Side chain methyl substitution in the δ-opioid receptor antagonist TIPP has an important effect on the activity profile

Article abstract of DOI:10.1021/jm981011u

The δ-opioid antagonist H-Tyr-Tic-Phe-Phe-OH (TIPP-OH) or its C- terminal amide analogue was systematically modified topologically by substitution of each amino acid residue by all stereoisomers of the corresponding β-methyl amino acid. The potency and se

Full text of DOI:10.1021/jm981011u

J . Med. Chem. 1998, 41, 5167-5176
5167
Sid e Ch a in Meth yl Su bstitu tion in th e δ-Op ioid Recep tor An ta gon ist TIP P Ha s
a n Im p or ta n t Effect on th e Activity P r ofile
Dirk Tourwe´,*,† Els Mannekens,† Trang Nguyen Thi Diem,† Patricia Verheyden,† Hendrika J aspers,† Ge´za To´th,‡
Antal Pe´ter,| Istva´n Kerte´sz,‡ Gabriella To¨ro¨k,| Nga N. Chung,§ and Peter W. Schiller§
Eenheid Organische Chemie, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium, Isotope Laboratory, Biological
Research Center, Hungarian Academy of Sciences, P.O. Box 521, H-6701 Szeged, Hungary, Department of Inorganic and
Analytical Chemistry, Attila J o´zsef University, P.O. Box 440, H-6701 Szeged, Hungary, Laboratory of Chemical Biology and
Peptide Research, Clinical Research Institute of Montreal, 110 Pine Avenue West, Montreal, Quebec, Canada H2W1R7
Received March 9, 1998
The δ-opioid antagonist H-Tyr-Tic-Phe-Phe-OH (TIPP-OH) or its C-terminal amide analogue
was systematically modified topologically by substitution of each amino acid residue by all
stereoisomers of the corresponding â-methyl amino acid. The potency and selectivity (δ- vs µ-
and κ-opioid receptor) were evaluated by radioreceptor binding assays. Agonist or antagonist
potency were assayed in the mouse vas deferens and in the guinea pig ileum. In the TIPP
analogues containing L-â-methyl amino acids the influence on δ-receptor affinity and on
δ-antagonist potency is limited, the [(2S,3R)-â-MePhe3]TIPP-OH analogue being among the
most potent δ-antagonists reported. In the D-â-methyl amino acid series, the [D-â-MeTic2]
analogues are δ-selective antagonists whereas [D-Tic2]TIPP-NH2 is a δ-agonist. NMR studies
did not indicate any influence of the â-methyl substituent on the conformation of the Tic residue.
The [(2R,3S)-â-MePhe3]TIPP-NH2 is a potent δ-agonist, its C-terminal carboxylic acid analogue
being more δ-selective but displaying partial agonism in both the δ- and µ-bioassay. These
results constitute further examples of a profound influence of â-methyl substitution on the
potency, selectivity, and signal transduction properties of a peptide.
In tr od u ction 1
of TIP(P) remains a challenging problem. The use of
â-methylated amino acids to constrain the conforma-
tional mobility of the side chain by biasing the popula-
tion of the ø1 torsion angle rotamers has been pioneered
by V. J . Hruby.7-10
The effects of methylation of the â-carbon of a side
chain on the biological properties of a peptide depends
on the chiralities of the stereoisomers. Important effects
on the receptor affinity, selectivity, and on the agonist/
antagonist character have been observed in opioid
peptides,9-14 analogues of somatostatin,15,16 R-MSH,10,17,18
CCK,19,20 oxytocin,21 substance P22 and angiotensin II,23
and glucagon.24
We now report a systematic study of the effect of
â-methyl substitution at each of the four amino acids
of the δ-antagonist TIPP-OH and of the mixed µ-agonist/
δ-antagonist TIPP-NH2 on the opioid activity profile. In
the latter case it was hoped that the â-methyl substitu-
tion might result in more balanced µ- and δ-affinities,
thereby leading to new analgesics with diminished
propensity to produce tolerance and dependence.25
The tetrapeptide H-Tyr-Tic-Phe-Phe-OH (TIPP-OH)
1 represents the prototype of a new class of potent and
selective δ-opioid antagonists.2 The δ-antagonist prop-
erties are a consequence of the conformational con-
straints imposed by the Tic residue. The aromatic
residues at position 3 and 4 are not essential for high
δ-antagonism since they can be replaced by lipophilic
residues3,4 or even omitted.5 Several theoretical models
of the receptor-bound conformation of TIP(P) peptides
have been proposed and compared to the naltrexone-
derived δ-antagonist naltrindole (NTI).6 An important
structural parameter in these models is the intramo-
lecular distance between the Tyr1 and Tic2 aromatic
rings which should be close to the distance between the
corresponding aromatic rings in naltrindole. In the
most plausible model, a close hydrophobic interaction
(collapse) between the Tyr1 and Tic2 aromatic rings is
prevented by intercalation of the Phe3 aromatic ring in
the tripeptide and of the Tic2-Phe3 peptide bond in the
tetrapeptide. The Phe3 and Phe4 aromatic rings are
then quite exposed and could engage in receptor inter-
actions that might explain the relatively higher antago-
nist potencies of the tetrapeptides as compared to the
tripeptides. It is clear that in such small peptides the
amino acid side chains exhibit considerable conforma-
tional flexibility. Therefore, the establishment of an
exact three-dimensional arrangement of the structural
moieties constituting the δ-antagonist pharmacophore
Resu lts a n d Discu ssion
Syn th esis. Since this study required all stereoiso-
mers of the â-methylated amino acids, a rapid synthetic
route resulting in racemates was chosen, rather than a
stereoselective synthesis for each isomer.26
Racemic erythro-(2S,3S and 2R,3R)-â-MePhe and
threo-(2S,3R and 2R,3S)-â-MePhe isomers were pre-
pared as described in the literature.9 The â-MeTic
isomers were prepared by a Pictet-Spengler reac-
tion,21,27 starting from pure erythro- or threo-â-MePhe.
For the preparation of the â-MeTyr stereoisomers, an
† Vrije Universiteit Brussel.
‡ Hungarian Academy of Sciences.
| Attila J ozsef University.
§ Clinical Research Institute of Montreal.
10.1021/jm981011u CCC: $15.00 © 1998 American Chemical Society
Published on Web 12/01/1998

5168 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 26
Tourwe´ et al.
Sch em e 1. Synthesis of Racemic erythro and threo Isomers of â-methyltyrosine
Ta ble 1. Influence of the Configuration of Aromatic â-Methyl
Ta ble 2. Analytical Data of TIPP Analogues
HPLCa
Rt(min)
Amino Acids on the Population of the Side Chains
TLCb
(2S,3S)
(2S,3R)
(2R,3R)
(2R,3S)
no.
peptide
TIPP-OH
TIPP-NH2
k′
Rf
Rf
Rf
C
A
B
gauche(-)
trans
gauche(+)
++
-
+
+
+
-
+
-
1
2
3
4
14.7
12.7
3.9 0.65 0.82 0.53
3.23 0.71 0.88 0.62
4.20 0.83 0.88 0.68
3.23 0.83 0.88 0.72
4.30 0.71 0.89 0.78
2.56 0.72 0.89 0.72
5.83 0.76 0.82 0.47
6.03 0.75 0.82 0.41
6.10 0.84 0.83 0.53
7.23 0.84 0.82 0.49
3.83 0.78 0.81 0.74
3.23 0.78 0.81 0.71
16.66 0.78 0.75 0.62
14.76 0.75 0.79 0.61
3.15 0.77 0.82 0.69
-
+
++
++
(SS)-âMeTyr1-TIPP-NH2 15.6
(SR)-âMeTyr1-TIPP-NH2 12.7
a
Legend: -, strongly disfavored; +, allowed; ++, favored.
12 (RR)-âMeTyr1-TIPP-NH2 15.9
13 (RS)-âMeTyr1-TIPP-NH2 10.7
Erlenmeyer condensation between 4-methoxyacetophe-
none and hippuric acid was performed using a modifica-
tion of the previously described protocol (Scheme 1).28-30
From the resulting 70/30 mixture of E- and Z-oxazolone,
the E isomer was easily isolated by crystallization.31 The
E-oxazolone can be isomerized to the Z isomer in
pyridine.28 Further catalytic hydrogenation and sub-
sequent deprotection steps afforded the erythro-â-MeTyr
and threo-â-MeTyr racemates.
After Boc protection all the â-methyl amino acids were
incorporated into the TIPP analogues as racemic pairs.
Peptide synthesis was performed by the solid-phase
method using either a Merrifield resin or a 4-methyl-
benzhydrylamine resin. After cleavage from the resin
by treatment with liquid HF, the resulting epimeric
peptides were separated by reversed phase HPLC (Table
2). A very good separation of the L-(2S) from the D-(2R)-
â-Me amino acid containing peptides was obtained.
However, a systematic study had shown that the
separation of epimers at the â-carbon (e.g. L-erythro-
(2S, 3S) from L-threo-(2S, 3R)) can be problematic.32
Therefore, in this synthetic strategy, special attention
should be paid to the diastereomeric purity of the
starting â-Me amino acids. The absolute configuration
at the R-carbon of the â-Me amino acid in each peptide
was determined after acid hydrolysis and derivatization
of the resulting amino acids with GITC or FDAA.32,33
5
6
(SS)-âMeTic2-TIPP-OH
(SR)-âMeTic2-TIPP-OH
20.5
21.1
21.3
24.7
15 (RR)-âMeTic2-TIPP-OH
16 (RS)-âMeTic2-TIPP-OH
7
8
(SS)-âMePhe3-TIPP-NH2 14.5
(SR)-âMePhe3-TIPP-NH2 12.7
19 (RR)-âMePhe3-TIPP-NH2 53.0
20 (RS)-âMePhe3-TIPP-NH2 47.3
9
(SR)-âMePhe3-TIPP-OH
13.28c
21 (RS)-âMePhe3-TIPP-OH
37.67c 10.77 0.77 0.88 0.55
10 (SS)-âMePhe4-TIPP-NH2 16.3
11 (SR)-âMePhe4-TIPP-NH2 28.5
22 (RR)-âMePhe4-TIPP-NH2 29.1
23 (RS)-âMePhe4-TIPP-NH2 12.0
4.43 0.77 0.79 0.70
8.5 0.76 0.76 0.67
8.7 0.76 0.75 0.67
3.0 0.75 0.82 0.76
a
Retention time and capacity factor for Vydac 218 TP54
reversed phase column (25 × 0.46 cm) with 0.1% trifluoroacetic
acid/acetonitrile (73:27 (v/v)). Flow rate 1 mL/min; to ) 3 min;
detection at λ ) 210 nm. b Kieselgel GF254 (Merck plates). Solvent
systems: (A) 1-butanol/acetic acid/water 4:1:1; (B) acetonitrile/
methanol/water 4:1:1; (c) ethyl acetate/pyridine/acetic acid/water
12/4/1.2/2.2. c Composition of eluent: 0.1% aqueous TFA/acetoni-
trile (70/30, v/v, isocratic).
those for the κ-opioid receptor using guinea pig cerebel-
lum membranes and [3H]U69593 (Tables 3 and 4).
The L-â-Me amino acid containing TIPP analogues
showed only relatively minor changes in δ-receptor
affinity as compared to the parent peptides TIPP-OH
or TIPP-NH2. The effect of changing the configuration
at the â-carbon on δ-receptor binding was generally
found to be quite small, except in the case of the (2S,3S)-
â-MeTic analogue 5 which showed 14-fold reduced
δ-receptor affinity. On the other hand the µ-receptor
affinity was affected to a somewhat larger extent by the
configurational change at the â-carbon, particularly at
the 4-position residue. This is indicated by the fact that
the (2S,3S)-â-MePhe4 analogue 10 showed a receptor
Biologica l Da ta . Affinities of the TIPP analogues
for µ-opioid receptors were determined in a binding
assay using rat forebrain membranes with [3H] sufen-
tanil as a µ-radioligand. Affinities for the δ-opioid
receptor were measured by displacement of [3H]DPDPE
from NxG108CC15 cell membrane binding sites and

Side Chain Methyl Substitution in TIPP
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 26 5169
Ta ble 3. Receptor Binding Affinities and Activities in MVD and GPI Bioassays of TIPP Analogues Containing L-â-Me Amino Acids
bioassay
receptor binding (Ki, nM)
GPI (IC50
agonist [nM]
)
MVD (Ke DPDPE)
antagonist [nM]
no.
µa N ) 3-5
δb N ) 3-6
κc N ) 2
1
2
3
4
H-Tyr-Tic-Phe-Phe-OH
H-Tyr-Tic-Phe-Phe-NH2
H-(2S,3S)-âMeTyr-Tic-Phe-Phe-NH2
H-(2S,3R)-âMeTyr-Tic-Phe-Phe-NH2
>10000
0.48 ( 0.07
0.83 ( 0.11
2.88 ( 0.25
1.21 ( 0.06
>10000
>10000
4.80 ( 0.20
178 ( 26
838 ( 116
>10000
1700 ( 220
>10000
18.0 ( 2.2
352 ( 100
284.0 ( 63.5
102 ( 6
>10000
1210 ( 260
partial agonist (max 50%)
IC25 ) 1030 ( 120
2.09 ( 0.10
5
6
7
8
9
H-Tyr-(2S,3S)âMeTic-Phe-Phe-OH
H-Tyr-(2S,3R)-âMeTic-Phe-Phe-OH
H-Tyr-Tic-(2S,3S)-âMePhe-Phe-NH2
H-Tyr-Tic-(2S,3R)-âMePhe-Phe-NH2
H-Tyr-Tic-(2S,3R)-âMePhe-Phe-OH
H-Tyr-Tic-Phe-(2S,3S)-âMePhe-NH2
H-Tyr-Tic-Phe-(2S,3R)-âMePhe-NH2
>10000
>10000
973 ( 309
149 ( 31
>10000
47.0 ( 12.4
2021 ( 202
6.81 ( 0.31
0.53 ( 0.08
2.91 ( 0.40
0.66 ( 0.04
0.38 ( 0.01
1.60 ( 0.18
1.68 ( 0.15
>10000
>10000
10000
>10000
1.61 ( 0.29
3396 ( 655
4324 ( 1978
>10000
5520 ( 640
5080 ( 700
>10000
18.2 ( 3.4
7.24 ( 0.11
0.192 ( 0.025
5.36 ( 1.05
10
11
1282 ( 87
253 ( 10
636 ( 62
2740 ( 360
5.37 ( 0.84
a
b
µ: [3H]sufentanil (rat forebrain). δ: [3H]DPDPE (NxG 108 CC 15 cells). c κ: [3H]U69596 (guinea pig cerebellum).
Ta ble 4. Receptor Binding Affinities and Activities in MVD and GPI and Bioassays of TIPP Analogues Containing D-â-Me Amino
Acids
bioassay
receptor binding (Ki, nM)
MVD (Ke DPDPE)
antagonist [nM]
no.
µa N ) 5
δb N ) 5
κc N ) 2
GPI (IC50) agonist [nM]
12 H-(2R,3R)-âMeTyr-Tic-Phe-Phe-NH2 1596 ( 412 28 ( 2
1180 ( 63.5 >10000
2509 ( 300 >10000
37.1 ( 2.6
1020 ( 150
13 H-(2R,3S)-âMeTyr-Tic-Phe-Phe-NH2 >10000
113 ( 14
317 ( 62
14 H-Tyr-D-Tic-Phe-Phe-NH2
7.30d ( 0.52 519e ( 46
agonist: IC50 ) 454 ( 72
1560 ( 120
401 ( 96
125 ( 6
partial agonist (max 45%)
54.4 ( 5.8
agonist: IC50 ) 1.78 ( 0.25
2
15 H-Tyr-(2R,3R)-âMeTic-Phe-Phe-OH >10000
543 ( 60
474 ( 81
7.83e
>10000
>10000
>10000
>10000
>10000
>10000
>10000
16 H-Tyr-(2R,3S)-âMeTic-Phe-Phe-OH >10000
17 H-Tyr-Tic-D-Phe-Phe-NH2
658d
60
18 H-Tyr-Tic-D-Phe-Phe-OH60
4000d
6.76e
19 H-Tyr-Tic-(2R,3R)-âMePhe-Phe-NH2 >10000
20 H-Tyr-Tic-(2R,3S)âMePhe-Phe-NH2 84 ( 21
21 H-Tyr-Tic-(2R,3S)âMePhe-Phe-OH 810 ( 185
74 ( 5.1
442 ( 52
0.50 ( 0.07 1190 ( 452 127 ( 19
0.76 ( 0.10 >10000
4.1 ( 0.3 766 ( 18
partial agonist (max 70.%) partial agonist max 60%)
IC35 ) 519 ( 62
partial agonist (max 70%) 20.5 ( 0.7
IC35 ) 1710 ( 270
IC30 ) 0.623 ( 0.12
22 H-Tyr-Tic-Phe-(2R,3R)-âMePhe-NH2 498 ( 150
23 H-Tyr-Tic-Phe-(2R,3S)-âMePhe-NH2 >10000
11.00 ( 1.01 2844 ( 519 4510 ( 750
27.7 ( 5.5
a
b
d
µ: [3H]sufentanil. δ: [3H]DPDPE (NXG 108CC15 cells). c κ: [3H]U69593 (guinea pig cerebellum). µ: [3H]DAGO (rat brain, see ref
2). e δ: [3H]DSLET (rat brain, see ref 2).
binding profile similar to that of the parent peptide 2,
whereas the epimeric (2S,3R)-â-MePhe4 analogue 11
displayed 11-fold reduced µ-receptor affinity and, con-
sequently, increased δ-selectivity.
cally evoked contractions of the GPI and of the MVD
(Tables 3 and 4). The GPI preparation contains mainly
µ-receptors, whereas in the MVD assay the opioid effects
are primarily mediated by δ-receptors.34 δ-Antagonist
potencies were determined against the agonist [D-Pen2,
D-Pen5]enkephalin (DPDPE).35 In the L-â-Me amino
acid series (Table 3), all analogues retained the δ-an-
tagonist properties of 1 and 2, with the exception of the
(2S,3R)-â-MeTyr1 analogue 4, which was a partial
δ-agonist. In general, the δ-antagonist potencies cor-
related quite well with the δ-receptor affinities deter-
mined in the binding assay. The (2S,3R)-â-MePhe3
analogue 9 has a very high δ-antagonist potency with
no µ-agonist or µ-antagonist effect at concentrations up
to 10 µM. In comparison with 2, compounds 10 and 11
are mixed µ-agonist/δ-antagonists with 3-fold increased
δ-antagonist potency and with 3-fold enhanced µ-agonist
potency in the case of 10 and 1.6-fold decreased µ-ago-
nist potency in the case of 11. Compounds 10 and 11
In the D-â-Me amino acid containing TIPP analogues,
the effects of the â-methyl substitution are much more
dramatic. Substitution at Tyr1 results in δ-selective
compounds, the (2R,3R) epimer 12 being more potent
than the (2R,3S) analogue 13. In contrast to [L-Tic2]-
TIPP-NH2 2 which is δ-selective, the [D-Tic2] analogue
14 is µ-selective.2 The â-methyl substitutions of the
D-Tic residues 15 and 16 did not appreciably affect the
moderate δ-affinity observed with the parent peptide,
but abolished the µ-affinity. [D-Phe3]TIPP-NH2 17 and
[D-Phe3]TIPP-OH 18 are both approximately 10 times
less potent at the δ-receptor than 1 or 2. Whereas the
(2R,3R)-â-Me-Phe3 analogue 19 showed 10 times lower
δ-affinity than its corresponding parent peptide and no
µ-affinity, the epimeric (2R,3S) analogue 20 displayed
subnanomolar δ-affinity. As expected, replacement of
the C-terminal carboxamide function in 20 with a
carboxylic acid group resulted in a compound 21 with
further improved δ-receptor selectivity. The â-methyl
substitutions performed at D-Phe4 resulted in the still
quite potent and very δ-selective analogues 22 and 23.
The binding to κ-opioid receptors remains low in all
cases.
are less potent and less balanced than other mixed
25
µ-agonist/δ-antagonists derived from TIPP-NH2.
In
the D-â-Me amino acid containing analogue series (Table
4), the â-MeTyr1 analogues 12 and 13 were found to be
weakly potent δ-antagonists, in agreement with their
relatively low δ-receptor affinities. The D-â-MeTic2
isomers 15 and 16 both turned out to be weak δ-antag-
onists, in contrast to compound 14 which is a δ-agonist.
Very interestingly, the (2R,3R)-â-MePhe3 analogue 19
is a moderately potent but quite selective δ-antagonist.
The in vitro opioid activities of the TIPP analogues
were tested in bioassays based on inhibition of electri-

5170 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 26
Tourwe´ et al.
F igu r e 1. NMR parameters of â-MeTic protons relevant for the conformation of the â-MeTic residue in TIPP-analogues.
On the other hand, the (2R,3S) epimer 20 is a very
potent full δ-agonist and a moderately potent µ-agonist.
The corresponding C-terminal carboxylic acid analogue
21 is a partial agonist at both receptors. The D-â-
MePhe4 analogues 22 and 23 maintain moderate δ-an-
tagonist potency. Whereas 23 is a very weak but full
µ-agonist, the epimeric 22 is a partial µ-agonist.
this peptide bond in the major conformer was assigned
the trans configuration on the basis of the characteristic
Tyr CRH nuclear Overhauser effect (NOE) with Tic C1H2
in the trans or with Tic C3H in the cis conformation.38
The chemical shifts were assigned using two-dimen-
sional homonuclear Hartmann-Hahn and rotating
frame NOE spectroscopy. A distinction between the
Phe3 and Phe4 signals was made on the basis of CRH-
NH NOEs. The NMR parameters which are relevant
for â-MeTic are presented in Figure 1. It is clear that
the â-methyl substituent does not change the side chain
conformation to an appreciable extent: both the vicinal
coupling constants and the NOEs indicate a predomi-
nant gauche(+) orientation in both (2S)-â-MeTic2 ana-
logues 5 and 6 and a gauche(-) orientation in both (2R)-
â-MeTic2 analogues 15 and 16, as observed previously
for L-Tic and D-Tic containing peptides.7,36,37
Con for m a tion a l Stu d ies. Previous conformational
studies on L-Tic containing peptides have indicated that
the conformation of the Tic heterocyclic ring corresponds
to a gauche(+) conformation of the side chain (gauche-
(-) for D-Tic) when the Tic residue is at an internal
position in the peptide sequence.7,36,37 The introduction
of a â-methyl substituent in the (2S,3R) or (2R,3S)
isomer is expected to stabilize this conformation because
of its pseudoequatorial position (Figure 1). In contrast,
in the (2S,3S) or (2R,3R) isomers the pseudoaxial
position of the methyl in the gauche(+) or, respectively,
gauche(-) configuration may shift the equilibrium
toward the conformation having the two substituents
in an equatorial position. Therefore, NMR studies in
DMSO solution were performed on the four â-MeTic
containing TIPP analogues 5, 6 and 15, 16. As in TIPP,
these four isomers all display a slow cis/ trans isomer-
ization around the Tyr-Tic peptide bond. In all cases
Discu ssion
The intramolecular distance between the aromatic
rings of Tyr1 and Tic2 is a crucial factor determining
the δ-antagonism in TI(PP). The Phe3 or Phe4 residues
may prevent the hydrophobic collapse of the Tyr-Tic
rings and/or can engage in additional receptor interac-
tions. The introduction of a â-methyl group on each of

Side Chain Methyl Substitution in TIPP
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 26 5171
the residues in the tetrapeptides may therefore have
profound effects on receptor affinity, on selectivity, and
on the agonist/antagonist character by biasing the side
chain topology to a particular gauche conformation.
NMR studies of CCK-8 analogues have indicated that
the introduction of a â-methyl substituent in the Phe
side chain virtually eliminates the trans ø1-rotamer in
the erythro isomers (2S,3S and 2R,3R) and strongly
reduces the population of the gauche(+) rotamer in the
(2S,3R) threo isomer and of the gauche(-) rotamer in
the (2R,3S) threo isomer. Both other rotamers are
populated to an appreciable extent (Table 1).39,40 These
results were confirmed by theoretical calculations,
indicating that there is a small (less than 2 kcal/mol)
energy preference to the ø1-rotamers which places both
Câ-substituents in a gauche conformation to HR and a
slight disfavor for the ø1-rotamer that places both Câ-
substituents gauche to the CO group.10 Overall, sub-
stitution of L-â-methyl amino acids in TIPP had a
minimal effect on δ-receptor affinity and on δ-antagonist
potency. The overlap studies of TIP(P) peptides with
naltrindole indicate a trans ø1 for Tyr1 in all models.6
Clearly, the small differences in opioid activities seen
between the two â-MeTyr1 isomers 3 and 4 do not allow
a conclusion with regard to the required side chain
conformer. The common allowed rotamer for both the
(2S,3R) and (2S,3S) isomers is gauche(-), which is not
consistent with the theoretical models and with the
Compound 9 ranks among the most potent δ-antag-
onists reported to date with a Ke-value (0.192 nM)
comparable to that of the highly δ-selective pseudopep-
tide H-Tyr-Ticψ(CH2NH)Cha-Phe-OH (Ke ) 0.219 nM)4
and to that of H-Dmt-Tic-Phe-Phe-OH (Ke ) 0.152
nM).45 In Tyr-Tic peptides, the substitution by 2′,6′-
dimethyltyrosine increases antagonist potency and se-
lectivity by several orders of magnitude.46 The Ke for
H-Dmt-Tic-OH of 5.7 nM and for the N,N-(Me)2-Dmt-
Tic-OH (Ke ) 0.25 nM)47 are also comparable to that of
9, as well as their receptor selectivity. In the â-MePhe4
analogues 10 and 11 the configuration at the â-carbon
has no effect on δ-affinity and potency. This can be
consistent with the fact that both isomers can adopt a
common gauche(-) orientation. Since µ-affinity is af-
fected to a larger extent, the (2S,3R)-â-MePhe4 isomer
11 becomes more δ-selective.
In the D amino acid containing analogue series, some
remarkable effects of the â-methyl substitution on opioid
activity profile are observed. The low potency of both
(2R,3R)- and (2R,3S)-â-MeTyr1 analogues 12 and 13 is
not surprising, since a D-Tyr1 residue is not tolerated
in opioid peptides. According to our NMR observations,
the conversion of the µ-selective agonist H-Tyr-D-Tic-
Phe-Phe-NH2 into the weak δ-selective antagonists 15
and 16 cannot be explained by a change in the side chain
(or ring) conformation of the Tic residue. The added
methyl group therefore either causes an overall confor-
mational change of the backbone or has an effect on the
transduction mechanism. The former is rather unlikely
in view of its orientation outside the cleft formed by the
Tyr1/Tic2/Phe3 aromatic rings, whereas precedent for the
latter has been reported.16
A similar but stereospecific effect of the â-methyl
substitution is observed in the (2R)-Phe3 analogues:
whereas the introduction of a â-methyl with (3R)
configuration in [D-Phe3]TIPP-NH2 17 to give analogue
19 does not change the antagonist character, the epimer
with (2R,3S) configuration 20 is a very potent δ-agonist
and a moderately potent µ-agonist. It is therefore
tempting to speculate that the trans ø1-rotamer, which
is highly disfavored in the (2R,3R) isomer but allowed
in the (2R,3S) isomer, is causing the effect. Very subtle
effects, even remote from the essential Tyr-Tic phar-
macophore can influence the antagonism: the change
from amide 17 to acid 18 results in partial δ-agonism,
which is maintained on (3S)-â-methyl substitution. In
contrast the D-â-MePhe4 analogues, 22 and 23 remain
moderate δ-antagonists, but in 22 partial µ-agonism is
observed. This observation is similar to the one ob-
served in the L-â-MePhe4 isomers 10 and 11.
observed solution conformation for Tyr-Tic dipeptide
41
analogues
and with the solid-state conformation of
1.42 The difference in δ-affinity is much higher in the
highly constrained dipeptide â-methyl-2′,6′-dimethylty-
rosine-Tic, for which a trans ø1 is proposed.43 For the
diketopiperazine c[Tyr-Tic] a gauche(-) orientation for
Tyr is observed in the NMR spectra and in the crystal.44
The cyclic compound displays, however, much weaker
δ-affinity and antagonism than the linear ones. The
(2S,3R)-â-MeTyr1 analogue 4 becomes a partial δ-ago-
nist (max ) 50%). A similar effect of â-methylation on
the transduction properties of somatostatin-derived
opioids has been observed and was interpreted as due
to differences in geometrical arrangement of the phar-
macophores.16
For the Tic2 residue, â-methylation does not change
the preferred gauche(+) orientation. The (2S,3S)-â-
MeTic2 analogue 5, having the â-methyl group in the
pseudoaxial orientation has, approximately 13 times
less δ-affinity than the (2S,3R)-â-MeTic2 analogue 6,
having the methyl group in the pseudoequatiorial
position, but both have very similar δ-antagonist po-
tency.
For Phe3, the (2S,3R)-â-MePhe isomer 8 is more
potent than its (2S,3S) analogue 7. As expected, the
receptor selectivity of 8 can be increased by changing
the C-terminal amide to the carboxylic acid 9. The high
antagonist potency of 9 suggests that models having the
side chain of Phe3 in a gauche(+) conformation6 are to
be considered unlikely candidates of the bioactive
conformation. The higher potency of the (2S,3R) epimer
8 compared to the (2S,3S) epimer 7 may indicate that
a trans ø1 is preferred. In the lowest energy conforma-
tion of H-Tyr-Ticψ(CH2NH)Phe-Phe-OH, the Phe3 takes
the trans orientation, which makes it more exposed and
accessible for interaction with the δ-receptor.6
Con clu sion s
The introduction of a â-methyl substituent on each
residue in TIPP can have important effects on receptor
selectivity and on agonist/antagonist properties. Al-
though it has been demonstrated that the dipeptides
Tyr-Tic and especially Dmt-Tic have the necessary
requirements for δ-antagonism, the present results
demonstrate that substitutions at Phe3 and Phe4 can
strongly influence the biological profile. Despite the fact
that a single â-methyl substituent is reported not to
dramatically constrain the conformational mobility of
aromatic amino acids in ø-space,10 the usefullness of

5172 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 26
Tourwe´ et al.
(E:Z ) 70:30) is crystallized from ethanol once again and gives
the oxazolone (E:Z ) 98/2) with a yield of 68%: NMR (CDCl3)
δ 8.08 (2H, aromatic, d, J ) 8.4 Hz), 7.50 (5H, aromatic), 6.93
(2H, aromatic, d, J ) 8.4 Hz), 3.85 (3H, s, OCH3), 2.64 (3H, s,
CH3); mp 123-127 °C; Rf 0.77 (E); MS (m/z, RI) 294 (M+ + 1).
2-P h en yl-4-[r-Z-p -m et h oxyp h en ylet h ylid en e]-5(4H )-
oxa zolon e. A solution of 0.3 g (1.02 mmol) of the oxazolone
(E:Z ) 70:30) in 15 mL of pyridine is stirred for 10 min at
room temperature. The solution is poured into a beaker which
contains 20 mL of 0.6 M hydrochloric acid and 30 g of ice water.
The oxazolone precipitates, and the solid is filtered as quickly
as possible over a layer of dicalite.
these topographically constrained amino acids for creat-
ing differences in biological activity of peptides is
demonstrated. As equally stated,10 the results of such
â-methyl substitutions are not easily interpreted in
terms of topographical requirements of the receptor to
the side chain conformations. More bulky substituents,
such as â-isopropyl, or combined substitutions, such as
â-methyl-2′,6′-dimethyl, may provide additional infor-
mation due to the higher energy differences between the
rotamers.10
The residue is dissolved in 300 mL of hot ethanol and the
dicalite is filtered off. By adding water to the solution, the
oxazolone (E:Z ) 4:96) crystallizes with a yield of 73% (0.22
g): NMR (CDCl3) δ 8.07 (2H, aromatic, d, J ) 8.2 Hz), 7.50
(5H, aromatic), 6.98 (2H, aromatic, d, J ) 8.2 Hz), 3.877 (3H,
s, OCH3), 2.77 (3H, s, CH3); mp 95-98 °C; Rf 0.77 (E); MS
(m/z, RI) 294 (M+ + 1).
E-2-ben zoyla m in o-3-p-m eth oxyp h en yl-2-bu ten oic Acid
Meth yl Ester . To a solution of 0.125 g (5.43 mmol) of sodium
in 50 mL of anhydrous methanol is added 5 g (17.06 mmol) of
oxazolone (E:Z ) 98/2) in portions. This mixture is stirred
for 2 h at room temperature. The precipitated solid is filtered
and washed with methanol. After neutralization of the filtrate
with concentrated hydrochloric acid, additional material pre-
cipitates and is collected by filtration. The remaining filtrate
is then evaporated in vacuo. The combined solids are crystal-
lized from ethanol/water and a yield of 4.77 g (86%) of the
E-isomer is obtained: NMR (CDCl3) δ 7.86 (2H, aromatic, d,
J ) 8.3 Hz), 7.35 (4H, aromatic), 7.16 (2H, aromatic, d, J )
8.7 Hz), 6.86 (2H, aromatic, d, J ) 8.7 Hz), 3.87 (3H, s,
COOCH3), 3.50 (3H, s, OCH3), 2.16 (3H, s, CH3); mp 147-148
°C; Rf 0.33 (E); MS (m/z, RI) 326 (M+ + 1, 9), 185 (30), 105
(70).
Exp er im en ta l Section
Gen er a l Meth od s. TLC was performed on 2.5 × 10 cm
plates precoated with Merck silica gel 60F254 using the
following solvent systems: (A) 1-butanol/acetic acid/water (4:
1:1), (B) acetonitrile/methanol/water (4:1:1), (C) ethyl acetate/
pyridine/acetic acid/water (12/4/1.2/2.2), (D) chloroform/metha-
nol/acetic acid (90:8:2), and (E) hexane/ethyl acetate (1:1). TLC
spots were detected under UV light and using iodine vapor.
Melting points were measured with a Bu¨chi 510 apparatus.
NMR spectra were obtained in either CDCl3, D2O, or D2O/TFA
on a Bruker AC 250-P 250 MHz spectrometer.
Mass spectra were recorded on a AEI/902S spectrometer
with fast atom bombardment (FAB) ionization using xenon
gas. Reversed phase HPLC (RP-HPLC) was performed on a
Vydac 218TP54 analytical column (25 × 0.46 cm) or on a Vydac
218TP510 semipreparative column (25 × 1.0 cm) using UV
detection at λ ) 210 nm.
Syn th esis of Nr-ter t-Bu tyloxyca r bon yl-er yth r o-â-m e-
th ylp h en yla la n in e a n d Nr-ter t-Bu tyloxyca r bon yl-th r eo-
â-m eth ylph en ylalan in e. The pure erythro-(2S,3S and 2R,3R)
racemate and the pure threo-(2S,3R and 2R,3S) racemate were
obtained by fractional crystallization 9 of the isomeric mixture
prepared by the method of Kataoka.48 A solution of 5 g (23
mmol) of erythro-â-methylphenylalanine hydrochoride salt or
5 g (23 mmol) of threo-(S,R and R,S)-â-methylphenylalanine
hydrochloride salt in 60 mL of dioxane/water (2:1) is cooled in
an ice bath. To this solution are added 30 mL of 1 N sodium
hydroxide and 5.22 g (24 mmol) of di-tert-butyldicarbonate. The
solution is stirred at room temperature for 25 h, whereby the
pH is continuously adjusted to 7.5 with 1 N sodium hydroxide.
After evaporation of the dioxane, the residue is diluted with
water and the pH is adjusted to 3 with a saturated KHSO4
solution under cooling in an ice bath. The aqueous phase is
extracted with 3 × 100 mL portions of ethyl acetate. The
combined organic phases are dried over MgSO4 and evapo-
rated. The residue is then dissolved in a minimum amount
of ethyl acetate, and petroleum ether is then added slowly to
give white crystalline products. NR-Boc-erythro-â-methylphe-
nylalanine: yield 6.42 g (75%); mp 109-112 °C (lit.9 mp 108
°C), Rf 0.82 (B). NR-Boc-threo-â-methylphenylalanine: yield
4.28 g (50%); mp 96-97 °C (lit.9 mp 96 °C); Rf 0.82 (B).
Syn th esis of er yth r o-(2S,3S a n d 2R,3R)-â-Meth ylty-
r osin e a n d th r eo-(2S,3R a n d 2R,3S)-â-Met h ylt yr osin e.
2-P h en yl-4-[r-E/Z-p -m et h oxyp h en ylet h ylid en e]-5(4H )-
oxa zolon e. A mixture of 15 g (0.08 mol) of hippuric acid and
23 mL (0.24 mol) of acetic anhydride is cooled to approximately
10 °C. Dropwise, 9 mL of sulfuric acid is added under stirring.
To the yellow solution is added 12.5 g (0.09 mol) of solid
p-methoxyacetophenone, and the reaction mixture is left
stirring until room temperature is reached. The solution is
then heated at 60 °C for 15 min and left stirring overnight at
room temperature.
Z-2-Ben zoyla m in o-3-p -m et h yloxyp h en yl-2-b u t en oic
Acid Meth yl Ester . The synthesis was started from Z-
oxalozone, and the procedure was the same for the E-
oxazolone: NMR (CDCl3) δ 7.86 (2H, aromatic), 7.35 (3H,
aromatic), 7.16 (2H, aromatic, d, J ) 8.7 Hz), 6.86 (2H,
aromatic, d, J ) 87 Hz), 3.8 (3H, s, COOCH3), 3.5 (3H, s,
OCH3), 2.29 (3H, s, CH3); mp 138-140 °C; Rf 0.33 (E); MS
identical that of the E isomer.
er yt h r o-N -Be n zoyl-â-m e t h yl-p -m e t h oxy-p h e n yla la -
n in e Meth yl Ester . A mixture of 4.73 g (14.55 mmol) of the
E isomer of the foregoing butenoic acid methyl ester in 190
mL of methanol and 0.4 g of Pd/C catalyst (10% type 90,
J ohnson Matthey, powder) is hydrogenated for 4 days at room
temperature at a pressure of 50 psi in a Parr-hydrogenator.
Then the catalyst is filtered over a layer of Celite, and the
filtrate is evaporated in vacuo. The yellow oil is dissolved in
hot ethanol and crystals appear on addition of water to the
solution: yield 3.77 g (76%); NMR (CDCl3) δ 7.67 (2H,
aromatic, d, J ) 8.7 Hz), 7.45 (3H, aromatic), 7.12 (2H,
aromatic, d, J ) 8.6 Hz), 6.86 (2H, aromatic, d, J ) 8.6 Hz),
6.28 (1H, NH, d, J ) 8.1 Hz), 5.00 (1H, HR, dd, J ) 8.1 and
5.2 Hz), 3.78 (3H, s, OCH3), 3.72 (3H, s, COOCH3), 3.46 (1H,
m, Hâ), 1.38 (3H, CH3, d, J ) 7.2 Hz); mp 80-83 °C; Rf 0.42
(E); MS (m/z, RI) 328(M+ + 1, 15), 268 (13), 135 (19), 105 (100).
th r eo-N-Ben zoyl-â-m et h yl-p -m et h oxy-p h en yla la n in e
Meth yl Ester . A mixture of the foregoing butenoic acid
methyl esters (Z:E ) 80:20) was submitted to the hydrogena-
tion as for the E isomer: NMR (CDCl3) (the spectrum contains
additional signals of the erythro isomer as described above,
for about 20%) δ 7.67 (2H, aromatic, d, J ) 8.7 Hz), 7.45 (3H,
aromatic), 7.12 (2H, aromatic, d, J ) 8.6 Hz), 6.86 (2H,
aromatic, d, J ) 8.6 Hz), 6.54 (1H, NH, d, J ) 8.2 Hz), 4.96
(1H, HR, d, J ) 5.2 Hz), 3.78 (3H, s, OCH3), 3.72 (3H, s,
COOCH3), 3.46 (1H, m, Hâ), 1.43 (3H, CH3, d, J ) 7.3 Hz);
mp 125-128 °C; Rf 0.42 (E); MS identical to that of the erythro
isomer.
The resulting dark brown oil is poured into 150 mL of ice
cold water, whereby the product precipitates. The water is
decanted, and the residue is washed 3 times with ice cold
water. Then, 100 mL of hot water is added under stirring.
The residue is filtered, dried over P2O5, and dissolved in 350
mL of hot ethanol. After cooling, the yellow crystals are
filtered to yield 20.37 g (83%) of product (E:Z ) 70:30 as
determined from the methyl signal in 1H NMR).
er yth r o-â-Meth yltyr osin e. To a solution of 5.2 mL of 48%
HBr (aqueous) and 10.4 mL of acetic acid is added 1 g (3 mmol)
of erythro-N-benzoyl-â-methyl-p-methoxy-phenylalanine meth-
Isola tion of 2-P h en yl-4-[r-E-p-m eth oxyp h en yleth -
ylid en e]-5(4H)-oxa zolon e. The E/ Z mixture of oxazolone

Side Chain Methyl Substitution in TIPP
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 26 5173
yl ester. The solution is refluxed for 24 h and evaporated in
vacuo. The resulting oil is dissolved in water and evaporated
again. To the residue is added 5 mL of water, and the flask is
placed in a refrigerator. Purple-colored crystals of benzoic acid
are filtered, and the remaining solution is extracted with ethyl
acetate. The aqueous phase is adjusted to pH 6 with am-
monium hydroxide, and erythro-â-methyltyrosine precipitates.
The precipitate is filtered, and a yield of 0.361 g (58%) is
obtained after recrystallization from hot water: NMR (D2O/
TFA) δ 13.6 (1H, s, COOH), 6.75 (2H, aromatic, d, J ) 8.6
Hz), 6.43 (2H, aromatic, d, J ) 8.6 Hz), 3.65 (1H, HR, d, J )
7.2 Hz), 3.07 (1H, Hâ, m), 0.94 (3H, CH3, d, J ) 7.2 Hz); mp
228-231 °C; Rf 0.47 (A); MS (m/z, RI) 196 (M+ + 1, 50), 110
(50).
th r eo-â-Met h ylt yr osin e. Threo-N-Benzoyl-â-methyl-p-
methoxy-phenylalanine methyl ester was hydrolyzed in 48%
HBr (aqueous) and acetic acid as above. Any erythro isomer
was removed by chromatography on a Lobar (Merck) LiChro-
prep RP8 (40-63) column, using 1% aqueous methanol as
eluent: NMR (D2O/TFA) δ 13.6 (1H, s, COOH), 6.81 (2H,
aromatic, d, J ) 8.6 Hz), 6.43 (2H, aromatic, d, J ) 8.6 Hz),
4.11 (1H, HR, d, J ) 7.2 Hz), 3.37 (1H, Hâ, m), 1.31 (3H, CH3,
d, J ) 7.0 Hz); mp 228-231 °C; Rf 0.47 (A); MS identical to
that of the erythro isomer.
Nr-ter t-Bu tyloxycar bon yl-er yth r o-â-m eth yltyr osin e an d
Nr-ter t-Bu tyloxyca r bon yl-th r eo-â-m eth yltyr osin e. A so-
lution of 1.41 g (5.13 mmol) of erythro-(2S,3S and 2R,3R)-â-
methyltyrosine or 1.41 g (5.13 mmol) of threo-(2S,3R and
2R,3S)-â-methyltyrosine in 30 mL of dioxane/water (2:1) is
cooled in an ice bath. To this solution is added a 4 N sodium
hydroxide solution under stirring until a pH of 10 is reached
and then 1.3 mL (1.1 equiv) of Boc2O is added. For 2 h this
solution is stirred at 0 °C while the pH is continuously adjusted
to 10. Then another 0.4 mL of Boc2O is added, and the mixture
is stirred overnight at room temperature.
After evaporation of the dioxane, the residue is diluted with
a few milliliters of water and the pH is adjusted to 2-3 with
a saturated KHSO4 solution under ice cooling. The aqueous
layer is then extracted 3 times with 50 mL of ethyl acetate.
The combined organic phases are washed with water, a
saturated NaCl solution, and again with water. After drying
over MgSO4 and evaporation in vacuo, the oil is diluted in a
minimum amount of ethyl acetate and slowly added to 130
prepared in the same way starting with 2 g of threo/ erythro
(8:2) â-Me-Phe: yield 1.60 (66%) g of threo-â-Me-Tic‚HCl (10%
erythro); NMR (D2O) δ 7.52-7.35 (4H, aromatic, m), 4.58 (2H,
H1, d, J ) 18 Hz), 4.47 (1H, H3, m), 3.74 (1H, H4, m), 1.39
(CH3, d, J ) 7.5 Hz); mp 253-257 °C; Rf 0.45 (A); MS (m/z)
192 (M+ + 1, 100).
Nr-Boc-er yth r o- a n d th r eo-â-Me-Tic. The procedure
described above for the preparation Boc-erythro- or threo-â-
Me-Phe was also used for the synthesis of erythro- or threo-
â-Me-Tic, starting with 1 g of amino acids. NR-Boc-erythro-â-
Me-Tic: yield 1.00 g (66.3%); NMR (CDCl3) (the spectrum
shows signals of the cis-trans isomers around the N-Boc bond
in a ration of 43/57) (7.2-7.0 (4H, aromatic, m), 4.9-4.45 (4H,
H1, H3, m), 3.45 (0.57 H, H4, m), 3.25 (0.43 H, H4, m), 1.57 and
1.43 (9H, t-Bu, s), 1.27 (3H, CH3, d, J ) 9 Hz); mp 80-82 °C;
Rf 0.48 (D); MS (m/z) 292 (M+ + 1, 86), 236 (100) and 192 (98).
Yield 0.84 g (55.7%) NR-Boc-threo-â-Me-Tic: NMR (CDCl3) δ
7.25-7.05 (4H, aromatic, m), 4.85-4.40 (3H, H1,H3, m), 3.30
(1H, H4, quintuplet, J ) 6.8 Hz), 1.47-1.27 (12H, t-Bu, CH3);
mp 149 °C; Rf 0.48 (D); MS identical to the one of the erythro
isomer.
Gen er a l Meth od s for th e Syn th esis of TIP P An a -
logu es. All the peptide analogues of TIPP were synthesized
by the solid-phase method using a semiautomatic Labortec
SP640 synthesizer. Chloromethylated (1.7 mmol/g resin)
polystyrene resin 1% cross-linked with divinylbenzene (Fluka)
was used for the C-terminal carboxylic acid analogues; 4-me-
thylbenzhydrylamine (0.53 mmol/g resin) polystyrene resin
(Neosystem, Strasbourg, France) was used for the C-terminal
amides. NR-Boc-protected amino acids were obtained from
Neosystem. The NR-tert-butyloxycarbonyl-phenylalanine was
attached to the Merrifield resin by the Gisin method.49
Removal of the Boc protecting group was performed by
washing with a TFA/DCM/anisole (33:65:2) solution during 5
and 20 min, followed by DCM washes (4 × 0.5 min), a
neutralization with 20% DIEA (3 × 1 min), and DCM washes
(4 × 0.5 min). Amino acids were coupled in 1.5 (â-Me amino
acids) to 2-fold (Phe, Tic, Tyr) excess using either diisopropy-
lcarbodiimide/HOBt or BOP/HOBt/DIEA. Completion of the
50
coupling reactions was monitored by the ninhydrin
or
chloranil test.51 Since important amounts of Tyr1-deletion
peptides were detected after purification, Boc-Tyr-OH or Boc-
Tyr(2,6-Cl2Bzl)-OH were used in either single or double
couplings, without, however, any significant improvement. The
peptides were cleaved from the resin with anhydrous HF (5
mL/g resin) with anisole added as scavenger (1 mL/g resin)
for 1 h at 0 °C. The peptide was extracted from the resin by
washing with anhydrous ethyl ether and then stirring of the
resin in 60 mL of a 50% aqueous solution of acetic acid for 0.5
h.
mL of ice-cooled petroleum ether with vigorous stirring.
A
white precipitate is formed, filtered, and immediately dried
in vacuo over P2O5: yield 1.40 g (92.3%) of NR-Boc-erythro-â-
methyltyrosine or 1.06 g (70%) of NR-Boc-threo-â-methylty-
rosine.
Nr-Boc-er yth r o-â-m eth yl-Tyr : NMR (DMSO-d6) δ 12.55
(1H, s, COOH), 9.17 (1H, s, OH), 7.06 (2H, aromatic, d, J )
8.3 Hz), 6.64 (2H, aromatic, d, J ) 8.3 Hz), 6.55 (1H, NH, d,
J ) 8.6 Hz), 3.98 (1H, HR, dd, J 1 ∼ J 2 ) 8.6 Hz), 2.94 (1H,
Hâ, m), 1.33 (9H, s, t-Bu), 1.13 (3H, CH3, d, J ) 7.0 Hz); mp
79-82 °C; Rf 0.80 (A); MS (m/z, RI) 296 (M+ + 1, 40), 240 (60),-
196 (55).
Nr-Boc-th r eo-â-m eth yl-Tyr : NMR (DMSO-d6) δ 12.35 (1H,
s, COOH), 9.16 (1H, s, OH), 7.26 (2H, aromatic, d, J ) 8.4
Hz), 7.16 (1H, NH, d, J ) 8.7 Hz), 7.00 (2H, aromatic, d, J )
8.4 Hz), 4.08 (1H, HR, dd, J 1 ∼ J 2 ) 8.7 Hz), 3.22 (1H, Hâ, m),
1.33 (9H, s, t-Bu), 1.15 (3H, CH3, d, J ) 7.1 Hz); mp 87-90
°C; Rf 0.80 (A); MS (m/z, RI) 296 (M+ + 1, 40), 240 (100), 194
(70), 185 (90).
The filtrate was lyophilized. Purity was determined by
analytical RP-HPLC. Purification and separation of the
diastereomeric peptides was performed by preparative RP-
HPLC (Gilson 712 HPLC system) using an acetonitrile/water
system with 0.1% TFA as eluent. The (M + 1)+ molecular ions
and fragmentation patterns were obtained by FAB-MS and
were in agreement with the calculated molecular weights for
each peptide (m/z ) 648 for peptide amides, 649 for peptide
acids). Yields varied from 2 to 12% for each isomer, and in
almost every peptide synthesis a similar amount of tyrosine-
deletion peptide isomers was isolated.
Id en tifica tion of En a n tiom er s of â-Meth yl Am in o
Acid s in P ep tid es.32,33 Identification of enantiomers of â-m-
ethyl amino acids in peptides involved three steps: acid
hydrolysis of peptides, derivatization of the resulting amino
acids with chiral derivatizing reagent GITC or FDAA, and
separation and identification of derivatized amino acids by
HPLC. The separation of derivatized amino acids was carried
out on a Vydac 218TP54 C18 column. The peaks of derivatized
â-methyl amino acids were identified by application of stan-
dards of (2R,3S)- and (2R,3R)-â-methyl amino acids, obtained
by L-AA oxidase digestion of the racemic erythro or threo
isomers.
er yth r o- a n d th r eo-â-Me-Tic. Pure erythro-(2S,3S and
2R,3R)-â-MePhe‚HCl (3.15 g) was suspended in 20 mL of
concentrated HCl, 5 mL of a formaldehyde solution (37%) was
added, and the mixture was refluxed for 30 min at 110 °C.
Then 5 mL of concentrated HCl and 2.5 mL of formaldehyde
solution were added to the reaction mixture, which was further
refluxed for an additional hour. The liquid was evaporated
and the remaining solid was crystallized from water. erythro-
â-Me-Tic‚HCl: yield 2.4 g (72.2%); NMR (D2O) δ 7.57-7.5 (4H,
aromatic, m), 4.53 (2H, H1, d, J ) 18 Hz), 3.89 (1H, H3, m),
3.54 (1H, H4, m), 1.59 (CH3, d, J ) 7.5 Hz); mp 278-281 °C;
Rf 0.47 (A); MS (m/z) 192 (M+ + 1, 100). Threo-â-Me-Tic was

5174 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 26
Tourwe´ et al.
R a d ioliga n d Bin d in g Met h od s. µ-Op ioid R ecep t or
Assa y.52 Wistar rats (150-200 g) are sacrificed, the brain is
removed and forebrain is dissected. The forebrain is homog-
enized in 10 volumes (v/w) of homogenization buffer (0.25 M
sucrose, 85.5 g/L) using a Dual homogenizer. The homogenate
is centrifuged at 0-4 °C for 10 min at 350g (2000 rpm), the
supernatant is carefully removed and the pellet is homogenized
again in 5 volumes (v/w) of buffer and centrifuged at 830g
(3000 rpm) for 10 min. The combined supernatants are diluted
to the double volume with incubation buffer (50 mM Tris-HCl,
pH 7.4, 6 g/L, containing 5 mM MgCl2, 5 mM EGTA, 0.1 mM
toluenesulfonyl fluoride, 0.1 mM bacitracin, and 0.1% bovine
albumin) and centrifuged at 23500g (16000 rpm) for 20 min.
The pellet is rehomogenized in fresh incubation buffer, incu-
bated at 37 °C for 30 min, and then centrifuged, and the
resulting pellet is suspended in 200 volumes of (v/w) incubation
buffer using a Dual homogenizer. Radioligand binding inhibi-
tion assay samples consisted of 0.4 mL of rat forebrain
membrane suspension, 0.05 mL of [3H]sufentanil (0.5 mM in
10% DMSO, specific activity 15 Ci/mmol, J anssen Research
Foundation, Belgium), and 0.05 mL of solvent (10% DMSO),
peptide analogue or blank. The blank consisted of dextro-
moramide (1.1 × 10-3 M in DMSO stock solution, diluted 1/50
into 10% DMSO). Peptide analogues were assayed over a
concentration range of 0.3 mM to 10 µM. Incubations were
performed at 25 °C for 60 min, after which 5 mL cold Tris-
HCl buffer was added and the samples were filtered through
GF/B glass fiber filters, presoaked in 0.1% poly(ethyleneimine)
for 1 h. The filters were washed two times with 5 mL of Tris-
HCl buffer and transferred to scintillation vials. The radio-
activity was measured using a Tri Carb 1500 liquid scintilla-
tion analyzer after adding 2 mL of Ultima Gold (Packard)
scintillation fluid, shaking for 5 min, and allowing the samples
to equilibrate over 12 h.
determined with [Leu5]enkephalin as standard for each ileum
and vas deferens preparation, and IC50 values of the com-
pounds being tested were normalized according to a published
procedure.59 Ke values for the antagonists were determined
from the ratio of IC50 values obtained in the presence and
absence of a fixed antagonist concentration, as described in
the literature.34
Ack n ow led gm en t. Supported by grants (to P.W.S.)
from the Medical Research Council of Canada (MI-5655)
and the National Institutes of Health (DA-04443) and
by grants (to D.T., G.T., and A.P) of Copernicus (CIPA-
CT94-0226), Flemish-Hungarian, Bilateral Intergov-
ernmental S&T Cooperation B-3/96. Dr. J . E. Leysen
and W. Gommeren (J anssen Research Foundation,
Belgium) are thanked for the receptor binding assays.
Refer en ces
(1) Abbreviations and definitions are those recommended by IUPAC-
IUB Commission on Nomenclature (J . Biol. Chem. 1972, 247,
977-983). All optically active amino acids are of L configuration
unless otherwise noted. Other abbreviations used are Tic,
tetrahydroisoquinoline-3-carboxylic acid; TFA, trifluoroacetic
acid; DCM, dichloromethane; DIEA, diisopropylethylamine;
DMF, dimethylformamide; DMSO, dimethyl sulfoxide; HOBt,
1-hydroxybenzotriazole; BOP, benzotriazole-1-yloxy-tris(dim-
ethylamino) phosphonium hexafluorophosphate; GITC, 2,3,4,6-
tetra-O-acetyl-â-D-glucopyranosylisothiocyanate; FDAA 1-fluoro-
2,4-dinitrophenyl-5-L-alanine amide; DPDPE, Tyr-D-Pen-Gly-
Phe-D-Pen-OH; DAGO, Tyr-D-Ala-Gly-NMe-Phe-Gly-ol; U69593,
(5a,7a,8a)-(-)-N-methyl-[7-1-pyrrolidinyl)-1-oxa spiro-[4,5]dec-
8-yl]benzeneacetamide; MVD, mouse vas deferens; GPI, guinea
pig ileum, HPLC, high-performance liquid chromatography,
TLC, thin layer chromatography.
(2) Schiller, P. W.; Nguyen, T. M.-D.; Weltrowska, G.; Wilkes, B.
C.; Marsden, B. J .; Lemieux, C.; Chung, N. N. Differential
stereochemical requirements of µ vs δ opioid receptors for ligand
binding and signal transduction: Development of a class of potent
and highly δ-selective peptide antagonists. Proc. Natl. Acad. Sci.
U.S.A. 1992, 89, 11871-11875.
(3) Mosberg, H. I.; Omnaas, J . R.; Sobczyk-Kojiro, K.; Dua, R.; Ho,
J . C.; Ma, W.; Bush, P.; Mousigian, C.; Lomize, A. Pharmacoph-
ore elements of the TIPP class of delta opioid receptor antago-
nists. Lett. Pept. Sci. 1994, 1, 69-72.
(4) Schiller, P. W.; Weltrowska, G.; Nguyen, T. M.-D.; Lemieux, C.;
Chung, N. N.; Zelent, B.; Wilkes, B. C.; Carpenter, K. A.
Structure-agonist/antagonist activity relationships of TIPP ana-
logs. In Peptides: Chemistry, Structure and Biology, Kaumaya,
P. T. P., Hodges, R. S., Eds., Mayflower: Kingswinford, England,
1996; pp 609-611.
(5) Temussi, P. A.; Salvadori, S.; Amodeo, P.; Bianchi, C.; Guerrini,
R.; Tomatis, R.; Lazarus, L. H.; Picone, D.; Tancredi, T. Selective
opioid dipeptides. Biochem. Biophys. Res. Commun. 1994, 198,
933-939.
(6) Wilkes, B. C.; Schiller, P. W. Comparative analysis of various
proposed models of the receptor-bound conformation of H-Tyr-
Tic-Phe-OH related δ-opioid antagonists. Biopolym. (Pept. Sci.)
1995, 37, 391-400.
(7) Hruby, V. J .; Al-Obeidi, F.; Kazmierski, W. Emerging approaches
in the molecular design of receptor-selective peptide ligands:
conformational, topographical and dynamic considerations. Bio-
chem. J . 1990, 268, 249-262.
Inhibition curves were plotted and were analyzed by least-
squares regression.
δ-Op ioid Recep tor Assa y.53 NXG108CC15 cells were
cultured in DMEM medium, supplemented with 10% (v/v)
foetal calf serum, 2 mM glutamine, antibiotics (100 µU/mL
penicillin and 0.1 mg/mL streptomycin), 0.1 mM hypoxantine,
0.001 mM aminopteridine, and 0.016 mM thymidine. After
at least two passages, cells are scraped off carefully and
centrifuged at 200g (1500 rpm) in a Sorvall RC-5B s-centrifuge.
The cells are washed once with 0.9% saline, and the cell pellet
is suspended in 0.9% saline in a concentration of 107 cells/
mL, which is divided into 2 mL aliquots and stored at -80 °C
for several weeks. For the receptor binding experiments, a
vial is thawed and homogenized in incubation buffer (see
µ-receptor assay). Radioligand binding inhibition assay samples
consisted of 0.4 mL of cell suspension, containing 5 × 105 cells,
0.05 mL of [3H]DPDPE (2 nM, specific activity 30-32 Ci/mmol,
New England Nuclear, Boston, MA), and 0.05 mL of solvent
(10% DMSO), peptide analogue or blank. The blank consisted
of naltrindole (1 µM). Assay conditions and analysis were as
described for the µ-receptor assay.
K-Op ioid Recep tor Assa y.54 Guinea pigs (200-250 g) are
sacrificed by decapitation, the brain is removed, and cerebel-
lum is dissected. The cerebellum is homogenized in 10
volumes (v/w) Tris-HCl buffer (50 mM, pH 7.4, 6.06 g/L) using
an Ultra Turrax homogenizer. The homogenate is centrifuged
at 0-4 °C at 23500g (16000 rpm) in a Sorvall RC-5B centri-
fuge. The pellet is washed twice by rehomogenization and
recentrifugation. Finally, the pellet is suspended in 100 v/w
incubation buffer (see µ-receptor assay) using a Dual homog-
enizer. Radioligand binding inhibition assay samples con-
sisted of 0.4 mL of guinea pig cerebellum membrane suspen-
sion, 0.05 mL of [3H]U69593 (0.5 mM in 10% DMSO, specific
activity 60-65 Ci/mmol, Amersham), and 0.05 mL of solvent
(10% DMSO), peptide analogue or blank. The blank consisted
of spiradoline (1 µM in 10% DMSO). Assay conditions and
analysis were as described for the µ-receptor.
(8) Dharanipragada, R.; Nicolas, E.; To´th, G.; Hruby, V. J . Asym-
metric synthesis of unusual amino acids: synthesis of optically
pure isomers of â-methylphenylalanine. Tetrahedron Lett. 1989,
49, 6841-6844.
(9) Hruby, V. J .; To´th, G.; Gehrig, C. A.; Kao, L.-F.; Knapp, R.; Lui,
G. K.; Yamamura, H. I.; Kramer, T. H.; Davis, P.; Burks, T. F.
Topographically designed analogues of [D-Pen2, D-Pen5]enkephalin.
J . Med. Chem. 1991, 34, 1823-1830.
(10) Hruby, V. J .; Li, G.; Haskell-Luevano, C.; Shenderovich, M.
Design of peptides, proteins, and peptidomimetics in chi space.
Biopolym. (Pept. Sci.) 1997, 43, 219-266.
(11) To´th, G.; Pe´ter, A.; Tourwe´, D.; J aspers, H.; Verheyden P. M.
F.; To´th, Z. New analogues of deltorphin C and enkephalin-
Val5,6-Gly7-NH2 with replacement of Phe by âMePhe. In Peptides
1994; Maia, H. L. S., Ed., ESCOM: Leiden, 1995; pp 335-336.
(12) Misicka, A.; Cavagnero, S.; Horvath, R.; Davis, P.; Porreca, F.;
Yamamura, H. I.; Hruby, V. J . Synthesis and biological proper-
ties of â-MePhe3 analogues of deltorphin I and dermenkepha-
lin: influence of biased ø1 of Phe3 residues on peptide recognition
for δ-opioid receptors. J . Peptide Res. 1997, 50, 48-54.
GP I a n d MVD Bioa ssa ys. In vitro opioid activities of the
55
compounds were tested in the GPI and MVD56 bioassays as
reported in detail elsewere.57,58 A log dose response curve was

Side Chain Methyl Substitution in TIPP
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 26 5175
(13) Mosberg, H. I.; Omnaas, J . R.; Lomize, A.; Heyl, D. L.; Nordan,
I.; Mousigian, C.; Davis, P.; Porreca, F. Development of a model
for the δ opioid receptor pharmacophore. 2. Conformationally
restricted Phe3 replacements in the cyclic δ receptor selective
tetrapeptide Tyr-c[D-Cys-Phe-D-Pen]OH (JOM-13). J . Med. Chem.
1994, 37, 4384-4391.
(14) Shenderovich, M. D.; Ko¨ver, K. E.; Nikiforovich, G. V.; J iao, D.;
Hruby, V. J . Conformational analysis of â-methyl-para-nitro-
phenylalanine stereoisomers of cyclo[D-Pen2, D-Pen5]enkephalin
by NMR spectroscopy and conformational energy calculations.
Biopolymers 1996, 38, 141-156.
(15) Huang, Z.; He, Y.-B.; Raynor, K.; Tallent, M.; Reisine, T.;
Goodman, M. Main chain and side chain chiral methylated
somatostatin analogs: syntheses and conformational analyses.
J . Am. Chem. Soc. 1992, 114, 9390-9401.
(16) Bonner, G. G.; Davis, P.; Stropova, D.; Ferguson, R.; Yamamura,
H. I.; Porreca, F.; Hruby, V. J . Opioid peptides: simultaneous δ
agonism and µ antagonism in somatostatin analogues. Peptides
1997, 18, 93-100.
(17) Haskell-Luevano, C.; Boteju, L. W.; Miwa, H.; Dickinson, C.;
Gantz, I.; Yamada, T.; Hadley, M. E.; Hruby, V. J . Topographical
modification of melanotropin peptide analogues with â-methyl-
tryptophan isomers at position 9 leads to differential potencies
and prolonged biological activities. J . Med. Chem. 1995, 38,
4720-4729.
(18) Haskell-Luevano, C.; Toth, K.; Boteju, L.; J ob, C.; Castrucci, A.
M. de L.; Hadley, M. E.; Hruby, V. J . â-Methylation of the Phe7
and Trp9 melanotropin side chain pharmacophores affects
ligand-receptor interactions and prolonged biological activity.
J . Med. Chem. 1997, 40, 2740-2749.
(19) Hruby, V. J .; Fang, S.; To´th, G.; Matsunaga, T. O.; Collins, N.;
Knapp, R.; Yamamura, H. I. Highly potent and selective chole-
cystokinin analogues for the CCK-B receptor. In Peptides 1990;
Giralt, E., Andreu, D., Eds.; ESCOM: Leiden, 1995; pp 707-
709.
(20) Boteju, W.; Nikiforovich, G. V.; Haskell-Luevano, C.; Fang, S.-
N.; Zalewska, T.; Stropova, D.; Yamamura, H. I.; Hruby, V. J .
The use of topographical constraints in receptor mapping:
investigation of the topographic requirements of the tryptophan
30 residue for receptor binding of Asp-Tyr-D-Phe-Gly-Trp-(N-
Me)Nle-Asp-Phe-NH2 (SNF 9007), a cholecystokinin (26-33)
analogue that binds to both CCK-B and δ-opioid receptors. J .
Med. Chem. 1996, 39, 4120-4124.
(21) Lebl, M.; To´th, G.; Slaninova, J .; Hruby, V. J . Conformationally
biased analogs of oxytocin. Int. J . Pept. Protein Res. 1992, 40,
148-151.
(22) Birney, D. M.; Cole, D. C.; Crosson, C. E.; Kahl, B. F.; Neff, B.
W.; Reid, T. W.; Ren, K.; Walkup, R. D. Use of â-methylpheny-
lalanine (âMeF) residues to probe the nature of the interaction
of Substance P with its receptor: effects of âMeF-containing
Substance P analogs on rabbit iris smooth muscle contraction.
J . Med. Chem. 1995, 38, 2478-2482.
(23) Samanen, J .; Narindray, D.; Cash, T.; Brandeis, E.; Adams, W.;
Yellin, T.; Eggleston, D.; DeBrosse, C.; Regoli, D. Potent angio-
tensin II antagonists with non-â-branched amino acids in
position 5. J . Med. Chem. 1989, 32, 466-472.
(24) Azizeh, B. Y.; Shenderovich, M. D.; Triverdi, D.; Li, G.; Sturm,
N. S. and Hruby, V. J . Topographical amino acid substitution
in position 10 of glucagon leads to antagonists/partial agonists
with greater binding differences. J . Med. Chem. 1996, 39, 2449-
2455.
(25) Schiller, P. W., Weltrowska, G.; Schmidt, R.; Nguyen, T. M. D.;
Berezowska, I.; Lemieux, C.; Chung, N. N.; Carpenter, K. A.;
Wilkes, B. C. Four different types of opioid peptides with mixed
µ agonist/δ antagonist properties. Analgesia 1995, 1, 703-706.
(26) Pasto, M.; Moyano, A.; Pericas, M. A.; Riera, A. A catalytic
asymmetric synthesis of N-Boc-â-Methylphenylalanines. J . Org.
Chem. 1997, 62, 8425-8431.
(27) Pictet, A.; Spengler, T. U¨ ber die Bildung von Isochinolin-
derivaten durch Einwirkung von Methylal auf Phenyl-a¨thy-
lamin, Phenyl-alanin und Tyrosin. (About the formation of
isoquinoline derivatives by reaction of methylol with phenyl-
ethylamine, phenylalanine and tyrosine.) Chem. Ber. 1911, 44,
2030-2046.
(28) Cativiela, C.; Mele´ndez, E. Geometric isomers of 2-phenyl-4-(R-
arylethylidene)-5(4H)-oxazolones. Synthesis 1978, 832-834.
(29) Cativiela, C.; Mele´ndez, E. Stereospecific synthesis of (Z/E)-
isomers of 2-benzoylamino-3-phenyl-2-butenoic acids. Synthesis
1980, 901-902.
(30) Cativiela, C.; Mele´ndez, E. Stereospecific synthesis of 2-amino-
3-arylbutanoic acids from 2-phenyl-4-(1-arylethylidene)-5-oxo-
4,5-dihydro-1,3-oxazoles. Synthesis 1981, 805-807.
(32) Pe´ter A.; To´th, G.; To¨ro¨k, G.; Tourwe´, D. Separation of enan-
tiomeric â-methyl amino acids and of â-methyl amino acid
containing peptides. J . Chromatogr. A, 1996, 728, 455-465.
(33) Pe´ter, A.; To´th, G.; Cserpan, E.; Tourwe´, D. Monitoring of optical
isomers of â-methylphenylalanine in opioid peptides. J . Chro-
matogr. A 1994, 660, 283-291.
(34) Kosterlitz, H. W.; Watt, A. J . Kinetic parameters of narcotic
agonist, with particular reference to N-allylnoroxymorphane
(naloxone). Br. J . Pharm. Chemother. 1968, 33, 266-276.
(35) Mosberg, H. I.; Hurst, R.; Hruby, V. J .; Gee, K.; Yamamura, H.
I.; Galligan, J . J .; Burks, T. F. Bis-penicillamine enkephalins
possess highly improved specificity towards δ opioid receptors.
Proc. Natl. Acad. Sci. U.S.A. 1983, 80, 5871-5874.
(36) Kazmierski, W. M.; Yamamura, H. I.; Hruby, V. J . Topographic
design of peptide neurotransmitters and hormones on stable
backbone templates: relation of conformation and dynamics to
bioactivity. J . Am. Chem. Soc. 1991, 113, 2275-2283.
(37) Valle, G.; Kazmierski, W. M.; Crisma, M.; Bonora, G. M.; Toniolo,
C.; Hruby, V. J . Constrained phenylalanine analogues. Preferred
conformation of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid
(Tic) residue. Int. J . Pept. Protein Res. 1992, 40, 222-232.
(38) Wu¨thrich, K. In NMR of Proteins and Nucleic Acids; J . Wiley:
New York, 1986; pp 123-125.
(39) Ko¨ve´r, K. E.; J iao, D.; Fang, S.; Hruby, V. J . Conformational
analysis of four â-methylphenylalanine stereoisomers in
a
bioactive peptide by Z-filtered relay NMR spectroscopy. Magn.
Reson. Chem. 1993, 31, 1072-1076.
(40) Ko¨ver, K. E.; J iao, D.; Fang, S.; Hruby, V. J . Conformational
properties of the unnatural amino acid â-methylphenylalanine
in a linear octapeptide system; correlations of 13C-NMR chemical
shifts with the side chain stereochemistry of these amino acid
residues. J . Org. Chem. 1994, 59, 991-998.
(41) Carpenter, K. A.; Wilkes, B. C.; Weltrowska, G.; Schiller, P. W.
Role of hydrophobic substituents in the interaction of opioid Tyr-
Tic dipeptide analogs with dodecylphosphocholine micelles. Eur.
J . Biochem. 1996, 241, 756-764.
(42) Flippen-Anderson, J . L.; George, C.; Deschamps, J . R.; Reddy
P. A.; Lewin, A. H.; Brine, G. A. X-ray structures of the δ opioid
antagonist TIPP and a protected derivative of the δ opioid
antagonist ICI 174,864. Lett. Pept. Sci. 1994, 1, 107-115.
(43) Liao, S.; Lin, J .; Shenderovich, M. D.; Han, Y.; Hasohata, K.;
Davis, P.; Qiu, W.; Porreca, F.; Yamamura, H. I.; Hruby, V. J .
The stereochemical requirements of the novel δ-opioid selective
dipeptide antagonist TMT-Tic. Bioorg. Med. Chem. Lett. 1997,
7, 3049-3052.
(44) Ciajolo, M. R.; Balboni, G.; Picone, D.; Salvadori, S.; Tancredi,
T.; Temussi, P. A.; Tuzi, A. Solution and solid-state structure of
the diketopiperazine of tyrosyl-tetrahydroisoquinoline-3-car-
boxylic acid. Int. J . Peptide Protein Res. 1995, 46, 134-138.
(45) Schiller, P. W.; Nguyen, T. M.-D.; Weltrowska, G.; Wilkes, B.
C.; Marsden, B. J .; Schmidt, R.; Lemieux, C.; Chung, N. N. TIPP
Opioid peptides: development of extraordinarily potent and
selective δ antagonists and observation of astonishing structure-
intrinsic activity relationships. In Peptides: Chemistry, Structure
and Biology, Hodges, R. S., Smith, J . A., Eds.; ESCOM: Leiden,
1994; pp 483-486.
(46) Bryant, S. D.; Salvadori, S.; Cooper, P. S.; Lazarus, L. H. New
δ-opioid antagonists as pharmacological probes. Trends. Phar-
macol. Sci. 1998, 19, 42-46.
(47) Salvadori, S.; Balboni, G.; Guerrini, R.; Tomatis, R.; Bianchi,
C.; Bryant, S. D.; Cooper, P. S.; Lazarus, L. H. Evolution of the
Dmt-Tic pharmacophore: N-terminal methylated derivatives
with extraordinary δ opioid antagonist activity. J . Med. Chem.
1997, 40, 3100-3108.
(48) Kataoka, Y.; Seto, Y.; Yamamoto, M.; Yamado, T.; Kuwata, S.;
Watanabe, H. Studies of unusual amino acids and their peptides.
VI. The synthesis and the optical resolution of â-methylpheny-
lalanine and its dipeptide present in Bottromycin. Bull. Chem.
Soc. J pn. 1976, 49, 1081-1084.
(49) Gisin, B. F. The preparation of MerrifielD-resins through total
esterification with cesium salts. Helv. Chim. Acta 1973, 56 (143),
1476-1482.
(50) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Color
test for detection of free amino groups in the solid-phase
synthesis of peptides. Anal. Biochem. 1970, 34, 595-598.
(51) Christensen, T. A chloranil test for monitoring coupling com-
pleteness in solid-phase peptide synthesis. In Peptides: Structure
and Biological Function; Gross, E., Meienhofer, J ., Eds.; Pierce
Chemical Co.: Rockford, IL, 1979; pp 385-388.
(52) Leysen, J . E.; Gommeren, W.; Niemegeers, C. J . E. [3H]-
sufentanil, a superior ligand for µ-opiate receptors: binding
properties and regional distribution in rat brain and spinal cord.
Eur. J . Pharmacol. 1983, 87, 209-225.
(31) Mannekens, E.; Tourwe´, D.; Vanderstichele, S.; Nguyen Thi
Diem, T.; To´th, G.; Pe´ter, A.; Chung, N. N.; Schiller, P. W.
Synthesis of the diastereomers of â-Me-Tyr and â-Me-Phe and
their effect on the biological properties of the delta opioid
receptor antagonist TIPP. Lett. Pept. Sci. 1995, 2, 190-192.
(53) Akiyama, K.; Gee, K. W.; Mosberg, H. I.; Hruby, V. J .; Yama-
mura, H. I. Characterization of [3H][2-D-penicillamine, 5-D-
penicillamine]-enkephalin binding to delta opiate receptors in
rat brain and neuroblastoma-glioma hybrid cell line (NG 108-
15). Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 2543-2547.

5176 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 26
Tourwe´ et al.
(54) Lahti, R. A.; Mickelson, M. M.; McCall, J . M.; Voightlander, P.
F. [3H]U-69593: a highly selective ligand for the opioid-κ
receptor. Eur. J . Pharmacol. 1985, 109, 281-284.
(55) Paton, W. D. M. The action of morphine and related substances
on contraction and on acetylcholine output of coaxially stimu-
lated guinea-pig ileum. Br. J . Pharmacol. 1957, 12, 119-127.
(56) Henderson, G.; Hughes, J .; Kosterlitz, H. W. A new example of
a morphine sensitive neuroeffector junction. Br. J . Pharmacol.
1972, 46, 7164-7166.
(57) DiMaio, J .; Nguyen, T. M.-D.; Lemieux, C.; Schiller, P. W.
Synthesis and pharmacological characterization in vitro of cyclic
enkephalin analogues: effects of conformational constraints on
opiate receptor selectivity. J . Med. Chem. 1982, 25, 1432-1438.
(58) Schiller, P. W.; Lipton, A.; Horrobin, D. F.; Bodanszky, M.
Unsulfated C-terminal 7-peptide of cholecystokinin: a new ligand
of the opiate receptor. Biochem. Biophys. Res. Commun. 1978,
85, 1332-1338.
(59) Waterfield, A. A.; Leslie, F. M.; Lord, J . A. H.; Ling, N.;
Kosterlitz, H. W. Opioid activities of fragments of â-endorphin
and of its leucine65-analogue. Comparison of binding properties
of methionine- and leucine-enkephalin. Eur. J . Pharmacol. 1979,
58, 11-18.
(60) Schiller, P. W. Results to be published.
J M981011U