Further studies on lead compounds containing the opioid pharmacophore Dmt-Tic

Article abstract of DOI:10.1021/jm800587e

Some reference opioids containing the Dmt-Tic pharmacophore, especially the δ agonists H-Dmt-Tic-GlyNH-Ph (1) and H-Dmt-TiC-NH-(S)CH(CH ₂-COOH)-Bid (4) (UFP-512) were evaluated for the influence of the substitution of Gly with aspartic acid, it

Full text of DOI:10.1021/jm800587e

J. Med. Chem. 2008, 51, 5109–5117
5109
Further Studies on Lead Compounds Containing the Opioid Pharmacophore Dmt-Tic
Gianfranco Balboni,*,†,‡ Stella Fiorini,‡ Anna Baldisserotto,‡ Claudio Trapella,‡ Yusuke Sasaki,§ Akihiro Ambo,§
Ewa D. Marczak,| Lawrence H. Lazarus,| and Severo Salvadori*,‡
Department of Toxicology, UniVersity of Cagliari, I-09124 Cagliari, Italy, Department of Pharmaceutical Sciences and Biotechnology Center,
UniVersity of Ferrara, I-44100 Ferrara, Italy, Department of Pharmacology, Tohoku Pharmaceutical UniVersity, 4-1 Komatsushima 4-chome,
Aoba-Ku, Sendai 981-8558, Japan, Medicinal Chemistry Group, Laboratory of Pharmacology, National Institute of EnVironmental Health
Sciences, Research Triangle Park, North Carolina 27709, USA
ReceiVed May 19, 2008
Some reference opioids containing the Dmt-Tic pharmacophore, especially the δ agonists H-Dmt-Tic-Gly-
NH-Ph (1) and H-Dmt-Tic-NH-(S)CH(CH2-COOH)-Bid (4) (UFP-512) were evaluated for the influence
of the substitution of Gly with aspartic acid, its chirality, and the importance of the -NH-Ph and N1H-Bid
hydrogens in the inductions of δ agonism. The results provide the following conclusions: (i) Asp increases
δ selectivity by lowering the µ affinity; (ii) -NH-Ph and N1H-Bid nitrogens methylation transforms the δ
agonists into δ antagonists; (iii) the substitution of Gly with L-Asp/D-Asp in the δ agonist H-Dmt-Tic-Gly-
NH-Ph gave δ antagonists; the same substitution in the δ agonist H-Dmt-Tic-NH-CH2-Bid yielded more
selective agonists, H-Dmt-Tic-NH-(S)CH(CH2-COOH)-Bid and H-Dmt-Tic-NH-(R)CH(CH2-COOH)-Bid;
(iv) L-Asp seems important only in functional bioactivity, not in receptor affinity; (v) H-Dmt-Tic-NH-
(S)CH(CH2-COOH)-Bid(N1-Me) (10) evidenced analgesia similar to 4, which was reversed by naltrindole
only in the tail flick. 4 and 10 had opposite behaviours in mice; 4 caused agitation, 10 gave sedation and
convulsions.
Introduction
Dmt-Tic-OH11andtheδinverseagonistN,N(Me)2-Dmt-Tic-NH212,13
as useful tools for pharmacological studies. Some other lead
compounds endowed with potential utility as therapeutic agents,
are represented by: the µ agonist/δ antagonist H-Dmt-Tic-Gly-
NH-Bzl (2); the µ agonist/δ agonist H-Dmt-Tic-Gly-NH-Ph (1);
and the potent δ agonists H-Dmt-Tic-NH-CH2-Bid (3) (UFP-
502)27 and H-Dmt-Tic-NH-(S)CH(CH2-COOH)-Bid (4) (UFP-
512).9 On the basis of different pharmacological studies, µ
agonists/δ antagonists14 and µ agonists/δ agonists15 may provide
new classes of analgesics with low propensity to induce
tolerance, physical dependence, constipation, and other side
effects. δ Opioid receptor agonists are known to produce many
pharmacological effects in rodents, including analgesia,16
antidepressant,10,17 neuroprotection/neurogenesis,18 and anti-
Parkinson19 activities. Moreover, peripheral δ-opioid receptors
seem to be involved in cancer,20 cardiovascular disease,21
gastrointestinal disorders,22 and newer paradigms for pain relief
that use peripherally restricted opioids.23 Starting from our
selected lead compounds, here we report some new attempts
for a better understanding of their biological profiles, especially
in the light of the fact that even minor modifications can change
their pharmacological characteristics.24,25 In particular, we
focused our attention once again on the importance of the
C-terminal Bid (1H-benzimidazole-2-yl) and the anilide function
(considered as an open ring surrogate of Bid); in fact, both
groups are important for δ agonist activity. From previous
studies, the hydrogen linked to the N1 of Bid24,25 (and probably
the corresponding hydrogen linked to the anilide function) seems
to be important for the induction of δ agonism. To ascertain
this hypothesis, we synthesized some N1-Bid and N-anilide
methylated analogues of our selected lead compounds. More-
over, to verify the importance of the negative charge in the
induction of δ selectivity and to confirm the ineffectiveness of
the C-terminal chiral center,8 we considered the substitution of
Gly with L-Asp or D-Asp residues. Furthermore, such modifica-
tion seems to be capable to influence the blood-brain barrier
The prototype opioid pharmacophore Dmt-Tic,a,1 which
evolved from H-Tyr-Tic-OH2 as a simplified form of TIP(P),3
represents the minimum sequence that selectively interacts with
δ opioid receptors as a potent δ antagonist. Extensive
structure-activity studies on this prototype revealed that even
minor chemical modifications changed its pharmacological
profile, including a wide range of properties such as: enhanced
δ antagonism,4 appearance of mixed µ agonism/δ agonism5 as
well as mixed µ agonism/δ antagonism,5 µ agonism,6
µ
antagonism,6 δ inverse agonism,7 and δ agonism.5,8-10 Among
all synthesized analogues, some lead compounds were obtained,
for example the potent and selective δ antagonist N,N(Me)2-
* To whom correspondence should be addressed. For G.B.: phone, (+39)-
70-675-8625;fax,(+39)-70-675-8612;E-mail:gbalboni@unica.it,bbg@unife.it.
For S.S.: phone, (+39)-532-455-918; fax, (+39)-532-455-953; E-mail,
sal@unife.it.
†
Department of Toxicology, University of Cagliari.
Department of Pharmaceutical Sciences and Biotechnology Center,
‡
University of Ferrara.
§
Department of Pharmacology, Tohoku Pharmaceutical University.
Medicinal Chemistry Group, Laboratory of Pharmacology, National
|
Institute of Environmental Health Sciences.
a Abbreviations. In addition to the IUPAC-IUB Commission on Bio-
chemical Nomenclature (J. Biol. Chem. 1985, 260, 14-42), this paper uses
the following additional symbols and abbreviations: AcOEt, ethyl acetate;
AcOH, acetic acid; Bid, 1H-benzimidazole-2-yl; Boc, tert-butyloxycarbonyl;
DAMGO, [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin; DEL C, deltorphin II (H-
Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2); DMF, N,N-dimethylformamide; DM-
SO-d6, hexadeuteriodimethyl sulfoxide; Dmt, 2′,6′-dimethyl-L-tyrosine;
DPDPE, (D-Pen2,D-Pen5)-enkephalin; Endomorphin-2, H-Tyr-Pro-Phe-Phe-
NH2; Et2O, diethyl ether GPI, guinea-pig ileum; HOBt, 1-hydroxybenzo-
triazole; HPLC, high performance liquid chromatography; IBCF, isobutyl
chloroformate; MALDI-TOF, matrix assisted laser desorption ionization time-
of-flight. MVD, mouse vas deferens; NMM, 4-methylmorpholine; NLX,
naloxone; NTI, naltrindole; pA2, negative log of the molar concentration required
to double the agonist concentration to achieve the original response; Pe,
petroleum ether; PL017, H-Tyr-Pro-(N-Me)Phe-D-Pro-NH2; TEA triethylamine;
TFA, trifluoroacetic acid; Tic, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid;
TLC, thin-layer chromatography; WSC, 1-ethyl-3-[3′-dimethyl)aminopropyl]-
carbodiimide hydrochloride; Z, benzyloxycarbonyl.
10.1021/jm800587e CCC: $40.75
2008 American Chemical Society
Published on Web 08/05/2008

5110 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Scheme 1. Synthesis of Compound 10
Balboni et al.
(BBB) penetration; in fact, both 326,27 and 410 show antidepres-
sant activities when administered icv, but only 4 after intrap-
eritoneus administration.
N1-Bid methylation was accomplished in DMF using K2CO3
as a base, followed after 1 h by the reaction with iodomethane.24
Subsequently, Boc deprotection (TFA) and the final condensa-
tion (WSC/HOBt) with Boc-Dmt-OH gave the fully protected
compounds. Final deprotections of Asp or D-Asp side chains
(by catalytic hydrogenation) and N-terminal amine functions
(by TFA treatment) gave the crude final compounds (5-13),
which were purified by preparative reverse phase HPLC.
Chemistry
All peptides and pseudopeptides (5-13) were prepared
stepwise in solution using conventional synthetic methods as
outlined in Scheme 1 for the more representative compound
(10). The intermediates containing Bid at their C-termini, Boc-
NH-(S)CH(CH2-COOBzl)-Bid8andBoc-NH-(R)CH(CH2-COOBzl)-
Bid were prepared according to the procedure of Nestor et al.28
Briefly, mixed carbonic anhydride coupling of Boc-Asp(OBzl)-
OH or Boc-D-Asp(OBzl)-OH with o-phenylendiamine gave the
crude intermediate amides, which were converted without
purification to the desired benzimidazole heterocycles (Bid) by
cyclization/dehydration in acetic acid at 60 °C for 1 h.. The
corresponding Boc-protected intermediate anilides, or N(Me)-
anilides, or benzylamides were prepared by condensation of
Boc-Gly-OH or Boc-Asp(OBzl)-OH or Boc-D-Asp(OBzl)-OH
with aniline, or N-methyl-aniline, or benzylamine, via mixed
carbonic anhydride. Each intermediate, after Boc deprotection
with TFA, was condensed with Boc-Tic-OH via WSC/HOBt.
Results and Discussion
Receptor Affinity Analysis. Receptor binding data for δ-
and µ-receptors and δ-selectivity (Kiµ/ Kiδ) are reported in Table
1. All new compounds (5-13) had subnanomolar affinities for
δ-opioid receptors (Kiδ ) 0.036-0.186 nM), which is in quite
good accordance with the reference compounds. As expected,
the introduction of a negative charge, derived from the substitu-
tion of Gly with L- or D-Asp, increased δ-selectivity essentially
by decreasing µ-affinity. In fact, compounds 6-13 with a Kiµ
) 7.49-364.3 nM exhibited a δ-selectivity ranging from 101
to 5730 in comparison with references and compound 5 lacking
the negative charge (δ-selectivity ) 4-14). The highest increase
in δ-selectivity derived from the substitution of Gly with aspartic

Lead Compounds Containing the Dmt-Tic
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 5111
Table 1. Receptor Binding Affinities and Functional Bioactivities of Compounds 1-13
receptor affinitya (nM)
Kiδ
functional bioactivity
MVD GPI
structure
Kiµ
Kiµ/ Kiδ MVD
c
b
b
selectivity pA2 IC50 (nM) IC50 (nM)
ref
1
2
3
H-Dmt-Tic-Gly-NH-Phd
0.042
0.031
0.035
0.443
0.16
0.16
0.50
53.9
4
5
14
3.02
2.57
2.69
26.92
1724
H-Dmt-Tic-Gly-NH-Bzld
9.25
H-Dmt-Tic-NH-CH2-Bidd
0.13
0.12
4
H-Dmt-Tic-NH-(S)CH(CH2-COOH)-Bide
122
compd
5
6
7
8
H-Dmt-Tic-Gly-N(Me)-Ph
H-Dmt-Tic-Asp-NH-Ph
0.143 ( 0.024 (4)
0.036 ( 0.002 (3)
0.058 (0.003 (3)
0.186 ( 0.008 (3)
0.084 ( 0.008 (4)
1.75 ( 0.067 (3)
10.8 ( 1.2 (5)
7.49 ( 0.57 (4)
364.3 ( 37 (3)
45.0 ( 4.3 (5)
5.93 ( 0.82 (5)
11.8 ( 1.4 (5)
16.1 ( 1.9 (6)
309.4 ( 24 (3)
12
300
129
1958
536
101
169
240
5730
8.80
9.40
8.62
8.75
8.06
9.90
9.65
1466 ( 629
265 ( 10.4
2655 ( 127.8
>10000
H-Dmt-Tic-D-Asp-NH-Ph
H-Dmt-Tic-Asp-N(Me)-Ph
H-Dmt-Tic-D-Asp-N(Me)-Ph
9
>10000
10
11
12
13
H-Dmt-Tic-NH-(S)CH(CH2-COOH)-Bid(N1-Me) 0.059 (0.008 (4)
2886 ( 983
>10000
179
H-Dmt-Tic-NH-(R)CH(CH2-COOH)-Bid(N1-Me) 0.070 (0.007 (4)
H-Dmt-Tic- NH-(R)CH(CH2-COOH)-Bid
H-Dmt-Tic-Asp-NH-Bzl
0.067 ( 0.008 (5)
0.054 ( 0.006 (4)
1.21
8.53
>10000
a The Ki values (nM) were determined according to Cheng and Prusoff.43 The mean ( SE with n repetitions in parenthesis is based on independent
duplicate binding assays with five to eight peptide doses using several different synaptosomal preparations. b Agonist activity was expressed as IC50 obtained
from dose-response curves. These values represent the mean ( SE for at least four tissue samples. DPDPE and PL017 were the internal standards for MVD
(δ-opioid receptor bioactivity) and GPI (µ-opioid receptor bioactivity) tissue preparation, respectively. c The pA2 values of opioid antagonists against the δ
and µ agonists (deltorphin II and endomorphin-2, respectively) were determined by the method of Kosterlitz and Watt.49 d Data taken from Balboni et al.5
e Data taken from Balboni et al.8
acid as seen in H-Dmt-Tic-Gly-NH-Bzl (2); in fact, its selectivity
(Kiµ/ Kiδ ) 5) rose over 3 orders of magnitude to 5730. The
same substitution gave lower increases in selectivity when
applied to the reference compounds 1 and 3. On the basis of
the C-terminal aromatic substituents, the best selectivity was
induced by -NH-Bzl > -N(Me)-Ph > -NH-Ph > -Bid g
-Bid(N1-Me). With regard to the aspartic acid chirality, no final
conclusions can be drawn about its influence on the observed
receptor selectivity.
analogues containing L-aspartic acid; however, this trend is not
supported by the corresponding affinity data.
In Vivo Biological Activity. In recallling the data reported
by Codd et al.,30 they demonstrated the in vivo biotrasformation
of a δ opioid agonist into a µ agonist by N-deethylation. Looking
at our new compounds (5-13), a similar behavior could be
expected from all N-methylated analogues (5, 8-11). On the
basis of this hypothesis, we chose the potent and selective δ
antagonist (10) as a potential prodrug of the potent and selective
δ agonist 4. Preliminary enzymatic degradation studies (Sup-
porting Information) failed to demonstrate this assumption. In
fact, both compounds 4 and 10 appeared stable to degradation
for 4 and 2 h in plasma and brain homogenate, respectively.
Notwithstanding the preceding negative results, we tested
compound 10 for in vivo analgesia in comparison with 4; a
positive result could be tentatively considered as indirect
evidence of the N-demethylation of 10 (δ antagonist) to the
corresponding 4 (δ agonist). The analgesic effects of both
compounds were determined by tail-flick and hot-plate tests.
Results reported in Figure 1 indicated a similar dose-dependent
analgesic effect for both compounds after intracerebroventricular
injection. Analgesia of both compounds was reversed by the δ
selective antagonist naltrindole and the nonselective antagonist
naloxone in the tail-flick test but not in the hot-plate (Figures 2
and 3.) Interestingly, at the same dose, 4 and 10 provided
opposite behavioural effects; namely, 4 caused excessive
grooming and agitation (constant, fast moving in the cage,
burrowing in the nesting material), while with 10, the mice
appeared sedated, quiet, easily handled, and moving slowly if
at all. Furthermore, 4 did not induce convulsions even at the
higher dosages, confirming our previous data on its antidepres-
sant and anxiolytic studies,9,10 which are in accord with
observations about the higher convulsive effects of the non-
peptidic δ agonists in comparison to opioid peptides.26,31,32
Functional Bioactivity. Compounds 5-13 were tested in the
electrically stimulated MVD and GPI pharmacological assays
for intrinsic functional bioactivity (Table 1). As quite usually
observed with compounds containing the Dmt-Tic pharmacoph-
ore, a close correlation between binding and functional bioac-
tivity data is often lacking. Recently, a similar lack of correlation
was observed by Hruby et al. with 4-anilidopiperidine ana-
logues.29 As expected, and partially demonstrated for 3,24,25 all
N-methylated analogues of anilides and N1-Bid (5, 8-11),
revealed potent and selective δ-opioid antagonist activity (MVD,
pA2 ) 8.06 - 9.90), confirming the importance of the hydrogen
of -NH-Ph and N1H-Bid in the induction of δ agonism.
Surprisingly, the substitution of Gly with L-Asp (6) or D-Asp
(7) in the reference compound 1 gave two potent and quite
selective δ antagonists (MVD, pA2 ) 9.40 and 8.62, respec-
tively) despite the presence of the -NH-Ph hydrogen. Com-
pound 12, the diastereoisomer containing the D-Asp side chain
of our best δ agonist 4, indicated for the first time that better
results can be obtained using L amino acids in the synthesis of
compounds containing a C-terminal Bid. In fact, it shows a δ
agonist activity of 1 order of magnitude lower than 4 and a µ
agonist activity of almost 1 order of magnitude higher than 4.
Interestingly, compound 10, the N1-Bid methylated analogue
of 4 gave the highest δ antagonism (pA2 ) 9.90) in this series
of compounds, associated with a µ agonism 1.7 fold higher than
4. The substitution of Gly with Asp (13) in the µ agonist/δ
antagonist 2 was detrimental in its activity profile; in fact, 13
showed a selective δ antagonist activity (5 fold lower than 2),
associated with a very weak µ antagonist activity (GPI, pA2 )
6.26, not reported in Table 1). Finally, in the 3 pairs of
compounds (6/7, 8/9, and 10/11) and in the pair consisting of
4 and 12, the best δ activities were always showed by the
Experimental Section
Chemistry. Boc-Gly-N(Me)-Ph. A solution of Boc-Gly-OH
(1.00 g, 5.71 mmol) and NMM (0.62 mL, 5.71 mmol) in DMF (10
mL) was treated at -20 °C with IBCF (0.74 mL, 5.71 mmol). After
10 min at -20 °C, N-methylaniline (0.62 mL, 5.71 mmol) was
added. The reaction mixture was allowed to stir while slowly

5112 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Balboni et al.
Figure 1. Dose dependent effect of icv injected 4 (A,B) and 10 (C,D)
in the hot-plate (A,C) and tail-flick (B,D) tests. Each point represents
the mean ( SEM (n ) 5 mice). The asterisks denote AUC values that
are significantly different from saline treated mice by Dunnett’s test
(*, p < 0.05; **, p < 0.01; ***, p < 0.001) following ANOVA ((A)
P < 0.0001: F ) 71.49, d.f. 4; (B) P < 0.0001: F ) 251.7, d.f. 4; (C):
P < 0.001, F ) 9.356, d.f. 3; (D) p < 0.0001: F ) 15.14, d.f. 3).
Figure 3. Effect of sc injected naltrindole (3 mg/kg) and naloxone
(10 mg/kg) on 4 induced antinociception in hot-plate (A,C) and tail-
flick (B,D) tests. 4 was administered icv (10 µg/mouse) 20 min after
administration of antagonists. (A,B) Time course; (C,D) area under
the curve (AUC). Each point represents the mean ( SEM (n ) 5 mice).
The asterisks denote AUC values that are significantly different from
saline treated mice by Dunnett’s test (**, p < 0.01; ***, p < 0.001)
following ANOVA ((C) p ) 0.0136: F ) 6.277, d.f. 2; (D) p < 0.0001:
F ) 87.13, d.f. 2).
Boc-Tic-Gly-N(Me)-Ph. To a solution of Boc-Tic-OH (0.20 g,
0.72 mmol) and TFA·H-Gly-N(Me)-Ph (0.20 g, 0.72 mmol) in
DMF (10 mL) at 0 °C, NMM (0.08 mL, 0.72 mmol), HOBt (0.12
g, 0.79 mmol), and WSC (0.15 g, 0.79 mmol) were added. The
reaction mixture was stirred for 3 h at 0 °C and 24 h at room
temperature. After DMF was evaporated, the residue was dissolved
in EtOAc and washed with citric acid (10% in H2O), NaHCO3 (5%
in H2O), and brine. The organic phase was dried (Na2SO4) and
evaporated to dryness. The residue was precipitated from Et2O/Pe
(1:9, v/v): yield 0.27 g (87%); Rf (B) 0.69; HPLC K′ 7.35; mp
133-135 °C; [R]20D -30.1; m/z 425 (M + H)+. 1H NMR (DMSO-
d6) δ 1.38-1.42 (d, 9H), 2.78 (s, 3H), 2.92-3.17 (m, 2H),
4.14-4.27 (m, 4H), 4.92 (m, 1H), 6.96-7.31 (m, 9H).
TFA·H-Tic-Gly-N(Me)-Ph. Boc-Tic-Gly-N(Me)-Ph (0.24 g,
0.57 mmol) was treated with TFA (1.5 mL) for 0.5 h at room
temperature. Et2O/Pe (1:1, v/v) were added to the solution until
the product precipitated: yield 0.24 g (95%); Rf (A) 0.39; HPLC
Figure 2. Effect of sc injected naltrindole (3 mg/kg) and naloxone
(10 mg/kg) on 10 induced antinociception in hot-plate (A,C) and tail-
flick (B,D) tests. Compound 10 was administered icv (10 µg/mouse)
20 min after administration of antagonists. (A,B) Time course; (C,D)
area under the curve (AUC). Each point represents the mean ( SEM
(n ) 5 mice). The asterisks denote AUC values that are significantly
different from saline treated mice by Dunett’s test (*, p < 0.05; ***,
p < 0.001) following ANOVA ((C) p ) 0.2138: F ) 1.759, d.f. 2; (D)
p < 0.001: F ) 13.61, d.f. 2).
K′ 6.22; mp 158-160 °C; [R]20 -30.6; m/z 324 (M + H)+.
D
Boc-Dmt-Tic-Gly-N(Me)-Ph. To a solution of Boc-Dmt-OH
(0.10 g, 0.32 mmol) and TFA·H-Tic-Gly-N(Me)-Ph (0.14 g, 0.32
mmol) in DMF (10 mL) at 0 °C, NMM (0.03 mL, 0.32 mmol),
HOBt (0.05 g, 0.35 mmol), and WSC (0.07 g, 0.35 mmol) were
added. The reaction mixture was stirred for 3 h at 0 °C and 24 h
at room temperature. After DMF was evaporated, the residue was
dissolved in EtOAc and washed with citric acid (10% in H2O),
NaHCO3 (5% in H2O), and brine. The organic phase was dried
(Na2SO4) and evaporated to dryness. The residue was precipitated
from Et2O/Pe (1:9, v/v): yield 0.17 g (87%); Rf (B) 0.68; HPLC K′
10.01; mp 141-43 °C; [R]20D -18.5; m/z 616 (M + H)+. 1H NMR
(DMSO-d6) δ 1.38-1.42 (d, 9H), 2.35 (s, 6H), 2.78 (s, 3H),
2.92-3.17 (m, 4H), 4.14-4.92 (m, 6H), 6.29 (s, 2H), 6.96-7.31
(m, 9H).
warming to room temperature (1 h) and was then stirred for an
additional 3 h. The solvent was evaporated, and the residue was
partitioned between EtOAc and H2O. The EtOAc layer was washed
with citric acid (10% in H2O), NaHCO3 (5% in H2O), and brine
and dried over Na2SO4. The solution was filtered, the solvent
evaporated, and the residual oil was precipitated from Et2O/Pe (1:
9, v/v): yield 1.39 g (92%); Rf (B) 0.52; HPLC K′ 6.01; mp 99-101
°C; m/z 265 (M + H)+. 1H NMR (DMSO-d6) δ 1.40 (s, 9H), 2.78
(s, 3H), 3.88-3.92 (m, 2H), 7.10-7.31 (m, 5H).
TFA·H-Dmt-Tic-Gly-N(Me)-Ph (5). Boc-Dmt-Tic-Gly-N(Me)-
Ph (0.14 g, 0.23 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 0.14 g (97%); Rf (A) 0.45;
TFA·H-Gly-N(Me)-Ph. Boc-Gly-N(Me)-Ph (1.36 g, 5.15 mmol)
was treated with TFA (2.5 mL) for 0.5 h at room temperature. Et2O/
Pe (1:1, v/v) were added to the solution until the product
precipitated: yield 1.36 g (95%); Rf (A) 0.49; HPLC K′ 5.12; mp
115-117 °C; m/z 165 (M + H)+.
HPLC K′ 7.25; mp 150-152 °C; [R]20 -20.3; m/z 516 (M +
D
H)+. 1H NMR (DMSO-d6) δ 2.35 (s, 6H), 2.78 (s, 3H), 2.92-3.17
(m, 4H), 3.95-4.92 (m, 6H), 6.29 (s, 2H), 6.96-7.31 (m, 9H);
Anal. C32H35F3N4O6: C; H; N.

Lead Compounds Containing the Dmt-Tic
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 5113
Boc-Asp(OBzl)-NH-Ph. This intermediate was obtained by
condensation of Boc-Asp(OBzl)-OH with aniline via mixed anhy-
dride as reported for Boc-Gly-N(Me)-Ph: yield 0.57 g (93%); Rf
(B) 0.73; HPLC K′ 7.02; mp 129-132 °C; [R]20D +12.5; m/z 399
(DMSO-d6) δ 1.38-1.42 (d, 9H), 2.35 (s, 6H), 2.70-3.20 (m, 6H),
4.40-4.53 (m, 2H), 4.93-5.32 (m, 5H), 6.29 (s, 2H), 6.96-7.64
(m, 14H).
Boc-Dmt-Tic-D-Asp-NH-Ph. Boc-Dmt-Tic-D-Asp(OBzl)-NH-
Ph was treated with Pd/C and H2 as reported for Boc-Dmt-Tic-
Asp-NH-Ph: yield 0.08 g (95%); Rf (B) 0.64; HPLC K′ 6.95; mp
1
(M + H)+. H NMR (DMSO-d6) δ 1.40 (s, 9H), 2.72-2.97 (d,
2H), 5.17-5.34 (m, 3H), 7.19-7.60 (m, 10H).
164-166 °C; [R]20 +12.8; m/z 660 (M + H)+.
TFA·H-Asp(OBzl)-NH-Ph. Boc-Asp(OBzl)-NH-Ph was treated
with TFA as reported for TFA·H-Gly-N(Me)-Ph: yield 0.54 g
(96%); Rf (A) 0.75; HPLC K′ 5.02; mp 138-140 °C; [R]20D +15.2;
m/z 299 (M + H)+.
D
TFA·H-Dmt-Tic-D-Asp-NH-Ph (7). Boc-Dmt-Tic-D-Asp-NH-
Ph was treated with TFA as reported for TFA·H-Dmt-Tic-Gly-
N(Me)-Ph: yield 0.04 g (93%); Rf (A) 0.64; HPLC K′ 4.15; mp
150-152 °C; [R]20D +12.5; m/z 560 (M + H)+. 1H NMR (DMSO-
d6) δ 2.35 (s, 6H), 2.70-3.19 (m, 6H), 3.96-4.53 (m, 3H),
4.85-4.90 (m, 2H), 6.29 (s, 2H), 6.96-7.66 (m, 9H); Anal.
C33H35F3N4O8: C; H; N.
Boc-Tic-Asp(OBzl)-NH-Ph. This intermediate was obtained by
condensation of Boc-Tic-OH with TFA·H-Asp(OBzl)-NH-Ph via
WSC/HOBt as reported for Boc-Tic-Gly-N(Me)-Ph: yield 0.21 g
(88%); Rf (B) 0.82; HPLC K′ 6.43; mp 147-149 °C; [R]20D +5.2;
Boc-Asp(OBzl)-N(Me)-Ph. This intermediate was obtained by
condensation of Boc-Asp(OBzl)-OH with N-methylaniline via
mixed anhydride as reported for Boc-Gly-N(Me)-Ph: yield 0.43 g
(90%); Rf (B) 0.75; HPLC K′ 7.11; mp 126-128 °C; [R]20D +10.3;
m/z 413 (M + H)+. 1H NMR (DMSO-d6) δ 1.40 (s, 9H), 2.72-2.97
(m, 5H), 5.17-5.34 (m, 3H), 7.10-7.31 (m, 10H).
1
m/z 559 (M + H)+. H NMR (DMSO-d6) δ 1.38-1.42 (d, 9H),
2.72-3.17 (m, 4H), 4.17-4.27 (m, 2H), 4.92-5.34 (m, 4H),
6.96-7.64 (m, 14H).
TFA·H-Tic-Asp(OBzl)-NH-Ph. Boc-Tic-Asp(OBzl)-NH-Ph was
treated with TFA as reported for TFA·H-Tic-Gly-N(Me)-Ph: yield
0.18 g (96%); Rf (A) 0.73; HPLC K′ 4.21; mp 145-147 °C; [R]20
TFA·H-Asp(OBzl)-N(Me)-Ph. Boc-Asp(OBzl)-N(Me)-Ph was
treated with TFA as reported for TFA·H-Gly-N(Me)-Ph: yield
D
+5.3; m/z 459 (M + H)+.
Boc-Dmt-Tic-Asp(OBzl)-NH-Ph. This intermediate was ob-
tained by condensation of Boc-Dmt-OH with TFA·H-Tic-As-
p(OBzl)-NH-Ph via WSC/HOBt as reported for Boc-Dmt-Tic-Gly-
N(Me)-Ph: yield 0.14 g (87%); Rf (B) 0.78; HPLC K′ 9.31; mp
165-167 °C; [R]20D -2.5; m/z 750 (M + H)+. 1H NMR (DMSO-
d6) δ 1.38-1.42 (d, 9H), 2.35 (s, 6H), 2.72-3.17 (m, 6H),
4.41-4.51 (m, 2H), 4.92-5.34 (m, 5H), 6.29 (s, 2H), 6.96-7.64
(m, 14H).
0.40 g (97%); Rf (A) 0.77; HPLC K′ 5.11; mp 135-139 °C; [R]20
D
+14.7; m/z 313 (M + H)+.
Boc-Tic-Asp(OBzl)-N(Me)-Ph. This intermediate was obtained
by condensation of Boc-Tic-OH with TFA·H-Asp(OBzl)-N(Me)-
Ph via WSC/HOBt as reported for Boc-Tic-Gly-N(Me)-Ph: yield
0.21 g (85%); Rf (B) 0.84; HPLC K′ 6.45; mp 144-146 °C; [R]20
D
1
+4.8; m/z 573 (M + H)+. H NMR (DMSO-d6) δ 1.38-1.42 (d,
9H), 2.73-3.19 (m, 7H), 4.17-4.27 (m, 2H), 4.92-5.34 (m, 4H),
6.96-7.31 (m, 14H).
Boc-Dmt-Tic-Asp-NH-Ph. To a solution of Boc-Dmt-Tic-
Asp(OBzl)-NH-Ph (0.11 g, 0.15 mmol) in methanol (30 mL) was
added Pd/C (10%, 0.07 g), and H2 was bubbled for 1 h at room
temperature. After filtration, the solution was evaporated to dryness.
The residue was crystallized from Et2O/Pe (1:9, v/v): yield 0.09 g
(95%); Rf (B) 0.67; HPLC K′ 7.19; mp 169-171 °C; [R]20D -6.4;
m/z 660 (M + H)+.
TFA·H-Tic-Asp(OBzl)-N(Me)-Ph. Boc-Tic-Asp(OBzl)-N(Me)-
Ph was treated with TFA as reported for TFA·H-Tic-Gly-N(Me)-
Ph: yield 0.18 g (97%); Rf (A) 0.75; HPLC K′ 4.25; mp 141-143
°C; [R]20 +4.6; m/z 473 (M + H)+.
D
Boc-Dmt-Tic-Asp(OBzl)-N(Me)-Ph. This intermediate was
obtained by condensation of Boc-Dmt-OH with TFA·H-Tic-
Asp(OBzl)-N(Me)-Ph via WSC/HOBt as reported for Boc-Dmt-
Tic-Gly-N(Me)-Ph: yield 0.13 g (88%); Rf (B) 0.81; HPLC K′ 9.36;
TFA·H-Dmt-Tic-Asp-NH-Ph (6). Boc-Dmt-Tic-Asp-NH-Ph
was treated with TFA as reported for TFA·H-Dmt-Tic-Gly-N(Me)-
Ph: yield 0.09 g (96%); Rf (A) 0.67; HPLC K′ 4.21; mp 158-160
°C; [R]20D -7.3; m/z 560 (M + H)+. 1H NMR (DMSO-d6) δ 2.35
(s, 6H), 2.72-3.17 (m, 6H), 3.95-4.51 (m, 3H), 4.86-4.92 (m,
2H), 6.29 (s, 2H), 6.96-7.64 (m, 9H); Anal. C33H35F3N4O8: C; H;
N.
mp 160-162 °C; [R]20 -3.5; m/z 764 (M + H)+. 1H NMR
D
(DMSO-d6) δ 1.38-1.42 (d, 9H), 2.35 (s, 6H), 2.70-3.17 (m, 9H),
4.43-4.51 (m, 2H), 4.92-5.34 (m, 5H), 6.29 (s, 2H), 6.96-7.31
(m, 14H).
Boc-Dmt-Tic-Asp-N(Me)-Ph. Boc-Dmt-Tic-Asp(OBzl)-N(Me)-
Ph was treated with Pd/C and H2 as reported for Boc-Dmt-Tic-
Asp-NH-Ph: yield 0.08 g (94%); Rf (B) 0.69; HPLC K′ 7.23; mp
Boc-D-Asp(OBzl)-NH-Ph. This intermediate was obtained by
condensation of Boc-D-Asp(OBzl)-OH with aniline via mixed
anhydride as reported for Boc-Gly-N(Me)-Ph: yield 0.38 g (92%);
165-167 °C; [R]20 -7.3; m/z 674 (M + H)+.
D
TFA·H-Dmt-Tic-Asp-N(Me)-Ph (8). Boc-Dmt-Tic-Asp-N(Me)-
Ph was treated with TFA as reported for TFA·H-Dmt-Tic-Gly-
N(Me)-Ph: yield 0.04 g (94%); Rf (A) 0.69; HPLC K′ 4.26; mp
152-154 °C; [R]20D -8.2; m/z 574 (M + H)+. 1H NMR (DMSO-
d6) δ 2.35 (s, 6H), 2.70-3.17 (m, 9H), 3.95-4.51 (m, 3H),
4.86-4.92 (m, 2H), 6.29 (s, 2H), 6.96-7.31 (m, 9H); Anal.
C34H37F3N4O8: C; H; N.
Rf (B) 0.73; HPLC K′ 7.02; mp 129-132 °C; [R]20 -12.5; m/z
D
1
399 (M + H)+. H NMR (DMSO-d6) δ 1.40 (s, 9H), 2.72-2.97
(d, 2H), 5.17-5.34 (m, 3H), 7.19-7.60 (m, 10H).
TFA·H-D-Asp(OBzl)-NH-Ph. Boc-D-Asp(OBzl)-NH-Ph was
treated with TFA as reported for TFA·H-Gly-N(Me)-Ph: yield
0.25 g (96%); Rf (A) 0.75; HPLC K′ 5.02; mp 138-140 °C; [R]20
D
-15.2; m/z 299 (M + H)+.
Boc-D-Asp(OBzl)-N(Me)-Ph. This intermediate was obtained by
condensation of Boc-D-Asp(OBzl)-OH with N-methylaniline via
mixed anhydride as reported for Boc-Gly-N(Me)-Ph: yield 0.42 g
(88%); Rf (B) 0.75; HPLC K′ 7.11; mp 126-128 °C; [R]20D -10.3;
m/z 413 (M + H)+. 1H NMR (DMSO-d6) δ 1.40 (s, 9H), 2.72-2.97
(m, 5H), 5.17-5.34 (m, 3H), 7.10-7.31 (m, 10H).
Boc-Tic-D-Asp(OBzl)-NH-Ph. This intermediate was obtained
by condensation of Boc-Tic-OH with TFA·H-D-Asp(OBzl)-NH-
Ph via WSC/HOBt as reported for Boc-Tic-Gly-N(Me)-Ph: yield
0.17 g (87%); Rf (B) 0.80; HPLC K′ 6.38; mp 147-149 °C; [R]20
D
1
+8.4; m/z 559 (M + H)+. H NMR (DMSO-d6) δ 1.38-1.42 (d,
TFA·H-D-Asp(OBzl)-N(Me)-Ph. Boc-D-Asp(OBzl)-N(Me)-Ph
was treated with TFA as reported for TFA·H-Gly-N(Me)-Ph: yield
9H), 2.73-3.19 (m, 4H), 4.21-4.29 (m, 2H), 4.90-5.31 (m, 4H),
6.96-7.64 (m, 14H).
TFA·H-Tic-D-Asp(OBzl)-NH-Ph. Boc-Tic-D-Asp(OBzl)-NH-
Ph was treated with TFA as reported for TFA·H-Tic-Gly-N(Me)-
Ph: yield 0.15 g (97%); Rf (A) 0.71; HPLC K′ 4.17; mp 146-148
0.39 g (97%); Rf (A) 0.77; HPLC K′ 5.11; mp 135-139 °C; [R]20
D
-14.7; m/z 313 (M + H)+.
Boc-Tic-D-Asp(OBzl)-N(Me)-Ph. This intermediate was ob-
tained by condensation of Boc-Tic-OH with TFA·H-D-Asp(OBzl)-
N(Me)-Ph via WSC/HOBt as reported for Boc-Tic-Gly-N(Me)-Ph:
yield 0.19 g (87%); Rf (B) 0.83; HPLC K′ 6.44; mp 145-147 °C;
[R]20D +7.8; m/z 573 (M + H)+. 1H NMR (DMSO-d6) δ 1.38-1.42
(d, 9H), 2.73-3.19 (m, 7H), 4.21-4.29 (m, 2H), 4.90-5.31 (m,
4H), 6.96-7.35 (m, 14H).
°C; [R]20 +10.1; m/z 459 (M + H)+.
D
Boc-Dmt-Tic-D-Asp(OBzl)-NH-Ph. This intermediate was ob-
tained by condensation of Boc-Dmt-OH with TFA·H-Tic-D-
Asp(OBzl)-NH-Ph via WSC/HOBt as reported for Boc-Dmt-Tic-
Gly-N(Me)-Ph: yield 0.11 g (89%); Rf (B) 0.75; HPLC K′ 8.98;
mp 160-162 °C; [R]20 +8.6; m/z 750 (M + H)+. 1H NMR
D

5114 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Balboni et al.
TFA·H-Tic-D-Asp(OBzl)-N(Me)-Ph. Boc-Tic-D-Asp(OBzl)-
N(Me)-Ph was treated with TFA as reported for TFA·H-Tic-Gly-
N(Me)-Ph: yield 0.16 g (96%); Rf (A) 0.73; HPLC K′ 4.24; mp
tert-Butyl-(R)-2-((benzyloxy)carbonyl)-1-(1H-benzo[d]imida-
zol-2-yl)ethylcarbamate [Boc-NH-(R)CH(CH2-COOBzl)-Bid].
A solution of Boc-D-Asp(OBzl)-OH (1 g, 3.1 mmol) and NMM
(0.34 mL, 3.1 mmol) in DMF (10 mL) was treated at -20 °C with
IBCF (0.4 mL, 3.1 mmol). After 10 min at -20 °C, o-phenylen-
diamine (0.33 g, 3.1 mmol) was added. The reaction mixture was
allowed to stir while slowly warming to room temperature (1 h)
and was then stirred for 3 h. The solvent was evaporated, and the
residue was partitioned between EtOAc and H2O. The AcOEt layer
was washed with NaHCO3 (5% in H2O) and brine and dried over
Na2SO4. The solution was filtered, the solvent was evaporated, and
the residual solid was dissolved in glacial acetic acid (10 mL). The
solution was heated at 60 °C for 1 h. After the solvent was
evaporated, the residue was precipitated from Et2O/Pe (1:9, v/v):
yield 1 g (82%); Rf (B) 0.52; HPLC K′ 6.50; mp 136-138 °C;
[R]20D -15.8; m/z 396 (M + H)+. 1H NMR (DMSO-d6) δ 1.40 (s,
9H), 2.67-2.92 (m, 2H), 5.34-5.51 (m, 3H), 7.19-7.70 (m, 9H).
142-144 °C; [R]20 +9.2; m/z 473 (M + H)+.
D
Boc-Dmt-Tic-D-Asp(OBzl)-N(Me)-Ph. This intermediate was
obtained by condensation of Boc-Dmt-OH with TFA·H-Tic-D-
Asp(OBzl)-N(Me)-Ph via WSC/HOBt as reported for Boc-Dmt-
Tic-Gly-N(Me)-Ph: yield 0.12 g (88%); Rf (B) 0.79; HPLC K′ 9.08;
mp 156-158 °C; [R]20 +7.8; m/z 764 (M + H)+. 1H NMR
D
(DMSO-d6) δ 1.38-1.42 (d, 9H), 2.35 (s, 6H), 2.70-3.20 (m, 9H),
4.40-4.53 (m, 2H), 4.93-5.32 (m, 5H), 6.29 (s, 2H), 6.96-7.35
(m, 14H).
Boc-Dmt-Tic-D-Asp-N(Me)-Ph. Boc-Dmt-Tic-D-Asp(OBzl)-
N(Me)-Ph was treated with Pd/C and H2 as reported for Boc-Dmt-
Tic-Asp-NH-Ph: yield 0.08 g (93%); Rf (B) 0.68; HPLC K′ 7.03;
mp 155-157 °C; [R]20 +11.3; m/z 674 (M + H)+.
D
TFA·H-Dmt-Tic-D-Asp-N(Me)-Ph (9). Boc-Dmt-Tic-D-Asp-
N(Me)-Ph was treated with TFA as reported for TFA·H-Dmt-Tic-
Gly-N(Me)-Ph: yield 0.04 g (96%); Rf (A) 0.68; HPLC K′ 4.23;
2TFA · H2N-(R)CH(CH2-COOBzl)-Bid.
Boc-NH-
(R)CH(CH2-COOBzl)-Bid was treated with TFA as reported for
TFA·H-Gly-N(Me)-Ph: yield 0.51 g (96%); Rf (A) 0.78; HPLC K′
mp 145-147 °C; [R]20 +11.7; m/z 574 (M + H)+. 1H NMR
D
4.20; mp 142-144 °C; [R]20 -17.3; m/z 296 (M + H)+.
D
(DMSO-d6) δ 2.35 (s, 6H), 2.70-3.19 (m, 9H), 3.96-4.53 (m, 3H),
4.85-4.90 (m, 2H), 6.29 (s, 2H), 6.96-7.35 (m, 9H); Anal.
C34H37F3N4O8: C; H; N.
Boc-Tic-NH-(R)CH(CH2-COOBzl)-Bid. To a solution of Boc-
Tic-OH (0.28 g, 1 mmol) and 2TFA·H2N-(R)CH(CH2-COOBzl)-
Bid (0.52 g, 1 mmol) in DMF (10 mL) at 0 °C, NMM (0.2 mL, 2
mmol), HOBt (0.17 g, 1.1 mmol), and WSC (0.21 g, 1.1 mmol)
were added. The reaction mixture was stirred for 3 h at 0 °C and
24 h at room temperature. After DMF was evaporated, the residue
was dissolved in EtOAc and washed with NaHCO3 (5% in H2O),
and brine. The organic phase was dried (Na2SO4) and evaporated
to dryness. The residue was precipitated from Et2O/Pe (1:9, v/v):
yield 0.48 g (87%); Rf (B) 0.72; HPLC K′ 5.92; mp 155-157 °C;
[R]20D -1.6; m/z 556 (M + H)+. 1H NMR (DMSO-d6) δ 1.38-1.42
(d, 9H), 2.80-3.17 (m, 4H), 4.17-4.27 (m, 2H), 4.92-5.51 (m,
4H), 6.96-7.70 (m, 13H).
(S)-tert-butyl 3-((S)-2-((benzyloxy)carbonyl)-1-(1-methyl-1H-
benzo[d]imidazol-2-yl)ethylcarbamoyl)-3,4-dihydroisoquinoline-
2(1H)-carboxylate [Boc-Tic-NH-(S)CH(CH2-COOBzl)-Bid(N1-
Me)]. To a solution of Boc-Tic-NH-(S)CH(CH2-COOBzl)-Bid8
(0.27 g; 0.40 mmol) in DMF (10 mL) at room temperature, K2CO3
(0.25 g; 1.8 mmol) and, after 1 h, iodomethane (0.03 mL; 0.42
mmol), were added. The reaction mixture was stirred for 24 h at
room temperature. After DMF was evaporated, the residue was
dissolved in EtOAc and washed with NaHCO3 (5% in H2O) and
brine. The organic phase was dried (Na2SO4) and evaporated to
dryness. The residue was precipitated from Et2O/Pe (1:9, v/v): yield
(S)-tert-Butyl 3-((R)-2-((Benzyloxy)carbonyl)-1-(1-methyl-1H-
benzo[d]imidazol-2-yl)ethylcarbamoyl)-3,4-dihydroisoquinoline-
2(1H)-carboxylate [Boc-Tic-NH-(R)CH(CH2-COOBzl)-Bid(N1-
Me)]. This intermediate was obtained by alkylation of Boc-Tic-
NH-(R)CH(CH2-COOBzl)-Bid with K2CO3 and iodomethane as
reported for Boc-Tic-NH-(S)CH(CH2-COOBzl)-Bid(N1-Me): yield
0.20 g (90%); Rf (B) 0.81; HPLC K′ 6.15; mp 151-153 °C; [R]20
D
1
+9.7; m/z 570 (M + H)+. H NMR (DMSO-d6) δ 1.38-1.42 (d,
9H), 2.80-3.17 (m, 4H), 3.63 (s, 3H), 4.17-4.27 (m, 2H),
4.92-5.51 (m, 4H), 6.96-7.70 (m, 13H).
2TFA·H-Tic-NH-(S)CH(CH2-COOBzl)-Bid(N1-Me). Boc-
Tic-NH-(S)CH(CH2-COOBzl)-Bid(N1-Me) was treated with TFA
as reported for TFA·H-Tic-Gly-N(Me)-Ph: yield 0.21 g (93%); Rf
(A) 0.71; HPLC K′ 5.52; mp 144-146 °C; [R]20D +10.1; m/z 470
(M + H)+.
0.22 g (88%); Rf (B) 0.77; HPLC K′ 6.07; mp 154-156 °C; [R]20
D
1
+4.3; m/z 570 (M + H)+. H NMR (DMSO-d6) δ 1.38-1.42 (d,
9H), 2.82-3.20 (m, 4H), 3.63 (s, 3H), 4.15-4.29 (m, 2H),
4.90-5.49 (m, 4H), 6.96-7.70 (m, 13H).
Boc-Dmt-Tic-NH-(S)CH(CH2-COOBzl)-Bid(N1-Me). To a
solution of Boc-Dmt-OH (0.08 g, 0.26 mmol) and 2TFA·H-Tic-
NH-(S)CH(CH2-COOBzl)-Bid(N1-Me) (0.18 g, 0.26 mmol) in
DMF (10 mL) at 0 °C, NMM (0.06 mL, 0.52 mmol), HOBt (0.04
g, 0.29 mmol), and WSC (0.05 g, 0.29 mmol) were added. The
reaction mixture was stirred for 3 h at 0 °C and 24 h at room
temperature. After DMF was evaporated, the residue was dissolved
in EtOAc and washed with NaHCO3 (5% in H2O), and brine. The
organic phase was dried (Na2SO4) and evaporated to dryness. The
residue was precipitated from Et2O/Pe (1:9, v/v): yield 0.17 g (86%);
2TFA·H-Tic-NH-(R)CH(CH2-COOBzl)-Bid(N1-Me). Boc-
Tic-NH-(R)CH(CH2-COOBzl)-Bid(N1-Me) was treated with TFA
as reported for TFA·H-Tic-Gly-N(Me)-Ph: yield 0.21 g (92%); Rf
(A) 0.68; HPLC K′ 5.39; mp 148-150 °C; [R]20 +5.4; m/z 470
D
(M + H)+.
Boc-Dmt-Tic-NH-(R)CH(CH2-COOBzl)-Bid(N1-Me). This
intermediate was obtained by condensation of Boc-Dmt-OH with
2TFA·H-Tic-NH-(R)CH(CH2-COOBzl)-Bid(N1-Me) via WSC/
HOBt as reported for Boc-Dmt-Tic-NH-(S)CH(CH2-COOBzl)-
Bid(N1-Me): yield 0.17 g (86%); Rf (B) 0.64; HPLC K′ 8.28; mp
169-171 °C; [R]20D -2.7; m/z 760 (M + H)+. 1H NMR (DMSO-
d6) δ 1.38-1.42 (d, 9H), 2.35 (s, 6H), 2.94-3.22 (m, 6H), 3.63 (s,
3H), 4.40-4.52 (m, 2H), 4.92-5.48 (m, 5H), 6.29 (s, 2H),
6.96-7.70 (m, 13H).
Rf (B) 0.68; HPLC K′ 9.23; mp 165-167 °C; [R]20 +3.8; m/z
D
1
760 (M + H)+. H NMR (DMSO-d6) δ 1.38-1.42 (d, 9H), 2.35
(s, 6H), 2.92-3.20 (m, 6H), 3.63 (s, 3H), 4.41-4.51 (m, 2H),
4.92-5.51 (m, 5H), 6.29 (s, 2H), 6.96-7.70 (m, 13H).
Boc-Dmt-Tic-NH-(S)CH(CH2-COOH)-Bid(N1-Me). Boc-
Dmt-Tic-NH-(S)CH(CH2-COOBzl)-Bid(N1-Me) was treated with
Pd/C and H2 as reported for Boc-Dmt-Tic-Asp-NH-Ph: yield 0.11 g
(94%); Rf (B) 0.55; HPLC K′ 7.26; mp 166-168 °C; [R]20D +4.6;
m/z 671 (M + H)+.
Boc-Dmt-Tic-NH-(R)CH(CH2-COOH)-Bid(N1-Me). Boc-
Dmt-Tic-NH-(R)CH(CH2-COOBzl)-Bid(N1-Me) was treated with
Pd/C and H2 as reported for Boc-Dmt-Tic-Asp-NH-Ph: yield 0.10 g
(92%); Rf (B) 0.50; HPLC K′ 6.98; mp 172-174 °C; [R]20D -1.9;
m/z 671 (M + H)+.
2TFA·H-Dmt-Tic-NH-(S)CH(CH2-COOH)-Bid(N1-Me) (10).
Boc-Dmt-Tic-NH-(S)CH(CH2-COOH)-Bid(N1-Me) was treated
with TFA as reported for TFA·H-Dmt-Tic-Gly-N(Me)-Ph: yield
2TFA·H-Dmt-Tic-NH-(R)CH(CH2-COOH)-Bid(N1-Me) (11).
Boc-Dmt-Tic-NH-(R)CH(CH2-COOH)-Bid(N1-Me) was treated
with TFA as reported for TFA·H-Dmt-Tic-Gly-N(Me)-Ph: yield
0.05 g (96%); Rf (A) 0.69 HPLC K′ 4.56; mp 152-154 °C; [R]20
0.07 g (96%); Rf (A) 0.58 HPLC K′ 4.02; mp 161-163 °C; [R]20
D
D
1
+7.3; m/z 571 (M + H)+. H NMR (DMSO-d6) δ 2.35 (s, 6H),
1
+2.2; m/z 571 (M + H)+. H NMR (DMSO-d6) δ 2.35 (s, 6H),
2.92-3.20 (m, 6H), 3.63-3.95 (m, 4H), 4.41-4.51 (m, 2H),
4.92-5.20 (m, 2H), 6.29 (s, 2H), 6.96-7.70 (m, 8H). Anal.
C36H37F6N5O9: C; H; N.
2.90-3.22 (m, 6H), 3.61-3.93 (m, 4H), 4.42-4.53 (m, 2H),
4.90-5.18 (m, 2H), 6.29 (s, 2H), 6.96-7.70 (m, 8H). Anal.
C36H37F6N5O9: C; H; N.

Lead Compounds Containing the Dmt-Tic
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 5115
2TFA·H-Tic-NH-(R)CH(CH2-COOBzl)-Bid. Boc-Tic-NH-
(R)CH(CH2-COOBzl)-Bid was treated with TFA as reported for
TFA·H-Tic-Gly-N(Me)-Ph: yield 0.18 g (93%); Rf (A) 0.66; HPLC
tions (n values are listed in Table 1 in parenthesis and results are
mean ( SE). Unlabeled peptide (2 µM) was used to determine
nonspecific binding in the presence of 1.9 nM [3H]deltorphin II
(45.0 Ci/mmol, Perkin-Elmer, Boston, MA; KD ) 1.4 nM) for
δ-opioid receptors and 3.5 nM [3H]DAMGO (50.0 Ci/mmol),
Amersham Bioscience, Buckinghamshire, UK; KD ) 1.5 nM) for
µ-opioid receptors. Glass fiber filters (Whatman GFC) were soaked
in 0.1% polyethylenimine in order to enhance the signal-to-noise
ratio of the bound radiolabeled-synaptosome complex, and the filters
were washed thrice in ice-cold buffered BSA,40 and the affinity
constants (Ki) were calculated according to Cheng and Prusoff..43
Biological Activity in Isolate Tissue Preparations. The my-
enteric plexus longitudinal muscle preparations (2-3 cm segments)
from the small intestine of male Hartley strain of guinea pigs (GPI)
measured µ-opioid receptor agonism, and a single mouse vas
deferens (MVD) was used to determine δ-opioid receptor agonism
as described previously.44 The isolated tissues were suspended in
organ baths containing balanced salt solutions in a physiological
buffer, pH 7.5. Agonists were tested for the inhibition of electrically
evoked contraction and expressed as IC50 (nM) obtained from the
dose-response curves. The IC50 values represent the mean ( SE
of five or six separate assays, and the δ-antagonist potencies in the
MVD assay were determined against the δ agonist deltorphin-II,
while µ antagonism (GPI assay) used the µ agonist endomorphin-
2. Antagonism is expressed as pA2 determined using the Schild
Plot.45
Animals for in Vitro and in Vivo Studies. Laboratory animals
were used under protocols approved and governed by the Animal
Care and Use Committees of Tohoku Pharmaceutical University,
University of Ferrara and the National Institute of Environmental
Health Sciences.
In Vivo Assessment of Opioid Bioactivity. Male Swiss-Webster
mice (20-25 g, Taconic, Germantown, NY) were housed on a 12 h
light/dark cycle with free access to food and water and experimental
protocol approved by the NIEHS Animal Care and Use Committee.
Intracerebroventricular injection (icv) used a Hamilton microsy-
ringe with disposable 26 gauge needle that was inserted 2.3-3 mm
into the ventricular sinus at the bregma as described;46 the total
volume administered was 5 µL (peptide in saline). Upon completion
of the study, mice were sacrificed according ACUC protocols and
those animals having needle tract 2 mm lateral from the midline
were counted as being valid.
Hot-plate test consisted of animals placed on an electrically
heated plate (55 ( 0.1 °C, IITC MODEL 39D Hot Plate analgesia
meter, IITC Inc., Woodland Hills, CA) 10 min after icv administra-
tion. Hot-plate latency (HPL) is the interval between placement of
mice onto the hot plate and observing movement consisting of either
jumping, licking, or shaking their hind paws; a baseline latency of
5-10 s (preresponse time) and maximal cut off time of 30 s.
Measurements were repeated every 10 min and testing was
terminated when the HPL was close to the preresponse time.
Spinal effects used of a tail-flick instrument (Columbus Instru-
ments, Columbus, OH). Radiant heat was applied on the dorsal
surface of the tail and the latency before removal of the tail from
the onset of the radiant heat is defined as the tail-flick latency. The
baseline was to 2-3 s (preresponse time) and a cut off time was
set at 8 s to avoid external heat-related damage. The time sequence
was the same as described for the HPL test47,48
Statistical Analysis. Statistical significance of the data was
estimated by one-way analysis of variance (ANOVA) followed by
Dunnett’s test using the computer software program JMP (SAS
Institute Inc., Cary, NC) and considered significant at p < 0.05.
Minimum effective dose (MED) is the minimum dose of compound
showing statistically significant antinociceptive effect expressed as
the area under the time-response curve (AUC) value compared to
a saline-treated group. The AUC was obtained by plotting the
response time (sec) on the ordinate and time (min) on the abscissa
after administration of the compounds. The percent maximum
possible effect (% MPE) was calculated as follows: ([postdrug
response latency - predrug response latency]/[cutoff time (30 s)
- predrug response latency]) × 100.
K′ 5.35; mp 149-151 °C; [R]20 +6.3; m/z 456 (M + H)+.
D
Boc-Dmt-Tic-NH-(R)CH(CH2-COOBzl)-Bid. This intermedi-
ate was obtained by condensation of Boc-Dmt-OH with 2TFA·H-
Tic-H2N-(R)CH(CH2-COOBzl)-Bid via WSC/HOBt as reported
for Boc-Dmt-Tic-NH-(S)CH(CH2-COOBzl)-Bid(N1-Me): yield
0.15 g (87%); Rf (B) 0.61; HPLC K′ 8.01; mp 174-176 °C; [R]20
D
1
-3.5; m/z 747 (M + H)+. H NMR (DMSO-d6) δ 1.36-1.40 (d,
9H), 2.35 (s, 6H), 2.63-3.17 (m, 6H), 4.42-4.53 (m, 2H),
4.92-5.51 (m, 5H), 6.29 (s, 2H), 6.96-7.70 (m, 13H).
Boc-Dmt-Tic-NH-(R)CH(CH2-COOH)-Bid. Boc-Dmt-Tic-
NH-(R)CH(CH2-COOBzl)-Bid was treated with Pd/C and H2 as
reported for Boc-Dmt-Tic-Asp-NH-Ph: yield 0.09 g (93%); Rf (B)
0.46; HPLC K′ 6.84; mp 176-178 °C; [R]20 -2.8; m/z 657 (M
D
+ H)+.
2TFA·H-Dmt-Tic-NH-(R)CH(CH2-COOH)-Bid (12). Boc-
Dmt-Tic-NH-(R)CH(CH2-COOH)-Bid was treated with TFA as
reported for TFA·H-Dmt-Tic-Gly-N(Me)-Ph: yield 0.06 g (96%);
Rf (A) 0.52; HPLC K′ 3.92; mp 166-168 °C; [R]20 +3.5; m/z
D
557 (M + H)+. H NMR (DMSO-d6) δ 2.35 (s, 6H), 2.90-3.17
1
(m, 6H), 3.95-4.52 (m, 3H), 4.90-5.21 (m, 2H), 6.29 (s, 2H),
6.96-7.70 (m, 8H). Anal. C35H35F6N5O9: C; H; N.
Boc-Asp(OBzl)-NH-Bzl. This intermediate was obtained by
condensation of Boc-Asp(OBzl)-OH with benzylamine via mixed
anhydride as reported for Boc-Gly-N(Me)-Ph: yield 0.45 g (90%);
Rf (B) 0.77; HPLC K′ 7.18; mp 126-128 °C; [R]20 -11.2; m/z
D
1
413 (M + H)+. H NMR (DMSO-d6) δ 1.40 (s, 9H), 2.63-2.88
(d, 2H), 4.46 (s, 2H), 5.17-5.34 (m, 3H), 7.06-7.19 (m, 10H).
TFA·H-Asp(OBzl)-NH-Bzl. Boc-Asp(OBzl)-NH-Bzl was treated
with TFA as reported for TFA·H-Gly-N(Me)-Ph: yield 0.42 g
(96%); Rf (A) 0.78; HPLC K′ 5.18; mp 135-137 °C; [R]20D +14.3;
m/z 313 (M + H)+.
Boc-Tic-Asp(OBzl)-NH-Bzl. This intermediate was obtained by
condensation of Boc-Tic-OH with TFA·H-Asp(OBzl)-NH-Bzl via
WSC/HOBt as reported for Boc-Tic-Gly-N(Me)-Ph: yield 0.22 g
(87%); Rf (B) 0.86; HPLC K′ 6.51; mp 144-146 °C; [R]20D +4.7;
1
m/z 573 (M + H)+. H NMR (DMSO-d6) δ 1.38-1.42 (d, 9H),
2.63-3.17 (m, 4H), 4.17-4.46 (m, 4H), 4.92-5.34 (m, 4H),
6.96-7.19 (m, 14H).
TFA·H-Tic-Asp(OBzl)-NH-Bzl. Boc-Tic-Asp(OBzl)-NH-Bzl
was treated with TFA as reported for TFA·H-Tic-Gly-N(Me)-Ph:
yield 0.18 g (97%); Rf (A) 0.77; HPLC K′ 4.29; mp 140-142 °C;
[R]20 +4.7; m/z 473 (M + H)+.
D
Boc-Dmt-Tic-Asp(OBzl)-NH-Bzl. This intermediate was ob-
tained by condensation of Boc-Dmt-OH with TFA·H-Tic-As-
p(OBzl)-NH-Bzl via WSC/HOBt as reported for Boc-Dmt-Tic-Gly-
N(Me)-Ph: yield 0.13 g (88%); Rf (B) 0.82; HPLC K′ 9.38; mp
160-162 °C; [R]20D -1.9; m/z 764 (M + H)+. 1H NMR (DMSO-
d6) δ 1.38-1.42 (d, 9H), 2.35 (s, 6H), 2.63-3.17 (m, 6H),
4.41-4.51 (m, 4H), 4.92-5.34 (m, 5H), 6.29 (s, 2H), 6.96-7.19
(m, 14H).
Boc-Dmt-Tic-Asp-NH-Bzl. Boc-Dmt-Tic-Asp(OBzl)-NH-Bzl
was treated with Pd/C and H2 as reported for Boc-Dmt-Tic-Asp-
NH-Ph: yield 0.09 g (96%); Rf (B) 0.71; HPLC K′ 7.24; mp
164-166 °C; [R]20 -5.2; m/z 674 (M + H)+.
D
TFA·H-Dmt-Tic-Asp-NH-Bzl (13). Boc-Dmt-Tic-Asp-NH-Bzl
was treated with TFA as reported for TFA·H-Dmt-Tic-Gly-N(Me)-
Ph: yield 0.05 g (96%); Rf (A) 0.69; HPLC K′ 4.29; mp 154-156 °C;
1
[R]20 -6.7; m/z 574 (M + H)+. H NMR (DMSO-d6) δ 2.35 (s,
D
6H), 2.63-3.17 (m, 6H), 3.95-4.51 (m, 5H), 4.86-4.92 (m, 2H), 6.29
(s, 2H), 6.96-7.14 (m, 9H). Anal. C34H37F3N4O8: C; H; N.
Pharmacology. Competitive Binding Assays. Opioid receptor
affinities were determined under equilibrium conditions [2.5 h room
temperature (23 °C)] in competition assays using brain P2 synap-
tosomal membranes prepared from Sprague-Dawley rats.40,41
Synaptosomes were preincubated to remove endogenous opioid
peptides and stored at -80 °C in buffered 20% glycerol.40,42 Each
analogue was analyzed in duplicate assays using 5-8 dosages and
3-5 independent repetitions with different synaptosomal prepara-

5116 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Balboni et al.
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.
Conclusions
Starting from the assumption that even minor chemical
modifications can change the pharmacological profile of opioids,
such as peptides and pseudopeptides containing the Dmt-Tic
pharmacophore or nonpeptide derivatives related to morphine,33
we selected some reference compounds, especially our δ
agonists 1 and 4, and evaluated the influence of aspartic acid
and its chirality and the importance of the -NH-Ph and N1H-
Bid hydrogens in the inductions of δ agonism. The results
obtained confirm some expectations: (i) Asp increases the δ
selectivity by lowering µ affinity; (ii) Methylation of -NH-Ph
and N1H-Bid nitrogens transforms potent δ agonists in potent
δ antagonists. However other conclusions are quite unexpected:
(iii) The substitution of Gly with L-Asp or D-Asp in the δ agonist
1 gave potent and selective δ antagonists; in contrast, the same
substitution made in the δ agonist 3, produced the more selective
δ agonists 4 and 12; (iv) Asp chirality seems to be important
only in terms of functional bioactivity because analogues 4, 6,
8, and 10, containing L-Asp, are more active than the corre-
sponding diastereoisomers 12, 7, 9, and 11; but the same is not
true for receptor affinity. Finally, and totally unexpected and in
our opinion of potential interest, the potent and selective δ
antagonist 10 yielded an analgesic effect similar to 4 that was
reversed by naltrindole only when it was tested by the tail flick
method, and not in the hot plate test. Furthermore, 4 and 10
gave opposite behavioral effects in mice; 4 caused grooming
and agitation (constant, fast moving in the cage, burrowing in
the nesting material), while with 10, mice appeared sedated,
quiet, easily handled, moving slowly if at all; convulsions were
reported only in animals treated with the δ antagonist at high
doses icv. Considering the novelty of such a compound, more
detailed pharmacological studies are in progress both in vivo
(as an analgesic for neuropatic and inflammatory pain, antide-
pressant. and anxiolytic) and in vitro, considering also its
potential interaction with different receptors,34 or with het-
erodimers involving δ receptors.35 The last consideration is
reserved to preliminary enzymatic degradation studies that failed
to demonstrate the N-demethylation of 10 to the corresponding
4; more detailed studies (involving the use of different methods36
and/or other tissue preparations, such as kidney and liver30) are
just planned, also in collaboration with other laboratories. If
we will be able to demonstrate such enzymatic N-demethylation,
10 could be considered a new lead compound of potential
pharmacological utility for in vivo studies connected to δ opioid
receptors trafficking at the membrane level.37-39
(4) 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.
(5) Balboni, G.; Guerrini, R.; Salvadori, S.; Bianchi, C.; Rizzi, D.; Bryant,
S. D.; Lazarus, L. H. Evaluation of the Dmt-Tic pharmacophore:
conversion of a potent δ-opioid receptor antagonist into a potent δ
agonist and ligands with mixed properties. J. Med. Chem. 2002, 45,
713–720.
(6) Balboni, G.; Onnis, V.; Congiu, C.; Zotti, M.; Sasaki, Y.; Ambo, A.;
Bryant, S. D.; Jinsmaa, Y.; Lazarus, L. H.; Trapella, C.; Salvadori, S.
Effect of lysine at C-terminus of the Dmt-Tic opioid pharmacophore.
J. Med. Chem. 2006, 49, 5610–5617.
(7) Tryoen-To´th, P.; De´caillot, F. M.; Filliol, D.; Befort, K.; Lazarus, L. H.;
Schiller, P. W.; Schmidhammer, H.; Kieffer, B. L. Inverse agonism
and neutral antagonism at wild-type and constitutively active mutant
delta opioid receptors. J. Pharmacol. Exp. Ther. 2005, 313, 410–421.
(8) Balboni, G.; Salvadori, S.; Guerrini, R.; Negri, L.; Giannini, E.;
Jinsmaa, Y.; Bryant, S. D.; Lazarus, L. H. Potent δ-opioid receptor
agonists containing the Dmt-Tic pharmacophore. J. Med. Chem. 2002,
45, 5556–5563.
(9) Aguila, B.; Coulbault, M.; Le´veille´, F.; Davis, A.; To´th, G.; Borsodi,
A.; Balboni, G.; Salvadori, S.; Jauzac, P.; Allouche, S. In vitro and in
vivo pharmacological profile of UFP-512, a novel selective delta-opioid
receptor agonist; correlations between desensitization and tolerance.
Br. J. Pharmacol. 2007, 152, 1312–1324.
(10) Vergura, R.; Balboni, G.; Spagnolo, B.; Gavioli, E.; Lambert, D. G.;
McDonald, J.; Trapella, C.; Lazarus, l. H.; Regoli, D.; Guerrini, R.;
Salvadori, S.; Calo´, G. Anxiolytic and antidepressant-like activities
of H-Dmt-Tic-NH-CH(CH2-COOH)-Bid (UFP-512), a novel selective
delta opioid receptor agonist. Peptides 2008, 29, 93–103.
(11) Ryu, E. K.; Wu, Z.; Chen, K.; Lazarus, L. H.; Marczak, E. D.; Sasaki,
Y.; Ambo, A.; Salvadori, S.; Ren, C.; Zhao, H.; Balboni, G.; Chen,
X. Synthesis of a potent and selective 18F-labeled δ-opioid receptor
antagonist derived from the Dmt-Tic pharmacophore for positron
emission tomograpgy imaging. J. Med. Chem. 2008, 51, 1817–1823.
(12) Snook, L. A.; Milligan, G.; Kieffer, B. L.; Massotte, D. µ-δ opioid
receptor functional interaction: insight using receptor-G protein fusions.
J. Pharmacol. Exp. Ther. 2006, 318, 683–690.
(13) Snook, L. A.; Milligan, G.; Kieffer, B. L.; Massotte, D. Co-expression
of mu and delta opioid receptors as receptor-G protein fusion enhances
both mu and delta signalling via distinct mechanisms. J. Neurochem.
2008, 105, 865–873.
(14) Salvadori, S.; Trapella, C.; Fiorini, S.; Negri, L.; Lattanzi, R.; Bryant,
S. D.; Jinsmaa, Y.; Lazarus, L. H.; Balboni, G. A new opioid designed
multiple ligand derived from the µ opioid agonist endomorphin-2 and
the δ opioid antagonist pharmacophore Dmt-Tic. Bioorg. Med. Chem.
2007, 15, 6876–6881.
(15) Lee, Y. S.; Petrov, R.; Park, C. K.; Ma, S. W.; Davis, P.; Lai, J.;
Porreca, F.; Vardanyan, R.; Hruby, V. J. Development of novel
enkephalin analogues that have enhanced opioid activities at both µ
and δ opioid receptors. J. Med. Chem. 2007, 50, 5528–5532.
(16) Dai, X.; Cui, S. G.; Li, S. R.; Chen, Q.; Wang, R. Melatonin attenuates
the development of antinociceptive tolerance to delta-, but not to mu-
opioid receptor agonist in mice. BehaV. Brain Res. 2007, 182, 21–27.
(17) Jutkiewicz, E. M. The antidepressant-like effects of delta-opioid
receptor agonists. Mol. InterV. 2006, 6, 162–169.
(18) Narita, M.; Kuzumaki, N.; Miyatake, M.; Sato, F.; Wachi, H.; Seyama,
Y.; Suzuki, T. R. Role of δ-opioid receptor function in neurogenesis
and neuroprotection. J. Neurochem. 2006, 97, 1494–1505.
(19) Hill, M. P.; Hille, C. J.; Brotchie, J. M. Delta-opioid receptor agonists
as a therapeutic approach in Parkinson’s disease. Drug News Perspect.
2000, 13, 261–268.
(20) Tegeder, I.; Geisslinger, G. Opioids as modulators of cell death and
survival-unravelling mechanism and revealing new indications. Phar-
macol. ReV. 2004, 23, 351–366.
(21) Watson, M. J.; Holt, J. D.; O’Neill, S. J.; Wei, K.; Pendergast, W.;
Gross, G. J.; Gengo, P. J.; Chang, K. J. ARD-353 [4-((2R,5S)-4-(R)-
(4-diethylcarbamoxylphenyl)(3-hydroxyphenyl)methyl-2,5dimethylpip-
erazin-1-ylmethyl)benzoic acid], a novel nonpeptide δ receptor agonist,
reduces myocardial infarct size without central effects. J. Pharmacol.
Exp. Ther. 2006, 316, 423–430.
Acknowledgment. This study was supported in part by the
University of Cagliari (G.B.), the University of Ferrara (S.S.),
and by the Intramural Research Program of the NIH and NIEHS
(L.H.L. and E.D.M.).
Supporting Information Available: Chemistry general methods,
enzymatic degradation general methods, elemental analysis, MS,
and HPLC data. This material is available free of charge via the
Internet at http://pubs.acs.org.
References
(1) 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.
(2) 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.
(22) Pol, O.; Palacio, J. R.; Puig, M. M. The expression of δ-and κ-opioid
receptors is enhanced during intestinal inflammation in mice. J. Phar-
macol. Exp. Ther. 2003, 306, 455–462.
(3) Schiller, P. W.; Nguyen, T. M.-D.; Weltrowska, G.; Wilkes, B. C.;
Marsden, B. J.; Lemieux, C.; Chung, N. N. Differential stereochemical
(23) Stein, C.; Scha¨fer, M.; Machelska, H. Attaching pain at its source:
new perspectives on opioids. Nat. Med. 2003, 9, 1003–1008.

Lead Compounds Containing the Dmt-Tic
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 5117
(24) Balboni, G.; Guerrini, R.; Salvadori, S.; Negri, L.; Giannini, E.; Bryant,
S. D.; Jinsmaa, Y.; Lazarus, L. H. Conversion of the potent δ-opioid
agonist H-Dmt-Tic-NH-CH2-Bid into δ-opioid antagonists by N1-
benzimidazole alkylation. J. Med. Chem. 2005, 48, 8112–8114.
(25) Salvadori, S.; Fiorini, S.; Trapella, C.; Porreca, F.; Davis, P.; Sasaki,
Y.; Ambo, A.; Marczak, E. D.; Lazarus, L. H.; Balboni, G. Role of
benzimidazole (Bid) in the δ-opioid agonist pseudopeptide H-Dmt-
Tic-NH-CH2-Bid (UFP-502). Bioorg. Med. Chem. 2008, 16, 3032–
3038.
(26) Torregrossa, M. M.; Jutkiewicz, E. M.; Mosberg, H. I.; Balboni, G.;
Watson, S. J.; Woods, J. H. Peptidic delta opioid receptor agonists
produce antidepressant-like effects in the forced swim test and regulate
BDNF mRNA expression in rats. Brain Res. 2006, 1069, 172–181.
(27) Vergura, R.; Valenti, E.; Hebbes, C. P.; Gavioli, E. C.; Spagnolo, B.;
McDonald, J.; Lambert, D. G.; Balboni, G.; Salvadori, S.; Regoli, D.;
Calo`, G. Dmt-Tic-NH-CH2-Bid (UFP-502), a potent DOP receptor
agonist: in vitro and in vivo studies. Peptides 2006, 27, 3322–3330.
(28) Nestor, J. J., Jr.; Horner, B. L.; Ho, T. L.; Jones, G. H.; McRae, G.;
Vickery, B. H. Synthesis of a novel class of heteroaromatic amino
acids and their use in the preparation of analogues of luteinizing
hormone-releasing hormone. J. Med. Chem. 1984, 27, 320–325.
(29) Lee, Y. S.; Nyberg, J.; Moye, S.; Agnes, R. S.; Davis, P.; Ma, S. W.;
Lai, J.; Porreca, F.; Vardanyan, R.; Hruby, V. J. Understanding the
structural requirements of 4-anilinopiperidine analogues for biological
activities at µ and δ opioid receptors. Bioorg. Med. Chem. Lett. 2007,
17, 2161–2165.
(30) Codd, E. E.; Carson, J. R.; Colburn, R. W.; Dax, S. L.; Desai-Krieger,
D.; Martinez, R. P.; McKown, L. A.; Neilson, L. A.; Pitis, P. M.;
Stahle, P. L.; Stone, D. J.; Streeter, A. J.; Wu, W. N.; Zhang, S. P.
The novel, orally active, delta opioid RWJ-394674 is biotransformed
to the potent mu opioid RWJ-413216. J. Pharmacol. Exp. Ther. 2006,
318, 1273–1279.
(31) Jutkiewicz, E. M.; Baladi, M. G.; Folk, J. E.; Rice, K. C.; Woods,
J. H. The convulsive and electroencephalographic changes produced
by nonpeptidic delta opioid agonists in rats: comparison with penty-
lenetetrazol. J. Pharmacol. Exp. Ther. 2006, 317, 1337–1348.
(32) Danielsson, I.; Gasior, M.; Stevenson, G. W.; Folk, J. E.; Rice, K. C.;
Negus, S. S. Electroencephalographic and convulsant effects of the
delta opioid agonist SNC80 in rhesus monkeys. Pharmacol., Biochem.
BehaV. 2006, 85, 428–434.
(36) Koda, Y.; Del Borgo, M.; Wessling, S. T.; Lazarus, L. H.; Okada, Y.;
Toth, I.; Blanchfield, J. T. Synthesis and in vitro evaluation of a library
of modified endomorphin 1 peptides. Bioorg. Med. Chem. 2008, 16,
6286–6296.
(37) Lesscher, H. M.; Bailey, A.; Burbach, J. P.; Van Ree, J. M.; Kitchen,
I.; Gerrits, M. A. Receptor-selective changes in mu-, delta-, and kappa-
opioid receptors after chronic naltrexone treatment in mice. Eur.
J. Neurosci 2003, 17, 1006–1012.
(38) Cahill, C. M.; Holdridge, S. V.; Morinville, A. Trafficking of δ-opioid
receptors and other G-protein-coupled receptors: implications for pain
and analgesia. Trends Pharmacol. Sci. 2007, 28, 23–31.
(39) Archer-Lahlou, E.; Audet, N.; Amraei, M. G.; Huard, K.; Paquin-
Gobeil, M.; Pineyro, G. Src promotes delta opioid receptor (DOR)
desensitization by interfering with receptor recycling. J. Cell. Mol.
Med. 2008, . in press, doi:, 10.1111/j.1582-4934.2008.00308.x.
(40) 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, 1350–1355.
(41) Lazarus, L. H.; Salvadori, S.; Attila, M.; Grieco, P.; Bundy, D. M.;
Wilson, W. E.; Tomatis, R. Interaction of deltorphin with opioid
receptors: Molecular determinants for affinity and selectivity. Peptides
1993, 14, 21–28.
(42) Lazarus, L. H.; Wilson, W. E.; de Castglione, R.; Guglietta, A.
Dermorphin gene sequence peptide with high affinity and selectivity
for δ-opioid receptors. J. Biol. Chem. 1989, 264, 3047–3050.
(43) Cheng, Y. C.; Prusoff, W. H. Relationship between the inhibition
constant (Ki) and the concentration of inhibitor which cause 50% (IC50)
of an enzymatic reaction. Biochem. Pharmacol. 1973, 22, 3099–3108.
(44) Sasaki, Y.; Sasaki, A.; Niizuma, H.; Goto, H.; Ambo, A. Endomorphin
2 analogues containing Dmp residue as an aromatic amino acid
surrogate with high µ-opioid receptor affinity and selectivity. Bioorg.
Med. Chem. 2003, 11, 675–678.
(45) Arunlakshana, Q.; Schild, H. O. Some quantitative uses of drug
antagonists. Br. J. Pharmacol. 1959, 14, 48–58.
(46) Laursen, S. E.; Belknap, J. K. Intracerebroventricular injections in mice.
Some methodological refinements. J. Pharmacol. Methods 1986, 16,
355–357.
(47) Jinsmaa, Y.; Marczak, E. D.; Fujita, Y.; Shiotani, K.; Miyazaki, A.;
Li, T.; Tsuda, Y.; Ambo, A.; Sasaki, Y.; Bryant, S. D.; Okada, Y.;
Lazarus, L. H. Potent in vivo antinociception and opioid receptor
preference of the novel analogue [Dmt1]endomorphin-1. Pharmacol.,
Biochem. BehaV. 2006, 84, 252–258.
(48) Marczak, E. D.; Jinsmaa, Y.; Li, T.; Bryant, S. D.; Tsuda, Y.; Okada,
Y.; Lazarus, L. H. [N-Allyl-Dmt1]-Endomorphins are µ-opioid receptor
antagonists lacking inverse agonist properties. J Pharmacol Exp Ther.
2007, 323, 374–380.
(33) Wentland, M. P.; Sun, X.; Cohen, D. J.; Bidlack, J. M. Redefining
the structure-activity relationships of 2,6-methano-3-benzazocines.
Bioorg. Med. Chem. 2008, 16, 5653–5664.
(34) Kosson, P.; Bonney, I.; Carr, D. B.; Lipkowski, A. W. Endomorphins
interact with tachykinin receptors. Peptides 2005, 26, 1667–1669.
(35) Pello, O. M.; Mart´ınez-Mun˜oz, L.; Parrillas, V.; Serrano, A.; Rod-
r´ıgues-Frude, J. M.; Toro, M. J.; Lucas, P.; Monterrubio, M.; Mart´ınez-
A, C.; Mellado, M. Ligand stabilization of CXCR4/δ-opioid receptor
heterodimers reveals a mechanism for immune response regulation.
Eur. J. Immunol. 2008, 38, 537–549.
(49) Kosterlitz, H. W.; Watt, A. J. Kinetic parameters of narcotic agonists
and antagonists, with particular reference to N-allylnoroxymorphone
(naloxone). Br. J. Pharmacol. 1968, 33, 266–276.
JM800587E