molecules

Communication
Synthesis and Evaluation of Aminothiazole-Paeonol
Derivatives as Potential Anticancer Agents
Chia-Ying Tsai 1,2,† , Mohit Kapoor 2,† , Ying-Pei Huang 2,† , Hui-Hsien Lin 3 , Yu-Chuan Liang 4 ,
Yu-Ling Lin 5,6 , Su-Chin Huang 7 , Wei-Neng Liao 7 , Jen-Kun Chen 7, *, Jer-Shing Huang 2 and
Ming-Hua Hsu 1, *
1 Nuclear Science & Technology Development Center, National Tsing Hua University, Hsinchu 30013, Taiwan;
s101000017@m101.nthu.edu.tw
2 Department of Chemistry, National Tsing Hua University, Hsinchu 30013, Taiwan;
mohitk48@gmail.com (M.K.); suzann800217@hotmail.com (Y.-P.H.); jshuang@mx.nthu.edu.tw (J.-S.H.)
3 Division of Radiotherapy, Department of Oncology, Taipei Veterans General Hospital, Taipei 11217, Taiwan;
twwarcgogo@gmail.com
4 Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan; ycliang@sinica.edu.tw
5 Department of Biological Science and Technology, National Chiao Tung University, Hsinchu 30010, Taiwan;
lyring@pchome.com.tw
6 Center for Bioinformatics Research, National Chiao Tung University, Hsinchu 30010, Taiwan
7 Institute of Biomedical Engineering and Nanomedicine, National Health Research Institutes, Miaoli 35053,
Taiwan; chin@nhri.org.tw (S.-C.H.); wei-neng@nhri.org.tw (W.-N.L.)
* Correspondence: jkchen@nhri.org.tw (J.-K.C.); mhhsu@mx.nthu.edu.twl (M.-H.H.);
Tel.: +886-3-724-6166 (ext. 38117) (J.-K.C.); +886-3-571-5131 (ext. 31180) (M.-H.H.);
Fax: +886-3-758-6440 (J.-K.C.); +886-3-572-5974 (M.-H.H.)
† These authors contributed equally to this work.

Academic Editors: Jean Jacques Vanden Eynde and Annie Mayence

Received: 20 November 2015 ; Accepted: 20 January 2016 ; Published: 26 January 2016

Abstract: In this study, novel aminothiazole-paeonol derivatives were synthesized and

characterized using 1 H-NMR, 13 C-NMR, IR, mass spectroscopy, and high performance liquid
chromatography. All the new synthesized compounds were evaluated according to their anticancer
effect on seven cancer cell lines. The experimental results indicated that these compounds
possess high anticancer potential regarding human gastric adenocarcinoma (AGS cells) and
human colorectal adenocarcinoma (HT-29 cells). Among these compounds, N-[4-(2-hydroxy-4-
methoxyphenyl)thiazol-2-yl]-4-methoxybenzenesulfonamide (13c) had the most potent inhibitory
activity, with IC50 values of 4.0 µM to AGS, 4.4 µM to HT-29 cells and 5.8 µM to HeLa cells.
The 4-fluoro-N-[4-(2-hydroxy-4-methoxyphenyl)thiazol-2-yl]benzenesulfonamide (13d) was the
second potent compound, showing IC50 values of 7.2, 11.2 and 13.8 µM to AGS , HT-29 and HeLa cells,
respectively. These compounds are superior to 5-fluorouracil (5-FU) for relatively higher potency
against AGS and HT-29 human cancer cell lines along with lower cytotoxicity to fibroblasts. Novel
aminothiazole-paeonol derivatives in this work might be a series of promising lead compounds to
develop anticancer agents for treating gastrointestinal adenocarcinoma.

Keywords: paeonol; 2-aminothiazole; anti-cancer; sulfonate; adenocarcenoma

1. Introduction
Paeonol, 2-hydroxy-4-methoxy acetophenone (1, Figure 1), is a major component of traditional
Chinese medicine. Moutan Cortex, the outer layer of the root of Moutan, is classified in the genus
Paeonia and has been used for more than 1000 years. Paeonol is categorized as a flavonoid derivative
and exhibits many remarkable biological effects, and it has been applied for anti-inflammatory [1,2],

Molecules 2016, 21, 145; doi:10.3390/molecules21020145 www.mdpi.com/journal/molecules

Molecules 2016, 21, 145 2 of 9

analgesic [2], antioxidant [3], antidiabetic [4], anticancer [5], and antiatherogenic purposes [6].
Paeonol was2016,
Molecules also21,found
145 to protect against memory loss after ischemic stroke by reducing2 of amyloid
9
precursor protein (APP), beta-site APP cleaving enzyme (BACE), and apoptosis [7]. Moreover, paeonol
was also found to protect against memory loss after ischemic stroke by reducing amyloid precursor
derivatives have been reported to show many attractive biological activities. For example, Pan
protein
Molecules (APP),
2016, 21,beta-site
145 APP cleaving enzyme (BACE), and apoptosis [7]. Moreover, paeonol derivatives 2 of 9
and Hui demonstrated that the donepezil-like paeonol derivative (2, Figure 1) exhibited strong
have been reported to show many attractive biological activities. For example, Pan and Hui demonstrated
metal-chelating
was the
that ability
alsodonepezil-like
found forpaeonol
to protect Alzheimer’s disease
againstderivative
memory loss (AD)
after
(2, Figure 1)treatment
ischemic stroke
exhibited [8]. Yang
by reducing
strong presented a copper
amyloid ability
metal-chelating for ion
precursor
chelating paeonol
Alzheimer’s Schiff-base
protein (APP),disease
beta-site derivative
APPtreatment
(AD) cleaving enzyme (3,
[8]. Yang Figure
(BACE), 1) complexes
and apoptosis
presented a copper ion that possessed
[7]. Moreover, high antioxidant
paeonol Schiff-base
chelating paeonol derivatives
activity and
have moderate
been
derivative (3, FigureDNA-binding
reported to1)show activity
many attractive
complexes that as well
biological
possessed as high For
highactivities.
antioxidanttumor celland
example,
activity cytotoxicity
Pan [9]. Moreover, Yu
and HuiDNA-binding
moderate demonstrated
that
reported athe
activity donepezil-like
as well as
paeonol paeonol
high tumor
thiosemicarbazone cellderivative (2,[9].
cytotoxicity
derivative Figure 1) exhibited
Moreover,
(4, Figure Yu1), strong
reported
which metal-chelating
aexhibited
paeonol ability
thiosemicarbazone
potential for
mushroom
Alzheimer’s
derivative
tyrosinase (4,disease
inhibitors [10].(AD)
Figure treatment
1),Recently,
which our[8].
exhibited Yangfound
presented
potential
group that aphenylsulfonyl
mushroom copper ion chelating
tyrosinase paeonol
inhibitors Schiff-base
[10]. Recently,
moieties-conjugated our
paeonol
derivative
group found(3, that
Figure 1) complexesmoieties-conjugated
phenylsulfonyl that possessed high paeonol
antioxidant activity and
derivatives weremoderate
potential DNA-binding
anti-Hepatitis
derivatives were potential anti-Hepatitis B virus leads [11] and could prevent lipid accumulation at
activity
B as well
virus leads as and
[11] highcould
tumorprevent
cell cytotoxicity [9]. Moreover,
lipid accumulation Yu reported
at lower a paeonol
doses, and thiosemicarbazone
they might be prominent
lower doses, and they might be prominent antiatherogenic agents [12].
derivative (4, Figure
antiatherogenic 1),[12].
agents which exhibited potential mushroom tyrosinase inhibitors [10]. Recently, our
group found that phenylsulfonyl moieties-conjugated paeonol derivatives were potential anti-Hepatitis
B virus leads [11] and could prevent lipid accumulation at lower doses, and they might be prominent
antiatherogenic agents [12].

Figure 1. Structures of paeonol, donepezil-like paeonol derivative, paeonol Schiff-base derivative, and
Figure 1. Structures of paeonol, donepezil-like paeonol derivative, paeonol Schiff-base derivative,
paeonol thiosemicarbazone derivative.
and paeonol thiosemicarbazone derivative.
The thiazole
Figure ring of
1. Structures (5,paeonol,
Figure donepezil-like
2), a five-membered heterocyclic
paeonol derivative, core Schiff-base
paeonol structure, derivative,
displays aandvariety
of biological
The thiazole effects, such as antibacterial,
2), a five-membered heterocyclic core structure, displaysanti-
ring (5, Figurederivative.
paeonol thiosemicarbazone antifungal, anti-Human immunodeficiency virus, a variety
inflammatory, antidiabetic, antioxidant,
of biological effects, such as antibacterial, and anticancer effectsanti-Human
antifungal, [13]. These heterocyclic rings, notably virus,
immunodeficiency
The thiazole(6,
2-aminothiazole ring (5, Figure
Figure 2), are 2), a five-membered
considered stable andheterocyclic core structure,
lipophilic bioisosteres displays
of phenol a variety
(7, Figure 2)
anti-inflammatory, antidiabetic, antioxidant, and anticancer effects [13]. These heterocyclic rings,
of catechol
or biological(8,effects, such
Figure 2) as antibacterial,
moieties, which might antifungal, anti-Human immunodeficiency
retain pharmacological action while havingvirus, anti-
improved
notably 2-aminothiazole
inflammatory,
oral bioavailability
(6, Figure
antidiabetic,
[14]. Talipexole
2), are
antioxidant, and
(9, Figure
considered
anticancer
2), a dopamine
stable
effects and
[13].
agonist
lipophilic
These
for
bioisosteres
heterocyclic
Parkinson’s rings,
disease
of phenol
notably
treatment,
(7, Figure
was 2) or catechol
2-aminothiazole
designed on (6, (8, Figure
theFigure
basis of2),the2) moieties,
arebioisosteric which
consideredeffectstable might
ofand retain
lipophilic
phenol pharmacological
bioisosteres of [15].
and 2-aminothiazole action
phenol while 2)having
(7, Figurethe
In addition,
improved oral bioavailability
or catechol (8, Figure
2-aminothiazole core 2)
was [14]. Talipexole
moieties,
found which
to act as the(9,pharmacophore
might Figure
retain 2), a dopamine
pharmacological agonist
action
for antitubercularwhile for Parkinson’s
having
agents, activitydisease
improved
the
oral
and bioavailability
treatment, was
the designed
cytotoxicity [14].
ofonTalipexole
the could
which (9, be
basis Figure
the 2),
of improved a dopamine
bioisosteric agonist
effect
and reduced offorphenol
with Parkinson’s disease
andmodification
appropriate treatment,
2-aminothiazole
[16]. [15].
was designed
Introducing a on the basis
phenylsulfonyl of the bioisosteric
moiety in some effect of
moleculesphenol
may and 2-aminothiazole
increase
In addition, the 2-aminothiazole core was found to act as the pharmacophore for antitubercular the [15].
solubility In
of addition,
the the
molecules
2-aminothiazole
and trigger core
antitumor was found
activity to act
[17–19]. as the pharmacophore for antitubercular
agents, the activity and the cytotoxicity of which could be improved and reduced with appropriate agents, the activity
and the cytotoxicity of which could be improved and reduced with appropriate modification [16].
modification [16]. Introducing a phenylsulfonyl moiety in some molecules may increase the solubility
Introducing a phenylsulfonyl moiety in some molecules may increase the solubility of the molecules
of the and
molecules and trigger antitumor activity [17–19].
trigger antitumor activity [17–19].

Figure 2. Structures of thiazole, 2-aminothiazole, phenol, catechol, talipexole and 2-aminothiazole derivative.

Figure 2. Structures of thiazole, 2-aminothiazole, phenol, catechol, talipexole and 2-aminothiazole derivative.
Figure 2. Structures of thiazole, 2-aminothiazole, phenol, catechol, talipexole and 2-aminothiazole derivative.
Molecules 2016, 21, 145 3 of 9

Herein, we present a new series of paeonol derivatives combined with the aminothiazole
ring Molecules
as the2016, 21, 145
core structure and further conjugated with the phenylsulfonyl side-chains. 3 of 9

With arylsulfonamidothiazole scaffold decoration, the anticancer activity of paeonol may be enhanced
Herein, we present a new series of paeonol derivatives combined with the aminothiazole ring as the
through additional hydrogen bonding
core structure and further conjugated withinteractions while retaining
the phenylsulfonyl or even
side-chains. improving the solubility of
With arylsulfonamidothiazole
paeonol itself [20–22]. This new series of aminothiazole-paeonol derivatives was determined
scaffold decoration, the anticancer activity of paeonol may be enhanced through additional hydrogen to have
potential anticancer
bonding effects
interactions in human
while retaininggastric
or evenadenocarcinoma (AGS),ofhuman
improving the solubility paeonolcervix adenocarcinoma
itself [20–22]. This
(HeLa),new series of
human aminothiazole-paeonol
pancreas adenocarcinoma derivatives was determined
(PaTu8988t), human to have potential
colorectal anticancer effects
adenocarcinoma (HT-29),
in human gastric adenocarcinoma (AGS), human cervix adenocarcinoma
human glioblastoma (U87-MG), human lung adenocarcinoma (A549) and mouse colon carcinoma (HeLa), human pancreas
adenocarcinoma (PaTu8988t), human colorectal adenocarcinoma (HT-29), human glioblastoma
(CT26.WT) cells. Simultaneously, the toxicity of aminothiazole-paeonol derivatives against normal
(U87-MG), human lung adenocarcinoma (A549) and mouse colon carcinoma (CT26.WT) cells.
cells was evaluated by embryonic fibroblast (BALB/3T3) cells. The newly synthesized compounds
Simultaneously, the toxicity of aminothiazole-paeonol derivatives against normal cells was evaluated
could by
be embryonic
structural fibroblast
templates(BALB/3T3)
for designing
cells.and
The developing novel anticancer
newly synthesized compounds agents.
could be structural
templates for designing and developing novel anticancer agents.
2. Results and Discussion
2. Results and Discussion
2.1. Chemistry
2.1. Chemistry
The synthetic methods of preparing the paeonol-2-aminothiazole-phenylsulfonyl derivatives are
outlined inThe synthetic
Scheme methods
1. The of preparing the
2-aminothiazole paeonol-2-aminothiazole-phenylsulfonyl
scaffold was obtained by treating paeonolderivatives are and
with thiourea
outlined in Scheme 1. The 2-aminothiazole scaffold was obtained by treating paeonol with
iodine; the condensation-cyclization of thiourea initiated by iodine afforded compound 11. To produce thiourea
and iodine; the condensation-cyclization of thiourea initiated by iodine afforded compound 11.
various paeonol-phenylsulfonyl derivatives, we treated 2-aminothiazole-paeonol 11 with substituted
To produce various paeonol-phenylsulfonyl derivatives, we treated 2-aminothiazole-paeonol 11 with
phenylsulfonyl chloride 12 to yield the final desired compounds 13. All these products were obtained
substituted phenylsulfonyl chloride 12 to yield the final desired compounds 13. All these products
in sufficient
were obtainedand
yield purifiedyield
in sufficient by using recrystallization
and purified for anticancerforassays.
by using recrystallization anticancer assays.

O O
S S
Cl
OH O
H2 N NH2 OH S 12 R
NH2
I2, EtOH, reflux N K2CO3, THF
O
O
1 11

OH S O
N S R a R=H e R = Cl
N H
O b R = Me f R = Br
O c R = OMe g R = NO2
13 d R=F

Scheme 1. Synthesis of the aminothiazole-paeonol derivatives.

Scheme 1. Synthesis of the aminothiazole-paeonol derivatives.
2.2. Anticancer Activity and Structure Activity Relationship Analysis
2.2. Anticancer Activity and Structure Activity Relationship Analysis
The antitumor effects of the new synthesized compounds against AGS, HeLa, PaTu8988t,
HT-29, U87-MG,effects
The antitumor A549, of
CT26.WT
the new and BALB/3T3 are
synthesized described in
compounds Table 1.AGS,
against Our results
HeLa,indicated
PaTu8988t, thatHT-29,
the aminothiazole-paeonol derivatives exhibited cytotoxic effects toward
U87-MG, A549, CT26.WT and BALB/3T3 are described in Table 1. Our results indicated that the the tested human cancer
cell lines. We observed
aminothiazole-paeonol that compound
derivatives 13c wascytotoxic
exhibited the most potent
effectscompound,
toward the withtested
IC50 values
human of 4.0 µM cell
cancer
to AGS, 4.4 µM to HT-29, 5.8 µM to HeLa, 10.0 µM to CT26.WT, 15.8 µM to PaTu8988t and 22.5 µM to
lines. We observed that compound 13c was the most potent compound, with IC50 values of 4.0 µM
U87-MG. Compound 13c was the only one providing efficient IC50 (less than 50 µM) against U87-MG
to AGS, 4.4 µM to Additionally,
glioblastoma. HT-29, 5.8 µM to HeLa,
compound 10.0
13c wasµM to CT26.WT,
relatively 15.8
less toxic µM to PaTu8988t
to BALB/3T3 and 22.5
(IC50: 32.7µM) in µM
to U87-MG. Compound 13c was the only one providing
comparison to 5-FU against BALB/3T3 (IC50: 1.0 µM). Compound 13d was efficient IC (less than 50 µM)
50 the second most potent against
U87-MG glioblastoma.
compound, showing Additionally,
IC50 values of 7.2,compound was
13c31.4
11.2, 13.8 and µMrelatively less toxic
to AGS, HT-29, HeLa to andBALB/3T3
PaTu8988t, (IC50 :
32.7 µM) in comparison
respectively. to 5-FU
However, against
compound 13dBALB/3T3
possessed lower(IC50water
: 1.0 µM). Compound
solubility than compound13d was the
13c second
did
(1.55 vs. 3.04 mmol/L, shown in Table 2), which arose from the F and OCH groups
most potent compound, showing IC50 values of 7.2, 11.2, 13.8 and 31.4 µM to AGS, HT-29, HeLa and
3 at the para-positions
of the phenylsulfonyl
PaTu8988t, terminals. On
respectively. However, the other hand,
compound compound 13g
13d possessed waswater
lower the most inactive compound.
solubility than compound
In the HT-29 treatment, the activity of the parent compound 13a was between those of the most potent
13c did (1.55 vs. 3.04 mmol/L, shown in Table 2), which arose from the F and OCH3 groups at the
compound 13c and the most inactive compound 13g. Comparing the structures of 13c and 13g,
para-positions of the phenylsulfonyl terminals. On the other hand, compound 13g was the most inactive
compound 13c was substituted with the OCH3 group and compound 13g was replaced with the NO2
compound. In
group at the thepara-positions
HT-29 treatment,
of the the activity of the
phenylsulfonyl parentIncompound
terminals. general, the13a NOwas between those of
2 group manifest
the most potent compound 13c and the most inactive compound 13g. Comparing the structures of
13c and 13g, compound 13c was substituted with the OCH3 group and compound 13g was replaced
Molecules 2016, 21, 145 4 of 9

with the NO2 group at the para-positions of the phenylsulfonyl terminals. In general, the NO2 group
manifest electron-withdrawing and lipophilic properties in the structure, which could conceivably alter
the pharmacokinetic behaviors of compounds. Moreover, the electron-donating OCH3 contributed
an additional oxygen atom to the structure, making it more hydrophilic and thus increasing the water
solubility. Some pharmacokinetic properties are listed in Table 2 [23,24]. All the compounds meet
Lipinski’s Rule of Five, with the molecular weights under 500 and the log p-values lower than five.
In summary, compound 13c presented the most potent activity against AGS and HT-29 cells with
IC50 values of 4.0 and 4.4 µM, respectively, which was superior to 5-FU (IC50 values: 43.8 and 7.2 µM
against AGS and HT-29). The compound 13c was less toxic against fibroblast cells than 5-FU; therefore,
compound 13c might be a promising lead compound for developing an anticancer agent against
gastrointestinal tract–related adenocarcinoma.

Table 1. Cytotoxicity of compounds toward various cell lines.

IC50 (µM)
Compounds
BALB/3T3 AGS HeLa PaTu8988t HT-29 U87-MG A549 CT26.WT
13a >50 >50 >50 31.1 20.7 >50 >50 >50
13b >50 22.0 27.4 38.7 14.1 >50 >50 >50
13c 32.7 4.0 5.8 15.8 4.4 22.5 >50 10.0
13d >50 7.2 13.8 31.4 11.2 >50 >50 >50
13e >50 > 50 >50 >50 13.4 >50 >50 >50
13f >50 > 50 16.4 22.8 11.0 >50 >50 >50
13g 32.2 > 50 >50 >50 47.8 >50 >50 30.0
5-Fu * 1.0 43.8 2.2 12.5 7.2 >50 >50 9.2
5-FU ** 13.0 79.5 0.232 11.3 19.3 4.9 10.3 61.0
[25–32] 72 h 24 h 48 h 48 h 48 h 48 h 48 h 72 h
*: the IC50 values were measured at 48 h after cells being treated with 5-FU in our study; **: the IC50 values of
5-FU against different cancer cells published in other references were measured at 24, 48 or 72 h after treatment.

Table 2. Calculation of lipophilicity and water solubility of paeonol derivatives library [23,24].

Molecular Weight, Lipophilicity and Water Solubility

Compounds
M.W. clogP clogS S (mmol/L)
13a 362.418 3.26 ´4.14 2.625486
13b 376.445 3.57 ´4.31 1.843748
13c 392.444 3.27 ´4.11 3.046335
13d 380.4804 3.76 ´4.39 1.550002
13e 396.86 3.83 ´4.43 1.474475
13f 441.314 4.07 ´4.57 1.187812
13g 407.026 3.21 ´4.33 1.903804

3. Experimental Section

3.1. General Experimental Procedures

All reactions were carried out in oven-dried glassware (120 ˝ C) under an atmosphere of nitrogen
unless indicated otherwise. Dichloromethane, ethanol, ethyl acetate, hexanes, methanol, and THF
were purchased from Mallinckrodt Chemical Co. Ethyl acetate was dried and distilled from CaH2 .
Tetrahydrofuran was dried by distillation from sodium and benzophenone under an atmosphere of
nitrogen. Benzenesulfonyl chloride, 4-bromobenzenesulfonyl chloride, 4-chlorobenzenesulfonyl chloride,
4-fluorobenzenesulfonyl chloride, 4-methoxybenzenesulfonyl chloride, 4-nitrobenzenesulfonyl chloride,
peaonol (21 -hydroxy-4-methoxyacetophenone), potassium carbonate, and p-toluenesulfonyl chloride
were purchased from Sigma-Aldrich China Inc., Shanghi, China.
Molecules 2016, 21, 145 5 of 9

Melting points were obtained with a Fargo MP-2D melting point apparatus (melting point
range up to ~400 ˝ C). Analytical thin layer chromatography (TLC) was performed on precoated
plates (silica gel 60 F-254), purchased from Merck Inc. Infrared (IR) spectra were measured on
a Perkin-Elmer Model Spectrum 100 spectrophotometer. Absorption intensities are recorded by the
following abbreviations: s = strong; m = medium; and w = weak. Proton NMR spectra were obtained
on a Varian Mercury-400 (400 MHz) spectrometer or Bruker AC-400 (400 MHz) spectrometer by
use of chloroform-d (CDCl3 ) and dimethylsulfoxide-d6 (DMSO-d6 ) as the solvents. Proton NMR
chemical shifts were referenced to residual protonated solvents (δ 7.24 for chloroform and δ 2.49
for dimethylsulfoxide). Carbon-13 NMR spectra were obtained on a Varian Mercury-400 (100 MHz)
spectrometer by use of chloroform-d (CDCl3 ) and dimethylsulfoxide-d6 (DMSO-d6 ) as the solvents.
Carbon-13 chemical shifts are referenced to the center of the CDCl3 triplet (δ 77.0 ppm) and DMSO
septet (δ 39.5 ppm). Multiplicities are recorded by the following abbreviations: s, singlet; d, doublet;
t, triplet; q, quartet; m, multiplet; J, coupling constant (hertz). High-resolution mass spectra were
obtained by means of a JEOL JMS-700 mass spectrometer.

3.2. Procedure for the Preparation of Aminothiazole-Paeonol (11)

To obtain aminothiazole-paeonol (11), paeonol (1) (1.0 equiv) was reacted with iodine (1.1 equiv)
and thiourea (3.0 equiv) in ethanol under reflux condition for 12.0–16.0 h. Then, the reaction mixture
was quenched with NaOH(aq) (2.0 equiv) and the ethanol was removed under reduced pressure.
The residue was extracted with ethyl acetate and the combined organic layer were washed the brine
and dried over MgSO4(s) . After being filtered and condensed under reduced pressure, the crude
product was purified by column chromatography on silica gel (ethyl acetate and hexane as eluent) to
give compound 11.
2-(2-Aminothiazol-5-yl)-5-methoxyphenol (11): 1 H-NMR (CDCl3 , 400 MHz): δ 7.40 (d, J = 8.4 Hz, 1 H,
H-3), 6.54 (s, 1 H, CH), 6.47 (s, 1 H, H-6), 6.42 (dd, J = 8.4, 2.0 Hz, 1 H, H-4), 5.05 (s, 2 H, NH2 ), 3.78 (s,
3 H, OMe) ppm. 13 C-NMR (CDCl3 , 100 MHz): δ 166.8, 161.0, 157.3, 148.9, 126.6, 111.0, 106.8, 101.6, 98.9,
55.2 (OMe) ppm; HRMS (ESI+) m/z [M + H]+ calculated for C16 H14 N2 O4 S2 , 223.0463, found 223.0462.

3.3. Standard Procedure for the Preparation of Aminothiazole-Paeonol Derivatives (13)

To a solution containing aminothiazole-paeonol (11, 1.0 equiv) in anhydrous THF (2.0–3.0 mL)
was added potassium carbonate (1.3 equiv) and a sulfonyl chloride 12 (1.1 equiv). After the reaction
mixture was stirred at 25 ˝ C for 2.0–3.0 h, it was diluted with dichloromethane (5.0 mL). Inorganic
solids were filtered off and the filtrate was concentrated under reduced pressure to afford the residue.
It was then purified by use of column chromatography on silica gel (various ratio of methanol to
dichloromethane) to give the desired conjugates 13.
N-[4-(2-Hydroxy-4-methoxyphenyl)thiazol-2-yl]benzenesulfonamide (13a): Yield 80%, green solid
product. IR (film): ν 3569.1, 2811.2, 1560.1, 1481.2, 1374.2, 1131.6, 853.4 cm´1 . 1 H-NMR (CDCl3 ,
400 MHz): δ 7.58–7.55 (m, 2 H, 2 ˆ ArH), 7.53–7.51 (m, 1 H, H-6), 7.43–7.33 (m, 3 H, 3 ˆ ArH), 6.84 (d,
J = 2.0 Hz, 1 H, H-3), 6.79–6.77 (m, 1 H, H-5), 6.55 (s, 1 H, SCH), 3.75 (s, 3 H, OMe) ppm. 13 C NMR
(CDCl3 , 100 MHz): δ 166.8, 163.9, 158.3, 147.2, 143.7, 131.5, 125.9, 121.1, 113.6, 113.2, 108.8, 106.2, 55.6
(OMe) ppm; HRMS (ESI+) m/z [M + H]+ calculated for C16 H14 N2 O4 S2 , 363.0395, found 363.0396.
N-[4-(2-Hydroxy-4-methoxyphenyl)thiazol-2-yl]-4-methylbenzenesulfonamide (13b): Yield 84%, off-white
solid product. IR (film): ν 3579.1, 2921.2, 1580.1, 1491.2, 1384.1, 1141.6, 843.4 cm´1 . 1 H-NMR (CDCl3 ,
400 MHz): δ 7.44 (d, J = 8.8 Hz, 1 H, H-6), 7.39 (d, J = 8.2 Hz, 2 H, 2 ˆ ArH), 7.06 (d, J = 8.2 Hz, 2 H,
2 ˆ ArH), 6.81 (d, J = 2.4 Hz, 1 H, H-3), 6.74 (dd, J = 8.8, 2.4 Hz, 1 H, H-5) 6.52 (s, 1 H, SCH), 3.72 (s,
3 H, OMe), 2.29 (s, 3 H, CH3 ) ppm. 13 C-NMR (CDCl3 , 100 MHz): δ 167.8, 159.3, 147.0, 145.3, 144.4,
132.1, 130.5, 129.1, 128.2, 120.9, 113.1, 108.7, 105.9, 55.4 (OMe), 21.4 (CH3 ) ppm; HRMS (ESI+) m/z
[M + H]+ calculated for C17 H16 N2 O4 S2 , 377.0551, found 377.0551.
Molecules 2016, 21, 145 6 of 9

N-[4-(2-Hydroxy-4-methoxyphenyl)thiazol-2-yl]-4-methoxybenzenesulfonamide (13c): Yield 83%,

off-white solid product. IR (film): ν 3672.1, 2931.1, 1560.6, 1473.2, 1388.1, 1142.7, 817.4 cm´1 . 1 H-NMR
(CDCl3 , 400 MHz): δ 7.46–7.42 (m, 3 H, H-6 + 2 ˆ ArH), 6.86 (d, J = 2.8 Hz, 1 H, H-3), 6.76–6.72 (m, 3 H,
H-5 + 2 ˆ ArH), 6.57 (s, 1 H, SCH), 3.75 (s, 3 H, OMe), 3.74 (s, 3 H, OMe) ppm. 13 C-NMR (CDCl3 ,
100 MHz): δ 166.8, 163.9, 159.4, 147.1, 144.7, 130.5, 125.9, 121.0, 113.7, 113.2, 108.8, 106.2, 55.6 (OMe),
55.5 (OMe) ppm; HRMS (ESI+) m/z [M + H]+ calculated for C17 H16 N2 O5 S2 , 393.0501, found 393.0498.
4-Fluoro-N-[4-(2-hydroxy-4-methoxyphenyl)thiazol-2-yl]benzenesulfonamide (13d): Yield 81%, white
solid product. IR (film): ν 3695.5, 2943.3, 1589.7, 1493.9, 1378.3, 1157.7, 837.7 cm´1 . 1 H-NMR (CDCl3 ,
400 MHz): δ 7.51–7.48 (m, 2 H, 2 ˆ ArH), 7.41 (d, J = 8.8 Hz, 1 H, H-6), 6.95–6.91 (m, 2 H, 2 ˆ ArH),
6.86 (d, J = 2.6 Hz, 1 H, H-3), 6.75 (dd, J = 8.8, 2.6 Hz, 1 H, H-5), 6.46 (s, 1 H, SCH), 3.74 (s, 3 H, OMe)
ppm. 13 C NMR (CDCl3 , 100 MHz): δ 167.3, 167.1, 164.5, 159.4, 146.8, 144.5, 131.2, 130.6, 120.9, 115.9,
115.6, 113.3, 108.9, 105.8, 55.5 (OMe) ppm; HRMS (ESI+) m/z [M + H]+ calculated for C16 H13 FN2 O4 S2 ,
381.0301, found 381.0302.
4-Chloro-N-[4-(2-hydroxy-4-methoxyphenyl)thiazol-2-yl]benzenesulfonamide (13e): Yield 81%, white
solid product. IR (film): ν 3708.8, 2796.7, 1566.74, 1459.55, 1314.15, 1256.54, 984.95 cm´1 . 1 H-NMR
(CDCl3 , 400 MHz): δ 7.53 (d, J = 8.8 Hz, 1 H, H-6), 7.48–7.46 (m, 2 H, 2 ˆ ArH), 7.29–7.27 (m, 2 H,
2 ˆ ArH), 6.94 (d, J = 2.4 Hz, 1 H, H-3), 6.82 (dd, J = 8.8, 2.4 Hz, 1 H, H-5), 6.56 (s, 1 H, SCH), 3.81 (s, 3 H,
OMe) ppm. 13 C-NMR (CDCl3 , 100 MHz): δ 167.2, 159.5, 146.8, 144.7, 140.7, 133.2, 130.7, 129.6, 128.7,
120.9, 113.5, 109.1, 106.0, 55.6 (OMe) ppm; HRMS (ESI+) m/z [M + H]+ calculated for C16 H13 ClN2 O4 S2 ,
397.0005, found 397.0006.
4-Bromo-N-[4-(2-hydroxy-4-methoxyphenyl)thiazol-2-yl]benzenesulfonamide (13f): Yield 83%, white
solid product. IR (film): ν 3691.2, 2838.7, 1591.3, 1479.1, 1362.1, 1143.7, 821.7 cm´1 . 1 H-NMR (CDCl3 ,
400 MHz): δ 7.36–7.33 (m, 3 H, H-5 + 2 ˆ ArH), 7.28–7.26 (m, 2 H, 2 ˆ ArH), 6.80 (d, J = 1.6 Hz, 1 H,
H-3), 6.71 (dd, J = 8.8, 1.6 Hz, 1 H, H-5), 6.35 (s, 1 H, SCH), 3.69 (s, 3 H, OMe) ppm. 13 C-NMR (CDCl3 ,
100 MHz): δ 167.2, 164.3, 158.4, 145.7, 143.6, 132.3, 121.2, 115.6, 113.2, 108.7, 105.2, 56.4 (OMe) ppm;
HRMS (ESI+) m/z [M + H]+ calculated for C16 H13 BrN2 O4 S2 , 440.9500, found 440.9502.
N-[4-(2-Hydroxy-4-methoxyphenyl)thiazol-2-yl]-4-nitrobenzenesulfonamide (13g): Yield 84%, orange
solid product. IR (film): ν 3607.7, 2685.6, 1554.65, 1360.65, 1325.25, 1267.54, 964.95 cm´1 . 1 H-NMR
(CDCl3 , 400 MHz): δ 8.06 (d, J = 8.0 Hz, 2 H, 2 ˆ ArH), 7.59 (d, J = 8.0 Hz, 2 H, 2 ˆ ArH), 7.29 (d,
J = 8.6 Hz, 1 H, H-6), 6.90 (s, 1 H, H-3), 6.77 (d, J = 8.6 Hz, 1 H, H-5), 6.32 (s, 1 H, SCH), 3.76 (s, 3 H,
OMe) ppm. 13 C-NMR (CDCl3 , 100 MHz): δ 167.2, 162.0, 156.2, 151.1, 147.8, 145.9, 132.9, 128.2, 124.2,
112.8, 107.4, 105.0, 104.2, 55.8 (OMe) ppm; HRMS (ESI+) m/z [M + H]+ calculated for C16 H13 N3 O6 S2 ,
408.0246, found 408.0247.

3.4. Cell Culture

Cell lines, including human gastric adenocarcinoma (AGS), human cervix adenocarcinoma (HeLa),
human pancreas adenocarcinoma (PaTu8988t), human colorectal adenocarcinoma (HT-29), human
glioblastoma (U87-MG), human lung adenocarcinoma (A549), mouse colon carcinoma (CT26.WT)
cells and embryonic fibroblast (BALB/3T3) were purchased from Bioresource Collection and Research
Center (BCRC, Taiwan) to evaluate anticancer activity of aminothiazole-paeonol derivatives. Five cell
lines were cultured in different media from Gibco (Life Technologies, Grand Island, NY, USA) and
Hyclone (GE Healthcare Life Science). Cells were cultured in 90% medium mixed with 10% serum at
37 ˝ C in humidified atmosphere with 5% CO2 and grown in T-75 flask with a feeding cycle of two to
three days. The composition and procedures of preparing culture media for different cell lines were
followed by instruction manual of suppliers [5,33].

3.5. Drug Treatment and Cell Viability Assay

The different cell lines were seeded into 96-well tissue culture plates at a concentration of
5 ˆ 103 cells/100 µL/well overnight. Subsequently, the cells were treated with serial concentrations of
Molecules 2016, 21, 145 7 of 9

seven paeonol derivatives. After 48 h of incubation, cell viability of each cell line was determined by
MTT colorimetric assay. Briefly, 100 µL of 2 mg/mL MTT reagent (Sigma-Aldrich, St. Louis, MO, USA)
was added to each well and incubated for 4 h at 37 ˝ C. Later, the medium was aspirated and 100 µL of
dimethyl sulphoxide (DMSO) added to each well; finally, the OD595 of each well was measured by
ELISA reader (TECAN, Wien, Austria).

4. Conclusions
Various phenylsulfonyl side-chains were directly conjugated to paeonol, the major component of
a traditional Chinese medicine, Moutan cortex, through chemical synthesis to generate a new series of
aminothiazole-paeonol derivatives. The substituents on the phenylsulfonyl side-chain included F, Cl,
Br, NO2 , Me, and OMe. All the synthesized compounds were characterized using 1 H-NMR, 13 C-NMR,
and mass spectra data. The cytotoxic effects of all compounds were evaluated against fibroblast cells
(BALB/3T3) and seven cancer cell lines, including AGS, HeLa, PaTu8988t, HT-29, U87-MG, A549 and
CT26.WT. We observed that the thiazole-paeonol-phenylsulfonyl derivatives demonstrated cytotoxic
effects against the tested cancer cell lines. We also discussed the structural activity relationship on the
basis of screening results and pharmacokinetic properties. Results indicated that 13c exhibited the most
potent activity against AGS and HT-29 cells with IC50 values of 4.0 and 4.4 µM, respectively, which is
superior to 5-FU (IC50 values: 43.8 and 7.2 µM against AGS and HT-29) and may be a promising lead
compound to develop an anticancer agent for gastrointestinal tract–related adenocarcinoma.

Acknowledgments: For financial support, we thank the Ministry of Science and Technology of the Republic
of China and National Tsing Hua University. Authors are grateful of financial supports from National
Health Research Institutes (NHRI-BN-104-PP-27 for JKC) and Ministry of Science and Technology (MOST
103-2113-M-400-001 for JKC) (MOST-103-2113-M-007-020 and MOST-104-2119-M-007-017 for MHH).
Author Contributions: Tsai C.Y., Huang Y.P., and Kapoor M. performed the synthesis and structure elucidation.
Tsai C.Y., Lin Y.L., Lin H.H., Huang S.C. and Liao W.N. contributed to evaluation of bioactivity. Huang J.S.,
Liang Y.C., Tsai, F.Y., Chen J.K. and Hsu M.H. prepared the manuscript and supervised whole research project.
Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds are available from the authors.

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