Brain delivery of HIV protease inhibitors

Article abstract of DOI:10.1002/ardp.200390003

To overcome the problems of peptidomimetic drug delivery to the specific organs, the use of dihydropyridine ? pyridinium chemical delivery systems to deliver peptides to the brain is considered in this work. An HIV protease inhibitor lead compound; KNI 279 was selected for the study. The N-alkylated dihydroisoquinoline derivatives of KNI-279 were synthesized and tested for their ability to be oxidized by brain homogenate and showed good results with reasonable half-life times specially for the N-alkoxycarbonyl-methyl derivative 8. The in-vivo distribution of compound 8 proved the brain delivery and locked in property of HIV PR inhibitors in the brain. All the prepared compounds (both quaternary and dihydro derivatives) showed between 51 and 86% HIV PR inhibitory activity compared to the parent compound.

Full text of DOI:10.1002/ardp.200390003

Arch. Pharm. Pharm. Med. Chem. 2003, 1, 47–52
HIV Protease Inhibitors 47
Mahmoud M. Sheha,
Nawal A. El-Koussi,
Hassan H. Farag
Brain Delivery of HIV Protease Inhibitors
To overcome the problems of peptidomimetic drug delivery to the specific organs,
the use of dihydropyridine ↔ pyridinium chemical delivery systems to deliver pep-
tides to the brain is considered in this work. An HIV protease inhibitor lead com-
pound; KNI 279 was selected for the study.The N-alkylated dihydroisoquinoline de-
rivatives of KNI-279 were synthesized and tested for their ability to be oxidized by
brain homogenate and showed good results with reasonable half-life times specially
for the N-alkoxycarbonyl-methyl derivative 8.The in-vivo distribution of compound 8
proved the brain delivery and locked in property of HIV PR inhibitors in the brain. All
the prepared compounds (both quaternary and dihydro derivatives) showed be-
tween 51 and 86 % HIV PR inhibitory activity compared to the parent compound.
Medicinal Pharmaceutical
Chemistry Department,
Faculty of Pharmacy,
Assiut University,
Assiut, Egypt
Keywords: brain-specific delivery; HIV PR inhibitors; KNI; Dihydroisoquinoline
Received: June 5, 2002 [FP706]
DHP-CDS, which retarded its development by the drug
industry, is its instability during formulation and storage
Introduction
Human immunodeficiency virus (HIV), a member of the
greater family of retroviruses, is the causative agent of
the debilitating and fatal acquired immune deficiency
syndrome (AIDS). During viral replication, the viral gag
and gag/pol polyproteins undergo enzymatic cleavage
by a viral encoded protease (PR) to generate the func-
tional proteins of the mature virus. Inhibition of HIV PR
results in the loss of proteolysis activity as well as pro-
duction of non-infectious virons.Due to this critical role in
viral maturation, HIV PR has become a prevalent target
in AIDS therapy and the design of new PR inhibitors has
escalated.As a consequence, a large number of specific
HIV PR inhibitors with various chemical structures have
been reported [1].The most effective inhibitors are pepti-
domimetic compounds containing transition-state in-
serts in place of the amino acids occupying the P1P1Ј
position of the substrate peptide. However, the use of
peptides as drugs is limited due to the poor brain bio-
availability of the peptide derived drugs [2, 3].
due to the hydration of the 5–6 double bond [5]. Certain
1,2-dihydroisoquinoline-CDS have been studied to over-
come this problem and proven to be efficient in brain-
specific delivery and shelf stable [6, 7]. Isoquinoline is
present as a structural element of many HIV PR inhibi-
tors [8]. Kynostatin compounds (KNI), potent HIV PR in-
hibitors, were selected as a model for a brain delivery
study. In this case the active compound will be the di-
hydroisoquinoline and the quaternary isoquinolinium
derivatives and no problem will be associated with the
rate of release of the active drug from the carrier (the car-
rier is part of the active compound).KNI 279 (1) contains
5-isoquinolineoxyacetic acid at the P3 position. Deriva-
tives of 1 were synthesized, and evaluated for brain de-
livery and HIV PR inhibitory activity.
The dihydropyridine ↔ pyridinium chemical delivery
system (CDS) has been used successfully for brain-spe-
cific delivery of many drugs since the first publication by
Bodor’s group [4].The drug carried on the dihydropyrid-
ine chemical delivery system (D-DHP) was shown to
cross the blood-brain barrier (BBB) easily.Some of these
dihydropyridines are oxidized in plasma but the major
part crosses the BBB and is oxidized by oxidizing en-
zymes in the brain to the corresponding quaternary form
(pyridinium) which is then retained (locked) in the brain
and can release the drug. The main disadvantage of
Investigations, results, and discussion
Synthesis of the dihydroisoquinoline
derivative of KNI-279
Correspondence: Mahmoud
Pharmaceutical Chemistry Department, Faculty of Pharmacy,
Assiut University, Assiut 71526, Egypt. Phone:
M. Sheha,
Medicinal
KNI-279 (1) was prepared by a solution method for pep-
tide synthesis using Boc-derivatives of thioproline (Thz),
allophenylnorstatine (Apns), and valine (Val) and the ter-
+20 12397 1632, fax: +20 8833 2776, e-mail:
Sheha@acc.aun.edu.eg.
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0365-6233/01/0047 $ 17.50+.50/0

48 Sheha et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 47–52
minal isoquinolineoxyacetic acid (iQoa)[9]. Quaterniza-
tion of the isoquinoline residue of 1 was performed by
treating 1 with alkyl halide (methyl iodide, ethyl bromide,
ethyl bromoacetate, or bromoacetic acid) at ambient
temperature, and afforded the derivatives 2–5 as shown
in Scheme1. Compound 5 was found as bromide salt,
not as zwitterion (positive test for bromide ion after wash-
ing the crystalline product with mixture of anhydrous di-
ethyl ether and methanol). Reduction of the quaternary
isoquinolinium salts 2–4 with sodium dithionite in sodium
bicarbonate under a stream of nitrogen afforded the di-
hydroisoquinoline derivatives 6–8.
Table 1. Kinetics of oxidation of dihydroisoquinoline de-
rivatives of KNI-279 (6–8) with silver nitrate.
Compound
kdisapp. [h–1]
t1/2 [h]
R
6
7
8
6.31 × 10–2
4.75 × 10–2
18.21 × 10–2
11
14.5
3.8
0.986
0.991
0.990
kdisapp is the apparent pseudo first-order rate constant of
disappearance of dihydroisoquinoline derivatives, t1/2 is
the half-time of the reaction, R is and regression coeffi-
cient.
In vitro studies
Chemical oxidation
Stability and oxidation in biological fluids
Silver nitrate and/or potassium ferricyanide were used
as oxidizing agent for studying the kinetics of oxidation of
dihydropyridine derivatives [10].The oxidation of the pre-
pared dihydro compounds 6–8 with silver nitrate solution
was studied under pseudo first-order conditions. The
rate constants of oxidation were determined by monitor-
ing the disappearance of the dihydroisoquinoline deriva-
tives, using HPLC, in alcoholic silver nitrate solution.The
results (Table 1) reflect the susceptibility of the dihydro-
isoquinoline derivatives for oxidation to the correspond-
ing quaternary isoquinolinium derivatives, which is es-
sential for the brain-specific delivery process. Com-
pound 8 is shown to undergo the fastest in oxidation
(t1/2 = 3.8 h), whereas compound 7 ungoes the slowest
(t1/2 = 14.5 h).
The rates of oxidation of the prepared dihydroisoquino-
line derivatives 6–8 were investigated in 20 % rat brain
homogenate (Table 2) and in fresh human plasma (Table
3). The rates of oxidation were found to follow pseudo
first-order kinetics with correlation coefficient higher
than 0.98.Compound 8 proved to undergo the fastest ox-
idation in 20 % brain homogenate (t1/2 = 1.5 h) which is
even faster than the rate of chemical oxidation by silver
nitrate. The corresponding acid 5 was found to be oxi-
dized in rat brain even faster than the ester 8, to an extent
that could not be monitored. Compound 7 was the slow-
est to be oxidized in brain homogenate yet also at a fast-
er rate than by chemical oxidation.These fast rates of en-
zymatic oxidation in brain homogenate satisfy the re-
quirements for efficient brain-specific delivery.
Scheme 1. Synthesis of the dihydroisoquinoline derivatives of KNI-279.

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 47–52
HIV Protease Inhibitors 49
Table 2. Kinetics of stability of dihydroisoquinoline deriv-
atives of KNI-279 (6–8) in rat brain homogenate.
Table 4. In vivo distribution (blood, and brain) of com-
pound 8 in rats (n = 3).
Compound
kdisapp. [h–1]
t1/2 [h]
R
Time Intervals (minutes) Blood*
Brain*
6
7
8
14.46 × 10–2
9.03 × 10–2
45.92 × 10–2
4.8
7.7
1.5
0.99
0.98
0.98
10
20
60
120
240
300
25.5 6.2
NA
18.17 2.4
12.69 3.3
6.6 1.2
NQ
2.23 1.5
6.05 2.7
10.6 0.9
9.8 1.8
7.62 2.1
kdisapp is the apparent pseudo first-order rate constant of
disappearance of dihydroisoquinoline derivatives, t1/2 is
the half-time of the reaction, R is and regression coeffi-
cient.
ND
NA, not available NQ;detected but cannot be quantified;
ND; not detected.
* Total concentrations ( standard deviation) of com-
pound 8 and its quaternary metabolite (compound 5,
without bromide) calculated in µg/g of rat brain or blood.
Table 3. Kinetics of stability of dihydroisoquinoline deriv-
atives of KNI-279 (6–8) in fresh human plasma.
Compound
kdisapp. [h–1]
t1/2 [h]
R
6
7
8
4.74 × 10–2
3.71 × 10–2
15.82 × 10–2
14.6
18.7
4.4
0.99
0.99
0.98
In vivo brain bioavailability
To validate the brain-specific delivery of the prepared di-
hydroisoquinoline derivatives, a brain delivery study for
the most in-vitro promising compound 8 was performed
on rats. A freshly prepared solution of compound 8 was
given by intra-jugular injection in a dose of 20 mg/kg
body weight of female Sprague Dawley rats. At specific
time intervals the animals were killed, and blood sam-
ples and brains were collected. The samples were ana-
lyzed by HPLC to determine the total quantity of the dihy-
droisoquinoline derivative 8, and its metabolites quater-
nary derivatives 4 and 5. The results of these experi-
ments are given in Table 4. Analysis of blood samples
taken after 10 minutes proved the presence of com-
pound 8 in high concentration in addition to its hydrolysis
and oxidation products;compound 5 (Figure 1).At 1 and
2 hours only the quaternary acid 5 could be detected.
Neither quaternary acid nor other oxidation or hydrolysis
products could be quantified after 4 and 5 hours.
kdisapp is the apparent pseudo first-order rate constant of
disappearance of dihydroisoquinoline derivatives, t1/2 is
the half-time of the reaction, R is and regression coeffi-
cient.
In human plasma, the dihydroisoquinoline derivatives
6–8 proved to be more stable than in the brain homo-
genate used in our exiperiment. They follow the same
trend as observed in the chemical oxidation and in the
brain homogenate, with compound 8 being the least
stable and compound 7 the more stable derivative.
The picture looked different in the brain samples. The
products detected after 20 minutes and 1hour are mainly
the quaternary acid 5 and some of dihydroisoquinoline
ester 8. After 2 hours only the quaternary acid could be
found. The concentration of HIV PR inhibitor was found
to increase with time (for about 4 hours) and to be main-
tained for more than 5 hours.
Figure 1. A representative HPLC chromatogram show-
ing the presence of both the quaternary isoquinolinium
derivative 5 (Rt = 4.46 min) and the dihydroisoquinoline
derivative 8 (Rt = 10.57 min) in blood after 20 minutes
from injection of compound 8 into rats.
HIV PR inhibitory activity
The prepared compounds 2–8 were evaluated for their
HIV PR inhibitory activity against a synthetic enzyme us-
ing a reported technique [11]. The activities were com-

50 Sheha et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 47–52
pared to that of the parent compound 1.Both the dihydro-
isoquinoline derivatives 6–8 and the quaternary isoqui-
nolinium derivatives 2–5 were found to be active as HIV
PR inhibitor at a concentration on the 50 ng scale. The
activities were found to be reduced (about 51–86 %)
compared to that of the parent compound 1, Table 5.The
more lipophilic compounds 6–8 were more active as HIV
PR inhibitors than the corresponding hydrophilic quater-
nary compounds 2–5.
Chemistry
Quaternization of KNI-279 with alkyl halides, general method
To a solution of KNI-279 (3.25 g, 5 mmol) in methanol (50 mL),
alkyl halide (as indicated, 6 mmol) solution in methanol (20 mL)
was added dropwise with stirring. The reaction mixture was
stirred at about 50 °C for 2–5 hours, and concentrated under
vacuum to one third of its volume. After cooling, the crystalline
product was filtered and washed with anhydrous diethyl ether
(3 × 50 mL) and dried in a desiccator over phosphorus pent-
oxide under vacuum.By the use of this procedure, the following
compounds were prepared.
Acknowledgment
3-{3(S)-[N-(N-Methyl-5-isoquinolinyloxyacetyl)-l-valinyl]-
amino-2(S)-hydroxy-4-phenylbutanoyl}-N-(tert-butyl)thiazoli-
dine-4(R)-carboxamide iodide (2)
The authors acknowledge professor Yoshiaki Kiso and
collaborators at Kyoto Pharmaceutical University, Ja-
pan, for conducting HIV PR inhibitory activity studies.
Methyl iodide (0.85 g), stirred for 2 hours to give 2 (3.5 g,
1
88.5 %): mp 124–126 °C, H NMR (DMSO-d6) δ 0.76 (d, 6 H),
1.26 (s, 9 H), 1.98 (m, 1 H), 2.67 (d, 2 H), 3.1 (m, 2 H), 4.28 (m,
2 H), 4.4 (s, 3 H), 4.66 (m, 2 H), 4.78 (m, 2 H), 4.92 (m, 3 H),
7.35–7.08 (m, 6 H), 7.53 (m, 1 H), 7.73 (s, 1 H), 7.93 (m, 1 H),
8.1 (m, 1 H), 8.25 (d, 1 H), 8.6 (d, 1 H), 8.69 (m, 1 H), 9.97 (s,
1 H); MS (HR FAB+) m/z 664.3181 (+1.9 ppm) observed for
C35H46N5O6S.
Experimental
Materials
Melting points were recorded (uncorrected) on a melting point
/MS (Stuart Scientific, UK).Infrared spectra were recorded on a
Shimadzu IR-470 Spectrometer (Shimadzu Corporation,
Tokyo, Japan) and UV spectra on Shimadzu UV-120-02 Spec-
trophotometer (Shimadzu Corporation.Kyoto, Japan).Thin lay-
er chromatography (TLC) was performed on precoated silica
gel plates (Merck, 0.25 MM, 60 F254). Column chromatogra-
phy was carried out on Merck silica gel 60 (Particle size 0.063–
0.200 mm). 1H-NMR spectra were recorded on a JEOL
JNM-EX 270 MHz Spectrometer (Jeol,Tokyo, Japan);all chem-
ical shifts are given in δ ppm, relative to tetramethylsilane
(TMS) as internal standard. High resolution FAB MS analyses
were recorded on JEOL JMS-SX 102 AQQ Hybrid Mass Spec-
trometer using thioglycerol as internal reference. All the ob-
tained new compounds gave satisfactory high resolution mass
spectra (error less than 3 ppm) and were fully characterized
spectroscopically.
3-{3(S)-[N-(N-Ethyl-5-isoquinolinyloxyacetyl)-l-valinyl]amino-
2(S)-hydroxy-4-phenylbutanoyl}-N-(tert-butyl)thiazolidine-
4(R)-carboxamide iodide (3)
Ethyl bromide (1.0 g), stirred for 3 hours to give 3 (3.2 g, 80 %):
1
mp 109–112 °C, H NMR (DMSO-d6) δ 0.78 (d, 6 H), 1.26 (s,
9 H), 1.78 (t, 3 H), 2.0 (m, 1 H), 2.67 (d, 2 H), 3.1 (m, 2 H), 4.29
(m, 2 H), 4.2 (q, 2 H), 4.58 (m, 2 H), 4.72 (m, 2 H), 4.92 (m, 3 H),
7.35–7.08 (m, 6 H), 7.5 (m, 1 H), 7.71 (s, 1 H), 7.81 (m, 1 H),
8.05 (m, 1 H), 8.25 (d, 1 H), 8.61 (d, 1 H), 8.66 (m, 1 H), 9.95 (s,
1 H); MS (HR FAB+) m/z 678.3274 (+1.5 ppm) observed for
C36H48N5O6S.
3-{3(S)-[N-(N-Ethoxycarbonylmethyl-5-isoquinolinyl-
oxyacetyl)-l-valinyl]-amino-2(S)-hydroxy-4-phenylbutanoyl}-
N-(tert-butyl)thiazolidine-4(R)-carboxamide bromide (4)
Ethyl bromoacetate (1.0 g), stirred for 5 hours at room tempera-
ture to give 4 (3.4 g, 85 %):mp 89–91 °C, 1H NMR (DMSO-d6) δ
0.76 (d, 6 H), 1.26 (s, 9 H), 1.32 (t, 3 H), 1.98 (m, 1 H), 2.67 (d,
2 H), 3.1 (m, 2 H), 4.25 (m, 4 H), 4.46 (m, 1 H), 4.67 (m, 2 H),
4.79 (m, 2 H), 4.92 (m, 4 H), 7.19–7.09 (m, 3 H), 7.37–7.31 (m,
2 H), 7.63 (m, 1 H), 7.70 (s, 1 H), 8.05 (m, 2 H), 8.31 (m, 2 H),
8.70 (d, 1 H), 8.82 (d, 1 H), 10.22 (s, 1 H); MS (HR FAB+) m/z
736.3401 (+2.9 ppm) observed for C38H50N5O8S.
The HPLC system consisted of a Knauer model 64 solvent de-
livery module (Knauer, Germany), a Knauer variable wave-
length UV detector a 20 µL sample loop, and a Shimadzu CR-
6A chromatopac integrator (Shimadzu, Tokyo, Japan).The col-
umn used was a Knauer C18 (250 mm × 4.6 mm ID, 5 µm).The
effluent monitored at 284 nm at flow rate of 1 mL/min.The mo-
bile phase used consisted of 40 volumes of acetonitrile and 60
volumes of 0.1 % trifluoroacetic acid. Dimethylformamide
(DMF) was purchased from El-Naser Chemical Industry Com-
pany (Cairo, Egypt), dried over phosphorus pentoxide and dis-
tilled under reduced pressure before use. All other chemicals
and solvents were of analytical grade.The starting compound,
KNI-279, was prepared in a pure crystalline form according to
the reported method [9].
3-{3(S)-[N-(N-Carboxymethyl-5-isoquinolinyloxyacetyl)-l-
valinyl]amino-2(S)-hydroxy-4-phenylbutanoyl}-N-(tert-
butyl)thiazolidine-4(R)-carboxamide bromide (5)
Bromoacetic acid (0.85 g), stirred for 5 hours at room tempera-
ture to give 5 (2.9 g, 74 %):mp 65–68 °C, 1H NMR (DMSO-d6) δ
Table 5. Percent of HIV PR inhibition by the prepared compounds (2–8) and parent compound 1, determined at con-
centration of 50 ng using peptidolytic assay.
Compound #
2
3
4
5
6
7
8
KNI-279 (1)
% HIV PR
inhibitory activity
48
50
51
45
76
72
65
88

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 47–52
HIV Protease Inhibitors 51
min–1) were determined by linear regression of the ln of the
mean peak area for 3 experiments against time in minutes.
0.76 (d, 6 H), 1.26 (s, 9 H), 1.98 (m, 1 H), 2.67 (d, 2 H), 3.1 (m,
2 H), 4.25 (m, 2 H), 4.44 (m, 1 H), 4.67 (m, 2 H), 4.79 (m, 2 H),
4.88 (m, 4 H), 7.19–7.09 (m, 3 H), 7.37–7.31 (m, 2 H), 7.63 (m,
1 H), 7.70 (s, 1 H), 8.05 (m, 2 H), 8.31 (m, 2 H), 8.70 (d, 1 H),
8.82 (d, 1 H), 10.22 (s, 1 H), 11.65 (s, 1 H); MS (HR FAB+) m/z
708.2175 (+2.6 ppm) observed for C36H46N5O8S.
Stability of dihydro-derivatives in biological fluids
In human plasma
Freshly collected heparinized human blood was centrifuged at
4000 rpm for 20 minutes; the supernatant (plasma) was then
collected by a Pasteur 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 37 1 °C for 5 minute, 300 µL of
0.2 mmol methanolic solution of freshly prepared dihydro com-
pound was added.The mixture was kept at 37 1 °C during the
experiment. At time intervals (5, 15, 30, 60, 90, 120, 180 240,
360, and 480 minutes) 200 µL was withdrawn from the tested
mixture, added immediately to 2 mL of ice cold methanol, vor-
texed, and kept frozen until analysis.When all the samples had
been collected, they were centrifuged, and the supernatants
were analyzed by HPLC for their content of the dihydro-com-
pounds and corresponding quaternary derivatives.The appar-
ent pseudo first-order rate constant of disappearance of dihy-
dro compounds (kdisapp., h–1) were determined by linear regres-
sion of the ln of the AUC against time in minutes.
Preparation of dihydroisoquinoline derivatives of KNI-279, gen-
eral method
To a solution of quaternary KNI-279 derivative (2.5 mmol) in
50 mL of degassed water, sodium bicarbonate (0.25 g;3 mmol)
and 50 mL of dichloromethane were added. The mixture was
stirred in an ice bath, and sodium dithionite (0.45 g;2.57 mmol)
was added portionwise over a period of 15 minutes, and stirring
was continued for 3 hours under a nitrogen steam.The organic
layer was then separated, washed with water, dried over anhy-
drous Na2SO4, and distilled under vacuum, to give the corre-
sponding dihydro-derivative as an oily material, which was
crystallized by trituration with hexane and preserved under
nitrogen protected form light in refrigerator. By the use of this
procedure, the following compounds were prepared.
3-{3(S)-[N-(N-Methyl-1,2-dihydroisoquinolin-5-yloxyacetyl)-l-
valinyl]-amino-2(S)-hydroxy-4-phenylbutanoyl}-N-(tert-
butyl)thiazolidine-4(R)-carboxamide (6)
In 20 % rat brain homogenate
About four grams of rat brain was taken, washed with ice-cold
saline solution, and homogenized in a tissue homogenizer with
about 20 mL of aqueous ice cold isotonic phosphate buffer (0.2
M, pH 7.4), while keeping the homogenizer tube in an ice bath.
To 5 mL of the freshly prepared brain homogenate, previously
equilibrated at 37 1 °C in a water bath for 5 minutes, 300 µL of
0.2 mmol methanolic solution of freshly prepared dihydro com-
pounds was added. The mixture was kept at 37 1 °C during
the experiment. Samples of 0.5 mL were withdrawn from the
tested mixture at different time intervals (0, 10, 20, 30, 60, 90,
120, 240, and 300 minutes) and immediately added to 2 mL ice
cold methanol, vortexed, and placed in the deep freezer
(–20 °C).When all the samples have been collected, they were
centrifuged, and the supernatants were analyzed by HPLC for
their content of the dihydro-compounds and corresponding
quaternary derivative. The apparent pseudo first-order rate
constants of disappearance of dihydro-derivative (kdisapp., h–1)
were determined by linear regression of the ln of the AUC
against time in minutes.
Yield (1.3 g, 78 %):mp 74–76 °C, 1H NMR (DMSO-d6) δ 0.77 (d,
6 H), 1.26 (s, 9 H), 2.0 (m, 1 H), 2.39 (s, 3 H), 2.64 (d, 2 H), 3.13
(m, 2 H), 3.69 (s, 2 H), 4.28 (m, 1 H), 4.44 (m, 2 H), 4.62 (m,
1 H), 4.78 (m, 2 H), 4.92 (m, 3 H), 5.8 (d, 1 H), 6.20 (d, 2 H),
6.98–6.67 (m, 4 H), 7.23–7.05 (m, 6 H); MS (HR FAB+) m/z
665.3079 (+2.7 ppm) observed for C35H47N5O6S.
3-{3(S)-[N-(N-Ethyl-1,2-dihydroisoquinolin-5-yloxyacetyl)-l-
valinyl]-amino-2(S)-hydroxy-4-phenylbutanoyl}-N-(tert-
butyl)thiazolidine-4(R)-carboxamide (7)
Yield (1.25 g, 73 %): mp 80–82 °C, 1H NMR (DMSO-d6) δ 0.76
(d, 6 H), 1.25 (s, 9 H), 1.65 (t, 3 H), 1.98 (m, 1 H), 2.25 (m, 2 H),
2.64 (d, 2 H), 3.09 (m, 2 H), 3.71 (s, 2 H), 4.28 (m, 1 H), 4.44 (m,
2 H), 4.62 (m, 1 H), 4.78 (m, 2 H), 4.92 (m, 3 H), 5.8 (d, 1 H),
6.20 (d, 2 H), 6.98–6.67 (m, 4 H), 7.26–7.02 (m, 6 H); MS (HR
FAB+) m/z 679.3516 (+2.3 ppm) observed for C36H49N5O6S.
3-{3(S)-[N-(N-Ethoxycarbonylmethyl-1,2-dihydroisoquinolin-
5-yloxy-acetyl)-l-valinyl]amino-2(S)-hydroxy-4-phenyl-
butanoyl}-N-(tert-butyl)-thiazolidine-4(R)-carboxamide (8)
In-vivo distribution studies
Six groups, each of three Sprague Dawley female rats of aver-
age weight of 120–140 g were anesthetized with urethane.The
freshly prepared solution of compound 8 at a concentration of
25 mg/mL in dimethyl sulfoxide (DMSO) was injected through
the external jugular vein at a dose level of 20 mg/kg of body
weight. At appropriate time intervals (10, 20, 60, 120, 240, and
360 minutes), 1 mL of blood was withdrawn from the eye and
added immediately to a centrifuge tube containing 4 mL of ace-
tonitrile, which was afterwards weighed to determine the
amount of blood added.The animal was then decapitated, and
the brain was collected, weighed, and kept frozen together with
the blood samples.The whole brain was homogenized in 1 mL
of water and mixed with 4 mL of 5 % DMSO in acetonitrile.The
mixture was homogenized again and centrifuged at 4000 rpm
for 10 minutes.
Yield (1.5 g, 81 %) of pale yellow oil, 1H NMR (DMSO-d6) δ 0.76
(d, 6 H), 1.28 (s, 9 H) , 1.31 (t, 3 H), 2.01 (m, 1 H), 2.65 (m, 2 H),
2.931 (m, 2 H), 3.55 (s, 2 H), 3.79 (m, 3 H), 4.14 (m, 2 H), 2.36
(m, 3 H), 4.50 (m, 1 H), 4.67 (m, 1 H), 4.79 (m, 1 H), 4.92 (s,
2 H), 5.78 (d, 1 H), 6.16 (d, 1 H), 6.65 (m, 2 H), 6.89 (d, 1 H),
7.25–7.08 (m, 6 H), 7.45 (m, 2 H); MS (HR FAB+) m/z 737.3475
(+2.4 ppm) observed for C38H51N5O8S.
Chemical oxidation of dihydro-derivatives with silver nitrate so-
lution
In a series of tubes, 1 mL of a 0.2 mmol methanolic solution of
the dihydro derivative (6, 7, and 8) was added to 5 mL of 10 %
methanolic silver nitrate solution and the mixture was agitated
for 3 minutes. At the specific time intervals (5, 15, 30, 60, 120,
240, and 360 minutes) one tube is vortexed, centrifuged, and
the supernatant was filtered on a 0.45 µm membrane filtration
disc and analyzed by HPLC. The rate of disappearance of the
dihydro-compounds and the appearance of the corresponding
quaternaries were determined.The apparent pseudo first order
The blood samples were also centrifuged at 4000 rpm for 10
minutes and the supernatants from both brain and blood sam-
ples were analyzed by HPLC. Control tests were performed on
six (one for every test time) Sprague Dawley female rats inject-
ed with DMSO at a dose of 1 mL/kg animal weight.
rate constant of disappearance of dihydro derivatives (kdisapp
,

52 Sheha et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 47–52
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2002.
Inhibition of HIV-1 PR was quantified using a peptidolytic assay
with a synthetic nonapeptide substrate Ac-Arg-Ala-Ser-Gln-
Asn-Tyr-Pro-Val-Val-NH2. The inhibitors were dissolved in
DMSO and assayed at 50 nM concentration.
[7] M. Abu-Elnile, Master Thesis, Assiut University, Egypt,
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