Further studies on the Dmt-Tic pharmacophore: Hydrophobic substituents at the C-terminus endow δ antagonists to manifest μ agonism or μ antagonism

Article abstract of DOI:10.1021/jm990165m

Twenty N- and/or C-modified Dmt-TiC analogues yielded similar K(i) values with either [³H]DPDPE (δ₁ agonist) or [³H]N,N(Me)₂-Dmt-Tic-OH (δ antagonist). N-Methylation enhanced δ antagonism while N-piperidine-1-yl

Full text of DOI:10.1021/jm990165m

5010
J . Med. Chem. 1999, 42, 5010-5019
F u r th er Stu d ies on th e Dm t-Tic P h a r m a cop h or e: Hyd r op h obic Su bstitu en ts a t
th e C-Ter m in u s En d ow δ An ta gon ists To Ma n ifest µ Agon ism or µ An ta gon ism
Severo Salvadori,† Remo Guerrini,† Gianfranco Balboni,† Clementina Bianchi,‡ Sharon D. Bryant,§
Peter S. Cooper,| and Lawrence H. Lazarus*,§
Department of Pharmaceutical Science and Biotechnology Center, University of Ferrara, I-441000 Ferrara, Italy,
Institute of Pharmacology, University of Ferrara, I-44100 Ferrara, Italy, Peptide Neurochemistry, LCBRA,
National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, and
National Center for Biotechnology Information, National Library of Medicine, Building 38A, 8600 Rockville Pike,
Bethesda, Maryland 20894
Received April 6, 1999
Twenty N- and/or C-modified Dmt-Tic analogues yielded similar Ki values with either [3H]-
DPDPE (δ1 agonist) or [3H]N,N(Me)2-Dmt-Tic-OH (δ antagonist). N-Methylation enhanced δ
antagonism while N-piperidine-1-yl, N-pyrrolidine-1-yl, and N-pyrrole-1-yl were detrimental.
Dmt-Tic-X (X ) -NHNH2, -NHCH3, -NH-1-adamantyl, -NH-tBu, -NH-5-tetrazolyl) had high
δ affinities (Ki ) 0.16 to 1 nM) with variable µ affinities to yield nonselective or weakly
µ-selective analogues. N,N-(Me)2Dmt-Tic-NH-1-adamantane exhibited dual δ and µ receptor
affinities (Kiδ ) 0.16 nM and Kiµ ) 1.12 nM) and potent δ antagonism (pA2 ) 9.06) with µ
agonism (IC50 ) 16 nM). H-Dmt-âHTic-OH (methylene bridge between CR of Tic and carboxylate
function) yielded a biostable peptide with high δ affinity (Ki ) 0.85 nM) and δ antagonism
(pA2 ) 8.85) without µ bioactivity. Dmt-Tic-Ala-X (X ) -NHCH3, -OCH3, -NH-1-adamantyl,
-NHtBu) exhibited high δ affinities (Ki ) 0.06 to 0.2 nM) and elevated µ affinities (Ki ) 2.5 to
11 nM), but only H-Dmt-Tic-Ala-NH-1-adamantane and H-Dmt-Tic-Ala-NHtBu yielded δ
receptor antagonism (pA2 ) 9.29 and 9.16, respectively). Thus, Dmt-Tic with hydrophobic
C-terminal substituents enhanced µ affinity to provide δ antagonists with dual receptor affinities
and bifunctional activity.
In tr od u ction 1
and acted systemically in vivo to reverse antinociception
by a δ agonist.6 Further enhancement of the hydropho-
bic properties of H-Dmt-Tic-OH through N-alkylation
by methyl groups not only retained high δ affinity and
δ receptor selectivity but also substantially enhanced
bioactivity (Ke ) 0.12 nM);7,8 other N-alkylating agents
affected Kiδ and decreased δ selectivity.9 In toto, those
observations demonstrated that the hydrophobicity
imparted by 2′,6′-dimethylation of Tyr and N-alkylation
was a substantial factor that influenced the interaction
between the Dmt-Tic pharmacophore and the δ-opioid
receptor ligand-binding domain in a tissue preparation.
Amidation of TIP(P),2 H-Tyr-Tic-OH,3 and H-Dmt-Tic-
OH5,7 explored the effect of charge on δ-receptor affinity.
Suppression of the anionic function led to elevated µ
receptor binding, a phenomenon also observed with the
deltorphins (δ-opioid agonists).10,11 Moreover, C-termi-
nal amidation with a change of configuration to D-Tic
produced weakly µ-selective compounds,2,5 suggesting
that discriminative modifications at the C-terminus
might result in opioids with new properties.
The development of δ-opioid antagonists were an
outgrowth of studies on truncated opioid peptides with
enhanced hydrophobic properties: H-Tyr-Tic-Phe (Phe)
[TIP(P)] and its reduced bond analogues, TIP(P)[ψ],
exhibited greater biological potency than non-peptide
opiate antagonists.2 Removal of the Phe residues in
those peptides engendered H-Tyr-Tic-OH and H-Tyr-
Tic-Ala-OH, which were the first opioid peptides without
Phe that had (albeit weak) δ-opioid selectivity and
antagonist bioactivity.3 In addition to the aromatic side
chain at the third position in Tyr-Tic-Phe peptides,2
compounds containing a residue with an aliphatic side
chain in Tyr-Tic-Xaa analogues also augmented δ recep-
tor binding.3 Studies with 2′,6′-dimethyl-L-tyrosyl-N-(3-
phenylpropyl)-D-alanine amide4 demonstrated the fea-
sibility of increasing the hydrophobicity by methylation
of the phenolic side chain of Tyr while maintaining
biological activity of the peptide. In fact, substitution
of Tyr in H-Tyr-Tic-OH by Dmt created the dipeptide
antagonist H-Dmt-Tic-OH which had exceptionally high
δ affinity (Kiδ ) 0.02 nM), ultraselectivity (Kiµ/Kiδ
)150 000), in vitro δ antagonist activity (Ke ) 5.7 nM),5
Recent data on the knock-out of the µ-opioid receptor
in mice confirmed that the µ receptor is primarily
involved in the appearance of analgesia induced by
morphine.12 However, it should be noted that δ receptor
agonists act as nonaddicting analgesic drugs,13 which
produce an antinociceptive response without the ap-
pearance of cross-tolerance to µ- or δ-opioid receptor
agonists14 and can lead to antinociception in the absence
of the µ receptor.15 The unique property of the δ receptor
permitted the application of moderately δ-selective
* To whom correspondence should be addressed: L. H. Lazarus,
Peptide Neurochemistry, MD C3-04, NIEHS, P.O. Box 12233, Research
Triangle Park, NC 27709, USA. Fax: +1-919-541-0696. E-mail:
lazarus@niehs.nih.gov.
† Department of Pharmaceutical Science and Biotechnology Center,
University of Ferrara.
‡ Institute of Pharmacology, University of Ferrara.
§ National Institute of Environmental Health Sciences.
| National Library of Medicine.
10.1021/jm990165m CCC: $18.00 © 1999 American Chemical Society
Published on Web 11/10/1999

Dmt-Tic Pharmacophore Studies
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24 5011
Interposing a methylene spacer (1) between the CR
of the Tic residue and the carboxylate function (Figure
1) to prevent diketopiperazine formation had minimal
effect on δ affinity, but it enhanced µ affinity (Table 1).
The same chemical approach employing a methylene
group between the amino group and CR of Dmt (2) was
more detrimental [compared to H-R(R,S)HDmt-Tic-OH].
The dipeptide analogues containing hydrazide (6), meth-
yl amide (7), and tetrazole-5-yl (10) exhibited high δ
affinities, but each with a marked gain in µ affinity,
which was also observed with the Ala-containing tri-
peptide methyl ester (18) relative to its title compound
(H-Dmt-Tic-Ala-NH2).
The largest increase in µ affinity occurred in the Dmt-
Tic analogues C-terminally substituted with either tert-
butyl amide (9 and 14) or 1-adamantyl amide (11, 12,
15, 16, and 20). In comparison to H-Dmt-Tic-OH, the µ
affinities of compounds 11 and 12 increased 4350- and
12 600-fold, respectively, while the µ affinity of peptide
15 rose nearly 2200-fold relative to N,N(Me)2-Dmt-Tic-
OH. Comparisons between the amidated parental pep-
tides to their C-terminal derivatives, however, indicated
smaller changes in µ affinities (Table 1).
F igu r e 1. Dmt-Tic pharmacophore and the R1 and R2 sub-
stituents. The bold numbers refer to the analogues in Table
1. Asterisks denote chiral centers.
alkaloid antagonists in clinical trials; for example, the
amelioration of the effects of alcoholism,16 autism,17 and
Tourette’s syndrome.18 Using animal models, the δ-opi-
ate antagonist naltrindole19 inhibited the reinforcing
properties of cocaine,20 moderated the behavioral effects
of amphetamines,21 and brought about immunosuppres-
sion22 suitable for organ transplantation.23 These trans-
plantation effects were also seen using another non-
peptide compound, 7-(benzylspiroindanyl)naltrexone.24
N-Alkylation of Dmt-Tic with piperidine-1-yl (3),
pyrrolidine-1-yl (4), or pyrrole-1-yl groups (5) (Figure
1) decreased δ affinity, particularly the latter compound
whose receptor binding was comparable to H-RDmt-Tic-
OH (2), H-Dmt-Tic-OMe (8), and H-Dmt-D-Tic-NH-1-
adamantane (12). Despite the bulky N-terminal sub-
stituents, the δ selectivities of 3 and 4 were analogous
to other modified peptides (Table 1).
Ra tion a le
The intractable membrane barriers, such as the blood-
brain barrier, must be circumvented in order for peptide
antagonists to express activity in vivo.25 The requisite
physicochemical properties of compounds capable of
passing through this barrier include low molecular
weight (<800 Da) and high octanol-water coefficient
characteristics. Although systemically injected H-Dmt-
Tic-OH was able to antagonize the analgesia of an icv
administered δ2 agonist (deltorphin B),6 the augmenta-
tion of the hydrophobic properties of [D-Ala2,Leu5]-
enkephalin with 1-adamantyl amide also permitted
passage through the blood-brain barrier.26 Thus, we
sought to enhance the hydrophobic properties of H-Dmt-
Tic-OH by altering the C-terminal function either with
or without substitution at the N-terminus (Figure 1) in
order to produce more potent δ-opioid antagonists.27
Recep tor Bin d in g. Com p etition a ga in st a High ly
Selective δ-Op ioid An ta gon ist. The receptor binding
using the δ antagonist [3H]N,N-(Me)2-Dmt-Tic-OH
yielded similar Ki values to those obtained with the
agonist [3H]DPDPE in over 80% of the peptides listed
in Table 1. Exceptions (peptides whose Ki values differed
by at least an order of magnitude) were the title peptide
H-Dmt-Tic-OH, which is relatively unstable and forms
a diketopiperazine,29,30 and analogues 2, 6, 8, and 10.
F u n ction a l Bioa ctivity. Dmt-Tic analogues 1, 9, 13,
15, 19, and 20 demonstrated the highest δ antagonist
functional bioactivities (Table 2) as well as some of the
highest δ receptor affinities (Table 1). Of greater inter-
est, however, was the observation that several ana-
logues acquired unusual bioactivity profiles. For ex-
ample, both compounds 13 and 15 elicited excellent δ
antagonism, yet the former was a weak µ antagonist
and the latter clearly displayed µ agonism (Table 2).
Inclusion of the D-Tic enantiomer (12 and 16) greatly
reduced bioactivity on MVD with micromolar activity
on GPI. Replacement of the N-terminal amine through
alkylation by piperidine-1-yl (3), pyrrolidine-1-yl (4), or
pyrrole-1-yl (5) was detrimental for all bioactivity
measurements on MVD (Table 2). N-Alkylation by
methyl groups to form secondary or tertiary amines was
the only N-terminal substitution tolerated9 (Table 2).
Interestingly, the nonalkylated, C-terminally modified
dipeptides 11 and 12 lacked δ antagonism; however, 11
surprisingly manifested a weak δ agonism and moderate
µ agonism despite its high δ affinity (Table 1) while
Resu lts
Recep tor Bin d in g. P r op er ties of N- a n d C-Ter -
m in a lly Mod ified An a logu es Usin g a δ1 Recep tor
Agon ist. Elimination of the carboxylate function of
H-Dmt-Tic-OH substantially increased the µ affinity of
di- and tripeptide derivatives (6-20) (Figure 1 and
Table 1). The net effect was the loss of δ-opioid selectiv-
ity and the appearance of compounds that were either
essentially nonselective (6, 9, 10, and 15), weakly
µ-selective (8, 11, and 16), or moderately µ-selective (12).
The δ affinity generally remained high for most C-
terminally modified analogues, with Ki values ranging
from 0.07 to 1 nM (Table 1). Nonetheless, the binding
data revealed that several analogues lost high δ affinity,
in particular the methyl ester derivative (8) and those
containing the D-Tic enantiomer (12 and 16) as seen
previously with amidated analogues of TIP(P)2 or Dmt-
Tic.5,7

5012 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24
Salvadori et al.
Ta ble 1. Membrane Receptor Binding of the Modified Dmt-Tic Pharmacophore
Kiδ (nM)
[3H]N,N(CH3)2-
Dmt-Tic-OHa
[3H]DAGO Kiµ
agonist
Kiµ/Kiδ
antagonist
Kiµ/Kiδ
no.
peptide
[3H]DPDPEa
(nM)a
Parental Compounds
0.022 ( 0.002 (6) 0.34 ( 0.05 (4)
H-Dmt-Tic-OH
H-Dmt-Tic-NH2
H-Dmt-Tic-Ala-OH
H-Dmt-Tic-Ala-NH2
N,N(Me)2-Dmt-Tic-OH
H-(R,S)Dmt-Tic-OH
3320 ( 440 (7)
277 ( 26 (3)
813 ( (4)
151 000b
227b
9 850
130
2 800
68
34 800
1 450
1.22 ( 0.09 (6)
0.29 ( 0.03 (6)
0.24 ( 0.02 (5)
0.12 ( 0.02 (3)
0.46 ( 0.001 (3)
2.13 ( 0.12 (3)
0.29 ( 0.05 (3)
0.69 ( 0.13 (3)
0.07 ( 0.01 (4)
0.80 ( 0.19 (4)
2 800b
195b
47 ( 3.4 (4)
2440 ( 460 (3)
1160 ( 330 (3)
20 300c
2 520
Dipeptide Derivatives
1
2
3
4
5
6
7
8
H-Dmt-âHTic-OH
0.85 ( 0.20 (5)
11.2 ( 3.5 (3)
1.18 ( 0.10 (3)
1.62 ( 0.19 (3)
16.6 ( 2.5 (5)
0.99 ( 0.04 (3)
0.47 ( 0.09 (3)
9.64 ( 2.2 (3)
0.43 ( 0.07 (5)
0.70 ( 0.03 (3)
0.26 ( 0.05 (4)
24.5 ( 4.9 (6)
0.54 ( 0.07 (3)
0.61 ( 0.02 (3)
0.16 ( 0.02 (3)
140 ( 30 (6)
0.71 ( 0.04 (3)
418 ( 86 (3)
1740 ( 16 (3)
2040 ( 260 (3)
814 ( 65 (3)
5590 ( 410 (3)
85.1 ( 7.3 (3)
85.5 ( 7.7 (3)
423 ( 25 (3)
5.96 ( 0.82 (4)
37.0 ( 4.5 (3)
0.76 ( 0.05 (4)
0.26 ( 0.08 (4)
359 ( 62 (3)
498
155
1 730
502
338
86
182
44
14
53
3
0.01
669
369
7
590
4
2 760
573
562
2
69
0.8
6
H-R(R,S)HDmt-Tic-OH
475 ( 37 (4)
0.74 ( 0.30 (3)
1.42 ( 0.14 (3)
9.94 ( 2.5 (3)
42.0 ( 7.9 (5)
1.24 ( 0.15 (4)
500 ( 90 (5)
[des-NH2-R-piperidine-1-yl]-Dmt-Tic-OH
[des-NH2-R-pyrrolidine-1-yl]-Dmt-Tic-OH
[des-NH2-R-pyrrole-1-yl]- Dmt-Tic-OH
H-Dmt-Tic-NHNH2
H-Dmt-Tic-NHMe
H-Dmt-Tic-OMe
9
H-Dmt-Tic-NH-tBu
0.93 ( 0.15 (4)
9.75 ( 1.73 (5)
1.01 ( 0.26 (3)
70.3 ( 7.25 (3)
0.28 ( 0.02 (3)
0.11 ( 0.04 (3)
0.12 ( 0.02 (3)
120 ( 26 (3)
10
11
12
13
14
15
16
H-Dmt-Tic-NH-tetrazole-5-yl
H-Dmt-Tic-NH-1-adamantane
H-Dmt-D-Tic-NH-1-adamantane
N,N(Me)2-Dmt-Tic-NHMe
N,N(Me)2-Dmt-Tic-NH-tBu
N,N(Me)2-Dmt-Tic-NH-1-adamantane
N,N(Me)2-Dmt-D-Tic-NH-1-adamantane
4
0.8
0.004
1270
2130
9
226 ( 21 (3)
1.12 ( 0.10 (3)
50.5 ( 2.6 (3)
0.36
0.42
Tripeptide Derivatives
17
18
19
20
H-Dmt-Tic-Ala-NHMe
H-Dmt-Tic-Ala-OMe
H-Dmt-Tic-Ala-NH-tBu
H-Dmt-Tic-Ala-NH-1-adamantane
0.058 ( 0.01 (3)
0.23 ( 0.09 (3)
0.066 ( 0.01 (3)
0.073 ( 0.02 (3)
0.12 ( 0.04 (3)
5.75 ( 0.72 (3)
11.3 ( 1.87 (3)
4.03 ( 0.21 (3)
2.52 ( 0.56 (4)
100
48
61
47
83
48
72
0.14 ( 0.04 (3)
0.08 ( 0.02 (3)
0.04 ( 0.01 (3)
35
a
The numeric value in the parentheses indicates the number (n) of repetitions of independent binding assays using different synaptosomal
preparations. Salvadori et al. (1995) (ref 5). c Salvadori et al. (1997) (ref 7).
b
Ta ble 2. Functional Bioactivity of the Modified Dmt-Tic Pharmacophore
MVD
GPI
Ke (µM)
no.
peptide
pA2 (range)a
8.2
7.2
8.4
8.0
9.4
Ke (nM)
ED50 (µM)
pA2 (range)
ED50 (µM)
Dmt-Tic-OH
Dmt-Tic-NH2
Dmt-Tic-Ala-OH
5.7
42
4.0
9.0
0.28
6.76
1.41
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
>10b
>10b
>10b
Dmt-Tic-Ala-NH2
N,N(Me)2-Dmt-Tic-OH
H-(R,S)Dmt-Tic-OH
4.74 ( 0.9
>10c
8.17 (7.8-8.6)
>10
1
3
4
5
7
9
H-Dmt-âHTic-OH
8.8 (8.6-9.1)
>10
[des-NH2-R-piperidine-1-yl]-Dmt-Tic-OH
7.31 (7.15-7.47) 49
>10
[des-NH2-R-pyrrolidine-1-yl]-Dmt-Tic-OH 6.9 (6.9-7.1)
121
408
-
-
>10
[des-NH2-R-pyrrole-1-yl]-Dmt-Tic-OH
H-Dmt-Tic-NHMe
6.39 (6.0-6.7)
>10
e
-
>10 000 21.2 (9.0-45)
1.74
36.3
5.94 (4.2-7.6)
-
-
-
1.15
-
-
-
>10
H-Dmt-Tic-NH-tBu
8.24 (8.2-8.5)
7.44 (7.1-7.7)
-
-
-
1.04 (0.69-1.5)
8.2 (2.7-24.9)
0.036 (0.019-0.068)
1.68 (1.23-2.3)
>10
10 H-Dmt-Tic-NH-tetrazole-5-yl
11 H-Dmt-Tic-NH-1-adamantane
12 H-Dmt-D-Tic-NH-1-adamantane
13 N,N(Me)2-Dmt-Tic-NHMe
14 N,N(Me)2-Dmt-Tic-NH-tBu
15 N,N(Me)2-Dmt-Tic-NH-1-adamantane
16 N,N(Me)2-Dmt-D-Tic-NH-1-adamantane
17 H-Dmt-Tic-Ala-NHMe
>10 000 0.87 (0.8-1.2)
-
>10 000
0.41
14.1
0.87
128
-
-
-
-
-
-
9.39 (8.8-9.9)
7.85 (7.5-8.1)
9.06 (8.6-9.5)
6.91 (6.8-7.0)
-
9.16 (8.7-9.5)
9.29 (9.0-9.5)
6.41 (6.3-6.5)
6.52 (6.2-6.7)
-
-
-
0.39
0.30
-
-
>10
0.016 (0.011-0.023)
4.48 (3.17-6.32)
0.29 (0.18-0.45)
>10
-
-
1.29 (1.18-1.42)
-
-
-
19 H-Dmt-Tic-Ala-NH-tBu
20 H-Dmt-Tic-Ala-NH-1-adamantane
0.69
0.51
6.76 (6.6-6.9)
-
0.17
-
1.0 (0.7-3.0)
a
b
d
pA2 is the mean, and the range is in parentheses. Salvadori et al. (1995) (ref 5). c Salvadori et al. (1997) (ref 7). Temussi et al.
(1994) (ref 3a). e A dash indicates the absence of pharmacological activity. Data were derived from at least four independent tissue samples
and dose-response curves.
other analogues (7, 13, 14, and 17) also produced
anomalous biological activities on MVD and GPI (Table
2) relative to their receptor binding parameters (Table
1). H-Dmt-Tic-NHMe (7) had extraordinarily weak δ
agonism with low µ antagonism, while its Ala-tripeptide
derivative (17) gave very weak δ and µ agonism. Despite
the high δ receptor affinity of compound 14, the bioac-
tivity data indicated only modest δ antagonism and
weak µ antagonism. The tetrazole-5-yl-amide analogue
(10) was a weak δ antagonist with very minimal activity
on GPI (Table 2).
Discu ssion
Our results support and extend the observations that
the Dmt-Tic pharmacophore represents a distinct class
of δ-opioid antagonists.5,7-9 The correlation between

Dmt-Tic Pharmacophore Studies
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24 5013
competitive binding studies using a δ1 agonist (DPDPE)
and a δ antagonist [N,N(Me)2-Dmt-Tic-OH] in 21 of 26
analogues appears to support the existence of a single
binding site for agonists and antagonists in the ligand-
binding domain of δ receptors. Further, the message
domain of these peptides probably presents a similar
conformation in order to fit the receptor cavity. [A series
of nine other Dmt-containing di- and tripeptides with
Tic or other heterocyclic residues at position 2 revealed
no discrepancy in the binding between these two labeled
ligands (Salvadori et al., unpublished data).] The mini-
mum size of that message domain constitutes the
dimensions of a dipeptide3,7 which has a specific spatial
geometry in solution8,28 as seen in the crystallographic
evidence for TIPP analogues34 and N,N(Me)2-Dmt-Tic-
OH (Flippen-Anderson, J .; George, G., unpublished
observations). Of course, until such time that a mem-
brane receptor has been crystallized containing a bound
agonist or antagonist, we are unable to absolutely verify
the nature of the interaction between these two types
of ligands within the δ receptor. However, the fact that
the δ receptor embedded in a membrane vesicle can be
equally labeled with either [3H]DPDPE or [3H]N,N(Me)2-
Dmt-Tic-OH and whose binding can be reversed by
graded dosages of unlabeled peptide at equilibrium
conditions constitutes the basis of the radioreceptor
assay. In contrast to ideal or hypothetical constructs,
experimental data can often reveal discrepancies. Once
such discrepancy was observed between the δ binding
affinities of H-Dmt-Tic-OH and peptide 8 in competition
against both labeled compounds that could be attributed
to the well-documented rapid formation of a diketopip-
erazine.29 Unfortunately, the assay of H-Dmt-Tic-OH
immediately after solubilization demonstrated a rapid,
time-dependent decline in δ binding, and even newly
synthesized preparations revealed an instability that
most likely led to the recorded incongruity between
assays that were carried out weeks apart. The intrinsic
δ binding capacity of cyclo(Dmt-Tic) (Ki ca. 10 nM) is
considerably lower than the linear (noncyclic) peptides,30
and increased levels of the diketopiperazine in the
sample would inherently decrease the observed values
for receptor affinity. The theoretical interpretation of
this observation was in fact substantiated by the
enhanced stability of peptide 1 (methylene bridge at CR
of Tic interposed before the carboxylate function; Figure
1) preventing the formation of the diketopiperazine to
yield binding data equivalent to that of the parent
peptide. An interesting observation relative to peptide
1 is the higher selectivity of H-Dmt-Tic-Ala-OH (paren-
tal peptide) that might indicate either a role for the
aliphatic side chain of Ala in ligand-receptor binding
mechanisms or in the modification of the solution
conformation of the peptide.31
nists.28a,b Furthermore, the spectrum of activity exhib-
ited by the Tyr-Tic cognates of 8, 17, and 18 differed
from the Dmt-Tic peptides.9,32 For example, H-Tyr-Tic-
Ala-OMe and H-Tyr-Tic-NH-1-adamantyl amide9,32
greatly exceeded the δ-receptor binding properties of
other Tyr-Tic di- and tripeptides3,5,9,32 except for the
TIP(P) series,2 suggesting that the C-terminal address
portion of the peptide can influence the message do-
main. In the peptides described herein, Dmt continued
to enhance δ binding and maintain bioactivity in some
compounds, even in the presence of the bulky C-
terminal substituents. The loss of δ affinity and MVD
bioactivity in 12 and 16 is comparable to the effect with
other D-Tic-containing peptides,2,5,7 implicating the
importance of the spatial orientation of Tic on the
interaction between aromatic rings in the peptide8 and
between peptide and receptor.
Enhancement of µ receptor binding occurred due to
the hydrophobicity of the substituents at the C-terminus
of the Dmt-Tic pharmacophore. Compounds containing
C-terminal tert-butyl amide and 1-adamantyl amide
were δ antagonists with high dual binding affinities and
biological activity toward both δ and µ receptor types
(Tables 1 and 2). 1-Adamantyl amide, with its high
hydrophobic constant, high steric constant, and van der
Waals volume,33 affects peptide activity and as a
consequence elevated µ affinity to yield nonselective
analogues (Table 1). Similarly, the elimination of the
negative charge in δ agonists10,11 or δ antagonists either
by amidation2,5 or reduction of the acid to an alcohol5
also augmented binding to µ receptors. Therefore, the
heightened activity of these analogues toward µ recep-
tors was not unexpected since the negatively charged
carboxylate anion accounts for δ selectivity due to
repulsion from the µ receptor.5,10,11,38 However, the
methyl amide moiety (7 and 17) appears to impart a
negative effect and an anomalous bioactive behavior to
the peptides. Thus, while high-affinity binding to µ
receptors increased in peptides devoid of a carboxylate
anion and which are physically larger than the Dmt-
Tic dipeptide, the di- or tripeptide analogues containing
either 1-adamantyl amide (11, 12, 15, and 20) or tert-
butyl amide (9, 14, and 19) also interacted effectively
with the δ receptor. Considering that both N,N(Me)2-
Dmt-Tic-NH-1-adamantane (15) and H-Dmt-Tic-Ala-
NH-1-adamantane (20) exhibited exceptional antagonist
activity on MVD and only 15 displayed µ agonism
demonstrated that different biological activities can
occur in the same, small opioid peptide, somewhat
analogous to that found with the non-peptide opiate
naltrindole.19 Furthermore, these two analogues disclose
the subtle interplay between the N- and C-termini in
the elicitation of bioactivty.
Earlier studies revealed that increased hydrophobicity
by N-methylation generally enhanced the biological
properties of the peptide analogue without significant
change in receptor affinity.7,9 For example, δ antagonism
of N,N(Me)2-Dmt-Tic-OH increased 20-fold (Ke ) 0.28
nM) while retaining high δ-receptor affinity (Ki ) 0.12
nM) and selectivity (Kiµ/Kiδ ) 20 600).7 On the other
hand, N-alkylation by piperidine-1-yl (3), pyrrolidine-
1-yl (4), and especially pyrrole-1-yl (5) led to substantial
losses in δ-receptor binding and bioactivity. Several
possible explanations can be attributed to this effect,
Since peptides 2, 6, and 10 have not been studied for
either diketopiperazine formation or other possible
intermediates, it would be inappropriate and premature
for us to speculate on their observed discrepancy.
The differences observed between the δ-opioid recep-
tor binding of Dmt-Tic peptides and their Tyr-Tic
cognates5,9,32 indicated that Dmt assumes a predomi-
nant role in the alignment or anchoring of the peptide
within δ-, µ-, and κ-opioid receptor binding sites8,28 or
affecting solution conformation of the dipeptide antago-

5014 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24
Salvadori et al.
dimethylphenyl)-propanoic acid was prepared from ethyl
cyanoacetate and O-carbethoxy-3,5-dimethyl-4-chlorometh-
ylphenol.41
such as the loss of the protonated nitrogen in compound
5 (comparable to that observed with the diketopipera-
zine derivative30), the derivatives exceeded the correct
spatial volume in the receptor pocket, an inability to
form nonionic bonds with side chains of residues in the
receptor, or a misalignment of Dmt that prevented
optimal orientation of the crucial hydroxyl group on the
phenol side chain. Thus, in support of other data,7,9
N-alkylation of Dmt-Tic was less favorably tolerated
than the covalent addition of a third amino acid (Ala)
or presence of bulky hydrophobic groups at the C-
terminus. This observation fits with the common mes-
sage domain discovered between deltorphins and der-
morphins, opioid peptides with bioactivities for δ and µ
receptors, respectively.11,27
Preparative reversed-phase HPLC was conducted with a
Waters Delta Prep 3000 Å (30 × 3 cm; 15 µm) column. Peptides
were eluted with a gradient of 0-60% B in 25 min at a flow
rate of 50 mL/min using the following mobile phases: solvent
A (10% acetonitrile in 0.1% TFA, v/v) and solvent B (60%
acetonitrile in 0.1% TFA, v/v). Analytical HPLC analyses were
carried out with a Bruker liquid chromatography LC 21-C
instrument using a Vydac 218 TP 5415 C18 column (250 ×
4.6 mm, 5 µm particle size) and equipped with a Bruker LC
313 UV variable wavelength detector. Recording and quanti-
fication were accomplished with
a chromatographic data
processor coupled to an Epson computer system (QX-10).
Analytical determinations were determined using HPLC con-
ditions in the above solvent systems programmed at a flow
rate of 1 mL/min in a linear gradient from 0 to 100% B in 25
min. All analogues showed less than 1% impurities when
monitored at 220 nm.
Con clu sion s
Our data provide additional evidence that δ-opioid
agonists and antagonists interact within the same
ligand-binding domain in opioid receptors and that
hydrophobic substituents at the C-terminus of the Dmt-
Tic pharmacophore augment µ-opioid receptor affinity.
The consequence of this alteration in some analogues
resulted in the formation of nonselective or µ-selective
analogues. Two analogues containing 1-adamantyl amide
(15 and 20) exhibited very high δ receptor binding and
potent δ antagonism with a MVD bioassay, and the
former was a µ-opioid agonist as well. Thus, a single
opioid peptide can possess antagonist and agonist
activity toward different receptor types through the
modification of the C-terminus; the dichotomy in the
unusual activity of this chimeric opioid molecule must
be related to the distinct physicochemical properties
imparted by 1-adamantyl amide.33 Considering the
effectiveness of non-peptide opiates in transplantation
studies,23,24 these biologically stable, dual activity opi-
oids should also be exploited for their potential phar-
macological and clinical effectiveness.9
TLC was performed on precoated plates of silica gel F254
(Merck, Darmstadt, Germany) using the following solvent
systems: (A) 1-butanol/AcOH/H2O (3:1:1, v/v/v) and (B) CH2-
Cl2/toluene/methanol (17:1:2, v/v/v). Ninhydrin (1%, Merck),
fluorescamine (Hoffman-La Roche) and chlorine reagents were
used as sprays. Open column chromatography (2 × 70 cm,
0.7-1 g material) was run on silica gel 60 (70-230 mesh,
Merck) using the same eluent systems.
Melting points were determined on a Kofler apparatus and
are uncorrected. Optical rotations were determined at 10 mg/
mL in methanol with a Perkin-Elmer 241 polarimeter with a
1
10 cm water-jacketed cell. All H NMR spectra were recorded
on a Bruker 200 MHZ spectrometer. MALDI-TOF analyses
(matrix-assisted laser desorption ionization time-of-flight mass
spectrometry) of peptides were conducted using a Hewlett-
Packard G 2025 A LD-TOF system. The samples were ana-
lyzed in the linear mode with 28 kV accelerating voltage,
mixing them with a saturated solution of R-cyano-4-hydroxy-
cinnamic acid matrix.
Boc-Dm t-Tic-OMe. To a solution of Boc-Dmt-OH (0.4 g,
1.29 mmol) and H-Tic-OMe42 (0.25 g, 1.29 mmol) in DMF (10
mL) at 0 °C were added HOBt (0.22 g, 1.42 mmol) and DCC
(0.29 g, 1.42 mmol). The reaction was stirred for 3 h at 0 °C
and 24 h at room temperature. After evaporation of DMF, the
residue was solubilized in EtOAc and washed with citric acid
(10%), NaHCO3 (5%), and brine. The organic phase was dried
and evaporated to dryness. The residue was crystallized from
Et2O/Pe (1:1, v/v): yield 0.52 g (83%); Rf (B) 0.81; HPLC K′ )
7.91; mp 135-137 °C; [R]20D -13.2; MH+ 483; 1H NMR (DMSO)
δ ) 1.37-1.46 (d, 9H), 2.16 (s, 6H), 2.96-3.01 (m, 2H), 3.08-
3.13 (m, 2H), 3.46-3.56 (m, 1H), 3.72 (s, 3H), 4.34-4.88 (m,
3H), 6.46 (s, 2H), 7.18-7.21 (m, 4H), 8.32 (bs, 1H).
Exp er im en ta l Section
Ma ter ia ls. H-Dmt-OH was synthesized as reported and
compared to a sample generously supplied by Dygos et al.37
Boc-Tic-OH was obtained from Bachem Feinchemikalien AG.
[3H]DPDPE (32.0 Ci/mol) was a product of NEN-DuPont
(Bilirica, MA), and [3H]DAGO (58.0 Ci/mmol) was obtained
from Amersham (Arlington Heights, IL).
Boc-Dm t-Tic-NHNH2. To a solution of Boc-Dmt-Tic-OMe
(0.26 g, 0.54 mmol) in MeOH (10 mL) was added NH2NH2‚
H2O (1 mL). The reaction mixture was stirred for 24 h at room
temperature. After evaporation of the solvent, the residue was
crystallized from Et2O/Pe (1:1, v/v): yield 0.25 g (94%); Rf (B)
0.75; HPLC K′ ) 6.83; mp 154-156 °C; [R]20D -14.5; MH+ 483;
1H NMR (DMSO) δ ) 1.35-1.44 (d, 9H), 2.16 (s, 6H), 3.07-
3.40 (s, 4H), 3.79 (m, 1H), 4.29-4.78 (m, 5H), 6.34 (m, 2H),
6.95 (bs, 1H), 7.14 (3, 4H), 8.22 (bs, 1H).
P ep tid e Syn th esis. All peptides were prepared by stan-
dard solution methods.38 Dipeptides were obtained by conden-
sation of Boc-Dmt-OH with Tic derivatives (H-Tic-OtBu, H-Tic-
NHMe, H-Tic-NH-1-adamantane, and H-âHTic-OMe) or by
condensation of Boc-Dmt-Tic-OH with tert-butylamine or
5-aminotetrazolyl via DCC/HOBt. Tripeptides were obtained
by condensation of Boc-L-Dmt-Tic-OH with Ala derivatives (H-
Ala-NHMe, H-Ala-NH-1-adamantane, H-Ala-NH-tBu, and H-
Ala-OMe) via DCC/HOBt. Final products were obtained, when
necessary, by ester function hydrolysis with 1 N NaOH and
removal of the Boc protecting group in TFA. N-Alkylated di-
and tripeptide derivatives were obtained by reductive alkyla-
tion of the corresponding deprotected linear peptides with
aldehydes (formaldehyde, glutaraldehyde, succinaldehyde) and
NaBH3CN in acetonitrile.39 HCl‚Pt-âHTic-OMe was prepared
by reduction of H-Tic-OMe with LiAlH4 to the corresponding
3-hydroxymethyl-Tic that was transformed first in 3-bromo-
methyl-Tic and then in 3-cyano-Tic. Treatment of the cyano
group with HCl-methanol gave the final product.40 H-R(R,S)-
HDmt-Tic-OH was obtained by condensation of (R,S)-2-cyano-
3-(4-hydroxy-2,6-dimethylphenyl)-propanoic acid with H-Tic-
OtBu via DCC/HOBt. In turn, (R,S)-2-cyano-3-(4-hydroxy-2,6-
TF A‚H-Dm t-Tic-NHNH2 (6). Boc-Dmt-Tic-NHNH2 (0.2 g,
0.41 mmol) was treated with TFA (1 mL) for 0.5 h at room
temperature. Et2O/Pe (1:1, v/v) were added to the solution until
the product precipitated: yield 0.18 g (91%); Rf (A) 0.69; HPLC
K′ ) 2.52; mp 143-145 oC; [R]20 +13.5; MH+ 382. Anal.
D
(C21H26N4O3‚TFA) C, H, N.
Z-Tic-NHMe. To a solution of Z-Tic-OH41 (1 g, 3.21 mmol)
and HCl‚H2NMe (0.22 g, 3.21 mmol) in DMF (10 mL) at 0 °C
were added NMM (0.35 mL, 3.21 mmol), HOBt (0.54 g, 3.53
mmol), and DCC (0.73 g, 3.53 mmol). The reaction mixture
was stirred for 3 h at 0 °C and 24 h at room temperature. After
evaporation of DMF, the residue was solubilized in EtOAC and
washed with citric acid (10%), NaHCO3 (5%), and brine. The

Dmt-Tic Pharmacophore Studies
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24 5015
organic phase was dried and evaporated to dryness. The
residue was crystallized from Et2O/Pe (1:1, v/v): yield 0.90 g
3.46-3.56 (m, 1H), 4.34-4.88 (m, 3H), 6.46 (s, 2H), 7.18-7.21
(m, 4H), 8.32 (bs, 1H), 8.45 (bs, 1H), 14.39 (bs, 1H).
(87%); Rf (B) 0.97; HPLC K′ ) 8.56; mp 137-139 °C; [R]20
D
TF A‚H -Dm t -Tic-NH -t et r a zole-5-yl (10). Boc-Dmt-Tic-
NH-tetrazole-5-yl was treated with TFA as reported for TFA‚
+13.5; MH+ 325; 1H NMR (DMSO) δ ) 2.44-2.46 (d, 3H),
3.12-3.24 (m, 2H), 4.36-4.46 (m, 3H), 5.17 (s, 2H), 7.18-7.21
(m, 5H), 7.36-7.39 (m, 4H), 7.74-7.77 (m, 1H).
H-Dmt-Tic-NHNH2 (6): yield 0.17 g (87%); Rf (A) 0.79; HPLC
1
K′ ) 3.75; mp 131-133 °C; [R]20 -18.3; MH+ 436; H NMR
D
H-Tic-NHMe. To a solution of Z-Tic-NHMe (0.90 g, 2.78
mmol) in MeOH (30 mL) was added C/Pd (5%, 0.05 g), and H2
was bubbled for 1 h at room temperature. After filtration, the
solution was evaporated to dryness. The residue was crystal-
lized form Et2O/Pe (1:1, v/v): yield 0.49 g (92%); Rf (B) 0.38;
(DMSO) δ ) 2.18 (s, 6H), 2.81-3.03 (m, 2H), 3.12-3.18 (m,
2H), 3.66-3.76 (m, 1H), 4.38-4.52 (m, 3H), 6.45 (s, 2H), 7.21-
7.25 (m, 4H), 8.27 (bs, 3H), 8.9 (bs, 3H), 14.39 (bs, 1H). Anal.
(C22H25N7O3‚TFA) C, H, N.
Boc-Dm t-Tic-Ala -OMe. To a solution of Boc-Dmt-Tic-OH
(0.2 g, 0.42 mmol) and HCl‚H-Ala-OMe (0.06 g, 0.42 mmol) in
DMF (10 mL) at 0 °C were added NMM (0.05 mL, 0.42 mmol),
HOBt (0.07 g, 0.46 mmol), and DCC (0.09 g, 0.46 mmol). The
reaction was stirred for 3 h at 0 °C and 24 h at room
temperature. After evaporation of DMF, the residue was
treated according to Boc-Dmt-Tic-NH-tetrazole-5-yl: yield 0.2
HPLC K′ ) 5.03; mp 123-125 °C; [R]20 +18.7; MH+ 191.
D
Boc-Dm t-Tic-NHMe. This product was obtained by con-
densation of Boc-Dmt-OH with H-Tic-NHMe via DCC/HOBt
as reported for Boc-Dmt-Tic-OMe: yield 0.13 g (85%); Rf (B)
0.73; HPLC K′ ) 6.34; mp 142-146 °C; [R]20D -15.3; MH+ 482;
1H NMR (DMSO) δ ) 1.35-1.44 (d, 9H), 2.16 (s, 6H), 2.44-
2.46 (d, 3H), 3.05-3.41 (m, 4H), 3.79 (m, 1H), 4.29-4.78 (m,
3H), 6.34 (s, 2H), 6.95 (bs, 1H), 7.14 (s, 4H), 8.22 (bs, 1H).
TFA‚H-Dm t-Tic-NHMe (7). Boc-Dmt-Tic-NHMe was treated
with TFA as reported for TFA‚H-Dmt-Tic-NHNH2 (6): yield
0.12 g (93%); Rf (A) 0.69; HPLC K′ ) 3.10; mp 152-154 °C;
g (85%); Rf (B) 0.77; HPLC K′ ) 7.68; mp 124-126 °C; [R]20
D
+40.1; MH+ 555; H NMR (DMSO) δ ) 1.37-1.46 (m, 12H),
1
2.16 (s, 6H), 2.96-3.01 (m, 2H), 3.08-3.13 (m, 2H), 3.46-3.56
(m, 1H), 3.73 (s, 3H), 4.03-4.07 (q, 1H), 4.34-4.88 (m, 3H),
6.46 (s, 2H), 7.18-7.21 (m, 4H), 8.32 (bs, 1H), 8.51 (bs, 1H).
TF A‚H-Dm t-Tic-Ala -OMe (18). Boc-Dmt-Tic-Ala-OMe was
treated with TFA as reported for TFA‚H-Dmt-Tic-NHNH2
(6): yield 0.19 g (92%); Rf (A) 0.64; HPLC K′ ) 3.72; mp 130-
[R]20 -22.0; MH+ 382. Anal. (C22H27N3O3‚TFA) C, H, N.
D
TF A‚N,N-(Me)2-Dm t-Tic-NHMe (13). This compound was
obtained by exhaustive methylation of TFA‚H-Dmt-Tic-NHMe
as reported for TFA‚N,N-(Me)2-Dmt-Tic-NH-1-adamantane
(15): yield 0.12 g (96%); Rf (A) 0.71; HPLC K′ ) 3.19; mp 156-
132 °C; [R]20 +90.1; MH+ 454. Anal. (C25H31N3O3‚TFA) C, H,
D
N.
158 °C; [R]20 -19.3; MH+ 410. Anal. (C24H31N3O3‚TFA) C, H,
Boc-Tic-NH-1-a d a m a n ta n e. To a solution of Boc-Tic-OH
(0.5 g, 1.8 mmol) and 1-amino adamantane hydrochloride (0.34
g, 1.8 mmol) in DMF (10 mL) at 0 °C were added NMM (0.20
mL, 1.8 mmol), HOBt (0.30 g, 1.98 mmol), and DCC (0.41 g,
1.98 mmol). The reaction was stirred for 3 h at 0 °C and 24 h
at room temperature. After evaporation of DMF, the residue
was treated as reported for Boc-Dmt-Tic-NH-tetrazole-5-yl:
D
N.
TF A‚H-Dm t-Tic-OMe (8). Boc-Dmt-Tic-OMe was treated
with TFA as reported for TFA‚H-Dmt-Tic-NHNH2 (6): yield
0.25 g (92%); Rf (A) 0.81; HPLC K′ ) 3.83; mp 118-120 °C;
[R]20 -24.0; MH+ 382. Anal. (C22H26N2O4‚TFA) C, H, N.
D
Boc-Dm t-âHTic-OMe. This substance was obtained by
condensation of Boc-Dmt-OH with HCl‚âHTic-OMe40 [Rf (B)
0.38, HPLC K′ ) 2.31, mp 153-155 °C, [R]20D -37.5; MH+ 206]
via DCC/HOBt as reported for Boc-Dmt-Tic-OMe: yield 0.42
yield 0.6 g (81%); Rf (B) 0.89; HPLC K′ ) 9.68; mp 127-129
1
°C; [R]20 +15.3; MH+ 412; H NMR (DMSO) δ ) 1.39-1.46
D
(d, 9H), 1.65 (s, 6H), 1.93-2.07 (m, 9H), 3.08-3.13 (m, 2H),
4.34-4.88 (m, 3H), 7.17-7.20 (m, 4H), 8.08 (bs, 1H).
g (97%); Rf (B) 0.93; HPLC K′ ) 9.27; mp 94-96 °C; [R]20
D
+33.9; MH+ 497; H NMR (DMSO) δ ) 1.82 (s, 9H), 2.35 (s,
1
TF A‚H -Tic-NH -1-a d a m a n t a n e. Boc-Tic-NH-1-adaman-
tane (0.6 g; 1.46 mmol) was treated with TFA (2 mL) for 0.5 h
at room temperature. The mixture Et2O/Pe (1:1, v/v) was added
to the solution until the product precipitated: yield 0.57 g
6H), 2.80-3.50 (m, 6H), 3.70 (s, 3H), 3.90 (m, 1H), 4.30 (m,
2H), 4.40 (dd, 1H), 6.43 (s, 2H), 7.20 (m, 6H), 9.50 (bs, 1H).
Boc-Dm t-âHTic-OH. To a solution of Boc-Dmt-âHTic-OMe
(0.42 g, 0.90 mmol) in MeOH (10 mL) was added 1 N NaOH
(1.34 mL). The reaction mixture was stirred for 24 h at room
temperature. After evaporation of the solvent, the residue was
solubilized in EtOAc and washed with citric acid (10%) and
brine. The organic phase was dried and evaporated to dryness.
The residue was crystallized from Et2O: yield 0.42 g (98%);
(92%); Rf (A) 0.73; HPLC K′ ) 5.80; mp 157-159 °C; [R]20
D
+18.4; MH+ 311.
Boc-Dm t-Tic-NH-1-a d a m a n ta n e. This peptide was ob-
tained by condensation of Boc-Dmt-OH with TFA‚H-Tic-NH-
1-adamantane via DCC/HOBt as reported for Boc-Dmt-Tic-
OMe: yield 0.16 g (85%); Rf (B) 0.93; HPLC K′ ) 9.28; mp
142-144 °C; [R]20 +28.1; MH+ 603; 1H NMR (DMSO) δ )
Rf (B) 0.35; HPLC K′ ) 7.54; mp 117-119 °C; [R]20 +36.2;
D
D
MH+ 483.
1.38-1.45 (d, 9H), 1.64 (s, 6H), 1.93-2.08 (m, 9H), 2.17 (s, 6H),
2.96-3.01 (m, 2H), 3.08-3.13 (m, 2H), 3.47-3.54 (m, 1H),
4.34-4.88 (m, 3H), 6.46 (s, 2H), 7.17-7.20 (m, 4H), 8.27 (bs,
1H), 8.47 (bs, 1H).
TFA‚H-Dm t-âHTic-OH (1). Boc-Dmt-âHTic-OH was treated
with TFA as reported for TFA‚H-Dmt-Tic-NHNH2 (6): yield
0.26 g (68%); Rf (A) 0.82; HPLC K′ ) 4.9; mp 118-120 °C; [R]20
D
+95.0; MH+ 383; H NMR (DMSO) δ ) 2.2 (s, 6H), 2.80-3.0
1
TF A‚H-Dm t-Tic-NH-1-a d a m a n ta n e (11). Boc-Dmt-Tic-
NH-1-adamantane was treated with TFA as reported for TFA‚
H-Dmt-Tic-NHNH2 (6): yield 0.15 g (94%); Rf (A) 0.64; HPLC
(m, 4H), 3.4 (s, 2H), 4.1 (m, 1H), 4.3 (m, 2H), 4.5 (m, 1H), 6.43
(s, 2H), 7.20 (m, 4H), 9.2 (bs, 3H). Anal. (C22H26N2O4‚TFA) C,
H, N.
K′ ) 6.98; mp 180-182 °C; [R]20 -2.7; MH+ 502. Anal.
D
(C31H39N3O3‚TFA) C, H, N.
Boc-Dm t-Tic-OH. To a solution of Boc-Dmt-Tic-OMe (0.52
g, 1.08 mmol) in MeOH (10 mL) was added 1 N NaOH (1.3
mL). The reaction mixture was stirred for 24 h at room
temperature and treated according to the methods reported
for Boc-Dmt-âHTic-OH: yield 0.45 g (89%); Rf (B) 0.36; HPLC
Boc-D-Tic-NH-1-a d a m a n ta n e. This compound was ob-
tained by condensation of Boc-D-Tic-OH with 1-amino ada-
mantane hydrochloride as reported for Boc-Tic-NH-1-adaman-
tane: yield 0.6 g (81%); Rf (B) 0.89; HPLC K′ ) 9.68; mp 127-
1
129 °C; [R]20 -15.3; MH+ 412; H NMR (DMSO) δ ) 1.39-
K′ ) 6.71; mp 147-149 °C; [R]20 -15.6; MH+ 469.
D
D
1.46 (d, 9H), 1.65 (s, 6H), 1.93-2.07 (m, 9H), 3.08-3.13 (m,
2H), 4.34-4.88 (m, 3H), 7.17-7.20 (m, 4H), 8.08 (bs, 1H).
TF A‚H -D-Tic-NH -1-a d a m a n t a n e. Boc-D-Tic-NH-1-ada-
mantane was treated with TFA as reported for TFA‚H-Tic-
NH-1-adamantane: yield 0.57 g (92%); Rf (A) 0.73; HPLC K′
Boc-Dm t-Tic-NH-tetr a zole-5-yl. To a solution of Boc-Dmt-
Tic-OH (0.4 g, 0.85 mmol) and 5-aminotetrazole monohydrate
(0.1 g, 0.85 mmol) in DMF (10 mL) at 0 °C were added HOBt
(0.14 g, 0.94 mmol) and DCC (0.19 g, 0.94 mmol). The reaction
mixture was stirred for 3 h at 0 °C and 24 h at room
temperature. After evaporation of the DMF, it was treated
according to the procedure reported for Boc-Dmt-âHTic-OH;
however, the residue was crystallized from Et2O/Pe (1:1, v/v):
) 5.80; mp 157-159 °C; [R]20 -18.4; MH+ 311.
D
Boc-Dm t-D-Tic-NH-1-a d a m a n ta n e. This substance was
obtained by condensation of Boc-Dmt-OH with TFA‚H-D-Tic-
NH-1-adamantane via DCC/HOBt as reported for Boc-Dmt-
Tic-OMe: yield 0.16 g (85%); Rf (B) 0.87; HPLC K′ ) 9.54; mp
yield 0.19 g (42%); Rf (B) 0.75; HPLC K′ ) 7.18; mp 146-148
1
°C; [R]20 -23.1; MH+ 537; H NMR (DMSO) δ ) 1.36-1.45
D
(d, 9H), 2.16 (s, 6H), 2.96-3.0 (m, 2H), 3.08-3.13 (m, 2H),
135-137 °C; [R]20 +14.2; MH+ 603; 1H NMR (DMSO) δ )
D

5016 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24
Salvadori et al.
1.39-1.46 (d, 9H), 1.63 (s, 6H), 1.94-2.09 (m, 9H), 2.16 (s, 6H),
2.97-3.03 (m, 2H), 3.05-3.14 (m, 2H), 3.49-3.56 (m, 1H),
4.41-4.91 (m, 3H), 6.41 (s, 2H), 7.18-7.20 (m, 4H), 8.31 (bs,
1H), 8.43 (bs, 1H).
TF A‚H-Dm t-D-Tic-NH-1-a d a m a n ta n e (12). Boc-Dmt-D-
Tic-NH-1-adamantane was treated with TFA as reported for
TFA‚H-Dmt-Tic-NHNH2 (6): yield 0.15 g (94%); Rf (A) 0.58;
HPLC K′ ) 7.24; mp 164-166 °C; [R]20D +27.5; MH+ 502. Anal.
(C31H39N3O3‚TFA) C, H, N.
in acetonitrile (10 mL) were added NMM (0.07 mL, 0.62 mmol),
50% aqueous glutaraldehyde (0.38 mL, 2.36 mmol), and
sodium cyanoborohydride (0.45 g, 0.73 mmol). Glacial acetic
acid (0.06 mL) was added over 10 min, and the reaction was
stirred at room temperature for 2 h. The reaction mixture was
evaporated in vacuo to give a crude product that was purified
by preparative HPLC: yield 0.16 g (90%); Rf (A) 0.74; HPLC
K′ ) 3.66; mp 205-207 °C; [R]20 -14.2; MH+ 437. Anal.
D
(C22H32N2O4‚TFA) C, H, N.
Boc-Ala -NH-1-a d a m a n ta n e. To a solution of Boc-Ala-OH
(0.34 g, 1.8 mmol) and 1-amino adamantane hydrochloride
(0.34 g, 1.8 mmol) in DMF (10 mL) at 0 °C were added NMM
(0.20 mL, 1.8 mmol), HOBt (0.30 g, 1.98 mmol), and DCC (0.41
g, 1.98 mmol). The reaction was stirred for 3 h at 0 °C and for
24 h at room temperature. After evaporation of DMF, the
residue was treated according to Boc-Dmt-Tic-NH-tetrazole-
5-yl: yield 0.52 g (89%); Rf (B) 0.85; HPLC K′ ) 7.73; mp 107-
TF A‚[d es-NH2-r-p yr r olid in e-1-yl]-Dm t-Tic-OH (4) a n d
TF A‚[d es-NH2-r-p yr r ole-1-yl]-Dm t-Tic-OH (5). These two
compounds were obtained by reductive alkylation of TFA‚H-
Dmt-Tic-OH with succinaldehyde as reported for TFA‚[des-
NH2-R-piperidine-1-yl]-Dmt-Tic-OH. Analytical data for TFA‚
[des-NH2-R-pyrrolidine-1-yl]-Dmt-Tic-OH (4): yield 0.016 g
(20%); Rf (A) 0.81; HPLC K′ ) 2.68; mp 168-170 °C; [R]20
D
+45.2; MH+ 423. Anal. (C25H30N2O4‚TFA) C, H, N.
109 °C; [R]20 +5.9; MH+ 324; 1H NMR (DMSO) δ ) 1.38-
D
Analytical data for TFA‚[des-NH2-R-pyrrole-1-yl]-Dmt-Tic-
1.45 (m, 12H), 1.65 (s, 6H), 1.93-2.07 (m, 9H), 4.04-4.07 (q,
1H), 8.08 (bs, 1H), 8.42 (bs, 1H).
OH (5): yield 0.1 g (80%); Rf (A) 0.67; HPLC K′ ) 5.85; mp
185-187 °C; [R]20 -56.4; MH+ 421. Anal. (C25H26N2O4‚TFA)
D
TF A‚H -Ala -NH -1-a d a m a n t a n e. Boc-Ala-NH-1-adaman-
tane (0.52 g, 1.6 mmol) was treated with TFA (2 mL) for 0.5 h
at room temperature. The mixture Et2O/Pe(1:1, v/v) was added
to the solution until the product precipitated: yield 0.5 g (92%);
Rf (A) 0.82; HPLC K′ ) 4.95; mp 131-133 °C; [R]20D +6.8; MH+
224.
Boc-Dm t-Tic-Ala -NH-1-a d a m a n ta n e. This compound was
obtained by condensation of Boc-Dmt-Tic-OH with TFA‚H-Ala-
NH-1-adamantane via DCC/HOBt as reported for Boc-Dmt-
C, H, N.
Boc-Dm t-Tic-NHtBu . This intermediate was obtained by
condensation of Boc-Dmt-Tic-OH with tert-butylamine via
DCC/NMM as reported for Boc-Dmt-Tic-NH-tetrazole-5-yl:
yield 0.26 g (87%); Rf (B) 0.88; HPLC K′ ) 7.02; mp 153-155
°C; [R]20D -8.2; MH+ 524; 1H NMR (DMSO) δ ) 1.23-1.44 (m,
18H), 2.16 (s, 6H), 3.05-3.41 (m, 4H), 3.79 (m, 1H), 4.29-4.78
(m, 3H), 6.34 (s, 2H), 6.95 (bs, 1H), 7.14 (s, 4H), 8.22 (bs, 1H).
TF A‚H -Dm t -Tic-NH t Bu (9). Boc-Dmt-Tic-NHtBu was
treated with TFA as reported for TFA‚H-Dmt-Tic-NHNH2
(6): yield 0.12 g (93%); Rf (A) 0.74; HPLC K′ ) 5.35; mp 150-
Tic-Ala-OMe: yield 0.12 g (87%); Rf (B) 0.91; HPLC K′ ) 9.32;
1
mp 137-139 °C; [R]20 +15.5; MH+ 674; H NMR (DMSO) δ
D
) 1.38-1.45 (d, 12H), 1.64 (s, 6H), 1.93-2.08 (m, 9H), 2.17 (s,
6H), 2.96-3.01 (m, 2H), 3.08-3.13 (m, 2H), 3.47-3.54 (m, 1H),
4.03-4.07 (m, 1H), 4.34-4.88 (m, 3H), 6.46 (s, 2H), 7.17-7.20
(m, 4H), 8.27 (bs, 1H), 8.47 (bs, 1H), 8.53 (bs, 1H).
152 °C; [R]20 -15.6; MH+ 424. Anal. (C25H33N3O3‚TFA) C, H,
D
N.
TF A‚N,N-(Me)2-Dm t-Tic-NHtBu (14). This N,N-alkylated
peptide was obtained by exhaustive methylation of TFA‚H-
Dmt-Tic-NHtBu (9) as reported for TFA‚H-Dmt-Tic-NH-1-
adamantane (11): yield 0.12 g (88%); Rf (A) 0.78; HPLC K′ )
6.01; mp 157-159 °C; [R]20D -18.7; MH+ 452. Anal. (C27H37N3O3‚
TFA) C, H, N.
TF A‚H-Dm t-Tic-Ala -NH-1-a d a m a n ta n e (20). Boc-Dmt-
Tic-Ala-NH-1-adamantane was treated with TFA as reported
for TFA‚H-Dmt-Tic-NHNH2 (6): yield 0.11 g (92%); Rf (A) 0.65;
HPLC K′ ) 6.91; mp 163-165 °C; [R]20D +20.1; MH+ 574. Anal.
(C34H44N4O4‚TFA) C, H, N: C, 71.3; H, 7.74; N, 9.78. Anal.
(C34H44N4O4‚TFA) C, H, N.
Boc-Dm t-Tic-Ala -NHtBu . This compound was obtained by
condensation of Boc-Dmt-Tic-OH with HCl‚H-Ala-NHtBu43 via
DCC/HOBt as reported for Boc-Dmt-Tic-Ala-OMe: yield 0.2 g
TF A‚N,N-(Me)2-Dm t -Tic-NH -1-a d a m a n t a n e (15). To a
stirred solution of TFA‚H-Dmt-Tic-NH-1-adamantane (0.7 g,
0.11 mmol) in acetonitrile (10 mL) were added NMM (0.01 mL,
0.11 mmol), 37% aqueous formaldehyde (0.07 mL, 0.83 mmol),
and sodium cyanoborohydride (0.016 g, 0.25 mmol). Glacial
acetic acid (0.02 mL) was added over 10 min, and the reaction
was stirred at room temperature for 2 h. The reaction mixture
was evaporated in vacuo to give a crude product that was
purified by preparative HPLC: yield 0.06 g (90%); Rf (A) 0.64;
(87%); Rf (B) 0.85; HPLC K′ ) 8.09; mp 150-152 °C; [R]20
D
+28.2; MH+ 595; H NMR (DMSO) δ ) 1.23-1.48 (m, 21H),
1
2.16 (s, 6H), 3.05-3.41 (m, 4H), 3.79 (m, 1H), 4.29-4.78 (m,
4H), 6.34 (s, 2H), 6.95 (bs, 1H), 7.14 (s, 4H), 8.22 (bs, 1H), 8.36
(bs, 1H).
TF A‚Dm t-Tic-Ala -NHtBu (19). Boc-Dmt-Tic-Ala-NH-tBu
was treated with TFA as reported for TFA‚H-Dmt-Tic-NHNH2
(6): yield 0.18 g (91%); Rf (A) 0.64; HPLC K′ ) 5.35; mp 150-
HPLC K′ ) 7.44; mp 118-120 °C; [R]20 -58.01; MH+ 530.
D
152 °C; [R]20 +25.7; MH+ 495. Anal. (C28H38N4O4‚TFA) C, H,
Anal. (C33H43N3O3‚TFA) C, H, N.
D
N.
TF A‚N,N-(Me)2-Dm t-D-Tic-NH-1-a d a m a n ta n e (16). The
peptide was obtained by exhaustive methylation of TFA‚H-
Dmt-D-Tic-NH-1-adamantane (12) as reported for TFA‚N,N-
(Me)2-Dmt-Tic-NH-1-adamantane (15): yield 0.06 g (90%); Rf
(R,S)-2-Cya n o-3-(4-h yd r oxy-2′,6′-d im eth ylp h en yl)-p r o-
p a n oic Acid Eth yl Ester . To a solution of sodium ethoxide
(Na, 0.17 g, 7.2 mmol; anhydrous EtOH, 12 mL) were added
ethyl cyanoacetate (0.62 mL, 7.08 mmol) and, after 10 min,
O-carbethoxy-3,5-dimethyl-4-chloromethylphenol (1.8 g, 7.42
mmol). The reaction mixture was refluxed for 2 h, cooled, and
filtered. The solution was evaporated in vacuo. The residue
was crystallized from H2O/acetone (5:1, v/v). The product was
purified by column chromatography [SiO2; Et2O/AcOEt (1:1,
v/v)]: yield 1.05 g (60%); Rf (B) 0.74; HPLC K′ ) 5.38; mp 132-
134 °C; MH+ 248; 1H NMR (DMSO) δ ) 1.25-1.81 (t, 3H),
2.23 (s, 6H), 3.08-3.14 (m, 2H), 4.14-4.27 (m, 3H), 6.44 (s,
2H), 9.17 (s, 1H).
(R,S)-2-Cya n o-3-(4-h yd r oxy-2′,6′-d im eth ylp h en yl)-p r o-
p a n oic Acid . To a solution of (R,S)-2-cyano-3-(4-hydroxy-2′,6′-
dimethylphenyl)-propanoic acid ethyl ester (1.05 g, 4.25 mmol)
in EtOH (10 mL) was added 1 N NaOH (4.68 mL, 4.68 mmol).
The reaction mixture was stirred for 24 h at room temperature.
After evaporation of the solvent, the residue was dissolved in
EtOAc and washed with citric acid (10%) and brine. The
organic phase was dried and evaporated to dryness. The
(A) 0.61; HPLC K′ ) 7.53; mp 134-136 °C; [R]20 +8.2; MH+
D
530. Anal. (C33H43N3O3‚TFA) C, H, N.
Boc-Dm t-Tic-Ala -NHMe. This compound was obtained by
condensation of Boc-Dmt-Tic-OH with HCl‚H-Ala-NHMe via
DCC/HOBt as reported for Boc-Dmt-Tic-Ala-OMe: yield 0.25
g (84%); Rf (B) 0.78; HPLC K′ ) 8.90; mp 131-133 °C; [R]20
D
+42.7; MH+ 553; H NMR (DMSO) δ ) 1.37-1.46 (m, 12H),
1
2.16 (s, 6H), 2.44-2.46 (d, 3H), 2.96-3.01 (m, 2H), 3.08-3.13
(m, 2H), 3.46-3.56 (m, 1H), 4.03-4.07 (q, 1H), 4.34-4.88 (m,
3H), 6.46 (s, 2H), 7.18-7.35 (m, 4H), 8.32 (bs, 1H), 8.43 (bs,
1H), 8.51 (bs, 1H).
TF A‚Dm t-Tic-Ala -NHMe (17). Boc-Dmt-Tic-Ala-NHMe
was treated with TFA as reported for TFA‚H-Dmt-Tic-NHNH2
(6): yield 0.24 g (91%); Rf (A) 0.58; HPLC K′ ) 4.29; mp 142-
144 °C; [R]20 +28.5; MH+ 453. Anal. (C25H32N4O4‚TFA) C, H,
D
N.
TF A‚[d es-NH2-r-p ip er id in e-1-yl]-Dm t-Tic-OH (3). To a
stirred solution of TFA‚H-Dmt-Tic-OH12 (0.15 g, 0.31 mmol)

Dmt-Tic Pharmacophore Studies
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24 5017
residue was crystallized from Et2O/PtEt (1:2, v/v): yield 0.79
g (85%); Rf (B) 0.31; HPLC K′ ) 3.54; mp 154-156 °C; MH+
220.
from at least four independent tissue samples and dose-
response curves from which the pA2 values were determined7
according to Arunlakshana and Schild.49
[Des-NH2-r-cya n o-(R,S)]-Dm t-Tic-OtBu . To a solution of
(R,S)-2-cyano-3-(4-hydroxy-2′,6′-dimethylphenyl)-propanoic acid
(0.12 g, 0.53 mmol) and H-Tic-OtBu (o.12 g, 0.53 mmol) in
DMF (10 mL) at 0 °C were added HOBt (0.09 g, 0.58 mmol)
and DCC (0.12 g, 0.58 mmol). The reaction mixture was stirred
for 3 h at 0 °C and 24 h at room temperature. After evaporation
of DMF, the residue was treated as reported for Boc-Dmt-Tic-
NH-tetrazole-5-yl: yield 0.18 g (80%); Rf (B) 0.82; HPLC K′ )
Ack n ow led gm en t. We appreciate the assistance of
F. T. Lyndon at the NIEHS library for her expeditious
retrieval of articles as well as the critical assessment
of the manuscript by Drs. Traci Hall and Ruth Lunn.
Refer en ces
(1) Abbreviations. In addition to the IUPAC-IUB Commission on
Biochemical Nomenclature (J . Biol. Chem. 1985, 260, 1442), this
paper uses the following symbols and abbreviations: Boc, tert-
butyloxycarbonyl; DAGO, [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin;
Dmt, 2′,6′-dimethyl-L-tyrosine; DPDPE, cyclo[D-Pen2,5]enkephalin;
GPI, guinea pig ileum; HOBt, 1-hydroxybenzotriazole; HPLC,
high-performance liquid chromatography; Ke, the antilog of pA2
in molar concentration; MeOH, methanol; MVD, mouse vas
deferens; pA2, negative log of the molar concentration required
to double the agonist concentration to achieve the original
response; tBu, tert-butyl; TFA, trifluoroacetic acid; Tic, 1,2,3,4-
tetrahydroisoquinoline-3-carboxylic acid; H-âTic, 1,2,3,4-tetrahy-
droisoquinoline-3-yl acetic acid; H-RDmt-OH, 2-methylamino-
3-(2′,6′-dimethyl-4-hydroxyphenyl)-propionic acid; TIP(P), H-Tyr-
Tic-Phe-(Phe)-OH; TLC, thin-layer chromatography; WSC, 1-ethyl-
3-[3′-dimethyl)aminopropyl]carbodiimide hydrochloride; Z, ben-
zyloxycarbonyl, TEA, triethylamine; NH-tBut, tert-butylamine;
Me, methyl; OMe, methyl ester; DCC, N,N′-dicyclohexylcarbo-
diimide; HOBt, 1-hydroxybenzotriazole; DMF, N,N-dimethyl-
formamide; LiAlH4, lithium aluminum hydride; NaBH3CN,
sodium cyanoborohydride; DMSO, dimethyl sulfoxide; EtPt,
petroleum ether; Et2O, diethyl ether.
(2) (a) Schiller, P. W.; Nguyen, T. M.-D.; Weltrowska, G.; Wilkes,
B. C.; Marsden, 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. (b) Schiller, P. W.;
Weltroska, G.; Nguyen, T. M.-D.; Wilkes, B. C.; Chung, N. N.;
Lemieux, C. TIPP[ψ]: a highly potent and stable pseudopeptide
δ opioid receptor antagonist with extraordinary δ selectivity. J .
Med. Chem. 1993, 36, 3182-3187. (c) 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: develop-
ment of extraordinarily potent and selective δ antagonists and
observation of astonishing structure-intrinsic activity relation-
ships. Peptides; Hodges, R. S., Smith, J . A., Eds.; ESCOM, 1994;
pp 483-486.
(3) (a) Temussi, P. A.; Salvadori, S.; Amodeo, P.; Bianchi, C.;
Guerrini, R.; Tomatis, R.; Lazarus, L. H.; Tancredi, T. Selective
opioid dipeptides. Biochem. Biophys. Res. Commun. 1994, 198,
933-939. (b) Mosberg, H. I.; Omnass, J . R.; Sobczyk-Kojiro, K.;
Dua, R.; Ho, J . C.; Ma, W.; Bush, P.; Mousigian, C.; Lomize, A.
Pharmacophore elements of the TIPP class of delta opioid
receptor antagonists. Lett. Pept. Sci. 1994, 1, 69-72.
(4) (a) Chandrakumar, N. S.; Yonan, P. K.; Stapelfeld, A.; Savage,
M.; Rorbacher, E.; Contreras, P. C.; Hammond, D. Preparation
and opioid activity of analogues of the analgesic dipeptide 2,6-
dimethyl-L-tyrosyl-N-(3-phenylpropyl)-D-alaninamide. J . Med.
Chem. 1992, 35, 223-233. (b) Chandrakumar, N. S.; Stapelfeld,
A.; Beardsley, P. M.; Lopez, O. T.; Drury, B.; Anthony, E.;
Savage, M. A.; Williamson, L. N.; Reichman, M. [2-D-Penicil-
lamine,5-D-Penicillamine]-enkephalin: 2′,6′-dimethyltyrosine and
Gly3-Phe4 amide bond isostere substitutions. J . Med. Chem.
1992, 35, 2928-2938. (c) Hansen, J ., D. W.; Stapelfeld, A.;
Savage, M. A.; Reichman, M.; Hammond, D. L.; Haaseth, R. C.;
Mosberg, H. I. Systemic analgesic activity and δ-opioid selectivity
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8.40; mp 140-142 °C; [R]20 -13.85; MH+ 435.
D
[Des-NH2-r-cyan o-(R,S)]-Dm t-Tic-OH. [Des-NH2-R-cyano-
(R,S)]-Dmt-Tic-OtBu (0.18, 0.42 mmol) was treated with TFA
(1 mL) for 0.5 h at room temperature. Et2O/Pe (1:1, v/v) was
added until the product precipitated: yield 0.15 g (96%); Rf
(A) 0.68; HPLC K′ ) 5.55; mp 142-144 °C; [R]20D -15.31; MH+
379; IR (KBr) 3420 (OH), 2360 (nitrile), 1636 (CdO, amide),
1734 (CdO, acid), 1142 (C-O, carboxylate anion) cm-1
.
H-r(R,S)HDm t-Tic-OH (2). To a solution of [des-NH2-R-
cyano-(R,S)]-Dmt-Tic-OH (0.15 g, 0.4 mmol) in EtOH (30 mL)
were added 1 N HCl (4.5 mL) and PtO2 (0.05 g), and H2 was
bubbled for 8 h at room temperature. After filtration, the
solution was evaporated to dryness. The residue was crystal-
lized from Et2O/Pe (1:1, v/v): yield 0.15 g (98%); Rf (A) 0.39;
HPLC K′ ) 2.51; mp 157-159 °C; [R]20 -18.7; MH+ 383; IR
D
(KBr) 3432 (NH3+), 1683 (CdO, amide), 1616 (CdO, carboxy-
late anion), 1203 (C-O, carboxylate anion) cm-1. Anal.
(C22H26N2O4‚TFA) C, H, N.
Recep tor Bin d in g Assa ys. Peptides were assayed with a
rat brain synaptosomal preparations (P2) previously preincu-
bated in 0.1 M NaCl, 0.4 mM GDP, 50 mM HEPES, pH 7.5,
and 50 µg/mL soybean trypsin inhibitor for 60 min at room
temperature to remove endogenous opioids.44 After extensive
washing in ice cold buffer containing protease inhibitor, the
material was resuspended in buffered inhibitor containing 20%
glycerol, aliquoted, and stored at -80 °C.44 The assays were
conducted as described in detail elsewhere.5,7,45 Briefly, the
agonists [3H]DPDPE (30-60 Ci/mmol, NEN-DuPont) and [3H]-
DAGO (30-60 Ci/mmol, Amersham) were used to label δ and
µ sites, respectively, under saturation binding conditions (2 h
at 22 °C). Excess unlabeled peptide (2 µM) established
nonspecific binding levels. The labeled membranes were
rapidly filtered on Whatman GF/C glass fiber filters, thor-
oughly washed, and dried, and the radioactivity was deter-
mined using CytoScint (ICN). The δ antagonist [3H]N,N-
(CH3)2-Dmt-Tic-OH was catalytically dehalogenated from a
diiodo intermediate to a specific activity of 59.88 Ci/mol and
binds to δ-receptors with a Kd ) 0.39 nM.46 The receptor
binding and biological properties of the unlabeled peptide were
described previously.7 All analogues were analyzed in duplicate
using 5-9 dosages, and at least three independent repetitions
using different synaptosomal preparations were conducted for
each peptide (actual n values are listed in Table 1 in paren-
theses), with results given as mean ( SEM. The affinity
constants (Ki) were calculated according to Cheng and Pru-
soff.47
P h a r m a cologica l Bioa ssa ys. The specifics of the standard
functional bioassays using mouse vas deferens (MVD) and
guinea pig ileum (GPI) for δ and µ activity, respectively, were
published.5,48 Briefly, a 2-3 cm segment of GPI was placed in
20 mL tissue bath containing Kreb’s solution, 70 µM hexa-
methonium bromide, and 0.125 µM mepyramine and aerated
with 95% O2/5% at 36 °C. Transmural stimulation of GPI was
by means of a square-wave electrical pulses of 0.5 ms duration
at a frequency of 0.1 Hz. A single MVD was suspended in 4
mL modified Kreb’s solution aerated with 95% O2/5% at 33
°C. An isometric transducer recorded the twitch induced by
field stimulation (0.1 Hz for 1 ms at 40 V). Dose-response
curves were obtained5 for both tissues. The µ agonist activity
was compared against dermorphin (IC50 ) 1.82 nM), and δ
antagonism was determined through the inhibition of deltor-
phin C (δ1 receptor agonist, IC50 ) 0.54 nM) in comparison to
the nonpeptide δ antagonist naltrindole. Data were derived
(5) Salvadori, S.; Attila, M.; Balboni, G.; Bianchi, C.; Bryant, S. D.;
Crescenzi, O.; Guerrini, R.; Picone, D.; Tancredi, T.; Temussi,
P. A.; Lazarus, L. H. δ Opioidmimetic antagonists: prototypes
for designing a new generation of ultraselective opioid peptides.
Mol. Med. 1995, 1, 678-689.
(6) Capasso, A.; Guerrini, R.; Balboni, G.; Sorrentino, L.; Temussi,
P.; Lazarus, L. H.; Bryant, S. D.; Salvadori, S. Dmt-Tic-OH, a
highly selective and potent δ-opioid dipeptide receptor antagonist
after systemic administration in the mouse. Life Sci. 1996, 59,
PL 93-98.
(7) 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, 42, 33100-3108.
(8) 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.

5018 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24
Salvadori et al.
(9) Lazarus, L. H.; Bryant, S. D.; Cooper, P. S.; Guerrini, R.; Balboni,
G.; Salvadori, S. Design of δ-opioid peptide antagonists for
emerging drug applications. Drug Dev. Today 1998, 284-294.
(10) Lazarus, L. H.; Salvadori, S.; Santagada, V.; Tomatis, R.; Wilson,
W. E. Function of negative charge in the “address domain” of
deltorphins. J . Med. Chem. 1991, 34, 1350-1359.
(11) Lazarus, L. H.; Bryant, S. D.; Cooper, P. S.; Salvadori, S. What
peptides these deltorphins be. Prog. Neurobiol. 1999, 57, 377-
420.
(12) Matthes, H.; Maldonado, R.; Simonin, F.; Valverde, O.; Slowe,
S.; Kitchen, I.; Befort, K.; Dierich, A.; Meur, M.; Dolle, P.;
Tzavara, E.; Hanoune, J .; Roques, B.; Kieffer, B. Loss of
morphine-induced analgesia reward effect and withdrawal
symptoms in mice lacking the µ opioid receptor. Nature 1996,
383, 819-823.
(13) Rapaka, R. S.; Porreca, F. Development of delta opioid peptides
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