Discovery of 2-phenyl-N-(5-(trifluoromethyl)-1,3,4-thiadiazol-2-yl) acetamide derivatives as apoptosis inducers via the caspase pathway with potential anticancer activity

Article abstract of DOI:10.1002/ardp.201300180

In the current research of medicinal chemistry, apoptosis induction is one of the novel strategies for the development and discovery of novel anticancer therapeutics. In the present study, a new series of 1,3,4-thiadiazole derivatives (4a-4p) were synthesized and their in vitro anticancer activities were evaluated against three cancer cell lines: PC3 (prostate cancer), MCF7 (breast cancer), and SKNMC (neuroblastoma). These cell lines were utilized in MTT assays and the obtained results were compared to doxorubicin. Apoptosis induction was also investigated through exploration of the activation of caspases 3, 8, and 9. According to the obtained results, compounds 4b (3-Cl) and 4c (4-Cl) demonstrated the best caspase activation. In fact, compounds 4b and 4c enhanced the activity of caspases 3 and 9 in the MCF7 cell line. A new series of 1,3,4-thiadiazole derivatives (4a-4p) were synthesized and their in vitro anticancer activities were evaluated against the cancer cell lines PC3 (prostate cancer), MCF7 (breast cancer), and SKNMC (neuroblastoma). Apoptosis induction was investigated with regard to activation of caspases 3, 8, and 9. Compounds 4b (3-Cl) and 4c (4-Cl) enhanced the activity of caspases 3 and 9 in the MCF7 cell line.

Full text of DOI:10.1002/ardp.201300180

812
Arch. Pharm. Chem. Life Sci. 2013, 346, 812–818
Full Paper
Discovery of 2-Phenyl-N-(5-(trifluoromethyl)-1,3,4-thiadiazol-
2-yl)acetamide Derivatives as Apoptosis Inducers via the
Caspase Pathway with Potential Anticancer Activity
Leila Hosseinzadeh1, Amanc Khorand2,3, and Alireza Aliabadi3
1
Faculty of Pharmacy, Department of Pharmacology, Toxicology and Medical Services, Kermanshah University
of Medical Sciences, Kermanshah, Iran
Students Research Committee, Kermanshah University of Medical Sciences, Kermanshah, Iran
Faculty of Pharmacy, Department of Medicinal Chemistry, Kermanshah University of Medical Sciences,
2
3
Kermanshah, Iran
In the current research of medicinal chemistry, apoptosis induction is one of the novel strategies for
the development and discovery of novel anticancer therapeutics. In the present study, a new series of
1,3,4-thiadiazole derivatives (4a–4p) were synthesized and their in vitro anticancer activities were
evaluated against three cancer cell lines: PC3 (prostate cancer), MCF7 (breast cancer), and SKNMC
(neuroblastoma). These cell lines were utilized in MTT assays and the obtained results were compared to
doxorubicin. Apoptosis induction was also investigated through exploration of the activation of
caspases 3, 8, and 9. According to the obtained results, compounds 4b (3-Cl) and 4c (4-Cl) demonstrated
the best caspase activation. In fact, compounds 4b and 4c enhanced the activity of caspases 3 and 9
in the MCF7 cell line.
Keywords: Anticancer / Apoptosis / Caspase enzymes / Synthesis / 1,3,4-Thiadiazole
Received: May 12, 2013; Revised: May 12, 2013; Accepted: August 7, 2013
DOI 10.1002/ardp.201300180
Introduction
anticancer drugs, current anticancer drugs still have major
limitations such as drug resistance, lack of selectivity, and
unwanted side effects (bone marrow depression, nausea,
vomiting, infertility, neuropathy, etc.) [2–7].
Cancer is a major global problem and is the second leading
cause of death in developed countries. Since many of the
current pharmacotherapeutic methods have problems with
toxicity and drug resistance, there is a strong demand for the
discovery and development of effective new anticancer
drugs [1]. Chemotherapy involves the use of low-molecular-
weight drugs (small molecules such as cyclophosphamide,
capecitabine, etoposide, etc.) to selectively destroy tumor cells
or at least prevent their proliferation or metastasis. The
research for identification and discovery of novel chemical
structures that can act as more effective and reliable
anticancer agents is still a major challenge to medicinal
chemists. Despite the important advances achieved over
recent decades in the research and development of various
Apoptosis or programmed cell death plays an important
role in promoting tissue homeostasis by eliminating damaged
or excessive cells. Defects in apoptosis signaling pathways
could result in uncontrolled tumor cell growth as well as
resistance to cancer treatment. Therefore, restoration of
normal apoptosis or promotion of apoptosis could lead to
cancer cell death as well as increase the response to
chemotherapeutics. In addition, it is known that many
chemotherapeutics kill cancer cells through the induction of
apoptosis [8]. It is known that the anti-tumor efficacy of many
chemotherapeutic agents is associated with their ability to
induce apoptosis. Novel approaches in medicinal chemistry
for design of effective anticancer agents are focused on
apoptosis induction in cancer cells. In fact, targeting
regulators of apoptosis could lead to the development of
new anticancer treatments. In addition, overcoming tumor
resistance is another advantage of these new agents over
conventional anti-cancer drugs [9].
Correspondence: Dr. Alireza Aliabadi, Faculty of Pharmacy, Department
of Medicinal Chemistry, Kermanshah University of Medical Sciences,
Kermanshah, Iran.
E-mail: aliabadi.alireza@gmail.com
Fax: þ98 831 4276493
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2013, 346, 812–818
1,3,4-Thiadiazole Derivatives as Apoptosis Inducers
813
O
O
O
H
C
O
N
N
N
N
N
S
N
N
O
S
S
N
O
F
S
S
N
O
A
B
Figure 1. Structures of two 1,3,4-thiadiazole-based compounds as apoptotic inducers.
During recent years a deep and vast exploration of different Results and discussion
pharmacophores and chemical structure containing 1,3,4-
thiadiazole ring has been carried out. Many of these
derivatives exhibited interesting and potential biological
effects such as antimicrobial, antitubercular, antiviral,
antiinflammatory, anticonvulsant, antihypertensive, antioxi-
dant, antifungal, and anticancer activity and now they are on
the market as commonly used drugs [10–19].
Diverse chemical structures containing the 1,3,4-thiadiazole
nucleus have been reported with potential anticancer activity
and as apoptotic inducers (Fig. 1). The 1,3,4-thiadiazole ring in
anticancer agents performs its role in pharmacophores of
apoptosis inducers and caspase activators, tyrosine kinase
inhibitors, carbonic anhydrase inhibitors, etc. [20–28]. Hence,
various mechanisms could be imagined for anticancer
chemical structures containing the 1,3,4-thiadiazole ring.
Structure–activity relationship (SAR)
A new series of 1,3,4-thiadiazole derivatives were synthesized
and their in vitro anticancer activities were evaluated against
three cancerous cell lines (Tables 1 and 2), namely, PC3
(prostate cancer), MCF7 (breast cancer), and SKNMC (neuro-
blastoma). These cell lines were utilized to test these
compounds using MTT assay. Totally, none of the tested
compounds 4a–4p showed significant potency toward these
cell lines. Various moieties with different electronic proper-
ties were applied to investigate the role of the electron-
withdrawing and electron-donating activity of the corre-
sponding substituents. Chlorine (Cl), fluorine (F), bromine
(Br), trifluoromethyl (CF3), and nitro (NO2) groups were
introduced on the phenyl ring as electron-withdrawing
Table 1. Cytotoxicity results (IC50, mM ꢀ SD) of compounds 4a–4p.
N
N
O
R
CF3
N
H
S
Compound
R
PC3
MCF7
SKNMC
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
2-Cl
3-Cl
4-Cl
2-F
3-F
4-F
2-OCH3
3-OCH3
4-OCH3
2-OH
3-OH
2-NO2
4-NO2
2-CH3
4-Br
>300
>300
>300
90 ꢀ 8.13
65 ꢀ 0.97
100 ꢀ 1.45
>300
107 ꢀ 9.50
210 ꢀ 6.69
>300
>300
49.5 ꢀ 6.001
56.2 ꢀ 1.30
>300
>300
>300
>300
90 ꢀ 4.33
63 ꢀ 1.08
>300
>300
>300
>300
140 ꢀ 14.03
105 ꢀ 6.20
70 ꢀ 6.72
80 ꢀ 1.91
61 ꢀ 4.45
>300
240 ꢀ 11.19
>300
119 ꢀ 3.07
80 ꢀ 4.62
>300
>300
>300
100 ꢀ 9.35
125 ꢀ 8.61
>300
>300
>300
>300
152 ꢀ 5.71
>300
>300
38 ꢀ 5.07
4-CF3
–
>300
75 ꢀ 2.34
Doxorubicin
3.7 ꢀ 0.5
7.2 ꢀ 0.4
1 ꢀ 0.03
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

814
L. Hosseinzadeh et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 812–818
Table 2. Properties of compounds 4a–4p.
Molecular
Melting
Compound
R
Molecular formula
weight (g/mol)
Yield (%)
point (°C)
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
2-Cl
3-Cl
4-Cl
2-F
3-F
C11H7ClF3N3OS
C11H7ClF3N3OS
C11H7ClF3N3OS
C11H7F4N3OS
C11H7F4N3OS
C11H7F4N3OS
C12H10F3N3O2S
C12H10F3N3O2S
C12H10F3N3O2S
C11H8F3N3O2S
C11H8F3N3O2S
C11H7F3N4O3S
C11H7F3N4O3S
C12H10F3N3OS
C11H7BrF3N3OS
C12H7F6N3OS
321.7
321.7
321.7
305.3
305.3
305.3
317.3
317.3
317.3
303.3
303.3
322.3
322.3
301.3
366.2
355.3
30
37
48
53
44
43
52
59
49
53
61
43
48
87
48
62
157
152
199
135
130
169
161
140
158–161
142–145
170–173
195
220
140
4-F
2-CH3O
3-CH3O
4-CH3O
2-OH
3-OH
2-NO2
4-NO2
2-CH3
4-Br
196
198
4-CF3
moieties. On the other hand, electron-donating moieties such
as hydroxyl (OH), methoxy (OCH3), and methyl (CH3) were also
substituted to improve the cytotoxic effects. Chlorine moiety
was applied at different positions of the phenyl ring. Chlorine
moiety caused the compounds 4b (IC50 ¼ 49.5 mM) and 4c
(IC50 ¼ 56.2 mM) to exert an acceptable activity against MCF7
cell line. Ortho positioning of the chlorine moiety was quite
detrimental for cytotoxic potency. Replacement of the
chlorine moiety with fluorine atom enhanced the anticancer
activity especially toward SKNMC cell line. Meta positioning of
the fluorine atom was so beneficial for in vitro anticancer
activity as observed in compound 4e. A moderate activity was
rendered with this compound against SKNMC cell line
(IC50 ¼ 63 mM). Probably, the increase in electron-withdrawing
activity of the moiety is beneficial for activity. Utilization of
methoxy group as an electron-donating moiety did not cause
a favorable enhancement in potency. Only compound 4h
(meta methoxy) demonstrated a moderate cytotoxic activity
toward MCF7 cell line (IC50 ¼ 80 mM). Increasing the electron-
donating activity of the corresponding moiety was detrimen-
tal for anticancer activity. In other words, replacement of the
methoxy group with hydroxyl moiety decreased the cytotoxic
effects. Compounds 4j (ortho hydroxyl, IC50 ¼ 140 mM) and 4k
(meta hydroxy, IC50 ¼ 105 mM) showed a weak activity against
SKNMC cell line only. Nitro substituent at positions ortho
(compound 4l) and para (compound 4m) of the phenyl ring
also caused a weak potency against tested compounds.
Introduction of the methyl group (compound 4n) as a weak
electron-donating and also lipophilic moiety increased the
potency against SKNMC cell line (IC50 ¼ 61 mM). In fact,
compound 4n was the most active compound against SKNMC
cell line. Compound 4o with para bromine substituent was the
most potent compound compared to other synthesized
derivatives in this series against PC3 cell line (IC50 ¼ 38 mM).
Compound 4p with para trifluoro (p-CF3) moiety was not so
active against the examined cell lines.
Caspase activity
Compounds 4b and 4c were evaluated by caspase activation
assay in MCF7 cell line. In fact, the ability of these compounds
was explored toward activation of caspases 3, 8, and 9.
According to Fig. 2, a remarkable activation of caspase
enzymes was recorded for both compounds 4b and 4c
especially caspases 3 and 9. Compound 4b (3-Cl) could cause a
better activation of caspase 3 as well as caspase 9. Compounds
4b and 4c did not render a significant activation of caspase 8.
According to the obtained results, it is obvious that
compounds 4b and 4c could be an apoptosis inducer via
intrinsic pathway. The obtained results of investigation about
the activation of caspase 8 implied that compounds 4b and 4c
do not have enough ability to activate caspase 8. In other
words, these compounds could not induce the apoptosis
through the extrinsic pathway.
***
150
100
50
**
Contro l
4-Cl (4c)
3-Cl (4b)
*
**
0
Caspase-3
Caspase-8
Caspase-9
Figure 2. Caspase activation of compounds 4b (3-Cl) and 4c (4-Cl).
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2013, 346, 812–818
1,3,4-Thiadiazole Derivatives as Apoptosis Inducers
815
H
O
O
N
NH2
N
N
+
H2N
a
F
3C
F
C
3
O
F
3C
NH2
S
S
3
2
1
N
N
O
R
b
F
C
3
N
H
Scheme 1. Synthetic pathway of compounds
4a–4p. Reagents and conditions: (a) solvent
free condition, rt, 5 h; (b) phenylacetic acid
derivatives, EDC, HOBt, CH3CN, rt, 24 h.
S
_
4a 4p
Experimental
dimethylaminopropyl carbodiimide (EDC), hydroxybenzotriazole
(HOBt), and appropriate phenylacetic acid derivative were stirred
in acetonitrile (CHCN) for 30 min. Then, equivalent amount of
compound 3 derivative was added and stirring was continued for
24 h. The progress of the reaction was checked using TLC.
After completion, the solvent was evaporated under reduced
pressure and equal portions of water and ethyl acetate were
added to the residue. The aqueous phase was removed and the
organic phase was treated two times with sodium bicarbonate 5%
solution, sulfuric acid 5%, and brine. Anhydrous sodium sulfate
was used for drying and then ethyl acetate was evaporated using
a rotary evaporator apparatus. The obtained product was
triturated by diethyl ether and n-hexane for eliminating the
impurities [30].
Chemistry
All chemical substances such as starter materials, reagents, and
solvents were purchased from commercial suppliers like Merck
and Sigma–Aldrich companies. The purity of the prepared
compounds was proved by thin layer chromatography (TLC)
using various solvents of different polarities. Merck silica gel 60
F254 plates were applied for analytical TLC. Column chromatog-
raphy was performed on Merck silica gel (70–230 mesh) for
purification of intermediate and final compounds. 1H NMR
spectra were recorded using a Varian 400 spectrometer, and
chemical shifts are expressed as d (ppm) with tetramethylsilane
(TMS) as internal standard. The IR spectra were obtained on a
Shimadzu 470 spectrophotometer (potassium bromide disks).
Melting points were determined using an elemental analyzer
apparatus and are uncorrected. The mass spectra were run
on a Finigan TSQ-70 spectrometer (Finigan, USA) at 70 eV. All
cell lines were purchased from Pasteur Institute of Iran. All
intermediate and final compounds were prepared according to
Scheme 1.
2-(2-Chlorophenyl)-N-(5-(trifluoromethyl)-1,3,4-thiadiazol-
2-yl)acetamide (4a)
1H NMR (DMSO-d6, 400 MHz) d (ppm): 4.05 (s, 2H, –CH2–), 7.33
(m, 2H, H4,6-2-chlorophenyl), 7.47 (m, 2H, H3,5-2-chlorophenyl). IR
(KBr, cmꢁ1) ꢀy : 3425 (N–H, stretch), 3143 (C–H, aromatic), 2924
(C–H, asymmetric, aliphatic), 2854 (C–H, symmetric, aliphatic),
–
1697 (C O, stretch), 1539 (N–H, bend), 1192 (C–N, stretch), 1153
–
(stretch, C–F). MS (m/z, %): 323 (Mþþ2, 1), 321 (Mþ, 3), 154 (10), 152
Synthesis of 5-(trifluoromethyl)-1,3,4-thiadiazol-2-amine (3)
In a flat bottom flask, 3 g (0.03 mmol) of thiosemicarbazide was
stirred under solvent-free conditions and 4.6 mL (0.03 mmol) of
trifluoroacetic anhydride was added dropwise via a dropping
(30), 127 (30), 125 (100), 113 (5), 99 (5), 89 (25), 69 (5), 63 (12).
2-(3-Chlorophenyl)-N-(5-(trifluoromethyl)-1,3,4-thiadiazol-
funnel during 5 min. After
a vigorous reaction, refluxing
2-yl)acetamide (4b)
condition was applied for 3 h in an oil bath and the temperature
was adjusted at 60–70°C. Continuation of the reaction was done
at room temperature for 1 h. Work-up procedure was performed
by adding cool water to the reaction medium and following
alkalinization by ammonia solution. Finally, the formed precipi-
tate was filtered and washed with cool water and recrystallized
from ethanol [29].
1H NMR (DMSO-d6, 400 MHz) d (ppm): 3.95 (s, 2H, –CH2–), 7.25 (d,
1H, J ¼ 8 Hz, H5-3-chlorophenyl), 7.37 (m, 2H, H4,6-3-chloro-
phenyl), 7.44 (s, 1H, H2-3-chlorophenyl), 13.33 (br s, NH). IR
(KBr, cmꢁ1) ꢀy : 3425 (N–H, stretch), 3147 (C–H, aromatic), 2927
(C–H, asymmetric, aliphatic), 2858 (C–H, symmetric, aliphatic),
–
–
1701 (C O, stretch), 1543 (N–H, bend), 1485 (C C, aromatic), 1195
–
–
(C–N, stretch), 1153 (stretch, C–F).
M.p. 224°C, yield: 73%, IR (KBr, cmꢁ1) ꢀy 3302 (N–H, stretch,
asymmetric), 3128 (N–H, stretch, symmetric), 2958 (C–H,
asymmetric, aliphatic), 2924 (C–H, asymmetric, aliphatic), 2854
(C–H, symmetric, aliphatic), 1639, 1519 (N–H, bend), 1327, 1192,
1149, 1037, 744, 686, 621. MS (m/z, %): 169 (Mþ, 100), 166 (8), 168
(45), 127 (5), 113 (10), 104 (12), 91 (10), 74 (30), 69 (65), 68 (35), 60
(50), 59 (35).
2-(4-Chlorophenyl)-N-(5-(trifluoromethyl)-1,3,4-thiadiazol-
2-yl)acetamide (4c)
1H NMR (DMSO-d6, 400 MHz) d (ppm): 3.85 (s, 2H, –CH2–), 7.35
(d, 2H, J ¼ 8 Hz,
H
2,6-4-chlorophenyl), 7.41 (d, 2H, J ¼ 8 Hz,
H3,5-4-chlorophenyl). IR (KBr, cmꢁ1) ꢀy : 3410 (N–H, stretch),
3151 (C–H, aromatic), 2924 (C–H, asymmetric, aliphatic), 2858
–
–
General procedure for the synthesis of compounds 4a–4p
All compounds 4a–4p (Table 2) were synthesized according to the
Scheme 1. In a flat bottom flask, equimolar quantities of N-ethyl-N-
(C–H, symmetric, aliphatic), 1701 (C O, stretch), 1545 (N–H,
–
bend), 1492 (C C, aromatic), 1199 (C–N, stretch), 1149 (stretch,
–
C–F).
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

816
L. Hosseinzadeh et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 812–818
–
–
2-(2-Fluorophenyl)-N-(5-(trifluoromethyl)-1,3,4-thiadiazol-
2-yl)acetamide (4d)
stretch), 1543 (C C, aromatic), 1516 (N–H, bend), 1489 (C C,
–
–
aromatic), 1192 (C–N, stretch), 1153 (stretch, C–F). MS (m/z, %): 317
(Mþ, 5), 149 (10), 148 (100), 121 (60), 120 (20), 91 (10), 78 (12), 77
(12), 69 (5), 51 (5).
1H NMR (DMSO-d6, 400 MHz) d (ppm): 3.95 (s, 2H, –CH2–), 7.16–
7.43 (m, 4H, 2-fluorophenyl). IR (KBr, cmꢁ1) ꢀy : 3410 (N–H, stretch),
3155 (C–H, aromatic), 2920 (C–H, asymmetric, aliphatic), 2858
–
(C–H, symmetric, aliphatic), 1708 (C O, stretch), 1550 (N–H,
–
2-(2-Hydroxyphenyl)-N-(5-(trifluoromethyl)-1,3,4-
–
bend), 1492 (C C, aromatic), 1199 (C–N, stretch), 1157 (stretch,
–
thiadiazol-2-yl)acetamide (4j)
C–F). MS (m/z, %): 305 (Mþ, 2), 303 (15), 277 (25), 252 (8), 196 (5), 169
1H NMR (DMSO-d6, 400 MHz) d (ppm): 3.72 (s, 2H, –CH2–), 3.79
(s, 1H, 7.40 (t, 1H, J ¼ 8 Hz, H3-2-hydroxyphenyl), 7.40 (d, 1H,
J ¼ 8 Hz, H3-2-hydroxyphenyl). IR (KBr, cmꢁ1) ꢀy : 3302 (O–H,
stretch), 3128 (C–H, aromatic), 2958 (C–H, asymmetric, aliphatic),
(12), 136 (45), 122 (10), 109 (100), 99 (5), 83 (10), 63 (12), 52 (5).
2-(3-Fluorophenyl)-N-(5-(trifluoromethyl)-1,3,4-thiadiazol-
–
–
–
–
1639 (C O, stretch), 1519 (N–H, bend), 1492 (C C, aromatic), 1192
(C–N, stretch), 1165 (C–F, stretch).
2-yl)acetamide (4e)
1H NMR (DMSO-d6, 400 MHz) d (ppm): 3.97 (s, 2H, –CH2–), 7.09
(t, 1H, J ¼ 8 Hz, H5-3-fluorophenyl), 7.20 (t, 1H, J ¼ 8 Hz, H6-3-
fluorophenyl), 7.42 (m, 2H, H2,4-3-fluorophenyl), 13.56 (br s, NH).
IR (KBr, cmꢁ1) ꢀy : 3414 (N–H, stretch), 3147 (C–H, aromatic), 2927
(C–H, asymmetric, aliphatic), 2858 (C–H, symmetric, aliphatic),
2-(3-Hydroxyphenyl)-N-(5-(trifluoromethyl)-1,3,4-
thiadiazol-2-yl)acetamide (4k)
1H NMR (DMSO-d6, 400 MHz) d (ppm): 3.81 (s, 2H, –CH2–),
6.64–6.76 (m, 4H, 3-hydroxyphenyl), 9.43 (br s, OH), 13.51 (br s,
NH). IR (KBr, cmꢁ1) ꢀy : 3460 (O–H, stretch), 3128 (C–H, aromatic),
–
–
–
1705 (C O, stretch), 1546 (N–H, bend), 1489 (C C, aromatic), 1188
–
–
(C–N, stretch), 1145 (stretch, C–F).
2920 (C–H, asymmetric, aliphatic), 1701 (C O, stretch), 1600
–
–
–
–
–
2-(4-Fluorophenyl)-N-(5-(trifluoromethyl)-1,3,4-thiadiazol-
2-yl)acetamide (4f)
(C C, aromatic), 1546 (N–H, bend), 1489 (C C, aromatic), 1157
(C–F, stretch).
1H NMR (DMSO-d6, 400 MHz) d (ppm): 3.90 (s, 2H, –CH2–), 7.18
(t, 2H, J ¼ 8 Hz, H2,6-4-fluorophenyl), 7.38 (t, 2H, J ¼ 8 Hz, H3,5-4-
fluorophenyl). IR (KBr, cmꢁ1) ꢀy : 3406 (N–H, stretch), 3151 (C–H,
aromatic), 2920 (C–H, asymmetric, aliphatic), 2858 (C–H,
–
2-(2-Nitrophenyl)-N-(5-(trifluoromethyl)-1,3,4-thiadiazol-2-
yl)acetamide (4l)
1H NMR (DMSO-d6, 400 MHz) d (ppm): 4.39 (s, 2H, –CH2–), 7.41
(t, 1H, J ¼ 8 Hz, H4-2-nitrophenyl), 7.54 (t, 1H, J ¼ 8 Hz, H5-2-
nitrophenyl), 7.64 (d, 1H, J ¼ 8 Hz, H6-2-nitrophenyl), 7.72 (d, 1H,
J ¼ 8 Hz, H3-2-nitrophenyl), 13.74 (br s, NH). IR (KBr, cmꢁ1) ꢀy : 3414
(N–H, stretch), 3159 (C–H, aromatic), 2920 (C–H, asymmetric,
aliphatic), 2893 (C–H, symmetric, aliphatic), 2854 (C–H, symmet-
–
symmetric, aliphatic), 1705 (C O, stretch), 1550 (N–H, bend),
–
–
1512 (C C, aromatic), 1199 (C–N, stretch), 1153 (stretch, C–F).
–
2-(2-Methoxyphenyl)-N-(5-(trifluoromethyl)-1,3,4-
thiadiazol-2-yl)acetamide (4g)
1H NMR (DMSO-d6, 400 MHz) d (ppm): 3.74 (s, 3H, OCH3), 3.85
(s, 2H, –CH2–), 6.87–7.00 (m, 2H, 2-methoxyphenyl), 7.23–7.28
(m, 2H, 2-methoxyphenyl). IR (KBr, cmꢁ1) ꢀy : 3410 (N–H, stretch),
3159 (C–H, aromatic), 2939 (C–H, asymmetric, aliphatic), 2855
–
ric, aliphatic), 1701 (C O, stretch), 1550 (N–H, bend), 1523
–
–
–
(stretch, asymmetric, NO ), 1492 (C C, aromatic), 1342 (stretch,
symmetric, NO2), 1195 (C–N, stretch), 1165 (C–F, stretch).
2
(C–H, symmetric, aliphatic), 1712 (C O, stretch), 1546 (N–H,
–
2-(4-Nitrophenyl)-N-(5-(trifluoromethyl)-1,3,4-thiadiazol-2-
yl)acetamide (4m)
–
bend), 1496 (C C, aromatic), 1195 (C–N, stretch), 1161 (stretch,
–
1H NMR (DMSO-d6, 400 MHz) d (ppm): 4.11 (s, 2H, –CH2–), 7.64
(d, 2H, J ¼ 8 Hz, H2,6-4-nitrophenyl), 8.21 (d, 2H, J ¼ 8 Hz, H2,6-4-
nitrophenyl). IR (KBr, cmꢁ1) ꢀy : 3429 (N–H, stretch), 3155 (C–H,
aromatic), 2924 (C–H, asymmetric, aliphatic), 2858 (C–H,
–
C–F).
2-(3-Methoxyphenyl)-N-(5-(trifluoromethyl)-1,3,4-
thiadiazol-2-yl)acetamide (4h)
1H NMR (DMSO-d6, 400 MHz) d (ppm): 3.75 (s, 3H, OCH3), 3.91
(s, 2H, –CH2–), 6.85 (d, 1H, J ¼ 8 Hz, H4-2-methoxyphenyl), 6.92
(d, 1H, J ¼ 8 Hz, H6-2-methoxyphenyl), 6.95 (s, 1H, H2-2-methox-
yphenyl), 7.26 (t, 1H, J ¼ 8Hz, H5-2-methoxyphenyl), 13.59 (br s,
NH). IR (KBr, cmꢁ1) ꢀy : 3398 (N–H, stretch), 3155 (C–H, aromatic),
2927 (C–H, asymmetric, aliphatic), 2889 (C–H, symmetric,
symmetric, aliphatic), 1697 (C O, stretch), 1554 (N–H, bend),
–
–
–
1519 (stretch, asymmetric, NO ), 1492 (C C, aromatic), 1350
2
(stretch, symmetric, NO2), 1199 (C–N, stretch), 1149 (C–F, stretch).
MS (m/z, %): 332 (Mþ, 15), 196 (10), 170 (10), 169 (10), 164 (15), 163
(100), 150 (8), 137 (12), 136 (80), 113 (10), 106 (30), 90 (25), 89 (40),
78 (40), 69 (12), 63 (20), 51 (5).
–
–
aliphatic), 1701 (C O, stretch), 1597 (C C, aromatic), 1550
–
–
–
–
(N–H, bend), 1492 (C C, aromatic), 1489 (C C, aromatic), 1184
–
–
2-o-Tolyl-N-(5-(trifluoromethyl)-1,3,4-thiadiazol-2-yl)-
(C–N, stretch), 1157 (stretch, C–F).
acetamide (4n)
1H NMR (DMSO-d6, 400 MHz) d (ppm): 2.27 (s, 3H, –CH3), 3.95 (s, 2H,
-CH2-), 7.10–7.24 (m, 4H, 2-methylphenyl), 13.52 (br s, NH). IR (KBr,
cmꢁ1) ꢀy : 3429 (N–H, stretch), 3140 (C–H, aromatic), 2924 (C–H,
asymmetric, aliphatic), 2858 (C–H, symmetric, aliphatic), 1697
2-(4-Methoxyphenyl)-N-(5-(trifluoromethyl)-1,3,4-
thiadiazol-2-yl)acetamide (4i)
1H NMR (DMSO-d6, 400 MHz) d (ppm): 3.74 (s, 3H, OCH3), 3.84 (s,
2H, –CH2), 6.91 (d, 2H, J ¼ 8 Hz, H3,5-4-methoxyphenyl), 7.26 (d, 2H,
J ¼ 8 Hz, H3,5-4-methoxyphenyl), 13.41 (br s, NH). IR (KBr, cmꢁ1) ꢀy :
3371 (N–H, stretch), 3147 (C–H, aromatic), 2927 (C–H, asymmet-
–
–
–
–
–
(C O, stretch), 1543 (N–H, bend), 1492 (C C, aromatic), 1192
(C–N, stretch), 1149 (C–F, stretch). MS (m/z, %): 301 (Mþ, 5), 196
(15), 169 (3), 150 (5), 132 (5), 105 (100), 90 (10), 77 (30), 69 (5), 63
(12), 52 (12).
ric, aliphatic), 2854 (C–H, symmetric, aliphatic), 1701 (C O,
–
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2013, 346, 812–818
1,3,4-Thiadiazole Derivatives as Apoptosis Inducers
817
2-(4-Bromophenyl)-N-(5-(trifluoromethyl)-1,3,4-thiadiazol-
2-yl)acetamide (4o)
Detection of caspases 3, 8, and 9 activation
The activity of caspase
3 was determined by the Sigma
1H NMR (DMSO-d6, 400 MHz) d (ppm): 3.92 (s, 2H, –CH2–), 7.31
(d, 2H, J ¼ 8 Hz, H2,6-4-bromophenyl), 7.56 (d, 2H, J ¼ 8 Hz, H3,5-4-
bromophenyl), 13.54 (br s, NH). IR (KBr, cmꢁ1) ꢀy : 3410 (N–H,
stretch), 3151 (C–H, aromatic), 2920 (C–H, asymmetric, aliphatic),
–
colorimetric caspase 3 kit according to the manufacturer’s
instrument. This assay is based on spectrophotometric detection
of the chromophore p-nitroanilide (pNA) after cleavage from the
labeled substrate DEVD-pNA in equal amount of cells protein
lysate. Briefly, 1 ꢂ 106 cells were collected and lysed with 50 mL of
chilled lysis buffer and incubated on ice for 10 min. Cell lysates
were centrifuged at maximum speed for 5 min at 4°C, after which
50 mL of 2ꢂ reaction buffer/DTT mix and 5 mL of 1 mM caspase 3
substrate (DEVD-pNA) were added to each reaction and incubated
at 37°C for 1 h. The pNA light emission was quantified using
a microplate reader at 400 or 405 nm. Comparison of the
absorbance of pNA from an apoptotic sample with an uninduced
control allowed determination of the fold increase in caspase 3
activity. The protein content was determined by the Bio-Rad
protein assay kit using the bovine serum albumin as a standard.
The above stages were also performed for determining the
activation of caspases 8 and 9 [33].
2862 (C–H, symmetric, aliphatic), 1701 (C O, stretch), 1546 (N–H,
–
–
bend), 1489 (C C, aromatic), 1203 (C–N, stretch), 1145 (C–F,
–
stretch). MS (m/z, %): 367 (Mþ2, 5), 365 (Mþ, 5), 198 (95), 196 (100),
171 (85), 169 (90), 150 (5), 136 (5), 113 (10), 90 (55), 77 (5), 69 (10), 63
(30), 52 (5).
N-(5-(Trifluoromethyl)-1,3,4-thiadiazol-2-yl)-2-(4-
(trifluoromethyl)phenyl)acetamide (4p)
1H NMR (DMSO-d6, 400 MHz) d (ppm): 4.08 (s, 2H, –CH2–), 7.58
(d, 2H, J ¼ 8 Hz, H2,6-4-trifluorophenyl), 7.72 (d, 2H, J ¼ 8 Hz, H3,5-4-
trifluorophenyl). IR (KBr, cmꢁ1) ꢀy : 3429 (N–H, stretch), 3155 (C–H,
aromatic), 2924 (C–H, asymmetric, aliphatic), 2862 (C–H,
–
symmetric, aliphatic), 1705 (C O, stretch), 1545 (N–H, bend),
–
–
1492 (C C, aromatic), 1327 (C–N, stretch), 1161 (C–F, stretch). MS
–
The authors appreciate the Research Council of Kermanshah University
of Medical Sciences for the financial supports. This work was performed
in partial fulfillment of the requirement for PharmD of Mr. Amanc
Khorand.
(m/z, %): 355 (Mþ, 10), 196 (3), 186 (45), 167 (5), 159 (100), 150 (3),
119 (5), 109 (15).
Cell culture conditions
PC3 (prostate carcinoma), MCF7 (breast cancer), and SKMNC
(neuroblastoma) cells were obtained from Pasteur Institute
The authors have declared no conflict of interest.
(Tehran, Iran) and maintained at 37°C in
a humidified
atmosphere (90%) containing 5% CO2. PC3, MCF7, and SKNMC
cell lines were cultured in Dulbecco’s modified Eagle’s medium
(DMEM) with 5% v/v fetal bovine serum, 100 U/mL penicillin, and
100 mg/mL streptomycin. Cells were seeded overnight and then
incubated with various concentrations of different derivatives 4a–
4p in a 95% CO2 humidified incubator. The medium was changed
every 2–3 days and subcultured when the cell population density
reached 70–80% confluence. Cells were seeded at an appropriate
density according to each experimental design [31].
References
[1] A. Lawen, Bioessays 2003, 25, 888–896.
[2] G. Kaushik, T. Kaushik, S. Khanduja, C. M. Pathak,
K. Khanduja, Cancer Lett. 2008, 270, 120–131.
[3] T. Albreht, M. Mckee, D. M. Alexe, M. P. Coleman, J. M. Martin-
Moreno, Eur. J. Cancer 2008, 44, 1451–1456.
[4] J. Ferlay, P. Autier, M. Boniol, M. Heanue, M. Colombet,
P. Boyle, Ann. Oncol. 2007, 18, 581–592.
Viability assay
[5] A. Jemal, R. Siegel, E. Ward, T. Murray, J. Xu, C. Smigal,
The cytotoxic effects of compounds 4a–4p were determined
against cell lines by a colorimetric assay using 3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and com-
pared with the untreated control. Cells were plated onto 96-well
plates at a density of 2.0 ꢂ 104 cells/well and in a volume of
200 mL. Stock solutions of synthesized compounds were prepared
in dimethyl sulfoxide (DMSO). The final concentration of the
vehicle in the medium was always 0.5%. One day after seeding,
2 mL of the DMSO containing intended derivative at different
concentrations was added to each well. At appropriate time
intervals, the medium was removed and replaced by 100 mL of
0.5 mg/mL MTT in growth medium and then the plates were
transferred to a 37°C incubator for 3–4 h. Supernatants were
removed and the reduced MTT dye was solubilized with DMSO
(100 mL/well). Absorbance was determined on an ELISA plate
M. J. Thun, Cancer J. Clin. 2006, 56, 106–130.
[6] H. A. Bhuva, S. J. Kini, J. Mol. Graph. Model. 2010, 29, 32–37.
[7] S. Madhusudan, S. Trivadi, T. S. Ganesan, Clin. Biochem. 2004,
37, 618–635.
[8] I. M. Ghobrial, T. E. Witzig, A. A. Adjei, CA Cancer J. Clin. 2005,
55, 178–194.
[9] D. Sunil, A. M. Isloor, P. Shetty, K. Satyamoorthy,
A. S. Bharath Prasad, Arab. J. Chem. 2010, 3, 211–217.
[10] H. Rajak, R. Deshmukh, N. Aggarwal, S. Kashaw,
M. D. Kharya, P. Mishra, Arch. Pharm. Chem. Life Sci. 2009,
342, 453–461.
[11] M. Abdel-Aziz, O. M. Aly, S. S. Khan, K. Mukherjee, S. Bane,
Arch. Pharm. Chem. Life Sci. 2012, 345, 535–548.
[12] X. Q. Deng, Z. Q. Dong, M. X. Song, B. Shu, S. B. Wang,
reader (Biotek, H1M) with
a test wavelength of 570 nm
Z. S. Qua, Arch. Pharm. Chem. Life Sci. 2012, 345, 565–573.
and a reference wavelength of 630 nm to obtain sample signal
(OD570-OD630). Percentage of proliferation was calculated
using the following formula: percent of control proliferation ¼
(OD test/OD control) ꢂ 100. IC50 values were calculated by
plotting the log 10 of the percentage of proliferation versus drug
concentration [32].
[13] S. A. F. Rostom, M. H. Badr, H. A. Abd El Razik, H. M.
A. Ashour, A. E. Abdel Wahab, Arch. Pharm. Chem. Life Sci. 2011,
344, 572–587.
[14] G. Mishra, A. K. Singh, K. Jyoti, Int. J. ChemTech Res. 2011, 3,
1380–1393.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

818
L. Hosseinzadeh et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 812–818
[15] A. K. Singh, G. Mishra, K. Jyoti, J. Appl. Pharm. Sci. 2011, 5,
44–49.
[24] W. Rzeski, J. Matysiakb, M. Kandefer-Szerszen, Bioorg. Med.
Chem. 2007, 15, 3201–3207.
[16] R. Kharb, P. Kaur, P. C. Sharma, M. S. Yar, Int. J. Res. Pharm.
Biomed. Sci. 2011, 4, 1520–1540.
[25] J. Y. Chou, S. Y. Lai, S. L. Pan, G. M. Jow, J. M. Chern, J. H. Guh,
Biochem. Pharmacol. 2003, 66, 115–124.
[17] U. Kalidhar, A. Kaur, Res. J. Pharm. Biol. Chem. 2011, 4, 1091–
[26] D. A. Ibrahim, Eur. J. Med. Chem. 2009, 44, 2776–2781.
1106.
[27] C. T. Supuran, A. Scozzafava, Eur. J. Med. Chem. 2000, 35, 867–
[18] N. Siddiqui, P. Ahuja, W. Ahsan, S. N. Pandeya, M. S. Alam, J.
Chem. Pharm. Res. 2009, 1, 19–30.
874.
[28] X. H. Yang, Q. Wen, T. T. Zhao, J. Sun, X. Li, M. Xing, X. Lu,
[19] S. K. Ardestani, F. Poorrajab, S. Razmi, A. Foroumadi,
S. Ajdary, B. Gharegozlou, M. Behrouzi-Fardmoghadam,
A. Shafiee, Exp. Parasitol. 2012, 32, 116–122.
H. L. Zhu, Bioorg. Med. Chem. 2012, 20, 1181–1187.
[29] I. Lalezari, N. Sharghi, J. Heterocyclic Chem. 1966, 3, 336–338.
[30] A. Aliabadi, F. Shamsa, S. N. Ostad, S. Emami, A. Shafiee,
J. Davoodi, A. Foroumadi, Eur. J. Med. Chem. 2010, 11, 5384–
5389.
[20] D. Kumar, B. Reddy, K. H. Vaddula, K. H. Chang, K. Shah,
Bioorg. Med. Chem. Lett. 2011, 21, 2320–2323.
[21] N. N. Farshori, R. M. Banday, A. Ahmad, A. U. Khan, A. Rauf,
[31] L. Hosseinzadeh, J. Behravan, F. Mosaffa, G. Bahrami,
A. Bahrami, G. Karimi, Food. Chem. Toxicol. 2011, 49, 1102–
1109.
Bioorg. Med. Chem. Lett. 2010, 20, 1933–1938.
[22] J. Matysiak, A. Opolski, Bioorg. Med. Chem. 2006, 14, 4483–
4489.
[32] D. Gerlier, N. Thomasset, J. Immunol. Methods 1986, 94, 57–63.
[33] S. J. Riedl, Y. Shi, Nat. Rev. Mol. Cell. Biol. 2004, 5, 897–907.
[23] M. Yusuf, P. Jain, Arab. J. Chem. 2011, in press.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com