Design, synthesis, and biological evaluation of a series of bifunctional ligands of opioids/SSRIs

Article abstract of DOI:10.1016/j.bmc.2015.01.047

A series of opioid and serotonin re-uptake inhibitors (SSRIs) bifunctional ligands have been designed, synthesized, and tested for their activities and efficacies at μ-, δ- and κ opioid receptors and SSRIs receptors. Most of the compounds showed high affinities for μ- and δ-opioid receptors and lower affinities for SSRIs and κ opioid receptors. A docking study on the μ-opioid receptor binding pocket has been carried out for ligands 3-11. The ligands 7 and 11 have displayed the highest binding profiles for the μ-opioid receptor binding site with ΔG_bind (-12.14 kcal/mol) and K_i value (1.0 nM), and ΔG_bind (-12.41 kcal/mol) and K_i value (0.4 nM), respectively. Ligand 3 was shown to have the potential of dual acting serotonin/norepinephrine re-uptake inhibitor (SNRI) antidepressant activity in addition to opioid activities, and thus could be used for the design of multifunctional ligands in the area of a novel approach for the treatment of pain and depression.

Full text of DOI:10.1016/j.bmc.2015.01.047

Accepted Manuscript
Design, synthesis, and biological evaluation of a series of bifunctional ligands
of Opioids/SSRIs
Mehr-un Nisa, Munawar A. Munawar, Yeon Sun Lee, David Rankin, Jawaria
Munir, Josephine Lai, Misbahul A. Khan, Victor J. Hruby
PII:
S0968-0896(15)00071-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.01.047
BMC 12050
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
14 November 2014
15 January 2015
26 January 2015
Please cite this article as: Nisa, M-u., Munawar, M.A., Lee, Y.S., Rankin, D., Munir, J., Lai, J., Khan, M.A., Hruby,
V.J., Design, synthesis, and biological evaluation of a series of bifunctional ligands of Opioids/SSRIs, Bioorganic
& Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.01.047
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Design, synthesis, and biological evaluation of a
series of bifunctional ligands of Opioids/SSRIs
Mehr-un-Nisaa,b, Munawar A. Munawara, Yeon Sun Leeb,*, David Rankinc, Jawaria Munira,d,
Josephine Laic, Misbahul A. Khana, Victor J. Hruby b,*
a Institute of Chemistry, University of the Punjab, Lahore 54590, Pakistan.
b Department of Chemistry and Biochemistry, University of Arizona, Tucson, Arizona 85721,
USA.
c Department of Pharmacology, University of Arizona, Tucson, Arizona 85721, USA.
d Institute of Molecular Sciences & Bioinformatics, Lahore 54000, Pakistan.
Keywords: Opioid, SSRIs, synergistic effect, bifunctional ligands, pain, depression.
* Corresponding Authors. Tel: +1 5206216332; fax: +1 520-621-8407.
E-mail addresses: yeon@email.arizona.edu (Y.S. Lee), hruby@email.arizona.edu (V.J. Hruby).
1

Abbreviations:AcOH, acetic acid; Boc, tert-butoxycarbonyl; CHO, Chinese hamster ovary;
DALEA, [D-Ala2, Leu5] enkephalin amide, DIEA, diisopropylethylamine; DMF, N,N-dimethyl
formamide; hDOR, human δ-opioid receptor; DPDPE, c[D-Pen2, D-Pen5]enkephalin; DAMGO,
[D-Ala2, NMePhe4, Gly5-ol]enkephalin; Dmt, 2,6-dimethyltyrosine; EtOAc, ethylacetate;
HBTU,O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium
hexafluorophosphate;5-HT,
5-
hydroxytryptamine/serotonin; hKOR, human κ-opioid receptor; MeOH, methanol; rMOR, rat μ-
opioid receptor; NE, norepinephrine; NET, norepinephrine transporter; RCSB, The Research
Collaboratory for Structural Bioinformatics; RP-HPLC, reversed-phase high performance liquid
chromatography. SAR, structure-activity relationship; SNRIs,serotonin/norepinephrine re-uptake
inhibitors; SSRIs, selective serotonin reuptake inhibitors; TMH3, transmembrane helix 3; TCA,
tricyclic antidepressants; TFA, trifluoroacetic acid; Tic, 1,2,3,4-tetrahydroisoquinoline-3-
carboxylic acid, TLC, thin layer chromatography.
2

ABSTRACT
A series of opioid and serotonin re-uptake inhibitors (SSRIs)bifunctional ligands have been
designed, synthesized, and tested for their activities and efficaciesat μ-, δ- andopioid receptors
and SSRIs receptors. Most of the compounds showed high affinities for μ- and δ-opioid
receptorsandlower affinities for SSRIs and  opioid receptors. A docking study on the µ-opioid
receptor binding pocket has been carried out for ligands 3-11. The ligands 7 and 11 have
displayed the highest binding profiles for the µ-opioid receptor binding site with ΔGbind (-12.14
kcal/mol) and Ki value (1.0 nM), and ΔGbind (-12.41 kcal/mol) and Ki value (0.4 nM),
respectively. Ligand 3 was shown to have the potential of dual acting serotonin/norepinephrine
re-uptake inhibitor (SNRI) antidepressant activity in addition to opioid activities, and thus could
be used for the design of multifunctional ligands in the area of a novel approach for the treatment
of pain and depression.
3

1. Introduction
Neuropathic pain is associated with disease or injury of the nervous system. In recent years, a
number of drugs have been approved for neuropathic pain treatment, including some
antidepressants. Antidepressants have been used for many years for the treatment of pain in
humans.1 Selective serotonin re-uptake inhibitors (SSRIs) are commonly safer as compared to
classical tricyclic antidepressants and are more often prescribed to treat chronic pain despite
weaker antinociceptive effects.2 The antinociceptive modes of action of antidepressants are not
completely understood. Enhanced availability of norepinephrine (NE) and serotonin (5-HT) at
neuronal terminals generated by antidepressant drugs3,4 may activate multiple monoamine
receptors, e.g., α1, α2A, 5-HT2 and 5-HT3 receptors.1,5,6 The opioid system has been shown to be
involved in the antinociceptive activity of antidepressants, including amitriptyline, paroxetine
sibutramine.7 In the same way activation of noradrenergic and serotonergic pathways occurs
during opioid analgesia.8 On the basis of this fact, we have hypothesized that the development of
novel ligands that possess properties of SSRIs at 5-HT receptors and of agonists at the opioid
receptors, within the same molecule, may present a significant therapeutic advantages in the
treatment of depression and pain states, and enhanced analgesics effects in chronic pain states
e.g., neuropathies and analgesia without the development of tolerance.
Among three (µ, δ and κ) opioid receptors, the activation of the µ-opioid receptors produces
the highest antinociception. Most clinically used opioid drugs, including morphine, are µ opioid
agonists. Previously, bifunctional compounds have shown concerted efficacy because of
successful combination of the functions e.g., chimera ESP6 (YPFFPLM-NH2) containing
overlapped pharmacophores of endomorphin-2 and substance-P.9-11 Our design is based on the
overlapping pharmacophore concept that has been developed for bifunctionalligands with opioid
4

agonists and cholecystokinin (CCK), neurokinin-1 (NK-1) or melanocortin antagonist activities
e.g., opioid and melanocortin bifunctional ligands formed by overlapping of 2,6-
dimethyltyrosine(Dmt)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) for δ-opioid
receptor and Tic-D-Phe(p-Cl)-piperidin-4-yl-N-phenyl-propionamide for the melanocortin
receptore.g..12-16 In our study, an enkephalin-based tetrapeptide, Tyr-D-Ala-Gly-Phe, and
desmethylescitalopram (1) were used as an opioid agonist pharmacophore, and SSRIs
pharmacophore, respectively.
2. Results and discussion
2.1. Design
The co-administration of opioid agonists and a type of antidepressants, SSRIs, is expected to
give a synergistic effect17,18 and we herein set out to design an strategy to this co-administration,
developing bifunctional ligands, an approach that has proven successful in the past for increasing
potency and selectivity of many drugs. The drug design was based on bifunctional ligands
interacting with two targets in a single molecule by combining two pharmacophores; opioid and
SSRIs (Figure1). The SSRIs pharmacophore desmethylescitalopram 1 was combined with an
opioid pharmacophore so that the designed ligands maintain the topographical structures of both
pharmacophores, bearing in mind the development of previously designed μ-opioid selective 4-
anilidopiperidine, and N-phenyl-N-piperidin-4-yl-propionamide analogues.18 For the opioid
agonist activity, enkephalin-like tetrapeptide structures (H-Tyr-D-Ala-Gly-Phe-) has been
selected and modified. For the SSRIs activity, escitalopram, one of the most efficient SSRIs drug,
has been selected and modified to get compound 1. Butler et al. demonstrated that escitalopram
has an N,N-dimethyl propylamine group, a primary aromatic moiety with a electronegative but
not hydrogen bond-donating cyano group, and a secondary aromatic ring with a fluorine
5

substituent.19 Structure-activity relationship (SAR) studies revealed that the aromatic CN
substituent is crucial for high serotonin transporter inhibitory activity, despite the fact that the
aromatic fluorine atom and the amino substitution are less significant factors for serotonin
transporter inhibition.20 Further studies recognized that (S)-enantiomers of escitalopram
analogues are also more selective and potent than (R)-enantiomers. In the meantime,
escitalopram remains the most potent and selective compound among phenyl-substituted
phthalanes as an inhibitor of serotonin transporter.20-23
2.2. Chemistry
The designed ligands (3-11) (Table 1) were synthesized by stepwise solution phase peptide
syntheses using Nα-Boc chemistry. During the peptide syntheses (chain elongation) the
intermediates were isolated by precipitation from appropriate organic solvents, generally diethyl
ether with high purity. The products were purified by preparative RP-HPLC to afford > 98 %
pure compounds in 45-56 % yields (Scheme 1). The ligands (3-11) were analyzed by 1H NMR,
13C NMR, Low Resolution and High Resolution Mass Spectroscopy and HPLC.
2.3. Biological Evaluation
2.3.1. Opioid receptors
Opioid binding were evaluated by radioligand competition analysis using [3H]DPDPE for the δ
opioid receptor, [3H]DAMGO for the μ-opioid receptor and [3H]U69,593 for the κ
opioidreceptor in cell membrane preparations from transfected cells that stably express the
respective receptor type. Ligand 3 in which a enkephalin-like tetrapeptide (H-Tyr-D-Ala-Gly-
Phe-) and desmethylescitalopram (1) are combined revealed nonselective moderate binding
affinities at the three opioids receptors (Ki= 88 nM, 57 nM, 86 nM for µ, δ and κ-opioid
receptors, respectively) (Table 2). Comparing to the binding affinities of tetrapeptide amide (H-
6

Tyr-D-Ala-Gly-Phe-NH2, Ki/Ki = 107), it is clear that the combination of the opioid
pharmacophore with the SSRIs reduced µ receptor selectivity over receptor in all cases.
As discussed by the formerly established SAR, the replacement of D-Ala at position 2 with D-
Nle and the halogenation at the para position of Phe aromatic ring at position 4 improved opioid
activities at both μ- and δ- opioid receptors in vitro, that was in agreement with the other
outcomes,24,25 in combination with increased lipophilicity (aLogP increase: 0.7 for D-Nle, 0.5 for
Cl).26 Herein, the modification in ligand 3 led to ligand 5, which demonstrated augmented opioid
binding affinity (MOR: Ki=16 nM, 5.5-fold increase; DOR: Ki=1.4 nM, 40-fold increase; KOR:
Ki = 68 nM). The D-Nle and the F-substitutions increased the δ-opioid binding affinity more than
the μ-opioid binding affinity. Likewise, Cl-replacement at the para position of Phe aromatic ring
in ligand 3 directed to ligand 4, which established augmented opioid binding affinities (MOR: Ki
= 7.0 nM, 13-fold increase; DOR: Ki = 1.6 nM, 37- fold increase; KOR: Ki = 43 nM). It is evident
from the literature that halogenation and methylation increase the lipophilicity and cell
permeability.27 It is also documented that substitution of a chlorine on the Phe residue of [D-
Pen2, D-Pen5]enkephalin (DPDPE) led to an important enhanced cell permeability in both in vivo
and in vitro trials.28 In our case, Cl-substitution on the aromatic ring and Dmt-substitution in
ligand 3 gave ligand 6, which validated better opioid affinities [Ki= 12 nM (7-fold increase), Ki=
4.0 nM (14-fold increase) and Ki= 37 nM (2-fold increase) at the µ, δ and κ-opioid receptors,
respectively] relative to 3. Correspondingly replacement of D-Ala with D-Nle and F-substitution
at the para position of Phe aromatic ring in ligand 3 led to ligand 7, which displayed non-
selective high opioid affinities [Ki= 1.0 nM (16-fold increase), Ki= 0.8 nM and Ki= 3.0 nM (23-
fold increase) at the µ, δ and κ-opioid receptors, respectively].
7

Previously it has been shown that Dmt-Tic is a highly selective δ-opioid antagonist, and its
modification lead to diverse changes in opioid activities and selectivities.29 The Tic residue was
changed to D-Tic in ligands 8-11. These ligands retained similar biological activities with the
other ligands, which suggested that the constrained and very hydrophobic D-Tic residue could be
replaced for the flexible D-Ala and D-Nle residues in the enkephalin structure. The F-
substitution in ligand 10 increased the opioid binding affinities [Ki= 7.0 nM (5-fold increase),
0.91 nM (22-fold increase), 9.0 nM (10-fold increase) for three the µ, δ and κ receptors,
respectively] relative to 8. However its δ receptor selectivity over µand κ receptors was
converted to µ receptor selectivity in ligand 11 by deleting the Gly at position 3.
With the aim of reducing the pharmacophore size but increasing the lipophilicity, the Gly3
residues in ligands 8 and 10 were deleted, achieving ligands 9 and 11, respectively. It has been
accepted that the comparative proximity of the Tyr aromatic ring and the Phe aromatic ring is
critical forthe opioid receptor selectivity.30 In our findings, the deletion of Gly3 reduced the δ-
opioid receptor activity while it increased the μ-opioid receptor affinity in one case (ligand 11).
The Gly3 deleted ligands exhibited increased δ-opioid receptor affinity over µ-opioid receptor
affinity in the binding assays in our second case (ligand 9). It is not clear how the F- and Cl-
substitutions could be responsible for the contradictory effects on the receptor selectivities in
ligands 9 and 11.
2.3.2. Serotonin, adrenergic, dopamine, and sigma receptors and monoamine transporters
Ligands were subjected to the primary radioligand binding assays at serotonin receptors (5HT1-
7) using CHO cells or HEK cells.31 In the primary binding assays, all ligands did not showmore
than 50% inhibition at 10 M concentration, when the percent inhibition was calculated as
follows: 100% - 0% radioactivity bound. In addition to the binding assays at opioid and serotonin
8

receptors, assays were also examined against a panel of neurotransmitters transporters and brain
receptors. While all serotonin receptors were devoid of sufficient inhibition, some of ligands
exhibited affinities for the other receptors: adrenergic receptors, dopamine receptors, sigma
receptors, and biogenic monoamine transporters (Table 3). Ligands 6 (Ki= 0.24 M) and 7 (Ki=
0.28 M) showed the submicromolar range of binding affinities at 2C receptor. Ligand 5
exhibited selective binding affinity (Ki = 47 nM) at the sigma1 receptor in the nanomolar range.
However, binding at the rest of the tested receptors and transporters was not found to give
significant affinity. Notably, ligand 3 was found to bind to NET (Ki= 0.75 M), SERT (Ki= 0.28
M), and  receptor (Ki= 0.41 M) in the submicromolar range. It is evident from the literature
that newer dual acting antidepressants e.g., SNRIs (e.g. venlafaxine and duloxetine) possesses
analgesic efficacy like TCAs, but have a more liable safety and tolerability profile and also have
an efficacy advantage over SSRIs in treating the painful physical symptoms of depression and in
achieving remission of all symptoms of depression.32 The ligand 3 emerged with the potential of
a dual acting serotonin /norepinephrine re-uptake inhibitor (SNRI) in addition to excellent opioid
activities. Therefore, the structure of ligand 3 can be considered as a good scaffold for designing
a novel series of bifunctional ligands for the treatment of pain and depression.
2.3.3. Functional Assays at 5HT2-family receptors
Functional assays of the ligands 6, 7, 10 and 11 were performed for 5HT2 receptors. Ligands 6
and 7, as we expected from the binding assay data, did not exhibit any significant functional
activities of 5HT2-family receptors (5HT2A, 5HT2B, 5HT2C) (Table 4). Ligands 10 and 11, which
showed 94% and 99% of antagonist activities, respectively, at 10 M in the primary functional
assays, were tested for secondary screening using concentration-response assays. From this assay,
9

it was confirmed that both ligands do not possess any functional activity for the 5HT2-family
receptors.
Our aim was to develop bifunctional ligands of SSRIs and opioid in one structure using two
known pharmacophores as a starting point. The bifunctional ligands showed excellent binding
affinities at µ, δ and κ opioid receptors while these ligands did not show any significant
interaction with 5-HT receptors. On the basis of the biological activity profile, these ligands are
considered to show good analgesic effects rather than bifunctional opioid and SSRIs properties.
It is likely that opioid decreases the release of the monoamines in the synaptic cleft while in case
of SSRIs it inhibits the reuptake of serotonin neurotransmitter.33,34 The Selective inhibition was
not possible and this may be the reason why ligands exhibited opioid but not SSRIs activities.
However, this idea could be helpful for further design and compatibility of two different
pharmacophores in the search of an approach for pain and depression management in a single
molecule. In our other findings, during a multitargeted screen of opioids/SSRIs ligands 3
exhibited the potential of dual acting SNRIs antidepressant properties in addition to opioid
activities.
2.3.4. Molecular Docking Studies
The 3D structure of Rattus norvegicus 5-HT2B receptor (PDB ID 4nc3) was retrieved from the
Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB). The 3D
structure of Rattus norvegicus µ-opioid receptor in active state was taken from Peptide Synthesis
and Molecular Recognition Lab35. Coordinate files were subjected to Discovery Studio
Visualizer for pre-docking receptor preparation. The pre-docking ligand preparation was
performed by Energy Minimize tool of MOE. It applied Merck Molecular Force Field 94x
(MMFF94x) to obtain the lowest energy conformation of the ligands. The optimized coordinates
10

of Ligands 3-11 were docked with the μ-opioid receptor and 5-HT2B receptor separately by
Molecular Operating Environment (MOE). The London dG and Affinity dG scoring functions
were applied by MOE to estimate the free binding energy in Kcal/mol. MOE Site Finder tool
estimated the potential binding sites for μ-opioid receptor and 5-HT2B receptor. The selected
binding pockets of both receptor proteins included the important amino acid residues as indicated
in the respective crystal structures.35, 36
To get qualitative assessment and to understand the molecular basis of the calculated biological
activities (Ki) the docked complexes of ligands 3-11 were analyzed. The docked structures were
evaluated by using the Ligand Interaction tool of MOE package. The binding affinity of the
docked ligands was given as S-score (free binding energy or ΔGbind kcal/mol) calculation.
Hydrogen bonding and hydrophobic interactions of each ligand were estimated within binding
pocket of receptor protein. The conformation of each compound which exhibits the highest
binding affinity has shown in Table 5 for the µ-opioid receptor and for the 5-HT2B receptor (See
Supporting Information: Table S9 and Figure S2). The results were used to suggest a preliminary
structure-activity correlation, bearing in mind that the assays have been performed in vitro,
enhancing the complexity of the biological response and, hence, of correlation to the theoretical
data.
In µ-opioid binding pocket, ligand 11 exhibited the highest binding affinity as indicated by
ΔGbind (-12.41 kcal/mol) which agrees with the experimentally determined binding affinity of this
ligand (Ki = 0.4 nM) (Table 5, Figure 2). Visual inspection of this complex shows that the
terminal amino group of the Dmt moiety potentially forms hydrogen bond with Asp216 (1.78 Å),
and the hydroxyl group of Dmt also forms two hydrogen bonds with Asn127 with bond length of
2.74 Å and 2.45 Å. It is worth noting that these hydrogen bond contacts are playing an important
11

role in giving ligand 11 superior binding affinity compared to other tested ligands. Also the free
rotation of the heterocyclic rings around the axis of the carbon linker allows the best orientation
inside the binding pocket, which is not allowed in the other ligands. All the benzo moieties of the
tetrahydroisoquinoline, isobenzofuran, 4-fluorophenyl and 4-fluoro-phenylalanine were within
Van der Waals contact distances with the hydrophobic side chains of the receptor amino acids.
Such contacts involve the hydrophobic amino acids of active site such as Val236,Leu232, Leu219,
Ile144, Met151, Trp293, Val300 and Trp318.
The binding affinity (Ki = 1.0 nM) of ligand 7 was correlated with ΔGbind (-12.14 kcal/mol)
(Table 5, Figure 2). Visual inspection of this complex exhibits that the terminal amino group of
the Dmt scaffold has a potential for hydrogen bond with Asp147 amino acid with bond length of
1.55 Å. In this study, the salt bridge between Asp147 residue in TMH3 of the MOR and terminal
amino group of peptide ligand is observed in 6 out of 9 ligands. For ligand 7, the benzo moieties
and rest of the molecule were found to lie within a hydrophobic contact to Ile144, Ile296, Trp226,
Val300 and Trp318. All the other ligands have shown decreased binding affinities (Table 5 and see
Supporting Information: Figure S1) relative to ligands 7 and 11.
If we correlate the ΔGbind and Ki values of ligand 3 and 4, it showed that in ligand 3
phenylalanine replacement with 4-Cl-phenylalanine ligand 4 formed and it exhibited higher
ΔGbind (-4.29 kcal/mol) and Ki (7.0 nM) than ligand 3 ΔGbind (-4.80 kcal/mol) and Ki (88 nM).
This may be due to the extensive hydrogen bonding between the amino group of Tyr and Asp147
(1.47 Å) and other hydrogen bonding between NH group of 4-Cl-phenylalanine and Glu229 (1.35
Å) amino acid. Similarly, oxygen atom of furan ring accepts a hydrogen bond from Lys209 (2.62
Å) amino acid residue while rest of the molecule was found to lie within a hydrophobic contact
to Val300, Trp293, Trp318, Ile296, Ile322 and Leu219 residues.
12

Similarly, correlation of ligand 6 and 9 revealed that substitution of alanine with
tetrahydroisoquinoline of ligand 6 and truncation of glycine it formed ligand 9, and exhibited the
greater ΔGbind (-9.34 kcal/mol) and Ki (10 nM) values as compared to ligand 6 ΔGbind (-6.01
kcal/mol) and Ki (12 nM). This is due to the hydrogen bonding between the terminal amino
group of Dmt and Asp147 (1.60 Å) and a hydrogen bond acceptance of most electronegative
oxygen of furan ring and Thr218 (3.27 Å) amino acid. Moreover, some hydrophobic contacts
could also be seen between the ligand and binding pocket such as Trp318, Trp293, Val300, Leu219,
Ile144, Ile322 and Ile296 amino acid residues and also showed arene-cation contact potential
between phenyl ring of Dmt and Tyr326.
It is worth noting that these ligands (3-11) showed lower binding affinities in vitro and higher
ΔGbind (-9.54 to -15.02 kcal/mol) within 5-HT2B receptor binding pocket in silico probably due to
bulky substitution at desmethylescitalopram moiety. This moiety was found to be oriented
differently (compared to µ-opioid binding pocket) in an unfavorable conformation within the
binding pocket.
3. Conclusion
In the current investigation, bifunctional ligands have been designed, synthesized and tested
for their activities at the opioid and SSRIs receptors for the treatment of pain and depression.
These bifunctional ligands produced potent δ, µ and κ-opioid activities and minimal SSRIs
activities. The rationale behind this opioid/SSRIs bifunctional ligands concept was that the
opioid would decrease the release of neurotransmitters in the synapse while the SSRIs moiety
increase the selective reuptake inhibition of serotonin only. The results showed that our novel
ligands exhibit excellent binding to opioid receptors relative to SSRIs. These ligands could be
good analgesics. To gain insight into the binding of these ligands at the µ-opioid receptor,
13

molecular modeling studies were performed. As the result, ligands 7 and 11 were found to have
the best µ-opioid binding profiles in the binding pocket with -12.14 kcal/mol of ΔGbind and -
12.41 kcal/mol of ΔGbind, respectively, which are a good match of in vitro binding affinities (Ki=
1.0 nM for ligand 7, 0.4 nM for ligand 11).
4. Material and Methods
All chemicals were purchased from Novabiochem, ChemImpex International, and RSP and
1
13
were used as received. H NMR and C spectra were recorded on a Bruker Advance-400 and
Bruker DRX-600 spectrometer equipped with 5-mm Narolac triple-resonance single z-axis
gradient probe at 25 °C with XWinNMR. All NMR chemical shifts are reported as ppm relative
to tetramethylsilane (TMS: δ=0.00). Coupling constants (J) are reported in hertz. Spectra were
processed with Mestre Nova and Mestre C softwares. Mass spectra were taken in the positive ion
mode under ESI methods. High resolution mass spectra were obtained on the 9.4 T Bruker FT-
ICR-MS spectrometer in CH3OH: CH3CN (1: 1). The purity of compounds was determined by
HPLC and TLC on silica gel plates (Analtech 02521), solvent system EtOAc: hexane (8: 2) and
CH2Cl2: MeOH: AcOH(9: 1: 0.1).
4.1.1
(S)-1-(4-Fluorophenyl)-1-[3-(methylamino)propyl]-1,3-dihydroisobenzofuran-5-
carbonitrile (desmethylescitalopram) (1)
(S)-1-(4-Fluorophenyl)-1-[3-(methylamino)propyl]-1,3-dihydroisobenzofuran-5-carbonitrile
(desmethylescitalopram) (1) was prepared by following the reported methods with a
modification.37,38 Escitalopram oxalate (12 mmol) was dissolved in EtOAc (50 mL) and stirred
for 5 min. Aqueous ammonia (30 %, 50 mL) was added and stirred for further 30 min. The
organic layer was separated and the aqueous layer was further extracted with EtOAc (2 x 25
mL). The combined organic extract was dried over anhydrous MgSO4 overnight. EtOAc was
14

evaporated under reduced pressure. The residue was dissolved in toluene (20 mL) and α-
o
chloroethylchloroformate (2.6 mL, 24 mmol) was added, and refluxed for 6 h at 110 C. After
cooling, the reaction mixture was diluted with CH3OH (10 mL) and refluxed for further 5 h. The
solvents were removed under reduced pressure. TLC (CH3OH: CHCl3 = 1:1) showed that the
residue is a mixture of two components. The two components were separated via column
chromatography using (CH3OH: CHCl3 = 1:1) as eluent. The component with Rf value (0.2) was
isolated as light brown oil (0.2 g, 4 %) and found to be dimer of desmethylescitalopram (1). IR
(Nujol/cm-1) νmax 1620 (C=N). MS (ESI): m/z 621 [M+H]+. HRMS calcd for C38H39F2N4O2:
621.2972 and found (ESI, [M+H]+): 621.2974. The component with Rf value (0.7) was the
desired product; light brown oil (4.8 g, 96 %). MS (ESI): m/z 311 [M+H]+. HRMS calcd for
C19H20FN2O: 311.1559 and found (ESI, [M+H]+): 311.1554. The desmethylescitalopram (1) was
kept under the atmosphere of argon in order to avoid oxidation. IR (Nujol/cm-1) νmax: 2260 (CN);
1H NMR (400 MHz, CDCl3) δH: 9.00 (br s, 1H), 7.70 (t, J = 12.1 Hz, 3H), 7.60 (q, J = 8.0 Hz,
2H), 7.10 (t, J = 12.1 Hz, 2H), 5.19 (dd, J = 12.0, 4.0 Hz, 2H), 2.74-2.70 (m, 2H), 2.25 (d, J =
4.0 Hz, 3H), 2.20 (t, J = 12.0 Hz, 2H), 1.51-1.59 (m, 2H). 13C NMR (100 MHz, CDCl3) δC:
163.20, 161.03, 149.03, 139.93, 132.10, 126.44, 125.22, 122.99, 118.33, 115.17, 112.08, 90.71,
71.46, 48.97, 38.00, 32.63, 20.46. MS-(ESI) m/z: [M+H]+, 311; HRMS: Calcd. for C19H20FN2O:
311.1559; found: 311.1554; Physical state: Light brown oil; Yield: 4.8 g (96 %).
4.2. Synthesis of (S)-1-(4-fluorophenyl)-1-(3-[methylamino)propyl]-1,3-dihydroisobenzo
furan-5-carbonitrile (1)-conjugated peptide ligands (3-11)
General Procedure
Ligands 3-11 were prepared by stepwise synthesis using Nα-Boc chemistry starting from (S)-1-
(4-fluorophenyl)-1-[3-(methylamino)propyl]-1,3-dihydroisobenzofuran-5-carbonitrile (1).12 (S)-
15

1-(4-Fluorophenyl)-1-[3-(methylamino)propyl]-1,3-dihydroisobenzofuran-5-carbonitrile (1, 1
mmol) and Nα-Boc-amino acid (1.1 mmol) were dissolved in DMF (15 mL) and cooled in an ice
bath for 10 minutes. HBTU (1.2 mmol) and DIEA (2 mmol) were added to the reaction mixture
and stirred for 1-2 h at room temperature. The disappearance of the starting amine (1) was
monitored by TLC. After completion of the reaction, saturated solution of NH4Cl (15 mL) was
added into the reaction mixture and extracted with EtOAc (3 x 15 mL) and washed with aqueous
NaHCO3 (5 %, 3 x 15 mL), brine (1 x 15 mL), and water (1 x 15 mL), consecutively. The
organic extract was dried over anhydrous MgSO4 and filtered. The EtOAc was evaporated under
reduced pressure and triturated with diethyl ether to give a solid. The Nα-Boc group was
o
deprotected by 80% TFA in dichloromethane at 0 C for 30 min. After completion of the chain
elongation by subsequent couplings and deprotections, the mixtures were evaporated under
reduced pressure. The residues were triturated with diethyl ether and purified by preparative RP-
HPLC (20-80 % acetonitrile in aqueous 0.1 % TFA within 20 min for ligands (3-11) to give pure
ligands as white powders in overall yields of 44-56 %. The purity of the ligands was determined
as ≥ 97 % by analytical HPLC (i) 10-90 % acetonitrile in aqueous 0.1 % TFA in aqueous 0.1 %
TFA within 40 min, ii) 25-65 % acetonitrile in aqueous 0.1 % TFA in aqueous 0.1 % TFA within
20 min). The purity of the ligands were also checked by TLC using three different solvent
systems (i) CHCl3/MeOH/AcOH = 90:10:3, (ii) EtOAc/BuOH/H2O/AcOH = 5:3:1:1, (iii)
BuOH/H2O/AcOH = 4:1:1. The sequence was repeated to insert other required amino acids in
the desired peptide.
(S)-2-(2-{(R)-2-[(S)-2-Amino-3-(4-hydroxyphenyl)propanamido]propanamido}acetamido)-
N-{3-[(S)-5-cyano-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-1-yl]propyl}-N-methyl-3-
phenylpropanamide (3)
16

1H NMR (400 MHz, DMSO-d6) δH: 8.44-8.34 (m, 3NH), 8.16-8.07 (m, 2H), 7.81-7.78 (m,
3H), 7.72-7.70 (m, 1H), 7.58-7.55 (m, 2H), 7.18-7.17 (m, 3H), 7.152 (s, 2H), 6.42 (d, J = 3.2 Hz,
3H), 5.14 (s, 1H), 2.77 (s, 2H), 2.67 (d, J = 1.6 Hz, 2H), 2.51-2.49 (m, 3H), 2.25-2.23 (m, 2H),
2.20-2.19 (m, 1H), 2.18 (s, 1H), 2.17-2.13 (m, 8H), 2.06-2.02 (m, 2H), 1.77 (s, 1H), 0.90-0.86
(m, 3H). Yield: 0.42 g (56 %).
(S)-2-(2-{(R)-2-[(S)-2-Amino-3-(4-hydroxyphenyl)propanamido]propanamido}acetamido)-
3-(4-chlorophenyl)-N-{3-[(S)-5-cyano-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-1-yl]
propyl}-N-methylpropanamide (4)
1H NMR (400 MHz, DMSO-d6) δH: 7.64-7.59 (m, 2H), 7.53-751 (d, J = 8.0 Hz, 1H), 7.47 (s,
2H), 7.44-7.42 (m, 1H), 7.40-7.38 (m, 2H), 7.28 (s, 1H), 7.27 (d, J = 7.2 Hz, 1H), 7.21-7.17 (m,
4H), 7.16 (s, 3H), 7.02-7.00 (m, 1H), 5.31 (s, 1H), 5.24-5.13 (m, 2H), 3.41-3.30 (m, 1H), 3.07-
3.06 (m, 1H), 3.02-3.00 (m, 1H), 2.89-2.88 (m, 4H), 2.70 (s, 3H), 2.76-2.72 (m, 2H), 2.58-2.51
(m, 2H), 2.26 (t, J = 6.8 Hz, 2H), 2.16-2.09 (m, 2H), 182-1.79 (m, 2H), 1.50-1.36 (m, 3H).
Yield: 0.35 g (47 %).
(R)-2-[(S)-2-Amino-3-(4-hydroxyphenyl)propanamido]-N-(2-{[(S)-1-({3-[(S)-5-cyano-1-(4-
fluorophenyl)-1,3-dihydroisobenzofuran-1-yl]propyl}(methyl)amino)-3-(4-fluorophenyl)-1-
oxopropan-2-yl]amino}-2-oxoethyl)hexanamide (5)
1H NMR (400 MHz, DMSO-d6) δH:7.65-7.60 (m, 2H), 7.53 (d, J = 6.4 Hz, 1H), 7.45 (s, 2H),
7.42-7.40 (m, 5H), 7.28-7.27 (m, 3H), 7.20-7.15 (m, 1H), 7.13-7.11 (m, 1H), 7.06-7.03 (m, 1H),
7.02-7.00 (m, 2H), 5.25-5.12 (m, 2H), 4.03-4.02 (m, 2H), 4.00-3.97 (m, 1H), 3.79 (s, 1H), 3.42-
3.37 (m, 1H), 3.05-3.00 (m, 1H), 2.88-2.86 (m, 6H), 2.81 (s, 3H), 2.75-2.55 (m, 2H), 2.22-2.13
(m, 2H), 2.09-1.81 (m, 8H), 1.54-1.40 (m, 3H). Yield: 0.34 g (48 %).
17

(S)-2-(2-{(R)-2-[(S)-2-Amino-3-(4-hydroxy-2,6-dimethylphenyl)propanamido]propanami
do}acetamido)-3-(4-chlorophenyl)-N-{3-[(S)-5-cyano-1-(4-fluorophenyl)-1,3-dihydroisobenz
ofuran-1-yl]propyl}-N-methylpropanamide (6)
1H NMR (400 MHz, DMSO-d6) δH: 8.78-8.71 (m, 1H), 8.37 (br s, 3H), 8.10-8.01 (m, 2H),
7.74-7.72 (m, 1H), 7.70-7.63 (m, 1H), 7.52-7.48 (m, 2H), 7.19-7.16 (m, 1H), 7.12-7.02 (m, 5H),
6.45-6.28 (m, 2H), 5.15-5.04 (m, 2H), 4.74-4.64 (m, 1H), 4.17-4.15 (m, 2H), 3.80-3.76 (m, 2H),
3.63-3.50 (m, 2H), 3.36-3.22 (m, 1H), 3.13-2.91 (m, 2H), 2.85-2.77 (m, 1H), 2.71 (s, 3H), 2.45-
13
2.43 (m, 1H), 2.11 (s, 6H), 2.07-1.94 (m, 2H), 1.29-1.16 (m, 2H), 0.84-0.57 (m, 3H). C NMR
(150 MHz, DMSO-d6) δC: 172.07, 171.23, 170.63, 168.55, 158.69, 156.14, 141.39, 140.34,
138.84, 134.43, 132.51, 132.31, 131.98, 131.60, 131.44, 129.90, 129.01, 128.78, 128.40, 117.67,
115.69, 114.78, 111.03, 90.90, 71.45, 59.86, 57.76, 56.49, 51.85, 47.68, 37.67, 36.45, 35.09,
21.85, 20.31, 18.30. Yield: 0.31 g (46 %).
(R)-2-[(S)-2-Amino-3-(4-hydroxy-2,6-dimethylphenyl)propanamido]-N-(2-{[(S)-1-({3-[(S)-
5-cyano-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-1-yl]propyl}(methyl)amino)-3-(4-
fluorophenyl)-1-oxopropan-2-yl]amino}-2-oxoethyl)hexanamide (7)
1H NMR (400 MHz, DMSO-d6) δH: 8.39 (br s, 3H), 8.27 (br s, 1H), 8.19-8.12 (m, 2H), 7.86-
7.82 (m, 3H), 7.76 (d, J = 8.0 Hz, 1H), 7.63-7.60 (m, 2H), 7.25-7.19 (m, 3H), 7.08-6.97 (m, 2H),
6.45 (br s, 2H), 5.25-5.16 (m, 2H), 4.86-4.77 (m, 1H), 4.18-4.15 (m, 2H), 3.72-3.63 (m, 2H),
3.45-3.32 (m, 1H), 3.23-3.06 (m, 2H), 2.95-2.86 (m, 2H), 2.82 (s, 3H), 2.72-270 (m, 1H), 2.57-
2.56 (m, 2H), 2.25 (s, 6H), 2.18-2.03 (m, 2H), 1.46-1.21 (m, 2H), 1.20-1.07 (m, 2H), 0.82-0.79
13
(m, 5H). C NMR (150 MHz, DMSO-d6) δC: 171.60, 170.76, 168.89, 168.51, 160.74, 159.14,
156.14, 141.23, 140.27, 138.71, 133.75, 133.67, 132.49, 131.63, 131.51, 131.35, 129.99, 128.80,
18

119.24, 117.51, 115.37, 114.70, 110.98, 90.89, 71.45, 53.59, 51.88, 50.46, 49.18, 47.52, 37.62,
35.07, 33.64, 31.88, 27.22, 22.33, 21.84, 20.41, 14.09. Yield: 0.35 g (47 %).
(S)-2-[(S)-2-Amino-3-(4-hydroxy-2,6-dimethylphenyl)propanoyl]-N-(2-{[(R)-3-(4-chloroph
enyl)-1-({3-[(S)-5-cyano-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-1-yl]propyl}(methyl)
amino)-1-oxopropan-2-yl]amino}-2-oxoethyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxam
ide (8)
1H NMR (400 MHz, DMSO-d6) δH: 8.35 (s, 2H), 7.30-7.29 (m, 2H), 7.27-7.26 (m, 1H), 7.23-
7.20 (m, 4H), 7.15-7.12 (m, 1H), 7.09-7.08 (m, 1H), 7.06-7.02 (m, 4H), 6.86-6.79 (m, 1H), 6.76-
6.75 (m, 1H), 6.57 (dt, J = 4.0, 8.0 Hz, 1H), 6.29-6.26 (m, 2H), 4.89 (s, 1H), 4.54-4.44 (m, 2H),
4.17-4.12 (m, 1H), 3.12-3.09 (m, 2H), 3.06-3.02 (m, 2H), 2.99-2.80 (m, 2H), 2.78-2.76 (m, 1H),
2.61-2.55 (m, 1H), 2.48 (s, 6H), 2.20-2.19 (m, 11H), 2.13-1.82 (m, 2H), 0.92-0.88 (m, 2H).
Yield: 0.34 g (46 %).
(S)-2-[(S)-2-Amino-3-(4-hydroxy-3,5-dimethylphenyl)propanoyl]-N-[(R)-3-(4-chlorophen
yl)-1-({3-[(S)-5-cyano-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-1-yl]propyl}(methyl)
amino)-1-oxopropan-2-yl]-1,2,3,4-tetrahydroisoquinoline-3-carboxamide (9)
1H NMR (400 MHz, DMSO-d6) δH: 8.30 (d, J = 12.0 Hz, 3H), 7.34-7.29 (m, 1H), 7.27-7.25
(m, 1H), 7.23-7.20 (m, 1H), 7.19-7.15 (m, 3H), 7.09-7.03 (m, 4H), 6.92-6.86 (m, 1H), 6.79-6.75
(m, 1H), 6.72-6.65 (m, 1H), 6.56-6.52 (m, 1H), 6.28 (d, J = 3.2 Hz, 2H), 6.22 (d, J = 5.6 Hz,
1H), 4.97 (s, 1H), 4.86-4.77 (m, 1H), 4.67-4.54 (m, 1H), 4.48-4.41 (m, 2H), 4.31-4.06 (m, 1H),
3.04-2.99 (m, 4H), 2.86-2.76 (m, 2H), 2.73-2.50 (m, 2H), 2.48 (s, 6H), 2.19-2.18 (m, 9H), 0.88-
.87 (m, 2H). Yield: 0.33 g (45 %).
(S)-2-[(S)-2-Amino-3-(4-hydroxy-3,5-dimethylphenyl)propanoyl]-N-(2-{[(R)-1-({3-[(S)-5-
cyano-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-1-yl]propyl}(methyl)amino)-3-(4-fluor
19

ophenyl)-1-oxopropan-2-yl]amino}-2-oxoethyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxam
ide (10)
1H NMR (400 MHz, DMSO-d6) δH: 8.36 (br s, 3NH), 8.33 (s, 2H), 8.12-8.09 (m, 1H), 7.80-
7.79 (m, 2H), 7.71-7.69 (m, 1H), 7.58-7.54 (m, 2H), 7.20-7.17 (m, 2H), 7.15 (s, 1H), 7.12-7.08
(m, 1H), 7.02-6.91 (m, 1H), 6.40-6.39 (m, 4H), 4.28 (s, 1H), 4.14-4.11 (m, 4H), 3.71-3.61 (m,
4H), 3.54 (s, 1H), 2.75-2.73 (m, 4H), 2.66-2.64 (m, 1H), 2.51-2.2.49 (m, 3H), 2.17-2.16 (m, 8H),
1.35-1.06 (m, 1H), 0.77-0.74 (m, 6H). Yield: 0.31 g (54 %).
(S)-2-[(S)-2-Amino-3-(4-hydroxy-3,5-dimethylphenyl)propanoyl]-N-[(R)-1-({3-[(S)-5-cyano-
1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-1-yl]propyl}(methyl)amino)-3-(4-fluorophen
yl)-1-oxopropan-2-yl]-1,2,3,4-tetrahydroisoquinoline-3-carboxamide (11)
1H NMR (400 MHz, DMSO-d6) δH: 8.35 (br s, 3NH), 7.92-7.70 (m, 1H), 7.23-7.20 (m, 3H),
7.06-7.02 (m, 7H), 6.87-6.83 (m, 1H), 6.75 (s, 1H), 6.56-6.53 (m, 1H), 6.29-6.27 (m, 3H), 5.08
(s, 1H), 4.89-4.85 (m, 4H), 4.55-4.44 (m, 2H), 3.11- 3.04 (m, 6H), 2.95-2.86 (m, 2H), 2.80-2.74
(m, 1H), 2.62-2.58 (m, 1H), 2.48 (s, 1H), 2.20-2.18 (m, 7H), 2.13-2.12 (m, 1H), 1.82-1.80 (m,
2H), 0.89-0.86 (m, 2H). Yield: 0.34 g (51 %).
4.3. Radioligand labeled binding assays and functional assays
Ligands were tested for their binding affinities at opioid receptors, as previously
described.12,14,39,40 Initial screens for various receptors and transporters and functional assays for
serotonin (5-HT) 5-HT2A, 5HT2B and 5HT2C receptors were determined in Roth lab.31,41 The
results of opioid binding affinities obtained from the Roth lab were consistent with our findings.
Supporting Information
1H NMR and 13C NMR spectra, and Mass spectrum of the ligands 1, 3-11 are provided.
Acknowledgement
20

We gratefully acknowledge a grant from the Higher Education Commission of Pakistan under
the IRSIP program to support Mehr-un-Nisa. This work was partially supported by grants from
The U.S. Public Health Services, NIH, and NIDA P01DA006284 and R01DA013449.
References
1. Ghelardinic, C.; Galeotti, N.; Bartolini, A. Jpn. J. Pharmacol. 2000, 82, 130.
2. Schreiber, S.; Pick, C. G. Eur. Neuropsychopharmacol. 2006, 16, 464.
3. Mico, J. A.; Ardid, D.; Berrocoso, E.; Eschalier, A. Trends Pharmacol. Sci. 2006, 27, 348.
4. Obata, H.; Saito, S.; Koizuca, S.; Nishikawa, K.; Goto, F. Anesth. Analg. 2005, 100, 1406.
5. Otsuka, N.; Kiuchi, Y.; Yokogawa, F.; Masuda, Y.; Oguchi, K.; Hosoyamada, A. J.
Anaesth. 2001, 15, 154.
6. Yokogawa, F.; Kiuchi, Y.; Ishikawa, Y.; Otsuka, N.; Masuda, Y.; Oguchi, K.;
Hosoyamada, A. Anesth. Analg. 2002, 95, 163.
7. Gray, A. M.; Pache, D. M.; Sewell, R. D. Eur. J. Pharmacol. 1999, 378, 161.
8. Eisenach, J. C.; Gebhart, G. F. Anesthesiology 1995, 583, 1036.
9. Foran, S. E.; Carr, D. B.; Lipkowski, A. W.; Maszczynska, I.; Marchand, J. E.; Misicka,
A.; Beinborn, M.; Kopin, A. S.; Kream, R. M. J. Pharmacol. Exp. Ther. 2000, 295, 1142.
10. Bonney, I. M.; Foran, S. E.; Marchand, J. E.; Lipkowski, A. W.; Carr, D. B. Eur. J.
Pharmacol. 2004, 488, 91.
11. Morphy, R.; Rankovic, Z. J. Med. Chem. 2005, 48, 6523.
21

12. Lee, Y. S.; Agnes, R. S.; Badghisi, H.; Davis, P.; Ma, S. W.; Lai, J.; Porreca, F.; Hruby, V.
J. J. Med. Chem. 2006, 49, 1773.
13. 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 2006, 8, E450.
14. Yamamoto, T.; Nair, P.; Davis, P.; Ma, S.-W.; Navratilova, E.; Moye, S.; Tumati, S.; Lai,
J.; Vanderah, T. W.; Yamamura, H. I.; Porreca, F.; Hruby, V. J. J. Med. Chem.2007, 50,
2778.
15. Lee, Y. S. ; Agnes, R. S.; Cain, J. P.; Kulkrani, V.; Cai, M.; Salibay, C.; Ciano, K.; Petrov,
R.; Mayorov, A.; Vagner, J.; Trivedi, D.; Davis, P.; Ma, S. M.; Lai, J.; Porreca, F.;
Vardanyan, R.; Hruby, V. J. Biopolymers: Peptide Science, 2007, 90, 433.
16. Horan, P. J.; Mattia, A.; Bilsky, E. J.; Weber, S.; Davis, T. P.; Yamamura, H. J.;
Malatynska, E.; Appleyard, S. M.; Slaninova, J.; Misicka, A.; Lipowski, A. W.; Hruby, V.
J.; Porreca, F. J. Pharmacol. Exp. Ther.1993, 265, 1446.
17. Eriksen, L. B.; Munro, G.; Nielsen, O. E.; Peters, D. WO 09/077585 A1 2009.
18. Lee, Y. S.; Petrov, R.; Park, C. K.; Ma, S.-w.; Davis, P.; Lai, J.; Porreca, F.; Vardanyan,
R.; Hruby, V. J. J. Med. Chem. 2007, 50, 5528–5532.
19. Butler, S. G.; Meegan, M. J. Curr. Med. Chem. 2008, 15, 1737.
20. Zhang, P.; Cyriac, G.; Kopajtic, T.; Zhao, Y.; Javitch, J. A.; Katz, J. L.; Newman, A. H. J.
Med. Chem. 2010, 53, 6112-6121.
22

21. Eildal, J. N.; Andersen, J.; Kristensen, A. S.; Jorgensen, A. M.; Bang-Andersen, B.;
Jorgensen, M.; Stromgaard, K. J. Med. Chem. 2008, 51, 3045.
22. Andersen, J.; Olsen, L.; Hansen, K. B.; Taboureau, O.; Jorgensen, F. S.; Jorgensen, A. M.;
Bang-Andersen, B.; Egebjerg, J.; Stromgaard, K.; Kristensen, A. S. J. Biol. Chem. 2010,
285, 2051.
23. Koldso, H.; Severinsen, K.; Tran, T. T.; Celik, L.; Jensen, H. H.; Wiborg, O.; Schiott, B.;
Sinning, S. J. Am. Chem. Soc. 2010, 132, 1311.
24. 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.
25. Weber, S. J.; Greene, D. L.; Sharma, S. D.; Yamamura, H. I.; Krammer, T. H.; Burks, T.
F.; Hruby, V. J.; Hersh, L. B.; Davis, T. P.J. Pharmacol. Exp. Ther. 1991, 259, 1109.
26. http://www.vcclab.org/lab/alogps/.
27. Weber, S. J.; Abbruscato, T. J.; Brownson, E. A.; Lipkowski, A. W.; Polt, R.; Misicka, A.;
Haaseth, R. C.; Bartosz, H.; Hruby, V. J.; Davis, T. P.J. Pharmacol. Exp. Ther. 1993, 266,
1649.
28. Weber, S. J.; Greene, D. L.; Sharma, S. D.; Yamamura, H. I.; Krammer, T. H.; Burks, T.
F.; Hruby, V. J.; Hersh, L. B.; Davis, T. P. J. Pharmacol. Exp. Ther.1991, 259, 1109.
29. Bryant, S. D.; Jinsmaa, Y.; Salvadori, S.; Okada, Y.; Lazarus, L. H.Biopolymers 2003, 71,
86.
23

30. Schiller, P. W. Opioid Peptides: Biology, Chemistry, and Genetics; in: Udentriend, S.;
Meienhotfer, J. (Eds.), The peptides: analysis, synthesis, biology, Vol. 6; Academic Press:
New York 1984, pp 220-268.
31. Ki determinations, receptor binding profiles, agonist and/or antagonist functional data,
were generously provided by the National Institute of Mental Health's Psychoactive Drug
Screening Program, Contract # HHSN-271-2013-00017-C (NIMH PDSP). The NIMH
PDSP is Directed by Bryan L. Roth MD, PhD at the University of North Carolina at
Chapel Hill and Project Officer Jamie Driscol at NIMH, Bethesda MD, USA.
32. Sussman, N. SNRIs versus SSRIs: Mechanisms of action in treating depression and
painful physical symptoms. J. Clin. Psychiatry 2003, 5, 19-26.
33. Davis, L. L.; Yonkers, K. A.; Trivedi, M.; Kramer, G. L.; Petty, F. The mechanism of
action of SSRIs: a new hypothesis. Clare, S. eds. R. J. Ranes Company, Standford Ch 11,
1999, pp 171-185.
34. Chahl, L. A. Aust. Prescr. 1996, 19, 63.
35. Anand, J. P.; Purington, L. C.; Pogozheva, I. D.; Traynor, J. R.; Mosberg, H. I. Chem.
Biol. Drug Des. 2012, 8(5), 763-770.
36. Liu, W.; Wacker, D.; Gati, C.; Han, G. W.; James, D.; Wang, D.; Nelson, G.; Weierstall,
U.; Katritch, V.; Barty, A.; Zatsepin, N. A.; Li, D.; Messerschmidt, M.; Boutet, S.;
Williams, G. J.; Koglin, J. E.; Seibert, M. M.; Wang, C.; Shah, S. T.; Basu, S.; Fromme,
R.; Kupitz, C.; Rendek, K. N.; Grotjohann, I.; Fromme, P.; Kirian, R. A.; Beyerlein, K. R.;
24

White, T. A.; Chapman, H. N.; Caffrey, M.; Spence, J. C.; Stevens, R. C.; Cherezov, V.
Science 2013, 342, 1521.
37. Vipin, K. K.; Umar, K. M.; Narsimha, R. B.; Ranjith, K. S.; Ramesh, D. and Sivakumaran,
M. EP 2017271 A1 2009.
38. Rao, D. R.; Kankan, R. N.; Bidari, D. R. and Singh, M. P. WO 142379 A2 2008.
39. Vanderah, T. W.; Largent-Milnes, T.; Lai, J.; Porreca, F.; Houghten, R. A.; Menzaghi,
F.; Wisniewski, K.; Stalewski, J.; Sueiras-Diaz, J.; Galyean, R.; Schteingart, C.; Junien, J.
L.; Trojnar, J.; Rivière, P. J. Eur. J. Pharmacol. 2008, 583, 62.
40. Lai, J.; Ma, S. W.; Zhu, R. H.; Rothman, R. B.; Lentes, K. U.; Porreca, F.Neuro Report
1994, 5, 2161.
41. http://pdsp.med.unc.edu
25

Table 1. Analytical data of opioid/SSRIs bifunctional ligands (3-11)
HPLC
Low MSa
Obsd.
HRMSb
TLC, Rfd
tR/purity (min/%)c
Molecular
Formula
no
Obsd.
Calcd.
(A)
(B)
(I)
(II)
(III)
3
4
C42H46FN6O6
C42H45ClFN6O6
C45H51F2N6O6
C44H49ClFN6O6
C47H55F2N6O6
C51H53ClFN6O6
C49H50ClFN5O5
C51H53F2N6O6
C49H50F2N5O5
749
783
809
811
837
899
842
883
826
749.3457
783.3807
809.3833
811.3379
837.4146
899.3449
842.3479
883.3989
826.3774
749.3455
783.3811
809.3831
811.3381
837.4156
899.3457
842.3479
883.3999
826.3785
22.4/98
23.1/99
20.3/100
24.6/100
21.6/99
23.2/100
24.1/98
20.2/100
20.7/100
34.2/98
35.2/99
32.2/100
35.2/100
32.1/99
34.3/100
33.2/98
31.5/100
31.2/100
0.82
0.79
0.75
0.74
0.72
0.77
0.83
0.78
0.80
0.47
0.39
0.31
0.37
0.21
0.41
0.44
0.25
0.43
0.77
0.61
0.59
0.55
0.56
0.67
0.75
0.49
0.62
5
6
7
8
9
10
11
a(M – TFA + H)+, ESI method [Finnigan, Thermoelectron, LCQ classic].
b(M – TFA + H)+, FAB-MS (JEOL HX110 sector instrument) or MALDI-TOF(Bruker
Ultraflex III).
cCompleted on a Hewlett Packard 1100 [C-18, Microsorb-MVTM, 4.6 mm x 250 mm, 5 μm].
(A) 10-90 % of acetonitrile with 0.1 % TFA within 30 min and up to 100 % within extra 5 min, 1
mL/min, (B) 25-65 % of acetonitrile having 0.1 % TFA within 40 min and equal to 100 % within
additional 5 min, 1 mL/min.
d(I) CHCl3/MeOH/AcOH = 90/10/3, (II) EtOAc/BuOH/water/AcOH = 5/3/1/1, (III)
BuOH/water/AcOH = 4:1:1.
26

Table 2.Binding affinities of the opioid/SSRIs bifunctional ligands at the opioid receptors.
rMORa, [3H]DAMGOb
hDORa [3H]DPDPEc
hKORa [3H]U69,593d
no
peptide structure
f, g
f,g
f,g
LogIC50
Kih(nM)
LogIC50
Kih(nM)
LogIC50
-6.52 ± 0.07
-6.82 ± 0.08
-6.61 ± 0.09
-6.81 ± 0.18
-7.92 ± 0.11
-6.49 ± 0.08
-7.34 ± 0.07
-7.50 ± 0.05
-6.09 ± 0.05
-
Kih(nM)
3
4
Tyr-D-Ala-Gly-Phe-
Tyr-D-Ala-Gly-Phe(4-Cl)-
Tyr-D-Nle-Gly-Phe(4-F)-
Dmt-D-Ala-Gly-Phe(4-Cl)-
Dmt-D-Nle-Gly-Phe(4-F)-
Dmt-D-Tic-Gly-Phe(4-Cl)-
Dmt-D-Tic-Phe(4-Cl)-
Dmt-D-Tic-Gly-Phe(4-F)-
Dmt-D-Tic-Phe(4-F)-
YaGF-NH2
-6.70 ± 0.13
-7.84 ± 0.10
-7.46 ± 0.03
-7.57 ± 0.35
-8.65 ± 0.23
-7.13 ± 0.09
-7.69 ± 0.08
-7.84 ± 0.05
-9.13 ± 0.14
-8.24 ± 0.13
-
88
7.0
16
-6.90 ± 0.03
-8.47 ± 0.06
-8.50 ± 0.05
-8.09 ± 0.09
-8.77 ± 0.12
-7.36 ± 0.03
-8.70 ± 0.06
-8.70 ± 0.04
-7.55 ± 0.06
-6.14 ± 0.16
-
57
1.6
1.4
4.0
0.8
20
86
43
68
37
3.0
91
13
9.0
230
-
5
6
12
7
1.0
35
8
9
10
0.88
0.91
13
10
11
7.0
0.4
2.8
7.7
300
DALEA
2.4
-
-
aCompetition analyses were determined using membrane preparations from transfected HN9.10
cells that constitutively expressed the respective receptor types.
bKd = 0.50 ± 0.1 nM.
cKd = 0.85 ± 0.2 nM.
dKd = 1.5 nM.
fLogarithmic values analyzed from the nonlinear regression analysis of data collected from as a
minimum two independent experiments.
gCompetition against radiolabeled ligand.
hCalculated by the Cheng-Prusoff equation.
27

Table 3.Binding affinities of the Opioid/SSRIs bifunctional ligands at various receptors and
transporters
Ki (M)a,b
Adrenergic receptorsc
Dopamine receptorsd
D2 D3
[3H]N-methylspiperone
Monoaminetransportersc
Sigma receptorc
No
α1A
α2A
α2C
NET
[3H]
SERT
[3H]
1
[
125I]
[
125I]
[3H]
[
125I]HEAT
iodoclonidine
iodoclonidine
niosoxetin
citalopram
pentazocine
3
5
6.8
6.6
n.d.
n.d.
n.d.
n.d.
2.2
>10
4.3
0.75
2.4
0.28
n.d.
n.d.
n.d.
5.7
0.41
0.047
>10
1.9
n.d.
4.7
n.d.
0.24
0.28
n.d.
n.d.
6
4.6
0.89
2.0
n.d.
n.d.
n.d.
n.d.
7
>10
n.d.
n.d.
n.d.
n.d.
n.d.
2.7
10
11
n.d.
n.d.
6.7
n.d.
n.d.
1.4
n.d.
aCompetition analyses were determined using membrane preparations from transiently or stably
transfected cell lines.
bStandards used for different receptors: chlorpromazine (Ki = 44 nM) forα1A;oxymetazoline (Ki =
2.0 nM) forα2A; prazosin (Ki = 50 nM) forα2C; diazepam (Ki = 1.2 nM) for Rat Brain Site BZP;
haloperidol (Ki = 4.5 nM) forD2; chlorpromazine(Ki = 3.2 nM) forD3;desipramine (Ki = 12 nM)
forNET; amitriptyline (Ki = 19 nM) for SERT; haloperidol (Ki = 7.0 nM) for Sigma1
cTris-HCl assaybuffers.
dHEPES-HCl assaybuffers.
n.d.: not determined due to a lower (<50%) inhibition in an initial screen at 10 M.
28

Table 4. Functional Assay results for the Opioid/SSRIs bifunctional ligands at serotonin
receptors (5-HT2).
a,b
a,b
5-HT2A (%)a,b
5-HT2B
5-HT2C
no
agonistc
antagonistd
agonistc
antagonistd
agonistc antagonistd
-0.1
-0.1
34
20
23
94
99
0.10
0.10
0.20
0.20
-6.1
4.7
49
-0.3
-0.3
2.8
6.0
11
6
7
5.2
10
10
11
0.7
43
0.2
aFunctional assays were performed on transiently or stably transfected cell lines using Hanks
balanced salt solution.
b5HT was used as a standard for 5HT2receptors: EC50 = 250 nM for 5HT2A receptor; EC50 = 1.6
nM for 5H2B receptor; EC50 = 1.7 nM for 5HT2Creceptor.
cNormalized and transformed to percentage values. The reference agonist activity at 10 M is set
as 100% and basal activity with buffer as 0%.
dThe basal activity with buffer only is set as 100% inhibition and the activity of the EC80 of the
reference agonist as 0% inhibition.
29