Effects of amino acid chirality and the chemical linker on the cell permeation characteristics of cyclic prodrugs of opioid peptides

Article abstract of DOI:10.1021/jm050277f

Previously, our laboratory showed that the oxymethyl-modified coumarinic acid (OMCA) cyclic prodrug of the opioid peptide DADLE ([D-Ala ²,D-Leu⁵]-Enk, H-Tyr-D-Ala-Gly-Phe-D-Leu-OH) exhibited low permeation across both the intestinal

Full text of DOI:10.1021/jm050277f

J. Med. Chem. 2006, 49, 1261-1270
1261
Effects of Amino Acid Chirality and the Chemical Linker on the Cell Permeation
Characteristics of Cyclic Prodrugs of Opioid Peptides
Bianca M. Liederer, Tarra Fuchs, David Vander Velde, Teruna J. Siahaan, and Ronald T. Borchardt*
Department of Pharmaceutical Chemistry, The UniVersity of Kansas, Lawrence, Kansas 66047
ReceiVed March 28, 2005
Previously, our laboratory showed that the oxymethyl-modified coumarinic acid (OMCA) cyclic prodrug of
the opioid peptide DADLE ([D-Ala2,D-Leu5]-Enk, H-Tyr-D-Ala-Gly-Phe-D-Leu-OH) exhibited low permeation
across both the intestinal mucosa and the blood-brain barrier (BBB). This low cell permeation arose from
its strong substrate activity for efflux transporters in these biological barriers. In an attempt to determine
whether the chirality of the amino acid asymmetric centers could influence the solution structure of the
cyclic prodrugs and thus their substrate activities for efflux transporters, we synthesized cyclic prodrugs of
the opioid peptides H-Tyr-Ala-Gly-Phe-D-Leu-OH ([Ala2,D-Leu5]-Enk), H-Tyr-D-Ala-Gly-Phe-Leu-OH ([D-
Ala2,Leu5]-Enk), and H-Tyr-Ala-Gly-Phe-Leu-OH ([Ala2,Leu5]-Enk). In an attempt to determine whether
the chemical linker (OMCA) bestowed efflux substrate activity on the cyclic prodrugs, we synthesized
capped linear derivatives (acetylated on the N-terminal and amidated on the C-terminal end) of [Ala2,D-
Leu5]-Enk, [D-Ala2,Leu5]-Enk, and [Ala2,Leu5]-Enk. The solution conformations of the cyclic prodrugs were
determined by molecular dynamics simulations using two-dimensional NMR data. The physicochemical
properties (molecular surface area, polar surface area, and cLogP) were estimated computationally using
Sybyl. Cell permeation characteristics were assessed using Caco-2 cells in the presence and absence of
known inhibitors of efflux transporters. Despite apparent differences in their solution conformations and
their physicochemical properties, the cyclic prodrugs of DADLE, [Ala2,D-Leu5]-Enk, [D-Ala2,Leu5]-Enk,
and [Ala2,Leu5]-Enk all exhibited strong substrate activity for efflux transporters in Caco-2 cells. In contrast,
the capped linear derivatives of [Ala2,D-Leu5]-Enk, [D-Ala2,Leu5]-Enk, and [Ala2,Leu5]-Enk exhibited very
poor substrate activity for efflux transporters in Caco-2 cells. Therefore, the substrate activities of the cyclic
prodrugs for efflux transporters in Caco-2 cells and in the intestinal mucosa and the BBB in vivo are most
likely due to the chemical linker used to prepare these molecules and/or its effect on solution structures of
the prodrugs.
Introduction
Opioid peptides such as DADLE ([D-Ala2,D-Leu5]-enkephalin,
H-Tyr-D-Ala-Gly-Phe-D-Leu-OH) have poor intestinal mucosal
and blood-brain barrier (BBB) permeability because they are
paracellular permeants.1 Therefore, their potential therapeutic
effects cannot be exploited clinically.2 To achieve better
membrane permeation, opioid peptides must possess physico-
chemical properties that allow them to diffuse across cellular
barriers (e.g., intestinal mucosa and BBB) via the transcellular
pathway rather than the paracellular pathway and not be
substrates for efflux transporters.3-6
One possible approach to solving this drug delivery problem
involves cyclization of opioid peptides using chemical linkers.7,8
These chemical linkers can be designed to be susceptible to
esterase hydrolysis, leading to a cascade of chemical reactions
that result in the eventual release of the peptide.9 Using this
prodrug strategy, opioid peptides can be converted from
paracellular to transcellular permeants.10-12 For example, oxy-
methyl-modified coumarinic acid (OMCA)-DADLE (1a), a
cyclic prodrug of DADLE that was developed earlier in our
laboratory, exhibited significantly better intrinsic cell permeation
characteristics than the parent linear peptide, DADLE, itself.10-12
However, despite its favorable physicochemical properties for
transcellular permeation, OMCA-DADLE (1a) showed low
permeation across both the intestinal mucosa and the BBB,
Figure 1. Structures of OMCA cyclic opioid prodrugs 1a-d and
N-acetylated and C-amidated derivatives of linear opioid peptides 2a-
c.
which could be attributed to its substrate activity for efflux
transporters, mainly MDR1 and MRP2.9,10,13
* To whom correspondence should be addressed. Phone: (785) 864-
3427. Fax: (785) 864-5736. E-mail: rborchardt@ku.edu.
10.1021/jm050277f CCC: $33.50 © 2006 American Chemical Society
Published on Web 02/02/2006

1262 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4
Liederer et al.
Scheme 1
The chirality of the amino acids in an opioid peptide like
DADLE can be manipulated while still retaining pharmacologi-
cal activity.14-16 For instance, it has been reported that the
residues in positions 2 and 5 of enkephalins, e.g., [Leu5]-Enk
(H-Tyr-Gly-Gly-Phe-Leu-OH) and [Met5]-Enk (H-Tyr-Gly-Gly-
Phe-Met-OH), are not critical for receptor binding interactions.
Consequently, analogues of these compounds including
DADLE, [Ala2,D-Leu5]-Enk (H-Tyr-Ala-Gly-Phe-D-Leu-OH),
[D-Ala2,Leu5]-Enk (H-Tyr-D-Ala-Gly-Phe-Leu-OH), and [Ala2,-
Leu5]-Enk (H-Tyr-Ala-Gly-Phe-Leu-OH) are biologically active
although they vary in their potencies.14-16 Since DADLE,
[Ala2,D-Leu5]-Enk, [D-Ala2,Leu5]-Enk, and [Ala2,Leu5]-Enk are
diastereomers, they would be expected to exhibit different
physicochemical properties. Similarily, cyclic prodrugs of these
opioid peptides might also be expected to have different
physicochemical characteristics.
Physicochemical factors, including polar surface area char-
acteristics, high hydrogen-bonding potential, and flexibility, have
all been shown to influence the ability of a molecule to permeate
cell membranes.3-5 For instance, as the polar surface area or
hydrogen-bonding potential of a transcellular permeant in-
creases, its cell membrane permeation will decrease. Thus,
changes in the chirality of the Ala and/or Leu residues in the
peptide portion of cyclic prodrugs could alter these physico-
chemical properties and thus influence their passive diffusion
across cell membranes.3,17
Polar surface characteristics are also very important for the
binding of molecules to efflux transporters.18-24 It has been

Cyclic Prodrugs of Opioid Peptides
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4 1263
Table 1. Chemical Shifts, Coupling Constants, and Dihedral Angles
(Calculated from Coupling Constants and Measured from
Energy-Minimized Average Structures) for Cyclic Prodrugs 1b-d
shown that a particular arrangement of electron donor groups
in the molecule is essential for substrate recognition by P-gp.
Again, altering the chirality of the Ala and/or Leu residues in
DADLE or in the cyclic prodrug of the opioid peptides could
change their polar surface area characteristics and, therefore,
affect their ability to interact with efflux transporters.
measured
chemical coupling
shift
constant
(ppm) NH 3JNHR (Hz)
calcd φ (deg)
φ (deg) ψ (deg)
OMCA-[Ala2,D-Leu5]-Enk (1b)
In an attempt to determine how the chirality of Leu and Ala
in the opioid peptide affects its solution conformation, physi-
cochemical properties, and cell permeation characteristics, we
synthesized cyclic prodrugs of the opioid peptides [Ala2,D-Leu5]-
Enk, [D-Ala2,Leu5]-Enk, and [Ala2,Leu5]-Enk (Figure 1, 1b-
d) and compared these to the properties of OMCA-DADLE (1a),
which had been reported earlier by our laboratory.8,10 Addition-
ally, capped derivatives (acetylated on the N-terminal and
amidated on the C-terminal end) (Scheme 1) of [Ala2,D-Leu5]-
Enk (2a), [D-Ala2,Leu5]-Enk (2b), and [Ala2,Leu5]-Enk (2c)
were synthesized and their cell permeation characteristics
determined in an attempt to elucidate whether the chemical
linker itself or conformations induced by the linker were
inducing the substrate activity for efflux transporters.
Tyr-1
7.94
8.32
8.07
7.36
8.42
5.7
5
11
7.5
7.1
-168, -60, 20, 120
172, -67, 31, 103
-135, -54, 54, 135
-157, -80, 35, 90
161, 79, -30, -91
114 -132
-69 -50
-88 41
Ala-2
Gly-3a
Phe-4
D-Leu-5
-150 -57
101 NA
OMCA-[D-Ala2,Leu5]-Enk (1c)
Tyr-1
8.47
8.66
8.00
7.55
8.32
5.5
4.6
11
7.9
7.2
-170, -71, 22, 97
175, 63, -18, -108
-135, -54, 54, 135
-157, -83, 35 to 90 -160 -71
-160, -78, 33, 90
-96 108
-27 -77
-89 55
D-Ala-2
Gly-3a
Phe-4
Leu-5
-153 NA
OMCA-[Ala2,Leu5]-Enk (1d)
Tyr-1
Ala-2
Gly-3a
Phe-4
Leu-5
7.10
8.48
8.36
7.70
8.62
2
2.3
12
8.7
5.8
-48, -2, 123, 155
117 112
71 -56
-50, 0, 120, 160
-128, -60, 60, 128 -162 -72
-175, -63, 18, 108
-165, -57, 23, 118
-72 -35
-85 NA
Results and Discussion
a ∑JNH-CH2
.
Synthesis. The N-acetylated, C-amidated peptides 2a-c were
synthesized using solution-phase methodologies8 beginning with
the tetrapeptide Boc-Tyr-(D,L)-Ala-Gly-Phe-OAll (3) (Scheme
1). Method A involved the Boc-deprotection of the tetrapeptide
(3), followed by acetylation of the N-terminal end (as well as
the Tyr phenol), deprotection of the C-terminal carboxylic group
(as well as the Tyr phenol), and, finally, coupling of the D-Leu-
amide or L-Leu-amide to Ac-Tyr-Ala-Gly-Phe-OH (5) to yield
Ac-[Ala2,D-Leu5]-Enk-NH2 (2a) and Ac-[Ala2,Leu5]-Enk-NH2
(2c), respectively. Method B involved initial deprotection of
the carboxylic acid of Boc-Tyr-D-Ala-Gly-Phe-OAll (3) using
LiOH.25 The resulting Boc-protected peptide, Boc-Tyr-D-Ala-
Gly-Phe-OH (6), was then coupled with Leu-amide. After Boc-
deprotection, acylation of the Tyr-D-Ala-Gly-Phe-Leu-NH2 (8)
yielded the desired Ac-[D-Ala2,Leu5]-Enk-NH2 (2b).
Conformational Characteristics. 1. NMR Spectroscopy.
NMR studies were performed to determine whether changes in
the chirality of the Ala or Leu amino acid residues of the opioid
peptide changed the solution conformations and, therefore, the
hydrogen-bonding interactions, polar surface characteristics, and
other physicochemical characteristics of the resulting cyclic
prodrugs 1b-d compared to 1a. These conformational/physi-
cochemical changes could then impact the intrinsic permeation
of the cyclic prodrugs via the transcellular pathway.3,26,27
Furthermore, these changes could affect the interaction of the
cyclic prodrugs with the efflux transporters that limit their
intestinal and BBB permeation.18-24 The populated conforma-
tions can be expected to vary with the nature of the environment,
from the very polar environment of water to the lipophilic
interior of the cell membrane. For these purposes, the most
relevant environment is the zone of reduced polarity at the
water-membrane interface. The slow step in transcellular
diffusion is likely to be entry into the membrane from this point,
because of the high energy cost of breaking hydrogen bonds
with water molecules. Evidence for intramolecular hydrogen
bonds in the peptide backbone is of particular interest, because
if these supplant hydrogen bonds with water, it could lower the
energy cost of desolvation.28,29 Although the membrane surface
environment is sometimes modeled in NMR experiments by
detergent micelles in water, these cyclic peptides were insuf-
ficiently soluble in water for such experiments. As an alternative,
a mixture of dimethyl sulfoxide (DMSO) and water (4:1 v/v)
was used instead, which dissolved the peptides in concentrations
suitable for the NMR experiments. The conformational results
we report for this solvent mixture will not necessarily hold for
water, but we have no experimental results in water to make
the comparison.
The proton chemical shifts of 1b-d were determined using
2D 1H-1H-correlation spectroscopy (COSY), 2D 1H-1H homo-
nuclear Hartmann-Hahn (HOHAHA), and 2D 1H-1H rotating
frame Overhauser effect spectroscopy (ROESY) (Table 1). The
amide-amide correlations in the ROESY are the most important
peaks in terms of understanding the peptide backbone confor-
mation and intramolecular hydrogen bonding.
Like all cyclic peptides, these compounds must formally
reverse the direction of the peptide backbone twice, but the chain
reversal may or may not take place in a normal â-turn
conformation. In all three cyclic peptides, ROE cross-peaks were
observed between the NH of Gly and the NH of Phe, and also
between the NH of Phe and the NH of Leu/D-Leu (Figure 2).
These through-space interactions are the expected markers for
a type I â-turn structure.30 Although in both 1b and 1d the â-turn
occurred at exactly the same residues (i.e., Ala, Gly, Phe, and
Leu), the first connectivity (Gly 3-Phe 4) was much stronger
than the second (Phe 4-Leu 5) for OMCA-[Ala2,Leu5]-Enk (1d),
while the reverse was true for OMCA-[Ala2,D-Leu5]-Enk (1b).
In two of the three compounds, a third cross-peak was also seen,
between Ala 2 and Gly 3; this is strongest in 1c, weaker in 1b,
and absent entirely in 1d. This third cross-peak would not be
observed in a normal type I â-turn and is indicative of varying
amounts of distortion away from typical â-turn geometry. A
long string of consecutive amide-amide cross-peaks is more
characteristic of an R-helix, but a conformation in R-space would
also have a series of low (∼4 Hz) coupling constants (3JHNR),30
which was not observed. It is unlikely that a short cyclic peptide
like 1c could adopt such an R-helix structure in any case. It
can also be appreciated from Figure 2 and Table 1 that the
chemical shifts of the amide protons vary significantly across
the series. A fourth related compound, the cyclic prodrug of
DADLE (1a), displayed no amide-amide cross-peaks at all and
thus adopts a very different nonstandard secondary structure.
These NMR data clearly show that changes in the stereochem-

1264 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4
Liederer et al.
Figure 2. Amide region of the ROESY spectrum of the cyclic prodrugs OMCA-[Ala2,D-Leu5]-Enk 1b (A), OMCA-[D-Ala2,Leu5]-Enk 1c (B), and
OMCA-[Ala2,Leu5]-Enk 1d (C).
istry of the Ala and Leu residue in DADLE can dramatically
alter the conformations of corresponding cyclic prodrugs.
On the basis of the large flexibility and, therefore, random
structure of short linear peptides, no NMR studies or MD
simulations were pursued for the capped linear opioid peptides
2a-c.26,31
2. Molecular Dynamics (MD) Simulations. To further
elucidate the solution conformations of cyclic prodrugs 1b-d,
MD simulations were done using distance constraints obtained
from the ROESY experiments described above.
The final energy-minimized average solution structures are
shown in Figure 3A-C. All dihedral angles (φ) of the residues
of these average solution structures except for Ala-2 in OMCA-
[Ala2,Leu5]-Enk (1d) are within 35° of those φ angles calculated
from the NMR coupling constant data (Table 1). Moreover, most
measured distances are consistent with the ROE distance
constraints. Cyclization represents a powerful constraint on the
accessible peptide backbone conformations for these molecules,
but little or no constraint on side chain conformations. Side
chain-backbone and side chain-side chain ROE’s were
minimal, e.g., compared with what is observed for a protein,
and overall there is little data to suggest preferred side chain
conformations are present. The side chains are represented in
Figure 3, but unlike the backbone conformation, the side chain
conformations shown only represent one low energy possibility.
All solution structures of cyclic prodrugs 1b-d appeared to
be stabilized at different locations by two to three intramolecular
hydrogen bonds involving mainly the backbone of the peptides.
These types of intramolecular hydrogen bonds would reduce
the extent to which these functional groups hydrogen bond
intermolecularly with water. Consequently, less desolvation
energy would be required for the molecules to enter and diffuse
across lipophilic cell membranes by the transcellular pathway.
The correlation of decreased hydrogen bonding potential of
peptides to increased permeability across Caco-2 cells has been
demonstrated previously and found to be the main driving force
for transcellular diffusion.28,29 The hydrogen bonds shown were
produced during the simulations, and they were not added as
constraints prior to the simulations.
In our simulations, OMCA-[Ala2,Leu5]-Enk (1d) displayed
a hydrogen bond between the NH of Gly and CdO of Tyr and
between the NH of Tyr and CdO of Leu. In contrast, OMCA-
[Ala2,D-Leu5]-Enk (1b) showed a hydrogen bond between the
NH of Leu and CdO of Tyr and between the NH of Tyr and
the phenolic oxygen of the promoiety. For OMCA-[D-Ala2,-
Leu5]-Enk (1c) three hydrogen bonds were observed, i.e.,
between the NH of Phe and CdO of Ala, the NH of Tyr and
CdO of Leu, and the NH of Leu and CdO of Tyr. For OMCA-
DADLE (1a) two possible hydrogen bonds have been reported
involving the NH of Gly and the oxygen of the phenol group

Cyclic Prodrugs of Opioid Peptides
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4 1265
Figure 3. Energy-minimized average solution structures of the cyclic prodrugs (A) OMCA-[Ala2,D-Leu5]-Enk 1b, (B) OMCA-[D-Ala2,Leu5]-Enk
1c, and (C) OMCA-[Ala2,Leu5]-Enk 1d; (- · -) intramolecular hydrogen bond.
Table 2. Calculated Physicochemical Properties of Cyclic Prodrugs
1a-d
Table 3. Calculated Physicochemical Properties of the Capped Linear
Opioid Peptides 2a-c
OMCA-
[Ala2,D-
Leu5]-
OMCA-
[D-Ala2,
Leu5]-
OMCA-
[Ala2,
Leu5]-
Enk 1d
Ac-
Ac-
Ac-
OMCA-
DADLE
1a
[Ala2,D-Leu5]- [D-Ala2,Leu5]- [Ala2,Leu5]-
Enk-NH2
Enk-NH2
2b
Enk-NH2
Enk 1b
Enk 1c
2a
2c
cLogP
5.4
571
170
30
5.4
589
139
24
5.4
560
150
27
5.4
650
150
23
cLogP
0.7
493
253
51
0.7
491
257
52
0.7
544
285
52
molecular surface area (Å2)
polar surface area (Å2)
polar surface area
(% of the total
molecular surface area (Å2)
polar surface area (Å2)
polar surface area
(% of the total molecular
surface area)
molecular surface area)
and/or the oxygen of the oxymethyl group.8 Accordingly, the
energy-minimized average structure of 1a obtained by MD
simulations shows one hydrogen bond between the NH of Gly
and the oxygen of the phenol group.
Physicochemical Properties. Certain physicochemical prop-
erties are typically predictive of the intrinsic cell membrane
permeation of a molecule via the transcellular pathway. For
instance, as the molecular surface area or polar surface area of
a transcellular permeant increases, its cell membrane permeation
will decrease.4,32-34 In contrast, the relationship between cLogP
and cell membrane permeation is nonlinear, i.e., the permeability
decreases and plateaus at both low (e.g., negative) and high
cLogP values.35
In summary, both the spectroscopic and MD simulation
studies suggest differences in solution structures among cyclic
prodrugs 1a-d. The apparent increase in the number of
intramolecular hydrogen bonds and the restricted conformational
flexibility of cyclic prodrugs 1b-d compared to 1a would
suggest that there may be differences in their physicochemical
properties and thus differences in their cell membrane perme-
ation characteristics.
To determine these relationships for the cyclic prodrugs (1a-
d) and the capped peptides (2a-c), their physicochemical
parameters were calculated using Sybyl 6.9; the results are

1266 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4
Liederer et al.
Table 4. Apparent Permeability Coefficients (Papp) of Cyclic Prodrugs 1a and 1b (A) and 1c and 1d (B) across Caco-2 Cell Monolayers in the Presence
and Absence of Known Inhibitors of P-gp and MRP2
A
P
app, cm/s (×107)a
OMCA-DADLE 1a
OMCA-[Ala2 ,D-Leu5]-Enk 1b
AP-to-BL
BL-to-AP
ratio
AP-to-BL
BL-to-AP
ratio
w/o inhibitor
0.80 ( 0.1
2.5 ( 0.2
5.1 ( 0.01
8.3 ( 1.9
2.0 ( 0.5
53.6 ( 10.9
33.1 ( 1.0
14.2 ( 1.1
40.8 ( 1.0
12.1 ( 1.8
67
13
2.8
4.9
6.1
0.61 ( 0.1
1.3 ( 0.4
6.4 ( 2.9
10.5 ( 1.3
1.4 ( 0.2
27.4 ( 0.5
7.0 ( 1.2
13.4 ( 1.5
30.0 ( 3.6
8.6 ( 0.9
45
GF120918 (2 µM)
GF120918 (10 µM)
MK 571 (50 µM)
CsA (20 µM)
5.4
2.1
2.9
6.1
B
P
app, cm/s (×107)a
OMCA-[D-Ala2,Leu5]-Enk 1c
OMCA-[Ala2,Leu5]-Enk 1d
AP-to-BL
BL-to-AP
ratio
AP-to-BL
BL-to-AP
ratio
w/o inhibitor
0.29 ( 0.05
2.0 ( 0.3
4.0 ( 0.9
5.1 ( 0.4
0.96 ( 0.2
28.1 ( 2.9
10.0 ( 0.9
9.0 ( 0.9
14.1 ( 1.9
13.2 ( 1.5
97
0.51 ( 0.05
2.4 ( 0.5
18.1 ( 2.8
5.9 ( 1.4
9.0 ( 1.3
34.5 ( 4.0
10.9 ( 2.2
35
GF120918 (2 µM)
GF120918 (10 µM)
MK 571 (50 µM)
CsA (20 µM)
5.0
2.3
2.8
14
2.5
1.9
3.3
12
4.8 ( 0.3
10.4 ( 1.9
0.94 ( 0.4
a Papp values are presented as mean ( SD (n ) 3).
shown in Tables 2 and 3, respectively. Cyclic prodrugs 1a-d
are more lipophilic than the capped peptides 2a-c as indicated
by their higher cLogP values (5.4 and 0.7, respectively). These
higher cLogP values for the cyclic prodrugs are not unexpected
because of the lipophilic nature of the promoiety.8,36 Addition-
ally, cyclization of the peptides positions the hydrophobic side
chains so that the polar backbone is shielded, which could have
a substantial effect on lipophilicity. Because Sybyl accounts only
for the two-dimensional (2D) structure, the calculated cLogP
values for cyclic prodrugs 1a-d are identical. A similar
observation was made for the capped peptides (2a-c). However,
based on high-performance liquid chromatography (HPLC)
retention times, differences in polarity were noted (OMCA-
[Ala2,Leu5]-Enk (most polar) > OMCA-[D-Ala2,Leu5]-Enk >
OMCA-[Ala2,D-Leu5]-Enk > OMCA-DADLE). Cyclic prodrugs
1a-d and capped peptides 2a-c have different molecular
surface areas ranging from 560 to 650 A2 for the cyclic prodrugs
and from 491 to 574 A2 for the capped peptides. Likewise, the
polar surface areas varied from 139 to 170 A2 and from 253 to
323 A2 for the cyclic prodrugs and capped peptides, respectively.
The data shown in Tables 2 and 3 clearly show the impact
that cyclization of a peptide has on its physicochemical
properties. While the molecular surface areas of cyclic prodrugs
1a-d are perhaps slightly greater than those of the capped linear
peptides 2a-c, the polar surface areas of 1a-d are significantly
lower than those of 2a-c. Significant differences in the cLogP
values were also observed, i.e., the cLogP values for cyclic
prodrugs 1a-d are 5.4 vs 0.7 for the capped linear peptides.
These differences in cLogP and polar surface area would suggest
that the intrinsic permeation of cyclic prodrugs 1a-d should
be significantly greater than the intrinsic permeation of the
capped linear peptides 2a-c.
Leu5)-Enk (1c), and OMCA-(Ala2,Leu5)-Enk (1d) across Caco-2
cell monolayers are provided in Table 4. Like OMCA-DADLE
(1a), the analogues 1b-d all exhibited low Caco-2 permeation.
In all cases, the BL-to-AP Papp values were significantly greater
(35 to 97 times) than the AP-to-BL Papp values, suggesting that
these diastereomers are all substrates for efflux transporters
in Caco-2 cells. Since MDR1 inhibitors (e.g., GF120918
(C34H33N3O5, N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-iso-
quinolyl)ethyl]phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acri-
dine carboxamide), cyclosporin A (CsA, C62H111N11O12, 3-ethyl-
6-(1-hydroxy-2-methyl-pent-3-enyl)-12,15,24,30-tetraisobutyl-
9,27-diisopropyl-1,4,7,10,13,16,18,21,25,31-decamethyl-
1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritriacontan-
2,5,8,11,14,17,20,23,26,29,32-undecaone)) and MRP2 inhibitors
(e.g., CsA, MK 571 (C26H27ClN2O3S2, 3-{3-[2-(7-chloro-
quinolin-2-yl)-vinyl]-phenyl}-3-(2-dimethylcarbamoyl-ethylsul-
fanylmethylsulfanyl)-propionic acid)) increase the AP-to-BL Papp
values and, thus, decrease the Papp BL-to-AP/Papp P-to-BL ratios,
the diastereomers 1b-d appear to be substrates for MDR1 and
MRP2. These data are very similar to the results observed with
OMCA-DADLE (1a).10
The cell permeation data suggest that despite differences in
the solution conformations, cyclic prodrugs 1a-d must all
present a structural feature(s) that is recognized by these efflux
transporters. One structural feature that has been shown to be
essential for substrate recognition by P-gp is at least one so-
called type I or type II unit. These type I and type II units are
entirely based on the particular arrangement of electron donor
groups and spatial separation distances (2.2 to 2.8 Å or 4.0 to
5.2 Å) in the molecule. These optimally arranged polar groups
are then recognized by P-gp.18,19,37 Since these diastereomers
all exhibit potential type I and type II units as determined
computationally (data not shown), it is not unexpected that they
would exhibit substrate activity for this efflux transporter.
On the basis of the differences in the preferred conformation
of cyclic prodrugs 1a-d as determined by NMR and MD
simulation, particularly the differences in molecular hydrogen
bonding interactions, one might expect to see differences in the
intrinsic permeabilities of these diastereomers. Unfortunately,
as shown in Table 4, complete inhibition of their efflux in
Caco-2 cells was never achieved; thus, we were unable to
determine accurately the intrinsic permeabilities of 1a-d.
Transport Studies. Previously, our laboratory had shown
that OMCA-DADLE (1a) was a substrate for efflux transporters
(e.g., P-gp, MRP2),9,10,13 which significantly limited its ability
to permeate cell membranes. In an attempt to modify this efflux
transporter substrate activity and thus alter its cell permeation
characteristics, we prepared analogues of DADLE that had
changes in the chirality of Ala and Leu in the 2 and 5 positions,
respectively, of this opioid peptide.
The Caco-2 cell permeation characteristics of OMCA-
DADLE (1a), OMCA-(Ala2,D-Leu5)-Enk (1b), OMCA-(D-Ala2,-

Cyclic Prodrugs of Opioid Peptides
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4 1267
Table 5. Apparent Permeability Coefficients (Papp) of the Capped Linear Opioid 2a-c Peptides across Caco-2 Cell Monolayers in the Presence and
Absence of Known Inhibitors of P-gp and MRP2
P
app, cm/ s (×107)a
Ac-[Ala2 ,D-Leu5]-Enk-NH2 2a
Ac-[D-Ala2,Leu5]-Enk-NH2 2b
Ac-[Ala2,Leu5]-Enk-NH2 2c
AP-to-BL
BL-to-AP
ratio
AP-to-BL
BL-to-AP
ratio
AP-to-BL
BL-to-AP
ratio
w/o inhibitor
0.17 ( 0.03
0.38 ( 0.01
0.29 ( 0.06
0.20 ( 0.01
0.42 ( 0.09
0.39 ( 0.07
1.1 ( 0.8
2.5
1.0
3.8
1.2
0.27 ( 0.08
0.54 ( 0.13
0.68 ( 0.13
1.1 ( 0.2
0.76 ( 0.08
0.57 ( 0.02
0.73 ( 0.16
2.7 ( 1.4
2.8
1.1
1.1
2.4
0.22 ( 0.03
0.14 ( 0.01
0.64 ( 0.06
0.21 ( 0.01
0.58 ( 0.26
0.29 ( 0.12
1.3 ( 0.1
2.6
2.1
2.0
1.8
GF120918 (10 µM)
MK 571 (50 µM)
CsA (20 µM)
0.23 ( 0.06
0.37 ( 0.01
a Papp values are presented as mean ( SD (n ) 3).
purchased from Aldrich Chemical Co. (Milwaukee, WI), Sigma
Chemical Co. (St. Louis, MO), Fluka Chemical Corp. (Milwaukee,
WI), Bachem Bioscience, Inc. (King of Prussia, PA), Fisher
Scientific (Pittsburgh, PA), and Novabiochem (La Jolla, CA) and
used as received.
In an attempt to resolve the importance of the OMCA linker
in cyclic prodrugs 1a-d as a determinant for efflux transporter
substrate activity, we assessed the Caco-2 cell permeation
characteristics of the capped linear opioid peptides 2a-c. As
shown in Table 5, all of these capped linear peptides exhibited
very low permeation across Caco-2 cell monolayers. Unlike
those of cyclic prodrugs 1a-d, the low AP-to-BL Papp values
observed for the capped linear peptides 2a-c are not the result
of extensive efflux. Instead, as predicted by their low cLogP
values and high polar surface areas, these capped linear peptides
appear to have low intrinsic cell permeation via the transcellular
pathway. While it was impossible to determine the intrinsic
permeability of cyclic prodrugs 1a-d, they appear (by compar-
ing their Papp values determined in the presence of 10 µM
GF120918 to the corresponding Papp values of the capped
peptides) qualitatively to be at least 7-fold more able to permeate
than the capped linear peptides 2a-c.
The poor substrate activity of the capped linear peptides 2a-c
for efflux transporters in Caco-2 cells would suggest that the
OMCA linker either makes cyclic prodrugs 1a-d sufficiently
lipophilic to gain access to efflux transporters in the lipid bilayer
and/or the cyclization creates solution structures (e.g., â-turns)
that are recognized by the efflux transporters, making these
cyclic prodrugs candidates for efflux.
Synthesis. Cyclic Prodrugs. The syntheses of cyclic prodrugs
1b-d (Figure 1) were carried out by following standard solution-
phase procedures described previously by our laboratory.8 Standard
Boc-amino acid solution-phase chemistry was employed to syn-
thesize the tetrapeptide fragment with the appropriate amino acids.
Unless specifically designated, amino acids employed were L-
configurations. The structural identity and purity of the cyclic
1
prodrugs were confirmed by H NMR, FAB-MS, and analytical
HPLC.
OMCA-[Ala2,D-Leu5]-Enk (1b). 1H NMR (500 MHz, DMSO-
d6) δ 0.72 (d, 3H, J ) 5.9 Hz), 0.78 (d, 3H, J ) 6.0 Hz), 1.18 (d,
3H, J ) 7.0 Hz), 1.34 (m, 2H), 1.48 (m, 1H), 2.69 (m, 2H), 2.88
(m, 2H), 3.18 (m, 1H), 3.76 (m, 1H), 4.03 (m, 1H), 4.10 (m, 1H),
4.31 (m, 1H), 4.52 (m, 1H), 5.72 (dd, 2H, J1 ) J2 ) 6.6 Hz), 6.05
(d, 1H, J ) 12.6), 6.71 (d, 2H, J ) 8.5 Hz), 6.91 (m, 1H), 6.80-
7.28 (m, 12H), 7.42 (d, 1H, J ) 7.7), 7.71 (d, 1H, J ) 8.4), 7.94
(d, 1H, J ) 7.9), 8.13 (m, 1H), 8.29 (d, 1H, J ) 8.3). FAB-MS:
m/z 728.3 (M + 1).
1
OMCA-[D-Ala2,Leu5]-Enk (1c). H NMR (500 MHz, DMSO-
d6) δ 0.82 (d, 3H, J ) 6.1 Hz), 0.87 (d, 3H, J ) 6.1 Hz), 1.09 (d,
3H, J ) 7.2 Hz), 1.54 (m, 3H), 2.85 (m, 3H), 3.58 (m, 2H), 3.98
(m, 1H), 4.23 (m, 2H), 4.39 (m, 2H), 5.65 (d, 1H, J ) 6.6 Hz),
5.78 (d, 1H, J ) 6.6 Hz), 6.04 (d, 1H, J ) 12.5 Hz), 6.70 (d, 2H,
J ) 8.5 Hz), 7.00-7.34 (m, 12 H), 7.37 (d, 1H, J ) 7.8), 7.49 (d,
1H, J ) 8.5), 8.22 (d, 1H, J ) 8.3), 8.33 (d, 1H, J ) 6.6), 8.49 (d,
1H, J ) 5.9). FAB-MS: m/z 728.5 (M + 1).
Conclusion
We have demonstrated that changes in the chirality of amino
acids in the peptide portion of cyclic prodrugs can have a
tremendous effect on the solution structure. However, despite
different solution conformations and physicochemical properties,
all molecules exhibited substrate activity for efflux transporters
that could severely limit their oral bioavailability and penetration
to the brain in vivo. This substrate activity for efflux transporters
is most likely due to the chemical linker used to prepare the
cyclic prodrugs and/or its effect on the solution structures of
these molecules.
1
OMCA-[Ala2,Leu5]-Enk (1d). H NMR (500 MHz, DMSO-
d6) δ 0.81 (d, 3H, J ) 5.8 Hz), 0.88 (d, 3H, J ) 5.9 Hz), 1.18 (d,
3H, J ) 6.9 Hz), 1.53 (m, 2H), 1.65 (m, 1H), 2.55 (m, 2H), 2.81
(m, 2H), 3.10 (dd, 1H, J1 ) J2 ) 4.7 Hz), 3.77 (m, 1H), 3.93 (m,
1H), 4.13 (m, 1H), 4.42 (m, 2H), 5.76 (d, 1H, J ) 6.7), 5.81 (d,
1H, J ) 6.8 Hz), 5.97 (d, 1H, J ) 12.6), 6.68 (d, 2H, J ) 8.4 Hz),
6.81 (m, 2H), 7.01 (d, 2H, J ) 8.4), 7.18 (m, 8H), 7.72 (d, 1H, J
) 8.7), 8.19 (s, 1H), 8.35 (d, 2H, J ) 6.6), 9.20 (s, 1H). FAB-MS:
m/z 728.3 (M + 1).
Capped PeptidessMethod A (Scheme 1). Method A was used
to synthesize the N-acetylated, C-amidated derivatives of [Ala2,D-
Leu5]-Enk (2a) and [Ala2,Leu5]-Enk (2c). Final products were all
purified by column chromatography and characterized by 1H NMR
and FAB-MS.
NH2-Tyr-Ala-Gly-Phe-OAll. Following a previously published
procedure,8 Boc-Tyr-Ala-Gly-Phe-OAll (3) (0.710 g, 1.0 mmol) was
dissolved in ethyl acetate (30 mL) and cooled to 0 °C. HCl (gas)
was bubbled into the solution for 10 min, and the solution was
then stirred for an additional 20 min. The ethyl acetate was removed
by rotary evaporation to give a white solid in quantitative yield.
This product was used in the next step without purification. Rf )
0.1 (100% ethyl acetate).8
Ac-Tyr(OAc)Ala-Gly-Phe-OAll (4). NH2-Tyr-Ala-Gly-Phe-
OAll (610 mg, 1.0 mmol) was dissolved in 10 mL of dioxane:
water (1:1, 10 mL) and acetic anhydride (3 mL). The solution was
stirred for 4 h at room temperature. The solvents were removed in
vacuo to give a white solid, which was used in the next step without
Experimental Section
Materials. L-Glutamine (200 mM (100×)), penicillin (10000
U/mL), streptomycin (10000 µg/mL), fetal bovine serum (FBS),
and nonessential amino acids (10 mM (100×) in 85% saline) were
obtained from Gibco BRL, Life Technologies (Grand Island, NY).
Dulbecco’s modified Eagle medium (DMEM) and trypsin/ethyl-
enediaminetetraacetic acid (EDTA) solution (0.25% and 0.02%,
respectively, in Ca2+- and Mg2+-free Hank’s balanced salt solution
(HBSS)) were purchased from JRH Bioscience (Lenexa, KS). D-[1-
14C]-Mannitol (specific activity 2.15 GBq/mmol) was purchased
from Amersham Biosciences (Uppsala, Sweden). Polyester mem-
branes (Transwell, 0.4 µm pore size, 24 mm diameter) were
obtained from Fisher Scientific (Pittsburgh, PA). MK 571 was
ordered from Biomol Research Laboratories, Inc. (Plymouth
Meeting, PA). GF120918 was donated by Dr. Kenneth Brouwer
(GlaxoSmithKline, Research Triangle Park, NC). OMCA-DADLE
(1a) was synthesized in our laboratory following procedures
described elsewhere.8 All other chemicals and solvents were

1268 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4
Liederer et al.
purification. Rf ) 0.4 (CHCl3/CH3OH, 10:1). 1H NMR (400 MHz,
CD3OD) δ 1.3 (d, 3H, J ) 7.2 Hz), 1.90 (s, 3H), 2.23 (s, 3H), 2.90
(dd, 1H, J1 ) 8.7 Hz, J2 ) 14.0 Hz), 3.30-3.34 (m, 3H), 3.75 (d,
1H, J ) 17.0 Hz), 3.90 (d, 1H, J ) 17.0 Hz), 4.25 (q, 1H, J ) 7.1
Hz), 4.54-4.60 (m, 3H), 4.68 (dd, 1H, J1 ) 6.1 Hz, J2 ) 8.2 Hz),
5.17 (dd, 1H, J1 ) 1.0, J2 ) 10.8 Hz), 5.26 (dd, 1H, J1 ) 1.5 Hz,
J2 ) 17.2 Hz), 5.79-5.87 (m, 1H), 6.99 (d, 2H, J ) 8.5 Hz), 7.18-
7.29 (m, 7 H).
Ac-Tyr-Ala-Gly-Phe-OH (5). Ac-Tyr(OAc)Ala-Gly-Phe-OAll
(4) was dissolved in CH3OH (75 mL), and aqueous LiOH (250 mg
dissolved into 25 mL of water) was added. LiOH is able to deprotect
the C-terminus and the O-acetyl ester of tyrosine.25 After the
reaction mixture was stirred at room temperature for 24 h, the CH3-
OH was removed by rotary evaporation. The aqueous solution was
then acidified by 2 N HCl and dried in vacuo. The resulting white
solid was suspended in CH3OH (50 mL) and filtered, and the CH3-
OH was evaporated to give a white solid containing the N-acetylated
peptide, which was used in the next step without purification. Rf )
0.4 (CHCl3/CH3OH, 1:1). 1H NMR (400 MHz, CD3OD) δ 1.34 (d,
3H, J ) 7.1 Hz), 1.95 (s, 3H), 2.85 (dd, 1H, J1 ) 8.4 Hz, J2 )
13.9 Hz), 3.01-3.09 (m, 2H), 3.19 (dd, 1H, J1 ) 5.1 Hz, J2 ) 6.3
Hz), 3.74 (d, 1H, J ) 13.2 Hz), 3.90 (d, 1H, J ) 16.9 Hz), 4.27 (q,
1H, J ) 7.3), 4.50 (t, 1H, J ) 7.3 Hz), 4.62 (dd, 1H, J1 ) 5.0 Hz,
J2 ) 8.1 Hz), 6.7 (d, 2H, J ) 8.5 Hz), 7.08 (d, 2H, J ) 8.4 Hz),
7.18-7.28 (m, 5H).
Ac-Tyr-Ala-Gly-Phe-Leu-NH2 (2c). To Ac-Tyr-Ala-Gly-Phe-
OH (5) (1.2 mmol) in dimethylformamide (DMF) (5 mL) was added
dry tetrahydrofuran (THF) (15 mL); the solution was cooled over
ice. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
(EDC‚HCl) (242 mg, 1.26 mmol) and 1-hydroxybenzotriazole
(HOBt) (17 mg, 1.26 mmol) were added, and the solution was
stirred for 30 min. Triethylamine (TEA) (256 mg, 352 µL, 2.53
mmol), 4-dimethylamino pyridine (DMAP) (15 mg, 0.126 mmol),
and Leu-NH2 (164 mg, 1.26 mmol) were added. The reaction
mixture was allowed to warm to room temperature and stirred under
N2. After the mixture was stirred overnight, the solvent was reduced
by rotary evaporation and dried in vacuo. The residue was purified
by silica gel column chromatography eluting with CHCl3/CH3OH
(9:1 followed by 3:1 followed by 1:1). Note: DMAP elutes in 9:1
CHCl3/CH3OH and the peptide elutes with increasing amounts of
CH3OH. This purification yielded a white solid (4.0 mg, 1.2%
yield). Contaminants (HOBt and TEA) remaining after purification
were removed by rinsing the white solid with minimal amounts of
ice-cold water until NMR spectroscopy showed that the peptide
was pure. Rf ) 0.6 (CHCl3/CH3OH, 8:2). 1H NMR (400 MHz, CD3-
OD) δ 0.91 (d, 3H, J ) 6.0 Hz), 0.95 (d, 3H, J ) 0.9 Hz), 1.33 (d,
3H, J ) 7.2 Hz), 1.70-1.80 (m, 3H), 1.95 (s, 3H), 2.86 (dd, 1H,
J1 ) 8.3, J2 ) 13.8 Hz), 3.00-3.06 (m, 2H), 3.22 (dd, 1H, J1 )
5.5 Hz, J2 ) 14.0 Hz), 3.69 (d, 1H, J ) 16.7 Hz), 3.83 (d, 1H, J
) 16.8 Hz), 4.22 (q, 1H, J ) 7.1 Hz), 4.35 (dd, 1H, J1 ) 4.3 Hz,
J2 ) 10.3 Hz), 4.55 (t, 1H, J ) 8.1 Hz), 4.62 (dd, 1H, J1 ) 4.4 Hz,
J2 ) 8.8 Hz), 6.70 (d, 2H, J ) 8.5 Hz), 7.06 (d, 2H, J ) 8.6 Hz),
7.21-7.31 (m, 5H). FAB-MS: m/z 611.4 (M + 1).
removed by rotary evaporation. The aqueous solution was then
neutralized (pH 4-5 as monitored by pH paper) with TFA and
dried in vacuo. The resulting white solid was used in the next step
without purification. Rf ) 0.4 (CHCl3/CH3OH, 1:1). 1H NMR (400
MHz, CD3OD) δ 1.22 (d, 3H, J ) 7.2 Hz), 1.41 (s, 9H), 2.84 (dd,
1H, J1 ) 7.3, J2 ) 13.5 Hz), 2.94 (dd, 1H, J ) 7.8, J2 ) 10.6 Hz),
3.05 (dd, 1H, J1 ) 7.9 Hz, J2 ) 13.6 Hz), 3.24 (dd, 1H, J1 ) 4.3
Hz, J2 ) 13.7 Hz), 3.78 (d, 1H, J ) 16.7 Hz), 3.85 (d, 1H, J )
16.9 Hz), 4.16 (t, 1H, J ) 7.5 Hz), 4.27 (dd, 1H, J1 ) J2 ) 7.1
Hz), 4.56 (t, 1H, J ) 7.2 Hz), 6.73 (d, 2H, J ) 8.3 Hz), 7.06 (d,
2H, J ) 8.3 Hz), 7.19-7.29 (m, 5H).
Boc-Tyr-D-Ala-Gly-Phe-Leu-NH2 (7). Boc-Tyr-D-Ala-Gly-Phe-
OH (6) (750 mg, 1.25 mmol) was dissolved in DMF (5 mL)
followed by dry THF (10 mL) and cooled to 0 °C. EDC‚HCl (251
mg, 1.31 mmol) and HOBt (177 mg, 1.31 mmol) were added and
the suspension stirred for 30 min. TEA (273 mg, 376 µL, 2.7 mmol),
DMAP (16 mg, 0.131 mmol), and Leu-NH2 (171 mg, 1.31 mmol)
were added, and the reaction mixture was allowed to warm to room
temperature and was stirred under N2. After being stirred overnight,
the suspension was reduced by rotary evaporation and dried in
vacuo. The residue was purified by silica gel column chromatog-
raphy with CHCl3/CH3OH (9:1, followed by 3:1, followed by 1:1).
Note: DMAP eluted in 9:1 CHCl3/CH3OH. Increasing amounts of
CH3OH lead to elution of the peptide. Rf ) 0.6 (CHCl3/CH3OH,
8:2). Contaminants (HOBt and TEA) were removed by rinsing the
white solid with minimal amounts of ice-cold water until NMR
spectroscopy showed that the peptide was pure (183 mg, 22% yield).
1H NMR (400 MHz, CD3OD) δ 0.90 (d, 3H, J ) 6.0 Hz), 0.96 (d,
3H, J ) 6.0 Hz), 1.22 (d, 3H, J ) 1.22 Hz), 1.41 (s, 9H), 3.74 (d,
1H, J ) 16.8 Hz), 3.91 (d, 1H, J ) 16.4 Hz), 4.17-4.21 (m, 2H),
4.24-4.32 (m, 1H), 4.56 (t, 1H, J ) 7.1 Hz), 6.73 (d, 2H, J ) 8.4
Hz), 7.04 (d, 2H, J ) 8.5 Hz), 7.21-7.32 (m, 5H).
NH2-Tyr-D-Ala-Gly-Phe-Leu-NH2 (8). Boc-Tyr-D-Ala-Gly-Phe-
Leu-NH2 (7) was dissolved in ethyl acetate (30 mL) cooled to 0
°C. HCl (gas) was bubbled into the solution for 10 min, and then
the solution was stirred for an additional 20 min. The ethyl acetate
was removed by rotary evaporation to give a white solid in
quantitative yield. This product was used in the next step without
1
purification. Rf ) 0.1 (100% ethyl acetate). H NMR (400 MHz,
CD3OD) δ 0.90 (d, 3H, J ) 7.4 Hz), 0.95 (d, 3H, J ) 6.3 Hz),
1.30 (d, 3H, J ) 7.2 Hz), 3.05 (dd, 2H, J1 ) 8.0, J2 ) 12.4 Hz),
3.15-3.20 (m, 2H), 3.82 (s, 2H), 4.08 (t, 1H, J ) 7.3 Hz), 4.22
(dd, 1H, J1 ) 7.2 Hz, J2 ) 14.3 Hz), 4.25-4.29 (m, 1H), 4.68 (dd,
1H, J1 ) 5.6 Hz, J2 ) 8.2 Hz), 6.82 (d, 2H, J ) 6.8 Hz), 7.14 (d,
2H, J ) 8.5 Hz), 7.25-7.36 (m, 5H).
Ac-Tyr-D-Ala-Gly-Phe-Leu-NH2 (2b). NH2-Tyr-D-Ala-Gly-Phe-
Leu-NH2 (8) was dissolved in dioxane:water (1:1, 10 mL) and acetic
anhydride (3 mL). The solution was stirred for 4 h at room
temperature. The solvents were removed in vacuo to give a white
solid. This solid was then dissolved in CH3OH (4 mL), water (2
mL), and saturated bicarbonate (2 mL) and stirred for 1 h in order
to remove any O-acylation from the tyrosine residue.38 The CH3-
OH was removed by rotary evaporation. The resulting aqueous
solution was neutralized with HCl, and the solvent was removed
in vacuo to afford a white solid. CHCl3/CH3OH (1:1) was used to
redesolve the peptide and the suspension of salts was gravity
filtered. After rotary evaporation, the residue was purified by silica
gel column chromatography with CHCl3/CH3OH (7% CH3OH
followed by 25% CH3OH, followed by 50% CH3OH to elute the
peptide) (4.3 mg, 2.4% yield). Rf ) 0.55 (CHCl3/CH3OH, 8:2). 1H
NMR (400 MHz, CD3OD) δ 0.89 (d, 3H, J ) 5.9 Hz), 0.94 (d,
3H, J ) 5.9 Hz), 1.24 (t, 3H, J ) 7.2 Hz), 1.59-1.68 (m, 3H),
1.99 (s, 3H), 2.94 (dd, 2H, J1 ) 4.9 Hz, J2 ) 7.4 Hz), 3.07 (dd,
1H, J1 ) 8.9 Hz, J2 ) 13.9 Hz), 3.21 (dd, 1H, J1 ) 5.6 Hz, J2 )
13.9 Hz), 3.44 (d, 2H, J ) 3.8 Hz), 4.13 (q, 1H, J ) 7.3 Hz), 4.34
(t, 1H, J ) 4.8 Hz), 4.43 (t, 1H, J ) 7.6 Hz), 4.60 (dd, 1H, J1 )
5.7 Hz, J2 ) 8.9 Hz), 6.74 (d, 2H, J ) 8.5 Hz), 7.06 (d, 2H, J )
8.5 Hz), 7.23-7.31 (m, 5H). FAB-MS: m/z 611.1 (M + 1).
NMR Spectroscopy. Cyclic prodrugs 1b-c (∼3.5 mg) were
dissolved in DMSO-d6 containing approximately 20% H2O (∼0.6
mL). One- and two-dimensional NMR data were acquired on a
Ac-Tyr-Ala-Gly-Phe-D-Leu-NH2 (2a). The D-Leu analogue (2a)
was synthesized as described above by coupling Ac-Tyr-Ala-Gly-
Phe-OH (5) to D-Leu-NH2. Rf ) 0.6 (CHCl3/CH3OH, 8:2). 1H NMR
(400 MHz, CD3OD) δ 0.73 (d, 3H, J ) 6.4 Hz), 0.79 (d, 3H, J )
6.6 Hz), 1.36 (d, 3H, J ) 7.2 Hz), 1.40-1.64 (m, 3H), 1.94 (s,
3H), 2.87 (dd, 1H, J1 ) 8.2 Hz, J2 ) 13.9 Hz), 3.00-3.09 (m,
3H), 3.75 (d, 1H, J ) 16.9 Hz), 3.87 (d, 1H, J ) 16.9 Hz), 4.14-
4.23 (m, 2H), 4.48-4.55 (m, 2H), 6.73 (d, 2H, J ) 6.7 Hz), 7.07
(d, 2H, J ) 8.5 Hz), 7.23-7.30 (m, 5H). FAB-MS: m/z 611.4 (M
+ 1).
Capped PeptidessMethod B (Scheme 1). Method B was used
to synthesize the N-acetylated, C-amidated derivatives of [D-Ala2,-
Leu5]-Enk (2b). The final product was purified by column chro-
1
matography and characterized by H NMR and FAB-MS.
Boc-Tyr-D-Ala-Gly-Phe-OH (6). Boc-Tyr-D-Ala-Gly-Phe-OAll
(3) (1.5 g) was dissolved in CH3OH (160 mL), and aqueous LiOH
(530 mg LiOH in 53 mL of water) was added.25 After the reaction
mixture was stirred at room temperature for 24 h, the CH3OH was

Cyclic Prodrugs of Opioid Peptides
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4 1269
Varian Inova 600 at 293 K. Coupling constants (3JHNR) were
measured from spectra obtained using double quantum filtered
correlated spectroscopy (DQF-COSY). HOHAHA and ROESY
experiments were performed with a 70 ms and a 300 ms spin-
lock time, respectively. On the basis of the intensity of the amide
ROE cross-peaks, the nature of the through space interproton
interactions were assigned as strong, medium, or weak with upper
and lower boundaries of distances of 1.9-2.5, 2.5-3.5, and 3.5-
5.0 Å, respectively. Side chain protons were not assigned stereospe-
cifically; hence, ROE restraints for side chains were calculated by
considering pseudo atoms.30 The NMR data were processed using
FELIX software (version 95, Biosym Technologies, San Diego, CA)
with a final matrix of 1 K × 1 K real datapoints.
MRP2 inhibitor CsA (20 µM), and the MRP inhibitor MK 571 (50
µM). Cell monolayers were washed 3 times with prewarmed HBSS,
pH 7.4. Peptide solutions (final concentration 20 µM) prepared in
HBSS containing 0.4% DMSO were applied to the donor compart-
ment (AP: 1.5 mL; BL: 2.6 mL). HBSS was added to the receiver
compartment and aliquots (donor: 10 µL; receiver: 100 µL) were
removed and replaced with prewarmed HBSS at various time
intervals up to 160 min. For transport experiments determining the
impact of efflux transporters on the cell permeation, GF120918,
CsA, or MK 571 was included in HBSS. The samples withdrawn
were diluted with HBSS containing 10% (v/v) acetonitrile and
immediately frozen and kept at -80 °C until analysis by high-
performance liquid chromatography with tandem mass spectrometric
detection (LC-MS-MS). Permeability coefficients (Papp) of the cyclic
and capped peptides were calculated using the following equation:
MD Simulations. Solution conformations for cyclic prodrugs
1b-d were determined by MD simulations using the 2D NMR
data as described previously by our laboratory.8 Briefly, MD
simulations using ROE constraints were carried out on a Silicon
Graphics Computer equipped with Insight II molecular modeling
software (Release 98.0, Molecular Simulations, Inc., San Diego,
CA). From initial MD simulations performed as described previ-
ously,8 four to nine distinct conformational families were obtained.
Average structures from each family were then energy-minimized,
solvated with a water layer of 8 Å, and subjected to MD simulations
at 300 K using ROE constraints. Several conformations were
selected for the final analysis using two criteria: (i) that the
conformation had an interproton distance error of less than 0.5 Å
compared to upper and lower bounds of distances from ROE data;
and (ii) that the conformation had angles within 35° of values
Papp ) (∆Q/∆t)/(A × C0)
where ∆Q/∆t is the linear appearance rate of mass in the receiver
solution, A is the cross-sectional area (4.71 cm2), and C0 is the
initial peptide concentration of the donor compartment at t ) 0.
Sample Analysis. LC-MS-MS was employed using a Quattro
Micro triple quadrupole mass spectrometer (Micromass, Beverly,
MA). The LC was conducted using a Waters 2690 HPLC system
(Waters, Milford, MA). The analytical column, which was kept at
25 °C, was a Vydac (Hesperia, CA) C18 HPLC column (50 × 1.0
mm i.d., 300 Å, Vydac 218TP) with a guard column (5 µm
microbore cartridge, Vydac 218GK51). Sample separation was
achieved utilizing a linear gradient from 5% to 100% B (mobile
phase: A, water with 0.1% FA (v/v); B, acetonitrile with 0.1% FA
(v/v)) and a flow rate of 0.2 mL/min. The total run time was 12
min (5 min gradient 5% to 100% B; 2 min 100% B, 1 min reduction
of 100% B to 5% B, 4 min equilibration with 5% B). The eluent
of the first two minutes and the last 3 min was directed to waste.
Samples were recorded by multiple reaction monitoring (cyclic
prodrugs: m/z 728.3 > 120.1; capped peptides: m/z 611.0 > 333.9).
The MS-MS conditions for the cyclic prodrugs were as follows:
capillary voltage 3.20 kV; cone voltage 30 V, source temperature
120 °C; desolvation gas flow 740 L/h; multiplier voltage 650 V;
dwell time 0.15 s; inter-channel delay 0.03 s. The MS-MS
conditions for the capped peptides were as follows: capillary
voltage 4.0 kV; cone voltage 25 V, source temperature 100 °C;
desolvation gas flow 740 L/h; multiplier voltage 650 V; dwell time
0.5 s; inter-channel delay 0.03 s. Data acquisition and analysis were
performed using Mass Lynx v3.5 and v4.0 software (Micromass).
Further information on analytical methods developed in our
laboratory for cyclic prodrugs can be found elsewhere.40
3
calculated from JNHR. These conformations were minimized with
solvent molecules using the conjugate gradient method without a
cross term until the rms derivation was 0.4 kcal/mol‚Å. Final
average energy-minimized solution structures were chosen based
on energy and their consistency with the NMR data (dihedral angles
and ROE distances).
Physicochemical Properties. Molecular surface area, polar
surface area, and cLogP estimations were performed on a Silicon
Graphics Computer using Sybyl Version 6.9 (Tripos Inc., St. Louis,
MO). The three-dimensional (3D) structures of the N-acetylated
and C-amidated opioid peptides were built and energy-minimized
using Sybyl. The cyclic opioid peptide prodrugs were energy-
minimized starting from the imported structures generated as
outlined above (see Molecular Dynamics Simulations). All mol-
ecules were in their neutral form. The cLogP values were obtained
from the Molecular Spreadsheet. The molecular surface areas and
polar surface areas were computed utilizing Connolly molecular
surfaces. The polar surface area was defined as the sum of the
surface areas of oxygen, nitrogen, and hydrogen atoms attached to
nitrogen or oxygen atoms.
Cell Culture. Caco-2 cells (passage 18) were obtained from
American Type Culture Collection (Rockville, MD). As described
previously,39 cells were grown in a controlled atmosphere of 5%
CO2 and 90% relative humidity at 37 °C in 150 cm2 culture flasks
using a culture medium consisting of DMEM supplemented with
10% heat-inactivated FBS, 1% nonessential amino acids, 100 µg/
mL streptomycin, 100 U/mL penicillin, and 1% L-glutamine. When
approximately 80% confluent (i.e., 3-5 days), cells were detached
from the plastic support by partial digestion using trypsin/EDTA
solution and either subcultured in new flasks or seeded in 6-well
cell culture plates on polyester membranes (Transwell, 0.4 µm pore
size, 24 mm diameter) at a density of 8.0 × 104 cells/cm2. Caco-2
cells were fed with culture medium the day after seeding and then
every other day until transport experiments were performed (apical
(AP) volume 1.5 mL; basolateral (BL) volume 2.6 mL). Cells used
in this study were between passages 30 and 39.
Acknowledgment. The authors thank Dr. Hui Ouyang for
helpful advice and discussions. Financial support was provided
by the United States Public Health Service (DA 09315).
Supporting Information Available: MS, HPLC, and NMR data
for the cyclic prodrugs 1b-d and the capped peptides 2a-c and
constraints used for MD simulations. This material is available free
of charge via the Internet at http://pubs.acs.org.
References
(1) Pauletti, G. M.; Gangwar, S.; Siahaan, T. J.; Aube´, J.; Borchardt, R.
T. Improvement of oral peptide bioavailability: Peptidomimetics and
prodrug strategies. AdV. Drug DeliVery ReV. 1997, 27, 235-256.
(2) Pardridge, W. M. Blood-brain barrier drug targeting: the future of
brain drug development. Mol. InterVentions 2003, 3, 90-105, 151.
(3) Veber, D. F.; Johnson, S. R.; Cheng, H. Y.; Smith, B. R.; Ward, K.
W.; Kopple, K. D. Molecular properties that influence the oral
bioavailability of drug candidates. J. Med. Chem. 2002, 45, 2615-
2623.
(4) Atkinson, F.; Cole, S.; Green, C.; van de Waterbeemd, H. Lipophi-
licity and other parameters affecting brain penetration. Curr. Med.
Chem.: Cent. NerV. Syst. Agents 2002, 2, 229-240.
(5) Pauletti, G. M.; Gangwar, S.; Knipp, G. T.; Nerkurkar, M. M.;
Okumu, F. W.; Tamura, K.; Siahaan, T. J.; Borchardt, R. T. Structural
requirements for intestinal absorption of peptide drugs. J. Controlled
Release 1996, 41, 3-17.
Transport Studies. For all transport studies, 21 to 28 day-old
Caco-2 cell monolayers grown on polyester membranes (Transwell)
were used. The integrity of each batch of cells was verified by
measuring [14C]-mannitol flux in representative monolayers. Bi-
directional transport experiments were conducted (n ) 3) at 37 °C
in a shaking water bath (30 rpm) in the absence and presence of
the MDR1 inhibitor GF120918 (2 µM and 10 µM), the MDR1 and

1270 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4
Liederer et al.
(6) Wacher, V. J.; Silverman, J. A.; Zhang, Y.; Benet, L. Z. Role of
P-glycoprotein and cytochrome P450 3A in limiting oral absorption
of peptides and peptidomimetics. J. Pharm. Sci. 1998, 87, 1322-
1330.
(7) Borchardt, R. T. Optimizing oral absorption of peptides using prodrug
strategies. J. Controlled Release 1999, 62, 231-238.
(8) Ouyang, H.; Vander Velde, D. G.; Borchardt, R. T.; Siahaan, T. J.
Synthesis and conformational analysis of a coumarinic acid-based
cyclic prodrug of an opioid peptide with modified sensitivity to
esterase-catalyzed bioconversion. J. Pept. Res. 2002, 59, 183-195.
(9) Yang, J. Z.; Chen, W.; Borchardt, R. T. In vitro stability and in vivo
pharmacokinetic studies of a model opioid peptide, H-Tyr-D-Ala-
Gly-Phe-D-Leu-OH (DADLE), and its cyclic prodrugs. J. Pharmacol.
Exp. Ther. 2002, 303, 840-848.
(10) Ouyang, H.; Tang, F.; Siahaan, T. J.; Borchardt, R. T. A modified
coumarinic acid-based cyclic prodrug of an opioid peptide: its
enzymatic and chemical stability and cell permeation characteristics.
Pharm. Res. 2002, 19, 794-801.
(11) Tang, F.; Borchardt, R. T. Characterization of the efflux transporter-
(s) responsible for restricting intestinal mucosa permeation of the
coumarinic acid-based cyclic prodrug of the opioid peptide DADLE.
Pharm. Res. 2002, 19, 787-793.
(12) Tang, F.; Borchardt, R. T. Characterization of the efflux transporter-
(s) responsible for restricting intestinal mucosa permeation of an
acyloxyalkoxy-based cyclic prodrug of the opioid peptide DADLE.
Pharm. Res. 2002, 19, 780-786.
(13) Chen, W.; Yang, J. Z.; Andersen, R.; Nielsen, L. H.; Borchardt, R.
T. Evaluation of the permeation characteristics of a model opioid
peptide, H-Tyr-D-Ala-Gly-Phe-D-Leu-OH (DADLE), and its cyclic
prodrugs across the blood-brain barrier using an in situ perfused rat
brain model. J. Pharmacol. Exp. Ther. 2002, 303, 849-857.
(14) Morley, J. S. Structure-activity relationships of enkephalin-like
peptides. Annu. ReV. Pharmacol. Toxicol. 1980, 20, 81-110.
(15) Ling, N. M. S.; Lazarus, L.; Rivier, J.; Guillemin, R. Structure-
activity relationships of enkephalin and endorphin analogs. Pept.,
Proc. Am. Pept. Symp., 5th 1977, 96-99.
(16) Eriksson, L.; Jonsson, J.; Hellberg, S.; Lindgren, F.; Skagerberg, B.;
Sjostrom M.; Wold, S. Peptide QSAR on substance P analogues,
enkephalins and bradykinins containing L- and D-amino acids. Acta
Chem. Scand. 1990, 44, 50-55.
(17) Bergstrom, C. A.; Strafford, M.; Lazorova, L.; Avdeef, A.; Luthman,
K.; Artursson, P. Absorption classification of oral drugs based on
molecular surface properties. J. Med. Chem. 2003, 46, 558-570.
(18) Seelig, A. A general pattern for substrate recognition by P-
glycoprotein. Eur. J. Biochem. 1998, 251, 252-261.
(19) Seelig, A.; Blatter, X. L.; Wohnsland, F. Substrate recognition by
P-glycoprotein and the multidrug resistance-associated protein
MRP1: a comparison. Int. J. Clin. Pharmacol. Ther. 2000, 38, 111-
121.
(20) Schwab, D.; Fischer, H.; Tabatabaei, A.; Poli, S.; Huwyler, J.
Comparison of in vitro P-glycoprotein screening assays: recom-
mendations for their use in drug discovery. J. Med. Chem. 2003, 46,
1716-1725.
(21) Pajeva, I. K.; Wiese, M. Pharmacophore model of drugs involved in
P-glycoprotein multidrug resistance: explanation of structural variety
(hypothesis). J. Med. Chem. 2002, 45, 5671-5686.
(24) O¨ sterberg, T.; Norinder, U. Theoretical calculation and prediction
of P-glycoprotein-interacting drugs using MolSurf parametrization
and PLS statistics. Eur. J. Pharm. Sci. 2000, 10, 295-303.
(25) Reid, R. C.; Kelso, M. J.; Scanlon, M. J.; Fairlie, D. P. Conforma-
tionally constrained macrocycles that mimic tripeptide beta-strands
in water and aprotic solvents. J. Am. Chem. Soc. 2002, 124, 5673-
5683.
(26) Gudmundsson, O. S.; Jois, S. D.; Vander Velde, D. G.; Siahaan, T.
J.; Wang, B.; Borchardt, R. T. The effect of conformation on the
membrane permeation of coumarinic acid- and phenylpropionic acid-
based cyclic prodrugs of opioid peptides. J. Pept. Res. 1999, 53, 383-
392.
(27) Gudmundsson, O. S.; Vander Velde, D. G.; Jois, S. D.; Bak, A.;
Siahaan, T. J.; Borchardt R. T. The effect of conformation of the
acyloxyalkoxy-based cyclic prodrugs of opioid peptides on their
membrane permeability. J. Pept. Res. 1999, 53, 403-413.
(28) Conradi, R. A.; Hilgers, A. R.; Ho, N. F.; Burton, P. S. The influence
of peptide structure on transport across Caco-2 cells. II. Peptide bond
modification which results in improved permeability. Pharm. Res.
1992, 9, 435-439.
(29) Conradi, R. A.; Hilgers, A. R.; Ho, N. F.; Burton, P. S. The influence
of peptide structure on transport across Caco-2 cells. Pharm. Res.
1991, 8, 1453-1460.
(30) Wu¨thrich, K. NMR of Protein and Nucleic Acids; John Wiley & Sons,
Inc.: New York, 1986.
(31) Rose, G. D.; Gierasch, L. M.; Smith, J. A. Turns in peptides and
proteins. AdV. Protein Chem. 1985, 37, 1-109.
(32) Clark, D. E. Rapid calculation of polar molecular surface area and
its application to the prediction of transport phenomena. 2. Prediction
of blood-brain barrier penetration. J. Pharm. Sci. 1999, 88, 815-
821.
(33) Kelder, J.; Grootenhuis, P. D.; Bayada, D. M.; Delbressine, L. P.;
Ploemen, J. P. Polar molecular surface as a dominating determinant
for oral absorption and brain penetration of drugs. Pharm. Res. 1999,
16, 1514-1519.
(34) He, Y. L.; Murby, S.; Gifford, L.; Collett, A.; Warhurst, G. et al.
Oral absorption of D-oligopeptides in rats via the paracellular route.
Pharm. Res. 1996, 13, 1673-1678.
(35) Wils, P.; Warnery, A.; Phung-Ba, V.; Legrain, S.; Scherman, D. High
lipophilicity decreases drug transport across intestinal epithelial cells.
J. Pharmacol. Exp. Ther. 1994, 269, 654-658.
(36) Lipinski, C. A. Drug-like properties and the causes of poor solubility
and poor permeability. J. Pharmacol. Toxicol. Methods 2000, 44,
235-249.
(37) Seelig, A. How does P-glycoprotein recognize its substrates? Int. J.
Clin. Pharmacol. Ther. 1998, 36, 50-54.
(38) Bu¨chi, G.; Weinreb, S. M. Total syntheses of aflatoxins M1 and G1
and an improved synthesis of aflatoxin B1. J. Am. Chem. Soc. 1971,
93, 746-752.
(39) Gao, J.; Hugger, E. D.; Beck-Westermeyer, M. S.; Borchardt, R. T.
Protocols in the application of Caco-2 cells in measuring permeability
coefficient of drugs. Current Protocols in Pharmacology; John Wiley
and Sons: New York, 2000; pp 1-23.
(22) Penzotti, J. E.; Lamb, M. L.; Evensen, E.; Grootenhuis, P. D. A
computational ensemble pharmacophore model for identifying sub-
strates of P-glycoprotein. J. Med. Chem. 2002, 45, 1737-1740.
(23) Ekins, S.; Kim, R. B.; Leake, B. F.; Dantzig, A. H.; Schuetz, E. G.;
Lan, L. B.; Yasuda, K.; Shepard, R. L.; Winter, M. A.; Schuetz, J.
D.; Wikel, J. H.; Wrighton, S. A. Application of three-dimensional
quantitative structure-activity relationships of P-glycoprotein inhibi-
tors and substrates. Mol. Pharmacol. 2002, 61, 974-981.
(40) Yang, J. Z.; Bastian, K. C.; Moore, R. D.; Stobaugh, J. F.; Borchardt,
R. T. Quantitative analysis of a model opioid peptide and its cyclic
prodrugs in rat plasma using high-performance liquid chromatography
with fluorescence and tandem mass spectrometric detection. J.
Chromatogr. B Anal. Technol. Biomed. Life Sci. 2002, 780, 269-281.
JM050277F