Synthesis and biological activity of a novel methylamine-bridged enkephalin analogue (MABE): A new route to cyclic peptides and peptidomimetics

Article abstract of DOI:10.1021/jm970861r

The synthesis and biological activity of a methylamine-bridged enkephalin analogue (MABE) is presented. The key step in the synthesis of the target compound involves the ring opening of Cbz-D-serine β-lactone with Boc-Phe-NHCH₂CH₂NHCH₃. Further synthetic elaboration of the resulting building block yielded compound 1 (MABE, Tyr-c[(N(β)CH₃)-D-A2pr-Gly-Phe- NHCH₂-CH₂-], where A₂pr is a 2,3-diaminopropionic acid residue). Utilizing a combination of NMR and molecular modeling, the structure-biological activity relationships for compound 1 were studied. Using an in vitro isolated receptor assay, MABE was found to have affinities for isolated μ, δ, and κ opioid receptors of 1.6, 2.1, and 340 nM, respectively. By an in vivo thermal escape assay, MABE was found to have an ED₅₀ of 0.027 μg in the rat when administered intrathecally. This effect was reversed by naloxone. By comparison, DAMGO, morphine, and DPDPE were found to yield ED₅₀ values of 0.14, 2.4, and 54 μg, respectively, in the same assay.

Full text of DOI:10.1021/jm970861r

J . Med. Chem. 1998, 41, 2631-2635
2631
Syn th esis a n d Biologica l Activity of a Novel Meth yla m in e-Br id ged En k ep h a lin
An a logu e (MABE): A New Rou te to Cyclic P ep tid es a n d P ep tid om im etics
Kevin Shreder,† Li Zhang,† Trunghau Dang,† Tony L. Yaksh,‡ Hiroshi Umeno,‡ Robert DeHaven,§
J effrey Daubert,§ and Murray Goodman*,†
Department of Chemistry and Biochemistry, 9500 Gilman Drive, University of California at San Diego,
La J olla, California 92093-0343, Department of Anesthesiology, 9500 Gilman Drive, University of California at San Diego,
La J olla, California 92093-0818, and Adolor Corporation, 395 Pheonixville Pike, Malvern, Pennsylvania 19355
Received December 29, 1997
The synthesis and biological activity of a methylamine-bridged enkephalin analogue (MABE)
is presented. The key step in the synthesis of the target compound involves the ring opening
of Cbz-D-serine â-lactone with Boc-Phe-NHCH2CH2NHCH3. Further synthetic elaboration of
the resulting building block yielded compound 1 (MABE, Tyr-c[(NâCH3)-D-A2pr-Gly-Phe-NHCH2-
CH2-], where A2pr is a 2,3-diaminopropionic acid residue). Utilizing a combination of NMR
and molecular modeling, the structure-biological activity relationships for compound 1 were
studied. Using an in vitro isolated receptor assay, MABE was found to have affinities for
isolated µ, δ, and κ opioid receptors of 1.6, 2.1, and 340 nM, respectively. By an in vivo thermal
escape assay, MABE was found to have an ED50 of 0.027 µg in the rat when administered
intrathecally. This effect was reversed by naloxone. By comparison, DAMGO, morphine, and
DPDPE were found to yield ED50 values of 0.14, 2.4, and 54 µg, respectively, in the same assay.
Ch a r t 1. Structure of
In tr od u ction
Tyr-c[(NâCH3)-D-A2pr-Gly-Phe-NHCH2CH2-] (MABE)
Enkephalins are a class of endogenous peptide opioids
first isolated and identified by Hughes in 1975.1 Since
this time, a variety of naturally occurring analogues has
been discovered, all of which contain the sequence Tyr-
X1-Y2-Phe, where X1 and Y2 are amino acid residues.
Many studies have noted the importance of enkephalins
in a variety of physiological functions ranging from the
modulation of pain to the regulation of the immune
system.2 To date, three major opioid receptor subtypes
have been identified: µ, δ, and κ, and each has been
associated with various physiological roles. Conse-
quently, the synthesis of potent and receptor-specific
enkephalin analogues is an area of active interest.3
Since the synthesis of the first cyclic enkephalin
analogue (H-Tyr-c[D-A2bu-Gly-Phe-Leu]) by Schiller,4
many analogues have been synthesized to yield deriva-
tives with enhanced selectivity, increased potency, and
extended biological stability when compared to their
parent enkephalins.5 Many cyclic analogues have been
synthesized that rely on the use of disulfide or lanthion-
ine (monosulfide)6 bridges. In general, a variety of
structures can be utilized as bridges in the synthesis of
cyclic peptides. Commonly used functionalities include
amide bonds that result from head-to-tail or main chain-
to-side chain cyclizations, disulfide bonds, urethanes,
and diaryl ethers. Other functionalities based on
phosphorus, silicon, and sulfur heteroatoms have also
been used.7,8 We have recently embarked on developing
the use of the nitrogen heteroatom as a bridge in the
design of cyclic peptides. More specifically, we synthe-
sized the cyclic enkephalin analogue 1 (MABE, Tyr-
c[(NâCH3)-D-A2pr-Gly-Phe-NHCH2CH2-]; Chart 1) with
a methylamine bridge. The resulting target MABE
exhibits a high affinity for the µ and δ receptors and a
modest affinity for the κ receptor. In addition, MABE
was found to be a potent opioid agonist as measured in
vivo.
Resu lts a n d Discu ssion
The key step in the synthesis of compound 1 (MABE;
see Scheme 1) relies on a tertiary amine-containing
tripeptide unit, 4, that becomes the cyclic peptide bridge.
In our previous synthesis of cyclic lanthionine enkepha-
lin analogues, the monosulfide bridging unit was de-
rived from the ring opening of [N-benzyloxycarbonyl
(Cbz)]-serine â-lactone (the Vederas lactone)9 with Boc-
Cys-OMe.10 Here, a similar strategy was employed
utilizing the secondary amine 2. This amine was
obtained from the ring opening of Boc-Phe-N-carboxy-
anhydride with N-methylethylenediamine. The result-
ing secondary amine nucleophile was used to ring-open
Cbz-D-serine â-lactone in DMF with Cs2CO3 as a base
to neutralize the proton derived from N-methylethyl-
enediamine reactant. Unlike the previous synthesis of
lanthionine dipeptide bridging units, the carboxylate
derivative 3 was not isolated but was coupled directly
to H-Gly-Ot-Bu using BOP as a coupling reagent.11 This
“one-pot” procedure avoided the difficult isolation of the
* Author for correspondence.
† Department of Chemistry and Biochemistry, UC San Diego.
‡ Department of Anesthesiology, UC San Diego.
§ Adolor Corp.
S0022-2623(97)00861-3 CCC: $15.00 © 1998 American Chemical Society
Published on Web 06/16/1998

2632 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14
Notes
Ta ble 1. In Vitro and in Vivo Bioactivity Data for MABE,
Sch em e 1. Synthesis of
Tyr-c[(NâCH3)-D-A2pr-Gly-Phe-NHCH2CH2-] (MABE)
DAMGO, Morphine, and DPDPEa
Ki (nM)
compd
MABE
µ
δ
κ
ED50 (µg)
0.027
1.6
2.1
340
(1.0-2.4) (1.3-3.5) (110-1020) (0.014-0.053)
n ) 5
n ) 3
n ) 2
>10000
260
n ) 30
DAMGO
14
0.14
(7.4-27) >10000
n ) 8
morphine 17
(0.094-0.20)
n ) 30
2.4
150
(6.8-43) (63-360) (100-690)
(1.8-3.1)
n ) 30
54
n ) 4
n ) 4
n ) 3
DPDPE
2.2
>10000
(1.0-3.9) >10000
n ) 5
(38-77)
n ) 24
a
The Ki and ED50 values are presented wth the 95% confidence
intervals and the number (n) of determinations.
F igu r e 1. Dose dependency of MABE, DAMGO, morphine,
and DPDPE on the thermal escape test.
an in vivo thermal escape assay, MABE was found to
have an ED50 of 0.027 µg. This effect was reversed by
naloxone. By comparison, DAMGO, morphine, and
DPDPE were found to have ED50 values of 0.14, 2.4, and
54 µg, respectively, in the same assay (Figure 1). Thus,
MABE was found to be a potent and broad-spectrum
opioid agonist.
1
Utilizing a combination of H NMR and molecular
modeling, MABE has been shown to exhibit both folded
and extended spatial arrangements of the aromatic
rings of phenylalanine and tyrosine which are critical
in defining selectivity between the µ and δ opioid
receptors (see Supporting Information).12-14 Further-
more, conformational analysis of MABE indicated that
its methlyamine bridge was highly flexible and confor-
mational exchange was observed on the NMR time
scale. Interestingly, MABE exhibits weak κ receptor
activity unlike its lanthionine enkephalin congener of
the sequence c[D-Cys2,D-Cys5].15 Consequently, the κ
activity of MABE may originate from the methylamine
bridge.
highly polar intermediate 3. The resulting tripeptide
4 was then readily purified using silica gel chromatog-
raphy in an overall yield of 72% from 2.
The resulting building block 4 was incorporated into
the target MABE as follows: Catalytic hydrogenolysis
of compound 4 yielded a free amine that was coupled
to Cbz-Tyr(Bzl)-OH to yield the branched intermediate
5. The acid-labile protecting groups of compound 5 were
removed using TFA/DCM (1:1), and the resulting crude
product was converted to its HCl salt. Cyclization under
dilute conditions using EDC, DIEA, and HOAt yielded
compound 6. This cyclic precursor to the final product
was deprotected using palladium black under 4 atm of
H2 to yield MABE.
Con clu sion s
The target compound was tested for its potency in
vitro and for its efficacy in vivo (Table 1). Using an in
vitro radioligand displacement assay, MABE was found
to have an affinity for isolated µ, δ, and κ opioid
receptors of 1.6, 2.1, and 340 nM, respectively. Using
MABE represents a new approach in the design of
cyclic peptides. Advantages of using an amine as a
bridging atom include the potential to construct a
diverse number of bridges based on commercially avail-
able or readily synthesized amines. Furthermore, amine

Notes
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14 2633
52 h. The DMF solvent was evaporated under high vacuum,
and the crude material was taken up in 10% K2CO3 (15 mL,
pH > 10) and extracted with EtOAc (5 × 25 mL). The organic
layers were combined, washed with brine (15 mL), and dried
with Na2SO4. The solution was filtered, and the resulting
filtrate was evaporated under reduced pressure to obtain an
oily yellow residue. The residue was chromatographed on
silica gel using a gradient of MeOH/CHCl3 (1-6%) to obtain
480 mg of a yellow solid (73% yield from compound 2): 1H
NMR (CDCl3) δ 1.36 (9H, s), 1.46 (9H, s), 2.26 (3H, s), 2.4-2.8
(4H, m), 2.97 (2H, m), 3.30 (2H, m), 3.83 (2H, m), 3.97 (2H,
m), 4.27 (1H, s), 5.10 (2H, s), 5.21 (1H, b), 5.82 (1H, b), 6.67
(1H, b), 7.1-7.34 (10H, m), 7.90 (1H, b); MS (FAB+) 656, 788
(M + Cs+); HRMS (FAB+) [M + Cs]+ calcd for C34H49CsN5O8
788.2635, found 788.2660.
bridges also contain a site for protonation or metal
chelation. Finally, the use of the trivalent amine offers
a “handle” by which to functionalize the bridge without
disturbing the pharmacophores. Such modifications
have the potential to modulate biological activity or
biodistribution.
The potency of MABE in vivo makes it a promising
new lead compound in our search for enkephalin
analogues with specific activities. Future efforts will
be focused on maintaining the high efficacy of this
derivative while improving its selectivity for the various
opioid receptors. The amine bridge offers several unique
possibilities to modify the activity of this class of
compounds. Because the methylamine bridge of MABE
was found to be highly flexible, bicyclic enkephalin
analogues derived from cyclic amine bridges might offer
a way to constrain the pharmacophores conformation-
ally to improve receptor selectivity. Additionally, modi-
fication of the bridgehead of the amine with charged or
hydrophobic functional groups might alter selectivity for
the various membrane-bound opioid receptors. Such
syntheses are now underway in our laboratories.
Cb z-Tyr (Bzl)-D-A2p r (Boc-P h e-NHCH 2CH2NâCH3)-Gly-
Ot-Bu t (5). Compound 4 (380 mg, 0.6 mmol) and 10% Pd/C
(90 mg) in EtOH (20 mL) were stirred under an atmosphere
of H2 for 19 h. The catalyst was filtered off using vacuum
filtration, and the resulting filtrate was evaporated under
reduced pressure to obtain a clear oil (71% yield). This crude
material were taken on without further purification. The oil
was taken up in DMF (1.5 mL), and to this solution were added
Cbz-Tyr(Bzl)-OH (216 mg, 0.618 mmol), DIEA (0.11 mL, 0.62
mmol), and BOP (274 mg, 0.62 mmol). The reaction mixture
was stirred for 2 days and the solvent removed under reduced
pressure. The crude material was partitioned between EtOAc
(25 mL) and 10% K2CO3 (25 mL, pH > 10). The layers were
separated, and the aqueous layer was extracted with EtOAc
(4 × 25 mL). The organic layers were combined, washed with
brine (15 mL), and dried with Na2SO4. The solution was
filtered, and the resulting filtrate was evaporated under
reduced pressure. Purification using a gradient of MeOH/CH2-
Cl2 (0-3%) yielded 242 mg of a clear oil (46% yield from
compound 4): 1H NMR (CDCl3) δ 1.36 (9H, s), 1.47 (9H, s),
2.19 (3H, s), 2.41 (2H, m), 2.58 (2H, m), 2.8-3.2 (4H, m), 3.32
(2H, m), 3.88 (2H, m), 4.25 (1H, b), 4.42 (2H, m), 5.02 (2H, s),
5.05 (2H, s), 5.47 (1H, b), 5.71 (1H, m), 6.88 (2H, d), 7.1-7.4
(18H, m), 7.91 (1H, b); MS (FAB+) 910, 1041 (M + Cs+); HRMS
(FAB+) [M + Cs]+ calcd for C50H64CsN6O10 1041.3738, found
1041.3738.
Cbz-Tyr (Bzl)-c[(NâCH3)-D-A2pr -Gly-P h e-NHCH2CH2-] (6).
Compound 5 (216 mg, 0.24 mmol) in CH2Cl2 (10 mL) and TFA
(6 mL) was stirred for 1 h. The solvent was evaporated under
reduced pressure, the residue was taken up in THF, and
concentrated HCL was added (∼5 drops) whereupon the HCl
salt precipitated out. The solvent was removed under reduced
pressure, and toluene (20 mL) was added to azeotrope off
traces of residual acid and water. The resulting foam was
dried overnight under high vacuum. To this residue were
added HOAt (95 mg, 0.70 mmol), DIEA (0.1 mL, 0.57 mmol),
DMF (3 mL), and CH2Cl2 (120 mL), and the reaction mixture
was cooled to 4 °C. EDC (181 mg, 0.95 mmol) was added, and
the reaction was stirred at 4 °C overnight and then at room
temperature for 1 week. The solution was then washed with
10% K2CO3 (10 mL, pH > 10), the organic layer was separated,
and the solvent was evaporated under reduced pressure.
Purification using a gradient of MeOH/CH2Cl2 (0-3%) yielded
86 mg of a solid (49% yield from compound 5): 1H NMR
(CDCl3) δ 2.12 (3H, s), 2.97 (4H, m), 3.26 (2H, m), 3.49 (2H,
m), 3.71 (2H, m), 4.12 (2H, m), 4.3 (2H, b), 5.27 (3H, m), 5.35
(2H, s), 5.38 (1H, b), 7.09 (2H, d), 7.14 (1H, b), 7.17-7.28 (17H,
m), 8.49 (1H, b); MS (ESI) 735, 757 (M + Na+).
Exp er im en ta l Section
Gen er a l P r oced u r es. Proton nuclear magnetic resonance
(1H NMR) and carbon nuclear magnetic resonance (13C NMR)
spectra were recorded on a QE-300 NMR spectrometer using
the residual peaks in the deuterated solvents as internal
standards. Fast atom bombardment (FAB) positive ion mass
spectra were obtained on a VG ZAB-VSE double focusing high-
resolution mass spectrometer equipped with a cesium ion gun.
3-Nitrobenzyl alcohol was used as the matrix for FAB mass
spectrometry. Electrospray ionization (ESI) mass spectra were
obtained on an API III Perkin-Elmer SCIEX triple quadrupole
mass spectrometer. Analytical reverse-phase high-perfor-
mance liquid chromatography (RP-HPLC) was conducted with
a C18 90 Å silica column (5 µm, 4.6 × 250 mm, Vydac).
Preparative RP-HPLC was conducted with a C18 90 Å silica
column (10 µm, 22 × 250 mm, Vydac). All reagents were of
the highest grade available and were purchased from the
Aldrich Chemical Co. unless indicated otherwise. Dichlo-
romethane was distilled from CaH2 under N2. Boc-Phe-N-
carboxyanhydride was prepared according to the method of
Fuller et al.16 Cbz-Tyr(Bzl)-OH was purchased from Bachem
California (Torrance, CA).
Boc-P h e-NHCH2CH2NHCH3 (2). To a solution of N-
methylethylenediamine (2.00 g, 6.87 mmol) in dry CH2Cl2 (15
mL) was added Boc-Phe-NCA. The resulting solution was
stirred for 1.5 h at room temperature under an atmosphere of
N2. The solvent was removed under reduced pressure to obtain
a yellow oil. The residue was dissolved in EtOAc (50 mL),
washed with brine (2 × 35 mL), dried with Na2SO4, and
filtered to remove inorganic solids. The filtrate was evaporated
under reduced pressure, and the resulting residue was purified
on a short column of silica gel using a gradient of MeOH/CH2-
Cl2 (1-10%) to obtain 1.10 g of a light-yellow solid (50%): 1H
NMR (CDCl3) δ 1.43 (9H, s), 2.36 (3H, s), 2.63 (2H, m), 3.08
(2H, b), 3.30 (2H, m), 4.34 (1H, quad), 5.41 (1H, d), 6.54 (1H,
b), 7.2-7.4 (5H, m); 13C NMR (CDCl3) δ 27.8, 35.2, 38.1, 38.5,
49.8, 55.5, 126.1, 127.8, 128.8, 136.7, 155.1, 171.6; MS (FAB+)
266, 322, 344 (M + Na+); HRMS (FAB+) [M + H]+ calcd for
Tyr -c[(NâCH3)-D-A2p r -Gly-P h e-NHCH2CH2-] (1, MABE).
Compound 6 (25 mg, 0.03 mmol) and Pd black (40 mg) in 20
mL of EtOH were shaken in a Parr hydrogenator overnight
under 4 atm of H2. The catalyst was removed using vacuum
filtration and the filtrate was evaporated under reduced
pressure. Purification of compound 1 using preparative RP-
HPLC yielded a peak at 20.0 min using an isocratic setting of
15% (0.1% TFA/CH3CN in 0.1% TFA/H2O) at 8.0 mL/min: 1H
NMR (DMSO-d6) δ 2.09 (4H, b, -CH3, D-A2pr2CâH), 2.29 (1H,
C
17H28N3O3 322.2131, found 322.2139.
Cbz-D-A2p r (Boc-P h e-NHCH2CH2NâCH3)-Gly-Ot-Bu t (4).
A suspension of compound 2 (321 mg, 1.0 mmol), Cbz-D-serine
â-lactone8 (250 mg, 1.13 mmol) and Cs2CO3 (180 mg, 0.6 mmol)
was dissolved in DMF (3 mL) and rapidly stirred for 24 h under
a blanket of N2 at room temperature. Thin-layer chromatog-
raphy (10% MeOH/CHCl3) indicated that compound 2 had been
completely consumed. To this flask were then added Gly-Ot-
Bu‚HCl (251 mg, 1.5 mmol) and BOP (531 mg, 1.2 mmol). The
reaction mixture was allowed to stir at room temperature for
b, CbH), 2.40 (2H, b, D-A2pr2CâH, CbH), 2.83 (3H, m, Tyr1Hâ2
Phe4Hâ), 3.01 (1H, b, CaH), 3.12 (3H, m, Gly3HR, Phe4Hâ, CaH),
,

2634 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14
Notes
3.97 (2H, m, Gly3HR, Tyr1HR), 4.12 (1H, quad, Gly3HR), 4.31
(1H, quint, Phe4HR), 4.48 (1H, b, D-A2pr2CR), 6.69 (2H, d, Tyr1-
H3,5), 6.94 (1H, b, NH5), 7.00 (2H, d, Tyr1-H2,6), 7.19-7.26 (5H,
m, Phe4HAr), 8.08 (3H, s, Tyr1NH3), 8.36 (1H, d, NH4), 8.47
(1H, d, NH2), 9.01 (1H, b, NH3), 9.19 (1H, s, -OH); MS (ESI-)
509 (M - H-), 531 (M + Cl-), 623 (M + TFA-); MS (ESI+)
511 (M + H+), 533 (M + Na+), 549 (M + K+); HRMS (FAB+)
[M + Cs]+ calcd for C26H34CsN6O5 643.1645, found 643.1665;
RP-HPLC 14.7 min using a gradient of 10-40% (0.1% TFA/
CH3CN in 0.1% TFA/H2O) over 20 min at 1 mL/min.
In Vitr o Ra d ioliga n d In h ibition Assa y. 1. P r ep a r a -
tion of Cell Mem br a n es Exp r essin g Op ia te Recep tor s.
This method is a modification of the method of Raynor et al.17
CHO cells stably expressing cloned human µ, δ, and κ receptors
were harvested by scraping from the culture flasks, centrifug-
ing at 1000g for 10 min, resuspending in assay buffer (50 mM
tris(hydroxymethyl)aminomethane HCl, pH 7.8, 1.0 mM eth-
ylene glycol bis(â-aminoethyl ether) N,N,N′,N′-tetraacetic acid
(EGTA free acid), 5.0 mM MgCl2, 10 mg/L leupeptin, 10 mg/L
pepstatin A, 200 mg/L bacitracin, 0.5 mg/L aprotinin), and
centrifuging again. The resulting pellet was resuspended in
assay buffer homogenized with a Polytron homogenizer (Brink-
mann, Westbury, NY) for 30 s at a setting of 1. The homo-
genate was centrifuged at 48000g for 10 min at 4 °C and the
pellet resuspended at 1 mg of protein/mL of assay buffer and
stored at -80 °C until use.
2. Assa y of Th er m a l Nocicep tion . This test was de-
scribed previously in detail.19 In brief, the animal was placed
on a glass surface which was maintained at 30 °C by a
feedback-controlled, under-glass, forced-air heating system.20
A projection bulb under the glass was focused on the foot pad
of the animal which induced an abrupt withdrawal. A cutoff
time was set at 20 s to prevent the tissue damage. The test
was started approximately 1 h after animal was placed in the
chamber for acclimation. After the baseline latency was
measured, the drugs were injected intrathecally and the test
was performed.
3. Dr u gs a n d In jection s. All agonists were injected i.t.
in a total volume 10 µL followed by 10 µL of saline to flush
the catheter. Naloxone was injected i.t. in a total volume of
10 µL followed by 10 µL of saline, 10 min before agonists were
injected. Naloxone hydrochloride (DuPont; Supporting Infor-
mation Figure 2), MABE (Supporting Information Figure 3),
DAMGO ([D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin; RBI, MA;
Supporting Information Figure 4), and morphine sulfate
(Merck, Sharpe and Dohme, West Point, PA) were dissolved
in physiological saline (0.9% NaCl w/v), and DPDPE ([D-Pen2,
D-Pen5]-enkephalin; RBI, MA) was dissolved in 10% 2-hydrox-
ypropyl-â-cyclodextrin (Research Biochemicals, Inc., Natick,
MA).
4. Da ta An a lysis. Response latency data from the hot box
test are presented as the mean ( SE at 0, 15, 30, 60, 90, and
120 min after drug injection and were converted to percent
maximum possible effect (% MPE) according to the formula:
2. [3H]Dip r en or p h in e Bin d in g to µ, δ, a n d K Op ia te
Recep tor s. After dilution in assay buffer and homogenization
as before, membrane proteins (50-100 mg) in 250 µL of assay
buffer were added to mixtures containing test compound and
radioligand (1.0 nM, 40 000-45 000 dpm) in 250 µL of assay
buffer in 96-well deep-well polystyrene titer plates (Beckman)
and incubated at room temperature for 60 min ([3H]diprenor-
phine). Reactions were terminated by vacuum filtration with
a Brandel MPXR-96T harvester through GF/B filters that had
been pretreated with a solution of 0.5% poly(ethylenimine) and
0.1% bovine serum albumin for at least 1 h. The filter-bottom
plates were washed four times with 1.0 mL of ice-cold 50 mM
Tris-HCl, pH 7.8, 30 µL of Microscint-20 (Packard Instrument
Co., Meriden, CT) was added to each filter, and radioactivity
on the filters was determined by scintillation spectrometry in
a Packard TopCount.
[3H]Diprenorphine was purchased from Amersham Life
Science, Inc. (Arlington Heights, IL) and had a specific activity
of 39-45 Ci/mmol. Preliminary experiments were performed
to show that no specific binding was lost during the wash of
the filters, that binding achieved equilibrium within the
incubation time and remained at equilibrium for at least an
additional 60 min, and that binding was linear with regard to
protein concentration. Nonspecific binding, determined in the
presence of 10 µM unlabeled naloxone, was less than 10% of
total binding.
The data from competition experiments were fit by nonlin-
ear regression analysis by Prism (GraphPad Software, Inc.,
San Diego, CA) using the 4-parameter equation for one-site
competition and subsequently calculating Ki from EC50 by the
Cheng-Prusoff equation.
In Vivo Th er m a l Esca p e Assa y. 1. Ra t P r ep a r a tion .
Animal surgeries and tests were approved by the institutional
animal care committee of the University of California, San
Diego. Male Sprague-Dawley rats (Harlan Industries, In-
dianapolis, IN), weighing 250-350 g, were housed in separate
plastic cages and maintained on a 12-h cycle (on, 7:00 a.m.;
off, 7:00 p.m.) with food and water given ad libitum.
% MPE ) (postdrug latency - predrug latency)/
(cutoff latency* - predrug latency) × 100%
where the cutoff latency is 20 s in this study.
Ack n ow led gm en t. This work was supported by the
National Institutes of Health (NIHDA 05539) and the
Adolor Corporation. We would also like to thank Drs.
Ralph Mattern and Elsa Locardi for their helpful
assistance in preparation of this manuscript.
Su p p or tin g In for m a tion Ava ila ble: NMR characteriza-
tion (1H chemical shifts, coupling constants, amide proton
temperature coefficients, and NOE data), conformational
analysis (preferred conformational families and torsional angle
data), and biological assay data (effects of naloxone on drug
analgesia, comparative intrathecal dose dependency of DAM-
GO and MABE on the thermal escape test) for MABE (12
pages). Ordering information is given on any current masthead
page.
Refer en ces
(1) Hughes, J . Isolation of An Endogenous Compound From the
Brain With Pharmacological Properties Similar To Morphine.
Brain Res. 1975, 88, 295-308.
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