The effect of substitution patterns on the release rates of opioid peptides DADLE and [Leu5]-enkephalin from coumarin prodrug moieties

Article abstract of DOI:10.1016/j.bioorg.2003.12.002

A coumarin-based prodrug system has been developed in our laboratory for the preparation of esterase-sensitive prodrugs of amines, peptides, and peptidomimetics. The drug release rates from this prodrug system were found to be dependent on the structural

Full text of DOI:10.1016/j.bioorg.2003.12.002

BIOORGANIC
CHEMISTRY
Bioorganic Chemistry 32 (2004) 109–123
www.elsevier.com/locate/bioorg
The effect of substitution patterns on the
release rates of opioid peptides DADLE
and [Leu5]-enkephalin from coumarin
prodrug moieties
Lindsay A. Zych,a Wenqian Yang,a Yuan Liao,a
Kellee R. Griffin,a and Binghe Wanga,b,
*
a
Department of Chemistry, North Carolina State University, Raleigh, NC 27695-8204, USA
Department of Chemistry, Georgia State University, 33 Gilmer St. S.E., Atlanta, GA 30303, USA
b
Received 1 November 2003
Abstract
A coumarin-based prodrug system has been developed in our laboratory for the prepara-
tion of esterase-sensitive prodrugs of amines, peptides, and peptidomimetics. The drug release
rates from this prodrug system were found to be dependent on the structural features of the
drug moiety. The effect of the phenyl ring substitutions on the release kinetics of such prodrugs
of model amines was examined recently and it was found that appropriately positioned alkyl
substituents on the phenyl ring could help to facilitate the release. Aimed at further under-
standing the structure–release rate relationship of the coumarin-based cyclic prodrugs, we syn-
thesized and examined a series of substituted coumarinic acid derivatives of opioid peptides,
DADLE, and [Leu5]-enkephalin.
Ó 2003 Elsevier Inc. All rights reserved.
Keywords: Coumarin; Cyclic prodrug; Opioid peptide; Esterase; Structural effect
1. Introduction
Many biologically active compounds do not make for good pharmaceutical can-
didates due to their lack of permeability across biological barriers such as the blood
* Corresponding author. Fax: 1-404-651-1416.
E-mail address: wang@gsu.edu (B. Wang).
0045-2068/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2003.12.002

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brain barrier or the intestinal mucosa [1,2]. The lack of permeability of these com-
pounds can be caused by such undesirable physicochemical properties as charge,
hydrogen bonding potential, and hydrophilicity. Temporarily masking these undesir-
able traits is one approach to facilitate drug transport across biological barriers. Pro-
drugs [3] are utilized for the purpose of aiding the facilitation of drug permeation
across biological barriers by optimizing the parent drugÕs physicochemical proper-
ties. Modification of these adverse properties, either by enhancing the chemical sta-
bility, altering the aqueous solubility, or improving upon the bioavailability, all while
maintaining the inherent pharmacological properties of the parent drug, allows for
increased membrane permeability across biological barriers.
An innovative coumarin-based prodrug system has been developed in our labora-
tory for the preparation of esterase-sensitive prodrugs of amines [4–8], peptides
[9–11], and peptidomimetics [12–14]. This design takes advantage of the facile
lactonization of coumarinic acid and its derivatives (Scheme 1). In such a strategy,
a latent nucleophile can be unmasked using an esterase triggering mechanism that
initiates a lactonization reaction to release the parent drug. Using this prodrug
strategy, we have prepared esterase-sensitive prodrugs of model amines [4–8], cyclic
prodrugs (Scheme 2) of opioid peptides [9–11], and peptidomimetic glycoprotein IIb/
IIIa antagonists [12–14] with greatly improved membrane permeability.
With the demonstration of the feasibility of this prodrug system, it became im-
portant for us to address one practical issue, i.e., the ability to fine-tune the release
rates so that the release time profile can be modulated to suit the need of individ-
ual drugs. In our earlier studies, we have found that the release rates of the cou-
marin-based prodrugs are largely dictated by the rate of lactonization, which in
turn depends on the structural features of the drug moieties [4,6]. The release rates
are affected by the pKa and the steric features of the amines to be released. Ideally,
one would like to be able to modulate the release rates independent of the struc-
tural properties of the drug moiety. To address this problem, we have examined
the effect of the phenyl ring substituents on the release rates of esterase-sensitive
prodrugs of model amines and found that introduction of methyl substitutions
ortho to the alkyl side chain and the phenol hydroxyl group results in significant
increases in release rates [7,8].
Opioid peptides are potential candidates for further development as therapeutic
agents [15,16]. However, in many cases the clinical development of these opioid
peptides has been hindered due to their metabolic lability and poor cell mem-
Scheme 1. A coumarin-based esterase-sensitive prodrug system.

L.A. Zych et al. / Bioorganic Chemistry 32 (2004) 109–123
111
Scheme 2. A coumarin-based cyclic prodrug system.
brane permeation characteristics [17]. Potentially the unfavorable biopharmaceuti-
cal properties of opioid peptides could be transiently modified using prodrug
strategies [3]. Recently, our laboratories have prepared cyclic prodrugs of peptides
[9–11] as a way to modify the physicochemical properties of the molecules suffi-
ciently to overcome these biopharmaceutical barriers. Aimed at further under-
standing the structure–release rate relationship of the coumarin-based cyclic
prodrugs, we synthesized a series of substituted coumarinic acid derivatives and
the release rates of these prodrugs were examined to investigate the substitution
effect.
2. Experimental
2.1. General methods
All 1H and 13C NMR spectra were recorded at 300 or 400 MHz with TMS as the
internal standard. Column chromatography was performed using silica gel (200–
400 mesh) from Aldrich. Commercially available starting materials and reagents
were purchased from Aldrich. Amino acids were purchased from Novabiochem. Tet-
rahydrofuran (THF) was distilled from sodium and benzophenone; dichloromethane
(DCM) was distilled from CaH2. A Shimadzu 1601 UV–Vis spectrophotometer was
used for the esterase kinetic studies. Atlantic Microlab, Norcross, Georgia per-
formed elemental analyses. The Mass Spectroscopy Laboratories at both University
of Kansas and North Carolina State University carried out mass spectral analyses.
Compounds 3a [18], 7a–b [8], and 8a–b [8] were prepared as reported in the literature.
The tetrapeptides 17 [11] and 18 [11] were synthesized following the standard peptide
synthesis procedure as reported previously.
2.2. Purified esterase kinetic studies
The esterase kinetic studies were carried out following procedures reported earlier
using PLE [4,6,9].

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2.3. 1-N-Boc-D -leucyl-2-[(Z)-3-(1-tert-butyl-1,1-dimethylsilyloxy)-1-propenyl]-3,
6-dimethylphenol (9a)
To a solution of Boc-D-Leu-OH hydrate (1.37 g, 5.50 mmol) in dry dichlorome-
thane (50 mL) was added N,N0-dicyclohexylcarbodiimide (1.16 g, 5.62 mmol) at
room temperature under N2 atmosphere. The reaction mixture was then put into
an ice bath (0 °C). To this cold solution was added 8a (1.34 g, 4.58 mmol) in
25 mL of dry dichloromethane and DMAP (0.29 g, 2.37 mmol). After stirring for
1 h, the reaction was taken out of the ice bath and stirred overnight at room temper-
ature. Dichloromethane (100 mL) was added to the residue, which was washed with
1 N HCl (2 ꢀ 40 mL), 5% NaHCO3 (35 mL), and water (35 mL). The organic layer
was then dried over MgSO4, filtered, and evaporated to afford the crude product.
The residue was purified by column chromatography (2:1 hexane/dichloromethane)
to give 9a as a white solid (1.96 g, 85%). 1H NMR (CDCl3) d 0.03 (6H, s), 0.86 (9H,
s), 0.99 (6H, d, J ¼ 6:1 Hz), 1.26 (1H, m), 1.46 (9H, s), 1.75 (2H, m), 2.13 (3H, s),
2.19 (3H, s), 4.00 (2H, d, J ¼ 4:9 Hz), 4.48 (1H, m), 5.11 (1H, brs), 5.86 (1H, m),
6.11 (1H, d, J ¼ 11:3 Hz), 6.98 (1H, d, J ¼ 7:8 Hz), 7.04 (1H, d, J ¼ 7:8 Hz). MS
(FAB) m=z 506.3 (Mþ + H). Anal. Calcd for C28H47NO5Si: C, 65.95; H, 9.22; N,
2.85. Found: C, 65.82; H, 9.26; N, 2.79.
2.4. 1-N-Boc-L -leucyl-2-[(Z)-3-(1-tert-butyl-1,1-dimethylsilyloxy)-1-propenyl]-3,
6-dimethylphenol (10a)
In a manner similar to the preparation of 9a, Boc-L-Leu-OH hydrate (0.88 g,
3.52 mmol, in 35 mL dry DCM), DCC (1.11 g, 5.38 mmol), 8a (0.93 g, 3.18 mmol,
in 20 mL dry DCM), and DMAP (0.20 g, 1.62 mmol) were treated to afford 10a as
1
a clear oil (1.52 g, 95%). H NMR (CDCl3) d 0.00 (6H, s), 0.86 (9H, s), 0.99 (6H,
d, J ¼ 6:3 Hz), 1.46 (9H, s), 1.64 (1H, m), 1.77 (2H, m), 2.13 (3H, s), 2.19 (3H, s),
4.00 (2H, d, J ¼ 5:1 Hz), 4.45–4.53 (1H, m), 5.12 (1H, brs), 5.82–5.90 (1H, m),
6.12 (1H, d, J ¼ 11:1 Hz), 7.01 (2H, dd, J1 ¼ 7:8 Hz, J2 ¼ 18:9 Hz).
2.5. 1-N-Boc-D-leucyl-2-[(Z)-3-hydroxy-1-propenyl]-3,6-dimethylphenol (11a)
To a solution of 9a (1.94 g, 3.84 mmol) in THF (30 mL) was added water (30 mL),
and then followed by addition of acetic acid (90 mL) dropwise. The mixture was
stirred at room temperature for 3 h and then evaporated to remove THF, water,
and acetic acid. Ethyl acetate (100 mL) was added to the residue, which was washed
with 5% NaHCO3 (3 ꢀ 40 mL), and water (2 ꢀ 40 mL). The ethyl acetate layer was
dried over MgSO4, concentrated, and purified by column chromatography (6:1 hex-
ane/ethyl acetate) to afford 11a as light yellow oil (1.38 g, 91%). 1H NMR (CDCl3) d
1.00 (6H, d, J ¼ 3:9 Hz), 1.45 (9H, s), 1.60 (1H, m), 1.81 (2H, m), 2.04 (3H, s), 2.13
(3H, s), 3.93 (2H, d, J ¼ 5:9 Hz), 4.49 (1H, m), 4.95 (1H, d, J ¼ 8:3 Hz), 5.96 (1H,
m), 6.22 (1H, d, J ¼ 11:2 Hz), 6.98 (1H, d, J ¼ 7:7 Hz), 7.05 (1H, d, J ¼ 7:2 Hz).
MS (FAB) m=z 392.2 (Mþ + H). Anal. Calcd for C22H33NO5: C, 67.49; H, 8.50; N,
3.58. Found: C, 67.54; H, 8.61; N, 3.52.

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113
2.6. 1-N-Boc-L-leucyl-2-[(Z)-3-hydroxy-1-propenyl]-3,6-dimethylphenol (12a)
In a manner similar to the preparation of 11a, 10a (1.52 g, 3.01 mmol, in 30 mL
THF), water (30 mL), and acetic acid (90 mL) were reacted to give 12a as a light yel-
low oil (1.12 g, 95%). 1H NMR (CDCl3) d 1.00 (6H, d, J ¼ 5:6 Hz), 1.45 (9H, s), 1.63
(1H, m), 1.79 (2H, m), 2.14 (6H, d, J ¼ 5:6 Hz), 3.93 (2H, d), 4.49 (1H, m), 4.96 (1H,
m), 5.97 (1H, m), 6.23 (1H, d, J ¼ 11:6 Hz), 7.02 (2H, dd, J1 ¼ 7:6 Hz, J2 ¼ 23 Hz).
2.7. 1-N-Boc-D-leucyl-3,6-dimethyl-2-[(Z)-3-oxo-1-propenyl]phenol (13a)
To a solution of 11a (1.36 g, 3.34 mmol) in dry dichloromethane (50 mL) was
added in one portion 85% activated manganese (IV) oxide (1.16 g, 11.6 mmol), which
was dried with a drying tube containing anhydrous CaSO4. Then every 1 h manga-
nese (IV) oxide (1.16 g, 11.6 mmol) was added in 1 portion for a period of 5 h. The
reaction mixture was filtered through a Celite plug, washed with dichloromethane,
1
and evaporated to afford 13a as yellow oil (1.20 g, 89%). H NMR (CDCl3) d 0.97
(6H, d, J ¼ 8:3 Hz), 1.44 (9H, s), 1.52–1.64 (1H, m), 1.71–1.79 (2H, m), 2.16 (3H,
s), 2.24 (3H, s), 4.45 (1H, m), 4.78 (1H, d, J ¼ 8:6 Hz), 6.15–6.22 (1H, m), 7.05
(1H, d, J ¼ 7:8 Hz), 7.15 (1H, d, J ¼ 7:8 Hz), 7.34 (1H, d, J ¼ 11:3 Hz), 9.49 (1H,
d, J ¼ 8:3 Hz). MS (FAB) m=z 390.3 (Mþ + H). Anal. Calcd for C22H31NO5: C,
67.84; H, 8.02; N, 3.60. Found: C, 67.91; H, 8.08; N, 3.65.
2.8. 1-N-Boc-L-leucyl-3,6-dimethyl-2-[(Z)-3-oxo-1-propenyl]phenol (14a)
In a manner similar to the preparation of 13a, 12a (1.12 g, 2.86 mmol, in 45 mL dry
DCM), and 85% activated manganese (IV) oxide (1.00 g ꢀ 6, 9.8 mmol ꢀ 6) were re-
1
acted to afford 14a as a yellow crystal (0.90 g, 81%). H NMR (CDCl3) d 0.97 (6H,
dd, J1 ¼ 2:4 Hz, J2 ¼ 6:4 Hz), 1.44 (9H, s), 1.61 (1H, s), 1.77 (2H, m), 2.16 (3H, s),
2.23 (3H, s), 4.43 (1H, m), 4.78 (1H, d, J ¼ 8:4 Hz), 6.18 (1H, m), 7.05 (1H, d, J ¼
8:0 Hz), 7.15 (1H, d, J ¼ 7:6 Hz), 7.34 (1H, d, J ¼ 10:8 Hz), 9.48 (1H, d, J ¼ 8:0 Hz).
2.9. (Z)-3-[2-(1-N-Boc-D-leucyl)-3,6-dimethylphenyl]-2-propenonic acid (15a)
A solution of 80% sodium chlorite (0.27 g, 3.0 mmol) in 3.9 mL water was added
dropwise over 2 h to a stirred solution of 13a (0.45 g, 1.17 mmol) in 1.8 mL acetoni-
trile, sodium phosphate (54 mg) in 1.8 mL water, and 30% hydrogen peroxide
(0.18 mL), while its temperature was kept at 10 °C with an ice-water bath. The reac-
tion mixture was then stirred at room temperature (ꢁ2 h) until there was no more
oxygen evolution seen (monitored with a bubbler). A small amount of sodium
thiosulfite was added to destroy the unreacted HOCl and H2O2. The solution was
acidified with 1 N HCl to pH 1–2. The mixture was extracted with ethyl acetate
(60 mL). The ethyl acetate layer was then washed with saturated NaCl (2 ꢀ 60 mL)
and dried over MgSO4. After removal of the solvent, the residue was purified by col-
umn chromatography (20:1 DCM/MeOH) to afford 15a as a white solid (0.46 g,
1
97%). H NMR (CDCl3) d 0.99 (6H, d, J ¼ 5:9 Hz), 1.45 (9H, s), 1.59 (1H, m),

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1.71–1.82 (2H, m), 2.12 (3H, s), 2.22 (3H, s), 4.50 (1H, m), 4.93 (1H, d, J ¼ 8:9 Hz),
6.13 (1H, d, J ¼ 12:2 Hz), 6.82 (1H, d, J ¼ 12:0 Hz), 7.01 (1H, d, J ¼ 7:7 Hz), and
7.08 (1H, d, J ¼ 7:8 Hz). MS (FAB) m=z 406.3 (Mþ + H). Anal. Calcd for
C22H31NO6: C, 65.17; H, 7.71; N, 3.45. Found: C, 65.21; H, 7.67; N, 3.40.
2.10. (Z)-3-[2-(1-N-Boc-L-leucyl)-3,6-dimethylphenyl]-2-propenonic acid (16a)
In a manner similar to the preparation of 15a, 80% sodium chlorite (0.46 g,
5.11 mmol) in 6.8 mL water, 14a (0.90 g, 2.31 mmol, in 3.2 mL acetonitrile), sodium
phosphate (94 mg) in 3.2 mL water, and 30% hydrogen peroxide (0.35 mL) were re-
1
acted to give 16a as light yellow solid (0.78 g, 83%). H NMR (CDCl3) d 0.99 (6H,
dd, J1 ¼ 1:5 Hz, J2 ¼ 6:3 Hz), 1.46 (9H, s), 1.60 (1H, m), 1.77 (2H, m), 2.17 (3H, s),
2.23 (3H, s), 4.51 (1H, m), 4.92 (1H, d, J ¼ 9:3 Hz), 6.13 (1H, d, J ¼ 12:3 Hz), 6.81
(1H, d, J ¼ 12:0 Hz), 7.01 (1H, d, J ¼ 7:8 Hz), 7.09 (1H, d, J ¼ 7:5 Hz).
2.11. Linear precursor (19a)
Under N2 atmosphere, an ice-cold (0 °C) solution of compound 15a (0.59 g,
1.43 mmol) in 12 mL of dry THF was reacted with DCC (0.85 g, 4.12 mmol). After
5 min, HOBt (0.37 g, 2.74 mmol) was added to the reaction mixture. After 20 min,
a solution of H2N-Tyr-D-Ala-Gly-Phe-OtBu 17 (0.81 g, 1.57 mmol) in 20 mL dry
THF was added, followed by the addition of DMAP (0.06 g, 0.49 mmol). After
the solution stirred at 0 °C for 1 h, the mixture was taken out of the ice bath and al-
lowed to stir at room temperature for 3 h. The resulting mixture was dissolved in
100 mL ethyl acetate. The ethyl acetate solution was extracted with 10% citric acid
(2 ꢀ 30 mL), 5% NaHCO3 (2 ꢀ 30 mL), and saturated NaCl (2 ꢀ 30 mL). The ethyl
acetate layer was dried and concentrated and the residue was purified by gradient
column chromatography (60:1 DCM/MeOH to 20:1 DCM/MeOH) to afford 19a
1
as a white solid (0.73 g, 57%). H NMR (CD3OD) d 0.99 (6H, dd, J1 ¼ 6:3 Hz,
J2 ¼ 8:1 Hz), 1.10 (3H, d, J ¼ 7:2 Hz), 1.36 (9H, s), 1.46 (9H, s), 1.71 (1H, m),
1.80 (2H, m), 2.08 (3H, s), 2.14 (3H, s), 2.81 (2H, m), 3.02 (2H, d, J ¼ 6:9 Hz),
3.64 (1H, m), 3.82 (2H, m), 4.17 (2H, m), 4.37 (1H, m), 4.51 (1H, m), 6.20 (1H, d,
J ¼ 11:7 Hz), 6.62–6.69 (3H, m), 6.92–6.98 (3H, m), 7.06 (1H, d, J ¼ 8:1 Hz),
7.21–7.27 (6H, m). MS (FAB) m=z 900.5 (Mþ + H). Anal. Calcd for C49H65N5O11:
C, 65.39; H, 7.28; N, 7.78. Found: C, 65.12; H, 7.36; N, 7.72.
2.12. Linear precursor (20a)
In a manner similar to the preparation of 19a, 16a (0.77 g, 1.92 mmol, in 16 mL
dry THF), DCC (1.13 g, 5.47 mmol), HOBt (0.48 g, 3.55 mmol), H2N-Tyr-Gly-Gly-
Phe-OtBu (18, 1.05 g, 2.12 mmol, in 30 mL dry THF), and DMAP (0.08 g,
0.66 mmol) were reacted to afford 20a as a yellow oil (0.98 g, 58%). 1H NMR
(CD3OD) d 0.99 (6H, dd, J1 ¼ 6:6 Hz, J2 ¼ 10:8 Hz), 1.37 (9H, s), 1.45 (9H, s),
1.70 (1H, m), 1.81 (2H, m), 2.08 (3H, s), 2.13 (3H, s), 2.85 (2H, m), 3.03 (2H, m),
3.59 (1H, m), 3.75–3.93 (2H, m), 4.29 (1H, m), 4.38 (1H, m), 4.53 (2H, m), 6.15

L.A. Zych et al. / Bioorganic Chemistry 32 (2004) 109–123
115
(1H, d, J ¼ 12:0 Hz), 6.65 (2H, d, J ¼ 8:1 Hz), 6.71 (1H, d, J ¼ 8:4 Hz), 6.90–6.99
(3H, m), 7.08 (2H, t, J ¼ 5:1 Hz), 7.21–7.27 (5H, m).
2.13. Cyclic prodrug of DADLE (21a)
Compound 19a (0.27 g, 0.30 mmol) was treated with a solution of 1:1 TFA:DCM
(45 mL) under N2 atmosphere for 5 h at room temperature. The mixture was concen-
trated and dried under vacuum pump for 1 h. The residue was then dissolved in
200 mL dry DCM and 4.2 mL DMF. Bop-Cl (0.72 g, 2.83 mmol) and 0.52 mL TEA
were added into the solution and the reaction was stirred for 18.5 h at room tempera-
ture. The reaction mixture was concentrated, dissolved in ethyl acetate (100 mL), and
extracted with water (30 mL), 10% citric acid (30 mL), 5% NaHCO3 (30 mL), and water
(3 ꢀ 30 mL). The ethyl acetate layer was then dried and concentrated to afford crude
product. Purification by column chromatography (20:1 DCM/MeOH) yielded final
1
product 21a as a white solid (55 mg, 25%). H NMR (CD3OD) d 1.02 (6H, dd,
J1 ¼ 6:8 Hz, J2 ¼ 18:0 Hz), 1.12 (3H, d, J ¼ 7:2 Hz), 1.72 (2H, m), 1.86 (1H, m), 2.19
(3H, s), 2.22 (3H, s), 3.21 (2H, m), 3.34 (1H, d, J ¼ 2:0 Hz), 3.39 (1H, s), 3.49 (1H,
m), 3.84 (1H, d, J ¼ 15:6 Hz), 4.09 (1H, m), 4.19 (1H, d, J ¼ 6:8 Hz), 4.39 (1H, m),
4.49 (1H, m), 6.19 (1H, m), 6.67 (1H, m), 6.79 (2H, t, J ¼ 12:4 Hz), 6.89 (1H, d,
J ¼ 8:4 Hz), 7.04 (1H, brs), 7.14–7.32 (7H, m). MS (FAB) m=z 726.3 (Mþ + H). Anal.
Calcd for C40H47N5O8: C, 66.19; H, 6.53; N, 9.65. Found: C, 66.40; H, 6.52; N, 9.57.
2.14. Cyclic prodrug of Leu-enkephalin (22a)
In a manner similar to the preparation of 21a, 20a (0.26 g, 0.29 mmol) was treated
with a solution of 1:1 TFA:DCM (45 mL) under N2 atmosphere for 3.5 h at room tem-
perature. Upon concentration, the residue (in 200 mL dry DCM and 4.2 mL DMF),
Bop-Cl (0.69 g, 2.69 mmol), and 0.5 mL TEA were reacted to afford 22a as a white solid
after column chromatography followed by recrystallization (0.028 g, 14%). 1H NMR
(CD3OD) d 1.01 (6H, dd, J1 ¼ 6:0 Hz, J2 ¼ 18:4 Hz), 1.77 (2H, m), 1.90 (1H, m),
2.12 (3H, s), 2.22 (3H, s), 2.52 (1H, m), 2.99 (1H, m), 3.13 (1H, m), 3.25 (1H, m),
3.41 (1H, d, J ¼ 3:2 Hz), 3.83 (2H, m), 4.32 (2H, m), 4.48 (1H, q, J1 ¼ 6:4 Hz, J2 ¼
9:2 Hz), 4.78 (1H, m), 6.21 (1H, d, J ¼ 12:0 Hz), 6.68 (1H, m), 6.72 (1H, d, J ¼ 12:0 Hz),
6.93–7.03 (3H, m), 7.06 (1H, d, J ¼8:0Hz), 7.13 (1H, d, J ¼8:0Hz), 7.21–7.30 (6H, m).
2.15. 1-N-Boc-D-leucyl-2-[(Z)-3-(1-tert-butyl-1,1-dimethylsilyloxy)-1-propenyl]-4-
methylphenol (9b)
In a manner similar to the preparation of 9a, Boc-D-Leu-OH hydrate (0.55 g,
2.2 mmol, in 30 mL dry DCM), DCC (0.90 g, 4.4 mmol), 8b (0.50 g, 1.8 mmol, in
10 mL dry DCM), and DMAP (0.11 g, 0.90 mmol) were treated to afford 9b as a clear
oil (0.69 g, 79%). 1H NMR (CDCl3) d 0.04 (6H, s), 0.89 (9H, s), 1.00 (6H, d,
J ¼ 6:4 Hz), 1.46 (9H, s), 1.62 (1H, m), 1.78 (2H, m), 2.34 (3H, s), 4.27 (2H, dd,
J1 ¼ 1:6 Hz, J2 ¼ 6:4 Hz), 4.51 (1H, m), 4.96 (1H, d, J ¼ 8:8 Hz), 5.86 (1H, m), 6.34
(1H, d, J ¼ 12:0 Hz), 6.94 (1H, d, J ¼ 8:0 Hz), 7.03 (1H, s), 7.08 (1H, d, J ¼ 8:4 Hz).

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2.16. 1-N-Boc-L-leucyl-2-[(Z)-3-(1-tert-butyl-1,1-dimethylsilyloxy)-1-propenyl]-4-
methylphenol (10b)
In a manner similar to the preparation of 9a, Boc-L-Leu-OH hydrate (0.55 g,
2.2 mmol, in 30 mL dry DCM), DCC (0.90 g, 4.4 mmol), 8b (0.44 g, 1.6 mmol, in
10 mL dry DCM), and DMAP (0.11 g, 0.90 mmol) were treated to afford 10b as a
1
clear oil (0.64 g, 83%). H NMR (CDCl3) d 0.04 (6H, s), 0.89 (9H, s), 1.00 (6H, d,
J ¼ 6:8 Hz), 1.46 (9H, s), 1.62 (1H, m), 1.78 (2H, m), 2.36 (3H, s), 4.27 (2H, dd,
J1 ¼ 1:6 Hz, J2 ¼ 6:4 Hz), 4.51 (1H, m), 4.96 (1H, d, J ¼ 8:4 Hz), 5.86 (1H, m),
6.35 (1H, d, J ¼ 11:6 Hz), 6.94 (1H, d, J ¼ 8:0 Hz), 7.03 (1H, s), 7.08 (1H, d,
J ¼ 8:0 Hz). 13C NMR (CDCl3) d 18.50, 21.15, 22.03, 23.12, 25.08, 26.12, 28.54,
41.83, 52.49, 60.40, 80.15, 121.92, 124.48, 129.28, 130.98, 133.97, 135.67, 146.11,
155.67, 172.06. MS (FAB) m=z 492.3 (Mþ + H). Anal. Calcd for C27H45NO5Si: C,
65.95; H, 9.22; N, 2.85. Found: C, 65.95; H, 9.32; N, 2.96.
2.17. 1-N-Boc-D-leucyl-2-[(Z)-3-hydroxy-1-propenyl]-4-methylphenol (11b)
In a manner similar to the preparation of 11a, 9b (0.62 g, 1.26 mmol, in 15 mL
THF), water (15 mL), and acetic acid (45 mL) were reacted to give 11b as yellow
1
oil (0.47 g, 99%). H NMR (CDCl3) d 1.00 (6H, d, J ¼ 5:7 Hz), 1.46 (9H, s), 1.61
(1H, m), 1.75 (2H, m), 2.34 (3H, s), 4.20 (2H, d, J ¼ 6:4 Hz) 4.48 (1H, m), 4.94
(1H, d, J ¼ 8:1 Hz), 5.94 (1H, m), 6.40 (1H, d, J ¼ 11:3 Hz), 6.92 (1H, s), 6.95
(1H, d, J ¼ 5:4 Hz), 7.10 (1H, d, J ¼ 8:3 Hz). 13C NMR (CDCl3) d 21.09, 21.96,
23.14, 25.08, 28.50, 41.48, 52.46, 59.67, 80.36, 121.78, 125.67, 128.73, 129.39,
131.11, 133.57, 135.90, 145.93, 155.78, 172.46. MS (FAB) m=z 378.3 (Mþ + H). Anal.
Calcd for C21H31NO5: C, 66.82; H, 8.28; N, 3.71. Found: C, 66.44; H, 7.94; N, 3.54.
2.18. 1-N-Boc-L-leucyl-2-[(Z)-3-hydroxy-1-propenyl]-4-methylphenol (12b)
In a manner similar to the preparation of 11a, 10b (0.57 g, 1.16 mmol, in 13.5 mL
THF), water (13.5 mL), and acetic acid (40 mL) were reacted to give 12b as a light
1
yellow oil (0.37 g, 85%). H NMR (CDCl3) d 1.00 (6H, d, J ¼ 5:8 Hz), 1.46 (9H,
s), 1.61 (1H, m), 1.79 (2H, m), 2.34 (3H, s), 4.20 (2H, d, J ¼ 6:1 Hz) 4.48 (1H, m),
4.93 (1H, d, J ¼ 8:4 Hz), 5.96 (1H, m), 6.40 (1H, d, J ¼ 11:5 Hz), 6.92 (1H, s),
6.95 (1H, d, J ¼ 5:4 Hz), 7.10 (1H, d, J ¼ 8:1 Hz).
2.19. 1-N-Boc-D-leucyl-4-methyl-2-[(Z)-3-oxo-1-propenyl]phenol (13b)
In a manner similar to the preparation of 13a, 11b (0.40 g, 1.06 mmol, in 15 mL
dry DCM) and 85% activated manganese (IV) oxide (0.40 g ꢀ 6, 4.0 mmol ꢀ 6)
1
were treated to afford 13b as a light yellow oil (0.35 g, 88%). H NMR (CDCl3)
d 1.00 (6H, dd, J1 ¼ 2:4 Hz, J2 ¼ 6:0 Hz), 1.45 (9H, s), 1.61 (1H, m), 1.78 (2H,
m), 2.37 (3H, s), 4.48 (1H, m), 4.86 (1H, d, J ¼ 8:0 Hz), 6.19 (1H, m), 7.03
(1H, d, J ¼ 8:0 Hz), 7.16 (1H, s), 7.23 (1H, m), 7.54 (1H, d, J ¼ 11:6 Hz), 9.82
(1H, d, J ¼ 8:0 Hz).

L.A. Zych et al. / Bioorganic Chemistry 32 (2004) 109–123
117
2.20. 1-N-Boc-L-leucyl-4-methyl-2-[(Z)-3-oxo-1-propenyl]phenol (14b)
In a manner similar to the preparation of 13a, 12b (0.31 g, 0.83 mmol, in 15 mL
dry DCM) and 85% activated manganese (IV) oxide (0.30 g ꢀ 6, 3.0 mmol ꢀ 6)
1
were reacted to afford 14b as a light yellow oil (0.25 g, 82%). H NMR (CDCl3)
d 1.00 (6H, dd, J1 ¼ 2:0 Hz, J2 ¼ 6:0 Hz), 1.45 (9H, s), 1.61 (1H, m), 1.78 (2H,
m), 2.37 (3H, s), 4.48 (1H, m), 4.86 (1H, d, J ¼ 7:6 Hz), 6.18 (1H, m), 7.03
(1H, d, J ¼ 8:0 Hz), 7.16 (1H, s), 7.23 (1H, m), 7.54 (1H, d, J ¼ 11:2 Hz), 9.82
(1H, d, J ¼ 8:0 Hz). 13C NMR (CDCl3) d 20.99, 21.99, 23.09, 25.15, 28.52,
41.45, 52.67, 80.43, 116.84, 122.48, 127.24, 131.69, 131.87, 132.30, 136.22,
143.49, 146.59, 155.68, 171.96, 192.69. MS (FAB) m=z 376.2 (Mþ + H). Anal.
Calcd for C21H29NO5: C, 67.18; H, 7.79; N, 3.73. Found: C, 67.40; H, 7.76; N,
3.63.
2.21. (Z)-3-[2-(1-N-Boc-D-leucyl)-4-methylphenyl]-2-propenonic acid (15b)
In a manner similar to the prepatation of 15a, 80% sodium chlorite (0.19 g,
2.10 mmol) in 2.6 mL water, 13b (0.35 g, 0.93 mmol, in 1.2 mL acetonitrile), so-
dium phosphate (36 mg) in 1.2 mL water, and 30% hydrogen peroxide
(0.12 mL) were reacted to give 15b as a yellow oil (0.27 g, 75%). 1H NMR
(CDCl3) d 0.99 (6H, d, J ¼ 4:8 Hz), 1.45 (9H, s), 1.61 (1H, m), 1.77 (2H, m),
2.32 (3H, s), 4.48 (1H, m), 4.95 (1H, d, J ¼ 8:8 Hz), 6.05 (1H, d, J ¼ 12:4 Hz),
6.86 (1H, d, J ¼ 12:8 Hz), 6.97 (1H, d, J ¼ 8:4 Hz), 7.13 (1H, d, J ¼ 8:8 Hz),
7.23 (1H, s).
2.22. (Z)-3-[2-(1-N-Boc-L-leucyl)-4-methylphenyl]-2-propenonic acid (16b)
In a manner similar to the preparation of 15a, 80% sodium chlorite (0.15 g,
1.66 mmol) in 2.2 mL water, 14b (0.19 g, 0.51 mmol, in 1.0 mL acetonitrile), sodium
phosphate (30 mg) in 1.0 mL water and 30% hydrogen peroxide (0.10 mL) were re-
1
acted to give 16b as a yellow solid (0.16 g, 82%). H NMR (CDCl3) d 1.00 (6H,
dd, J1 ¼ 2:0 Hz, J2 ¼ 6:0 Hz), 1.46 (9H, s), 1.61 (1H, m), 1.78 (2H, m), 2.33 (3H,
s), 4.49 (1H, m), 4.95 (1H, d, J ¼ 8:8 Hz), 6.07 (1H, d, J ¼ 12:0 Hz), 6.89 (1H, d,
J ¼ 12:4 Hz), 6.98 (1H, d, J ¼ 9:0 Hz), 7.15 (1H, d, J ¼ 8:0 Hz), 7.22 (1H, s). 13C
NMR (CDCl3) d 21.00, 21.96, 23.12, 25.14, 28.54, 41.46, 52.64, 80.51, 121.69,
122.34, 127.94, 130.75, 130.95, 135.68, 138.95, 145.96, 155.94, 169.38, 172.19. MS
(FAB) m=z 392.2 (Mþ + H). Anal. Calcd for C21H29NO6: C, 64.43; H, 7.47; N,
3.58. Found: C, 64.36; H, 7.37; N, 3.52.
2.23. Linear precursor (19b)
In a manner similar to the preparation of 19a, 15b (0.37 g, 0.93 mmol, in 8 mL dry
THF), DCC (0.54 g, 2.6 mmol), HOBt (0.14 g, 1.03 mmol), H2N-Tyr-D-Ala-Gly-Phe-
OtBu (17, 0.53 g, 1.03 mmol, in 16 mL dry THF), and DMAP (0.04 g, 0.28 mmol)

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L.A. Zych et al. / Bioorganic Chemistry 32 (2004) 109–123
were reacted to afford 19b as a white solid (0.34 g, 41%). 1H NMR (CD3OD) d 0.99
(6H, dd, J1 ¼ 6:6 Hz, J2 ¼ 9:0 Hz), 1.14 (3H, d, J ¼ 7:2 Hz), 1.35 (9H, s), 1.46 (9H,
s), 1.69 (1H, m), 1.78 (2H, m), 2.26 (3H, s), 2.81 (2H, m), 3.02 (2H, d, J ¼ 7:2 Hz),
3.73 (2H, d, J ¼ 16:5 Hz), 4.17–4.27 (1H, m), 4.34 (1H, m), 4.48 (1H, t, J ¼ 7:2 Hz),
6.13 (1H, d, J ¼ 12:0 Hz), 6.67 (2H, d, J ¼ 8:4 Hz), 6.76 (1H, d, J ¼ 12:3 Hz), 6.94
(3H, t, J ¼ 8:7 Hz), 7.12–7.27 (7H, m). 13C NMR (CD3OD) d 17.29, 21.05, 23.49,
26.25, 28.39, 28.94, 37.76, 38.87, 41.50, 43.54, 54.01, 56.12, 58.08, 80.97, 83.22,
116.51, 122.83, 126.84, 127.95, 128.47, 129.59, 130.65, 131.39, 131.60, 132.08,
134.14, 137.22, 138.25, 157.58, 171.46, 172.10, 172.20, 173.81, 173.88, 174.91,
175.36. MS (FAB) m=z 887.5 (Mþ + H).
2.24. Linear precursor (20b)
In a manner similar to the preparation of 19a, 16b (0.42 g, 1.1 mmol, in 10 mL dry
THF), DCC (0.67 g, 3.2 mmol), HOBt (0.17 g, 1.3 mmol), H2N-Tyr-Gly-Gly-Phe-
OtBu (18, 0.59 g, 1.2 mmol, in 16 mL dry THF), and DMAP (0.04 g, 0.32 mmol) were
1
reacted to afford 20b as a light yellow oil (0.31 g, 33%). H NMR (CD3OD) d 0.99
(6H, dd, J1 ¼ 6:3 Hz, J2 ¼ 9:0 Hz), 1.36 (9H, s), 1.46 (9H, s), 1.71 (1H, m), 1.81
(2H, m), 2.24 (3H, s), 2.77 (1H, m), 2.91 (1H, m), 3.02 (2H, dd, J1 ¼ 4:8 Hz,
J2 ¼ 7:8 Hz), 3.53 (1H, d, J ¼ 16:8 Hz), 3.64 (1H, d, J ¼ 16:8 Hz), 3.76 (1H, d,
J ¼ 16:4 Hz), 3.87 (1H, m), 4.34 (2H, t, J ¼ 7:5 Hz), 4.51 (1H, t, J ¼ 7:2 Hz), 6.08
(1H, d, J ¼ 12:3 Hz), 6.65 (2H, d, J ¼ 8:7 Hz), 6.75 (1H, d, J ¼ 12:3 Hz), 6.93
(3H, t, J ¼ 9:9 Hz), 7.11–7.29 (7H, m). 13C NMR (CD3OD) d 21.06, 22.00, 23.48,
26.26, 28.38, 28.95, 37.69, 38.84, 41.50, 43.35, 43.96, 54.04, 56.13, 57.72, 80.97,
83.26, 116.59, 122.83, 126.76, 128.00, 128.64, 129.61, 130.02, 130.64, 131.34,
131.62, 134.26, 137.26, 138.28, 147.63, 157.52, 158.25, 169.16, 171.33, 172.12,
172.18, 174.41. MS (FAB) m=z 873.4 (Mþ + H).
2.25. Cyclic prodrug of DADLE (21b)
In a similar manner to the preparation of 21a, 19b (0.329 g, 0.371 mmol) was trea-
ted with a solution of 1:1 TFA:DCM (45 mL) under N2 atmosphere for 3.5 h at room
temperature. Upon concentration, the residue (in 200 mL dry DCM and 4.2 mL
DMF), Bop-Cl (0.81 g, 3.15 mmol), and 0.57 mL TEA were reacted to afford 21b
1
as a white solid (0.126 g, 48%). H NMR (CD3OD) d 0.93 (6H, dd, J1 ¼ 5:7 Hz,
J2 ¼ 13:2 Hz), 1.18 (3H, d, J ¼ 7:3 Hz), 1.72 (3H, m), 2.28 (3H, s), 2.79 (2H, m),
3.06 (1H, m), 3.42 (1H, m), 3.70 (1H, d, J ¼ 17:3 Hz), 3.96 (1H, q, J ¼ 7:3 Hz),
4.36 (1H, t, J ¼ 7:5 Hz), 4.60 (2H, m), 6.15 (1H, d, J ¼ 12:0 Hz), 6.67 (2H, d,
J ¼ 8:3 Hz), 6.73 (1H, d, J ¼ 12:2 Hz), 6.91 (3H, t, J ¼ 7:2 Hz), 7.02 (1H, s), 7.13–
7.32 (6H, m). 13C NMR (CD3OD) d 17.05, 21.06, 21.77, 23.44, 26.02, 36.73, 37.85,
41.05, 43.96, 50.00, 51.62, 52.46, 56.29, 57.06, 116.37, 122.31, 127.58, 127.79,
128.56, 129.58, 130.36, 130.93, 131.30, 131.47, 131.57, 136.23, 137.42, 139.00,
146.68, 157.47, 167.77, 172.24, 172.64, 173.35, 174.40, 175.79. MS (FAB) m=z
712.4 (Mþ + H).

L.A. Zych et al. / Bioorganic Chemistry 32 (2004) 109–123
119
2.26. Cyclic prodrug of Leu-enkephalin (22b)
In a manner similar to the preparation of 21a, 20b (0.13 g, 0.15 mmol) was treated
with a solution of 1:1 TFA:DCM (15 mL) under N2 atmosphere for 3.5 h at room
temperature. Upon concentration, the residue (in 144 mL dry DCM and 3 mL
DMF), Bop-Cl (0.39 g, 1.53 mmol), and 0.23 mL TEA were reacted to afford 22b as
a white solid (0.026 g, 25%). 1H NMR (CD3OD) d 1.01 (6H, dd, J1 ¼ 6:0 Hz,
J2 ¼ 20:0 Hz), 1.75 (2H, m), 1.88 (1H, t, J ¼ 10:0 Hz), 2.30 (3H, s), 2.63 (1H, m),
2.73 (1H, m), 3.08 (1H, m), 3.25 (1H, dd, J1 ¼ 5:2 Hz, J2 ¼ 14:0 Hz), 3.41 (1H, d,
J ¼ 2:0 Hz), 3.83 (2H, dd, J1 ¼ 1:6 Hz, J2 ¼ 16:8 Hz), 4.34 (1H, t, J ¼ 8:4 Hz),
4.50–4.60 (3H, m), 6.19 (1H, d, J ¼ 12:0 Hz), 6.67 (2H, d, J ¼ 8:8 Hz), 6.77 (1H, d,
J ¼ 12:0 Hz), 6.93 (3H, t, J ¼ 8:4 Hz), 7.03 (1H, s), 7.14 (1H, d, J ¼ 9:6 Hz), 7.20–
7.30 (5H, m). 13C NMR (CD3OD) d 21.02, 21.84, 23.57, 26.17, 37.92, 38.12, 40.88,
43.85, 44.34, 53.07, 57.20, 57.29, 116.44, 122.80, 127.78, 127.98, 128.47, 129.69,
130.40, 130.78, 130.96, 131.31, 131.53, 135.45, 137.36, 138.68, 146.84, 157.57,
168.48, 171.62, 171.94, 172.53, 174.23, 174.83. MS (FAB) m/z 704.3 (Mþ + Li).
3. Results and discussion
For this study, we designed a series of compounds with methyl substitutions at
different positions of the phenyl ring to achieve a thorough understanding of the
effect of alkyl substitution on the release kinetics. The opioid peptides [Leu5]-enkeph-
alin and DADLE were used as the drug moieties for the study.
3.1. Synthesis
The coumarin-based cyclic prodrugs were synthesized by the approach that uses
the commercially or synthetically available coumarins with different substituents
(Scheme 3). Coumarin 3b is commercially available. However, coumarin 3a had to
be synthesized. Starting from the substituted phenol 6a, by heating with propiolic
acid in the presence of methanesulfonic acid, coumarin 3a [18] was obtained. Then
the substituted coumarins 3 were reduced to the corresponding diols 7 [8] using lith-
ium aluminum hydride (LAH) at 0 °C. The primary hydroxyl groups of diols 7 were
selectively protected using tert-butyldimethylsilyl (TBDMS) chloride in the presence
of 4-dimethylaminopyridine (DMAP) at 0 °C to afford 8 [8]. The free hydroxyl
groups of 8 were then coupled with either D-Boc-Leu-OH or L-Boc-Leu-OH using
N,N0-dicyclohexylcarbodiimide (DCC) as the activating agent, in the presence of
DMAP to give compounds 9 and 10. The free allylic hydroxyl groups of 11 and
12, after deprotection of the silyl groups using acetic acid, were converted to the car-
boxyl groups in two steps. The oxidation to the aldehydes 13 and 14 was accom-
plished using manganese (IV) oxide (MnO2) in dichloromethane (DCM).
Conversion of the aldehydes 13 and 14 to the carboxylic acids 15 and 16 was accom-
plished by oxidation with sodium chlorite in the presence of hydrogen peroxide un-
der acidic conditions. The free acids were then coupled with either tetrapeptide 17

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L.A. Zych et al. / Bioorganic Chemistry 32 (2004) 109–123
Scheme 3. Synthesis of coumarinic acid derivatives.
[11] (H2N-Tyr-D-Ala-Gly-Phe-OtBu) or 18 [11] (H2N-Tyr-Gly-Gly-Phe-OtBu) in the
presence of DCC, 1-hydroxybenzotriazole (HOBt), and DMAP to give the linear
precursor compounds 19 and 20 (Scheme 4). After deprotection of the Boc- and
t-BuO- groups with 50% trifluoroacetic acid (TFA) in dichloromethane, the peptides
were cyclized in the presence of N,N-bis[2-oxo-3-oxazolidinyl]phosphorodiamidic
chloride (Bop-Cl) and triethylamine (TEA) to afford cyclic prodrugs of DADLE
(21) and [Leu5]-enkephalin (22).
3.2. Esterase kinetics
The esterase kinetic studies were carried out following procedure reported earlier
using porcine liver esterases (PLE) [4,6,9]. Briefly the reaction was monitored via
UV–Vis spectroscopy following the appearance of the coumarin starting materials
3. Upon incubation with PLE in phosphate buffer (0.5 M, pH 7.4, and 37 ꢂ 0.5 °C),
all cyclic prodrugs released the opioid peptide drug as designed.

L.A. Zych et al. / Bioorganic Chemistry 32 (2004) 109–123
121
Scheme 4. Synthesis of DADLE and [Leu5]-enkephalin cyclic prodrugs.
The objectives of this study were to see how alkyl substitutions on the phenyl ring
of coumarin would affect the release rates and whether such substitutions could help
to enhance the release rates as previously reported with the coumarin-based amine
prodrugs. As shown in Table 1, the release rates of the phenyl substituted cyclic pro-
drugs were found to be slower than the non-substituted one. This is opposite to what
was observed with the amine prodrugs [7,8].
It is known in our earlier studies with model amides [7,8] that methyl groups ortho
to either the side chain alkenyl group or the phenol carboxyl group can greatly
enhance the rate of the rate-limiting lactonization step through the introduction of
steric congestion. However, compounds 21a and 22a with methyl substituents ortho
to either the side chain alkenyl group or the phenolic carboxyl group have longer
Table 1
Esterase-catalyzed release rates of opioid peptides
Compound
R1
R2
R3
R4
t1=2 (min)
21a
21b
21ca
22a
22b
22ca
CH3
H
H
H
H
H
H
H
H
CH3
H
5636 ꢂ 1300
235 ꢂ 11
761 ꢂ 69
1950 ꢂ 58
668 ꢂ 35
317 ꢂ 27
CH3
H
H
H
CH3
H
H
CH3
H
CH3
H
H
H
a Refs. [9] and [11].

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L.A. Zych et al. / Bioorganic Chemistry 32 (2004) 109–123
half-lives compared with those of the corresponding unsubstituted ones (21c and
22c) [9,11]. Most likely, the bulkiness of the peptides in cyclic prodrugs makes it
more difficult for the esterase to reach the ester bond of the peptides and to catalyze
the initial hydrolysis. In such an event, the rate-limiting step in cyclic prodrugs must
be the esterase-catalyzed hydrolysis. Thus the steric congestion created by the sub-
stituents on the phenyl ring further hindered the access of the ester bond by porcine
liver esterase. Since the steric effect imposed by the ortho substitution is unfavorable
to the eventual release of the peptides, we synthesized the cyclic prodrugs where an
electron-donating methyl group was introduced to the position para to the hydroxyl
group (R3). The rationale was that the increased nucleophilicity of the hydroxyl
group might help to facilitate the cyclization rate. However, the anticipated results
were not observed. While the release rate of DADLE prodrug 21b increased com-
pared to the unsubstituted one, the release of the [Leu5]-enkephalin prodrug 22b de-
creased. Overall, the release rates of the cyclic prodrugs of peptides cannot be
predicted based on the substitution patterns alone. The idiosyncratic structural
features of each peptide prodrug seem to play a more prominent role in determining
the release kinetics in the coumarin-based prodrug system.
4. Conclusion
In order to fully understand the capabilities of the coumarin prodrug system
and the substitution effect upon the release rates of opioid peptides DADLE and
[Leu5]-enkephalin, we have designed and synthesized a series of substituted coumarin
derivatives to study their release kinetics. It was found that the release rates of
coumarin-based cyclic prodrugs of opioid peptides are determined by the structural
property of individual compounds. The substitution patterns on the phenyl ring of
coumarins did not affect the release rates of opioid peptides the same way that they
affected the release rate of model amines as previously reported.
Acknowledgments
Financial support from the American Heart Association (#9740117N) is grate-
fully acknowledged.
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