Synthesis, characterization, and biological evaluations of substituted phenethylamine-based urea as anticancer and antioxidant agents

Article abstract of DOI:10.1007/s00706-021-02830-7

Cancer is one of the most important diseases. It is the second leading cause of death worldwide following cardiovascular disease. There is a need to develop new chemotherapeutic agents for the treatment of cancer and it is still a significant task for med

Full text of DOI:10.1007/s00706-021-02830-7

Monatshefte für Chemie - Chemical Monthly
https://doi.org/10.1007/s00706-021-02830-7
ORIGINAL PAPER
Synthesis, characterization, and biological evaluations of substituted
phenethylamine‑based urea as anticancer and antioxidant agents
Fatma Betül Özgeriş1 · Bünyamin Özgeriş2
Received: 4 April 2021 / Accepted: 1 August 2021
© Springer-Verlag GmbH Austria, part of Springer Nature 2021
Abstract
Cancer is one of the most important diseases. It is the second leading cause of death worldwide following cardiovascular
disease. There is a need to develop new chemotherapeutic agents for the treatment of cancer and it is still a signifcant task for
medicinal chemists. Symmetrical and unsymmetrical urea derivatives have been reported to play a signifcant role in biologi-
cal systems. Phenethylamine is a precursor of dopamine and related compounds. In this study, we disclose the frst examples
of phenethylamine-based urea derivatives bearing fuoro, methoxy, and methyl groups. We also report cytotoxicity and tox-
icity profles of novel urea derivatives on HeLa (human cervical cancer), A549 (non-small cell lung carcinoma), and HDF
(human dermal fbroblast) cell lines. Moreover, we demonstrate antioxidant properties of synthesized compounds by DPPH,
ABTS, and CUPRAC method. The results revealed that 1,3-bis(4-methylphenethyl)urea was up to eightfold more potent than
cisplatin against HeLa cell line and also displayed very low toxic efect on HDF cell line compared to cisplatin. On the other
hand, the anticancer activity of 1-(3,4-dimethoxyphenethyl)-3-(4-fuorophenethyl)urea and 1-(3,4-dimethoxyphenethyl)-
3-(4-methylphenethyl)urea were close to that of cisplatin on A549 cell line, although 1-(3,4-dimethoxyphenethyl)-3-(4-
fuorophenethyl)urea was more toxic than compound 1-(3,4-dimethoxyphenethyl)-3-(4-methylphenethyl)urea on HDF cell
line. All tested compounds displayed remarkable activity compared to the standard antioxidants. Considering these properties,
they may be a lead compounds for the treatment of human cervical and non-small cell lung cancer. These fndings may be
helpful for the discovery of more biologically active phenethylamine-based urea for clinical studies.
Graphic abstract
Keywords Cytotoxicity · Toxicity · Antioxidant properties · HeLa · A549 · HDF
Introduction
Cancer is one of the most important health problems of
* Fatma Betül Özgeriş
worldwide. It is also a public health problem because of
its high lethality. Cancer is the second considerable cause
of death worldwide [1]. Lung [2] and cervical [3] cancers
are the most important threats to human health among vari-
ous types of cancer. Non-small cell lung carcinoma forms
80% of lung cancers [2]. In recent years, chemotherapy has
been disappointing because of multidrug-resistant tumors
and systemic toxicity [4]. However, cancer treatment is still
betul.ozgeris@atauni.edu.tr
* Bünyamin Özgeriş
bunyamin.ozgeris@erzurum.edu.tr
1
Department of Nutrition and Dietetics, Faculty of Health
Science, Ataturk University, 25240 Erzurum, Turkey
2
Department of Basic Sciences, Faculty of Sciences, Erzurum
Technical University, 25100 Erzurum, Turkey
Vol.:(0123456789)
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F. B. Özgeriş, B. Özgeriş
a signifcant task for medicinal chemists. Therefore, it is a
fact that new chemotherapeutic agents have also been tried
to be developed which may possess diferent mechanism of
action, and minimum toxicity and also will give the least
damage to the healthy tissue close to the tumor cell.
Urea plays an important role in agricultural, supramolec-
ular, and medicinal chemistry [5–8]. Recently, urea deriva-
tives have found applications in medicinal chemistry and
they have been explored many drug-like properties such as
antibacterial, antiviral, analgesic, and HDL elevating efects
[9]. They have also a wide range of biological activities such
as antimalarial [10, 11], cardiac-specifc myosin activators
[12], anaplastic lymphoma kinase inhibitors [13], tyrosine
inhibitors [14], antidiabetic, anti-infammatory, and antihy-
pertension [15, 16].
derivatives have been shown to have anticancer activity
[25]. For example, unsymmetrical urea compound 4 con-
taining a phenethylamine ring has been reported to have
anticancer activities against KB, MCF-7, NCI-H187, and
Vero cell lines [26]. In another study, it has been reported
that unsymmetrical urea compound 5 has been exhibited a
signifcant activity against HT-29 cell line [27]. Symmetrical
urea compound 6 containing a phenethylamine ring has been
shown to promote adipocyte diferentiation in 3T3-L1 cells
[28] (Fig. 1).
Over-production of reactive oxygen species (ROS) in the
body causes oxidative damage associated with many health
problems related to aging such as cancer, diabetes, cardio-
vascular diseases, and neurodegenerative diseases [29].
Antioxidants prevent the formation of ROS and delay the
progression of mentioned diseases [30]. To date, the cor-
relation between cancer and antioxidant activity has been
extensively discussed. Evidence from studies indicate that
elevated rates of intracellular reactive oxygen species are
existed in almost all cancers, and this situation has been
contributed to cancer initiation and progression [31, 32].
Therefore, antioxidant agents are needed to clean the tumor
cells from ROS.
Phenethylamine is a trace amine and a natural monoamine
alkaloid, which is widely known as a neurotransmitter in the
human central nervous system and plays signifcant role in
neuromodulation [17]. Determination of trace amines in the
brain is very important to understand their role in psychiat-
ric, and neurological disorders such as depression, schizo-
phrenia, Parkinson, epilepsy, attention defcit hyperactivity
disorder, and Reye’s syndrome [18].
There are many studies on the synthesis and biological
activity of urea derivatives in the literature. Notably, a recent
report demonstrates that the incorporation of phenethyl-
amine in urea based derivatives furnishes potential cardiac
myosin activators for the treatment of systolic heart failure
[19]. Another report demonstrates that urea based deriva-
tives AR-A014418 1 and 2 [20] have been investigated for
the treatment of Alzheimer’s disease [21] and depression
[22]. Sorafenib (3) is an unsymmetrical urea and is used
in the treatment of metastatic renal cell [23] and unresect-
able hepatocellular carcinoma [24]. Many aromatic urea
Prodrug compound 7 containing a phenethylamine ring
can be used in the treatment of malignant melanoma [33].
In another study, the authors report that urea derivative
compound 8 containing a phenethylamine ring can be used
to reduce hyperpigmentation [34]. Yang and coworkers
report that coumarin derivatives 9 comprising a pheneth-
ylamine ring have strong antioxidant activity to protect
DNA against hydroxyl radical [35]. In our earlier stud-
ies, we have also reported that phenethylamine-based
urea derivatives 10 and 11 have human carbonic anhy-
drase, acetylcholinesterase, butyrylcholinesterase enzyme
Fig. 1 Urea derivatives 1–6 used and investigated for various diseases
1 3

Synthesis, characterization, and biological evaluations of substituted phenethylamine‑based…
inhibitory activities and antioxidant properties [29, 36].
Rani and coworkers have investigated the anticancer activ-
ity of derivatives 12 and 13 containing 1-phenylethylamine
ring with halogen and methyl groups in para position on
the aromatic ring against breast cancer and neuroblastoma.
According to the results of their study, they have reported
that halogens on the aromatic ring contributed positively
to anticancer and anti-infammatory activity [37] (Fig. 2).
As can be seen from literature, urea and phenethyl-
amine moieties are important scafolds to develop novel
biologically active agents in the treatment of many dis-
eases. Although chemotherapy has been disappointing
due to multidrug-resistant tumors and systemic toxicity,
new chemotherapeutic agents still need to be developed
for the treatment of cancer. In this study, the main skeleton
was designed in the light of the literature data mentioned
above. It is aimed to investigate the efects on activity by
positioning methyl and fuoro atoms in para position of
the right phenethylamine ring and methoxy, methyl and
fuoro atoms in ortho, meta, and para positions of the left
phenethylamine ring according to the study of Rani and
coworkers. Moreover, to the best of our knowledge, no
comparative work has been published synthesis of these
compounds as well as anticancer and antioxidant proper-
ties. Therefore, the objectives of this study were to syn-
thesize a series of novel symmetrical/unsymmetrical urea
derivatives. In addition, the anticancer activity against
HeLa (human cervical carcinoma), A549 (non-small cell
lung carcinoma), and HDF (Human dermal fbroblast)
cell lines and antioxidant activities by DPPH, ABTS, and
CUPRAC methods of compounds were assessed. The
results were also compared on the basis of the efect of
substituents on the anticancer and antioxidant activities.
Results and discussion
Chemistry
There are many methods for the synthesis of symmetrical
and unsymmetrical ureas in the literature. Although, there
are various methods sufer from lack of functional group
tolerance, Padiya and coworkers have reported that the
preparation of ureas can easily be with CDI in aqueous
conditions [38]. In our previous study, we were synthesized
some unsymmetrical ureas containing phenethylamine ring
[39]. For this aim, substituted phenethylamines 14–17 were
reacted with CDI and obtained N-substituted imidazolide
intermediates. Then, these intermediates were reacted with
4-fuorophenethylamine (18) and 4-methylphenethylamine
(19) in situ. As a result, symmetrical and unsymmetrical
urea compounds 20–26 were synthesized in yields between
11 and 76%. The synthetic route to the target compounds is
given in Scheme 1.
The chemical structures of the synthesized compounds
were characterized with 1H NMR, 13C NMR, IR, and ele-
1
mental analysis. In the H NMR spectra of synthesized
compounds, the characteristic signals due to the –HN(CO)
NH– protons appeared between 4.31 and 4.81 ppm as
a broad singlet. In addition, chemical shifts appearing at
around 3.82 ppm and 2.30 ppm were assigned to the –OCH3
and –CH3 protons, respectively. In the 13C NMR spectra of
synthesized compounds, –CO, and –OCH3 signals appeared
at around 160.0 ppm and 56.0 ppm, respectively. In addition
to these, –CH3 signals belonging to phenethylamine ring
appeared at around 21.0 ppm. All these spectral data con-
frm the structures of symmetrical and unsymmetrical urea
derivatives.
Fig. 2 Some anticancer, antioxidant, and biologically active substances 7–13 containing phenethylamine ring
1 3

F. B. Özgeriş, B. Özgeriş
Scheme 1
Anticancer studies of compounds 20–26 against A549
cell line demonstrated that compound 23 (IC50 21 µM) and
22 (IC50 22 µM) were found to have good cytotoxic activ-
ity compared to other compounds. On the other hand, these
compounds were less active than cisplatin. While compound
22 has methoxy groups in meta and para positions of left
phenethylamine ring and one fuoro group in para position
of right phenethylamine ring, compound 23 has methoxy
groups in meta and para positions of left phenethylamine
ring and one methyl group in para position of right pheneth-
ylamine ring.
Biological assay
The cytotoxicity of urea compounds 20–26 was evalu-
ated against HeLa (human cervical carcinoma) and A549
(non-small cell lung carcinoma) cell lines by MTT, LDH,
and SRB assays. Anticancer activities of synthesized
compounds 20–26 were compared with cisplatin using
the MTT assay. Cisplatin, one of the most commonly
used anticancer drug in cancer, was used as a positive
control (IC50 value for HeLa cell line: 16 µM, for A549
cell line: 10 µM) [40]. When examining the IC50 values
of newly synthesized compounds, compound 25 (IC50
2 µM) showed highly strong activity compared to cispl-
atin against HeLa cell line. Its activity against HeLa cell
was up to eightfold more potent than cisplatin. Compound
24 (IC50 9 µM) and 22 (IC50 10 µM) showed less activity
than compound 25 while it showed stronger activity than
cisplatin. Even though other compounds showed weaker
activity than cisplatin, they showed activity against HeLa
cell line in micromolar range. Compound 25 is a symmet-
rical urea and it has one methyl group in para position of
phenethylamine ring. Notably, recent report demonstrates
that most of the EWG (electron withdrawing groups) such
as fuoro group showed stronger activity than those with
EDG (electron donating groups) such as methyl and meth-
oxy groups [41]. Contrary to this, although our compound
contain methyl (EDG) group in para position of pheneth-
ylamine ring, it has highly strong activity against HeLa
cell line. This could be due to the efect of steric hindrance
and can also be indicated as the reason why para substi-
tution in terms of the position of the substituents better
inhibits the growth of cancer cells [41]. This report sup-
ports our results.
Similar to MTT and SRB assay, LDH assay also gave
similar results on HeLa and A549 cells. Our results revealed
that cell viability decreased with the increase in concentra-
tions of synthesized compounds. The cytotoxicity values
against all tested cell lines in MTT, SRB, and LDH assays
were in accordance with each other (Table 1, Figs. 3 and 4).
On the other hand, the cytotoxic efect of compounds
20–26 on non-cancerous human dermal fibroblast cell
(HDF) was also assessed. According to the results, com-
pound 25 (IC50 on HDF: 33 µM) was up to eightfold more
potent than cisplatin against HeLa cell line while less toxic
than cisplatin (IC50 on HDF: 4 µM) against HDF cell line.
On the other hand, the anticancer activity of compound 22
and 23 were close to that of cisplatin on A549 cell line,
although compound 22 (IC50 on HDF: 17 µM) was more
toxic than compound 23 (IC50 on HDF: 76 µM) against HDF
cell line. Additionally, all tested compounds 20–26 exhibited
low toxic efect against non-cancerous cell line. The toxic-
ity values against non-cancerous cell line in MTT, and SRB
assays were in accordance with each other (Table 1).
Carcinogenesis is a multistage process comprise of ini-
tiation, promotion, and progression. These are very closely
1 3

Synthesis, characterization, and biological evaluations of substituted phenethylamine‑based…
Table 1 IC50 values of compounds 20–26 on HeLa, A549, HDF, DPPH, ABTS, and CUPRAC values of compounds 20–26 (p<0.05)
Compounds
IC50/µM
HeLa
λ450*
DPPH
89
ABTS
48
CUPRAC
A549
MTT
HDF
MTT
MTT
SRB
57
SRB
125
SRB
1149
53
135
265
0.078 0.003
57
52
65
62
22
17
149
22
87
49
0.079 0.002
0.091 0.004
10
15
22
24
110
45
29
35
21
25
76
33
27
19
78
50
46
0.093 0.001
0.076 0.002
9
12
489
501
95
2
5
121
100
124
93
33
20
100
90
48
46
0.081 0.002
0.083 0.002
72
79
163
227
Cisplatin
Trolox
BHT
Ascorbic acid
Β-carotene
16
–
–
–
–
25
–
–
–
–
10
–
–
–
–
15
–
–
–
–
4
–
–
–
–
2
–
–
–
–
–
–
–
96
105
67
68
58
86
35
81
0.173 0.002
0.151 0.001
0.960 0.002
0.087 0.001
Cells were treated with concentrations ranging from 6.25 to 200 μM for 48 h
HeLa LDH
A549 LDH
120
100
80
60
40
20
0
120
200
100
50
100
80
60
40
20
0
200
100
50
25
12,5
6,25
25
12,5
6,25
pc nc 13 14 15 16 17 18 19
pc nc 13 14 15 16 17 18 19
Fig. 4 Viability percentage measured by LDH assay on A549 cell
exposed to 6.25–200 µM concentrations of novel ureas 20–26 for 48 h
(nc negative control, pc positive control)
Fig. 3 Viability percentage measured by LDH assay on HeLa cell
exposed to 6.25–200 µM concentrations of novel ureas 20–26 for 48 h
(nc negative control, pc positive control)
linked to each other. Promotion is associated with oxidative
tissue damage. Conversely, substances with potent antioxida-
tive activities are expected to show chemopreventive efects
on carcinogenesis, especially in the promotion stage and act
1 3

F. B. Özgeriş, B. Özgeriş
as antitumor promoters [42]. Taking under consideration the
above mentioned facts, antioxidative potential of new urea
derivatives 20–26 was studied and compared to the well-
known antioxidant agents. Antioxidant properties, espe-
cially radical scavenging activities, are very important due
to removal of harmful efects of reactive oxygen species in
biological systems. DPPH• (2,2-diphenyl-1-picrylhydrazyl)
and ABTS•+ (2,2′-azino-bis(3-ethylbenzothiazoline-6-sul-
fonic acid)) have been widely used to determine the free
radical scavenging efcacy of various antioxidant materials.
In this study, antioxidant activity of the newly synthesized
compounds 20–26 and standard antioxidants such as trolox,
BHT, β-carotene, and ascorbic acid were determined using
DPPH•, ABTS•+, and CUPRAC methods.
profles on ABTS assay compared to standard antioxidants.
Similar to DPPH assay, compound 23 and 25 can be evalu-
ated as possible cytostatic agents due to their low toxicity
profles against non-cancerous human dermal fbroblast.
The values of the CUPRAC (cupric reducing antioxidant
capacity) method, in which the chromogenic neocuproine
was used as the oxidizing agent, are shown in Table 3. The
highest CUPRAC value was observed in the ascorbic acid
(0.960 0.002) solution. Compounds 22 and 23 showed
higher activity than β-carotene, while less active than
ascorbic acid, trolox, and BHT. On the other hand, other
compounds showed weaker activity than standard antioxi-
dants, compounds 22, and 23. At the same concentration
(200 μg cm−3), reducing capacities decreased in the order
of ascorbic acid>trolox>BHT>23>22>β-carotene>26
>25>21>20>24.
The DPPH• scavenging effect of all compounds and
standards decreased in the following order: ascorbic acid>
β-carotene>23>21>20>26>24>trolox>25>BHT>2
2. A lower IC50 value indicates a higher DPPH• free radi-
cal scavenging efect (Table 1). According to these values,
ascorbic acid (67 μg cm−3) has highest DPPH• free radical
scavenging efect. Compound 23 (78 μg cm−3) has closest
free radical scavenging capabilities to ascorbic acid. Other
urea compounds have close or weak free radical scaveng-
ing capabilities compared to standard antioxidants. On the
other hand, compound 25 having the best anticancer activ-
ity against HeLa cell line has high antioxidant capability
on DPPH assay compared to BHT, while less antioxidant
capability compared to ascorbic acid, β-carotene, and trolox.
In addition to this, compound 23 having the best anticancer
activity against A549 cell line has high antioxidant capa-
bility on DPPH assay compared to trolox and BHT, while
less antioxidant capability compared to ascorbic acid and
β-carotene. Moreover, compounds 23 and 25 have exhib-
ited low toxicity profles. Considering these their proper-
ties, these compounds can be evaluated as possible cytostatic
agents for the treatment of human cervical and non-small
cell lung cancer.
When examining the results of methods such as DPPH,
ABTS, and CUPRAC applied to determine the antioxidant
activity, it is seen that all tested compounds have remarkable
activity compared to the standards. It is also seen that com-
pounds 22 and 23 which have dimethoxy group in meta and
para positions at the left phenethylamine ring and methyl or
fuoro group in para positions at the right phenethylamine
ring have highest antioxidant activities in all antioxidant
assays. Notably, a recent report demonstrates that the anti-
oxidant potential of urea derivatives containing methoxy
or halogen was increased [43]. It has been reported in the
literature that urea compounds containing halogen atoms
in the para position have a positive efect on antioxidant
results [42]. Our results support these literature fndings
since our skeletal structure contains a fuoro or methoxy
group in the para position. However, compounds 22, 23,
and 25 have exhibited both highly most anticancer activities
than cisplatin and remarkable antioxidant profles all antioxi-
dant assays. Moreover, these compounds have exhibited low
toxicity profles. In this aspect, these compounds, especially
compound 25, can be evaluated as possible cytostatic agent
for the treatment of human cervical and non-small cell lung
cancer.
The ABTS•+ scavenging efect of all compounds and
standards decreased in the following order: ascorbic acid>
22>24>26>20>25>21>23>trolox>β-carotene>BH
T. A lower IC50 value indicates a higher ABTS•+ free radi-
cal scavenging efect (Table 1). According to these values,
ascorbic acid (35 μg cm−3) has highest ABTS•+ free radical
scavenging efect. Compound 22 (45 μg cm−3) has closest
free radical scavenging capabilities to ascorbic acid. Other
urea compounds have weaker free radical scavenging capa-
bilities to ascorbic acid. On the other hand, these compounds
have higher free radical scavenging capabilities compared
to trolox, β-carotene, and BHT. Compound 22 have exhib-
ited the best antioxidant profle on ABTS assay, while com-
pounds 23 and 25 with the highest anticancer activities have
exhibited the best antioxidant profles after compound 22. In
addition to this, all compounds have remarkable antioxidant
Conclusion
In conclusion, we have prepared a set of phenethylamine-
based urea derivatives bearing methoxy, fuoro, and methyl
substituents at diferent positions of phenethylamine ring.
The syntheses of symmetrical and unsymmetrical urea of
substituted phenethylamines 20–26 were carried out for the
frst time. The principal aim of this study was to evaluate
these compounds in terms of their anticancer, antioxidant,
and toxicity properties. In addition, it was aimed to com-
pare the anticancer and antioxidant activities on the basis
of substituents. Anticancer activities of newly synthesized
1 3

Synthesis, characterization, and biological evaluations of substituted phenethylamine‑based…
compounds were investigated against HeLa and A549 cell
lines. The results revealed that compound 25 was up to eight-
fold more potent than cisplatin against HeLa cell line and
also displayed very low toxic efect on HDF cell line com-
pared to cisplatin. On the other hand, the anticancer activ-
ity of compound 22 and 23 were close to that of cisplatin
on A549 cell line, although compound 22 was more toxic
than compound 23 on HDF cell line. We also investigated
for antioxidant properties of synthesized urea by DPPH,
ABTS, and CUPRAC methods. According to the results,
all compounds showed remarkable antioxidant properties
compared to standard antioxidants. The relative ease in the
synthesis of the cytotoxic substituted phenethylamine-based
urea derivatives 20–26 makes these compounds suitable can-
didates for further study. In particular, compound 25 may be
a lead compound for the treatment of human cervical cancer,
while compound 22 and 23 may be a lead compounds for the
treatment of non-small cell lung cancer. Considering these
properties, compound 22, 23, and 25 can be evaluated as
possible cytostatic agents for the treatment of human cervi-
cal and non-small cell lung cancer. However, further phar-
macokinetic and pharmacodynamics studies are needed to
determine their efects. The products described here provide
the foundation for the discovery of more biologically active
phenethylamine-based urea.
the mixture was fltered. Precipitate obtained was washed
with cold water and dried. Recrystallization or column
chromatography from EtOAc/Hexane aforded symmetri-
cal or unsymmetrical urea compounds 20–26.
1 ‑ ( 4 ‑ F l u o r o p h e n e t h y l ) ‑ 3 ‑ p h e n e t h y l u r e a ( 2 0 ,
C17H19FN2O) White powder; yield 70%; m.p.: 136–138 °C;
1H NMR (400 MHz, CDCl3): δ=7.42–7.24 (m, 3H, ArH),
7.24–7.05 (m, 5H, ArH), 7.01–6.92 (m, 1H, ArH), 4.43 (bs,
2H, NH), 3.55–3.21 (m, 4H, CH2), 2.91–2.59 (m, 4H, CH2)
ppm; 13C NMR (100 MHz, CDCl3): δ=158.0 (CO), 139.1
(C), 134.8 (C), 130.2 (C), 128.8 (2CH), 128.6 (2CH), 126.4
(CH), 115.4 (2CH), 115.2 (2CH), 41.7 (CH2), 41.6 (CH2),
36.4 (CH2), 35.6 (CH2) ppm; IR: ꢀ = 3340, 2889, 2362,
2315, 1670, 1558 cm−1.
1 ‑ ( 4 ‑ M e t h y l p h e n e t h y l ) ‑ 3 ‑ p h e n e t h y l u r e a ( 2 1 ,
C18H22N2O) White powder; yield 76%; m.p.: 121–123 °C;
1H NMR (400 MHz, CDCl3): δ=7.31–7.23 (m, 2H, ArH),
7.23–6.97 (m, 7H, ArH), 4.81 (bs, 2H, NH), 3.43–3.21 (m,
4H, CH2), 2.81–2.62 (m, 4H, CH2), 2.31 (s, 3H, CH3) ppm;
13C NMR (100 MHz, CDCl3): δ = 158.4 (CO), 139.3 (C),
136.1 (C), 135.9 (C), 129.3 (2CH), 128.8 (2CH), 128.7
(2CH), 128.6 (2CH), 126.4 (CH), 41.7 (CH2), 41.6 (CH2),
36.5 (CH2), 36.0 (CH2), 21.0 (CH3) ppm; IR: ꢀ=3339, 2894,
2372, 2318, 1689, 1555 cm−1.
Experimental
1‑(3,4‑Dimethoxyphenethyl)‑3‑(4‑fuorophenethyl)urea (22,
C19H23FN2O3) White powder; yield 28%; m.p.: 123–125 °C;
1H NMR (400 MHz, CDCl3): δ=7.09 (dd, J=8.5, 5.5 Hz,
2H, ArH), 6.98–6.89 (m, 2H, ArH), 6.76 (d, J=8.5 Hz, 1H,
ArH), 6.72–6.64 (m, 2H, ArH), 4.55 (bs, 2H, NH), 3.82 (s,
6H, 2OCH3), 3.41–3.28 (m, 4H, 2CH2), 2.71 (q, J=6.9 Hz,
4H, 2CH2) ppm; 13C NMR (100 MHz, CDCl3): δ = 160.5
(CO), 158.3 (C), 149.1 (C), 147.8 (C), 135.0 (C), 131.9 (C),
130.4 (2CH), 120.9 (2CH), 115.4 (CH), 112.1 (CH), 111.5
(CH), 56.1 (OCH3), 56.0 (OCH3), 41.9 (2CH2), 36.2 (CH2),
35.8 (CH2) ppm; IR: ꢀ = 3332, 2875, 2358, 2260, 1661,
1514 cm−1.
All chemicals and solvents are commercially available and
used without further purifcation. 1H and 13C NMR spectra
were recorded a Varian 400 MHz in CDCl3 and NMR shifts
are presented as δ in ppm. FT-IR spectra were recorded on
a Shimadzu IRTracer-100 spectrophotometer with single-
refection ATR accessory. Melting point was determined on
Electrothermal IA9100 capillary melting point apparatus.
Elemental analysis was performed on a Leco CHNS-932
apparatus.
General procedure for the synthesis of symmetrical
and unsymmetrical ureas 20–26
1‑(3,4‑Dimethoxyphenethyl)‑3‑(4‑methylphenethyl)urea
(23, C20H26N2O3) White powder; yield 51%; m.p.: 122–
124 °C; 1H NMR (400 MHz, CDCl3): δ=7.07 (dd, J=15.6,
7.2 Hz, 4H, ArH), 6.80–6.74 (m, 1H, ArH), 6.73–6.64 (m,
2H, ArH), 4.53 (bs, 1H, NH), 3.85 (s, 3H, OCH3), 3.82 (s,
3H, OCH3), 3.42–3.21 (m, 4H, 2CH2), 2.79–2.55 (m, 4H,
2CH2), 2.33 (s, 3H, CH3), 1.25 (bs, 1H, NH) ppm; 13C NMR
(100 MHz, CDCl3): δ=158.3 (CO), 149.1 (C), 147.8 (C),
136.2 (C), 136.1 (C), 131.9 (C), 129.5 (2CH), 128.9 (2CH),
120.9 (CH), 112.1 (CH), 111.5 (CH), 56.1 (OCH3), 56.0
(OCH3), 41.9 (2CH2), 36.2 (2CH2), 21.2 (CH3) ppm; IR:
ꢀ =3332, 2875, 2358, 2260, 1661, 1514 cm−1.
Substituted phenethylamine 14–17 (1.0 mmol) was taken
in water at room temperature and stirred at 0 °C. CDI
(1.2 mmol) was added to the reaction mixture at the same
temperature. Reaction mixture was stirred at 0 °C for 1 h
and then warm up to room temperature. Reaction was
monitored with TLC. After complete formation of carbon-
ylimidazolide intermediate, 4-fuorophenethylamine (18)
or 4-methylphenethylamine (19) (1.2 mmol) were added.
Reaction mixture was stirred at room temperature for 4 h
and monitored by TLC. At the end of the reaction time,
1 3

F. B. Özgeriş, B. Özgeriş
1‑(4‑Fluorophenethyl)‑3‑(4‑methylphenethyl)urea (24,
C18H21FN2O) White crystal; yield 16%; m.p.: 149–151 °C;
1H NMR (400 MHz, CDCl3): δ=7.17–7.08 (m, 4H, ArH),
7.08–7.02 (m, 2H, ArH), 7.01–6.93 (m, 2H, ArH), 4.30
(bs, 2H, NH), 3.47–3.17 (m, 4H, CH2), 2.82–2.60 (m, 4H,
CH2), 2.32 (s, 3H, CH3) ppm; 13C NMR (100 MHz, CDCl3):
δ=160.6 (CO), 158.1 (C), 136.2 (C), 136.1 (C), 135.0 (C),
130.4 (2CH), 129.5 (2CH), 128.9 (2CH), 115.7 (2CH),
41.9 (2CH2), 36.1 (CH2), 35.8 (CH2), 21.2 (CH3) ppm; IR:
ꢀ =3334, 2882, 2361, 2279, 1660, 1525 cm−1.
for 48 h. After incubation periods, 20 mm3 of MTT solu-
tion (MTT stock solution: 5 mg/cm3 in phosphate-bufered
saline) was added to each well and the cells were incubated
at 37 °C, 5% CO2 containing incubator for 3 h. Then, plates
were centrifuged at 1800 rpm for 10 min. Supernatant was
removed and 150 mm3 DMSO (Sigma–Aldrich ®, USA)
was added into each well to dissolve the formazan crystals.
Finally, the absorbance values were measured at 570 nm by
a spectrophotometer [44]. The viability was determined as
the percentage of absorbance of compounds 20–26 treated
cultures compared with those of untreated control cultures.
Cisplatin (Sigma–Aldrich®) was used as a reference com-
pound. Whereas used as a negative control, cells were stud-
ied without compounds 20–26 [44].
1,3‑Bis(4‑methylphenethyl)urea (25, C19H24N2O) White
powder; yield 58%; m.p.: 175–177 °C; 1H NMR (400 MHz,
CDCl3): δ=7.00 (dd, J=18.7, 8.0 Hz, 8H, ArH), 4.31 (bs,
2H, NH), 3.32–3.25 (m, 4H, CH2), 2.65 (t, J=6.9 Hz, 4H,
CH2), 2.24 (s, 6H, CH3) ppm; 13C NMR (100 MHz, CDCl3):
δ=158.0 (CO), 136.0 (2C), 135.9 (2C), 129.3 (4CH), 128.7
(4CH), 41.7 (2CH2), 35.9 (2CH2), 21.0 (2CH3) ppm; IR:
ꢀ =3341, 2894, 2359, 2313, 1670, 1539 cm−1.
Measurement of cell growth by SRB assay
The SRB assay is used for cell density determination, based
on the measurement of cellular protein content. The method
described here has been optimized for the toxicity screening
of compounds to adherent cells in a 96-well format. After
an incubation period, cell monolayers were fxed with 10%
trichloroacetic acid and stained for 30 min; then, excess dye
was removed by washing the cells repeatedly with 1% ace-
tic acid. The protein-bound dye was dissolved in 10 mM
Tris base solution for optical density (OD) determination at
510 nm using a microplate reader [45].
1,3‑Bis(4‑fluorophenethyl)urea (26, C17H18F2N2O) White
crystal; yield 11%; m.p.: 138–140 °C; 1H NMR (400 MHz,
CDCl3): δ = 7.06–7.00 (m, 4H, ArH), 6.93–6.85 (m, 4H,
ArH), 4.48 (bs, 2H, NH), 3.26 (t, J=6.9 Hz, 4H, CH2), 2.65
(t, J=6.9 Hz, 4H, CH2) ppm; 13C NMR (100 MHz, CDCl3):
δ=162.8 (CO), 160.4 (2C), 134.8 (2C), 130.1 (4CH), 115.4
(4CH), 41.6 (2CH2), 35.6 (2CH2) ppm; IR: ꢀ =3345, 2897,
2360, 2311, 1675, 1549 cm−1.
LDH release assay
Culture of HeLa, A549, and HDF cells
Cell membrane integrity is evaluated by LDH (lactate
dehydrogenase) method. For this purpose, the level of
cytoplasmic lactate dehydrogenase (LDH) released from
dead or damaged cells is determined. In this study, LDH
cytotoxicity assay kit (Cayman Chemical Company®, Ann
Arbor, MI, USA) was applied according to the manufac-
turer’ instructions for LDH assay application. The cells were
seeded to 96-well plates and from 6.25 to 200 µM of com-
pounds 20–26 were applied to cell culture as triplicates for
48 h. Then, 100 mm3 supernatant were transferred to a fresh
96-well plates and 100 mm3 of reaction mixture was added
to the samples and incubated for 30 min at room tempera-
ture. Finally, a microplate reader was used to analyze the
absorbance of the cultures at 490 nm [44].
The human cervical (HeLa), non-small cell lung (A549),
and human dermal fbroblast (HDF) cell line was purchased
from ATCC (American Type Culture Collection, Manas-
sas, VA, USA). The cells were grown and maintained in
Dulbecco’s Modifed Eagle Medium/Nutrient Mixture F12
(DMEM-F12) medium containing 10% fetal bovine serum
(FBS) and 1% penicillin–streptomycin (Gibco, Life Tech-
nologies GmbH, Germany). Cells were cultured in a humidi-
fed atmosphere at 37 °C in 5% CO2. Medium was refreshed
in every 2 days.
Measurement of cell growth by MTT assay
Anti-proliferative efects of compounds 20–26 on HeLa,
A549, and HDF were determined by MTT cell prolifera-
tion assay. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphe-
nyltetrazolium bromide) is a yellow auxiliary agent that
reduced formazan crystals in living cells. MTT was pur-
chased from Sigma Chemical Company (St. Louis, Missouri,
USA). 2×104 cells were inoculated in each well of 96-well
plates. The cells were incubated in the absence or presence
of compounds 20–26 (6.25, 12.5, 25, 50, 100, 200 µM)
Evaluation of antioxidant activity
The total radical scavenging capacity of the tested com-
pounds was determined by the DPPH• scavenging method
as per the reported procedure and compared to that of BHT,
β-carotene, trolox, and ascorbic acid [46]. The solution of
DPPH• was daily prepared, stored in a fask coated with
aluminum foil and kept in the dark at 4 °C. In brief, fresh
1 3

Synthesis, characterization, and biological evaluations of substituted phenethylamine‑based…
solution of DPPH• (0.1 mM) was prepared in ethanol. Then,
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