Blood-brain barrier penetration by two dermorphin tetrapeptide analogues: Role of lipophilicity vs structural flexibility

Article abstract of DOI:10.1021/jm701404s

Two dermorphin analogues having an almost identical structure but different structural flexibility were compared for opioid activity. In 1 the aromatic side chains were incorporated into a lactam structure, while in 2 N-amide alkylation was retained but the side chains were flexible. Both compounds produced comparable antinociceptive effects in the mouse tail flick test after peripheral administration. This indicates that lipophilicity, rather than side chain flexibility, is the key determinant for blood-CNS barrier penetration.

Full text of DOI:10.1021/jm701404s

J. Med. Chem. 2008, 51, 2571–2574
2571
Brief Articles
Blood-Brain Barrier Penetration by Two Dermorphin Tetrapeptide Analogues: Role of
Lipophilicity vs Structural Flexibility
Steven Ballet,† Aleksandra Misicka,‡,§ Piotr Kosson,§ Carole Lemieux,# Nga N. Chung,# Peter W. Schiller,#
Andrzej W. Lipkowski,§ and Dirk Tourwé*,†
Organic Chemistry Department, Vrije UniVersiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium, Department of Chemistry, Warsaw
UniVersity, Pasteura 1, 02-093 Warsaw, Poland, Medical Research Centre, Polish Academy of Sciences, Pawinskiego 5,
02-106 Warsaw, Poland, and Laboratory of Chemical Biology and Peptide Research, Clinical Research Institute of Montreal,
Montreal, PQ H2W 1R7, Canada
ReceiVed NoVember 8, 2007
Two dermorphin analogues having an almost identical structure but different structural flexibility were
compared for opioid activity. In 1 the aromatic side chains were incorporated into a lactam structure, while
in 2 N-amide alkylation was retained but the side chains were flexible. Both compounds produced comparable
antinociceptive effects in the mouse tail flick test after peripheral administration. This indicates that
lipophilicity, rather than side chain flexibility, is the key determinant for blood-CNS barrier penetration.
Introduction
Endogenous opioid peptides have properties that secure their
local and short duration actions. Structure–activity studies of
opioid peptides have resulted in the development of a large series
of analogues that are very potent as analgesics only after direct
application to their site of action.1 The major sites of pain signal
modulation are located in the central nervous system (CNSa).
Therefore, systemically administered drugs have to penetrate
into the CNS. The permeation through biological barriers,
especially the blood-brain barrier (BBB), is still one of the
major challenges in the development of peptide analogues as
drugs. The BBB is located at the level of endothelial cells in
brain capillaries. It utilizes selective molecular mechanisms to
prevent certain substances in the blood stream from penetrating
into the cerebrospinal fluid. High proteolytic activity at the BBB
and the selectivity of active and passive transporters limit the
Figure 1. Structures of 1 and 2.
designed and synthesized.2 N-Methylation of amide bonds is
another well-known structural modification to increase the
enzymatic stability of peptides and to improve their oral
bioavailability.3,4 Recently, we synthesized opioid tetrapeptide
analogues structurally derived from dermorphin,5 in which the
peptide segments around Tyr1 and Phe3 were conformationally
constrained and stabilized by cyclization into 4-aminotetrahydro-
2-benzazepin-3-one rings.6 The analogue H-Hba-D-Ala-Aba-
Gly-NH2 1 (SB0304, Figure 1) displayed the highest potency
in vitro in the guinea pig ileum (GPI) assay and was selected
for in vivo evaluation. After intrathecal injection in rats,
analogue 1 produced an antinociceptive effect in the tail-flick
assay that was 40–50 times more potent than that of morphine
and was also of longer duration.6
We now describe the analgesic effect of 1 after intravenous
administration. Moreover, we examined the effect of the
increased structural rigidity of 1 due to incorporation of the Tyr1
and Phe3 residues into a lactam ring by comparing its antinoci-
ceptive properties to those of the structurally related, more
flexible analogue 2 (SB0306, Figure 1). The importance of the
rigidity of a compound on the activity is an intriguing question.
The opioid peptide sequence H-Tyr-D-Ala-Phe-Gly-NH2 3 has
permeation of endogenous and exogenous active compounds
from the periphery into the CNS. For the development of active
opioid peptide analogues as potential analgesics, high resistance
to enzymatic degradation and ability to cross the BBB are
essential requirements.
Aromatic amino acids (Tyr, Phe, Trp) are key structural
elements of opioid peptides. Conformational restriction of such
residues has been a successful strategy to increase the potency,
selectivity, and metabolic stability of peptides. Therefore, many
constrained analogues of these natural amino acids have been
* To whom correspondence should be addressed. Phone: 32 2 629 3295.
Fax: 32 2 629 3304. E-mail: datourwe@vub.ac.be.
†
Vrije Universiteit Brussel.
Warswaw University.
Polish Academy of Sciences.
Clinical Research Institute of Montreal.
‡
§
#
a Abbreviations: Aba, 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one;
AUC, area under the curve; BBB, blood-brain barrier; CNS, central nervous
system; DIPEA, diisopropylamine; GPI; guinea pig ileum; Hba, 8-hydroxy-
4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one; MBHA, 4-methylbenzhy-
drylamine; MPE, maximum possible effect; MVD, mouse vas deferens;
PSA, polar surface area; RP, reversed phase; TBTU, O-(benzotriazol-1-
yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate; TFA, trifluoracetic acid.
10.1021/jm701404s CCC: $40.75
2008 American Chemical Society
Published on Web 03/28/2008

2572 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8
Brief Articles
Figure 2. Superimposition of [Hba-D-Ala]amide (in green) with oxy-
morphone (in purple), showing spatial overlap of the N-amino groups
and phenyl rings.
been constrained by linking the aromatic rings of the Tyr and
Phe residues to the amide nitrogen of the succeeding amino
acid through a methylene bridge, resulting in 1.6 This compound
was a highly potent analgesic after intrathecal administration.
To distinguish the effect of cyclization from that of nitrogen
alkylation, we synthesized the “ring-opened” analogue 2, having
N-methyl-D-Ala and N-methyl-Gly (Sar) residues in the se-
quence (Figure 1). The in vitro and in vivo activities of both
compounds were compared.
Figure 3. Analgesic effect as % MPE ( SEM of 1 and 2 in the mouse
tail-flick assay at different times after intravenous administration.
Table 1. HPLC Retention Time, ClogP, and tPSA Values of
Compounds
a
compd
tR (min)
ClogPb
tPSAb
1
2
3
8.87
8.50
6.25
0.6808
0.6912
-0.4752
159.06
159.06
176.64
Results and Discussion
a RP-HPLC was performed using a RP C-18 column: Supelco Discovery
BIO Wide Pore, 25 cm × 4.6 mm, 5 µm. System 1: solvent A, 0.1% TFA
in water; solvent B, 0.1% TFA in acetonitrile; gradient, 3–97% B in A
over 20 min; flow rate, 1.2 mL/min. System 2: solvent A, 0.1% TFA in
water; solvent B, 0.1% TFA in MeOH; gradient, 3–97% B in A over 20
min; flow rate, 1.2 mL/min. b Calculated using the BioByte estimator and
the tPSA calculation algorithm, as implemented in the ChemDraw Ultra
software, version 10.0. tPSA ) topological polar surface area.
Evolution has selected flexible linear neuropeptides, including
endogenous opioid peptides, to act locally on cell membrane
receptors for a very limited time. These peptides are very quickly
metabolized and have very limited ability to permeate biological
barriers. Therefore, increase of the stability against enzymatic
degradation and improvement of membrane permeability are
major goals in the development of peptide-derived therapeutic
agents. In the case of the opioids, morphine is the gold standard
of an exogenous ligand that is able to penetrate the gastro-
intestinal-blood barrier and the BBB to effectively interact with
central opioid receptors. In contrast to the structurally flexible
endogenous opioid peptides, morphine is very rigid and has a
well defined three-dimensional structure. This led to studies on
the development of structurally more rigid opioid peptide
analogues. During the interaction with its receptor, the N-
terminal fragment of an opioid peptide adopts a proper
conformation necessary for the stabilization of the active
ligand–receptor complex. Therefore, the main challenge of such
an approach is to create rigid tyramine structures that correspond
to the “active conformation”. Our previous studies identified
the 8-hydroxy-4-aminotetrahydro-2-benzazepin-3-one (Hba)
structure as a conformationally constrained analogue of tyrosine
that when incorporated into an opioid peptide sequence, is well
recognized by µ- and δ-opioid receptors.6 The results of
molecular modeling studies indicated that the conformation of
the tyramine moiety in Hba has a high similarity to that
contained in the benzomorphan skeleton (Figure 2).
Tyr and Phe side chains into the 2-benzazepinone ring involved
the alkylation of the amide nitrogens of the D-Ala and Gly
residues of the parent opioid peptide.
N-Alkylation increases the lipophilicity of the peptide ana-
logue, which raises the question of whether the increase of BBB
permeation seen with 1 is the result of a conformational
stabilization or of an increased lipophilicity due to N-alkylation.
To answer this question, a “ring opened” peptide analogue 2
with N-methylated amino acids in positions 2 and 4 was
synthesized (Figure 1). This restores the flexibility of the Tyr
and Phe side chains while maintaining the N-alkyl (methyl)
substitutions. It is mentioned in this context that N-alkylation
of a peptide bond restricts the conformational space of the amino
acid residue to extended conformations, as was also observed
for the benzazepine residue,8 and permits the cis-peptide bond
conformation.9 The calculated lipophilicities (ClogP) of 1 and
2 are very similar (ClogP ) 0.6809 and 0.6912, respectively)
and higher than that of the parent peptide 3 (ClogP ) -0.4752)
(Table 1).
Additionally, we were able to show that constraining the Phe3
residue by formation of the 4-aminotetrahydro-2-benzazepin-
3-one (Aba) ring resulted in opioid peptides with high affinity
for the δ-opioid receptor.7 The synthetic peptidomimetic 1, in
which the conformations of the tyrosine and the phenylalanine
residues of the original peptide H-Tyr-D-Ala-Phe-Gly-NH2 3
are constrained (Figure 1), showed high binding affinity for
opioid receptors and also high antinociceptive activity after
intrathecal application.6 We now show that this compound also
produces a potent analgesic effect when administered intrave-
nously (Figure 3). This indicates that 1 is able to penetrate the
BBB to a similar extent as morphine. The incorporation of the
This is confirmed by the similarity of the HPLC retention
times (Table 1), indicating that 1 is only slightly more lipophilic
than 2 but considerably more lipophilic than 3. The opioid
receptor binding affinities of both compounds are high and quite
similar (Table 2). Interestingly, the compound with flexible side
chains (2) showed moderate preference for δ- over µ-receptors
δ
(IC50µ/IC50 ) 3.96), whereas the rigid 1 was nonselective
δ
(IC50µ/IC50 ) 1.17). Neither compound displayed κ-receptor
binding affinity at concentrations up to 10 µM. Like 1, the
peptide 2 displayed agonist activity in the GPI and mouse vas
deferens (MVD) assays, albeit with lower potency (Table 2).
While in general there is good agreement between the receptor

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8 2573
Table 2. Opioid Receptor Binding Affinities, in Vitro Activities of Compounds, and in Vivo AUC Values for the Analgesic Effect
IC50 ( SEM (nM)
compd
µ
δ
GPI
MVD
AUC ( SEMa
1
2
20.8 ( 3.6
28.1 ( 6.1
9.77 ( 3.45
17.8 ( 5.0
7.1 ( 5.9
112.2 ( 5.7
3.64 ( 0.2
22.0 ( 1.6
29.3 ( 2.2
30.3 ( 4.4
79.7 ( 6.1
155 ( 31
7820 ( 551
8732 ( 750
9921 ( 589
MF/morphine
a AUC values for the analgesic effect after intravenous administration of a dose of 2 mg/kg for each compound.
Table 3. Physicochemical Properties of Opioid Tetrapeptides 1 and 2
m/z (M+H)+ a
HPLC tR (min) (% purity)c
compd
sequence
calcd M + H
obsd M + H
TLC Rfb
system 1
system 2
1
2
H-Hba-D-Ala-Aba-Gly-NH2
H-Tyr-NMe-D-Ala-Phe-Sar-NH2
480.22
484.25
480.3
484.3
0.59
0.62
8.87 (97%)
8.50 (98%)
12.37 (97%)
11.85 (98%)
a Observed by ESI MS+ ionization. b TLC system: EtOAc/n-BuOH/AcOH/H2O, 1:1:1:1. c HPLC: RP C-18 column, Supelco Discovery BIO Wide Pore,
25 cm × 4.6 mm, 5 µm. System 1: solvent A, 0.1% TFA in water; solvent B, 0.1% TFA in acetonitrile; gradient, 3–97% B in A over 20 min; flow rate, 1.2
mL/min. System 2: solvent A, 0.1% TFA in water; solvent B, 0.1% TFA in MeOH; gradient, 3–97% B in A over 20 min; flow rate, 1.2 mL/min.
Cl2Bn)-OH were from Senn Chemicals (Dielsdorf, Switzerland).
Boc-NMe-D-Ala-OH was purchased from Bachem (Bubendorf,
Switzerland). Trifluoroacetic acid (TFA), N-methylmorpholine, and
N,N-dimethylformamide were obtained from Fluka (Bornem,
Belgium).
binding data and the functional assay data of the compounds,
some quantitative discrepancies were observed. Such quantita-
tive discrepancies have often been observed and could be due
to possible differences in the structural requirements or receptor
access between central and peripheral receptors.
Solid-phase peptide synthesis was performed on a manual
synthesizer. Analytical RP-HPLC was carried out using an Agilent
1100 series system with a RP C-18 column: Supelco Discovery
BIO Wide Pore, 25 cm × 4.6 mm, 5 µm. Purification of the peptides
was carried out on a semipreparative (HPLC) system (Gilson) using
a RP C-18 column: Supelco Discovery BIO Wide Pore, 25 cm ×
2.1 cm, 5 µm with a linear gradient (3-80% CH3CN, containing
0.1% TFA, in 30 min), a flow rate of 13 mL/min, and UV detection
at 215 nm. The peptides were analyzed by HPLC using the
conditions indicated in Table 3. Mass spectra were recorded on a
VG Quatro II spectrometer (electrospray ionization, ESI MS) using
MassLynx 2.22 software for data analysis. For thin-layer chroma-
tography (TLC) plastic silica coated plates with F254 indicator were
used (Merck, Darmstadt, Germany).
After intravenous application, both compounds produced a
similarly high antinociceptive effect in the tail-flick test (Figure
3). This result indicates that both compounds have similar ability
to cross the BBB. Whereas 1 produces a significant analgesic
response after 5 min, it takes 2 about 15 min. This might reflect
a difference in the speed of crossing the BBB between the two
analogues. A comparison of the area under the curve (AUC) at
the same dose reveals that 1 and 2 have a very similar
antinociceptive potency as morphine (Table 2).
A number of physicochemical properties have been identified
that influence the ability of drugs to penetrate the BBB.10
Analysis of CNS and non-CNS drugs revealed that the CNS
drugs had fewer hydrogen bond donors, lower polar surface
areas (PSA), higher calculated log P (ClogP) values, and fewer
rotatable bonds.11 Comparison of 1 and 2 reveals that both
compounds have the same number of hydrogen bond donors,
very similar ClogP values, and identical calculated PSA values
(Table 1). However, the main difference between the two
compounds is the number of rotatable bonds. It is obvious that
in this case the latter is not a determining factor influencing
BBB permeability.
It is widely accepted that the structural rigidity of compounds
affects receptor binding affinity and selectivity. The results
presented here support the hypothesis that the Hba structure in
1 has a conformation that closely resembles the conformation
of the tyrosine residue in the receptor-bound conformation of
structurally flexible opioid peptide analogues. In this particular
comparison of a rigid and a structurally flexible molecule, both
compounds showed similar agonist activities. Thus, these
findings also support the hypothesis that the 4-aminotetrahydro-
2-benzazepinon-3-one core fulfills the conformational require-
ments for complex formation with the receptor in the “active”
conformation. Finally, it is mentioned that opioids with a mixed
µ-agonist/δ-agonist profile, such as the peptide analogues
described here, are actually of therapeutic interest as potential
analgesics with low propensity to produce tolerance and physical
dependence.12
The molecular modeling calculations were carried out as
described previously using Macromodel 5.0 with Maestro 8.0 as a
graphic interface.8
Synthesis of 2. Analogue 2 was prepared on a 0.4 mmol scale
by manual solid phase synthesis using MBHA resin (loading 1.1
mmol/g) as a solid support and following standard Boc amino
protection procedures. The Boc deprotection was performed in a
mixture of TFA/CH2Cl2/2% anisole (5 min + 20 min). After
filtration of the TFA mixture and neutralization with 20% DIPEA/
CH2Cl2, the couplings were performed by using a 3-fold excess of
the amino acids and activating agent (TBTU) and a 9-fold excess
of N-methylmorpholine. The completeness of the couplings was
checked with the ninhydrin or NF 31 color tests.13,14 Cleavage of
the peptide from the resin and side chain deprotection was
accomplished by treatment with HFliq for 1 h at 0 °C. The crude
peptide was purified by preparative HPLC. Analytical data of the
compound are presented in Table 3.
Receptor Binding Assay. Opioid receptor binding assays were
performed as described in detail elsewhere.15,16 Binding affinities
for µ- and δ-receptors were determined by displacing, respectively,
[3H]naloxone and [3H]deltorphin II from rat brain membrane
binding sites, and κ-opioid receptor binding affinities were measured
by displacement of [3H]U69,593 from guinea pig brain membrane
binding sites.
Functional Bioassays (GPI and MVD). The GPI and MVD
bioassays were carried out as reported in detail elsewhere.17
A
dose–response curve was determined with [Leu5]enkephalin as
standard for each ileum and vas preparation, and IC50 values of
the compounds were normalized according to a published proce-
dure.18
Experimental Section
General. 1 was prepared as described before.6 4-Methylben-
zhydrylamine (MBHA) resin and Boc-Sar-OH were purchased from
Neosystem (Strasbourg, France). O-(Benzotriazol-1-yl)-N,N,N′,N′-
tetramethyluronium tetrafluoroborate (TBTU) and Boc-Tyr(2,6-
In Vivo Antinociception Test. Male C57 mice (20–25 g, from
Medical Research Centre Animal House) were housed in groups
of five, maintained on a 12 h light-dark cycle, and allowed free

2574 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8
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access to food and water. Ten mice were used for each dose-effect
test. All procedures were approved by the Local Animal Rights
Committee. Peptides were dissolved in saline, injected into the tail
vein, and flushed with saline solution. The total volumes injected
were 10 mL/kg. The respective amounts of saline were injected in
the control group. Analgesia was measured by the warm water (55
°C) tail-flick latency test.19 Three trials with a maximum exposure
to heat (cutoff) of 15 s were performed. The degree of analgesia
was expressed as a percentage of maximum possible effect (MPE)
calculated as
posttreatment latency - control
MPE (%) )
× 100
cut-off latency - control
The MPE was measured at baseline, 5, 15, 30, 60, and 120 min
after administration of the drug or saline. Differences between
groups were analyzed using one-way ANOVA for comparison at
each time point. The t test was used to compare % MPE between
treatment groups. P < 0.05 was considered statistically significant.20
Acknowledgment. This work has been supported by Euro-
pean Grant “Normolife” (Grant LSHC-CT-2006-037733), by
the Fund for Scientific Research Flanders (FWO), and by the
U.S. National Institutes of Health (Grants DA-004443 and DA-
008924). The authors thank the Institute for the Promotion of
Innovation by Science and Technology in Flanders (IWT) for
financial support, and Rien De Wachter for help with Figure 2.
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Supporting Information Available: HPLC tracings of 1and2.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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