Synthesis and anticancer activity evaluation of N-[4-(2-methylthiazol-4-yl) phenyl]acetamide derivatives containing (benz)azole moiety

Article abstract of DOI:10.3109/14756366.2013.763253

A new class of novel thiazole-(benz)azole derivatives was synthesized to investigate their anticancer activity. The structure of the compounds was confirmed by IR, ¹H-NMR, and MS spectral data and elemental analyses. Anticancer effect of the compounds was evaluated against A549 and C6 tumor cell lines. MTT, analysis of DNA synthesis, acridine orange/ethidium bromide staining method and analysis of caspase-3 activation assays were performed for anticancer activity investigations. Compounds 6f and 6g, which carry 5-chloro and 5-methylbenzimidazole groups showed significant anticancer activity. Potential of these compounds to direct tumor cells to apoptotic pathway, which is a precondition of anticancer action, was also observed.

Full text of DOI:10.3109/14756366.2013.763253

http://informahealthcare.com/enz
ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, 2014; 29(2): 175–184
2014 Informa UK Ltd. DOI: 10.3109/14756366.2013.763253
!
Synthesis and anticancer activity evaluation of N-[4-(2-methylthiazol-4-
yl)phenyl]acetamide derivatives containing (benz)azole moiety
1
1
2
Leyla Yurttas¸ , Yusuf Ozkay , Gu¨ls¸en Akalın-C¸iftc¸i , and S¸afak Ulusoylar-Yıldırım
3
¨
2
3
1Department of Pharmaceutical Chemistry, Department of Biochemistry, and Department of Pharmacology, Faculty of Pharmacy, Anadolu
University, Eski¸sehir, Turkey
Abstract
Keywords
A new class of novel thiazole-(benz)azole derivatives was synthesized to investigate their
anticancer activity. The structure of the compounds was confirmed by IR, 1H-NMR, and MS
spectral data and elemental analyses. Anticancer effect of the compounds was evaluated
against A549 and C6 tumor cell lines. MTT, analysis of DNA synthesis, acridine orange/ethidium
bromide staining method and analysis of caspase-3 activation assays were performed for
anticancer activity investigations. Compounds 6f and 6g, which carry 5-chloro and
5-methylbenzimidazole groups showed significant anticancer activity. Potential of these
compounds to direct tumor cells to apoptotic pathway, which is a precondition of anticancer
action, was also observed.
A549, anticancer activity, benz(azole), C6,
thiazole
History
Received 11 September 2012
Revised 12 December 2012
Accepted 31 December 2012
Published online 7 February 2013
Introduction
topoisomerase I and many other cellular processes32. Natural
bis(benzoxazole) product UK-11 shows cytotoxic action on cancer
cells, selectively33. Benzothiazole derivative phortress is another
selective anticancer compound that shows anticancer activity by
inducing protein expression and forming a DNA adduct34.
As described above, in general, azole compounds causes a
DNA-damage and exhibit anticancer activity by varying mechan-
isms of action. Prompted from these observations, we synthesized a
series of azole and benzazole compounds and investigated their
anticancer activity profiles in a recent study35. In such study, N-
phenyl-2-(benz)azolylthio acetamide substructure indicated essen-
tial impact on cytotoxic action. Hence, in the present study we
synthesized novel compounds that bear thiazole and N-phenyl-2-
(benz)azolylthio acetamide substructures and investigated their
effect on DNA synthesis of carcinogenic cell lines and apoptosis.
Malignancy is caused by abnormalities in cells, which might be
due to inherited genes or caused by outside exposure of the body
to chemicals, radiation or even infectious agents1,2. Chemotherapy
is one of the techniques adopted for the treatment and eradication
of cancerous cells. DNA-damaging agents displaying various
mechanisms of action have achieved widespread use as che-
motherapeutics and still constitute a basic tool set in the treatment
of cancer3,4. On the other hand, significant side-effects, such as
nausea, vomiting, diarrhea, hair loss, serious infections and
growth of tumor-cell population are often encountered during
chemotherapy5. Thus, the limitations of current anticancer drugs
highlight the need for the discovery of new anticancer agents.
Thiazoles represent a class of heterocyclic compounds of great
importance in anticancer drug research. Interest in the anti-
neoplastic activity of thiazoles has increased after discovery of the
thiazole-based cytotoxic agents tiazofurin6–8, distamycin9, bleo-
mycin10,11, netropsin and thia-netropsin12. Several studies includ-
ing the anticancer activity of certain thiazole analogs were
Experimental section
Chemistry
reported by medicinal chemists13–20
.
All chemicals were purchased from Merck (Nottingham, UK) or
Sigma-Aldrich Chemical (Poole, UK) companies. All melting
points (m.p.) were determined by Electrothermal 9100 digital
melting point apparatus (Electrothermal, Essex, UK) and were
In addition to thiazoles, some other azole compounds have also
attracted much attention due to their prominent utilization as
antitumor agents21. For instance, derivatives of triazole22, imid-
azole23, benzimidazole24,25, benzoxazole26–28 and benzothiazole29
have shown anticancer properties. Triazole derivatives anastrozole
and letrozole are the aromatase inhibitors, currently finding
widespread application in the clinic30. Dacarbazine, an imidazole
compound, inhibits DNA, RNA and protein synthesis by alkylation
mechanism31. Bisbenzimidazole compounds such as Hoechst
33342 and Hoechst 33258 have anticancer ability to inhibit DNA
1
uncorrected. H-NMR data were recorded by Bruker 500 MHz
spectrometer (Bruker Bioscience, Billerica, MA). M þ 1 peaks
were determined by AB Sciex-3200 Q-TRAP LC/MS/MS system
(500 Old Connecticut Path, Framingham, MA). Elemental
analyses were performed on a Perkin Elmer EAL 240 (Perkin
Elmer, Norwalk, CT) elemental analyzer.
40-Acetaminoacetophenone (1)
40-Aminoacetophenone (0.05 mol, 6.75 g) and triethylamine
(0.06 mol, 8.34 mL) were dissolved in THF (100 mL) with a
constant stirring at 0–5 ꢀC, then acetyl chloride (0.06 mol,
4.78 mL) was added dropwise to this solution. The reaction
Address for correspondence: Leyla Yurttas¸, Department of Pharmaceu-
tical Chemistry, Faculty of Pharmacy, Anadolu University, Eskis¸ehir
26470, Turkey. Tel: þ902223350580/3783. E-mail: lyurttas@anadolu.
edu.tr

176 L. Yurttas¸ et al.
J Enzyme Inhib Med Chem, 2014; 29(2): 175–184
mixture was allowed to stirring for 1 h at room temperature. After For C12H11ClN2OS calculated: 54.03% C, 4.16% H and 10.50%
evaporation of solvent, the obtained solid was washed with water, N; found: 54.65% C, 4.15% H and 10.47% N. MS [M þ 1]þ:
filtered, dried and recrystallized from ethanol. Yield: 82%; m.p. m/z 267.5.
168 ꢀC (reference 168–170 ꢀC)36. IR: (KBr) ꢀmax (cmꢁ1): 3350
(amide N–H), 3068 (aromatic C–H), 1692 (ketone C¼O), 1664
General procedure for synthesis of 2-(substituted-
sulfanyl)-N-[4-(2-methylthiazol-4-yl)phenyl]acetamide
derivatives (6a–n)
(amide C¼O), 1369–1180 (C–N) and 841 (1,4-disubstituted
benzene).
A mixture of compound 5 (0.001 mol, 0.27 g), the appropriate
mercapto-(benz)azole derivative (0.001 mol) and K2CO3
(0.001 mol, 0.14 g) in acetone was refluxed for 2 h. The cooled
mixture was filtered, dried and recrystallized from ethanol to
afford target compounds 6a–n.
4-(2-Bromoacetyl)acetanilide (2)
Compound 1 (0.04 mol, 7.08 g) and HBr (0.5 mL) were dissolved
in acetic acid (30 mL) and bromine (0.044 mol, 2.27 mL) was
added dropwise at room temperature. After completion of the
addition of bromine, reaction mixture was allowed to additional
stirring for 1 h and then poured into ice-water (100 mL).
Precipitated product was filtered, washed with water, dried and
2-[(Thiazoline-2-yl)sulfanyl]-N-[4-(2-methylthiazol-4-yl)
pheny])acetamide (6a)
then recrystallized from ethanol. Yield: 86%; m.p. 188 ꢀC
Yield 81%; m.p. 105 ꢀC. IR (KBr) ꢀmax (cmꢁ1): 3252 (amide
N–H), 3064 (aromatic C–H), 2978 (aliphatic C–H), 1661 (amide
C¼O), 1608–1412 (C¼C and C¼N) and 1335–981 (C–N).
1H-NMR (500 MHz, DMSO-d6) ꢁ (ppm): 2.70 (s, 3H, CH3), 3.48
(t, 2H, J ¼ 7.30 Hz, thiazoline C5–H), 4.10 (s, 2H, S–CH2), 4.14
(t, 2H, J ¼ 7.30 Hz, thiazoline C4–H), 7.63 (d, 2H, J ¼ 7.50 Hz,
Ar-H), 7.81 (s, 1H, thiazole C5–H), 7.87 (d, 2H, J ¼ 7.50 Hz, Ar-
H) and 10.34 (s, 1H, N–H). For C15H15N3OS3 calculated: 51.55%
C, 4.33% H and 12.02% N; found: 51.56% C, 4.35% H, and
12.01% N. MS [M þ 1]þ: m/z 350.
(reference 185–187 ꢀC)37. IR: (KBr) ꢀmax (cmꢁ1): 3363 (amide
N–H), 3062 (aromatic C–H), 1698 (ketone C¼O), 1665 (amide
C¼O), 1378–1196 (C–N) and 848 (1,4-disubstituted benzene).
4-(2-Methyl-4-thiazolyl)acetanilide (3)
Method A: Compound 2 (0.03 mol, 7.68 g) and thioacetamide
(0.03 mol, 2.25 g) in ethanol (100 mL) were stirred at room
temperature for 48 h. The precipitated product was filtered, dried
and recrystallized from ethanol. Yield: 78%; m.p. 144 ꢀC
(reference 141–142 ꢀC)38. IR: (KBr) ꢀmax (cmꢁ1): 3365 (amide
N–H), 3062 (aromatic C–H), 1665 (amide C¼O), 1367–1211
(C–N) and 843 (1,4-disubstituted benzene).
2-[(1-Methylimidazole-2-yl)sulfanyl]-N-[4-(2-methylthia-
zol-4-yl)phenyl]acetamide (6b)
Method B: Compound 2 (0.003 mol, 0.77 g) and thioacetamide
(0.03 mol, 2.25 g) were dissolved in ethanol (30 mL). This
solution was transferred into a beaker (150 mL), a bigger beaker
(400 mL) was closed onto the reaction mixture, and put into a
domestic microwave oven. Reaction mixture was left under
microwave irradiation (180 W) 2 min intervals and ethanol
(10 mL) was added before each microwave irradiations.
Yield 85%; m.p. 193 ꢀC. IR (KBr) ꢀmax (cmꢁ1): 3281 (amide
N–H), 3024 (aromatic C–H), 2945 (aliphatic C–H), 1670 (amide
C¼O), 1597–1399 (C¼C and C¼N) and 1298–1021 (C–N).
1H-NMR (500 MHz, DMSO-d6) ꢁ (ppm): 2.70 (s, 3H, CH3), 3.61
(s, 3H, N–CH3), 3.89 (s, 2H, S–CH2), 6.98 (d, 1H, J ¼ 7.17 Hz,
imidazole C4–H), 7.26 (d, 1H, J ¼ 7.14 Hz, imidazole C5–H), 7.61
(d, 2H, J ¼ 8.30 Hz, Ar-H), 7.81 (s, 1H, thiazole C5–H), 7.87 (d,
2H, J ¼ 8.10 Hz, Ar-H) and 10.47 (s, 1H, N–H). For
C16H16N4OS2 calculated: 55.79% C, 4.68% H and 16.27% N;
found: 55.74% C, 4.69% H and 16.25% N. MS [M þ 1]þ: m/z 345.
Reaction
was
routinely
controlled
by
thin-layer
chromatography. After third microwave irradiation, the residue
was recrystallized from ethanol. Yield: 89%; m.p. 144 ꢀC
(reference 141–142 ꢀC)38.
2-[(4-Methyl-4H-1,2,4-triazole-3-yl)sulfanyl]-N-[4-(2-
methylthiazol-4-yl)phenyl]acetamide (6c)
4-(2-Methyl-4-thiazolyl)aniline (4)
Compound 3 (0.025 mol, 5.8 g) was refluxed in 10% HCl
(100 mL) for 1 h. The mixture was cooled down, poured into
iced water (100 mL) and made basic with 10% NaOH solution.
The precipitated product was filtered, dried and recrystallized
from ethanol. Yield: 92%; m.p. 136 ꢀC (reference m.p. 133–
135 ꢀC)38. IR: (KBr) ꢀmax (cmꢁ1): 3365 (amine N–H), 3361
(amine N–H), 3062 (aromatic C–H), 1367–1211 (C–N) and 843
(1,4-disubstituted benzene).
Yield 82%; m.p. 187 ꢀC. IR (KBr) ꢀmax (cmꢁ1): 3276 (amide
N–H), 3028 (aromatic C–H), 2990 (aliphatic C–H), 1672 (amide
C¼O), 1569–1402 (C¼C and C¼N) and 1315–1007 (C–N).
1H-NMR (500 MHz, DMSO-d6) ꢁ (ppm): 2.70 (s, 3H, CH3), 3.61
(s, 3H, N–CH3), 4.07 (s, 2H, S–CH2), 7.61 (d, 2H, J ¼ 8.50 Hz,
Ar-H), 7.81 (s, 1H, thiazole C5–H), 7.88 (d, 2H, J ¼ 8.20 Hz,
Ar-H), 8.56 (s, 1H, triazole C5–H) and 10.47 (s, 1H, N–H).
For C15H15N5OS2 calculated: 52.15% C, 4.38%
20.27% N; found: 52.16% C, 4.40% H and 20.29% N. MS
H and
2-Chloro-N-[4-(2-methyl-4-thiazolyl)phenyl]acetamide (5)
[M þ 1]þ: m/z 346.
Chloroacethyl chloride (0.02 mol, 1.6 mL) was added dropwise
over 15 min to a magnetically stirred solution of compound 4
(0.02 mol, 3.8 g) and triethylamine (0.02 mol, 2.8 mL) in dry THF
(15 mL). After completion of reaction, the solvent was evaporated
under reduced pressure. Water was added to wash the resulting
solid and the mixture was filtered, dried and recrystallized from
ethanol to give compound 5. Yield: 83%; m.p. 156 ꢀC. IR: (KBr)
ꢀmax (cmꢁ1): 3367 (amide N–H), 3053 (aromatic C–H), 1676
(amide C¼O), 1605–1403 (C¼C, C¼N), 1369–1214 (C–N) and
838 (1,4-disubstituted benzene). 1H-NMR (500 MHz, dimethyl
sulphoxide, DMSO-d6) ꢁ (ppm): 2.69 (3H, s, CH3), 4.32 (2H, s,
CO–CH2), 7.76 (2H, d, J ¼ 8.15 Hz, Ar-H), 7.81 (1H, s, thiazole
C5-H), 7.87 (d, 2H, J ¼ 8.10 Hz, Ar-H) and 10.50 (s, 1H, N–H).
2-[(5-Methyl-1,3,4-thiadiazole-3-yl)sulfanyl]-N-[4-(2-
methylthiazol-4-yl)phenyl]acetamide (6d)
Yield 91%; m.p. 186 ꢀC. IR (KBr) ꢀmax (cmꢁ1): 3269 (amide
N–H), 3029 (aromatic C–H), 2964 (aliphatic C–H), 1686 (amide
C¼O), 1566–1410 (C¼C and C¼N) and 1324–1012 (C–N).
1H-NMR (500 MHz, DMSO-d6) ꢁ (ppm): 2.68 (s, 3H, CH3), 2.71
(s, 3H, CH3), 4.29 (s, 2H, S–CH2), 7.64 (d, 2H, J ¼ 8.60 Hz,
Ar-H), 7.82 (s, 1H, thiazole C5–H), 7.89 (d, 2H, J ¼ 8.30 Hz,
Ar-H) and 10.47 (s, 1H, N–H). For C15H14N4OS3 calculated:
49.70% C, 3.89% H and 15.46% N; found: 49.71% C, 3.86% H
and 14.48% N. MS [M þ 1]þ: m/z 363.

DOI: 10.3109/14756366.2013.763253
N-[4-(2-methylthiazol-4-yl)phenyl]acetamide derivatives 177
2-[(1H-Benzimidazole-2-yl)sulfanyl]-N-[4-(2-methylthiazol-
4-yl)phenyl]acetamide (6e)
10.56 (s, 1H, N–H). For C19H15N3O2S2 calculated: 59.82% C,
3.96% H and 11.02% N; found: 59.82% C, 3.98% H and 11.05%
N. MS [M þ 1]þ: m/z 382.
Yield 76%; m.p. 241 ꢀC. IR (KBr) ꢀmax (cmꢁ1): 3274 (amide
N–H), 3012 (aromatic C–H), 2973 (aliphatic C–H), 1681 (amide
C¼O), 1549–1399 (C¼C and C¼N) and 1268–984 (C–N).
1H-NMR (500 MHz, DMSO-d6) ꢁ (ppm): 2.70 (s, 3H, CH3), 4.30
2-[(5-Chlorobenzoxazole-2-yl)sulfanyl]-N-[4-(2-methylthia-
zol-4-yl)phenyl]acetamide (6j)
(s, 2H, S–CH2), 7.46 (brs, 2H, Ar-H), 7.12–7.15 (m, 2H, Ar-H), Yield 82%; m.p. 129 ꢀC. IR (KBr) ꢀmax (cmꢁ1): 3276 (amide
7.65 (d, 2H, J ¼ 8.15 Hz, Ar-H), 7.81 (s, 1H, thiazole C5-H), 7.89 N–H), 3053 (aromatic C–H), 2949 (aliphatic C–H), 1676 (amide
(d, 2H, J ¼ 8.32 Hz, Ar-H), 10.62 (s, 1H, N–H) and 12.67 (s, 1H, C¼O), 1598–1422 (C¼C and C¼N) and 1310–1005 (C–N and
benzimidazole N–H). For C19H16N4OS2 calculated: 59.98% C, C–O). 1H-NMR (500 MHz, DMSO-d6): 2.71 (s, 3H, CH3), 4.39 (s,
4.24% H and 14.73% N; found: 59.96% C, 4.23% H and 14.75% 2H, S–CH2), 7.38 (d, 1H, J ¼ 8.55 Hz, Ar-H), 7.65 (d, 2H,
N. MS [M þ 1]þ: m/z 381.
J ¼ 8.80 Hz, Ar-H), 7.72–7.75 (m, 2H, Ar-H), 7.81 (s, 1H,
thiazole C5–H), 7.89 (d, 2H, J ¼ 8.40 Hz, Ar-H) and 10.59 (s, 1H,
N–H). For C19H14ClN3O2S2 calculated: 54.87% C, 3.39% H and
10.10% N; found: 54.89% C, 3.40% H and 10.13% N. MS
[M þ 1]þ: m/z 416.5.
2-[(5-Chloro-1H-benzimidazole-2-yl)sulfanyl]-N-[4-(2-
methylthiazol-4-yl)phenyl]acetamide (6f)
Yield 86%; m.p. 233 ꢀC. IR (KBr) ꢀmax (cmꢁ1): 3269 (amide
N–H), 3053 (aromatic C–H), 2953 (aliphatic C–H), 1682 (amide
C¼O), 1576–1395 (C¼C and C¼N) and 1305–996 (C–N).
1H-NMR (500 MHz, DMSO-d6) ꢁ (ppm): 2.71 (s, 3H, CH3), 4.31
2-[(5-Methylbenzoxazole-2-yl)sulfanyl]-N-[4-(2-
methylthiazol-4-yl)phenyl]acetamide (6k)
(s, 2H S-CH2), 7.16 (dd, 1H, J ¼ 8.35 Hz and 7.92 Hz, Ar-H), 7.46 Yield 79%; m.p. 158 ꢀC. IR (KBr) ꢀmax (cmꢁ1): 3281 (amide
(d, 1H, J ¼ 7.96 Hz, Ar-H), 7.52 (s, 1H, Ar-H), 7.65 (d, 2H, N–H), 3024 (aromatic C–H), 2976 (aliphatic C–H), 1678 (amide
J ¼ 8.20 Hz, Ar-H), 7.81 (s, 1H, thiazole C5–H), 7.89 (d, 2H, C¼O), 1598–1409 (C¼C and C¼N) and 1299–1006 (C–N and
J ¼ 8.35 Hz, Ar-H), 10.56 (s, 1H, N–H) and 12.86 (s, 1H, C–O). 1H-NMR (500 MHz, DMSO-d6) ꢁ (ppm): 2.40 (s, 3H,
benzimidazole N–H). For C19H15ClN4OS2 calculated: 55.00% C, CH3), 2.70 (s, 3H, CH3), 4.39 (s, 2H, S–CH2), 7.13 (d, 1H,
3.64% H and 13.50% N; found: 55.04% C, 3.63% H and 13.51% J ¼ 8.45 Hz, Ar-H), 7.43 (s, 1H, Ar-H), 7.52 (d, 1H, J ¼ 8.50 Hz,
N. MS [M þ 1]þ: m/z 415.5.
Ar-H), 7.65 (d, 2H, J ¼ 8.30 Hz, Ar-H), 7.81 (s, 1H, thiazole C5–
H), 7.89 (d, 2H, J ¼ 8.35 Hz, Ar-H) and 10.54 (1H, s, N–H). For
C20H17N3O2S2 calculated: 60.74% C, 4.33% H and 10.62% N;
found: 60.73% C, 4.36% H and 10.61% N. MS [M þ 1]þ: m/z 396.
2-[(5-Methyl-1H-benzimidazole-2-yl)sulfanyl]-N-[4-(2-
methylthiazol-4-yl)phenyl]acetamide (6g)
Yield 90%; m.p. 106 ꢀC. IR (KBr) ꢀmax (cmꢁ1): 3268 (amide
N–H), 3042 (aromatic C–H), 2969 (aliphatic C–H), 1686 (amide
C¼O), 1546–1419 (C¼C and C¼N) and 1279–1022 (C–N).
2-[(5-Nitrobenzoxazole-2-yl)sulfanyl]-N-[4-(2-methylthia-
zol-4-yl)phenyl]acetamide (6l)
1H-NMR (500 MHz, DMSO-d6) ꢁ (ppm): 2.39 (s, 3H, CH3), 2.70 Yield 84%; m.p. 218 ꢀC. IR (KBr) ꢀmax (cmꢁ1): 3268 (amide
(s, 3H, CH3), 4.27 (s, 2H, S–CH2), 6.96 (d, 1H, J ¼ 8.50 Hz, Ar- N–H), 3046 (aromatic C–H), 2994 (aliphatic C–H), 1669 (amide
H), 7.25 (s, 1H, Ar-H), 7.35 (d, 1H, J ¼ 8.15 Hz, Ar-H), 7.65 (d, C¼O), 1596–1400 (C¼C and C¼N) and 1302–1015 (C–N and
2H, J ¼ 8.55 Hz, Ar-H), 7.81 (s, 1H, thiazole C5–H), 7.87 (d, 2H, C–O). 1H-NMR (500 MHz, DMSO-d6) ꢁ (ppm): 2.70 (s, 3H,
J ¼ 8.20 Hz, Ar-H), 10.59 (s, 1H, N–H) and 12.54 (s, 1H, CH3), 4.48 (s, 2H, S–CH2), 7.65 (d, 2H, J ¼ 8.35 Hz, Ar-H), 7.82
benzimidazole N–H). For C20H18N4OS2 calculated: 60.89% C, (s, 1H, thiazole C5–H), 7.89–7.93 (m, 3H, Ar-H), 8.26 (dd, 1H,
4.60% H and 14.20% N; found: 60.87% C, 4.63% H and 14.22% J ¼ 8.25 Hz and J ¼ 4.15 Hz, Ar-H), 8.48 (d, 1H, J ¼ 4.60 Hz, Ar-
N. MS [M þ 1]þ: m/z 395.
H) and 10.54 (s, 1H, N–H). For C19H14N4O4S2 calculated:
53.51% C, 3.31% H and 13.14% N; found: 53.54% C, 3.35% H
and 13.17% N. MS [M þ 1]þ: m/z 427.
2-[(5-Nitro-1H-benzimidazole-2-yl)sulfanyl]-N-[4-(2-
methylthiazol-4-yl)phenyl]acetamide (6h)
2-[(Benzothiazole-2-yl)sulfanyl]-N-[4-(2-methylthiazol-4-
yl)phenyl]acetamide (6m)
Yield 78%; m.p. 207 ꢀC (dekomp.). IR (KBr) ꢀmax (cmꢁ1): 3296
(amide N–H), 3042 (aromatic C–H), 2976 (aliphatic C–H), 1689
(amide C¼O), 1604–1412 (C¼C and C¼N) and 1330–1064 Yield 87%; m.p. 130 ꢀC. IR (KBr) ꢀmax (cmꢁ1): 3277 (amide
(C–N). 1H-NMR (500 MHz, DMSO-d6) ꢁ (ppm): 2.70 (s, 3H, N–H), 3039 (aromatic C–H), 2975 (aliphatic C–H), 1676 (amide
CH3), 4.38 (s, 2H, S–CH2), 7.62 (d, 1H, J ¼ 7.96 Hz, Ar-H), 7.66 C¼O), 1569–1408 (C¼C and C¼N) and 1295–1011 (C–N).
(d, 2H, J ¼ 8.20 Hz, Ar-H), 7.81 (s, 1H, thiazole C5–H), 7.89 (d, 1H-NMR (500 MHz, DMSO-d6) ꢁ (ppm): 2.70 (s, 3H, CH3), 4.42
2H, J ¼ 8.35 Hz, Ar-H), 8.07 (dd, 1H, J ¼ 8.10 Hz and 7.97 Hz, (s, 2H, S–CH2), 7.37 (t, 1H, J ¼ 7.56 Hz, Ar-H), 7.47 (t, 1H,
Ar-H), 8.32 (s, 1H, Ar-H), 10.63 (s, 1H, N–H) and 12.63 (s, 1H, J ¼ 7.52 Hz, Ar-H), 7.66 (d, 2H, J ¼ 7.82, Ar-H), 7.82 (s, 1H,
benzimidazole N–H). For C19H15N5O3S2 calculated: 53.63% C, thiazole C5–H), 7.84 (d, 1H, J ¼ 8.50 Hz, Ar-H), 7.90 (d, 2H,
3.55% H and 16.46% N; found: 53.61% C, 3.57% H and 16.45% J ¼ 8.35 Hz, Ar-H), 8.03 (d, 1H, J ¼ 8.32 Hz, Ar-H) and 10.54 (s,
N. MS [M þ 1]þ: m/z 426.
1H, N–H). For C19H15N3OS3 calculated: 57.40% C, 3.80% H and
10.57% N; found: 57.42% C, 3.83% H and 10.54% N. MS
[M þ 1]þ: m/z 427.
2-[(Benzoxazole-2-yl)sulfanyl]-N-[4-(2-methylthiazol-4-
yl)phenyl]acetamide (6i)
2-[(5-Chlorobenzothiazole-2-yl)sulfanyl]-N-[4-(2-
methylthiazol-4-yl)phenyl]acetamide (6n)
Yield 83%; m.p. 148 ꢀC. IR (KBr) ꢀmax (cmꢁ1): 3269 (amide
N–H), 3037 (aromatic C–H), 2989 (aliphatic C–H), 1667 (amide
C¼O), 1602–1450 (C¼C and C¼N) and 1301–1055 (C–N and Yield 75%; m.p. 195 ꢀC. IR (KBr) ꢀmax (cmꢁ1): 3294 (amide
C–O). 1H-NMR (500 MHz, DMSO-d6): 2.71 (s, 3H, CH3), 4.41 (s, N–H), 3015 (aromatic C–H), 2986 (aliphatic C–H), 1659 (amide
2H, S–CH2), 7.33–7.37 (m, 2H, Ar-H), 7.62–7.67 (m, 4H, Ar-H), C¼O), 1593–1402 (C¼C and C¼N) and 1296–1023 (C–N).
7.81 (s, 1H, thiazole C5–H), 7.90 (d, 2H, J ¼ 8.35 Hz, Ar-H) and 1H-NMR (500 MHz, DMSO-d6) ꢁ (ppm): 2.71 (s, 3H, CH3), 4.44

178 L. Yurttas¸ et al.
J Enzyme Inhib Med Chem, 2014; 29(2): 175–184
(s, 2H, S–CH2), 7.42 (dd, 1H, J ¼ 8.30 Hz and 4.55 Hz, Ar-H), Kit was designed to measure caspase-3 or DEVD-cleaving
7.67 (d, 2H, J ¼ 8.40 Hz, Ar-H), 7.82 (s, 1H, thiazole C5–H), activity, an early marker of cells undergoing apoptosis40. First,
7.90–7.91 (m, 3H, Ar-H), 8.07 (s, 1H, Ar-H) and 10.54 (s, 1H, cells (1 ꢂ 106 cells/mL) were washed with phosphate-buffered
N–H). For C19H14ClN3OS3 calculated: 52.83% C, 3.27% H and saline (PBS), resuspended in cold cell lysis buffer and incubated
9.73% N; found: 52.87% C, 3.29% H and 9.75% N. MS [M þ 1]þ: for 30 min on ice. After 24 h incubation period with various
m/z 432.5.
concentrations of compounds 6f, 6g and mitoxantrone, cell lysates
were prepared. For each reaction, 5 mL of reconstituted Ac-
DEVD-AMC (synthetic tetrapeptide fluorogenic substrate for
Caspase-3 activity) was added to a well containing 0.2 mL of
1 ꢂ HEPES buffer. Cell lysate (20 mL) was added to each well/
reaction. Reaction mixtures were incubated for 1 h at 37 ꢀC. The
amount of AMC liberated from Ac-DEVD-AMC was measured
using microplate reader (Perkin Elmer/Victor/X3) with an exci-
tation wavelength of 380nm and an emission wavelength of
460 nm. Apoptotic cell lysates containing active Caspase-3
yielded a considerable emission as compared to controls. Also
non-apoptotic control cell lysates AMC emission was accepted as
100% and other cell lysates emissions were measured according to
control cells emissions. All experiments were repeated twice. For
all doses, duplicate wells were used.
Anticancer screening
Cell culture and drug treatment
C6 glioma cells were incubated in Dulbecco’s modified Eagle’s
medium (Sigma, Deisenhofen, Germany) supplemented with 10%
fetal calf serum (Gibco, Paisley, Scotland). A549 cells were
incubated in 90% Roswell Park Memorial Institute
medium(Roswell Park Memorial Institute, Buffalo, NY) supple-
mented with 10% fetal bovine serum (Gibco). All media were
supplemented with 100 IU/mL penicillin–streptomycin (Gibco)
and cells were incubated at 37 ꢀC in a humidified atmosphere of
95% air and 5% CO2. Exponentially growing cells were plated at
2 ꢂ 104 cells/mL into 96-well microtiter tissue culture plates
(Nunc, Roskilde, Denmark) and incubated for 24 h before the
addition of the test compounds (the optimum cell number for
cytotoxicity assays was determined in preliminary experiments).
Stock solutions of the compounds were prepared in DMSO
(Sigma-Aldrich) and further dilutions were made with a fresh
culture medium (the concentration of DMSO in the final culture
medium was 50.1% which had no effect on the cell viability).
Acridine orange/ethidium bromide staining methods
C6 glioma cells were cultured at 1 ꢂ 105 cells/well onto six wells
plate until 50% confluent. The medium was replaced with a fresh
medium containing different concentrations of compounds 6f and
6g and mitoxantrone. The cells, without treatment, were used as
negative controls. Cells were then incubated for 24 h in
humidified atmosphere at 37 ꢀC in CO2 5%. The medium was
removed and cells were washed thrice with PBS. Cells were fixed
with 70% ethanol and incubated for 5 min and then washed with
PBS. A mixture of ethidium bromide:acridin orange (1:1)
(100 mg/mL) was added and incubated for 5 min. Cells were
washed with PBS, they were removed and covered on the object
glass. Then assessment was carried out under fluorescence
microscope (Olympus, Tokyo, Japan)41. Acridine orange/eth-
idium bromide combination was used to visualize cells with
aberrant chromatin organization. Acridine orange was used to
visualize the number of cells which has undergone apoptosis.
MTT assay for cytotoxicity of compounds
The level of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide (MTT) (Sigma-Aldrich) reduction was quanti-
fied as previously described in the literature39 with small
modifications. After 24 h of preincubation, the tested compounds
(6a–n) were added to give final concentration in the range of
6–200 mg/mL and the cells were incubated for 24 h. At the end of
this period, MTT was added to final concentration of 0.5 mg/mL
and the cells were incubated for 4 h at 37 ꢀC. After removal of
medium, the formazan crystals formed by MTT metabolism were
solubilized by the addition of 200 mL DMSO to each well and
absorbance was read at 540 nm with a microplate spectropho-
tometer (Bio-Tek, Winooski, VT). Every concentration was
repeated in three wells and IC50 values (mM) were defined as
the compound concentrations that reduced absorbance to 50% of
control values.
Statistical analyses
SPSS (Chicago, IL) for Windows 15.0 was used for statistical
analysis. Data were expressed as mean ꢃ SD. Comparisons were
performed by one-way analysis of variance test for normally
distributed continuous variables and post hoc analyses of group
differences were expressed by the Tukey test.
Analysis of DNA synthesis
Results and discussions
Analysis of DNA synthesis was measured by, BrdU (bromodeox-
yuridine) cell proliferation colorimetric kit (Roche, Mannheim,
Germany). First, cells were seeded into 96-well flat-bottomed
microtiter plates at a density of 2 ꢂ 103. The tumor cells cultured
in the presence of various concentrations of compounds 6b, 6c,
6e, 6f, 6g and mitoxantrone. Microtiter plates were incubated at
37 ꢀC in a 5% CO2/95% air humidified atmosphere for 24 and
48 h. At the end of each day, the cells were labeled with 10 mL
BrdU solution for 2 h and then fixed. Anti-BrdU-POD (100 mL)
was added and incubated for 90 min. Finally, wells were washed
with BPS and cells were incubated with substrate. Absorbance of
the samples was measured with an ELX808-IU Bio-Tek apparatus
at 492 nm. All experiments were repeated twice. For all
compound doses, duplicate wells were used.
Chemistry
Present study was undertaken to synthesize some novel thiazole–
(benz)azole derivatives and investigate their probable anticancer
effects. Synthetic way for compounds 6a–n was outlined in
Scheme 1. Initially, 40-aminoacetophenone was acetylated to
prevent the occurrence of probable impurities, which may appear
due to free amino group in further reaction steps, and 40-
acetaminoacetophenone (1) was obtained. Bromination of com-
pound 1 in acetic acid gave 4-(2-bromoacetyl)acetanilide (2),
which was then reacted with thioacetamide to afford 4-(2-methyl-
4-thiazolyl)acetanilide (3) via the Hantzsch reaction. Hydrolysis
of acetamide group of compound 3 in 10% HCl solution provided
4-(2-methyl-4-thiazolyl)aniline (4). Compound 4 in THF was
acetylated with chloroacetyl chloride in the presence of
triethylamine (5). Finally, compound 5 was reacted with corres-
ponding (benz)azole-thiol derivatives under basic conditions to
achieve the target compounds (6a–n). Hantzsch reaction was also
Spectrofluorometric analysis of caspase-3 activation
Caspase-3 activation was analyzed by Spectrofluorometric
Caspase-3 Assay kit (BD Pharmingen, Franklin Lakes, NJ).

DOI: 10.3109/14756366.2013.763253
N-[4-(2-methylthiazol-4-yl)phenyl]acetamide derivatives 179
Scheme 1. The synthetic protocol of the compounds (6a–n).
Reagents: (i) acetyl chloride, TEA, THF, 0–5 ꢀC; (ii) Br2, AcOH; (iii) thioacetamide, EtOH, room temperature or MWI; (iv) 10% HCl, EtOH, reflux;
(v) chloroacetyl chloride, TEA, THF, room temperature; and (vi) appropriate thiol-benz(azole), K2CO3, acetone, reflux.
carried out by microwaves irradiation (180 W, 2 min intervals; The structures of target compounds were assigned on the basis
total 6 min) in a kitchen-type microwave oven. It was observed of spectroscopic and analytical data. Characteristic stretching
that yield (87%) of compound 3 was notably increased along with absorptions for C¼O and N–H bonds were observed at 1659–
short reaction time.
1689 and 3252–3296 cmꢁ1, respectively. The stretching absorp-
All the intermediates were analyzed in IR spectra to confirm tion at about 1608–1395 and 1335–981 cmꢁ1 was recorded for
each reaction step. During the analysis of compound 1, two C¼C, C¼N double bonds and C–O, C–N bonds, respectively.
significant carbonyl bands at 1692 and 1664 cmꢁ1 were recorded Disappearance of stretching absorption for the S–H bond at about
for ketone and amide groups, respectively. Compound 2 gave 2550 cmꢁ1 was an evidence for the occurrence of target
same bands at 1698 and 1665 cmꢁ1, whereas in the IR spectra of compounds. In the 1H-NMR spectra, methyl substituent at the
compound 3 disappearance of stretching band about 1700 cmꢁ1 second position of thiazole ring appeared at 2.70–2.71 as a
belonging to ketone carbonyl was an evidence for ring closure. singlet. Aromatic proton (C5–H) of thiazole ring gave peak at
Similarly, disappearance of amide carbonyl stretching bond at 7.81–7.82 ppm as a singlet. Aromatic protons of phenyl moiety at
about 1660–1670 cmꢁ1 showed that compound 4 was successfully 4th position of thiazole observed at about 7.61 and 7.87 were
obtained via deacetylation. On the other hand, for compound 5 double doublets. Acetamide (NHCOCH2) peaks were observed at
such stretching bond was observed again at 1676 cmꢁ1 due to the 3.89–4.48 ppm as a singlet and at 10.34–10.62 ppm as a broad
acetylation reaction of compound 4 with chloroacetyl chloride.
singlet for methylene (CH2) and amide (NHCO) protons,

180 L. Yurttas¸ et al.
J Enzyme Inhib Med Chem, 2014; 29(2): 175–184
respectively. The other peaks belonging to aromatic and aliphatic chemical structure cause significant changes in cytotoxicity.
protons of variable side chains were recorded at estimated areas. According to cytotoxicity test results, for the azole side groups it
In the mass spectra, M þ 1 peaks agreed well with the calculated can be declared that, 1-methylimidazol-2-yl and 1-methyl-1,2,4-
molecular weight of the target compounds. Elemental analysis triazol-2-yl enhance the cytotoxic activity more than thiazolin-2-
results for C, H and N elements were satisfactory within yl and 5-methyl-1,3,4-thiadiazol-2-yl. In varying benzazole side
calculated values of the compounds.
groups, benzimidazol-2-yl has a greater effect than benzoxazol-2-
yl and benzothiazol-2-yl. In addition, electronic characters of the
substituents on fifth position of benzimidazole have an essential
impact on cytotoxic effect. The cytotoxic effect of compounds 6f
and 6g, which carry electron-donating chloro and methyl
substituents on benzimidazole ring, was at the same level with
that of nonsubstituted benzimidazole-bearing compound 6e. On
the other hand, in compound 6h substitution of benzimidazole
with electron withdrawing nitro group reduced the cytotoxicity.
This result suggests that, substitution of benzimidazole ring with
an electron-donating group improves the cytotoxicity.
Biochemistry
The synthesized compounds 6a–n were tested against rat glioma
(C6) and human lung (A549) cancer cell lines. Anticancer activity
screening was carried out at four steps. Initially, cytotoxic
potential of compounds on carcinogenic cells was tested by the
MTT method. Mitoxantrone, a cytotoxic anticancer agent, was
used as appositive control. Second, the compounds, which
displayed comparable cytotoxicity to mitoxantrone were selected
and tested for their inhibitory potency on DNA synthesis of
carcinogenic cells. In the third step, Caspase-3 activation test was
performed in order to observe carcinogenic cells undergoing
Analysis of DNA synthesis
apoptosis. Finally, acridine orange/ethidium bromide staining This immune staining procedure depends on measuring the
method was applied to visualize cells which have undergone incorporation of BrdU into nuclear DNA in place of thymidine
apoptosis.
during the S-phase of the cell cycle using specific anti-BrdU
antibodies31. Thus, such method provides a colorimetric meas-
urement for DNA synthesis inhibition ratio of the carcinogenic
cells.
Cytotoxicity (MTT) method
MTT, based on the ability of metabolically active cells to convert
the pale yellow MTT dye to a spectrophotometrically quantifiable
blue formazan product, is one of the most preferred cytotoxicity
tests applied to investigate potential of new compounds against
cancer cell lines29. In such test, C6 and A549 cell lines were
incubated with various concentrations (6–200 mg/mL) of 6a–n
that are maintained via serial dilutions. After completion of
incubation period (24 h), cytotoxic activity of the compounds was
examined and IC50 values were calculated (Table 1). Cytotoxic
agent mitoxantrone was used as a positive control.
As seen in Table 1, on both cell type, the compounds 6b, 6c
and 6e–g were more cytotoxic than the other derivatives due to
their IC50 values about 122.6 mM and below. A549 cell line found
to be more sensitive to the synthesized compounds than C6 cell
line. The cytotoxic effect of the compounds 6b, 6c and 6e–g on
A549 cells was comparable to that of mitoxantrone, whereas
control agent showed higher cytotoxicity on C6 cells than the
synthesized compounds.
DNA synthesis of the carcinogenic cell lines was analyzed for
compounds 6b, 6c and 6e–g, which indicated significant cytotoxic
activity in the MTT test. Mitoxantrone was used as a positive
control. For 24 and 48 h time periods, A549 and C6 cells were
incubated with three different concentrations (IC50/2, IC50 and
2 ꢂ IC50) of the compounds. Tested compounds showed time- and
dose-dependent inhibitory activity on the DNA synthesis of both
cell lines (Figures 1 and 2). In all DNA synthesis inhibition
results, C6 cells were the most resistant cells against all tested
compounds. Compounds 6f and 6g had significant antiprolifera-
tive activity against both of cell lines.
Figures 1 and 2 demonstrate the effect of the selected
compounds on DNA synthesis of A549 and C6 cells. As seen in
the figures, 1-methylimidazole and 1-methyltriazole carrying
compounds 6b and 6c have low inhibitory potency than
benzimidazole carrying compounds 6e–g on the DNA synthesis
of carcinogenic cells. It is clear that benzimidazole side provides
more lipophilic character to compounds 6e–g, while imidazole
and triazole sides in compounds 6b and 6c cannot facilitate
similar lipophilicity. Thus, increasing lipophilic character in
compounds 6e–g may enhance the DNA synthesis inhibition
profile due to hydrophobic interaction with DNA. The inhibitory
effect of the compounds 6f and 6g on DNA synthesis is higher
than that of compound 6e. Such result refers that chloro or methyl
substitution of benzimidazole side contributes the inhibition of
DNA synthesis. Due to their comparable inhibitory potential to
reference drug, compounds 6f and 6g were selected for further
assays, which were performed to observe carcinogenic cells
undergoing apoptosis.
MTT test results demonstrate that there are important differ-
ences in the cytotoxic activity of compounds 6a–n. Thus, it can be
mentioned that varying azole or benzazole side groups on the
Table 1. IC50 (mM) values of the compounds 6a–n on A549 and C6 cells.
Compound
A549 cells
C6 cells
6a
6b*
6c*
6d
6e*
6f*
6g*
6h
6i
6j
6k
6l
6m
117.5 ꢃ 24.4
43.0 ꢃ 6.7
315.2 ꢃ 28.7
101.7 ꢃ 5.8
122.6 ꢃ 7.2
488.1 ꢃ 69.6
107.9 ꢃ 26.3
120.1 ꢃ 15.9
73.6 ꢃ 2.5
83.2 ꢃ 15.9
119.6 ꢃ 16.0
63.2 ꢃ 9.5
Analysis of caspase-3 activation
112.7 ꢃ 18.3
65.2 ꢃ 5.3
The understanding of the cellular signaling processes leading to
programmed cell death, called as apoptosis, is of utmost
importance in cancer because damaged cells that do not enter
the apoptotic pathway may proliferate unchecked and become a
cancerous cell mass. Thus, letting tumor cells to apoptosis is a
requirement for an anticancer agent. It is generally accepted that
cytotoxic drugs eliminate malignant cells by inducing apop-
tosis32,33. The activation of caspases, a family of important
signaling molecules with various tasks depending on subtype and
organ, is a marker for apoptosis34. The detection of activated
152.2 ꢃ 11.8
284.3 ꢃ 27.3
220.7 ꢃ 45.5
282.8 ꢃ 69.6
167.4 ꢃ 2.8
242.5 ꢃ 35.9
143.7 ꢃ 21.3
45.8 ꢃ 6.2
251.1 ꢃ 59.3
223.1 ꢃ 13.1
148.5 ꢃ 18.3
95.4 ꢃ 27.8
133.1 ꢃ 6.8
176.1 ꢃ 51.2
258.9 ꢃ 17.6
19.9 ꢃ 1.2
6n
Mitoxantrone
Data represent means ꢃ SD of three independent experiments.
*The compounds selected for the analysis of DNA synthesis assay.

Figure 1. DNA synthesis inhibitory activity
of compounds 6b, 6c, 6e, 6f, 6g and
mitoxantrone on A549 cells.
Mean percent absorbance of untreated control
cells was assumed 0% and three different
concentrations (a ¼ IC50/2, b ¼ IC50
,
c ¼ 2 ꢂ IC50) of test compounds and mitox-
antrone were given. Data points represent
means for two independent experiments ꢃSD
of four independent wells. p50.05.
Figure 2. DNA synthesis inhibitory activity
of compounds 6b, 6c, 6e, 6f, 6g and
mitoxantrone on C6 cells.
Mean percent absorbance of untreated control
cells was assumed 0% and different concen-
trations (a ¼ IC50/2, b ¼ IC50, c ¼ 2 ꢂ IC50) of
test compounds and mitoxantrone were given.
Data points represent means for two inde-
pendent experiments ꢃSD of four independ-
ent wells. p50.05.
a
b
c
a
b
c
a
b
c
a
b
c
a
b
c
a
b
c
Comp 6b
Comp 6c
Comp 6e
Comp 6f
Comp 6g Mitoxantrone
Figure 3. Effect of Ac-DEVD-amc on the activity of caspase-3 induced by compounds 6f, 6g and mitoxantrone in A549 cell line.
A549 cells were maintained in cultures for 24 h and then exposed to Ac-DEVD-amc (1.0 mM) 30 min before exposure to two different concentrations
(a ¼ IC50/2 and b ¼ IC50) of compounds 6f, 6g and mitoxantrone. Values represent mean ꢃ SD from duplicate samples for each experiment.
Significantly different from respective control cells: ***p50.001.

182 L. Yurttas¸ et al.
J Enzyme Inhib Med Chem, 2014; 29(2): 175–184
Figure 4. Effect of Ac-DEVD-amc on the
activity of caspase-3 induced by compounds
6f, 6g and mitoxantrone in C6 cell line.
C6 cells were maintained in cultures for 24 h
and then exposed to Ac-DEVD-amc
(1.0 mM) 30 min before exposure to two
different concentrations (a ¼ IC50/2 and
b ¼ IC50) of compounds 6f, 6g and mitoxan-
trone. Values represent mean ꢃ SD from
duplicate samples for each experiment.
Significantly different from respective con-
trol cells: *p50.05, ** p50.01 and
***p50.001.
(a)
(b)
(c)
(d)
Figure 5. Cellular and nuclear morphological changes of A549 cells following exposure to IC50 of compounds 6f, 6g and mitoxantrone for 24 h: (a)
A549 control cells, (b) IC50 of mitoxantrone, (c) IC50 of compound 6f and (d) IC50 of compound 6g. Cells were distinguished according to the
fluorescence emission and the morphological aspect of chromatin condensation in the stained nuclei. Viable cells have uniform bright green nuclei with
organized structure. Early apoptotic cells have green nuclei, but perinuclear chromatin condensation is visible as bright green patches or fragments.
White arrows indicate apoptotic cells. Colored version of the figure can be sought in the online version of this manuscript.
caspase-3, the main effector caspase in the apoptotic enzyme cell lines. However, activation of caspase-3 seemed to be stronger
cascade, by using fluorogenic substrates is a method commonly on A549 cell line. When compared with control, compounds 6f
employed to observe the apoptosis level35.
and 6g and mitoxantrone caused 2.5-fold higher caspase-3 activity
The compounds 6f and 6g, which displayed notable anticancer induction at IC50 concentrations. Besides, induction potent of
profile in both MTT and analysis of DNA synthesis assays, were compound 6g on caspase-3 activation was greater than both
tested for their caspase-3 activation potencies. Mitoxantrone was compound 6f and reference drug. These findings suggest that, as
used as a control agent. A549 and C6 cells were maintained in well as reference mitoxantrone, compounds 6f and 6g have
cultures for 24 h and then exposed to Ac-DEVD-amc (1.0 mM) potencies to direct tumor cells to apoptotic pathway, which is a
30 min before exposure to three different concentrations (IC50/2 precondition of anticancer action.
and IC50) of the compounds.
Figures 3 and 4 present the caspase-3 activation percent of
Acridine orange/ethidium bromide staining method
A549 and C6 cells that are induced by compounds 6f and 6g at
different concentrations (IC50/2 and IC50). Synthesized com-
pounds and control agent induced the caspase-3 activation in both
This method facilitates to visualize the changes in nuclear
morphology induced by test compounds and thus observation of

DOI: 10.3109/14756366.2013.763253
N-[4-(2-methylthiazol-4-yl)phenyl]acetamide derivatives 183
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While the untreated A549 cells (control) were well spread with
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Conclusions
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In the present study, novel N-[4-(2-methylthiazol-4-
yl)phenyl]acetamide derivatives were synthesized and evaluated
for their anticancer activity. Consecutive anticancer tests includ-
ing cytotoxicity, analysis of DNA synthesis and induction of
apoptosis revealed that compounds 6f and 6g, which carry
benzimidazole and 5-chlorobenzimidazole side groups, were the
most potent members in the series. In conclusion, result of this
work has encouraged us to synthesize novel compounds that bear
N-phenyl-2-(5-substitutedbenzimidazol-2-yl)thio acetamide sub-
structure and investigate their anticancer profiles in further
studies.
N-substituted thiazole sulphonamides.
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Declaration of interest
23. Boiani M, Gonzalez M. Imidazole and benzimidazole derivatives as
chemotherapeutic agents. Mini-Rev Med Chem 2005;5:409–24.
24. Ramla MM, Omar MA, Tokuda H, El-Diwani H. Synthesis and
The authors report no conflicts of interest.
inhibitory
activity
of
new
benzimidazole
derivatives
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