Synthesis and anticancer activity of some pyrimidine derivatives with aryl urea moieties as apoptosis-inducing agents

Article abstract of DOI:10.1016/j.bioorg.2020.104028

A new series of pyrimidine derivatives containing aryl urea moieties was designed and synthesized. The anticancer activities of all compounds were evaluated in vitro against colon and prostat cancer cell lines by MTT assay. Among these compounds, 4b exhibited the highest cytotoxic activity against SW480 cancer cell line with IC₅₀ value of 11.08 μM. Mechanistic studies showed that compound 4b arrested cell cycle at G2/M phase and induced apoptosis through upregulating Bax, Ikb-α and cleaved PARP and downregulating Bcl-2 expression levels. Moreover, compound 4b induced loss of mitochondrial membrane potential in SW480 cells. These results suggest that pyrimidine with urea moieties could be a template for designing new anticancer agents.

Full text of DOI:10.1016/j.bioorg.2020.104028

BioorganicChemistry101(2020)104028
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Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis and anticancer activity of some pyrimidine derivatives with aryl
urea moieties as apoptosis-inducing agents
⁎
Zühal Kilic-Kurta, ,1, Nuri Ozmenb, Filiz Bakar-Atesb
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara University, Ankara, Turkey
b Department of Biochemistry, Faculty of Pharmacy, Ankara University, Ankara, Turkey
A R T I C L E I N F O
A B S T R A C T
Keywords:
A new series of pyrimidine derivatives containing aryl urea moieties was designed and synthesized. The an-
ticancer activities of all compounds were evaluated in vitro against colon and prostat cancer cell lines by MTT
assay. Among these compounds, 4b exhibited the highest cytotoxic activity against SW480 cancer cell line with
IC50 value of 11.08 µM. Mechanistic studies showed that compound 4b arrested cell cycle at G2/M phase and
induced apoptosis through upregulating Bax, Ikb-α and cleaved PARP and downregulating Bcl-2 expression
levels. Moreover, compound 4b induced loss of mitochondrial membrane potential in SW480 cells. These results
suggest that pyrimidine with urea moieties could be a template for designing new anticancer agents.
Pyrimidines
Urea
Anticancer
Apoptosis mechanism
Synthesis
1. Introduction
lines. Among them, compound II (Fig. 2) was found the most active
derivative with IC50 values ranging from 1.42 to 6.52 µM against all the
Pyrimidine derivatives have attracted great interest in medicinal
chemistry because of their wide range of biological and pharmacolo-
gical activities [1,2]. To date, a large array of pyrimidine and pyr-
imidine-fused heterocyclic compounds having anticancer activity via
multiple different mechanisms and targets have been reported [3–7]. A
representative pyrimidine derivative Imatinib (Fig. 1) is the first kinase
inhibitor approved for the treatment of chronic myeloid leukemia
(CML) [8]. Clinical success of Imatinib has prompted substantial in-
terest in the further development of novel pyrimidines. Nilotinib
(Fig. 1), structurally related to Imatinib, is developed for the treatment
of imatinib-resistant chronic myelogenous leukemia [9]. Infigratinib
(Fig. 1), a pan-FGFR kinase inhibitor has a manageable safety profile in
phase 2 trial for cholangiocarcinoma treatment [10]. 2,4-Pyrimidine-
based drugs such as Rociletinib [11] and Osimertinib [12] (Fig. 1) are
presently used for EGFR triggered NSCLC.
cancer cell lines. Also, further mechanism studies of compound II
identified that cytotoxicity in EC-109 was mediated through inducing
apoptosis and arresting cell cycle at G2/M phase [14]. Munikrishnapp
et al. have reported in vitro cytotoxic activity of 5-bromo-pyrimidine
derivatives against HCT116, A549, K562 and U937 cell lines. Com-
pound III (Fig. 2) showed strong cytotoxicity against K562 cells and
comparable Bcr/Abl antiproliferation activity with IC50 value of
0.012 mg/mL [15]. El-Deeb et al. have reported the synthesis of N-
substituted-2-aminopyrimidines and their anticancer profiles. All com-
pounds tested over a panel of 60 cancer cell lines and 54 kinases. The
compound IV (Fig. 2) showed the strong cytotoxicity against the breast
cancer cells lines (T-47D and MDA-MB-468) with IC50 values of < 1 µM
and the high inhibition against multiple kinases including ABL1, AKT1,
LCK, C-SRC, PIM1, FLT3, FYN, and KDR [16]. Chikhale et al. have re-
ported the aminobenzazolyl pyrimidines and their anticancer activity.
Compound V (Fig. 2) showed highest potency against leukaemia, renal
and prostate cell lines with IC50 values of 0.7244, 0.8511 and
0.7932 µM, respectively. Also, PTK assay exhibited that the compound
V had promising inhibitory activity with IC50 value of 0.0711 µM [17].
On the other hand, diarylurea scaffold is also of wide interest due to
their diverse anticancer properties. Sorafenib (Fig. 3) is the first ex-
ample of kinase inhibitor bearing urea moiety that is used for treatment
of advanced renal cancer [18]. To date many diarylurea devivatives
In recent years, many substituted pyrimidine derivatives possessing
anticancer activity have been reported. For example, Luo et al. have
reported the in vitro anticancer activity of N-trisubstituted pyrimidine
derivatives against various human tumor cell lines. The compound I
(Fig. 2) showed the potent growth inhibitory activity in the solid CNE-2
tumor cell and the arrested cell-cycle at the G2/M phase [13]. Ma et al.
have reported that synthesis of some 1,2,3-triazole-pyrimidine hybrids
and evaluation of their anticancer activity against several cancer cell
⁎ Corresponding author.
E-mail address: zkurt@ankara.edu.tr (Z. Kilic-Kurt).
1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara University, Ankara, Turkey.
https://doi.org/10.1016/j.bioorg.2020.104028
Received 28 December 2019; Received in revised form 14 April 2020; Accepted 2 June 2020
Availableonline18June2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

Z. Kilic-Kurt, et al.
BioorganicChemistry101(2020)104028
Fig.1. The structures of several anticancer agents with pyrimidine ring.
Fig. 2. Reported pyrimidine derivatives with anticancer activity.
were reported as potential anticancer agents against various cancer cell
lines [19–22].
R2 position (Fig. 4). Anticancer activities of designed compounds
against colon (SW480) and prostat (PC3) cancer cell lines are presented
and discussed.
Previously, we reported some pyrrolopyrimidine derivatives de-
signed by merging substituted phenylurea moieties. Among them,
compounds bearing 3-trifluoromethyl- (VI, Fig. 3) [23] and 4-chloro-3-
trifluoromethylphenyl urea (VII, Fig. 3) [24] groups showed strong
cytotoxic effect through activation of the mitochondrial apoptosis
pathway in A549 and PC3 cells, respectively. Moreover, some analo-
gues of compound VI and VII bearing same R1 fragments used in this
work showed submicromolar cytotoxic activity on colon cancer cell line
and induced mitochondrial apoptosis through activation of p53-in-
dependent apoptotic signaling pathways (unpublished data). In this
work, we designed novel pyrimidine derivatives in the light of the
findings of previous compounds bearing pyrimidine ring. In order to
compare the activity and optimize the scaffold, fragments which were
previously used and showed promising results were selected as R1 and
2. Result and discussion
2.1. Chemistry
Synthesis of the target compounds (Scheme 1) started with the
chlorination reaction of 2,4-diamino-6-hydroxypyrimidine using POCl3
[25]. The urea linker at 4-position of pyrimidine ring was formed by the
reaction of 6-chloropyrimidine-2,4-diamine (1) or 6-phenylpyrimidine-
2,4-diamine (4) with isocyanate derivatives in the moderate yield [26].
For the preparation of the 6-phenylpyrimidine-2,4-diamine (4), 6-
chloropyrimidine-2,4-diamine underwent a Suzuki coupling reaction
with phenylboronic acid in DME in the presence of Pd(PPh3)4 and 2 M
2

Z. Kilic-Kurt, et al.
BioorganicChemistry101(2020)104028
Fig. 3. Chemical structures of sorafenib and pyrrolopyrimidine based urea derivatives VI [23] and VII [24].
Na2CO3 [27]. Final compounds (2a-e, 3a-e) were prepared via reaction
of the chloropyrimidines (2 and 3) with appropriate amine derivatives
in the mixture of DMF:H2O:IPA (1:1:1) under reflux [28].
2.2.2. Cell cycle analysis
In order to obtain a better understanding of the cytotoxic activity of
compound 4b, cell cycle assay was performed by treating SW480 cells
with IC50 concentration of 11.08 µM for 24 h. As shown in Fig. 5, the
cell population percent at G2/M phase increased to 45.3% in compound
4b treated group, whereas it was 21.6% in untreated control group,
suggesting that compound 4b induced a significant G2/M phase arrest
(p < 0.05).
2.2. Biological evaluation
2.2.1. Cell proliferation assay
The synthesized compounds (2a-e, 3a-e, 4a-b) were screened for in
vitro cytotoxicity against human colon cancer and human prostate
cancer cell lines using MTT assay. The results are given in Table 1. 3-
(Trifluoromethyl)phenyl) urea derivatives containing piperazine
moiety at the 4-position of pyrimidine ring (2a-d) showed cytotoxicity
in the range of 24.27–58.48 µM against SW480 cells. While the re-
placement of the piperazine moiety phenyl (4a) moiety did not change
cytotoxicity, morpholine ring (2e) decreased the activity against
SW480 cell line. Among the 4-chloro-3-(trifluoromethyl)phenyl)urea
derivatives, compounds bearing p-fluorophenyl (3b), 3-fluorobenzyl
(3c), phenethyl (3d) substituted piperazine ring have exhibited better
activity than corresponding methyl substituted derivative (3a) against
both SW480 and PC3 cell lines. Substitution of phenyl group (4b) in-
stead of N-methyl piperazine has remarkably increased the cytotoxicity.
Introduction of a chloro atom at the 4-position of phenylurea moiety
(3c, 3d and 4b) resulted in approximately 2-fold increase and drama-
tically improvement in cytotoxicity against SW480 and PC3 cell lines,
respectively. Compound 4b substituted with the rigid and hydrophobic
phenyl ring at the 6-position of pyrimidine ring showed the best cyto-
toxic effect against SW480 cell line with the IC50 value of 11.08 µM.
Based on the cytotoxicity profile of the synthesized pyrimidine deri-
vatives, compound 4b can be taken as lead to explore the effect of the
bulky and hydrophobic groups at the 4-position of pyrimidine ring for
the design of better compounds.
2.2.3. Apoptosis and possible mechanism
The evaluation of apoptotic effect of compound 4b was performed
through Annexin V binding assay. The SW480 cells were treated with
compound 4b at IC50 concentration and the annexin V bound cell po-
pulation were measured after a 24 h incubation. As shown in Fig. 6, the
percentage of cells in early apoptosis were recorded as to
5.69
0.16% while it was 0.7
0.28% in nontreated cells. The late
apoptotic cell population were also significantly increased to
3.52
0.30% in compound 4b treated group when compared to
control (p < 0.01).
The apoptotic efficiency of compound 4b on SW480 cells were also
shown through fluorescein staining (Fig. 7). The annexin V binding of
cells treated with compound 4b at IC50 concentration were observed
under a fluorescence microscope. The annexin V/FITC staining of
SW480 cells showed that the cells had undergone remarkable apoptotic
changes by compound 4b treatment.
Western blot analysis were displayed the alteration on expression of
apoptosis-related proteins. As shown in Fig. 8, the expression of anti-
apoptotic protein Bcl-2 significantly decreased, while pro-apoptotic Bax
expression increased by the treatment of SW480 cells with compound
4b (p
< 0.05). Caspases have important roles in many forms of
apoptosis [29]. They are synthesized as inactive zymogens in the cells
and following the receipt of apoptotic stimuli, they are cleaved into
Fig. 4. Design strategy of the target compounds.
3

Z. Kilic-Kurt, et al.
BioorganicChemistry101(2020)104028
Scheme 1. Synthesis method of the target compounds. Reagents and conditions: (a) POCl3, reflux, 3 h; (b) phenylisocyanate derivatives, ACN, rt; (c) appropriate
amine derivatives, DMF:H2O:IPA (1:1:1) mixture, reflux, 24–48 h; (d) PhB(OH)2, Pd(PPh3)4, 2 M Na2CO3, DME, 48 h.
Table 1
The IC50 values of the target compounds (2a-e, 3a-e, 4a-b) on SW480 and PC3 cells. The cells were treated with different concentrations of compounds ranged
between 0.1 and 50 μM and the cytotoxicity was determined by MTT assay
.
Compd.
R1
R2
R3
X
Inhibition of cell growth (IC50, μM)
SW480
PC3
2a
2b
2c
2d
2e
3a
3b
3c
3d
3e
4a
4b
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
H
CH3
N
N
N
N
O
N
N
N
N
O
N
N
25.82
24.27
58.48
24.55
90.33
134.38
35.33
24.90
12.61
73.13
25.90
11.08
117.38
79.51
190.19
141.39
162.82
91.46
47.86
16.43
25.16
404.71
75.82
17.39
H
p-F-Ph
H
-CH2-m-F-Ph
H
-CH2CH2Ph
H
–
Cl
Cl
Cl
Cl
Cl
H
CH3
p-F-Ph
-CH2-m-F-Ph
-CH2CH2Ph
–
–
–
Cl
their active substrates [30]. In order to determine whether caspase 3
activation was induced by compound 4b treatment, SW480 cells were
exposed to 11.08 μM of compound 4b for 24 h and pro-caspase-3 and
−9. The result showed that procaspase-3 and −9 expression have
significantly decreased (p < 0.05), confirming the caspase-activating
effect of Bax protein [31].
In accordance with these data, the caspase (+) cell population has
significantly increased to 9.17
0.21% in compound 4b treated group
4

Z. Kilic-Kurt, et al.
BioorganicChemistry101(2020)104028
Fig. 5. The effect of compound 4b on inhibition of cell cycle. The SW480 cells were treated with compound 4b with IC50 value of 11.08 μM for 24 h and the cell
population were detected by Muse Cell Analyzer (Millipore, Germany). The nontreated cells were used as control. The results indicate mean
independent experiments. The difference is * p < 0.05, compared to control.
S.D. of two
(p
<
0.0001) which was determined through multicaspase assay
compound-4b induced apoptosis occurs through mitochondria-medi-
(Fig. 9).
ated intrinsic apoptosis pathway. These findings demonstrate that
compound 4b could be used as a lead for designing new anticancer
compounds for further studies.
NF-κB is a key protein in regulating DNA transcription and con-
sidered as an apoptosis inhibitor [32]. TNF-α-induced Nf-κB activity is
reduced by overexpression of Ikb-alfa [33]. Treatment of SW480 cells
with compound 4b resulted in a decreased expression of Nf-κB and
increased expression of Ikb-α. Also, the expression of cleavage of PARP
considered to be a hallmark of apoptosis [34] increased when compared
to control (p < 0.05). Expression of PI3K which is a vital anti-apop-
totic protein in the growth factor signaling pathway decreased by
treatment of compound 4b (p < 0.05). Western blot results indicated
that the Bcl-2 expression was downregulated in SW480 cells after
treatment with compound 4b. Since the activation of caspase 9 and
caspase 3 suggest that the molecular mechanism of apoptosis might be
via the mitochondrial pathway [35], we also evaluated the effect of
compound 4b on mitochondrial membrane potential of SW480 cells.
So, compound 4b-treated SW480 cells were stained with Muse Mito-
potential assay dye. As shown in Fig. 10, treatment with 11.08 μM of
compound 4b resulted in an increase at depolarized/live cell popula-
tion percentage, indicating loss of mitochondrial membrane potential.
4. Experimental
4.1. General
Commercial reagents were used without further purification. The
NMR spectra (400 MHz) were recorded on Varian Mercury 400 NMR
spectrometer (Varian Inc., Palo Alto, CA, USA) using tetramethylsilane
as internal standard. TLC was performed on F254 (Merck) silica gel
plates. The ESI spectra were recorded on a Waters ZQ Micromass LC-MS
spectrometer (Waters Corporation, Milford, MA, USA) with ESI source
as ionization. Elemental analysis was performed using a Leco-932
CHNS-O analyzer (Leco, St. Joseph, MI, USA) within 0.4% of the
theoretical values.
4.2. Chemistry
3. Conclusion
4.2.1. Synthesis of 6-chloropyrimidine-2,4-diamine (1)
In conclusion, among the synthesized compounds, 4b bearing the
rigid and hydrophobic phenyl moiety at the 4-position of pyrimidine
ring showed the most cytotoxic activity against SW480 cell line with
IC50 value of 11.08 µM. Compound 4b also induced apoptosis in SW480
cells by upregulating Bax, Ikb-α and cleaved PARP and downregulating
Bcl-2 expression. Moreover, compound 4b has potent effect on mi-
tochondrial membrane potential in SW480 cells, indicating that
2,4-diamino-6-hydroxypyrimidine (2.25 g, 0.017 mol) was refluxed
in POCl3 (17 mL) for 3 h. The reaction mixture was added to ice-water
and stirred at 90 °C for 1 h. The pH of the mixture was adjusted to 7–9
with aqueous ammonia and then it was extracted with EtOAc. Organic
phase was dried over anhydrous Na2SO4 and evaporated to obtain pure
product 1.88 g, yield 73%. MS (ESI) m/z: 144.9 [M]+, 146.8 [M+2]
[25].
Fig. 6. The effect of compound 4b on Annexin V binding. The cells were treated with 11.08 μM of compound 4b for 24 h and the apoptosis was detected by Annexin
V binding assay. Nontreated cells were used as control. The figure represents four different cell population % as follows: Live, early apoptotic, late apoptotic and
dead. The differences are ** and *** from control; p < 0.01, p < 0.0001, respectively.
5

Z. Kilic-Kurt, et al.
BioorganicChemistry101(2020)104028
Fig. 7. Fluorescence imaging of compound 4b in SW480 cells. The cells were left nontreated (control), and treated with 11.08 μM of compound 4b and the
fluorescence images were obtained by annexin V staining protocol through fluorescence microscope (Olympus, Germany).
4.2.2. General procedures for synthesis of compound 2 and 3
4.2.3. Synthesis of 6-phenylpyrimidine-2,4-diamine (4)
This compound was similarly prepared, as described in lit [27].
Yield: 37%
To a solution of 6-chloropyrimidine-2,4-diamine (1, 1 eq) in acet-
onitrile, the phenylisocynate derivatives (1 eq) was added. The reaction
mixture was stirred at room temperature for overnight. After comple-
tion of the reaction; the precipitate formed was filtered, washed with
ethanol, and dried to obtain the pure product 2 and 3.
4.2.4. General procedures for synthesis of compound 4a and 4b
To a solution of 6-phenylpyrimidine-2,4-diamine (4, 1 eq) in acet-
onitrile, the phenylisocynate derivatives (2 eq) was added. The reaction
mixture was stirred at room temperature for overnight. After comple-
tion of the reaction; the precipitate formed was filtered and crystallized
by the mixture of ethanol and ethylacetate to obtain the pure product
4a and 4b.
4.2.2.1. 1-(2-Amino-6-chloropyrimidin-4-yl)-3-(3-(trifluoromethyl)
phenyl)urea (2). Yield: 45%; Mp 267 °C; 1H NMR (400 MHz, DMSO‑d6)
δ: 6.13 (s, 1H), 7.38 (d, 1H, J = 7.6 Hz), 7.54 (t, 1H), 7.70 (s, 2H, NH2),
7.90 (d, 1H, J = 8.4 Hz), 8.15 (s, 1H), 9.95 (s, 1H, NH), 11.63 (s, 1H,
NH). 13C NMR (100 MHz, DMSO‑d6) δ: 96.55, 115.56 (q, J = 3.8 Hz),
119.27 (q, J = 4.5 Hz), 123.41, 124.15 (q, J = 270.6 Hz), 129.52 (q,
J = 30.8 Hz), 129.74, 139.42, 151.54, 157.28, 158.02, 164.02. MS
4.2.4.1. 1-(2-Amino-6-phenylpyrimidin-4-yl)-3-(3-(trifluoromethyl)
phenyl)urea (4a). Yield: 40%; Mp 275 °C; 1H NMR (400 MHz,
DMSO‑d6) δ: 6.59 (s, 1H), 7.28 (s, 2H), 7.37 (d, 1H, J = 8.0 Hz),
7.51–7.58 (m, 4H), 7.88 (d, 1H, J = 8.4 Hz), 7.94–7.97 (m, 2H), 8.10
(s, 1H), 9.57 (s, 1H, NH), 12.34 (s, 1H, NH). 13C NMR (100 MHz,
DMSO‑d6) δ: 94.73, 115.14 (q, J = 3.9 Hz), 118.86 (q, J = 4.5 Hz),
122.77 (q, J = 272.7 Hz), 126.37 (2C), 128.70 (2C), 129.57 (q,
J = 31.2 Hz), 129.73, 129.87, 130.27, 136.93, 139.68, 152.10, 157.79,
161.52, 164.44. MS (ESI) m/z: 374.50 [M+1]. Anal. calcd. for
(ESI) m/z: 332.4 [M+H]+
.
4.2.2.2. 1-(2-Amino-6-chloropyrimidin-4-yl)-3-(4-chloro-3-
(trifluoromethyl)phenyl)urea (3). Yield: 51%; Mp 277 °C; 1H NMR
(400 MHz, DMSO‑d6) δ: 6.14 (s, 1H), 7.65 (d, 1H, J = 8.8 Hz), 7.70
(s, 2H, NH2), 7.93 (t, 1H), 8.28 (s, 1H), 10.03 (s, 1H, NH), 11.69 (s, 1H,
NH). 13C NMR (100 MHz, DMSO‑d6) δ: 96.63, 118.20 (q, J = 5.3 Hz),
122.77 (q, J = 271.2 Hz), 123.68, 124.73, 126.71 (q, J = 30.5 Hz),
131.81, 138.12, 151.54, 157.17, 158.05, 163.99. MS (ESI) m/z: 366.4
[M]+, 368.4 [M+2].
C
18H14F3N5O·0.25H2O (%): C, 57.21; H, 3.86; N, 18.53. Found (%):
C, 57.25; H, 4.23; N, 18.15.
4.2.4.2. 1-(2-Amino-6-phenylpyrimidin-4-yl)-3-(4-chloro-3-
(trifluoromethyl)phenyl)urea (4b). Yield: 38%; Mp 288 °C; 1H NMR
Fig. 8. Effects of compound 4b on apoptotic protein expression in SW480 cells. The cells were treated with 11.08 μM of compound 4b for 24 h and total cell lysates
were prepared and immunblotted to detect Bax, Bcl-2, caspase 3/-9, Ikbα, NfκB, PARP, cleaved PARP and PI3K. β-actin served as a loading control. Results were
expressed as the mean
S.D. *p < 0.05 as compared with control.
6

Z. Kilic-Kurt, et al.
BioorganicChemistry101(2020)104028
156.85, 162.66, 163.63. MS (ESI) m/z: 396.8 [M+1]. Anal. calcd. for
C17H20F3N7O·0.2H2O0.2CH3COOC2H5 (%): C, 51.26; H, 5.41; N, 23.51.
Found (%): C, 50.97; H, 5.26; N, 23.11.
4.2.5.2. 1-(2-Amino-6-(4-(4-fluorophenyl)piperazin-1-yl)pyrimidin-4-yl)-
3-(3-(trifluoro methyl)phenyl)urea (2b). Yield: 32%; Mp: 265 °C. 1H
NMR (400 MHz, DMSO‑d6) δ: 3.13 (t, 4H, piperazine protons), 3.60 (t,
4H, piperazine protons), 5.39 (s, 1H), 6.70 (s, 2H, NH2), 6.97–7.08 (m,
4H), 7.35 (d, 1H, J = 7.6 Hz), 7.52 (t, 1H), 7.89 (d, 1H, J = 8.4 Hz),
8.20 (s, 1H), 9.19 (s, 1H, NH), 12.23 (s, 1H, NH). 13C NMR (100 MHz,
DMSO‑d6) δ: 43.50 (2C), 48.75 (2C), 76.61, 115.13 (q, J = 3.8 Hz),
115.35 (2C, d, J = 22.1 Hz), 117.50 (2C, d, J = 7.6 Hz), 118.69 (q,
J
= 4.5 Hz), 122.97, 124.15 (q, J = 270.6 Hz), 129.46 (q,
Fig. 9. The effect of compound 4b on caspase levels. The cells were treated with
11.08 μM of compound 4b and the total caspase levels were detected through
Muse Cell Analyzer (Millipore, Germany). The results were given as
J = 31.3 Hz), 129.67, 139.80, 147.71 (d, J = 2.3 Hz), 152.29,
156.16 (d, J = 234.7 Hz), 156.83, 162.56, 163.60. MS (ESI) m/z:
476.56 [M+1]. Anal. calcd. for C22H21F4N7O·0.04H2O (%): C, 55.49;
H, 4.46; N, 20.59. Found (%): C, 55.10; H, 4.72; N, 20.37.
mean
S.D. of cell population % including caspase+, caspase+/dead and
dead cells. The differences are ** and *** from control; p < 0.01, p < 0.0001,
respectively.
4.2.5.3. 1-(2-Amino-6-(4-(3-fluorobenzyl)piperazin-1-yl)pyrimidin-4-yl)-
3-(3-(trifluoro methyl)phenyl)urea (2c). Yield: 44%; Mp: 201 °C. 1H
NMR (400 MHz, DMSO‑d6) δ: 2.40 (t, 4H, piperazine protons), 3.43 (t,
4H, piperazine protons), 3.51 (s, 2H, CH2), 5.30 (s, 1H), 6.62 (s, 2H,
NH2), 7.04–7.15 (m, 3H), 7.31–7.38 (m, 2H), 7.49 (t, 1H), 7.87 (d, 1H,
J = 8.4 Hz), 8.13 (s, 1H), 9.10 (s, 1H, NH), 12.20 (s, 1H, NH). 13C NMR
(100 MHz, DMSO‑d6) δ: 43.66 (2C), 52.11 (2C), 61.18, 76.56, 113.76
(d, J = 20.6 Hz), 115.12 (q, J = 3.8 Hz), 115.23 (d, J = 21.4 Hz),
118.72 (q, J = 4.5 Hz), 122.97, 124.18 (q, J = 270.6 Hz), 124.77,
129.50 (q, J = 31.2 Hz), 129.72, 130.08 (d, J = 8.4 Hz), 139.85,
141.10 (d, J = 6.8 Hz), 152.31, 156.84, 162.22 (d, J = 245.5 Hz),
162.59, 163.64. MS (ESI) m/z: 490.69 [M+1]. Anal. calcd. for
(400 MHz, DMSO‑d6) δ: 6.57 (s, 1H), 7.31 (s, 2H), 7.51 (t, 3H), 7.66 (d,
1H, J = 8.8 Hz), 7.91–7.95 (m, 3H), 8.17 (d, 1H, J = 2.4 Hz), 9.71 (s,
1H, NH), 12.43 (s, 1H, NH). 13C NMR (100 MHz, DMSO‑d6) δ: 94.83,
117.76 (q, J = 4.5 Hz), 121.41 (q, J = 272 Hz), 123.23 (q, J = 3.8 Hz),
124.06, 126.44 (2C), 126.70 (q, J = 30.5 Hz), 128.80 (2C), 130.37,
132.04, 136.93, 138.45, 152.17, 157.70, 161.56, 164.44. MS (ESI) m/z:
408.49 [M+1], 410.45 [M+2]. Anal. calcd. for C18H13ClF3N5O (%): C,
53.01; H, 3.21; N, 17.17. Found (%): C, 52.93; H, 3.52; N, 17.14.
4.2.5. General procedures for synthesis of compound 2a-f and 3a-f
The compound 2 or 3 (1 eq) and an appropriate amine derivative
(2 eq) was refluxed in the mixture of DMF:H2O:IPA (1:1:1) for 24–48 h.
After completion of the reaction; the solvent was evaporated under
reduced pressure. Water was added and extracted with EtOAc (3x20
ml). The organic layer was dried over anhydrous Na2SO4 and evapo-
rated under reduced pressure. The residue was crystallized with ethanol
to obtain pure compound 2a-f and 3a-f.
C23H23F4N7O (%): C, 56.43; H, 4.73; N, 20.03. Found (%): C, 56.12;
H, 5.04; N, 20.07.
4.2.5.4. 1-(2-Amino-6-(4-phenethylpiperazin-1-yl)pyrimidin-4-yl)-3-(3-
(trifluoromethyl) phenyl)urea (2d). Yield: 35%; Mp: 260 °C. 1H NMR
(400 MHz, DMSO‑d6) δ: 2.49 (t, 4H, piperazine protons), 2.55 (t, 2H,
CH2), 2.75 (q, 2H, CH2), 3.45 (t, 4H, piperazine protons), 5.38 (s, 1H),
6.61 (s, 2H, NH2), 7.15–7.35 (m, 5H), 7.34 (d, 1H, J = 7.2 Hz), 7.52 (t,
1H), 7.87 (d, 1H, J = 8.0 Hz), 8.16 (s, 1H), 9.09 (s, 1H, NH), 12.21 (s,
1H, NH). 13C NMR (100 MHz, DMSO‑d6) δ: 32.61, 43.67 (2C), 52.20
(2C), 59.58, 76.54, 115.10 (q, J = 3.8 Hz), 118.73 (q, J = 4.5 Hz),
122.94, 124.36 (q, J = 270.5 Hz), 125.76, 128.16 (2C), 128.56 (2C),
129.31 (q, J = 31.2 Hz), 129.68, 139.82, 140.29, 152.27, 156.80,
162.60, 163.60. MS (ESI) m/z: 486.64 [M+1]. Anal. calcd. for
4.2.5.1. 1-(2-Amino-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)-3-(3-
(trifluoromethyl)phenyl) urea (2a). Yield: 30%; Mp: 256 °C. 1H NMR
(400 MHz, DMSO‑d6) δ: 2.19 (s, 3H, CH3), 2.34 (t, 4H, piperazine
protons), 3.44 (t, 4H, piperazine protons), 5.32 (s, 1H), 6.65 (s, 2H,
NH2), 7.35 (d, 1H, J = 8.0 Hz), 7.52 (t, 1H), 7.89 (d, 1H, J = 8.4 Hz),
8.18 (s, 1H), 9.15 (s, 1H, NH), 12.23 (s, 1H, NH). 13C NMR (100 MHz,
DMSO‑d6) δ: 43.57 (2C), 45.76, 54.17 (2C), 76.57, 115.10 (q,
J
=
3.8 Hz), 118.75 (q,
J
=
4.5 Hz), 122.98, 125.57 (q,
C
24H26F3N7O·C3H7NO·0.05H2O (%): C, 57.96; H, 5.96; N, 20.02.
J = 272.5 Hz), 129.51 (q, J = 31.2 Hz), 129.75, 139.88, 152.35,
Found (%): C, 57.55; H, 5.64; N, 20.54.
Fig. 10. The effect of compound 4b on mitochondrial membrane potential in SW480 cells. The cells were treated with 11.08 μM of compound 4b and the cells were
stained with Muse Mitopotential assay kit. The histogram obtained from Muse Cell Analyzer showed the percentage of depolarization in SW480 cells following
treatment. Data represent of three independent experiments with similar results. **p < 0.001 as compared with control.
7

Z. Kilic-Kurt, et al.
BioorganicChemistry101(2020)104028
4.2.5.5. 1-(2-Amino-6-morpholinopyrimidin-4-yl)-3-(3-(trifluoromethyl)
phenyl)urea (2e). Yield: 15%; Mp: 235 °C. 1H NMR (400 MHz,
DMSO‑d6) δ: 3.39 (t, 4H, morpholine protons), 3.62 (t, 4H,
morpholine protons), 5.30 (s, 1H), 6.67 (s, 2H, NH2), 7.33 (d, 1H,
J = 8.0 Hz), 7.50 (t, 1H), 7.87 (d, 1H, J = 8.4 Hz), 8.16 (s, 1H), 9.14 (s,
1H, NH), 12.17 (s, 1H, NH). 13C NMR (100 MHz, DMSO‑d6) δ: 44.07
(2C), 65.76 (2C), 76.61, 115.18 (q, J = 3.8 Hz), 118.79 (q, J = 4.5 Hz),
123.05, 124.21 (q, J = 271.2 Hz), 129.52 (q, J = 31.4 Hz), 129.74,
139.84, 152.33, 156.82, 162.96, 163.65. MS (ESI) m/z: 383.51 [M+1].
Anal. calcd. for C16H17F3N6O2 (%): C, 50.26; H, 4.48; N, 21.98. Found
(%): C, 49.89; H, 4.88; N, 22.11.
[M+3]. Anal. calcd. for C24H25ClF3N7O·0.1H2O (%): C, 55.24; H, 4.86;
N, 18.79. Found (%): C, 54.85; H, 5.18; N, 18.83.
4.2.5.10. 1-(2-Amino-6-morpholinopyrimidin-4-yl)-3-(4-chloro-3-
(trifluoromethyl)phenyl) urea (3e). Yield: 37%; Mp: 262 °C. 1H NMR
(400 MHz, DMSO‑d6) δ: 3.42 (t, 4H, morpholine protons), 3.65 (t, 4H,
morpholine protons), 5.34 (s, 1H), 6.66 (s, 2H, NH2), 7.62 (d, 1H,
J = 8.8 Hz), 7.91 (dd, 1H, J = 8.8 Hz, 2.4 Hz), 8.28 (d, 1H,
J = 2.4 Hz), 9.14 (s, 1H, NH), 12.25 (s, 1H, NH). 13C NMR
(100 MHz, DMSO‑d6) δ: 44.04 (2C), 65.74 (2C), 76.62, 117.78 (q,
J = 5.3 Hz), 122.8 (q, J = 272 Hz), 123.09 (q, J = 3.8 Hz), 124.32,
126.68 (q, J = 30.5 Hz), 131.79, 138.54, 152.30, 156.70, 162.93,
163.59. MS (ESI) m/z: 417.48 [M+1], 419.51 [M+3]. Anal. calcd. for
4.2.5.6. 1-(2-Amino-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl)-3-(4-
chloro-3-(trifluoro methyl)phenyl)urea (3a). Yield: 25%; Mp: 263 °C. 1H
NMR (400 MHz, DMSO‑d6) δ: 2.18 (s, 3H, CH3), 2.31 (t, 4H, piperazine
protons), 3.43 (t, 4H, piperazine protons), 5.23 (s, 1H), 6.64 (s, 2H,
NH2), 7.62 (d, 1H, J = 8.8 Hz), 7.91 (dd, 1H, J = 8.0 Hz, 2.4 Hz), 8.28
(d, 1H, J = 2.4 Hz), 9.20 (s, 1H, NH), 12.30 (s, 1H, NH). 13C NMR
(100 MHz, DMSO‑d6) δ: 43.56 (2C), 45.72, 54.14 (2C), 76.61, 117.73
(q, J = 6.1 Hz), 122.81 (q, J = 271.3 Hz), 123.08 (q, J = 3.8 Hz),
124.27, 126.53 (q, J = 30.4 Hz), 131.81, 138.57, 152.31, 156.72,
162.64, 163.59. MS (ESI) m/z: 430.70 [M+1]. Anal. calcd. for
C
16H16ClF3N6O2·0.3H2O (%): C, 45.51; H, 3.96; N, 19.90. Found (%): C,
45.26; H, 4.09; N, 19.86.
4.3. Biological evaluation
4.3.1. Cell culture
Human SW480 colon adenocarcinoma (CCL-228) and human PC3
prostate carcinoma cell lines were purchased from American Type
Culture Collection. Cells were cultured in DMEM (Sigma, Germany)
media supplemented with 10% FBS (Sigma, Germany), 1% penicillin/
streptomycin (Sigma, Germany) and 1% L-glutamine (Sigma, Germany)
and incubated in a 5% CO2 humidified atmosphere at 37 °C. The syn-
thesized compounds were dissolved in 0.01% dimethylsulfoxide and
cells were treated with 0.1–50 μM of compounds.
C
17H19ClF3N7O·0.7H2O (%): C, 46.14; H, 4.64; N, 22.16. Found (%):
C, 45.83; H, 4.66; N, 21.91.
4.2.5.7. 1-(2-Amino-6-(4-(4-fluorophenyl)piperazin-1-yl)pyrimidin-4-yl)-
3-(4-chloro-3-(trifluoromethyl)phenyl)urea (3b). Yield: 24%; Mp: 270 °C.
1H NMR (400 MHz, DMSO‑d6) δ: 3.12 (t, 4H, piperazine protons), 3.59
(t, 4H, piperazine protons), 5.39 (s, 1H), 6.71 (s, 2H, NH2), 6.96–7.0
(m, 2H), 7.05 (t, 2H), 7.63 (d, 1H, J = 8.8 Hz), 7.92 (dd, 1H,
J = 8.8 Hz, 2.4 Hz), 8.32 (d, 1H, J = 2.4 Hz), 9.27 (s, 1H, NH),
12.31 (s, 1H, NH). 13C NMR (100 MHz, DMSO‑d6) δ: 43.50 (2C), 48.75
(2C), 76.64, 115.24 (2C, d, J = 22.1 Hz), 117.50 (2C, d, J = 7.6 Hz),
117.69 (q, J = 4.5 Hz), 122.77 (q, J = 272.1 Hz) 123.05 (q,
J = 3.8 Hz), 124.27, 126.64 (q, J = 30 Hz), 131.75, 138.52, 147.71
(d, J = 2.3 Hz), 152.28, 156.16 (d, J = 234.7 Hz) 156.72, 162.55,
163.57. MS (ESI) m/z: 510.63 [M+1], 512.51 [M+2]. Anal. calcd. for
4.3.2. MTT assay
The cytotoxic effects of synthesized compounds on SW480 and PC3
cells were evaluated by MTT assay. The cells were treated with the
synthesized compounds at 0.1, 1, 12.5, 25 and 50 μM concentrations for
24 h in a CO2 incubator. The cells were than treated with MTT reagent
(5 mg/ml) for 4 h and the reduced formazan crystals were dissolved in
DMSO and the absorbance at 540 nm was measured by a spectro-
photometer (Thermo, Germany). The untreated cells were used as
control. Each experiment was performed in triplicate and the IC50 va-
lues were calculated by using linear regression equations obtained from
the concentration vs viable cell amount % graphs.
C
22H20ClF4N7O (%): C, 51.82; H, 3.95; N, 19.22. Found (%): C, 51.46;
H, 4.26; N, 19.08.
4.2.5.8. 1-(2-Amino-6-(4-(3-fluorobenzyl)piperazin-1-yl)pyrimidin-4-yl)-
3-(4-chloro-3-(trifluoromethyl)phenyl)urea (3c). Yield: 35%; Mp: 230 °C.
1H NMR (400 MHz, DMSO‑d6) δ: 2.39 (t, 4H, piperazine protons), 3.43
(t, 4H, piperazine protons), 3.50 (s, 2H, CH2), 5.30 (s, 1H), 6.64 (s, 2H,
NH2), 7.03–7.13 (m, 3H), 7.32–7.38 (m, 1H), 7.60 (d, 1H, J = 8.4 Hz),
7.90 (dd, 1H, J = 8.8 Hz, 2.4 Hz), 8.24 (d, 1H, J = 2.8 Hz), 9.19 (s, 1H,
NH), 12.28 (s, 1H, NH). 13C NMR (100 MHz, DMSO‑d6) δ: 43.67 (2C),
52.11 (2C), 61.19, 76.61, 113.75 (d, J = 20.5 Hz), 115.23 (d,
J = 20.6 Hz), 117.77 (q, J = 4.5 Hz), 122.80 (q, J = 272 Hz),
123.07 (q, J = 3.8 Hz), 124.27, 124.77, 126.68 (q, J = 30.5 Hz),
130.08 (d, J = 8.4 Hz), 131.81, 138.56, 141.10 (d, J = 6.9 Hz), 152.31,
156.74, 162.23 (d, J = 242 Hz), 162.59, 163.60. MS (ESI) m/z: 524.73
[M+1], 526.75 [M+3]. Anal. calcd. for C23H22ClF4N7O·1.5H2O (%): C,
51.64; H, 4.71; N, 18.32. Found (%): C, 51.32; H, 4.71; N, 18.40.
4.3.3. Cell cycle assay
Cell cycle analysis was performed by Muse Cell Cycle Assay Kit
(Merck Millipore, Germany). The SW480 cells were treated with
11.08 μM of compound 4b for 24 h and the nontreated cells were used
as control. The cells were then fixed with ethanol and prepared for
analysis according to the instructions. The cell population at G0/G1, S
and G2/M stages of the cell cycle were measured by Muse Cell Analyzer
(Millipore, Germany).
4.3.4. Annexin V binding assay
In order to evaluate the apoptotic effects of synthesized compounds,
Annexin V binding assay was performed by using Annexin V/Dead Cell
assay kit (Merck Millipore), according to the manufacturer’s instruc-
tions. The cells were treated with compound 4b at 11.08 μM, which was
the IC50 value determined from the MTT measurements. Annexin V and
7-aminoactinomycin (7-AAD) dye were applied for 20 min, at room
temperature. Four different populations were monitored by using
Annexin V and/or 7-AAD positivity on Muse Cell Analyzer (Merck,
Millipore): non-apoptotic live (7-AAD negative, Annexin V negative),
non-apoptotic dead (7-AAD positive, Annexin V negative), apoptotic
live (7-AAD negative, Annexin V positive), and apoptotic dead (7-AAD
positive, Annexin V positive) cells.
4.2.5.9. 1-(2-Amino-6-(4-phenethylpiperazin-1-yl)pyrimidin-4-yl)-3-(4-
chloro-3-(trifluoro methyl)phenyl)urea (3d). Yield: 30%; Mp: 260 °C. 1H
NMR (400 MHz, DMSO‑d6) δ: 2.47 (t, 4H, piperazine protons), 2.52 (t,
2H, CH2), 2.74 (t, 2H, CH2), 3.43 (t, 4H, piperazine protons), 5.31 (s,
1H), 6.64 (s, 2H, NH2), 7.13–7.27 (m, 5H), 7.61 (d, 1H, J = 8.8 Hz),
7.90 (dd, 1H, J = 8.8 Hz, 2.4 Hz), 8.28 (d, 1H, J = 2.4 Hz), 9.20 (s, 1H,
NH), 12.30 (s, 1H, NH). 13C NMR (100 MHz, DMSO‑d6) δ: 32.61, 43.64
(2C), 52.20 (2C), 59.61, 76.56, 117.74 (q, J = 4.5 Hz), 122.77 (q,
J = 272 Hz) 123.02 (q, J = 3.8 Hz), 124.24, 125.78, 126.65 (q,
J = 30.5 Hz), 128.17 (2C), 128.57 (2C), 131.78, 138.54, 140.28,
152.29, 156.70, 162.60, 163.55. MS (ESI) m/z: 520.77 [M+1], 522.75
4.3.5. Fluorescence imaging
The SW480 cells were treated with compound 4b and the apoptotic
cells were visualized through staining of cells with Annexin V-FITC
8

Z. Kilic-Kurt, et al.
BioorganicChemistry101(2020)104028
Fluorescence microscopy kit (BD, Germany). Following treatment of
cells with compound 4b for 24 h, the cells were incubated with Annexin
V-FITC dye for 20 min at room temperature in dark. Images were ob-
tained using an inverted fluorescence microscope (Olympus, Germany).
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Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.104028.
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