Synthesis and molecular docking of 1,2,3-triazole-based sulfonamides as aromatase inhibitors

Article abstract of DOI:10.1016/j.bmc.2015.04.036

Abstract A series of 1,4-disubstituted-1,2,3-triazoles (13-35) containing sulfonamide moiety were synthesized and evaluated for their aromatase inhibitory effects. Most triazoles with open-chain sulfonamide showed significant aromatase inhibitory activity

Full text of DOI:10.1016/j.bmc.2015.04.036

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and molecular docking of 1,2,3-triazole-based
sulfonamides as aromatase inhibitors
b
Ratchanok Pingaew a, , Veda Prachayasittikul , Prasit Mandi b,c, Chanin Nantasenamat b,c
,
⇑
Supaluk Prachayasittikul c, Somsak Ruchirawat d,e, Virapong Prachayasittikul b,
⇑
a Department of Chemistry, Faculty of Science, Srinakharinwirot University, Bangkok 10110, Thailand
b Department of Clinical Microbiology and Applied Technology, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand
c Center of Data Mining and Biomedical Informatics, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand
d Laboratory of Medicinal Chemistry, Chulabhorn Research Institute, and Program in Chemical Biology, Chulabhorn Graduate Institute, Bangkok 10210, Thailand
e Center of Excellence on Environmental Health and Toxicology, Commission on Higher Education (CHE), Ministry of Education, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 1,4-disubstituted-1,2,3-triazoles (13–35) containing sulfonamide moiety were synthesized
Received 12 February 2015
Revised 9 April 2015
Accepted 10 April 2015
Available online xxxx
and evaluated for their aromatase inhibitory effects. Most triazoles with open-chain sulfonamide showed
significant aromatase inhibitory activity (IC50 = 1.3–9.4
benzene-sulfonamide (34) bearing 6,7-dimethoxy substituents on the isoquinoline ring displayed the
most potent aromatase inhibitory activity (IC50 = 0.2 M) without affecting normal cell. Molecular dock-
ing of these triazoles against aromatase revealed that the compounds could snugly occupy the active site
of the enzyme through hydrophobic, stacking, and hydrogen bonding interactions. The potent com-
lM). Interestingly, the meta analog of triazole-
l
Keywords:
Triazole
Sulfonamide
Tetrahydroisoquinoline
Anti-aromatase activity
Structure–activity relationships
Molecular docking
p–p
pound 34 was able to form hydrogen bonds with Met374 and Ser478 which were suggested to be the
essential residues for the promising inhibition. The study provides compound 34 as a potential lead mole-
cule of anti-aromatase agent for further development.
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
(1) and anastrozole (2), both containing 1,2,4-triazole ring, were
approved by the Food and Drug Administration (FDA) and using
Breast cancer is one of the leading causes of cancer-related mor-
tality among women worldwide from different age groups. The
vast majority of breast cancers in postmenopausal women are
deriving from estrogens production.1–3 Estrogens are biosynthe-
sized from androgens catalyzed by aromatase (CYP19), an enzyme
belonging to the P450 family of monooxygenase heme proteins.
Two main strategies to control or block breast cancer progression
include binding of the estrogen receptors (ERs) with receptor
antagonists (ERAs such as tamoxifen), and inhibiting the produc-
tion of estrogen with aromatase inhibitors (AIs).3 AIs were found
to have less side effects than ERAs owing to the the lack of estro-
genic activity on uterus and vasculature.3
as the first-line therapy in the treatment of breast cancer in post-
menopausal women since they have been shown to be superior
to tamoxifen.3 Based on the AIs, the triazole ring plays a pivotal
role in chelation with heme iron.5 Along the line, Touaibia group
has studied on an aromatase inhibitory activity of various substi-
tuted-1,2,3-triazole letrozole-based analogs.6 The results revealed
that 1,2,3-trizole (3) analog of letrozole showed equipotent activity
to the parent compound. In addition, the 1,4-disubstituted-1,2,3-
triazole (4) was shown to be the most potent compound
(IC50 = 1.36 lM) among the tested 1,4-disubstituted-1,2,3-triazole
series. Aromatase inhibitors 1–4 are shown in Figure 1. However,
the interaction mode of the 1,4-disubstituted-1,2,3-triazole series
with the target enzyme remains to be explored.
Recently, 1,4-disubstituted-1,2,3-triazoles bearing 1,2,3,4-te-
trahydroisoquinoline (THIQ) and its open-chain derivatives 5
(Fig. 2) with cytotoxic activity against four cancer cell lines (e.g.,
HuCCA-1, HepG2, A549 and MOLT-3) have been reported by our
group.7,8 Based on the molecular docking study, an aldoketo reduc-
tase 1C3 (AKR1C3) has been identified to be a plausible target
responsible for anticancer activity of the THIQ analogs.8
Triazoles are common pharmacophore found in a diverse range
of biologically active molecules due to their potential structural
features (i.e., capability of hydrogen bonding, stable to metabolic
degradation and less undesired effects).4 Among the AIs, letrozole
⇑
Corresponding authors. Tel.: +66 2 649 5000x18253; fax: +66 2 260 0128 (R.P.);
tel.: +66 2 441 4376; fax: +66 2 441 4380 (V.P.).
E-mail addresses: ratchanok@swu.ac.th (R. Pingaew), virapong.pra@mahidol.ac.
th (V. Prachayasittikul).
http://dx.doi.org/10.1016/j.bmc.2015.04.036
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
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2
R. Pingaew et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
N
sulfonamide, triazole, ether and carbonyl moieties would partici-
N
pate in hydrogen bonding formation. Moreover, molecular docking
of the synthesized compounds against the aromatase was also per-
formed to give insights into their binding modes governing the
investigated aromatase inhibitory activities.
N
N
N
N
N
NC
NC
NC
CN
CN
NC
1
2
4
2. Results and discussion
2.1. Chemistry
N
N
N1
N
4
The synthesis of triazoles (e.g., types I and II) has been previ-
ously reported by our group.7,8 The open chain THIQ analogs of tri-
azoles 13–24 (type I) were prepared through
N
3
O
a sequential
CN
sulfonation/reduction/diazotization/cycloaddition reactions (route
a; steps i–iv) as outlined in Scheme 1. In the same manner, the syn-
thesis of triazoles type II 25–31 and type III 32–35 was carried out
via route b (steps i–v) in which an additional step (i.e., step v) was
performed using the Pictet–Spengler reaction to form isoquinoline
ring (10) prior to steps ii–iv.
Structures of the novel 1,2,3-triazoles 32–35 were confirmed
based on their 1H NMR, 13C NMR, HRMS and IR spectra. For
instance the triazole 34, its 1H NMR spectra revealed two triplets
at d 2.86 and 3.47 ppm which were assigned to the methylene pro-
tons of C4- and C3-THIQ, respectively. The methylene protons at
C1-THIQ ring appeared as a singlet at d 4.31 ppm whereas two
methoxy protons at C6- and C7-positions of the THIQ part were
noted as a singlet at d 3.83 ppm. In addition, the methylene protons
of –CH2O– group were found to be displayed as a singlet at d
5.39 ppm. Aromatic protons of THIQ ring (H-5 and H-8) displayed
as two singlets at d 6.54 ppm and 6.56 ppm. Two methine protons
of coumarin ring appeared as a multiplet at d 6.92–6.98 (H-6 and
H-8). The rest of three methine protons of coumarin moiety were
Figure 1. Aromatase inhibitors containing triazole 1–4.
R1
N
1
N
N
4
N
R1
S
R2
n
O
O
5
R1 = H, OMe
R2= Ph, -CH2OPh
n = 0, 1
Figure 2. Cytotoxic agents containing triazole 5.
In general, two structural features of the aromatase active site
associate with highly hydrophobic and H-bonding interactions.9
Therefore, to design and seek for a novel class of aromatase inhibi-
tor, many in-house 1,2,3-triazoles (series I and II) and four novel
1,2,3-triazoles of THIQ (series III) were synthesized through the
Click reaction, and evaluated for their aromatase inhibitory effects.
Herein, the molecules of rational designed inhibitors (Fig. 2) bear-
ing THIQ, benzene, naphthalene and coumarin rings might be
anticipated in forming hydrophobic interaction. In addition, vari-
ous functional groups of the designed compounds such as
observed as three doublets at
d 6.30 (J = 9.5 Hz), d 7.39
(J = 9.2 Hz) and d 7.63 (J = 9.5 Hz) ppm, which were assigned to
methine protons of C3, C5 and C4, respectively. A triplet at d
7.70 ppm (J = 8.0 Hz), two doublets at d 7.87 (J = 7.8 Hz) and
7.99 ppm (J = 7.6 Hz) and a singlet at d 8.17 ppm were attributed
R1
R1
R1
R1
R1
(v)
(i)
NO2
NO2
NH2
HN
N
(
m
-or
p
-)
(
m
- or
p
p
-)
S
R1
S
O
O
O
O O
6
7
10
(ii)
(ii)
R1
R1
R1
R1
N3
R1
R1
NH2
(iii)
NH2
N
HN
HN
(
m
- or
-)
S
S
S
O
O
O
O
O
8
11
9
(iv)
(iii)
N
R1
H
NO2/NH2/N3
para
N
N
R1
R1
6-12
R2
R1
R1
a
b
c
d
N3
OMe para
meta
OMe meta
N
O
(
m
- or
p
-)
S
O
HN
H
S
O
O
12
type I
13-35 : R1, R2 (see table 1)
(iv)
(13-24)
R2
N
N
N
N
N
N
R2
R1
R1
R1
R1
N
N
S
O
S
O
O O
type III
type II
(32-35)
(25-31)
Scheme 1. Synthesis of 1,2,3-triazole-based sulfonamides 13–35 through the Click reaction. Reagents and conditions: (i) 3- or 4-benzenesulfonyl chloride, Na2CO3, CH2Cl2, rt;
(ii) SnCl2ꢀ2H2O, EtOH, reflux; (iii) NaNO2, HCl/CH3COOH, 0 °C, NaN3, rt; (iv) „–R2, CuSO4ꢀ5H2O, sodium ascorbate, t-BuOH/H2O, rt; (v) (CH2O)n, HCOOH, reflux; route a = steps
i–iv; route b = steps i–v.
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R. Pingaew et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
to four aromatic protons of benzenesulfonyl moiety. A singlet of a
methine proton of the triazole ring appeared down field chemical
shift at d 8.13 ppm.
triazole was substituted with 7-coumarinoxymethyl substituent
(R2) led to the compound 21 with loss of the activity as compared
to the 4-coumarinyl group of compound 22 (IC50 = 1.8
Inhibition potency was distinctively appeared as noted in com-
pound 23 (IC50 = 4.6
M) when dimethoxy groups (R1) were intro-
duced to 3,4-positions on phenethyl moiety of analog 13 (R1 = H,
lM).
In the 13C NMR spectra, three methylene carbons (C1, C3 and
C4) of THIQ ring were visible at d 28.2, 43.8 and 47.2 ppm whereas
a methylene carbon of –CH2O– group was observed at d 62.2 ppm.
Two methoxy carbons (at C6 and C7) of THIQ ring were noted at
55.9 and 56.0 ppm. Two tertiary aromatic carbons of THIQ ring
appeared at chemical shift 109.0 (C8) and 111.5 ppm (C5), and
C5 of triazole ring was observed at d 121.2 ppm. Five tertiary car-
bons (C3, C4, C5, C6 and C8) of coumarin ring were seen at chem-
ical shift 102.2, 112.7, 113.7, 129.0 and 143.2 ppm. The rest of
tertiary aromatic carbons of phenyl ring were noted at chemical
shift 119.4, 124.4, 127.6 and 130.8 ppm. Four quaternary aromatic
carbons (C4a, C8a, C6 and C7) of THIQ ring resonated at corre-
sponding chemical shift 122.9, 124.8, 147.9 and 148.1 ppm, and
C4 of triazole ring was observed at d 144.5 ppm. Two quaternary
aromatic carbons (C1 and C7) of phenyl ring were appeared at
chemical shift 137.3 and 139.4 ppm whereas three quaternary aro-
matic carbons (C4a, C8a, C7) and one carbonyl carbon (C2) were
observed at chemical shift 113.2, 155.8, 160.9 and 161.1 ppm.
The HRMS-TOF experiment showed molecular ion [M+H]+ peak
at 575.1589 corresponding to the molecular formula of
l
IC50 >12.5 lM). However, no aromatase inhibition was observed
for triazole type II (25–31) containing R1 = H and OMe on the
THIQ. Both triazole types I and II constituting sulfonyl group at
para position on phenyl ring, anti-aromatase activity was observed
for type I compounds, except for compounds 13 and 21 (IC50
>12.5 lM). Surprisingly, when the sulfonyl group on the phenyl
ring was moved to meta position, the activity of compounds was
remarkably manifested. Promisingly, 7-coumarinyl analog 34
(R1 = OMe) was shown to be the most potent compound with
IC50 value of 0.2
l
M. Potent activity was also found in dimethoxy
THIQ of 4-coumarinyl analog 35 (R1 = OMe, IC50 1.8
lM).
However, without 6,7-dimethoxy groups on the THIQ ring, 4-cou-
marinyl analog 33 (R1 = H) was shown to be an inactive aromatase
inhibitor.
The SAR results imply that lipophilic effect of dimethoxy groups
(R1) enhances the activity of triazoles type I and type III. Our results
are in-line with earlier studies, in which the lipophilicity is respon-
sible for high aromatase inhibition of xanthone10 and coumarin11
derivatives. Even with or without dimethoxy groups, the triazoles
type II with restricted THIQ were shown to be inactive compounds.
This could be possibly due to their structural features, that is, flex-
ible or rigid conformation, isomeric effect and binding interaction
with the target site of action. Therefore, the aid of molecular dock-
ing may provide insight into their mechanism of action.
To determine the safety index, these compounds were also
tested against the noncancerous (Vero) cell line derived from
African green monkey kidney (Table 1). It was found that the
potent analogs (14–20, 22–24, 34 and 35) were non-cytotoxic
toward normal cell, except for compound 35. However, the triazole
35 had high safety index with a selective index value of 48.
C
29H27N4O7S. The IR spectra exhibited the vibration absorption
bands of C@O group at 1723 cmꢁ1 and S@O moiety at 1347 and
1163 cmꢁ1
.
2.2. Biological activity
A series of 1,2,3-triazole-based sulfonamides types I–III (13–
35), bearing disubstituents at position 1 (phenyl sulfonamide)
and position 4 (R2), were evaluated for their aromatase inhibitory
activities. All tested triazoles had inhibition effect at 12.5 lM in
the range of 14–97% (data not shown), except for compound 32
(could not be evaluated due to its insolubility in the assay). The tri-
azoles type I (14–20, 22–24) and type III (34 and 35) with inhibi-
tion >50% were further explored to determine their IC50 values as
summarized in Table 1. The derivatives with the inhibition 650%
2.3. Molecular docking
were identified as inactive compound (IC50 >12.5
Ketoconazole (IC50 = 2.6 M) and letrozole (IC50 = 3.3 nM) were
used as the reference drugs.
lM).
l
Molecular docking of triazoles 13–35 to the aromatase enzyme
was performed to investigate their binding modes. The most
potent compound 34 (Fig. 3) was able to form hydrogen bonds
with Met374 and Ser478 using the sulfonyl oxygen (bond dis-
tance = 2.3 Å) and the oxycoumarinyl group (bond dis-
tance = 2.0 Å), respectively. The 2D ligand–protein interaction
map of the co-crystallized ligand ASD (Fig. S1) displayed
hydrophobic interaction involving steroidal backbone with amino
acids residues (i.e., Ile133, Phe134, Trp224 and Leu477), and
hydrogen bonding of the CO group at position 17 with the amino
group of Met374.
Results showed that sulfonamide substituents as open-chain
(type I) and restricted-THIQ analogs on the phenyl ring (types II
and III); and R2 substituents as phenyl, phenoxymethyl, naph-
thalene oxymethyl and coumarin oxymethyl on the triazole core
play crucial roles in governing their anti-aromatase activities.
Obviously, most of the triazoles in types I and III displayed aro-
matase inhibition activity (IC50 = 0.2–9.4
lM) whereas the tria-
zoles in type II were shown to be inactive (IC50 >12.5
lM).
Structure–activity relationship (SAR) studies of the tested com-
pounds are discussed hereafter.
Interestingly, isomeric coumarinyl and naphthalenyl triazoles
play crucial roles in exerting more potent aromatase inhibitory
activity than other tested compounds. This could be attributed to
their binding interactions with the aromatase enzyme. Docking
results (Figs. S2 and S3) of 4-coumarinyl triazoles (22 and 35) with
No significant inhibition effect was observed for triazole 13
(R2 = phenyl substituent); however, triazoles bearing R2 as phe-
noxymethyls (14–18) displayed the inhibition effect with IC50
values in the range of 2.9–9.4
4-nitrophenoxymethyl (R2) analog 17 (IC50 = 2.9
highest activity having comparable IC50 value with that of the keto-
conazole (IC50 = 2.6 M). When the phenyl group of compound 14
l
M. Among these compounds,
lM) exerted the
the same potency (IC50 = 1.8 lM) showed that compound 22 occu-
pied the binding cavity in a more straight extended form when
compared with that of compound 35. The binding of compound
22 (Fig. S2) facilitated hydrophobic interaction of phenethyl
phenylsulfonamide moiety with Ile133, Val370, Ser478, Thr310
and Trp224 along with the interaction of 4-coumarinyl ring with
l
was replaced with naphthalenyl rings as found in compounds 19
and 20, and with a 4-coumarinyl ring as found in compound 22,
the enhanced inhibitory potency was observed. In comparison
between 4-coumarinyl of open-chain (22) and restricted THIQ
(26) analogs (R1 = H), the activity was noted for compound 22
(IC50 = 1.8 lM) but not for compound 26 (IC50 >12.5 lM).
Apparently, the triazoles 20 and 22 exerted inhibition activity
higher than that of the ketoconazole. On the other hand, the
Phe221;
p–p stacking interaction of triazole and 4-coumarinyl
rings with Phe221; and hydrogen bonding of coumarin-2-one with
the amino group of Arg192. Docking of triazole 35 (Fig. S3) involves
hydrophobic interactions of dimethoxy THIQ with Ile133, of
sulfonyl phenyl with Asp309 and Thr310 as well as of the
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R. Pingaew et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Table 1
Aromatase inhibitory activity and cytotoxic activity (IC50
,
l
M) of triazoles (13–35)
Inhibitory activity (aromatase)
Series
I
Compound
R1
R2
Cytotoxic activity (Vero cell line)
Non-cytotoxic
Selective indexa
—
13
H
>12.5
O
O
14
15
H
H
9.4 1.6
Non-cytotoxic
Non-cytotoxic
>12.24
>13.93
8.0 0.2
7.9 0.7
2.9 0.1
3.4 0.1
Me
O
O
16
17
18
H
H
H
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
>13.68
>35.96
>32.79
CHO
NO2
O
Me
O
19
H
3.4 1.5
Non-cytotoxic
>30.35
O
O
O
20
21
H
H
1.3 0.4
>12.5
Non-cytotoxic
Non-cytotoxic
>79.37
—
O
O
O
O
22
H
1.8 0.5
Non-cytotoxic
>55.27
23
24
OMe
OMe
4.6 2.0
2.7 0.1
Non-cytotoxic
Non-cytotoxic
>23.40
>34.32
O
O
NO2
Non-cytotoxicb
28.58b
—
—
O
25
26
H
H
>12.5
>12.5
II
Me
O
O
O
O
O
27
28
OMe
OMe
>12.5
>12.5
Non-cytotoxicb
Non-cytotoxicb
—
—
NO2
Me
O
O
29
30
OMe
OMe
>12.5
2.82b
—
—
OMe
O
O
>12.5
>12.5
37.23b
Me
O
CHO
31
32
OMe
H
Non-cytotoxicb
91.54
—
—
MeO
O
O
O
O
O
c
—
III
O
O
O
33
34
H
>12.5
Non-cytotoxic
Non-cytotoxic
—
O
OMe
0.2 0.1
>435.09
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R. Pingaew et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
Table 1 (continued)
Series
Compound
R1
R2
Inhibitory activity (aromatase)
Cytotoxic activity (Vero cell line)
86.32
Selective indexa
47.96
O
O
O
35
OMe
1.8 0.2
Ketoconazoled
Letrozoled
2.6 0.7
0.0033 0.0004
—
—
—
1.94
—
—
—
Ellipticined
Vero cell line = African green monkey kidney cell line.
Non-cytotoxic = IC50 >50 g/mL.
l
a
Selective index = IC50 for Vero cells/IC50 for aromatase.
Data from reference number 8.
Insoluble in testing medium
b
c
d
Ketoconazole, letrozole and ellipticine were used as reference drugs.
Figure 3. Molecular docking of 1,2,3-triazole sulfonamides to aromatase enzyme. Redocking of co-crystallized androstenedione (ASD) yielded RMSD of 0.711 Å (A). Docking
pose of the most potent compound 34 (B) is shown where green dashed lines indicate distances of hydrogen bonds.
4-coumarinyl ring with Phe221 and His480. This molecular
arrangement induced stacking of phenylsulfonyl, triazole
compound 35 with Ile133 in exerting its activity (IC50 = 1.8
while compound 33 without dimethoxy groups was shown to be
an inactive compound (IC50 >12.5 M).
lM)
p–p
and 4-coumarinyl rings with Phe221; and hydrogen bonding of tri-
azole (N3 position) with Ser478, and oxycoumarinyl moiety with
Arg192. Such interactions involve molecules in the bent form
(35), which arises from m-substitution of the sulfonyl group and
triazole ring on the phenyl ring of sulfonamide. The results sug-
gested that the Ser478 and Arg192 residues were essential for
the inhibition of aromatase. It should be noted that both Ser478
and Arg192 played crucial roles in anti-aromatase activity of the
natural substrate ASD.9 Accordingly, such amino acids participated
in a water-mediated network of hydrogen bonding, thereby allow-
ing the interaction between the aromatase and the C3-keto oxygen
of androstenedione to undergo enolization.9 Furthermore, Ser478
was capable of promoting the aromatase inhibition confirmed by
the mutagenesis study.12 In addition, such amino acid was the
residue responsible for the inhibition and selectivity.13,14
l
Considering the most potent dimethoxy THIQ bearing 7-cou-
marinyl analog 34 (Fig. 4), additional hydrophobic interactions
with Val370, Ser478, Leu477 and Trp224 were observed as com-
pared to compound 35 (Fig. S3). Furthermore, the compound 34
was arranged in such a way to appropriately form hydrogen bond-
ing of the sulfonyl group with the amino group of Met374 as well
as hydrogen bonding of oxycoumarinyl moiety with Ser478. These
hydrophobic and hydrogen bonding interactions were suggested to
play pertinent roles contributing to the most potent activity of
The triazole 26 (Fig. S4), a restricted THIQ analog of 22, similarly
showed hydrophobic and
p–p stacking interactions with the aro-
matase. The triazole 21 (Fig. S5), an isomeric analog of 22, showed
that both terminal ends (phenethyl and 7-coumarinyl) could inter-
act with the hydrophobic pockets. However, no hydrogen bonding
with Arg192 was noted for compounds 26 and 21. This could likely
result in the absence of inhibitory activity for the compounds 26
and 21 (IC50 >12.5
(IC50 1.8 M).
lM) as compared to that of compound 22
l
Comparison of THIQs with 4-coumarinyl moiety 33 (R1 = H,
Fig. S6) and 35 (R1 = OMe, Fig. S3) revealed that both compounds
displayed similar hydrophobic and hydrogen bonding interactions
with the binding pockets of the aromatase. It was observed that
lipophilic dimethoxy groups were essential for the interaction of
Figure 4. 2D-ligand–protein interaction scheme of compound 34.
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R. Pingaew et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
compound 34 (IC50 = 0.2
lM). Importantly, Leu477 and Trp224
the most potent inhibitor (IC50 = 0.2 lM) without affecting the nor-
were the same hydrophobic residues interacting with steroidal
skeleton of ASD (Fig. S1). The H-bonding formations with Ser478
and Met374 (bond distance = 3.11 and 2.31 Å) were also noted in
the antiestrogenic letrozole in which it used two benzonitrile
groups to form such interactions.15 However, the compound 34
showed lower potency than letrozole. This may be due to the lack
of chelation with heme iron. Notably, the Met374 played a crucial
role in anti-aromatase activity of the natural substrate ASD
through H-bonding interaction with the C17-keto oxygen.9 In addi-
tion, Met374 and/or Ser478 were suggested to be the crucial resi-
dues found in many classes of aromatase inhibitors such as
coumarin,11 acridone,13 xanthone14 and flavonoids.16,17
mal cell line. Moreover, the molecular docking study revealed that
the investigated triazoles could snugly occupy the active site of
aromatase through the interactions of hydrophobic,
p–p stacking
and H-bonding. The most potent compound (34) was the only
one that displayed H-bonding interactions with both Met374 and
Ser478 which were suggested to be the essential amino acid resi-
dues for the inhibitory activity.
4. Experimental
4.1. Chemistry
Unfortunately, 7-coumarinyl analog 32 (R1 = H) was not tested
for the activity due to its insolubility in the testing system.
Therefore, the activity of 7-coumarinyl 32 cannot be compared
with 7-coumarinyl 34 (R1 = OMe, Fig. 4). However, the 2D
ligand–protein interaction of 32 (Fig. S7) showed hydrophobic
Column chromatography was carried out using silica gel 60
(70–230 mesh ASTM). Analytical thin-layer chromatography
(TLC) was performed with silica gel 60 F254 aluminum sheets. 1H
and 13C NMR spectra were recorded on a Bruker AVANCE 300
NMR spectrometer (operating at 300 MHz for 1H and 75 MHz for
13C). The following standard abbreviations were used for signal
multiplicities: singlet (s), doublet (d), triplet (t), quartet (q) and
multiplet (m). FTIR spectra were obtained using a universal atten-
uated total reflectance attached on a Perkin–Elmer Spectrum One
spectrometer. Mass spectra were recorded on a Bruker Daltonics
(microTOF). Melting points were determined using a Griffin melt-
ing point apparatus and were uncorrected.
and p–p stacking interactions but without hydrogen bond forming.
In addition, the analysis of triazole type I analog of naphthalenyl
(R2), particularly the highly potent 2-naphthalenyl compound 20
(Fig. S8), was found to be in the extended form when occupying
the binding site via hydrophobic interaction of phenethyl with
Ile133, Val370 and Thr310, phenyl sulfonyl with Leu477 and
Ser478 as well as 2-naphthalenyl with Phe221, Asp309 and
His480; through
p–p stacking of triazole and naphthalenyl rings
Data associated with the compounds 13–31 were reported in
our previous work.7,8
with Phe221; and hydrogen bonding of oxy-naphthalenyl moiety
with Ser478. On the other hand, the less extended form of 1-naph-
thalenyl compound 19 (Fig. S9) showed hydrophobic interaction of
phenethyl phenyltriazole with Ile133, Thr310, Phe221, Ser478 and
4.2. General procedure for the synthesis of nitrobenzene-
sulfonamides (7c–d)
Asp309 as well as 1-naphthalenyl ring with Gln218; p–p stacking
of triazole and 1-naphthalenyl rings with Phe221; but no hydrogen
bonding was observed. The results implied that the presence of H-
bonding interaction with Ser478 contributed to the better activity
A solution of phenylethylamine 6 (10 mmol) in dichloro-
methane (50 mL) was added dropwise to a stirred mixture of 3-ni-
trobenzenesulfonyl chloride (10 mmol) and sodium carbonate
(14 mmol) in dichloromethane (20 mL). The reaction mixture was
stirred at room temperature overnight, and added distilled water
(20 mL). The organic phase was separated and the aqueous phase
was extracted with dichloromethane (2 ꢂ 30 mL). The organic
extracts were combined and washed with water (30 mL). The
organic layer was dried over anhydrous sodium sulfate (anhyd
Na2SO4), filtered and evaporated to dryness under reduced pres-
sure. The crude product was further purified by recrystallization.
1H NMR of 3-nitro-N-phenethylbenzenesulfonamide (7c) was
consistent with that reported in the literature.18
of compound 20 (IC50 = 1.3 lM) over compound 19 (IC50 = 3.4 lM).
Taken together, the molecular requirements for the most potent
triazole inhibitor (34) included the restricted THIQ with 6,7-
dimethoxy groups (R1), 7-coumaryloxymethyl (R2) at position 4
of the triazole ring, and m-substitution of triazole and sulfonyl
moieties on the phenyl ring. Such structural features were essen-
tial for engaging in hydrophobic,
p–p stacking and H-bonding
interactions with the aromatase enzyme, particularly, hydrogen
bond forming with Met374 and Ser478. Obviously, triazole 34
was the only compound that engaged in the hydrogen bonding
interactions. In comparison with letrozole and ASD, the sulfoyl
and coumarinyl ether moieties of compound 34 could act as hydro-
gen bond acceptors to mimic the benzonitrile groups of the letro-
zole whereas mimicking the steroidal backbone and the C17-keto
oxygen of ASD was responsible by the hydrophobic moieties
(6,7-dimethoxy THIQ and phenyl rings) and the sulfonyl oxygen
of compound 34, respectively.
4.2.1. N-(3,4-Dimethoxyphenethyl)-3-nitrobenzenesulfonamide
(7d)
Pale yellow solid. 87%. Mp 103–104 °C. IR (UATR) cmꢁ1: 3233,
1607, 1592, 1534, 1332, 1162 cmꢁ1 1H NMR (300 MHz, CDCl3) d
.
2.77 (t, J = 6.6 Hz, 2H, ArCH2), 3.31 (q, J = 6.6 Hz, 2H, CH2NH),
3.82, 3.86 (s, 6H, 2ꢂ OCH3), 4.65 (br t, 1H, NH), 6.58 (s, 1H, ArH),
6.64 (d, J = 8.1 Hz, 1H, ArH), 6.76 (d, J = 8.1 Hz, 1H, ArH), 7.70 (t,
J = 8.0 Hz, 1H, ArH), 8.10 (d, J = 8.0 Hz, 1H, ArH), 8.43 (d,
J = 8.1 Hz, 1H, ArH), 8.62 (s, 1H, ArH). 13C NMR (75 MHz, CDCl3):
d 35.5, 44.5, 55.8, 55.9, 111.4, 111.7, 120.8, 122.2, 127.0, 130.0,
130.3, 132.4, 142.3, 148.1, 148.3, 149.2. HRMS-TOF: m/z [M+H]+
367.0957 (Calcd for C16H19N2O6S: 367.0958).
3. Conclusions
A series of 1,2,3-triazole-based sulfonamides (13–35) have been
synthesized using the Click reaction as a key step. Their aromatase
inhibitory activities and molecular docking were explored. It was
observed that most open-chain sulfonamide triazoles (Type I)
exerted aromatase inhibitory activity (IC50 = 1.3–9.4
l
M). All tria-
4.3. General procedure for the synthesis of 1,2,3,
4-tetrahydroisoquinolines (10c–d)
zoles (Type II) with restricted THIQ analogs were shown to be inac-
tive compounds. The triazoles 34 and 35 displayed potent activity
where they contained 6,7-dimethoxy substituents (R1) on the THIQ
core, and coumarinyloxymethyl (R2) on the triazole ring; and m-
substitution of sulfonyl group and triazole ring on the phenyl moi-
ety of sulfonamide. Particularly, the triazole 34 was shown to be
A mixture of sulfonamide 7 (6.7 mmol) and paraformaldehyde
(7.2 mmol) in formic acid (30 mL) was refluxed for 2 h, and then
allowed to cool to room temperature. The reaction mixture was
added to 30 mL of water, and the product was extracted with
Please cite this article in press as: Pingaew, R.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.04.036

R. Pingaew et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
CH2Cl2 (2 ꢂ 30 mL). Combined extracts were washed with satu-
rated aqueous NaHCO3, dried (anhyd Na2SO4) and evaporated to
dryness under reduced pressure. The crude product was recrystal-
lized from methanol.
J = 6.0 Hz, 2H, C4-H), 3.42 (t, J = 6.0 Hz, 2H, C3-H), 4.31 (s, 2H, C1-
H), 7.02-7.18 (m, 4H, ArH), 7.22 (d, J = 7.9 Hz, 1H, ArH), 7.47 (s,
1H, ArH), 7.51 (t, J = 7.9 Hz, 1H, ArH), 7.60 (d, J = 7.8 Hz, 1H, ArH).
13C NMR (75 MHz, CDCl3) d 28.7, 43.7, 47.5, 118.0, 123.1, 123.7,
126.3, 126.5, 126.9, 128.8, 130.5, 131.4, 133.0, 138.8, 141.6.
HRMS-TOF: m/z [M+Na]+ 337.0737 (Calcd for C15H14N4NaO2S:
337.0730).
1H NMR of 2-((3-nitrophenyl)sulfonyl)-1,2,3,4-tetrahydroiso-
quinoline (10c) was consistent with that reported in the
literature.19
4.3.1. 6,7-Dimethoxy-2-((3-nitrophenyl)sulfonyl)-1,2,3,4-
tetrahydroisoquinoline (10d)
4.5.2. 2-((3-Azidophenyl)sulfonyl)-6,7-dimethoxy-1,2,3,
4-tetrahydroisoquinoline (12d)
Pale yellow solid. 80%. Mp 133–134 °C. IR (UATR) cmꢁ1: 1611,
1532, 1519, 1351, 1175, 1117. 1H NMR (300 MHz, CDCl3) d 2.86
(t, J = 5.8 Hz, 2H, C4-H), 3.47 (t, J = 5.8 Hz, 2H, C3-H), 3.84 (s, 6H,
2ꢂ OCH3), 4.30 (s, 2H, C1-H), 6.54, 6.55 (2s, 2H, C-5, C-8), 7.76 (t,
J = 8.1 Hz, 1H, ArH), 8.16 (d, J = 7.7 Hz, 1H, ArH), 8.44 (d,
J = 8.0 Hz, 1H, ArH), 8.68 (s, 1H, ArH). 13C NMR (75 MHz, CDCl3) d
28.1, 43.8, 47.2, 55.9, 56.0, 108.9, 111.4, 122.7, 124.6, 127.2,
130.5, 132.9, 139.4, 147.9, 148.1, 148.3. HRMS-TOF: m/z [M+Na]+
401.0768 (Calcd for C17H18N2NaO6S: 401.0778).
Pale yellow solid. 85%. Mp 86–87 °C. IR (UATR) cmꢁ1: 2155,
2106, 1592, 1518, 1465, 1346, 1167, 1116. 1H NMR (300 MHz,
CDCl3) d 2.81 (t, J = 5.8 Hz, 2H, C4-H), 3.37 (t, J = 5.8 Hz, 2H, C3-
H), 3.80 (s, 6H, 2ꢂ OCH3), 4.20 (s, 2H, C1-H), 6.50, 6.53 (s, 2H,
ArH), 7.21 (d, J = 7.7 Hz, 1H, ArH), 7.44 (s, 1H, ArH), 7.48 (t,
J = 7.9 Hz, 1H, ArH), 7.56 (d, J = 7.7 Hz, 1H, ArH). 13C NMR
(75 MHz, CDCl3) d 28.3, 43.8, 47.2, 55.9, 56.0, 109.0, 111.5, 118.0,
123.1, 123.7, 124.9, 130.5, 141.5, 147.9, 148.1. HRMS-TOF: m/z
[M+Na]+ 397.0925 (Calcd for C17H18N4NaO4S: 397.0941).
4.4. General procedure for the synthesis of 1,2,3,4-
tetrahydroisoquinolines (11c–d)
4.6. General procedure for the synthesis of triazoles (32–35)
To a stirred solution of azido 12 (0.2 mmol) and the correspond-
ing alkyne (0.2 mmol) in t-BuOH/H2O (3:3 mL), CuSO4ꢀ5H2O
(0.2 mmol) and ascorbic acid (0.5 mmol) were added. The reaction
mixture was stirred at room temperature for 2–12 h (monitored by
TLC), then concentrated under reduced pressure. The residue was
added water (10 mL) and extracted with dichloromethane
(3 ꢂ 20 mL). The combined organic phases were washed with
water (20 mL), dried over anhyd Na2SO4 and evaporated to dry-
ness. The crude product was purified using silica gel column chro-
matography and eluted with methanol/dichloromethane (1:50).
A mixture of nitroisoquinoline 10 (4 mmol) and SnCl2ꢀ2H2O
(20 mmol) in absolute ethanol (20 mL) was stirred under reflux
for 4 h then concentrated under reduced pressure. Water (20 mL)
was added and extracted with EtOAc (3 ꢂ 20 mL). The organic
extracts were combined and washed with water (20 mL) and brine
(20 mL). The organic layer was dried over anhyd Na2SO4, filtered
and concentrated. The crude product was recrystallized from
methanol.
1H NMR of 2-((3-aminophenyl)sulfonyl)-1,2,3,4-tetrahydroiso-
quinoline (11c) was consistent with that reported in the
literature.19
4.6.1. 7-((1-(3-((3,4-Dihydroisoquinolin-2(1H)-yl)sulfonyl)
phenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one
(32)
4.4.1. 2-((3-Aminophenyl)sulfonyl)-6,7-dimethoxy-1,2,3,4-
tetrahydroisoquinoline (11d)
Pale yellow solid. 80%. Mp 170–171 °C. IR (UATR) cmꢁ1: 3468,
3374, 1626, 1599, 1519, 1317, 1158, 1117. 1H NMR (300 MHz,
DMSO-d6) d 2.76 (t, J = 5.8 Hz, 2H, C4-H), 3.18 (t, J = 5.8 Hz, 2H,
C3-H), 3.64 (s, 6H, 2ꢂ OCH3), 4.00 (s, 2H, C1-H), 5.62 (s, 2H, NH2),
6.67, 6.72 (2s, 2H, C-5, C-8), 6.80 (dd, J = 8.0, 1.3 Hz, 2H, ArH),
6.85 (d, J = 8.0 Hz, 1H, ArH), 6.97 (s, 1H, ArH), 7.22 (t, J = 7.8 Hz,
1H, ArH). 13C NMR (75 MHz, DMSO-d6) d 28.2, 44.2, 47.4, 56.0,
110.0, 110.3, 112.1, 112.3, 114.5, 118.4, 123.7, 125.3, 130.2,
147.8, 148.0, 150.0. HRMS-TOF: m/z [M+Na]+ 371.1039 (Calcd for
White solid. 88%. Mp 180–181 °C. IR (UATR) cmꢁ1: 1725, 1616,
1486, 1342, 1229, 1163, 1127. 1H NMR (300 MHz, CDCl3) d 2.95 (t,
J = 5.9 Hz, 2H, C4-THIQH), 3.50 (t, J = 5.9 Hz, 2H, C3-THIQH), 4.38 (s,
2H, C1-THIQH), 5.39 (s, 2H, CH2O), 6.30 (d, J = 9.5 Hz, 1H, ArH),
6.96–7.02 (m, 2H, ArH), 7.03–7.18 (m, 4H, ArH), 7.44 (d,
J = 9.3 Hz, 1H, ArH), 7.68 (d, J = 9.5 Hz, 1H, ArH), 7.74 (t, J = 8.0 Hz,
1H, ArH), 7.93 (d, J = 8.0 Hz, 1H, ArH), 8.06 (d, J = 8.0 Hz, 1H, ArH),
8.17 (s, 1H, CHN), 8.20 (s, 1H, ArH). 13C NMR (75 MHz, CDCl3) d
28.6, 43.8, 47.5, 62.2, 102.2, 112.7, 113.2, 113.7, 119.3, 121.2,
124.5, 126.3, 126.5, 127.0, 127.6, 128.9, 129.0, 130.9, 131.1,
132.8, 137.4, 139.3, 143.2, 144.4, 155.8, 161.0, 161.1. HRMS-TOF:
m/z [M+H]+ 515.1390 (Calcd for C27H23N4O5S: 515.1384).
C17H20N2NaO4S: 371.1036).
4.5. General procedure for the synthesis of 2-((3-azidophenyl)
sulfonyl)-1,2,3,4-tetrahydroisoquinolines (12c–d)
4.6.2. 4-((1-(3-((3,4-Dihydroisoquinolin-2(1H)-yl)sulfonyl)
phenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one
(33)
To a cold solution of amine 11 (3 mmol) in HCl/CH3COOH
(3:3 mL) at 0 °C, a solution of sodium nitrite (9 mmol) in water
(5 mL) was added. The stirred reaction mixture was maintained
for 15 min and then added dropwise a solution of sodium azide
(9 mmol) in water (5 mL). The reaction mixture was allowed to stir
at room temperature for 0.5 h, then the precipitate was filtered and
washed with cold water. The crude product was recrystallized
from methanol.
White solid. 91%. Mp 168–170 °C. IR (UATR) cmꢁ1: 1712, 1623,
1566, 1494, 1455, 1372, 1336, 1248, 1191, 1153. 1H NMR
(300 MHz, CDCl3) d 2.95 (t, J = 5.9 Hz, 2H, C4-THIQH), 3.51 (t,
J = 5.9 Hz, 2H, C3-THIQH), 4.40 (s, 2H, C1-THIQH), 5.47 (s, 2H,
CH2O), 5.93 (s, 1H, CHCO), 7.04–7.20 (m, 4H, ArH), 7.30 (t,
J = 7.6 Hz, 1H, ArH), 7.35 (d, J = 7.6 Hz, 1H, ArH), 7.59 (dt, J = 7.8,
1.6 Hz, 1H, ArH), 7.76 (t, J = 8.0 Hz, 1H, ArH), 7.85 (dd, J = 7.9,
1.5 Hz, 1H, ArH), 7.95 (d, J = 8.1 Hz, 2H, ArH), 8.08 (d, J = 8.1 Hz,
2H, ArH), 8.22, 8.23 (2s, 2H, ArH, CHN). 13C NMR (75 MHz, CDCl3)
d 28.6, 43.8, 47.5, 62.5, 91.4, 115.4, 116.9, 119.4, 121.6, 123.1,
4.5.1. 2-((3-Azidophenyl)sulfonyl)-1,2,3,4-tetrahydroiso-
quinoline (12c)
Pale yellow solid. 94%. Mp 120–121 °C. IR (UATR) cmꢁ1: 2154,
2109, 1595, 1337, 1169. 1H NMR (300 MHz, CDCl3) d 2.93 (t,
Please cite this article in press as: Pingaew, R.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.04.036

8
R. Pingaew et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
124.0, 124.6, 126.3, 126.6, 127.0, 127.8, 128.9, 131.0, 131.1, 132.7,
132.8, 137.3, 153.4, 162.5, 164.9. HRMS-TOF: m/z [M+H]+ 515.1385
(Calcd for C27H23N4O5S: 515.1384).
4.8. Cytotoxicity assay: primate cell line (Vero)
The cytotoxicity was carried out by using the Green Fluorescent
Protein (GFP) detection method.21 The GFP-expressing Vero cell
line was generated in-house by stably transfecting the African
green monkey kidney cell line (Vero, ATCC CCL-81), with pEGFP-
4.6.3. 7-((1-(3-((6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-
yl)sulfonyl)phenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-
chromen-2-one (34)
N1 plasmid (Clontech). The assay was performed by adding 45 lL
of cell suspension at 3.3 ꢂ 104 cells/mL to each well of 384-well
White solid. 92%. Mp 180–181 °C. IR (UATR) cmꢁ1: 1723, 1613,
1522, 1466, 1347, 1226, 1163, 1120. 1H NMR (300 MHz, CDCl3) d
2.86 (t, J = 5.8 Hz, 2H, C4-THIQH), 3.47 (t, J = 5.9 Hz, 2H, C3-
THIQH), 3.83 (s, 6H, 2ꢂ OCH3), 4.31 (s, 2H, C1-THIQH), 5.39 (s,
2H, CH2O), 6.30 (d, J = 9.5 Hz, 1H, ArH), 6.54, 6.56 (2s, 2H, ArH),
6.92–6.98 (m, 2H, ArH), 7.39 (d, J = 9.2 Hz, 1H, ArH), 7.63 (d,
J = 9.5 Hz, 1H, ArH), 7.70 (t, J = 8.0 Hz, 1H, ArH), 7.87 (d, J = 7.8 Hz,
1H, ArH), 7.99 (d, J = 7.6 Hz, 1H, ArH), 8.13 (s, 1H, CHN), 8.17 (s,
1H, ArH). 13C NMR (75 MHz, CDCl3) d 28.2, 43.8, 47.2, 55.9, 56.0,
62.2, 102.2, 109.0, 111.5, 112.7, 113.2, 113.7, 119.4, 121.2, 122.9,
124.4, 124.8, 127.6, 129.0, 130.8, 137.3, 139.4, 143.2, 144.5,
147.9, 148.1, 155.8, 160.9, 161.1. HRMS-TOF: m/z [M+H]+
575.1589 (Calcd for C29H27N4O7S: 575.1595).
plates containing 5 lL of test compounds diluted in 0.5% DMSO.
After incubation for 4 days in 37 °C incubator with 5% CO2.
Fluorescence signals were measured using SpectraMax M5 micro-
plate reader (Molecular Devices, USA) with excitation and emission
wavelengths of 485 and 535 nm, respectively. IC50 values were
deduced from dose-response curves, using 6 concentrations of 3-
fold serially diluted samples, by the SOFTMax Pro software
(Molecular device). Ellipticine and 0.5% DMSO were used as a pos-
itive and a negative control, respectively.
4.9. Molecular docking
Molecular docking was performed to elucidate interactions of
investigated compounds toward its target protein namely the
aromatase enzyme. Initially, the crystal structure of the human
placental aromatase (cytochrome P450, member 19A1) co-
crystallized with the androstenedione (ASD) substrate was
retrieved from the Protein Data Bank (PDB ID: 3EQM).
Investigated compounds (13–35) were constructed using Marvin
Sketch version 6.0 and were geometrically optimized by Gaussian
0922 using Becke’s three-parameter hybrid method with the
Lee–Yang–Parr correlation functional (B3LYP) together with the
6-31 g(d) basis set. Prior to docking, the protein structure was
prepared by addition of H atoms and side chain repair using the
WHAT IF software.23 Subsequently, the Gasteiger and Kollman
charges were added to the ligands (the co-crystallized ligand ASD
and optimized compounds 13–35) and protein structures, respec-
tively, using the PyRx 0.6 software.24 A grid box with a size of
62.06 ꢂ 71.95 ꢂ 51.46 Å was created by the AutoGrid software to
cover all area of aromatase. The center of the grid box was allo-
cated using x, y, z coordinates of 83.4375, 50.1006 and 46.3803,
respectively. Molecular docking was performed using AutoDock
Vina, which is a part of the PyRx 0.6 software.24 The co-crystallized
ligand ASD was re-docked to aromatase as to validate the docking
protocol. The re-docking was evaluated by the calculation of root
mean standard deviation (RMSD) between the original and re-
docked position of the co-crystallized ligand using the Chimera
software.25 Docking poses of investigated compounds (13–35)
were visualized using the PyMoL software26 and the ligand–pro-
tein interactions were generated using online available tool,
PoseViewWeb version 1.97.0.27
4.6.4. 4-((1-(3-((6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-
yl)sulfonyl)phenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-
chromen-2-one (35)
White solid. 90%. Mp 211–213 °C. IR (UATR) cmꢁ1: 1717, 1622,
1518, 1457, 1384, 1343, 1238, 1156, 1114. 1H NMR (300 MHz,
CDCl3) d 2.86 (t, J = 5.7 Hz, 2H, C4-THIQH), 3.48 (t, J = 5.7 Hz, 2H,
C3-THIQH), 3.83 (s, 6H, 2ꢂ OCH3), 4.35 (s, 2H, C1-THIQH), 5.48
(s, 2H, CH2O), 5.94 (s, 1H, CHCO), 6.54, 6.56 (2s, 2H, ArH), 7.29 (t,
J = 7.7 Hz, 1H, ArH), 7.35 (d, J = 8.3 Hz, 1H, ArH), 7.59 (t, J = 7.6 Hz,
1H, ArH), 7.76 (t, J = 7.8 Hz, 1H, ArH), 7.85 (d, J = 7.9 Hz, 1H, ArH),
7.94 (d, J = 7.7 Hz, 1H, ArH), 8.07 (d, J = 7.6 Hz, 2H, ArH), 8.24 (s,
2H, ArH, CHN). 13C NMR (75 MHz, CDCl3) d 28.2, 43.9, 47.3, 55.9,
56.0, 62.5, 91.4, 108.9, 111.4, 115.4, 116.9, 119.5, 121.6, 122.9,
123.1, 124.0, 124.5, 124.8, 127.8, 130.9, 132.7, 137.2, 139.5,
142.9, 147.9, 148.1, 153.4, 162.5, 164.9. HRMS-TOF: m/z [M+H]+
575.1597 (Calcd for C29H27N4O7S: 575.1595).
4.7. Aromatase inhibition assay
Aromatase inhibitory effect was performed using the modified
method reported by Stressor et al.20 This method was carried out
according to the Gentest kit using CYP19 enzyme and O-benzyl flu-
orescein benzyl ester (DBF) as a fluorometric substrate. DBF was
dealkylated by aromatase and then hydrolyzed to give the fluores-
cein product.
Briefly, 100
phate buffer (pH 7.4); 20
(26 mM NADP+, 66 mM glucose-6-phosphate, and 66 mM MgCl2);
and 1.6 L of 100 U/mL glucose-6-phosphate dehydrogenase, were
l
L of cofactor, containing 78.4
lL of 50 mM phos-
lL of 20ꢂ NADPH-generating system
Acknowledgments
l
pipetted into a 96-well black plate and preincubated in 37 °C
(water bath) for 10 min. The reaction was initiated by addition of
This project is financially supported from Srinakharinwirot
University and the Higher Education Research Promotion (HERP).
R.P. is grateful for the Thailand Research Fund (MRG5680001).
Great supports from the Office of Higher Education Commission
and Mahidol University under the National Research Universities
Initiative are appreciated. We are also indebted to Chulabhorn
Research Institute for recording mass spectra and bioactivity
testing.
100
50 mM phosphate buffer (pH 7.4); 12.5
0.2 L of 0.2 mM DBF, and 10 L of tested sample or 10% DMSO
lL of enzyme/substrate (E/S) mixture containing 77.3
lL of
l
L of 16 pmol/mL CYP19;
l
l
as a negative control or ketoconazole/letrozole as a positive con-
trol. Fluorescence signal was recorded using an excitation wave-
length of 490 nm and emission wavelength of 530 nm with cutoff
515 nm. Percentage of inhibition (% inhibition) was calculated as
shown in Eq. 1. Samples with % inhibition greater than 50 were fur-
ther calculated according to Eq. 1 to obtain their IC50 values.
Supplementary data
%inhibition ¼ 100 ꢁ ½ðsample ꢁ blankÞ=ðDMSO ꢁ blankÞ ꢂ 100ꢃ
ð1Þ
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.04.036.
Please cite this article in press as: Pingaew, R.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.04.036

R. Pingaew et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
9
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