Synthesis and biological evaluation of new coumarins bearing 2,4-diaminothiazole-5-carbonyl moiety

Article abstract of DOI:10.1016/j.ejmech.2018.06.015

A series of new coumarin-containing compounds 3a-l and 4a-c was designed and synthesized based on the chalcone-type 4-amino-5-cinnamoylthiazole scaffold 2, and screened for their in vitro anticancer and antioxidant activities. Representatively, the 2-thiomorpholinothiazole derivative 3k with IC₅₀ values of 7.5–16.9 μg/ml demonstrated good cytotoxic effects against tested cell lines MCF-7, HepG2 and SW480. Further investigation by flow cytometric analysis confirmed that this compound induces apoptotic cell death in MCF-7 cells and cause G1-phase arrest in the cell cycle. Moreover, most of compounds had intrinsic potential for radical scavenging activity and ferric-reducing power as investigated by DPPH and FRAP assays.

Full text of DOI:10.1016/j.ejmech.2018.06.015

Accepted Manuscript
Synthesis and biological evaluation of new coumarins bearing 2,4-diaminothiazole-5-
carbonyl moiety
Adileh Ayati, Tayebeh Oghabi Bakhshaiesh, Setareh Moghimi, Rezvan Esmaeili,
Keivan Majidzadeh-A, Maliheh Safavi, Loghman Firoozpour, Saeed Emami, Alireza
Foroumadi
PII:
S0223-5234(18)30506-3
10.1016/j.ejmech.2018.06.015
EJMECH 10485
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 January 2018
Revised Date: 23 April 2018
Accepted Date: 5 June 2018
Please cite this article as: A. Ayati, T.O. Bakhshaiesh, S. Moghimi, R. Esmaeili, K. Majidzadeh-A, M.
Safavi, L. Firoozpour, S. Emami, A. Foroumadi, Synthesis and biological evaluation of new coumarins
bearing 2,4-diaminothiazole-5-carbonyl moiety, European Journal of Medicinal Chemistry (2018), doi:
10.1016/j.ejmech.2018.06.015.
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ACCEPTED MANUSCRIPT
Graphical Abstract
NH2
O
O
R
N
S
O
NH2
S
O
N
3a-l
N
R
N
NH2
S
O
R
N
4-Amino-5-cinnamoylthiazoles
(Chalcone-like scaffold)
O
O
NH
R1
4a-c

ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of new coumarins bearing 2,4-
diaminothiazole-5-carbonyl moiety
a
b
a
b
Adileh Ayati , Tayebeh Oghabi Bakhshaiesh , Setareh Moghimi , Rezvan Esmaeili ,
b
a
d,
Keivan Majidzadeh-A , Maliheh Safavi c, Loghman Firoozpour , Saeed Emami *, Alireza
Foroumadi a,e**
a The Institute of Pharmaceutical Sciences (TIPS), Tehran University of Medical Sciences, Tehran, Iran
b Genetics Department, Breast Cancer Research Center, Motamed Cancer Institute, ACECR, Tehran, Iran
c
Department of Biotechnology, Iranian Research Organization for Science and Technology, P. O. Box 3353-
5111, Tehran, Iran
d
Department of Medicinal Chemistry and Pharmaceutical Sciences Research Center, Faculty of Pharmacy,
Mazandaran University of Medical Sciences, Sari, Iran
e
Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran,
Iran
Abstract: A series of new coumarin-containing compounds 3a-l and 4a-c was designed and
synthesized based on the chalcone-type 4-amino-5-cinnamoylthiazole scaffold 2, and
screened for their in vitro anticancer and antioxidant activities. Representatively, the 2-
thiomorpholinothiazole derivative 3k with IC50 values of 7.5-16.9 µg/ml demonstrated good
cytotoxic effects against tested cell lines MCF-7, HepG2 and SW480. Further investigation
by flow cytometric analysis confirmed that this compound induces apoptotic cell death in
MCF-7 cells and cause G1-phase arrest in the cell cycle. Moreover, most of compounds had
intrinsic potential for radical scavenging activity and ferric-reducing power as investigated by
DPPH and FRAP assays.
Keywords: Anticancer; Antioxidant; coumarin; cytotoxic activity; thiazole
* Corresponding author.
Tel: 98 11 33543082; Fax: 98 11 33543084. E-mail: sd_emami@yahoo.com (S. Emami)
** Corresponding author.
Tel.: 98 21 66406757; Fax: 98 21 66461178. E-mail: aforoumadi@yahoo.com (A. Foroumadi)
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1. Introduction
Nowadays cancer is recognized as one of the major threats against human health, which in
many cases leads to the death of patients [1]. Cancer is a class of diseases resulted from
uncontrolled growth and division of cells due to the dysregulation of essential enzymes and
proteins in cell division and proliferation [2]. Since chemotherapy is still considered as a
main treatment approaches among the others, many attempts have been devoted to find potent
anticancer agents [3]. Despite prominent progress in the field of anticancer agents and their
cellular and molecular aspects, still many patients suffer from resistance to currently
available anticancer drugs and diverse adverse effects [4].
Coumarin (2H-chromen-2-one or 2H-1-benzopyran-2-one) is a fused heterocyclic system,
belongs to flavonoid group of plant secondary metabolite. To date, many coumarin bearing
compounds have been reported in the literature with significant anticancer activities, working
through different mechanisms. The underlying mechanisms of these compounds appear to
depend on the substitution pattern of the coumarin core structure [5,6]. Moreover,
hybridization of coumarin with varied pharmacophores led to the diverse pharmacologically
active
derivatives
exhibiting
antioxidant,
antituberculosis,
antinflammatory,
anticholinesterase, antiviral, antihyperlipidemic, antimicrobial and antidepressant activities
[7].
In the field of medicinal chemistry, 1,3-thiazole is an important five-membered heterocyclic
nucleus, found in many biologically active natural and synthetic compounds [8]. Among the
thiazole derivatives, 2-aminothiazole is the most widely used pharmacophore for drug design
and discovery [9]. Dasatinib is a significant example of 2-aminothiazole containing drugs
with anticancer activity [10]. The affinity of 2-aminothiazole derivatives to different targets
led to focusing on this class of heterocyclic molecules in the design of new anticancer agents
[11-14]. As an example, 2-pyrrolidinyl-4-amino-5-aroyl thiazole 1 (Fig.1) showed substantial
antiproliferative activity against broad spectrum of tumor cell lines, with potent antimitotic
effects at submicromolar concentrations [15].
Due to importance of chalcone type compounds in the field of anticancer drug discovery [16
-
22], we have recently described 4-amino-5-cinnamoylthiazoles 2 as chalcone-like anticancer
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agents [23]. This series of heterocycle-based chalcones were designed by insertion of a
double bound in the benzoylthiazole lead structure 1 (Fig. 1). According to these findings and
in continuation of our studies on the synthesis of chromene-based anticancer agents [24
herein, we describe coumarin-containing hybrid compounds namely 3-(4-amino-2-(cyclic
amino-1-yl)thiazole-5-carbonyl)-2H-chromen-2-ones and 3-(4-amino-2-(arylamino)
-30],
3
thiazole-5-carbonyl)-2H-chromen-2-ones 4 as new anticancer agents. The target compounds
3 and 4 can be considered as confromationally constrained analogs of 4-amino-5-
cinnamoylthiazoles 2 (Fig. 1). The effect of different amino groups (arylamino or cyclic
amino) at C-2 of thiazole core was investigated for structure-activity relationship analysis.
2. Chemistry
The final compounds 3a-l and 4a-c were prepared through synthetic pathways shown in
Scheme 1. The commercially available 3-acetylcoumarins 5 were brominated using Br2 in
CHCl3 to give bromoacetyl derivatives 6. In order to prepare compounds 3a-l, dimethyl N-
cyanodithioimidocarbonate (7) was treated with appropriate cyclic amine and sodium sulfide
to produce intermediates 8, which subsequently reacted with 3-(bromoacetyl)coumarins 6 in
N,N-dimethyl formamide (DMF). After TLC monitoring and upon consumption of the
bromoacetyl derivatives 6, the precipitated solid was filtered and purified by column
chromatography to obtain final compounds 3a-l. On the other hand, the reaction between
phenylisothiocyanates 9 and cyanamide in the presence of sodium methoxide afforded
intermediate 10. The resulting intermediate 10 was then treated with 3-
(bromoacetyl)coumarin 6a to give 3-(4-amino-2-(arylamino)thiazole-5-carbonyl)-2H-
chromen-2-ones (4a-c).
3. Results and discussion
3.1. Antiproliferative activity
The antiproliferative activity of compounds 3a-l and 4a-c was investigated against breast
carcinoma (MCF-7), human colon adenocarcinoma (SW480) and human liver cancer
(HepG2) cell lines using MTT assay. The results are presented as IC50 values in comparison
with etoposide (Table 1).
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As seen in Table 1, all the target compounds have moderate to good activities against all the
tested cancer cells, displaying IC50 values in the range of 7.5-48.7 µg/ml. Most of compounds
showed lower IC50 values against MCF-7 compared to other cell lines. In particular,
morpholine and thiomorpholine derivatives (3i and 3k) with IC50 values of 9.8 and 7.5 µg/ml
found to be the most potent compounds against MCF-7 cells. Besides these derivatives,
compounds 3a-c, 3e, 3f, 3j and 3l showed substantial cytotoxic activity against MCF-7 cell
line (IC50s <15 µg/ml). In the case of HepG2, compound 3h followed by 3k had superior
activity. Compound 3k with IC50 value of 13.0 µg/ml was also the best one against human
colon adenocarcinoma cells (SW480).
Based on different amino groups attached to the C-2 of thiazole nucleus, the synthesized
compounds could be categorized into 2-(cyclic amino) and 2-anilino series. The structure-
activity relationship (SAR) studies revealed that 2-(cyclic amino) derivatives 3 exhibited
better cytotoxic effects rather than 2-arylamino analogs 4. The comparison of unsubstituted-
piperidine derivative 3c with 4-benzylpiperidine analog 3e revealed that the benzyl moiety
can be tolerated or even can improve antiproliferative activity. The six-membered cyclic
amine analogs 3c, 3i, and 3k were more potent than five-membered cyclic amine congener 3a
against MCF-7 and HepG2 cells. The effect of bromo- substituent on the coumarin ring
depended on the type of cyclic amine attached to the thiazole core and the type of cell line
tested. For example, while the bromo analog 3b was more potent than its parent compound
3a against MCF-7 and HepG2, but its activity toward SW480 was less than that of
unsubstituted congener 3a. The bromo-substitution in compound 3g resulted in compound 3h
with highest activity against HepG2 and lesser activity against MCF-7. Replacement of
oxygen in morpholine derivative 3i by sulfur resulted in compound 3k with greater potency
against all tested cell lines. All aniline derivatives 4a-c exhibited modest activity against all
cell lines in the same range. The insertion of methoxy or poly-methoxy groups on aniline
moiety cannot improve the cytotoxicity.
3.2. Apoptosis inducing activity
Flow cytometry analysis was performed to determine the mechanism of cell death which
observed by the most active compound 3k, in MCF7 cells by Annexin V-FITC/PI (Propidium
iodide) dual staining assay [31]. In this regard, non-treated cells were used as negative control
and etoposide was used as standard drug. The cells were treated with two different
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concentrations of compound 3k, 7.5 µg/ml (IC50) and 11.2 µg/ml (1.5 fold IC50) for 48 h.
After that, the cells were harvested and stained with FITC Annexin-V and Propidium iodide
solutions, respectively and the percentages of apoptotic cells were determined by flow
cytometry. As shown in Figure 2, the treatment of MCF-7 cells with compound 3k at 7.5 and
11.2 µg/ml concentrations resulted in 19.2 and 41.7% of cell apoptosis, whereas non-treated
cells and etoposide showed 15.6 and 23%. These results confirmed that the cytotoxicity of
compound 3k is associated with cell apoptosis in a concentration-dependent manner.
3.3. Cell cycle arrest
The cell cycle analysis was performed to investigate the prevention of proliferation in cancer
cells (MCF-7 cells) by the most potent compound 3k. MCF-7 cells were treated with
compound 3k at two concentrations (7.5 and 11.2 µg/ml) for 48 h then the treated cells were
harvested, stained with propidium iodide (PI) and analyzed by flow cytometry [32]. The
obtained results were compared with non-treated MCF-7 cells, as control. As shown in Figs.
3 and 4, treatment of MCF-7 cells with 3k at 7.5 and 11.2 µg/ml concentrations increased the
percentage of G1-phase cells from 68.5 % (as control group) to 83.9 and 89.3 % respectively.
These results confirmed that compound 3k significantly caused G1-phase arrest in MCF-7
cells.
3.4. In vitro antioxidant activity
Since antioxidant activity has been reported for numerous natural and synthetic coumarins
[33], thus the antioxidant activity of newly synthesized coumarin-based compounds 3a-l and
4a-c was evaluated by using two distinct method; ferric reducing antioxidant power (FRAP)
assay and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay.
3.4.1. Ferric-reducing antioxidant power (FRAP)
In this experiment, ferric tripyridyl triazine [TPTZ-Fe(III)] complex is converted into ferrous
tripyridyl triazine [TPTZ-Fe(II)] complex and the absorbance of the blue color [TPTZ-Fe(II)]
complex was measured at 593 nm [34]. The experiments were conducted at different
concentrations (from 5 to 100 µg/ml) and the results were shown by FRAP values and
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compared with ascorbic acid as a positive control (Table 2). Based on the obtained results at
the lowest concentration (5 µg/ml), a majority of compounds showed higher FRAP values
compared to ascorbic acid as the standard antioxidant. Notably, morpholino derivative 3i and
3,4,5-trimethoxyanilino analog 4c showed the highest ferric-reducing power at low
concentrations. All 6-bromocoumarin derivatives exhibited lower FRAP values compared to
unsubstituted counterparts. In the other words, the introduction of bromine at the C-6 of
coumarin decreased the reducing power of the synthesized compounds. Compounds with
morpholine, piperidine and pyrrolidine moieties at C-2 of the thiazole ring, showed higher
antioxidant activities rather than derivatives bearing cyclic amines with pendent groups
(phenyl or benzyl).
3.4.2. Radical-scavenging activity on DPPH
The radical scavenging activity of synthesized compounds was measured by DPPH assay.
This method evaluates the hydrogen donating ability of synthesized compounds, converting
the purple DPPH radical to the yellow hydrazine. The results were illustrated as IC50 values
and listed in Table 2. The ascorbic acid was used as the positive control for the experiment.
As displayed in Table 2, all the tested compounds showed moderate radical scavenging
activity with IC50 values less than 50 µg/ml with the exception of 3h. The most active
compound was thiomorpholine derivative 3k with IC50 = 23.9 µg/ml which was about 2.5
fold less potent than ascorbic acid. Compounds with morpholine, pyrrolidine, piperidine
moieties showed good radical scavenging activity better than other derivatives with larger
substituents. Furthermore, the introduction of bromine into the C-6 of coumarin decreased the
radical scavenging activity of synthesized compounds. Among the 2-anilino derivatives, the
3,4,5-trimethoxy derivative (4c) showed the highest antioxidant activity (IC50 =26.3 µg/ml).
4. Conclusions
In conclusion, we have synthesized and evaluated 3-(4-aminothiazole-5-carbonyl)-2H-
chromen-2-ones 3a-l and 4a-c bearing cyclic amine, substituted cyclic amine, aniline or
substituted aniline moieties, as antiproliferative and antioxidant agents. In vitro MTT assay of
synthesized compounds revealed that most of them had significant cytotoxic activity against
MCF-7, HepG2 and SW400 cell lines. The best results were obtained for thiomorpholine
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derivative 3k with IC50 values of 7.5-16.9 µg/ml. Flow cytometric analysis on the
representative compound 3k against MCF-7 cells revealed the induction of apoptosis and
blockage of the cell proliferation at the G1-phase. Further biological evaluations by DPPH
and FRAP assays indicated the potential of target compounds for antioxidant activity.
5. Experimental section
5.1. General chemistry
Starting materials, reagents and solvents were purchased from Merck and Sigma Aldrich
companies and were used without any purification. The 3-(bromoacetyl)coumarins 6 were
prepared according to the previously reported method [35]. The reactions progress and the
purity of the synthesized compounds were monitored by thin-layer chromatography (TLC)
using silica gel 250 micron F254 plastic sheets. Flash chromatography was performed for the
purification of the synthesized compounds by using 230-400 mesh silica gel and the indicated
solvent system. Melting points were determined with a Kofler hot-plate microscope apparatus
and are uncorrected. IR spectra were recorded in KBr disks, using a Nicolet FT-IR Magna
1
550 spectrophotometer. H and 13C NMR spectra were recorded on Bruker 500 MHz
spectrometer using DMSO-d6 and CDCl3 as solvent. Chemical shifts (δ) are given in ppm
relative to tetramethyl silane (TMS) as internal standard and coupling constant (J) values are
presented in Hz. The elemental analysis for C, H, N was carried out with an Elementar
Analysen system GmbH VarioEL.
5.2. General procedure for the synthesis of compound 3a-l
An appropriate cyclic amine (1 mmol) was added to the mixture of dimethyl N-
cyanodithioimidocarbonate 7 (1 mmol) in DMF (5 ml) and heated at 70° C for 1 h. Then,
Na2S·9H2O 60% (1 mmol) was added and the mixture was heated for another 2 h. After this
time, the reaction mixture was cooled down to 0-5 °C and proper 3-(bromoacetyl)coumarin
derivative 6 was added over 10 min. After the consumption of compound 6 (checked by
TLC), the reaction mixture was poured into cold water (15 ml). The precipitated solid was
separated by filtration, washed with cold water and dried. The crude product was purified by
flash chromatography on silica gel using ethyl acetate-petroleum ether as eluent to give
corresponding target compound 3.
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5.2.1. 3-(4-amino-2-(pyrrolidin-1-yl)thiazole-5-carbonyl)-2H-chromen-2-one (3a)
Yield 71%; mp 192-194 °C, IR (KBr, 1/cm): 3333, 2924, 1708, 1609, 1542, 1442, 1351,
1
1282, 1172, 1101, 1049, 901, 801, 751. H-NMR (DMSO-d6, 500 MHz) δ: 1.96 (s, 4H,
2×CH2 pyrrolidine), 3.56 (bs, 4H, 2×CH2 pyrrolidine), 7.39 (t, 1H, H-6 coumarin, J = 7.5
Hz), 7.44 (d, 1H, H-8 coumarin, J = 8.0 Hz), 7.67 (t, 1H, H-7 coumarin, J =7.5 Hz), 7.79 (d,
13
1H, H-5 coumarin, J = 7.5 Hz), 8.07 (bs, 2H, NH2), 8.22 (s, 1H, H-4 coumarin). C-NMR
(DMSO-d6, 125 MHz) δ: 24.88, 49.16, 116.06, 118.22, 124.72, 127.82, 127.96, 129.06,
132.65, 141.48, 153.37, 157.39, 166.34, 168.38, 175.54. MS (m/z, %): 341 (M+, 100), 324
(29), 284 (21), 173 (25), 97 (27), 55 (19). Anal. calcd for C17H15N3O3S: C, 59.81; H, 4.43;
N, 12.31. Found: C, 60.01; H, 4.45; N, 12.36.
5.2.2. 3-(4-amino-2-(pyrrolidin-1-yl)thiazole-5-carbonyl)-6-bromo-2H-chromen-2-one (3b)
Yield 53%; mp 134-136 °C, IR (KBr, 1/cm): 3375, 3269, 1732, 1619, 1558, 1446, 1282, 822,
619. 1H-NMR (DMSO-d6, 500 MHz) δ: 1.86-2.07 (m, 4H, 2×CH2 pyrrolidine), 3.56 (bs, 4H,
2×CH2 pyrrolidine), 7.42 (d, 1H, H-8 coumarin, J = 8.5 Hz), 7.82 (d, 1H, H-7 coumarin, J =
13
8.5 Hz), 8.06 (s, 1H, H-5 coumarin), 8.11 (bs, 2H, NH2), 8.18 (s, 1H, H-4 coumarin). C-
NMR (DMSO-d6, 125 MHz,) δ: 24.97, 49.26, 116.31, 118.35, 120.20, 130.21, 131.15,
135.01, 139.70, 140.19, 153.70, 157.03, 166.53, 168.47, 175.01. MS (m/z, %): 421 (M+2,
100), 419 (M+, 100), 391 (39), 362 (46), 221 (86), 196 (57), 89 (54), 55 (47). Anal. calcd for
C17H14BrN3O3S: C, 48.58; H, 3.36; N, 10.00. Found: C, 48.76; H, 3.24; N, 10.09.
5.2.3. 3-(4-amino-2-(piperidin-1-yl) thiazole-5-carbonyl)-2H-chromen-2-one (3c)
Yield 66%; mp 186-188 °C, IR (KBr, 1/cm): 3370, 3268, 2942, 1726, 1614, 1569, 1523,
1
1470, 1437, 1311, 1252, 1164, 1022, 881, 775, 753. H-NMR (CDCl3, 500 MHz) δ: 1.48-
1.71 (m, 6H, 3×CH2 piperidine), 3.51 (bs, 4H, 2×CH2 piperidine), 7.30 (t, 1H, H-6 coumarin,
J = 7.5 Hz), 7.35 (d, 1H, H-8 coumarin, J = 8.0 Hz), 7.54-7.58 (m, 2H, H-5,7 coumarin), 8.00
(s, 1H, H-4 coumarin). 13C-NMR (CDCl3, 125 MHz) δ: 23.85, 25.20, 49.17, 116.62, 118.66,
124.56, 127.34, 128.66, 129.67, 132.48, 142.38, 154.21, 158.28, 166.96, 172.56, 176.66. MS
(m/z, %): 355 (M+, 100), 338 (27), 324 (21), 297 (15), 173 (21), 111 (25). Anal. calcd for
C18H17N3O3S: C, 60.83; H, 4.82; N, 11.82. Found: C, 60.59; H, 4.67; N, 11.70.
5.2.4. 3-(4-amino-2-(piperidin-1-yl)thiazole-5-carbonyl)-6-bromo-2H-chromen-2-one (3d)
Yield 59%; mp 138-140 °C, IR (KBr, 1/cm): 3437, 3327, 2923, 1709, 1619, 1534, 1464,
1
1433, 1322, 1241, 1016, 827, 514. H-NMR (DMSO-d6, 500 MHz) δ: 1.51-1.68 (m, 6H,
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3×CH2 piperidine), 3.50 (bs, 4H, 2×CH2 piperidine), 7.42 (d, 1H, H-8 coumarin, J = 9.0 Hz),
7.82 (d, 1H, H-7 coumarin, J = 9.0 Hz), 8.01 (s, 1H, H-5 coumarin), 8.05 (bs, 2H, NH2), 8.18
13
(s, 1H, H-4 coumarin). C-NMR (DMSO-d6, 125 MHz,) δ: 23.44, 25.36, 48.23, 116.61,
120.44, 121.65, 129.12, 130.64, 134.21, 139.78, 141.39, 152.12, 159.47, 167.35, 170.42,
175.21. MS (m/z, %): 435 (M+2, 100) 433 (M+, 100), 404 (41), 377 (57), 363 (23), 350 (25),
55 (38), 41 (76). Anal. calcd for C18H16BrN3O3S: C, 49.78; H, 3.71; N, 9.68. Found: C,
49.90; H, 3.82; N, 9.70.
5.2.5. 3-(4-amino-2-(4-benzylpiperidin-1-yl)thiazole-5-carbonyl)-2H-chromen-2-one (3e)
Yield 49%; mp: 134-136 °C, IR (KBr, 1/cm): 3446, 3299, 2918, 1717, 1607, 1539, 1447,
1
1322, 1253, 1169, 755. H-NMR (CDCl3, 500 MHz) δ: 1.26-1.31 (m, 2H, CH2 piperidine),
1.55-1.66 (m, 1H, piperidine), 1.72-1.75 (m, 2H, CH2 piperidine), 2.55-2.57 (m, 2H, CH2-
Ph), 2.97-3.02 (m, 2H, CH2 piperidine), 4.01-4.06 (m, 2H, CH2 piperidine), 7.12 (d, 2H, H-
2,6 Ph, J = 7.5 Hz), 7.20 (t, 1H, H-6 coumain, J = 8.5 Hz), 7.26-7.29 (m, 3H, H-3,4,5 Ph),
7.35 (d, 1H, H-8 coumarin, J = 8.5 Hz), 7.54 (m, 2H, H-5,7 coumarin), 8.01 (s, 1H, H-4
coumarin). 13C-NMR (CDCl3-d6, 125 MHz) δ: 31.34, 37.67, 42.73, 48.49, 116.68, 118.71,
124.59, 126.21, 127.62, 128.38, 128.69, 129.04, 132.53, 139.54, 142.50, 157.72, 159.53,
166.94, 172.24, 176.80. MS (m/z, %): 445 (M+, 100), 354 (28), 298 (67), 285 (35), 271 (18),
91 (38), 55 (14). Anal. calcd for C25H23N3O3S: C, 67.40; H, 5.20; N, 9.43. Found: C, 67.43;
H, 5.02; N, 9.49.
5.2.6. 3-(4-amino-2-(4-benzylpiperazin-1-yl)thiazole-5-carbonyl)-2H-chromen-2-one (3f)
Yield 57%; mp: 128-130 °C, IR (KBr, 1/cm): 3403, 3290, 1724, 1608, 1536, 1466, 1432,
1
1323, 1246, 1167, 1040, 996, 879, 804, 746. H-NMR (DMSO-d6, 500 MHz) δ: 2.35-2.46
(m, 4H, 2×CH2 piperazine), 3.16-3.29 (m, 2H, CH2-Ph), 3.32-3.58 (m, 4H, 2×CH2
piperazine), 7.26 (d, 2H, H-2,6 Ph, J = 6.5 Hz), 7.31-7.32 (m, 3H, H-3,4,5 Ph), 7.39 (t, 1H,
H-6 coumarin, J = 7.5 Hz), 7.43 (d, 1H, H-8 coumarin, J = 8.0 Hz), 7.67 (t, 1H, H-7
coumarin, J = 7.5 Hz), 7.79 (d, 1H, H-5 coumarin, J = 7.5 Hz), 8.03 (bs, 2H, NH2), 8.23 (s,
1H, H-4 coumarin). 13C-NMR (DMSO-d6, 125 MHz) δ: 47.39, 51.45, 61.51, 116.17, 118.21,
124.69, 127.01, 127.98, 128.24, 128.62, 128.86, 129.32, 132.67, 137.54, 141.54, 153.39,
157.37, 166.16, 171.47, 175.93. MS (m/z, %): 446 (M+, 100), 298 (28), 160 (42), 146 (77),
91 (89), 56 (21). Anal. calcd for C24H22N4O3S: C, 64.56; H, 4.97; N, 12.55. Found: C, 64.33;
H, 5.09; N, 12.50.
5.2.7. 3-(4-amino-2-(4-phenylpiperazin-1-yl)thiazole-5-carbonyl)-2H-chromen-2-one (3g)
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Yield 76%; mp: 136-138 °C, IR (KBr, 1/cm): 3365, 2923, 1712, 1609, 1522, 1463, 1431,
1
1315, 1229, 1018, 927, 759. H-NMR (DMSO-d6, 500 MHz) δ: 3.15-3.26 (m, 4H, 2×CH2
piperazine), 3.51-3.68 (m, 4H, 2×CH2 piperazine), 6.82 (t, 1H, H-4 phenyl, J = 7.5 Hz), 6.97
(d, 2H, H-2,6 phenyl, J = 8.0 Hz), 7.23 (t, 2H, H-3,5 phenyl, J = 7.5 Hz), 7.40 (t, 1H, H-6
coumarin, J =7.5 Hz), 7.45 (d, 1H, H-8 coumarin, J = 8.0 Hz), 7.68 (t, 1H, H-7 coumarin, J =
7.5 Hz), 7.81 (d, 1H, H-5 coumarin, J = 7.5 Hz), 8.09 (bs, 2H, NH2), 8.26 (s, 1H, H-4
coumarin). 13C-NMR (DMSO-d6, 125 MHz) δ: 47.27, 47.78, 114.36, 116.12, 118.28, 119.57,
124.71, 127.86, 128.32, 129.16, 132.58, 141.70, 150.40, 153.46, 157.40, 166.15, 171.75,
176.08. MS (m/z, %): 432 (M+, 100), 311 (37), 298 (19), 132 (85), 104 (23), 77 (21). Anal.
calcd for C23H20N4O3S: C, 63.87; H, 4.66; N, 12.95. Found: C, 64.12; H, 4.53; N, 13.11.
5.2.8. 3-(4-amino-2-(4-phenylpiperazin-1-yl)thiazole-5-carbonyl)-6-bromo-2H-chromen-2-
one (3h)
Yield 61%; mp 173-175 °C, IR (KBr, 1/cm): 3438, 3287, 1729, 1600, 1537, 1465, 1440,
1
1338, 1307, 1232, 1159, 1107, 1023, 924, 820. H-NMR (DMSO-d6, 500 MHz) δ: 3.04-3.09
(m, 4H, 2×CH2 piperazine), 3.66-3.73 (m, 4H, 2×CH2 piperazine), 6.82 (t, 1H, H-4 phenyl, J
= 7.5 Hz), 6.97 (d, 2H, H-2,6 phenyl, J = 8.0 Hz), 7.24 (t, 2H, H-3,5 phenyl, J = 7.5 Hz), 7.43
(d, 1H, H-8 coumarin, J = 8.5 Hz), 7.83 (d, 1H, H-7 coumarin, J = 8.5 Hz), 8.06 (s, 1H, H-5
coumarin), 8.10 (bs, 2H, NH2), 8.21 (s, 1H, H-4 coumarin). 13C-NMR (DMSO-d6, 125 MHz)
δ: 46.45, 48.23, 114.53, 116.48, 119.64, 119.89, 120.43, 127.45, 129.16, 129.89, 133.74,
137.56, 143.36, 149.47, 152.26, 157.54, 166.48, 171.27, 176.80. MS (m/z, %): 512 (M+2,
100), 510 (M+, 100), 391 (34), 295 (51), 223 (97), 197 (98), 171 (67), 145 (73). Anal. calcd
for C23H19BrN4O3S: C, 54.02; H, 3.74; N, 10.96. Found: C, 53.99; H, 3.75; N, 10.72.
5.2.9. 3-(4- amino-2-morpholinothiazole-5-carbonyl)-2H-chromen-2-one (3i)
Yield 62%; mp 172-174 °C, IR (KBr, 1/cm): 3459, 3394, 2922, 1724, 1610, 1533, 1464,
1319, 1270, 1242, 1114, 890, 762. 1H-NMR (CDCl3, 500 MHz) δ: 3.45-3.56 (m, 4H, 2×CH2
morpholine), 3.64- 3.82 (m, 4H, 2×CH2 morpholine), 7.31 (t, 1H, H-6 coumarin, J = 7.5 Hz),
7.36 (d, 1H, H-8 coumarin, J = 8.0 Hz), 7.54-7.59 (m, 2H, H-5,7 coumarin), 8.04 (s, 1H, H-4
coumarin). 13C-NMR (CDCl3, 125 MHz) δ: 47.86, 65.96, 116.68, 118.63, 124.66, 127.45,
128.77, 129.38, 132.70, 142.93, 154.28, 158.30, 166.52, 173.04, 177.27. MS (m/z, %): 357
(M+, 100), 298 (44), 167 (31), 149 (87), 97 (48), 86 (87), 57 (63), 43 (51). Anal. calcd for
C17H15N3O4S: C, 57.13; H, 4.23; N, 11.76. Found: C, 57.11; H, 4.31; N, 11.53.
5.3.10. 3-(4-amino-2-morpholinothiazole-5-carbonyl)-6-bromo-2H-chromen-2-one (3j)
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Yield 67%; mp 138-140 °C, IR (KBr, 1/cm): 3392, 3287, 2923, 1728, 1607, 1531, 1466,
1
1435, 1320, 1239, 1113, 892, 819. H-NMR (DMSO-d6, 500 MHz) δ: 3.44-3.55 (m, 4H,
2×CH2 morpholine), 3.57-3.86 (m, 4H, 2×CH2 morpholine), 7.42 (d, 1H, H-8 coumarin, J =
8.5 Hz), 7.81 (d, 1H, H-7 coumarin, J = 8.5 Hz), 8.05 (s, 1H, H-5 coumarin), 8.09 (bs, 2H,
NH2), 8.20 (s, 1H, H-4 coumarin). 13C-NMR (DMSO-d6, 125 MHz,) δ: 45.48, 66.47, 116.94,
119.83, 120.23, 128.92, 131.14, 137.52, 139.48, 140.50, 153.34, 159.71, 166.85, 172.62,
176.12. MS (m/z, %): 437 (M+2, 100), 435 (M+, 100), 377 (24), 340 (23), 222 (46), 165 (23),
54 (63). Anal. calcd for C17H14BrN3O4S: C, 46.80; H, 3.23; N, 9.63. Found: C, 46.98; H,
3.14; N, 9.70.
5.3.11. 3-(4-amino-2-thiomorpholinothiazole-5-carbonyl)-2H-chromen-2-one (3k)
Yield 69%; mp 174-176 °C, IR (KBr, 1/cm): 3444, 1697, 1608, 1531, 1461, 1427, 1351,
1
1279, 1170, 945, 777, 749. H-NMR (CDCl3, 500 MHz) δ: 2.56-2.78 (m, 4H, 2×CH2
thiomorpholine), 3.75-4.03 (m, 4H, 2×CH2 thiomorpholine), 7.31 (t, 1H, H-6 coumarin, J =
7.5 Hz), 7.36 (d,1H, H-8 coumarin, J = 8.0 Hz), 7.55-7.60 (m, 2H, H-5,7 coumarin), 8.04 (s,
13
1H, H-4 coumarin). C-NMR (CDCl3, 125 MHz,) δ: 26.80, 50.80, 116.71, 118.67, 124.67,
127.23, 128.77, 132.70, 142.95, 155.83, 158.36, 166.62, 172.47, 177.22. MS (m/z, %): 373
(M+, 100), 356 (18), 298 (87), 228 (26), 173 (38), 129 (22), 69 (29). Anal. calcd for
C17H15N3O3S2: C, 54.67; H, 4.05; N, 11.25. Found: C, 55.10; H, 4.06; N, 11.29.
5.3.12. 3-(4-amino-2-thiomorpholinothiazole-5-carbonyl)-6-bromo-2H-chromen-2-one (3l)
Yield 70%; mp 178-180 °C, IR (KBr, 1/cm): 3421, 2924, 1735, 1606, 1536, 1465, 1244,
1
1190, 1080, 955, 854, 820. H-NMR (DMSO-d6, 500 MHz) δ: 2.67 (bs, 4H, 2×CH2
thiomorpholine), 3.80 (bs, 4H, 2×CH2 thiomorpholine), 7.41 (bs, 1H, H-8 coumarin), 7.81
(bs, 1H, H-7 coumarin), 8.08 (bs, 3H, H-5 coumarin and NH2), 8.18 (bs, 1H, H-4 coumarin).
13C-NMR (DMSO-d6, 125 MHz) δ: 26.87, 51.27, 116.82, 119.35, 120.86, 128.96, 130.44,
137.25, 138.68, 141.56, 153.94, 159.23, 166.70, 170.24, 176.52. MS (m/z, %): 453 (M+2,
100), 451 (M+, 100), 436 (28), 378 (24), 272 (23), 222 (25), 128 (29), 67 (48). Anal. calcd for
C17H14BrN3O3S2: C, 45.14; H, 3.12; N, 9.29. Found: C, 45.32; H, 3.21; N, 9.33.
5.3. General procedure for the synthesis of compounds 4a-c
Cyanamide (1 mmol) was added to the solution of arylisothiocyanate 9 (1 mmol) in methanol
at 0-5 °C. A solution of sodium methoxide (0.8 M), obtained by dissolving sodium (1 mmol)
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in MeOH (1.2 ml), was slowly added at 0 °C to the reaction mixture over 10 min. After 2 h,
3-(bromoacetyl)coumarin 6 (1 mmol) was added portion by portion over 10 min and the
reaction color turned to yellow. After the consumption of 3-(bromoacetyl)coumarin, the
mixture was diluted with cold water (15 ml) and the obtained yellow solid was filtered and
washed with cold water three times. The crude product was dried and purified by column
chromatography on silica gel using ethyl acetate-petroleum ether as eluent system.
5.3.1. 3-(4-amino-2-(phenylamino)thiazole-5-carbonyl)-2H-chromen-2-one (4a)
Yield 66%; mp 178-180 °C, IR (KBr, 1/cm): 3275, 2923, 1721, 1606, 1549, 1439, 1171, 755.
1H-NMR (CDCl3, 500 MHz) δ: 7.16 (t, 1H, H-4 Ph, J = 7.5 Hz), 7.31-7.36 (m, 6H, H-Ph, H-
6,8 coumarin), 7.54-7.60 (m, 2H, H-5,7 coumarin), 8.03 (s, 1H, H-4 coumarin). 13C-NMR
(CDCl3, 125 MHz) δ: 116.09, 117.36, 118.26, 119.11, 123.54, 124.77, 129.14, 131.32,
132.92, 139.33, 140.23, 142.06, 153.02, 159.63, 167.96, 172.62, 176.70. MS (m/z, %): 363
(M+, 100), 339 (13), 323 (16), 198 (22), 180 (21), 152 (36), 77 (34), 51 (28). Anal. calcd for
C19H13N3O3S: C, 62.80; H, 3.61; N, 11.56. Found: C, 62.66; H, 3.62; N, 11.39.
5.3.2. 3-(4-amino-2-((4-methoxyphenyl)amino)thiazole-5-carbonyl)-2H-chromen-2-one (4b)
Yield 76%; mp 180-182 °C, IR (KBr, 1/cm): 3424, 1727, 1608, 1546, 1429, 1308, 1248,
1214, 1173, 1081, 1028, 766. 1H-NMR (DMSO-d6, 500 MHz) δ: 3.73 (s, 3H, OCH3), 6.93 (d,
2H, H-3,5 Ph, J = 8.5 Hz), 7.38-7.45 (m, 4H, H-2,6 Ph, H-6,8 coumarin), 7.67 (t, 1H, H-7
coumarin, J = 7.5 Hz), 7.80 (d, 1H, H-5 coumarin, J = 7.5 Hz), 8.13 (bs, 2H, NH2), 8.26 (s,
13
1H, H-4 coumarin), 10.63 (s, 1H, NH). C-NMR (DMSO-d6, 125 MHz) δ: 55.23, 114.32,
115.97, 116.21, 121.80, 124.72, 127.23, 128.65, 129.07, 132.27, 132.74, 141.81, 153.44,
157.37, 159.92, 166.89, 170.94, 176.24. MS (m/z, %): 393 (M+, 100), 173 (43), 149 (24), 122
(23), 97 (36), 83 (33), 69 (24), 57 (26), 43 (39). Anal. calcd for C20H15N3O4S: C, 61.06; H,
3.84; N, 10.68. Found: C, 59.88; H, 4.00; N, 10.64.
5.3.3. 3-(4-amino-2-((3,4,5-trimethoxyphenyl)amino)thiazole-5-carbonyl)-2H-chromen-2-one
(4c)
Yield 68%; mp 186-188 °C, IR (KBr, 1/cm): 3413, 3313, 1710, 1607, 1561, 1508, 1457,
1
1425, 1234, 1178, 1129, 997, 987, 762. H-NMR (DMSO-d6, 500 MHz) δ: 3.64 (s, 3H,
OCH3), 3.78 (s, 6H, 2×OCH3), 6.96 (bs, 2H, H-2,6 Ph), 7.40 (t, 1H, H-6 coumarin, J = 7.5
Hz), 7.45 (d, 1H, H-8 coumarin, J = 8.0 Hz), 7.68 (t, 1H, H-7 coumarin, J = 7.5 Hz), 7.81 (d,
1H, H-5 coumarin, J = 7.5 Hz), 8.18 (bs, 2H, NH2), 8.29 (s, 1H, H-4 coumarin), 10.69 (s,
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13
1H, NH). C-NMR (DMSO-d6, 125 MHz) δ: 55.83, 59.99, 97.70, 116.22, 118.27, 124.72,
127.32, 128.91, 129.08, 132.77, 133.90, 135.30, 141.97, 152.95, 153.48, 157.38, 167.56,
170.64, 176.51. MS (m/z, %): 453 (M+, 100), 368 (23), 194 (26), 147 (29), 98 (33), 85 (46),
71 (74), 57 (93), 43 (89). Anal. calcd for C22H19N3O6S: C, 58.27; H, 4.22; N, 9.27. Found: C,
58.50; H, 4.23; N, 9.31.
5.4. MTT assay
MTT assay was used to evaluate the cytotoxic activity of synthesized compounds (3a-l and
4a-c) against three different tumor cell lines MCF-7, HepG2 and SW400 which were grown
in RPMI-1640 medium and DMEM with 10% FBS (Gibco, Milano, Italy). All the cell lines
were purchased from National Cell Bank of Iran (Pastor Institute, Tehran, Iran). Different
concentrations of target compounds were prepared by dissolving synthetic compounds in
DMSO. Each wells of a 96-well microtiter plate had 190 µl of complete medium containing 8
× 103 cells, incubated at 37°C in a humidified 5% CO2 incubator overnight. The cells were
treated with different concentrations of test compounds and further incubated at 37°C for 48
h. After that, the cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide (MTT) and the absorbance of each well was measured by
microplate reader (Bio-Rad microplate reader, Model 680) at 570 nm wavelengths [36]. The
negative control was 0.1% DMSO. The reference drug etoposide was used as positive
control. The data were expressed by IC50 which means the compound concentration required
to inhibit cell proliferation by 50% and were depicted as the mean± SE from the dose-
response curves of at least three independent experiments.
5.5. Analysis of cellular apoptosis
Annexin V-FITC/PI (Propidium iodide) dual staining assay was used to determine the
induction of apoptosis by the most potent compound 3k, in comparison with etoposide (tested
at IC50 concentration). MCF-7 cells were seeded into 6-well plates and incubated overnight at
37 °C under 5% CO2. The cells were treated with two concentrations of test compound 3k at
IC50 and 1.5×IC50 concentrations and incubated for 48 h. Then, the treated cells were
trypsinized, washed with PBS twice and centrifuged at 1200 rpm to collect the cells. After
that, 500 µl of binding buffer and 5 µl of Annexin V-FITC and PI were added to suspend
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cells. After 5−15 min incubation at room temperature in dark, cell apoptosis was analyzed by
flow cytometry (FACS Calibur Bectone-Dickinson) [31].
5.6. Cell cycle assessment
Propidium iodide (PI) staining assay was used to investigate the cell cycle distributions in the
MCF-7 cells by flow cytometry analysis. PI can bind to DNA and emit a fluorescent, the
intensity of which is in proportion to the DNA content. The cells were treated with IC50 and
1.5×IC50 concentrations of test compound (3k), after 48 h, the treated cells were trypsinized,
washed with PBS and centrifuged at 1000 rpm for 5 min. After incubation with PBS, the
obtained MCF-7 cells were fixed with 70% cold ethanol (-20 °C). The fixed cells were
washed with PBS, treated with RNase A (0.1 mg/ml) and incubated for half an hour,
sequentially treated with 50 mg/ml of PI and incubated for 15 min more. The cell cycle
distribution was calculated using a Novocyte flow cytometer (ACEA Biosciences) and the
data were analyzed by NovoExpress 1.1.0 software. All the experiments were performed on
three samples in parallel [32].
5.7. FRAP assay
FRAP assay was used as a standard method to evaluate antioxidant activity of target
compounds. This experiment measured the reduction capacity of compounds which
converting ferric tripyridyl triazine (Fe (III)-TPTZ) complex into a blue color ferrous
tripyridyl triazine (Fe (II)-TPTZ) complex at low pH. An 1 ml TPTZ solution (10 mM) in 40
mM HCl was mixed with 1 ml FeCl3.6H2O solution (20 mM) and 10 ml acetate buffer (300
mM) at pH =3.6 to make Fe (II)-TPTZ (2,4,6-tripyridyl-s-triazine) reagent. 20 µl of sample
solution in different concentration was mixed with 150 µl of Fe (II)-TPTZ reagent and
incubated at 37°C for 25 min. The absorbance of mixture was measured at 593 nm. The
ascorbic acid was used as the standard solution. The results were expressed in µM equivalent
to FeSO4.7H2O by calculating from calibration curve [37].
5.8. DPPH radical scavenging activity
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The ability of target compounds to capture free radicals was evaluated by DPPH radical
scavenging method [38]. Different concentrations of synthesized compounds were prepared
by dissolving in DMSO. The working solution was obtained by mixing 0.5 mM stock
solution of DPPH in methanol to obtain an absorbance of 9.1±0.02 units at 517 nm using the
spectrophotometer. A mixture of 20 µl of each sample solution and 250 µl of DPPH in
methanol was incubated at room temperature for 30 min in dark. Then the absorbance of the
mixture was measured at 517 nm. The control group had the same content without any
compound and the ascorbic acid solution was used as standard. The experiments were carried
out in triplicates. The average values were used to determine the radical scavenging activity
as the percentage of inhibition according to the following equation: % inhibition =
[(absorbance of control- absorbance of sample)/absorbance of control]× 100. The data were
exhibited as IC50 which define as compound concentration required capturing free radicals by
50%.
Acknowledgments
This work was supported and funded by a grant from Iran National Science Foundation
(INSF); Grant no: 96011863.
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Captions:
Figure 1. Design of compounds 3 and 4 based on the thiazole-derived lead 1, as new
anticancer agents.
Figure 2. Flow cytometric analysis of MCF-7 cells treated with compounds 3k. (A) Non-
treated cells as negative control group; (B) treated with etoposide as positive control; (C)
treated with 3k at IC50 concentration (7.5 µg/ml); (D) treated with 3k at 1.5×IC50
concentration (11.2 µg/ml).
20

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Figure 3. Cell cycle analysis of MCF-7 cells by flow cytometric analysis. (A) Non-treated
cells as control group; (B) treated with 3k at IC50 concentration (7.5 µg/ml); (C) treated with
3k at 1.5×IC50 concentration (11.2 µg/ml)
Figure 4. Quantitative analysis of cell cycle distributions; (1) Non-treated cells as control
group; (2) treated with 3k at IC50 concentration (7.5 µg/ml); (3) treated with 3k at 1.5×IC50
concentration (11.2 µg/ml)
Scheme 1. Synthesis of compounds 3a-l and 4a-c. Reagents and conditions: a) Br2, CHCl3,
rt.; b) appropriate cyclic amine, DMF, 70 °C, 1 h; c) Na2S·9H2O 60%, 70 °C, 2 h; d) 0-5 °C,
2 h; e) NH2CN, CH3OH, 0-5 °C, 1h; f) Na/CH3OH, 0-5 °C, 2h; g) 0-5 °C, 2h.
Table 1
In vitro cytotoxic activity (IC50 values, µg/ml) of compounds 3a-l and 4a-c against cancer cell lines.
Compound
A
R
R1
MCF-7
HepG2
SW480
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pyrrolidin-1-yl
pyrrolidin-1-yl
piperidin-1-yl
piperidin-1-yl
4-benzylpiperidin-1-yl
4-benzylpiperazin-1-yl
4-phenylpiperazin-1-yl
4-phenylpiperazin-1-yl
morpholino
H
-
14.1 ± 2.3 40 ± 0.7
10.9 ± 3.4 27.7 ± 1.8
10.7 ± 0.9 37.1 ± 3.2
29.1 ± 1.2 19.8 ± 2.3
11.1 ± 1.7 21.7 ± 1.1
11.6 ± 2.4 32 ± 0.6
17.9 ± 3.2 34.9 ± 1.5
28.6 ± 2.6 12.2 ± 2.3
13.5 ± 3.1
33.4 ± 2.1
16.3 ± 1.4
18.3 ± 0.6
14.5 ± 3.1
23.4 ± 2.3
22.5 ± 0.7
17.1 ± 1.3
20.5 ± 1.9
48.7 ± 0.9
13.0 ± 0.6
44.5 ± 4.3
19.2 ± 0.9
20.9 ± 2.3
24.4 ± 2.5
7.7 ± 1.5
3a
Br
H
-
3b
-
3c
Br
H
-
3d
-
3e
H
-
3f
H
-
3g
Br
H
-
3h
-
9.8 ± 0.8
14.6 ± 1.2 19.3 ± 2.1
7.5 ± 0.7 16.9 ± 0.7
34.8 ± 2.6
3i
morpholino
Br
H
-
3j
thiomorpholino
thiomorpholino
-
-
3k
Br
H
-
12.5 ± 1.3 29.7 ± 3.1
26.6 ± 2.1 20.6 ± 2.1
30.3 ± 0.5 23.1 ± 1.3
24.2 ± 1.6 26.8 ± 0.8
3l
H
4a
-
H
4-MeO
3,4,5-(MeO)3
4b
-
H
4c
3.1 ± 0.6
6.2 ± 0.5
Etoposide
Table 2
In vitro antioxidant activity of compounds 3a-l and 4a-c determined by ferric-reducing antioxidant power
(FRAP) and DPPH methods
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Compound FRAP values at different concentrations
DPPH
(IC50, µg/ml)
5 µg/ml
10 µg/ml
20 µg/ml
40 µg/ml
80 µg/ml
100 µg/ml
93.3±0.6
116.5±1.6
155.3±0.7
272.5±3.9
275.8±3.5
542.3±3.4
28.8±1.3
31.7±0.8
27.3±2.1
46.6±1.4
37.7±1.2
31.7±2.8
38.3±0.9
>50
3a
3b
3c
3d
3e
3f
72.3±2.3
103.5±4.6
88.8±2.4
54.3±3.9
88.5±2.4
91±1.7
97.5±1.7
134.5±3.8
118.8±4.1
111.3±1.3
134±2.5
102.8±1.2
237 ±2.5
151.3±2.7
136.5±3.6
197.5±0.3
154±1.9
147.5±2.2
445 ±2.6
194.3±2.8
179.8±2.7
226.5±3.5
217±1.3
205.5±0.6
463.5±1.9
243.3±1.6
236.8±3.8
228.8±2.2
326±2.7
243.3±1.3
676 ±2.5
243.8±3.5
263.3±3.2
443.8±0.7
400±2.2
108±3.1
3g
3h
3i
63.5±2.1
110.8±5.6
76.55±2.4
76.3±3.5
57±0.7
72.3±1.4
142 ±0.4
84.3±3.8
86.3±0.9
70.5±0.7
92.5±3.8
121.3±0.7
173.3±1.6
123.6±3.1
73.3±2.1
205.3±3.4
110 ±2.1
148±2.3
82±1.4
98.3±4.1
383.8±1.8
211.5±3.1
454.8±2.9
164±1.5
121.3±2.3
605.3±1.4
247.8±1.6
564.5±2.4
171.3±3.1
289.8±4.8
350.3±3.4
372.3±3.8
484±2.3
344.8±3.5
162.3±4.2
266±3.8
25.8±1.5
34.6±0.9
23.9±1.4
27.7±1.8
49.9±3.7
39.7±1.6
26.3±1.3
9.6±1.5
3j
3k
3l
78.5±3.1
158.8±2.7
184.1 ±3.5
270.5±4.7
215.8±1.7
94.3±2.3
176.3±1.1
220 ±3.1
346.8±1.7
309.7±0.6
65.5±3.7
88.3±2.1
131.8±4.4
74.9±3.4
246.8±3.6
300±3.3
4a
4b
4c
372.3±1.7
397.4±3.7
Ascorbic
acid
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Figure 1. Design of compounds 3 and 4 based on the thiazole-derived lead 1, as new anticancer agents.
24

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Figure 2. Flow cytometric analysis of MCF-7 cells treated with compounds 3k. (A) Non-treated cells as
negative control group; (B) treated with etoposide as positive control; (C) treated with 3k at IC50 concentration
(7.5 µg/ml); (D) treated with 3k at 1.5×IC50 concentration (11.2 µg/ml).
25

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Figure 3. Cell cycle analysis of MCF-7 cells by flow cytometric analysis. (A) Non-treated cells as control
group; (B) treated with 3k at IC50 concentration (7.5 µg/ml); (C) treated with 3k at 1.5×IC50 concentration (11.2
µg/ml)
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Figure 4. Quantitative analysis of cell cycle distributions; (1) Non-treated cells as control group; (2) treated with
3k at IC50 concentration (7.5 µg/ml); (3) treated with 3k at 1.5×IC50 concentration (11.2 µg/ml)
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O
O
O
R
R
Br
a
O
O
O
6
5
NH2 O
CN
CN
N
Compound 6
R
b, c
N
N
H3C
CH3
S
S
d
S
N
S
Na
O
O
N
7
8
3a-l
R: H, Br
Cyclic amine:
pyrrolidin-1-yl, piperidin-1-yl,
4-benzylpiperidin-1-yl, 4-benzylpiperazin-1-yl,
4-phenylpiperazin-1-yl,
morpholino, thiomorpholino
NH2
S
O
O
R
CN
S
N
N
e,f
Compound 6
R1
Na
NCS
g
O
NH
10
R1
NH
9
4a-c
R: H
R1: H, 4-OMe,
3,4,5-(OMe)3
R1
Scheme 1. Synthesis of compounds 3a-l and 4a-c. Reagents and conditions: a) Br2, CHCl3, rt.; b) appropriate
cyclic amine, DMF, 70 °C, 1 h; c) Na2S·9H2O 60%, 70 °C, 2 h; d) 0-5 °C, 2 h; e) NH2CN, CH3OH, 0-5 °C, 1h;
f) Na/CH3OH, 0-5 °C, 2h; g) 0-5 °C, 2h.
28