Novel prodrug approach for tertiary amines: Synthesis and preliminary evaluation of N-phosphonooxymethyl prodrugs

Article abstract of DOI:10.1021/jm980539w

The synthesis and preliminary evaluation of a novel prodrug approach for improving the water solubility of drugs containing a tertiary amine group are reported. The prodrug synthesis involves a nucleophilic substitution reaction between the parent tertiary amine and a novel derivatizing reagent, di-tert- butyl chloromethyl phosphate, resulting in formation of the quaternary salt. The tertiary butyl groups are easily removed under acidic conditions with trifluoroacetic acid giving the N-phosphonooxymethyl prodrug in the free phosphoric acid form, which can subsequently be converted to the desired salt form. The synthesis was successfully applied to a model compound (quinuclidine) and to three tertiary amine-containing drugs (cinnarizine, loxapine, and amiodarone). The prodrugs were designed to undergo a two-step bioreversion process. The first step was an enzyme-catalyzed rate-determining dephosphorylation followed by spontaneous chemical breakdown of the N- hydroxymethyl intermediate to give the parent drug. Selected prodrugs were shown to be substrates for alkaline phosphatase in vitro. A preliminary in vivo study confirmed the ability of the cinnarizine prodrug to be rapidly and completely converted to cinnarizine in a beagle dog following iv administration.

Full text of DOI:10.1021/jm980539w

3094
J . Med. Chem. 1999, 42, 3094-3100
Novel P r od r u g Ap p r oa ch for Ter tia r y Am in es: Syn th esis a n d P r elim in a r y
Eva lu a tion of N-P h osp h on ooxym eth yl P r od r u gs
J effrey P. Krise,† J an Zygmunt,‡ Gunda I. Georg,‡ and Valentino J . Stella*,†
Department of Pharmaceutical Chemistry, The University of Kansas, 2095 Constant Avenue, Lawrence, Kansas 66047, and
Department of Medicinal Chemistry, The University of Kansas, Malott Hall, Lawrence, Kansas 66045
Received September 25, 1998
The synthesis and preliminary evaluation of a novel prodrug approach for improving the water
solubility of drugs containing a tertiary amine group are reported. The prodrug synthesis
involves a nucleophilic substitution reaction between the parent tertiary amine and a novel
derivatizing reagent, di-tert-butyl chloromethyl phosphate, resulting in formation of the
quaternary salt. The tertiary butyl groups are easily removed under acidic conditions with
trifluoroacetic acid giving the N-phosphonooxymethyl prodrug in the free phosphoric acid form,
which can subsequently be converted to the desired salt form. The synthesis was successfully
applied to a model compound (quinuclidine) and to three tertiary amine-containing drugs
(cinnarizine, loxapine, and amiodarone). The prodrugs were designed to undergo a two-step
bioreversion process. The first step was an enzyme-catalyzed rate-determining dephosphory-
lation followed by spontaneous chemical breakdown of the N-hydroxymethyl intermediate to
give the parent drug. Selected prodrugs were shown to be substrates for alkaline phosphatase
in vitro. A preliminary in vivo study confirmed the ability of the cinnarizine prodrug to be
rapidly and completely converted to cinnarizine in a beagle dog following iv administration.
Sch em e 1. Illustration of the Prodrug Strategy
In tr od u ction
The synthesis and preliminary evaluation of a novel
prodrug strategy for improving the water solubility of
tertiary amine-containing drugs is described. A repre-
sentation of the prodrug strategy is shown in Scheme
1. The tertiary amine 1 is chemically derivatized to
produce the polar, water-soluble prodrug 2. The prodrug
is designed to release the parent tertiary amine in vivo
through a two-step bioreversion process. The first step
(k1, rate-determining step) in prodrug bioreversion
involves a phosphatase-catalyzed dephosphorylation to
give the resultant hydroxymethyl quaternary ammo-
nium intermediate 4 and inorganic phosphate (3). The
intermediate 4, at physiological pH, is highly unstable
and spontaneously breaks down (k2) to give the parent
tertiary amine 1 plus formaldehyde (5).
Many drugs, including tertiary amines, have poor
aqueous solubilities, which can create obstacles to their
safe and effective delivery. Formulation-related toxici-
ties can occur when a drug is given parenterally.1,2 In
addition, poor and/or erratic bioavailability can occur
when a drug is given orally.3 Prodrugs of tertiary amines
have received little attention in the literature. Bodor
et al. has previously described acyloxyalkyl quaternary
ammonium derivatives of tertiary amines as “soft
drugs”,4-7 while others, albeit few, have explored qua-
ternary ammonium derivatives as water-soluble pro-
drugs for tertiary amines.8-11 Quaternary amine pro-
drugs resulting from N-phosphonooxymethyl derivitiza-
tion of the tertiary amine functionality of problematic
drugs represent a novel approach for improving the
water solubility.
Three drugs (loxapine, cinnarizine, and amiodarone)
and one model compound (quinuclidine), each containing
a tertiary amine group, were chosen to demonstrate the
synthetic feasibility of this prodrug strategy. Cinnariz-
ine is a calcium channel blocker that is clinically used
as a vasodilator and antihistaminic and antiallergic
agent.12 Cinnarizine has a pKa1 of 1.9513 and pKa2 of
7.47.14 The poor, pH-dependent, aqueous solubility of
cinnarizine is believed to be responsible for the erratic
oral bioavailability observed in dogs and in humans.15,16
Loxapine is a tricyclic dibenzoxazepine antipsychotic
agent, which is used in the treatment of schizophrenic
and psychotic disorders.17 The loxapine pKa2 is 7.5, and
the intrinsic solubility of the free base is 12.6 µg/mL.18
The drug is currently formulated for im administration
* To whom correspondence should be addressed.
† Department of Pharmaceutical Chemistry.
‡ Department of Medicinal Chemistry.
10.1021/jm980539w CCC: $18.00 © 1999 American Chemical Society
Published on Web 07/17/1999

Novel Prodrug Approach for Tertiary Amines
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 16 3095
Ch a r t 1. N-Phosphonooxymethyl Derivatives of
Quinuclidine (6), Cinnarizine (7), Loxapine (8), and
Amiodarone (9)
Sch em e 2. General Synthetic Scheme for
N-Phosphonooxymethyl Prodrugs of Tertiary Amines
with di-tert-butyl chloromethyl phosphate (11), which
results in the formation of the quaternary ammonium
phosphate-protected prodrug 12. The solvent selection
for this quaternization reaction was important. It was
found that a relatively polar aprotic solvent such as
acetonitrile worked best. The derivatizing reagent, di-
tert-butyl chloromethyl phosphate (11), was not com-
pletely stable under the elevated temperature conditions
required by less reactive amines, such as cinnarizine,
and would slowly degrade to produce hydrochloric acid
(HCl). The generation of HCl protonated any unreacted
tertiary amine and catalyzed the removal of tertiary
butyl protecting groups from both the product (12) and
the unreacted derivatizing reagent (11). This led to the
formation of multiple polar products, which made the
purification of some prodrugs difficult. To limit this
occurrence, a nonnucleophilic proton scavenger (1,2,2,6,6-
pentamethylpiperidine) was used in excess in all reac-
tions, except for quinuclidine which was sufficiently
reactive to proceed at 37 °C in a relatively short time.
Even in the presence of the proton scavenger, cinnariz-
ine and loxapine reaction products were isolated as the
monotertiary butyl-protected derivatives as opposed to
the ditertiary butyl-protected intermediates that were
isolated with quinuclidine and amiodarone. The mono-
or diester prodrugs 12 were then treated with trifluo-
roacetic acid in benzene at room temperature to remove
the tertiary butyl group(s). The resulting prodrugs in
their free acid form, 13, were isolated and characterized.
In the cases of cinnarizine and loxapine, more than one
molecule of trifluroacetic acid might have been associ-
ated with the products since they contain other basic
centers. The prodrugs can be converted to their sodium
salt by neutralization; however, the compounds were
somewhat hygroscopic, so materials were stored in the
quaternary, free acid form and all initial studies were
on these materials.
in a cosolvent consisting of propylene glycol (70% v/v)
and polysorbate 80 (5% v/v) which has been shown to
cause muscle damage.19 Amiodarone is a widely used
antiarrhythmic agent which is currently approved for
life-threatening ventricular arrhythmias.20-22 Amio-
darone has a pKa of 6.56 and also has poor aqueous
solubility.23 This necessitates an iv formulation to have
lowered pH and nonaqueous cosolvent addition.24 The
development of venous phlebitis has been reported with
the iv infusion of this formulation which is thought to
be caused through precipitation of amiodarone at the
injection site.25 When given orally, the absolute bio-
availability of amiodarone ranges from 22-86% which
has been partially attributed to the poor water solubil-
ity.26
The purpose of the current studies was to evaluate
the potential usefulness of the novel prodrug strategy
in improving the water solubility of these drugs. To meet
this goal the prodrug must have ample water solubility,
sufficient chemical stability, and a rapid and complete
bioreversion process. These issues were topics of sepa-
rate reports.18,27 At pH 7.4, the loxapine prodrug had
over a 15 000-fold improvement in solubility relative to
loxapine, and the predicted shelf life of an aqueous
formulation was nearly 2 years. Both the loxapine and
cinnarizine prodrugs appeared to be rapidly and quan-
titatively converted to their respective parent compound
following iv dosing. The specific aim of this work was
to describe the method of synthesis of the prodrugs. Also
included are preliminary in vitro reversion kinetics of
7 and 8 in the presence of isolated human placental
alkaline phosphatase and an in vivo evaluation of 7 in
a beagle dog.
The synthesis of derivatives of 11 with an improved
leaving group, compared to chlorine (i.e., p-toluene-
sulfonate, triflate), were attempted; however, these
compounds were too unstable for isolation. Derivatives
of 11 with phosphate protecting groups other than tert-
butyl may allow for these substitutions. A derivatizing
reagent with improved reactivity could potentially lead
to enhanced yields and less difficult purifications (par-
ticularly important when derivatizing weakly basic
tertiary amines).
Resu lts
Syn th esis. The structures of the quaternary N-
phosphonooxymethyl prodrugs of quinuclidine (6), cin-
narizine (7), loxapine (8), and amiodarone (9) are shown
in Chart 1. The general mechanism for the synthesis of
prodrugs is depicted in Scheme 2. The parent tertiary
amine 10 undergoes a nucleophilic substitution reaction
In Vitr o a n d in Vivo Eva lu a tion . In order for the
derivatives to behave as prodrugs they must undergo a
chemical or enzymatic bioreversion process. Alkaline

3096 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 16
Krise et al.
F igu r e 1. In vitro alkaline phosphatase-catalyzed reversion
of 7 (b) to cinnarizine free base (O) at 37 °C and pH 10.4. As
a control, levels of 7 (2) are plotted as a function of time under
the same conditions in which the buffer is devoid of alkaline
phosphatase.
F igu r e 3. Plot of cinnarizine plasma concentration versus
time following equimolar (3 µmol/kg) iv doses of cinnarizine
and 7 to a beagle dog: (b) cinnarizine levels following
cinnarizine administration; (O) cinnarizine levels following
administration of 7.
titration) and some minor impurities based on relative
peak area HPLC analysis of the sample. Similarly, in
Figure 2, the disappearance of 8 led to near-quantitative
94% molar recovery of loxapine. The 6% not recovered
could be accounted for by a 4.2% water content and
2-3% impurities based on relative peak area HPLC
analysis of the sample. NMR spectra (hydrogen, carbon,
and phosphorus) and MS as well as HPLC chromatog-
raphy all suggested that the prodrugs were quite pure;
however, in hindsight the less than quantitative recov-
ery from the alkaline phosphatase study suggested the
presence of some unaccounted for impurities in the
samples. The most likely impurities are small quantities
of chloride ion and trifluroacetic acid species.
The ability of the prodrugs to revert to parent
compound in the presence of alkaline phosphatase
clearly indicates that these prodrugs are substrates for
the enzyme. The promising in vitro reversion profiles
of the prodrugs warranted further investigation in
animals. In vivo, ideally, these prodrugs should be
nontoxic and be rapidly and quantitatively converted
to the parent compound. The ability of 7 to revert to
cinnarizine was evaluated in a single beagle dog,
crossover study. Neither injection (cinnarizine or 7)
caused observable signs of discomfort or toxicity to the
dog. Figure 3 shows the concentration versus time
profile for cinnarizine following equimolar (3 µmol/kg)
iv doses of cinnarizine and 7. A comparison of the
cinnarizine area under the plasma concentration versus
time curve was used to assess the extent of bioreversion
in the dog. The [AUC]0-8h values were 456.0 and 449.5
ngh/mL after cinnarizine and 7, respectively. Dividing
the resulting cinnarizine [AUC]0-8h after administration
of 7 by the [AUC]0-8h following cinnarizine administra-
tion gives a value of 0.98, which represents the extent
of reversion of the prodrug to parent drug. This, along
with the superimposability of the profiles, suggests that
the prodrug is both rapidly and quantitatively converted
to parent drug in dog.
F igu r e 2. In vitro alkaline phosphatase-catalyzed reversion
of 8 (b) to loxapine free base (O) at 37 °C and pH 10.4. As a
control, levels of 8 (2) are plotted as a function of time under
the same conditions in which the buffer is devoid of alkaline
phosphatase.
phosphatase is an enzyme that is found throughout
various tissues, membranes, etc.28 The described pro-
drugs were designed to be substrates for this enzyme,
and the ability of the prodrugs to generate parent drug
in the presence of the enzyme was assessed.
Figures 1 and 2 show the depletion of 7 and 8 in the
presence of alkaline phosphatase along with the simul-
taneous formation of cinnarizine and loxapine, respec-
tively. The closed triangles represent the prodrug levels
under identical conditions without the addition of
enzyme, which serves to demonstrate the chemical
stability of the prodrugs under the conditions. As seen
in Figure 1, complete disappearance of 7 led to ap-
proximately 88% apparent molar recovery of cinnarizine
free base. The 12% not recovered could be partially
accounted for by a 3.7% water content (Karl Fisher

Novel Prodrug Approach for Tertiary Amines
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 16 3097
mixture was stirred at 60 °C for 15 min and filtered. The filter
cake was washed three times with water (5 mL); all filtrates
were combined, mixed with decolorizing carbon (1.0 g), and
again stirred at 60 °C for 20 min. After filtration, the resulting
colorless solution was cooled to 0 °C (ice bath) and carefully
acidified with concentrated hydrochloric acid (7 mL) with
stirring. The precipitated di-tert-butyl phosphate was filtered,
washed with ice-cold water (10 mL), and dissolved in acetone
(100 mL). To this stirred solution in an ice bath was added 1
mol equiv of a 10% water solution of tetramethylammonium
hydroxide (24.2 mmol), and the resulting, homogeneous solu-
tion was evaporated under reduced pressure to give 7.16 g of
solid. Crystallization from refluxing dimethoxyethane gave
6.52 g of the pure tetramethylammonium di-tert-butyl phos-
phate (6.52 g, 57%) as white hygroscopic crystals. To a
refluxing solution of this phosphate (3.59 g, 12.70 mmol) in
dimethoxyethane (70 mL) was added chloroiodomethane (25
g, 141.74 mmol), and the resulting reaction mixture was
refluxed for 1.5 h. The mixture was filtered, and the filtrate
was placed under reduced pressure to remove excess chlor-
oiodomethane and solvent to yield a viscous yellow oil, which
was a mixture of 11 and bis-di-tert-butyl methyl phosphate.
The bis-di-tert-butyl methyl phosphate product was identified
by NMR and mass spectral analysis (data not shown). The two
products were easily separated via flash column chromatog-
raphy (eluent 30% ethyl acetate and 70% hexane). 11 was
isolated as a pale-gold oil (2.07 g, 35%): 1H NMR (CDCl3, 300
MHz) δ 1.51 (s, 12H), 5.63 (d, 2H, J ) 15); 31P NMR (CDCl3,
300 MHz) δ -13.9 (s); MS (FAB+, GLY) m/z 259 (M + 1). Anal.
(C9H20ClO4P) C, H: calcd 41.79, 7.79; found 41.60, 7.68.
Con clu sion
The reaction of selected tertiary amine-containing
drugs with di-tert-butyl chloromethyl phosphate fol-
lowed by deprotection led to the formation of the
N-phosphonooxymethyl prodrugs. When the parent
tertiary amine is highly nucleophilic (e.g., quinuclidine)
the prodrug can be obtained in high yield with little
purification necessary. When the amine is less nucleo-
philic (e.g., cinnarizine), the required elevated temper-
ature and long reaction times led to complex product
formations, difficult purification, and reduced yields.
Prodrugs 7 and 8 were shown to be substrates for
alkaline phosphatase, an enzyme ubiquitous to the
human body. In a preliminary in vivo dog study, 7 was
shown to produce cinnarizine rapidly and quantitatively
following intravenous administration. Further in-depth
studies evaluating this prodrug concept are reported in
separate papers.18,27 Ongoing chemical studies include
improved synthesis and purification procedures for
these polar molecules.
Exp er im en ta l Section
Syn t h et ic Ma t er ia ls. Anhydrous acetonitrile, chloro-
iodomethane, anhydrous dimethoxyethane, 1,2,2,6,6-penta-
methylpiperidine, quinuclidine, tetramethylammonium hy-
droxide 10% (w/v) aqueous solution, trifluoroacetic acid,
magnesium chloride hexahydrate, tetrabutylammonium di-
hydrogen phosphate, and zinc chloride were obtained from
Aldrich Chemical Co. (Milwaukee, WI). Cinnarizine and amio-
darone hydrochloride were obtained from Sigma Chemical Co.
(St. Louis, MO). Loxapine succinate was obtained from Re-
search Biochemicals Inc. (Natick, MA). Di-tert-butyl phosphite
was obtained from Lancaster Synthesis, Inc. (Windham, NH),
while potassium bicarbonate and glycine were obtained from
Fischer Scientific (Pittsburgh, PA). Potassium permanganate
was purchased from Mallinckrodt Chemical Works (St. Louis,
MO). Normal phase silica gel, particle size 32-63 µm, was
obtained from Selecto Scientific (Norcross, GA), while prepara-
tive TLC plates (1000-µm thickness, 20 × 20 cm) were obtained
from Alltech Associates, Inc. (Deerfield, IL). Amiodarone
hydrochloride and loxapine succinate were converted to their
free base form prior to use. All water was distilled in an all-
glass still prior to use. All other chemicals and solvents were
of reagent grade obtained from conventional sources and used
without further purification.
N-(P h osp h on ooxym eth yl)qu in u clid in iu m Tr iflu or o-
a ceta te (6). To a solution of 11 (0.16 g, 0.64 mmol) in
anhydrous acetonitrile (5 mL) maintained under argon was
added quinuclidine (0.07 g, 0.636 mmol). The reaction mixture
was capped with a rubber septum and stirred at 37 °C for 12
h. The mixture was then placed under reduced pressure to
remove solvent. To the residue was added anhydrous ethyl
ether (5 mL) with stirring to create a suspension that was
centrifuged, and the supernatant was removed. This process
was repeated three times. The remaining solid was dried under
vacuum to give di-tert-butyl N-(phosphonooxymethyl)quinu-
clidinium chloride (0.18 g, 78%) isolated as a white amorphous
1
solid: mp 88-105 °C; H NMR (CDCl3, 300 MHz) δ 1.54 (s,
18H), 2.07 (m, 6H), 2.27 (m, 1H), 3.86 (t, 6H, J ) 7.9), 5.36 (d,
2H, J ) 8.4); 31P NMR (CDCl3, 500 MHz) δ -14.97 (t, J )
19.4); MS (FAB+, GLY) m/z 334 (M+).
Deprotection of di-tert-butyl N-(phosphonooxymethyl)qui-
nuclidinium chloride (0.17 g, 0.46 mmol) was by the addition
of trifluoroacetic acid (40 µL, 0.52 mmol) in benzene (5 mL)
with stirring at room temperature for 24 h. The reaction vessel
was then placed under reduced pressure to remove excess
trifluoroacetic acid, HCl, and benzene to afford the title
compound (0.14 g, 94% yield) as a white amorphous solid: 1H
NMR (D2O, 300 MHz) δ 1.97 (m, 6H), 2.18 (m, 1H), 3.40 (t,
6H, J ) 7.9), 4.68 (d, 2H, J ) 6.8); 19F NMR (D2O, 500 MHz)
δ -77.98 (s); 31P NMR (D2O, 500 MHz) δ -2.6 (s); MS (FAB+,
GLY) 222 (M+).
An a lytica l Equ ip m en t. Nuclear magnetic resonance spec-
tra were obtained using either a Bruker AM 500 or a Varian
XL 300 instrument. Chemical shifts are reported as parts per
million downfield from tetramethylsilane or 3-(trimethylsilyl)-
1
propanesulfonic acid as internal standards for H NMR spectra
and from 85% H3PO4 as an external standard in 31P NMR
spectra. J values are given in hertz (Hz). Elemental mi-
croanalyses were performed by Desert Analytics, Tuscon, AZ.
Melting points obtained were determined in capillary tubes
on a Mel-Temp II apparatus. Mass spectral analyses were
performed by the Mass Spectral Laboratory at The University
of Kansas (Lawrence, KS). Water content was determined on
a Metrohm AG Karl Fischer coulometer (Herisam, Switzer-
land).
N-(P h osp h on ooxym eth yl)cin n a r izin iu m Tr iflu or oa c-
eta te (7). To a solution of 11 (0.178 g, 0.74 mmol) in anhydrous
acetonitrile (4 mL) maintained under argon were added
cinnarizine (0.227 g, 0.62 mmol) and 1,2,2,6,6-pentamethylpi-
peridine (125 µL, 0.74 mmol). The reaction mixture was capped
with a rubber septum and stirred at 70 °C for 6 days. The
mixture was then placed under reduced pressure to remove
the solvent. To the residue was added anhydrous ethyl ether
(5 mL) with stirring to create a suspension, which was then
centrifuged, and the supernatant was removed. This process
was repeated three times. The product was then purified using
preparative thin-layer chromatography (eluent 75% methylene
chloride and 25% methanol). The Rf value of the intermediate
was 0.7. The mono-tert-butyl N-(phosphonooxymethyl)cin-
narizinium chloride (0.033 g, 8%) was isolated as a white
amorphous solid: 1H NMR (acetonitrile-d3, 300 MHz) δ 1.35
Di-ter t-bu tyl Ch lor om eth yl P h osp h a te (11). The conver-
sion of di-tert-butyl phosphite into the corresponding phosphate
was performed using a modification of the method reported
by Zwierzak and Kluba.29 To a stirred solution, in an ice bath,
of di-tert-butyl phosphite (7.84 g, 40.36 mmol) and potassium
bicarbonate (2.42 g, 24.20 mmol) in water (35 mL) was added,
in three equal portions, finely powdered potassium perman-
ganate (4.46 g, 28.30 mmol) over 1 h followed by continued
stirring at room temperature for an additional 30 min.
Decolorizing carbon (0.6 g) was added, and the resulting

3098 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 16
Krise et al.
(s, 9H), 2.70 (m, 4H), 3.39 (m, 2H), 3.56 (m, 2H), 4.12 (d, 2H,
J ) 7.8), 4.46 (s, 1H), 5.01 (d, 2H, J ) 8.4), 6.4 (m, 1H), 6.95
(d, 1H, J ) 16), 7.3 (m, 15H); 31P NMR (acetonitrile-d3, 500
MHz) δ -4.9 (s); MS (FAB+, GLY) m/z 535 (M+).
The reaction was then placed under reduced pressure to
remove excess trifluoroacetic acid, HCl, and benzene to yield
9 as a yellow oil. This was dissolved in water (5 mL) containing
a two molar excess of sodium bicarbonate (0.026 g, 0.31 mmol)
to form the disodium salt. The aqueous solution was then
lyophilized to remove the water to yield N-(phosphonooxy-
methyl)amiodaronium sodium salt (0.16 g, 94%) as a white
hygroscopic solid. Residual water content was variable due to
the hygroscopic nature of the sample but corresponded ap-
proximately to a pentahydrate: 1H NMR (DMSO-d6, 300 MHz)
δ 0.84 (t, 3H, J ) 7.2), 0.98 (m, 2H), 1.31 (m, 6H), 1.68 (m,
2H), 2.74 (m, 2H), 3.54 (m, 4H), 3.84 (m, 2H), 4.36 (m, 2H),
4.95 (d, 2H, J ) 8.7), 7.22-7.65 (m, 4H), 8.18 (s, 2H); 31P NMR
(D2O, 500 MHz) δ 4.77 (s); MS (FAB+, NBA) m/z 756 (M+).
In Vitr o/in Vivo Eva lu a tion . Ma ter ia ls: 7 and 8 were
synthesized using the previously described procedure. Human
placental alkaline phosphatase, type XVII (14 units/mg), and
meclizine were obtained from Sigma Chemical Co. (St. Louis,
MO). The synthesis and characterization for (SBE)4M-â-CD has
been previously described.30 Magnesium chloride hexahydrate,
tetrabutylammonium dihydrogen phosphate, and zinc chloride
were obtained from Aldrich Chemical Co. (Milwaukee, WI).
Glycine was obtained from Fisher Scientific (Pittsburgh, PA).
All other chemicals and solvents were of reagent grade and
were used without further purification.
This product (0.027 g, 0.048 mmol) was mixed with trifluo-
roacetic acid (20 µL, 0.26 mmol) in benzene (1 mL) with
stirring at room temperature for 24 h to remove the tert-butyl
protecting group. The reaction was then placed under reduced
pressure to remove excess trifluoroacetic acid, HCl, and
benzene to yield 7 (0.025 g, 87%) as a white amorphous solid
(sample had 3.7% residual water): mp 140-146 °C; 1H NMR
(D2O, 300 MHz) δ 2.98 (m, 4H), 3.58 (m, 4H), 4.23 (d, 2H, J )
7.7), 4.72 (s, 1H), 4.98 (d, 2H, J ) 6.2), 6.3 (m, 1H), 7.01 (d,
1H, J ) 15) 7.2-7.6 (m, 15H); 31P NMR (acetonitrile-d3, 500
MHz) δ 2.1 (s); MS (FAB+, GLY) m/z 479 (M+). HPLC analysis
showed a single major peak accounting for the majority of the
total peak peak. There was a small peak corresponding to
cinnarizine which accounted for <1% of the total peak area.
N-(P h osp h on ooxym et h yl)loxa p in iu m Tr iflu or oa ce-
ta te (8). To a solution of 11 (0.24 g, 0.91 mmol) maintained
under argon in anhydrous acetonitrile (1 mL) were added
loxapine (0.20 g, 0.61 mmol) and 1,2,2,6,6-pentamethylpiperi-
dine (500 µL, 2.76 mmol), and the reaction mixture was capped
with a rubber septum and stirred at 50 °C for 64 h. The
mixture was then placed under reduced pressure to remove
the solvent. To the residue was added anhydrous ethyl ether
(5 mL) with stirring to create a suspension that was centri-
fuged, and the supernatant was removed. This process was
repeated three times. The product was then purified using
preparative thin-layer chromatography (eluent 90% methylene
chloride and 10% methanol). The product had an Rf value of
0.3. The mono-tert-butyl N-(phosphonooxymethyl)loxapinium
chloride was isolated as a white solid (0.08 g, 25%): 1H NMR
(D2O, 300 MHz) δ 1.41 (s, 9H), 3.22 (s, 3H), 3.4-3.8 (m, 6H),
3.85 (m, 2H), 5.02 (d, 2H, J ) 7.6) 7.20 (m, 5H), 7.42 (m, 2 H);
31P NMR (D2O, 500 MHz) δ -5.7 (t, J ) 17.5); MS (FAB+,
NBA) m/z 494 (M+).
In Vitr o En zym a tic Eva lu a tion . 1. P r oced u r e: All
experiments involving alkaline phosphatase were performed
in a pH 10.4 glycine buffer at 37 °C. The glycine buffer solution
contained 1 mM ZnCl2, 1 mM MgCl2, and 0.1 M glycine. The
final pH of the buffer was adjusted to pH 10.4 with additions
of a 2 N NaOH solution. An alkaline phosphatase stock
solution was prepared at a concentration of 1.54 units/mL in
the glycine buffer. The enzyme solution was brought to 37 °C
and spiked with a prodrug stock solution to give an initial
prodrug concentration of 264 and 230 µM for 8 and 7,
respectively. Aliquots (0.2 mL) were separated into individual
4-mL glass tubes, capped, and placed in a 37 °C shaking water
bath. Samples were removed from the bath at predetermined
time points (0, 5, 10, 15, 30, 45, 60, 75, and 90 min) and spiked
with acetonitrile (0.2 mL) which served to quench the enzy-
matic reaction and to solubilize the reaction products. As a
test for the chemical stability of the prodrugs under these
experimental conditions, the previously described procedure
was repeated with the removal of enzyme from the media. All
samples were analyzed by HPLC for both parent and prodrug
concentrations.
The mono-tert-butyl-protected N-(phosphonooxymethyl)loxa-
pinium chloride (0.08 g, 0.153 mmol) was treated with a
solution of trifluoroacetic acid (60 µL, 0.78 mmol) in benzene
(4 mL) at room temperature for 24 h to remove the remaining
tert-butyl protecting group. The reaction was then placed under
reduced pressure to remove excess trifluoroacetic acid, HCl,
and benzene to yield 8 (0.066 g, 76%) as a white amorphous
1
solid (sample had 4.2% residual water): mp 114-145 °C; H
NMR (D2O, 300 MHz) δ 3.27 (s, 3H) 3.4-4.2 (m, 8H), 5.08 (d,
2H, J ) 7.2), 7.10-7.45 (m, 7H); 31P NMR (D2O, 500 MHz) δ
-1.77 (m); MS (FAB+, TG) m/z 438 (M+). HPLC analysis
showed a single major peak accounting for 97% of the total
peak area.
2. An a lytica l: The HPLC system for all compounds con-
sisted of a Waters model 510 pump (Milford, MA), a Waters
717 autosampler (Milford, MA), an LDC analytical spec-
tromonitor 3100 variable wavelength detector, a Waters C18
symmetry column (3.9 × 150 mm; Milford, MA), a Shimadzu
CR6A integrator (Kyoto, J apan), and a column heater (Tim-
berline Instruments Inc., Boulder, CO).
N-(P h osp h on ooxym eth yl)a m iod a r on iu m Tr iflu or oa c-
eta te (9). To a solution of amiodarone (0.275 g, 0.42 mmol),
11 (0.217 g, 0.84 mmol), and 1,2,2,6,6-pentamethylpiperidine
(152 µL, 0.84 mmol) maintained under argon in anhydrous
acetonitrile (3 mL) was added sodium iodide (5 mg, 33 µmol),
and the resulting reaction mixture was capped with a rubber
septum and stirred at 40 °C for 24 h with protection from light.
The mixture was then placed under reduced pressure to
remove the solvent. To the residue was added anhydrous ethyl
ether (5 mL) with stirring to create a suspension that was
centrifuged, and the supernatant was removed. This process
was repeated three times. After vacuum-drying the di-tert-
butyl-protected N-(phosphonooxymethyl)amiodaronium chlo-
ride was obtained as a white amorphous solid (0.180 g, 47%):
1H NMR (CDCl3, 300 MHz) δ 0.92 (t, 3H, J ) 7.3), 1.30-1.85
(m, 28H), 2.89 (t, 2H, J ) 7.7), 3.88 (q, 4H, J ) 4.3), 4.4-4.6
(c, 4H), 5.47 (d, 2H, J ) 7.4), 7.3 (m, 2H), 7.49 (d, 2H, J )
8.1), 8.21 (s, 2H); 31P NMR (CDCl3, 500 MHz) δ -12.34 (t, J )
17.2); MS (FAB+, NBA) m/z 868 (M+).
For the analysis of cinnarizine and 7, the mobile phase
consisted of acetonitrile (55% v/v) and a 10 mM ammonium
dihydrogen phosphate buffer adjusted to pH 3 with phosphoric
acid (45% v/v) and was pumped at a flow rate of 0.9 mL/min.
The injection volume was 20 µL, and the detection was
ultraviolet using a wavelength of 254 nm. Under these
conditions, the retention times were 5 and 9 min for 7 and
cinnarizine, respectively.
For the analysis of loxapine and 8, the mobile phase
consisted of acetonitrile (28% v/v) and a 10 mM ammonium
dihydrogen phosphate buffer adjusted to pH 3 with phosphoric
acid (72% v/v) and was pumped at a flow rate of 0.9 mL/min.
The injection volume was 20 µL, and the detection was
ultraviolet using a wavelength of 251 nm. Under these
conditions, the retention times were 8 and 12 min for 8 and
loxapine, respectively.
The di-tert-butyl-protected N-(phosphonooxymethyl)amio-
daronium chloride (0.154 g, 0.17 mmol) was treated with
trifluoroacetic acid (60 µL, 0.78 mmol) in benzene (5 mL) at
room temperature for 24 h to remove the tert-butyl groups.
In Vivo Dog Stu d y. 1. P r ep a r a tion of for m u la tion s:
Cinnarizine for iv injection was prepared in a 10 mM phos-
phate buffer solution at pH 4.5 at a concentration of 12.5 mg/
10 mL (3.39 mM) along with 37.5 mM (SBE)4M-â-CD as a

Novel Prodrug Approach for Tertiary Amines
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 16 3099
solubilizing excipient. The cyclodextrin solution (10 mL) was
prepared first and the pH adjusted to 3.5 with HCl. Cinnariz-
ine was then added, and the solution was then sonicated for 3
h and subsequently stirred overnight. The pH was then
adjusted to 4.5 with NaOH along with the addition of 29 mg
of NaCl to adjust for isotonicity. The solution was subsequently
passed through a 0.22-µm nylon membrane filter just prior to
iv administration. The prodrug injection was prepared by
dissolving 16.97 mg of 7 in 10 mL of 0.9% NaCl sterile solution
for injection (3.39 mM). The solution was passed through a
0.22-µm nylon membrane filter just prior to injection.
2. Exp er im en ta l p r oced u r e: The evaluation of the cin-
narizine plasma concentration versus time was conducted
using a male beagle dog weighing 11.1 kg. The dog received
the cinnarizine injection followed by a 2-week washout period
and then received the prodrug injection in an equal molar
quantity. Samples were taken from the dog prior to dosing (10-
mL blank plasma) and 2, 6, 10, 20, and 40 min and 1, 2, 4, 6,
and 8 h postdosing (3 mL each). Blood samples were drawn
from either cephalic, saphenous, or jugular vein. The samples
were centrifuged for 10 min, and 1 mL of plasma was
separated, collected, and frozen at -20 °C prior to extraction.
The dog received a regular diet between experiments and was
fasted the day of the experiment. The trapezoidal calculation
was used to approximate the area under the plasma concen-
tration versus time profile from 0 to 8 h.
Technology Enterprise Corp. through the Centers of
Excellence program.
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by fluorescence using an excitation wavelength of 260 nm and
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