Synthesis and pharmacological characterization of ethylenediamine synthetic opioids in human μ-opiate receptor 1 (OPRM1) expressing cells

Article abstract of DOI:10.1002/prp2.511

Opioids are powerful analgesics acting via the human μ-opiate receptor (hMOR). Opioid use is associated with adverse effects such as tolerance, addiction, respiratory depression, and constipation. Two synthetic opioids, AH-7921 and U-47700 that were developed in the 1970s but never marketed, have recently appeared on the illegal drug market and in forensic toxicology reports. These agents were initially characterized for their analgesic activity in rodents; however, their pharmacology at hMOR has not been delineated. Thus, we synthesized over 50 chemical analogs based on core AH-7921 and U-47700 structures to assess for their ability to couple to Gα_i signaling and induce hMOR internalization. For both the AH-7921 and U-47700 analogs, the 3,4-dichlorobenzoyl substituents were the most potent with comparable EC₅₀ values for inhibition of cAMP accumulation; 26.49?±?11.2?nmol L^?1 and 8.8?±?4.9?nmol L^?1, respectively. Despite similar potencies for Gα_i coupling, these two compounds had strikingly different hMOR internalization efficacies: U-47700 (10?μmol L^?1) induced ~25% hMOR internalization similar to DAMGO while AH-7921 (10?μmol L^?1) induced ~5% hMOR internalization similar to morphine. In addition, the R, R enantiomer of U-47700 is significantly more potent than the S, S enantiomer at hMOR. In conclusion, these data suggest that U-47700 and AH-7921 analogs have high analgesic potential in humans, but with divergent receptor internalization profiles, suggesting that they may exhibit differences in clinical utility or abuse potential.

Full text of DOI:10.1002/prp2.511

Received: 18 June 2019ꢀ ꢀ Revised: 12 July 2019ꢀ ꢀ Accepted: 13 July 2019
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DOI: 10.1002/prp2.511
O R I G I N A L A R T I C L E
Synthesis and pharmacological characterization of
ethylenediamine synthetic opioids in human μ‐opiate receptor
1 (OPRM1) expressing cells
Tom Hsuꢀ| Jayapal R. Mallareddyꢀ| Kayla Yoshidaꢀ| Vincent Bustamanteꢀ| Tim Leeꢀ|
John L. Krstenansky ꢀ| Alexander C. Zambon
Department of Biopharmaceutical
Sciences, School of Pharmacy and Health
Abstract
Sciences, Keck Graduate Institute,
Claremont, California
Opioids are powerful analgesics acting via the human μ‐opiate receptor (hMOR).
Opioid use is associated with adverse effects such as tolerance, addiction, respira‐
Correspondence
John L. Krstenansky and Alexander C.
tory depression, and constipation. Two synthetic opioids, AH‐7921 and U‐47700 that
were developed in the 1970s but never marketed, have recently appeared on the
illegal drug market and in forensic toxicology reports. These agents were initially
characterized for their analgesic activity in rodents; however, their pharmacology
at hMOR has not been delineated. Thus, we synthesized over 50 chemical analogs
based on core AH‐7921 and U‐47700 structures to assess for their ability to cou‐
ple to Gαi signaling and induce hMOR internalization. For both the AH‐7921 and
U‐47700 analogs, the 3,4‐dichlorobenzoyl substituents were the most potent with
comparable EC50 values for inhibition of cAMP accumulation; 26.49 ± 11.2 nmol L−1
and 8.8 ± 4.9 nmol L−1, respectively. Despite similar potencies for Gαi coupling, these
two compounds had strikingly different hMOR internalization efficacies: U‐47700
(10 μmol L−1) induced ~25% hMOR internalization similar to DAMGO while AH‐7921
(10 μmol L−1) induced ~5% hMOR internalization similar to morphine. In addition, the
R, R enantiomer of U‐47700 is significantly more potent than the S, S enantiomer at
hMOR. In conclusion, these data suggest that U‐47700 and AH‐7921 analogs have
high analgesic potential in humans, but with divergent receptor internalization pro‐
files, suggesting that they may exhibit differences in clinical utility or abuse potential.
Zambon, Department of Biopharmaceutical
Sciences, School of Pharmacy and Health
Sciences, Keck Graduate Institute,
Claremont, CA 91711.
Email: john_krstenansky@kgi.edu (J. L. K.)
and azambon@kgi.edu (A. C. Z.)
Funding information
National Institute of Justice, Grant/Award
Number: 2016‐R2‐CX‐0059; Office of
Justice Programs; US Department of Justice
K E Y W O R D S
biased signaling, Gαi signaling, internalization, synthetic opioids, μ‐opioid receptor (OPRM1)
Abbreviations: GPCRs, G protein‐coupled receptors; GRK, G protein‐coupled receptor kinase; hMOR, human μ‐opiate receptor; HRMS, high resolution mass spectrometry; HPLC,
high‐performance liquid chromatography; MOR, μ‐opiate receptor; NLX, naloxone; NMR, nuclear magnetic resonance.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
© 2019 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for
Pharmacology and Experimental Therapeutics.
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1ꢀ|ꢀINTRODUCTION
using mouse models with targeted mutations in the OPRM1 gene
that prevent MOR internalization, suggest that this may not be the
best approach for developing safer opiates with fewer side effects.
Mutations of carboxyl tail serine and threonine residues, including
Ser375, that are phosphorylated by GRKs reveal that respiratory de‐
pression and constipation are significantly exacerbated when MOR
receptors fail to internalize after agonist binding14 while analgesic
tolerance is reduced. Thus, perhaps the pursuit of new opioids that
better mimic the endogenous system might result in novel more
“balanced” agents with improved clinical utility over morphine‐like
derivatives.
The ability of opiates to suppress painful stimuli is undisputed.
Opiates are a class of small molecules or peptides regardless of
structure that can bind and activate opiate receptors. Morphine is an
opioid extracted from the opium poppy that has been used as early
as the third century BC to treat dysentery, pain, and suffering.1 It
relieves pain by binding to and activating any of the three gene prod‐
ucts encoding for cell surface G protein‐coupled receptors (GPCRs).
These receptors are the μ‐, δ‐, or κ‐opiate receptors and they are
encoded by the OPRM1, OPRD1, and OPRK1 genes, respectively.2,3
Opioid analgesic properties stem from opioid receptor gene expres‐
sion in sensory neurons of the brain and peripheral CNS and their
coupling to intracellular heterotrimeric G proteins. Opiate binding
induces a conformational change in opiate receptors and signals to
rapidly suppress neuronal excitability by G protein‐dependent mod‐
ulation of Ca2+, K+, and Na+ currents resulting in a profound reduced
perception of pain. Of the three main opiate receptor subtypes, only
compounds with relatively high selectivity for the μ‐opiate recep‐
tor (MOR) have achieved widespread clinical utility due, in part, to
increased adverse effects such as dysphoria, convulsions, or poor
selectivity of agents that have been developed to selectively target
the δ‐ or κ‐ opiate receptors.1,2
In light of this, we sought to synthesize and pharmacologically
characterize a series of structural analogs based on the ethylenedi‐
amine structural analogs AH‐7921 and U‐47700 and compare their
pharmacology to morphine and the endogenous opioid mimetic
DAMGO. These two synthetic opioids, first synthesized and pat‐
ented in the early 1970s, have naloxone (NLX)‐reversible analgesic
potential in rodent models15,16 in the potency range of morphine.
However, their pharmacological properties, including their ability
to cause internalization of human μ‐opioid receptors (hMORs), are
unknown. Thus, they represent a potentially useful series of core
structures that are relatively easy to synthesize from where new
“balanced” or “biased” opiates could be designed.
Agonist binding‐induced conformational changes of the MOR,
in addition to activating inhibitory Gαi proteins, cause the phos‐
phorylation of intracellular residues such as Ser375 by a number of
kinases (eg G protein‐coupled receptor kinases [GRKs], PKC).4 The
MOR phosphorylation sites and the efficacy of phosphorylation can
differ based on the agonist structure. MOR phosphorylation leads
to β‐arrestin recruitment, receptor desensitization, and internaliza‐
tion which are all regulatory processes central to the development
of opiate tolerance.5,6 While many synthetic and naturally occurring
MOR agonists have high potency and efficacy for coupling to Gαi,
they can diverge significantly in their ability to promote β‐arrestin re‐
cruitment and receptor internalization.7‐9 For example, despite mor‐
phine's high potency and efficacy for Gαi coupling and widespread
clinical use, it has very low efficacy for β‐arrestin recruitment and
causes very little MOR internalization.7 Conversely, endogenously
produced opioids, such as the enkephalins and β‐endorphins, are
within a 10‐fold range of morphine in terms of Gαi coupling potency;
however, they are far more efficacious than morphine for recruiting
β‐arrestin and inducing receptor internalization.9
The concept that agonists could be designed to preferentially
couple to Gαi vs β‐arrestin recruitment, referred to as “biased sig‐
naling,” was a driving force behind the development of new opiates
such as oliceridine.10 This idea was bolstered by reports showing
that β‐arrestin‐2 knockout mice display increased analgesia, de‐
creased tolerance, and have less respiratory depression after mor‐
phine administration.11‐13 Hence new opiates, such as oliceridine,
were designed and selected for the ability to couple strongly to the
Gαi pathway but with low efficacies for β‐arrestin recruitment and
internalization, similar to morphine. However, recent events, such as
the failure of FDA approval for oliceridine in 2018 and new studies
The design of analogs was to assess both compounds described
in the patents, as well as related novel analogs, for their pharma‐
cological selectivity and efficacy for causing hMOR internalization.
Additionally, while the prior literature on the U‐series compounds
indicated that stereoisomers differ in their selectivity for κ‐ vs μ‐
opioid receptors, we sought to more clearly define the impacts of
these differences on hMOR pharmacology by synthesizing and test‐
ing single stereoisomers of the U‐series compounds. We present
herein findings related to structure activity relationships of these
two compounds and over 50 structural analogous to provide new
insights on how chemical structures affect potency for Gαi signaling
and efficacy for hMOR internalization.
2ꢀ|ꢀMATERIALS AND METHODS
2.1ꢀ|ꢀSynthesis of compounds
The achiral amine precursor 1‐aminomethyl‐1‐cyclohexanedi‐
methylamine for the AH‐series compounds, A01‐17, was prepared
from cyclohexanone according to the original patent US4049663
Example 1b.17 (1R,2R)‐N,N,N′‐trimethyl‐1,2‐diaminocyclohexane,
the precursor for the R,R enantiomer of the U‐series compounds,
U01‐17, (1R,2R)‐N,N‐dimethyl‐1,2‐diaminocyclohexane, the pre‐
cursor for the amide desmethyl U‐series compounds, Udes01‐09,
and
(1S,2S)‐N,N,N'‐trimethyl‐1,2‐diaminocyclohexane,
the
precursor for the S,S enantiomer of the U‐series compounds,
US01‐09, were obtained from LabNetwork (San Diego, CA). The
acid chlorides used were purchased from Fisher Scientific (analog
code numbers 01‐04, 07‐17) or Sigma Aldrich (analog code num‐
bers 05 and 06).

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vector containing EF1α promoter and the lentiviral 2k7bsd destina‐
tion vector with blasticidin resistance coding sequence, as described
previously.18 The resulting lentiviral plasmid vector is referred to
herein as EF1α‐3xHA‐OPRM1‐2k7bsd.
2.2ꢀ|ꢀAcylation of starting amines
The respective acid chloride (1.05 eq) was added to a solution of
the appropriate precursor amine (1.0 eq) and triethylamine (1.0 eq)
in 5 mL of dry diethyl ether and stirred at room temperature for
16 hour. The reaction mixture was extracted with ethyl acetate (3×),
washed with brine, dried over Na2SO4, and concentrated in vacuo.
The crude product was recrystallized from dichloromethane or pre‐
cipitated with ethyl acetate upon sonication to give desired product
as a solid. Reported hydrochloride salts of final compounds were
made using 2.0 N HCl in diethyl ether solution.
2.5ꢀ|ꢀCell lines
Human fibrosarcoma HT1080 cells (ATCC CCL‐121) are main‐
tained in DMEM high glucose, supplemented with 10% fetal bovine
serum, 1% penicillin/streptomycin, 1% L‐glutamine, 1% nonessen‐
tial amino acid, and 1% sodium pyruvate. Wild type (WT) HT1080
cells were transduced with lentivirus generated with EF1α‐3xHA‐
OPRM1‐2k7bsd lentiviral plasmid and transduced cells were selected
for using DMEM supplemented media with 10 µg mL−1 blasticidin.
2.3ꢀ|ꢀValidation of synthesized compound
purity and structure
Nuclear magnetic resonance (NMR) spectra were recorded on a
Varian 400MR spectrometer (proton frequency 399.765 MHz)
equipped with an AutoX DB broadband probe. Pulse sequences,
acquisition, and data processing were accomplished using VnmrJ
software (VnmrJ 4.2, Agilent Technologies, Santa Clara, CA). The
spectrometer was locked on to D2O and spectra were acquired
at 28°C without spinning. Water suppression suitability studies
were carried out using the presaturation (presat), WET (WET1D),
and excitation sculpting (water_ES) pulse sequences (VnmrJ 4.2,
Agilent Technologies, Santa Clara, CA) with automatic suppres‐
sion of the tallest peak (water at δ 4.86 ppm), an observation pulse
of 90° (10.8 μs), a spectral width of 6410.3 Hz, a relaxation time
of 30 second, and an acquisition time of 5.112 second. Eight scans
were taken. Three replicates were taken for each sample.
High Resolution Mass Spectrometry spectra were collected on
a Agilent Technologies 6530 Accurate‐Mass Q‐TOF LC/MS spec‐
trometer using direct infusion into the nanoelectronspray source.
Samples were dissolved in high‐performance liquid chromatography
(HPLC)‐grade methanol to a final concentration of ∼0.01 mg mL−1.
Spectra were run with 0.1% (v/v) formic acid/ HPLC‐grade methanol
as solvent.
2.6ꢀ|ꢀWestern blot
5 × 105 OPRM1‐expressing HT1080 cells were lysed with 1xLDS
buffer directly and ~25 μg of protein lysate is loaded into NuPAGE
4%‐12% Bis‐Tris Protein Gels (Thermo Fisher NP0335BOX) and
transferred onto PVDF membrane. Membrane was blocked in 1X
TBST with 5% w/v nonfat dry milk for 1 hour and probed with either
rabbit anti‐HA antibody (Cell Signaling 3724S) or rabbit anti‐GAPDH
(Cell Signaling 2118S) overnight at 4°C. The membrane was washed
3x with 1X TBST and probed with goat anti‐rabbit HRP antibody
(Santa Cruz SC2004), then visualized using SuperSignal West Femto
Maximum Sensitivity Substrate (Thermo Fisher 34095) in Azure
Biosystems c500 imaging system.
2.7ꢀ|ꢀImmunocytochemistry
OPRM1‐expressing or WT HT1080 cells were seeded at 7.5 × 105
onto cover glass precoated with 0.1 mg mL−1 Poly‐D‐Lysine (Sigma
P7280). Cells were first washed with Hank's Balanced Salt Solution
(Fisher Scientific MT21022CV), and then fixed with 4% paraformal‐
dehyde/PBS for 15 minutes followed by quenching with 0.1 mol L−1
glycine for 15 minutes. Afterward, the cells are washed 3x with 1X
PBS and then blocked in 1%BSA/TBST for 1 hour. Rabbit monoclo‐
nal anti‐HA antibody (Cell Signaling 3724S) is used to stain the cells
overnight, followed by goat anti‐rabbit Alexa Fluor 488 (Thermo
Fisher A11070) and counterstained with Draq5 (Biolegend 424101)
at 1:200 dilution. Slides were mounted and visualized using Leica
DMI6000 confocal microscope using 63× oil‐immersion objective,
with image processing using ImageJ software.
Purity determinations were performed by GCMS using
a
Shimadzu GC/MS 2010 SE with an Rtx‐5MS column (a DB‐5 MS
equivalent); 30 m × 0.25 mm × 0.25 m. The carrier gas was helium at
1 mL min−1, with the injector at 280°C, MSD transfer line at 280°C,
and ion source at 200°C. Injection Parameters: Split Ratio = 1:15,
1 μL injected. MS Parameters: Mass scan range: 34‐550 amu &
Threshold: 100. Acquisition mode: scan. The oven programs were
as follows: (a) 90°C initial temperature for 2.0 minutes; (b) Ramp to
300°C at 14°C min−1; (c) Hold final temperature for 10.0 minutes.
2.8ꢀ|ꢀCatchpoint cAMP assay
2.4ꢀ|ꢀDNA constructs
OPRM1‐expressing or WT HT1080 cells were seeded at 2 × 104
in 96‐well plates in DMEM high glucose, supplemented with 5%
fetal bovine serum, 1% penicillin/streptomycin, 1% L‐glutamine,
1% nonessential amino acid, and 1% sodium pyruvate. Culture
media was removed and cells were washed with Krebs‐Ringer
Bicarbonate Buffer (Sigma K4002), pH 7.4, then 1 mmol L−1
Human µ‐opioid receptor, OPRM1, with three sequential hemag‐
glutinin antigen (3xHA) tags at the N‐terminus (cDNA Resource
Center OPRM10TN00) was subcloned into pENTR/D‐TOPO vec‐
tor (Thermo Fisher K240020). The subcloned plasmid underwent
a three‐way Gateway LR recombination along with pENTR‐EF1α

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IBMX (Sigma I5879) was added to cells, and incubated at 37°C
for 30 minutes. Compounds were added and incubated at 37°C
for 15 minutes, followed by addition of 100 µmol L−1 forskolin
(FSK) (Sigma F3917). Selected samples were treated with 10 μmol
L−1 NLX (Sigma N7758) in addition to analogs that significantly
altered FSK‐induced cAMP levels to validate that any observed
changes in cAMP levels were mediated directly by hMOR. Cells
were further incubated at 37°C for 15 minutes. A solution of 1X
PBS was used for untreated cells in place of opioid compounds,
FSK, and NLX as a vehicle control. All solutions were removed
and the cells were lysed with Catchpoint Cyclic‐AMP Fluorescent
Assay Kit Lysis Buffer (Molecular Devices R8088) and shaken
on a plate shaker for 15 minutes at room temperature. Lysed
cells are processed according to manufacturer's protocol in the
Catchpoint Cyclic‐AMP Fluorescent Assay Kit and the cAMP lev‐
els were measured by SpectraMax Gemini EM Microplate Reader.
to FSK‐only (Dunnett's adjusted P < 0.05) dose, were then tested
with expanded dose ranges (in triplicate) to compute the average
EC50 value ± SEM from three independent expanded dose range
experiments (n = 3). The EC50 values for each experimental rep‐
licate were computed by fitting dose response data for each ex‐
perimental replicate to a variable‐slope sigmoidal curve (GraphPad
Prism 7). Receptor internalization data were analyzed in GraphPad
Prism 7 using unpaired t tests.
3ꢀ|ꢀRESULTS
3.1ꢀ|ꢀChemical syntheses, stereochemical
considerations, and structural analog design
AH‐7921 (Figure 1) is an achiral compound made from cyclohex‐
anone via a Strecker reaction as described in the original patents
and in (Figure 2A).17,19,20 The key intermediate of aminomethyl‐1‐
cyclohexanedimethylamine was used to synthesize AH‐7921
(Compound A01) and derivatives. U‐47700 (Figure 1) has two chiral
centers but is commercially available only as a racemic trans‐1,2‐
diaminocyclohexane mixture. The goal of this study was to study
the stereoselective pharmacology of single (R,R) or (S,S) isomers of
U‐47700 and related analogs, which had not previously been de‐
termined. Thus, for the synthesis of (R,R)‐isomer U‐series (U01‐17,
Figure 2B) and the Udes‐series (Udes01‐09, Figure 2B) com‐
pounds, the (1R,2R)‐N,N,N'‐trimethyl‐1,2‐diaminocyclohexane and
(1R,2R)‐N,N‐dimethyl‐1,2‐diaminocyclohexane were used, respec‐
tively, as the starting material. The (S,S)‐isomer US‐series (US01‐09)
was synthesized using (1S,2S)‐N,N,N'‐trimethyl‐1,2‐diaminocy‐
clohexane as the starting material. (Analytical data of all compounds
available in Supplementary information).
2.9ꢀ|ꢀQuantification of agonist‐mediated receptor
internalization
OPRM1‐expressing cells were seeded at 7.5 × 105 in 6‐well plates.
Culture media was removed and drug compounds diluted in 10% FBS
DMEM high glucose media (fully supplemented) were added to the
cells. The drug compound media was removed following 60 minutes
incubation at 37°C. Cells were washed with ice‐cold PBS (Ca2+ and
Mg2+ free) and kept cold (4°C) for the remainder of the assay to pre‐
vent further receptor trafficking. Washed cells were detached from
the plate by incubating with 2.9 mmol L−1 EDTA in PBS for 30 min‐
utes. Detached cells were pelleted and washed with cold PBS before
incubation with rabbit anti‐HA antibody (Cell Signaling 3724S) at
4°C. Cells are centrifuged down and washed with cold PBS before in‐
cubation with goat anti‐rabbit Alexa Fluor 488 antibody (Invitrogen
A11070) at 4°C. Cells are then centrifuged and washed with cold
PBS before analysis on BD Accuri C6 Flow Cytometer. Flow cytom‐
etry data were analyzed using FlowJo V10 software and gated for
single, viable cells.
3.2ꢀ|ꢀValidation of 3xHA‐tagged hMOR expression
in HT1080 fibrosarcoma cells
A lentiviral construct (Figure S1) was used to generate stable trans‐
duced human 3x HA‐tagged OPRM1‐expressing HT1080 cells as
described in Materials and Methods. Western blot was used to
facilitate identification of antibodies suitable for immunohisto‐
chemistry and receptor internalization. The hMOR receptor was
detected at the expected 65 kDa size in the hMOR but not in WT
cells (Figure 3A). Confocal microscopy was also performed to further
2.10ꢀ|ꢀData analysis and statistics
All data are presented as mean ± SEM. Due to the high number of
compounds tested, for the initial screening of compounds, each
compound was tested at three different concentrations (0.01,
0.1, and 1 μmol L−1) in triplicate using the Catchpoint cAMP assay
on two independent experiments (n = 2). In addition, a morphine
sulfate (MS) group was included as an internal positive control in
each independent experiment (data not shown) in addition to a
1 μmol L−1 compound plus 10 μmol L−1 NLX group to validate that
any changes in cAMP levels were mediated by hMOR. Cyclic AMP
responses for each of the three single doses were compared to
FSK alone by one‐way ANOVA with Dunnett multiple compari‐
son (GraphPad Prism 7). An adjusted P < 0.05 being considered
significant. All high potency agonists, that is, those which showed
significant suppression of cAMP at the 0.01 µmol L−1 compared
F I G U R E 1 ꢀ Structures of AH‐7921 and U‐47700

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F I G U R E 2 ꢀ Core analog synthesis and
structure overview (A) Synthetic routes
to all analogs (R′ = Me for U‐ & US‐series,
R′ = H for Udes‐series): (a) KCN, Me2NH.
HCl, H2O, rt, 16 hour; (b) LiAlH4, Et2O,
rt, 16 hour; (c) Et3N, RCOCl, Et2O,
rt, 16 hour. US starts with the (1S,2S)
diamine. (B) General structures of the
analogs in the four series tested: A, U,
Udes, and US
assess for hMOR expression at the subcellular level in the stable cell
line. Human MOR expression was detected to be enriched at the cell
membrane and in some endosomal compartments (Figure 3B).
3.3ꢀ|ꢀPharmacological validation of EF1α‐3xHA‐
OPRM1‐expressing HT1080 cell (hMOR) line
Human MOR‐expressing and WT cells were assessed for opiate‐me‐
diated inhibition of FSK‐stimulated adenylyl cyclase activity to assess
for hMOR Gαi coupling in response to the prototypic opioid agonist
MS to ensure that the 3x HA amino terminal tag did not interfere
with receptor function. Treatment with MS (1 µmol L−1) resulted in
a significant (P < 0.005) decrease in FSK‐induced cAMP accumula‐
tion in hMOR‐expressing cells but not in WT cells, validating surface
expression and functional coupling of the epitope‐tagged hMOR in
HT1080 cells and the lack of endogenous hMOR expression in WT
HT1080 cells. Coadministration of MS with NLX completely inhibited
the hMOR‐mediated Gαi response and restored cAMP to FSK‐only
levels (Figure 4).
Expanded MS dose ranges were assessed to determine an EC50
value for MS (39.3 nmol L−1), which is comparable to previously
published EC50 values of MS in heterologous OPRM1‐expressing
cells.7,8,21 The EC50 values for AH‐7921 (A01) and U‐47700 (U01)
found to be 26.9 ± 11.2 nmol L−1 and 8.8 ± 4.9 nmol L−1, respectively,
(Figure 4C) in hMOR‐expressing cells.
F I G U R E 3 ꢀ Validation of hMOR expression in HT1080 cells.
(A) Western blot of HT1080 Wild Type (WT) and 3xHA‐OPRM1
lentivirus transduced cells as detected with an anti‐HA monoclonal
antibody. (B) Confocal microscopy images detecting amino‐terminal
3x HA‐tagged hMOR using an anti‐HA primary antibody and
Alexa488 conjugated secondary. Nucleus is counterstained with
Draq5 and shown in red
3.4ꢀ|ꢀPotency of AH‐7921 and structural analogs
at hMOR
further increasing dose‐dependent decrease of cAMP levels. Thus,
of the AH‐series analogs we synthesized and tested, A01 (Figure 4C;
EC50 26.9 ± 11.2 nmol L−1) and A02 (Figure S6; EC50 59.3 ± 2.0 nmol
L−1) were classified as high potency hMOR agonists followed only
by A04, which we classified as a low potency agonist (Figure 5). The
remainder of the AH‐7921 compound series demonstrated either no
activity or activity that was not reversible by NLX treatment (Table
S1, Figure S2A).
Seventeen analogs based on the AH‐7921 core structure were syn‐
thesized and assessed for hMOR pharmacological activity (Table
S1). In addition to A01 (AH‐7921), two analogs were found to result
in significant suppression of FSK‐induced cAMP accumulation that
was reversed by NLX coadministration (Figure 5). A02 significantly
decreased FSK‐induced cAMP levels at (0.1 µmol L−1) and showed

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F I G U R E 4 ꢀ Pharmacological validation
of HT1080 hMOR cells. (A) HT1080
hMOR cells are treated with 100 μmol L−1
of forskolin (FSK) only, FSK with 1 μmol
L−1 morphine sulfate (MS), FSK with
MS plus 10 μmol L−1 NLX, or PBS only
(Untreated). (B) HT1080 WT is treated
similarly. Data from representative
experiment are shown. Statistical analysis
is performed using one‐way ANOVA with
Dunnett multiple comparison test with
FSK only as standard. *P < 0.05. (C) Dose
response curve and EC50 values table for
morphine, AH‐7921 (A01), and U‐47700
(U01) in the HT1080 hMOR cell line. Each
data point was performed in triplicate and
repeated three times (n = 3). Data from
representative experiment are shown
F I G U R E 5 ꢀ Drug potency of active
AH‐7921 series compounds. HT1080
hMOR cells are treated with 100 μmol
L−1 FSK only, or FSK with 0.01 μmol L−1
(low), 0.1 μmol L−1 (mid), 1 μmol L−1 (high)
of the indicated compound, and high
concentration dose of the compound
with 10 μmol L−1 NLX. Each treatment
dose was performed in triplicate (n = 2;
data from representative experiment
shown) and data were analyzed using
one‐way ANOVA with Dunnett multiple
comparison test and FSK only as standard.
*P < 0.05
S2). Of these, U01 and U04 (Figure 6) were classified as hMOR
selective high potency agonists with EC50 values 8.8 ± 4.9 nmol
L−1 and 26.0 ± 11.1 nmol L−1, respectively (Figure 4C, Figure S6).
Pharmacological profiling also revealed U02, U05, and U08 as low
potency agonists. Analogous to findings with AH‐7921 analogs,
3.5ꢀ|ꢀPotency of U‐47700 and structural analogs
at hMOR
Sixteen different U‐47700‐related analogs with (R,R) stereochem‐
istry were synthesized and assessed for hMOR activity (Table

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F I G U R E 6 ꢀ Drug potency of active
U‐47700 series compounds. HT1080
hMOR cells are treated with 100 μmol
L−1 FSK only, or FSK with 0.01 μmol L−1
(low), 0.1 μmol L−1 (mid), 1 μmol L−1 (high)
of the indicated compound, and high
concentration dose of the compound
with 10 μmol L−1 NLX. Each treatment
dose was performed in triplicate (n = 2;
data from representative experiment
shown) and data were analyzed using
one‐way ANOVA with Dunnett multiple
comparison test and FSK only as standard.
*P < 0.05
most of the U‐47700 analogs were found to have no activity at
hMOR at 1 μmol L−1 or lower concentrations. U06‐07 and U10‐17
were inactive based on comparison to FSK‐only control (Figure S3A).
U03 and U09 showed significant decreases in cAMP levels in the
range of concentrations tested, but their regulation of cAMP was not
NLX reversible (Figure S3B). The summary of the U‐47700 series of
opioid compound analogs is shown in Table S2.
Initial screening of Udes01 resulted in a significant decrease of FSK‐
induced cAMP levels at 10 nmol L−1, and specificity for hMOR was
confirmed by NLX reversibility (Figure 7). The EC50 value of Udes01
was subsequently determined to be 3.0 ± 0.3 nmol L−1 (Figure S6)
characterizing it as the most potent analog that we discovered. The
summary of the pharmacological activity of Udes‐series of com‐
pound analogs at the hMOR is shown in Table S3. The remaining
Udes‐series (Udes02‐09) did not significantly alter FSK‐induced
cAMP accumulation at any of the tested concentrations (Figure S4).
The US‐series was synthesized to assess for pharmacological
activity of the S,S enantiomer of the U‐47700 analogs. The nine US‐
series analogs were synthesized and screened for hMOR agonism
(Table S4, Figure S5). Of these nine, only US01 (3,4‐dichlorobenzoyl
substituent) demonstrated significant decrease in cAMP levels at
3.6ꢀ|ꢀPotency of Udes‐ and US‐series of analogs
at hMOR
To study the effect of removing N‐methyl group on the amide in
(R,R)‐U‐series, Udes‐series (compounds Udes01‐09) was synthe‐
sized and screened for hMOR signaling (Figure S4 and Table S3).

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F I G U R E 7 ꢀ Drug potency of Udes01
and US01. HT1080 hMOR cells are
treated with 100 μmol L−1 FSK only, or
FSK with 0.01 μmol L−1 (low), 0.1 μmol L−1
(mid), 1 μmol L−1 (high) of the indicated
compound, and high concentration dose
of the compound with 10 μmol L−1 NLX.
Each treatment dose was performed in
triplicate (n = 2; data from representative
experiment shown) and data were
analyzed using one‐way ANOVA with
Dunnett multiple comparison test and
FSK only as standard. *P < 0.05
the highest screening dose of 1 μmol L−1 but not at the lower (0.1
or 0.01 µmol L−1) concentration ranges (Figure 7, Figure S5) indicat‐
ing that the S,S enantiomer of U‐47700 was significantly less potent
than the R,R enantiomer.
3.7ꢀ|ꢀEfficacy of hMOR internalization by high
potency AH‐7921, U‐47700 analogs
Agonist‐induced receptor endocytosis remains a hallmark feature of
GPCR activation and regulation and provides insights into the level of
receptor desensitization that occurs after agonist stimulation.8 Thus,
the AH‐7921, U‐47700, and Udes‐series compounds that demon‐
strated high potency were tested for their ability to induce hMOR in‐
ternalization. The hMOR‐expressing cells were treated with saturating
concentrations of 10 µmol L−1 [D‐Ala2,N‐MePhe4,Gly5‐ol] enkephalin
(DAMGO) or 10 µmol L−1 morphine, which are reported to cause high
levels or low levels of hMOR internalization, respectively.8,21,22 Similar
results were found for DAMGO and morphine in our hMOR‐express‐
ing cells (Figure 8). Interestingly, A01 and A02 (10 µmol L−1) resembled
morphine treatment with only ~5% of cell‐surface receptors internal‐
izing after 1 hour. In contrast, U01 (10 µmol L−1) resulted in 6‐fold
more hMOR internalization than morphine and A01, to levels similar
to DAMGO (10 µmol L−1) treatment (~30% of cell‐surface receptors).
U04 and Udes01 demonstrated hMOR internalization similar to mor‐
phine and the two AH‐series compounds (Figure 8).
F I G U R E 8 ꢀ Measuring hMOR internalization levels after
treatment with the high potency compounds. HT1080 hMOR
cells were treated with the indicated concentration of drugs
for 1 hour, then analyzed using flow cytometry to evaluate the
changes in surface hMOR compared to untreated cells. The average
internalization percentage is shown (n = 3). Data were analyzed
using unpaired t test. *P < 0.05
hMOR agonists that we found, the rank order of potencies
for functional Gαi coupling and cAMP inhibition is as follows:
Udes01 > U01 > U04 = A01 ≥ morphine sulfate > A02. We also
classified A04, U02, U05, U08, and US01 as low potency hMOR
agonists. Our findings are somewhat aligned with findings de‐
tailed in the original patents and early studies, however, with some
key differences. In the literature, compounds A01 (AH‐7921),
A03, and A13 demonstrated 100% inhibition in the mouse hot
plate test when dosed at 100 mg kg−1 sc, but only A01 and A03
4ꢀ|ꢀDISCUSSION
We present a synthesis and screening strategy that enabled us
to identify the pharmacological activity of over 50 ethylenedi‐
amine‐containing compounds at the hMOR. Of the high potency

HSU et al.
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demonstrated activity greater than that of aspirin in a phenylqui‐
not the single (1R,2R) stereoisomer (personal communication with
Cayman Chemical customer service 12/10/2018).
none test when orally dosed at 100 mg kg−1 15
The determination
.
of ED50 (mg kg−1 sc) gave the following rank order or potency: A01
2.5 ≥ morphine 2.8 > A02 5.0 > A15 9.5 > A13 15.5.23 They also
demonstrated that AH‐7921 had a high addictive liability in rats
and rhesus monkeys. In our system, A03, A13, and A15 failed to
significantly suppress FSK‐induced cAMP accumulation based at
0.01, 0.1, or 1 μmol L−1 dosages in human OPRM1‐expressing cells
(Figure S2) possibly reflecting differences in human and rodent
OPRM1 receptor structures and underscoring the importance of
validating SAR with human receptors.
It has been proposed that hMOR endocytosis is closely associated
with the development of drug tolerance in the opiate user8,30 and in
mouse models.14 Our data reveal that A01 and A02 demonstrated low
levels (~15%) of hMOR internalization with similar drug potency for
Gαi coupling. However, U01 and U04/Udes01 significantly (P < 0.05)
diverged in their desensitization capacities: U01 induced high levels
of internalization (~26%) while U04 and Udes01 induced between
8% and 12% of cell surface hMORs to internalize. Thus, the levels of
hMOR internalization for A01, A02, U04, and Udes01 are similar to
the reported internalization levels of well‐characterized opioids, such
as morphine and fentanyl, while U01 is much more similar to the en‐
dogenous opioids, the endorphins and the enkephalins.7,9,31 Thus, if
the goal was to design new opiates with safer and improved pharma‐
codynamic properties that mimic endogenously produced “balanced/
natural” opiate responses at the hMOR, further assessment of the
(1R,2R) single isomer U01 is likely to produce internalization and po‐
tentially receptor desensitization effects more aligned with endoge‐
nous opiates although further studies are needed.
In 1973, a group at Upjohn began looking at conformationally
restrained analogs of phenampromide and by 1975 had developed
the κ‐selective opioid analog, U‐50,488.24 Moving away from the
N‐arylpropanamide structure, they developed the chemical series
to which U‐47700 belongs. Rat tail flick (ED50 mg kg−1) and MOR‐
related behavioral data (Straub tail, arched back, and increased lo‐
comotor activity) were reported.25,26 A number of the compounds
originally described are functionally characterized in this current
study. The rank order of potency for analgesia (tail flick response
time‐ED50 mg kg−1) is as follows: U01 (U‐47700) 0.2 > U04 0.4 > U02
1.0 > morphine sulfate 1.5 > Udes01 11.25,26 Each of these analogs
demonstrated MOR selectivity based on NLX reversibility. Our rank
order potency data for cAMP inhibition are similar to these trends
showing U01 to be the most potent U‐series analog followed by U04,
then U02, U05, and U08; however, our data indicate that Udes01
was ~3X more potent than U01, which is much more potent than
indicated in the aforementioned studies. This could be explained by
differences in human vs rodent MOR protein‐coding sequences or if
the Udes01 tested contained a racemic mixture, which is quite likely
and could explain its much lower potency.
In summary, our results demonstrate that this in vitro functional
assay for hMOR pharmacology provides a foundation from which
effects of these agents in humans can be anticipated and they pro‐
vide a scaffold for the rational design of potentially superior analge‐
sics that better mimic the endogenous opioid system.14,32 Through
our combinatorial approach, we discovered some novel moderately
potent hMOR agonists. Whether these novel compounds have dif‐
ferent tolerance profiles or addictive potential needs to be further
investigated in vivo.
ACKNOWLEDGEMENTS
In light of this, our U‐series analog synthesis approach benefit‐
ted from the availability of the single stereoisomers of the advanced
amine intermediates. This allowed for the synthesis of single stereo‐
isomer analogs rather than the racemic versions of the analogs. Our
findings, the (1R,2R) stereoisomer of U‐47700 (U01), were substan‐
tially more potent than the (1S,2S) stereoisomer (US01). The influ‐
ence of stereochemistry in the U‐series compounds on μ‐ vs κ‐opioid
receptor selectivity has been reported25,27 indicating that the (1S,2S)
isomer has high κ‐opioid receptor selectivity, while the (1R,2R) iso‐
mer has high μ‐opioid receptor selectivity.
This project was supported by Award No. [2016‐R2‐CX‐0059],
awarded by the National Institute of Justice, Office of Justice
Programs, US Department of Justice. The opinions, findings, and con‐
clusions or recommendations expressed in this publication are those
of the authors and do not necessarily reflect those of the Department
of Justice.
DISCLOSURE
None to declare.
Differentiating the effects of single isomers vs racemic U‐se‐
ries compounds may help delineate in vivo effects in humans me‐
diated by μ‐ and κ‐opioid receptors. One report of a seized sample
of U‐49900, a diethyl amine version of U‐47700 which is a dimethyl
amine, demonstrated that it was a racemic mixture, as determined
by circular dichroism.28 A study reporting the murine μ‐opioid recep‐
tor affinity and activity of U‐47700 provided by Cayman Chemicals,
reports a binding affinity of 57 nmol L−1 for U‐47700 at the mouse
μ‐opioid receptor vs 5 nmol L−1 for MS and an EC50 of 214 ± 23 nmol
L−1 in a [35S]‐GTPγS binding assay,29 which is less potent than we
found for (1R,2R)‐U‐47700 (U01). To date, Cayman Chemicals has
confirmed that they supply racemic trans isomers of U‐47700 and
AUTHOR CONTRIBUTIONS
ACZ, JLK, TH participated in research design; ACZ, JLK, TH, JRM, KY
participated in writing and preparation of the manuscript, KY, VB,
TL, TH, and JRM contributed to data acquisition.
ORCID
John L. Krstenansky
Alexander C. Zambon
https://orcid.org/0000‐0003‐3773‐2523
https://orcid.org/0000‐0001‐7874‐4462

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