Design, synthesis and molecular docking studies of novel N-benzenesulfonyl-1,2,3,4-tetrahydroisoquinoline-based triazoles with potential anticancer activity

Article abstract of DOI:10.1016/j.ejmech.2014.05.019

A novel series of N-benzenesulfonyl-1,2,3,4-tetrahydroisoquinolines (14-33) containing triazole moiety were designed and synthesized through rational cycloadditions using the modified Pictet-Spengler reaction and the Click chemistry. Antiproliferative act

Full text of DOI:10.1016/j.ejmech.2014.05.019

European Journal of Medicinal Chemistry 81 (2014) 192e203
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and molecular docking studies of novel N-
benzenesulfonyl-1,2,3,4-tetrahydroisoquinoline-based triazoles with
potential anticancer activity
,
b
,
,c
Ratchanok Pingaew a , Prasit Mandi c, Chanin Nantasenamat b
,
*
Supaluk Prachayasittikul b, Somsak Ruchirawat d f, Virapong Prachayasittikul c
,e,
a Department of Chemistry, Faculty of Science, Srinakharinwirot University, Bangkok 10110, Thailand
b Center of Data Mining and Biomedical Informatics, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand
c Department of Clinical Microbiology and Applied Technology, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand
d Laboratory of Medicinal Chemistry, Chulabhorn Research Institute, Bangkok 10210, Thailand
e Program in Chemical Biology, Chulabhorn Graduate Institute, Bangkok 10210, Thailand
f 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 novel series of N-benzenesulfonyl-1,2,3,4-tetrahydroisoquinolines (14e33) containing triazole moiety
were designed and synthesized through rational cycloadditions using the modified PicteteSpengler
reaction and the Click chemistry. Antiproliferative activity against four cancer cell lines (e.g., HuCCA-1,
HepG2, A549 and MOLT-3) revealed that many substituted triazole analogs of benzoates (20, 29) and
benzaldehydes (30, 32) exhibited anticancer activity against all of the tested cancer cell lines in which the
Received 23 December 2013
Received in revised form
1 May 2014
Accepted 4 May 2014
Available online 6 May 2014
ester analog 20 was shown to be the most potent compound against HuCCA-1 (IC50 ¼ 0.63
mM) and A549
(IC50 ¼ 0.57
m
M) cell lines. Triazoles bearing phenyl (15, 24), tolyl (26, 27), acetophenone (19), benzoate
Keywords:
Isoquinoline
Triazole
PicteteSpengler reaction
Antiproliferative activity
Molecular docking
AKR1C3
(20, 29), benzaldehyde (21, 30) and naphthalenyl (25) substituents showed stronger anticancer activity
against HepG2 cells than that of the etoposide. Interestingly, the p-tolyl analog (27) displayed the most
potent inhibitory activity (IC50 ¼ 0.56
mM) against HepG2 cells without affecting normal cells. Of the
investigated tetrahydroisoquinoline-triazoles, the promising compounds 20 and 27 were selected for
molecular docking against AKR1C3, which was identified to be a plausible target site.
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
[(4-methylphenyl)sulfonyl]-1-(2-hydroxyphenylisoquinoline)
(1)
and 1-acetyl-6,7-dimethoxy-N-4-methoxybenzenesulfonyl-1,2,3,4-
tetrahydroisoquinoline thiosemicarbazone (2) exhibited cytotoxic
activity reported by our group as shown in Fig. 1 [6,7]. Furthermore,
N-sulfonyl-THIQ (3) and N-sulfonyl-THIQs with hydroxamate (4) and
carboxylate (5) have been revealed to act as carbonic anhydrase and
matrix-matalloproteinase inhibitors, respectively (Fig. 1) [8e10].
Isoquinoline alkaloid is a class of natural products that possess a
broad spectrum of pharmacological actions, particularly, anticancer
activity [1e3]. A variety of bioactive 1,2,3,4-tetrahydroisoquinolines
(THIQs) have been reported. Generally, THIQs are synthesized by
the PicteteSpengler reaction involving the cyclization of iminium
ions derived from the condensation of
b-arylethylamines with al-
Additionally,
3-(3,4-dihydroisoquinolin-2(1H)-ylsulfonyl)benzoic
dehydes [4,5]. The modified PicteteSpengler protocol was accom-
plished by increasing the electrophilicity of the iminium
intermediates using N-sulfonylphenethylamines as starting mate-
rials to yield N-sulfonyl-1,2,3,4-tetrahydroisoquinoline products.
Such N-sulfonyl-THIQs have been proven to be interesting scaffolds
for medical applications [6,7]. 1,2,3,4-Tetrahydro-6,7-dimethoxy-2-
acid (6) was shown to be selective and potent inhibitor of an aldo-
keto reductase 1C3 (AKR1C3) [11]. Replacement of the carboxyl
group with pyrrolidin-2-one core as compound 7 retained the ac-
tivity, but with 3-fold less potent than the parent compound 6. The
structureeactivity relationship (SAR) results of THIQs 6 and 7
showed that the sulfonamide moiety was critical for AKR1C3
inhibitory activity [12].
AKR1C3, a member of the aldo-keto reductase superfamily of
enzymes, plays an important role in stereospecific reduction of
carbonyl groups on both steroid and prostaglandin substrates using
* Corresponding author.
E-mail address: ratchanok@swu.ac.th (R. Pingaew).
http://dx.doi.org/10.1016/j.ejmech.2014.05.019
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

R. Pingaew et al. / European Journal of Medicinal Chemistry 81 (2014) 192e203
193
Fig. 1. Representative structures of bioactive N-sulfonyl-1,2,3,4-tetrahydroisoquinolines 1e8.
NADPH as a cofactor [13e16]. The reduction products of AKR1C3-
promoted reaction such as testosterone, 17 -estradiol, 20 -hy-
part of our ongoing research, a new series of hybrid molecules
containing both THIQ and triazole were designed and synthesized.
Thus, target molecules 14e33 (Fig. 3) were obtained by the
replacement of a phenethylamine moiety of compound 13 with a
restricted THIQ ring and by the replacement of a carboxyl group of
compound 6 with a triazole ring. A variety of expected novel hy-
brids were then evaluated for their in vitro antiproliferative activity
against cancer cell lines and normal cells. Molecular docking of
these compounds against AKR1C3 has been carried out.
b
a
droxy progesterone and PGF2 can stimulate tumor growth,
a
therefore AKR1C3 expression levels are correlated with both
hormone-dependent and hormone-independent cancers [15].
AKR1C3 is over-expressed in a number of cancers, particularly, in
the prostate and mammary gland where it is responsible for the
production of a series of growth-stimulatory steroid hormones.
Consequently, inhibition of the catalytic activity renders AKR1C3 as
an attractive therapeutic target for treating various types of cancer
and for the development of novel anticancer drugs.
2. Results and discussion
Triazole moiety is a common pharmacophore found in a diverse
range of biologically active molecules [17,18]. Potential structural
features of bioactive triazole include capability of hydrogen
bonding, stable to metabolic degradation, high selectivity and less
unfavorable reactions. Molecules linking with triazole scaffold are
considered to improve their pharmacological activities [19,20].
Importantly, substituted triazole analogs 9e12 (Fig. 2) have been
used in clinical trials for cancer therapy [17,18]. Recently, our group
reported that an open chain analog of N-sulfonyl-THIQ bearing
triazole, methyl 2-((1-(4-(N-phenethylsulfamoyl)phenyl)-1H-1,2,3-
triazol-4-yl)methoxy)benzoate (13), displayed antiproliferative
activity against cancer cell lines (e.g., HepG2 and MOLT-3) [20].
The reported bioactive pharmacophores lead to the rational
design of new THIQ rigid analogs based on the triazole moiety. As
2.1. Chemistry
Several new N-benzenesulfonyl-1,2,3,4-tetrahydroisoquinolines
(14e33) were synthesized using the modified PicteteSpengler re-
action and the Click chemistry approaches as key steps shown in
Scheme 1. Initially, treatments of nitrobenzenesulfonamides 34 [7]
with paraformaldehyde in refluxing formic acid were smoothly
cyclized via the modified PicteteSpengler reaction [6] to furnish
1,2,3,4-tetrahydroisoquinolines 35 [21,22] in good yields. Reduction
of the nitro derivatives 35 was performed using stannous chloride
in refluxing ethanol to give aminobenzenesulfonamides 36. Con-
version of the amino compounds 36 to the corresponding azido-
benzensulfonamides 37 was readily achieved through diazotization
Fig. 2. Representative structures of bioactive triazoles 9e13.

194
R. Pingaew et al. / European Journal of Medicinal Chemistry 81 (2014) 192e203
at
d 6.67 ppm and 6.76 ppm. Two doublets at d 7.99 and 8.19 ppm
with J value of 8.8 Hz were attributed to four aromatic protons of
benzenesulfonyl moiety at position 20(60) and 30(50). A singlet of a
methine proton of the triazole ring appeared down field chemical
shift at
bons (C-1, C-3 and C-4) of THIQ ring were visible at
47.4 ppm whereas a methylene carbon of eCH2OH group was
observed at 55.4 ppm. Two methoxy carbons (at C-6 and C-7) of
d
8.83 ppm. In the 13C NMR spectra, three methylene car-
d
28.0, 44.1 and
d
THIQ ring were noted at 55.9 and 56.0 ppm. Seven quaternary ar-
omatic carbons (ArC) were observed at chemical shift 123.6, 125.2,
136.0,140.2,147.8,148.1 and 150.1 ppm, and seven tertiary aromatic
carbons (ArCH) appeared at chemical shift 110.3, 112.3, 120.8
(2ArCH), 121.7 and 129.8 (2ArCH) ppm.
Fig. 3. Synthetic N-benzenesulfonyl-1,2,3,4-tetrahydroisoquinoline based triazoles
14e33.
reaction using sodium nitrite in a mixture of glacial acetic acid and
concentrated hydrochloric acid in the presence of sodium azide.
Finally, cycloaddition reaction (the Click chemistry) of the azides 37
with various alkynes 38 obtaining from alkylation of the appro-
priate phenol derivatives with propargyl bromide afforded a variety
of the desired triazoles 14e33 (Scheme 1) in moderate to good
yields (45e94%).
Structures of the desired 1,2,3-triazoles 14e33 were identified
based on their HRMS, IR and NMR spectra. For example, the HRMS-
TOF of hydroxymethyltriazole 23 showed its molecular ion [MþH]þ
peak at 431.1389 corresponding to the molecular formula of
2.2. Biological activity
A series of N-benzenesulfonyl-1,2,3,4-tetrahydroisoquinolines
(14e33) (i.e., with or without 6,7-dimethoxy substituents) were
preliminarily evaluated in vitro as antiproliferative agents against
four human cancer cell lines including HuCCA-1 (chol-
angiocarcinoma), HepG2 (hepatocellular carcinoma), A549 (lung
carcinoma) and MOLT-3 (lymphoblastic leukemia) cell lines as
summarized in Table 1. These compounds were also tested against
the noncancerous (Vero) cell line derived from African green
monkey kidney (Table 1). Results showed that substituents (R1) on
the isoquinoline ring and substituents (R2) on the triazole core play
important roles in governing their cytotoxicities. SAR studies of the
tested compounds are discussed hereafter.
It was observed that in a series of 1,2,3,4-tetrahydroisoquinoline
(14e22) without 6,7-dimethoxy substituents (R1 ¼ H), the triazole
(14) bearing hydroxymethyl substituent (R2 ¼ H) showed selective
inhibition against HuCCA-1 cells. The cytotoxic activity of com-
pound 14 in HuCCA-1 cells was lost when the H atom (R2) of the OH
group was replaced with a phenyl group as seen for compound 15.
On the other hand, the cytotoxic activity of compound 15 showed
C
20H23N4O5S. The IR spectra of the compound 23 exhibited ab-
sorption bands of OeH group at 3280 cmꢀ1 and of C]C moiety at
1599 cmꢀ1. The 1H NMR spectra revealed two triplets at
2.78 and
d
3.33 ppm which were assigned to the methylene protons of C4- and
C3-THIQ, respectively. The methylene protons at C1-THIQ ring
appeared as a singlet at
at C6- and C7-positions of the THIQ part appeared as two singlets at
3.67 and 3.68 ppm. In addition, the methylene protons of eCH2OH
d 4.16 ppm whereas two methoxy protons
d
group were found to be displayed as a doublet at
the hydroxyl proton was observed as a triplet at
d
4.62 ppm, and
5.41 ppm. Aro-
d
matic protons of THIQ ring (H-5 and H-8) displayed as two singlets
Scheme 1. Synthesis of N-benzenesulfonyl-1,2,3,4-tetrahydroisoquinoline based triazoles 14e33 through the PicteteSpengler reaction and the Click reaction.

R. Pingaew et al. / European Journal of Medicinal Chemistry 81 (2014) 192e203
195
Table 1
Cytotoxic activity (IC50, mM) of compounds (14e33) against four cancer cell lines and Vero cell line.
Compound
R1
R2
Cancer cell linesa
HuCCA-1
Vero cell line
HepG2
A549
MOLT-3
14
15
H
H
H
51.35 ꢁ 5.66
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
6.50 ꢁ 0.14
Non-cytotoxic
Non-cytotoxic
66.30 ꢁ 0.70
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
16
17
H
H
60.48 ꢁ 14.14
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
18
19
H
H
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
30.16 ꢁ 4.07
19.12 ꢁ 3.06
14.90 ꢁ 1.02
21.86 ꢁ 3.67
20
H
0.63 ꢁ 0.04
12.36 ꢁ 1.97
0.57 ꢁ 0.02
18.63 ꢁ 1.62
4.98
21
22
H
H
Non-cytotoxic
5.27 ꢁ 0.71
59.07 ꢁ 11.31
25.29 ꢁ 10.78
Non-cytotoxic
Non-cytotoxic
28.58
24.80 ꢁ 2.19
Non-cytotoxic
80.78 ꢁ 10.23
23
24
OMe
OMe
H
72.0 ꢁ 10.54
31.79 ꢁ 2.89
2.57 ꢁ 0.99
41.04 ꢁ 9.40
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
25
26
OMe
OMe
Non-cytotoxic
1.26 ꢁ 0.42
1.48 ꢁ 0.61
Non-cytotoxic
36.35 ꢁ 1.36
Non-cytotoxic
Non-cytotoxic
39.71 ꢁ 1.48
27.21 ꢁ 1.77
Non-cytotoxic
27
28
OMe
OMe
Non-cytotoxic
Non-cytotoxic
0.56 ꢁ 0.01
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
37.23
Non-cytotoxic
29
30
OMe
OMe
4.79 ꢁ 0.28
3.37 ꢁ 0.96
8.43 ꢁ 2.79
11.74 ꢁ 4.97
34.12 ꢁ 0.97
2.82
31.09 ꢁ 8.91
12.49 ꢁ 2.47
31.84 ꢁ 8.13
Non-cytotoxic
(continued on next page)

196
R. Pingaew et al. / European Journal of Medicinal Chemistry 81 (2014) 192e203
Table 1 (continued )
Compound
R1
R2
Cancer cell linesa
Vero cell line
Non-cytotoxic
HuCCA-1
HepG2
A549
MOLT-3
31
32
33
OMe
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
OMe
OMe
76.15 ꢁ 1.77
39.98 ꢁ 4.03
41.36 ꢁ 2.89
31.91 ꢁ 9.76
5.82 ꢁ 0.85
5.50 ꢁ 0.61
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
Non-cytotoxic
Etoposideb
Doxorubicinb
Ellipticineb
ND
30.16 ꢁ 0.50
0.79 ꢁ 0.08
ND
ND
0.051 ꢁ 0.002
ND
ND
1.94
0.83 ꢁ 0.07
0.44 ꢁ 0.01
ND
ND
ND
ND
Non-cytotoxic ¼ IC50 > 50
mg/mL.
Vero cell line ¼ African green monkey kidney cell line.
ND not determined.
a
Cancer cell lines comprise the following: HuCCA-1 human cholangiocarcinoma cell line, HepG2 human hepatocellular carcinoma cell line, A549 human lung carcinoma cell
line, MOLT-3 human lymphoblastic leukemia cell line.
b
Etoposide, doxorubicin and ellipticine were used as reference drugs.
remarkably enhanced effect (IC50 ¼ 6.50
m
M) in HepG2 cells.
activity than etoposide and doxorubicin, respectively). Obviously, it
was found that triazoles bearing the phenoxymethyl (24) moiety
and phenoxymethyl (26, 27, 29, 30) with substituents (CH3, CO2Me,
CHO) on the phenyl ring as well as naphthalenoxymethyl (25)
exhibited superior inhibitory potency toward HepG2 cells than that
of the etoposide. The order of decreasing cytotoxic potency of these
cytotoxic triazoles was shown by the following trend:
27 > 25 > 26 > 29 > 30. Inhibition effect in HepG2 cells of p-tol-
yloxymethyltriazole (27) was found to decrease when methyl
group on the phenyl ring was replaced with polar substituents (i.e.,
COMe, CO2Me, CHO and NO2). At this point, it was suggested that
lipophilic groups, thus seems to be important sites responsible for
strong cytotoxic activity as observed in HepG2 cells. Phenox-
ymethyl compounds constituting methyl ester (29) and formyl (30)
groups on the phenyl ring were found to be active towards all of the
tested cancer cell lines whereas compounds with acetylphenyl (28)
and nitrophenyl (31) moieties displayed no cytotoxic activity to-
ward all of the tested cells. Furthermore, additional introduction of
methoxyl to the phenyl moiety containing m- and p-formyl groups
as indicated by compounds 32 and 33 led to enhanced cytotoxic
activity against MOLT-3 cell line.
Interestingly, most triazole compounds were shown to be non-
cytotoxic toward normal cells except for triazole derivatives having
methyl esters (20, 29), coumaryl (22) and acetyl (28) substituents.
SAR analysis revealed that substituents (R1 and R2) on the target
triazoles play a crucial role in governing their anticancer activity.
The strongest activities against HuCCA-1 and A549 cells were noted
for compounds bearing polar substituents (R2) such as triazole ester
20 (R1 ¼ H, R2 ¼ C6H4eCO2Me-o). Promisingly, compounds having
high lipophilic substituents (R1 and R2), particularly p-tolyl triazole
27 (R1 ¼ OMe, R2 ¼ C6H4eMe-p) provided the most potent anti-
cancer activity against HepG2 cells without affecting the normal
cell line. It was conceivable that such difference in potency and
selectivity of compounds in exerting anticancer activity was
dependent upon the functionality of their R1 and R2 substituents.
The strongest activity (HuCCA-1 and A549 cells) may require the
compound (20) to have polar (CO) electrophilic center for
Replacement of the phenyl ring (R2) with the naphthalenyl ring as
observed in compound 16 afforded a decrease in the cytotoxic
potency against HepG2. o-Tolyloxymethyl analog (17) was shown to
be a selective cytotoxic compound against A549 cells. However,
isomeric effect of tolyl group on the cytotoxic activity was seen in
triazole 18 bearing p-tolyl group (R2) in which its activity was
totally lost. Significantly, phenyl groups (R2) possessing ortho-
substituents on the phenyl ring such as COMe (19) and CO2Me (20)
displayed cytotoxic activities toward all tested cancer cell lines in
which methylbenzoate (20) was shown to be the most potent
compound having comparable IC50 value (0.57
control drug, doxorubicin (IC50 ¼ 0.44 M), in A549 cells. It was also
found that the ester analog 20 was the most active compound
(IC50 ¼ 0.63 M) against HuCCA-1 cells and exerted higher activity
mM) with that of the
m
m
than that of the doxorubicin. Enhancement in anticancer activity of
the phenoxytriazole compound 15 (R2 ¼ C6H5) against HepG2 and
A549 cells was observed when a formyl group was introduced at
the para position of the phenyl moiety as noted for compound 21.
Apparently, the phenoxy compound (15) and its derivatives bearing
ketone (19), ester (20) and aldehyde (21) groups displayed high
cytotoxic activity against HepG2 cells as compared to the reference
drug, etoposide. When the phenyl group of compound 15 was
replaced with a coumaryl ring as found in compound 22, the
enhanced cytotoxic potency was observed against HuCCA-1, A549
and MOLT-3 cell lines whereas the activity was lost in HepG2 cells.
In a 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline series (23e
33, R1 ¼ OMe), the hydroxymethyltriazole (23) (R2 ¼ H) was shown
to be active against HuCCA-1, HepG2 and A549 cell lines. Such
cytotoxic activity in HuCCA-1 and A549 cells of compound 23 was
lost when the H atom was replaced with a phenyl group as seen for
compound 24. Cytotoxic activity against HepG2 cells was distinc-
tively enhanced when the H atom of 23 was substituted with
phenyl (24), naphthalenyl (25) and tolyl (26, 27) groups. Significant
results showed that p-tolyloxy compound (27) displayed selective
inhibition and exerted the highest cytotoxic activity against HepG2
cells with IC50 of 0.56
mM (i.e., 53.9-fold and 1.4-fold stronger

R. Pingaew et al. / European Journal of Medicinal Chemistry 81 (2014) 192e203
197
Table 2
interacting with cellular nucleophiles. For higher potency in HepG2
Binding energy of 1,2,3,4-tetrahydroisoquinoline-triazole derivatives (14e33).
cells, higher hydrophobic effect such as those afforded by the tolyl
group (27) may be involved in binding with the hydrophobic area of
the target site.
Compound
Binding energy
(Kcal/mol)
Compound
Binding energy
(kcal/mol)
14
15
16
17
18
19
20
21
22
23
8a
ꢀ11.1
ꢀ12.8
ꢀ14.5
ꢀ13.2
ꢀ10.8
ꢀ13.2
ꢀ13.1
ꢀ12.9
ꢀ14.5
ꢀ9.5
24
25
26
27
28
29
30
31
32
33
ꢀ10.1
ꢀ12.7
ꢀ11.7
ꢀ11.5
ꢀ10.0
ꢀ11.2
ꢀ11.3
ꢀ11.7
ꢀ11.5
ꢀ11.1
2.3. Molecular docking
A
search
for
the
putative
target
of
1,2,3,4-
tetrahydroisoquinoline-triazoles investigated herein was carried
out using the promising triazole analogs 20 and 27 as the query
molecules in PubChem. It was revealed that 4-phenylpyrrolidine-2-
one analog of 1,2,3,4-tetrahydroisoquinoline (7) provided nano-
molar potency against AKR1C3 with IC50 values of 42 and 52 nM as
elucidated by two separate bioactivity experiments [11,12]. Mo-
lecular docking was performed as to evaluate the binding modal-
ities of the 1,2,3,4-tetrahydroisoquinoline-triazoles against
ꢀ11.0
a
3-(3,4-Dihydroisoquinolin-2(1H)-ylsulfonyl)-N-methylbenzamide (8) is the co-
crystallized ligand found in the crystal structure of AKR1C3.
AKR1C3.
The
results
suggested
that
all
1,2,3,4-
Molecular modeling analysis of the crystal structure revealed
that the co-complexed inhibitor 8, binds the active site of AKR1C3
via hydrogen bonding of amide oxygen to Tyr55 and His117 while
the phenyl group engages in hydrophobic interaction with Leu54
tetrahydroisoquinoline-triazole analogs (14e33) could snugly
occupy the active site of AKR1C3 (Fig. 4) with binding energies in
the range of ꢀ9.5 to ꢀ14.5 kcal/mol (Table 2). It was observed that
many of the 1,2,3,4-tetrahydroisoquinoline-triazole analogs affor-
ded binding energy lower than the co-complexed ligand found in
the crystal structure, 3-(3,4-dihydroisoquinolin-2(1H)-ylsulfonyl)-
N-methylbenzamide 8, which provided binding energy
of ꢀ11.0 kcal/mol indicating possibly higher potency for AKR1C3
inhibition.
and forms
p
-
p
stacking with Trp227. The 1,2,3,4-
tetrahydroisoquinoline moiety of 8 occupies the hydrophobic
pocket inside the active site bounded by Met120, Phe306, Phe311
and Tyr317 residues (Fig. 5A). The triazole 20 could plausibly bind
the active site of AKR1C3 with a similar modality as deduced from
its
docked
conformer,
particularly
the
1,2,3,4-
tetrahydroisoquinoline moiety of compound 20 sits inside the
hydrophobic pocket formed by Met120, Phe311 and Phe306 res-
idues whereas the phenyl ring (next to the sulfonyl group) is
located in a pocket defined by Phe306 and Tyr216 residues. It was
observed that the methoxy group of ester (R2) at the other end of
the compound is responsible for hydrogen bond formation with
Tyr55. Moreover, compound 20 was found to also engage in
hydrogen bonding with Gln222 (using phenoxy O-atom),
p-
stacking with Tyr24 (using the triazole ring) and hydrophobic
interaction with Leu268 and Lys270 (using phenyl ring of R2
substituent) as shown in Fig. 5B. Interestingly, compound 27,
which exerts specific activity towards the HepG2 cell line, was
shown to interact with AKR1C3 through a different mechanism
(Fig. 5C). Particularly, the methoxy substituent at the R1 position
sterically hinders the 1,2,3,4-tetrahydroisoquinoline moiety in
occupying the hydrophobic pocket as defined by Met120, Phe306
and Phe311. However, the p-tolyl substituent at the R2 position
counters the previously mentioned moiety by allowing the ligand
to occupy the aforementioned pocket. This instance enabled the
triazole ring and the adjacent phenyl ring of sulfonamide 27 to
form p-p stacking with Phe306 and Tyr216. Such interaction was
strengthen by a hydrogen bonding network formed between the
sulfonyl oxygen and Tyr55 as well as the between the oxygen
atom of two methoxy substituents at R1 positions to Lys270 and
Ser221.
Taken together, the docking results support the viewpoint that
AKR1C3 is a plausible target of 1,2,3,4-tetrahydroisoquinoline-tri-
azoles for which it was shown to interact strongly. It is notable that
substituents (R1 and R2) on the triazoles (14e33) are crucial for
binding the active site of AKR1C3. Most THIQs without 6,7-
dimethoxy substituents (R1 ¼ H) had lower binding energy than
the corresponding compounds with 6,7-dimethoxy substituents
(R1 ¼ OMe). However, the lowest binding energy (ꢀ14.5 kcal/mol)
was observed for compounds having more bulky planar lipophilic
groups (R2) such as those found in naphthalenyl (16) and coumaryl
(22). Such results could be reasonably explained that the chemical
properties of R1 and R2 governed the compounds in interacting or
fitting inside the hydrophobic pocket areas of AKR1C3.
Fig. 4. Molecular docking of tetrahydroisoquinoline-triazole derivatives to AKR1C3.
Re-docking of the co-crystallized ligand 3-(3,4-dihydroisoquinolin-2(1H)-ylsulfonyl)-
ꢀ
N-methylbenzamide
8
yielded RMSD of 0.692
A
(A). Poses of docked
tetrahydroisoquinoline-triazole derivatives are shown inside the binding cavity (B).

198
R. Pingaew et al. / European Journal of Medicinal Chemistry 81 (2014) 192e203
Fig. 5. 2D ligandeprotein interaction schemes of the co-crystallized ligand 3-(3,4-dihydroisoquinolin-2(1H)-ylsulfonyl)-N-methylbenzamide 8 (A), the docked pose of compound
20 (B) and the docked pose of compound 27 (C).
3. Conclusions
compound against HuCCA-1 (IC50
¼
0.63 mM) and A549
(IC50 ¼ 0.57
mM) cell lines. The triazole 27 containing 6,7-
A novel series of tetrahydroisoquinoline-triazole hybrids (14e
33) have been synthesized via two key steps using the modified
PicteteSpengler reaction and the click reaction. Their anti-
proliferative activity against four cancer (HuCCA-1, HepG2, A549
and MOLT-3) cell lines and molecular docking with AKR1C3 were
studied. It was observed that triazole esters (20 and 29) with
R2 ¼ methylbenzoate were active toward all of the tested cell lines
in which the ester analog 20 was shown to be the most potent
dimethoxy substituent (R1) on the isoquinoline core and p-tolyl
group (R2) on the triazole part was a promising compound showing
the strongest and selective cytotoxicity in HepG2 cells (IC50
0.56 mM) without affecting the normal cell line. The potent and
selective action of compounds may be a direct function of their
substituents at R1 and R2 positions. Activities against HuCCA-1 and
A549 cells may require the compound (20) to have polar electro-
philic center in interacting with cellular nucleophiles. The potent

R. Pingaew et al. / European Journal of Medicinal Chemistry 81 (2014) 192e203
199
activity against HepG2 cells may be rationalized to be due to the
4.3.2. 2-((4-Aminophenyl)sulfonyl)-6,7-dimethoxy-1,2,3,4-
tetrahydroisoquinoline (36b)
Pale yellow solid. 80%. mp 185e186 ꢃC. IR (UATR) cmꢀ1: 3468,
3372, 1596, 1518, 1316, 1153. 1H NMR (300 MHz, DMSO-d6)
2.75 (t,
hydrophobic group (27) in which it binds the hydrophobic region of
the target site of action. Furthermore, molecular docking study
indicated that all of the investigated triazoles could snugly occupy
the active site of AKR1C3, which had been identified to be a plau-
sible target site of such compounds.
d
J ¼ 5.9 Hz, 2H, C4eH), 3.12 (t, J ¼ 5.9 Hz, 2H, C3eH), 3.68 (s, 6H,
2 ꢂ OCH3), 3.92 (s, 2H, C1eH), 6.65 (d, J ¼ 8.4 Hz, 2H, ArH), 6.66 (s,
1H, ArH), 6.73 (s, 1H, ArH), 7.42 (d, J ¼ 8.4 Hz, 2H, ArH). 13C NMR
(75 MHz, DMSO-d6) d 28.2, 44.2, 47.6, 55.9, 110.3, 112.1, 113.2, 120.4,
123.9, 125.2, 130.0, 147.7, 147.9, 153.6. HRMS-TOF: m/z [MþNa]þ
4. Experimental section
371.1035 (Calcd for C17H20N2NaO4S: 371.1036).
4.1. Chemistry
4.4. General procedure for the synthesis of 2-((4-azidophenyl)
sulfonyl)-1,2,3,4-tetrahydroisoquinolines (37)
Column chromatography was carried out using silica gel 60
(70e230 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 multi-
plicities: singlet (s), doublet (d), triplet (t), quartet (q), multiplet
(m), and broad (br). FTIR spectra were obtained using a universal
attenuated total reflectance attached on a PerkineElmer Spectrum
One spectrometer. Mass spectra were recorded on a Bruker Dal-
tonics (microTOF). Melting points were determined using a Griffin
melting point apparatus and were uncorrected.
To a cold solution of amine 36 (3 mmol) in HCl:CH3COOH
(3:3 mL) at 0 ꢃC, 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 purified using silica
gel column chromatography and eluted with acetone:hexane (2:8).
4.4.1. 2-((4-Azidophenyl)sulfonyl)-1,2,3,4-tetrahydroisoquinoline
(37a)
4.2. General procedure for the synthesis of 1,2,3,4-
tetrahydroisoquinolines (35)
Pale yellow solid. 94%. mp 126e127 ꢃC. IR (UATR) cmꢀ1: 2129,
2099, 1589, 1489, 1337, 1287, 1163. 1H NMR (300 MHz, CDCl3)
d 2.90
(t, J ¼ 6.0 Hz, 2H, C4eH), 3.36 (t, J ¼ 6.0 Hz, 2H, C3eH), 4.25 (s, 2H,
A
mixture of sulfonamide 34 (0.67 mmol) and para-
C1eH), 6.96e7.18 (m, 6H, ArH), 7.80 (d, J ¼ 8.7 Hz, 2H, ArH). 13C NMR
formaldehyde (0.72 mmol) in formic acid (15 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 CH2Cl2 (2 ꢂ 30 mL). Combined extracts were
washed with saturated aqueous NaHCO3, dried (anh. Na2SO4) and
evaporated to dryness under reduced pressure. The crude product
was recrystallized from methanol.
(75 MHz, CDCl3) d 28.8, 43.7, 47.5, 119.4, 126.3, 126.4, 126.9, 128.8,
129.6, 131.4, 132.9, 133.0, 144.9. HRMS-TOF: m/z [MþNa]þ 337.0726
(Calcd for C15H14N4NaO2S: 337.0730).
4.4.2. 2-((4-Azidophenyl)sulfonyl)-6,7-dimethoxy-1,2,3,4-
tetrahydroisoquinoline (37b)
Pale yellow solid. 85%. mp 145e146 ꢃC. IR (UATR) cmꢀ1: 2126,
1H
tetrahydroisoquinoline
nitrophenyl)sulfonyl)-1,2,3,4-tetrahydroisoquinoline (35b) were
NMR
of
2-((4-nitrophenyl)sulfonyl)-1,2,3,4-
(35a) and 6,7-dimethoxy-2-((4-
2100, 1588, 1519, 1345, 1284, 1163. 1H NMR (300 MHz, CDCl3)
d 2.81
(t, J ¼ 5.9 Hz, 2H, C4eH), 3.33 (t, J ¼ 5.9 Hz, 2H, C3eH), 3.80 (s, 6H,
2 ꢂ OCH3), 4.17 (s, 2H, C1eH), 6.48 (s, 1H, ArH), 6.53 (s, 1H, ArH),
7.12 (d, J ¼ 8.7 Hz, 2H, ArH), 7.79 (d, J ¼ 8.7 Hz, 2H, ArH). 13C NMR
consistent with those reported in the literatures [21,22].
(75 MHz, CDCl3)
d 28.3, 43.7, 47.2, 55.9, 56.0, 108.9, 111.4, 119.4,
123.2, 124.9, 129.5, 132.8, 144.9, 147.8, 148.0. HRMS-TOF: m/z
4.3. General procedure for the synthesis of 1,2,3,4-
tetrahydroisoquinolines (36)
[MþNa]þ 397.0946 (Calcd for C17H18N4NaO4S: 397.0941).
4.5. General procedure for the synthesis of propynyloxy derivatives
(38)
A mixture of nitroisoquinoline 35 (4 mmol) and SnCl2$2H2O
(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 anhydrous sodium sulfate, filtered
and concentrated. The crude product was purified using silica gel
column chromatography and eluted with acetone:hexane (3:7).
A propargyl bromide (2.4 mmol) was added to a suspension of
an appropriate phenol (2 mmol) and potassium carbonate (4 mmol)
in acetone (15 mL). The suspension was heated to reflux for 2e6 h
(monitored by TLC). The reaction was allowed to cool and then
concentrated under reduced pressure. Water (30 mL) was added
and extracted with CH2Cl2 (3 ꢂ 30 mL). The organic extracts were
combined and washed with water (20 mL) and brine (20 mL). The
organic layer was dried over anhydrous sodium sulfate, filtered and
concentrated. The crude product was purified by column
chromatography.
4.3.1. 2-((4-Aminophenyl)sulfonyl)-1,2,3,4-tetrahydroisoquinoline
(36a)
Pale yellow solid. 82%. mp 175e176 ꢃC (175.0e175.5 ꢃC [23]). IR
(UATR) cmꢀ1
:
3454, 3362, 1591, 1503, 1324, 1155. 1H NMR
1H NMR spectra of propynyloxy derivatives (38) including (2-
propynyloxy)benzene [24], 2-(2-propynyloxy)naphthalene [25], 1-
(300 MHz, DMSO-d6)
d
2.84 (t, J ¼ 5.8 Hz, 2H, C4eH), 3.16 (t,
J ¼ 5.8 Hz, 2H, C3eH), 4.06 (s, 2H, C1eH), 6.08 (s, 2H, NH2), 6.64 (d,
J ¼ 8.7 Hz, 2H, ArH), 7.05e7.18 (m, 4H, ArH), 7.44 (d, J ¼ 8.7 Hz, 2H,
methyl-2-(2-propynyloxy)benzene
[26],
1-methyl-4-(2-
propynyloxy)benzene [27], 1-(2-(prop-2-ynyloxy)phenyl)ethanone
[28], methyl 2-(2-propynyloxy)benzoate [29], 4-(2-propynyloxy)
benzaldehyde [28], 4-(propynyloxy)-2H-chromen-2-one [30], 1-
ArH). 13C NMR (75 MHz, DMSO-d6)
d 28.1, 43.6, 47.4, 112.7, 120.0,
126.0, 126.4, 126.5, 128.6, 129.5, 131.9, 133.1, 153.2. HRMS-TOF: m/z
[MþNa]þ 311.0829 (Calcd for C15H16N2NaO2S: 311.0825).
nitro-4-(2-propynyloxy)benzene
[25],
4-methoxy-3-(2-

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R. Pingaew et al. / European Journal of Medicinal Chemistry 81 (2014) 192e203
propynyloxy)benzaldehyde [31] and 3-methoxy-4-(2-propynyloxy)
[31] were consistent with those reported in the literatures.
4.6.5. 2-((4-(4-((p-Tolyloxy)methyl)-1H-1,2,3-triazol-1-yl)phenyl)
sulfonyl)-1,2,3,4-tetrahydroisoquinoline (18)
White solid. 88%. mp 196e197 ꢃC. IR (UATR) cmꢀ1: 1598, 1512,
1338, 1244, 1162. 1H NMR (300 MHz, CDCl3)
d 2.27 (s, 3H, CH3), 2.92
4.6. General procedure for the synthesis of 4-(4-(substituted)-1H-
1,2,3-triazol-1-yl)-N-phenethylbenzenesulfonamides (14e33)
(t, J ¼ 6.0 Hz, 2H, C4eH), 3.43 (t, J ¼ 6.0 Hz, 2H, C3eH), 4.32 (s, 2H,
C1eH), 5.26 (s, 2H, CH2O), 6.89 (d, J ¼ 8.5 Hz, 2H, ArH), 7.00e7.16
(m, 6H, ArH), 7.90 (d, J ¼ 8.8 Hz, 2H, ArH), 7.98 (d, J ¼ 8.8 Hz, 2H,
To a stirred solution of azido 37 (0.2 mmol) and alkyne 38
(0.2 mmol) in t-BuOH:H2O (3:3 mL), CuSO4$5H2O (0.2 mmol) and
ascorbic acid (0.5 mmol) were added. The reaction mixture was
stirred at room temperature for 2e12 h (monitored by TLC), then
concentrated under reduced pressure. The residue was added wa-
ter (10 mL) and extracted with dichloromethane (3 ꢂ 20 mL). The
combined organic phases were washed with water (20 mL), dried
over anhydrous sodium sulfate and evaporated to dryness. The
crude product was purified using silica gel column chromatography
and eluted with methanol:dichloromethane (1:50).
ArH), 8.09 (s, 1H, CHN). 13C NMR (75 MHz, CDCl3)
d 20.5, 28.7, 43.7,
47.5, 62.1, 114.6, 120.5, 126.3, 126.5, 126.6, 128.9, 129.4, 130.1, 130.9,
131.2, 132.9, 137.1, 139.9, 146.1, 155.9. HRMS-TOF: m/z [MþH]þ
461.1646 (Calcd for C25H25N4O3S: 461.1653).
4.6.6. 1-(2-((1-(4-((3,4-Dihydroisoquinolin-2(1H)-yl)sulfonyl)
phenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)ethanone (19)
White solid. 78%. mp 185e186 ꢃC. IR (UATR) cmꢀ1: 1655, 1595,
1450, 1358, 1161. 1H NMR (300 MHz, CDCl3)
d 2.58 (s, 3H, COCH3),
2.91 (t, J ¼ 6.0 Hz, 2H, C4eH), 3.42 (t, J ¼ 6.0 Hz, 2H, C3eH), 4.31 (s,
2H, C1eH), 5.38 (s, 2H, CH2O), 6.99e7.15 (m, 6H, ArH), 7.46 (dt,
J ¼ 7.7, 1.7 Hz, 1H, ArH), 7.69 (dd, J ¼ 7.7, 1.7 Hz, 1H, ArH), 7.92 (d,
J ¼ 8.8 Hz, 2H, ArH), 7.99 (d, J ¼ 8.8 Hz, 2H, ArH), 8.15 (s, 1H, CHN).
4.6.1. (1-(4-((3,4-Dihydroisoquinolin-2(1H)-yl)sulfonyl)phenyl)-
1H-1,2,3-triazol-4-yl)methanol (14)
White solid. 45%. mp 111e112 ꢃC. IR (UATR) cmꢀ1: 3280, 1597,
13C NMR (75 MHz, CDCl3)
d 28.7, 31.7, 43.7, 47.5, 62.4, 113.1, 120.7,
1503, 1336, 1243, 1161. 1H NMR (300 MHz, DMSO-d6)
d 2.87 (br t,
120.8, 121.5, 126.3, 126.5, 127.0, 128.9, 129.0, 129.5, 130.5, 131.2,
132.9, 133.6, 137.2, 139.7, 145.1, 157.0, 199.7. HRMS-TOF: m/z
[MþNa]þ 511.1391 (Calcd for C26H24N4NaO4S: 511.1410).
2H, C4eH), 3.35 (br t, 2H, C3eH), 4.27 (s, 2H, C1eH), 4.62 (d,
J ¼ 5.4 Hz, 2H, CH2OH), 5.41 (t, J ¼ 5.4 Hz,1H, CH2OH), 7.05e7.20 (m,
4H, ArH), 8.03 (d, J ¼ 8.7 Hz, 2H, ArH), 7.97 (d, J ¼ 8.6 Hz, 2H, ArH),
8.85 (s, 1H, CHN). 13C NMR (75 MHz, DMSO-d6)
d 28.4, 44.0, 47.7,
55.4, 120.8, 121.7, 126.6, 126.9, 127.2, 129.1, 129.8, 132.0, 133.4, 136.0,
4.6.7. Methyl 2-((1-(4-((3,4-dihydroisoquinolin-2(1H)-yl)sulfonyl)
phenyl)-1H-1,2,3-triazol-4-yl)methoxy)benzoate (20)
140.2, 150.1. HRMS-TOF: m/z [MþNa]þ 393.0988 (Calcd for
White solid. 91%. mp 162e163 ꢃC. IR (UATR) cmꢀ1: 1696, 1599,
C
18H18N4NaO3S: 393.0992).
1453, 1306, 1254, 1160. 1H NMR (300 MHz, CDCl3)
d 2.92 (t,
J ¼ 6.0 Hz, 2H, C4eH), 3.43 (t, J ¼ 6.0 Hz, 2H, C3eH), 3.88 (s, 3H,
CO2CH3), 4.32 (s, 2H, C1eH), 5.39 (s, 2H, CH2O), 7.00e7.18 (m, 6H,
ArH), 7.48 (dt, J ¼ 7.8, 1.7 Hz, 1H, ArH), 7.82 (dd, J ¼ 7.8, 1.7 Hz, 1H,
ArH), 7.93 (d, J ¼ 8.9 Hz, 2H, ArH), 7.99 (d, J ¼ 8.9 Hz, 2H, ArH), 8.31
4.6.2. 2-((4-(4-(Phenoxymethyl)-1H-1,2,3-triazol-1-yl)phenyl)
sulfonyl)-1,2,3,4-tetrahydroisoquinoline (15)
White solid. 84%. mp 186e187 ꢃC. IR (UATR) cmꢀ1: 1598, 1497,
1337, 1243, 1162. 1H NMR (300 MHz, CDCl3)
d
2.92 (t, J ¼ 6.0 Hz, 2H,
(s, 1H, CHN). 13C NMR (75 MHz, CDCl3)
d 28.7, 43.7, 47.5, 52.0, 63.4,
C4eH), 3.43 (t, J ¼ 6.0 Hz, 2H, C3eH), 4.32 (s, 2H, C1eH), 5.29 (s, 2H,
CH2O), 6.94e7.33 (m, 9H, ArH), 7.91 (d, J ¼ 8.8 Hz, 2H, ArH), 7.98 (d,
J ¼ 8.8 Hz, 2H, ArH), 8.10 (s, 1H, CHN). 13C NMR (75 MHz, CDCl3)
114.1, 120.6, 121.0, 121.3, 126.3, 126.5, 127.0, 128.9, 129.4, 131.2, 131.8,
132.9, 133.8, 137.0, 139.9, 146.0, 157.8, 166.1. HRMS-TOF: m/z
[MþNa]þ 527.1365 (Calcd for C26H24N4NaO5S: 527.1360).
d
28.7, 43.7, 47.5, 61.9, 114.7, 120.6, 121.5, 126.3, 126.5, 127.0, 128.9,
129.4, 129.7, 131.2, 132.9, 137.1, 139.9, 145.9, 158.0. HRMS-TOF: m/z
[MþNa]þ 469.1292 (Calcd for C24H22N4NaO3S: 469.1305).
4.6.8. 4-((1-(4-((3,4-dihydroisoquinolin-2(1H)-yl)sulfonyl)phenyl)-
1H-1,2,3-triazol-4-yl)methoxy)benzaldehyde (21)
White solid. 81%. mp 189e190 ꢃC. IR (UATR) cmꢀ1: 1686, 1605,
4.6.3. 2-((4-(4-((Naphthalen-2-yloxy)methyl)-1H-1,2,3-triazol-1-
yl)phenyl)sulfonyl)-1,2,3,4-tetrahydroisoquinoline (16)
1578, 1500, 1357, 1243, 1160. 1H NMR (300 MHz, CDCl3)
d 2.92 (t,
White solid. 52%. mp 241e242 ꢃC. IR (UATR) cmꢀ1: 1597, 1508,
J ¼ 5.9 Hz, 2H, C4eH), 3.43 (t, J ¼ 5.9 Hz, 2H, C3eH), 4.32 (s, 2H, C1e
H), 5.37 (s, 2H, CH2O), 7.00e7.18 (m, 6H, ArH), 7.84 (d, J ¼ 8.8 Hz, 2H,
ArH), 7.92 (d, J ¼ 8.8 Hz, 2H, ArH), 7.99 (d, J ¼ 8.8 Hz, 2H, ArH), 8.14
1337, 1243, 1161. 1H NMR (300 MHz, CDCl3)
d
2.91 (t, J ¼ 5.6 Hz, 2H,
C4eH), 3.43 (t, J ¼ 5.8 Hz, 2H, C3eH), 4.32 (s, 2H, C1eH), 5.26 (s, 2H,
CH2O), 6.85e7.18 (m, 11H, ArH), 7.91 (d, J ¼ 8.6 Hz, 2H, ArH), 7.98 (d,
J ¼ 8.6 Hz, 2H, ArH), 8.09 (s, 1H, CHN). 13C NMR (75 MHz, CDCl3)
(s,1H, CHN), 9.88 (s, 3H, CHO). 13C NMR (75 MHz, CDCl3)
d 28.7, 43.7,
47.5, 62.0, 115.1, 120.7, 120.9, 126.3, 126.5, 127.0, 128.9, 129.5, 130.6,
131.2, 132.2, 132.8, 137.4, 139.7, 144.8, 162.9, 190.7. HRMS-TOF: m/z
[MþH]þ 475.1450 (Calcd for C25H23N4O4S: 475.1446).
d
28.7, 43.7, 47.5, 62.1, 114.6, 120.6, 126.3, 126.5, 127.0, 128.9, 129.4,
130.1, 130.9, 131.2, 132.9, 137.2, 139.9, 146.1, 156.0. HRMS-TOF: m/z
[MþH]þ 497.1647 (Calcd for C28H25N4O3S: 497.1653).
4.6.9. 4-((1-(4-((3,4-Dihydroisoquinolin-2(1H)-yl)sulfonyl)
phenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (22)
White solid. 94%. mp 150e151 ꢃC. IR (UATR) cmꢀ1: 1687, 1618,
4.6.4. 2-((4-(4-((o-Tolyloxy)methyl)-1H-1,2,3-triazol-1-yl)phenyl)
sulfonyl)-1,2,3,4-tetrahydroisoquinoline (17)
White solid. 77%. mp 168e169 ꢃC. IR (UATR) cmꢀ1: 1596, 1495,
1456, 1354, 1248, 1161. 1H NMR (300 MHz, CDCl3)
d 2.96 (t,
1339, 1240, 1164. 1H NMR (300 MHz, CDCl3)
d
2.28 (s, 3H, CH3), 2.96
J ¼ 5.9 Hz, 2H, C4eH), 3.47 (t, J ¼ 5.9 Hz, 2H, C3eH), 4.36 (s, 2H, C1e
H), 5.46 (s, 2H, CH2O), 5.92 (s, 1H, CHCO), 7.04e7.36 (m, 6H, ArH),
7.57 (dt, J ¼ 7.7, 1.4 Hz, 1H, ArH), 7.82 (dd, J ¼ 7.9, 1.4 Hz, 1H, ArH),
7.99 (d, J ¼ 8.7 Hz, 2H, ArH), 8.05 (d, J ¼ 8.7 Hz, 2H, ArH), 8.27 (s, 1H,
(t, J ¼ 5.9 Hz, 2H, C4eH), 3.47 (t, J ¼ 5.9 Hz, 2H, C3eH), 4.36 (s, 2H,
C1eH), 5.34 (s, 2H, CH2O), 6.90e7.24 (m, 8H, ArH), 7.95 (d,
J ¼ 8.9 Hz, 2H, ArH), 8.03 (d, J ¼ 8.8 Hz, 2H, ArH), 8.11 (s, 1H, CHN).
13C NMR (75 MHz, CDCl3)
d
16.3, 28.7, 43.7, 47.5, 62.2, 111.5, 120.4,
CHN). 13C NMR (75 MHz, CDCl3)
d 28.7, 43.7, 47.5, 62.4, 91.4, 115.4,
120.6, 121.3, 126.3, 126.5, 127.0, 128.9, 129.4, 131.0, 131.2, 132.9,
116.8, 120.8, 121.5, 123.1, 124.0, 126.3, 126.6, 127.0, 128.9, 129.5,
131.2, 132.7, 132.8, 139.6, 142.9, 153.4, 162.4, 164.8. HRMS-TOF: m/z
[MþH]þ 515.1384 (Calcd for C27H23N4O5S: 515.1384).
139.9, 146.3, 156.2. HRMS-TOF: m/z [MþH]þ 461.1637 (Calcd for
C
25H25N4O3S: 461.1653).

R. Pingaew et al. / European Journal of Medicinal Chemistry 81 (2014) 192e203
201
4.6.10. (1-(4-((6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)
sulfonyl)phenyl)-1H-1,2,3-triazol-4-yl)methanol (23)
43.8, 47.2, 55.9, 56.0, 62.0, 109.0, 111.4, 114.6, 120.6, 123.0, 129.4,
130.1, 130.9, 137.2, 139.9, 146.1, 147.9, 148.1, 155.9. HRMS-TOF: m/z
[MþH]þ 521.1842 (Calcd for C27H29N4O5S: 521.1853).
Pale yellow solid. 58%. mp 159e160 ꢃC. IR (UATR) cmꢀ1: 3280,
1599,1523,1342,1226, 1158. 1H NMR (300 MHz, DMSO-d6)
d 2.78 (t,
J ¼ 5.5 Hz, 2H, C4eH), 3.33 (t, J ¼ 5.8 Hz, 2H, C3eH), 3.67, 3.68 (2s,
6H, 2 ꢂ OCH3), 4.16 (s, 2H, C1eH), 4.62 (d, J ¼ 5.5 Hz, 2H, CH2OH),
5.41 (t, J ¼ 5.4 Hz, 1H, CH2OH), 6.67 (s, 1H, ArH), 6.76 (s, 1H, ArH),
7.99 (d, J ¼ 8.8 Hz, 2H, ArH), 8.19 (d, J ¼ 8.8 Hz, 2H, ArH), 8.83 (s, 1H,
4.6.15. 1-(2-((1-(4-((6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-
yl)sulfonyl)phenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)ethanone
(28)
White solid. 70%. mp 171e172 ꢃC. IR (UATR) cmꢀ1: 1670, 1596,
CHN). 13C NMR (75 MHz, DMSO-d6)
d 28.0, 44.1, 47.4, 55.4, 55.9,
1519, 1450, 1347, 1226, 1161. 1H NMR (300 MHz, CDCl3)
d 2.58 (s, 3H,
56.0, 110.3, 112.3, 120.8, 121.7, 123.6, 125.2, 129.8, 136.0, 140.2, 147.8,
COCH3), 2.81 (t, J ¼ 5.7 Hz, 2H, C4eH), 3.39 (t, J ¼ 5.7 Hz, 2H, C3eH),
3.79, 3.80 (2s, 6H, 2 ꢂ OCH3), 4.23 (s, 2H, C1eH), 5.38 (s, 2H, CH2O),
6.49 (s, 1H, ArH), 6.52 (s, 1H, ArH), 7.03 (t, J ¼ 8.0 Hz, 1H, ArH), 7.12
(d, J ¼ 8.2 Hz, 1H, ArH), 7.46 (dt, J ¼ 8.2, 1.8 Hz, 1H, ArH), 7.69 (dd,
J ¼ 7.7, 1.8 Hz, 1H, ArH), 7.91 (d, J ¼ 8.9 Hz, 2H, ArH), 7.98 (d,
J ¼ 8.9 Hz, 2H, ArH), 8.15 (s, 1H, CHN). 13C NMR (75 MHz, CDCl3)
148.1, 150.1. HRMS-TOF: m/z [MþH]þ 431.1389 (Calcd for
C
20H23N4O5S: 431.1384).
4.6.11. 6,7-Dimethoxy-2-((4-(4-(phenoxymethyl)-1H-1,2,3-triazol-
1-yl)phenyl)sulfonyl)-1,2,3,4-tetrahydroisoquinoline (24)
White solid. 91%. mp 158e159 ꢃC. IR (UATR) cmꢀ1: 1597, 1519,
d
28.3, 31.7, 43.8, 47.2, 55.9, 56.0, 62.4,108.9,111.4,113.1,120.7,120.8,
1348, 1227, 1163. 1H NMR (300 MHz, CDCl3)
d
2.82 (t, J ¼ 6.0 Hz, 2H,
121.5,122.9,124.8,129.0,129.5,130.5,133.6,137.3,139.7,145.1,147.9,
C4eH), 3.40 (t, J ¼ 6.0 Hz, 2H, C3eH), 3.80 (s, 6H, 2 ꢂ OCH3), 4.24 (s,
2H, C1eH), 5.29 (s, 2H, CH2O), 6.49 (s, 1H, ArH), 6.53 (s, 1H, ArH),
6.94e7.02 (m, 3H, ArH), 7.24e7.34 (m, 2H, ArH), 7.91 (d, J ¼ 8.8 Hz,
2H, ArH), 7.98 (d, J ¼ 8.8 Hz, 2H, ArH), 8.10 (s, 1H, CHN). 13C NMR
148.1, 157.0, 199.7. HRMS-TOF: m/z [MþNa]þ 571.1608 (Calcd for
C28H28N4NaO6S: 571.1622).
(75 MHz, CDCl3)
d
28.2, 43.8, 47.2, 55.9, 56.0, 61.9, 109.0, 111.4, 114.7,
4.6.16. Methyl 2-((1-(4-((6,7-dimethoxy-3,4-dihydroisoquinolin-
2(1H)-yl)sulfonyl)phenyl)-1H-1,2,3-triazol-4-yl)methoxy)benzoate
(29)
120.6, 121.6, 123.0, 124.8, 129.4, 129.7, 137.2, 139.8, 145.9, 147.9,
148.1, 158.0. HRMS-TOF: m/z [MþH]þ 507.1706 (Calcd for
C
26H27N4O5S: 507.1697).
White solid. 94%. mp 142e143 ꢃC. IR (UATR) cmꢀ1: 1723, 1598,
1519, 1451, 1347, 1258, 1162. 1H NMR (300 MHz, CDCl3)
d 2.83 (t,
4.6.12. 6,7-Dimethoxy-2-((4-(4-((naphthalen-2-yloxy)methyl)-1H-
1,2,3-triazol-1-yl)phenyl)sulfonyl)-1,2,3,4-tetrahydroisoquinoline
(25)
J ¼ 5.8 Hz, 2H, C4eH), 3.40 (t, J ¼ 5.8 Hz, 2H, C3eH), 3.80 (s, 6H,
2 ꢂ OCH3), 3.87 (s, 3H, CO2CH3), 4.24 (s, 2H, C1eH), 5.38 (s, 2H,
CH2O), 6.50 (s, 1H, ArH), 6.53 (s, 1H, ArH), 7.02 (t, J ¼ 8.2 Hz, 1H,
ArH), 7.12 (d, J ¼ 8.3 Hz, 1H, ArH), 7.48 (dt, J ¼ 8.2, 1.8 Hz, 1H, ArH),
7.82 (dd, J ¼ 7.8,1.7 Hz,1H, ArH), 7.94 (d, J ¼ 8.9 Hz, 2H, ArH), 7.99 (d,
J ¼ 8.9 Hz, 2H, ArH), 8.31 (s, 1H, CHN). 13C NMR (75 MHz, CDCl3)
Pale yellow solid. 83%. mp 210e211 ꢃC. IR (UATR) cmꢀ1: 1598,
1519, 1463, 1347, 1257, 1163. 1H NMR (300 MHz, CDCl3)
d 2.86 (t,
J ¼ 5.7 Hz, 2H, C4eH), 3.43 (t, J ¼ 5.7 Hz, 2H, C3eH), 3.83, 3.84
(2s, 6H, 2 ꢂ OCH3), 4.27 (s, 2H, C1eH), 5.45 (s, 2H, CH2O), 6.53
(s, 1H, ArH), 6.56 (s, 1H, ArH), 7.22 (dd, J ¼ 8.9, 2.5 Hz, 1H, ArH),
7.31 (d, J ¼ 2.4 Hz, 1H, ArH), 7.38 (t, J ¼ 8.1 Hz, 1H, ArH), 7.47 (t,
J ¼ 8.1 Hz, 1H, ArH), 7.75e7.82 (m, 3H, ArH), 7.95 (d, J ¼ 8.9 Hz,
2H, ArH), 8.01 (d, J ¼ 8.9 Hz, 2H, ArH), 8.17 (s, 1H, CHN). 13C NMR
d
28.3, 43.8, 47.2, 52.0, 55.9, 56.0, 63.4, 108.9, 111.4, 114.0, 120.5,
120.6, 121.0, 121.3, 123.0, 124.8, 129.4, 131.9, 133.8, 137.0, 139.9,
146.0, 147.8, 148.0, 157.8, 166.1. HRMS-TOF: m/z [MþH]þ 565.1737
(Calcd for C28H29N4O7S: 565.1752).
(75 MHz, CDCl3)
d 28.2, 43.8, 47.2, 55.9, 56.0, 61.9, 107.3, 108.9,
4.6.17. 4-((1-(4-((6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)
sulfonyl)phenyl)-1H-1,2,3-triazol-4-yl)methoxy)benzaldehyde (30)
White solid. 80%. mp 186e187 ꢃC. IR (UATR) cmꢀ1: 1692, 1604,
111.4, 118.6, 120.6, 120.7, 123.0, 124.1, 124.8, 126.6, 126.9, 127.7,
129.3, 129.4, 129.7, 134.4, 137.2, 139.8, 145.8, 147.9, 148.1, 155.9.
HRMS-TOF: m/z [MþH]þ 557.1843 (Calcd for C30H29N4O5S:
557.1853).
1519, 1348, 1231, 1162. 1H NMR (300 MHz, CDCl3)
d 2.86 (t,
J ¼ 5.8 Hz, 2H, C4eH), 3.45 (t, J ¼ 5.8 Hz, 2H, C3eH), 3.84 (s, 6H,
2 ꢂ OCH3), 4.29 (s, 2H, C1eH), 5.42 (s, 2H, CH2O), 6.53 (s, 1H, ArH),
6.56 (s, 1H, ArH), 7.15 (d, J ¼ 8.7 Hz, 2H, ArH), 7.88 (d, J ¼ 8.7 Hz, 2H,
ArH), 7.95 (d, J ¼ 8.8 Hz, 2H, ArH), 8.04 (d, J ¼ 8.8 Hz, 2H, ArH), 8.17
4.6.13. 6,7-Dimethoxy-2-((4-(4-((o-tolyloxy)methyl)-1H-1,2,3-
triazol-1-yl)phenyl)sulfonyl)-1,2,3,4-tetrahydroisoquinoline (26)
White solid. 87%. mp 138e139 ꢃC. IR (UATR) cmꢀ1: 1596, 1519,
(s,1H, CHN), 9.92 (s,1H, CHO). 13C NMR (75 MHz, CDCl3)
d 28.2, 43.8,
1463,1346, 1237,1160. 1H NMR (300 MHz, CDCl3)
d 2.28 (s, 3H, CH3),
47.2, 55.9, 56.0, 62.0, 109.0, 111.5, 115.1, 120.6, 120.9, 123.0, 124.8,
129.5, 130.6, 132.1, 137.5, 139.7, 144.8, 147.9, 148.1, 162.9, 190.6.
HRMS-TOF: m/z [MþH]þ 535.1645 (Calcd for C27H27N4O6S:
535.1646).
2.86 (t, J ¼ 5.9 Hz, 2H, C4eH), 3.44 (t, J ¼ 5.9 Hz, 2H, C3eH), 3.84 (s,
6H, 2 ꢂ OCH3), 4.28 (s, 2H, C1eH), 5.34 (s, 2H, CH2O), 6.53 (s, 1H,
ArH), 6.57 (s, 1H, ArH), 6.90e7.02 (m, 2H, ArH), 7.16e7.23 (m, 2H,
ArH), 7.96 (d, J ¼ 8.9 Hz, 2H, ArH), 8.02 (d, J ¼ 8.9 Hz, 2H, ArH), 8.11
(s, 1H, CHN). 13C NMR (75 MHz, CDCl3)
d 16.3, 28.2, 43.8, 47.2, 55.9,
56.0, 62.1, 109.0, 111.5, 120.6, 121.3, 123.0, 124.2, 124.8, 127.0, 129.4,
129.5, 131.0, 137.2, 139.9, 146.3, 147.9, 148.1, 156.2. HRMS-TOF: m/z
[MþH]þ 521.1859 (Calcd for C27H29N4O5S: 521.1853).
4.6.18. 6,7-Dimethoxy-2-((4-(4-((4-nitrophenoxy)methyl)-1H-
1,2,3-triazol-1-yl)phenyl)sulfonyl)-1,2,3,4-tetrahydroisoquinoline
(31)
White solid. 77%. mp 173e174 ꢃC. IR (UATR) cmꢀ1: 1592, 1518,
4.6.14. 6,7-Dimethoxy-2-((4-(4-((p-tolyloxy)methyl)-1H-1,2,3-
triazol-1-yl)phenyl)sulfonyl)-1,2,3,4-tetrahydroisoquinoline (27)
White solid. 92%. mp 168e169 ꢃC. IR (UATR) cmꢀ1: 1596, 1509,
1464, 1341, 1257, 1162. 1H NMR (300 MHz, CDCl3)
d 2.86 (t,
J ¼ 5.9 Hz, 2H, C4eH), 3.45 (t, J ¼ 5.9 Hz, 2H, C3eH), 3.84 (s, 6H,
2 ꢂ OCH3), 4.29 (s, 2H, C1eH), 5.42 (s, 2H, CH2O), 6.53 (s, 1H, ArH),
6.56 (s, 1H, ArH), 7.12 (d, J ¼ 7.3 Hz, 2H, ArH), 7.96 (d, J ¼ 8.8 Hz, 2H,
ArH), 8.03 (d, J ¼ 8.8 Hz, 2H, ArH), 8.19 (s, 1H, CHN), 8.25 (d,
1464, 1346,1225,1162. 1H NMR (300 MHz, CDCl3)
d 2.31 (s, 3H, CH3),
2.86 (t, J ¼ 5.7 Hz, 2H, C4eH), 3.44 (t, J ¼ 5.7 Hz, 2H, C3eH), 3.84 (s,
6H, 2 ꢂ OCH3), 4.28 (s, 2H, C1eH), 5.30 (s, 2H, CH2O), 6.53 (s, 1H,
ArH), 6.56 (s, 1H, ArH), 6.92 (d, J ¼ 8.5 Hz, 2H, ArH), 7.12 (d,
J ¼ 8.5 Hz, 2H, ArH), 7.94 (d, J ¼ 8.7 Hz, 2H, ArH), 8.01 (d, J ¼ 8.7 Hz,
J ¼ 7.3 Hz, 2H, ArH). 13C NMR (75 MHz, CDCl3)
d 28.2, 43.8, 47.2, 55.9,
56.0, 62.3, 108.9, 111.4, 114.8, 120.7, 121.0, 122.9, 124.8, 126.0, 129.5,
137.5, 139.5, 142.1, 144.3, 148.1, 162.8. HRMS-TOF: m/z [MþH]þ
552.1539 (Calcd for C26H26N5O7S: 552.1548).
2H, ArH), 8.13 (s, 1H, CHN). 13C NMR (75 MHz, CDCl3)
d 20.5, 28.2,

202
R. Pingaew et al. / European Journal of Medicinal Chemistry 81 (2014) 192e203
4.6.19. 3-((1-(4-((6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)
sulfonyl)phenyl)-1H-1,2,3-triazol-4-yl)methoxy)-4-
methoxybenzaldehyde (32)
wavelengths of 485 and 535 nm. Fluorescence signal at day 4 was
subtracted with background fluorescence at day 0. IC50 values were
derived from doseeresponse curves, using 6 concentrations of 3-
fold serially diluted samples, by the SOFTMax Pro software (Mo-
lecular device). Ellipticine and 0.5% DMSO were used as a positive
and a negative control, respectively.
Pale yellow solid. 76%. mp 141e142 ꢃC. IR (UATR) cmꢀ1: 1682,
1587, 1519, 1464, 1346, 1260, 1161. 1H NMR (300 MHz, CDCl3)
d 2.86
(t, J ¼ 5.8 Hz, 2H, C4eH), 3.44 (t, J ¼ 5.8 Hz, 2H, C3eH), 3.83, 3.84,
3.95 (3s, 9H, 3 ꢂ OCH3), 4.27 (s, 2H, C1eH), 5.48 (s, 2H, CH2O), 6.53
(s, 1H, ArH), 6.56 (s, 1H, ArH), 7.24 (d, J ¼ 7.9 Hz,1H, ArH), 7.46 (s, 1H,
ArH), 7.47 (d, J ¼ 8.0 Hz, 1H, ArH), 7.94 (d, J ¼ 8.8 Hz, 2H, ArH), 8.01
(d, J ¼ 8.8 Hz, 2H, ArH), 8.21 (s, 1H, CHN), 9.88 (s, 1H, CHO). 13C NMR
4.9. Identification of putative targets of 1,2,3,4-
tetrahydroisoquinolines
(75 MHz, CDCl3)
d
28.2, 43.8, 47.2, 55.9, 56.0, 62.7, 109.0,109.5,111.5,
A structure similarity search was performed in PubChem using
compounds 20 and 27 as the query molecules, which identified 1-
[4-(3,4-dihydro-1H-isoquinolin-2-ylsulfonyl)phenyl]pyrrolidin-2-
one 7 (CID 18096680) as homologous structure. Analysis on the
112.6, 120.6, 121.2, 123.0, 124.8, 126.6, 129.4, 130.9, 137.4, 139.7,
144.8, 147.9, 148.1, 150.0, 152.8, 190.8. HRMS-TOF: m/z [MþH]þ
565.1759 (Calcd for C28H29N4O7S: 565.1752).
previously tested bioactivity of this compound revealed 17b-
4.6.20. 4-((1-(4-((6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)
sulfonyl)phenyl)-1H-1,2,3-triazol-4-yl)methoxy)-3-
methoxybenzaldehyde (33)
hydroxysteroid dehydrogenase type 5, belonging to the aldo-keto
reductase family 1 member C3 (AKR1C3), as a putative target
affording potent nanomolar inhibitory activity with IC50 values of
42 and 52 nM as verified by two separate bioactivity experiments
[11,12]. In addition, the X-ray crystallographic structure of AKR1C3
as obtained from the Protein Data Bank (PDB id 4FAL) was co-
complexed with 3-(3,4-dihydroisoquinolin-2(1H)-ylsulfonyl)-N-
methylbenzamide (8), which displayed 2-(benzenesulfonyl)-
1,2,3,4-tetrahydroisoquinoline as a similar moiety to compounds
investigated herein.
Pale yellow solid. 77%. mp 116e117 ꢃC. IR (UATR) cmꢀ1: 1683,
1596, 1518, 1437, 1346, 1264, 1162. 1H NMR (300 MHz, CDCl3)
d 2.87
(t, J ¼ 5.7 Hz, 2H, C4eH), 3.46 (t, J ¼ 5.9 Hz, 2H, C3eH), 3.84, 3.99 (2s,
9H, 3 ꢂ OCH3), 4.28 (s, 2H, C1eH), 5.44 (s, 2H, CH2O), 6.54 (s, 1H,
ArH), 6.57 (s, 1H, ArH), 7.04 (d, J ¼ 8.3 Hz, 1H, ArH), 7.55 (dd, J ¼ 8.2,
1.8 Hz, 1H, ArH), 7.61 (d, J ¼ 1.8 Hz, 1H, ArH), 7.95 (d, J ¼ 8.8 Hz, 2H,
ArH), 8.02 (d, J ¼ 8.8 Hz, 2H, ArH), 8.20 (s, 1H, CHN), 9.88 (s, 1H,
CHO). 13C NMR (75 MHz, CDCl3)
d 28.2, 43.8, 47.2, 56.0, 56.2, 62.8,
108.9, 111.1, 111.4, 112.0, 120.7, 121.0, 123.0, 124.8, 127.2, 129.4, 130.1,
137.3, 139.8, 144.9, 148.1, 155.0, 190.6. HRMS-TOF: m/z [MþH]þ
565.1753 (Calcd for C28H29N4O7S: 565.1752).
4.10. Molecular docking
Molecular docking was performed as to shed light on the
binding modalities of investigated ligands toward its putative
target AKR1C3 using a similar protocol as previously described [36].
Preparation of the AKR1C3 protein structure prior to docking was
performed by adding essential hydrogen atoms using Kollman
united atom charges and solvation parameters as provided by
AutoDock Tools in PyRx 0.6 [37]. 1,2,3,4-Tetrahydroisoquinoline-
triazole structures were constructed using Marvin Sketch Version
6.0 [38] and geometrically optimized with Gaussian 09 [39] using
the B3LYP/6-31G(d) method. Ligand structures were prepared for
docking by merging non-polar hydrogen atoms, adding Gasteiger
partial charges and defining rotatable bonds. A maximum grid box
4.7. Cytotoxic assay: cancer cell lines
Cell lines suspended in the corresponding culture medium were
inoculated in 96-well microtiter plates (Corning Inc., NY, USA) at a
density of 10,000e20,000 cells per well and incubated at 37 ꢃC in a
humidified atmosphere with 95% air and 5% CO2. After 24 h, an
equal volume of additional medium containing either the serial
dilutions of the test compounds, positive control (etoposide and/or
doxorubicin) or negative control (DMSO) was added to the desired
final concentrations, and the microtiter plates were further incu-
bated for an additional 48 h. The number of surviving cells in each
well was determined using either MTT assay [32,33] (for adherent
cells: HuCCA-1, HepG2, and A549 cells) or XTT assay [34] (for sus-
pended cells: MOLT-3 cells) in order to determine the IC50, which is
defined as the concentration that inhibits cell growth by 50%
(relative to negative control) after 48 h of continuous exposure to
each test compound.
ꢀ
size of 47.27 ꢂ 49.52 ꢂ 59.94 A was generated to cover the entire
AKR1C3 protein using Auto Grid. The grid box was allocated at the
center of the protein using x,y,z coordinates of 7.1616, 5.6316,
11.0790, respectively. Molecular docking simulations were per-
formed using AutoDock Vina as part of the PyRx 0.6 software. The
co-crystallized ligand was re-docked as validation of the docking
protocol. Docked structures were visualized using PyMOL [40]. A 2-
dimensional schematic representation of protein-ligand interaction
was generated using PoseViewWeb version 1.97.0 [41].
4.8. Cytotoxicity assay: primate cell line (Vero)
The cytotoxicity was performed by using the Green Fluorescent
Protein (GFP) detection method [35]. 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-N1
plasmid (Clontech). The cell line was maintained in a minimal
essential medium supplemented with 10% heat-inactivated fetal
Acknowledgments
This project was financially supported from Srinakharinwirot
University under the Government Budget (B.E. 2558) and the
Thailand Research Fund under the Young Scholars Research
Fellowship (MRG5680001 to R.P.). 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.
bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 1.5 g/L
sodium bicarbonate and 0.8 mg/mL geneticin, at 37 ꢃC in a hu-
midified incubator with 5% CO2. The assay was carried out by
adding 45
384-well plates containing 5
m
L of cell suspension at 3.3 ꢂ 104 cells/mL to each well of
mL of test compounds previously
diluted in 0.5% DMSO, and then incubating for 4 days in 37 ꢃC
incubator with 5% CO2. Fluorescence signals were measured by
using SpectraMax M5 microplate reader (Molecular Devices, USA)
in the bottom reading mode with excitation and emission
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.05.019.

R. Pingaew et al. / European Journal of Medicinal Chemistry 81 (2014) 192e203
203
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