Design, in silico, one-pot synthesis, and biological evaluations of novel bis-urea analogs

Article abstract of DOI:10.1007/s11164-020-04134-7

Abstract: The structure of urea has received special attention due to its biological activity. A new and efficient one-pot three-component reaction for the synthesis of bis-urea compounds from a variety of substituted diamino derivatives and isocyanate derivatives at room temperature with suitable yield is reported. Seven novel bis-urea derivatives were designed, synthesized, isolated, purified, and characterized (3a–g) with a variety of aromatic and aliphatic linkers. All compounds were evaluated for their cytotoxic and antibacterial properties. Most of the synthesized compounds exhibited reasonable activity compared to the positive control group. Graphic abstract: Development of a novel one-pot three-component reaction (3-CR) for the preparation of bis-urea scaffolds based on in silico studies was done. Evaluations of cytotoxic and antibacterial activities of all compounds were done. Most of the compounds exhibited good activities.[Figure not available: see fulltext.].

Full text of DOI:10.1007/s11164-020-04134-7

Research on Chemical Intermediates
https://doi.org/10.1007/s11164-020-04134-7
Design, in silico, one‑pot synthesis, and biological
evaluations of novel bis‑urea analogs
Sajede Shoja, et al. [full author details at the end of the article]
Received: 5 December 2019 / Accepted: 19 March 2020
© Springer Nature B.V. 2020
Abstract
The structure of urea has received special attention due to its biological activity.
A new and efcient one-pot three-component reaction for the synthesis of bis-urea
compounds from a variety of substituted diamino derivatives and isocyanate deriva-
tives at room temperature with suitable yield is reported. Seven novel bis-urea deriv-
atives were designed, synthesized, isolated, purifed, and characterized (3a–g) with
a variety of aromatic and aliphatic linkers. All compounds were evaluated for their
cytotoxic and antibacterial properties. Most of the synthesized compounds exhibited
reasonable activity compared to the positive control group.
Graphic abstract
Development of a novel one-pot three-component reaction (3-CR) for the prepara-
tion of bis-urea scafolds based on in silico studies was done. Evaluations of cyto-
toxic and antibacterial activities of all compounds were done. Most of the com-
pounds exhibited good activities.
Keywords Synthesis · Design · Sorafenib · Cytotoxicity · Antibacterial · One-pot
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s1116
4-020-04134-7) contains supplementary material, which is available to authorized users.
Vol.:(0123456789)
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S. Shoja et al.
Introduction
One of the areas of interest in synthetic organic chemistry is fnding suitable tech-
niques for C–N bonding. It is widely used in natural product synthesis and indus-
trial production [1, 2]. These functional groups are used as building blocks of sev-
eral pharmaceuticals [3, 4] and agrochemicals [5]. Initial study indicates that the
structure of urea is very important because NH groups can play an important role
in forming hydrogen bonds with amino acids [6] and lead to its biological activity.
Many urea analogs have also shown positive antiproliferative activity [7–10]. For
example, sorafenib (Nexavar®), lenvatinib (Lenvima®), regorafenib (Stivarga®), and
linifanib (ABT-869) include this functional group (Fig. 1).
Multicomponent reactions (MCRs) are considered as important methods in
organic synthesis. These types of reactions have many advantages over conventional
reactions such as higher chemical yields, operational simplicity, reduced purifcation
and isolation processes and minimized time, energy, costs, and waste generation.
MCRs are also valuable for the production of chemical libraries with a high diver-
sity of bioactive compounds and high levels of molecular complexity. Thus, they
help drug discovery programs to identify and optimize potential intelligent mole-
cules [11–20].
Liver cancer is the sixth leading cause of cancer death in women and the second
leading cause of cancer deaths in men [21]. Sorafenib (Nexavar®) is a diaryl urea
multikinase inhibitor accepted by the FDA for the treatment for advanced hepatocel-
lular carcinoma (HCC) in 2007 [22, 23]. It is also intended for testing other cancers,
including clinical trials of brain, ovarian, leukemia, metastatic colorectal, glioblas-
toma, Hodgkin’s lymphoma, metastatic breast, renal cancer, thyroid, non-small cell
lung cancer (NSCLC), prostate cancers, melanoma, and bladder [24, 25]. Not only
Fig. 1 Several drugs containing urea moiety with antiproliferative activity
1 3

Design, in silico, one-pot synthesis, and biological…
it can block the Raf/MEK/ERK signaling pathway, but also can inhibit more kinases
involved in tumor angiogenesis and proliferation, such as VEGFR-2, PDGFR, Flit-3,
and c-KIT [26].
The method of this work is to dimerize part of the molecular structure of
sorafenib, which has been confrmed as a potential target based on in silico studies.
For this suggestion, a variety of linkers such as aromatic and aliphatic groups were
used to connect the urea moieties.
Results and discussion
Chemistry
Conferring on in silico analyses and docking studies on sorafenib drug concluded
that unit of [1-(4-chloro-3-(trifuoromethyl)phenyl)-3-urea-] is the core active site.
This active site encouraged us to synthesize bis-urea compound that contains two
urea units in its structure. Based on our previous works related to the two-directional
synthesis, and with a focus on exploring convenient synthetic methods for the pro-
duction of compounds with pharmacological activities [27–35], a one-pot 3-CR for
the preparation of bis-urea scafolds (Scheme 1) from the reaction of 2 eq. isocy-
anate derivatives 1 to 1 eq. of diamino-compounds 2 was developed. A total of seven
new bis-urea derivatives 3a–g were designed, synthesized, isolated, purifed, and
characterized.
All compounds described in Scheme 1 were characterized by spectroscopic
1
methods FTIR, H NMR, and 13C NMR and elemental analysis. The FTIR spec-
tra of bis-urea derivatives 3a–g reveal the presence of carbonyl C=O stretching
Scheme 1 Synthesis of bis-urea derivatives 3a–g
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S. Shoja et al.
vibration bands at υ 1639–1696 cm−1, absorption bands in the 3277–3389 cm−1
and 1536–1566 cm−1 regions corresponding to N–H stretching and bending peaks,
respectively, due to urea formation.
1
The H NMR spectra of bis-ureas 3a–g showed sharp bands at δ 6.5–9.5 ppm
attributed to the N–H proton of urea moiety. Aromatic ring protons were observed
in the expected chemical shift region and exhibited the estimated integral values for
all compounds. Chemical shifts and the integral values of aliphatic protons in 3a,
3c, 3d, and 3f referred to their structures. The 13C NMR spectra of bis-ureas 3a–g
showed signals at 152.77–155.66 ppm assigned to the carbonyls of urea groups.
The carbon of CF3 substituent displayed signals at 119–128 ppm as a quartet with
1JFC =271–272 Hz for compounds 3a–e. The coupling of fuorine with the second
carbon (2JFC) was observed in 3b–e as a small quartet with J value 30 Hz (Fig. 2;
Table 1). Signals related to aromatic and aliphatic carbons resonate at their predicted
locations (see the “Supplementary material” fle).
The 3a–g were designed and synthesized based on the predicted active sites
and stabilized their molecular structure in the feld of energy minimization using
HyperChem software and using NCBI PubChem [36]. The importance of each por-
tion of the molecule in relation to the properties of sorafenib as typical urea analog
drug was investigated (Fig. 1). The structure of the synthesized compounds is
described in Table 1.
A
B
Fig. 2 Splitting patterns of 1JFC and 2JFC in 13C NMR for a 3b and b 3c
1 3

Design, in silico, one-pot synthesis, and biological…
Table 1 Synthesis of bis-urea derivatives 3a–g with fve diferent linkers
Entry
1
Product
Name
Time (h)
12
Yielda (%)
90
3a
2
3
4
3b
3c
3d
15
15
15
86
88
85
5
6
7
3e
3f
14
12
14
80
86
84
3g
All of the reactions were carried out with 1 (9.6 mmol) and 2 (4.5 mmol) in CH2Cl2 (8+8 mL)
aIsolated yield
Biology
In silico analyses
According to Lipinski’s Rule of Five [37], 74 analogs are designed to fnd bet-
ter compounds than the structure of sorafenib as typical urea analog drugs. As
a frst step, these analogs were made by little in silico modifcations. Substitute
groups included are OH, CH3, Cl, NH2, cyclopentane, and pyrrole.
The results showed that for minor modifcation, the best potential atoms of
sorafenib might be N11 and C23.
The results of this analysis are used to potentially initiate the main research
idea for the synthesis of new compounds with further modifcations of sorafenib.
(For more information, please see the “Supplementary material” fle, in particu-
lar, Figs. S1 and S2.)
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S. Shoja et al.
120
100
80
60
40
20
0
3a
3b
3c
3d
3e
3f
3g
0
50
100
150
Concentraꢀons (µg/ml)
Fig. 3 Antiproliferative activity of compounds 3a–g against HT-29 cells using MTT assays
120
100
80
60
40
20
0
Compounds
Fig. 4 IC50 values of derivatives 3a–g, sorafenib and cisplatin against the HT-29 cells
Cytotoxic activity
In the present work, we investigated the cytotoxic efects of 3a–g on human
colon adenocarcinoma cell line proliferation (HT-29) and human normal adult
dermal fbroblasts (HDF). Cells were treated with diferent concentrations of the
3a–g, sorafenib, and cisplatin in diferent amounts ranging from 0 to 175 μM
for 24 h. Cells were incubated with 3a–g and cell viability was measured 18 h
later by MTT assay. Inhibition of cell proliferation in each treatment is reported
as a percentage of the number of cells treated in relation to the untreated con-
trol cells (Fig. 3). Figure 4 shows the IC50 values of 3a–g. The IC50 of cisplatin
and sorafenib, as the positive control group, under all identical conditions, was
approximately 4.49 and 5.25 µM, respectively. MTT assay results showed that 3a
had higher cytotoxicity than other compounds. However, all IC50 values showed
approximately reasonable activity for the compounds. Among the compounds
tested, 3a, 3b, and 3c exhibited antiproliferative activity with IC50 of 85.37,
88.92, and 92.46 µM, respectively.
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Design, in silico, one-pot synthesis, and biological…
Relatively, a higher IC50 value (>450 µM) for 3a–g on the normal fbroblast cells
compared to the colon adenocarcinoma cells of HT29 indicates that the 3a–g are
nontoxic to normal human cells.
It appears that the presence of substituents in the phenyl rings led to changes in
activity. As shown, chlorine and trifuoromethyl substituents, similar in structure to
sorafenib, are efective in the activity of 3a–e compared to positive control groups
(cisplatin and sorafenib). However, in the case of 3d, it seems that decreasing the
length of the linkage between two urea moieties resulted in decreased activity.
Antibacterial activity
The in vitro antibacterial activities of compounds were measured against Gram-neg-
ative (GN) and Gram-positive (GP) bacteria. Four standard microorganisms were
used: Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa) as
GN; and Staphylococcus aureus (S. aureus) and Micrococcus luteus (M. luteus) as
GP. The results are reported in Table 2. As shown, most of the compounds exhibit
strong activities toward GP bacteria (Micrococcus luteus and Staphylococcus
aureus) and no reactivity was found against GN bacteria. Changes in activity are
due to the efect of substituents on the phenyl ring. As shown, compounds 3f and 3g
showed no activity in either bacterial group. The presence of Cl and CF3 is highly
efective in the antibacterial activity of 3a–e in comparison with the positive con-
trols (ciprofoxacin and ampicillin).
GN bacteria are surrounded by a thin murein cell wall that is surrounded by an
outer membrane containing lipopolysaccharide. GP bacteria lack the outer mem-
brane but are often surrounded by murein layers thicker than those found in GN
ones. As can be seen, S. aureus and M. luteus were generally sensitive, unlike two
Table 2 Antibacterial activity of
Entry Compound
Antibacterial activity (zone of inhibition in
mm)
the compounds (3a–g)
Gram-negative
Gram-positive
E. coli P. aeruginosa S. aureus M. luteus
1
2
3
4
5
6
7
8
9
3a
3b
3c
3d
3e
3f
–
–
–
–
–
–
–
–
–
–
–
–
–
–
28
–
–
14
15
17
18
20
–
17
21
14
24
25
–
3g
–
–
Ciprofoxacina 13
13
22
–
11
27
–
Ampicillina
DMSOb
8
–
10
aPositive control
bNegative control
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S. Shoja et al.
GN isolates. However, the synthesized compounds displayed great activity on this
bacterium relative to positive controls. This may be explained by the fact that
although some transport mechanisms in cell membranes are quite general, others
require specifc proteins for transportation to take place. In general, due to the
presence of the two distinct membranes in GN bacteria, they are more efective in
expelling harmful molecules, or inhibiting their entry into the cytosol. The order
of increased activity 3c, 3d, and 3e exhibited strong activities against S. aureus,
and 3a, 3b, 3d, and 3e showed strong activity against M. luteus.
Conclusions
In conclusion, a new and efcient one-pot 3MCR based on the in silico analyses
for the synthesis of bis-urea compounds from a variety of diamino-substituted
derivatives and isocyanate derivatives at r.t. with good yields was developed. The
di-substituted aryl urea residue similar to the part of the molecular structure of
sorafenib is practically present in 3a–g products. Simplicity, easy execution, high
atom economy, simple workup and good yields are aspects of this synthetic pro-
cedure. In vitro antibacterial activity of the compounds showed that the presence
of Cl and CF3 substitutions is very efective in the high activity of compounds in
comparison with positive controls. The cytotoxic efects of compounds against
the HT-29 colorectal cell line using a modifed MTT assay are studied. The pres-
ence of substituents in phenyl rings and the use of diferent linkers result in a
change in activity.
Experimental section
Chemistry
Materials and methods
Chemical materials were purchased from Aldrich, Merck, and Fluka and were
used without further purifcation. The purity defnition of the substrate and reac-
tion monitoring were done using thin-layer chromatography (TLC) on silica-gel
polygram SILG/UV 254 plates. The FTIR spectra were determined on a Shi-
madzu IR-470 spectrometer. Melting points were recorded on a Büchi B-545
apparatus in open capillary tubes and are uncorrected. All 1H NMR and 13C NMR
data were recorded using a Bruker Avance 400 and 100-MHz spectrometer at
293 K and dimethyl sulfoxide (DMSO)-d6, and spectra were internally referenced
to tetramethylsilane (TMS). Chemical shifts are reported in ppm (δ) using deuter-
ated solvents as internal references. Elemental analysis was made by a Carlo-Erba
EA1110 CNNO-S analyzer and agreed with the calculated values.
1 3

Design, in silico, one-pot synthesis, and biological…
General procedure for the synthesis of bis‑urea derivatives (3a–g)
Appropriate derivatives of diamino-substituted compounds (2, 4.5 mmol, 1 eq)
were dissolved in CH2Cl2 (8 mL) at r.t. A solution of isocyanate derivatives (1,
9.6 mmol, 2.05 eq) in CH2Cl2 (8 mL) was added dropwise to the mixture under
vacuum/argon condition. The mixture was stirred at r.t. for a period of times as
shown in Table 1; the reaction progress was monitored by TLC. Then, the reac-
tion mixture was fltered and washed with CH2Cl2 (2 mL × 2) to result in the com-
pounds (3a–g) as powdered solid yields shown in Table 1. The products were
established by their 1H NMR, 13C NMR, FTIR, and elemental analysis data.
1,1′‑(Methylenebis(4,1‑phenylene))bis(3‑(4‑chloro‑3‑(trifluoromethyl)phenyl)
1
urea) (3a) Creamy powder, yield 90%. Mp: 244–247 °C. H NMR (400 MHz,
DMSO) (δ/ppm): 9.12 (2H, s, N–H), 8.77 (2H, s, N–H), 8.11 (2H, s, Ar–H), 7.62
(4H, m, Ar–H), 7.38 (4H, d, J = 8.4 Hz, Ar–H), 7.15 (4H, d, J = 8.4 Hz, Ar–H),
3.84 (2H, s, –CH2–). 13C NMR (100 MHz, DMSO) (δ/ppm): 152.57 (C=O of
1
urea), 119.24–127.39 (q, JFC = 272 Hz, –CF3), 118.91–139.34 (Ar–C), 40.06 (–
CH2–). IR (νmax/cm−1): 1647 (C=O stretching), 3277 (N–H stretching), 1552 (N–H
bending). Anal Calcd. for C29H20Cl2F6N4O2: C, 54.31; H, 3.14; N, 8.74%. Found
C, 54.33; H, 3.11; N, 8.70%; exact mass: (M+): Calcd 640.0868; found 640.0871.
1,1′‑(Sulfonylbis(4,1‑phenylene))bis(3‑(4‑chloro‑3‑(trifuoromethyl)phenyl)urea)
1
(3b) White powder, yield 86%. Mp: 175–178 °C. H NMR (400 MHz, DMSO)
(δ/ppm): 9.39 (2H, s, N–H), 9.31 (2H, s, N–H), 8.11 (2H, s, Ar–H), 7.86 (2H, d,
J = 8.8 Hz, Ar–H), 7.76 (2H, d, J = 8.8 Hz, Ar–H), 7.69 (4H, d, J = 8.8 Hz, Ar–H),
7.62–7.65 (4H, m, Ar–H). 13C NMR (100 MHz, DMSO) (δ/ppm): 152.63 (C=O of
urea), 126.77–127.67 (q, 2JFC = 30 Hz, –C–CF3), 119.17–127.33 (q, 1JFC = 272 Hz,
–CF3), 118.86–139.34 (Ar–C). IR (νmax/cm−1): 1665 (C=O stretching), 3338 (N–H
stretching), 1544 (N–H bending). Anal Calcd. for C28H18Cl2F6N4O4S: C, 48.64;
H, 2.62; N, 8.10%. Found C, 48.61; H, 2.61; N, 8.14%. Exact mass: (M+): Calcd
690.0330; found 690.0328.
1,1′‑(Hexane‑1,6‑diyl)bis(3‑(4‑chloro‑3‑(trifuoromethyl)phenyl)urea) (3c) White
powder,yield88%.Mp:189–192°C.1HNMR(400MHz,DMSO)(δ/ppm):8.95(2H,
s, N–H), 8.07 (2H, d, 4JHH = 2.4 Hz, Ar–H), 7.57 (2H, dd, J = 8.8 Hz, 4JHH = 2.4 Hz,
3
Ar–H), 7.53 (2H, d, J = 8.8 Hz, Ar–H), 6.35 (2H, t, JH–N–C–H = 5.6 Hz, N–H),
3.07–3.12 (4H, m, –CH2–), 1.43–1.45 (4H, m, –CH2–), 1.31–1.32 (4H, m, –CH2–).
13C NMR (100 MHz, DMSO) (δ/ppm): 155.33 (C=O of urea), 126.58–127.48 (q,
1
2JFC = 30 Hz, –C–CF3), 119.29–127.42 (q, JFC = 271 Hz, –CF3), 116.47–140.70
(Ar–C), 26.56–31.17 (–CH2–). IR (νmax/cm−1): 1651 (C=O stretching), 3324 (N–H
stretching), 1566 (N–H bending). Anal Calcd. for C22H22Cl2F6N4O2: C, 47.24; H,
3.96; N, 10.02%. Found C, 47.20; H, 3.95; N, 10.06%. Exact mass: (M+): Calcd
558.1024; found 558.1018.
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S. Shoja et al.
1,1′ ‑(Ethane‑1,2‑diyl)bis(3‑(4‑chloro‑3‑(trifluoromethyl)phenyl)urea)
1
(3d) White powder, yield 85%. Mp: 192–194 °C. H NMR (400 MHz, DMSO)
4
(δ/ppm): 9.29 (2H, s, N–H), 8.08 (2H, d, JHH = 2.4 Hz, Ar–H), 7.58 (2H, dd,
4
J = 9.0 Hz, JHH = 2.4 and 2.0 Hz, Ar–H), 7.53 (2H, d, J = 8.8 Hz, Ar–H), 6.60
(2H, s, N–H), 3.21–3.23 (4H, m, –CH2–). 13C NMR (100 MHz, DMSO) (δ/ppm):
155.66 (C=O of urea), 126.57–127.47 (q, 2JFC = 30 Hz, –C–CF3), 119.28–127.41
(q, 1JFC = 271 Hz, –CF3), 116.54–140.67 (Ar–C), 35.63 (–CH2–). IR (νmax/cm−1):
1646 (C=O stretching), 3333 (N–H stretching), 1565 (N–H bending). Anal Calcd.
for C18H14Cl2F6N4O2: C, 42.96; H, 2.80; N, 11.13%. Found C, 42.99; H, 2.78; N,
11.14%. Exact mass: (M+): Calcd 502.0398; found 502.0392.
(2E,2′E)‑2,2′‑(1,4‑Phenylenebis(methanylylidene))bis(N‑(4‑chloro‑3‑(trifluorom
ethyl)phenyl)hydrazinecarboxamide) (3e) Pale yellow powder, yield 80%. Mp:
1
289–291 °C. H NMR (400 MHz, DMSO/Acetone 1:1) (δ/ppm): 11.10 (2H,
4
s, N–H), 9.42 (2H, s, N–H), 8.37 (2H, d, JHH = 2.4 Hz, Ar–H), 8.12 (2H, d,
J = 8.8 Hz, 4JHH = 2.0 Hz, Ar–H), 8.07 (2H, s, HC = N), 7.89–7.97 (4H, m, Ar–H),
7.65 (2H, d, J = 8.8 Hz, Ar–H). 13C NMR (100 MHz, DMSO/Acetone 1:1) (δ/
ppm): 153.77 (C=O of urea), 141.83 (–C=N–), 127.11–128.02 (q, 2JFC = 30.5 Hz,
1
–C–CF3), 119.70–127.83 (q, JFC = 271 Hz, –CF3), 119.21–139.84 (Ar–C). IR
(νmax/cm−1): 1696 (C=O stretching), 3389 (N–H stretching), 1536 (N–H bend-
ing). Anal Calcd. for C24H16Cl2F6N6O2: C, 47.62; H, 2.66; N, 13.88%. Found C,
47.60; H, 2.65; N, 13.83%. Exact mass: (M+): Calcd 604.0616; found 604.0611.
1,1′‑(Methylenebis(41,1‑phenylene))bis(3‑phenylurea) (3f) White powder, yield
86%. Mp: > 300 °C. H NMR (400 MHz, DMSO) (δ/ppm): 9.42 (2H, s, N–H),
8.80 (2H, s, N–H), 7.97 (4H, d, J = 8.8 Hz, Ar–H), 7.72–7.78 (2H, m, Ar–H),
7.58 (4H, d, J = 8.4 Hz, Ar–H), 7.10–7.18 (4H, m, Ar–H), 6.72–6.89 (4H, m,
Ar–H), 3.83 (2H, s, –CH2–). 13C NMR (100 MHz, DMSO) (δ/ppm): 153.69
(C=O of urea), 120.91–138.07 (Ar–C), 44.26 (–CH2–). IR (νmax/cm−1): 1639
(C=O stretching), 3303 (N–H stretching), 1558 (N–H bending). Anal Calcd. for
C27H24N4O2: C, 74.29; H, 5.54; N, 12.84%. Found C, 74.30; H, 5.51; N, 12.82%.
Exact mass: (M+): Calcd 436.1899; found 436.1904.
(2E,2′E)‑2,2′‑(1,4‑Phenylenebis(methanylylidene))bis(N‑phenylhydrazinecar‑
boxamide) (3g) Lemon yellow powder, yield 84%. Mp: > 300 °C. 1H NMR
(400 MHz, DMSO) (δ/ppm): 10.86 (2H, s, N–H), 10.12 (2H, s, N–H), 8.68 (2H,
s, HC=N), 8.00 (4H, s, Ar–H), 7.47 (4H, d, J = 7.6 Hz, Ar–H), 7.27–7.34 (4H,
m, Ar–H), 6.98 (2H, t, J = 7.4 Hz, Ar–H). 13C NMR (100 MHz, DMSO) (δ/ppm):
153.01 (C=O of urea), 145.25 (–C=N–), 118.65–140.19 (Ar–C). IR (νmax/cm−1):
1677 (C=O stretching), 3362 (N–H stretching), 1541 (N–H bending). Anal
Calcd. for C22H20N6O2: C, 65.99; H, 5.03; N, 20.99%. Found C, 66.02; H, 5.06;
N, 20.97%. Exact mass: (M+): Calcd 400.1648; found 400.1646.
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Design, in silico, one-pot synthesis, and biological…
Biology
In silico analyses
The molecular structure of sorafenib was obtained from NCBI PubChem [36] and
then stabilized regarding energy minimization by HyperChem software. Based on
the predicted active sites, the grid box was designed for every six candidate recep-
tors as 100×100×100 for XYZ. The in silico results indicated no remarkable can-
didate among 74 in silico synthesized analogs from sorafenib. The results demon-
strated that little alterations make no diference in sorafenib analogs to bind more
powerful with their targets compared to sorafenib. (For more information, please see
the “Supplementary material” fle, in particular, Figs. S1 and S2.)
Cytotoxic activity
Cell culture The human colon adenocarcinoma (HT-29) cell line and normal human
adult dermal fbroblasts (HDF) were obtained from the Pasteur Institute of Iran
(Tehran, I.R. Iran). The cells were maintained at 37 °C in a humidifed atmosphere
(90%) containing 5% CO2 and then cultured in DMEM (Dulbecco’s Modifed Eagle’s
Medium) with 10% (v/v) FBS (fetal bovine serum), 100 units/mL penicillin, and
100 μM streptomycin. The cells were seeded overnight and then incubated with dif-
ferent concentrations of the 3a–g, sorafenib, and cisplatin.
Cell viability The cell viability was determined using a modifed MTT (3-(4,5-dimeth-
ylthiazol-2-y1)-2,5-diphenyltetrazolium bromide) assay [38]. In brief, the cells
(1×104 cells/well) were seeded in 96-well plates and exposed to the indicated con-
centration of the 3a–g, sorafenib, and cisplatin for 24 h. The samples including 3a–
g, sorafenib, and cisplatin were tested at 25, 50, 75, 100, 125, 125, 150, 150, and
175 µM concentrations. The samples were dissolved in dimethyl sulfoxide (DMSO)
and further diluted with cell culture medium. The fnal concentration of DMSO was
adjusted to 1% of the total volume of the medium in all treatment, including the
blank. A control medium without DMSO was also incubated. After the treatment,
5 mg/mL of MTT solution was added and incubated for 3 h at 37 °C in a dark place.
The absorbance of formazan creation was measured at a wavelength of 540 nm with
an enzyme-linked immunosorbent assay (ELISA) reader (Molecular Devices, Sun-
nyvale, CA, USA). The cell viability by MTT assay was calculated as a percentage of
the control value (untreated cells) (Fig. 3).
Antibacterial activity
Antibacterial activity was determined using the good difusion technique; 50 µl of
bacterial culture, grown in nutrient broth (Merck) overnight at 37 °C, was inoculated
onto nutrient agar (Merck) plates. Wells, which are 0.5 mm in diameter, were bored
in the agar using the ends of sterile Pasteur pipettes; 0.2 mg of samples was dis-
solved in 0.2 mL DMSO to a fnal concentration of 1 mg/mL. To each well, 30 µl
of each sample was added and the plates were incubated at 37 °C overnight. For
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S. Shoja et al.
comparison, ciprofoxacin and ampicillin were used as positive controls and DMSO
as a negative control. Zones of growth inhibition were measured and the results are
presented in Table 2.
Acknowledgements The authors are grateful to the Research Council of the University of Guilan for
fnancial support of this research work.
Compliance with ethical standards
Confict of interest There are no conficts of interest to declare.
References
1. R. Hili, A.K. Yudin, Nat. Chem. Biol. 2, 284 (2006)
2. K. Shin, H. Kim, S. Chang, Acc. Chem. Res. 48, 1040 (2015)
3. E.A. Terefenko, J. Kern, A. Fensome, Bioorg. Med. Chem. Lett. 15, 3600 (2005)
4. Q. Li, T.M. Li, K.W. Woods, Bioorg. Med. Chem. Lett. 15, 2918 (2005)
5. A.Y. Guan, C.L. Liu, X.P. Yang, M. Dekeyser, Chem. Rev. 114, 7079 (2014)
6. M. Wang, S. Xu, H. Lei, C. Wang, Z. Xiao, S. Jia, J. Zhi, P. Zheng, W. Zhu, Bioorg. Med. Chem. 25,
5754 (2017)
7. D. Moreno, B. Díaz de Greñu, B. García, S. Ibeas, T. Torroba, Chem. Commun. 48, 2994 (2012)
8. C.R. Zhao, R.Q. Wang, G. Li, X.X. Xue, C.J. Sun, X.J. Qu, W.B. Li, Bioorg. Med. Chem. Lett. 23,
1989 (2013)
9. L. Ren, N. Jiao, Chem. Commun. 50, 3706 (2014)
10. M.M. Gamal El-Din, M.I. El-Gamal, M.S. Abdel-Maksoud, K.H. Yoo, C.H. Oh, Eur. J. Med. Chem.
119, 122 (2016)
11. J. Zhu, H. Bienayme, Multicomponent Reactions (Wiley, Weinheim, 2005), pp. 199–223
12. J.D. Sunderhaus, S.F. Martin, Chem. Eur. J. 15, 1300 (2009)
13. I. Ugi, Pure Appl. Chem. 73, 187 (2001)
14. M. Syamala, Org. Prep. Proced. Int. 41, 1 (2009)
15. B. Jiang, L.Y. Xue, X.H. Wang, M.S. Tu, Y.P. Liu, S.J. Tu, Tetrahedron Lett. 53, 1261 (2012)
16. M. Sarohaa, J.M. Khurana, New J. Chem. 43, 8644 (2019)
17. S.S. Sajadikhah, M.T. Maghsoodlou, N. Hazeri, S. Mohamadian-Souri, Res. Chem. Intermed. 42,
2805 (2016)
18. I. Ryu, H. Kuriyama, S. Minakata, M. Komatsu, J.Y. Yoon, S. Kim, J. Am. Chem. Soc. 121, 12190
(1999)
19. S. Srivastava, New J. Chem. 43, 6469 (2019)
20. A. Farhadi, J. Noei, R.H. Aliyari, M. Albakhtiyari, M.A. Takassi, Res. Chem. Intermed. 42, 1401
(2016)
21. A. Jemal, F. Bray, M.M. Center, J. Ferlay, E. Ward, D. Forman, CA Cancer J. Clin. 61, 69 (2011)
22. Sorafenib, BAY43-9006 (Nexavar®, Bayer) NDA 21923 Medical Review, FDA (2005)
23. Nexavar® (Sorafenib) Scientifc Discussion EMEA WC500027707 (2007)
24. L. Liu, Y. Cao, C. Chen, X. Zhang, A. McNabola, D. Wilkie, S. Wilhelm, M. Lynch, C. Carter, Can-
cer Res. 66, 11851 (2006)
25. G. Ranieri, G. Gadaleta-Calodarola, V. Gofredo, R. Patruno, A. Mangia, A. Rizzo, R.L. Sciorsci,
C.D. Gadaleta, Curr. Med. Chem. 19, 938 (2012)
26. S.M. Wilhelm, L. Adnane, P. Newell, A. Villanueva, J.M. Llovet, M. Lynch, Mol. Cancer Ther. 7,
3129 (2008)
27. N.O. Mahmoodi, S. Shoja, K. Tabatabaeian, B. Sharifzadeh, Ultrason. Sonochem. 23, 31 (2015)
28. N.O. Mahmoodi, S. Shoja, B. Sharifzadeh, M. Rassa, Med. Chem. Res. 23, 1207 (2014)
29. R. Sharif Aliabadi, N.O. Mahmoodi, J. Clean. Prod. 179, 235 (2018)
30. R. SharifAliabadi, N.O. Mahmoodi, H. Ghafoori, H. Roohi, V. Pourghasem, Res. Chem. Intermed.
44, 2999 (2018)
31. N.O. Mahmoodi, B. Khalili, O. Rezaeianzade, A. Ghavidast, Res. Chem. Intermed. 42, 6531 (2016)
1 3

Design, in silico, one-pot synthesis, and biological…
32. N.O. Mahmoodi, I. Nikokar, M. Farhadi, A. Ghavidast, Z. Naturforsch. B 69, 715 (2014)
33. N.O. Mahmoodi, M. Namroudi, F. Ghanbari Pirbasti, H. Roohi, I. Nikokar, Res. Chem. Intermed.
42, 3625 (2016)
34. N.O. Mahmoodi, N.K. Ahmadi, A. Ghavidast, J. Mol. Struct. 1160, 463 (2018)
35. A. Sheykhi-Estalkhjani, N.O. Mahmoodi, A. Yahyazadeh, M. Pasandideh Nadamani, Tetrahedron
74, 4868 (2018)
36. https://pubchem.ncbi.nlm.nih.gov/compound/216239
37. C.A. Lipinski, F. Lombardo, B.W. Dominy, P.J. Feeney, Adv. Drug Deliv. Rev. 64, 4 (2012)
38. M.V. Berridge, P.M. Herst, A.S. Tan, Biotechnol. Annu. Rev. 11, 127 (2005)
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Afliations
Sajede Shoja1 · Nosrat Ollah Mahmoodi1 · Hossein Ghafouri2 · Mehdi Rassa2 ·
Alireza Sharafshah3 · Esmaeel Panahi Kokhdan4
*
Nosrat Ollah Mahmoodi
mahmoodi@guilan.ac.ir
1
2
3
4
Department of Chemistry, Faculty of Sciences, University of Guilan, PO Box 41335-1914,
Rasht, Islamic Republic of Iran
Department of Biology, Faculty of Sciences, University of Guilan, PO Box 41335-1914, Rasht,
Islamic Republic of Iran
Cellular and Molecular Research Center, School of Medicine, Guilan University of Medical
Sciences, Rasht, Islamic Republic of Iran
Medicinal Plants Research Center, Yasuj University of Medical Sciences, Yasuj,
Islamic Republic of Iran
1 3