Synthesis and Investigation of Mixed μ-Opioid and δ-Opioid Agonists as Possible Bivalent Ligands for Treatment of Pain

Article abstract of DOI:10.1002/jhet.2696

Several studies have suggested functional association between μ-opioid and δ-opioid receptors and showed that μ-activity could be modulated by δ-ligands. The general conclusion is that agonists for the δ-receptor can enhance the analgesic potency and effi

Full text of DOI:10.1002/jhet.2696

Month 2016
Synthesis and Investigation of Mixed μ-Opioid and δ-Opioid Agonists as
Possible Bivalent Ligands for Treatment of Pain
a
a
a
a
b
*
Ruben S. Vardanyan, James P. Cain, Saghar Mowlazadeh Haghighi, Vlad K. Kumirov, Mary I. McIntosh,
Alexander J. Sandweiss,b Frank Porreca,b and Victor J. Hrubya
aDepartment of Chemistry and Biochemistry, University of Arizona, Tucson, Arizona 85721, USA
bDepartment of Pharmacology, University of Arizona Health Sciences Center, Tucson, Arizona 85724, USA
*
E-mail: vardanyr@email.arizona.edu
Additional Supporting Information may be found in the online version of this article.
Received February 11, 2016
DOI 10.1002/jhet.2696
Published online 00 Month 2016 in Wiley Online Library (wileyonlinelibrary.com).
Several studies have suggested functional association between μ-opioid and δ-opioid receptors and
showed that μ-activity could be modulated by δ-ligands. The general conclusion is that agonists for the δ-
receptor can enhance the analgesic potency and efficacy of μ-agonists. Our preliminary investigations dem-
onstrate that new bivalent ligands constructed from the μ-agonist fentanyl and the δ-agonist enkephalin-like
peptides are promising entities for creation of new analgesics with reduced side effects for treatment of neu-
ropathic pain. A new superposition of the mentioned pharmacophores led to novel μ-bivalent/δ-bivalent
compounds that demonstrate both μ-opioid and δ-opioid receptor agonist activity and high efficacy in
anti-inflammatory and neuropathic pain models with the potential of reduced unwanted side effects.
J. Heterocyclic Chem., 00, 00 (2016).
INTRODUCTION
agonists [18–20] while others state that μ-agonist signaling
can be enhanced by co-treatment with δ-selective antago-
nists [21,22]. Others indicate differential localizations and
pain relieving profiles of the μ-receptors and δ-receptors
in dorsal root ganglion cells [23].
One of the promising new approaches to answer the
questions raised could be the creation and investigation
of bivalent ligands. Bivalent ligands contain two
pharmacophores that are fused or variably separated by a
chemical spacer, and there are several interesting examples
of them in the literature [24–41]. Finding the “correct” pair
of two ligands and their “correct” superposition is an at-
tractive yet challenging problem.
Our preliminary investigations demonstrate that new
bivalent ligands with mixed μ-profile/δ-profile, which rep-
resents one attempt at combining fentanyl 1 and
enkephalin-like peptides 2 (Fig. 1), are promising com-
pounds with high binding affinities to both μ-receptors
and δ-receptors [42–46].
Recently, we have designed and synthesized compounds
of types 3 and 4 (Fig. 2), which differ mainly in super-
position of the μ/δ constituents. Compounds in series 3
display binding affinities in the range of 30–60 nM for
μ-Opioid analgesics are the mainstay for treatment of
moderate to severe pain, but they have significant side
effects including constipation, respiratory depression, tol-
erance, addiction, and even death [1]. Convincing data
(biochemical, pharmacological, and studies using geneti-
cally modified animals) regarding the modulatory interac-
tions between opioid receptors exist in the literature.
Several studies indicate that δ-receptor agonists as well as
δ-receptor antagonists provide significant modulation of
the pharmacological effects of μ-agonists. It has been
shown that agonists at the δ-receptor can enhance the anal-
gesic potency and efficacy of μ-agonists while
δ-antagonists can prevent or diminish the development of
tolerance and physical dependence by μ-agonists; multiple
and controversial interpretations of the observed phenom-
ena have been made [2–16].
Functional association between μ-opioid and δ-opioid
receptors was first suggested by studies showing that μ-
activity could be modulated by δ-ligands [17], but the true
role of δ-ligands remains obscure. Some authors insist that
δ-agonists increase antinociceptive responses to μ-receptor
© 2016 Wiley Periodicals, Inc.

R. S. Vardanyan, J. P. Cain, S. M. Haghighi, V. K. Kumirov, M. I. McIntosh,
A. J. Sandweiss, F. Porreca, and V. J. Hruby
Vol 000
Figure 1. Structures of fentanyl 1 and enkephalin-like peptides 2.
Figure 3. Structures of novel μ-bivalent/δ-bivalent ligands.
both receptors [44–46]. Compounds in series 4 display
higher affinity (~0.4 nM) for both receptors with
increased hydrophobicity (aLogP 3.01–4.74). Binding
affinities of series 4 exceed desired affinities and cross
the threshold of nanomolar into picomolar range at both
μ-opioid and δ-opioid receptors [42,43]. The bivalent
ligands with synergistic action on different subtypes
of the same (opioid) receptors could be called concor-
dant ligands.
Another novel attempt to create μ-bivalent/δ-bivalent
compounds using both a different superposition of ligands
and a different linker (carboxy group) and spacer
(hydrazino group) is presented in Figure 3 and is the
subject of the present publication.
perchlorates chemistry”—preparation of succinisoimidium
perchlorates from succinamic acids with acetic anhydride
and perchloric acid [49–52]. Previously, this method was
extended and simplified by us, implementing it for the cre-
ation of new type of isoimidium compounds—
glutarisoimidium and 3-oxaglutarisoimidium perchlorates
(8b, c) [47]. All three isoimidium perchlorates smoothly
produce appropriate hydrazides (9a, b, c) when treated
with N-Boc-phenylalanine hydrazide, which on further
Boc deprotection with trifluoroacetic acid gave (10a, b,
c). Coupling of the obtained products with Boc-protected
Tyr-D-Ala-Gly-OH followed by deprotection gave the
desired bivalent ligands with mixed μ-profile/δ-profile
(11a, b, c).
Another potentially convenient method for the creation
of compounds of the formula (11) by direct coupling of hy-
drazides (12) (Fig. 4) with enkephalin-like peptides was
undertaken, but our various attempts to develop a protocol
for their preparative synthesis failed. Direct hydrazination
of both (7) and (8) gave very poor yields of an unstable
product (12). However, use of Boc or benzylhydrazines
gave excellent yields of protected products (13) and (14).
But upon deprotection, Boc hydrazides (13) decomposed
in different ways depending on conditions, and the nature
of the linker between the two carbonyl groups. Bn-
hydrazides (14) decomposed on H2/Pd-C hydrogenation
to give 1-alkyl-1,2,3,6-tetrahydropyridines (16) (Fig. 4).
The bivalent ligands with mixed μ-/δ-profile (11a, b, c)
demonstrated good binding affinity to both μ-opioid and
δ-opioid receptors (Table 1).
RESULTS AND DISCUSSION
Chemistry. The gram scale synthesis of functionalized
fentanyls—carboxyfentanyl (7a), carboxymethyl-fentanyl
(7b), and fentanyl derivative (7c) starting from 1-
phenethyl-N-phenylpiperidin-4-amine (6)—was previously
described by our group for the synthesis of μ-agonist/
NK1-antagonist bivalent ligands [47,48]. The synthetic
Scheme 1 ensures good yields of the desired compounds.
A “peptide chemistry” method with carbodiimide
activation of the carboxyl function of (7a, b, c)
with EDAC/HOBt [1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide/hydroxybenzotriazole] was successfully
implemented for the creation of N-Boc-phenylalanine
hydrazides (9a, b, c) acylated with fentanyl carboxylic
acids (7a, b, c).
Compound (11b) was selected for testing in several
in vivo pain models, including inflammatory pain, using
the formalin flinch assay and antiallodynia, in a nerve
Another convenient method for creation of substituted
fentanyls has been developed using “succinisoimidium
Figure 2. Structures of μ-bivalent/δ-bivalent ligands already proposed by our group.
Journal of Heterocyclic Chemistry
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Synthesis and Investigation of Mixed μ-Opioid and δ-Opioid Agonists as Possible
Month 2016
Bivalent Ligands for Treatment of Pain
Scheme 1. Synthesis of novel μ-bivalent/δ-bivalent ligands. Reagents and conditions: (a) succinic, glutaric, or diglycolic anhydride in CH2Cl2; (b) Ac2O,
HClO4; (c) N-Boc-PheNHNH2, EDAC, HOBt, CHCl3; (d) N-Boc-PheNHNH2, Et3N, CHCl3; (e) CF3COOH/CH2Cl2; (f) 1. Boc-Tyr(Boc)-D-Ala-Gly-OH,
BOP, HOBt, DMF; 2. CF3COOH/CH2Cl2. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
ligation model with no significant sedation, or motor
impairment and demonstrates potent and efficacious activ-
ity. This is the first report of a fentanyl-based structure with
δ-opioid and μ-opioid receptor activity that exhibits out-
standing antinociceptive efficacy in neuropathic pain,
reducing the propensity of unwanted side effects that occur
with current μ-opioid agonist therapies.
administration. The three doses (1, 3, and 10μg) produced
significant dose-dependent antinociception in non-injured
mice compared with vehicle-treated mice 15min after intra-
thecal injection (21 6.4%, 36 15.8%, and 77 10.7%,
respectively). The A50 dose was calculated to be 3.92μg.
These effects were blocked by naloxone, indicating that
they were opioid receptor-mediated effects. Furthermore,
the significant attenuation by the μ-selective antagonist, (D-
Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2) CTAP, and by
Compound (11b) demonstrated significant antinocice-
ption using a warm water tail-flick test after the spinal
Figure 4. Failed attempts of the synthesis of hydrazides (12).
Journal of Heterocyclic Chemistry
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R. S. Vardanyan, J. P. Cain, S. M. Haghighi, V. K. Kumirov, M. I. McIntosh,
A. J. Sandweiss, F. Porreca, and V. J. Hruby
Vol 000
Table 1
agonist constituents reveal high efficacy in anti-
inflammatory and neuropathic pain models with the
potential of reduced unwanted side effects. In addition,
μ-agonists/δ-agonists have been shown to significantly
decrease anxiety in mice and rats. This latter aspect needs
to be further investigated. Detailed pharmacological
investigation on (11b) is in progress.
Results in the MVD and GPI/LMMP assays.
Compound MVD GPI/LMMP
Agonist activity (nM IC50 sem)
a
11a
11b
11c
69 12
39 8.2
48 16
44 9.7
63 13
42 8.3
MVD, mouse vas deferens; GPI, guinea pig isolated ileum; LMMP, lon-
gitudinal muscle with myenteric plexus; sem, standard error of mean.
aConcentration of compounds at 50% specific binding.
EXPERIMENTAL
1
Chemistry. The compounds were characterized by H
the δ-selective antagonist, naltrindole, suggests that the
antinociception produced by (11b) is indeed mediated by
both the μ-opioid and δ-opioid receptors. In order to deter-
mine whether (11b) has anti-inflammatory effects, it was
tested in a murine formalin flinch model. Compound (11b)
resulted in significant inhibition of formalin-induced
flinching in both the first and second phases, suggesting
antinociception by inhibiting both δ-fiber and C-fiber activity.
Implementing the Kim and Chung model for peripheral
neuropathy [53,54], it was shown that (11b) resulted in
significant mechanical antiallodynia and thermal
antihypersensitivity in the nerve injured animal. Such stud-
ies suggest that a fentanyl-based, mixed mu-delta opioid
agonist (11b) can act to inhibit neuropathic pain. Rotorod
experiments were performed and demonstrated that the
lack of (11b) induced motor paralysis and/or signs of seda-
tion because such behavior would mask paw withdrawal
results in our pain behavior tests. Owing to the added anal-
gesic effects of δ-opioid receptor occupation, less μ-
receptor occupation is required, essentially decreasing the
dose and unwanted side effects driven by μ-opioid receptor
agonists. Ultimately, by using a combination of acute and
chronic pain models, we demonstrate the efficacy of the
novel μ-bivalent/δ-bivalent compound (11b) in treating
neuropathic pain without sedation and motor impairment.
Overall, these studies demonstrate a novel fentanyl-
based structure that contains both μ-opioid and δ-opioid
receptor agonist activity resulting in high efficacy in anti-
inflammatory and neuropathic pain models; they also have
the potential to reduce unwanted side effects. There may
also be the added potential of being anxiolytic, which will
need to be further investigated. Detailed pharmacological
investigations on (11b) are already published [1].
and 13C nuclear magnetic resonance (NMR). NMR
spectra were recorded on a Bruker (Billerica, MA) DRX-
600 spectrometer using tetramethylsilane as an internal
standard. The compounds were analyzed by electrospray
mass spectrometry (MS) on
a
Thermo Electron
(Waltham, MA) (Finnigan) LCQ classic ion trap mass
spectrometer. Direct infusion (10 μL/min) was applied to
about 50 μM solutions of the samples in MeOH/H2O 1:1
2% AcOH. Standard electrospray ionization conditions
were applied, and the masses of protonated molecules
[MH]+ were measured. Optical rotations were measured
on a Rudolph Research Analytical (Hackettstown, NJ)
AutoPol III polarimeter using the Na-D line. The purity
of the compounds was determined by thin-layer
chromatography on silica gel plates (Analtech, Newark,
DE; 02521), solvent system MeOH/CHCl3 1:4. Melting
points are uncorrected. The purity of the compounds was
also checked by analytical reversed-phase high-
performance liquid chromatography using an Amersham
Pharmacia Biotech (Piscataway, NJ) Äkta Basic 10F with
a ODS-A C18 (120Å, 5 μm, 250 mm× 4.6 mm) column
(Omnicrom; YMC Europe GmbH, Dinslaken, Germany)
monitored at 220 and 254 nm and by high-resolution
mass spectral analysis (Supporting Information).
General method for the synthesis of the 4-oxo-4-((1-
phenethylpiperidin-4-yl)(phenyl)amino)alcanoic acids (7a, b,
c).
A mixture of [1-(2-phenyl)-ethyl-piperidin-4-yl]-
phenyl-amine (2.8g, 10mmol), appropriate acid anhydride
(succinic, glutaric, or diglycolic, 11mmol) and two to three
drops of acetic acid in dichloromethane (30mL) was heated
in a closed pressure-proof flask in a boiling water bath for
5h and left overnight at room temperature. The solvent was
evaporated; ethyl acetate (50mL) was added, and the
mixture was heated to dissolve the content of the flask. For
the cases (7b) and (7c), it is necessary to add some ethanol
(5–10mL). Crystals of the appropriate acid separated on
cooling with 80–90% yield.
CONCLUSION
The result of these studies indicate that our design and
synthesis of novel non-peptide μ-agonists/δ-agonists
resulted in new series of compounds such as (11), which
have potent analgesic effects. Novel fentanyl-based struc-
tures that contain both μ-opioid and δ-opioid receptor
4-Oxo-4-((1-phenethylpiperidin-4-yl)(phenyl)amino)
butanoic acid (7a). Crystalline solid; yield: 3.4 g (89%);
mp 119–120°C; 1H-NMR (600 MHz, methanol-d4):
δ= 7.50 (t, J= 7.3 Hz, 2H), 7.48–7.43 (m, 1H), 7.32–7.25
(m, 4H), 7.25–7.18 (m, 3H), 4.70 (tt, J = 12.0, 3.5 Hz,
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Synthesis and Investigation of Mixed μ-Opioid and δ-Opioid Agonists as Possible
Month 2016
Bivalent Ligands for Treatment of Pain
1H), 3.42 (d, J= 12.2 Hz, 2H), 3.00 (m, 2H), 2.90 (dd,
J =10.5, 6.1Hz, 2H), 2.80 (t, J =12.0 Hz, 2H), 2.42 (t,
J =6.8 Hz, 2H), 2.18 (t, J =6.8 Hz, 2H), 2.00 (d,
J =12.2 Hz, 2H), 1.62ppm (dq, J = 3.3, 13.1 Hz, 2H);
13C-NMR (150 MHz, methanol-d4): δ= 172.77, 138.08,
137.48, 130.16, 129.40, 128.65, 128.31, 126.44, 58.09,
51.92, 50.76, 30.85, 30.47, 30.25, 28.14ppm; MS [electron
impact (EI), 70 eV] m/z (%): 381; high-resolution MS fast
atom bombardment (HRMS-FAB) m/z [M+H]+ calcd for
C23H29N2O3:381.2178; found: 381.2183.
General method for the synthesis of the N-Boc-phenylalanine
hydrazides acylated with fentanyl carboxylic acids (9a, b, c).
Peptide chemistry method. To the stirred mixture of acid
(7a, b, c, 0.25 mmol) in CHCl3 (25 mL) cooling in ice
bath, EDAC (0.048 mL, 0.275 mmol) was added. After
15 min, HOBt (0.04 g, 0.275 mmol) was added, and
after stirring for additional 15 min, N-Boc-PheNHNH2
(0.07 g, 0.275 mmol) was added. The reaction mixture
was allowed to come to room temperature and left
overnight. The solvent was removed in vacuo, and
acetone (15 mL) was added to the remaining mixture,
which was then refluxed for 5–10 min and allowed to
cool. Precipitate of the obtained acetone hydrazone
formed from excess of N-Boc-PheNHNH2 was filtered,
acetone was removed in vacuo, CHCl3 (25 mL) was
added to remaining solid, and the solution was washed
twice with 5% NaHCO3 (10 mL) and with water and
dried on MgSO4. Solvent evaporation gave pure (7a, b,
c) with 70–80% yield. If necessary, product can be
purified on silica gel column with the 4:1
chloroform/methanol solvent mixture.
5-Oxo-5-((1-phenethylpiperidin-4-yl)(phenyl)amino)
pentanoic acid (7b).
Crystalline solid; 3.42g (87%); mp
1
104–105 °C; H-NMR (600MHz, methanol-d4): δ=7.49 (t,
J=7.3Hz, 2H), 7.45 (d, J= 7.2Hz, 1H), 7.29 (t, J=7.2Hz,
2H), 7.24–7.19 (m, 5H), 4.72 (tt, J= 12.0, 3.6Hz, 1H), 3.42
(d, J=12.3Hz, 2H), 2.99 (m, 2H), 2.89 (m, 2H), 2.79 (t,
J= 11.9 Hz, 2H), 2.10 (t, J=7.3Hz, 2H), 2.05–1.95 (m,
4H), 1.79 (dd, J=14.7, 7.3Hz, 2H), 1.61ppm (dd, J= 12.8,
3.2Hz, 2H); 13C-NMR (150MHz, methanol-d4): δ =173.27,
138.07, 137.56, 130.05, 129.44, 128.66, 128.32, 126.43,
58.11, 51.87, 50.78, 34.52, 33.99, 30.89, 28.19, 21.05ppm;
MS (EI, 70eV) m/z (%): 395; HRMS-FAB m/z [M+H]+
calcd for C24H31N2O3: 395.2325; found: 395.2323.
Isoimidium perchlorates method.
N-Boc-PheNHNH2
(0.07g, 0.275mmol) was added to the stirred suspension of
perchlorate (8) (0.25mmol) in CHCl3 (25mL), and the
solution was cooled in an ice bath before a dropwise
addition of triethylamine (0.13mL, 1mmol) in CHCl3
(5mL). The reaction mixture was allowed to come to room
temperature and left overnight. The solvent was removed in
vacuo, and acetone (15mL) was added to the remaining
mixture, which was then refluxed for 5–10min and allowed
to cool. Precipitate of the obtained acetone hydrazone was
filtered, acetone was removed in vacuo, CHCl3 (25mL) was
added to the remaining solid, and the solution was washed
twice with 5% NaHCO3 (10mL) and with water before it
was dried on MgSO4. Solvent evaporation gave pure (7a, b,
c) with 65–70% yield. If necessary, the product can be
purified on silica gel column with the 4:1
chloroform/methanol mixture.
2-(2-Oxo-2-((1-phenethylpiperidin-4-yl)(phenyl)amino)
ethoxy)acetic acid (7c).
Crystalline solid; 3.12g (79%);
mp 143–144°C; 1H-NMR (600 MHz, methanol-d4):
δ= 7.55–7.45 (m, 3H), 7.35–7.26 (m, 4H), 7.25–7.22 (m,
3H), 4.74 (tt, J = 12.1, 3.6 Hz, 1H), 3.86 (d, J= 6.0 Hz,
4H), 3.56 (d, J= 12.3Hz, 2H), 3.15 (dd, J= 10.3, 6.6 Hz,
2H), 3.01 (t, J = 12.2Hz, 2H), 2.95 (dd, J= 10.3, 6.6 Hz,
2H), 2.09 (d, J = 12.7Hz, 2H), 1.72 ppm (dd, J =12.8,
2.8Hz, 2H); 13C-NMR (150 MHz, methanol-d4):
δ= 174.52, 170.05, 136.78, 136.17, 129.90, 129.70,
129.28, 128.46, 128.33, 126.67, 69.40, 68.52, 57.60,
51.69, 50.81, 30.36, 27.41, 19.81 ppm; MS (EI, 70eV)
m/z (%): 397; HRMS-FAB m/z [M+H]+ calcd for
C23H29N2O4: 397.2127; found: 397.2123.
General method for the synthesis of the isoimidium
perchlorates (8a, b, c). The stirred mixture of acid (7a,
b, c, 0.5mmol) in acetic anhydride (1.5 mL) was slightly
heated until the beginning of dissolution of the starting
material. Then, the heater was turned off, but stirring was
continued for 1 h. The mixture was cooled (ice bath), and
70% HClO4 (0.15 mL) was carefully added dropwise.
The obtained solution was allowed to come to room
temperature overnight, and 5mL of dry ether was added
with stirring. After 15–20 min, the liquid was separated
from the precipitated perchlorate, and the solid residue
was washed with three portions of ether (5 mL). For
analytical purposes, the residue was dried overnight in a
vacuum desiccator over phosphorous pentoxide. For
synthetic purposes, it was dissolved in CHCl3/CH3CN
(5 mL/1 mL) mixture and added to a solution of N-Boc-
phenylalanine hydrazide.
Tert-butyl (1-oxo-1-(2-(4-oxo-4-((1-phenethylpiperidin-4-yl)
(phenyl)amino)butanoyl)hydrazinyl)-3-phenylpropan-2-yl)
carbamate (9a).
Viscous oil; 0.12 g (75%); 1H-NMR
(600 MHz, CDCl3): δ= 7.42–7.39 (m, 2H), 7.27–7.09
(m, 13H), 5.69 (d, J = 8.8 Hz, 1H), 5.39 (d, J = 8.8 Hz,
1H), 5.38 (d, J = 9.6 Hz, 1H), 4.65 (m, 2H), 4.10 (s,
2H), 3.83 (s, 2H), 3.16 (m, 2H), 3.01 (d, J = 10.3 Hz,
2H), 2.72 (m, 2H), 2.54 (m, 2H), 2.16 (m, 2H), 1.82
(m, 2H), 1.46 (qd, J = 12.3, 3.8 Hz, 2H), 1.36 ppm (s,
9H); 13C-NMR (150 MHz, CDCl3) δ= 171.64, 169.01,
167.63, 155.49, 139.80, 136.95, 136.61, 130.28,
129.62, 129.49, 129.48, 128.70, 128.53, 128.49,
126.84, 126.22, 77.37, 60.12, 54.06, 52.88, 52.83,
38.66, 33.38, 30.55, 30.07, 29.18, 28.36; MS (EI,
70 eV) m/z (%): 641; HRMS-FAB m/z [M+H]+ calcd for
C37H47N5O5: 641.3577; found: 641.3583.
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R. S. Vardanyan, J. P. Cain, S. M. Haghighi, V. K. Kumirov, M. I. McIntosh,
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Vol 000
Tert-butyl (1-oxo-1-(2-(5-oxo-5-((1-phenethylpiperidin-4-yl)
(phenyl)amino)pentanoyl)hydrazinyl)-3-phenylpropan-2-yl)
carbamate (9b).
chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium
hexafluorophosphate] (2.06g, 5.0 mmol), and DIEA
(1.74 mL, 10.0 mmol) in DMF (12 mL) were added, and
the mixture was shaken for 30min, followed by washing
with DMF and CH2Cl2. After subsequent Fmoc
deprotection 5 and 13min and washing, Boc-Tyr(tBu)-
OH (1.69g, 5.0 mmol), HCTU (2.06 g, 5.0 mmol), and
DIEA (1.74 mL, 10.0mmol) in DMF (12 mL) were added,
followed by mixing for 30min and washing with DMF
and CH2Cl2. The protected tripeptide was cleaved from
the resin with 12mL of 20% hexafluoroisopropanol in
CH2Cl2 for 1.5 h, which was evaporated to provide
(0.406 g, 87.1%) of the product. Boc-Tyr(tBu)-D-Ala-Gly-
OH (0.122 g, 0.24mmol), 10 (a–c) (0.22 mmol), BOP
(0.115 g, 0.26 mmol), HOBt (0.035 g, 0.26 mmol), and
DIEA (114 μL, 0.65mmol) in DMF (1.5 mL) were stirred
overnight. The reaction mixture was diluted with ethyl
acetate and washed with 10% (aq.) potassium carbonate
and then dried over solid potassium carbonate. After
filtering, the solvent was evaporated. The resulting residue
was dissolved in 1:1 CH2Cl2/CF3COOH (4 mL) and
stirred for 30min. After evaporating the reaction solvent
and coevaporation of excess CF3COOH with toluene and
diethyl ether, the residue was purified by reversed-phase
high-performance liquid chromatography (C18) using a
30–70% gradient of acetonitrile in 0.1% aq. CF3COOH
and lyophilized. Owing to the limited sensitivity of
natural-abundance 13C and the relatively large size of
compounds 11a–c, the 13C spectra were not conclusive.
Instead, the 1H spectra were resolved using 2D total
correlation spectroscopy and 2D rotational Overhauser
effect spectroscopy to confirm the connectivity.
Viscous oil; 0.12g (73%); 1H-NMR
(600 MHz, CDCl3): δ= 7.41–7.34 (m, 2H), 7.29–7.05 (m,
13H), 5.57 (d, J= 8.8 Hz, 1H), 5.41 (d, J= 8.8Hz, 1H),
5.33 (d, J = 9.6Hz, 1H), 4.64 (m, 2H), 3.13 (m, 2H), 3.01
(d, J = 10.3Hz, 2H), 2.72 (m, 2H), 2.52 (m, 2H), 2.26 (t,
J =7.3 Hz), 2.13 (m, 2H), 2.03 (m, 2H), 1.84 (quintet,
J =7.3 Hz), 1.79 (m, 2H), 1.42 (m, 2H), 1.35ppm (s, 9H);
13C-NMR (150 MHz, CDCl3) δ =172.25, 168.65, 167.09,
155.61, 140.29, 136.96, 136.53, 130.37, 129.56, 129.49,
129.48, 128.72, 128.62, 128.49, 126.96, 126.14, 77.37,
60.55, 54.15, 53.18, 53.14, 38.61, 33.91, 33.72, 33.15,
30.61, 28.40, 21.37; MS (EI, 70 eV) m/z (%): 655;
HRMS-FAB m/z [M+H]+ calcd for C38H49N5O5:
655.3734 found: 655.3743
Tert-butyl (1-oxo-1-(2-(2-(2-oxo-2-((1-phenethylpiperidin-4-
yl)(phenyl)amino)ethoxy)acetyl)hydrazinyl)-3-phenylpropan-2-
1
yl)carbamate (9c).
Viscous oil; 0.11g (67%); H-NMR
(600MHz, CDCl3): δ=7.39–7.33 (m, 2H), 7.27–7.07 (m,
13H), 5.58 (d, J= 8.8Hz, 1H), 5.36 (d, J=8.8 Hz, 1H), 5.33
(d, J=9.6Hz, 1H), 4.63 (m, 2H), 3.14 (m, 2H), 3.04 (d,
J= 10.3 Hz, 2H), 2.74 (m, 2H), 2.59 (m, 2H), 2.50 (t,
J= 7.3 Hz), 2.26 (t, J= 7.3 Hz), 2.19 (m, 2H), 1.79 (m, 2H),
1.47 (qd, J= 12.3, 3.8Hz, 2H), 1.34ppm (s, 9H); 13C-NMR
(150MHz, CDCl3) δ= 169.34, 168.93, 167.50, 155.62,
140.11, 136.59, 136.29, 130.14, 129.79, 129.51, 129.46,
128.64, 128.50, 128.42, 126.80, 126.10, 77.37, 71.23,
70.68, 60.38, 54.04, 52.93, 52.87, 38.66, 33.76, 30.16,
28.33; MS (EI, 70 eV) m/z (%): 657; HRMS-FAB m/z [M
+H]+ calcd for C37H47N5O6: 657.3526; found: 657.3543
General method for the Boc deprotection of the
4-(2-((R)-2-(2-((S)-2-((R)-2-Amino-3-(4-hydroxyphenyl)
propanamido)propanamido) acetamido)-3-phenylpropanoyl)
hydrazinyl)-4-oxo-N-(1-phenethylpiperidin-4-yl)-N-phenyl-
N-Boc-phenylalanine hydrazides acylated with fentanyl
carboxylic acids (10a, b, c).
To the stirred mixture of
hydrazide (10, 0.25mmol) in CH2Cl2 (10mL), on cooling, a
solution of CF3COOH (7mL) in CH2Cl2 (7mL) was added
dropwise. The reaction mixture was allowed to come to
room temperature overnight. The solvent and excess of
CF3COOH were removed in vacuo, and CH2Cl2 (25 mL)
was added to the remaining mixture. The mixture was
cooled in an ice bath and basified with 28–30% NH4OH
solution. The organic layer was separated and dried on
MgSO4, and obtained (10a, b, c) were acylated with Boc-
Tyr(Boc)-D-Ala-Gly-OH.
butanamide trifluoroacetate (11a).
Viscous oil; 0.13g
(71%); 1H-NMR (600MHz, DMSO-d6): δ=10.03 (d,
J=11.1Hz, 1H), 9.82 (d, J=13.7Hz, 1H), 9.36 (s, 1H),
8.53 (d, J=7.6Hz, 1H), 8.15 (t, J=6.4Hz, 1H), 8.09 (d,
J=8.2Hz, 1H), 7.51 (t, J=7.5Hz, 2H), 7.46 (t, J=7.5Hz,
1H), 7.31 (t, J= 7.5 Hz, 2H), 7.29–7.15 (m, 10H), 7.00 (d,
J=8.5Hz, 2H), 6.69 (d, J=8.5Hz, 2H), 4.69 (m, 1H), 4.56
(m, 1H), 4.30 (m, 1H), 3.97 (m, 1H), 3.72 (m, 1H), 3.58
(m, 1H), 3.51 (m, 2H), 3.16 (m, 2H), 3.06 (m, 2H), 2.99
(m, 1H), 2.87 (m, 4H), 2.76 (m, 1H), 2.33 (m, 2H), 2.07 (t,
J=7.1Hz, 2H), 1.92 (m, 2H), 1.49 (m, 2H), 1.03ppm (d,
General method for the synthesis of ligands with mixed
μ-profile/δ-profile (11a, b, c).
Fmoc-Gly-OH (0.35 g,
20
J=7.3Hz, 3H); ½αꢀD =+57° (c 0.2; DMSO); MS (EI,
1.2mmol) and diisopropylethylamine (DIEA) (0.84mL,
4.8mmol) was added to a stirred suspension of 2-chlorotrityl
chloride resin (0.834g, 1.0 mmol) in CH2Cl2 (10 mL). After
2h, the resin was filtered and washed with
dimethylformamide (DMF) and CH2Cl2. The resin was
treated twice with 20% piperidine in DMF with mixing for 5
and 13min and washed with DMF and CH2Cl2.
>Fmoc-D-Ala-OH (1.558 g, 5.0 mmol), HCTU [2-(6-
70eV) m/z (%): 832; HRMS-FAB m/z [M+H]+ calcd for
C46H56N8O7: 832.4272; found: 832.4283.
5-(2-((R)-2-(2-((S)-2-((R)-2-Amino-3-(4-hydroxyphenyl)
propanamido)propanamido)acetamido)-3-phenylpropanoyl)
hydrazinyl)-5-oxo-N-(1-phenethylpiperidin-4-yl)-N-phenyl-
pentanamide trifluoroacetate (11b).
(75%); H-NMR (600MHz, DMSO-d6): δ =10.06 (s, 1H),
Viscous oil; 0.14g
1
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Synthesis and Investigation of Mixed μ-Opioid and δ-Opioid Agonists as Possible
Month 2016
Bivalent Ligands for Treatment of Pain
[13] Gupta, A.; Decaillot, F. M.; Devi, L. A. AAPS J 2006, 8,
E153.
[14] Kabli, N.; Martin, N.; Fan, T.; Nguyen, T.; Hasbi, A.; Balboni,
G.; O’Dowd, B. F.; George, S. R. Brit J Pharm 2010, 161, 1122.
[15] George, S. R.; Fan, T.; Xie, Z.; Tse, R.; Tam, V.; Varghese, G.;
O’Dowd, B. F. J. Biol Chem 2000, 275, 26128.
[16] He, S.-Q.; Zhang, Z.-N.; Guan, J.-S.; Liu, H.-R.; Zhao, B.;
Wang, H.-B.; Yang, Q.; Li, H.; Luo, J.; Li, Z.-Y.; Wang, Q.; Lu, Y.-J.;
Bao, L.; Zhang, X. Neuron 2011, 69, 120.
[17] Lee, N. M.; Leybin, L.; Chang, J. K.; Loh, H. H. Eur J
Pharmacol 1980, 68, 181.
[18] Porreca, F.; Takemori, A. E.; Sultana, M.; Portoghese, P. S.;
Bowen, W. D.; Mosberg, H. I. J. Pharm Exp Therapeutics 1992, 263, 147.
[19] Heyman, J. S.; Vaught, J. L.; Mosberg, H. I.; Haaseth, R. C.;
Porreca, F. Eur J Pharm 1989, 165, 1.
[20] Heyman, J. S.; Jang, Q.; Rothman, R. B.; Mosberg, H. I.;
Porreca, F. Eur J Pharm 1989, 169, 43.
[21] Rozenfeld, R.; Devi, L. A. FASEB J 2007, 21, 2455.
[22] Gomes, I.; Gupta, A.; Filipovska, J.; Szeto, H. H.; Pintar, J. E.;
Devi, L. A Proc Natl Acad Sci USA 2004, 101, 5135.
[23] Scherrer, G.; Imamachi, N.; Cao, Y. Q.; Contet, C.;
Mennicken, F.; O’Donnell, D.; Kieffer, B. L.; Basbaum, A. I. Cell 2009,
137, 1148.
[24] Dietis, N.; Guerrini, R.; Calo, G.; Salvadori, S.; Rowbotham,
D. J.; Lambert, D. G. Brit J Anaesth 2009, 103, 38.
[25] Hruby, V. J.; Porreca, F.; Yamamura, H. I.; Tollin, G.; Agnes,
R. S.; Lee, Y. S.; Cai, M.; Alves, I.; Cowell, S.; Varga, E.; Davis, P.;
Salamon, Z.; Roeske, W.; Vanderah, T.; Lai, J. AAPS J 2006, 8, E450.
[26] Schiller, P. W. Life Sci 2010, 86, 598–603.
9.89 (s, 1H), 9.47 (s, 1H), 8.59 (d, J=7.1Hz, 1H), 8.20 (t,
J=6.5Hz, 1H), 8.16 (d, J=8.2Hz, 1H), 7.49 (t, J=6.8Hz,
2H), 7.44 (t, J=6.8Hz, 1H), 7.31 (t, J=7.2Hz, 2H), 7.27–
7.15 (m, 10H), 7.00 (d, J=8.2Hz, 2H), 6.69 (d, J=8.2Hz,
2H), 4.72 (m, 1H), 4.55 (m, 1H), 4.29 (m, 1H), 3.97 (m,
1H), 3.70 (m, 1H), 3.57 (m, 1H), 3.50 (m, 2H), 3.16 (m,
2H), 3.08 (m, 2H), 2.98 (m, 1H), 2.87 (m, 4H), 2.75 (m,
1H), 2.01 (t, J=7.1Hz, 2H), 1.94 (m, 2H), 1.87 (t,
J=7.1Hz, 2H), 1.66 (quintet, J=7.1Hz, 2H) 1.48 (m,
20
2H), 1.02 ppm (d, J=7.3Hz, 3H); ½αꢀD =+58° (c 0.2;
DMSO); MS (EI, 70eV) m/z (%): 846; HRMS-FAB m/z
[M+H]+ calcd for C47H58N8O7: 846.4418; found: 846.4428.
(R)-2-Amino-N-((9R,15S)-9-benzyl-1,5,8,11,14-pentaoxo-1-
((1-phenethylpiperidin-4-yl)(phenyl)amino)-3-oxa-6,7,10,13-
tetraazahexadecan-15-yl)-3-(4-hydroxyphenyl)propanamide
trifluoroacetate (11c).
Viscous oil; 0.14g (75%); 1H-
NMR (600 MHz, DMSO-d6): δ = 10.09 (s, 1H), 9.89 (s,
1H), 9.47 (s, 1H), 8.59 (d, J= 7.3 Hz, 1H), 8.20 (m, 1H),
8.16 (d, J = 8.4 Hz, 1H), 7.52–7.44 (m, 3H), 7.30 (t,
J =7.3 Hz, 2H), 7.29–7.15 (m, 10H), 7.00 (d, J= 8.1 Hz,
2H), 6.69 (d, J= 8.1 Hz, 2H), 4.71 (m, 1H), 4.56 (td,
J =9.3, 4.3Hz, 1H), 4.28 (m, 1H), 4.01 (s, 2H), 3.97 (m,
1H), 3.76 (s, 1H), 3.71 (m, 1H), 3.57 (m, 1H), 3.53 (m,
2H), 3.16 (m, 2H), 3.10 (m, 2H), 2.98 (m, 1H) 2.87 (m,
4H), 2.76 (m, 1H), 1.96 (m, 2H), 1.51 (m, 2H), 1.02ppm
[27] Ballet, S.; Pietsch, M.; Abell, A. D. Protein & Peptide Lett
2008, 15, 668.
[28] Liu, Z.; Zhang, J.; Zhang, A. Curr Pharm Design 2009, 15,
682.
[29] Zhang, A.; Liu, Z.; Kan, Y. Curr Top Med Chem 2007, 7, 343.
[30] Messer, J. R.; William, S. Curr Pharm Design 2004, 10, 2015.
[31] Pasternak, G. W Neuropharmacol 2004, 47(Suppl. 1), 312.
[32] Vance, D.; Shah, M.; Joshi, A.; Kane, R. S. Biotechnol Bioen-
gineer 2008, 101, 429.
(d, J = 6.8Hz, 3H); ½αꢀD20 = +61° (c 0.2; DMSO); MS (EI,
70eV) m/z (%): 848; HRMS-FAB m/z [M+H]+ calcd for
C46H56N8O7: 848.4221; found: 848.4233.
[33] Zhang, S.; Yekkirala, A.; Tang, Y.; Portoghese, P. S. Bioorg
Med Chem Lett 2009, 19, 6978.
[34] Zheng, Y.; Akgun, E.; Harikumar, K. G.; Hopson, J.; Powers,
M. D.; Lunzer, M. M.; Miller, L. J.; Portoghese, P. S. J. Med Chem 2009,
52, 247.
Acknowledgments. This work was supported by the U.S. Public
Health Service National Institute of Health DA 13449, DA
06284, and DA 06789.
[35] Yekkirala, A. S.; Lunzer, M. M.; McCurdy, C. R.; Powers,
M. D.; Kalyuzhny, A. E.; Roerig, S. C.; Portoghese, P. S Proc Natl Acad
Sci USA 2011, 108, 5098.
[36] Balboni, G.; Salvadori, S.; Marczak, E. D.; Knapp, B. I.;
Bidlack, J. M.; Lazarus, L. H.; Peng, X.; Si, Y. G.; Neumeyer, J. L. Eur
J Med Chem 2011, 46, 799.
[37] Valant, C.; Lane, J. R.; Sexton, P. M.; Christopoulos, A. Ann
Rev Pharmacol Toxicol 2012, 52, 153.
[38] Halazy, S. Exp Opin Ther Pat 1999, 9, 431.
[39] Shonberg, J.; Scammells, P. J.; Capuano, B. ChemMedChem
2011, 6, 963.
[40] Erlanson, D. A. ACS Chem Biol 2007, 2, 779.
[41] Hruby, V. J.; Agnes, R. S.; Davis, P.; Ma, S. W.; Lee, Y. S.;
Vanderah, T. W.; Lai, J.; Porreca, F. Life Sci 2003, 73, 699.
[42] Lee, Y. S.; Nyberg, J.; Moye, S.; Agnes, R. S.; Davis, P.; Wu,
S.; Ma, S.; Lai, J.; Porreca, F.; Vardanyan, R.; Hruby, V. J. Bioorg Med
Chem Lett 2007, 17, 2161.
[43] Lee, Y. S.; Kulkarani, V.; Cowell, S. M.; Ma, S. W.; Davis, P.;
Hanlon, K. E.; Vanderah, T. W.; Lai, J.; Porreca, F.; Vardanyan, R.;
Hruby, V. J. J Med Chem 2011, 54, 382.
[44] Lee, Y. S.; Petrov, R.; Kulkarni, V.; Min, B. J.; Ma, S.; Davis,
P.; Oyarzo, J.; Vanderah, T.; Lai, J.; Porreca, F.; Vardanyan, R.; Hruby,
V. J. Adv Exp Med Biol 2009, 611, 517.
[45] Lee, Y. S.; Agnes, R. S.; Cain, J. P.; Kulkarni, V.; Cai, M.;
Salibay, C.; Ciano, K.; Petrov, R.; Mayorov, A.; Vagner, J.; Trivedi, D.;
Davis, P.; Ma, S.; Lai, J.; Porreca, F.; Vardanyan, R.; Hruby, V. J. Pept
Sci 2008, 90, 433.
REFERENCES AND NOTES
[1] Podolsky, A. T.; Sandweiss, A.; Hu, J.; Bilsky, E. J.; Cain,
J. P.; Kumirov, V. K.; Lee, Y.; Hruby, V. J.; Vardanyan, R. S.; Vanderah,
T. W. Life Sci 2013, 93, 1010–1016.
[2] Ananthan, S. AAPS J 2006, 8, E118.
[3] Hruby, V. J.; Porreca, F.; Yamamura, H. I.; Tollin, G.; Agnes,
R. S.; Lee, Y.-S.; Cai, M.; Alves, I.; Cowell, S.; Varga, E.; Davis, P.;
Salamon, Z.; Roeske, W.; Vanderah, T.; Lai, J. AAPS J 2006, 8, E450.
[4] Dietis, N.; Guerrini, R.; Calo, G.; Salvadori, S.; Rowbotham,
D. J.; Lambert, D. G. Brit J Anaesth 2009, 103, 38.
[5] Balboni, G.; Salvadori, S.; Trapella, C.; Knapp, B. I.; Bidlack,
J. M.; Lazarus, L. H.; Peng, X.; Neumeyer, J. L. ACS Chem Neurosci
2010, 1, 155.
[6] Rozenfeld, R.; Devi, L. A. Trends Pharm Sci 2010, 31, 124.
[7] Pasternak, G. W.; Pan, Y.-X. Neuron 2011, 69, 6.
[8] Tao, P. L.; Law, P.-Y.; Loh, H. H. IUBMB Life 2010, 62, 103.
[9] Rozenfeld, R.; Abul-Husn, N. S.; Gomes, I.; Devi, L. A.
ScientificWorld 2007, 7(Suppl. 2), 64.
[10] Kabli, N.; Martin, N.; Fan, T.; Nguyen, T.; Hasbi, A.; Balboni,
G.; O’Dowd, B. F.; George, S. R. Brit J Pharmacol 2010, 161,
1122–1136.
[11] Gomes, I.; Gupta, A.; Filipovska, J.; Szeto, H. H.; Pintar, J. E.;
Devi, L. A Proc Natl Acad Sci USA 2004, 101, 5135.
[12] Zhang, Z.; Pan, Z. Z. J. Neurosci 2010, 30, 4735.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

R. S. Vardanyan, J. P. Cain, S. M. Haghighi, V. K. Kumirov, M. I. McIntosh,
A. J. Sandweiss, F. Porreca, and V. J. Hruby
Vol 000
[46] Lee, Y. S.; Petrov, R.; Park, C. K.; Ma, S.; Davis, P.; Lai,
J.; Porreca, F.; Vardanyan, R.; Hruby, V. J. J. Med Chem 2007, 50,
5528.
[47] Vardanyan, R.; Kumirov, V. K.; Nichol, G. S.; Davis, P.;
Liktor-Busa, E.; Rankin, D.; Varga, E.; Vanderah, T.; Porreca, F.; Lai,
J.; Hruby, V. J. Bioorg Med Chem 2011, 19, 6135.
[50] Baydar, A. E.; Boyd, G. V. J Chem Soc, Perkin Trans 1 1978,
11, 1360.
[51] Boyd, G. V.; Monteil, R. L. J Chem Soc, Perkin Trans 1 1978,
11, 1338.
[52] Balaban, A. R.; Balaban, T. S.; Boyd, G. V. Synthesis 1987, 6, 577.
[53] Kim, S. H.; Chung, J. M. Pain 1992, 50, 355.
[48] Nichol, G. S.; Kumirov, V. K.; Vardanyan, R.; Hruby, V. J.
CrystEngComm 2010, 12, 3651.
[54] Schiller, P. W.; Fundytus, M. E.; Merovitz, L.; Weltrowska,
G.; Nguyen, T. M.; Lemieux, C.; Chung, N. N.; Coderre, T. J. J. Med
Chem 1999, 42, 3520.
[49] Hoogewerff, S.; van Dorp, W. A. Rec Trav Chim 1892, 11, 84.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet