Structural determinants of diphenethylamines for interaction with the κ opioid receptor: Synthesis, pharmacology and molecular modeling studies

Article abstract of DOI:10.1016/j.bmcl.2016.08.031

The κ opioid (KOP) receptor crystal structure in an inactive state offers nowadays a valuable platform for inquiry into receptor function. We describe the synthesis, pharmacological evaluation and docking calculations of KOP receptor ligands from the class of diphenethylamines using an active-like structure of the KOP receptor attained by molecular dynamics simulations. The structure–activity relationships derived from computational studies was in accordance with pharmacological activities of targeted diphenethylamines at the KOP receptor established by competition binding and G protein activation in vitro assays. Our analysis identified that agonist binding results in breaking of the Arg156-Thr273 hydrogen bond, which stabilizes the inactive receptor conformation, and a crucial hydrogen bond with His291 is formed. Compounds with a phenolic 4-hydroxy group do not form the hydrogen bond with His291, an important residue for KOP affinity and agonist activity. The size of the N-substituent hosted by the hydrophobic pocket formed by Val108, Ile316 and Tyr320 considerably influences binding and selectivity, with the n-alkyl size limit being five carbon atoms, while bulky substituents turn KOP agonists in antagonists. Thus, combination of experimental and molecular modeling strategies provides an initial framework for understanding the structural features of diphenethylamines that are essential to promote binding affinity and selectivity for the KOP receptor, and may be involved in transduction of the ligand binding event into molecular changes, ultimately leading to receptor activation.

Full text of DOI:10.1016/j.bmcl.2016.08.031

Bioorganic & Medicinal Chemistry Letters 26 (2016) 4769–4774
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structural determinants of diphenethylamines for interaction with
the
j opioid receptor: Synthesis, pharmacology and molecular
modeling studies
Elena Guerrieri a,y, Marcel Bermudez b,y, Gerhard Wolber b, Ilona P. Berzetei-Gurske c,
Helmut Schmidhammer a, Mariana Spetea a,
⇑
a Department of Pharmaceutical Chemistry, Institute of Pharmacy and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innrain 80-82, A-6020
Innsbruck, Austria
b Institute of Pharmacy, Freie Universität Berlin, D-14195 Berlin, Germany
c Biosciences Division, SRI International, 333 Ravenswood Avenue, Menlo Park, CA 94025, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 July 2016
The
j opioid (KOP) receptor crystal structure in an inactive state offers nowadays a valuable platform for
inquiry into receptor function. We describe the synthesis, pharmacological evaluation and docking calcu-
lations of KOP receptor ligands from the class of diphenethylamines using an active-like structure of the
KOP receptor attained by molecular dynamics simulations. The structure–activity relationships derived
from computational studies was in accordance with pharmacological activities of targeted diphenethy-
lamines at the KOP receptor established by competition binding and G protein activation in vitro assays.
Our analysis identified that agonist binding results in breaking of the Arg156-Thr273 hydrogen bond,
which stabilizes the inactive receptor conformation, and a crucial hydrogen bond with His291 is formed.
Compounds with a phenolic 4-hydroxy group do not form the hydrogen bond with His291, an important
residue for KOP affinity and agonist activity. The size of the N-substituent hosted by the hydrophobic
pocket formed by Val108, Ile316 and Tyr320 considerably influences binding and selectivity, with the
n-alkyl size limit being five carbon atoms, while bulky substituents turn KOP agonists in antagonists.
Thus, combination of experimental and molecular modeling strategies provides an initial framework
for understanding the structural features of diphenethylamines that are essential to promote binding
affinity and selectivity for the KOP receptor, and may be involved in transduction of the ligand binding
event into molecular changes, ultimately leading to receptor activation.
Revised 10 August 2016
Accepted 11 August 2016
Available online 12 August 2016
Keywords:
j
opioid receptor
Diphenethylamine
Agonist
Antagonist
Partial agonist
SAR
Molecular dynamics
Ó 2016 Elsevier Ltd. All rights reserved.
As a prominent member of the large family of seven transmem-
brane G protein-coupled receptors (7TM-GPCRs), the kappa opioid
(KOP) receptor has been the subject of intense focus for drug dis-
covery efforts over the past years. The KOP receptor is activated
by the endogenous peptide-ligands, the dynorphins, and it signals
through the heterotrimeric Gi/o proteins.1 Given the importance of
the KOP receptor/dynorphin system as a powerful regulator of a
multitude of neurophysiological and behavioral responses, modu-
lation of this system is considered as a promising strategy for the
treatment of neuropsychiatric and other human disorders, includ-
ing pain, drug addiction, mood disorders (e.g. depression and anx-
iety), neurological conditions (e.g. epilepsy) and itching skin
diseases.2–13 Numerous lines of evidence have been accumulated
pointing to the KOP receptor as an important substrate in comor-
bidity between addictive and depressive disorders, or chronic pain
and mood disorders.9,10
Differential strategies in modulating the downstream effects of
KOP receptor signaling involve development of selective ligands
that can either activate or block the receptor. KOP agonists attract
considerable attention for their ability to produce analgesia with-
out abuse potential. On the other hand, KOP agonists are limited
by side effects (e.g. dysphoria, sedation, psychotomimetic
effects),1,5,14 whereas KOP antagonists and partial agonists have
potential to emerge as antidepressants, anxiolytics and anti-addic-
tive medications.15–19
Ligand–receptor interactions represent the basis of the
mediation of many neuro-behavioral responses by both
endogenous and exogenous ligands, and their tuning represents
the goal of a large variety of pharmacotherapies. Molecular details
of these interactions are nowadays emerging, with the recently
⇑
Corresponding author. Tel.: +43 512 507 58277; fax: +43 512 507 58299.
E-mail address: Mariana.Spetea@uibk.ac.at (M. Spetea).
y
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmcl.2016.08.031
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.

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E. Guerrieri et al. / Bioorg. Med. Chem. Lett. 26 (2016) 4769–4774
solved crystal structure of the human KOP receptor,20 therefore
providing a novel platform for inquiry into receptor function and
mechanism(s) of activation, and structural features promoting
ligand binding affinity and selectivity.21–26 Elucidation of ligand-
receptor interactions and structure–function relationships of the
KOP receptor represent important steps towards the development
of improved pharmacotherapies for human disorders, where the
KOP receptor plays a key role.
R1
CBM
CPM
n-C3H7
n-C4H9
n-C5H11
n-C6H13
phenylethyl OH
CBM
R2
R3
H
H
H
H
H
H
H
OH
R2
R3
1 (HS665)
OH
OH
OH
OH
OH
OH
3
2 (HS666)
N
4
3
4
5
6
7
8
9
R1
H
H
CPM
OH
Hence, an important objective of our laboratories is the design
and biological evaluation of structurally-distinct KOP receptor-tar-
geted ligands in order to specifically assess the contribution of the
KOP receptor in opioid system neurobiology, and furthermore, to
provide novel opportunities for the discovery of potential thera-
peutic agents.27–29 We recently disclosed a diphenethylamine ser-
ies of KOP ligands, exemplified by 1 (HS665, Fig. 1), as one of the
most interesting derivatives in terms of very high binding affinity
and selectivity for the KOP receptor,28,29 complemented by high
potency and efficacy in vivo.28 The N-cyclopropylmethyl (N-CPM)
substituted analogue 2 (HS666, Fig. 1) also displayed favorable
properties related to the activity at the KOP receptor as a potent
partial agonist. We have described that cyclobutylmethyl (CBM)
and CPM groups (1 and 2, respectively) at the nitrogen are more
suitable for interaction with the KOP receptor than n-alkyl sub-
stituents (derivatives 3–6, Fig. 1).28
On the basis of the currently available crystal structure of the
human KOP receptor in an inactive conformation,20 the present
study was undertaken to attain an active-like receptor state apply-
ing molecular dynamics (MD) simulations. The generated receptor
model was next used to characterize the binding mode of KOP
receptor-targeting diphenethylamine derivatives 1–6 (Fig. 1).
Additionally, the importance of the substitution pattern at the
nitrogen and the position of the phenolic hydroxyl group on the
interaction with the KOP receptor were investigated after synthesis
of the new derivatives 7–9 (Fig. 1). We herein report the in vitro
binding and G protein activation of the KOP receptor by 7–9 in
comparison to the previously reported analogues 1 and 2, paral-
leled by binding mode investigations of these KOP ligands.
The new diphenethylamines 7–9 were prepared as depicted in
Schemes 1 and 2. (3-Methoxyphenyl)-N-phenethaneamine (10)
and (4-methoxyphenyl)-N-phenethaneamine (11) were prepared
from 2-(3-methoxyphenyl)ethaneamine (12) and 2-(4-methoxy-
phenyl)ethaneamine (13), respectively by alkylation with
phenethylbromide.30 Next, 10 and 11 were alkylated with the
respective alkyl bromide in DMF to afford 14 and 15, and then con-
verted into the respective phenols 7 and 9 by ether cleavage with
sodium ethanethiolate in DMF (Scheme 1). Compound 8 was syn-
thetized starting from 4-hydroxyphenylacetic acid (16), which was
reacted with phenethylamine (17) in CH2Cl2 in the presence of
EDCI and HOAt to afford amide 18. BH3 reduction in THF yielded
amine 19, which was N-alkylated with CBM bromide in CH3CN in
presence of NaHCO3 to give the phenol 8 (Scheme 2).
Figure 1. Structures of investigated diphenethylamines 1–9.
R1
R2
R1
R2
R1
R2
c
a
b
NH2
NH
NR
7, 9
12 R1 = OCH3; R2 = H
13 R1 = H; R2 = OCH3
10
11
1
1 = OCH3; R2 = H; R = phenethyl
1 = H; R2 = OCH3; R = CPM
R
= OCH3; R2 = H
= H; R2 = OCH3
14
15
R
R
1
R
Scheme 1. Synthesis of diphenethylamine derivatives
7 and 9. Reagents and
conditions: (a) phenethyl bromide, K2CO3, DMF, 80 °C; (b) respective alkyl bromide,
K2CO3, DMF, 80 °C; (c) sodium ethanethiolate, DMF, 130 °C.
H
N
NH2
O
a
+
OH
HO
O
HO
16
17
18
b
c
NH
8
HO
19
Scheme 2. Synthesis of diphenethylamine derivative 8. Reagents and conditions:
(a) EDCI, HOAt, CH2Cl2, rt; (b) 1 M BH3ꢀTHF, THF, reflux; (c) CBM bromide, NaHCO3,
CH3CN, reflux.
was about 430- and 35-fold lower than that of the N-CBM and
N-CPM substituted 1 and 2, respectively. It was also evident that
the presence of the N-phenethyl moiety in 7 has major conse-
quences on the interaction with MOP and DOP receptors, by com-
pletely abolishing binding at these two receptors. In contrast to
earlier developed diphenethylamines 1–6,28 the N-phenethyl
derivative 7 exhibited no appreciable agonist activity at the KOP
receptor, but it rather antagonized the stimulation of [35S]GTP
cS
binding by the reference agonist U69,593 with relatively low
potency (Ke = 1311 nM).
Another interesting outcome of our expanded SAR in the
diphenethylamine series relates to the effect on the interaction
with the KOP receptor upon shifting the position of the phenolic
hydroxyl group. Switching the hydroxyl group from position 3 to
4 significantly decreased affinity and selectivity for the KOP recep-
tor of 8 and 9, in comparison to the 3-OH derivatives 1 and 2,
respectively (Table 1). Differential functional activity at the KOP
receptor was also noted regarding G protein activation for the
N-CBM, 3-hydroxy substituted 1 and its 4-hydroxy analogue 8, as
well as for the N-CPM, 3-hydroxy substituted 2 and its 4-hydroxy
analogue 9. While 1 is a highly potent and full KOP agonist,28 the
4-hydroxy modification in 8 results in ca. 30-fold lower KOP
potency, demonstrating properties of a low efficacy partial KOP
agonist (Table 1). Furthermore, the presence of the phenolic 4-
hydroxy group in 9 drastically altered the KOP-mediated G protein
activation when compared to the corresponding analogue 2, previ-
ously reported as a potent partial agonist at the KOP receptor.28
Derivative 9 did not show any agonist activity at the KOP receptor,
The new target diphenethylamines 7–9 were examined for
binding affinity and selectivity for KOP, MOP and DOP receptors
in competition binding assays using membranes from Chinese
hamster ovary (CHO) cells stably expressing one of the human opi-
oid receptors, according to the reported procedure.28 Their func-
tional activity was evaluated in G protein activation assays using
guanosine 50-O-(3-[35S]thio)-triphosphate ([35S]GTP
cS) binding
and CHO cells expressing the human KOP receptors, as described
earlier.28 Their in vitro opioid activity profile is summarized in
Table 1. To facilitate comparison to our previous SAR efforts, the
diphenethylamines 1–6 were included. Receptor binding studies
demonstrated that the introduction of a phenethyl group at the
nitrogen led to a considerable reduction in KOP receptor affinity
(7: Ki = 211 nM) when compared to the previously reported ana-
logues (Table 1). It was found that the KOP receptor affinity of 7

E. Guerrieri et al. / Bioorg. Med. Chem. Lett. 26 (2016) 4769–4774
4771
Table 1
Binding affinities and functional activities of diphenethylamines 1–9 at the human opioid receptors
Receptor binding (Ki, nM)a
Functional activityb
35S]GTP
S KOP
Affinityb
MOP
Selectivity
[
c
d
KOP
DOP
MOP/KOP
DOP/KOP
EC50 (nM)
% stim.c
Ke (nM)
1 (HS665)e
0.49 0.20
5.90 3.00
8.13 0.32
10.9 2.4
12.6 1.9
141 42
211 106
36.3 11.3
218 27
542 239
826 98
594 101
412 19
325 26
788 175
>10,000
>10,000
>10,000
1106
140
73
38
26
5.6
>47
20
>20000
>1700
457
223
104
25
>47
59
10
3.62 1.87
35.0 5.3
49.1 8.8
46.2 11.4
86.4 4.6
647 88
>10,000
90.0 3.7
53.4 8.1
21.2 0.1
45.5 5.9
36.2 2.7
24.0 1.3
2 (HS666)e
3e
4e
5e
6e
7
3713 1266
2429 837
1315 364
3572 222
>10,000
f
—
1311 593
32.1 8.4
8
9
731 235
1750 893
2129 266
2187 1113
109 18
>10,000
43.8 6.2
f
8
—
a
Determined in competition binding assays against [3H]U69,593 (KOP receptor), [3H][
D
D
-Ala2,Me-Phe4,Gly-ol5]enkephalin ([3H]DAMGO, MOP receptor) and [3H][
-Pen5]enkephalin ([3H]pCl-DPDPE, DOP receptor) with CHO cell membranes expressing human opioid receptors.
S binding experiments using CHO-hKOP cell membranes.
D
-Pen2,pCl-
Phe4,
b
Determined in [35S]GTP
c
c
d
e
f
Percentage stimulation (% stim.) relative to the reference KOP agonist U69,593.
Determined by inhibition of U69,593-stimulated [35S]GTP
Data taken from Ref. 28.
cS binding to CHO-hKOP cell membranes.
No simulation up to 10 lM. Experimental data were analyzed using the GraphPad Prism Software. Values are the mean SEM of at least three independent experiments.
therefore it was tested for antagonism against U69,593-stimulated
35S]GTP
S binding. In this study, 9 exhibited moderate KOP antag-
conformations that present breaking of the Arg156-Thr273 hydro-
gen bond (Fig. 2B).
[
c
onist activity with a Ke value of 32.1 nM, which was about 40-fold
higher than the antagonist potency of the N-phenethyl substituted
7 (Table 1).
A re-docking of 1 (Fig. 2C) and the first four amino acids of the
endogenous KOP ligand dynorphin (Tyr-Gly-Gly-Phe) was carried
out for the validation of the active-like properties. The essential
polar interaction with Asp138, that was reported by O’Conner
et al.,45 played also an essential role for diphenethylamine 1. We
observed that the phenolic group of 1 forms a hydrogen bond with
His291 and it was projected into a pocket formed by residues
Trp287, Ile294, and Tyr139. The positively ionizable nitrogen forms
The crystal structure of the human KOP receptor in complex
with the selective antagonist JDTic (PDB code: 4DJH)20 (see Sup-
porting information, Fig. S3A) provides a valuable platform for in
silico investigating binding characteristics of diphenethylamines
1–9 by means of molecular docking, and 3D-pharmacophores.31
It is well-recognized that GPCRs share a common molecular
activation mechanism, although their ligands are chemically
diverse and vary in their binding site locations. In particular, ligand
binding results in molecular switches disrupting stabilized
intramolecular interactions.32 Prominent examples are (i) the
Trp287 toggle-switch,33 (ii) the ionic lock mimicking hydrogen
bond between Arg156 and Thr27320, and (iii) the so-called 3–7
lock, a hydrogen bond between residues Asp138 and Tyr320.34
Thus, GPCRs could exhibit significant structural changes between
inactive and active states.35–37 With respect to opioid receptors,
so far, the elucidated crystal structures of KOP,20 MOP38 and
DOP39 receptors represent the inactive receptor states, exception
being the recently resolved crystal structure of the murine MOP
receptor bound to a morphinan agonist.40
a salt bridge to Asp138, and additionally showed a p–cation inter-
action with Tyr139. The N-CBM group is projected into a pocket
formed by the residues Val108, Ile316 and Tyr320, while the other
phenyl moiety deeply interacts with the residues Trp287, Val108
and Ile290 (Fig. 2C). The binding orientation of 1 and dynorphin
turned out to be comparable (Fig. 2D) regarding the essential inter-
actions. The subsequent binding mode investigations explain (i)
the agonist activity of the diphenethylamine derivatives (Table 1),
where the hydrogen bond to His291 is linked to the active-like
conformation of the receptor, and (ii) the subtype selectivity for
the KOP receptor, as the residues Val108 and Ile294 differ in the
other closely related opioid receptors (MOP38 and DOP39, and are
thought to contribute to the subtype selectivity of several) KOP
selective ligands.20,21
To begin our computational approach, the binding mode of the
most active diphenethylamine of the series, 1 (Fig. 1 and Table 1),
to the human KOP receptor was docked in the crystal structure to
find a plausible starting point for a subsequent MD-based sampling
to obtain a receptor model that resembles active-like properties.41
During the MD simulation process, the hydrogen bond between
residues Arg156 and Thr273 was broken (Fig. 2A), which repre-
sents a molecular switch linked to the activation of the KOP recep-
tor. For comparison, this hydrogen bond is present in the crystal
structure of the KOP receptor in complex with the antagonist JDTic
(distance of 3.5 Å), and it is proposed to stabilize the inactive state
of the receptor.20 Due to our focus on the ligand binding site, devi-
ations of heavy atoms were calculated for key residues in ligand
binding (Val108, Asp138, Tyr139, Trp287, Ile290, His291, Ile316
and Tyr320). After an initial structural rearrangement, we only
noted smaller conformational changes (Fig. 2B). Since previous
studies suggested a contribution of His291 to the interaction pat-
tern of KOP ligands,20,22,34,42–44 we interestingly observed a hydro-
gen bond between His291 and the hydroxyl group of 1 in nearly all
The position of the phenolic hydroxyl group strongly influences
the KOP receptor affinity, as it was experimentally observed for 1
and 8, and 2 and 9 (Fig. 1 and Table 1). The 3-OH substituted 1 is
the most active compound of the series, and the only one that
shows full agonist behavior,28 indicating that a N-CBM substitution
is favorable for KOP receptor binding and activation. Its analogue 8,
in which the phenolic hydroxyl group is located at position 4,
shows reduced KOP affinity and selectivity, and acts as a partial
agonist (Table 1). A similar phenomenon was observed for com-
pounds 2 and 9 having an N-CPM moiety. In this case shifting
the position of the phenolic hydroxyl group from 3 to 4, a partial
KOP agonist (2) was converted into an antagonist (9) that has
reduced affinity and selectivity for the KOP receptor. Our 3D-phar-
macophores indicate that the 4-OH substituted compounds (8 and
9) are not likely to form a hydrogen bond with residue His291,
which is important for KOP affinity and agonist activity (Fig. 3).
The hydrophobic pocket formed by the residues Val108, Ile316
and Tyr320 is important for hosting the N-substituent. The N-CBM
(in 1) and N-CPM (in 2) groups appear to have the optimal size.

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E. Guerrieri et al. / Bioorg. Med. Chem. Lett. 26 (2016) 4769–4774
Figure 2. MD-guided generation of an active-like KOP receptor model. (A) A hydrogen bond between Arg156 with Thr273 is proposed to stabilize the inactive state of the
human KOP receptor (grey). The breakage of this ‘ionic lock’ could be found in a representative conformation of the active-like KOP receptor model (green). (B) The upper plot
shows the atomic deviation of key residues of the binding site. The plot below shows the distance between the nitrogen of His291 and the phenolic hydroxyl group of the
diphenethylamine 1. (C and D) 3D-pharmacophore analysis of 1 (C) and dynorphin (D) in the active-like KOP receptor model. Yellow spheres indicate lipophilic contacts,
green arrows hydrogen bond donors, red arrows hydrogen bond acceptors and positive ionizable centres are shown as blue spheres.
Concerning the linear substituents (from n-C3H7 to n-C6H13),
increasing the chain length results in a decrease of the KOP affinity
(Ki = 8.3 nM (3) < 10.9 nM (4) < 12.6 nM (5) < 141 nM (6), Table 1).
While the KOP affinities of the n-propyl, n-butyl, and n-pentyl
N-substituted derivatives are in the same range, the presence of
the n-hexyl chain at the nitrogen in 6 produces a markedly
decreased KOP affinity (Table 1), indicating that five carbon atoms
in linear alkyl substituents is the critical size for agonist activation
of the KOP receptor (Fig. 3A). The N-phenethyl group in analogue 7
is rather bulky to be hosted by the hydrophobic pocket, which
results in a different orientation of the phenolic moiety (Fig. 3B),
and making this compound a weak KOP antagonist (Table 1).
In order to investigate structural features for KOP receptor
selectivity of diphenethylamines, we built mutant receptor models
using MOE (Molecular Operating Environment 2014.09; Chemical
Computing Group Inc.). The amino acid residue Val108 was virtu-
ally mutated into Ala, which represents the corresponding residue
in MOP and DOP receptors.38,39 Docking of 1 into the mutant
receptor indicated that the crucial hydrogen bond of the phenolic
group with His291 was lost, and 1 showed a reduction of the
hydrophobic contact with the receptor (see Supporting informa-
tion, Fig. S3B). Thus, a reduction of Val108 by two carbon atoms
(Ala) leads to an extension of the hydrophobic pocket, in which
volume and shape are important for hosting the N-substituent.
Interestingly, we observed decreasing MOP/KOP and DOP/KOP
selectivity ratios for diphenethylamines 1–6 (Table 1) with increas-
ing the chain length from C3 to C6, which is in accordance with our
computational outcomes for the mutation of Val108 to Ala, show-
ing more space in the hydrophobic pocket (binding site volume:
KOwildtype: 521.3 Å3, KOPVal108/Ala: 549.8 Å3).
Based on our computational studies applying molecular dock-
ing and MD simulations, we can draw a SAR pattern on the inves-
tigated diphenethylamines 1–9 as KOP receptor ligands
summarized in Table 2. The main ligand–receptor interactions
are represented by the hydrogen bond between the phenolic
hydroxyl group and His291, the salt bridge formed by the proton-
able nitrogen with Asp138, and the projection of the N-substituent
into the hydrophobic pocket formed by Val108, Ile316, and Tyr320.

E. Guerrieri et al. / Bioorg. Med. Chem. Lett. 26 (2016) 4769–4774
4773
Figure 3. Binding mode analysis of diphenethylamines. (A) Superimposition of diphenethylamines 1–5 (light grey) and 6 (dark grey). (B–D) 3D-pharmacophore analysis of
diphenethylamines. Yellow spheres indicate lipophilic contacts, green arrows hydrogen bond donors, red arrows hydrogen bond acceptors and positive ionizable centres are
shown as blue spheres. (B) Diphenethylamine derivative 7 (light grey) in superimposition with 1 (dark grey) indicating the different orientation of the phenolic hydroxyl
group of 7 compared to 1 due to the bulky N-substituent. (C and D) Binding mode comparison of diphenylethylamines 1 and 8 (C), and 2 and 9. (D) Compounds without the
hydroxyl group in position 3 cannot form the crucial hydrogen bond with residue His291, which results in lower KOP receptor affinity of 8 and 9.
Table 2
Overview of the ligand-receptor interactions observed for diphenethylamines 1–9 with the human KOP receptor
Moiety
Interactions with the KOP receptor
Relevance
Phenolic hydroxyl group at position 3 Hydrogen bond with His291
KOP receptor affinity
Protonable nitrogen
N-substituents
Salt bridge with Asp138
Hydrophobic interactions with Val108, Ile316, Tyr320
Essential for KOP receptor binding
KOP receptor selectivity (linear substituents and small
rings)
Lipophilic moiety
Phenyl ring: Hydrophobic interactions with Trp287, Val108 and Ile KOP receptor selectivity
290
Concerning the N-substituent, the n-alkyl size limit is five carbon
atoms, and bulky substituents turn KOP receptor agonists in antag-
onists, since they prevent the interaction between the protonable
nitrogen and Asp138, essential in anchoring positively ionizable
KOP receptor ligands.
In summary, our current findings expanded the SAR exploration
in the series of diphenethylamines as KOP receptor ligands. The
SAR pattern derived from molecular modeling is supported by
the in vitro pharmacological profile of the target ligands. The size
of the N-substituent hosted by the hydrophobic pocket formed
by the residues Val108, Ile316 and Tyr320 influences ligand bind-
ing and selectivity. The hydrogen bond formed by the phenolic
3-OH group of 1 with His291 is essential for binding affinity and
agonist activity at the KOP receptor. Shifting the phenolic hydroxyl
group from position 3 to 4 resulted in reduced KOP binding affinity
due to the absence of a hydrogen bond with His291. Potency and
efficacy to activate the KOP receptor was strongly dependent on
the 3-OH?4-OH switch, with remarkable consequences as this
modification turned a highly potent full agonist (1) into a much
less potent partial agonist (8) or even eradicated essentially all
KOP activity by converting a potent partial agonist (2) into a mod-
erate antagonist (9).

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21. Martinez-Mayorga, K.; Byler, K. G.; Yongye, A. B.; Giulianotti, M. A.; Dooley, C.
Combination of experimental and molecular modeling strate-
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B.; Aubé, J.; Stevens, R. C. J. Biol. Chem. 2013, 288, 34470.
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Papadopoulos, M. G. J. Chem. Inf. Model. 2014, 54, 2294.
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A. J. Phys. Chem. B 2015, 119, 824.
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gies offers fundamental insights into the structural features of
diphenethylamines that are necessary to promote binding affinity,
agonist efficacy and selectivity for the KOP receptor. These struc-
tural characteristics may be also involved in transduction of the
ligand binding event into molecular changes, ultimately leading
to receptor activation. The present results are useful for guiding
drug design, and the structural simplicity and readily accessible
synthesis of these scaffolds compared to those of currently utilized
KOP agents make them attractive for further exploration.
Supplementary data
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