New opioid designed multiple ligand from Dmt-Tic and morphinan pharmacophores

Article abstract of DOI:10.1021/jm0605785

Here, we report the synthesis of a designed multi-pharmacophore ligand derived from the linkage of a delta selective peptide antagonist (Dmt-Tic) and a mu/kappa morphinan agonist butorphan (MCL 101) through a two methylene spacer. The new compound MCL 450

Full text of DOI:10.1021/jm0605785

5640
J. Med. Chem. 2006, 49, 5640-5643
New Opioid Designed Multiple Ligand from Dmt-Tic and Morphinan Pharmacophores
John L. Neumeyer,† Xuemei Peng,† Brian I. Knapp,‡ Jean M. Bidlack,‡ Lawrence H. Lazarus,§ Severo Salvadori,|
Claudio Trapella,| and Gianfranco Balboni*,|,
Alcohol and Drug Abuse Research Center, McLean Hospital, HarVard Medical School, 115 Mill Street, Belmont, Massachusetts 02478,
Department of Pharmacology and Physiology, School of Medicine and Dentistry, UniVersity of Rochester, Rochester, New York 14642,
Medicinal Chemistry Group, Laboratory of Pharmacology and Chemistry, National Institute of EnVironmental Health Sciences, Research
Triangle Park, North Carolina 27709, Department of Pharmaceutical Sciences and Biotechnology Center, UniVersity of Ferrara,
I-44100 Ferrara, Italy, and Department of Toxicology, UniVersity of Cagliari, I-09124 Cagliari, Italy
ReceiVed May 16, 2006
Here, we report the synthesis of a designed multi-pharmacophore ligand derived from the linkage of a delta
selective peptide antagonist (Dmt-Tic) and a mu/kappa morphinan agonist butorphan (MCL 101) through
a two methylene spacer. The new compound MCL 450 maintains the same characteristics as those the two
reference compounds. MCL 450 represents a useful starting point for the synthesis of other multiple opioid
ligands endowed with analgesic properties with low tolerance and dependence.
Introduction
at the same time of administration, cleavable conjugates are
single molecules, which is one potential advantage that this
approach has over drug cocktails.2,3 The prevalence in the
literature of designing in new activities, that is, the synthesis
of designed multiple (DM) ligands from selective ligands,
indicates that this approach is certainly more popular and
probably more feasible than designing out activities from
nonselective ligands. For example, a mixed µ agonist/δ antago-
nist pseudopeptide was obtained by linking tail to tail a selective
δ antagonist (H-Tyr-TicΨ[CH2-NH]Cha-Phe-OH) with a selec-
tive µ agonist (H-Dmt-D-Arg-Phe-Lys-NH2).4 Recently, Neu-
meyer et al. reported the synthesis and pharmacological
evaluation of bivalent ligands containing homo and het-
erodimeric pharmacophores related to morphinans.5 Ligands
having two pharmacophores connected by a spacer have the
potential for binding vicinal receptors.6-8 Such bridging should
be manifested by a substantial increase in potency due to the
high concentration of the pharmacophore in the vicinity of the
recognition site when the ligand is bound in a monovalent mode.
Portoghese et al. reported a range of homo and heterodimeric
ligands with varying linker lengths designed to investigate
pharmacodynamic and organizational features of opioid recep-
tors.9 For example, recently reported heterodimeric ligands
containing δ antagonist (naltrindole) and κ1 agonist (ICI-
199,441) pharmacophores joined by variable length oligoglycyl-
based linkers were demonstrated to possess significantly greater
potency and selectivity compared to their monomer congeners,
providing further evidence for the opioid receptor hetero-
oligomerization phenomena.10 Considering earlier data in this
field, we report the synthesis and pharmacological evaluation
of the first opioid designed multiple ligand obtained through
the linkage of the δ-selective peptidic Dmt-Tica pharmacophore
and the κ agonist/µ partial agonist morphinan compound MCL
101 (butorphan).
For many years, clinicians have treated unresponsive patients
by combining therapeutic mechanisms with a cocktail of drugs.
The design of ligands that act on specific multiple targets
(multiple ligands) is a more recent trend, as indicated by the
substantial increase over the past few years in the number of
publications describing such approaches. Numerous terms are
currently used to describe ligands that have multiple activities.
The terms dual, binary, bivalent, dimeric, mixed, triple, or
balanced are used in conjunction with numerous suffixes, such
as ligand, inhibitor, agonist, antagonist, conjugate, or blocker.
To improve communication and awareness of this emerging field
within the drug discovery community, some authors propose
using the term designed multiple (DM) ligands as a generic
phrase to describe compounds that are rationally designed to
modulate multiple targets of relevance to a disease, with the
overall goal of enhancing efficacy and/or improving safety.1-3
Compared with drug combinations, there are some advantages
associated with multiple ligands, such as the more predictable
pharmacokinetic and pharmacodynamic relationship, which is
a consequence of the administration of a single drug, as well
as improved patient compliance. The molecular starting point
for a multiple-ligand project is generated by using one of two
distinct approaches: either rational design by a combination of
pharmacophores or the screening of compounds libraries of
known drugs. The methodical combination of pharmacophores
from selective ligands is currently the predominant technique
used for the generation of multiple ligands. The pharmacophores
are joined together by a cleavable or noncleavable linker (termed
conjugates), or more commonly, they are overlapped by taking
advantage of structural commonalities (overlapping pharma-
cophore). The majority of reported examples of cleavable
conjugates contain an ester linker that is cleaved by plasma
esterases to release two individual drugs that then act indepen-
dently. Although the pharmacokinetic-pharmacodynamic re-
lationship could become complex after the cleavage of the linker,
Chemistry
The synthesis of compound MCL 450 is reported in Scheme
1. Boc-â-Ala-OH was condensed with MCL 10111 via EDC/
DMAP12 to yield crude MCL 438, which after solvent evapora-
tion was purified by column chromatography as reported in the
Experimental Section. After N-terminal Boc deprotection with
TFA/CH2Cl2,13 the product was condensed with Boc-Dmt-Tic-
OH14 via EDC/HOBt. Boc deprotection with TFA/CH2Cl2 gave
* To whom correspondence should be addressed. Tel.: +39-532-291-
275. Fax: +39-532-91-296. E-mail: gbalboni@unica.it or bbg@unife.it.
† Harvard Medical School.
‡ University of Rochester.
§ National Institute of Environmental Health Sciences.
| University of Ferrara.
University of Cagliari.
10.1021/jm0605785 CCC: $33.50 © 2006 American Chemical Society
Published on Web 07/27/2006

Brief Articles
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18 5641
Scheme 1
GTPγS binding mediated by the µ, δ, and κ opioid receptors.
Binding assays shown in Table 1 for MCL 450 confirm the
pharmacological behaviors of its constituents MCL 101 (µ
partial agonist/κ agonist) and MCL 451 (δ antagonist). The data
show a decrease of EC50 for µ and κ receptors of 5.6 and 4.6-
fold, respectively, in comparison to that of MCL 101; at the
same time, the corresponding Emax (%) values increase 1.58 and
1.63-fold. However, MCL 450 is characterized by a δ antagonist
activity contributed by MCL 451 and is slightly more potent
(2.6-fold) than the δ selective counterpart MCL 451. As
expected, MCL 451 results in a potent and selective δ antagonist,
in line with all other δ antagonists containing the Dmt-Tic
pharmacophore.14,16-18
Conclusion
We demonstrate the possibility of obtaining a multiple ligand
in the opioid peptide field incorporating the Dmt-Tic pharma-
cophore. We selected a two methylene spacer because Schiller
et al. reported the same spacer between a µ agonist and a δ
antagonist (H-Dmt f D-Arg f Phe f Lys-NH-CH2-CH2-NH-
Phe r Cha[NH-CH2]ΨTic r Tyr-H).5 We synthesized a similar
compound incorporating a δ antagonist and a µ agonist (H-
Dmt f Tic-NH-CH2-CH2-NH-Phe r Phe r Pro r Tyr-H)
and obtained similar pharmacological results (personal data).
However, when the tripeptide H-Dmt-Tic-Glu-NH2 containing
the Dmt-Tic pharmacophore is linked to a fluorophore through
a five methylene spacer (H-Dmt-Tic-Glu-NH-(CH2)5-NH-fluo-
rophore), it maintains the selectivity and δ antagonist properties
characteristic of this pharmacophore.19,20 MCL 450 could
represent a useful starting point in the synthesis of other designed
multiple ligands from peptidic and morphinan opioid pharma-
cophores.
MCL 450. Condensation of Boc-Dmt-Tic-OH with HCl‚H-â-
Ala-OMe via EDC/HOBt gave the corresponding protected
tripeptide, which was hydrolyzed at the C-terminus methyl ester
by NaOH and deprotected at the Boc N-terminus with TFA to
yield crude MCL 451. Final compounds were purified by reverse
phase preparative HPLC.
Pharmacological Results and Discussion
Affinity and Selectivity of the Synthesized Ligands. MCL
450 and MCL 451 were evaluated for their affinity at and
selectivity for µ, δ, and κ opioid receptors with Chinese hamster
ovary (CHO) cell membranes stably expressing the opioid
receptors. Data are summarized in Table 1. For comparison
purposes, opioid binding affinity data for MCL 101and Dmt-
Tic are included. The designed multiple ligand MCL 450 derives
from the ester formation between MCL 451 and MCL 101. As
expected, its selectivity derives from a combination of affinity
data from MCL 101 (essentially a κ/µ ligand) and MCL 451 (δ
ligand). In fact, µ and κ affinity drop 3- and 3.5-fold from MCL
101 to MCL 450, respectively, whereas δ affinity increases 3.9-
fold. A comparison of MCL 450 with MCL 451 shows that µ
affinity increases about 1600-fold and δ affinity unexpectedly
increases 2.8-fold. Moreover, MCL 451 confirms once again
the importance of the Dmt-Tic pharmacophore in the induction
of δ affinity and selectivity, especially if a free carboxylic
function is present at the C-terminus.15 Starting from a κ/µ ligand
(MCL 101) and a selective δ ligand (MCL 451), a nonselective
MCL 450 was obtained.
Experimental Section
(-)-3-Hydroxy-N-cyclobutylmethylmorphinan (MCL 101).
(-)-3-Hydroxy-N-cyclobutylmethylmorphinan free base was made
from commercially available levorphanol tartrate (Mallinckrodt,
Inc.) by a procedure previously reported.12
1-((-)N-Cyclobutylmethylmorphinan-3-yl)-N-Boc-â-Ala-
nine. (Boc-â-Ala-O-MCL 101). (MCL 438). MCL-101 (0.16 g,
0.5 mmol) and Boc-â-Ala-OH (0.11 g, 0.6 mmol) were dissolved
in anhydrous dichloromethane (10 mL) under nitrogen. A catalytic
amount of 4-(dimethylamino)pyridine (0.006 g, 0.05 mmol) was
added, followed by EDC (0.12 g, 0.6 mmol). The mixture was
stirred at room-temperature overnight. After solvent evaporation,
the crude product was purified by column chromatography on silica
gel (EtOAc/Et3N, 100:1, v/v) to afford a colorless oil: yield 0.12
g (50%); Rf(B) 0.77; HPLC K′ 8.68; oil; [R]20D -22.6; MH+ 484;
1H NMR (CDCl3) δ 1.05-2.83 (m, 35H), 3.02 (d, J ) 18.6 Hz,
1H), 3.47-3.53 (m, 2H), 6.85(dd, J ) 8.1 Hz, 2.4 Hz, 1H), 6.94
(d, J ) 2.4 Hz, 1H), 7.11(d, J ) 8.1 Hz, 1H).
2TFA‚H-â-Ala-O-MCL 101. Boc-â-Ala-O-MCL 101 (0.12 g,
0.25 mmol) was treated with 50% TFA in CH2Cl2 (2 mL) for 2 h
at room temperature. After solvent evaporation, Et2O/Pe (1:5, v/v)
was added to the solution until the product precipitated: yield 0.14
g (90%); Rf(A) 0.68; HPLC K′ 7.26; mp 121-123 °C; [R]20D -15.1;
MH+ 384.
Boc-Dmt-Tic-â-Ala-O-MCL 101. To a solution of Boc-Dmt-
Tic-OH (0.05 g, 0.11 mmol) and 2TFA‚H-â-Ala-O-MCL 101 (0.07
g, 0.11 mmol) in DMF (10 mL) at 0 °C, NMM (0.02 mL, 0.22
mmol), HOBt (0.02 g, 0.12 mmol), and EDC (0.02 g, 0.12 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 0.07 g (83%); Rf(B) 0.75; HPLC K′ 9.52; mp 112-114 °C;
Functional Activity. Table 2 shows the agonist and antago-
nist properties of MCL 450 and MCL 451 in stimulating [35S]-
a Abbreviations: Boc, tert-butyloxycarbonyl; CHO, Chinese hamster
ovary; DMAP, 4-(dimethylamino)pyridine; DAMGO, [D-Ala2,N-Me-
Phe4,Gly ol5]-enkephalin; DMF,N,N-dimethylformamide; DMSO-d6, hexa-
deuteriodimethyl sulfoxide; Dmt, 2′,6′-dimethyl-L-tyrosine; EDC, 1-ethyl-
3-[3′-dimethyl)aminopropyl]-carbodiimide hydrochloride; EtOAc, ethyl
acetate; Et3N, triethylamine; Et2O, diethyl ether; HOBt, 1-hydroxybenzo-
triazole; HPLC, high performance liquid chromatography; MALDI-TOF,
matrix assisted laser desorption ionization time-of-flight; MeOH, methanol;
NMM, 4-methylmorpholine; Pe, petroleum ether; TFA, trifluoroacetic acid;
Tic, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; TLC, thin-layer chro-
matography; U69,593, (5R,7R,8â)-(+)-N-methyl-N-[7-(1-pyrrolidinyl)-1-
oxaspiro[4.5]dec-8-yl]-benzeneacetamide. These abbreviations and symbols
are used in addition to those by the IUPAC-IUB Commission on
Biochemical Nomenclature (J. Biol. Chem. 1985, 260, 14-42).

5642 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18
Brief Articles
Table 1. Ki Values of the Inhibition of µ, δ, and κ Opioid Binding to CHO Membranes
a Data from Peng, X., et al. J. Med. Chem. 2006, 49, 256-262. b Data from Page´, D., et al. J. Med. Chem. 2001, 41, 2387-2390. c Selectivity Kiµ/Kiδ.
d Selectivity Kiκ/Kiδ. e Selectivity Kiκ/Kiµ. f Maximum inhibition of [3H]U69,593 binding in the presence of 10 µM MCL 451.
Table 2. Functional Activity at the µ, δ, and κ Receptorsa
[35S]GTP-γ-S Binding (Agonism)
µ
δ
κ
EC50 (nM)
Emax (%)
EC50 (nM)
Emax (%)
NT
18 ( 1.7
0.95 ( 2.2
EC50 (nM)
Emax (%)
MCL 101b
MCL 450
MCL 451
1.6 ( 0.2
8.9 ( 0.76
NT
50 ( 2.5
79 ( 2.3
NT
NT
2.8 ( 0.75
NA
1.3 ( 0.44
6.0 ( 1.0
NT
80 ( 6.8
130 ( 5.1
NT
[35S]GTP-γ-S Binding (Antagonism)
µ
δ
κ
IC50 (nM)
Imax (%)
IC50 (nM)
Imax (%)
IC50 (nM)
NT
no inhibition
NT
Imax (%)
MCL 101b
MCL 450
MCL 451
20 ( 3
97 ( 21
NT
50 ( 3
47 ( 1.7
NT
NT
3.9 ( 0.34
10 ( 1.5
NT
84 ( 5.2
93 ( 1.0
NT
no inhibition
NT
a Membranes from CHO that stably expressed only the µ, δ, or κ receptor were incubated with various concentrations of the compounds. The stimulation
of [35S]GTPγS binding was measured as described in the Experimental Section. The Emax value is the maximal percent stimulation obtained with the
compound. The EC50 value is the concentration of compound needed to produce 50% of the Emax value. When the Emax value was 30% or lower, it was not
possible to calculate an EC50 value. To determine the antagonistic properties of a compound, the membranes were incubated with the 100 nM κ agonist
U50,488 or 200 nM µ agonist DAMGO or 100 nM δ agonist SNC-80 in the presence of varying concentrations of the conpound. The Imax value is the
maximal percent inhibition obtained with the compound. The IC50 value is the concentration of compound needed to produce half-maximal inhibition. NT,
not tested; NA, not active. b Data from Peng, X. et al. J. Med. Chem. 2006, 49, 256-262.
1
[R]20 -15.6; MH+ 834; H NMR (DMSO-d6) δ 1.05-3.17 (m,
temperature. After DMF was evaporated, and 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 0.15 g (88%); Rf(B) 0.62; HPLC K′
8.21; mp 124-126 °C; [R]20D +7.2; MH+ 555; 1H NMR (DMSO-
d6) δ 1.40-3.17 (m, 21H), 3.59-4.92 (m, 9H), 6.29 (s, 2H), 6.96-
7.02 (m, 4H).
Boc-Dmt-Tic-â-Ala-OH. To a solution of Boc-Dmt-Tic-â-Ala-
OMe (0.15 g, 0.27 mmol) in MeOH (10 mL) was added 1 N NaOH
(0.32 mL). The reaction mixture was stirred for 24 h at room
temperature. After solvent evaporation, the residue was dissolved
in EtOAc and washed with citric acid (10% in H2O) and brine.
The organic phase was dried (Na2SO4) and evaporated to dryness.
D
46H), 3.47-4.92 (m, 6H), 6.29-7.02 (m, 9H).
TFA‚H-Dmt-Tic-â-Ala-O-MCL 101 (MCL 450). Boc-Dmt-
Tic-â-Ala-O-MCL 101 (0.07 g, 0.08 mmol) was treated with 50%
TFA in CH2Cl2 (2 mL) for 2 h at room temperature. After solvent
evaporation, Et2O/Pe (1:1, v/v) was added to the solution until the
product precipitated: yield 0.07 g (92%); Rf(A) 0.65; HPLC K′
7.21; mp 128-130 °C; [R]20D -17.9; MH+ 734; 1H NMR (DMSO-
d6) δ 1.05-3.17 (m, 37H), 3.47-4.92 (m, 6H), 6.29-7.02 (m, 9H).
Boc-Dmt-Tic-â-Ala-OMe. To a solution of Boc-Dmt-Tic-OH
(0.14 g, 0.31 mmol) and HCl‚H-â-Ala-OMe (0.04 g, 0.31 mmol)
in DMF (10 mL) at 0 °C, NMM (0.03 mL, 0.31 mmol), HOBt
(0.05 g, 0.34 mmol), and EDC (0.06 g, 0.34 mmol) were added.
The reaction mixture was stirred for 3 h at 0 °C and 24 h at room

Brief Articles
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18 5643
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The residue was precipitated from Et2O/Pe (1:9, v/v): yield 0.12 g
(82%); Rf(B) 0.58; HPLC K′ 7.92; mp 138-140 °C; [R]20D +9.1;
MH+ 541.
TFA‚H-Dmt-Tic-â-Ala-OH (MCL 451). Boc-Dmt-Tic-â-Ala-
OH (0.12 g, 0.22 mmol) was treated with TFA (1 mL) for 30 min
at room temperature. After solvent evaporation, Et2O/Pe (1:1, v/v)
was added to the solution until the product precipitated: yield 0.11
g (92%); Rf(A) 0.46; HPLC K′ 6.12; mp 145-147 °C; [R]20D +12.3;
1
MH+ 441; H NMR (DMSO-d6) δ 2.35 (s, 6H), 2.51-3.59 (m,
8H), 3.95-4.92 (m, 4H), 6.29 (s, 2H), 6.96-7.02 (m, 4H).
Acknowledgment. This work was supported in part by NIH
Grants RO1-DA14251 (to J.L.N.), K05-DA 00360 (to J.M.B.),
University of Cagliari (G.B., PRIN 2004), University of Ferrara,
and the Intramural Research Program of the NIH and NIEHS.
We appreciate the professional services of the library staff of
the NIEHS. Levorphanol tartrate was generously donated by
Mallinckrodt, Inc.
Note Added after ASAP Publication. This manuscript was
released ASAP on July 27, 2006, with incorrect citations of
reference numbers in the text. The correct version was posted
on August 9, 2006.
Supporting Information Available: Chemistry general meth-
ods, biological general methods, and elemental analysis. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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JM0605785