previously [18]. Briefly, a compound’s
ability to inhibit STS activity was determined using the lysate of JEG-3, a human placenta
choriocarcinoma cell line which has high STS activity. To ascertain STS inhibition, enzyme
activity was measured in the absence and presence of the inhibitor (0.001–10ꢀμM) using [3H]
estrone sulfate (E1S; 4ꢀ×ꢀ105 dpm, Perkin Elmer) adjusted to 20ꢀμM with unlabelled E1S substrate.
After incubation of the substrate and inhibitor with JEG-3 lysate (125ꢀμg of protein/mL) for 1ꢀh,
the product formed, estrone (E1), was separated from the mixture by extraction with toluene. [4–
14C]E1 (American Radiolabelled Chemicals) was also used throughout the assay to monitor
3
14
procedural losses. An organic phase aliquot was added to scintillation fluid and the H and C
content measured by scintillation spectrometry. The mass of E1S hydrolyzed was calculated from
3
the H counts detected (corrected for the volume of medium and organic solvent used and for
recovery of 14C counts) and the specific activity of the substrate.
Page 24 of 31

4.8.
Whole-cell STS enzyme assay
To determine if compounds could pass through the cell lipid bilayer, intact monolayers of JEG-
3ꢀcells were incubated for 20ꢀhꢀat 37ꢀ°C with [3H]E1S (5ꢀpmol, 7ꢀ×ꢀ105 dpm, 60ꢀCi/mmol) in serum-
free Eagle's Minimal Essential Medium (1.0ꢀmL) with or without inhibitors (10ꢀμM). After
incubation, medium (0.5ꢀmL) was removed and product E1 separated from E1S by solvent
partition using toluene. [14C] Estrone (7ꢀ×ꢀ103 dpm, 52ꢀmCi/mmol) was used to correct for
procedural losses. The mass of E1S hydrolyzed was calculated as outlined in section 4.7 above.
4.9.
Screening of antiproliferative activity
T-47D cells were grown in Eagle’s minimum essential medium, supplemented with sodium
pyruvate (110 mg/mL), gentamycin sulfate (50 mg/L), sodium bicarbonate (2.2 g/L) and 10% fetal
bovine serum. The serum was sterilized through a 0.20 μM filter and stored at -20 °C. The cells
were grown in a humidified incubator in 5% carbon dioxide at 37 °C and harvested with 0.05%
trypsin/0.02% EDTA in 0.15 M sodium chloride solution. The cell suspension was transferred to
96-well microplates (100 μL per well) at the beginning of the experiment. After growing them for
3 days in a humidified incubator with 5% carbon dioxide at 37 °C, the medium was replaced by
one containing the test compound, 100 nM estradiol sulfate (E2S), and 10% charcoal-stripped fetal
bovine serum. The initial cell density was determined by addition of glutaric dialdehyde (1% in
phosphate-buffered saline; 100 μL per well). After incubation for 5 days, the medium was removed
and 100 μL of glutaric aldehyde in phosphate-buffered saline (1%) was added for fixation. After
15 min, the solution of aldehyde was decanted. Cells were stained by treating them for 25 min with
100 μL of an aqueous solution of crystal violet (0.02%). After decanting, cells were washed several
times with water to remove adherent dye. After addition of 100 μL of ethanol (70%), plates were
gently shaken for 2 h. Optical density of each well was measured in a microplate autoreader EL
309 at 578 nm.
Acknowledgments
Page 25 of 31

The authors are thankful to Al-Jalila Foundation, United Arab Emirates, for funding this
project (grant No. AJF201744).
Supplementary File
1H NMR, 13C NMR, and LC-MS charts are available at XXXX
References
[1] J.W. Mueller, L.C. Gilligan, J. Idkowiak, W. Arlt, P.A. Foster, The regulation of steroid action
by sulfation and desulfation. Endocr. Rev. 36 (2015) 526-563.
[2] S.-O. Zaraei, A.R. Abduelkarem, H.S. Anbar, S. Kobeissi, M. Mohammad, A. Ossama, M.I.
El-Gamal, Sulfamates in drug design and discovery: Pre-clinical and clinical investigations. Eur.
J. Med. Chem. 179 (2019) 257-271.
[3] M.P. Thomas, B.V.L. Potter, Discovery and development of the aryl O-sulfamate moiety for
oncology and women’s health. J. Med. Chem. 58 (2015) 7634-7658.
[4] M.P. Thomas, B.V.L. Potter, Estrogen O-sulfamates and their analogues: clinical steroid
sulfatase inhibitors with broad potential. J. Steroid Biochem. Mol. Biol. 153 (2015) 160-169.
[5] B.V.L. Potter, Steroid sulfatase inhibition via aryl sulfamates: Clinical progress, mechanism
and future prospects. J. Mol. Endocrinol. 61 (2018) T233-T252.
[6] P.A. Foster, S.P. Newman, S.K. Chander, C. Stengel, R. Jhalli, L.W.L. Woo, B.V.L. Potter,
M.J. Reed, A. Purohit, In vivo efficacy of STX213, a second generation steroid sulfatase inhibitor,
for hormone-dependent breast cancer therapy. Clin. Cancer Res. 12 (2006) 5543-5549.
[7] L.W.L. Woo, D.S. Fischer, C.M. Sharland, M. Trusselle, P.A. Foster, S.K. Chander, A.D.
Fiore, C.T. Supuran, G.D. Simone, A. Purohit, M.J. Reed, B.V.L. Potter, Anticancer steroid
sulfatase inhibitors: synthesis of a potent fluorinated second-generation agent, in vitro and in vivo
activities, molecular modeling, and protein crystallography. Mol. Cancer Ther. 7 (2008) 2435-
2444.
Page 26 of 31

[8] L.W.L. Woo, D. Ganeshapillai, M.P. Thomas, O.B. Sutcliffe, B. Malini, M.F. Mahon, A.
Purohit, B.V.L. Potter, Structure–Activity Relationship for the First‐in‐Class Clinical Steroid
Sulfatase Inhibitor Irosustat (STX64, BN83495). ChemMedChem 6 (2011) 2019-2034.
[9] C. Phan, Y. Liu, B. Kim, Y. Mostafa, S. Taylor, Inhibition of steroid sulfatase with 4-
substituted estrone and estradiol derivatives. Bioorg. Med. Chem. 19 (2011) 5999-6005.
[10] A. Purohit, P.A. Foster, Steroid sulfatase inhibitors for estrogen- and androgen-dependent
cancers. J. Endocrinol. 212 (2012) 99-110.
[11] L.W.L. Woo, B. Leblond, A. Purohit, B.V.L. Potter, Synthesis and evaluation of analogues
of estrone-3-O-sulfamate as potent steroid sulfatase inhibitors. Bioorg. Med. Chem. 20 (2012)
2506-2519.
[12] M.I. El-Gamal, M.H. Semreen, P.A. Foster, B.V.L. Potter, Design, synthesis, and biological
evaluation of new arylamide derivatives possessing sulfonate or sulfamate moieties as steroid
sulfatase enzyme inhibitors. Bioorg. Med. Chem. 24 (2016) 2762-2767.
[13] C. Quellet, R. Maltais, E. Ouellet, X. Barbeau, P. Lague, D. Poirier, Discovery of a sulfamate-
based steroid sulfatase inhibitor with intrinsic selective estrogen receptor modulator properties.
Eur. J. Med. Chem. 119 (2016) 169-182.
[14] M. Dasko, M. Przybylowska, J. Rachon, M. Maslyk, K. Kubinski, M. Misiak, A.
Skladanowski, S. Demkowicz, Synthesis and biological evaluation of fluorinated N-benzoyl and
N-phenylacetoyl derivatives of 3-(4-aminophenyl)-coumarin-7-O-sulfamate as steroid sulfatase
inhibitors. Eur. J. Med. Chem. 128 (2017) 79-87.
[15] D. Ganeshapillai, L.W.L. Woo, M.P. Thomas, A. Purohit, B.V.L. Potter, C-3- and C-4-
substituted bicyclic coumarin sulfamates as potent steroid sulfatase inhibitors. ACS Omega 3
(2018) 10748-10772.
Page 27 of 31

[16] D. Moi, P.A. Foster, L.G. Rimmer, A. Jaffri, A. Deplano, G. Balboni, V. Onnis, B.V.L. Potter,
Synthesis and in vitro evaluation of piperazinyl-ureido sulfamates as steroid sulfatase inhibitors.
Eur. J. Med. Chem. 182 (2019) 111614.
[17] S.J. Stanway, A. Purohit, L.W. Woo, S. Sufi, D. Vigushin, R. Ward, R.H. Wilson, F.Z.
Stanczyk, N. Dobbs, E. Kulinskaya, M. Elliott, B.V.L. Potter, M.J. Reed, R.C. Coombes, Phase I
study of STX 64 (667 Coumate) in breast cancer patients: the first study of a steroid sulfatase
inhibitor. Clin. Cancer Res. 12 (2006) 1585-1592.
[18] A. Purohit, G.J. Williams, N.M. Howarth, B.V.L. Potter, M.J. Reed, Inactivation of steroid
sulfatase by an active site-directed inhibitor, estrone-3-O-sulfamate. Biochemistry 34 (1995)
11508-11514.
Page 28 of 31

Graphical abstract
O
H
N
N
O
O
O
O
H
S
O
S
H2N
O
H2N
O
1h
Cl
1q
% inhibition against STS at 10 µM = 93.90%
IC50 (STS, cell lysate) = 109.5 nM
IC50 (STS, whole-cell) = 13.6 nM
IC50 (T-47D cell line) = 5.78 µM
% inhibition against STS at 10 µM = 98.30%
IC50 (STS, cell lysate) = 25.8 nM
IC50 (STS, whole-cell) = 150.0 nM
IC50 (T-47D cell line) = 1.04 µM
Page 29 of 31