Synthesis and characterization of new types of halogenated and alkylated imidazolidineiminothiones and a comparative study of their antitumor, antibacterial, and antifungal activities

Article abstract of DOI:10.1016/j.ejmech.2011.03.009

A series of twenty novel imidazolidineiminothiones (4-8) with various substituents at N-(1) and N-(3) were synthesized by various permutations of halogenated and alkylated N-arylcyanothioformanilides (1) with aromatic isocyanates (2). Preliminary screenin

Full text of DOI:10.1016/j.ejmech.2011.03.009

European Journal of Medicinal Chemistry 46 (2011) 2280e2289
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and characterization of new types of halogenated and alkylated
imidazolidineiminothiones and a comparative study of their antitumor,
antibacterial, and antifungal activities
,
b
,
c,
Ziad Moussa a , Marwa A.M.Sh. El-Sharief , Ahmed M.Sh. El-Sharief
*
*
*
a Department of Chemistry, Faculty of Science, Taibah University, P.O. Box 30002, Almadinah Almunawarrah, Saudi Arabia
b Applied Organic Chemistry Department, National Research Center, Dokki, Cairo, Egypt
c Department of Chemistry, Faculty of Science, Al-Azhar University, Nasr City, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of twenty novel imidazolidineiminothiones (4e8) with various substituents at N-(1) and N-(3)
were synthesized by various permutations of halogenated and alkylated N-arylcyanothioformanilides (1)
with aromatic isocyanates (2). Preliminary screening of all compounds against Ehrlich ascites carcinoma
cells (EAC) indicated that 5d, and 8aec were the most active compounds as they displayed the highest
percent inhibition of cell viability (80%, 70%, 80%, and 70%, respectively). Thus, they were further sub-
jected to in vitro biological evaluation against other tumor cancer cell lines (HEPG2, HEP2, MCF7, HELA,
Received 12 December 2010
Received in revised form
27 February 2011
Accepted 1 March 2011
Available online 10 March 2011
and HCT116). The IC50 values ranged from 3.12 to 12.1
thoxyphenyl; N-(3): 4-methoxyphenyl) was markedly active against all cell lines and consistently
produced low IC50 values in all cases (ranging from 3.12 to 4.34 g/mL). This underscored the synergistic
effect of the suitably positioned methoxy groups on the aromatic rings of N-(1) and N-(3) of the imi-
dazolidineiminothiones. All compounds were also tested against microbial organisms (Escherichia coli,
Sarcina lutea, Bacillus subtilis, and Staphylococcus aureus), and fungal strains (Candida albicans and
Aspergillus flavus). Most tested compounds showed significant activities which could be optimized with
the appropriate selection of matching aromatic substituents on N-(1) and N-(3).
mg/mL where compound 8b (N-(1): 2,4-dime-
Keywords:
N-Arylcyanothioformanilides
Halogenated imidazolidineiminothiones
Alkylated imidazolidineiminothiones
Antitumor
Antimicrobial
Antifungal
m
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
tert-butylamine [27], hydroxylamine [28], tryptamine [29], o-phe-
nylenediamine, and o-aminophenethylamine [30] (Fig. 1).
Aromatic cyanothioformanilides (1) are effective herbicides [1],
pecticides [2], fungicides, and bactericides [3]. Such compounds
have been most commonly prepared by treating an N-aryl iso-
thiocyanate with cyanide [4e13], and to a lesser extent by other
methods which include Reissert’s dethiohydration of N-aryldi-
thiooxamides [13,14], thionationedehydration of aryloxalamides
[14], and thionationedethiohydration of N-arylthiooxamides [14]
(Fig. 1). Some of the latest synthetic approaches to cyanothio-
formanilides employ environmentallyefriendly N-arylimino-1,2,3-
dithiazoles which convert readily to the former species by oxidation
with m-chloroperoxybenzoic acid (m-CPBA) [15], reduction with
sodium cyanoborohydride (NaBH3(CN)) [16], or reaction with
nucleophilic reagents which include DBU [17], Ph3P [18e24], eth-
ylmagnesium bromide [22,25], triphenylphosphoraneylidenes [26],
In addition to the aforementioned agricultural and pharma-
ceutical applications of N-arylcyanothioformanilides, they have
proven extremely versatile reagents in organic synthesis. Thus the
aniline nitrogen may be aroylated [9,31], the thione moiety can
participate in cycloadditions with 1,3-dipoles [31], as well as in
DielseAlder [9,32,33] and ene [34] reactions, and the nitrile group
may be hydrated with H2O, thiohydrated with H2S or reacted
with NH2OH to afford aminooxothioacetylanilines, aminothioxo-
thioacetylanilines [35,36] or amidinothioformylanilines [5,37],
respectively. Aside from the preceding trivial reactions of arylcya-
nothioformanilides and more appropriate to our longstanding
interest in heterocyclic synthesis, we and others have used such
intermediates for the synthesis of various heterocycles which
exhibit interesting medicinal and pharmacological properties. In
this context, they undergo a variety of “one-pot” ring-closure
reactions [7,12,36,38,39,40] giving rise to various heterocyclic cores
which include pyrroles [8,41e43], pyrrolothiazoles [44], imidaz-
oles [4,7,45e53], benzimidazoles [54], oxazoles [5,37,39,40], ben-
zoxazoles [37,39,40,54], thiazoles [47,55], quinazolinonethiones
* Corresponding author. Tel./fax: þ966 4 8611190.
E-mail addresses: zmousa@taibahu.edu.sa (Z. Moussa), melsheref.2005@gmail.
com (M.A.M.Sh. El-Sharief), alshraef_2008@hotmail.com (A.M.Sh. El-Sharief).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.03.009

Z. Moussa et al. / European Journal of Medicinal Chemistry 46 (2011) 2280e2289
2281
Fig. 1. Synthetic approaches to the preparation of N-arylcyanothioformanilides.
Fig. 2. Preparation of
a new series of imidazolidineiminothiones with differing
aromatic substituents at N-(1) and N-(3).
[37,54], quinoxaline [37], imidazoquinoxalines [55,56], benzo-
thiazoloquinazolines [56], quinazolinones [41e43], thienoimida-
zoles [8,57], 1,3,4-thiadiazoles [58], quinazolines [6,54,59], and
other types of heterocycles [60e63].
N-Arylcyanothioformanilides 1aee were prepared according to
literature procedures and their physical and spectral data were in
accord to those reported [64e69]. It is noteworthy to mention that
analysis of the cyanothioformanilides by NMR (1H and 13C) indi-
cated the presence of tautomeric mixtures of thiol and thione
forms, in which the latter predominates. For example, the 1H NMR
spectrum of the iodo derivative 1d (tautomeric mixture; major:-
As part of our effort to examine the full range of possible
heterocyclic ring cores that could be constructed from N-arylcya-
nothioformanilides and derivatives thereof, we have recently
directed our attention to the preparation, derivatization, and in vitro
evaluation of imidazolidineiminothiones [8,44,45,60,64]. Thus, as
a synthetic entry point to access such compounds, a heterocyclic
ring closing reaction of isocyanates with N-arylcyanothioformani-
lides affords imidazolidineiminothiones which contain adjacent
imino and thione functions in the 4 and 5 positions, respectively.
This arrangement has been shown to serve as a reactive template in
a number of subsequent ring closing reactions in which one or both
of the exocyclic heteroatoms can be incorporated into additional
fused heterocyclic rings. Imidazolidineiminothiones and the various
heterocycles derived from them were shown to exhibit an inter-
esting and a wide range of pharmacological effects against anti-
tumor, antiviral, antimicrobial and antifungal strains.
minor; 64:36) revealed at
(broad s, 1H, NH). The aromatic ring displayed a set of two signals
d
¼ 9.44 ppm an exchangeable proton
for the major tautomer as follows: AreH (2&6) as a doublet at
d
7.78 ppm (2H, J ¼ 8.8 Hz), and AreH (3&5) as a doublet at
d
7.56 ppm (2H, J ¼ 8.8 Hz). Protons for the minor tautomer
exhibited well separated chemical shifts and also appeared as a set
of two signals with the same multiplicity pattern and J-values as
the major one (d
8.11 & 7.16 ppm). The 13C spectrum showed six
signals for the major form which were assigned to the structure
with the aid of DEPT 90/135 as well as COSYDQF and HSQC 2D
techniques. It showed the furthest downfield signal for the thione
2. Chemistry
group (C]S) at
groups, another two signals stemming from the quaternary
aromatic carbons with the CeI shift at 92.7 ppm, and an upfield
signal resulting from the nitrile group ( 119.7 ppm). The minor
d 160.4 ppm, two signals corresponding to the CH
Encouraged by the precedent bioactivity of imidazolidineimino-
thiones and their wide scope of reactivity which allows facile
chemselective structural modifications, the present investigation
reportson the preparationandbiological activityofa series of twenty
novel imidazolidineiminothiones (4e8) which were synthesized by
various permutations of N-arylcyanothioformanilides (1) with
aromatic isocyanates (2) (Fig. 2). Whereas the latter contains 4-
methoxyphenyl (eC6H4.OMe-4), 4-S-methyl-phenyl (eC6H4.SMe-4),
4-tolyl, and benzyl groups, and contributes to substituent variability
at N-(3), the former bears 4-chlorophenyl, 4-bromophenyl, 4-iodo-
phenyl, and 2,4-dimethoxyphenyl functions, leading to variability at
N-(1). Owing to the significant bioactivity of imidazolidineimino-
thiones which presumably stems from the iminothione function, in
this report we address the evaluation of the series of analogs 4e8 as
potential antitumor, antimicrobial, and antifungal agents that
contain a combination of pharmacophores of the former compounds
and variants at N-(1) and N-(3). Thus, a comparative study of activity
within each group will serve to illustrate the effect exerted by the N-
(3) substituent, whereas a similar parallel study of activity for a given
substituent throughout the five groups will shed light on the role
played by the N-(1) substituent. Finally, a structureeactivity rela-
tionship study was anticipated to reveal any potential synergistic or
opposing effect between the N-(1) and N-(3) substituents which will
ultimately assist in the design of highly active derivatives.
d
d
isomer showed five signals, although all six were observed when
CD3CN was used instead of CDCl3. It is noted that the ratio may
change depending on the deuterated solvent used, although the
thione is maintained as the major isomer. The tautomeric nature of
the cyanothioformanilides reflects their ambident nucleophilic
character in which they may react via the sulfur or nitrogen atom
leading to different heterocylic cores in ring closing reactions. This
is consistent with Ketcham’s observation that N-substituted cya-
nothioformanilides react with alkyl or aryl isothiocyanates to give
5-imino-l,3-disubstituted-2,4-imidazolidinedithiones or 5-(phe-
nylimino)-4-imino-2-thiazolidinethiones depending upon the
reactants and reaction conditions [27]. Interestingly, the combina-
tion of cyanothioformanilides with isocyanates affords 5-imino-4-
thioxo-2-imidazolidinones as the sole products, indicating that the
ring closing reaction proceeds via a single path which involves
attack via the nitrogen atom. A typical procedure for the prepara-
tion of compounds 4e8 involves treating an ethereal solution of the
N-cyanothioformanilide with an ethereal solution of an equimolar
amount of the corresponding isocyanate, followed by addition of
a few drops of triethylamine as a catalyst to furnish the corre-
sponding 5-imino-4-thioxo-2-imidazolidinone as shown in Fig. 2.
These products were purified by recrystallization from chloroform/

2282
Z. Moussa et al. / European Journal of Medicinal Chemistry 46 (2011) 2280e2289
Table 1
n-hexane and obtained in 75e90% yields (Table 7, see
Supplementary material section). These products are all stable and
could be stored at room temperature for prolonged period of time
without showing any signs of decomposition or developing odors
which indicate instability. It is noted that compound 8d contained
a significant amount of an impurity and therefore was not included
in biological testing (vide infra).
Inhibition of Ehrlich ascites carcinoma cells (EAC) viability.
%Inhibition of cell viability
Concentration
Compound no.
100mg/mL
Compound no.
4a
4b
4c
4d
5a
5b
5c
5d
6a
6b
1
6c
6d
7a
7b
7c
7d
8a
8b
8c
8d
20
45
10
20
40
30
70
80
70
*ND
50
10
60
20
0.5
10
80
40
10
3. Results and discussion
3.1. Antitumor properties
Imidazolidineiminothiones which contain adjacent imino and
thione functions in the 4 and 5 positions show broad spectrum
pharmacological properties. In our previous work, many com-
pounds containing these moieties were synthesized [64] and sub-
jected to in vitro biological evaluation against tumor, viral,
microbial, and fungal strains. Several heterocycles were also deriv-
atized for biological evaluation purposes by functionalizing either of
the imino and thione groups, or both. Most compounds displayed
antitumor, antiviral, antibacterial and antifungal activities and
could therefore serve as lead chemical entities for further structural
modification and optimization to render them clinically useful drug
agents. Indeed this is a major objective of the work reported herein,
where the most promising leads disclosed earlier have been sub-
jected to further structural optimization by varying the substituents
on N-(1) and N-(3). Subsequent investigation of the bioactivity of
such derivatives should clarify the effect exerted by the imino, thi-
one, N-(1), and N-(3) groups on bioactivity. Eventually, this should
enable us to predict the most effective and synergetic combination
of functional groups required for optimal biological activity.
*ND: not determined
substituents on bioactivity, a comparative study of activity was
carried out within and between each of the five groups, respec-
tively. Fig. 3 shows that while the methoxy and benzyl groups
resulted in high % inhibition values in the 4aed series, the former
group formed a mismatch pair with the chlorine atom in the 5aed
series (0.5% inhibition), while the latter function was reinforced to
give the highest % inhibition value (80% inhibition). With the benzyl
group as invariant, % inhibition dropped significantly as the chlo-
rine was replaced with bromine and iodine in compounds 6aed
and 7aed (45 & 30% inhibition, respectively). On the other hand,
keeping the methoxy unchanged, % inhibition was optimized from
0.5% to 10%, 20%, and 80% as the aromatic substituent at N-(1) was
changed from Cl, to Br, I, and 2,4-dimethoxy groups, respectively.
The remaining compounds in the first four groups show almost
random combination of synergetic and opposing effects between
the substituents at N-(1) and N-(3). However, the most interesting
and informative trend is observed in the last series (8aec) which
points to the dominant role played by the substituent at N-(1).
Thus, with the 2,4-dimethoxyphenyl moiety at N-(1), a complete
match with the N-(3) substituents was realized as clearly seen by
the high % inhibition values achieved within 8aec.
Thus, all of the prepared compounds (4aed, 5aed, 6aed, 7aed,
and 8aec) underwent a preliminary screening test against Ehrlich
Ascites Carcinoma cells (EAC). The antitumor activity was assessed
by cell viability with trypan blue dye exclusion assay. The protocol
involved treating an EAC suspension (2.5 ꢀ 105/mL) with 0.8 mL
RPMI 1640 media and 0.1 mL of each tested compound (corre-
sponding to 100 m
g) with subsequent incubation at 37 ꢁC for 2 h.
Trypan blue exclusion test was carried out to calculate the
percentage of non-viable cells as follows: % of non-viable cells ¼ (No.
of non-viable cells/No. of cells) ꢀ 100. Compounds producing more
than 70% non-viable cells are considered active. Those displaying the
highest percent inhibition of cells were then further evaluated for
cytotoxic activity against the following tumor cancer cell lines:
hepatocellular carcinoma cell line (HEPG2), human laryngeal carci-
noma cell line (HEP2), human breast adenocarcinoma cell line
(MCF7), human epithelial carcinoma cell line (HELA), and colon
carcinoma cell line (HCT116).
3.1.2. Cytotoxic activity of compounds 5d and 8aec against various
tumor cell lines
Since 5d and 8aec were the most active compounds against
EAC, we were interested in exploring their full potential and
100
90
80
70
60
50
40
30
20
10
0
3.1.1. Screening of compounds 4aed, 5aed, 6aed, 7aed, and 8aec
against EAC cells
Compounds 4aed, 5aed, 6aed, 7aed, and 8aec were screened
against EAC cells at a concentration of 100
mg/mL as shown in
Table 1 and represented graphically in Fig. 3. Compounds 5d (N-(1):
4-chlorophenyl; N-(3): benzyl), and 8aec (N-(1): 2,4-dimethox-
yphenyl; N-(3): 4-methylthiophenyl, 4-methoxyphenyl, 4-tolyl,
respectively) were the most active as they displayed the highest %
inhibition of cell viability (80%, 70%, 80%, and 70%, respectively).
Compounds 4b,d (50 & 60%, respectively; N-(1): phenyl; N-(3):
4-methoxyphenyl; N-(1): phenyl; N-(3): benzyl, respectively), 6a,d,
(40 & 45%, respectively; N-(1): 4-bromophenyl; N-(3): 4-methyl-
thiophenyl; N-(1): 4-bromophenyl; N-(3): benzyl), and 7c (40%;
N-(1): 4-iodophenyl; N-(3): 4-tolyl) produced lower, but reasonable
% inhibition of cell viability values which ranged from 40 to 60%. In
order to assess the effects exerted by the N-(1) and N-(3)
Compound number
Fig. 3. Cytotoxicity of compounds 4a-d, 5a-d, 6a-d, 7a-d, and 8a-c against Ehrlich
ascites carcinoma cells (EAC).

Z. Moussa et al. / European Journal of Medicinal Chemistry 46 (2011) 2280e2289
2283
Table 2
Table 3
Evaluation of compounds 5d and 8aec against human hepatocellular carcinoma cell
line (HEPG2), human laryngeal carcinoma cell line (HEP2), human breast adeno-
carcinoma cell line (MCF7), human epithelial carcinoma cell line (HELA), and colon
carcinoma cell line (HCT116).
IC50 values of compounds 5d and 8aec obtained against human hepatocellular
carcinoma cell line (HEPG2), human laryngeal carcinoma cell line (HEP2), human
breast adenocarcinoma cell line (MCF7), human epithelial carcinoma cell line
(HELA), and colon carcinoma cell line (HCT116).
cell line
Sample conc. (
mg/mL)
Surviving fraction
Sample no
IC50(mg/mL)
5d
8a
8b
8c
Cell line
HEPG2
HEPG2
0
5.0
12.5
25.0
50.0
0
1.00
0.45
0.22
0.21
0.30
1.00
0.24
0.22
0.16
0.28
1.00
0.49
0.12
0.16
0.20
1.00
0.44
0.18
0.29
0.33
1.00
0.24
0.22
0.16
0.28
1.00
0.69
0.47
0.19
0.24
1.00
0.71
0.49
0.20
0.27
1.00
0.68
0.49
0.19
0.28
1.00
0.74
0.37
0.20
0.22
1.00
0.74
0.42
0.24
0.35
1.00
0.42
0.28
0.19
0.23
1.00
0.36
0.20
0.21
0.28
1.00
0.25
0.17
0.18
0.27
1.00
0.21
0.16
0.17
0.23
1.00
0.35
0.26
0.19
0.29
1.00
0.60
0.22
0.23
0.24
1.00
0.60
0.31
0.16
0.30
1.00
0.43
0.21
0.21
0.29
1.00
0.24
0.14
0.22
0.26
1.00
0.38
0.15
0.20
0.26
HEP2
MCF7
HELA
HCT116
5d
8a
8b
8c
4.34
11.8
4.34
6.78
3.28
12.1
3.89
7.09
4.65
12.0
3.28
4.34
9.68
9.83
3.12
2.97
4.19
10.6
3.73
4.04
HEP2
5.0
12.5
25.0
50.0
0
However, under mild basic conditions it can be extracted from cells
and dissolved for measurement. The SRB assay possesses a colori-
metric end point, is indefinitely stable, and is nondestructive. These
practical advantages render the assay an appropriate and sensitive
assay to measure drug-induced cytotoxicity.
Due to the encumbered and overlapping nature of the dose
response curve profiles, Fig. 4 was rendered impractical for the
purpose of a comparative study. Thus, we opted to use the median
inhibition concentration (IC50) values determined from Fig. 4 as
they comprise a convenient means for comparing activity of various
compounds as antitumor agents. The IC50 values measured in this
manner are tabulated in Table 3 and plotted in Fig. 5.
The IC50 values shown in Table 3 and the dose response curves of
Fig. 4 clearly showcase the superiority of imidazolidineiminothione
5d and 8aec against HEPG2, HEP2, MCF7, HELA, and HCT116 and
underscore their general effectiveness. Generally, all cell lines were
responsive to dose increase as indicated by the ensuing reduction in
surviving fraction, although dose response was less pronounced
MCF7
HELA
5.0
12.5
25.0
50.0
0
5.0
12.5
25.0
50.0
0
HCT116
5.0
12.5
25.0
50.0
general applicability to other tumor cell lines. Thus, the cytotoxicity
was further evaluated against human hepatocellular carcinoma cell
line (HEPG2), human laryngeal carcinoma cell line (HEP2), human
breast adenocarcinoma cell line (MCF7), human epithelial carci-
noma cell line (HELA), and colon carcinoma cell line (HCT116). The
between 25 and 50
m
g/mL as shown in Table 2 and Fig. 4. The IC50
values ranged from 3.12 to 12.1
mg/mL and were consistently higher
for group 8a (N-(1): 2,4-dimethoxyphenyl; N-(3): 4-methythio-
phenyl). Notably, group 8b (N-(1): 2,4-dimethoxyphenyl; N-(3): 4-
methoxyphenyl) was markedly active against all cell lines and
consistently produced low IC50 values in all cases (ranging from
compounds were tested at concentrations between 5 and 50 mg/mL
using the sulforhodamine B (SRB) assay (Table 2) and the rela-
tionship between surviving fraction and drug concentration is
plotted in Fig. 4 to get the survival curve of each tumor cell line for
the specified compound. The sulforhodamine B (SRB) assay was
developed by Skehan to measure drug-induced cytotoxicity for
large-scale drug-screening applications. Under mild acidic condi-
tions, the protein dye sulforhodamine B binds electrostatically on
protein basic amino acid residues of trichloroacetic acid-fixed cells.
3.12 to 4.34 mg/mL). This clearly underlines the synergistic effect of
the suitably positioned methoxy groups on the aromatic rings of N-
(1) and N-(3) of the imidazolidineiminothiones. The dominant role
of the N-(1) substituent is further seen within groups 5d and 8c
which show uniform bioactivity against all cell lines with very
negligible disparity in median inhibition concentration values.
1.1
1
14
12
10
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
8
HEPG2
HEP2
6
MCF7
HELA
4
HCT116
0
5
12.5
Concentration (µg/mL)
25
50
2
0
5d-HEPG2
8a-HEPG2
8b-HEPG2
8c-HEPG2
5d-HEP2
8a-HEP2
8b-HEP2
8c-HEP2
5d-MCF7
8a-MCF7
8b-MCF7
8c-MCF7
5d-HELA
5d-HCT116
8a-HCT116
8b-HCT116
8c-HCT116
8a-HELA
8b-HELA
8c-HELA
5d
8a
8b
8c
Compound number
Fig. 4. Cytotoxicity of compounds 5d and 8a-c against human hepatocellular carci-
noma cell line HEPG2, human laryngeal carcinoma cell line (HEP2), human breast
adenocarcinoma cell line MCF7, human epithelial carcinoma cell line HELA, and colon
carcinoma cell line HCT116.
Fig. 5. Minimum inhibitory concentration (MIC) values obtained for compounds 5d
and 8a-c against human hepatocellular carcinoma cell line HEPG2, human laryngeal
carcinoma cell line (HEP2), human breast adenocarcinoma cell line MCF7, human
epithelial carcinoma cell line HELA, and colon carcinoma cell line HCT116.

2284
Z. Moussa et al. / European Journal of Medicinal Chemistry 46 (2011) 2280e2289
3.2. Antimicrobial properties
25
E. coli
B. subtilis
S. lutea
S. aureus
The entire series of compounds 4aed, 5aed, 6aed, 7aed, and
8aec were tested under the same conditions for antimicrobial
activity against Gram negative and Gram positive bacteria.
20
15
10
5
3.2.1. Antimicrobial activities of compounds 4aed, 5aed, 6aed,
7aed, and 8aec against Gram negative and Gram positive bacteria
All of the synthesized compounds 4aed, 5aed, 6aed, 7aed, and
8aec were tested in vitro for antibacterial activity against the
following bacterial strains: Gram negative bacteria, Escherichia coli
NCTC-10416, Sarcina lutea ATCC-934, and Gram positive bacteria,
Bacillus subtilis NCTC-1040, Staphylococcus aureus NCTC-7447. The
results are summarized in Table 4 and represented graphically in
Fig. 6. The compounds were dissolved in N, N-dimethylformamide
(5 mg/mL) and tested for antimicrobial activity by the agar disk
diffusion technique using a 1 cm microplate well diameter and
0
Compound number
Fig. 6. Antibacterial activities of compounds 4aed, 5aed, 6aed, 7aed, and 8aec
against Gram negative and Gram positive bacteria.
a 100
mL of each concentration. Chloroamphenicol was used as
benzyl) exhibited the highest and lowest antibacterial activities in
the series, respectively (25 & 12 mm, respectively). All heterocycles
were effective against Gram positive and Gram negative bacteria,
displaying negligible disparity in antibacterial activity for indi-
vidual compounds across all strains of bacterial organisms under
study. However, a clear difference in antibacterial activity can be
observed between groups which can be attributed to the rein-
forcing or opposing effects induced by N-(1) and N-(3) substituents.
a standard antibacterial agent. In the disk diffusion method for
susceptibility testing, compound-impregnated disks are placed on
an agar plate previously containing a standard suspension of
microorganism. The compound contained in the disk diffuses 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 diameters of the zones of inhibition are
measured with calipers or automated scanners and are compared
with standard zone size ranges that determine susceptibility,
intermediate susceptibility, or resistance to the antimicrobials that
were examined.
3.3. Antifungal properties
The emergence of resistance among fungi against common
known antifungal agents is a major concern. Thus, the discovery of
newer antifungal agents is an important field of research. Very
often clinical isolates of standard strains of fungi are screened for
their susceptibility against new antifungal agents by broth dilution
and disc diffusion methods, whereby the former is often performed
following the standard method of NCCLS M38A. The emergence of
resistance among Aspergillus has been noted as a major problem.
Invasive aspergillosis has emerged worldwide as a cause of infec-
tion among immunocompromised patients. In specific, aspergil-
losis is a mycotic infection caused by the genus Aspergillus majorly
by Aspergillus flavus, which rationalized our judicious choice of such
a strain in the antifungal susceptibility tests. Besides invasive
aspergillosis, the spectrum of aspergillosis includes colonization of
air sacs, pulmonary, aspergillosis in solid organs transplantation
and invasive aspergillosis in AIDS patients. Treatment of invasive
aspergillosis has been poorly addressed where mortality rate can be
reduced if the disease is diagnosed and treated at an early stage.
Although various novel broad spectrum antifungal agents such as
triazoles, ravuconazole, voriconazole, and posaconazole were
reported in recent time, concurrent emergence of resistance has
become an issue. Thus, new classes of such agents are desired. As
such, imidazolidineiminothiones comprise a new class of agents
exhibiting promising antifungal activity. Compounds 4aed, 5aed,
6aed, 7aed, and 8aec were all tested under the same conditions
for antifungal activity using Candida albicans IMRU-3669 and
Aspergillus flavus as unicellular and filamentous fungi, respectively.
The mean values of inhibition zone diameter summarized in
Table 4 and plotted in Fig. 6 show that all imidazolidineimino-
thiones tested in the antimicrobial assays possess significant anti-
bacterial activities against the growth of E. coli, S. lutea, B. subtilis,
and S. aureus on solid media, where compounds 4a (N-(1): phenyl;
N-(3): 4-methythiophenyl) and 7d (N-(1): 4-iodophenyl; N-(3):
Table 4
Antimicrobial activity of compounds 4aed, 5aed, 6aed, 7aed, and 8aec against
Gram negative and Gram positive bacteria.
Compound no
Mean values of inhibition zone diameter (mm)
Test organism
Grams negative
bacteria
Grams positive bacteria
E. coli
S. lutea
B. subtilis
S. aureus
4a
4b
4c
4d
5a
5b
5c
5d
6a
6b
6c
6d
7a
7b
7c
7d
8a
8b
8c
8d
25.5
25.0
20.0
23.5
15.5
23.5
17.0
22.0
15.5
16.0
16.5
18.5
14.5
19.0
14.0
14.0
19.0
24.5
25.5
*ND
25.0
25.0
18.0
20.0
15.0
22.0
21.0
20.0
15.0
19.0
14.0
18.0
14.0
18.0
13.0
12.0
18.0
24.0
25.0
*ND
23.5
19.0
21.0
21.5
16.5
20.0
16.0
21.0
19.5
18.0
18.0
17.0
16.0
19.5
16.5
15.0
18.0
20.0
22.5
*ND
24.0
18.0
21.5
22.0
16.0
19.5
12.0
21.0
19.0
13.0
18.0
15.5
16.5
18.5
17.0
15.0
18.0
20.0
22.0
*ND
3.3.1. Antifungal activities of compounds 4aed, 5aed, 6aed, 7aed,
and 8aec against C. albicans and A. flavus
Compounds4aed, 5aed, 6aed, 7aed, and8aecweresubjected to
in vitro testing of antifungal activity against the following fungal
strains: C. albicans IMRU-3669 and A. flavus. The results are displayed
in Table 5 and represented graphically in Fig. 7. The compounds were
tested for antifungal activity by the agar disk diffusion technique
*ND: Not determined
using a 1 cm microplate well diameter and a 100 mL of each

Z. Moussa et al. / European Journal of Medicinal Chemistry 46 (2011) 2280e2289
2285
Table 5
Table 6
Antifungal activity of compounds 4aed, 5aed, 6aed, 7aed, and 8aec against
C. albicans and A. flavus.
Minimum inhibitory and bactericidal concentrations of compounds 4d, 5b, and 8b
obtained against various strains of Gram positive and Gram negative bacteria and
fungi.
Compound no
Mean values of inhibition zone diameter
(mm)
Minimum inhibitory concentration MIC (
mg/mL)
C. albicans
A. flavus
Test organism
4a
4b
4c
4d
5a
5b
5c
5d
6a
6b
6c
6d
7a
7b
7c
7d
8a
8b
8c
8d
24.0
21.0
22.0
24.0
16.5
22.0
12.0
22.0
16.0
12.0
14.0
17.0
16.5
23.0
17.0
13.0
20.0
22.0
17.5
*ND
18.0
18.0
15.0
17.0
15.0
17.5
14.0
18.0
15.0
18.0
15.0
15.0
15.0
18.0
14.0
15.0
14.0
17.0
14.0
*ND
Compound no Gram negative Gram positive
bacteria bacteria
Fungus
E. coli S. lutea B. subtilis S. aureus C. albicans A. flavus
4d
5d
8d
16.1
64.5
16.1
32.3
64.5
32.3
64.5
64.5
64.5
64.5
125
64.5
64.5
250
125
250
250
250
Minimum bactricidal concentration MBC (
mg/mL)
Test organism
Compound no Gram negative Gram positive
bacteria bacteria
E. coli S. lutea B. subtilis S. aureus C. albicans A. flavus
Fungus
4d
5d
8d
16.1
64.5
16.1
32.3
64.5
32.3
64.5
125
64.5
64.5
64.5
64.5
64.5
250
125
250
250
250
concentrations (MIC) and minimum bactericidal concentrations
(MBC). The former technique determines the lowest antimicrobial
concentration of the assayed agent required to inhibit visible
growth of the organism being investigated after approximately
24 h of incubation in a specific growth medium, whereas the latter
determines the lowest concentration of the drug that results in
99.9% or greater reduction of the concentration of organisms after
incubation relative to the initial concentration. The MIC quantita-
tively determines in vitro antimicrobial activity. Classically, MICs
were measured through the macrotube dilution method, which
uses liquid growth medium (broth), doubling serial dilutions of
antimicrobials in test tubes, and a standard antimicrobial inoc-
ulum. In certain infections such as meningitis and endocarditis, the
bactericidal activity may be more predictive of a favorable infection
outcome than the MIC. The MBC can be carried out in conjunction
with the broth microtiter MIC test by taking aliquots of broth from
microtiter wells that demonstrate no visible growth and applying
them onto antibiotic-free agar plates for subsequent incubation.
For certain antibiotic classes like the aminoglycosides and the
quinolones, the MIC often approximates the MBC. However, for
concentration. Grisofluvin was used as a standard antifungal agent.
AllcompoundsweredissolvedinN, N-dimethylformamide(5mg/mL)
which itself was inactive in the above assay.
All of the tested compounds were found to possess strong
antifungal activity against the two microorganisms where the
mean value of the inhibition zone diameter fluctuated from 12 to
24 mm. In general, most compounds showed slightly higher anti-
fungal activity toward C. albicans. In particular, imidazolidineimi-
nothiones 4a,d (N-(1): phenyl; N-(3): 4-methlthiophenyl; N-(1):
phenyl; N-(3): benzyl, respectively) and 7b (N-(1): iodophenyl;
N-(3): methoxyphenyl) exhibited the highest antifungal activities
and were almost equally as effective against C. albicans (24, 24, and
33 mm, respectively). It is noted that all tested compounds were
almost equally as effective against A. flavus, producing a consistent
and a narrow range of inhibition zone diameter values (14e18 mm).
3.4. MIC and MBC
glycopeptides and
b-lactam antibiotics, the MBC can exceed the
3.4.1. Minimum inhibitory concentrations and minimum
bactericidal concentrations of compounds 4d, 5b, and 8b against
various strains of Gram positive and Gram negative bacteria and
fungi
MIC significantly, resulting in an overestimation of in-vivo bacte-
ricidal activity. When the MBC exceeds the MIC by 32-fold or more,
an organism is labeled tolerant to the antimicrobial’s killing
activity. Although tolerance has been documented for b-lactams
In addition to the preceding tests of the potential antimicrobial,
and antifungal activities of heterocycles 4aede8aec by the agar
disk diffusion technique, we performed a more quantitative
measure of the antimicrobial and antifungal activity of a narrow
selection of potential leads by evaluating their minimum inhibitory
and glycopeptides against certain staphylococci, streptococci, and
enterococci, its impact on the outcome of infections caused by
organisms other than those just oulined appears to be limited. MIC
and MBC techniques, although fundamentally not much different,
are reliable means to determine the susceptibilities of bacteria and
other organisms to drugs and also to monitor the activity of new
potential agents. Thus, the antibacterial and antifungal properties
of compounds 4d, 5b, and 8b were evaluated by the macrotube
dilution procedure (Table 6) following the guidelines of the Clinical
and Laboratory Standard Institute (CLSI) [70,71], formerly National
Committee for Clinical and Laboratory Standards (NCCLS), against
a series of 4 bacterial strains and 2 fungal species. To carry out the
antimicrobial and antifungal evaluation, concentrations of
30
C. albicans
25
A. flavus
20
15
10
5
0
compounds up to 250 mg/mL were incorporated to growth media
4a 4b 4c 4d 5a 5b 5c 5d 6a 6b 6c 6d 7a 7b 7c 7d 8a 8b 8c
according to published procedures [70,71]. As shown in Fig. 8, all
compounds tested showed inhibitory effect, ranging from as low as
Compound number
16.125
mg/mL to as high as 250 mg/mL. In certain cases, such values
Fig. 7. Antifungal activities of compounds 4aed, 5aed, 6aed, 7aed, and 8aec against
C. albicans and A. flavus.
are low enough to render such agents as potential candidates for

2286
Z. Moussa et al. / European Journal of Medicinal Chemistry 46 (2011) 2280e2289
5. Experimental
300
250
200
150
100
50
E. coli
5.1. Chemistry
S. lutea
IR spectra were recorded (KBr) on a Perkin Elmer 1650 spec-
B. subtilis
S. aureus
C. albicans
A. flavus
trophotometer. 1H NMR and 13C NMR spectra were recorded on
Avance II Bruker FT-NMR spectrometer 400 (400 MHz) using 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 GCeMS QP 100 EX (70 eV) at the Micro Analytical Center
at Cairo University. Found: C, H, N & S for all compounds were
within ꢂ0.4 from the theoretical value. Melting points were
obtained on a Fisher-Johns melting points apparatus and are
uncorrected. The physical data of the synthesized compounds are
shown in Table 7 (see Supplementary material section).
0
Compound number
Fig. 8. MIC values of compounds 4d, 5b, and 8b obtained against various strains of
Gram positive and Gram negative bacteria and fungi.
5.1.1. For the preparation of the known cyanothioformanilides
1aeg, see ref. [64e69]
For a general procedure for the preparation of cyanothio-
formanilides see ref. [65]; 1a,b are commercially available; For the
preparation of 1b and 1c, see ref. [66]; Also see the ref. [67] for 1c;
See the ref. [68] for 1bed.
further studies. It is noted that high MIC values were observed for
C. albicans and A. flavus (MIC 64.5e250
mg/mL) pointing to the
relative ineffectiveness of tested compounds as antifungal agents.
On the other hand, low MIC values were obtained with Gram
negative bacteria (MIC 16.125e64.5
values were obtained with Gram positive bacteria (MIC
64.5e125 g/mL). In relation to structure-activity trends, compar-
mg/mL), whereas modest MIC
5.1.2. Typical procedure for the preparation of compounds 4aed,
5aed, 6aed, 7aed, and 8aec
m
A solution of the cyanothioformanilide (5 mmol) and the cor-
responding isocyanate (5 mmol) in dry ether (30 mL) was treated
with three drops of triethylamine. The reaction mixture was
magnetically stirred for 15 min. The obtained product was filtered
off, washed with ether, air-dried and recrystallized from chloro-
form/n-hexane to give compounds 4aed, 5aed, 6aed, 7aed, and
8aec (Table 7, see Supplementary material section) inyields ranging
from 75 to 90%.
ison is only valid between compounds 6b (N-(1): 4-bromophenyl;
N-(3): 4-methoxyphenyl) and 8b (N-(1): 2,4-dimethoxyphenyl;
N-(3): 4-methoxyphenyl). Thus, a clear trend can be seen as the
bromine atom of 6b is replaced by the 2,4-dimethoxy group in 8b.
A sharp drop in MIC values was observed for E. coli, S. lutea,
S. aureus, and C. albicans in which they were reduced in half. This
again points to the dominant role played by the substituent on N-
(1) and to the enhancement of activity by the 2,4-dimethoxy group
which is in line with earlier observations. The MBC values shown in
Table 6 matched well with their MIC counterpart except in the case
of 5b. Thus, similar conclusions can be drawn as was done earlier
for the MIC data.
5.1.3. 4-Imino-3-(4-(methylthio)phenyl)-1-phenyl-5-
thioxoimidazolidin-2-one (4a)
IR (KBr) 3428 (NH), 1760 (C]O), 1663 (C]N), 1120 (C]S) cmꢃ1
;
1H NMR (CDCl3, 400 MHz)
d 9.56 (s, 1H, NH), 7.59e7.45 (m, 7H,
AreH), 7.38 (d, J ¼ 8.8 Hz, 2H, AreH), 2.51 (s, 3H, SCH3); 13C NMR
4. Conclusion
(CDCl3, 100 MHz) d 181.4 (C]S), 154.1 (C]O), 153.7 (C]N), 139.7
(C), 132.7 (C), 129.7 (CH), 129.5 (CH), 128.7 (C), 127.1 (CH), 126.9
(CH), 126.7 (CH), 15.7 (CH3); MS (m/z, %) 329 (10, Mþ þ 2), 328 (17,
Mþ þ 1), 327 (40, Mþ), 165 (100, CH3eSeC6H4-NCO-p), 135 (53,
C6H5NCS), 77 (77, C6H5þ).
In summary, the synthesis and characterization of a new series of
imidazolidineiminothiones with variable aromatic substituents at
N-(1) and N-(3) has been described. Most compounds displayed
antitumor, antibacterial and antifungal activities. Structureeactivity
relationship studies revealed several matching pairs of substituents
that could serve to optimize structural features for optimal activity
that would eventually render such compounds clinically useful drug
agents. Initial screening of all compounds against EAC cells indicated
that 5d, and 8aec werethe most activecompoundsas theyexhibited
the highest percent inhibition of cell viability (80%, 70%, 80%, and
70%, respectively). Consequently, they were further assayed against
other tumor cancer cell lines (HEPG2, HEP2, MCF7, HELA, and
5.1.4. 4-Imino-3-(4-methoxyphenyl)-1-phenyl-5-
thioxoimidazolidin-2-one (4b)
IR (KBr) 3439 (NH), 1788 (C]O), 1665 (C]N), 1246 (CeO), 1171
(CeO),1121 (C]S)cmꢃ1;1HNMR(DMSO, 400MHz)
d 9.58(s,1H, NH),
7.61e7.50 (m, 5H, AreH), 7.47 (d, J ¼ 9.0 Hz, 2H, AreH), 7.08 (d,
J ¼ 9.0 Hz, 2H, AreH), 3.80 (s, 3H, OMe); 13C NMR (DMSO, 100 MHz)
d
183.2 (C]S),159.1 (CeO),154.7 (C]O),154.1 (C]N),133.6 (C),129.6
(CH), 129.4 (CH), 128.9 (CH), 127.9 (CH), 125.2 (C), 114.4 (CH), 55.6
(OCH3); MS (m/z, %) 314 (1, Mþ þ3), 313 (6, Mþ þ2), 312 (14, Mþ þ1),
311 (60, Mþ), 149 (100, CH3eOeC6H4eNCO-p), 135 (82, C6H5NCS).
HCT116). The IC50 values ranged from 3.12 to 12.1
mg/mL where
compound 8b (N-(1): 2,4-dimethoxyphenyl; N-(3): 4-methox-
yphenyl) was markedly active against all cell lines and consistently
produced low IC50 values in all cases (ranging from 3.12 to 4.34
m
g/
5.1.5. 4-Imino-1-phenyl-5-thioxo-3-p-tolylimidazolidin-2-one (4c)
mL). This points to the synergistic effect of the suitably positioned
methoxy substituents on the aromatic rings of N-(1) and N-(3) of the
imidazolidineiminothiones. All compounds were also tested against
microbial organisms (E. coli, S. lutea, B. subtilis, and S. aureus), and
fungal strains (C. albicans and A. flavus). Most tested compounds
showed significant activities which could be optimized with the
appropriate combination of matching aromatic substituents on N-
(1) and N-(3).
IR (KBr) 3451 (NH), 1771 (C]O), 1667 (C]N), 1125 (C]S) cmꢃ1
;
1H NMR (CDCl3, 400 MHz)
AreH), 7.35e7.30 (m, 2H, AreH), 2.41 (s, 3H, ArCH3); 13C NMR
(CDCl3, 100 MHz) 181.6 (C]S), 154.3 (C]O), 153.8 (C]N), 138.8
d 9.54 (s, 1H, NH), 7.59e7.41 (m, 7H,
d
(C), 132.8 (C), 130.0 (CH), 129.7 (CH), 129.5 (CH), 129.3 (C), 127.1
(CH), 126.3 (CH), 21.2 (CH3); MS (m/z, %) 296 (13, Mþ þ 1), 295 (48,
Mþ), 135 (100, C6H5eNCS), 133 (57, CH3eC6H4eNCO-p), 91 (54,
C6H4eCH3þ), 77 (58, C6H5þ).

Z. Moussa et al. / European Journal of Medicinal Chemistry 46 (2011) 2280e2289
2287
5.1.6. 3-Benzyl-4-imino-1-phenyl-5-thioxoimidazolidin-2-one (4d)
IR (KBr) 3430 (NH), 1769 (C]O), 1665 (C]N), 1092 (C]S) cmꢃ1
1H NMR (CDCl3, 400 MHz)
9.37 (s, 1H, NH), 7.54e7.41 (m, 5H,
AreH), 7.40e7.20 (m, 5H, AreH), 4.98 (s, 2H, ArCH2); 13C NMR
(CDCl3, 100 MHz) 182.0 (C]S), 154.7 (C]N), 153.6 (C]O), 135.4
(C), 132.8 (C), 129.5 (CH), 129.4 (CH), 129.1 (CH), 128.8 (CH), 128.3
(CH), 127.0 (CH), 44.4 (CH2); MS (m/z, %) 296 (11, Mþ þ 1), 295 (30,
Mþ), 135 (23, C6H5eNCS), 133 (7, C6H5eCH2eNCO), 91 (100,
C6H5eCH2þ), 77 (87, C6H5þ).
5.1.12. 1-(4-Bromophenyl)-4-imino-3-(4-methoxyphenyl)-5-
thioxoimidazolidin-2-one (6b)
IR (KBr) 3439 (NH), 1769 (C]O), 1665 (C]N), 1256 (CeO), 1179
(CeO), 1117 (C]S) cmꢃ1 1H NMR (CDCl3, 400 MHz)
9.53 (s, 1H,
;
d
;
d
d
NH), 7.68 (d, J ¼ 8.8 Hz, 2H, AreH), 7.45 (d, J ¼ 9.0 Hz, 2H, AreH),
7.38 (d, J ¼ 8.8 Hz, 2H, AreH), 7.03 (d, J ¼ 9.0 Hz, 2H, AreH), 3.85 (s,
3H, OMe); 13C NMR (CDCl3, 100 MHz)
d 181.2 (C]S), 159.7 (CeO),
154.3 (C]O), 153.6 (C]N), 132.7 (CH), 131.7 (C), 128.7 (CH), 127.9
(CH), 124.3 (C), 123.7 (CeBr), 114.7 (CH), 55.6 (CH3); MS (m/z, %) 392
(3, Mþ þ 3), 391 (14, Mþ þ 2), 390 (5, Mþ þ 1), 389 (13, Mþ), 149
(100, CH3.O.C6H4.NCO-p).
5.1.7. 1-(4-Chlorophenyl)-4-imino-3-(4-(methylthio)phenyl)-5-
thioxoimidazolidin-2-one (5a)
IR (KBr) 3424 (NH), 1771 (C]O), 1665 (C]N), 1093 (C]S) cmꢃ1
;
5.1.13. 1-(4-Bromophenyl)-4-imino-5-thioxo-3-p-
tolylimidazolidin-2-one (6c)
1H NMR (CDCl3, 400 MHz)
d
9.57 (s, 1H, NH), 7.52 (d, J ¼ 9.0 Hz, 2H,
AreH), 7.47 (d, J ¼ 8.8 Hz, 2H, AreH), 7.42 (d, J ¼ 9.0 Hz, 2H, AreH),
IR (KBr) 3455 (NH),1773 (C]O),1663(C]N),1126(C]S)cmꢃ1;1H
7.37 (d, J ¼ 8.8 Hz, 2H, AreH), 2.51 (s, 3H, SCH3); 13C NMR (CDCl3,
NMR(CDCl3, 400MHz)
7.45e7.35 (m, 4H, AreH), 7.34e7.30 (m, 2H, AreH), 2.41 (s, 3H,
ArCH3); 13C NMR (CDCl3,100 MHz)
181.2 (C]S),154.2 (C]O),153.5
d
9.55(s,1H, NH), 7.68(d, J¼ 8.8Hz, 2H, AreH),
100 MHz) d 181.1 (C]S), 154.0 (C]O), 153.4 (C]N), 139.8 (C), 135.6
(CeCl), 131.0 (C), 129.7 (CH), 128.5 (C), 128.4 (CH), 126.9 (CH), 126.7
(CH), 15.6 (CH3); MS (m/z, %) 365 (4, Mþ þ4), 364 (9, Mþ þ3), 363
(37, Mþ þ2), 362 (21, Mþ þ1), 361 (79, Mþ), 75 (100).
d
(C]N),138.9(C),132.7(CH),131.7(C),130.0(CH),129.1 (C),128.7(CH),
126.3 (CH), 123.7 (CeBr), 21.2 (CH3); MS (m/z, %) 375 (34, Mþ þ 2), 374
(43, Mþ þ 1), 373 (40, Mþ), 215 (63), 213 (49, Br.C6H4.NCS-p),155 (20),
133 (100, C6H5.CH2.NCO), 91 (46, C6H4eCH3þ), 107 (27).
5.1.8. 1-(4-Chlorophenyl)-4-imino-3-(4-methoxyphenyl)-5-
thioxoimidazolidin-2-one (5b)
IR (KBr) 3439 (NH), 1769 (C]O), 1665 (C]N), 1256 (CeO), 1179
5.1.14. 3-Benzyl-1-(4-bromophenyl)-4-imino-5-
(CeO), 1126 (C]S) cmꢃ1
;
1H NMR (CDCl3, 400 MHz)
d
9.53 (s, 1H,
thioxoimidazolidin-2-one (6d)
NH), 7.52 (d, J ¼ 8.9 Hz, 2H, AreH), 7.47e7.41 (m, 4H, AreH), 7.03 (d,
IR (KBr) 3430 (NH), 1771 (C]O), 1665 (C]N), 1072 (C]S) cmꢃ1
;
J ¼ 8.9 Hz, 2H, AreH), 3.85 (s, 3H, OMe); 13C NMR (CDCl3, 100 MHz)
1H NMR (CDCl3, 400 MHz)
AreH), 7.53e7.48 (m, 2H, AreH), 7.39e7.21 (m, 5H, AreH), 4.98 (s,
2H ArCH2); 13C NMR (CDCl3, 100 MHz)
181.5 (C]S), 154.3 (C]O),
d
9.40 (s, 1H, NH), 7.63 (d, J ¼ 8.8 Hz, 2H,
d
181.8 (C]S), 159.7 (CeO), 154.3 (C]O), 153.7 (C]N), 135.6 (CeCl),
131.2 (C), 129.7 (CH), 128.4 (CH), 127.9 (CH), 124.3 (C), 114.7 (CH),
55.5 (CH3); MS (m/z, %) 347 (19, Mþ þ 2), 346 (12, Mþ þ 1), 345 (27,
Mþ),169 (40, Cl.C6H4NCS-p),168 (43),149 (100, CH3.O.C6H4.NCO-p),
107 (27), 91 (25).
d
153.5 (C]N), 135.2 (C), 132.6 (CH), 131.6 (C), 129.1 (CH), 128.8 (CH),
128.6 (CH), 128.3 (CH), 123.5 (CeBr), 44.5 (CH2); MS (m/z, %) 372
(16, Mþ e 1), 168 (43), 91 (100, C6H5CH2þ), 106 (61).
5.1.9. 1-(4-Chlorophenyl)-4-imino-5-thioxo-3-p-tolylimidazolidin-
2-one (5c)
5.1.15. 4-Imino-1-(4-iodophenyl)-3-(4-(methylthio)phenyl)-5-
thioxoimidazolidin-2-one (7a)
IR (KBr) 3460 (NH), 1771 (C]O), 1663 (C]N), 1125 (C]S)
IR (KBr) 3430 (NH), 1777 (C]O), 1661 (C]N), 1094 (C]S) cmꢃ1
;
cmꢃ1
;
1H NMR (CDCl3, 400 MHz)
d
9.55 (s, 1H, NH), 7.52 (d,
J ¼ 9.0 Hz, 2H, AreH), 7.47e7.40 (m, 4H, AreH), 7.35e7.30 (m, 2H,
AreH), 2.41 (s, 3H, ArCH3); 13C NMR (CDCl3, 100 MHz)
181.3
1H NMR (DMSO, 400 MHz)
d
9.67 (s, 1H, NH), 7.95 (d, J ¼ 8.7 Hz, 2H,
AreH), 7.48 (d, J ¼ 8.7 Hz, 2H, AreH), 7.41 (d, J ¼ 8.7 Hz, 2H, AreH),
d
7.35 (d, ¼ 8.7 Hz, 2H, AreH), 2.51 (s, 3H, SCH3); 13C NMR (DMSO,
(C]S), 154.2 (C]O), 153.6 (C]N), 138.9 (C), 135.6 (CeCl), 131.1
(C), 130.0 (CH), 129.7 (CH), 129.1 (C), 128.4 (CH), 126.3 (CH), 21.2
(CH3); MS (m/z, %) 332 (6, Mþ þ 3), 331 (20, Mþ þ 2), 330 (15,
Mþ þ 1), 329 (46, Mþ), 169 (100, Cl.C6H4.NCS-p), 133 (88,
CH3.C6H4.NCO-p).
100 MHz) d 182.8 (C]S), 154.3 (C]O), 153.7 (C]N), 138.9 (C), 138.4
(CH), 133.3 (C), 130.2 (CH), 129.3 (C), 127.9 (CH), 126.2 (CH), 96.2
(CeI), 14.8 (CH3); MS (m/z, %) 455 (8, Mþ þ 2), 454 (19, Mþ þ 1), 453
(61, Mþ), 261 ( 20, I.C6H4NCS-p), 165 (100, CH3SC6H4NCO-p).
5.1.16. 4-Imino-1-(4-iodophenyl)-3-(4-methoxyphenyl)-5-
thioxoimidazolidin-2-one (7b)
5.1.10. 3-Benzyl-1-(4-chlorophenyl)-4-imino-5-thioxoimidazolidin-
2-one (5d)
IR (KBr) 3417 (NH), 1777 (C]O), 1661 (C]N), 1250 (CeO), 1175
IR (KBr) 3433 (NH), 1777 (C]O), 1665 (C]N), 1094 (C]S) cmꢃ1
;
(CeO), 1126 (C]S) cmꢃ1 1H NMR (DMSO, 400 MHz)
; d 9.60 (s, 1H,
1H NMR (CDCl3, 400 MHz)
AreH), 7.40e7.28 (m, 5H, AreH), 4.98 (s, 2H, ArCH2); 13C NMR
(CDCl3, 100 MHz) 181.6 (C]S), 154.4 (C]N), 153.5 (C]O), 135.4
d
9.40 (s, 1H, NH), 7.55e7.45 (m, 4H
NH), 7.95 (d, J ¼ 8.7 Hz, 2H, AreH), 7.44 (d, J ¼ 9.0 Hz, 2H, AreH),
7.34 (d, J ¼ 9.0 Hz, 2H, AreH), 7.08 (d, J ¼ 8.7 Hz, 2H, AreH), 3.80 (s,
d
3H, OMe); 13C NMR (DMSO, 100 MHz)
d 182.7 (C]S), 159.1 (CeO),
(CeCl), 135.2 (C), 131.1 (C), 129.6 (CH), 129.1 (CH), 128.8 (CH), 128.4
(CH), 128.3 (CH), 44.5 (CH2); MS (m/z, %) 332 (2, Mþ þ 3), 331 (9,
Mþ þ 2), 330 (10, Mþ þ 1), 329 (21, Mþ), 91 (100, C6H5CH2þ).
154.6 (C]O), 153.8 (C]N), 138.3 (CH), 133.0 (C), 130.1 (CH), 128.8
(CH),125.2 (C),114.4 (CH), 96.1 (CeI), 55.6 (CH3); MS (m/z, %) 439 (2,
M
þ þ 2), 438 (6, Mþ þ 1), 437 (26, Mþ), 261 ( 32, I.C6H4.NCS-p), 149
(100, CH3.O.C6H4.NCO-p).
5.1.11. 1-(4-Bromophenyl)-4-imino-3-(4-(methylthio)phenyl)-5-
thioxoimidazolidin-2-one (6a)
5.1.17. 4-Imino-1-(4-iodophenyl)-5-thioxo-3-p-tolylimidazolidin-
2-one (7c)
IR (KBr) 3428 (NH), 1769 (C]O), 1663 (C]N), 1094 (C]S) cmꢃ1
;
1H NMR (CDCl3, 400 MHz)
AreH), 7.48 (d, J ¼ 8.9 Hz, 2H, AreH), 7.40e7.35 (m, 4H, AreH), 2.51
(s, 3H, SCH3); 13C NMR (CDCl3, 100 MHz)
181.0 (C]S), 154.0 (C]
d
9.58 (s, 1H, NH), 7.68 (d, J ¼ 8.8 Hz, 2H,
IR (KBr) 3450 (NH), 1773 (C]O), 1663 (C]N), 1126 (C]S) cmꢃ1
;
1H NMR (CDCl3, 400 MHz)
d
9.55 (s, 1H, NH), 7.88 (d, J ¼ 8.8 Hz, 2H,
d
AreH), 7.42 (d, J ¼ 8.4 Hz, 2H, AreH), 7.32 (d, J ¼ 8.4 Hz, 2H, AreH),
O), 153.4 (C]N), 139.9 (C), 132.7 (CH), 131.6 (C), 128.7 (CH), 128.5
(C), 126.9 (CH), 126.7 (CH), 123.7 (C), 15.6 (CH3); MS (m/z, %) 408 (1,
7.24 (d, J ¼ 8.8 Hz, 2H, AreH), 2.41 (s, 3H, ArCH3); 13C NMR (CDCl3,
100 MHz) d 181.1 (C]S), 154.2 (C]O), 153.4 (C]N), 138.9 (C), 138.6
M
þ þ 3), 407 (2, Mþ þ 2), 406 (4, Mþ þ 1), 405 (2, Mþ), 139 (100),
(CH), 132.4 (C), 130.0 (CH), 129.1 (C), 128.8 (CH), 126.3 (CH), 95.4
165 (82, CH3.S.C6H4.NCO-p).
(CeI), 21.2 (CH3); MS (m/z, %) 423 (7, Mþ þ 2), 422 (10, Mþ þ 1), 421

2288
Z. Moussa et al. / European Journal of Medicinal Chemistry 46 (2011) 2280e2289
(59, Mþ), 288 (11, I.C6H4.NH.CS.CN-p), 261 (68, I.C6H4.NCS-p), 203
preinoculated mice. The ascetic fluid was diluted to 1:10 with
saline to contain 2.5 ꢀ 106 cells on a hemocytometer. In a set of
test tubes, 0.1 mL of tumor cells suspension, 0.8 mL RPMI 1640
media, and 0.1 mL of each tested compound (corresponding to
(8), 133 (78, CH3.C6H4.NCO-p), 107 (100), 91 (61, C6H4eCH3þ).
5.1.18. 3-Benzyl-4-imino-1-(4-iodophenyl)-5-thioxoimidazolidin-
2-one (7d)
100 m
g) were mixed and incubated at 37 ꢁC for 2 h. Trypan blue
IR (KBr) 3430 (NH), 1773 (C]O), 1663 (C]N), 1072 (C]S) cmꢃ1
;
exclusion test was carried out to calculate the percentage of non-
viable cells [72].
1H NMR (CDCl3, 400 MHz)
d
9.39 (s, 1H, NH), 7.84 (d, J ¼ 8.7 Hz, 2H,
AreH), 7.55e7.48 (m, 2H, AreH), 7.40e7.20 (m, 3H, AreH), 7.15 (d,
J ¼ 8.7 Hz, 2H, AreH), 4.98 (s, 2H, ArCH2); 13C NMR (CDCl3,
5.2.2. Cytotoxic activity studies of 5d and 8aec against various
tumor cell lines
100 MHz)
d 181.4 (C]S), 154.3 (C]O), 153.5 (C]N), 138.6 (CH),
135.2 (C), 132.0 (C), 129.1 (CH), 128.8 (CH), 128.7 (CH), 128.3 (CH),
95.2 (CeI), 44.5 (CH2); MS (m/z, %) 422 (35, Mþ þ 1), 421 (29, Mþ),
261 (10, I.C6H4.NCS-p), 106 (71), 91 (100, C6H5eCH2þ), 65 (86).
Compounds 5d and 8aec were tested at concentrations
between 1 and 10
mg/mL using SRB assay for cytotoxic activity
against HEPG2, HEP2, MCF7, HELA, and HCT116 tumor cell lines.
5.1.19. 1-(2,4-Dimethoxyphenyl)-4-imino-3-(4-(methylthio)
phenyl)-5-thioxoimidazolidin-2-one (8a)
5.2.3. Measurement of potential cytotoxicity by SRB assay
Potential cytotoxicity of compounds 5d and 8aec was tested
using the method of Skehan et al. [73]. Cells were plated in 96
multiwell plate (104 cells/well) for 24 h before treatment with the
compounds to allow attachment to the wall of the plate. Different
IR (KBr) 3455 (NH), 1778 (C]O), 1661 (C]N), 1167 (CeO), 1142
(CeO), 1090 (C]S) cmꢃ1; 1H NMR (DMSO, 400 MHz)
d 9.72 (s, 1H,
NH), 7.49 (d, J ¼ 8.8 Hz, 2H, 2H, AreH), 7.41 (d, J ¼ 8.8 Hz, 2H, 2H,
AreH), 7.36 (d, J ¼ 8.8 Hz, 1H, 2H, AreH), 6.77 (d, J ¼ 2.6 Hz, 1H, 2H,
AreH), 6.68 (dd, J ¼ 8.8, 2.6 Hz, 1H, 2H, AreH), 3.83 (s, 3H, OMe),
3.77 (s, 3H, OMe), 2.51 (s, 3H, SCH3); 13C NMR (DMSO, 100 MHz)
concentration of the compounds (5.0, 12.5, 25 and 50 mg/mL) 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 deter-
d
183.2 (C]S), 161.9 (CeO), 155.8 (CeO), 154.2 (C]O), 153.7 (C]N),
139.0 (C), 130.3 (CH), 129.2 (C), 127.9 (CH), 126.2 (CH), 114.4 (C),
105.8 (CH), 99.6 (CH), 56.2 (CH3), 55.8 (CH3), 14.8 (CH3); MS (m/z, %)
388 (10, Mþ þ 1), 387 (37, Mþ), 195 (64, (CH3O)2C6H3NCS-2,4), 165
(100, CH3SC6H4NCO-p).
5.1.20. 1-(2,4-Dimethoxyphenyl)-4-imino-3-(4-methoxyphenyl)-5-
thioxoimidazolidin-2-one (8b)
mine the IC50.
IR (KBr) 3425 (NH), 1777 (C]O), 1667 (C]N), 1252 (CeO), 1163
5.2.4. Antimicrobial and antifungal studies of compounds
4aede8aec
(CeO), 1125 (C]S) cmꢃ1 1H NMR (DMSO, 400 MHz)
; d 9.63 (s, 1H,
NH), 7.44 (d, J ¼ 9.0 Hz, 2H, AreH), 7.35 (d, J ¼ 8.8 Hz, 1H, AreH),
7.07 (d, J ¼ 9.0 Hz, 2H, AreH), 6.76 (d, J ¼ 2.6 Hz, 1H, AreH), 6.68
(dd, J ¼ 8.8, 2.6 Hz, 1H, AreH), 3.82 (s, 3H, OMe), 3.80 (s, 3H, OMe),
Antimicrobial and antifungal studies were carried out by the
diffusion agar technique [74] using a 1 cm microplate well diameter
and a 100 mL of a sample concentration of 5 mg/mL, unless other-
3.77 (s, 3H, OMe); 13C NMR (DMSO, 100 MHz)
d
183.3 (C]S), 161.9
wise indicated. The tested organisms were Gram negative bacteria
(Escherichia coli, NCTC-10416 & Sarcina lutea ATCC-934), Gram
positive bacteria (Staphylococcus aureus, NCTC-7447 & Bacillus
subtilis, NCTC-1040), and fungi (Candida albicans, IMRU-3669 &
Aspergillus flavus). The bacteria and fungi were maintained on
nutrient agar and Czapek’s Dox agar medium, respectively. All
chemical compounds were dissolved in N, N-dimethylformamide
(DMF) (5 mg/mL), except if noted otherwise. DMF showed no
inhibition activity. The results are tabulated in Tables 4 and 5.
(CeO), 159.1 (CeO), 155.8 (CeO), 154.5 (C]O), 153.9 (C]N), 130.3
(CH), 128.9 (CH), 125.1 (C), 114.4 (C), 114.3 (CH), 105.8 (CH), 99.6
(CH), 56.2 (CH3), 55.8 (CH3), 55.6 (CH3); MS (m/z, %) 373 (8, Mþ þ 2),
372 (32, Mþ þ 1), 371 (100, Mþ), 195 (53, ( CH3O)2.C6H3.NCS-2,4),
165 (100, CH3.S.C6H4.NCO-p).
5.1.21. 1-(2,4-Dimethoxyphenyl)-4-imino-5-thioxo-3-p-
tolylimidazolidin-2-one (8c)
IR (KBr) 3435 (NH), 1784 (C]O), 1668 (C]N), 1211 (CeO), 1163
(CeO), 1123 (C]S) cmꢃ1
;
1H NMR (CDCl3, 400 MHz)
d
9.46 (s, 1H,
5.2.5. Antimicrobial and antifungal susceptibility testing of
NH), 7.45 (d, J ¼ 8.5 Hz, 2H, AreH), 7.31 (d, J ¼ 8.5 Hz, 2H, AreH),
7.22 (d, J ¼ 9.0 Hz, 1H, AreH), 6.63e6.59 (m, 2H, AreH), 3.85 (s, 3H,
OMe), 3.80 (s, 3H, OMe), 2.40 (s, 3H, ArCH3); 13C NMR (CDCl3,
compounds 4d, 5b, and 8b
The Minimum Inhibitory Concentration (MIC) of the four tested
compounds was determined by the macrotube dilution technique
following the guidelines of the National Committee for Clinical
Laboratory Standards for bacteria [70] and for filamentous fungi
[71]. The four Compounds were tested for their MIC against the
Gram negative bacteria (Escherichia coli, NCTC-10416 & Sarcina
lutea ATCC-934), Gram positive bacteria (Staphylococcus aureus,
NCTC-7447 & Bacillus subtilis, NCTC-1040), and fungi (Candida
albicans, IMRU-3669 & Aspergillus flavus). The results are summa-
rized in Table 6. The bacteria and fungi were maintained on
nutrient broth medium and Czapek’s Dox medium, respectively.
Procedure: 15 test tubes were utilized for each microorganism,
where the first one was charged with 2.0 mL of DMF and 2 mg of the
tested compound. To the remaining tubes was added 1.0 mL of
sterile broth medium. Subsequently, 1.0 mL was transfered from the
first tube to the second one, followed by continueous dilutions in
this manner to the 14th tube, discarding 1.0 mL from the latter. The
15th tube served as a control. Several colonies of tested culture were
100 MHz)
d 182.6 (C]S), 162.2 (CeO), 155.7 (CeO), 154.7 (C]O),
153.7 (C]N), 138.5 (C), 129.9 (CH), 129.7 (CH), 129.5 (C), 126.3 (CH),
114.4 (C), 105.0 (CH), 99.9 (CH), 55.9 (CH3), 55.6 (CH3), 21.2 (CH3);
MS (m/z, %) 357 (10, Mþ þ 2), 356 (15, Mþ þ 1), 355 (73, Mþ), 152
(100), 133 (28, CH3eC6H4eNCO-p).
5.2. Biological evaluation
5.2.1. Screening studies of compounds 4a-d-8a-c against EAC cells
Antitumor activities were carried out at the Biotechnology Lab
of the Egyptian Petroleum Research Institute using RPMI 1640
medium (Sigma), Ehrlich ascites carcinoma cells (EAC) suspension
(2.5 ꢀ105/mL), and trypan blue dye. A stock solution was prepared
from 1.0 g of the dye and 100 mL of H2O. Subsequent dilution of
1 mL with 9 mL of H2O was done. The stain was used to stain dead
EAC cells obtained by needle aspiration of ascetic fluid from the

Z. Moussa et al. / European Journal of Medicinal Chemistry 46 (2011) 2280e2289
[34] K. Friedrich, M. Zamkanei, Chem. Ber. 112 (1979) 1916e1918.
2289
suspended to an appropriate turbidity in 5.0 mL of broth medium.
Further dilution by transferring 0.2 mL of the suspension into 40 mL
was done.1.0 mL of the diluted culture suspension was then added
to each of the tubes. Finally, Incubation at 37 ꢁC overnight for
bacteria and 30 ꢁC and 72 h for fungi was performed. The tubes
were examined for visible signs of microbial and fungal growth,
where the highest dilution without growth is the MIC.
[35] A.D. Grabenko, M.N. Danchenko, P.S. Pel’kis, J. Org. Chem. (USSR) 8 (1972)
528e531.
[36] R. Ketcham, E. Schaumann, J. Org. Chem. 45 (1980) 3748e3750.
[37] M.S.A. El-Gaby, Y.A. Ammar, A.M.Sh. El-Sharief, M.A. Zahran, A.A. Khames,
Heteroat. Chem. 13 (2002) 611e616.
[38] Sq.G. Kömürcü, S. Rollas, N. Yilmaz, A. Çevikbas, Drug Metab. Drug Interact.
12 (1995) 161e169.
[39] R. Ketcham, E. Schaumann, T. Niemer, Synthesis 11 (1980) 869e870.
[40] A.M. Sh. El-Sharief, Y.A. Ammar, Y.A. Mohamed, M.S.A. El-Gaby, Phosphorus,
Sulfur Silicon Relat. Elem. 148 (1999) 215e226.
[41] K. Tanaka, K. Matsuo, A. Nakanishi, M. Jo, H. Shiota, M. Yamaguchi, S. Yoshino,
K. Kawaguchi, Chem. Pharm. Bull. 32 (1984) 3291e3298.
[42] A.M.Sh. El-Sharief, Y.A. Ammar, Y.A. Mohamed, M.S.A. El-Gaby, Phosphorus,
Sulfur Silicon Relat. Elem. 148 (1999) 117e130.
[43] A.A. Elagamey, F.M.A. El-Taweel, R.A.N.A. El-Enein, Phosphorus, Sulfur Silicon
Relat. Elem. 181 (2006) 2155e2176.
Acknowledgments
This work was supported by the Deanship of Scientific Research
of Taibah University (project number: 54). Ziad Moussa (principle
investigator) and Ahmed M. Sh. El-Sharief greatly acknowledge this
generous financial support.
[44] A.M.Sh. El-Sharief, F.F. Mahmoud, N.M. Taha, E.M. Ahmed, Phosphorus, Sulfur
Silicon Relat. Elem. 180 (2005) 573e589.
[45] S.Y. Al-Raqa, A.M.Sh. El-Sharief, S.M.E. Khalil, A.M. Al-Amri, Heteroat. Chem.
17 (2006) 634e647.
[46] M.S. Singer. U.S. Patent 3,655,689, 1972.
Appendix. Supplementary material
[47] H. Huang, M.D. Graves, J. Heterocycl. Chem. 24 (1987) 1781e1785.
[48] P. Kinzel, M. Wilhelm. Ger Offen. 3,000,130, 1981.
[49] P. Kinzel. Ger Offen. 3,437,853, 1986.
Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2011.03.009.
[50] Y.A. Ammar, A.M.Sh. El-Sharief, M.M. Aly, Y.A. Mohamed, Sh.I. Mohamed,
Phosphorus, Sulfur Silicon Relat. Elem. 166 (2000) 173e185.
[51] A.M.Sh. El-Sharief, Y.A. Ammar, Y.A. Mohamed, M.M. Aly, M.S.A. El-Gaby,
A.S. Aly, Phosphorus, Sulfur Silicon Relat. Elem. 173 (2001) 39e58.
[52] A.M.Sh. El-Sharief, Y.A. Ammar, M.S.A. El-Gaby, M.A. Zahran, A.A. Khames,
Afinidad 60 (2003) 47e54.
[53] Y. Kobayashi-Matsunaga, T. Ishiia, T. Hamaguchia, H. Osadab, M. Sato, Lett.
Drug Des. Discovery 2 (2005) 224e227.
[54] A.M.Sh. El-Sharief, Y.A. Ammar, Y.A. Mohamed, M.S.A. El-Gaby, Heteroat.
Chem. 13 (2002) 291e298.
References
[1] J.F. Olin. U.S. Patent 3,287,102, 1966.
[2] J.F. Olin, W.A. Darlington, U.S. Patent 3,592,911, 1971.
[3] CIBA Ltd. NL. Patent 6500321, 1965.
[4] M.S.A. El-Gaby, A.M.Sh. El-Sharief, A.A. Atalla, A.-B.A.A.M. El-Adasy, J. Chin.
Chem. Soc. 51 (2004) 327e333.
[5] A.M.Sh. El-Sharief, Y.A. Ammar, M.A. Zahran, H. Kh. Sabet, J. Chem. Res. Synop.
3 (2003) 162e167.
[6] L.M. Deck, E.P. Papadopoulos, K.A. Smith, J. Heterocycl. Chem. 40 (2003)
885e893.
[7] A.M.Sh. El-Sharief, M.S.A. El-Gaby, A.A. Atalla, A.-B.A.A.M. El-Adasy, Heteroat.
Chem. 16 (2005) 218e225.
[8] A.M.Sh. El-Sharief, A.M. Al-Amri, S.Y. Al Raqa, J. Sulfur Chem. 27 (2006) 1e19.
[9] J.D. Friedrich, J. Org. Chem. 52 (1987) 2442e2446.
[10] J. Gonda, P. Kristian, Collect. Czech. Chem. Commun. 53 (1988) 1761e1769.
[11] W. Walter, P.-M. Hell, Liebigs Ann. 727 (1969) 22e34.
[12] W. Walter, K.-D. Bode, Liebigs Ann. 698 (1966) 131e144.
[13] A. Reissert, K. Brüggemann, Chem. Ber. 57 (1924) 981e989.
[14] A. Reissert, Chem. Ber. 37 (1904) 3708e3733.
[55] I. Khattak, R. Ketcham, E. Schaumann, G. Adiwidjaja, J. Org. Chem. 50 (1985)
3431e3434.
[56] A.M.Sh. El-Sharief, A.A. Atalla, A.M. Hussein, M.S.A. El-Gaby, A.A. Hassan,
Phosphorus, Sulfur Silicon Relat. Elem. 160 (2000) 141e158.
[57] A.M.Sh. El-Sharief, A.M. Hussein, M.S.A. El-Gaby, A.A. Atalla, A.A. Ahmed,
Phosphorus, Sulfur Silicon Relat. Elem. 170 (2001) 47e63.
[58] A.M. Mahran, N.A. Hassan, Arch. Pharm. Res. 29 (2006) 46e49.
[59] T. Besson, C.W. Rees, J. Chem. Soc. Perkin Trans. 1 (1996) 2857e2860.
[60] A.M.Sh. El-Sharief, S.Y. Al-Raqa, Phosphorus, Sulfur Silicon Relat. Elem.
182 (2007) 1557e1580.
[61] A.M.Sh. El-Sharief, R. Ketcham, E. Schaumann, Phosphorus, Sulfur Silicon
Relat. Elem. 46 (1989) 83e86.
[62] A.M.Sh. El-Sharief, Y.A. Ammar, M.S.A. El-Gaby, Afinidad 61 (2004) 240e255.
[63] A.M.Sh. El-Sharief, M.S.A. El-Gaby, A.A. Atalla, B.-B.A.A.M. El-Adasy, Afinidad
60 (2003) 475e481.
[64] A.M.Sh. El-Sharief, Z. Moussa, Eur. J. Med. Chem 44 (2009) 4315e4334.
[65] E.P. Papadopoulos, J. Org. Chem. 44 (1979) 3858e3861.
[66] B. Kumelj, M. Tisler, Vestn. Slov. Kem. Drus 5 (1958) 69e73.
[67] A.M.Sh. El-Sharief, A. Al-Amri, S.Y. Al-Raqa, J. Sulfur Chem. 27 (2006) 245e263.
[68] A.D. Grabenko, P.S. Pel’kis, Inst. Org. Chem.. (Kiev, Zh. Obshch. Khim.)
31 (1961) 2739e2743.
[15] T. Besson, C.W. Rees, J. Chem. Soc. Perkin Trans. 1 (1995) 1659e1662.
[16] H. Lee, K. Kim, Bull. Korean Chem. Soc. 13 (1992) 107e108.
[17] S.S. Michaelidou, P.A. Koutentis, Synthesis (2009) 4167e4174.
[18] T. Besson, K. Emayan, C.W. Rees, J. Chem. Soc. Chem. Commun. (1995)
1419e1420.
[19] T. Besson, K. Emayan, C.W. Rees, J. Chem. Soc. Perkin Trans.
1 (1995)
2097e2102.
[20] G. Cottenceau, T. Besson, V. Gautier, C.W. Rees, A.-M. Pons, Bioorg. Med. Chem.
Lett. 6 (1996) 529e532.
[69] A.M.Sh. El-Sharief, R. Ketcham, M. Ries, E. Schaumann, G. Adiwidjaja,
J. Heterocycl. Chem. 47 (2010) 425e429.
[21] T. Besson, C.W. Rees, G. Cottenceau, A.-M. Pons, Bioorg. Med. Chem. Lett.
6 (1996) 2343e2348.
[70] NCCLS, National Committee for Clinical Laboratory Standards, Methods for
Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically
Approved Standard, fourth ed. NCCLS, 1997, M7-A4.
[71] NCCLS, National Committee for Clinical Laboratory Standards, in: Wayne (Ed.),
second ed., Method M27 A2, Vol. 22(15) NCCLS, Pennsylvania, 2002, pp. 1e29;
NCCLS, National Committee for Clinical Laboratory Standards, in: Wayne (Ed.),
second ed., Method M-38A, Vol. 22(16) NCCLS, Pennsylvania, 2002, pp. 1e27.
[72] W.F. Mclimans, E.V. Davis, F.L. Glover, G.W. Rake, J. Immunol. 79 (1957)
428e433.
[73] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica,
J.T. Warren, H. Bokesch, S. Kenny, M.R. Boyd, J. Natl. Cancer Inst. 82 (1990)
1107e1112.
[74] R.E. Cooper, in: F.W. Kavangeh (Ed.), Analytical Microbiology, vol. 1&2,
Academic Press, New York and London, 1972.
[22] T. Besson, J. Guillard, C.W. Rees, V.J. Thiéry, Chem. Soc. Perkin Trans. 1 (1998)
889e892.
[23] V.-D. Le, C.W. Rees, S. Sivadasan, Tetrahedron Lett. 41 (2000) 9407e9411.
[24] T. Besson, J. Guillard, C.W. Rees, J. Chem. Soc. Perkin Trans. 1 (2000) 563e566.
[25] T. Besson, J. Guillard, C.W. Rees, M. Thérisod, J. Chem. Soc. Chem. Commun.
(1997) 881e882.
[26] H.-S. Lee, K. Kim, Tetrahedron Lett. 37 (1996) 869e872.
[27] H.-S. Lee, Y.-G. Chang, K. Kim, J. Heterocycl. Chem. 35 (1998) 659e668.
[28] Y.-G. Chang, K. Kim, Heterocycles 51 (1999) 2653e2666.
[29] P.K. Mohanta, K. Kim, Tetrahedron Lett. 43 (2002) 3993e3996.
[30] M.-F. Pereira, V. Thiéry, T. Besson, Tetrahedron 63 (2007) 847e854.
[31] K. Friedrich, M. Zamkanei, Chem. Ber. 112 (1979) 1873e1878.
[32] K. Friedrich, M. Zamkanei, Tetrahedron Lett. 18 (1977) 2139e2140.
[33] K. Friedrich, M. Zamkanei, Chem. Ber. 112 (1979) 1867e1872.