1-Malonyl-1,4-dihydropyridine as a novel carrier for specific delivery of drugs to the brain

Article abstract of DOI:10.1016/j.bmc.2008.12.051

A group of 1-malonyl-1,4-dihydropyridine derivatives were synthesized as novel carrier systems for site-specific and sustained drug delivery to the brain. Such carriers are expected to be stable against air oxidation due to the presence of the carbonyl group close to nitrogen of the dihydropyridine. These carrier systems were attached to a group of different aldehydes to afford novel quaternary pyridinium derivatives 9a-e, 11a-d, 13 and 18a-b. Reduction of the prepared quaternary pyridinium derivatives with sodium dithionite afforded a novel group of 1-malonyl-1,4-dihydropyridine chemical delivery systems (CDSs) 10a-e, 12a-d, 14 and 19a-b. The synthesized 1-malonyl-1,4-dihydropyridine CDSs were subjected to various chemical and biological investigations to evaluate their ability to cross the blood-brain barrier, and to be oxidized biologically into their corresponding quaternary derivatives. The in vitro oxidation studies showed that most of the 1-malonyl-1,4-dihydropyridine CDSs could be oxidized into their corresponding quaternary derivatives at an adequate rate. The in vivo studies showed that compounds 10c and 14 were able to cross the blood-brain barrier at detectable concentrations. Moreover, the pyridinium quaternary intermediates 9a, 9c, 13, 18a and their corresponding dihydro derivatives 10a, 10c, 14 and 19a were screened for their antidepressant activity using tail suspension behavioral despair test compared to imipramine as a reference at a dose level of 10 mg/kg. The results indicated that compounds 13, 14 and 19a induced remarkable antidepressant activity comparable to imipramine. Compounds 10a, 10c and 18a exhibited good antidepressant activity, their activities nearly equal to 92.8%, 86.7% and 90.20% of the activity of imipramine, respectively. The other derivatives 9a and 9c exhibited moderate antidepressant activity compared with imipramine.

Full text of DOI:10.1016/j.bmc.2008.12.051

Bioorganic & Medicinal Chemistry 17 (2009) 1681–1692
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
1-Malonyl-1,4-dihydropyridine as a novel carrier for specific delivery of drugs
to the brain
a
b
Heba A. Hassan a, Mohamed Abdel-Aziz a, , Gamal El-Din A. A. Abuo-Rahma , Hassan H. Farag
*
a Department of Medicinal Chemistry, Faculty of Pharmacy, Minia University, 61519-Minia, Egypt
b Department of Pharmaceutical Medicinal Chemistry, Faculty of Pharmacy, Assiut University, 71526-Assiut, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
A group of 1-malonyl-1,4-dihydropyridine derivatives were synthesized as novel carrier systems for site-
specific and sustained drug delivery to the brain. Such carriers are expected to be stable against air oxi-
dation due to the presence of the carbonyl group close to nitrogen of the dihydropyridine. These carrier
systems were attached to a group of different aldehydes to afford novel quaternary pyridinium deriva-
tives 9a–e, 11a–d, 13 and 18a–b. Reduction of the prepared quaternary pyridinium derivatives with
sodium dithionite afforded a novel group of 1-malonyl-1,4-dihydropyridine chemical delivery systems
(CDSs) 10a–e, 12a–d, 14 and 19a–b. The synthesized 1-malonyl-1,4-dihydropyridine CDSs were sub-
jected to various chemical and biological investigations to evaluate their ability to cross the blood–brain
barrier, and to be oxidized biologically into their corresponding quaternary derivatives. The in vitro oxi-
dation studies showed that most of the 1-malonyl-1,4-dihydropyridine CDSs could be oxidized into their
corresponding quaternary derivatives at an adequate rate. The in vivo studies showed that compounds
10c and 14 were able to cross the blood–brain barrier at detectable concentrations. Moreover, the pyrid-
inium quaternary intermediates 9a, 9c, 13, 18a and their corresponding dihydro derivatives 10a, 10c, 14
and 19a were screened for their antidepressant activity using tail suspension behavioral despair test
compared to imipramine as a reference at a dose level of 10 mg/kg. The results indicated that compounds
13, 14 and 19a induced remarkable antidepressant activity comparable to imipramine. Compounds 10a,
10c and 18a exhibited good antidepressant activity, their activities nearly equal to 92.8%, 86.7% and
90.20% of the activity of imipramine, respectively. The other derivatives 9a and 9c exhibited moderate
antidepressant activity compared with imipramine.
Received 28 September 2008
Revised 17 December 2008
Accepted 22 December 2008
Available online 29 December 2008
Keywords:
1,4-Dihydropyridine
CDS
INH
Malonic acid
Antidepressant
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
in vivo to the ionic pyridinium salt prevents its elimination from
the brain (locked-in), while elimination from the general circula-
The blood–brain barrier is a major impediment for the treat-
ment of central nervous system diseases. Since many drugs are un-
able to reach this organ at therapeutic concentrations.1 Various
attempts have been made to overcome the limiting access of drugs
to the brain and at the same time, reduce the systemic side effects
that are common with general treatments.2–7 One of these at-
tempts was the linking of the active compound to a brain-specific
carrier to give a chemical delivery system (CDS) which delivers the
active compound specifically into the brain after its cleavage from
the carrier at the site of action.8–13
The dihydropyridineMpyridinium salt redox system was de-
scribed as a general and flexible method for site-specific and sus-
tained delivery of drugs to the brain.14,15 The biologically active
compound linked to a lipoidal dihydropyridine carrier easily pene-
trates the blood–brain barrier; oxidation of the carrier moiety
tion is accelerated. By time, the drug is released from the carrier
in the brain at a rate depending on the connecting group. The main
obstacle faced the industrial development of the idea is the very
short life time of the compound, mainly due to their fast air oxida-
tion and/or hydration of the 5,6-double bond, which is a common
problem with all the 1,4-dihydropyridineMpyridinium salt redox
CDSs.16,17
In the present proposal, a novel carrier was investigated
keeping in mind all the requirements for a safe and useful
brain-specific chemical delivery system including; reasonable
shelf life time, a good rate of oxidation, not be neurotoxic and
should be excreted from the brain in a reasonable rate. The sys-
tem will be used to deliver certain drugs specifically to the
brain. The suggested carrier is 1-malonyl-1,4-dihydropyridine,
such carrier is expected to be stable against air oxidation due
to the presence of the carbonyl group close to nitrogen of the
dihydropyridine. Such group will act as electron withdrawing
group, whether by resonance or induction, which will result in
* Corresponding author. Tel./fax: +20 86 2369075.
E-mail address: abulnil@hotmail.com (M. Abdel-Aziz).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.12.051

1682
H. A. Hassan et al. / Bioorg. Med. Chem. 17 (2009) 1681–1692
decreasing the electron density on the ring nitrogen. It is well
known that the lone pair of electrons on the ring nitrogen is
the initiator of oxidation and/or hydration of the dihydropyri-
dine.16,17 Accordingly, reduction of the electron density on the
ring nitrogen may result in shelf-stable carrier system. On
administration, hydrolysis of the ester function follows distribu-
tion to give the corresponding 1,4-dihydropyridine-1-malonyl
monoanion derivatives at the physiological pH (7.4). The rate
of oxidation of the anion is expected to be accelerated, firstly
by destabilization of the ground state, secondly by stabilization
of the oxidation transition state, and thirdly by stabilization of
the final quaternary product. The acid anion function at the
vicinity of the nitrogen atom of the dihydro derivatives reduces
the energy of the transition state of the enzymatic oxidation to
the corresponding quaternary. One of the carboxylic groups of
malonic acid may be utilized to connect selected drugs through
an amide or ester function, the CONHNH2– group at C3 or C4
may be also used to connect a drug moiety (Fig. 1).
Another strategy was designed in this work is the evaluation
of the antidepressant activity for some of the novel synthesized
dihydro derivatives and their corresponding quaternary deriva-
tives using tail suspension behavioral despair test compared to
imipramine as a reference drug at a dose level of 10 mg/kg.
The aim is to target the potential antidepressant agents to the
brain in order to avoid the serious peripheral side effects associ-
ated with their use such as cardiovascular dysfunctions, sexual
disturbances, and central nervous system side effects.18 In the
future, the designed 1-malonyl-1,4-dihydropyridine derivatives
may be utilized to deliver isonicotinic acid hydrazide (INH) into
the brain aiming in treatment of the brain TB in the same time
avoiding the systemic side effects associated with the use of
INH.
2. Results and discussion
2.1. Chemistry
Bromomalonate 3 and bromomalonic acid 5 were prepared
according to Scheme 1. Partial hydrolysis of diethylmalonate 1
with alcoholic potassium hydroxide in a ratio of 2:1, respectively,
under dry condition followed by acidification of the obtained solu-
tion with HCl afforded the corresponding malonic acid monoethyl
ester 2 in 40% yield.19 Treatment of the malonic monoester 2 and
malonic acid 4 with Br2 in glacial acetic acid to afford the corre-
sponding 2-bromomalonic acid monoethyl ester 3 and 2-bromo-
malonic acid 5 in 60% and 48% yield, respectively.20,21
The first group of the designed malonic acid monoethyl ester
pyridine chemical delivery systems MEPCDSs 10a-e was success-
fully synthesized as illustrated in Scheme 2. Heating at reflux of
isonicotinic acid hydrazide 6 with different aromatic aldehydes
including 2-hydroxybenzaldehyde 7a22, 4-methoxybenzaldehyde
7b23, 2-chlorobenzaldehyde 7c24, 4-hydroxy-3-methoxybenzalde-
hyde 7d25 and 4-(N,N-dimethylamino)benzaldehyde 7e26, respec-
tively, in absolute ethanol for 6–8 h to afford the corresponding
benzylideneisonicotinic acid hydrazide derivatives 8a–e in 60–
82% yield. The quaternary pyridinium derivatives 9a–e were pre-
pared by heating at reflux of compounds 8a–e with 2-bromoma-
lonic acid monoethyl ester
affording 9a–e in 40–70% yield, Scheme 2. The IR spectra of the
prepared compounds 9a–e showed an absorption band at 1730–
3 in absolute ethanol for 30 h
1745 cmÀ1, due to the
m
(C@O) stretching of the carboxylic acid
moiety and strong absorption band in the region of 1739–
1755 cmÀ1 related to the
(C@O) stretching of the ester moiety.
A sharp absorption band at 1668–1682 cmÀ1 due to the
(C@O)
stretching of the amide moiety, in addition to broad bands in the
region of 3000–3450 cmÀ1 due to
(NH) and (OH) stretching.
m
m
m
The 1H NMR spectra recorded for the prepared quaternary com-
pounds 9a–e showed prominent triplet signal corresponding to
the CH3 protons at d 1.1–1.3 ppm and quartet signals appear at d
4.2–4.4 ppm indicating the 2 protons of CH2. A characteristic
downfield shift of the CH protons attached to the nitrogen of pyrid-
inium moiety was observed to appear at d 5.7–5.9 ppm. The pro-
tons belonging to the aromatic system and azomethine were
observed at the expected chemical shifts and integral values.
The designed MEPCDSs 10a–e were prepared in 60–70% yield
through reduction of the corresponding quaternary derivatives
9a–e using sodium dithionite in the presence of sodium bicarbon-
ate in CH2Cl2 under atmosphere of nitrogen.27 Methylene chloride
serves to extract the MEPCDSs 10a–e as soon as it is formed to pro-
tect it from degradation in the alkaline aqueous medium.
CONHNH2
CONHNH2
CONHNH2
N
N
N
CH
COOH
CH
COOH
CH
COOH
COOH
COOCH2CH3
COOCH2CH3
Another group of N-methyl PCDSs 12a–d and malonyl pyridine
chemical delivery system MPCDS 14 were prepared as illustrated
Figure 1. Different forms of the designed 1-malonyl-1,4-dihydropyridine CDS.
COOCH2CH3
COOH
COOH
b
a
H2C
H2C
HC
Br
COOCH2CH3
COOCH2CH3
COOCH2CH3
3
2
1
COOH
COOH
b
H2C
HC
Br
COOH
COOH
5
4
Scheme 1. Synthesis of bromomalonate derivatives 3 and 5. Reagents: (a) KOH, EtOH, HCl; (b) Br2, HOAc.

H. A. Hassan et al. / Bioorg. Med. Chem. 17 (2009) 1681–1692
1683
in Scheme 3. Heating at reflux of different Schiff bases 8a–d with
bromomalonic acid 5 in absolute ethanol for about 30 h afforded
the corresponding N-methyl quaternary pyridinium derivatives
11a–d in 40–61% yield. In this reaction, quaternization occurs first
then followed by decarboxylation. The IR spectra of the prepared
compounds 11a–d showed an absorption bands in the region of
and 12.4 ppm, which are related to the presence of OH and NH pro-
tons, respectively. The protons belonging to the aromatic system
and azomethine were observed at the expected chemical shifts
and integral values. Mass spectrum and elemental analysis clearly
supports the proposed structure. The decarboxylation process did
not occur during the preparation of compound 13 and this may
be attributed to the basic characters of the dimethyl amino group
in compound 8e.
Reduction of N-methyl quaternary pyridinium derivatives 11a–
d and MPQ+ 13 with sodium dithionite in CH2Cl2 in the presence of
NaHCO3 under atmosphere of N2 afforded the corresponding N-
methyl PCDSs 12a–d and MPCDS 14, respectively in 40–50% yield
(Scheme 3).27
Another group of the designed quaternary compounds
MEPCDSs (19a,b Scheme 4) were successfully synthesized. Heating
at reflux of nicotinamide 15 with hydrazine monohydrate in abso-
lute ethanol for 5 h affording the corresponding nicotinic acid
hydrazide 16 in 85% yield.28 Heating at reflux of the hydrazide
16 with different aldehydes including 2-hydroxybenzaldehyde
7a29 and 4-methoxybenzaldehyde 7b30 in absolute ethanol for
5 h to afford the corresponding benzylidenenicotinic acid hydra-
zide derivatives 17a–b in 60–77% yield.
1617–1664 cmÀ1 due to
In addition, an absorption bands in the region of 3423–3441cmÀ1
related to
(NH) stretching. The 1H NMR spectra recorded for
m (C@O) stretching of the amide moiety.
m
the prepared compounds 11a–d showed a prominent singlet signal
at d 4.4 ppm related to the protons of CH3 attached to pyridinium
nitrogen, the protons belonging to the aromatic system and azo-
methine were observed at the expected chemical shifts and inte-
gral values. Mass spectra and elemental analyses clearly support
the proposed structures.
Under the same conditions, heating at reflux of compound 8e
with 5 in absolute ethanol afforded the corresponding malonic acid
pyridinium derivative MPQ+ 13 in 42% yield. The IR spectra of the
prepared MPQ+ derivative 13 exhibited a strong stretching band at
1690 cmÀ1, which is attributed to the
m
(C@O) stretching of the acid
and another absorption band at 1650 cmÀ1, related to the
m
(C@O)
of the amide moiety. The 1H NMR spectra for compound 13 showed
a singlet signal at d 6.4 ppm, related to the CH proton of malonic
acid moiety attached to the pyridinium nitrogen. The high deshiel-
ding character of this CH proton is attributed to the formation of
quaternary pyridinium salt and its adjacent to two electronegative
COOH groups. Broad signals appear in the offset region at d 11.8
Heating at reflux of compounds 17a,b with 2-bromomalonic
acid monoethyl ester 3 in absolute ethanol for 30 h afforded the
corresponding MEPQ+ 18a,b in 50–75% yield. The IR spectra of
the prepared compounds 18a,b showed an absorption band at
1730 cmÀ1, which is related to
m (C@O) stretching of the acid moi-
CONHNH2
CONHN
CH Ar
CONHN
C
H
Ar
CONHN
C
H
Ar
b
a
3
a
Ar
CHO
7a-e
+
N
N
N
N
Br
COOH
8a-e
6
a, Ar= 2-Hydroxyphenyl
b, Ar= 4-Methoxyphenyl
c, Ar= 2-Chlorophenyl
HC COOH
HC
COOCH2CH3
COOCH2CH3
d, Ar= 4-Hydroxy-3-methoxyphenyl
e, Ar= 4-(N,N-Dimethylamino)phenyl
9a-e
10a-e
Scheme 2. Synthesis of MEPCDSs 10a–e. Reagents: (a) EtOH, reflux; (b) Na2S2O4, NaHCO3, CH2Cl2, N2 gas.
CONHN
CH
Ar
CONHN
CH
Ar
CONHN
CH
Ar
5
b
a
N
N
8a-d
N
Br
CH3
CH3
a, Ar= 2-Hydroxyphenyl
b, Ar= 4-Methoxyphenyl
c , Ar= 2-Chlorophenyl
12a-d
11a-d
d , Ar= 4-Hydroxy-3-methoxyphenyl
CH3
CH3
CONHN CH
N
CONHN CH
N
CH3
CH3
5
b
8e
N
a
Br
N
HC
COOH
CH
COOH
COOH
COOH
13
14
Scheme 3. Synthesis of N-methyl PCDSs derivatives 12a–d and MPCDS 14. Reagents: (a) EtOH, reflux; (b) Na2S2O4, NaHCO3, CH2Cl2, N2 gas.

1684
H. A. Hassan et al. / Bioorg. Med. Chem. 17 (2009) 1681–1692
CONHNH2
CONH2
CONHN
CH Ar
3
a
7a-b
b
b
N
N
N
15
17a-b
16
CONHN
CH Ar
CONHN
CH Ar
c
N
N
Br
COOH
HC
COOH
HC
COOCH2CH3
COOCH2CH3
19a-b
18a-b
Scheme 4. Synthesis of MEPCDSs 19a,b. Reagents: (a) NH2NH2ÁH2O, EtOH, reflux; (b) EtOH, reflux; (c) Na2S2O4, NaHCO3, CH2Cl2, N2 gas.
ety, and another stretching band at 1744 cmÀ1 which is related to
the (C@O C@O) of ester moiety. A strong absorption band at
1671 cmÀ1, which is attributed to the
(C@O) of the amide moiety,
another broad bands in the region of 3000–3402 cmÀ1 which are
related to (NH) and
(OH) stretching. The 1H NMR spectra re-
2.2.1. Chemical oxidation
m
2.2.1.1. Oxidation with ferricyanide31
.
The ferricyanide oxida-
m
tion of dihydropyridine derivatives is commonly used to study
their sequential electron–proton–electron transfer mechanism.32
In the present study, ferricyanide oxidation rates of the prepared
MEPCDSs 10a, 10c, 12c, 14, 19a and 19b were investigated spec-
trophotometrically by monitoring the absorbance of MEPCDSs
against time. The test was carried out by mixing freshly prepared
m
m
corded for the prepared quaternary compounds 18a,b showed
prominent triplet signals of the CH3 protons at d 1.1–1.3 ppm
and quartet signals at d 4.2–4.3 ppm indicating the 2 protons of
CH2. A characteristic downfield shifted signal corresponding to
the CH protons directly attached to the pyridinum nitrogen was
observed at d 5.7 ppm. The protons belonging to the aromatic sys-
tem and azomethine were observed at the expected chemical shifts
and integral values.
MEPCDSs with ferricyanide solution in
a concentration of
(2 Â 10À1 mmol). Either the decrease of absorption of the dihydro
derivative (MEPCDSs) or the increase of the absorption of the
quaternary ones (MEPQ+) was monitored in the UV region at spe-
cific kmax (330, 335, 300, 370, 290 and 295) for MEPCDSs 10a,
10c, 12c, 14, 19a and 19b, respectively, and kmax (370, 380,
345, 395, 345 and 350) for their corresponding MEPQ+ 9a, 9c,
11c, 13, 18a and 18b, respectively. Pseudo first order rate con-
stants and their t1/2 were determined by calculation from linear
regression of Ln of absorbance against time; the results were
illustrated in Table 1.
Reduction of the prepared MEPQ+ 18a,b with sodium dithionite
in CH2Cl2 in the presence of NaHCO3, under atmosphere of nitrogen
afforded the corresponding MEPCDSs 19a,b in 60–70% yield27
(Scheme 4).
2.2. Biological investigations
Data in Table 1 show that the prepared MEPCDSs have high rate
of oxidation in ferricyanide solution and that may be attributed to
the presence of carboxylic acid function group. 1-Malonyl-1,4-
A group of the prepared 1-malonyl-1,4-dihydropyridine deriva-
tives including; 10a, 10c, 12c, 14, 19a and 19b was subjected to
various chemical and biological investigations including; chemical
oxidation with ferricyanide and 30% H2O2, stability in phosphate
buffer (0.2 M, of pHs 7.4 and 5.8), in vitro studies with 80% mice
plasma and 20% brain homogenate and in vivo studies to explore
these compounds as potential carrier model for brain-specific
delivery. Moreover, to evaluate the ability of these compounds to
cross the blood–brain barrier, and to be oxidized into their corre-
sponding quaternary derivatives. In this study, high performance
liquid chromatography (HPLC) was used to detect and monitor
the oxidation of the tested 1-malonyl 1,4-dihydropyridine deriva-
tives into their corresponding quaternary derivatives either chem-
ically or in biological fluids. MeOH/H2O (50:50) and 0.1%
triflouroacetic acid was found to be the most suitable system for
running. Experimental parameters including mobile phase, flow
rate, type of column and the linearity range were studied in order
to determine the optimal conditions for the assay procedure. The
HPLC analysis showed that 1-malonyl-1,4-dihydropyridine deriva-
tives were separated from plasma and brain homogenate at reten-
tion times (9.16–15.96 min). Their corresponding quaternary
derivatives were separated at (3.55–4.98 min). The chromato-
graphic system used allowed a complete base line separation with
good resolution of the peaks. The mean calibration curve was plot-
ted; the mean best-fit linear regression equation was derived, and
used to estimate the concentrations of the quaternary salts.
dihydropyridine derivatives have an N-substituted-a-amino acid
anion moiety, most -amino acids exist as zwitterions in biological
a
fluids, which are stabilized by the close locations of the opposite
charges and the expected ionic interactions. This may result in
reduction of the oxidation potential due to decrease of the energy
of the transition state and/or the overall activation energy needed
for the reaction.
Upon comparison between different forms of the tested
MEPCDSs, compound 14 showed the highest rate of oxidation
(t1/2 = 1.14 min), which may be attributed to the presence of two
carboxylic acid function groups in the tested compound. MEPCDSs
10a, 19a and 19b showed good rate of oxidation but less than
Table 1
The rate of oxidation of prepared MEPCDSs 10a, 10c, 12c, 14, 19a and 19b with
ferricyanide solution
Compound
n
Kdisapp. (minÀ1
)
t1/2 (min)
r
10a
10c
12c
14
19a
19b
6
6
6
6
6
6
0.28
0.23
0.21
0.61
0.32
0.33
2.48
3.01
3.3
1.14
2.17
2.1
0.994
0.995
0.984
0.958
0.999
0.999

H. A. Hassan et al. / Bioorg. Med. Chem. 17 (2009) 1681–1692
1685
MPCDS 14 (t1/2 = 2.48, 2.17 and 2.10 min, respectively) that could
be attributed to the presence of only one carboxylic acid function
group in these compounds. MEPCDSs 10c and 12c showed the low-
est rate of oxidation of the tested compounds due to the absence of
carboxylic acid function group in these compounds (t1/2 = 3.01 and
3.30 min, respectively).
buffer (0.2 M, pHs 7.4 and 5.8) at room temperature (32 2 °C).
Solutions of different MEPCDSs 10c, 14 and 19b with a concentra-
tion (2 Â 10À1 mmol) in phosphate buffers of different pHs (0.2 M,
pHs 7.4 and 5.8) were monitored for 4 days using HPLC for both
concentrations of the parent MEPCDSs and for their oxidation or
degradation products. Pseudo first order rate constants of disap-
pearance of different MEPCDSs was calculated by linear regression
of Ln of its peak areas against time, t1/2 was calculated and the re-
sults are listed in Table 3.
The results of the stability of different MEPCDS 10c, 14 and 19b
in phosphate buffers of different pHs revealed that; all of the tested
MEPCDSs were stable in both phosphate buffers of pH 7.4 and 5.8,
but the stability was higher at phosphate buffer of pH 7.4 com-
pared to pH of 5.8 and that could be attributed to acid catalyzed
hydration of the prepared MEPCDS in acidic pH.
2.2.1.2. Oxidation with hydrogen peroxide (30%). Hydrogen
peroxide oxidizes the dihydropyridines by a free radical mecha-
nism, which is the mechanism of air oxidation of these derivatives.
Accordingly; this test may be used in accelerated stability studies
to compare between different forms of dihydropyridines with re-
spect to their shelf-life stability against air oxidation. In this test,
an aliquot of 30% solution of hydrogen peroxide is added to meth-
anolic solution of the MEPCDSs 10a, 10c, 12c, 14, 19a and 19b with
a concentration of (2 Â 10À1 mmol). The decrease of absorption of
the MEPCDSs derivatives or the increase of the absorption of the
MEPQ+ was monitored spectrophotometrically in the UV region
at specific kmax (330, 335, 300, 370, 290 and 295) for MEPCDS
10a, 10c, 12c, 14, 19a and 19b, respectively and kmax (370, 380,
345, 395, 345 and 350) for their corresponding MEPQ+ 9a, 9c,
11c, 13, 18a and 18b, respectively. Pseudo first order rate constants
and their t1/2 were determined by calculation from linear regres-
sion of Ln of absorbance against time; the results were illustrated
in Table 2.
The results of oxidation with hydrogen peroxide showed high
rate of oxidation of the prepared MEPCDSs in 30% H2O2 but lower
than ferricyanide oxidation. MPCDS 14 exhibited the highest rate
of oxidation (t1/2 = 3.15 min) that could be attributed to the pres-
ence of dicarboxylic acid function group. Compounds 10a, 10c,
19a and 19b exhibited moderate rate of oxidation (t1/2 = 3.35,
3.85, 4.08, 5.30 min, respectively) that may be attributed to the
presence of only one carboxylic acid function group. Compound
12c exhibited the lowest rate of oxidation of the tested MEPCDSs
(t1/2 = 6.30 min) that may be attributed to the absence of carboxylic
acid function group.
2.2.3. In vitro stability in biological fluids
The in vitro stability studies of the redox chemical delivery sys-
tems and their corresponding quaternary derivatives in biological
fluids, for example, plasma, blood, brain homogenate, are routinely
carried out to investigate the transformation pathways of the car-
rier system in these media and if there will be in vivo site-specific
conversion of the dihydro derivative into the corresponding qua-
ternary. In this investigation, the model MEPCDSs were subjected
to in vitro stability studies in 80% mice plasma, and 20% mice brain
homogenate. Scheme 5 illustrates the expected metabolic path-
ways of the tested MEPCDSs.
The study of the stability of different MEPCDSs and its corre-
sponding quaternary derivatives MEPQ+s in plasma gives an idea
about their comparative stability against enzymatic hydrolysis
(K2 and K3) by esterase predominant in plasma, since minimal oxi-
dation is expected. On the other hand, investigation of their stabil-
ities in brain homogenate demonstrates the susceptibility of the
system components to oxidation by flavin dehydrogenases (K1
and K4). From the obtained results, it is possible to determine
which comes first, in vivo in the brain, whether hydrolysis of the
MEPCDSs or its oxidation to the corresponding MEPQ+.
2.2.2. Stability in buffer solution (shelf-life stability)
Stability of redox-carrier system in buffers of different pHs is
usually carried out to investigate the suitable pH for their formula-
tions and to expect their shelf life stability. The stability of
MEPCDSs 10c, 14 and 19b was investigated in different phosphate
2.2.3.1. Stability in 80% mice plasma. The stability of both
MEPCDSs 10c, 14 and 19b and their corresponding MEPQ+ 9c, 13
and 18b was investigated in 80% mice plasma at 37 °C, with respect
to the tested MEPCDSs, Results revealed that, no quaternary ester
(MEPQ+) peak was detected, but only a peak corresponding to
the quaternary acid (MPQ+) was observed, no other peaks were de-
tected in the spectrum. These results suggest that the possible
metabolic pathway in plasma is hydrolysis prior to oxidation.
Pseudo first order rate constants of disappearances (Kdisapp.) of
the tested MEPCDSs, mainly due to hydrolysis (K2), was found to
be Kdisapp. 2.97 Â 10À2 minÀ1 and its t1/2 = 23.33 min; while hydro-
Table 2
The rate of oxidation of prepared MEPCDSs 10a, 10c, 12c, 14, 19a and 19b with 30%
hydrogen peroxide
Compound
n
Kdisapp. (minÀ1
)
t1/2 (min)
r
10a
10c
12c
14
19a
19b
6
6
6
6
6
6
0.17
0.13
0.11
0.22
0.18
0.21
4.08
5.30
6.30
3.15
3.85
3.35
0.996
0.997
0.974
0.993
0.994
0.998
lysis of the quaternary (K3) was 4.44 Â 10À2 minÀ1 and its t1/2
=
15.75 min.
Several conclusions could be deduced from these results; first,
the rate of hydrolysis of the tested MEPCDSs in plasma is about
17 times faster than rate of its hydrolysis in phosphate buffer of
Table 3
Stability of different MEPCDSs 10c, 19 and 25b in phosphate buffer (0.2 M, pHs 7.4 and 5.8)
Phosphate buffer (0.2 M, pHs)
pH 7.4
Compound
n
Kdisapp SD (hÀ1
)
t1/2 (min)
r
10c
14
19b
4
4
4
0.1034 0.003
0.1677 0.001
0.160 0.004
402.13
247.94
259.88
0.997
0.999
0.997
pH 5.8
10c
14
19b
4
4
4
0.165 0.001
0.2465 0.003
0.2117 0.003
252
169.29
196.4
0.999
0.999
0.999

1686
H. A. Hassan et al. / Bioorg. Med. Chem. 17 (2009) 1681–1692
CONHN
C
H
Ar
investigated for their stabilities in 20% mice brain homogenates.
Mice were sacrificed by decapitation and their brains were isolated
and homogenized with isotonic phosphate buffer (0.2 M, pH 7.4).
Methanolic solution of MEPCDSs 10c, 14 and 19b were added to
the homogenate at different time intervals. The supernatants were
analyzed by HPLC for their contents of MEPCDSs and their corre-
sponding MEPQ+s. Both of the Kdisapp. and t1/2 of oxidation of the
MEPCDSs were calculated by linear regression of Ln of peak areas
against time in min. Table 5 shows the results of the test with
MEPCDS 10c, 14 and 19b.
N
HC COOH
COOCH2CH3
k1
k2
CH2ONHN
C
H
Ar
CH2ONHN
C
H
Ar
In mice brain homogenate, the MEPQ+ was hydrolyzed with a
pseudo first order rate constant of disappearance K3 of Kdisapp.
3.4 Â 10À2 minÀ1 and its t1/2 = 20.38 min and the rate of appear-
ance of the quaternary acid (MPQ+) (K3–K5) of Kdisapp. 6.49 Â
10À2 minÀ1 and its t1/2 = 10.68 min). The above data showed that
the tested MEPCDSs exhibited fast rate of oxidation in brain than
in plasma and these results emphasize the ‘lock-in’ mechanism
of the tested MEPCDSs. As the drug reaches the brain, it is readily
oxidized to give its corresponding MEPQ+, which is hydrophilic en-
ough not to pass the BBB and so become locked in the brain afford-
ing its expected effect.
Br
N
N
HC COOH
HC COOH
COO
COOCH2CH3
k4
k3
CONHN
C
Ar
H
From the above data it is clear that all of the tested MEPCDSs
10c, 14 and 19b exhibited high rate of oxidation and their t1/2 were
found to be around 10 min. With respect to the stability of the
tested MEPCDSs, it was interesting to observe that the system is
more stable in 20% mice brain homogenate than that in 80% mice
plasma (t1/2 = 9.58 and 18.88 min, respectively) for compound 14.
Hydrolysis (K2) may be the main route of its transformation in
brain and that is why its rate of hydrolysis in plasma is higher since
the concentration of esterase in plasma is higher. No dihydroacid
derivative could be detected during the investigation. This proves
that once the ester is hydrolyzed, the product is immediately oxi-
dized to the corresponding quaternary acid. Accordingly, the
sequential distribution, hydrolysis and oxidation scenario may be
accomplished.
Br
N
HC COOH
COO
k5
CH2ONHN
C
H
Ar
N
CH3
Br
Scheme 5. The expected metabolic pathways of the tested MEPCDSs.
2.2.4. In vivo distribution study
Compounds 10c and 14 were selected for in vivo study, the pur-
pose of this study is to prove that the tested compounds would be
able to deliver the corresponding MPQ+ in a good concentration to
the brain and as a result of its hydrophilic character, it will be
‘locked-in’ there.33
Freshly prepared MEPCDSs 10c or 14 were injected through the
external jugular vein as solution in DMF to a group of male Spra-
gue-Dawley rats at a dose level of 20 mg/kg body weight. At spe-
cific time intervals, the injected animals were sacrificed and both
of their brains and blood samples were collected, and analyzed
by HPLC for the appearance of the quaternary acid derivatives
MPQ+. The results of the in vivo experiments of MEPCDS 10c and
14 are given in Figures 2 and 3.
Neither MEPCDSs nor its hydrolysis product could be detected
in blood or brain at any time point, only the quaternary acid could
be detected. The concentration of the quaternary acid in blood was
found to decrease very quickly to undetectable levels within the
first 45 min for both MEPCDSs 10c and 14, while its concentration
in brain was highest at 20 min and decrease slowly for both
MEPCDSs 10c and 14. This trend is typical with the ‘locking-in’
pH 7.4, which illustrates the importance of enzymatic hydrolysis
studies on ester type CDSs. Second, close enzymatic rates of hydro-
lysis of both MEPCDSs and its corresponding MEPQ+, in spite of the
activating positive charge on the quaternary molecule. Finally,
although the rate of hydrolysis of the MEPCDSs in plasma is fast
enough for hydrolysis to precede oxidation, it is relatively reason-
able to give enough time for the dihydro-ester to be distributed
and reach the brain before complete hydrolysis (t1/2 = 18.88–
23.33 min). Pseudo first order rate constants of disappearances
(Kdisapp.) of the MEPCDSs and their t1/2 were calculated by linear
regression of Ln of peak area against time in minutes. Table 4
shows the results of the test with MEPCDSs 10c, 14 and 19b.
The results showed that MEPCDS 14 exhibited the fastest rate of
oxidation in plasma (t1/2 = 18.88 min) followed by MEPCDS 19b
(t1/2 = 21.81 min) and finally MEPCDS 10c with a rate of oxidation
(t1/2 = 23.33 min).
2.2.3.2. Stability in 20% brain homogenate. MEPCDSs 10c, 14
and 19b and their corresponding MEPQ+ 9c, 13 and 18b were
Table 4
Table 5
Stability of different MEPCDSs 10c, 14 and 19b in 80% mice plasma
Stability of different MEPCDSs 10c, 14 and 19b in 20% mice brain homogenate
Compound
n
Kdisapp SD (minÀ1
)
t1/2 (min)
r
Compound
n
Kdisapp SD (minÀ1
)
t1/2 (min)
r
10c
14
19b
4
4
4
0.0297 0.0013
0.0367 0.0011
0.03178 0.0004
23.33
18.88
21.81
0.997
0.998
0.999
10c
14
19b
4
4
4
0.06489 0.0023
0.07234 0.0106
0.0683 0.00265
10.679
9.58
10.146
0.997
0.959
0.997

H. A. Hassan et al. / Bioorg. Med. Chem. 17 (2009) 1681–1692
1687
Table 6
35
30
25
20
15
10
5
Comparison of the result of the in vivo distribution studies for different nicotinic acid,
3,5-pyridine dicarboxylic acid carriers, and 1-malonyl-1,4-dihydropyridine CDS
Brain
Blood
Compound
Dose
(mg/kg)
Theoretical max. conc.
g/g)
Exp. max. conc.
(lg/g)
Ratio
(l
DA-CDSa34
T-CDSb35
50
30
20
20
20
20
50
30
20
20
20
20
22
15
9.6
6.0
23.23
29.17
0.44
0.50
0.48
0.30
1.16
1.46
Ph-CDSc36
Try-CDSd36
MEPCDS 10c
MEPCDS 14
a
Dopamine CDS.
Testosterone CDS.
Phenethylamine CDS.
Tryptamine CDS.
b
c
0
d
0
10
20
30
40
50
Time (min.)
Figure 2. In vivo distribution of MEPCDS 10c in blood and rat brain homogenate.
treated with CMC solution (0.5% w/v in water). One hour later,
the mice were suspended by the tail to the edge of a shelf 80
cm above the floor. The tail was secured to the shelf by adhesive
tape placed approximately 1 cm from the tip of the tail. The dura-
tion of the immobility was recorded over a period of 6 min. Mice
were considered immobile only when they hung passively and
completely motionless.
35
Brain
Blood
30
25
20
15
10
5
Results of the antidepressant activity of the tested compounds
and reference drug are given in Table 7. MEPCDSs 19a and 14
and the MEPQ+ 13 induced remarkable antidepressant activity
compared to imipramine. Compounds 13, 14 and 19a significantly
reduced the duration of immobility times to be 85.30, 108.40 and
118.50 s, respectively, compared to 132.00 s reduction of the dura-
tion of immobility for imipramine. MEPCDSs 10a, 10c and MEPQ+
18a induce good antidepressant activity, their activities nearly
equal to 92.8%, 86.7% and 90.20% of the activity of imipramine.
Other MEPQ+ including 9a and 9c exhibited moderate antidepres-
sant activity, their activities nearly equal to 78.80% and 79.60% of
the activity of imipramine. Moreover, the results of the antidepres-
sant activities of the tested MEPCDSs revealed that the MEPCDS
19a exhibited the highest antidepressant activity compared to
other tested MEPCDSs (Figure 4A). The MEPQ+ 13 exhibited the
highest antidepressant activity compared to other tested MEPQ+
(Figure 4B). The results of the antidepressant activities also re-
vealed that MEPCDS 10a, 10c, 14 and 19a exhibited good antide-
pressant activities better than their corresponding MEPQ+ 9a, 9c,
13 and 18a (Figure 4C) and that reflects the importance of the
chemical delivery system for delivering drugs to the brain. The re-
sults of comparison between different MEPCDS 10a, 10c, 14 and
19a and their corresponding MEPQ+ 9a, 9c, 13 and 18a are listed
in Table 7 and shown in Figures 4A–C
0
0
10
20
30
40
50
Time (min.)
Figure 3. In vivo distribution of MPCDS 14 in blood and rat brain homogenate.
property of the brain-specific chemical delivery systems. Without
the involvement of active transport systems or the use of the re-
dox-CDSs, no drug can reach significantly higher concentrations
in brain than blood.
To test the efficiency of the tested MEPCDSs 10c and 14, the ob-
tained results were compared with reported results of selected effi-
cient PCDSs including dopamine, testosterone, phenethylamine
and tryptamine-PCDSs, in which the concentration of the quater-
nary end products in brain were determined. The overall efficiency
of the carrier system can be measured by calculating the ratio of
highest concentration of the quaternary in brain to the dose
administered, considering the body weight as the theoretical vol-
ume of distribution. Table 6 illustrates the results of these
calculations.
Table 7
Effect of the tested compounds 10a, 10c, 14, 19a, 9a, 9c, 13 and 18a and imipramine
on the duration of immobility in mice using tail suspension technique
2.2.5. Antidepressant screening
MEPCDS 10a, 10c, 14 and 19a and their corresponding quater-
nary MEPQ+ 9a, 9c, 13 and 18a were evaluated for their antidepres-
sant activity using tail suspension behavioral despair test.37–43
This test is used effectively in predicting the activity of a wide
variety of antidepressants such as MAO inhibitors and tricyclic
antidepressants.37,38 The synthesized compounds 10a, 10c, 14,
19a, 9a, 9c, 13 and 18a (10 mg/kg), and the tricyclic antidepres-
sant imipramine as a reference drug (10 mg/kg) were dissolved
in carboxymethylcellulose (CMC) solution (0.5% w/v in water)
and were injected ip in a standard volume of 0.5 mL per 100 g
body weight, 1 h prior to the test. Control animals were similarly
Compound
Duration of immobility (s)
(mean S.E.M.)
% Deviation from
control
Control
Imipramine
9a
9c
13
18a
10a
10c
14
240.6 14.93
132 0.0
0.00
À45.14
À30.34
À31.42
À50.75
À39.15
À36.7
167.6 3.225
165.8 5.87
118.5 10.13
146.4 3.44
152.3 4.66
142.2 15.9
108.4 4.24
85.3 17.24
À40.9
À54.95
À64.55
19a

1688
H. A. Hassan et al. / Bioorg. Med. Chem. 17 (2009) 1681–1692
control
300
Imipramine
10a
19a
10c
14
control
Imipramine
9a
9c
13
18a
A
B
300
control
control
250
200
150
100
50
250
200
150
100
50
9a
9c
10a 10c
18a
13
14
19a
0
0
Compounds
Compounds
C
10a
10c
9a
13
Imipramine
14
Control
300
9c
Control
250
200
150
100
50
9a
9c
10c
10a
13
14
0
Compounds
Figure 4. The antidepressant activities of different MEPCDSs 10a, 10c, 14, 19a and MEPQ+s 9a, 9c, 13, 18a compared to imipramine at a dose level of 10 mg/kg. (A) The
antidepressant activities of different MEPCDSs 10a, 10c, 14, 19a compared to imipramine at a dose level of 10 mg/kg. (B) The antidepressant activities of different MEPQ+s 9a,
9c, 13, 18a compared to imipramine at a dose level of 10 mg/kg. (C) The antidepressant activities of MEPCDSs 10a, 10c, 14 and their corresponding MEPQ+s 9a, 9c, 13
compared to imipramine at a dose level of 10 mg/kg.
3.1.3. General procedure for synthesis of
benzylideneisonicotinic acid hydrazides 8a–e
3. Experimental
3.1. Chemistry
To a stirred solution of isonicotinic acid hydrazide 6 (1.37 g,
10.0 mmol) in 30 mL absolute ethanol, the aromatic aldehyde
7a–e was added (10.0 mmol). The mixture was heated at reflux
for 5–7 h. The obtained precipitate was filtered off and crystallized
from ethanol.
Reactions were monitored by TLC analysis using Merck 9385
pre-coated aluminum plate Silica Gel (Kieselgel 60) with F254
indicator thin layer plates. Melting points were determined on Stu-
art electrothermal melting point apparatus and were uncorrected.
IR spectra were recorded as KBr disks on a Brukar Vector 22 IR spec-
trophotometer. 1H NMR spectra were carried out on 200 MHz GEM-
INI-200 NMR spectrophotometer and on 60 MHz Varian EM-360L
NMR spectrophotometer using TMS as internal reference. Chemical
shifts (d values are given in parts per million (ppm) using CDCl3
(7.29) or DMSO-d6 (2.5) as solvents and coupling constants (J) in
Hertz. Splitting patterns are designated as follows: s, singlet; d,
doublet; t, triplet; q, quartet; dd, doublet of doublet; m, multiplet.
Accurate masses were obtained on HP mass spectrometer. Ele-
mental analysis was performed on Perkin Elmer 2400 CHN Ele-
mental analyzer and on Vario EL III, Elemental analyzer, GMBH.
D-63452 HANAU.
3.1.3.1. (2-Hydroxybenzylidene)isonicotinic acid hydrazide
8a22
.
Yellowish white crystals in (1.93 g, 80% yield); mp 253–
255 °C. 1H NMR (60 MHz, DMSO-d6) d (ppm): 7.3–9.7 (m, 9H, Ar-
H, –N@CH-Ar), 12.3 (br s, 1H, OH), 13.1 (br s, 1H, NH).
3.1.3.2. (4-Methoxybenzylidene)isonicotinic acid hydrazide
8b23
.
Yellowish white crystals in (1.78 g, 70% yield); mp 171–
172 °C. 1H NMR (60 MHz, DMSO-d6) d (ppm): 3.8 (s, 3H, OCH3),
7.3–9.8 (m, 9H, Ar-H, –N@CH-Ar), 12.1 (br s, 1H, NH).
3.1.3.3. (2-Chlorobenzylidene)isonicotinic acid hydrazide
8c24
.
White crystals in (2.13 g, 82.2% yield); mp 216–
217 °C. 1H NMR (60 MHz, DMSO-d6) d (ppm): 7.7–9.9 (m, 9H,
Ar-H, –N@CH-Ar), 13.1 (br s, 1H, NH).
3.1.1. Synthesis of 2-bromomalonic acid monoethyl ester 3 and
2-bromomalonic acid 520,21
3.1.2. Synthesis of nicotinic acid hydrazide 16.28
3.1.3.4. (4-Hydroxy-3-methoxybenzylidene)isonicotinic
acid
hydrazide 8d25
.
Yellow crystals in (1.84 g, 68% yield); mp
To a stirred solution of nicotinamide 15 (1.22 g, 10.0 mmol) in
20 mL absolute ethanol, hydrazine monohydrate 98% (0.98 g,
20.0 mmol) was added. The mixture was heated at reflux for about
6 h, the solvent was evaporated under reduced pressure and the
resulting solid was filtered off and crystallized from ethanol afford-
ing 16 as white crystals in (1.16 g, 85% yield), mp 102 °C.
238–240 °C. 1H NMR (60 MHz, DMSO-d6) d (ppm): 4.3 (s, 3H,
OCH3), 7.3–9.8 (m, 9H, Ar-H, –N@CH-Ar), 12.5 (br s, 1H, NH).
3.1.3.5. (4-Dimethylaminobenzylidene)isonicotinic acid hydra-
zide 8e26
.
Yellow crystals in (1.61 g, 60% yield); mp 189–190 °C.

H. A. Hassan et al. / Bioorg. Med. Chem. 17 (2009) 1681–1692
1689
1H NMR (60 MHz, DMSO-d6) d (ppm): 3.4 [s, 6H, N–(CH3)2], 7.1–9.6
(m, 9H, Ar-H, –N@CH-Ar), 12.6 (br s, 1H, NH).
3122 (OH), 3445 (NH). 1H NMR (200 MHz, DMSO-d6) d (ppm):
1.26 (t, 3H, J = 7.2 Hz, CH3), 4.27 (q, 2H, J = 7.2 Hz, CH2), 5.75(s,
1H, CH), 7.4–9.3 (m, 9H, Ar-H, –N@CH-Ar), 12.3 (br s, 1H, –COOH),
12.8 (br s, 1H, NH). MS: m/z (%) 259 (36.8) [M+À211], 122 (40.0),
106 (100.0), 78.3 (58.6). Anal. Calcd for C18H17BrClN3O5: C, 45.93;
H, 3.64; N, 8.93. Found: C, 45.75; H, 3.53; N, 9.05.
3.1.4. General procedure for synthesis of benzylidenenicotinic
acid hydrazides 17a,b
An equimolar mixture of nicotinic acid hydrazide 16 (1.37 g,
10.0 mmol) and different aromatic aldehydes 7a,b (10.0 mmol) in
30 mL absolute ethanol was heated at reflux for 10 h. The resulting
solid was filtered off and crystallized from ethanol.
3.1.5.4. 1-(Carboxyethoxycarbonylmethyl)-4-[(4-hydroxy-3-
methoxybenzylidene)hydrazinocarbonyl]pyridinium bromide
9d. Yellow crystals in (2.41 g, 50% yield); mp 178–180 °C. IR
(KBr) mmax (cmÀ1): 1668 (C@O amide), 1730 (C@O acid), 1739
(C@O ester), 3207 (OH), 3411 (NH). 1H NMR (200 MHz, DMSO-
d6) d (ppm): 1.28 (t, 3H, J = 7.0 Hz, CH3), 3.84 (s, 3H, OCH3), 4.26
(q, 2H, J = 7.0 Hz, CH2), 5.75 (s, 1H, CH), 6.7–9.3 (m, 8H, Ar-H, –
N@CH-Ar), 9.8 (s, 1H, OH), 11.9 (br s, 1H, COOH), 12.4 (br s, 1H,
NH). MS: m/z (%) 270 (26.4) [M+À211], 149 (100), 123 (26.2), 106
(56.5), 78 (74.6). Anal. Calcd for C19H20BrN3O7: C, 47.32; H, 4.18;
N, 8.71. Found: C, 47.29; H, 4.10; N, 8.24.
3.1.4.1. (2-Hydroxybenzylidene)nicotinic
acid
hydrazide
17a29
.
Yellowish white crystals in (1.86 g, 77.2% yield); mp
185–186 °C. 1H NMR (60 MHz, DMSO-d6) d (ppm): 7.3–10.2 (m,
9H, Ar-H, –N@CH-Ar), 12.5 (br s, 1H, OH), 13.0 (br s, 1H, NH).
3.1.4.2. (4-Methoxybenzylidene)nicotinic
acid
hydrazide
17b30 Yellow crystals in (1.53 g, 60% yield); mp 177–180 °C. 1H
.
NMR (60 MHz, DMSO-d6) d (ppm): 4.2 (s, 3H, OCH3), 7.3–9.8 (m, 9H,
Ar-H, –N@CH-Ar), 12.1 (br s, 1H, NH).
3.1.5.5. 1-(Carboxyethoxycarbonylmethyl)-4-[(4-dimethylami-
nobenzylidene)hydrazinocarbonyl]pyridinium bromide 9e.
Reddish brown crystals in (2.40 g, 50% yield); mp 216–217 °C. IR
(KBr) mmax (cmÀ1): 1671 (C@O amide), 1740 (C@O acid), 1748
(C@O ester), 3165 (OH), 3429 (NH). 1H NMR (200 MHz, DMSO-
d6) d (ppm): d 1.32 (t, 3H, J = 7.2 Hz, CH3), 3.24 (s, 3H, CH3), 3.35
(s, 3H, CH3), 4.4 (q, 2H, J = 7.2 Hz, CH2), 5.76 (s, 1H, CH), 6.8–9.49
(m, 9H, Ar-H, –N@CH-Ar), 12.29 (br s, 1H, CO–OH), 12.52 (br s,
1H, NH). MS: m/z (%) 268 (61.1) [M+À211], 146 (100), 106 (13.6),
78 (35.1). Anal. Calcd for C20H23BrN4O5: C, 50.12; H, 4.84; N,
11.69. Found: C, 50.01; H, 4.78; N, 11.66.
3.1.5. General procedure for synthesis of 1-(carboxyethoxycar-
bonylmethyl)-4-(benzylidenehydrazino-carbonyl)pyridinium
bromide derivatives 9a–e
To a stirred solution of the prepared benzylideneisonicotinic
acid hydrazides 8a–e (10.0 mmol) in 30 mL absolute ethanol, a
solution of 2-bromomalonic acid monoethyl ester 3 (20.0 mmol)
in 30 mL absolute ethanol was added and the mixture was heated
at reflux for 48 h. The solution was concentrated under reduced
pressure and the resulting solid was filtered off and crystallized
from ethanol/diethyl ether.
3.1.5.1. 1-(Carboxyethoxycarbonylmethyl)-4-[(2-hydroxyben-
zylidene)hydrazinocarbonyl]pyridinium bromide 9a. Orange
crystals in (1.81 g, 40% yield); mp 210–211 °C. IR (KBr) mmax
(cmÀ1): 1680 (C@O amide), 1735 (C@O acid), 1755 (C@O ester),
3050 (OH), 3450 (NH). 1H NMR (200 MHz, DMSO-d6) d (ppm):
1.29 (t, 3H, J = 7.4 Hz, CH3), 4.3 (q, 2H, J = 7.4 Hz, CH2), 5.76
(s, 1H, CH), 6.9–9.4 (m, 9H, Ar-H, –N@CH-Ar), 10.8 (br s, 1H,
COOH), 12.8 (br s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) d
13.63 (CH3), 63.72 (CH2), 72.1 (CH), 116.35 (CH), 118.59 (C–
CH@N), 119.33 (CH), 121.45 (CH), 129.12 (CH), 131.63 (CH),
139.99 (C–CO), 148.91 (CH), 149.29 (C), 150.19 (CH), 157.88
(CO), 160.38 (CO), 162.09 (CO). MS: m/z (%) 241 (25.9)
[M+À211], 123 (79.2), 106 (100), 78.3 (67.9). Anal. Calcd for
C18H18BrN3O6: C, 47.80; H, 4.01; N, 9.29. Found: C, 48.06; H,
3.95; N, 9.56.
3.1.6. General procedure for synthesis of 1-(carboxyethoxycar-
bonylmethyl)-3-(benzylidenehydrazino carbonyl)pyridinium
bromide derivatives 18a,b
To a stirred solution of the prepared benzylidenenicotinic acid
hydrazides 17a,b (10.0 mmol) in 30 mL absolute ethanol, a solu-
tion of 2-bromomalonic acid monoethyl ester 3 (20.0 mmol) in
30 mL absolute ethanol was added and the mixture was heated
at reflux for 48 h. The solution was concentrated under reduced
pressure and the resulting solid was filtered off and crystallized
from ethanol/diethyl ether.
3.1.6.1. 1-(Carboxyethoxycarbonylmethyl)-3-[(2-hydroxyben-
zylidene)hydrazinocarbonyl]pyridinium bromide 18a. Yel-
lowish white crystals in (3.39 g, 75% yield); mp 218–219 °C. IR
(KBr) mmax (cmÀ1): 1671 (C@O amide), 1738 (C@O acid), 1744
(C@O ester), 3137 (OH), 3467 (NH). 1H NMR (200 MHz, DMSO-
d6) d (ppm): 1.28 (t, 3H, J = 7.1 Hz, CH3), 4.27 (q, 2H, J = 7.1 Hz,
CH2), 5.77 (s, 1H, CH), 6.95–9.64 (m, 9H, Ar-H, –N@CH-Ar), 10.8
(br s, 1H, COOH), 12.3 (br s, 1H, OH), 12.66 (br s, 1H, NH). MS:
m/z (%) 241 (25.9) [M+À211], 123 (79.2), 106 (100), 78.3 (67.9).
Anal. Calcd for C18H18BrN3O6: C, 47.80; H, 4.01; N, 9.29. Found:
C, 48.00; H, 3.88; N, 9.38.
3.1.5.2. 1-(Carboxyethoxycarbonylmethyl)-4-[(4-methoxyben-
zylidene)hydrazinocarbonyl]pyridinium bromide 9b. Yellow
crystals in (3.27 g, 70.1% yield); mp 186–188°C. IR (KBr) mmax
(cmÀ1): 1678 (C@O amide), 1745 (C@O acid), 1755 (C@O ester),
3127 (OH), 3451 (NH). 1H NMR (200 MHz, DMSO-d6) d (ppm):
1.28 (t, 3H, J = 7.0 Hz, CH3), 3.83 (s, 3H, OCH3) 4.27 (q, 2H,
J = 7.0 Hz, CH2), 5.74 (s, 1H, CH), 6.9–9.4 (m, 9H, Ar-H, –N@CH-
Ar), 12.2 (br s, 1H, COOH), 12.44 (br s, 1H, NH). 13C NMR
(100 MHz, DMSO-d6) d 14.1 (CH3), 55.77 (OCH3), 64.19 (CH2),
72.51 (CH), 114.85 (CH), 125.81 (CH), 126.47 (C), 129.61 (CH),
148.42 (CH), 150.12 (C), 150.88 (CH), 152.52 (C), 158.85 (CO),
161.83 (CO), 162.57 (CO). MS: m/z (%) 255.2 (11.7) [M+À211],
133.2 (100), 106 (37.3), 78.3 (48.4). Anal. Calcd for C19H20BrN3O6:
C, 48.94; H, 4.32; N, 9.01. Found: C, 48.48; H, 3.91; N, 9.14.
3.1.6.2. 1-(Carboxyethoxycarbonylmethyl)-3-[(4-methoxyben-
zylidene)hydrazinocarbonyl]pyridinium bromide 18b. Orange
red crystals in (2.33 g, 50% yield); mp 147–148 °C. IR (KBr) mmax
(cmÀ1): 1673 (C@O amide), 1730 (C@O acid), 1738 (C@O ester),
3213 (OH), 3402 (NH). 1H NMR (200 MHz, DMSO-d6) d (ppm): d
1.26 (t, 3H, J = 7.1 Hz, CH3), 3.85 (s, 3H, OCH3), 4.25 (q, 2H,
J = 7.0 Hz, CH2), 5.78 (s, 1H, CH), 6.9–9.7 (m, 9H, Ar-H,
–
N@CH-Ar), 12.1 (br s, 1H, COOH), 12.48 (br s, 1H, NH). MS:
m/z (%) 255 (10.2) [M+À211], 133 (100), 106 (63.9), 78 (50.8).
Anal. Calcd for C19H20BrN3O6: C, 48.94; H, 4.32; N, 9.01. Found:
C, 48.62; H, 3.91; N, 9.24.
3.1.5.3. 1-(Carboxyethoxycarbonylmethyl)-4-[(2-chlorobenzyli-
dene)hydrazinocarbonyl]pyridinium bromide 9c. Yellowish
white crystals in (2.64 g, 56% yield); mp 183–184 °C. IR (KBr) mmax
(cmÀ1): 1682 (C@O amide), 1746 (C@O acid), 1752 (C@O ester),

1690
H. A. Hassan et al. / Bioorg. Med. Chem. 17 (2009) 1681–1692
3.1.7. General procedure for synthesis of 1-methyl-4-(benzylid-
enehydrazinocarbonyl)pyridinium bromide derivatives 11a–d
To a stirred solution of the prepared benzylideneisonicotinic acid
hydrazides 8a–e (10.0 mmol) in 30 mL absolute ethanol, a solution
of 2-bromomalonic acid 5 (20.0 mmol) in 20 mL absolute ethanol
was added. The mixture was heated at reflux for 30 h. The solution
was concentrated under reduced pressure and the resulting solid
was filtered off and crystallized from ethanol/diethyl ether.
and sodium bicarbonate (1.1 g; 12.00 mmol) were added. Sodium
dithionite (2.1 g; 12.00 mmol) was added gradually to the mixture
while stirring, the mixture was stirred under atmosphere of nitro-
gen gas for 3 h. The red organic layer was separated, washed with
2 Â 30 mL water, dried over anhydrous sodium sulfate, filtered and
evaporated under reduced pressure to give 10a–e, 12a–d, 14 and
19a,b as yellow orange viscous oily products in 60–70% yield.
The obtained products were directly protected from light and kept
at (À10 °C) and used directly for further biological investigations.
3.1.7.1. 1-Methyl-4-[(2-hydroxybenzylidene)hydrazinocarbon-
yl]pyridinium bromide 11a. White crystals in (1.34 g, 40%
yield); mp 244–245 °C. IR (KBr) mmax (cmÀ1): 1682 (C@O amide),
3181 (OH), 3441 (NH). 1H NMR (200 MHz, DMSO-d6) d (ppm): d
4.39 (s, 3H, CH3), 7.48–9.27 (m, 9H, Ar-H, –N@CH-Ar), 11.4 (br s,
1H, OH), 12.2 (br s, 1H, NH). MS: m/z (%) 241 (16.4) [M+À211],
123 (87.1), 106 (83.4), 78 (100). Anal. Calcd for C14H14BrN3O2: C,
50.02; H, 4.20; N, 12.50. Found: C, 50.08; H, 4.30; N, 11.95.
3.2. Biology
3.2.1. Chemical oxidation
3.2.1.1. Oxidation with ferricyanide. To a series of tubes, each
containing 3 mL of freshly prepared ferricyanide reagent, 1 mL of
(2 Â 10À1 mmol) freshly prepared methanolic solution of MEPCDSs
10a, 10c, 12c, 14, 19a and 19b was added, the mixture was shaken
and set aside at room temperature. At a suitable time intervals (5,
10, 20, 45, and 60 min) the tubes were vortexed, filtered and mon-
itored spectrophotometrically for the disappearance of the
MEPCDSs and the appearance of their corresponding MEPQ+s at
their specific kmax. The rate of disappearance of MEPCDSs and the
appearance of their corresponding MEPQ+s were determined. The
apparent pseudo first order rate constant of disappearance of
MEPCDS (Kdisapp., minÀ1) was determined by calculation from lin-
ear regression of absorbance against time in min.
3.1.7.2. 1-Methyl-4-[(4-methoxybenzylidene)hydrazinocarbon-
yl]pyridinium bromide 11b. Yellowish white crystals in (1.93 g,
55.1% yield); mp 150–152 °C. IR (KBr) m
max (cmÀ1): 1649 (C@O amide),
3441 (NH). 1H NMR (200 MHz, DMSO-d6) d (ppm): 3.84 (s, 3H, OCH3),
4.44 (s, 3H, CH3), 6.49–8.81 (m, 9H, Ar-H, –N@–N@CH-Ar), 11.96 (br s,
1H, NH). MS: m/z (%) 349 (8.1) [M+], 255 (7.0) [M+À94], 133 (43.0),
106 (23.3), 93 (100), 78 (34.9). Anal. Calcd for C15H16BrN3O2: C,
51.44; H, 4.60; N, 12.00. Found: C, 51.51; H, 4.83; N, 12.86.
3.1.7.3. 1-Methyl-4-[(2-chlorobenzylidene)hydrazinocarbon-
yl]pyridinium bromide 11c. Yellowish white crystals in (1.88 g,
53% yield); mp 250–252 °C. IR (KBr) mmax (cmÀ1): 1658 (C@O
amide), 3423 (NH). 1H NMR (200 MHz, DMSO-d6) d (ppm): 4.44
(s, 3H, CH3), 7.48–9.27 (m, 9H, Ar-H, –N@CH-Ar), 12.76 (br s, 1H,
NH). MS: m/z (%) 356 (9.3) [M+], 224 (14.7), 122 (52), 106 (100),
78 (70.0). Anal. Calcd for C14H13BrClN3O: C, 47.42; H, 3.69; N,
11.85. Found: C, 47.49; H, 3.56; N, 12.14.
3.2.1.2. Oxidation with hydrogen peroxide (30%). In a series of
tubes, 1 mL of (2 Â 10À1 mmol) freshly prepared methanolic solu-
tion of MEPCDSs 10a, 10c, 12c, 14, 19a and 19b was added to
10 mL of 30% H2O2 solution. The mixtures were mixed and set
aside at room temperature. At the specific time intervals (5, 10,
20, 45, and 60 min) the tubes were vortexed, filtered and moni-
tored spectrophotometrically for the disappearance of the
MEPCDSs and the appearance of their corresponding MEPQ+s at
their specific kmax. The apparent pseudo first order rate constants
of disappearance of MEPCDS (Kdisapp., minÀ1) was determined by
calculation from linear regression of absorbance against time in
minutes.
3.1.7.4. 1-Methyl-4-[(4-hydroxy-3-methoxybenzyli-
dene)hydrazinocarbonyl]pyridinium bromide 11d. Yellow
crystals in (2.23 g, 61% yield); mp 228–229 °C. IR (KBr) mmax
(cmÀ1): 1664 (C@O amide), 3021 (OH), 3423 (NH). 1H NMR
(200 MHz, DMSO-d6) d (ppm): 3.84 (s, 3H, OCH3), 4.43 (s, 3H,
CH3), 6.89–9.6 (m, 9H, Ar-H, –N@CH-Ar), 11.9 (br s, 1H, OH),
12.33 (br s, 1H, NH). MS: m/z (%) 271 (35.8) [M+À94], 149 (83.0),
123 (26.4), 106 (77.4), 78 (86.6). Anal. Calcd for C15H16BrN3O3: C,
49.20; H, 4.40; N, 11.47. Found: C, 48.98; H, 4.28; N, 11.58.
3.2.1.3. Stability of the prepared MEPCDSs in buffer sys-
tems. Two series each of four tubes, each tube contains 5 mL
of phosphate buffer of (pH 5.8 or 7.4), 0.5 mL of (2 Â 10À1 mmol)
freshly prepared methanolic solution of MEPCDSs 10c, 14 and
19b was added. The mixtures were kept in dark at room tempera-
ture all the time of experiment (4 days). At appropriate time inter-
3.1.8. Synthesis of 1-dicarboxymethyl-4-(4,4-dimethylaminob-
enzylidenehydrazinocarbonyl)pyridinium bromide 13
vals (2, 24, 48, 72, and 96 h), 20 lL of the mixture was taken and
analyzed by HPLC for their contents of the MEPCDSs and their cor-
Compound 13 was prepared according to the general procedure
described above for synthesis of compounds 11a–d with a refluxing
time of 15 h resulting in a brownish red crystals in (2.4 g, 42% yield),
responding metabolites. The apparent pseudo first order rate con-
stant of disappearance of MEPCDSs (Kdisapp.
,
minÀ1
)
was
mp 180–182 °C; m
max/cmÀ1 1642 (C@O amide), 1689 (C@O acid),
determined by calculation from linear regression of the Ln of the
MEPCDS peak area against time in hours.
3422 (OH), 3495 (NH); 1H NMR (200 MHz, DMSO-d6): d 3.2 (s, 6H,
2CH3), 5.64 (s, 1H, CH), 6.52–8.82 (m, 9H, Ar-H, –N@CH-Ar), 11.83
(br s, 1H, NH); MS: m/z (%) 449 (7.4) [M2+], 289 (3.7), 105 (18.5),
79 (7.4), 56 (100). Anal. Calcd for C18H19BrN4O5: C, 47.91; H, 4.24;
N, 12.42. Found: C, 47.49; H, 4.56; N, 12.35.
3.2.2. In vitro stability in biological fluids
3.2.2.1. Stability in 80% mice plasma. A freshly collected hepa-
rinized mice blood was centrifuged at 4000 rpm for 20 min, and
the supernatant (plasma) was collected by a Pasteur pipette. To
3 mL of 80% freshly collected plasma diluted with (phosphate buf-
fer 0.11 mol, pH 7.4) pre-warmed in a water bath at 37 °C for
3.1.9. General procedure for synthesis 2-[4-(benzylidenehydra-
zinocarbonyl)-2-(carboxyethoxycarbonylmethyl)-1,4-dihydro-
pyridine 10a–e, 4-(benzylidenehydrazino carbonyl)-1,4-dihydro-
N-methylpyridine derivatives 12a–d, N-(dicarboxymethyl)-1,
4-dihydro-4-[(4,4-dimethylamino)benzylidenehydrazinocarbo
nyl] pyridine 14 and 2-[3-(benzylidenehydrazinocarbonyl)-2-
(carboxyethoxycarbonylmethyl)-1,4-dihydropyridine 19a,b27
To an ice cooled stirred mixture of compounds 9a–e, 11a–d, 13
or 18a,b (3.00 mmol) in 100 mL deareated water, 200 mL CH2Cl2
5 min, 300
l
L of (2 Â 10À1 mmol) methanolic solution of freshly
prepared MEPCDSs 10c, 14 and 19b was added and mixed thor-
oughly. The mixture was kept at 37 °C all the time of the experi-
ment. Samples of 200 lL were withdrawn from the tested
mixture at different time intervals (1, 2, 4, 8, 16, 32, and 64 min)
and added immediately to 2 mL of ice-cooled methanol, vortexes,
and places in a deep freezer (À80 °C). When all the samples have

H. A. Hassan et al. / Bioorg. Med. Chem. 17 (2009) 1681–1692
1691
been collected, they were centrifuged, and the supernatants were
filtered through nitrocellulose membrane filter (0.22 m) and ana-
(imipramine) were dissolved in carboxymethylcellulose (CMC)
solution (0.5% w/v in water). The prepared MEPCDSs 10a, 10c, 14
and 19a and their corresponding quaternary MEPQ+ 9a, 9c, 13
and 18a (10 mg/kg) and imipramine (10 mg/kg) were injected ip
to mice at a volume of 0.5 mL per 100 g body weight. Control ani-
mals were similarly treated with CMC solution (0.5% w/v in water).
One hour later, the mice were suspended by the tail to the edge of a
shelf 80 cm above the floor. The tail was secured to the shelf by
adhesive tape placed approximately 1cm from the tip of the tail.
The duration of the immobility was recorded for a period of
6 min. Mice were considered immobile only when they hung pas-
sively and completely motionless.
l
lyzed by HPLC for their contents of the MEPCDSs 10c, 14 and 19b
and their corresponding MEPQ+s derivatives. The apparent pseudo
first order rate constant of disappearance (Kdisapp., minÀ1) of the
MEPCPS was determined by linear regression of the Ln of the
MEPCPS peak area against time in minutes.
3.2.2.2. Stability in 20% mice brain homogenate. Ten adult
male albino Swiss-Webster mice were sacrificed by decapitation
and their brains were removed, washed with ice cold saline solu-
tion, weighed (total weight ꢀ 4 g) and homogenized in a tissue
homogenizer with 20 mL of aqueous ice-cold isotonic phosphate
buffer (0.2 mol, pH 7.4), while keeping the homogenizer tube in
ice bath during homogenization. To 5 mL of the freshly prepared
brain homogenate, previously equilibrated at 37 °C in a water bath
Acknowledgment
Authors introduce their great thanks to Dr. Al-Shimaa Faisel,
Pharmacology Department, Faculty of Pharmacy, Minia University
for her valuable help in measuring the antidepressant activity.
for 5 min, 400
l
L of (2 Â 10À1 mmol) methanolic solution of
freshly prepared MEPCDSs 10c, 14 and 19b was added, the pre-
pared mixture was kept at 37 °C all the time of the experiment.
Samples of 0.5 mL were withdrawn from the mixture at different
time intervals (1, 2, 4, 8, 16, 32, and 64 min) and immediately
added to 3 mL of ice-cooled methanol, vortexes, and places in a
deep freezer (À80 °C). When all the samples have been collected,
they were centrifuged, and the supernatants were filtered through
References and notes
1. Rapoport, S. In Blood–Brain Barrier in Physiology and Medicine; Rapoport, S., Ed.;
Raven Press: New York, 1976. pp 43–86.
2. Chem, P.; Bodor, N.; Wu, W.; Prokai, L. J. Med. Chem. 1998, 41, 3773.
3. Rana, K.; Bodor, N.; Elkoussi, A.; Raad, I.; Miyake, A.; Houck, H.; Gilder-Sleeve,
N. J. Med. Virol. 1986, 20, 1.
4. Anderson, W.; Simpkins, J.; Woodard, P.; Winood, D.; Stern, W.; Bodor, N.
Psychopharmacology (Berl.) 1987, 92, 157.
5. Greig, N.; Daly, E.; Sweeney, D.; Rapoport, S. Cancer Chemother. Pharmacol.
1990, 25, 320.
nitrocellulose membrane filter (0.22 lm) and analyzed by HPLC for
their contents of the MEPCDSs 10c, 14 and 19b and their corre-
sponding MEPQ+s derivatives. The apparent pseudo first order rate
constant of appearance (Kdisapp., minÀ1) of the MEPCDS was deter-
mined by linear regression of the Ln of the MEPCDS peak area
against time in minutes.
6. Krogsgaard-Larsen, P.; Christiansen, A. Eur. J. Med. Chem. 1979, 14, 157.
7. Brewster, M.; Raghavan, K.; Pop, E.; Bodor, N. Antimicrob. Agents Chemother.
1994, 38, 817.
8. Pop, E.; Bodor, N. Review-Epilepsy Res. 1992, 13, 1.
9. Raghavan, K.; Loftsson, T.; Brewster, M.; Bodor, N. Pharm. Res. 1992, 9, 743.
10. Brewster, M.; Pop, E.; Braunstein, A.; Pop, A.; Druzgala, A.; Drinculescu, A.;
Anderson, W.; Elkoussi, A.; Bodor, N. Pharm. Res. 1993, 10, 1356.
11. Brewster, M.; Bartruff, M.; Anderson, W.; Druzgala, P.; Bodor, N.; Pop, E. J. Med.
Chem. 1994, 37, 4237.
3.2.3. In vivo distribution studies
Four groups, each of three Sprague-Dawley male rats of average
weight of 120–150 g were anesthetized with 0.7 mL solution of
25% urethane in water. A freshly prepared solution of MEPCDSs
10c or 14 in DMF (25 mg/mL) was injected through the jugular
vein in a dose level of 20 mg/kg of animal body weight. At appro-
priate time intervals (10, 20, and 45 min), 1 mL of blood was with-
drawn and added immediately to a tarred tube containing 4 mL of
acetonitrile, which was afterwards weighed to determine the
amount of the blood added. The samples kept freezing (À80 °C) un-
til analysis. The animal was then decapitated, and the brain was
12. Eisner, U.; Kuthan, J. Chem. Rev. 1972, 72, 1.
13. Bodor, N.; Farrag, H.; Omar, F. Pharm. Sci. 1995, 1, 301.
14. Bodor, N.; Farag, H.; Brewster, M. Science 1981, 214, 1370.
15. Bodor, N.; Farag, H. J. Med. Chem. 1983, 26, 313.
16. Pop, E.; Loftsson, T.; Bodor, N. Pharm. Res. 1991, 8, 1044.
17. Bodor, N.; Farrag, H.; Omar, F. Bull. Pharm. Sci. 1993, 16, 175. Assiut University.
18. Mai, A.; Artico, M.; Esposito, M.; Ragno, R.; Sbardella, G.; Massa, S. Farmaco
2003, 58, 231.
19. Kuyl-Yeheskiely, E.; Tromp, C. M.; Van der Marel, G. A.; Boom, J. H. V.
Tetrahedron Lett. 1987, 28, 4461.
collected, weighed and immediately kept in
a deep freezer
20. Solomons, T. W. G. In Solomons Organic Chemistry; Magida, S., Ed., 6th ed.; John
Wiley & Sons: New York, 1996. pp 833–834.
21. Francis, A.; Sundberg, J. In Advanced Organic Chemistry Part B: Reactions and
Synthesis, 5th ed.; Springer Sceince + Business media: Virginia, 2007. pp 1008–
1010.
22. Xu, Y.-J.; Zhao, S.; Bi, S. Acta Crystallogr., Sect. E 2007, 63, o4633.
23. Lin, M.; Liu, S.-X. Acta Crystallogr., Sect. E 2007, 63, o3974.
24. Sinha, N.; Jain, S.; Tilekar, A.; Upadhayaya, R. S.; Kishore, N.; Jana, G. H.; Arora,
S. K. Bioorg. Med. Chem. Lett. 2005, 15, 1573.
25. Shi, X.-F.; He, L.; Ma, G.-Z.; Yuan, C.-C. Acta Crystallogr., Sect. E 2007, 63,
o1119.
(À80 °C). The whole brain homogenized in 1 mL of water, 4 mL of
5% DMF in acetonitrile was added to the homogenate, and the mix-
ture was homogenized again and centrifuged at 4000 rpm for
10 min. Blood samples were mixed with 4 mL of acetonitrile, for
protein precipitation, and vortexes at 4000 rpm for 10 min. The
supernatants from the brain and the blood samples were filtered
through nitrocellulose membrane filter (0.22 lm) and analyzed
by HPLC. The amount of the quaternary derivatives was deter-
mined from the HPLC spectrum in relation to a recovery experi-
ment made by adding a specific amount of the MEPQ+s 9c or 13
to a blank rat brain and blood and treated under the same manner.
26. Yang, D.-S. J. Chem. Crystallogr. 2007, 37, 343.
27. Fryer, R.; Earley, J.; Schneider, J.; Sternbach, L. Org. Chem. 1970, 35, 2455.
28. Khan, K. M.; Rasheed, M.; Zia, U.; Hayat, S.; Kaukab, F.; Choudhary, M. I.; Atta
ur, R.; Perveen, S. Bioorg. Med. Chem. 2003, 11, 1381.
29. Melnyk, P.; Leroux, V.; Sergheraert, C.; Grellier, P. Bioorg. Med. Chem. Lett. 2006,
16, 31.
30. Fun, H.-K.; Sivakumar, K.; Lu, Z.-L.; Duan, C.-Y.; Tian, Y.-P.; You, X.-Z. Acta
Crystallogr., Sect. C 1996, 52, 986.
3.2.4. Antidepressant screening using tail suspension
technique
Adult male albino Swiss-Webster mice (22 2 g) were obtained
from the animal house, Cairo. The mice were housed in a quiet and
temperature and humidity-controlled room (22 3 °C and 60 5%,
respectively) in which a 12 h light/dark cycle was maintained
(08:00–20.00 h light). The animals were acclimated to their envi-
ronment for at least 2 days before the experiments and were al-
lowed free access to food and water before being tested.
On the testing day, mice were assigned into different groups
(n = 6 for each group). The tested compounds and reference drug
31. Powell, M. F.; Wu, J. C.; Bruice, T. C. Am. Chem. Soc. 1984, 106, 3850.
32. Brewster, M. E.; Kaminski, J.; Gabanyi, Z.; Czako, K.; Simay, A.; Bodor, N.
Tetrahedron 1989, 45, 4395.
33. Bodor, N.; El-Kommos, M. E. Bull. Pharm. Sci. 1986, 9, 14. Assiut University.
34. Woodard, P. A.; Winwood, D.; Brewster, M. E.; Estes, K. S.; Bodor, N. Drug Des.
Delivery 1990, 6, 15.
35. Rahimy, M. H.; Simpkins, J. W.; Bodor, N. In Trends in Medicinal Chemistry; Sarel,
S., Mechoulam, R., Agranat, I., Eds.; Blackwell Scientific: New York, 1992. pp
369–376.
36. Sheha, M.; Al-Tayeb, A.; El-Sherief, H.; Farag, H. Bioorg. Med. Chem. 2003, 11,
1865.

1692
H. A. Hassan et al. / Bioorg. Med. Chem. 17 (2009) 1681–1692
37. Xu, Q.; Pan, Y.; Yi, L. T.; Li, Y. C.; Mo, S. F.; Jiang, F. X.; Qiao, C. F.; Xu, H. X.; Lu, X.
B.; Kong, L. D.; Kung, H. F. Biol. Pharm. Bull. 2008, 31, 1109.
38. Sakakibara, H.; Yoshino, S.; Kawai, Y.; Terao, J. Biosci. Biotechnol. Biochem. 2008,
72, 94.
39. Bhattamisra, S. K.; Khanna, V. K.; Agrawal, A. K.; Singh, P. N.; Singh, S. K. J.
Ethnopharmacol. 2008, 117, 51.
40. Wei, X. H.; Cheng, X. M.; Shen, J. S.; Wang, Z. T. J. Ethnopharmacol. 2008, 117,
339.
41. Schroeder, F. A.; Lin, C. L.; Crusio, W. E.; Akbarian, S. Biol. Psychiat. 2007, 62, 55.
42. Mineur, Y. S.; Picciotto, M. R.; Sanacora, G. Biol. Psychiat. 2007, 61, 250.
43. Liu, X.; Stancliffe, D.; Lee, S.; Mathur, S.; Gershenfeld, H. K. Biol. Psychiat. 2007,
62, 81.