Synthesis and antiproliferative activity evaluation of new thiazole-benzimidazole derivatives using real-time cell analysis (RTCA DP)

Article abstract of DOI:10.1007/s00044-016-1507-0

A series of new 2-[(5-substituted-1H-benzimidazol-2-yl)thio]-N-[4-[2-phenylthiazol-4-yl]phenyl]acetamide derivatives (4a-p) were synthesized according to the reported literature, and anticancer activity of the compounds was evaluated. Cytotoxic activity w

Full text of DOI:10.1007/s00044-016-1507-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1507-0
ORIGINAL RESEARCH
Synthesis and antiproliferative activity evaluation of new thiazole–
benzimidazole derivatives using real-time cell analysis (RTCA DP)
1
Yusuf Ozkay Leyla Yurttas¸1 Miris¸ Dikmen2 Selin Engu¨r2
¨
•
•
•
Received: 9 March 2015 / Accepted: 6 January 2016
Ó Springer Science+Business Media New York 2016
Abstract A series of new 2-[(5-substituted-1H-benzimi-
dazol-2-yl)thio]-N-[4-[2-phenylthiazol-4-yl]phenyl]acetamide
derivatives (4a–p) were synthesized according to the
reported literature, and anticancer activity of the com-
pounds was evaluated. Cytotoxic activity was confirmed by
real-time cytotoxicity analysis system determining half
maximal inhibitory concentrations (IC50) of the title com-
pounds based on the dose–response curves derived by
xCELLigence measurements against NIH/3T3, A549 and
Caco2 cell lines for 24, 48 and 72 h exposure. Compound
4c was found to be as the most efficient molecule that
exhibited selective antiproliferative activity against both of
the cancer cells.
Introduction
Cytotoxic agents constitute an important class of anticancer
drugs. They act by damaging DNA, inhibiting its synthesis
or interfering with the mechanics of cell division, for
instance, by blocking topoisomerases or binding to
microtubules (Chabner and Roberts, 2005; Workman,
2005). Many of the cytotoxic agents were discovered by
screening for chemical compounds that were capable to
destroy cancer cells, as with a natural product like the
microtubule inhibitor paclitaxel (Rowinsky et al., 1992).
DNA-alkylating agents, originally based on sulphur and
nitrogen mustards, were structurally modified to control
their rates of chemical reactivity, leading to drugs like
cyclophosphamide and ifosphamide (Colvin, 1999). There
have been many remarkable successes with cytotoxic drug
treatments for cancer. The cancer exists in a large number
of forms, as defined anatomically under the light micro-
scope and at the molecular level. The efficacy of drug
treatment varies across these different anatomical, histo-
logical and molecular types. Significant progresses have
been reached in the treatment of leukaemias, lymphomas,
testicular cancer and children’s malignancies with cyto-
toxic drugs, leading to marked increases in survival
(Workman and Colins, 2008). The benzimidazole ring
system is an essential pharmacophore in medicinal chem-
istry and modern drug discovery. 2-Substitutedbenzimida-
zoles have been recognized to act as potential anticancer
(Prudhomme, 2006; Kumar et al., 2002; Hranjec et al.,
2007; Piskin et al., 2009). For instance, bis-benzimidazole
derivatives (Kim et al., 1996; Neidle et al., 1999; Le Sann
et al., 2006; Alpan et al., 2009; Huang et al., 2006) were
remarkably active compounds in interfering with DNA
topoisomerase I (Kim et al., 1996) and were also found to
be cytotoxic against breast adenocarcinoma (MCF7) (Le
Keywords Thiazole Á Benzimidazole Á
Cytotoxic activity Á Anticancer activity Á
Antiproliferative activity Á
Real-time cell analysis (RTCA) Á xCELLigence
All cell lines were purchased from ATCC with account number
20033704 on behalf of Miris¸ Dikmen.
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-016-1507-0) contains supplementary
material, which is available to authorized users.
& Leyla Yurttas¸
lyurttas@anadolu.edu.tr
1
Department of Pharmaceutical Chemistry, Faculty of
Pharmacy, Anadolu University, 26470 Eskisehir, Turkey
2
Department of Pharmacology, Faculty of Pharmacy, Anadolu
University, 26470 Eskisehir, Turkey
123

Med Chem Res
Sann et al., 2006) and skin epidermoid carcinoma (A431)
(Le Sann et al., 2006; Alpan et al., 2009; Huang et al.,
2006). Also, methyl-2-benzimidazole carbamate (carben-
dazim, FB642) is an anticancer agent that induces apop-
tosis of cancer cells (Hao et al., 2002; Hammond et al.,
2001). The next generation of rationally designed inhibi-
tors, benzimidazole carboxamides, for example, 2-(4-hy-
Results and discussion
Chemistry
The synthesis of 2-[(5-substituted-1H-benzimidazol-2-
yl)thio]-N-[4-[2-(4-substituted-phenyl)thiazol-4-yl]phenyl]
acetamide derivatives (4a–p) was carried out as shown in
Scheme 1. All reactions were carried out according to our
previous study (Yurttas et al. 2014) starting from 4-(2-
bromoacetyl)acetanilide which was prepared from
4-aminoacetophenone via acetylation and bromination
reactions. Compounds 1a–d were gained from the reaction
of the obtained a-halo ketone and thioacetamide derivatives
to get 4-(2-aryl-4-thiazolyl)acetanilides. The hydrolysation
reaction of the acetamide group of compounds 1a–d pro-
vided 4-(2-aryl-4-thiazolyl)aniline derivatives (2a–d).
After acetylation reaction with chloroacetyl chloride, the
obtained amide compounds (3a–d) were reacted to acquire
final compounds (4a–p) in yields ranging from 66 to 81 %.
The structures of all final compounds were demonstrated
droxyphenyl)benzimidazole-4-carboxamide
(NU1085)
(Bryant and Helleday, 2004) and 2-(4-oxadiazolylphenyl)
analogue (Tong et al., 2009), were vastly more potent as
poly(ADP-ribose)polymerase inhibitor (PARP inhibitor)
and improved the effects of chemotherapy and radiation
therapy in vitro (Bryant and Helleday, 2004) and in vivo
(Tong et al., 2009). Also, the tricyclic benzimidazole
(AG14361), a PARP inhibitor, has been developed and
used in vivo at non-toxic doses to augment the effect of the
DNA-damaging agents irinotecan (topoisomerase I poi-
sons), g-irradiation or temozolomide (DNA-alkylating
agent) (Calabrese et al., 2004).
In addition to benzimidazoles, thiazole derivatives con-
cerned the interest of medicinal chemists due to their syn-
thetic feasibility and their incorporation into variety of
therapeutically active agents. Meanwhile, DNA is one of the
major targets of anticancer drugs since the development of
the nitrogen mustards. Targeting DNA of tumour cells has
been one of the most effective clinical strategies for many
DNA intercalators such as groove binders and anticancer
antibiotics (Hurley, 2002; Cai et al., 2009; Bailly et al.,
1997; Yamamoto and Kawanishi, 1992). The clinical effi-
cacy of the groove binders tiazofurin, distamycin, netropsin,
thia-netropsin and bleomycin pointed out the importance of
the 1,3-thiazole moieties and their contribution to enhance
the antitumour activity (Chen and Pankiewicz, 2007; Pop-
savin et al., 2007, Foy et al., 2008; Wolter et al., 2009;
Silvermann, 1992; Nelson et al., 2007; Plouvier et al., 1995;
Delgado and Remers, 1998; Abraham et al., 2003; Da Rocha
et al., 2001). The antitumour activity of 2,4-disubstituted
1,3-thiazole analogues was reported and well documented
(Schnur et al., 1991; Kumar et al., 1993; El-Subbagh et al.,
1994, 1999, 2001; El-Subbagh and Al-Obaid, 1996; Al-
Omary et al., 2012, El-Messery et al., 2012).
1
using IR, H-NMR, 13C-NMR and MS spectral data. The
characteristic stretching bands in the IR spectra of the
compounds were determined at 3276–3264, 1662–1649,
1568–1279 and 1266–972 cm-1 that belong to N–H; C=O;
C=C; C=N double bonds; and C–O, C–N single bond. In
1
the 500-MHz H-NMR of the compounds, singlet peaks
at about 8.08–8.12 ppm and 12.52–12.89 ppm were
assigned which belong to C5 proton of thiazole ring and
nitrogen proton of the benzimidazole ring in suit-
able compounds. Aromatic ring protons, belonging to
phenyls at second and fourth positions of thiazole and
the protons of benzimidazole, overlapped due to the
multiplicity and were assigned between 6.76 and
8.10 ppm. The CH2 and NH protons of acetamide residue
were observed at about 4.25–4.37 and 10.61–10.81 ppm,
respectively. The aliphatic protons of CH3 and OCH3
groups were observed at 2.38–2.39 ppm and 3.75–3.84,
respectively. In the 100 MHz 13C-NMR, peaks at about
36.03–36.71 ppm were assigned for the acetyl carbon
bonded to sulphur atom and the signals which were seen
in the down field of the spectrum over 149.26 ppm were
defined for carbonyl carbons and vicinal aromatic car-
bons to heteroatoms as expected. Mass spectra of the
compounds M ? 1 peaks agreed well with the calculated
molecular weight of the target compounds, and ele-
mental analysis results were found within the calculated
values for the C, H, N elements of the compounds
(Figs. 1, 2, 3).
The need for anticancer agents that selectively kill or
inhibit the growth of neoplastic cells without affecting non-
cancerous host tissues is high and persistent. Thus, the aim
of the current study was the synthesis of novel benzimi-
dazole derivatives that incorporated thiazole ring system,
which have been expected to exhibit potent cytotoxic
activity against cancer cells.
123

Med Chem Res
Scheme 1 Synthesis of the compounds (4a–p). Reactions and conditions: i 4-(2-bromoacetyl)acetanilide, EtOH, r.t., 8 h; ii 10 % HCl, EtOH,
reflux; iii chloroacetyl chloride, TEA, THF, r.t.; iv 2-mercaptobenzimidazole derivatives, K2CO3, acetone, reflux, 2 h
Cytotoxic properties of the compounds
the compounds to test in xCELLigence system. The indi-
cated method provides dynamic cell response profiles and
temporal IC50 histograms, through cell index (CI) term
which is based on the changes of measured electrical
impedance of viable cells depending on applied test com-
pounds at various concentrations (12.5, 50 and 200 lM) in
The xCELLigence RTCA DP system was used for real-
time and time-dependent analysis of the cellular response
of NIH/3T3, A549 and Caco2 cells. Conventional methods
(MTT) were used to select appropriate concentrations of
123

Med Chem Res
Fig. 1 Real-time monitoring of cell proliferative effects of compound 4c at 12.5, 50 and 20 lM, cisplatin at 70 lM concentrations, test medium
and 0.1 % solution of control group in DMSO on NIH/3T3 cells using RTCA DP System (n = 6)
Fig. 2 Real-time monitoring of cell proliferative effects of compound 4c at 12.5, 50 and 20 lM, cisplatin at 70 lM concentrations, test medium
and 0.1 % solution of control group in DMSO on A549 cells using RTCA DP System (n = 6)
a certain period of time (Moe et al., 2013). The starting CI
values, as a measure of cell proliferation, were determined
as 2.0, 2.5 and 1.0 on NIH/3T3, A549 and Caco2 cells,
respectively, and the values decreased in a time- and
concentration-dependent manner after the application of
test compounds, while the CI values of the controls in
0.1 % dimethylsulphoxide kept rising to the end of the test
except a little decrease, initially. The test compounds did
not show any significant changes in the cell index during
the first 5 h of their addition on NIH/3T3 cells. After this
time, there was a slight increase or a prominent decrease in
the CI values of treated cells due to concentration
increasing. In order to compare the cytotoxic effects of the
sixteen thiazole compounds (4a–p), their IC50 values were
calculated after 24, 48 and 72 h exposure. Compounds 4c,
4d, 4f, 4g, 4h, 4j, 4k, 4l, 4n, 4o and 4p caused A549 cell
death at the highest concentration (200 lM) due to viewing
the cell index value as zero in various time periods to the
end of test time (102 h) in contrast to standard drug, cis-
platin. This situation was also observed only in compounds
4b and 4l at same concentration against Caco2 cell line
although most of IC50 values were found lower than cis-
platin’s IC50 values, especially after 48-h incubation.
Therefore, this finding can be claimed that tested two lower
doses of the compounds were found to be cytostatic not
cytotoxic.
As shown in Tables 1, 2 and 3, most of the compounds
exhibited significant cytotoxicity against A549 and Caco2
tumour cell lines at lower doses. Among them, compound
4c caused antiproliferation more efficiently than the other
123

Med Chem Res
Fig. 3 Real-time monitoring of cell proliferative effects of com-
pound 4c at 12.5, 50 and 20 lM, cisplatin at 70 lM concentrations,
test medium and 0.1 % solution of control group in DMSO on Caco2
cells as a function of time 72 h using RTCA DP System (n = 6). In
the graph the black line shows the time of drug addition at which the
CI values were normalized
Table 1 IC50 values (lM) of the compounds 4a–p against NIH/3T3,
A549 and Caco2 cell lines calculated via xCELLigence analysis after
24-h incubation
yl]phenyl]acetamide derivatives (4a–p) differ from each
other with substituents methyl, methoxy and chloro at the
fifth position of the benzimidazole ring and the substituents
methoxy, chloro and fluoro at para position of phenyl
residue. Compound 4c is the most active compound that
possesses methoxy substitution on benzimidazole ring. In
evaluating substituent effect of the title compounds,
methoxy and chloro substituents cause higher increase in
activity than the other substituents.
Compound
NIH/3T3
A549
Caco2
4a
33.45
33.87
74.56
66.94
35.29
37.86
37.47
186.04
57.37
30.33
36.93
48.26
44.98
43.50
71.00
23.13
95.42
42.26
38.84
27.90
18.69
38.05
35.16
44.14
42.29
25.54
35.09
36.57
18.22
50.17
16.79
36.67
32.75
20.33
103.75
164.59
65.21
68.83
70.38
57.30
71.58
105.14
71.35
86.20
60.56
68.89
33.06
76.39
113.17
75.61
69.10
4b
4c
4d
4e
4f
4g
Conclusion
4h
4i
New 2-[(5-substituted-1H-benzimidazol-2-yl)thio]-N-[4-
[2-phenylthiazol-4-yl]phenyl]acetamide (4a–p) derivatives
were synthesized, and their antiproliferative activities were
investigated using RTCA system on NIH/3T3, A549 and
Caco2 cell lines. The measurements were carried out via
xCELLigence system which is a reliable and accurate
method. Most of the compounds showed significant cyto-
toxic activity with low IC50 values. However, compound 4c
exhibited selective cytotoxicity against both of the cancer
cells and determined as the most efficient molecule.
4j
4k
4l
4m
4n
4o
4p
Cisplatin
compounds against both of cancer cell lines and in three
measurement periods. The IC50 values were found to be
74.56, 82.24 and 128.96 lM against NIH/3T3 for com-
pound 4c which were higher than the inhibition values
against the tumour cell lines of the same compound;
therefore, it can be claimed that the compound 4c showed
selectivity against tumour cells and it also exhibited more
similar responses to cisplatin.
Materials and methods
Chemistry
All chemicals were purchased from Sigma-Aldrich
Chemical Co., (Sigma-Aldrich Corp., St. Louis, MO, USA)
and Merck (Merck, Darmstadt, Germany). All melting
points (m.p.) were determined by Electrothermal 9300
The structures of the 2-[(5-substituted-1H-benzimida-
zol-2-yl)thio]-N-[4-[2-(4-substituted-phenyl)thiazol-4-
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Med Chem Res
Table 2 IC50 values (lM) of the compounds 4a–p against NIH/3T3,
A549 and Caco2 cell lines calculated via xCELLigence analysis after
48-h incubation
Table 3 IC50 values (lM) of the compounds 4a–p against NIH/3T3,
A549 and Caco2 cell lines calculated via xCELLigence analysis after
72-h incubation
Compound
NIH/3T3
A549
Caco2
Compound
NIH/3T3
A549
Caco2
4a
44.79
43.68
82.24
34.57
45.25
44.41
43.49
50.63
39.99
41.98
44.53
39.59
40.62
27.91
60.32
40.31
106.90
58.82
35.94
45.27
29.30
45.63
33.17
52.26
38.55
20.00
20.02
18.48
22.60
73.04
36.57
45.39
41.68
13.60
68.12
78.21
77.05
108.74
42.46
42.14
82.36
86.88
63.30
90.31
49.95
81.60
43.53
93.00
71.85
76.20
86.72
4a
26.27
37.68
128.96
30.80
35.35
34.98
16.92
32.01
45.15
30.68
44.75
40.94
41.87
36.52
55.39
43.58
139.06
93.87
32.94
50.18
30.59
62.56
30.99
97.95
41.68
31.21
19.64
35.29
36.58
72.09
36.57
44.27
42.41
14.17
48.50
54.95
85.84
65.69
96.87
44.96
143.14
51.75
123.02
71.16
53.77
54.74
32.90
55.33
71.55
90.68
45.14
4b
4c
4b
4c
4d
4e
4d
4e
4f
4f
4g
4g
4h
4i
4h
4i
4j
4j
4k
4l
4k
4l
4m
4n
4o
4m
4n
4o
4p
Cisplatin
4p
Cisplatin
C=N), 1228–975 (C–O, C–N); 1H-NMR (500 MHz,
DMSO-d6, ppm): d 4.32 (s, 2H, COCH2), 7.13–7.16 (m,
2H, Ar–H), 7.47–7.56 (m, 5H, Ar–H), 7.71 (d, 2H, J:
8.50 Hz, Ar–H), 8.01–8.04 (m, 4H, Ar–H), 8.12 (s, 1H,
thiazole C5–H), 10.68 (s, 1H, CONH), 12.67 (s, 1H, ben-
zimidazole N–H); 13C NMR (100 MHz, DMSO-d6, ppm):
d 36.71 (CH2, COCH2), 114.02 (CH, thiazole C5), 117.54
(2 9 CH, benzimidazole C4 ? C7), 119.66 (2 9 CH,
benzene C2 ? C6), 122.00 (2 9 CH, benzimidazole
C5 ? C6), 126.67 (2 9 CH, benzene C3 ? C5), 127.21 (C,
digital melting point apparatus (Electrothermal, Essex,
UK) and are uncorrected. All the reactions were monitored
by thin-layer chromatography (TLC) using Silica gel 60
F254 TLC plates (Merck, Darmstadt, Germany). Spectro-
scopic data were recorded with the following instruments:
IR, Shimadzu 8400S spectrophotometer (Shimadzu,
1
Tokyo, Japan); H-NMR, Bruker 500 MHz spectrometer
(Bruker Bioscience, Billerica, MA, USA); 13C-NMR,
VARIAN Mercury 100 FT spectrometer (Varian Inc., Palo
Alto, CA, USA); VG Quattro mass spectrometer (VG
BioTech, Altrincham, UK), and elemental analyses were
performed on a Perkin Elmer EAL 240 elemental analyser
(Perkin Elmer, Norwalk, CT, USA).
0
benzene C4), 129.074 (CH, benzene C4 ), 129.78 (2 9 CH,
0
0
0
benzene C3 ? C5 ), 130.82 (2 9 CH, benzene C2 ? C6 ),
133.50 (2 9 C, benzimidazole C3a ? C7a), 139.34 (C,
0
benzene C1), 143.82 (C, benzene C1 ), 150.31 (C, benz-
imidazole C2), 155.39 (C, thiazole C4), 166.72 (C=O,
COCH2), 167.33 (C, thiazole C2); ESMS m/z 442 [M]?
(100); Anal. Calcd. for C24H18N4OS2: C, 65.13; H, 4.10; N,
12.66. Found: C, 65.14; H, 4.14; N, 12.71.
General procedure for the synthesis 2-[(5-
substituted-1H-benzimidazol-2-yl)thio]-N-[4-[2-(4-
substituted-phenyl)thiazol-4-yl]phenyl]acetamide
derivatives (4a–p)
2-[(5-Methyl-1H-benzimidazol-2-yl)thio]-N-[4-[2-phenylth
iazol-4-yl]phenyl]acetamide (4b) It was obtained as
brown powder, yield 75 %; m.p. 189–192 °C; IR (KBr,
cm-1): m 3269 (amide N–H), 1662 (C=O), 1554–1302
(C=C, C=N), 1264–975 (C–O, C–N); 1H-NMR (500 MHz,
DMSO-d6, ppm): d 2.38 (s, 3H, CH3), 4.37 (s, 2H,
COCH2), 6.95 (d, 1H, J: 8.0 Hz, Ar–H), 7.24 (brs, 1H, Ar–
H), 7.34 (brs, 1H, Ar–H), 7.51–7.57 (m, 3H, Ar–H), 7.71
(d, 2H, J: 8.5 Hz, Ar–H), 7.98–8.05 (m, 4H, Ar–H), 8.09
(s, 1H, thiazole C5–H), 10.71 (s, 1H, CONH), 12.65 (s, 1H,
benzimidazole N–H); 13C NMR (100 MHz, DMSO-d6,
A mixture of compound 3a–d (1 mmol), the appropriate
mercapto-benzimidazole derivative (1 mmol) and K2CO3
(1 mmol) in acetone was refluxed for 2 h. After the solvent
was evaporated, the obtained residue was treated with
water, filtered, dried and then recrystallized from ethanol to
afford target compounds 4a–p.
2-[(1H-benzimidazol-2-yl)thio]-N-[4-[2-phenylthiazol-4-
yl]phenyl]acetamide (4a) It was obtained as brown
powder, yield 71 %; m.p. 233–234 °C; IR (KBr, cm-1):
mmax 3269 (amide N–H), 1659 (C=O), 1568–1279 (C=C,
123

Med Chem Res
ppm): d 21.10 (CH3), 36.17 (CH2, COCH2), 113.44 (CH,
thiazole C5), 117.29 (CH, benzimidazole C7), 117.98 (CH,
benzimidazole C4), 119.06 (2 9 CH, benzene C2 ? C6),
122.73 (CH, benzimidazole C6), 126.09 (2 9 CH, benzene
C3 ? C5), 126.62 (C, benzene C4), 129.17 (CH, benzene
C2 ? C6), 121.53 (CH, benzimidazole C6), 125.80 (C,
benzimidazole C5), 126.09 (2 9 CH, benzene C3 ? C5),
0
126.62 (C, benzene C4), 129.017 (CH, benzene C4 ), 129.23
0
(2 09 CH, benzene C3 ? C5 ), 130.25 (2 9 CH, benzene
0
C2 ? C6 ), 132.93 (C, benzimidazole C7a), 138.73 (C,
0
0
0
C4 ), 130.24 (2 9 CH, b0enzene C3 ? C5 ), 132.93
benzimidazole C3a), 138.98 (C, benzene C1), 143.11 (C,
0
0
(2 9 CH, benzene C2 ? C6 ), 133.45 (C, benzimidazole
benzene C1 ), 151.65 (C, benzimidazole C2), 154.81 (C,
C5), 135.29 (C, benzimidazole C7a), 138.77 (C, benzimi-
dazole C3a), 138.96 (C, benzene C1), 143.56 (C, benzene
thiazole C4), 165.9 1(C=O, COCH2), 166.75 (C, thiazole
C2); ESMS m/z 476 [M]?: (100); Anal. Calcd. for C24-
H17ClN4OS2: C, 60.43; H, 3.59; N, 11.75. Found: C, 60.55;
H, 3.51; N, 11.63.
0
C1 ), 149.89 (C, benzimidazole C2), 154.81 (C, thiazole
C4), 166.20 (C=O, COCH2), 166.74. (C, thiazole C2);
ESMS m/z 456 [M]? (100); Anal. Calcd. for C25H20N4OS2:
C, 65.76; H, 4.42; N, 12.27. Found: C, 65.71; H, 4.54; N,
12.35.
2-[(1H-Benzimidazol-2-yl)thio]-N-[4-[2-(4-methoxyphenyl)
thiazol-4-yl]phenyl]acetamide (4e) It was obtained as
cream powder, yield 68 %; m.p. 238–239 °C; IR (KBr,
cm-1): mmax 3275 (amide N–H), 1651 (C=O), 1541–1296
(C=C, C=N), 1256–972 (C–O, C–N); 1H-NMR (500 MHz,
DMSO-d6, ppm): d 3.84 (s, 3H, OCH3), 4.30 (s, 2H,
COCH2), 7.08 (d, 2H, J: 8.50 Hz, Ar–H), 7.13–7.15 (m,
2H, Ar–H), 7.46 (brs, 1H, Ar–H), 7.69 (d, 2H, J: 9 Hz, Ar–
H), 7.95–7.99 (m, 6H, Ar–H), 10.68 (s, 1H, CONH), 12.66
(s, 1H, benzimidazole N–H); 13C NMR (100 MHz, DMSO-
d6, ppm): d 36.03 (CH2, COCH2), 55.19 (OCH3), 112.24
2-[(5-Methoxy-1H-benzimidazol-2-yl)thio]-N-[4-[2-phenyl
thiazol-4-yl]phenyl]acetamide (4c) It was obtained as
brown powder, yield 70 %; m.p. 182–184 °C; IR (KBr,
cm-1): mmax 3267 (amide N–H), 1654 (C=O), 1568–1288
(C=C, C=N), 1234–975 (C–O, C–N); 1H-NMR (500 MHz,
DMSO-d6, ppm): d 3.76 (s, 3H, OCH3), 4.25 (s, 2H,
COCH2), 6.76 (d, 1H, J: 8.5 Hz, Ar–H), 6.98 (brs, 1H, Ar–
H), 7.34 (brs, 1H, Ar–H), 7.49–7.56 (m, 3H, Ar–H), 7.70
(d, 2H, J: 9 Hz, Ar–H), 8.01–8.03 (m, 4H, Ar–H), 8.08 (s,
1H, thiazole C5–H), 10.67 (s, 1H, CONH), 12.55 (s, 1H,
benzimidazole N–H); 13C NMR (100 MHz, DMSO-d6,
ppm): d 36.26 (CH2, COCH2), 55.38 (OCH3), 101.70 (CH,
benzimidazole C4), 110.39 (CH, benzimidazole C6), 113.44
(CH, thiazole C5), 117.52 (CH, benzimidazole C7), 119.09
(2 9 CH, benzene C2 ? C6), 126.10 (2 9 CH, benzene
C3 ? C5), 126.63 (C, benzene C4), 129.17 (CH, benzene
0
0
(CH, thiazole C5), 114.38 (2 9 CH, benzene C3 ? C5 ),
118.94 (2 9 CH, benzimidazole C4 ? C7), 121.34
(2 9 CH, benzene C2 ? C6), 125.67 (2 9 CH, benzimi-
dazole C5 ? C6), 126.46 (2 9 CH, benzene C3 ? C5),
127.56 (C, benzene C4), 129.22 (2 9 CH, benzene
0
0
C2 ? C6 ), 132.27 (20 9 C, benzimidazole C3a ? C7a),
136.75 (C, benzene C1 ), 138.55 (C, benzene C1), 149.62
(C, benzimidazole C2), 154.40 (C, thiazole C4) 160.72 (C,
0
0
0
0
C4 ), 129.20 (2 9 CH, b0enzene C3 ? C5 ), 130.25
benzene C4 ), 165.99 (C=O, COCH2), 166.54 (C, thiazole
C2); ESMS m/z 472 [M]?: (100); Anal. Calcd. for C25-
H20N4O2S2: C, 63.54; H, 4.27; N, 11.86. Found: C, 63.61;
H, 4.30; N, 11.71.
0
(2 9 CH, benzene C2 ? C6 ), 132.93 (C, benzimidazole
C7a), 138.79 (C, benzimidazole C3a), 138.99 (C, benzene
0
C1), 143.07 (C, benzene C1 ), 148.68 (C, benzimidazole
C2), 154.83 (C, thiazole C4), 155.27 (C, benzimidazole C5),
166.25 (C=O, COCH2), 166.77 (C, thiazole C2); ESMS m/z
472 [M]?: (100); Anal. Calcd. for C25H20N4O2S2: C,
63.54; H, 4.27; N, 11.86. Found: C, 63.59; H, 4.24; N,
11.75.
2-[(5-Methyl-1H-benzimidazol-2-yl)thio]-N-[4-[2-(4-meth
oxyphenyl)thiazol-4-yl]phenyl]acetamide (4f) It was
obtained as brown powder, yield 66 %; m.p. 201–202 °C.
IR (KBr, cm-1): mmax 3268 (amide N–H), 1661 (C=O),
1567–1312 (C=C, C=N), 1254–975 (C–O, C–N); 1H-NMR
(500 MHz, DMSO-d6, ppm): d 2.39 (s, 3H, CH3), 3.84 (s,
3H, OCH3), 4.28 (s, 2H, COCH2), 6.95 (d, 1H, J: 8.5 Hz,
Ar–H), 7.08 (d, 2H, J: 8.5 Hz, Ar–H), 7.22 (brs, 1H, Ar–
H), 7.33 (brs, 1H, Ar–H), 7.69 (d, 2H, J: 9 Hz, Ar–H),
7.95–7.99 (m, 5H, Ar–H), 10.68 (s, 1H, CONH), 12.65 (s,
1H, benzimidazole N–H); 13C NMR (100 MHz, DMSO-d6,
ppm): d 21.17 (CH3), 36.23 (CH2, COCH2), 55.38 (OCH3),
112.43 (0CH, thiazole C5), 114.56 (2 9 CH, benzene
2-[(5-Chloro-1H-benzimidazol-2-yl)thio]-N-[4-[2-phenylth
iazol-4-yl]phenyl]acetamide (4d) It was obtained as
white powder, yield 75 %; m.p. 241–243 °C; IR (KBr,
cm-1): mmax 3269 (amide N–H), 1656 (C=O), 1568–1305
(C=C, C=N), 1264–975 (C–O, C–N); 1H-NMR (500 MHz,
DMSO-d6, ppm): d 4.32 (s, 2H, COCH2), 7.15 (d, 1H, J:
8.5 Hz, Ar–H), 7.45 (d, 1H, J: 7.50 Hz, Ar–H), 7.51–7.55
(m, 4H, Ar–H), 7.69 (d, 2H, J: 8.0 Hz, Ar–H), 8.0–8.03 (m,
4H, Ar–H), 8.08 (s, 1H, thiazole C5–H), 10.62 (s, 1H,
CONH), 12.75 (s, 1H, benzimidazole N–H); 13C NMR
(100 MHz, DMSO-d6, ppm): d 36.14 (CH2, COCH2),
113.46 (CH, thiazole C5), 115.62 (CH, benzimidazole C4),
116.54 (CH, benzimidazole C7), 119.11 (2 9 CH, benzene
0
C3 ? C5 ), 117.26 (CH, benzimidazole C7), 117.92 (CH,
benzimidazole C4), 119.11 (2 9 CH, benzene C2 ? C6),
125.85 (CH, benzimidazole C6), 126.65 (2 9 CH, benzene
C3 ? C5), 127.75 (C, benzene C4), 129.38 (2 9 CH, ben-
0
0
zene C2 ? C6 ), 133.41 (C, benzimidazole C5), 134.93 (C,
123

Med Chem Res
0
0
benzene C1 ), 135.32 (C, benzimidazole C7a), 138.74 (C,
C2), 154.59 (C, thiazole C4), 160.91 (C, benzene C4 ),
benzimidazole C3a), 138.97 (C, benzene C1), 149.81 (C,
benzimidazole C2), 154.58 (C, thiazole C4), 160.91 (C,
165.99 (C=O, COCH2), 166.73 (C, thiazole C2); ESMS m/z
506 [M]?: (100); Anal. Calcd. for C25H19ClN4O2S2: C,
59.22; H, 3.78; N, 11.05. Found: C, 59.25; H, 3.71; N,
11.13.
0
benzene C4 ), 166.24 (C=O, COCH2), 166.73. (C, thiazole
C2); ESMS m/z 486 [M]?: (100); Anal. Calcd. for C26
H22N4O2S2: C, 64.17; H, 4.56; N, 11.51. Found: C, 64.11;
H, 4.39; N, 11.61.
2-[(1H-Benzimidazol-2-yl)thio]-N-[4-[2-(4-chlorophenyl)
thiazol-4-yl]phenyl]acetamide (4i) It was obtained as
white powder, yield 68 %; m.p. 233–235 °C; IR (KBr,
cm-1): mmax 3275 (amide N–H), 1658 (C=O), 1552–1287
(C=C, C=N), 1266–975 (C–O, C–N); 1H-NMR (500 MHz,
DMSO-d6, ppm): d 4.30 (s, 2H, COCH2), 7.12–7.15 (m,
2H, Ar–H), 7.48 (brs, 2H, Ar–H), 7.61 (d, 2H, J: 8 Hz, Ar–
H), 7.70 (d, 2H, J: 8.5 Hz, Ar–H), 7.97–8.06 (m, 4H, Ar–
H), 8.12 (s, 1H, thiazole C5–H), 10.71 (s, 1H, CONH),
12.69 (s, 1H, benzimidazole N–H); 13C NMR (100 MHz,
DMSO-d6, ppm): d 36.19 (CH2, COCH2), 113.99 (CH,
thiazole C5), 117.59 (2 9 CH, benzimidazole C4 ? C7),
119.14 (2 9 CH, benzene C2 ? C6), 121.46 (2 9 CH,
benzimidazole C5 ? C6), 126.72 (2 9 CH, benzene
C3 ? C5), 127.085 (C, benzene C4), 129.10 (2 9 CH, ben-
2-[(5-Methoxy-1H-benzimidazol-2-yl)thio]-N-[4-[2-(4-meth
oxyphenyl)thiazol-4-yl]phenyl]acetamide (4g) It was
obtained as cream powder, yield 69 %; m.p. 191–193 °C.
IR (KBr, cm-1): mmax 3269 (amide N–H), 1658 (C=O),
1558–1288 (C=C, C=N), 1245–976 (C–O, C–N); 1H-NMR
(500 MHz, DMSO-d6, ppm): d 3.79 (s, 3H, OCH3), 3.88 (s,
3H, OCH3), 4.25 (s, 2H, COCH2), 6.76 (d, 1H, J: 8.50 Hz,
Ar–H), 6.98 (brs, 1H, Ar–H), 7.09 (d, 2H, J: 8.0 Hz, Ar–
H), 7.35 (d, 1H, J: 8.5 Hz, Ar–H), 7.69 (d, 2H, J: 9.0 Hz,
Ar–H), 7.95–8.0 (m, 5H, Ar–H), 10.68 (s, 1H, CONH),
12.65 (s, 1H, benzimidazole N–H); 13C NMR (100 MHz,
DMSO-d6, ppm): d 36.39 (CH2, COCH2), 55.46 (OCH3),
55.54 (OCH3), 101.73 (CH, benzimidazole C4), 110.60
(CH, benzimidazole C6), 112.52 (CH, thiazole C5), 114.66
0
0
0
zene C2 ? C6 ), 129.209 (2 9 CH, benzene C3 ? C5 ),
131.83 (C, benzene C4 ), 134.82 (2 9 C, benzimidazole
C3a ? C7a), 138.91 (C, benzene C1), 143.89 (C, benzene
0
0
(2 9 CH, benzene C3 ? C5 ), 117.58 (CH, benzimidazole
C7), 119.26 (2 9 CH, benzene C2 ? C6), 125.94 (2 9 CH,
benzene C3 ? C5), 126.740 (C, benzene C4), 127.85
0
C1 ), 149.83 (C, benzimidazole C2), 155.00 (C, thiazole
0
(2 9 CH, benzene C2 ? C6 ), 129.51 (C, benzimidazole
C4), 165.46 (C=O, COCH2), 166.22 (C, thiazole C2);
ESMS m/z 476 [M]?: (100); Anal. Calcd. for C24H17
ClN4OS2 calculated: C, 60.43; H, 3.59; N, 11.75. Found: C,
60.45; H, 3.61; N, 11.68.
0
C7a), 136.12 (C, benzene C1 ), 138.80 (C, benzimidazole
C3a), 139.05 (C, benzene C1), 148.72 (C, benzimidazole
C2), 154.66 (C, thiazole0C4), 155.45 (C, benzimidazole C5),
161.00 (C, benzene C4 ), 166.40 (C=O, COCH2), 166.86
(C, thiazole C2); ESMS m/z 502 [M]?: (100); Anal. Calcd.
for C26H22N4O3S2: C, 62.13; H, 4.41; N, 11.15. Found: C,
62.18; H, 4.39; N, 11.21.
2-[(5-Methyl-1H-benzimidazol-2-yl)thio]-N-[4-[2-(4-chloro
phenyl)thiazol-4-yl]phenyl]acetamide (4j) It was obtained
as white powder, yield 70 %; m.p. 202–204 °C; IR (KBr,
cm-1): mmax 3276 (amide N–H), 1659 (C=O), 1548–1299
(C=C, C=N), 1263–978 (C–O, C–N); 1H-NMR (500 MHz,
DMSO-d6, ppm): d 2.39 (s, 3H, CH3), 4.28 (s, 2H,
COCH2), 7.61 (d, 2H, J: 9.0 Hz, Ar–H), 7.71 (d, 2H, J:
9.0 Hz, Ar–H), 8.01 (d, 2H, J: 8.0 Hz, Ar–H), 8.06 (d, 2H,
J: 8.50 Hz, Ar–H), 8.12 (s, 1H, thiazole C5–H), 10.72 (s,
1H, CONH), 12.52 (s, 1H, benzimidazole N–H); 13C NMR
(100 MHz, DMSO-d6, ppm): d 21.18 (CH3), 36.24 (CH2,
COCH2), 113.98 (CH, thiazole C5), 117.12 (CH, benzim-
idazole C7), 117.84 (CH, benzimidazole C4), 119.12
(2 9 CH, benzene C2 ? C6), 122.74 (CH, benzimidazole
C6), 126.72 (2 9 CH, benzene C3 ? C5),0 127.85 (C, ben-
2-[(5-Chloro-1H-benzimidazol-2-yl)thio]-N-[4-[2-(4-meth
oxyphenyl)thiazol-4-yl]phenyl]acetamide (4h) It was
obtained as white powder, yield 77 %; m.p. 238–240 °C;
IR (KBr, cm-1): mmax 3267 (amide N–H), 1658 (C=O),
1555–1287 (C=C, C=N), 1231–976 (C–O, C–N); 1H-NMR
(500 MHz, DMSO-d6, ppm): d 3.84 (s, 3H, OCH3), 4.33 (s,
2H, COCH2), 7.08 (d, 2H, J: 8.5 Hz, Ar–H), 7.15 (d, 1H, J:
7.5 Hz, Ar–H), 7.46 (d, 1H, J: 8.5 Hz, Ar–H), 7.53 (s, 1H,
Ar–H), 7.69 (d, 2H, J: 8.0 Hz, Ar–H), 7.93–8.0 (m, 5H,
Ar–H), 10.62 (s, 1H, CONH), 12.88 (s, 1H, benzimidazole
N–H); 13C NMR (100 MHz, DMSO-d6, ppm): d36.22
(CH2, COCH2), 55.37 (OCH3), 112.043 (CH, thiazole C5),
0
zene C4), 129.09 (2 9 CH, 0 benzene C3 ? C5 ), 129.29
0
0
114.56 (2 9 CH, benzene C3 ? C5 ), 115.58 (CH, benz-
(2 9 CH, benzene C2 ? C6 ), 130.58 (C, benzimidazole
imidazole C4), 116.81 (CH, benzimidazole C7), 119.15
(2 9 CH, benzene C2 ? C6), 121.56 (CH, benzimidazole
C6), 125.86 (C, benzimidazole C5), 126.64 (2 9 CH,
benzene C3 ? C5), 127.740 (C, benzene C4), 129.42
C5), 131.83 (C, benzimidazole C7a), 134.83 (C, benzene
0
C4 ), 138.94 (C, benzimidazole C3a), 139.15 (C, benzene
0
C1), 144.24 (C, benzene C1 ), 149.26 (C, benzimidazole
C2), 155.02 (C, thiazole C4), 165.46 (C=O, COCH2),
166.32. (C, thiazole C2); ESMS m/z 490 [M]?: (100); Anal.
Calcd. for C25H19ClN4OS2: C, 61.15; H, 3.90; N, 11.41.
Found: C, 61.24; H, 3.78; N, 11.33.
0
(2 9 CH, benzene C2 ? C6 ), 132.98 (C, benzimidazole
C7a), 138.71 (C, benzimidazole C3a), 139.13 (C, benzene
0
C1), 143.24 (C, benzene C1 ), 151.78 (C, benzimidazole
123

Med Chem Res
2-[(5-Methoxy-1H-benzimidazol-2-yl)thio]-N-[4-[2-(4-
chlorophenyl)thiazol-4-yl]phenyl]acetamide (4k) It
was obtained as brown powder, yield 69 %; m.p.
200–203 °C; IR (KBr, cm-1): mmax 3268 (amide N–H), 1654
(C=O), 1537–1298 (C=C, C=N), 1213–976 (C–O, C–N);
1H-NMR (500 MHz, DMSO-d6, ppm): d 3.75 (s, 3H,
OCH3), 4.25 (s, 2H, COCH2), 6.76 (d, 2H, J: 8.50 Hz, Ar–
H), 6.98 (s, 1H, Ar–H), 7.35 (d, 1H, J: 9.0 Hz, Ar–H), 7.60
(d, 2H, J: 8.5 Hz, Ar–H), 7.70 (d, 2H, J: 8.50 Hz, Ar–H),
8.01 (d, 2H, J: 8.5 Hz, Ar–H), 8.4 (d, 2H, J: 8.5 Hz, Ar–H),
8.11 (s, 1H, thiazole C5–H), 10.71 (s, 1H, CONH), 12.88 (s,
1H, benzimidazole N–H); 13C NMR (100 MHz, DMSO-d6,
ppm): d36.31 (CH2, COCH2), 55.45 (OCH3), 101.74 (CH,
benzimidazole C4), 110.41 (CH, benzimidazole C6), 114.00
(CH, thiazole C5), 117.61 (CH, benzimidazole C7), 119.14
(2 9 CH, benzene C2 ? C6), 126.72 (2 9 CH, benzene
brown powder, yield 67 %; m.p. 242–245 °C; IR (KBr,
cm-1): mmax 3272 (amide N–H), 1657 (C=O), 1531–1296
(C=C, C=N), 1214–975 (C–O, C–N); 1H-NMR (500 MHz,
DMSO-d6, ppm): d 4.30 (s, 2H, COCH2), 7.12–7.14 (m,
2H, Ar–H), 7.37 (t, 2H, J: 9 Hz, Ar–H), 7.49 (brs, 2H, Ar–
H), 7.69 (d, 2H, J: 8.50 Hz, Ar–H), 7.99 (d, 2H, J: 8.0 Hz,
Ar–H), 8.06–8.09 (m, 3H, Ar–H), 10.67 (s, 1H, CONH),
12.65 (s, 1H, benzimidazole N–H); 13C NMR (100 MHz,
DMSO-d6, ppm): d 36.20 (CH2, COCH2), 113.59 (CH,
thi0azole C5), 116.26 (2 9 CH, J: 22.0 Hz, benzene
0
C3 ? C5 ), 117.65 (2 9 CH, benzimidazole C4 ? C7),
119.14 (2 9 CH, benzene C2 ? C6), 121.48 (2 9 CH,
benzimidazole C5 ? C6), 126.71 (2 9 CH, benzene
C3 ? C5), 128.44 (C, benzene C4), 129.19 (2 9 C, benz-
im0idazole C3a ? C7a), 129.69 (2 9 CH, J: 3.0 Hz benzene
0
C2 ? C6 ), 138.86 (C, benzene C1), 143.67 (C, benzene
0
0
C3 ? C5), 127.86 (C, benzene C4), 129.09 (C, benzene C4 ),
0
C1 ), 149.82 (C, benzimidazole C2), 154.87 (C, thiazole
0
0
129.30 (2 9 CH,0 benzene C3 ? C5 ), 131.83 (2 9 CH,
C4), 163.23 (C, J: 247.0 Hz benzene C4 ), 165.64 (C=O,
benzene C2 ? C6 ), 134.83 (C, benzimidazole C7a), 138.93
COCH2), 166.22 (C, thiazole C2); ESMS m/z 459 [M]?:
(100); Anal. Calcd. for C24H17FN4OS2: C, 62.59; H, 3.72;
N, 12.17. Found: C, 62.68; H, 3.62; N 12.28.
0
(C, benzimidazole C3a), 139.07 (C, benzene C1), 143.16 (C,
0
benzene C1 ), 148.08 (C, benzimidazole C2), 155.02 (C,
thiazole C4), 155.30 (C, benzimidazole C5), 165.47 (C=O,
COCH2), 166.34 (C, thiazole C2); ESMS m/z 506 [M]?:
(100); Anal. Calcd. for C25H19ClN4O2S2: C, 59.22; H, 3.78;
N, 11.05. Found: C, 59.29; H, 3.72; N, 11.13.
2-[(5-Methyl-1H-benzimidazol-2-yl)thio]-N-[4-[2-(4-fluo-
rophenyl)thiazol-4-yl]phenyl]acetamide (4n) It was
obtained as white powder, yield 71 %; m.p. 204–206 °C;
IR (KBr, cm-1): mmax 3267 (amide N–H), 1649 (C=O),
1516–1311 (C=C, C=N), 1232–972 (C–O, C–N); 1H-NMR
(500 MHz, DMSO-d6, ppm): d 2.38 (s, 3H, CH3), 4.27 (s,
2H, COCH2), 6.95 (d, 2H, J: 8.50 Hz, Ar–H), 7.37 (t, 3H,
J: 8.0 Hz, Ar–H), 7.69 (d, 2H, J: 8.0 Hz, Ar–H), 8.0 (d, 2H,
J: 8.5 Hz, Ar–H), 8.06–8.09 (m, 4H, Ar–H), 10.75 (s, 1H,
CONH), 12.64 (s, 1H, benzimidazole N–H); 13C NMR
(100 MHz, DMSO-d6, ppm): d21.18 (CH3), 36.25 (CH2,
COCH2), 113.59 (CH, thia0zole C5), 116.27 (2 9 CH, J:
2-[(5-Chloro-1H-benzimidazol-2-yl)thio]-N-[4-[2-(4-
chlorophenyl)thiazol-4-yl]phenyl]acetamide (4l) It
was obtained as white powder, yield 72 %; m.p.
210–213 °C; IR (KBr, cm-1): mmax 3266 (amide N–H),
1654 (C=O), 1558–1292 (C=C, C=N), 1264–976 (C–O, C–
1
N); H-NMR (500 MHz, DMSO-d6, ppm): d 4.32 (s, 2H,
COCH2), 7.16 (d, 2H, J: 9.0 Hz, Ar–H), 7.49 (brs, 2H, Ar–
H), 7.61 (d, 1H, J: 8.5 Hz, Ar–H), 7.70 (d, 2H, J: 8.5 Hz,
Ar–H), 8.01 (d, 2H, J: 8.5 Hz, Ar–H), 8.05 (d, 2H, J:
8.0 Hz, Ar–H), 8.12 (s, 1H, thiazole C5–H), 10.61 (s, 1H,
CONH), 12.89 (s, 1H, benzimidazole N–H); 13C NMR
(100 MHz, DMSO-d6, ppm): d 36.20 (CH2, COCH2),
114.01 (CH, thiazole C5), 115.08 (CH, benzimidazole C4),
116.98 (CH, benzimidazole C7), 119.18 (2 9 CH, benzene
C2 ? C6), 121.58 (CH, benzimidazole C6), 125.86 (C,
benzimidazole C5), 126.72 (2 9 CH, benzene C3 ? C5),
0
21.0 Hz, benzene C3 ? C5 ), 117.11 (CH, benzimidazole
C7), 117.82 (CH, benzimidazole C4), 119.13 (2 9 CH,
benzene C2 ? C6), 122.42 (CH, benzimidazole C6), 126.71
(2 9 CH, benzene C3 ? C5), 128.44 (C, benzene C4),
0
0
129.70 (2 9 CH, J: 2.0 Hz benzene C2 ? C6 ), 132.41 (C,
benzimidazole C5), 135.36 (C, benzimidazole C7a), 138.87
(C, benzimidazole C3a), 139.11 (C, benzene C1), 143.22
0
(C, benzene C1 ), 149.91 (C, benzimidazole C2), 154.88 (C,
0
0
127.85 (C, benzene C04), 129.13 (C, benzene C4 ), 129.29
thiazole C4), 163.23 (CH, J: 247.0 Hz benzene C4 ), 165.65
0
(2 09 CH, benzene C3 ? C5 ), 131.83 (2 9 CH, benzene
(C=O, COCH2), 166.28. (C, thiazole C2); ESMS m/z 473
[M]?: (100); Anal. Calcd. for C25H19FN4OS2: C, 63.27; H,
4.04; N, 11.81. Found: C, 63.20; H, 3.98; N, 11.73.
0
C2 ? C6 ), 134.83 (C, benzimidazole C7a), 138.87 (C,
benzimidazole C3a), 139.06 (C, benzene C1), 144.15 (C,
0
benzene C1 ), 151.73 (C, benzimidazole C2), 155.00 (C,
2-[(5-Methoxy-1H-benzimidazol-2-yl)thio]-N-[4-[2-(4-flu-
orophenyl)thiazol-4-yl]phenyl]acetamide (4o) It was
obtained as white powder, yield 67 %; m.p. 181–185 °C;
IR (KBr, cm-1): mmax 3264 (amide N–H), 1662 (C=O),
1515–1282 (C=C, C=N), 1233–975 (C–O, C–N); 1H-NMR
(500 MHz, DMSO-d6, ppm): d 3.76 (s, 3H, OCH3), 4.26 (s,
2H, COCH2), 6.75 (d, 1H, J: 9.0 Hz, Ar–H), 6.98 (s, 1H,
thiazole C4), 165.47 (C=O, COCH2), 165.99 (C, thiazole
C2); ESMS m/z 510 [M]?: (100); Anal. Calcd. for C24
H16Cl2N4OS2: C, 56.36; H, 3.15; N, 10.95. Found: C,
56.39; H, 3.02; N, 10.83.
2-[(1H-Benzimidazol-2-yl)thio]-N-[4-[2-(4-fluorophenyl)
thiazol-4-yl]phenyl]acetamide (4m) It was obtained as
123

Med Chem Res
Ar–H), 7.34–7.37 (m, 3H, Ar–H), 7.71 (d, 2H, J: 9.0 Hz,
Ar–H), 8.0 (d, 2H, J: 9.0 Hz, Ar–H), 8.05–8.07 (m, 3H,
Ar–H), 10.81 (s, 1H, CONH), 12.87 (s, 1H, benzimidazole
N–H); 13C NMR (100 MHz, DMSO-d6, ppm): d 36.35
(CH2, COCH2), 55.46 (OCH3), 96.97 (CH, benzimidazole
C4), 110.39 (CH, benzimidazole C6), 113.57 (CH, thiazole
C5), 114.71 (CH, benzimidazole C7), 116.26 (2 9 CH, J:
cancer cell line (A549; ATCC Number: CCL-185) were
obtained from American Type Culture Collection. The
cells were grown in RPMI 1640 medium supplemented
with 2 mM L-glutamine and 10 % foetal bovine serum,
1 % penicillin/streptomycin at a temperature of 37 °C in a
humidified incubator with a 5 % CO2 atmosphere. The
cells were harvested at confluence with 0.05 % trypsin—
0.02 % EDTA—and plated in tissue culture dishes and
grown with fresh medium. Compounds were dissolved in
DMSO solution.
0
0
23.0 Hz, benzene C3 ? C5 ), 119.16 (2 9 CH, benzene
C2 ? C6), 126.72 (2 9 CH, benzene C3 ? C5), 128.44 (C,
benzene C4), 129.20 (C, benzimidazole C7a), 129.71
0
0
(2 9 CH, J: 3.0 Hz, benzene C2 ? C6 ), 138.51 (C, ben-
zimidazole C3a), 138.92 (C, benzene C1), 143.47 (C, ben-
0
Cell growth and proliferation assay using Real-Time Cell
Analysis System (RTCA DP)
zene C1 ), 148.88 (C, benzimidazole C2), 154.90 (C,
thiazole C4), 155.31 (C, benzimidazole C5), 162.24 (C, J:
0
247.0 Hz, benzene C4 ), 165.67 (C=O, COCH2), 166.41 (C,
thiazole C2); ESMS m/z 489 [M]?: (100); Anal. Calcd. for
C25H19FN4O2S2: C, 61.21; H, 3.90; N, 11.42. Found: C,
61.28; H, 3.82; N, 11.33.
Kinetics of cell adhesion and spreading were experimented
using the Real-Time Cell Analyser (RTCA DP). The
growth characteristics following seeding were monitored to
determine the optimum seeding density and time to
beginning of the death. The system measures electrical
impedance across interdigitated microelectrodes incorpo-
rated on the bottom of tissue culture e-plates. The impe-
dance measurement, which is indicated as cell index (CI)
value, provides quantitative information about the condi-
tion of the cells, involving cell number, viability and
morphology (Bird and Kirstein, 2009; Solly et al., 2004;
Urcan et al., 2010).
2-[(5-Chloro-1H-benzimidazol-2-yl)thio]-N-[4-[2-(4-fluo-
rophenyl)thiazol-4-yl]phenyl]acetamide (4p) It was
obtained as brown powder, yield 71 %; m.p. 142–147 °C;
IR (KBr, cm-1): mmax 3268 (amide N–H), 1658 (C=O),
1529–1305 (C=C, C=N), 1247–1012 (C–O, C–N); 1H-
NMR (500 MHz, DMSO-d6, ppm): d 4.32 (s, 2H, COCH2),
7.16 (d, 1H, J: 9.0 Hz, Ar–H), 7.38 (t, 2H, J: 8.50 Hz, Ar–
H), 7.46 (d, 1H, J: 8.50 Hz, Ar–H), 7.53 (s, 1H, Ar–H),
7.70 (d, 2H, J: 8.50 Hz, Ar–H), 8.02 (d, 2H, J: 9.0 Hz, Ar–
H), 8.07–8.10 (m, 3H, Ar–H), 10.63 (s, 1H, CONH), 12.89
(s, 1H, benzimidazole N–H); 13C NMR (100 MHz, DMSO-
d6, ppm): d 36.19 (CH2, COCH2), 113.61 (CH, thiazole
C5), 115.21 (CH, benzimidazole C4), 116.03 (CH, benz-
im0idazole C7), 116.26 (2 9 CH, J: 18.0 Hz benzene
Background of the e-plates was measured in 100 ll
medium in the Real-Time Cell Analyser (RTCA DP) sta-
tion (xCELLigence, Roche/ACEA Biosciences, Man-
nheim, Germany). Afterwards 100 ll of a 3T3, Caco2 and
A549 cell suspension was added (10.000 cells/well). Plates
were incubated for 30 min at room temperature, e-plates
were placed into the Real-Time Cell Analyser, and impe-
dance was measured every hour. When the cells were in the
log growth phase, the cells were treated to 100 ll of
medium containing different concentrations of compounds
4a–p (12.5, 50 and 200 lM) and 70 lM Cisplatin and
impedance monitoring continued for another 72 h. Control
was received medium ? DMSO with a final concentration
of 0.1 %. Impedance was demonstrated as cell index (CI)
values. Dose–response curves at 72 h were generated to
determine IC50 values during the incubation time. The
electrical impedance was analysed by the RTCA DP
system.
0
C3 ? C5 ), 119.18 (2 9 CH, benzene C2 ? C6), 121.59
(CH, benzimidazole C6), 125.87 (C, benzimidazole C5),
126.70 (2 9 CH, benzene C3 ? C5), 128.44 (C, benzene
C4), 129.202 (C, 0 benzimidazole C7a), 129.59(2 9 CH,
benzene C2 ? C6 ), 138.52 (C, benzimidazo0le C3a), 138.82
(C, benzene C1), 143.51 (C, benzene C1 ), 151.73 (C,
benzimidazole C2), 154.86 (C, thiazole C4), 163.22 (C, J:
0
198.0 Hz benzene C4 ), 165.65 (C=O, COCH2), 165.99 (C,
thiazole C2); ESMS m/z 493 [M]?: (100); Anal. Calcd. for
C24H16ClFN4OS2: C, 58.23; H, 3.26; N, 11.32. Found: C,
58.29; H, 3.29; N, 11.43.
Cytotoxicity assay
Compliance with ethical standards
Cell culture and treatment
Conflict of interests The authors report no conflict of interest.
Mouse embryonic fibroblast cell line (3T3; ATTC Number
CRL-1685), human colorectal adenocarcinoma cell line
(Caco2; ATCC number: HTB-37), non-small cell lung
Ethical statement For this type of study formal consent is not
required. Informed consent was obtained from all individual partici-
pants included in the study.
123

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