Synthesis and in vitro evaluation of novel morpholinyl- and methylpiperazinylacyloxyalkyl prodrugs of 2-(6-methoxy,2-naphthyl)propionic acid (naproxen) for topical drug delivery

Article abstract of DOI:10.1021/jm991149s

Various novel morpholinyl- (3a,b) and methylpiperazinylacyloxyalkyl (3c- f) esters of 2-(6-methoxy-2-naphthyl)propionic acid were synthesized and evaluated in vitro for topical drug delivery as potential prodrugs of naproxen (1). Compounds 3a-f were prepared by coupling the corresponding naproxen hydroxyalkyl ester with the morpholinyl- or (4-methyl-1- piperazinyl)acyl acid in the presence of dicyclohexylcarbodiimide (DCC) and 4-(dimethylamino)-pyridine (DMAP) and quantitatively hydrolyzed (t(1/2) = 1- 26 min) to naproxen in human serum. Compounds 3c-f showed higher aqueous solubility and similar lipophilicity, determined by their octanol-buffer partition coefficients (log P(app)), at pH 5.0 when compared to naproxen. At pH 7.4 they were significantly more lipophilic than naproxen. The best prodrug 3c led to a 4-and 1.5-fold enhancement of skin permeation when compared to naproxen at pH 7.4 and 5.0, respectively. The present study indicates using a methylpiperazinyl group yields prodrugs that are partially un-ionized under neutral and slightly acidic conditions, and thus, a desirable combination is achieved in terms of aqueous solubility and lipophilicity. Moreover, the resulting combination of biphasic solubility and fast enzymatic hydrolysis of the methylpiperazinylacyloxyalkyl derivatives gave improved topical delivery of naproxen.

Full text of DOI:10.1021/jm991149s

J . Med. Chem. 2000, 43, 1489-1494
1489
Syn th esis a n d in Vitr o Eva lu a tion of Novel Mor p h olin yl- a n d
Meth ylp ip er a zin yla cyloxya lk yl P r od r u gs of 2-(6-Meth oxy-2-n a p h th yl)p r op ion ic
Acid (Na p r oxen ) for Top ica l Dr u g Deliver y
J arkko Rautio,*,† Tapio Nevalainen,† Hannu Taipale,† J ouko Vepsa¨la¨inen,‡,# J ukka Gynther,†,#
Krista Laine,† and Tomi J a¨rvinen†,#
Departments of Pharmaceutical Chemistry and Chemistry, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland,
and Finncovery Ltd., Kuopio, Finland
Received August 17, 1999
Various novel morpholinyl- (3a ,b) and methylpiperazinylacyloxyalkyl (3c-f) esters of 2-(6-
methoxy-2-naphthyl)propionic acid were synthesized and evaluated in vitro for topical drug
delivery as potential prodrugs of naproxen (1). Compounds 3a -f were prepared by coupling
the corresponding naproxen hydroxyalkyl ester with the morpholinyl- or (4-methyl-1-piper-
azinyl)acyl acid in the presence of dicyclohexylcarbodiimide (DCC) and 4-(dimethylamino)-
pyridine (DMAP) and quantitatively hydrolyzed (t1/2 ) 1-26 min) to naproxen in human serum.
Compounds 3c-f showed higher aqueous solubility and similar lipophilicity, determined by
their octanol-buffer partition coefficients (log Papp), at pH 5.0 when compared to naproxen. At
pH 7.4 they were significantly more lipophilic than naproxen. The best prodrug 3c led to a 4-
and 1.5-fold enhancement of skin permeation when compared to naproxen at pH 7.4 and 5.0,
respectively. The present study indicates using a methylpiperazinyl group yields prodrugs that
are partially un-ionized under neutral and slightly acidic conditions, and thus, a desirable
combination is achieved in terms of aqueous solubility and lipophilicity. Moreover, the resulting
combination of biphasic solubility and fast enzymatic hydrolysis of the methylpiperazinylacyl-
oxyalkyl derivatives gave improved topical delivery of naproxen.
In tr od u ction
are needed for topical prodrugs: e.g., enzymatic biocon-
version to the parent drug and 3-fold greater in vitro
skin permeation than naproxen itself from aqueous
suspensions.11
The topical application of drugs has recently received
considerable attention due to its advantages over other
drug delivery methods. Development of an efficient
means of topical delivery can increase local soft-tissue
and joint-drug concentrations while reducing the sys-
temic distribution of a drug, thereby offsetting certain
limitations of its oral use.1-3 However, the barrier
function of the skin limits the use of topical administra-
tion for most drugs. This limitation has led to the
development of various strategies to enhance drug-skin
permeation, such as modifying the lipophilicity of parent
molecules to optimize partitioning into the skin and to
maximize skin permeation.
2-(6-Methoxy-2-naphthyl)propionic acid (naproxen) is
a nonsteroidal antiinflammatory drug (NSAID) that is
widely used for the treatment of rheumatic diseases and
related painful conditions.4 Because the bioavailability
of topically applied naproxen is only 1-2% in
humans,2,5-7 the temporary masking of the carboxylic
acid via simple esterification has been investigated as
a promising means of increasing the dermal permeation
of naproxen.8-10 These prodrugs permeate the skin in
vitro markedly better than the parent drug, but they
release naproxen very slowly in both human serum and
skin-serum homogenate.8 1-Alkylazacycloalkan-2-one
esters of naproxen possess the main requirements that
Recently, we synthesized and evaluated a series of
acyloxyalkyl and aminoacyloxyalkyl esters of naproxen,
which readily hydrolyzed to naproxen in vitro and
possessed highly variable aqueous solubilities and li-
pophilicities.12,13 The highly lipophilic acyloxyalkyl es-
ters did not enhance dermal permeation of naproxen,
most probably due to the low aqueous solubilities.12
However, the aminoacyloxyalkyl esters of naproxen had
fast rates of enzymatic hydrolysis, in addition to a
combination of adequate aqueous solubility and lipo-
philicity that resulted in a 3-fold increase in the in vitro
skin permeation of naproxen.13
Amino acid esters bearing nitrogen heterocycles, e.g.
the morpholine group attached to different drugs, have
been prepared for oral drug delivery and demonstrated
both high aqueous solubility and lipophilicity, with
adequate chemical stability and a high susceptibility to
undergo enzymatic hydrolysis.14-17 On the basis of our
earlier results12,13 and the recent work of others,14-18
we synthesized and evaluated a series of novel mor-
pholinyl- and methylpiperazinylacyloxyalkyl esters as
potential prodrugs of naproxen for topical drug delivery.
Resu lts a n d Discu ssion
Ch em istr y. Morpholinyl- (3a ,b) and methylpipera-
zinylacyloxyalkyl (3c-f) prodrugs of naproxen were
prepared by coupling the corresponding hydroxyalkyl
ester of naproxen 2a ,b with the morpholinyl- and (4-
* Corresponding author: J arkko Rautio. Tel: 358-17-163445. Fax:
358-17-162456. E-mail: jarkko.rautio@uku.fi.
† Department of Pharmaceutical Chemistry, University of Kuopio.
‡ Department of Chemistry, University of Kuopio.
#
Finncovery Ltd.
10.1021/jm991149s CCC: $19.00 © 2000 American Chemical Society
Published on Web 03/31/2000

1490 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8
Rautio et al.
Sch em e 1a
a
Reagents: (a) NaOH; (b) Br-R1-OH, DMF; (c) HOOCR2N(CH2CH2)2X, DCC, DMAP, CH2Cl2.
Ta ble 1. Aqueous Solubility (mean ( SD; n ) 2-4) and
Apparent Partition Coefficient (log Papp, mean ( SD; n ) 2-3)
of Naproxen and Prodrugs
a
aqueous solubility (mM)
pH 7.4 pH 5.0
naproxen 101.9 ( 1.3 0.40 ( 0.04 0.30 ( 0.03 2.38 ( 0.02
log Papp
compd
pH 7.4
pH 5.0
3a
3b
3c
3d
3e
3f
0.07 ( 0.00 0.05 ( 0.00 2.14 ( 0.08 2.62 ( 0.00
b
b
b
b
4.1( 0.4
5.3 ( 0.9 1.16 ( 0.08 0.43 ( 0.05
8.8 ( 0.9
16.2 ( 1.2 1.30 ( 0.05 0.94 ( 0.05
432.4 ( 27.5 141.6 ( 27.0 3.04 ( 0.07 1.20 ( 0.06
50.0 ( 1.9
61.0 ( 8.8 2.69 ( 0.06 1.41 ( 0.02
a
Papp is the apparent partition coefficient between 1-octanol and
b
phosphate buffer (pH 7.4 and 5.0) at room temperature. Not
determined.
methyl-1-piperazinyl)acyl acids in the presence of dicy-
clohexylcarbodiimide (DCC) and 4-(dimethylamino)-
pyridine (DMAP) in dry dichloromethane (Scheme 1).
Aqu eou s Solu bility a n d Lip op h ilicity. Drug lipo-
philicity is a very important property for topical drug
delivery because the stratum corneum, the major bar-
rier to drug permeation, is lipophilic in nature and
generally favors the permeation of lipophilic drugs. The
aqueous solubility, or hydrophilicity, of a drug molecule
has also been suggested to be as important a property
as lipophilicity, especially for very lipophilic com-
pounds.19 Thus, a drug molecule should possess both
hydrophilic and lipophilic properties to readily diffuse
across the skin.13,20-22 The two characteristics (ex-
F igu r e 1. Time courses for naproxen 2-[(4-methyl-1-piper-
azinyl)acetyloxy]ethyl ester (3c) (b) and naproxen (O) during
hydrolysis of the prodrug in 80% human serum (pH 7.4) at 37
°C.
Ta ble 2. Hydrolysis Rates of Naproxen Prodrugs in Phosphate
Buffer Solutions (pH 7.4 and 5.0) and 80% Human Serum (pH
7.4) at 37 °C
t1/2 (days)
t1/2 (min)
f50% (min)a
buffer buffer 80% human serum 80% human serum
compd pH 7.4 pH 5.0
(mean; n ) 2)c
(mean; n ) 2)c
pressed as the apparent partition coefficients (log Papp
)
3a
3b
3c
3d
3e
3f
1.4
b
2.4
7.6
0.7
2.0
6.7
b
66.5
79.9
18.0
37.2
20 (24, 15)
13 (13, 12)
26 (30, 22)
3 (3, 3)
4 (3, 4)
1 (1, 1)
25 (33, 17)
11 (11, 11)
22 (26, 19)
7 (8, 6)
5 (6, 4)
3 (4, 1)
between 1-octanol and phosphate buffer) are shown in
Table 1 at pH 7.4 and 5.0 for naproxen and prodrugs.
Because of the acidic character of naproxen (pKa 4.15),
it is more water soluble at pH 7.4 than at pH 5.0. In
contrast, the prodrugs 3c-d ,f are more soluble in acidic
aqueous solutions due to the ionizable basic group in
the promoiety. At pH 7.4, the prodrugs possess lower
aqueous solubility (except 3e) when compared to naprox-
en and showed a higher lipophilicity than naproxen, as
indicated by the log Papp values (Table 1). The aqueous
solubility of all methylpiperazine prodrugs (3c-f) was
significantly greater at pH 5.0 compared to that of
naproxen, while they maintained a log Papp value near
1. In the design of drugs for topical and transdermal
use, it is often appropriate to have a log P value in the
range 1-3.23 The only tested morpholinylacyloxyalkyl
prodrug (3a ) was more lipophilic than naproxen and
was poorly soluble at both pH values.
a
f50% is the time by which 50% of total naproxen is formed
(mean of two determinations). Not determined. c Individual val-
b
ues are shown in parentheses.
prodrug was determined in 80% human serum, because
human serum or plasma is a commonly used medium
to determine the ester hydrolysis of prodrugs for topical
drug delivery.8,24
The prodrugs were highly susceptible to enzymatic
hydrolysis in serum and hydrolyzed quantitatively to
naproxen (Figure 1) with half-lives (t1/2) ranging from
1 to 26 min. The formation of naproxen is represented
by the f50% values (Table 2), indicating pseudo-first-order
times at which 50% of total naproxen was formed.25
These mean values also ranged from 3 to 25 min. The
chemical stability of prodrugs in aqueous solutions was
substantially higher at pH 5.0 than at pH 7.4, with half-
lives ranging from 6.7 to 79.9 days and from 0.7 to 7.6
Ch em ica l a n d En zym a tic Hyd r olyses. Kinetics of
the chemical and enzymatic hydrolyses of each naproxen
prodrug followed pseudo-first-order kinetics over several
half-lives. The rate of enzymatic hydrolysis of each

Prodrugs of Naproxen for Topical Drug Delivery
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8 1491
Sch em e 2
days, respectively. Because the esters proved to be
chemically stable at pH 5.0, they fulfill an important
criterion for becoming an ideal prodrug: i.e., that of
maintaining chemical stability while providing fast
enzymatic degradation. However, the stability of these
prodrugs at pH 5.0 is not good enough to prepare a
commercial product at pH 5.0. Further studies are
under way to stabilize the prodrugs.
The hydrolysis of acyloxymethyl esters is a two-step
process (Scheme 2).26,27 The first step, which is rate-
determining, is the enzymatic hydrolysis of the terminal
ester group with the formation of the hydroxymethyl
ester as an unstable intermediate, which spontaneously
dissociates to the parent drug. However, the hydroxy-
ethyl and -butyl esters (2a ,b), which are intermediates
in the hydrolysis of 3a -f, are stable in aqueous solution
but hydrolyze to naproxen in human serum.12 Moreover,
the hydrolysis rates of 2a ,b (t1/2 ) 224 and 147 min,
respectively) are much slower than those of 3a -f (t1/2
) 1-26 min) in human serum. A similar order of
magnitude in t1/2 and f50% values of 3a -f (Table 2)
indicates that the formation of naproxen takes place at
a similar rate as the loss of prodrug in human serum.
Therefore, prodrugs 3a -f may hydrolyze to naproxen
without formation of the hydroxyalkyl ester intermedi-
ate, and thus hydrolysis of prodrugs 3a -f may result
from enzymatic attack on the carbonyl of the parent
drug, rather than the carbonyl of the promoiety (Scheme
2).
In Vitr o Sk in P er m ea tion Stu d y. Excised post-
mortem human skin was used to examine the perme-
ation of naproxen and prodrugs 3a ,c-f. Prodrug sus-
pensions in phosphate buffer were applied to maintain
constant diffusion and maximum flux, and each com-
pound was applied in isotonic phosphate buffer (0.05
M) at both pH 7.4 and 5.0.
Cumulative amounts of permeated naproxen (in nmol/
cm2), intermediate, or intact prodrug are shown in
Figure 2. The steady-state flux (J ss) was calculated from
the slope of the linear portions of these plots, and the
values are presented in Table 3. The permeability
coefficients (Kp) for the steady-state delivery were
obtained by dividing the steady-state flux by the solu-
bilities of the compounds in the corresponding vehicle
(Table 3).
F igu r e 2. Permeation profiles (mean ( SE; n ) 3-12) for
naproxen (0) and prodrugs 3c (b) and 3f (2) through excised
human skin in 0.05 M phosphate buffer (pH 7.4) vehicle and
for naproxen (O) in 0.05 M phosphate buffer (pH 5.0) vehicle.
The data represent the sum of naproxen, the intermediate,
and its prodrug.
Ta ble 3. Steady-State Fluxes (J ss) and Permeability
Coefficients (Kp) (mean ( SE; n ) 3-12) for Delivery of Total
Naproxen Species through Excised Human Skin in Vitro in
Isotonic Phosphate Buffer (0.05 M, pH 7.4 and 5.0) at 37 °C
J ss (nmol/cm2‚h)
pH 7.4 pH 5.0
6.5 ( 0.6 1.6 ( 0.2 0.06 ( 0.01
Kp × 106 (cm/h)
compd
pH 7.4
pH 5.0
naproxen
4.07 ( 0.47
3a
3c
3d
3e
3f
0.7 ( 0.0a 0.6 ( 0.0a 9.59 ( 0.27a 12.56 ( 0.77a
24.6 ( 1.0a 2.2 ( 0.1 6.00 ( 0.25a
7.2 ( 0.1 0.2 ( 0.0a 0.82 ( 0.01a
0.41 ( 0.02a
0.02 ( 0.00a
7.7 ( 1.3 0.6 ( 0.0a 0.02 ( 0.00a 0.004 ( 0.000a
13.2 ( 1.6a 1.2 ( 0.2 0.27 ( 0.03a
0.02 ( 0.00a
a
p < 0.05 compared to naproxen (ANOVA, Fisher’s PLSD test).
applied vehicle. Naproxen afforded a 4-fold greater flux
at pH 7.4 (6.5 ( 0.6 nmol/cm2‚h) than at pH 5.0 (1.6 (
0.2 nmol/cm2‚h) despite decreased ionization and an
increased partition coefficient of naproxen at pH 5.0.
This increased flux is attributed to a 250-fold greater
aqueous solubility of naproxen at pH 7.4 than at pH
5.0.
The methylpiperazinylacyloxyalkyl prodrugs of naprox-
en (3c-f) permeated better from saturated solutions at
pH 7.4 than at pH 5.0. Because the aqueous solubilities
of prodrugs 3c-f generally showed small differences at
different pH values except for 3e, the higher partition
coefficients at pH 7.4 favor permeation at this pH rather
than at pH 5.0 (Table 1). At pH 7.4 the best prodrug
(3c) afforded almost a 4-fold higher flux when compared
to naproxen itself. At pH 5.0 the methylpiperazinyla-
cyloxyalkyl prodrugs 3c-f permeated the skin with an
order of magnitude similar to that of naproxen itself at
pH 5.0. The only tested morpholinylacyloxyalkyl pro-
drug (3a ) exhibited similar fluxes at pH 5.0 and 7.4
which were, however, lower than the flux of naproxen.
This is most probably due to the poor aqueous solubility
of 3a .
Passive diffusion is driven by high drug concentration
in the aqueous vehicle, and therefore, naproxen itself
showed a 28-fold greater flux (6.5 ( 0.6 nmol/cm2‚h)
across human skin from saturated suspension than from
a 5.0 mM solution at pH 7.4 (0.23 ( 0.03 nmol/cm2‚h).12
Because different pH values in donor or receptor sides
do not cause different degrees of damage to the skin,28
the higher flux of naproxen from a saturated solution
of pH 7.4, compared to that from a saturated solution
of pH 5.0, is due to increased drug concentration in the
It is interesting to note that the flux of naproxen at
pH 7.4 is lower than that of 3c, although a 25 times
higher concentration of naproxen was used in the donor
compartment, due to a higher intrinsic aqueous solubil-

1492 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8
Rautio et al.
from 2b (0.96 g, 3.2 mmol) and 4-morpholinylacetic acid (0.46
g, 3.2 mmol). Flash chromatography (5% MeOH in CH2Cl2)
gave 3b as a viscous oil (0.27 g, 20%): TLC Rf 0.68 (5% MeOH
ity of naproxen. The permeability coefficients of the
prodrugs which, unlike flux, are independent of donor
concentration were 0.3-160- and 0.001-3-fold higher
than that of naproxen at pH 7.4 and 5.0, respectively.
The permeability of these compounds in aqueous solu-
tions increased as the solubility decreased. Thus, 3a ,
having the lowest aqueous solubility among all pro-
drugs, showed the highest permeability coefficient while
its value for flux was much lower than that for any other
compound. Therefore, the flux, which measures the
mass of material transported through the skin, is a more
relevant parameter, therapeutically, than the perme-
ability coefficient. Finally, comparing the flux and
solubilities of these compounds confirms earlier obser-
vations which indicated that an effective prodrug for
topical drug delivery must possess good biphasic solu-
bility characteristics: i.e., adequate aqueous solubility
as well as lipophilicity over the parent drug.21,22,29
In conclusion, the present study shows that the
permeation of naproxen through human skin can be
markedly improved by using bioreversible methylpip-
erazinylacyloxyalkyl prodrugs of naproxen. The ioniz-
able basic prodrugs combine the desirable aqueous
solubility and lipophilicity for skin permeation, which
can vary widely by changing the acyl group. Further-
more, these prodrugs were rapidly bioconverted to the
parent drug in human serum. These properties make
these novel methylpiperazinylacyloxyalkyl esters prom-
ising prodrugs for improved topical delivery of naproxen.
1
in CH2Cl2); H NMR (CDCl3, 400 MHz) δ 7.72-7.11 (6H, m,
aromatic), 4.10 (2H, t, J ) 6.1 Hz, OCH2-), 4.06 (2H, t, J )
6.1 Hz, OCH2-), 3.92 (3H, s, CH3O), 3.85 (1H, q, J ) 7.1 Hz,
CHMe), 3.74 (4H, t, J ) 4.7 Hz, CH2OCH2), 3.15 (2H, s, -CH2-
COO), 2.55 (4H, t, J ) 4.6 Hz, CH2NCH2), 1.62 (4H, m,
CCH2CH2C), 1.58 (3H, d, J ) 7.2 Hz, CH3C); HRMS m/z
429.2281, calcd for C24H31NO6 429.2151. Anal. (C24H31NO6‚
0.5H2O) C, H, N.
2-[(4-Meth yl-1-piper azin yl)acetyloxy]eth yl (6-Meth oxy-
2-n a p h th yl)p r op a n oa te (3c). 3c was prepared as described
for 3a from 2a (0.39 g, 1.4 mmol) and (4-methyl-1-piperazinyl)-
acetic acid (0.24 g, 1.5 mmol), DMAP (70 mg, 0.6 mmol), and
DCC (0.44 g, 2.1 mmol). The flash chromatography (20%
MeOH in CH2Cl2) provided 3c as a viscous oil (0.41 g, 71%):
TLC Rf 0.45 (50% MeOH in CH2Cl2); 1H NMR (CDCl3, 400
MHz) δ 7.71-7.11 (6H, m, aromatic), 4.29 (4H, m, OCH2CH2O,),
3.91 (3H, s, CH3O), 3.84 (1H, q, J ) 7.1 Hz, CHMe), 3.02 (2H,
s, -CH2COO), 2.54 (8H, bm, N(CH2CH2)2N), 2.33 (3H, s,
NCH3), 1.56 (3H, d, J ) 7.1 Hz, CH3C); HRMS m/z 414.2208,
calcd for C24H31NO6 414.2155. Anal. (C23H30N2O5‚0.8H2O) C,
H; N: calcd, 6.53; found, 5.97.
4-[(4-Meth yl-1-p ip er a zin yl)a cetyloxy]bu tyl 2-(6-Meth -
oxy-2-n a p h th yl)p r op a n oa te (3d ). 3d was prepared as de-
scribed for 3a from 2b (0.85 g, 2.8 mmol) and (4-methyl-1-
piperazinyl)acetic acid (0.44 g, 2.8 mmol). Flash chromatography
(10% MeOH in CH2Cl2) gave 3d (0.50 g, 40%) as a viscous oil:
TLC Rf 0.42 (50% MeOH in CH2Cl2); 1H NMR (CDCl3, 400
MHz) δ 7.71-7.10 (6H, m, aromatic), 4.07 (4H, m, OCH2-),
3.91 (3H, s, CH3O), 3.84 (1H, q, J ) 7.1 Hz, CHMe), 3.16 (2H,
s, -CH2COO), 2.6-2.5 (8H, bm, N(CH2CH2)2N), 2.33 (3H, s,
NCH3), 1.62 (4H, m, CCH2CH2C), 1.56 (3H, d, J ) 7.1 Hz,
CH3C); HRMS m/z 442.2569, calcd for C25H34N2O5 442.2468.
Anal. (C25H34N2O5‚0.5H2O) C, H, N.
Exp er im en ta l Section
Gen er a l P r oced u r es. 1H and 13C NMR spectra were
recorded on a Bruker AM 400 WB operating at 400.1 MHz,
and chemical shifts are reported in parts per million (δ) using
TMS as the internal standard. The splitting pattern abbrevia-
tions are as follows: s ) singlet, d ) doublet, t ) triplet, q )
quartet, qui ) quintet, m ) multiplet, dt ) doublet of triplets,
bs ) broad singlet, bm ) broad multiplet. Electron impact (EI)
mass spectra of the prodrugs were determined by a VG 70-
250SE magnetic sector mass spectrometer (VG Analytical,
Manchester, U.K.). Flash chromatography was accomplished
using silica gel (30-60 µm, J . T. Baker 7024-02). Thin-layer
chromatography (TLC) analyses of reactions were run on
aluminum foil plates coated with silica gel 60 F254 (Merck).
Elemental analysis was carried out by a Perkin-Elmer Series
II CHNS/O Analyzer 2400.
4-[3-(4-Meth yl-1-p ip er a zin yl)p r op ion yloxy]bu tyl 2-(6-
Meth oxy-2-n a p h th yl)p r op a n oa te (3e). 3e was prepared as
described for 3a from 2b (2.0 g, 6.6 mmol) and 3-(4-methyl-
1-piperazinyl)propionic acid (1.2 g, 6.9 mmol). Flash chroma-
tography (50% MeOH in CH2Cl2) gave 3e (2.45 g, 54%) as a
viscous oil: TLC Rf 0.39 (50% MeOH in CH2Cl2); 1H NMR
(CDCl3, 400 MHz) δ 7.72-7.11 (6H, m, aromatic), 4.10 (1H, t,
J ) 6.4 Hz, CO2CH2-), 4.09 (1H, t, J ) 6.1 Hz, CO2CH2-),
4.02 (2H, t, J ) 6.1 Hz, CH2O2C-), 3.91 (3H, s, CH3O) 3.85
(1H, q, J ) 7.2 Hz CHMe), 2.66 (2H, t, J ) 7.3 Hz, -CH2-
COO), 2.45 (2H, t, J ) 7.5 Hz, CH2N), 2.6-2.3 (8H, bm, N(CH2-
CH2)2N), 2.27 (3H, s, NCH3), 1.7-1.5 (4H, m, CCH2CH2C), 1.57
(3H, d, J ) 7.0 Hz, CH3C); HRMS m/z 456.2728, calcd for
C
26H36N2O5 456.2624. Anal. (C26H36N2O5‚0.7H2O) C, H, N.
4-[4-(4-Met h yl-1-p ip er a zin yl)b u t yr yloxy]b u t yl 2-(6-
All reagents were obtained from commercial suppliers and
were used without further purification.The hydroxyalkyl esters
of naproxen (2a ,b) were synthesized and identified as de-
scribed earlier.12
Meth oxy-2-n a p h th yl)p r op a n oa te (3f). 3f was prepared as
described for 3a from 2b (1.0 g, 3.3 mmol) and 4-(4-methyl-
1-piperazinyl)butyric acid (0.61 g, 3.3 mmol). Flash chroma-
tography (MeOH) gave 3f (1.17 g, 75%) as a viscous oil: TLC
Rf 0.35 (50% MeOH in CH2Cl2); 1H NMR (CDCl3, 400 MHz) δ
7.72-7.11 (6H, m, aromatic), 4.10 (1H, t, J ) 6.4 Hz, CO2CH2-
), 4.09 (1H, t, J ) 6.1 Hz, CO2CH2-), 4.00 (2H, t, J ) 6.2 Hz,
CO2CH2-), 3.91 (3H, s, CH3O) 3.85 (1H, q, J ) 7.2 Hz, CHMe),
2.6-2.3 (8H, bm, N(CH2CH2)2N), 2.33 (2H, t, J ) 7.3 Hz, -CH2-
COO), 2.29 (2H, m, CH2N), 2.27 (3H, s, NCH3), 1.78 (2H, m, J
) 7.4 Hz, CCH2C), 1.7-1.5 (4H, m, CCH2CH2C), 1.57 (3H, d,
J ) 7.3 Hz, CH3C); HRMS m/z 470.2593, calcd for C27H38N2O5
470.2781. Anal. (C27H38N2O5‚0.5H2O) C, H, N.
4-Mor p h olin yla cetic Acid . Morpholine (5.5 g, 63 mmol)
in 10 mL of benzene was added dropwise to a solution of ethyl
bromoacetate (5.5 g, 33 mmol) in 10 mL of benzene and the
solution was refluxed for 30 min.30 After cooling, morpholine
hydrobromide was filtered and the filtrate was evaporated to
provide ethyl 4-morpholinylacetate (5.2 g, 91%) as a yellowish
liquid: 1H NMR (CDCl3, 400 MHz) δ 1.28 (3H, t), 2.58 (4H, t),
3.21 (2H, s), 3.75 (4H, t), 4.19 (2H, q). A portion of the above
ester (3.3 g, in 75 mL of water) was refluxed for 30 h. Water
was evaporated and the residue was recrystallized form
2-[(4-Mor ph olin yl)acetyloxy]eth yl 2-(6-Meth oxy-2-n aph -
th yl)p r op a n oa te (3a ). To a solution of 2-hydroxyethyl 2-(6-
methoxy-2-naphthyl)propanoate (2a ) (0.32 g, 1.2 mmol), 4-mor-
pholinylacetic acid (0.18 g, 1.2 mmol), and DMAP (40 mg, 0.2
mmol) in dry CH2Cl2 (15 mL) was added DCC (0.41 g, 2.0
mmol) and the mixture was stirred at 30 °C for 3 days. The
precipitated dicyclohexylurea (DCU) was filtered, and the
filtrate was evaporated. The resulting residue was purified by
flash silica gel column chromatography, eluting with 5% MeOH
in CH2Cl2 and affording 3a as a white solid (0.19 g, 40%): mp
84.9-85.6 °C; TLC Rf 0.56 (5% MeOH in CH2Cl2); 1H NMR
(CDCl3, 400 MHz) δ 7.72-7.11 (6H, m, aromatic), 4.27 (4H,
m, OCH2CH2O), 3.91 (3H, s, CH3O) 3.86 (1H, q, J ) 7.1 Hz,
CHMe), 3.69 (4H, m, CH2OCH2), 3.00 (2H, s, -CH2COO), 2.43
(4H, m, CH2NCH2), 1.58 (3H, d, J ) 7.1 Hz, CH3C); HRMS
m/z 401.1918, calcd for C22H27NO6 401.1838. Anal. C: calcd,
65.82; found, 66.74; H, N.
4-[(4-Mor ph olin yl)acetyloxy]bu tyl 2-(6-Meth oxy-2-n aph -
th yl)p r op a n oa te (3b). 3b was prepared as described for 3a

Prodrugs of Naproxen for Topical Drug Delivery
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8 1493
dichloromethane-methanol providing (2.42 g, 87%) white
crystals: mp 161.5-161.9 °C (lit. mp 160-162 °C);31 1H NMR
(CD3OD) δ 3.31 (4H, bs), 3.61 (2H, s), 3.92 (4H, bm).
(4-Meth yl-1-p ip er a zin yl)a cetic a cid was prepared from
4-methylpiperazine and ethyl bromoacetate using the proce-
dure described above. The product was recrystallized from
methanol-ether: mp 160-161 °C (lit. mp 159.5-161 °C);32
1H NMR (CD3OD) δ 2.56 (3H, s), 2.95 (4H, bm), 3.09 (4H, bm),
3.37 (2H, s).
3-(4-Meth yl-1-p ip er a zin yl)p r op a n oic a cid was prepared
from methyl acrylate and 4-methylpiperazine by utilizing the
same procedure as for 4-morpholinylpropanoic acid:33 1H NMR
(CD3OD) δ 2.36 (3H, s), 2.52 (2H, t), 2.61 (4H, bm), 2.79 (4H,
bm), 2.83 (2H, t).
3-(4-Meth yl-1-p ip er a zin yl)bu tyr ic a cid was prepared by
utilizing the same procedure as for 4-morpholinylacetic acid
from 4-methylpiperazine and ethyl 4-bromobutyrate: mp 98
°C; 1H NMR (CDCl3) δ 1.82 (2H, qui) 2.35 (3H, s), 2.41 (2H, t),
2.56 (2H, t), 2.65 (8H, bm).
HP LC An a lysis. The analytical high-performance liquid
chromatography (HPLC) system consisted of a Merck Hitachi
L-6200A intelligent pump, a Hewlett-Packard HP1046A pro-
grammable fluorescence detector (exitation 226 nm; emission
368 nm), a Merck Hitachi D-6000A interface module, a Merck
Hitachi AS-2000 autosampler, and a Merck LaChrom column
oven L-7350. For all sample separations a Purospher RP-C18
column (125 × 4 mm, 5 µm) was used. A mobile phase mixture
of acetonitrile and a 0.02 M phosphate buffer solution of pH
5.0-5.5 at a flow rate of 1.2 mL/min were used. The ratio of
solvents varied according to the compound.
was analyzed for remaining prodrug and released naproxen
by the HPLC. Pseudo-first-order half-time (t1/2) for the hy-
drolysis of prodrug was calculated from the slope of the linear
portion of the plotted logarithm of remaining prodrugs against
time. The pseudo-first-order times, at which 50% of total
parent compound had been formed (f50%), were determined
from the linear slope of the logarithm of unformed parent
compound (log(parent compoundmax - parent compoundt)) over
time.23
In Vitr o Sk in P er m ea tion . Samples of human skin were
obtained from the abdominal region of adult human cadavers
from the Kuopio University Hospital (Kuopio, Finland). The
epidermis was isolated from the underlying dermis by heat
separation at 60 °C in distilled water for 2 min, after which
the skin specimens were dried and frozen prior to use. The
permeation studies were carried out using the Franz-type
diffusion cell (PermeGear, Inc., Riegel, PA) as previously
described.12 Skin specimens were rehydrated before being
mounted in the diffusion cell. The receptor medium (0.05 M
isotonic phosphate buffer solution of pH 7.4) was stirred and
kept at 37 °C throughout the study. The compounds were
applied as suspensions in 0.05 M phosphate buffer of pH 5.0
and 7.4 which had been prepared in the same way that the
suspensions for determining the aqueous solubility had been
prepared. At specified time intervals 250-µL aliquots were
withdrawn from the receptor compartment and replaced with
fresh buffer. The drug concentrations were assayed by HPLC.
The steady-state flux for naproxen and its selected prodrug
(3a ,c-f) was determined by plotting the cumulative amount
(in nmol) of the parent drug, intermediates and intact prodrug
as measured in the receptor phase against time and dividing
the slope of the steady-state position by the surface area of
the diffusion cell (0.71 cm2). The permeability coefficients of
naproxen and selected prodrugs were calculated by dividing
the steady-state flux by the saturation solubility of the
compound in the corresponding vehicle.
Aqu eou s Solu bility. The aqueous solubility of naproxen
and its prodrugs was determined at room temperature in
phosphate buffer (0.16 M) at the physiological pH 7.4 and at
pH 5.0. The pH of 5.0 was selected for the aqueous solubility
and partition coefficient determinations due to acidic condi-
tions (pH ∼ 5)34 on the outer surface of the skin. Excess
amounts of each compound were added to 1 mL of buffer; the
mixtures were stirred for either 60 min (pH 5.0) or 30 min
(pH 7.4), filtered (Millipore 0.45 µm), and analyzed by the
HPLC. The pH of the mixtures was held constant throughout
the study.
Sta tistica l An a lysis. A one-factor analysis of variance
(ANOVA factorial) was used to test the statistical significance
of differences between the fluxes of naproxen and prodrugs.
Significance in the differences in the means was tested using
Fisher’s protected least significance difference (PLSD) at 95%
confidence.
Ap p a r en t P a r tition Coefficien ts. The apparent partition
coefficients (log Papp) of naproxen and its prodrugs were
determined at room temperature by a 1-octanol-phosphate
buffer system at both pH 5.0 and 7.4. Before use, the 1-octanol
was saturated with phosphate buffer for 24 h by stirring
vigorously. A known concentration of compound in phosphate
buffer was shaken for either 30 min (pH 5.0) or 15 min (pH
7.4), with a suitable volume of 1-octanol. After shaking, the
phases were separated by centrifugation at 14000 rpm for 4
min. The concentrations of the compounds in the buffer phase
before and after partitioning were determined by HPLC.
Hyd r olysis in Aqu eou s Solu tion . The rates of chemical
hydrolysis of prodrugs were studied in aqueous phosphate
buffer solutions of pH 7.4 and 5.0 (0.16 M, ionic strength 0.5)
at 37 °C. An appropriate amount of prodrug was dissolved in
10 mL of preheated buffer and the solutions were placed in a
thermostatically controlled water bath at 37 °C. At appropriate
intervals, samples were taken and analyzed for remaining
prodrug by the HPLC. Pseudo-first-order half-time (t1/2) for the
hydrolysis of prodrug was calculated from the slope of the
linear portion of the plotted logarithm of remaining prodrugs
versus time.
Hyd r olysis in Hu m a n Ser u m . The rates of enzymatic
hydrolysis for naproxen prodrugs were studied in human
serum at 37 °C (Institute of Public Health, University of
Kuopio) which was diluted to 80% with 0.16 M phosphate
buffer of pH 7.4. The reactions were initiated by dissolving
an appropriate amount of prodrug in phosphate buffer, and
preheated human serum was added. The solutions were kept
in a water bath at 37 °C, and 0.5-mL aliquots of serum/buffer
mixture were withdrawn and added to 1.0 mL of ethanol to
precipitate protein from the serum. After immediate mixing
and centrifugation for 10 min at 14000 rpm, the supernatant
Ack n ow led gm en t. The authors thank Mrs. Helly
Rissanen, Mrs. Tarja Ihalainen, and Mr. J ukka
Knuutinen for their skillful technical assistance and the
Academy of Finland and Technology Development Cen-
tre (Finland) for financial support.
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