1,2-Dihydroisoquinoline-N-acetic acid derivatives as new carriers for brain-specific delivery II: Delivery of phenethylamine as model drug

Article abstract of DOI:10.1002/ardp.200300764

N-alkyloxycarbonylmethyl-1,2-dihydroisoquinolin-4-carboxylic acid derivatives 7 a-c were synthesized as new carriers for brain specific delivery. The design of the carrier systems are based on sequential hydrolysis at the acetic acid ester group linked to dihydroisoquinoline nitrogen followed by ring oxidation and formation of quaternary isoquinolinium derivatives which are then hydrolyzed to release the drug. Once the carrier system is administered, a sequential enzymatic process will take place resulting in significant increase in its rate of oxidation, the key factor in brain specific delivery. The chemical stability of the synthesized carrier system was investigated in aqueous buffer solutions and ferricyanide reagent and proofed to be quite stable against hydration and oxidation during formulation and storage. Furthermore, enzymatic stability was also investigated in 80% human plasma and 20% rabbit brain homogenate. Both oxidation and hydrolysis were found to take place; however, hydrolysis was the major route. In vivo distribution of the ethyl ester derivative 7 b was studied in rats and showed that the concentration of the quaternary product is increasing in the brain and cleared from blood with time.

Full text of DOI:10.1002/ardp.200300764

258 Mahmoud et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 258–263
Sahar Mahmoud,
Mahmoud Sheha,
Tarek Aboul-Fadl,
Hassan Farag
1,2-Dihydroisoquinoline-N-acetic Acid Derivatives
as New Carriers for Brain-specific Delivery II:
Delivery of Phenethylamine as Model Drug
Medicinal Pharmaceutical
Chemistry Department,
Faculty of Pharmacy, Assiut
University, Assiut 71526,
Egypt
N-alkyloxycarbonylmethyl-1,2-dihydroisoquinolin-4-carboxylic acid derivatives
7 a–c were synthesized as new carriers for brain specific delivery.The design of the
carrier systems are based on sequential hydrolysis at the acetic acid ester group
linked to dihydroisoquinoline nitrogen followed by ring oxidation and formation of
quaternary isoquinolinium derivatives which are then hydrolyzed to release the
drug. Once the carrier system is administered, a sequential enzymatic process will
take place resulting in significant increase in its rate of oxidation, the key factor in
brain specific delivery.The chemical stability of the synthesized carrier system was
investigated in aqueous buffer solutions and ferricyanide reagent and proofed to be
quite stable against hydration and oxidation during formulation and storage.Further-
more, enzymatic stability was also investigated in 80 % human plasma and 20 %
rabbit brain homogenate. Both oxidation and hydrolysis were found to take place;
however, hydrolysis was the major route.In vivo distribution of the ethyl ester deriva-
tive 7 b was studied in rats and showed that the concentration of the quaternary
product is increasing in the brain and cleared from blood with time.
Keywords:Brain-specific delivery;Dihydroisoquinoline;In vitro Stability;In vivo Dis-
tribution
Received: January 7, 2003; Accepted: March 19, 2003 [FP764]
DOI 10.1002/ardp.200300764
cule depend on predictable sequential enzymatic and/or
chemical transformations of the prodrug after adminis-
tration. This leads to preferential delivery to the brain;
smaller doses can be used, more prolonged effects
reached and peripheral activity avoided.
Introduction
The brain is a delicate organ which has efficient ways to
protect itself against invasive chemicals.The blood brain
barrier (BBB) is an unique membranous barrier that
tightly segregates the brain from the circulating blood.
Therefore, only lipid-soluble solutes can freely diffuse
through the endothelium of the brain capillary. However,
delivery of poorly lipid-soluble compounds, such as pep-
tides and ionized drugs, to the brain, requires some al-
ternative ways of getting past the BBB [1].There are sev-
eral possible strategies, such as transient osmotic open-
ing of the BBB [2], exploiting natural chemical transport-
ers [3], high-dose chemotherapy, or even biodegradable
implants [4, 5]. But these methods have major limita-
tions: they may be invasive, have toxic side effects, low
efficiency, and are not sufficiently safe.
The most commonly used approach involves the use of
the dihydropyridine moiety as the carrier of the drug to
the brain. Bodor et al. developed a novel redox chemical
delivery system (dihydropyridine ↔ pyridinum salt)
which delivers drugs specifically to the brain [8].This sys-
tem was applied successfully for the delivery of many es-
sential drugs to the brain [9]. However, two major prob-
lems hindered their pharmaceutical application, short
shelf life stability due to hydration of the 5,6-double bond
and oxidation and isomerization during reduction of the
quaternary salt [10].1,2-Dihdroisoquinoline was tried as
alternative carrier to overcome those problems and re-
vealed sufficient stability in formulation and enhanced
stability toward oxidation to their corresponding quater-
naries compared with dihydropyridine CDS [11].
Chemical delivery systems (CDSs) represent novel and
systematic ways of targeting biologically active mole-
cules to specific target sites or organs where they exert
their action.They depend on a certain modified prodrug
approach, called retrometabolic drug design [6, 7]. The
site-specific or site-enhanced CDSs of the active mole-
In order to increase the efficiency of brain specific deliv-
ery of the 1,2-dihydroisoquinoline CDSs, more chemical
modifications are needed to optimize the new carrier
system.The modifications are mainly aiming at increas-
ing the rate of oxidation through the use of N-carboxy-
methyl derivatives (I) instead of N-methyl derivatives (Fig-
Correspondence: Mahmoud
Sheha,
Med. Chem.
Department, Faculty of Pharmacy, Assiut University, Assiut
71526, Egypt. Phone: +20 88 314724, Fax: +20 88 332776,
e-mail: Sheha@acc.aun.edu.eg, Sheha@mailcity.com
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0365-6233/04–05/0258 $ 17.50+.50/0

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 258–263
New Carriers for Brain-specific Delivery II 259
of ethyl chloroformate and triethylamine resulted in iso-
quinoline-4-(β-phenthyl)amide (5). Quaternization of 5
with bromoacetic acid or its ester in acetone yielded the
quaternary derivatives (6 a–c).The structures of the pre-
1
pared quaternaries (6 a–c) were confirmed by their H
NMR, IR, and elemental analyses.
Different reported methods are used for the preparation
of 1,2-dihydropyridine CDSs from their corresponding
quaternaries e.g., reduction with sodium borohydride
[12], lithium aluminium hydride [13], sodium dithionite in
alkaline medium [14], and 1-benzyl 1,2-dihydroisonico-
tinamide [15]. The quaternary compounds (6 a–c) were
reduced to their corresponding dihydro derivatives
(7 a–c) either by using sodium dithionite or 1-benzyl-4-
carbamoyl-1,2-dihydropyridine.
Figure 1. Structure of N-carboxymethyl derivatives.
ure 1).The drug was conjugated through ester or amide
bond to the 4-carboxy group of the carrier system. The
prepared derivatives were investigated for their chemical
stability in buffers, plasma, and brain homogenate and in
an in-vivo distribution study.
In vitro stability studies
Results and discussion
Chemistry
Chemical stability of 1,2-dihydroisoquinoline-4-dicarb-
oxylic acid derivatives 7 a–c were investigated using oxi-
dizing agent (ferricyanide reagent), aqueous phosphate
buffer solutions (pH 5.8 and 7.4), plasma (80 % in isoton-
ic phosphate buffer pH 7.4), and brain homogenate
(20 % in saline). To carry out these investigations, re-
verse phase HPLC method of analysis of these com-
pounds and their metabolites in solutions, plasma and
brain homogenate was developed, and validated before
use.
The designed isoquinoline CDSs 7 a–c, were synthe-
sized through multi-step pathways as illustrated by
Scheme 1, starting with isoquinoline (1), which was sub-
jected to bromination to give 4-bromoisoquinoline (2).
Fusion of 2 with cuprous cyanide at 250 °C afforded the
cyano derivative (3), which was hydrolyzed to give iso-
quinoline-4-carboxylic acid (4). Reaction of 4 with
β-phenethylamine, used as model drug, in the presence
Scheme 1. Synthesis of dihydroisoquinoline chemical delivery system 7 a–c.
Derivatives: a, R = H; b, R = C2H5; c, R = iso-C3H7
Reagents:i, bromine at 180 °C in nitrobenzene for 5 h;ii, cuprous cyanide fusion at 250 °C for 45 min;iii, reflux with 20 %
sodium hydroxide; iv, glacial acetic acid; v, ethyl chloroformate and triethylamine in chloroform; vi, phenethylamine; vii,
bromoacetic acid (ester) and acetone reflux overnight; viii, sodium dithionite, sodium carbonate in ethylacetate-water
mixture, or 1-benzyl-1,2-dihydro-isonicotinamide in methanol.

260 Mahmoud et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 258–263
The ester group stabilizes the dihydroisoquinoline deriv-
atives against prompt oxidation.Furthermore, the amidic
linkage at position 4 seemed to be quite stable at the in-
vestigated conditions, that means reasonable concen-
tration of the drug will be released from the carrier after
being locked in the brain.
Chemical oxidation study
The kinetics of the oxidation of dihydroisoquinoline de-
rivatives with 0.01 M ferricyanide reagent was found to
obey pseudo first order reaction and their oxidation data
are given in Table 1. 1,2-Dihydroisoquinoline-N-acetic
acid esters 7 b, c were found to be quite stable towards
oxidation with ferricyanide reagent compared with the
corresponding acid 7 a.Oxidation rate of 7 a was too fast
to be monitored. Furthermore, isopropyl ester 7 c is al-
most 18 times more stable against oxidation than the
ethyl ester 7 b.This observation indicates the occurrence
of hydrolysis prior to oxidation.
In vitro enzymatic studies
In order to estimate the stability of the tested carrier sys-
tem in biological media, the tested compounds were in-
vestigated in 80 % human plasma and 20 % rabbit brain
homogenate at 37 °C. The reaction of the tested com-
pounds with the investigated enzymes was found to
obey pseudo first-order kinetics and the results are given
in Table 3. The obtained data revealed that the tested
compounds have greater susceptibility toward the plas-
ma enzymes compared to that observed in the tested
buffer solutions.The ethyl ester is about 3 times more la-
bile in plasma than the isopropyl one. Quaternary acid
6 a (major) and quaternary esters (minor) 6 b and 6 c
were detected as reaction products, which indicate oc-
currence of both hydrolysis and oxidation at the same
time.
Table 1. The kinetics of oxidation of compounds 7 a–c
with ferricyanide reagents at room temperature.
Compound
Kobs (min–1)
T1/2 min
r
7 a
7 b
7 c
Too fast to be monitored
(2.112 0.143) × 10–2
(1.518 0.002) × 10–3
–
–
32.53 0.99
546.46 0.98
Table 3. The stability study of the dihydroisoquinoline
CDSs 7 b and 7 c in 80 % human plasma and 20 % rabbit
brain homogenate.
Stability studies in aqueous buffer solutions
Stability of the prepared dihydroisoquinoline CDS was
investigated in aqueous buffer solutions of pH 7.4 (physi-
ological pH) and 5.8. the rates of disappearance of the
dihydroisoquinoline esters were determined as first-or-
der kinetics (Kdisapp) at the investigation conditions as
shown in Table 2. It is clear from the obtained data that
these compounds are more stable in the investigated
acidic buffer solution compared to the physiological envi-
ronment. Furthermore, the ethyl ester 7 b is more labile
for chemical hydrolysis in the tested buffer solutions
compared to the corresponding isopropyl one 7 c. The
main hydrolytic product of the tested compounds was
the quaternary acid 6 a.This is another confirmation for
the prompted oxidation of the product of hydrolysis.
Enzymatic com-
(Kdisapp. ± SD)
× 10–3 min–1
t hr
½
r
system
pound
80 %
Human
plasma
7 b
7 c
5.62 0.351
1.87 0.002
2.05 0.99
6.15 0.96
20 %
Rabbit
7 b
7 c
1.799 0.020
0.665 0.015
6.40 0.99
17.35 0.98
brain
homogenate
As observed with human plasma, isopropyl ester 7 c is
more stable toward brain homogenate enzymes than the
ethyl ester 7 b with half-life times of 17.3 and 6.4 h, re-
spectively. However, both compounds are less labile in
rabbit brain homogenate compared with plasma.In spite
of the occurrence of both oxidation and hydrolysis pro-
cess, the hydrolytic pathway was the major route. Both
processes will result in formation of the quaternary forms
which will be locked-in the brain followed by the release
of the target drug in the brain.The results were consist-
ent with the expected results calculated by the molecular
orbital calculation (S. Mahmoud, unpublished results).
Table 2. The stability studies of the dihydroisoquinoline
CDSs 7 b and 7 c at different pH values.
pH
Com-
pound
Kdisapp ± SD
[10–4 min–1]
t
½
(hr)
r
5.8
7.4
7 b
7 c
7.50 0.277
3.42 0.532
15.4
33.7
0.99
0.98
7 b
7 c
11.28 0.005
6.38 0.242
10.2
18.0
0.99
0.99

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 258–263
New Carriers for Brain-specific Delivery II 261
In vivo distribution
Experimental
The dihydroisoquinoline derivative 7 b was selected for
the in vivo study due to its reasonable stability in the in
vitro 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 points (15, 30,
60, 180, and 300 min); their blood and brains were ana-
lyzed for the quaternary precursors 6 a and 6 b. Results
of blood and brain distribution are presented in Figure 2.
Concentrations of the tested compound in blood and
brain were reversibly proportional to each other with time
progress.The results showed that the quaternary acid of
the tested CDS was detected at higher concentrations in
blood than in brain within the first hour.The concentration
of the quaternary product was below detection limit in
blood after 5 h, while it increases in the brain.The pres-
ence of the quaternary acid as the sole product in brain
proves the main idea of the new CDS. Dihydroisoquino-
line CDS 7 b penetrates the BBB and rapidly hydrolyzed
by brain esterase to the corresponding acid, present as
anion at pH 7.4. Oxidation of the resultant anion by brain
dehydrogenases to the corresponding quaternary is ac-
celerated due to the negative charge on the carboxylate
anion [S. Mahmoud, unpublished results]. Accordingly,
the results prove the following sequential processes af-
ter brain penetration, the D-CDS is sequentially; (i) hy-
drolyzed to give the dihydro acid anion (ii) oxidized to the
quaternary acid anion “the locked-in moiety”, that (iii) hy-
drolyzed by time progress to release the parent drug.
General
Melting points were determined on Electrothermal Melting
Point Apparatus (Stuart Scientific, Staffordshire, UK) and are
uncorrected. Column chromatography is done by silica gel for
column. Reactions are monitored by TLC using DC Alufolien,
Kieselgel 60 F 254 percolated plates (E. Merck, Darmstadt,
Germany) and visualized by UV. IR spectra were recorded as
KBr disk on a Shimadzu IR 200-91527 Spectrophotometer
(Shimadzu Corp., Kyoto, Japan). 1H-NMR spectra were run on
Varian Em-360L NMR Spectrophotometer (60 MHZ) Varian
(Varian, Palo Alto, CA, USA). Chemical shifts are expressed in
ppm relative to tetramethylsilane. Elemental microanalyses
were performed on Elementaranalysen Systeme GmbH
VarioEL (Hanau, Germany).
HPLC system consisted of a Knauer model 64, solvent delivery
module (Knauer, Berlin, Germany), Knauer variable wave-
length UV detector, 20 µL sample loop, and a Shimadzu CR-6A
chromatopac integrator (Shimadzu,Tokyo, Japan).The column
used was Knauer C18 (250 mm × 4.6 mm ID, 5 µm).The efflu-
ent monitored at 254 nm at flow rate of 1 mL/min. The mobile
phase used consisted of 75 volumes of methanol and 25 vol-
umes of water, containing 1 % hexanesulfonic acid sodium salt.
Methanol, hexanesulfonic acid sodium salt, and water used in
chromatography were of HPLC grade. All other chemicals and
solvents are of analytical grade and were used without further
purification.
Chemistry
4-( N-β-phenetheylcarbamoyl )isoquinoline (5)
Ethyl chloroformate (1.1 mL, 0.014 mol) was added dropwise to
an ice-cooled stirred suspension of isoquinoline-4-carboxylic
acid (1.7 g, 0.01 mol) and triethylamine (2 mL, 0.014 mol) in dry
CHCl3 (20 mL). Stirring was continued for 30 min at 5–10 °C,
then β-phenethylamine (1.3 mL, 0.01 mol) was added together
with an equivalent amount of triethylamine (1.4 mL, 0.01 mol).
The mixture was stirred for further 5 hr under anhydrous condi-
tions at ambient temperature. The solvent was evaporated to
give an oily residue purified by column chromatography eluted
with CHCl3 : CH3OH (40:1) mixture. The appropriate fraction
evaporated to afford 1.96 g (71 %) of 5 with m.p 96–99 °C. 1H-
NMR (CDCl3) 2.9–3.1 (t, 2 H), 3.6–4 (q, 2 H), 6.3–6.6 (broad,
1 H), 7.2–7.3 (s, 5 H), 7.4–8.3 (m, 4 H), 8.4 (s, 1 H, C3), 9.0 (s,
1 H); elemental analysis for C18H16N2O.1/4 H2O, Calcd: C,
76.98; H, 6.23; N, 9.97; Found: C, 77.10; H, 6.27; N, 9.8.
Preparation of 2-alkoxycarbonylmethyl-4-(phenethylcarbamo-
yl)-isoquinolinium bromide (6 a–c)
To a solution of compound 5 (3.3 g, 0.012 mol) in 30 mL of ace-
tone, 0.02 mol of bromoacetic acid (ester) was added. The re-
action mixture was refluxed overnight and then cooled to room
temperature. The formed crystals were filtered, washed with
acetone, dried to give the corresponding isoquinolinium bro-
mide derivative. The following compounds were prepared by
this procedure.
2-(Carboxymethyl)-4-(phenethylcarbamoyl)isoquinolinium bro-
mide (6 a)
Yield: 3.7 g (75 %) of pale rose powder; m.p. 205–207 °C. 1H-
NMR (DMSO-d6) 3.0–3.4 (t, 2 H), 3.8–4.2 (m, 2 H), 6 (s, 2 H),
7.5–7.8 (m, 5 H), 8.2–9 (m, 5 H), 9.1–9.2 (s, 1 H), 10.5 (s, 1 H).
Elemental analysis, C20H19BrN2O3; Calcd: C, 57.84; H, 4.61; N,
6.75. Found: C, 57.39; H, 4.68; N, 6.72.
Figure 2.In vivo distribution of dihydroisoquinoline-CDS
(7 b) in blood and brain homogenate of rats.

262 Mahmoud et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 258–263
2-(Ethoxycarbonylmethyl)-4-(N-phenythylcarbamoyl)isoquino-
Chemical oxidation and in vitro studies
linum bromide (6 b)
Chemical oxidation with ferricyanide reagent
Yield: 3.9 g (75 %) of brown semisolid material. 1H-NMR
(DMSO-d6) 1.2–1.5 (t, 3 H), 2.8–3.3 (m, 2 H), 3.6–3.9 (m, 2 H),
4.1–4.5 (m, 2 H), 6 (s 2 H), 7.5–7.6 (m, 5 H), 8–8.9 (m, 4 H),
9–9.1 (s, 1 H), 10.2–10.3 (s, 1 H). Elemental analysis,
C22H23BrN2O3.1/2 H2O; Calcd: C, 58.41; H, 5.347; N, 6.1929.
Found: C, 57.93; H, 5.821; N, 6.332.
RLD1To a series of tubes, each containing 4 mL of freshly pre-
pared ferricyanide reagent [16], 1 mL of the freshly prepared
1,2-dihydroisoquinoline CDS (7 a–c) in concentration of (2 ×
10–4 M) was added and mixed at room temperature. At suitable
time intervals (0, 0.5, 1, 2, 3, 5, 8, 12, and 24 h), 20 µl of the so-
lution were withdrawn, filtered, and analyzed by HPLC. The
rates of disappearance of the dihydroisoquinoline derivative
and the appearance of the corresponding quaternaries were
determined. The apparent pseudo first-order rate constants of
oxidation (Kdisapp min–1) of the tested compounds 7 a–c were
calculated as the average of 5 experiments.
2-(Isopropyloxycarbonylmethyl)-4-(N-phenethylcarbamoyl)iso-
quinolinum bromide (6 c)
Yield: 3.5 g (63 %) of yellow oily product. 1H-NMR (CDCl3) 1.1–
1.5 (d, 6 H), 3–4.1 (m, 4 H), 5–5.5 (m, 1 H), 6–6.3 (s, 2 H), 7.3–
7.8 (m, 5 H), 7.9–9.3 (m, 6 H) 10.5 (s, 1 H).
Stability of the prepared 1,2-dihydroisoquinoline CDS (7 b and
7 c) in buffer systems
Preparation of 2-alkoxycarbonylmethyl-4-(β-phenethylcarb-
amoyl)-1,2-dihydroisoquinoline (7 a–c)
In screw caped tubes, each tube contains 5 mL of phosphate
buffers (pH 5.8 or 7.4), 0.5 mL of freshly prepared solution (2 ×
10–4 M) of dihydroisoquinoline derivative 7b or 7c was added.
The mixtures were kept stirring in dark at room temperature all
the time of experiment (5 d). At appropriate time intervals (0, 2,
12, 24, 48, 72, and 96 h), 20 ìl of the mixture was withdrawn and
analyzed by HPLC for the dihydroisoquinoline derivative (7b or
7c) and corresponding hydrolytic products. The apparent pseu-
do first-order rate constants of disappearance of 7b and 7c
(kdisapp., min–1) were calculated as the average of 3 experiments.
Method A
To a stirred solution of isoquinolinium bromide (0.4 mmol) in an-
hydrous methanol (40 mL), 1-benzyl-1,2-dihydroisonicotin-
amide (0.1 g; 0.5 mmol) was added. The solution was conti-
nously stirred for 4 h at 0 °C under nitrogen.The separated solid
was filtered and washed with methanol/ether mixture. The fil-
trate was evaporated under stream of nitrogen to give semisolid
yellow residue which was identified as the 1,2-dihydroisoquino-
line derivatives.
In vitro stability in biological fluids
Stability in 80 % human plasma
Method B
To a solution of compound 6 a–b (0.1 mmol) in 50 mL of deaer-
ated water, sodium bicarbonate (1 g; 0.012 mol) and 50 mL of
dichloromethane (DCM) 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, stirring was continued for
3 h under nitrogen stream. The DCM layer was separated,
washed with water, dried over anhydrous Na2SO4, and evapo-
rated under vacuum, to give the corresponding dihydroisoquin-
oline derivative. The following compounds were prepared by
the above-mentioned procedures:
To 5 mL of freshly collected plasma (diluted with isotonic buffer
pH 7.4 to 80 %), pre-warmed in a water bath at 37 1 °C for
5 min, 300 µl of 1.2 × 10–4 M methanolic solution of freshly pre-
pared dihydro compounds (7 b or 7 c) were added and mixed
thoroughly. At appropriate time intervals (0.25, 0.5, 1, 1.5, 2, 3,
6, 10, and 24 h), 200 µl were withdrawn, mixed immediately
with 2 mL of ice-cold methanol, centrifuged, and the superna-
tants were filtered through nylon membrane (0.22 µm pore
size) and analyzed by HPLC to determine the amount of the di-
hydroisoquinoline derivatives 7 b or 7 c and their oxidation or
degradation products. The apparent pseudo first order rate
constants (kdisapp min–1) of compounds 7 b and 7 c were calcu-
lated as the average of 3 experiments.
2-(Ethoxycarbonyl methyl)-4-(N-phenethylcarbamoyl)-1,2-dihy-
droisoquinoline (7 b)
Compound 6 b (0.239 g) was used in preparation of 7b by
method A to yield 0.29 g (78 %) of pale buff crystalline solid with
m.p.147–150 °C. 1H-NMR (CDCl3) 1.2–1.5 (t, 3 H), 2.9–3.2 (m,
2 H), 3.6–3.9 (m, 4 H), 4.3–4.7 (m, 4 H), 5.8–6.1 (broad, 1 H),
7.1–7.8 (m, 10 H). Elemental analysis, C22H24N2O3.1/4 H2O;
Calcd:C, 71.62;H, 6.69;N, 7.592.Found:C, 71.85;H, 7.011;N,
7.598.
Stability in rabbit brain homogenate
About 300 µl of 2.4 × 10–4 M methanolic solution of freshly pre-
pared dihydro derivative (7 b or 7 c) were mixed with 10 mL of
rabbit brain homogenate (20 % in saline), previously equilibrat-
ed at 37 1 °C. Samples of 200 ml were withdrawn from the
tested mixture at different time intervals (0.25, 0.5, 1, 2, 4, 6, 10,
and 16 h), mixed with 2 mL ice-cold methanol and centrifuged
at 4000 rpm for 10 min. 20 µl of the clear supernatant was ana-
lyzed by HPLC to determine the amount of the dihydroisoquin-
oline derivative and the corresponding oxidation or degradation
products. The apparent pseudo first order rate constants
(Kdisapp min–1) of compounds 7 b and 7 c were determined as
the average of 3 experiments.
2-(Carboxymethyl)-4-(N-phenethylcarbamoyl)1,2-dihydroiso-
quinoline (7 a)
Compound 6 a (0.415 g, 0.1 mmol) was used in preparation of
7 a by method B to yield 0.225 g (65 %) of white crystals with
m.p. 136–138 °C. H-NMR (CDCl3) 3.2–3.6 (m, 4 H), 3.7 (s,
1
2 H), 4.3–4.7 (m, 4 H), 5.8–6.1 (s, 1 H), 7.1–7.8 (m, 9 H).
2-(Isopropyloxy carbonylmethyl)-4-(N-phenethylcarbamoyl)1,2-
isoquinoline (7 c)
Stability in whole human blood
Mix 3 mL of freshly collected heparinized human blood sample
at 37 °C with 100 µl of freshly prepared methanolic solution of
dihydroisoquinoline derivative 7 c (0.1 mmol). At appropriate
time intervals (0, 2, 5, 15, 30, 60, and 120 min), samples of
(200 µl) were withdrawn, mixed with ice-cooled acetonitrile
(0.2 mL) and centrifuged at 4000 rpm for 10 min.The clear su-
pernatant was analyzed by HPLC to determine the amount of
7 c and the corresponding oxidation or degradation products.
Compound 6 c (0.457 g, 0.1 mmol) was used in preparation of
7 c by method A to yield 0.28 g (72 %) of faint yellow crystals
with m.p 151–153 °C. 1H-NMR (CDCl3) 1.2–1.5 (d, 6 H), 2.8–
3.1 (t, 2 H), 3.4–3.9 (t, 2 H), 4 (s, 2 H), 4.4–4.5 (s, 2 H), 5–5.3 (m,
1 H), 7.1–7.9 (m, 13 H). Elemental analysis, C23H26N2O3.1/4
H2O; Calcd: C, 72.13; H, 6.974; N, 7.314. Found: C, 72.18; H,
7.032; N, 7.423.

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 258–263
New Carriers for Brain-specific Delivery II 263
The apparent pseudo first order rate constant of hydrolysis
(Kdisapp min–1) of compound 7c was calculated as the average of
3 experiments.
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Ther. Drug Carrier Syst., 1991, 8, 1–17.
In vivo distribution study
Five groups, each of four Sprague Dawley male rats of average
weight of 60–65 g were anesthetized with Urethane. A freshly
prepared solution (25 mg/mL) of compound 7 b in DMSO (dilut-
ed to 40 % with water) was injected through jugular vein at a
dose of 20 mg/Kg. At time intervals (15, 30, 60, 180, and
300 min), blood samples were withdrawn from the eyeball, im-
mediately added to previously weighted centrifuge tubes con-
taining acetonitrile (4 mL) and weighed to determine the
amount of blood added. Blood samples were centrifuged at
4000 rpm for 10 min and the supernatant analyzed by HPLC.
The animals were decapitated and the brains were taken,
weighed and immediately homogenized with 1 mL saline and
diluted with 4 mL of 5 % DMSO in acetonitrile, homogenized
again and centrifuged at 4000 rpm for 10 min. The superna-
tants were analyzed by HPLC.The apparent pseudo first order
rate constants (Kdisapp min–1) of 7 b and 7 c were calculated as
average of 4 experiments.
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