Bioreversible quaternary N-acyloxymethyl derivatives of the tertiary amines bupivacaine and lidocaine - Synthesis, aqueous solubility and stability in buffer, human plasma and simulated intestinal fluid

Article abstract of DOI:10.1016/j.ejps.2004.12.007

Design of water-soluble prodrugs may constitute a means to improve the oral bioavailability of drugs suffering from dissolution rate-limited absorption. The model drug bupivacaine containing a tertiary amine function has been converted into bioreversible quaternary N-acyloxymethyl derivatives. The pH-independent solubility of the N-butanoyloxymethyl derivate exceeded 1000 mg ml^-1 corresponding approximately to a 10,000-fold increase in water solubility compared to that of bupivacaine base. The kinetics of hydrolysis of the prodrugs was studied in the pH range 0.1-9.8 (37°C). Decomposition was found to follow first-order kinetics and U-shaped pH-rate profiles were constructed. The observed differences between the hydrolytic lability of the derivatives might most likely be ascribed to steric effects. In most cases, the prodrugs were quantitatively converted into bupivacaine. However, for the hydrolysis of the N-butanoyloxymethyl derivative at neutral to slightly alkaline pH parallel formation of bupivacaine (～80%) and an unknown compound X (～20%) was observed. LC-MS analysis of the latter compound suggests that an aromatic imide structure has been formed from an intramolecular acyl transfer reaction involving a nucleophilic attack of the amide nitrogen atom on the ester carbonyl carbon atom. Whereas the derivatives were poor substrates for plasma enzymes; they were hydrolyzed rapidly to parent bupivacaine in the presence of pancreatic enzymes (simulated intestinal fluid) at 37°C. The data indicate that such prodrugs possess sufficient stability in the acidic environment of the stomach to reach the small intestine in intact form where they can be cleaved efficiently by action of pancreatic enzymes prior to drug absorption. Thus, the N-acyloxymethyl approach might be of potential utility to enhance oral bioavailability of tertiary amines exhibiting pK_a values below approximately 6 and intrinsic solubilities in the low μM range.

Full text of DOI:10.1016/j.ejps.2004.12.007

European Journal of Pharmaceutical Sciences 24 (2005) 433–440
Bioreversible quaternary N-acyloxymethyl derivatives of the
tertiary amines bupivacaine and lidocaine—synthesis,
aqueous solubility and stability in buffer, human
plasma and simulated intestinal fluid
Anders Bach Nielsena, Anders Buurb, Claus Larsena, ∗
a
Department of Pharmaceutics, The Danish University of Pharmaceutical Sciences,
Universitetsparken 2, DK-2100 Copenhagen, Denmark
H. Lundbeck A/S, Ottiliavej 9, DK-2500 Copenhagen Valby, Denmark
b
Received 20 August 2004; received in revised form 25 November 2004; accepted 20 December 2004
Abstract
Design of water-soluble prodrugs may constitute a means to improve the oral bioavailability of drugs suffering from dissolution rate-limited
absorption. The model drug bupivacaine containing a tertiary amine function has been converted into bioreversible quaternary N-acyloxymethyl
derivatives. The pH-independent solubility of the N-butanoyloxymethyl derivate exceeded 1000 mg ml−1 corresponding approximately to a
10,000-fold increase in water solubility compared to that of bupivacaine base. The kinetics of hydrolysis of the prodrugs was studied in the pH
range 0.1–9.8 (37 ◦C). Decomposition was found to follow first-order kinetics and U-shaped pH-rate profiles were constructed. The observed
differences between the hydrolytic lability of the derivatives might most likely be ascribed to steric effects. In most cases, the prodrugs were
quantitatively converted into bupivacaine. However, for the hydrolysis of the N-butanoyloxymethyl derivative at neutral to slightly alkaline
pH parallel formation of bupivacaine (∼80%) and an unknown compound X (∼20%) was observed. LC–MS analysis of the latter compound
suggests that an aromatic imide structure has been formed from an intramolecular acyl transfer reaction involving a nucleophilic attack of
the amide nitrogen atom on the ester carbonyl carbon atom. Whereas the derivatives were poor substrates for plasma enzymes; they were
hydrolyzed rapidly to parent bupivacaine in the presence of pancreatic enzymes (simulated intestinal fluid) at 37 ◦C. The data indicate that
such prodrugs possess sufficient stability in the acidic environment of the stomach to reach the small intestine in intact form where they can
be cleaved efficiently by action of pancreatic enzymes prior to drug absorption. Thus, the N-acyloxymethyl approach might be of potential
utility to enhance oral bioavailability of tertiary amines exhibiting pKa values below approximately 6 and intrinsic solubilities in the low ␮M
range.
© 2005 Elsevier B.V. All rights reserved.
Keywords: Quaternary N-acyloxymethyl derivatives; Bupivacaine; Lidocaine; Solubility; Stability; Prodrugs
1. Introduction
to enhance aqueous solubility of tertiary amines (Davidsen
et al., 1994; Vinogradova et al., 1980). Together with the
more recent N-phosphonooxymethyl approach (Krise et al.,
1999a,b,c) such quaternary prodrugs, potentially feasible for
parenteral administration, have been reported to exhibit ex-
cellent solubility properties and rapid conversion to the par-
ent drug in the blood. In analogy, this type of transient en-
hancement of water solubility might also, in theory, be ex-
ploited to improve oral absorption of poorly soluble tertiary
Although bioreversible derivatisation of drugs possess-
ing tertiary amine functionality in the form of quaternary
N-acyloxyalkyl derivatives is well known (Bodor, 1985) this
prodrug strategy has gained modest recognition as a means
∗
Corresponding author. Tel.: +45 3530 6466; fax: +45 3530 6012.
E-mail address: csl@dfuni.dk (C. Larsen).
0928-0987/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejps.2004.12.007

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A.B. Nielsen et al. / European Journal of Pharmaceutical Sciences 24 (2005) 433–440
Scheme 1.
amines suffering from dissolution rate-limited bioavailabil-
ity. In contrast to the parent amine, the quaternary ammonium
derivatives are likely to show pH-independent aqueous solu-
bility (Nielsen et al., 2005). The permanently charged com-
pounds are expected to possess poor biomembrane transport
properties and the strategy (Scheme 1) therefore, relies on
prodrug bioconversion in the GI-tract preferably affected by
brush border or pancreatic enzymes prior to drug absorption.
Fast enzyme-mediated regeneration may create transient su-
persaturated drug solutions at the apical membrane favour-
ing passive drug absorption (Heimbach, 2003). Also in case
of drug precipitation in the GI-tract facile dissolution and
subsequently absorption may result from formation of amor-
phousmicroparticleshavingalargesurfacearea(Graneroand
Amidon, 2003).
Recently, we have investigated the combined approach
of N-alkylation and salt formation to enhance aqueous
solubility of tertiary amines using bupivacaine as a model
drug (Nielsen et al., 2005). As compared to the parent drug
substantial improvement of the water solubility of these
non-prodrug derivatives of bupivacaine was achieved. In
this report, we want to present the synthesis of bioreversible
quaternary N-acyloxymethyl derivatives of bupivacaine,
their solubility properties and pH-dependent hydrolysis rates
together with regeneration rates of bupivacaine in plasma and
simulated intestinal fluid. Further, for comparison reasons
the chemical stability of a quaternary prodrug derivative
of lidocaine (containing a terminal aliphatic tertiary amine
function) has been determined.
2.2. Methods
2.2.1. Procedure for the synthesis of alkanoyloxymethyl
chlorides
Alkanoyloxymethyl chlorides were synthesized as
previously desribed (Nudelman et al., 2001; Rasmussen
and Leonard, 1967) with minor modifications. In short, a
mixture of alkanoyl-chloride (2 mol), paraformaldehyde
(60.1 g, 2 mol) and zinc chloride (0.5 g, cat.) were stirred
under nitrogen for a 40 min period at room temperature after
which the temperature was raised to 50–60 ◦C for 24 h. The
crude product was purified by fractional distillation at about
100 mbar. Progress of reactions was followed by NMR.
Acetyloxymethyl chloride: 1H NMR (CDCl3): δ 2.19 (s,
3H, CH3), 5.72 (s, 2H, CH2); propanoyloxymethyl chlo-
ride: 1H NMR (CDCl3): δ 1.20 (t, 3H, CH3), 2.45 (q,
2H, CO–CH2–C), 5.75 (s, 2H, Cl–CH2–O); isobutanoy-
loxymethyl chloride: 1H NMR (CDCl3): δ 1.15 (d, 6H,
2 × CH3), 2.55 (h, 1H, CH), 5.65 (s, 2H, CH2).
2.2.2. Procedure for the synthesis of quaternary
N-acyloxymethyl chloride salts of bupivacaine and
lidocaine
Racemic bupivacaine base was obtained from bupivacaine
hydrochloride as previously described (Larsen et al., 2002).
In essence the N-acyloxymethyl derivatives of bupivacaine
and lidocaine were synthesised according to Bodor et al.
(1980) and Waranis and Sloan (1987). To a stirred solu-
tion of bupivacaine base (5.0 g, 17.3 mmol) or lidocaine base
(5.0 g, 21.3 mmol) in an organic solvent (20 ml) was added
alkanoyloxymethyl chloride (2 eqv.). The reaction mixture
was refluxed under nitrogen at 50 ◦C. After cooling the iso-
lated crude product was purified by recrystallization in most
cases. No efforts have been given to optimize the syntheses.
Progress of reactions was followed by LC–MS. The chemical
structures of the synthesized prodrug derivatives are depicted
in Fig. 1a and b.
2. Materials and methods
2.1. Materials
Reagents (reagent grade) used for the syntheses: bupiva-
caine hydrochloride and lidocaine, Unikem (Copenhagen,
Denmark). Zinc chloride >98%, propionyl chloride 99%,
chloromethyl pivaloate 97%, Aldrich (Steinheim, Germany).
Paraformaldehyde, acetyl chloride, isobutanoyl chloride,
Fluka (Buchs, Switzerland). Chloromethyl butyrate 99%,
Acros Organics (New Jersey, USA). Further, Pancreatin P-
1625 was purchased from Sigma–Aldrich Chemie Gmbh
(Steinheim, Germany). All other chemicals were of analyt-
ical grade. Demineralised water was used throughout the
study.
2.2.2.1. N-Butanoyloxymethyl bupivacaine chloride salt.
Solvent: acetonitrile, reflux time: 14 days. After filtration the
supernatant was evaporated in vacuo and the oil residue was
recrystallized from ethyl acetate. The crystals were treated
with small amounts of ethyl acetate containing triethylamine
to remove unreacted bupivacaine and dried under vacuum.
Yield: 15%; mp 135–137 ◦C; H NMR (CDCl3): δ 0.9–1.1
(dt, 6H, CH3 in butyl and CH3 in butanoyl), 1.4–1.5 (m,
1

A.B. Nielsen et al. / European Journal of Pharmaceutical Sciences 24 (2005) 433–440
435
1
obtained as an amorphous powder. Yield: 10%; H NMR
(CDCl3): δ 1.3 (s, 9H, 3 × CH3 in pivaloyl), 1.5–1.6 (t, 6H,
2 × N–C–CH3), 2.3 (s, 6H, 2 × CH3 on phenyl), 3.6–3.7
(m, 4H, 2 × N–CH2–C), 5.1 (s, 2H, N–CH2–CO), 5.7 (s,
2H, N–CH2–O), 7.0–7.1 (m, 3H, phenyl), 11.1–11.2 (s, 1H,
NH), LC–MS: m/z 349.5, calculated molecular weight of
cation: 349.1.
Fig. 1. Structure of N-acyloxymethyl prodrugs of (a) bupivacaine and (b)
lidocaine.
2.2.2.5. Cyclic N-Mannich base like structures formed from
reactingbupivacaine with acetyl- and propanoyloxymethyl
chloride. Solvent: acetone, reflux time: 11 days. The pre-
cipitate formed was isolated, washed with ethyl ether, and
triturated with ethyl acetate containing triethylamine. After
filtration the solid was recrystallized from acetonitrile. Yield:
15%; mp 129–135 ◦C; 1H NMR (MeOD): δ 1.1 (t, 3H, CH3
in butyl), 1.4–1.6 (m, 2H, CH2 next to CH3 in butyl), 1.8–2.2
(m, 6H, 3 × CH2), 2.3 (s, 6H, 2 × CH3 on phenyl), 2.4–2.6
(m, 2H, CH2 next to CH), 3.5–4.1 (m, 4H, N(CH2)2), 4.8 (m,
1H, CH), 5.8–6.0 (m, 2H, NCH2N), 7.1–7.2 (m, 3H, phenyl);
LC–MS: m/z 301.2, calculated molecular weight of cation:
301.5.
2H, CH2 next to CH3 in butyl), 1.7–1.8 (m, 2H, CH2 next
to CH3 in butanoyl), 1.9–2.1 (m, 6H, 3 × CH2), 2.3 (s,
6H, 2 × CH3 on phenyl), 2.3–2.6 (m, 4H, CH2 next to CH
and CO–CH2–C), 3.3–3.9 (m, 4H, N(CH2)2), 5.8–5.9 (dd,
2H, NCH2O), 6.3 (m, 1H, CH), 7.0–7.1 (m, 3H, phenyl),
11.2–11.3 (s, 1H, NH); LC–MS: m/z 389.2, calculated molec-
ular weight of cation: 389.6. HPLC analysis showed a single
major peak accounting for 98% of the total peak area.
2.2.2.2. N-Isobutanoyloxymethyl bupivacaine chloride salt.
Solvent: 2-butanone, reflux time: 14 days. The precipitate
formed was isolated, washed with ethyl ether and dispersed
in ethyl acetate containing triethylamine. After filtration,
the solid was taken up in acetone, filtered to remove rem-
nants of triethylammonium chloride. The crystalline deriva-
tive was obtained from evaporation of the acetone solution
under reduced pressure. Yield: 20%; mp 78–80 ◦C; 1H NMR
(CDCl3): δ 1.0–1.1 (t, 3H, CH3 in butyl), 1.2–1.3 (t, 6H,
2 × CH3 in isobutanoyl), 1.4–1.5 (m, 2H, CH2 next to CH3
in butyl), 1.9–2.1 (m, 6H, 3 × CH2), 2.3 (s, 6H, 2 × CH3 on
phenyl), 2.3–2.6 (m, 2H, CH2 next to CH), 2.8 (m, 1H, CH
in isobutanoyl), 3.4–3.9 (m, 4H, N(CH2)2), 5.8–6.0 (dd, 2H,
NCH2O), 6.3 (m, 1H, CH), 7.0–7.1 (m, 3H, phenyl), 11.2 (s,
1H, NH); LC–MS: m/z 389.3, calculated molecular weight
of cation: 389.6. HPLC analysis showed a single major peak
accounting for 98% of the total peak area.
2.2.3. Solubility studies
Estimation of the solubility of the N-acyloxymethyl bupi-
vacaine chloride salts was done in a semi-quantitative manner
in duplicate, due to low stability. To 10.0 mg of the respective
derivatives in 1.5 ml glass vials water was added stepwise in
small portions until a clear solution was obtained. After each
addition of water the solutions were allowed to equilibrate
for 1 h at room temperature.
The buffer free pH-solubility profile (37 ◦C) was con-
structed from two parallel experiments by addition of ap-
proximately 5 mg bupivacaine base to 5 ml 0.1 M KCl. After
equilibration for 24 h pH was measured and 25 ␮l of the su-
pernatant was withdrawn for HPLC analysis. 0.1 M HCl (0.1
equivalent relative to the initial amount of bupivacaine) was
added and the suspension was allowed to equilibrate for ad-
ditional 24 h. Successive additions of hydrochloric acid were
continued until pH of the suspension reached 7.
2.2.2.3. N-Pivaloyloxymethyl bupivacaine chloride salt.
Solvent: acetone, reflux time: 14 days. The precipitate formed
was washed with ethyl ether and recrystallized from acetone.
The crystals were treated with small amounts of ethyl ac-
etate containing triethylamine to remove remaining bupiva-
caine and dried under vacuum. Yield: 20%; mp 163–164 ◦C;
1H NMR (CDCl3): δ 1.0–1.1 (t, 3H, CH3 in butyl), 1.3 (s,
9H, 3 × CH3 in pivaloyl), 1.4–1.5 (m, 2H, CH2 next to CH3
in butyl), 1.9–2.2 (m, 6H, 3 × CH2), 2.3 (s, 6H, 2 × CH3
on phenyl), 2.3–2.5 (m, 2H, CH2 next to CH), 3.3–3.9 (m,
4H, N(CH2)2), 5.8–6.0 (dd, 2H, NCH2O), 6.4 (m, 1H, CH),
7.0–7.1 (m, 3H, phenyl), 11.2–11.3 (s, 1H, NH); LC–MS: m/z
403.3, calculated molecular weight of cation: 403.6. HPLC
analysis showed a major single peak accounting for 99% of
the total peak area.
2.2.4. Stability measurements in aqueous solutions
The hydrolytic lability of the N-acyloxymethyl derivatives
of bupivacaine and lidocaine was investigated in buffer so-
lutions covering the pH range 0.1–9.8 at 37 0.5 ◦C. The
0.02 M buffers used were acetate (pH 5.0), phosphate (pH
3.0, 6.9 and 7.4) and borate (pH 8.5 and 9.75). Below pH 1.1,
hydrochloric acid was used. A constant ionic strength of 0.5
was maintained for each buffer, wherever possible, by adding
a calculated amount of potassium chloride. In case of the N-
isobutanoyloxymethyl bupivacaine derivative, the influence
of buffer concentration (0.02–0.10 M) on the degradation rate
was studied in the pH range 6.90–8.50 at 37 0.5 ◦C. The
reactions were initiated by adding 15 ␮l of a 10−2 M stock
solution of the respective derivative in acetonitrile to 1.5 ml
of preheated buffer solution in auto sampler vials resulting
2.2.2.4. N-Pivaloyloxymethyl lidocaine chloride salt.
Solvent: acetonitrile, reflux time: 14 days. The salt was

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A.B. Nielsen et al. / European Journal of Pharmaceutical Sciences 24 (2005) 433–440
in a final concentration of 10−4 M. The reaction solutions
were kept at constant temperature by using a Peltier ele-
ment and at appropriate times samples were withdrawn and
chromatographed immediately. Pseudo-first-order rate con-
stants for the hydrolytic degradation were in most cases de-
termined from the slope of linear plots of the logarithm of
intact prodrug against time. For slowly proceeding reactions
of the N-pivaloyloxymethyl bupivacaine salt (pH 1.14–5.00)
the pseudo-first-order rate constants were obtained from em-
ploying the initial-rate method (Connors, 1990).
HPLC column: Symmetry C-18 (3.5 ␮m, 4.6 mm ×
30 mm). Mobile phase A: water containing 0.05% TFA. Mo-
bile phase B: acetonitrile containing 5% water and 0.035%
TFA. Gradient: 10–100% B in 4 min; 10% B in 1 min with a
total run time of 5 min. The injection volume was 10 ␮l and
the flow rate was 2 ml/min. The UV detection was performed
at 254 nm using a Shimatsu LC10ADDvp UV-detector.
3. Results and discussion
3.1. Synthesis
2.2.5. Stability measurements in human plasma and
simulated intestinal fluid
Parent bupivacaine possesses a chiral centre. By N-
acyloxymethylation, resulting in quaternary ammonium
salts, another chiral centre is introduced allowing the pres-
ence of four stereoisomers. Reversed-phase HPLC analyses
revealed only one major peak strongly suggesting that the ob-
tained N-acyloxymethyl derivatives consisted of one of the
two enantiomeric pairs. The presence of only one of the four
isomers has been excluded since the compounds gave op-
tical rotation angles almost equal to zero. This observation
may indicate that formation of one of the pairs is favoured
or might be related to recrystallization process. To this end,
recrystallization of N-methyl bupivacaine iodide from a mix-
ture containing all four isomers resulted in isolation of one of
the enantiomeric pairs (Nielsen et al., 2005). HPLC analyses
revealed salt purities exceeding 98% with a content of par-
ent bupivacaine corresponding to about 1.5% or less. Upon
total hydrolysis in 0.1N NaOH almost equimolar amounts of
bupivacaine were recovered in case of the isobutanoyl- and
pivaloyloxymethyl derivatives. For the butanoyloxymethyl
salt liberated parent drug only accounted for 90% of the ex-
pected amount due to contamination with triethylammonium
chloride as observed by NMR.
Reacting bupivacaine with acetyl- and propanoy-
loxymethyl chloride, respectively, under conditions similar
to those of the other alkyl halides resulted in the formation
of the 4-imidazolidinone shown in Fig. 2 as evidenced
by NMR. In a prodrug perspective the latter compound
appeared uninteresting due to high hydrolytic stability in the
pH range 1–13. Prilocaine, containing a secondary amine
function, reacts easily with formaldehyde or acetaldehyde
to yield the corresponding 4-imidazolidinones (Larsen et
The rates of regeneration of bupivacaine and lidocaine
from the N-acyloxymethyl prodrugs were studied in hu-
man plasma 67 mM phosphate buffer pH 7.4 (4:1, v/v) at
37 0.5 ◦C. In addition, hydrolysis rates of the bupivacaine
derivatives were determined in simulated intestinal fluid pH
7.50 at 37 0.5 ◦C. The simulated intestinal fluid was pre-
pared according to USP (The United States Pharmacopeia)
and centrifuged prior to use. Reactions were initiated by ad-
dition of 60 ␮l of a 10−2 M stock solution in acetonitrile to
1.5 ml of the latter two matrices. At appropriate times sam-
ples were taken, deproteinized by addition of two volumes
of acetonitrile. After centrifugation at 14,500 rpm for 5 min
the supernatant was diluted with mobile phase (1:1, v/v) and
analysed by HPLC. Pseudo-first-order rate constants for the
degradation were calculated from slopes of linear plots of the
logarithm of intact prodrug against time. Average rate con-
stants were calculated from experiments done in duplicate.
2.2.6. HPLC analysis
The Merck–Hitachi LaChrom system consisted of a L-
7300columnoven, aL-7400UVdetector, aD-7000interface,
a L-7200 auto sampler and a L-7100 pump equipped with
a Waters reversed-phase SymmetryShieldTM RP18 column
(5 ␮m, 4.6 mm × 150 mm) (Milford, Massachusetts, USA).
Mobile phases consisted of acetonitrile 0.1% phosphoric acid
(20:80 or 25:75, v/v), which were made 1 mM with respect
to triethylamine. The flow rate was maintained at 1.0 ml/min
and the column effluent was monitored at 220 nm.
2.2.7. 1H NMR analysis
1H NMR spectra were recorded on a 500-MHz instrument
(Bruker-Spectrospin 500). Chemical shifts are expressed in
part per million (δ) without internal standard.
2.2.8. LC–MS analysis
The mass spectra were recorded on an Applied Biosys-
tems (Foster city, USA) API150ex single quadrupole mass
spectrometer with atmospheric pressure photoionization ion-
source (APPI). The mass spectrometer was operated with
positive polarity with ion monitoring between 100 and
1000 amu. Further setting: OR/RNG 20/200 V; OR/RNG
5/100 V; temperature 450 ◦C.
Fig. 2. Structure of the cyclic N-Mannich base like product formed from
reacting bupivacaine with acetyl- and propanoyloxymethyl chloride.

A.B. Nielsen et al. / European Journal of Pharmaceutical Sciences 24 (2005) 433–440
437
Table 1
Rate constants for the hydrogen ion catalysed (kH), the spontaneous (k0), and the hydroxide ion catalysed (kOH) hydrolysis of various N-acyloxymethyl
bupivacaine prodrugs and N-pivaloyloxymethyl lidocaine prodrug at 37 0.5 ◦C (µ = 0.5) and aqueous solubility at room temperature
Compound
kH (M−1 h−1
)
k0 (h−1
)
kOH (M−1 h−1
)
Solubilityb (mM)
N-Butanoyloxymethyl bupivacaine
N-Isobutanoyloxymethyl bupivacaine
N-Pivaloyloxymethyl bupivacaine
N-Pivaloyloxymethyl lidocaine
Bupivacaine base
0.209
0.178
0.0197
0.0271
n.d.
1.36 × 105
1.18 × 105
2.53 × 104
4.62 × 104
>2350
>2350
91–114
n.d.
9.24 × 10−4
4.37 × 10−4
n.d.
0.24
Bupivacaine (pH = 7.4)
Bupivacaine (pH < 6)
1.45
90a
n.d., not determined.
a
˚
Average from two different reports (Friberger and Aberg, 1971; Shah and Maniar, 1993).
Average values obtained from experiments done in duplicate.
b
al., 2003). The N-alkylations of bupivacaine were performed
the aqueous solubility of the model drug bupivacaine
possessing a tertiary amine function (Nielsen et al., 2005).
For the iodide salts of these chemically stable quaternary
ammonium derivatives the solubility decreased moderately
(about a factor of 4) with increasing chain length of the alkyl
substituent going from methyl to butyl. In case of N-methyl
bupivacaine, the choice of anionic counterion significantly
influenced the water solubility that exceeded 1000 mg ml−1
(above 2.7 M) for the mesylate, acetate and chloride salts.
Solubilities of the same order of magnitude have been de-
termined for the chloride salts of the N-butanoyloxymethyl
and N-isobutanoyloxymethyl derivatives of bupivacaine
with St values corresponding to about 2.3 M whereas that of
the quaternary N-pivaloyloxymethyl chloride salt amounted
to approximately 100 mM (Table 1). Although based on
a limited number of observations the data may suggest
that N-alkylation of a tertiary amine introducing either
simple alkyl or acyloxymethyl substituents of comparable
molecular size may result in permanently charged derivatives
exhibiting quite similar pH-independent aqueous solubili-
ties. For pyrrolothiazole, an unsaturated nitrogen-containing
compound, the corresponding N-methyl pyridinium salt and
various N-acyloxyalkyl pyridinium salts all exhibited a water
solubility at pH 3 of about 32 mM or greater (Davidsen et al.,
1994). The solubility of the starting tertiary amine (pKa of
about 5.2) at pH 7 was reported to be of the order of 2 ␮M. As-
suming that the latter value is close to the intrinsic solubility
of pyrrolothiazole, N-acyloxyalkylation imparts an approxi-
mately 16,000-fold increase in water solubility relative to the
unprotonated parent amine. In the present study, an intrinsic
solubility of bupivacaine of 0.24 mM (0.05 M carbonate
buffer pH 10.5, 37 ◦C) was calculated which is in fair agree-
ment with the So value of 0.31 mM obtained under slightly
different conditions (Nakano, 1979; Nakano et al., 1978).
Thus, for the most soluble water-soluble N-acyloxymethyl
derivatives of bupivacaine the ratio St/So amounts at least
to 9800. Similar St/So ratios in the range of 10,000–15,000
have been found for a N-acyloxymethyl derivative of an an-
timaleria drug (Bodor, 1981) and a N-phosphonooxymethyl
prodrug of loxapine (Krise et al., 1999b). In general, the
solubility of amines increases with decreasing pH, as long
as the solubility product of the protonated amine and the
under dry conditions and therefore, formaldehyde should
not be formed during the syntheses. In a separate experi-
ment, reacting bupivacaine free base with 10 times excess
of formaldehyde under conditions comparable to those
employed in the alkylation reactions with the ␣-halo esters,
the 4-imidazolidinone (Fig. 2) was not formed as evidenced
by HPLC analysis. The latter observation suggests that the
cyclic structure arises from the respective N-acyloxymethyl
bupivacaine derivatives through an intramolecular reaction.
Intramolecular cyclization is favoured by formation of five-
and six-membered rings and since the nucleophilic character
of the anilide nitrogen atom is increased by the two methyl
groups of the benzene ring, a possible mechanistical pathway
might involve nucleophilic attack of the amide nitrogen
atom on the N–CH2–O–carbon atom with abstraction of
carboxylate (Scheme 2a). This intramolecular cyclization is
expected to be sensitive to steric hindrance that might explain
why 4-imidazolidinone only is formed from N-acetyl- and
N-propanoyloxymethyl bupivacaine.
3.2. Solubility
Recently, we have shown that the combined approach of
N-alkylation and salt formation was effective in improving
Scheme 2.

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A.B. Nielsen et al. / European Journal of Pharmaceutical Sciences 24 (2005) 433–440
Fig. 4. pH-rate profiles for the hydrolysis of N-butanoyloxymethyl (ꢀ), N-
Fig. 3. Buffer free pH-solubility profile of bupivacaine (37 ◦C).
isobutanoyloxymethyl (ꢀ), N-pivaloyloxymethyl bupivacaine chloride (᭹)
and N-pivaloyloxymethyl lidocaine chloride (ꢁ) in aqueous buffer solutions
at 37 0.5 ◦C and µ = 0.5. The solid lines have been calculated from Eq.
(3). k(obs) refers to the observed pseudo-first-order rate constant (min−1).
particular counterion is not yet reached, according to
St = So (1 + 10pK − pH
)
(1)
a
degradation of the compounds, as followed by monitoring
the concentration of intact prodrug as a function of time,
displayed strict pseudo-first-order kinetics in most cases for
more than two half-lives. For the slowly proceeding hydrol-
ysis reactions of N-pivaloyloxymethyl bupivacaine chloride
(pH 1–5) first-order rate constants were derived by using the
initial rate method (Connors, 1990). At pH 1.14 identical rate
constants for the latter derivative were obtained by employing
the two kinetic approaches.
The pH-dependent rate of hydrolysis of the N-
acyloxymethyl derivatives is shown in Fig. 4. The typical
U-shaped profiles can be accounted for in terms of specific
acid catalysis (kH), water catalysis (k0) and specific base-
catalysis (kOH) of the hydrolysis
where St refers to the total solubility of the compound at a
given pH value. Thus, in the pH range 5–7 prevailing in the
small intestine the determinants of St is expected to be the
intrinsic solubility and pKa. The buffer free pH–solubility
profile (37 ◦C) shown in Fig. 3 was constructed from two
parallel experiments by addition of approximately 5 mg
bupivacaine base to 5 ml 0.1 M KCl (see Section 2.2.3). With
the knowledge of So the ionization constant of bupivacaine
might be calculated from the expression
St
aH
Ka
− 1 =
(2)
So
where aH refers to the hydrogen ion activity. From the linear
plot of the left side of Eq. (2) against the hydrogen activity a
Ka of 10−8.12 was calculated which is in excellent agreement
kobs = kHaH + k0 + kOHaOH
(3)
a value of 10−8.1 previously reported (Friberger and Aberg,
˚
where aH and aOH refer to the hydrogen and hydroxide ion
activities, respectively. Slopes of the pH-rate profiles close
to −1 and +1 in the acidic and the weakly alkaline region,
respectively, indicate that general acid–base catalysis is neg-
ligible or are of equal size under the experimental condi-
tions used. The rate constants were obtained by fitting to Eq.
(3) using SigmaPlot 2000 (SPSS Inc., Chicago, IL, USA)
(Table 1). In Fig. 4, the solid curves for N-isobutanoyl- and
N-pivaloyloxymethyl bupivacaine were constructed from Eq.
(3). The observed agreement between the fitted curve and
experimental data points strongly supports that Eq. (3) ad-
equately describes the degradation kinetics of the prodrugs.
Similar pH-rate profiles have previously been reported for
N-acyloxyalkyl derivatives of 5-fluorouracil (Buur et al.,
1985) and thiabendazole (Nielsen et al., 1992). The hy-
drolysis of N-acyloxyalkyl derivatives is proposed to take
place via a two-step reaction (Taylor and Sloan, 1998) with
1971). Using Eq. (1) the total solubility of bupivacaine
at pH 6 corresponds to about 32 mM far exceeding the
solubility limits of about 1 mg ml−1 indicative of potential
occurrence of oral absorption problems (Wells, 1988). It
appears therefore, plausible to suggest that circumvention of
poor oral bioavailability of tertiary amines due to dissolution
rate-limited absorption by using the N-acyloxymethyl
prodrug approach only is feasible for such amine-containing
drugs characterized by pKa values below approximately 6
and intrinsic solubilities in the low ␮M range.
3.3. Stability in aqueous solution
The kinetics of hydrolysis of the N-acyloxymethyl bupiva-
caine derivatives was studied in the pH range 0.1–9.8 (37 ◦C).
0.02 M buffer solutions were used whenever possible. The

A.B. Nielsen et al. / European Journal of Pharmaceutical Sciences 24 (2005) 433–440
439
Table 2
Rate data for the decomposition of various N-acyloxymethyl bupivacaine prodrugs and N-pivaloyloxymethyl lidocaine prodrug in buffer pH 7.40, 80% human
plasma and simulated intestinal fluid pH 7.5 (SIF) at 37 0.5 ◦C (half-lives (t1/2)a are in min)
Compound
Buffer, pH = 7.4 (t1/2
)
80% Human plasma (t1/2
)
SIF (t1/2)
N-Butanoyloxymethyl bupivacaine
N-Isobutanoyloxymethyl bupivacaine
N-Pivaloyloxymethyl bupivacaine
N-Pivaloyloxymethyl lidocaine
a
464
558
3022
1253
66
96
660
280
6
24
192
102
Average values obtained from experiments done in duplicate.
the rate-determining step being the cleavage of the ester
group to give an N-hydroxymethyl derivate, which decom-
poses spontaneously into formaldehyde and the parent com-
pound. The rate-determining cleavage of the ester bonds is
controlled by steric and/or electronic factors. The polar ef-
fects of the selected acyl groups are almost identical. Thus,
the observed differences between the reactivity of the N-
acyloxymethyl bupivacaine derivatives can primarily be as-
cribed to steric effects in the accordance with the fact that the
N-butanoyloxymethyl derivative of bupivacaine degrades six
to seven times faster than the N-pivaloyloxymethyl deriva-
tive at pH 7.4 (Table 2). Similar difference in reactivity has
been observed for corresponding bioreversible derivatives of
5-fluorouracil (Mollgaard et al., 1982). In contrast to the
bupivacaine prodrugs, the quaternary nitrogen atom of N-
pivaloyloxymethyl lidocaine is not locked up in a ring sys-
tem. The hydrolytic lability of the latter compound is slightly
enhanced in comparison to the corresponding bupivacaine
prodrug (Table 1).
In most hydrolysis reactions, the disappearance of the
prodrug was accompanied by simultaneous and quantita-
tive formation of parent bupivacaine. However, by the de-
composition of N-butanoyloxymethyl bupivacaine at neu-
tral or slightly alkaline pH parallel formation of bupiva-
caine (about 80%) and an unidentified compound (about
20%) was observed. The latter compound eluted with a
greater retention time by HPLC than the starting deriva-
tive and 4-imidazolidinone (Fig. 2). From the time courses
for disappearance and formation of the various species at
pH 7.4 (Fig. 5) it is seen that compound X slowly decom-
poses to parent bupivacaine. Treating the terminal part of
the descending curvature representing degradation of com-
pound X according to first-order kinetics a rough estimate
of kobs of 0.00022 min−1 was calculated (t1/2 = 53 h). By
LC–MS a molecular mass of compound X of 359.1 was
found suggesting that an intramolecular acyl transfer reaction
has taken place (Scheme 2b) leading to an aromatic imide
structure that is expected to be slowly hydrolyzed to par-
ent bupivacaine at slightly alkaline conditions. Formation of
seven-membered cyclic transition states is less favourable
in accordance with the observation that hydrolysis of the
N-butanoyloxymethyl ester bond is the major degradation
pathwayforN-butanoyloxymethylbupivacaine. Formationof
such aromatic imide type structures was not observed during
the degradation of the two other N-acyloxymethyl derivatives
most likely reflecting steric hindrance of the intramolecular
reaction.
3.4. Bupivacaine regeneration rates in biological media
The stability of the N-acyloxymethyl derivatives was stud-
ied in 80% human plasma and simulated intestinal fluid (SIF,
pH = 7.5) at 37 ◦C. Pseudo-first-order kinetics was observed
for all the degradation experiments. Half-lives for regen-
eration of bupivacaine are listed in Table 2 together with
t1/2 values calculated for the hydrolysis rates in buffer solu-
tion pH = 7.4. Whereas all the N-acyloxymethyl bupivacaine
prodrugs are susceptile to undergo significant SIF enzyme-
mediatedcleavagethederivativesappeartobepoorsubstrates
for plasma hydrolases taken into consideration that the lot of
80% human plasma used, for unknown reasons, had a pH
value of about 7.8. As expected the less bulky derivatives are
more readily hydrolysed than N-pivaloyloxymethyl bupiva-
caine. Similar tendency has been found for the conversion
of N-acyloxyalkyl pyridinium salts to the parent unsaturated
amine effected by plasma serine esterases (Davidsen et al.,
1994). In comparison to plasma the rate of bupivacaine and
lidocaine liberation from the prodrugs in SIF proceeds 3–10
times faster.
The presented data imply that substantial improvement of
water solubility of tertiary amines can be accomplished by N-
acyloxymethylation and that such prodrugs possess sufficient
stability in the acidic environment of the stomach to reach
Fig. 5. Decomposition of N-butanoyloxymethyl bupivacaine chloride in
0.02 M phosphate buffer pH 7.40 (37 0.5 ◦C and µ = 0.5). Time courses
for disappearance/formation of N-butanoyloxymethyl bupivacaine chloride
(ꢀ), compound X (᭹) and bupivacaine (ꢂ).

440
A.B. Nielsen et al. / European Journal of Pharmaceutical Sciences 24 (2005) 433–440
the small intestine in intact form where they can be cleaved
efficiently by action of pancreatic enzymes prior to drug ab-
sorption. Studies along these lines including bioavailability
studies in the dog are presently carried out in this laboratory
employing a tertiary amine possessing an intrinsic solubility
in the nanomolar range.
N-phosphonooxymethyl prodrugs in rats and dogs. J. Pharm. Sci. 88,
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Krise, J.P., Narisawa, S., Stella, V.J., 1999b. A novel prodrug approach
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Krise, J.P., Zygmunt, J., Georg, G.I., Stella, V.J., 1999c. Novel prodrug
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Dr. Claus Cornett is acknowledged for conducting and
analysing the NMR spectra. Professor Mikael Begtrup is ac-
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mechanisms. The Department of Research and Development
at H. Lundbeck A/S is acknowledged for their help with syn-
theses. The study has been supported by the Drug Research
Academy, Copenhagen Denmark.
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