New imidazolone derivatives comprising a benzoate or sulfonamide moiety as anti-inflammatory and antibacterial inhibitors: Design, synthesis, selective COX-2, DHFR and molecular-modeling study

Article abstract of DOI:10.1016/j.bioorg.2019.103438

New imidazol-5-one derivatives 12a,b and 12e, f, 14a,b and 16a,b were synthesized and screened for their in vivo anti-inflammatory activity using a standard acute carrageenan-induced rat paw oedema method. All the tested compounds exhibited good anti-infl

Full text of DOI:10.1016/j.bioorg.2019.103438

Journal Pre-proofs
New imidazolone derivatives comprising a benzoate or sulfonamide moiety as
anti-inflammatory and antibacterial inhibitors: Design, synthesis, selective
COX-2, DHFR and molecular-modeling study
Nadia Hanafy Metwally, Mona Said Mohamed
PII:
S0045-2068(19)31009-0
DOI:
https://doi.org/10.1016/j.bioorg.2019.103438
YBIOO 103438
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
24 June 2019
7 November 2019
12 November 2019
Please cite this article as: N. Hanafy Metwally, M. Said Mohamed, New imidazolone derivatives comprising a
benzoate or sulfonamide moiety as anti-inflammatory and antibacterial inhibitors: Design, synthesis, selective
COX-2, DHFR and molecular-modeling study, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/
j.bioorg.2019.103438
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New imidazolone derivatives comprising a benzoate or sulfonamide
moiety as anti-inflammatory and antibacterial inhibitors: Design,
synthesis, selective COX-2, DHFR and molecular-modeling study
Nadia Hanafy Metwally1* and Mona Said Mohamed1
1Chemistry Department, Faculty of Science, Cairo University.
Email: nhmmohamed@yahoo.com
Abstract
New imidazol-5-one derivatives 12a,b and 12e, f, 14a,b and 16a,b were synthesized
and screened for their in vivo anti-inflammatory activity using a standard acute
carrageenan-induced rat paw oedema method. All the tested compounds exhibited good
anti-inflammatory activity; especially compound 12f which produced the maximum
effect of 49.0 % compared to the standard drug, celecoxib, (43.1%). The most active
anti-inflammatory agents 12a, 12e, and 12f were studied for their interactions with
enzyme COX-2 compared to celecoxib. The study showed that, compound 12e
exhibited a high selectivity towards COX-2 inhibition with IC50 = 0.087 μM. Moreover,
the antibacterial screening indicated that some synthesized compounds showed good
antibacterial activity toward the Gram-negative bacteria Escherichia coli. Additionally,
compounds 7, 12a, 12f, and 12 showed a good binding affinity with enzyme
dihydrofolate reductase (DHFR) whereas compound 12f has a higher inhibitory effect
on DHFR than the tested compounds 7, 12a and 12h. On the other hand, the
combination between these tested compounds and sulfadiazine as a reference drug (10
μM compound + 1 μM reference), showed that compound 12h has higher potency
(0.078±0.002) than sulfadiazine (0.135±0.004). In addition, docking analysis was
performed and it confirmed the presented results.
Keywords: Imidazol-5-ones; Synthesis; COX-2 inhibitors; Anti-inflammatory; DHFR
inhibitors, Antibacterial, DHFR.
1

1. Introduction
Inflammation is a process which occurs when the tissues are injured by foreign
organisms such as bacteria, viruses, trauma, toxins, heat, or any other cause[1,2]. It is
an essential immune response by the host that allows for the removal of harmful stimuli
as well as the healing of damaged tissue. Inflammation can be acute and chronic.
However, acute inflammation is short-duration and its response in the body is not
dangerous. It represents the early physical reaction followed by healing. Chronic
inflammation, on the other hand, is longer in duration than acute inflammation and is
caused by a variety of diseases especially cancer, neurodegenerative disorders and
cardiovascular diseases [3,4]. The function of inflammation is that the damaged cells
release chemicals including histamine, bradykinin, leukotrienes, prostaglandins (PGs),
serotonin and others. These chemicals cause the blood vessels to leak fluid into the
tissues causing swelling. This helps to isolate the foreign substance and prevent further
contact with body tissues [5,6].
Steroid drugs prevent the synthesis of leukotrienes and PGs through the
phospholipase A2 pathway. Also they are highly effective for chronic inflammatory
conditions [7-10]. On the other hand, non-steroidal anti-inflammatory drugs (NSAIDs)
can block specific prostaglandins (PGs) synthesis through the cyclooxygenase enzymes
(COX-1 and COX-2) and lipoxygenase (5-LOX) inhibition [11,12]. NSAIDs are
frequently used for arthritis, rheumatism and to alleviate pain in daily life but they have
side effects such as gastric ulcer and renal impairment [13,14]. Selective COX-2
inhibitors were marketed as safer agents with no gastric ulcer risks compared to
NSAIDs [15-17].
Celecoxib was the first selective COX-2 inhibitor (coxisb) to appear as a safer
replacement for NSAIDs (non-selective COX-1/COX-2 inhibitors) but it has some side
effects. So some celecoxib analogs were synthesized with higher therapeutic value and
lower side effects through increasing specificity toward COX-2 rather than COX-1 [18-
20].
Thus, it is important to find new anti-inflammatory agents to inhibit a selective
COX-2 with a higher potential for clinical use and lesser adverse effects like Celecoxib.
It's worth mentioning that antibacterial as well as anti-infammatory agents have a
2

significant function in pharmacological and medicinal chemistry. Both
chemotherapeutic and anti-inflammatory agents are required to treat bacterial infections
with inflammatory disorders.
Additionally, the antibacterial activity is one of the most important activities for
inspecting novel antibacterial agents that control the most resistant pathogenic types of
bacteria due to the over-use of antibiotics [21,22]. A remarkable example of Gram-
negative bacteria is pseudomonas aeruginosa (P. aeruginasa) which is found
abundantly in nature, for instance: plants, water, and soil. Pseudomonades are fairly
common pathogens involved in infections acquired in a hospital setting by patients who
are already hospitalized with another illness or condition, or people who have a weak
immune system. Infections can occur in any part of the body and the symptoms depend
on which part of the body is infected [23-27]. On the other hand, Staphylococcus aureus
(S. aureus) is an example of Gram-positive bacteria which is a normal inhabitant of the
respiratory tract and on the skin. S. aureus at skin infection causes sinusitis and food
poisoning besides producing virulence factors such as potent toxins [28]. Moreover,
Streptococcus mutans (S. mutans) is another type of Gram-positive in cocci bacteria.
These arbitrary anaerobes are usually found in the human oral cavity and are a major
contributor to tooth decay. S. mutans are mesophiles that grow at temperatures between
o
18-40 C. In addition, they are cariogenic microorganisms that break down sugar to
produce an acid that demineralizes teeth. The result of this conversion is that the coating
of the tooth breaks down then the calcium molecule dissolves generating a hole [29].
It was reported that several imidazolone derivatives have a wide range of
biological activities like analgesic, anti-inflammatory [30-34] and antibacterial
activities [35-38] among others [39-43]. Based on the above-mentioned information,
and extension to synthesize bioactive molecules of interest biological activity [44-50]
motivated us to find compounds having dual antimicrobial and anti-inflammatory
activities. Thus, in the present study, we synthesize some new substituted imidazol-5-
one analogs with comparable antibacterial and anti-inflammatory potencies. Also, we
investigated them regarding their in vivo anti-inflammatory activity against acute
carrageenan-induced paw oedema in wistar albino rats and determined their ability to
inhibit selective cyclooxygenase (COX-2) to develop new anti-inflammatory drugs
with greater activity and lesser side effects. Additionally, we evaluated them against
3

some types of pathogen bacteria and determine dihydrofolate reductase (DHFR) at
different concentrations to provide a biochemical mechanism of action.
2. RESULTS AND DISCUSSION
2.1. Chemistry
Cyclocondensation of benzoylglycine (3) with 4-formyl arylbenzoates 2a, b
[which is prepared by alkylating 4-hydroxybenzaldehyde derivatives 1a, b with
benzoyl chloride and triethylamine in dichloromethane] in acetic anhydride containing
anhydrous sodium acetate in water bath at 100 oC afforded products 4a, b (Scheme 1).
The IR spectrum of 4a showed absorption bands at 1738 and 1647 cm-1 corresponding
to ester and carbonyl groups, respectively. Compound 4a is characterized by the
presence of a singlet signal at δ = 8.18 ppm assigned to vinylic proton in the 1H NMR
spectrum. Also, the mass spectrum of 4a exhibited a correct molecular ion peak at m/z
= 369 (M+) corresponding to the molecular formula C23H15NO4.
O
Cl
H
O
O
O
X
TEA / CH2Cl2
O
X
OH
2
a
X
H
1a,b
2a,b
b
OMe
O
H
O
N
O
OH
O
Ac2O
O
O
N
2a,b
+
AcONa
100 oC, 6h.
X
3
4a,b
4
a
X
H
b
OMe
Scheme 1. Synthetic routes of 4-[(5-oxo-2-aryloxazol-4(5H) ylidene) methyl]-phenyl
benzoate 4a,b.
Moreover, cyclocondensation of compound
3
with 2-formylphenyl
benzenesulfonate (6) [which is prepared by alkylating 2-hydroxybezaldehyde with
4

benzenesulfonyl chloride and triethylamine in tetrahydrofuran (THF)] in acetic
anhydride concerning anhydrous sodium acetate afforded the new product 7 (Scheme
2). The 1H NMR spectrum of 7 showed a singlet signal at δ = 8.76 ppm due to vinylic
proton, besides the signals corresponding to aryl protons in the expected region. Also
the IR spectrum implied the presence of carbonyl group at 1653 cm-1.
O
O
O
S
SO2Cl
TEA / THF
OH
O
O
6
5
O
O
H
O
Ac2O
O
O
N
OH
O
S
AcONa
100 oC, 6h.
N
O
6
+
3
7
Scheme 2. Synthesis of 2-[(5-oxo-2-phenyloxazol-4(5H)-ylidene) methyl] phenyl
benzenesulfonate 7.
Additionally, compound 3 was condensed with arylazosalicyladehydes 8a-d
under the reaction conditions to produce compounds 10a-d (Scheme 3). The structures
of the prepared compounds were screened by spectral data and elemental
microanalyses. The IR spectrum of 10d taken as a representative example of the
prepared series showed characteristic peaks at 3330, 1721 and 1670 cm-1 corresponding
to NH and two carbonyl groups, respectively. The 1H NMR spectrum of 10d revealed
two singlet signals at δ = 8.78 and 9.76 ppm attributed to chromene and NH protons,
along with signals corresponding to aryl protons. Elemental analysis and spectral data
are in agreement with the suggested structure 10d.
5

O
H
O
O
N
OH
OH
+
N
N
3
8a-d
Ar
Ac2O
AcONa
100 oC, 6h.
O
O
O
O
O
N
OH
N
Ar
N
H
N
N
N
Ar
10a-d
9
8, 10
Ar
C6H5
4-MeC6H4
4-OMeC6H4
4-FC6H4
a
b
c
d
Scheme 3. Synthetic route of N-(2-oxo-6-(aryldiazenyl)-2H-chromen-3-yl)-
benzamides 10a-d.
Heating of compounds 4a, b with different aromatic amines 11a-d in glacial
o
acetic acid and anhydrous sodium acetate in a water bath at 100 C produced
compounds 12a-h (Scheme 4). The chemical structure of the products was confirmed
on the bases of their analytical and spectroscopic data. The IR spectrum of 12e taken as
a typical example of the prepared series showed absorption bands at 3332, 1725
6

O
O
glacial AcOH
4a,b + R
N
R
NH2
O
N
AcONa
100 oC, 24h.
X
11a-d
12a-h
11 R
12 X
R
12 X
R
a
b
c
d
SO2NH2
a H SO2NH2
b H COOH
c H COMe
d H OH
e OMe SO2NH2
f OMe COOH
g OMe COMe
h OMe OH
COOH
COMe
OH
Scheme 4. Synthesis of 5-oxo-2-phenylimidazol-4-ylidenes 12a-h.
and 1639 cm-1 revealed to amino and two carbonyl groups, respectively. The 1H NMR
spectrum displayed a signal at δ = 3.86 ppm assigned to methoxy protons and a D2O
exchangeable signal at δ = 7.37 ppm attributed to NH2 protons. Moreover, a singlet
signal at δ = 8.32 ppm due to vinylic proton. Its 13C NMR spectrum showed
characteristic signals at δ = 55.8, 116.1, 163.7 and 169.1 ppm owing to methoxy, vinylic
and carbonyl carbons, respectively. Also, the mass spectrum of 12e presented a correct
molecular ion peak at m/z = 553 (M+) which is consistent with the molecular formula
C30H23N3O6S.
Similarly, compounds 4a,b were heated with each of 5-aminopyrimidine-
2,4(1H,3H)-dione (13) and thiazol-2-amine (15) under the same reaction conditions and
this produced compounds 14a, b and 16a, b, respectively (Scheme 5). The structure of
the products was confirmed via their elemental analysis and spectral data (see exp.).
7

NH2
O
HN
NH
O
O
O
O
NH
NH
13
N
O
O
N
glacial AcOH
AcONa
X
100 oC, 24h.
14a,b
14
a
b
X
H
OMe
4a,b
S
NH2
N
O
O
S
N
15
N
O
N
glacial AcOH
AcONa
X
100 oC, 24h.
16a,b
16
a
X
H
b
OMe
Scheme 5. Synthesis of compounds 14 and 16.
On the other hand, the reaction of compounds 4a, b with each of benzene-1,4-
diamine (17) and benzidine (19) in glacial acetic acid containing anhydrous sodium
o
acetate in a water bath at 100 C produced bis-compounds 18a, b, and 20a, b
respectively, (Scheme 6). The structure of the new products was deduced from their
elemental analyses and spectral data. For example, the IR spectrum of 20b showed
characteristic absorption bands at 1737 and 1640 cm-1 corresponding to four carbonyl
groups. Furthermore, two singlet signals at δ = 3.87 and 8.12 ppm due to the two
1
methoxy and vinylic protons appeared in its H NMR spectrum besides the expected
signals of aryl protons. The elemental analyses together with the spectral data are
provided in the proposed structure 20.
8

X
O
O
X
N
N
O
N
N
O
O
O
18a,b
18
a
b
X
H
OMe
NH2
glacial AcOH
AcONa
100 oC, 24h
H2N
17
4a,b
glacial AcOH
AcONa
H2N
NH2
100 oC, 24h
19
O
O
N
O
O
O
N
N
O
X
N
X
20a,b
20
a
X
H
b
OMe
Scheme 6. Synthesis of bis-imidazol-5-ones 18 and 20.
9

2.2. Screening of in vivo anti-inflammatory activity: Carrageenan-induced paw
edema in rats
The new imidazol-5-one derivatives 12a,b, 12e, 12f, 14a, 14b, 16a and 16b
were evaluated in vivo anti-inflammatory activity using acute carrageenan-induced rat
paw edema and celecoxib (as a reference drug) according to the reported procedure [51]
using a dose of compounds (50 mg/Kg b.w.). The anti-inflammatory activity was
measured based on a change in paw thickness (mm) at 0, 1, 2, 3, 4 and 5 hours after
injection of carrageen as shown in Table 1.
At the beginning of the experiment at 0 and 0.5h, all the tested compounds were
rather weak and ineffective, but after the first hour the effect of the tested compounds
increased throughout all the time intervals. Thus, compounds 12e and 12f showed more
potent anti-inflammation efficiency among the rest of the compounds where compound
12e proved to be very close in inhibition (41.8%) to the reference drug celecoxib after
the five hours. On the other hand, compound 12f showed remarkable anti-inflammatory
activity (49.0%) compared to the standard celecoxib (43.1%). Compounds 12b and 14a
exhibited weak anti-inflammatory activity (18.9 and 10.3%) whereas the rest of the
tested compounds namely 12a, 14b, 16a and 16b exhibited moderate anti-inflammatory
activity (34.0, 27.3, 34.6 and 22.4%) respectively, compared to celecoxib (43.1%) at
the end of experiment time (Table 2 and Fig. 1).
10

Table 1: The anti-inflammatory activity of tested compounds 12a, 12b, 12e, 12f, 14a, 14b, 16a, 16b and celecoxib (50 mg/Kg b.w)
Compound no. Mean value of paw edema thickness (mm) ± SEM
Zero time
0.5 h
1 h
2 h
3 h
4 h
5 h
Control –Ve
Celecoxib
12a
3.89 ± 0.10**
4.79 ± 0.11
6.19 ± 0.09***
7.80 ± 0.10***
9.48 ± 0.10***
8.95 ± 0.10***
8.79 ± 0.07***
4.35 ± 0.17
4.05 ± 0.12
4.33 ± 0.12
4.18 ± 0.09
4.09 ± 0.11
4.20 ± 0.13
4.46 ± 0.14
4.45 ± 0.25
3.95 ± 0.08
4.74 ± 0.17
4.76 ± 0.12
4.95 ± 0.13
4.38 ± 0.09
4.57 ± 0.11
4.98 ± 0.13
4.86 ± 0.15
4.72 ± 0.24
4.84 ± 0.09
5.63 ± 0.18
5.63 ± 0.11
5.73 ± 0.13
4.89 ± 0.09***
5.29 ± 0.10
6.01 ± 0.14
5.47 ± 0.15
5.25 ± 0.23
6.06 ± 0.08**
6.51 ± 0.21
6.86 ± 0.12
5.82 ± 0.15
6.35 ± 0.12**
7.96 ± 0.16***
6.67 ± 0.08***
5.44 ± 0.10
5.31 ± 0.18
5.00 ± 0.16
5.80 ± 0.10***
7.13 ± 0.11***
5.12 ± 0.06
6.04 ± 0.12***
7.43 ± 0.12***
5.87 ± 0.09***
4.79 ± 0.14**
8.20 ± 0.12***
6.69 ± 0.13***
6.23 ± 0.25***
6.94 ± 0.08***
12b
6.74 ± 0.14
12e
5.71 ± 0.10***
6.28 ± 0.12
12f
4.48 ± 0.1**
14a
7.27 ± 0.14***
6.34 ± 0.014
5.94 ± 0.24**
7.54 ± 0.10***
8.67 ± 0.13***
7.46 ± 0.16***
6.83 ± 0.23***
7.14 ± 0.08***
7.88 ± 0.13***
6.39 ± 0.13***
5.75 ± 0.26***
6.82 ± 0.10***
14b
16a
16b
Data analyzed using ANOVA and expressed as Mean ± SEM of four animals for each group
**Significant difference with celecoxib at P< 0.05
*** Highly significant difference with celecoxib at P< 0. 01
11

Table 2: Percentage of inhibition of oedema % in rat using all tested compounds at time
interval 1,3 and 5h
Compound no.
Percentage of inhibition of oedema %
3h
1h
9.1
5h
Celecoxib
38.6
33.0
16.0
29.6
42.6
8.5
43.1
34.0
18.9
41.8
49.0
10.3
27.3
34.6
22.4
12a
12b
12e
12f
14a
14b
16a
16b
9.1
7.4
21.0
14.5
2.9
11.6
15.2
2.1
21.3
28.0
24.7
The anti-inflammatory activity (inhibition %) is expressed according to the equation %
inhibition = (Mc-Mt/Mc) x 100, where Mt represents the mean increase in paw thickness in rats
treated with the tested compound and Mc represents the mean increase in paw thickness in
control group.
Fig. 1. The percentage of oedema inhibition of the tested compounds and standard
drug celecoxib
12

2.3. Structure activity relationship (SAR)
The results presented in Table 1 show that the anti-inflammatory activity of
compound 12e is very close to the reference drug (celecoxib) due to the similarity in
their structures which contain sulfonamide (SO2NH2) moiety, but compound 12f
showed a higher activity than celecoxib and this may be due to the presence of
carboxylic moiety (COOH), which is a more ionizable group, instead of sulfonamaide
(SO2NH2) moiety (Fig. 2).
O
OCH3
H2N
S
N
N
O
O
O
CH3
O
O
S
H2N
O
(12e)
N
N
F
F
OCH3
F
N
O
N
HOOC
(Celecoxib)
O
O
(12f)
Fig. 2. Structure activity relationship (SAR) of compounds 12e and 12f with standard
drug celecoxib
2.4. Enzyme inhibition of cyclo-oxygenase-2 (COX-2)
Additionally, compounds 12a, 12e and 12f were tested for inhibiting COX-2
enzyme and inhibiting activity was determined by measuring IC50. It was observed that
compounds 12a, 12e and 12f inhibit COX-2 enzyme more than the reference drug
celecoxib. Compound 12e has more inhibiting activity compared to the rest of the tested
compounds and this may be due to the similarity in structure with celecoxib (Table 3).
13

Table 3: Inhibition of some tested compounds 12a, 12e, 12f and celecoxib on cyclo-
oxygenase enzyme (COX-2)
Compound no.
COX-2 IC50 (μM)
12a
12e
0.09
0.087
0.092
0.11
12f
Celecoxib
2.5. Antibacterial evaluation
Some selected newly synthesized compounds 4a, b, 7, 12a, b, 12e, f, 12h, 14a,
14b, and 16a were screened in vitro for their antibacterial activity against
Staphylococcus aureus and Streptococcus mutans (Gram-positive bacteria),
Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumonia (Gram-negative
bacteria) using nutrient agar medium. Ampicillin and gentamicin were used as standard
drugs for Gram-positive and Gram-negative, respectively. The antimicrobial activity of
the synthesized compounds was determined using agar well diffusion method [52], and
the diameters (mm) of inhibition zones are shown in Table 4.
The results depicted in Table 4 show that the toxicity of most of the tested
compounds against Staphylococcus aureus and Streptococcus mutans is higher than
Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumonia. Compound 12a
is the most toxic compound against Staphylococcus aureus and Streptococcus mutans
with activity index (184 and 94%) respectively, compared to standard drug ampicillin.
It's also the only tested compound to exhibit toxicity against Pseudomonas aeruginosa
which showed moderate activity with activity index (48%) compared to standard drug
gentamicin. Compounds 7, 12f and 12h showed good toxicity against Staphylococcus
aureus with activity index (80, 84 and 80%) respectively, whereas compounds 12b and
16a showed moderate toxicity against Staphylococcus aureus. Also, compounds 12e,
12f, 12h, 14a, 14b, and 16a showed moderate activity against Streptococcus mutans.
Table 4: Antibacterial activity of some tested compounds 4a, 4b, 7, 12a, 12b, 12e, 12f,
12h, 14a, 14b and 16a against Gram-positive and negative bacteria.
14

Diameter inhibition zone in mm (% activity index)
Gram-negative bacteria Gram-positive bacteria
Pseudomonas Staphylococcus Streptococcus
aeruginosa aureus mutans
(ATCC:6538) (ATCC:25175)
Escherichia
coli
Klebsiella
pneumonia
Compound
no.
(ATCC:9637) (ATCC:10031) (ATCC:27853)
4a
4b
7
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
17.6 ± 0.6
(80%)
12a
12b
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
14.6 ± 0.5
(48%)
NA
40.6 ± 0.6
(184%)
11.3 ± 0.5
(51%)
28.3 ± 0.5
(94%)
NA
12e
NA
NA
NA
NA
NA
NA
NA
15.3 ± 0.5
(51%)
16.6 ± 0.6
(55%)
14.3 ± 0.5
(47%)
21.6 ± 0.6
(72%)
17.6 ± 0.6
(58%)
13.6 ± 0.5
(45%)
12f
18.6 ± 0.6
(84%)
17.6 ± 0.6
(80%)
12h
14a
NA
14b
NA
16a
10.6 ± 0.5
(48%)
-
Gentamicin
Ampicilin
27±0.5
(100%)
-
25±0.5
(100%)
-
30±0.5
(100%)
-
-
22±0.1
(100%)
30±0.5
(100%)
-
-
-
-
Zone of inhibition is expressed in the form of mean± standard deviation (mm).
NA: No activity
Well diameter (6mm).
100 µ l was tested.
2.6. Minimum inhibitory concentration (MIC)
Minimum inhibitory concentration (MIC) of compounds 7, 12a, 12e, 12f, 12h,
14a and 14b with high antibacterial activity showed that compound 12a has both a
higher toxicity (40.6±0.6, 184%) and a minimum inhibitory concentration (MIC = 62.5
μg/ml) among all the tested compounds against Staphylococcus aureus compared to
the standard drug ampicillin.
Table 5: Minimum inhibitory concentration of some tested compounds 7, 12a, 12e, 12f,
12h, 14a and 14b
15

Minimum inhibitory concentration
(MIC) in μg/ml
Compound
no.
Gram positive bacteria
Staphylococcus
aureus
Streptococcus
mutans
(ATCC:6538)
Stock
(ATCC:25175)
-
7
12a
62.5
250
stock
250
-
12e
-
250
250
-
12f
12h
14a
250
250
62.5
-
14b
Ampicilin
62.5
Stock concentration 1 mg/ml
Concentrations unit of MIC are represented as μg/ml
2.7. Enzyme inhibition of dihydrofolate reductase (DHFR)
Compounds 7, 12a, 12f and 12h which have good MIC values were introduced
to enzyme assay against dihydrofolate reductase (DHFR) enzyme to determine
inhibitory activity expressed as IC50 using sulfadiazine as a reference drug. The results
showed that compound 12f has an inhibitory effect on DHFR higher than the other
tested compounds 7, 12a and 12h. Combining some synthesized compounds with
sulfadiazine (10 μM compound + 1 μM reference) indicated that all tested compounds
have higher inhibitory effect toward DHFR. Compound 12f showed the highest
inhibiting activity compared to the standard drug and the other tested compounds. This
may be due to presence of carboxylic group. Also, compounds 12a and 12h have an
inhibiting effect higher than the standard drug sulfadiazine.
Table 6: Inhibition of some tested compounds 7, 12a, 12f, 12h and sulfadiazine on
dihydrofolate reductase (DHFR)
16

Compound no. DHFR IC50 (μM) Combination (10 μM compound+ 1 μM
reference)
7
0.593 ± 0.013
0.306 ± 0.003
12a
12f
0.792 ± 0.019
0.285 ± 0.006
0.347 ± 0.009
0.135 ± 0.004
0.133 ± 0.003
0.078 ± 0.002
0.124 ± 0.003
-
12h
Sulfadiazine
2.8. In silico studies
2.8.1. Molecular docking study
Molecular docking study was performed to identify the most preferred binding
mode of compound into the enzyme binding active site. Celecoxib is known as an anti-
inflammatory standard drug by inhibiting COX-2 enzyme, so the synthesized new
target compounds 12e and 12f were docked with COX-2 enzyme (pdb code: 4cox)
compared to celecoxib utilizing molecular operating environment (MOE. 2009.10)
which showed the possible interactions of the compounds with active site of enzyme
(Table 7).
From the results presented in table 7, it was found that the standard drug
celecoxib showed two hydrogen bond interactions one of them between oxygen of SO2
group and Ser530 with bond length 1.83 Å and the other between SO2 and Tyr385 with
bond length 2.51 Å with binding energy (S = -13.6661) Kcal/mol (Fig. 3). Moreover,
compound 12e also showed two bond interactions, the first is hydrogen bond between
NH2 which is attached to SO2 moiety in 12e and OH of Tyr385 (3.67 Ǻ), the other bond
is arene-cation between phenyl ring of 12e and Arg120 with bonding energy equal to -
16.1076 Kcal/mol (Fig. 4). In spite of this, compound 12f has the highest potency
against COX-2 than each of compound 12e and the standard drug celecoxib as clarified
by five interactions one of them for arene-arene interaction between phenyl ring of 12f
and Arg120 and the remaining four are hydrogen bonds. The first hydrogen bond is
between oxygen of methoxy group in 12f and
17

Table 7: The protein- ligand interactions of compounds 12e, 12f and celecoxib
(standard drug) with 4cox active site pocket.
Compound
no.
Binding
energy (S)
Kcal/mol
Groups of the
ligand involved
in the
Amino acids
involved in
the
Type of the
bond and length of
hydrogen bonds
interaction
interaction
Ǻ
Celecoxib
12e
-13.6661
SO2
SO2
Ser530
Tyr385
Tyr385
Arg120
Polar 1.83 Ǻ
Polar 2.51 Ǻ
Polar 3.67 Ǻ
-16.1076
NH
Phenyl ring
Basic
(Arene-cation
interaction)
12f
-15.2095
OCH3
OCH3
CO
Arg120
Tyr 355
Arg513
His90
Basic 2.50 Ǻ
Polar 2.13 Ǻ
Basic 2.19 Ǻ
Basic 2.69 Ǻ
CO
Phenyl ring
Arg120
Basic (arene-arene
interaction)
Arg120 (2.50 Å), and the second is also between the oxygen atom of methoxy group
and Tyr355 (2.13 Å). The remaining two hydrogen bonds are between carbonyl
function of benzoate moiety in 12f with each of Arg513 (2.19 Å) and His90 (2.69 Å)
with binding energy S = -15.2095 Kcal/mol (Fig. 5). The results obtained in these
docking studies suggested that the synthesized compounds 12e and 12f were fit on the
active site of the enzyme to form the binding interactions as indicated by their docking
pattern compared to that of celecoxib. Overall, these interactions and binding patterns
into 4cox active site explain the remarkable COX-2 inhibitory activity of these
compounds.
18

2D structure
3D structure
Fig. 3. The binding pattern of celecoxib (2D and 3D) structure with the active site of
cyclo-oxygenase enzyme (PDB code = 4cox).
2D structure
3D structure
Fig. 4. The binding pattern of compound 12e (2D and 3D) structure with the active
site of cyclo-oxygenase enzyme (PDBcode = 4cox).
19

2D structure
3D structure
Fig. 5. The binding pattern of compound 12f (2D and 3D) structure with the active
site of cyclo-oxygenase enzyme (PDBcode = 4cox).
2.8.2. Lipiniski's rule of five (the effect of lipophilic and steric parameters)
Compounds 12a, 12e, 12f and 12h have topological polar surface area (TPSA) of
127.51, 136.74, 105.50 and 88.43 Ǻ2, respectively. These values denoted that the
studied compounds can permeate cell membranes [53] (Table 8). Compound 12h is
20

more capable of permeating cell membrane than the other studied compounds and this
is due to its containing the hydroxyl moiety (more polar group) which is highly
absorbable through gastrointestinal (GI) tract.
Table 8: Lipiniski's parameters and TPSA of the more potent cytotoxic compounds
Compound
no.
Lipinisk's Parameters
TPSA Log MW nHB
(Ǻ2)
nHB
A
GI
absorption
Low
No. of viol
P
D
12a
12e
12f
12h
127.51 4.09 523.5
1
1
1
1
7
1 violation:
MW > 500
1 violation:
MW > 500
0
6
136.74 4.03 553.5
8
7
6
Low
High
High
9
105.50 4.46 494.4
9
88.43 4.76 490.5
1
0
nHBD = number of hydrogen bond donor
nHBA = number of hydrogen bond acceptor
GI absorption = Gastrointestinal absorption
3. Conclusion:
The present study investigates the synthesis of novel 5-imidazol-5-one analogs with
comparable antibacterial and anti-inflammatory potencies which were analyzed by the
1
suitable methods such as IR, H NMR, 13C NMR and MS. All spectral data were
following postulated structures. All the tested compounds exhibited good anti-
inflammatory activity, especially compound 12f which produced the maximum effect
of 49.0 % compared to the standard drug, celecoxib (43.1%). Also, compounds 12a,
12e and 12f were studied for their interactions with enzyme COX-2. Additionally, the
most active antibacterial agents 7, 12a, 12f and 12h exhibited good binding affinity
with enzyme dihydrofolate reductase (DHFR). The inhibitory effect of compound 12f
on DHFR is higher than the other tested compounds 7, 12a and 12h. On the other hand,
combining these tested compounds with sulfadiazine as a reference drug indicated that
compounds 12a, 12f and 12h have more potency than sulfadiazine.
21

22

4. Experimental
4.1. Chemistry
4.1.1. General
Melting points were determined on an Electrothermal (9100) apparatus and
are uncorrected. The IR spectra were recorded as KBr pellets on a Perkin Elmer
1
1430 spectrophotometer. H NMR and 13C NMR spectra were recorded in
deuterated dimethylsulfoxide at 300 and 75 MHz, respectively, on a Varian Gemini
NMR spectrometer using tetramethylsilane as an internal reference and the results
are expected as δ value. Mass spectra were taken on a Shimadzu GCMS-QP 1000
Ex mass spectrometer at 70 eV. Elemental analyses were carried out at the
Microanalyses Center of Cairo University, Giza, Egypt. Anti-inflammatory carried
out in Mansoura pharmacy, Egypt. Antibacterial activity was performed at the
central laboratory of biochemistry at Cairo University, Giza, Egypt. Compounds
2a,b and 6 were prepared according to the procedure in the literature [54, 55].
4.1.2. Synthesis of compounds 4a,b.
A mixture of benzoylglycine (3) (0.01 mol), aromatic aldehydes 2a,b and
fused sodium acetate (0.015 mol) in acetic anhydride (5 ml) was heated in a water
o
bath (100 C), for 2 hours. The reaction mixture was allowed to cool at room
temperature and poured on cold water. The solid was collected by filtration and
recrystallized from an ethanol-dioxane mixture.
4.1.2.1. 4-((5-Oxo-2-phenyloxazol-4(5H)-ylidene)methyl)phenyl benzoate
(4a).
Yellow crystals; yield 71%; m.p = 218 °C; IR (KBr) 1738 (CO), 1647 (CO) cm-1;
1H NMR (DMSO-d6) δ = 7.47 (d, 2H, J = 8.7 Hz, Ar), 7.60-8.16 (m, 10H, Ar),
8.18 (s, 1H, CH), 8.40 (d,2H, J = 8.7 Hz, Ar); m/z = 369 (M+, 2.5%), 106 (7.7%),
105 (100%), 77 (34.6%); Anal. Calcd for C23H15NO4: C, 74.79; H, 4.09; N, 3.79.
Found: C, 74.62; H, 4.25; N, 3.56%.
4.1.2.2.
2-Methoxy-4-((5-oxo-2-phenyloxazol-4(5H)-ylidene)methyl)phenyl
benzoate (4b).
Yellow crystals; yield 67%; m.p = 230 °C; IR (KBr) 1784 (CO), 1653 (CO) cm-1;
1H NMR (DMSO-d6) δ = 3.85 (s, 3H, OCH3), 7.25-7.80 (m, 10H, Ar), 7.82 (d, 1H,
23

J = 7.5 Hz, Ar), 8.03 (s, 1H, CH), 8.09 (d,1H, J = 7.2 Hz, Ar), 8.18 (s, 1H, CH,
Ar); m/z = 399 (M+, 4.6%), 398 (17.4%), 106 (10.7%), 105 (100%), 77 (34.9%);
Anal. Calcd for C24H17NO5: C, 72.17; H, 4.29; N, 3.51. Found: C, 72.34; H, 4.13;
N, 3.72%.
4.1.3. Synthesis of 2-((5-oxo-2-phenyloxazol-4(5H)-ylidene)methyl)phenyl
benzenesulfonate (7).
A mixture of compound 3 (0.01 mol), 2-formylphenyl benzenesulfonate 6 (0.01
o
mol) and fused sodium acetate (0.015 mol) was heated at 100 C in acetic
anhydride (5 ml) for 2 hours. The mixture was allowed to cool at room temperature
and poured on cold water. The solid so formed was filtrated off and recrystallized
from a mixture of ethanol-dioxane.
2-((5-Oxo-2-phenyloxazol-4(5H)-ylidene)methyl)phenyl benzenesulfonate (7).
1
Yellow crystals; yield 54%; m.p = 150 °C; IR (KBr) 1653 (CO) cm-1; H NMR
(DMSO-d6) δ = 6.96-8.13 (m, 14H, Ar), 8.76 (s, 1H, CH); Anal. Calcd for
C22H15NO5S: C, 65.18; H, 3.73; N, 3.45; S, 7.91. Found: C, 65.35; H, 3.57; N,
3.68; S, 7.73%.
4.1.4. Synthesis of compounds 10a-d.
A mixture of compound 3 (0.01 mol), arylazosalicyaldehdes 8a-d (0.01 mol) and
o
fused sodium acetate (0.015 mol) was heated in acetic anhydride at 100 C for 3
hours. The reaction mixture was allowed to cool at room temperature and poured
onto cold water. The solid which collected by filtration was recrystallized from an
ethanol-dioxane mixture to give compounds 10a-d.
4.1.4. 1. N-(2-Oxo-6-(phenyldiazenyl)-2H-chromen-3-yl)benzamide (10a).
Yellow crystals; yield 66%; m.p > 300 °C; IR (KBr) 3327 (NH), 1717 (CO), 1664
(CO) cm-1; 1H NMR (DMSO-d6) δ = 7.42-8.07 (m, 12H, Ar), 8.32 (s, 1H, CH, Ar),
8.90 (s, 1H, CH, Ar), 9.81 (s, 1H, NH); Anal. Calcd for C22H15N3O3: C, 71.54; H,
4.09; N, 11.38. Found: C, 71.71; H, 4.25; N, 11.16%.
4.1.4. 2. N-(2-Oxo-6-(p-tolyldiazenyl)-2H-chromen-3-yl)benzamide(10b).
Yellow crystals; yield 56%; m.p > 300 °C; IR (KBr) 3320 (NH), 1725 (CO), 1658
1
(CO) cm-1; H NMR (DMSO-d6) δ = 2.32 (s, 3H, CH3), 7.21-8.06 (m, 11H, Ar),
24

8.41 (s, 1H, CH, Ar), 8.72 (s, 1H, CH, Ar), 9.64 (s, 1H, NH); Anal. Calcd for
C23H17N3O3: C, 72.05; H, 4.47; N, 10.96. Found: C, 72.21; H, 4.30; N, 10.73%.
4.1.4. 3. N-(6-((4-Methoxyphenyl)diazenyl)-2-oxo-2H-chromen-3-yl) benza-
mide (10c).
Yellow crystals; yield 64%; m.p > 300 °C; IR (KBr) 3335 (NH), 1716 (CO), 1668
(CO) cm-1; 1H NMR (DMSO-d6) δ = 3.81 (s, 3H, OCH3), 7.32-8.11 (m, 11H, Ar),
8.21 (s, 1H, CH, Ar), 8.68 (s, 1H, CH, Ar), 9.62 (s, 1H, NH); Anal. Calcd for
C23H17N3O4: C, 69.17; H, 4.29; N, 10.52. Found: C, 69.01; H, 4.46; N, 10.30%.
4.1.4. 4. N-(6-((4-Fluorophenyl)diazenyl)-2-oxo-2H-chromen-3-yl)benzamide
(10d).
Yellow crystals; yield 62%; m.p > 300 °C; IR (KBr) 3330 (NH), 1721 (CO), 1670
(CO) cm-1; 1H NMR (DMSO-d6) δ = 7.44-8.05 (m, 11H, Ar), 8.34 (s, 1H, CH, Ar),
8.78 (s, 1H, CH, Ar), 9.76 (s, 1H, NH); Anal. Calcd for C22H14FN3O3: C, 68.21;
H, 3.64; N, 10.85. Found: C, 68.36; H, 3.46; N, 10.64%.
4.1.5. Synthesis of compounds 12a-h
A solution of the appropriate compound 4a,b (0.01 mol) was refluxed in glacial
acetic acid (10 ml) with aromatic amine derivatives 11a-d (0.01 mol) containing
a catalytic amount of fused sodium acetate for 3 hours. The solid product that
precipitated by cooling was filtered off and recrystallized from ethanol-dioxane
mixture.
4.1.5.1. 4-((5-Oxo-2-phenyl-1-(4-sulfamoylphenyl)-1,5-dihydro-4H-imidazol-
4-ylidene)-methyl)phenyl benzoate (12a).
Yellow crystals; yield 66%; m.p > 300 °C; IR(KBr) 3316 (NH2), 1701 (CO), 1637
(CO) cm-1; 1H NMR (DMSO-d6) δ = 7.01 (s, 2H, NH2), 7.42 (d, 2H, J = 8.4 Hz,
Ar), 7.52-7.81 (m, 10H, Ar), 7.82 (d, 2H, J = 8.1 Hz, Ar), 8.01 (d,2H, J = 8.7 Hz,
Ar), 8.22 (d, 2H, J = 8.1 Hz, Ar), 8.31 (s, 1H, CH); Anal. Calcd for C29H21N3O5S:
C, 66.53; H, 4.04; N, 8.03; S, 6.12.Found: C, 66.36; H, 4.20; N, 8.26; S, 6.29%.
4.1.5.2.
4-(4-(4-(Benzoyloxy)benzylidene)-5-oxo-2-phenyl-4,5-dihydro-1H-
imidazol-1-yl)-benzoic acid (12b).
25

White crystals; yield 68%; m.p = 292 °C; IR (KBr) 3431 (OH), 1732 (CO), 1684
(CO), 1637 (CO) cm-1; 1H NMR (DMSO-d6) δ = 7.31 (d, 2H, J = 7.8 Hz, Ar), 7.44-
7.61 (m, 10H, Ar), 7.74 (d, 2H, J = 8.1 Hz, Ar), 8.11 (d, 2H, J = 8.4 Hz, Ar), 8.20
(d, 2H, J = 7.8 Hz, Ar), 8.26 (s, 1H, CH), 10.31 (s, 1H, OH); m/z = 488 (M+, 1.0%),
487 (2.7%), 368 (9.1%), 264 (2.3%), 223 (2.6%), 120 (8.5%), 106 (7.8%), 105
(100%), 77 (30.0%); Anal. Calcd for C30H20N2O5: C, 73.76; H, 4.13; N, 5.73.
Found: C, 73.59; H, 4.30; N, 5.51%.
4.1.5.3. 4-((1-(4-Acetylphenyl)-5-oxo-2-phenyl-1,5-dihydro-4H-imidazol-4-
ylidene)-methyl)phenyl benzoate (12c).
Yellow crystals; yield 62%; m.p = 264 °C; IR (KBr) 1737 (CO), 1715 (CO), 1680
1
(CO) cm-1; H NMR (DMSO-d6) δ = 2.60 (s, 3H, CH3), 7.37-7.65 (m, 10H, Ar),
7.75 (d, 2H, J = 6.6 Hz, Ar), 8.01 (d, 2H, J = 8.4 Hz, Ar), 8.02 (s, 1H, CH), 8.15
(d,2H, J = 7.8 Hz, Ar), 8.46 (d, 2H, J = 8.7 Hz, Ar); Anal. Calcd for C31H22N2O4:
C, 76.53; H, 4.56; N, 5.76. Found: C, 76.69; H, 4.39; N, 5.53%.
4.1.5.4. 4-((1-(4-Hydroxyphenyl)-5-oxo-2-phenyl-1,5-dihydro-4H-imidazol-4-
ylidene)-methyl)phenyl benzoate (12d).
Yellow crystals; yield 67%; m.p = 269 °C; IR (KBr) 3435 (OH), 1747 (CO), 1677
(CO) cm-1; 1H NMR (DMSO-d6) δ = 7.10 (d, 2H, J = 8.7 Hz, Ar ), 7.21 (d, 2H, J
= 8.7 Hz, Ar ), 7.34-7.71 (m, 10H, Ar), 7.82 (d, 2H, J = 8.4 Hz, Ar), 8.12 (s, 1H,
CH), 8.22 (d, 2H, J = 8.1 Hz, Ar), 9.87 (s, 1H, OH); Anal. Calcd for C29H20N2O4:
C, 75.64; H, 4.38; N, 6.08. Found: C, 75.46; H, 4.55; N, 6.31 %.
4.1.5.5.
2-Methoxy-4-((5-oxo-2-phenyl-1-(4-sulfamoylphenyl)-1,5-dihydro-
4H-imidazol-4-ylidene)methyl)phenyl benzoate (12e).
Yellow crystals; yield 70%; m.p = 284 °C; IR(KBr) 3332 (NH2), 1725 (CO), 1639
(CO) cm-1; 1H NMR (DMSO-d6) δ = 3.86 (s, 3H, OCH3), 7.37 (s, 2H, NH2), 7.39-
7.57 (m, 10H, Ar), 7.59 (d, 2H, J = 7.8 Hz, Ar), 7.65 (s, 1H, Ar), 7.89 (d, 1H, J =
7.8 Hz, Ar), 8.05 (d, 2H, J = 8.1 Hz, Ar), 8.13 (d, 1H, J = 7.8 Hz, Ar), 8.32 (s, 1H,
CH); 13C NMR (DMSO-d6) δ = 55.8, 66.2, 116.1, 120.2, 123.3, 125.4, 126.5,
127.2, 128.0, 128.3, 128.8, 128.9, 129.7, 131.5, 133.0, 134.0, 137.1, 138.1, 141.1,
143.6, 150.8, 160.2, 163.7, 169.1; m/z = 553 (M+, 3.9%), 449 (2.1%), 258 (18.2%),
179 (5.5%), 106 (7.5%), 105 (100%), 77 (16.5%); Anal. Calcd for C30H23N3O6S:
C, 65.09; H, 4.19; N, 7.59; S, 5.79. Found: C, 65.26; H, 4.01; N, 7.36; S, 5.63%.
26

4.1.5.6.
4-(4-(4-(Benzoyloxy)-3-methoxybenzylidene)-5-oxo-2-phenyl-4,5-
dihydro-1H-imidazol-1-yl)benzoic acid (12f).
Yellow crystals; yield 58%; m.p > 300 °C; IR (KBr) 3433 (OH), 1730 (CO), 1691
(CO), 1638 (CO) cm-1; 1H NMR (DMSO-d6) δ = 3.86 (s, 3H, OCH3), 7.29 (d, 2H,
J = 8.1 Hz, Ar), 7.35-7.43 (m, 10H, Ar), 7.53 (s, 1H, Ar), 7.62 (d, 2H, J = 7.8 Hz,
Ar), 7.96 (d, 1H, J = 8.1 Hz, Ar), 8.13 (d, 1H, J = 8.1 Hz, Ar), 8.33 (s, 1H, CH),
10.22 (s, 1H, OH); Anal. Calcd for C31H22N2O6: C, 71.81; H, 4.28; N, 5.40. Found:
C, 71.63; H, 4.45; N, 5.18%.
4.1.5.7. 4-((1-(4-Acetylphenyl)-5-oxo-2-phenyl-1,5-dihydro-4H-imidazol-4-
ylidene)-methyl)-2-methoxyphenyl benzoate (12g).
Yellow crystals; yield 66%; m.p = 273 °C; IR (KBr) 1740 (CO), 1720 (CO), 1687
(CO) cm-1; 1H NMR (DMSO-d6) δ = 2.61 (s, 3H, CH3), 3.85 (s, 3H, OCH3), 7.37
(d, 2H, J = 8.1 Hz, Ar ), 7.42-7.74 (m, 10H, Ar), 7.77 (d, 2H, J = 7.5 Hz, Ar), 7.89
(s, 1H, Ar), 8.02 (d, 1H, J = 8.1 Hz, Ar), 8.13 (d,1H, J = 7.5 Hz, Ar), 8.32 (s, 1H,
CH); Anal. Calcd for C32H24N2O5: C, 74.41; H, 4.68; N, 5.42. Found: C, 74.59; H,
4.51; N, 5.63%.
4.1.5.8. 4-((1-(4-Hydroxyphenyl)-5-oxo-2-phenyl-1,5-dihydro-4H-imidazol-4-
ylidene)-methyl)-2-methoxyphenyl benzoate (12h).
Yellow crystals; yield 69%; m.p = 230 °C; IR (KBr) 3434 (OH), 1744 (CO), 1684
(CO) cm-1; 1H NMR (DMSO-d6) δ = 3.85 (s, 3H, OCH3), 6.82 (d, 2H, J = 8.7 Hz,
Ar ), 7.09 (d, 2H, J = 8.7 Hz, Ar ), 7.29 (s, 1H, Ar), 7.38-7.60 (m, 10H, Ar), 7.62
(d, 1H, J = 7.8 Hz, Ar), 8.13 (d, 1H, J = 8.1 Hz, Ar), 8.32 (s, 1H, CH), 9.81 (s, 1H,
OH); Anal. Calcd for C30H22N2O5: C, 73.46; H, 4.52; N, 5.71. Found: C, 73.29; H,
4.70; N, 5.49%.
4.1.6. Synthesis of compounds 14a,b and 16a,b.
A solution of the appropriate compound 4a,b (0.01 mol) was refluxed in glacial
acetic acid (10 ml) with each of aromatic amine 13 and 15 (0.01 mol) containing
a catalytic amount of fused sodium acetate for 4 hours. The solid so formed by
cooling at room temperature was filtered off and recrystallized from ethanol-
dioxane mixture.
27

4.1.6.1. 4-((1-(2,4-Dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-5-oxo-2-phenyl-
1,5-dihydro-4H-imidazol-4-ylidene)methyl)phenyl benzoate (14a).
Yellow crystals; yield 62%; m.p > 300 °C; IR(KBr) 3233 (NH), 3120 (NH), 1735
(CO), 1685 (CO), 1650 (2CO) cm-1; 1H NMR (DMSO-d6) δ = 7.44 (d, 2H, J = 8.7
Hz, Ar), 7.53-7.84 (m, 10H, Ar), 8.01 (s, 1H, CH), 8.14 (s, 1H, CH), 8.43 (d, 2H,
J = 8.7 Hz, Ar), 11.41 (s, 1H, NH), 11.52 (s, 1H, NH); m/z = 478 (M+, 14.7%),
374 (3.0%), 214 (5.7%), 171 (4.9%), 116 (7.4%), 106 (7.6%), 105 (100%), 89
(4.3%), 77 (31.3%); Anal. Calcd for C27H18N4O5: C, 67.78; H, 3.79; N,
11.71.Found: C, 67.60; H, 3.62; N, 11.93%.
4.1.6.2. 4-((1-(2,4-Dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-5-oxo-2-phenyl-
1,5-dihydro-4H-imidazol-4-ylidene)methyl)-2-methoxyphenyl
benzoate
(14b).
Yellow crystals; yield 66%; m.p > 300 °C; IR (KBr) 3280 (NH), 3115 (NH), 1737
(CO), 1717 (CO), 1663 (2CO) cm-1; 1H NMR (DMSO-d6) δ = 3.86 (s, 3H, OCH3),
7.31-7.76 (m, 10H, Ar), 7.82 (d, 1H, J = 7.5 Hz, Ar), 7.90 (s, 1H, Ar), 8.01 (s, 1H,
CH), 8.11 (s, 1H, CH), 8.21 (d,1H, J = 7.5 Hz, Ar), 11.21 (s, 2H, 2NH); Anal.
Calcd for C28H20N4O6: C, 66.14; H, 3.96; N, 11.02. Found: C, 66.31; H, 3.80; N,
11.25%.
4.1.6.3.
4-((5-Oxo-2-phenyl-1-(thiazol-2-yl)-1,5-dihydro-4H-imidazol-4-
ylidene)-methyl)-phenyl benzoate (16a).
Yellow crystals; yield 68%; m.p = 216 °C; IR (KBr) 1737 (CO), 1660 (CO) cm-1;
1H NMR (DMSO-d6) δ = 7.48 (d, 2H, J = 8.7 Hz, Ar), 7.61-7.78 (m, 10H, Ar),
8.14 (d, 1H, J = 8.1 Hz, Ar), 8.18 (s, 1H, CH), 8.26 (d, 1H, J = 7.8 Hz, Ar), 8.41
(d, 2H, J = 9 Hz, Ar); Anal. Calcd for C26H17N3O3S: C, 69.17; H, 3.80; N, 9.31; S,
7.10.Found: C, 69.35; H, 3.63; N, 9.54; S, 7.27%.
4.1.6.4.
2-Methoxy-4-((5-oxo-2-phenyl-1-(thiazol-2-yl)-1,5-dihydro-4H-
imidazol-4-ylidene)methyl)-phenyl benzoate (16b).
Yellow crystals; yield 64%; m.p = 210 °C; IR (KBr) 1735 (CO), 1649 (CO) cm-1;
1H NMR (DMSO-d6) δ = 3.87 (s, 3H, OCH3), 7.36 (s, 1H, Ar), 7.39 (d, 1H, J = 8.4
Hz, Ar), 7.59-7.69 (m, 10H, Ar), 7.71 (d, 1H, J = 8.4 Hz, Ar), 7.91 (d, 1H, J = 8.4
Hz, Ar), 8.09 (d, 1H, J = 8.4 Hz, Ar), 8.21 (s, 1H, CH); Anal. Calcd for
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C27H19N3O4S: C, 67.35; H, 3.98; N, 8.73; S, 6.66. Found: C, 67.19; H, 3.82; N,
8.52; S, 6.49%.
4.1.7. Synthesis of compounds 18a,b and 20a,b.
A solution of the appropriate compound 4a,b (0.02 mol) was refluxed in glacial
acetic acid (10 ml) with each of benzene-1,4-diamine (17) (0.01 mol) and
benzidine (19) (0.01 mol) containing fused sodium acetate (0.015 mol). The solid
product that precipitated was filtered off and recrystallized from DMF.
4.1.7.1. 1,4-Phenylenebis(5-oxo-2-phenyl-1,5-dihydro-4H-imidazole-1-yl-4-
ylidene))-bis(methanylylidene))bis(4,1-phenylene) dibenzoate (18a).
Yellow crystals; yield 56%; m.p > 300 °C; IR(KBr) 1731 (2CO), 1644 (2CO) cm-1;
1H NMR (DMSO-d6) δ = 7.24-8.06 (m, 32H, Ar), 8.14 (s, 2H, 2CH); Anal. Calcd
for C52H34N4O6: C, 77.03; H, 4.23; N, 6.91. Found: C, 77.19; H, 4.05; N, 6.69%.
4.1.7.2. 1,4-Phenylenebis(5-oxo-2-phenyl-1,5-dihydro-4H-imidazole-1-yl-4-
ylidene))-bis(methanylylidene))bis(2-methoxy-4,1-phenylene)
(18b).
dibenzoate
Yellow crystals; yield 61%; m.p > 300 °C; IR(KBr) 1725 (2CO), 1640 (2CO) cm-1;
1H NMR (DMSO-d6) δ = 3.86 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 7.37-8.16 (m,
30H, Ar), 8.21 (s, 2H, 2CH); Anal. Calcd for C54H38N4O8: C, 74.47; H, 4.40; N,
6.43. Found: C, 74.29; H, 4.23; N, 6.66%.
4.1.8.1.
4-(-(1-(4'-(4-(-4-(Benzoyloxy)benzylidene)-5-oxo-2-phenyl-4,5-
dihydro-1H-imidazol-1-yl)-[1,1'-biphenyl]-4-yl)-5-oxo-2-phenyl-1,5-dihydro-
4H-imid-azol-4-ylidene)methyl)phenyl benzoate (20a).
Yellow crystals; yield 60%; m.p > 300 °C, IR(KBr) 1731 (2CO), 1645 (2CO) cm-1;
1H NMR (DMSO-d6) δ = 7.31-8.11 (m, 36H, Ar), 8.22 (s, 2H, 2CH); Anal. Calcd
for C58H38N4O6: C, 78.54; H, 4.32; N, 6.32. Found: C, 78.38; H, 4.14; N, 6.55%.
4.1.8.2. 4-(-(1-(4'-(4-(4-(Benzoyloxy)-3-methoxybenzylidene)-5-oxo-2-phenyl-
4,5-dihydro-1H-imidazol-1-yl)-[1,1'-biphenyl]-4-yl)-5-oxo-2-phenyl-1,5-
dihydro-4H-imidazol-4-ylidene)methyl)-2-methoxyphenyl benzoate (20b).
Yellow crystals; yield 64%; m.p > 300 °C; IR(KBr) 1737 (2CO), 1640 (2CO) cm-1;
1H NMR (DMSO-d6) δ = 3.87 (s, 6H, 2OCH3), 7.31-8.11 (m, 34H, Ar), 8.12 (s,
29