Synthesis and antioxidant and antitumor activity of novel pyridine, chromene, thiophene and thiazole derivatives

Article abstract of DOI:10.1002/ardp.201100335

2-Tosylacetonitrile (1) when reacted with α,β-unsaturated nitriles 2a-c or a mixture of formaldehyde and 3-amino-2-substituted-pent-2- endinitriles 6a,b yielded pyridine derivatives 3a-c and 9a,b, respectively, while when subjected to react with salicylal

Full text of DOI:10.1002/ardp.201100335

Arch. Pharm. Chem. Life Sci. 2011, 000, 1–8
1
Full Paper
Synthesis and Antioxidant and Antitumor Activity of Novel
Pyridine, Chromene, Thiophene and Thiazole Derivatives
Ahmed A. Fadda, Moged A. Berghot, Fathy A. Amer, Doria S. Badawy, and Nesma M. Bayoumy
Department of Chemistry, Faculty of Science, Mansoura University, Mansoura, Egypt
2-Tosylacetonitrile (1) when reacted with a,b-unsaturated nitriles 2a–c or a mixture of formaldehyde
and 3-amino-2-substituted-pent-2-endinitriles 6a,b yielded pyridine derivatives 3a–c and 9a,b,
respectively, while when subjected to react with salicylaldehyde yielded chromene derivatives 4
and 5, subsequently. The behavior of thiocarbamoyl derivative 10 derived from 1 towards some a-
halogenated compounds have been investigated as well as its behavior towards elemental sulfur and
phenyl isothiocyanate. Newly synthesized compounds were screened for their antioxidant activity,
erythrocytes haemolysis and bleomycin-independent DNA damage. Some of the tested compounds
exhibited promising activities.
Keywords: 2-Tosylacetonitrile / Arylidenes / Antioxidant and antitumor activities / a-Halo compounds / Thiocarbamoyl
Received: September 22, 2011; Revised: October 18, 2011; Accepted: October 21, 2011
DOI 10.1002/ardp.201100335
Introduction
[6–8]. Several new approaches for the synthesis of five and six
membered rings and their fused heterocyclic derivatives have
been developed during this work [9, 10]. In the present work
we explore the synthetic potential of 2-tosylacetonitrile (1) to
form poly-substituted pyridines and coumarin heterocycles
via the reaction of 1 with a,b-unsaturated nitriles 2a–c or
salicylaldehyde, respectively. Thus, compound 1 reacted
under triethylamine (TEA) catalysis with a,b-unsaturated
nitriles 2a–c in refluxing ethanol to afford 6-amino-4-substi-
tuted-5-tosylnicotinonitriles 3a–c [11]. The IR spectra of these
products showed in each case absorption bands at y 3337,
This work is the continuation of a program aiming to develop
new simple methods for the synthesis of functionally sub-
stituted heterocycles with anticipated biological activity. We
have recently extended it to include the investigation of the
pharmacological aspects of the newly synthesized hetero-
cycles based on the finding that some heterocycles can
achieve activity in both the pharmacological and pesticidal
areas, e.g. thiabenzdazole, the well known human and vet-
erinary anthelmintic, is also used as a fungicide [1, 2].
2257 and 1610 cmꢀ1 corresponding to NH , CN and C N
–
–
2
Results and discussion
group, respectively. Structures 3a–c were assigned to these
products on the basis of the spectral as well as analytical data.
The 1H-NMR spectrum of 3a revealed a broad signal at d
6.5 ppm assigned to the amine protons and a multiplet at
d 7.8–8.4 ppm assigned to the pyridine-2H and aromatic
protons.
Chemistry
Pyridines and coumarin derivatives have recently received
considerable attention due to their synthetic and pharma-
ceutical importance and different approaches for their syn-
thesis have been developed [3–5]. In the last few years the
authors have been exploring the synthetic potential, scope
and limitations of activated nitriles in heterocyclic synthesis
Compound 1 reacted with salicylaldehyde in refluxing
ethanol catalyzed by TEA to give 3-tosyl-2H-chromen-2-imine
(4) in high yield. Mass spectral measurements and analytical
data were in complete agreement with structure 4 which
showed the molecular ion peak at m/z 300 (Mþþ1). Moreover,
the resulting coumarin derivatives have latent functional
substituents which have the potential for further chemical
transformations giving new routes for the preparation of
substituted coumarin derivatives with possible biological
Correspondence: Prof. Ahmed A. Fadda, Department of Chemistry,
Faculty of Science, Mansoura University, El-Gomhoria Street, Mansoura,
35516, Egypt.
E-mail: afadda2@yahoo.com
Fax: þ20502246781
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A. A. Fadda et al.
Arch. Pharm. Chem. Life Sci. 2011, 000, 1–8
H3C
activity. 3-Tosyl-2H-chromen-2-one (5) was synthesized via
hydrolysis of 4 in a mixture of conc. hydrochloric acid and
ethanol (Scheme 1). The mass spectrum of 5 showed the
molecular ion peak at m/z 300 (Mþ) which is in agreement
with the expected molecular formula C16H12O4S.
CN
O
H2N
S
CN
EtOH/ TEA
R
NH2
R
1
+ CH2O +
O
CN
CN
6a,b
CN
H+
7a,b
_
Aldehydes condense with active methylene nitriles to yield
the corresponding ylidene derivatives [12]. The reaction of
these ylidenes with active methylene and active methyl
reagents has been extensively utilized for the synthesis
of 4-pyrans, pyridines and benzene derivatives. Although
a-functionally substituted acrylonitriles are expected to
react similarly with active methylene reagents affording
substituted pyran, pyridine and benzene derivatives,
reactions of this type have not been reported, to our
knowledge, with the exception of our recent report [13].
The difficulty of preparing a-functionally substituted
acrylonitriles might explain the lack of reports on their
utility. In the present paper we report the results of our work
aiming at an investigation of the synthetic potentialities of
this kind of reagent and related functionally substituted
acrylonitriles.
+H+
H3C
H3C
NH
NH2
N
O
O
S
O
S
O
NH
CN
CN
CN
R
CN
R
8a,b
H3C
NH2
O
S
O
a, R = CN
b, R =
N
N
S
CN
CN
9a,b
R
Scheme 2. Synthesis of 6-amino-2-(substituted (cyano)methyl)-
5-tosylnicotine-nitriles 9a,b.
It was found that compound 1 reacts with a mixture of
formaldehyde and 3-aminoprop-1-en-1,1,3-tricarbonitriles
6a,b in boiling ethanol containing a catalytic amount of
TEA to give 6-amino-2-(substituted (cyano)methyl)-5-tosylnico-
tinonitriles 9a,b. Structure 9a was established by elemental
and spectroscopic analysis. The IR spectrum of 9a
showed absorption bands at y 3450, 2207 and 1628 cmꢀ1
the product 9b was established as described above for 9a
(Scheme 2).
Previously, we have investigated the reaction of phenyl
isothiocyanate with active methylene compounds in alkaline
medium, which has been proved to be a convenient route for
the synthesis of thiazole, pyrazole, oxazine and pyrimidine
ring systems [14]. Now, we have extended our synthetic pro-
gram to the synthesis of otherwise inaccessible heterocyclic
ring systems utilizing phenyl isothiocyanate as a key starting
material. It is known that a great variety of reactants bearing
–
corresponding to NH , CN and C N groups, respectively.
2
–
The mass spectroscopic and analytical data were in
agreement with structure 9a (c.f. Experimental Part).
The formation of 9a is assumed to proceed via in situ
formation of the methylene derivative of the nitrile 1 which
was added to the dimer 6a yielding the intermediate
Michael adduct 7. These were spontaneously cyclized to
the intermediates 8a,b giving the sensitive product 9a.
In the same way, 1 can react with a mixture of formaldehyde
and 6b to yield the pyridine derivative 9b. The structure of
–
–
–
the N C S fragment undergo cyclization on reaction with
–
a-halocarbonyl compounds to afford thiazoles, 2,3-dihydro-
thiazoles and thiazolidines [15], which have been shown to
exhibit local anaesthetic [16], antiprotozoal [17] and fungici-
dal properties [18]. In this paper, we describe a generally
applicable extension of this synthetic approach, first
H3C
CN
CN
O
S
Ar
N
Ar
CN
_
O
2a c
a, Ar = C6H2-2,3,4-OCH3
b, Ar = C6H5
c, Ar = p-N(CH3)2-C6H4
EtOH/ TEA
H2N
CN
O
_
3a c
O
S
CHO
OH
CH3
CH3
H3C
1
O
O
S
S
O
O
EtOH
Conc. HCl
EtOH/ TEA
Scheme 1. Reactions of 2-tosylacetonitrile
(1) with arylidenes and salicylaldehyde.
O
4
NH
O
5
O
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Arch. Pharm. Chem. Life Sci. 2011, 000, 1–8
Bioactive Heterocycles Derived from 2-Tosylacetonitrile
3
H3C
H3C
O
NaOEt/ DMF
rt
O
Ph
N
C S
S
CN
S-Na+
+
S
O
CN
O
PhHN
1
10
O
Br
Ph
EtOH/ TEA
H3C
H3C
O
S
O
NH2
O
S
O
O
CN
S
Ph
PhHN
Ph
PhHN
S
Scheme 3. Synthesis of 3-tosylthiophene
derivative 12.
12
O
11
reported by Hantzsch and Weber [19]. Thus, the base-
prompted reaction of the acidic methylene compound 1 with
phenyl isothiocyanate in dry dimethylformamide (DMF) at
room temperature yields the non-isolable intermediate 10.
The intermediate 10 undergoes in situ cyclization upon the
reaction with equimolar amounts of phenacyl bromide in
boiling ethanol containing a catalytic amount of TEA, giving
product 12. (3-Amino-5-(phenylamino)-4-tosylthiophen-2-
yl)(phenyl)methanone (12) was suggested for this product
on the basis of analytical and spectral data. The mass spec-
trum of 12 showed the ion peak at m/z 448 (Mþ) which is in
agreement with the molecular formula C24H20N2O3S2. The
reaction may occur through the non-isolated intermediacy of
acyclic derivative 11. Attempts to isolate 11 by refluxing
phenacyl bromide with the intermediate 10 in ethanol were
unsuccessful. Compound 12 is assumed to be formed via the
acyclic intermediate 11 (Scheme 3).
thiophene derivative 16. Structure 16 was established based
on its IR spectrum which showed bands related to NH2, NH
and CN functions. Its H-NMR spectrum revealed a multiplet
signal at d 7.31–7.56 ppm (9H, aromatic), broad signals at
d 6.5 (2H, NH2) and d 8.7 ppm (1H, NH). On the other hand,
it was found that it is directly formed by refluxing 10 and
chloroacetonitrile in DMF.
When 10 was treated with an equimolar amount of chloro-
acetyl chloride or with chloroacetic acid in boiling ethanol,
a product that analyzed for C20H20N2O4S2 was isolated in
each case in good yield. The acyclic structure 17 was estab-
lished based on its IR spectrum that showed bands related to
NH, CN and CO function groups. Its H-NMR spectrum
revealed a muliplet at (d ppm) 7.50–8.56 (9H, aromatic), a
triplet signal at d 1.3 (3H, CH3), singlet at d 1.9 (3H, CH3),
singlet at d 3.7 (2H), quartet at d 4.3 (2H, CH2) and
a D2O exchangeable NH at 8.91 ppm. Alternatively, treat-
ment of 10 with ethyl bromoacetate in refluxing ethanol
gave a single product, which is identical in all respects to
17 (m.p., mixed m.p. and IR spectrum). The mass spectrum of
17 showed the molecular formula C20H20N2O4S2 (Mþ ¼ 416).
Refluxing of 17 in ethanol with a catalytic amount of
TEA afforded the corresponding thiophene derivative 18
(Scheme 4).
Compound 10 reacted readily with chloroacetone in boil-
ing ethanol to afford the acyclic intermediate 13 by NaCl
elimination. Refluxing 13 in ethanol with a catalytic amount
of TEA gave the thiophene derivative 14 whose structure was
confirmed by elemental analysis and spectral data (c.f.
Experimental Part). The structure of 14 was further con-
firmed by its alternative synthesis. Thus, refluxing 10 with
chloroacetone in DMF affords the thiophene derivative 14 in
a good yield. Similarly, when the intermediate 10 was treated
with chloroacetonitrile in refluxing ethanol containing a
catalytic amount of TEA, the corresponding acyclic inter-
mediate 15 is exclusively isolated in good yield. The structure
15 was confirmed on the basis of elemental and spectral data,
e.g. the IR spectrum exhibits bands at y 3200 (NH), 2220,
2195 cmꢀ1 (2 CN). Its 1H-NMR spectrum indicated a CH2 signal
at d 3.23 ppm. Furthermore, heating of the intermediate 15
in ethanol containing a catalytic amount of TEA afforded the
The thiazoline heterocyclic ring incorporating a tosyl
moiety was prepared from compound 1. Thus, treatment
of 1 with a mixture of sulfur metal and phenyl isothiocyanate
in ethanol and excess of TEA and stirring overnight gave the
corresponding N-phenylthiazole derivative 19 that is in
complete agreement with mass spectral measurements and
analytical data (Scheme 5). The structure of 19 could be
established for the reaction product based on the IR spectrum
which showed absorption bands at y 3480 and 3390 cmꢀ1
(NH2). Its 1H-NMR spectrum revealed a D2O exchangeable
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A. A. Fadda et al.
Arch. Pharm. Chem. Life Sci. 2011, 000, 1–8
CH3
Cl
H3C
H3C
O
O
O
NH2
EtOH/ TEA
O
S
CN
S
S
O
EtOH
O
O
CH3
S
CH3
PhHN
PhHN
O
14
13
CN
H3C
H3C
O
S
NH2
EtOH/ TEA
Cl
[10]
CN
S
S
O
EtOH
O
CN
S
PhHN
PhHN
CN
16
15
R2
R1
O
H3C
H3C
R1 = R2 = Cl
O
O
NH2
EtOH/ TEA
or R1 = Cl, R2 = OH
or R1 = Br, R2 = OEt
EtOH
S
CN
S
S
O
O
O
OEt
S
OEt
PhHN
PhHN
Scheme 4. Synthesis of thiophene derivatives
14, 16 and 18.
O
17
18
H3C
H3C
O
NH2
Ph
EtOH/ TEA
Stirring
O
S
Ph
+
N
C S
+
S
O
S
O
CN
N
S
1
Scheme 5. Synthesis of 4-amino-3-phenyl-5-
tosylthiazole-2(3H)-thione (19).
S
19
Table 1. Antioxidant assay for the prepared new compounds.
proton at d 6.1 ppm due to NH2 protons besides a multiplet at
d 7.2–8.1 ppm due to 9 aromatic protons.
Compd. no.
ABTS
(Inhibition %)
Erythrocytes
haemolysis (%)
Pharmacology
Control
L-Ascorbic acid
1
–
–
88.61
39.70
41.60
35.55
40.90
38.20
83.30
72.30
76.20
78.20
80.30
0.85
1.40
0.86
5.55
1.50
3.65
0.95
1.10
1.90
0.86
0.85
Antioxidant activity for ABTS and erythrocytes
haemolysis
3a
3c
9a
9b
12
14
16
18
19
All compounds were tested for antioxidant activity as
reflected in the ability to inhibit lipid peroxidation in rat
brain and kidney homogenates and the rate of erythrocyte
haemolysis. The pro-oxidant activities of the compounds
were assayed for their antioxidant effects using ABTS assay.
Compounds 12 and 19 proved to exhibit potent antioxidative
activity. On the other hand, compounds 14, 16, and 18
showed a very good activity. In the meantime, compounds
1, 3a, 3c, 9a and 9b showed moderate activity, while com-
pounds 3a, 12, 18 and 19 showed high activity in case of
erythrocyte haemolysis.
the pyridine moiety incorporating three methoxy groups
(electron donating groups) enhances the activity in erythro-
cytes haemolysis (Fig. 1).
From Table 1 and the above-mentioned results, we may
include the following structure activity relationships (SARs)
and by comparing the tested compounds: (1) The presence of
thiophene and thiazole ring systems incorporated with the
tosyl moiety enhances the activity. (2) Introducing acyclic
chains or pyridine moieties into the tosyl moiety did not
affect the antioxidant activity (ABTS method). (3) Introducing
Bleomycin-dependent DNA damage
The bleomycins are a family of antitumor antibiotics, which
are used routinely as antitumor agent. The bleomycin assay
has been adopted for assessing the pro-oxidant effect of food
antioxidants. The antitumor antibiotic bleomycin binds iron
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Arch. Pharm. Chem. Life Sci. 2011, 000, 1–8
Bioactive Heterocycles Derived from 2-Tosylacetonitrile
5
Figure 1. Antioxidant activity screening assay.
University, the results were found to be in good agreement
(ꢁ0.03%) with the calculated values.
ions and DNA. The bleomycin-iron complex degrades DNA
that, upon heating with thiobarbituric acid, yields a pink
chromogen. Added suitable reducing agent ‘‘antioxidants’’
compete with DNA and diminish chromogen formation. We
select the best results of antioxidant activity for ABTS, eryth-
rocytes haemolysis and evaluate their bleomycin dependent-
DNA damage. Among the tested compounds (Table 2), com-
pound 12 showed the highest protection activity against DNA
damage induced by bleomycin-iron complex, thus diminish-
ing chromogen formation between the damaged DNA and
thiobarbituric acid (TBA).
General procedure for the synthesis of 6-amino-4-
substituted-5-tosylnicotinonitrile derivatives 3a–c
A mixture of 2-tosylacetonitrile (1) (1.95 g, 0.01 mol) and a,b-
unsaturated nitriles, namely 2a (2.44 g, 0.01 mol) or 2b (1.54 g,
0.01 mol) or 2c (1.97 g, 0.01 mol) in ethanol (15 mL), containing
a catalytic amount of TEA (0.4 mL) were refluxed for 6 h. The
solid products obtained after cooling were filtered off, dried and
recrystallized from ethanol to give pyridine derivatives 3a–c,
respectively.
6-Amino-5-tosyl-4-(2,3,4-trimethoxyphenyl)nicotinonitrile
(3a)
Table 2. Result of bleomycin-dependent DNA damage assay of
compound 1 as blank and compound 12 as the best antioxidant
agent.
White crystals, yield 80%, mp: 908C, IR (KBr): nmax, cmꢀ1: 3350–
3450 (NH ), 2220 (CN), 1610 (C N), 1210, 1230 (SO ); 1H NMR
–
–
2
2
Compd. no.
Absorbance
(DMSO-d6): d 2.34 (s, 3H, CH3), 3.8 (s, 3H, OCH3), 3.9 (s, 3H, OCH3),
4.0 (s, 3H, OCH3), 7.1 (d, 2H, NH2, exchangeable with D2O), 7.7–8.0
(m, 6H, Ar–H), 8.5 (s, 1H, O-py-H). MS (m/z, %): 439 (Mþ, 11.2%), 378
(100.0), 217 (64.7), 203 (15.4), 155 (30.5), 86 (49.2). Anal. Calcd.
for C22H21N3O5S (439.48): C, 60.12; H, 4.82; N, 9.56%. Found: C,
60.20; H, 4.87; N, 9.63%.
Vit. C
1
12
0.100
0.196
0.088
6-Amino-4-phenyl-5-tosylnicotinonitrile (3b)
White crystals, yield 75%, mp: 1438C, IR (KBr): nmax, cmꢀ1: 3366
Experimental
Chemistry
–
–
(broad, NH ), 3030 (CH), 2217 (CN), 1615 (C C), 1597 (C N), 1255,
2
–
–
1208 (SO2); 1H NMR (DMSO-d6): d 2.34 (s, 3H, CH3þ), 6.5 (s, 2H, NH2),
All melting points are uncorrected in degree centigrade and
determined on Gallenkamp electric melting point apparatus.
The infrared (IR) spectra were recorded (KBr disk) with a
Mattson 5000 FTIR spectrometer at the Faculty of Science,
Mansoura University. The 1H-NMR spectra were determined on
a Bruker WPSY 200 MHz spectrometer with TMS as internal
standard and the chemical shifts are in d ppm using dimethyl-
sulfoxide (DMSO) as solvent. The mass spectra were recorded at
70 eV with a Varian MAT 311 at the Microanalytical Center,
Faculty of Science, Cairo University. Elemental analyses (C, H
and N) were carried out at the Faculty of Science, Cairo
–
7.8–8.4 (m, 5H, py-c -HþAr). MS (m/z, %): 283 [M –(CN–C–CH N),
–
2
14.6%], 219 (0.9), 195 (8.9), 155 (69.2), 128 (3.2), 91 (100.0). Anal.
Calcd. for C19H15N3O2S (349.41): C, 65.31; H, 4.33; N, 12.03%.
Found: C, 65.38; H, 4.41; N, 12.11%.
6-Amino-4-(4-(dimethylamino)phenyl)-5-tosylnicotinonitrile
(3c)
Orange crystals, yield 84%, mp: 2208C, IR (KBr): nmax, cmꢀ1: 33361–
1
3428 (NH ), 2197 (CN), 1612 (C N); H NMR (DMSO-d ): d 1.2 (s, 3H,
–
–
CH3), 7.1–8.2 (m, 8H, Ar–H), 8.2 (s, 1H, py-C2-H). MS (m/z, %): 392
2
6
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Arch. Pharm. Chem. Life Sci. 2011, 000, 1–8
(Mþ, 100.0), 326 (52.2), 197 (60.2), 170 (42.3), 155 (63.4), 134 (43.7),
86 (100.0). Anal. Calcd. for C21H20N4O2S (392.47): C, 64.27; H, 5.14;
N, 14.28%. Found: C, 64.18; H, 5.09; N, 14.34%.
(20 mL), 2-tosylacetonitrile (1) (1.95 g, 0.01 mol) was added. To
the resulting solution the phenyl isothiocyanate (1.2 mL,
0.01 mol) was added and the reaction mixture stirred for 24 h
at room temperature.
Synthesis of 3-tosyl-2H-chromen-2-imine (4)
Synthesis of (3-amino-5-(phenylamino)-4-tosylthiophen-2-
yl)(phenyl)methanone (12)
A mixture of 1 (1.95 g, 0.01 mol) and salicyaldehyde (1.07 mL,
0.01 mol) in ethanol (15 mL) containing a catalytic amount of
TEA (0.5 mL) was refluxed for 2 h. The product iminochromene 4
was obtained after cooling, filtered off, dried and recrystallized
from ethanol. Orange crystals, yield 80%, mp: 1908C, IR (KBr):
nmax, cmꢀ1: 3344 (NH), 1211, 1148 (SO2); 1H NMR (DMSO-d6): d 2.3
(s, 3H, CH3), 7.3–8.5 (m, 8H, Ar–H), 8.8 (s, 3H, NH). MS (m/z, %): 300
(Mþþ1, 1.12), 248 (0.96), 234 (100.0), 208 (9.4), 165 (4.1), 139 (5.0),
89 (32.1). Anal. Calcd. for C16H13NO3S (299.34): C, 64.20; H, 4.38;
N, 4.68%. Found: C, 64.26; H, 4.41; N, 4.72%.
A mixture of equimolar amounts of 10 (0.01 mol) and phenacyl
bromide (1.99 g, 0.01 mol) was refluxed in ethanol (20 mL) con-
taining a catalytic amount of TEA (0.5 mL) for 6 h. The reaction
mixture was cooled, filtered off, dried and recrystallized from
ethanol to give 12. Black powder, yield 78%, mp: 1408C, IR (KBr):
nmax, cmꢀ1: 3430 (NH2), 3309 (NH), 1710 (CO); 1H NMR (DMSO-d6):
d 2.3 (s, 3H, CH3), 6.5 (broad, 2H), 7.3–8.1 (m, 14H, Ar–H), 9.22
(s, 1H, NH). MS (m/z, %): 448 (Mþ, 12.2), 164 (100.0), 120 (83.7),
86 (14.3). Anal. Calcd. for C24H20N2O3S2 (448.56): C, 64.26; H, 4.49;
N, 6.25%. Found: C, 64.33; H, 4.53; N, 6.30%.
Synthesis of 3-tosyl-2H-chromen-2-one (5)
Compound 4 (2.99 g, 0.01 mol) was heated in a mixture of conc.
HCl and ethanol (1:1, 20 mL) for 15 min. The reaction mixture
was left to stand at room temperature overnight and the solid
product was filtered off, dried and recrystallized from ethanol to
give corresponding chromenone derivative 5. Red crystals, yield
76%, mp: 2208C, IR (KBr): nmax, cmꢀ1: 3036 (CH), 1740 (CO), 1212,
1151 (SO2); 1H NMR (DMSO-d6): d 2.3 (s, 3H, CH3), 7.55–8.25 (m, 8H,
Ar–H). MS (m/z, %): 300 (Mþ, 3.0), 235 (97.0), 208 (33.7), 165 (10.8),
139 (15.1), 89 (100.0), 63 (61.5). Anal. Calcd. for
C16H12O4S (300.33): C, 63.99; H, 4.03%. Found: C, 63.95; H, 3.98%.
General procedure for the synthesis of the acyclic 3-
(substituted-methylthio)-3-(phenylamino)-2-
tosylacrylonitriles 13, 15 and 17
Equimolar amounts of 10 (0.01 mol) in ethanol and a-haloke-
tones, namely chloroacetone (0.93 g, 0.01 mol) or chloroacetoni-
trile (0.76 g, 0.01 mol) or [chloroacetyl chloride (0.8 mL,
0.01 mol) or chloroacetic acid (0.95 g, 0.01 mol) or ethyl bromo-
acetate (1.11 mL, 0.01 mol)], were stirred for 6 h at room
temperature, then left to stand at the same temperature for
24 h. The separated solid material was washed with water, dried
and crystallized from ethanol to give 13, 15 and 17, respectively.
Synthesis of 6-amino-2-(substituted (cyano)methyl)-5-
tosylnicotinonitriles 9a,b
Equimolar amounts of 1 (0.78 g, 4 mmol), formaldehyde (1 mL,
30% aqueous solution) and 3-amino-2-substituted-pent-2-enedini-
triles 6a (0.53 g, 4 mmol) or 6b (0.96 g, 4 mmol) in ethanol
(25 mL) in the presence of a catalytic amount of TEA (0.4 mL).
The reaction mixture was refluxed for 3 h and the obtained solid
product while hot was filtered off, dried and recrystallized from
ethanol.
3-(2-Oxopropylthio)-3-(phenylamino)-2-tosylacrylonitrile
(13)
White crystals, yield 94%, mp: 2208C, IR (KBr): nmax, cmꢀ1: 3287
(NH), 2220 (CN), 1730 (CO), 1603 (Ph); 1H NMR (DMSO-d6): d 1.9
(s, 3H, CH3), 2.3 (s, 3H, CH3), 3.7 (s, 2H, CH2), 7.12–7.97 (m, 10H,
Ar–H), 9.25 (s, 1H, NH). MS (m/z, %): 386 (Mþ, 100.0), 386 (100.0),
321 (64.8), 231 (45.7), 143 (35.2), 86 (19.9). Anal. Calcd.
for C19H18N2O3S2 (386.49): C, 59.05; H, 4.69; N, 7.25%. Found:
C, 59.11; H, 4.76; N, 7.31%.
2-(6-Amino-3-cyano-5-tosylpyridin-2-yl)malononitrile (9a)
Red crystals, yield 82%, mp: 2008C, IR (KBr): nmax, cmꢀ1: 3450
(NH ), 2207 (CN), 1628 (C N); 1H NMR (DMSO-d6): d 2.36 (s, 3H,
–
–
2
3-(Cyanomethylthio)-3-(phenylamino)-2-tosylacrylonitrile
CH3), 4.72 (s, 1H, CH), 6.43 (s, 2H, NH2), 7.35–8.62 (m, 5H, Ar–H).
MS (m/z, %): 337 (Mþ, 65.2), 278 (76.1), 246 (52.6), 155 (38.0), 139
(100.0), 123 (58.9), 86 (65.2). Anal. Calcd. for C16H11N5O2S (337.36):
C, 56.96; H, 3.29; N, 20.76%. Found: C, 57.04; H, 3.36; N, 20.83%.
(15)
White powder, yield 89%, mp: 2708C, IR (KBr): nmax, cmꢀ1: 3200
(NH), 2220, 2195 (CN), 1603 (Ph); 1H NMR (DMSO-d6): d 2.2 (s, 3H,
CH3), 3.23 (s, 2H, CH2), 7.12–7.87 (m, 9H, Ar), 9.25 (s, 1H, NH). MS
(m/z, %): 367 (Mþꢀ2, 1.4), 279 (50.0), 246 (40.3), 192 (20.8), 155
(40.3), 123 (50.0), 84 (100.0). Anal. Calcd. for C18H15N3O2S2
(369.46): C, 58.52; H, 4.09; N, 11.37%. Found: C, 58.47; H, 4.03;
N, 11.43%.
6-Amino-2-(benzo[d]thiazol-2-yl(cyano)methyl)-5-
tosylnicotinonitrile (9b)
White crystals, yield 80%, mp: 2908C, IR (KBr): nmax, cmꢀ1: 3358
(NH ), 2190 (CN), 1601 (C N); 1H NMR (DMSO-d6): d 2.33 (s, 3H,
–
–
2
CH3), 4.68 (s, 1H, CH), 6.47 (s, 2H, NH2), 7.28–8.71 (m, 9H, Ar–H).
MS (m/z, %): 445 (Mþ, 61.7), 251 (100.0), 156 (23.9), 124 (16.3), 86
(61.7). Anal. Calcd. for C22H15N5O2S2 (445.52): C, 59.31; H, 3.39%.
Found: C, 59.37; N, 15.72%; H, 3.42; N, 15.79%.
Ethyl 2-(2-cyano-1-(phenylamino)-2-tosylvinylthio)acetate
(17)
White crystals, yield 87%, mp: 2908C, IR (KBr): nmax, cmꢀ1: 3278
(NH), 2220 (CN), 1730 (CO); 1H NMR (DMSO-d6): d 1.3 (t, 3H, CH3),
2.3 (s, 3H, CH3), 3.7 (s, 2H, CH2), 4.3 (q, 2H, CH2), 7.5–8.56 (m, 9H,
Ar), 9.25 (s, 1H, NH). MS (m/z, %): 416 (Mþ, 94.3), 278 (10.8),
246 (21.3), 155 (46.3), 123 (20.2), 84 (100.0). Anal. Calcd.
for C20H20N2O4S2 (416.51): C, 57.67; H, 4.84; N, 6.73%. Found:
C, 57.74; H, 4.87; N, 6.77%.
Preparation of sodium 2-cyano-1-(phenylamino)-2-
tosylethenethiolate (10)
To a stirred suspension of sodium ethoxide [prepared from
0.23 g, 0.01 mol sodium metal in 10 mL ethanol) in DMF
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Arch. Pharm. Chem. Life Sci. 2011, 000, 1–8
Bioactive Heterocycles Derived from 2-Tosylacetonitrile
7
General procedure for the synthesis of thiophene
derivatives 14, 16 and 18
Antioxidant screening assay (ABTS method) [20]
L-Ascorbic acid was obtained from Sigma, 2,20-azinobis-(3-ethyl-
benzthiazoline-6-sulphonic acid) (ABTS) was purchased from
Wak and all other chemicals were of the highest quality avail-
able. For each of the investigated compounds, 2 mL of ABTS
solution (60 mM) was added to 3 M magnesium oxide (MnO2)
solution (25 mg/mL) all prepared in phosphate buffer (pH 7,
0.1 M). The mixture was shaken, centrifuged, filtered, and the
absorbance (Acontrol) of the resulting green-blue solution (ABTS
radical solution) was adjusted at ca. 0.5 at l 734 nm. Then, 50 mL
of (2 mM) solution of the test compound in spectroscopic grade
methanol/phosphate buffer (1:1) was added. The absorbance
(Atest) was measured and the reduction in color intensity was
expressed as % inhibition. The inhibition for each compound was
calculated from the following equation.
Method A: A mixture of equimolar amounts of 10 (0.01 mol) and a-
haloketones, namely chloroacetone (0.93 g, 0.01 mol) or chlor-
oacetonitrile (0.76 g, 0.01 mol) or [chloroacetyl chloride (0.8 mL,
0.01 mol) or chloroacetic acid (0.95 g, 0.01 mol) or ethyl bromo-
acetate (1.11 mL, 0.01 mol)], was refluxed in DMF (20 mL) for 6 h.
The reaction mixture was cooled, filtered off, dried and recrystal-
lized from ethanol to give the corresponding thiophene deriva-
tives 14, 16 and 18, respectively.
Method B: Refluxing of 13 (3.86 g, 0.01 mol) or 15 (3.69 g,
0.01 mol) or 17 (4.17 g, 0.01 mol) in ethanol (20 mL) containing
a catalytic amount of TEA (0.5 mL) for 3 h afforded the corre-
sponding substituted thiophene derivatives 14, 16 and 18,
respectively.
% Inhibition ¼ ½AðcontrolÞ ꢀ AðtestÞ=AðcontrolÞꢂ ꢃ 100
1-(3-Amino-5-(phenylamino)-4-tosylthiophen-2-yl)-
ethanone (14)
White powder, yield 88%, mp: 1958C, IR (KBr): nmax, cmꢀ1: 3438
(NH2), 3286 (NH), 1650 (CO), 1602 (Ph); H NMR (DMSO-d6): d 1.9
Ascorbic acid (vitamin C) was used as standard antioxidant
(positive control). Blank sample was run without ABTS and using
methanol/phosphate buffer (1:1) instead of sample. Negative
control sample was run with methanol/phosphate buffer (1:1)
instead of tested compound.
1
(s, 3H, CH3), 2.4 (s, 3H, CH3), 7.2–7.9 (m, 9H, Ar), 8.2 (d, 2H, NH2),
9.5 (s, 1H, NH). MS (m/z, %): 386 (Mþ, 100.0), 386 (100.0), 321 (64.8),
231 (45.7), 143 (35.2), 86 (19.9). Anal. Calcd. for C19H18N2O3S2
(386.49): C, 59.05; H, 4.69; N, 7.25%. Found: C, 59.01; H, 4.62; N,
7.19%.
Antioxidant activity screening assay for erythrocyte
haemolysis
3-Amino-5-(phenylamino)-4-tosylthiophene-2-carbonitrile
(16)
The blood was obtained from rats by cardiac puncture and
collected in heparinized tubes. Erythrocytes were separated from
plasma and the buffy coat was washed three times with 10
volumes of 0.15 M NaCl. During the last wash, the erythrocytes
were centrifuged at 2500 rev./min for 10 min to obtain a con-
stantly packed cell preparation. Erythrocyte haemolysis was
mediated by peroxyl radicals in this assay system. A 10% suspen-
sion of erythrocytes in phosphate buffered saline pH 7.4 (PBS) was
added to the same volume of 200 mM 2,20-azobis(2-amidinopro-
pane) dihydrochloride (AAPH) solution (in PBS) containing
samples to be tested at different concentrations. The reaction
mixture was shaken gently while being incubated at 378C for 2 h.
The reaction mixture was then removed, diluted with eight
volumes of PBS and centrifuged at 1500 g for 10 min. The absorb-
ance of the supernatant was read at 540 nm. Similarly, the
reaction mixture was treated with 8 volumes of distilled water
to achieve complete haemolysis, and the absorbance of the
supernatant obtained after centrifugation was measured at
540 nm. The data percentage haemolysis was expressed as
mean-standard deviation. L-Ascorbic acid was used as a positive
control.
Black powder, yield 80%, mp: 2408C, IR (KBr): nmax, cmꢀ1: 3456
(NH2), 3266 (NH), 2220 (CN), 1603 (Ph); H NMR (DMSO-d6): d 2.3
1
(s, 3H, CH3), 6.5 (br, 2H, NH2), 7.31–7.96 (m, 9H, Ar–H), 8.7 (s, 1H,
NH). MS (m/z, %): 367 (Mþ, 1.4), 279 (50.0), 246 (40.3), 192 (20.8),
155 (40.3), 123 (50.0), 84 (100.0). Anal. Calcd. for C18H15N3O2S2
(369.46): C, 58.52; H, 4.09; N, 11.37%. Found: C, 58.61; H, 4.13;
N, 11.44%.
Ethyl 3-amino-5-(phenylamino)-4-tosylthiophene-2-
carboxylate (18)
Black powder, yield 76%, mp: 2408C, IR (KBr): nmax, cmꢀ1: 3459
(NH2), 3301 (NH), 1743 (CO), 1598 (Ph); 1H NMR (DMSO-d6): d 1.26
(t, 3H, CH3), 2.3 (s, 3H, CH3), 4.22 (q, 2H, CH2), 6.55 (br, 2H, NH2),
7.18–7.96 (m, 9H, Ar–H), 19.25 (s, 1H, NH). MS (m/z, %): 416
(Mþ, 94.3), 278 (10.8), 246 (21.3), 155 (46.3), 123 (20.2), 84
(100.0). Anal. Calcd. for C20H20N2O4S2 (416.51): C, 57.67;
H, 4.84; N, 6.73%. Found: C, 57.74; H, 4.87; N, 6.75%.
Synthesis of 4-amino-3-phenyl-5-tosylthiazole-2(3H)-
thione (19)
Bleomycin-dependent DNA damage assay
A mixture of equimolar amounts of 1 (1.95 g, 0.01 mol), elemen-
tal sulfur (0.32 g, 0.01 mol) and phenyl isothiocyanate (1.2 mL,
0.01 mol) in ethanol (10 mL) containing a catalytic amount of
TEA (0.5 mL) was stirred overnight, then refluxed for 2 h. The
reaction mixture was poured on ice-water, then filtered off, dried
and recrystallized from ethanol to give the thiazole derivative 19.
Green powder, yield 90%, mp: 2708C, IR (KBr): nmax, cmꢀ1: 3480,
3390 (NH2); 1H NMR (DMSO-d6): d 2.2 (s, 3H, CH3), 6.1 (d, 2H, NH2),
7.2–8.1 (m, 9H, Ar-H). MS (m/z, %): 362 (Mþ, 7.5), 321 (8.8), 291
(12.5), 274 (20.0), 194 (12.5), 167 (16.3), 135 (52.5), 77 (100.0). Anal.
Calcd. for C16H14N2O2S3 (362.49): C, 53.01; H, 3.89; N, 7.73%.
Found: C, 52.92; H, 3.85; N, 7.69%.
The reaction mixtures contained, in a final volume of 1.0 mL, the
following reagents at the final concentrations stated: DNA
(0.2 mg/ mL), bleomycin (0.05 mg/mL), FeCl3 (0.025 mM), mag-
nesium chloride (5 mM), KH2PO4–KOH buffer pH 7.0 (30 mM)
and ascorbic acid (0.24 Mm) or the compounds tested in MeOH to
give a concentration of (0.1 mg/mL). The reaction mixtures were
incubated in a water-bath at 378C for 1 h. At the end of the
incubation period, 0.1 mL of 0.1 M ethylenediaminetetraacetic
acid (EDTA) was added to stop the reaction (the iron-EDTA com-
plex is unreactive in the bleomycin assay). DNA damage was
assessed by adding 1 mL 1% (w/v) thiobarbituric acid (TBA) and
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8
A. A. Fadda et al.
Arch. Pharm. Chem. Life Sci. 2011, 000, 1–8
1 mL 25% (v/v) hydrochloric acid (HCl) followed by heating in a
water-bath maintained at 808C for 15 min. The chromogen
formed was extracted into butan-1-ol and the absorbance was
measured at 532 nm [21, 22].
[8] F. M. Abdelrazek, A. A. Fadda, A. N. Elsayed, Synth. Commun.
2011, 41, 1119–1126.
[9] A. A. Fadda, E. H. E. Youssif, Synth. Commun. 2011, 41, 677–694.
[10] A. A. Fadda, S. Bondock, T. Abdel-Gaber, Curr. Org. Chem. 2011,
15, 753–781.
The authors have declared no conflict of interest.
[11] L. Fuentes, J. J. Vaquero, M. I. Ardid, J. C. Del Castillo, J. L.
Soto, Synthesis 1984, 1984, 768–770.
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