Brief Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 385
and 30 μg. A dose-response curve was generated at peak effect
which occurred 30 min after ith administration. The A50 values
at this time point were 11.0 (6.5-21.1 μg; 95% CI for allodynia)
and 1.04 (1.0-1.2 μg/5 μL; 95% CI for hyperalgesia). No effect
was seen in vehicle treated animals.
12/12 h light/dark cycle (lights on at 07:00 a.m.) and provided
food and water ad libitum except as noted during the experi-
mental procedures. All the experiments were performed under a
protocol approved by Institutional Animal Care and Use Com-
mittee (IACUC) of the University of Arizona and in accordance
with policies and guidelines for the care and use of laboratory
animals as adopted by International Association for the Study
of Pain (IASP) and the National Institutes of Health (NIH).
L5/L6 Spinal Nerve Ligation Surgery. SNL injury was induced
as described by Chung and colleagues.13
Intrathecal Catheter Surgery. All rats were prepared for in-
trathecal drug administration by placing anesthetized (ketamine/
xylazine 100 mg/kg, ip) animals in a stereotaxic head holder.
The cisternum magnum was exposed, an incision was made,
and animals were implanted with a catheter (PE: 10, 8 mm) that
terminated in the lumbar region of the spinal cord. The animals
were allowed to recover 5-7 days after surgery before any phar-
macological manipulations were made.
Drug Administration. 6 was dissolved in 100% methanol. For
ith drug administration, 5 μL of drug was injected followed by
a 9 μL saline flush. Testing took place 15, 30, 45, 60, 75, and 90
min after drug injection, and dose-response curves were gener-
ated from data gathered at the time of peak effect.
Behavioral Assessment. Thermal Hypersensitivity. Thermal
hypersensitivity was assessed using the rat plantar test (Ugo Basile,
Italy) following a modified method of Hargreaves et al.15 Paw
withdrawal latencies were recorded in seconds. An automatic
cut-off point of 33 s was set to prevent tissue damage. The appa-
ratus was calibrated to give a paw withdrawal latency of approx-
imately 20 s on the uninjured paw. The radiant heat source was
activated with a timer and focused onto the plantar surface of
the hindpaw. Paw-withdrawal latency was determined by a
motion detector that halted heat source and timer when the
paw was withdrawn.
Conclusions
Inthe presentstudy, a systematic SAR studyon the enkeph-
alin analogue 12 with conservation of the propionamide moiety
and Dmt1 residue resulted in the development of nonselective
potent opioid agonists for μ and δ receptors with high lipophi-
licity. These analogues can produce the desired physiological
effects without many of the undesirable side effects of selective
μ opioid agonist, for example, 5 (Ki of 0.40 and 0.02 nM at
hDOR and rMOR; IC50 of 0.37 and 0.26 nM in MVD and
GPI, respectively). Insummary, the substitution ofD-Nle2 and
the halogenation of the aromatic ring of Phe4 increased opioid
activities at both receptors along with the lipophilicity. Ligand
6 which possessed highly potent opioid activities at both re-
ceptors (Ki = 0.14 nM at hDOR and rMOR; IC50 of 0.70 and
2.6 nM in MVD and GPI assays, respectively) showed potent
antihyperalgesic and antiallodynic effects in the in vivo assays,
demonstrating that increase of lipophilicity can be a good
approach to develop a potent ligand with good bioavailability.
Experimental Section
All amino acid derivatives were purchased from Novabio-
chem, ChemImpex International, and RSP. N-Phenyl-N-piper-
idin-4-ylpropionamide was prepared as previously described.14
Coupling reactions were monitored by TLC using the following
solvent systems: (1) CHCl3/MeOH/AcOH = 90:10:3 with ninhy-
drin spray used for detection. Analytical HPLC was performed
on a Hewlett-Packard 1090 [C-18, Vydac, 4.6 mm ꢀ 250 mm, 5 μm]
and preparative RP-HPLC on Hewlett-Packard 1100 [C-18,
Microsorb-MV, 10 mm ꢀ 250 mm, 10 μm]. 1H NMR spectra were
recorded on a Bruker Advance-300 spectrometer. Mass spectra
were taken in the positive ion mode under ESI methods.
Ligands 1-12. These ligands were prepared by stepwise syn-
thesis using NR-Boc chemistry starting from N-phenyl-N-piper-
idin-4-ylpropionamide. N-Phenyl-N-piperidin-4-ylpropionamide
(1 equiv) andNR-Boc-Phe-OH (1.1 equiv) were dissolved in DMF
and cooled in an ice bath for 10 min. Bop (1.1 equiv), HOBt (1.1
equiv), and NMM (2 equiv) were added to the reaction mixture
and stirred for 2 h at room temp. After a check for the disap-
pearance of the starting amine by TLC, the reaction mixture was
concentrated under reduced pressure. The concentrated mixture
was diluted with EtOAC and washed with 5% NaHCO3, 5%
citric acid, brine, and water consecutively. The organic layer was
dried under anhydrous Na2SO4 and filtered. The solution was
concentrated under reduced pressure and triturated with diethyl
ether to give a solid. The NR-Boc group was deprotected by 100%
TFA at 0 °C for 20 min. After completion of the chain elongation
by subsequent coupling and deprotection, the mixtures were eva-
porated under reduced pressure. The residues were solidified by
diethyl ether and purified by preparative RP-HPLC (20-50%
acetonitrile within 20 min for 1, 6, and 7, 20-60% acetonitrile
within 15 min for 5 and 10) to give pure ligands as white powders
in overall yields of 20-42%. The purity of the ligands was deter-
mined as g95% by analytical HPLC (10-90% acetonitrile in
40 min, 25-65% acetonitrile in 20 min). For analytical data, see
Supporting Information.
Mechanical Hypersensitivity. The assessment of mechanical
hypersensitivity consisted of measuring the withdrawal thresh-
old of the paw ipsilateral to the site of nerve injury in response to
probing with a series of calibrated von Frey filaments. Each
filament was applied perpendicularly to the plantar surface of
the ligated paw of rats kept in suspended wire-mesh cages. Mea-
surements were taken before and after administration of drug or
vehicle. The withdrawal threshold was determined by sequen-
tially increasing and decreasing the stimulus strength (“up-
down” method) analyzed using a Dixon nonparametric test16
and expressed as the mean withdrawal threshold.
Acknowledgment. The work was supported by grants from
the USPHS and National Institutes of Health (Grants DA-
13449 and DA-06284). We thank Margie Colie for assistance
with the manuscript.
Supporting Information Available: 1H NMR data of the
ligands 1-12. This material is available free of charge via the
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Lee, Yeon Sun
