Synthesis and Functional Characterization of 2-(2,5-Dimethoxyphenyl)-N-(2-fluorobenzyl)ethanamine (25H-NBF) Positional Isomers

Article abstract of DOI:10.1021/acschemneuro.1c00124

Serotonergic psychedelics, substances exerting their pharmacological action through activation of the serotonin 2A receptor (5-HT2AR), have continuously comprised a substantial fraction of the over 1000 reported New Psychoactive Substances (NPS) so far. Within this category, N-benzyl derived phenethylamines, such as NBOMes and NBFs, have shown to be of particular relevance. As these substances remain incompletely characterized, this study aimed at synthesizing positional isomers of 25H-NBF, with two methoxy groups placed on different positions of the phenyl group of the phenethylamine moiety. These isomers were then functionally characterized in an in vitro bioassay monitoring the recruitment of β-Arrestin 2 to the 5-HT2AR through luminescent readout via the NanoBiT technology. The obtained results provide insight into the optimal substitution pattern of the phenyl group of the phenethylamine moiety of N-benzyl derived substances, a feature so far mostly explored in the phenethylamines underived at the N-position. In the employed bioassay, the most potent substances were 24H-NBF (EC50 value of 158 nM), 26H-NBF (397 nM), and 25H-NBF (448 nM), with 23H-NBF, 35H-NBF, and 34H-NBF yielding μM EC50 values. A similar ranking was obtained for the compounds' efficacy: Taking as a reference LSD (lysergic acid diethylamide), 24H-, 26H-, and 25H-NBF had an efficacy of 106-107%, followed by 23H-NBF (96.1%), 34H-NBF (75.2%), and 35H-NBF (58.9%). The stronger activity of 24H-, 25H-, and 26H-NBF emphasizes the important role of the methoxy group at position 2 of the phenethylamine moiety for the in vitro functionality of NBF substances.

Full text of DOI:10.1021/acschemneuro.1c00124

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Research Article
Synthesis and Functional Characterization of 2‑(2,5-
Dimethoxyphenyl)‑N‑(2-fluorobenzyl)ethanamine (25H-NBF)
Positional Isomers
Eline Pottie, Olga V. Kupriyanova, Vadim A. Shevyrin, and Christophe P. Stove*
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ABSTRACT: Serotonergic psychedelics, substances exerting their
pharmacological action through activation of the serotonin 2A
receptor (5-HT2AR), have continuously comprised a substantial
fraction of the over 1000 reported New Psychoactive Substances
(NPS) so far. Within this category, N-benzyl derived phenethyl-
amines, such as NBOMes and NBFs, have shown to be of
particular relevance. As these substances remain incompletely
characterized, this study aimed at synthesizing positional isomers
of 25H-NBF, with two methoxy groups placed on different
positions of the phenyl group of the phenethylamine moiety.
These isomers were then functionally characterized in an in vitro
bioassay monitoring the recruitment of β-arrestin 2 to the 5-
HT2AR through luminescent readout via the NanoBiT technology.
The obtained results provide insight into the optimal substitution pattern of the phenyl group of the phenethylamine moiety of N-
benzyl derived substances, a feature so far mostly explored in the phenethylamines underived at the N-position. In the employed
bioassay, the most potent substances were 24H-NBF (EC50 value of 158 nM), 26H-NBF (397 nM), and 25H-NBF (448 nM), with
23H-NBF, 35H-NBF, and 34H-NBF yielding μM EC50 values. A similar ranking was obtained for the compounds’ efficacy: taking as
a reference LSD (lysergic acid diethylamide), 24H-, 26H-, and 25H-NBF had an efficacy of 106−107%, followed by 23H-NBF
(96.1%), 34H-NBF (75.2%), and 35H-NBF (58.9%). The stronger activity of 24H-, 25H-, and 26H-NBF emphasizes the important
role of the methoxy group at position 2 of the phenethylamine moiety for the in vitro functionality of NBF substances.
KEYWORDS: Psychedelic, new psychoactive substances, synthesis, bioassay, structure−activity relationship, serotonin receptor
in psychotherapy, mainly with LSD (lysergic acid diethyla-
mide) and psilocybin.5,6
INTRODUCTION
■
At the beginning of 2021, a cumulative total of more than 1000
New Psychoactive Substances (NPS) had been reported to the
United Nations Office on Drugs and Crime (UNODC).1
When looking at the classification of NPS based on their
pharmacological mechanism, one of the consistently larger
groups is that of the classic hallucinogens.1 Classic
hallucinogens, or serotonergic psychedelics, are substances
that exert their main pharmacological effects through activation
of the serotonin 2A receptor (5-HT2AR), a G protein-coupled
receptor (GPCR).2 Effects typically sought for by users of
psychedelics include, inter alia, mystical experiences, empathic
feelings, alterations in consciousness, and sensory and somatic
effects. However, the use of these substances can result in
severe side effects, such as tachycardia, hyperthermia, agitation,
rhabdomyolysis, hypertension, and seizures, and with the more
recent group of NBOMes even deaths have been reported.3,4
On the other hand, interest in the potential therapeutic
benefits of psychedelics is increasing. This translates into
clinical trials in, e.g., the context of treatment of addiction and
Serotonergic psychedelics (not limited to the NPS) can be
categorized into three main structural classes, more specifically
the ergolines (such as the prototypical psychedelic substance
LSD), tryptamines (such as psilocybin), and phenylalkylamines
(with mescaline as the prototypical substance). This latter
group comprises subcategories such as the phenethylamines
(among which 2C-X substances), the phenylisopropylamines
(DOx substances) and, more recently, N-benzylderivatives of
the former group (e.g., N-methoxybenzylderivatives or
NBOMes).3,7 Certain representatives of the N-benzyl derived
substances, such as 25B-NBOMe, have been explored as
Received: March 6, 2021
Accepted: April 19, 2021
Published: April 28, 2021
https://doi.org/10.1021/acschemneuro.1c00124
© 2021 American Chemical Society
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Figure 1. Structures of compounds 1−6 investigated in the present study.
neuroimaging tools.8 For the group of phenethylamine
psychedelics, several Risk Assessment Reports have been
issued by the European Monitoring Centre for Drugs and Drug
Addiction (EMCDDA). These reports specifically emphasize
the relevance and risk of the specific substances 2C-I, 2C-T-2
and 2C-T-7, and 25I-NBOMe.9,10
Despite their relevance, psychedelic NPS still remain
incompletely characterized. One example of a scarcely
described group is that of the 25X-NBF substances, structurally
related to the NBOMes, only differing in a fluorine substituent
instead of a methoxy group at position 2 of the N-benzyl
group. The recreational use of these substances is suggested by
the identification of 25B/C-NBF on the illegal drug market in
Japan and by the mention of 25I-NBF on drug forums.11,12
Additionally, several 25X-NBF substances are subject to
legislative control, for example, in Sweden, Japan, and the
United Kingdom.11,13,14 However, little is known about the
pharmacology and toxicology of NBF substances. In rodent in
vivo studies, 25C-NBF was shown to initiate neurotoxicity and
addiction potential. Therefore, Hur et al. emphasize the danger
that lies with the use of 25C-NBF.15
far as we know, synthesis, analytical data, and biological studies
for compounds 2, 3, 4, and 6 have not been previously
described in the scientific literature. For compound 5, data on
testing its antibacterial activity were published.16 Compounds
1 and 5 are commercially available. The activation potential of
each of these isomers was determined in this study by means of
their ability to induce recruitment of the cytosolic scaffolding
protein β-arrestin 2 (βarr2) to the activated target receptor 5-
HT2AR.
RESULTS AND DISCUSSION
■
Chemistry. Synthesis of compounds 2−6 was achieved in
three steps. The first two steps (synthesis of β-nitrostyrene
derivatives 8a−e by nitroaldol condensation reaction (Henry
reaction17) with subsequent reduction to substituted phene-
thylamines 9a−e) were carried out using previously reported
procedures18 (Scheme 1). Target compounds were obtained
Scheme 1. Two Step Synthesis of Phenethylamines 9a−e
As the NBF substances, either with 4-substituent (25X-
NBF) or without 4-substituent (25H-NBF) on the phenyl ring
of the phenethylamine moiety, are pharmacologically scarcely
described, this study aimed to provide an estimation of what
substitution patterns are favorable for their in vitro receptor
activation. Therefore, we here describe the synthesis and
functional characterization of five positional isomers of 2-(2,5-
dimethoxyphenyl)-N-(2-fluorobenzyl)ethanamine (25H-NBF
(1)), namely, 2-(2,3-dimethoxyphenyl)-N-(2-fluorobenzyl)-
ethanamine (23H-NBF (2)), 2-(2,4-dimethoxyphenyl)-N-(2-
fluorobenzyl)ethanamine (24H-NBF (3)), 2-(2,6-dimethox-
yphenyl)-N-(2-fluorobenzyl)ethanamine (26H-NBF (4)), 2-
(3,4-dimethoxyphenyl)-N-(2-fluorobenzyl)ethanamine (34H-
NBF (5)), and 2-(3,5-dimethoxyphenyl)-N-(2-fluorobenzyl)-
ethanamine (35H-NBF (6)). The structures of these
compounds are shown in Figure 1. In these isomers, the two
methoxy groups on the phenylethylamine moiety, which are
situated at positions 2 and 5 in the original structure (hence
25H-NBF), are switched to different positions on the benzene
ring. Typically, the X in the 25X-NBF conventional
nomenclature (in this case, H for hydrogen) points at the
substituent at position 4 of the phenethylamine moiety. As
24H- and 34H-NBF carry a methoxy group at position 4, these
substances can theoretically also be denoted as 24-NBF and
34-NBF. In this study, we opted to refer to these compounds
as 24H-NBF and 34H-NBF, to emphasize that no other
substituents are introduced on the phenyl ring of the
phenethylamine moiety, besides the two methoxy groups. As
by a stepwise reductive N-amination from dimethoxyphene-
thylamines 9a−e via the formation of the corresponding imines
10a−e (Scheme 2), using the reported earlier method18 which
we adapted to the synthesized class of compounds. In the final
version, the synthesis of compounds 2−6 was carried out
according to Schemes 1 and 2.
Functional Evaluation. As this is the first report
describing the synthesis of this full set of positional isomers
of 25H-NBF, the pharmacological profile of these substances
was also unknown. Therefore, they were functionally
characterized by means of their potential to induce recruitment
of the cytosolic protein β-arrestin 2 (βarr2) to the target
receptor 5-HT2AR. To this end, a NanoBiT functional
complementation assay was employed, of which the assay
components were stably expressed in a previously developed
stable cell system.19 In this assay, each of two (inactive) parts
of a split-nanoluciferase (LgBiT and SmBiT) is fused to one of
two potentially interacting proteins, in this case the receptor
and βarr2. When the receptor (C-terminally fused to LgBit) is
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Scheme 2. Synthesis of Substituted N-(2-
Fluorobenzyl)phenylethanamines (2−6) via Reductive
Amination
Table 1. Quantitative Data Obtained for the Potency (EC50)
and Efficacy (Emax) for Each of the Tested Substances and
a
for the Reference Agonist LSD
compd
LSD
25H-NBF
23H-NBF
24H-NBF
26H-NBF
EC50 (nM, 95% CI)
Emax (%, 95% CI)
6.23 (4.78−7.94)
448 (304−647)
1175 (978−1533)
158 (120−219)
397 (261−596)
8382 (6566−12 000)
2566 (1889−3477)
100 (96.5−104)
106 (98.0−115)
96.1 (90.1−105)
107 (101−114)
107 (98.7−116)
75.2 (67.0−89.0)
58.9 (54.6−64.9)
b
34H-NBF
35H-NBF
a
Data are from three independent experiments, each performed in
duplicate. The maximal response in each individual experiment is
normalized to the maximal response of LSD (set at 100%). CI:
b
confidence interval. As the maximum of the sigmoidal curve was not
reached in the tested concentration range, care must be taken when
interpreting these values.
activated, βarr2 (N-terminally fused to SmBiT) is recruited,
leading to a reassociation of the split fragments. In the
presence of the enzyme’s substrate, this can then be monitored
via a luminescent readout, in which the luminescence is related
to the activity of the substance in a concentration-dependent
manner.20 This approach enabled the establishment of
concentration−response curves, as depicted in Figure 2, and
looking at the EC50 values, the most potent substance is 24H-
NBF, with 25H-NBF and 26H-NBF having potencies that are
2−3 times lower. Substantially lower potencies are then found
in 23H-NBF and 35H-NBF. 34H-NBF had the lowest
observed potency in this set of substances, not reaching the
maximal plateau of the concentration−response curve in the
tested concentration range. The Emax values, as a measure of
the efficacy of the substances, show values that are similar for
25H-, 24H-, and 26H-NBF, lying in the same range as that of
the reference agonist LSD. 23H-NBF tended to have a slightly
lower efficacy, while substantially lower values were obtained
for 35H- and 34H-NBF. The data obtained for the latter
substance have to be interpreted with caution, as no maximal
response was reached in the tested concentration range.
Overall, it can be concluded that 24H-NBF is the most active
substance in the in vitro bioassay, followed by 25H-NBF and
26H-NBF, with similar potencies and efficacies. Of the
substances carrying a methoxy group at position 2 of the
phenethylamine moiety, 23H-NBF is the least active substance.
The overall least active substances are 34H-NBF and 35H-
NBF.
The positioning of the two methoxy groups on the
phenethylamine moiety has been described mostly in
substances unsubstituted at the N-position and recently also
in their N-methoxybenzyl counterparts, the NBOMes.23 For
the former group, PiHKAL describes the 2,4,5-substitution of
the phenyl ring to be more effective than the 3,4,5-substitution
pattern, with the suggestion of a 2,4,6-substitution pattern to
be effective as well. In this first substitution pattern, positions 2
and 5 ideally carry a methoxy group, with more flexibility
possible at position 4.24 When testing desmethoxy analogues of
2C-B and DOB, the omission of either group appeared to more
dramatically impact the in vivo effects than the in vitro effects of
the substances. In in vitro functional assays, the removal of the
2-methoxy group resulted in a stronger decrease in activity
than that of the 5-methoxy group.25
Figure 2. Concentration−response curves of each of the tested 25H-
NBF isomers, together with that of the reference psychedelic
substance LSD. Each point represents the mean of three independent
experiments, each performed in duplicate SEM (standard error of
the mean).
the calculation of EC50 (as a measure of potency) and Emax
values (as a measure of efficacy, relative to the reference
agonist LSD). These latter values can be found in Table 1.
Data obtained for LSD and 25H-NBF are in the same range
as those previously reported employing our βarr2 recruitment
assays (in both transiently transfected and stably expressing
cells). For the reference agonist LSD, we have previously
reported EC50 values between 5.95 and 7.43 nM for the
recruitment of βarr2 to the 5-HT2AR.19,21−23 The value
obtained in this study, 6.23 nM, perfectly aligns with those
results. For 25H-NBF, we have previously reported values in
the high nanomolar range, with an efficacy that was similar
(104−107%) to that of the reference psychedelic substance
LSD.19,22 Also here, the EC50 value of the conventionally
substituted 25H-NBF lies in the higher nanomolar range (448
nM), with an efficacy of 106%, where that of LSD is set at
100%.
In previous work, we have evaluated the potencies and
efficacies of five positional isomers of 25H-NBOMe, where the
positions of the methoxy groups on the phenyl group of the
phenethylamine moiety were explored similarly as in this set of
substances.23 Overall, the observed trends of the -NBOMe
isomers were largely the same, irrespective of the reference
agonist (LSD versus serotonin), the calculation time point (2 h
versus 30 min AUC), or the bioassay used (recruitment of
When looking at the data in Table 1, several trends can be
observed concerning the activity of the isomers, considering
either the potency or the efficacy of the substances. When
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βarr2 versus miniGαq). The ranking based on potency was
24H > 25H − 26H > 23H > 35H − 34H-NBOMe, and 24H-,
25H-, 26H-, and 34H-NBOMe were categorized as more
efficacious substances, with 35H- and 23H-NBOMe being less
efficacious. Therefore, the data in the present study are in line
with our earlier findings, hinting at the importance of the
methoxy group at position 2 in both NBOMe and NBF
substances. However, combining this methoxy group at
position 2 with a 3-methoxy group negatively impacts activity
at the 5-HT2AR. Overall, our data also hint at the fact that
introducing a methoxy group at position 3 strongly decreases
the activity of a substance, likely owing to an altered
interaction with specific 5-HT2AR residues, as suggested by
molecular docking of the corresponding panel of -NBOMes
into a 5-HT2AR model, based on the cryo-EM structure of the
receptor with 25CN-NBOH.23,26
In terms of structure−activity relationship, introducing an
N-benzyl substituent at the amine function of a phenethyl-
amine has been reported to increase the activity of
substances.27,28 The best explored groups having this
substitution are -NBOMe, -NBMD, -NBOH, and -NBF
derivatives of 2C-X substances as N-unsubstituted counter-
parts. Consistent with our previously reported data, also here
25H-NBF is more potent and efficacious than 2C-H
(comparison with our historical data from 2C-H),19,22 the
counterpart that does not have the N-fluorobenzyl group at its
amine function. On the other hand, the -NBF isomers tested
here were consistently less potent than the corresponding
-NBOMe isomers. Jensen et al. ascribes this low potency and
efficacy for the -NBF substances to the absence of an oxygen
atom at position 2 on the N-benzyl group, that is present in the
corresponding -NBOMe, -NBMD, and -NBOH derivatives.29
Analogous to the NBOMe substances, O-demethylation of
methoxy groups has been reported to be an important
metabolic pathway in the degradation of 25X-NBF substances.
At this moment, it is unknown what the impact on metabolism
is of the altered positions of the methoxy groups, compared to
the 2,5-dimethoxy pattern.30 This is one of the limitations that
complicates the interpretation of data obtained in in vitro
functional assays and their potential translation to in vivo
outcomes. Additionally, the presented data do not allow for the
assessment of the binding affinities of the synthesized isomers,
which could provide more insight on how these ligands behave
in the presence of other (endogenous) ligands. The use of
other in vitro bioassays, monitoring different events along the
signaling cascade (such as Ca2+, miniGαq, PLC or PLA assays),
could potentially yield additional insights into the molecular
pharmacology of these substances. As the precise mechanism
of action of psychedelics at their (main) pharmacological
target, 5-HT2AR, has not been fully elucidated, it remains
unclear how the obtained results correlate with the in vivo
situation.31
24H − 25H − 26H ≥ 23H > 34H and 35H-NBF. Together
with data from previously published NBOMe positional
isomers, these data emphasize the importance of the methoxy
group at position 2 of the phenyl group of the phenethylamine
moiety for the in vitro activity of N-benzyl derived substances.
EXPERIMENTAL PROCEDURES
■
Materials, Solvents, and Reagents. The following starting
materials, reagents, and solvents were used for the synthesis of
compounds 2−6: 2-fluorobenzaldehyde (97%), 2,4-dimethoxybenzal-
dehyde (98%), 2,6-dimethoxybenzaldehyde (97%), 3,4-dimethoxy-
benzaldehyde (99%), and 3,5-dimethoxybenzaldehyde (98%), all
from Alfa Aesar (Kandel, Germany); 2,3-dimethoxybenzaldehyde
(97%), nitromethane (96%), lithium aluminum hydride (95%), and
sodium borohydride (99%), all from Acros Organics (Fair Lawn, NJ);
solvents: methylene chloride (≥99%, TU 2631-019-44493179-98),
acetone (≥99%, TU 2633-018-44493179-98), propanol-2 (≥99%, TU
2632-181-44493179-14), all from EKOS-1 (Moscow, Russia); diethyl
ether (≥99%, TU 2600-001-45286126-11) from Medhimprom
(Moscow, Russia); and methanol (≥99%, Russian GOST 6995-77)
from Vekton (St. Petersburg, Russia). Other reagents used were as
follows: glacial acetic acid (≥99%, Russian GOST 61-75), hydro-
chloric acid (≥99%, Russian GOST 3118-77), sodium chloride
(≥99%, Russian GOST 4233-77), ammonium acetate (≥98.5%,
Russian GOST 3117-78), sodium hydroxide (≥99%, Russian GOST
4328-77), all from Reachem (Moscow, Russia); and anhydrous
magnesium sulfate (≥99.0%, Bioshop, Canada), purchased commer-
cially and used without further purification. Tetrahydrofuran (99.8%,
Tathimprodukt, Kazan, Russia) was dried and purified using standard
procedures.32 Flash chromatography was carried out using high-purity
grade Merck silica gel (9385, 60 Å, 230−400 mesh). The analytical
standard of 25H-NBF hydrochloride was from Chiron AS
(Trondheim, Norway). Poly-D-lysine hydrobromide and the analytical
standard of LSD were from Sigma-Aldrich (Overijse, Belgium).
Dulbecco’s modified Eagle’s medium (DMEM, GlutaMAX), fetal
bovine serum (FBS), Hank’s balanced salt solution (HBSS),
penicillin/streptomycin, and amphotericin B were purchased from
Thermo Fisher Scientific (Pittsburgh, PA). Nano-Glo Live Cell
Reagent was from Promega (Madison, WI).
Instrumentation. NMR Spectroscopy. 1H, 13C, and 19F NMR
spectra were acquired in CD3OD at 400, 101, and 376 MHz,
respectively, using a Bruker Avance II spectrometer (Switzerland).
1
For H and 13C spectra, signals of residual protons and carbons from
the solvent were used as internal standards. To record the 19F spectra,
trichlorofluoromethane was used as an internal standard. For NMR
spectroscopy, CD3OD of isotopic purity ≥ 99.5% (Russian TU 95-
669-79, FSUE RSC “Applied Chemistry,” St. Petersburg, Russia) was
used.
Melting Point. The melting points of compounds 2−6 were
obtained using a Stuart SMP 30 (Bibby Scientific, Ltd., UK) digital
melting temperature measuring instrument at a rate of 1 °C/min.
Gas Chromatography−Mass Spectrometry (GC−MS). GC−MS
analysis was performed using an Agilent 7890B/5977A system
(Agilent Technologies, USA) with a HP-5 ms (30.0 m × 0.25 mm
× 0.25 μm; 19091S-433; Agilent) capillary column. The oven
temperature was maintained at 70 °C for 1.0 min and then
programmed at 15 °C/min to 295 °C, which was maintained for
15 min. The injector temperature and the interface temperature were
280 and 290 °C, respectively. Helium with flow rate of 1.0 mL/min
was used as the carrier gas.
Ultrahigh-Performance Liquid Chromatography−High Resolu-
tion Mass Spectrometry (UHPLC−HRMS). High resolution mass
spectra were recorded using an Agilent 1290 Infinity II UHPLC
system with a tandem quadrupole time-of-flight (Q-TOF) accurate
mass detector Agilent 6545 Q-TOF LC−MS (Agilent Technologies,
USA). The Q-TOF instrument was operated with an electrospray ion
source in positive ion mode. The mobile phase was prepared from
0.1% aqueous formic acid (solvent A) and 0.1% formic acid in
acetonitrile (solvent B). A gradient of 5% solvent B to 100% solvent B
In conclusion, this study is the first report describing the
synthesis of all five 25H-NBF isomers with different positions
of two methoxy groups in the phenethylamine moiety.
Functional evaluation of these isomers in an in vitro βarr2
recruitment assay yielded marked differences, based on either
the EC50 or Emax values. Focusing on the potencies of the
substances, 24H-NBF was found to be the most potent,
followed by 25H- and 26H-NBF, with 23H-NBF being the
least potent substance with a 2-methoxy group. 34H- and 35H-
NBF are the least potent substances in the tested panel.
Looking at the efficacies, a highly similar ranking is obtained:
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at 5 min, with a flow rate of 0.4 mL/min, was employed. UHPLC
analysis was performed on a Zorbax Eclipse Plus C18 RRHD (2.1 mm
× 50 mm × 1.8 μm) column with additional 5 mm guard column.
Acetonitrile (HPLC-gradient grade, Panreac, Barcelona, Spain), water
(for GC, HPLC, and spectrophotometry grade, Honeywell, Burdick,
and Jackson, Muskegon, MI) and formic acid (≥98.0%) from Sigma-
Aldrich (Steinheim, Germany) were used for UHPLC-HRMS.
Synthetic Procedures. General Procedure for the Synthesis of
2-Dimethoxyphenyl-N-(2-fluorobenzyl)ethanamines (2−6). To a
solution of substituted phenethylamine 9a−e (1.0 g, 5.52 mmol) in
CH2Cl2 (40 mL) with cooling to 0 °C was added a solution of 2-
fluorobenzaldehyde (0.788 g, 6.35 mmol) in CH2Cl2 (40 mL)
dropwise over 2 h. Stirring with cooling was continued for another 2
h. The completeness of the reaction and identification of the formed
imines 10a−e were monitored using GC−MS. Then the solvent was
evaporated, the residue was dissolved in MeOH abs. (80 mL),
followed by the addition of NaBH4 (0.839 g, 22.08 mmol) over 1 h.
The reaction was quenched with distilled H2O (20 mL), the organic
phase was evaporated to dryness, and the pH of the residual solution
was adjusted to 11 with 3 M aqueous NaOH. Products-bases 2−6
were extracted with CH2Cl2 (3 × 40 mL), the combined extracts were
washed with distilled H2O (2 × 30 mL) and brine (40 mL), dried
over anhydrous MgSO4, and evaporated. Then, with stirring, 5 mL of
propan-2-ol saturated with 5 M hydrochloric acid was added
dropwise. After filtering off, washing with diethyl ether, and drying,
hydrochlorides 2−6 were obtained as white powders. Compounds 2−
6 were purified by flash chromatography and extensively analytically
characterized, with no detectable impurities present.
2-(2,6-Dimethoxyphenyl)-N-(2-fluorobenzyl)ethanamine Hydro-
chloride (26H-NBF, (4)). White powder, yield 83%; 1H NMR
(CD3OD) δ, ppm: 3.06−3.22 (m, 4H), 3.85 (s, 6H), 4.34 (s, 2H),
3
6.67 (d, J = 8.4 Hz, 2H), 7.19−7.35 (m, 3H), 7.48−7.62 (m, 2H);
13C NMR (CD3OD) δ, ppm: 20.85, 45.38 (d, 3JC−F = 3.9 Hz), 47.76;
56.26 (2C), 104.95 (2C), 113.07, 116.96 (d, 2JC−F = 21.5 Hz), 119.85
2
3
(d, JC−F = 14.7 Hz), 126.20 (d, JC−F = 3.7 Hz), 130.01, 133.20 (d,
3JC−F = 8.4 Hz), 133.28 (d, JC−F = 2.8 Hz), 159.75 (2C), 162.72 (d,
4
1JC−F = 247.6 Hz); 19F NMR (CD3OD) δ, ppm: −118.16 (1F);
HRMS (ESI, [M + H]+), m/z: accurate mass 290.1555, exact mass
290.1551, Δ = −1.27 ppm, C17H20FNO2; melting point 118−120 °C.
2-(3,4-Dimethoxyphenyl)-N-(2-fluorobenzyl)ethanamine Hydro-
chloride (34H-NBF, (5)). White powder, yield 82%; 1H NMR
(CD3OD) δ, ppm: 2.99−3.09 (m, 2H), 3.28−3.38 (m, 2H), 3.82
3
(s, 3H), 3.85 (s, 3H), 4.35 (s, 2H), 6.86 (d, J = 8.0 Hz, 1H), 6.89−
6.97 (m, 2H), 7.22−7.34 (m, 2H), 7.48−7.57 (m, 1H), 7.65 (td, 3J =
7.6 Hz/4J = 1.6 Hz, 1H); 13C NMR (CD3OD) δ, ppm: 32.76, 45.59
3
(d, JC−F = 4.0 Hz), 50.13; 56.60 (2C), 113.50, 113.80, 116.96 (d,
2JC−F = 21.5 Hz), 119.84 (d, JC−F = 14.8 Hz), 122.25, 126.23 (d,
2
3JC−F = 3.7 Hz), 130.50, 133.32 (d, 3JC−1F = 8.4 Hz), 133.50 (d, 4JC−F
=
2.7 Hz), 149.79, 150.84, 162.71 (d, JC−F = 247.7 Hz); 19F NMR
(CD3OD) δ, ppm: −118.04 (1F); HRMS (ESI, [M + H]+), m/z:
accurate mass 290.1553, exact mass 290.1551, Δ = −0.87 ppm,
C17H20FNO2; melting point 163−165 °C.
2-(3,5-Dimethoxyphenyl)-N-(2-fluorobenzyl)ethanamine Hydro-
chloride (35H-NBF, (6)). White powder, yield 77%; 1H NMR
(CD3OD) δ, ppm: 2.98−3.06 (m, 2H), 3.29−3.36 (m, 2H), 3.78
(s, 6H), 4.34 (s, 2H), 6.40 (t, 3J = 2.2 Hz, 1H), 6.48 (d, 3J = 2.2 Hz,
2H), 7.22−7.34 (m, 2H), 7.49−7.56 (m, 1H), 7.64 (td, 3J = 7.6 Hz/4J
Analytical data. 23H-NBF Imine (10a). MS (EI, 70 eV), m/z
(%): 287 (M+•), 256 (100), 109 (60), 136 (51), 257 (20), 91 (12),
107 (8), 135 (8), 65 (6), 108 (6), 77 (5).
= 1.6 Hz, 1H); 13C NMR (CD3OD) δ, ppm: 33.40, 45.60 (d, 3JC−F
=
=
2
4.0 Hz), 49.82; 56.87 (2C), 100.11, 107.85 (2C), 116.97 (d, JC−F
24H-NBF Imine (10b). MS (EI, 70 eV), m/z (%): 287 (M+•), 151
(100), 109 (28), 121 (28), 91 (12), 152 (10), 136 (9), 77 (8), 108
(7), 78 (7), 107 (7).
2
3
21.4 Hz), 119.79 (d, JC−F = 14.8 Hz), 126.23 (d, JC−F = 3.7 Hz),
133.33 (d, 3JC−F = 8.4 Hz), 133.49 (d, 4JC−F = 2.8 Hz), 139.96, 162.71
(d, JC−F = 247.8 Hz), 162.80 (2C); 19F NMR (CD3OD) δ, ppm:
1
26H-NBF Imine (10c). MS (EI, 70 eV), m/z (%): 287 (M+•), 151
(100), 256 (78), 109 (62), 136 (43), 91 (39), 257 (15), 77 (11), 107
(11), 108 (11), 152 (11).
−118.14 (1F); HRMS (ESI, [M + H]+), m/z: accurate mass
290.1553, exact mass 290.1551, Δ = −0.88 ppm, C17H20FNO2;
melting point 126−128 °C.
34H-NBF Imine (10d). MS (EI, 70 eV), m/z (%): 287 (M+•), 151
(100), 109 (51), 136 (35), 287 (18), 107 (15), 152 (10), 108 (8),
135 (7), 106 (6), 78 (6).
Routine Cell Culture and NanoBiT βarr2 Recruitment Assay.
The generation of human embryonic kidney (HEK) 293T cells that
stably express the components of the NanoBiT functional
complementation assay has been previously described.19 The cells
were routinely cultured in Dulbecco’s modified Eagle’s medium
(DMEM, GlutaMAX) supplemented with 10% heat-inactivated FBS,
100 IU/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL
amphotericin B and maintained in a humidified atmosphere at 37 °C
and 5% CO2. For the implementation of the in vitro functional assay,
the cells were seeded in poly-D-lysine coated 96-well plates and
incubated overnight. The cells were then rinsed twice with HBSS, and
100 μL of HBSS was added to each of the wells, followed by the
addition of 25 μL of the Nano-Glo Live Cell Reagent (diluted 1/20 in
the provided LCS buffer, according to the manufacturer’s protocol)
and equilibration in the Tristar2 LB 942 multimode microplate reader
(Berthold Technologies GmbH & Co, Germany). After stabilization
of the luminescent signal, a series of 13.5× concentrated agonist
dilutions was added to the wells, followed by a continuous readout for
2 h. The measured concentrations were (100 μM) − 25 μM − 10 μM
− 1 μM − 10−7 M − 10−8 M − 10−9 M − (10−10 M). In total, three
independent experiments were carried out, each performed in
duplicate, including LSD as a reference agonist for the purpose of
normalization and the appropriate solvent controls for blank
correction.
35H-NBF Imine (10e). MS (EI, 70 eV), m/z (%): 287 (M+•), 109
(100), 136 (94), 192 (30), 287 (29), 166 (17), 137 (11), 77 (10),
108 (10), 151 (9), 91 (9).
2-(2,3-Dimethoxyphenyl)-N-(2-fluorobenzyl)ethanamine Hydro-
chloride (23H-NBF, (2)). White powder, yield 87%; 1H NMR
(CD3OD) δ, ppm: 3.05−3.14 (m, 2H), 3.23−3.31 (m, 2H), 3.85
(s, 3H), 3.86 (s, 3H), 4.36 (s, 2H), 6.87 (d, 3J = 7.6 Hz, 1H), 6.97 (d,
3J = 7.2 Hz), 7.05 (t, 3J = 8.0 Hz, 1H), 7.21−7.34 (m, 2H), 7.48−7.56
(m, 1H); 7.61−7.68 (m, 1H); 13C NMR (CD3OD) δ, ppm: 28.19,
3
45.48 (d, JC−F = 4.0 Hz), 49.05, 56.35, 61.25, 113.38, 116.97 (d,
2JC−F = 21.5 Hz), 119.79 (d, 2JC−F = 14.9 Hz), 123.23, 125.64, 126.24
3
3
(d, JC−F = 3.7 Hz), 131.05, 133.30 (d, JC−F = 8.3 Hz), 133.48 (d,
4JC−F = 2.8 Hz), 148.66, 154.28, 162.71 (d, JC−F = 247.8 Hz); 19F
1
NMR (CD3OD) δ, ppm: −117.96 (1F); HRMS (ESI, [M + H]+), m/
z: accurate mass 290.1552, exact mass 290.1551, Δ = −0.57 ppm,
C17H20FNO2; melting point 153−155 °C.
2-(2,4-Dimethoxyphenyl)-N-(2-fluorobenzyl)ethanamine Hydro-
chloride (24H-NBF, (3)). White powder, yield 72%; 1H NMR
(CD3OD) δ, ppm: 2.96−3.04 (m, 2H), 3.19−3.28 (m, 2H), 3.80
(s, 3H), 3.83 (s, 3H), 4.33 (s, 2H), 6.50 (dd, 3J = 8.2 Hz/4J = 2.4 Hz,
1H), 6.56 (d, 4J = 2.4 Hz, 1H), 7.13 (d, 3J = 8.2 Hz, 1H), 7.22−7.35
(m, 2H), 7.48−7.56 (m, 1H), 7.62 (td, 3J = 7.6 Hz/4J = 1.6 Hz, 1H);
13C NMR (CD3OD) δ, ppm: 27.91, 45.45 (d, 3JC−F = 4.0 Hz), 48.67,
Data Analysis. The data were analyzed as previously described in
more detail.33 In brief, the obtained activation profiles are plotted,
data are corrected for interwell variability, and the corresponding
AUC (area under the curve) values are calculated. After subtraction of
the AUC value of the corresponding solvent control (“blank”), the
data are normalized in GraphPad (San Diego, CA) and sigmoidal
curves are fit through the four-parameter nonlinear regression model.
The data of the three independent experiments are then used to
calculate potency (EC50) and efficacy (Emax) parameters.
2
55.90, 55.93, 99.62, 105.96, 116.95 (d, JC−F = 21.6 Hz), 117.73,
2
3
119.85 (d, JC−F = 14.8 Hz), 126.21 (d, JC−F = 3.7 Hz), 132.04,
3
4
133.27 (d, JC−F = 8.4 Hz), 133.42 (d, JC−F = 2.8 Hz), 159.90,
162.13, 162.72 (d, JC−F = 247.8 Hz); 19F NMR (CD3OD) δ, ppm:
1
−118.31 (1F); HRMS (ESI, [M + H]+), m/z: accurate mass
290.1553, exact mass 290.1551, Δ = −0.79 ppm, C17H20FNO2;
melting point 123−124 °C.
1671
https://doi.org/10.1021/acschemneuro.1c00124
ACS Chem. Neurosci. 2021, 12, 1667−1673

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
A Randomized Clinical Trial. JAMA psychiatry, DOI: 10.1001/
jamapsychiatry.2020.3285.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acschemneuro.1c00124.
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Relationships of Psychedelics. Curr. Top. Behav. Neurosci. 36, 1−43.
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Electrospray Ionization Mass Spectrometry Imaging of Cimbi-36, a 5-
HT2A Receptor Agonist, with Direct Comparison to Autoradiog-
raphy and Positron Emission Tomography. Mol. Imaging Biol.,
DOI: 10.1007/s11307-021-01592-2.
EI spectra of imine intermediates, the 1H, 13C, 19F NMR
spectra, data of elemental analysis and LC-MS
chromatograms for the final synthesized compounds
(PDF)
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AUTHOR INFORMATION
■
Corresponding Author
Christophe P. Stove − Laboratory of Toxicology, Department
of Bioanalysis, Faculty of Pharmaceutical Sciences, Ghent
University, B-9000 Ghent, Belgium; orcid.org/0000-
0001-7126-348X; Phone: +32 9 264 81 35;
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Email: christophe.stove@ugent.be; Fax: +32 9 264 81 83
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cessed February 2021).
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(Ketamine etc.) (Amendment) Order 2014. https://www.legislation.
gov.uk/uksi/2014/1106/made (accessed February 2021).
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K., Ma, S. X., Kim, Y. J., Jeong, Y., Pham, D. T., Trinh, Q. D., Shin, E.
J., Kim, H. C., Lee, Y. S., Lee, S. Y., and Jang, C. G. (2020) 25C-NBF,
a New Psychoactive Substance, has Addictive and Neurotoxic
Potential in Rodents. Arch. Toxicol. 94, 2505−2516.
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Verkman, A. S. (2006) Small Molecules with Antimicrobial Activity
against E. coli and P. aeruginosa Identified by High-Throughput
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(18) Kupriyanova, O. V., Shevyrin, V. A., Shafran, Y. M., Lebedev, A.
T., Milyukov, V. A., and Rusinov, V. L. (2020) Synthesis and
Determination of Analytical Characteristics and Differentiation of
Positional Isomers in the Series of N-(2-Methoxybenzyl)-2-
(Dimethoxyphenyl)ethanamine using Chromatography-Mass Spec-
trometry. Drug Test. Anal. 12, 1154−1170.
(19) Pottie, E., Cannaert, A., and Stove, C. P. (2020) In Vitro
Structure-activity Relationship Determination of 30 Psychedelic New
Psychoactive Substances by means of Beta-arrestin 2 Recruitment to
the Serotonin 2A Receptor. Arch. Toxicol. 94, 3449.
(20) Dixon, A. S., Schwinn, M. K., Hall, M. P., Zimmerman, K.,
Otto, P., Lubben, T. H., Butler, B. L., Binkowski, B. F., Machleidt, T.,
Kirkland, T. A., Wood, M. G., Eggers, C. T., Encell, L. P., and Wood,
K. V. (2016) NanoLuc Complementation Reporter Optimized for
Accurate Measurement of Protein Interactions in Cells. ACS Chem.
Biol. 11, 400−408.
(21) Pottie, E., Cannaert, A., Van Uytfanghe, K., and Stove, C. P.
(2019) Setup of a Serotonin 2A Receptor (5-HT2AR) Bioassay:
Demonstration of Its Applicability To Functionally Characterize
Hallucinogenic New Psychoactive Substances and an Explanation
Why 5-HT2AR Bioassays Are Not Suited for Universal Activity-Based
Screening of Biofluids for New Psychoactive Substances. Anal. Chem.
91, 15444−15452.
Authors
Eline Pottie − Laboratory of Toxicology, Department of
Bioanalysis, Faculty of Pharmaceutical Sciences, Ghent
University, B-9000 Ghent, Belgium
Olga V. Kupriyanova − Institute of Fundamental Medicine
and Biology, Kazan (Volga Region) Federal University,
420008 Kazan, Russian Federation; Kazan State Medical
University, 420012 Kazan, Russian Federation
Vadim A. Shevyrin − Institute of Chemistry and Technology,
Ural Federal University, 620002 Ekaterinburg, Russian
Federation; orcid.org/0000-0002-0369-0786
Complete contact information is available at:
https://pubs.acs.org/10.1021/acschemneuro.1c00124
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
C.P.S. acknowledges funding by the Research Foundation-
Flanders (FWO) [G069419N and G0B8817N] and the Ghent
University − Special Research Fund (BOF) [01J15517].
O.V.K. acknowledges support by the subsidy allocated to the
Kazan Federal University for the state assignment in the field
of scientific activity (Project 0671-2020-0058); the research
was performed using the equipment of the Interdisciplinary
centre for shared use of Kazan Federal University. V.A.S.
acknowledges Russian Science Foundation for financially
supporting this work, Project 20-13-00089.
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