Synthesis and in vitro opioid activity profiles of DALDA analogues

Article abstract of DOI:10.1016/S0223-5234(00)01171-5

The tetrapeptide DALDA (H-Tyr-D-Arg-Phe-Lys-NH₂) is a polar and selective μ agonist showing poor penetration of the placental and blood-brain barriers. In an effort to enhance the potency of DALDA, analogues containing 2',6'-dimethyltyrosine (Dmt), N,2',6'-trimethyltyrosine (Tmt), 2'-methyltyrosine (Mmt) or 2'-hydroxy,6'-methyltyrosine (Hmt) in place of Tyr¹, or Orn or α,γ-diaminobutyric acid (A₂bu) in place of Lys⁴, were synthesized. All compounds displayed high it receptor selectivity in the rat and guinea pig brain membrane binding assays and most of them were more potent μ agonists than DALDA in the μ receptor-representative guinea pig ileum assay, with [Dmt¹]DALDA showing the highest potency. Because of its extraordinary μ agonist potency, high μ selectivity, polar character (charge of 3 +) and metabolic stability, [Dmt¹]DALDA has potential for use in obstetrical or peripheral analgesia. (C) 2000 Editions scientifiques et medicales Elsevier SAS.

Full text of DOI:10.1016/S0223-5234(00)01171-5

Eur. J. Med. Chem. 35 (2000) 895–901
´
© 2000 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved
Original article
PII: S0223-5234(00)01171-5/FLA
Synthesis and in vitro opioid activity profiles of DALDA analogues
Peter W. Schiller*, Thi M.-D. Nguyen, Irena Berezowska, Se´bastien Dupuis,
Grazyna Weltrowska, Nga N. Chung, Carole Lemieux
Laboratory of Chemical Biology and Peptide Research, Clinical Research Institute of Montreal, 110 Pine Avenue West,
Montreal, Quebec, Canada H2W 1R7
Received 11 April 2000; revised 5 May 2000; accepted 5 May 2000
Abstract – The tetrapeptide DALDA (H-Tyr- -Arg-Phe-Lys-NH2) is a polar and selective m agonist showing poor penetration of
D
the placental and blood–brain barriers. In an effort to enhance the potency of DALDA, analogues containing 2%,6%-dimethyltyrosine
(Dmt), N,2%,6%-trimethyltyrosine (Tmt), 2%-methyltyrosine (Mmt) or 2%-hydroxy,6%-methyltyrosine (Hmt) in place of Tyr1, or Orn or
a,g-diaminobutyric acid (A2bu) in place of Lys4, were synthesized. All compounds displayed high m receptor selectivity in the rat and
guinea pig brain membrane binding assays and most of them were more potent m agonists than DALDA in the m receptor-represen-
tative guinea pig ileum assay, with [Dmt1]DALDA showing the highest potency. Because of its extraordinary m agonist potency, high
m selectivity, polar character (charge of 3+) and metabolic stability, [Dmt1]DALDA has potential for use in obstetrical or
´
peripheral analgesia. © 2000 Editions scientifiques et me´dicales Elsevier SAS
m opioid agonists / DALDA / DALDA analogues / [Dmt1]DALDA / opioid receptor binding assays / GPI and MVD bioassays / opoid
peptides
1. Introduction
efforts to develop centrally acting opioid analgesics
are aimed at developing compounds with different
opioid activity profiles (e.g. d agonists or mixed m
agonist/d antagonists (for a review, see ref. [1]). How-
ever, very hydrophilic m opioid agonists that are un-
able to cross certain biological barriers still have
considerable therapeutic potential for some analgesic
applications. For example, m opioid compounds that
cannot penetrate the blood–brain barrier (BBB) may
elicit peripheral analgesic effects without producing
centrally mediated side effects. Also, some m opioid
agonists with polar character may not be able to cross
the placental barrier (PB) and, therefore, may have
potential for use in obstetrical analgesia.
In general, opioid peptides are more hydrophilic
than non-peptide opiates. Opioid peptides with high m
receptor binding affinity and high m receptor selectiv-
ity include several enkephalin analogues as well as
morphiceptin and its analogues (for a review, see ref.
[2]). The putative endogenous m opioid receptor lig-
ands endomorphin-1 and endomorphin-2 are also po-
tent and very selective m agonists [3], but are subject
to enzymatic degradation [4, 5]. Among various ana-
Centrally acting m opioid agonists (e.g. morphine)
are still the most frequently used analgesics for the
relief of severe pain, but their usefulness is limited by
a number of well known side-effects, including toler-
ance, physical dependence, respiratory depression, ad-
verse gastrointestinal effects, etc. Therefore, current
Abbre6iations: A2bu, a,g-diaminobutyric acid; BBB, blood–brain
barrier; Boc, tert-butyloxycarbonyl; (Boc)2O, di-tert-butyl dicarbon-
ate; DALDA, H-Tyr-D-Arg-Phe-Lys-NH2; DAMGO, H-Tyr-D-Ala-
Gly-NaMePhe-Gly-ol; DIC, 1,3-diisopropylcarbodiimide; DIEA,
diisopropylethylamine; DMAP, 4-dimethylaminopyridine; Dmt, 2%,6%-
dimethyltyrosine;
DSLET, H-Tyr-
[Dmt1]DALDA,
H-Dmt-
D-Arg-Phe-Lys-NH2;
D-Ser-Gly-Phe-Leu-Thr-OH; FAB-MS, fast atom
bombardment mass spectrometry; Fmoc, (fluoren-9-ylmethoxy)-
carbonyl; GPI, guinea pig ileum; Hmt, 2%-hydroxy,6%-methyltyrosine;
HOBt, 1-hydroxybenzotriazole; HPLC, high performance liquid chro-
matography; Mmt, 2%-methyltyrosine; MVD, mouse vas deferens; Orn,
ornithine; PB, placental barrier; Tmt, N,2%,6%-trimethyltyrosine; TAPS,
H-Tyr-D-Arg-Phe-Ser-OH; TEA, triethylamine; TFA, trifluoroacetic
acid; THF, tetrahydrofuran; Tic, 1,2,3,4-tetrahydroisoquinoline-3-car-
boxylic acid; Tic(OH), 7-hydroxy,1,2,3,4-tetrahydroisoquinoline-3-car-
boxylic acid; TLC, thin layer chromatography; U69,593,
(5a,7a,8b)-(—)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-
yl]benzeneacetamide.
* Correspondence and reprints:
E-mail address: schillp@ircm.qc.ca (P.W. Schiller).

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P.W. Schiller et al. / European Journal of Medicinal Chemistry 35 (2000) 895–901
logues of the N-terminal tetrapeptide segment of der-
morphin, the compounds TAPS (H-Tyr- -Arg-Phe-
Ser-OH) [6] and DALDA (H-Tyr- -Arg-Phe-Lys-
NH2) [7] were reported to be potent and selective m
agonists.
2. Chemistry
D
Peptides were synthesized by the solid-phase
method using tert-butyloxycarbonyl (Boc)-protected
amino acids and 1,3-diisopropylcarbodiimide (DIC)/
1-hydroxybenzotriazole (HOBt) as coupling agents.
Boc-Tmt was prepared as described elsewhere [11].
Mmt was first synthesized by hydrogenolysis of 7-
hydroxy,1,2,3,4-tetrahydroisoquinoline-3-carboxylic
acid [Tic(OH)] using Pd-black as catalyst and a reac-
tion temperature of 90 °C [12]. Under these con-
ditions extensive racemization occurred. Van Bets-
brugge prepared 2%-methylphenylalanine from 1,2,3,4-
tetrahydroisoquinoline-3-carboxylic acid (Tic) with
minimal racemization (8%) by carrying out the hy-
drogenolysis at 60 °C using 10% Pd/C and 4 atm H2
pressure [13]. We used the latter conditions to prepare
Mmt from Tic(OH) in good yield (48%) and with
minimal racemization (B10%).
sized in racemic form essentially as described in the
literature [14]. The bis-Boc derivative of Mmt and the
tris-Boc derivative of
tion with di-tert-butyl dicarbonate in the presence of
triethylamine (TEA) and 4-dimethylaminopyridine
(DMAP). The tris-Boc derivative of
D
Among these selective m agonist peptides, DALDA
has the most polar character because it carries a net
positive charge of 3+ at physiological pH. DALDA
was shown to be metabolically stable and to have
very limited distribution across the BBB [8]. The
results of a recent study indicated that DALDA did
not cross the PB to a significant extent in the preg-
nant sheep model [9]. In agreement with this finding,
DALDA had no effect on fetal cardiorespiratory
parameters when given to pregnant sheep [10]. Fur-
thermore, DALDA produced only a transient, minor
increase in blood pressure and did not affect the
maternal respiratory pattern. Taken together, these
results suggest that DALDA or DALDA analogues
may have potential for use in obstetrical analgesia or
peripheral analgesia.
In the present paper we describe a series of
DALDA analogues that were designed with the goal
to develop highly polar m opioid agonists with in-
creased potency. The design was mainly based on
structural modification of the Tyr1 residue. Tyr1 was
replaced with 2%,6%-dimethyltyrosine (Dmt), N,2%,6%-
trimethyltyrosine (Tmt), 2%-methyltyrosine (Mmt) or
2%-hydroxy,6%-methyltyrosine (Hmt). Analogues con-
taining an a,v-diamino acid with a shorter side chain
(ornithine [Orn] or a,g-diaminobutyric acid [A2bu]) in
place of Lys4 were also prepared.
D
,L-Hmt was synthe-
D
, -Hmt were prepared by reac-
L
D
, -Hmt was
L
incorporated into the peptide in racemic form. Pep-
tides were cleaved from the resin by HF/anisole treat-
ment in the usual manner. Peptide purification and
separation of the diasteromeric peptides in the case of
the Hmt1-analogue were achieved by preparative re-
versed-phase HPLC. Analytical data of the peptides
are presented in table I.
Table I. Analytical parameters of DALDA analogues.
No.
Compound
TLC
HPLC
FAB-MS
Rf (II)
Rf (III)
k%a
(M+H)+
1
H-Tyr-D-Arg-Phe-Lys-NH2 (DALDA)
0.097
0.111
0.118
0.111
0.112
0.115
0.125
0.125
0.470
0.482
0.482
0.487
0.465
0.476
0.465
0.467
1.60
3.34
3.05
2.87
3.63
2.33
2.21
2.02
612
640
626
612
654
626
642
642
2
H-Dmt-
H-Dmt-
H-Dmt-
D
D
D
-Arg-Phe-Lys-NH2 ([Dmt1]DALDA)
-Arg-Phe-Orn-NH2
3
4
-Arg-Phe-A2bu-NH2
5
Tmt-
H-Mmt-
D
-Arg-Phe-Lys-NH2
-Arg-Phe-Lys-NH2
6
D
7a
7b
H-Hmt-
H-Hmt-
D
-Arg-Phe-Lys-NH2 I
-Arg-Phe-Lys-NH2 II
D
a Capacity factor.

P.W. Schiller et al. / European Journal of Medicinal Chemistry 35 (2000) 895–901
897
3. In vitro bioassays and opioid receptor binding
assays
4. Results and discussion
DALDA (1) is a full m agonist in the m receptor
representative GPI assay (table II). It has high m
receptor binding affinity and very weak binding affini-
ties for d and k receptors (table III). Therefore,
DALDA has very high selectivity for m receptors vs. d
and k receptors with selectivity ratios of Kdi /Kim=
11400 and Kki /Kmi =2500.
For the determination of their in vitro opioid activ-
ities, analogues were tested in bioassays based on
inhibition of electrically evoked contractions of the
guinea pig ileum (GPI) and the mouse vas deferens
(MVD). The GPI assay is usually considered as being
representative for m opioid receptor interactions, even
though the ileum does also contain k opioid recep-
tors. In the MVD assay opioid effects are primarily
mediated by d opioid receptors but m and k receptors
also exist in this tissue. Binding affinities for m and d
receptors were determined by displacing, respectively,
[3H]DAMGO and [3H]DSLET from rat brain mem-
brane binding sites, and k receptor affinities were
measured by displacement of [3H]U69,593 from
guinea pig brain membrane binding sites.
Replacement of Tyr1 in DALDA with Dmt pro-
duced a 180-fold potency enhancement in the GPI
assay and a somewhat less pronounced (34-fold) po-
tency increase in the MVD assay. In the rat brain
membrane binding assay, [Dmt1]DALDA (2) showed
12 times higher m receptor affinity than its parent
compound and nine times higher affinity for d
receptors, thus retaining very high m vs. d selectivity
(Kdi /Kmi =14700). These data confirm previous
Table II. Guinea pig ileum (GPI) and mouse vas deferens (MVD) assay of DALDA analogues and related compounds.
No.
Compound
GPI
MVD
MVD/GPI
IC50 [nM]a
IC50 [nM]a
IC50 ratio
1
H-Tyr-
D
-Arg-Phe-Lys-NH2 (DALDA)
254927
1.4190.29
2.0890.38
7.5691.12
7.8690.88
10.990.8
189925
11209140
7.7191.47
29.392.2
246939
7819146
3.07
16.4
14.6
16.7
7.04
2
H-Dmt-
H-Dmt-
H-Dmt-
D
-Arg-Phe-Lys-NH2 ([Dmt1]DALDA)
-Arg-Phe-Orn-NH2
23.192.0
30.394.6
126932
3
D
D
4
-Arg-Phe-A2bu-NH2
5
Tmt-
H-Mmt-
D
-Arg-Phe-Lys-NH2
-Arg-Phe-Lys-NH2
55.3915.3
21.291.5
6
D
1.94
7a
7b
H-Hmt-
H-Hmt-
D
-Arg-Phe-Lys-NH2 I
-Arg-Phe-Lys-NH2 II
\10000
\10000
\52.9
\8.93
1.98
D
H-Tyr-Pro-Phe-Phe-NH2
Morphine
15.391.8
155931
5.29
0.0463
[Leu5]enkephalin
11.491.1
a Mean of three determinations9SEM.
Table III. Opioid receptor binding affinities of DALDA analogues and related compounds.
No. Compound
Kmi [nM]a
Kdi [nM]a
Kki [nM]a
Kdi /Kmi
11400
14700
8210
7630
2830
10500
\5680
\885
1910
Kki /Kmi
1
H-Tyr-
D
-Arg-Phe-Lys-NH2(DALDA)
1.6990.25
1920092000
42309360
2500
156
174
59.2
240
869
1050
816
\4850
217
2
H-Dmt-
H-Dmt-
H-Dmt-
D
D
D
-Arg-Phe-Lys-NH2([Dmt1]DALDA) 0.14390.015
21009310
12409240
61309850
543975
22.394.2
26.392.4
47.592.6
46.199.0
193943
3
-Arg-Phe-Orn-NH2
-Arg-Phe-A2bu-NH2
0.15190.018
0.80390.076
0.19290.043
0.22290.028
1.7690.02
11.390.9
2.0690.19
1.0090.04
9.4392.07
4
5
Tmt-
H-Mmt-
D
-Arg-Phe-Lys-NH2
-Arg-Phe-Lys-NH2
6
D
23209360
7a
7b
H-Hmt-
H-Hmt-
D
-Arg-Phe-Lys-NH2 I
-Arg-Phe-Lys-NH2 II
\10000
\10000
18409160
922091470
D
H-Tyr-Pro-Phe-Phe-NH2
Morphine
39409460 \10000
32.693.7
2.5390.35
217949
45709550
32.6
0.268
[Leu5]enkephalin
485
a Mean of three to six determinations9SEM.

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P.W. Schiller et al. / European Journal of Medicinal Chemistry 35 (2000) 895–901
observations indicating that substitution of Dmt for
Tyr1 in opioid peptides generally resulted in a higher
increase in m receptor affinity than in d receptor
affinity [1, 15]. The fact that the potency increase of
this Dmt1-analogue in the GPI assay as compared to
DALDA was much higher than expected on the basis
of its m receptor binding affinity enhancement may be
due to differences between central and peripheral m
receptors with regard to their structural requirements
for ligand binding. Interestingly, [Dmt1]DALDA also
displayed 190-fold increased k receptor binding
affinity as compared to DALDA but still exhibited
pronounced m vs. k selectivity (Kki /Kim =156).
Substitution of
D
,
-Hmt for Tyr1 in DALDA re-
L
sulted in one diastereoisomer (I) (7a) which showed
about the same m agonist potency as DALDA in the
GPI assay and a similar opioid receptor binding
profile. Diastereoisomer II (7b) exhibited substantially
lower m agonist potency in the GPI assay and lower m
and k receptor binding affinities than 7a. Since it is
well known that
-configuration at the Tyr1 residue of
L
opioid peptides is generally required for high opioid
activity, we assume that diastereoisomer I (7a) repre-
sents the
-Hmt1-analogue.
L
Shortening of the side chain of Lys4 in analogue 2
by one methylene group resulted in a compound,
5. Conclusions
H-Dmt-
D
-Arg-Phe-Orn-NH2 (3), with slightly re-
Most of the prepared DALDA analogues displayed
higher m receptor binding affinity and higher m ago-
nist potency than the DALDA parent peptide and
they were all m receptor-selective. The most potent m
agonist was [Dmt1]DALDA (2), which also showed
highest m receptor binding affinity, highest m vs. d
receptor selectivity and substantial preference for m
receptors over k receptors. This compound was 5
times more potent in the GPI assay and had 14 times
higher m receptor binding affinity than endomorphin-
2 (tables II and III). In comparison with morphine, it
turned out to be a 20 times more potent m agonist in
the functional in vitro assay and displayed 7 times
higher m receptor binding affinity. Both DALDA and
[Dmt1]DALDA carry a net positive charge of 3+.
Inspection of the capacity factors k% determined by
HPLC (table I) revealed that [Dmt1]DALDA is still a
quite hydrophilic compound, even though it is not
quite as polar as the DALDA parent peptide due to
the presence of the two additional methyl groups in
the Dmt1 residue. Therefore, [Dmt1]DALDA is also
likely to have very limited distribution across the BBB
or the PB. The much more lipophilic peptide endo-
morphin-2 did not elute from the column under the
isocratic conditions used for the determination of the
k% values of the DALDA analogues. The recently
duced agonist potency in the GPI assay and with
slightly lower m receptor binding affinity. It retained
high m vs. d and m vs. k receptor selectivities similar to
those of analogue 2. An analogue with an even
shorter side chain in the 4-position of the peptide
sequence, H
-
Dmt- -Arg-Phe-A2bu-NH2 (4), was a
D
somewhat less potent m agonist in the GPI assay and
bound somewhat less tightly to m receptors. Com-
pound 4 still showed high preference for m receptors
over d receptors but lower preference for m receptors
over k receptors.
Methylation of the N-terminal amino group of
[Dmt1]DALDA led to a compound, Tmt-
-Arg-Phe-
D
Lys-NH2 (5), which was an about 5 times less potent
m agonist in the GPI assay as compared to
[Dmt1]DALDA and was about half as potent in the
MVD assay. In comparison with [Dmt1]DALDA, the
Tmt1-analogue showed slightly lower m receptor bind-
ing affinity, four times higher d receptor affinity and
about half the k receptor affinity in the receptor
binding assays. Consequently, compound 5 displayed
lower m vs. d selectivity than [Dmt1]DALDA but
higher m vs. k selectivity.
Introduction of only one methyl group at the 2%-po-
sition of the Tyr1 aromatic ring of DALDA led to a
compound, H-Mmt-
D
-Arg-Phe-Lys-NH2 (6), which
reported tetrapeptide amide H-Dmt- -Arg-Phe-b-
D
was 23 times more potent than DALDA and 6 times
less potent than [Dmt1]DALDA in the GPI assay. In
comparison with DALDA, compound 6 displayed
about half as much binding affinity for m receptors,
similar affinity for d receptors and 15-fold reduced k
affinity. Its m vs. d receptor selectivity is similar to
that of the DALDA parent peptide, whereas its m vs.
k selectivity is lower.
Ala-NH2 [16] also contains an N-terminal Dmt
residue and carries a positive charge of 2+, thus
being somewhat less polar than the DALDA com-
pounds. This compound was reported to have ex-
traordinary m agonist potency and quite high m vs. d
receptor selectivity; however, its k receptor binding
affinity had not been determined. Since [Dmt1]-
DALDA is structurally very similar to its parent

P.W. Schiller et al. / European Journal of Medicinal Chemistry 35 (2000) 895–901
899
DALDA, it can be expected to have the same high
stability against enzymatic degradation. On the other
hand, endomorphin-1 and -2 were reported to pro-
duce only a short-lasting analgesic effect after in-
trathecal (i.th.) administration due to rapid break-
down in the spinal cord [17] and there is evidence to
indicate that the endomorphins are degraded by
dipeptidyl peptidase IV [4, 5]. Because of its extraor-
dinary m agonist potency, high m receptor selectivity,
polar character and stability against enzymatic degra-
dation, [Dmt1]DALDA is of potential interest for use
in obstetrical analgesia (e.g. i.th. administration) or in
peripheral analgesia.
6.1.2.1. Preparation of 2%-methyltyrosine (Mmt)
500 mg (2.76 mmol) of Tic(OH), 65 mL of AcOH and
1.0 g of Pd/C (10%) were placed into a Parr stainless
steel mini-reactor equipped with a mechanical stirrer.
After purging with argon the reactor was closed, the H2
pressure was adjusted to 5 atm and the temperature was
raised to 60 °C [13]. The reaction was continued for 24
h under vigorous stirring. At the end of the reaction the
catalyst was removed by filtration and the AcOH was
evaporated in vacuo. The oily residue was dissolved in
10 mL H2O and the resulting solution was decolorized
with charcoal, filtered and lyophilized to yield 415 mg
(82%) Mmt: TLC Rf 0.42 (I). To assess the extent of
racemization, the compound was run on an HPTLC-
Chir plate (Merck) using the solvent system (v/v) aceto-
nitrile/MeOH/H2O (6:1:1): L-Mmt, Rf 0.77, D-Mmt, Rf
0.82 (faint spot, B10% of total product).
6. Experimental protocols
6.1. Chemistry
6.1.2.2. Preparation of Boc-Mmt(Boc)-OH
To a solution of H-Mmt-OH (309 mg, 1.6 mmol) in
150 mL of dioxane/H2O (2:1, v/v) were added 870 mg (4
mmol) of di-tert-butyl dicarbonate [(Boc2O)]. The pH of
the solution was adjusted to 9 with 1.0 N NaOH and the
reaction was carried out for 16 h under stirring. A
second portion of (Boc)2O (870 mg, 4 mmol) was then
added and the reaction was continued for another 24 h.
After evaporation of the dioxane, the pH of the aqueous
phase was adjusted to 2 with 1N HCl and three extrac-
tions with 50-mL portions of EtOAc were performed.
The combined extracts were dried over MgSO4. After
filtration and solvent evaporation the residue was crys-
tallized from EtOAc/hexane to yield 550 mg (85%)
6.1.1. General methods
Precoated plates (silica gel 60 F254, 250 mm; Merck,
Darmstadt, Germany) were used for ascending TLC in
the following systems (all v/v): (I) CHCl3/AcOH/MeOH
(85:10:15); (II) n-BuOH/AcOH/H2O (4:1:1); (III) n-
BuOH/pyridine/AcOH/H2O (15:10:3:12).
A
Varian
VISTA 5500 liquid chromatograph was used for the
purification and the purity control of the peptides.
Preparative reversed-phase HPLC was performed on a
Vydac 218-TP column (22×250 mm) with a linear
gradient of 10–40% MeOH in 0.1% TFA at a flow rate
of 7 mL/min. Analytical reversed-phase HPLC was car-
ried out on a Vydac 218-TP column (10×250 mm) with
a linear gradient of 10–40% MeOH in 0.1% TFA at a
flow rate of 1.5 mL/min. The same column was also
used for the determination of the capacity factors k%
under isocratic conditions (15% MeOH in 0.1% TFA at
a flow rate of 2 mL/min). Proton magnetic resonance
spectra were recorded at 25 °C on a Varian VXR-400S
spectrometer using tetramethylsilane as internal stan-
dard. Molecular weights of compounds were determined
by FAB mass spectrometry on an MS-50 HMTCTA
mass spectrometer interfaced to a DS-90 data system
(Dr M. Evans, Department of Chemistry, University of
Montreal).
1
Boc-Mmt(Boc)-OH: TLC Rf 0.80 (I); H-NMR (CDCl3)
l: 1.40 (m, 9H, C(CH3)3), l: 1.56 (s, 9H, C(CH3)3), l:
2.26 (s, 3H, CH3 ar), l: 2.83–3.00 (m, 2H, CH2), l:
4.00–4.06 (m, 1H, CH), l: 6.53 (dd, 1H, ar), l: 6.60 (d,
1H, ar), l: 7.02 (d, ar), l: 7.18 (d, 1H, NH); FAB-MS
m/e 410.
6.1.2.3. Preparation of Boc-D,L-Hmt(Boc)2-OH
To a solution of H-D,L-Hmt-OH [14] (1.669 g, 7.9
mmol) in 50 mL of THF/H2O (1:1, v/v) were added
(Boc)2O (6.024 g, 26.4 mmol), TEA (2.88 mL, 20.6 mM)
and DMAP (214 mg, 1.78 mmol). The solution was
stirred at room temperature for 18 h. After evaporation
of the THF, the aqueous phase was acidified to pH 4
with a 5% KHSO4 solution and was then extracted with
three 50-mL portions of EtOAc. The combined extracts
were washed with water and dried over MgSO4. After
filtration and solvent evaporation 3.78 g (94%) of Boc-
6.1.2. Amino acids and deri6ati6es
Amino acids and Boc amino acid derivatives were
purchased from Bachem Bioscience. Boc-L-Dmt-OH
was obtained from RSP Amino Acid Analogues, Inc.

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P.W. Schiller et al. / European Journal of Medicinal Chemistry 35 (2000) 895–901
D,L-Hmt(Boc)2-OH was obtained in pure form: TLC Rf
0.77 (II), H-NMR (CDCl3) l: 1.38 (s, 9H, C(CH3)3), l:
1.55 (s, 18H, C(CH3)3), l: 2.62 (s, 3H, CH3 ar), l:
2.82–2.95 (m, 2H, CH2), l: 4.07–4.13 (m, 1H, CH), l:
6.98 (d, 2H, ar); FAB-MS m/e 512.
sponse curve was determined with [Leu5]enkephalin as
standard for each ileum and vas preparation, and IC50
values of the compounds being tested were normalized
according to a published procedure [22].
1
6.2.2. Opioid receptor binding assays
6.1.3. Peptide synthesis
Opioid receptor binding studies were performed as
described in detail elsewhere [20]. Binding affinities for m
and d receptors were determined by displacing, respec-
tively, [3H]DAMGO (Multiple Peptide Systems, San
Diego, CA) and [3H]DSLET (Multiple Peptide Systems)
from rat brain membrane binding sites, and k opioid
receptor binding affinities were measured by displace-
ment of [3H]U69,593 (Amersham) from guinea pig brain
membrane binding sites. Incubations were performed
for 2 h at 0 °C with [3H]DAMGO, [3H]DSLET and
[3H]U69,593 at respective concentrations of 0.72, 0.78
and 0.80 nM. IC50 values were determined from log
dose-displacement curves, and Ki values were calculated
from the obtained IC50 values by means of the equation
of Cheng and Prusoff [23], using values of 1.3, 2.6 and
2.9 nM for the dissociation constants of [3H]DAMGO,
[3H]DSLET and [3H]U69,593, respectively.
Peptide synthesis was performed by the manual solid-
phase technique using a p-methylbenzhydrylamine resin
(1% cross-linked, 100–200 mesh, 0.54 mequiv/g of titrat-
able amine) obtained from Bachem Bioscience. Peptides
were assembled using Boc-protected amino acids and
Dic and HOBt as coupling agents. The side chains of
Dmt, Tmt, Mmt, Hmt were also Boc-protected. Other
side chain protection was as follows: tosyl (Arg), 2-
chlorobenzyloxycarbonyl (Lys), (fluoren-9-yl-methoxy)-
carbonyl (Fmoc) (Orn, A2bu). The following steps were
performed in each cycle: (1) addition of Boc amino acid
in CH2Cl2 (2.5 equiv); (2) addition of HOBt (2.5 equiv);
(3) addition of DIC (2.5 equiv) and mixing for 2–3 h;
(4) washing with CH2Cl2 (3×1 min); (5) washing with
EtOH (1 min); (6) monitoring completion of the reac-
tion with the ninhydrin test; (7) Boc deprotection with
50% (v/v) TFA in CH2Cl2 (30 min); (8) washing with
CH2Cl2 (5×1 min); (9) neutralization with 10% (v/v)
DIEA in CH2Cl2 (2×5 min); and (10) washing with
CH2Cl2 (5×1 min).
Acknowledgements
In the case of the Fmoc side chain protected Orn4-
and A2bu4-analogues, the Fmoc group was removed
with 20% (v/v) piperidine in DMF after complete pep-
tide assembly. After final deprotection with 50% (v/v)
TFA in CH2Cl2 (30 min), the resin was washed with
CH2Cl2 (3×1 min) and EtOH (3×1 min) and was
dried in a desiccator. Peptides were cleaved from the
resin by treatment with HF for 60 min at 0 °C (20 mL
of HF plus 1 mL of anisole/g of resin). After evapora-
tion of the HF, the resin was extracted three times with
Et2O and, subsequently, three times with glacial AcOH.
The crude peptide was obtained in solid form through
lyophilization of the acetic acid extract. Crude peptides
were purified by preparative HPLC. Each peptide was at
least 98% pure as assessed by analytical reversed-phase
HPLC and by TLC. Molecular weights were confirmed
by FAB-MS.
This work was supported by a Multicenter Consor-
tium Grant (PO1-DA08924) from the National Institute
on Drug Abuse, NIH, USA.
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