Synthesis and serotonin antagonist and antianexity activities of pyrrolidine derivatives from 4-hydrazinyl-1-p-substituted phenyl-2,5-dihydro-1H- pyrrole-3-carbonitriles

Article abstract of DOI:10.1007/s00706-008-0012-7

N-(p-substituted phenyl)-4-cyanopyrrolidin-3-ones and their corresponding hydrazines were prepared and used as starting materials to synthesize heterocyclic candidates as serotonin antagonist and antianexity agents. Condensation of hydrazines with selecte

Full text of DOI:10.1007/s00706-008-0012-7

Monatsh Chem (2009) 140:129–137
DOI 10.1007/s00706-008-0012-7
ORIGINAL PAPER
Synthesis and serotonin antagonist and antianexity activities
of pyrrolidine derivatives from 4-hydrazinyl-1-p-substituted
phenyl-2,5-dihydro-1H-pyrrole-3-carbonitriles
Mohamed M. Abdalla Æ Bakr F. Abdel-Wahab Æ
Abdel-Galil E. Amr
Received: 25 April 2008 / Accepted: 20 May 2008 / Published online: 30 September 2008
Ó Springer-Verlag 2008
Abstract N-(p-substituted phenyl)-4-cyanopyrrolidin-3-
ones and their corresponding hydrazines were prepared and
used as starting materials to synthesize heterocyclic candi-
dates as serotonin antagonist and antianexity agents.
Condensation of hydrazines with selected aromatic
aldehydes afforded the corresponding Schiff bases. The
hydrazines were treated with phenyl isothiocyanate to afford
the corresponding thiosemicarbazides, which were cyclized
with ethyl bromoacetate to N-phenylthiazolidinones. The
hydrazine was reacted with 1,2,4,5-tetrachlorophthalic
anhydride to give the tetrachloroimide derivative. It was
reacted with benzoyl acetonitrile, 2-(bismethylsulfanyl-
methylene)malononitrile, 2-ethoxymethylenemalononitrile,
or 2-cyano-3-ethoxyacrylic acid ethyl ester to afford the
corresponding pyrazoline derivatives. Schiff bases were
obtained by simple condensation of the hydrazine with
different carbonyl compounds. All the compounds were
screened for their serotonin antagonistic and antianexity
activities, and they showed high activities compared to
buspirone and diazepam as controls.
Introduction
A literature survey reveals that the pyrrolidine ring is
important for antimycobacterial activity [1]. In addition,
many hetero-nitrogen derivatives exhibit a wide variety of
biological activities, such as antimicrobial [2], anti-
inflammatory [3], antihistaminic [4], antihypertensive [5],
hypnotic [6] and anticonvulsant [7] activity. From previous
work, Schiff bases are known to possess antimicrobial
[8–11] and anti-inflammatory [12, 13] activities. Also,
thiosemicarbazide and triazole derivatives have been found
to be of interest with potential activities including antimi-
crobial, analgesic and anticonvulsant [14–17]. In addition,
we have reported that certain of our new heterocyclic
compounds exhibited antiparkinsonian [18], antitumor
[19–21], antimicrobial [22] and anti-inflammatory [23]
activities. In view of these observations and in continuation
of our previous work in heterocyclic chemistry, we herein
synthesized some new substituted pyrrolidine derivatives
for their pharmacological screening.
Keywords Pyrrolidinone Á Thiosemicarbazide Á
Schiff bases Á Pharmacological activities
Results and discussion
Chemistry
N-(p-substituted phenyl)-4-cyano-pyrrolidin-3-ones 1a and
1b were synthesized according to the reported procedure
[24] and used as starting materials. They were treated with
hydrazine hydrate to afford the corresponding hydrazine
derivatives 2a and 2b [25], which were reacted with
p-chlorobenzaldehyde or p-nitrobenzaldehyde in refuxing
ethanol to afford the corresponding Schiff bases 3a–3c.
Condensation of hydrazines 2a and 2b with phenyl iso-
thiocyanate afforded the corresponding thiosemicarbazide
M. M. Abdalla
Research Units, Hi-Care Pharmaceutical Co., Cairo, Egypt
B. F. Abdel-Wahab Á A.-G. E. Amr (&)
Applied Organic Chemistry Department,
National Research Center, Dokki, Cairo, Egypt
e-mail: aamr1963@yahoo.com
123

130
M. M. Abdalla et al.
Ph
derivatives 4a and 4b, which were cyclized with ethyl
bromoacetate in the presence of sodium acetate to afford
the corresponding N-phenyl thiazolidinone derivatives 5a
and 5b (Scheme 1).
SMe
N
N
N
NC
NC
N
CN
NH2
NH2
N
N
The hydrazine 2b was reacted with 1,2,4,5-tetrachlor-
ophthalic anhydride in refluxing glacial acetic acid to give
the corresponding imide 6. Compounds 2a and 2b were
treated with benzoyl acetonitrile to afford the correspond-
ing 3,5-disubstituted pyrazole derivatives 7a and 7b.
Compound 2b was reacted with 2-(bis-methylsulfanyl-
methylene)malononitrile, 2-ethoxy-methylenemalononitrile,
or 2-cyano-3-ethoxyacrylic acid ethyl ester to afford the
corresponding 3,4,5-tri- and 4,5-disubstituted pyrazolines
8, 9a and 9b (Scheme 2).
R
OMe
7a, R = Me
7b, R = OMe
8
NC
NC
SMe
SMe
PhCOCH2CN
2b
Cl
Cl
O
H
O
Cl
Cl
Schiff bases 10–13 were obtained via simple conden-
sation of the hydrazine 1b with 1-(4-methoxyphenyl)-
4-oxopyrrolidine-3-carbonitrile, indan-1,3-dione, acetyl
derivatives, or cycloalkanones in refluxing ethanol con-
taining a catalytic amount of acetic acid, thus affording the
Schiff bases 10–13 (Scheme 3).
NC
N
O
O
Cl
N
N
Cl
O
2a, R = Me
2b, R = OMe
2b
Cl
Cl
6
EtO
X
N
OMe
NC
N
2b
X
CN
NH2
N
H
NC
N
N
OMe
ArCHO
N
R
X
9a, X = CN
9b, X = COOEt
Scheme 2
3a, R = Me, X = Cl
3b, R = Me, X = NO
Pharmacological screening
2
3c, R = OMe, X = Cl
Serotonin antagonist activity
H
N
H
N
S
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 receptors were
designed as the 5HT1A subtype and the insensitive recep-
tors were referred as the 5HT1B subtype. Schlegel and
Peroutka identified [3H] DPAT as a selective ligand for
5HT1 receptors [26].
NC
O
NC
NC
Ph
NH2
PhNCS
N
H
N
H
N
N
N
R
NH2NH2.H2O
EtOH
R
R
4a, R = Me
4b, R = OMe
1a, R = Me
1b, R = OMe
2a, R = Me,
2b, R = OMe
H
S
Specific binding is defined as the difference between
total binding and binding in the presence of 10 lM 5HT
[26, 27]. IC50 values are calculated from the percent spe-
cific binding at each drug concentration (Table 1).
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 rel-
atively higher affinity for the 5HT1A receptor than other
receptors and no effect on the benzodiazepine site, their
NC
N
BrCH2COOEt
NaOAc
N
N
O
Ph
N
R
5a, R = Me
5b, R = OMe
Scheme 1
123

Synthesis and serotonin antagonist and antianexity activities of pyrrolidine derivatives
131
OMe
Table 1 Serotonin antagonist activities of the new compounds
Compound
Concentration/mg kg-1
IC50/mg kg-1
H
N
N
2 9 10-7
2 9 10-8
2 9 10-7
2 9 10-8
2 9 10-7
2 9 10-8
2 9 10-7
2 9 10-8
2 9 10-7
2 9 10-8
2 9 10-7
2 9 10-8
2 9 10-7
2 9 10-8
2 9 10-7
2 9 10-8
2 9 10-7
2 9 10-8
2 9 10-7
2 9 10-8
2 9 10-7
2 9 10-8
2 9 10-7
2 9 10-8
2 9 10-7
2 9 10-8
2 9 10-7
2 9 10-8
2 9 10-7
2 9 10-8
2 9 10-7
2 9 10-8
2 9 10-7
2 9 10-8
2 9 10-7
2 9 10-8
2 9 10-7
2 9 10-8
2 9 10-7
2 9 10-8
2 9 10-7
2 9 10-8
63
77
58
80
62
75
62
75
63
87
63
87
39
67
66
77
66
87
63
86
39
65
61
75
60
73
47
56
62
75
38
67
68
79
83
89
66
77
77
96
63
87
NC
O
CN
3a
N
CN
N
N
3b
4a
10
OMe
O
O
OMe
4b
H
N
H
N
NC
NC
NH2
N
4c
N
N
O
5a
5b
OMe
11
OMe
O
2b
6
H
N
Me
Ar
Me
O
NC
N
Ar
7a
(
)n
N
7b
H
N
NC
(
)n
N
8
OMe
N
12a, Ar = 2-Thienyl
12b, Ar = 2-Furyl
12c, Ar = 2-Pyrrolyl
9a
9b
OMe
13a, n = 1
13b, n = 2
13c, n = 3
10
11
Scheme 3
12a
12b
12c
13b
13c
Buspirone
anxiolytic properties are attributed to activity at the 5HT1A
receptor. So all derivatives were subjected to anxiolytic
screening. From the results in Table 1, all the tested
compounds showed potent serotonin antagonist activities;
the order of descending activities is 13c, 12c, (5a, 4c and
Buspirone), 7a, 7b, 3b, 12b, (1, 3b and 6), 3a, (11, 4b and
4a), 9a, 9b, 12a, 5b, 8 and 10.
Structure activity relationship
–
Schiff’s bases with alicyclic compounds give the
highest activity (as the carbon atoms of the alicyclic
ring system increase the activity increases).
The p-methoxy group sharply increases the activities.
Steric hindrance tend to diminish the activity to
moderate levels.
Antianexity test in mice
–
–
Crawley and colleagues have described a simple behavioral
model in mice to detect compounds with anxiolytic effects.
Mice tend to explore a novel environment, but to retreat
–
5-Amino-3-phenyl pyrazol is more active than its
bioisosteric methylthiol.
123

132
M. M. Abdalla et al.
Table 2 Antianexity activities
of new compounds
Table 3 Acute toxicity (LD50)
of the synthesized compounds
Compound Relative potencies
to diazepam
Compound
LD50/mg/kg
3a
321 0.10
294 0.19
243 0.18
376 0.18
344 0.11
235 0.17
252 0.12
124 0.18
324 0.18
136 0.16
497 0.15
221 0.67
342 0.18
524 0.18
308 0.19
387 0.18
303 0.18
236 0.15
346 0.16
434 0.17
274 0.18
113 0.18
102 0.10
3a
2
4
3b
3b
4a
4a
3
4b
4b
2
4c
4c
1
5a
5a
12
3
5b
5b
6
6
4
7a
7a
4
7b
7b
24
6
8
8
9a
9a
7
9b
9b
8
10
10
3
11
11
5
12a
12b
12c
13a
13b
13c
Buspirone
Diazepam
12a
12b
12c
13a
13b
13c
Diazepam
22
5
8
6
9
5
1
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 crossings between the two chambers and more
locomotor activity after treatment with anxiolyties [28–30].
Dose response curves were 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-made decreases in total activity, such
as that made by chlorpromazine 10 mg/kg s.c., and the
relative potencies to chlorpromazine were determined.
From the results in Table 2, all the tested compounds
showed potent antianexity activities; the order of descend-
ing activities is 7b, 12a, 5a, 13b, (12c and 9b), 9a, (8 and
13a), (13c, 12b and 11), (6, 7a and 3b), (5b, 4a and 10),
3a and 3b.
–
–
Schiff’s bases with an alicyclic are lees active than
those containing heteroatoms or aromatic systems.
Steric hindrance tend to diminish the activity to
moderate levels
Determination of acute toxicity (LD50)
The LD50 was determined by using rats. They were injected
with a range of increasing doses of the synthesized com-
pounds. The dose that killed 50% of the animals was
calculated according to Austen and Brocklehurst [31]
(Table 3).
Experimental
Melting points were determined on open glass capillaries
using an Electrothermal IA 9000 digital melting point
apparatus. Elemental analyses were performed on Ele-
mentar Vario EL (Microanalytical Unit, National Research
Center, Cairo, Egypt), and were found within 0.4% of the
theoretical values. Infrared spectra were recorded on a
Carl Zeiss Spectrophotometer (model UR 10) using the
Structure activity relationship
–
–
The pyrazol ring is essential for high antianexity
activity.
(5-Amino-3-phenyl-1H-pyrazol-1-yl) provides higher
activity than the (4-cyano-1-(4-methoxyphenyl)-2,5-
dihydro-1H-pyrrol-3-yl), while the later is more active
than the [4-cyano-1-(4-methoxyphenyl)-2,5-dihydro-
1H-pyrrol-3-yl] residue.
1
KBr disc technique. H-NMR spectra were recorded on a
Varian Gemini 270 MHz spectrometer (DMSO-d6) and the
123

Synthesis and serotonin antagonist and antianexity activities of pyrrolidine derivatives
133
1-(4-Cyano-2,5-dihydro-1-p-substitued phenyl-1H-pyrrol-
3-yl)-4-phenylthiosemicarbazide (4a and 4b)
A mixture of 5 mmol 2a or 2b and 0.67 g phenylisothi-
ocyanate (5 mmol) in 30 cm3 absolute ethanol was
refluxed for 1.5 h. The formed solid was filtered off, dried
and crystallized to give 1.46 g 4a (84%) and 1.48 g 4b
(81%).
chemical shifts are given in d (ppm) downfield from tet-
ramethylsilane as an internal standard. The mass spectra
were measured using a Finnigan SSQ 7000 mass spec-
trometer. Follow up of the reactions and checking the
purity of the compounds was made by TLC on silica gel-
precoated aluminum sheets (Type 60 F254
; Merck,
Darmstadt, Germany). The starting materials 1, 2a, and 2b
were prepared according to Southwich et al. [24] and Amer
et al. [25].
2-(4-Cyano-1-p-tolyl-2,5-dihydro-1H-pyrrol-3-yl)-N-phen-
ylhydrazinecarbothioamide (4a, C19H19N5S)
ꢀ
Mp 195–196 °C (DMF/EtOH); IR (film): m = 1,257 (C=S)
4-(2-Arylidenehydrazinyl)-2,5-dihydro-1-(4-substituted
phenyl)-1H-pyrrole-3-carbonitriles (3a–3c)
cm-1, 2,249 (CN) cm-1, 3,205 (NH) cm-1 1H NMR
;
(DMSO-d6): d = 2.11 (s, CH3), 4.08, 4.15 (2s, 2CH2),
6.39–7.11 (m, Ar-H), 8.01, 9.11, 10.01 (3s, 3 NH,
exchangeable with D2O) ppm; MS (EI, 70 eV): m/
z = 350 (M? ?1, 2) and at 108 (100, base peak).
A mixture of 5 mmol 2a or 2b and 5 mmol p-chloro-
benzaldehyde or p-nitrobenzaldehyde in 30 cm3 absolute
ethanol in the presence of few drops of AcOH was
refluxed for 1 h. The reaction mixture was cooled and
the formed solid was filtered off, dried and crystallized
to give 1.48 g 3a (88%), 1.6 g 3b (94%) and 1.37 g
3c (78%).
2-(4-Cyano-1-(4-methoxyphenyl)-2,5-dihydro-1H-pyrrol-
3-yl)-N-phenylhydrazine-carbothioamide
(4b, C19H19N5OS)
ꢀ
Mp 186–188 °C (DMF/EtOH); IR (film): m = 1,268 (C=S)
cm-1, 2,251 (CN) cm-1, 3,169–3,343 (NH) cm-1 1H
;
4-(2-(4-Chlorobenzylidene)hydrazinyl)-1-p-tolyl-2,5-dihy-
dro-1H-pyrrole-3-carbonitrile (3a, C19H17ClN4)
NMR (DMSO-d6): d = 3.61 (s, OCH3), 4.10, 4.18 (2s,
2CH2), 6.42–7.19 (m, Ar-H), 8.12, 9.03, 10.12 (3s, 3 NH,
exchangeable with D2O) ppm; MS (EI, 70 eV): m/z = 366
(M? ?1, 5) and at 108 (100, base peak).
ꢀ
Mp 210–211 °C (DMF/EtOH); IR (film): m = 1,660 (C=O)
cm-1, 2,224 (CN) cm-1, 3,213 (NH) cm-1 1H NMR
;
(DMSO-d6): d = 2.21 (s, CH3), 4.08, 4.20 (2s, 2 CH2),
7.49 (d, J = 7.90 Hz Ar-H), 7.56 (d, J = 7.90 Hz Ar-H),
7.72 (d, J = 7.92 Hz Ar-H), 7.86 (d, J = 7.92 Hz Ar-H),
8.10 (s, olefinic CH), 8.72 (s, NH, exchangeable with D2O)
ppm; MS (EI, 70 eV): m/z = 338 (M? ?1, 12) and at 336
(100, base peak).
4-(2-(4-Oxo-3-phenylthiazolidin-2-ylidene)hydrazinyl)-1-
(p-substituted phenyl)-2,5-dihydro-1H-pyrrole-3-carboni-
trile (5a and 5b)
A mixture of 1 mmol 3a or 3b and 0.17 g ethyl bromo-
acetate (1 mmol) in 30 cm3 absolute ethanol in the
presence of 0.33 g anhydrous sodium acetate (4 mmol)
was refluxed for 6 h. The reaction mixture was cooled,
diluted with water and allowed to stand overnight, and the
obtained solid was filtered off, washed with water, dried
and crystallized to give 0.25 g 5a (64%) and 0.24 g 5b
(61%).
4-(2-(4-Nitrobenzylidene)hydrazinyl)-1-p-tolyl-2,5-dihy-
dro-1H-pyrrole-3-carbo-nitrile (3b, C19H17N5O2)
ꢀ
Mp 229–230 °C (DMF/EtOH); IR (film): m = 1,673 (C=N)
cm-1, 2,210 (CN) cm-1, 3,198 (NH) cm-1 1H NMR
;
(DMSO-d6): d = 2.19 (s, CH3), 4.06, 4.19 (2s, 2 CH2),
6.89 (d, J = 7.92 Hz Ar-H), 7.12 (d, J = 7.92 Hz Ar-H),
7.41 (d, J = 7.90 Hz Ar-H), 7.75 (d, J = 7.90 Hz Ar-H),
8.12 (s, olefinic-CH), 9.13 (s, NH, exchangeable with D2O)
ppm; MS (EI, 70 eV): m/z = 348 (M? ?1, 10) and at 347
(100, base peak).
4-(2-(4-Oxo-3-phenylthiazolidin-2-ylidene)hydrazinyl)-1-
p-tolyl-2,5-dihydro-1H-pyrrole-3-carbonitrile
(5a, C21H19N5OS)
ꢀ
Mp 185–186 °C (EtOH); IR (film): m = 1,605 (C=N)
cm-1, 1,735 (C=O) cm-1, 2,224 (CN), 3,206 (NH) cm-1
;
4-(2-(4-Chlorobenzylidene)hydrazinyl)-1-(4-methoxy-
phenyl)-2,5-dihydro-1H-pyrrole-3-carbonitrile
(3c, C19H17ClN4O)
1H NMR (DMSO-d6): d = 2.13 (s, CH3), 3.91 (s, SCH2),
4.12, 4.19 (2s, 2CH2), 6.51, 6.87 (2d, J = 7.95 Hz Ar-H),
11.12 (s, NH exchangeable with D2O) ppm; MS (EI,
70 eV): m/z = 389 (M?, 11) and at 77 (100, base peak).
ꢀ
Mp 200–202 °C (DMF/EtOH); IR (film): m = 1,641 (C=N)
cm-1, 2,195 (CN) cm-1, 3,196 (NH) cm-1 1H NMR
;
(DMSO-d6): d = 3.53 (s, OCH3), 4.11, 4.23 (2s, 2 CH2),
7.11 (d, J = 7.94 Hz Ar-H), 7.43 (d, J = 7.94 Hz Ar-H),
7.51 (d, J = 7.92 Hz Ar-H), 7.60 (d, J = 7.92 Hz Ar-H),
8.13 (s, olefinic-CH), 8.99 (s, NH, exchangeable with D2O)
ppm; MS (EI, 70 eV): m/z = 353 (M?, 5) and at 108 (100,
base peak).
1-(4-Methoxyphenyl)-4-(2-(4-oxo-3-phenylthiazolidin-2-
ylidene)hydrazinyl)-2,5-dihydro-1H-pyrrole-3-carbonitrile
(5b, C21H19N5O2S)
ꢀ
Mp 209–210 °C (EtOH); IR (film): m = 1,601 (C=N)
cm-1, 1,733 (C=O) cm-1, 2,219 (CN) cm-1, 3,198 (NH)
123

134
M. M. Abdalla et al.
1
cm-1; H NMR (DMSO-d6): d = 3.49 (s, OCH3), 4.01 (s,
SCH2), 4.11, 4.20 (2s, 2CH2), 6.53, 6.88 (2d, J = 7.93 Hz
Ar-H), 11.25 (s, NH, exchangeable with D2O) ppm; MS
(EI, 70 eV): m/z = 405 (M?, 10) and at 77 (100, base
peak).
(65%). Mp 230–231 °C (MeOH); IR (film): ꢀm = 1,653
(C=N) cm-1, 2,214, 2,249 (2CN) cm-1, 3,263, 3,220
1
(NH2) cm-1; H NMR (DMSO-d6): d = 2.41 (s, SCH3),
3.62 (s, OCH3), 4.15, 4.23 (2s, 2 CH2), 6.51, 6.83 (2d,
J = 7.95 Hz Ar-H), 8.21 (s, NH2, exchangeable with D2O)
ppm; MS (EI, 70 eV): m/z = 352 (M?, 2) and at 134 (100,
base peak).
1-(4-Methoxyphenyl)-4-(4,5,6,7-tetrachloro-1,3-dioxoiso-
indolin-2-yl-amino)-2,5-dihydro-1H-pyrrole-3-carbonitrile
(6, C20H12Cl4N4O3)
Synthesis of pyrazolocarbonitrile derivatives
(9a and 9b)
A mixture of 0.23 g 2b (1 mmol) and 0.29 g 3,4,5,6-
tetrachlorophthalic anhydride (1 mmol) in 50 cm3 AcOH
was heated under reflux for 6 h. The reaction mixture was
concentrated under reduced pressure, the obtained solid
was filtered off and crystallized to yield 0.41 g 6 (83%).
A mixture of 0.23 g 2b (5 mmol) and 2-(ethoxymethyl-
ene)malononitrile or ethyl 2-cyano-3-ethoxyacrylate
(5 mmol) in 25 cm3 absolute ethanol was refluxed for 4 h.
After cooling, the formed solid was filtered off, dried and
crystallized to give 0.88 g 9a (58%) and 0.92 g 9b (52%).
ꢀ
Mp [ 300 °C (AcOH/H2O); IR (film): m = 1,749, 1,779 (2
C=O) cm-1, 2,206 (CN) cm-1 1H NMR (DMSO-d6):
;
d = 3.65 (s, OCH3), 4.10 (s, CH2), 4.14 (s, CH2), 6.56,
6.86 (2d, J = 7.95 Hz Ar-H), 10.25 (s, NH, exchangeable
with D2O) ppm; MS (EI, 70 eV): m/z = 500 (M? ?2, 12)
and at 277 (100, base peak).
5-Amino-1-(4-cyano-1-(4-methoxyphenyl)-2,5-dihydro-1H-
pyrrol-3-yl)-1H-pyrazole-4-carbonitrile
(9a, C16H14N6O)
ꢀ
Mp 193–195 °C (EtOH); IR (film): m = 1,660 (C=N)
4-(5-Amino-3-phenyl-1H-pyrazol-1-yl)-2,5-dihydro-1-p-
substitued phenyl-1H-pyrrole-3-carbonitrile (7a and 7b)
A mixture of 5 mmol 2a or 2b and 0.73 g benzoylaceteo-
nitrile (5 mmol) in 20 cm3 absolute ethanol was refluxed
for 4 h. The reaction mixture was cooled, the formed solid
was filtered off, dried and crystallized to afford 0.75 g 7a
(44%) and 0.86 g 7b (48%).
cm-1, 2,213, 2,252 (2 CN) cm-1, 3,281, 3,240 (NH2)
cm-1; H NMR (DMSO-d6): d = 3.39 (s, OCH3), 4.48 (s,
CH2), 4.85 (s, CH2), 6.30 (s, pyrazole-CH), 6.62, 6.92 (2d,
J = 7.90 Hz Ar-H), 8.52 (s, NH2, exchangeable with D2O)
ppm; MS (EI, 70 eV): m/z = 306 (M?, 100, base peak).
1
Ethyl 5-amino-1-(4-cyano-1-(4-methoxyphenyl)-2,5-dihy-
dro-1H-pyrrol-3-yl)-1H-pyrazole-4-carboxylate
(9b, C18H19N5O3)
4-(5-Amino-3-phenyl-1H-pyrazol-1-yl)-1-p-tolyl-2,5-dihy-
dro-1H-pyrrole-3-carbonitrile (7a, C21H19N5)
ꢀ
Mp 166–167 °C (EtOH); IR (film): m = 1,652 (C=N)
Mp 150–151 °C (EtOH); IR (film): ꢀm = 1,598 (C=N)
cm-1, 1,686 (C=O) cm-1, 2,215 (CN) cm-1, 3,235, 3,219
1
cm-1, 2,198 (CN) cm-1, 3,198, 3,218 (NH2) cm-1; H
(NH2) cm-1 1H NMR (DMSO-d6): d = 1.41 (t, CH3),
;
NMR (DMSO-d6): d = 2.15 (s, CH3), 3.92, 4.25 (2s, 2
CH2), 6.38 (s, pyrazole-CH), 6.61–7.08 (m, Ar-H), 8.31
(s, NH2, exchangeable with D2O) ppm; MS (EI, 70 eV):
m/z = 341 (M?, 6) and at 321 (100, base peak).
3.39 (s, OCH3), 3.96 (q, CH2), 4.27 (s, CH2), 4.41 (s, CH2),
6.19 (s, pyrazole-CH), 6.70, 6.91 (2d, J = 7.92 Hz Ar-H),
8.63 (s, NH2, exchangeable with D2O) ppm; MS (EI,
70 eV): m/z = 353 (M?, 100, base peak).
4-(5-Amino-3-phenyl-1H-pyrazol-1-yl)-1-(4-methoxy-
phenyl)-2,5-dihydro-1H-pyrrole-3-carbonitrile
(7b, C21H19N5O)
Synthesis of Schiff bases (10 and 11)
ꢀ
Mp 177–179 °C (EtOH); IR (film): m = 1,619 (C=N)
A mixture of 0.46 g 2b (2 mmol) and 1-(4-methoxy-
phenyl)-4-oxopyrrolidine-3-carbonitrile or 1, 3-indanedione
(5 mmol) in 50 cm3 absolute ethanol in the presence of drops
of acetic acid was refluxed for 1 h. The formed solid was
filteredoff, dried and recrystallized to afford 0.75 g 10 (88%)
and 0.60 g 11 (84%).
1
cm-1, 2,203 (CN) cm-1, 3,218, 3,201 (NH2) cm-1; H
NMR (DMSO-d6): d = 3.54 (s, OCH3), 3.92, 4.25 (2s, 2
CH2), 6.38 (s, pyrazole-CH), 6.61–7.08 (m, Ar-H), 8.31 (s,
NH2, exchangeable with D2O) ppm; MS (EI, 70 eV): m/
z = 357 (M?, 4) and at 321 (100, base peak).
5-Amino-1-(4-cyano-1-(4-methoxyphenyl)-2,5-dihydro-1H-
pyrrol-3-yl)-3-(methylthio)-1H-pyrazole-4-carbonitrile
(8, C17H16N6OS)
4-(2-(4-Cyano-1-(4-methoxyphenyl)pyrrolidin-3-yli-
dene)hydrazinyl)-1-(4-methoxy-phenyl)-2,5-dihydro-1H-
pyrrole-3-carbonitrile (10, C24H24N6O2)
A mixture of 0.46 g 2b (2 mmol) and 0.34 g 2-[bis(meth-
ylthio)methylene]malononitrile (2 mmol) in 50 cm3
absolute ethanol was refluxed for 3 h. The formed solid
was filtered off, dried and crystallized to give 0.46 g 8
Mp 249–50 °C (DMF/EtOH); IR (film): ꢀm = 1,656 (C=N)
1
cm-1, 2,205, 2,254 (2CN) cm-1, 3,198 (NH) cm-1; H
NMR (DMSO-d6): d = 3.31, 3.52 (s, 2 OCH3), 3.97- 4.10
(m, CHCN and CH2), 4.21, 4.28, 4.32 (3s, 3CH2), 6.46–
123

Synthesis and serotonin antagonist and antianexity activities of pyrrolidine derivatives
135
6.88 (m, Ar-H), 9.67 (s, NH, exchangeable with D2O) ppm;
MS (EI, 70 eV): m/z = 428 (M?, 15) and at 316 (100, base
peak).
Synthesis of 4-(2-cycloalkylidenehydrazinyl)-1-(4-
methoxyphenyl)-2,5-dihydro-1H-pyrrole-3-carbonitrile
(13a–13c)
1-(4-Methoxyphenyl)-4-(2-(3-oxo-2,3-dihydroinden-1-yli-
dene)hydrazinyl)-2,5-dihydro-1H-pyrrole-3-carbonitrile
(11, C21H18N4O2)
A mixture of 0.46 g 2b (2 mmol) and cycloalkanone deriva-
tive, namely, cyclopentanone, cyclohexanone, or
cycloheptanone (2 mmol) in 20 cm3 absolute ethanol in the
presence of AcOH as a catalyst was refluxed for 1 h. The
formed solid was filtered off, dried and recrystallized to afford
0.5 g 13a (85%), 0.54 g 13b (88%) and 0.54 g 13c (83%).
ꢀ
Mp 230–31 °C (DMF/EtOH); IR (film): m = 1,648 (C=N)
cm-1, 1,713 (C=O) cm-1, 2,197 (CN) cm-1, 3,220 (NH)
1
cm-1; H NMR (DMSO-d6): d = 2.39 (s, CH2), 3.35 (s,
OCH3), 4.12 (s, CH2), 4.22 (s, CH2), 6.52–7.80 (m, Ar-H),
10.42 (s, NH, exchangeable with D2O) ppm; MS (EI,
70 eV): m/z = 359 (M? ?1, 18) and at 212 (100, base
peak).
4-(2-Cyclopentylidenehydrazinyl)-1-(4-methoxyphenyl)-
2,5-dihydro-1H-pyrrole-3-carbonitrile
(13a, C17H20N4O)
ꢀ
Mp 209–210 °C (DMF/EtOH); IR (film): m = 1,634 (C=N)
1
Synthesis of 1-(4-methoxyphenyl)-4-(2-(1-aryl-
ethylidene)hydrazinyl)-2,5-dihydro-1H-pyrrole-3-
carbonitrile (12a–12c)
cm-1, 2,209 (CN) cm-1; H NMR (DMSO-d6): d = 1.38
(s, cyclopentane), 2.10 (s, cyclopentane), 2.27 (s, cyclo-
pentane), 3.35 (s, OCH3), 4.12 (s, CH2), 4.23 (s, CH2),
6.57, 6.82 (2d, J = 7.95 Hz Ar-H), 10.12 (s, NH,
exchangeable with D2O) ppm; MS (EI, 70 eV):
m/z = 296 (M?, 28) and at 160 (100, base peak).
A mixture of 0.46 g 2b (2 mmol) and appropriate methyl
ketone, namely, 2-acetylthiophene, 2-acetylfurane or 2-
acetylpyrrole (2 mmol) in 20 cm3 absolute ethanol con-
taining a few drops of AcOH was heated under reflux for
1 h. The formed solid was filtered off, dried and recrys-
tallized to afford 0.58 g 12a (86%), 0.56 g 12b (87%) and
0.52 g 12c (82%).
4-(2-Cyclohexylidenehydrazinyl)-1-(4-methoxyphenyl)-2,5-
dihydro-1H-pyrrole-3-carbonitrile (13b, C18H22N4O)
ꢀ
Mp 204–205 °C (DMF/EtOH); IR (film): m = 1,643 (C=N)
cm-1, 2,198 (CN) cm-1; 1H NMR (DMSO-d6): d = 1.50–
2.40 (m, cyclohexane), 3.29 (s, OCH3), 4.08 (s, CH2), 4.11
(s, CH2), 6.55, 6.88 (2d, J = 7.95 Hz Ar-H), 10.03 (s, NH,
exchangeable with D2O) ppm; MS (EI, 70 eV): m/z = 310
(M?, 30) and at 160 (100, base peak).
1-(4-Methoxyphenyl)-4-(2-(1-(thiophen-2-yl)ethylidene)hy-
drazinyl)-2,5-dihydro-1H-pyrrole-3-carbonitrile
(12a, C18H18N4OS)
ꢀ
Mp 226–227 °C (DMF/EtOH); IR (film): m = 1,644 (C=N)
4-(2-Cycloheptylidenehydrazinyl)-1-(4-methoxyphenyl)-
2,5-dihydro-1H-pyrrole-3-carbonitrile
(13c, C19H24N4O)
1
cm-1, 2,193 (CN) cm-1, H NMR (DMSO-d6): d = 2.13
(s, CH3), 3.65 (s, OCH3), 4.12, 4.24 (2s, 2 CH2), 6.51–6.98
(m, Ar-H), 10.28 (s, NH, exchangeable with D2O) ppm;
MS (EI, 70 eV): m/z = 338 (M?, 100, base peak).
ꢀ
Mp 242–243 °C (DMF/EtOH); IR (film): m = 1,628 (C=N)
1
cm-1, 2,231(CN) cm-1; H NMR (DMSO-d6): d = 1.50–
4-(2-(1-(Furan-2-yl)ethylidene)hydrazinyl)-1-(4-methoxy-
phenyl)-2,5-dihydro-1H-pyrrole-3-carbonitrile
(12b, C18H18N4O2)
2.38 (m, cycloheptane), 3.33 (s, OCH3), 4.11 (s, CH2), 4.19
(s, CH2), 6.61, 6.95 (2d, J = 7.94 Hz Ar-H), 10.13 (s, NH,
exchangeable with D2O) ppm; MS (EI, 70 eV): m/z = 324
(M?, 41) and at 160 (100, base peak).
ꢀ
Mp 228–230 °C (DMF/EtOH); IR (film): m = 1,631 (C=N)
1
cm-1, 2,197 (CN) cm-1; H NMR (DMSO-d6): d = 2.08
(s, CH3), 3.57 (s, OCH3), 4.14, 4.27 (2s, 2 CH2), 6.53–7.11
(m, Ar-H), 10.21 (s, NH, exchangeable with D2O) ppm;
MS (EI, 70 eV): m/z = 323 (M? ?1, 3) and at 322 (M?,
100, base peak).
Pharmacological screening
Serotonin antagonist
Procedure:
4-(2-(1-(1H-Pyrrol-2-yl)ethylidene)hydrazinyl)-1-(4-
methoxyphenyl)-2,5-dihydro-1H-pyrrole-3-carbonitrile
(12c, C18H19N5O)
1. Tris buffers pH 7.7
a. 57.29 Tris–HCl, 16.2 g Tris base, q. s. 1 dm3 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)
ꢀ
Mp 236–238 °C (DMF/EtOH); IR (film): m = 1,637 (C=N)
1
cm-1, 2,203 (CN) cm-1, H NMR (DMSO-d6): d = 2.11
(s, CH3), 3.62 (s, OCH3), 4.10, 4.22 (2s, 2 CH2), 6.11 (s,
NH), 6.52–6.95 (m, Ar-H), 10.28 (s, NH, exchangeable
with D2O) ppm; MS (EI, 70 eV): m/z = 321 (M?, 88) and
at 160 (100, base peak).
c. 0.05 M Tris buffer, pH 7.7 containing 10 lM
paragyline 4 mM CaCl2 and 0.1% ascorbic acid.
123

136
M. M. Abdalla et al.
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)
animal cage, 44 cm 9 21 cm, is darkened with black spray
over one-third. A partition containing a 13-cm long 9 5-
cm high opening separates the dark one-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 photocells across the
partition automatically counts movements through the
partition and clocks the time spent in the light and dark
compartments. Naive male albino mice with a body weight
of 18–25 g are placed into the cage. The animals are
treated 30 min before the experiment with the test drugs or
the vehicle intraperitoneally and are then observed for
10 min.
2. [3H]-DPAT(2-N,N-Di[2,3(n) -3propylamino)-8-hydro-
xy-1,2,3,4-tetrahydronaphthalene) (160–206 ci/mmol)
was obtained from Amersham. For IC50 determina-
tion = a, 10 nM stock solution is made up and
50 mm3 are added to each tube (final concentration =
0.5 nM).
3. Serotonin sulphate. 0.5 nM stock solution is made
up in 0.01 N HCl and 20 mm3 added to three tubes
for determination of non-specific binding (final
concentration = 10 lM).
4. Test compound
One millimolar stock solution is made up in a suitable
solvent and serially diluted, such that the final concentra-
tions in the assay range from 2 9 10-5 to 2 9 10-8 M.
Seven concentrations are used for each assay. Higher or
lower concentrations may be used based on the potency of
the drug
References
1. Bonde CG, Nadkarni BA, Khadse BG (2001) Indian J Heterocycl
Chem 10:271
2. Sihgh SP, Parmar SS, Raman K, Stenherb VI (1981) Chem Pharm
Bull 81:197
3. Patel PB, Trivedi JT (1987) Ind Chem Soc 54:765
4. Vittoria DM, Mazzoni O, Piscopo E, Caligmano A, Bolognese A
(1992) J Med Chem 35:2910
5. Omar AM, Eshba NH (1984) J Pharm Sci 73:1166
6. Chaudhary M, Parmar SS, Chaudhari SK, Ramasastry BV (1976)
J Pharm Sci 65:443
7. Ragab FA, Hussain MM, Hanna MM, Hassan GS (1993) J Pharm
Sci 34:387
8. Attia A, Abd El-Salam OI, Amr AE (1997) Egypt J Chem 40:
317
9. Amr AE, Abdel-Salam OI, Attia A, Stibor I (1999) Collect Czech
Chem Commun 64:288
10. Amr AE, Mohamed AM, Ibrahim AA (2003) Z Naturforsch
58b:861
Tissue preparation
Male Wister rats are sacrificed by decapitation. Hippo-
campus are removed, weighed and homogenized in 20
volumes of 0.05 M Tris buffer, pH 7.7. The homogenate is
centrifuged at 48,000g 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 °C for 10 min and re-
centrifuged at 48,000g for 10 min. The final membrane
pellet is re-suspended in 0.05 M Tris buffer containing
4 mM CaCl2, 0.1% vitamin C and 10 lM pargyline.
11. Amr AE, Abo Ghalia MH, Abdalah MM (2006) Z Naturforsch
61b:1335
12. Abo Ghalia MH, Amr AE, Abdalah MM (2003) Z Naturforsch
58b:903
13. Amr AE, Abo Ghalia MH, Abdalah MM (2007) Arch Pharm
Chem Life Sci 340:304
14. Amr AE (2005) Z Naturforsch 60b:990
15. Nehad AA, Amr AE, Ibrahiem AA (2007) Monatsh Chem
138:559
16. Attia A, Abdel-Salam OI, Abo Ghalia MH, Amr AE (1995) Egypt
J Chem 38:543
Assay
–
–
800 mm3 Tissue
130 mm3 0.05 M Tris ? CaCl2 ? pargyline ? vita-
min C
–
–
20 mm3 Vehicle/5HT/drug
50 mm3 [3H] DPAT
17. Attia A, Abdel-Salam OI, Amr AE (2000) Egypt J Chem 43:297
18. Amr AE, Hegab MI, Ibrahim AA, Abdalah MM (2003) Monatsh
Chem 134:1395
19. Abo Ghalia MH, Amr AE (2004) Amino Acids 26:283
20. Hammam AG, Fahmy AFM, Amr AE, Mohamed AM (2003)
Indian J Chem 42B:1985
21. Hammam AG, Sharaf MA, Abdel Hafez NA (2001) Indian J
Chem 40B:213
22. Attia A, Abdel-Salam OI, Amr AE, Stibor I, Budesinsky M
(2000) Egypt J Chem 43:187
Tubes are incubated for 15 min at 25 °C. The assay is
stopped by vacuum filtration through Whatman GF/B fil-
ters, which are then washed twice with 5 cm3 of ice-cold
0.05 M Tris buffer. The filters are then placed into scin-
tillation vials with 10 cm3 of liquescent scintillation
cocktail and counted.
Antianexity test in mice
23. Amr AE, Abdulla MM (2002) Indian J Heterocycl Chem 12:129
24. Southwich PL, Madhav R, Filzgerald JA (1969) J Heterocycl
Chem 6:507
Procedure
25. Amer FA, Hammouda M, El-Ahl AS, Abdel-Wahab BF (2007)
J Chin Chem Soc 54:1543
26. Schlegel JR, Peroutka SJ (1986) Biochem Pharmacol 35:1943
The testing apparatus consists of a light and a dark chamber
divided by a photo cell-equipped zone, A polypropylene
123

Synthesis and serotonin antagonist and antianexity activities of pyrrolidine derivatives
137
27. Pedigo NW, Yammamura HI, Nelson DL (1981) J Neurochem
36:220
29. Crawley J, Goodwin KK (1980) Pharmacol Biochem Behav
13:167
28. Costall B, Hendrie CA, Kelly ME, Naylor R (1987) J Neuro-
pharmacol 26:195
30. Crawley JN (1981) Pharmacol Biochem Behav 15:695
31. Austen KF, Brocklehurst WE (1961) J Exp Med 113:521
123