Design, synthesis, and anticancer activity of novel 4-thiazolidinone-phenylaminopyrimidine hybrids

Article abstract of DOI:10.1007/s11030-020-10087-1

Abstract: 4-Thiazolidinones and phenylaminopyrimidines are known as anticancer agents. Imatinib is the pioneer phenylaminopyrimidine derivative kinase inhibitor, which is used for the treatment of chronic myeloid leukemia. With a hybrid approach, a novel series of 5-benzylidene-2-arylimino-4-thiazolidinone derivatives containing phenylaminopyrimidine core were designed, synthesized, and tested for their anticancer activity on K562 (chronic myeloid leukemia), PC3 (prostat cancer), and SHSY-5Y (neuroblastoma) cells. Since superior anticancer activity was observed on K562 cells, further biological studies of selected compounds (8, 15, and 34) were performed on K562 cells. For the synthesis of designed compounds, thiourea compounds were converted to 2-imino-1,3-thiazolidin-4-ones with α-chloroacetic acid in the presence of sodium acetate. 5-Benzylidene-2-imino-1,3-thiazolidin-4-one derivatives were obtained by Knoevenagel condensation of 2-imino-1,3-thiazolidin-4-ones with related aldehydes. Compounds 8, 15, and 34 were evaluated for cell viability, apoptosis studies, cell cycle experiments, and DNA damage assays. IC₅₀ values of compounds 8, 15, and 34 were found as 5.26 ± 1.03, 3.52 ± 0.91, and 8.16 ± 1.27?μM, respectively, in K562 cells. Preferably, these compounds showed less toxicity towards L929 cells compared to imatinib. Furthermore, compounds 8 and 15 significantly induced early and late apoptosis in a time-dependent manner. Compounds 15 and 34 induced cell cycle arrest at G0/G1 phase and compound 8 caused cell cycle arrest at G2/M phase. Based on DNA damage assay, compounds 8 and 15 were found to be more genotoxic than imatinib towards K562 cells. To put more molecular insight, possible Abl inhibition mechanisms of most active compounds were predicted by molecular docking studies. In conclusion, a novel series of 5-benzylidene-2-arylimino-4-thiazolidinone derivatives and their promising anticancer activities were reported herein. Graphic abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s11030-020-10087-1

Molecular Diversity
https://doi.org/10.1007/s11030-020-10087-1
ORIGINAL ARTICLE
Design, synthesis, and anticancer activity of novel
4‑thiazolidinone‑phenylaminopyrimidine hybrids
Aslı Türe1 · Mustafa Ergül2 · Merve Ergül3 · Ahmet Altun4 · İlkay Küçükgüzel1
Received: 28 February 2020 / Accepted: 8 April 2020
© Springer Nature Switzerland AG 2020
Abstract
4-Thiazolidinones and phenylaminopyrimidines are known as anticancer agents. Imatinib is the pioneer phenylaminopyrimi-
dine derivative kinase inhibitor, which is used for the treatment of chronic myeloid leukemia. With a hybrid approach, a novel
series of 5-benzylidene-2-arylimino-4-thiazolidinone derivatives containing phenylaminopyrimidine core were designed,
synthesized, and tested for their anticancer activity on K562 (chronic myeloid leukemia), PC3 (prostat cancer), and SHSY-5Y
(neuroblastoma) cells. Since superior anticancer activity was observed on K562 cells, further biological studies of selected
compounds (8, 15, and 34) were performed on K562 cells. For the synthesis of designed compounds, thiourea compounds
were converted to 2-imino-1,3-thiazolidin-4-ones with α-chloroacetic acid in the presence of sodium acetate. 5-Benzylidene-
2-imino-1,3-thiazolidin-4-one derivatives were obtained by Knoevenagel condensation of 2-imino-1,3-thiazolidin-4-ones with
related aldehydes. Compounds 8, 15, and 34 were evaluated for cell viability, apoptosis studies, cell cycle experiments, and
DNA damage assays. IC50 values of compounds 8, 15, and 34 were found as 5.26 1.03, 3.52 0.91, and 8.16 1.27 μM,
respectively, in K562 cells. Preferably, these compounds showed less toxicity towards L929 cells compared to imatinib.
Furthermore, compounds 8 and 15 signifcantly induced early and late apoptosis in a time-dependent manner. Compounds
15 and 34 induced cell cycle arrest at G0/G1 phase and compound 8 caused cell cycle arrest at G2/M phase. Based on
DNA damage assay, compounds 8 and 15 were found to be more genotoxic than imatinib towards K562 cells. To put more
molecular insight, possible Abl inhibition mechanisms of most active compounds were predicted by molecular docking stud-
ies. In conclusion, a novel series of 5-benzylidene-2-arylimino-4-thiazolidinone derivatives and their promising anticancer
activities were reported herein.
This work was presented as oral and poster presentation in 5th
Bahçeşehir University Drug Design Congress on November
13–15, 2017, in İstanbul and selected as best poster presentation in
mentioned congress.
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11030-020-10087-1) contains
supplementary material, which is available to authorized users.
* İlkay Küçükgüzel
ikucukguzel@marmara.edu.tr
1
Department of Pharmaceutical Chemistry, Faculty
of Pharmacy, Marmara University, P.O. Box: 34668,
Istanbul, Turkey
2
Department of Biochemistry, Faculty of Pharmacy, Sivas
Cumhuriyet University, Sivas, Turkey
3
Department of Pharmacology, Faculty of Pharmacy, Sivas
Cumhuriyet University, Sivas, Turkey
4
Department of Medical Pharmacology, Faculty of Medicine,
Sivas Cumhuriyet University, Sivas, Turkey
Vol.:(0123456789)
1 3

Molecular Diversity
Graphic abstract
Keywords Phenylaminopyrimidines · 2-Arylimino-4-thiazolidinone derivatives · Anticancer agents · Apoptosis · Molecular
docking
Abbreviations
is the pioneer kinase inhibitor, approved for chronic
myeloid leukemia (CML) and gastrointestinal stromal
tumors [8]. In the related literature, PAP derivatives are
recorded as straight nominees for anticancer drug devel-
opment [9–12]. Heterocycles are one of the important
classes of organic molecules. In particular, nitrogenous
heterocyles have proven to be prominent drug candidates.
In this context, the synthesis of nitrogenous heterocycles
has attracted the attention of many researchers in the
community of synthetic chemistry and medicinal chem-
istry [13–18]. As of mentioned heterocyclic compounds,
2-imino-4-thiazolidinones has attracted great attention due
to the diversity of their biological efects [19]. 2-Imino-
4-thiazolidinones were reported as potential anticancer
agents [20–22] as well as kinase inhibitors [23, 24]. Based
on a hybrid approach of these mentioned ideas, novel PAP
derivatives containing 5-substituted benzylidene-2-imino-
4-thiazolidinone were designed as anticancer agents in this
study. The design approach of the molecules is presented
in Fig. 1.
PAP Phenylaminopyrimidine
CML Chronic myeloid leukemia
Introduction
According to the report published by the World Health
Organization, being the second leading cause of death
worldwide after cardiovascular diseases, cancer is respon-
sible for 9.6 million deaths in the year of 2018. The hole
between the number of people sufering from cardiovascular
diseases and cancer is getting narrow year after year. Cancer
represents to be a global health problem that needs continu-
ing development of novel treatment modalities [1, 2].
The disrupted equilibrium, aberrant signalization, abnor-
mal cellular activity, increased proliferation, and decreased
apoptosis levels are well-known drivers of tumorigenesis.
This biological equilibrium is largely controlled by protein
kinase enzymes in cells. Kinase proteins are established as
cellular switches that are regulators of cells. For the regula-
tion of biological reactions, 518 kinase enzymes have been
identifed in the human genome thus far [3]. Activation of
any receptor kinase or cytosolic kinase commences difer-
ent cellular signalization pathways [4]. However, unusual
activity or overexpression of a kinase protein may role in
cancer [5–7].
Anticancer activity of the compounds was evaluated
on K562, PC3, and SHSY-5Y cells, initially. As the com-
pounds demonstrated better anticancer activity towards
K562 cells compared to PC3 and SHSY-5Y cells; IC50
calculations, apoptotic, cell cycle, and DNA damage pro-
fles of the most active compounds were evaluated on
K562 cells as further studies. Moreover, because of the
link between CML and Abl kinase [25], molecular dock-
ing studies were performed with Abl kinase domain and
compounds 8, 15 and 34.
Kinase proteins are featured as outstanding molecular
targets for cancer treatment. Imatinib, which has phe-
nylaminopyrimidine (PAP) core in its molecular structure,
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Molecular Diversity
Fig. 1 Design of some novel 5-substituted benzylidene-2-arylimino-4-thiazolidinone derivatives containing pharmacophoric PAP core
preparation of benzoyl isothiocyanate, ammonium thiocy-
anate was reacted with benzoyl chloride in dry acetone. To
this medium that contains benzoyl isothiocyanate, 4-methyl-
N3-[4-(pyridin-3-yl)pyrimidin-2-yl]benzene-1,3-diamine
was then added. The reaction was monitored with TLC.
The precipitate was obtained by fltration after the TLC
spot of the above-mentioned diamine was disappeared. The
resultant crude was washed with hot methanol (1). For the
synthesis of the targeted mono-substituted thiourea 2, com-
pound 1 was partially hydrolyzed using methanolic solution
of 1 N NaOH (aq.). For the synthesis of the di-substituted
thiourea compounds 3–4, 4-methyl-N3-[4-(pyridin-3-yl)
pyrimidin-2-yl]benzene-1,3-diamine was refluxed with
substituted ethyl and methyl isothiocyanates in dry acetone
[12]. Thioureas 2–4 were converted to 2-imino-1,3-thiazo-
lidin-4-ones 5–7 with α-chloroacetic acid in the presence of
sodium acetate. Excess amounts of α-chloroacetic acid and
sodium acetate were used for the cyclization. And lastly,
synthesis of 5-benzylidene-2-imino-1,3-thiazolidin-4-one
derivatives was performed by Knoevenagel condensation of
2-imino-1,3-thiazolidin-4-ones with related benzaldehydes
[28, 29]. Scheme 1 represents the route to synthesize com-
pounds 1–49.
Results and discussion
Chemistry
It is known that 2-imino-1,3-thiazolidin-4-one ring sys-
tem can be synthesized via diferent methods. One of them
includes cyclization of chloroacetamide compounds in
the presence of ammonium thiocyanate [26–33] or potas-
sium thiocyanate [34]. In other respects, the treatment of
α-halo carboxylic acid derivatives with thiourea compounds
afords 2-imino-1,3-thiazolidin-4-ones [35]. Reaction of
chloroacetyl chloride [36], ethyl chloroacetate [20], ethyl
bromoacetate [37–39], or ethyl chloroacetate [40] reagents
with thiourea compounds were reported to give 2-imino-
1,3-thiazolidin-4-one derivatives. In this study, we fol-
lowed the latter method to obtain 2-imino-1,3-thiazolidin-
4-one compounds. We carried out cyclization reaction
with α-chloroacetic acid owing to the preferable yield after
the purifcation procedure. Furthermore, whether hydro-
gen substitution of third position nitrogen is necessary for
structure–activity relationship or not, we obtained 3-substi-
tuted-2-imino-1,3-thiazolidin-4-ones as well by starting the
reaction with di-substituted thioureas.
The purity of all the synthesized compounds was checked
with thin layer chromatography (TLC) and reverse phase-
high performance liquid chromatography (RP-HPLC).
In previous communications reported for the synthesis of
designed compounds, thioureas were produced by the reac-
tion of amines with isothiocyanates [41, 42]. For the in situ
1 3

Molecular Diversity
Scheme 1 Synthetic route to compounds 1–49. i Acetone, methyl or ethyl isothiocyanate, refux. ii Acetone, NH4SCN benzoyl chloride, refux.
iii MeOH, 1 N NaOH, refux. iv EtOH, ClCH2COOH, NaOAc, refux. v NaOMe, ArCHO, refux
Structures of the compounds were confrmed by FTIR, 1H-
NMR, 13C-NMR, HMBC, and mass spectral data besides
elemental analysis.
in the 1H-NMR spectra of the compounds when CDCl3 was
used as a solvent. D2O exchange method was also used to
identify exchangeable protons. Moreover, it was observed
that 2-amino(/imino)-4-thiazol(idin)one NH protons in
addition to secondary NH protons exchange with deute-
rium of D2O as well as CDCl3 [48, 49]. Unless 2-amino(/
imino)-4-thiazol(idin)one compounds 8–21 NH protons are
changed with deuterium, they were observed between 11.40
and 12.90 ppm in DMSO-d6 as broad singlets that confrm
imino structures of the compounds. 2-Amino-4-thiazolone
protons would appear at much higher feld around 9.00 ppm
[31]. Amino-imino tautomeric equilibrium for compounds
5 and 8–21 was monitored in their both 1H- and 13C-NMR
data. Detailed spectral data are presented in supporting
information.
We have previously published the chemistry of thioureas
[12]. In FTIR spectra, C=S stretching bends of thioureas 1–4
were observed at 1232–1288 cm−1. Discriminately, C=O
stretching bend of compound 1 was identifed at 1666 cm−1.
C=O stretching bends of 1,3-thiazolidin-4-ones 5–7 were
detected at 1708–1712 cm−1. C=O stretching bends of
5-benzylidene-1,3-thiazolidin-4-ones 8–49 were identifed
at 1697–1720 cm−1.
In 1H-NMR spectra of compounds 1,3-thiazolidin-4-one
rings 5–7, –SCH2– protons were observed 3.92–4.01 ppm.
In 13C-NMR spectra of compounds 5–6, –SCH2– signals
were observed at 33–35 ppm and C=O carbon signals of the
compounds were identifed at ppm 171–188 ppm [35, 42].
Benzylidene substitution to 1,3-thiazolidin-4-one rings was
confrmed with the 1H- and 13C-NMR results of compounds
8–49. Absence of -SCH2- signals at 32–36 ppm and merge of
signals at 127–130 ppm demonstrated the structures of fnal
compounds 8–49 [43, 44]. Similarly, in 1H-NMR spectra,
while -SCH2- protons were disappeared, methylidene proton
signals of compounds 8–49 were observed between 7.58 and
7.99 ppm which confrms Z isomer of methylidene structure
[29, 31, 45, 46]. Methylidene carbon signals of =CH–Ar
structures were identifed at 134–135 ppm [47].
In the literature, keto/enol and amino/imino tautomeric
forms of 2-amino(/imino)-4-thiazol(idin)one rings were
investigated elaborately [50–53]. As can be seen in Fig. 2,
2-phenylimino-1,3-thiazolidin-4-one structure can be found
in eight diferent forms depending on keto/enol tautomerism,
amino/imino tautomerism in addition to E/Z geometrical
isomers. In light of this information, modeling studies of
compounds 5 and 8–21, possessing amino/imino tautomer-
ism, were held with both forms of the compounds.
Biological activity
In 1H-NMR studies, secondary NH proton signals of the
compounds that belong to PAP core were detected in the
range of 8.90 and 9.10 ppm. These NH signals disappeared
Anticancer activity of compounds 5–49 was evaluated
against K562, PC3 and SHSY-5Y cells. For preliminary
1 3

Molecular Diversity
Fig. 2 Keto/enol and amino/
imino tautomeric forms of
2-phenylimino-1,3-thiazolidin-
4-one ring
screening, cytotoxic bioactivity of synthesized compounds
was evaluated in vitro against K562, PC3 and SHSY-5Y
cells with the XTT assay. To evaluate the anticancer potency
of target compounds, the cancer cells were treated with the
compounds at 10 µM constant concentration. Cell viability
percentages were calculated after the treatment of cells for
24 and 48 h. Imatinib was used as a positive control. Prelimi-
nary anticancer activity results of compounds 5–49 against
K562, PC3, and SHSY-5Y are presented in Tables 1, 2 and
3 respectively. It was observed that while compounds 5–49
showed moderate antiproliferative activity against K562
cells, compounds 5–49 demonstrated weak antiproliferative
activity against PC3 and SHSY-5Y cells. Since compounds
5–49 displayed better anticancer activity towards K562 cells
compared to PC3 and SHSY-5Y cells, further biological
studies were held with K562 cells. As of compounds, 8,
15 and 34 exhibited better anticancer activity and further
activity studies were carried on with compounds 8, 15 and
34. It was also observed that the introduction of substituted
benzylidene substitution at the 5th position of 2-imino-4-thi-
azolidinone ring signifcantly enhances anticancer activity
on K562 cells.
other compounds demonstrated poor activity against K562
cells. Herewith, compounds 8, 15 and 34, which revealed out
cell viability% values less than 50.0% on K562 cells, were
selected for further biological studies. These three com-
pounds were also tested for the following IC50 calculations,
apoptosis studies, cell cycle experiments, and DNA damage
assay on K562 cells.
Afterward, to determine IC50 values of the compounds
8, 15 and 34, K562 cells were treated with 0.1, 1, 10, 25
and 50 µM concentrations of these compounds for 24
and 48 h and cell viability assay was performed. Results
obtained from IC50 calculations were demonstrated in Fig. 3
and Table 4. As shown in Fig. 3, compound 15 was found
to be the most potent compound towards K562 cells (IC50
values of 4.86 0.73 and 3.52 0.91 μM for 24 and 48 h,
respectively). For 24 and 48 h, the IC50 values were calcu-
lated as 9.97 1.14 and 8.16 1.27 μM for compound 34,
8.79 1.09 and 5.26 1.03 μM for compound 8 in K562
cells, respectively. IC50 values of imatinib as positive control
were calculated as 0.73 0.17 for 24 h and 0.59 0.11 μM
for 48 h in K562 cells. These IC50 values were used in sub-
sequent apoptosis and cell cycle assays.
As seen in Table 1, the percentage of viability values of
compounds 8, 15 and 34 were determined as 34.1, 31.1 and
49.8 for 24 h and 31.8, 24.6 and 41.7 for 48 h, respectively.
On the other hand, compounds 19, 39 and 48 displayed
moderate antiproliferative activity, while nearly all of the
Later on, the most active compounds 8, 15 and 34 were
evaluated against mouse subcutaneous connective tissue
cells (L929) to determine whether the synthesized com-
pounds display selective cytotoxicity between normal and
tumor cells or not. Assertively, compounds 8, 15 and 34
1 3

Molecular Diversity
Table 1 Cell viability % SD values of synthesized compounds 5–49 on K562 cells for 24 and 48 h
R2
R1
–H
–Me
–Et
Comp. no.
Comp. no.
Comp. no.
24 h
48 h
24 h
48 h
24 h
48 h
Viab%
Viab%
Viab%
Viab%
Viab%
Viab%
–
2-F
3-F
4-F
2,6-F2
2-Cl
4-Cl
5
8
97.4 5.32
34.1 3.46 31.8 5.46 22
83.1 4.56
6
101.1 4.22
77.9 3.04
97.4 3.78
91.3 5.11
63.4 4.16 36
89.5 1.07 37
7
101.1 3.33 95.7 3.99
57.3 3.47 49.3 4.13
85.1 1.35 73.4 3.48
77.3 2.34 70.1 1.79
54.5 3.19 46.9 4.11
61.5 5.79 59.4 4.19
71.8 1.59 63.9 3.46
73.5 4.65 68.1 6.15
57.3 4.16 47.9 4.78
59.3 4.69 50.2 5.05
73.4 3.48 62.5 5.09
85.7 1.99 73.9 3.31
81.5 2.11 72.7 3.89
55.6 3.12 44.6 3.21
63.1 3.19 51.6 4.13
9
77.4 4.96
82.6 1.99
63.5 5.13
75.2 3.74
86.1 6.11
74.8 3.12
68.1 6.11 23
63.4 4.93 24
52.3 1.89 25
67.5 4.19 26
70.6 5.98 27
62.2 3.47 28
10
11
12
13
14
15
16
17
18
19
20
112.8 4.12 101.3 3.79 38
83.7 5.13
94.3 5.63
98.8 3.15
92.6 3.89
83.8 4.29
85.5 1.07
67.2 4.17
73.2 5.11
68.6 4.13
49.8 5.97
91.9 1.02
66.7 5.12 39
83.9 4.13 40
92.7 5.09 41
85.8 5.93 42
67.9 3.49 43
79.2 3.97 44
60.4 4.37 45
66.6 4.78 46
59.7 2.33 47
41.7 5.11 48
83.2 1.03 49
4-Br
4-CF3
4-OCF3
2-OH
2-OMe
3-OMe
4-OMe
31.1 3.97 24.6 2.45 29
81.1 4.19
56.5 4.17
61.1 1.95
54.7 3.47
58.6 3.11
66.9 1.11
73.2 1.79 30
49.3 3.47 31
55.9 5.13 32
48.1 2.44 33
50.1 4.19 34
57.1 3.47 35
4-N(Me)2 21
24 h
Viab%
48 h
Viab%
Imatinib
22.1 4.13
16.7 2.76
Compounds were implemented at 10 µM concentration on K562 cell line
exhibited a weak cytotoxic efect on L929 cells with IC50 val-
ues of 87.3 4.96, 73.7 5.13 and 69.1 3.49 µM, respec-
tively, in comparison with imatinib (IC50 =19.6 1.33 µM).
Results that are presented in Table 2 suggest that these com-
pounds possess relatively low cytotoxicity towards L929
cells. It is noteworthy to emphasize that compounds 8, 15
and 34 had lower toxicity according to imatinib on L929
cells.
the amount of total apoptotic cells in all groups, especially
for compounds 8 and 15 when compared to control cells.
Percentages of total apoptotic cells were 27.27%, 19.18%,
14.59% and 24.88% for compounds 8, 15, 34 and imatinib
at 24 h, respectively. For 48 h, total apoptotic cell amount
was signifcantly increased to 50.24%, 57.72%, 19.70%, and
62.67% for compounds 8, 15, 34 and imatinib, respectively.
These data indicate that compounds 8 and 15 induce mainly
early and late apoptosis in K562 cells in a time-dependent
manner.
Imatinib constitutively eliminates K562 cells by inducing
apoptosis [54]. In our study, it was also investigated whether
the three most efective compounds have apoptotic efects
on K562 cells. Before apoptosis experiments, the cells were
treated with IC50 concentrations of the most active com-
pounds and imatinib for 24 and 48 h. Later, Annexin V bind-
ing assay was performed to evaluate the apoptotic efects of
the compounds and imatinib on K562 cells. As shown in
Fig. 4, step-up of incubation time from 24 to 48 h increased
Above-mentioned studies exhibited that the three com-
pounds inhibited cell proliferation and signifcantly induced
apoptosis compared to control cells. In resuming studies, to
investigate whether the compounds induce growth inhibition
of cells via alterations in cell cycle arrest, we determined
the efect of the compounds on cell cycle distribution by
the cell cycle kit. The cell cycle results showed that, after
1 3

Molecular Diversity
Table 2 Cell viability % SD values of synthesized compounds 5–49 on PC-3 cells for 24 and 48 h
5-7
8-49
R2
R1
–H
–Me
–Et
Comp. no.
Comp. no.
Comp. no.
24 h
48 h
24 h
48 h
24 h
48 h
Viab%
Viab%
Viab%
Viab%
Viab%
Viab%
–
2-F
3-F
4-F
2,6-F2
2-Cl
4-Cl
5
8
87.4 2.72
45.6 3.23
68.8 3.56
50.7 2.46
55.2 4.07
73.4 1.08
80.6 4.18
86.2 4.53
46.1 1.21
59.2 1.12
87.0 3.94
69.0 1.10
62.6 2.86
63.4 5.64
93.7 2.11
80.1 2.94
6
79.7 1.02
83.0 3.27
92.1 1.09
76.3 4.95
98.9 4.01
99.5 1.25
73.4 2.13
75.2 2.98
62.7 1.83
94.6 3.90
55.0 4.87
69.9 5.13
61.4 2.46
81.0 2.19
88.9 3.09
77.1 2.81
7
59.4 3.92
59.0 2.24
98.3 1.91
69.4 2.13
52.7 2.17
79.2 3.08
68.9 2.28
65.1 1.89
58.9 3.07
55.7 3.11
63.7 4.15
68.0 1.78
52.1 3.09
55.3 4.26
72.6 1.47
50.3 5.13
53.8 3.12
92.4 1.76
63.3 2.78
47.6 1.97
76.5 2.91
63.8 3.11
59.6 4.96
52.9 2.77
48.7 5.07
58.6 3.46
57.8 1.78
48.1 2.47
49.1 2.12
67.2 1.01
40.9 1.05 22
60.1 3.12 23
43.7 1.78 24
47.9 2.94 25
67.1 2.36 26
78.6 1.96 27
78.2 2.04 28
40.1 1.08 29
56.7 2.07 30
77.8 2.97 31
65.2 2.71 32
51.9 3.09 33
59.7 1.01 34
89.2 5.10 35
78.1 2.09 36
88.9 4.22 37
69.3 1.09 38
89.9 5.01 39
95.5 3.13 40
67.1 1.57 41
66.1 2.39 42
57.1 4.16 43
91.7 4.19 44
51.6 3.45 45
62.9 1.54 46
58.3 5.29 47
74.5 2.09 48
81.4 2.23 49
9
10
11
12
13
14
15
16
17
18
19
20
21
4-Br
4-CF3
4-OCF3
2-OH
2-OMe
3-OMe
4-OMe
4-N(Me)2
24 h
Viab%
48 h
Viab%
Imatinib
74.3 2.69
67.1 4.12
Compounds were implemented at 10 µM concentration on PC-3 cell line
48 h, G0/G1 phase population of control cells was 32.8% and
the percentages signifcantly increased to 52.0%, 47.5% and
57.6% for compounds 15, 34 and positive control imatinib,
respectively. As several studies have reported, imatinib
arrests K562 and diferent cells at G0/G1 phase of the cell
cycle [55, 56] and our fndings showed consistency with
these works of literature. Assertively, compound 8 dramati-
cally induced G2/M phase arrest (69.2%) compared with
control cells (33.0%). After the treatment of K562 cells with
compounds 8, 15 and 34, the cell cycle results demonstrated
that the growth inhibition was observed in all groups mainly
associated with G0/G1 (compound 15 and 34) and G2/M
phase arrest (compound 8). Figure 5 represents the cell cycle
analysis of K562 cells exposed to compounds 8, 15, 34, and
imatinib.
It is well known that DNA damage and apoptosis are
closely related processes and in most cases, DNA dam-
age in the cells results in apoptosis. Therefore, DNA is
the major target of most cytotoxic anticancer drugs and
inducing DNA damage is an important anticancer strategy
[57]. Many studies have reported that inducing DNA dam-
age results in apoptotic cell death in many types of cancer
[58]. In this study, having compared to the control cells,
while compound 34 exhibited low DNA damage response,
compounds 8 and 15 signifcantly promoted DNA damage
response (increased phosphorylation of ATM and H2AX)
in K562 cells. As given in Fig. 6, total DNA damage per-
centage of control cells was 8.70% and the percentages
were increased to 32.90%, 37.20%, and 10.40%, after treat-
ment of compounds 8, 15 and 34, respectively. For the
1 3

Molecular Diversity
Table 3 Cell viability % SD values of synthesized compounds 5–49 on SHSY-5Y cells for 24 and 48 h
5-7
8-49
R2
R1
–H
–Me
–Et
Comp. no.
Comp. no.
Comp. no.
24 h
48 h
Viab%
24 h
48 h
24 h
48 h
Viab%
Viab%
Viab%
Viab%
Viab%
–
2-F
3-F
4-F
2,6-F2
2-Cl
4-Cl
5
8
83.7 1.09 72.4 4.02
6
73.9 3.78 65.1 3.21
7
63.3 5.96 54.3 3.12
46.8 2.02 43.1 2.28
109.1 3.28 97.1 2.76
46.8 4.75 42.8 3.12
49.2 5.47 41.8 3.17
52.2 1.79 42.2 2.08
66.7 2.18 56.2 1.92
71.8 3.27 60.9 1.86
59.9 3.48 47.9 2.67
57.8 2.97 43.7 1.26
49.3 3.56 42.8 3.87
65.4 3.47 51.4 3.86
54.6 2.85 41.9 2.09
67.6 2.18 53.1 5.13
76.8 5.03 64.8 2.99
42.3 3.08 33.9 1.56 22
75.5 2.48 62.9 3.19 23
47.7 3.24 42.1 4.21 24
49.0 2.76 42.3 2.75 25
58.6 1.09 46.4 2.09 26
83.0 5.15 70.1 3.17 27
87.0 3.45 74.3 4.13 28
50.4 2.91 42.6 1.09 29
63.1 4.12 50.9 3.56 30
84.3 3.58 69.1 4.13 31
70.2 4.18 59.2 1.76 32
51.8 4.10 42.8 1.25 33
67.8 1.08 51.6 3.72 34
104.2 5.02 91.2 3.02 35
81.3 4.25 72.9 3.14 36
101.1 4.12 93.1 2.91 37
69.9 4.19 61.9 3.12 38
54.1 2.19 41.8 5.21 39
55.5 1.07 42.5 3.09 40
77.8 2.24 59.8 1.23 41
63.0 2.78 71.8 3.27 42
66.6 4.56 53.1 3.02 43
59.5 5.24 47.4 2.24 44
68.4 1.45 56.4 2.56 45
72.2 2.09 61.2 1.43 46
63.6 2.47 50.6 2.76 47
78.5 3.25 64.5 4.07 48
98.5 2.19 86.4 2.12 49
9
10
11
12
13
14
15
16
17
18
19
20
4-Br
4-CF3
4-OCF3
2-OH
2-OMe
3-OMe
4-OMe
4-N(Me)2 21
24 h
Viab%
48 h
Viab%
Imatinib
93.2 1.25
89.3 2.19
Compounds were implemented at 10 µM concentration on SHSY-5Y cell line
Fig. 3 The antiproliferative
activity of compounds 8, 15
and 34 on K652 cells (The cells
were treated with the com-
pounds at various concentra-
tions (0.1–50 µM) for 24 and
48 h and the cell viability
was evaluated using the XTT
assay. All data are expressed as
mean SD in three replicates.
The diferences are identifed
as *p<0.05 from the control
cells.)
1 3

Molecular Diversity
Table 4 IC50 values of compounds 8, 15, 34 and imatinib on K562
Molecular modeling studies
and L929 cell lines
To gain more molecular insight on the activity of the three
compounds, molecular modeling studies were conducted
with Abl enzyme. It is well known that imatinib owes its
clinical success against CML by inhibiting Abl kinase pro-
tein [59]. Figure 7 represents the binding of imatinib and
hydrogen bond interactions in the active site of Abl kinase.
Evaluating possible binding mechanisms of the most active
compounds is crucial in this respect. Imatinib makes hydro-
gen bond contacts with Glu286, Thr315, Met318, Ile360,
His361, and Asp381 amino acid residues.
Compound
IC50 (µM)
K562_24 h
K562_48 h
L929_48 h
8
15
34
Imatinib
8.79 1.09
4.86 0.73
9.97 1.14
0.73 0.17
5.26 1.03
3.52 0.91
8.16 1.27
0.59 0.11
73.7 5.13
69.1 3.49
87.3 4.96
19.6 1.33
positive control imatinib, total DNA damage was meas-
ured as 17.20%. These results demonstrate that compounds
8 and 15 possess more genotoxic efect than imatinib.
Binding poses of the three most active compounds were
studied as representative conformations after docking calcu-
lations. Firstly, it is substantial to indicate that PAP cores did
not place in the Abl binding site as it places in the structure
Fig. 4 a Apoptotic efects of compound 8, 15, 34 and imatinib, on
K562 cells (The cells were treated with the IC50 concentrations of
compounds 8, 15, 34 and imatinib for 24 and 48 h. After the incuba-
tion time, the cells were stained with Muse Annexin V & Dead Cell
reagent and then % gated values evaluated by Muse Cell Analyzer
(Merck Millipore). Quantitative data demonstrate that total apoptosis
signifcantly increased in compound 8 and compound 15-treated cells
in a time-dependent manner compared with control.) b Early and late
apoptotic cell percentages signifcantly (p<0.05) increased following
compounds treatment (All experiments were carried out in triplicate
and obtained similar results. Results are expressed as the mean SD.
Statistically signifcant diferences are *p<0.05 from values com-
pared to control cells.)
1 3

Molecular Diversity
Fig. 5 a Efects of compounds 8, 15, 34 and imatinib on cell cycle
distribution in K562 cells (K562 cells were treated with their IC50
concentrations of compounds and imatinib for 48 h. While, com-
pounds 15 and 34 arrest G0/G1 phase similar to imatinib, compound
8 induces cell cycle arrest G2/M phase.) b Histogram display the per-
centage of cell cycle phases of K562 cells (Results are expressed as
the mean SD. The diferences are given as compared to the control
cells, *p<0.05.)
of imatinib after 5-arylidene-2-imino-4-thiazolidinone intro-
duction to the PAP core.
that while amino tautomers of 8 and 15 replace binding
region of imatinib, imino tautomers of 8 (Fig. 8) and 15
(Fig. 9) replaces allosteric sites away from hinge region.
It was also observed that amino tautomers of ring systems
form hydrogen bonds with Lys271 and Glu286. Amongst
the most active compounds, the amino group of 2-amino-
4-thiazolidinone ring behaves as a hydrogen bond donor
making hydrogen bond interaction with Glu286. Oxygen
atom as hydrogen bond acceptor interacts with Lys271.
Moreover, CF3 substitution of compound 15 interacts with
Met318. This interaction is rather important since Met318
amino acid residue takes place hinge region of Abl kinase.
Due to the bulky substitution, an additional methyl
group to the nitrogen atom of 3-methyl-2-imino-4-thi-
azolidinone ring results with another conformational
In detail, amino-imino tautomerization of 2-amino(/
imino)-4-thiazol(idin)one rings were needed to be consid-
ered for molecular modeling studies when R1 is hydrogen in
the chemical structure of synthesized compounds [49]. As
stated in the chemistry part and supporting fle, amino-imino
tautomerization of compounds 5 and 8–21 was demonstrated
by spectral studies. According to NMR results, compounds 5
and 8–21 possess both amino and imino isomers. For dock-
ing calculations, both confgurations of the compounds were
considered.
Out of the most active three compounds, 8 and 15 have
H at R1 substitution. Tautomeric forms of compound 8 and
15 were evaluated during docking studies. It was detected
1 3

Molecular Diversity
Fig. 6 K562 cells were treated with the IC50 concentrations of com-
pounds 8, 15, 34 and positive control imatinib for 48 h to induce
DNA damage (Activation of ATM and H2AX were determined
using the Muse™ Cell Analyzer (Merck Millipore). Bars represent
mean SD, *p<0.05 compared to the control cells.)
Fig. 7 Imatinib in the Abl binding region and hyrdogen bond interactions between Abl and imatinib
replacement in the Abl binding site. Fig 10 depicts the
possible conformation of compound 34 in Abl binding
region. Compound 34 also forms hydrogen bond interac-
tions with Asp381 and His361.
seems more reasonable and rational compared to 34. Tak-
ing into account all these biological and molecular fndings,
biological data and docking studies support each other.
Apart from docking calculations, biological results
revealed out that compounds 8 and 15 are more active than
34. Coordination of compounds 8 and 15 in Abl binding site
1 3

Molecular Diversity
Fig. 8 Interactions of amino tautomer of compound 8 in the Abl binding region
Fig. 9 Interactions of amino tautomer of compound 15 in the Abl binding region
Conclusion
In conclusion, starting from 4-methyl-N3-[4-(pyridin-
3-yl)pyrimidin-2-yl]benzene-1,3-diamine, a novel series
of PAP core containing 5-benzylidene-2-arylimino-4-thi-
azolidinone derivatives were designed and synthesized
in this study. For the synthesis of designed compounds,
starting material was frst converted to thiourea deriva-
tives 1–4. Thiourea compounds were later reacted with
α-chloroacetic acid to obtain 2-arylimino-4-thiazolidinone
compounds 5–7. 5-Substituted benzylidene-2-arylimino-
4-thiazolidinone compounds 8–49 were achieved from
related 2-arylimino-4-thiazolidinones and benzaldehydes
in the presence of sodium methoxide via Knoevenagel con-
densation. After the confrmation of the structures of the
compounds with spectral studies, compounds were tested
for their anticancer activity on CML cell line. Based on the
viability assay, the introduction of arylmethylene group
to 2-arylimino-4-thiazolidinones prominently increased
inhibitory activity on K562 cells. Compounds inhibiting
Fig. 10 Interactions of compound 34 in the Abl binding region
1 3

Molecular Diversity
50.0% of the cells at 10 µM concentration were selected
for follow-up studies to analyze induction of apoptosis,
cell cycle arrest and DNA damage profles. Prior to these
further studies, IC50 values were found as 5.26, 3.52 and
8.16 µM for compound 8, 15 and 34, respectively. After
IC50 calculation, it was detected that compounds 8 and
15 induced early and late apoptosis in a time-dependent
manner. In depth, 48 h after the implementation of the
compounds to K562 cells, the total apoptotic cell amount
was 50.24%, 57.72%, 19.70% and 62.67% for 8, 15, 34
and imatinib, respectively. In addition, cell cycle stud-
ies revealed out that compound 8 dramatically induced
G2/M phase arrest (69.2%) compared with control cells
(33.0%), while compound 15, 34 and imatinib caused an
increment of the cell population to 52.0, 47.5 and 57.6%,
respectively, at G0/G1 phase. Moreover, DNA damage
assay results which are notably connected with cellular
apoptotic results executed that compound 8 (32.90%) and
15 (37.20%) possess more genotoxicity than imatinib
(17.20%). It is better to state that inhibition of Abl protein
results with apoptosis. From this point of view, in further-
ance compounds have PAP structure as so imatinib, molec-
ular modeling studies were held with Abl protein kinase.
Finally, in light of biological results as well as docking
studies, we purpose that our compounds may induce pro-
grammed cell death by inhibiting Abl kinase in CML cells.
were implied by wavenumber ν (cm−1). 1H- and 13C-NMR
spectra were recorded on Brüker AVANCE-DPX instrument.
DMSO-d6 or CDCl3 was used as a solvent and chemical
shifts were specifed in δ (ppm) downfeld from tetramethyl-
silane (TMS). Low-resolution mass spectra were acquired
with AB SCIEX API 2000 LC–MS/MS instrument.
Documented spectral and chromatographic data of the
compounds are presented as the supplementary fle.
General procedure for the synthesis of thiourea derivatives
(1–4)
Synthesis of thiourea derivatives (1–4) and structure charac-
terization of the compounds was published in our previous
study [12].
N-(Benzoyl)-N′-(4-methyl-3-{[4-(pyridin-3-yl)pyrimidin-
2-yl]amino}phenyl)thiourea 1
White solid. M.p.: 204–205 °C [12].
N-(4-Methyl-3-{[4-(pyridin-3-yl)pyrimidin-2-yl]amino}
phenyl)thiourea 2
White solid. M.p.: 210–212 °C [12].
N-(4-Methyl)-N′-(4-methyl-3-{[4-(pyridin-3-yl)pyrimidin-
2-yl]amino}phenyl)thiourea 3
White solid. M.p.: 201–202 °C [12].
N-(4-Ethyl)-N′-(4-methyl-3-{[4-(pyridin-3-yl)pyrimidin-
2-yl]amino}phenyl)thiourea 4
White solid. M.p.: 187–188 °C [12].
Materials and methods
General procedure for the synthesis of thiazolidin‑4‑one
Chemistry
derivatives (5–7)
Solvents, reagents and starting materials except from
4-methyl-N3-[4-(pyridin-3-yl)pyrimidin-2-yl]benzene-
1,3-diamine were purchased from Sigma-Aldrich or Merck.
To monitor reactions, TLC studies were run on silica gel 60
F254 plates. The RP-HPLC was used to prove the purity of
the compounds. Agilent technologies 1100 series instrument
equipped with a quaternary solvent delivery system, a model
Agilent series G1315, a photodiode array detector, a Rheo-
dyne syringe loading sample injector with a 50-µL sample
loop and Agilent ChemStation Plus software was used for
chromatographic analysis. All synthesized compounds were
signifcantly separated from the starting materials and chro-
matographic purities of the compounds were found above
95% based on the peak area values obtained from chroma-
tograms. Chromatographic systems (CS1–7) which were used
in RP-HPLC studies and chromatograms were presented in
supplementary data. Melting points (°C) of the compounds
were measured with Schmelzpunktbestimmer SMP II basic
model melting point apparatus. Elemental analysis studies
were held with LECO CHNS-932 instrument. Infrared spec-
tra were performed on a Shimadzu FTIR 8400S and data
Corresponding thiourea derivative compounds 1–4
(0.01 mol) were dissolved in ethanol and heated in the
presence of sodium acetate (0.03 mol) and α-chloroacetic
acid (0.02 mol) for 8–10 h. Ethanol was evaporated under
vacuum at the end of the reaction. For the purifcation, the
precipitate was washed with water frst. The solid material
obtained was washed with petroleum ether later and crystal-
lized from ethanol.
2‑[(4‑Methyl‑3‑{[4‑(pyridin‑3‑yl)pyrimidin‑2‑yl]amino}phe-
nyl)imino]‑1,3‑thiazolidin‑4‑one 5 Salmon color solid.
Yield 42%, 1.52 g. TLC Rf: 0.43 (S1). HPLC tR (min): 3.19
(CS1). M.p.: 200–201 °C. IR υmax (cm−1): 3435, 3229 (N–H
str), 3031 (aromatic C–H str), 2981–2897 (aliphatic C–H
str), 1708 (thiazolidin-4-one C=O str), 1647, 1585, 1553
(C=N str, N–H bending, C=C str), 1406 (aliphatic C–H
1
bending), 810–798 (aromatic C–H bending). H-NMR δ
ppm (300 MHz, DMSO-d6): 9.27 and 9.24 (s and s, 1H),
9.02 and 8.90 (s and s, 1H), 8.69 (s, 1H), 8.52 (d, 1H,
J = 4.8 Hz), 8.42 (d, 1H, J = 7.8 Hz), 7.99 (s, 0.5H), 7.54
(m, 1H), 7.43 (d, 1H, J = 5.1 Hz), 7.39 (m, 1H), 7.25 (m,
1 3

Molecular Diversity
1H), 6.75 (d, 0.5H, J=7.8 Hz), 3.98 and 3.92 (s and s, 2H,
SCH2CO), 2.24 and 2.22 (s and s, 3H, ArCH3). 13C-NMR
δ ppm (75 MHz, DMSO-d6): 188.08 and 177.04 (thiazoli-
din-4-one C=O), 161.68, 160.94, 159.43, 151.33, 148.14,
138.44, 136.79, 134.39, 132.15, 130.44, 128.66, 127.92,
123.77, 117.52, 116.34, 107.83, 35.26 (thiazolidin-4-one
CH2) 17.63. (ArCH3). LC/MS ESI+ m/z (%): 399.154
([M+Na]+, 77), 377.162 ([M+H]+, 100); ESI− m/z (%):
375.394 ([M−H]−, 7), 97.027 (100). Elemental analysis cald
for C19H16N6OS·5/4H2O, C 57.20, H 4.67, N 21.07, S 8.04;
found C 57.21, H 4.64, N 21.18, S 7.61.
3H, NCH2CH3, J = 6.9 Hz, J = 7.2 Hz). 13C-NMR δ ppm
(75 MHz, DMSO-d6): 172.24 (thiazolidin-4-one C=O),
162.02, 161.43, 160.05, 155.17, 152.02, 148.78, 146.57,
138.98, 134.78, 132.61, 131.38, 127.66, 124.31, 117.24,
117.10, 108.29, 39.81 (thiazolidin-4-one NCH2CH3), 33.10
(thiazolidin-4-one CH2), 18.17 (ArCH3), 12.83 (thiazo-
lidin-4-one NCH2CH3). LC/MS APCI+ m/z (%): 419.155
([M+CH3]+, 100), 405.259 ([M+H]+, 29); APCI− m/z (%):
403.275 ([M−H]−, 6), 97.034 (100). Elemental analysis cald
for C21H20N6OS·1/2EtOH, C 61.81, H 5.42, N 19.66, S 7.50;
found C 61.54, H 5.03, N 19.99, S 6.82.
3‑Methyl‑2‑[(4‑methyl‑3‑{[4‑(pyridin‑3‑yl)pyrimidin‑2‑yl]
amino}phenyl)imino]‑1,3‑thiazolidin‑4‑one 6 Yellow-orange
solid. Yield 80%, 3.12 g. TLC Rf: 0.49 (S1). HPLC tR (min):
6.12 (CS1). M.p.: 182–184 °C. IR υmax (cm−1): 3352 (N–H
str), 3031 (aromatic C–H str), 2945–2915 (aliphatic C–H
str), 1708 (thiazolidin-4-one C=O str), 1627, 1581, 1519
(C=N str, N–H bending, C=C str), 1415, 1400 (aliphatic
General procedure for the synthesis of 5‑substituted
benzylidene‑1,3‑thiazolidin‑4‑one derivatives (8–49)
Compounds 2–4 (1.0 mmol) were dissolved in methanolic
solution of sodium methoxide (1.0 mmol). Corresponding
aldehyde (1.1 mmol) was added into the reaction medium.
The mixture was refuxed for 4–24 h. At the end of the reac-
tion, the product was cooled, washed with ice-cold water,
and neutralized by 10% acetic acid. The precipitate was fl-
tered and the crude product was washed with hot ethanol to
obtain pure compounds (8–49).
1
C–H bending), 802 (aromatic C–H bending). H-NMR δ
ppm (300 MHz, DMSO-d6): 9.30 (d, 1H, J=1.5 Hz), 8.90
(s, 1H, sec. NH), 8.70–8.68 (dd, 1H, J=1.5 Hz, J=3.2 Hz,
J=1.5 Hz), 8.53 (d, 1H, J=5.3 Hz), 8.43 (d, 1H, J=8.5 Hz),
7.55–7.51 (q, 1H), 7.44 (d, 1H, J = 5.1 Hz), 7.28 (1H, d,
J = 2.1 Hz), 7.21–7.19 (1H, d, J = 8.1 Hz), 6.68–6.64
(dd, 1H, J = 2.1 Hz, J = 6.0 Hz, J = 2.3 Hz), 4.01 (s, 2H,
SCH2CO), 3.16 (s, 3H, thiazolidin-4-one NCH3), 2.25 (s,
3H, ArCH3). 13C-NMR δ ppm (75 MHz, DMSO-d6): 171.93
(thiazolidin-4-one C=O), 161.53, 160.89, 159.41, 155.48,
151.41, 148.18, 146.00, 138.40, 134.26, 132.03, 130.81,
127.18, 123.75, 116.76, 116.62, 107.72, 32.60 (thiazoli-
din-4-one CH2), 29.14 (thiazolidin-4-one NCH3), 17.58
(ArCH3). LC/MS APCI+ m/z (%): 390.927 ([M+H]+, 100);
APCI− m/z (%): 389.285 ([M−H]−, 100), 347.083 (87),
318,059 (27). Elemental analysis cald for C20H18N6OS, C
61.52, H 4.65, N 21.52, S 8.21; found C 61.33, H 4.63, N
21.35, S 7.95.
5‑(2‑Fluorobenzylidene)‑[(4‑methyl‑3‑{[4‑(pyridin‑3‑yl)pyrimi-
din‑2‑yl]amino}phenyl)imino]‑1,3‑thiazolidin‑4‑one 8 Yellow
solid. Yield 80%, 0.385 g. TLC Rf: 0.49 (S1). HPLC tR (min):
6.12 (CS1). M.p.: 183–186 °C. IR υmax (cm−1): 3352 (N–H
str), 3031 (aromatic C–H str), 2945–2915 (aliphatic C–H
str), 1708 (thiazolidin-4-one C=O str), 1627, 1581, 1519
(C=N str, N–H bending, C=C str), 1415, 1400 (aliphatic
1
C–H bending), 802 (aromatic C–H bending). H-NMR δ
ppm (300 MHz, DMSO-d6): 9.30 (d, 1H, J=1.5 Hz), 8.90
(s, 1H, sec. NH), 8.70–8.68 (dd, 1H, J=1.5 Hz, J=3.2 Hz,
J=1.5 Hz), 8.53 (d, 1H, J=5.3 Hz), 8.42 (d, 1H, J=8.5 Hz),
7.55–7.51 (q, 1H), 7.44 (d, 1H, J = 5.1 Hz), 7.28 (1H, d,
J = 2.1 Hz), 7.21–7.19 (1H, d, J = 8.1 Hz), 6.68–6.64
(dd, 1H, J = 2.1 Hz, J = 6.0 Hz, J = 2.3 Hz), 4.01 (s, 2H,
SCH2CO), 3.16 (s, 3H, thiazolidin-4-one NCH3), 2.25 (s,
3H, ArCH3). LC/MS APCI+ m/z (%): 390.927 ([M+H]+,
100); APCI− m/z (%): 389.285 ([M−H]−, 100), 347.083
(87), 318,059 (27). Elemental analysis cald for C20H18N6OS,
C 64.14, H 4.39, N 16.62, S 6.34; found C 63.78, H 4.06, N
16.68, S 6.28.
3‑Ethyl‑2‑[(4‑methyl‑3‑{[4‑(pyridin‑3‑yl)pyrimidin‑2‑yl]amino}
phenyl)imino]‑1,3‑thiazolidin‑4‑one 7 Orange solid; yield
51%, 2.06 g. TLC Rf: 0.52 (S1). HPLC tR (min): 6.03 (CS1).
M.p.: 159–160 °C. IR υmax (cm−1): 3340 (N–H str), 2983–
2947 (aliphatic C–H str), 1712 (thiazolidin-4-one C=O
str), 1635, 1606, 1587, 1519 (C=N str, N–H bending, C=C
str), 1402, 1386 (aliphatic C–H bending), 1230 (C–N str),
1
896, 786, 702 (aromatic C–H bending). H-NMR δ ppm
5‑(3‑Fluorobenzylidene)‑[(4‑methyl‑3‑{[4‑(pyridin‑3‑yl)pyrimi-
din‑2‑yl]amino}phenyl)imino]‑1,3‑thiazolidin‑4‑one 9 Yellow
solid. Yield 47%, 0.226 g. TLC Rf: 0.57 (S1). HPLC tR (min):
4.30 (CS1). M.p.: 254–256 °C. IR υmax (cm−1): 3437 (N–H
str), 2978 (aliphatic C–H str), 1691 (thiazolidin-4-one C=O
str), 1654, 1602, 1581, 1564, 1523 (C=N str, N–H bending,
C=C str), 1446, 1415, 1402 (aliphatic C–H bending), 1224
(C–N str), 1147 (C–F str), 794, 777, 700 (aromatic C–H
(300 MHz, DMSO-d6): 9.30 (d, 1H, J=1.8 Hz), 8.90 (s, 1H,
sec. NH), 8.70–8.68 (dd, 1H, J=1.8, J=3.0 Hz, J=4.8 Hz),
8.53 (d, 1H, J=5.7 Hz), 8.42 (d, 1H, J=8.4 Hz), 7.55–7.50
(q, 1H), 7.45 (d, 1H, J=5.1 Hz), 7.31 (1H, d, J=2.1 Hz),
7.22 (1H, d, J = 8.4 Hz), 6.67–6.64 (dd, 1H, J = 2.3 Hz,
J=5.6 Hz, J=2.1 Hz), 4.01 (s, 2H, -SCH2CO), 3.79–3.72
(q, 2H, NCH2CH3), 2.25 (s, 3H, ArCH3), 1.19–1.15 (t,
1 3

Molecular Diversity
bending). 1H-NMR δ ppm (300 MHz, DMSO-d6): 12.80–
11.52 (bs, 3H-thiazolidin-4-one), 9.26 (s, 1H), 9.10 and 8.97
(s and s, 1H, sec. NH), 8.69–8.68 (dd, 0.5H, J = 1.5 Hz,
J = 4.8 Hz, J = 3.3 Hz), 8.65–8.63 (dd, 0.5H, J = 1.5 Hz,
J = 4.8 Hz, J = 3.3 Hz), 8.54–8.50 (q, 1H), 8.47–8.39 (m,
1H), 8.12 (s, 0.5H), 7.71 and 7.63 (s and s, 1H, =CH–Ar),
7.61–7.22 (m, 8H), 6.81–6.79 (d, 0.5H, J=7.5 Hz), 2.28 and
2.26 (s, s, 3H, ArCH3). LC/MS APCI+ m/z (%): 482.892
([M+H]+ (100), 376.084 (48), 278.108 (22); APCI− m/z
(%): 481.136 ([M−H]−, 100). Elemental analysis cald for
C26H19FN6OS, C 64.72, H 3.97, N 17.42, S 6.65; found C
64.13, H 4.09, N 17.30, S 6.37.
12 Yellow solid. Yield 68%, 0.338 g. TLC Rf: 0.52 (S1).
HPLC tR (min): 5.68 (CS1). M.p.: 240–242 °C. IR υmax
(cm−1): 3444 (N–H str), 3051 (aromatic C–H str), 2978 (ali-
phatic C–H str), 1691 (thiazolidin-4-one C=O str), 1643,
1573, 1552, 1533, 1514 (C=N str, N–H bending, C=C str),
1471, 1454, 1435, 1427, 1394 (aliphatic C–H bending),
1222 (C–N str), 1190 (C–Cl str), 881, 808, 756 (aromatic
C–H bending). 1H-NMR δ ppm (300 MHz, DMSO-d6): 9.25
(s, 1H), 9.08 and 8.95 (s, 1H, sec. NH), 8.68–8.67 (d, 0.5H,
J=3.5 Hz), 8.65–8.63 (d, 0.5H, J=3.5 Hz), 8.54–8.52 (d,
0.5H, J=5.0 Hz), 8.49–8.48 (d, 0.5H, J=5.0 Hz), 8.46–8.38
(m, 1H), 8.12 (s, 0.5H), 7.88 and 7.78 (s, s, 1H, =CH–Ar),
7.68–7.24 (m, 8H), 6.75–6.72 (d, 0.5H, J=7.7 Hz), 2.27 and
2.26 (s, 3H, ArCH3). 13C-NMR δ ppm (75 MHz, DMSO-d6):
179.74 and 170.22, 161.71 and 161.55, 160.91 and 160.80,
159.44 and 159.37, 151.33, 148.12 and 148.08, 138.59 and
138.28, 134.39 and 134.03 (=CH–Ar), 134.28 and 134.22,
132.10 and 132.01, 131.44, 131.16 and 131.06, 130.65,
130.22 and 130.13, 128.64, 128.57, 128.00, 127.87, 125.11,
124.40, 123.77 and 123.64, 117.33 and 117.16, 116.96 and
116.68, 107.91, 17.70 and 17.63. LC/MS APCI+ m/z (%):
500.991 ([M+2]+, 62), 498.938 ([M+H]+, 98), 463.087
(54), 376.07 (100); APCI− m/z (%): 497.103 ([M−H]−,
100), 461.311 (54), 139.17 (47). Elemental analysis cald
for C26H19ClN6OS·1/2H2O, C 61.47, H 3.97, N 16.54, S
6.31; found C 61.77, H 4.05, N 16.77, S 6.43.
5‑(4‑Fluorobenzylidene)‑[(4‑methyl‑3‑{[4‑(pyridin‑3‑yl)pyrimi-
din‑2‑yl]amino}phenyl)imino]‑1,3‑thiazolidin‑4‑one 10 Yel-
low solid. Yield 43%, 0.207 g. TLC Rf: 0.52 (S1). HPLC tR
(min): 5.48 (CS2). M.p.: 240–243 °C. IR υmax (cm−1): 3452
(N–H str), 3071 (aromatic C–H str), 2918 (aliphatic C–H str
1653 (thiazolidin-4-one C=O str), 1577, 1539, 1516 (C=N
str, N–H bending, C=C str), 1456, 1417 (aliphatic C–H
bending), 1261 (C–N str), 1159 (C–F str), 887, 827, 788,
771 (aromatic C–H bending). 1H-NMR δ ppm (300 MHz,
DMSO-d6): 12.50–11.40 (bs, 3H-thiazolidin-4-one), 9.26
(s, 1H), 9.09 and 8.98 (s, 1H, sec. NH), 8.69–8.67 (d, 0.5H,
J=3.3 Hz), 8.65–8.64 (d, 0.5H, J=4.8 Hz), 8.54–8.51 (t,
1H, J = 4.8 Hz, J = 4.2 Hz), 8.46–8.40 (m, 1H), 8.11 (s,
0.5H), 7.46 and 7.44 (s and s, 1H, =CH–Ar), 7.61–7.22
(m, 8H), 6.80–6.77 (d, 0,5H, J = 8.4 Hz), 3.83–3.77 (s, s,
2H), 2.28 and 2.26 (s, s, 3H, ArCH3). LC/MS ESI+ m/z (%):
505.192 ([M+Na]+, 100), 483.101 ([M+H]+, 77), ESI− m/z
(%): 481.268 ([M−H]−, 7), 89.203 (100). Elemental analysis
cald for C26H19FN6OS·MeOH, C 63.02, H 4.51, N 16.33, S
6.32; found C 63.42, H 4.35, N 16.74, S 6.03.
5‑(4‑Chlorobenzylidene)‑[(4‑methyl‑3‑{[4‑(pyridin‑3‑yl)pyrimi-
din‑2‑yl]amino}phenyl)imino]‑1,3‑thiazolidin‑4‑one 13 Yel-
low solid. Yield 13%, 0.065 g. TLC Rf: 0.54 (S1). HPLC
tR (min): 2.08 (CS3). M.p.: 227–231 °C. IR υmax (cm−1):
3443 (N–H str), 1691 (thiazolidin-4-one C=O str), 1637,
1612, 1571, 1564, 1523, 1518 (C=N str, N–H bending, C=C
str), 1454, 1445, 1415, 1398 (aliphatic C–H bending), 1089
1
5‑(2,6‑Difluorobenzylidene)‑[(4‑methyl‑3‑{[4‑(pyridin‑3‑yl)
pyrimidin‑2‑yl]amino}phenyl)imino]‑1,3‑thiazolidin‑4‑one
11 Yellow solid. Yield 41%, 0.205 g. TLC Rf: 0.47 (S1).
HPLC tR (min): 1.56 (CS6). M.p.: 161–163 °C. IR υmax
(cm−1): 3446 (N–H str), 2935 (aliphatic C–H str), 1639 (thi-
azolidin-4-one C=O str), 1604, 1573, 1523 (C=N str, N–H
bending, C=C str), 1473, 1452, 1423, 1321 (aliphatic C–H
bending, C–N str), 1091 (C–F str), 779, 748, 702 (aromatic
C–H bending). 1H-NMR δ ppm (300 MHz, DMSO-d6): 9.20
(s, 1H), 8.66 (d, 1H, J=3.3 Hz), 8.50 (d, 1H, J=5.1 Hz),
8.34 (d, 1H, J = 8.1), 8.02 (s, 1H), 7.83 and 7.70 (s, 1H,
=CH–Ar), 7.38–7.17 (m, CHCl3 and 3.5H), 6.90–6.82 (m,
3H), 6.91–6.82 (m, 0.5H), 2.32 and 2.28 (s, s, 3H, ArCH3).
Elemental analysis cald for C26H18F2N6OS·2H2O, C 58.20,
H 4.13, N 15.66, S 5.98; found C 58.58, H 4.16, N 15.53,
S 5.32.
(C–Cl str), 783, 700 (aromatic C–H bending). H-NMR δ
ppm (300 MHz, DMSO-d6): 12.09 (bs, 3H-thiazolidin-4-one
0.6H), 9.26 (s, 1H), 9.02 and 8.97 (s, s 1H, sec. NH), 8.69–
8.65 (dd, 1H, J=3.3 Hz, J=6.6 Hz, J=1.5 Hz), 8.54–8.51
(t, 1H, J=4.5 Hz, J=5.1 Hz), 8.44–8.40 (m, 1H), 8.09 (s,
0.4H), 7.69–7.25 (m, 9H), 6.79–6.77 (d, 0.6H, J=7.8 Hz),
2.28 and 2.25 (s, s, 3H, ArCH3). LC/MS APCI+ m/z (%):
521.127 ([M+Na]+, 100), 523.066 ([M+Na+2]+, 40),
499.099 ([M+H]+, 37), 343.18 (37), 303.133 (40), 259.180
(43), 131.173 (83); APCI− m/z (%): 497.068 ([M−H]−,
7), 181.15 (34), 155.209 (41), 97.039 (46), 59.118 (100).
Elemental analysis cald for C26H19ClN6OS·2H2O, C 58.37,
H 4.33, N 15.71, S 5.99; found C 58.94, H 3.98, N 16.25,
S 5.58.
5‑(4‑Bromobenzylidene)‑[(4‑methyl‑3‑{[4‑(pyridin‑3‑yl)pyrimi-
din‑2‑yl]amino}phenyl)imino]‑1,3‑thiazolidin‑4‑one 14 Yel-
low solid. Yield 59%, 0.320 g. TLC Rf: 0.55 (S1). HPLC
tR (min): 3.28 (CS4). M.p.: 198–200 °C. IR υmax (cm−1):
5‑(2‑Chlorobenzylidene)‑[(4‑methyl‑3‑{[4‑(pyridin‑3‑yl)
pyrimidin‑2‑yl]amino}phenyl)imino]‑1,3‑thiazolidin‑4‑one
1 3

Molecular Diversity
3446 (N–H str), 1649 (thiazolidin-4-one C=O str), 1587,
1548, 1535 (C=N str, N–H bending, C=C str), 1415 (ali-
phatic C–H bending), 1003 (C–Br str), 812–798 (aromatic
m/z (%): 548.663 (M+, 100); APCI− m/z (%): 547,095
([M−H]−, 100). Elemental analysis cald for C27H19F3N6O2S,
C 59.12, H 3.49, N 15.32, S 5.85; found C 58.52, H 3.72, N
15.09, S 5.80.
1
C–H bending). H-NMR δ ppm (300 MHz, DMSO-d6):
9.25–9.23 (dd, 1H, J=1.5 Hz, J=5.5 Hz, J=1.8 Hz), 8.83
(s, 1H, sec. NH), 8.68–8.67 (dd, 0.5H, J=1.5 Hz, J=4.8 Hz,
J = 1.5 Hz), 8.66–8.63 (dd, 0.5 H, J = 1.5 Hz, J = 4.7 Hz,
J = 1.8 Hz), 8.50 (d, 1H, J = 5.1 Hz), 8.48–8.42 (m, 1H),
7.64–7.35 (m, 6.5H), 7.18–7.03 (m, 3H), 6.75–6.72 (dd,
0,5H, J=2.1 Hz, J=6.0 Hz, J=8.1 Hz), 2.23 and 2.17 (s,
s, 3H, ArCH3). LC/MS ESI+ m/z (%): 564.962 ([M+Na]+,
100), 347.165 (47); ESI− m/z (%): 541.276 ([M−H]−, 100).
Elemental analysis cald for C26H19BrN6OS·MeOH, C 63.02,
H 4.51, N 16.33, S 6.23; found C 63.42, H 4.35, N 16.74,
S 6.03.
5‑(2‑Hydroxybenzylidene)‑[(4‑methyl‑3‑{[4‑(pyridin‑3‑yl)
pyrimidin‑2‑yl]amino}phenyl)imino]‑1,3‑thiazolidin‑4‑one
17 Yellow solid. Yield 27%, 0.130 g. TLC Rf: 0.49 (S1).
HPLC tR (min): 2.61 (CS2). M.p.: 217–218 °C (dec.). IR
υmax (cm−1): 3450–3100 (O–H str, N–H str), 3066 (aro-
matic C–H str), 2974 (aliphatic C–H str), 1680 (thiazoli-
din-4-one C=O str), 1573, 1525 (C=N str, N–H bending,
C=C str), 1444, 1417, 1402 (aliphatic C–H bending, C–N
str), 1193 (C–O str), 800, 758 (aromatic C–H bending).
1H-NMR δ ppm (300 MHz, DMSO-d6): 9.26–6.77 (14H,
ArH), 2.35–2.15 (3H, ArCH3). Elemental analysis cald for
C26H20N6O2S·3/2EtOH, C 63.37, H 5.32, N 15.29, S 5.83;
found C 62.74, H 4.56, N 14.40, S 3.83.
5‑(4‑Trifuoromethylbenzylidene)‑[(4‑methyl‑3‑{[4‑(pyridin‑3
‑yl)pyrimidin‑2‑yl]amino}phenyl)imino]‑1,3‑thiazolidin‑4‑one
15 Yellow solid. Yield 71%, 0.377 g. TLC Rf: 0.57 (S1).
HPLC tR (min): 11.94 (CS1). M.p.: 257–262 °C (dec.). IR
υmax (cm−1): 3441 (N–H str), 3055 (aromatic C–H str), 1699
(thiazolidin-4-one C=O str), 1639, 1612, 1583, 1537, 1514
(C=N str, N–H bending, C=C str), 1456, 1438 (aliphatic
C–H bending), 1224 (C–N str), 1114 (C–F str), 810, 783,
752 (aromatic C–H bending). 1H-NMR δ ppm (300 MHz,
DMSO-d6): 13.05–11.20 (bs, 3H-thiazolidin-4-one), 9.26 (s,
1H), 9.10 and 8.97 (s, 1H, sec. NH), 8.68 (d, 1H, J=3.4 Hz),
8.63 (d, 1H, J = 3.4 Hz), 8.55–8.51 (t, 1H, J = 5.1 Hz,
J=9.1 Hz, J=6.0 Hz), 8.47–8.39 (m, 1H), 8.12 (s, 0.5H),
7.93–7.71 (m, 5H), 7.53–7.26 (m, 4H), 6.81–6.79 (d, 0.5H,
J=7.7 Hz), 2.29 and 2.26 (s, s, 3H, ArCH3). LC/MS APCI+
m/z (%): 532.659 ([M+H]+, 100), 338.199 (17); APCI− m/z
(%): 531.087 ([M−H]−, 100). Elemental analysis cald for
C27H19F3N6OS·3/2H2O, C 57.95, H 3.96, N 15.02, S 5.73;
found C 58.17, H 3.07, N 15.00, S 5.82.
5‑(2‑Methoxybenzylidene)‑[(4‑methyl‑3‑{[4‑(pyridin‑3‑yl)
pyrimidin‑2‑yl]amino}phenyl)imino]‑1,3‑thiazolidin‑4‑one
18 Yellow-orange solid. Yield 29%, 0.145 g. TLC Rf:
0.49 (S1). HPLC tR (min): 1.56 (CS5). M.p.: 228–230 °C.
IR υmax (cm−1): 3443 (N–H str), 2902 (aliphatic C–H str),
1687 (thiazolidin-4-one C=O str), 1645, 1581, 1521 (C=N
str, N–H bending, C=C str), 1479, 1442, 1415 (aliphatic
C–H bending), 1330 (C–N str), 1209 (C–O str), 833, 798,
723 (aromatic C–H bending). 1H-NMR δ ppm (300 MHz,
D2O Exchange, DMSO-d6): 9.25 (s, 1H), 8.69–8.65–6.80
(m, 14H), 3.86 (D2O), 3.90–3.86 (s, 3H, OCH3), 2.29
and 2.27 (s, 3H, ArCH3). Elemental analysis cald for
C27H22N6O2S·EtOH, C 64.97, H 4.87, N 16.24, S 6.19;
found C 64.86, H 4.60, N 16.47, S 6.14.
5‑(3‑Methoxybenzylidene)‑[(4‑methyl‑3‑{[4‑(pyridin‑3‑yl)
pyrimidin‑2‑yl]amino}phenyl)imino]‑1,3‑thiazolidin‑4‑one
19 Yellow-orange solid. Yield 56%, 0.275 g. TLC Rf:
0.49 (S1). HPLC tR (min): 1.69 (CS3). M.p.: 160–161 °C.
IR υmax (cm−1): 3453 (N–H str), 2962 (aliphatic C–H str),
1691 (thiazolidin-4-one C=O str), 1649, 1606, 1564, 1552,
1523 (C=N str, N–H bending, C=C str), 1446, 1415 (ali-
phatic C–H bending), 1398 (C–N str), 1147 (C–F str), 877,
5‑(4‑Trifuoromethoxybenzylidene)‑[(4‑methyl‑3‑{[4‑(pyridin‑3
‑yl)pyrimidin‑2‑yl]amino}phenyl)imino]‑1,3‑thiazolidin‑4‑one
16 Yellow-orange solid. Yield 47%, 0.260 g. TLC Rf: 0.57
(S1). HPLC tR (min): 4.30 (CS1). M.p.: 250–256 °C (dec.).
IR υmax (cm−1): 3444 (N–H str), 3063 (aromatic C–H str),
2978 (aliphatic C–H str), 1697 (thiazolidin-4-one C=O
str), 1641, 1583, 1541, 1516 (=N str, N–H bending, C=C
str), 1458, 1438, 1400 (aliphatic C–H bending), 1249 (C–O
str), 1224 (C–N str), 1157 (C–F str), 810, 783, 702 (aro-
matic C–H bending). 1H-NMR δ ppm (300 MHz, DMSO-
d6): 12.90–11.40 (bs, 3H-thiazolidin-4-one), 9.26 (d, 1H,
J = 1.8 Hz), 9.10 and 8.98 (s, 1H, sec. NH), 8.69–8.75
(dd, 0.5 H, J = 1.5 Hz, J = 4.8 Hz, J = 1.5 Hz), 8.70–8.62
(dd, 0.5 H, J = 1.5 Hz, J = 4.7 Hz, J = 1.8 Hz), 8.55–8.50
(t, 1H, J=5.2 Hz, J=11.4 Hz, J=5.2 Hz), 8.47–8.39 (m,
1H), 8.12 (s, 0.5H), 7.78–7.25 (m, 9H), 6.75–6.72 (d, 0.5 H,
J=5.4 Hz), 2.28 and 2.25 (s, s, 3H, ArCH3). LC/MS APCI+
1
785, 763, 752 (aromatic C–H bending). H-NMR δ ppm
(300 MHz, CDCl3): 9.24 and 9.20 (s, s, 1H), 8.63–8.33 (m,
3H), 8.00 (s, 1H), 7.77–6.57 (m, CHCl3 and 9H), 3.87–3.75
(m, 3H, OCH3), 2.37 and 2.33 (s, s, 3H, ArCH3). Elemen-
tal analysis cald for C27H22N6O2S·H2O·3/2H2O, C 62.17,
H 5.41, N 15.04, S 6.15; found C 62.15, H 4.95, N 15.84,
S 5.66.
5‑(4‑Methoxybenzylidene)‑[(4‑methyl‑3‑{[4‑(pyridin‑3‑yl)
pyrimidin‑2‑yl]amino}phenyl)imino]‑1,3‑thiazolidin‑4‑one
20 Yellow-orange solid. Yield 61%, 0.300 g. TLC Rf: 0.49
1 3

Molecular Diversity
(S1). HPLC tR (min): 1.47 (CS6). M.p.: 235–238 °C. IR υmax
(cm−1): 3450 (N–H str), 3012 (aromatic C–H str), 2972 (ali-
phatic C–H str), 1710 (thiazolidin-4-one C=O str), 1649,
1600, 1573, 1537, 1514 (C=N str, N–H bending, C=C str),
1454, 1417, 1402, 1336 (aliphatic C–H bending, C–N str),
1180, 1157 (C–O str), 825, 790, 769, 700 (aromatic C–H
bending). 1H-NMR δ ppm (300 MHz, DMSO-d6): 12.5–11.5
(bs), 9.26 (d, 1H, J =1.5 Hz), 9.08 and 8.96 (s, and s, 1H,
sec. NH), 8.68–8.64 (m, 1H), 8.52 (d, 1H, J=5,1 Hz), 8.46–
8.41 (m, 1H), 8.11 (s, 0.4H), 7.67–7.38 (m, 6H), 7.31–7.25
(t, 1H, J=7.5 Hz, J=7.8 Hz), 7.13 (d, 1H, J=8.4 Hz), 7.03
(d, 1H, J=8.7 Hz), 6.80 (d, 1H, J=7.2 Hz), 6.77 (d, 0.6H,
J=7.2 Hz), 3.83–3.78 (s, s, 3H, OCH3), 2.28 and 2.25 (s,
s, 3H, ArCH3). Elemental analysis cald for C27H22N6O2S,
C 65.57, H 4.48, N 16.99, S 6.47; found C 65.12, H 4.53, N
16.82, S 5.77.
thiazolidin-4-one NCH3), 2.28 (s, 3H, ArCH3). 13C-NMR
δ ppm (75 MHz, DMSO-d6): 166.15 (thiazolidin-4-one
C=O), 162.11, 161.42, 159.94, 159.57, 151.93, 150.34,
148.68, 146.17, 139.16, 134.79 (=CH–Ar), 132.73, 132.58,
131.66, 129.20, 128.53, 125.83, 125.00, 124.25, 121.80,
121.28, 117.45, 117.18, 116.76, 116.55, 108.42, 30.24 (thi-
azolidin-4-one NCH3), 18.22 (ArCH3). LC/MS APCI+ m/z
(%): 496.757 (M+, 100), 498.002 ([M+H]+), 338.187 (38);
LC/MS APCI− m/z (%): 495.228 ([M−H]−, 100), 219.358
(26). Elemental analysis cald for C27H21FN6OS·1/2MeOH,
C 64.44, H 4.52, N 16.40, S 6.26; found C 64.60, H 4.44, N
16.64, S 6.43.
5‑(3‑Fluorobenzylidene)‑3‑methyl‑[(4‑methyl‑3‑{[4‑(pyridin‑3
‑yl)pyrimidin‑2‑yl]amino}phenyl)imino]‑1,3‑thiazolidin‑4‑one
23 Yellow solid. Yield 56%, 0.277 g. TLC Rf: 0.60 (S1).
HPLC tR (min): 12.49 (CS6). M.p.: 184–185 °C. IR υmax
(cm−1): 3446 (N–H str), 3010 (aromatic C–H str), 1720
(thiazolidin-4-one C=O str), 1641, 1577, 1533 (C=N str,
N–H bending, C=C str), 1415 (aliphatic C–H bending),
5‑(N,N‑Dimethylaminobenzylidene)‑[(4‑methyl‑3‑{[4‑(pyridin‑
3‑yl)pyrimidin‑2‑yl]amino}phenyl)imino]‑1,3‑thiazolidin‑4‑one
21 Yellow solid. Yield 43%, 0.220 g. TLC Rf: 0.45 (S1).
HPLC tR (min): 3.28 (CS3). M.p.: 246–251 °C. IR υmax
(cm−1): 3446 (N–H str), 2899 (aliphatic C–H str), 1649 (thi-
azolidin-4-one C=O str), 1620, 1573, 1548 (C=N str, N–H
bending, C=C str), 1473, 1444, 1413 (aliphatic C–H bend-
ing), 1338 (C–N str), 1195 (C–Cl str), 802, 754, 705 (aro-
matic C–H bending). 1H-NMR δ ppm (300 MHz, DMSO-
d6): 12.12 and 11.51 (bs and bs 0.9 H, 3H-thiazolidin-4-one),
9.27 (s, 1H, sec. NH), 9.08 and 8.97 (s, s 1H), 8.66 (s, 1H),
8.53–8.52 (d, 1H, J=5.1 Hz), 8.44–8.40 (m, 1H), 8.11 (s,
0.5H), 7.60–7.26 (m, 7H), 6.85–6.72 (m, 2.5H), 3.01 and
3.95 (s and s, 6H), 2.28 and 2.26 (s and s, 3H, ArCH3). LC/
MS APCI+ m/z (%): 482.892 [M+H]+, 100), 376.084 (48),
278.108 (22); APCI− m/z (%): 481.136 ([M−H]−, 100).
Elemental analysis cald for C28H25N7OS·3/2H2O, C 62.90,
H 5.28, N 18.34, S 6.00; found C 62.65, H 5.00, N 18.56,
S 5.63.
1
1120 (C–F str), 792–783 (aromatic C–H e.b). H-NMR
δ ppm (300 MHz, DMSO-d6): 9.26 (d, 1H, J = 1.5 Hz),
8.96 (s, 1H, sec. NH), 8.64–8.62 (dd, 1H, J = 1.5 Hz,
J=3.0 Hz, J=1.5 Hz), 8.51 (d, 1H, J=5.27 Hz), 8.41 (d,
1H, J=7.9 Hz), 7.75 (s, 1H, =CH–Ar), 7.54–7.23 (m, 8H),
6.77 (d, 1H, J = 5.6 Hz), 3.37 (s, 3H, thiazolidin-4-one
NCH3), 2.29 (s, 3H, ArCH3). 13C-NMR δ ppm (75 MHz,
DMSO-d6): 165.66 (thiazolidin-4-one C=O), 163.77,
161.61, 160.87, 160.53, 159.33 151.33, 149.71, 148.09,
145.59, 138.58, 135.77, 135.66, 134.22 (=CH–Ar), 132.03,
131.15, 128.40, 127.92, 125.20, 125.16, 123.65, 123.29,
116.82, 116.68, 116.27, 107.86, 29.62 (thiazolidin-4-one
NCH3), 17.62 (ArCH3). LC/MS APCI+ m/z (%): 498.081
([M+H]+, 100), 496.897 (M+, 70). LC/MS APCI− m/z (%):
495.312 ([M−H]−, 100), 347.148 (24). Elemental analysis
cald for C27H21FN6OS, C 65.31, H 4.26, N 16.92, S 6.46;
found C 65.20, H 4.26, N 16.89, S 6.29.
5‑(2‑Fluorobenzylidene)‑3‑methyl‑[(4‑methyl‑3‑{[4‑(pyridin‑3
‑yl)pyrimidin‑2‑yl]amino}phenyl)imino]‑1,3‑thiazolidin‑4‑one
22 Yellow solid. Yield 65%, 0.322 g. TLC Rf: 0.56 (S1).
HPLC tR (min): 13.47 (CS6). M.p.: 210–213 °C. IR υmax
(cm−1): 3410 (N–H str), 3030 (aromatic C–H str), 2912
(aliphatic C–H str), 2858 (aliphatic C–H str sim.), 1703
(thiazolidin-4-one C=O str), 1631, 1604, 1583, 1556, 1516
(C=N str, N–H bending, C=C str), 1435, 1411, 1398, 1363
(aliphatic C–H bending), 1232 (C–N str), 1118 (C–F str,),
5‑(4‑Fluorobenzylidene)‑3‑methyl‑[(4‑methyl‑3‑{[4‑(pyridin‑3
‑yl)pyrimidin‑2‑yl]amino}phenyl)imino]‑1,3‑thiazolidin‑4‑one
24 Yellow solid. Yield 65%, 0.320 g. TLC Rf: 0.60 (S1).
HPLC tR (min): 14.17 (CS6). M.p.: 217–219 °C. IR υmax
(cm−1): 3410 (N–H str), 3053 (aromatic C–H str), 2912 (ali-
phatic C–H str); 1699 (thiazolidin-4-one C=O str), 1629,
1581, 1558, 1519,1508 (C=N str, N–H bending, C=C str),
1437, 1402, 1363 (aliphatic C–H bending), 1116 (C–F
str), 802 (aromatic C–H e.b). 1H-NMR δ ppm (300 MHz,
DMSO-d6): 9.25 (d, 1H, J=2.0 Hz), 8.94 (s, 1H, sec. NH),
8.63–8.61 (dd, 1H, J = 1.6 Hz, J = 3.2 Hz, J = 1.5 Hz),
8.51 (d, 1H, J = 5.27 Hz), 8.43–8.39 (d, 1H, J = 7.9 Hz),
7.74 (s, 1H, =CH–Ar), 7.36 (d, 1H, J = 2.0 Hz), 7.60–
7.25 (m, 8H), 6.76–6.72 (dd, 1H, J = 2.0 Hz, J = 5.9 Hz,
J=2.1 Hz), 3.33 (s, 3H, thiazolidin-4-one NCH3), 2.28 (s,
1
825, 806, 752, 700 (aromatic C–H bending). H-NMR δ
ppm (300 MHz, DMSO-d6): 9.27 (d, 1H, J = 1.5 Hz),
8.95 (s, 1H, sec. NH), 8.65–8.63 (dd, 1H, J = 1.5 Hz,
J = 3.2 Hz, J = 1.6 Hz), 8.51 (d, 1H, J = 5.0 Hz), 8.42 (d,
1H, J=8.5 Hz), 7.75 (s, 1H, =CH–Ar), 7.52–7.26 (m, 6H),
7.37 (d, J=2.7 Hz, 1H), 7.28 (d, 1H, J=8.1 Hz), 6.76–6.73
(dd, 1H, J = 2.1 Hz, J = 5.8 Hz, J = 2.1 Hz), 3.35 (s, 3H,
1 3

Molecular Diversity
3H, ArCH3).13C-NMR δ ppm (75 MHz, DMSO-d6): 166.43
(thiazolidin-4-one C=O), 162.14, 161.79, 161.42, 159.95,
151.93, 150.58, 148.68, 146.30, 139.16, 134.81 (=CH–Ar),
132.85, 132.66, 132.59, 131.65, 130.59, 129.33, 128.42,
124.25, 121.86, 117.45, 117.13, 117.03, 116.81, 108.43,
29.66 (thiazolidin-4-one NCH3), 18.22 (ArCH3). LC/MS
APCI+ m/z (%): 519.242 ([M+Na]+, 100); LC/MS APCI−
m/z (%): 495.398 ([M−H]−, 92), 219, 369 (71), 78.972
(100). Elemental analysis cald for C27H21FN6OS·H2O, C
63.02, H 4.51, N 16.33, S 6.24; found C 63.06, H 4.37, N
16.26, S 6.24.
1H, J=8.3 Hz), 6.75–6.72 (dd, 1H, J=2.3 Hz, J=5.6 Hz,
J = 2.1 Hz), 3.34 (s, 3H, thiazolidin-4-one NCH3), 2.27
(s, 3H, ArCH3). 13C-NMR δ ppm (75 MHz, DMSO-d6):
166.05 (thiazolidin-4-one C=O), 162.13, 162.02, 161.41,
159.94, 151.93, 150.38, 148.69, 146.16, 139.15, 134.79
(=CH–Ar), 134.59, 132.81, 132.59, 131.96, 131.87, 130.77,
129.33, 128.55, 125.95, 125.50, 124.23, 117.44, 117.17,
108.43, 30.23 (thiazolidin-4-one NCH3), 18.21 (ArCH3).
LC/MS ESI+ m/z (%): 513.125 ([M+H]+, 34), 515.129
([M+H+2]+, 29), 325.376 (69), 288.347 (100). LC/MS
ESI− m/z (%): 78.95 (100). Elemental analysis cald for
C27H21ClN6OS·3/4H2O, C 61.59, H 4.31, N 15.96, S 6.09;
found C 61.98, H 4.28, N 16.06, S 6.08.
5‑(2,6‑Difuorobenzylidene)‑3‑methyl‑[(4‑methyl‑3‑{[4‑(pyr
idin‑3‑yl)pyrimidin‑2‑yl]amino}phenyl)imino]‑1,3‑thiazoli-
din‑4‑one 25 Yellow solid. Yield 64%, 0.330 g. TLC Rf:
0.55 (S1). HPLC tR (min): 11.94 (CS6). M.p.: 172–174 °C.
IR υmax (cm−1): 3429 (N–H str), 3041, 3022 (aromatic C–H
str), 2939 (aliphatic C–H str), 1710 (thiazolidin-4-one C=O
str), 1629, 1602, 1583, 1519 (C=N str, N–H bending, C=C
g.b), 1452, 1437, 1415, 1398, 1369 (aliphatic C–H bending),
1257 (C–N str), 1114 (C–F str), 877, 792, 740 (aromatic
5‑(4‑Chlorobenzylidene)‑[(4‑methyl‑3‑{[4‑(pyridin‑3‑yl)pyrimi-
din‑2‑yl]amino}phenyl)imino]‑1,3‑thiazolidin‑4‑one 27 Yel-
low solid. Yield 63%, 0.323 g. TLC Rf: 0.60 (S1). HPLC tR
(min): 3.44 (CS4). M.p.: 190–193 °C. IR υmax (cm−1): 3435
(N–H str), 3093, 3049 (aromatic C–H str), 1712 (thiazoli-
din-4-one C=O str), 1643, 1606, 1575, 1548, 1523 (C=N
str, N–H bending, C=C str), 1413 (aliphatic C–H bending),
1093 (C–Cl str), 798 (aromatic C–H e.b). 1H-NMR δ ppm
(300 MHz, DMSO-d6): 9.25 (d, 1H, J = 2.0 Hz), 8.94 (s,
1H, sec. NH), 8.63–8.61 (dd, 1H, J = 1.5 Hz, J = 3.2 Hz,
J=1.7 Hz), 8.50 (d, 1H, J=5.3 Hz), 8.41 (d, 1H, J=8.1 Hz),
7.71 (s, 1H, =CH–Ar), 7.53–7.41 (m, 6H), 7.36 (d, 1H,
J = 2.1 Hz), 7.27 (d, 1H, J = 8.1 Hz), 6.75–6.72 (dd, 1H,
J=2.3 Hz, J=5.8 Hz, J=2.1 Hz), 3.33 (s, 3H, thiazolidin-
4-one NCH3), 2.28 (s, 3H, ArCH3). Elemental analysis cald
for C27H21ClN6OS, C 63.21, H 4.13, N 16.38, S 6.25; found
C 64.24, H 4.42, N 16.72, S 6.55.
1
C–H bending). H-NMR δ ppm (300 MHz, DMSO-d6):
9.26 (d, 1H, J=1.5 Hz), 8.92 (s, 1H, sec. NH), 8.66–8.64
(dd, 1H, J = 1.5 Hz, J = 3.2 Hz, J = 1.6 Hz), 8.48 (d, 1H,
J=5.1 Hz), 8.40 (d, 1H, J=8.5 Hz), 7.60 (s, 1H, =CH–Ar),
7.57–7.43 (m,3H), 7.32 (d, 1H, J=2.1 Hz), 7.25–7.16 (m,
3H), 6.72–6.69 (dd, J=2.1 Hz, 1H, J=5.8 Hz, J=8.1 Hz),
3.34 (s, 3H, thiazolidin-4-one NCH3), 2.26 (s, 3H, ArCH3).
13C-NMR δ ppm (75 MHz, DMSO-d6): 166.59 (thiazoli-
din-4-one C=O), 162.11, 161.40, 161.05, 159.91, 158.56,
151.91, 150.31, 148.67, 146.19, 139.10, 134.76 (=CH–Ar),
133.14, 132.55, 131.55, 129.54, 128.52, 124.20, 117.83,
117.40, 117.18, 112.89, 112.79, 111.39, 108.36, 30.27
(thiazolidin-4-one NCH3), 18.17 (ArCH3). LC/MS APCI+
m/z (%): 515.893 ([M+H]+, 100), 514.655 (M+, 90), APCI−
m/z (%): 513.128 ([M−H]−, 100), 219.338 (17). Elemental
analysis cald for C27H20F2N6OS·H2O, C 60.89, H 4.16, N
15.78, S 6.02; found C 60.83, H 4.05, N 15.80, S 6.17.
5‑(4‑Bromobenzylidene)‑3‑methyl‑[(4‑methyl‑3‑{[4‑(pyridin‑3
‑yl)pyrimidin‑2‑yl]amino}phenyl)imino]‑1,3‑thiazolidin‑4‑one
28 Yellow solid. Yield 58%, 0.325 g. TLC Rf: 0.58 (S1).
HPLC tR (min): 14.17 (CS6). M.p.: 205–207 °C. IR υmax
(cm−1): 3433 (N–H str), 3095(aromatic C–H str), 1714
(thiazolidin-4-one C=O str), 1641 1604, 1575, 1548, 1523
(C=N str, N–H e.b), (C=C str), 1413 (aliphatic C–H bend-
ing), 1003 (C–Br str), 798 (aromatic C–H e.b). 1H-NMR δ
ppm (300 MHz, DMSO-d6): 9.25 (d, 1H, J=1.8 Hz), 8.94
(s, 1H, sec. NH), 8.63–8.61 (dd, 1H, J=1.5 Hz, J=3.2 Hz,
J=1.7 Hz), 8.51 (d, 1H, J=5.3 Hz), 8.41 (d, 1H, J=8.1 Hz),
7.69 (s, 1H, =CH–Ar), 7.65 (d, 2H, J=8.4 Hz), 7.45–7.42
(m, 3H), 7.36 (d, 1H, J=2.1 Hz), 7.28 (d, 1H, J=8.1 Hz),
6.75–6.72 (dd, 1H, J=2.1 Hz, 1H, J=5.8 Hz, J=2.3 Hz),
3.33 (s, 3H, thiazolidin-4-one NCH3), 2.28 (s, 3H, ArCH3).
13C-NMR δ ppm (75 MHz, DMSO-d6): 166.37 (thiazoli-
din-4-one C=O), 162.14, 161.42, 159.96, 151.96, 150.43,
148.69, 146.26, 139.19, 134.80 (=CH–Ar), 133.11, 132.86,
132.59, 132.74, 132.56, 132.10, 131.66, 129.17, 128.43,
124.25, 123.86, 123.01, 117.45, 117.06, 108.43, 30.20 (thi-
azolidin-4-one NCH3), 18.23 (ArCH3). LC/MS ESI+ m/z
5‑(2‑Chlorobenzylidene)‑3‑methyl‑[(4‑methyl‑3‑{[4‑(pyrid
in‑3‑yl)pyrimidin‑2‑yl]amino}phenyl)imino]‑1,3‑thiazoli-
din‑4‑one 26 Yellow solid. Yield 55%, 0.282 g. TLC Rf:
0.56 (S1). HPLC tR (min): 4.08 (CS1). M.p.: 205-210 °C.
IR υmax (cm−1): 3443 (N–H str), 3070 (aromatic C–H str),
2976 (aliphatic C–H str), 1708 (thiazolidin-4-one C=O str),
1663, 1600, 1583, 1518 (C=N str, N–H bending, C=C str),
1437, 1417, 1398, 1365 (aliphatic C–H bending), 1259 (C–N
str), 1105 (C–Cl str), 881, 798, 759, 702 (aromatic C–H
bending). 1H-NMR δ ppm (300 MHz, DMSO-d6): 9.26 (d,
1H, J=1.7 Hz), 8.95 (s, 1H, sec. NH), 8.66–8.63 (dd, 1H,
J=1.5 Hz, J=3.2 Hz, J=1.7 Hz), 8.50 (d, 1H, J=5.0 Hz),
8.41 (d, 1H, J=8.5 Hz), 7.78 (s, 1H, =CH–Ar), 7.62–7.60
(m, 1H),7.49–7.38 (m, 5H), 7.35 (d, 1H, J=2.0 Hz), 7.27 (d,
1 3

Molecular Diversity
(%): 580.097 ([M+Na]+, 100); ESI− m/z (%): 75.12 (100).
Elemental analysis cald for C27H21BrN6OS·H2O, C 56.35,
H 4.03, N 14.60, S 5.57; found C 56.92, H 3.86, N 14.80,
S 5.74.
for C28H21F3N6O2S, C 59.78, H 3.76, N 14.94, S 5.70; found
C 59.49, H 3.89, N 14.84, S 5.62.
5‑(2‑Hydroxybenzylidene)‑3‑methyl‑[(4‑methyl‑3‑{[4‑(pyri
din‑3‑yl)pyrimidin‑2‑yl]amino}phenyl)imino]‑1,3‑thiazoli-
din‑4‑one 31 Yellow solid. Yield 32%, 0.158 g. TLC Rf:
0.53 (S1). HPLC tR (min): 1.72 (CS7). M.p.: 195–198 °C.
IR υmax (cm−1): 3387 (O–H str, N–H str), 3041 (aromatic
C–H str), 2951 (aliphatic C–H str), 1716 (thiazolidin-4-one
C=O str), 1633, 1593, 1560, 1527 (C=N str, N–H bending,
C=C str), 1444, 1415 (aliphatic C–H bending, C–N str),
1122 (C–O str), 896, 758, 704 (aromatic C–H bending). 1H-
NMR δ ppm (300 MHz, DMSO-d6): 10.47 (s, 0.7 H), 9.27
(d, 1H, J = 1.5 Hz), 8.98 (s, sec. NH), 8.66–8.64 (dd, 1H,
J=1.8 Hz, J=3.0 Hz, J=1.5 Hz), 8.51 (d, 1H, J=5.1 Hz),
8.44–8.40 (m, 1H), 7.98 (s, 1H, =CH–Ar), 7.47–7.43 (m,
2H), 7.33 (d, 1H, J=2.1 Hz), 7.27–7.23 (m, 3H), 7.21 (d,
1H, J=8.1 Hz), 6.87 (t, 1H, J=7.8 Hz, J=7.5 Hz), 6.76–
6.73 (dd, 1H, J=2.1 Hz, J=6.0 Hz, J=2.1 Hz), 3.34 (s, 3H,
thiazolidin-4-one NCH3), 2.27 (s, 3H, ArCH3). 13C-NMR
δ ppm (75 MHz, DMSO-d6): 166.68 (thiazolidin-4-one
C=O), 162.11, 161.46, 159.94, 151.93, 151.17, 148.68,
146.45, 139.12, 134.80 (=CH–Ar), 132.61, 132.65, 131.60,
128.74, 128.39, 125.65, 124.26, 120.85, 120.55, 120.05,
117.59, 117.17, 116.52, 110.33, 108.35, 30.03 (thiazolidin-
4-one NCH3), 18.21 (ArCH3). Elemental analysis cald for
C27H22N6O2S·H2O, C 63.27, H 4.72, N 16.40, S 6.26; found
C 62.97, H 4.44, N 15.97, S 6.04.
5 ‑ ( 4 ‑ T r i f l u o r o m e t h y l b e n z y l i d e n e ) ‑ 3 ‑ m e t h y l ‑ ‑
[(4‑methyl‑3‑{[4‑(pyridin‑3‑yl)pyrimidin‑2‑yl]amino}phenyl)
imino]‑1,3‑thiazolidin‑4‑one 29 Yellow solid. Yield 40%,
0.218 g. TLC Rf: 0.58(S1). HPLC tR (min): 4.49 (CS4).
M.p.: 196–200 °C. IR υmax (cm−1): 3435 (N–H str), 3030
(aromatic C–H str), 2945 (aliphatic C–H str), 1703 (thia-
zolidin-4-one C=O str), 1639, 1602, 1579, 1566, 1554,
1523 (C=N str N–H bending, C=C str), 1448, 1415, 1400,
1319 (aliphatic C–H bending), 1263 (C–N str), 1114
1
(C–F str), 794, 702 (aromatic C–H bending). H-NMR
δ ppm (300 MHz, DMSO-d6): 9.27 (d, 1H, J = 1.5 Hz),
8.95 (s, 1H, sec. NH), 8.65–8.63 (dd, 1H, J = 1.5 Hz,
J = 3.2 Hz, J = 1.6 Hz), 8.51 (d, 1H, J = 5.0 Hz), 8.42 (d,
1H, J=8.5 Hz), 7.75 (s, 1H, =CH–Ar), 7.52–7.26 (m, 6H),
7.37 (d, 1H, J=2.7 Hz), 7.29 (d, 1H, J=8.1 Hz), 6.76–6.73
(dd, J = 2.1 Hz, 1H, J = 5.8 Hz, J = 8.1 Hz), 3.35 (s, 3H,
thiazolidin-4-one NCH3), 2.28 (s, 3H, ArCH3). 13C-NMR
δ ppm (75 MHz, DMSO-d6): 166.20 (thiazolidin-4-one
C=O), 162.15, 162.42, 159.94, 151.93, 150.25, 148.68,
146.19, 139.20, 137.88, 134.77 (=CH–Ar), 132.58,
131.66, 130.74, 129.86, 129.54, 128.56, 128.49, 126.52,
125.75, 125.21, 124.20, 123.03, 117.42, 117.02, 108.43,
30.24 (thiazolidin-4-one NCH3), 18.23 (ArCH3). LC/MS
APCI+ m/z (%): 547.860 ([M+H]+, 100), 546.547 (M+,
94), 514.953 (21), 338.200 (22); APCI− m/z (%): 545.146
([M−H]−, 100), 219.351 (11). Elemental analysis cald for
C28H21F3N6OS·H2O, C 61.53, H 3.87, N 15.38, S 5.87;
found C 61.26, H 3.90, N 15.27, S 5.93.
5‑(2‑Methoxybenzylidene)‑3‑methyl‑[(4‑methyl‑3‑{[4‑(pyr
idin‑3‑yl)pyrimidin‑2‑yl]amino}phenyl)imino]‑1,3‑thiazoli-
din‑4‑one 32 Yellow solid. Yield 67%, 0.340 g. TLC Rf:
0.59 (S1). HPLC tR (min): 3.09 (CS3). M.p.: 177–180 °C.
IR υmax (cm−1): 3450 (N–H str), 3051, 3016 (aromatic C–H
str), 2897, 2845(aliphatic C–H str), 1716 (thiazolidin-4-one
C=O str), 1643, 1612, 1577, 1558, 1523 (C=N str, N–H
bending, C=C str), 1454, 1410, 1396 (aliphatic C–H bend-
ing, C–N str), 1128 (C–O str), 879, 796, 746 (aromatic C–H
bending). 1H-NMR δ ppm (300 MHz, DMSO-d6): 9.27 (d,
1H, J=1.5 Hz), 8.96 (s, 1H, sec. NH), 8.66–8.63 (dd, 1H,
J=1.5 Hz, J=3.3 Hz, J=1.5 Hz), 8.51 (d, 1H, J=5.1 Hz),
8.43–8.40 (m, 1H), 7.95 (s, 1H, =CH–Ar), 7.45–7.27 (m,
6H), 7.13 (d, 1H, J=8.4 Hz), 7.02–6.97 (t, 1H, J=7.5 Hz,
J = 7.8 Hz), 6.76–6.72 (dd, 1H, J = 2.1 Hz, J = 3.0 Hz,
J = 2.1 Hz), 3.87 (s, 3H, OCH3), 3.34 (s, 3H, thiazolidin-
4-one NCH3, covered by DMSO peak), 2.28 (s, 3H, ArCH3).
13C-NMR δ ppm (75 MHz, DMSO-d6): 166.50 (thiazoli-
din-4-one C=O), 162.12, 161.45, 159.93, 158.33, 151.96,
150.93, 148.67, 146.35, 139.12, 134.79 (=CH–Ar), 132.60,
132.42, 131.61, 128.62, 128.44, 124.81, 124.24, 122.36,
122.05, 121.37, 117.54, 117.28, 112.29, 108.37, 56.33
(2-OCH3), 30.07 (thiazolidin-4-one NCH3), 18.21 (ArCH3).
Elemental analysis cald for C28H24N6O2S·1/2MeOH, C
5 ‑ ( 4 ‑ Tr i f l u o r o m e t h o x y b e n z y l i d e n e ) ‑ 3 ‑ m e t h y l ‑ ‑
[(4‑methyl‑3‑{[4‑(pyridin‑3‑yl)pyrimidin‑2‑yl]amino}phenyl)
imino]‑1,3‑thiazolidin‑4‑one 30 Yellow solid. Yield 53%,
0.296 g. TLC Rf: 0.58 (S1). HPLC tR (min): 4.59 (CS1).
M.p.: 184–187 °C. IR υmax (cm−1): 3446 (N–H str), 3038
(aromatic C–H str), 1713 (thiazolidin-4-one C=O str), 1644,
1581, 1567, 1556, 1529 (C=N str, N–H bending, C=C str),
1412 (aliphatic C–H bending), 1153 (C–F str), 794 (aro-
1
matic C–H e.b). H-NMR δ ppm (300 MHz, DMSO-d6):
9.27 (d, 1H, J=1.5 Hz), 8.96 (s, 1H, sec. NH), 8.64–8.62
(dd, 1H, J = 1.7 Hz, J = 3.2 Hz, J = 1.7 Hz), 8.52 (d, 1H,
J = 5.1 Hz), 8.42 (d, 1H, J = 8.5 Hz), 7.78 (s, 1H, =CH–
Ar), 7.68–7.64 (m, 2H), 7.47–7.42 (m, 4H), 7.37 (d, 1H,
J = 2.0 Hz), 7.28 (d, 1H, J = 8.3 Hz), 6.76–6.73 (dd, 1H,
J=2.3 Hz, J=5.8 Hz, J=2.1 Hz), 3.35 (s, 3H, thiazolidin-
4-one NCH3), 2.29 (s, 3H, ArCH3). LC/MS APCI+ m/z (%):
585.155 ([M+Na]+, 23), 563.111 ([M+H]+, 100), 360.380
(53); APCI− m/z (%): 97.036 (100). Elemental analysis cald
1 3

Molecular Diversity
65.25, H 5.00, N 16.02, S 6.11; found C 65.41, H 4.53, N
16.38, S 5.57.
5‑(N,N‑Dimethylaminobenzylidene)‑3‑methyl‑[(4‑methyl‑3‑‑
{[4‑(pyridin‑3‑yl)pyrimidin‑2‑yl]amino}phenyl)imino]‑1,3‑thi-
azolidin‑4‑one 35 Yellow solid. Yield 55%, 0.285 g. TLC
Rf: 0.57 (S1). HPLC tR (min): 3.35, 3.95 (CS3). M.p.: 203–
205 °C. IR υmax (cm−1): 3448 (N–H str), 3024 (aromatic
C–H str), 2893, 2818 (aliphatic C–H str), 1699 (thiazoli-
din-4-one C=O str), 1641, 1612, 1579, 1554, 1523 (C=N
str, N–H bending, C=C str), 1442, 1400, 1402, 1363 (ali-
phatic C–H bending, C–N str), 877, 790 (aromatic C–H
bending). 1H-NMR δ ppm (300 MHz, DMSO-d6): 9.28 (d,
1H, J=1.5 Hz), 8.96 (s, 1H, sec. NH), 8.66–8.64 (dd, 1H,
J=1.5 Hz, J=3.3 Hz, J=1.5 Hz), 8.53 (d, 1H, J=5.1 Hz),
8.45–8.41 (m, 1H), 7.62 (s, 1H, =CH–Ar), 7.45–7.36 (m,
2H), 7.36–7.34 (m, 3H), 7.28–7.25 (d, 1H, J = 8.1 Hz),
6.77–6.72 (m, 3H), 3.32 (s, 3H, thiazolidin-4-one NCH3),
2.96 (s, 6H, N(CH3)2), 2,28 (s, 3H, ArCH3). 13C-NMR δ
ppm (75 MHz, DMSO-d6): 166.84 (thiazolidin-4-one C=O),
162.07, 161.49, 159.97, 151.96, 151.58, 151.44, 151.40,
148.67, 146.72, 139.11, 134.76 (=CH–Ar), 132.61, 132.23,
131.56, 131.47, 128.27, 124.26, 120.83, 117.68, 117.39,
114.63, 112.50, 108.30, 59.38 (4-N(CH3)2), 29.91 (thiazo-
lidin-4-one NCH3), 18.22 (ArCH3). Elemental analysis cald
for C29H27N7OS·1/2MeOH, C 65.90, H 5.44, N 18.24, S
5.96; found C 65.82, H 4.92, N 18.68, S 5.55.
5‑(3‑Methoxybenzylidene)‑3‑methyl‑[(4‑methyl‑3‑{[4‑(pyr
idin‑3‑yl)pyrimidin‑2‑yl]amino}phenyl)imino]‑1,3‑thiazoli-
din‑4‑one 33 Yellow solid. Yield 56%, 0.285 g. TLC Rf:
0.59 (S1). HPLC tR (min): 3.02 (CS3). M.p.: 158–161 °C.
IR υmax (cm−1): 3448 (N–H str), 3043, 3001 (aromatic C–H
str), 2893, 2978 (aliphatic C–H str), 1712 (thiazolidin-4-one
C=O str), 1641, 1608, 1579, 1558, 1554, 1525 (C=N str,
N–H bending, C=C str), 1475, 1410 (aliphatic C–H bend-
ing), 1396, 1300 (C–N str), 1147 (C–F str), 875, 790, 705
1
(aromatic C–H bending). H-NMR δ ppm (300 MHz,
CDCl3): 9.21 (d, 1H, J = 1,8 Hz), 8.65–8.63 (dd, 1H,
J=1.8 Hz, J=3.0 Hz, J=1.8 Hz), 8.51 (d, 1H, J=5.1 Hz),
8.38–8.34 (m, 1H), 8.01 (m, 1H), 7.72 (s, 1H, =CH–Ar),
7.35–7.22 (m, 3H), 7.17 (d, 1H, J=8.1 Hz), 7.08–6.86 (m,
3H), 6.73–6.70 (dd, 1H, J=2.1 Hz, J=5.7 Hz, J=2.1 Hz),
3.77 (s, 3H, OCH3), 3.49 (s, 3H, thiazolidin-4-one NCH3),
2.39 (s, 3H, ArCH3), 1.70 (H2O). 13C-NMR δ ppm (75 MHz,
DMSO-d6): 166.38 (thiazolidin-4-one C=O), 162.14,
161.45, 160.04, 159.93, 151.93, 150.67, 148.67, 146.24,
139.13, 135.25, 134.76 (=CH–Ar), 132.60, 131.63, 130.88,
130.33, 128.51, 124.24, 122.54, 121.85, 117.48, 117.31,
116.39, 115.88, 108.38, 55.75 (3-OCH3), 30.12 (thiazolidin-
4-one NCH3), 18.23 (ArCH3). Elemental analysis cald for
C28H24N6O2S·1/2H2O, C 64.97, H 4.87, N 16.24, S 6.19;
found C 65.25, H 4.49, N 16.27, S 5.64.
5‑(2‑Fluorobenzylidene)‑3‑ethyl‑[(4‑methyl‑3‑{[4‑(pyridin‑3
‑yl)pyrimidin‑2‑yl]amino}phenyl)imino]‑1,3‑thiaz olidin‑4‑one
36 Yellow solid. Yield 75%, 0.380 g. TLC Rf: 0.59 (S1).
HPLC tR (min): 4.42 (CS5). M.p.: 170–171 °C. IR υmax
(cm−1): 3443 (N–H str), 3036, 2976, 2933 (aliphatic C–H
str), 1708 (thiazolidin-4-one C=O str), 1639, 1608, 1581,
1558, 1554, 1518 (C=N str, N–H bending, C=C str), 1431,
1390, 1371, 1340 (aliphatic C–H bending, C–N str), (1118
(C–F str), 875, 796, 769, 707 (aromatic C–H bending). 1H-
NMR δ ppm (300 MHz, D2O Exchange, DMSO-d6): 9.24 (d,
1H, J=1.5 Hz), 8.65–8.63 (dd, 1H, J=1.5 Hz, J=3.3 Hz,
J = 1.5 Hz), 8.51 (d, 1H, J = 5.1 Hz), 8.43–8.39 (m, 1H),
7.72 (s, 1H, =CH–Ar), 7.50–7.21 (m, 8H), 6.77–6.74 (dd,
1H, J=2.1 Hz, J=5.7 Hz, J=2.1 Hz), 3.98 (q, 2H, thiazo-
lidin-4-one NCH2CH3), 2.56 (H2O), 2.29 (s, 3H, ArCH3),
1.30–1.25 (t, 3H, thiazolidin-4-one NCH2CH3, J=6.9 Hz,
J=7.2 Hz). Elemental analysis cald for C28H23FN6O2S·H2O,
C 63.62, H 3.59, N 15.90, S 6.07; found C 63.69, H 4.30, N
15.85, S 5.94.
5‑(4‑Methoxybenzylidene)‑3‑methyl‑[(4‑methyl‑3‑{[4‑(pyr
idin‑3‑yl)pyrimidin‑2‑yl]amino}phenyl)imino]‑1,3‑thiazoli-
din‑4‑one 34 Yellow solid. Yield 51%, 0.260 g. TLC Rf:
0.56 (S1). HPLC tR (min): 2.28 (CS3). M.p.: 195–198 °C.
IR υmax (cm−1): 3446 (N–H str), 3010 (aromatic C–H str),
2947 (aliphatic C–H str), 1708 (thiazolidin-4-one C=O
str), 1635, 1577, 1533, 1508 (C=N str, N–H bending, C=C
str), 1435, 1410, 1402, 1365 (aliphatic C–H bending, C–N
str), 1101 (C–O str), 885, 785, 700 (aromatic C–H bend-
ing). 1H-NMR δ ppm (300 MHz, DMSO-d6): 9.26 (d, 1H,
J=1.5 Hz), 8.95 (s, sec. NH), 8.65–8.63 (dd, 1H, J=1.8 Hz,
J=3.0 Hz, J=1.5 Hz), 8.52 (d, 1H, J=5.4 Hz), 8.45–8.41
(m, 1H), 7.70 (s, 1H, =CH–Ar), 7.49–7.42 (m, 4H), 7.29
(d, 1H, J=8.1 Hz), 7.04 (d, 2H, J=8.7 Hz), 6.77–6.74 (dd,
1H, J=2.1 Hz, J=5.7 Hz, J=2.1 Hz), 3.77 (s, 3H, OCH3),
3.33 (s, 3H, thiazolidin-4-one NCH3), 2.28 (s, 3H, ArCH3).
13C-NMR δ ppm (75 MHz, DMSO-d6): 166.62 (thiazoli-
din-4-one C=O), 162.11, 161.45, 160.06, 159.95, 151.94,
150.94, 148.67, 146.46, 143.49, 139.14, 134.76 (=CH–Ar),
132.59, 132.27, 132.25, 131.62, 130.38, 128.36, 126.37,
124.24, 118.97, 117.55, 117.25, 115.31, 108.37, 55.92
(4-OCH3), 30.04 (thiazolidin-4-one NCH3), 18.32 (ArCH3).
Elemental analysis cald for C28H24N6O2S, C 66.12, H 4.76,
N 16.52, S 6.30; found C 65.68, H 4.50, N 16.43, S 5.39.
5‑(3‑Fluorobenzylidene)‑3‑ethyl‑[(4‑methyl‑3‑{[4‑(pyridin‑3
‑yl)pyrimidin‑2‑yl]amino}phenyl)imino]‑1,3‑thiazolidin‑4‑one
37 Yellow solid. Yield 56%, 0.285 g. TLC Rf: 0.62 (S1).
HPLC tR (min): 4.49 (CS4). M.p.: 184 °C. IR υmax (cm−1):
3446 (N–H str), 3068 (aromatic C–H str), 2970 (aliphatic
C–H str), 1712 (thiazolidin-4-one C=O str), 1647, 1606,
1573, 1529 (C=N str, N–H bending, C=C str), 1438, 1413,
1386 (aliphatic C–H bending), 1249 (C–N str), 1120 (C–F
1 3

Molecular Diversity
str), 840, 808, 783, 707 (aromatic C–H e.b). 1H-NMR δ ppm
(300 MHz, DMSO-d6):): 9.26 (d, 1H, J=1.8 Hz), 8.96 (s,
1H, sec. NH), 8.65–8.63 (dd, 1H, J = 1.5 Hz, J = 3.0 Hz,
J = 1.7 Hz), 8.52 (d, 1H, J = 5.4 Hz), 8.43–8.39 (d, 1H,
J = 8.4 Hz), 7.75 (s, 1H, =CH–Ar), 7.54–7.22 (m, 6H),
7.41 (d, 1H, J=2.1 Hz), 7.29 (d, 1H, J=8.3 Hz), 6.78–6.74
(dd, 1H, J= 2.1 Hz, J = 5.7 Hz, J = 2.1 Hz), 3.98–3.91 (q,
2H), 2.29 (s, 3H, ArCH3), 1.29–1.25 (t, 3H, J = 6.9 Hz,
J=7.2 Hz). 13C-NMR δ ppm (75 MHz, DMSO-d6): 166.89
(thiazolidin-4-one C=O), 163.93, 162.11, 161.50, 161.40,
159.96, 151.95, 149.42, 148.68, 146.14, 139.16, 136.26,
134.73 (=CH–Ar), 132.59, 131.63, 129.06, 128.44, 125.73,
124.19, 123.81, 117.33, 117.17, 117.12, 116.97, 108.43,
38.55 (thiazolidin-4-one NCH2CH3), 18.22 (ArCH3), 12.99
(thiazolidin-4-one NCH2CH3). LC/MS APCI+ m/z (%):
511.997 ([M+H]+, 95), 510.786 (M+, 100); APCI− m/z
(%): 509.275 ([M−H]−, 100). Elemental analysis cald for
C28H23FN6OS, C 65.87, H 4.54, N 16.46, S 6.28; found C
65.51, H 4.67, N 16.42, S 6.11.
J=1.5 Hz, J=3.0 Hz, J=1.6 Hz), 8.48 (d, 1H, J=5.1 Hz),
8.41–8.37 (d, 1H, J = 6.3 Hz), 7.58 (s, 1H, =CH–Ar),
7.56–7.42 (m, 3H), 7.35 (d, 1H, J=2.1 Hz), 7.25–7.16 (m,
3H), 6.72–6.69 (dd, 1H, J=2.4 Hz, J=5.7 Hz, J=2.3 Hz),
3.97–3.90 (q, 2H), 2,25 (s, 3H, ArCH3), 1.30–1.25 (t, 3H,
J = 6.9 Hz, J = 7.2 Hz). LC/MS APCI+ m/z (%): 529.896
([M+H]+, 66), 528.564 (M+, 100); APCI− m/z (%): 527.271
([M−H]−, 100), 219.363 (18). Elemental analysis cald for
C28H22F2N6OS, C 63.62, H 4.20, N 15.90, S 6.07; found C
63.36, H 4.31, N 15.80, S 5.75.
5‑(2‑Chlorobenzylidene)‑3‑ethyl‑[(4‑methyl‑3‑{[4‑(pyridi
n‑3‑yl)pyrimidin‑2‑yl]amino}phenyl)imino]‑1,3‑thiazoli-
din‑4‑one 40 Yellow solid. Yield 60%, 0.315 g. TLC Rf:
0.55 (S1). HPLC tR (min): 6.00 (CS4). M.p.: 160–162 °C.
IR υmax (cm−1): 3444 (N–H str), 3036 (aromatic C–H str),
2974, 2933 (aliphatic C–H str), 1705 (thiazolidin-4-one
C=O str), 1633, 1581, 1554, 1518 (C=N str, N–H bending,
C=C str), 1431, 1390, 1371, 1342 (aliphatic C–H bending),
1284 (C–N str), 1093 (C–Cl str), 796, 763, 704 (aromatic
C–H e.b). 1H-NMR δ ppm (300 MHz, DMSO-d6): 9.26 (d,
1H, J=1.8 Hz), 8.95 (s, 1H, sec. NH), 8.65–8.64 (dd, 1H,
J=1.5 Hz, J=3.0 Hz, J=1.7 Hz), 8.50 (d, 1H, J=5.1 Hz),
8.43–8.39 (d, 1H, J=8.5 Hz), 7.88 (s, 1H, =CH–Ar), 7.62–
7.59 (m, 1H), 7.49–7.41 (m, 5H), 7.38 (d, 1H, J=2.1 Hz),
7.27 (d, 1H, J = 8.4 Hz), 6.76–6.72 (dd, 1H, J = 2.4 Hz,
J = 5.7 Hz, J = 2.3 Hz), 3.99–3.91 (q, 2H), 2.27 (s, 3H,
ArCH3), 1.30–1.25 (t, 3H, J=6.9 Hz, J=7.2 Hz). LC/MS
APCI+ m/z (%): 528.789 ([M+2]+, 75), 526.787 ((M+H]+,
100); APCI− m/z (%): 525.172 ([M−H]−, 100). Elemental
analysis cald for C28H23ClN6OS·MeOH, C 62.30, H 4.87,
N 15.03, S 5.74; found C 62.14, H 4.39, N 15.54, S 5.37.
5‑(4‑Fluorobenzylidene)‑3‑ethyl‑[(4‑methyl‑3‑{[4‑(pyridin‑3
‑yl)pyrimidin‑2‑yl]amino}phenyl)imino]‑1,3‑thiazolidin‑4‑one
38 Yellow solid. Yield 55%, 0.280 g. TLC Rf: 0.57 (S1).
HPLC tR (min): 3.98, 4.30 (CS4). M.p.: 139 °C. IR υmax
(cm−1): 3437 (N–H str), 3026 (aromatic C–H str), 2974
(aliphatic C–H str) 1707 (thiazolidin-4-one C=O str),
1637, 1575, 1533, 1506 (C=N str, N–H bending, C=C
str), 1442, 1417, 1386, 1367, 1338 (aliphatic C–H bend-
ing), 1228 (C–N str), 1124 (C–F str), 885, 825, 796 (aro-
1
matic C–H e.b). H-NMR δ ppm (300 MHz, DMSO-d6):
9.26 (d, 1H, J=1.8 Hz), 8.96 (s, 1H, sec. NH), 8.65–8.63
(dd, 1H, J = 1.5 Hz, J = 3.0 Hz, J = 1.7 Hz), 8.52 (d, 1H,
J = 5.4 Hz), 8.43–8.39 (d, 1H, J = 8.4 Hz), 7.75 (s, 1H,
=CH–Ar), 7.60–7.56 (m, 2H), 7.45–7.43 (m, 1H), 7.40
(d, 1H, J=2.1 Hz), 7.33–7.25 (m, 3H), 6.76–6.73 (dd, 1H,
J=2.1 Hz, J=5.7 Hz, J=2.2 Hz), 3.97–3.91 (q, 2H), 2.29
(s, 3H, ArCH3), 1.29–1.24 (t, 3H, J =6.9 Hz, J=7.2 Hz).
LC/MS APCI+ m/z (%):510.877 ([M+H]+, 100); APCI− m/z
(%): 509.250 ([M−H]−, 100). Elemental analysis cald for
C28H23FN6OS·1/2 MeOH, C 65.00, H 4.79, N 15.96, S 6.09;
found C 64.84, H 4.77, N 16.09, S 6.07.
5‑(4‑Chlorobenzylidene)‑3‑ethyl‑[(4‑methyl‑3‑{[4‑(pyridin‑3
‑yl)pyrimidin‑2‑yl]amino}phenyl)imino]‑1,3‑thiazolidin‑4‑one
41 Yellow solid. Yield 25%, 0.130 g. TLC Rf: 0.59 (S1).
HPLC tR (min): 5.35 (CS4). M.p.: 162–163 °C. IR υmax
(cm−1): 3441 (N–H str), 3022 (aromatic C–H str), 2991 (ali-
phatic C–H str), 1703 (thiazolidin-4-one C=O str), 1641,
1573, 1535 (C=N str, N–H bending, C=C str), 1450, 1423,
1388, 1373, 1333 (aliphatic C–H bending), 1234 (C–N str),
1093 (C–Cl str), 829, 802, 707 (aromatic C–H e.b). 1H-NMR
δ ppm (300 MHz, DMSO-d6): 9.26 (d, 1H, J = 1.8 Hz),
8.96 (s, 1H, sec. NH), 8.65–8.63 (dd, 1H, J = 1.8 Hz,
J=3.0 Hz, J=1.7 Hz), 8.50 (d, 1H, J=5.1 Hz), 8.43–8.39
(d, 1H, J=8.1 Hz), 7.74 (s, 1H, =CH–Ar), 7.45–7.39 (m,
6H), 7.40 (d, 1H, J = 2.1 Hz), 7.28 (d, 1H, J = 8.1 Hz),
6.76–6.73 (dd, 1H, J = 2.4 Hz, J = 5.7 Hz, J = 2.4 Hz),
3.98–3.91 (q, 2H), 2.28 (s, 3H, ArCH3), 1.29–1.24 (t, 3H,
J = 6.9 Hz, J = 7.2 Hz). LC/MS APCI+ m/z (%): 548.917
([M+Na]+, 34), 526.873 (M+, 100), 363.16 (27); APCI− m/z
(%): 525.189 ([M−H]−, 100). Elemental analysis cald for
5‑(2,6‑Difuorobenzylidene)‑3‑ethyl‑[(4‑methyl‑3‑{[4‑(pyridin‑
3‑yl)pyrimidin‑2‑yl]amino}phenyl)imino]‑1,3‑thiazolidin‑4‑one
39 Yellow solid. Yield 27%, 0.142 g. TLC Rf: 0.57 (S1).
HPLC tR (min): 4.03 (CS4). M.p.: 151–153 °C. IR υmax
(cm−1): 3431 (N–H str), 3095, 3730 (aromatic C–H str),
2976, 2947 (aliphatic C–H str), 1708 (thiazolidin-4-one C=O
str), 1633, 1604, 1581, 1554, 1519 (C=N str, N–H bending,
C=C str), 1435, 1390, 1371, 1342 (aliphatic C–H bending),
1253 (C–N str), 1118 (C–F g.b), 792, 744, 707 (aromatic
C–H e.b). 1H-NMR δ ppm (300 MHz, DMSO-d6): 9.25 (d,
1H, J=1.5 Hz), 8.92 (s, 1H, sec. NH), 8.63–8.63 (dd, 1H,
1 3

Molecular Diversity
C28H23ClN6OS, C 63.81, H 4.40, N 15.95, S 6.08; found C
63.32, H 4.55, N 15.88, S 5.71.
2931, 2872 (aliphatic C–H str), 1701 (thiazolidin-4-one
C=O str), 1637, 1602, 1583, 1558, 1523 (C=N str, N–H
bending, C=C str), 1413, 1338 (aliphatic C–H bending),
1234 (C–N str), 1163 (C–O str), 1112 (C–F str), 800, 705
(aromatic C–H e.b). 1H-NMR δ ppm (300 MHz, DMSO-d6):
9.26 (d, 1H, J=1.5 Hz), 8.96 (s, 1H, sec. NH), 8.64–8.62
(dd, 1H, J = 1.5 Hz, J = 3.0 Hz, J = 1.8 Hz), 8.52 (d, 1H,
J = 5.1 Hz), 8.43–8.39 (d, 1H, J = 6.3 Hz), 7.78 (s, 1H,
=CH–Ar), 7.78–7.70 (dd, 4H), 7.45–7.43 (m, 2H), 7.41 (d,
1H, J=2.1 Hz), 7.29 (d, 2H, J=8.1 Hz), 6.77–6.74 (dd, 1H,
J=2.1 Hz, J=5.7 Hz, J=2.1 Hz), 3.99–3.92 (q, 2H), 2.29
(s, 3H, ArCH3), 1.30–1.26 (t, 3H, J =6.9 Hz, J=7.8 Hz).
LC/MS APCI+ m/z (%): 576.564 (M+, 100); APCI− m/z
(%): 575.247 ([M−H]−, 100). Elemental analysis cald for
C29H23FN6O2S, C 60.41, H 4.02, N 14.58, S 5.56; found C
61.25, H 4.29, N 14.75, S 5.48.
5‑(4‑Bromobenzylidene)‑3‑ethyl‑[(4‑methyl‑3‑{[4‑(pyridi
n‑3‑yl)pyrimidin‑2‑yl]amino}phenyl)imino]‑1,3‑thiazoli-
din‑4‑one 42 Yellow solid. Yield 62%, 0.355 g. TLC Rf:
0.60 (S1). HPLC tR (min): 6.98 (CS4). M.p.: 167–170 °C.
IR υmax (cm−1): 3309 (N–H str), 3084 (aromatic C–H str),
2974 (aliphatic C–H str), 1701 (thiazolidin-4-one C=O str),
1635, 1604, 1573, 1548, 1533 (C=N str, N–H bending,
C=C str), 1452, 1421, 1384, 1369 (aliphatic C–H bending),
1240 (C–N str), 1114 (C–Br str), 812, 794, 705 (aromatic
C–H e.b). 1H-NMR δ ppm (300 MHz, DMSO-d6): 9.26 (d,
1H, J=1.8 Hz), 8.96 (s, 1H, sec. NH), 8.65–8.63 (dd, 1H,
J=1.5 Hz, J=3.0 Hz, J=1.8 Hz), 8.52 (d, 1H, J=5.4 Hz),
8.43–8.39 (d, 1H, J=8.5 Hz), 7.71 (s, 1H, =CH–Ar), 7.76–
7.73 (d, 2H, J= 9.0 Hz), 7.47–7.41 (m, 4H), 7.40 (d, 1H,
J = 4.2 Hz), 7.28 (d, 1H, J = 8.1 Hz), 6.76–6.73 (dd, 1H,
J=2.4 Hz, J=5.7 Hz, J=2.3 Hz), 3.98–3.91 (q, 2H), 2.28
(s, 3H, ArCH3), 1.29–1.24 (t, 3H, J =6.9 Hz, J=7.2 Hz).
LC/MS APCI+ m/z (%): 572.796 ([M+2]+, 96), 570.700
(M+, 100); APCI− m/z (%): 569.078 ([M−H]−, 100),
219.352 (21). Elemental analysis cald for C28H23BrN6OS,
C 57.93, H 4.17, N 14.48, S 5.52; found C 58.05, H 4.06, N
14.59, S 4.92.
5‑(2‑Hydroxybenzylidene)‑3‑ethyl‑[(4‑methyl‑3‑{[4‑(pyridin‑3
‑yl)pyrimidin‑2‑yl]amino}phenyl)imino]‑1,3‑thiazolidin‑4‑one
45 Yellow solid. Yield 40%, 0.202 g. TLC Rf: 0.54 (S1).
HPLC tR (min): 2.46 (CS7). M.p.: 250–255 °C. IR υmax
(cm−1): 3228 (O–H str, N–H str), 3039 (aromatic C–H str),
2970, 2928 (aliphatic C–H str), 1701 (thiazolidin-4-one
C=O str), 1622, 1597, 1587, 1554 (C=N str, N–H bending,
C=C str), 1448, 1388, 1371 (aliphatic C–H bending, C–N
str), 1244 (C–O str), 788, 704 (aromatic C–H bending). 1H-
NMR δ ppm (300 MHz, DMSO-d6): 10.49 (s, 0.9 H), 9.27
(d, 1H, J = 1.5 Hz), 8.97 (s, sec. NH), 8.66–8.64 (dd, 1H,
J=1.2 Hz, J=3.3 Hz, J=1.5 Hz), 8.52 (d, 1H, J=5.1 Hz),
8.44–8.40 (m, 1H), 7.99 (s, 1H, =CH–Ar), 7.46–7.43 (m,
2H), 7.38 (d, 1H, J = 1.8 Hz), 7.27–7.23 (m, 3H), 7.22
(d, 1H, J = 8.1 Hz), 6.87 (t, 1H, J = 7.5 Hz, J = 7.5 Hz),
6.77–6.74 (dd, 1H, J = 2.1 Hz, J = 6.0 Hz, J = 2.1 Hz),
3.97 (q, 2H, thiazolidin-4-one NCH2CH3), 3.43 (H2O),
2.29 (s, 3H, ArCH3), 1.29–1.24 (t, 3H, thiazolidin-4-one
NCH2CH3, J=6.9 Hz, J=6.9 Hz). Elemental analysis cald
for C28H24N6O2S, C 66.12, H 4.76, N 16.52, S 6.30; found
C 65.97, H 4.58, N 16.07, S 6.07.
5‑(4‑Trifuoromethylbenzylidene)‑3‑ethyl‑[(4‑methyl‑3‑{[4‑(p
yridin‑3‑yl)pyrimidin‑2‑yl]amino}phenyl)imino]‑1,3‑thiazoli-
din‑4‑one 43 Yellow solid. Yield 22%, 0.125 g. TLC Rf:
0.62 (S1). HPLC tR (min): 6.91 (CS4). M.p.: 152–153 °C. IR
υmax (cm−1): 3267 (N–H str), 2997, 2972 (aliphatic C–H str),
1707 (thiazolidin-4-one C=O str), 1637, 1579, 1556, 1525
(C=N str, N–H bending, C=C str), 1417, 1386, 1373 (ali-
phatic C–H bending), 1253 (C–N str), 1165 (C–F str), 798,
786, 705 (aromatic C–H e.b). 1H-NMR δ ppm (300 MHz,
DMSO-d6): 9.26 (d, 1H, J=1.5 Hz), 8.96 (s, 1H, sec. NH),
8.64-8.62 (dd, 1H, J=1.5 Hz, J=3.0 Hz, J=1.8 Hz), 8.52
(d, 1H, J=5.1 Hz), 8.43–8.39 (d, 1H, J=6.3 Hz), 7.78 (s,
1H, =CH–Ar), 7.68–7.63 (d, 2H), 7.47–7.42 (m, 4H), 7.41
(d, 1H, J = 2.4 Hz), 7.29 (d, 2H, J = 8.4 Hz), 6.77–6.74
(dd, 1H, J= 2.1 Hz, J = 5.7 Hz, J = 2.1 Hz), 3.99–3.92 (q,
2H), 2.28 (s, 3H, ArCH3), 1.30–1.25 (t, 3H, J = 6.9 Hz,
J = 7.8 Hz). LC/MS APCI+ m/z (%): 560.584 (M+, 100),
562.870 ([M+2]+, 66); APCI− m/z (%): 559.241 ([M−H]−,
100). Elemental analysis cald for C29H23FN6O2S, C 60.41,
H 4.02, N 14.58, S 5.56; found C 61.25, H 4.29, N 14.75,
S 5.48.
5‑(2‑Methoxybenzylidene)‑3‑ethyl‑[(4‑methyl‑3‑{[4‑(pyridin‑3
‑yl)pyrimidin‑2‑yl]amino}phenyl)imino]‑1,3‑thiazolidin‑4‑one
46 Yellow solid. Yield 81%, 0.422 g. TLC Rf: 0.57 (S1).
HPLC tR (min): 4.16 (CS7). M.p.: 205–208 °C. IR υmax
(cm−1): 3221 (N–H str), 3039–2841 (aliphatic C–H str),
1703 (thiazolidin-4-one C=O str), 1637, 1593, 1579, 1529
(C=N str, N–H bending, C=C str), 1419, 1384 (aliphatic
C–H bending, C–N str), 1244 (C–O str), 800, 705 (aromatic
C–H bending). 1H-NMR δ ppm (300 MHz, D2O Exchange,
DMSO-d6): 9.25 (d, 1H, J = 1.8 Hz), 8.65–8.63 (dd, 1H,
J=1.8 Hz, J=3.0 Hz, J=1.5 Hz), 8.51 (d, 1H, J=5.1 Hz),
8.43–8.39 (m, 1H), 7.93 (s, 1H, =CH–Ar), 7.46–7.42 (m,
4H), 7.29 (d, 1H, J=7.8 Hz), 7.13 (d, 1H, J=8.7 Hz), 7.01
(t, 1H, J=7.5 Hz, J=7.5 Hz), 6.77–6.74 (dd, 1H, J=2.1 Hz,
5‑(4‑Trifuoromethoxybenzylidene)‑3‑ethyl‑[(4‑methyl‑3‑{[4‑(
pyridin‑3‑yl)pyrimidin‑2‑yl]amino}phenyl)imino]‑1,3‑thiazo-
lidin‑4‑one 44 Yellow solid. Yield 36%, 0.205 g. TLC Rf:
0.62 (S1). HPLC tR (min): 6.29 (CS4). M.p.: 191–193 °C.
IR υmax (cm−1): 3452 (N–H str), 3034 (aromatic C–H str),
1 3

Molecular Diversity
J = 5.7 Hz, J = 2.1 Hz), 3.98 (q, 2H, thiazolidin-4-one
NCH2CH3), 3.86 (D2O), 3.73 (s, 3H, OCH3), 3.18 (MeOH),
2.29 (s, 3H, ArCH3), 1.29–1.25 (t, 3H, thiazolidin-4-one
NCH2CH3, J=7.2 Hz, J=6.9 Hz). Elemental analysis cald
for C29H26N6O2S·1/7MeOH, C 66.39, H 5.08, N 15.94, S
6.08; found C 65.71, H 5.37, N 16.03, S 5.78.
2893 (aliphatic C–H str), 1697 (thiazolidin-4-one C=O str),
1639, 1610, 1579, 1554, 1523 (C=N str, N–H bending, C=C
str), 1452, 1443, 1398 (aliphatic C–H bending), 1365, 1344
1
(C–N str), 792, 700 (aromatic C–H bending). H-NMR δ
ppm (300 MHz, DMSO-d6): 9.28 (d, 1H, J=1.8 Hz), 8.95
(s, 1H, sec. NH), 8.66–8.64 (dd, 1H, J=1.5 Hz, J=3.3 Hz,
J=1.5 Hz), 8.53 (d, 1H, J=5.1 Hz), 8.45–8.41 (m, 1H), 7.62
(s, 1H, =CH–Ar), 7.46–7.41 (m, 2H), 7.38–7.33 (m, 3H),
7.26 (d, 1H, J=8.1 Hz), 6.77–6.72 (m, 3H), 3.95–3.89 (q,
2H, thiazolidin-4-one NCH2CH3), 2.95 (s, 6H, N(CH3)2),
2.29 (s, 3H, ArCH3), 1.28–1.23 (t, 3H, thiazolidin-4-one
NCH2CH3, J=6.9 Hz, J=7.2 Hz). Elemental analysis cald
for C30H29N7OS·1/2MeOH, C 66.40, H 5.66, N 17.77, S
5.81; found C 66.38, H 5.31, N 18.12, S 5.70.
5‑(3‑Methoxybenzylidene)‑3‑methyl‑[(4‑methyl‑3‑{[4‑(pyridin‑
3‑yl)pyrimidin‑2‑yl]amino}phenyl)imino]‑1,3‑thiazolidin‑4‑one
47 Yellow solid. Yield 31%, 0.162 g. TLC Rf: 0.57 (S1).
HPLC tR (min): 4.08 (CS7). M.p.: 164–165 °C. IR υmax
(cm−1): 3490 (N–H str), 2949, 2901, 2829 (aliphatic C–H
str), 1716 (thiazolidin-4-one C=O str), 1639, 1608, 1579,
1554, 1523 (C=N str, N–H bending, C=C str), 1446, 1427,
1386 (aliphatic C–H bending, C–N str), 1242 (C–O str), 781,
682 (aromatic C–H bending). 1H-NMR δ ppm (300 MHz,
DMSO-d6): 9.27 (d, 1H, J=1.5 Hz), 8.97 (s, sec. NH), 8.66–
8.64 (dd, 1H, J = 1.5 Hz, J = 3.3 Hz, J = 1.5 Hz), 8.52 (d,
1H, J=5.1 Hz), 8.44–8.40 (m, 1H), 7.72 (s, 1H, =CH–Ar),
7.46–7.36 (m, 4H), 7.29 (d, 1H, J=8.1 Hz), 7.10–6.89 (m,
3H), 6.78–6.74 (dd, 1H, J=1.5 Hz, J=5.7 Hz, J=2.1 Hz),
3.98 (q, 2H, thiazolidin-4-one NCH2CH3), 3.74 (s, 3H,
OCH3) 3.45 (H2O), 2.29 (s, 3H, ArCH3), 1.29–1.25 (t, 3H,
thiazolidin-4-one NCH2CH3, J = 7.2 Hz, J = 6.9 Hz). Ele-
mental analysis cald for C29H26N6O2S·1/2EtOH, C 66.03,
H 5.36, N 15.40, S 5.88; found C 65.69, H 5.19, N 15.78,
S 5.98.
Anticancer activity
Cell culture conditions and reagents
K562 (CCL-243), PC3 (CRL-1435), SHSY-5Y (CRL-
2266) and L929 (CRL-6364) cells were acquired from
American Type Culture Collection (Manassas, VA, USA).
PC3, SHSY-5Y and L929 cells were cultured in DMEM
and K562 cells were grown in RPMI-1640 (Gibco Thermo
Fisher Scientifc) medium supplemented with 10% (v/v)
heat-inactivated FBS (Sigma-Aldrich) and 1% penicil-
lin/streptomycin (Gibco Thermo Fisher Scientifc). Cells
were cultured in a 25 cm2 cell culture fask and incubated
at 37 °C in a 5% CO2 humidifed atmosphere until they
reached approximately 80% confuence.
5‑(4‑Methoxybenzylidene)‑3‑ethyl‑[(4‑methyl‑3‑{[4‑(pyridin‑3
‑yl)pyrimidin‑2‑yl]amino}phenyl)imino]‑1,3‑thiazolidin‑4‑one
48 Yellow solid. Yield 58%, 0.305 g. TLC Rf: 0.56 (S1).
HPLC tR (min): 3.58 (CS7). M.p.: 170–174 °C. IR υmax
(cm−1): 3295 (N–H str), 2966–2829 (aliphatic C–H str),
1697 (thiazolidin-4-one C=O str), 1637, 1595, 1579, 1525
(C=N str, N–H bending, C=C str), 1421,1384 (aliphatic
C–H bending, C–N str), 1249 (C–O str), 798, 705 (aromatic
C–H bending). 1H-NMR δ ppm (300 MHz, DMSO-d6): 9.25
(d, 1H, J= 1.5 Hz), 8.95 (s, sec. NH), 8.64–8.62 (dd, 1H,
J=1.5 Hz, J=3.3 Hz, J=1.5 Hz), 8.51 (d, 1H, J=5.1 Hz),
8.43–8.39 (m, 1H), 7.68 (s, 1H, =CH–Ar), 7.47–7.38 (m,
4H), 7.28 (d, 1H, J = 8.1 Hz), 7.02 (d, 2H, J = 9.0 Hz),
6.76–6.73 (dd, 1H, J=2.1 Hz, J=5.7 Hz, J=2.1 Hz), 3.96
(q, 2H, thiazolidin-4-one NCH2CH3), 3.76 (s, 3H, OCH3),
2.28 (s, 3H, ArCH3), 1.27–1.23 (t, 3H, thiazolidin-4-one
NCH2CH3, J=6.9 Hz, J=7.2 Hz). Elemental analysis cald
for C29H26N6O2S, C 66.65, H 5.01, N 16.08, S 6.14; found
C 66.24, H 4.77, N 15.87, S 5.84.
Cell viability assay
Cell viability was assessed using the XTT (2,3-bis-
(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-
5-carboxanilide) colorimetric assay (Roche Diagnostic,
Germany). Imatinib and compounds 5–49 were dissolved
in dimethyl sulfoxide (DMSO concentration did not exceed
0.1%) and diluted in DMEM or RPMI-1640 prior to treat-
ment. The cells were incubated with 10 µM constant con-
centration of compounds for 24 and 48 h. At the end of the
incubation period, for determination of living cells, 50-μL
XTT labeling mixture was added to each well and then
the plates were incubated at 37 °C for another 4 h. After
mixing, the absorbance of XTT-formazan was measured
using an ELISA microplate reader (Thermo, Germany) at
450 nm. All experiments were performed in three inde-
pendent experiments and the cell viability was expressed
in percentage related to control (100% of viability).
5 ‑ ( N , N ‑ D i m e t h y l a m i n o b e n z y l i d e n e ) ‑ 3 ‑ e t h y l ‑ ‑
[(4‑methyl‑3‑{[4‑(pyridin‑3‑yl)pyrimidin‑2‑yl]amino}phenyl)
imino]‑1,3‑thiazolidin‑4‑one 49 Yellow solid. Yield 40%,
0.215 g. TLC Rf: 0.55 (S1). HPLC tR (min): 4.61, 5.34 (CS5).
M.p.: 187–190 °C. IR υmax (cm−1): 3454 (N–H str), 2972,
1 3

Molecular Diversity
Annexin V binding assay
of DNA double-strand breaks were determined using the
Muse™ Multi-Color DNA Damage software module.
After treatment of K562 cells with IC50 concentrations
of the compounds (8, 15 and 34) and imatinib, the extent
apoptosis was examined using the Muse Annexin V/Dead
Cell (Merck Millipore, Germany) assay, as described in
the manufacturer’s instructions. Briefy, K562 cells were
treated with selected compounds and imatinib for 24 and
48 h. After that, the cells were collected, diluted with PBS
containing 1% FBS and incubated with Muse™ Annexin
V & Dead Cell reagent for 20 min at room temperature in
the dark. The events for live, dead, early and late apoptotic
cells were analyzed by Muse™ Cell Analyzer (Merck Mil-
lipore, Germany). Data of apoptosis induction by com-
pounds and imatinib were calculated from three independent
experiments.
Statistical analyses
Statistical signifcances for the assays were assessed using
the GraphPad Prism 7.0 (GraphPad Software, Inc.). The
data obtained from the experiments were expressed as the
mean standard deviation (SD) and one-way ANOVA test
was performed for multiple comparisons. p values less
than 0.05 were considered to be statistically signifcant.
Molecular modeling studies
Molecular modeling studies with Abl kinase protein were
carried out to simulate the potential inhibition profle of
designed compounds. Protein crystal structure of the Abl
enzyme was obtained from protein data bank with the
1IEP PDB ID. Prior to molecular modeling studies, water
was removed from the crystal structure and protein was
prepared using the Schrödinger Protein Preparation Wiz-
ard. Ligands were subsequently prepared as not charged
by using the Schrödinger LigPrep tool. Energy minimiza-
tion was held using the OPLC force feld. Prepared ligand
structures were docked into protein binding site at pH:
7.0 1.0 using the Schrödinger Glide-Standard Precision
docking procedure considering induced ft approach [60].
In the meantime, imatinib which was extracted from the
1IEP structure was re-docked for the validation of the
docking procedure. RMSD between co-crystallized and
re-docked conformations of imatinib was found less than
3.0 Å that exists under the limitations. Superposition of
co-crystallized and re-docked conformations of imatinib
is presented in the supplementary fle. Detailed analyses
of the most active compounds are visualized in Figs. 7, 8,
9 and 10. Figures are prepared using the PyMOL software.
Cell cycle analysis
Cell cycle arrest was evaluated by Muse Cell Cycle Assay
Kit (Merck Millipore, Germany) according to the user’s
guide. The cells were treated with IC50 concentrations of the
compounds (8, 15 and 34) and imatinib frst and then incu-
bated for 48 h at 37 °C. After completion of the incubation,
the cells were collected by centrifugation (300×g, 5 min),
washed with cold PBS and fxed in chilled 70% (v/v) ethanol
for at least 3 h at − 20 °C before staining. Next, the fxed
cells were washed once with PBS and incubated with 200 μL
of assay solution for 30 min in the dark at room temperature
according to the manufacturer’s protocol. After staining, the
percentages of the cells at diferent stages of the cell cycle
(G0/G1, S and G2/M phases) were analyzed by the Muse
Cell Analyzer (Merck Millipore, Germany).
DNA damage assay
Acknowledgements This work was supported by the Research
Fund of Marmara University with the project number: SAĞ-C-
DRP-111115-0502. This work was also financially supported by
the Research Fund of Sivas Cumhuriyet University (CÜBAP) with
the project number: ECZ-046. During this research, Aslı Türe was
granted by the Scientifc and Technological Research Council of Tur-
key (TÜBİTAK) under the National Scholarship Programme for PhD
Students in Natural Sciences, Medical Sciences, Social Sciences and
Humanities and Engineering and Technological Sciences. The authors
are grateful to Prof. Dr. Habil. Wolfgang Sippl for giving the oppor-
tunity to use Schrödinger software. Authors also would like to thank
DNA damage induced by the compounds (8, 15 and 34)
was evaluated using Muse™ Multi-Color DNA Damage kit
(Merck Millipore, Germany) according to the manufacturer’s
instructions. Initially, K562 cells were treated with IC50 con-
centrations of the compounds and imatinib and then they
were incubated for 48 h at 37 °C. Next, the cells were centri-
fuged, washed once with 1X PBS and fxed in fxation bufer
for 10 min on ice, followed by washing and permeabilization
in the ice-cold bufer. Ten µL antibody cocktail solution was
added to each tube containing the cell suspension and incu-
bated again for 30 min in dark at room temperature. After the
fnal centrifugation and washing steps, the cells were resus-
pended in 200 µL 1X assay bufer and percentages of nega-
tive cells (no DNA damage), percentages of ATM activated
cells, percentages of H2AX activated cells and percentages
DEVA Pharmaceuticals for donation of im
atinib mesylate
and 4-methyl-N3-[4-(pyridin-3-yl)pyrimidin-2-yl]benzene-1,3-diamine
specimens for our research.
Compliance with ethical standards
Conflict of interest The authors declared no confict of interest.
1 3

Molecular Diversity
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