Synthesis and biological evaluation of bis-imidazolidineiminothiones: A comparative study

Article abstract of DOI:10.1002/ardp.201300097

A series of 15 novel symmetrical and non-symmetrical bis- imidazolidineiminothiones (6a-g, 7a-e, 8a,b, and 9) with various substituents at N-(1) (p-tolyl, p-methoxyphenyl, p-ethoxyphenyl, p-chlorophenyl, p-bromophenyl, p-iodophenyl, and 3,5-dichlorophenyl) and different linkers between the N-(3) atoms [4,4′-oxybis(4,1-phenylene), 2,2′-dimethoxybiphenyl, and (1,3,3-trimethylcyclohexyl)methyl)] were prepared in 65-75% yields from substituted N-arylcyanothioformanilides and various bis-isocyanates. Screening for cytotoxicity against the HEPG2, HEP2, MCF7, and HCT116 tumor cell lines gave IC₅₀ values ranging from 6.3 to 84.6 μM, where compounds 6b,d,e,g and 7a were markedly active against a least one cell line, underlining the matching effect of properly positioned substituents on N-(1) and the appropriate N-(3)-N-(3) linker. Likewise, all heterocyles were tested against microbial organisms (Pseudomonas aeruginosa, Sarcina lutea, Bacillus pumilus, and Micrococcus luteus) and fungal strains (Candida albicans and Penicilium chrysogenum). Most compounds showed significant antibacterial and antifungal activities, reaching in certain cases the same level of antimicrobial activity as the standard antibacterial agent erythromycin and the antifungal agent metronidazole. The antimicrobial activity was further supported by quantitative assessment of susceptibilities of a selection of the preceding microorganisms using minimum inhibitory concentration and minimum bactericidal concentration techniques. Finally, the antiviral properties of all compounds were investigated against the viral strains HAV, HSV1, and CoxB4, where 6c,d,f and 7a,c,e were markedly active against one or two viral strains, reducing the virus plaque count of various viral strains by 66 to 88%. Structure activity relationship studies revealed several matching pairs of aromatic substituents on N-(1) and the N-(3)-N-(3) linkers, which could serve to optimize structural features for high activity to eventually render such compounds clinically useful drug agents. Bis-imidazolidineiminothiones with various substituents at N-(1) and differing linkers between the N-(3) atoms were synthesized. The in vitro biological evaluation of these compounds showed significant antitumor, antiviral, antibacterial, and antifungal activities. Copyright

Full text of DOI:10.1002/ardp.201300097

542
Arch. Pharm. Chem. Life Sci. 2013, 346, 542–555
Full Paper
Synthesis and Biological Evaluation of
Bis-Imidazolidineiminothiones: A Comparative Study
Marwa A. M. Sh. El-Sharief1, Ziad Moussa2, and Ahmed M. Sh. El-Sharief3
1
Applied Organic Chemistry Department, National Research Center, Dokki, Cairo, Egypt
Department of Chemistry, Faculty of Science, Taibah University, Almadinah Almunawarrah, Saudi Arabia
Department of Chemistry, Faculty of Science, Al-Azhar University, Nasr City, Cairo, Egypt
2
3
A series of 15 novel symmetrical and non-symmetrical bis-imidazolidineiminothiones (6a–g, 7a–e, 8a,b,
and 9) with various substituents at N-(1) (p-tolyl, p-methoxyphenyl, p-ethoxyphenyl, p-chlorophenyl,
p-bromophenyl, p-iodophenyl, and 3,5-dichlorophenyl) and different linkers between the N-(3) atoms
[4,40-oxybis(4,1-phenylene), 2,20-dimethoxybiphenyl, and (1,3,3-trimethylcyclohexyl)methyl)] were
prepared in 65–75% yields from substituted N-arylcyanothioformanilides and various bis-isocyanates.
Screening for cytotoxicity against the HEPG2, HEP2, MCF7, and HCT116 tumor cell lines gave IC50
values ranging from 6.3 to 84.6 mM, where compounds 6b,d,e,g and 7a were markedly active against
a least one cell line, underlining the matching effect of properly positioned substituents on N-(1) and
the appropriate N-(3)-N-(3) linker. Likewise, all heterocyles were tested against microbial organisms
(Pseudomonas aeruginosa, Sarcina lutea, Bacillus pumilus, and Micrococcus luteus) and fungal strains (Candida
albicans and Penicilium chrysogenum). Most compounds showed significant antibacterial and antifungal
activities, reaching in certain cases the same level of antimicrobial activity as the standard antibacterial
agent erythromycin and the antifungal agent metronidazole. The antimicrobial activity was further
supported by quantitative assessment of susceptibilities of a selection of the preceding microorganisms
using minimum inhibitory concentration and minimum bactericidal concentration techniques.
Finally, the antiviral properties of all compounds were investigated against the viral strains HAV, HSV1,
and CoxB4, where 6c,d,f and 7a,c,e were markedly active against one or two viral strains, reducing the
virus plaque count of various viral strains by 66 to 88%. Structure activity relationship studies revealed
several matching pairs of aromatic substituents on N-(1) and the N-(3)-N-(3) linkers, which could serve to
optimize structural features for high activity to eventually render such compounds clinically useful
drug agents.
Keywords: Antibacterial / Antifungal / Antitumor / Antiviral / Bis-imidazolidineiminothiones / N-Arylcyanothioformanilides
Received: March 20, 2013; Revised: April 29, 2013; Accepted: April 30, 2013
DOI 10.1002/ardp.201300097
Additional Supporting information may be found in the online version of this article at the publishers web-site.
:
Introduction
novel bioactive heterocycles under mild conditions. Interest-
ingly, aromatic cyanothioformanilides are themselves bioac-
tive and have been reported to exhibit a wide spectrum of
bioactivity [1–3]. Several classical methods [4–14] as well as
more recent eco-friendly protocols [15–30] have been reported
in the literature for the preparation of such compounds.
N-Arylcyanothioformanilides are versatile intermediates that
have been used in heterocyclic annulation reactions with
various electrophilic coupling partners to rapidly assemble
Correspondence: Ziad Moussa, Department of Chemistry, Faculty of
Science, Taibah University, P.O. Box 30002, Almadinah Almunawarrah,
Saudi Arabia.
Additional corresponding authors: Marwa A. M. Sh. El-Sharief,
E-mail: melsheref.2005@gmail.com
Ahmed M. Sh. El-Sharief,
E-mail: zmousa@taibahu.edu.sa
E-mail: alshraef_2008@hotmail.com
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2013, 346, 542–555
Bioactive Bis-Imidazolidineiminothiones
543
Aside from the agricultural and pharmaceutical uses of
N-arylcyanothioformanilides, they have found wide utility in
organic synthesis [5, 9, 31–37]. For our purposes and in the
context of our ongoing effort in the preparation of bioactive
molecules, we have employed N-arylcyanothioformanilides to
prepare various heterocycles with promising pharmacologi-
cal properties. N-Arylcyanothioformanilides are ambident
nucleophiles and may react via the sulfur or nitrogen atom
[38, 39], with the nitrile group serving as an electrophilic
tether that may participate in subsequent transformations.
These inherent features have been employed to design several
“one-pot” annulation reactions to produce various heterocy-
clic cores [4, 5, 7, 8, 40–65].
as they were markedly more active in the 1–5 mg/mL range
relative to imidazolidineiminothiones and related derivatives
as indicated by the median inhibition concentration (IC50)
values [38]. Thus, in view of the scarcity of bis-imidazolidinei-
minothiones and encouraged by their superior bioactivity and
the presence of adjacent imino and thione groups in the 4 and
5 positions which serve as a template for further derivatiza-
tion, the present investigation reports on the preparation and
biological activities (antitumor, antibacterial, antifungal, and
antiviral properties) of a series of fifteen symmetrical and non-
symmetrical novel bis-imidazolidineiminothiones. These con-
tain various substituents at N-(1) and different linkers between
the N-(3) atoms. They were synthesized by the reaction of one
equivalent of various symmetrical and non-symmetrical
diisocyanate linkers with two equivalents of substituted
N-(1)-arylcyanothioformanilides.
We have recently described the preparation of imidazoli-
dineiminothiones (3) and derivatives thereof [8, 38, 39, 46, 47,
62, 63] and reported a detailed investigation of their in vitro
biological activities. These were constructed in one step from
N-arylcyanothioformanilides (1) via a heterocyclic ring closing Results and discussion
reaction with various aromatic isocyanates (2) (Scheme 1). The
resulting heterocyles, which contain adjacent imino and
thione groups in the 4 and 5 positions, respectively, exhibited
a wide range of bioactivity against tumor cell lines and
various viral, microbial, and fungal strains. The preceding
functional groups have been suggested as being the
pharmacophore responsible for the broad spectrum of
biological activities shown by these heterocycles. The above
studies were extended to include the synthesis and a brief
preliminary bioactivity studies of some bis-imidazolidineimi-
nothiones (4) (Scheme 1) containing various substituents at
N-(1) and linkers between the N-(3) atoms as a prelude to a
more elaborate future investigation.
Bis-imidazolidineiminothiones (4) are virtually unknown
and remain unexplored in many respects. It is noted that
literature search returned no references detailing the
preparation and bioactivity of such species besides seven
bis-imidazolidineiminothione compounds reported earlier by
our group [38, 63, 64]. Interestingly, cytotoxic activity studies
against various carcinoma cell lines (brain tumor cell line,
human hepatocellular carcinoma cell line, human breast
adenocarcinoma cell line, human epithelial carcinoma cell
line, and colon carcinoma cell line) underscored the
effectiveness and superiority of bis-imidazolidineiminothiones
Chemistry
The preparation of bis-imidazolidineiminothiones 6a–g with
various aromatic substituents at N-(1) and a n-hexane linker
between N-(3) atoms is illustrated in Scheme 2. Thus, one
equivalent of commercial hexamethylene diisocyanate (5) was
reacted with two equimolar amounts of cyanothioformani-
lides 1a–g containing various aromatic substituents (p-tolyl,
p-methoxyphenyl, p-chlorophenyl, p-bromophenyl, p-iodo-
phenyl, and 3,5-dichlorophenyl) in ether, followed by the
addition of a few drops of triethylamine as a catalyst to
furnish the corresponding 3,30-(hexane-1,6-diyl)bis(1-aryl-4-
imino-5-thioxoimidazolidin-2-ones 6a–g as shown in
Scheme 2. Arylcyanothioformanilides 1a–g were prepared by
either treating an ethanolic solution of the requisite commer-
cial arylisothiocyanate with an aqueous solution of KCN
according to literature protocols [4–13, 38, 39, 63] or through
the preparation of dithiocarbamates from aromatic amines,
followed by conversion to the corresponding isothiocyanates,
and a subsequent cyanation to afford the desired aryl
cyanothioformanilides. The annulation reaction proceeded
smoothly and cleanly by attack of the nitrogen atom of the
arylcyanothioformanilide onto the isocyanate carbons of
hexamethylene diisocyanate, where the ensuing ring closing
Scheme 1. Previous preparation of imidazol-
idineiminothiones with various aromatic sub-
stituents at N-(1) and N-(3).
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544
M. A. M. Sh. El-Sharief et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 542–555
Scheme 2. Preparation of bis-imidazolidine-
iminothiones with various aromatic substitu-
ents at N-(1) and a n-hexane linker between the
N-(3) atoms.
step proceeded by attack of the resulting nitrogen anion onto
the electrophilic nitrile group. The products were obtained as
crystalline solids in 65–75% yields and were purified by
recrystallization from chloroform/n-hexane. These could be
stored at room temperature for prolonged periods of time
without showing any signs of instability as corroborated by
NMR analysis or developing odors indicative of decomposi-
tion. Encouraged by the precedent broad spectrum bioactivity
of imidazolidineiminothiones and the preliminary promising
biological activity of the corresponding bis-imidazolidineimi-
nothiones reported earlier [38, 63, 64], herein we investigate
bis-iminothiones 6a–g as potential antitumor, antibacterial,
antifungal, and antiviral agents that contain the pharmaco-
phore iminothione units separated by a flexible n-hexane
linker between the N-(3) atoms and contain variously
substituted phenyl rings at N-(1). Therefore, a comparative
structure-activity study should uncover the effect exerted by
the N-(1) substituents and highlight the most appropriate
group for optimum bioactivity which will ultimately enable
structural optimization and the design of highly active bis-
imidazolidineiminothione heterocycles. On the other hand,
evaluating the activities of 6a–g with bis-imidazolidineimi-
nothione (vide infra) containing various other linkers between
the N-(3) atoms should reveal any synergetic or opposing
effects with the N-(1) substituents.
Preparation of the second series of symmetrical (7a–e and
8a,b) and unsymmetrical (9) bis-imidazolidineiminothiones
with various substituents on N-(1) atoms (p-tolyl, p-ethoxy-
phenyl, p-chlorophenyl, p-bromophenyl, p-iodophenyl, and
3,4-dichlorophenyl) and different linkers between N-(3) [4,40-
oxybis(4,1-phenylene), 2,20-dimethoxybiphenyl, and (1,3,3-
trimethylcyclohexyl)methyl)] is shown in Scheme 3. Thus,
commercial 4,40-oxybis(isocyanatobenzene) was reacted with
ethereal solutions containing two equivalents of variously
substituted aromatic cyanothioformanilides (p-tolyl, p-ethox-
yphenyl, p-bromophenyl, p-iodophenyl, and 3,4-dichloro-
phenyl). As before, the reactions were catalyzed by the
addition of a few drops of triethylamine to furnish the
corresponding 3,30-(4,40-oxybis(4,1-phenylene))bis(4-imino-1-
aryl-5-thioxoimidazolidin-2-one) products 7a–e as shown in
Scheme 3. The products were obtained as pure crystalline
solids in 65–75% yields and were fully characterized as
before by standard spectroscopic and analytical methods (see
Experimental section). It is noted that this series contains a
rigid 4,40-oxybis(4,1-phenylene) linker between the N-(3) atoms
and variants at N-(1) which is anticipated to illustrate the
Scheme 3. Preparation of symmetrical and
non-symmetrical bis-imidazolidineiminothiones
with various aromatic substituents at N-(1) and
different linkers between the N-(3) atoms.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 542–555
Bioactive Bis-Imidazolidineiminothiones
545
effect exerted by the linker and reveal any potential matching
and mismatching enhancement effect with the N-(1) sub-
stituents. In addition, comparing the bioactivity with that of
the corresponding analogs of series 6a–g will reveal the effect
of the linker unit.
human laryngeal carcinoma cell line (HEP2), human breast
adenocarcinoma cell line (MCF7), and colon carcinoma cell
line (HCT116). The compounds were tested at concentrations
between 10 and 100 mM to test the effect of different
concentrations using the sulforhodamine B (SRB) assay
(Table 1) and the relationship between surviving fraction
and drug concentration was plotted to get the survival curve
of each tumor cell line for each compound (see Supporting
Information section). The sulforhodamine B (SRB) assay was
developed by Skehan et al. [65] to evaluate drug-induced
cytotoxicity for large-scale drug-screening applications. The
SRB assay features a colorimetric end point, is very stable, and
is nondestructive to cells. Under mild acidic conditions, the
protein dye sulforhodamine B binds electrostatically on
protein basic amino acid residues of trichloroacetic acid-fixed
cells. However, under mild basic conditions it can be extracted
from cells and dissolved for measurement. These practical
advantages render the assay an effective and sensitive assay to
measure drug-induced cytotoxicity.
Generally, all cell lines were responsive to dose increase
between 10 and 50 mM as indicated by the ensuing reduction
in surviving fraction, while dose–response was less pro-
nounced between 50 and 100 mM (Table 1). Due to the
encumbered and overlapping nature of the dose–response
curve profiles (see Supporting Information section) and the
large number of data, we opted to use the median inhibition
concentration (IC50) values extracted from the plotted dose–
The synthesis of the next series of symmetrical bis-
imidazolidineiminothiones (8a,b) in which an even more
rigid linker unit (2,20-dimethoxybiphenyl) was used is also
outlined in Scheme 3. These were prepared according to the
conditions outlined for the other bis-imidazolidineimino-
thiones where commercial 2,20-dimethoxy-4,40-biphenylene
diisocyanate was reacted with ethereal solutions containing
two equivalents of the cyanothioformanilide. The physical,
spectroscopic, and microanalytical data are summarized in
the Experimental and Supporting Information sections.
Finally, an unsymmetrical bis-imidazolidineiminothione (9)
was similarly prepared under the usual standard conditions
from isophorone diisocyanate and two molar equivalents of
p-chlorocyanothioformanilide (1d). In this case, the impact of
the new linker, conformation, and any potential advantages
of the unsymmetrical analog will be uncovered.
Biological in vitro evaluation
Antitumor properties
Compounds 6a–g, 7a–e, 8a,b, and 9 were all evaluated for
cytotoxic activity against the following tumor cancer cell
lines: human hepatocellular carcinoma cell line (HEPG2),
Table 1. Evaluation of compounds 6a–g, 7a–e, 8a,b, and 9 against human hepatocellular carcinoma cell line (HEPG2), human
laryngeal carcinoma cell line (HEP2), human breast adenocarcinoma cell line (MCF7), and colon carcinoma cell line (HCT116).
Cell line Sample
Surviving fraction
7a 7b 7c
conc.
(mM)
6a
6b
6c
6d
6e
6f
6g
7d
7e
8a
8b
9
a)Std.
HEPG2
HEP2
0
10
25
50
100
0
10
25
50
100
0
10
25
50
100
0
10
25
50
100
1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
0.66 0.63 0.73 0.47 0.68 0.64 0.69 0.70 0.79 0.73 0.79 0.84 0.78 0.70 0.70 0.33
0.43 0.44 0.56 0.41 0.44 0.56 0.39 0.51 0.60 0.61 0.59 0.63 0.61 0.56 0.59 0.21
0.28 0.29 0.32 0.32 0.34 0.38 0.35 0.30 0.36 0.40 0.44 0.38 0.40 0.35 0.35 0.19
0.29 0.30 0.37 0.35 0.36 0.42 0.34 0.32 0.31 0.36 0.35 0.32 0.36 0.32 0.32 0.26
1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
0.77 0.48 0.53 0.80 0.73 0.68 0.54 0.61 0.87 0.76 0.92 0.75 0.69 0.70 0.92 0.34
0.50 0.36 0.35 0.62 0.48 0.52 0.42 0.51 0.42 0.57 0.80 0.61 0.50 0.39 0.62 0.29
0.29 0.24 0.27 0.36 0.29 0.33 0.34 0.30 0.23 0.41 0.44 0.31 0.32 0.30 0.43 0.22
0.26 0.29 0.22 0.28 0.28 0.30 0.20 0.18 0.20 0.20 0.33 0.23 0.22 0.20 0.26 0.27
1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
0.70 0.59 0.65 0.63 0.67 0.68 0.66 0.67 0.78 0.79 0.84 0.74 0.83 0.72 0.82 0.19
0.50 0.48 0.51 0.49 0.45 0.54 0.47 0.54 0.65 0.59 0.71 0.56 0.51 0.63 0.59 0.17
0.35 0.39 0.36 0.37 0.36 0.42 0.34 0.37 0.44 0.39 0.62 0.39 0.45 0.42 0.44 0.19
MCF7
0.38 0.34 0.43 0.39 0.36 0.4
0.36 0.34 0.37 0.33 0.41 0.37 0.38 0.42 0.43 0.20
HCT116
1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
0.73 0.71 0.80 0.67 0.57 0.62 0.67 0.50 0.84 0.70 0.78 0.72 0.62 0.75 0.85 0.35
0.50 0.48 0.55 0.49 0.59 0.60 0.43 0.44 0.63 0.61 0.62 0.62 0.37 0.56 0.66 0.28
0.37 0.38 0.47 0.41 0.39 0.44 0.38 0.26 0.48 0.41 0.41 0.43 0.43 0.43 0.39 0.24
0.43 0.28 0.45 0.42 0.37 0.30 0.39 0.29 0.42 0.45 0.43 0.39 0.42 0.40 0.43 0.28
a)
Std.: Doxorubicin was used as a standard antiproliferative agent and a positive control.
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546
M. A. M. Sh. El-Sharief et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 542–555
response curves as they comprise a more convenient means
for comparing the activities of 6a–g, 7a–e, 8a,b, and 9 as
antitumor agents. The IC50 values measured are tabulated in
Table 2 (see the Supporting Information for a graphical
representation).
6d,e rendered these bis-imidazolidineiminothiones in the 6a-g
series highly active against three carcinoma cell lines, the
bulkier iodine group of 6f formed a mismatch pair with the
N-(3) n-hexyl linker as it consistently produced much higher
IC50 values across all four cell lines. On the other hand and as
for the effect of the N-(3) linkers, the most interesting and
informative trend observed between the four groups of bis-
imidazolidineiminothiones points to the dominant role
played by the n-hexyl linker. For instance, the IC50 values of
6a,b,e,f against HEPG2 cell line where the N-(3) linker is the
n-hexyl are significantly lower that those measured for
the corresponding analogs 7a,b,d,e where the N-(3) linker is
the rigid 4,40-oxybis(4,1-phenylene). This trend was consistent
across at least two other cell lines where 6a,b,e,f proved
superior to the 7a,b,d,e analogs. Thus, the IC50 values
obtained for 6a,b,e,f against HEP2 and MCF7 were in all
cases lower than those observed for the 7a,b,d,e analogs
against HEP2 and MCF7. The enhancing effect of the n-hexyl
linker is also apparent when a comparison is made with the
analogs of the 2,20-dimethoxybiphenyl linker unit. As follows,
the IC50 values realized for 6d against HEPG2, HEP2, MCF7,
HCT116 are significantly lower and better across three cell
lines than those achieved with its cloro analog 8b against
HEPG2, HEP2, MCF7, and HCT116. Finally, a comparison of
the IC50 values obtained for 6d against those observed with
the unsymmetrical bis-imidazolidineiminothione 9, which
contains an isophorone linker unit, clearly underlines the
synergetic attribute of the n-hexyl linker unit and points to an
opposing effect of the more rigid isophorone linker.
The IC50 values shown in Table 2 and represented
graphically (Supporting Information section) clearly demon-
strate the effectiveness of all bis-imidazolidineiminothiones
against all carcinoma cell lines (with the exception of
compound 7d against MCF7 cell line). In particular, 6d [N-
(1): 4-chlorophenyl; N-(3)-N-(3): n-hexyl], 6e [N-(1): 4-bromo-
phenyl; N-(3)-N-(3): n-hexyl], and 7a [(N-(1): phenyl; N-(3)-N-(3):
4,40-oxybis(4,1-phenylene)] were the most active as they
displayed the lowest IC50 values which ranged from as low
as 6.30–9.60 mM against HEPG2, HEP2, and HCT116 carcino-
ma cell lines. Although 6e exhibited the lowest IC50 values
against two cell lines (HEP2 and MCF7; 6.30 and 21.0 mM,
respectively), it was relatively less effective against MCF7. This
was generally observed across cell lines with other species like
6d and 7a where they showed optimum activity against one
particular cell line but were not consistently as effective
against others.
In order to evaluate the effects exerted by the N-(1)
substituents and N-(3) linkers on bioactivity, a comparative
study of activity was carried out, where applicable, within and
between each of the four groups, respectively. As for the effect
of the N-(1) substituents, while the chloro and bromo groups of
Table 2. IC50 values of compounds 6a–g, 7a–e, 8a,b, and 9
obtained against human hepatocellular carcinoma cell line
(HEPG2), human laryngeal carcinoma cell line (HEP2), human
breast adenocarcinoma cell line (MCF7), and colon carcinoma cell
line (HCT116).
Antimicrobial properties
The heterocycles 6a–g, 7a–e, 8a,b, and 9 were all tested in vitro
for antibacterial activity against the following bacterial
strains: Gram-negative bacteria, Pseudomonas aeruginosa ATCC-
10145, and Sarcina lutea ATCC-9341, and Gram-positive
bacteria, Bacillus pumilus NCTC-8214, Micrococcus luteus ATCC-
25922, and the results are summarized in Table 3 and
represented graphically in the Supporting Information
section. All compounds were dissolved in N,N-dimethylform-
amide at a concentration of 1 ꢀ 10ꢁ2 M and tested for
antimicrobial activity by the agar disk diffusion method [66]
using a 10 cm microplate well diameter and a 100 mL volume
of each concentration. The antibacterial agent erythromycin
was used as a standard. In the disk diffusion technique for
susceptibility testing, compound-impregnated disks are
placed on an agar plate containing a standard suspension
of the desired microorganism. The heterocycle being tested
then diffuses from the disk in a concentration gradient out
into the agar. The plate is incubated for 24 h at 35°C and
visual bacterial growth is observed only in areas in which the
drug concentrations are below those required for growth
inhibition. The inhibition zone diameters are measured with
Sample no.
IC50 (mM)
Carcinoma cell line
HEPG2
HEP2
MCF7
HCT116
6a
6b
6c
6d
6e
6f
6g
7a
7b
7c
7d
7e
8a
8b
9
19.8
20.2
30.6
9.30
20.4
33.0
19.5
25.2
34.8
38.2
40.6
37.8
37.0
31.8
35.2
7.46
25.0
9.30
12.6
36.0
6.30
27.6
14.1
25.2
22.2
37.0
46.0
33.6
25.0
19.5
40.0
7.46
25.0
22.0
25.2
23.8
21.0
34.2
22.2
31.0
43.0
36.6
84.6
34.2
25.0
40.8
40.2
5.94
25.0
23.6
38.8
23.4
35.2
39.0
20.4
9.60
45.6
37.6
38.8
40.6
16.4
35.4
39.4
7.46
Std.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 542–555
Bioactive Bis-Imidazolidineiminothiones
547
Table 3. Antimicrobial activities of compounds 6a–g, 7a–e, 8a,b,
and 9 against Gram negative and Gram positive bacteria.
luteus. It is noted that the same level of antibacterial activity
was realized with 9 against M. luteus as was measured for
erythromycin, underlining the effectiveness of such species.
In general, the enhancing effect of the N-(1) substituent for
each series is clearly reflected in the significant disparity of
antibacterial activity obtained against a given bacterial strain.
For instance, the mean values of inhibition zone diameter
obtained with series 6a–g against P. aeruginosa ranged from as
low as 17 mm with 6b (N-(1): 4-tolyl; N-(3)-N-(3): n-hexyl) and 6f
(N-(1): 4-iodophenyl; N-(3)-N-(3): n-hexyl) to as high as 23 mm
with 6a (N-(1): phenyl; N-(3)-N-(3): n-hexyl) and 6c (N-(1): 4-
methoxyphenyl; N-(3)-N-(3): n-hexyl). Consistent with these
results are those obtained with the same series against S. lutea,
B. pumilus, and M. luteus, although the disparity in mean values
of inhibition zone diameter varied between bacterial strains
and may be less apparent in certain cases.
Compound
no.
Mean values of inhibition zone
diameter (mm)
Test organism
Gram negative
bacteria
Gram positive
bacteria
P.
S.
B.
M.
aeruginosa
lutea
pumilus
luteus
6a
6b
6c
6d
6e
6f
6g
7a
7b
7c
23.0
17.0
23.0
22.0
20.0
17.0
20.0
20.0
17.0
18.0
16.0
0.00
0.00
14.0
27.0
30.0
26.0
27.0
24.0
26.0
28.0
26.0
21.0
23.0
23.0
21.0
21.0
24.0
21.0
23.0
23.0
44.0
18.0
17.0
21.0
18.0
19.0
18.0
19.0
19.0
16.0
16.0
15.0
16.0
19.0
20.0
28.0
32.0
16.0
18.0
17.0
18.0
17.0
17.0
15.0
16.0
16.0
17.0
17.0
16.0
17.0
21.0
32.0
32.0
Regarding the effect of the N-(3)-N-(3) linker, it is evident that
varying such
a unit may have a dramatic effect on
antimicrobial activity, which may be augmented or reduced
depending on whether a matching or mismatching relation-
ship exists with the N-(1) group. Thus, comparing the zone
inhibition diameter values obtained with 9 (N-(1): 4-chloro-
phenyl; N-(3)-N-(3): isophorone) with those obtained with its n-
hexyl and 2,20-dimethoxybiphenyl analogs 6d and 8b,
respectively, against P. aeruginosa, B. pumilus, and M. luteus
indicates the superiority of 9 and much improved activity.
Whereas 6d and 8b produced values ranging from 18.0 to
22.0 mm and 14.0 to 21.0 mm, respectively, compound 9
afforded zone inhibition diameter values ranging from 27 to
32 mm. This clear difference in antibacterial activity observed
between the preceding compounds is a direct reflection of the
reinforcing effect induced by the N-(3)-N-(3) linker. A similar
comparison of relevant compounds from each series with the
appropriate analogs with variant linkers clearly demonstrates
the influence of the N-(3)-N-(3) group, although the optimum
effect of a synergetic combination against a particular strain
may not necessarily extend to other strains.
7d
7e
8a
8b
9
a)Std.
a)
Erythromycin was used as positive control and standard,
whereas DMF was used as negative control.
calipers or automated scanners and are compared with
standard zone size ranges that determine susceptibility,
intermediate susceptibility, or resistance to the screened
compounds.
The mean values of inhibition zone diameter summarized
in Table 3 and represented graphically (see Supporting
Information) show that all bis-imidazolidineiminothiones
tested in the antimicrobial assay possess significant antibac-
terial activity against the growth of P. aeruginosa, S. lutea,
B. pumilus, and M. luteus on solid media, with the exception of
7e and 8a which proved inactive against P. aeruginosa. Whereas
compound 6e (N-(1): 4-bromophenyl; N-(3)-N-(3): n-hexyl)
exhibited the highest antibacterial activity against the Gram
negative bacteria S. lutea, the remaining bis-imidazolidinei-
minothiones with the exception of 9 were particularly
effective against the same bacterial strain, more so than
the other strains, with mean values of inhibition zone
diameter ranging from 21.0 to 28.0 mm. Interestingly,
the antibacterial agent erythromycin was significantly more
effective against S. lutea than the remaining bacterial strains
as well. Among all compounds, the unsymmetrical bis-
imidazolidineiminothione 9 (N-(1): 4-chlorophenyl; N-(3)-N-(3):
isophorone) consistently produced the highest antibacterial
activity against the growth of P. aeruginosa, B. pumilus, and M.
Antifungal properties
The search for newer antifungal agents continues due to the
rapid development of resistance among fungi to the existing
antifungal agents. Potential antifungal agents are evaluated
against clinical isolates of standard strains of fungi by the
broth dilution and disc diffusion methods, where the former
technique follows the standard method of NCCLS M38A [67,
68]. We chose Penicilium chrysogenum as a filamentous fungus,
and Candida albicans as a unicellular fungus in the antifungal
susceptibility tests. Even though novel broad-spectrum
antifungal agents such as triazoles among others were
reported in recent years, the emergence of resistance has
become an obstacle. Thus, more effective classes of such
agents are desired. As such, bis-imidazolidineiminothiones
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548
M. A. M. Sh. El-Sharief et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 542–555
may comprise a new class of antifungal agents. Thus,
compounds 6a–g, 7a–e, 8a,b, and 9 were all tested under
the same conditions for antifungal activity using the
preceding filamentous and unicellular fungi. The results
are tabulated in Table 4 and represented graphically in the
Supporting Information section. The compounds were tested
for antifungal activity by the agar disk diffusion method using
a 10 cm microplate well diameter and a 100 mL volume of
each concentration [68]. The antifungal agent griseofulvin
was used as a standard at the same concentration outlined
above and was tested under the same conditions. All
compounds were dissolved in the negative standard N,N-
dimethylformamide (at a concentration of 1 ꢀ 10ꢁ2 M),
which was inactive in the above assay. All of the above tested
compounds exhibited strong antifungal activity against the
two fungi where the mean value of the inhibition zone
diameter ranged from as high as 27 mm [7a: (N-(1): phenyl; N-
(3)-N-(3): 4,40-oxybis(4,1-phenylene)] to as low as 15.0 mm [7e:
(N-(1): p-bromophenyl; N-(3)-N-(3): 4,40-oxybis(4,1-phenylene)].
In general, most compounds were very effective antifungal
agents and in several cases achieved similar levels of activity
as the standard antifungal agent griseofulvin. It is noted
though that some fluctuation in antibacterial activity for
individual heterocycles was observed across the two strains of
tested fungi. For instance, compounds 7a,c,e proved more
effective against C. albicans than the related filamentous
fungus P. chrysogenum. As anticipated, a clear difference in
antifungal activity is noted between compounds within and
between each series, pointing to the reinforcing and opposing
effects of the N-(1) and N-(3)-N-(3) groups. Most notably, series
7a–e with the 4,40-oxybis(4,1-phenylene) as the N-(3)-N-(3)
linker consistently produced relatively higher inhibition zone
diameter values against C. albicans. The dominant effect of the
N-(3)-N-(3) linker is also illustrated in the high inhibition zone
diameter values obtained by 9 (N-(1): 4-chlorophenyl; N-(3)-N-
(3): isophorone) against C. albicans and P. chrysogenum. The
heterocycle was comparable to the antifungal agent griseo-
fulvin, achieving the highest activity against P. chrysogenum. In
addition, it proved more effective than its n-hexyl 6d and 2,20-
dimethoxybiphenyl 8b analogs, producing 22 mm and
25 mm inhibition zone diameter values. In comparison, 6d
produced 19 mm and 19 mm inhibition zone diameter values
against the two fungal strains and 8b gave 20 mm and
18 mm. Thus, these compounds comprise a promising new
class of promising and potential broad-spectrum antifungal
agents.
MIC and MBC
The minimum inhibitory concentration (MIC) is standard
technique used to measure the lowest concentration of a
compound required to inhibit visible growth of a microor-
ganism being investigated after approximately 24 h of
incubation in the appropriate growth medium, whereas
the minimum bactericidal concentration (MBC) determines
the lowest concentration of a drug that results in 99.9% or
greater reduction of the concentration of microorganisms
after incubation relative to the initial concentration. MIC and
MBC techniques are reliable means to determine the
susceptibilities of microorganisms to drugs and also to
monitor the activity of new ones. These techniques provide a
quantitative assessment of in vitro antimicrobial activity.
Hence, further to the preceding tests of the potential
antimicrobial and antifungal activities of heterocycles 6a–g,
7a–e, 8a,b, and 9 by the agar disk diffusion method, we
conducted a more quantitative assessment of the antimicro-
bial and antifungal activity of a narrow selection of potential
leads by evaluating their minimum inhibitory and bacteri-
cidal concentrations. Usually, MICs are measured through the
macrotube dilution method, which uses broth growth
medium, double serial dilutions of microorganisms, and a
standard antimicrobial inoculum. It is noteworthy that the
MBC test may be more predictive of a favorable infection
outcome than the MIC as noted in certain infections such as
meningitis and endocarditis. MBC measurements can be
conducted conjointly with the MIC test by taking aliquots of
broth from MIC microtiter wells that show no visible growth
and applying them onto antibiotic-free agar plates for
subsequent incubation. Whereas the MIC often approximates
the MBC for certain antibiotic classes, the latter can exceed
Table 4. Antifungal activity of compounds 6a–g, 7a–e, 8a,b, and
9 against C. albicans and P. chrysogenum.
Compound
no.
Mean values of inhibition zone
diameter (mm)
Test organism
C. albicans
P. chrysogenum
6a
6b
6c
6d
6e
6f
6g
7a
7b
7c
20.0
20.0
21.0
19.0
21.0
21.0
18.0
27.0
21.0
26.0
23.0
22.0
17.0
20.0
22.0
27.0
21.0
20.0
20.0
19.0
22.0
17.0
16.0
22.0
19.0
17.0
19.0
15.0
19.0
18.0
25.0
25.0
7d
7e
8a
8b
9
a)Std.
a)
Griseofulvin was used as positive control and standard,
whereas DMF was used as negative control.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 542–555
Bioactive Bis-Imidazolidineiminothiones
549
the MIC significantly, resulting in an overestimation of in vivo
bactericidal activity. If the difference exceeds 32-fold or more,
a microorganism is classified as tolerant to the antimicrobial’s
killing activity.
subtle in some cases like those involving compounds 6a,c,e
against Gram negative bacteria, the impact was clearly
detectable against Gram positive bacteria. However, the effect
of the N-(3)-N-(3) linkers is much more discernible and
dominant as clearly reflected in the significant disparity of
MIC values obtained against a given bacterial strain for
compounds with different linkers. The MBC values shown in
Table 5 matched well with those obtained by the MIC
technique. Thus, similar conclusions can be drawn as was
done earlier for the MIC data.
The MIC and MBC values of compounds 6a,c,e, 7a, and 9
were measured by the macrotube dilution method (Table 5)
following the guidelines of the Clinical and Laboratory
Standard Institute (CLSI) [67, 68] against one fungal species
(C. albicans) and the same four bacterial strains used earlier. To
carry out the tests, concentrations of compounds up to
625 mg/mL were incorporated into growth media according
to published procedures [67, 68]. As shown in Table 5 and
represented graphically in the Supporting Information
section, all compounds tested showed inhibitory effect,
ranging from as low as 78 mg/mL to as high as 625 mg/mL.
In certain cases, such values are low enough to render such
agents as potential candidates for further studies. It is noted
that high MIC values were observed for C. albicans, in line with
what had been reported earlier for the related imidazolidi-
neiminothiones [39]. On the other hand, the lowest MIC values
were obtained against the Gram-negative bacteria P. aerugi-
nosa, whereas modest MIC values were obtained for the
remaining microorganisms. Compound 9 was consistently
the most effective against all bacterial strains, producing MIC
values ranging from 78 to 156 mg/mL. In relation to structure
activity trends, while the effect of the N-(1) substituents was
Antiviral properties
Unlike antibiotics for bacteria, which may exhibit broad
spectrum activity, antivirals are known to specifically target
viral infections and specific viruses. Whereas most antibiotics
destroy their target pathogen, antivirals inhibit their replica-
tion and development. Most common antiviral agents have
been designed to treat influenza A and B viruses, HIV, herpes
viruses, and the hepatitis B and C viruses. Most antivirals are
prone to drug resistance as viruses mutate over time,
becoming less susceptible or even develop complete immuni-
ty. Consequently, designing effective antivirals is challenging
as viruses employ the host’s cells to multiply, thus introduc-
ing a risk of harming the host cells. Currently, there is some
urgency to develop newer agents to address the preceding
issues of resistance and selectivity.
Table 5. Minimum inhibitory and bactericidal concentrations of compounds 6a,c,e, 7a, and 9 obtained against P. aeruginosa, S. lutea,
B. pumilus, M. luteus bacterial strains and the fungus C. albicans.
Compound no.
Minimum inhibitory concentration, MIC (mg/mL)
Test organism
Gram negative bacteria
Gram positive bacteria
Fungus
P. aeruginosa
S. lutea
B. pumilus
M. luteus
C. albicans
6a
6c
6e
7a
9
78
78
78
90
78
312
312
312
625
156
312
156
156
625
78
625
625
312
625
156
625
625
625
625
625
Compound no.
Minimum bactericidal concentration, MBC (mg/mL)
Test organism
Gram negative bacteria
Gram positive bacteria
Fungus
P. aeruginosa
S. lutea
B. pumilus
M. luteus
C. albicans
6a
6c
6e
7a
9
78
78
78
90
78
312
312
312
625
156
312
156
156
625
78
625
625
312
625
156
625
625
625
625
625
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550
M. A. M. Sh. El-Sharief et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 542–555
Initially, the maximum non-toxic concentration (MNTC) of
compounds 6a–g, 7a–e, 8a,b, and 9 on the uninfected African
green monkey kidney (Vero) host cells was determined using
three 10-fold serial dilutions of each compound starting from
10ꢁ1 until 10ꢁ3 dilution of the initial sample concentration of
1 ꢀ 10ꢁ2 M (DMSO) [69]. Cytotoxicity was determined by
microscopic examination of cell morphology, where cells
were monitored for any physical signs of toxicity including
partial or complete loss of the monolayer, rounding,
shrinkage, cell granulation, or lysis for up to 3 days. The
MNTC data is shown in Table 6 and the appropriate non-toxic
concentration dilution for each compound (dilution number:
6a,d,e: 10ꢁ2; 6b,c,f,g: 10ꢁ3; 7a–e: 10ꢁ2; 8a,b: 10ꢁ2; 9: 10ꢁ3) was
selected for further anti-infectivity studies.
Heterocycles 6a–g, 7a–e, 8a,b, and 9 were subjected to
in vitro testing of antiviral activity against the following three
viral strains: hepatitis A virus (HAV), human herpes simplex
virus 1 (HSV1) and Coxsackie B4 (CoxB4) viral strain. Viral
infectivity assay was carried out using the plaque formation
method [70, 71]. A plaque is a localized focus of virus-infected
cells, which under optimal conditions originates from a single
infectious virus particle. Counting of these foci for serial
dilution of virus suspension is a highly quantitative method
for assay of viral infectivity. Under these conditions, reduction
in virus plaque counts provides a very sensitive mean for
measuring antiviral activity of potential antivirals. The results
of the plaque reduction assay are summarized in Table 7.
While the non-substituted bis-imidazolidineiminothione 6a
(N-(1): phenyl; N-(3)-N-(3): n-hexyl) and its tolyl (6b), p-bromo
(6e), and 3,5-dichloro (6g) analogs showed insignificant
activities against all three viral strains, their p-methoxy, p-
chloro, and p-iodo (6c, 6d, and 6f, respectively) analogs were
markedly active against at least one viral strain. Thus, 6f (N-(1):
p-iodophenyl; N-(3)-N-(3): n-hexyl) was one of the most effective
in series 6a–g, reducing virus plaque count of HAV and CoxB4
Table 7. Anti-infectivity effect of compounds 6a–g, 7a–e, 8a,b,
and 9 against HAV, HSV1, and COxB4 viral strains.
Sample
no.
Selected
Anti-infectivity effect
Viral strain
dilutiona)
HAV
HSV1
CoxB4
6a
6b
6c
6d
6e
6f
6g
7a
7b
7c
7d
7e
8a
8b
9
10ꢁ2
10ꢁ3
10ꢁ3
10ꢁ2
10ꢁ2
10ꢁ3
10ꢁ3
10ꢁ2
10ꢁ2
10ꢁ2
10ꢁ2
10ꢁ2
10ꢁ2
10ꢁ2
10ꢁ3
ꢁveb)
ꢁve
ꢁve
78%
ꢁve
82%
6%
87%
ꢁve
ꢁve
3%
ꢁve
1%
ꢁve
1%
79%
4%
ꢁve
77%
2%
86%
ꢁve
ꢁve
2%
88%
ꢁve
ꢁve
ꢁve
66%
ꢁve
ꢁve
ꢁve
ꢁve
ꢁve
ꢁve
73%
ꢁve
ꢁve
ꢁve
ꢁve
ꢁve
ꢁve
ꢁve
ꢁve
ꢁve
a)
Sample dilution at which no cytotoxicity on Vero cells was
detected for 3 days.
b)
No anti-infectivity effect was observed.
by 82 and 77%, respectively. Compound 6c reduced virus
plaque count of HSV1 and CoxB4 by 66 and 79%, whereas an
anti-infectivity effect of 78% was realized against HAV with 6d.
The sharp difference in activity among compounds in the
same series is
a testament to the effect exerted by
manipulating the N-(1) substituent and how subtle differences
in substitution pattern may have a marked effect on anti-
infectivity effect. With regards to the remaining compounds,
series 7a–e contained a number of compounds that were
highly active against one or two viral strains. Hence, 7a [(N-(1):
Table 6. Cytotoxicity of compounds 6a–g, 7a–e, 8a,b, and 9 on Vero cells.
Sample no.
Dilution
6a
6b
6c
6d
6e
6f
6g
7a
7b
7c
7d
7e
8a
8b
9
Days
Cytotoxicity on Vero cell
ꢁ1
ꢁ1
ꢁ1
ꢁ2
ꢁ2
ꢁ2
ꢁ3
ꢁ3
ꢁ3
1st
2nd
3rd
1st
2nd
3rd
1st
þ4a)
þ4
þ4
þ4
þ4
þ4
þ4
þ4
ꢁ
þ4
þ4
þ4
þ4
þ4
þ4
ꢁ
þ4
þ4
þ4
þ4
þ4
þ4
ꢁ
þ4
þ4
þ4
ꢁ
þ4
þ4
þ4
ꢁ
þ4
þ4
þ4
þ4
þ4
þ4
ꢁ
þ4
þ4
þ4
ꢁ
þ4
þ4
þ4
ꢁ
þ4
þ4
þ4
ꢁ
þ4
þ4
þ4
ꢁ
þ4
þ4
þ4
ꢁ
þ4
þ4
þ4
ꢁ
þ4
þ4
þ4
ꢁ
þ4
þ4
þ4
þ4
þ4
þ4
ꢁ
þ4
b)
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
2nd
3rd
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
a)
Sample dilution at which cytotoxicity on Vero cells was significant; þ1 partial or complete loss of cell monolayer; þ2 rounding; þ3
shrinkage; þ4 cell granulation or lysis.
b)
Sample dilution at which no cytotoxicity on Vero cells was detected.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 542–555
Bioactive Bis-Imidazolidineiminothiones
551
phenyl; N-(3)-N-(3): 4,40-oxybis(4,1-phenylene)] was the most
active among the series, displaying an anti-infectivity effect of
87 and 86% against HAV and CoxB4, respectively. Interesting-
ly, the n-hexyl analog 6a was completely inactive against all
three viral strains, pointing to the enhancing effect of the
several matching pairs of aromatic substituents on N-(1) and
appropriate N-(3)-N-(3) linker which could serve to optimize
structural features for optimal activity to eventually render
such compounds clinically useful drug agents.
N-(3)-N-(3) 4,40-oxybis(4,1-phenylene) linker in 7a. Compounds Experimental
7c [(N-(1): p-ethoxyphenyl; N-(3)-N-(3): 4,40-oxybis(4,1-phenyl-
ene)] and 7e [(N-(1): 4-iodophenyl; N-(3)-N-(3): 4,40-oxybis(4,1-
phenylene)] were also outstanding, giving anti-infectivity
effects of 73 and 88% against HSV1 and CoxB4, respectively.
On the other hand, series 8a,b and compound 9 were
completely inactive against all three viral strains, pointing to
the opposing effect between the N-(3)-N-(3) linkers and N-(1)
substituents and underscoring the precedent specificity of
antivirals.
General
IR spectra were recorded (KBr) on
a Perkin–Elmer 1650
spectrophotometer. 1H NMR, 13C NMR, 19F, 13C-DEPT135, COSY,
and HSQC spectra were recorded on a Avance II Bruker FT-NMR
spectrometer 400 (400 MHz) using d1-TFA/CDCl3 (1/1), CDCl3, or
DMSO-d6 as solvents and TMS as an internal standard. Chemical
shifts are expressed as d ppm units. Mass spectra were recorded on
Shimadzu GC–MS QP 100 EX (70 eV) at the Micro Analytical
Center at Cairo University. The microanalyses were in satisfactory
agreement with the calculated values (C, H, N, S values were
within ꢂ0.4%), and the mass spectra produced the desired
molecular ion peaks with the appropriate isotopic distributions
where applicable. Melting points were obtained on a Fisher-Johns
melting points apparatus and are uncorrected. The physical
properties data of the synthesized compounds are shown in the
Supporting Information section.
Conclusion
In summary, the synthesis, characterization, and biological
activity of a new series of bis-imidazolidineiminothiones with
variable aromatic substituents at N-(1) and linkers between
the N-(3)-N-(3) atoms have been described. The products were
obtained in 65–75% yields as yellow/orange solids. Most
compounds displayed antitumor, antibacterial, antifungal,
and antiviral activities. Screening of all compounds for
cytotoxic activity against HEPG2, HEP2, MCF7, and HCT116
tumor cell lines was carried out, where the IC50 values ranged
from 6.30 to 84.6 mM. Several compounds proved markedly
active against a least one cell line (e.g. 6b,d,e,g, 7a),
underlining the matching effect of suitably positioned
substituents on N-(1) with the proper N-(3)-N-(3) linker.
Likewise, all heterocycles were tested against bacterial
organisms (P. aeruginosa, S. lutea, B. pumilus, and M. luteus),
and fungal strains (C. albicans and P. chrysogenum). All bis-
imidazolidineiminothiones showed significant antibacterial
activities against the growth of P. aeruginosa, S. lutea, B. pumilus,
and M. luteus, with the exception of 7e and 8a, which proved
inactive against P. aeruginosa. It is noteworthy that the same
level of antibacterial activity was realized with 9 against M.
luteus as was measured for the standard antibacterial agent
erythromycin, underscoring the effectiveness of 9. As for the
antifungal activity, most compounds were very effective
antifungal agents and in several cases achieved similar levels
of activity as the standard antifungal agent griseofulvin
(27 mm). The antimicrobial activity was further supported by
quantitative assessment of susceptibilities of the preceding
microorganisms using MIC and MBC techniques. Finally, the
antiviral effect of all compounds were investigated against
HAV, HSV1, and CoxB4 viral strains, where 6c,d,f, and 7a,c,e
were markedly active against one or two viral strains,
reducing the virus plaque count of various viral strains by
66 to 88%. Structure activity relationship studies revealed
Chemistry
For the preparation of the known cyanothioformanilides 1a–g,
see [4–13]. Also see [38, 39, 63] and references therein.
The physical and spectral data of 1b,c–g were in accord to those
reported [38]. 1a,d are commercially available.
Typical procedure for the preparation of bis-
imidazolidineiminothiones 6a–g, 7a–e, 8a,b, and 9
To a solution of the arylcyanothioformanilide (1) (10 mmol,
2.0 equiv.) in ether (20 mL), a solution of the corresponding bis-
isocyanate (5 mmol, 1.0 equiv.) in ether (10 mL) was added,
followed by three drops of triethylamine. The reaction mixture
was stirred for 25 min. The obtained product was filtered off,
washed with a minimum amount of ether, air-dried and re-
crystallized from chloroform/n-hexane to give 6a–g, 7a–e, 8a,b,
and 9 in yields ranging from 65 to 75%.
3,30-(Hexane-1,6-diyl)bis(4-imino-1-phenyl-5-
thioxoimidazolidin-2-one) (6a)
IR (KBr): n 3420 (NH), 1771 (C O), 1663 (C N), 1086 (C S) cmꢁ1; 1H
–
–
–
–
–
–
NMR (d1-TFA/CDCl3 1:1, 400 MHz) d 11.67 (s, 1H, NH), 7.68–7.54
(m, 3H), 7.43–7.31 (m, 2H), 4.11 (d, J ¼ 7.6 Hz, 2H, NCH2), 1.98–
1.81 (m, 2H), 1.62–1.48 (m, 2H); 13C NMR (d1-TFA/CDCl3 1:1,
–
–
–
–
100 MHz) d 176.9 (C¼S), 153.5 (C O), 150.2 (C N), 131.4 (CH),
130.9 (C), 130.4 (CH), 126.7 (CH), 43.6 (CH2), 26.9 (CH2), 25.7 (CH2);
MS (m/z, %) 493 (20, Mþþ1), 492 (24, Mþ), 168 (28, OCN(CH2)6NCO),
135 (84, PhNCS), 77 (100, C6H5þ).
3,30-(Hexane-1,6-diyl)bis(4-imino-5-thioxo-1-p-
tolylimidazolidin-2-one) (6b)
IR (KBr): n 3420 (NH), 1775 (C O), 1663 (C N), 1090 (C S) cmꢁ1; 1H
–
–
–
–
–
–
NMR (d1-TFA/CDCl3 1:1, 400 MHz) d 11.62 (s, 1H, NH), 7.41 (d,
J ¼ 8.5 Hz, 2H), 7.24 (d, J ¼ 8.5 Hz, 2H), 4.11 (d, J ¼ 7.6 Hz, 2H,
NCH2), 2.47 (s, 3H), 1.95–1.81 (m, 2H), 1.61–1.50 (m, 2H); 13C NMR
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552
M. A. M. Sh. El-Sharief et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 542–555
–
–
–
(d1-TFA/CDCl ; 1:1, 100 MHz) d 177.1 (C S), 153.9 (C O), 150.3
3,30-(4,40-Oxybis(4,1-phenylene))bis(4-imino-1-phenyl-5-
–
3
–
(C N), 142.5 (C), 131.1 (CH), 128.3 (C), 126.5 (CH), 43.6 (CH ), 27.0
–
2
thioxoimidazolidin-2-one) (7a)
(CH2), 25.8 (CH2), 20.9 (CH3); MS (m/z, %) 521 (14, Mþþ1), 520 (40,
Mþ), 519 (39, MþꢁH), 91 (100, CH3.C6H4þ).
IR (KBr): n 3432 (NH), 1771 (C O), 1662 (C N), 1118 (C S) cmꢁ1; 1H
–
–
–
–
–
–
NMR (DMSO, 400 MHz) d 9.66 (br s, 2H, NH), 7.64–7.50 (m, 14H),
7.24 (d, J ¼ 8.8 Hz, 4H); 13C NMR (DMSO, 100 MHz) d 183.2 (C S),
–
–
–
–
–
156.4 (C–O), 154.7 (C O), 154.3 (C N), 133.7 (C–N), 130.0 (CH),
–
3,30-(Hexane-1,6-diyl)bis(4-imino-1-(4-methoxyphenyl)-5-
129.7 (CH), 129.5 (CH), 128.4 (C–N), 128.1 (CH), 119.5 (C–H); MS (m/
z, %) 576 (5, Mþ), 575 (3, Mþþ1), 252 (58, p-OCN–C6H4–O–C6H4.
NCO), 135 (98, C6H5NCS), 77 (100, C6H5þ).
thioxoimidazolidin-2-one) (6c)
IR (KBr): n 3420 (NH), 1771 (C O), 1665 (C N), 1086 (C S) cmꢁ1; 1H
–
–
–
–
–
–
NMR (d1-TFA/CDCl3 1:1, 400 MHz) d 11.57 (s, 1H, NH), 7.34 (d,
J ¼ 9.1 Hz, 2H), 7.17 (d, J ¼ 9.1 Hz, 2H), 4.11 (d, J ¼ 7.6 Hz, 2H,
NCH2), 3.99 (s, 3H), 1.96–1.82 (m, 2H), 1.60–1.51 (m, 2H); 13C NMR
3,30-(4,40-Oxybis(4,1-phenylene))bis(4-imino-5-thioxo-1-p-
tolylimidazolidin-2-one) (7b)
–
–
–
–
(d1-TFA/CDCl 1:1, 100 MHz) d 177.3 (C S), 161.0 (C), 153.9 (C O),
3
IR (KBr): n 3430 (NH), 1770 (C O), 1661 (C N), 1117 (C S) cmꢁ1; 1H
–
–
–
–
–
–
–
–
150.5 (C N), 128.5 (CH), 124.3 (C), 116.0 (CH), 56.1 (CH ), 43.7
3
(CH2), 27.0 (CH2), 25.9 (CH2); MS (m/z, %) 554 (23, Mþþ2), 553 (31,
NMR (DMSO, 400 MHz) d 9.63 (br s, 2H, NH), 7.59 (d, J ¼ 8.8 Hz,
4H), 7.40 (d, J ¼ 8.6 Hz, 4H), 7.36 (d, J ¼ 8.6 Hz, 4H), 7.22 (d,
J ¼ 8.8 Hz, 4H), 2.36 (s, 6H, CH3); 13C NMR (DMSO, 100 MHz) d
Mþþ1), 552 (100, Mþ), 204 (90).
–
–
–
–
183.3 (C S), 156.3 (C–O), 154.7 (C O), 154.3 (C N), 139.7 (C–Me),
–
–
3,30-(Hexane-1,6-diyl)bis(1-(4-chlorophenyl)-4-imino-5-
131.1 (C–N), 130.1 (CH), 129.7 (CH), 128.4 (C–N), 127.8 (CH), 119.5
(C–H), 21.2 (CH3); MS (m/z, %) 604 (5, Mþ), 603 (2, Mþꢁ1), 252 (100,
OCN-C6H4–O-C6H4.NCO), 149 (62, p-CH3.C6H4NCS), 91 (93, CH3.
C6H4þ).
thioxoimidazolidin-2-one) (6d)
IR (KBr): n 3420 (NH), 1761 (C O), 1661 (C N), 1090 (C S) cmꢁ1; 1H
–
–
–
–
–
–
NMR (d1-TFA/CDCl3 1:1, 400 MHz) d 11.59 (s, 1H, NH), 7.58 (d,
J ¼ 8.9 Hz, 2H), 7.34 (d, J ¼ 8.9 Hz, 2H), 4.11 (d, J ¼ 7.6 Hz, 2H,
NCH2), 1.98–1.77 (m, 2H), 1.61–1.48 (m, 2H); 13C NMR (d1-TFA/
3,30-(4,40-Oxybis(4,1-phenylene))bis(1-(4-ethoxyphenyl)-
–
–
–
–
–
CDCl 1:1, 100 MHz) d 176.8 (C S), 153.3 (C O), 150.4 (C N), 138.0
–
3
4-imino-5-thioxoimidazolidin-2-one) (7c)
(C–Cl), 130.8 (CH), 129.3 (C), 128.1 (CH), 43.7 (CH2), 27.0 (CH2), 25.8
(CH2); MS (m/z, %) 565 (12, Mþþ5), 563 (11, Mþþ3), 562 (42, Mþþ2),
560 (30, Mþ), 170 (100), 168 (36, OCN(CH2)6NCO).
IR (KBr): n 3429 (NH), 1776 (C O), 1664 (C N), 1118 (C S) cmꢁ1; 1H
–
–
–
–
–
–
NMR (DMSO, 400 MHz) d 9.61 (br s, 2H, NH), 7.58 (d, J ¼ 8.8 Hz,
4H), 7.42 (d, J ¼ 9.0 Hz, 4H), 7.22 (d, J ¼ 8.8 Hz, 4H), 7.07 (d,
J ¼ 9.0 Hz, 4H), 4.07 (q, J ¼ 7.1 Hz, 4H), 1.33 (t, J ¼ 7.1 Hz, 6H);
13
–
C NMR (DMSO, 100 MHz) d 183.4 (C S), 159.4 (C–O), 156.3 (C–O),
3,30-(Hexane-1,6-diyl)bis(1-(4-bromophenyl)-4-imino-5-
–
–
–
154.7 (C O), 154.4 (C N), 129.7 (CH), 129.4 (CH), 128.4 (C–N), 126.0
–
–
thioxoimidazolidin-2-one) (6e)
(C–N), 119.5 (CH), 115.2 (C–H), 63.9 (CH2), 14.9 (CH3); MS (m/z, %)
664 (11, Mþ), 663 (18, Mþꢁ1), 206 (17, p-C2H5–O–C6H4–NH–CS–
CN), 179 (49, p-C2H5–O–C6H4–NCS), 109 (100).
IR (KBr): n 3444 (NH), 1761 (C O), 1661 (C N), 1072 (C S) cmꢁ1; 1H
–
–
–
–
–
–
NMR (d1-TFA/CDCl3 1:1, 400 MHz) d 11.63 (s, 1H, NH), 7.73 (d,
J ¼ 8.7 Hz, 2H), 7.25 (d, J ¼ 8.7 Hz, 2H), 4.26–4.09 (m, 2H), 2.06–
1.80 (m, 2H), 1.67–1.49 (m, 2H); 13C NMR (d1-TFA/CDCl3 1:1,
3,30-(4,40-Oxybis(4,1-phenylene))bis(1-(4-bromophenyl)-
–
–
–
–
–
–
100 MHz) d 177.1 (C S), 153.7 (C O), 150.7 (C N), 134.1 (CH),
130.1 (C), 128.4 (CH), 126.1 (C), 43.9 (CH2), 27.1 (CH2), 26.0 (CH2);
MS (m/z, %) 655 (3, Mþþ7), 654 (4, Mþþ6), 652 (7, Mþþ4), 651 (11,
Mþþ3), 650 (13, Mþþ2), 648 (7, Mþ), 214 (15), 55 (100).
4-imino-5-thioxoimidazolidin-2-one) (7d)
IR (KBr): n 3430 (NH), 1773 (C O), 1664 (C N), 1118 (C S) cmꢁ1; 1H
–
–
–
–
–
–
NMR (DMSO, 400 MHz) d 9.69 (br s, 2H, NH), 7.79 (d, J ¼ 8.8 Hz, 4H),
13
7.59 (d, J ¼ 8.9 Hz, 4H), 7.51 (d, J ¼ 8.8 Hz, 4H), 7.23 (d, J ¼ 8.9 Hz,
–
4H); C NMR (DMSO, 100 MHz) d 183.0 (C S), 156.4 (C–O), 154.6
–
3,30-(Hexane-1,6-diyl)bis(4-imino-1-(4-iodophenyl)-5-
–
–
(C O), 154.0 (C N), 132.9 (C–N), 132.8 (CH), 130.3 (CH), 129.7 (CH),
–
–
128.3 (C–N), 123.1 (C–Br), 119.5 (C–H); MS (m/z, %) 734 (12, Mþþ2),
thioxoimidazolidin-2-one) (6f)
IR (KBr): n 3420 (NH), 1771 (C O), 1665 (C N), 1086 (C S) cmꢁ1; 1H
–
–
–
–
–
252 (57, p-OCN–C6H4–O–C6H4.NCO), 240 (27), 136 (100).
–
NMR (d1-TFA/CDCl3 1:1, 400 MHz) d 11.63 (s, 1H, NH), 7.97 (d,
J ¼ 8.1 Hz, 2H), 7.13 (d, J ¼ 8.1 Hz, 2H), 4.19–4.02 (m, 2H),
1.99–1.77 (m, 2H), 1.61–1.48 (m, 2H); 13C NMR (d1-TFA/CDCl3 1:1,
3,30-(4,40-Oxybis(4,1-phenylene))bis(4-imino-1-
–
–
–
100 MHz) d 176.6 (C S), 153.2 (C O), 150.4 (C N), 140.0 (CH),
(4-iodophenyl)-5-thioxoimidazolidin-2-one) (7e)
–
–
–
IR (KBr): n 3432 (NH), 1773 (C O), 1664 (C N), 1119 (C S) cmꢁ1; 1H
–
–
–
–
–
–
130.6 (C), 128.2 (CH), 97.2 (C–I), 43.7 (CH2), 27.0 (CH2), 25.8 (CH2);
MS (m/z, %) 746 (30, Mþþ2), 745 (41, Mþþ1), 744 (100, Mþ), 261 (56,
p-I-C6H4.NCS).
NMR (DMSO, 400 MHz) d 9.68 (br s, 2H, NH), 7.95 (d, J ¼ 8.8 Hz,
13
4H), 7.58 (d, J ¼ 8.9 Hz, 4H), 7.35 (d, J ¼ 8.8 Hz, 4H), 7.23 (d,
–
–
J ¼ 8.9 Hz, 4H); C NMR (DMSO, 100 MHz) d 182.9 (C S), 156.4
–
–
–
(C–O), 154.6 (C O), 154.0 (C N), 138.6 (CH), 133.4 (C–N), 130.3 (CH),
–
3,30-(Pentane-1,5-diyl)bis(1-(3,5-dichlorophenyl)-4-imino-
129.7 (CH), 128.3 (C–N), 119.5 (C–H), 96.3 (C–I); MS (m/z, %) 828 (6,
Mþ), 261 (11, p-I–C6H4–NCS), 245 (38, p-I–C6H4–NCO), 64 (100).
5-thioxoimidazolidin-2-one) (6g)
IR (KBr): n 3449 (NH), 1771 (C O), 1665 (C N), 1092 (C S) cmꢁ1; 1H
–
–
–
–
–
–
NMR (CDCl3, 400 MHz) d 9.33 (s, 2H, NH), 7.47 (t, J ¼ 1.9 Hz, 2H),
3,30-(2,20-Dimethoxybiphenyl-4,40-diyl)bis(1-
7.35 (d, J ¼ 1.9 Hz, 4H), 3.84 (t, J ¼ 7.3 Hz, 4H), 1.83–1.74 (m, 4H),
1.51–1.43 (m, 4H); 13C NMR (CDCl , 100 MHz) d 181.3 (C S), 154.1
–
(4-ethoxyphenyl)-4-imino-5-thioxoimidazolidin-2-one) (8a)
–
3
–
–
–
–
–
IR (KBr): n 3443 (NH), 1778 (C O), 1668 (C N), 1250 (C–O), 1171
(C N), 153.5 (C O), 135.6 (C–Cl), 134.2 (C–N), 129.8 (CH), 125.8
–
–
–
(C–O), 1115 (C S) cmꢁ1; H NMR (DMSO, 400 MHz) d 9.63 (s, 1H,
1
–
(CH), 40.9 (CH2), 27.3 (CH2), 26.0 (CH2).
–
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Arch. Pharm. Chem. Life Sci. 2013, 346, 542–555
Bioactive Bis-Imidazolidineiminothiones
553
NH), 7.60 (d, J ¼ 7.9 Hz, 1H), 7.54–7.50 (m, 1H), 7.49–7.41 (m, 3H),
13
The median inhibition concentration (IC50) which is the required
concentration to reduce the survival to 50% is then determined
from the survival curve.
7.10 (d, J ¼ 9.0 Hz, 2H), 4.09 (q, J ¼ 7.0 Hz, 2H), 3.93 (s, 3H), 1.35 (t,
–
J ¼ 7.0 Hz, 3H); C NMR (DMSO, 100 MHz) d 183.2 (C S), 159.2
–
–
–
–
(C–O), 155.7 (C–O), 154.1 (C O), 153.8 (C N), 142.8 (C), 130.7 (CH),
–
129.1 (CH), 125.7 (C), 120.6 (C), 119.7 (CH), 115.1 (CH), 111.8 (CH),
63.7 (CH2), 56.4 (CH3), 14.7 (CH3); MS (m/z, %) 708 (4, Mþ), 706 (7,
Mþꢁ2), 206 (31, p-C2H5–O–C6H4–NH–CS–CN), 179 (24, p-C2H5–O–
C6H4–NCS), 86 (100).
Antibacterial and antifungal susceptibility testing of
compounds 6a–g, 7a–e, 8a,b, and 9
Antimicrobial and antifungal studies were carried out by the
diffusion agar technique [66] using a 10 cm microplate well
diameter and 100 mL of a sample concentration of 1 ꢀ 10ꢁ2 M,
unless otherwise indicated. The tested organisms were Gram-
negative bacteria, Pseudomonas aeruginosa ATCC-10145, and Sarcina
lutea ATCC-9341, and Gram-positive bacteria, Bacillus pumilus
NCTC-8214, Micrococcus luteus ATCC-25922, and fungi (Candida
albicans, IMRU-3669, and Penicilium chrysogenum ATCC-9179). The
bacteria and fungi were maintained on nutrient agar and
Czapek’s Dox agar medium, respectively. All chemical com-
pounds were dissolved in N,N-dimethylformamide (DMF), except
if noted otherwise. DMF showed no inhibition activity. The results
are tabulated in Tables 3 and 4.
3,30-(2,20-Dimethoxybiphenyl-4,40-diyl)bis(1-
(4-chlorophenyl)-4-imino-5-thioxoimidazolidin-2-one) (8b)
–
–
–
IR (KBr): n 3431 (NH), 1778 (C O), 1668 (C N), 1258 (C–O), 1194
–
(C–O), 1113 (C S) cmꢁ1; H NMR (DMSO, 400 MHz) d 9.71 (s, 1H,
1
–
–
NH), 7.70–7.65 (m, 2H), 7.64–7.56 (m, 3H), 7.55–7.44 (m, 2H), 3.93
13
–
(s, 3H); C NMR (DMSO, 100 MHz) d 182.8 (C S), 155.7 (C–O),
–
–
–
153.9 (C N), 153.4 (C O), 142.8 (C), 134.3 (C–Cl), 132.1 (C), 130.6
–
–
(CH), 129.8 (CH), 129.6 (CH), 120.4 (C), 119.7 (CH), 111.8 (CH), 56.4
(CH3); MS (m/z, %) 690 (17, Mþþ2), 296 (18, p-OCN–OCH3–C6H3–
C6H3–OCH3.NCO), 197 (21), 169 (86, p-Cl–C6H4–NCS), 153 (33, p-Cl–
C6H4–NCO).
1-(4-Chlorophenyl)-3-((5-(3-(4-chlorophenyl)-5-imino-2-
oxo-4-thioxoimidazolidin-1-yl)-1,3,3-trimethylcyclohexyl)-
methyl)-4-imino-5-thioxoimidazolidin-2-one (9)
Virucidal activity studies
Heterocycles 6a–g, 7a–e, 8a,b, and 9 were tested against HAV,
HSV1 and CoxB4 viral strains where viral activity was assayed by
the plaque formation method.
IR (KBr): n 3441 (NH), 1777 (C O), 1665 (C N), 1092 (C S) cmꢁ1; 1H
–
–
–
–
–
–
NMR (CDCl3, 400 MHz) d 9.42 (s, 1H, NH), 9.39 (s, 1H, NH), 7.50–
7.45 (m, 4H), 7.40–7.33 (m, 4H), 4.72–4.61 (m, 1H), 3.67 (s, 2H),
2.42–2.32 (m, 1H), 2.26–2.17 (m, 1H), 1.69–1.62 (m, 1H), 1.61–1.55
(m, 1H), 1.41–1.37 (m, 2H), 1.26 (s, 3H), 1.15 (s, 3H), 1.03 (s, 3H); 13C
Determination of cytotoxicity of 6a–g, 7a–e, 8a,b, and 9
on Vero cells
Initially, the cytotoxicity of compounds 6a–g, 7a–e, 8a,b, and 9 on
Vero cells was determined. The method used was that described
by Vanden Berghe et al. [69]. Propagation of healthy Vero cell line
by enzyme treatment and assessment of cytotoxicity effect of 6a–
g, 7a–e, 8a,b, and 9 was carried out as follows:
–
–
NMR (CDCl , 100 MHz) d 181.5 (C¼S), 181.4 (C S), 155.4 (C O),
–
–
3
–
–
–
155.2 (C O), 154.0 (C N), 153.6 (C¼N), 135.3 (C–Cl), 135.2 (C–Cl),
–
131.1 (C–N), 131.0 (C–N), 129.5 (CH), 129.4 (CH), 128.3 (CH), 128.2
(CH), 54.4 (N-CH2), 48.2 (N-CH), 47.1 (CH2), 40.9 (CH2), 38.3 (C), 37.9
(CH2), 35.0 (CH3), 32.2 (C), 27.4 (CH3), 23.5 (CH3); MS (m/z, %) 619 (1,
Mþþ5), 618 (3, Mþþ4), 617 (3, Mþþ3), 616 (8, Mþþ2), 615 (3,
Mþþ1), 614 (8, Mþ).
1. The media overlaying cell monolayer was decanted and cells
were released from tissue culture flask by treatment with
5 mL of pre-warmed trypsin–EDTA solution.
Biological in vitro evaluation
Cytotoxic activity studies of compounds 6a–g, 7a–e, 8a,b,
and 9 against various tumor cell lines
Compounds 6a–g, 7a–e, 8a,b, and 9 were tested at concentrations
between 10 and 100 mM using SRB assay for cytotoxic activity
against HEPG2, HEP2, MCF7, and HCT116 tumor cell lines.
2. The trypsin was aspirated with a pipette, then 2 mL of
trypsin was dispensed, the bottle rocked, and was incubated
at 37°C for more than 10 min.
3. Cells were suspended in about 8 mL of growth media.
4. Cells were counted using a hemocytometer and using trypan
blue vital stain.
5. About 10 mL of 2 ꢀ 105 Vero cell suspension was transferred
to a 50 cm3 TC bottle (Falcon) tightly closed then was
incubated at 37°C. Cells were sub-cultured once weekly.
6. For seeding 96-well plates, 0.1 mL (2 ꢀ 105 cells) was
transferred to each flat-bottom well and incubated at 37°C
for 24–48 h to develop a complete monolayer sheet.
7. The 6- or 12-well plates used for plaque assay and for
cytotoxicity study were seeded with 2–3 mL cell suspension
per well. Confluent monolayer was obtained after 24 h
incubation at 37°C.
Measurement of potential cytotoxicity by SRB assay
Potential cytotoxicity of heterocycles 6a–g, 7a–e, 8a,b, and 9 was
tested using the method of Skehan et al. [65]. Cells were plated in a
96 multiwell plate (104 cells/well) for 24 h before treatment with
the compounds to allow attachment to the wall of the plate.
Different concentrations of the compounds (0, 10, 25, 50, and
100 mM) were added to the cell monolayer, where triplicate wells
were prepared for each individual dose. Monolayer cells were
incubated with the compounds for 48 h at 37°C in an atmosphere
of 5% CO2. After 48 h, cells were fixed, washed and stained with
SRB stain, where excess stain was washed with acetic acid and
attached stain was recovered with Tris EDTA buffer. Finally, color
intensity was measured in an ELISA reader and the relationship
between surviving fraction and drug concentration was plotted to
get the survival curve of each tumor cell line for each compound.
8. Compounds 6a–g, 7a–e, 8a,b, and 9 were each dissolved in
1 mL of DMSO to give an initial concentration of 1 ꢀ 10ꢁ2 M.
9. Growth medium was decanted from 96-well micro titer
plates after confluent sheet of Vero cell was formed, cell
monolayer was washed twice with wash media, then about
1 mL of wash media was added and the plates were
incubated at room temperature for 5–10 min.
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www.archpharm.com

554
M. A. M. Sh. El-Sharief et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 542–555
10. Ten-fold serial dilutions of different compounds were made
in Eagle’s minimum essential medium (MEM), starting from
10ꢁ1 till 10ꢁ3 dilution.
11. 0.2 mL of each dilution was tested in different wells
leaving six wells as control, receiving only maintenance
medium.
[8] A. M. Sh. El-Sharief, A. M. Al-Amri, S. Y. Al Raqa, J. Sulfur Chem.
2006, 27, 1–19.
[9] J. D. Friedrich, J. Org. Chem. 1987, 52, 2442–2446.
[10] J. Gonda, P. Kristian, Collect. Czech. Chem. Commun. 1988, 53,
1761–1769.
12. The plate was incubated at 37°C and examined frequently
for up to 3 days. Cells were checked for any physical signs of
toxicity, e.g. partial or complete loss of the monolayer,
rounding, shrinkage/retraction, cell granulation, and lysis.
13. The MNTC of each compound on the cultured cells was
determined and was used in antiviral tests.
[11] W. Walter, P.-M. Hell, Liebigs Ann. 1969, 727, 22–34.
[12] W. Walter, K.-D. Bode, Liebigs Ann. 1966, 698, 131–144.
[13] A. Reissert, K. Brüggemann, Chem. Ber. 1924, 57, 981–989.
[14] A. Reissert, Chem. Ber. 1904, 37, 3708–3733.
[15] T. Besson, C. W. Rees, J. Chem. Soc. Perkin Trans. 1 1995, 1659–
1662.
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This work was supported by the Deanship of Scientific Research of
Taibah University (project number: 162). Ziad Moussa (principle
investigator) and Ahmed M. Sh. El‐Sharief greatly acknowledge this
generous financial support. We thank Lena Moussa for helpful
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