Synthesis and Biological Evaluation of Fentanyl Analogues Modified at Phenyl Groups with Alkyls

Article abstract of DOI:10.1021/acschemneuro.8b00363

A series of fentanyl analogues modified at the phenyl group of the phenethyl with alkyl and/or hydroxyl and alkoxy, and the phenyl group in the anilido moiety replaced with benzyl or substituted benzyl, were synthesized. The in vitro opioid receptor functional activity of these compounds was evaluated by assessment of their ability to modulate forskolin-stimulated cAMP accumulation and by their ability to induce β-arrestin2 recruitment. Compound 12 is a potent μ-opioid (MOP) receptor agonist, a potent κ-opioid (KOP) receptor antagonist with weak β-arrestin2 recruitment activity. Compounds 10 and 11 are potent MOP receptor agonists with weak δ-opioid (DOP) receptor antagonist activity and moderate KOP receptor antagonist activity as well as weak β-arrestin2 recruitment activity at the MOP receptor. These compounds are promising leads for discovery of potent opioid analgesics with reduced side effects relative to clinically available strong opioid analgesics.

Full text of DOI:10.1021/acschemneuro.8b00363

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Letter
Synthesis and biological evaluation of fentanyl
analogues modified at phenyl groups with alkyls
Yajuan Qin, Luofan Ni, Jiawei Shi, Zhiying Zhu, Saijian Shi, Ai Leen Lam,
Julia Magiera, Sunderajhan Sekar, Andy Kuo, Maree T. Smith, and Tingyou Li
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00363 • Publication Date (Web): 04 Sep 2018
Downloaded from http://pubs.acs.org on September 4, 2018
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Synthesis and biological evaluation of fentanyl analogues modified at
phenyl groups with alkyls
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Yajuan Qin†, Luofan Ni†, Jiawei Shi†, Zhiying Zhu†, Saijian Shi†, Aiꢀleen Lam‡, Julia
Magiera‡, Sunderajhan Sekar‡, Andy Kuo‡, Maree T. Smith*,‡, Tingyou Li*,†,§
† School of Pharmacy, Nanjing Medical University, Nanjing 211166, China
‡ School of Biomedical Sciences, Faculty of Medicine, The University of Queensland,
Brisbane, QLD 4072, Australia
§Key Laboratory of Targeted Intervention of Cardiovascular Disease, Collaborative
Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing
Medical University, Nanjing 211166, China
*Corresponding authors:
T
Li, Tel& Fax: +86ꢀ25ꢀ8686ꢀ8467; eꢀmail:
l_tingyou@njmu.edu.cn; MT Smith, Tel: +61ꢀ7ꢀ3365ꢀ2554; Fax: +61ꢀ3346ꢀ7391;
eꢀmail: maree.smith@uq.edu.au
Abbreviations: cAMP, Cyclic Adenosine monophosphate; MOP, ꢁ opioid; KOP, κ
opioid; DOP, δ opioid; HEK, human embryonic kidney; Dmt, 2,6ꢀdimetylꢀLꢀtyrosine;
Tmp, 2,3,6ꢀtrimethylꢀLꢀphenylalanine; Dmp, 2,6ꢀdimethylꢀLꢀphenylalanine；DAMGO,
[
D
ꢀAla2, NꢀMePhe4, Glyꢀol] enkephalin; DPDPE, [
D
ꢀPen2, ꢀPen5] enkephalin; NTB,
D
naltriben; NX, naloxone; Tic, 1,2,3,4ꢀtetrahydroisoquinolineꢀ3ꢀcarboxyl acid.
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Abstract:
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A series of fentanyl analogues modified at the phenyl group of the phenethyl
with alkyl and/or hydroxyl and alkoxy, and the phenyl group in the anilido moiety
replaced with benzyl or substituted benzyl, were synthesized. The in vitro opioid
receptor functional activity of these compounds was evaluated by assessment of their
ability to modulate forskolinꢀstimulated cAMP accumulation and by their ability to
induce βꢀarrestin2 recruitment. Compound 12 is a potent ꢀꢀopioid (MOP) receptor
agonist, a potent κꢀopioid (KOP) receptor antagonist with weak βꢀarrestin2
recruitment activity. Compounds 10 and 11 are potent MOP receptor agonists with
weak δꢀopioid (DOP) receptor antagonist activity and moderate KOP receptor
antagonist activity as well as weak βꢀarrestin2 recruitment activity at the MOP
receptor. These compounds are promising leads for discovery of potent opioid
analgesics with reduced side effects relative to clinically available strong opioid
analgesics.
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Keywords: fentanyl, analgesics, opioid, βꢀarrestin2
Introduction
Fentanyl is one of the most commonly used strong opioid analgesics for the
relief of acute and chronic nociceptive pain. After its discovery, scientists have
synthesized a huge number of fentanyl analogues,1, 2 which led to discovery of
sufentanil, alfentanil, lofentanil and remifentanil. However, fentanyl formulated as
transdermal patches is the most prescribed analgesic of this group for the relief of
chronic nociceptive pain.1
Fentanyl has a rapid onset of analgesia and it shows more potent activity than
morphine. Longꢀterm administration of fentanyl, like morphine, for relief of
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cancerꢀrelated pain, produces a series of undesired side effects with analgesic
tolerance and constipation being particularly troublesome.1, 2 Therefore, the synthesis
of new fentanyl analogs with high potency but reduced opioidꢀrelated side effects is
desirable.3ꢀ8
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Recent advances in understanding opioid receptor signal transduction have
suggested new possibilities regarding opioid receptor activation to evoke analgesia,
while reducing or eliminating unwanted side effects.9ꢀ11 Pharmacological studies on
opioid receptors have found that δꢀopioid (DOP) receptors modulate the function of
ꢀꢀopioid (MOP) receptors.12,13 Administration of a MOP agonist in conjunction with a
DOP antagonist14 or a MOP agonist mixed with DOP antagonism exhibit analgesia
but with reduced tolerance and physical dependence liabilities.15ꢀ19 Within this context,
fentanyl was used to synthesize bivalent ligands comprising fentanyl conjugated to a
DΟΡ receptor antagonist, DmtꢀTic. However, this bivalent ligand showed a
substantial loss in MOP receptor functional activity.20
Opioid receptors are Gꢀproteinꢀcoupled receptors. Apart from activating
Gi/oꢀGꢀproteins to inhibit downstream cAMP formation, they also activate the
βꢀarrestin2ꢀcoupled downstream signaling system. Recent research by others suggests
that MOP receptor agonists biased to signal primarily via Gi/o proteins with minimal
to no βꢀarrestin2 signaling, induce analgesic activity but with less constipation and
respiratory depression relative to morphine.9ꢀ11
TRV130, a novel MOP receptor G proteinꢀbiased ligand, is reportedly a potent
analgesic. It induces reduced gastrointestinal dysfunction and respiratory depression
than morphine at equiꢀanalgesic doses both in rats and mice.21ꢀ22 Recently, TRV130
completed Phase 3 clinical trials for the relief of moderate to severe acute pain, and
the New Drug Application has been filed with FDA.23ꢀ26 Very recently, several novel
G proteinꢀbiased ligands, such as PZM2127 and the SRꢀseries compounds,28 have been
reported as potent MOPꢀreceptor mediated analgesic agents that show no respiratory
depression and no hyperlocomotive response in mice at analgesic doses.27,28 These
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advances in the opioid pharmacology field have provided renewed impetus for the
discovery of novel opioid analgesics with a reduced propensity to evoke respiratory
depression, gastrointestinal discomfort and/or analgesic tolerance and dependence.
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In our previous work, we found that endomorphin analogues modified at the
1,3ꢀposition of their aromatic side chains with alkyls, such as
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DmtꢀProꢀTmp/Dmp/TrpꢀPheꢀNH2
and
DmtꢀProꢀTmpꢀTmpꢀNH2
(Dmt:
Dmp:
2,6ꢀdimetylꢀLꢀtyrosine, Tmp:
2,3,6ꢀtrimethylꢀLꢀphenylalanine,
2,6ꢀdimethylꢀLꢀphenyl alanine)， are potent MOP agonists with DOP antagonism.
Furthermore, DmtꢀProꢀTmpꢀTmpꢀNH2 exhibited no βꢀarrestin2 recruitment activity.30
Opioids with such a pharmacological profile are expected to have fewer
gastrointestinal side effects, and less respiratory depression, with a low propensity to
evoke tolerance and physical dependence compared with an equiꢀanalgesic dose of
morphine. The in vivo biological activities of these compounds are currently under
investigation. Opioid peptides generally have low oral bioavailability, primarily due to
their inability to be absorbed across the gastrointestinal mucosa, their poor capacity to
go through the bloodꢀbrain barrier and their low stability towards peptidases. In
previous work on the structureꢀactivity relationships of endomorphins, it was found
that the side chains of the 1,3ꢀposition aromatic amino acids in these opioid peptides
play a critical role in binding to and/or activating opioid receptors.30 Interestingly,
fentanyl also has two phenyl groups, although they may have different binding modes
with the MOP receptor compared with Tyr containing opioid peptides.31 Nevertheless,
they provide two possible modifying moieties that have not been extensively studied.1
We applied the modification strategy used successfully for the endomorphin
analogues to fentanyl to investigate if we could induce DOPꢀreceptor antagonistic
activity in fentanyl derivatives and inhibit the recruitment of βꢀarrestin2. We now
report on a series of fentanyl analogues derived by modification of fentanyl’s two
phenyl groups with alkyl groups, as well as their MOP, DOP and KOP functional
activities in the forskolinꢀstimulated cAMP assay, and βꢀarrestin2 recruitment activity.
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Results and discussion
Chemical synthesis.
Three series of fentanyl analogues modified at the two
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phenyl groups were synthesized (Scheme 1). 2ꢀArylbromoethanes (3) were prepared
from the corresponding arylbromides (1) through Grignard reaction subsequent to a
bromination reaction (Scheme 2). 3,5ꢀDimethylbenzylamine was synthesized from
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3,5ꢀdimethylbenzoic
acid
as
reported.32
The
preparation
of
Nꢀ3,5ꢀdimethylbenzylꢀNꢀ(4ꢀpiperidinyl)ꢀpropanamide (7b) was achieved following
the procedure reported for the NꢀphenylꢀNꢀ(4ꢀpiperidinyl) propanamide (7a)7 starting
from Nꢀbenzypiperidineꢀ4ꢀone (4) (Scheme 3). Briefly, Nꢀbenzypiperidineꢀ4ꢀone (4)
reacted with aniline or 3,5ꢀdimethylbenzylamine to give the corresponding Schiff
base which was then reduced with NaBH4 to yield the corresponding
Nꢀbenzylꢀ4ꢀanilinopiperidine
(5a)
or
Nꢀbenzylꢀ4ꢀ(3,5ꢀdimetnylbenzylamino)ꢀpiperidine (5b). Compound 5 was acylated
using propionyl chloride. The benzyl group of the resulting compound 6 was removed
by hydrogenation to give NꢀphenylꢀNꢀ(4ꢀpiperidinyl) propanamide (7a) or Nꢀ(3,
5ꢀdimethylbenzyl)ꢀNꢀ(4ꢀpiperidinyl)ꢀpropanamide (7b). NꢀBenzylꢀNꢀ(4ꢀpiperidinyl)
propanamide (19) (Scheme 4) was synthesized starting from NꢀBocꢀpiperidineꢀ4ꢀone
(16) with a series of reactions analogous to those used in the preparation of compound
5. The synthesized propanamide derivatives (7a, 7b and 19) were then Nꢀalkylated
with 2ꢀarylbromoethane to yield the desired fentanyl analogues (Scheme 3 and 4).
The structures of the analogues were verified by MS and NMR. The purities were
determined by HPLC, all the compounds showed purities greater than 98%.
Scheme 1. Structural formulae of fentanyl analogues.
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Scheme 2. Synthesis of 2ꢀarylbromoethane. Reagents and conditions: (i) Mg,
CH3CH2Br, THF, 30℃, 6.5 h, then ethylene oxide, ꢀ15℃ to rt, 12 h; (ii) PBr3, CCl4,
60℃, 1 h; (iii) BBr3, CH2Cl2, ꢀ15℃ to rt, 2 h.
Scheme 3. Synthesis of fentanyl analogues. Reagents and conditions: (i) RNH2,
pꢀTsOH, 4Å MS, toluene, reflux 12 h; (ii) NaBH4, MeOH, reflux 3 h; (iii) propionyl
chloride, CHCl3, ꢀ15℃ to rt, 12 h; (iv) Pd/C, 1 atm H2, MeOH, 50℃, 3d; (v)
ArCH2CH2Br, Et3N, toluene, reflux 24 h.
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Scheme 4. Synthesis of fentanyl analogues. Reagents and conditions: (i) PhCH2NH2,
pꢀTsOH, 4Å MS, toluene, reflux 12 h; (ii) NaBH4, MeOH, reflux 3 h; (iii) propionyl
chloride, CHCl3, ꢀ15℃ to rt, 12 h; (iv) 6.4 mol/L HCl/dioxane, rt, 1 h; (v)
ArCH2CH2Br, Et3N, toluene, reflux 48 h.
Inhibition of forskolinꢀstimulated cAMP formation in HEKꢀ293 cells stably
transfected with MOP, DOP or KOP receptors. Following opioid agonist binding at
HEKꢀMOP, HEKꢀDOP and HEKꢀKOP cells, forskolinꢀstimulated cAMP formation is
inhibited. Hence, the agonist and antagonist potencies of the new fentanyl analogues
were evaluated using this assay as previously described.30 (Figures 1ꢀ6). For these
cell based assays undertaken in 384ꢀwell plates, the final concentration of DMSO in
each well was <1%, to prevent cellular toxicity. As compounds 13, 20 and 21 were not
soluble in vehicle that contained 8% DMSO, the in vitro data of these three
compounds were unavailable in the present cell based assays.
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Figure 1. Effect of fentanyl analogues in three concentrations (0.1, 1 and 10 ꢀM)
relative to the reference control, fentanyl, the positive control, DAMGO, and the
negative controls (DPDPE and U69593) on mean (±SEM) forskolinꢀstimulated
cAMP formation in HEKꢀMOP cells. Data (n=3 independent experiments) are shown
in nM (A) and as relative percentage (B). Basal levels of cAMP in the cells were
assessed using buffer (SB) not containing forskolin.
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Consistent with expectations, the positive control, DAMGO at 1 ꢀM, was a
potent inhibitor of forskolinꢀstimulated cAMP formation in HEKꢀMOP cells. Fentanyl
at 1 ꢀM also inhibited production of cAMP albeit less potently than DAMGO. Of the
tested fentanyl analogues, compounds 8ꢀ12 were equiꢀpotent with DAMGO (1 µM) as
agonists at the MOP receptor. Compounds 8ꢀ11 are fentanyl analogues that retain the
framework of fentanyl, but with substituents on the phenyl group of the phenethyl
moiety. In order to mimic the phenolic side chain of Tyr, a common element in
naturally occurring and many synthetic opioids, a hydroxyl group was introduced to
the 4’ꢀposition of the phenethyl moiety (10). Compounds 8ꢀ11 showed similar potent
MOP receptor agonism that is independent of the properties of the introduced group
(ꢀH, ꢀOH, ꢀOMe, ꢀMe) at the 4’ꢀposition of the phenethyl group, suggesting that the
phenethyl is different from the phenolic side chain of Tyr in activating the MOP. This
observation is consistent with the findings of Portorghese.33, 34 Many other docking
results also indicate that fentanyl has a different binding mode with the MOP receptor
compared with Tyr containing opioid peptides.31,35
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Compounds 22 and 23 have Nꢀbenzyl groups instead of the Nꢀphenyl group in
fentanyl. This modification reduced MOP agonist bioactivity substantially. This
observation is in agreement with previous work.36 Interestingly, this reduction was
countered by introducing methyl groups to the phenyl group (compounds 12, 14 and
15). As shown in Fig. 1, compounds 12, 14 and 15 showed improved MOP agonist
bioactivities compared with that of 22 and 23. This is especially so for compound 12
that was equipotent with DAMGO at 1 ꢀM. Compounds 14 and 15 exhibited weak
MOP receptor agonist activity, for inhibition of forskolinꢀstimulated cAMP
accumulation.
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Figure 2. Assessment of MOP receptor antagonist activity of the fentanyl analogues
at 100 ꢀM in HEKꢀMOP cells. This was done by measuring mean (±SEM) inhibition
of forskolinꢀstimulated cAMP levels (n=3 independent experiments) expressed in nM
(A) or as a relative percentage (B) in the presence of the MOP receptor agonist,
DAMGO (1 ꢀM), and relative to the control antagonist, naloxone (NX) at 10 ꢀM. The
basal level of cAMP formation in HEKꢀMOP cells was assessed using buffer that did
not contain forskolin (SB).
As anticipated, the inhibitory effect of DAMGO on forskolinꢀstimulated cAMP
formation in HEKꢀMOP cells was antagonized by the classical opioid antagonist,
naloxone. Compounds 8ꢀ11 are full MOPꢀreceptor agonists, as they did not
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antagonize the inhibitory agonist effects of DAMGO on forskolinꢀstimulated cAMP
formation. On the contrary, the Nꢀbenzyl substituted compounds 14, 15, 22 and 23
showed partial to full reversal of DAMGO’s inhibitory effects at the MOPꢀreceptor,
indicating that these compounds are partial MOPꢀreceptor agonists. Of these,
compound 15 at 100 µM was equipotent with naloxone at 10 µM in inhibiting the
MOP receptor agonist bioactivity of DAMGO at 1 µM.
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Figure 3. Effect of fentanyl analogues in three concentrations (0.1, 1 and 10 ꢀM)
relative to the reference control, fentanyl, the positive control, DPDPE, and the
negative controls (DAMGO and U69593) on mean (±SEM) forskolinꢀstimulated
cAMP levels in HEKꢀDOP cells. Data (n=3 independent experiments) are in nM (A)
and as relative percentage (B). The basal level of cAMP in HEKꢀDOP cells was
assessed using buffer not containing forskolin (SB).
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With the exception of compound 8, the new fentanyl analogues were either
inactive or exhibited only weak DOP receptor agonist activity when assessed using
the forskolinꢀstimulated cAMP accumulation assay in HEKꢀDOP cells (Fig. 3A and
3B). The IC50 of compound 8 as an agonist at the DOP receptor was less than 1 ꢀM.
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Figure 4. DOP receptor antagonist activity of the new fentanyl analogues at 100 ꢀM
in HEKꢀDOP cells was assessed by measuring mean ( ± SEM) inhibition of
forskolinꢀstimulated cAMP levels (n=3 independent experiments) in the presence of
the DOP receptor agonist, DPDPE (1 ꢀM). The data are expressed in nM (A) or as
relative percentage (B) relative to the control DOP receptor antagonist, naltriben
(NTB) at 10 ꢀM. The basal level of cAMP formed in HEKꢀDOP cells was determined
using buffer that did not contain forskolin (SB).
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As shown in Figure 4A and 4B, the DOPꢀreceptor antagonism of compounds
8ꢀ12 and 14 was negligible when assessed using the forskolinꢀstimulated cAMP assay.
Of the tested compounds, compound 15 showed moderate DOPꢀreceptor antagonism
at approximately 64% that of naltriben (96% vs 150%).
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A primary purpose of the present study was discovery of fentanyl analogues that
were MOPꢀreceptor agonists and DOPꢀreceptor antagonists. Compounds 8 and 12 are
potent MOP receptor agonists, but they are devoid of DOP antagonism. Compounds
9ꢀ11 are opioid ligands exhibiting potent MOP receptor agonism equal to that of
DAMGO but with insignificant DOP receptor antagonism. Compound 15 is a weak
agonist and a full antagonist at the MOP receptor and a moderate antagonist at the
DOP receptor.
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Figure 5. Effect of fentanyl analogues in three concentrations (0.1, 1 and 10 ꢀM)
relative to the reference control, fentanyl, the positive control, U69593, and the
negative controls (DAMGO and DPDPE) on mean (±SEM) forskolinꢀstimulated
cAMP levels in HEKꢀKOP cells. Data (n=3 independent experiments) are mean
(±SEM) cAMP levels expressed in nM (A) or as relative percentage (B). The basal
level of cAMP in HEKꢀKOP cells was assessed using buffer that did not contain
forskolin (SB).
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Most of the fentanyl analogues were inactive as agonists at the KOP receptor
when assessed using the forskolinꢀstimulated cAMP formation assay. The exception
was compound 8 that showed moderate bioactivity at 1 ꢀM and that was about half as
potent as the positive control, U69593 at 1 ꢁM.
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Figure 6. KOP receptor antagonist activity of the novel fentanyl analogues at 100 µM
in HEKꢀKOP cells was evaluated by quantifying the mean (±SEM) inhibition of
forskolinꢀstimulated cAMP levels (n=3 independent experiments) in the presence of
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the KOP receptor agonist, U69593 (1 µM). The data are in nM (A) or as relative
percentage (B) in comparison with the opioid antagonist, naloxone (NTB) at 100 µM.
The basal level of cAMP formed in HEKꢀKOP cells was determined using buffer that
did not contain forskolin (SB).
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Of the fentanyl analogues herein, only compound 10 exhibited KOPꢀreceptor
antagonism in a manner mimicking naloxone at 100 µM (Fig. 6A and 6B). Thus,
compound 10’s in vitro profile shows that it is a potent MOPꢀreceptor agonist and a
KOPꢀreceptor antagonist with negligible activity at the DOPꢀreceptor. By comparison,
compound 8 exhibited weak agonism at the KOP receptor and full agonism at both
MOPꢀ and DOPꢀreceptors. For compounds 12, 14, 15, 22 and 23, their KOPꢀreceptor
antagonism was more pronounced than that of naloxone at 100 µM.
βꢀArrestin2 recruitment assay. Internationally, there is a large collective research
effort aimed at identifying opioid agonists that are Gꢀprotein biased with negligible
ability to recruit βꢀarrestin2. Hence, we used a BRET ratio assay (see Experimental
section), to assess the ability of our novel fentanyl analogues to induce βꢀarrestin2
recruitment to MOP, DOP and KOP receptors transiently expressed in HEK cells. The
intrinsic efficacy (Emax) and potency (EC50) of the nine fentanyl analogues were
derived from concentrationꢀresponse curves, in comparison to the corresponding data
for the control opioid ligands.
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Figure 7. Mean (±SEM) concentrationꢀresponse curves (n=3 independent
experiments) for the BRET ratio observed in HEK cells expressing MOPꢀRluc8.1 and
GFP2ꢀβArr2 following incubation of cells with the novel fentanyl analogues or with
control opioid ligands. All data are relative to fentanyl and expressed as a fraction of
its Emax
.
For recruitment of βꢀarrestin2 to the MOPꢀreceptor, compound 8 was the most
potent (EC50 = 160 nM) of the new fentanyl analogues such that it was 2.3ꢀfold more
potent than DAMGO (375 nM) and 1.3ꢀfold more potent than fentanyl (210 nM). Of
the ligands assessed, fentanyl had the highest efficacy (Emax = 1.0) whereas that for
compound 8 was 0.61. Although DADLE is sometimes reported as a selective DOP
receptor ligand, this is not the case as its EC50 was 1.1 ꢀM, being only 3ꢀfold less
potent than DAMGO (375 nM). Compound 9 showed a similar potency (1 ꢀM) to
DADLE but with lower efficacy (Emax = 0.57 vs 0.69 respectively).
In the present work, compounds 10ꢀ12 exhibited significantly lower efficacy and
potency for βꢀarrestin2 recruitment at the MOP receptor. For efficacy, their Emax
values were 20%, 37% and 37% of the efficacy of fentanyl (Emax = 1.0) respectively.
Regarding EC50 values, they were 4.8ꢀ (1 ꢀM), 12ꢀ (2.5 ꢀM) and 13ꢀ (2.7 ꢀM) fold
less potent than fentanyl (210 nM), respectively. For the forskolinꢀstimulated cAMP
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assay (Fig. 1), compounds 10ꢀ12 are potent MOPꢀreceptor agonists with potency
equal to that of DAMGO at 1 ꢀM. However, for βꢀarrestin2 recruitment to the MOP
receptor, their efficacy and potency was much lower than that of DAMGO. Together
these data indicate that compounds 10ꢀ12 are Gꢀprotein biased opioid ligands at the
MOP receptor.
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Herein, compounds 14, 15, 22 and 23 were inactive for recruitment of βꢀarrestin2
to the MOP receptor. By comparison for inhibition of forskolinꢀstimulated cAMP
formation, compounds 14 and 15 are weak MOP receptor agonists as well as weak
and potent MOP receptor antagonists respectively (Fig. 1). In view of the fact that
they are inactive for recruitment of βꢀarrestin2 to the MOP receptor, they are also
Gꢀprotein biased opioid ligands.
Our finding that fentanyl is more potent than DAMGO for recruiting βꢀarrestin2
to the MOP receptor (210 and 375 nM), is in agreement with previous work by others
(53 nM vs 229 nM respectively).28 With the exception of compound 8, all of the new
fentanyl analogues had significantly lower (P≤0.05) βꢀarrestin2 recruitment activities
relative to fentanyl and DAMGO. These data are encouraging based upon previous
work by others suggesting that decreased βꢀarrestin2 recruitment activity is correlated
with lower respiratory depression liability and an improved therapeutic index28.
Hence, compounds 10ꢀ12 that are potent MOP receptor agonists in the
forskolinꢀstimulated cAMP assay (Fig. 1) but with low efficacy for recruitment of
βꢀarrestin2 to the MOP receptor relative to fentanyl, may potentially show decreased
respiratory depression and an improved therapeutic index compared with fentanyl, but
this needs to be assessed in vivo in future work.
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Figure 8. Mean (±SEM) concentrationꢀresponse curves (n=3 independent
experiments) for the BRET ratio observed in HEK cells expressing DOPꢀRluc8.1 and
GFP2ꢀβArr2 following incubation of cells with the novel fentanyl analogues or with
control opioid ligands. All data are relative to DADLE and expressed as a fraction of
its Emax
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Figure 9. Mean (±SEM) concentrationꢀresponse curves (n=3 independent
experiments) for the BRET ratio observed in HEK cells expressing KOPꢀRluc8.1 and
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GFP2ꢀβArr2 following incubation of cells with the novel fentanyl analogues or with
control opioid ligands. All data are relative to U69593 and expressed as a fraction of
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The in vitro efficacy (Emax) and potency (EC50) of the new fentanyl analogues
assessed in concentrations spanning six log units for recruitment of βꢀarrestin2 to the
DOPꢀreceptor in transiently transfected HEKꢀcells were insignificant relative to that
of the DOP receptor agonist, DADLE (Emax =1, EC50 = 100 nM) (Fig. 8). Similarly,
the ability of the new fentanyl analogues to recruit βꢀarrestin2 to the KOPꢀreceptor in
transiently transfected HEKꢀcells, was insignificant compared with U69593 (Emax
=1.0, EC50 = 758 nM) (Fig. 9.)
Conclusion
Herein, we showed that methyl, hydroxyl or methoxy substitution of the phenyl
group of the phenethyl moiety of fentanyl, is tolerable. The modified compounds 8ꢀ11
retained high MOP receptor agonist activity in the cAMP assay. However, 4ꢀNꢀbenzyl
is intolerable (22 and 23), although this could be reversed by substitution of the
benzyl group, such as with a 3,5ꢀdimethyl moiety (12). Indeed, for the cAMP assay,
Compound 12 is a potent MOP receptor agonist and a full KOP receptor
antagonist with weak βꢀarrestin2 recruitment activity at the MOP receptor.
Additionally in the cAMP assay, compounds 10 and 11 are potent MOP receptor
agonists with weak DOPꢀreceptor antagonism and moderate KOP receptor
antagonism with weak βꢀarrestin2 recruitment activity at the MOP receptor. In the
cAMP assay, compound 15 is a weak MOP receptor agonist, a full MOP receptor
antagonist, a moderate antagonist at the DOP receptor with no βꢀarrestin2 recruitment
activity.
In view of the Gꢀprotein bias of our new nonpeptide fentanyl analogues, 10ꢀ12,
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these compounds are worthy of further investigation as promising leads for discovery
of novel opioid analgesics with improved adverse event profiles, compared with
currently available strong opioid analgesics.
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METHODS
Chemistry. The fentanyl analogues were synthesized as illustrated in Schemes 2ꢀ4.
Qingdao F254 silica gel plates were used for TLC detection. A Waters Delta 600 was
used for analytical and semiꢀpreparative HPLC by using a Sunfire C18 analytical
column (4 × 150 mm) or a Sunfire C18 semiꢀpreparative column (20 × 150 mm),
respectively. 1H NMR spectra were recorded on a Bruker AVANCE ꢀ300 spectrometer
in deuterated solvent. The samples were dissolved in deuterated solvent that
contains tetramethylsilane (TMS) as internal standard. Mass spectra were recorded
with an Agilent 1100 mass spectrometer.
Inhibition of forskolinꢀstimulated cAMP accumulation assay. The cAMP assays
were performed in HEKꢀ293 cells stably expressing MOP, DOP or KOP receptors as
previously described.30
βꢀArrestin2–recruitment assay. Bioluminescence Resonance Energy Transfer
(BRET) detection assays were used to assess the intrinsic efficacy of the fentanyl
analogues for βꢀarrestin2 recruitment at murine MOP, DOP and KOP receptors.
HEKꢀ293 cells that were coꢀtransfected with MOPꢀRluc8, DOPꢀRluc8 or KOPꢀRluc8
together with GFP2ꢀβꢀArrestin2 (Perkin Elmer, Victoria, Australia) were used as
previously described.30
Supporting Information
Experimental procedures for the synthesis of fentanyl analogues.
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Author Contributions:
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Y.Q. synthesized and characterized the compounds, L.N., J.S., Z.Z., and S.S.
synthesized the compounds. A.L., J.M., S.S. and A.K. performed the biological assays.
M.S. analyzed the data. T.L. designed the target compounds. T.L., M.S., and Y.Q.
wrote the manuscript.
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Acknowledgments
This work was supported in part by grants from the National Nature Science
Foundation of China (NSFC No. 81573280) to T.L, and supported in part by grants to
Y.Q. from Jiangsu Province Funds for Young Scientists (No. BK20171048). Research
done at The University of Queensland was supported by a Smart Futures Research
Industry Partnership Program.The authors acknowledge the Queensland Government
Smart State Research Programme for supporting Centre for Integrated Preclinical
Drug Development (CIPDD) research infrastructure. CIPDD is also supported by
Therapeutic Innovation Australia (TIA). TIA is supported by the Australian
Government through the National Collaborative Research Infrastructure Strategy
(NCRIS) program.We thank A Loening and S Gambhir of Stanford University (CA,
USA) for the kind gift of pcDNA3.1ꢀRluc8.
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Compounds 10ꢀ12 are potent ꢁꢀopioid receptor agonists with weak βꢀarrestin2 recruitment activity
89x35mm (300 x 300 DPI)
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