Synthesis and anticholinesterase activity and cytotoxicity of novel amide derivatives

Article abstract of DOI:10.1002/ardp.201100124

In the present study, some amide derivatives were synthesized and their potential anticholinesterase properties were investigated. N-(Benzothiazol-2-yl) -2-[(5-amino/methyl-1,3,4-thiadiazol-2-yl)thio]acetamide derivatives were obtained by nucleophilic substitution of 2-chloro-N-(benzothiazole-2-yl) acetamide derivatives with appropriate 1,3,4-thiadiazole-2-thioles. The chemical structures of the compounds were elucidated by ¹H-NMR, ¹³C-NMR and FAB⁺-MS spectral data and elemental analyses. Each amide derivative was evaluated for its ability to inhibit AChE and BuChE using a modification of Ellman's spectrophotometric method. The compounds were also investigated for their cytotoxic properties using MTT assay. 2-(5-Amino-1,3,4-thiadiazol-2-yl)thio-N-(benzothiazol-2-yl)acetamide derivatives have anticholinesterase activity, whereas 2-(5-methyl-1,3,4-thiadiazol-2-yl) thio-N-(benzothiazol-2-yl)acetamide derivatives have no inhibitory effect on enzyme activity. Among these compounds, it is clear that compound IIh is the most potent derivative. Among these new compounds, compound IIh is the most potent inhibitor of AChE and BuChE with an IC₅₀ value of 66±0.71μg/mL and 48.75±2.48μg/mL, respectively.

Full text of DOI:10.1002/ardp.201100124

112
Arch. Pharm. Chem. Life Sci. 2012, 345, 112–116
Full Paper
Synthesis and Anticholinesterase Activity and Cytotoxicity
of Novel Amide Derivatives
Mehlika Dilek Altintop1, Zafer Asim Kaplancikli1, Ahmet Ozdemir1, Gu¨lhan Turan-Zitouni1,
Halide Edip Temel2, and Gu¨ls¸en Akalın2
1 Anadolu University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Eskis¸ehir, Turkey
2 Anadolu University, Faculty of Pharmacy, Department of Biochemistry, Eskis¸ehir, Turkey
In the present study, some amide derivatives were synthesized and their potential anticholinesterase
properties were investigated. N-(Benzothiazol-2-yl)-2-[(5-amino/methyl-1,3,4-thiadiazol-2-yl)thio]acetamide
derivatives were obtained by nucleophilic substitution of 2-chloro-N-(benzothiazole-2-yl)acetamide
derivatives with appropriate 1,3,4-thiadiazole-2-thioles. The chemical structures of the compounds
were elucidated by 1H-NMR, 13C-NMR and FABþ-MS spectral data and elemental analyses. Each amide
derivative was evaluated for its ability to inhibit AChE and BuChE using a modification of Ellman’s
spectrophotometric method. The compounds were also investigated for their cytotoxic properties
using MTT assay. 2-(5-Amino-1,3,4-thiadiazol-2-yl)thio-N-(benzothiazol-2-yl)acetamide derivatives have
anticholinesterase
activity,
whereas
2-(5-methyl-1,3,4-thiadiazol-2-yl)thio-N-(benzothiazol-2-
yl)acetamide derivatives have no inhibitory effect on enzyme activity. Among these compounds, it
is clear that compound IIh is the most potent derivative.
Keywords: Amide / Anticholinesterase activity / Cytotoxicity / Thiadiazole
Received: April 5, 2011; Revised: September 5, 2011; Accepted: September 5, 2011
DOI 10.1002/ardp.201100124
Introduction
Acetylcholine, which is the physiologic cholinergic neuro-
transmitter, is a poor therapeutic agent due to its fleeting
pharmacological action and chemical instability as a result of
the rapid hydrolysis catalysed by cholinesterases [10].
Two cholinesterases are present in humans: acetylcholin-
esterase (AChE), which selectively hydrolyses acetylcholine,
and butyrylcholinesterase (BuChE), which is a non-specific
cholinesterase. The main difference between the two types of
cholinesterase is the respective preferences for substrates: the
former hydrolyses acetylcholine more quickly; the latter
hydrolyses butyrylcholine more quickly. The main function
of acetylcholinesterase is the termination of cholinergic
neurotransmission, but the function of butyrylcholinester-
ase is not so clear [9, 10].
Alzheimer’s disease is the most common age-related neuro-
degenerative disorder characterized by progressive deficits in
memory and cognition, together with impairment in the
ability to perform basic activities of daily living.
Alzheimer’s disease has become an urgent public health
problem due to the prevalence of Alzheimer’s disease world-
wide [1–9].
Over the last 20 years, scientists have carried out consider-
able research for deciphering the underlying mechanisms of
this devastating disease. Substantial evidence demonstrates
that Alzheimer’s disease is associated with the deficiency in
cholinergic neurotransmission and therefore current
research interest in the design and synthesis of new choli-
nomimetics is focused on discovering agents that might be
effective in the treatment of Alzheimer’s disease [1–9].
Cholinomimetics can be divided into two groups: direct
cholinomimetics and indirect cholinomimetics. Direct chol-
inomimetics act directly on cholinergic receptors, whereas
indirect cholinomimetics act as inhibitors of acetylcholin-
esterase [10].
Correspondence: Zafer Asim Kaplancikli, Anadolu University, Faculty of
Pharmacy, 26470 Eskis¸ehir, Turkey.
E-mail: zakaplan@anadolu.edu.tr
Fax: þ90-222-3350750
Acetylcholinesterase inhibitors (AChEIs), which are classi-
fied as indirect cholinomimetics, have found use in the treat-
ment of symptoms of Alzheimer’s disease [10].
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2012, 345, 112–116
Anticholinesterase Activity of Novel Amide Derivatives
113
Carbamates, which are the most widely studied class of
anticholinesterase agents, have attracted a great deal of
interest among medicinal chemists and considerable
research on them in relation to Alzheimer’s disease has been
accomplished. Rivastigmine, which is the dual AChE and
thiazole and thiadiazole and focused on the potential anti-
cholinesterase properties of these compounds. The prepared
compounds were also investigated for their cytotoxic
properties.
BuChE inhibitor, is one of the most widely used anticholin- Results and discussion
esterase agents bearing carbamate group [1–10].
Many researchers have also investigated amides exten-
sively due to the fact that new effective compounds can be
obtained by the bioisosteric replacement of carbamate group
with amide group. Some studies have confirmed that amide
derivatives possess anticholinesterase activity [11–14].
Acetylcholine undergoes rapid hydrolysis in the gastroin-
testinal tract associated with its ester and quaternary
ammonium salt functional groups and therefore acetyl-
choline cannot be administered orally [10]. In order to syn-
thesize more metabolically stable cholinomimetic ligands, it
is possible to replace the ester group with a series of five-
membered rings like oxadiazoles, thiadiazoles, triazoles and
tetrazoles [12]. Some researchers have studied thiadiazoles
extensively and compounds bearing thiadiazole moiety have
been reported to exhibit significant anticholinesterase
activity [15–17].
Initially, 2-chloro-N-(benzothiazol-2-yl)acetamides (Ia–e) were
obtained by reacting 2-aminobenzothiazoles with chloroace-
tyl chloride in the presence of triethylamine, which was
responsible for the removal of hydrogen chloride from the
reaction mixture.
The desired compounds (IIa–j) were synthesized via nucle-
ophilic substitution reaction of 2-chloro-N-(benzothiazol-2-yl)
acetamides with appropriate 1,3,4-thiadiazole-2-thioles in
the presence of potassium carbonate. These reactions are
summarized in Scheme 1 and some properties of the com-
pounds are given in Table 1.
The structures of these compounds (IIa–j) were confirmed
by 1H-NMR, 13C-NMR and FABþ-MS spectral data and
elemental analyses. In the 1H-NMR spectra of the synthesized
compounds, the signal due to the amide proton appears at
12–13 ppm as a broad peak. The signal due to the –S–CH2–
protons gives rise to a singlet peak at 4.0–4.5 ppm. The
benzothiazole protons of all derivatives are observed in the
In this study, we described the synthesis of some amide
derivatives bearing two functional moieties, namely benzo-
R
R
H
S
R
O
S
N
N(C2H5)3
N
+
+
Cl
NH2
Cl
Cl
N
O
Ia-e
N
N
N
N
H
N
K2CO3
S
N
R'
I
S
S
HS
R'
S
O
IIa-j
Scheme 1. The synthetic protocol of the title compounds (IIa–j).
Table 1. Some properties of the synthesized compounds (IIa–j)
Compound
R
R0
Yield (%)
M.p. (8C)
Molecular formula
Molecular weight
IIa
IIb
IIc
IId
IIe
IIf
IIg
IIh
IIi
H
Cl
CH3
OCH3
OC2H5
H
CH3
CH3
CH3
CH3
CH3
NH2
NH2
NH2
NH2
NH2
80
85
82
84
84
85
90
85
88
88
228–232
258–261
252–255
244–248
245–246
250–253
277–279
248–250
260–262
254–255
C12H10N4OS3
C12H9ClN4OS3
C13H12N4OS3
C13H12N4O2S3
C14H14N4O2S3
C11H9N5OS3
C11H8ClN5OS3
C12H11N5OS3
C12H11N5O2S3
C13H13N5O2S3
322
356
336
352
366
323
357
337
353
367
Cl
CH3
OCH3
OC2H5
IIj
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M. D. Altintop et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 112–116
Table 2. The anticholinesterase activities of the compounds (IIa–j) as IC50 values (mg/mL)
Compound
AChE
80 mg/mL (% inhibition)
BuChE
IC50 mg/mL
80 mg/mL (% inhibition)
IC50 mg/mL
IIf
IIg
IIh
IIi
29,35 ꢀ 0,28
76,23 ꢀ 1,67
84,65 ꢀ 2,97
70,53 ꢀ 2,60
45,21 ꢀ 2,08
>80
70,33 ꢀ 0,35
66 ꢀ 0,71
6,98 ꢀ 4,09
60,34 ꢀ 5,19
90,24 ꢀ 1,17
26,47 ꢀ 2,50
13,46 ꢀ 2,87
>80
72,50 ꢀ 0,71
48,75 ꢀ 2,48
>80
72,75 ꢀ 0,35
>80
IIj
Eserine
Galanthamine
>80
4,7 ꢁ 10ꢂ2 ꢀ 0,08
6,3 ꢁ 10ꢂ3 ꢀ 0,03
0,28 ꢀ 0,04
7 ꢀ 1,41
region 7.0–8.5 ppm. In the 1H-NMR spectra of the compounds
IIf–j the signal due to the amine protons appears at 7.35–
7.38 ppm as a broad peak. Other aromatic and aliphatic
protons were observed at expected regions.
have a considerable influence on anticholinesterase activity.
When compared with reference drugs and other derivatives
(IIg–IIj), compound IIf exhibited the lowest anticholinester-
ase activity.
In their 13C-NMR spectra, the signal due to the amide
carbon is observed at 165–170 ppm as a proof of the for-
mation of amide moiety. The signal due to the –S–CH2–
carbon appears at 36–38 ppm.
In the mass spectra of all compounds, the [MþH]þ peak is
observed. All compounds gave satisfactory elemental
analysis.
The compounds were also evaluated for their cytotoxic
properties using MTT assay (Table 3). The biological study
indicated that compound IIf possessed the highest
cytotoxicity, whereas compound IIi exhibited the lowest
cytotoxicity in vitro against NIH/3T3 cells among the title
compounds.
The anticholinesterase activities of the compounds were Conclusion
determined by a modification of Ellman’s spectrophotomet-
ric method (Table 2). All compounds showed less anticholin-
esterase potency than reference drugs.
In conclusion, we synthesized some amide derivatives and
evaluated their anticholinesterase activities and cytotoxic
properties. The results indicate that compound IIh is the
most potent inhibitor of AChE and BuChE among these
compounds. Furthermore, the cytotoxicity of compound
IIh is lower than its effective dose (66 ꢀ 0.71 mg/mL).
This outcome indicates that the amino group on thiadiazole
ring and the methyl substituent on benzothiazole ring
have a considerable influence on the ability to inhibit
cholinesterases.
5-Amino-1,3,4-thiadiazole derivatives (IIf–j) have anticholin-
esterase activity, whereas 5-methyl-1,3,4-thiadiazole deriva-
tives (IIa–e) have no inhibitory effect on enzyme activity.
The results indicate that compound IIh is the most effec-
tive inhibitor of AChE and BuChE among these compounds.
This outcome confirms that the amino group on thiadiazole
ring and the methyl substituent on benzothiazole ring may
Table 3. In vitro cytotoxicity of the compounds (IIa–j)
Experimental
Compound
IC50 (mg/mL)a
Chemistry
IIa
IIb
IIc
IId
IIe
IIf
IIg
IIh
IIi
60 ꢀ 10
76,7 ꢀ 5,8
45,7 ꢀ 14,0
80 ꢀ 10
All reagents were purchased from commercial suppliers and
were used without further purification. Melting points (m.p.)
were determined on a Electrothermal 9100 melting point appar-
atus (Weiss-Gallenkamp, Loughborough, UK) and are uncor-
rected. Proton nuclear magnetic resonance (1H-NMR) spectra
were recorded on a Bruker 400 MHz spectrometer (Bruker,
Billerica, MA, USA). Carbon nuclear magnetic resonance
66,6 ꢀ 15,27
33,3 ꢀ 5,8
63,3 ꢀ 5,7
70 ꢀ 8,7
90 ꢀ 10
(
13C-NMR) spectra were recorded on a Bruker 100 MHz spec-
IIj
70 ꢀ 10
trometer (Bruker, Billerica, MA, USA). Chemical shifts were
expressed in parts per million (ppm) and tetramethylsilane
was used as an internal standard. Mass spectra were recorded
on a VG Quattro Mass spectrometer (Agilent, Minnesota, USA).
Elemental analyses were performed on a Perkin Elmer EAL 240
elemental analyser (Perkin-Elmer, Norwalk, CT, USA).
a
Cytotoxicity of compounds to mouse fibroblast (NIH/3T3) cell
line. Incubation for 24 h. IC50 is the drug concentration required
to inhibit 50% of the cell growth. The values represent
mean ꢀ standard deviation of triplicate determinations.
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Arch. Pharm. Chem. Life Sci. 2012, 345, 112–116
Anticholinesterase Activity of Novel Amide Derivatives
115
133.65 (C), 141.20 (C), 149.95 (C), 156.29 (C), 157.21 (C), 167.42
(C), 169.99 (C).
General procedure for the synthesis of the compounds
N-(Benzothiazol-2-yl)-2-chloroacetamides (Ia–e)
Chloroacetyl chloride (0,1 mol) was added dropwise with stirring
to a mixture of 2-aminobenzothiazole derivatives (0,1 mol) and
triethylamine (0,1 mol) in toluene (50 mL) at 0–58C. The solvent
was evaporated under reduced pressure. The residue was washed
with water to remove triethylamine hydrochloride and crystal-
lized from ethanol [18].
For C13H12N4O2S3 calculated: C, 44.30; H, 3.43; N, 15.90; found:
C, 44.29; H, 3.42; N, 15.91.
MS (FAB) [Mꢀ1]þ: m/z 353
2-(5-Methyl-1,3,4-thiadiazol-2-yl)thio-N-
(6-ethoxybenzothiazol-2-yl)acetamide (IIe)
1H NMR (400 MHz, DMSO-d6) d (ppm): 1.36 (3H, t, J ¼ 7.1 Hz), 2.72
(3H, s), 4.06 (2H, q, J ¼ 7.1 Hz), 4.51 (2H, s), 7.05 (1H, dd, J ¼ 8.8,
1.8 Hz), 7.63 (1H, d, J ¼ 1.8 Hz), 7.72 (1H, d, J ¼ 8.8 Hz), 12.60
(1H, br).
N-(Benzothiazol-2-yl)-2-[(5-amino/methyl-1,3,4-thiadiazol-
2-yl)thio]acetamide derivatives (IIa–j)
A mixture of N-(benzothiazol-2-yl)-2-chloroacetamide derivatives
(Ia–e) (2 mmol) and 5-amino/methyl-1,3,4-thiadiazole-2-thiole
(2 mmol) in acetone (10 mL) was stirred at room temperature
for 8 hours in the presence of potassium carbonate (2 mmol) and
filtered. The residue was washed with water and crystallized
from ethanol.
13C NMR (100 MHz, DMSO-d6) d (ppm): 14.60 (CH3), 15.18 (CH3),
37.45 (CH2), 63.66 (CH2), 105.95 (CH), 114.36 (CH), 122.25 (CH),
134.64 (C), 143.11 (C), 147.12 (C), 155.45 (C), 155.60 (C), 166.75 (C),
169.90 (C).
For C14H14N4O2S3 calculated: C, 45.88; H, 3.85; N, 15.29; found:
C, 45.84; H, 3.88; N, 15.30.
MS (FAB) [Mþ1]þ: m/z 367
2-(5-Methyl-1,3,4-thiadiazol-2-yl)thio-N-(benzothiazol-2-yl)-
acetamide (IIa)
2-(5-Amino-1,3,4-thiadiazol-2-yl)thio-N-
1H NMR (400 MHz, DMSO-d6) d (ppm): 2.67 (3H, s), 4.44 (2H, s), 7.32
(1H, t, J ¼ 7.7 Hz), 7.45 (1H, t, J ¼ 7.7 Hz), 7.77 (1H, d, J ¼ 7.7 Hz),
7.98 (1H, d, J ¼ 7.7 Hz), 12.75 (1H, br).
(benzothiazol-2-yl)acetamide (IIf)
1H NMR (400 MHz, DMSO-d6) d (ppm): 4.21 (2H, s), 7.33 (1H, t,
J ¼ 7.8 Hz), 7.37 (2H, br), 7.48 (1H, t, J ¼ 7.8 Hz), 7.83 (1H, d,
J ¼ 7.8 Hz), 8.02 (1H, d, J ¼ 7.8 Hz), 12.71 (1H, br).
13C NMR (100 MHz, DMSO-d6) d (ppm): 36.80 (CH2), 105.48 (CH),
114.90 (CH), 122.14 (CH), 131.96 (CH), 141.92 (C), 147.15 (C),
154.61 (C), 155.82 (C), 167.02 (C), 169.85 (C).
13C NMR (100 MHz, DMSO-d6) d (ppm): 15.17 (CH3), 36.80 (CH2),
120.66 (CH), 121.74 (CH), 123.68 (CH), 126.18 (CH), 131.42 (C),
148.47 (C), 157.65 (C), 163.80 (C), 165.91 (C), 166.74 (C).
For C12H10N4OS3 calculated: C, 44.70; H, 3.13; N, 17.38; found:
C, 44.72; H, 3.12; N, 17.36.
MS (FAB) [Mþ1]þ: m/z 323
For C11H9N5OS3 calculated: C, 40.85; H, 2.80; N, 21.65; found:
C, 40.88; H, 2.84; N, 21.63.
MS (FAB) [Mꢀ1]þ: m/z 324
2-(5-Methyl-1,3,4-thiadiazol-2-yl)thio-N-
(6-chlorobenzothiazol-2-yl)acetamide (IIb)
1H NMR (400 MHz, DMSO-d6) d (ppm): 2.67 (3H, s), 4.43 (2H, s), 7.47
(1H, dd, J ¼ 8.6, 2.1 Hz), 7.77 (1H, t, J ¼ 8.6 Hz), 8.14 (1H, d,
J ¼ 2.1 Hz), 12.83 (1H, br).
2-(5-Amino-1,3,4-thiadiazol-2-yl)thio-N-
(6-chlorobenzothiazol-2-yl)acetamide (IIg)
1H NMR (400 MHz, DMSO-d6) d (ppm): 4.21 (2H, s), 7.26 (1H, dd,
J ¼ 8.5, 2.0 Hz), 7.37 (2H, br), 7.60 (1H, t, J ¼ 8.5 Hz), 7.75 (1H, d,
J ¼ 2.0 Hz), 12.62 (1H, br).
13C NMR (100 MHz, DMSO-d6) d (ppm): 15.17 (CH3), 36.72 (CH2),
121.49 (CH), 121.88 (CH), 126.55 (CH), 127.74 (C), 133.12 (C),
147.37 (C), 158.52 (C), 163.74 (C), 165.96 (C), 166.99 (C).
For C12H9ClN4OS3 calculated: C, 40.39; H, 2.54; N, 15.70; found:
C, 40.40; H, 2.56; N, 15.69.
13C NMR (100 MHz, DMSO-d6) d (ppm): 36.82 (CH2), 122.09 (CH),
122.90 (CH), 126.75 (CH), 127.82 (C), 133.42 (C), 148.07 (C), 158.90
(C), 163.68 (C), 166.06 (C), 167.05 (C).
MS (FAB) [Mꢀ1]þ: m/z 357
For C11H8ClN5OS3 calculated: C, 36.92; H, 2.25; N, 19.57; found:
C, 36.90; H, 2.28; N, 19.60.
MS (FAB) [Mꢀ1]þ: m/z 358
2-(5-Methyl-1,3,4-thiadiazol-2-yl)thio-N-
(6-methylbenzothiazol-2-yl)acetamide (IIc)
1H NMR (400 MHz, DMSO-d6) d (ppm): 2.49 (3H, s), 2.71 (3H, s), 4.48
(2H, s), 7.51 (1H, dd, J ¼ 8.6, 2.1 Hz), 7.71 (1H, t, J ¼ 8.6 Hz), 8.20
(1H, d, J ¼ 2.1 Hz), 12.89 (1H, br).
2-(5-Amino-1,3,4-thiadiazol-2-yl)thio-N-
(6-methylbenzothiazol-2-yl)acetamide (IIh)
1H NMR (400 MHz, DMSO-d6) d (ppm): 2.45 (3H, s), 4.16 (2H, s), 7.09
(1H, dd, J ¼ 8.7, 1.9 Hz), 7.38 (2H, br), 7.61 (1H, d, J ¼ 1.9 Hz), 7.69
(1H, d, J ¼ 8.7 Hz), 12.51 (1H, br).
13C NMR (100 MHz, DMSO-d6) d (ppm): 15.11 (CH3), 20.99 (CH3),
36.60 (CH2), 121.89 (CH), 122.11 (CH), 127.15 (CH), 127.99 (C),
134.01 (C), 148.02 (C), 159.02 (C), 164.02 (C), 166.52 (C), 167.52 (C).
For C13H12N4OS3 calculated: C, 46.41; H, 3.59; N, 16.65; found:
C, 46.40; H, 3.60; N, 16.64.
13C NMR (100 MHz, DMSO-d6) d (ppm): 21.10 (CH3), 37.11 (CH2),
105.48 (CH), 115.21 (CH), 122.01 (CH), 131.99 (C), 141.95 (C),
147.12 (C), 156.05 (C), 156.99 (C), 167.12 (C), 169.12 (C).
For C12H11N5OS3 calculated: C, 42.71; H, 3.29; N, 20.75; found:
C, 42.70; H, 3.30; N, 20.72.
MS (FAB) [Mꢀ1]þ: m/z 337
MS (FAB) [Mꢀ1]þ: m/z 338
2-(5-Methyl-1,3,4-thiadiazol-2-yl)thio-N-
(6-methoxybenzothiazol-2-yl)acetamide (IId) [19]
1H NMR (400 MHz, DMSO-d6) d (ppm): 2.70 (3H, s), 3.83 (3H, s), 4.49
(2H, s), 7.00 (1H, dd, J ¼ 8.7, 1.8 Hz), 7.62 (1H, d, J ¼ 1.8 Hz), 7.69
(1H, d, J ¼ 8.7 Hz), 12.53 (1H, br).
2-(5-Amino-1,3,4-thiadiazol-2-yl)thio-N-(6-
methoxybenzothiazol-2-yl)acetamide (IIi) [19]
1H NMR (400 MHz, DMSO-d6) d (ppm): 3.81 (3H, s), 4.14 (2H, s), 7.04
(1H, dd, J ¼ 8.8, 1.9 Hz), 7.35 (2H, br), 7.58 (1H, d, J ¼ 1.9 Hz), 7.65
(1H, d, J ¼ 8.8 Hz), 12.50 (1H, br).
13C NMR (100 MHz, DMSO-d6) d (ppm): 15.16 (CH3), 36.80
(CH2), 55.99 (CH3), 105.85 (CH), 114.21 (CH), 122.45 (CH),
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M. D. Altintop et al.
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13C NMR (100 MHz, DMSO-d6) d (ppm): 37.40 (CH2), 55.59 (CH3),
104.68 (CH), 115.00 (CH), 121.24 (CH), 132.76 (C), 142.52 (C),
148.75 (C), 155.59 (C), 156.20 (C), 166.95 (C), 170.09 (C).
For C12H11N5O2S3 calculated: C, 40.78; H, 3.14; N, 19.81; found:
C, 40.75; H, 3.18; N, 19.79.
MTT assay for cytotoxicity of the compounds
It is widely used as a measure of cytotoxicity. After 24 h of
preincubation, the tested compounds were added to give final
concentration in the range 0.5–500 mg/mL and the cells were
incubated for 24 h. At the end of this period, MTT was added to a
final concentration of 0.5 mg/mL and the cells were incubated
for 4 h at 378C. After the medium was removed, the formazan
crystals formed by MTT metabolism were solubilized by addition
of 200 mL DMSO to each well and absorbance was read at 540 nm
with a microtitre plate spectrophotometer (Bio-Tek plate reader).
Every concentration was repeated in three wells and IC50 values
were defined as the drug concentrations that reduced absorb-
ance to 50% of control values.
MS (FAB) [Mþ1]þ: m/z 354
2-(5-Amino-1,3,4-thiadiazol-2-yl)thio-N-
(6-ethoxybenzothiazol-2-yl)acetamide (IIj)
1H NMR (400 MHz, DMSO-d6) d (ppm): 1.34 (3H, t, J ¼ 7.0 Hz), 4.05
(2H, q, J ¼ 7.0 Hz), 4.14 (2H, s), 7.02 (1H, dd, J ¼ 8.8, 2.6 Hz), 7.36 (2H,
br), 7.54 (1H, d, J ¼ 2.6 Hz), 7.64 (1H, d, J ¼ 8.8 Hz), 12.48 (1H, br).
13C NMR (100 MHz, DMSO-d6) d (ppm): 14.65 (CH3), 37.41 (CH2),
63.56 (CH2), 105.30 (CH), 115.33 (CH), 121.23 (CH), 132.74 (C),
142.44 (C), 148.73 (C), 155.43 (C), 155.55 (C), 166.93 (C), 170.10 (C).
For C13H13N5O2S3 calculated: C, 42.49; H, 3.57; N, 19.06; found:
C, 42.48; H, 3.56; N, 19.05.
The authors have declared no conflict of interest.
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