Selectivity of N-aroyl-N′-arylthioureas towards 2-(1,3-Dioxo-1H- inden-2(3H)-ylidene)malononitrile. New synthesis of (Z)-N-((E)-4-amino-1-aryl-5- cyano-6-oxo-1H-indeno[1,2-d][1,3]-thiazepin-2(6H)-ylidene)-4-arylamides of antitumor and antioxidant activities

Article abstract of DOI:10.1002/jhet.344

The reaction between N-aroyl-N′-arylthioureas with 2-(1,3-dioxoindan-2-ylidene)malononitrile furnished indeno[1,2-d][1,3] thiazepines in 70-85% yields. The mechanism of the products' formation is discussed. Some of the products showed effective antitumor and antioxidant activities. The results revealed that compound indenothiazepine derivative showed a high inhibition of the cell growth of Hep-G2 cells is compared with the growth of untreated control cells, as concluded from their low IC₅₀ value 21.73 μM. On the other hand, two indenothiazepine derivatives have an effective antioxidant activity with SC₅₀ values of 62.5 mM and 87.4 mM, respectively.

Full text of DOI:10.1002/jhet.344

May 2010
Selectivity of N-Aroyl-N0-arylthioureas towards 2-(1,3-Dioxo-1H-
inden-2(3H)-ylidene)malononitrile. New Synthesis of (Z)-N-((E)-
4-Amino-1-aryl-5-cyano-6-oxo-1H-indeno[1,2-d][1,3]-
thiazepin-2(6H)-ylidene)-4-arylamides of Antitumor and
Antioxidant Activities
503
Ashraf A. Aly,a* Alan B. Brown,b Mohamed Ramadan,c
Mohamed Abdel-Aziz,c Gamal El-Din A. A. Abuo-Rahma,c
Mohamed F. Radwan,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, Giza, Egypt
*E-mail: ashrafaly63@yahoo.com
Received August 2, 2009
DOI 10.1002/jhet.344
Published online 31 March 2010 in Wiley InterScience (www.interscience.wiley.com).
The reaction between N-aroyl-N0-arylthioureas with 2-(1,3-dioxoindan-2-ylidene)malononitrile fur-
nished indeno[1,2-d][1,3]thiazepines in 70–85% yields. The mechanism of the products’ formation is
discussed. Some of the products showed effective antitumor and antioxidant activities. The results
revealed that compound indenothiazepine derivative showed a high inhibition of the cell growth of
Hep-G2 cells is compared with the growth of untreated control cells, as concluded from their low IC50
value 21.73 lM. On the other hand, two indenothiazepine derivatives have an effective antioxidant
activity with SC50 values of 62.5 mM and 87.4 mM, respectively.
J. Heterocyclic Chem., 47, 503 (2010).
INTRODUCTION
ways of substituted thioureas vary from one reagent to
another. Our synthetic program uses cycloadditions as
efficient methods of preparation of novel heterocycles,
rather than those suffering from low yields because of
the multiple steps described in their preparation [4]. Aly
et al. [5] reported the synthesis of a series of 1,3-thia-
zines by the reaction of N-aroyl-N0-substituted thioureas
with ethyl propiolate, dimethyl but-2-ynedioate, and (E)-
1,4-diphenyl-but-2-ene-1,4-dione. Herein we report on
our findings for the synthesis of various novel thiaze-
pines, during the reaction of various N-aroyl-N0-aryl-thi-
oureas 1a–e [6] with 2-(1,3-dioxo-1H-inden-2(3H)-
ylidene)-malononitrile (2).
The importance of N-aroyl-N0-arylthioureas is found
largely in heterocyclic syntheses. Many of these sub-
strates have interesting biological activities and are used
as a rich source for materials for development of agro-
chemical and pharmaceutical products [1]. Additionally,
it was known that the reaction of amidinothioureas, imi-
doylthioureas, thioacylamidines, amidino-thioureas, o-
methyl-1-aryl-2-thioisobiurets, and 1-aryl-isodithiobiur-
ets with diethyl azodicarboxylate gave the corresponding
thiadiazoles by the oxidative cyclic SAN bond forma-
tion [2]. A series of 3-alkyl-5-methylene-2-arylimino-
1,3-thiazolidin-4-ones were obtained from the reaction
of N-alkyl-N0-arylthioureas with dimethyl but-2-yne-
dioate [3]. Hyrazino-thioureas, such as 1-acylthiosemi-
carbazides reacted with phenyl propiolate in acetic acid
under reflux to afford triazolothiazines [3]. In light of
the aforementioned, it appears that the reaction path-
RESULTS AND DISCUSSION
Chemistry. Scheme 1 outlines the reaction of 1a–e
with 2 in dry ethyl acetate under N2 atmosphere. The
C
V
2010 HeteroCorporation

504
A. A. Aly, A. B. Brown, M. Ramadan, M. Abdel-Aziz, G. E.-D. A. A. Ab-Rahma,
M. F. Radwan, and A. M. Gamal-Eldeen
Vol 47
Scheme 1. Reactions of aroyl thioureas 1a–e with 2; synthesis of (Z)-N-((E)-4-amino-1-aryl-5-cyano-6-oxo-1H-indeno[1,2-d][1,3]thiazepin-2(6H)-
ylidene)-4-arylamides 3a–e. 3a: 75%; 3b: 80%; 3c: 85%; 3d: 82%; 3e: 70%.
reaction proceeded to yield, after chromatographic puri-
fication and recrystallization, compounds 3a–e (70–
85%). We chose N-aroyl-N0-arylthioureas 1a–e having
aryl groups with electron donating and withdrawing sub-
stituents on the benzene ring to examine their effect on
the course of reaction. The structure of 3a–e was estab-
lished on the basis of mass, IR, 1H NMR, 13C NMR
spectra, and elemental analyses. Mechanistically, the
formation of compounds 3a–e can be explained as
because of addition of the sulfur atom nonbonded pair
of 1a–e to the nitrile group in 2 (Scheme 2). The
formed intermediate 4 undergoes a hydrogen shift to
give 5. Cyclization then occurs via addition of the NH
lone-pair to the carbonyl group, followed by elimination
of water, to give the stable heterocyclic compounds 3a–
e. The NMR spectra excluded the formation of spiro-
indolothiazines 7a–e, but all the data are consistent with
indeno[1,2-d][1,3]thiazepines 3a–e (Scheme 2).
Compounds 3a–e show IR absorptions at m ¼ 3120–
3350, 2222–2200, 1733–1665, and 1649–1595 cmꢀ1
corresponding to the NH2, nitrile, carbonyl, and azome-
thine groups, respectively. The spectra are strikingly
similar: clearly the products are of the same general
type, and share some substructures. The spectra show
Scheme 2. Suggested mechanism of the reaction of 1a–e with 2.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

May 2010
Selectivity of N-Aroyl-N0-arylthioureas towards
2-(1,3-Dioxo-1H-inden-2(3H)-ylidene)malononitrile
505
local symmetry only in the aryl rings, which can appa-
rently rotate about their axes; the indanedione-derived
substructures show individual signals for each proton
and each carbon, requiring these substructures to lack
the plane of symmetry present in 2 and the alternative
(HMQC: dC ¼ 125.0 ppm) is assigned as C-10. H-10 also
gives COSY correlation with one of the two unresolved
protons at dH ¼ 7.76 ppm, which therefore is H-8, 9.
These protons give HMQC correlation with the carbons at
dC ¼ 136.6 and 132.2 ppm. The latter carbons give HMBC
correlation to H-10; the former does not. C-9 is two bonds
from H-10, C-8 is three bonds away; as three-bond CAH
couplings usually give stronger HMBC correlations than
two-bond couplings, dC ¼ 132.2 ppm is assigned as C-8
and dC ¼ 136.6 ppm is assigned as C-9. C-9 gives HMBC
correlation with the signal at dH ¼ 7.98 ppm, which thus is
assigned as H-7 and its attached carbon (dC ¼ 125.5 ppm)
as C-7. In 3b, H-7 appears as a double of doublet with
large and small coupling constants (J ¼ 6.6, 1.4 Hz), as
expected in this position. The remaining 13C signals are
those at dC ¼ 193.2, 170.2, 165.9, 143.4, 133.4, 105.0,
70.9, and 53.1 ppm. The remaining carbon atoms are C-6,
2, 4, 10a, 6a, 10b, 5a. and 5; they are assigned in the order
stated. In 3b and 3c, the signal at dC ¼ 105.2 ppm gives
HMBC correlation to H-10, so it was assigned as C-10b.
Also in 3b and 3c, the signal at dC ¼ 143.5 ppm gives
HMBC correlation to H-7 and either H-8 or H-9; this is
expected for C-10a, as which this signal is therefore
assigned. In 3e and 3b, the signal at dC ¼ 53.2 ppm gives
HMBC correlation to the remaining proton signal at dH ¼
8.22 ppm, which has an integral of 2H, and gives no
HMQC correlation in any of the compounds; its only other
HMBC correlation is in 3b to dC ¼ 165.9. This idea is con-
sistent with chemical-shift simulation using ChemNMR,
which predicts for 3b that C4, 5, and 5a will resonate at dC
¼ 152, 9, and 107.0 ppm, respectively.
1
products 7a–e. Each H NMR spectrum showed a broad,
2H signal near dH ¼ 8.20 ppm, which gives no hetero-
nuclear multiple quantum coherence (HMQC) or hetero-
nuclear single quantum coherence (HSQC) correlation,
suggesting that these protons are not attached to carbon.
In 3e, the p-tolyl CACH3 group is distinctive at dH
2.31 and dC ¼ 21.2 ppm. The proton signal at dH
¼
¼
2.31 ppm gives heteronuclear multiple bond correlation
correlation (HMBC) correlation with one of the signals
at dC ¼ 143.3 ppm, which is assigned as C-40. The
CACH3 1H signal gives COSY correlation with dH
7.21 ppm, and C-40 gives HMBC correlation with dH
¼
¼
7.69 ppm; these correlations suggest that dH ¼ 7.21 and
7.69 ppm are H-30 and H-20, respectively. These signals
showed HMQC correlation with dC ¼ 129.0 and 129.3
ppm, respectively. The p-toluamide carbonyl at dC
¼
175.2 ppm gives HMBC with dH ¼ 7.69 but not 7.21
ppm, which is consistent with the foregoing. The spectra
of 3b contain signals identical to those just described
within dH ¼ 0.02, dC ¼ 0.07 ppm, and J ¼ 0.1 Hz;
these signals are assigned to the toluamide substructure
of 3b. In the HMBC spectrum of 3b, correlation is
observed between H-30 and dC ¼ 132.6 not 133.3 ppm;
therefore, the former is assigned as C-10. By analogy
with the chemical shifts of 3e, dC ¼ 132.6 is assigned
as C-10, and dC ¼ 143.3 ppm is assigned as C-40. Simi-
larly, in 3c the methoxy group is distinctive at dH
¼
The most surprising thing here is the predicted chemi-
cal shift of C-5, which is unusually far upfield for an
sp2-hybridized carbon. The rationale would be that the
electron density at C-5 is higher than normal, due to res-
onance donation by the nitrogen on C-4. However, the
assigned experimental shifts are very close to those of
1,1-dimethoxyethene, and enamines behave similarly to
enol ethers (Fig. 1). The dC values for C-4 and C-5 are
in accordance with the observed trends in the dC values
in push–pull system in alkenes [8]. It is consistent with
these assignments, however, that the amine protons give
HMBC correlation to C-4 and C-5. The remaining
upfield signal at dC ¼ 70.9 ppm is assigned as C-5a.
3.77 and dC ¼ 55.4 ppm. This proton signal gives
HMBC correlation with a signal at dC ¼ 163.0 ppm,
which is assigned as C-40. C-40 gives HMBC correlation
with proton signals at dH ¼ 7.75 and 6.91 ppm, which
thus must be H-20 and H-30. The amide carbonyl at dC
¼ 174.6 gives HMBC correlation with dH ¼ 7.75 ppm,
which leads to assignment of this proton signal as H-20.
By elimination, dH ¼ 6.91 ppm must be H-30. H-20 and
H-30 give HSQC correlation with dC ¼ 132.6 and 113.7
ppm, which therefore are assigned as C-20 and C-30,
respectively. H-30 gives HMBC correlation with the
nonprotonated carbon at dC ¼ 127.4 ppm, which is
assigned as C-10. The signals at dC ¼ 115.0–116.0 ppm
are assigned as nitrile carbons [7]. The carbons at dC
¼ 136.6, 132.2, 125.5, and 125.0 give HMQC correlation
with proton signals at dH ¼ 7.76, 7.76, 7.98, and 6.69
ppm, again with little variation in the chemical shifts.
The most distinctive of these proton signals is that at
dH ¼ 6.69 ppm, which is a doublet in 3b and 3c, sug-
gesting that it is one of the end protons of the four-spin
system (either H-7 or H-10). For the moment arbitrarily
assigning this proton as H-10, the attached carbon
Biological section.
Anticancer activity. The cytotoxicity testing of com-
pounds 3b–d was carried out using solid tumor (Hep-
G2) cells, which were treated with different doses of the
tested compounds and submitted to MTT assay [9]. The
yellow tetrazolium salt is reduced by mitochondrial
enzyme succinate dehydrogenase, present in living cells,
to form insoluble purple formazan crystals, which are
solubilized by the addition of detergent. The relative
viable cells were determined by the amount of MTT
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

506
A. A. Aly, A. B. Brown, M. Ramadan, M. Abdel-Aziz, G. E.-D. A. A. Ab-Rahma,
M. F. Radwan, and A. M. Gamal-Eldeen
Vol 47
Figure 1. Assigned chemical shift values of the carbon signals in 1,1-
dimethoxyethene and enamines.
converted to the insoluble formazan crystals. The data
were expressed as the mean percentage of the viable
cells as compared with the respective control cultures
treated with solvent. Half-maximal growth inhibitory
concentration (IC50) values were calculated from the
line equation of the dose-dependent curve of each com-
pound. Compound 3b resulted in a high inhibition of the
cell growth of Hep-G2 cells compared with the growth
of untreated control cells, as concluded from the IC50
value of 21.73 lM. on the other hand, compounds 3c,d
led to insignificant change in the growth of Hep-G2
cells as indicated from their IC50 values (>100 lM).
Results are represented as percentage of control
untreated cells as shown in Figures 2–4.
Figure 3. The effect of compound 3c on the growth of Hep-G2 cells.
As measured by MTT assay. As measured by MTT assay. Results are
represented as percentage of control untreated cells.
oindan-2-ylidene)-malononitrile (2) was prepared according to
literature [8]. IR spectra were measured of KBr pellets;
absorption frequencies (m) are stated in cmꢀ1. NMR spectra
were measured in DMSO-d6 solution, at 400.13 MHz for 1H
and 100.6 MHz for 13C; chemical shifts are stated in ppm (d),
and coupling constants are stated in Hz.
Chemistry
Reaction between N-aroyl-N0-arylthioureas 1a–e and 2-
Antioxidant activity. 1,1-Diphenyl-2-picrylhydrazyl
(DPPH) is a stable nonphysiological radical, which could
provide a relative figure of the radical scavenging activity
of the tested compounds [10]. The DPPH assay showed
that 3c possessed no scavenging activity to DPPH with
high SC50 values (>100 lM) compared with the scaveng-
ing activity (SC50 8.41) of the well-known antioxidant
(ascorbic acid); on the other hand, compounds 3b and 3d
had effective antioxidant activity with SC50 values of 62.5
and 87.4 lM, respectively (Fig. 5).
(1,3-dioxo-1H-inden-2(3H)-ylidene)malononitrile
(CNIND,
2). To a solution of 2 (0.208 g, 1 mmol) in dry ethyl acetate
(10 mL) a solution of 1a–e (1 mmol) in dry ethyl acetate (10
mL) was added over 10 min at room temperature with stirring.
The reaction mixture was continued with stirring at refluxing
temperature for 24–20 h. The reaction mixture was concen-
trated and the residue was separated by preparative TLC (silica
gel) using toluene:ethyl acetate (2:1) as eluant. The major
zones were extracted with acetone. The isolated products 3a–e
were recrystallized from the stated solvents.
(Z)-N-(E)-4-Amino-5-cyano-6-oxo-1-phenyl-1H-indeno[1,2-
d][1,3]thiazepin-2(6H)-ylidene)benzamide (3a). Yellowish
white crystals (DMF/H2O, 10:1), 336 mg (75%), m.p. 257–
259ꢁC. IR: 3284, 3123 (m, NH2), 3048–3000 (m, Ar-CH),
2989–2913 (m, aliph.-CH), 2213 (s, CN), 1729, 1667 (s,
EXPERIMENTAL
Chemistry: General methods. N-Aroyl-N0-arylthioureas 1a–e
were prepared according to literature [6], whereas 2-(1,3-diox-
Figure 2. The effect of compound 3b on the growth of Hep-G2 cells.
As measured by MTT assay. Results are represented as percentage of
control untreated cells.
Figure 4. The effect of 3d on the growth of Hep-G2 cells. As mea-
sured by MTT assay. Results are represented as percentage of control
untreated cells.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

May 2010
Selectivity of N-Aroyl-N0-arylthioureas towards
2-(1,3-Dioxo-1H-inden-2(3H)-ylidene)malononitrile
507
105.2 (C-11), 70.8 (C-5a), 55.4 (OCH3), 53.2 (C-5). MS (70
eV): m/z (%) ¼ 478 [Mþ] (24), 451 (20), 387 (13), 285 (20),
119 (100), 91 (32), 65 (14). Anal. Calcd. for C27H18N4O3S
(478.52): C, 67.77; H, 3.79; N, 11.71; S, 6.70. Found: C,
67.97; H, 3.70; N, 11.79; S, 6.85.
(Z)-N-(E)-4-Amino-5-cyano-1-(4-metoxylphenyl)-6-oxo-1H-
indeno[1,2-d][1,3]thiazepin-2(6H)-ylidene)-4-methylbenzamide
(3d). Yellowish white crystals (DMF/H2O, 10:1), yield ¼ 404
mg, (82%), m.p. 278–280ꢁC. IR (KBr): 3296, 3132 (m, NH2),
3048–3011 (m, Ar-CH), 2992–2914 (m, aliph.-CH), 2212 (s,
CN), 1731, 1670 (s, C¼¼O), 1645, 1605 (s, C¼¼N). 1H NMR:
8.21 (bs, 2H; NH2), 7.98 (d, J ¼ 8.5, 1H; H-7), 7.78 (t, J ¼
6.7, 2H; H-8,9), 7.72 (d, J ¼ 8.0, 2H; H-20), 7.35 (bs, 2H; H-
200), 7.21 (d, J ¼ 8.0, 2H; H-30), 7.16 (d, J ¼ 8.2, 2H; H-300),
6.65 (d, J ¼ 5.1, 1H; H-10), 3.89 (s, 3H; OCH3), 2.31 (s, 3H;
CACH3). 13C NMR: dC 193.4 (C-6), 175.2 (benzamide C¼¼O),
170.3 (C-2), 165.9 (C-4), 159.6 (C-400), 143.6 (C-40), 143.2 (C-
10a), 136.5 (C-9), 133.4 (C-6a), 132.5 (C-8), 132.3 (C-100),
130.7 (C-200), 129.3 (C-20), 129.0 (C-30), 128.7 (C-10), 125.4 (C-
7), 125.1 (C-10), 116.0 (C-300), 114.5 (CN), 105.4 (C-11), 70.5
(C-5a), 55.5 (OCH3), 53.2 (C-5), 21.1 (CACH3). MS (70 eV):
m/z (%) ¼ 492 [Mþ] (25), 465 (10), 342 (12), 300 (18), 266
(14), 208 (12), 183 (20), 165 (36), 119 (100), 91 (34), 65 (12).
Anal. Calcd. for C28H20N4O3S (492.55): C, 68.28; H, 4.09; N,
11.37; S, 6.51. Found: C, 68.00; H, 4.12; N, 11.20; S, 6.45.
(Z)-N-(E)-4-amino-5-cyano-1-(4-iodophenyl)-6-oxo-1H-
indeno[1,2-d][1,3]thiazepin-2(6H)-ylidene)-4-methylbenzamide
(3e). Yellowish white crystals (ethyl acetate), yield ¼ 412 mg
(70%), m.p. 294–295ꢁC. IR (KBr): 3275, 3150 (m, NH2),
3063–3013 (m, Ar-CH), 2988–2917 (m, aliph.-CH), 2202 (s,
CN), 1732, 1669 (s, C¼¼O), 1643, 1605 (s, C¼¼N). 1H NMR:
8.22 (bs, 2H; NH2), 7.99 (d, J ¼ 8.2, 2H; H-200), 7.98–7.94
(m, 1H; H-7), 7.76–7.72 (m, 2H; H-8,9), 7.69 (d, J ¼ 7.7, 2H;
H-20), 7.28 (d, J ¼ 7.6, 2H; H-300), 7.21 (d, J ¼ 7.7, 2H; H-
30), 6.69–6.65 (m, 1H; H-10), 2.31 (s, 3H; CACH3). 13C
NMR: 193.2 (C-6), 175.2 (benzamide C¼¼O), 170.2 (C-2),
165.9 (C-4), 143.4 (C-10a), 143.3 (C-40), 138.3 (C-200), 136.6
(C-9), 136.1 (C-100), 133.4 (C-6a), 132.6 (C-10), 132.2 (C-8),
131.5 (C-300), 129.3 (C-20), 129.0 (C-30), 125.5 (C-7), 125.0
(C-10), 115.9 (CN), 105.0 (C-11), 96.2 (C-400), 70.9 (C-5a),
53.1 (C-5), 21.2 (CACH3). MS (70 eV): m/z (%) ¼ 588 [Mþ]
(12), 119 (100), 91 (12), 65 (11). Anal. Calcd. for
C27H17IN4O2S (588.42): C, 55.11; H, 2.91; N, 9.52; S, 5.45.
Found: C, 55.24; H, 2.88; N, 9.28; S, 5.30.
Figure 5. The antioxidant activity of 3b, 3c, and 3d was investigated
using DPPH assay. The results are represented as SC50 values (lM) as
(Mean6 S.E, n ¼ 4).
C¼¼O), 1649, 1602 (s, C¼¼N). 1H NMR: 8.21 (bs, 2H; NH2),
7.97 (dd, J ¼ 6.8, 1.6, 1H; H-7), 7.92–7.29 (m, 10H; Ar-H),
6.63 (d, J ¼ 6.5, 1H; H-10). 13C NMR: 193.2 (C-6), 175.3
(benzamide C¼¼O), 170.3 (C-2), 165.7 (C-4), 142.9 (C-10a),
136.7 (C-40), 136.2 (C-9), 134.7 (C-10), 134 (C-100), 133.5 (C-
6a), 131.8 (C-8), 129.9 (C-20), 128.7 (C-30), 127.6 (C-300),
125.5 (C-7), 125.4 (C-10), 125.1 (C-200), 124.6 (C-400), 116.3
(CN), 104.9 (C-11), 70.9 (C-5a), 54.1 (C-5). FAB MS: m/z
(%)
¼
448 [Mþ] (22). Anal. Calcd. for C26H16N4O2S
(448.10): C, 69.63; H, 3.60; N, 12.49; S, 7.15. Found: C,
69.79; H, 3.49; N, 12.52; S, 7.28.
(Z)-N-(E)-4-Amino-5-cyano-1-(4-methylphenyl)-6-oxo-1H-
indeno[1,2-d][1,3]thiazepin-2(6H)-ylidene)-4-methylbenzamide
(3b). Yellowish white crystals (DMF/H2O, 10:1), yield ¼ 381
mg (80%), m.p. 271–272ꢁC. IR (KBr): 3295, 3120 (w, m, NH2),
3070–3000 (m, Ar-CH), 2990–2910 (m, aliph.-CH), 2210 (s,
CN), 1733, 1669 (s, s, C¼¼O), 1645, 1608 (s, s, C¼¼N). 1H
NMR: 8.22 (bs, 2H; NH2), 7.98 (dd, J ¼ 6.6, 1.4, 1H; H-7),
7.75 (t, J ¼ 5.7, 2H; H-8,9), 7.7 (d, J ¼ 8.1, 2H; H-20), 7.43 (d,
J ¼ 8.2, 2H; H-300), 7.33 (bd, J ¼ 7.2, 2H; H-200), 7.19 (d, J ¼
8.0, 2H; H-30), 6.61 (d, J ¼ 6.8, 1H; H-10), 2.47 (s, 3H; H-4a00),
2.3 (s, 3H; H-4a0). 13C NMR: 193.4 (C-6), 175.2 (benzamide
C¼¼O), 170.1 (C-2), 165.9 (C-4), 143.6 (C-10a), 143.2 (C-40),
139.1(C-400), 136.5 (C-9), 133.7 (C-100), 133.3 (C-6a), 132.6 (C-
10), 132.3 (C-8), 129.9 (C-300), 129.2 (C-20), 129.1 (C-200), 129.0
(C-30), 125.4 (C-7), 125.0 (C-10), 116.0 (CN), 105.3 (C-11),
70.6 (C-5a), 53.2 (C-5), 21.1 (C-4a0), 20.9 (C-4a00). MS (70 eV):
m/z (%) ¼ 476 [Mþ] (24), 449 (23), 342 (14), 284 (12), 183
(10), 149 (20), 119 (100), 91 (40), 65 (18). Anal. Calcd. for
C28H20N4O2S (476.55): C, 70.57; H, 4.23; N, 11.76; S, 6.73.
Found: C, 70.69; H, 4.29; N, 11.52; S, 6.58.
(Z)-N-(E)-4-Amino-5-cyano-6-oxo-1-phenyl-1H-indeno[1,2-
d][1,3]thiazepin-2(6H)-ylidene)-4-methoxybenzamide (3c).
Yellowish white crystals (acetone), yield ¼ 407 mg, (85%),
m.p. 270–271ꢁC. IR (KBr): 3350, 3150 (w, NH2), 3050–3013
(m, Ar-CH), 2996–2923 (m, aliph.-CH), 2200 (s, CN), 1727,
1665 (s, C¼¼O), 1608 (s, C¼¼N). 1H NMR: 8.22 (bs, 2H;
NH2), 7.97 (d, J ¼ 7.4, 1H; H-7), 7.75 (d, J ¼ 8.4, 2H; H-20),
7.71–7.68 (m, 2H; H-8,9), 7.63 (bs, 3H; H-300,400), 7.45 (bs,
2H; H-200), 6.91 (d, J ¼ 8.7, 2H; H-30), 6.57 (d, J ¼ 7.5, 1H;
H-10), 3.77 (s, 3H; OCH3). 13C NMR: 193.4 (C-6), 174.6
(benzamide C¼¼O), 169.6 (C-2), 165.9 (C-4), 163 (C-40), 143.5
(C-10a), 136.5 (C-9), 136.4 (C-100), 133.5 (C-6a), 132.6 (C-20),
131.4 (C-8), 129.6 (C-200), 129.5 (C-400), 129.4 (C-300), 127.4
(C-10), 125.4 (C-7), 125.0 (C-10), 116.0 (CN), 113.7 (C-30),
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 serum, 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 dis-
solved in dimethyl sulphoxide (DMSO). All cell culture material
was obtained from Cambrex BioScience (Copenhagen, Den-
mark). All chemicals were obtained from Sigma/Aldrich, USA,
except mentioned. All experiments were repeated three times,
unless mentioned.
Cytotoxicity assay. Cytotoxicity of tested samples was
measured using the MTT cell viability assay. MTT (3-[4,5-
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

508
A. A. Aly, A. B. Brown, M. Ramadan, M. Abdel-Aziz, G. E.-D. A. A. Ab-Rahma,
M. F. Radwan, and A. M. Gamal-Eldeen
Vol 47
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 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 liberation of crystals, which are then solubi-
lized. 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 [9].
using serial dilutions of ascorbic acid in concentrations ranging
from 0 to 2.5 lM in distilled water.
Procedure. In a flat bottom 96 well-microplate, 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 80 lL of ethanolic DPPH were
mixed and incubated for 30 min at 37ꢁC. Triplicate wells were
prepared for each concentration and the average was calcu-
lated. Then photometric determination of absorbance at 515
nm was done using a microplate ELISA reader.
Calculations. The half-maximal scavenging capacity (SC50)
values for each tested compounds and ascorbic acid was esti-
mated via two competitive dose curves.
Reagents preparation. MTT solution: 5 mg/mL of MTT dis-
solved in 0.9% NaCl. Acidified isopropanol: 0.04 N HCl in
absolute isopropanol.
Procedure. Cells (0.5 ꢂ 105 cells/well) in serum-free media
were plated 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 the plate was shaken at room tempera-
ture, followed by photometric determination of the absorbance
at 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.
Abs50 of ascorbic acid ¼ ðAbs100 ꢀ Abs0Þ=2
SC50 of ascorbic acid was calculated using the curve equa-
tion. SC50 of each compound was determined using the curve
equation using Abs50 of ascorbic acid.
REFERENCES AND NOTES
[1] Danilkina, N. A.; Mikhailov, L. E.; Ivin, B. A. Russ J Org
Chem 2006, 42, 783.
[2] Kihara, Y.; Kabashima, S.; Uno, K.; Okawara, T.; Yama-
saki, T.; Furukawa, M. Synthesis 1970, 1020.
[3] Danilkina, N. A.; Mikhailov, L. E.; Ivin, B. A. In the 3rd
Euro-Asian Heterocyclic Meeting ‘‘Heterocycles in Organic and Com-
binatorial Chemistry’’ (EAHM-2004), Novosibirsk, Russia, September
12–17, 2004.
Calculations. Percentage of relative viability was calculated
using the following equation:
[4] (a) Aly, A. A. Org Biomol Chem 2003, 1, 756; (b) Aly, A.
A. Tetrahedron 2003, 59, 1739; (c) Aly, A. A.; Ehrhardt, S.; Hopf, H.;
Dix, I.; Jones, P. G. Eur J Org Chem 2006, 335; (d) Hopf, H.; Aly, A.
A.; Swaminathan, V. N.; Ernst, L.; Dix, I.; Jones, P. G. Eur J Org
Chem 2005, 68; (e) Aly, A. A.; Hopf, H.; Ernst, L.; Dix, I.; Jones, P.
G. Eur J Org Chem 2006, 3001; (f) Aly, A. A.; Hopf, H.; Dix, I.;
Jones, P. G. Tetrahedron 2006, 62, 4498.
½Absorbance of treated cells=Absorbance of control cells
ꢂ100
Then the half maximal inhibitory concentration IC50 was
calculated from the equation of the dose-response curve.
Antioxidant activity (scavenging of DPPH). DPPH is a
stable deep violet radical because of its unpaired electron. In
the presence of an antioxidant radical scavenger, which can
donate an electron to DPPH, the deep violet color decolorizes
to the pale yellow nonradical form [10]. The change of color
and the subsequent fall in absorbance are monitored spectro-
photometrically at m ¼ 520 nm.
[5] Aly, A. A.; Ahmed, E. Kh.; El-Mokadem, K. M. J Hetero-
cycl Chem 2007, 44, 1431.
[6] Sarkis, G. Y.; Faisal. E. D. J Heterocycl Chem 1985, 22,
137.
[7] Breitmaier, E.; Voelter, W. Carbon-13 NMR Spectroscopy,
3rd ed.; VCH: Weinheim, 1987, 242.
[8] Gewald, K.; Schindler, R. S. Prakt Chem 1990, 332, 223.
[9] Hansen, M. B.; Nielsen, S. E.; Berg, K. J Immunol Methods
1989, 119, 203.
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 to 2.5
lM in distilled water. A standard calibration curve was plotted
[10] Van Amsterdam, F. T. M.; Roveri, A.; Maiorino, M.; Ratti,
E.; Ursini, F. Free Radic Biol Med 1992, 12, 183.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet