Synthesis of new 4-oxo-thiazolidine-5-ylidenes of antitumor and antioxidant activities

Article abstract of DOI:10.1002/jhet.290

(Chemical Equation Presented) Reaction of diacyl thiocarbohydrazides with dimethyl but-2-ynedioate in refluxing ethanol led to 4-oxa-thiazolidine-5- ylidene-acetates in good yields. Reaction of the newly prepared N-(2-(propan-2-ylidene) hydrazine-carbonot

Full text of DOI:10.1002/jhet.290

May 2010
Synthesis of New 4-Oxo-thiazolidine-5-ylidenes of Antitumor and
Antioxidant Activities
547
Ashraf A. Aly,a* Alan B. Brown,b Mohamed Abdel-Aziz,c
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, El-Minia 61519, Egypt
bChemistry Department, Florida Institute of Technology, Melbourne, Florida 32901
cDepartment of Medicinal Chemistry, Faculty of Pharmacy, El-Minia University, El-Minia 61519, Egypt
dDepartment of Biochemistry, Division of Genetic Engineering and Biotechnology,
National Research Centre, Cairo, Egypt
*E-mail: ashrafaly63@yahoo.com
Received August 11, 2009
DOI 10.1002/jhet.290
Published online 14 April 2010 in Wiley InterScience (www.interscience.wiley.com).
Reaction of diacyl thiocarbohydrazides with dimethyl but-2-ynedioate in refluxing ethanol led to 4-
oxa-thiazolidine-5-ylidene-acetates in good yields. Reaction of the newly prepared N-(2-(propan-2-ylidene)
hydrazine-carbonothioyl)arylhydrazides with dimethyl but-2-ynedioate gave the corresponding (Z)-methyl-
2-arylhydrazide-4-oxo-3-(propan-2-ylideneamino)thiazolidine-5-ylidene)-acetates. The mechanism is dis-
cussed. Antitumor and antioxidant activities have been also investigated.
J. Heterocyclic Chem., 47, 547 (2010).
INTRODUCTION
thiazolidine-4-ones [20]. The reaction of thioureas with
acetylenic esters has been reported to give a thiazolin-4-
one, an imidazolinthion, or a 1,3-thiazin-4-one [21].
Recent reports by Aly et al. [22] demonstrated that the
reaction of N-aroyl thioureas with of dimethyl but-2-
ynedioate under reflux in acetic acid yielded the corre-
sponding 1,3-thizinones. Additionally, diethyl maleate
reacts with N-substituted-hydrazino-carbothioamides to
form ethyl [1,2,4]triazolo[3,4-b][1,3]thiazine-5-carboxy-
lates [23]. Reaction proceeds via bicyclization and oxi-
dation processes [23]. Whilst 2,3-diphenylcycloprope-
none reacts with ylidene-N-phenylhydrazine-carbothioa-
mides to form the pyrrolo[2,1-b]-1,3,4-oxadiazoles via
formal [2 þ 3]cycloaddition [24]. On the other side, we
reported on one pot synthesis of 1,3-thiazin-2-ylidene-
substituted hydrazides via one-pot reaction of N-substi-
tuted-hydrazino-carbothioamides with 1,4-diphenylbut-2-
The development of simple synthesis routes for
widely used organic compounds from readily available
reagents is one of the major tasks in organic synthesis.
Recent report has shown that various N-ethyl hydrazine-
carbothioamides can undergo different cyclization reac-
tions to give five member heterocycles, which showed a
general stimulation effect on B cell’s response [1]. Thia-
zolidine-4-one ring systems are known to possess anti-
bacterial [2,3], antituberculosis [4–6], antiviral [7–14],
anticancer [15–18], and antioxidant [19]. In view of the
various physiological activities of thiazolidinones, many
thiazolidinone derivatives have been prepared. 4-Phenyl-
thiosemicarbazide reacts smoothly with dimethyl but-2-
ynedioate in the presence of aldehydes or ketones under
solvent free conditions to produce highly functionalized
C
V
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548
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 47
Scheme 1. Synthesis of new 1,3-thiazolidine-4-ones 3a–c.
yne-1,4-dione [25]. On the basis of aforementioned
encouraged results, we investigate the reaction of acyl
thiocarbohydrazides with dimethyl but-2-ynedioate.
Moreover antitumor and antioxidant activities of the iso-
lated products have been investigated.
vinylic-proton are mutually cis. Under gated decoupling,
the ring carbonyl (C-4) couples to vinylic-proton with J
¼ 5.2 Hz, a value which requires a three- not two-bond
coupling as depicted in structures 3b and 3bIII and
excluded the formation of other regioisomers (3bI and
3bII). It was reported, if the coupling constant for the
vinylic-proton and endocyclic carbon atom in a conden-
sation product is about ꢀ5 Hz (vicinal-coupling), this
product has a five-membered ring; if the coupling con-
stant approaches a value of 1 Hz (geminal-coupling),
the product should be assigned six-membered thiazine
structure [27,28]. Most of the CAH coupling constants
are within conventional ranges [29], except that the JC–H
values for C-20 and C-30 are unusually small for ben-
zenes. Presumably this arises from restricted rotation. In
compound 3b there are two p-toluoyl units, one slightly
broadened, which is presumably due to restricted rota-
tion. Toluamide rotation and NH exchange are inde-
pendent processes, which in general occur at different
RESULTS AND DISCUSSION
Chemistry. We have now reacted diacyl thiocarbohy-
drazides 1a–c [26] with dimethyl but-2-ynedioate (2);
the reactions gave mainly the corresponding (Z)-methyl-
2-[(Z)-3-aroylamido-2-(2-arylhydrazono)]-4-oxa-thiazoli-
dine-5-ylidene)-acetates (3a–c, Scheme 1). For structure
prevalent, we choose one derivative identified as 3b and
investigate its NMR in comparative with its expected
regioisomers 3bI–III (Fig. 1). As IR and 13C NMR did
not reveal any absorbance of the C¼¼S group. Moreover,
the five C¼¼S in 13C chemical shifts are all too far
upfield for a C¼¼S. Therefore the upfield five carbon
signals in the 13C NMR spectra of compound 3b must
represent three carbon signals of four C¼¼O and one for
the C¼¼N carbon (see the EXPERIMENTAL SEC-
TION). Accordingly the structure of the regioisomer
3bI is excluded. The magnitude of the coupling constant
(J ¼ 5.2 Hz) further argues that ring carbonyl (C-4) and
rates. The methoxyl protons are distinctive at dH
¼
3.81; this signal gives HMQC correlation with the
attached carbon at dC ¼ 52.7 and HMBC correlation
with the ester carbonyl at dC ¼ 165.6. The signal (dC ¼
160.9) giving HMBC correlation to vinylic-H (dH
¼
6.96) is assigned as C-4. The carbon (dC ¼ 116.8) giv-
ing HMQC correlation to vinylic-H is assigned as
Figure 1. Structure of some commercial triazolopyrimidine-2-sulfonamide herbicides.
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Synthesis of New 4-Oxo-thiazolidine-5-ylidenes of Antitumor and Antioxidant Activities
549
Table 1
NMR spectroscopic data of compound 3b.
COSY
HMQC
HMBC
Assignment
1H NMR (ppm)
11.67 (bs; 1H)
11.34 (bs; 1H)
7.88 (d, J ¼ 7.7; 2H)
7.77 (bd, J ¼ 6.4; 2H)
7.39 (d, J ¼ 8.0; 2H)
7.31 (bd, J ¼ 6.1; 2H)
6.96 (s; 1H)
3.81 (s; 3H)
2.41 (s; 3H)
2.37 (s; 3H)
13C NMR (ppm)
11.34
11.67
7.39
7.31
7.88
7.77
benzamido-NH
hydrazono-NH
H-20
H-200
H-30
H-300
vinylic-H
OCH3
benzamido-CH3
hydrazono-CH3
165.6 (q, J ¼ 4.3)
164.3 (dt, Jd ¼ 8.6, Jt ¼ 4.2)
163.4 (b)
3.81
7.88
ester C¼O
benzamido-C¼O
11.34
6.96
11.34
7.88, 2.41
7.77, 2.37
6.96
hydrazono-C¼O
160.9 (d, J ¼ 5.2)
C-4
C-20
152.0 (b)
143.0 (q, J ¼ 7.5)
141.8 (bq)
137.5 (s)
C-4
C-400
C-5
C-10
129.8 (t, J ¼ 7.7)
129.2 (ddq, Jd ¼ 165.8, 5.5; Jq ¼ 5.5)
128.9 (bd, J ¼ 136.9)
127.8 (t, J ¼ 7.6)
127.7 (dd, J ¼ 160.9, 6.4)
127.5 (bd, J ¼ 136.3)
116.8 (d, J ¼ 173.7)
52.7 (q, J ¼ 148.2)
21.0 (tq, Jt ¼ 4.9, Jq ¼ 126.7)
20.9 (tq, Jt ¼ 4.9, Jq ¼ 126.7)
7.39
7.31
7.39, 2.41
2.37
C-30
C-300
C-100
7.88
7.77
6.96
3.81
2.41
2.37
7.88
C-20
C-200
vinylic-CH
OCH3
7.39
benzamido-CH3
hydrazono-CH3
vinylic-CH. One other carbon (dC ¼ 137.5) gives
HMBC correlation to vinylic-H, and is assigned as C-5.
The benzamido- and hydrazono-C¼¼O appear distinc-
tively at dC ¼ 164.3 and 163.4, respectively. They give
HMBC correlation to the ortho protons on the attached
tolyl rings at dH ¼ 7.88 and 7.77, respectively.
bon signals and their d values as well as the correspond-
ing coupling constants of compound 3b are as shown in
Table 1.
On the basis of well established chemistry of electro-
philic acetylenes [21], it is reasonable to assume that
compounds 4 resulted from the initial conjugate addition
of the sulfur atom of 2 to the acetylenic ester. Then, the
ester group of intermediate 4 was attacked by the amino
moiety to yield 5 by elimination of methanol molecule
(Scheme 2). Hydrogen shift is then proposed to be
The signals at dC ¼ 143 and 141.8 give HMBC corre-
lation with the ortho protons and CH3 (dH ¼ 7.88, 2.41
and 7.77, 2.37) are assigned as C-40 and C-400, respec-
tively. The assignment of distinctive hydrogen and car-
Scheme 2. Plausible mechanism of 1,3-thiazolidine-4-ones 3a–c.
Journal of Heterocyclic Chemistry
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550
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 47
Scheme 3. Synthesis of 1,3-thiazolidine-4-ones 8a–c.
occurred in 5 to produce the stable heterocycle 3
(Scheme 2). Previously it was reported that thiocarbohy-
drazide (6) condensed with acetone to form the corre-
sponding mono-condensed products [30] likewise in
case of 7a–c (Scheme 3). Herein we reacted compound
2 with aroyl chlorides in presence of acetone. The reac-
tion proceeds successfully to give compounds 7a–c in
good yields (Scheme 3). Interestingly, on reacting the
newly prepared compounds 7a–c with dimethyl but-2-
ynedioate ethyl ester (2), the reaction gave the corre-
sponding thiazolidines 8a–c in good yields (Scheme 3).
a high inhibition of the cell growth of Hep-G2 cells
compared with the growth of untreated control cells, as
concluded from their low IC50 value 36.14 lM. How-
ever, compounds 8a and 3b represents a moderate anti-
tumor agent against Hep-G2 cells. Figure 2 shows the
effect of compounds 3a–c and 8a,b on the growth of
Hep-G2 cells. As measured by MTT assay, results are
represented as percentage of control untreated cells.
Antioxidant activity. DPPH is a stable nonphysiological,
radical, which could provide a relative figure of the radi-
cal scavenging activity of the tested compounds. The
DPPH assay showed that some of the tested compounds
possessed no scavenging activity to DPPH with high
SC50 values (>100 lM) compared to the scavenging ac-
tivity (SC50 8.41) of the well-known antioxidant (ascor-
bic acid, A.A), except compounds 3b, 3c, and 8a which
1
In compound 8a, the H NMR spectrum showed the
two methyl protons are distinctive at dH ¼ 2.06 and
1.94; this signals gives HMQC correlation with the
attached carbon at dC ¼ 25.0 and 18.7 and HMBC cor-
relation with the carbon at dC ¼ 168.9 which is assigned
as C(CH3)2. The methoxyl protons are distinctive at dH
¼ 3.87; this signal gives HMQC correlation with the
attached carbon at dC ¼ 52.6 and HMBC correlation
with the ester carbonyl at dC ¼ 166.1. The signal (dC ¼
162.1) giving HMBC correlation to vinylic-H (dH
¼
6.96) is assigned as C-4. The carbon (dC ¼ 117.3) giv-
ing HMQC correlation to vinylic-H is assigned as
vinylic-CH. One other carbon (dC ¼ 139.4) gives
HMBC correlation to vinylic-H, and is assigned as C-5.
The benzoyl C¼¼O appear at dC ¼166.0 gives HMBC
correlation with ortho protons at (dH ¼ 7.91).
Biological section.
Cytotoxicity against Hep-G2 cells. Using MTT assay, we
studied the effect of the compounds on the proliferation
of human hepatocellular carcinoma after 48 h incuba-
tion. 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 val-
ues (>100 lM), except, compound 3c, which resulted in
Figure 2. The effect of compounds 3a,c and 8a,b on the growth Hep-
G2 cells. As measured by MTT assay. Results are represented as per-
centage of control untreated cells.
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Synthesis of New 4-Oxo-thiazolidine-5-ylidenes of Antitumor and Antioxidant Activities
551
zoyl C¼¼O), 164.5 (C-4), 160.8 (ester C¼¼O), 151.9 (C-2),
137.4 (C-5), 132.8 (C-40), 132.6 (C-10), 131.7 (C-400), 130.6
(C-100), 128.7 (C-30), 128.4 (C-300), 127.7 (C-20), 127.5 (C-200),
116.9 (vinylic-CH), 52.7 (OCH3) ppm. MS (70 eV, EI); m/z
(%) ¼ 424 [Mþ] (24), 312 (20), 283 (32), 281 (100), 138
(20), 104 (63), 91 (25), 77 (96), 69 (24), 57 (14), 51 (24).
Anal. Calcd. for C20H16N4O5S (424.43): C, 56.60; H, 3.80; N,
13.20; S, 7.55. Found: C, 56.50; H, 3.82; N, 13.28; S, 7.86.
(Z)-Methyl-2-[(Z)-3-(4-methylbenzamido)-2-(2-(4-methylbenzoyl)-
hydrazono)-4-oxo-1,3-thiazolidin-5-ylidene]-acetate (3b). Yellow
crystals (methanol), yield ¼ 353 mg (78%), m.p. 270–271ꢁC.
IR (potassium bromide): m ¼ 3070–3005 (Ar-CH), 2990–2850
(aliph.-CH), 1740, 1680, 1640 (C¼¼O), 1612 (C¼¼N) cmꢂ1
.
The NMR: Table 1. MS (70 eV, EI); m/z (%) ¼ 452 [Mþ]
(24), 375 (18), 343.23 (30), 119 (100), 91 (27), 65 (30). Anal.
Calcd. for C22H20N4O5S (452.48): C, 58.40; H, 4.46; N, 12.38;
S, 7.09. Found: C, 58.38; H, 4.63; N, 12.43; S, 7.23.
Figure 3. The antioxidant activity of 3b, 3c, and 8a was investigated
using DPPH assay. The results are represented as SC50 values (lM) as
(mean 6 SE, n ¼ 4)
had effective antioxidant activity with SC50 values of
86.4, 79.2, and 92.8 lM, respectively (Fig. 3).
(Z)-Methyl-2-[(Z)-3-(1-napthamido)-2-(2-(1-naphthoyl)hydra-
zono)-4-oxo-1,3-thiazolidin-5-ylidene]-acetate (3c). Yellow crys-
tals (methanol), yield ¼ 430 mg (82%), m.p. 259–260ꢁC. IR
(potassium bromide): m ¼ 3240, (NH), 3035–3005 (Ar-CH),
2985–2910 (aliph.-CH), 1740, 1691, 1670, 1953 (C¼¼O), 1610
1
(C¼¼N) cmꢂ1. H NMR (400.13 MHz, DMSO-d6): dH ¼ 11.81
EXPERIMENTAL
(b, s, 1H, napthamido-NH), 11.70 (b, s, 1H, hydrazino-NH),
8.55 (d, 1H, H-80, J ¼ 8.1 Hz), 8.22 (d, 1H, H-800, J ¼ 6.4
Hz), 8.17 (d, 1H, H-40, J ¼ 7.8 Hz), 8.12 (d, 1H, H-400, J ¼
8.0 Hz), 8.06–7.95 (m, 2H, H-30,300), 7.89 (d, 1H, H-20, J ¼
6.2 Hz), 7.78 (d, 1H, H-200, J ¼ 6.6 Hz), 7.69 (d, 2H, H-50,500,
J ¼ 7.4 Hz), 7.64–7.56 (m, 4H, H-60,700, 600,700), 7.02 (s, 1H,
vinylic-H), 3.84 (s, OCH3) ppm. 13C NMR (100.6 MHz,
DMSO-d6): dC ¼ 166.9 (napthamido-C¼¼O), 165.7 (ester-
C¼¼O), 165.1 (C¼¼O), 160.6 (C-4), 147.8 (C-2), 137.6 (C-5),
133.2 (C-8a0), 133.1 (C-4a0), 132.3 (C-8a00), 131.2 (C-40),
130.8 (C-10), 130.5 (C-400), 130 (C-30), 129.8 (C-300), 128.3 (C-
4a00), 128.2 (C-100), 127.4 (C-70), 127.0 (C-700), 126.6 (C-60, C-
600), 126.4 (C-20), 126.2 (C-200), 126.1 (C-80), 125.4 (C-800),
124.9 (C-50, C-500), 116.8 (vinylic-CH), 52.7 (OCH3) ppm. MS
(FAB, 70 eV); m/z (%) ¼ 524 [Mþ] (100). Anal. Calcd. for
C28H20N4O5S (524.55): C, 64.11; H, 3.84; N, 10.68; S, 6.11.
Found: C, 63.87; H, 3.95; N, 10.73; S, 6.21.
Synthesis of N-(2-propan-2-ylidene)-hydrazine-carbonothio-
nyl)arylhydrazides 7a–c. To a suspension solution of 6 (0.106
g, 1 mmol) and NaHCO3 (0.126 g, 1.5 mmol) in dry acetone
(20 mL) was stirred at room temperature, the corresponding
acid chloride (1 mmol) in dry acetone (5 mL) was added drop-
wise over a period of 20 min. The reaction mixture was stirred
for further continued 3 h at room temperature then at refluxing
temperature for 15 min. The reaction mixture was filtered and
the salt precipitate was washed three times with chloroform
(20 mL). The solvent of the filtrate was removed under vac-
uum. The obtained precipitate was then washed three times
with 0.1N HCl (5 mL) followed by three times with water (30
mL). The obtained products 7a–c were recrystallized from gla-
cial acetic acid.
Chemistry. TLC analysis was performed on analytical
Merck 9385 silica aluminium sheets (Kiselgel 60) with PF254
indicator. Melting points were determined on Stuart electro-
thermal melting point apparatus and were uncorrected. The IR
spectra were recorded as KBr disks on Shimadzu-408 infrared
spectrophotometer, Faculty of Science, El-Minia University.
The NMR spectra were measured using Bruker AV-400, Flor-
ida Institute of Technology, USA. Chemical shifts were
expressed as d (ppm) with tetramethylsilane as internal refer-
ence. The samples were dissolved in chloroform-d6 and/or di-
methyl sulphoxide (DMSO)-d6, s ¼ singlet, d ¼ doublet, dd ¼
doublet of doublet, and t ¼ triplet. Mass spectra were recorded
on Varian MAT 312 instrument in E1 mode (70 eV), Techni-
¨
sche Universitat Braunschweig, Germany. Elemental analyses
were performed using Varian Elementary device in National
Research Center (Dokki, Giza, Egypt).
Materials. Dimethyl but-2-ynedioate (2) and thiocarbohy-
drazide (6) were bought from Fluka. Diaroyl thiocarbohydra-
zides 1a–c were prepared according to the literature [26].
Reactions between diaroyl thiocarbohyhydrazides 1a–c
with 2. An equal mixture of 1a–c (1 mmol) and 2 (0.142 g, 1
mmol) was heated at reflux in absolute ethanol for 1–5 h (the
reaction was followed by TLC analysis). The solvent was
evaporated under vacuum and the obtained yellow precipitates
were dissolved in dichloromethane and applied on column
chromatography (dichloromethane, silica gel). The obtained
products 3a–c were recrystallized from the stated solvents.
(Z)-Methyl-2-[(Z)-3-benzamido-2-(2-benzoylhydrazono)-4-oxo-
1,3-thiazolidin-5-ylidene]-acetate (3a). Yellow crystals (tolu-
ene), yield ¼ 323 mg (76%), m.p. 261–263ꢁC. IR (potassium
bromide): m ¼ 3240, (NH), 3070–3010 (Ar-CH), 2985–2875
1
(aliph.-CH), 1742, 1696, 1663 (C¼¼O), 1618 (C¼¼N) cmꢂ1. H
4-Methyl-N-(2-propan-2-ylidene)hydrazinecarbono-thionyl)-
benzamide (7a). White crystals, yield ¼ 225 mg (90%), m.p.
172–174ꢁC. IR (potassium bromide): m ¼ 3230–3315 (NH),
3041–3009 (Ar-CH), 2981–2915 (aliph.-CH), 1674 (C¼¼O),
NMR (400.13 MHz, DMSO-d6): dH ¼ 11.78 (b, s, 1H, benza-
mido-NH), 11.44 (b, s, 1H, hydrazino-NH), 7.99 (d, 2H, H-20,
J ¼ 7.5 Hz), 7.87 (d, 2H, H-200, J ¼ 7.0 Hz), 7.71 (t, 1H, H-
40, J ¼ 7.4 Hz), 7.61 (t, 3H, H-300,400, J ¼ 7.4, 7.7 Hz), 7.53 (t,
2H, H-30, J ¼ 7.2 Hz), 6.98 (s, 1H, vinylic-H), 3.81 (s, OCH3)
ppm. 13C NMR (100.6 MHz, DMSO-d6): dC ¼ 165.6 (2 ben-
1617 (C¼¼N), 1365 (C¼¼S) cmꢂ1
.
1H NMR (400.13 MHz,
chloroform-d3): dH ¼ 9.98 (b, s, 1H, NH-2), 9.76 (b, s, 1H,
NH-1), 9.24 (b, s, 1H, NH-3), 7.92 (d, 2H, H-2, J ¼ 7.8 Hz),
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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 47
7.57 (t, 1H, H-4, J ¼ 7.5 Hz), 7.41 (t, 2H, H-3, J ¼ 7.5 Hz),
1.97 (s, 3H, CHa3), 1.89 (s, 3H, CHb3) ppm. 13C NMR (100.6
MHz, chloroform-d3): dC ¼ 132.8 (C-4), 131.5 (C-1), 128.6
(C-3), 128.4 (C-2), 24.8 (CHa3), 19.4 (CH3b) ppm. MS (70 eV,
EI); m/z (%) ¼ 250 [Mþ] (40), 105 (100), 77 (42), 56 (31).
Anal. Calcd. for C11H14N4OS (250.32): C, 52.78; H, 5.64; N,
22.38; S, 12.81. Found: C, 53.03; H, 5.50; N, 22.54; S,
12.97.
CHb3) ppm. 13C NMR (100.6 MHz, chloroform-d3): dC
¼
168.9 (C(CH3)2), 166.1 (ester C¼¼O), 166 (benzoyl C¼¼O),
162.1 (C-4), 152.5 (C-2), 139.4 (C-5), 133 (C-40), 130.9 (C-
10), 128.9 (C-30), 127.7 (C-20), 117.3 (vinylic-CH), 52.6
(OCH3), 25.0 (CHa3), 18.7 (CHb3) ppm. MS (70 eV, EI); m/z
(%) ¼ 360 [Mþ] (30), 217 (28), 105 (100), 77 (32), 56 (20).
Anal. Calcd. for C16H16N4O4S (360.39): C, 53.32; H, 4.47;
N, 15.55; S, 8.90. Found: C, 53.50; H, 4.50; N, 15.34; S,
8.97.
4-Methyl-N-(2-(propan-2-ylidene)hydrazine-carbonothioyl)-
benzohydrazide (7b). White crystals, yield ¼ 243 mg (92%),
m.p. 179–181ꢁC. IR (potassium bromide): m ¼ 3234–3321
(NH), 3033–3005 (Ar-CH), 2978–2914 (aliph.-CH), 1679
(Z)-Methyl-2-[(Z)-2-(2-(4-methylbenzoyl)-hydrazono)-4-oxo-
3-(propan-2-ylidene-amino)-1,3-thiazolidin-5-ylidene]-acetate
(8b). Yellow crystals (methanol), yield ¼ 315 mg (84%),
m.p. 245–247ꢁC. IR (potassium bromide): m ¼ 3200 (NH),
3070–3019 (Ar-CH), 2976–2873 (aliph.-CH), 1735, 1695,
(C¼¼O), 1619 (C¼¼N), 1359 (C¼¼S) cmꢂ1
.
1H NMR (400.13
MHz, chloroform-d3): dC ¼ 9.96 (b, s, 1H, NH-2), 9.72 (b, s,
1H, NH-1), 9.44 (b, s, 1H, NH-3), 7.83 (d, 2H, H-2, J ¼ 7.6
Hz), 7.23 (d, 2H, H-3, J ¼ 7.6 Hz), 2.33 (3H, Ar-CH3), 1.96
(s, 3H, CHa3), 1.90 (s, 3H, CHb3) ppm. 13C NMR (100.6 MHz,
chloroform-d3): dC ¼ 176.4 (C¼¼S), 164.7 (benzoyl C¼¼O),
156.5 (C(CH3)2), 143.4 (C-4), 132.8 (C-1), 129.4 (C-3), 127.6
(C-2), 24.9 (CHa3), 21.5 (Ar-CH3), 19.6 (CHb3) ppm. MS (70
eV, EI); m/z (%) ¼ 264 [Mþ] (30), 119 (100), 91 (32), 56
(27). Anal. Calcd. for C12H16N4OS (264.35): C, 54.52; H,
6.10; N, 21.19; S, 12.13. Found: C, 54.63; H, 6.23; N, 21.41;
S, 12.27.
1665 (C¼¼O), 1645, 1607 (C¼¼N) cmꢂ1
.
1H NMR (400.13
MHz, chloroform-d3): dH ¼ 8.45 (b, s, 1H, hydrazino-NH),
7.80 (d, 2H, H-20, J ¼ 8.0 Hz), 7.27 (d, 2H, H-30, J ¼ 7.9
Hz), 6.93 (s, 1H, vinylic-H), 3.86 (s, OCH3), 2.41 (benzoyl
CH3), 2.04 (s, CH3a), 1.92 (s, CHb3) ppm. 13C NMR (100.6
MHz, chloroform-d3): dC ¼ 168.9 (N¼¼C(CH3)2), 166.1
(ester C¼¼O), 165.3 (benzoyl C¼¼O), 162.2 (C-4), 152.6 (C-
2), 143.7 (C-40), 139.5 (C-5), 129.5 (C-30), 128.0 (C-10),
127.7 (C-20), 117.2 (vinylic-CH), 52.5 (OCH3), 25.0 (CHa3),
21.6 (benzoyl CH3) 18.7 (CHb3) ppm. MS (70 eV, EI); m/z
(%) ¼ 374 [Mþ] (24), 275 (18), 119 (100), 91 (17), 56 (12).
Anal. Calcd. for C17H18N4O4S (374.41): C, 54.53; H, 4.85;
N, 14.96; S, 8.56. Found: C, 54.24; H, 5.07; N, 14.82; S,
8.67.
4-Methyl-N-(2-(propan-2-ylidene)hydrazine-carbono-thioyl)-
naphthamide (7c). White crystals, yield ¼ 288 mg (96%),
m.p. 181–183ꢁC. IR (potassium bromide): m ¼ 3242–3316
(NH), 3030–3012 (Ar-CH), 2981–2919 (aliph.-CH), 1669
(C¼¼O), 1624 (C¼¼N), 1341 (C¼¼S) cmꢂ1 1H NMR (400.13
MHz, chloroorm-d3): dH ¼ 10.7 (b, s, 1H, NH-3), 10 17 (b, s,
1H, NH-1), 10.11 (b, s, 1H, NH-2), 9.0 (d, 1H, H-8, J ¼ 8.1
Hz), 8.05 (d, 1H, H-2, J ¼ 7.4 Hz), 7.98 (d, 1H, H-4, J ¼ 8.0
Hz), 7.91 (d, 1H, H-5, J ¼ 8.0 Hz), 7.79 (dd, 1H, H-7, J ¼
7.7, 7.5 Hz), 7.55 (dd, 1H, H-H-6, J ¼ 7.6, 7.4 Hz), 7.48 (t,
1H, H-3, J ¼ 8.0 Hz), 1.98 (s, 3H, CHa3), 1.91 (s, 3H, CH3b)
ppm. 13C NMR (100.6 MHz, chloroform-d3): dC ¼ 76.4
(C¼¼S), 165.3 (napthoyl C¼¼O), 159.7 (N¼¼C(CH3)2), 137.2
(C-4), 136.8 (C-4a), 133.5 (C-1), 132.4 (C-2), 131.6 (C-8a),
131.1 (C-3), 129.3 (C-5), 129.2 (C-7), 128.6 (C-8), 127.8 (C-
6), 25.1 (CHa3), 19.9 (CHb3) ppm. MS (70 eV, EI); m/z (%) ¼
300 [Mþ] (36), 155 (100), 127 (23), 56 (19). Anal. Calcd. for
C15H16N4OS (300.38): C, 59.98; H, 5.37; N, 18.65; S, 10.67.
Found: C, 59.73; H, 5.23; N, 18.41; S, 10.47.
(Z)-Methyl-2-[(Z)-2-(2-(1-napthoyl)hydrazono)-4-oxo-3-(propan-
2-ylideneamino)-1,3-thiazolidin-5-ylidene]-acetate (8c). Yellow
crystals (methanol), yield ¼ 361 mg (88%), m.p. 224–225ꢁC.
IR (potassium bromide): m ¼ 3064–3006 (Ar-CH), 2968–2879
(aliph.-CH), 1729, 1698, 1671 (C¼¼O), 1648, 1612 (C¼¼N)
cmꢂ1
.
1H NMR (400.13 MHz, chloroform-d3): dH ¼ 9.60 (b,
s, 1H, hydrazino-NH), 9.14 (d, 1H, H-80, J ¼ 8.4 Hz), 8.23 (d,
1H, H-20, J ¼ 7.2 Hz), 7.95 (d, 1H, H-40, J ¼ 8.0 Hz), 7.82
(d, 1H, H-50, J ¼ 8.1 Hz), 7.65 (dd, 1H, H-70, J ¼ 7.7, 7.5
Hz), 7.51 (dd, 1H, H-60, J ¼ 7.5, 7.3 Hz), 7.49 (t, 1H, H-30,
J ¼ 7.9 Hz), 6.95 (s, 1H, vinylic-H), 3.94 (s, OCH3), 2.07 (s,
CHa3), 1.96 (s, CHb3) ppm. 13C NMR (100.6 MHz, chloro-
form-d3): dC ¼ 169.3 (N¼¼C(CH3)2), 167.4 (ester C¼¼O),
167.2 (napthoyl C¼¼O), 164.7 (C-4), 153.9 (C-2), 141.2
a
(C-40), 140.3 (C-5), 134.6 (C-40a), 133.2 (C-20), 132.5 (C-80 ),
131.3 (C-10), 130.2 (C-50), 129.6 (C-70), 128.8 (C-80), 128.1
(C-60), 127.8 (C-30), 118.3 (vinylic-CH), 52.8 (OCH3), 25.5
(CHa3), 19.3 (CHb3) ppm. MS (70 eV, FAB); m/z (%) ¼ 410
[Mþ] (100). Anal. Calcd. for C20H18N4O4S (410.45): C,
58.53; H, 4.42; N, 13.65; S, 7.81. Found: C, 58.28; H, 4.67;
N, 13.82; S, 7.67.
Reactions between aroyl thiocarbohyhydrazides 7a–c with
2. As previously mentioned before: an equal mixture of 7a–c
(1 mmol) and 2 (0.142 g, 1 mmol) was heated at reflux in
absolute ethanol for 10–14 h (the reaction was followed by
TLC analysis). The solvent was evaporated under vacuum.
The obtained products were then dissolved in dichloromethane
and applied on column chromatography (dichloromethane,
silica gel). The obtained pure products were recrystallized
from the stated solvents.
Biological section.
Cell culture. Human hepatocellular carcinoma (HepG2)
cells were routinely cultured in Dulbeco’s Modified Eagle’s
Medium. Media were supplemented with 10% fetal bovine se-
rum, 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 were dissolved in DMSO. All cell culture ma-
terial was obtained from Cambrex BioScience (Copenhagen,
(Z)-Methyl-2-[(Z)-2-(2-benzoylhydrazono)-4-oxo-3-(propan-
2-ylideneamino)-1,3-thiazolidin-5-ylidene]-acetate (8a). Yellow
crystals (methanol), yield ¼ 296 mg (82%), m.p. 216–218ꢁC.
IR (potassium bromide): m ¼ 3235 (NH), 3063–3015 (Ar-CH),
2995–2905 (aliph.-CH), 1736, 1698, 1672 (C¼¼O), 1642, 1608
(C¼¼N) cmꢂ1
.
1H NMR (400.13 MHz, chloroform-d3): dH
¼
8.30 (b, s, 1H, hydrazino-NH), 7.91 (d, 2H, H-20, J ¼ 7.6 Hz),
7.60 (t, 1H, H-40, J ¼ 7.4 Hz), 7.49 (t, 2H, H-30, J ¼ 7.6 Hz),
6.96 (s, 1H, vinylic-H), 3.87 (s, OCH3), 2.06 (s, CHa3), 1.94 (s,
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

May 2010
Synthesis of New 4-Oxo-thiazolidine-5-ylidenes of Antitumor and Antioxidant Activities
553
Denmark). All chemicals were from Sigma/Aldrich, except
mentioned. All experiments were repeated three times, unless
mentioned.
Calculations. The half-maximal scavenging capacity (SC50)
values for each tested compounds and ascorbic acid was esti-
mated via two competitive dose curves.
Cytotoxicty assay. Cytotoxicity of tested samples was meas-
ured using the MTT cell viability assay. MTT (3-[4,5-dimethylth-
iazole-2-yl]-2,5-diphenyltetrazolium bromide) assay is based on
the ability of active mitochondrial dehydrogenase enzyme of liv-
ing cells to cleave the tetrazolium rings of the yellow MTT and
form a dark blue insoluble formazan crystals which is largely
impermeable to cell membranes, resulting in its accumulation
within healthy cells. Solubilization of the cells results in the liber-
ation 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 by
measuring the absorbance at 570 nm [31].
Abs50 of ascorbic acid ¼ (Abs100 ꢂ Abs0)/2.
SC50 of ascorbic acid was calculated using the curve
equation.
SC50 of each compound was determined using the curve
equation using Abs50 of ascorbic acid.
REFERENCES AND NOTES
[1] Mavrova, A. T.; Wesselinova, D.; Tsenov, Y. A.; Denkova,
P. Eur J Med Chem 2009, 44, 63.
[2] Andres, C. J.; Bronson, J. J.; D’Andrea, S. V.; Deshpande,
M. S.; Falk, P. J.; Grant-Young, K. A.; Harte, W. E.; Ho, H.; Misco,
P. F.; Robertson, J. G.; Stock, D.; Sun, Y.; Walsh, A. W. Bioorg Med
Chem Lett 2000, 10, 715.
Reagents preparation. MTT solution: 5 mg/mL of MTT in
0.9% NaCl. Acidified isopropanol: 0.04N HCl in absolute
isopropanol.
[3] Bonde, C. G.; Gaikwad, N. J. Bioorg Med Chem 2004, 12, 2151.
[4] Karalı, N.; Gu¨rsoy, A.; Kandemirli, F.; Shvets, N.; Kaynak,
¨
F. B.; Ozbey, S.; Kovalishyn, V.; Dimoglo, A. Bioorg Med Chem
Procedure. Cells (0.5 ꢃ 105 cells/well) in serum-free media
were placed in a flat bottom 96-well microplate and treated
with 20 lL of different concentrations of each tested com-
pound for 20 h at 37ꢁC, in a humidified 5% CO2 atmosphere.
After incubation, media were removed and 40 lL MTT solu-
tion/well were added and incubated for an additional 4 h.
MTT crystals were solubilized by adding 180 lL of acidified
isopropanol/well and plate was shacked at room temperature,
followed by the photometric determination of the absorbance
at 570 nm using microplate ELISA reader. Triplicate repeats
were performed for each concentration and the average was
calculated.
2007, 15, 5888.
[5] Ku¨c¸u¨kgu¨zel, Sꢀ. G.; Oruc¸, E. E.; Rollas, S.; Sꢀahin, F.;
Ozbek, A. Eur J Med Chem 2007, 37, 197.
¨
[6] Ku¨c¸u¨kgu¨zel, Sꢀ. G.; Kocatepe, A.; De Clercq, E.; Sꢀahin, F.;
Gu¨llu¨ce, M. Eur J Med Chem 2006, 41, 353.
[7] Barreca, M. L.; Balzarini, J.; Chimirri, A.; De Clercq, E.;
¨ ¨
De Luca, L.; Holtje, H. D.; Holtje, M.; Monforte, A. M.; Monforte, P.;
`
Pannecouque, C.; Rao, A.; Zappala, M. J Med Chem 2002, 45, 5410.
[8] Barreca, M. L.; Chimirri, A.; De Luca, L.; Monforte, A. M.;
`
Monforte, P.; Rao, A.; Zappala, M.; Balzarini, J.; De Clercq, E.; Panne-
couque, C.; Witvrouw, M. Bioorg Med Chem Lett 2001, 11, 1793.
[9] Rao, A.; Carbone, A.; Chimirri, A.; De Clercq, E.; Mon-
`
forte, A. M.; Monforte, P.; Pannecouque, C.; Zappala, M. Farmaco
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.
Calculations. Percentage of relative viability was calculated
using the following equation: [Absorbance of treated cells/Ab-
sorbance of control cells)] ꢃ 100.
2003, 58, 115.
[10] Barreca, M. L.; Chimirri, A.; De Clercq, E.; De Luca, L.;
`
Monforte, A. M.; Monforte, P.; Rao, A.; Zappala, M. Farmaco 2003,
58, 259.
[11] Rao, A.; Balzarini, J.; Carbone, A.; Chimirri, A.; De Clercq,
`
E.; Monforte, A. M.; Monforte, P.; Pannecouque, C.; Zappala, M.
Then the half maximal inhibitory concentration IC50 was
calculated from the equation of the dose response curve.
Antioxidant activity (scavenging of DPPH). 1,1-Diphenyl-
2-picrylhydrazyl 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 decolorize to the pale yellow nonradical form
[32]. The change in colorization and the subsequent fall in
absorbance are monitored spectrophotometrically at m ¼ 520
nm.
Antiviral Res 2004, 63, 79.
[12] Rawal, R.; Yenamandra, S. P.; Katti, S. B.; De Clercq, E.
Bioorg Med Chem 2005, 13, 6771.
[13] Kaushik-Basu, N.; Bopda-Waffo, A.; Talele, T. T.; Basu,
A.; Chen, Y.; Ku¨c¸u¨kgu¨zel, Sꢀ. G. Front Biosci 2008, 13, 3857.
[14] Rawal, R.; Tripathi, R.; Katti, S. B.; Pannecouque, C.; De
Clercq, E. Bioorg Med Chem 2007, 15, 1725.
[15] Ramla, M. M.; Omar, M. A.; Tokuda, H.; El-Diwani, H. I.
Bioorg Med Chem 2007, 15, 6489.
`
[16] Ottana, R.; Carotti, S.; Maccari, R.; Landini, I.; Chiricosta,
Reagents preparation. Ethanolic DPPH: 0.1 mM DPPH/
absolute ethanol, standard ascorbic acid solution. Serial dilu-
tions of ascorbic acid in concentrations ranging from 0–2.5
lM in distilled water. A standard calibration curve was plotted
using serial dilutions of ascorbic acid in concentrations ranging
from 0–2.5 lM in distilled water.
G.; Caciagli, B.; Vigorita, M. G.; Mini, E. Bioorg Med Chem Lett
2005, 15, 3930.
[17] Gududuru, V.; Hurh, E.; Dalton, J. T.; Miller, D. D. J Med
Chem 2005, 48, 2584.
[18] Gududuru, V.; Hurh, E.; Dalton, J. T.; Miller, D. D. Bioorg
Med Chem Lett 2004, 14, 5289.
Procedure. In a flat bottom 96-well microplates, a total test
volume of 200 lL was used. In each well, 20 lL of different
concentrations (0–100 lg/mL final concentration) of tested
compounds were mixed with 180 lL of ethanolic DPPH and
incubated for 30 min at 37ꢁC. Triplicate wells were prepared
for each concentration and the average was calculated. Then,
the photometric determination of absorbance at m ¼ 515 nm
was done using microplate ELISA reader.
[19] Clemens, J. A.; Ho, P. P.; Panetta, J. A. Stroke 1991, 22,
1048.
[20] Yavari, I.; Hosseini, N.; Moradi, L. Mon Chem 2008, 139,
133.
[21] George, M. V.; Khetan, S. K.; Gupta, R. K. Adv Heterocycl
Chem 1976, 19, 273.
[22] Aly, A. A.; Ahmed, E. K.; El-Mokadam, K. M. J Hetero-
cycl Chem 2007, 44, 1431.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

554
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 47
[23] Aly, A. A.; Hassan, A. A.; Ibrahim, Y. R.; Abdel-Aziz, M.
[29] (a) Breitmaier, E.; Voelter, W. Carbon-13 NMR Spectros-
copy, 3rd ed.; VCH: Weinheim, 1987; p 134. (b) Pretsch, E.; Bu¨hl-
mann, P.; Badertscher, M. Structure Determination of Organic
Compounds, 4th ed.; Springer: Berlin, 2009; p 80, 93.
[30] Dvorko, M.; Albanov, A.; Chipanina, N.; Sherstyannikova,
L.; Samoilov, V.; Komarova, T.; Glotova, T. Chem Heterocycl Compd
2006, 42, 1421.
J Heterocycl Chem 2009, 46, 687.
[24] Aly, A. A.; Hassan, A. A.; Ameen, M. A.; Brown, A. B.
Tetrahedron Lett 2008, 49, 4060.
[25] Aly, A. A.; Hassan, A. A.; Ibrahim, Y. R. J Chem Res 2008, 699.
[26] Li, Z.; Zhao, Y.; Yang, J. Phosphorus Sulfur Silicon Relat
Elem 2007, 182, 79.
[27] Danilkina, N. A.; Mikhailov, L. E.; Ivin, B. A. Russ J Org
Chem 2006, 42, 783.
[31] Hansen, M. B.; Nielsen, S. E.; Berg, K. J Immunol Methods
1989, 119, 203.
[28] Vo¨geli, U.; von Philipsborn, W.; Nagarajan, K.; Nair, M. D.
Helv Chim Acta 1978, 61, 607.
[32] Van Amsterdam, F. T.; Roveri, A.; Maiorino, M.; Ratti, E.;
Ursini, F. Free Radical Biol Med 1992, 12, 183.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet