Synthesis and evaluation of novel biased μ-opioid-receptor (μOR) agonists

Article abstract of DOI:10.3390/molecules24020259

‘Biased’ ligands of G protein-coupled receptors (GPCRs) represent a type of promising analgesic with reduced on-target side effects. PZM21, a potent μ-opioid-receptor (μOR)-biased agonist with a new chemical scaffold compared to classic opioids, has been identified as a therapeutic lead molecule for treating pain. In the current study, novel PZM21 analogues were synthesized and evaluated for their in vitro and in vivo efficacy. Novel compound 7a and PZM21 demonstrated undetectable β-arrestin-2 recruitment, however, their analgesic effects need to be further confirmed. Compounds 7b, 7d, and 7g were stronger analgesics than PZM21 in both the mouse formalin injection assay and the writhing test. Compound 7d was the most potent analogue, requiring a dose that was 1/16th to 1/4th of that of PZM21 for its analgesic activity in the two assays, respectively. Therefore, compound 7d could serve as a lead to develop new biased μOR agonists for treating pain.

Full text of DOI:10.3390/molecules24020259

molecules
Article
Synthesis and Evaluation of Novel Biased
µ-Opioid-Receptor (µOR) Agonists
Mengjun Ma 1,†, Jialin Sun 1,†, Menghua Li 2, Zixing Yu 1, Jingchao Cheng 1, Bohua Zhong 1,3,
*
and Weiguo Shi 1,
*
1
State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of Pharmacology &
Toxicology, 27 Tai-Ping Road, Beijing 100850, China; mengjunma@126.com (M.M.); monkeysjl@163.com (J.S.);
yuyuzx1990@163.com (Z.Y.); chengjingchao3017@126.com (J.C.)
National Glycoengineering Research Center and Shandong Provincial Key Laboratory of Carbohydrate
Chemistry and Glycobiology, Shandong University, 27 Shanda Nan Lu, Jinan 250100, China;
lmh0920@126.com
TaizhouHuayuan Med Tech Company LTD, 1 Yaocheng Avenue, Taizhou 225312, China
Correspondence: bohuazhong@yahoo.com (B.Z.); shiweiguo@bmi.ac.cn (W.S.); Tel.: +86-10-669-316-39 (B.Z.);
+86-10-668-746-12 (W.S.)
2
3
*
†
These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 13 December 2018; Accepted: 10 January 2019; Published: 11 January 2019
Abstract: ‘Biased’ ligands of G protein-coupled receptors (GPCRs) represent a type of promising
analgesic with reduced on-target side effects. PZM21, a potent µ-opioid-receptor (µOR)-biased
agonist with a new chemical scaffold compared to classic opioids, has been identified as a therapeutic
lead molecule for treating pain. In the current study, novel PZM21 analogues were synthesized
and evaluated for their in vitro and in vivo efficacy. Novel compound 7a and PZM21 demonstrated
undetectable β-arrestin-2 recruitment, however, their analgesic effects need to be further confirmed.
Compounds 7b, 7d, and 7g were stronger analgesics than PZM21 in both the mouse formalin injection
assay and the writhing test. Compound 7d was the most potent analogue, requiring a dose that was
1/16th to 1/4th of that of PZM21 for its analgesic activity in the two assays, respectively. Therefore,
compound 7d could serve as a lead to develop new biased µOR agonists for treating pain.
Keywords: µOR-biased agonist; PZM21; Gi signaling pathway; β-arrestin-2 recruitment; structure–
activity relationship
1. Introduction
The discovery of safer and more effective analgesics without the drawbacks of classic opioids
is urgently needed. ‘Biased’ ligands of G protein-coupled receptors (GPCRs) represent a type of
promising molecule with a specific ability to cherry pick the beneficial rather than the deleterious
signaling pathways activated by the
Recent studies have suggested that some GPCR ligands exhibit an “unbalanced effect” when activating
signaling pathways. They can bind to specific receptor forms [ ] or selectively bind to different types
of G protein subunits or even -arrestin, thereby biasing the cytoplasmic signal to a certain pathway.
These ligands are named ‘biased agonists’ or ‘biased ligands’ [ ]. Previous research has shown that
opioid-induced analgesia results from OR signaling through the G protein Gi; while many side effects,
µ-opioid-receptor (µOR) so that on-target toxicity is reduced [1–3].
4,5
β
6–8
µ
including respiratory depression and constipation, may be transmitted through β-arrestin pathway
signaling, which occurs downstream of µOR activation [9–11].
PZM21 (Figure 1) is a potent biased
unrelated to known opioids. It was initially identified from over three million molecules by
computational docking against the OR structure; therefore, it is a promising therapeutic lead for pain
µOR agonist with a structurally distinct chemical scaffold
µ
Molecules 2019, 24, 259; doi:10.3390/molecules24020259
www.mdpi.com/journal/molecules

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relief [12,13]. Although the potency and biased action of PZM21 have been partially confirmed [13,14],
the detailed structure–activity relationship of this type of compound needs to be further investigated.
Figure 1. The chemical structure of PZM21.
Here, synthetic structure optimization of PZM21 was performed by introducing an aromatic
naphthalene scaffold to replace the benzene ring of PZM21, leaving the specific dimethyl amino and
urea pharmacophore groups unchanged. Additionally, the position of the thiophene ring substituent
and the chirality of the methyl group were investigated. A series of PZM21 analogues (Figure 2) were
synthesized and assessed for their in vitro and in vivo activities.
Figure 2. The design of novel PZM21 analogues.
2. Results and Discussion
2.1. Synthesis
As shown in Scheme 1, target compounds 7a
acids were converted to amino amides, which were then subjected to reductive amination with
formaldehyde. Next, the carbonyl group was reduced by borane to obtain intermediates 4a and 4b
The raw materials 5a were, respectively, reacted with 4-nitrophenyl chloroformate to obtain 6a
which were reacted with 4a or 4b to obtain final products 7a–g.
–g were synthesized in five steps. First, the amino
.
–d
–d,

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◦
Scheme 1. Synthesis of compounds 7a–g. Reagents and Conditions: (i) SOCl2, CH3OH, 0 C to rt,
12 h, NH4OH, 12 h; (ii) CH2O, H2/Pd-C, CH3OH, rt, 0.4 MPa, 5 h; (iii) BH3, THF, reflux, 15 h; (iv)
4-nitrophenyl chloroformate, triethylamine, THF, 0 ◦C to rt, 6 h; (v) triethylamine, CH3CN, 60 ◦C to
−
80 ◦C, 2 h; (vi) nitroethane, HCOOH/ethanolamine, 90 ◦C; (vii) LiAlH4, THF, reflux, 30 min; (viii)
di-p-anisoyl-D-tartaric acid, di-p-anisoyl-L-tartaric acid, acetonitrile, rt.
2.2. The Selective Gi-Biased µOR Agonist Activities and the β-Arrestin Recruitment Assay In Vitro
2.2.1. µOR Gi/o-Mediated cAMP Inhibition
The
OR by the Gi/o-mediated signaling pathway, we detected the changes of intracellular cAMP
content as the activity of adenosine cyclase can be inhibited by the combination of OR with its agonist,
which leads to a decrease in the intracellular cAMP concentration. The Gi/o signaling assay showed
potent OR agonist activity for new compounds 7b 7d 7e, and 7g, as well as PZM21 (Table 1). The
µOR agonist activities of 7a–g were tested in a Gi/o signaling assay. To measure the activation
of
µ
µ
µ
,
,
potencies of 7b (EC50 = 91.14) and 7e (EC50 = 82.43) are similar to that of PZM21 (EC50 = 52.41), while
7d and 7g are weaker than PZM21.
Table 1. EC50 and Max Response of the tested compounds in the Gi activation assay.
Substituted
Position of the
-CH2- in the A Ring
Substituted
Position of the
-CH2- in the B Ring
R and Configuration
of Chiral Carbon
Atom b
%Max Response
Compound
EC50(nM)
(100 µM)
0
7a
7b
7c
7d
7e
7f
7g
1
2
2
1
2
1
2
-
3
3
3
3
3
2
2
CH3 (S)
CH3 (S)
CH3 (R)
455.6
91.14
>100,000
203.4
82.43
1448
242.8
52.41
8.17
60
80
-
50
60
40
60
60
100
0
0
0
H
H
H
H
-
0
0
0
PZM21
DAMGO
-
-
-
-
2.2.2. β-Arrestin Recruitment Assay
To determine whether these novel compounds activate
recruitment assays. The results are shown in Table 2. PZM21 and 7a showed undetectable
µOR, we tested them in β-arrestin-2

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β
-arrestin-2 recruitment in the NanoBit assay. Unfortunately, 7b
,
7d
,
7e, and 7g showed potent
β-arrestin recruitment.
Table 2. EC50 and Max Response of the tested compounds in the β-arrestin recruitment assay.
Substituted
Position of the
-CH2- in the A Ring
Substituted
Position of the
-CH2- in the B Ring
R and Configuration
of Chiral Carbon
Atom b
%Max Response
Compound
EC50 (µM)
(100 µM)
0
0
0
0
0
7a
7b
7d
7e
7g
1
2
1
2
2
-
3
3
3
3
2
CH3 (S)
CH3 (S)
N.A.a
0.7239
1.621
1.128
2.162
N.A.a
1.237
0
50
70
25
12
0
H
H
H
-
PZM21
DAMGO
-
-
-
-
100
a
No measurable activity.
2.3. Analgesic Activities of the Novel Compounds in a Formalin Injection Nociception Assay
Analgesia to a formalin injection was carried out as described previously [13]. The tested
compounds and PZM21, at dosages of 20 mg/kg and 40 mg/kg, were injected subcutaneously,
respectively. As shown in Table 3, all tested compounds produced sustained analgesia in phase 2 of the
formalin injection assay (p < 0.001; compared with the vehicle group). The percentages of analgesia of
7b, 7d, 7e, and 7g were higher than that of PZM21 at both dosages. In particular, 7d showed the most
potent analgesic effect of 100% at both dosages, demonstrating that a lower dosage should be tested.
Table 3. In vivo activities of the synthesized compounds in the formalin injection assay.
Substituted
Position of the
-CH2- in the A Ring
Substituted
Position of the
-CH2- in the B Ring
R and Configuration
of Chiral Carbon
Atom b
%Analgesia
(20 mg/kg)
%Analgesia (40
mg/kg)
Compound
0
7a
7b
7c
7d
7e
7f
7g
PZM21
1
2
2
1
2
1
2
-
3
3
3
3
3
2
2
CH3 (S)
CH3 (S)
CH3 (R)
53.7 ± 17.8 **
82.4 ± 15.8 **
79.6 ± 17.2 **
100 **
84.1 ± 12.5 **
69.6 ± 21.7 **
89.8 ± 10.6 **
77.9 ± 14.6 **
76.9 ± 19.1 **
96.7 ± 3.4 **
53.5 ± 19.9 **
100 **
92.3 ± 12.9 **
86.7 ± 9.0 **
91.7 ± 7.4 **
92.8 ± 11.2 **
0
0
0
H
H
H
H
-
0
0
0
-
Values are the mean ± standard deviation (n = 8). ** p < 0.001 versus control.
Further investigation revealed that compound 7d exhibited 100% and 93.9% analgesia upon
subcutaneous injection at 5 mg/kg and 2.5 mg/kg, respectively (Figure 3). These effects were more
potent than that of PZM21, which showed 92.8% analgesia upon subcutaneous injection at 40 mg/kg.

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Figure 3. Activities of compound 7d in the formalin injection assay. Values are the mean
deviation (n = 8). **p < 0.001 versus control.
±
standard
2.4. Analgesic Activities of the Novel Compounds in the Writhing Test
The analgesic results of the novel compounds in the writhing test are shown in Table 4. Consistent
with their activities in the formalin injection assay, 7b 7d, and 7g displayed stronger analgesia in
,
the mouse writhing assay than PZM21. Additionally, compound 7a showed a higher percentage of
analgesia than PZM21. The low dose of 7d (2.5 mg/kg) yielded a higher (92.7%) analgesic response
than 10 mg/kg PZM21 (45.7%).
Table 4. In vivo activities of the synthesized compounds in the writhing test.
Substituted
Position of the
-CH2- in the A Ring
Substituted
Position of the
-CH2- in the B Ring
R and Configuration
of Chiral Carbon
Atom b
Start Time of
Writhing (s)
Compound
%Analgesia
0
7a
7b
7c
7d
7e
7f
7g
PZM21
1
2
2
1
2
1
2
-
3
3
3
3
3
2
2
CH3 (S)
CH3 (S)
CH3 (R)
307
525
261
891
320
317
334
254
60.6 ± 10.5 **
65.1 ± 13.4 **
27.1 ± 10.6 **
92.7 ± 9.7 **
42.4 ± 13.4 **
44.4 ± 8.6 **
50.3 ± 10.5 **
45.7 ± 15.0 **
0
0
0
H
H
H
H
-
0
0
0
-
Values are the mean
dose of 2.5 mg/kg; the others were administered at a dose of 10 mg/kg.
±
standard deviation (n = 8). ** p < 0.001 versus control. Compound 7d was administered at a
2.5. Discussion
The OR-biased agonist PZM21 represents an attractive new chemical scaffold, which is unrelated
µ
to known opioids, with reduced on-target toxicity. Therefore, its structure-activity relationship needs
to be investigated in detail. The tertiary amine and urea groups are unique to these novel compounds
and are not present in other opioid agonists like morphine and fentanyl. Thus, we kept these core
groups unchanged and introduced a naphthalene group to replace the phenolic hydroxyl of PZM21 in
order to exploit the effects of steric bulk and hydrophobicity. Moreover, the requirement of specific
stereoisomers for efficacy and the chemical position of the thiophene ring were also studied.

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First, we tested the in vitro activities of the designed compounds and PZM21. Similar to PZM21,
the novel compounds strongly activated Gi/o. Unfortunately, most of the novel compounds also
showed high levels of β-arrestin recruitment; only 7a and PZM21 showed undetectable β-arrestin-2
recruitment in the assay. Next, we measured the analgesic responses of the novel compounds using
different animal models for pain. Intriguingly, we observed no analgesic effect for PZM21 or the
novel compounds in a mouse hotplate assay, even at a high dose of 100 mg/kg. These findings were
inconsistent with those reported for PZM21 [
Therefore, we assessed the analgesic effects of the novel compounds using the formalin injection
nociception assay and the writhing test. Consistent with their OR agonist activity, PZM21 and
7]. They also showed no analgesia in the tail flick assay.
µ
the novel compounds displayed dose-dependent analgesia in these two assays. The most potent
compound 7d exhibited 93.9% and 92.7% analgesia, respectively, at the low dose of 2.5 mg/kg. These
results were higher than those of PZM21 at doses of 40 mg/kg and 10 mg/kg in the formalin injection
and writhing assays, respectively.
The structure–activity relationship studies showed that replacing the phenolic hydroxyl group
of PZM21 with a larger naphthalene group could maintain the
µOR agonist activity but decrease
the selectivity of -arrestin-2 recruitment. Additionally, the position of both the naphthalene and
β
thiophene rings affected the potency and efficacy. The (R) stereoisomer of 7c had a potency that
was reduced by 1000-fold, while the most potent compound7d was not chiral, suggesting a specific
stereochemical requirement for both potency and efficacy.
Our studies support minimal β-arrestin-2 signaling in vitro by PZM21; however, its analgesic
effect still needs to be further confirmed. PZM21 did not show an analgesic effect in the mouse hotplate
assay, a traditional pain model; therefore, the painkilling activity of PZM21 remains ambiguous.
Although novel compound 7b displayed a higher efficacy in vivo than PZM21, respiratory depression
induced by this compound still needs to be further investigated.
3. Experimental Section
3.1. General Information
Reagents were obtained from Beijing Chemical Works (Beijing, China), Acros Organics
(Geel, Belgium), and Alfa-Aesar (Ward Hill, MA, USA) and were used without further
purification. Proton nuclear magnetic resonance (1H-NMR, 400 MHz) spectra were measured with a
JNM-ECA-400spectrometer (JEOL Co. Ltd., Tokyo, Japan). Mass spectra were measured by using an
API-150 mass spectrometer (ABI Inc., Foster City, CA, USA) with an 1100-HPLC electrospray ionization
source (Agilent Technologies Inc., Palo Alto, CA, USA).
3.2. Chemistry
3.2.1. Synthesis 2a and 2b
Thionyl chloride (11.05 g, 92.92 mmol) was slowly added to a suspension of 1a or 1b (10.00 g,
46.46 mmol) in methanol (100 mL) in an ice bath. The mixture was allowed to warm up to room
temperature and stirred for 12 h. The solvent was removed under reduced pressure. The obtained
yellow solid was dissolved in ammonia solution (120 mL) and methanol (30 mL), and stirred for 12 h.
The mixture was concentrated under reduced pressure. The obtained yellow solid was purified by
silica gel column chromatography using dichloromethane-methanol (12:1) as the eluent, yielding 2a
(8.83 g, 83.82%) or 2b (8.10 g, 76.89%) as a white powdery solid.
3.2.2. Synthesis of 3a and 3b
Aqueous formaldehyde (22.00 mL, 294.00 mmol, 37%) was added to a suspension of 2a or 2b
(5.30 g, 24.75 mmol) in methanol (20 mL), followed by the addition of 10% palladium carbon (2.00 g).
The hydrogenation reaction was carried out for 5 h at a pressure of 0.4 MPa and room temperature.

Molecules 2019, 24, 259
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After the reaction was completed, palladium carbon was removed by suction filtration, and the filtrate
was concentrated under reduced pressure. The crude residue was purified by silica gel column
chromatography using dichloromethane-methanol (1:20) as the eluent, yielding 3a (4.30 g, 71.78%) or
3b (4.53 g, 75.62%) as a white solid.
3.2.3. Synthesis of 4a and 4b
A 1M solution of borane-tetrahydrofurane complex (107.00 mL, 107.00 mmol) was slowly added
to solution of 4a or 4b (4.30 g, 17.76 mmol) in anhydrous THF (50 mL). The mixture was refluxed
for 8 h under a nitrogen atmosphere. The reaction was quenched with anhydrous methanol (50 mL,
dropwise). The solvent was removed under reduced pressure. The obtained crude residue was purified
by silica gel column chromatography using dichloromethane-methanol (50:1–20:1) as the eluent. The
obtained oily residue was resuspended in methanol (20 mL) with the addition of excess HCl/diethyl
ether and concentrated under reduced pressure, yielding 4a (3.10 g, 58.16%) or 4b (3.33 g, 62.48%) as a
white solid.
3.2.4. Synthesis of 6a–c
Compounds 5a–c (6.09 g, 34.40 mmol) and triethylamine (9.60 mL, 68.80 mmol) in anhydrous
THF (150 mL) were mixed under a nitrogen atmosphere in an ice bath. A solution of 4-nitrophenyl
chloroformate (6.93 g, 34.40 mmol) in anhydrous THF (60 mL) was added dropwise. The reaction
mixture was allowed to warm up to room temperature and stirred for 2 h. The slurry was diluted
with dichloromethane (100 mL) and filtered. The filtrate was washed and concentrated under
reduced pressure. The crude residue was purified by silica gel column chromatography using
petroleumether-dichloromethane (4:1–1:1) as the eluent, yielding 6a–c as a white foam (6.04–6.44 g,
57.36–61.16%).
3.2.5. Synthesis of 9
Ethanolamine (17.00 mL, 280.00 mmol) was added dropwise to formic acid (15.00 mL, 400.00
mmol) in an ice bath. Nitroethane (26.00 mL, 360.00 mmol) and (3.9 mL, 45 mmol) were added to the
8
mixture. The reaction mixture was stirred for 7 h. The resulting solution was poured into cold water
(500 mL), and the slurry was filtered. The precipitated product was recrystallized from ethanol/water
(4:1 v/v, 50 mL), yielding 9 as a yellow crystalline solid (9.80 g, 64.47%).
3.2.6. Synthesis of 10
Compound 9 (9.00 g, 53.25 mmol) in anhydrous THF (120 mL) was added dropwise to a slurry
of LiAlH4 (10.02 g, 266.25 mmol) in anhydrous THF (250 mL) in an ice bath. The mixture was stirred
for 12 h under a nitrogen atmosphere. The reaction was quenched by the dropwise addition of water
(10 mL) and 15% NaOH (10 mL). After filtration, the filter cake was washed with ethyl acetate. The
residue was dissolved in diethyl ether (100 mL), cooled to 0 ◦C, and the product was precipitated by
excess HCl/diethyl ether and filtered off. The precipitate was recrystallized from acetonitrile (200 mL),
yielding 10 as a gray crystalline solid. 49 g, 79.01%).
3.2.7. Synthesis of 5a and 5b
Racemic 10 (3.15 g, 17.70 mmol) was dissolved in water (100 mL), and the solution was basified
by aqueous ammonia and extracted with dichloromethane (3
×
80 mL). The liquid was added to a
hot solution of di-p-anisoyl-D-tartaric acid (7.50 g, 17.70 mmol) in acetonitrile (60 mL). The slurry
was diluted with water (30 mL). The obtained crystalline precipitate was dissolved in 1N NaOH and
extracted with dichloromethane. The organic layers were dried over anhydrous Na2SO4. The liquid
residue was dissolved in diethyl ether (70 mL), cooled to 0 ◦C, and the product was precipitated by

Molecules 2019, 24, 259
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excess HCl/diethyl ether, yielding a white solid (2.13 g, 67.62%, ee = 34.0%). The above step was
repeated three times, yielding 5a as a white solid (0.8 g, 26.99%, ee = 98.4%).
Compound 5b was obtained by the same procedures as those for 5a except that di-p-anisoyl-
L-tartaric acid was used as a chiral resolving agent, yielding 5b as a white solid (0.7 g, 25.23%,
ee = 98.0%).
3.2.8. Synthesis of 7a–g
To a suspension of 4a or 4b (0.60 g, 1.99 mmol) in acetonitrile (15 mL), triethylamine (0.90 mL,
5.97 mmol) was added. Next, a solution of 6a–c (0.67 g, 2.19 mmol) in acetonitrile (15 mL) was added.
The mixture was stirred for 2 h, filtered, and concentrated under reduced pressure. The residue was
dissolved in ethyl acetate (20 mL) and filtered. The crude residue was purified by silica gel column
chromatography using dichloromethane-methanol (100:1–30:1) as the eluent, yielding the desired
product 7a–g as a yellow oil (0.47–0.52 g, 59.49–65.82%).
1-((S)-2-(Dimethylamino)-3-(naphthalen-1-yl)propyl)-3-((S)-1-(thiophen-3-yl)propan-2-yl)urea
1
(
7a): H-NMR δH (400MHz, DMSO-d6, ppm): 0.91–0.93 (d, J = 6.44Hz, 3H, CH-CH3), 2.38 (s, 6H,
N-CH3), 2.50–2.54 (m, 2H, Ar-CH2thiophene), 2.61–2.87 (m, 2H, Ar-CH2), 2.90–3.05 (m, 2H, CH2-NH),
3.34–3.49 (m, 1H, CH-N), 3.63–3.74 (m, 1H, NH-CH), 5.65–5.66 (d, J = 4.76Hz, 1H, NH-CO), 6.01–6.03
(d, J = 7.84Hz, 1H, NH-CO), 6.88–6.90 (dd, J = 1.12, 5.04Hz, 1H, HAr), 7.07–7.08 (d, J = 1.96Hz, 1H,
HAr), 7.36–7.46 (m, 3H, HAr), 7.50–7.61 (m, 2H, HAr), 7.78–7.80 (d, J = 8.12Hz, 1H, HAr), 7.92–7.94 (m,
1H, HAr), 8.06–8.08 (d, J = 8.16Hz, 1H, HAr). MS-HR (ESI) m/z: 396.2104 [M + H].
1-((S)-2-(Dimethylamino)-3-(naphthalen-2-yl)propyl)-3-((S)-1-(thiophen-3-yl)propan-2-yl)urea
1
(
7b): H-NMR δH (400MHz, DMSO-d6, ppm): 0.91–0.92 (d, J = 6.44Hz, 3H, CH-CH3), 2.30 (s, 6H,
N-CH3), 2.54–2.67 (m, 2H, Ar-CH2thiophene), 2.75–2.89 (m, 2H, Ar-CH2), 2.98–3.10 (m, 2H, CH2-NH),
3.38–3.41 (m, 1H, CH-N), 3.67–3.73 (m, 1H, NH-CH), 5.63–5.65 (d, J = 5.04Hz, 1H, NH-CO), 6.00–6.02
(d, J = 7.84Hz, 1H, NH-CO), 6.90–6.91 (dd, J = 1.12, 4.76Hz, 1H, HAr), 7.09 (d, J = 1.68Hz, 1H, HAr),
7.36–7.40 (m, 2H, HAr), 7.50–7.61 (m, 2H, HAr), 7.70 (s, 1H, HAr), 7.83–7.88 (m, 3H, HAr). MS-HR (ESI)
m/z: 396.2104 [M + H].
1-((S)-2-(Dimethylamino)-3-(naphthalen-2-yl)propyl)-3-((R)-1-(thiophen-3-yl)propan-2-yl)urea
1
(
7c): H-NMR δH (400MHz, DMSO-d6, ppm): 0.90–0.91 (d, J = 6.44Hz, 3H, CH-CH3), 2.30 (s, 6H,
N-CH3), 2.46–2.69 (m, 2H, Ar-CH2thiophene), 2.74–2.92 (m, 2H, Ar-CH2), 2.98–3.13 (m, 2H, CH2-NH),
3.37–3.47 (m, 1H, CH-N), 3.68–3.76 (m, 1H, NH-CH), 5.65–5.66 (d, J = 4.76Hz, 1H, NH-CO), 6.02–6.04
(d, J = 7.88Hz, 1H, NH-CO), 6.93–6.95 (dd, J = 1.12, 4.76Hz, 1H, HAr), 7.12 (d, J = 1.96Hz, 1H, HAr),
7.36–7.43 (m, 2H, HAr), 7.45–7.51 (m, 2H, HAr), 7.70 (s, 1H, HAr), 7.83–7.88 (m, 3H, HAr). MS-HR (ESI)
m/z: 396.2105 [M + H].
(S)-1-(2-(Dimethylamino)-3-(naphthalen-1-yl)propyl)-3-(2-(thiophen-3-yl)ethyl)urea
(7d):
1H-NMR δH (400MHz, DMSO-d6, ppm): 2.38 (s, 6H, N-CH3), 2.58–2.62 (t, J = 7.14Hz, 2H,
Ar-CH2thiophene), 2.78–2.83 (m, 2H, Ar-CH2), 2.94–3.05 (m, 2H, CH2-NH), 3.10–3.15 (q, 2H, CH2-NH),
3.30–3.40 (m, 1H, CH-N), 5.75 (s, 1H, NH-CO br), 6.09–6.12 (t, J = 5.20Hz, 1H, NH-CO), 6.94–6.95 (d,
J = 4.8Hz, 1H, HAr), 7.13 (s, 1H, HAr), 7.37–7.46 (m, 3H, HAr), 7.50–7.58 (m, 2H, HAr), 7.78–7.80 (d,
J = 8.12Hz, 1H, HAr), 7.92–7.94 (d, J = 7.28Hz, 1H, HAr), 8.06–8.08 (d, J = 8.12Hz, 1H, HAr). MS-HR
(ESI) m/z: 382.1948 [M + H].
(S)-1-(2-(Dimethylamino)-3-(naphthalen-2-yl)propyl)-3-(2-(thiophen-3-yl)ethyl)urea
(7e):
1H-NMR δH (400MHz, DMSO-d6, ppm): 2.30 (s, 6H, N-CH3), 2.60–2.67 (t, J = 7.14Hz, 2H,
Ar-CH2thiophene), 2.77–2.91 (m, 2H, Ar-CH2), 2.98–3.17 (m, 4H, CH2-NH), 3.30–3.40 (m, 1H, CH-N),
5.73–5.75 (d, J = 4.76Hz, 1H, NH-CO), 6.10–6.13 (t, J = 5.46Hz, 1H, NH-CO), 6.95–6.96 (d, J = 5.04Hz,
1H, HAr), 7.14 (s, 1H, HAr), 7.36–7.50 (m, 4H, HAr), 7.70 (s, 1H, HAr), 7.83–7.88 (m, 3H, HAr). MS-HR
(ESI) m/z: 382.1949 [M + H].
(S)-1-(2-(Dimethylamino)-3-(naphthalen-1-yl)propyl)-3-(2-(thiophen-2-yl)ethyl)urea
(7f):
1H-NMR δH (400MHz, DMSO-d6, ppm): 2.38 (s, 6H, N-CH3), 2.78–2.87 (m, 4H, Ar-CH2), 2.91–3.05
(m, 2H, CH2-NH), 3.12–3.17 (q, 2H, CH2-NH), 3.32–3.36 (m, 1H, CH-N), 5.77–5.79 (m, 1H, NH-CO),

Molecules 2019, 24, 259
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6.19–6.22 (t, J = 5.74Hz, 1H, NH-CO), 6.81–6.82 (q, 1H, HAr), 6.90–6.93 (q, 1H, HAr), 7.29–7.30 (dd,
J = 1.12, 5.04Hz, 1H, HAr), 7.37–7.39 (q, 1H, HAr), 7.42–7.46 (t, J = 7.42Hz, 1H, HAr), 7.50–7.58 (m,
2H, HAr), 7.78–7.80 (d, J = 8.12Hz, 1H,HAr), 7.92–7.94 (q, 1H,HAr), 8.06–8.08 (d, J = 8.44Hz, 1H, HAr).
MS-HR (ESI) m/z: 382.1948 [M + H].
(S)-1-(2-(Dimethylamino)-3-(naphthalen-2-yl)propyl)-3-(2-(thiophen-2-yl)ethyl)urea
(7g):
1H-NMR δH (400MHz, DMSO-d6, ppm): 2.30 (s, 6H, N-CH3), 2.74–2.92 (m, 4H, Ar-CH2), 2.97–3.10 (m,
2H, CH2-NH), 3.13–3.20 (m, 2H, CH2-NH), 3.35–3.47 (m, 1H, CH-N), 5.77–5.78 (d, J = 4.48Hz, 1H,
NH-CO), 6.19–6.22 (t, J = 5.74Hz, 1H, NH-CO), 6.82–6.83 (q, 1H, HAr), 6.91–6.94 (dd, J = 3.36, 5.04Hz,
1H, HAr), 7.30–7.31 (dd, J = 1.12, 5.04Hz, 1H, HAr), 7.36–7.39 (q, 1H, HAr), 7.43–7.51 (m, 2H, HAr), 7.70
(s, 1H, HAr), 7.81–7.88 (m, 3H, HAr). MS-HR (ESI) m/z: 382.1948 [M + H].
3.3. µOR Gi/o-Mediated cAMP Inhibition
Human embryonic kidney (HEK) 293 cells in the logarithmic growth phase were washed
with phosphate-buffered saline and dissociated with trypsin. Then, the cells were centrifuged,
resuspended in serum-free culture media (containing 0.1% bovine serum albumin and 0.5 mM
3-isobutyl-1-methylxanthine), plated at a density of 2000 cells per 5-
plates, and incubated at 37 C with 5% CO2. Drug dilutions (the final drug concentrations were 100
µ
Laliquotin 384-well cell culture
M,
M) were prepared in saline or DMSO in triplicate.
L aliquot of each drug solution was added to each well, and the plates were incubated for 30 min
in the dark at room temperature. Then, to increase the level of intracellular cAMP, 5 L of forskolin
(final concentration of 10 M) was added to each well, and the plate was incubated for 30 min in the
◦
µ
10
µM, 1
µM, 100 nM, 10 nM, 1 nM, 100 pM, and 0
µ
A 5
µ
µ
µ
dark at room temperature. At the end of the reaction, the cAMP assay substrate was added to each
well, and the cells were again incubated in the dark at room temperature for 60 min. The luminescence
intensity was quantified using an EnVision 2104 microplate reader (PerkinElmer Inc., Waltham, CA,
USA). Data were normalized to [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO)-induced cAMP
inhibition and analyzed with GraphPad Prism 6.0 software (Graphpad Software Inc., San Diego,
CA, USA).
3.4. β-Arrestin Recruitment Assay
β-Arrestin recruitment was measured by a NanoBit assay, which is a double subunit system
based on NanoLuc luciferase that can be used to detect intracellular protein interactions. The plasmids
expressing MOR or ARRB1/2 and fused with the subunit LgBiT (17.6 kDa) or SmBiT(11 amino acids),
respectively, were provided by the National Center for Drug Screening (Shanghai, China). HEK293
cells were cotransfected with the plasmids stated above by electric shock for at least 24 h. Next, the
transfected cells were plated into 96-well, white, opaque bottom, cell culture plates in culture media at
a density of 2
40 L of phenol-red-free medium and 10 Ml of substrate were added to each well, and the plate was
incubated for 10 min in the dark. Finally, 50 L of drug solution was added to three wells, and the plate
×
105 cells in 50 L of medium and incubated at 37 ◦C with 5% CO2 overnight. Then,
µ
µ
µ
was incubated for 10 min in the dark. The plates were read using an EnVision 2104 micro plate reader
(PerkinElmer Inc., Waltham, CA, USA). The data were normalized to DAMGO-induced stimulation
and analyzed using nonlinear regression in GraphPad Prism 6.0 software (Graphpad Software Inc.,
San Diego, CA, USA).
3.5. Formalin Injection Assay
Analgesia to formalin injection was carried out as described previously [15]. Male adult ICR
mice, weighing 25
were used. The mice had free access to water and food, and were kept at 21
humidity of 60 10% and illumination for 12 h/day (8 a.m. to 8 p.m.). The mice were acclimated for
±
2 g, from Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China)
±
2 ◦C with a relative
±
2 days and randomly divided into nine groups. Each group of mice was first habituated for 20 min
in our homemade polyvinyl chloride cage without bedding, food, or water. Then, vehicle (0.1 mL of

Molecules 2019, 24, 259
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saline/10 g of body weight, n = 8), PZM21 (40 mg/kg, n = 8), or 7a
–g
(40 mg/kg, n = 8) was injected
subcutaneously. After 1 h, 20 µL of 2.7% formalin in saline was injected under the skin of the dorsal
surface of the right hind paw, and the mice were returned to their cage. Nociception was estimated
by measuring the cumulative amount of time spent by the animals licking the formalin-injected
paw during the late phase (20–30 min). The analgesic effect of the compounds was presented as the
percentage of the inhibition ratio, according to the following formula:
Average Time(Vehicle) − Average Time(Drug)
%Analgesia =
× 100%
(1)
Average Time(Vehicle)
3.6. Analgesic Activitiesin the Writhing Test
The writhing test methods described by Souza et al. [16] were followed, with a few modifications.
Male adult ICR mice, weighing 25 2 g, from Vital River Laboratory Animal Technology Co. Ltd.
(Beijing, China) were used. The mice had free access to water and food, and were kept at 21
2 ◦C
with a relative humidity of 60 10% and illumination for 12 h/day (8 a.m. to 8 p.m.). The mice were
acclimated for 2 days and randomly divided into nine groups. Then vehicle (0.1 mL of saline/10 g of
body weight, n = 8), PZM21 (10 mg/kg, n = 8), 7a (10 mg/kg, n = 8), 7e (10 mg/kg, n = 8), or 7d
±
±
±
–c
–g
(2.5 mg/kg, n = 8) was injected subcutaneously. After 0.5 h, 1% acetic acid solution (10 mL/kg) was
injected intraperitoneally, and the mice were returned to their cage. The number of writhes exhibited
by each mouse was counted for 20 min. The analgesic effect of the compounds was presented as the
percentage of the inhibition ratio, according to the following formula:
Average Number(Vehicle) − Average Number(Drug)
%Analgesia =
× 100%
(2)
Average Number(Vehicle)
4. Conclusions
In conclusion, novel PZM21 analogues were synthesized and evaluated for their specificity and
efficacy as OR-biased agonists. Novel compound 7a and PZM21 were confirmed to show undetectable
-arrestin-2 recruitment. However, several novel compounds exhibited significant analgesic activity in
µ
β
the formalin injection assay and in the writhing test. Compound 7d was more potent than PZM21 and
exhibited the most potent analgesic activity of all of the novel compounds in the formalin injection
and writhing assays, respectively. Therefore, compound 7d should be used as lead to develop new
biased µOR agonists for treating pain.
Author Contributions: W.S. and B.Z. conceived and designed the experiments; M.M. and J.S. performed the
experiments; M.M. and M.L. analyzed the data; Z.Y. and J.C. contributed reagents/materials/analysis tools; M.M.
and W.S. wrote the paper.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the
decision to publish the results. The authors agreed to submit the manuscript to the journal unanimously.
References
1.
Raehal, K.M.; Walker, J.K.; Bohn, L.M. Morphine side effects in beta-arrestin 2 knockout mice. J. Pharmacol.
Exp. Ther. 2005, 314, 1195–1201. [CrossRef] [PubMed]
2.
Dewire, S.M.; Yamashita, D.S.; Rominger, D.H.; Liu, G.; Cowan, C.L.; Graczyk, T.M.; Chen, X.T.; Pitis, P.M.;
Gotchev, D.; Yuan, C. AG protein-biased ligand at the µ-opioid receptor is potently analgesic with reduced
gastrointestinal and respiratory dysfunction compared with morphine. J. Pharmacol. Exp. Ther. 2013, 344,
708–717. [CrossRef] [PubMed]

Molecules 2019, 24, 259
11 of 11
3.
Soergel, D.G.; Subach, R.A.; Burnham, N.; Lark, M.W.; James, I.E.; Sadler, B.M.; Skobieranda, F.; Violin, J.D.;
Webster, L.R. Biased agonism of the -opioid receptor by TRV130 increases analgesia and reduces on-target
µ
adverse effects versus morphine: A randomized, double-blind, placebo-controlled, crossover study in
healthy volunteers. Pain 2014, 155, 1829–1835. [CrossRef] [PubMed]
4.
5.
6.
7.
Kingwell, K. Pioneering biased ligand offers efficacy with reduced on-target toxicity. Nat. Rev. Drug Discovery
2015, 14, 809. [CrossRef] [PubMed]
Vaidehi, N.; Kenakin, T. The role of conformational ensembles of seven transmembrane receptors in
functional selectivity. Curr. Opin. Pharmacol. 2010, 10, 775–781. [CrossRef] [PubMed]
Violin, J.D.; Crombie, A.L.; Soergel, D.G.; Lark, M.W. Biased ligands at G-protein-coupled receptors: Promise
and progress. Trends Pharmacol. Sci. 2014, 35, 308–316. [CrossRef] [PubMed]
Manglik, A.; Lin, H.; Aryal, D.K.; Mccorvy, J.D.; Dengler, D.; Corder, G.; Levit, A.; Kling, R.C.; Bernat, V.;
Hübner, H. Structure-based discovery of opioid analgesics with reduced side effects. Nature 2016, 537, 185.
[CrossRef] [PubMed]
8.
9.
Violin, J.D.; Lefkowitz, R.J. Beta-arrestin-biased ligands at seven-transmembrane receptors. Trends Pharmacol.
Sci. 2007, 28, 416–422. [CrossRef] [PubMed]
Bohn, L.M.; Lefkowitz, R.J.; Gainetdinov, R.R.; Peppel, K.; Caron, M.G.; Lin, F.T. Enhanced Morphine
Analgesia in Mice Lacking β-Arrestin 2. Science 1999, 286, 2495–2498. [CrossRef] [PubMed]
10. Dohlman, H.G. Thematic Minireview Series: New Directions in G Protein-coupled Receptor Pharmacology.
J. Biol. Chem. 2015, 290, 19469–19470. [CrossRef] [PubMed]
11. Bohn, L.M.; Gainetdinov, R.R.; Lin, F.; Lefkowitz, R.J.; Caron, M.G.
µ-Opioid receptor desensitization by
β-arrestin-2 determines morphine tolerance but not dependence. Nature 2000, 408, 720. [CrossRef] [PubMed]
12. Schneider, J.P.; Kirsh, K.L. Defining clinical issues around tolerance, hyperalgesia, and addiction: A
quantitative and qualitative outcome study of long-term opioid dosing in a chronic pain practice. J. Opioid.
Manag. 2010, 6, 385–395. [CrossRef] [PubMed]
13. Irwin, J.J.; Sterling, T.; Mysinger, M.M.; Bolstad, E.S.; Coleman, R.G. ZINC: A Free Tool to Discover Chemistry
for Biology. J. Chem. Inf. Model. 2012, 52, 1757–1768. [CrossRef] [PubMed]
14. Kingwell, K. Analgesia: Screening for cleaner pain relief. Nat. Rev. Drug Discovery 2016, 15, 677. [CrossRef]
[PubMed]
15. Veneroni, O.; Maj, R.; Calabresi, M.; Faravelli, L.; Fariello, R.G.; Salvati, P. Anti-allodynic effect of NW-1029,
a novel Na+ channel blocker, in experimental animal models of inflammatory and neuropathic pain. Pain
2003, 102, 17–25. [CrossRef]
16. De Souza, E.T.; de Lira, D.P.; de Queiroz, A.C.; da Silva, D.J.; de Aquino, A.B.; Mella, E.A.; Lorenzo, V.P.;
de Miranda, G.E.; de Araujo-Junior, J.X.; Chaves, M.C.; et al. The antinociceptive and anti-inflammatory
activities of caulerpin, a bisindole alkaloid isolated from seaweeds of the genus Caulerpa. Mar. Drugs 2009
7, 689–704. [CrossRef] [PubMed]
,
Sample Availability: Samples of the compounds 7a–g are available from the authors.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).