Discovery of 3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyphenyl)-N-phenylpiperidine-1-carboxamide as novel potent analgesic

Article abstract of DOI:10.1016/j.ejmech.2020.112070

Management of moderate to severe pain by clinically used opioid analgesics is associated with a plethora of side effects. Despite many efforts have been dedicated to reduce undesirable side effects, moderate progress has been made. In this work, starting from Tramadol, a series of 3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyphenyl)piperidine-1-carboxamide derivatives were designed and synthesized, and their in vitro and in vivo activities were evaluated. Our campaign afforded selective μ opioid receptor (MOR) ligand 2a (K_iMOR: 7.3 ± 0.5 nM; K_iDOR: 849.4 ± 96.6 nM; K_iKOR: 49.1 ± 6.9 nM) as potent analgesic with ED₅₀ of 3.1 mg/kg in 55 °C hot plate model. Its antinociception effect was blocked by opioid antagonist naloxone. High binding affinity toward MOR of compound 2a was associated with water bridge, salt bridge, hydrogen bond and hydrophobic interaction with MOR. The high selectivity of compound 2a for MOR over δ opioid receptor (DOR) and κ opioid receptor (KOR) was due to steric hindrance of compound 2a with DOR and KOR. 2a, a compound with novel scaffold, could serve as a lead for the development of novel opioid ligands.

Full text of DOI:10.1016/j.ejmech.2020.112070

Journal Pre-proof
Discovery of 3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyphenyl)-N-
phenylpiperidine-1-carboxamide as novel potent analgesic
Huoming Huang, Wenli Wang, Xuejun Xu, Chen Zhu, Yujun Wang, Jinggen Liu, Wei
Li, Wei Fu
PII:
S0223-5234(20)30037-4
DOI:
https://doi.org/10.1016/j.ejmech.2020.112070
EJMECH 112070
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 December 2019
Revised Date: 12 January 2020
Accepted Date: 13 January 2020
Please cite this article as: H. Huang, W. Wang, X. Xu, C. Zhu, Y. Wang, J. Liu, W. Li, W. Fu, Discovery
of 3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyphenyl)-N-phenylpiperidine-1-carboxamide as
novel potent analgesic, European Journal of Medicinal Chemistry (2020), doi: https://doi.org/10.1016/
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Discovery of
3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyphenyl)-N-pheny
lpiperidine-1-carboxamide as Novel Potent Analgesic
Huoming Huang a, Wenli Wang a, Xuejun Xu b, Chen Zhu a, Yujun Wang b, Jinggen Liu b*, Wei Li
a*, Wei Fu a*
Affiliation:
a. Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai, 201203,
China.
b. Shanghai Institute of Materia Medica, Chinese Academy of Science, Shanghai, 201203, China.
Graphical Abstract

Discovery of
3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyphenyl)-N-pheny
lpiperidine-1-carboxamide as Novel Potent Analgesic
Huoming Huang a, Wenli Wang a, Xuejun Xu b, Chen Zhu a, Yujun Wang b, Jinggen Liu b*, Wei Li
a*, Wei Fu a*
Affiliation:
a. Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai, 201203,
China.
b. Shanghai Institute of Materia Medica, Chinese Academy of Science, Shanghai, 201203, China.
Abstract:
Management of moderate to severe pain by clinically used opioid analgesics is associated
with a plethora of side effects. Despite many efforts have been dedicated to reduce undesirable
side effects, moderate progress has been made. In this work, starting from Tramadol, a series of
3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyphenyl)piperidine-1-carboxamide derivatives
were designed and synthesized, and their in vitro and in vivo activities were evaluated. Our
campaign afforded selective µ opioid receptor (MOR) ligand 2a (Ki MOR: 7.3±0.5 nM; Ki
:
DOR
849.4±96.6 nM; Ki KOR: 49.1±6.9 nM) as potent analgesic with ED50 of 3.1 mg/kg in 550C hot
plate model. Its antinociception effect was blocked by opioid antagonist naloxone. High binding
affinity toward MOR of compound 2a was associated with water bridge, salt bridge, hydrogen
bond and hydrophobic interaction with MOR. The high selectivity of compound 2a for MOR over
δ opioid receptor (DOR) and κ opioid receptor (KOR) was due to steric hindrance of compound
2a with DOR and KOR. 2a, a compound with novel scaffold, could serve as a lead for the
development of novel opioid ligands.
Keywords: Selective MOR agonists; Antinociception; Analgesic; Opioids; Molecular
mechanism
Introduction
Management of moderate to severe pain in clinics relied heavily on opioid analgesics due to
their effectiveness in relieving pain. Clinically used opioids were divided into three categories,
naturally derived morphinans such as Morphine, semi-synthetic morphinan analogues such as
Oxycodone, Hydromorphone, and synthetic narcotic analgesics, such as Fentanyl, Tramadol and
Pethidine (Figure 1)[1]. However, despite their desirable effectiveness in amelioration of pain,
continued use of opioids was associated with a plethora of undesirable effects including, but not
limited to, respiratory depress[2, 3], tolerance[4], dependence or abuse[5], constipation[6], nausea and
vomiting[7]. In addition, overdose related mortality in America increased exponentially in recent
years mainly due to prescription or illegal use of opioids[8-10]. Thus, analgesics with potent
antinociception effect and without or with reduced side effects were in great demand in clinical
practice. Many efforts have been devoted to the development of analgesics with reduced
1

undesirable effects, such as combination therapy of agonist and antagonist[11], new formulation
strategy[12, 13]. These efforts led to moderate improvement.
Opioids elicited its analgesic effect via activating µ opioid receptor (MOR)[14, 15]. The opioid
receptors belonged to G protein coupling receptor (GPCR) and generally were categorized into
three subtypes: MOR, δ opioid receptor (DOR) and κ opioid receptor (KOR)[16]. Recently, it was
found that Gi-biased MOR agonists that do not recruit β-arrestin 2 displayed improved
antinociception effect and less side effects such as constipation, respiratory inhibition and
tolerance[17-21]. The most advanced drug was biased MOR agonist TRV130 (Figure 1), which was
not approved by FDA due to its safety concern[22-26], presumably its addiction liability[27]. In
addition, it has been reported that Gi-biased KOR agonists were superior to balanced KOR
agonists in antinociception and do not induce sedation and anhedonia-like actions[28, 29]. Several
biased KOR agonists have been reported but none has entered clinical trial to date[30-36]. Many
strategies targeting none opioid receptors such as ion channel, enzyme, for the management of
moderate to severe pain, have been reported, but no new treatments were available in the market
to date[37, 38]
.
As part of our continued interest in developing analgesics, Tramadol was selected as lead
compound for its moderate side effects and pain-relieving effects. Recently, modification of
Tramadol afforded selective MOR agonists and DOR agonists[39, 40]. Herein, we reported the
design,
synthesis,
structure
activity
relationship
and
antinociception
of
3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyphenyl)-N-phenylpiperidine-1-carboxamide
(2a). Also, binding pose of compound 2a with MOR was proposed.
Figure 1 Structure of clinically used opioids and TRV130.
Compound design
Tramadol’s moderate analgesic effect and metabolically labile property were associated with
large oral dosing (100 mg) and relatively short duration (every 4~6 hours), respectively[41]
.
Furthermore, the maximum dose of tramadol was 400 mg per day, suggesting its potential
relatively high toxicity[41]. “Message-address concept”, proposed by Schwyzer in 1977[42], has
been extensively used in the design of opioid ligands with varied selectivity over opioid
receptors[43-46]. For example, the upper part of compound 1, a balanced MOR/DOR ligand,
corresponds to “message”, while the bottom part belongs to “address” that determined its
2

selectivity (Figure 2)[47]. By carefully examining the structure of Tramadol, it was proposed that
meta-methoxyphenyl and dimethylamine correspond to “message” part (Figure 2). Therefore, it
would be interesting to probe where the “address” part of tramadol derivatives is and position 4 of
cyclohexane ring of tramadol would be a good start. For the ease of synthesis, carbon atom of
position 4 was replaced by nitrogen. Then phenyl was employed to probe the “address” part and
urea group was selected as linker to add hydrogen donor and receptor with an aim to enhance its
activity and hydrophilicity. As a result, compound 2a was designed (Figure 2). To confirm our
preliminary hypothesis, compound 2a was docked into the active site of MOR and the binding
mode was shown in Figure 3A. Compound 2a fitted into the binding pocket of MOR well.
Multiple interactions including water bridge, hydrogen bond and salt bridge interaction were
observed for compound 2a, suggesting that it would bind to MOR tightly and hence enhance its
activity. In addition, 2a aligned with Tramadol well and phenyl attaching to nitrogen extended into
a hydrophobic pocket (Figure 3B). Based on above hypothesis and docking studies, nitrogen
substitution patterns were explored and finally compound 2a~2r were designed (Figure 2).
O
HO
O
O
Message
OH
O
O
OH
N
N
OH
1
H
N
N
2
N
N
N
HN
4
R1
N
O
???
Address
R2
Tramadol
2a~2r
2a
Cl
cLogP: 3.1
cLogP: 2.367
1
Cl
Cl
Cl
Cl
Cl
R1=H, R2=
Cl
Cl
2a
2b
2c
2d
F3C
2f
2g
2h
COOMe
F
F
Cl
F
COOMe
F
S
S
CF3
2i
2j
2k
2l
2m
2n
2o
OH
2r
2p
Cl
2q
R1=CH3, R2=
2e
Figure 2 Design of target compounds
3

Figure 3 Binding mode of 2a with MOR and alignment of Tramadol with 2a. (A: Binding mode
of 2a with MOR. 2a was shown in sticks, green. B: Alignment of Tramadol with 2a. Tramadol
was shown in sticks, light blue.)
Chemistry
Synthesis of target compounds were shown in Figure 4, Figure 5 and Figure 6. Initially,
synthetic route shown in Figure 4 was employed. Hydroxyl in compound 3 was protected by
triethylsilyl (TES) using triethylchlorosilane (TESCl) in the presence of imidazole to afford 4,
which reacted with isocyanate 5 to give 6. Compound 6 was deprotected in tetrabutylammonium
fluoride (TBAF) to give free base, which underwent salt formation to get hydrochloride 2d.
However, several disadvantages were obvious. Protecting group TES was not stable, making 4
undergo deprotection at room temperature. Isocyanates are highly toxic and many are not
commercially available. Therefore, synthetic route in Figure 5 was devised and implemented.
Phenyl chloroformate or para-nitrophenyl chloroformate reacted with amines to give 8[48]
,
followed by reaction with 3 to obtain 9. Free base 9 reacted with HCl to give 2a~2c, 2e~2r.
Compound 12 reacted with phenyl phenylcarbamate to give 11 as free base, which underwent salt
formation with HCl to give 12 (Figure 6). All final compounds were confirmed by 1H NMR, 13
C
NMR and HRMS.
O
O
O
O
OTES
OH
O
N
N
C
HCl
c, d
a
b
OH
Cl
N
OTES
N
N
N
N
O
NH
O
NH
N
HCl
N
H
H
5
4
3
Cl
Cl
6
2d
Figure 4 Initial synthesis of compound 2d. (a) TESCl, imidazole, DCM; b) TEA, DCM, 00C~R.T.;
c) TBAF, THF, R.T. d) HCl/Dioxane.)
4

Figure 5 Modified route for the synthesis of compound 2a~2c, 2e~2r. (a) NaHCO3, THF/H2O,
R.T.; b) TEA, DMF, R.T.; c) HCl/Dioxane.)
Figure 6 Synthesis of 12 (a) TEA, DCM, R.T.; b) HCl/Dioxane, R.T.)
Results and Discussion
0
55 C hot plate model is a reliable animal model extensively used in the evaluation of
compounds acting on pain perception via central nervous systems[49]. It’s readily available in our
lab. Therefore, we employed a combination strategy of in vitro binding affinity and in vivo 55 0C
hot plate model screening simultaneously. Evaluation on animal model was first tested at dosage
10 mg/kg via intraperitoneal injection. Compounds that displayed full antinociception (Maximum
possible effect% (MPE%) was 100%) was further studied in detail. Compounds that showed
partial antinociception (defined as MPE% <100%) at 10 mg/kg will not investigate further.
Binding affinities of target compounds were evaluated in radioligand binding assay and
summarized in Table 1. Tramadol showed weak binding affinities for three opioid receptors and is
in consistent with literature[50]. Compound 2a with phenyl ring attached to nitrogen displayed high
affinity (Ki = 7.3 ± 0.5 nM) and selectivity for MOR over DOR and KOR. Its high binding for
MOR further confirmed our hypothesis. Compound 2a displayed full antinociception at dose of 10
mg/kg in hot plate model. Substitution of phenyl with benzyl group afforded 2b, which showed
sharply decreased binding for three receptors. This indicated that benzyl with higher flexibility
and longer chain was incompatible with three receptors in the sub-pocket. Adding methyl to the
metabolically labile benzyl position gave 2c. Compound 2c also showed lower binding for MOR
as well as KOR in compare with 2b and abolished affinity for DOR. Compound 2b and 2c showed
partial antinociception in vivo.
We next turn our attention to explore substitution patterns on phenyl ring. Meta-chloro
bearing 2d displayed one-fold decrease in affinity for MOR and 15-fold decrease for KOR, but
increase for DOR in comparison to 2a. Also, compound 2d was a selective ligand for MOR.
Deleting hydrogen bond donor in 2d by adding methyl group yielded 2e. Compound 2e showed
5

substantially decreased binding for three opioid receptors, suggesting hydrogen bonding was
favorable in this position. Moving meta-chloro to para position (2f), or multi-substitution in
phenyl (2g and 2h) decreased their binding for MOR and KOR greatly in comparison to 2d, and
abolished affinity for DOR. Compound 2d~2h showed partial antinociception in vivo. Substitution
of meta-chloro with fluoro lead to 2i. Compound 2i showed decreased binding for MOR and DOR
as well as abolished activity for KOR. However, 2i showed potent antinociception in vivo. In
consistent with 2g and 2h, multi-substitution 2j and 2k showed low binding for three opioid
receptors. Meta-bis(trifluoromethyl) and β-naphthyl substitution gave 2l and 2m, respectively.
Compound 2l and 2m showed abolished activities for MOR and low binding for DOR and KOR.
Adding hydrogen bond acceptor ester group to thienyl (2o and 2p) displayed no improvement in
affinities for three receptors.
It has been reported that binding pockets of opioid receptors were exposed to extracellular
side[51-54]. The nitrogen substitution corresponded to the “address” part (Figure 2). We next
explored the “address” pocket by adding bulky adamantyl and gave 2p, 2q and 2r. Unfortunately,
it turned out that 2p~2r showed decreased or abolished binding for opioid receptors, indicating
that adamantyl is too bulky for these receptors. Overall, 2j~2r showed partial antinociception in
vivo.
Table 1 Binding affinities of compound 2a~2r
Ki (nM) or Inhibition at 1 µM (%) a
In vivo
Compounds
R
activity e
MOR b
6.0±0.4%
2.30±0.02
ND
DOR c
KOR d
Tramadol
DAMGO
DPDPE
--
--
--
--
0%
ND f
0%
--
--
--
--
ND
2.30±0.69
ND
ND
U50488
ND
0.91±0.20
2a
2b
2c
2d
2e
2f
7.3±0.5
35.1±4.4%
20.5±5.5%
14.1±2.7
849.4±96.6
2.6±2.9%
0%
49.1±6.9
5.1±4.0%
100%
partial
partial
partial
partial
partial
4.1±6.7%
537±69.3
0%
718.6±197.0
14.0±9.7%
22.1±10.7%
8.7±3.7%
48.8±3.9%
0%
6

2g
2h
46.0±5.4%
13.2±8.0%
0%
0%
18.2±1.9%
0%
partial
partial
2i
2j
36.7±7.4%
13.6±13.1%
25.0±13.3%
9.2±7.8%
16.4±10.6%
21.6±13.4%
0%
0%
100%
partial
partial
2k
7.1±5.0%
2l
2m
2n
0%
0.5%
0.9%
0%
8.6±4.8%
3.9%
partial
partial
partial
10.2±9.0%
0%
0%
2o
2p
2q
2.3%
16.0±12.4%
9.2±3.1%
17.9±4.8%
10.4±10.2%
partial
partial
partial
13.5±4.8%
32.8±5.5%
0%
0%
2r
0%
1.9%
0%
partial
a. Values are expressed as the mean ± SEM for three independent experiments performed in triplicate. b.
Displacement of [3H] DAMGO from the CHO cell membrane expressing the µ opioid receptor. c. Displacement of
[3H] DPDPE from the CHO cell membrane expressing the δ opioid receptor. D. Displacement of [3H] U69593
0
from the CHO cell membrane expressing the κ opioid receptor. e. 55 C hot plate model, i.p., mice, n=4~5. f. Not
determined.
0
Compounds 2a and 2i were potent in 55 C hot plate model at 10 mg/kg dose after
intraperitoneal injection (Table 1). Lowering dose to 5 mg/kg, 2a was more potent than 2i (Figure
7). In addition, compound 2a produced longer duration action than 2i in this model. Therefore, 2a
was selected as candidate for further study. Compound 2a dose dependently prolonged latency of
0
mice to noxious stimuli in 55 C hot plate model (Figure 8), and ED50 was determined as 3.1
mg/kg. In contrast, Tramadol showed partial antinociception activity up to 50 mg/kg. Furthermore,
analgesic effect of compound 2a was blocked by nonselective opioid antagonist naloxone (Figure
9). Taken high selectivity of compound 2a for MOR and its antagonism effect by naloxone
together, we assumed that 2a produced its antinociception mainly via MOR.
7

Figure 7 Comparison of antinociception of 2a and 2i at 5 mg/kg. (55 hot plate model, mice, i.p.,
n=4)
Figure 8 Antinociception of 2a (550C hot plate model, mice, i.p., n= 9~14; shown was 60 min for
2a and 15 min for tramadol.)
Figure 9 Antinociception of 2a was antagonized by naloxone. Naloxone was injected
intraperitoneally 10 min before dosing of 2a. (550C hot plate, mice, i.p., n=7~8; p<0.0001: ****;
p<0.001: ***)
Tramadol acted as a prodrug and its active metabolite is O-demethyl product M1[50, 55, 56]. As
tramadol does, though 2a binds to MOR tightly (Ki = 7.3±0.5 nM), the O-demethyl product of 2a
might be active to MOR. To verify it, its O-demethyl metabolite 12 was synthesized and its in
8

vitro activities were evaluated. As shown in Table 2, compound 12 showed high binding affinity
and selectivity for MOR (Ki MOR: 1.2±0.14 nM) and its activity for MOR was ten-fold potent than
that of M1. In addition, compound 12 showed seven-fold potent in binding activity for MOR than
compound 2a (Table 1). Also, compound 12 showed high potency for MOR and moderate
potency for DOR and KOR, rendering it to be a selective MOR agonist (Table 3). These in vitro
data gave us hint that 2a may elicits its in vivo antinociception via both 2a and 12.
Table 2 Binding affinities of compound 12 for three opioid receptors
Inhibition at 1 µM (%) or Ki (nM) a
Compounds
DOR/MOR
KOR/MOR
MOR b
6.0±0.4%
13.0±0.5
1.2±0.14
DOR c
KOR d
Tramadol
M1
0%
0%
--
--
--
--
19.0±2.3%
103.60±0.90
20.2±0.1%
71.38±4.03
12
86.3
59.5
a. Values are expressed as the mean ± SEM for three independent experiments performed in triplicate. b.
Displacement of [3H] DAMGO from the CHO cell membrane expressing the µ opioid receptor. c. Displacement of
[3H] DPDPE from the CHO cell membrane expressing the δ opioid receptor. D. Displacement of [3H] U69593
from the CHO cell membrane expressing the κ opioid receptor.
Table 3 Functional activity in stimulating [35S] GTPγS binding of compound 12
[S35] GTPγS assay
Compounds
MOR
EC50 (nM) a
DOR
EC50 (nM)
KOR
EC50 (nM)
Emax (%)
225.7%
205.0±2.05
ND
Emax (%)
ND
Emax (%)
ND
M1
DAMGO
DPDPE
U50488
12
244.7 nM
8.06±0.75
ND
ND b
ND
ND
ND
ND
ND
ND
1.43±0.15
ND
231.3±7.05
ND
ND
ND
ND
2.77±0.08 199.5±6.30
110.4±5.3 196.6±3.8
2.3±0.12
240.0±0.8
112.2±7.2
254.1±5.3
35
a. Potency and efficacy in stimulating [ S] GTPγS binding to CHO cell membranes stably expressing opioid
receptors. Data are expressed as the mean ± SEM of independent experiments performed in triplicate. b. Not
determined due to low stimulation.
Binding modes and selectivity of 2a on MOR
As shown in Table 1, compound 2a showed high binding affinity and selectivity for MOR
over DOR and KOR (Ki MOR: 7.3±0.5 nM; Ki DOR: 849.4±96.6 nM; Ki KOR: 49.1±6.9 nM). We next
investigated the intrinsic reasons of its high binding and selectivity. Engagement of H6.52 and D3.32
has been reported to be involved in the activation process of MOR. As shown in Figure 3A, in
consistent with morphine and its derivative, where the phenol hydroxyl engaged in hydrogen
bonding with H6.52 via two water molecules, oxygen of methoxy group interacted with H6.52
.
Protonated nitrogen of dimethylamine participated in salt bridge interaction with D3.32. As
suggested in structure activity relationship exploration, NH in urea group also engaged in the
interaction with D3.32 via hydrogen bonding, highlighting its importance for its high binding
affinity. Carbonyl oxygen in urea group hydrogen bonded with S57. Phenyl attaching to nitrogen
extended into the hydrophobic pocket flanked by Val3.28, Gln3.09, Thr3.04, Phe3.08 and Trp3.18
(Figure 10A). All these forces contributed to its high binding for MOR. To further explain the
selectivity of 2a to MOR over KOR and DOR, the sequences alignment in binding site of MOR
with DOR and KOR were conducted. As shown in Figure 10E, three residues in positions 3.11,
9

7.15 and 7.35 among three opioid receptors are different and thus makes the shapes and
electrostatic potential of active sites different (Figure 10C and 10D), leading to different
pharmacological profile of 2a in three opioid receptor subtypes. It underlies the high selectivity of
2a for MOR over KOR and DOR.
Figure 10 Binding modes of compound 2a with opioid receptors. (A: Dimensional diagram of 2a
with MOR. B: Electrostatic potential map of 2a with MOR. 2a was shown in sticks, green. C:
Electrostatic potential map of 2a with DOR. D: Electrostatic potential map of 2a with KOR. E:
The sequences alignment in active binding site of MOR with KOR and DOR.)
Conclusion
In
summary,
using
Tramadol
as
a
lead
compound,
a
series
of
3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyphenyl)-N-phenylpiperidine-1-carboxamide
derivatives were designed, synthesized and their activities were evaluated in vitro and in vivo.
Furthermore, the structure activity relationship was extensively explored. In vitro and in vivo
screening afforded highly selective MOR ligand 2a (Ki MOR: 7.3±0.5 nM; Ki DOR: 849.4±96.6 nM;
0
Ki KOR: 49.1±6.9 nM) as potent analgesic in 55 C hot plate model in mice and ED50 was 3.1
mg/kg. Its antinociception was antagonized by nonselective antagonist naloxone. Water bridge,
salt bridge, hydrogen bond and hydrophobic interaction with MOR contributed to compound 2a’s
high affinity for MOR. After alignment of sequences at binding sites of three opioid receptors,
steric hindrance of 2a with DOR and KOR explained its high selectivity for MOR. Due to its
potent in vivo analgesic effect, as well as ease of synthesis in comparison to naturally-derived
morphinans, compound 2a could serve as a novel scaffold for the development of novel opioid
ligands that might combat morphine-like side effects.
Experimental section
General aspects
10

All chemicals and solvents were supplied by Tansoole or Adamas or other suppliers and were
1
1
used without further purification. HNMR and CNMR spectra were recorded on Varian-400
instrument, Bruker-600 MHz instrument, respectively. Proton coupling patterns were described as
singlet, doublet, triplet, quartet, multiple, and broad. Mass spectra were generated with electric
spray ionization (ESI) produced by an analytical Agilent 1200 liquid chromatography-mass
spectrometer tandem instrument. High-resolution mass spectrometry (HRMS) spectra were
recorded with an AB 5600+ Q TOF instrument.
Chemical synthesis
1-(4-(3-methoxyphenyl)-4-((triethylsilyl)oxy)piperidin-3-yl)-N,N-dimethylmethanamine (4)
To a 250 ml three neck flask equipped with nitrogen gas balloon, constant pressure dropping
funnel and thermometer was charged with 3 (10 g, 33.22 mmol, 1 eq.), imidazole (20.35 g, 298.98
mmol, 9 eq.), DCM (100 ml). The resulting clear solution was cooled with ice water bath and
triethylchlorosilane (35.06 g, 232.6 mmol, 7 eq.) was added under 10oC. After the addition, the
coolant was removed and the mixture was stirred overnight. Reaction mixture was added slowly to
sodium bicarbonate solution (100 ml) and stirred for 10 min. Then the aqueous phase was
extracted with DCM three times (75+25+25 ml). The combined organic layers were washed with
water (20 ml), dried over anhydrous magnesium sulfate, filtered and evaporated under reduced
pressure to give 36.7 g yellow oil, which was subjected to Al2O3 fast column chromatography to
yield 10.2 g pale yellow oil and used without further purification. LC-MS: ESI+ [M + H]+ 379.1.
N-(3-chlorophenyl)-3-((dimethylamino)methyl)-4-(3-methoxyphenyl)-4-((triethylsilyl)oxy)pip
eridine-1-carboxamide (6)
To a 50 ml three neck flask equipped with nitrogen gas balloon, constant pressure dropping funnel
and thermometer was added with 4 (0.6 g, 1.59 mmol, 1 eq.), DCM (6 ml), TEA (0.193 g, 1.91
mmol, 1.2 eq.). The mixture was cooled to -50C and a solution of 1-chloro-3-isocyanatobenzene (5,
0.244 g, 1.59 mmol, 1 eq.) in DCM (2 ml) was added dropwise under 00C. The reaction mixture
was stirred for 3 h. After the completion of reaction, water (25 ml) and DCM (10 ml) were added.
The organic layer was separated by funnel. Then the aqueous phase was extracted with DCM (15
ml). The organic layers were combined, washed with water (10 ml) and evaporated under reduced
pressure to give 1.1 g pale yellow oil, which underwent fast column chromatography to afford 0.8
g pale yellow oil and used without further purification.
N-(3-chlorophenyl)-3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyphenyl)piperidine-1-
carboxamide hydrochloride (2d)
To a 50 ml flask was added 6 (0.75 g, 1.41 mmol, 1 eq.), THF (8 ml) and TBAF-3H2O (0.67 g,
2.114 mmol, 1.5 eq.). The mixture was stirred at room temperature overnight. The reaction was
monitored by TLC (DCM: MeOH = 15:1, v/v). After the reaction, the THF was removed by
evaporation under reduced pressure and NaHCO3 solution (30 ml) was added. The aqueous phase
was extracted with DCM (15+15 ml). The combined organic layers were washed with water (20
ml), dried over MgSO4, filtered and evaporated under reduced pressure to give 0.8 g residue,
which was purified by column chromatography to give 0.66 g off-white solid. The resulting solid
was recrystallized in IPA: EA: PE (0.5 ml: 0.5 ml: 2.5 ml) to obtain 0.47 g off-white solid. Then
0.35 g off-white solid, DCM (3.5 ml) and Methanol (0.5 ml) were added to 50 ml flask. To the
clear solution was added HCl/dioxane (0.25 ml, 1.008 mmol, 1.2 eq.) and then MTBE (5 ml) was
added dropwise. After stirring for 4 hours, 0.31 g off-white solid was collected by filtration (yield
1
64%). H NMR (400 MHz, CD3OD), δ 7.64~7.65 (m, 1H), 7.37~7.39 (m, 1H), 7.33 (t, J = 8 Hz,
11

1H), 7.22~7.26 (t, 1H), 7.4~7.15 (m, 1H), 7.09 (d, J = 4 Hz, 1H), 7.01 (dd, J = 8 Hz, J = 4 Hz,
1H), 6.86 (dd, J = 8 Hz, J = 4 Hz, 1H), 4.43~4.68 (m, 1H), 4.08~4.12 (m, 1H), 3.81 (s, 3H),
3.38~3.45 (m, 1H), 3.24~3.34 (m, 1H), 3.01~3.06 (m, 1H), 2.72~2.75 (m, 4H), 2.57 (s, 3H),
2.38~2.44 (m, 1H), 2.17~2.25 (m, 1H), 1.71~1.76 (m, 1H). 13C NMR (150 MHz, CD3OD) δ
159.50, 155.24, 146.62, 140.56, 133.13, 128.86, 128.76, 121.67, 119.48, 117.71, 116.39, 111.47,
110.50, 72.41, 56.62, 53.68, 42.90, 40.53, 39.93, 38.49. LC-MS-ESI+: [M + H]+ 418.2. HRMS
(ESI), calcd for C22H28ClN3O3 [M + H]+, 418.1892; found, 418.1888.
General procedure for the synthesis of 8
Take the synthesis of phenyl naphthalen-2-ylcarbamate (8m) as example
To a 100 ml three neck flask equipped with thermometer was added naphthalen-2-amine (2 g,
13.97 mmol, 1 eq.), THF (20 ml), NaHCO3 (1.41 g, 14.67 mmol, 1.2 eq.) and cooled to 00C. Then
a solution of phenyl chloroformate (1.84 g, 14.67 mmol, 1.05 eq.) in THF (10 ml) was added
dropwise under 50C. The reaction was monitored by TLC. After completion of the reaction, THF
was removed by evaporation under reduced pressure. The aqueous phase was extracted with ethyl
acetate (50+25 ml). The combined organic layers were washed with brine (20 ml), dried over
anhydrous magnesium sulfate, filtered and evaporated under reduced pressure to give 3.5 g brown
solid, which was subjected to crystallization in EA:PE (1:2 / v:v, 45 ml) to give 3 g off-white solid
(yield 81.5%). 1H NMR (400 MHz, CDCl3), δ 8.07 (s, 1H), 7.76~7.86 (m, 3H), 7.39~7.50 (m, 5H),
7.22~7.29 (m, 3H), 7.13 (m, 1H). LC-MS-ESI+: [M + H]+ 264.1, [M + Na]+ 286.1.
General procedure for the synthesis of 2 and 12
Take
the
synthesis
of
3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyphenyl)-N-(naphthalen-2-yl)piperidine-1-car
boxamide hydrochloride (2m) as example.
To a 100 ml flask was added 3 (0.6 g, 2 mmol,1 eq.), DCM (10 ml), TEA (0.71 g, 7 mmol, 3.5 eq.)
and the resulting mixture was stirred till the completion of reaction. Water (30 ml) was added and
extracted with DCM (10+10 ml). Organic layers were combined, washed with water (10 ml), dried
over anhydrous magnesium sulfate, filtered and evaporated under reduced pressure to give 0.72 g
brown oil. The free base was purified by silica gel column chromatography to obtain 0.45 g foam
(yield 51%). To a 100 ml flask was added the free base (.4 g, 0.923 mmol, 1 eq.), DCM (3 ml),
MeOH (1 ml) and to the resulting clear solution was added HCl/dioxane (0.35 ml, 1.384 mmol,
1.5 eq.). Then MTBE (20 ml) was added dropwise. After stirring for 3 hours, the solid was filtered,
1
dried under reduced pressure to 0.38 g off-white soli (87.6%). H NMR (400 MHz, CD3OD), δ
7.83 (s, 1H), 7.59~7.65 (m, 3H), 7.47~7.51 (m, 1H), 7.26~7.30 (m, 1H), 7.17~7.23 (m, 2H),
6.94~7.02 (m, 1H), 6.73 (d, J = 8 Hz, 1H), 4.36 (d, J = 12 Hz, 1H), 4.03 (d, J = 12 Hz, 1H), 3.68
(s, 3H), 3.29~3.36 (m, 1H), 3.06~3.21 (m, 1H), 2.92 (t, J = 12 Hz, 1H), 2.51~2.63 (m, 7H), 2.30
(m, 1H), 2.08~2.16 (m, 1H), 1.63 (d, J = 8 Hz, 1H). 13C NMR (150 MHz, CD3OD) δ 159.51,
155.80, 146.69, 136.52, 133.35, 129.64, 128.87, 127.15, 126.50, 126.19, 125.22, 123.51, 120.64,
116.41, 116.02, 111.47, 110.53, 72.46, 56.66, 53.69, 43.08, 40.60, 39.93, 38.52. LC-MS-ESI+: [M
+ H]+ 434.3. LC-MS-ESI-: [M + H]- 432.4, [M + Cl-]- 468.3. HRMS (ESI), calcd for C26H31N3O3
[M + H]+, 434.2438; found, 434.2437.
3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyphenyl)-N-phenylpiperidine-1-carboxa
mide hydrochloride (2a)
Phenyl phenylcarbamate (8a)
12

Off-white solid, yield: 95.2%. 1H NMR (400 MHz, DMSO-d6), δ 10.26 (s, 1H), 7.52 (d, J = 8 Hz,
2H), 7.43 (t, J = 8 Hz, 2H), 7.33 (t, J = 8 Hz, 2H), 7.22~7.28 (m, 3H), 7.05 (d, J = 8 Hz, 1H).
3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyphenyl)-N-phenylpiperidine-1-carboxamide
hydrochloride (2a)
Off-white solid, yield: 81.4%. 1H NMR (400 MHz, CD3OD), δ 7.44~7.47 (m, 2H), 7.25~7.35 (m,
3H), 7.01~7.15 (m, 2H), 6.68~6.88 (m, 1H), 4.43~4.48 (m, 1H), 4.09~4.14 (m, 1H), 3.81 (s, 3H),
3.37~3.45 (m, 1H), 3.20~3.30 (m, 1H), 3.00~3.06 (m, 1H), 2.65~2.75 (m, 7H), 2.37~2.44 (m, 1H),
2.18~2.26 (m, 1H), 1.72~1.76 (m, 1H). 13C NMR (150 MHz, CD3OD) δ 159.49, 155.76, 146.69,
138.84, 128.85, 127.56, 122.20, 120.20, 116.39, 111.46, 110.51, 72.45, 56.66, 53.69, 43.00, 40.57,
39.86, 38.49. LC-MS-ESI+: [M + H]+ 384.3. HRMS (ESI), calcd for C22H29N3O3 [M + H]+,
384.2282; found, 384.2282.
N-benzyl-3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyphenyl)piperidine-1-carboxam
ide hydrochloride (2b)
Phenyl benzylcarbamate (8b)
Off-white solid, yield: 92%. 1H NMR (400 MHz, CDCl3), δ 7.31~7.41 (m, 7H), 7.11~7.26 (m, 3H),
5.37 (s, 1H), 4.44~4.51 (m, 2H). LC-MS-ESI+: [M + H]+ 227.1, [M + Na]+ 250.1, [2M + Na]+
477.2.
N-benzyl-3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyphenyl)piperidine-1-carboxamide
hydrochloride (2b)
Off-white solid, yield: 83.5%. 1H NMR (400 MHz, CD3OD), δ 7.15~7.21 (m, 5H), 7.07~7.10 (m,
1H), 6.95 (d, J = 4 Hz, 1H), 6.87 (d, J = 8 Hz, 1H), 6.71 (d, J = 8 Hz, 1H), 4.26 (q, 2H), 4.13 (dd,
J1 = 12 Hz, J2 = 4 Hz, 1H), 3.78 (d, J = 8 Hz, 1H), 3.66 (s, 3H), 3.15~3.25 (m, 1H), 3.03 (t, J =
12 Hz, 1H), 2.82~2.89 (m, 1H), 2.48~2.56 (m, 7H), 2.12~2.15 (m, 1H), 1.99 (m, J1 = 16 Hz, J2 =
4 Hz, 1H), 1.52~1.56 (m, 1H). 13C NMR (150 MHz, CD3OD) δ 159.48, 157.95, 146.69, 139.56,
128.84, 127.37, 126.33, 125.90, 116.32, 111.40, 110.50, 72.43, 56.60, 53.69, 43.28, 42.94, 40.40,
39.70, 38.28. LC-MS-ESI+: [M + H]+ 398.3. HRMS (ESI), calcd for C23H31N3O3 [M + H]+,
398.2438; found, 398.2435.
3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyphenyl)-N-((S)-1-phenylethyl)piperidine
-1-carboxamide hydrochloride (2c)
Phenyl (S)-(1-phenylethyl)carbamate (8c)
1
Off-white solid, yield: 94%. H NMR (400 MHz, CDCl3), δ 7.26~7.41 (m, 7H), 6.97~7.21 (m,
3H), 5.34 (s, 1H), 4.89~4.97 (m, 1H), 1.55~1.60 (m, 3H). LC-MS-ESI+: [M + H]+ 242.2, [M +
Na]+ 264.2, [2M + Na]+ 505.3.
3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyphenyl)-N-((S)-1-phenylethyl)piperidine-1-c
arboxamide hydrochloride (2c)
1
Off-white solid, yield: 83.3%. H NMR (400 MHz, CD3OD), δ 7.22~7.24(m, 2H), 7.13~7.18(m,
3H), 7.05(s, 1H), 6.93(d, J = 12 Hz, 1H), 6.85(dd, J1 = 16 Hz, J2 = 4 Hz, 1H), 6.68~6.70(m, 1H),
4.75~4.81(m, 1H), 4.14(d, J = 16 Hz, 1H), 3.83(d, J = 16 Hz, 1H), 3.65(s, 3H), 3.14~3.17(m, 1H),
2.95~3.04(m, 1H), 3.83(t, J = 12 Hz, 1H), 2.50~2.58(m, 4H), 2.36~2.38(d, 3H), 1.94~2.10(m, 2H),
1.53(d, J = 16 Hz, 1H), 1.33~1.38(m, 3H). 13C NMR (150 MHz, CD3OD) δ 159.47, 157.31,
157.23, 146.74, 144.86, 144.78, 128.82, 127.39, 127.34, 125.75, 125.72, 125.12, 125.02, 116.35,
116.32, 111.41, 110.49, 110.46, 72.40, 56.62, 53.68, 49.96, 49.94, 43.04, 40.42, 40.37, 39.66,
39.58, 38.46, 38.23, 21.18, 21.05. LC-MS-ESI+: [M + H]+ 412.3. LC-MS-ESI-: [M + Cl-]- 446.2.
HRMS (ESI), calcd for C24H33N3O3 [M + H]+, 412.2595; found, 412.2591.
13

N-(3-chlorophenyl)-3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyphenyl)-N-methylpi
peridine-1-carboxamide hydrochloride (2e)
4-nitrophenyl (3-chlorophenyl)(methyl)carbamate (8e)
1
Off-white solid, yield: 99%. H NMR (400 MHz, DMSO-d6), δ 8.28 (d, J = 8 Hz, 2H), 7.67 (s,
1H), 7.44~7.51 (m, 4H), 7.37 (d, J = 8 Hz, 1H), 3.38 (s, 3H). LC-MS-ESI+: [M + H]+ 262.2, [M +
Na]+ 284.1.
N-(3-chlorophenyl)-3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyphenyl)-N-methylpiperid
ine-1-carboxamide hydrochloride (2e)
1
Off-white solid, yield: 79%. H NMR (400 MHz, CD3OD), δ 7.24~7.29 (m, 1H), 7.15~7.19 (m,
2H), 7.05 (d, J = 4 Hz, 1H), 6.92 (s, 1H), 6.87 (s, J = 8 Hz, 1H), 6.89~6.73 (m, 1H), 3.94 (d, J =
12 Hz, 1H), 3.66 (s, 3H), 3.37 (d, J = 12 Hz, 1H), 3.12 (s, 3H), 2.85~2.99 (m, 3H), 2.51~2.28 (m,
4H), 2.41 (s, 3H), 2.18 (s, 1H), 1.80~1.88 (m, 1H), 1.37 (d, J = 16 Hz, 1H), 1.15~1.19 (m, 1H).
13C NMR (150 MHz, CD3OD) δ 160.55, 159.49, 147.00, 146.50, 134.00, 130.02, 128.87, 123.83,
122.58, 120.89, 116.24, 111.43, 110.44, 72.21, 56.32, 53.70, 43.80, 43.69, 41.78, 40.35, 39.96,
38.12, 37.51. LC-MS-ESI+: [M + H]+ 432.2. HRMS (ESI), calcd for C23H30ClN3O3 [M + H]+,
432.2048; found, 432.2039.
N-(4-chlorophenyl)-3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyphenyl)piperidine-1-
carboxamide hydrochloride (2f)
Phenyl (4-chlorophenyl)carbamate (8f)
1
Off-white solid, yield: 54%. H NMR (400 MHz, CDCl3), δ 7.34~7.45 (m, 4H), 7.23~7.30 (m,
3H), 7.11~7.20 (m, 2H). LC-MS-ESI+: [M + H]+ 248.1, [M + Na]+ 270.1.
N-(4-chlorophenyl)-3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyphenyl)piperidine-1-carb
oxamide hydrochloride (2f)
Off-white solid, yield: 91%. 1H NMR (400 MHz, CD3OD), δ 7.33 (d, J = 4 Hz, 2H), 7.18 (t, J = 8
Hz, 1H), 7.11 (d, J = 4 Hz, 2H), 6.99 (s, 1H), 6.94 (d, J = 4 Hz, 1H), 6.72 (dd, J1 = 8 Hz, J2 = 4
Hz, 1H), 4.29 (dd, J1 = 12 Hz, J2 = 4 Hz, H), 3.93~3.97 (m, 1H), 3.66 (s, 3H), 3.27 (t, J = 12 Hz,
1H), 3.08~3.16 (m, 1H), 2.86~2.92 (m, 1H), 2.57~2.60 (m, 4H), 2.4 (s, 3H), 2.25~02.28 (m, 1H),
2.03~2.11 (m, 1H), 1.59 (d, J = 8 Hz, 1H). 13C NMR (150 MHz, CD3OD) δ 159.50, 155.39,
146.65, 137.85, 128.85, 127.45, 126.94, 121.26, 116.38, 111.46, 110.52, 72.42, 56.64, 53.68,
42.99, 40.57, 39.84, 38.47. LC-MS-ESI+: [M + H]+ 418.2. LC-MS-ESI-: [M + H]- 416.2. HRMS
(ESI), calcd for C22H28ClN3O3 [M + H]+, 419.1923; found, 419.1929.
N-(3,4-dichlorophenyl)-3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyphenyl)piperidin
e-1-carboxamide hydrochloride (2g)
Phenyl (3,4-dichlorophenyl)carbamate (8g)
1
Off-white solid, yield: 92.5%. H NMR (400 MHz, DMSO-d6), δ 10.57 (s, 1H), 7.80~7.82 (m,
1H), 7.29~7.62 (m, 1H), 7.42~7.49 (m, 3H), 7.24~7.30 (m, 2H). LC-MS-ESI+: [M+H]+ 282.0.
N-(3,4-dichlorophenyl)-3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyphenyl)piperidine-1-
carboxamide hydrochloride (2g)
1
Off-white solid, yield:82%. H NMR (400 MHz, CD3OD), δ 7.67~7.69 (m, 1H), 7.23~7.29 (m,
2H), 7.16~7.23 (m, 1H), 7.00 (s, 1H), 6.95 (d, J = 16 Hz, 1H), 6.72 (d, J = 16 Hz, 1H), 4.30 (d, J
= 12 Hz, 1H), 3.96 (d, J = 12 Hz, 1H), 3.67 (s, 3H), 3.28 (t, J = 12 Hz, 1H), 2.52~2.61 (m, 7H),
2.26 (m, 1H), 2.04~2.11 (m, 1H), 1.60 (d, J = 12 Hz, 1H). 13C NMR (150 MHz, CD3OD) δ 159.50,
154.89, 146.62, 139.27, 130.95, 129.13, 128.85, 124.44, 120.94, 119.07, 116.40, 111.49, 110.51,
72.41, 56.63, 53.69, 43.09, 40.59, 39.78, 38.45. LC-MS-ESI+: [M + H]+452.2,454.2. LC-MS-ESI-:
14

[M + H]-450.2, 452.1. HRMS (ESI), calcd for C22H27Cl2N3O3 [M + H]+, 452.1502; found,
452.1500.
3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyphenyl)-N-(3,4,5-trichlorophenyl)piperi
dine-1-carboxamide hydrochloride (2h)
Phenyl (3,4,5-trichlorophenyl)carbamate (8h)
1
Off-white solid, yield: 71.4%. H NMR (400 MHz, CDCl3), δ 7.96~8.00 (m, 2H), 7.42~7.43 (m,
2H), 7.25~7.30 (m, 2H), 7.11~7.15 (m, 1H), 7.02~7.06 (m, 2H), 6.87 (s, 1H). LC-MS-ESI+: [M +
Na]+ 338.0, 340.0.
3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyphenyl)-N-(3,4,5-trichlorophenyl)piperidine-
1-carboxamide hydrochloride (2h)
Off-white solid, yield: 81%. 1H NMR (400 MHz, CD3OD), δ 7.64 (s, 2H), 7.18 (d, J = 8 Hz, 1H),
6.99 (s, 1H), 6.94 (d, J = 4 Hz, 1H), 6.71 (d, J = 4 Hz, 1H), 3.3 (d, J = 4 Hz, 1H), 3.96 (dd, J1 =
16 Hz, J2 = 4 Hz, 1H), 3.68 (s, 3H), 3.26 (t, J = 12 Hz, 1H), 3.10~3.16 (m, 1H), 2.86~2.91 (m,
1H), 2.43~2.60 (m, 7H), 2.23~2.29 (m, 1H), 2.07 (m, J1 = 12 Hz, J2 = 4 Hz, 1H), 1.59 (d, J = 12
Hz, 1H). 13C NMR (150 MHz, CD3OD) δ 159.51, 154.52, 146.55, 139.39, 132.57, 128.86, 122.74,
119.14, 116.38, 111.48, 110.51, 72.38, 56.57, 53.68, 42.89, 40.52, 39.92, 38.46. LC-MS-ESI+: [M
+ H]+ 486.1, 488.1. LC-MS-ESI-: [M + H]- 484.1, 486.1. HRMS (ESI), calcd for C22H26Cl3N3O3
[M + H]+, 486.1113; found, 486.1100.
3-((dimethylamino)methyl)-N-(3-fluorophenyl)-4-hydroxy-4-(3-methoxyphenyl)piperidine-1-
carboxamide hydrochloride (2i)
Phenyl (3-fluorophenyl)carbamate (8i)
1
Off-white solid, yield: 85%. H NMR (400 MHz, CDCl3), δ 7.21~7.28 (m, 3H), 7.08~7.14 (m,
2H), 7.02~7.05 (m, 2H), 6.91~6.95 (m, 2H), 6.62~6.68 (m, 1H). LC-MS-ESI+: [M + H]+ 232.1,
[M + Na]+ 254.1.
3-((dimethylamino)methyl)-N-(3-fluorophenyl)-4-hydroxy-4-(3-methoxyphenyl)piperidine-1-carb
oxamide hydrochloride (2i)
Off-white solid, yield: 84%. 1H NMR (400 MHz, CD3OD), δ 7.265 (d, J = 8 Hz, 1H), 7.16~7.20
(m, 1H), 7.09~7.12 (m, 2H), 6.99 (s, 1H), 6.94 (d, J = 4 Hz, 1H), 6.72 (dd, J1 = 8 Hz, J2 = 4 Hz,
1H), 6.58~6.61 (m, 1H), 4.28~4.31 (m, 1H), 3.94~3.97 (m, 1H), 3.67 (s, 3H), 3.24~3.31 (m, 1H),
3.08~3.17 (m, 1H), 2.86~2.92 (m, 1H), 2.52~2.61 (m, 7H), 2.62 (m, 1H), 2.03~2.11 (m, 1H),
1.57~1.61 (m, 1H). 13C NMR (150 MHz, CD3OD) δ 162.31 (d, J = 241.5 Hz) 159.50, 155.25,
146.63, 141.01, 140.93, 128.85, 128.76, 116.38, 114.94, 111.47, 110.51, 108.14 (d, J = 22.5 Hz),
106.46 (d, J = 25.5 Hz), 72.42, 56.63, 53.68, 42.88, 40.54, 39.93, 38.49. LC-MS-ESI+: [M + H]+
402.2. LC-MS-ESI-: [M + H]- 400.3, [M + Cl-]- 436.2. HRMS (ESI), calcd for C22H28FN3O3 [M +
H]+, 402.2187; found, 402.2184.
N-(3,4-difluorophenyl)-3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyphenyl)piperidin
e-1-carboxamide hydrochloride (2j)
Phenyl (3,4-difluorophenyl)carbamate (8j)
1
Off-white solid, yield: 87%. H NMR (400 MHz, CDCl3), δ 7.32~7.38 (m, 1H), 7.25~7.30 (m,
2H), 7.11~7.17 (m, 1H), 7.03~7.09 (m, 2H), 6.96~7.00 (m, 1H), 6.86~6.93 (m, 2H). LC-MS-ESI+:
[M + H]+ 250.1, [M + Na]+ 272.1.
N-(3,4-difluorophenyl)-3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyphenyl)piperidine-1-
carboxamide hydrochloride (2j)
Off-white solid, yield: 81%. 1H NMR (400 MHz, CD3OD), δ 7.51~7.56 (m, 1H), 7.33 (t, J = 8 Hz,
15

1H), 7.17~7.19 (m, 1H), 7.09 (d, J = 8 Hz, 1H), 6.86~6.88 (m, 1H), 4.39~4.43 (m, 1H), 4.05~4.10
(m, 1H), 3.81 (s, 3H), 3.41~3.48 (m, 1H), 3.25 (t, J = 12 Hz, 1H), 3.02~3.07 (m, 1H), 2.66~2.75
(m, 7H), 2.39~2.42 (m, 1H), 2.18~2.26 (m, 1H), 1.74 (d, J = 8 Hz, 1H). 13C NMR (150 MHz,
CD3OD) δ 159.49, 155.25, 146.64, 136.06, 128.84, 116.36, 115.74 (d, J = 18 Hz), 115.56, 111.43,
110.51, 108.95 (d, J = 21 Hz), 72.42, 56.62, 53.67, 42.78, 40.57, 39.92, 38.50. LC-MS-ESI+: [M +
H]+ 420.3. LC-MS-ESI-: [M + H]- 418.3, [M+Cl-]- 454.3. HRMS (ESI), calcd for C22H27F2N3O3
[M + H]+, 420.2093; found, 420.2091.
N-(3-chloro-4-fluorophenyl)-3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyphenyl)pip
eridine-1-carboxamide hydrochloride (2k)
Phenyl (3-chloro-4-fluorophenyl)carbamate (8k)
1
Off-white solid, yield: 95.9%. H NMR (400 MHz, CDCl3), δ 7.45~7.46 (m, 1H), 7.23~7.30 (m,
2H), 7.06~7.16 (m, 2H), 7.00~7.08 (m, 2H), 6.91~6.99 (m, 1H), 6.82 (s, 1H).
N-(3-chloro-4-fluorophenyl)-3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyphenyl)piperidi
ne-1-carboxamide (free base of 2k)
Off-white solid, yield: 79%. 1H NMR (400 MHz, CD3OD), δ 7.629 (dd, J1 = 6.8 Hz, J2 = 2.8 Hz,
1H), 7.244~7.326 (m, 2H), 7.070~7.122 (t, 2H), 7.026 (d, J = 7.6 Hz, 1H), 6.80 (dd, J1 = 8.4 Hz,
J2 = 2.4 Hz, 1H), 4.307 (dd, J1 = 13.6 Hz, J2 = 4 Hz, 1H), 4.036 (d, J = 13.6 Hz, 1H), 3.755 (s,
3H), 3.356 (m, J1 = 13.2 Hz, J2 = 2.8 Hz, 1H), 3.168~3.230 (t, 1H), 2.803~2.860 (m, 1H),
2.289~2.514 (m, 7H), 2.258~2.309 (m, 1H), 2.137 (m, J1 = 13.6 Hz, J2 = 4.8 Hz, 1H),
1.644~1.693 (m, 1H). LC-MS-ESI+: [M + H]+ 436.2; LC-MS-ESI-: [M-H]- 434.2.
N-(3-chloro-4-fluorophenyl)-3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyphenyl)piperidi
ne-1-carboxamide hydrochloride (2k)
Off-white solid, yield: 76%. 1H NMR (400 MHz, CD3OD), δ 7.706 (dd, J1 = 6.8 Hz, J2 = 2.8 Hz,
1H), 7.314~7.401 (m, 2H), 7.132~7.184 (m, 2H), 7.097 (d, J = 7.6 Hz, 1H), 6.778 (dd, J1 = 8 Hz,
J2 = 2.4 Hz, 1H), 4.411~4.458 (m, 1H), 4.087 (d, J = 6.6 Hz, 1H), 3.819 (s, 3H), 3.403~3.472 (m,
1H), 3.230~3.351 (m, 1H), 3.018~3.075 (m, 1H), 2.720~2.758 (m, 4H), 2.585 (s, 3H),
2.378~2.432 (m, 1H), 2.183~2.262 (m, 1H), 1.719~1.767 (m, 1H). 13C NMR (150 MHz, CD3OD)
δ 159.50, 155.27, 146.61, 136.09, 128.85, 121.75, 119.75, 116.37, 115.23, 115.09, 111.45, 110.51,
72.41, 56.61, 53.68, 42.84, 40.53, 39.89, 38.47. LC-MS-ESI+: [M + H]+ 436.2. LC-MS-ESI-: [M +
H]- 434.2. HRMS (ESI), calcd for C22H27ClFN2O3 [M + H]+, 436.1798; found, 436.1794.
N-(3,5-bis(trifluoromethyl)phenyl)-3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyphen
yl)piperidine-1-carboxamide hydrochloride (2l)
Phenyl (3,5-bis(trifluoromethyl)phenyl)carbamate (8l)
1
Off-white solid, yield: 85%. H NMR (400 MHz, CDCl3), δ 7.96~8.00 (m, 2H), 7.61~7.63 (m,
1H), 7.40~7.46 (m, 2H), 7.26~7.34 (m, 2H), 7.18~7.22 (m, 2H).
N-(3,5-bis(trifluoromethyl)phenyl)-3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyphenyl)pi
peridine-1-carboxamide hydrochloride (2l)
1
Off-white solid, yield: 96.3%. H NMR (400 MHz, CD3OD), δ 8.22 (s, 2H), 7.55 (s, 1H),
7.31~7.35 (m, 1H), 7.10~7.12 (m, 1H), 6.87 (d, J = 8 Hz, 1H), 4.51~4.54 (m, 1H), 4.13~4.17 (m,
1H), 3.82 (s, 3H), 3.42~3.49 (m, 1H), 3.28~3.35 (m, 1H), 3.18~3.24 (m, 1H), 3.03~3.08 (m, 1H),
2.74~2.78 (m, 4H), 3.58 (s, 3H), 2.44 (m, 1H), 2.21~2.29 (m, 1H). 13C NMR (150 MHz, CD3OD)
δ 159.50, 154.52, 146.57, 141.57, 130.4 (q, J = 33 Hz), 128.86, 123.76, 121.96, 118.71, 116.42,
114.06, 111.53, 110.51, 72.41, 66.10, 56.62, 53.69, 45.88, 43.17, 40.59, 39.74, 38.41, 7.18.
LC-MS-ESI+: [M + H]+ 520.3. LC-MS-ESI-: [M + H]- 518.3; [M + Cl-]- 554.3. HRMS (ESI), calcd
16

for C24H27F6N3O3 [M + H]+, 520.2029; found, 520.2017.
3-(3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyphenyl)piperidine-1-carboxamido)thi
ophene-2-carboxylate hydrochloride (2n)
Methyl 3-((phenoxycarbonyl)amino)thiophene-2-carboxylate (8n)
1
Off-white solid, yield: 88%. H NMR (400 MHz, CDCl3), δ 9.90 (s, 1H), 7.89~7.91 (m, 1H),
7.47~7.52 (m, 1H), 7.39~7.46 (m, 2H), 7.24~7.31 (m, 1H), 7.19~7.23 (m, 2H), 2.93 (s, 3H).
3-(3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyphenyl)piperidine-1-carboxamido)thiophe
ne-2-carboxylate hydrochloride (2n)
Off-white solid, yield: 90.8%. 1H NMR (400 MHz, CD3OD), δ 7.77 (d, J = 8 Hz, 1H), 7.54 (d, J =
8 Hz, 1H), 7.18 (t, J = 8 Hz, 1H), 6.99 (s, 1H), 6.94 (d, J = 8 Hz, 1H), 7.72 (d, J = 8 Hz, 1H), 4.20
(d, J = 12 Hz, 1H), 3.85 (d, J = 12 Hz, 1H), 3.74 (s, 3H), 3.67 (s, 3H), 3.44 (t, J = 12 Hz, 1H),
3.15~3.17 (m, 2H), 2.93~2.99 (m, 1H), 2.59~2.62 (m, 4H), 2.53 (s, 3H), 2.31 (m, 1H), 2.09~2.16
(m, 1H), 1.65 (t, J = 12 Hz, 1H). 13C NMR (150 MHz, CD3OD) δ 164.77, 159.52, 152.98, 146.40,
145.84, 131.31, 128.90, 120.51, 116.35, 111.56, 110.47, 107.31, 72.30, 56.32, 53.69, 50.49, 42.43,
40.21, 38.26. LC-MS-ESI+: [M + H]+ 448.3. HRMS (ESI), calcd for C22H29N3O5S [M + H]+,
448.1901; found, 448.1896.
2-(3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyphenyl)piperidine-1-carboxamido)thi
ophene-3-carboxylate hydrochloride (2o)
Methyl 2-((phenoxycarbonyl)amino)thiophene-3-carboxylate (8o)
Off-white solid, yield: 79%. 1H NMR (400 MHz, CDCl3), δ 10.57 (s, 1H), 7.39~7.43 (m, 2H),
7.19~7.29 (m, 4H), 6.75 (d, J = 4 Hz, 1H), 3.91 (s, 3H).
2-(3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyphenyl)piperidine-1-carboxamido)thiophe
ne-3-carboxylate hydrochloride (2o)
Off-white solid, yield: 97.5%. 1H NMR (400 MHz, CD3OD), δ 7.19 (t, J = 8 Hz, 1H), 7.05 (d, J =
4 Hz, 1H), 7.00 (s, 1H), 6.95 (d, J = 8 Hz, 1H), 6.73 (d, J = 8 Hz, 1H), 6.64 (d, J = 8 Hz, 1H),
4.19 (d, J = 12 Hz, 1H), 3.80 (d, J = 12 Hz, 1H), 3.74 (s, 3H), 3.67 (s, 3H), 3.44~3.47 (m, 1H),
3.19~3.26 (m, 1H), 2.95~3.10 (m, 1H), 2.58~2.63 (m, 7H), 2.33 (m, 1H), 2.11~2.19 (m, 1H), 1.68
(d, J = 16 Hz, 1H). 13C NMR (150 MHz, CD3OD) δ 165.94, 159.53, 152.33, 151.24, 146.31,
128.91, 122.63, 116.36, 114.15, 111.64, 110.44, 110.04, 72.22, 66.10, 56.23, 53.70, 50.24, 42.43,
40.08, 38.13. LC-MS-ESI+: [M + H]+ 448.3. HRMS (ESI), calcd for C22H29N3O5S [M + H]+,
448.1901; found, 448.1896.
N-((3s,5s,7s)-adamantan-1-yl)-3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyphenyl)pi
peridine-1-carboxamide hydrochloride (2p)
Phenyl ((3s,5s,7s)-adamantan-1-yl)carbamate (8p)
1
Off-white solid, yield: 75%. H NMR (400 MHz, CDCl3), δ 7.18~7.25 (m, 2H), 6.96~7.15 (m,
3H), 4.75 (s, 1H), 1.97 (s, 3H), 1.86~1.89 (m, 6H), 1.53~1.58 (m, 6H). LC-MS-ESI+: [M + H]+
300.2, [M + Na]+ 322.2, [2M + Na]+ 621.4.
N-((3s,5s,7s)-adamantan-1-yl)-3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyphenyl)piperi
dine-1-carboxamide hydrochloride (2p)
1
Off-white solid, yield: 99%. H NMR (400 MHz, CD3OD), δ 7.17~7.22 (m, 1H), 6.90~6.97 (m,
2H), 6.71~6.75 (m, 1H), 4.06 (d, J = 12 Hz, 1H), 3.73 (d, J = 16 Hz, 1H), 3.67 (s, 3H), 3.07~3.12
(m, 1H), 2.95 (t, J = 12 Hz, 1H), 2.83~2.89 (m, 1H), 2.56~2.62 (m, 4H), 2.42 (s, 3H), 2.18 (m,
1H), 1.99~2.04 (m, 1H), 1.94 (s, 9H), 1.59 (s, 6H), 1.54 (d, J = 12 Hz, 1H), 1.17 (t, J = 8 Hz, 1H).
13C NMR (150 MHz, CD3OD) δ 159.66, 159.47, 157.08, 146.85, 145.24, 129.16, 128.83, 116.35,
17

116.13, 112.02, 111.41, 110.49, 110.40, 72.34, 70.60, 56.71, 56.01, 53.77, 53.67, 50.82, 50.65,
45.87, 42.95, 42.52, 41.15, 40.36, 39.70, 39.47, 39.33, 38.28, 37.99, 35.61, 35.09, 34.39, 29.11,
28.38, 7.19. LC-MS-ESI+: [M + H]+ 442.4. HRMS (ESI), calcd for C26H39N3O3 [M + H]+,
442.3064; found, 442.3060.
N-(1-((3r,5r,7r)-adamantan-1-yl)ethyl)-3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyp
henyl)piperidine-1-carboxamide hydrochloride (2q)
Phenyl (1-((3r,5r,7r)-adamantan-1-yl)ethyl)carbamate (8q)
Off-white solid, yield: 93.5%. 1H NMR (400 MHz, CDCl3), δ 7.333~7.394 (m, 2H), 7.128~7.200
(m, 3H), 4.883 (d, J = 5 Hz, 1H), 3.436~3.477 (m, 1H), 2.018 (s, 3H), 1.506~1.746 (m, 12H),
1.109~1.158 (m, 3H). LC-MS-ESI+: [M + H]+ 300.2, [M + Na]+ 322.2, [2M + Na]+ 621.4.
N-(1-((3r,5r,7r)-adamantan-1-yl)ethyl)-3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxypheny
l)piperidine-1-carboxamide(free base of 2q)
Off-white solid, yield: 24.3%. 1H NMR (400 MHz, CD3OD), δ 7.28~7.37 (m, 1H), 7.01~7.11 (m,
2H), 6.84~6.87 (m, 1H), 4.18 (d, J = 16 Hz,1H), 3.97 (d, J = 12 Hz, 1H), 3.80~3.85 (m, 3H),
3.60~3.65 (m, 1H), 3.32~3.39 (m, 1H), 3.09~3.17 (m, 1H), 2.84~2.87 (m, 1H), 2.50~2.59 (m, 7H),
2.24 (m, 1H), 2.09~2.17 (m, 1H), 1.96~2.01 (m, 3H), 1.59~1.76 (m, 14H), 1.08~1.13 (m, 3H).
LC-MS-ESI+: [M + H]+ 470.3.
N-(1-((3r,5r,7r)-adamantan-1-yl)ethyl)-3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxypheny
l)piperidine-1-carboxamide hydrochloride (2q)
Off-white solid, yield: 78%. 1H NMR (400 MHz, CD3OD), δ 7.17(t, J = 8 Hz, 1H), 6.94(d, J = 4
Hz, 1H), 6.86~6.91(m, 1H), 6.71(dd, J1 = 8 Hz, J2 = 4 Hz, 1H), 4.15(d, J = 12 Hz, 1H), 3.80 (t, J
= 16 Hz, 1H), 3.65 (s, 3H), 3.43~3.50 (m, 1H), 3.15~3.23 (m, 1H), 3.00 (t, J = 12 Hz, 1H),
2.82~2.91 (m, 1H), 2.53~2.59 (m, 4H), 2.39~2.43 (d, 3H), 2.15~2.17 (m, 1H), 2.01 (m, J1 = 16
Hz, J2 = 4 Hz, 1H), 1.84 (d, J = 4 Hz, 3H), 1.50~1.62 (m, 7H), 1.45 (s, 6H), 0.94~0.97 (m, 1H).
13C NMR (150 MHz, CD3OD) δ 159.49, 158.10, 157.97, 146.80, 128.84, 116.30, 116.25, 111.38,
110.52, 72.46, 72.41, 56.61, 56.55, 53.98, 53.86, 53.69, 43.74, 43.69, 43.35, 43.09, 40.44, 40.40,
40.31, 40.08, 39.86, 38.42, 38.38, 37.86, 37.81, 36.59, 36.25, 35.86, 35.74, 35.59, 29.10, 27.93,
27.46, 12.88, 12.67. LC-MS-ESI+: [M + H]+ 470.4. HRMS (ESI), calcd for C28H43N3O3 [M + H]+,
470.3377; found, 470.3371.
3-((dimethylamino)methyl)-4-hydroxy-N-((1r,3s,5R,7S)-3-hydroxyadamantan-1-yl)-4-(3-met
hoxyphenyl)piperidine-1-carboxamide hydrochloride (2r)
Phenyl ((1r,3s,5R,7S)-3-hydroxyadamantan-1-yl)carbamate (8r)
Off-white solid, yield: 87.8%. 1H NMR (400 MHz, CDCl3), δ 7.35 (t, J = 8 Hz, 2H), 7.19 (m, J =
8 Hz, 1H), 7.11 (d, J = 8 Hz, 2H), 4.99 (s, 1H), 2.30 (t, J = 4 Hz, 2H), 1.99 (s, 2H), 1.93 (s, 4H),
1.70 (s, 4H), 1.52~1.61 (m, 3H).
3-((dimethylamino)methyl)-4-hydroxy-N-((1r,3s,5R,7S)-3-hydroxyadamantan-1-yl)-4-(3-methoxy
phenyl)piperidine-1-carboxamide hydrochloride (2r)
Off-white solid, yield: 94%. 1H NMR (400 MHz, CD3OD), δ 7.19 (d, J = 8 Hz, 1H), 6.91~9.74 (m,
1H), 6.71~6.74 (m, 1H), 4.07 (d, J = 16 Hz, 1H), 3.75 (d, J = 12 Hz, 1H), 3.67 (s, 3H), 3.06~3.14
(m, 2H), 2.96 (t, J = 12 Hz, 1H), 2.83~2.88 (m, 1H), 2.56~2.62 (m, 4H), 2.42 (s, 3H), 2.18 (m,
1H), 2.10 (s, 2H), 1.96~2.04 (m, 1H), 1.79~1.89 (m, 6H), 1.41~1.55 (m, 8H), 1.16~1.19 (m, 1H).
13C NMR (150 MHz, CD3OD) δ 159.47, 157.04, 146.83, 128.83, 116.35, 111.40, 110.50, 72.34,
67.83, 56.72, 53.68, 53.15, 48.19, 45.88, 42.99, 42.86, 40.37, 39.91, 39.78, 38.28, 34.23, 30.17,
7.18. LC-MS-ESI+: [M + H]+ 458.4. LC-MS-ESI-: [M + Cl-]-492.4. HRMS (ESI), calcd for
18

C26H39N3O4 [M + H]+, 458.3013; found, 458.3004.
3-((dimethylamino)methyl)-4-hydroxy-4-(3-hydroxyphenyl)-N-phenylpiperidine-1-carboxam
ide (11)
Foam-like solid, yield: 60%. 1H NMR (400 MHz, CD3OD), δ 7.39 (d, J = 8 Hz, 2H), 7.27 (t, J =
8 Hz, 2H), 7.16 (t, J = 8 Hz, 2H), 7.02 (t, J = 8 Hz, 1H), 6.93 (d, J = 8 Hz, 2H), 6.66 (d, J = 8 Hz,
2H), 4.15 (dd, J1 = 12 Hz, J2 = 4 Hz, 2H), 3.22~3.34 (m, 3H), 2.27~2.33 (q, 1H), 2.02~2.08 (m,
9H), 1.93 (d, J = 16 Hz, 1H), 1.66 (d, J = 16 Hz, 1H).
3-((dimethylamino)methyl)-4-hydroxy-4-(3-hydroxyphenyl)-N-phenylpiperidine-1-carboxam
ide hydrochloride (12)
Off-white solid, yield: 94%. 1H NMR (400 MHz, CD3OD), δ 7.44 (d, J = 8 Hz, 2H), 7.28 (d, J = 8
Hz, 2H), 7.23 (d, J = 8 Hz, 1H), 7.04 (d, J = 8 Hz, 1H), 6.985 (d, J = 4 Hz, 2H), 6.71~6.73 (m,
1H), 4.38~4.42 (m, 1H), 4.07~4.11 (m, 1H), 3.44 (t, J = 12 Hz, 1H), 3.22~3.34 (m, 2H),
3.01~3.07 (q, 1H), 2.76~2.79 (m, 4H), 2.59 (s, 3H), 2.34 (m, 1H), 2.21 (m, J1 = 12 Hz, J2 = 4 Hz,
1H), 1.75 (d, J = 16 Hz, 1H). 13C NMR (150 MHz, CD3OD) δ 159.00, 157.93, 148.69, 140.88,
130.90, 129.82, 129.66, 124.32, 122.28, 117.29, 115.28, 113.47, 74.39, 58.70, 45.78, 44.92, 42.66,
42.13, 40.46. HRMS (ESI), calcd for C21H27N3O3 [M + Na]+, 392.1945; found, 392.1936.
Pharmacology
Receptor binding assay
Cell membrane preparation followed the reported procedure[57]. Ligand competition binding
experiments used [3H]DAMGO (50.1 Ci/mmol) for rat µ opioid receptor, [3H]DPDPE (52.7
Ci/mmol) for rat δ opioid receptor, [3H]U69593 (39.1 Ci/mmol) for human κ opioid receptor,
respectively. Binding assay was conducted in 50 mM Tris-HCl buffer solution (pH 7.4) at 37 °C
for 30 min in triplicate in a final volume of 0.5 ml with 30 µg of membrane protein. Nonspecific
binding employed U50488H, DAMGO, or DPDPE at a concentration of 10 µM. Radio ligand
bound membrane protein was separated by fast filtration via GF/B filter paper under reduce
pressure. Then the radioactivity of filter paper was dried under oven and was determined by liquid
scintillation counting. Binding data and Ki values were calculated using GraphPad Prism 6.0
program.
[35S]GTPγS Binding assay
[35S]GTPγS binding assay was performed according to procedure reported before[56]. Membranes
(15 µg/sample) were incubated with 0.1 nM [35S]GTPγS (1030 Ci/mmol, PerkinElmer) in a
binding buffer consisting of 50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 5 mM MgCl2, 100 mM NaCl,
and 40 µM GDP at 30 °C for 1 h in the presence of increasing concentrations of compounds.
Nonspecific binding was determined in the presence of nonradioactive GRPγS (10 µM).
Incubations were terminated by rapid filtration, and bound radioactivity was determined by liquid
scintillation counting. The percentage of stimulated [35S]GTPγS binding was calculated according
to formula: 100×(cpmsample−cpmnonspecific)/(cpmbasal−cpmnonspecific). EC50 and Emax were analyzed
and calculated with GraphPad prism 6.0 program.
Animals
Kunming strain female mice (18 ~ 22 g) were obtained from the Animal Holding Unit, School of
Pharmacy of Fudan University. Mice were housed in groups (6 mice/group) and raised in an
environment of 12 h light/dark cycle in a constant temperature (20 ~ 26 0C). Mice were free access
to food and sterilized water. All animal experiments were conducted under the guidelines of the
National Institutes of Health Guide for the Care and Use of Laboratory Animals using an
19

approved animal protocol.
Hot plate antinociception test.
The hot plate test conducted according to reported procedure[58]. Hot plate was maintained for 55.0
0C for mice. Before dosing a drug, animal was placed on the smooth hot plate surface to test its
latency threshold defined as time interval after animal was put on the heated surface to licking
back paws and a cutoff time of 30 seconds was maintained to prevent from mice injury. Mice with
latency threshold of 5~30 s were selected to further dosing. Latency was measured twice before
dosing and mean value was defined as basal latency. After dosing of drugs, latency threshold was
tested and a cutoff time of 60 seconds was maintained to prevent mice injury. Antinociception was
calculated by the following formula: maximum possible effect (MPE)%=100× (test latency–basal
latency)/ (cutoff time–basal latency). ED50 (dose of drug that produce 50% MPE) were determined
using a computer-assisted linear analysis of the dose-response curve. Antagonism effect was
conducted by dosing naloxone after 10 min before drug injection.
Molecular modeling
Homology modeling
Active homo sapiens MOR (sequence NO.: P35372) was built from murine active MOR
(PDB NO.: 5C1M, resolution: 2.1 Å) as template using Discovery Studio 3.5. Sequence identity
and sequence similarity of both receptors were 97.3%, 98.3%, respectively. Active homo sapiens
DOR (sequence NO.: P41143) was built from homo sapiens active KOR (PDB NO.: 6B73,
resolution: 3.1 Å) as template using Discovery Studio 3.5. Sequence identity and sequence
similarity of both receptors were 93.9%, 98.1%, respectively. Ramachandran plot of active homo
sapiens MOR and DOR were shown below. Homo sapiens KOR (PDB NO.: 6B73, resolution: 3.1
Å) was employed.
Ramachandran plot of Active homo sapiens MOR and DOR (A: MOR, optimum region 95.2%,
acceptance region 99.6%; B: DOR, optimum region 93.9%, acceptance region 98.1%））
Docking and plotting
Docking studies were performed in induce-fitting module of Schrödinger 3.5. Pymol 0.99 was
employed in plotting.
Acknowledgement
20

This work is supported by grants from National Natural Science Foundation of China (NO.
81773635) and Shanghai Science and Technology Development Funds (14431900500).
Conflict of interest
The authors declared no conflict of interest.
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24

Discovery of
3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyphenyl)-N-pheny
lpiperidine-1-carboxamide as Novel Potent Analgesic
Huoming Huang a, Wenli Wang a, Xuejun Xu b, Chen Zhu a, Yujun Wang b, Jinggen Liu b*, Wei Li
a*, Wei Fu a*
Affiliation:
a. Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai, 201203,
China.
b. Shanghai Institute of Materia Medica, Chinese Academy of Science, Shanghai, 201203, China.
Highlights
1. A series of
3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyphenyl)piperidine-1-carboxamide
derivatives were designed based on “Address-message concept”.
2. In vitro and in vivo evaluation afforded compound 2a as potent analgesic.
3. Compound 2a was potent in hot plate model (ED50 = 3.1 mg/kg).
4. Antinociception of compound 2a was blocked by naloxone.
5. Binding mode of compound 2a was proposed.

Discovery of
3-((dimethylamino)methyl)-4-hydroxy-4-(3-methoxyphenyl)-N-pheny
lpiperidine-1-carboxamide as Novel Potent Analgesic
Huoming Huang a, Wenli Wang a, Xuejun Xu b, Chen Zhu a, Yujun Wang b, Jinggen Liu b*, Wei Li
a*, Wei Fu a*
Affiliation:
a. Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai, 201203,
China.
b. Shanghai Institute of Materia Medica, Chinese Academy of Science, Shanghai, 201203, China.
Conflict of interest
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.