Highly water-soluble derivatives of the anesthetic agent propofol: In vitro and in vivo evaluation of cyclic amino acid esters

Article abstract of DOI:10.1016/S0928-0987(03)00161-1

Cyclic amino acid esters of propofol were synthesized in an attempt to develop new water-soluble anesthetic agents. Their solubility and stability in aqueous solution, and their ability to release propofol in vitro under physiological conditions were determined. L-Proline (6a) and racemic nipecotic acid (6c) esters were found to be highly soluble in water. Sufficiently stable at physiological pH (half-lives >6 h), the α-amino acid esters, 6a and 6b, were found to be quantitatively hydrolyzed in plasma and liver esterase solutions within a few minutes, showing prodrug behavior. The in vitro activity of the esters, determined either by the [ ³⁵S]tert-butylbicyclophosphorothionate ([³⁵S]TBPS) binding assay or electrophysiological measurements of the action at cloned human receptors, proved to be a mechanism involving allosteric modulation of GABA_A receptors. Indeed, L-proline (6a), and racemic pipecolinate (6b) and nipecotate (6c), like propofol, reduced [³⁵S]TBPS binding, whereas isonipecotate (6d) showed bicuculline-like behavior, increasing [ ³⁵S]TBPS binding. A nonlinear relation between GABA_A receptor binding affinity and lipophilicity, as assessed by reversed-phase high-performance liquid chromatography, emerged as a trend. The in vivo anticonvulsant and anesthetic activities of prolinate 6a, intraperitoneally administered in water solution, showed that is a water-soluble propofol prodrug candidate for developing formulations useful for parenteral administration.

Full text of DOI:10.1016/S0928-0987(03)00161-1

European Journal of Pharmaceutical Sciences 20 (2003) 17–26
www.elsevier.com/locate/ejps
Highly water-soluble derivatives of the anesthetic agent propofol: in vitro
and in vivo evaluation of cyclic amino acid esters
a
b
b
Cosimo Altomarea, Giuseppe Trapania , , Andrea Latrofa , Mariangela Serra , Enrico Sanna ,
*
Giovanni Biggiob, Gaetano Lisoa
Dipartimento Farmaco-Chimico, Facolta di Farmacia, Universita degli Studi di Bari, Via Orabona 4, 70125 Bari, Italy
a
`
`
b
`
Dipartimento di Biologia Sperimentale, Sezione di Neuroscienze, Universita di Cagliari, Cittadella Universitaria Monserrato, SS 554 Km 4.5,
Monserrato (Cagliari), Italy
Received 20 February 2003; received in revised form 21 May 2003; accepted 6 June 2003
Abstract
Cyclic amino acid esters of propofol were synthesized in an attempt to develop new water-soluble anesthetic agents. Their solubility
and stability in aqueous solution, and their ability to release propofol in vitro under physiological conditions were determined. L-Proline
(6a) and racemic nipecotic acid (6c) esters were found to be highly soluble in water. Sufficiently stable at physiological pH (half-lives .6
h), the a-amino acid esters, 6a and 6b, were found to be quantitatively hydrolyzed in plasma and liver esterase solutions within a few
minutes, showing prodrug behavior. The in vitro activity of the esters, determined either by the [35S]tert-butylbicyclophosphorothionate
([35S]TBPS) binding assay or electrophysiological measurements of the action at cloned human receptors, proved to be a mechanism
involving allosteric modulation of GABAA receptors. Indeed, L-proline (6a), and racemic pipecolinate (6b) and nipecotate (6c), like
propofol, reduced [35S]TBPS binding, whereas isonipecotate (6d) showed bicuculline-like behavior, increasing [35S]TBPS binding. A
nonlinear relation between GABAA receptor binding affinity and lipophilicity, as assessed by reversed-phase high-performance liquid
chromatography, emerged as a trend. The in vivo anticonvulsant and anesthetic activities of prolinate 6a, intraperitoneally administered in
water solution, showed that is a water-soluble propofol prodrug candidate for developing formulations useful for parenteral
administration.
2003 Elsevier B.V. All rights reserved.
Keywords: Propofol; Water-soluble prodrugs; Stability; Lipophilicity; Anesthetic activity
1. Introduction
1% (w/v) solution in the presence of Cremophor EL (a
solubilizing surfactant), but the anaphylactic reactions
associated with its administration led to a search for
alternative formulations (Trapani et al., 2000; Franks and
Lieb, 1994). Currently, propofol is formulated as an oil-in-
water emulsion (1%, w/v) of soya bean oil, glycerol and
purified egg phosphatide (Diprivan , Zeneca, UK). In-
travenous (i.v.) injection of Diprivan rapidly (usually
within 40 s) and smoothly produces hypnosis with minimal
excitation, but pain at the site of injection is a major
adverse effect (Prankerd and Stella, 1990). As a lipid-
based emulsion, it suffers from a number of limitations,
such as poor physical stability, potential for embolism, and
need for strictly aseptic handling (Bennett et al., 1995).
Particular care is required for patients with disorders of fat
metabolism (Dollery, 1991). These drawbacks have stimu-
lated an active search for safe alternative dosage forms,
and, in particular, aqueous formulations. Several ap-
proaches have been exploited, including complexation with
Propofol, 2,6-diisopropylphenol (1, Fig. 1), is an in-
travenous agent used for anesthesia. Due to its very low
solubility in water, propofol was initially formulated as a
Fig. 1. Structure of propofol and related prodrugs (Trapani et al., 1998b).
*
Corresponding author. Tel.: 139-080-544-2764; fax: 139-080-544-
2754.
E-mail address: trapani@farmchim.uniba.it (G. Trapani).
0928-0987/03/$ – see front matter
doi:10.1016/S0928-0987(03)00161-1
2003 Elsevier B.V. All rights reserved.

18
C. Altomare et al. / European Journal of Pharmaceutical Sciences 20 (2003) 17–26
hydroxypropyl-b-cyclodextrin (Brewster, 1991; Trapani et
al., 1996, 1998c) and chemical delivery systems, the main
goals being an increase of the hydrosolubility, an improve-
ment in patient acceptance (e.g., reduced pain at injection),
a decrease in side-effects and a prolonged duration of
action (Pop et al., 1992). The prodrug approach has also
been used to increase the solubility of propofol in water
(Banaszczyk et al., 2002; Morimoto and Barker, 2000;
Stella et al., 2000; Sagara et al., 1999; Hendler et al.,
1999), and of the formulations available for parenteral
administration, Aquavanீ injection is under Phase II
clinical trials in patients undergoing coronary artery
surgery (Guilford Pharmaceuticals Inc., 2002).
In previous studies, we investigated a number of a-
amino acid ester derivatives of propofol (2, Fig. 1)
(Trapani et al., 1998b), which showed low-to-moderate
solubility and high stability in water solution, but, in
contrast to data recently reported by others (Anderson et
al., 2001), exhibited resistance in plasma and brain
homogenate that was too high for them to be considered as
true prodrugs. However, interestingly, some of them have
been found to interact with subtype A of the g-amino-
butyric acid (GABAA) receptor, a major target mediating
the pharmacological actions of propofol and other general
anesthetics (Trapani et al., 2000; Franks and Lieb, 1994).
In the framework of our research program aimed at
developing new anesthetic formulations for parenteral
administration, we prepared a number of potentially highly
water-soluble derivatives of propofol (6a–d, Fig. 2) by
coupling, via an ester bond, the drug with four cyclic
amino acids, namely L-proline (3a) and the three positional
isomers of piperidine carboxylic acids (3b–d) (i.e.,
pipecolinic, nipecotic, and isonipecotic acids), and investi-
gated their solubility and stability in aqueous solution,
their lipophilicity, susceptibility to enzymatic hydrolysis in
animal plasma and liver, as well as their ability to
modulate GABAA receptors. Two esters representing dif-
ferent rates of hydrolysis and profiles of in vitro activity
were selected and evaluated for their in vivo anticonvul-
sant and anesthetic activities.
It is worth noting that two of the hydrophilic aminoacyl
promoieties chosen are endowed with activities involving
GABAergic transmission. Nipecotic acid (3c), as its (R)-
enantiomer, and isonipecotic acid (3d) are an in vitro
GABA uptake inhibitor and a GABAA agonist, respective-
ly (Krogsgaard-Larsen et al., 1977). Their pharmacological
value is strongly restricted because of their poor bioavail-
ability in the central nervous system (CNS), but a number
of alkyl and aryl esters have shown anticonvulsive effects
in animals that reflect the rates of hydrolysis to produce the
active amino acid in vivo (Altomare et al., 1988; Krog-
sgaard-Larsen et al., 1988; Wermuth et al., 1982). With this
in mind, it cannot be excluded that propofol itself can
function as a promoiety of the GABAmimetic agents 3c
and 3d; hence, the respective esters could be dual acting
prodrugs, provided that normally effective doses of the two
Fig. 2. Synthetic pathway for preparing cyclic amino acid ester prodrugs.
drugs are attained in the CNS following peripheral ad-
ministration. These aspects have also been taken into
account in evaluating the pharmacological potential of the
propofol derivatives described.
2. Materials and methods
2.1. Chemicals
Propofol (1), dicyclohexylcarbodiimide (DCC), L-pro-
line (3a), racemic pipecolinic acid (3b) and nipecotic acid
(3c), isonipecotic acid (3d), and all other reagents were
purchased from Sigma–Aldrich S.r.l. (Milan, Italy). Rat
serum (lyophilized powder) and porcine liver esterase
(suspension in 3.2 M (NH4)2SO4 solution, pH 8) were also
purchased from Sigma–Aldrich. Reagents used for the
preparation of the buffers were of analytical grade. Fresh
deionized water was used in the preparation of all solu-
tions.
2.1.1. Apparatus
Melting points were determined by the capillary method
¨
on a Buchi apparatus and are uncorrected. IR spectra were
recorded as Nujol films for liquids and KBr pellets for
solids on a Perkin-Elmer 283 spectrophotometer. 1H-NMR
spectra were recorded on a Varian EM 390 spectrometer
operating at 90 MHz (Varian, Milan, Italy). Chemical

C. Altomare et al. / European Journal of Pharmaceutical Sciences 20 (2003) 17–26
19
shifts are expressed as d values downfield from tetra-
methylsilane (TMS) used as internal standard. Mass spec-
tra were recorded on a Hewlett-Packard 5995c GC–MS
low-resolution spectrometer (Hewlett-Packard, Milan,
Italy) operating in electron impact mode. The ESI (elec-
tron-spray ionization) mass spectra of compounds 6a–d
were obtained using an Agilent 1100 LC–MSD trap
system VL instrument operating in positive ionization
mode. Elemental analyses were performed on a Hewlett-
Packard 185 C, H, N analyzer and agreed with theoretical
values to within 60.40%. High-performance liquid chro-
matography (HPLC) analyses were carried out on a Waters
Associates Model 600 pump equipped with a Waters 990
variable-wavelength UV detector and a 20 ml loop in-
jection valve (Waters, Milan, Italy). HPLC mobile phase
was prepared using HPLC-grade methanol. For analysis, a
Phenomenex C18 column (25 cm33.9 mm; 5 mm par-
ticles) was used as the stationary phase. A flow rate of
1 ml/min was maintained and the column effluent was
monitored continuously at 210 or 270 nm for the propofol
esters 6a–d and propofol 1, respectively. Quantification of
the compounds was carried out by measuring the peak
areas in relation to those of external standards. Stability
studies were carried out at a controlled temperature of
3760.2 8C in a water bath.
2.1.2. Syntheses
The propofol esters 6a–d were prepared, according to
the procedure illustrated in Fig. 2, by reacting BOC-
protected cyclic amino acids 4a–d with propofol 1 in the
presence of DCC to give the corresponding esters 5a–d,
which, deprotected with HCl gas, yielded derivatives 6a–d
as hydrochlorides (physical and spectral data of newly
synthesized compounds 4a, 5a–d, and 6a–d are given in
Table 1), the purity of which was mainly checked by
elemental analysis and HPLC.
Table 1
Physical and spectral characteristics of newly synthesized compounds
Compound
Yield
(%)
M.p.
(8C)
IR (KBr)
1H-NMR (CDCl3)
d (ppm)
MS
(m/z)
(n max)
(cm21
)
5a
5b
72
68
69–71
1770,
1700
1.17 (d, 12H, CH3), 1.46 (s, 9H, CH3), 1.9–2.1 (m, 2H, CH2),
2.2–2.4 (m, 2H, CH2), 2.8–3.1 (m, 2H, CH), 3.4–3.7
(m, 2H, CH2), 4.6–4.7 (m, 1H, CH), 7.1– 7.2 (m, 3H, Ar)
274 (19),
178 (base)
100–104
80–84
1760,
1700
1.18 (d, 12H, CH3), 1.47 (s, 9H, CH3), 1.6–1.9 (m, 2H, CH2),
2.4–2.5 (m, 1H, CH), 2.8–3.1 (m, 3H, CH2 1CH), 3.1–3.2
(m, 1H, CH), 3.9–4.1 (m, 1H, CH), 4.1–4.2 (m, 1H, CH),
5.0–5.2 (m, 2H, CH2), 7.1–7.2 (m, 3H, Ar)
389 (1, M1),
178 (41),
128 (base)
5c
70
1755,
1700
1.17 (d, 12H, CH3), 1.47 (s, 9H, CH3), 1.7–1.9 (m, 2H, CH2),
2.1–2.3 (m, 1H, CH), 2.7–3.0 (m, 5H, CH2 1CH), 3.0–3.2
(m, 1H, CH), 3.9–4.1 (m, 1H, CH), 4.3–4.6 (m, 1H, CH),
7.1–7.2 (m, 3H, Ar)
389 (1, M1),
178 (base)
5d
6a
67
90
94–97
1750,
1700
1.17 (d, 12H, CH3), 1.47 (s, 9H, CH3), 1.7–1.9 (m, 2H, CH2),
2.0–2.1 (m, 2H, CH2), 2.6–3.0 (m, 5H, CH2 1CH), 4.0–4.2
(m, 2H, CH2), 7.1–7.2 (m, 3H, Ar)
389 (1, M1),
178 (60),
112 (base)
189–192
1760
1.14 (dd, 12H, CH3), 1.9–2.2 (m, 2H, CH2), 2.3–2.5
(m, 1H, CH), 2.5–2.7 (m, 1H, CH), 2.7–2.9 (m, 2H, CH),
3.4–3.6 (m, 2H, CH2), 4.7–4.8 (m, 1H, CH), 7.0–7.2
(m, 3H, Ar), 9.2–10.0 (bs, 1H, NH), 11.1–11.4 (bs, 1H, NH)
276.2a
6b
6c
6d
87
95
90
225–228
154–156
234–236
1770
1.14 (d, 12H, CH3), 1.5–1.7 (m, 1H, CH), 1.7–2.0
(m, 3H, CH2 1CH), 2.1–2.3 (m, 1H, CH), 2.4–2.6
(m, 1H, CH), 2.8–3.1 (m, 3H, CH), 3.5–3.7 (m, 1H, CH),
4.1–4.2 (m, 1H, CH), 7.0–7.2 (m, 3H, Ar), 9.8
(bs, 1H, NH), 10.5 (bs, 1H, NH)
290.2a
1760
1740
1.14 (d, 12H, CH3), 1.6–1.9 (m, 1H, CH), 1.9–2.0
(m, 1H, CH), 2.1–2.3 (m, 1H, CH), 2.4–2.5 (m, 1H, CH),
2.6–3.0 (m, 3H, CH), 3.0–3.2 (m, 1H, CH), 3.3–3.5
(m, 1H, CH), 3.5–3.7 (m, 1H, CH), 3.7–3.9 (m, 1H, CH),
7.0–7.2 (m, 3H, Ar), 9.8 (bs, 2H, NH2)
290.2a
290.2a
1.14 (d, 12H, CH3), 2.1–2.5 (m, 4H, CH2), 2.7–2.8
(m, 2H, CH), 2.8–3.0 (m, 1H, CH), 3.0–3.2 (m, 2H, CH2),
3.4–3.6 (m, 2H, CH2), 7.1–7.2 (m, 3H, Ar), 9.5–9.9
(bs, 2H, NH2)
a [M1235], from ESI mass spectra of hydrochlorides 6a–d.

20
C. Altomare et al. / European Journal of Pharmaceutical Sciences 20 (2003) 17–26
2.1.2.1. BOC-protected amino acids: preparation of 1-
(tert-butoxycarbonyl)proline (4a)
2.1.2.3. Removal of the tert-butoxycarbonyl group: prepa-
ration of 2,6-diisopropylphenyl pyrrolidine-2-carboxylate
hydrochloride (6a) as a typical procedure
HCl gas was bubbled for 5 min through a stirred and
ice-cooled solution of ester 5a (0.50 g, 1.33 mmol) in
chloroform (20 ml). Evaporation of the solvent under
reduced pressure gave compound 6a as a white solid.
To a stirred mixture of L-proline (4.60 g, 40 mmol) in
H2O (25 ml) containing triethylamine (8.3 ml, 60 mmol),
a solution of 2-(tert-butoxycarbonyloxyimino)-2-phenyl-
acetonitrile (BOC-ON, 10.58 g, 43 mmol) in acetone (25
ml) was added. Stirring was prolonged for 12 h, and then
125 ml of a mixture of ethyl acetate–water (1:1, v/v) was
added. The aqueous phase, combined with water (55 ml),
used for washing the organic phase, was further washed
with 50 ml of ethyl acetate, and then acidified with cold
0.1 N HCl (pH 2) to give compound 4a as a white
precipitate.
2.1.3. Physicochemical measurements
2.1.3.1. Solubility
N-BOC-piperidine-carboxylic acids 4b–d were prepared
in 87–89% yields according to the above procedure
(analytical data in agreement with those reported in the
literature) (Ho et al., 1998; Bonina et al., 1998; Freund and
Mederski, 2000).
The solubility of the propofol derivatives 6a–d in
deionized water at 25 8C was determined by adding an
excess amount of compound to 1–2 ml of water in a
screw-capped test tube. The resulting mixture was vortex-
ed for 10 min and then mechanically shaken in a thermo-
static bath shaker (100 rpm) for 72 h to attain equilibrium.
Next, the mixture was filtered through a 0.45 mm mem-
brane filter (Millipore , cellulose acetate) and an aliquot
was diluted with an appropriate amount of water and
analyzed for the amino acid ester prodrug content spectro-
photometrically at 210 nm. All manipulations were made
without removal of the test tubes from the water bath,
using thermostated pipettes, syringes, and buffer solutions.
Solubility data in Table 2 are compared with those
previously determined for propofol derivatives 2a–c
(Trapani et al., 1998b).
2.1.2.2. Esterification of BOC-protected cyclic amino
acids: preparation of 2,6-diisopropylphenyl-1-(tert-butoxy-
carbonyl)pyrrolidine-2-carboxylate (5a) as a typical pro-
cedure
To a stirred solution of 1 (0.40 g, 2.25 mmol), com-
pound 4a (0.57 g, 2.50 mmol), and dimethylaminopyridine
(0.1 g, 0.82 mmol) in dry dichloromethane (15 ml), a
solution of DCC (1.4 g, 6.8 mmol) in dry dichloromethane
(10 ml) was added dropwise over 10 min. Stirring was
continued at room temperature for 24 h, and then the
dicyclohexylurea (DCU) precipitate was filtered off. The
solution was evaporated under reduced pressure to give a
residue which was purified by column chromatography on
silica gel (petroleum ether–ethyl acetate (98:2, v/v) as
eluent) to give compound 5a.
2.1.3.2. Lipophilicity parameters
The lipophilicity of the propofol esters 6a–d and 2a–c
was assessed by a reversed-phase (RP)-HPLC method.
Table 2
Aqueous solubility, lipophilicity, stability in physiological media, and GABAA receptor binding of amino acid esters (as hydrochloride salts) of propofol
Compound
Solubility
(mmol/ml)
in watera
Lipophilicity
CLOG Pb
Half-lives at 37 8Ca
[
35S]TBPS
binding
9 c
Log kw
pH 7.4
buffer
50% rat
serum
PLEd
(13 U/ml)
IC50 (mM)e
1
8.631024
1.1
4.33
4.34
4.90
4.64
3.96
6.39
5.04
4.38
4.17
31.7
39.5
6a
6b
6c
6d
2a
2b
2c
2.40
2.93
3.19
3.29
5.28
3.05
3.95
6 h
7 h
s
17 min
2.5 min
17 min
13 min
3 h
4.131022
1.6
6 h
s
152
9.131022
,2.031024f,g
s
45 h
h
f
sf
sf
n.d.
2.431023
sf
sf
6.52f
f
6.531024
sf
sf
n.d.
f
f
s, stable up to 48 h; n.d., no displacement.
a Data are means of two determination (less than 10% difference).
b Calculated 1-octanol–water partition coefficients of the free bases by CLOGP software; for propofol the experimental log P is 3.83 (pH 6–8.5).
c
9
Polycratic capacity factor (log kw) determined on an octylsilane stationary phase (see Materials and methods for definitions).
d Porcine liver esterase.
e
[
35S]TBPS binding in unwashed rat brain.
f Previously reported data (Trapani et al., 1998b).
g Determined as free base.
h No displacement, but an increase in [35S]TBPS binding was observed.

C. Altomare et al. / European Journal of Pharmaceutical Sciences 20 (2003) 17–26
21
Retention data were measured using a Phenomenex C8
2.1.5. Pharmacological studies
2.1.5.1. Animals
column (15 cm33.2 mm; 5 mm particles) as the non-polar
stationary phase, at 0.05 increments of the volume fraction
of methanol (f) in the aqueous mobile phase (0.025 M
phosphate buffer, pH 4.5). The flow rate was maintained at
0.8 ml/min. Capacity factors (k9) of each solute for
different mobile phase compositions (0.4 , f , 0.7) were
calculated as k9 5 (tR 2 t0)/t0, where tR is the retention
time of the solute and t0 is the column dead time. The
log k9 values increased linearly (r2 . 0.95) with decreasing
Male Sprague–Dawley CD rats (Charles River, Como,
Italy) weighing 180–200 g were used. The animals were
kept on a controlled light–dark cycle (light period between
8:00 a.m. and 8:00 p.m.) in a room with constant tempera-
ture (2262 8C) and humidity (65%). Upon arrival at the
animal facilities there was a minimum of 7 days of
acclimation during which the animals had free access to
food and water.
9
methanol volume fraction. Thus, the polycratic log kw
values (i.e., the capacity factors extrapolated to 100%
water in the mobile phase) were calculated by using the
Animal care and handling throughout the experimental
procedure were performed in accordance with the Euro-
pean Communities Council Directive of 24 November
1986 (86/609/EEC). The experimental protocols were
approved by the Animal Ethical Committee of the Uni-
versity of Cagliari.
9
following linear relationship: log k9 5 log kw 2 Sf, where
S (slope) is a constant for the solute–eluent combination.
Octanol–water partition coefficients were calculated by the
CLOGP software (CLOG P for Windows Vers. 4, BioByte
Corp., Claremont, CA, USA). Lipophilicity parameters are
reported in Table 2.
2.1.5.2. In vitro [35S]TBPS binding assay
Rats were killed by decapitation and their brains rapidly
removed on ice. The cerebral cortex was dissected out and
homogenized in 50 volumes of ice-cold 50 mM Tris–
citrate buffer (pH 7.4 at 25 8C) containing 100 mM CaCl2
using a Polytron PT 10 (setting 5, for 20 s) and centrifuged
at 20,0003g for 20 min. The resulting pellet was re-
suspended in 50 volumes of 50 mM Tris–citrate buffer
(pH 7.4 at 25 8C) and used for the assay. [35S]TBPS
binding was determined in a final volume of 500 ml
consisting of 200 ml of tissue homogenate (0.20–0.25 mg
protein), 50 ml of [35S]TBPS (final assay concentration
2 nM), 50 ml 2 M NaCl, 50 ml of drugs or solvent and
buffer to volume. Incubations (25 8C) were initiated by
addition of tissue and terminated 90 min later by rapid
filtration through glass-fiber filter strips (Whatman GF/B,
Clifton, NJ, USA), which were rinsed twice with a 4 ml
portion of ice-cold Tris–citrate buffer using a Cell Harves-
ter filtration manifold (model M-24m Brandel, Gaithers-
burg, MD, USA). Filter-bound radioactivity was quanti-
tated by liquid scintillation spectrometry. Nonspecific
binding was defined as binding in the presence of 100 mM
picrotoxin and represented about 10% of total binding.
Protein content was determined by the method of Lowry et
al. (1951) using bovine serum albumin as a standard.
2.1.4. Kinetic measurements
2.1.4.1. Chemical hydrolysis
Hydrolysis of the propofol esters 6a–d was studied in
aqueous buffer solutions (0.05 M phosphate buffers; ionic
strength 0.5 maintained by adding a calculated amount of
KCl) at pH 4, 6, and 7.4 and temperatures of 3760.2 8C.
The reactions were initiated by adding 100 ml of a stock
solution of the ester (13 mg/ml methanol) to 20 ml of the
buffer solution preheated at 37 8C, in screw-capped test
tubes (final concentration about 2.031024 M). The solu-
tions were kept in a water bath at a constant temperature,
and at appropriate intervals aliquots of 20 ml were with-
drawn and analyzed by HPLC. Pseudo-first-order rate
constants for the hydrolysis were determined from the
slopes of linear plots of the logarithm of residual propofol
ester against time.
2.1.4.2. Stability in physiological media
The susceptibility of the derivatives 6a–d to undergo
conversion to the parent propofol was studied in 0.05 M
phosphate buffer (pH 7.4) containing 50% rat serum at 37
8C. Each reaction was initiated by adding 100 ml of the
methanolic stock solution of the compound under examina-
tion to 1.6 ml of preheated serum solution (final con-
centration about 131023 M) and the mixture was main-
tained in a water bath at 37 8C. At appropriate times, 100
ml samples were withdrawn and added to 500 ml of cold
acetonitrile in order to deproteinize the serum. After
mixing and centrifugation (10 min at 4000 rpm), 20 ml of
the clear supernatant was filtered through a 0.2 mm
membrane filter (Waters, PTFE 0.2 mm) and analyzed by
HPLC.
2.1.5.3. Functional in vitro studies: electrophysiological
measurements using Xenopus oocytes
Complementary DNAs encoding the human a1, b2 and
,
g2 GABAA receptor subunits were subcloned into the
pCDM8 expression vector (Invitrogen, San Diego, CA,
USA). The cDNAs were purified using a Promega Wizard
Plus Miniprep DNA Purification System (Madison, WI,
USA) and then resuspended in sterile distilled water,
divided into portions, and stored at 220 8C until used for
injection. Stage V and VI oocytes were manually isolated
from sections of Xenopus laevis ovary, placed in modified
Barth’s saline (MBS) containing 88 mM NaCl, 1 mM KCl,
Hydrolysis of compounds 6a–d in the presence of
porcine liver esterase was followed using a reported
procedure (Bonina et al., 1998).

22
C. Altomare et al. / European Journal of Pharmaceutical Sciences 20 (2003) 17–26
10 mM Hepes–NaOH buffer (pH 7.5), 0.82 mM MgSO4,
2.4 mM NaHCO3, 0.91 mM CaCl2, and 0.33 mM
Ca(NO3)2 and treated with 0.5 mg/ml of collagenase Type
IA (Sigma) in collagenase buffer (83 mM NaCl, 2 mM
KCl, 1 mM MgCl2, 5 mM Hepes–NaOH buffer, pH 7.5)
for 10 min at room temperature, to remove the follicular
3. Results and discussion
Water-soluble derivatives, including prodrugs, provided
preparations for the parenteral administration of propofol
(Banaszczyk et al., 2002; Morimoto and Barker, 2000;
Stella et al., 2000; Sagara et al., 1999; Hendler et al.,
1999), a highly lipophilic anesthetic agent sparingly solu-
ble in water. A few years ago (Trapani et al., 1998b), we
reported on N,N-disubstituted-a-amino acid esters (com-
pounds 2a–c in Fig. 1 are representative derivatives; data
in Table 2) that showed a low-to-moderate increase of
solubility in water and very high stability in aqueous
solution at physiological pH (t1 / 2 4 1 day). In apparent
contrast with a recent report (Anderson et al., 2001), they
did not show ready conversion into the parent propofol,
either in plasma or in brain homogenate. Nevertheless, due
to their binding affinity to the GABAA receptors, similar to
that of the parent propofol, some of them were suggested
as promising candidates for in vivo pharmacological
evaluation.
In this work, we investigated four esters (6a–d) of
propofol with two cyclic a- (L-proline and pipecolinic
acid), b- (nipecotic acid) and g- (isonipecotic acid) amino
acids, which according to the classical pathway (Fig. 2)
were synthesized in satisfactory yield (Table 1).
The amino acids 3b–c were used in synthesis as
racemates, since investigation of the activity dependence
upon stereochemistry is out of the scope of the present
work.
layer. A mixture of GABAA receptor a1, b2 and g2
,
subunit cDNAs (1.5 ng/30 nl) was injected into the oocyte
nucleus using a 10 ml glass micropipette (10–15 mm tip
diameter). The injected oocytes were cultured at 19 8C in
sterile MBS supplemented with streptomycin (10 mg/ml),
penicillin (10 U/ml), gentamicin (50 mg/ml), 0.5 mM
theophylline, and 2 mM sodium pyruvate. Electrophysio-
logical recordings began approximately 24 h following
cDNA injection. Oocytes were placed in a 100-ml rectan-
gular chamber and continuously perfused with MBS
solution at a flow rate of 2 ml/min at room temperature.
The animal pole of the oocytes was impaled with two glass
electrodes (0.5–3 MV) filled with filtered 3 M KCl and the
voltage was clamped at 270 mV with an Axoclamp 2-B
amplifier (Axon Instruments, Burlingame, CA, USA).
Currents were continuously recorded on a strip-chart
recorder. The resting membrane potential usually ranged
from 230 to 250 mV. Drugs were perfused for 20 s (7–10
s were required to reach equilibrium in the recording
chamber). Intervals of 5 to 10 min were allowed between
drug applications.
2.1.5.4. In vivo screening of anticonvulsant and anesthetic
activities
3.1. Aqueous solubility and lipophilicity
Rats received an intraperitoneal (i.p.) administration of
propofol 1 (40 mg/kg, suspended in saline with one drop
of Tween 80 per 5 ml) and equimolar doses of compounds
6a and 6d. Pentylenetetrazole (PTZ) was administered (55
mg/kg) 20 min after the drug in groups of 10 rats and the
activity against PTZ-induced seizures assessed. Rats
treated with PTZ were considered ‘‘protected’’ when
clonic or tonic seizures and death did not occur during 2 h
of observation.
The loss and re-establishment of righting responses,
time of anesthesia induction, and sleeping time were also
assessed. Rats (five per group) were treated with propofol
and Diprivan , both at a dose of 60 mg/kg, and an
equimolar dose of compound 6a (105 mg/kg) and were
continuously monitored for the loss of righting reflex
(onset and duration). Propofol and its derivative 6a were
dissolved in saline with one drop of Tween 80 per 5 ml
and administered i.p. in a volume of 0.3 ml per 100 g body
mass. Anesthesia induction (sleep onset) was defined as
the time from drug administration to loss of righting reflex,
whereas the sleeping time was the time from the loss of the
righting reflex until the animals were plantigrade on all
four legs. The significance of differences in behavioral data
was analyzed by the ANOVA test.
The solubility of the hydrochloride salts of 2,6-diiso-
propylphenyl pyrrolidine-2-carboxylate (6a) and piper-
idinecarboxylate isomers (6b–d) was determined in deion-
ized water at a temperature of 2560.5 8C. Solubility data
are summarized in Table 2, along with those relative to
N-substituted glycinates (2a–c) (Trapani et al., 1998b).
Compared with propofol, the solubility of which under
the same conditions was about 8.631024 M (0.15 mg/
ml), two derivatives, 6a and 6c, the solubilities of which
were 1.1 and 1.6 M, respectively, afforded a very strong
increase in the aqueous solubility of the anesthetic drug.
More than a 50-fold increase in water solubility over
propofol was observed for pipecolinate 6b and isonipeco-
tate 6d. No mathematical model used to compute intrinsic
solubilities gave accurate predictions (Peterson and Yal-
kowsky, 2001; Teitko et al., 2001), whereas the differences
in solubility of derivatives 6 could be accounted for, at
least in part, by the large differences in crystal lattice
energies. As a matter of fact, the most soluble, 6a and 6c,
have melting points lower than 200 8C, and the less soluble
pipecolinate 6b and isonipecotate 6d melt at 225 and 234
8C, respectively.
The lipophilicity of propofol amino acid esters 6a–d and

C. Altomare et al. / European Journal of Pharmaceutical Sciences 20 (2003) 17–26
23
2a–c was assessed by calculating the 1-octanol/water
partition coefficients with the CLOGP software and
measuring retention using a reversed-phase high-perform-
ance liquid chromatography (RP-HPLC) method (data in
Table 2). Log P values, calculated for the free bases, span
a range of more than two log units. RP-HPLC retention
data were measured using an octylsilane (OS) non-polar
stationary phase and aqueous mobile phases (0.02 M
phosphate buffer, pH 4.5) containing different volume
fractions of methanol. At the pH of the mobile phase, the
amino groups of all the examined derivatives should be
mainly in the protonated form (calculated pKa values range
between ca. 6.5 for compound 2c and ca. 11 for compound
6d) (Perrin et al., 1981). From the linear relationships
between capacity factors (log k9) and methanol/aqueous
withdrawing effect of the protonated amino group, which
activates the ester linkage toward OH2 attack.
A relevant finding of this study is that prolinate 6a,
highly soluble and stable in water at physiological pH and
rapidly hydrolyzed in plasma, could have potential as a
water-soluble propofol prodrug for parenteral administra-
tion.
3.3. In vitro pharmacological studies
3.3.1. Receptor binding
GABAA receptors are sensitive targets for the action of
propofol and other general anesthetics (Trapani et al.,
2000; Franks and Lieb, 1994). The binding assay of
[35S]tert-butylbicyclophosphorothionate ([35S]TBPS),
a
9
buffer ratios, log kw values (i.e., the capacity factors
cage convulsant which binds in close proximity to the
chloride channel portion of the GABAA receptor at the
level of the picrotoxin binding site, constitutes a tool for
studying the function of the GABAA receptor complex
(Squires et al., 1983). Propofol, mimicking the action of
other general anesthetics, such as alphaxalone and pen-
tobarbital (Concas et al., 1991), reduces [35S]TBPS bind-
ing in a concentration-dependent manner. In contrast, it is
well established that benzodiazepine receptor inverse
agonists and inhibitors of GABAergic transmission, such
as bicuculline, enhance in a dose-dependent manner the
total number of [35S]TBPS binding sites, an effect opposite
to that produced by benzodiazepine and GABA agonists.
The in vitro effects of compounds 6a–d at GABAA
receptors were measured by the determination of their
abilities to inhibit [35S]TBPS binding to unwashed rat
cortical membranes. Binding data, expressed as IC50
values (Table 2), showed that compounds 6a and 6b are
able to reduce the [35S]TBPS binding, with IC50 values
one magnitude order higher than the IC50 value for
propofol (4.17 mM). A similar effect, at doses higher than
100 mM, was shown by nipecotate 6c, whereas 6d
displayed an increase of [35S]TBPS binding, an effect
similar to that of bicuculline (Concas et al., 1990). Fig. 3
shows the competitive inhibition curves of the examined
derivatives.
extrapolated to 100% water mobile phase) were calculated.
No significant correlation was found between calculated
log P and log kw. As far as ester derivatives 6 are
concerned, piperidine-2-carboxylate (6b), expected from
the CLOGP calculation to be about one log unit more
lipophilic than its isomeric piperidine-4-carboxylate (6d),
was in fact found to be less lipophilic in RP-HPLC.
9
3.2. Hydrolysis in physiological media
The kinetics of hydrolysis of derivatives 6a–d was
determined in phosphate buffer at pH 4.0, 6.0 and 7.4, as
well as in rat serum solution and in the presence of porcine
liver esterase, at 37 8C.
All the examined derivatives were stable at pH values of
4.0 and 6.0 for 48 h, whereas at physiological pH the
hydrolysis of prolinate 6a and pipecolinate 6b followed
first-order kinetics with half-lives of 6 and 7 h, respective-
ly. The derivatives 6a and 6b, but not 6c and 6d, were
found to be cleaved quantitatively to the parent drug in rat
serum and porcine liver esterase solutions at 37 8C. The
observed half-lives are reported in Table 2.
Kinetic data showed that 6a and 6b are stable enough in
solution buffered at pH 7.4, their half-lives exceeding 6 h,
but undergo a fast cleavage under conditions similar to
those prevailing in vivo, providing propofol within a few
minutes. Conversely, compounds 6c and 6d were found to
be stable in buffer solution and more resistant than the
a-amino acid esters to esterase-catalyzed hydrolysis. The
observed high stability of all the propofol esters with
respect to chemical hydrolysis can be ascribed to the steric
protection of the C(O)O– bond by bulky flanking diiso-
propyl groups on the phenyl ring. The fact that the proline
(6a) and pipecolinic acid (6b) esters, similarly to a-amino
acid esters or related short-chain aliphatic amino acid
esters (Bundgaard et al., 1984), are less resistant than
compounds 6c and 6d to chemical and enzyme-catalyzed
hydrolysis could most likely result from the electron-
3.3.2. Electrophysiology of Xenopus oocytes expressing
human GABAA receptors
Further proof of their different behavior was obtained by
investigating compounds 6a and 6d for their modulatory
action at cloned human a1b2g2 GABAA receptors by
means of electrophysiological measurements. Expression
of a1b2g2 GABAA receptor subunit constructs in Xenopus
laevis oocytes was utilized in a voltage-clamp electro-
physiological assay, and Fig. 4 shows the profiles de-
termined for prolinate 6a and isonipecotate 6d. Consistent
with the binding data, GABA-evoked chloride currents
elicited at cloned GABAA receptors were enhanced by 6a
and diminished by 6d, both in a concentration-dependent

24
C. Altomare et al. / European Journal of Pharmaceutical Sciences 20 (2003) 17–26
Fig. 3. Effect of propofol 1 and compounds 6a–d on [35S]TBPS binding
to unwashed rat cortical membranes. Rat cortical membranes were
incubated with 2 nM [35S]TBPS for 90 min in the presence of different
concentrations of propofol 1 (d), or compounds 6a (j), 6b (,), 6c (s),
and 6d (.). Data are expressed as a percentage of binding measured in
the presence of solvent and are means of two experiments.
manner, with their maximal effects apparent at concen-
trations of 50 and 100 mM, respectively.
Taken together, our in vitro results demonstrate that all
the ester derivatives 6a–d modulate GABAA receptors and
possess intrinsic activity, although lower than that of the
parent compound 1. Three amino acid esters, 6a–c,
behaved like propofol, whereas the isonipecotate 6d re-
vealed a bicuculline-like profile. We cannot rule out partial
hydrolysis in receptor preparation, at least for prolinate 6a
and pipecolinate 6b, which could also account for their
results.
Fig. 4. Modulatory action of compounds 6a (a) and 6d (b) at human
a1b2g2 GABAA receptors expressed in Xenopus laevis oocytes. Values
are expressed as mean (six to 13 different oocytes) percentages (6S.E.M.)
of the potentiation of the control response to GABA (EC20, 2–10 mM).
3.3.3. Implications of lipophilicity in the binding at
GABAA receptors
3.4. Anticonvulsant and anesthetic activities
Some insight into the structure–affinity correlations can
be obtained by comparing [35S]TBPS binding data and
lipophilicity parameters of the propofol derivatives.
Besides compounds 6a–c, we also considered compounds
2a–c previously tested (the pyrrolidinyl acetate 2b showed
an IC50 of 6.52 mM, a value close to that of propofol,
whereas the other two derivatives were found to be
inactive up to 131024 M concentration). No general
correlation between [35S]TBPS binding data and lipo-
philicity parameters emerged, but it is worth noting that,
within each series of derivatives, the compounds with the
Compounds 6a and 6d, displaying different rates of
esterase-catalyzed hydrolysis (readily hydrolyzed and high-
ly stable, respectively) and in vitro activity (propofol-like
and bicuculline-like, respectively), were selected and test-
ed in vivo at a dose of 0.22 mmol/kg for antagonism of
convulsions induced by pentylenetetrazole (PTZ).
As shown in Table 3, compound 6a, like propofol,
completely protected the animals from the PTZ-induced
convulsions. In contrast to the in vitro GABAA receptor
activity profile (Figs. 3 and 4), which exhibited biculline-
like behavior, isonipecotate 6d appeared to protect 60% of
the animals against convulsions. Clarification of this point
deserves more elaborate pharmacodynamic and phar-
macokinetic studies. However, of the hypotheses that may
be formulated to explain such a result, we cannot rule out
9
lowest lipophilicity (log kw) are those displaying the
highest binding affinity (compounds 2b and 6a). Such a
trend might suggest the existence of a non-linear lipo-
philicity–affinity relationship within each series of prop-
ofol amino acid esters.

C. Altomare et al. / European Journal of Pharmaceutical Sciences 20 (2003) 17–26
25
Table 3
derivative having the properties of a water-soluble pro-
drug. Compound 6a was found to protect animals against
PTZ-induced convulsions, and to induce in a shorter time
an anesthetic action of duration comparable to that of the
marketed emulsion Diprivan . Its high solubility and
stability in water at physiological pH would allow freeze-
dried formulations to be developed for parenteral adminis-
tration. The susceptibility of 6a to enzymatic cleavage by
ester hydrolases in plasma and liver should afford a rapid
conversion in vivo to the parent drug. Therefore, a 17
mg/ml aqueous solution, equivalent to the commercial
oil-in-water emulsion Diprivan containing 10 mg/ml of
propofol, could be a candidate as a new anesthetic dosage
form.
The in vitro effects at GABAA receptors were also
assessed. They demonstrated that prolinate 6a, and also
piperidine-2-carboxylate 6b, behave similarly to propofol,
with IC50 values of 30–40 mM (one log unit less than
propofol). The results reported herein, together with other
data on propofol congeners (Trapani et al., 1998a; James
and Glen, 1980) and also structurally diverse compounds
(Krasowski et al., 2001), may be of significance in the
design and development of propofol-like anesthetics.
In vivo anticonvulsant and anesthetic activities of propofol ester deriva-
tives
Compound
Anticonvulsant
Loss of righting reflex, LRR
(s)b
activitya. No. of rats
protected/tested
Onset
Duration
1
6a
6d
Diprivan
10/10
10/10
6/10
114.469.5
22456252
24036592
162.764.3c,d
289614.8c
389561113
a Protection against clonic and tonic seizures induced by pentylenetet-
razole (55 mg/kg, i.p.). Compounds 6a and 6d were tested at doses
equimolar to 40 mg/kg propofol.
b Anesthetic activity measured as onset and duration of LRR
(mean6S.E.M.). Compound 6a was administered i.p. at a dose of 105
mg/kg, equimolar to a 60 mg/kg dose of propofol.
c P,0.01 vs. propofol-treated animals.
d P,0.01 vs. Diprivan -treated animals.
that propofol isonipecotate 6d, stable in vitro in rat serum
solution, could instead release in vivo into the CNS
propofol and isonipecotic acid (Anderson et al., 2001), a
well established GABAA agonist, in pharmacologically
effective amounts, thus behaving as a dual prodrug. On the
other hand, proof of the anticonvulsive activity (PTZ test)
at doses (ED50 50.32 mmol/kg; i.p. administration in
mice) close to that tested by us for 6d has recently been
obtained for a potential dual prodrug, obtained by coupling
isonipecotic acid and vigabatrin with an amide bond (Kim
et al., 2000).
Only the proline ester 6a, which is the derivative
showing the best properties as a water-soluble prodrug,
was compared to propofol (administered either as an oil/
water emulsion or as the commercial formulation
Diprivan ) for in vivo anesthetic activity at a dose of 0.34
mmol/kg. The anesthetic activity was investigated by
measuring the onset and duration of loss of the righting
reflex, in comparison with that elicited by the clinical
propofol formulation (Diprivan ), and an oil/water emul-
sion of 1 in the presence of Tween 80 (Table 3). The
induction time for loss of the righting reflex subsequent to
i.p. administration of compound 1 was notably shorter than
that observed for Diprivan . Compound 6a showed an
induction time intermediate between the emulsion formula-
tion and Diprivan , whereas the duration of anesthesia
followed the order propofol emulsion,6a¯Diprivan .
Therefore, compound 6a can be considered an efficacious
anesthetic with the same duration of action as Diprivan
but with a considerably shorter induction time than the
marketed formulation.
Acknowledgements
This research was supported financially by the Italian
Ministry of Education, Universities, Scientific and Tech-
nological Research, and by the Universities of Bari and
Cagliari.
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