Design, synthesis and biological evaluation of aminobenzyloxyarylamide derivatives as selective κ opioid receptor antagonists

Article abstract of DOI:10.1016/j.ejmech.2017.02.029

Opioid receptors play an important role in both behavioral and mood functions. Based on the structural modification of LY2456302, a series of aminobenzyloxyarylamide derivatives were designed and synthesized as κ opioid receptor antagonists. The κ opioid receptor binding ability of these compounds were evaluated with opioid receptors binding assays. Compounds 1a-d showed high affinity for κ opioid receptor. Especially for compound 1c, exhibited a significant K_ivalue of 15.7?nM for κ opioid receptor binding and a higher selectivity over μ and δ opioid receptors compared to (±)LY2456302. In addition, compound 1c also showed potent κ antagonist activity with κ IC₅₀?=?9.32?nM in [³⁵S]GTP-γ-S functional assay. The potential use of the representative compounds as antidepressants was also investigated. The most potent compound 1c not only exhibited potent antidepressant activity in the mice forced swimming test, but also displayed the effect of anti-anxiety in the elevated plus-maze test.

Full text of DOI:10.1016/j.ejmech.2017.02.029

Accepted Manuscript
Design, synthesis and biological evaluation of aminobenzyloxyarylamide derivatives
as selective κ opioid receptor antagonists
Junwei Wang, Qiao Song, Anhua Xu, Yu Bao, Yungen Xu, Qihua Zhu
PII:
S0223-5234(17)30094-6
10.1016/j.ejmech.2017.02.029
EJMECH 9225
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 November 2016
Revised Date: 8 February 2017
Accepted Date: 11 February 2017
Please cite this article as: J. Wang, Q. Song, A. Xu, Y. Bao, Y. Xu, Q. Zhu, Design, synthesis and
biological evaluation of aminobenzyloxyarylamide derivatives as selective κ opioid receptor antagonists,
European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.02.029.
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ACCEPTED MANUSCRIPT
Graphical Abstract

ACCEPTED MANUSCRIPT
synthesis and biological
Design,
evaluation
of
aminobenzyloxyarylamide derivatives as selective κ opioid receptor
antagonists
Junwei Wanga,£, Qiao Song a,£, Anhua Xua, Yu Baoa, Yungen Xua,b,*, Qihua Zhua,b,*
aDepartment of Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, China
bJiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University,
Nanjing 210009, China
£Both authors contributed equally to this work
Abstract:
Opioid receptors play an important role in both behavioral and mood functions.
Based on the structural modification of LY2456302,
a
series of
aminobenzyloxyarylamide derivatives were designed and synthesized as κ opioid
receptor antagonists. The κ opioid receptor binding ability of these compounds were
evaluated with opioid receptors binding assays. Compounds 1a-d showed high
affinity for κ opioid receptor. Especially for compound 1c, exhibited a significant Ki
value of 15.7 nM for κ opioid receptor binding and a higher selectivity over µ and δ
opioid receptors compared to (±)LY2456302. In addition, compound 1c also showed
potent κ antagonist activity with κ IC50 = 9.32 nM in [35S]GTP-γ-S functional assay.
The potential use of the representative compounds as antidepressants was also
investigated. The most potent compound 1c not only exhibited potent antidepressant
activity in the mice forced swimming test, but also displayed the effect of anti-anxiety
in the elevated plus-maze test.
Keywords: κ opioid receptor antagonists, aminobenzyloxyarylamides, LY2456302,
selectivity, antidepressants.
1. Introduction
Major depression or depression, characterized by negative mood, reduced
motivation and decreased energy, affects nearly 5% of people worldwide each year
[1,2]. The first line of drugs for the treatment of depression is normally selective
serotonin reuptake inhibitors (SSRIs) such as fluoxetine and escitalopram. However,
60% of patients treated with these medications fail to achieve remission [3]. The
clinical treatment of depression is limited due to the drawbacks of existing
antidepressants such as the slow onset time, low tolerance and common relapse [4].
Therefore, new methods of treatment that work through alternative mechanisms are
urgent to be developed.

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Opioid receptors belong to the super family of G-protein-coupled receptors
(GPCRs) which include mu (µ), delta (δ), kappa (κ) and the opioid-like receptor
(ORL-1). They are involved in multiple physiological activities [5,6]. The three opioid
receptors, µ, δ and κ opioid receptors, regulate major functions including pain,
emotional tone, appetite and reward circuitry [7]. Each of these three types of opioid
receptors has its specific endogenous ligand and exerts different biological effect. The
κ opioid receptor is closely associated to the action of dynorphin (DYN) peptides as
specific endogenous ligands. The role of κ opioid receptor ligands in the modulation
of mood has attracted great interest in recent years [8,9]. The function of cyclic
adenosine monophosphate (cAMP) response element binding protein (CREB) can be
disrupted by antagonizing κ opioid receptor, thus increasing the dopamine release,
resulting in antidepressant-like effects [10,11]. Recently, accumulating evidence
indicates that κ opioid receptor antagonists: nor-BNI [12,13] and JDTic [14,15] (Fig.
1), have anxiolytic and antidepressant-like activity in rodents [16]. In addition, the
short-acting κ opioid receptor antagonists PF-04455242 [17] and LY2456302 [18]
(Fig. 1) also show antidepressant-like effects. LY2456302 has passed phase I clinical
trial and is currently in phase II trial for adjunctive treatment of major depressive
disorders and for the treatment of substance abuse disorders [19].
OH
CH3
CH3
CH3
N
H3C
NH
CH3
CH
3
HO
O
N
OH
O
HO
N
O
N
CH3
CH3
NH2
O
O
O
OH
N
O
S
H
N
NH
F
N
H
OH
nor-BNI
JDTic
PF-04455242
LY2456302
Fig. 1. Structures of nor-BNI, JDTic, PF-04455242 and LY2456302
LY2456302 is a structurally-unique, high-affinity and selective κ opioid receptor
antagonist with 24- and 175-fold selectivity over µ and δ opioid receptors,
respectively. In contrast to the prototypical κ opioid receptor antagonists nor-BNI and
JDTic, LY2456302 exhibits rapid absorption, good oral bioavailability (F = 25%, tmax
=1-2 h) and a more typical rate of clearance (T1/2 = 2-4 h) [20]. This unique
biopharmacological profile makes it as an attractive lead compound in the search for
new κ opioid receptor antagonists. Recent study indicates that selective κ opioid
receptor antagonism is a promising experimental strategy for the treatment of
depression [20]. Selective κ opioid receptor antagonists can reduce both ethanol
intake and reinstatement in a number of preclinical paradigms [21,22], while
nonselective κ opioid antagonists produce neither reliable antidepressant- nor
anxiolytic-like effects in animals or humans, which may be caused by the functional
opposition between µ and κ receptors [10,23]. In this paper, we describe details of our
discovery of potent and selective κ opioid receptor antagonists in an
aminobenzyloxyarylamide scaffold. We also describe the evaluation of these selective

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κ opioid receptor antagonists for potential use as antidepressants.
2. Results and discussion
2.1. Design of selective κ opioid receptor antagonists
In order to search novel and efficient antidepressants, LY2456302 which is a
selective κ opioid receptor antagonist was chosen as the lead compound. According to
previous studies [24,25], some modifications have been taken in part I (Fig. 2)
including different substitution on the benzene ring, the replacement of benzene ring
with N-heterocycle, the replacement of pyrrolidine ring and the chiral isomers of
pyrrolidine with other cyclic amine. However, the modifications of part II have been
rarely reported, with modification only on aromatic ring B. For example, the
substitution on benzene ring B was changed or the benzene ring was replaced with
pyridine ring [24,25]. Thus, in this work, we focus on the modification of part II (Fig.
2). Firstly, fluorine or chlorine was introduced into the ring A to investigate the
influence on activity and selectivity towards κ opioid receptor. Secondly, the benzene
ring B was replaced by naphthalene ring and the aromatic ring bound to the
pyrrolidine was modified according to previous study [24] to study the
structure-activity relationship.
Fig. 2. Process of rational design of aminobenzyloxyarylamide derivatives as κ opioid receptor
antagonists.
2.2. Chemistry
The synthetic routes of the proposed aminobenzyloxyarylamide derivatives were
illustrated in Scheme 1-3. Treatment of 3a-h with thionyl chloride produced the
corresponding acid chloride which reacted with ethanol giving ethyl benzoate
derivatives 4a-h. Intermediates 4a-h was coupled with N-Vinylpyrrolidone under
sodium-hydrogen condition to form 5a-h which were heated in hydrochloric acid to
give 5-phenyl-3,4-dihydro-2H-pyrrole derivatives 6a-h. Reduction of 6a-h with
sodium borohydride provided 2-phenylpyrrolidine derivatives 7a-h (Scheme 1). The
synthesis of the designed compounds 1a-d stemmed from the reductive amination of
aromatic aldehyde derivatives with 7h. Obtained reductive amination 8a-b was
etherified with benzonitrile derivatives 9a-b to give etherification products 10a-d.

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The target compounds 1a-d were finally obtained by the hydrolysis of 10a-d (Scheme
2). Substitution of bromine with cyano-group on 1-bromo-4-fluoronaphthalene 11
gave intermediate 12. Etherification of 12 with 4-hydroxybenzaldehyde afforded ether
13 which was further hydrolyzed by hydrogen peroxide and gave
4-(4-formylphenoxy)-1-naphthamide 14. Compounds 2a-h were obtained by reductive
amination of 14 with 2-aryl substituted pyrrolidine 7a-h (Scheme 3). All the target
compounds have been confirmed by IR, 1H-NMR, 13C-NMR and HR-MS.
Scheme 1. Synthetic route of intermediates 7a-h. Regents and conditions: (a) SOCl2, EtOH, reflux, 2 h; (b)
N-Vinylpyrrolidone, NaH, THF, r.t. to 60 oC, 3 h; (c) HCl, THF, reflux, 12 h; (d) NaBH4, AcOH, MeOH, r.t., 1 h.
Scheme 2. Synthetic route of target compounds 1a-d. Regents and conditions: (e) Aldehyde, NaBH3CN, AcOH,
MeOH, r.t., 12 h; (f) K2CO3, DMF, N2, 120 oC, 10 h. (g) H2O2, DMSO, 10 oC to r.t., 3 h.
Scheme 3. Synthetic route of target compounds 2a-h. Regents and conditions: (h) K4Fe(CN)6, K2CO3, Pd(OAc)2,
DMAC, N2, 120 oC, 12 h; (i) 4-Hydroxybenzaldehyde, K2CO3, DMF, N2, 120 oC, 12 h; (j) H2O2, DMSO, 10 oC to
r.t, 3 h; (k) NaBH3CN, AcOH, MeOH, r.t. 12 h.

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2.3. Molecular modeling studies
In order to investigate the potential activity and selectivity of the designed
compounds against κ opioid receptors, modeling studies of compound 1c in the ligand
binding pocket of κ opioid receptor were carried out. The docking model (PDB code:
4DJH) revealed that comparing with LY2456302, compound 1c could form an
additional halogen bond with Tyr139 by the introduction of chlorine atom (Fig. 3).
The σ-hole interaction shift the whole molecule to the left, thus the oxygen atom of
diphenyl ether could form an additional hydrogen bond interaction with HOH1311,
which was speculated to improve the selectivity for κ opioid receptor. As shown in
figure 3, the optimum conformation of compound 1c is (R)-isomer. We will isolate
this isomer in the following work and the binding affinity is expected to be improved.
Fig. 3. Docked conformation of compound 1c (A) and LY2456302 (B) in the ligand κ opioid
receptor binding pocket. The interaction mode was obtained through molecular docking (PDB ID:
4DJH) and depicted using MOE 2013.08. (A) Best docking pose of (R)-isomer of 1c, the carbon
atoms and the key residues in the active site of κ opioid receptor were colored in blue and purple
red, respectively. (B) Best docking pose of (S)-isomer of LY2456302, the carbon atoms and the
key residues were colored in yellow and purple red, respectively. The H-bonds were shown as
black dot lines.
2.4. In vitro opioid receptor binding and [35S] GTP-γ-S binding assays
In vitro receptor binding affinity of compounds for µ, κ and δ opioid receptors
were first evaluated with radioligand binding assays and (±)LY2456302 was used as
the positive control. As shown in Table 1, the results indicated that compounds 1a-d
showed similar affinities as (±)LY2456302 with κ opioid receptor inhibition ratio > 90%
at the concentration of 1 µM. The results of compounds 2a-h were summarized in
Table 2. Compounds 2b, 2c, 2d and 2h showed moderate binding affinities with κ
opioid receptor inhibition ratio > 50% at the concentration of 10 µM. However, their
binding affinities for µ and δ opioid receptors were weak with µ opioid receptor
inhibition ratio < 20% and δ opioid receptor inhibition ratio < 10% at the
concentration of 10 µM. Among 2-aryl pyrrolidine derivatives, the binding affinity of
3-methyl substituted compound 2h was stronger than that of 2-methyl substituted
compound 2e and 3,5-dimethyl substituted compound 2a (2h > 2e > 2a), and the

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binding affinities of 3-fluorinated compound 2b was stronger than that of
4-fluorinated compound 2g.
Table 1
In vitro receptor binding affinity of compounds 1a-d and (±)LY2456302 for µ, κ and δ opioid receptors with the
competitive inhibition ratio.
µ Inhibition (%)a
κ Inhibition (%)b
δ Inhibition (%)c
Compd
1x10-5 M
1x10-6 M
78.8±0.8
71.0±1.0
51.4±0.7
73.4±0.3
56.1±1.0
1x10-5 M
1x10-6 M
99.3±0.4
98.8±0.1
98.5±0.3
95.8±0.5
97.0±0.3
1x10-5 M
1x10-6 M
36.5±1.6
45.5±2.2
54.1±2.3
37.4±3.4
45.5±0.7
99.1±0.6
95.3±0.3
94.2±0.2
96.8±0.5
93.6±0.2
100±0.0
100±0.0
100±0.0
98.7±0.2
100±0.0
87.5±0.6
97.1±0.2
92.4±0.1
89.2±0.2
87.2±0.7
1a
1b
1c
1d
(±)LY2456302
aCompetitive binding inhibition ratio of µ opioid receptor, [3H]-DAMGO was used.
bCompetitive binding inhibition ratio of κ opioid receptor, [3H]-U69,593 was used.
cCompetitive binding inhibition ratio of δ opioid receptor, [3H]-DPDPE was used.
Table 2
In vitro receptor binding affinity of compounds 2a-h and (±)LY2456302 for µ, κ and δ opioid receptors with the
competitive inhibition ratio.
µ Inhibition (%)a
κ Inhibition (%)b
δ Inhibition (%)c
Compd
1x10-5 M
1x10-6 M
1x10-5 M
1x10-6 M
12.2±2.1
27.7±1.0
31.4±0.1
46.5±1.5
13.9±2.7
1x10-5 M
1x10-6 M
0
0
0
0
0
0
23.7±0.8
57.9±0.4
64.1±2.5
80.5±0.0
34.0±0.2
0
0
2a
2b
2c
2d
2e
0
4.0±0.2
0
2.3±0.2
0
15.0±2.0
0
0
0
0
9.0±0.8
2.9±0.9

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52.2±1.0
0
11.3±1.2
12.0±0.1
39.0±1.5
86.9±0.1
100±0.0
5.1±0.8
13.8±0.4
39.9±2.1
97.0±0.3
0
0
2f
0
0
0
0
0
0
2g
2h
16.3±1.1
93.6±0.2
56.1±1.0
87.2±0.7
45.5±0.7
(±)LY2456302
aCompetitive binding inhibition ratio of µ opioid receptor, [3H]-DAMGO was used.
bCompetitive binding inhibition ratio of κ opioid receptor, [3H]-U69,593 was used.
cCompetitive binding inhibition ratio of δ opioid receptor, [3H]-DPDPE was used.
Together with compounds 1a-d, compounds 2d and 2h were selected to
determine the Ki values of opioid receptors binding affinities. As shown in table 3,
compounds 1a-d exhibited potent binding affinities for κ opioid receptor with the Ki
values in the same order of magnitude as (±)LY2456302. Among these derivatives,
compound 1c showed the strongest binding affinity for κ opioid receptor, which was
almost as potent as (±)LY2456302. Notably, we found that the selectivity of 1c over µ
and δ opioid receptors was 14- and 12.2-fold, respectively, which was almost twice as
much as that of (±)LY2456302. The docking results above explained the reason why
compound 1c showed higher κ opioid receptor selectivity over µ and δ opioid
receptors compared with (±)LY2456302. The cause of the affinity was not obviously
improved may be the chiral problem. The chiral separation of the active compounds
would be continued in the further work based on the optimum conformation in the
molecular docking studies.
Table 3
The binding affinities Ki values of 1a-d, 2d, 2h and (±)LY2456302 for opioid receptorsa
Ki (nM)
κc
Selectivity
Compd
µb
δd
µ/κ
δ/κ
32.3±0.1
62.8±4.7
220.0±1.9
27.9±0.4
>10000
>10000
90.9±4.2
37.6±0.6
29.4±0.3
15.7±0.5
42.7±1.4
4603±484.1
1738±309.5
11.9±0.09
489.4±11.5
266.6±4.0
191.4±4.2
429.3±5.8
>10000
0.85
2.13
14.0
0.65
>2.2
>5.8
7.6
13.0
9.0
1a
1b
12.2
10.1
>2.2
>5.8
7.6
1c
1d
2d
2h
>10000
91.5±8.8
(±)LY2456302
aBinding assays were carried out in duplicate.
b[3H-DAMGO was used.
c[3H]-U69,593 was used.
d[3H]-DPDPE was used.
Compound 1c was evaluated for in vitro antagonist activity, utilizing inhibition of

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agonist stimulated [35S]GTP-γ-S binding with cloned human opioid receptors
expressed in Chinese hamster ovary cells (CHO) cells (µ and κ) or human embryonic
kidney 293 (HEK293) cells (δ). Results are shown in Table 4. Compound 1c showed
good κ antagonist potency and selectivity in functional GTP-γ-S binding assays, with
κ IC50 = 9.32 nM, µ IC50 = 144.2 nM, and δ IC50 = 15.82 nM. This demonstrates that
compound 1c was an antagonist for each of the types of opioid receptors.
Table 4
In vitro [35S]GTP-γ-S binding assays for κ, µ, and δ opioid receptors.
Compd
1c
µ IC50 (nM)a
144.2±5.0
κ IC50 (nM)b
9.32±1.09
6.68±1.83
δ IC50 (nM)c
15.82±3.35
95.50±0.33
µ/κ
15.5
3.4
δ/κ
1.7
22.61±6.07
14.3
(±)LY2456302
aMu (µ) antagonism was determined in a human [35S] GTP-γ-S assay using DAMGO as the agonist stimulus. The
IC50 is reported as the mean ± SEM of at least three independent experiments.
bKappa (κ) antagonism was determined in a human [35S] GTP-γ-S assay using U69,593 as the agonist stimulus.
The IC50 is reported as the mean ± SEM of at least three independent experiments.
cDelta (δ) antagonism was determined in a human [35S] GTP-γ-S assay using DPDPE as the agonist stimulus. The
IC50 is reported as the mean ± SEM of at least three independent experiments.
2.5. Biological activity in vivo
2.5.1. Mice forced swimming test
Compounds 1b, 1c and 2h were evaluated for in vivo antidepressant activity in
mice forced swimming model and (±)LY2456302 was used as the positive control.
The results indicated that all the test compounds could shorten the motionless-time of
mice in the forced swimming test at the dosage of 30 mg/kg (Fig. 4). Among them,
compounds 1c, 2h and LY2456302 can significantly shorten the immobility time of
mice in the forced swimming test compared with the normal saline group (P < 0.05),
suggesting that they have similar antidepressant effect.
Fig. 4. Mice forced swimming test of compounds 2h, 1c, 1b, (±)LY2456302 and vehicle (dosages

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30 mg/kg, i.p.). Each bar represents mean±SEM of immobility time (s) obtained from 10 mice
(*p < 0.05 vs. vehicle).
2.5.2. Mice elevated plus-maze test
Based on the above result, compounds 1c and 2h were chosen to evaluate their
anti-anxiety effect in the elevated plus-maze test (EPM) which is the international
common-used anxiety animal model. The results showed that compounds 1c, 2h and
(±)LY2456302 can significantly increase the number proportion of mice going into
the open arms [OE / (CE + OE)] and the retention time proportion in the open arms
[OT / (OT + CT)] compared with the blank control group (P < 0.01) (Fig. 5). Based on
these profiles, compounds 1c and 2h showed some anti-anxiety effect.
Fig. 5. Representative images show typical examples of compounds 2h, 1c, (±)LY2456302 and
vehicle (dosages 30 mg/kg, i.p.) mice exploring in the elevated plus maze apparatus. (A) Each bar
represents mean±SEM of the number proportion of mice going into the open arms [OE/(CE+OE)]
obtained from 10 mice (*p < 0.01 vs. vehicle). (B) Each bar represents mean±SEM of the
retention time proportion in the open arms [OT/(OT+CT)] obtained from 10 mice (*p < 0.01 vs.
vehicle).
3. Conclusions
In conclusion, highly selective antagonists for κ opioid receptor have been found
from the aminobenzyloxyarylamide scaffold. All the compounds were biologically
evaluated for their binding affinities for opioid receptors. Representative compounds
were evaluated in vitro for opioid receptors antagonist potency and in vivo for their
antidepressant and anti-anxiety effects. The highest affinity and selectivity for κ
receptor was found for compound 1c, with κ Ki = 15.7 nM. The selectivity of 1c over
µ and δ opioid receptors was improved nearly 2-fold in comparison to that of
(±)LY2456302. Compound 1c also showed good in vitro κ antagonist potency in
GTP-γ-S functional assays, with κ IC50 = 9.32 nM. In mice forced swimming and
elevated plus-maze tests, compound 1c showed good effect on antidepressant and
anti-anxiety, which indicated that it could be a promising candidate for the treatment
of depression.
4. Experimental section

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4.1. General procedures
Reactions were monitored by thin-layer chromatography on silica gel plates
(60F-254) visualized under UV light. Melting points were determined on a
1
Mel-TEMP II melting point apparatus without correction. HNMR and 13CNMR
spectra were recorded in CDCl3 on a Bruker Avance 300 MHz spectrometer at 300
MHz and 75 MHz, respectively. Chemical shifts (δ) are reported in parts per million
(ppm) from tetramethylsilane (TMS) using the residual solvent resonance (CDCl3:
7.26 ppm for 1H NMR, 77.16 ppm for 13C NMR. Multiplicities are abbreviated as
follows: s = singlet, d = doublet, t = traplet, q = quartet, m = multiplet). IR spectra
were recorded on a Nicolet iS10 Avatar FT-IR spectrometer using KBr film. MS
spectra were recorded on a LC/MSD TOF HR- MS Spectrum.
All chemicals purchased from commercial suppliers were used as received unless
otherwise stated. Reactions and chromatography fractions were monitored by Merck
silica gel 60F-254 glass TLC plates. All solvents were reagent grade and, when
necessary, were purified and dried by standard methods.
4.2. Preparation of the compounds
4.2.1. General procedure for the preparation of intermediates 7a-h.
To a solution of substituted benzoic acid 3a-h (0.07 mol) in absolute ethyl alcohol
(100 mL) was slowly added thionyl chloride (15 mL). The mixture was heated to
reflux for 2 h, and then cooled to room temperature. The solvent was removed and the
solid residue was dissolved in ethyl acetate (100 mL), and washed with saturated
NaHCO3 solution (100 mL × 2). The organic layer was dried and concentrated to
give the intermediates 4a-h as pale yellow oily matter in 94.4-99.4% yield. They were
used in the next step without additional purification.
To a solution of NaH (5.6 g, 0.14 mol, 60%) in anhydrous THF (250 mL) was
o
added 4a-h (0.07 mol) under the mechanical agitation. After heating to 60 C,
N-Vinylpyrrolidone (0.07 mol) was added by dropwise and the mixture was heated to
72 oC for 3 h. Then the reaction mixture was cooled to room temperature, then poured
into ice water (500 mL), extracted with ethyl acetate (100 × 3 mL), and washed with
saturated NH4Cl solution (300 mL) and brine (300 mL). The organic layer was dried
and concentrated to give the intermediates 5a-h as brown oily matter in 82.3-94.2%
yield, which was used in the next step without additional purification.
A solution of 5a-h (0.06 mol) in THF (15 mL) was added to the 5N HCl (60 mL)
under reflux. After the reaction mixture was refluxed for 3-5 h, then the reaction
mixture was cooled to room temperature and concentrated in vacuo to remove the
solvent THF. The pH was adjusted to 11 with saturated NaOH solution, then extracted
with ethyl acetate (100 mL × 3), and washed with brine (150 mL× 2). The organic
layer was dried and concentrated to give the intermediates 6a-h as brown oily matter
in 82.8-87.2% yield, which was used in the next step without additional purification.
To a solution of 6a-h (0.05 mol) in methanol (80 mL) was added acetic acid two
drops, and then NaBH4 (3.8 g, 0.1mol) was added slowly under the condition of ice
salt bath. After reaction at room temperature for 3 h, the solvent was removed and the
residue was dissolved in water. The pH was adjusted to 1 with 6N HCl, washed with

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methyl tertiary butyl ether (50 mL × 3), and then the pH was adjusted to 12 with
NaOH solution. The mixture was extracted with dichloromethane (100 mL × 3), and
washed with brine (150 mL× 2). The organic layer was dried and concentrated to give
the brown oil, which was dissolved in acetone to form oxalic acid salt. Intermediates
7a-h were obtained as white solid in 42.3-55.6% yield.
4.2.2. General procedure for the preparation of compounds 1a-d.
To a solution of 7h (0.8 g, 4.8 mmol) in methanol (20 mL) was added substituted
benzaldehyde (4.8 mmol) and acetic acid (0.8 mL). After stirring at room temperature
for 1 h, NaBH3CN (0.38 g, 9.6 mmol) was added in batches under the condition of ice
bath. After reaction at room temperature for 12 h, the solvent was removed and water
(20 mL) was added. The pH was adjusted to 9~10 with saturated Na2CO3 solution,
then extracted with ethyl acetate (50 mL × 3), and washed with brine (100 mL× 2).
The organic layer was dried and concentrated to give the intermediates 8a-b as light
yellow oil in 81.1-83.4% yield.
To a solution of 8a-b (1.58 mmol) in anhydrous DMF (10mL) was added K2CO3
powder (0.44 g, 3.17 mmol). After stirring at room temperature for 0.5 h, intermediate
o
9a-b (1.58 mmol) was added and the mixture was heated to 140 C for 10 h under
nitrogen protection. After cooling to room temperature, water (50 mL) was added,
then the mixture was extracted with ethyl acetate (50 mL × 3) and washed with water
(100 mL × 2) and brine (100 mL × 2). The organic layer was dried and concentrated,
the residue was dissolved in DMSO (10 mL), and then 30% H2O2 (3 mL) was added
o
in at 10 C. After stirring at room temperature for 3 h, water (50 mL) was added and
then the mixture was extracted with ethyl acetate (30 mL × 3), and washed with with
water (50 mL × 2) and brine (50 mL × 2). The organic layer was dried and
concentrated, and the pure products 1a-d were obtained with further purified by
column chromatograph on silica gel (petroleum ether /ethyl acetate = 5/1) as white
solid in 38.2-45.5% yield.
4.2.2.1
4-[2-Chloro-4-[2-(3,5-dimethylphenyl)pyrrolidin-1-yl]methylphenoxyl]
1
benzamide (1a). Yield: 42.1%. White solid. M.p.:146-147oC. H-NMR (300 MHz,
CDCl3) δ (ppm): 7.77 (d, J = 8.8 Hz, 2H, ArH), 7.40 (s, 1H, ArH), 7.18 (d, J = 8.3 Hz,
1H, ArH), 7.10-6.96 (m, 3H, ArH), 6.91 (d, J = 8.7 Hz, 3H, ArH), 6.23-5.51 (br, 2H),
3.79 (d, J = 13.1 Hz, 1H, 1/2CH2), 3.28 (t, J = 7.9 Hz, 1H, CH), 3.14 (t, J = 7.8 Hz,
1H, 1/2CH2), 3.04 (d, J = 13.2 Hz, 1H, 1/2CH2), 2.33 (s, 6H, 2CH3), 2.23-2.14(m, 2H,
CH2), 1.98-1.69 (m, 3H, CH2, 1/2CH2). 13C-NMR (75 MHz, CDCl3) δ (ppm): 168.32,
160.13, 148.92, 142.88, 137.90, 137.48, 130.50, 128.90, 128.34, 127.94, 127.12,
125.79, 124.87, 121.44, 115.97, 76.99, 76.56, 76.14, 69.39, 56.91, 53.17, 34.54, 21.83,
20.88. HRMS (ESI): m/z [M+H]+. Calcd for C26H27ClN2O2: 435.1839; Found:
435.1841. IR (cm-1): 3391.98, 3184.63, 2967.84, 2911.73, 2793.11, 1645.07, 1609.17,
1580.04, 1509.50, 1488.85, 1419.49, 1401.87, 1364.10, 1299.40, 1259.32, 1213.34,
1171.14, 1147.14, 1115.57, 1058.30, 900.92, 845.06, 794.84, 688.13, 641.34, 622.12,
547.10, 482.50.

ACCEPTED MANUSCRIPT
4.2.2.2
4-[2-Fluoro-4-[2-(3,5-dimethylphenyl)pyrrolidin-1-yl]methyl
phenoxyl]
benzamide (1b). Yield: 38.2%. White solid. M.p.:122-124oC. H-NMR (300 MHz,
CDCl3) δ (ppm): 7.77 (d, J = 8.7 Hz, 2H, ArH), 7.17 (d, J = 11.7 Hz, 1H, ArH),
7.10-6.98 (m, 4H), 6.99-6.84 (m, 3H, ArH), 5.91 (br, 2H), 3.81 (d, J = 13.3 Hz, 1H,
1/2CH2), 3.29 (t, J = 7.9 Hz, 1H, CH), 3.14 (t, J = 7.7 Hz, 1H, 1/2CH2), 3.03 (d, J =
13.3 Hz, 1H, 1/2CH2), 2.33 (s, 6H, 2CH3), 2.24-2.10 (m, 2H, CH2), 1.98-1.64 (m, 3H,
1
13
CH2, 1/2CH2). C-NMR (75 MHz, CDCl3) δ (ppm): 168.51, 160.41, 155.4, 152.11,
142.92, 137.48, 128.87, 128.36, 127.15, 124.84, 124.36, 121.90, 116.93, 116.69,
115.63, 77.01, 76.59, 76.16, 69.34, 57.01, 53.13, 34.54, 21.85, 20.89. HRMS (ESI):
m/z [M+H]+. Calcd for C26H27FN2O2: 419.2135; Found: 419.2136. IR (cm-1): 3392.39,
3191.20, 2966.28, 2914.71, 2787.19, 1647.79, 1604.00, 1578.12, 1505.66, 1417.67,
1396.24, 1279.23, 1224.83, 1167.19, 1111.29, 951.01, 862.85, 847.45, 703.50, 620.15.
4.2.2.3
4-[2-Chloro-4-[2-(3,5-dimethylphenyl)pyrrolidin-1-yl]methylphenoxyl]-3-fluorobenza
1
mide (1c). Yield: 45.5%. White solid. M.p.:68-70oC. H-NMR (300 MHz, CDCl3) δ
(ppm): 7.67 (dd, J = 11.2, 2.1 Hz, 1H , ArH), 7.53-7.43 (m, 1H, ArH), 7.22-7.13 (m,
1H, ArH), 7.10-6.95 (m, 4H, ArH), 6.91-6.80 (m, 2H, ArH), 5.90 (br, 2H), 3.81 (d, J =
13.3 Hz, 1H, 1/2CH2), 3.35-3.26 (m, 1H, CH), 3.15-3.01 (m, 2H, CH2), 2.32 (s, 6H,
2CH3), 2.26-2.15 (m, 2H, CH2), 2.00-1.70 (m, 3H, CH2, 1/2CH2). 13C-NMR (75 MHz,
CDCl3) δ (ppm): 154.85, 153.51, 151.55, 150.20, 137.50, 128.39, 124.84, 124.32,
123.25, 123.20, 120.83, 117.12, 116.96, 116.72, 116.12, 115.86, 114.50, 76.96, 76.54,
76.12, 69.35, 56.92, 53.09, 34.47, 21.82, 20.87. HRMS (ESI): m/z [M+H]+. Calcd for
C26H26ClFN2O2: 453.1745; Found: 453.1742. IR (cm-1): 3411.14, 3200.42, 2965.59,
2916.15, 2872.37, 2788.93, 1663.46, 1618.21, 1578.28, 1511.59, 1489.05, 1438.41,
1382.50, 1277.29, 1231.61, 1201.20, 1153.73, 1095.54, 1054.84, 934.02, 897.91,
846.71, 756.54, 704.05, 614.80.
4.2.2.4
4-[2-Fluoro-4-[2-(3,5-dimethylphenyl)pyrrolidin-1-yl]methylphenoxyl]-3-fluorobenza
1
mide (1d). Yield: 43.8%. White solid. M.p.:70-72oC. H-NMR (300 MHz, CDCl3) δ
(ppm): 7.70 (dd, J = 11.1, 2.1 Hz, 1H, ArH), 7.54-7.47 (m, 1H, ArH), 7.42 (d, J = 1.8
Hz, 1H, ArH), 7.19 (dd, J = 8.3, 1.7 Hz, 1H, ArH), 7.07 (s, 2H, ArH), 7.00-6.87 (m,
2H, ArH), 6.79 (t, J = 8.3 Hz, 1H, ArH), 5.91 (br, 2H), 3.81 (d, J = 13.2 Hz, 1H,
1/2CH2), 3.30 (t, J = 7.9 Hz, 1H, CH), 3.15-2.97 (m, 2H, CH2), 2.34 (s, 6H, 2CH3),
2.29-2.15 (m, 2H, CH2), 2.02-1.68 (m, 3H, CH2, 1/2CH2). 13C-NMR (75 MHz,
DMSO-d6) δ (ppm): 165.91, 153.23, 149.96, 149.11, 146.12, 145.97, 143.30, 137.94,
137.17, 130.59, 130.52, 130.14, 128.51, 128.36, 124.98, 124.73, 123.76, 120.31,
118.30, 116.36, 116.11, 68.81, 56.30, 53.00, 34.74, 21.94, 20.92. HRMS (ESI): m/z
[M+H]+. Calcd for C26H26F2N2O2: 437.2041; Found: 437.2040. IR (cm-1): 3411.19,
3200.95, 2965.54, 2915.44, 2788.75, 1662.82, 1618.36, 1587.04, 1489.02, 1437.98,
1382.56, 1277.13, 1231.45, 1200.92, 1139.32, 1095.95, 1054.96, 934.24, 898.46,
846.87, 763.69, 703.61, 615.50.

ACCEPTED MANUSCRIPT
4.2.3. General procedure for the preparation of compounds 2a-h.
To a solution of 1-bromo-4-fluoronaphthalene 11 (0.5 g, 2.22 mmol) and
K4Fe(CN)6 (0.23 g, 0.62 mmol) in DMAc (20 mL) was added anhydrous Na2CO3
(0.24 g, 1.69 mmol) and Pd(OAc)2 (0.02 g, 0.1 mmol). The mixture was heated to 120
oC for 12 h under nitrogen protection, then the reaction mixture was cooled to room
temperature. Water (50 mL) was added and then the mixture was extracted with ethyl
acetate (50 mL × 3), and washed with brine (100 mL× 2). The organic layer was dried
and concentrated to give the intermediate 12 (0.34 g) as off-white solid in 88.4%
yield.
To a solution of 4-hydroxybenzaldehyde (4.78 g, 39.1 mmol) in DMF (100 mL)
was added K2CO3 powder (10.8 g, 78.3 mmol). After stirring at room temperature for
0.5 h, intermediate 12 (6.7 g, 39.1 mmol) was added and the mixture was heated to
o
140 C for 12 h under nitrogen protection. After cooling to room temperature, the
reaction mixture was filtered and washed with ethyl acetate (50 mL × 3), then the
filtrate was washed with water (100 mL × 2) and brine (100 mL × 2). The organic
layer was dried and concentrated to give the crude product, which was recrystallized
in ethanol-water mixed solvent to give intermediate 13 (8.78 g) as white solid in 81.6%
yield.
To a solution of 13 (8.26 g, 30.22 mmol) in DMSO (100 mL) was added K2CO3
powder (2.1 g, 15.2 mmol), and then 30% H2O2 (13.7 mL) was added at 10 oC. After
stirring at room temperature for 3 h, water (300 mL) was added and then the mixture
was extracted with ethyl acetate (100 mL × 3), and washed with with water (200 mL ×
2) and brine (200 mL × 2). The organic layer was dried and concentrated to give the
intermediate 14 (7.6 g) as light yellow solid in 81.4% yield.
To a solution of 14 (0.5 g, 1.72 mmol) in the mixture of absolute methanol (20
mL) and anhydrous dichloromethane (10 mL) was added 7a-h (1.72 mmol). After
stirring at room temperature for 0.5 h, acetic acid (0.8 mL) and NaBH3CN (0.22 g,
3.44 mmol) was added. After reaction at room temperature for 8 h, the solvent was
removed and water (30 mL) was added. The pH was adjusted to 9~10 with saturated
Na2CO3 solution, then extracted with ethyl acetate (50 mL × 3), and washed with
brine (100 mL× 2). The organic layer was dried and concentrated, and the pure
products 2a-h were obtained with further purified by column chromatograph on silica
gel (dichloromethane /ethyl acetate = 5/1) as white solid in 58.7-81.2% yield.
4.2.3.1 4-[4-[2-(3,5-Dimethylphenyl)pyrrolidin-1-yl]methylphenoxyl]-1-naphthamide
o
1
(2a). Yield: 81.2%. White solid. M.p.:164-166 C. H-NMR (300 MHz, CDCl3) δ
(ppm): 8.54 (d, J = 8.6 Hz, 1H, ArH), 8.39 (d, J = 7.8 Hz, 1H, ArH), 7.67-7.57 (m, 3H,
ArH), 7.32 (d, J = 8.3 Hz, 2H, ArH), 7.10 (s, 2H, ArH), 7.04 (d, J = 8.4 Hz, 2H, ArH),
6.92 (s, 1H, ArH), 6.78 (d, J = 7.9 Hz, 1H, ArH), 5.88 (br, 2H, NH2), 3.87 (d, J = 13.0
Hz, 1H , 1/2CH2), 3.31 (t, J = 8.0 Hz, 1H , CH), 3.15 (t, J = 8.1 Hz, 1H, 1/2CH2), 3.06
(d, J = 12.8 Hz, 1H, 1/2CH2), 2.35 (s, 6H, 2CH3), 2.27-2.13 (m, 2H, CH2), 1.99-1.67
(m, 3H, 1/2CH2, CH2). 13C-NMR (75 MHz, CDCl3) δ (ppm): 170.95, 155.91, 154.61,
143.13, 137.42, 135.40, 131.31, 129.87, 128.24, 127.54, 126.91, 126.08, 125.88,
125.74, 125.03, 124.84, 121.88, 118.99, 109.28, 69.28, 57.23, 53.01, 34.61, 21.78,

ACCEPTED MANUSCRIPT
20.90. HRMS (ESI): m/z [M+H]+. Calcd for C30H30N2O2: 451.23846; Found:
451.2384. IR (cm-1): 3380.46, 3187.62, 2964.48, 2920.45, 2779.77, 1644.93, 1614.12,
1585.52, 1504.30, 1464.24, 1428.47, 1365.01, 1321.52, 1264.12, 1242.33, 1217.22,
1161.26, 1116.02, 1098.52, 1053.21, 1018.78, 989.32, 847.38, 830.81, 769.17, 706.10,
647.10, 530.19, 515.19, 515.08, 419.27.
4.2.3.2 4-[4-[2-(3-fluorophenyl)pyrrolidin-1-yl]methylphenoxyl]-1-naphthamide (2b).
1
Yield: 76.2%. White solid. M.p.:116-118oC. H-NMR (300 MHz, CDCl3) δ (ppm):
8.54 (d, J = 8.5 Hz, 1H, ArH), 8.38 (d, J = 8.4 Hz, 1H, ArH), 7.75-7.52 (m, 3H, ArH),
7.32 (d, J = 8.4 Hz, 3H, ArH), 7.23 (d, J = 7.6 Hz, 2H, ArH), 7.04 (d, J = 8.5 Hz, 2H,
ArH), 6.96 (t, J = 8.2 Hz, 1H, ArH), 6.77 (d, J = 7.9 Hz, 1H, ArH), 5.91 (br, 2H, NH2),
3.84 (d, J = 12.9 Hz, 1H , 1/2CH2), 3.42 (t, J = 8.1 Hz, 1H, CH), 3.15 (t, J = 11.4 Hz,
2H, 1/2CH2), 2.40-2.13 (m, 2H, CH2), 2.00-1.64 (m, 3H, CH2, 1/2CH2). 13C-NMR (75
MHz, CDCl3) δ (ppm): 171.11, 164.32, 161.07, 155.81, 154.68, 146.53, 135.14,
131.31, 129.69, 129.33, 127.53, 127.01, 125.07, 125.88, 125.74, 125.04, 122.68,
121.86, 119.01, 113.45, 109.33, 68.63, 57.16, 53.00, 34.75, 21.95. HRMS (ESI): m/z
[M+H]+. Calcd for C28H25FN2O2: 441.1978; Found: 441.1979. IR (cm-1): 3386.48,
3181.29, 2964.51, 2799.19, 1641.12, 1588.32, 1502.76, 1462.52, 1447.77, 1428.50,
1322.91, 1361.55, 1266.66, 1242.45, 1241.56, 1160.27, 1137.38, 1094.06, 1054.79,
1015.69, 988.92, 864.35, 838.06, 785.44, 768.39, 741.41, 712.44, 695.12, 650.46,
613.25, 512.80, 464.66.
4.2.3.3 4-[4-[2-(3-Chlorophenyl)pyrrolidin-1-yl]methylphenoxyl]-1-naphthamide (2c).
1
Yield: 71.7%. White solid. M.p.:148-149oC. H-NMR (300 MHz, CDCl3) δ (ppm):
8.54 (d, J = 8.3 Hz, 1H , ArH), 8.38 (d, J = 8.0 Hz, 1H , ArH), 7.72-7.52 (m, 3H, ArH),
7.41 (d, J = 8.5 Hz, 2H, ArH), 7.32 (d, J = 8.4 Hz, 3H , ArH), 7.03 (d, J = 8.4 Hz, 2H,
ArH), 6.76 (d, J = 7.9 Hz, 1H, ArH), 5.90 (br, 2H, NH2), 3.80 (d, J = 13.1 Hz, 1H,
1/2CH2), 3.39 (t, J = 8.0 Hz, 1H, CH), 3.20-3.08 (m, 2H, CH2), 2.31-2.12(m, 2H,
CH2), 2.01-1.66 (m, 3H, CH2, 1/2CH2). 13C-NMR (75 MHz, CDCl3) δ (ppm): 171.09,
155.81, 154.69, 142.08, 135.13, 131.99, 131.31, 129.69, 128.39, 128.09, 127.54,
127.01, 126.07, 125.89, 125.78, 125.04, 121.86, 118.99, 109.34, 68.47, 57.11, 53.05,
34.81, 21.92. HRMS (ESI): m/z [M+H]+. Calcd for C28H25ClN2O2: 457.1683; Found:
457.1680. IR (cm-1): 3380.49, 3181.36, 2963.70, 2803.77, 1641.70, 1611.22, 1588.66,
1502.19, 1462.70, 1428.56, 1412.96, 1362.03, 1323.21, 1266.30, 1243.73, 1215.28,
1159.57, 1118.37, 1088.90, 1054.90, 1013.85, 989.30, 824.93, 767.90, 713.15, 662.99,
606.18, 536.97, 513.66.
4.2.3.4 4-[4-[2-(4-Methoxylphenyl)pyrrolidin-1-yl]methylphenoxyl]-1-naphthamide
1
(2d). Yield: 68.3%. White solid. M.p.:120-122oC. H-NMR (300 MHz, CDCl3) δ
(ppm): 8.53 (d, J = 8.6 Hz, 1H, ArH), 8.38 (d, J = 8.3 Hz, 1H, ArH), 7.72-7.49 (m, 3H,
ArH), 7.38 (d, J = 8.1 Hz, 2H, ArH), 7.32-7.26 (m, 2H, ArH), 7.01 (d, J = 8.5 Hz, 2H,
ArH), 6.90 (d, J = 8.6 Hz, 2H, ArH), 6.74 (d, J = 8.0 Hz, 1H, ArH), 5.91 (br, 2H ,
NH2), 3.88-3.81 (m, 4H, 1/2CH2, CH3), 3.38-3.30 (m, 1H, CH), 3.22-2.97 (m, 2H,

ACCEPTED MANUSCRIPT
CH2), 2.33-2.12 (m, 2H, CH2), 2.01-1.66 (m, 3H, 1/2CH2, CH2). 13C-NMR (75 MHz,
CDCl3) δ (ppm): 171.18, 158.21, 155.85, 154.57, 135.48, 135.19, 131.30, 129.76,
128.12, 127.50, 126.98, 126.98, 126.04, 125.85, 125.76, 125.05, 121.86, 119.00,
113.35, 109.27, 68.68, 57.01, 54.79, 52.95, 34.66, 21.72. HRMS (ESI): m/z [M+H]+.
Calcd for C29H28N2O3: 453.2178; Found: 453.2179. IR (cm-1): 3380.20, 3193.37,
2950.87, 2904.06, 2807.96, 1731.86, 1641.80, 1612.90, 1580.69, 1512.02, 1503.30,
1464.57, 1428.32, 1362.20, 1319.76, 1299.70, 1263.85, 1240.47, 1214.34, 1158.97,
1119.81, 1106.34, 1051,52, 1033.59, 851.34, 827.35, 813.07, 767.44, 736.22, 671.13,
649.36, 544.04, 530.84, 514.99.
4.2.3.5 4-[4-[2-(2-Methylphenyl)pyrrolidin-1-yl]methylphenoxyl]-1-naphthamide (2e).
1
Yield: 75.8%. White solid. M.p.:160-162oC. H-NMR (300 MHz, CDCl3) δ (ppm):
8.54 (d, J = 8.5 Hz, 1H, ArH), 8.39 (d, J = 8.1 Hz, 1H, ArH), 7.66 (d, J = 7.4 Hz, 1H,
ArH), 7.65-7.51 (m, 3H, ArH), 7.33 (d, J = 8.4 Hz, 2H, ArH), 7.25-7.19 (m, 1H, ArH),
7.14 (d, J = 3.9 Hz, 2H, ArH), 7.02 (d, J = 8.5 Hz, 2H, ArH), 6.76 (d, J = 8.0 Hz, 1H,
ArH), 5.92 (br, 2H, NH2), 3.87 (d, J = 13.3 Hz, 1H, 1/2CH2), 3.62 (t, J = 8.4 Hz, 1H,
CH), 3.21-3.12 (m, 1H, 1/2CH2), 3.07 (d, J = 12.7 Hz, 1H, CH2), 2.37 (s, 3H, CH3),
2.36-2.23 (m, 2H, CH2), 2.01-1.75 (m, 2H, CH2), 1.74-1.60 (m, 1H, 1/2CH2).
13C-NMR (75 MHz, CDCl3) δ (ppm): 171.17, 155.86, 154.61, 141.35, 135.48, 135.41,
131.31, 129.72, 127.53, 126.99, 126.07, 125.87, 125.80, 125.77, 125.05, 121.88,
119.03, 109.30, 64.97, 57.35, 52.98, 32.67, 22.00, 18.95. HRMS (ESI): m/z [M+H]+.
Calcd for C29H28N2O2: 437.2229; Found: 437.2229. IR (cm-1): 3365.73, 3179.23,
2951.42, 2905.54, 2806.40, 2775.12, 1646.83, 1614.36, 1584.65, 1465.05, 1430.75,
1367.22, 1321.02, 1241.64, 1265.18, 1216.11, 1161.65, 1122.47, 1094.42, 1053.26,
987.87, 857.68 815.94, 768.48, 753.23, 726.40, 712.32, 684.38, 515.16.
4.2.3.6 4-[4-[2-(Pyridine-3-yl)pyrrolidin-1-yl]methylphenoxyl]-1-naphthamide (2f).
1
Yield: 58.7%. White solid. M.p.:168-170oC. H-NMR (300 MHz, CDCl3) δ (ppm):
8.65 (s, 1H, ArH), 8.54-8.44 (m, 2H, ArH), 8.34 (d, J = 8.1 Hz, 1H, ArH), 7.88-7.55
(m, 1H, ArH), 7.68-7.48 (m, 3H, ArH), 7.29 (s, 3H, ArH), 7.00 (d, J = 8.5 Hz, 2H,
ArH), 6.74 (d, J = 7.9 Hz, 1H, ArH), 5.92 (br, 2H, NH2), 3.77 (d, J = 13.1 Hz, 1H ,
1/2CH2), 3.50-3.38 (m, 1H, CH), 3.24-3.12 (m, 2H, CH2), 2.35-2.16 (m, 2H, CH2),
2.00-1.62 (m, 3H, 1/2CH2, CH2). 13C-NMR (75 MHz, CDCl3) δ (ppm): 170.76,
154.77, 149.12, 148.15, 138.89, 134.89, 134.50, 131.32, 129.67, 128.35, 127.56,
126.99, 126.07, 125.90, 125.72, 125.04, 123.14, 121.84, 119.00, 109.37, 66.58, 57.13,
53.17, 34.78, 22.06. HRMS (ESI): m/z [M+H]+. Calcd for C27H25N3O2: 424.2025;
Found: 424.2023. IR (cm-1): 3351.44, 3159.19, 2967.57, 2804.48, 1667.44, 1614.80,
1579.34, 1504.63, 1463.49, 1428.41, 1407.80, 1362.18, 1320.17, 1262.67, 1230.67,
1206.39, 1162.78, 1097.27, 1050.32, 1027.51, 1018.04, 978.53, 866.86, 843.66,
832.34, 798.44, 773.71, 713.24, 639.28, 600.12, 513.91.
4.2.3.7 4-[4-[2-(4-Fluorophenyl)pyrrolidin-1-yl]methylphenoxy]-1-naphthamide (2g).
1
Yield: 66.2%. White solid. M.p.:160-162oC. H-NMR (300 MHz, CDCl3) δ (ppm):
8.54 (d, J = 8.7 Hz, 1H, ArH), 8.38-8.30 (m, 1H, ArH), 7.69-7.52 (m, 3H, ArH),

ACCEPTED MANUSCRIPT
7.50-7.36 (m, 2H, ArH), 7.32-7.24 (m, 2H, ArH), 7.09-6.97 (m, 4H, ArH), 6.80-6.70
(m, 1H, ArH), 5.91 (br, 2H, NH2), 3.88-3.70 (m, 1H, 1/2CH2), 3.45-3.29 (m, 1H, CH),
3.22- 3.01 (m, 2H, CH2), 2.33-2.12 (m, 2H, CH2), 2.04-1.64 (m, 3H, 1/2CH2, CH2).
13C-NMR (75 MHz, CDCl3) δ (ppm): 170.91, 163.05, 159.81, 155.87, 154.64, 139.03,
135.25, 131.31, 129.68, 128.50, 128.40, 127.55, 126.94, 126.07, 125.89, 125.74,
125.03, 121.86, 119.00, 114.85, 114.57, 109.29, 68.49, 57.07, 53.04, 34.84, 21.82.
HRMS (ESI): m/z [M+H]+. Calcd for C28H25FN2O2: 441.1978; Found: 441.1978. IR
(cm-1): 3388.51, 3183.94, 2965.35, 2806.55, 1641.05, 1611.26, 1588.39, 1503.26,
1462.94, 1428.44, 1362.46, 1323.09, 1266.64, 1242.70, 1215.96, 1159.62, 1095.05,
1054.78, 1015.02, 833.39, 768.44, 512.16.
4.2.3.8 4-[4-[2-(3-Methylphenyl)pyrrolidin-1-yl]methylphenoxyl]-1-naphthamide (2h).
1
Yield: 72.1%. White solid. M.p.:158-159oC. H-NMR (300 MHz, CDCl3) δ (ppm):
8.51 (d, J = 8.1 Hz, 1H, ArH), 8.35 (d, J = 8.2 Hz, 1H, ArH), 7.72-7.49 (m, 3H, ArH),
7.37-7.22 (m, 6H, ArH), 7.08 (d, J = 6.4 Hz, 1H, ArH), 7.01 (d, J = 8.5 Hz, 2H, ArH),
6.75 (d, J = 7.9 Hz, 1H, ArH), 5.97 (br, 2H, NH2), 3.85 (d, J = 12.4 Hz, 1H, 1/2CH2),
3.43-3.24 (m, 1H, CH), 3.33-3.31 (m, 1H, 1/2CH2), 3.25-2.96 (m, 2H, CH2), 2.37 (s,
13
3H, CH3), 2.31-2.10 (m, 2H, CH2), 1.98-1.69 (m, 3H, 1/2CH2, CH2). C-NMR (75
MHz, CDCl3) δ (ppm): 171.11, 155.87, 154.62, 143.24, 137.51, 135.42, 131.32,
129.81, 127.88, 127.77, 127.52, 127.33,126.99, 126.08, 125.87, 125.75, 125.05,
124.14, 121.88, 118.98, 109.31, 69.26, 57.17, 53.02, 34.69, 21.85, 21.04. HRMS
(ESI): m/z [M+H]+. Calcd for C29H28N2O2: 437.2229; Found: 437.2229. IR (cm-1):
3385.50, 3185.50, 2951.39, 2783.35, 1641.40, 1613.01, 1582.72, 1504.14, 1427.01,
1409.71, 1358.42, 1263.21, 1319.48, 1238.06, 1216.91, 1160.87, 1121.85, 1096.97,
1051.48, 779.99, 701.08, 629.75, 606.39.
4.3. Docking studies
The molecular modeling study was performed using the published crystal
structure of κ opioid receptor in complex with JDTic (Protein Data Bank code: 4DJH)
[26] and the structure was editor according to provide a monomer of the protein and
protonated use GOLD 5.1. The ligand was then removed to leave the receptor
complex, which was used for the subsequent docking studies. For preparation of
ligand structures, the selected analogs were energy minimized using MOE 2013.08
with a MMFF94x forcefield using gas phase calculations and a cutoff of 0.01.
Charges were then fixed using an MMFF94 forcefield. For computational docking,
GOLD 5.1 software was used in combination with ChemScore scoring function. The
active site was defined as being any volume within 10 Å of the scaffold of ligand A in
its crystal pose in 4DJH. The number of genetic algorithm (GA) run was set to 10, and
scoring of the docked poses was performed with the ChemScore scoring function.
Each GOLD run was saved and the strongest scoring binding pose of each ligand was
compared to that of the reference ligand position observed in the crystal structure. The
best output poses of the ligands generated were analyzed on the basis of ChemScore,
feasibility of hydride transfer process, and H-bonding to the enzyme. The best poses
were visualized with MOE 2013.08.

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4.4. Chemicals and reagents for in vitro assays
Membranes from Chinese hamster ovary (CHO) cells expressing the human κ and
µ opioid receptor and membranes from human embryonic kidney (HEK) 293 cells
expressing the human δ opioid receptor were obtained from Shanghai Institute of
Materia Medica, Chinese Academy of Sciences. [3H]-DAMGO, [3H]-DPDPE and
[3H]-U69,593 were purchased from Perkin Elmer Italia. DAMGO, DPDPE and
U50488 were purchased from Sigma Aldrich. Scintillation reagents PPO and POPOP
were purchased from Sigma Aldrich. Tris was purchased from Amresco company.
4.5. Radioligand binding methods
Radioligand binding assays were carried out according to previously reported
procedures [27,28]. Briefly, radioligand displacement studies with [3H]-U69,593,
[3H]-DAMGO and [3H]-DPDPE were carried out using membranes prepared from
CHO cells expressing cloned human κ and µ opioid receptors or HEK293 cells
expressing the cloned δ opioid receptor. Membranes were incubated with the
appropriate concentration of [3H]-U69,593 or [3H]-DAMGO or [3H]-DPDPE in
Tris-HCl buffer at 25 oC for 60 min in the absence or presence of naloxone (1µM) (for
µ and δ receptors) or U69,593 (10 µM) (for κ receptor). The binding reaction was
stopped by rapid filtration under vacuum through glass-fiber filters (Whatman GF/B)
using a Brandell 36-sample harvester (Millipore) and thereafter the filters were
washed with 4×5 mL ice-cold 50 mM Tris-HCl buffer (pH 7.4). Filter-bound
radioactivity was counted in a liquid scintillation counter (Pri-carb 2910; PerkinElmer)
using 4 mL of scintillation fluid (Packard Ultima). Displacement curves were carried
out using serial dilutions ranging from 100 µM to 0.1 nM of the compounds.
Compounds were dissolved in DMSO. Concentrations causing 50% inhibition (IC50)
of [3H]-ligand binding were determined from 11-point concentration response curves
in assay buffer. All receptor binding experiments were performed in triplicate and
results were confirmed in at least four independent experiments. Data from
radioligand inhibition experiments were analyzed by nonlinear regression analysis of
a Sigmoid Curve using GraphPad Prism program. Ki values were calculated from the
obtained IC50 values by means of the equation of Cheng and Prusoff [29]. Values of
0.68 nM, 0.92 nM and 1.34 nM for the dissociation constants of [3H]-U69,593,
[3H]-DAMGO and [3H]-DPDPE were used respectively.
4.6. GTP-γ-S antagonist functional assays.
In vitro [35S] GTP-γ-S binding experiments were conducted as previously
described [24]. Reagent dispensing into plates was conducted with Beckman
Multimek 96-channel robots and Titertek Multidrops. Stock solutions of test
compound or naltrexone were diluted in buffer (50 HEPES, pH 7.4, 5 mM MgCl2)
and pipetted into a 96-well polypropylene plate. Then 11 point concentration curves
that bracketed the IC50 (a concentration range of 0.01 nM to 10 µM) were prepared in
this plate using a Tecan Evo liquid handler. Membranes were thawed and diluted with
assay buffer (20 mM HEPES, pH 7.4, 5 mM MgCl2, 100 mM NaCl, 1 mM EDTA, 1

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mM dithiothreitol) containing 200 µM GDP and homogenized using a Polytron. A
GTP-γ-35S mixture (0.5 nM final) was prepared for each receptor with the addition of
an agonist specific for each opioid receptor. For κ, U69,693 was added (300 nM final
assay concentration), for µ, DAMGO was added (1 µM final), and for δ, DPDPE was
added (30 nM final). The final agonist concentrations are equal to concentrations that
stimulate approximately 80% of the maximum GTP-γ-S binding in the absence of
antagonist. Finally, 50 µL of each compound dilution, 50 µL of GTP-γ-35S/agonist
solution, 50 µL of diluted membranes (15-30 µg protein), and 50 µL of WGA SPA
beads (1 mg) prepared in assay buffer were added to the assay plate in the order listed.
Following an incubation of 2 h (δ) or 4 h (κ and µ) at room temperature, the assay
plates were placed at 4 oC overnight to allow the SPA beads to settle to the bottom of
the well. Radioactivity in the plates was determined using a Trilux Microbeta
scintillation counter (Perkin-Elmer) and reported in counts per minute (CPM). For
total GTP-γ-S binding, assay buffer was used in place of added compound. For
nonspecific GTP-γ-S binding, the following antagonists were used: κ (NorBNI at 10
µM final), µ (naltrexone at 10 µM final), and δ (naltriben at 10 µM final). Data were
analyzed using four-parameter (curve maximum, curve minimum, IC50, Hill slope)
nonlinear regression routines (XLFit version 4.0; ActivityBase, IDBS), where IC50 is
the concentration of added compound that results in 50% inhibition of GTP-γ-35S
binding.
4.7. Mice forced swimming test.
Male ICR mice weighing 20-25 g were housed in plastic cages with 10-12
mice/cage in a vivarium for at least 7 days. 50 mice were divided into 5 groups
randomly and removed from the vivarium to the testing area in their home cages, and
allowed to adapt to the new environment for at least 15 min before testing. The groups
were administrated with compounds 1b, 1c, 2h, LY2456302 and normal saline as
vehicle, respectively (30 mg/kg i.p., 30 min before the test) [19]. The forced swim test
was performed using the original method described by Porsolt et al. [30]. Briefly,
mice were placed individually in clear plastic cylinders (10 cm in diameter, 25 cm in
o
height) filled to 6 cm with 25 C water for 6 min. The duration of immobility was
recorded during the last 4 min of a 6-min trial. A mouse was regarded as immobile
when floating motionless or making only those movements necessary to keep its head
above the water.
4.8. Mice elevated plus-maze test.
Elevated plus-maze test apparatus consisted of two open arms (length 30 × width
5 cm) and two closed arms of the same size, along with a semi-transparent wall
(height 15 cm) and central platform (length 5 × width 5 cm). These arms and central
platform were elevated 50 cm above the floor. Male ICR mice weighing 18-22 g were
housed in plastic cages with 10-12 mice/cage in a vivarium for at least 3 days. 40
mice were divided into 4 groups randomly and removed from the vivarium to the
testing area and allowed to adapt to the new environment for at least 1 h before testing.
The mice elevated plus-maze test was performed according to published procedures

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[31]. The groups were administrated with compounds 1c, 2h, LY2456302 and normal
saline as vehicle, respectively (30 mg/kg i.p., 30 min before the test). Thirty-minutes
after the drug administration, each mouse was placed in the central platform, facing
one of the open arms. During a 5 min test session, mouse behavior was recorded using
EthoVision XT. The number of entries into the open (OE) and closed (CE) arms, and
the time spent in the open (OT) and closed (CT) arms were scored. The number
proportion into the open arms [OE / (CE + OE)] and the retention time proportion in
the open arms [OT / (OT + CT)] were calculated from the above indicators.
Acknowledgments
This work was supported by Excellent Science and Technology Innovation Team
Projects of Jiangsu Province Universities in 2015, China.
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List of captions
Title
Abstract
Keywords
1. Introduction
2. Results and discussion
2.1. Design of selective κ opioid receptor antagonists
2.2. Chemistry
2.3. Molecular modeling studies
2.4. In vitro opioid receptor binding and [35S] GTP-γ-S binding assays
2.5. Biological activity in vivo
2.5.1. Mice forced swimming test
2.5.2. Mice elevated plus-maze test
3. Conclusions
4. Experimental section
4.1. General procedures
4.2. Preparation of the compounds
4.2.1. General procedure for the preparation of intermediates 7a-h.

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4.2.2. General procedure for the preparation of compounds 1a-d.
4.2.3. General procedure for the preparation of compounds 2a-h.
4.3. Docking studies
4.4. Chemicals and reagents for in vitro assays
4.5. Radioligand binding methods
4.6. GTP-γ-S antagonist functional assays.
4.7. Mice forced swimming test.
4.8. Mice elevated plus-maze test.
Acknowledgments
References and notes