Design and synthesis of 3-benzylaminocoumarin-7-O-sulfamate derivatives as steroid sulfatase inhibitors

Article abstract of DOI:10.1016/j.bioorg.2020.103618

Steroid sulfatase (STS) is a sulfatase enzyme that catalyzes the conversion of sulfated steroid precursors to free steroid. The inhibition of STS could abate estrogenic steroids that stimulate the proliferation and development of breast cancer, and therefore STS is a potential target for adjuvant endocrine therapy. In this study, a series of 3-benzylaminocoumarin-7-O-sulfamate derivatives targeting STS were designed and synthesized. Structure-relationship activities (SAR) analysis revealed that attachment of a benzylamino group at the 3-position of coumarin improved inhibitory activity. Compound 3j was found to have the highest inhibition activity against human placenta isolated STS (IC₅₀ 0.13 μM) and MCF-7 cell lines (IC₅₀ 1.35 μM). Kinetic studies found compound 3j to be an irreversible inhibitor of STS, with K_I and k_inact value of 86.9 nM and 158.7 min^?1, respectively.

Full text of DOI:10.1016/j.bioorg.2020.103618

Journal Pre-proofs
Design and Synthesis of 3-Benzylaminocoumarin-7-O-Sulfamate Derivatives
as Steroid Sulfatase Inhibitors
Yue Hng, Mei-Hsiang Lin, Tzung-Sheng Lin, I-Chen Liu, I-Chun Lin, Yeh-
Lin Lu, Chiao-Nien Chang, Pei-Fang Chu, Keng-Chang Tsai, Mei-Jou Chen,
Pi-Hui Liang
PII:
S0045-2068(19)32077-2
DOI:
https://doi.org/10.1016/j.bioorg.2020.103618
YBIOO 103618
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
4 December 2019
22 January 2020
22 January 2020
Please cite this article as: Y. Hng, M-H. Lin, T-S. Lin, I-C. Liu, I-C. Lin, Y-L. Lu, C-N. Chang, P-F. Chu, K-C.
Tsai, M-J. Chen, P-H. Liang, Design and Synthesis of 3-Benzylaminocoumarin-7-O-Sulfamate Derivatives as
Steroid Sulfatase Inhibitors, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.103618
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Design and Synthesis of 3-Benzylaminocoumarin-7-O-Sulfamate Derivatives as Steroid
Sulfatase Inhibitors
1
2
1
1
1
1, 2
Yue Hng, Mei-Hsiang Lin, Tzung-Sheng Lin, I-Chen Liu, I-Chun Lin, Yeh-Lin Lu,
Chiao-Nien
1
1
3
4
1, 5, *
Chang, Pei-Fang Chu, Keng-Chang Tsai, Mei-Jou Chen, Pi-Hui Liang
Y. Hng, Dr. T.-S. Lin, I.-C. Liu, I.-C. Lin, Dr. Y.-L., Lu, C.-N. Chang, P.-F. Chu, Prof. Dr. P.-H. Liang
. School of Pharmacy, College of Medicine, National Taiwan University, Taipei 100, Taiwan.
1
Prof. Dr. M.-H. Lin, Dr. Y.-L. Lu
. School of Pharmacy, College of Pharmacy, Taipei Medical University, Taipei 110, Taiwan.
2
Prof. Dr. K.C Tsai
. National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei 112, Taiwan
3
Prof. Dr. M.-J. Chen
. Department of Obstetrics and Gynecology and Livia Shangyu Wan Scholar, National Taiwan University
4
Hospital, National Taiwan University, College of Medicine, Taipei 100, Taiwan
Prof. Dr. P.-H. Liang
5
. Genomics Research Center, Academia Sinica, Taipei 128, Taiwan
Corresponding author
P.-H. Liang, Ph.D.
School of Pharmacy, College of Medicine, National Taiwan University,
No. 17, Xu-Zhou Road, Taipei 100, Taiwan
Tel: +886-233668695
E-mail: phliang@ntu.edu.tw
Key Words: Coumarin; Steroid Sulfatase; Sulfamate; Hormone dependent cancer; Breast Cancer;
Irreversible Inhibitors

Abstract
Steroid sulfatase (STS) is a sulfatase enzyme that catalyzes the conversion of sulfated steroid precursors
to free steroid. The inhibition of STS could abate estrogenic steroids that stimulate the proliferation and
development of breast cancer, and therefore STS is a potential target for adjuvant endocrine therapy. In this
study, a series of 3-benzylaminocoumarin-7-O-sulfamate derivatives targeting STS were designed and
synthesized. Structure-relationship activities (SAR) analysis revealed that attachment of a benzylamino group
at the 3-position of coumarin improved inhibitory activity. Compound 3j was found to have the highest
inhibition activity against human placenta isolated STS (IC = 0.13 μM) and MCF-7 cell lines (IC 1.35
5
0
50
µM). Kinetic studies found compound 3j to be an irreversible inhibitor of STS, with K and k
value of 86.9
inact
I
-1
nM and 158.7 min , respectively.

1
. Introduction
Steroid sulfatase (STS) is a potential target for hormone-dependent breast cancer
treatment [1]. STS is a sulfatase enzyme with a molecular mass of 63 kDa that is involved
in the metabolism of steroids. It plays a crucial role in the regulation of steroid hormone
concentration, and is responsible for the conversion of estrone sulfate (E1S) to estrone
(E1), as well as dehydroepiandrosterone sulfate (DHEAS) to dehydroepiandrosterone
(DHEA). After STS hydrolysis, E1 and DHEA undergo further reduction to give estradiol
(E2) and adiol, which can bind to the estrogen receptor (ER) and cause mitogenesis. STS
is present in several human tissues and organs. However, in ER-positive breast cancer
tumors, STS is overexpressed and estrogen concentrations are increased [2]. Therefore,
inhibition of STS could diminish the production of the estrogenic steroids that stimulate
the proliferation and development of breast cancer [3-5].
Irreversible steroidal and non-steroidal STS inhibitors have been under development
since the 1990s [6-10]. Estrone-O-sulfamate (EMATE, Figure 1), a mimic of natural E1S,
exhibited high inhibitory potential (IC = 80 nM) against STS placental microsome.
5
0
However, it was withdrawn from clinical trials due to its high estrogenic properties [7].
In 2000, sulfamoylated tricyclic coumarin derivatives were developed, one of which,
STX64 (also called 667Coumate, Irosustat, BN83495, 2, Figure 1), was found to be a
highly potent (IC = 8 nM) STS inhibitor, without exhibiting significant estrogenicity.
5
0
STX64 became the first non-steroidal STS inhibitor to enter clinical trials for hormone-
dependent breast cancer [11-15].
Known STS inhibitors shared two structural common features: a non-steroidal aryl
core and a sulfate congener. However, coumarin-based sulfamates have also proved
capable of inhibiting STS enzyme activities [16-19]. In this study, we synthesized a series
of 3-benzylaminocoumarin-7-O-sulfamate derivatives (3, Figure 1) incorporating a
1

conserved coumarin sulfamate core and bearing a benzyl ring at 3-position, and studied
their structure relationship activities (SAR). A simplified structure (4, Figure 1) was also
studied.
O
H
N
RR
O
S
O
S
H N
2
O
H N
2
O
O
O
R = F, Cl, CH3,
O
O
OCH , NO
3
2
3
EMATE (1)
H
N
O
O
n
S
S
H N
2
O
O
O
H N
2
O
O
O
n = 1~3
4
STX64 (2)
Figure 1. Structure of estrone-O-sulfamate (EMATE, 1), STX64 (2) and rational design
of 3-aminocoumarin provided a series of compounds 3 and 4.
2
. Chemistry
2
.1 Synthesis of compound 3a-q and 8
The synthesis of 3-benzylaminocoumarin-7-O-sulfamate derivatives is depicted in
Scheme 1. 2,4-Dihydroxybenzaldehyde, N-acetylglycine, sodium acetate (NaOAc), and
acetic anhydride (Ac O) were refluxed together to give 3-amino-7-hydroxy-2H-chromen-
2
2
-one (5) [20]. Reductive amination with a series of benzaldehydes and sodium
cyanoborohydride (NaBH CN) gave the 7-hydroxy-3- benzylaminocoumarin derivatives
3
(6a-q). Finally, compounds 6a-q were treated with triethylamine (TEA) followed by
sulfamoyl chloride, and 3-benzylaminocoumarin-7- O-sulfamate derivatives (3a-q) were
obtained. 3-Phenylethylamino congener 8 was synthesized using the similar process.
2

R2
R5
O
R1
R3
R4
a
NH2
O
b
H
N
H
HO
O
n
HO
OH
HO
O
O
5
6
a-q (n = 1)
7
(n = 2)
R2
R5
R1
R3
R4
c
H
N
O
n
S
H N
2
O
O
O
O
3
8
a-q (n = 1)
(n = 2)
Scheme 1. Synthesis of compounds 3a-q and 8. Reagents and conditions: (a) i. N-
acetylglycine (1 eq), NaOAc (1.5 eq), Ac O, reflux; ii. Conc. HCl, EtOH, reflux; (b)
2
o
acetaldehyde or benzaldehyde (1.5 eq), EtOH, 5% (v/v) acetic acid, 80 C, NaBH CN (2
3
o
eq); (c) sulfamoyl chloride (2 eq), TEA (1 eq), anhydrous THF, 0 C-rt.
2
.2. Synthesis of 4-amino-phenyl-O-sulfamates 4
Sulfamoylations of 13a-c were achieved by adding an in situ preparation of
sulfamoyl chloride by formic acid and chlorosulfonyl isocyanate to afford 4a-c (Scheme
).
2
H
N
H
a
N
O
S
n
n
H N
2
O
HO
O
4a, n = 1
4b, n = 2
4c, n = 3
13a, n = 1
13b, n = 2
13c, n = 3
Scheme 2. Synthesis of 4a-c. Reagents and conditions: (a) i. formic acid, chlorosulfonyl
o
isocyanate, anhydrous DCM/DMA, 40 C, 3 h; ii. DMA, rt, 1 h.
2
.3. Synthesis of amide containing compound 12
Synthesis of 3-(2,3-dimethoxybenzamido)coumarin-7-O-sulfamate (12) is depicted in
3

Scheme 3. 2,3-Dimethoxybenzoic acid (9) was reacted with thionyl chloride (SOCl ) to
2
form 2,3-dimethoxybenzoyl chloride (10), which was reacted with compound 5 to give
N-(7-hydroxy-2-oxochromen-3-yl)-2,3-dimethoxybenzamide (11). Sulfamoylation of 11
was accomplished using TEA and sulfamoyl chloride to afford 12.
O
O
O
O
O
O
O
O
O
O
O
O
H
N
H
N
a
b
c
O
S
HO
Cl
HO
O
O
H2N
O
O
O
O
9
10
11
12
Scheme 3. Synthesis of compound 12. Reagents and conditions: (a) SOCl (2 eq), DCM,
2
0
0
°C; (b) 5, ACN/THF (1:1); (c) sulfamoyl chloride (2 eq), TEA (1 eq), anhydrous DMF,
°C - rt.
3
. Results and Discussion
3
.1 Para-position inductive effects of compounds on STS inhibition
STS inhibitory activities of the synthetic compounds were measured using purified
STS from human placenta [21, 22]. The results are presented in Table 1. The influence
of the inductive and mesomeric effects of the para-position of the adjacent benzyl ring of
compound 3a-f on STS inhibition was first studied. Compound 3f (R = OCH ) was found
3
3
to be the most potent inhibitor, followed by compound 3c (R = Cl), their IC values on
3
50
purified human placental STS were 1.2 µM and 2.6 µM, respectively. The inhibitory
potencies of compound 3a (R = H) and compound 3e (R = F) were almost identical;
3
3
their IC on purified human placental STS were found to be 3.9 µM and 4.1 µM,
50
respectively, 3-fold less potent than compound 3f (R = OCH ). Compound 3d (R = NO )
3
3
3
2
did not measurably inhibit STS at all, from which it can be inferred that a strongly
electron-withdrawing group on the benzyl ring was unfavorable. The STS inhibition trend
of these compounds on STS from MCF-7 cells are roughly consistent, compound 3f (R3
4

=
OCH ) still shows the highest inhibition activity (IC = 4.9 µM), while secondly is
3 50
compound 3c (R = Cl) with IC of 8.9 µM.
3
50
For compounds bearing a single methoxy group (compound 3f, 3g, and 3h),
compound 3f showed highest inhibitory potential, while compounds 3g and 3h had no
significant inhibitory effect. Of the compounds bearing two methoxy groups (compound
3
i-n), compound 3j had the highest inhibitory potential with an IC of 0.13 μM; ahead
50
of compound 3m, with an IC of 1.2 µM, making it about 9-fold less potent of an
5
0
inhibitor than compound 3j. For compounds bearing three methoxy groups (compound
o-q), compound 3o had the highest inhibitory potency (IC50 1.8 µM), ahead of
compound 3q (IC 23 µM). These STS inhibitory trends are roughly consistent with
3
5
0
those observed for MCF-7 cells, although absolute IC50 values between the two
experiments vary by about a factor of ten.
Compound 3j was the most potent inhibitor of human placental STS (IC 0.13
5
0
μM), still 13-fold less potent than STX64 (IC 10 nM). The methoxy substituents on the
5
0
phenyl ring were crucial for STS inhibition. Compound 3f, 3m and 3o (bearing methoxy
groups at para position; ortho and para; otho, meta and para positions, respectively)
exhibited similar STS inhibition, with IC of 1.2 µM, 1.2 µM and 1.8 µM; 9.5-fold, 9-
5
0
fold and 14-fold less potent in comparison with compound 3j, respectively. All the other
compounds did not significantly inhibit STS at all, in either the placenta or MCF-7
experiments. The results suggest that methoxy substitution at the para-position was not
necessary for good inhibitory potency. However, methoxy substitution at the ortho and
meta position (compound 3j) was significant; removal of either (compounds 3g and 3h)
resulted in loss of inhibitory activity. Compound 3o (R = R = R = OCH ), which retains
1
2
3
3
methoxy groups at the otho and meta positions, had a similar inhibitory potency to
compound 3f (R = OCH ), but was much less potent than compound 3j, showing that the
3
3
5

presence of a methoxy group at the para-position reduces the inhibitory potency. The
exact meacahnsim of aryl sulfamate bearing inhibitors for STS were still unknown [5,
2
3]. One potential mechanism was so-called pre-inhibition binding mode. After docking
into the catalytic site, the sulfamate ester bound to the catalytical formyl form of STS and
phenol bond was activated to eliminate a reactive sulphonlamine that attacked FGly [5].
In our study, the number of substitutions and their positions at the phenyl of compound
3
did influence STS inhibition, but subtle changes of them would loss of activity. It was
presumably that steric factor and position of substitutents [24] played important roles for
bind with STS. This remains to be investigated furhure after much higher-resolution
crystallographic structure of STS and sulfamate inhibitor is available.
Table 1. Potential inhibitory compounds 3, 4, 8, and 12 on STS from purified human
placenta microsome and MCF-7 cells.
R2
OCH3
R1
R3
H CO
H
N
3
H
N
H
N
O
S
n
O
S
R4
O
S
n
R5
H N
2
O
O
O
H N
2
O
O
O
O
H N
2
O
O
O
O
3
8
a-q (n = 1)
4a, n = 1
4b, n = 2
(n = 2)
12
4c, n = 3
a
IC of STS inhibition (μM)
5
0
Compds
R1
R2
R3
R4
R5
Placental
microsome
3.9 ± 1.0
30 ± 5.0
2.6 ± 0.8
74 ± 7.3
4.1 ± 1.1
1.2 ± 0.9
55 ± 7.5
NIb
MCF-7 cell
3
a
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
15 ± 2.3
49 ± 8.8
8.9 ± 1.5
NIb
3
b
3
c
3
d
NO2
F
3
e
25 ± 3.2
4.9 ± 1.3
NIb
3
f
OCH3
H
3
g
H
OCH3
H
3
h
OCH3
H
H
NIb
NIb
3
i
OCH3 OCH3
NIb
6

3
j
OCH3 OCH3
H
H
H
OCH3
OCH3
H
H
0.13 ± 0.3
53 ± 3.7
NIb
1.3 ± 0.9
NIb
3
k
H
OCH3
H
H
3
l
OCH3
OCH3
OCH3
H
H
NIb
3
m
H
OCH3
H
H
1.2 ± 0.53
NIb
3.3 ± 2.5
NIb
3
n
H
H
OCH3
3
o
OCH3 OCH3 OCH3
H
H
H
H
-
1.82 ± 0.37
NIb
3.6 ± 1.7
NIb
3
3
p
q
OCH3
H
OCH3 OCH3
H
-
OCH3 OCH3 OCH3
23 ± 3.5
NIb
35 ± 5.6
NIb
4
a
-
-
-
-
-
-
-
4
b
-
-
NIb
NIb
NIb
NIb
4
c
-
-
-
-
8
OCH3 OCH3
OCH3 OCH3
H
H
-
H
H
-
H
H
-
30 ± 2.5
0.16 ± 0.02
0.01 ± 0.002
NIb
1
2
8.2 ± 3.3
0.07 ± 0.01
STX64
-
-
a
b
Each value is the mean ± S.E.M, n = 3; no inhibition at 50 μM.
Compound 8, an elongated version of compound 3j wherein the 2,3-
dimethoxybenzyl group was lengthened by one CH unit, was synthesized to investigate
2
the effect of rigidity and rotational freedom on inhibitory activity. It was not found to
have any inhibitory effect at 30 μM. Compound 12, wherein the benzylamino group of
compound 3j was substituted by a benzamide group (to increase conformational rigidity),
was found to exhibit similar inhibitory activity to compound 3j. Simply structures 4a-c
were all found not to have any inhibitory activities, suggesting that the coumarin core was
essential for STS interaction.
3
.2 Quantification of STS inactivation by compound 3j
To investigate whether compound 3j might be irreversible inhibitor, a Kitz-Wilson
analysis was undertaken, to determine the inhibition constant (K ) and the maximal STS
I
inactivation rate (kinact) [25]. Human placental STS enzyme was pre-incubated with
different concentrations of compound 3j, followed by the addition of drug-free reaction
7

buffer containing the substrate 4-methylumbelliferyl sulfate (4-MUS) at various time
intervals. The dose-dependent inhibitory curve shifted to the left (Figure 2) and the
calculated inhibition activity (IC ) of compound 3j increased from 180 nM to 84 nM, as
5
0
the pre-incubation time increased from 0 to 40 min (Table 2). The relative STS activity
at various concentration of compound 3j was plotted as time-dependent graph (Figure
3
A). The slope for each concentration (the reciprocal of reaction rate constant, Kobs-1) was
-1
then calculated and plotted against the reciprocal of inhibitor concentration ([3j] )
(
Figure 3B). The inverse of x-intercept represented K and the slope represented kinact. The
I
-1
determined K and k
values were 86.9 ± 29 nM and 158.7 ± 32.8 min , respectively.
inact
I
These dada supported the capability of compound 3j to be an irreversible inhibitor for
STS.
Table 2. Inhibition activity (IC ) of compound 3j on human placental STS after 0, 10,
5
0
2
0 and 40 min of pre-incubation time with different inhibitor concentrations.
O
O
H
N
O
S
H N
2
O
O
O
O
3j
a
Time (min)
IC (nM)
50
8

0
180 ± 50
171 ± 41
111 ± 30
84 ± 1
1
0
0
0
2
4
aEach value is the mean ± S.E.M., n = 3.
Figure 2. Dose-dependent curve of inhibitory effects of compound 3j on human
placental STS after 0, 10, 20, and 40 min of pre-incubation time.
B
A
Figure 3. Kitz-Wilson analysis of compound 3j on human placental STS. (A)
Extrapolation of the time depenent change in STS activity at a given concentration of 3j.
(
B) The slope (Kobs-1) calculated from each concentration was plotted against inhibitor
-1
concentration ([3j] ). K and k
values are the mean ± S.E.M., n = 3.
I
inact
4
. Conclusion
A series of coumarin sulfamate derivatives (3a-q) were designed and synthesized,
and tested for their ability to inhibit the activity of STS. SAR of different substitution
patterns on the benzyl side chain were studied. Inhibition assays were conducted using
9

purified human placental STS, and MCF-7 breast cancer cells. It was found that the
presence of a methoxy group at the otho and meta positions of the benzyl side chain (3j)
was found to be essential for good potency (IC on human placental STS, 0.13 μM); the
5
0
removal of either of these methoxy groups (to give compounds 3g and 3h) resulted in a
loss of the inhibitory effect. In addition, extension of the side chain (8) of compound 3 or
simply structures (phenyl-O-sulfamates, 4) resulted in a loss of inhibitory activities,
suggesting that increased flexibility on this side chain disfavored binding with STS. This
assumption was verified by way of compound 12 – the rigid nature (and appropriately
located methoxy groups) of this molecule are thought to account for its good inhibitory
activity. Finally, kinetic studies found compound 3j to be an irreversible inhibitor of STS,
-1
with K and k
value of 86.9 nM and 158.7 min , respectively. The modification on the
inact
I
amine side chain of compound 3 might be worth exploring in the future studies.
5
. Materials and methods
5
.1 Chemistry
Most chemicals and solvents were of analytical grade and, when necessary, were
purified and dried by standard methods. All reactions were monitored by thin layer
chromatography (TLC) using Merck (Art. 5715) silica gel plates and visualized under UV
light (254 nm), upon treatment with iodine vapor, or upon heating after treatment with
1
13
5
% phosphomolybdic acid in ethanol. H and C NMR spectra were recorded on either
a Bruker AV-400 or AVIII-600-Cry spectrometer. Chemical shifts are referenced to
1
13
1
13
residual solvent peaks (CDCl : H δ = 7.24, C δ = 77.0; CD OD: H δ = 3.30, C δ =
3
3
4
9.0). Splitting patterns are reported as s (singlet), d (doublet), t (triplet), q (quartet), dd
double of doublets), m (multiplet). Coupling constants (J) are given in Hertz. The purities
of all of the biologically tested compounds were confirmed to be higher than 95% using
(
1
0

an analytical HPLC with a dual pump Shimadzu LC-20AT system. Mass spectra were
recorded on a Bruker bioTOF III. Melting points were measured on a FARGO melting
point apparatus MP-1D (Mandarin Scientific Co., Ltd.).
5
.2. General procedure for the synthesis of 3-Benzylaminocoumarin derivatives 6a-q
-Amino-7-hydroxy-2H-chromen-2-one (5, 500 mg, 2.82 mmol) was dissolved in
3
EtOH (20 mL) with 5% (v/v) acetic acid. Benzaldehyde was added (4.23 mmol), and the
mixture was heated at 80 °C for 10 minutes, with stirring. Sodium cyanoborohydride (354
mg, 5.64 mmol) was added to the mixture and stirring continued for 12 h. EtOH was
removed in vacuo and followed by extraction with dichloromethane (DCM)/H O. The
2
organic layers were combined and concentrated in vacuo. For compounds 6a-b, 6f-l, 6m-
p, the residues were re-crystallized from EtOAc; for compounds 6c-e, they were purified
by flash column chromatography (silica gel; EtOAc/Hexane = 3:7).
5
.2.1. 7-Hydroxy-3-benzylaminocoumarin (6a)
1
White solid; 75% yield; mp: 170-175 °C; H-NMR (200 MHz, DMSO-d ) δ 9.90 (s, 1H,
6
OH), 7.35-7.28 (m, 4H), 7.26-7.14 (m, 2H), 6.64 (dd, J = 4.2, 2.0 Hz, 2H), 6.37 (br, 2H),
1
3
4
1
.32 (d, J = 6.4 Hz, 2H, CH ) ; C-NMR (50 MHz, DMSO-d ) δ 158.9, 156.1, 148.6,
2 6
38.9, 130.0, 128.4, 127.0, 126.8, 126.0, 113.4, 113.1, 106.3, 101.9, 45.8; HRMS (ESI)
-
calcd. for C H NO [M-H] : 266.0812, found: 266.0831.
1
6
12
3
5
.2.2. 7-Hydroxy-3-(4-methylbenzyl)aminocoumarin (6b)
1
White solid; 51% yield; mp: 180-182 °C; H-NMR (200 MHz, DMSO-d ) δ 9.81 (s, 1H,
6
OH), 7.24 (d, J = 7.8 Hz, 2H), 7.18-7.10 (m, 3H), 6.66-6.63 (m, 2H), 6.35 (s, 1H), 6.29
1
3
(
t, J = 6.4 Hz, 1H, NH), 4.27 (d, J = 6.4 Hz, 2H, CH ), 2.25 (s, 3H, CH ); C-NMR (50
2 3
MHz, DMSO-d ) δ 158.9, 156.1, 148.6, 135.9, 135.8, 130.0, 129.0, 127.1, 126.0, 113.5,
6
-
1
13.1, 106.4, 101.9, 45.7, 20.7; HRMS (ESI) calcd. for C H NO [M-H] : 280.0968,
1
7
14
3
found: 280.0988.
1
1

5
.2.3. 7-Hydroxy-3-(4-chlorobenzyl)aminocoumarin (6c)
1
Lacteous solid; 37% yield; mp: 178-182 °C; H NMR (200 MHz, DMSO-d ) δ 10.01 (s,
6
1
1
1
4
5
H, OH), 7.38 (s, 4H), 7.16 (d, J = 9.0 Hz, 1H), 6.67-6.62 (m, 2H), 6.43 (t, J = 6.4 Hz,
1
3
H, NH), 6.35 (s, 1H), 4.31 (d, J = 6.4 Hz, 2H, CH ); C NMR (50 MHz, DMSO-d ) δ
2
6
58.8, 156.2, 148.6, 138.0, 131.3, 129.9, 128.9, 128.3, 126.0, 113.35, 113.1, 106.5, 101.9,
-
5.1; HRMS (ESI) calcd. for C H ClNO [M-H] : 300.0422, found: 300.0445.
1
6
11
3
.2.4. 7-Hydroxy-3-(4-nitrobenzyl)aminocoumarin (6d)
1
Orange solid; 46% yield; mp: 174-177 °C; H NMR (200 MHz, DMSO) δ 8.23-8.17 (m,
2
4
1
H), 7.61 (d, J = 8.6 Hz, 2H), 7.14 (d, J = 9.0 Hz, 1H), 6.67-6.56 (m, 3H), 6.34 (s, 1H),
1
3
.47 (d, J = 6.2 Hz, 2H, CH ); C NMR (50 MHz, DMSO-d ) δ 158.7, 156.2, 148.7,
2
6
47.4, 146.5, 129.7, 128.0, 126.0, 123.6, 113.2, 113.1, 106.6, 101.8, 45.3; HRMS (ESI)
-
calcd. for C H N O [M-H] : 311.0662, found: 311.0682.
1
6
11
2
5
5
.2.5. 7-Hydroxy-3-(4-fluorobenzyl)aminocoumarin (6e)
1
Yellowish solid; 46% yield; mp: 175-178 °C; H NMR (200 MHz, DMSO-d ) δ 10.00 (s,
6
1
4
1
H, OH), 7.44-7.36 (m, 2H), 7.20-7.08 (m, 3H), 6.68-6.62 (m, 2H), 6.41-6.35 (m, 2H),
1
3
.30 (d, J = 6.2 Hz, 2H, CH ); C NMR (50 MHz, DMSO-d ) δ 158.9, 156.2, 148.6,
2
6
47.9, 135.1, 129.9, 129.1, 128.9, 126.0, 115.3, 114.9, 113.3, 113.1, 106.4, 101.9, 45.1;
-
HRMS (ESI) calcd. for C H FNO [M-H] : 284.0717, found: 284.0743.
1
6
11
3
5
.2.6. 7-Hydroxy-3-(4-methoxybenzyl)aminocoumarin (6f)
1
White solid; 72% yield; mp: 180-182 °C; H NMR (200 MHz, DMSO-d ) δ 9.83 (s, 1H,
6
OH), 7.29 (d, J = 8.4 Hz, 2H), 7.17 (d, J = 9.2 Hz, 1H), 6.88 (d, J = 8.4 Hz, 2H), 6.66 (dd,
J = 6.6, 2.2 Hz, 2H), 6.38 (s, 1H), 6.25 (t, J = 6.2 Hz, 1H, NH), 4.24 (d, J = 6.2 Hz, 2H,
1
3
CH ), 3.71 (s, 3H, OCH ); C NMR (50 MHz, DMSO-d ) δ 158.9, 158.2, 156.0, 148.6,
2
3
6
1
30.6, 130.0, 128.4, 125.9, 113.8, 113.5, 113.1, 106.3, 101.9, 55.0, 45.3; HRMS (ESI)
-
calcd. for C H NO [M-H] : 296.0917, found: 296.0934.
1
7
13
4
1
2

5
.2.7. 7-Hydroxy-3-(3-methoxybenzyl)aminocoumarin (6g)
1
Lacteous solid; 66% yield; mp: 175-177 °C; H NMR (200 MHz, DMSO-d ) δ 7.27-
6
7
2
.15 (m, 2H), 6.94-6.92 (m, 2H), 6.81-6.76 (m, 1H), 6.68-6.62 (m, 2H), 6.37-6.30 (m,
1
3
H), 4.29 (d, J = 6.4 Hz, 2H, CH ), 3.72 (s, 3H, OCH ); C NMR (50 MHz, DMSO-d )
2
3
6
δ 159.4, 158.9, 156.1, 148.6, 140.6, 130.0, 129.5, 126.0, 119.2, 113.4, 113.1, 112.7, 112.1,
-
1
2
5
06.4, 101.9, 55.0, 45.8; HRMS (ESI) calcd. for C H NO [M-H] : 296.0917, found:
1
7
14
4
96.0933.
.2.8. 7-Hydroxy-3-(2-methoxybenzyl)aminocoumarin (6h)
1
White solid; 64% yield; mp: 176-177 °C; H NMR (200 MHz, DMSO-d ) δ 9.87 (s, 1H,
6
OH), 7.22-7.17 (m, 3H), 7.01 (d, J = 7.8 Hz, 1H), 6.91-6.84 (m, 1H), 6.69-6.64 (m, 2H),
6
.36 (s, 1H), 6.05 (t, J = 6.2 Hz, 1H, NH), 4.28 (d, J = 6.2 Hz, 2H, CH ), 3.85 (s, 3H,
2
13
OCH ); C NMR (50 MHz, DMSO-d ) δ 158.9, 156.8, 156.1, 148.6, 130.2, 128.1, 127.4,
3
6
1
26.0, 125.9, 120.3, 113.4, 113.1, 110.6, 106.2, 101.9, 55.3, 41.1; HRMS (ESI) calcd. for
-
C H NO [M-H] : 296.0917, found: 296.0938.
1
7
14
4
5
.2.9. 7-Hydroxy-3-(3,4-dimethoxybenzyl)aminocoumarin (6i)
1
Orange solid; 50% yield; mp: 181-183 °C; H NMR (400 MHz, DMSO-d ) δ 9.85 (s, 1H,
6
OH), 7.18 (d, J = 8.8 Hz, 1H), 7.00 (s, 1H), 6.88 (s, 2H), 6.68-6.65 (m, 2H), 6.41 (s, 1H),
6
3
1
.23 (t, J = 6.4 Hz, 1H, NH), 4.23 (d, J = 6.4 Hz, 2H, CH ), 3.73 (s, 3H, OCH ), 3.70 (s,
2 3
1
3
H, OCH ); C NMR (100 MHz, DMSO-d ) δ 158.9, 156.1, 148.8, 148.6, 147.7, 131.1,
3
6
30.0, 126.0, 119.2, 113.5, 113.1, 111.7, 111.2, 106.5, 101.9, 55.5, 55.4, 45.8; HRMS
-
(
ESI) calcd. for C H NO [M-H] : 326.1023, found: 326.1044.
1
8
16
5
5
.2.10. 7-Hydroxy-3-(2,3-dimethoxybenzyl)aminocoumarin (6j)
1
Lacteous solid; 63% yield; mp: 190-192 °C; H NMR (400 MHz, CD OD) δ 7.08 (d, J =
3
8
.4 Hz, 1H), 6.97 (dd, J = 8.4, 6.8 Hz, 1H), 6.88 (d, J = 8.0 Hz, 2H), 6.65 (dd, J = 8.4, 2.4
Hz, 1H), 6.62 (d, J = 2.4 Hz, 1H), 6.37 (s, 1H), 4.32 (s, 2H, CH ), 3.84 (s, 3H, OCH ),
2
3
1
3

1
3
3
1
.82 (s, 3H, OCH ); C NMR (100 MHz, CD OD) δ 161.5, 157.7, 154.0, 150.4, 148.2,
3 3
32.9, 131.5, 127.2, 125.2, 121.4, 115.3, 114.2, 112.9, 108.5, 103.0, 61.2, 56.2, 42.8;
-
HRMS (ESI) calcd. for C H NO [M-H] : 326.1023, found: 326.1042.
1
8
16
5
5
.2.11. 7-Hydroxy-3-(3,5-dimethoxybenzyl)aminocoumarin (6k)
1
Yellowish solid; 68% yield; mp: 178-180 °C; H NMR (400 MHz, DMSO-d ) δ 9.86 (s,
6
1
2
H, OH), 7.18 (d, J = 9.2 Hz, 1H), 6.67 (dd, J = 6.0, 2.0 Hz, 2H), 6.54 (d, J = 2.0 Hz,
H), 6.38-6.30 (m, 3H), 4.25 (d, J = 6.4 Hz, 2H, CH ), 3.72 (s, 3H, OCH ), 3.70 (s, 3H,
2
3
1
3
OCH ); C NMR (100 MHz, DMSO-d ) δ 160.6, 160.4, 158.9, 156.2, 148.6, 141.5, 130.0,
3
6
1
26.0, 113.4, 113.1, 106.5, 105.0, 104.0, 101.9, 98.4, 55.1, 46.0; HRMS (ESI) calcd. for
-
C H NO [M-H] : 326.1023, found: 326.1054.
1
8
16
5
5
.2.12. 7-Hydroxy-3-(2,5-dimethoxybenzyl)aminocoumarin (6l)
1
Orange solid; 61% yield; mp: 180-183 °C; H NMR (400 MHz, DMSO-d ) δ 9.87 (s, 1H,
6
OH), 7.19 (d, J = 9.2 Hz, 1H), 6.91 (d, J = 8.8 Hz, 1H), 6.80 (d, J = 3.2 Hz, 1H), 6.76 (dd,
J = 8.8 Hz, 3.2, 1H), 6.69-6.66 (m, 2H), 6.38 (s, 1H), 6.08 (t, J = 6.4 Hz, 1H, NH), 4.25
1
3
(
d, J = 6.4 Hz, 2H, CH ), 3.80 (s, 3H, OCH ), 3.63 (s, 3H, OCH ); C NMR (100 MHz,
2 3 3
DMSO-d ) δ 158.9, 156.2, 153.2, 150.9, 148.7, 130.1, 127.3, 126.1, 114.3, 113.5, 113.1,
6
-
1
3
5
11.6, 111.5, 106.3, 101.9, 55.8, 55.3, 41.1; HRMS (ESI) calcd. for C H NO [M-H] :
1
8
16
5
26.1023, found: 326.1046.
.2.13. 7-Hydroxy-3-(2,4-dimethoxybenzyl)aminocoumarin (6m)
1
Orange solid; 65% yield; mp: 181-185 °C; H NMR (400 MHz, DMSO-d ) δ 9.85 (s, 1H,
6
OH), 7.19 (d, J = 9.2 Hz, 1H), 7.12 (d, J = 8.4 Hz, 1H), 6.68 (dd, J = 8.4, 2.4 Hz, 2H),
6
1
.56 (d, J = 2.4 Hz, 1H), 6.45 (dd, J = 8.4, 2.4 Hz, 1H), 6.38 (s, 1H), 5.89 (t, J = 6.0 Hz,
1
3
H, NH), 4.19 (d, J = 6.0 Hz, 2H, CH ), 3.83 (s, 3H, OCH ), 3.71 (s, 3H, OCH ); C
2
3
3
NMR (100 MHz, DMSO-d ) δ 159.8, 159.0, 157.9, 156.1, 148.6, 130.2, 128.6, 126.0,
6
1
18.0, 113.5, 113.1, 106.3, 104.4, 101.9, 98.4, 55.4, 55.1, 40.9; HRMS (ESI) calcd. for
1
4

-
C H NO [M-H] : 326.1023, found: 326.1048.
1
8
16
5
5
.2.14. 7-Hydroxy-3-(2,6-dimethoxybenzyl)aminocoumarin (6n)
1
Lacteous solid; 75% yield; mp: 188-190 °C; H NMR (400 MHz, DMSO-d ) δ 9.89 (s,
6
1
8
=
H, OH), 7.28 (d, J = 8.4 Hz, 1H), 7.23 (d, J = 8.4 Hz, 1H), 6.77 (s, 1H), 6.72 (dd, J =
.4, 2.4 Hz, 1H), 6.69 (s, 1H), 6.67-6.66 (m, 2H), 5.01 (t, J = 6.0 Hz, 1H, NH), 4.24 (d, J
1
3
6.0 Hz, 2H, CH ), 3.83 (s, 6H, OCH ); C NMR (100 MHz, DMSO-d ) δ 159.0, 158.2,
2
3
6
1
56.3, 148.7, 130.0, 129.3, 126.2, 113.4, 113.3, 113.2, 107.1, 104.2, 101.9, 55.8, 35.51;
-
HRMS (ESI) calcd. for C H NO [M-H] : 326.1023, found: 326.1044.
1
8
16
5
5
.2.15. 7-Hydroxy-3-(2,3,4-trimethoxybenzyl)aminocoumarin (6o)
1
Greenish solid; 61% yield; mp: 193-195 °C; H NMR (400 MHz, DMSO-d ) δ 9.85 (s,
6
1
6
H, OH), 7.20 (d, J = 9.2 Hz, 1H), 6.95 (d, J = 8.4 Hz, 1H), 6.72 (d, J = 8.4 Hz, 1H),
.69-6.67 (m, 2H), 6.42 (s, 1H), 5.94 (t, J = 6.0 Hz, 1H, NH), 4.24 (d, J = 6.0 Hz, 2H,
1
3
CH ), 3.85 (s, 3H, OCH ), 3.75 (s, 3H, OCH ), 3.74 (s, 3H, OCH ); C NMR (100 MHz,
2
3
3
3
DMSO-d ) δ 158.9, 156.1, 152.6, 151.2, 148.6, 141.7, 130.0, 126.0, 123.7, 122.4, 113.5,
6
1
13.1, 107.6, 106.3, 101.9, 60.9, 60.4, 55.8, 41.0; HRMS (ESI) calcd. for C H NO [M-
19 18 6
-
H] : 356.1129, found: 356.1162.
5
.2.16. 7-Hydroxy-3-(2,4,5-trimethoxybenzyl)aminocoumarin (6p)
1
Yellowish solid; 55% yield; mp: 183-185 °C; H NMR (400 MHz, DMSO-d ) δ 9.84 (s,
6
1
H, OH), 7.21 (d, J = 8.4 Hz, 1H), 6.93 (s, 1H), 6.70-6.65 (m, 3H), 6.46 (s, 1H), 5.90 (t,
J = 6.0 Hz, 1H, NH), 4.20 (d, J = 6.0 Hz, 2H, CH ), 3.84 (s, 3H, OCH ), 3.77 (s, 3H,
2
3
1
3
OCH ), 3.63 (s, 3H, OCH ); C NMR (100 MHz, DMSO-d ) δ 158.9, 156.1, 151.2, 148.7,
3
3
6
1
4
5
48.6, 142.6, 130.1, 126.0, 117.1, 113.6, 113.5, 113.1, 106.4, 101.9, 98.1, 56.4, 56.2, 55.8,
-
0.7; HRMS (ESI) calcd. for C H NO [M-H] : 356.1129, found: 356.1149.
1
9
18
6
.2.17. 7-Hydroxy-3-(3,4,5-trimethoxybenzyl)aminocoumarin (6q)
1
Lacteous solid; 66% yield; mp: 190-192 °C; H NMR (400 MHz, DMSO-d ) δ 9.84 (s,
6
1
5

1
H, OH), 7.21 (d, J = 8.0 Hz, 1H), 6.72 (s, 2H), 6.68-6.66 (m, 2H), 6.46 (s, 1H), 6.26 (t,
J = 6.0 Hz, 1H, NH), 4.22 (d, J = 6.0 Hz, 2H, CH ), 3.75 (s, 6H, OCH ), 3.62 (s, 3H,
2
3
1
3
OCH ); C NMR (100 MHz, DMSO-d ) δ 158.9, 156.2, 152.9, 148.7, 136.2, 134.7, 130.1,
3
6
1
26.0, 113.5, 113.1, 106.6, 104.4, 101.9, 60.0, 55.8, 46.4; HRMS (ESI) calcd. for
-
C H NO [M-H] : 356.1129, found: 356.1156.
1
9
18
6
5
.3. General procedure for the synthesis of 3-Benzylaminocoumarin-7-O-sulfamate
derivatives (3a-q)
Formic acid (45 L, 1.2 mmol) was slowly added to a stirred solution of
chlorosulfonyl isocyanate (104 L, 1.2 mmol) at 0 °C. Stirring was continued until
sulfamoyl chloride (white solid) was formed, whereupon anhydrous THF (5 mL) was
added to dissolve the solid. Meanwhile, TEA (84 L, 0.6 mmol) was added to 7-hydroxy-
3
-benzyl aminocoumarin derivatives 6a-q (0.6 mmol) in anhydrous THF (10 mL). The
mixture was stirred at room temperature for 1 h, and then cooled to 0 °C. The freshly-
prepared sulfamoyl chloride was added drop-wise at 0 °C, and the mixture stirred for 2 h
at 0 °C. After the temperature was raised to room temperature, the reaction mixture was
stirred for another 12 h. A solution of sodium bicarbonate (NaHCO ) was added into the
3
stirred mixture to adjust the pH to 5-7. The mixture was evaporated and the residue was
extracted with DCM (10 mL × 2). The organic layers were collected and evaporated. The
crude mixture was purified by flash column chromatography (silica gel; EtOAc/hexane =
1
:1) to afford 3-benzylaminocoumarin-7-O-sulfamate derivatives 3a-q.
.3.1. 3-Benzylaminocoumarin-7-O-sulfamate (3a)
5
The purity of product 3a was 95.3% as shown by HPLC on a RP18 column, t = 8.6 min
R
1
(
ACN/H O = 1:1). Lacteous solid; 34% yield; H NMR (200 MHz, DMSO-d ) δ 7.58 (s,
2
6
1
6
H), 7.42 (d, J = 8.4 Hz, 1H), 7.28 (s, 1H), 7.26-7.23 (m, 3H), 6.77 (d, J = 2.2 Hz, 1H),
1
3
.71 (dd, J = 3.2, 2.2 Hz, 1H), 6.14 (s, 2H), 4.62 (s, 2H, CH ); C NMR (50 MHz, DMSO-
2
1
6

d6) δ 161.2, 157.7, 154.7, 142.6, 138.8, 129.8, 128.2, 128.0, 126.9, 123.9, 113.1, 111.6,
-
1
01.9, 49.9; HRMS (ESI) calcd. for C H N O S-H [M-H] : 345.0540, found: 345.0569.
1
6
14
2
5
5
.3.2. 3-(4-Methylbenzyl)aminocoumarin-7-O-sulfamate (3b)
The purity of product 3b was 98.9% as shown by HPLC on a RP18 column, t = 11.9 min
R
1
(
ACN/H O = 1:1). White solid; 35% yield; H NMR (200 MHz, DMSO-d ) δ 7.52 (s,
2
6
1
H), 7.36 (d, J = 8.6 Hz, 1H), 7.16-7.05 (m, 4H), 6.71 (dd, J = 8.4, 2.4 Hz, 1H), 6.66 (d,
1
3
J = 2.4 Hz, 1H), 6.09 (s, 2H, NH ), 4.57 (s, 2H, CH ), 2.24 (s, 3H, CH ); C NMR (50
2
2
3
MHz, DMSO-d ) δ 161.8, 157.7, 154.7, 142.5, 135.8, 135.7, 129.7, 128.8, 128.0, 123.5,
6
-
1
13.3, 111.2, 101.8, 49.6, 20.7; HRMS (ESI) calcd. for C H N O S-H [M-H] : 359.0696,
1
7
16
2
5
found: 359.0714.
5
.3.3. 3-(4-Chlorobenzyl)aminocoumarin-7-O-sulfamate (3c)
The purity of product 3c was 98.3% as shown by HPLC on a RP18 column, t = 8.2 min
R
1
(
ACN/H O = 1:1). Yellow solid; 17% yield; H NMR (400 MHz, DMSO-d ) δ 7.62 (s,
2
6
1
H), 7.44 (d, J = 8.8 Hz, 1H), 7.35-7.29 (m, 4H), 6.75 (dd, J = 8.4, 2.0 Hz, 1H), 6.70 (d,
1
3
J = 2.0 Hz, 1H), 6.16 (s, 2H, NH ), 4.60 (s, 2H, CH ); C NMR (100 MHz, DMSO-d ) δ
2
2
6
1
4
5
61.2, 159.0, 157.7, 154.7, 142.5, 137.9, 131.4, 129.9, 128.2, 123.9, 113.1, 111.6, 101.8,
-
9.4; HRMS (ESI) calcd. for C H ClN O S-H [M-H] : 379.0150, found: 379.0185.
1
6
13
2
5
.3.4. 3-(4-Nitrobenzyl)aminocoumarin-7-O-sulfamate (3d)
The purity of product 3d was 96.4% as shown by HPLC on a RP18 column, t = 8.3 min
R
1
(
ACN/H O = 1:1). Lacteous solid; 28% yield; H NMR (200 MHz, DMSO-d ) δ 8.16 (d,
2
6
J = 8.4 Hz, 2H), 7.74 (s, 1H), 7.59 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 8.4 Hz, 1H), 6.74 (dd,
1
3
J = 8.6, 2.0 Hz, 1H), 6.70 (d, J = 2.0 Hz, 1H), 6.24 (s, 2H, NH ), 4.73 (s, 2H, CH ); C
2
2
NMR (50 MHz, DMSO-d ) δ 161.5, 157.7, 154.8, 147.3, 146.5, 142.6, 129.9, 128.9,
6
-
1
3
23.9, 123.4, 113.2, 111.5, 101.9, 50.0; HRMS (ESI) calcd. for C H N O S-H [M-H] :
1
6
13
3
7
90.0390, found: 390.0384.
1
7

5
.3.5. 3-(4-Fluorobenzyl)aminocoumarin-7-O-sulfamate (3e)
The purity of product 3e was 98.8% as shown by HPLC on a RP18 column, t = 3.5 min
R
1
(
ACN/H O = 1:1). Yellow solid; 36% yield; H NMR (200 MHz, DMSO-d ) δ 7.55 (s,
2
6
1
8
H), 7.41 (d, J = 8.4 Hz, 1H), 7.30 (dd, J = 8.2, 6.0 Hz, 2H), 7.09 (m, 2H), 6.72 (dd, J =
1
3
.4, 2.0 Hz, 1H), 6.67 (d, J = 2.0 Hz, 1H), 6.12 (s, 2H, NH ), 4.58 (s, 2H, CH ); C NMR
2
2
(
50 MHz, DMSO-d ) δ 161.2, 159.0, 157.6, 154.7, 142.6, 135.0, 130.1, 130.0, 123.8,
6
1
22.3, 115.1, 114.7, 113.1, 111.6, 101.8, 49.3; HRMS (ESI) calcd. for C H FN O S-H
16 13 2 5
-
[
M-H] : 363.0445, found: 363.0499.
5
.3.6. 3-(4-Methoxybenzyl)aminocoumarin-7-O-sulfamate (3f)
The purity of product 3f was 99.4% as shown by HPLC on a RP18 column, t = 8.2 min
R
1
(
ACN/H O = 1:1). Yellow solid; 22% yield; H NMR (200 MHz, DMSO-d ) δ 7.45 (s,
2
6
1
H), 7.39 (d, J = 8.6 Hz, 1H), 7.16 (d, J = 8.4 Hz, 2H), 6.82 (d, J = 8.4 Hz, 2H), 6.67 (dd,
J = 8.4, 2.2 Hz, 1H), 6.61 (d, J = 2.2 Hz, 1H), 6.05 (s, 2H, NH ), 4.54 (s, 2H, CH ), 3.70
2
2
1
3
(
s, 3H, OCH ); C NMR (50 MHz, DMSO-d ) δ 162.7, 159.1, 158.2, 157.7, 155.0, 142.7,
3
6
1
30.7, 129.7, 129.4, 122.9, 113.6, 110.8, 101.9, 55.0, 49.2; HRMS (ESI) calcd. for
-
C H N O S-H [M-H] : 375.0645, found: 375.0675.
1
7
16
2
6
5
.3.7. 3-(3-Methoxybenzyl)aminocoumarin-7-O-sulfamate (3g)
The purity of product 3g was 99.2% as shown by HPLC on a RP18 column, t = 8.2 min
R
1
(
ACN/H O = 1:1). Lacteous oil; 25% yield; H NMR (200 MHz, DMSO-d ) δ 7.60 (s,
2
6
1
H), 7.43 (d, J = 8.4 Hz, 1H), 7.18 (t, J = 8.0 Hz, 1H), 6.84 (d, J = 1.2 Hz, 1H), 6.80 (d,
J = 3.2 Hz, 1H), 6.75 (s, 1H), 6.71-6.68 (m, 2H), 6.14 (s, 2H, NH ), 4.60 (s, 2H, CH ),
2
2
1
3
3
1
.70 (s, 3H, OCH ); C NMR (50 MHz, DMSO-d ) δ 161.4, 159.2, 157.7, 154.7, 142.4,
3 6
40.5, 129.9, 129.3, 123.9, 120.1, 113.4, 113.2, 112.4, 111.5, 101.9, 54.9, 50.0; HRMS
-
(
ESI) calcd. for C H N O S-H [M-H] : 375.0645, found: 375.0682.
1
7
16
2
6
5
.3.8. 3-(2-Methoxybenzyl)aminocoumarin-7-O-sulfamate (3h)
1
8

The purity of product 3h was 98.2% as shown by HPLC on a RP18 column, t = 9.5 min
R
1
(
ACN/H O = 1:1). Lacteous oil; 24% yield; H NMR (200 MHz, DMSO-d ) δ 7.53 (s,
2
6
1
8
3
1
H), 7.37-7.30 (m, 2H), 7.19 (td, J = 7.8, 1.4 Hz, 1H), 6.93-6.84 (m, 2H), 6.68 (dd, J =
.4, 2.0 Hz, 1H), 6.63 (d, J = 2.0 Hz, 1H), 6.06 (s, 2H, NH ), 4.57 (s, 2H, CH ), 3.59 (s,
2
2
1
3
H, OCH ); C NMR (50 MHz, DMSO-d ) δ 161.7, 159.2, 156.9, 154.7, 142.0, 129.7,
3
6
29.0, 128.2, 126.2, 123.9, 120.2, 113.3, 111.3, 110.4, 101.8, 55.1, 45.1; HRMS (ESI)
-
calcd. for C H N O S-H [M-H] : 375.0645, found: 375.0682.
1
7
16
2
6
5
.3.9. 3-(3,4-Dimethoxybenzyl)aminocoumarin-7-O-sulfamate (3i)
The purity of product 3i was 98.1% as shown by HPLC on a RP18 column, t = 6.2 min
R
1
(
ACN/H O = 1:1). Orange ampholous solid; 20% yield; H NMR (400 MHz, CD OD) δ
2
3
7
.57 (s, 1H), 7.31 (d, J = 8.8 Hz, 1H), 7.00 (s, 1H), 6.80 (d, J = 1.2 Hz, 2H), 6.73 (dd, J
8.8, 2.0 Hz, 1H), 6.66 (d, J = 2.0 Hz, 1H), 4.67 (s, 2H, CH ), 3.78 (s, 3H, OCH ), 3.74
=
2
3
1
3
(
s, 3H, OCH ); C NMR (100 MHz, CD OD) δ 163.6, 161.9, 156.3, 150.5, 150.2, 146.4,
3 3
1
31.0, 130.4, 123.3, 122.7, 114.9, 113.6, 112.6, 112.5, 103.1, 56.4, 56.3, 53.7; HRMS
-
(
ESI) calcd. for C H N O S-H [M-H] : 405.0751, found: 405.0790.
1
8
18
2
7
5
.3.10. 3-(2,3-Dimethoxybenzyl)aminocoumarin-7-O-sulfamate (3j)
The purity of product 3j was 98.7% as shown by HPLC on a RP18 column, t = 8.3 min
R
1
(
ACN/H O = 1:1). Orange solid; 25% yield; H NMR (400 MHz, CD OD) δ 7.49 (s, 1H),
2
3
7
1
3
1
5
5
.21 (d, J = 8.8 Hz, 1H), 6.99 (s, 1H), 6.97 (d, J = 4.4 Hz, 1H), 6.85 (dd, J = 6.4, 3.2 Hz,
H), 6.69 (dd, J = 8.8, 2.4 Hz, 1H), 6.64 (d, J = 2.4 Hz, 1H), 4.78 (s, 2H, CH ), 3.71 (s,
2
1
3
H, OCH ), 3.70 (s, 3H, OCH ); C NMR (100 MHz, CD OD) δ 163.3, 161.9, 156.2,
3
3
3
53.9, 148.8, 146.3, 131.0, 130.1, 125.2, 123.5, 123.4, 114.8, 113.6, 112.6, 103.1, 61.2,
-
6.2, 48.0; HRMS (ESI) calcd. for C H N O S-H [M-H] : 405.0751, found: 405.0781.
1
8
18
2
7
.3.11. 3-(3,5-Dimethoxybenzyl)aminocoumarin-7-O-sulfamate (3k)
The purity of product 3k was 99.7% as shown by HPLC on a RP18 column, t = 8.1 min
R
1
9

1
(
ACN/H O = 1:1). Yellowish oil; 23% yield; H NMR (200 MHz, CD OD) δ 7.65 (s,
2
3
1
6
H), 7.35 (d, J = 8.6 Hz, 1H), 6.76 (dd, J = 8.6, 2.2 Hz, 1H), 6.68 (d, J = 2.2 Hz, 1H),
.52 (d, J = 2.2 Hz, 2H), 6.33 (t, J = 2.2 Hz, 1H), 4.67 (s, 2H, CH ), 3.71 (s, 6H, OCH );
2
3
1
3
C NMR (50 MHz, CD OD) δ 163.6, 162.4, 161.8, 156.4, 146.3, 140.2, 131.0, 123.3,
3
1
14.9, 112.6, 107.6, 105.9, 103.2, 100.7, 55.7, 53.9; HRMS (ESI) calcd. for
-
C H N O S-H [M-H] : 405.0751, found: 405.0786.
1
8
18
2
7
5
.3.12. 3-(2,5-Dimethoxybenzyl)aminocoumarin-7-O-sulfamate (3l)
The purity of product 3l was 98.6% as shown by HPLC on a RP18 column, t = 8.8 min
R
1
(
ACN/H O = 1:1). Yellowish oil; 22% yield; H NMR (200 MHz, DMSO-d ) δ 7.59 (s,
2
6
1
H), 7.46 (d, J = 8.2 Hz, 1H), 6.93 (d, J = 2.6 Hz, 1H), 6.82-6.71 (m, 5H), 6.11 (s, 2H,
1
3
NH ), 4.57 (s, 2H, CH ), 3.68 (s, 3H, OCH ), 3.53 (s, 3H, OCH ); C NMR (50 MHz,
2
2
3
3
DMSO-d ) δ 161.0, 157.6, 154.6, 153.0, 150.9, 141.6, 129.8, 127.4, 124.4, 115.2, 113.0,
6
1
12.2, 111.6, 111.4, 101.8, 55.6, 55.3, 45.3; HRMS (ESI) calcd. for C H N O S-H [M-
18 18 2 7
-
H] : 405.0751, found: 405.0786.
5
.3.13. 3-(2,4-Dimethoxybenzyl)aminocoumarin-7-O-sulfamate (3m)
The purity of product 3m was 96.3% as shown by HPLC on a RP18 column, t = 8.2 min
R
1
(
ACN/H O = 1:1). Orange solid; 25% yield; H NMR (400 MHz, CD OD) δ 7.38 (s, 1H),
2
3
7
6
.26 (dd, J = 9.6, 8.8 Hz, 2H), 6.74 (dd, J = 8.4, 2.4 Hz, 1H), 6.69 (d, J = 2.4 Hz, 1H),
.47 (dd, J = 8.4, 2.4 Hz, 1H), 6.34 (d, J = 2.4 Hz, 1H), 4.63 (s, 2H, CH ), 3.73 (s, 3H,
2
1
3
OCH ), 3.54 (s, 3H, OCH ); C NMR (100 MHz, CD OD) δ 163.5, 162.6, 162.4, 160.2,
3
3
3
1
56.3, 145.5, 133.1, 130.9, 123.8, 117.6, 114.9, 112.6, 105.8, 103.1, 99.0, 55.7, 55.7, 48.2;
-
HRMS (ESI) calcd. for C H N O S-H [M-H] : 405.0751, found: 405.0781.
1
8
18
2
7
5
.3.14. 3-(2,6-Dimethoxybenzyl)aminocoumarin-7-O-sulfamate (3n)
The purity of product 3n was 99.5% as shown by HPLC on a RP18 column, t = 6.9 min
R
1
(
ACN/H O = 1:1). Lacteous oil; 13% yield; H NMR (200 MHz, DMSO-d ) δ 7.23 (d, J
2
6
2
0

=
4.6 Hz, 1H), 7.15 (d, J = 4.2 Hz, 1H), 6.93 (s, 1H), 6.67 (s, 2H), 6.50 (d, J = 4.2 Hz,
1
3
2
1
3
5
H), 5.91 (s, 2H, CH ), 3.53 (s, 6H, OCH ); C NMR (50 MHz, DMSO-d ) δ 161.0,
2 3 6
59.1, 158.4, 157.6, 154.6, 141.5, 129.4, 123.3, 112.9, 111.2, 103.8, 101.7, 101.5, 55.5,
-
7.7; HRMS (ESI) calcd. for C H N O S-H [M-H] : 405.0751, found: 405.0781.
1
8
18
2
7
.3.15. 3-(2,3,4-Trimethoxybenzyl)aminocoumarin-7-O-sulfamate (3o)
The purity of product 3o was 98.7% as shown by HPLC on a RP18 column, t = 7.9 min
R
1
(
ACN/H O = 1:1). Orange oil; 29% yield; H NMR (400 MHz, CD OD) δ 7.40 (s, 1H),
2
3
7
1
.26 (d, J = 8.4 Hz, 1H), 7.06 (d, J = 8.4 Hz, 1H), 6.73-6.69 (m, 2H), 6.68 (d, J = 2.4 Hz,
1
3
H), 4.66 (s, 2H, CH ), 3.77 (s, 3H, OCH ), 3.74 (s, 3H, OCH ), 3.52 (s, 3H, OCH ); C
2
3
3
3
NMR (100 MHz, CD OD) δ 163.4, 162.2, 156.3, 155.1, 153.6, 146.0, 143.1, 130.9, 126.9,
3
1
23.5, 123.1, 114.8, 112.7, 108.7, 103.1, 61.5, 60.9, 56.4, 48.2; HRMS (ESI) calcd. for
-
C H N O S-H [M-H] : 435.0857, found: 435.0868.
1
9
20
2
8
5
.3.16. 3-(2,4,5-Trimethoxybenzyl)aminocoumarin-7-O-sulfamate (3p)
The purity of product 3p was 95.8% as shown by HPLC on a RP18 column, t = 6.7 min
R
1
(
ACN/H O = 1:1). Orange oil; 19% yield; H NMR (400 MHz, CD OD) δ 7.43 (s, 1H),
2
3
7
1
.24 (d, J = 8.4 Hz, 1H), 6.98 (s, 1H), 6.70 (dd, J = 8.4, 2.4 Hz, 1H), 6.65 (d, J = 2.4 Hz,
H), 6.43 (s, 1H), 4.64 (s, 2H, CH ), 3.73 (d, J = 1.6 Hz, 6H, OCH ), 3.52 (s, 3H, OCH );
2
3
3
1
3
C NMR (100 MHz, CD OD) δ 163.3, 162.3, 156.2, 153.7, 151.2, 145.3, 144.1, 130.9,
3
1
23.7, 116.6, 116.5, 114.8, 112.6, 103.1, 98.3, 57.2, 56.5, 56.4, 48.0; HRMS (ESI) calcd.
-
for C H N O S-H [M-H] : 435.0857, found: 435.0877.
1
9
20
2
8
5
.3.17. 3-(3,4,5-Trimethoxybenzyl)aminocoumarin-7-O-sulfamate (3q)
The purity of product 3q was 98.8% as shown by HPLC on a RP18 column, t = 8.2 min
R
1
(
ACN/H O = 1:1). Orange oil; 36% yield; H NMR (400 MHz, CD OD) δ 7.27 (d, J =
2
3
8
1
.4 Hz, 1H), 7.22 (d, J = 2.4 Hz, 1H), 7.12 (dd, J = 8.4, 2.4 Hz, 1H), 6.71 (s, 2H), 6.41 (s,
1
3
H), 4.35 (s, 2H, CH ), 3.82 (s, 6H, OCH ), 3.74 (s, 3H, OCH ); C NMR (100 MHz,
2
3
3
2
1

CD OD) δ 160.9, 154.7, 151.9, 149.2, 138.0, 135.9, 133.4, 126.5, 120.0, 119.5, 109.9,
3
-
1
07.0, 105.3, 61.1, 56.6, 48.0; HRMS (ESI) calcd. for C H N O S-H [M-H] : 435.0857,
1
9
20
2
8
found: 435.0877.
5
.4. 4-(Benzylamino)phenyl sulfamate (4a)
After stirring formic acid (57 μL, 1.51 mmol) and chlorosulfonyl isocyanate (131 μL,
.51 mmol) for 10 min at rt under N , DMA (1 mL) was added dropwise under ice bath,
1
2
and then stirred at rt. Upon the cessation of effervescence, a solution of 13a (200 mg, 1.00
mmol) in anhydrous DCM (2 mL) was added dropwise. The mixture was extracted with
DCM/saturated NaHCO3 (aq). The organic layer was dried over MgSO4 (s) and concentrated
to dryness in vacuo. The residue was purified by column chromatography (silica gel;
Hexane/EtOAc, 100/0 to 60/40) to give 4a (41 mg, 15%) as a brown solid. R 0.43
f
(Hexane/EtOAc = 6:4); HPLC purity (RP18; phosphate buffer/ACN, 80/20 to 10/90, 25
1
min) 100.0%; t 16.11 min. H NMR (200 MHz, CD OD) δ 7.38-7.16 (m, 5H, benzyl),
R
3
1
3
7
.07-6.99 (m, 2H, phenyl), 6.64-6.56 (m, 2H, phenyl), 4.30 (s, 2H, CH Ph); C NMR (50
2
MHz, CD OD) δ 148.8, 142.6, 141.2, 129.4 (2C), 128.3 (2C), 127.9, 124.0 (2C), 114.0
3
+
(
2C), 48.6; HRMS (ESI) calcd. for C H N O S [M+H] : 279.0798, found : 279.0807.
1
3
14
2
3
5
.5. 4-(Phenethylamino)phenyl sulfamate (4b)
After stirring formic acid (42 μL, 1.11 mmol) and chlorosulfonyl isocyanate (96 μL, 1.10
mmol) for 10 min at rt under N atmosphere, DMA (1 mL) was added dropwise under ice
2
bath, and then stirred at rt. Upon the cessation of effervescence, a solution of 13b (157
mg, 0.74 mmol) in anhydrous DCM (2 mL) was added dropwise into the reaction. The
mixture was extracted with DCM/saturated NaHCO3 (aq), the organic layer was dried over
MgSO and concentrated to dryness in vacuo. The residue was purified by column
4
,
chromatography (silica gel; Hexane/EtOAc, 100/0 to 60/40) to give 71b (17 mg, 8%) as
a brown oil: R 0.13 (Hexane/EtOAc = 7:3); HPLC purity (RP18; phosphate buffer/ACN,
f
2
2

1
7
0/30 to 10/90, 25 min) 96.9%; t 14.29 min. H NMR (200 MHz, CD OD) δ 7.27-7.15
R 3
(m, 5H, benzyl), 7.08-7.03 (m, 2H, phenyl), 6.62-6.58 (m, 2H, phenyl), 3.29 (t, J = 7.4
1
3
Hz, 2H), 2.84 (t, J = 7.4 Hz, 2H); C NMR (50 MHz, CD OD) δ 148.8, 142.7, 141.0,
3
1
29.8 (2C), 129.5 (2C), 127.2, 124.1 (2C), 114.0 (2C), 46.6, 36.4 ppm; HRMS (ESI) calcd.
+
for C H N O S [M+H] : 293.0954, found : 293.0962.
1
4
16
2
3
5
.6.4-((3-Phenylpropyl)amino)phenyl sulfamate (4c)
4
c (68 mg, 33%) was synthesized by using formic acid (125 μL, 3.31 mmol), DMA (1
μL), chlorosulfonyl isocyanate (287 μL, 3.30 mmol) in anhydrous DCM (3 mL), then 13c
150 mg, 0.66 mmol) in anhydrous DMA (1 mL) was added. To a stirred solution of
(
saturated NaHCO3 (aq). The mixture was extracted with DCM/saturated NaHCO3 (aq), the
organic layer was dried over MgSO and concentrated to dryness in vacuo. The residue
4
,
was crystallized by DCM/Hexane to give 4c: R 0.18 (Hexane/EtOAc = 7:3); HPLC purity
f
(RP18; phosphate buffer/ACN, 90/10 to 50/50, 5 min, 50/50 to 5/95, 20 min) 100.0%; tR
1
1
3.25 min. H NMR (400 MHz, CD OD) δ 7.28-7.14 (m, 5H, benzyl), 7.05 (d, J = 8.8
3
Hz, 2H, phenyl), 6.58 (d, J = 8.8 Hz, 2H, phenyl), 3.08 (t, J = 6.9 Hz, 2H), 2.72 (t, J = 7.5
1
3
Hz, 2H), 1.94-1.89 (m, 2H); C NMR (100 MHz, CD OD) δ 149.2, 143.3, 142.6, 129.5
3
(2C), 129.4 (2C), 126.8, 124.1 (2C), 113.8 (2C), 44.4, 34.3, 32.1 ppm; HRMS (ESI) calcd.
+
for C H N O S [M+H] : 307.1111, found : 307.1115.
1
5
18
2
3
5
.7. 7-Hydroxy-3-(2,3-dimethoxyphenethyl)aminocoumarin (7)
(2,3-dimethoxyphenyl)acetic acid (1.82 g, 10.0 mmol) was treated with DIBAL (20% in
toluene, 8.9 mL, 10 mmol) under DCM at -60 °C. After 6 h, it was diluted with
deionized water and filtered. The organic layer was collected and purified by flash silica
gel column chromatography (EA/hexane = 1:9) to give (2,3-dimethoxyphenyl)-
acetaldehyde. Next, 3-amino-7-hydroxy-2H-chromen-2-one (5) (500 mg, 2.82 mmol)
was dissolved in EtOH (with 5% (v/v) acetic acid, 20 mL), and (2,3-dimethoxyphenyl)
2
3

acetaldehyde (762 mg, 4.23 mmol) was added. The mixture was heated in a water bath
to 80 °C and stirred for 10 min Sodium cyanoborohydride (354 mg, 5.64 mmol) was
added to the mixture and stirring was continued for 12 h. EtOH was removed by
concentration in vacuo, and the residue was extracted with DCM/H O. The organic
2
layers were collected and concentrated in vacuo. The residue was purified by flash
column chromatography (silica gel; EtOAc/Hexane = 3:7) to give compound 7 (732 mg,
1
7
8
6%) as transparent oil. H NMR (200 MHz, DMSO-d ) δ 9.86 (s, 1H, OH), 7.28 (d, J =
6
.2 Hz, 1H), 7.04-6.93 (m, 3H), 6.89-6.82 (m, 2H), 6.59 (s, 1H), 5.65 (t, J = 5.6 Hz, 1H,
1
3
NH), 3.79 (s, 6H, OCH ), 3.27-3.17 (m, 2H, CH ), 2.90-2.83 (m, 2H, CH ); C NMR
3
2
2
(
50 MHz, DMSO-d ) δ 158.9, 156.1, 152.4, 148.6, 146.9, 132.8, 130.2, 126.0, 124.0,
6
1
22.1, 113.7, 113.2, 111.3, 105.8, 101.9, 60.3, 55.6, 43.7, 28.4; HRMS (ESI) calcd. for
-
C H NO -H [M-H] : 340.1179, found: 340.1204.
1
9
19
5
5
.8. 3-(2,3-Dimethoxyphenethyl)aminocoumarin-7-O-sulfamate (8)
Formic acid (45 L, 1.2 mmol) was added to a stirred solution of chlorosulfonyl
isocyanate (104 L, 1.2 mmol) at 0 °C. Stirring was continued until sulfamoyl chloride
(white solid) was form, and then anhydrous THF (5 mL) was added to dissolve the solid.
TEA (84 L, 0.6 mmol) was added to a solution of compound 7 (189 mg, 0.6 mmol) in
anhydrous THF (10 mL). The mixture was stirred at room temperature for 1 h, and then
cooled to 0 °C. A solution of sulfamoyl chloride (prepared as above) was added dropwise,
at 0 °C, and the mixture stirred for 2 h. The reaction mixture was allowed to warm to
room temperature and stirred for another 12 h. A solution of saturated sodium bicarbonate
(
NaHCO ) was added into the stirred mixture to adjust the pH to 5-7. The crude product
3
was purified by flash column chromatography (silica gel; EtOAc/hexane = 1:1) to give
compound 8 (63 mg, 25%) as yellowish oil. The purity of product 8 was 99.7% as shown
1
by HPLC on a RP18 column, t = 9.4 min (ACN/H O = 1:1). H NMR (200 MHz, DMSO-
R
2
2
4

d6) δ 7.28 (d, J = 8.2 Hz, 1H), 6.96-6.93 (m, 2H), 6.89-6.82 (m, 2H), 6.74-6.66 (m, 3H),
6
2
1
4
5
2
.59 (s, 1H), 5.65 (t, J = 5.6 Hz, 1H, NH), 3.79 (s, 6H, OCH ), 3.27-3.17 (m, 2H, CH ),
3 2
1
3
.90-2.83 (m, 2H, CH ); C NMR (50 MHz, DMSO-d ) δ 158.9, 156.2, 152.3, 148.6,
2
6
46.3, 131.9, 130.1, 126.0, 124.0, 122.1, 113.7, 113.4, 111.7, 106.2, 101.9, 60.3, 55.6,
-
3.7, 28.4; HRMS (ESI) calcd. for C H N O S-H [M-H] : 419.0871, found: 419.0899.
1
9
20
2
7
.9. N-(7-Hydroxy-2-oxochromen-3-yl)-2,3-dimethoxybenzamide (11)
,3-Dimethoxybenzoic acid (9) (648 mg, 3.56 mmol) in DCM (20 mL) was treated with
thionyl chloride (SOCl , 517 μL, 7.12 mmol) to give 2,3-dimethoxy benzoyl chloride (10)
2
at 0 °C. After the reaction was complete, 2,3-dimethoxybenzoyl chloride (10) was dried
by evaporation in vacuo. Next, the residue containing 2,3-dimethoxybenzoyl chloride (10)
was dissolved in anhydrous THF (10 mL) and added to a solution of 3-amino-7-hydroxy-
2
H-chromen-2-one (5, 500 mg, 2.82 mmol) in anhydrous ACN/THF (20 mL, 1:1). The
mixture was stirred at room temperature for 8 h. The resulting solids were collected by
filtration and washed with methanol. The solid was recrystallized from EtOAc to afford
1
1
1 (866 mg, 90%) as a white solid. mp: 170-172 °C; H NMR (200 MHz, DMSO-d ) δ
6
8
.71 (s, 1H), 7.63-7.54 (m, 2H), 7.35-7.20 (m, 2H), 6.82 (dd, J = 8.4, 2.2 Hz, 1H), 6.77
1
3
(
d, J = 2.2 Hz, 1H), 3.98 (s, 3H, OCH ), 3.88 (s, 3H, OCH ); C NMR (50 MHz, DMSO-
3 3
d6) δ 162.7, 159.7, 158.5, 152.6, 151.3, 147.2, 129.2, 125.0, 124.6, 124.5, 121.7, 121.2,
-
1
3
5
17.1, 113.8, 111.5, 102.2, 61.4, 56.2; HRMS (ESI) calcd. for C H NO -H [M-H] :
1
8
15
6
40.0816, found: 340.0837.
.10. 3-(2,3-Himethoxybenzamido)coumarin-7-O-sulfamate (12)
Formic acid (45 L, 1.2 mmol) was slowly added to a stirred solution of chlorosulfonyl
isocyanate (104 L, 1.2 mmol) at 0 °C. Stirring was continued until sulfamoyl chloride
(white solid) was formed, whereupon anhydrous DMF (5 mL) was added to dissolve the
solid. Meanwhile, TEA (84 L, 0.6 mmol) was added to compound 11 (205 mg, 0.6 mmol)
2
5

in anhydrous DMF (10 mL). The mixture was stirred at room temperature for 1 h, and
then cooled to 0 °C. Prepared sulfamoyl chloride was added to the mixture drop wise at
0
°C and stirred for 2 h. After warming to room temperature, the reaction was stirred for
another 12 h. A solution of saturated sodium bicarbonate (NaHCO ) was added into the
3
stirred mixture to adjust the pH value to 5-7. The crude product was purified by flash
column chromatography (silica gel; EtOAc/Hexane = 1:1) to give compound 12 (45 mg,
1
8%) as lacteous oil. The purity of product 12 was 99.6% as analyzed by HPLC on a
1
RP18 column, t = 9.4 min (ACN/H O = 1:1). H NMR (400 MHz, DMSO-d ) δ 8.73 (s,
R
2
6
1
1
H), 7.62 (dd, J = 8.0, 1.2 Hz, 1H), 7.58 (d, J = 8.4 Hz, 1H), 7.34 (dd, J = 8.0, 1.2 Hz,
H), 7.26 (t, J = 8.0 Hz, 1H), 6.83 (dd, J = 8.4, 2.0 Hz, 1H), 6.79 (d, J = 2.0 Hz, 1H), 3.98
1
3
(
s, 3H, OCH ), 3.89 (s, 3H, OCH ); C NMR (100 MHz, DMSO-d ) δ 162.9, 159.8,
3 3 6
1
1
4
5
58.6, 152.7, 151.4, 147.3, 129.3, 125.1, 124.7, 124.7, 121.8, 121.3, 117.2, 113.9, 111.6,
-
02.3, 61.6, 56.4; HRMS (ESI) calcd. for C H N O S-H [M-H] : 419.0515, found:
1
8
16
2
8
19.0537.
.11. Purification of human placental STS
Human placenta was obtained from the Department of Obstetrics and Gynecology of the
NTUH, with the consent of the NTUH Research Ethics Committee (201712232RINB).
3
The human placenta was cut into 1 cm cubes and immersed in phosphate-buffered saline
(
PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na HPO and 1.8 mM KH PO ) at 0-4 ˚C.
2 4 2 4
The placenta tissue was granulated with Ultra-Turrax T-18 homogenizer (IKA) and
subjected to centrifugation (2000 × g, 4 ˚C, 20 mins) to remove the tissue debris. The
supernatant was collected and centrifuged (10,000 x g, 4 ˚C, 1 h); the supernatant was
collected, and then subjected to ultracentrifugation (100,000 x g, 4 ˚C, 1 h). After removal
of the supernatant, the microsomal pellets were resuspended in Tris-HCl buffer (20 mM,
pH 7.4) supplemented with 0.3% (v/v) Triton X-100 auxiliary with sonication. The
2
6

protein concentrations were determined by Bradford assay with bovine serum albumin
BSA) as a standard. The prepared microsomes were stored at 4 ˚C until assay.
.12. MCF-7 cell culture and subculture
(
5
®
TM
The MCF-7 (ATCC HTB-22 ) cell line was kindly provided by Prof. Ji-Wang Chern
(National Taiwan University, Taipei, Taiwan (R.O.C). MCF-7 cells were cultivated with
TM
RPMI 1640 medium (Gibco , Thermo Fisher) supplemented with 10% fetal bovine
TM
serum (FBS; Gibco , Thermo Fisher) and 1% antibiotic-antimycotic (100 U/mL
penicillin and 100 µg/mL streptomycin) in a T-75 cell culture flask [26]. The cell culture
was maintained at 37 ˚C in a humidified atmosphere of 5% CO and 95% air. When the
2
cells had grown to 80-90% confluence, they were harvested with 2 mL of Trypsin-EDTA
(2.5X). After the cell layer was dispersed from the flask, 8 mL of complete growth
medium was added and pipetting the cells gently. The cell suspensions were transferred
into the centrifuge tube and centrifuged at 800-1000 rpm for 5-10 minutes. After removal
of the supernatant, the cell pellet was resuspended with complete growth medium. The
cells were sub cultured or prepared for further biological assays.
5
.13. STS inhibition assay on human placental microsome
The sample drugs were prepared into 0.0001, 0.001, 0.01, 0.1, 1, 10 and 100 µM with
Tris-HCl (20 mM, pH 7.4) buffer supplemented with 0.3 mM potassium 4-nitrophenyl
sulfate (NPS). Purified human placental STS enzyme (10 µg/mL) was prepared with Tris-
HCl (20 mM, pH 7.4) buffer and added into a 96-well plate. Prepared compounds and
NPS were added into purified human placental STS and incubated at 37 ˚C for 1 h. The
reaction was terminated with 0.1 M NaOH. The absorption of 4-nitrophenol (NP) at 405
nm was detected with multi-mode microplate reader (SpectraMax® Paradigm®,
Molecular Devices, USA). The inhibition activity of the drugs (IC ) on STS was
5
0
calculated with GraphPad Prism 5 (GraphPad Software, Inc., USA). Data was collected
2
7