New imidazolidineiminothione derivatives: Synthesis, spectral characterization and evaluation of antitumor, antiviral, antibacterial and antifungal activities

Article abstract of DOI:10.1016/j.ejmech.2016.06.051

A series of new imidazolidineiminothione derivatives with various halogenated and alkylated aromatic substituents at N-(1) and at N-(3) was synthesized through the reaction of N-arylcyanothioformamides with arylisocyanate derivatives. Structure of imidazo

Full text of DOI:10.1016/j.ejmech.2016.06.051

Accepted Manuscript
New imidazolidineiminothione derivatives: Synthesis, spectral characterization and
evaluation of antitumor, antiviral, antibacterial and antifungal activities
Ziad Moussa, Marwa A.M.Sh El-Sharief, Samir Y. Abbas
PII:
S0223-5234(16)30541-4
10.1016/j.ejmech.2016.06.051
EJMECH 8712
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 March 2016
Revised Date: 28 May 2016
Accepted Date: 28 June 2016
Please cite this article as: Z. Moussa, M.A.M.S. El-Sharief, S.Y. Abbas, New imidazolidineiminothione
derivatives: Synthesis, spectral characterization and evaluation of antitumor, antiviral, antibacterial
and antifungal activities, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.06.051.
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ACCEPTED MANUSCRIPT
New imidazolidineiminothione derivatives: Synthesis, spectral characterization and
evaluation of antitumor, antiviral, antibacterial and antifungal activities
Ziad Moussa, Marwa A. M. Sh. El-Sharief
,
Samir Y. Abbas
Cl
Cl
R
O
O=C=N
S
R
N
N
Cl
Et3N (cat), Et2O
Cl
N
H
CN
S
NH
Cytotoxicity IC50 (µg):-
R= 4-CH3: 0.014 for HEPG2 and 0.009 for MCF7
R = 2,4,5-Cl3: 0.015-0.017 for HEPG2, MCF7, HCT116, PC3
Antiviral effect (%):-
R = 4-Cl: HAV (100), CoxB4 (41.6) and HSV1 (36.4)
R = 2,4,5-Cl3: HAV (80.3), CoxB4 (20.6) and HSV1(96.7)
Antibacterial and antifungal activities (MIC values µg/mL):-
0.78-6.25 for R = 3-OCH3, 4-SCH3 and 2-CH3,5-F
A series of new imidazolidineiminothiones with various substituents were synthesized. In
vitro biological evaluation of such compounds showed significant antitumor, antiviral,
antibacterial and antifungal activities

ACCEPTED MANUSCRIPT
New imidazolidineiminothione derivatives: Synthesis, spectral characterization and
evaluation of antitumor, antiviral, antibacterial and antifungal activities
Ziad Moussaa,*, Marwa A. M. Sh. El-Shariefb,c,* Samir Y. Abbasd,*
,
aDepartment of Chemistry, Faculty of Science, Taibah University, Almadinah
Almunawarrah, Saudi Arabia
bApplied Organic Chemistry Department, National Research Centre, Cairo, Egypt
cFaculty of Science and Arts, Mohail Asser, King Khalid University, Saudi Arabia
dOrganometallic and Organometalloid Chemistry Department, National Research Centre,
Cairo, Egypt
*Correspondence may be addressed to all authors: zmousa@taibahu.edu.sa (Z. Moussa);
melsheref.2005@gmail.com (M. El-Sharief); samiryoussef98@yahoo.com ( Abbas)
S.
Abstract: A series of new imidazolidineiminothione derivatives with various halogenated
and alkylated aromatic substituents at N-(1) and at N-(3) was synthesized through the
reaction of N-arylcyanothioformamides with arylisocyanate derivatives. Structure of
1
imidazolidineiminothione derivatives were established based on spectroscopic IR, H NMR,
1
19
13C NMR, H,1H-COSY, HSQC, F NMR, MS and elemental analyses data. Evaluation of
antitumor, antiviral, antibacterial and antifungal activities for the synthesized compounds
were carried out to probe their activities. Most of the synthesized compounds displayed
antitumor activity. The presence of 3,5-dichlorophenyl moiety at N-(1) and trichlorophenyl
moiety on N-(3) (2f) resulted the highest cytotoxic activity. The presence of 9H-fluorenyl
moiety on N-(3) resulted in the lowest cytotoxic activity. The antiviral screening displayed
that 2d and 2f were markedly active against one or two viral strains. Compound 2d (3,5-
dichlorophenyl moiety at N-(1) and 4-chlorophenyl moiety on N-(3)) showed 100 % antiviral
effect toward HAV. Compound 2f showed 96.7 % antiviral effect toward HSV1 and 80.3 %
antiviral effect toward HAV. The antimicrobial activity suggested that all of the
imidazolidineiminothione derivatives possess significant antimicrobial activity against most
of the test organisms. Some imidazolidineiminothione derivatives showed MIC values of
antibacterial and antifungal activities ranged from 0.78 to 6.25 µg/mL.
Keywords: N-Arylcyanothioformamides; Imidazolidineiminothiones; NMR Spectra;
Antitumor; Antiviral; Antibacterial; Antifungal activities
1. Introduction
Cancer is one of the major health problems in all countries. Numerous advances
chemotherapeutic management have been made for some patients, but the discovering of new
anticancer agents remain critically important. Conventional chemotherapy does not
discriminate between tumour cells and rapidly dividing normal cells. Targeted therapy is
promising approach to cancer therapy which leading to beneficial clinical effects [1-3].
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Imidazole derivatives are key components of great many bioactive compounds of both natural
and synthetic origin [4, 5]. Also, imidazoles are intermediates in the biosynthesis of
nucleotides, and some of their metal complexes are found to be active as cytotoxic
metallopharmaceuticals [6].
Also, viral infection diseases are one of the major health problems. Hepatitis A virus (HAV)
infections remain a major cause of acute hepatitis [7], acute kidney injury [8] and
hemophagocytic syndrome [9]. HAV is usually transmitted by the fecal-oral route [10].
Despite the successful results of hepatitis A vaccination programs, the death of HAV infected
children were increased [10]. Although there are safe and effective vaccines against HAV, it
is also important to discover new and potential drugs for the treatment of HAV. Infection
with Cox B can cause fever, headache, chest pain and other problems. Currently, there is no
specific treatment or vaccine available for Coxsackie virus infections [11]. Herpes simplex
viruses (HSV-1) infection may result several diseases such as genital herpes, labial herpes
and chronic mucocutaneous ulceration [12,13]. HSV infections are usually treated with
nucleoside analogues such as acyclovir [14]. However, the efficacy of these drugs is limited
by the recent increase in the resistance of virus and recurrence of latent virus [15]. Thus, the
development of new anti-HSV agents is highly desirable. As general, the medical community
faces a serious problem against virus infections and needs search for effective antiviral agents
[16].
The search for newer antibacterial and antifungal agents continues due to the rapid
development the resistance among bacteria and fungi to the existing antimicrobial agents [17-
20]. Even though novel broad spectrum antimicrobial agents were reported in recent years,
the emergence of resistance has become an obstacle. Imidazolidineiminothione derivatives
and the various heterocycles derived from them were shown to exhibit an interesting and a
wide range of pharmacological effects [21-27]. In view of these facts, it was of interest to
synthesize a novel series of imidazolidineiminothiones, and preliminary evaluation of the
antitumor, antiviral, antimicrobial and antifungal properties of the synthesized compounds.
2. Results and discussion
2.1. Chemistry
N-Arylcyanothioformamide derivatives 1a-e were prepared by treating arylisothiocyanate
derivatives with potassium cyanide according to literatures procedure [20, 21, 28, 29]. It is
noteworthy to mention that analysis of the N-arylcyanothioformamides by NMR (1H and 13C)
indicated the presence of tautomeric mixtures of thiol and thione forms, in which the latter
predominates. The tautomeric nature of the N-arylcyanothioformamides reflects their
nucleophilic character in which they may react via the sulfur or nitrogen atom leading to
different heterocylic cores in ring closing reactions. Comprehensive study on the tautomeric
nature of the N-arylcyanothioformamide was carried out by El-Sharief et al [21]. Treating an
ethereal solution of various N-arylcyanothioformamides 1a-e with an equimolar amount of
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various isocyanate derivatives, followed by addition of a few drops of triethylamine as
catalyst afforded imidazolidineiminothione derivatives 2-6. 5-Imino-4-thioxo-2-
imidazolidinone derivatives 2-6 were furnished as the sole products, indicating that the ring
closing reaction proceeds via a single path which involves attack via the nitrogen atom. These
products were purified by crystallization and obtained in 75-90 % yields. The structure of
compounds 2-6 was inferred from their correct elemental analyses and spectral data.
Scheme 1: Synthesis of imidazolidineiminothione derivatives
2.1.1. IR spectral analysis
Infrared measurements of 5-imino-4-thioxo-2-imidazolidinone derivatives 2-6 showed
diagnostic absorbance bands in region 3244-3221 cm-1 for the imino NH functional group,
1773-1766 cm-1 for the C=O functional group, and 1667-1658 cm-1 for the C=N functional
group. The thione stretch appeared at its expected frequency from 1126 to 1065 cm-1.
Aliphatic C-H stretching vibration bands are observed in the region of 2832-3063 cm-1.
Aromatic C-H bands are obtained in the region of 3018-3063 cm-1. Moreover, IR spectra
showed disappearance of bands for cyano group. IR spectrum of 2a, as representative
example, exhibited characteristic bands at 3231, 1772, 1665 and 1110 for NH, C=O, C=N
and C=S functional group, respectively.
2.1.2. 1H NMR spectral analysis
1H NMR spectrum of compound 2f as representative example (Fig. 1) exhibited four signals
for aromatic protons in the region of 7.42 - 7.72 ppm. Douplet signal appeared at 7.42 ppm
with two proton integral value is assigned to proton at C-2 and C-6 carbons of 3,5-
dichlorophenyl moiety. The latter two protons resonated as douplet signal not two singlet
signals due to the meta-coupling of each proton at C-2 and C-6 carbons of 3,5-dichlorophenyl
moiety with the proton at C-4 carbon. Triplet signal appeared at 7.51 ppm with one proton
integral value is assigned to proton at C-4 carbon of 3,5-dichlorophenyl moiety. By similar
manner, The latter proton resonated as a triplet signal not singlet signal due to the meta-
coupling of this proton with the two protons at C-2 and C-6 carbons of 3,5-dichlorophenyl
moiety. The remaining two singlet signals at 7.61 and 7.72 ppm are assigned to two protons
at C-3 and C-6 carbons of 2,4,5-trichlorophenyl moiety. Beside the latter four aromatic
1
signals, a broad exchangeable single at 9.56 ppm are assigned for the imine proton. All H
NMR spectra of 5-imino-4-thioxo-2-imidazolidinone derivatives 2-6 exhibited broad
1
exchangeable signal for the imine proton about 9.60 ppm. 1H, H-COSY spectra of 5-imino-
4-thioxo-2-imidazolidinone derivatives supported these assignments.
Fig. 1: 1H NMR spectrum of compound 2f.
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2.1.3. 1H, 1H-COSY spectra spectral analysis
1H,1H-COSY supported the interpretations of structure elucidation of 5-imino-4-thioxo-2-
1
imidazolidinone derivatives 2-6. H,1H-COSY of compound 2f as representative example
(Fig. 2) revealed the following information. The two protons at C-2 and C-6 carbons of 3,5-
dichlorophenyl moiety appeared at 7.42 ppm are meta-coupled with the proton at C-4 carbon.
The proton at C-4 carbon appeared at 7.51 ppm is meta-coupled with the two protons at C-2
1
and C-6 carbons of 3,5-dichlorophenyl moiety. Moreover H,1H-COSY showed that the two
protons at C-3 and C-6 carbons of 2,4,5-trichlorophenyl moiety do not coupled with any other
protons.
Fig. 2: 1H, 1H-COSY spectrum of compound 2f
2.1.4. 13C NMR spectral analysis
13C NMR spectra (on-resonance & DEPT) spectral assignment has been made based on
characteristic signal positions of the functional groups. 13C NMR spectra were most
informative and displayed the expected three signals for the 4-imino-5-thioxoimidazolidine-
2-one core 153-161 ppm for C=N, 153-163 ppm for C=O and 180-182 ppm for C=S. In
general, the aromatic carbons could be readily distinguished from the other carbons due to
their characteristic absorption in the region of 110-150 ppm. 13C NMR spectrum of
compound 3c as representative example is shown in Fig. 3. 13C NMR spectrum of compound
3c showed three signals in the aliphatic region. The signals at 15.7, 55.9 and 56.4 ppm are
due to SCH3 and 2OCH3 carbons, respectively. The signals appeared in the range 113.5-148.9
ppm are due to aromatic carbons. The signals resonated in the deshielded region of 153.4,
153.6, 154.4 and 181.9 ppm are assigned to C=N, C=O, C-O and C=S carbons, respectively.
1H, 13C Heteronuclear Single Quantum Coherence (HSQC) spectra of 5-imino-4-thioxo-2-
imidazolidinone derivatives 2-6 supported these assignments and added a strong evidence for
these interpretations.
Fig. 3: 13C NMR spectrum of compound 3c
2.1.5. 1H, 13C Heteronuclear Single Quantum Coherence (HSQC) spectral analysis
1
From the H-13C HSQC spectrum of 3c, as representative example, (Fig. 4), the following
important assignments can be made: Singlet signal at 2.51 ppm showed cross peak with the
signal at 15.7 ppm. This suggests that the carbon signal is due to SCH3 carbon. Two singlet
signal at 3.78 and 3.79 ppm show cross peak with the signal at 55.9 and 56.4 ppm. This
suggests that the carbon signals are due to two OCH3 carbons. Doublet signal at 6.88 ppm
showed cross peak with the signal at 113.5 ppm. This suggested that this carbon signal is due
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to C-6 carbon of 2,5-dimethoxyphenyl moiety. Multiplet signal at 7.04-7.01 ppm with two
proton integral value showed cross peak with the carbon signals at 114.7 and 116.9 ppm; it
suggested that these carbon signals is due to C-3,4 carbons of 2,5-dimethoxyphenyl moiety.
Two doublet signals at 7.37 and 7.51 ppm showed cross peak with the signals at 126.7 and
126.9 ppm. This confirmed that these carbons signals are due to C-2,6 and C-3,5 of 4-
(methylthio)phenyl moiety. The assignment of NH proton is confirmed as there are no
correlations for NH by HSQC spectral analysis. In the higher frequency region of the 13C
spectrum, apart from the C=S signal at 181.9 ppm there are seven signals without correlations
respectively at 121.6 (C-N), 129.0 (C-N), 139.4 (C-S), 148.9 (C-O), 153.4 (C=N), 153.6
(C=O) and 154.4 (C-O) ppm.
Fig. 4: HSQC spectrum of compound 3c
2.1.6. 19F NMR spectral analysis
19F NMR spectral measurement of 5-imino-4-thioxo-2-imidazolidinone derivative 2g showed
diagnostic signal at -62.9 for fluorine atom in trifluoromethyl moiety (Fig 5).19F NMR
spectral measurements of 5-imino-4-thioxo-2-imidazolidinone derivative 2h and 6 showed
signals at -114.6 and -114.6, respectively. These signals are attributed to fluorine atom at 5-
fluoro-2-methylphenyl moiety.
Fig. 5: 19F NMR spectra of compounds 2g and 2h
2.1.7. Mass spectral analysis
Additional structural information can be deduced from mass spectroscopy. The electron
impact mass spectra of the 4-imino-5-thioxoimidazolidine-2-one derivatives 2-6 confirmed
the proposed formula by showing a peak at: m/z value corresponding to the 4-imino-5-
thioxoimidazolidine-2-one moiety. Also, their mass spectra showed two characterized peaks
attributable to the raw starting materials fragments of the 4-imino-5-thioxoimidazolidine-2-
one isothiocyanate and isocyanate derivatives as shown in the fragmentation pattern in Fig. 6.
Beside the two beaks, there are other beaks attributed to fragments of aryl or phenyl moieties.
Fig 6: Fragmentation pattern of 4-imino-5-thioxoimidazolidine-2-one
2.2. Biological activity
The aim of the present investigation is to synthesize series of imidazolidineiminothiones
which bearing various substituent at N-(1) and others at N-(3). The antitumor, antiviral,
antibacterial and antifungal activities of these derivatives were measured. The effect of each
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substituent at N-(1) and those at N-(3) on these activities was studied and make a comparative
study between them to deduce a structure activity relationship. A series of
imidazolidineiminothione 2a-h which contain 3,5-dichlorophenyl moiety at N-(1) and
various moieties at N-(3) was synthesized. The effect of each substituent on N-(3) of the
imidazolidine ring on the activities of the imidazoslidines 2a-h was studied. Thus, changing
the substituent on N-(3) from 4-tolyl to 4-anizyl to 4-metylthiophenyl (2a → 2b → 2c) was
carried out to show the difference between CH3, OCH3 and SCH3 on the effect of the
biological activities. Changing the substituent on N-(3) from momo- to di- to trichlorophenyl
(2d → 2e → 2f) was carried out for the same reason. Also, changing the substituent on N-(3)
from 3-(CF3)-4-Cl-phenyl to 2-(CH3)-5-F-phenyl (2g → 2h) was carried out for the same
study. Another imidazolidineiminothiones 3a-d which contain 2,5-dimethoxyphenyl moiety
at N-(1) and various moiety at N-(3) was synthesized. Changing the substituent on N-(3)
from p-tolyl to p-anizyl to p-metylthiophenyl to 9H-fluorenyl ( 3a → 3b → 3c → 3d) was
carried out to show the difference between each substituent on the effect of the biological
activities. Changing the substituent on N-(1) from dichlorophenyl to dimethoxyphenyl and let
the substituent on N-(3) not changed was carried out such as 2a → 3a, 2b → 3b and 2c → 3c.
Moreover, substituent on N-(1) was changed from 3,5-dichlorophenyl to 2,4-
dimethoxyphenyl while the substituent at N-(3) is not changed (3-chlorophenyl) this in order
to study the difference between 2d and 4. Finally, the same study was carried out between 2e
and 5, and 2h and 6 where substituent at N-(3) not changed but that at N-(1) was changed
2.2.1. Antitumor properties
The cytotoxic activity was evaluated against human hepatocellular carcinoma cell line
(HEPG2), human breast cancer cell line (MCF7), colon carcinoma cell line (HCT116) and
prostate carcinoma cell line (PC3). Doxorubicin (CAS-23214-92-8) was used as the reference
drug, which considers one of the most effective anticancer agents. Potential cytotoxicity of
the compounds was tested using the method of Skehan et al. [30]. IC50 value was
summarized in Table 1. The obtained IC50 values for these compounds suggest that all of the
evaluated imidazolidineiminothione derivatives possess significant cytotoxic activity against
most of the tested cell lines used in these assays. A moderate difference in cytotoxic activity
is noted between the tested compounds, this indicate that the main effect related to the
presence of the imidazolidineiminothione moiety which contain adjacent imino and thione
functions groups. Certain aspects of the structure activity relationships for these compounds
can be more clearly highlighted.
Regarding the effect of each substituent on N-(3) (4-tolyl (2a), 4-anizyl (2b) and 4-
metylthiophenyl (2c)); the presence of 4-tolyl (2a) resulted in the highest cytotoxic activity
followed by 4-metylthiophenyl. Compound 2a showed half potency of doxorubicin in
inhibiting the growth of HEPG2 and MCF7 cell lines. Regarding the effect of changing the
substituent on N-(3) from momo- to di- to trichlorophenyl (2d → 2e → 2f); the presence of
trichlorophenyl resulted in the highest cytotoxic activity among all of the tested compounds.
Compound 2f showed IC50 about 0.015 µg toward all the tested cell lines.
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Imidazolidineiminothiones 3a-d which contain 2,5-dimethoxyphenyl moiety at N-(1) and
different moieties on N-(3), like structure 2a-c, the presence of 4-tolyl (3a) resulted in the
highest cytotoxic activity. Increasing the size of the substituent, as in compound 3d had a
detrimental effect on cytotoxic activity. The presence of 9H-fluorenyl moiety resulted in the
lowest cytotoxic activity among all of the tested compounds. Changing the substitution on N-
(1) from 3,5-dichlorophenyl to 3,5-dimethoxyphenyl on N-(1) while the substituent at N-(3)
is not changed (2d to 4) had a good effect on cytotoxic activity.
Table 1
2.2.2. Antiviral activities.
The synthesized compounds 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 [31]. 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 antiviral. The results of the plaque
reduction assay are summarized in Table 2. The percentage of antiviral effect obtained for the
synthesized compounds suggested that most of the imidazolidineiminothione derivatives
evaluated possess significant antiviral activity against most of the test virus used in these
assays. Certain aspects of the structure activity relationships for these compounds can be
more clearly highlighted.
Regarding the effect of changing the substituent on N-(3) from p-tolyl to p-anizyl to p-
metylthiophenyl (2a → 2b → 2c); the presence of p-tolyl (2a) resulted in the highest antiviral
activity. Regarding the effect of changing the substituent on N-(3) from momo- to di- to
trichlorophenyl (2d → 2e → 2f); the presence of momochlorophenyl and trichlorophenyl
moieties resulted in the highest antiviral activity among all of the tested compounds.
Compound 2d showed 100 % antiviral effect toward HAV. Compound 2f showed 80.3 %
antiviral effect toward HAV and 96.7 % antiviral effect toward HSV1.
Imidazolidineiminothiones 3a-d which contain 2,5-dimethoxyphenyl moiety at N-(1) and
different moieties on N-(3). The presence of 4-metylthiophenyl (3c) resulted in the highest
antiviral activity followed by 4-anizyl moiety. Compound 3c showed 85.7 % antiviral effect
toward HSV1. Increasing the size of the substituent, as in compound 3d had a detrimental
effect on antiviral activity. Changing substitution on N-(1) from 3,5-dichlorophenyl to 3,5-
dimethoxyphenyl on N-(1) while the substituent at N-(3) is not changed (2d → 4) had a
detrimental effect on antiviral activity. Also, Changing the substitution on N-(1) from 3,5-
dichlorophenyl to phenyl on N-(1) while the substituent at N-(3) is not changed (2e → 5) had
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detrimental effect on antiviral activity. Finally, Changing the substitution on N-(1) from 3,5-
dichlorophenyl to bromophenyl on N-(1) while the substituent at N-(3) is not changed (2h
and 6) had slight effect on antiviral activity.
Table 2
2.2.3. Antibacterial activities
The synthesized compounds were tested in vitro for antibacterial activity against the
following bacterial strains: three Gram-positive bacteria, Micrococcus luteus (ATCC10240),
B. subtilis (NCTC-10400) and B. Pumilus and three Gram-negative bacteria, Pseudomonas
aeruginosa (ATCC25619), E. coli (ATCC10536) and S. lutea, and the results are summarized
in Table 3. Antimicrobial tests were carried out by the agar well diffusion method [32] using
100 ꢀL of tested compound solution prepared by dissolving 5 mg of the chemical compound
in 1 ml of dimethyl sulfoxide (DMSO). The inoculated plates were then incubated for 24 h at
37 °C. The antibacterial agent Erythromycin (5 mg/ml) was used as a standard. After
incubation time, antimicrobial activity was evaluated by measuring the inhibition zone
diameters against the test organisms and compared with standard zone size ranges that
determine susceptibility, intermediate susceptibility, or resistance to the screened compounds.
Visual bacterial growth is observed only in areas in which the drug concentrations are below
those required for growth inhibition. The experiment was carried out in triplicate and the
average zone of inhibition was calculated. The mean values of the inhibition zone diameter
obtained for these compounds suggest that all of the imidazolidineiminothione derivatives
evaluated possess significant antibacterial activity against most of the test organisms used in
these assays. A moderate difference in antibacterial activity is noted between the tested
compounds, this indicate that the main effect related to the presence of the
imidazolidineiminothione moiety which contain adjacent imino and thione functions groups.
The results of the antibacterial screening demonstrated the following assumptions about the
structural activity relationship (SAR).
Regarding the effect of changing the substituent on N-(3) from p-tolyl to p-anizyl to p-
metylthiophenyl (2a → 2b → 2c); the presence of p-anizyl (2b) resulted in the highest
antibacterial activity followed by p-metylthiophenyl. The presence of p-tolyl moiety resulted
in the lowest antibacterial activity. Regarding the effect of changing the substituent on N-(3)
from momo- to di- to trichlorophenyl (2d → 2e → 2f); the presence of momochlorophenyl
resulted in the highest antibacterial activity followed by trichlorophenyl. The lowest
antibacterial activity was exhibited by dichlorophenyl moiety. Compounds 2g,h had the
same activity. Imidazolidineiminothiones 3a-d which contain 2,5-dimethoxyphenyl group at
N-(1) and different substituent on N-(3). Also, like structure 2a-c, the presence of p-anizyl
(3b) resulted in the highest antibacterial activity followed by p-metylthiophenyl, the presence
of p-anizyl and p-metylthiophenyl moieties resulted in the highest antimicrobial activity
among all the compounds investigated in this study. Increasing the size of the substituent, as
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in compound 3d had a detrimental effect on antibacterial activity. The presence of to 9H-
fluorenyl moiety resulted in the lowest antibacterial activity among all of the tested
compounds. Changing the substitution on N-(1) from 3,5-dichlorophenyl to 3,5-
dimethoxyphenyl while the substituent at N-(3) is not changed (2d → 4) had a detrimental
effect on antibacterial activity. Also, changing the substitution on N-(1) from 3,5-
dichlorophenyl to phenyl while substituent at N-(3) is not changed (2e → 5) had no effect on
antibacterial activity. Finally, changing the substitution on N-(1) from 3,5-dichlorophenyl to
bromophenyl while the substituent at N-(3) is not changed (2h → 6) had slight effect on
antibacterial activity.
Table 3
2.2.4. Antifungal activity
The synthesized compounds were tested in vitro for antifungal activity against the following
three fungal strains: Candida albicans (IMRU3669), A. niger and P. chrysogenum in the
antifungal susceptibility tests. Antifungal agents are evaluated against clinical isolates of
standard strains of fungi by the agar well diffusion method [32] using 100 ꢀL of tested
compound solution prepared by dissolving 5 mg of the chemical compound in 1 ml of
dimethyl sulfoxide. The antifungal agent Metronidazole was used as a standard at
concentration 5 mg/mL and was tested under the same conditions. The inoculated plates were
then incubated. After incubation time, antifungal activity was evaluated by measuring the
inhibition zone against the test organisms and compared with that of the standard. Antifungal
activities were expressed as inhibition zone diameter in millimeters (mm). The experiment
was carried out in triplicate and the average zone of inhibition was calculated. The results are
tabulated in Table 3. In general, some compounds were very effective antifungal agents and
in several cases achieved similar level of activity as the standard antifungal agent
Metronidazole. A moderate difference in antifungal activity is noted between the tested
compounds, this indicate that the main effect related to the presence of the
imidazolidineiminothione moiety which contain adjacent imino and thione functions groups.
Regarding the effect of changing the substituents on N-(1) and N-(3), the results of screening
demonstrated the presence of good correlation between the antifungal and antibacterial
activities.
2.2.5. Minimum inhibitory concentrations against Gram positive bacteria, Gram
negative bacteria and fungi
Minimum inhibitory concentrations of the more active synthesized compounds 2b, 2c, 2h, 3b
and 3c was then evaluated in vitro using the twofold serial dilution technique [33] against
various strains of Gram positive and Gram negative bacteria and fungi. These techniques
provide a quantitative assessment of in vitro antimicrobial activity. Hence, further to the
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preceding tests of the potential antibacreial and antifungal activities. Minimum inhibitory
concentration (MIC) The lowest concentration showing no growth was taken as the minimum
inhibitory concentration (MIC). The results of minimum inhibitory concentration were
reported in Table 4.
Table 4
As shown in Table 4, most of the tested compounds revealed MIC 0.78 µg/ml against
Pseudomonas aeroginosa. Most of the tested compounds showed MIC 6.25 µg/ml against
most of the tested organisms.
3. Conclusion
The synthesis, characterization and biological activity of
a
new series of
imidazolidineiminothiones with variable aromatic substituents at N-(1) and N-(3) atoms have
been described. Most compounds displayed antitumor, antiviral, antibacterial and antifungal
activities. A moderate difference in cytotoxic activity is noted between the tested compounds,
this indicate that the main effect related to the presence of the imidazolidineiminothione
moiety which contain adjacent imino and thione functions groups. Regarding the effect of
changes the substituents on N-(3); the presence of trichlorophenyl resulted in the highest
cytotoxic activity followed among all of the tested compounds. The presence of to 9H-
fluorenyl moiety resulted in the lowest cytotoxic activity among all of the tested compounds.
The antiviral effect of all compounds were investigated, where 2a,d,f were markedly active
against one or two viral strains. Structure activity relationship studies revealed several
matching pairs of aromatic substituents on N-(1) and N-(3) which could serve to optimize
structural features for optimal activity to eventually render such compounds clinically useful
drug agents. The antimicrobial activity obtained for these compounds suggest that all of the
imidazolidineiminothione derivatives evaluated possess significant antimicrobial activity
against most of the test organisms used in these assays. A moderate difference in
antimicrobial activity is noted between the tested compounds, this indicate that the main
effect related to the presence of the imidazolidineiminothione moiety which contain adjacent
imino and thione functions groups.
4. Experimental
IR spectra were recorded (KBr) on a Perkin Elmer 1650 spectrophotometer. 1H NMR and 13C
NMR spectra were recorded on Avance II Bruker FT NMR spectrometer 400 (400 MHz)
using CDCl3 as solvents and TMS as an internal standard. CFCl3 was used as an internal
standard for all 19F NMR measurements. Chemical shifts are expressed as δ ppm units. NMR
spectra were recorded at Department of Chemistry, Faculty of Science, Taibah University,
Almadinah lmunawarrah, Saudi Arabia Mass spectra were recorded on Shimadzu GC-MS QP
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100 EX (70 eV) at the Micro Analytical Center at Cairo University. Melting points were
obtained on a Fisher–Johns melting points apparatus and are uncorrected.
4.1. General procedure for the synthesis of imidazolidineiminothione derivatives 2a-j,
3a-d, 4, 5 and 6
A solution of the cyanothioformanilide derivative (0.01 mol) and the corresponding
isocyanate derivative (0.01 mol) in dry ether (30 mL) was treated with three drops of
triethylamine. The reaction mixture was stirred for 15 min. The obtained product was filtered
off, washed with ether, air-dried and recrystallized from chloroform/ n-hexane to give
imidazolidineiminothione derivatives 2a-j, 3a-d, 4, 5 and 6.
.
4.1.1. 1-(3,5-Dichlorophenyl)-4-imino-5-thioxo-3-p-tolylimidazolidin-2-one (2a)
Yield 73 %; mp 146-147 °C; IR: ν/cm-1: 3231 (NH), 1772 (C=O), 1665 (C=N), 1110 (C=S);
1H NMR: δ/ppm = 2.41 (s, 3H, CH3), 7.33 (d, 2H, J = 8.3 Hz, Ar-H), 7.40 (d, 2H, J = 8.3
Hz, Ar-H), 7.43 (d, 2H, J = 1.8 Hz, Ar-H), 7.49 (t, 1H, J = 1.8 Hz, Ar-H), 9.57 (br, 1H, NH);
13C NMR: 21.3 (CH3); 126.0 (2CH), 126.3 (2CH), 128.9 (C-N), 129.9 (CH), 130.1 (2CH),
134.2 (C-N), 135.7 (2C-Cl), 139.1 (C-CH3), 153.1 (C=N), 154.0 (C=O), 180.8 (C=S); MS
(m/z, %): 363 (M+, 40), 364 (M+1, 16), 365 (M+2, 29), 366 (M+3, 10), 362 (M-1, 16), 203
(Cl2C6H3NCS, 51), 133 (CH3C6H4NCO, 100); Anal. Calcd for C16H11Cl2N3OS (364.25): C,
52.76; H, 3.04; N, 11.54; Found: C, 52.84; H, 3.08; N, 11.37
4.1.2. 1-(3,5-Dichlorophenyl)-4-imino-3-(4-methoxyphenyl)-5-thioxoimidazolidin-2-one
(2b)
Yield 71 %; mp 140-141°C; IR: ν/cm-1: 3240 (NH), 1767 (C=O), 1664 (C=N), 1125 (C=S);
1H NMR: δ/ppm = 3.85 (s, 3H, OCH3), 7.04 (d, 2H, J = 8.9 Hz, Ar-H), 7.41-7.45 (m, 4H, Ar-
H), 7.50 (t, 1H, J = 1.9 Hz, Ar-H), 9.55 (br, 1H, NH); 13C NMR: 55.6 (OCH3), 114.8 (CH),
124.1 (2C), 126.0 (CH), 127.9 (2C), 129.9 (2C), 134.2 (C-N), 135.7 (2C-Cl), 153.2 (C=N),
154.1 (C=O), 159.7 (C-O), 180.8 (C=S); MS, m/z (%): 379 (M+, 10.4), 380 (M+1, 4.1), 381
(M+2, 9.1), 378 (M-1, 5.8), 203 (Cl2C6H3NCS, 27.0), 149 (OCH3C6H4NCO, 100); Anal.
Calcd for C16H11Cl2N3O2S (380.25): C, 50.54; H, 2.92; N, 11.05; Found: C, 50.49; H, 2.88;
N, 11.13
4.1.3. 1-(3,5-Dichlorophenyl)-4-imino-3-(4-(methylthio)phenyl)-5-thioxoimidazolidin-2-
one (2c)
Yield 71 %; mp 191-192 °C; IR: ν/cm-1: 3241 (NH), 1772 (C=O), 1666 (C=N), 1118 (C=S);
1H NMR: δ/ppm = 2.54 (s, 3H, SCH3), 7.34 (d, 2H, J = 8.9 Hz, Ar-H), 7.41 - 7.44 (m, 4H,
Ar-H), 7.63 (t, 1H, J = 1.9 Hz, Ar-H), 11.55 (br, 1H, NH); 13C NMR: 14.8 (CH3), 123.5 (C-
N), 126.1 (CH), 127.1 (CH), 127.9 (CH), 132.2 (CH), 132.8 (C-N), 137.5 (C-Cl), 147.2 (C-
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S), 151.0 (C=N), 152.2 (C=O), 176.4 (C=S); MS, m/z (%): 395 (M+, 8.5), 396 (M+1, 12.2),
203 (Cl2C6H3NCS, 47.6), 165 (SCH3C6H4NCO, 100); Anal. Calcd for C16H11Cl2N3OS2
(396.31): C, 48.49; H, 2.80; N, 10.60; Found: C, 48.42; H, 2.76; N, 10.73
4.1.4.
(2d)
1-(3-Chlorophenyl)-3-(3,5-dichlorophenyl)-5-imino-4-thioxoimidazolidin-2-one
Yield 74 %; mp 162-163 °C; IR: ν/cm-1: 3219 (NH), 1778 (C=O), 1663 (C=N), 1128 (C=S);
1H NMR: δ/ppm = 7.39-7.49 (m, 5H, Ar-H), 7.51 (s, 1H, Ar-H), 7.60 (t, 1H, J = 1.9 Hz, Ar-
13
H), 9.66 (br, 1H, NH); C NMR: 124.5 (CH), 125.9 (CH), 126.6 (CH), 130.0 (2CH), 130.1
(CH), 130.3 (CH), 132.7 (C-N), 134.0 (C-N), 134.9 (C-Cl), 135.8 (2C-Cl), 152.8 (C=N),
153.3 (C=O), 180.2 (C=S); MS, m/z (%): 385 (M+, 33.8), 386 (M+1, 11.7), 387 (M+2, 11.5),
384 (M-1, 23.9), 203 (Cl2C6H3NCS, 100), 153 (ClC6H4NCO, 43.6); Anal. Calcd for
C15H8Cl3N3OS (384.67): C, 46.84; H, 2.10; N, 10.92; Found: C, 46.72; H, 2.04; N, 10.82
4.1.5. 3-(2,6-Dichlorophenyl)-1-(3,5-dichlorophenyl)-4-imino-5-thioxoimidazolidin-2-one
(2e)
Yield 74 %; mp 220-222 °C; IR: ν/cm-1: 3232 (NH), 1769 (C=O), 1658 (C=N), 1118 (C=S);
1H NMR: δ/ppm = 7.44-7.45 (m, 3H, Ar-H), 7.50 (d, 2H, J = 1.9 Hz, Ar-H), 7.53 (t, 1H, J =
1.9 Hz, Ar-H), 9.54 (br, 1H, NH); 13C NMR: 126.0 (2CH), 127.6 (C-N), 129.0 (2CH), 130.1
(CH), 131.9 (CH), 134.0 (C-N), 135.4 (2C-Cl), 135.7 (2C-Cl), 151.6 (C=N), 151.9 (C=O),
180.4 (C=S); MS, m/z (%): 419 (M+, 29.9), 420 (M+1, 15.9), 418 (M-1, 8.2), 203
(Cl2C6H3NCS, 100), 187 (Cl2C6H3NCO, 30.0); Anal. Calcd for C15H7Cl4N3OS (419.11): C,
42.99; H, 1.68; N, 10.03; Found: C, 42.84; H, 1.73; N, 10.10
4.1.6. 1-(3,5-Dichlorophenyl)-4-imino-5-thioxo-3-(2,4,5-trichlorophenyl)imidazolidin-2-
one (2f)
Yield 75 %; mp 173-175 °C; IR: ν/cm-1: 3217 (NH), 1777 (C=O), 1661 (C=N), 1126 (C=S);
1H NMR: δ/ppm = 7.42 (d, 2H, J = 1.6 Hz, Ar-H), 7.51 (t, 1H, J = 1.6 Hz, Ar-H), 7.61 (s,
13
1H, Ar-H), 7.72 (s, 1H, Ar-H), 9.56 (br, 1H, NH); C NMR: 125.9 (2CH), 128.5 (C-N),
130.2 (CH), 131.7 (CH), 131.8 (CH), 132.0 (C-N), 132.4 (C-Cl), 133.8 (C-Cl), 135.6 (C-Cl),
135.8 (2C-Cl), 151.9 (C=N), 152.6 (C=O), 180.1 (C=S); MS, m/z (%): 453 (M+, 5.7), 454
(M+1, 3.1), 452 (M-1, 2.1), 203 (Cl2C6H3NCS, 70), 195 (Cl3C6H2NCO, 100); Anal. Calcd for
C15H6Cl5N3OS (453.56): C, 39.72; H, 1.33; N, 9.26; Found: C, 39.64; H, 1.37; N, 9.28
4.1.7.
1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(3,5-dichlorophenyl)-5-imino-4-
thioxoimidazolidin-2-one (2g)
Yield 69 %; mp 152-153 °C; IR: ν/cm-1: 3255 (NH), 1773 (C=O), 1663 (C=N), 1126 (C=S);
1H NMR: δ/ppm = 7.42 (d, 2H, J = 1.7 Hz, Ar-H), 7.52 (t, 1H, J = 1.7 Hz, Ar-H), 7.68 (d,
1H, J = 8.7 Hz, Ar-H), 7.78 (dd, 1H, J = 8.7, 2.4 Hz, Ar-H), 8.01 (d, 1H, J = 2.4 Hz, Ar-H),
9.72 (br, 1H, NH); 13C NMR: 122.2 (q, J = 273.4 Hz, CF3), 125.5 (q, J = 5.9 Hz, CH), 125.9
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(2CH), 126.3 (C-N), 129.5 (q, J = 32.2 Hz, C-CF3), 130.2 (CH), 130.3 (CH), 130.5 (C-N),
132.4 (CH), 133.8 (C-Cl), 135.9 (2C-Cl), 152.6 (C=N), 152.9 (C=O), 180.0 (C=S); 19F NMR:
-62.98; MS, m/z (%): 453 (M+, 34.6), 454 (M+1, 9.2), 452 (M-1, 14.6), 203 (Cl2C6H3NCS,
100), 221 (Cl(CF3)C6H3NCO, 12.2); Anal. Calcd for C16H7Cl3F3N3OS (452.67): C, 42.45; H,
1.56; N, 9.28; Found: C, 42.41; H, 1.52; N, 9.34
4.1.8. 1-(3,5-Dichlorophenyl)-3-(5-fluoro-2-methylphenyl)-4-imino-5-thioxoimidazolidin-
2-one (2h)
Yield 69 %; mp 125-126 °C; IR: ν/cm-1: 3445 (NH), 1766 (C=O), 1661 (C=N), 1120 (C=S);
1H NMR: δ/ppm = 2.25 (s, 3H, CH3), 7.08 (dd, 1H, J = 8.5, 2.6 Hz, Ar-H), 7.14 (td, 1H, J =
8.3, 2.6 Hz, Ar-H), 7.38-7.33 (m, 1H, Ar-H), 7.44 (d, 2H, J = 1.8 Hz, Ar-H), 7.51 (t, 1H, J =
1.8 Hz, Ar-H), 9.53 (br, 1H, NH); 13C NMR: 17.4 (CH3), 115.8 (d, J = 23.4 Hz, CH), 117.3
(d, J = 20.5 Hz, CH), 125.9 (2CH), 130.0 (CH), 131.0 (d, J = 9.6 Hz, C-N), 132.2 (d, J = 3.6
Hz, C), 132.5 (d, J = 8.1 Hz, C-H), 134.0 (C-N), 135.8 (2C-Cl), 153.3 (C=O), 152.6 (C=N),
19
161.2 (d, J = 246 Hz, C-F), 181.6 (C=S); F NMR: -114.6; MS, m/z (%): 381 (M+, 76.1),
382 (M+1, 25.2), 383 (M+2, 52.8), 203 (Cl2C6H3NCS, 20), 150 (F(CH3)C6H3NCO, 100);
Anal. Calcd for C16H10Cl2FN3OS (382.24): C, 50.28; H, 2.64; N, 10.99; Found: C, 50.32; H,
2.61; N, 10.86
4.1.9. 1-(2,5-Dimethoxyphenyl)-4-imino-5-thioxo-3-p-tolylimidazolidin-2-one (3a)
Yield 64 %; mp 135-136 °C; Yield 77 %; mp 131-132 °C; IR: ν/cm-1: 3439 (NH), 1769
1
(C=O), 1665(C=N), 1117 (C=S) cm-1; H NMR: δ/ppm = 2.40 (s, 3H, CH3), 3.77 (s, 3H,
OCH3), 3.78 (s, 3H, OCH3), 6.88 (dd, 1H, J = 2.4, 0.8 Hz, Ar-H), 7.03-7.01 (m, 2H, Ar-H),
7.31 (d, 2H, J = 8.4 Hz, Ar-H), 7.45 (d, 2H, J = 8.4 Hz, Ar-H), 9.47 (br, 1H, NH); 13C NMR:
21.2 (CH3), 55.9 (OCH3), 56.4 (OCH3), 113.5 (CH), 114.7 (CH), 116.8 (CH), 121.7 (C-N),
126.3 (2CH), 129.4 (C-N), 129.9 (2CH), 138.6 (C), 148.9 (C-O), 153.5 (C=N), 153.6 (C=O),
154.6 (C-O), 182.1 (C=S); Anal. Calcd for C18H17N3O3S (355.41): C, 60.83; H, 4.82; N,
11.82; Found: C, 60.87; H, 4.79; N, 11.90
4.1.10. 1-(2,5-Dimethoxyphenyl)-4-imino-3-(4-methoxyphenyl)-5-thioxoimidazolidin-2-
one (3b)
Yield 67 %; mp 205-206 °C; IR: ν/cm-1: 3439 (NH), 1769 (C=O), 1665(C=N), 1117 (C=S)
cm-1; 1H NMR: δ/ppm = 3.79 (s, 3H, OCH3), 3.80 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 6.90-
6.87 (m, 1H, Ar-H), 7.05-7.00 (m, 4H, Ar-H), 7.48 (d, 2H, J = 9.3 Hz, Ar-H), 9.44 (br, 1H,
13
NH); C NMR: 55.4 (OCH3), 55.9 (OCH3), 56.4 (OCH3), 113.4 (CH), 114.6 (2CH), 114.7
(CH), 116.8 (CH), 121.7 (C-N), 124.6 (C-N), 127.9 (2CH), 148.9 (C-O), 153.6 (C=O and
C=N), 154.7 (C-O), 159.4 (C-O), 182.1 (C=S); Anal. Calcd for C18H17N3O4S (371.41): C,
58.21; H, 4.61; N, 11.31; Found: C, 58.17; H, 4.58; N, 11.23
4.1.11. 1-(2,5-Dimethoxyphenyl)-4-imino-3-(4-(methylthio)phenyl)-5-thioxoimidazolidin-
2-one (3c)
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Yield 60 %; mp 170-171 °C; IR: ν/cm-1: 3439 (NH), 1769 (C=O), 1665 (C=N), 1117 (C=S)
cm-1; 1H NMR: δ/ppm = 2.51 (s, 3H, SCH3), 3.78 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 6.88 (d,
1H, J = 2.5 Hz, Ar-H), 7.04-7.01 (m, 2H, Ar-H), 7.37 (d, 2H, J = 8.8 Hz, Ar-H), 7.51 (d,
2H, J = 8.8 Hz, Ar-H), 9.50 (br, 1H, NH); 13C NMR: 15.7 (SCH3), 55.9 (OCH3), 56.4
(OCH3), 113.5 (CH), 114.7 (CH), 116.9 (CH), 121.6 (C-N), 126.7 (2CH), 126.9 (2CH), 129.0
(C-N), 139.4 (C-S), 148.9 (C-O), 153.4 (C=N), 153.6 (C=O), 154.4 (C-O), 181.9 (C=S);
Anal. Calcd for C18H17N3O3S2 (387.48): C, 55.80; H, 4.42; N, 10.84; Found: C, 55.93; H,
4.37; N, 10.74
4.1.12. 1-(2,5-Dimethoxyphenyl)-3-(9H-fluoren-2-yl)-4-imino-5-thioxoimidazolidin-2-one
(3d)
Yield 66 %; mp 205-206 °C; IR: ν/cm-1: 3422 (NH), 1779 (C=O), 1666 (C=N), 1132 (C=S);
1H NMR: δ/ppm = 3.77 (s, 3H, OCH3), 3.78 (s, 3H, OCH3), 6.88 - 7.45 (m, 12H, Ar-H), 9.47
(br, 1H, NH); Anal. Calcd for C24H19N3O3S (429.49): C, 67.12; H, 4.46; N, 9.78; Found: C,
67.34; H, 4.49; N, 9.41
4.1.13. 1-(3-Chlorophenyl)-3-(2,4-dimethoxyphenyl)-5-imino-4-thioxoimidazolidin-2-one
(4)
Yield 62 %; mp 162-163 °C; IR: ν/cm-1: 3439 (NH), 1769 (C=O), 1665 (C=N), 1117 (C=S)
1
cm-1; H NMR: δ/ppm = 3.79 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 6.63-7.58 (m, 2H, Ar-H),
7.20 (d, 1H, J = 9.3 Hz, Ar-H), 7.39-7.35 (m, 1H, Ar-H), 7.43 (t, 1H, J = 7.9 Hz, Ar-H),
7.55-7.51 (m, 1H, Ar-H), 7.67-7.65 (m, 1H, Ar-H), 9.55 (br, 1H, NH); 13C NMR: 55.6
(OCH3), 55.9 (OCH3), 99.8 (CH), 105.0 (CH), 114.1 (C-N), 124.5 (CH), 126.6 (CH), 128.5
(CH), 129.7 (CH), 130.1 (CH), 133.3 (C-N), 134.7 (C-Cl), 153.4 (C=N), 154.0 (C=O), 155.7
(C-O), 162.3 (C-O), 182.1 (C=S); Anal. Calcd for C17H14ClN3O3S (375.83): C, 54.33; H,
3.75; N, 11.18; Found: C, 54.27; H, 3.64; N, 11.23
4.1.14. 1-(2,6-Dichlorophenyl)-5-imino-3-phenyl-4-thioxoimidazolidin-2-one (5)
Yield 65 %; mp 125-126 °C; IR: ν/cm-1: 3439 (NH), 1769 (C=O), 1665 (C=N), 1117 (C=S)
1
cm-1; H NMR: δ/ppm = 7.45-7.40 (m, 1H, Ar-H), 7.61-7.47 (m, 7H, Ar-H), 9.49 (br, 1H,
13
NH); C NMR: 127.2 (2CH), 127.9 (C-N), 129.0 (2CH), 129.5 (2CH), 129.8 (CH), 131.7
(CH), 132.6 (C-N), 135.5 (2C-Cl), 152.3 (C=O and C=N), 181.3 (C=S); MS, m/z (%): 349.9
(M+, 8.9), 350.9 (M+1; 13.4), 351.9 (M+2; 5.4), 348.9 (M-1; 18.9), 135 (C6H5NCS, 100), 77
(C6H5; 81.0); Anal. Calcd for C15H9Cl2N3OS (350.22): C, 51.44; H, 2.59; N, 12.00; Found:
C, 51.53; H, 2.62; N, 12.11
4.1.15. 1-(4-Bromophenyl)-3-(5-fluoro-2-methylphenyl)-4-imino-5-thioxoimidazolidin-2-
one (6)
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Yield 68 %; mp 122-123 °C; IR: ν/cm-1: 3439 (NH), 1769 (C=O), 1665 (C=N), 1117 (C=S)
1
cm-1; H NMR: δ/ppm = 2.26 (s, 3H, CH3); 7.17-7.07 (m, 2H, Ar-H), 7.36-7.33 (m, 1H, Ar-
13
H), 7.39 (d, 2H, J = 8.8 Hz, Ar-H), 7.70 (d, 2H, J = 8.8 Hz, Ar-H), 9.51 (br, 1H, NH); C
NMR: 17.5 (CH3), 115.8 (d, J = 23.5 Hz, CH), 117.3 (d, J = 20.4 Hz, CH), 123.8 (C-Br),
128.6 (2CH), 131.2 (d, J = 9.7 Hz, C-N), 131.5 (C-N), 132.2 (d, J = 3.7 Hz, C), 132.5 (d, J
= 8.7 Hz, CH), 132.8 (2CH), 153.0 (C=N), 153.5 (C=O), 161.1 (d, J = 247 Hz, C-F), 181.0
19
(C=S); F NMR: -114.7; Anal. Calcd for C16H11BrFN3OS (392.25): C, 48.99; H, 2.83; N,
10.71; Found: C, 48.84; H, 2.86; N, 10.61
4.2. Cytotoxic activity studies of synthesized compounds against various tumor cell lines
Cytotoxicity of synthesized compounds was tested using the method of Skehan et al. [28].
Cells were plated in a 96 multiwell plate (104 cells/well) for 24 hrs before treatment with the
compounds to allow attachment to the wall of the plate. Different concentrations of the
compounds (0, 5, 12.5, 25, and 50 ꢀg/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 hrs at 37 °C in an atmosphere of 5% CO2. After 48 hrs, 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. The median
inhibition concentration (IC50) which is the required concentration to reduce the survival to
50% is then determined from the survival curve.
4.3. Virucidal activity studies
The synthesized compounds were tested against HAV, HSV1 and CoxB4 viral strains where
viral activity was assayed by the plaque formation method. Initially, the cytotoxicity of
synthesized compounds on Vero cells was determined. The method used was that described
by Vanden Berghe et al [31]. Viral infectivity was assayed by titration of viruses by the
plaque formation method [34].
4.4. Antimicrobial activity
Chemical compounds were individually tested against a panel of gram positive and gram
negative bacterial pathogens and fungi. Antimicrobial tests were carried out by the agar well
diffusion method [32] using 100 ꢀL of suspension containing 1 x108 CFU/mL of pathological
tested bacteria and 1 x106 CFU/ml of yeast spread on nutrient agar (NA) and Sabouraud
dextrose agar (SDA) media, respectively. After the media had cooled and solidified, wells (10
mm in diameter) were made in the solidified agar and loaded with 100 ꢀL of tested
compound solution prepared by dissolving 5 mg of the chemical compound in one ml of
dimethyl sulfoxide (DMSO). The inoculated plates were then incubated for 24 h at 37 °C.
Negative controls were prepared using DMSO employed for dissolving the tested compound.
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Erythromycin and Metronidazole (5 mg/ml) were used as standard for antibacterial and
antifungal activity, respectively. After incubation time, antimicrobial activity was evaluated
by measuring the zone of inhibition against the test organisms and compared with that of the
standard. The observed zone of inhibition is presented in Table 3. Antimicrobial activities
were expressed as inhibition diameter zones in millimeters (mm). The experiment was carried
out in triplicate and the average zone of inhibition was calculated.
4.5. Minimal inhibitory concentration (MIC) measurement
Screening was performed following the procedure outlined in the Manual of Clinical
Microbiology [33]. All the bacteria were incubated and activated at 30 °C for 24 h
inoculation into nutrient broth and the fungi were incubated in malt extract broth for 48 h.
The compounds were dissolved in DMSO and then diluted using cautiously adjusted Mueller-
Hinton broth. Two-fold serial concentrations of the compounds were employed to determine
the (MIC). The final concentrations of the solutions were 100, 50, 25, 12.5, 6.25, 3.12, 1.56
and 0.78 ꢀg/ml. In each case triplicate tests were performed and the average was taken as the
final reading. The tubes were then inoculated with the test organisms, grown in their suitable
broth at 37 °C for 24 hours for tested microorganisms (1×108 CFU/ml for bacteria and 1 x106
CFU/ml of fungi), each 5 ml received 0.1 ml of the above inoculum and incubated at 37 °C
for 24 hours. The lowest concentration showing no growth was taken as the minimum
inhibitory concentration (MIC).
References
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2739-2743
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Figure captions:
Scheme 1: Synthesis of imidazolidineiminothione derivatives
Figure 1: 1H NMR spectrum of compound 2f
Figure 2: 1H, 1H-COSY spectrum of compound 2f
Figure 3: 13C NMR spectrum of compound 3c
Figure 4: HSQC spectrum of compound 3c
Figure 5: 19F NMR spectra of compounds 2g and 2h
Figure 6: Fragmentation pattern of 4-imino-5-thioxoimidazolidine-2-one
Table 1: IC50 values of synthesized compounds obtained against tumour cell lines
Table 2: The percentage of anti-infectivity effect of the synthesized against viral strains.
Table 3: Antimicrobial activity of the synthesized compounds against the pathological
organisms expressed as inhibition diameter zones in millimeters (mm) based on well
diffusion assay
Table 4: Minimum inhibitory concentration MIC (µg/mL) of the most potent synthesized
compounds against the pathological organisms
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Table 1: IC50 values of synthesized compounds obtained against tumour cell lines
IC50 µg of Cytotoxicity
Cell lines
MCF7
0.009
Compd. No.
HEPG2
0.014
HCT116
0.053
PC3
0.035
0.056
0.049
0.031
0.064
0.017
0.039
0.064
0.048
0.051
0.047
0.056
0.031
0.008
2a
0.037
0.029
0.025
0.039
0.017
0.028
0.050
0.037
0.032
0.029
0.048
0.012
0.007
0.027
0.018
0.011
0.020
0.015
0.024
0.011
0.015
0.031
0.028
0.052
0.020
0.005
0.056
0.031
0.041
0.039
0.015
0.044
0.034
0.014
0.051
0.050
0.047
0.023
0.007
2b
2c
2d
2e
2f
2g
2h
3a
3b
3c
3d
4
Doxorubicin
Table 2: The percentage of anti-infectivity effect of the synthesized against viral strains.
Anti viral effect (%)
Viral strain
Compd. No.
HAV
66.0
0
CoxB4
HSV1
71.6
13.3
3.4
43.6
0
2a
2b
2c
2d
2e
2f
2g
2h
3a
3b
3c
3d
4
0
0
100
70.2
80.3
0
41.6
0
36.4
33.6
96.7
9.6
20.6
0
20.6
30.7
60.1
0
31.1
0
45.3
50.2
42.0
85.7
32.4
47.4
33.3
43.1
0
18.5
0
29.9
36.2
0
2.3
0
5
36.2
0
6
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Table 3: Antimicrobial activity of the synthesized compounds against the pathological
organisms expressed as inhibition diameter zones in millimeters (mm) based on well
diffusion assay
Mean values of inhibition zone diameter in millimeters (mm)
Gram +ve
Gram -ve
Fungi
Compd. No.
M l
17
B s
B p
P a
16
E c
16
25
23
30
19
27
18
18
16
28
29
18
20
18
20
37
----
S l
28
C a A n
P c
19
16
25
24
30
19
27
20
20
16
28
29
12
20
18
22
40
----
20
21
18
21
19
16
15
15
12
22
16
19
2a
2b
2c
2d
2e
2f
2g
2h
3a
3b
3c
3d
4
20
18
16
16
16
21
20
17
17
17
16
14
14
22
26
25
15
17
21
21
21
20
23
20
17
17
18
21
32
----
20
21
0
30
26
24
27
21
23
24
26
27
31
24
28
26
23
44
27
25
24
23
21
22
18
17
29
27
14
27
23
22
----
23
20
21
18
20
20
21
17
20
17
18
21
22
14
26
25
0
20.0 22
14.0 18
18.0 20
17.0 16
20.0 21
16
15
21
30
----
5
6
Erythromycin 32
Metronidazole ----
----
33
----
25
---- 27
where, m l = m. luteus, b s = b. subtilis , b p = b. pumilus, p a = p aeruginosa, e c = e. coli, s
l = s. lutea, c a = c. albicans, a n = a. niger and p c = p. chrysogenum
Table 4: Minimum inhibitory concentration MIC (µg/mL) of the most potent synthesized
compounds against the pathological organisms
Compd. No.
P. aeruginosa S. lutea
B. pumilus M. luteus
C. albicans
1.56
0.78
0.78
3.12
0.78
0.78
3.12
6.25
3.12
6.25
6.25
3.12
6.25
6.25
3.12
6.25
6.25
6.25
6.25
6.25
6.25
2b
2c
2h
3b
3c
6.25
6.25
6.25
6.25
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Scheme 1: Synthesis of imidazolidineiminothione derivatives
O
X
NCS
3
N
1
N
S
Ar
Ar-N=C=O
i, KCN
ii,HCl
X
X
N
CN
Et3N (cat), Et2O
H
S
NH
1a-e
2-6
2a
3a
X = 2,5-(OCH3)2 Ar = C6H4CH3-4
3b X = 2,5-(OCH3)2 Ar = C6H4OCH3-4
3c X = 2,5-(OCH3)2 Ar = C6H4SCH3-4
1a X = 3,5-Cl2
1b
X = 3,5-Cl2 Ar = C6H4-CH3-4
2b X = 3,5-Cl2 Ar = C6H4OCH3-3
2c
2d
2e X = 3,5-Cl2 Ar = C6H3Cl2-2,6
2f
X = 2,5-(OCH3)2
1c X = 2,4-(OCH3)2
X = 3,5-Cl2 Ar = C6H4SCH3-4
X = 3,5-Cl2 Ar = C6H4Cl-3
3d
1d
1e
X = 2,5-(OCH3)2 Ar = 9H-fluoren-2-yl
X = H
X = 4-Br
4
X = 2,4-(OCH3)2 Ar = C6H4Cl-3
X = 3,5-Cl2 Ar = C6H2Cl3-2,4,5
X = 3,5-Cl2 Ar = C6H3Cl-4, CF3-3
5 X = H
Ar = C6H3Cl2-2,6
2g
6
X = 4-Br
Ar = C6H3CH3-2,F-5
2h X = 3,5-Cl2 Ar = C6H3CH3-2, F-5
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Fig. 1: 1H NMR spectrum of compound 2f.
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Fig. 2: 1H, 1H-COSY spectrum of compound 2f
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Fig. 3: 13C NMR spectrum of compound 3c
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Fig. 4: HSQC spectrum of compound 3c
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Fig. 5: 19F NMR spectra of compounds 2g and 2h
Fig. 6: Fragmentation pattern of 4-imino-5-thioxoimidazolidine-2-one
b
a
O
-a
-b
R
Ar
N C S
Ar-N=C=O
-NCO
N
N
R
S
NH
-R
-NCS
Ar
NCS
R
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•
•
•
•
•
Synthesis of N-arylcyanothioformamides derivatives
Using the cyanothioformamides for synthesizing imidazolidineiminothiones.
Antitumor activity was determined.
Antiviral activity was determined.
Antibacterial and antifungal activities were determined.