Design, synthesis, and In-vivo pharmacological screening of N,3-(Substituted Diphenyl)-5-phenyl-1 H-pyrazoline-1- carbothioamide derivatives

Article abstract of DOI:10.1002/ardp.200800130

Various 3,5-(substituted diphenyl)-4,5-dihydro-pyrazole-1-carbothioic acid phenylamides were synthesized starting from substituted acetophenones. Structures of the compounds were confirmed on the basis of spectral data. The compounds were evaluated for their anticonvulsant and antidepressant activity. Interestingly, out of 26 compounds, four (3f, 3g, 3t, and 3u) were found to protect 100% of the animals in the MES screen at a dose of 25 mg/kg. They were also found to have appreciable anticonvulsant activity in scPTZ screen. Two compounds, 3j and 3o, significantly reduced the duration of the immobility time at 25 mg/kg dose, when compared to control.

Full text of DOI:10.1002/ardp.200800130

Arch. Pharm. Chem. Life Sci. 2009, 342, 173–181
N. Siddiqui et al.
173
Full Paper
Design, Synthesis, and In-vivo Pharmacological Screening
of N,3-(Substituted Diphenyl)-5-phenyl-1H-pyrazoline-1-
carbothioamide Derivatives
Nadeem Siddiqui, Perwaiz Alam, and Waquar Ahsan
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hamdard University, Hamdard Nagar, New
Delhi, India
Various 3,5-(substituted diphenyl)-4,5-dihydro-pyrazole-1-carbothioic acid phenylamides were
synthesized starting from substituted acetophenones. Structures of the compounds were con-
firmed on the basis of spectral data. The compounds were evaluated for their anticonvulsant
and antidepressant activity. Interestingly, out of 26 compounds, four (3f, 3g, 3t, and 3u) were
found to protect 100% of the animals in the MES screen at a dose of 25 mg/kg. They were also
found to have appreciable anticonvulsant activity in scPTZ screen. Two compounds, 3j and 3o,
significantly reduced the duration of the immobility time at 25 mg/kg dose, when compared to
control.
Keywords: Anticonvulsant activity / Antidepressant activity / Lipophilicity / Pyrazole /
Received: July 17, 2008; accepted: November 14, 2008
DOI 10.1002/ardp.200800130
part of our continuous research in this area, several new
pyrazoline derivatives were synthesized. Previously, pyra-
zoline moieties showed a broad spectrum of biological
activities such as anticonvulsant [9], antidepressant [10],
antibacterial [11], analgesic [12], antimicrobial [13], and
antiandrogenic [14] activities.
Keeping the structural requirements as mentioned in
the anticonvulsant model [15] in view, several 3,5-(substi-
tuted diphenyl)-4,5-dihydro-pyrazole-1-carbothioic acid
phenylamides were designed, synthesized, and tested for
their anticonvulsant and antidepressant activities. Inter-
estingly, these compounds were found to have appreci-
able anticonvulsant activity with lower neurotoxicity. All
the titled compounds fulfilled the pharmacophoric
requirements as shown in Fig. 1.
Introduction
Epilepsy is a group of disorders of the central nervous sys-
tem (CNS) characterized by paroxysmal cerebral dysryth-
mia, manifesting itself as brief episodes (seizures) of loss
or disturbances of consciousness, with or without charac-
teristic body movements (convulsions), sensory or psychi-
atric phenomena [1]. Use of estimates suggests that avail-
able medication controls the seizures in only 50% of the
patients or decreases the incidence in only 75% of the
patients [2]. Thus, the search for anti-epileptic agents
with more selectivity and lower toxicity continues to be
an area of investigation in medicinal chemistry [3].
In the effort to get those agents, we have reported [4–
8] several five-membered heterocyclic compounds which
have shown considerable anticonvulsant activities. As
Results and discussion
Correspondence: Nadeem Siddiqui, Department of Pharmaceutical
Chemistry, Faculty of Pharmacy, Hamdard University, Hamdard Nagar,
New Delhi 110062, India.
Synthesis
The synthesis of 3,5-(substituted diphenyl)-4,5-dihydro-
pyrazole-1-carbothioic acid phenylamides was carried
out as presented in Scheme 1. Various substituted aceto-
phenones were mixed with benzaldehyde in the presence
E-mail: nadeems_03@rediffmail.com
Fax: +91 11 26059688 x 5307
Abbreviations: hydrogen bonding domain (HBD): maximal electroshock
seizure (MES); subcutaneous pentylenetetrazole (scPTZ)
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Arch. Pharm. Chem. Life Sci. 2009, 342, 173–181
Table 1. Physicochemical properties of the titled compounds (3a–z).
Compound
R
R9
Molecular
formulaa)
Log Pb)
Rfc) value
Elemental analysesd) % found (calculated)
C
H
N
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
-H
-H
-H
-H
-H
-H
-H
-Br
-Br
-Br
-Br
-Br
-Br
-Br
-Cl
-Cl
-Cl
-Cl
-Cl
-H
C22H19N3S
C23H21N3S
C23H21N3S
C23H21N3S
4.614
5.216
5.220
5.219
4.386
5.354
5.509
5.518
5.984
6.020
6.019
5.248
6.314
6.394
5.354
5.853
5.898
5.868
5.124
6.106
6.241
4.406
4.849
4.864
5.124
5.238
0.82
0.91
0.73
0.90
0.57
0.71
0.82
0.77
0.63
0.92
0.77
0.69
0.87
0.91
0.83
0.61
0.57
0.71
0.77
0.93
0.88
0.61
0.57
0.81
0.83
0.69
73.5 (73.77)
5.89 (6.01)
11.69 (11.57)
11.31 (11.40)
11.31 (11.42)
11.31 (11.37)
13.92 (14.01)
10.72 (11.20)
9.63 (9.87)
9.63 (9.32)
9.33 (9.41)
9.33 (9.41)
9.33 (9.41)
11.64 (11.73)
8.93 (9.01)
8.16 (8.32)
10.72 (11.10)
10.35 (10.23)
10.35 (10.23)
10.35 (10.23)
12.82 (13.21)
9.86 (9.93)
2-CH3
3-CH3
4-CH3
4-NO2
4-Cl
74.36 (74.44) 5.70 (5.72)
74.36 (75.01) 5.70 (5.72)
74.36 (75.01) 5.70 (5.72)
65.65 (65.88) 4.51 (4.81)
67.42 (67.92) 4.63 (4.92)
60.55 (61.60) 4.16 (4.31)
60.55 (61.60) 4.16 (4.31)
61.33 (62.01) 4.48 (4.71)
61.33 (62.01) 4.48 (4.71)
61.33 (62.01) 4.48 (4.71)
54.89 (55.03) 3.56 (3.41)
56.12 (55.90) 3.64 (3.57)
51.28 (51.70) 3.33 (3.41)
67.42 (68.00) 4.63 (4.73)
68.05 (67.99) 4.97 (4.99)
68.05 (67.99) 4.97 (4.99)
68.05 (67.99) 4.97 (4.99)
60.48 (61.31) 3.92 (4.02)
61.97 (62.33) 4.02 (4.21)
56.12 (55.89) 3.64 (3.77)
65.65 (66.21) 4.51 (4.63)
66.33 (66.73) 4.84 (4.73)
66.33 (66.73) 4.84 (4.73)
60.48 (60.81) 3.92 (4.030
54.89 (55.03) 3.56 (3.61)
C22H18N4O2S
C22H18ClN3S
C22H18BrN3S
C22H18BrN3S
C23H20BrN3S
C23H20BrN3S
C23H20BrN3S
C22H17BrN4O2S
C22H17BrClN3S
C22H17Br2N3S
C22H18ClN3S
C23H20ClN3S
C23H20ClN3S
C23H20ClN3S
C22H17ClN4O2S
C22H17Cl2N3S
C22H17ClBrN3S
C22H18N4O2S
C23H20N4O2S
C23H20N4O2S
C22H17ClN4O2S
C22H17BrN4O2S
4-Br
-H
2-CH3
3-CH3
4-CH3
4-NO2
4-Cl
4-Br
-H
2-CH3
3-CH3
4-CH3
4-NO2
4-Cl
4-Br
-H
3-CH3
4-CH3
4-Cl
3t
-Cl
-Cl
3u
3v
3w
3x
3y
3z
8.93 (9.10)
-NO2
-NO2
-NO2
-NO2
-NO2
13.92 (14.02)
13.45 (13.57)
13.45 (13.57)
12.82 (12.60)
11.64 (11.88)
4-Br
a)
Solvent of crystallization: ethanol.
Log P was calculated using absorbance data (chloroform / phosphate buffer at 288C).
Solvent system: benzene / acetone (8 : 2), toluene / ethyl acetate / formic acid (5 : 4 : 1).
Elemental analysis for C, H, N were within l 0.4% of the theoretical value.
b)
c)
d)
of 40% NaOH in ethanol to yield chalcones 1a–d, which
were cyclized in the presence of hydrazine hydrate to
afford the substituted pyrazoline derivatives 2a–d. These
derivatives were treated with substituted phenylisothio-
cyanates to yield the titled compounds 3a–z. The physi-
cal properties of the synthesized compounds are pre-
sented in Table 1. The structures and purity of the com-
pounds were confirmed by spectral data, elemental anal-
yses, and thin layer chromatography (Table 1).
A: Hydrophobic domain; HBD: hydrogen bonding domain; D: distal hydrophobic
domain; C: electron donor moiety in basic structure of titled compounds 3a – z.
Figure 1. Pharmacophoric requirements in the anticonvulsant
model.
Pharmacology
Anticonvulsant activity
The pharmacological testing of the synthesized com- (scPTZ) tests. Neurological impairment (NT) was assessed
pounds was carried out according to the protocol given by rotorod apparatus and the findings are presented in
by the Epilepsy Section of the National Institute of Neuro- Table 2.
logical Disorders and Stroke (NINDS) using a standard
protocol adopted by the Antiepileptic Drug Development (R = -H, R9 = 4-CH3), 3f (R = -H, R9 = 4-Cl), 3g (R = -H, R9 = 4-Br),
(ADD) program [16]. 3m (R = -Br, R9 = 4-Cl), 3n (R = -Br, R9 = 4-Br), 3s (R = -Cl, R9 = 4-
In the preliminary (phase I) screening, compounds 3d
The anticonvulsant activity was evaluated by using the NO2), 3t (R = -Cl, R9 = 4-Cl), 3u (R = -Cl, R9 = 4-Br), 3y (R = -NO2,
two most adopted models: the maximal electroshock R9 = 4-Cl) showed potent anticonvulsant activity in the
seizure (MES) and the subcutaneous pentylenetetrazole MES test.
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Arch. Pharm. Chem. Life Sci. 2009, 342, 173–181
Synthesis and Pharmacology of Pyrazoline Derivatives
175
Table 2. Phase-I anticonvulsant and toxicity data in mice (i.p.)a).
Compound
Anticonvulsant activity (% Pro- Neurotoxicity
tection)
screening (NT)b)
MESc)
scPTZc)
d)
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
3t
3u
3v
3w
3x
50
25
25
75**
50
100***
100***
50
25
50
50
25
75**
75**
25
50
25
–
–
–
–
–
NDe)
ND
ND
(+)
ND
(+)
75**
75**
(+)
–
–
–
–
–
–
–
–
–
–
–
–
ND
ND
ND
ND
ND
(+)
(–)
ND
ND
ND
ND
(–)
(–)
(+)
ND
ND
ND
(–)
ND
ND
(–)
50
75**
100***
100***
50
50
25
75**
50
00
100***
75**
–
–
–
–
–
–
–
3y
3z
Control
Phenytoinf)
100**
n = 8, dose = 25 mg/kg (i.p.); * P a 0.05; ** P a 0.01; *** P a 0.001
a)
Solvent used: propylene glycol (0.1 mL, i.p.).-
The tests were performed 30 min after administration of the
Reactions and conditions: a) Ethanol, 40% NaOH; b) NH2-NH2 N H2O, ethanolic
NaOH, reflux; c) arylisothiocyanate, ethanol, reflux.
b)
test compounds.
Neurotoxicity was measured after 45 min. The (+) indicates
50% or more passed the neurotoxicity screening, i. e. not
showing neurological deficit. The (–) indicates 50% or more
failed the neurotoxicity screening.
- Absence of activity at 25 mg/kg.
ND: Not determined.
Data from references [17].
Scheme 1. Synthesis of 3,5-(substituted diphenyl)-4,5-dihydro-
pyrazole-1-carbothioic acid phenylamides.
c)
d)
The four most active compounds (3f, 3g, 3t, and 3u) in
the MES test were also found to be active in the pentyle-
netetrazole-induced seizure (scPTZ) test. In scPTZ tests, 3t
(R = -Cl, R9 = 4-Cl) was found to be 100% active as it was in
e)
f)
the MES test. Other compounds (3f, 3g, and 3u) have 3d, 3f, 3g, 3m, and 3u successfully passed the rotorod test
shown 75% protection in PTZ while in MES they were without any sign of neurological deficit while the com-
100% protective. Introduction of halogens (-Cl, -Br) at the pounds 3n, 3s, 3t, and 3y exhibited neurological deficit
para-position of the phenyl group (3f, 3g, 3t, and 3u) at the anticonvulsant dose of 25 mg/kg (i.p.).
showed maximum activity (P a 0.001) as compared to the
In the phase-II pharmacology test, the four most active
standard drug phenytoin sodium at a dose level of compounds 3f, 3g, 3t, 3u were quantitatively evaluated
25 mg/kg (i.p.). Significant results (P a 0.01) were observed for their anticonvulsant activity (ED50) and neurotoxicity
with compounds 3d, 3m, 3n, 3s, and 3y. The other com- (TD50). All the four compounds have shown an encourag-
pounds of the series exhibited maximal seizures with sig- ing anticonvulsant activity (Table 3). These were compa-
nificant / less activity at the same dose level as compared rable to the ED50 of the standard drug phenobarbital and
to standard drug phenytoin sodium.
better than that of valproate. Toxicity measurements
All nine compounds showing good biological activity (TD50, PI) were also found to be better than some standard
were screened for their neurotoxicity. The compounds drugs.
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Table 3. Phase-II quantitative anticonvulsant data in mice.
PIc)
a)
b)
Compound
ED50
TD50
MES
scPTZ
MES
scPTZ
3f
3g
3t
3u
14.8 (12.9–16.6)d)
18.3 (14.9–22.4)
11.6 (9.5–13.9)
19.6 (17.8–23.9)
9.5 (8.1–10.4)
8.8 (5.5–14.1)
21.8 (21.8–25.5)
272 (247–338)
21.3 (18.6–25.7)
247.6 (201.5–278.4)
316.4 (254.9–398.2)
58.6 (46.7–74.9)
146.3 (122.6–174.4)
65.5 (52.5–72.9)
71.6 (45.9–135)
69 (62.8–72.9)
16.7
17.2
5.0
7.4
6.9
8.1
3.2
1.6
11.62
13.2
3.1
23.9 (19.7–29.5)
18.6 (17.5–20.8)
22.6 (20.2–24.6)
> 300
> 100
13.2 (5.8–15.9)
149 (123–177)
6.4
Phenytoine)
Carbamazepinee)
Phenobarbitale)
Valproatee)
a0.22
a0.22
5.2
426 (369–450)
2.9
Number of animals used: 10; Solvent used: propylene glycol (0.1 mL, i.p.).
a)
Dose in milligrams per kilogram body mass.
Minimal toxicity which was determined by the rotorod test 30 min after the test drug was administered.
PI = Protective index (TD50/ED50).
Data in parentheses are the 95% confidence limits.
Data from references [18].
b)
c)
d)
e)
Substitutions to the aryl ring attached to hydrogen central nervous system (CNS) is obtained with congeners
bonding domain (HBD) had a marked effect on the anti- having an optimum lipophilicity (log Po). In this study,
convulsant and toxicity profile of the compound. Com- we attempted to correlate the anticonvulsant activity of
pounds 3f, 3g, 3m, 3n, 3t, and 3u with substitution at the substituted 3,5-diphenyl-pyrazolines with the log P value.
fourth position by a halogen considerably increased the This experimental log P values were determined using
activity. Substitutions at the distal phenyl ring also play octanol-phosphate buffer method for compounds 3a–z
an important role in the activity as the unsubstituted 3f The compounds 3f, 3g, 3t, and 3u having log P of 5.35,
and 3g and the one-possessing chloro substitution 3t and 5.50, 6.10, and 6.24, respectively, are found to be more
3u at the fourth position showed maximum activity. Sub- lipophilic and having potent anticonvulsant activity
stitutions by a halogen atom on the two rings of com- (100%) in the MES test. Due to their high lipophilicity,
pounds 3m, 3n, 3t, and 3u were more active than the they are expected to have rapid onset and short duration
other compounds of the series.
of action.
Antidepressant activity
In-silico studies
The forced-swim test is a well validated and extensively N,3,5-Triphenyl-pyrazoline-1-carbothioamide 3a, which
used screen for compounds with antidepressant activity. represents the prototype structure, was studied in silico.
Compounds 3a–z were evaluated for their antidepres- Fig. 2 depicts the 3D structure of the compound showing
sant activity and compared with the standard imipr- solvent-accessible surfaces all around it, which may have
amine at the dose level of 25 mg/kg. Antidepressant activ- contributed to its increased lipophilicity. Fig. 3 shows
ity was assessed as mean average immobility time in sec- the energy minima conformer of the compound 3c
onds and data are presented as Mean l SEM (Table 4). where the N-H bonding interaction was represented.
Compounds 3j and 3o significantly reduced the duration There was a strong hydrogen-bonding between the vinyl
of immobility time at 25 mg/kg dose when compared to protons and nitrogen atoms.
control (*P a 0.05).
Log-P determination
The dependence of biological activity in the set of conge-
Conclusion
neric agents or lipophilic character has been shown in A new series of anticonvulsant agents were synthesized
many types of drug action in particular, the reports by and evaluated for their pharmacological properties. All
Lien and co-workers indicate that the anticonvulsant compounds have shown some degree of protection
activity of different types of compounds were correlated against the MES-induced seizures with four compounds
with lipophilicity [19]. However, it has been observed showing very promising activity against both MES-
that the maximum potency of the drugs which act on the induced as well as scPTZ-induced seizures. These com-
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Arch. Pharm. Chem. Life Sci. 2009, 342, 173–181
Synthesis and Pharmacology of Pyrazoline Derivatives
177
Table 4. Antidepressant activities of the compounds 3a–z.
Compound
Antidepressant activities
Duration of
immobility (s)
Change from
control (%)
(Mean l S.E.M.)
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
3t
3u
3v
3w
3x
160 l 6.2
136 l 3.5
142 l 9.8
180 l 6.1
180 l 7.2
130 l 12.2
170 l 22
176 l 3.8
168 l 7.9
113 l 11.8
180 l 5.7
172 l 8.8
140 l 11.3
180 l 5.1
113 l 19
160 l 12.2
180 l 6.1
170 l 12.6
172 l 20.1
180 l 6.2
173 l 5.9
178 l 12.7
147 l 9.1
180 l 5.2
176 l 11
112 l 7.9
60 l 13
–0.0155
–14.96
–11.35
12.49
12.47
–18.91
–0.042
10.10
5.53
–29.02*
12.5
7.93
–12.63
12.52
–43.0*
–0.16
12.5
11.0
7.15
10.0
4.7
10.9
–7.5
12.5
9.3
Figure 3. Energy minima conformer of N,3,5-triphenyl-pyrazo-
line-1-carbothioamide 3a showing N…H bonding interaction as
dotted line.
nytoin, phenobarbital, carbamazepine, and valproate.
These agents have strong future commitments.
We are thankful to the Department of Pharmaceutical Chemis-
try, Faculty of Pharmacy, Jamia Hamdard, New Delhi, India
for providing necessary research facilities.
3y
3z
–2.8
–62.0*
Imipramine sulfate
(10 mg/kg, i.p.)
Control
160 l 5.5
–
The authors have declared no conflict of interest.
n = 8, dose = 25 mg/kg; * P a 0.05.
Experimental
Chemistry
Melting points were determined in open capillary tubes and are
uncorrected. IR spectra were recorded in KBr pellets on FT-IR
(BioRad FTs) spectrophotometer (Browser Morner, USA.). The
homogen elemental analyses (C, H, N) of all compounds were
performed on the CHNS-Elementaranalysator (Analysensysteme
GmbH, Germany) Vario EL III. The Proton Magnetic Resonance
spectra (1H-NMR) were recorded on DRX-300 NMR spectrometric
and BRUKER 400 Ultra ShieldTM (Bruker Bioscience, USA). Chemi-
cal shifts (d) are expressed in parts per million (ppm) using TMS
(tetramethylsilane, Me4Si) as an internal reference. All TLCs were
carried out using silica gel.
General procedure for the synthesis of 1,3-(substituted
diphenyl)-propenones 1a–d
Substituted acetophenones (0.43 mol) and benzaldehyde
(0.43 mol) were added to a solution of 40% NaOH (200 mL) and
rectified spirit (99.8%, 12.5 mL) at 5 to 108C and stirred for 4 h
while maintaining the temperature to 258C. The solid mass
obtained was kept on the ice chest over night, washed with
water, dried, and was recrystallized using ethanol to afford the
target compounds 1a–d.
Figure 2. 3D structure of N,3,5-triphenyl-pyrazoline-1-carbo-
thioamide 3a showing solvent-accessible surfaces.
pounds also showed high lipophilicity, low neurotoxic-
ity, minimal CNS depressant activity and, therefore,
lesser side effects compared with the standard drugs phe-
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Arch. Pharm. Chem. Life Sci. 2009, 342, 173–181
11.5 Hz, 1H, HB), 5.56–5.64 (dd, JAX = 4.2 Hz, JBX = 4.5 Hz, 1H, HX),
General procedure for the synthesis of 3,5-(substituted
diphenyl)-4,5-dihydro-1H-pyrazoles 2a–d
7.46–7.54 (m, 4H, ArH), 7.62–7.71 (m, 5H, ArH), 7.82–7.94 (m,
5H, ArH), 8.12 (bs, 1H, NH, D2O exchangeable). IR (KBr) cm– 1
3239, 3089, 1646, 1419.
:
The solution of appropriate chalcones (1a–d, 0.01 mol) and
hydrazine hydrate (0.02 mol) in ethanolic sodium hydroxide
(0.025 mol, 20 mL) was refluxed for 4 h. The reaction mixture
was poured into ice-cold water and the crude product which sep-
arated out was filtered and recrystallized using ethanol to
receive the target compounds 2a–d.
N-(4-Chlorophenyl)-3,5-diphenyl-1H-pyrazoline-1-
carbothioamide 3f
Yield: 63%, m.p.: 858C. H-NMR (DMSO-d6) d: 3.11–3.19 (dd, JAB
11.3 Hz, JAX = 3.5 Hz, 1H, HA), 3.64–3.78 (dd, JAB = 4.5 Hz, JBX
1
=
=
4.5 Hz, 1H, HB), 5.55–5.68 (dd, JAX = 13.5, JBX = 11.4 Hz, 1H, HX),
General procedure for the synthesis of 3,5-(substituted
diphenyl)-4,5-dihydro-pyrazole-1-carbothioic acid
phenylamides 3a–z
To the stirred solution of compounds (2a–d, 0.02 mol) in abso-
lute ethanol, was added substituted phenyl isothiocyanates and
refluxed for 4–6 h. The reaction mixture was concentrated and
kept over night. The crude product was filtered, washed with
petroleum ether, dried, and was recrystallized from methanol
to afford the title compounds 3a–z.
7.42–7.58 (m, 4H, ArH), 7.67–7.79 (m, 5H, ArH), 7.93–8.16 (m,
5H, ArH), 8.67 (bs, 1H, NH, D2O exchangeable). IR (KBr) cm– 1
3238, 3013, 1649, 1442.
:
N-(4-Bromophenyl)-3,5-diphenyl-1H-pyrazoline-1-
carbothioamide 3g
Yield: 70%, m.p.: 788C. H-NMR (DMSO-d6) d: 3.15–3.22 (dd, JAB
4.5 Hz, JAX = 4.5 Hz, 1H, HA), 3.72–3.82 (dd, JAB = 12.0 Hz, JBX
1
=
=
11.7 Hz, 1H, HB), 5.61–5.66 (dd, JAX = 4.2 Hz, JBX = 4.5 Hz, 1H, HX),
7.38–7.49 (m, 4H, ArH), 7.51–7.67 (m, 5H, ArH), 7.74–7.89 (m,
N,3,5-Triphenyl-pyrazoline-1-carbothioamide 3a
5H, ArH), 8.61 (bs, 1H, NH, D2O exchangeable). IR (KBr) cm– 1
3312, 3079, 1670, 1426.
:
Yield: 73%, m.p.: 1058C. 1H-NMR (DMSO-d6) d: 3.12–3.18 (dd, JAB
4.7 Hz, JAX = 4.5 Hz, 1H, HA), 3.69–3.79 (dd, JAB = 12.4 Hz, JBX
=
=
13.3 Hz, 1H, HB), 5.58–5.62 (dd, JAX = 4.5 Hz, JBX 4.3 Hz, 1H, HX),
7.23–7.30 (m, 5H, ArH), 7.36–7.48 (m, 5H, ArH), 7.68–7.77 (m,
3-(4-Bromophenyl)-N-5-diphenyl-1H-pyrazoline-1-
5H, ArH), 8.66 (bs, 1H, NH, D2O exchangeable). IR (KBr) cm– 1
3230, 3009, 1658, 1418.
:
carbothioamide 3h
1
Yield: 75%, m.p.: 818C. H-NMR (DMSO-d6) d: 3.14–3.21 (dd, JAB
4.5 Hz, JAX = 3.7 Hz, 1H, HA), 3.64–3.69 (dd, JAB = 4.5 Hz, JBX
=
=
N-(2-Methylphenyl)-3,5-diphenyl-1H-pyrazoline-1-
4.6 Hz, 1H, HB), 5.64–5.76 (dd, JAX = 12.3 Hz, JBX = 14.5 Hz, 1H, HX),
7.57–7.71 (m, 4H, ArH), 7.78–7.91 (m, 5H, ArH), 8.13–8.39 (m,
carbothioamide 3b
5H, ArH), 8.49 (bs, 1H, NH, D2O exchangeable). IR (KBr) cm– 1
3312, 3079, 1670, 1426.
:
Yield: 78%, m.p.: 1198C. 1H-NMR (DMSO-d6) d: 3.14–3.22 (dd, JAB
5.1 Hz, JAX = 4.5 Hz, 1H, HA), 3.41 (s, 3H, -CH3), 3.72-3.82 (dd, JAB
=
=
4.5 Hz, JBX = 4.5 Hz, 1H, HB), 5.61-5.67 (dd, JAX = 4.5 Hz, JBX = 4.5 Hz,
1H, HX), 7.23–7.32 (m, 4H, ArH), 7.37–7.45 (m, 5H, ArH), 7.64–
7.79 (m, 5H, ArH), 8.53 (bs, 1H, NH, D2O exchangeable). IR (KBr)
cm– 1: 3123, 3006, 1656, 1424.
3-(4-Bromophenyl)-N-(2-methylphenyl)-5-phenyl-1H-
pyrazoline-1-carbothioamide 3i
Yield: 78%, m.p.: 828C. 1H-NMR (DMSO-d6) d: 3.14–3.19 (dd, , JAB
4.2 Hz, JAX = 3.6 Hz, 1H, HA), 3.26 (s, 3H, -CH3), 3.54–3.69 (dd, JAB
4.2 Hz, JBX = 4.6 Hz, 1H, HB), 5.56–5.77 (dd, JAX = 11.2 Hz, JBX
=
=
=
N-(3-Methylphenyl)-3,5-diphenyl-1H-pyrazoline-1-
13.6 Hz, 1H, HX), 7.21–7.41 (m, 4H, ArH), 7.48–7.63 (m, 5H, ArH),
7.62–7.77 (m, 4H, ArH), 8.38 (bs, 1H, NH, D2O exchangeable). IR
(KBr) cm– 1: 3240, 3064, 1558, 1398.
carbothioamide 3c
Yield: 80%, m.p.: 788C. 1H-NMR (DMSO-d6) d: 3.10–3.16 (dd, JAB
4.5 Hz, JAX = 4.5 Hz, 1H, HA), 3.24 (s, 3H, -CH3), 3.67–3.83 (dd, JAB
4.5 Hz, JBX = 4.5 Hz, 1H, HB), 5.53–5.61 (dd, JAX = 4.5 Hz, JBX
=
=
=
4.5 Hz, 1H, HX), 6.12 (bs, 1H, NH, D2O exchangeable), 7.22–7.34
(m, 4H, ArH), 7.37–7.49 (m, 5H, ArH), 7.59–7.70 (m, 5H, ArH). IR
(KBr) cm– 1: 3233, 3019, 1648, 1416.
3-(4-Bromophenyl)-N-(3-methylphenyl)-5-phenyl-1H-
pyrazoline-1-carbothioamide 3j
Yield: 65%, m.p.: 1078C. 1H-NMR (DMSO-d6) d: 3.13–3.19 (dd, JAB
4.7 Hz, JAX = 4.6 Hz, 1H, HA), 3.31 (s, 3H, -CH3), 3.56–3.68 (dd, JAB
13.6 Hz, JBX = 14.6 Hz, 1H, HB), 5.56–5.67 (dd, JAX = 13.2 Hz, JBX
=
=
=
N-(4-Methylphenyl)-3,5-diphenyl-1H-pyrazoline-1-
carbothioamide 3d
14.6 Hz, 1H, HX), 7.23–7.47 (m, 4H, ArH), 7.51–7.64 (m, 5H, ArH),
7.64–7.78 (m, 4H, ArH), 8.40 (bs, 1H, NH, D2O exchangeable). IR
(KBr) cm– 1: 3258, 3069, 1570, 1402.
Yield: 63%, m.p.: 858C. 1H-NMR (DMSO-d6) d: 3.06–3.12 (dd, JAB
4.2 Hz, JAX = 4.5 Hz, 1H, HA), 3.27 (s, 3H, -CH3), 3.70–3.81 (dd, JAB
11.5 Hz, JBX = 15.4 Hz, 1H, HB), 5.49–5.58 (dd, JAX = 11.5 Hz, JBX
=
=
=
11.5 Hz, 1H, HX), 5.94 (bs, 1H, NH, D2O exchangeable), 7.18–7.31
(m, 4H, ArH), 7.36–7.45 (m, 5H, ArH), 7.65–7.71 (m, 5H, ArH),. IR
(KBr) cm– 1: 3231, 3109, 1678, 1428.
3-(4-Bromophenyl)-N-(4-methylphenyl)-5-phenyl-1H-
pyrazoline-1-carbothioamide 3k
Yield: 71%, m.p.: 938C. 1H-NMR (DMSO-d6) d: 3.12–3.18 (dd, JAB
4.5 Hz, JAX = 4.5 Hz, 1H, HA), 3.28 (s, 3H, -CH3), 3.56–3.67 (dd, JAB
13.2 Hz, JBX = 11.6 Hz, 1H, HB), 5.51–5.64 (dd, JAX = 17.2 Hz, JBX
=
=
=
N-(4-Nitrophenyl)-3,5-diphenyl-1H-pyrazoline-1-
carbothioamide 3e
13.3 Hz, 1H, HX), 7.24–7.44 (m, 4H, ArH), 7.48–7.60 (m, 5H, ArH),
7.67–7.76 (m, 4H, ArH), 8.32 (bs, 1H, NH, D2O exchangeable). IR
(KBr) cm– 1: 3248, 3211, 1586, 1424.
Yield: 57%, m.p.: 1438C. 1H-NMR (DMSO-d6) d: 3.17-3.24 (dd, JAB
4.5 Hz, JAX = 4.5 Hz, 1H, HA), 3.69–3.81 (dd, JAB = 13.5 Hz, JBX
=
=
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Arch. Pharm. Chem. Life Sci. 2009, 342, 173–181
Synthesis and Pharmacology of Pyrazoline Derivatives
179
4.5 Hz, JBX = 4.5 Hz, 1H, HB), 5.36–5.46 (dd, JAX = 11.4 Hz, JBX
=
3-(4-Bromophenyl)-N-(4-nitrophenyl)-5-phenyl-1H-
11.2 Hz, 1H, HX), 7.35–7.49 (m, 4H, ArH), 7.68–7.81 (m, 5H, ArH),
7.89–7.97 (m, 4H, ArH), 8.16 (bs, 1H, NH, D2O exchangeable). IR
(KBr) cm– 1: 3313, 3079, 1670, 1243.
pyrazoline-1-carbothioamide 3l
1
Yield: 57%, m.p.: 908C. H-NMR (DMSO-d6) d: 3.10–3.16 (dd, JAB
=
=
4.5 Hz, JAX = 4.4 Hz, 1H, HA), 3.55–3.67 (dd, JAB = 4.2 Hz, JBX
3.9 Hz, 1H, HB), 5.50–5.63 (dd, JAX = 17.6 Hz, JBX = 11.3 Hz, 1H, HX),
7.39–7.52 (m, 4H, ArH), 7.67–7.79 (m, 5H, ArH), 7.86–7.98 (m,
3-(4-Chlorophenyl)-N-(4-nitrophenyl)-5-phenyl-1H-
4H, ArH), 8.51 (bs, 1H, NH, D2O exchangeable). IR (KBr) cm– 1
3113, 3012, 1556, 1398.
:
pyrazoline-1-carbothioamide 3s
1
Yield: 53%, m.p.: 978C. H-NMR (DMSO-d6) d: 3.15–3.26 (dd, JAB
14.4 Hz, JAX = 13.4 Hz, 1H, HA), 3.51–3.65 (dd, JAB = 13.6 Hz, JBX
=
=
13.7 Hz, 1H, HB), 5.48–5.56 (dd, JAB = 8.2 Hz, JAX = 8.6 Hz, 1H, HX),
3-(4-Bromophenyl)-N-(4-chlorophenyl)-5-phenyl-1H-
7.51–7.68 (m, 4H, ArH), 7.73–7.85 (m, 5H, ArH), 7.93–8.13 (m,
pyrazoline-1-carbothioamide 3m
4H, ArH), 8.42 (bs, 1H, NH, D2O exchangeable). IR (KBr) cm– 1
3230, 3059, 1628, 1408.
:
1
Yield: 75%, m.p.: 958C. H-NMR (DMSO-d6) d: 3.16–3.24 (dd, JAB
4.5 Hz, JAX = 4.5 Hz, 1H, HA), 3.51–3.64 (dd, JAB = 14.2 Hz, JBX
=
=
13.7 Hz, 1H, HB), 5.54–5.59 (dd, JAX = 4.2 Hz, JBX = 4.6 Hz, 1H, HX),
7.41–7.55 (m, 4H, ArH), 7.63–7.77 (m, 5H, ArH), 7.89–7.90 (m,
N-3-bis(4-Chlorophenyl)-5-phenyl-1H-pyrazoline-1-
4H, ArH), 8.21 (bs, 1H, NH, D2O exchangeable). IR (KBr) cm– 1
3216, 3019, 1596, 1428.
:
carbothioamide 3t
Yield: 77%, m.p.: 1198C. 1H-NMR (DMSO-d6) d: 3.11–3.16 (dd, JAB
4.5 Hz, JAX = 4.5 Hz, 1H, HA), 3.70–3.80 (dd, JAB = 11.7 Hz, JBX
=
=
N-3-bis(4-Bromophenyl)-5-phenyl-1H-pyrazoline-1-
12.0 Hz, 1H, HB), 5.50–5.56 (dd, JAX = 4.5 Hz, JBX = 4.5 Hz, 1H, HX),
7.49–7.64 (m, 4H, ArH), 7.71–7.83 (m, 5H, ArH), 7.89–8.03 (m,
carbothioamide 3n
4H, ArH), 8.34 (bs, 1H, NH, D2O exchangeable). IR (KBr) cm– 1
3470, 3017, 1663, 1342.
:
1
Yield: 50%, m.p.: 908C. H-NMR (DMSO-d6) d: 3.11–3.15 (dd, JAB
4.5 Hz, JAX = 4.4 Hz, 1H, HA), 3.50–3.63 (dd, JAB = 13.2 Hz, JBX
=
=
13.5 Hz, 1H, HB), 5.50–5.61 (dd, JAX = 13.5 Hz, JBX = 13.9 Hz, 1H,
HX), 7.37–7.54 (m, 4H, ArH), 7.64–7.76 (m, 5H, ArH), 7.84–7.93
N-(4-Bromophenyl)-3-(4-chlorophenyl)-5-phenyl-1H-
(m, 4H, ArH), 8.43 (bs, 1H, NH, D2O exchangeable). IR (KBr) cm– 1
3018, 3132, 1548, 1406.
:
pyrazoline-1-carbothioamide 3u
Yield: 58%, m.p.: 1058C. 1H-NMR (DMSO-d6) d: 3.10–3.15 (dd, JAB
4.5 Hz, JAX = 4.5 Hz, 1H, HA), 3.70–3.79 (dd, JAB = 11.7 Hz, JBX
=
=
3-(4-Chlorophenyl)-N-5-diphenyl-1H-pyrazoline-1-
11.1 Hz, 1H, HB), 5.60–5.65 (dd, JAX = 4.5 Hz, JBX = 4.5 Hz, 1H, HX),
7.21–7.39 (m, 4H, ArH), 7.79–7.85 (m, 5H, ArH), 7.91–7.97 (m,
carbothioamide 3o
4H, ArH), 8.59 (bs, 1H, NH, D2O exchangeable). IR (KBr) cm– 1
3312, 3079, 1670, 1393.
:
Yield: 67%, m.p.: 1438C. 1H-NMR (DMSO-d6) d: 3.12–3.18 (dd, JAB
4.7 Hz, JAX = 4.6 Hz, 1H, HA), 3.69–3.79 (dd, JAB = 12.3 Hz, JBX
=
=
12.1 Hz, 1H, HB), 5.58–5.62 (dd, JAX = 4.1 Hz, JBX = 3.7 Hz, 1H, Hx),
7.39–7.56 (m, 4H, ArH), 7.59–7.76 (m, 5H, ArH), 7.81–7.96 (m,
3-(4-Nitrophenyl)-N,5-diphenyl-1H-pyrazoline-1-
5H, ArH), 8.34 (bs, 1H, NH, D2O exchangeable). IR (KBr) cm– 1
3124, 3035, 1543, 1421.
:
carbothioamide 3v
Yield: 53%, m.p.: 1268C. 1H-NMR (DMSO-d6) d: 3.12–3.18 (dd, JAB
=
5.3 Hz, JAX = 5.2 Hz, 1H, HA), 3.69–3.79 (dd, J = 12.1 Hz, 12.3 Hz,
1H, HB), 5.58–5.62 (dd, J = 4.5 Hz, 4.5 Hz, 1H, HX), 7.61–7.78 (m,
4H, ArH), 7.80–7.91 (m, 5H, ArH), 8.03–8.24 (m, 5H, ArH), 8.66
(bs, 1H, NH, D2O exchangeable). IR (KBr) cm– 1: 3320, 3009, 1638,
1428.
3-(4-Chlorophenyl)-N-(2-methylphenyl)-5-phenyl-1H-
pyrazoline-1-carbothioamide 3p
Yield: 75%, m.p.: 928C. 1H-NMR (DMSO-d6) d: 3.11–3.15 (dd, JAB
4.1 Hz, JAX = 3.8 Hz, 1H, HA), 3.26 (s, 3H, -CH3), 3.50–3.63 (dd, JAB
14.2 Hz, JBX = 13.7 Hz, 1H, HB), 5.50–5.61 (dd, JAX = 11.5 Hz, JBX
=
=
=
11.6 Hz, 1H, HX), 7.37–7.54 (m, 4H, ArH), 7.64–7.76 (m, 5H, ArH),
7.84–7.93 (m, 4H, ArH), 8.43 (bs, 1H, NH, D2O exchangeable). IR
(KBr) cm– 1: 3018, 3132, 1548, 1406.
N-(3-Methylphenyl)-3-(4-nitrophenyl)-5-phenyl-1H-
pyrazoline-1-carbothioamide 3w
Yield: 75%, m.p.: 1298C. 1H-NMR (DMSO-d6) d: 3.11–3.19 (dd, JAB
6.4 Hz, JAX = 6.2 Hz, 1H, HA), 3.28 (s, 3H, -CH3), 3.53–3.66 (dd, JAB
13.3 Hz, JBX = 13.4 Hz, 1H, HB), 5.54-5.59 (dd, JAX = 4.5 Hz, JBX
=
=
=
3-(4-Chlorophenyl)-N-(3-methylphenyl)-5-phenyl-1H-
pyrazoline-1-carbothioamide 3q
4.2 Hz, 1H, HX), 7.31–7.47 (m, 4H, ArH), 7.60–7.73 (m, 5H, ArH),
7.85–7.93 (m, 4H, ArH), 8.36 (bs, 1H, NH, D2O exchangeable). IR
(KBr) cm– 1: 3231, 3007, 1655, 1428.
Yield: 68%, m.p.: 1078C. 1H-NMR (DMSO-d6) d: 3.14–3.19 (dd, JAB
4.5 Hz, JAX = 4.3 Hz, 1H, HA), 3.26 (s, 3H, -CH3), 3.51–3.61 (dd, JAB
11.2 Hz, JBX = 11.4 Hz, 1H, HB), 5.47–5.52 (dd, JAX = 4.2 Hz, JBX
=
=
=
4.4 Hz, 1H, HX), 7.33–7.50 (m, 4H, ArH), 7.59–7.70 (m, 5H, ArH),
7.89–7.98 (m, 4H, ArH), 8.38 (bs, 1H, NH, D2O exchangeable). IR
(KBr) cm– 1: 3298, 3026, 1643, 1461.
N-(4-Methylphenyl)-3-(4-nitrophenyl)-5-phenyl-1H-
pyrazoline-1-carbothioamide 3x
Yield: 56%, m.p.: 1438C. 1H-NMR (DMSO-d6) d: 3.11–3.16 (dd, JAB
4.5 Hz, JAX = 4.6 Hz, 1H, HA), 3.35 (s, 3H, -CH3), 3.51–3.64 (dd, JAB
13.3 Hz, JBX = 13.2 Hz, 1H, HB), 5.52–5.59 (dd, JAX = 6.3 Hz, JBX
=
=
=
3-(4-Chlorophenyl)-N-(4-methylphenyl)-5-phenyl-1H-
pyrazoline-1-carbothioamide 3r
6.6 Hz, 1H, HX), 7.35–7.49 (m, 4H, ArH), 7.63–7.71 (m, 5H, ArH),
7.89–7.89 (m, 4H, ArH), 8.41 (bs, 1H, NH, D2O exchangeable). IR
(KBr) cm– 1: 3230, 3109, 1598, 1438.
Yield: 73%, m.p.: 1138C. 1H-NMR (DMSO-d6) d: 3.21–3.34 (dd, JAB
14.2 Hz, JAX = 13.6 Hz, 1H, HA), 3.41 (s, 3H, -CH3), 3.56-3.61 (dd, JAB
=
=
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180
N. Siddiqui et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 173–181
calculated by means of the computer program by Litchfield and
Wilcoxon's method [23].
N-(4-Chlorophenyl)-3-(4-nitrophenyl)-5-phenyl-1H-
pyrazoline-1-carbothioamide 3y
Yield: 69%, m.p.: 1058C. 1H-NMR (DMSO-d6) d: 3.16–3.23 (dd, JAB
6.6 Hz, JAX = 6.8 Hz, 1H, HA), 3.48–3.53 (dd, JAB = 4.5 Hz, JBX
=
=
Antidepressant study
4.5 Hz, 1H, HB), 5.50–5.63 (dd, JAX = 13.5 Hz, JBX = 13.4 Hz, 1H, HX),
Forced swim pool method
7.56–7.69 (m, 4H, ArH), 7.73–7.84 (m, 5H, ArH), 7.95–8.24 (m,
4H, ArH), 8.16 (bs, 1H, NH, D2O exchangeable). IR (KBr) cm– 1
3230, 3010, 1649, 1418.
:
Albino mice were placed in a chamber (diameter: 45 cm; height:
20 cm) containing water up to a height of 15 cm at 25 l 28C. Two
swim sessions were conducted, an initial 15 min pre-test, fol-
lowed by a 5 min test after 24 h. The animals were administered
an i.p. injection (30 mg/kg) of the test compounds after the pre-
test sessions. The period of immobility (passive floating without
struggling and making only those movements which are neces-
sary to keep its head above the surface of water) during the
5 min test period was measured [24].
N-(4-Bromophenyl)-3-(4-nitrophenyl)-5-phenyl-1H-
pyrazoline-1-carbothioamide 3z
Yield: 71%, m.p.: 1298C. 1H-NMR (DMSO-d6) d: 3.08–3.14 (dd, JAB
6.0 Hz, JAX = 5.8 Hz, 1H, HA), 3.79–3.89 (dd, JAB = 12.0 Hz, JBX
=
=
12.0 Hz, 1H, HB), 5.52–5.58 (dd, JAX = 7.2 Hz, JBX = 7.1 Hz, 1H, HX),
7.61–7.76 (m, 4H, ArH), 7.84–7.99 (m, 5H, ArH), 8.11–8.29 (m,
4H, ArH), 8.41 (bs, 1H, NH, D2O exchangeable). IR (KBr) cm– 1
3228, 3029, 1658, 1416.
:
Neurotoxicity screening (NT)
The minimal motor impairment was measured in mice by the
rotorod test. The mice were trained to stay on an accelerating
rotorod (diameter: 3.2 cm) that rotates at 10 rpm. Trained ani-
mals were injected intraperitoneally with the test compounds at
a dose of 25 mg/kg. Neurotoxicity was indicated by the inability
of the animal to maintain equilibration on the rod for at least
1 min in each of the three trials [25].
Pharmacology
The MES, scPTZ, and rotorod tests were carried out according to
the protocol issued by the Antiepileptic Drug Development Pro-
gram (ADD), Epilepsy Branch, National Institute of Health,
Bethesda, MD, USA [20]. The investigations were conducted with
albino mice of either sex (25–30 g). The albino mice were kept
under standard conditions at an ambient temperature of
25 l 28C. Food and water were withdrawn prior to the experi-
ments.
LogP determination
The partition coefficient between octanol and phosphate buffer
was determined at room temperature (298C). Octanol (10 mL)
and phosphate buffer (10 mL, pH = 7.4) were taken in a glass
stoppered graduated tube and 5 mg of accurately weighed drug
was added. The mixture was then shaken with the help of
mechanical shaker for 24 h at room temperature. The mixture
was then transferred to a separating funnel and allowed to equi-
librate for 6 h. The aqueous and octanol phase were separated
and filtered through membrane filter and drug content in aque-
ous phase was analyzed by UV spectroscopy.
All the experimental protocols were carried out with the per-
mission from Institutional Animal Ethics committee (IAEC),
form no. 416. Animals were obtained from the Central Animal
House Facility., Hamdard University, New-Delhi-62. (Registration
no. and date of registration, 173/CPCSEA, 28 Jan., 2000).
Anticonvulsant activity
The partition coefficient was calculated by
MES test (Maximal electroshock seizure method)
The compounds were screened for their anticonvulsant activity
by electroshock seizure method. A supra-maximal electroshock
of a current intensity of 50 mA, 60 Hz for 0.2 s duration was
given to mice, administered with 25 mg/kg of the test com-
pounds. The abolition of the hind limb tonic extensor spasm
was recorded as an increased of anticonvulsant activity [21].
PC = (Ct –Ca)/Ca
(1)
PC = Partition coefficient, Ct = Concentration of total drug, Ca =
Concentration of the drug in aqueous phase.
In-silico studies
The in-silico studies were carried out using the computer pro-
grams Chem3D Pro 6.0 version and ACD Chem3D Freeware.
Pentylenetetrazole induced seizures (scPTZ) test
Pentylenetetrazole (85 mg/kg) that produces seizures in >95% of
mice, is administered as a 0.5% solution subcutaneously in the
posterior midline. The animal was observed for 30 min, failure
to observe even a threshold seizure (a single episode of clonic
spasms of at least 5 s duration) was defined as protection [22].
The pharmacological parameters estimated in phase-I screen-
ing were quantified for compound 3a–z in phase-II screening
(Table 3). Anticonvulsant activity was expressed in terms of the
median effective dose (ED50), and neurotoxicity was expressed as
the median toxic dose (TD50). For the determination of ED50 and
TD50 values, groups of ten mice were given a range of intraperito-
neal (i.p.) doses of the test drug until at least three points were
established in the range of 10–90% seizure protection or mini-
mal observed neurotoxicity. From the plot of these data, the
respective ED50 and TD50 values, 95% confidence intervals, slope
of the regression line, and the standard error of the slope were
Statistical analysis
All the statistical analyses were performed by using the software
SigmaStat 2.03.
References
[1] C. Wasterlain in “Basic Neurochemistry: Molecular, Cellular
and Medical Aspects”, (Eds.: G. Siegel, G. Agranoff, R. W. Alb-
ers, P. Molinof), Raven Press, New York, 4th ed, 1989, pp.
797–810.
[2] J. J. Coatsworth, NINDS Monograph No. 12 HEW Publica-
tion No. (NIH) 73–51, U. S. Government Printing Office,
1971.
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