Design and biological evaluation of phenyl-substituted analogs of β-phenylethylidenehydrazine

Article abstract of DOI:10.1016/j.bmc.2005.04.072

β-Phenylethylidenehydrazine (PEH) has been demonstrated previously to be an inhibitor of γ-aminobutyric acid transaminase (GABA-T) and to cause a marked increase in rat brain levels of GABA, a major neurotransmitter. A group of PEH analogs, possessing a variety of substituents (Me, OMe, Cl, F, and CF₃) at the 2-, 3-, and 4-positions of the phenyl ring, were synthesized for evaluation as inhibitors of GABA-T. The details of the synthesis and chemical characterization of the analogs are described. Preliminary in vitro screening for GABA-T inhibition showed that all the analogs possessed activity against this enzyme, although substitution of CF₃ at the 2- and 4-positions caused reduced activity. One of the drugs, 4-fluoro-β- phenylethylidenehydrazine, was investigated further ex vivo, where it was shown to inhibit GABA-T, elevate brain levels of GABA, and decrease levels of glutamine, similar to the profile observed previously for PEH.

Full text of DOI:10.1016/j.bmc.2005.04.072

Bioorganic & Medicinal Chemistry 13 (2005) 4389–4395
Design and biological evaluation of phenyl-substituted
analogs of b-phenylethylidenehydrazine
a
Bernard Sowa, Gillian Rauw, Asghar Davood, Afshin Fassihi,
a
b
b
b
Edward E. Knaus and Glen B. Baker
a,b,
*
a
Neurochemical Research Unit, Department of Psychiatry, University of Alberta, Edmonton, Canada T6G 2R7
b
Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Canada T6G 2N8
Received 31 March 2005; revised 19 April 2005; accepted 20 April 2005
Available online 31 May 2005
Abstract—b-Phenylethylidenehydrazine (PEH) has been demonstrated previously to be an inhibitor of c-aminobutyric acid trans-
aminase (GABA-T) and to cause a marked increase in rat brain levels of GABA, a major neurotransmitter. A group of PEH ana-
logs, possessing a variety of substituents (Me, OMe, Cl, F, and CF ) at the 2-, 3-, and 4-positions of the phenyl ring, were
3
synthesized for evaluation as inhibitors of GABA-T. The details of the synthesis and chemical characterization of the analogs
are described. Preliminary in vitro screening for GABA-T inhibition showed that all the analogs possessed activity against this
3
enzyme, although substitution of CF at the 2- and 4-positions caused reduced activity. One of the drugs, 4-fluoro-b-phen-
ylethylidenehydrazine, was investigated further ex vivo, where it was shown to inhibit GABA-T, elevate brain levels of GABA,
and decrease levels of glutamine, similar to the profile observed previously for PEH.
ꢀ
2005 Elsevier Ltd. All rights reserved.
1
. Introduction
preparations, which may contain sympathomimetic
amines; the possibility of such interactions has severely
restricted its use as a psychiatric drug. Studies by us
Phenelzine (PLZ) (Fig. 1) is a monoamine oxidase
MAO)-inhibiting antidepressant, which is also effective
1
,10
(
and others
have provided indirect evidence for a
for treating panic disorder and social anxiety disorder.
PLZ is also an inhibitor of the c-aminobutyric acid
transaminase (GABA-T) and causes a marked elevation
metabolite of PLZ that might contribute to the GABA-
elevating action of PLZ. We have synthesized one such
putative metabolite, namely b-phenylethylidenehydra-
zine (PEH) (Fig. 1), and found that it retained the GAB-
1
–3
of brain levels of GABA in rats and gerbils.
1
1
Aergic actions of PLZ but had minimal effect on MAO,
Because of its GABAergic properties, PLZ was tested in
our laboratories to verify if it had neuroprotective prop-
suggesting that it could be an effective neuroprotective
drug without the adverse effects of PLZ. To investigate
the structure–activity relationships with PEH, we have
prepared a number of phenyl ring-substituted analogs
and conducted preliminary studies on their effects on
4
–8
erties similar to those of other GABAergic drugs.
PLZ was shown to reduce neuronal cell death if given
prior to or within a few hours of induction of global
3
,9
ischemia in gerbils (an animal model of stroke). These
findings suggested that PLZ should be investigated fur-
ther for its effects on stroke and other neurodegenerative
disorders. However, because PLZ inhibits MAO irre-
versibly, there is the potential danger of adverse interac-
tions of PLZ with foods containing sympathomimetic
amines such as tyramine or with drugs such as cold
Keywords: b-Phenylethylidenehydrazine; c-Aminobutyric acid; GABA
transaminase; Glutamine.
*
Corresponding author. Tel.: +1 7804076503; fax: +1 7804076804;
e-mail: glen.baker@ualberta.ca
Figure 1. Structures of phenelzine (1) and b-phenylethylidenehydr-
azine (2).
0
968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.04.072

4
390
B. Sowa et al. / Bioorg. Med. Chem. 13 (2005) 4389–4395
Table 1. Inhibition of GABA-T in vitro
GABA metabolism. One of these drugs, 4-FPEH, was
chosen for initial ex vivo studies on GABA-T activity
and levels of GABA and related amino acids in rat brain
since a substituent in the 4-position of the ring would be
expected to reduce the likelihood of ring hydroxylation,
perhaps increasing the availability of the drug in the
brain. In addition, replacement of a hydrogen atom by
a fluorine atom is known to enhance the pharmacologi-
cal activity of many classes of drugs.
a
Compound
R
% Inhibition of GABA-T
PEH
H
75.4 ± 2.7
51.3 ± 0.6
60.1 ± 0.4
56.1 ± 2.8
62.9 ± 0.2
46.3 ± 1.8
75.4 ± 0.2
77.6 ± 0.7
74.9 ± 0.1
72.5 ± 0.3
76.0 ± 0.3
70.4 ± 0.4
66.6 ± 0.9
54.2 ± 0.2
61.6 ± 0.7
33.2 ± 2.4
59.0 ± 0.7
À4
4
4
4
a
b
c
2-Me
2-OMe
2-Cl
2-F
4d
4e
4f
2-CF
3
3-Me
3-OMe
3-Cl
4
4
4
4
4
4
4
g
h
i
3-F
2. Results
j
3-CF
3
k
l
4-Me
4-OMe
4-Cl
A group of b-phenylethylidenehydrazines (4a–p), pos-
sessing a variety of substituents (Me, OMe, Cl, F, and
m
CF ) at the 2-, 3-, and 4-positions of the phenyl ring,
3
4n
4o
4p
4-F
4-CF3
2,4-Cl
were synthesized in 67–96% yield by condensation of
the respective phenylacetaldehydes (3a–p) with hydra-
zine hydrate using a procedure similar to that reported
for the preparation of the parent compound b-phen-
2
All drugs were present at a concentration of 10 M.
a
Values are presented as means (% of control) ± standard error of the
mean (n = 4).
1
1,12
ylethylidenehydrazine (2)
(Fig. 2).
Compounds 4a–p were obtained as a mixture of the (E)-
4
and (Z)-4 stereoisomers, in a ratio of approximately
The ratio of the (E)- and (Z)-stereoisomers 4a–p was
calculated from the integrals for the dual CH
3
ety. The H NMR spectra for compounds 4a–p exhib-
:1, which differ in stereochemistry about the C@N moi-
2
1
1
resonances in the H NMR spectrum.
ited dual resonances for the methylene (CH ), methine
2
(
@CH), and amino (NH ) groups. In this regard, the
The ability of this group of b-phenylethylidenehydra-
zines (4a–p) to inhibit GABA-T was determined using
an in vitro assay, and the results are summarized in
Table 1.
2
@
NH resonance was shielded, in the (E)-isomer relative
to the (Z)-isomer. These shielding effects are consistent
1
with those reported for acetaldehyde hydrazones.
CH and CH resonances were deshielded, and the
2
2
3
The in vitro screening data acquired indicate that all the
analogs have the ability to inhibit GABA-T, but none
was more effective than PEH at the concentration
À4
(
10 M) tested. As illustrated in Figures 3 and 4,
administration of 4-FPEH (4n) to rats was associated
with time-dependent elevations in brain levels of GABA
as well as inhibition of GABA-T.
Figure 3. Brain GABA levels (mean ± SEM) in rats at varying times
following a single injection of 4-FPEH (30 mg/kg, ip). Mean control
(vehicle-treated) levels were 255 ± 50 lg/g (n = 15). *p < 0.05 versus
control group. n = 4–5 animals per group.
Figure 2. Reagents and conditions: (a) H
2 2 2
NNH ÆH O, 25 ꢁC, 2 h (4a–
b, d, and f–o), or 110 ꢁC, 2 h (c, e, and p).

B. Sowa et al. / Bioorg. Med. Chem. 13 (2005) 4389–4395
4391
Drug treatment was also associated with a significant
reduction in brain glutamine content, but with no sig-
nificant changes in brain glutamate levels (Fig. 5).
3. Discussion
While the in vitro screening data acquired indicate that
all the analogs have the ability to inhibit GABA-T, com-
pounds having a C-3 phenyl substituent (4f–j) were al-
ways more efficacious than the respective isomeric C-2
(
4a–e) or C-4 (4k–o) compounds. The addition of a
CF3 group in the C-2 and C-4 positions reduced
GABA-T-inhibiting activity rather markedly (Table 1).
The observed elevation in brain GABA levels following
the administration of 4-FPEH (4n) (Fig. 3) is explained,
at least in part, by inhibition of GABA-T (Fig. 4). The
reason for the reduction in brain glutamine levels com-
bined with maintenance of brain glutamate levels is
not yet known but it is possible that these effects are a
result of inhibition of glutamine synthetase concurrent
with continued glutaminase activity. Such actions will
now be studied.
Figure 4. Inhibition of GABA-T (% of control, mean ± SEM) follow-
ing a single injection of 4-FPEH (30 mg/kg, ip). *p < 0.05 versus
control group. n = 4–8 animals per group.
The effects of 4-FPEH (4n) on GABA-T and levels of
GABA, glutamate and glutamine are similar in direction
1
1
and magnitude to those seen by Paslawski et al. with
the parent compound, PEH; similar results have now
been obtained in gerbil brain (Sowa et al., unpublished).
Based on the findings in vitro with all of the analogs syn-
thesized and ex vivo with 4-FPEH, several of these ana-
logs are now being investigated for their effects on brain
levels of GABA, glutamate, and glutamine in rat and
gerbil brain, and their actions in the global ischemia
model of stroke.
4. Experimental
4.1. Materials
The following chemicals were obtained from Sigma
Chemical Company (St. Louis, MO, USA): GABA,
glutamate, glutamine, alanine, a-ketoglutaric acid,
2-aminoethylisothiouronium, and tri-n-octylamine. o-
Phthalaldehyde reagent solution was obtained from
Pierce Chemicals (Rockford, IL, USA).
4.2. Spectroscopy
Infrared (IR) spectra were recorded using a Nicolet 550
Series II Magna FT-IR spectrometer. Nuclear magnetic
1
13
resonance ( H NMR, C NMR) spectra were recorded
on a Bruker AM-300 spectrometer. The assignment of
exchangeable protons (NH ) was confirmed by the addi-
2
1
3
tion of D O. C NMR were acquired using the J mod-
2
ulated spin-echo technique where methyl and methine
carbon resonances appear as positive peaks, and meth-
ylene and quaternary carbon resonances appear as neg-
ative peaks. Silica gel column chromatography was
performed using silica gel (70–230 mesh) purchased
from Silicycle (Quebec, Canada). The phenylacetalde-
Figure 5. Brain glutamate and glutamine levels (mean ± SEM) in rats
at varying times following a single injection of 4-FPEH (30 mg/kg, ip).
Mean control (vehicle-treated) levels for glutamate and glutamine
were 1924 ± 365 lg/g (n = 14) and 623 ± 148 lg/g (n = 14), respec-
tively. *p < 0.05 versus control group. n = 3–5 animals per group.

4392
B. Sowa et al. / Bioorg. Med. Chem. 13 (2005) 4389–4395
hydes 3a–p were prepared by oxidation of the corre-
sponding phenylethyl alcohols using dipyridine
chromium (VI) oxide (Collinꢀs reagent) in dry dichloro-
CH@N), 5.42 (br s, 2H, NH ), 3.84 (s, 3H, OCH ), 3.46
13
2
3
(d, J = 5.1 Hz, 2H, CH CH); C NMR (CDCl ): d
2
3
157.19 (C-2), 144.20 (CH@N), 130.06 (C-6), 128.03
(C-4), 125.86 (C-1), 120.67 (C-5), 110.39 (C-3), 55.30
(OCH ), 27.45 (CH CH).
1
4,15
methane at 25 ꢁC according to reported procedures.
4
-Trifluoromethyl- and 2,4-dichlorophenethyl alcohol,
3
2
which were not commercially available from Aldrich,
were prepared by the lithium aluminum hydride reduc-
tion of the respective 4-trifluoromethyl- and 2,4-dichlor-
4.3.3. [2-(2-Chlorophenyl)ethylidene]hydrazine (4c). Yield,
82%; (E):(Z) = 70:30; IR (film): m 3369, 3207 (NH ),
2
16,17
À1
1
ophenylacetic acids using reported procedures.
other reagents were purchased from Aldrich Chemicals
Milwaukee, WI).
All
1616 (CH@N) cm ; (E)-isomer, H NMR (CDCl ): d
3
7.35–7.42 (m, 1H, H-3), 7.15–7.26 (m, 4H, H-4, H-5, H-
6, CH@N), 5.20 (br s, 2H, NH ), 3.64 (d, J = 5.4 Hz,
(
2
1
3
2
H, CH CH); C NMR (CDCl ): d 142.89 (CH@N),
2
3
4
.3. General procedure for the syntheses of phenyl-
139.30 (C-1), 135.36 (C-2), 130.79 (C-6) 129.45 (C-3),
128.03 (C-4), 126.92 (C-5), 36.49 (CH CH); (Z)-isomer,
substituted analogs of b-phenylethylidenehydrazine (4a–p)
2
1
H NMR CDCl ): d 7.35–7.42 (m, 1H, H-3), 7.15–7.26
3
The phenylacetaldehyde (3a–p, 1 mmol) was added
dropwise to a solution of hydrazine monohydrate
(m, 3H, H-4, H-5, H-6), 6.67 (t, J = 4.5 Hz, 1H, CH@N),
5.40 (br s, 2H, NH ), 3.57 (d, J = 4.5 Hz, 2H, CH CH);
2
2
1
3
(
10 mmol) with vigorous stirring at 25 ꢁC, and the reac-
C NMR (CDCl ): d 142.20 (CH@N), 139.30 (C-1),
3
tion was allowed to proceed with stirring for 2 h at 25 ꢁC
for the preparation of 4a–b, d, and f–o, or heating at
135.36 (C-2), 130.38 (C-6), 129.66 (C-3), 128.29 (C-4),
127.09 (C-5), 30.55 (CH CH).
2
1
10 ꢁC for 2 h for the preparation of 4c, e, and p. Chlo-
roform (5 mL), and then water (3 mL), was added to the
reaction mixture at 25 ꢁC. This mixture was extracted
with chloroform (3 · 5 mL), the combined chloroform
extracts were washed with water (2 · 1 mL), and the
chloroform fraction was dried (Na SO ). Removal of
4.3.4. [2-(2-Fluorophenyl)ethylidene]hydrazine (4d). Yield,
71%; (E):(Z) = 73:27; IR (film): m 3368, 3214 (NH ), 1622
2
À1
1
(CH@N) cm ; (E)-isomer, H NMR (CDCl ): d 7.01–
3
7.27 (m, 5H, phenyl hydrogens, CH@N), 5.20 (br s, 2H,
1
NH ), 3.54 (d, J = 5.7 Hz, 2H, CH CH); C NMR
3
2
4
2
2
1
the solvent in vacuo at 25 ꢁC afforded the respective b-
phenylethylidenehydrazine products 4a–p as a pale yel-
low oil that consisted of a mixture of (E)-4 and (Z)-4
stereoisomers. Products 4a–p were stored at À78 ꢁC
prior to their use in biological studies. The % chemical
(CDCl ): d 160.84 (d, JC,F = 246.1 Hz, C-2), 143.07
3
3
(CH@N), 130.85 (d, J = 4.39 Hz, C-6), 128.32 (d,
C,F
3
2
J
= 8.79 Hz, C-4), 124.34 (d, J = 15.38 Hz, C-1),
C,F
C,F
4
124.10 (d, JC,F = 3.3 Hz, C-5), 115.27 (d, JC,F
2
=
21.97 Hz, C-3), 32.07 (CH CH); (Z)-isomer, H NMR
1
2
1
yield, (E):(Z) ratio at 25 ꢁC as determined from the H
(CDCl ): d 7.01–7.27 (m, 4H, phenyl hydrogens), 6.70 (t,
3
NMR integrals for the CH resonances of the two iso-
J = 4.5 Hz, 1H, CH@N), 5.40 (br s, 2H, NH ), 3.49 (d,
2
2
1
mers, and spectral data (IR, H NMR, and C NMR)
13
13
J = 4.5 Hz, 2H, CH CH); C NMR (CDCl ): d 160.84
2
3
1
for compounds 4a–p are listed below.
(d, JC,F = 246.1 Hz, C-2), 142.32 (CH@N), 130.52
4
d, JC,F = 3.29 Hz, C-6), 128.56 (d,
3
J
(
C-4), 124.34 (d, JC,F = 15.38 Hz, C-1), 124.32 (d,
= 8.78 Hz,
C,F
2
4
9
.3.1. [2-(2-Methylphenyl)ethylidene]hydrazine(4a). Yield,
6%; (E):(Z) = 72:28; IR (film): m 3382, 3214 (NH ), 1649
4
2
JC,F = 3.3 Hz, C-5), 115.48 (d, J = 20.88 Hz, C-3),
C,F
2
À1
1
(
7
CH@N) cm ; (E)-isomer, H NMR (CDCl ): d 7.16–
25.79 (CH CH).
2
3
.21 (m, 4H, phenyl hydrogens), 7.13 (t, J = 5.7 Hz, 1H,
CH@N), 5.15 (br s, 2H, NH ), 3.52 (d, J = 5.7 Hz, 2H,
4.3.5. [2-(2-Trifluoromethylphenyl)ethylidene]hydrazine
(4e). Yield, 91%; (E):(Z) = 73:27; IR (film): m 3369, 3201
2
1
3
CH CH), 2.34 (s, 3H, CH ); C NMR (CDCl ): d
2
3
3
À1
1
1
44.06 (CH@N), 136.50, 135.55 (phenyl C-1, C-2),
30.21, 129.26 (phenyl C-3, C-6), 126.69, 126.00 (phenyl
(NH ), 1609 (CH@N) cm ; (E)-isomer, H NMR
2
1
C-4, C-5), 36.51 (CH CH), 19.47 (CH ); (Z)-isomer, H
(CDCl ): d 7.30–7.65 (m, 4H, phenyl hydrogens), 7.14
3
1
(t, J = 5.4 Hz, 1H, CH@N), 5.20 (br s, 2H, NH ), 3.69
2
3
2
1
(d, J = 5.4 Hz, 2H, CH CH); (Z)-isomer, H NMR
NMR (CDCl ): d 7.16–7.21 (m, 4H, phenyl hydrogens),
3
2
6
3
.67 (t, J = 4.8 Hz, 1H, CH@N), 5.35 (br s, 2H, NH ),
(CDCl ): d 7.30–7.65 (m, 4H, phenyl hydrogens), 6.64
2
3
1
3
.44 (d, J = 4.8 Hz, 2H, CH CH), 2.31 (s, 3H, CH );
C
(t, J = 4.5 Hz, 1H, CH@N), 5.35 (br s, 2H, NH ), 3.63
2
3
2
NMR (CDCl ): d 143.47 (CH@N), 136.50, 135.55 (phenyl
(d, J = 4.5 Hz, 2H, CH CH).
3
2
C-1, C-2), 130.37, 128.97 (phenyl C-3, C-6), 126.92, 126.22
phenyl C-4, C-5), 30.55 (CH CH), 19.47 (CH ).
(
4.3.6. [2-(3-Methylphenyl)ethylidene]hydrazine (4f). Yield,
2
3
6
7%; (E):(Z) = 72:28; IR (film): m 3373, 3212 (NH ), 1607
2
1
À1
4.3.2. [2-(2-Methoxyphenyl)ethylidene]hydrazine (4b). Yield,
(CH@N) cm ; (E)-isomer, H NMR (CDCl ): d 7.12–
3
8
8%; (E):(Z) = 64:36; IR (film): m 3381, 3199 (NH ), 1597
7.29 (m, 2H, H-5, CH@N), 7.07–7.09 (m, 3H, H-2,
2
À1
1
(CH@N) cm ; (E)-isomer, H NMR (CDCl ): d 7.15–
H-4, H-6), 5.20 (br s, 2H, NH ), 3.48 (d, J = 5.7 Hz,
3
2
1
3
7.28 (m, 3H, phenyl H-4, H-6, CH@N), 6.88–6.95 (m,
2H, CH CH), 2.34 (s, 3H, CH ); C NMR (CDCl ): d
2 3 3
2H, phenyl H-3, H-5), 5.13 (br s, 2H, NH ), 3.84 (s, 3H,
OCH ), 3.52 (d, J = 5.7 Hz, 2H, CH CH); C NMR
144.81 (CH@N), 138.16, 137.18 (C-1, C-3), 129.51
(C-2), 128.42, 127.23 (C-4, C-5), 125.75 (C-6), 38.73
2
13
3
2
1
(CH CH), 21.32 (CH ); (Z)-isomer, H NMR (CDCl ):
(
1
5
CDCl ): d 157.19 (C-2), 144.93 (CH@N), 130.17 (C-6),
3
2
3
3
27.82 (C-4), 125.86 (C-1), 120.53 (C-5), 110.29 (C-3),
d 7.12–7.29 (m, 1H, H-5), 6.98–7.09 (m, 3H, H-2, H-4,
H-6), 6.77 (t, J = 4.8 Hz, 1H, CH@N), 5.31 (br s, 2H,
NH ), 3.43 (d, J = 4.8 Hz, 2H, CH CH), 2.35 (s, 3H,
1
5.30 (OCH ), 33.30 (CH CH); (Z)-isomer, H NMR
3
2
(
CDCl ): d 7.15–7.28 (m, 2H, phenyl H-4, H-6), 6.88–
3
2
2
1
3
6.95 (m, 2H, phenyl H-3, H-5), 6.69 (t, J = 5.1 Hz, 1H,
CH3); C NMR (CDCl ): d 144.06 (CH@N), 138.42,
3

B. Sowa et al. / Bioorg. Med. Chem. 13 (2005) 4389–4395
4393
3
123.42 (q, J = 3.3 Hz, C-4), 38.54 (CH CH); (Z)-
136.44 (C-1, C-3), 129.29 (C-2), 128.64, 127.37 (C-4,
C-5), 125.52 (C-6), 32.34 (CH CH), 21.32 (CH ).
C,F
2
1
isomer, H NMR (CDCl ): d 7.39–7.55 (m, 4H, phenyl
2
3
3
hydrogens), 6.73 (t, J = 5.1 Hz, 1H, CH@N), 5.32
1
3
4
.3.7. [2-(3-Methoxyphenyl)ethylidene]hydrazine (4g).
(br s, 2H, NH ), 3.54 (d, J = 5.1 Hz, 2H, CH CH);
C
2
2
Yield, 74%; (E):(Z) = 73:27; IR (film): m 3385, 3205
NMR (CDCl ): d 142.30 (CH@N), 138.33 (C-1), 133.36
3
À1
1
2
(q, J = 29.66 Hz, C-3), 131.93 (C-6), 129.26 (C-5),
(
(
NH ), 1600 (CH@N) cm ; (E)-isomer, H NMR
2
C,F
3
125.32 (q, JC,F = 3.3 Hz, C-2), 124.02 (q, JC,F =
1
CDCl ): d 7.21–7.27 (m, 1H, H-5), 7.16 (t, J = 5.7 Hz,
3
3
1
s, 2H, NH ), 3.80 (s, 3H, OCH ), 3.49 (d, J = 5.7 Hz,
H, CH@N), 6.77–6.82 (m, 3H, H-2, H-4, H-6), 5.16 (br
272.46 Hz, CF ) 123.64 (q, J = 3.3 Hz, C-4), 32.14
3 C,F
(CH CH).
2
2
3
1
3
2
1
6
H, CH CH); C NMR (CDCl ): d 159.71 (C-3),
2 3
44.42 (CH@N), 138.87 (C-1), 129.50 (C-5), 121.08 (C-
4.3.11. [2-(4-Methylphenyl)ethylidene]hydrazine (4k).
Yield, 85%; (E):(Z) = 73:27; IR (film): m 3373, 3207
), 114.38 (C-2), 111.95 (C-4), 55.13 (OCH ), 38.84
3
1
À1
1
(
(
6
CH CH); (Z)-isomer, H NMR (CDCl ): d 7.21–7.27
m, 1H, H-5), 6.77–6.82 (m, 4H, CH@N, H-2, H-6, H-
), 5.28 (br s, 2H, NH ), 3.81 (s, 3H, OCH ), 3.45 (d,
(NH ), 1614 (CH@N) cm ; (E)-isomer, H NMR
2
3
2
(CDCl ): d 7.12–7.16 (m, 5H, CH@N, phenyl hydro-
3
gens), 5.15 (br s, 2H, NH ), 3.48 (d, J = 5.7 Hz, 2H,
2
3
2
1
3
13
J = 4.8 Hz, 2H, CH CH); C NMR (CDCl ): d 159.71
CH CH), 2.33 (s, 3H, CH ); C NMR (CDCl ): d
2
3
2
3
3
(
1
3
C-3), 143.68 (CH@N), 138.87 (C-1), 129.74 (C-5),
144.96 (CH@N), 134.17 (C-1, C-4), 129.22 (C-2, C-6),
20.83 (C-6), 114.25 (C-2), 112.04 (C-4), 55.13 (OCH ),
2.46 (CH CH).
128.65 (C-3, C-5), 38.38 (CH CH), 21.00 (CH ); (Z)-iso-
3
2
3
1
mer, H NMR (CDCl ): d 7.12–7.14 (m, 4H, phenyl
2
3
hydrogens), 6.77 (t, J = 4.8 Hz, 1H, CH@N), 5.35 (br
4
9
.3.8. [2-(3-Chlorophenyl)ethylidene]hydrazine (4h). Yield,
5%; (E):(Z) = 73:27; IR (film): m 3361, 3203 (NH ), 1602
s, 2H, NH ), 3.44 (d, J = 5.7 Hz, 2H, CH CH), 2.35 (s,
2
2
1
3
3H, CH3); C NMR (CDCl ): d 144.32 (CH@N),
2
3
À1
1
(
CH@N) cm ; (E)-isomer, H NMR (CDCl ): d 7.08–
134.17 (C-1, C-4), 129.44 (C-2, C-6), 128.40 (C-3, C-5),
32.01 (CH CH), 21.00 (CH ).
3
7
NH ), 3.49 (d, J = 5.7 Hz, 2H, CH CH); C NMR
.25 (m, 5H, phenyl hydrogens, CH@N), 5.20 (br s, 2H,
2
3
1
3
2
2
(
CDCl ): d 143.43 (CH@N), 139.37 (C-1), 134.32 (C-3),
4.3.12. [2-(4-Methoxyphenyl)ethylidene]hydrazine (4l).
Yield, 87%; (E):(Z) = 73:27; IR (film): m 3388, 3220
3
1
3
7
29.76 (C-5), 128.89 (C-2), 126.96, 126.73 (C-4, C-6),
1
À1
1
8.41 (CH CH); (Z)-isomer, H NMR (CDCl ): d 7.08–
(NH ), 1609 (CH@N) cm ; (E)-isomer, H NMR
2
3
2
.25 (m, 4H, phenyl hydrogens), 6.72 (t, J = 4.5 Hz, 1H,
(CDCl ): d 7.11–7.15 (m, 3H, H-2, H-6, CH@N), 6.84–
3
CH@N), 5.30 (br s, 2H, NH ), 3.45 (d, J = 4.5 Hz, 2H,
CH CH); C NMR (CDCl ): d 142.60 (CH@N), 139.37
6.89 (m, 2H, H-3, H-5), 5.15 (br s, 2H, NH ), 3.78 (s,
3H, OCH ), 3.45 (d, J = 5.7 Hz, 2H, CH CH);
2
2
1
3
13
C
2
3
3
2
(
1
C-1), 134.32 (C-3), 130.02 (C-5), 128.71 (C-2), 126.96,
26.73 (C-4, C-6), 32.02 (CH CH).
NMR (CDCl ): d 158.40 (C-4), 144.96 (CH@N), 129.69
3
(C-2, C-6), 129.23 (C-1), 113.93 (C-3, C-5), 55.19
(
2
1
OCH ), 37.87 (CH CH); (Z)-isomer, H NMR (CDCl ):
3
2
3
4
8
1
7
.3.9. [2-(3-Fluorophenyl)ethylidene]hydrazine (4i). Yield,
5%; (E):(Z) = 73:27; IR (film): m 3375, 3207 (NH ),
d 7.11–7.15 (m, 2H, H-2 and H-6 aryl), 6.84–6.89 (m, 2H,
H-3 and H-5), 6.74 (t, J = 4.8 Hz, 1H, CH@N), 5.33 (br s,
2H, NH ), 3.80 (s, 3H, OCH ), 3.41 (d, J = 4.8 Hz, 2H,
2
À1
1
622 (CH@N) cm ; (E)-isomer, H NMR (CDCl ): d
3
2
3
1
3
.23–7.31 (m, 1H, H-5), 7.15 (t, J = 5.4 Hz, 1H,
CH CH); C NMR (CDCl ): d 158.40 (C-4), 144.32
2 3
CH@N), 6.90–7.02 (m, 3H, H-2, H-4, H-6), 5.21 (br s,
(CH@N), 129.44 (C-2, C-6), 129.23 (C-1), 114.15 (C-3,
C-5), 55.19 (OCH ), 31.48 (CH CH).
1
3
2
H, NH ), 3.51 (d, J = 5.4 Hz, 2H, CH CH);
C
2
2
3
2
1
NMR (CDCl ): d 162.87 (d, JC,F = 246.1 Hz, C-3),
3
3
1
1
2
43.56 (CH@N), 139.87 (d, JC,F = 7.69 Hz, C-1),
4.3.13. [2-(4-Chlorophenyl)ethylidene]hydrazine (4m).
Yield, 83%; (E):(Z) = 75:25; IR (film): m 3365, 3182
3
29.96 (d, JC,F = 7.69 Hz, C-5), 124.40 (d, JC,F
4
=
.2 Hz, C-6), 115.67 (d, J = 20.88 Hz, C-2), 113.43
2
À1
1
(NH ), 1601 (CH@N) cm ; (E)-isomer, H NMR
C,F
2
2
(
d, J = 20.88 Hz, C-4), 38.47 (CH CH); (Z)-isomer,
C,F
H NMR (CDCl ): d 7.23–7.31 (m, 1H, H-5), 6.90–
(CDCl ): d 7.22–7.32 (m, 3H, H-3, H-5, CH@N),
2
3
1
7.08–7.16 (m, 2H, H-2, H-6), 5.20 (br s, 2H, NH ),
3
2
1
3
3.47 (d, J = 5.7 Hz, 2H, CH CH); C NMR (CDCl3):
7
.02 (m, 3H, H-2, H-4, H-6), 6.73 (t, J = 5.1 Hz, 1H,
2
CH@N), 5.34 (br s, 2H, NH ), 3.47 (d, J = 5.1 Hz, 2H,
d 143.74 (CH@N), 141.10 (C-1), 136.70 (C-4), 130.12
2
1
3
1
CH CH); C NMR (CDCl ): d 162.87 (d,
J
=
=
(C-2, C-6), 128.65 (C-3, C-5), 38.12 (CH CH); (Z)-iso-
2
3
C,F
2
3
1
2
7
46.1 Hz, C-3), 142.70 (CH@N), 139.87 (d, JC,F
mer, H NMR (CDCl ): d 7.22–7.32 (m, 2H, H-3, H-
3
3
.69 Hz, C-1), 130.26 (d, J = 7.69 Hz, C-5), 124.19
5), 7.08–7.16 (m, 2H, H-2, H-6), 6.70 (t, J = 4.8 Hz,
1H, CH@N), 5.32 (br s, 2H, NH ), 3.43 (d, J = 4.8 Hz,
C,F
4
2
d, J = 3.3 Hz, C aryl), 115.53 (d, J = 20.87 Hz,
(
C-2), 113.74 (d, J = 20.88 Hz, C-4), 32.11 (CH CH).
C,F
6
C,F
2
2
13
2H, CH CH); C NMR (CDCl ): d 142.70 (CH@N),
C,F
2
2
3
141.10 (C-1), 136.70 (C-4), 129.88 (C-2, C-6), 128.93
(C-3, C-5), 31.76 (CH CH).
4
.3.10. [2-(3-Trifluoromethylphenyl)ethylidene]hydrazine
2
(
(
(
(
(
1
4j). Yield, 89%; (E):(Z) = 75:15; IR (film): m 3369, 3214
À1
1
NH ), 1622 (CH@N)cm ; (E)-isomer, H NMR
4.3.14. [2-(4-Fluorophenyl)ethylidene]hydrazine (4n).
Yield, 70%; (E):(Z) = 75:25; IR (film): m 3368, 3189
2
CDCl ): d 7.39–7.55 (m, 4H, phenyl hydrogens), 7.17
3
À1
1
t, J = 5.7 Hz, 1H, CH@N), 5.24 (br s, 2H, NH ), 3.58
(NH ), 1602 (CH@N) cm ; (E)-isomer, H NMR
2
2
1
3
d, J = 5.7 Hz, 2H, CH CH); C NMR (CDCl ): d
(CDCl ): d 7.14–7.24 (m, 3H, H-2, H-6, CH@N), 6.97–
2
3
3
2
43.17 (CH@N), 138.33 (C-1), 133.36 (q, JC,F
=
9.66 Hz, C-3), 132.22 (C-6), 128.96 (C-5), 125.48 (q,
7.06 (m, 2H, H-3, H-5), 5.18 (br s, 2H, NH ), 3.48 (d,
2
1
3
2
J = 5.7 Hz, 2H, CH CH); C NMR (CDCl ): d 161.64
2 3
1
(d, JC,F = 243.89 Hz, C-4), 144.23 (CH@N), 132.98
3
1
JC,F = 3.3, C-2), 124.02 (q, JC,F = 272.46 Hz, CF3),

4394
B. Sowa et al. / Bioorg. Med. Chem. 13 (2005) 4389–4395
4
d, J = 2.19 Hz, C-1), 130.22 (d, J = 7.69 Hz, C-2,
3
(
C-6), 115.35 (d, JC,F = 20.88 Hz, C-3, C-5), 37.99
4.6. HPLC assay for amino acids
C,F
C,F
2
1
(
(
CH CH); (Z)-isomer, H NMR (CDCl ): d 7.14–7.24
Rat brain tissue was homogenized in 5 volumes of dou-
ble-distilled water. The homogenates were then diluted
in 5 volumes of 100% methanol, centrifuged, and the
supernatants were further diluted in 10 volumes of dou-
ble-distilled water. Once loaded into the HPLC, immedi-
ately prior to injection amino acids were derivatized to
fluorescent thioalkyl-substituted isoindoles by reaction
with o-phthaldialdehyde in alkaline medium, with 2-
2
3
m, 2H, H-2, H-6), 6.97–7.06 (m, 2H, H-3, H-5), 6.73 (t,
J = 4.8 Hz, 1H, CH@N), 5.28 (br s, 2H, NH ), 3.44 (d,
2
1
3
J = 4.8 Hz, 2H, CH CH); C NMR (CDCl ): d 161.64
2
3
1
(
d, J = 243.89 Hz, C-4), 143.55 (CH@N), 132.98 (d,
C,F
3
JC,F = 2.19 Hz, C-1), 130.21 (d, J = 7.69 Hz, C-2,
4
C,F
2
C-6), 115.56 (d, JC,F = 20.88 Hz, C-3, C-5), 31.60
(
CH CH).
2
2
0
mercaptoethanol serving as a reducing agent. Fluores-
cence emitted by the thioalkyl derivatives was detected
using a Shimadzu RF10A fluorescence detector (excita-
tion wavelength 260 nm and emission wavelength
455 nm) following elution of the derivatized amino acids
from the column.
4
.3.15. [2-(4-Trifluoromethylphenyl)ethylidene]hydrazine
(
4o). Yield, 90%; (E):(Z) = 73:27; IR (film): m 3363,
À1
1
3
198 (NH ), 1623 (C@N)cm ; (E)-isomer, H NMR
2
(
CDCl ): d 7.55–7.61 (m, 2H, H-3 and H-5), 7.31–7.36
3
(
5
m, 2H, H-2 and H-6), 7.16 (t, J = 5.4 Hz, 1H, CH@N),
.29 (br s, 2H, NH ), 3.57 (d, J = 5.4 Hz, 2H, CH CH);
2 2
1
(Z)-isomer, H NMR (CDCl ): d 7.55–7.61 (m, 2H, H-3
and H-5), 7.31–7.36 (m, 2H, H-2 and H-6), 6.72 (t,
J = 4.8 Hz, 1H, CH@N), 5.39 (br s, 2H, NH ), 3.53 (d,
J = 4.8 Hz, 2H, CH CH).
3
Acknowledgments
2
2
We are grateful to the Canadian Institutes of Health
Research and the Canada Research Chairs Program
for financial support of this research, and to the
Government of Tehran for an Iranian Ministry of
Health & Medical Education for a six-month scholar-
ship to two of us (A.D. and A.F.). Bernard Sowa is
the recipient of an Alberta Heritage Foundation
for Medical Research MD/PhD studentship. The
technical assistance of Ms. Gail Rauw is gratefully
acknowledged.
4
.3.16. [2-(2,4-Dichlorophenyl)ethylidene]hydrazine (4p).
Yield, 71%; (E):(Z) = 72:28; IR (film): m 3369, 3201
À1
1
(
(
NH ), 1589 (CH@N) cm ; (E)-isomer, H NMR
2
CDCl ): d 7.16–7.42 (m, 4H, H-3, H-5, H-6, CH@N),
3
5
.22 (br s, 2H, NH ), 3.59 (d, J = 5.1 Hz, 2H, CH CH);
2 2
1
3
C NMR (CDCl ): d 142.02 (CH@N), 134.58, 133.97,
3
1
1
33.03 (C-1, C-2, C-4), 131.52 (C-6), 129.21 (C-3),
27.15 (C-5), 35.87 (CH CH); (Z)-isomer, H NMR
1
2
(
CDCl ): d 7.16–7.42 (m, 3H, H-3, H-5, H-6), 6.61 (t,
3
J = 4.8 Hz, 1H, CH@N), 5.35 (br s, 2H, NH ), 3.52 (d,
2
1
3
J = 4.8 Hz, 2H, CH CH); C NMR (CDCl ): d 141.30
2
3
References and notes
(
CH@N), 134.58, 133.97, 133.03 (C-1, C-2, C-4), 131.08
(
C-6), 129.44 (C-3), 127.36 (C-5), 29.97 (CH CH).
2
1
2
3
4
. Popov, N.; Matthies, H. J. Neurochem. 1969, 16, 899–
07.
. Baker, G. B.; Wong, J. T. F.; Yeung, J. M.; Coutts, R. T.
J. Affect. Dis. 1991, 21, 207–211.
. Todd, K. G.; Banigesh, A. I.; Baker, G. B.; Coutts, R. T.;
Shuaib, A. J. Neurochem. 1999, 73, S202B.
. Shuaib, A.; Ijaz, S.; Kalra, J.; Code, W. Brain Res. 1992,
574, 120–124.
5. Shuaib, A.; Ijaz, S.; Kalra, J.; Code, W. Can. J. Neurol.
Sci. 1992, 19, 428–432.
9
4
.4. Injection of animals
Male Sprague–Dawley rats (200–250 g) were injected
intraperitoneally with 4-FPEH or vehicle (corn oil)
and killed at various time intervals after injection by
decapitation. Brains were immediately frozen in isopen-
tane on solid carbon dioxide, removed to another set of
containers, and frozen at À80 ꢁC until the time of anal-
ysis. All procedures were approved by the Health Sci-
ences Animal Welfare Committee, Faculty of Medicine
and Dentistry, University of Alberta.
6
7
8
9
. Shuaib, A.; Ijaz, S.; Hasan, S.; Kalra, J. Brain Res. 1992,
5
90, 13–17.
. Shuaib, A.; Ijaz, S.; Miiyashita, H.; Hussain, S.; Kanthan,
R. Exp. Neurol. 1997, 147, 311–315.
. Green, A. R.; Hainsworth, A. H.; Jackson, D. M.
Neuropharmacology 2000, 39, 1483–1494.
. Tanay, V. A.-M. I.; Baker, G. B.; Todd, K. G., Proc.
Congr. Collegium Internationale Neuro-Psychopharmaco-
logicum, Montr e´ al, Canada, 2002.
4
.5. GABA transaminase assay
In the initial in vitro screen for GABA-T activity, a
modification of the colorimetric method of Sethi
(
1993) was used. In the ex vivo study of homogenates
10. Todd, K. G.; Baker, G. B. J. Affect. Dis. 1995, 35, 125–
129.
from brains of rats treated with 4n ([2-(4-fluorophen-
yl)ethylidine]-hydrazine; 4-FPEH), GABA-T activity
was measured using the radiochemical procedure of
1
1. Paslawski, T.; Knaus, E.; Iqbal, N.; Coutts, R. T.; Baker,
G. B. Drug Dev. Res. 2001, 54, 35–39.
1
8
12. Pross, A.; Sternell, S. Aust. J. Chem. 1970, 23, 989–
1003.
Sterri and Fonnum, as modified by McManus et
19
al. Briefly, homogenates were incubated with radiola-
belled GABA in the presence of pyridoxal phosphate
and the resultant products were isolated using a liquid
anion exchanger (tri-n-octylamine). The radioactivity
was then counted using a liquid scintillation counter
1
3. Karabatsos, G. J.; Osborne, C. E. Tetrahedron 1968, 24,
361–3368.
4. Li, M.; Jobson, M. E. Synth. Commun. 1995, 25, 533–
37.
3
1
5
15. Ratcliffe, R.; Rodehorst, R. J. Org. Chem. 1970, 35, 4000–
4002.
(
Beckman LS 6000SC).

B. Sowa et al. / Bioorg. Med. Chem. 13 (2005) 4389–4395
4395
1
1
1
6. Cameron, D. W.; Feutrill, G. I.; Perlmutter, P. Aust. J.
Chem. 1982, 35, 1469–1480.
7. Brown, H. C.; Weissman, P. M.; Yoon, N. M. J. Am.
Chem. Soc. 1966, 88, 1458–1463.
8. Sterri, S.H.;Fonnum,F.Eur. J.Biochem. 1978,91,215–222.
19. McManus, D. J.; Baker, G. B.; Martin, I. L.; Greenshaw,
A. J.; McKenna, K. F. Biochem. Pharmacol. 1992, 43,
2486–2489.
20. Parent, M.; Bush, D.; Rauw, G.; Master, S.; Vaccarino,
F.; Baker, G. Methods 2001, 23, 11–20.