Novel cyanothiouracil and cyanothiocytosine derivatives as concentration-dependent selective inhibitors of U87MG glioblastomas: Adenosine receptor binding and potent PDE4 inhibition

Article abstract of DOI:10.1016/j.ejmech.2020.113125

Thiouracil and thiocytosine are important heterocyclic pharmacophores having pharmacological diversity. Antitumor and antiviral activity is commonly associated with thiouracil and thiocytosine derivatives, which are well known fragments for adenosine receptor affinity with many associated pharmacological properties. In this respect, 33 novel compounds have been synthesized in two groups: 24 thiouracil derivatives (4a-x) and 9 thiocytosine derivatives (5a-i). Antitumor activity of all the compounds was determined in the U87 MG glioblastoma cell line. Compound 5e showed an anti-proliferative IC₅₀ of 1.56 μM, which is slightly higher activity than cisplatin (1.67 μM). The 11 most active compounds showed no signficant binding to adenosine A₁, A_2A or A_2B receptors at 1 μM. Brain tumors express high amounts of phosphodiesterases. Compounds were tested for PDE4 inhibition, and 5e and 5f showed the best potency (5e: 3.42 μM; 5f: 0.97 μM). Remakably, those compounds were also the most active against U87MG. However, the compounds lacked a cytotoxic effect on the HEK293 healthy cell line, which encourages further investigation.

Full text of DOI:10.1016/j.ejmech.2020.113125

European Journal of Medicinal Chemistry 212 (2021) 113125
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Novel cyanothiouracil and cyanothiocytosine derivatives as
concentration-dependent selective inhibitors of U87MG
glioblastomas: Adenosine receptor binding and potent PDE4
inhibition
,
*
a
b
a
c
Zafer Sahin a , Sevde Nur Biltekin , Leyla Yurttas , Barkin Berk , Yagmur Ozhan ,
€
ꢀ
Hande Sipahi c, Zhan-Guo Gao d, Kenneth A. Jacobson d, S¸ eref Demirayak a
a Istanbul Medipol University, School of Pharmacy, Department of Pharmaceutical Chemistry, Istanbul, Turkey
b Anadolu University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Eskisehir, Turkey
c Yeditepe University, Faculty of Pharmacy, Department of Toxicology, Istanbul, Turkey
d Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes
of Health, Bethesda, MD 20892-0810, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Thiouracil and thiocytosine are important heterocyclic pharmacophores having pharmacological di-
versity. Antitumor and antiviral activity is commonly associated with thiouracil and thiocytosine de-
rivatives, which are well known fragments for adenosine receptor affinity with many associated
pharmacological properties. In this respect, 33 novel compounds have been synthesized in two groups:
24 thiouracil derivatives (4a-x) and 9 thiocytosine derivatives (5a-i). Antitumor activity of all the com-
pounds was determined in the U87 MG glioblastoma cell line. Compound 5e showed an anti-proliferative
Received 6 November 2020
Received in revised form
7 December 2020
Accepted 20 December 2020
Available online 31 December 2020
IC50 of 1.56
showed no signficant binding to adenosine A1, A2A or A2B receptors at 1
amounts of phosphodiesterases. Compounds were tested for PDE4 inhibition, and 5e and 5f showed the
best potency (5e: 3.42 M; 5f: 0.97 M). Remakably, those compounds were also the most active against
U87MG. However, the compounds lacked a cytotoxic effect on the HEK293 healthy cell line, which en-
courages further investigation.
m
M, which is slightly higher activity than cisplatin (1.67
m
M). The 11 most active compounds
Keywords:
Thiouracil
Thiocytosine
Adenosine
U87
Antitumor
Phosphosdiesterase
PDE
m
M. Brain tumors express high
m
m
© 2020 Elsevier Masson SAS. All rights reserved.
HEK293
MCF7
1. Introduction
these chemical groups. Especially, effects of cyanothiouracil de-
rivatives, such as anti-inflammatory [2], antidiabetic [3,4], anti-
Purine and pyrimidine heterocycles are important chemical
moieties that constitute structural components of nucleotides. Both
of these bases themselves and their nucleoside phosphate struc-
tures have functions on various receptors and thus on physiological
processes. Many of these substances themselves cannot be used as
drugs, as they do not remain in circulation for a sufficient time.
However, through development based on these structures, purine
and pyrimidine receptors have been considered as drug targets [1].
Different pharmacological activities have been investigated on
tumor [5e13] and antifungal [14,15] activities, have been
discovered in some recent studies. Previously, thiouracil derivatives
have been investigated on different cancer cell lines in vitro, but
there is need to study their effects on brain tumor cells. As shown in
Fig. 1, cyanothiouracil derivatives compound I, II and III showed
moderate inhibition on MCF7, HOP92 and HC116 cell lines. Purine
and pyrimidine bases are found to be effective on phosphodies-
terases, adenosine receptors and kinases. Glioblastomas expresses
high amounts of these macromolecules, so purine and pyrimidine
derivatives can be considered as a potential source of inhibitors of
proliferation. These studies have encouraged efforts on cyano-
thiouracils and cyanothiocytosines. The 4-oxo forms are preferred
over hydroxy forms when representing cyanothiouracils; whereas,
* Corresponding author.
E-mail address: zsahin@medipol.edu.tr (Z. Sahin).
https://doi.org/10.1016/j.ejmech.2020.113125
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

Z. Sahin, S.N. Biltekin, L. Yurttas et al.
European Journal of Medicinal Chemistry 212 (2021) 113125
Fig. 1. Synthesized compounds and pharmacologically active molecules bearing the same fragments.
amino forms are preferred over imino forms for cyanothiocyto-
sines, with respect to tautomeric states described for 4-oxo and 4-
amino pyrimidines [16,17].
adenosine receptor agonists and partial agonists that do not
contain ribose moieties (non-ribose nucleoside analogs) have been
introduced in the literature in recent years [20,22,23]. Adenosine A1
and A3 agonists and A2A and A2B antagonists show antitumor ac-
tivity, with respect to their secondary messengers. Selective in-
hibitors of each subtype have been investigated for their antitumor
potential in vitro and/or in vivo.
Glioblastomas are responsible for almost 75% of aggressive
malignant brain tumors. In the United States, approximately ten
thousand people are diagnosed with malignant glioma annually,
and survival of only 1 year is expected for 50% of these patients [24].
U87MG (Uppsala 87 Malignant Glioma) human glioma primary
brain tumor cells are from an aggressive type of brain tumor and
express high levels of adenosine receptors (especially A2 and A3),
phosphodiesterase enzymes (especially subtype 4A) and kinases,
and only a few drugs are being used to treat this tumors. Temo-
zolomide is one of the primary options, which becomes ineffective
upon the development of resistance, and U87 glioblastomas may
also produce resistance against cisplatin and other effective drugs
[25e27]. Thus, combination therapy or discovery of novel potent
molecules is required for an effective treatment [28,29]. Kinases
(mainly epidermal growth factor receptor, EGFR) and phosphodi-
esterases are potential targets for inhibition of U87MG cell line
Compounds similar to cyanothiocytosine, where the 4-oxo
group of thiouracil is replaced with an amino group, have mainly
been investigated for adenosine (A1, A2A, A2B, A3) receptor binding
affinities and pharmacological activities in such conditions as
cancer, heart failure, etc. [1,18e20]. Capadenoson (IV), a selective
adenosine A1 agonist, was evaluated for heart failure but with-
drawn from clinical trials due to side effects. LUF6941 (V) is a se-
lective A1 agonist with enhanced residence time. VI is another
selective A1 agonist with a binding Ki value of 1.9 nM. Furthermore,
these derivatives have potential as antitumor agents, as they are
bearing a pyrimidine core, which is commonly studied on different
cell lines, but not extensively studied on brain tumor cells.
Adenosine receptor agonist and antagonist ligands typically
contain a nitrogenous ring and an exocyclic amino group. This NH
or NH2 group forms a hydrogen bond with an Asn amino acid
residue in the binding site of adenosine receptors, as was shown for
adenosine itself in an X-ray crystallograpic structure [21]. Also,
most of the early generation of adenosine agonists contain a ribose
sugar that will interact with a hydrophilic region of the binding site
in the center of the transmembrane helical bundle. In addition,
2

Z. Sahin, S.N. Biltekin, L. Yurttas et al.
European Journal of Medicinal Chemistry 212 (2021) 113125
proliferation. Rolipram, a potent phosphodiesterase 4 inhibitor,
potently inhibited U87 cell proliferation, but the mechanism of
action is still unknown. It is still unclear whether the activity is
related to phosphodiesterase or kinases [30e32].
2. Results
2.1. Chemistry
In this study, a series of novel cyanothiouracil and cyanothio-
cytosine derivatives (4a-x, 5a-i) (Table 1) were synthesized, char-
acterized and evaluated for their antiproliferative potential against
glioblastoma tumor (U87MG) and human embryonic kidney
(HEK293) healthy cell lines. The 11 most potent compounds (4d, 4l,
5a-i) were subjected to adenosine A1, A2A and A2B receptor binding
There are several structures that have characteristic peaks in the
FT-IR spectra of the final compounds. A C^N triple bond stretch
band was observed in all compounds in the range of
2200e2210 cmꢀ1. Bands belonging to the carbonyl group were
observed in the 1600-1700 cmꢀ1 range for all final compounds. In
compounds with methoxy and methylenedioxy groups, character-
istic CeO single bond stretch bands were observed between 1200
and 1300 cmꢀ1. First aliphatic and then aromatic CeH stretch bands
were observed in the range of 2800e3000 cmꢀ1. The NH group of
ring A, as seen in one tautomeric representation, could be observed
in some of the IR spectra. The NeH stretch bands of the NH2 group
assays at a 1 mM concentration to determine if there is an interac-
tion with these receptors. Antitumor activity could not be associ-
ated with adenosine receptors, and subsequently the active
compounds were subjected to a phosphodiesterase 4 assay and an
additional MTT assay in MCF7 cells, a common breast cancer cell
line that does not express high levels of adenosine receptors or the
same type of phosphodiesterases as U87MG cells [33]. Briefly, we
evaluated in vitro whether the 4-oxo and/or 4-amino type novel
non-nucleoside analogs display antitumor activity, and mechanism
of action was investigated in PDE4 and adenosine receptor assays.
in the group of compounds 5a-i were observed around 3300 cmꢀ1
.
The hydrogens in the compounds can be seperated in 3 main
groups as aliphatic, aromatic and those bound to heteroatoms. The
first aliphatic group is a methyl group attached to the thiazole ring.
Proton NMR peaks belonging to this group were observed in all
Table 1
IC50 (mean SD) values of synthesized compounds and cisplatin on HEK293 and U87MG cells.
IC50 (mM)
Compound
Ring
R1
R2
U87MG
HEK293
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
B
B
B
B
B
B
B
B
B
H
H
H
H
H
H
H
H
H
56.61 0.02
59.72 0.04
48.36 0.01
15.33 0.03
28.21 0.06
43.35 0.05
65.04 0.01
33.69 0.02
57.25 0.02
15.34 0.06
25.27 0.03
12.47 0.03
78.90 0.02
45.93 0.01
68.47 0.02
65.28 0.02
27.57 0.03
40.97 0.02
35.49 0.03
38.64 0.05
40.13 0.05
53.01 0.01
83.89 0.01
55.53 0.01
28.95 0.02
13.98 0.03
10.80 0.08
8.54 0.04
1.56 0.09
4.54 0.06
11.42 0.04
10.57 0.04
3.42 0.06
1.67 0.04
93.54 5.10
90.24 3.63
76.24 6.59
91.77 3.29
ND
ND
85,07 0.33
ND
3-OMe
4-OMe
3,4-diOMe
3-Cl
4-Cl
3,4-diCl
3,4-OCH2O-
H
3-OMe
4-OMe
3,4-diOMe
3-Cl
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
H
ND
95.39 5.16
ND
23.30 3.14
ND
63.01 0.46
45.60 2.51
ND
ND
77.93 0.41
ND
4-Cl
3,4-diCl
3,4-OCH2O-
H
3-OMe
4-OMe
3,4-diOMe
3-Cl
4s
4t
ND
4u
4v
4w
4x
5a
5b
5c
5d
5e
5f
30.05 1.92
75.07 9.24
46.89 1.72
81.44 4.41
46.69 4.65
ND
ND
ND
ND
39.16 1.38
ND
ND
72.96 2.33
8.44 0.86
4-Cl
3,4-diCl
3,4-OCH2O-
H
4-OMe
4-Cl
H
H
H
4-OMe
4-OMe
4-OMe
4-Cl
4-Cl
4-Cl
4-OMe
4-Cl
H
4-OMe
4-Cl
5g
5h
5i
cisplatin
(ND: Not determined. Means viability is higher than 50% at 100 mM).
3

Z. Sahin, S.N. Biltekin, L. Yurttas et al.
European Journal of Medicinal Chemistry 212 (2021) 113125
compounds in the range of 2.6e2.7 ppm as singlets with a 3H
integration. Other aliphatic hydrogens observed belong to the
methylene (CH2) group in the neighborhood of sulfur and carbonyl.
The hydrogens of this group were observed for all compounds as
singlets with a 2H integration in the range of 4.45e4.7 ppm. The
compounds 4b-d, 4i, 4r-t, 5b, 5d-5f, and 5h carry a methoxy (OCH3)
group. These methyl groups attached to oxygen were observed in
the range of 3.6e3.85 ppm as a singlet with 3H integration (Fig. 2).
In the compounds 4d, 4l and 4t carrying 3,4-dimethoxyphenyl
group, two of the methoxy groups overlapped, and a singlet was
observed with 6H integration. CH2 hydrogens in compounds 4h, 4p
and 4x carrying methylenedioxy groups were observed in the
5.85e5.92 ppm range with 2H integration.
Aromatic hydrogens belonging to the A and B rings (Fig. 2) were
observed in the range of 6.7e8.0 ppm. While the peaks in the
compounds with a methoxy (OCH3) group are approaching 6.7 ppm
due to their electron donor property, in the compounds containing
a Cl substituent approached 8 ppm due to the electron withdrawing
effect. In 4-substituted phenyl derivatives, peaks were observed as
doublet with 2H integration, representing 1e4 splitting, except for
overlapping signals. Since the compounds 4f, 4n, 4q-x, 5c and 5f-i
carry 4-Cl substituents, two peaks were observed as a doublet in
the aromatic region and with 2H integration in these derivatives. In
some compounds, peaks could not be differentiated due to over-
laps, and multiplets were observed. NH2 or OH protons defined as X
in the Figure, were not observed possibly due to the tautomeric
structure. The aliphatic and aromatic hydrogen numbers were
provided as observed for all compounds, and the structure of the
compounds was assured.
positioning. Finally, the peak of the carbonyl group, one of the
important determining groups of our compounds, was detectable
in the range of 180e190 ppm. High resolution mass spectra gave
the Mþ1 peaks accurately.
2.2. Biological activity
2.2.1. U87MG and HEK293 cellular antitumor and cytotoxic activity
measurement
The compounds’ antitumor and cytotoxic activity is summarized
in Table 1. Accordingly, most of the compounds showed anti-
proliferative activity on U87MG aggresive glioblastoma. Further-
more, almost all compounds seem did not attenuate the prolifer-
ation of healthy, noncancerous human embyonic kidney (HEK)
293 cell line.
As seen in the effects of the compounds over a concentration
range, the degree of inhibition increased with concentration
(Table 2). At a concentration of 5 mM, compounds 5b-i were able to
reduce viability by 70%. Compounds 5b-i at a concentration of
Table 2
% Viability values SD of synthesized compounds (5
cisplatin against U87MG tumor cells.
mM, 10 mM, 50 mM, 100 mM) and
Compound No.
5
mM
10
mM
50
m
M
100 mM
4a
4b
4c
4d
4e
4f
4g
4 h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
92.10 4.14
99.00 3.33
97.30 0.62
80.80 6.19
86.90 1.67
90.90 2.11
98.60 6.68
93.40 4.44
99.00 3.48
71.30 1.10
97.00 6.31
81.20 3.45
95.7 3.53
102.00 9.35
101.00 6.46
100.00 4.02
88.9 3.72
96.6 4.09
92.1 2.73
93.4 0.80
80.2 5.63
99.00 4.97
101.00 3.46
101.00 3.76
103.00 3.08
68.8 3.46
62.9 1.93
61.8 3.38
35.6 2.95
51.4 1.25
71.1 3.57
69.7 0.78
46.1 1.95
31.7 1.53
87.5 2.12
90.2 2.20
90.4 0.77
69.8 0.37
83.7 3.58
85.2 4.29
92.5 2.32
84.2 2.61
90.9 0.84
66.7 1.54
80.3 0.96
57.6 3.49
90.6 3.17
92.7 2.95
88.6 1.97
88.3 2.76
83.1 2.62
89.7 1.45
88.3 3.64
83.3 3.18
95.2 3.05
98.4 0.59
97.1 1.02
95.0 3.09
93.0 2.95
60.8 3.66
58.8 1.24
57.8 2.40
28.7 2.26
44.0 2.67
67.9 2.28
64.5 3.08
37.3 3.52
18.2 1.47
77.5 2.17
80.3 4.10
52.1 1.86
21.4 3.52
50.6 2.63
63.4 3.37
75.3 1.84
42.6 2.39
72.5 2.83
43.2 1.18
32.6 2.25
22.2 2.57
83.4 3.72
48.0 2.64
78.4 3.04
83.2 3.65
41.6 1.47
47.6 1.30
46.3 3.06
59.8 3.56
52.6 2.77
60.0 1.71
93.9 1.11
68.3 2.86
26.3 3.17
33.8 2.51
44.3 3.90
16.5 1.27
13.0 1.75
23.1 3.02
15.2 2.87
17.8 2.40
15.5 2.85
6.71 1.64
9.23 1.09
21.8 2.77
1.08 0.45
6.48 0.60
10.0 1.79
22.5 2.25
24.4 2.11
18.7 2.53
10.9 4.02
12.4 0.78
11.5 3.86
5.39 1.22
40.6 3.08
2.85 0.87
31.1 2.80
22.9 2.28
11.4 1.58
8.54 2.66
16.6 1.40
18.7 1.54
12.5 2.26
12.8 3.65
36.5 3.66
9.91 1.20
6.48 1.70
17.2 1.21
17.1 2.84
5.25 0.37
0.26 0.45
3.90 0.79
4.59 1.06
0.79 0.79
1.65 0.87
2.25 1.86
For interpretation of 13C NMR spectral results, it is possible to
group the carbon atoms in compounds into several groups. The first
of these is the CH3 carbon in thiazole ring at position 4. The peak for
this carbon was observed at 18.7 ppm in all compounds. It is ex-
pected that the methylene group between the sulfur and carbonyl
group in the structure would be observed in the 38e40 ppm range.
Since the spectra are taken in DMSO, the solvent peaks are also
observed in this range in proximity to the CH2 carbon. The peak of
OCH3 carbon was observed around 55 ppm in all compounds
containing methoxy groups. In compounds containing more than
one methoxy, these peaks coincided in some compounds, and it
was possible to observe them separately in others. The carbon atom
to which the cyano group is bound in the A ring, is expected and
found in the range of 80e100 ppm. In compounds 4h, 4p and 4x
containing methylenedioxy groups attached to the aromatic ring,
the OeCH2eO carbon was observed in the range of 102e108 ppm.
The carbon atom belonging to the CN group was observed at
~115 ppm. There are 4 aromatic rings in the compounds, two of
which are phenyl and the other two are heteroaromatic rings. As
expected, phenyl ring carbons were detected between 110 and
148 ppm. The carbon atoms of the heteroaromatic pyrimidine and
thiazole rings were observed in the range of 150e170 ppm. These
carbons can sometimes be observed overlapping due to their
symmetrical state and the shading effect in three-dimensional
4s
4t
4u
4v
4w
4x
5a
5 b
5c
5d
5e
5f
5g
5 h
5i
Cisplatin
Fig. 2. 1H NMR interpretation summary of the compounds.
4

Z. Sahin, S.N. Biltekin, L. Yurttas et al.
European Journal of Medicinal Chemistry 212 (2021) 113125
Table 3
10
tration, while only 4d, 4l and 4j from the 4a-x group caused <70%
viability. Although compounds 4a-x and 5a at 50 M showed a
significantly increased antiproliferative effect, on average it
remained above 45% (excluding 4d, 4l and 4j), while compounds
between 5d-i reduced the viability to <20% on average. A signifi-
cant increase in the efficacy of all compounds was observed at a
mM showed a modest increase compared to the 5 mM concen-
IC50 (mean SD) values of compounds and cisplatin on
MCF7.
m
Compound
IC50 (mM)
4d
4l
5a
5b
5c
5d
5e
5f
63.71 1.92
24.02 0.95
30.14 1.62
27.15 0.95
20.17 0.75
72.12 2.33
24.31 1.95
11.25 0.09
25.22 1.17
10.09 0.03
12.52 1.13
2.51 0.29
100 mM concentration, and compounds 4d, 5e, 5h and 5i produced
an even greater effect than the standard cisplatin (Fig. 3, Table 2).
In general, compounds 5b-i were significantly more active than
the restofseries (4a-x) except for 4d, 4j, and 4l. Unlike the 4a-xgroup,
these compounds (5a-i) carry the amino group instead of the oxo at
the 4thposition of the pyrimidinering. Compound5aalsocontains an
amino group at the 4 position, but its aromatic rings at both ends of
the compound are unsubstituted. This compound showed reduced
inhibition compared to 5b-i. Concentration dependent viability re-
sults for all synthesized molecules are summarized in Table 2.
With respect to the effects of the compounds on the HEK293
healthy cell line, 50% inhibition was not observed for 16 of the 33
5g
5h
5i
Cisplatin
The MCF7 cell line is known to express kinases and phospho-
diesterases, but the range of isoforms present may change from a
cell line to the other. For example, brain tumors like glioblastomas
usually expresses higher amounts of PDE4B and PDE7 than MCF7
cells. The lower anticancer activity observed is possibly due to this
situation. The choice of a cell line to study after U87MG will require
consideration of the mechanism of action. Among the compounds
tested on MCF7 cells, 5f, 5h and 5i showed higher activity than the
other 8 compounds.
synthesized compounds even at 100
mM. The IC50 values of the
remaining molecules were quite high, that is, the cytotoxic effects
of the molecules were very low. This situation is remarkable and
important in terms of antitumor drug properties. Of the com-
pounds, in particular 5e showed an IC50 of 1.56
while the IC50 of cisplatin was 1.67 M (Table 1). Cisplatin also
showed an IC50 of 8.44 M on the healthy HEK293 cell line. Com-
pound 5e had no cytotoxic effect even at high concentrations. Apart
from that, while 5i: 3.42 M, 5f: 4.54 M and 5d had an IC50 of
8.54 M against U87MG cells, these molecules did not show any
cytotoxic effect in HEK293 cells. Although the compounds do not
have significant effects in most of the series at 5 M concentration,
mM against U87MG,
m
m
2.2.3. Measurement of adenosine receptor binding affinity
The relationship of adenosine ligands with antitumor activity
is mainly on glioblastomas. It is known that adenosine concen-
m
m
m
tration (1.5
than normal cells (3
all subtypes. The agonism of A3 receptors and the antagonism of
2A may provide antitumor activity, but the antitumor effect of A3
m
M) in extracellular fluid of glioblastomas is lower
m
m
M). Antitumor effect studies are ongoing in
as seen in Figs. 3, 5b-i compounds showed higher effects compared
to the 4a-x group. Although there was no significant increase in the
A
effect at 10
mM, the efficiency of compound 5e became evident and
receptor agonists is most evident in vivo models [34]. U87MG
glioblastoma tumor cells were selected for the reasons described
above to determine the pharmacological activity basis of the
compounds. The degree of binding of compounds 4d, 4l, 5a-i to
adenosine A1 and A2A receptor subtypes was measured. The most
antitumor compound 5e additionally subjected to an A2B assay to
probe the basis of its activity. According to the data given in
Table 4, the values are not high enough to be considered receptor
became closer to cisplatin. At a concentration of 50
m
M, the active
and non-active compounds were clearly delineated. Accordingly, 11
compounds, 4d, 4l, and 5a-i, were considered active and were
tested for adenosine receptor binding affinity.
2.2.2. MCF7 cellular antitumor activity measurement
As shown in Table 3, the compounds displayed an anti-
proliferative effect on MCF7 cell line. Although the IC50 values were
considerable, the anticancer activity was not high as U87MG cell
line. Different cell lines have different levels of specific proteins
such as enzymes and receptors that may affect proliferation. In
general, cancer cell lines typically highly expresss proteins that are
considered as cancer drug targets.
blockers, at least at a fixed concentration of 1 mM. Compounds 4d,
5a, 5e and 5g are the compounds showing the most binding in-
hibition (8.7e10%) at the A1 subtype. The highest degree of inhi-
bition at the A2A receptor was observed for 4d and 4l, with 12%
and 8.5% inhibition, respectively. Compound 5e showed 18%
binding at 1 mM.
Fig. 3. Effect of increasing concentrations of 5b-i on viability of U87MG cells.
5

Z. Sahin, S.N. Biltekin, L. Yurttas et al.
European Journal of Medicinal Chemistry 212 (2021) 113125
Table 4
Inhibition of human A1 and A2A receptor radioligand binding by the synthesized substances (at 1 mM, expressed as percent of specific binding).
Cpd No.
A1 [3H]DPCPX
A2A [3H]ZM241385
Cpd No
A1 [3H]DPCPX
A2A/A2B [3H]ZM241385
4d
4l
5a
5b
5c
5d
10
2.5
9.5
ꢀ4.6
ꢀ23
ꢀ2.8
12
8.5
6.5
0.8
5e
5f
5g
5h
5i
9.0
1.1
8.7
1.2
ꢀ18/18a
ꢀ7.1
ꢀ1.4
ꢀ22
ꢀ9.9
ꢀ3.7
1.8
ꢀ2.6
a
Inhibition of A2B receptor binding by 10 mM 5e was 49%. Only 5e was measured at the A2B receptor.
It seems that some of these values are negative values. In this
radioligand binding method, negative (ꢀ) values are not meaningful
and will be considered as zero. It was observed that compounds 4d,
4l and 5a showed weak binding at both A1 and A2A subtypes. Among
the other compounds, 5g and 5h showed weak binding only on A1
receptors, 5e showed weak binding on A2A and A2B, and 5b and 5i
were found to show only weak binding on A2A receptors.
Adenosine receptor binding was only evaluated at a concen-
tration of 1 mM, and none of the compounds at this concentration
showed high levels of binding. In the literature, adenosine ligands
have been reported act at the nanomolar (nM) level. In addition, the
A1 receptor activities of some compounds at a concentration of
10
mM were found to be similar [35,36]. For a molecule to be
Fig. 4. Logaritmic curves of active compounds on PDE assay.
considered an adenosine receptor ligand, some inhibition at the
submicromolar level would be expected, so full concentration
curves have not been determined. Therefore, the anticancer effect
of the compounds is not associated with adenosine A1 or A2A re-
ceptor subtypes.
pyrimidine ring. Compound 4l and 4j also has a 4-methoxy group
on the other phenyl ring attached to the thiazole. It is noteworthy
that compound 5e, which is the most active in the 5a-i group, also
carries 4-methoxyphenyl groups at both ends, parallel to the
structures of 4d and 4l. In addition, compounds 4c, 4n, 5h, which
2.2.4. Measurement of PDE4B assay
act at 100 mM, also carry a methoxy group on one of the phenyl
Table 5 summarizes the IC50 values of the compounds tested for
PDE4 inhibition. 3-Isobutyl-1-methylxanthine (IBMX), a general
rings. The activity of structures bearing methylenedioxy and chlo-
rine substituents remained relatively low. The IC50 value of temo-
zolomide, the drug used most frequently against glioblastoma
tumors, affected the U87MG cell line only in the range of
130e171 mM [37]. From this view, our compounds are important
hits as they are potent and not cytotoxic.Fig. 5.
Compounds with the highest antitumor activity did not show
any parallel in adenosine A1 or A2A receptor results. The lack of
correlation of adenosine receptor results and the antitumor effect
precludes those receptors as the biochemical basis of the high
antitumor effect. U87MG cells are in the class of glioblastomas, an
important type of brain tumors. This group of tumors express
phosphodiesterase 4 in particularly high amounts. Apart from that,
it expresses kinase enzymes in high amounts as in all tumor types.
MCF7 cells do not express PDE4 as much as U87MG. Considering
the MCF7 cellular activity and phosphodiesterase enzyme results,
the effect seems likely to occur through the phosphodiesterase
enzyme. Compounds 5e, 5f and 5i that showed the highest effect in
the U87MG cell line, were similarly the most effective compounds
of the series in phosphodiesterase enzyme activity.
phosphodiesterase standard, had an IC50 value of 14.31
pram, a potent PDE4B inhibitor, showed a 0.32 M IC50, which is
consistent with the literature data. Compound 5e and 5f showed
good potency on PDE4B, with 3.15 M and 0.97 M IC50 values
mM. Roli-
m
m
m
respectively. These inhibitory potencies are considerably high, i.e.
close to the most potent PDE4B inhibitor rolipram (Fig. 4). There is a
close correspondence between the most active compounds against
U87MG cells and in the PDE4B assay. This parallelism is remarkable
in the context of suggesting a basis of antitumor activity on U87MG.
When comparing the chemical structures of the compounds
with U87 antitumor activity, it seems that compounds 5a-i (thio-
cytosine derivatives) contain exocyclic amino groups in common.
4d, 4l and 4j were the only active compounds in the cyanothiouracil
series 4a-x, and they have methoxyphenyl groups on the
Table 5
IC50 (mean SD) values of compounds, IBMX and rolipram
on PDE4B.
Cpd
PDE4B IC50 (mM)
4d
4l
5a
>100
17.11 0.32
>100
3. Conclusion
The importance of pyrimidine and thiouracil derivatives in drug
development studies has been clearly demonstrated in recent
studies. Developing resistance to temozolomide drug encourages
the discovery of effective new drugs and the development of
combination therapy for glioblastoma tumors [38,39]. Thus, the
current results are relevant to others working on similar chemo-
types and/or pharmacological effects.
5b
5c
5d
5e
5f
5g
5h
5i
33.14 0.13
32.26 0.37
11.17 0.12
3.15 0.12
0.97 0.35
22.13 0.56
18.11 0.34
24.14 2.15
0.32 0.01
14.31 0.02
Rolipram
IBMX
The studies conducted within the scope of this article have been
important in terms of evaluating the thiouracil and thiocytosine
6

Z. Sahin, S.N. Biltekin, L. Yurttas et al.
European Journal of Medicinal Chemistry 212 (2021) 113125
Fig. 5. Graphical representation of IC50 values on all tested cell lines. An HEK293 bar is not given for 5d and 5e because the viability was more than 50%, even at 100 mM, and
therefore IC50 values could not be calculated.
derivatives together, investigating the biochemical bases of the
antitumor effect and determining in vitro antitumor activity close to
FDA approved drugs. In addition, further studies are planned to
determine antiproliferative activity on other brain tumor cell lines,
different healthy cell lines, the kinase phosphorylation levels in the
presence of the active compounds, and subsequent in vivo experi-
ments with a brain tumor model.
4.1.1. General synthesis of 1-(4-methyl-2-(40-
substitutedphenylthiazole-5-yl)ethane-1-one derivatives (method
A)
According to the Hantzsch method, 3-chloro-2,4-pentandione is
added to the hot solution of aryl thioamide in absolute ethanol
(Scheme 1) and boiled for 8 h. After being controlled with TLC
where the reaction is completed, it was left to cool, poured into cold
water and neutralized by sodium acetate solution. After the pre-
cipiation was completed, it is taken and dried, crystallized from
ethanol [40].
4. Experimental
4.1. Chemistry
4.1.1.1. 1-(4-Methyl-2-phenylthiazole-5-yl)ethane-1-one.
Obtained according to Method A by the reaction of 3-chloro-2,4-
pentanedione (110 mmol, 14.80 g) and thiobenzamide (100 mmol,
13.72 g). Yield: %90, m.p.: 68e69 ꢁC, m.p. reference: 68e70 ꢁC
(Ethanol) [41].
All chemicals and biological materials were purchased from
Sigma-Aldrich Chemical Corp and Honeywell Corp. All intermedi-
ate compounds were checked for their purities using thin-layer
chromatography (TLC) with a Silica Gel 60 F254 TLC plate (Merck
KGaA, Darmstadt, Germany). Melting points were measured by a
Stuart Melting Point Apparatus SMP30 (Staffordshire, UK) and
defined by recording the point where compound started to melt.
Spectroscopic data were determined with the following in-
struments: Fourier transform IR (FT-IR), PerkinElmer Spectrum
Two FT-IR Spectrometer (PerkinElmer, Inc, Waltham, Massachu-
setts, USA); 1H NMR, Bruker 300- MHz UltraShield NMR Spectro-
photometer (Bruker Corp, Billerica, Massachusetts, USA); and 13C
NMR, Bruker 75- MHz UltraShield NMR Spectrophotometer (Bruker
Corp, Billerica, Massachusetts, USA) in DMSO-d6 using standard
TMS. The high resolution mass spectra of the compounds were
determined on a Shimadzu 8040 LC/MS/MS ITTOF system (Shi-
madzu, Tokyo, Japan) using a mass spectrometer with the electron
spray method (ESI).
4.1.1.2. 1-(4-Methyl-2-(40-methoxyphenyl)thiazole-5-yl)ethane-1-
one. Obtained according to Method A by the reaction of 3-chloro-2,4-
pentanedione (110 mmol, 14.80 g) and 4-methoxythiobenzamide
(100 mmol, 16.72 g). Yield: 85%, m.p.: 88.8e90 ꢁC, m.p. reference:
89 ꢁC (Ethanol) [41].
4.1.1.3. 1-(4-Methyl-2-(40-chlorophenyl)thiazole-5-yl)ethane-1-one.
Obtained according to Method A by the reaction of 3-chloro-2,4-
pentanedione (110 mmol, 14.80 g) and 4-chlorothiobenzamide
(100 mmol, 17.16 g). Yield: 90%, m.p.: 113e115 ꢁC, m.p. reference:
114e115 ꢁC (Ethanol) [42].
4.1.2. General synthesis of 2-bromo-1-(4-methyl-2-(40-
substitutedphenylthiazole-5-yl)ethane-1-one derivatives (1a-c)
The appropriate 1-(4-methyl-2-(4’substitutedphenylthiazole-5-
yl)ethan-1-one derivative dissolved in chloroform and the reaction
environment is brought to 0 ꢁC. After 10 min of stirring, bromine
solution in chloroform added dropwise. Then it is stirred at room
temperature for 12 h. Then it is heated to 40 ꢁC and stirred for
30 min for 1b and 1c. After being controlled with TLC, where the
reaction is completed, the saturated sodium bicarbonate solution is
added and stirred for 10 min. Phases are separated and the aqueous
phase is re-extracted with chloroform. After organic phase is dried
using sodium sulphate, the solvent is evaporated at low pressure
and then the product is obtained [43]. Since the reaction was not
completed in the first trials, it was determined that there was a
second bromine entry when it was heated for a long time. Bromine
addition should be carried out at 0 ꢁC and the reaction should be
controlled frequently, especially after heating. When the starting
Scheme 1. Synthesis of the compounds 1a-c (i: aryl thioamide, 3-chloro-2,4-
pentandione, EtOH, reflux 8 h, NaOAc; ii: product of i, CHCl3,0 ꢁC, Br2, r.t. 12 h,
30 min at 40 ꢁC.
7

Z. Sahin, S.N. Biltekin, L. Yurttas et al.
European Journal of Medicinal Chemistry 212 (2021) 113125
material was consumed the most and a second product started to
form, the reaction was terminated and the product crystallized
from ethanol.
carbonate (20 mmol, 2.76 g) and ethyl cyanoacetate (20 mmol,
2.26 g), obtained according to Method C. Yield: 78%, m.p.:
246e247 ꢁC, m.p. reference: 244 ꢁC (Ethanol) [48].
4.1.2.1. 2-Bromo-1- (4-methyl-2-phenyl-thiazol-5-yl) ethan-1-one
(1a). Obtained according to Method B by reaction of 1- (4-
Methyl-2-phenylthiazol-5-yl) ethan-1-one (75 mmol, 16.30 g) and
bromine (Br2) (75 mmol, 11.99 g). Yield: 76%, m.p.: 117.5e119 ꢁC,
m.p. reference: 117e118 ꢁC (Ethanol) [44].
4.1.3.3. 4-Hydroxy-2-mercapto-6-(4-methoxyphenyl)pyrimidine-5-
carbonitrile (2c). By reaction of 4-methoxybenzaldehyde (20 mmol,
2.72 g), thiourea (20 mmol, 1.5224 g), potassium carbonate
(20 mmol, 2.76 g) and ethyl cyanoacetate (20 mmol, 2.26 g), ob-
tained according to Method C. Yield: 70%, m.p.: 280e282 ꢁC, m.p.:
281e283 ꢁC (Ethanol) [49].
4.1.2.2. 2-Bromo-1-(4-methyl-2-
(40-methoxyphenylthiazole-5-yl)
ethan-1-one (1b). By reaction of 1- (4-Methyl-2- (40-methox-
yphenylthiazol-5-yl) ethan-1-one (75 mmol, 18.55 g) and bromine
(Br2) (75 mmol, 11.99 g), was obtained according to Method B.
Yield: 80%, m.p.: 90.2e91 ꢁC, m.p. reference: 90e91 ꢁC (Ethanol)
[45].
4.1.3.4. 4-(3,4-dimethoxyphenyl)-6-hydroxy-2-mercaptopyrimidine-
5-carbonitrile (2d). By reaction of 3,4-dimethoxybenzaldehyde
(20 mmol, 3.32 g), thiourea (20 mmol, 1.5224 g), potassium car-
bonate (20 mmol, 2.76 g) and ethyl cyanoacetate (20 mmol, 2.26g),
obtained according to Method C. Yield: 80%, m.p.: 281e283 ꢁC, m.p.
reference: 280e282 ꢁC (Acetic acid) [50].
4.1.2.3. 2-Bromo-1-(4-methyl-2-
(40-chlorophenylthiazole-5-yl)
ethan-1-one (1c). By reaction of 1-(4-methyl-2- (40-chlor-
ophenylthiazole-5-yl) ethan-1-one (75 mmol,18.88 g) and bromine
(Br2) (75 mmol, 11.99 g), was obtained according to Method B.
Yield: 70%, m.p.: 117e118 ꢁC, m.p. reference: 117e118 ꢁC (ethanol)
[45].
4.1.3.5. 4-(3-chlorophenyl)-6-hydroxy-2-mercaptopyrimidine-5-
carbonitrile (2e). By reaction of 3-chlorobenzaldehyde (20 mmol,
2.81 g), thiourea (20 mmol, 1.5224 g), potassium carbonate
(20 mmol, 2.76 g) and ethyl cyanoacetate (20 mmol, 2.26 g), ob-
tained according to Method C. Yield: 65%, m.p.: 227e228 ꢁC, m.p.
reference: 229 ꢁC [51].
4.1.3. General synthesis of 2-mercapto-4-hydroxy-6-
4.1.3.6. 4-(4-chlorophenyl)-6-hydroxy-2-mercaptopyrimidine-5-
carbonitrile (2f). By reaction of 4-chlorobenzaldehyde (20 mmol,
2.81 g), thiourea (20 mmol, 1.5224 g), potassium carbonate
(20 mmol, 2.76 g) and ethyl cyanoacetate (20 mmol, 2.26 g), ob-
tained according to Method C. Yield: 85%, m.p.: 269.6e271 ꢁC, m.p.
reference: 270e271 ꢁC (Acetic acid) [46].
substitutedphenypyrimidine-5-carbonitrile derivatives (method C)
(2a-h)
In this method, ethyl cyanoacetate was used instead of methy-
lene ketone in the Biginelli method. In addition, the reaction is not
acid catalyzed, but base catalyzed (Scheme 2). Solvents such as
dimethylformamide, acetone and acetonitrile are used as solvents
[44]. Potassium carbonate is most commonly used as a base. The
appropriate aldehyde, thiourea and ethyl cyanoacetate were dis-
solved in anhydrous ethanol and potassium carbonate was added.
The reaction was cooled after boiling for 16 h. The precipitated
product was dissolved in 0.5 M NaOH solution. It was extracted 3
times and washed with ethyl acetate. It was then brought to pH 2
with the slow addition of a 1 M HCl and the product precipitated.
The product was crystallized from ethanol [2,46,47].
4.1.3.7. 4-(3,4-Dichlorophenyl)-6-hydroxy-2-mercaptopyrimidine-5-
carbonitrile (2g). By reaction of 3,4-dichlorobenzaldehyde
(20 mmol, 3.50 g), thiourea (20 mmol, 1.5224 g), potassium car-
bonate (20 mmol, 2.76 g) and ethyl cyanoacetate (20 mmol, 2.26 g),
obtained according to Method C. Yield: 77%, m.p.: 267e269 ꢁC, m.p.
reference: 268 ꢁC [52].
4 .1. 3. 8 . 4- (3 , 4- m e thyle n e di ox y ph e nyl) - 6- hy d ro x y-2 -
mercaptopyrimidine-5-carbonitrile (2h). By reaction of 3,4-
methylenedioxybenzaldehyde (20 mmol, 3.00 g), thiourea
(20 mmol, 1.5224 g), potassium carbonate (20 mmol, 2.76 g) and
ethyl cyanoacetate (20 mmol, 2.26 g), obtained according to
Method C. Yield: 87%, m.p.: 250.5e252 ꢁC, m.p. reference:
248e250 ꢁC (Ethanol) [50].
4.1.3.1. 4-Hydroxy-2-mercapto-6-phenylpyrimidine-5-carbonitrile
(2a). By reaction of benzaldehyde (20 mmol, 2.12 g), thiourea
(20 mmol, 1.5224 g), potassium carbonate (20 mmol, 2.76 g) and
ethyl cyanoacetate (20 mmol, 2.26 g), was obtained according to
Method C. Yield: %75, m.p.: 299e301 ꢁC, m.p. reference:
300e302 ꢁC (Acetic acid) [46].
4.1.4. General synthesis of compounds 4a-x (method D)
4.1.3.2. 4-Hydroxy-2-mercapto-6-(3-methoxyphenyl)pyrimidine-5-
carbonitrile (2b). By reaction of 3-methoxybenzaldehyde
(20 mmol, 2.72 g), thiourea (20 mmol, 1.5224 g), potassium
2a-h derivatives (100 mmol) and thiazolyl acetylbromide com-
pounds (1a-c) (110 mmol) are dissolved in acetone and potassium
carbonate (100 mmol) was added. The reaction was mixed for 12 h
at room temperature (Scheme 3). If it was not completed in this
time, the reaction was boiled for 1 h. After the reaction was
completed, it was poured into cold water. The product was ex-
pected to be precipitated thoroughly. In the meantime, thorough
solubility of potassium carbonate should be ensured in water. The
precipitated product was filtered away. Compounds are crystallized
from 80% DMSO (Dimethyl sulfoxide) - 20% water mixture. The
resulted products are kept in vacuum survey seizing phosphorus
pentaoxide (P2O5) at 45 ꢁC for overnight and ensured complete
drying before analysis [15,53].
4.1.4.1. 2-((2-(4-methyl-2-phenylthiazol-5-yl)-2-oxoethyl)thio)-6-
Scheme 2. Synthesis of the compounds 2a-h and 3a-c (i: aldehyde, thiourea, K2CO3,
ethyl cyanoacetate, EtOH, reflux 16 h; ii: aldehyde, thiourea, K2CO3, malononitrile,
EtOH, reflux 5 h).
oxo-4-phenyl-1,6-dihydropyrimidine-5-carbonitrile
(4a). Yield:
17.21% m.p.: 210 ꢁC (decomposed). FT-IR ymax (cmꢀ1): 3380.8 (NH),
8

Z. Sahin, S.N. Biltekin, L. Yurttas et al.
European Journal of Medicinal Chemistry 212 (2021) 113125
Scheme 3. Synthesis of the compounds 4a-x and 5a-i (i: 2a-h, 1a-c, acetone, K2CO3, r.t. 12 h, reflux 1 h, i: 3a-c, 1a-c, acetone, K2CO3, r.t. 8 h, reflux 1 h).
3056e2914 (CeH), 2205 (C^N), 1680.33 (C]O), 1555e1462 (C]C,
C]N). 1H NMR (300 MHz) DMSO‑d6)
(ppm): 2.70 (3H, s, CH3),
112.18, 120.85, 121.65, 127.02, 129.83, 130.21, 130.55, 131.91, 132.57,
148.42, 150.65, 159.50, 166.65, 168.98, 170.82, 170.99, 188.36 (C]O).
d
4.52 (2H, s, CH2), 7.29e7.37 (3H, m, Ar), 7.54 (3H, m, Ar), 7.65 (2H, d,
HRMS (M
505.0988.
þ
H): For C25H20N4O4S2 calcd: 505.0999, found:
J: 6.57 Hz, Ar), 7.98 (2H, d, J: 7.77 Hz, Ar). 13C NMR (75 MHz)
DMSO‑d6)
d (ppm): 18.75 (CH3), 39.18 (CH2), 90.02 (CeCN), 119.72,
127.08, 128.37, 128.50, 129.89, 130.38, 130.77, 131.97, 132.58, 137.51,
4.1.4.5. 4-(3-chlorophenyl)-2-((2-(4-methyl-2-phenylthiazol-5-yl)-
159.44, 167.41, 169.03, 170.28, 188.04 (C]O). HRMS (M þ H): For
2-oxoethyl)thio)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile
(4e).
C
23H16N4O2S2 Calcd: 445.0787, found: 445.0781.
Yield: 61.86% m.p.: 231 ꢁC. FT-IR ymax (cmꢀ1): 3374.8 (NH),
2990.9e2902.3 (CeH), 2203.3 (C^N), 1673.95 (C]O),
1560.23e1423.97 (C]C, C]N). 1H NMR (300 MHz) DMSO‑d6)
4.1.4.2. 4-(3-methoxyphenyl)-2-((2-(4-methyl-2-phenylthiazol-5-
yl)-2-oxoethyl)thio)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile
(4b). Yield: 8.92% m.p.: 283 ꢁC (decomposed). FT-IR ymax (cmꢀ1):
3397.8 (NH), 3056.5e2899 (CeH), 2206.6 (C^N), 1675 (C]O),
1548e1446 (C]C, C]N), 1235.85 (CeO). 1H NMR (300 MHz)
d
(ppm): 2.70 (3H, s, CH3), 4.47 (2H, s, CH2), 7.37e7.41 (2H, m, Ar),
7.51e7.54 (3H, m, Ar), 7.55e7.64 (2H, m, Ar), 7.96e7.98 (2H, m, Ar).
13C NMR (75 MHz) DMSO‑d6)
(ppm): 18.75 (CH3), 39.44 (CH2),
d
89.87 (CeCN), 120.09, 127.05, 128.12, 129.82, 129.96, 130.28, 131.89,
132.64, 133.34, 139.87, 159.39, 165.57, 169.00, 170.41, 171.35, 188.21
(C]O). HRMS (M þ H): For C23H15ClN4O2S2 calcd: 479.0398, found:
479.0391.
DMSO‑d6)
CH2), 6.94e6.95 (1H, m, Ar), 7.22e7.26 (3H,m, Ar), 7.50e7.56 (3H,
m, Ar), 7.97e8.00 (2H, m, Ar). 13C NMR (75 MHz) DMSO‑d6)
(ppm):
d (ppm): 2.70 (3H, s, CH3), 3.66 (3H, s, OCH3), 4.50 (2H, s,
d
18.72 (CH3), 39.16 (CH2), 55.46 (OCH3), 90.04 (CeCN), 114.04,
115.79, 120.02, 120.77, 120.97, 126.54, 126.84, 127.05, 129.44, 129.68,
129.85, 130.64, 131.92, 132.62, 139.07, 159.26, 159.40, 167.10, 168.96,
188.16 (C]O). HRMS (M þ H): For C24H18N4O3S2 calcd: 475.0893,
found: 475.0890.
4.1.4.6. 4-(4-chlorophenyl)-2-((2-(4-methyl-2-phenylthiazol-5-yl)-
2-oxoethyl)thio)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile
(4f).
Yield: 70.36% m.p.: 314 ꢁC. FT-IR ymax (cmꢀ1): 3371.6 (NH),
2987.6e2895.7 (CeH), 2209.8 (C^N), 1658.5 (C]O), 1583e1417.96
(C]C, C]N). 1H NMR (300 MHz) DMSO‑d6)
d (ppm): 2.68 (3H, s,
4.1.4.3. 4-(4-methoxyphenyl)-2-((2-(4-methyl-2-phenylthiazol-5-
yl)-2-oxoethyl)thio)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile
(4c). Yield: 10.89% m.p.: 124.3 ꢁC. IR ymax (cmꢀ1): 3394.5 (NH),
3000e2836 (CeH), 2206.6 (C^N), 1671.6 (C]O), 1543.13e1413
(C]C, C]N), 1253.63 (CeO). 1H NMR (300 MHz) DMSO‑d6)
CH3), 4.43 (2H, s, CH2), 7.35 (2H, dd, J: 6.72 Hz, j: 1.86 Hz, Ar),
7.51e7.53 (3H, m, Ar), 7.65 (2H, dd, J: 6.70 Hz, j: 1.88 Hz, Ar),
7.93e7.96 (2H, m, Ar). 13C NMR (75 MHz) DMSO‑d6)
d (ppm): 18.72
(CH3), 39.37 (CH2), 89.75 (CeCN), 120.18, 127.02, 128.42, 129.83,
130.18, 130.76, 131.89, 132.54, 134.91, 136.58, 159.42, 166.14, 168.98,
170.64, 171.36, 188.45 (C]O). HRMS (M þ H): For C23H15ClN4O2S2
calcd: 479.0386, found: 479.0386.
d
(ppm): 2.71 (3H, s, CH3), 3.67 (3H, s, OCH3), 4.45 (2H, s, CH2), 6.85
(2H, d, J: 8.62 Hz, Ar), 7.52e7.56 (3H, m, Ar), 7.65 (2H, d, J: 8.56 Hz,
Ar), 7.98e8.01 (2H, m, Ar). 13C NMR (75 MHz) DMSO‑d6)
(ppm):
d
18.76 (CH3), 39.16 (CH2), 55.53 (OCH3), 88.90 (CeCN), 113.63,
120.80, 127.05, 129.87, 130.01, 130.21, 130.86, 131.92, 132.62, 159.36,
160.81, 166.60, 168.92, 170.78, 170.85, 188.52 (C]O). HRMS
(M þ H): For C24H18N4O3S2 calcd: 475.0893, found: 475.0885.
4.1.4.7. 4-(3,4-dichlorophenyl)-2-((2-(4-methyl-2-phenylthiazol-5-
yl)-2-oxoethyl)thio)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile
(4g). Yield: 61.20% m.p.: 231.5 ꢁC. FT-IR ymax (cmꢀ1): 3384.7 (NH),
2990.9e2902.3
1567.83e1420.31 (C]C, C]N). 1H NMR (300 MHz) DMSO‑d6)
(ppm): 2.69 (3H, s, CH3), 4.45 (2H, s, CH2), 7.48e7.56 (3H, m, Ar),
7.59 (1H, s, Ar), 7.66 (2H, dd, J: 8.4 Hz, j: 2.04 Hz, Ar), 7.75e7.80 (1H,
m, Ar), 7.94e7.97 (2H, m, Ar). 13C NMR (75 MHz) DMSO‑d6)
(ppm):
(CeH),
2209.8
(C^N),
1661.8
(C]O),
4.1.4.4. 4-(3,4-dimethoxyphenyl)-2-((2-(4-methyl-2-phenylthiazol-
5-yl)-2-oxoethyl)thio)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile
(4d). Yield: 39.17% m.p.: 244 ꢁC. FT-IR ymax (cmꢀ1): 2977.8e2899
(CeH), 2206.6 (C^N), 1658.5 (C]O), 1568.75e1416.32 (C]C, C]
d
d
N), 1255.71 (CeO). 1H NMR (300 MHz) DMSO‑d6)
d
(ppm): 2.70 (3H,
18.75 (CH3), 39.40 (CH2), 89.87 (CeCN), 119.94, 127.02, 128.46,
128.82, 129.80, 130.19, 130.53, 130.64, 130.70, 131.45, 131.87, 132.59,
132.89, 138.23, 159.36, 164.52, 168.99, 170.34, 171.50, 188.26 (C]O).
HRMS (M þ H): For C23H14Cl2N4O2S2 calcd: 513.0008, found:
513.0011.
s, CH3), 3.70 (6H, m, OCH3), 4.49 (2H, s, CH2), 6.88 (1H, d, J: 8.47 Hz,
Ar), 7.32e7.38 (2H, m, Ar), 7.48e7.53 (3H, m, Ar), 7.96 (2H, d, J:
7.80 Hz, Ar). 13C NMR (75 MHz) DMSO‑d6)
d
(ppm): 18.75 (CH3),
39.42 (CH2), 55.78 (OCH3), 55.84 (OCH3), 89.09 (CeCN), 111.24,
9

Z. Sahin, S.N. Biltekin, L. Yurttas et al.
European Journal of Medicinal Chemistry 212 (2021) 113125
4.1.4.8. 4-(benzo[d] [1,3]dioxol-5-yl)-2-((2-(4-methyl-2-
phenylthiazol-5-yl)-2-oxoethyl)thio)-6-oxo-1,6-dihydropyrimidine-
5-carbonitrile (4h). Yield: 23.64% m.p.: 240.8 ꢁC. FT-IR ymax (cmꢀ1):
3594.7 (NH), 2977.8e2915.4 (CeH), 2209.8 (C^N), 1648.64 (C]O),
1503.41e1415.91 (C]C, C]N), 1254.60, 1242.78 (CeO). 1H NMR
Ar), 7.92 (2H, d, J: 8.87 Hz, Ar). 13C NMR (75 MHz) DMSO‑d6)
d
(ppm): 18.77 (CH3), 39.15 (CH2), 55.77 (OCH3), 55.86 (OCH3), 55.94
(OCH3), 89.03 (CeCN), 111.26, 112.17, 115.21, 120.89, 121.64, 125.33,
128.81, 129.60, 130.26, 148.42, 150.63, 159.52, 162.35,166.61, 169.04,
170.81, 170.90, 188.12 (C]O). HRMS (M þ H): For C26H22N4O5S2
calcd: 535.1104, found: 535.1084.
(300 MHz) DMSO‑d6)
d (ppm): 2.67 (3H, s, CH3), 4.76 (2H, s, CH2),
5.85 (2H, s, CH2), 6.85 (1H, d, J: 8.23 Hz, Ar), 7.16, (1H, s, Ar), 7.36
(1H, dd, J: 8.19 Hz, j: 1.61 Hz, Ar), 7.52e7.59 (3H, m, Ar), 7.99 (2H, dd,
4.1.4.13. 4-(3-chlorophenyl)-2-((2-(2-(4-methoxyphenyl)-4-
methylthiazol-5-yl)-2-oxoethyl)thio)-6-oxo-1,6-dihydropyrimidine-
5-carbonitrile (4m). Yield: 83.67% m.p.: 245 ꢁC. FT-IR ymax (cmꢀ1):
3676.8, 3532.4 (NH), 2990.9e2908.8 (CeH), 2213.1 (C^N), 1670.98
(C]O), 1606e1406.68 (C]C, C]N), 1259.41 (CeO). 1H NMR
J: 7.52 Hz, j: 1.58 Hz, Ar). 13C NMR (75 MHz) DMSO‑d6)
d (ppm):
18.74 (CH3), 39.18 (CH2), 102.22 (OeCH2eO), 92.01 (CeCN), 108.26,
108.83, 116.55, 124.29, 127.08, 129.20, 129.86, 130.48, 132.07, 132.52,
147.77, 150.55, 159.83, 161.94, 165.38, 166.59, 169.55, 186.51 (C]O).
HRMS (M þ H): C24H16N4O2S2 calcd: 489.0686, found: 489.0685.
(300 MHz) DMSO‑d6)
4.46 (2H, s, CH2), 7.07 (2H, brs, Ar), 7.42 (2H, brs, Ar), 7.65 (2H, brs,
Ar), 7.92 (2H, brs, Ar). 13C NMR (75 MHz) DMSO‑d6)
(ppm): 18.78
d (ppm): 2.68 (3H, s, CH3), 3.84 (3H, s, CH3),
4.1.4.9. 2-((2-(2-(4-methoxyphenyl)-4-methylthiazol-5-yl)-2-
oxoethyl)thio)-6-oxo-4-phenyl-1,6-dihydropyrimidine-5-carbonitrile
(4i). Yield: 82.93% m.p.: 288 ꢁC. FT-IR ymax (cmꢀ1): 3404.4 (NH),
d
(CH3), 39.13 (CH2), 55.97 (OCH3), 89.90 (CeCN), 115.20, 119.95,
125.39, 127.06, 128.16, 128.85, 129.69, 130.05, 130.30, 133.36, 139.76,
159.44, 162.32, 165.56, 169.08, 170.29, 171.31, 187.88 (C]O). HRMS
(M þ H): For C24H17ClN4O3S2 calcd: 509.0503, found: 509.0493.
3004e2872.7
1553.10e1411.21 (C]C, C]N), 1265.51 (CeO). 1H NMR (300 MHz)
DMSO‑d6) (ppm): 2.68 (3H, s, CH3), 3.84 (3H, s, CH3), 4.45 (2H, s,
(CeH),
2209.8
(C^N),
1672.11
(C]O),
d
CH2), 7.08 (2H, d, J: 8.88 Hz, Ar), 7.32e7.35 (3H, m, Ar), 7.66 (2H, dd,
4.1.4.14. 4-(4-chlorophenyl)-2-((2-(2-(4-methoxyphenyl)-4-
methylthiazol-5-yl)-2-oxoethyl)thio)-6-oxo-1,6-dihydropyrimidine-
5-carbonitrile (4n). Yield: 51.98% m.p.: 278 ꢁC. FT-IR ymax (cmꢀ1):
3371.6 (NH), 2971.2e2902.3 (CeH), 2213.1 (C^N), 1652 (C]O),
1606e1411.64 (C]C, C]N), 1257.08 (CeO). 1H NMR (300 MHz)
J: 7.8 Hz, j: 1.65 Hz, Ar), 7.93 (2H, d, J: 8.83 Hz, Ar). 13C NMR (75 MHz)
DMSO‑d6)
d (ppm): 18.77 (CH3), 39.15 (CH2), 55.95 (OCH3), 89.73
(CeCN), 115.23, 120.40, 125.35, 128.33, 128.45, 128.84, 129.77,
130.11, 137.91, 159.46, 162.34, 167.33, 169.02, 170.58, 171.07, 188.11
(C]O). HRMS (M þ H): For C24H18N4O3S2 calcd: 475.0893, found:
475.0884.
DMSO‑d6)
d (ppm): 2.67 (3H, s, CH3), 3.83 (3H, s, CH3), 4.42 (2H, s,
CH2), 7.07 (2H, d, J: 8.88 Hz, Ar), 7.38 (2H, d, J: 8.54 Hz, Ar), 7.67 (2H,
d, J: 8.55 Hz, Ar), 7.91 (2H, d, J: 8.83 Hz, Ar). 13C NMR (75 MHz)
4.1.4.10. 4-(3-methoxyphenyl)-2-((2-(2-(4-methoxyphenyl)-4-
methylthiazol-5-yl)-2-oxoethyl)thio)-6-oxo-1,6-dihydropyrimidine-
5-carbonitrile (4j). Yield: 36.75% m.p.: 212.5 ꢁC. FT-IR ymax (cmꢀ1):
3322.3 (NH), 2977.8e2902.3 (CeH), 2213.1 (C^N), 1670.63 (C]O),
1562.11e1442.88 (C]C, C]N), 1257.20, 1236.30 (CeO). 1H NMR
DMSO‑d6) d (ppm): 18.76 (CH3), 39.13 (CH2), 55.98 (OCH3), 89.69
(CeCN), 115.21, 120.23, 125.32, 128.44, 128.82, 129.79, 130.22,
134.88, 136.65, 159.46, 162.33, 166.09, 169.03, 170.52, 171.33, 188.19
(C]O). HRMS (M þ H): For C24H17ClN4O3S2 calcd: 509.0503, found:
509.0488.
(300 MHz) DMSO‑d6) d (ppm): 2.68 (3H, s, CH3), 3.68 (3H, s, OCH3),
3.85 (3H, s, CH3), 4.46 (2H, s, CH2), 6.93e6.97 (1H, m, Ar), 7.08 (2H,
dd, J: 6.84 Hz, j: 2.07 Hz, Ar), 7.22e7.26 (3H, m, Ar), 7.94 (2H, dd, J:
4.1.4.15. 4-(3,4-dichlorophenyl)-2-((2-(2-(4-methoxyphenyl)-4-
methylthiazol-5-yl)-2-oxoethyl)thio)-6-oxo-1,6-dihydropyrimidine-
5-carbonitrile (4o). Yield: 36.22% m.p.: 243 ꢁC. FT-IR ymax (cmꢀ1):
3565.2, 3378.1 (NH), 2977.8e2836.6 (CeH), 2209.8 (C^N), 1665
(C]O), 1556.59e1410.08 (C]C, C]N), 1262.69 (CeO). 1H NMR
6.81 Hz, j: 2.08 Hz, Ar). 13C NMR (75 MHz) DMSO‑d6)
d (ppm): 18.75
(CH3), 39.16 (CH2), 55.45 (OCH3), 55.96 (OCH3), 89.74 (CeCN),
113.97, 115.22, 115.71, 120.41, 120.77, 125.39, 128.84, 129.42, 129.68,
139.31, 159.26, 159.43, 162.33, 167.04, 168.99, 170.57, 171.04, 188.08
(C]O). HRMS (M þ H): For C25H20N4O4S2 calcd: 505.0999, found:
505.0985.
(300 MHz) DMSO‑d6)
d (ppm): 2.68 (3H, s, CH3), 3.84 (3H, s, CH3),
4.44 (2H, s, CH2), 7.06 (2H, d, J: 8.91 Hz, Ar), 7.60 (1H, d, J: 8.38 Hz,
Ar), 7.68 (1H, dd, J: 8.4 Hz, j: 2.03 Hz, Ar), 7.81 (1H, d, J: 1.98 Hz, Ar),
7.91 (2H, dd, J: 6.81 Hz, j: 2.01 Hz, Ar). 13C NMR (75 MHz) DMSO‑d6)
4.1.4.11. 4-(4-methoxyphenyl)-2-((2-(2-(4-methoxyphenyl)-4-
methylthiazol-5-yl)-2-oxoethyl)thio)-6-oxo-1,6-dihydropyrimidine-
5-carbonitrile (4k). Yield: 58.55% m.p.: 242.9 ꢁC. FT-IR ymax (cmꢀ1):
3680 (NH), 2971.2e2840 (CeH), 2203.3 (C^N), 1642.1 (C]O),
1606e1416.86 (C]C, C]N), 1288.33, 1242.77 (CeO). 1H NMR
d (ppm): 18.78 (CH3), 39.14 (CH2), 55.97 (OCH3), 89.82 (CeCN),
115.19, 119.97, 125.36, 128.49, 128.82, 129.73, 130.21, 130.66, 131.45,
132.87, 138.28, 159.40, 162.31, 164.48, 169.06, 170.26, 171.50, 188.00
(C]O). HRMS (M þ H): For C24H16Cl2N4O3S2 calcd: 543.0114,
found: 543.0101.
(300 MHz) DMSO‑d6) d (ppm): 2.68 (3H, s, CH3), 3.69 (3H, s, OCH3),
3.84 (3H, s, CH3), 4.43 (2H, s, CH2), 6.85 (2H, d, J: 8.89 Hz, Ar), 7.07
(2H, d, J: 8.88 Hz, Ar), 7.66 (2H, d, J: 8.83 Hz, Ar), 7.93 (2H, d, J:
4.1.4.16. 4-(benzo[d] [1,3]dioxol-5-yl)-2-((2-(2-(4-methoxyphenyl)-
4 - m e t h y l t h i a z o l - 5 - y l ) - 2 - o x o e t h y l ) t h i o ) - 6 - o x o - 1, 6 -
dihydropyrimidine-5-carbonitrile (4p). Yield: 44.09% m.p.: 216 ꢁC.
FT-IR ymax (cmꢀ1): 2899 (CeH), 2213.1 (C^N), 1650 (C]O), 1643.63
(C]O), 1538.68e1429.88 (C]C, C]N), 1251.39 (CeO). 1H NMR
8.82 Hz, Ar). 13C NMR (75 MHz) DMSO‑d6)
d (ppm): 18.79 (CH3),
39.16 (CH2), 55.53 (OCH3), 56.00 (OCH3), 88.86 (CeCN), 113.65,
115.23, 120.82, 125.36, 128.82, 129.83, 130.03, 130.19, 159.45, 160.84,
162.35, 166.56, 169.00, 170.92, 188.28 (C]O). HRMS (M þ H): For
(300 MHz) DMSO‑d6) d (ppm): 2.66 (3H, s, CH3), 3.85 (3H, s, OCH3),
C
25H20N4O4S2 calcd: 505.0999, found: 505.0986.
4.59 (2H, s, CH2), 5.92 (2H, s, CH2), 6.86 (1H, d, J: 8.21 Hz, Ar), 7.08,
(2H, d, J: 8.86 Hz, Ar), 7.17 (1H, d, J: 1.65 Hz, Ar), 7.32 (1H, dd, J:
8.18 Hz, j: 1.73 Hz, Ar), 7.93 (2H, d, J: 8.78 Hz, Ar). 13C NMR (75 MHz)
4.1.4.12. 4-(3,4-dimethoxyphenyl)-2-((2-(2-(4-methoxyphenyl)-4-
methylthiazol-5-yl)-2-oxoethyl)thio)-6-oxo-1,6-dihydropyrimidine-
5-carbonitrile (4l). Yield: 48.37% m.p.: 243.4 ꢁC. FT-IR ymax (cmꢀ1):
3673.5 (NH), 2977.8e2836.6 (CeH), 2206.6 (C^N), 1642.1 (C]O),
1606e1417.63 (C]C, C]N), 1255.24, 1228.20 (CeO). 1H NMR
DMSO‑d6)
d (ppm): 18.73 (CH3), 39.09 (CH2), 55.54 (OCH3), 89.82
(CeCN),120.30, 128.33,128.43, 128.78, 129.97, 130.12, 131.25, 131.41,
136.52, 137.83, 159.35, 167.38, 167.48, 170.22, 170.88, 188.35 (C]O).
HRMS (M þ H): For C25H18N4O5S2 calcd: 519.0791, found: 519.0779.
(300 MHz) DMSO‑d6) d (ppm): 2.68 (3H, s, CH3), 3.71 (6H, s, OCH3),
3.84 (3H, s, CH3), 4.47 (2H, s, CH2), 6.90 (1H, d, J: 8.45 Hz, Ar), 7.06
(2H, d, J: 8.90 Hz, Ar), 7.35 (1H, d, J: 8.33 Hz, Ar), 7.37e7.38 (1H, m,
4.1.4.17. 2-((2-(2-(4-chlorophenyl)-4-methylthiazol-5-yl)-2-
oxoethyl)thio)-6-oxo-4-phenyl-1,6-dihydropyrimidine-5-carbonitrile
10

Z. Sahin, S.N. Biltekin, L. Yurttas et al.
European Journal of Medicinal Chemistry 212 (2021) 113125
(4q). Yield: 53.49% m.p.: 160 ꢁC. FT-IR ymax (cmꢀ1): 3365 (NH),
2964.6e2912.1 (CeH), 2206.6 (C^N), 1670.81 (C]O), 1654.91 (C]
O) 1535e1432.94 (C]C, C]N). 1H NMR (300 MHz) DMSO‑d6)
5-carbonitrile (4v). Yield: 70.51% m.p.: 287 ꢁC. FT-IR ymax (cmꢀ1):
3370 (NH), 2990.9e2908.8 (CeH), 2209.8 (C^N), 1668.95 (C]O),
1537.86e1396.57 (C]C, C]N). 1H NMR (300 MHz) DMSO‑d6)
d
(ppm): 2.70 (3H, s, CH3), 4.46 (2H, s, CH2), 7.28e7.38 (3H, m, Ar),
7.59e7.64 (4H, m, Ar), 8.01 (2H, d, J: 8.60 Hz, Ar). 13C NMR (75 MHz)
DMSO‑d6) (ppm): 18.73 (CH3), 39.09 (CH2), 90.01 (CeCN), 120.30,
d
(ppm): 2.69 (3H, s, CH3), 4.47 (2H, s, CH2), 7.36 (2H, d, J: 8.59 Hz,
Ar), 7.57e7.64 (4H, m, Ar), 7.98 (2H, d, J: 8.58 Hz, Ar). 13C NMR
(75 MHz) DMSO‑d6) (ppm): 18.74 (CH3), 39.08 (CH2), 85.43
d
d
128.33, 128.43, 128.78, 129.97, 130.12, 131.25, 131.41, 136.52, 137.83,
159.35, 167.38,167.48,170.22,170.88, 188.35 (C]O). HRMS (M þ H):
For C23H15ClN4O2S2 calcd: 479.0398, found: 479.0387.
(CeCN), 127.10, 128.16, 128.79, 129.94, 130.22, 130.34, 131.12, 131.44,
133.35, 136.5158, 159.38, 165.65, 170.38, 188.36 (C]O). HRMS
(M þ H): For C23H14Cl2N4O2S2 calcd 513.0008, found: 513.0008.
4.1.4.18. 2-((2-(2-(4-chlorophenyl)-4-methylthiazol-5-yl)-2-
oxoethyl)thio)-4-(3-methoxyphenyl)-6-oxo-1,6-dihydropyrimidine-
5-carbonitrile (4r). Yield: 21.41% m.p.: 195 ꢁC (decomposed). FT-IR
ymax (cmꢀ1): 3381.4 (NH), 2994.2e2908.8 (CeH), 2206.6 (C^N),
1675.57 (C]O), 1551.61e1433.78 (C]C, C]N), 1231.89 (CeO). 1H
4.1.4.23. 2-((2-(2-(4-chlorophenyl)-4-methylthiazol-5-yl)-2-
oxoethyl)thio)-4-(3,4-dichlorophenyl)-6-oxo-1,6-dihydropyrimidine-
5-carbonitrile (4w). Yield: 68.84% m.p.: 258 ꢁC. FT-IR ymax (cmꢀ1):
3370 (NH), 2990.9e2908.8 (CeH), 2213.1 (C^N), 1665 (C]O),
1567.59e1432.79 (C]C, C]N). 1H NMR (300 MHz) DMSO‑d6)
NMR (300 MHz) DMSO‑d6)
d
(ppm): 2.69 (3H, s, CH3), 3.67 (3H, s,
d
(ppm): 2.70 (3H, s, CH3), 4.44 (2H, s, CH2), 7.57e7.66 (4H, m, Ar),
7.77 (1H, d, J: 1.90 Hz, Ar), 7.98 (2H, dd, J: 6.6 Hz, j: 1.98 Hz, Ar). 13
NMR (75 MHz) DMSO‑d6) (ppm): 18.70 (CH3), 39.72 (CH2), 89.90
OCH3), 4.46 (2H, s, CH2), 6.91e6.95 (1H, m, Ar), 7.18e7.25, (3H, m,
C
Ar), 7.61 (2H, d, J: 8.53 Hz, Ar), 8.01 (2H, d, J: 8.52 Hz, Ar). 13C NMR
d
(75 MHz) DMSO‑d6)
d
(ppm): 18.67 (CH3), 39.72 (CH2), 55.47
(CeCN), 119.90, 128.47, 128.74, 129.91, 130.17, 130.64, 131.26, 131.40,
(OCH3), 105.06 (CeCN), 113.97, 115.65, 120.30, 120.74, 128.77,
129.40, 129.93, 131.49, 136.48, 159.23, 159.33, 167.09, 188.35 (C]O).
HRMS (M þ H): For C24H17ClN4O3S2 calcd: 509.0503, found:
509.0494.
131.44, 132.80, 136.48, 138.29, 159.24, 164.56, 167.48, 170.06, 171.37,
188.34 (C]O). HRMS (M
þ
H): For C23H13Cl3N4O2S2 calcd:
548.0220, found: 548.0220.
4.4.5.24. 4-(benzo[d] [1,3]dioxol-5-yl)-2-((2-(2-(4-chlorophenyl)-4-
methylthiazol-5-yl)-2-oxoethyl)thio)-6-oxo-1,6-dihydropyrimidine-
5-carbonitrile (4x). Yield: 65.26% m.p.: 291.1 ꢁC. FT-IR ymax (cmꢀ1):
3667 (NH), 2990.9e2905.9 (CeH), 2223 (C^N), 1675 (C]O), 1642
(C]O), 1550e1373.04 (C]C, C]N), 1248.26 (CeO). 1H NMR
4.1.4.19. 2-((2-(2-(4-chlorophenyl)-4-methylthiazol-5-yl)-2-
oxoethyl)thio)-4-(4-methoxyphenyl)-6-oxo-1,6-dihydropyrimidine-
5-carbonitrile (4s). Yield: 32.05% m.p.: 215 ꢁC. FT-IR ymax (cmꢀ1):
3338.1 (NH), 2963.7e2838.9 (CeH), 2201.6 (C^N), 1669.02 (C]O),
1537.92e1470 (C]C, C]N), 1253.44 (CeO). 1H NMR (300 MHz)
(300 MHz) DMSO‑d6)
d (ppm): 2.65 (3H, s, CH3), 4.74 (2H, s, CH2),
DMSO‑d6)
CH2), 6.83 (2H, d, J: 8.83 Hz, Ar), 7.59e7.64 (4H, m, Ar), 8.00 (2H, d, J:
8.57 Hz, Ar). 13C NMR (75 MHz) DMSO‑d6)
(ppm): 18.74 (CH3),
d
(ppm): 2.70 (3H, s, CH3), 3.66 (3H, s, OCH3), 4.49 (2H, s,
5.87 (2H, s, CH2), 6.85 (1H, d, J: 8.23 Hz, Ar), 7.13 (1H, d, J: 1.67 Hz,
Ar), 7.35 (1H, dd, J: 8.26 Hz, j: 1.64 Hz, Ar), 7.60 (2H, d, J: 8.60 Hz, Ar),
d
7.98 (2H, d, J: 8.57 Hz, Ar). 13C NMR (75 MHz) DMSO‑d6)
d (ppm):
39.35 (CH2), 55.50 (OCH3), 89.56 (CeCN), 113.66, 128.76, 129.67,
129.96, 130.13, 131.32, 131.38, 136.54, 159.39, 161.02, 166.68, 167.58,
188.22 (C]O). HRMS (M þ H): For C24H17ClN4O3S2 calcd: 509.0503,
found: 509.0486.
18.68 (CH3), 39.73 (CH2), 92.40 (CeCN), 102.26 (OeCH2eO), 108.27,
108.80, 116.48, 124.29, 128.77, 129.15, 129.93, 130.92, 131.33, 136.68,
147.76, 150.55, 159.81, 161.86, 165.30, 166.56, 168.11, 186.49 (C]O).
HRMS (M þ H): For C24H15ClN4O4S2 calcd: 523.0296, found:
523.0288.
4.1.4.20. 2-((2-(2-(4-chlorophenyl)-4-methylthiazol-5-yl)-2-
o x o e t h yl ) t h i o ) - 4 - ( 3 , 4 - d i m e t h o x y p h e n yl ) - 6 - o x o - 1, 6 -
dihydropyrimidine-5-carbonitrile (4t). Yield: 48.59% m.p.: 243 ꢁC.
FT-IR ymax (cmꢀ1): 3568 (NH), 3091.8e2842.2 (CeH), 2205 (C^N),
1653.76 (C]O), 1556.74e1433.31 (C]C, C]N), 1298.61, 1257.95
4.1.5. General synthesis of 4-amino-2-mercapto-6-
substitutedphenylpyrimidine-5-carbonitrile derivatives (method E)
(3a-c)
Appropriate aldehyde, thiourea and malononitrile were dis-
solved in anhydrous ethanol and potassium carbonate was added.
The reaction was controlled with TLC after boiling for 5 h (ethyl
acetate 1:2 petroleum ether). The precipitated product was washed
with ethanol. The raw product was mixed with hot water and
filtered. After the filtrate cools, it was acidified with acetic acid. It
was taken and washed with water. Crystallized from ethanol.
(CeO). 1H NMR (300 MHz) DMSO‑d6)
d (ppm): 2.70 (3H, s, CH3),
3.69 (6H, s, OCH3), 4.49 (2H, s, CH2), 6.89 (1H, d, J: 8.42 Hz, Ar),
7.29e7.34 (2H, m, Ar), 7.59 (2H, d, J: 8.55 Hz, Ar), 7.99 (2H, d, J:
8.55 Hz, Ar). 13C NMR (75 MHz) DMSO‑d6)
d (ppm): 18.70 (CH3),
39.72 (CH2), 55.80 (OCH3), 89.20 (CeCN), 111.24, 112.18, 120.67,
121.62, 128.75, 129.93, 130.18, 131.11, 131.42, 136.52, 148.41, 150.64,
159.37, 166.69, 167.52, 170.45, 188.35 (C]O). HRMS (M þ H): For
C
25H19ClN4O4S2 calcd: 539.0609, found: 539.0612.
4.1.5.1. 4-Amino-2-mercapto-6-phenylpyrimidine-5-carbonitrile
(3a). It was synthesized by Method E, using benzaldehyde
(98.00 mmol, 10.4 g), thiourea (98.00 mmol, 7.46 g), potassium
carbonate (98.00 mmol, 13.54 g) and malondinitrile (98.00 mmol,
6.47 g). Yield: 55%, m.p.: 255 ꢁC, No reference value.
4.1.4.21. 4-(3-chlorophenyl)-2-((2-(2-(4-chlorophenyl)-4-
methylthiazol-5-yl)-2-oxoethyl)thio)-6-oxo-1,6-dihydropyrimidine-
5-carbonitrile (4u). Yield: 50.71% m.p.: 245 ꢁC. FT-IR ymax (cmꢀ1):
3397.3 (NH), 2980.1e2904.6 (CeH), 2214.8 (C^N), 1671.69 (C]O),
1580.10e1434.03 (C]C, C]N). 1H NMR (300 MHz) DMSO‑d6)
4.1.5.2. 4-Amino-6-(4-methoxyphenyl)-2-mercaptopyrimidine-5-
d
(ppm): 2.69 (3H, s, CH3), 4.50 (2H, s, CH2), 7.37e7.42 (2H, m, Ar),
7.58e7.66 (4H, m, Ar), 8.00 (2H, d, J: 8.60 Hz, Ar). 13C NMR (75 MHz)
DMSO‑d6) (ppm): 18.74 (CH3), 39.08 (CH2), 85.43 (CeCN), 127.10,
128.16, 128.79, 129.94, 130.22, 130.34, 131.13, 131.44, 133.35, 136.52,
159.38, 165.65, 170.40, 188.42 (C]O). HRMS (M H): For
23H14Cl2N4O2S2 calcd: 513.0008, found: 513.0010.
carbonitrile (3b). It was synthesized by Method E, using 4-
methoxybenzaldehyde
(132.21
mmol, 18
g),
thiourea
d
(132.21 mmol, 10.06 g), potassium carbonate (132.21 mmol,
36.65 g) and malondinitrile (132.21 mmol, 8.73 g). Yield: 35%, m.p.:
261e262 ꢁC, m.p. reference: 258e260 ꢁC (N,N-DMF) [54].
þ
C
4.1.5.3. 4-Amino-6-(4-chlorophenyl)-2-mercaptopyrimidine-5-
carbonitrile (3c). It was synthesized by Method E, using 4-
chlorobenzaldehyde (177.85 mmol, 25 g), thiourea (177.85 mmol,
4.1.4.22. 4-(4-chlorophenyl)-2-((2-(2-(4-chlorophenyl)-4-
methylthiazol-5-yl)-2-oxoethyl)thio)-6-oxo-1,6-dihydropyrimidine-
11

Z. Sahin, S.N. Biltekin, L. Yurttas et al.
European Journal of Medicinal Chemistry 212 (2021) 113125
13.54 g), potassium carbonate (177.85 mmol, 24.58 g) and malon-
dinitrile (177.85 mmol, 11.75 mmol). Yield: 45%, m.p.: 268e269 ꢁC,
m.p. reference: 268e270 ꢁC (1,4-dioxane) [54].
8.84 Hz, Ar), 7.99 (2H, dd, J: 7.35 Hz, j: 1.85 Hz, Ar). 13C NMR
(75 MHz) DMSO‑d6)
d (ppm): 18.82 (CH3), 39.17 (CH2), 55.64
(OCH3), 82.38 (CeCN), 114.02, 116.87, 127.07, 128.20, 129.91, 130.62,
130.91, 132.04, 132.54, 159.58, 161.92, 163.88, 166.88, 169.26, 172.34,
187.48 (C]O). HRMS (M þ H): For C24H19N5O2S2 calcd: 474.1053,
found: 474.1050.
4.1.6. General synthesis of compounds 5a-i (method F)
3a-c derivatives (50 mmol) and thiazolyl acetylbromide com-
pounds 1a-c (55 mmol) are dissolved in acetone and then potas-
sium carbonate (50 mmol) was added. The reaction was mixed for
12 h at room temperature. If it was not completed in this time, the
reaction was boiled for 1 h. After the reaction was completed, it was
poured into cold water. The product was expected to be precipi-
tated thoroughly. In the meantime, thorough solubility of potas-
sium carbonate should be ensured in water. The precipitated
product was filtered away. Compounds are crystallized from
ethanol twice. The resulted products are kept in vacuum survey
seizing phosphorus pentaoxide (P2O5) at 45 ꢁC for overnight and
ensured complete drying before analysis [15,53].
4.1.6.5. 4-Amino-6-(4-methoxyphenyl)-2-((2-(2-(4-methoxyphenyl)-
4-methylthiazol-5-yl)-2-oxoethyl)thio)pyrimidine-5-carbonitrile
(5e). Yield: 64.28% m.p.: 199.3 ꢁC. FT-IR ymax (cmꢀ1): 3667 (NH2),
3476.6, 3235.5 (NH), 2987.51e2901.05 (CeH), 2206.6 (C^N), 1665
(C]O), 1530.15e1393.63 (C]C, C]N), 1250.25 (CeO). 1H NMR
(300 MHz) DMSO‑d6) d (ppm): 2.68 (3H, s, CH3), 3.67 (3H, s, OCH3),
3,85 (3H, s, OCH3), 4.60 (2H, s, CH2), 6.87 (2H, d, J: 8.96 Hz, Ar), 7.08
(2H, d, J: 8.93 Hz, Ar), 7.69 (2H, d, J: 8.90 Hz, Ar), 7.95 (2H, d, J:
8.86 Hz, Ar). 13C NMR (75 MHz) DMSO‑d6)
d (ppm): 18.86 (CH3),
39.17 (CH2), 55.67 (OCH3), 56.03 (OCH3), 82.34 (CeCN), 114.03,
115.27, 116.89, 125.29, 128.22, 128.87, 129.93, 130.64, 159.65, 161.94,
162.44, 163.88, 166.85, 169.33, 172.39, 187.23 (C]O). HRMS
(M þ H): For C25H21N5O3S2 calcd: 504.1159, found: 504.1150.
4.1.6.1. 4-Amino-2-((2-(4-methyl-2-phenylthiazol-5-yl)-2-oxoethyl)
thio)-6-phenylpyrimidine-5-carbonitrile (5a). Yield: 41.04% m.p.:
196 ꢁC. FT-IR ymax (cmꢀ1): 3676.8 (NH), 3299 (NH), 3145e2901.05
(CeH), 2216.4 (C^N), 1638.8 (C]O), 1525.27e1393.71 (C]C, C]
4.1.6.6. 4-Amino-6-(4-chlorophenyl)-2-((2-(2-(4-methoxyphenyl)-4-
methylthiazol-5-yl)-2-oxoethyl)thio)pyrimidine-5-carbonitrile (5f).
Yield: 48.48% m.p.: 216.5 ꢁC. FT-IR ymax (cmꢀ1): 3670.2 (NH2),
3328.9, 3181.2 (NH), 2987.54e2901.04 (CeH), 2209.8 (C^N),1658.5
(C]O), 1513.68e1371.62 (C]C, C]N), 1249.71 (CeO). 1H NMR
N). 1H NMR (300 MHz) DMSO‑d6)
d (ppm): 2.69 (3H, s, CH3), 4.64
(2H, s, CH2), 7.32e7.37 (2H, m, Ar), 7.42 (1H, d, J: 7.25 Hz, Ar),
7.54e7.58 (3H, m, Hz, Ar), 7.68 (2H, d, J: 7.07 Hz, Ar), 7.98 (2H, dd, J:
7.22 Hz, j: 1,78 Hz, Ar). 13C NMR (75 MHz) DMSO‑d6)
d (ppm): 18.79
(CH3), 39.17 (CH2), 83.37 (CeCN), 116.52, 127.09, 128.65, 128.79,
129.90, 130.81, 131.43, 132.03, 132.54, 136.11, 159.59, 163.73, 167.71,
169.2, 172.61, 187.31 (C]O). HRMS (M þ H): For C23H17N5OS2 calcd:
444.0947, found: 444.0940.
(300 MHz) DMSO‑d6)
4.62 (2H, s, CH2), 6.85 (2H, d, J: 8.96 Hz, Ar), 7.60e7.68 (4H, m, Ar),
8.01 (2H, d, J: 8.60 Hz, Ar). 13C NMR (75 MHz) DMSO‑d6)
(ppm):
18.78 (CH3), 39.17 (CH2), 55.66 (OCH3), 82.42 (CeCN),114.02,116.85,
128.21, 128.78, 129.98, 130.60, 131.37, 136.64, 159.53, 161.91, 163.86,
166.92, 167.81, 187.53 (C]O). HRMS (M þ H): For C24H18ClN5O2S2
calcd: 508.0663, found: 508.0638.
d (ppm): 2.70 (3H, s, CH3), 3.67 (3H, s, OCH3),
d
4.1.6.2. 4-Amino-6-(4-methoxyphenyl)-2-((2-(4-methyl-2-
phenylthiazol-5-yl)-2-oxoethyl)thio)pyrimidine-5-carbonitrile (5b).
Yield: 23.52% m.p.: 218 ꢁC. FT-IR ymax (cmꢀ1): 3689.9e3663.6
(NH2), 3378, 3309.2, (NH), 3168e2901.11 (CeH), 2213.1 (C^N),
1671.6 (C]O), 1648.7 (C]O), 1541.13e1394.06 (C]C, C]N),
4.1.6.7. 4-Amino-2-((2-(2-(4-chlorophenyl)-4-methylthiazol-5-yl)-2-
oxoethyl)thio)-6-phenylpyrimidine-5-carbonitrile
(5g). Yield:
1251.39 (CeO). 1H NMR (300 MHz) DMSO‑d6)
d
(ppm): 2.67 (3H, s,
88.79% m.p.: 228 ꢁC. FT-IR ymax (cmꢀ1): 3420.3, 3334.9 (NH2), 3233
(NH), 3000e2898 (CeH), 2211.5 (C^N), 1637.40 (C]O),
1521.65e1493.19 (C]C, C]N). 1H NMR (300 MHz) DMSO‑d6)
CH3), 3.85 (3H, s, CH3), 4.62 (2H, s, CH2), 7.08 (2H, d, J: 8.01 Hz, Ar),
7.40 (3H, m, Ar), 7.69 (2H, d, J: 6.71 Hz, Ar), 7.94 (2H, d, J: 8.08 Hz,
Ar). 13C NMR (75 MHz) DMSO‑d6)
d
(ppm): 18.83 (CH3), 39.45 (CH2),
d
(ppm): 2.69 (3H, s, CH3), 4.62 (2H, s, CH2), 7.40 (2H, d, J: 8.60 Hz,
Ar), 7.54e7.57 (3H, m, Ar), 7.67 (2H, d, J: 8.58 Hz, Ar), 7.98 (2H, dd, J:
7.64 Hz, j: 1.93 Hz, Ar). 13C NMR (75 MHz) DMSO‑d6)
(ppm): 18.82
56.01 (OCH3), 85.08 (CeCN), 115.27, 116.54, 125.29, 128.66, 128.81,
128.88, 131.46, 136.12, 159.64, 162.43, 163.73, 167.68, 172.65, 187.07
(C]O). HRMS (M þ H): For C24H19N5O2S2 calcd: 474.1053, found:
474.1047.
d
(CH3), 39.09 (CH2), 83.46 (CeCN), 116.35, 127.06, 128.76, 129.92,
130.60,130.93,132.06, 132.47,134.85,136.28,159.53,163.56,166.62,
169.25, 172.70, 187.49 (C]O). HRMS (M þ H): For C23H16ClN5OS2
calcd: 478.0558, found: 478.0573.
4.1.6.3. 4-Amino-6-(4-chlorophenyl)-2-((2-(4-methyl-2-
phenylthiazol-5-yl)-2-oxoethyl)thio)pyrimidine-5-carbonitrile (5c).
Yield: 28.20% m.p.: 231 ꢁC. FT-IR ymax (cmꢀ1): 3397.3, 3325 (NH2),
3223.2 (NH), 2990e2894.7 (CeH), 2214.8 (C^N), 1669.52 (C]O),
4.1.6.8. 4-Amino-2-((2-(2-(4-chlorophenyl)-4-methylthiazol-5-yl)-2-
oxoethyl)thio)-6-(4-methoxyphenyl)pyrimidine-5-carbonitrile (5h).
Yield: 31.72% m.p.: 247 ꢁC. FT-IR ymax (cmꢀ1): 3670.2(NH2), 3325.6,
3164.8 (NH), 2987.52e2900.47 (CeH), 2216.4 (C^N), 1652 (C]O),
1518.50e1373.97 (C]C, C]N), 1248.10 (CeO). 1H NMR (300 MHz)
1525.40e1400 (C]C, C]N). 1H NMR (300 MHz) DMSO‑d6)
d (ppm):
2.69 (3H, s, CH3), 4.62 (2H, s, CH2), 7.34e7.43 (3H, m, Ar), 7.61 (2H, d,
J: 8.62 Hz, Ar), 7.66 (2H, d, J: 7.06 Hz, Ar), 7.98 (2H, d, J: 8.61 Hz, Ar).
13C NMR (75 MHz) DMSO‑d6)
d
(ppm): 18.75 (CH3), 39.17 (CH2),
DMSO‑d6) d (ppm): 2.70 (3H, s, CH3), 3.65 (3H, s, OCH3), 4.61 (2H, s,
83.39 (CeCN), 116.50, 128.65, 128.77, 129.97, 131.25, 131.36, 131.41,
136.10, 136.63, 159.57, 163.71, 167.73, 167.79, 172.57, 187.35 (C]O).
HRMS (M þ H): For C23H16ClN5OS2 calcd: 478.0534, found:
478.0534.
CH2), 6.84 (2H, d, J: 8.91 Hz, Ar), 7.61 (2H, d, J: 8.61 Hz, Ar), 7.65 (2H,
d, J: 8.88 Hz, Ar), 8.01 (2H, d, J: 8.61 Hz, Ar). 13C NMR (75 MHz)
DMSO‑d6)
d (ppm): 18.81 (CH3), 39.09 (CH2), 55.61 (OCH3), 82.38
(CeCN), 113.99, 116.89, 128.18, 128.77, 129.99 130.60, 131.33, 131.42,
136.64, 159.51, 161.86, 163.83, 166.92, 167.81, 172.31, 187.57 (C]O).
HRMS (M þ H): For C24H18ClN5O2S2 calcd: 508.0663, found:
508.0664.
4.1.6.4. 4-Amino-2-((2-(2-(4-methoxyphenyl)-4-methylthiazol-5-
yl)-2-oxoethyl)thio)-6-phenylpyrimidine-5-carbonitrile
(5d).
Yield: 46.38% m.p.: 206 ꢁC. FT-IR ymax (cmꢀ1): 3670.2 (NH2), 3407,
3225.6 (NH), 2987.53e2900 (CeH), 2209.8 (C^N), 1675 (C]O),
1579.8e1315.72 (C]C, C]N), 1249.42 (CeO). 1H NMR (300 MHz)
4.1.6.9. 4-Amino-6-(4-chlorophenyl)-2-((2-(2-(4-chlorophenyl)-4-
methylthiazol-5-yl)-2-oxoethyl)thio)pyrimidine-5-carbonitrile (5i).
Yield: 8.60% m.p.: 230 ꢁC. FT-IR ymax (cmꢀ1): 3673.5 (NH2), 3430.6,
3335.5 (NH), 2987.50e2899 (CeH), 2219.7 (C^N), 1661.8 (C]O),
DMSO‑d6)
d (ppm): 2.70 (3H, s, CH3), 3,64 (3H, s, OCH3), 4.62 (2H, s,
CH2), 6.85 (2H, d, J: 8.89 Hz, Ar), 7.54e7.56 (3H, m, Ar), 7.68 (2H, d, J:
12

Z. Sahin, S.N. Biltekin, L. Yurttas et al.
European Journal of Medicinal Chemistry 212 (2021) 113125
1518.24e1368.60 (C]C, C]N). 1H NMR (300 MHz) DMSO‑d6)
50 mM tris HCl. The suspension was homogenized for 10 s with an
electrical homogenizer and immediately centrifuged at 20 kg for
20 min at 4 ꢁC. The resulting pellets are resuspended in 3 U/mL
adenosine deaminase buffer and stored at ꢀ80 ꢁC until the time of
use.
d
(ppm): 2.69 (3H, s, CH3), 4.61 (2H, s, CH2), 7.40 (2H, d, J: 8.60 Hz,
Ar), 7.61 (2H, d, J: 8.62 Hz, Ar), 7.65 (2H, d, J: 8.59 Hz, Ar), 7.99 (2H, d,
J: 8.62 Hz, Ar). 13C NMR (75 MHz) DMSO‑d6)
(ppm): 18.75 (CH3),
d
39.17 (CH2), 87.84 (CeCN), 116.28, 128.76, 129.97, 130.56, 131.34,
134.86, 136.25, 136.62, 159.49, 163.58, 166.66, 167.79, 172.69, 187.52
(C]O). HRMS (M þ H): For C23H15Cl2N5OS2 calcd: 512.0168, found:
512.0151.
4.2.2.2. Measuring A1 and A2A adenosine receptor binding inhibition.
50 mL each of 1 mM concentrations of test compounds and standard
[3H]DPCPX radioligand (2 nM, PerkinElmer, Boston, MA) for hA1AR
binding measurement with A1AR-expressing CHO cell membranes
4.2. Biological activity
(40
volume of 200
For measurement of A2AAR and A2BAR binding, 50
M concentrations of test compounds and [3H]ZM241385 con-
m
g/tube) 50 mM Tris- Incubate for 60 min at 25 ꢁC in a total
4.2.1. Cellular U87MG antitumor MTT activity measurement
mL in HCl buffer (pH: 7.4; MgCl2, 10 mM) [60,61].
4.2.1.1. Subcultures of the cells. HEK293 cells were maintained in
Dulbecco’s Modified Eagle medium (Gibco, UK) supplemented with
10% (v/v) fetal bovine serum (Gibco, UK), 10.000 U/mL penicillin,
mL each of
1
m
centrations to 1 and 15 nM for a2a and a2b binding, respectively.
The mixture was incubated for 60 min at 25 ꢁC in a total volume of
10.000 mg/mL of streptomycin. The human glioblastoma cell lines
U87MG were cultured in Dulbecco’s Modified Eagle’s medium/high
glucose (Gibco, UK) supplemented with 10% heat inactivated fetal
bovine serum (Gibco, UK), 1% (v/v) antibiotic-antimycotics solution
200 mL in 50 mM Tris-HCl buffer (pH: 7.4; MgCl2, 10 mM). The re-
action was terminated by filtration through GF/B filters. Filters used
for A1 and A2A measurement are placed in scintillation vials con-
taining 5 mL of Hydrofluor scintillation buffer and counted with the
help of a PerkinElmer Tricarb 2810 TR Liquid Scintillation counter
[62].
(10.000 U/mL penicillin, 10.000 mg/mL streptomycin and 0.25 mg/
mL amphotericin B; Gibco, UK), and 1% (v/v) non-essential amino
acids (Gibco, UK) at 37 ꢁC in a humidified 5% CO2 atmosphere. MCF7
cells were maintained in Dulbecco’s Modified Eagle medium-
Nutrient Mixture F-12 (Gibco, UK) supplemented with 10% (v/v)
fetal bovine serum (Gibco, UK), 10.000 U/mL penicillin, 10.000 mg/
mL of streptomycin and %10 L-glutamine. The cells were passaged
every 3 days [55].
4.2.3. Phosphodiesterase enzyme (PDE) inhibition assay
Experiments were carried out following the instructions pro-
vided by the “PDE Activity Assay Kit” (ab139460, Abcam, Camb-
drige, UK) using the modified method detailed by Araiz et al. [63].
Stock solutions of the compounds were prepared with DMSO (Less
4.2.1.2. Cytotoxic and anticancer activity. Cytotoxic activities of the
tested compounds were determined by cell proliferation analysis
using the standard 3- (4,5-dimethylthiazole-2 yl) ꢀ2,5-
diphenyltetrazolium bromide (MTT) assay [55e57]. In order to
determine the toxic and proliferative effects of compounds, cells
were seeded in 96-well plates add a density of 5.104 (HEK293 hu-
man embryonic kidney cell line (ATCC, USA), 4.104 (MCF7), 105
(U87MG) and per well 24 h prior to treatment. The compounds to
be tested were first dissolved in DMSO (less than 1%) and diluted
with DMEM medium for use in MTT assay. After 24 and 48 h in-
cubation times, the medium on the cells was removed, washed
than 1%). Recombinant human PDE4 was incubated with 0.5
m
M 50-
cGMP and 10
m
L 50-nucleotidase under 30 ꢁC for 90 min. The kit
based on the sequential hydrolysis of cyclic nucleotides by PDE4
and 50-nucleotidase. The released phosphate by enzymatic cleavage
was directly proportional to PDE4 activity. The reaction was
terminated by incubation with termination buffer under room
temperature for 20 min. OD 620 nm were measured on a plate
reader to determine the amount of GMP generated. For measure-
ment of IC50, 6 concentrations of inhibitors were used under the
substrate concentration the suitable enzyme concentration. The
PDE4B and PDE4D inhibitor rolipram and PDE inhibitor IBMX were
used as a positive control.
with D-PBS and 30
mL, prepared in D-PBS). At the end of the 4 h incubation period,
150 L DMSO was added and the culture vessel was shaken at room
mL of MTT was added from stock solution (5 mg/
m
4.2.4. Statistical analysis
temperature for homogeneity of the solution. The absorbance of
the solution at a wavelength of 540 nm was measured in a micro-
plate reader (EON, BioTek Instruments Inc.). Three independent
experiments were performed at least [58,59].
Data are presented as mean standard deviation. All experi-
ments were performed at least in triplicates. Statistical compari-
sons were performed through the Student’s t-test or one-way
ANOVA followed by Tukey test, for 2 or more experimental groups,
respectively. Statistical significance was set at p < 0.05. Statistical
analysis and artwork were performed using Graph Pad Prism 7.0d
(Graph Pad Software, La Jolla, USA). Specific tests are detailed in the
figure legends.
% Viability ¼ (Absorbance experiment group) / (Absorbance
control) ꢂ 100%
Declaration of competing interest
4.2.2. Measuring adenosine receptor binding inhibition
4.2.2.1. Cell culture and membrane preparation. The degree of
binding of the synthesized compounds to adenosine A1 and A2A
receptor subtypes was tested. For this purpose, competitive
attachment experiments NIH (American National Institute of
Health) NIDDK unit. CHO cells (Chinese Hamster Ovary cells) pro-
ducing recombinant hA1 (human adenosine A1) receptor and
HEK293 (human embryonic kidney cell line) cells producing
hA2AAR (human adenosine A2A) receptor, 10% fetal bovine serum in
There is not any conflict of interest by the authors.
Acknowledgements
This project is supported by NIDDK Intramural Research Pro-
gram (ZIADK031117).
DMEM: F12 (1: 1) mixture, 100 U/mL penicillin, 100
mg/mL strep-
Appendix A. Supplementary data
tomycin, and 2 mol/mL glutamine. Cells are seeded by trypsina-
m
tion. After homogenization and suspension, cells are centrifuged at
500 g for 10 min and the precipitate formed was suspended in
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.113125.
13

Z. Sahin, S.N. Biltekin, L. Yurttas et al.
European Journal of Medicinal Chemistry 212 (2021) 113125
681e691, https://doi.org/10.1016/j.ejmech.2015.07.023.
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