Synthesis of some 5-arylidene-2-(4-acetamidophenylimino)-thiazolidin-4-one derivatives and exploring their breast anticancer activity

Article abstract of DOI:10.1002/jhet.3906

Ten 2-(4-acetamidophenylimino)-5-arylidenethiazolidin-4-one derivatives 6a-k were synthesized and evaluated for their anticancer activity against MCF-7 cell line (breast adenocarcinoma). The synthetic approach involves cyclocondensation of N,N′-bis(4-acet

Full text of DOI:10.1002/jhet.3906

Received: 5 December 2019
DOI: 10.1002/jhet.3906
Revised: 27 December 2019
Accepted: 9 January 2020
A R T I C L E
Synthesis of some 5-arylidene-2-(4-acetamidophenylimino)-
thiazolidin-4-one derivatives and exploring their breast
anticancer activity
Hana M. A. Abumelha1
| Ali Saeed2
1Department of Chemistry, Faculty of
Science, Princess Nourah Bint
Abdulrahman University, Riyadh, Saudi
Arabia
2Department of Chemistry, Faculty of
Science, Mansoura University, Mansoura,
Egypt
Abstract
Ten 2-(4-acetamidophenylimino)-5-arylidenethiazolidin-4-one derivatives 6a-k
were synthesized and evaluated for their anticancer activity against MCF-7 cell
line (breast adenocarcinoma). The synthetic approach involves cyclocondensation
of N,N0-bis(4-acetamidophenyl)-thiourea (3) with ethyl bromoacetate in ethanol
and sodium acetate to furnish the 2-(4-acetamidophenylimino)-4-thiazolidinone
derivative 4, which underwent Knoevenagel condensation reaction with some
substituted aldehydes to afford the targeted 2-(4-acetamidophenylimino)-
5-arylidenethiazolidin-4-ones 6a-k. The 4-chlorobenzylidene-thiazolidin-4-one
compound 6h exhibited strong inhibitory effect on the growth of breast cancer
cell with IC50 (58.33 1.74μM), very close to that of the reference drug doxorubi-
cin (IC50 48.06 0.36μM).
Correspondence
Hana M. A. Abumelha, Department of
Chemistry, Faculty of Science, Princess
Nourah Bint Abdulrahman University,
84428 Riyadh, Saudi Arabia.
Email: hmaabumelha@gmail.com
Funding information
Deanship of Scientific Research at Princess
Nourah Bint Abdelulrahman University
1 | INTRODUCTION
recent decades, one of the most promising molecules in the
anticancer drug discovery process. They have displayed sig-
Sulfur and nitrogen heterocycles are very important due to
their physicochemical properties, particularly in the sense
of developing new drugs and materials.[1–4] Especially,
thiazolidin-4-one–containing compounds have gained sig-
nificant attention since they were promising scaffolds in
the area of medicinal chemistry.[5,6] The literature survey of
thiazolidin-4-ones demonstrated a wide range of pharmaco-
logical properties, including antimicrobial,[7,8] anti-
inflammatory,[9,10] antioxidant,[11,12] antitubercular,[13,14]
antidiabetic,[15] human immunodeficiency virus (HIV)
nificant antitumor activity against a variety of cancer cells
such as breast cancer, lung cancer, and leukemia.[26] Con-
sidering the biological importance of 4-thiazolidinones as
anticancer agents (Figure 1), the present work focuses on
the design and synthesis of 2-(4-acetamidophenylimino)-
2-arylidene-4-thiazolidin-4-ones and the evaluation of their
anticancer activity.
2 | RESULTS AND DISCUSSION
2.1 | Chemistry
inhibitors,[16]
antihypertensive,[17]
anticonvulsant,[18]
antihepatitis,[19] antiproliferative,[20] and anticancer
activities.[21–24] Published data showed that anticancer
activity of thiazolidin-4-ones may be correlated with their
sensitivity to various targets including non-membrane pro-
tein tyrosine phosphatase (SHP-2), JNK-stimulating
phosphatase-1 (JSP-1), tumor necrosis factor (TNF-α), anti-
apoptotic biocomplex Bcl-XL-BH3, and inregrin.[25] Among
the thiazolidinone scaffolds, 5-alkenylthiazolidinones are in
The nucleophilic addition 4-aminoacetanilide (1) to phe-
nyl isothiocyanate proceeded as previously described
in the literature[27] in boiling ethanol to furnish N-
(4-acetamidophenyl)-N0-phenylthiourea (2) (Scheme 1).
Reaction of unsymmetrical thiourea derivative 2 with
4-aminoacetanilide (1) affected transamination through
J Heterocyclic Chem. 2020;1–9.
wileyonlinelibrary.com/journal/jhet
© 2020 Wiley Periodicals, Inc.
1

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ABUMELHA AND SAEED
FIGURE 1 Selected examples of potent anticancer thiazolidine-4-ones
SCHEME 1 Synthesis of 2-(4-acetamidophenylimino)-4-thiazolidinone derivative 4
replacement the aniline moiety by 4-aminoacetanilide
and produced the symmetric thiourea, N,N0-bis
(4-acetamidophenyl)-thiourea (3). Heterocyclization of
signals for six protons of two methyl groups at 2.05 and
2.07 ppm and singlet for two protons of methylene group
(thiazolidinone ring) at 4.13 ppm. The aromatic protons
(AA`BB` system) resonated as four doublet signals at
down-fielded chemical shift 6.79, 7.28, 7.50, and
7.66 ppm. In addition, the singlet signals at 9.88 and
10.10 ppm indicated the two protons of acetamide moie-
ties (MeCONH–). The 13C NMR spectrum clearly demon-
strated the signals of carbon atoms related to the
methylene and carbonyl part (thiazolidine-4-one ring) at
32.70 and 171.73 ppm, respectively.
3
with ethyl bromoacetate has been successfully
achieved in ethanol containing fused sodium acetate
to afford the precursor, 3-(4-acetamidophenyl)-
2-(4-acetamidophenylimino)-thiazolidin-4-one (4). The
infrared (IR) spectrum of 4 clearly demonstrated absorp-
tions at 3362, 3327, and 1705 cm−1 to indicate the pres-
ence of two NH and carbonyl (C O) of thiazolidine ring,
1
respectively. The H NMR spectrum showed two singlet

ABUMELHA AND SAEED
3
SCHEME 2 Synthesis of 2-(4-acetamidophenylimino)-5-arylidenethiazolidin-4-one derivatives 6a-k
In an attempt to prepare the pyranothiazole deriva-
tives 5 through treatment of thiazolidine-4-one deriva-
tive 4 with arylidene-malononitrile derivatives in hot
ethanol containing drops of piperidine, the reaction did
not afford our target pyranothiazoles 5, and the product
was assigned as the 5-arylidenethiazolidin-4-ones
6 (Scheme 2). The synthesis of all 5-arylidenethiazolidin-
9.95 and 10.14 ppm. The 13C NMR spectrum of 6c dis-
played signals for the aliphatic carbon atoms in the
expected regions, 23.93 (CH3), 24.01 (CH3), and
55.40 ppm (OCH3). The signals for aromatic carbon
atoms were indicated by their chemical shifts, 114.90
(2C), 119.28 (2C), 119.91 (2C), 121.13 (2C), 125.88,
128.82 (2C), 129.59, 130.32, 131.85 (2C), 136.22, 139.46,
and 150.56 ppm. The signal at 118.12 ppm indicated the
fifth carbon of thiazolidinone ring. The signal of olefinic
carbon was observed at 143.09 ppm. The signal of imine
group (C N) was observed at 160.63 ppm. In addition,
the signals of carbonyl groups were resonated at 165.81
(C O, amide), 168.13 (C O, amide), and 168.53 ppm
(C O, thiazolidinone ring).
4-one derivatives
the
6
was performed by heating
2-(4-acetamidophenylimino)-thiazolidine-4-one
with different aromatic aldehydes
derivative
4
(particularly benzaldehyde, 4-methylbenzaldehyde,
4-methoxybenzaldehyde, 2,5-dimethoxybenzaldehyde,
4-N,N-dimethylaminobenzaldehyde,
4-hydroxybenza-
ldehyde, 4-nitrobenzaldehyde, 4-chlorobenzaldehyde,
5-methylfurfural, and 2-thiophenecarboxaldehyde) in
ethanol containing five drops of piperidine (Scheme 2).
The chemical structures of 5-arylidenethiazolidin-4-one
derivatives 6 were secured based on their compatible
spectroscopic (IR and NMR) data. The IR spectrum of 6c
(as an example) clearly demonstrated absorptions at
3289 and 3175 cm−1 for the N–H groups along with the
characteristic carbonyl absorptions at 1714 and
2.2 | In vitro anticancer activity
Breast cancer is the most commonly occurring cancer in
women and the second leading cause of cancer death in
women. The chance that a woman will die from breast
cancer is about one in 38 (about 2.6%). Based on American
Cancer Society's estimates for 2019, approximately 268 600
new cases of invasive breast cancer will be diagnosed in
women, and about 41 760 women will die from breast can-
cer.[28,29] Thus, it is imperative to search for new alterna-
tives to breast cancer prevention agents. The aim of the
present investigation is to explore the anticancer activity of
the chemically synthesized 2-(4-acetamidophenylimino)-
1
1658 cm−1. The H NMR signals of 6c were presented at
their expected regions, singlet signals resonated at 2.03,
2.06, and 3.78 ppm due to the protons of three methyl
groups. The aromatic protons appeared as six double sig-
nals at 6.90, 7.07, 7.41, 7.53, 7.56, and 7.70 ppm. The ole-
finic proton was observed as singlet at 7.74 ppm. The
protons of NH groups were observed as singlet signals at

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ABUMELHA AND SAEED
TABLE 1 In vitro cytotoxic activity of the synthesized
2-(4-acetamidophenylimino)-thiazolidine-4-ones against MCF-7
cell line
molecules, which is considered as potentially responsible
for enhancement of the anticancer activity of the
thiazolidinones 6. Furthermore, the replacement of the
phenyl ring with different heterocyclic rings such as;
5-methyl-2-furyl and 2-thienyl derivatives 6i and 6k did
not enhance the anticancer activity. However, it encour-
aged an increase in the anticancer activity and contrib-
uted to a decrease in the IC50 value from 192.89μM to
101.26 and 95.79μM, respectively.
Upon treatment of MCF-7 cells with various concen-
trations of 6h at (12.5, 25, and 50 μg/mL), no appreciable
changes were observed. At higher concentration of 6h at
(100 μg/mL), marked changes in the cell morphology,
were evident after 24 hours of incubation (Figure 2).
MCF-7 cells were smaller, distorted, shrunken, and had
condensed nuclei.
Compound
IC50, μM
Doxorubicin
48.06 0.36
216.19 8.47
213.74 8.63
192.89 9.14
138.51 6.19
147.92 5.40
166.88 7.35
139.39 9.35
147.13 8.23
75.92 4.07
58.33 1.74
101.26 4.64
95.79 5.88
3
4
6a
6b
6c
6d
6e
6f
6g
6h
6i
3 | CONCLUSION
6k
A
new
series
of
2-(4-acetamidophenylimino)-
5-arylidenethiazolidin-4-ones have been synthesized and
1
5-arylidene-thiazolidin-4-ones 6a-k against breast cancer
(MCF-7). Accordingly, the anticancer activity of the new
thiazolidin-4-one derivatives was assessed using MTT
assay.[30–32]
characterized by IR, H- and 13C-NMR tools. All synthe-
sized thiazolidin-4-ones were subjected to anticancer
evaluation against breast cancer growth by MTT tech-
nique using doxorubicin as a standard drug. The
best activity was obtained by 4-chlorobenzylidene-
thiazolidinone derivative 6h (IC50 = 58.33 1.74μM).
The inhibitory effect of the synthesized thiazolidin-
4-ones on breast cancer cells was studied at different con-
centrations (12.5, 25, 50, and 100 μg/mL). IC50 values for
the investigated thiazolidine-4-ones 4 and 6a-k are listed
in Table 1 and compared with standard anticancer drug
doxorubicin. The SAR was generated by cyclization of
thiourea derivative 3 into its corresponding thiazolidine-
4-one 4 that did not improve the anticancer activity.
Introduction of benzylidene and substituted benzylidene
moieties to the thiazolidine-4-one ring encouraged an
increase the anticancer activity. The obtained results
indicated that the significance of the potency relates to
the type of substituents on the phenyl ring hybridized
with the thiazolidinone ring. It was observed that the
presence of electron donating groups (methyl, methoxy,
dimethylamino, and hydroxyl) at the benzylidene moiety
of compounds 6b-f resulted into moderate activity, their
IC50 values ranged from 138.51 to 166.88μM. The most
potent activity was gained by the 4-chlorobenzylidene-
thiazolidinone hybrid 6h, its IC50 (58.33 1.74μM) was
very close to that obtained by the reference doxorubicin
(IC50 = 48.06 0.36μM). The next potent hybrid was
4-nitrobenzylidene-thiazolidinone hybrid 6g, which dis-
play anticancer activity with IC50 = 75.92 4.07μM. The
presence of the chlorine or nitro substituents on the phe-
nyl ring yields an electron withdrawing effect. Even so,
the halogen atom increased the lipophilicity of the
4 | EXPERIMENTAL
All melting points (uncorrected) were measured on Gal-
lenkamp electric melting point apparatus. IR spectra were
determined on Nicolete IS10 spectrometer (KBr discs).
NMR spectra were measured in DMSO-d6 as a solvent at
500 MHz (1H NMR) and 125 MHz (13C NMR) on JEOL's
NMR spectrometer. Elemental analyses (C, H, and N)
were determined on Perkin-Elmer 2400 analyzer. Antican-
cer activity evaluation was performed at the Bioassay-Cell
Culture Laboratory, National Research Centre, El-Tahrir
St., Dokki, Cairo 12622, Egypt.
4.1 | Synthesis of N,N0-bis
(4-acetamidophenyl)-thiourea (3)
N-(4-Acetamidophenyl)-N0-phenylthiourea (2) (2.85 g,
10 mmol) was suspended in 20 mL tetrahydrofuran
(THF), and then 4-aminoacetanilide (1.50 g, 10 mmol) was
added. The reaction mixture was refluxed for 7 to 8 hours.
The solid that obtained upon cooling was filtered and
dried to afford N,N0-bis(4-acetamidophenyl)-thiourea (3).

ABUMELHA AND SAEED
5
FIGURE 2 Treatment of
MCF-7 cells with various
concentrations of 6h
Gray crystals; yield 71%; m.p. = 248-250ꢀC. IR
(υ/cm−1): 3297, 3219 (N–H), 1663 (C O). 1H NMR
(DMSO-d6) δ: 2.05 (s, 6H, 2CH3), 7.36 (d, J = 8.4 Hz, 4H,
Ar–H), 7.54 (d, J = 8.4 Hz, 4H, Ar–H), 9.58 (s, 2H, 2NH),
9.94 (s, 2H, 2NH). Anal. Calcd. for C17H18N4O2S (342): C,
59.63; H, 5.30; N, 16.36%. Found: C, 59.77; H,
5.33; N, 16.42%.
171.73. Anal. Calcd. for C19H18N4O3S (382): C, 59.67; H,
4.74; N, 14.65%. Found: C, 59.52; H, 4.79; N, 14.56%.
4.3 | General procedure for the synthesis
of 3-(4-acetamidophenyl)-2-phenylimino-
5-(4-substitutedbenzylidene)thiazolidin-
4-one derivatives 6a-k
4.2 | Synthesis of 3-(4-acetamidophenyl)-
2-(4-acetamidophenylimino)-thiazolidin-
4-one (4)
Thiazolidine-4-one derivative 4 (0.76 g, 2 mmol) was dis-
solved in hot ethanol 30 mL, and then the appropriate alde-
hyde, namely, benzaldehyde, 4-methylbenzaldehyde,
4-methoxybenzaldehyde, 2,5-dimethoxybenzaldehyde, 4-N,
N-dimethylaminobenzaldehyde, 4-hydroxybenzaldehyde,
4-nitrobenzaldehyde, 4-chlorobenzaldehyde, 5-methyl-
furfural, and 2-thiophenecarboxaldehyde (2 mmol), and
five drops of piperidine was added. The reaction compo-
nents were refluxed for 3 to 4 hours. After cooling to 25ꢀC,
the precipitate that formed was picked up by filtration and
recrystallized from acetic acid.
To a suspension of thiourea derivative 3 (1.71 g, 5 mmol)
and fused sodium acetate (1.0 g) in 30 mL ethanol, ethyl
bromoacetate (5 mmol) was added. The reaction compo-
nents were refluxed for 3-4 hours, and then cooled in air
to 25ꢀC. The precipitate that formed was filtered, and rec-
rystallized from ethanol/DMF mixture (5:1) to afford the
conforming thiazolidin-4-one derivative 4.
White crystals; yield 75%; m.p. = 288-290ꢀC. IR (υ
/cm−1): 3362, 3327 (N–H), 1705, 1675 (C O), 1636 (C N).
1H NMR (DMSO-d6) δ: 2.00 (s, 3H, CH3), 2.05 (s, 3H,
CH3), 4.13 (s, 2H, CH2), 6.79 (d, J = 8.5 Hz, 2H, Ar–H),
7.28 (d, J = 9.0 Hz, 2H, Ar–H), 7.50 (d, J = 9.0 Hz, 2H,
Ar–H), 7.66 (d, J = 8.0 Hz, 2H,Ar–H), 9.88 (s, 1H, NH),
10.10 (s, 1H, NH). 13C NMR (DMSO-d6) δ: 23.93, 24.01,
32.7, 119.28 (2C), 119.79 (2C), 120.93 (2C), 128.76 (2C),
129.87, 135.78, 139.29, 143.22, 155.79, 168.04, 168.52,
4.3.1 | 5-(Benzylidene)-
3-(4-acetamidophenyl)-
2-(4-acetamidophenylimino)-thiazolidin-
4-one (6a)
Golden crystals; yield 77%; m.p. = 288-289ꢀC. IR
(υ/cm−1): 3352, 3322 (N–H), broad centered at 1678

6
ABUMELHA AND SAEED
1
(C O), 1624 (C N). H NMR (DMSO-d6) δ: 2.03 (s, 3H,
CH3), 2.07 (s, 3H, CH3), 6.90 (d, J = 9.0 Hz, 2H, Ar–H),
7.41-7.51 (m, 5H, Ar–H), 7.57 (d, 4H, J = 8.5 Hz, Ar–H),
7.70 (d, J = 8.5 Hz, 2H, Ar–H), 7.78 (s, 1H, CH C), 9.95
(s, 1H, NH), 10.14 (s, 1H, NH). 13C NMR (DMSO-d6) δ:
23.96, 24.03, 119.31 (2C), 119.92 (2C), 121.14 (2C), 121.34,
128.84 (2C), 129.33 (2C), 129.52, 129.85 (2C), 130.02,
130.28, 133.38, 136.33, 139.53, 142.93, 150.34, 165.66,
168.17, 168.58. Anal. Calcd. for C26H22N4O3S (470): C,
66.37; H, 4.71; N, 11.91%. Found: C, 66.47; H,
4.66; N, 11.95%.
4.3.4 | 5-(2,5-Dimethoxybenzylidene)-
3-(4-acetamidophenyl)-
2-(4-acetamidophenylimino)-thiazolidin-
4-one (6d)
Yellow crystals; yield 82%; m.p. = 301-302ꢀC. IR
(υ/cm−1): 3287, 3258 (N–H), 1721, 1668 (C O), 1641
1
(C N). H NMR (DMSO-d6) δ: 2.02 (s, 3H, CH3), 2.07 (s,
3H, CH3), 3.68 (s, 3H, OCH3), 3.80 (s, 3H, OCH3), 6.89 (d,
J = 6.0 Hz, 2H, Ar–H), 7.05 (s, 2H, Ar–H), 7.27 (s, 1H,
Ar–H), 7.41 (d, J = 6.5 Hz, 2H, Ar–H), 7.56 (d, J = 6.0 Hz,
2H, Ar–H), 7.69 (d, J = 6.5 Hz, 2H, Ar–H), 7.90 (s, 1H,
CH C), 9.95 (s, 1H, NH), 10.14 (s, 1H, NH). 13C NMR
(DMSO-d6) δ: 23.95, 24.01, 55.49, 56.13, 112.84, 114.25,
115.93, 119.29 (2C), 119.81 (2C), 120.91, 121.16 (2C),
121.96, 122.59, 124.75, 128.80 (2C), 129.49, 136.32, 139.50,
142.85, 150.37, 152.10, 165.56, 168.13, 168.56. Anal.
Calcd. for C28H26N4O5S (530): C, 63.38; H, 4.94; N,
10.56%. Found: C, 63.54; H, 4.88; N, 10.47%.
4.3.2 | 5-(4-Methylbenzylidene)-
3-(4-acetamidophenyl)-
2-(4-acetamidophenylimino)-thiazolidin-
4-one (6b)
Yellow crystals; yield 80%; m.p. = 315-317ꢀC. IR
(υ/cm−1): 3347, 3315 (N–H), broad centered at 1677
1
(C O), 1623 (C N). H NMR (DMSO-d6) δ: 2.03 (s, 3H,
CH3), 2.07 (s, 3H, CH3), 2.31 (s, 3H, CH3), 6.90 (d,
J = 8.0 Hz, 2H, Ar–H), 7.31 (d, J = 7.5 Hz, 2H, Ar–H),
7.41 (d, J = 8.5 Hz, 2H, Ar–H), 7.46 (d, J = 7.5 Hz, 2H,
Ar–H), 7.57 (d, J = 8.5 Hz, 2H, Ar–H), 7.70 (d, J = 8.5 Hz,
2H, Ar–H), 7.75 (s, 1H, CH C), 9.95 (s, 1H, NH), 10.14
(s, 1H, NH). 13C NMR (DMSO-d6) δ: 21.11, 23.97, 24.05,
119.32 (2C), 119.94 (2C), 120.07, 121.15 (2C), 128.55 (2C),
129.58, 129.93 (4C), 130.41, 130.63, 136.32, 139.52, 140.21,
142.99, 150.40, 165.74, 168.18, 168.59. Anal. Calcd. for
C27H24N4O3S (484): C, 66.92; H, 4.99; N, 11.56%.
Found: C, 66.84; H, 4.95; N, 11.50%.
4.3.5 | 5-(4-(Dimethylamino)
benzylidene)-3-(4-acetamidophenyl)-
2-(4-acetamidophenyl-imino)-thiazolidin-
4-one (6e)
Orange crystals; yield 75%; m.p. > 330ꢀC. IR (υ/cm−1):
1
3299, 3217 (N–H), 1699 (C O), 1635 (C N). H NMR
(DMSO-d6) δ: 2.03 (s, 3H, CH3), 2.06 (s, 3H, CH3), 2.96 (s,
6H, –N(CH3)2), 6.79 (d, J = 9.0 Hz, 2H, Ar–H), 6.89 (d,
J = 8.0 Hz, 2H, Ar–H), 7.39 (d, J = 8.5 Hz, 4H, Ar–H),
7.56 (d, J = 8.0 Hz, 2H, Ar–H), 7.65 (s, 1H, CH C), 7.69
(d, J = 8.5 Hz, 2H, Ar–H), 9.94 (s, 1H, NH), 10.13 (s, 1H,
NH). 13C NMR (DMSO-d6) δ: 23.92, 24.01, 39.50, (2C),
112.06 (2C), 113.64, 119.25 (2C), 119.89 (2C), 120.32,
121.17 (2C), 128.83 (2C), 129.78, 131.39, 131.83 (2C),
136.07, 139.32, 143.35, 151.02, 151.12, 166.00, 168.08,
168.49. Anal. Calcd. for C28H27N5O3S (513): C, 65.48; H,
5.30; N, 13.64%. Found: C, 65.58; H, 5.26; N, 13.75%.
4.3.3 | 5-(4-Methoxybenzylidene)-
3-(4-acetamidophenyl)-
2-(4-acetamidophenylimino)-thiazolidin-
4-one (6c)
White crystals; yield 86%; m.p. = 310-311ꢀC. IR (υ/cm−1):
1
3289, 3175 (N-H), 1714, 1658 (C O), 1632 (C N). H
NMR (DMSO-d6) δ: 2.03 (s, 3H, CH3), 2.06 (s, 3H, CH3),
3.78 (s, 3H, OCH3), 6.90 (d, J = 9.0 Hz, 2H, Ar–H), 7.07
(d, J = 8.0 Hz, 2H, Ar–H), 7.41 (d, J = 9.0 Hz, 2H, Ar–H),
7.53 (d, J = 8.5 Hz, 2H, Ar–H), 7.56 (d, J = 9.0 Hz, 2H,
Ar–H), 7.70 (d, J = 9.0 Hz, 2H, Ar–H), 7.74 (s, 1H,
CH C), 9.95 (s, 1H, NH), 10.14 (s, 1H, NH); 13C NMR
(DMSO-d6) δ: 23.93, 24.01, 55.40, 114.90 (2C), 118.12,
119.28 (2C), 119.91 (2C), 121.13 (2C), 125.88, 128.82 (2C),
129.59, 130.32, 131.85 (2C), 136.22, 139.46, 143.09, 150.56,
160.63, 165.81, 168.13, 168.53. Anal. Calcd. for
C27H24N4O4S (500): C, 64.78; H, 4.83; N, 11.19%.
Found: C, 64.91; H, 4.88; N, 11.08%.
4.3.6 | 5-(4-Hydroxybenzylidene)-
3-(4-acetamidophenyl)-
2-(4-acetamidophenylimino)-thiazolidin-
4-one (6f)
Yellow crystals; yield 89%; m.p. > 330ꢀC. IR (υ/cm−1): 3464
(O-H), 3352, 3296 (N–H), broad centered at 1678 (C O),
1624 (C N). 1H NMR (DMSO-d6) δ: 2.03 (s, 3H, CH3), 2.07
(s, 3H, CH3), 6.90 (d, J = 9.0 Hz, 2H, Ar–H), 7.42 (d,
J = 8.5 Hz, 3H, Ar–H and OH), 7.50 (t, J = 7.5 Hz, 2H, Ar–
H), 7.57 (d, J = 8.0 Hz, 4H, Ar–H), 7.70 (d, J = 8.5 Hz, 2H,
Ar–H), 7.78 (s, 1H, CH C), 9.95 (s, 1H, NH), 10.14 (s, 1H,

ABUMELHA AND SAEED
7
NH). 13C NMR (DMSO-d6) δ: 23.97, 24.05, 119.32 (2C),
119.93 (2C), 121.15 (2C), 121.35, 128.85 (2C), 129.35 (2C),
129.52, 129.86 (2C), 130.03, 130.29, 133.39, 136.34, 139.54,
142.94, 150.35, 165.68, 168.18, 168.59. Anal. Calcd. for
C26H22N4O4S (486): C, 64.18; H, 4.56; N, 11.52%. Found: C,
64.07; H, 4.58; N, 11.44%.
(DMSO-d6) δ: 2.02 (s, 3H, CH3), 2.06 (s, 3H, CH3), 2.31 (s,
3H, CH3), 6.35 (d, J = 2.0 Hz, 1H, furan-H4), 6.88 (d,
J = 8.5 Hz, 2H, Ar–H), 6.95 (d, J = 3.0 Hz, 1H, furan-H3),
7.39 (d, J = 8.5 Hz, 2H, Ar–H), 7.52 (s, 1H, CH C), 7.56
(d, J = 8.5 Hz, 2H, Ar–H), 7.68 (d, J = 8.5 Hz, 2H, Ar–H),
9.94 (s, 1H, NH), 10.13 (s, 1H, NH). 13C NMR (DMSO-d6)
δ: 13.88, 23.96, 24.03, 110.19, 116.29, 117.20, 119.24 (2C),
119.57, 119.88 (2C), 121.11 (2C), 128.83 (2C), 129.62,
136.15, 139.41, 142.97, 148.33, 151.11, 156.98, 165.56,
168.14, 168.55. Anal. Calcd. for C25H22N4O4S (474): C,
63.28; H, 4.67; N, 11.81%. Found: C, 63.38; H,
4.73; N, 11.73%.
4.3.7 | 5-(4-Nitrobenzylidene)-
3-(4-acetamidophenyl)-
2-(4-acetamidophenylimino)-thiazolidin-
4-one (6g)
Brown crystals; yield 71%; m.p. > 330ꢀC. IR (υ/cm−1):
1
3294 (N–H), 1716, 1666 (C O), 1637 (C N). H NMR
4.3.10 | 5-(Thiophen-2-ylmethylene)-
3-(4-acetamidophenyl)-
2-(4-acetamidophenylimino)-thiazolidin-
4-one (6k)
(DMSO-d6) δ: 2.03 (s, 3H, CH3), 2.07 (s, 3H, CH3), 6.91
(d, J = 7.5 Hz, 2H, Ar–H), 7.44 (d, J = 8.5 Hz, 2H, Ar–H),
7.57 (d, J = 7.5 Hz, 2H, Ar–H), 7.71 (d, J = 8.0 Hz, 2H,
Ar–H), 7.82 (d, J = 7.5 Hz, 2H, Ar–H), 7.89 (s, 1H,
CH C), 8.31 (d, J = 8.0 Hz, 2H, Ar–H), 9.96 (s, 1H, NH),
10.15 (s, 1H, NH). Anal. Calcd. for C26H21N5O5S (515): C,
60.57; H, 4.11; N, 13.58%. Found: C, 60.47; H,
4.06; N, 13.51%.
Orange crystals; yield 81%; m.p. > 330ꢀC. IR (υ/cm−1):
3311 (N–H), broad centered at 1675 (C O), 1626 (C N).
1H NMR (DMSO-d6) δ: 2.03 (s, 3H, CH3), 2.07 (s, 3H,
CH3), 6.91 (d, J = 8.5 Hz, 2H, Ar–H), 7.23-7.25 (dd,
J = 5.0, 3.5 Hz, 1H, thiophene-H4), 7.42 (d, J = 8.5 Hz,
2H, Ar–H), 7.57 (d, J = 9.0 Hz, 2H, Ar–H), 7.64 (d,
J = 3.0 Hz, 1H, thiophene-H3), 7.70 (d, J = 9.0 Hz, 2H,
Ar–H), 7.90 (d, J = 5.0 Hz, 1H, thiophene-5), 8.06 (s, 1H,
CH C), 9.96 (s, 1H, NH), 10.14 (s, 1H, NH). 13C NMR
(DMSO-d6) δ: 23.9, 24.03, 118.80, 119.29 (2C), 119.95
(2C), 121.14 (2C), 123.77, 128.82 (3C), 129.61, 132.48,
133.92, 136.34, 137.48, 139.50, 143.05, 150.03, 165.45,
168.18, 168.57. Anal. Calcd. for C24H20N4O3S2 (476): C,
60.49; H, 4.23; N, 11.76%. Found: C, 60.71; H,
4.31; N, 11.64%.
4.3.8 | 5-(4-Chlorobenzylidene)-
3-(4-acetamidophenyl)-
2-(4-acetamidophenylimino)-thiazolidin-
4-one (6h)
Yellow crystals; yield 73%; m.p. = 308-309ꢀC. IR
(υ/cm−1): 3291 (N–H), 1713, 1673 (C O), 1635 (C N).
1H NMR (DMSO-d6) δ: 2.03 (s, 3H, CH3), 2.07 (s, 3H,
CH3), 6.90 (d, J = 7.5 Hz, 2H, Ar–H), 7.42 (d, J = 7.5 Hz,
2H, Ar–H), 7.57 (broad s, 6H, Ar–H), 7.70 (d, J = 7.5 Hz,
2H, Ar–H), 7.78 (s, 1H, CH C), 9.95 (s, 1H, NH), 10.14
(s, 1H, NH). 13C NMR (DMSO-d6) δ: 23.94, 24.01, 119.30
(2C), 119.92 (2C), 121.10 (2C), 122.11, 128.80 (2C),
128.91, 129.39 (2C), 131.49 (2C), 132.27, 134.52, 136.35,
139.54, 142.86, 150.02, 165.54, 168.15, 168.54. Anal.
Calcd. for C26H21ClN4O3S (504): C, 61.84; H, 4.19; N,
11.09%. Found: C, 61.71; H, 4.12; N, 11.00%.
4.4 | Cytotoxicity assay
Cell viability was assessed by the mitochondrial depen-
dent reduction of yellow MTT (3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyl tetrazolium bromide) to purple
formazan.[30] All the procedures were done in a sterile
area using a Laminar flow cabinet biosafety class II level
(Baker, SG403INT, Sanford, ME, USA). Cells were
suspended in DMEM medium for MCF-7, 1% antibiotic-
antimycotic mixture (10 000 U/mL potassium penicillin,
10 000 μg/mL streptomycin sulfate and 25 μg/mL
amphotericin B) and 1% L-glutamine at 37ꢀC under 5%
CO2. Cells were batch cultured for 10 days, then seeded
at concentration of 10 × 103 cells/well in fresh complete
growth medium in 96-well microtiter plastic plates at
4.3.9 | 5-((5-Methylfuran-2-yl)
methylene)-3-(4-acetamidophenyl)-
2-(4-acetamidophenyl-imino)-thiazolidin-
4-one (6i)
Orange crystals; yield 87%; m.p. > 330ꢀC. IR (υ/cm−1):
3328 (N–H), broad centered at 1673 (C O). 1H NMR

8
ABUMELHA AND SAEED
37ꢀC for 24 hours under 5% CO2 using a water-jacketed
carbon dioxide incubator (Sheldon, TC2323, Cornelius,
OR, USA). Media were aspirated, fresh medium (without
serum) was added, and cells were incubated either alone
(negative control) or with different concentrations of
sample to give a final concentration of (100, 50, 25, and
12.5 μg/mL). After 48 hours of incubation, medium was
aspirated, 40 μL MTT salt (2.5 μg/mL) were added to each
well and incubated for further 4 hours at 37ꢀC under 5%
CO2. To stop the reaction and dissolving the formed crys-
tals, 200 μL of 10% sodium dodecyl sulphate (SDS)
in deionized water was added to each well and incubated
overnight at 37ꢀC. A positive control, which composed
of 100 μg/mL was used as a known cytotoxic natural
agent who gives 100% lethality under the same
conditions.[31,32]
The absorbance was then measured using a micro-
plate multi-well reader (Bio-Rad Laboratories Inc., model
3350, Hercules, California, USA) at 595 nm and a refer-
ence wavelength of 620 nm. A statistical significance was
tested between samples and negative control (cells with
vehicle) using independent t test by SPSS 11 program.
Dimethyl sulfoxide (DMSO) is the vehicle used for disso-
lution of plant extracts, and its final concentration on the
cells was less than 0.2%. The percentage of change in via-
bility was calculated according to the formula: ([Reading
of extract/Reading of negative control] − 1) × 100.
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FUNDING INFORMATION
This research was funded by the Deanship of Scientific
Research at Princess Nourah Bint Abdelulrahman
University through the Fast-track Research Funding
Program.
ORCID
Hana M. A. Abumelha https://orcid.org/0000-0001-
8697-1585
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103035.
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How to cite this article: Abumelha HMA,
Saeed A. Synthesis of some 5-arylidene-2-(4-
acetamidophenylimino)-thiazolidin-4-one
derivatives and exploring their breast anticancer
activity. J Heterocyclic Chem. 2020;1–9. https://doi.
org/10.1002/jhet.3906
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