ACS Chemical Neuroscience
Research Article
reagents used during the chromatographic analyses were at least of
high performance liquid chromatography (HPLC) grade.
with 10% heat inactivated FBS, 100 IU/mL penicillin, 100 mg/L
streptomycin, and 0.25 mg/L amphotericin B. The cells were cultured
at 37 °C in a humidified atmosphere containing 5% CO2. The stability
of the cell lines was routinely monitored via flow cytometric analysis
of coexpressed markers.67
Synthesis. The generic nitazene synthesis pathway, as outlined in
the Results and Discussion section, was based on published synthesis
routes, utilizing commercially available starting materials where
available.46,48 This yielded isotonitazene (1), N desethylisotonitazene
HCl (2), 4′ OH nitazene(3), 5 aminoisotonitazene (4), metonitazene
(5), N desethyletonitazene (7), protonitazene HCl (8), butonitazene
(9), clonitazene (10), flunitazene HCl (11), isotodesnitazene citrate
(12), metodesnitazene HCl (13), and etodesnitazene citrate (14). All
final products were purified to >98% HPLC−UV purity by standard
purification techniques. The structure and purity of all synthesized
materials were confirmed via nuclear magnetic resonance spectrosco
py (1H NMR) (pp S5 and S6) and extensive analytical character
ization, as outlined below. 1H NMR spectra were obtained on either a
Varian Unity Inova instrument (400 MHz) or a JEOL ECZ 400S
(400 MHz). Samples were dissolved and recorded in DMSO d6.
Analytical Characterization. Analytical characterization via
HPLC coupled to diode array detection (HPLC DAD), gas
chromatography mass spectrometry (GC MS) (except for 3, 7, and
9), and liquid chromatography coupled to time of flight mass
spectrometry (LC QTOF MS) was done as described before.7 The
details of each technique are briefly summarized below.
High-Performance Liquid Chromatography Coupled to Diode
Array Detection (HPLC-DAD). Reversed phase separation was
performed on a LaChrom HPLC system from Merck Hitachi
(Tokyo, Japan), using a Merck Purospher Star RP 8 end capped
column (5 μm, 125 mm × 4.6 mm) fitted with a Merck Purospher
Star RP 8 end capped guard column (5 μm, 4 mm × 4 mm).
Detection was done via DAD, monitoring a wavelength from 220 to
350 nm with a slit of 1 nm, a spectral bandwidth of 1 nm, and a
spectral interval of 200 ms. Concentrations of the injected dilutions
ranged from 8 to 40 μg/mL.
Gas Chromatography−Mass Spectrometry (GC-MS). An amount
of 1 μL of a 1 mg/mL solution was injected on an Agilent 7890A GC
system coupled to a 5975 XL mass selective detector operated by
MSD Chemstation software, as described in ref 7. The mass
spectrometer operated in SCAN mode, scanning the range of 50−
700 Da. For 4′ OH nitazene (3), N desethyletonitazene (7), and
butonitazene (9), 1 μL of a 1 mg/mL solution was injected on an
Agilent 8890 GC system coupled to a 5977 mass selective detector
operated by OpenLabs CDS software. Injections with a split ratio of
15:1 were performed automatically at an injection temperature of 300
°C, with helium as the carrier gas at a constant flow rate of 2 mL/min.
A 30 m × 0.32 mm i.d. × 0.5 μm Restek Rtx 5MS column was used.
For 3 and 7, the temperature program started at 50 °C for 1 min and
was ramped at 30 °C/min to 300 °C, which was held for 16 more
minutes. For 9, the temperature program started at 240 °C for 1 min
and was ramped at 30 °C/min to 300 °C, which was held for 27 more
minutes. The transfer line temperature and ion source temperature
were set at 300 and 280 °C, respectively. MS quadrupole temperature
was set at 150 °C, and an ionization energy of 70 eV was used. The
mass spectrometer operated in SCAN mode, scanning the range of
40−650 Da.
Liquid Chromatography Coupled to Time-of-Flight Mass
Spectrometry (LC-QTOF-MS). Using a 1 μg/mL solution, spectra
were recorded after infusion or after chromatographic separation. The
latter was accomplished with an Agilent 1290 Infinity LC system and
a Phenomenex Kinetex C18 column (2.6 μm, 3 mm × 50 mm),
maintained at 30 °C. The high resolution mass spectrometry system
was a 5600+ QTOF from Sciex, with an electrospray ionization (ESI)
source and Analyst TF 1.7.1 software, from the same provider.
Settings for QTOF MS were the same as published before.7 The LC−
QTOF MS settings resulted in TOF MS full scan spectra combined
with data dependent acquisition of product ion spectra (both
scanning from 5 to 450 Da).
NanoBiT MOR-β-arrestin2/mini-Gi Recruitment Bioassays. Two
stable cell based reporter assays were used to assess the in vitro
biological MOR activity of 14 different nitazenes, fentanyl, morphine,
and hydromorphone. The employed assays have been described
before.63,67 In short, activation of human MOR, fused to one part of a
split nanoluciferase (NanoLuc Binary Technology, Promega), results
in the recruitment of either β arrestin2 (βarr2) (in the presence of
coexpressed G protein coupled receptor kinase 2, GRK2) or mini Gi
(GTPase domain of the Gαi subunit), fused to the complementing
part of the split nanoluciferase. The resulting functional comple
mentation of the nanoluciferase restores its enzymatic activity, which,
upon addition of the substrate furimazine, yields a measurable
bioluminescent signal.
Cells expressing either MOR βarr2 GRK2 (for simplicity, referred
to as MOR βarr2) or MOR mini Gi were seeded on poly D lysine
coated 96 well plates (5 × 104 cells/well) 1 day prior to the
experiments. Following overnight incubation, the cells were washed
twice with Opti MEM I reduced serum medium before adding 90 μL
of OptiMEM. Nano Glo Live Cell Reagent was then prepared by 20
fold dilution of Nano Glo Live Cell Substrate with Nano Glo LCS
Dilution Buffer, and 25 μL was added to each well. The plate was
subsequently placed into a TriStar2 LB 942 multimode microplate
reader (Berthold Technologies GmbH & Co., Bad Wildbad,
Germany), and luminescence was continuously monitored until
stabilization of the signal (10−15 min). Next, 20 μL of a 6.75 fold
concentrated stock solution of each test compound (in Opti MEM/
MeOH or Opti MEM/ACN) was added per well and luminescence
was monitored for 2 h. All compounds were tested in both assays in
concentrations ranging between 1 pM and 100 μM, with appropriate
solvent controls included in each experiment. Each compound was
evaluated in five independent experiments (n = 5), with duplicates run
for each concentration within an experiment to ensure the reliability
of single values.
Data and Statistical Analysis. Absolute time−luminescence
profiles obtained during the 2 h readout were corrected for inter
well variability and used for calculation of the area under the curve
(AUC), as previously detailed by Pottie et al.71 Solvent controls were
performed by subtraction of the mean AUC of the corresponding
blank. Concentration−response curves were subsequently generated
via GraphPad Prism 8 software (San Diego, CA, USA) via three
parametric nonlinear regression, which implies a fixed Hill slope of 1.
The use of this model is required for the implementation of the ligand
bias calculation described below.72,73 To facilitate interpretation and
comparison between different studies, the data were normalized to the
maximum response of hydromorphone (arbitrarily set at 100%) for
each experiment. Hydromorphone was selected as a reference agonist
for normalization based on previous experience.7−10,67 To facilitate
interpretation of the data, efficacies were also calculated relative to
fentanyl. A part of the data for fentanyl (n = 3 out of the total of n = 5
experiments) were also reported in Vandeputte et al.9 Morphine was
included for comparison, further facilitating interpretation of the data.
It was defined a priori that AUC values from the highest
concentration(s) were excluded in the case of a reduction of 20%
or more compared to the AUC of the next dilution. As previously
hypothesized for different receptor systems,9,68 high concentrations
may potentially lead to cell toxicity or solubility issues, with a rapid
drop of the signal resulting in a lower AUC. Inclusion of such data
points could inadvertently skew the obtained concentration−response
graph. By use of the standard Grubbs’ test, the complete data set
(2349 data points) was screened for outliers, resulting in a total of 17
outliers (0.72%) that were subsequently omitted from the data set. All
duplicate data points were considered separately in the Grubbs’ test.
This means that data points were only excluded if a single value (e.g.,
out of n = 5 × 2) was considered an outlier in the Grubbs’ test, as in
this case the reliability of this single value could not be ensured. For
Determination of in Vitro Biological Activity at the μ-Opioid
Receptor (MOR). Cell Culture. HEK 293T cells stably expressing
either the MOR βarr2 GRK2 or MOR mini Gi system (see below)
were routinely maintained in DMEM (GlutaMAX) supplemented
1248
ACS Chem. Neurosci. 2021, 12, 1241−1251
Vandeputte, Marthe M.
