Investigation of the 2,5-Dimethoxy Motif in Phenethylamine Serotonin 2A Receptor Agonists

Article abstract of DOI:10.1021/acschemneuro.0c00129

The 2,5-dimethoxyphenethylamine (2,5-PEA) scaffold is recognized as a motif conferring potent agonist activity at the serotonin 2A receptor (5-HT_2AR). The 2,5-dimethoxy motif is present in several classical phenethylamine psychedelics such as 2

Full text of DOI:10.1021/acschemneuro.0c00129

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Letter
Investigation of the 2,5-dimethoxy motif in
phenethylamine Serotonin 2A receptor agonists
Emil Marcher-Rørsted, Adam L Halberstadt, Adam K Klein, Muhammad
Chatha, Simon Jademyr, Anders A. Jensen, and Jesper L. Kristensen
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.0c00129 • Publication Date (Web): 26 Mar 2020
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Investigation of the 2,5-dimethoxy motif in phenethylamine
Serotonin 2A receptor agonists
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Emil Marcher-Rørsted1, Adam L Halberstadt2, Adam K. Klein2, Muhammad Chatha2, Simon
Jademyr1, Anders A. Jensen1, Jesper L. Kristensen1*
Institute of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen,
Denmark.1
Department of Psychiatry, University of California San Diego, 9500 Gilman Dr. La Jolla, California2
KEYWORDS: Phenethylamine, Serotonin 2A receptor, agonists, Structure activity relationship.
ABSTRACT: The 2,5-dimethoxyphenethylamine (2, 5-PEA) scaffold is recognized as a motif conferring potent agonist
activity at the serotonin 2A receptor (5-HT2AR). The 2,5-dimethoxy motif is present in several classical phenethylamine
psychedelics such as mescaline, TMA-2, DOM, DOI, DOB, 2C-B and 2C-I, and it has previously been suggested that this
structural motif is essential for 5-HT2AR activation. In the present study we present data that challenges this assumption.
The 2- and 5-desmethoxy derivatives of 2C-B and DOB were synthesized and their pharmacological profiles evaluated in
vitro at 5-HT2AR and 5-HT2CR in binding and functional assays and in vivo by assessing their induction of Head Twitch
Response in mice. Elimination of either the 2- or 5-methoxy leads to a modest drop in binding affinity and functional
potency at 5-HT2AR and 5-HT2CR, which was more pronounced upon removal of the 5-methoxy. However, this trend was
not mirrored in vivo, as removal of either methoxy group resulted in significant reduction in the compounds ability to
induce the Head Twitch Response in mice. Thus, the 2,5-dimethoxyphenethylamine motif appears to be important for in
vivo potency of phenethylamine 5-HT2AR agonists, but this does not correlate to the relative affinity and potency of the
ligands at the recombinant 5-HT2AR.
For the past five decades, agonistic activity at the serotonin 2A receptor (5-HT2AR) has been viewed primarily as an
undesirable off-target effect within the field of drug-development. This subclass of serotonin receptors has been linked to
(and is thought to be primarily responsible for inducing) - the psychedelic experience induced by hallucinogenic drugs in
man1,2. Compounds known to work as agonists at 5-HT2AR include the classic psychedelics lysergic acid diethylamide ((+)-
LSD), psilocybin and 2C-B, each representing one of the three primary structural classes interacting with this target
receptor, ergolines, the tryptamines and phenethylamines respectively3.
Recently several classic psychedelic drugs have been investigated as potential therapeutic agents for treating multiple
psychiatric disorders4–6. In light of this recent development the effects and mechanisms of action of 5-HT2AR agonists are
now receiving considerable attention. As this new avenue of treatment is explored to address several psychiatric illnesses,
including treatment-resistant depression, interest in the development of new 5HT2AR ligands is gaining momentum.
Therefore elucidating the key structural elements required for agonist activity at this receptor target is of high importance.
Among 5HT2AR ligands one of the most prevalent chemical motifs is the 2, 5-dimethoxyphenthylamine scaffold, which is
present in many well characterized high potency compounds7.
The current study analyzes the role that the 2,5-dimethoxy motif plays in the activity of the classic 5-HT2AR agonists 2,5-
dimethoxy-4-bromophenethylamine (2C-B, 1, Fig 1) and 4-bromo-2,5-dimethoxyamphetamine (DOB, 2, Fig 1). Specifically,
compounds 3-6 (Fig 1), the desmethoxy derivatives of DOB and 2C-B, were synthesized, analyzed, and characterized.
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O
O
1
2
NH2
NH2
NH2
3
4
5
Br
Br
Br
O
O
6
7
2C-B (1)
3
5
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O
O
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NH2
NH2
NH2
Br
Br
Br
O
O
DOB (2)
4
6
Figure 1. Structures of the phenethylamines 1-6 investigated in the present study.
Inspired by the prototypical phenethylamine psychedelic mescaline (7, Fig 2), many studies have examined the structure-
activity relationships (SAR) of phenethylamine hallucinogens at 5-HT2 receptors8. Phenethylamines containing a 2,5-
dimethoxy-substitution pattern and a lipophilic 4-substituent (such as halogen, alkyl, alkoxy or alkylthio) have relatively
high potency at those receptor subtypes. Methylation of the α-position further increases in vivo potency, with
amphetamines such as, 2, 2,4,5-trimethoxyamphetamine (TMA-2, 8, Fig 2), and 2,5-dimethoxy-4-methylamphetamine
(DOM, 9, Fig 2) being prominent examples9,10
.
Numerus potent analogs of 2 have been synthesized by varying the 4-position substituent11,12. The presence of methoxy
groups in the 2- and 5-positions has been reported to be important for 5-HT2AR activation and thus has been preserved in
most investigations. Shulgin observed that replacement of either the 2- (10, Fig 2) or the 5-position (11, Fig 2) oxygen in 9
with a sulfur atom reduces its hallucinogenic potency by approximately 15- or 10-fold, respectively13. Replacing both oxygen
atoms with sulfur (12, Fig 2) completely abolished activity. Removal of the 2- or 5-position methoxy-group in 2,4,5-
substituted compounds is also detrimental for their in vivo activity. For example, 2,4-dimethoxyamphetamine(13, Fig 2) and
3,4-dimethoxyamphetamine(14, Fig 2) are less potent than 8 in humans and in rats trained to discriminate 9.14,15 Likewise,
6 is significantly less potent than 2 and produces only partial generalization in DOM-trained rats.16 Other studies examining
the stimulus effects of 9 and 2-desmethoxy-DOM(15, Fig 2) in rats trained to discriminate LSD has found the former to
produce full substitution with an ED50 of 0.148 mg/kg, whereas the latter is almost an order of magnitude less potent and
does not fully substitute for LSD.15,17
Evidence strongly indicates that the psychedelic effects produced by serotonergic hallucinogens are mediated by activation
of 5-HT2AR18,19. Due to a high degree of homology between 5-HT2A, 5-HT2B and 5-HT2C receptors, compounds active at 5-
HT2AR often show little to no selectivity at the two other subtypes. 2 displays high affinity for 5-HT2ARs in rat cortical
homogenates in a [3H]ketanserin binding assay (Ki = 63 nM), whereas 4 (Ki > 25,000 nM) and 6 (Ki = 4,870 nM) displayed
markedly lower 5-HT2AR affinity 16,20 Nichols and collaborators found that the affinity and potency of 9 at 5-HT2AR is reduced
if one of the methoxy groups is replaced with an ethyl group, giving 2-Et-DOM (16, Fig 2) and 5-Et-DOM (17, Fig 2), which
is further corroborated by the very low 5-HT2AR affinity exhibited by the DOB analog 4-bromo-2,5-diflouroamphetamine
(18, Fig 2) 21,22
.
Westkaemper and Glennon have proposed that the 2,5-dimethoxy substitution pattern may enhance 5-HT2AR affinity
because of hydrogen bond interactions between the methoxy groups and serine residues in the binding pocket, and
subsequent mutagenesis and homology modelling studies identified Ser-159 and Ser-239 as likely candidates to interact
with the 2- and 5-methoxy groups, respectively21,23,24
.
Molecular modeling studies published in 2017 by Roth et al. based on the 5-HT2BR receptor crystal structure (-which shares
significant homology to the 2A subtype), have led to significant insight into the binding mode of ligands in the orthosteric
pocket of 5-HT2AR25. However, a crystal structure of the 5-HT2AR bound to a phenethylamine-based agonist ligand remains
elusive; -therefore relevant interactions within the orthosteric binding pocket have yet to be fully elucidated.
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O
O
1
2
3
4
NH2
O
O
NH2
NH2
O
O
O
O
5
6
7
Mescaline (7)
TMA-2 (8)
DOM (9)
8
9
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O
S
NH2
NH2
NH2
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S
S
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O
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NH2
NH2
NH2
O
O
O
O
13
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O
F
NH2
NH2
NH2
Br
F
O
2-Et-DOM (16)
5-Et-DOM (17)
18
O
R
H
N
O
H
N
O
OH
Br
N
O
20
21
22
: R = OCH3
: R = OH
: R = H
25CN-NBOH (19)
Figure 2. Structure and analogs of prototypical phenethylamine derived 5-HT2AR agonists.
The search for 5-HT2AR selective compounds, using the 2,5 dimethoxy phenethylamine scaffold as a starting point, led to
the discovery of the NBOMe class of benzylated phenethylamines26–28. While N-methylation of phenethylamines causes a
significant reduction of 5-HT2AR binding affinity, N-benzylation yields highly potent 5-HT2AR agonists29–32. 25CN-NBOH(19,
Fig 2) emerged from this compound class as a selective 5-HT2AR agonist amenable for in vivo investigations33–37
.
Investigations into the metabolism of the NBOMe class of ligands using 25B-NBOMe (20, Fig 1) lead us to conclude that the
5-position methoxy-group is a metabolic soft spot with 2-bromo-4-methoxy-5-(2-((2-methoxybenzyl)amino)ethyl)phenol
(21, Fig 2) being the primary phase 1 metabolite38. This prompted us to synthesize and evaluate the 5-desmethoxy analog 2-
(4-bromo-2-methoxyphenyl)-N-(2-methoxybenzyl)ethan-1-amine (22, Fig 2)39. To our surprise, removal of the 5-methoxy-
group from the NBOMe-scaffold resulted in only a 10-fold decrease in 5-HT2AR binding affinity compared to the parent
compound 20 (pKi 9.68 and 8.68, respectively), whereas the effect on efficacy was even smaller (20: pEC50 = 9.04, Rmax
83%; 22: pEC50 = 8.87, Rmax = 70%). This observation motivated us to re-investigate the importance of the 2,5-dimethoxy
=
motif on the activity of the parent phenethylamine and amphetamine scaffold.
Due to the large degree of homology between 5HT2 subtypes numerous scientific studies have investigated 5HT2AR over
5HT2CR selectivity in this drug class33,40,41. Historically, the functional properties of 5HT2AR agonists have been characterized
by their selectivity for the 2A over the 2C subtype. For this reason we were prompted to assess the implications of modifying
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this structural motif, with regards to the selectivity profile of these compounds. Thus, in the present study, the des-methoxy
analogs of 1 and 2 were synthesized and evaluated pharmacologically in vitro and in vivo.
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2
3
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5
6
7
8
9
Synthesis of 1 and 2 was carried out using the procedures previously reported by Heim and Aldous, respectively26,42
.
Synthesis of the des-methoxy analogs 3-6 was achieved in two steps by condensing the appropriate brominated
anisaldehyde with either nitromethane or nitroethane. Subsequent reduction of the formed nitroalkenes (23-26, Fig 3) using
diisopropylaluminium hydride yielded the desired phenethylamine and amphetamines (3-6, Fig 3)(see supporting
information for full experimental details). The chiral amphetamine compounds were used as racemic mixture and not
separated further as it has been established that the activity of 2 and several other chiral amphetamines resides primarily
at in the R-enantiomer, which boasts 6-fold higher potency at the 5HT2AR as compared to its corresponding S-
enantiomere.43,44
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O
O
NH2
NH2
a
1
2
: R1 = H
: R1 = CH3
R1
R1
Br
O
O
1, 2
R1
R1
O
R1
NH2
NO2
d
b/c
R3
R3
Br
Br
Br
R2
R2
R2
23-26
3-6
3, 23
4, 24
5, 25
6, 26
: R1 = H; R2 = OMe; R3 = H
: R1 = H; R2 = OMe; R3 = CH3
: R1 = OMe; R2 = H; R3 = H
: R1 = OMe; R2 = H; R3 = CH3
Figure 3. (a)Br2, AcOH, 3 h, rt; (b) MeNO2, AcOH, Et(NH2)2, i-PrOH, 75 ⁰C, 1 h; (c) EtNO2, AcOH, Et(NH2)2, i-PrOH, 75⁰C, 1 h;
(d) DIBAL-H, THF, 60 ⁰C, 2 h.
The binding properties of compounds 1-6 at human 5-HT2AR, 5-HT2BR and 5-HT2CR were characterized using membranes
from tsA201 cells transiently expressing the three receptors in a [3H]LSD competition binding assay (Table 1.). 1 and 2 were
found to display comparable Ki values at the three receptors in the assay. For 5-HT2AR, removal of the 2-methoxy(4) and
the 5-methoxy group(6) in 2 resulted in 9- and 26-fold lower binding affinity respectively, whereas removal of the 2-
methoxy(3) and the 5-methoxy(5) group in 1 resulted in 17- and 31-fold lower binding affinities, respectively. Similar
reductions in binding affinity were observed for the des-methoxy analogs compared to their parent compounds at 5-HT2BR
and 5-HT2CR (Table 1.).
5-HT2A
R
5-HT2B
R
5-HT2CR
Binding
selectivity
(2A/2B)
3
Binding
selectivity
(2A/2C)
1
Ki (nM) [pKi ± SEM]
32 [7.50 ± 0.04]
Ki (nM) [pKi ± SEM]
97 [7.01 ± 0.08]
Ki (nM) [pKi ± SEM]
32 [7.49 ± 0.12]
Compound
1 (2C-B)
1.9
0.6
0.5
1.3
1.4
0.4
0.5
1.4
2 (DOB)
25 [7.61 ± 0.07]
48 [7.32 ± 0.04]
590 [6.23 ± 0.11]
300 [6.52 ± 0.07]
710 [6.15 ± 0.08]
260 [6.58 ± 0.13]
34 [7.47 ± 0.07]
3
4
5
6
1,000 [5.98 ± 0.04]
650 [6.19 ± 0.09]
540 [6.27 ± 0.12]
230 [6.63 ± 0.10]
380 [6.42 ± 0.09]
330 [6.48 ± 0.06]
760 [6.12 ± 0.05]
1,100 [5.97 ± 0.11]
1.1
4.7
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Table 1. Binding affinities of compounds 1-6 at 5-HT2AR, 5-HT2BR and 5-HT2CR, based on[3H]LSD competition binding assays
(affinities are provided as Ki values in nM, with pKi ± SEM in brackets). The receptors were expressed in tsA201 cells. The data
are based on 3 independent experiments.
1
2
3
4
The functional agonist activities of compounds 1-6 were next characterized at human 5-HT2AR and 5-HT2CR stably expressed
in HEK293 cells in a fluorescence-based calcium flux assay (Table 2).45 Overall, the four des-methoxy analogs exhibited
significantly lower agonist potencies compared to their respective parent compounds at both 5-HT2R subtypes, whereas
their agonist efficacy was largely unaffected, with all analogs acting as high-efficacy partial agonists, bordering to full
agonism, at both receptors (Table 2.). For example, analogs 3 and 5 activated 5HT2AR with 27- and 6-fold lower potency
than 1, and analogs 4 and 6 activated 5HT2AR with 70- and 17-fold lower potency than 2. Thus, in both series of compounds
removal of the 2-methoxy group had a notably greater effect on 5-HT2AR potency compared to removal of the 5-methoxy
group, which aligns well with the binding affinity data. Neither the modest 2A/2C selectivity exhibited by 2 nor the
equipotent activity of 1 at the two receptors were influenced substantially by the removal of the methoxy groups (Table 2.).
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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25
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32
33
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36
37
38
39
40
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42
43
44
45
46
47
48
49
50
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52
53
54
55
56
57
58
59
60
5-HT2A
R
5-HT2C
R
Functional
selectivity
(2A/2C)
n
4
n
4
Compound
EC50 (nM) [ pEC50]
1.6 [8.79 ± 0.08]
0.2 [9.71 ± 0.16]
42 [7.38 ± 0.08]
14 [7.86 ± 0.05]
11 [7.97 ± 0.04]
3.4 [8.47± 0.09]
Rmax
EC50 (nM) [pEC50]
4.1 [8.36 ± 0.02]
4.1 [8.38 ± 0.18]
64 [7.19 ± 0.07]
91 [7.04 ± 0.04]
21 [7.68 ± 0.11]
Rmax
1 (2C-B)
68 (± 6)
94 (± 6)
80 (± 3)
93 (± 7)
72 (± 4)
94 (± 5)
74 (± 3)
92 (± 7)
85 (± 3)
81 (± 3)
79 (± 2)
97 (± 4)
2.8
20.7
1.5
3
4
3
4
4
3
2 (DOB)
4
4
4
4
3
4
5
6
6.5
1.9
38 [7.42± 0.06]
11.2
Table 2. Functional properties of compounds 1-6 at stable 5-HT2AR- and 5-HT2CR-HEK293 cell lines in the Ca2+/Fluo-4 assay
given as EC50 values in nM (with pEC50 ± SEM in brackets) and Rmax ± SEM values (in % of Rmax for 5-HT tested at the same
plate).
Next, the functional properties of the compounds were assessed by investigation of their ability to induce the head twitch
response (HTR) in mice, a well-established measure of 5-HT2AR activation in vivo (Figure 4 and Table S1).46,47 When tested
previously under equivalent conditions, 1 and 2 induced the HTR with ED50 values of 0.75 and 2.43 µmol/kg,
respectively48. In concordance with the reduced 5-HT2AR agonist potency displayed by analogs 3-6 in the Ca2+ flux assay,
removal of the 2- or the 5-position methoxy groups reduced the potency of 1 and 2 in mice. Both 4 (ED50 = 2.26 µmol/kg)
and 5 (ED50 = 2.55 µmol/kg) were threefold less potent than the parent compound, meaning they induce the HTR with
about the same potency as 1.
In addition to reducing the potency of 2, loss of the 2- or 5-methoxy also produced a threefold reduction in the magnitude
of the HTR (Figure 2). In comparison to 4 and 6, the reduction of potency was more pronounced in the 2C-B derivatives 3
and 5. Removal of the 2-methoxy-group in 1 resulted in a 25-fold reduction in potency (3: ED50 = 61.1 µmol/kg). In contrast,
removal of the 5-methoxy-group was not tolerated; 5 did not induce the HTR at doses up to 100 mg/kg. In fact, 5 actually
reduced the HTR below baseline levels, displaying an ID50 = 36.0 mg/kg (95% CI 17.0–76.3 mg/kg), which is equivalent to
135 µmol/kg.
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Figure 4. Time-course of the head-twitch response (HTR) induced by compounds 1-6 in C57BL/6J mice. The mean response
during successive 2-minute time blocks is shown.
In the present study, we have shown that removing either the 2- or 5-methoxy group in the 2,5-dimethoxyphenethylamine
scaffold produces a substantial loss of potency at 5-HT2AR in vivo, in contrast to the more modest effect on 5-HT2AR affinity
and agonist potency. The results of the study show that removing the 5-methoxy substituent is more detrimental to 5-
HT2AR activity compared to removing the 2-methoxy group. This supports anecdotal accounts regarding the influence of
the 5-position O-alkyl group on potency and duration of action.49 These results, however, are not consistent with evidence
that the potency of 5-HT2AR agonists in vivo is correlated with their in vitro activity at the receptor level50–52. Therefore,
caution is warranted when evaluating the importance of this structural motif for potent in vivo agonist activity at 5-HT2AR.
Although the phenethylamine scaffold still merits further investigation as a starting point for developing potent 5-HT2AR
compounds, this study shows that our understanding of the SAR for 5-HT2AR is not complete. There is clearly considerable
room for variation within the classic 5-HT2AR agonist structural motifs.
Methods.
[3H]LSD binding assay. Binding affinity was measured in competition binding assays using [3H]LSD (PerkinElmer, Waltham,
MA) and membranes from tsA201 cells transiently transfected with cDNA for human 5-HT2AR, 5-HT2BR and 5-HT2CR. 40-48
h after transfection, the transfected tsA201 cells were scraped into ice-cold assay buffer (50 mM Tris–HCl, 10 mM MgCl2, 0.1
mM EDTA, pH 7.4), homogenized on ice using an IKA T18 Basic Ultra-Turrax for 10 s, and centrifuged for 20 min at 50.000 g
at 4 °C. Cell pellets were re-suspended in fresh assay buffer, homogenized on ice, and centrifuged at 50.000 g at 4 °C for
another 20 min, after which the membranes were stored at −80 °C. On the day of the assay, the cell membranes were re-
suspended in assay buffer and incubated with a fixed concentration of [3H]LSD (0.4-0.6 nM for 5-HT2AR and 1.0-1.5 nM for
5-HT2BR and 5-HT2CR) and various concentrations of the test compounds in a 96-well plate at 37 C for 2 h. The filters in
UniFilter 96-well GF/C plates (PerkinElmer, Waltham, MA) were presoaked for 1h in assay buffer supplemented with 0.5%
BSA, and the binding reactions were terminated by rapid filtration through the plates using a FilterMate Harvester
(PerkinElmer), followed by four washes with 300 L of ice-cold wash buffer (0.9% w/v NaCl, 50 mM Tris-HCl, pH 7.4).
Next, the filters were dried for at least 1 h at 50°C, 25 L Microscint-0 (PerkinElmer) was added to each well in the filter,
which were then shaken gently over-night. The following day, the amount of bound radioactivity on the filters was
determined in a TopCount NXT scintillation counter (PerkinElmer). Bound radioligand was plotted as a function of log[ligand],
and the data were analyzed using a one-site Ki model built into Graphpad Prism 7.0 (Graphpad, La Jolla, CA). The determined IC50
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values were used to calculate the Ki values for the compounds using the Chen-Prusoff equation: Ki = IC50/(1+([RL]/KD)), where
[RL] and KD are the [3H]LSD concentration used in that experiment and the dissociation constant for [3H]LSD determined
in a saturation binding experiment performed with the respective receptor.
1
2
3
4
5
6
7
8
9
Ca2+/Fluo-4 assay. The agonist activity of the compounds was assessed using stable 5-HT2AR- and 5-HT2CR-HEK293 cell lines
in a fluorescence-based Ca2+/Fluo-4 assay, essentially as previously described45. Briefly, the cells were split into poly-D-
lysine-coated black 96-well plates with clear bottoms (6 x 104 cells/well). The following day, the culture medium was
aspirated and the cells were incubated in 50 µL assay buffer [Hanks Buffered Saline Solution containing 20 mM HEPES, 1
mM CaCl2, 1 mM MgCl2 and 2.5 mM probenecid, pH 7.4] supplemented with 6 mM Fluo–4/AM at 37 °C for 1 h. Next, the
buffer was aspirated, the cells were washed once with 100 µL assay buffer, and then 100 µL assay buffer was added to the
cells. The 96-well plates was assayed in a FLEXStation3 (Molecular Devices, Crawley, UK) measuring emission [in
fluorescence units (FU)] at 525 nm in response to excitation at 485 nm before and up to 90 s after addition of 33.3 µL of
assay buffer containing the test compound. Each compound was tested at each cell line in three independent experiments
performed in duplicate. Data were normalized to the Rmax value for 5-HT from the same plate and analyzed using log
[agonist] versus response in Graphpad Prism 7.0, and the EC50 and Rmax values for test compounds were estimated based on
the concentration-response curves.
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Animals. Male C57BL/6J mice (6-8 weeks old) obtained from Jackson Laboratories (Bar Harbor, ME, USA) were housed in
a vivarium at the University of California San Diego, an AAALAC-approved animal facility that meets all Federal and State
requirements for care and treatment of laboratory animals. Mice were housed up to four per cage in a climate-controlled
room on a reverse-light cycle (lights on at 1900 h, off at 0700 h) and were provided with ad libitum access to food and water,
except during behavioral testing. Testing was conducted between 1000 and 1800 h. All animal experiments were carried
out in accordance with NIH guidelines and were approved by the UCSD animal care committee.
Head-Twitch Response Studies. The head-twitch response (HTR) was assessed using a head-mounted magnet and a
magnetometer detection coil53,54. Briefly, mice were anesthetized, a small incision was made in the scalp, and a small
neodymium magnet was attached to the dorsal surface of the cranium using dental cement. Following a two-week recovery
period, HTR experiments were carried out in a well-lit room with at least 7 days between sessions to avoid carryover effects.
Test substances were dissolved in saline and injected intraperiotoneally (IP) at a volume of 5 mL/kg. Mice (n = 5-7/group)
were injected with drug or vehicle and then HTR activity was recorded in a glass cylinder surrounded by a magnetometer
coil for 30 min. Coil voltage was low-pass filtered (2–10 kHz cutoff frequency), amplified, and digitized (20 kHz sampling
rate) using a Powerlab/8SP with LabChart v 7.3.2 (ADInstruments, Colorado Springs, CO, USA), then filtered off-line (40–
200 Hz band-pass). Head twitches were identified manually based on the following criteria: 1) sinusoidal wavelets; 2)
evidence of at least three sequential head movements (usually exhibited as bipolar peaks) with frequency ≥ 40 Hz; 3)
amplitude exceeding the level of background noise; 4) duration < 0.15 s; and 5) stable coil voltage immediately preceding
and following each response.
Data Analysis. Head-twitch counts were analyzed using one-way analyses of variance (ANOVA). Post hoc pairwise
comparisons between selected groups were performed using Tukey's studentized range method. Median effective doses
(ED50 values) and 95% confidence intervals (95% CI) for HTR dose-response experiments were calculated by nonlinear
regression (Prism 7.00, GraphPad Software, San Diego, CA, USA). A gaussian distribution was used to fit biphasic HTR
dose-response data48,55
.
ASSOCIATED CONTENT
Supporting Information. Experimental procedures for the synthesis of compounds 1-6 and extended pharmacological data are
available free of charge at http://pubs.acs.org
AUTHOR INFORMATION
Corresponding Author
*E-mail: jesper.kristensen@sund.ku.dk. Phone: (+45) 35336487
Author Contributions
All authors have contributed to the writing of and given approval to the final version of the manuscript.
Funding Sources
Generous support from the Lundbeck Foundation and the National Institute on Drug Abuse (R01 DA041336) is gratefully
acknowledged.
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ACKNOWLEDGMENT
Raimond Stroe is thanked for his assistance in collection of in-vitro data on DOB.
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ABBREVIATIONS
2,5-PEA 2,5-dimethoxyphenethylamine, 5-HT2AR serotonin 2A receptor, , LSD (+)-Lysergic Acid diethylamide, TMA-2 2,4,5-
trimethoxyamphetamine, DOM 2,5-dimethoxy-4-methylamphetamine, DOI 2,5.dimethoxy-4-iodoamphetamine, DOB 2,5-
dimethoxy-4-bromoamphetamine, 2C-B 2,5-dimethoxy-4-bromophenethylamine, 2C-I 2,5-dimethoxy-4-iodophenethylamine SAR
6
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9
Structure activity relationship2-Et-DOM 2-ethyl,5-methoxy-4-methylamphetamine,
,
5-Et-DOM 2-methoxy,5-ethyl-4-
methylamphetamine, Ser-159 Serine 159, Ser-239 Serine 239, NBOMe N-benzylphenethylamine, 25CN-NBOH 4-[2-[[(2-
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Hydroxyphenyl)methyl]amino]ethyl]-2,5-dimethoxybenzonitril,
methoxybenzyl)ethan-1-amine, HTR Head-twitch Response
25B-NBOMe
2-(4-bromo-2,5-dimethoxyphenyl)-N-(2-
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SYNOPSIS TOC. The 2,5-dimethoxy phenethylamine scaffold is widely associated with agonistic activity at 5-HT2AR
and other 5-HT2Rs. Herein we challenge this dogma via the synthesis and in vitro and in vivo evaluation of ligands
where the methoxy groups are systematically eliminated and the effects of this on their pharmacological profiles at
5-HT2AR and 5-HT2CR are evaluated along with their binding affinity for the 5-HT2AR, 5-HT2BR and 5-HT2CR.
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