New carrier for specific delivery of drugs to the brain

Article abstract of DOI:10.1016/S0968-0896(03)00038-5

1,4-Dihydropyridines were thoroughly investigated as carriers for specific drug delivery to the brain and were found very efficient. The main problem which arises in their application in pharmaceutical preparations, is the short shelf-life time due to hydration and/or oxidation. To overcome these problems, a new carrier system is suggested. Many of 1,4-dihydropyridine-3,5-dicarboxylate derivatives are used as calcium channel blockers which are known to have long shelf-life time, and at the same time, they are safe, with no reported neurotoxicity. Since efficiency of brain specific delivery depends on the rate of oxidation of the dihydropyridine carrier, (the faster the rate, the higher the efficiency), these 3,5-dicarbonyl compounds have almost of no brain-specific delivery properties. N-alkoxycarbonylmethyl derivatives of 1,4-dihydropyridine-3,5-dicarboxylate, a new carrier system is suggested and expected to be stable enough for formulation and storage. The drug moiety will be connected to the 3,5-dicarbonyl groups in the form of esters or amides. The suggested drug-carrier once delivered and distributed in the body, will be subjected to several sequential enzymatic hydrolytic and oxidative processes. The D-carrier is designed so that, the rate of hydrolysis of the ester group at the nitrogen atom should be faster than that of the hydrolysis and release of the drug moiety. The stability and efficiency of brain specific delivery of model drugs were investigated. The in vitro and in vivo studies proved the efficiency of brain specific delivery of the carrier and at the same time its shelf life stability. The results suggest that the designed carrier is promising to be used in application of pharmaceutical formulation.

Full text of DOI:10.1016/S0968-0896(03)00038-5

Bioorganic & Medicinal Chemistry 11 (2003) 1865–1872
New Carrier for Specific Delivery of Drugs to the Brain
Mahmoud Sheha,a,* Ali Al-Tayeb,b Hosny El-Sheriefb and Hassan Faraga
aMedicinal Chemistry Department, Faculty of Pharmacy, Assiut University, Assuit 71526, Egypt
bMedicinal Chemistry Department, Faculty of Pharmacy, Al-Azhar University, Assiut 71526, Egypt
Received 8 August 2002; accepted 19 November 2002
Abstract—1,4-Dihydropyridines were thoroughly investigated as carriers for specific drug delivery to the brain and were found very
efficient. The main problem which arises in their application in pharmaceutical preparations, is the short shelf-life time due to
hydration and/or oxidation. To overcome these problems, a new carrier system is suggested. Many of 1,4-dihydropyridine-3,5-
dicarboxylate derivatives are used as calcium channel blockers which are known to have long shelf-life time, and at the same time,
they are safe, with no reported neurotoxicity. Since efficiency of brain specific delivery depends on the rate of oxidation of the
dihydropyridine carrier, (the faster the rate, the higher the efficiency), these 3,5-dicarbonyl compounds have almost of no brain-
specific delivery properties. N-alkoxycarbonylmethyl derivatives of 1,4-dihydropyridine-3,5-dicarboxylate, a new carrier system is
suggested and expected to be stable enough for formulation and storage. The drug moiety will be connected to the 3,5-dicarbonyl
groups in the form of esters or amides. The suggested drug-carrier once delivered and distributed in the body, will be subjected to
several sequential enzymatic hydrolytic and oxidative processes. The D-carrier is designed so that, the rate of hydrolysis of the ester
group at the nitrogen atom should be faster than that of the hydrolysis and release of the drug moiety. The stability and efficiency of
brain specific delivery of model drugs were investigated. The in vitro and in vivo studies proved the efficiency of brain specific
delivery of the carrier and at the same time its shelf life stability. The results suggest that the designed carrier is promising to be used
in application of pharmaceutical formulation.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
successfully used for targeting drugs to specific organs
like the brain, lung and within the eye.3
One of the most important goals of pharmaceutical
research and development is the targeted drug delivery,
which is the most efficient way to improve the drug
therapeutic index (the ratio of safety to efficacy). This
goal could be achieved by physical, biological, or molec-
ular systems that result in producing high concen-
trations of pharmacologically active agents at the
pathologically relevant site.1
The delivery of drugs to the brain is often seriously
limited by the blood–brain barrier (BBB).4 It is gen-
erally accepted that the ability of the molecule to cross
the BBB is a function of its partition coefficient. The
ionized compounds fail to achieve cerebrospinal fluid
over plasma distribution ratio, unless they are actively
transported. The approach of derivatizing the com-
pound and forming a prodrug that exhibits improved
physicochemical properties for the transport through
the BBB should be treated with caution. This approach
may improve the delivery of the drug to the brain and
simultaneously improves transport to other tissues, thus
increasing the incidence of systemic side effects.
The chemical approaches of drug delivery are the most
advanced chemical-enzymatic-based drug targeting sys-
tems or, better stated, ‘retro-metabolic drug design
approaches’.2 Chemical drug delivery systems (CDS)
are defined as compounds that are produced by syn-
thetic chemical reactions forming covalent bonds
between the drug and the so-called ‘carrier’ and other
moieties. At least one chemical bond needs to be broken
for release of the active component. This concept was
A general method for brain specific delivery with sus-
tained release of the drug was developed based on
dihydroazine $ azinium salt redox chemical delivery
systems.5,6 This delivery system reduces the systemic
toxicity by accelerating the elimination of the hydro-
philic drug-quaternary carrier from the general circu-
lation. On the other hand, it provides a low-level
*Corresponding author. Tel.: +20-12-3971-632; fax: +20-88-332-776;
e-mail: sheha@acc.aun.eun.eg
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00038-5

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M. Sheha et al. / Bioorg. Med. Chem. 11 (2003) 1865–1872
faster the rate, the higher the efficiency). These
3,5-dicarbonyl compounds were almost of no brain-
specific delivery properties.
The rationale of the present work based on insertion of
an alkoxycarbonylmethyl group on the nitrogen of
1,4-dihydropyridine-3,5-dicarboxylic acid to give a car-
rier system with expected brain locked in properties and
enough stability during formulation and storage. The
suggested drug-carriers I once delivered and distributed
in the body, will be subjected to several enzymatic
hydrolytic and oxidative processes (Scheme 1). The
D-carrier I is designed so that, the rate of hydrolysis of
the alkyl ester (site 1) is faster than that of the hydro-
lysis and release of the drug moiety (site 3): K1>K6.
Selecting the suitable linkages of the drug and type of R
group can control this difference.12 Once the ester is
hydrolyzed, compound I will be transformed to the
corresponding anion II. The negative charge on the acid
anion is expected to decrease the energy of activation of
oxidation (ÁE) of the 1,4-dihydropyridine to the
corresponding quaternary, since the quaternary product
III, with the close location of opposite charges, resem-
bles the zwitterions of a-amino acids. The quaternary
ion formed inside the brain will be locked-in, while that
formed in blood and other tissues will be cleared easily.
Two simple model drugs, phenethylamine and trypt-
amine, were chosen, each to be linked through amide
bonds on both C3 and C5 of pyridine-3,5-dicarboxylic
acid. Representative compounds will be used in the in
vitro and in vivo studies to investigate their stabilities
and efficiency of brain-specific delivery.
Scheme 1. Representative outline for the chemical delivery system
design and work rationale; D=drug; X=NH, or O; R=alkyl.
sustained release of the active species in the brain that
even reduces the central toxicity. The main factor is the
choice of the quaternary carrier, which must be of low
toxicity alone and when conjugated with the drug.7
A class of organ-targeting systems that use dihydro-
pyridine conjugates to enhance brain delivery of drugs
with low toxicity has been described.8 These chemical
delivery systems rely on the conversion of dihydro-
pyridine to pyridinium salts, biomimicking the
NADH!NAD+ oxidation, to achieve selective CNS
sequestration. The lipophilicity of the designed pyridine
chemical delivery systems (PCDS) is essential for sig-
nificant brain uptake. In the brain the PCDS should be
relatively rapidly oxidized to the polar quaternary salt
to prevent efflux. The release of the active drug occurs
subsequently in a sustained manner at a rate that allows
reaching active concentrations at the site of action. Two
main problems arise in the application of the above
approach in pharmaceutical preparations. (1) Short
shelf-life stability due to acid-catalyzed hydration at
C5¼C6 double bond that gives 6-hydroxy-1,4,5,6-tetra-
hydropyridine derivatives.9,10 This hydrated product
will not be oxidized enzymatically in vivo to the corre-
sponding quaternary derivative. Accordingly, the che-
mical delivery system loses the most important step in
its specific delivery pathway, and hence loses its brain
lock-in property. (2) Isomerization into 1,2-dihydro-
derivatives during preparation.11
Results and Discussion
The 3,5-di(N-b-phenethyl)carbamoylpyridine (3a–c) and
3,5-di(N-2-[3-indolyl]ethyl)carbamoylpyridine
(4a–c)
reported in this work were prepared by coupling pyri-
dine-3,5-dicarboxylic acid with the appropriate amine as
shown in Scheme 2. The reaction proceeded at very mild
conditions, was less time consuming and afforded the
corresponding pure amide (3 and 4). The structures of
the prepared amides were ascertained by spectral ana-
1
lysis (IR and H NMR) and elemental analysis. The
quaternary derivatives (5,6:a–c) were obtained by treat-
ing the derivatives (3 and 4) with the appropriate alkyl
halide (ethyl bromoacetate, isopropyl bromoacetate and
bromoacetic acid) under mild condition. The quaterni-
zation process was observed to be easier in the case of
tryptamine derivatives than that of phenethylamine
derivatives, where reaction times were shorter and with
better yields. The spectral analysis (IR and 1H NMR) as
well as elemental analysis confirmed the structures of
the synthesized compounds (5,6:a–c). It was reported
that reduction of 3-substituted or 3,5-disubstituted pyr-
idinium salts with sodium dithionite in mildly basic
solution affords only the corresponding 1,4-dihydro-
pyridines.6 Reduction of the quaternaries (5,6:a,b) was
accomplished by using sodium dithionite in aqueous
solution of NaHCO3. The use of dithionite for reduc-
tion of quaternaries is limited by water solubility of the
quaternary salt and/or stability of the dihydropyridine
Many of N-substituted-1,4-dihydropyridine-3,5-dicarb-
oxylate derivatives are used as calcium channel blockers
which are known to have long shelf-life time, and at the
same time, they are safe, with no reported neurotoxicity.
Previous studies on these compounds as brain-specific
chemical delivery systems showed that they are very
stable toward oxidation and require a very long time to
get oxidized to the corresponding quaternary salts.6
Since efficiency of brain specific delivery depends on the
rate of oxidation of the dihydropyridine carrier, (the

M. Sheha et al. / Bioorg. Med. Chem. 11 (2003) 1865–1872
1867
Scheme 2. Preparation of the carrier system with tryptamine (Ar=3-indolyl) or b-phenethylamine (Ar=phenyl) as model drugs.
in the aqueous alkali solution. A novel method for the
reduction of pyridinium salts to the corresponding
dihydropyridine using 1-benzyl-1,2-dihydroisonicotin-
amide was described by Nuvole et al.13 A dis-
proportionation reaction occurs in organic solvent
resulting in reduction of the pyridinium salt to the corres-
ponding dihydropyridine derivative, while 1,2-dihydro-
isonicotinamide is oxidized to the corresponding
quaternary isonicotinamide that is easily removed by
filtration.14 This method was successfully used in the
reduction processes of drug-carrier quaternaries that are
particularly sensitive to aqueous basic media. Attempts
to prepare the 1,4-dihydropyridine derivatives (1c and
2c) were successful but the product could not be
obtained in analytical pure form. Trials for purification
of the product from 1-benzylisonicotinamide were
unsuccessful, the purity was found to be about 90–95%
as checked by HPLC. The structure of dihydropyridine
these compounds and their metabolites in solutions,
plasma and brain homogenate was developed, and vali-
dated before use.
The 1,4-dihydropyridine derivatives (1,2:a–b) with
N-acetic acid esters (ethyl and isopropyl) were found to
be relatively stable towards oxidation with silver nitrate
(Table 1). While their corresponding acids (1c and 2c)
were found to be oxidized much faster, to the extent
that oxidation of compound 1c could not be monitored
and compound 2c was found to have t1/2 of oxidation of
about 0.5 h. The ferricyanide oxidation of dihydropyr-
idine is commonly used to study their sequential elec-
tron–proton–electron transfer mechanism.15 During
oxidation study with ferricyanide, it was found that the
ethyl esters (1a and 2a) are partially hydrolyzed to the
corresponding acid (minor quantity) in addition to the
oxidation product. The isopropyl ester 1b showed a
slower rate of disappearance, by both hydrolysis and
oxidation, throughout the time of the experiment. These
results prove the idea that the acid anion catalyzed the
oxidation process of the dihydro compounds to the
corresponding quaternaries.
1
derivatives (1,2:a–c) were confirmed based on their H
NMR and UV-spectra.
The stabilities of the 1,4-dihydropyridine-3,5-dicarb-
oxylic acid derivatives (1 and 2) were determined in the
presence of the oxidizing agents (alcoholic AgNO3 and
potassium ferricyanide), buffer solution (stability after
reconstitution), 80% plasma, and 20% brain homo-
genate. The dihydropyridine derivatives (1a and 2a) were
selected for the in vivo study. To carry out these investi-
gations, a reverse-phase HPLC method of analysis of
The stability in both slightly alkaline or acidic buffers of
the prepared dihydropyridine CDS was investigated to
help in selecting the buffer system to be used as the sol-
vent, if needed in solution during its formulation. The
stabilities of tryptamine CDS ester 2a, and its corres-
ponding acid 2c were investigated in phosphate buffers
(0.2M, pHs 7.4 and 5.8) at room temperature ( Table 2).
The concentration of the parent CDS and its oxidation
or degradation products were monitored by HPLC for 4
days. Pseudo first order rate of disappearance of each
CDS was determined and its t1/2 was calculated (Table
2). It was observed that the tryptamine CDS ester 2a
disappears more than twice as fast at pH 7.4 than at pH
5.8 (t1/2=97 and 246 h, respectively). Hydrolysis was
observed in both buffers, where the hydrolysis product
was detected after 24 h at pH 7.4 and after 48 h at pH
5.8. The acid 2c was found only as quaternary acid 6c at
both pHs. The results showed that these CDS should be
stored in solid forms to be reconstituted in slightly
acidic buffer prior to administration.
Table 1. The rates of oxidation of prepared phenethylamine-CDS
(1a–c) and tryptamine-CDS (2a and 2c) (n=3)
a
Oxidizing
agent
Compd
kdisapp
(minꢀ1
T1/2
(h)
r
)
Silver
nitrate
1a
1b
1c
2a
2c
8.03ꢁ10ꢀ4
6.83ꢁ10ꢀ4
14.4
17
0.94
0.99
Too fast to be monitored
8.4ꢁ10ꢀ4
13.8
0.5
0.97
0.99
2.23ꢁ10ꢀ2
Ferricyanide
reagent
1a
1b
1c
2a
2c
1.15ꢁ10ꢀ2
1.17ꢁ10ꢀ3
2.36ꢁ10ꢀ3
8.67ꢁ10ꢀ3
1.17ꢁ10ꢀ3
1.0
9.9
4.9
1.33
9.9
0.98
0.98
0.98
0.96
0.95
akdisapp is pseudo first order rate constant for the dihydropyridine
derivatives (1a–c, 2a, and 2c) calculated from three replicate determi-
nation at each time.
To know the possible metabolic pathways of the syn-
thetized CDS in human plasma, the stability of the ester

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M. Sheha et al. / Bioorg. Med. Chem. 11 (2003) 1865–1872
importance of the steric factor on the rates of enzymatic
oxidation. According to the results of the in vitro study,
the ethyl ester has enough stability in plasma to reach
the brain and at the same time it is hydrolysed at a rea-
sonable rate to give the acid anion, in the brain, which is
needed to enhance the rate of oxidation of the CDS to
the corresponding quaternary. The dihydropyridine
derivatives (1a and 2a) were selected for the in vivo
study. A solution in DMSO was injected through the
jugular vein to a group of male Sprague–Dawley rats
which were then sacrificed at various time intervals;
their blood and brains were analyzed for the quaternary
precursors of phenethylamine (5a and 5c) and trypt-
amine (6a and 6c). The results of these experiments are
presented in Figures 1 and 2. With respect to phenthyl-
amine-CDS 1a, analysis of blood samples taken after 10
min proved the presence of 1a in high concentration
(17.14 mg/g) in addition to its products of hydrolysis. At
20 min only the dihydro and quaternary acids (1c and
5c) could be detected. Neither quaternary acid nor other
oxidation or hydrolysis products could be detected after
2h. The picture looked different in the brain samples.
The only product detected after 20 min was the quaternary
acid and this increased with time. With respect to trypt-
amine-CDS 2a, almost the same trend of delivery was
observed, while delivery to the brain was slower than that
of Ph-CDS 1a. The amount of 2a delivered to the brain
Table 2. Stability of tryptamine CDSs in buffers at various pHs
(n=3)
pH
7.4
Compd
(kdisappꢃSD)aꢁ10ꢀ3 hꢀ1
t1/2 (h)
r
2a
2c
7.14 ꢃ0.304
97
0.97
0.99
11.66ꢃ0.4259.5
5.8
2a
2c
2.87ꢃ0.468
246
45.6
0.96
0.98
15.56ꢃ2.99
akdisapp is pseudo first order rate constant for the dihydropyridine
derivatives (2a and 2c) calculated from three replicate determination at
each time.
1a and 1b in 80% human plasma was determined. The
pseudo first order rate constant and t1/2 of dis-
appearance of the CDS was calculated (Table 3). It was
observed that only the quaternary acid was found in
plasma as a metabolite in addition to the dihydropyr-
idine 1a or 1b. The isopropyl ester is more stable than
the ethyl ester that may be due to the difference in the
rate of hydrolysis of the different esters.
The stabilities of the synthesized dihydropyridines (1a–c
and 2c) were investigated in 20% rabbit brain homo-
genate. The homogenate was equilibrated at 37 ꢂC and
freshly prepared solution of 1,4 dihydro compound was
added. The concentration of the spiked compound and
its oxidation or degradation products were monitored
by HPLC. As shown in Table 4, the isopropyl ester 1b is
more stable toward enzymatic oxidation in brain
homogenate than the ethyl ester 1a and the corres-
ponding acid derivative 1c, with half-life times of 24.0,
9.5 and 5.6 h, respectively. The results were concurrent
with the expected results calculated by the molecular
orbital calculation.12
The results prove the idea that these types of esters are
more stable even against enzymatic oxidation, once the
ester is hydrolyzed to the corresponding acid, which will
exist as the anion, the rate of oxidation will be acceler-
ated to give the corresponding quaternary compound.
The tryptamine-acid derivative 2c is oxidized at a slower
rate than the phenethyl-acid derivative 1c; t1/2=7.7 and
5.6 h, respectively. These results may indicate the
Figure 1. In vivo distribution of phenethylamine-CDS (1a) in blood
and in rat brain homogenate (concd in mg/g against time in minutes).
Table 3. The rate of hydrolysis phenethylamine CDSs 1a and 1c in
80% human plasma
Compd
(kdisappꢃSD)a ꢁ 10ꢀ3 minꢀ1
t1/2 (h)
r
1a
1b
1.35ꢃ0.0145
8.56
23.1
0.95
0.92
0.5ꢃ0.00428
akdisapp is pseudo first order rate constant for the dihydropyridine
derivatives (1a and 1b) calculated from three replicate determination
at each time.
Table 4. The rate of disappearance of CDSs (1a–c and 2c) in 2 0%
rabbit brain homogenate
Compd
(kdisappꢃSD)ꢁ10ꢀ3 minꢀ1
t1/2 (h)
r
1a
1b
1c
2c
1.213ꢃ0.0956
0.480ꢃ0.0866
2.058ꢃ0.0971
1.499ꢃ0.1066
9.5
24.0
5.6
0.98
0.81
0.96
0.96
Figure 2. In vivo distribution of tryptaine-CDS (2a) in blood and in
rat brain homogenate (concd in mg/g against time in minutes).
7.7

M. Sheha et al. / Bioorg. Med. Chem. 11 (2003) 1865–1872
1869
after 5 h was about two-thirds that of 1a. This may be due
to the rapid clearance of 2a from blood so its concentration
necessary for BBB penteration was less than 1a.
loop was 20 mL. The chromatographic peaks were UV-
scanned with the use of a photodiode array detector
(TSP Thermo Separation Products, Spectromonitor1
5000, CT, USA). The system controlled with LC talk
program (LC talk Version 2.03 Software, Copyright
1993 by Thermo Separation Products, CT, USA). An
isocratic solvent system was prepared by mixing 0.1%
aqueous solution of trifluoroacetic acid with acetonitrile
in a ratio of 45:55, v/v. The mobile phase was degassed
and filtered through a 0.22 mm membrane filter (Milli-
pore, Bedford, MA, USA) and pumped at a flow rate of
1.0 mL/min. Acetonitrile (ACN), methanol (MeOH)
and trifluoroacetic acid (TFA) used were of HPLC
grade and obtained from E-Merck, Darmstadt, Ger-
many. All other chemicals and solvents were of analy-
tical reagent grade and were used without further
purification.
In general, the results show in both CDSs that, the
corresponding quaternary acid was detected at higher
concentrations in blood than in brain within the first
hour. The concentration of this quaternary was below
detection limit in blood after 2h from injection while it
increases in the brain until 5 h, that is, increase in the
brain and decrease in the blood with time until 5 h. The
presence of the quaternary acid as the only product in
brain, proves the main idea of the new CDS. The lipoi-
dal CDS 1a or 2a penetrates the BBB and rapidly
hydrolyzed by brain esterases to the corresponding acid,
present as anion in pH 7.4. Oxidation of the resultant
anion, by brain dehydrogenases, to the corresponding
quaternary is accelerated due to the negative charge on
the carboxylate anion. Accordingly, the results prove
the following sequential processess after brain penetra-
tion, the D-CDS is sequentially; (i) hydrolyzed to give
the dihydro acid anion (ii) oxidized to the quaternary
acid anion ‘the locked-in moiety’, which (iii) hydrolyzed
in a timely manner to release the parent drug.
Preparation of 3,5-di-(N-[aryl]ethyl)carbamoylpyridine
(3 and 4). To an ice-cold solution of 3,5-pyri-
dinedicarboxylic acid (1.67 g; 0.01 mol) in 30 mL di-
chloromethane, triethylamine (10 mL; 0.072mol) was
added, while stirring. To the mixture, ethyl chloro-
formate (4.8 mL; 0.05 mol) was added drop wise over a
period of 10 min, and stirring was continued for 1 h.
The amine (0.03 mol) was then gradually added within
10 min and stirring was continued for an additional 6 h.
The solvent was then distilled under reduced pressure,
and the residue was extracted with ethyl acetate. The
extract was washed with 0.01 N HCl, dried, distilled,
and the crude solid product was crystallized from ethanol
to yield the diamide derivatives of pyridine-3,5-dicarb-
oxylic acid. The following compounds were prepared
according to this procedure.
Conclusions
1,4-Dihydropyridine-3,5-dicarboxylic acid with alkoxy-
carbonylmethyl substituent on the nitrogen atom was
investigated as a novel shelf-stable carrier system to
deliver biologically active compounds to the brain. The
results proved that these types of esters-CDS, specially
N-ethoxycarbonylmethyl substituent, are more stable
even against enzymatic oxidation. Once the ester is
hydrolyzed to the corresponding acid, the rate of oxi-
dation will be accelerated to give the corresponding
quaternary acid that has fast clearance from the blood
and lock-in property in the brain to release the drug.
3,5-Di(N-ꢀ-phenethyl)carbamoylpyridine (3). b-Phenethyl-
amine (3.8 mL; 0.03 mol) was used as the amine to yield
3 g (80%) of white crystals; mp 189–190 ꢂC. H NMR
1
(CDCl3) d 2.8 (t, 4H, NHCH2CH2), 3.5 (q, 4H,
NHCH2CH2), 7.2(s, 10H, Ar–H ), 8.3 (t, 2H, NH), 8.6
(s, 1H, C4–H), 9.1 (s, 2H, C2–H, C6–H).
5
Experimental
3,5-Di(N-2-[3-indolyl]ethyl)carbamoylpyridine (4). Tryp-
tamine hydrochloride (5.89 g; 0.03 mol) was used as
amine to yield 4 g (73%) of white crystals; mp 198–
General methods
Melting points were determined on electrothermal
melting point apparatus and are uncorrected. Elemental
microanalysis for carbon, hydrogen, and nitrogen were
performed on Elementar Analysensysteme GmbH
VarioEL (Germany). IR spectra were recorded as KBr
disks on a Shimadzu IR 200-91527 spectrophotometer.
200 ꢂC. 1H NMR (DMSO-d6)
d
3.0 (t, 4H,
NHCH2CH2), 3.6 (t, 4H, NHCH2CH2), 6.9–7.5 (m,
10H, indole protons), 8.5 (s, 2H, NH of indole), 8.6 (s,
1H, C4–H of pyridine), 9.1(s, 2H, C2-H, C6-H of pyri-
dine), 10.3 (s, 2H, CONH). Elemental analysis,
C27H25N5O2 (451.52); calcd C, 71.82; H, 5.58; N, 15.51.
Found C, 71.45; H, 5.46; N, 15.22.
1H NMR spectra were run on Varian Em-360L NMR
spectrophotometer (60 MHz) (Varian, USA) and on
Jeol, Lambda, Oxford NMR YH (400 MHz, Japan)
using TMS as internal standard, chemical shifts are
expressed in d (ppm).
Preparation of 1-(alkyl)-pyridinium bromide derivatives
(5,6:a–c)
To a solution of compound 3 or 4 (0.003 mol) in 30 mL
of methanol, (0.004 mol) of bromoacetic acid (ester) was
added, the reaction mixture was refluxed overnight, and
then cooled to room temperature. The formed crystals
were filtered, washed with methanol, dried to give the
corresponding pyridinium bromide derivative. The fol-
lowing compounds were prepared by this procedure.
The high performance liquid chromatographic system
consisted of Pump (TSP Thermo Separation Products,
ConstaMetric1 3500, CT, USA). The column used was
an Eurosepher-100 C18 (Knauer) (25 cmꢁ4.6 mm
internal diameter, 5 mm particle diameter). The sample

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M. Sheha et al. / Bioorg. Med. Chem. 11 (2003) 1865–1872
1-(Ethoxycarbonylmethyl) - 3,5 - bis(N - ꢀ - phenethylcarb-
amoyl)-pyridinium bromide (5a). Compound 3 and ethyl
bromoacetate (0.22 mL) were used to yield 1.2 g (76%)
used to give 0.45 g (76%) of yellow crystal mp 213–
216 ꢂC. 1H NMR (DMSO-d6)
3.0 (t, 4H,
d
NHCH2CH2), 3.6 (t, 4H, NHCH2CH2), 5.6 (s, 2H, N–
CH2COO), 6.9–7.5 (m, 10H, indole protons), 9.4 (s,
2H, NH of indole), 9.5 (s, 1H, C4-H of pyridine), 9.6
(s, 2H, C2-H C6-H of pyridine), 10.8 (s, 2H, CONH).
Elemental analysis, C29H28BrN5O4 (590.47); calcd C,
58.99; H, 4.78; N, 11.86. Found C, 58.60; H, 4.67; N,
11.61.
of white crystals; mp 212–215 ꢂC. H NMR (DMSO-d6/
1
CDCl3)
d
1.2(t, 3H, CH 2CH3), 2.8 (t, 4H,
NHCH2CH2), 3.5 (t, 4H, NHCH2CH2), 4.1 (m, 2H,
CH2CH3), 5.7 (s, 2H, NCH2COO), 7.2(s, 10H, Ar–H 5),
9.2(s, 1H, C ), 9.8 (s, 2H, C2, C6). Elemental analysis,
4
C27H30BrN3O4 (540.45); calcd C, 60.00; H, 5.60; N,
7.78. Found C, 60.17; H, 5.68; N, 7.59.
Preparation of 1-(alkyl)-1,4-dihydropyridine derivatives
(1,2:a–b)
1-(Isopropyloxycarbonylmethyl)-3,5-bis(N-ꢀ-phenethyl-
carbamoyl)-pyridinium bromide (5b). Compound 3 and
isopropyl 2-bromoacetate (1 mL) were used to yield 1.1
To a solution of compound 5,6:a–b (0.4 mmol) in 200
mL of deaerated water, sodium bicarbonate (1 g; 0.012
mol) and 200 mL of ether were added. The mixture was
stirred in an ice bath, and sodium dithionite (0.7 g; 4
mmol) was added portion-wise over a period of 5 min
and stirring was continued for 3 h under nitrogen
stream. The ether layer was then separated, washed with
water, dried over anhydrous Na2SO4 and evaporated
under vacuum, to give the corresponding dihydro-
pyridine derivative. The following compounds were
prepared by the abovementioned procedure.
g (81%) of white crystalline solid; mp 217–219 ꢂC. H
1
NMR (DMSO-d6/CDCl3) d 1.3 (d, 6H, CH3), 2.8 (t,
4H, NHCH2CH2), 3.5 (t, 4H, NHCH2CH2), 5.0 (m,
1H, CHCH3), 5.7 (s, 2H, NCH2COO), 7.3 (s, 10H, Ar–
H5), 9.3 (s, 1H, C4-H), 9.8 (s, 2H, C2–H, C6H). Ele-
mental analysis, C28H32BrN3O4 (554.48); calcd C,
60.65; H, 5.82; N, 7.58. Found C, 60.77; H, 5.81; N,
7.48.
1-(Carboxymethyl)-3,5-bis(N-ꢀ-phenethylcarbamoyl)-
pyridinium bromide (5c). Compound 3 (0.75 g; 0.002
mol) and bromoacetic acid (0.8 g, 0.006 mol) were used
to yield 0.7 g (68%) of yellow crystals; mp 140–142 ꢂC.
1H NMR (DMSO-d6) d 2.8 (t, 4H, NHCH2CH2), 3.5 (t,
4H, NHCH2CH2), 5.2(s, 2H, NCH 2COO), 7.2(s, 10H,
Ar-H5), 9.2(s, 1H, C 4–H), 9.4 (s, 2H, C2-H, C6-H).
Elemental analysis, C25H26BrN3O4 (512.40); calcd C,
58.60; H, 5.11; N, 8.20. Found C, 57.88; H, 5.26; N,
8.16.
1-(Ethoxycarbonylmethyl)-3,5-bis(N-ꢀ-phenethylcarb-
amoyl)-1,4-dihydropyridine (1a). Compound 5a (0.216
g) was used to yield 0.1 g (54%) of yellow solid, mp
160–162 ꢂC. 1H NMR (DMSO-d6) d 1.2(t, 3H,
CH2CH3), 2.7 (t, 4H, NHCH2CH2), 3.0 (s, 2H, C4–H),
3.3 (t, 4H, NHCH2CH2), 4.1 (s, 2H, NCH2COO), 4.2
(m, 2H, CH2CH3), 6.8 (s, 2H, C2-H, C6-H), 7.1–7.3 (m,
10H, Ar-H5).
1-(Ethoxycarbonylmethyl)-3,5-bis{N-[2-(3-indolyl)ethyl]-
carbamoyl}pyridinium bromide (6a). Compound
1-(Isopropyloxycarbonylmethyl)-3,5-bis(N-ꢀ-phenethyl-
carbamoyl)-1,4-dihydropyridine (1b). Compound 5b
(0.22 g) was used to give 0.17 g (66%) of yellow semi-
solid, which could not be crystallized. 1H NMR
(DMSO-d6) d 1.2(d, 6H, CHCH 3), 2.7 (t, 4H,
NHCH2CH2), 3.0 (s, 2H, C4–H), 3.3 (t, 4H,
NHCH2CH2), 4.1 (s, 2H, NCH2COO), 4.9 (m, 1H,
CHCH3), 6.8 (s, 2H, C2–H, C6–H), 7.1–7.3 (m, 10H,
Ar–H5).
4
(0.451 g, 0.001 mol) and ethyl bromoacetate (0.55 mL;
0.005 mol) were used to yield 0.5 g (81%) of yellow
ꢂ
1
crystals, mp 194–196 C. H NMR (DMSO-d6) d 1.2(t,
3H, CH2CH3), 3.0 (t, 4H, NHCH2CH2), 3.6 (t, 4H,
NHCH2CH2), 4.2(m, 2H, CH 2CH3), 5.7 (s, 2H, N–
CH2COO), 6.9–7.5 (m, 10H, indole protons), 9.4 (s, 2H,
NH of indole), 9.5 (s, 1H, C4-H of pyridine), 9.6 (s, 2H,
C2-H, C6-H of pyridine), 10.8 (s, 2H, CONH). Ele-
mental analysis, C31H32BrN5O4 (618.52); calcd C, 60.20;
H, 5.21; N, 11.32. Found C, 59.97; H, 5.08; N, 11.20.
1-(Ethoxycarbonylmethyl)-3,5-bis{N-[2-(3-indolyl)ethyl]-
carbamoyl}-1,4-dihydropyridine (2a). Compound 6a
(0.246 g) was used to yield 0.119 g (55%) of pale-yellow
solid of mp 178–80 ꢂC. H NMR (DMSO-d6) d 1.2(t,
1
1-(Isopropyloxycarbonylmethyl)-3,5-bis{N-[2-(3-indolyl)-
ethyl]-carbamoyl}pyridinium bromide (6b). Compound 4
(0.451 g; 0.001 mol) and 1 mL of isopropyl-2-bromo-
acetate were used to yield 0.55 g (87%) of yellow crystal;
mp 218–220 ꢂC. 1H NMR (DMSO-d6) d 1.2(d, 6H,
CHCH3), 3.0 (t, 4H, NHCH2CH2), 3.6 (t, 4H,
NHCH2CH2), 5.0 (m, 1H, CHCH3), 5.7 (s, 2H, N–
CH2COO), 6.9–7.5 (m, 10H, indole protons), 9.4 (s, 2H,
NH of indole), 9.5 (s, 1H, C4–H of pyridine), 9.6 (s, 2H,
C2–H, C6–H of pyridine), 10.8 (s, 2H, CONH). Ele-
mental analysis, C32H34BrN5O4 (632.55); calcd C, 60.76;
H, 5.42; N, 11.07. Found C, 60.49; H, 5.40; N, 10.94.
3H, CH2CH3), 2.8 (t, 4H, NHCH2CH2), 3.1 (s, 2H, C4-
H), 3.3 (t, 4H, NHCH2CH2), 4.1 (s, 2H, NCH2COO),
4.2(m, 2H, CH 2CH3), 6.8 (s, 2H, C2-H, C6-H), 6.9–7.5
(m, 10H, indole protons).
1-(Carboxymethyl)-3,5-bis(N- ꢀ-phenethylcarbamoyl)-
1,4-dihydropyridine (1c). To a solution of 5c (0.2g; 0.4
mmol) and the 1-benzyl-1,2-dihydroisonicotinamide
(0.1 g; 0.5 mmol) in anhydrous methanol (20 mL) was
stirred at 0 ꢂC for 4 h under nitrogen. The solid that
separated was filtered and washed with methanol and
ether. The solid was identified as 1-benzyl-4-carbamoyl-
pyridinium bromide. The filtrate was evaporated by
stream of nitrogen to give semisolid yellow residue,
which was identified as 1,4-dihydro acid (1c).
1-(Carboxymethyl)-3,5-bis{N-[2-(3-indolyl)ethyl]-carb-
amoyl}pyridinium bromide (6c). Compound 4 (0.451g;
0.001 mol) and bromoacetic acid (1.1 g; 0.008 mol) were

M. Sheha et al. / Bioorg. Med. Chem. 11 (2003) 1865–1872
1871
1-(Carboxymethyl)-3,5-bis{N-[2-(3-indolyl)ethyl]carb-
amoyl}-1,4-dihydropyridine (2c). The same procedure
used for the preparation of compound 1c was followed
to give semisolid yellow residue, which was identified to
be the 1,4-dihydro acid (2c) with a little impurities of
1-benzyl-4-carbamoylpyridinium bromide (about 5% by
HPLC) and could not be separated.
teur pipette. To 5 mL of 80% freshly collected plasma
(diluted with phosphate buffer (0.11 M, pH 7.4), pre-
warmed in a water bath at 37ꢃ1 ꢂC for 5 min, 300 mL of
2ꢁ10ꢀ4 M methanolic solution of freshly prepared
PCDSs (1a or 1b) was added and mixed thoroughly.
The mixture was kept at 37ꢃ1 ꢂC the whole time of the
experiment. At time intervals (5, 15, 30, 60, 90, 120, 180,
and 240 min) 200 mL withdrawn from the tested mix-
ture, added immediately to 2mL of ice-cold methanol,
vortexed, and placed in deep freezer (ꢀ20 ꢂC). When all
samples were collected, they were centrifuged, and
supernatants were filtered through nylon membrane
0.22 mm pore size and analyzed by HPLC for their con-
tents of the CDSs (1a or 1b) and corresponding qua-
ternary derivatives.
Chemical oxidation and in vitro studies
Chemical oxidation of the prepared dihydropyridine
CDSs (1,2:a–c). Oxidation with silver nitrate. In a series
of tubes, 1 mL of 2ꢁ10ꢀ4 M methanolic solution of the
PCDSs (1,2:a–c) was added to 5 mL of 10% methanolic
silver nitrate solution and the mixture was mixed for 3
min. At the specific time intervals (5, 15, 30, 60, and 120
min), one tube is vortexed, centrifuged and the super-
natant was filtered by using 0.22 mm nylon-membrane fil-
tration disc and analyzed by HPLC. The rate of
disappearance of the PCDSs and the appearance of the
corresponding quaternaries was determined. The appar-
ent pseudo first order rate constant of disappearance of
PCDSs (kdisapp, minꢀ1) was determined by linear regres-
sion of the log of the mean peak area of three experiments
against time in minutes.
In 20% rabbit brain homogenate (1a–c and 2c). About 4
g of rabbit brain were taken, washed with ice-cold saline
solution and homogenized in a tissue homogenizer with
about 20 mL of aqueous ice-cold isotonic phosphate
buffer (0.2M, pH 7.4), while keeping the homogenizer
tube in ice bath. Methanolic solution (300 mL of 2ꢁ10ꢀ4
M) of freshly prepared PCDSs (1a–c and 2c) were mixed
with 10 mL of homogenate, previously equilibrated at
37ꢃ1 ꢂC in a water bath. Samples of 0.5 mL were
withdrawn from the tested mixture at different time
intervals (15, 60, 120, and 300 min) and immediately
added to 2mL ice-cold methanol, vortexed, and placed
in the deep freezer (ꢀ20 ꢂC). When all the samples have
been collected, centrifuged, and the supernatants were
filtered through nitrocellulose membrane filter (0.22
mm) and analyzed by HPLC for their content of the
PCDSs (1a, 1c, and 2c) and corresponding quaternary
derivatives.
Oxidation with ferricyanide reagent. To a series of
tubes, each containing 3 mL of freshly prepared ferri-
cyanide reagent,16 1 mL of the freshly prepared PCDSs
(1,2:a–c) in concentration of (2ꢁ10ꢀ4 M) were added at
room temperature. The mixture was shaken and set
aside. At a suitable time interval (5, 15, 30, 60, and 120
min) one tube is vortexed, filtered, and analyzed by
HPLC. The rate of disappearance of the PCDSs and the
appearance of the corresponding quaternaries was
determined. The apparent pseudo first order rate con-
stant of disappearance of PCDSs (kdisapp, minꢀ1) was
determined by linear regression of the log of the mean
peak area three experiments against time in min.
In vivo distribution. Five groups, each of three Sprague–
Dawley female rats of average weight of 120–140 g were
anesthetized with urethane. A freshly prepared solution
of dihydropyridine (1a or 2a) in DMSO (25 mg/mL)
was injected through the jugular vein in a dose level of
20 mg/kg of animal body weight. After the appropriate
time intervals, 1 mL of blood was withdrawn from the
eye and added immediately to a centrifuge tube con-
taining 4 mL of acetonitrile, which was afterwards
weighed to determine the amount of blood added. The
sample kept freezing till analysis. The animal was then
decapitated, and the brain was weighed and kept in the
freezer overnight then homogenized in 1 mL of water.
Four milliliter of 5% DMSO in acetonitrile was added
and the mixture homogenized again then centrifuged at
4000 rpm for 10 min. The amount of the quaternary
derivatives was determined from the HPLC spectrum in
relation to a recovery experiment made by adding a
specific amount of the quaternary to a blank brain and
hybrid in the same manner of homogenization and
extraction.
Stability of the prepared 1,4-dihydropyridine CDSs (2a
and 2c) in buffer systems (shelf stability). Two series;
each of three tubes, each tube contain 5 mL of phos-
phate buffers (pH 5.8 or 7.4), 0.5 mL of freshly prepared
dihydropyridine derivatives solution (2ꢁ10ꢀ4 M) was
added. The mixtures were kept stirring in a dark at
room temperature all the time of experiment (4 days).
At appropriate time intervals (2, 24, 48, 72, and 96 h),
20 mL of the mixture was taken and analyzed by HPLC
for the dihydropyridine derivative (2a or 2c) and corre-
sponding metabolites. The apparent pseudo first order
rate constant of disappearance of PCDSs (kdisapp
,
minꢀ1) was determined by linear regression of the log
peak area of the corresponding dihydropyridine against
time in minutes.
Acknowledgements
In vitro stability in biological fluids
In human plasma (1a and 1b). A freshly collected hepar-
inized human blood was centrifuged at 4000 rpm for 20
min. The supernatant (plasma) was collected by a Pas-
The authors thank the T3A Pharma group (Egypt) for
financial support of the presentation of this work in
AAPS, 2001 (Denver, USA).

1872
M. Sheha et al. / Bioorg. Med. Chem. 11 (2003) 1865–1872
9. Pop, E.; Loftsson, T.; Bodor, N. Pharm. Res. 1991, 8,
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