An efficient synthesis of thiazolidine-4-ones with antitumor and antioxidant activities

Article abstract of DOI:10.1002/jhe.641

Reaction of 3-aroyl-1-arylthioureas with dimethyl but-2-ynedioate in dichloromethane and catalyzed by triphenylphosphine at -5°C led to (Z)-methyl 2-[(Z)-2-(4-aroylimino)-4-oxo-3-aryl-1,3-thiazolidin-5-ylidene] acetates in good yields. The mechanism is di

Full text of DOI:10.1002/jhe.641

726
An Efficient Synthesis of Thiazolidine-4-ones with Antitumor
Vol 49
and Antioxidant Activities
a
b
c
*
Ashraf A. Aly [1], Alan B. Brown, Mohamed Abdel-Aziz,
Gamal El-Din A. A. Abuo-Rahma,c Mohamed F. Radwan,c
Mohamed Ramadan,c and Amira M. Gamal-Eldeend
aChemistry Department, Faculty of Science, El-Minia University, 61519-El-Minia, Egypt
bChemistry Department, Florida Institute of Technology, Melbourne, Florida, 32901
cDepartment of Medicinal Chemistry, Faculty of Pharmacy,
El-Minia University, 61519-El-Minia, Egypt
dDepartment of Biochemistry, Division of Genetic Engineering and Biotechnology,
National Research Centre, Cairo, Egypt
*
E-mail: ashrafaly63@yahoo.com
Received May 4, 2010
DOI 10.1002/jhe.641
View this article online at wileyonlinelibrary.com.
Reaction of 3-aroyl-1-arylthioureas with dimethyl but-2-ynedioate in dichloromethane and catalyzed by tri-
phenylphosphine at ꢀ5ꢁC led to (Z)-methyl 2-[(Z)-2-(4-aroylimino)-4-oxo-3-aryl-1,3-thiazolidin-5-ylidene]
acetates in good yields. The mechanism is discussed. X-ray structure analysis of one thiazolidine derivative
is described. Antitumor and antioxidant activities have been investigated. One derivative of 1,3-thiazolidine
showed moderate antiproliferative in vitro activity against hepatocellular carcinoma Hep-G2, whereas another
1,3-thiazolidine introduced effective antioxidant activity compared to ascorbic acid.
J. Heterocyclic Chem., 49, 726 (2012).
INTRODUCTION
afford triazolothiazines [16]. Aly et al. recently demonstrated
that the reaction of N-aroyl-N0-arylthioureas 1a-c with
dimethyl but-2-ynedioate (2) under reflux in acetic acid yields
the corresponding 1,3-thiazinones 3a-c (Scheme 1) [17].
Thiazolidin-4-one ring systems are known to act as
analgesic, antibacterial, anticonvulsant, antiparasitic,
anti-inflammatory, herbicidal agents [2–7], and potent
anti-HIV agents [8]. In view of the biological activities
of thiazolidinones, several approaches for their synthesis
have been described [9]. Imidazolidine-2-thiones were
obtained from the oxidative cyclization of 1-benzoyl-3-
aryl-thioureas with bromine and enolizable carbonyl
compounds in the presence of excess triethylamine
[10,11]. Manaka [12] reported the one-pot condensation
of aroyl-arylthiourea with a-halocarbonyl derivatives to
afford 2-acylimino-3-alkyl-3H-thiazolines. Aly et al. have
demonstrated a convenient synthesis of fused thiazoles
from the reaction of aroylphenyl thioureas with p-acceptor
quinones [13], but the reactions of N-aroyl-N0-arylthioureas
with 2,3-diphenyl-cyclopropenone afford E/Z mixtures
of 3-(30-aroylthioureido)-2,3-diphenyl-cinnamicacids [14].
It is also known that the reactions of amidinothioureas,
imidoylthioureas, and thioacylamidines with diethyl azodi-
carboxylate give the corresponding thiadiazoles by oxidative
cyclic S-N bond formation [15]. 1-Acylthiosemicarbazides,
react with phenyl propiolate in acetic acid under reflux to
RESULTS AND DISCUSSION
Chemistry. The protocol (Scheme 2) was as follows. A
solution of 2 was mixed together with triphenylphosphine
at ꢀ5–0ꢁC for 4 h. N-Aroyl-N0-arylthiourea 1a-f was then
added at 0ꢁC, and the reaction mixture was stirred at
room temperature for 12–24 h. TLC indicated that the
products differ in Rf from authentic samples [17] of
thiazin-4-ones 3a-c. The isolated products were identified
as thiazolidin-4-ones 4a-f by NMR, IR, and mass spectra
along with elemental analyses (Scheme 2). Figure 1
shows numbering of compounds 4d (exemplifying 4a-e)
and 4f. The 13C-NMR shifts in the new ring are similar
to each other throughout the series (Table 1), and are
quite different from those of 3a-c [17]. In 1H-coupled
13C-NMR spectra of 4c, 4d, and 4f, the lactam carbonyls
C-4 appear as doublets, whose coupling constants (5.8–5.9
Hz) require that the couplings span three bonds and that C-4
© 2012 HeteroCorporation

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An Efficient Synthesis of Thiazolidine-4-ones with Antitumor and Antioxidant Activities
727
Scheme 1. Synthesis of 1,3-thiazin-4-ones 3a-c.
and the vinylic H be mutually cis [18–20]. The structure
of 4d was unambiguously proved by X-ray structure
analysis (Fig. 2). The mechanism depends upon the
known nucleophilic character of triphenylphosphine,
which catalyzes the conjugate addition of the sulfur lone
pair to the acetylenic ester [21].
the scavenging activity (SC50 = 8.41 mM) of the well-
known antioxidant ascorbic acid. On the other hand
compound 4e has effective antioxidant activity, with an
SC50 value of 99.3 mM.
EXPERIMENTAL
Crystals of general formula, C20H15IN2O4S, were
obtained by slow evaporation of dichloromethane, Mr
506.31, orthorhombic Pbca, Z = 8, a = 15.2205(3) Å,
b = 10.7102(2) Å, c = 24.6618(5) Å, a = 90ꢁ, b = 90ꢁ, g = 90ꢁ,
V = 4020.23(14) Å3, Dx = 1.673 Mg m-3, θ = 2.910–22.986ꢁ,
m = 1.73 mm-1, Mo (l = 0.71073 Å), T = 298 K. The
crystallographic data of 4d have been deposited with
the Cambridge Crystallographic Data Centre as supple-
Chemistry. TLC was performed on analytical Merck 9385
silica aluminium sheets (Kieselgel 60) with PF254 indicator. TLCs
were viewed under n = 254 nm. Column chromatography (CC)
was packed with silica gel. Melting points were determined on a
Stuart electrothermal melting point apparatus and are uncorrected.
IR spectra were recorded as KBr disks on a Shimadzu-408
infrared spectrophotometer, Faculty of Science, Minia University.
1
NMR spectra were measured in CDCl3, at 400 MHz for H and
100 MHz for 13C, using a Bruker AV-400 spectrometer at
Florida Institute of Technology. Chemical shifts are expressed as
δ (ppm) vs. tetramethylsilane (TMS) = 0. Coupling constants are
mentary publication number CCDC 705627.
Biological investigation. Cytotoxicity of compounds 4a-f
against Hep-G2 cells. It was found that 4f showed moderate
1
stated in Hz; H-coupled 13C spectra were measured using gated
antiproliferative in vitro activity against hepatocellular
carcinoma Hep-G2, Using the MTT Cell Viability Assay
[22], we studied the effect of compounds 4d-f on the
proliferation of human hepatocellular carcinoma after 48 h
incubation. Incubation of Hep-G2 cell line with gradual
doses of the compounds led to insignificant change in the
growth of Hep-G2 cells as indicated from their IC50 values
(>100 mM). Figure 3 indicates the effect of compounds
4d-f on the growth of Hep-G2 cells.
Antioxidant activity. 1,1-Diphenyl-2-picrylhydrazyl
(DPPH) is a stable deep violet radical due to its
unpaired electron. In the presence of an antioxidant
radical scavenger, which can donate an electron to
DPPH, the deep violet color decolourizes to the pale
yellow nonradical form [23]. The DPPH assay showed
that both 4d and 4f possessed no scavenging activity to
DPPH with high SC50 values (>100 mM) compared to
decoupling. Correlations were established using 1H-1H COSY,
HMQC, HSQC, and HMBC experiments. Mass spectral data
were recorded on Varian MAT 312 instrument in EI mode (70 eV)
at the Technische Universität Braunschweig, Germany. Elemental
analyses were carried out using Varian El Elementar in National
Research Center, Giza, Egypt. X ray analysis was carried on
Bruker Nonius, Delft & MacScience, National Research Center,
Giza, Egypt.
Materials. Dimethyl but-2-ynedioate ethyl ester (DMAD, 2)
was bought from Fluka. N-Aroyl-N0-arylthioureas 1a-f were
prepared according 0to the literature [24].
N-(4-Iodophenyl)-N -(4-methylbenzoyl)thiourea (1d). White crystals
(EtOH) 333 mg (84%), m.p. 195–197ꢁC. IR: n = 3310, 3340 (2NH),
3090–3009 (Ar―CH), 2960–2830 (aliph. ―CH), 1720 (C¼O)
0
1
cmꢀ1. H-NMR (CDCl3, 400 MHz): δ = 12.66 (bs, 1H, N H), 9.05
(bs, 1H, NH), 7.78 (d, J = 8.2, 2H; H-20), 7.73 (d, J = 8.5, 2H;
H-3), 7.52 (d, J = 8.6, 2H; H-2), 7.35 (d, J = 8.0, 2H; H-30),
2.46 (s, 3H; CH3). 13C-NMR CDCl3, 100 MHz): δ 178.4 (C¼S),
Scheme 2. Reactions of N-aroyl-N0 -arylthioureas with DMAD; synthesis of new 1,3-thiazolidin-4-ones 4a-f. Yields of 4a (82%), 4b (83%), 4c (85%),
4d (74%), 4e (84%), 4f (80%).
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

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A. A. Aly, A. B. Brown, M. Abdel-Aziz, G. E. A. A. Abuo-Rahma, M. F. Radwan, M. Ramadan,
and A. M. Gamal-Eldeen
Vol 49
Figure 1. Numbering of thiazolidine derivatives 4d–f.
166.9 (C¼O), 145.1 (C-40), 138.0 (C-1), 137.4 (C-3), 130.0 (C-30),
128.5 (C-10), 127.6 (C-20), 125.7 (C-2), 91.1 (C-4), 21.7 (CH3).
MS; m/z (70 eV, %) = 396 (24), 395 [M+] (28), 390 (22), 305
(24), 303 (44), 302 (60), 230 (14), 229 (18), 128 (42), 127 (100),
92 (38), 91 (40). Anal. Calcd. for C15H13IN2OS (396.25): C,
45.47; H, 3.31; N, 7.07; S, 8.09. Found: C, 45.66; H, 3.49; N,
6.88; S, 8.22.
Reactions between N-aroyl-N0-aryl-thioureas and dimethyl
but-2-ynedioate (2). To an ice cooled (ꢀ5ꢁC), stirred
suspension of triphenylphosphine (1 mmol) in absolute dry
dichloromethane (10 mL) was added over 15 min a solution of
2 (1 mmol) in the same solvent (10 mL). At 0oC, a solution of
aroyl-arylthioureas (1, 1 mmol) in absolute dichloromethane
(25 mL) was added over 4 h. The reaction mixture was then
stirred at room temperature for 12–24 h (the reaction was
followed by TLC). The solvent was evaporated under vacuum
and the residue was applied to a small column packed (silica
gel) using dichloromethane as eluent.
(EtOAc), 310 mg (81.5%), m.p. 236–237. IR: n = 3097–3080
(Ar―CH), 2990–2870 (aliph.―CH), 1711, 1700, 1645 (C¼O),
1
1602 (C¼N), 1596 (C¼C) cmꢀ1. H-NMR (CDCl3, 400 MHz):
δ = 7.80 (d, J = 6.6, 2H; H-2″), 7.70–7.50 (m, 5H; aromatic H),
7.25 (d, J = 6.6, 2H; H-3″), 6.93 (s, 1H; vinylic-H), 3.85 (s, 3H;
OCH3), 2.34 (s, 3H, Ar-CH3). 13C-NMR: (CDCl3, 100 MHz):
δ = 173.4 (benzoyl―CO), 167.6 (C-4), 165.5 (ester―CO),
158.2 (C-2), 149.2 (C-5), 138.0 (C-4″), 137.8 (C-2″0), 135.4 (C-1″0),
130.4 (C-3″), 128.2 (C-40), 127.6 (C-30), 127.2 (C-1 ), 126.8 (C-2 ),
119.5 (vinylic-CH), 50.4 (OCH3), 32.8 (Ar―CH3). MS; m/z
(70 eV, %) = 380 [M+] (20), 265 (30), 349 (24), 303 (12), 124
(21), 119 (100), 91 (42), 77 (32), 59 (19), 31 (23). Anal.
Calcd. (C20H16N2O4S) C, 63.14; H, 4.24; N, 7.36. Found: C,
63.05; H, 4.20; N, 7.24.
(Z)-Methyl-2-[(Z)-2-(4-methoxybenzoylimino)-4-oxo-3-phenyl-
1,3-thiazolidin-5-ylidene]-acetate (4c). Yellow crystals (EtOAc),
325 mg (82%), m.p. 196–198ꢁC. IR: n = 3080–2980 (Ar―CH),
2980–2870 (aliph.―CH), 1731, 1707, 1665 (C¼O), 1613 (C¼N),
1
1587 (C¼C) cmꢀ1. H-NMR (CDCl3, 400 MHz): δ = 7.98 (d,
(Z)-Methyl-2-[(Z)-2-(benzoylimino)-4-oxo-3-phenyl-1,3-thiazo-
lidine-5-ylidene]acetate (4a). Yellowish white crystals (EtOAc),
325 mg (88.7%), m.p. 222–224ꢁC (lit. [25], 223ꢁC). IR: n = 3080–
2990 (Ar―CH), 1715, 1705, 1655 (C¼O), 1605 (C¼N), 1590
J = 8.9, 2H; H-2″), 7.59 (t, J = 7.3, 2H; H-30), 7.55 (t, J =7.1,
1H; H-40), 7.40 (d, J = 7.1, 2H; H-20), 7.08 (s, 1H; vinylic-H),
6.86 (d, J = 9.0, 2H Hz; H-3″), 3.91 (s, 3H; CO2CH3), 3.84
(s, 3H; ArOCH3). 13C-NMR: (CDCl3, 100 MHz): δ = 176.2
(t, J = 3.7; benzoyl―CO), 165.5 (dq, Jd = 1.6, Jq = 4.0; C-4),
165.1 (s; ester―C¼O), 165.0 (d, J = 5.8 Hz; C-4), 164.1 (m; C-4″),
141.1 (s; C-5), 143.2 (tt, J = 9.5, 2.9; C-10), 132.7 (dd, J = 163.2,
6.9; C-2″), 129.4 (dt, Jd = 160.7, Jt = 8.0; C-40), 129.3 (dd, J =
163.7, 7.8; C-30), 128.6 (ddd, J = 164.0, 6.7, 6.7; C-10), 127.5
(t, J = 7.3; C-1″), 120.3 (d, J = 172.4; vinylic-CH=), 113.7
(dd, J = 161.2, 4.7; C-3″), 55.5 (q, J = 144.5; ArOCH3), 52.8
(q, J = 147.9; CO2CH3). MS; m/z (70 eV, %) = 396 (34, M+),
365 (21), 319 (18), 262 (23), 135 (100), 124 (19), 107 (13), 91
(24), 77 (27), 59 (20), 31 (18). Anal. Calcd. (C20H16N2O5S):
C, 60.60; H, 4.07; N, 7.07. Found: C, 60.31; H, 4.56; N, 6.84.
1
(C¼C) cmꢀ1. H-NMR (CDCl3, 400 MHz): δ = 8.04–7.36 (m,
10H; aromatic H), 7.09 (s, 1H; vinylic H), 3.92 (s, 3H; OCH3).
13C-NMR: (CDCl3, 100 MHz): δ = 172.6 (benzoyl C¼O), 167.0
(C-4), 165.8 (ester C¼O), 0158.0 (C-2), 139.0 (C-4″), 138.6 (C-5),
134.2 (C-1″), 130.0 (C-1 ), 128.2 (C-2″), 128.0 (C-3″), 127.6
(C-4″), 126.8 (C-40), 125.8 (C-30), 125.0 (C–20), 119.0
(vinylic―CH¼), 50.2 (OCH3). MS; m/z (70 eV, %) = 366 [M+]
(38), 334 (40), 307 (18), 289 (28), 261 (24), 145 (14), 124 (29),
105 (100), 77 (37), 59 (17), 31 (22). Anal. Calcd. (C19H14N2O4S):
C, 62.28; H, 3.85; N, 7.65. Found: C, 62.05; H, 3.83; N, 7.67.
(Z)-Methyl-2-[(Z)-2-(4-methylbenzoylimino)-4-oxo-3-phenyl-
1,3-thiazolidin-5-ylidene]acetate (4b). Yellowish white crystals
Table 1
13C-NMR shifts in the new rings of 4a-f and 3a-c.
Compound
C2
C4
C5
=CH
Compound
C2
C4
C6
=CH
4a
4b
4c
4d
4e
4f
158.0
158.2
165.0
164.7
159.6
165.2
167.0
167.6
165.5
165.4
164.7
165.7
138.6
149.2
141.1
140.7
141.1
141.2
119.0
119.5
120.3
120.8
121.0
120.3
3a [16]
3b [16]
3c [16]
158.0
158.2
158.4
172.6
173.4
171.4
155.0
155.2
155.6
134.2
135.4
133.8
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An Efficient Synthesis of Thiazolidine-4-ones with Antitumor and Antioxidant Activities
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7.34 (d, 2H, J = 7.7 Hz; H-3″), 7.24 (d, 2H, J = 7.9 Hz; H-30),
7.09 (s, 1H; vinylic-H), 3.92 (s, 3H; CO2CH3), 2.34 (s, 3H; Aroyl-
CH3), 2.32 (s, 3H; Ar-CH3). 13C-NMR: (CDCl3, 100 MHz): δ =
176.3 (benzoyl-CO), 164.7 (C-4), 163.8 (ester-CO), 159.6 (C-2),
141.1 (C-5), 140.2 (C-4″), 139.8 (C-40), 135.2 (C-1″), 132.4 (C-10),
130.2 (C-30), 129.6 (C-20), 129.2 (C-2″), 128.5 (C-3″), 121.0
(vinylic-CH), 51.4 (ester-CH3), 30.8 (Aroyl-CH3), 28.7 (Ar-CH3).
MS; m/z (70 eV, %) = 394 (69, M+), 379 (13), 363 (18), 275 (19),
124 (25), 105 (11), 91 (100), 59 (14), 31 (19). Anal. Calcd.
(C21H18N2O4S): C, 63.94; H, 4.60; N, 7.10. Found: C, 63.59; H,
5.04; N, 7.15.
(Z)-Methyl-2-[(Z)-3-(4-methoxyphenyl)-2-(1-napthoylimino)-
4-oxo-1,3-thiazolidin-5-ylidene]-acetate (4f).Canary yellow crystals
(EtOAc), 390 mg (87%), m.p. 170–171ꢁC. IR: n = 3080–2960
(Ar―CH), 2980–2870 (aliph.―CH), 1720, 1705, 1650 (C¼O),
1
1615 (C¼N), 1595 (C¼C) cmꢀ1. H-NMR (CDCl3, 400 MHz):
δ = 9.26 (d, J = 8.6, 1H; H-8″), 8.30 (d, J = 7.1, 1H; H-2″), 8.01
(d, J = 8.1, 1H; H-4″), 7.86 (d, J = 8.0, 1H; H-5″), 7.61 (dd, J = 7.4,
7.2, 1H; H-7″), 7.53 (dd, J = 7.3, 7.2, 1H; H-6″), 7.41 (dd, J = 7.8,
7.8, 1H; H-3″), 7.34 (d, J = 8.9, 2H; H-20), 7.089 (s, 1H; vinylic-H),
7.086 (d, J = 8.9, 2H; H-30), 3.92 (s, 3H, CO2CH3), 3.90 (s, 3H;
ArOCH3). 13C-NMR: (CDCl3, 100 MHz): δ = 178.4 (d, J = 5.2;
naphthoyl-CO), 165.7 (dq, Jd = 1.5, Jq = 3.7; C-4), 165.2 (d, J = 5.8;
C-2), 165.1 (s; ester―C¼O), 160.1 (b; C-40), 141.2 (s; C-5), 134.8
(ddd, J = 160.0, 6.5, 6.5; C-4″), 133.9 (ddd, J = 6.5, 6.5, 6.5; C-4″a),
132.9 (ddd, J = 163.2, 8.3, 2.0; C-2″), 131.9 (bdd, J = 7.4, 7.4;
C-8″a), 130.5 (m; C-1″), 128.9 (dd, J = 162.0, 6.1; C-20), 128.6
(ddd, J = 159.1, 6.1, 6.1; C-5″), 128.3 (dd, J = 160.3, 8.3; C-7″),
126.7 (tt, J = 9.9, 2.9; C-10), 126.4 (dd, J = 166.2, 7.8; C-8”/6”),
126.3 (dd, J = 160.1, 9.0; C-6”/8”), 124.6 (d, J = 162.4; C-3″),
120.3 (d, J = 172.6; vinylic-CH), 114.6 (dd, J = 161.0, 5.1; C-30),
55.6 (q, J = 144.2; Ar-OCH3), 52.8 (q, J = 147.9; ester-CH3). MS
m/z (70 eV, %) = 446 (27, M+), 415 (48), 387 (21), 319 (18), 291
(12), 155 (42), 127 (100), 124 (31), 59 (48), 31 (14). Anal. Calcd.
(C24H18N2O5S): C, 64.56; H, 4.06; N, 6.27. Found: C, 64.69; H,
4.28; N, 6.35.
Figure 2. X-ray structure analysis of compound 4d.
(Z)-Methyl-2-[(Z)-3-(4-iodophenyl)-2-(4-methylbenzoylimino)-
4-oxo-1,3-thiazolidin-5-ylidene]-acetate (4d). Pale yellow crystals
(ethyl acetate), 410 mg (81%), m.p. 239–240ꢁC. IR: n = 3080–3009
(Ar―CH), 2986–2850 (aliph.―CH), 1722, 1705, 1645 (C¼O),
1
1610 (C¼N), 1538 (C¼C) cmꢀ0 1. H-NMR (CDCl3, 400 MHz):
δ = 7.91 (d, J = 8.4, 4H; H-2 ,2″), 7.21 (d, J = 8.0, 2H; H-3″),
7.17 (d, J = 8.6, 2H; H-30), 7.06 (s, 1H; vinylic-H), 3.90 (s, 3H;
CO2CH3), 2.40 (s, 3H; ArCH3). 13C-NMR: (CDCl3, 100 MHz):
δ = 176.8 (t, J = 4.1; benzoyl―CO), 165.4 (dq, Jd = 1.2,
Jq = 3.6; C‐4), 165.3 (s; ester―C¼O), 164.7 (d, J = 5.9; C-2),
144.8 (q, J = 6.9; C-4″), 140.7 (s; C-5), 138.5 (dd, J = 168.5,
6.6; C-20), 133.7 (tt, J = 9.7, 2.6; C-10), 131.9 (t, J = 7.5;
C-1″), 130.5 (dd, J = 162.5, 6.1; C-2″), 129.5 (dd, J = 165.5,
5.9; C-30), 129.3 (ddq, Jd = 159.2, 5.2, Jq = 5.2; C-3″),
120.8 (d, J = 172.6; vinylic―CH), 95.2 (tt, J = 10.4, 2.6;
C-40), 52.8 (q, J = 147.9; OCH3), 30.9 (tq, Jt = 4.4, Jq = 127.0;
ArCH3). MS: m/z (70 eV, %) = 508 [M+2], (15), 506 [M+]
(40), 490 (17), 474 (23), 447 (32), 415 (29), 379 (42), 245 (25),
174 (14), 124 (32), 119 (100), 91 (30), 65 (11), 59 (30), 43
(11), 31 (17). Anal. Calcd. (C20H15IN2O4S): C, 47.44; H, 2.99;
N, 5.53. Found: C, 47.51; H, 2.94; N, 5.47.
Biology. Cell culture. Hepatocellular carcinoma (HepG2)
cells were routinely cultured in DMEM (Dulbeco’s
Modified Eagle’s Medium). Media were supplemented
with 10% fetal bovine serum (FBS), 2 mM L-glutamine,
containing 100 units/mL penicillin G sodium, 100 units/mL
streptomycin sulphate, and 250 ng/mL amphotericin B. Cells
were maintained at subconfluency at 37ꢁC in humidified air
containing 5% CO2. For subculturing, monolayer cells were
harvested after trypsin/EDTA treatment at 37ꢁC. Cells were
used when confluence had reached 75%. Tested samples
(Z)-Methyl-2-[(Z)-2-(4-methylbenzoylimino)-3-(4-methylphenyl)-
4-oxo-1,3-thiazolidin-5-ylidene]-acetate (4e). Cottony white crystals
(EtOAc), 335 mg (85%), m.p. 240–241ꢁC. IR: n = 3086–2995
(Ar―CH), 2890–2860 (aliph.―CH), 1722, 1705, 1647 (C¼O),
1610 (C¼N), 1530 (C¼C) cmꢀ1
.
1H-NMR (CDCl3, 400 MHz):
δ = 7.93 (d, J =7.7, 2H; H-2″), 7.42 (d, 2H, J = 7.9 Hz; H-20),
Figure 3. The effect of (a) 4d, (b) 4e, and (c) 4f on the growth of Hep-G2 cells, as measured by MTT assay. Results are represented as percentage of
control untreated cells.
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DOI 10.1002/jhet

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A. A. Aly, A. B. Brown, M. Abdel-Aziz, G. E. A. A. Abuo-Rahma, M. F. Radwan, M. Ramadan,
and A. M. Gamal-Eldeen
Vol 49
were dissolved in dimethyl sulphoxide (DMSO). All cell
culture material was obtained from Cambrex BioScience
(Copenhagen, Denmark). All chemicals were from Sigma/
Aldrich, except mentioned. All experiments were repeated
three times, unless mentioned.
was plotted using serial dilutions of ascorbic acid in
concentrations ranging from 0–2.5 mM in distilled water.
Procedure. In a flat bottom 96-well microplate, a total test
volume of 200 mL was used. In each well, 20 mL of different
concentrations (0–100 mg/mL final concentration) of tested
compounds were mixed with 180 mL of ethanolic DPPH
were mixed and incubated for 30 min at 37ꢁC. Triplicate
wells were prepared for each concentration and the average
was calculated. Then photometric determination of
absorbance at n = 515 nm was made, using microplate
Cytotoxicity assay. Cytotoxicity of tested samples against
Hepatocellular carcinoma (HepG2) was measured using the
MTT Cell Viability Assay. MTT (3-[4,5-dimethylthiazole-
2-yl]-2,5-diphenyltetrazolium bromide) assay is based on
the ability of active mitochondrial dehydrogenase enzyme
of living cells to cleave the tetrazolium rings of the yellow
MTT and form dark blue insoluble formazan crystals to
which cell membranes are largely impermeable, resulting
in its accumulation within healthy cells. Solubilization of
the cells results in the liberation of crystals, which are then
solubilized. The number of viable cells is directly
proportional to the level of soluble formazan dark blue
color. The extent of the reduction of MTT was quantified
ELISA reader.
Calculations. The half maximal scavenging capacity
(SC50) values for each tested compounds and ascorbic acid
was estimated via two competitive dose curves. Abs50 of
ascorbic acid = (Abs100ꢀAbs0)/2. The SC50 of ascorbic
acid was calculated using the curve equation. SC50 of each
compound was determined using the curve equation
utilizing Abs50 of ascorbic acid.
by measuring the absorbance at 570 nm [22].
Reagents preparation. MTT solution: A solution of 5 mg/mL
of MTT in 0.9% of NaCl. Acidified isopropanol: 0.04 N HCl
CONCLUSION
in absolute isopropanol.
In conclusion, we have prepared novel thiazolidin-4-one
derivatives via one-pot reaction between 3-aroyl-1-arylcar-
bonylthioureas with dimethyl but-2-ynedioate. The present
method carries the advantage that the reaction is performed
under neutral conditions. Since in general thiazolidines
have shown antitumor activity, it is valuable to continue
that investigation with the newly synthesized thiazolidines
4a-e.
Procedure. Cells (0.5 X 105 cells/well) in serum-free
media were plated in a flat bottom 96-well microplate, and
treated with 20 mL of different concentrations of each
tested compound for 20 h at 37ꢁC, in a humidified 5% CO2
atmosphere. After incubation, media were removed and
40 mL MTT solution/well were added and Incubated for an
additional 4 h. MTT crystals were solubilized by adding
180 mL of acidified isopropanol/well and the plate was
shaken at room temperature, followed by photometric
determination of the absorbance at n = 570 nm using a
microplate ELISA reader. Triplicate repeats were
performed for each concentration and the average was
calculated. Data were expressed as the percentage of
relative viability compared with the untreated cells
compared with the vehicle control, with cytotoxicity
indicated by <100% relative viability.
Acknowledgments. Purchase of an NMR spectrometer was
assisted by the National Science Foundation (CHE 03-42251).
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