Antiarrhythmic, serotonin antagonist and antianxiety activities of novel substituted thiophene derivatives synthesized from 2-amino-4,5,6,7-tetrahydro-N- phenylbenzo[b]thiophene-3-carboxamide

Article abstract of DOI:10.1016/j.ejmech.2010.09.059

A series of novel thiophene derivatives 3-17 were synthesized by initial reactions of 2-amino-4,5,6,7-tetrahydro-N-phenylbenzo[b]thiophene-3-carboxamide 1 and 2-amino-4,5,6,7-tetrahydro-benzo[b]thiophene-3-carbonitrile 7 with different organic reagents. T

Full text of DOI:10.1016/j.ejmech.2010.09.059

European Journal of Medicinal Chemistry 45 (2010) 5935e5942
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Antiarrhythmic, serotonin antagonist and antianxiety activities of novel
substituted thiophene derivatives synthesized from 2-amino-4,5,6,7-tetrahydro-
N-phenylbenzo[b]thiophene-3-carboxamide
,
b
b
a
b
Abd El-Galil E. Amr a , Mohamed H. Sherif , Mohamed G. Assy , Mohamed A. Al-Omar , Islam Ragab
*
a Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, El-Tahrir, St., Dokki, Riyadh 11451, Saudi Arabia
b Department of Chemistry, Faculty of Science, Zagazig University, Zagazig, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel thiophene derivatives 3e17 were synthesized by initial reactions of 2-amino-4,5,6,7-
tetrahydro-N-phenylbenzo[b]thiophene-3-carboxamide and 2-amino-4,5,6,7-tetrahydro-benzo[b]
thiophene-3-carbonitrile with different organic reagents. The structures of newly synthesized
Received 26 April 2010
Received in revised form
21 August 2010
Accepted 24 September 2010
Available online 14 October 2010
1
7
compounds were confirmed by IR, 1H NMR, MS spectral data and elemental analysis. Initially the acute
toxicity of the compounds was assayed via the determination of their LD50. All the compounds were
screened for their antiarrhythmic, serotonin antagonist and antianexiety activities and they showed high
activity compared with procaine amide, lidocaine, diazepam and buspirone as positive controls. The
detailed synthesis, spectroscopic data, LD50 and pharmacological activities of the synthesized
compounds were reported.
Keywords:
Thiophenes
Thiazoles
Oxazinones
Ó 2010 Elsevier Masson SAS. All rights reserved.
Antiarrhythmic
Serotonin antagonist and antianxiety
activities
1. Introduction
reported that certain of our newly substituted heterocyclic
compounds exhibited antiandrogenic [26], anti-inflammatory [27],
Aromatic thiophenes play on part in animal metabolism; for
examples, in Fig. 1, BiotinÒ, one of the vitamins (Vitamin H), is
a tetrahydrothiophene, however aromatic thiophenes do occur in
some plants, in association with polyacetylenes with which they
are biogenetically linked and BanminthÒ (pyrantel), available
anthelmintic used in animal husbandry, is one of the thiophene
compounds in chemotherapy.
Thiophenes with a wide spectrum of biological activities are
known, several of these derivatives possess potent analgesic [1,2],
anticonvulsant, anti-inflammatory and antibacterial [3e6], anti-
pyretics [7], antitumor [8,9], antiparasitic [10], antimicrobial [11],
antihistaminic (H1) [12], antianexiety test in mice [13], antiar-
rhythmic [14] and serotonin antagonist [15]. In previous work we
have found that certain substituted pyrimidine and their hetero-
cyclic derivatives show antimicrobial and anti-inflammatory
[16e19] and antitumor activities [20e22]. On the other hand, thi-
oxopyrimidine and thiazolopyrimidine derivatives have promising
biological and anticancer activities [23e25]. In addition, we have
anticancer [28], anticonvulsant [29] and antiarrhythmic [30]
activities. Recently, some new thienopyrimidinone and p-methox-
yphenyl pyrrolidine derivatives have been synthesized and tested
as analgesic, antiparkinsonian, anti-inflammatory, serotonin
antagonist and antianexiety activities [31e33].
Arrhythmia is a term for any of a large and heterogeneous group
of conditions in which there is abnormal electrical activity in the
heart. The heart beat may be too fast or too slow, and may be regular
or irregular [34]. Serotonin is a monoamine neurotransmitter, bio-
chemically derived from tryptophan, that is primarily found in the
gastrointestinal tract, platelets, and central nervous system of
humans and animals. It regulates mood, appetite, sleep, muscle
contraction, and some cognitive functions including memory and
learning. It is actively taken up by blood platelets, which store it.
When the platelets bind to a clot, they disgorge serotonin, where it
serves as a vasoconstrictor and helps to regulate hemostasis and
blood clotting [35]. Anxiety is a psychological and physiological state
characterized by cognitive, somatic, emotional, and behavioral
components. These components combine to create an unpleasant
feeling that is typically associated with uneasiness, apprehension,
fear, orworry. Anxiety isa generalized mood conditionthatcanoften
* Corresponding author. Fax: þ966 1 4676220.
E-mail address: aamr1963@yahoo.com (A.E.-G.E. Amr).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.09.059

5936
A.E.-G.E. Amr et al. / European Journal of Medicinal Chemistry 45 (2010) 5935e5942
O
O
N
O
Ar
N
Ar
N
N
NH
HN
Me
S
N
S
N
H
S
4
H
H
3
H
S
N
NaOEt
CS2
(CH2)4CO2H
S
O
O
Ar
(+)-Biotin (Vitamin H)
Banminth
N
NHAr
NaNO2 / HCl
H
Fig. 1. Chemical structures of (þ)-Biotin and Banminth.
N
N
S
NH
S
2
occur without an identifiable triggering stimulus [36]. In view of the
aforementioned facts, it seemed most interesting to synthesize
some condensed tetrahydro-N-phenylbenzo[b]thiophene-3-car-
boxamide derivatives with the aim to evaluate their antiarrhythmic,
serotonin antagonist and antianxiety activities.
Cl
1
2
RCOCl
O
Ar
CONHAr
NHCOR
N
Ac2O
2. Results and discussion
Reflux
S
N
R
S
6a,b
Ar =
2.1. Chemistry
5a,b
OMe
b; R = -Ph
c; R = -CH=CHPh
In the present work, a series of condensed thiophenes via the
heterocyclization functionalized thiophene using several reagents.
2-Amino-4,5,6,7-tetrahydro-N-phenylbenzo[b]thiophene-3-car-
boxamide 1 and 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-
carbonitrile 7 were synthesized as starting materials according to
Gewald procedures [37e39] (Scheme 1).
Nitrosation of thiophene derivative 1 by using NaNO2 in the
presence of HCl afforded the corresponding thienotriazine deriva-
tive 3. The formation of 3 from nitrosation of 1 may proceed via the
formation of diazonium salt 2 followed by the attack of nucleophilic
nitrogen of carboxamide to the electrophilic nitrogen of diazonium
salt. Compound 1 undergoes heterocyclization via the addition of
amino function of thiophene to carbon disulfide in the presence of
sodium ethoxide followed by cycloaddition to produce thienopyr-
imidine 4. Also, thiophene derivative 1 was reacted with acid
chloride at room temperature to give the acylated derivatives 5a,
b which were refluxed with acetic anhydride to afford the cyclized
thienopyrimidines 6a, b (Scheme 2). The structures of the synthe-
sized compounds were assigned on the bases of its spectral data
and elemental analysis (cf. Section 4).
Scheme 2. Synthetic routes of compounds 3e6.
acid in xylene to give cyclized oxazine derivatives 9a, b. Compound 8c
was reacted with ammonium thiocyanate to produce N-thienylth-
iazole derivative 11 presumably via the non isolable thiocyanate
derivative 10. The reaction of aminocyanothiophene 7 with maleic
anhydride afforded the acylated product 12, which was cyclized with
acetic anhydride to give N-thienylmalimide 13 (Scheme 3). The
structures of the synthesized compounds were assigned on the bases
of its spectral data and elemental analysis (cf. Section 4).
The author investigated the behavior of aminocyanothiophene
towards the aroylisothio cyanate. Thus the addition of amino
function of thiophene to the electrophilic carbon of isothiocyanate
afforded thiourea derivative 14. The latter compound was cyclized
via the addition of nucleophilic nitrogen of thiourea function to the
cyano function to afford thienopyrimidine 15. Condensation of 7
and benzaldehyde afforded Schiff base 16, which was treated with
benzoylisothiocyanate to give the corresponding oxadiazine
derivative 17 (Scheme 4). The structures of the synthesized
Treatment of aminocyanothiophene derivative 7 with acid chlo-
rides afforded the derivatives 8aec, which were refluxed sulphuric
CONHAr
CN
CONHAr
H
O
NCH2C
N
Ar
C
N
S / Base
+
O
S
CONHAr
CONHAr
NH
OMe
Ar =
S
NH2
1
S
H
Scheme 1. Synthetic routes of compound 1.

A.E.-G.E. Amr et al. / European Journal of Medicinal Chemistry 45 (2010) 5935e5942
5937
CN
CN
N
O
O
Ac2O
COOH
reflux
NH
S
O
S
12
13
O
O
O
O
AcOH
NH
CN
CN
O
H2SO4 /
Xylene
RCOCl
NHCOR
R
NH2
S
N
S
S
9a,b
8a-c
7
a; R = Ph
b; R = CH=CHPh
c; R = CH2Cl
a; R = Ph
b; R = CH=CHPh
8c
NH4SCN
CN
CN
OH
N
S
NHCOCH2NCS
S
HN
10
S
11
Scheme 3. Synthetic routes of compounds 8e13.
compounds were assigned on the bases of its spectral data and
elemental analysis.
receptors were designed as the 5HT1A subtype and the insensitive
receptors were referred as the 5HT1B subtype. Schlegel and Peroutka
identified [3H] DPAT as a selective ligand for 5HT1 receptors [40].
Specific binding is defined as the difference between total
2.2. Pharmacological screening
binding and binding in the presence of 10 mM 5HT [40,41]. IC50
2.2.1. Antiarrhythmic activity
Procaine amide, 5 mg/kg iv and lidocaine 5 mg/kg iv. lead to an
increase in LD100 by 65%, which corresponds to a LD100 of approx-
values are calculated from the percent specific binding at each drug
concentration (Table 2). Serotonin may play a role in anxiety, since
drugs, which reduce serotoninergic function, have anxiolytic effects
in animal models. Since buspirone and its analogs have relatively
higher affinity for the 5HT1A receptor than other receptor and no
effect on the benzodiazepine site, their anxiolytic properties are
attributed to activity at the 5HT1A receptor. So all derivatives were
subjected to anxiolytic screening.
imately 9 mg/100 mg (Fig. 2).
From Table 1, all the tested compounds showed potent antiar-
rhythmic activities, where the compounds 6b, 9b, 11, 14, 15 and 17
are more potent than procaine amide and lidocaine.
2.2.2. Serotonin antagonist activity
From the results in Table 2 all the tested compounds showed
potent serotonin antagonist activities, the order of descending
activities is 15, 6b, (9b, 11 & Buspirone), 14, 17, 8c, 9a, (8a & 8b), 12,
(4, 5b & 13), 3, 5a, 6a, 16, 7 and 1.
Determination of the affinity of tested compounds for the 5HT1A
receptor in brain may be useful for predicating compounds with
novel anxiolytic or atypical antipsychotic profiles. The existence of
at least two populations of 5HT1 receptors in rat brain was shown
by differential sensitivity to spiroperdiol. The spiroperdiol-sensitive
2.2.3. Antianxiety test in mice [42e44]
Crawley has described a simple behavior model in mice to detect
compounds with anxiolytic effects. Mice tend to explore a novel
environment, but to retreat from the aversive properties of
a brightly-lit open field. In a two chambered system, where mice
can freely move between a brightly-lit open field and a dark corner,
animals show more crossing between the two chambers and more
locomotors activity after treatment with anxiolytics.
Dose response curves are obtained and the number of crossings
through the partition between the light and the dark chambers are
compound with total activity compound during 10 min. Dose makes
decrease intotal activityasthat made by diazepam 10 mg/kg s.c. were
determinedandthe relative potencies to diazepamwere determined.
From the results in Table 3, all the tested compounds showed
potent antianxiety activities, the order of descending activities is 17,
11, 14, 6b, 9b, (5b, 6a, 8b & 15), (1, 3 & 8a), (4, 8c 9a, 12, & 13), (5a &
16), and 7.
CN
S
PhCONCS
Ph
7
N
NH
14
H
S
O
PhCOH
NH
N
O
C
CN
N
N
Ph
CHPh
SH
S
S
16
15
CN
N
Ph
PhCONCS
O
S
S
Ph
N
17
Scheme 4. Synthetic routes of compounds 14e17.

5938
A.E.-G.E. Amr et al. / European Journal of Medicinal Chemistry 45 (2010) 5935e5942
Anti-arrythmic activities
(Intravenous adminstration)
90
80
70
60
50
40
30
20
10
0
Compound in (5 mg/kg)
Fig. 2. Antiarrhythmic activities of the newly synthesized compounds.
2.2.4. Structure activity relationship (SAR)
3. Conclusion
ꢀ Thiophene nucleus is essential for antiarrhythmic, serotonin
antagonist and antianxiety activities.
A
series of novel substituted thiophene derivatives were
synthesized by initial reaction of 2-amino-4,5,6,7-tetrahydro-N-
phenylbenzo[b]thiophene-3-carboxamide 1 and 2-amino-4,5,6,7-
tetrahydro-benzo[b]thiophene-3-carbonitrile
ꢀ Increasing the number of sulfur and nitrogen atoms sharply
increases the activities.
7
with different
ꢀ The side chains containing olefinic bonds are more active than
non-olefinic bond substituted (for example compounds 5b, 6b,
8b and 9b more active than 5a, 6a, 8a and 9a).
organic. All the compounds were screened for their antiarrhythmic,
serotonin antagonist and antianexiety activities and they showed
high activity compared with procaine amide, lidocaine, diazepam
and buspirone as positive controls. Thiophene nucleus is essential
for activity and increasing the number of sulfur or nitrogen atoms
sharply increases the activities. The olefinic bond in the substituted
side chain led to increase the activities (for examples, 5b, 6b, 8b and
9b more active than 5a, 6a, 8a and 9a).
2.2.5. Determination of acute toxicity (LD50
)
The LD50 was determined by using rats. They were injected with
different increasing doses of the synthesized compounds. The dose
that killed 50% of the animal was calculated according to Austen
et al. [45] (Table 4).
4. Experimental
4.1. Chemistry
Table 1
Antiarrhythmic activities of the newly synthesized compounds.
All melting points are uncorrected and measured using Electro-
thermal IA 9100 apparatus (Shimadzu, Tokyo, Japan). IR spectrawere
recorded as potassium bromide pellets on a PerkineElmer 1650
spectrophotometer (PerkineElmer, Norwalk, CT, USA). 1H NMR was
determined on a Jeol-Ex-270NMR spectrometer (JEOL, Tokyo, Japan)
Compound in (5 mg/kg)
Percentage increase
in LD100
Lidocaine
Procaine amide
3
4
65
65
75.00
76.00
67.90
74.60
70.10
82.00
77.50
73.10
68.15
80.10
82.10
69.00
84.00
84.30
73.60
84.00
and chemical shifts were expressed as part per million; ppm (
d
5a
5b
6a
6b
8b
8c
9a
9b
11
12
14
15
16
17
values) against TMS as internal reference. Mass spectra were
recorded on VG 2AM-3F mass spectrometer (Thermo electron
corporation, USA) Microanalyses were operated using Mario El
Mentar apparatus, Organic Microanalysis Unit, and the results were
within the accepted range (ꢁ0.2%) of the calculated values. Follow
up of the reactions and checking the purity of the compounds was
made by TLC on silica gel-coated aluminum sheets (Type 60 F254,
Merck, Darmstadt, Germany). 2-Amino-4,5,6,7-tetrahydro-N-
(3-methoxyphenyl)benzo[b]-thiophene-3-carboxamide (1) and 2-
amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile (7) were
synthesized according to the previous procedures [37e39] as starting
materials.

A.E.-G.E. Amr et al. / European Journal of Medicinal Chemistry 45 (2010) 5935e5942
5939
Table 2
Table 4
Serotonin antagonist activities of known compounds (1, 7) and newly synthesized
Acute toxicity (LD50) of known compounds (1, 7) and newly
compounds.
synthesized compounds.
Compound No.
(mg kgꢃ1
Concentration
IC50
Compound No
LD50/mg/kg
)
(mg kgꢃ1
)
(mg kgꢃ1
)
Buspirone
Diazepam
1
3
113 ꢁ 0.18
102 ꢁ 0.10
121 ꢁ 0.10
124 ꢁ 0.18
210 ꢁ 0.19
236 ꢁ 0.15
276 ꢁ 0.18
136 ꢁ 0.16
243 ꢁ 0.18
152 ꢁ 0.12
185 ꢁ 0.17
186 ꢁ 0.18
194 ꢁ 0.19
224 ꢁ 0.18
244 ꢁ 0.11
274 ꢁ 0.18
224 ꢁ 0.18
234 ꢁ 0.17
175 ꢁ 0.18
221 ꢁ 0.67
246 ꢁ 0.16
297 ꢁ 0.15
Buspirone
2 ꢄ 10ꢃ7
2 ꢄ 10ꢃ8
2 ꢄ 10ꢃ7
2 ꢄ 10ꢃ8
2 ꢄ 10ꢃ7
2 ꢄ 10ꢃ8
2 ꢄ 10ꢃ7
2 ꢄ 10ꢃ8
2 ꢄ 10ꢃ7
2 ꢄ 10ꢃ8
2 ꢄ 10ꢃ7
2 ꢄ 10ꢃ8
2 ꢄ 10ꢃ7
2 ꢄ 10ꢃ8
2 ꢄ 10ꢃ7
2 ꢄ 10ꢃ8
2 ꢄ 10ꢃ7
2 ꢄ 10ꢃ8
2 ꢄ 10ꢃ7
2 ꢄ 10ꢃ8
2 ꢄ 10ꢃ7
2 ꢄ 10ꢃ8
2 ꢄ 10ꢃ7
2 ꢄ 10ꢃ8
2 ꢄ 10ꢃ7
2 ꢄ 10ꢃ8
2 ꢄ 10ꢃ7
2 ꢄ 10ꢃ8
2 ꢄ 10ꢃ7
2 ꢄ 10ꢃ8
2 ꢄ 10ꢃ7
2 ꢄ 10ꢃ8
2 ꢄ 10ꢃ7
2 ꢄ 10ꢃ8
2 ꢄ 10ꢃ7
2 ꢄ 10ꢃ8
2 ꢄ 10ꢃ7
2 ꢄ 10ꢃ8
2 ꢄ 10ꢃ7
2 ꢄ 10ꢃ8
2 ꢄ 10ꢃ7
2 ꢄ 10ꢃ8
63
87
47
56
61
75
62
75
60
73
62
75
38
67
83
89
39
65
66
77
66
78
58
80
68
79
63
87
63
87
63
77
62
75
66
87
77
96
39
67
63
86
1
4
3
5a
5b
6a
6b
7
8a
8b
8c
9a
9b
11
12
13
14
15
16
17
4
5a
5b
6a
6b
7
8a
8b
8c
9a
9b
11
12
13
14
15
16
17
4.1.1. 3-(3-Methoxyphenyl)-4,5,6,7-tetrahydrobenzo[b]thieno[2,3-
d][1,2,3]-triazin-4(3H)-one (3)
To a mixture of compound 1 (0.01 mol) in concentrated HCl
(20 ml), sodium nitrite (0.01 mol) was added upon stirring for 2 h at
10 ꢂC. The separated solid was filtrated off, dried and crystallized
from acetic acid to give brown crystals of 3. Yield (95%), m.p. 220 ꢂC,
IR (KBr, n
, cmꢃ1): 1654 (C]O), 1602 (C]C). 1H NMR (DMSO-d6):
d
¼ 1.20e2.90 (m, 8H, cyclohexane), 3.90 (s, 3H, OCH3), 6.90e8.40
(m, 4H, Ar-H) ppm. MS: 313 (Mþ, 8), 282 (32), 206 (24),164 (15),136
(12), 108 (6), 79 (100). Elemental analysis for C16H15O2N3S (313.37)
Calcd: C, 61.32; H, 4.82; N, 13.41; S, 10.23. Found: C, 61.22; H, 4.76;
N, 13.34; S, 10.18
4.1.2. 3(3-MethoxyPhenyl)-2-thio-5,6,7,8,-tetrahydrobenzo[b]
thieno[2,3-d]pyrimidin-4-(3H)-one (4)
A mixture of compound 1 (0.01 mol), carbon disulfide (0.01 mol)
and sodium ethoxide (0.01 mol) in ethanol (20 ml) was refluxed for
6 h. The reaction mixture was acidified with HCl (10 ml, 20%) and
diluted with water (20 ml). The obtained solid was filtered off, dried
and crystallized from acetic acid to give dark brown crystals of 4.
Table 3
Antianxiety activities of known compounds (1, 7) and newly
synthesized compounds.
Yield (94%), m.p. 190 ꢂC, IR (KBr,
n
, cmꢃ1): 3428 (NH), 1690 (C]O).
¼ 1.20e2.90 (m, 8H, cyclohexane), 3.90 (s,
1H NMR (DMSO-d6):
d
Compound No.
Relative potencies
to Diazepam
3H, OCH3), 7.00e7.88 (m, 4H, Ar-H), 8.15 (s, 1H, NH) ppm. MS: 344
(Mþ, 32), 223 (100). Elemental analysis for C17H16N2O2S2 (344.45):
C, 59.28; H, 4.68; N, 8.13; S, 18.62. Found: C, 59.22; H, 4.63; N, 8.08;
S, 18.56.
Diazepam
1
3
4
1
4
4
3
5a
5b
6a
6b
7
8a
8b
8c
9a
9b
11
12
13
14
15
16
17
2
5
5
7
1
4
5
3
3
6
12
3
3
10
5
2
4.1.3. 2-(2-Substituted)-4,5,6,7-tetrahydro-N-(3-methoxyphenyl)
benzo[b]thiophene-3-carboxamide (5a, b)
A mixture of compound 1 (0.01 mol) and aroyl chloride, namely,
benzoyl chloride or cinamoyl chloride (0.0l mol) in acetic acid
(20 ml) was stirred at room temperature for 2 h. The reaction
mixture was concentrated under reduced pressure and then poured
onto water. The formed solid was filtered off, dried and crystallized
from acetic acid to give brown crystals of 5a, b respectively.
4.1.3.1. 2-Benzamido-N-(3-methoxyphenyl)-4,5,6,7-tetrahydrobenzo[b]
thiophene-3-carboxamide (5a). Yield (90%), m.p. 190 ꢂC, IR (KBr,
n,
cmꢃ1): 3426 (NH), 1666 (C]O). 1H NMR (DMSO-d6):
(m, 8H, cyclohexane), 3.76 (s, 3H, OCH3), 7.16e7.86 (m, 9H, Ar-H),
d
¼ 1.70e2.82
16
10.65, 12.30 (2s, 2H, 2 NH, exchangeable with D2O) ppm. MS: 406

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A.E.-G.E. Amr et al. / European Journal of Medicinal Chemistry 45 (2010) 5935e5942
(Mþ, 85), 329 (100). Elemental analysis for C23H22N2O3S (406.50):
Calcd.: C, 67.96; H, 5.46; N, 6.89; S, 7.89. Found: C, 67.90; H, 5.40; N,
6.84; S, 7.85.
(NH), 1680 (C]O). 1H NMR (DMSO-d6):
d
¼ 1.72e2.86 (m, 8H,
cyclohexane), 4.18 (s, 2H, CH2), 11.65 (s, 1H, NH exchangeable with
D2O) ppm. MS: 256 (Mþþ2, 32), 254 (Mþ, 100); Elemental analysis
for C11H11ClN2OS (254.74): Calcd.: C, 51.86; H, 4.35; Cl, 13.92; N,
11.00; S, 12.59. Found: C, 51.76; H, 4.30; Cl, 13.84; N, 10.88; S, 12.53.
4.1.3.2. 2-Cinnamamido-N-(3-methoxyphenyl)-4,5,6,7-tetrahydrobenzo
[b]thiophene-3-carboxamide (5b). Yield (85%), m.p.: 220 ꢂC, IR (KBr,
n,
cmꢃ1): 3432 (NH), 1668 (C]O). 1H NMR (DMSO-d6):
d
¼ 1.68e2.90
4.1.6. 2-Aryl-4,5,6,7-tetrahydrobenzo[b]-4H-thieno[2,3-d][1,3]
oxazin-4-imine (9a, b)
(m, 8H, cyclohexane), 3.70 (s, 3H, OCH3), 6.65e8.00 (m, 11H,
Ar-H þ CH]CH), 10.52, 11.85 (2s, 2H, 2 NH, exchangeable with
D2O) ppm. MS: 432 (Mþ, 14), 310 (100). Elemental analysis for
C25H24N2O3S (432.53): Calcd.: C, 69.42; H, 5.59; N, 6.48; S, 7.41. Found:
C, 69.34; H, 5.55; N, 6.42; S, 7.35.
A mixture of compounds 8a, b (0.01 mol) and conc. H2SO4
(0.01 mol) in xylene (20 ml) was refluxed for 8 h. After cooling, the
reaction mixture was poured onto ice-water, neutralized with
sodium carbonate, the obtained solid was filtered off, washed with
water, dried and crystallized from acetic acid to give brown crystals
of 9a, b respectively.
4.1.4. 2-(Supstituted)-3-(3-methoxyphenyl)-4,5,6,7-
tetrahydrobenzo[b]thieno[2,3-d]pyrimidin-4(3H)-one (6a, b)
A solution of compounds 5a, b (0.01 mol) in acetic anhydride
(20 ml) was refluxed for 2 h. The reaction mixture was concentrated
under reduced pressure, the solid obtained was collected by
filtration, dried and crystallized from acetic acid to give brown
crystals of 6a, b respectively.
4.1.6.1. 2-Phenyl-4,5,6,7-tetrahydrobenzo[b]-4H-thieno[2,3-d][1,3]
oxazin-4-imine (9a). Yield (80%), m.p. >360 ꢂC, IR (KBr, , cmꢃ1):
n
3434 (NH). 1H NMR (DMSO-d6):
d
¼ 1.70e2.92 (m, 8H, cyclo-
hexane), 7.05e8.00 (m, 5H, Ar-H), 12.15 (s, 1H, NH exchangeable
with D2O) ppm. MS: 282 (Mþ, 18), 179 (100); Elemental analysis
for C16H14N2OS (282.36): Calcd.: C, 68.06; H, 5.00; N, 9.92; S,
11.36. Found: C, 68.00; H, 4.96; N, 9.86; S, 11.30.
4.1.4.1. 2-(Phenyl)-3-(3-methoxyphenyl)-4,5,6,7-tetrahydrobenzo[b]
thieno[2,3-d]pyrimidin-4(3H)-one (6a). Yield (80%), m.p. >300 ꢂC,
IR (KBr,
n
, cmꢃ1): 1680 (C]O). 1H NMR (DMSO-d6):
d
¼ 1.73e2.92
4.1.6.2. 2-Cinnamoyl-4,5,6,7-tetrahydrobenzo[b]-4H-thieno[2,3-d]
(m, 8H, cyclohexane), 3.68 (s, 3H, OCH3), 7.24e8.02 (m, 9H, Ar-
H) ppm. MS: 390 (Mþþ2, 8), 204 (100); Elemental analysis for
C23H20N2O2S (388.48): Calcd.: C, 71.11; H, 5.19; N, 7.21; S, 8.25.
Found: C, 71.05; H, 5.14; N, 7.16; S, 8.20
[1,3]oxazin-4-imine (9b). Yield (78%), m.p. >360 ꢂC, IR (KBr,
n,
cmꢃ1): 3438 (NH). 1H NMR (DMSO-d6):
d
¼ 1.72e2.90 (m, 8H,
cyclohexane), 6.88e7.78 (m, 7H, Ar-H þ CH]CH), 12.18 (s, 1H, NH
exchangeable with D2O) ppm. MS: 308 (Mþ, 6), 129 (100);
Elemental analysis for C18H16N2OS (308.40): Calcd.: C, 70.10; H,
5.23; N, 9.08; S, 10.40. Found: C, 70.01; H, 5.17; N, 9.00; S, 10.35.
4.1.4.2. 2-(Cinnamoyl)-3-(3-methoxyphenyl)-4,5,6,7-tetrahydrobenzo
[b]thieno[2,3-d]pyrimidin-4(3H)-one (6b). Yield (78%), m.p. >300 ꢂC,
IR (KBr,
n
, cmꢃ1): 1676 (C]O). 1H NMR (DMSO-d6):
d
¼ 1.70e2.88
4.1.7. 4,5,6,7-Tetrahydro-2-(4-hydroxy-2-iminothiazol-3(2H)-yl)
benzo[b]thiophene-3-carbonitrile (11)
(m, 8H, cyclohexane), 3.70 (s, 3H, OCH3), 7.00e8.18 (m, 11H, Ar-
H þ CH]CH) ppm. MS: 414 (Mþ, 28), 103 (100); Elemental analysis
for C25H22N2O2S (414.52): Calcd.: C, 72.44; H, 5.35; N, 6.76; S, 7.74.
Found: C, 72.36; H, 5.30; N, 6.71; S, 7.68.
A
mixture of 8c (0.01 mol) and ammonium thiocynate
(0.01 mol) in absolute ethanol (20 ml) was refluxed for 3 h. The
reaction mixture was concentrated under reduced pressure, then
poured onto water, the separated solid was filtered off, dried and
crystallized from acetic acid to give brown crystals of 11. Yield
4.1.5. 2-N-Substituted-4,5.6.7-tetrahydrobenzo[b]thiophene-3-
carbonitrile (8aec)
(92%), m.p. >300 ꢂC, IR (KBr,
n
, cmꢃ1): 3648 (OH), 3365 (NH), 2208
¼ 1.70e2.87 (m, 8H,
A mixture of compound 7 (0.01 mol) and halo compounds,
namely, aroyl chloride and/or chloroacetylchloride (0.01 mol) in
dry acetone (20 ml) was refluxed for 3 h. The solvent was evapo-
rated under reduced pressure, the obtained residue was solidified
with diethyl ether, filtered off, washed with ether, dried and crys-
tallized from acetic acid to give white crystals of 8a, light green of
8b and light yellow crystal of 8c respectively.
(CN), 1660 (C]N). 1H NMR (DMSO-d6):
d
cyclohexane), 6.84 (s, 1H, thiazole-H), 10.65 (s, 1H, NH exchange-
able with D2O), 12.05 (s, 1H, OH exchangeable with D2O) ppm. MS:
277 (Mþ, 16), 114 (100); Elemental analysis for C12H11N3OS2
(277.37): Calcd.: C, 51.96; H, 4.00; N, 15.15; S, 23.12. Found: C, 51.90;
H, 3.92; N, 15.08; S, 23.06.
4.1.8. 4-(3-Cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-
4.1.5.1. N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)benza-
ylcarbamoyl)-2-oxobut-3-enoic acid (12)
mide (8a). Yield (95%), m.p. 196 ꢂC, IR (KBr,
n
, cmꢃ1): 3464 (NH),
A mixture of 7 (0.01 mol) and malic anhydride (0.01 mol) in
glacial acetic acid (50 ml) was heated under reflux for 6 h. The
reaction mixture was concentrated under reduced pressure, after
cooling, the obtained solid was filtered off and crystallized from
acetic acid to gave yellow crystals of 12. Yield (85%), m.p. 230 ꢂC, IR
2220 (CN), 1678 (C]O). 1H NMR (DMSO-d6):
d
¼ 1.65e2.86 (m, 8H,
cyclohexane), 7.23e8.00 (m, 5H, Ar-H), 11.54 (s, 1H, NH exchange-
able with D2O) ppm. MS: 282 (Mþ, 32), 205 (100); Elemental
analysis for C16H14N2OS (282.36): Calcd. C, 68.06; H, 5.00; N, 9.92; S,
11.36. Found: C, 68.00; H, 4.96; N, 9.85; S, 11.30.
(KBr,
NMR (DMSO-d6):
n H
, cmꢃ1): 3464 (OH), 2560 (NH), 2210 (CN), 1692 (C]O). 1
d
¼ 1.68e2.84 (m, 8H, cyclohexane), 6.08, 7.10
4.1.5.2. N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)cinna-
(2d, 2H, J ¼ 12.6 Hz, CH]CH), 10.55 (s, 1H, NH exchangeable with
D2O), 12.12 (s, 1H, OH exchangeable with D2O) ppm. MS: 304 (Mþ,
12), 259 (100); Elemental analysis for C14H12N2O4S (304.32): Calcd.:
C, 55.25; H, 3.97; N, 9.21; S, 10.54. Found: C, 55.19; H, 3.92; N, 9.16;
S, 10.50.
mamide (8b). Yield (95%), m.p. 196 ꢂC, IR (KBr, , cmꢃ1): 3464 (NH),
n
2220 (CN), 1678 (C]O). 1H NMR (DMSO-d6):
d
¼ 1.68e2.90 (m, 8H,
cyclohexane), 6.90e7.76 (m, 7H, Ar-H þ CH]CH), 11.42 (s, 1H, NH
exchangeable with D2O) ppm. MS: 310 (Mþþ2, 8), 131 (100);
Elemental analysis for C18H16N2OS (308.40): Calcd.: C, 70.10; H,
5.23; N, 9.08; S, 10.40. Found: C, 70.00; H, 5.18; N, 9.00; S, 10.35.
4.1.9. 4,5,6,7-Tetrahydro-2-(2,5-dioxo-2H-pyrrol-1(5H)-yl)benzo[b]
thiophene-3-carbonitrile (13)
4.1.5.3. 2-Chloro-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-
A solution of compound 12 (0.01 mol) in acetic anhydride
(20 ml) was refluxed for 2 h. The solid obtained on concentration
yl)acetamide (8c). Yield (68%); m.p. 287 ꢂC, IR (KBr, , cmꢃ1): 3426
n

A.E.-G.E. Amr et al. / European Journal of Medicinal Chemistry 45 (2010) 5935e5942
5941
and cooling was collected by filtration and crystallized from acetic
4.2. Pharmacological screening
acid to give brown crystals of 13. Yield (80%), m.p. >300 ꢂC, IR (KBr,
n
,
cmꢃ1): 2208 (CN), 1680 (C]O). 1H NMR (DMSO-d6):
¼ 1.65e2.90 (m, 8H, cyclohexane), 6.75 (d, 2H, pyrrol-H) ppm.
4.2.1. Antiarrhythmic activity [46e50]
d
4.2.1.1. Purpose and rational. The plant alkaloid aconitine persis-
tently activates sodium channel. Infusion of aconitine in the anes-
thetizedratcauses ventriculararrhythmias. Drugsconsideredtohave
antiarrhythmic properties can be tested in aconitine-intoxicated rats.
MS: 277 (Mþ, 16), 114 (100); Elemental analysis for C13H10N2O2S
(258.30): Calcd.: C, 60.45; H, 3.90; N, 10.85; S, 12.41. Found: C,
60.40; H, 3.85; N, 10.80; S, 12.35.
4.1.10. N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)
benzoylthiourea (14)
4.2.1.2. Procedure. Male Ivanovas rats weighing 300e350 g are
used. The animals are anesthetized by intra peritoneal injection of
1.25 g/kg urethane: 5 mg/kg aconitine dissolved in 0.1 N HNO3 is
adminstered by continuous infusion into the saphenous vein of
0.1 ml/min and the electrocardiogram (ECG) in lead II is recorded
every 30 s. The test compound is injected IV at a screening dose of
5 mg/kg 5 min before the start of the aconitine infusion, 24 animals
are used per compound.
A mixture of compound 7 (0.01 mol) and benzoylisothiocyanate
(0.01 mol) in dry acetone (20 ml) was refluxed for 1 h. The reaction
mixture was poured onto water, the separated solid was filtered off,
dried and crystallized from acetic acid to give brown crystals of 14.
Yield (92%), m.p. 180 ꢂC, IR (KBr, , cmꢃ1): 3290 (NH), 2210 (CN),
n
1678 (C]O). MS: 343 (Mþþ2, 6), 264 (100); Elemental analysis for
C17H15N3OS2 (341.45): Calcd.: C, 59.80; H, 4.43; N, 12.31; S, 18.78.
Found: C, 59.74; H, 4.36; N, 12.25; S, 18.70.
4.2.1.3. Evaluation. The antiarrhythmic effect of a test compound is
measured by the amount of aconitine/100 g animal. (Duration of
infusion) which induces.
4.1.11. (4-Imino-2-mercapto-4,5,6,7-terahydrobenzo[b]thieno[2,3-
d]pyrimidin-3(4H)-yl)(phenyl) methanone (15)
To a solution of compound 14 (0.01 mol) in dry acetone (20 ml),
anhydrous potassium carbonate (0.5 g) was added. The reaction
mixture was refluxed for 3 h, after cooling, it was poured onto
water. The formed solid was filtered off, dried and crystallized from
acetic acid to give yellow crystals of 15. Yield (78%), m.p. 134 ꢂC, IR
- Ventricular extra systoles.
- Ventricular tachycardia.
- Ventricular fibrillation.
Higher doses of aconitine in the treated group as compared to an
untreated control group are an indication of antiarrhythmic
activity.
Statistical significance between the groups is assessed by the
student’s t-test.
(KBr,
¼ 1.66e2.92 (m, 8H, cyclohexane), 7.15e7.96 (m, 5H, Ar-H), 9.90
(s, 1H, NH exchangeable with D2O), 14.10 (s, 1H, SH exchangeable
with D2O), ppm. 13C NMR (DMSO-d6):
n
, cmꢃ1): 3344 (NH), 1670 (C]O). 1H NMR (DMSO-d6):
d
d
¼ 23.10, 23.15, 23.65, 24.55
(4CH2), 127.34, 129.05, 136.85, 143.40 (thiophene ring), 126.12,
128.20, 131.10, 131.86 (Ph-C), 165.75 (C]NH), 164.10 (C-SH), 172.04
(C]O) ppm. MS: 341 (Mþ, 6), 164 (100); Elemental analysis for
C17H15N3OS2 (341.45): Calcd.: C, 59.80; H, 4.43; N, 12.31; S, 18.78.
Found: C, 59.74; H, 4.36; N, 12.24; S, 18.70.
4.2.2. Serotonin antagonist
4.2.2.1. Procedure.
1. Tris buffer pH 7.7
a. 57.29 Tris HCl, 16.2 g Tris base, q. s to/liter with distilled
water (0.5 M Tris buffer, pH 7.7)
b. Make a 1:10 dilution in deionized H2O (0.05 M Tris buffer,
pH 7.7).
4.1.12. 2-(Benzylideneamino)-4,5,6,7-tetrahydrobenzo[b]
thiophene-3-carbonitrile (16)
A mixture of compound 7 (0.01 mol) and benzaldehyde
(0.01 mol) in absolute ethanol (20 ml) was refluxed for 3 h. The
separated solid formed upon dilution with water (20 ml) was
filtered off, dried and crystallized from ethanol to give brown
c. 0.05 M Tris buffer, pH 7.7 containing 10
mM paragyline
4 mM CaCl2 and 0.1% ascorbic acid. 0.49 mg paragyline HCl,
111 mg CaCl2, 20 mg vitamin C, q. s. to 250 ml with 0.05 M
Tris buffer, pH 7.7 (reagent 1b).
crystals of 16. Yield (95%), m.p. 104 ꢂC, IR (KBr,
n
, cmꢃ1): 2204 (CN),
¼ 1.65e2.88 (m, 8H, cyclo-
1660 (C]N). 1H NMR (DMSO-d6):
d
hexane), 3.60 (s,1H, CH]N), 6.98e7.88 (m, 5H, Ar-H) ppm; MS: 268
(Mþþ2, 16), 104 (100); Elemental analysis for C16H14N2S (266.36):
Calcd.: C, 72.15; H, 5.30; N, 10.52; S, 12.04. Found: C, 72.08; H, 5.23;
N, 10.46; S, 11.95.
2. [3H]-DPAT(2-N,N-Di[2,3(n)-3propylamino)-8-hydroxy-1,2,3,4-
tetrahydronaphth-alene). (160e206 ci/mmol) was obtained
from Amersham. For IC50 determination ¼ a 10 nM stock
solution is made up and 50 mm3 are added to each tube (final
concentration ¼0.5 nM).
4.1.13. 4,5,6,7-Tetrahydro-2-(2,6-diphenyl-4-thioxo-2H-1,3,5-
oxadiazin-3(4H)-yl)benzo[b] thiophene-3-carbonitrile (17)
3. Serotonin sulphate. 0.5 nM stock solution is made up in 0.01 N
HCl and 20 mm3 added to 3 tubes for determination of non
A mixture of compound 16 (0.01 mol) and phenyl isothiocyanate
(0.01 mol) in dry acetone (20 ml) was refluxed for 6 h. The reaction
mixture was evaporated under reduced pressure, the obtained
residuewas trituratedwith n-hexane, the solid obtained was filtered
off, washed with n-hexane and diethyl ether, dried and crystallized
from acetic acid to give yellow crystals of 17. Yield (82%), m.p.156 ꢂC,
specific binding (final concentration ¼10
mM).
4. Test compound 1 mM stock solution is made up in a suitable
solvent and serially diluted, such that the final concentrations
in the assay range from 2 ꢄ 10ꢃ5 to 2 ꢄ 10ꢃ8 M. Seven
concentrations are used for each assay. Higher or lower
concentrations may be used based on the potency of the drug.
IR (KBr,
¼ 1.62e2.90 (m, 8H, cyclohexane), 5.62 (s, 1H, CH), 7.12e7.86 (m,
10H, Ar-H) ppm. 13C NMR (DMSO-d6):
n
, cmꢃ1): 2206 (CN), 1670 (C]N). 1H NMR (DMSO-d6):
d
d
¼ 22.75, 23.18, 23.65, 24.34
(4CH2), 82.80, 135.50, 136.10, 140.22 (thiophene ring), 116.56 (CN),
125.05,126.15,126.45,128.00,128.12,130.60,134.80,139.76 (2Ph-C),
86.65 (CH), 179.55 (C]S) ppm. MS: 429 (Mþ, 24), 267 (100);
Elemental analysis for C24H19N3OS2 (429.56): Calcd.: C, 67.11; H,
4.46; N, 9.78; S, 14.93. Found: C, 67.01; H, 4.39; N, 9.70; S, 14.86.
4.2.2.2. Tissue Preparation. Male Wister rats are sacrificed by
decapitation. Hippocampus are removed, weighed, and homoge-
nized in 20 volumes of 0.05 M Tris buffer, pH 7.7. The homogenate is

5942
A.E.-G.E. Amr et al. / European Journal of Medicinal Chemistry 45 (2010) 5935e5942
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centrifuged at 48000g for 10 min and the supernatant is discarded.
The pellet is re-suspended in an equal volume of 0.05 M Tris buffer,
incubated at 37 ꢂC for 10 min and re-centrifuged at 48000g for
10 min. The final membrane pellet is re-suspended in 0.05 M Tris
[8] K. Starcevic, M. Krali, I. Piantanida, L. Suman, K. Pavelic, G. Karminski-Zamola,
Eur. J. Med. Chem. 41 (2006) 925e939.
buffer containing 4 mM CaCl2, 0.1% vitamin C and 10
mM paragyline.
[9] Y.D. Wang, S. Johnson, D. Powell, J.P. McGinnis, M. Miranda, S.K. Rabindran,
Bioorg. Med. Chem. 15 (2007) 3763e3766.
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4.2.2.3. Assay.
- 800 mm3 Tissue
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223e227.
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- 130 mm3 0.05 M Tris þ CaCl2 þ paragyline þ vitamin C
- 20 mm3 vehicle/5HT/drug.
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Pharmacol. 14 (1992) 183e188.
[15] S.J. Peroutka, J. Neurochem. 47 (1986) 925e932.
- 50 mm3 [3H] DPAT.
[16] A.E. Amr, Indian J. Heterocycl. Chem. 10 (2000) 49e58.
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433e440.
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Tubes are incubated for 15 min at 25 ꢂC. The assay is stopped by
vacuum filtration through whatman GF/B filters, which are then
washed 2 times with 5 cm3 of ice-cold 0.05 M Tris buffer. The filters
are then placed into scintillation vials with 10 cm3 of liquiscint
scintillation cocktail and counted.
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(2003) 1985e1993.
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1825e1833.
4.2.3. Antianxiety test in mice
4.2.3.1. Procedure. The testing apparatus consists of a light and
a dark chamber that was divided by a photo cell-equipped zone, A
polypropylene animal cage 44 ꢄ 21 cm, is darkened with black
spray over one-third. A partition containing a 13 cm long x5 cm
high opening separates the dark on third from the bright two thirds
of the cage. The cage rests on an animex activity monitor, which
counts total locomotor activity. An electronic system using four sets
of photo cells across the partition automatically counts movements
through the partition and clocks the time spent in the light and
dark compartments. Nanive male albino mice with a body weight of
18e25 g are placed into the cage. The animals are treated (30 min
before the experiment) with the tested drugs or the vehicle intra-
peritoneally and are then observed for 10 min.
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Acknowledgement
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195e200.
The kind help of Dr. M. M. Abdalla, Research Unit, Hi-Care
Pharmaceutical Co., Cairo, Egypt, for carrying out the pharmaco-
logical screening is highly appreciated.
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