Synthesis, antimicrobial, anti-cancer and in silico studies of new urea derivatives

Article abstract of DOI:10.1016/j.bioorg.2021.104953

The reaction of an alkyl or aryl isocyanates with some primary amines in acetonitrile at room temperature afforded the corresponding alkyl- and aryl-urea derivatives. All the prepared urea compounds have been elucidated by FTIR, NMR, and elemental analysis. The compounds 1 and 3 were confirmed by single-crystal X-ray diffraction. The 4-tolylsulfonyl isocyanate reacted with the aryl amines 1, 2, 3, and 2,4-dichloroaniline to afford the corresponding sulfonylurea derivatives 5–8. Likewise, the reaction of the isocyanates with 2,4-dichloroaniline, 5-methyl isoxazole-3-amine, and 2-aminothiazole derivatives gave the corresponding urea derivatives 9–17. All the prepared compounds 5–17 were tested in vitro as anti-microbial and anti-HepG2 agents. Moreover, analyzing gene expression of TP53-exon4 and TP53-exon7, DNA damage values, and DNA fragmentation percentages have been discussed. The compounds 5 and 8 recorded the highest activity against the tested microbial strains with maximum activity against C. albicans (50 mm) and B. mycoides (40 mm), respectively. The compounds 5 inhibited the growth of E. coli, S. aureus, and C. Albicans at the MIC level of 0.0489 μM, while the compound 8 was able to inhibit the visible growth of E. coli and C. albicans at MIC value of 3.13 μM and S. aureus at 0.3912 μM. In the same line, compound 5 showed the best cytotoxic activity against the HepG2 cell line (IC₅₀ = 4.25 μM) compared to 5 fluorouracil with IC₅₀ = 316.25 μM. Expression analysis of liver cancer related to a gene including TP53-exon4 and TP53-exon7 was used in HepG2 Liver cancer cell lines using RT-qPCR. The expression values of TP53-exon4 and TP53-exon7 genes were decreased. The DNA damage values and DNA fragmentation percentages were increased significantly (P < 0.01) in the treated HepG2 (5) sample compared with the negative control. Docking studies were performed for the synthetic compounds against 2 bacterial proteins (DNA gyrase subunit B, and penicillin binding protein 1a) that are known targets for some antibiotics, and one cell division protein kinase 2 (CDK2) as target for anticancer drugs.

Full text of DOI:10.1016/j.bioorg.2021.104953

Bioorganic Chemistry 112 (2021) 104953
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis, antimicrobial, anti-cancer and in silico studies of new
urea derivatives
,
Farid M. Sroora *, Abdelmageed M. Othmanb, Mohamed A. Tantawyc, Karima F. Mahrousd,
Mostafa E. El-Naggare
a Organometallic and Organometalloid Chemistry Department, National Research Centre, 12622 Cairo, Egypt
b Microbial Chemistry Department, Genetic Engineering and Biotechnology Division, National Research Centre, Cairo, Egypt
c Hormones Department, Medical Research Division, National Research Centre, Cairo, Egypt
d Cell Biology Department, National Research Centre, 12622 Dokki, Egypt
e Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Sadat City, Menoufia, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
The reaction of an alkyl or aryl isocyanates with some primary amines in acetonitrile at room temperature
afforded the corresponding alkyl- and aryl-urea derivatives. All the prepared urea compounds have been
elucidated by FTIR, NMR, and elemental analysis. The compounds 1 and 3 were confirmed by single-crystal X-
ray diffraction. The 4-tolylsulfonyl isocyanate reacted with the aryl amines 1, 2, 3, and 2,4-dichloroaniline to
afford the corresponding sulfonylurea derivatives 5–8. Likewise, the reaction of the isocyanates with 2,4-dichlor-
oaniline, 5-methyl isoxazole-3-amine, and 2-aminothiazole derivatives gave the corresponding urea derivatives
9–17. All the prepared compounds 5–17 were tested in vitro as anti-microbial and anti-HepG2 agents. Moreover,
analyzing gene expression of TP53-exon4 and TP53-exon7, DNA damage values, and DNA fragmentation per-
centages have been discussed. The compounds 5 and 8 recorded the highest activity against the tested microbial
strains with maximum activity against C. albicans (50 mm) and B. mycoides (40 mm), respectively. The com-
pounds 5 inhibited the growth of E. coli, S. aureus, and C. Albicans at the MIC level of 0.0489 µM, while the
compound 8 was able to inhibit the visible growth of E. coli and C. albicans at MIC value of 3.13 µM and S. aureus
at 0.3912 µM. In the same line, compound 5 showed the best cytotoxic activity against the HepG2 cell line (IC50
= 4.25 µM) compared to 5 fluorouracil with IC50 = 316.25 µM. Expression analysis of liver cancer related to a
gene including TP53-exon4 and TP53-exon7 was used in HepG2 Liver cancer cell lines using RT-qPCR. The
expression values of TP53-exon4 and TP53-exon7 genes were decreased. The DNA damage values and DNA
fragmentation percentages were increased significantly (P < 0.01) in the treated HepG2 (5) sample compared
with the negative control. Docking studies were performed for the synthetic compounds against 2 bacterial
proteins (DNA gyrase subunit B, and penicillin binding protein 1a) that are known targets for some antibiotics,
and one cell division protein kinase 2 (CDK2) as target for anticancer drugs.
Urea derivatives
Anti-microbial
Anti-HepG2
Gene expression
DNA damage
DNA fragmentation
Molecular docking
1. Introduction
such as Staphylococcus aureus, Enterococcus faecium, Streptococcus pneu-
monia, Mycobacterium tuberculosis (M. tuberculosis), nontuberculous
The progress of drug resistance among human pathogens continues
to be a serious health problem and leads to the importance of discov-
ering and developing new drugs and synthesizing new compounds with
active pharmacophore. The incidence of antimicrobial resistance (AMR)
is increasing and a growing serious threat to public health burden
worldwide [1]. In the recent few years, the expression of resistance-
related complications and therapeutic difficulties by microorganisms
(atypical) mycobacteria, and gram-positive and gram-negative bacteria
against the marketed antibacterial agents, therefore, there is a great
need for providing an effective approach in drug design of novel anti-
microbial molecules and modification of their mechanisms against mi-
crobes [2,3].
Urea derivatives are a well-known class of compounds which present
in many biologically active compounds. In recent years, several
* Corresponding author.
E-mail addresses: faridsroor@gmx.de, fm.sroor@nrc.sci.eg (F.M. Sroor).
https://doi.org/10.1016/j.bioorg.2021.104953
Received 4 September 2020; Received in revised form 22 March 2021; Accepted 23 April 2021
Available online 29 April 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

F.M. Sroor et al.
Bioorganic Chemistry 112 (2021) 104953
publications derived from sorafenib have been reported on topics about
the design, synthetic approach, mechanistic aspects, structure–activity
relationships (SAR), and the interactions with biological systems of urea
derivatives as potential anticancer [4], antidiabetic [5], antiviral [6],
anticonvulsan [7], antifungal activities [8] and antibacterial [9]. It is
worth noting at this point that no univocal data about sulfonylureas’
effects on cancer growth are available. Limited previous articles about
the same topic have been published; therefore, there is conflicting evi-
dence about the real role of different compounds of the sulfonylurea
family on cancer cell growth [10].
combination of the previous moieties will increase the biological profile
of the targeted compounds as anti-microbial and anti-HepG2 agents.
2. Results and discussion
2.1. Chemistry
The title compounds were prepared straightforwardly according to
Schemes 4 and 5 by treatment of commercially available isocyanates, 4-
tolylsulfonylisocyanate, benzoylisocyanat, 4-tolylisocyanate and ethyl
isocyanate with the primary aryl amines 1, 2, 3 or 2,4-dichloroaniline
and aminothiazole or aminoisoxazole derivatives in dry acetonitrile at
room temperature. The key starting material 1, 2, 3, and 4 were pre-
pared according to the previously described in the literatures [24–26]
and shown in Schemes 1, 2, and 3.
Given these observations and based on these facts we are encouraged
to design and synthesize a series of new alkyl- and aryl-based urea de-
rivatives and evaluate their biological activity as potential antimicrobial
and anti-HepG2 agents. “Molecular hybridization” is one of the impor-
tant concepts in drug design and development by a combination of
pharmacophoric moieties of different bioactive compounds, either
linked with one another or fused to produce a new hybrid structure with
improved biological efficacy and affinity. As illustrated in Fig. 1, several
drugs and biologically active compounds are containing the isoxazole,
thiazol, thiophene, and 2,4-dichloroaniline moieties that are designed to
treat infections and diseases of different etiologies. Likewise, recently
several sulfonamide-based compounds have been designed, synthesized,
and evaluated as potential antibacterial agents [11]. On the other hand,
benzoylurea, 4-tolylsulfonylurea, 4-tolylurea, and ethyl urea containing
structures exhibited a high biological activity as anti-diabetic [12] anti-
cancer [13,14] and anti-tumor agents [15].
The starting materials 1 (Ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b]
thiophene-3-carboxylate) and 3 (4-amino-N-(4,6-dimethylpyrimidin-2-
yl)benzenesulfonamide) were elucidated by single crystal X-ray
diffraction as depicted in Fig. 2.
As illustrated in scheme 4, the 4-tolylsufonyl isocyanate reacted with
the primary amines 1, 2, 3, or 2,4-dichloroaniline via nucleophilic
addition reaction to afford the corresponding sulfonylurea derivatives
5–8 respectively, in excellent yields. The reaction of 4-tolylsufonyl iso-
cyanate with the amines as described in scheme 4 was carried out under
mild conditions and variated times 5 (2 h), 6 (3 h), 7 (25 min), and 8 (5
min). The new compounds 5–8 were produced in moderate to excellent
yields, 87% (5), 74% (6), 62% (7), and 96% (8). It is worth to mention
that, the urea derivative 8 was prepared by Howbert et al by the reaction
of a substituted amine with a sulfonyl isocyanate in an inert and aprotic
solvent under worming but without full characterization of compound 8
Consequently and in continuation of our previous work to prepare
biologically active organic and organometallic compounds
[12,14,16–23] in this study we decided to design and prepare a new
series of alkyl- and aryl- urea derivatives with the aim that the
Fig. 1. Design concept of the newly synthesized alkyl- and aryl-urea compounds 5–17 using molecular hybridization approach.
2

F.M. Sroor et al.
Bioorganic Chemistry 112 (2021) 104953
Scheme 1. Synthesis of ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate 1.
Scheme 2. Synthesis of 4-amino-N-carbamimidoylbenzenesulfonamide 2 and 4-amino-N-(4,6-dimethylpyrimidin-2-yl)benzenesulfonamide 3.
Scheme 3. Synthesis of 2-amino-4-(4-bromophenyl)thiazol 4.
Scheme 4. Synthesis of sulfonylurea derivatives 5–8.
3

F.M. Sroor et al.
Bioorganic Chemistry 112 (2021) 104953
Scheme 5. Synthesis of benzoyl, alkyl- and aryl- urea derivatives 9–17.
Fig. 2. X-ray structures of the starting materials 1 (left) and 3 (right).
[27]. Unfortunately, the primary amines 1, 2 and 3 did not react with
the other isocyanates (benzoylisocyanat, 4-tolylisocyanate and ethyl
isocyanate) under the same conditions of acetonitrile at room temper-
ature for a long time.
characterized [28–33]. The IR spectrum of compound 8 indicated the
presence of the NH group at 3322 and 3278 cmꢀ 1. Besides, the carbonyl
band observed at 1680 cmꢀ 1. The strong bands at 1330 and 1161 cmꢀ 1
assigned to the SO2 group. The 1H NMR spectrum of 8 showed the
presence of a single signal at δ 2.37 ppm assigned to the methyl group.
NH groups appear as two broad singlet signals at δ 8.48 and 11.33 ppm.
Furthermore, the 13C NMR spectrum revealed the methyl signal at δ
21.3 ppm. All other signals appear at their expected positions.
The reaction of 2,6-dichloroaniline with benzoylisocyanat, 4-tolyli-
socyanate, or ethyl isocyanate in acetonitrile at room temperature
afforded the urea derivatives 9–11 respectively, as shown in scheme 5.
On the other hand, the reaction of 2-aminothiazole derivatives (thiazol-
2-amine and 4-(4-bromophenyl)thiazol-2-amine) gave the correspond-
ing urea derivatives 12–15 respectively, in excellent yield (82, 95, 97
and 89%, respectively). Likewise, the reaction of 5-methylisoxazol-3-
amine with benzoylisocyanat and 4-tolylisocyanate under the same
conditions gave the urea derivatives 16 and 17 in 87% and 96% yields,
respectively.
2.2. Antimicrobial activity
E. coli, B. mycoides, S. aureus, and C. albicans were applied as repre-
sentatives for Gram-negative, Gram-positive bacterial strains and a non-
filamentous fungal strain respectively, to express different microbial
inhabitants, to assess the antimicrobial effectiveness of the synthesized
compounds.
The chemical structures of the compounds 5–17 were proved spec-
troscopically based on the elemental analysis and spectral data.
Although some of these compounds have been reported in previous lit-
eratures by preparation with different methods, they are not
The well diffusion assay was carried out for the synthesized urea
compounds using four representative microorganisms namely (E. coli, B.
4

F.M. Sroor et al.
Bioorganic Chemistry 112 (2021) 104953
mycoides, S. aureus, and C. albicans). The resistance of microbial strains
Table 2
MIC values of 5 and 8 compounds against selected microorganisms.
to the synthesized compounds, in general, varies considerably with the
compositions and functional groups present in each35, 36
.
compound
MIC value (µM)
The results obtained were cited in Table 1 and presented in Figure S1
(supplementary data), which indicate the different responses of various
tested microorganisms toward different compounds. The highest anti-
microbial activity was obtained in the case of compounds 5 and 8 fol-
lowed by compounds 12, 6, 15, 14, and 7, respectively. In contrast,
compounds 10 and 16 have not offered any antimicrobial activity to-
ward all tested microorganisms, whereas other compounds (9, 11, 13,
and 17) showed antimicrobial activity against some represented mi-
croorganisms. Compounds 5 and 8 recorded the highest activity among
tested derivatives against the tested microbial strains with maximum
activity against C. albicans (50 mm) and B. mycoides (40 mm), respec-
tively. It is worthy to mention that the antimicrobial activities by these
two compounds are also higher than those obtained by the standard
applied antibiotics as presented in Table 1. Consequently, these two
compounds (5 and 8) were selected to determine their MIC values
against the four tested microorganisms.
E. coli
B. mycoides
S. aureus
C. albicans
5
8
0.0489
3.13
>0.0122
>0.0122
0.0489
0.3912
0.0489
3.13
MIC: minimal inhibition concentration.
E. coli
B. mycoides
S. aureus
40
30
20
10
C. albicans
In order to test the antimicrobial activity durability of both 5 and 8
compounds against the represented microorganisms, different concen-
trations of each compound were tested regarding to their antimicrobial
activity for an incubation period of 72 h. The results obtained were
illustrated in Figure S2a, b (supplementary data) which indicates that
the inhibition zones caused by compound 8 starts to be diminished of
after 24 h of incubation. This could be due to the adaptability of tested
microorganisms to compound 8 that means this compound has bacte-
riostatic properties. In contrast, compound 5 was able to prevent the
microbial growth for a longer time (72 h) which confirms the suscepti-
bility of tested strains to compound 5. This behavior was also clear from
the obtained MIC values as shown in Table 2, where the MIC values of
compound 5 are much lower than (has more ability to inhibit the mi-
crobial growth) that of compound 8 with exception of B. mycoides which
was inhibited by both of them at values<0.0122 µM as appears in Figs. 3
and 4. The compounds 5 inhibited the growth of E. coli, S. aureus, and
C. albicans at the MIC level of 0.0489 µM, whereas compound 8 was able
to inhibit the visible growth of E. coli and C. albicans at MIC value of
5
3
.1
5
56
.5
12
50
25
.2
6
3
.
1
0.3912 0.1956 0.0489 0.0122
Concentration (μM)
Fig. 3. Antimicrobial activity of different concentrations from compound 5.
Each sample (100 µl) containing the specified concentration was loaded in a 12
mm agar well and then culture plates were incubated overnight at 37 ◦C. The
experiment was performed in triplicate and the mean values were cited ±
error values.
40
E. coli
B. mycoides
S. aureus
C. albicans
30
Table 1
Antimicrobial activity assessment of compounds 5–17 by agar diffusion method.
Compound
Inhibition zones diameter (mm)
E. coli
B. mycoides
S. aureus
C. albicans
20
10
5
47 ± 1.25
48 ± 0.75
20 ± 2.05
16 ± 1.33
40 ± 0.35
0
49 ± 0.00
21 ± 1.86
17 ± 0.33
35 ± 1.13
13 ± 0.25
0
50 ± 0.85
19 ± 1.20
16 ± 1.10
36 ± 1.05
0
6
20 ± 1.33
7
17 ± 0.00
8
37 ± 0.45
9
0
10
0
0
0
11
0
14 ± 0.13
20 ± 0.45
17 ± 0.09
15 ± 0.00
18 ± 0.45
0
16 ± 1.05
22 ± 1.70
0
0
5
3
.1
5
2
.5
12
50
25
12
21 ± 1.00
0
24 ± 1.30
17 ± 1.23
18 ± 0.00
19 ± 1.27
0
.2
56
.
1
6
3
012
.
0.3912 0.1956 0.0489
0
13
14
17 ± 0.76
18 ± 1.08
0
21 ± 1.15
19 ± 0.63
0
Concentration (μM)
15
16
Fig. 4. Antimicrobial activity of different concentrations from compound 8.
Each sample (100 µl) containing the specified concentration was loaded in a 12
mm agar well and then culture plates were incubated overnight at 37 ◦C. The
experiment was performed in triplicate and the mean values were cited ±
error values.
17
0
0
18 ± 0.26
0
0
DMSO (control)
Colstin 10 mcg
Tobramycin 10 mcg
Gentamicin 10 mcg
Ampicillin 10 mcg
Streptomycin 10 mcg
Erythromycin 15 mcg
0
0
0
0
0
0
0
13 ± 0.55
12 ± 0.00
20 ± 1.05
15 ± 0.00
29 ± 2.13
10 ± 0.08
11 ± 0.35
12 ± 0.93
17 ± 0.75
17 ± 0.15
12 ± 1.00
12 ± 0.33
22 ± 0.00
15 ± 1.33
25 ± 0.76
12 ± 0.75
12 ± 0.50
14 ± 0.25
17 ± 0.13
25 ± 0.00
3.13 µM and S. aureus at 0.3912 µM (Table 2).
100 μl of dissolved compounds (0.1 mM) in DMSO were applied to 12 mm holes
2.3. Anti-cancer activity
prepared in the inoculated agar plates. Standard antibiotic discs (7 mm) from
Bioanalyse® were used as standard antibiotics for comparison with the syn-
thesized compounds. Culture plates were incubated overnight at 37 ◦C. The
experiment was performed in triplicate and the mean values were cited ± error
values.
The cytotoxicity of each compound was investigated against the liver
cancer cell line (HepG2) and normal Skin fibroblast (BJ1) at concen-
trations (0.1, 1, 10, and 100 μM) using MTT colorimetric assay (Table S1
5

F.M. Sroor et al.
Bioorganic Chemistry 112 (2021) 104953
Morphological assessment
Table 3
IC50 (μM) of the tested compounds against HepG2 and BJ1cells.
2.3.1. Gene expression analysis
Comp.
HepG2
BJ1
Expression analysis of liver cancer-related genes including the
Tumor protein p53 exon4 gene (TP53-exon4) and Tumor protein p53
exon7 gene (TP53-exon7) was used in HepG2 Liver cancer cell lines
using RT-qPCR as summarized in Fig. 7. The results revealed that the
expression of TP53-exon4 and TP53-exon7 genes were significantly
increased (P < 0.01) in negative samples of liver cancer cell lines
(HepG2 control (-ve) compared with other treated or positive control of
HepG2 cell lines (Fig. 6). In contrast, the expression values of TP53-
exon4 and TP53-exon7 genes were decreased significantly (P < 0.05)
in HepG2 positive control and treated HepG2 (5) sample compared with
negative control cell lines. These results observed that the expression of
TP53-exon4 and TP53-exon7 genes were expressed in a descending
manner from HepG2 negative control > HepG2 positive control >
HepG2 (5) treated sample (Fig. 6).
5
4.29
7.45
5.48
16.56
ND
25.3
75.3
64.5
24.5
23.2
28.6
21.9
45.1
23.2
70.2
ND
6
7
8
9
10
ND
11
ND
12
11.58
54.60
12.84
8.52
ND
13
14
15
16
21.1
13.2
13.5
ND
17
ND
Doxorubicin
5-Fluorouracil
21.6
316.25
ND: not detected.
2.3.2. Determination of DNA damage using the comet assay
in supplementary data). Data illustrated in (Table 3 and Fig. 5) show the
IC50 values and the percentage of viability of HepG2 and normal cell
lines (BJ1) after 48 h treatment with different concentrations of the
tested compounds versus control. Compounds 5, 7, 6, 15, 12, 14, and 8
showed a very promising cytotoxic effect against HepG2, which was
depicted by low IC50 values (4.29, 5.48, 7.45, 8.52, 11.58. 12.84, and
16.56 µM, respectively) compared to 5-fluorouracil as standard refer-
ence drug (IC50 = 316.25 µM), while compound 13 showed mild cyto-
toxicity (IC50 = 54.60 µM). We could not observe any cytotoxic effect for
compounds 9, 10, 11, 16, and 17. The cytotoxic effect was not a
concentration-dependent, as HepG2 cell growth increased at higher
concentration upon treatment with some of the tested compounds, one
explanation for this could be the cancer cells use a high concentration of
compounds as the carbon source for their metabolic activity. Based on
this result, compound 5 has been chosen as a promising compound to
further validate it. Morphological assessment depicted increasing in cell
lysis, shrinkage, and loose of confluency of HepG2 cells upon treatment
with compound 5 (Photo 1).
The DNA damage in HepG2 Liver cancer cell lines was determined
using comet assay as shown in Table 4 and Fig. 7. The results exhibited
that negative HepG2 liver cancer cell lines showed a significant decrease
(P < 0.01) in DNA damage values (7.78 ± 0.48) compared with the
positive control (25.75 ± 0.86). However, the DNA damage values were
increased significantly (P < 0.01) in the treated HepG2 (5) sample
(23.28 ± 0.63) compared with the negative control. Furthermore, the
DNA damage values increased in ascending manner form HepG2 nega-
tive control < HepG2 positive control < HepG2 (5) treated cell lines
(Table 4).
2.3.3. Measurement of DNA fragmentation
Assessment of the rate of DNA fragmentation in HepG2 Liver cancer
cell lines is summarized in Table 5 and Figs. 8 and 9. The results found
that negative HepG2 liver cancer cell lines exhibited a significant
decrease (P < 0.01) in DNA fragmentation rates (7.9 ± 0.38) compared
with those in the positive control (21.6 ± 0.35). However, the DNA
Fig. 5. Cytotoxic effect of the tested compounds against liver cancer HepG2 cells: 1x104 cell/well of were treated with serial dilution of the tested compounds (0.1, 1,
10, and 100 µM) for 48 h cytotoxic effect was detected by MTT assay. The figure shows the % of cell growth viability compared to control which were untreated cell
(n = 3).
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F.M. Sroor et al.
Bioorganic Chemistry 112 (2021) 104953
4.00
3.50
3.00
2.50
2.00
1.50
1.00
0.50
0.00
a
b
b
HepG2 control (-ve)
HepG2 (5)
HepG2 control (+ve)
Treatment
4.00
a
3.50
3.00
2.50
2.00
1.50
1.00
0.50
0.00
b
b
HepG2 control (-ve)
HepG2 (5)
HepG2 control (+ve)
Treatment
Fig. 6. The alterations of TP53-exon7 and TP53-exon4 genes in liver cancer cell lines treated with 5 (4.29 µM). Data are presented as mean ± SEM. a,bMean values
within tissue with unlike superscript letters were significantly different (a: P < 0.01, b P < 0.05).
Table 4
Visual score of DNA damage in HepG2 (liver tumor cell lines) treated with 5
(4.29 µM).
Treatment
No. of cells
Class**
DNA damaged
cells % (Mean
± SEM)
Normal cell
(Class 0)
Class 2
Analyzed
*
Comets
31
0
1
2
8
3
0
7.78 ± 0.48b
Class 3
HepG2
control
(-ve)
400
369
23
HepG2 (5)
HepG2
control
(+ve)
400
400
93
307
297
35
36
38
42
20
25
23.28 ± 0.63a
25.75 ± 0.86a
103
Class 1
*
Number of cells examined per a group,
Fig. 7. A visual score of normal DNA (class 0, class 1, 2 and class 3) using
comet assay in liver cancer cell lines exposed to 5.
**
Class 0 = no tail; 1 = tail length < diameter of nucleus; 2 = tail length
between 1X and 2X the diameter of nucleus; and 3 = tail length > 2X the
diameter of nucleus.
7

F.M. Sroor et al.
Bioorganic Chemistry 112 (2021) 104953
(Table S6 (see supplementary data). In addition, Compound 5 formed 3
hydrogen bond interactions with the key amino acid residues with CDK2
protein (LYS33: 3.132 Å, ASN132: 2.143 Å, LYS129: 3.963 Å) (Table S4
(see supplementary data), Fig. 12, and hydrophobic interaction through
ILE10, VAL18, VAL64, LEU148, LEU134, LEU83, PHE82 (Table S7 (see
supplementary data).
Table 5
DNA fragmentation detected in liver cancer cell lines treated with 5 (4.29 µM).
Treatment
DNA Fragmentation % M ± SEM
Change
Inhibition
HepG2 control (-ve)
HepG2 (5)
7.9 ± 0.38b
19.8 ± 0.55a
21.6 ± 0.35a
0
0
12.5
14.3
12.587
0
HepG2 control (+ve)
Based on these in silico studies, we are proposing two main targets
(DNAG, and PBP1) as mode of action of compound 5 as promising
antibacterial agents, while compound 5 may act as promising anticancer
agent via inhibition of CDK2. Further wet lab work should be done for
validation of these molecular studies.
Means with different superscripts (a and b) between locations in the same col-
umn are significantly different at P < 0.05.
fragmentation rates were increased significantly (P < 0.01) in the
treated HepG2 (5) sample (19.8 ± 0.55) compared with the negative
control. Moreover, the DNA 19.8 ± 0.55 increased in ascending manner
form HepG2 negative control < HepG2 positive control < HepG2 (5)
treated cell lines (Table 5).
3. Conclusions
In summary, the purpose of this article was to prepare a new alkyl
and aryl urea derivatives and study their biological activity as anti-
microbial and anti-cancer agents. The targeted urea compounds were
prepared in facile protocol and free-catalyst condition by the reaction of
2.4. Molecular docking
To gain insights the mode of action of the tested compounds,
molecular-docking study was employed to determine the binding modes
against (DNAG), and (PBP1a) which are important targets to develop
antibacterial agents. These targets were selected based on its key roles in
bacterial cell formation, therefore, targeting of these proteins provides
potential benefits in killing bacteria. The cocrystal ligand was redocked
to assure the validity of the docking parameters and methods using
AutoDockvina to represent the position and orientation of the ligand
detected in the crystal structure. The difference of RMSD value between
cocrystal ligands to the original cocrystal ligand was < 2 Å which
approved the accuracy of the docking protocols and parameters.
Compound 5 considered to be one of the best docked compounds
against the tested proteins, as it has the lowest free energy of binding
(-8.5 Kcal/mol, ꢀ 8.6 Kcal/mol, and ꢀ 9.3 Kcal/mol) against DNAG,
PBP1a, and CDK2 proteins respectively, compared to reference ligands
(-9.3 Kcal/mol, ꢀ 7.2 Kcal/mol, and ꢀ 8.3 Kcal/mol) respectively
(Table S8, see supplementary data). As depicted in (Table S2, see sup-
plementary data), and Fig. 10, Compound 5 formed 3 hydrogen bond
interactions with the active site of DNAG protein (ASN46: 3.074 Å,
ASN46: 2.930 Å, VAL120: 3.063 Å), and hydrophobic interaction
through VAL43, VAL71, VAL93, VAL118, VAL120, VAL167, ILE90,
ILE78, ILE52 (Table S5 (see supplementary data). Moreover, Compound
5 formed 4 hydrogen bond interactions like co-crystalized ligand with
the active site of PBP1 protein (THR670: 2.718 Å, SER487: 3.206 Å,
SER487: 3.136 Å, SER434: 3.138 Å) (Table S3, see supplementary data),
Fig. 11, and hydrophobic interaction through ILE710, LEU486, LEU526
M
1
2
3
1500
bp
1000
bp
500
bp
250
bp
50
bp
Fig. 9. DNA fragmentation detected with Agarose gel in Liver cancer cell lines
(HepG2) treated with 5 (4.29 µM). M: represent DNA marker, Lanes 1: repre-
sents HepG2 control (-ve), Lane 2: represents HepG2 (5), Lane 3: represents
HepG2 control (+ve).
25
20
15
a
a
10
5
b
0
Treated groups
Fig. 8. The alterations of DNA fragmentation in liver cancer cell lines treated with 5 (4.29 µM). Data are presented as mean ± SEM. a,bMean values within tissue with
unlike superscript letters were significantly different (a: P < 0.01, b P < 0.05).
8

F.M. Sroor et al.
Bioorganic Chemistry 112 (2021) 104953
RL
5
Fig. 10. 3D interaction, and hydrogen bond formation, between reference ligand (RL), and tested compound 5 with DNAG protein.
the isocyanates with the primary amines in acetonitrile at room tem-
perature to produce the required products. The synthesized derivatives
confirm effective antimicrobial activities in opposition to a variety of
microbial strains. Likewise, the prepared compounds showed very good
activity against liver cancer HepG2 cell line. Further on, to gain insight
the mode of action of the synthetic compounds as antibacterial and
anticancer agents, docking studies were performed for the synthetic
compounds against 2 bacterial proteins (DNA gyrase subunit B, and
penicillin binding protein 1a) that are known targets for some antibi-
otics, and one cell division protein kinase 2 (CDK2) as target for anti-
cancer drugs. Our results revealed that compound 5 is one of the best
docked compound against the three target protein. The data obtained
results may well assist in the drug design of novel antimicrobial and
anticancer agents to face rising microbial resistance problems to treat
various microbial and cancer diseases types.
starting materials 1, 2, 3, and 4 have been prepared as described in the
literatures [24–26]. All melting points are uncorrected and measured
using Electro-Thermal IA 9100 apparatus (Shimadzu, Japan). The
Infrared spectra were recorded as potassium bromide pellets on a JASCO
spectrophotometer between 4000 cmꢀ 1 and 400 cmꢀ 1. 1H NMR and 13
C
NMR spectra were recorded in deuterated dimethyl sulfoxide (DMSO‑d6)
on a Brucker spectrometer (400 MHz) at 25 ◦C. The chemical shifts were
expressed as part per million (δ values, ppm) against TMS as an internal
reference. Microanalyses were operated using Mario Elmentar appa-
ratus, Organic Microanalysis Unit, National Research Centre, Cairo,
Egypt.
4.1. General procedure of synthesis of urea derivatives (5–17)
To a solution of requisite amines (0.01 mol) in acetonitrile, the
corresponding isocyanate derivatives (0.01 mol) was added while stir-
ring at room temperature. The reaction mixture was stirred to the
desired time. The completion of reactions was monitored by TLC on
silica gel coated aluminum sheets. The obtained precipitate was filtered
off, washed with cold (two times) dried well, and recrystallized from
ethyl acetate/acetonitrile (3:1) to give:
4. Experimental section
All reactions were carried out in aerobic conditions at room tem-
perature. Acetonitrile was distilled and kept under an inert atmosphere.
All glassware was oven-dried at 120 ◦C for at least 24 h before use. The
4-tolylsulfonylisocyanate, 4-tolylisocyanate, benzoylisocyanat, and
ethyl isocyanate were purchased from Aldrich and used as received. The
9

F.M. Sroor et al.
Bioorganic Chemistry 112 (2021) 104953
RL
5
Fig. 11. 3D interaction, and hydrogen bond formation, between reference ligand (RL), and tested compound 5 with PBP1a protein.
4.1.1. 4-methyl-N-{[3-propionyl-4,5,6,7-tetrahydrobenzo[b]thiophen-2-
Ar-H), 2.38 (3H, s, CH3), 2.23 (6H, s, CH3); 13C NMR (400 MHz,
DMSO‑d6) δ ppm: 149.8, 144.5, 142.3, 141.9, 137.3, 130.8, 130.0,
129.8, 128.0, 126.1, 118.3, 112.3, 23.6, 21.5. EI/MS (m/z) 475 (M).
Anal. For C20H21N5O5S2: C, 50.52; H, 4.45; N, 14.73. Found: C, 50.68; H,
4.62; N, 15.02.
yl]carbamoyl}-benzene-sulfonamide (5)
White solid; m.p.: 130–132 ◦C; IR (KBr, cmꢀ 1): 3331, 3275 (NH),
1
–
1684 (C O), 1342, 1163 (SO ); H NMR (400 MHz, DMSO‑d ) δ ppm:
–
10.80 (1H, s, NH), 7.86 (2H, d,2J = 8 Hz, Ar-H), 7.46 (2H, d, J =68 Hz, Ar-
H), 4.20 (2H, m, CH2, Et), 3.86 (4H, m, CH2, C6H8), 2.61 (4H, m, CH2,
C6H8), 2.40 (3H, s, CH3), 1.26 (3H, t, CH3). Anal. For C19H22N2O4S2:
Calcd. C, 56.14; H, 5.46; N, 6.89. Found: C, 55.70; H, 6.00; N, 6.98.
4.1.4. N-{[2,4-dichlorophenyl]carbamoyl}-4-methylbenzenesulfonamide
(8) [27]
White solid; m.p.: 203–204 ◦C; IR (KBr, cmꢀ 1): 3322, 3278 (NH),
1
–
4.1.2. N-carbamimidoyl-4-{3-tosylureido}benzenesulfonamide (6)
1680 (C O), 1330, 1161 (SO ); H NMR (400 MHz, DMSO‑d ) δ ppm:
–
2
White solid; m.p.: 206–208 ◦C; IR (KBr, cmꢀ 1): 3317, 3282 (NH),
11.33 (1H, s, br, NH, exchangeable in D2O), 8.48 (1H6, s, NH,
exchangeable in D2O), 7.87 (3H, m, Ar-H), 7.58 (1H, s, Ar-H), 7.28–7.43
(3H, m, Ar-H), 2.37 (3H, s, CH3); 13C NMR (400 MHz, DMSO‑d6) δ ppm:
144.1, 142.6, 141.4, 129.8, 128.5, 128.1, 126.1, 119.7, 118.0, 116.7,
21.3. Anal. For C14H12Cl2N2O3S: Calcd. C, 46.81; H, 3.37; N, 7.80.
Found: C, 47.10; H, 4.12; N, 7.96.
1
–
1680 (C O), 1350, 1162 (SO ); H NMR (400 MHz, DMSO‑d ) δ ppm:
–
2
9.14 (1H, s, NH), 7.87 (2H, d, J = 8 Hz, Ar-H), 7.72 (2H, d, J =68 Hz, Ar-
H), 7.65 (2H, d, J = 8 Hz, Ar-H), 7.30–7.45 (5H, m, Ar-H + NH), 6.67
(3H, s, br, NH + NH2), 2.40 (3H, s, CH3); 13C NMR (400 MHz, DMSO‑d6)
δ ppm: 158.2, 151.8, 149.8, 142.3, 141.9, 139.3, 137.4, 131.3, 130.0,
129.8, 128.0, 127.7, 127.1, 126.1, 118.7, 112.8, 21.4. Anal. For
C
15H17N5O5S2: Calcd. C, 43.79; H, 4.16; N, 17.02. Found: C, 44.01; H,
4.1.5. N-{[2,4-dichlorophenyl]carbamoyl}benzamide (9)
4.55; N, 17.82.
White solid; m.p.: 225–227 ◦C; IR (KBr, cmꢀ 1): 3314, 3282 (NH),
1
–
1683 (C O), 1341, 1162 (SO ); H NMR (400 MHz, DMSO‑d ) δ ppm:
–
2
6
4.1.3. N-{4,6-dimethylpyrimidin-2-yl}-4-{3-tosylureido}
11.88 (1H, s, NH), 11.50 (1H, s, NH), 8.00 (3H, m, Ar-H), 7.47–7.73 (5H,
m, Ar-H); 13C NMR (400 MHz, DMSO‑d6) δ ppm: 134.5, 133.8, 133.0,
129.4, 129.2, 129.1, 128.9, 128.4, 128.1, 124.0, 122.9. EI/MS (m/z) 408
(M). Anal. For C14H10Cl2N2O2: Calcd. C, 54.39; H, 3.26; N, 9.06. Found:
C, 54.66; H, 3.90; N, 8.94.
benzenesulfonamide (7)
White solid; m.p.: 212–214 ◦C; IR (KBr, cmꢀ 1): 3320, 3262 (NH),
1
–
1681 (C O), 1341, 1160 (SO ); H NMR (400 MHz, DMSO‑d ) δ ppm:
–
2
6
11.54 (1H, s, br, NH), 10.95 (1H, s, br, NH), 9.23 (1H, s, NH), 7.84–7.90
(3H, m, Ar-H), 7.69 (1H, m, Ar-H), 7.30–7.51 (4H, m, Ar-H), 6.43 (1H, s,
10

F.M. Sroor et al.
Bioorganic Chemistry 112 (2021) 104953
RL
5
Fig. 12. 3D interaction, and hydrogen bond formation, between reference ligand (RL), and tested compound 5 with CDK2 protein.
4.1.6. 1-{2,4-dichlorophenyl}-3-{4-tolyl}urea (10) [29]
132.1, 131.7, 128.8, 128.7, 128.2, 127.9, 121.3, 109.6. Anal. Calcd. for
C17H12BrN3O2S (402.27): C, 50.76; H, 3.01; N, 10.45; Found: C, 50.85;
H, 2.96; N, 10.33%. Anal. For C17H12BrN3O2S: Calcd. C, 50.76; H, 3.01;
N, 10.45. Found: C, 50.93; H, 3.65; N, 10.95.
White solid; m.p.: 240–241 ◦C; IR (KBr, cmꢀ 1): 3312, 3280 (NH),
1682 (C O); 1H NMR (400 MHz, DMSO‑d6) δ ppm: 9.34 (1H, s, NH),
–
–
8.34 (1H, s, NH), 8.21 (1H, d, J = 8 Hz, Ar-H), 7.60 (1H, s, Ar-H), 7.36
(3H, m, Ar-H), 7.1 (2H, d, J = 8 Hz, Ar-H), 2.25 (3H, s, CH3); 13C NMR
(400 MHz, DMSO‑d6) δ ppm: 152.5, 137.1, 136.8, 131.7, 129.8, 129.6,
129.0, 128.1, 126.4, 123.0, 122.5, 118.8, 20.8. Anal. For C14H12Cl2N2O:
Calcd. C, 56.97; H, 4.10; N, 9.49. Found: C, 57.13; H, 4.44; N, 9.82.
4.1.10. 1-{4-[4-bromophenyl]thiazol-2-yl}-3-{p-tolyl}urea (14)
White solid; m.p.: 190–192 ◦C; IR (KBr, cmꢀ 1): 3315, 3285 (NH),
1
–
1683 (C O); H NMR (400 MHz, DMSO‑d ) δ ppm: 10.65 (1H, s, NH),
–
6
8.81 (1H, s, NH), 7.84 (2H, d, J = 8 Hz, Ar-H), 7.61 (3H, d, J = 8 Hz, Ar-
H), 7.37 (2H, d, J = 8 Hz, Ar-H), 7.13 (1H, d, J = 8 Hz, Ar-H), 2.26 (3H, s,
CH3); 13C NMR (400 MHz, DMSO‑d6) δ ppm: 159.8, 152.0, 148.0, 136.3,
134.0, 132.1, 129.8, 128.1, 121.2, 119.3, 108.4, 20.8. Anal. For
4.1.7. 1-{2,4-dichlorophenyl}-3-ethylurea (11) [34]
White solid; m.p.: 214–215 ◦C; IR (KBr, cmꢀ 1): 3322, 3271 (NH),
1
–
1685 (C O); H NMR (400 MHz, DMSO‑d ) δ ppm: 8.19 (1H, d, J = 8
–
6
Hz, Ar-H), 8.06 (1H, s, NH), 7.54 (1H, s, Ar-H), 7.31 (1H, d, J = 8 Hz, Ar-
H), 7.03 (1H, s, br, NH) 3.12 (2H, m, CH2), 1.07 (3H, t, J = 4 Hz, CH3).
EI/MS (m/z) 232 (M). Anal. For C9H10Cl2N2O: Calcd. C, 46.38; H, 4.32;
N, 12.02. Found: C, 46.13; H, 4.61; N, 12.22.
C17H14BrN3OS: Calcd. C, 52.59; H, 3.63; N, 10.82. Found: C, 53.00; H,
3.84; N, 10.98.
4.1.11. 1-ethyl-3-{thiazol-2-yl}urea (15) [14]
White solid; m.p.: 180–182 ◦C; IR (KBr, cmꢀ 1): 3312, 3283 (NH),
1
–
4.1.8. N-{thiazol-2-ylcarbamoyl}benzamide (12)
1684 (C O); H NMR (400 MHz, DMSO‑d ) δ ppm: 10.33 (1H, s, NH),
–
White solid; m.p.: 198–199 ◦C; IR (KBr, cmꢀ 1): 3319, 3282 (NH),
7.28 (1H, d, J = 4 Hz, CH-thiazole), 7.00 (16H, d, J = 4 Hz, CH-thiazole),
1
1684 (C O); H NMR (400 MHz, DMSO‑d ) δ ppm: 11.91 (1H, s, NH),
6.50 (1H, s, NH), 3.14 (2H, m, CH2), 1.06 (3H, t, J = 4 Hz, CH3); 13
C
–
–
6
11.48 (1H, s, NH), 8.05 (2H, d, J = 12 Hz, Ar-H), 7.48–7.70 (4H, m, Ar-
H), 7.30 (1H, s, Ar-H). Anal. For C11H9N3O2S: Calcd. C, 53.43; H, 3.67;
N, 16.99. Found: C, 53.48; H, 3.81; N, 17.11.
NMR (101 MHz, DMSO‑d6) δ ppm: 160.1, 153.7, 137.2, 111.6, 34.1,
15.2. Anal. Calcd. for C6H9N3OS (171.22): C, 42.09; H, 5.30; N, 24.54;
Found: C, 41.95; H, 4.99; N, 24.17%. Anal. For C6H9N3OS: Calcd. C,
42.09; H, 5.30; N, 24.54. Found: C, 42.81; H, 5.45; N, 25.00.
4.1.9. N-{[4-(4-bromophenyl)thiazol-2-yl]carbamoyl}benzamide (13)
[14]
4.1.12. N-{[5-methylisoxazol-3-yl]carbamoyl}benzamide (16) [14]
White solid; m.p.: 182–183 ◦C; IR (KBr, cmꢀ 1): 3310, 3284 (NH),
White solid; m.p.: 208–210 ◦C; IR (KBr, cmꢀ 1): 3318, 3286 (NH),
1
1
–
–
–
–
1686, 1696 (C O); H NMR (400 MHz, DMSO‑d ) δ ppm: 9.82 (1H, s,
1685, 1695 (C O); H NMR (400 MHz, DMSO‑d ) δ ppm: 11.30 (1H, s,
6
6
br, NH), 8.85 (1H, s, br, NH), 7.37–8.16 (10H, m, Ar-H);13C NMR (400
MHz, DMSO‑d6) δ ppm: 174.8, 168.4, 158.6, 149.5, 135.0, 134.7, 134.0,
NH), 11.19 (1H, s, NH) , 8.02 (2H, m, Ar-H), 7.67 (1H, m, Ar-H), 7.55
(2H, m, Ar-H), 6.66 (1H, s, CH-oxazole), 2.41 (3H, s, CH3); 13C NMR
11

F.M. Sroor et al.
Bioorganic Chemistry 112 (2021) 104953
Contro
0.1μM
10μM
1μM
100 μM
Photo 1. Effect of compound 5 versus control on HepG2 cell line morphology. Black arrows indicate shrinkage, and cell lysis.
(101 MHz, DMSO‑d6) δ ppm: 170.8, 169.6, 157.8, 151.0, 133.8, 132.5,
129.1, 96.7, 12.6. EI/MS (m/z) 245 (M). Anal. For C12H11N3O3: Calcd. C,
58.77; H, 4.52; N, 17.13. Found: C, 59.14; H, 4.81; N, 17.96.
agar medium was prepared by suspending 28 g of the ready medium in
1.0 L of distilled water, and then the pH value was adjusted at 7.0. The
previously mentioned media were sterilized by autoclaving under 1.5
atmosphere for 20 min at 121 ◦C.
4.1.13. 1-{5-methylisoxazol-3-yl}-3-{4-tolyl}urea (17) [14]
White solid; m.p.: 190–192 ◦C; IR (KBr, cmꢀ 1): 3314, 3289 (NH),
4.2.2. Antimicrobial activity assessment
1686 (C O); 1H NMR (400 MHz, DMSO‑d6) δ ppm: 9.40 (1H, s, NH),
The efficiency of the synthesized compounds as antimicrobial agents
was assessed applying the agar diffusion method in opposition to four
pathogenic microorganisms [35]. The microbial cultures were seeded in
–
–
8.72 (1H, s, NH), 7.34 (2H, d, J = 8 Hz, Ar-H), 7.10 (2H, d, J = 8 Hz, Ar-
H), 6.54 (1H, s, CH-oxazole), 2.36 (3H, s, CH3), 2.25 (3H, s, CH3); 13
C
NMR (101 MHz, DMSO‑d6) δ ppm: 169.6, 159.2, 151.8, 136.8, 131.9,
the nutrient agar medium (70148 Nutrient agar, Fluka) using 100
μ
l of
129.7, 119.1, 97.0, 20.8, 12.5. EI/MS (m/z) 232 (M+). Anal. Calcd. for
re-suspended overnight culture at 37 ◦C (1 × 107 CFU/100
μl) as follow:
C
12H13N3O2 (231.26): C, 62.33; H, 5.67; N, 18.17; Found: C, 62.03; H,
The nutrient agar medium was poured in a set of Petri dishes after
adding the specified microbial inoculums to the melted medium at
5.41; N, 17.84%.
around 45 ◦C. The plates left to solidify at room temperature then 100
μl
of dissolved compounds (0.1 mM) in DMSO were applied to 12 mm holes
prepared in the inoculated agar plates. Standard antibiotic discs (7 mm)
(Colstin 10 mcg, Tobramycin 10 mcg, Gentamicin 10 mcg, Ampicillin 10
mcg, Streptomycin 10 mcg, and Erythromycin 15 mcg) from Bio-
analyse® were also used as standard antibiotics for comparison with the
synthesized compounds. Culture plates were then incubated at 37 ◦C for
24 h, then the developed inhibition zones were measured [36–38]. To
determine the minimum inhibitory concentration (MIC), the test sam-
ples were serially diluted using DMSO to get a range of concentrations
from 50 to 0.0122 µM. The MIC was determined as the lowest concen-
tration able to inhibit the microbial visible growth [39].
4.2. Anti-microbial evaluation
4.2.1. Microorganisms and media
Escherichia coli, Bacillus mycoides, Staphylococcus aureus, and Candida
albicans were maintained on modified nutrient agar medium slants (g/
L): Peptone, 3; yeast extract, 1.5; meat extract, 1.5; Glucose, 0.5; NaCl,
0.25 and Agar, 20.0 at pH 7.0. Through applying agar diffusion method
for antimicrobial assessment, the microbial strains were seeded and
grown at 37 ◦C on nutrient agar medium (70148 Nutrient agar, Fluka,
Spain) with the following components (g/L): Peptone, 5.0; yeast extract,
2.0; meat extract, 1.0; sodium chloride, 5.0; and agar, 15.0. Nutrient
12

F.M. Sroor et al.
Bioorganic Chemistry 112 (2021) 104953
4.3. Anti-cancer evaluation
Table 6
Primers sequence used for RT-qPCR.
4.3.1. Cell cultures
Gene
Forward
Reverse
A human hepatocellular carcinoma (HepG2) cells were propagated
in RPMI-1640 medium L-Glutamine (Lonza Verviers SPRL, Belgium,
cat#12-604F), and supplemented with 10% fetal bovine serum (FBS)
(Seralab, UK, cat# EU-000-H), and 1% antibiotic (Antibiotic anti-
mycotic, Biowest, cat#). The cells were incubated in 5% CO2 humidified
at 37 ◦C for growth.
TP53-
5′- TGA GGA CCT GGT CCT CTG
5′- AGA GGA ATC CCA AAG TTC
exon4
AC -3′
CA -3′
TP53-
exon7
β-actin
5′- CTT GCC ACA GGT CTC CCC
5′- AGG GGT CAG AGG CAA GCA
AA -3′
GA-3′
5′-CTG GAA CGG TGA AGG TGA
CA-3′
5′-CGG CCA CAT TGT GAA CTT
TG-3′
TP53-exon4: Tumor protein p53 exon4; TP53-exon7: Tumor protein p53 exon7.
4.3.2. Evaluation of cell proliferation by MTT assay
The cytotoxic effect of the tested compounds on two cancer cell lines
was evaluated by the MTT (3-[4,5-methylthiazol-2-yl]-2,5-diphenyl-
tetrazolium bromide) assay as reported previously with slight modifi-
cation [40]. In brief, after evaluation of cell count and viability by try-
pan blue dye, cancer cells (1x104 cells/well) were seeded in a 96-well
plate in triplicate and were allowed to adhere for 24 h. The tested
compounds were dissolved in 500 µl Dimethyl sulfoxide (DMSO) to have
a stock solution of 100 mM, as the final concentration of DMSO in the
culture medium never exceeded 0.2% (v/v)[41] and then various con-
centrations of tested compounds were prepared by further diluting in
complete medium to have a final concentration of (0.1, 1, 10, and 100
µM). On the next day, the medium was replaced with fresh medium with
the indicated concentrations of tested compounds, and cells were
allowed to grow for 48 h. Four hours before completion of incubation,
Homogenized liver tumor cell lines (HepG2) samples of treated groups
were mixed with low melting point agarose and loaded in small pieces
on slides which pre-coated with normal melting agarose. The loaded
samples were kept on the slides in a horizontal position for a half-hour in
a dark environment at 4 ◦C. Low melting point agarose was then
pipetting above the slides including samples and the slides were left for
30 min at 4 ◦C to harden and put afterward in lysis buffer for one hour.
Then, a fresh alkaline unwinding buffer was used to submerge the slides
in a dark place for one hour at 20 ◦C. Afterward, electrophoresis (0.8 V/
cm, 300mAmps) for the slides were carried out for 30 min to assess the
DNA damage in the form of tail migration in 100 cell per each animal.
Specific software (TriTek corp., Comet Score, Sumerduck, VA22742)
was used to determine the rate of the DNA damage per sample [44].
10
μl of MTT (5 mg/mL in PBS w/o Ca, Mg, Lonza Verviers SPRL
4.3.6. DNA fragmentation assay
Belgium, cat#17-516F) was added in each well. After completing the
Fragmentation of apoptotic DNA was carried out by detecting the
laddering pattern according to Yawata [45] with some modifications.
After obtaining the isolated DNA from liver cancer cell lines, the elec-
trophoresis of the DNA was done on 1.5% agarose gel with ethidium
bromide dye. A DNA ladder (Invitrogen, USA) was used as a molecular
size marker and the fragments of the DNA were visualized by exposing
the gels to UV transillumination.
incubation, 100 μl of Dimethyl sulfoxide (DMSO) was added to each
well, then the 96 well plates were centrifuged for 5 min at 4000 rpm to
precipitate the formazan crystals. The color developed after the reaction
was measured at 490 nm using a Bio-Tek microplate reader. The
experiment was conducted in triplicate.
Data were calculated as a percent of cell viability by the following
formula: % cell viability = (Mean absorbance in test wells / Mean
absorbance in control wells) × 100. The effect of tested compounds on
the morphology of treated lung cancer cells was investigated by the light
microscope and then photographed by SONY CYBER-SHORT [42].
4.4. Molecular docking study
The structures of all tested compounds were modeled using the
Chemsketch software (http://www.acdlabs.com/resources/freeware/).
The structures were optimized and energy minimized using the VEGAZZ
software [46]. The optimized compounds were used to perform molec-
ular docking. The three-dimensional structures of the molecular target
were obtained from Protein Data Bank (PDB) (www.rcsb.org): DNA
gyrase subunit b Center (DNAG) (PDB: 1KZN, https://www.rcsb.
org/structure/1KZN), and penicillin binding protein 1a (PBP1a) (PDB:
3UDI, https://www.rcsb.org/structure/3UDI). Cell division protein ki-
nase 2 (CDK2) (PDB: 1DI8, https://www.rcsb.org/structure/1di8). The
steps for receptor preparation included the removal of heteroatoms
(water and ions), the addition of polar hydrogen, and the assignment of
charge. The active sites were defined using grid boxes of appropriate
sizes around the bound cocrystal ligands. The docking study was per-
formed using Autodock vina [47] and Chimera for visualization [48].
4.3.3. Gene expression analysis
TRIzol® extraction Chemical (Invitrogen) was utilized to isolate the
total genomic RNA of liver cancer cell lines (HepG2) of all treated
groups. After completion of the isolation procedures, the RNA pellet was
stored in DEPC treated water. To digest the potential DNA residues the
pellet of isolated RNA was treated with an RNAse-free DNAse kit
(Invitrogen, Germany). RNA aliquots were stored at ꢀ 20 ◦C or utilized
immediately for reverse transcription.
First Strand cDNA Synthesis Kit (RevertAidTM, MBI Fermentas) was
used to synthesize the cDNA copy from liver cancer cell lines (HepG2)
via reverse transcription reaction (RT). ART reaction program of 25 ◦C
for 10 min, then one hour at 42 ◦C then 5 min at 95 ◦C was used to obtain
the cDNA copy of the liver genome. Finally, tubes of a reaction con-
taining cDNA copy were collected on ice up to use for cDNA
amplification.
4.3.4. Quantitative real Time- PCR (qPCR)
4.5. Single crystal X-ray diffraction
SYBR® Premix Ex TaqTM kit (TaKaRa, Biotech. Co. Ltd.) was used to
perform the qRT-PCR analyses using the synthesized cDNA copies from
liver cancer cell lines (HepG2). For each reaction, a melting curve profile
was conducted. The quantitative values of the target genes were
normalized on the expression of the housekeeping gene (Table 6) ac-
cording to Qiu et al. (2016). The 2ꢀ ΔΔCT method was used to determine
the quantitative values of the specific genes to the β-actin gene.
A single Crystals suitable for X-ray diffraction analysis for com-
pounds 1 and 3 were obtained from a saturated ethyl acetate/acetoni-
trile solution at room temperature. The X-ray crystal structures were
determined by using a Rigaku R-AXISRAPID diffractometer and Bruker
X8 Prospector. The collection of single-crystal data was made at room
temperature by using Cu-Kα radiation (see supplementary data). The
structures were solved by using direct methods and expanded using
Fourier techniques. The non-hydrogen atoms were refined anisotropi-
cally [49]. The CCDC numbers of 1 and 3 are 1,896,799 and 1896796,
respectively. More details could be found in supplementary data.
4.3.5. DNA damage using the comet assay
The DNA damage in treated liver tumor cell lines (HepG2) samples
using comet assay was performed according to Olive et al. [43]
13

F.M. Sroor et al.
Bioorganic Chemistry 112 (2021) 104953
ligand set, including a “giant neodymium wheel”, Dalton T 45 (2016)
Declaration of Competing Interest
13332–13346.
[21] F.M. Sroor, C.G. Hrib, L. Hilfert, P.G. Jones, F.T. Edelmann, Lanthanide(III)-bis
(cyclopropylethinylamidinates): Synthesis, structure, and catalytic activity,
J Organomet Chem 785 (2015) 1–10.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
[22] F.M. Sroor, C.G. Hrib, L. Hilfert, L. Hartenstein, P.W. Roesky, F.T. Edelmann,
Synthesis and structural characterization of new bis(alkynylamidinato)lanthanide
(III)-amides, J. Organomet. Chem. 799–800 (2015) 160–165.
[23] F.M. Sroor, C.G. Hrib, L. Hilfert, S. Busse, F.T. Edelmann, Synthesis and catalytic
activity of homoleptic lanthanide-tris(cyclopropylethinyl)amidinates, New J.
Chem. 39 (2015) 7595–7601.
Acknowledgments
This work was supported by the National Research Centre (NRC)
under project number 12010102.
[24] E.F. Silva-Júnior, E.P.S. Silva, P.H.B. França, J.P.N. Silva, E.O. Barreto, E.B. Silva,
R.S. Ferreira, C.C. Gatto, D.R.M. Moreira, J.L. Siqueira-Neto, F.J.B. Mendonça-
Júnior, M.C.A. Lima, J.H. Bortoluzzi, M.T. Scotti, L. Scotti, M.R. Meneghetti, T.
M. Aquino, J.X. Araújo-Júnior, Design, synthesis, molecular docking and biological
evaluation of thiophen-2-iminothiazolidine derivatives for use against
Trypanosoma cruzi, Bioorg. Med. Chem. 24 (2016) 4228–4240.
[25] Z. Abedi-Jazini, J. Safari, Z. Zarnegar, M. Sadeghi, A Simple and Efficient Method
for the Synthesis of 2-Aminothiazoles Under Mild Conditions, Polycyclic Aromat.
Compd. 38 (2016) 231–235.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104953.
[26] C.T. Supuran, A. Scozzafava, F. Briganti, B.W. Clare, Protease Inhibitors: Synthesis
and QSAR Study of Novel Classes of Nonbasic Thrombin Inhibitors Incorporating
Sulfonylguanidine andO-Methylsulfonylisourea Moieties at P1, J. Med. Chem. 43
(2000) 1793–1806.
References
[1] I. Chopra, J. Hodgson, B. Metcalf, G. Poste, The search for antimicrobial agents
effective against bacteria resistant to multiple antibiotics, Antimicrob. Agents Ch.
41 (1997) 497–503.
[27] J.J. Howbert, C.S. Grossman, T.A. Crowell, B.J. Rieder, R.W. Harper, K.E. Kramer,
E.V. Tao, J. Aikins, G.A. Poore, Novel agents effective against solid tumors: the
diarylsulfonylureas. Synthesis, activities, and analysis of quantitative structure-
activity relationships, J. Med. Chem. 33 (1990) 2393–2407.
´
ˇ
´
´
ˇ
´
[2] M. Kratký, J. Vinsova, M. Volkova, V. Buchta, F. Trejtnar, J. Stolaríkova,
Antimicrobial activity of sulfonamides containing 5-chloro-2-hydroxybenzalde-
hyde and 5-chloro-2-hydroxybenzoic acid scaffold, Eur. J. Med. Chem. 50 (2012)
433–440.
[28] C.B. Mishra, S. Kumari, A. Angeli, S.M. Monti, M. Buonanno, A. Prakash, M. Tiwari,
C.T. Supuran, Design, synthesis and biological evaluation of N-(5-methyl-isoxazol-
3-yl/1,3,4-thiadiazol-2-yl)-4-(3-substitutedphenylureido) benzenesulfonamides as
human carbonic anhydrase isoenzymes I, II, VII and XII inhibitors, J. Enzyme Inhib.
Med. Chem. 31 (2016) 174–179.
[3] T.S. Huang, C.M. Kunin, B.S. Yan, Y.S. Chen, S.S.J. Lee, W. Syu, Susceptibility of
Mycobacterium tuberculosis to sulfamethoxazole, trimethoprim and their
combination over a 12 year period in Taiwan, J. Antimicrob. Chemother. 67 (2011)
633–637.
[29] Y. Zhang, M. Anderson, J.L. Weisman, M. Lu, C.J. Choy, V.A. Boyd, J. Price,
M. Sigal, J. Clark, M. Connelly, F. Zhu, W.A. Guiguemde, C. Jeffries, L. Yang,
A. Lemoff, A.P. Liou, T.R. Webb, J.L. DeRisi, R.K. Guy, Evaluation of Diarylureas
for Activity Against Plasmodium falciparum, ACS Med. Chem. Lett. 1 (2010)
460–465.
[4] D.-Q. Song, Y. Wang, L.-Z. Wu, P. Yang, Y.-M. Wang, L.-M. Gao, Y. Li, J.-R. Qu, Y.-
H. Wang, Y.-H. Li, N.-N. Du, Y.-X. Han, Z.-P. Zhang, J.-D. Jiang, Benzoylurea
Derivatives as a Novel Class of Antimitotic Agents: Synthesis, Anticancer Activity,
and Structureꢀ Activity Relationships, J. Med. Chem. 51 (2008) 3094–3103.
[5] L.S. Abovyan, V.A. Shkulev, N.O. Stepanyan, A.S. Agaronyan, O.L. Mndzhoyan,
Synthesis and hypoglycemic activity of N-(5-carboalkoxyfuran-2-sulfonyl)-N?-
butyl urea, Pharm. Chem. J. 17 (1983) 410–413.
[30] S.M. Shahnaz Perveen, K.M. Khan, An unplanned synthesis of symmetrical 1,3-
disubstituted ureas, J. Chem. Soc. Pak. 32 (2010) 229–234.
[31] X. Zhang, Z. Tang, X. Niu, Z. Li, X. Fan, G. Zhang, Selenium-catalyzed
carbonylation of 2-aminothiazole with nitro aromatics to N-aryl-N′-2-
thiazolylureas, Tetrahedron Lett. 57 (2016) 5266–5270.
[6] P.P. Huang, J.T. Randolph, L.L. Klein, S. Vasavanonda, T. Dekhtyar, V.S. Stoll, D.
J. Kempf, Synthesis and antiviral activity of P1′ arylsulfonamide azacyclic urea HIV
protease inhibitors, Bioorg. Med. Chem. Lett. 14 (2004) 4075–4078.
[7] J.A. Shimshoni, M. Bialer, B. Wlodarczyk, R.H. Finnell, B. Yagen, Potent
Anticonvulsant Urea Derivatives of Constitutional Isomers of Valproic Acid, J. Med.
Chem. 50 (2007) 6419–6427.
[32] F. Mjambili, M. Njoroge, K. Naran, C. De Kock, P.J. Smith, V. Mizrahi, D. Warner,
K. Chibale, Synthesis and biological evaluation of 2-aminothiazole derivatives as
antimycobacterial and antiplasmodial agents, Bioorg. Med. Chem. Lett. 24 (2014)
560–564.
[33] M. Hadianawala, B. Datta, Design and development of sulfonylurea derivatives as
zinc metalloenzyme modulators, RSC Adv. 6 (2016) 8923–8929.
[34] S. Perveen, K.M. Khan, M.A. Lodhi, M.I. Choudhary, R. Atta ur, W. Voelter, Urease
[8] R.H. Tale, A.H. Rodge, G.D. Hatnapure, A.P. Keche, The novel 3,4-dihydropyri-
midin-2(1H)-one urea derivatives of N-aryl urea: Synthesis, anti-inflammatory,
antibacterial and antifungal activity evaluation, Bioorg. Med. Chem. Lett. 21
(2011) 4648–4651.
and α-Chymotrypsin Inhibitory Effects of Selected Urea Derivatives, Lett. Drug Des.
Discovery 5 (2008) 401–405.
[9] K. Pandurangan, J.A. Kitchen, S. Blasco, F. Paradisi, T. Gunnlaugsson,
Supramolecular pyridyl urea gels as soft matter with antibacterial properties
against MRSA and/or E. coli, Chem. Commun. 50 (2014) 10819–10822.
[10] G. Pasello, L. Urso, P. Conte, A. Favaretto, Effects of Sulfonylureas on Tumor
Growth: A Review of the Literature, The Oncologist 18 (2013) 1118–1125.
[35] R. Jenkins, S. Maddocks, Antimicrobial. Test. (2019) 73–97.
[36] S.A.A. Mohamed, M. El-Sakhawy, E.L.S.H.A. Nashy, A.M. Othman, Novel natural
composite films as packaging materials with enhanced properties, Int. J. Biol.
Macromol. 136 (2019) 774–784.
[37] A.M. El Nahrawy, A.B.A. Hammad, A.M. Youssef, A.M. Mansour, A.M. Othman,
Thermal, dielectric and antimicrobial properties of polystyrene-assisted/ITO: Cu
nanocomposites, Appl. Phys. A 125 (2019).
¨
˘
[11] N. Ozbek, H. Katırcıoglu, N. Karacan, T. Baykal, Synthesis, characterization and
antimicrobial activity of new aliphatic sulfonamide, Bioorg. Med. Chem. 15 (2007)
5105–5109.
[38] A.M. Othman, M.A. Elsayed, A.M. Elshafei, M.M. Hassan, Application of response
surface methodology to optimize the extracellular fungal mediated nanosilver
green synthesis, J. Genet. Eng. Biotechnol. 15 (2017) 497–504.
[39] R.K. Jacobson, M.J. Notaro, G.J. Carr, Comparison of Neisseria gonorrhoeae
minimum inhibitory concentrations obtained using agar dilution versus microbroth
dilution methods, J. Microbiol. Methods 157 (2019) 93–99.
[12] F.M. Sroor, S.Y. Abbas, W.M. Basyouni, K.A.M. El-Bayouki, M.F. El-Mansy, H.
F. Aly, S.A. Ali, A.F. Arafa, A.A. Haroun, Synthesis, structural characterization and
in vivo anti-diabetic evaluation of some new sulfonylurea derivatives in normal
and silicate coated nanoparticle forms as anti-hyperglycemic agents, Bioorg Chem
92 (2019) 103290.
[13] H.-Q. Li, P.-C. Lv, T. Yan, H.-L. Zhu, Urea Derivatives as Anticancer Agents, Anti-
Cancer Agents Med. Chem. 9 (2009) 471–480.
[40] D.K. Maurya, N. Nandakumar, T.P.A. Devasagayam, Anticancer property of gallic
acid in A549, a human lung adenocarcinoma cell line, and possible mechanisms,
J. Clin. Biochem. Nutrit. 48 (2010) 85–90.
[14] F.M. Sroor, A.M. Abdelmoniem, I.A. Abdelhamid, Facile Synthesis,Structural
Activity Relationship, Molecular Modeling and In Vitro Biological Evaluation of
New Urea Derivatives with Incorporated Isoxazole and Thiazole Moieties as
Anticancer Agents, ChemistrySelect 4 (2019) 10113–10121.
[41] P.K. Singh, S. Ranganathan, D. Halagowder, N.D. Sivasithambaram, Quercetin
Suppresses Twist to Induce Apoptosis in MCF-7 Breast Cancer Cells, Plos One 10
(2015) e0141370.
´
[15] D. Warszycki, S. Mordalski, J. Staron, A.J. Bojarski, Bioisosteric Matrices for
[42] M. El-Far, G.A. Elmegeed, E.F. Eskander, H.M. Rady, M.A. Tantawy, Novel
modified steroid derivatives of androstanolone as chemotherapeutic anti-cancer
agents, Eur. J. Med. Chem. 44 (2009) 3936–3946.
Ligands of Serotonin Receptors, ChemMedChem 10 (2015) 601–605.
[16] F.M. Sroor, M.M. Aboelenin, K.F. Mahrous, K. Mahmoud, A.H.M. Elwahy, I.
A. Abdelhamid, Novel 2-cyanoacrylamido-4,5,6,7-tetrahydrobenzo[b]thiophene
derivatives as potent anticancer agents, Arch. Pharm. (2020), https://doi.org/
10.1002/ardp.202000069.
´
[43] P.L. Olive, J.P. Banath, R.E. Durand, Heterogeneity in Radiation-Induced DNA
Damage and Repair in Tumor and Normal Cells Measured Using the “Comet”
Assay, Radiat. Res. 178 (2012) AV35–AV42.
[17] F.M. Sroor, T.K. Khatab, W.M. Basyouni, K.A.M. El-Bayouki, Synthesis and
molecular docking studies of some new thiosemicarbazone derivatives as HCV
polymeraseinhibitors, Synthetic Commun 49 (2019) 1444–1456.
[44] A. Collins, M. Dusinska, M. Franklin, M. Somorovska, H. Petrovska, S. Duthie,
L. Fillion, M. Panayiotidis, K. Raslova, N. Vaughan, Comet assay in human
biomonitoring studies: Reliability, validation, and applications, Environ. Mol.
Mutagen 30 (1997) 139–146.
[18] F.M. Sroor, W.M. Basyouni, W.M. Tohamy, T.H. Abdelhafez, M.K. El-awady, Novel
pyrrolo[2,3-d]pyrimidine derivatives: Design, synthesis, structure elucidation and
in vitro anti-BVDV activity, Tetrahedron 75 (2019) 130749.
[45] A. Yawata, M. Adachi, H. Okuda, Y. Naishiro, T. Takamura, M. Hareyama,
S. Takayama, J.C. Reed, K. Imai, Prolonged cell survival enhances peritoneal
dissemination of gastric cancer cells, Oncogene 16 (1998) 2681–2686.
[19] F.M. Sroor, L. Vendier, M. Etienne, Cyclooctatetraenyl calcium and strontium
amido complexes, Dalton Trans. 47 (2018) 12587–12595.
[20] F.M. Sroor, C.G. Hrib, P. Liebing, L. Hilfert, S. Busse, F.T. Edelmann, Five different
types of eta(8)-cyclooctatetraenyl-lanthanide half-sandwich complexes from one
14

F.M. Sroor et al.
Bioorganic Chemistry 112 (2021) 104953
[46] A. Pedretti, L. Villa, G. Vistoli, VEGA – An open platform to develop chemo-bio-
informatics applications, using plug-in architecture and script programming,
J. Comput. Aided Mol. Des. 18 (2004) 167–173.
[48] E.F. Pettersen, T.D. Goddard, C.C. Huang, G.S. Couch, D.M. Greenblatt, E.C. Meng,
T.E. Ferrin, UCSF Chimera?A visualization system for exploratory research and
analysis, J. Comput. Chem. 25 (2004) 1605–1612.
[47] O. Trott, A.J. Olson, AutoDock Vina: Improving the speed and accuracy of docking
with a new scoring function, efficient optimization, and multithreading, J. Comput.
Chem. (2009).
[49] G.M. Sheldrick, A short history ofSHELX, Acta Crystallographica Sect. A Found.
Crystallogr. 64 (2007) 112–122.
15