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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