Synthesis and pharmacological evaluation of N-benzyl substituted 4-bromo-2,5-dimethoxyphenethylamines as 5-HT2A/2C partial agonists

Article abstract of DOI:10.1016/j.bmc.2014.12.011

N-Benzyl substitution of phenethylamine 5-HT_2A receptor agonists has dramatic effects on binding affinity, receptor selectivity and agonist activity. In this paper we examine how affinity for the 5-HT_2A/2C receptors are influenced by N-benzyl substitution of 4-bromo-2,5-dimethoxyphenethylamine derivatives. Special attention is given to the 2′ and 3′-position of the N-benzyl as such compounds are known to be very potent. We found that substitutions in these positions are generally well tolerated. The 2′-position was further examined using a range of substituents to probe the hydrogen bonding requirements for optimal affinity and selectivity, and it was found that small changes in the ligands in this area had a profound effect on their affinities. Furthermore, two ligands that lack a 2′-benzyl substituent were also found to have high affinity contradicting previous held notions. Several high-affinity ligands were identified and assayed for functional activity at the 5-HT_2A and 5-HT_2C receptor, and they were generally found to be less efficacious agonists than previously reported N-benzyl phenethylamines.

Full text of DOI:10.1016/j.bmc.2014.12.011

Accepted Manuscript
Synthesis and pharmacological evaluation of N-benzyl substituted 4-bromo-2,5-
dimethoxyphenethylamines as 5-HT
partial agonists
2A/2C
Martin Hansen, Stine Engesgaard Jacobsen, Shane Plunkett, Gudrun Eckhard
Liebscher, John D. McCorvy, Hans Bräuner-Osborne, Jesper Langgaard
Kristensen
PII:
S0968-0896(14)00859-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.12.011
BMC 11943
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
9 October 2014
6 December 2014
10 December 2014
Please cite this article as: Hansen, M., Jacobsen, S.E., Plunkett, S., Liebscher, G.E., McCorvy, J.D., Bräuner-
Osborne, H., Kristensen, J.L., Synthesis and pharmacological evaluation of N-benzyl substituted 4-bromo-2,5-
dimethoxyphenethylamines as 5-HT
partial agonists, Bioorganic & Medicinal Chemistry (2014), doi: http://
2
A/2C
dx.doi.org/10.1016/j.bmc.2014.12.011
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Synthesis and pharmacological evaluation of
N-benzyl substituted 4-bromo-2,5-
dimethoxyphenethylamines as 5-HT2A/2C
partial agonists
Leave this area blank for abstract info.
Martin Hansen, Stine Engesgaard Jacobsen, Shane Plunkett, Gudrun Eckhard Liebscher, John D. McCorvy,
Hans Bräuner-Osborne, Jesper Langgaard Kristensen

Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com
Synthesis and pharmacological evaluation of N-benzyl substituted 4-bromo-2,5-
dimethoxyphenethylamines as 5-HT2A/2C partial agonists
a,b
a
a,
a
Martin Hansen , Stine Engesgaard Jacobsen , Shane Plunkett Gudrun Eckhard Liebscher , John D.
c
a
a,b,*
McCorvy , Hans Bräuner-Osborne , Jesper Langgaard Kristensen
a
Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, 2100 København Ø,
Denmark.
Center for Integrated Molecular Brain Imaging (CIMBI), Rigshospitalet and University of Copenhagen, Blegdamsvej 9, 2100 Copenhagen, Denmark
b
c
Pharmacology Department, School of Medicine, University of North Carolina at Chapel Hill, 120 Mason Farm Road. 4009 Genetic Medicine Bldg, Campus
Box 7365, Chapel Hill, NC 27599-7365, United States
*
Corresponding Author: Jesper L. Kristensen, e-mail: jesper.kristensen@sund.ku.dk, Phone: (+45) 35336487.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
N-benzyl substitution of phenethylamine 5-HT2A receptor agonists has dramatic effects on
binding affinity, receptor selectivity and agonist activity. In this paper we examine how affinity
for the 5-HT2A/2C receptors are influenced by N-benzyl substitution of 4-bromo-2,5-
dimethoxyphenethylamine derivatives. Special attention is given to the 2’ and 3'-position of the
N-benzyl as such compounds are known to be very potent. We found that substitutions in these
positions are generally well tolerated. The 2’-position was further examined using a range of
substituents to probe the hydrogen bonding requirements for optimal affinity and selectivity, and
it was found that small changes in the ligands in this area had a profound effect on their
affinities. Furthermore, two ligands that lack a 2’-benzylsubstituent was also found to have high
affinity contradicting previous held notions. Several high-affinity ligands were identified and
assayed for functional activity at the 5-HT2A and 5-HT2C receptor, and they were generally found
to be less efficacious agonists than previously reported N-benzyl phenethylamines.
Available online
Keywords:
Serotonin
5
-HT2A agonists
N-Benzyl phenethylamines
Selectivity
Structure activity relations
2
009 Elsevier Ltd. All rights reserved.
1
. Introduction
HT2A agonists have also been investigated as potent inhibitors of
tumor necrosis factor alpha (TNF-α) mediated inflammation and
may one day lead to novel treatments of inflammatory diseases
The 5-HT2A receptor plays a significant role in the regulation
and control of various physiological mechanisms in both the
central and peripheral nervous system. In the CNS, 5-HT2A
receptors are implicated in the regulation of cognitive states,
associative learning, mood and circadian rhythm. Peripheral 5-
HT2A receptors mediate processes such as vasoconstriction,
8
and conditions. Despite this fact, legislative impediments still
hinder the exploration of the clinical potential of 5-HT
agonists.
2
A
9
1
The 5-HT receptor subtypes 2A, 2B and 2C share a high
2
2
platelet aggregation and intra-ocular pressure. Drugs targeting
degree of sequence homology which makes the development of
selective ligands difficult. Selective antagonists exist for all three
the 5-HT2A receptor are used in the treatment of various
neurological disorders. Atypical antipsychotics such as
risperidone and clozapine are inverse agonists at the 5-HT
receptor. Neutral antagonists such as 2-bromolysergic acid
diethylamide (BOL-148) have been investigated for the treatment
of cluster headaches. Peripherally selective agonists are being
investigated for the treatment of glaucoma and other afflictions
related to intra-ocular pressure. Agonism of central 5-HT2A
receptors is generally accepted to be responsible for the
psychopharmacological effects of the hallucinogens lysergic acid
5
-HT2 receptor subtypes, while selective agonists have been
10,11
discovered only recently for the 5-HT2A
receptor.
and 5-HT2C
2
A
3
12.
Of the 5-HT2A agonists developed so far, only a few are
selective for the 5-HT2A receptor. 5-HT2A agonists from the
4
ergoline family (e.g. LSD) generally have
a
broad
5
pharmacological profile that encompasses most of the 5-HT
receptor subtypes as well as dopaminergic and adrenergic
receptors. Tryptamines (e.g. psilocybin) are slightly more
selective, but are known to also activate 5-HT1A receptors. The
most selective group of compounds is the phenethylamines,
6
diethylamide (LSD) and psilocybin. Some of these compounds
have in the past and the present been explored in the
psychotherapy of people with fear and anxiety related to terminal
which bind preferentially to 5-HT receptor subtypes A, B, and
7
2
illness or post-traumatic stress disorder (PTSD). Recently, 5-
13
C.

N-substitution of the phenethylamine series of 5-HT2A
agonists has a dramatic effect on the affinity for the 5-HT2A
receptor. Simple alkylation generally results in a decrease of
The final compounds were all synthesized by indirect
reductive amination of 4-bromo-2,5-dimethoxyphenethylamine
with the required aldehydes, as detailed in the supporting
information. The aldehydes were either commercially available
or synthesized as detailed below.
1
4,15
affinity,
the unsubstituted derivative. Introduction of an ortho methoxy
or hydroxyl) group on the N-benzyl substituent had a dramatic
effect on the affinity giving access to the most potent family of 5-
whereas benzylation restores affinity to the level of
16
(
Chemistry
1
7
HT2A agonists reported to date – often referred to as NBOMe’s.
This discovery paved the way for the development of selective
agonists: Juncosa et al. reported that conformational restriction of
the known agonist 25B-NBOMe lead to a 100 fold selective 5-
1
0
HT2A agonist. As part of a research program directed towards
the development of selective agonist PET-ligands for the 5-HT
2
A
1
8-21
receptor
combinations of 4- and 2’-substituents of the N-benzyl
phenethylamine chemotype wherein 12 different
phenethylamines (with different 4-substituents) were joined with
we recently reported a systematic survey of different
1
1
Scheme 1. a) NaH, BrCH
C. c) Mg at 80 °C then DMF at 0 °C, THF. d) K
DMF, rt.
2
CH(OEt)
2
, DMF, 100 °C b) PPA, PhCl, 130
four different benzyl groups. As previous studies indicated that
the 2’-position of the N-benzyl substituent is an important
determinant for 5-HT2A/2C affinity and functional potency, we
limited our investigation to include substituents of that nature.
Although we discovered a new potent and selective 5-HT2A
ligand we also saw an erratic picture with respect to the structure
activity relationships for the entire ligand set as no correlation
between the substitution pattern and affinity/selectivity could be
identified. To shed further light on this issue we wish to report a
more in-depth investigation into the effect of changing the
substituent on the N-benzyl group while keeping the
phenethylamine core static.
°
2
CO , BrCH CH(OEt)
3
2
2
,
Benzofuran 2 and benzothiophene 3 were synthesized using a
similar sequence; alkylation with 2-bromoacetaldehyde diethyl
acetal followed by Friedel-Craft style ring closure to give the
benzofuran and benothiophene. Halogen-metal exchange with n-
BuLi followed by a DMF quench led to products formylated in
the 2-position as the organolithium intermediate rapidly
rearranges. However, reductive metallation with Mg followed by
DMF resulted in the desired 7-formylated products.
In our previous study we noticed a general trend that 2’,3’-
methylenedioxy substitution gave rise to a moderate selectivity
for the 5-HT2A receptor, see figure 1 for a selected example.
Compound 1a is a very potent agonist both in binding and
functional assays. To explore this in more detail we targeted
ligands with various heteroatoms occupying the 2’ and/or 3’
position, see Figure 1.
Scheme 2. a) (1) NH
HCOOH, HCl, H O (5) H
MnO , CH Cl /DMF.
3
, H
2
O (2) Br , Pd(C), MeOH. b) (4)
2
, KOH, H
2
O (3) H
2
2
2
SO , MeOH (6) LiAlH , THF/1,4-dioxane (7)
4
4
2
2
2
Aldehyde 4 was synthesized from 3-nitrophthalic anhydride in
seven steps in 8 % overall yield. Regioselective ammoniolysis
afforded the phthalamic acid which was converted to anthranilic
acid by Hofmann rearrangement. The nitro group was reduced by
catalytic hydrogenation to give the diaminobenzoic acid.
Treatment with formic acid in aqueous HCl assembled the
benzimidazole. The carboxylic acid was converted to the methyl
ester to improve solubility and then reduced with LiAlH to give
4
the benzylic alcohol. Subsequent oxidation to the required
aldehyde 4 was accomplished with MnO2.
1
1
Figure 1. Structure of previously reported 5-HT2A/C agonist and ligands
targeted in this study.
We wanted to examine other benzylic positions in
combination with the 2’-position in order to probe the binding
pocket for favorable interactions that may give rise to higher
selectivity. We chose 4-bromo-2,5-dimethoxyphenethylamine as
our phenethylamine template since this is known to give very
potent, albeit non-selective agonist activity at the 5-HT2A/2C
receptor.
Scheme 3. a) (1) NaNO
LiAlH , THF (4) MnO , CH
2
, aq. HBF
4 3
(2) KOAc, 18-crown-6, CHCl . b) (3)
4
2
2
Cl /DMF.
2
The indazole aldehyde was accessed by diazotation followed
by ring closure of methyl 2-amino-3-methylbenzoate. A two-step
reduction-oxidation sequence furnished aldehyde 5.
2
. Results and discussion
All compounds were prepared as detailed below and screened
for binding affinity at 5-HT2A and 5-HT2C receptors at the NIMH
2
2
Psychoactive Drug Screening Program (PDSP). Selected
compounds were subsequently evaluated in functional assays
measuring Gq-mediated inositol monophosphate production.
Scheme 4. a) (1) H
2
SO
4
, MeOH (2) H
2
, Pd(C), THF/EtOAc (3) Yb(OTf)
3
,
HC(OEt) , Toluene. b) (3) LiAlH
3
4
, THF (4) MnO
2
, CH Cl /DMF.
2
2

Benzoxazole 6 was synthesized in five steps from 3-
nitrosalicylic acid. Fischer esterification gave the methyl ester
and then the nitro group was reduced by catalytic hydrogenation
to give the corresponding aminophenol. Cyclization was
accomplished using triethyl orthoformate in the presence of a
catalytic amount of Lewis acid to yield the benzoxazole. LiAlH4
reduction of the methyl ester followed by final oxidation with
TPAP/NMO gave the desired aldehyde 6.
moiety to give coumaran analogue 1e gave a N-benzyl
phenethylamine with one of the highest affinities we have
assayed to date with similar selectivity towards 5-HT2C as for 1a.
We then analyzed derivatives that in principle could act as
combined hydrogen bond donors and acceptors comparable to the
2
’-hydroxybenzyl substituted compound which in previous
studies also resulted in potent agonists.
The benzimidazole 1f, indazole 1g and indole 1h all retain
affinity for the 5-HT2A but they compare unfavorably with 1a
with a ten-fold loss of affinity and negligible selectivity for either
receptor subtype.
The pyridine derivatives were included to determine the effect
of a ring nitrogen in the benzylic substituent. First, we examined
derivatives of 1i and 1j with nitrogen in the 3’-position.
2 2
Scheme 5. a) (1) LiTMP, HCOOEt, THF, -78 °C. b) cat. H O , aq. HCl c)
MeONa, MeOH.
Aldehydes 7 and 8 were both made from 2-chloropyridine.
Lithiation with LiTMP followed by a quench with ethyl formate
yielded the 2-chloronicotinaldehyde. Treatment with either HCl
and a catalytic amount of H O or MeONa gave pyridone 7 and
2
2
2
-methoxypyridine 8 respectively.
Pharmacology - binding studies
Initially we focused on heterocyclic derivatives where a
heteroatom was preserved in the 2’-position on the N-benzyl
core.
Figure 3. Structure of pyridine substituted ligands 1i-m and their
i
affinities for 5-HT2A/2C (pK ).
Both derivatives exhibited a significant loss of affinity (>100
fold) at both 5-HT2A and 5-HT2C receptor subtypes. The reason
for this is unclear; one explanation could be a diminished
capability of forming hydrogen bonds due to the electron
withdrawing effect of the nitrogen-containing ring.
The unsubstituted regioisomeric pyridines 1k-m were tested
for comparison and they performed in a similar fashion - despite
lacking the hydrogen bond acceptor in the 2’ position. Taken
together, the presence of a nitrogen ring likely dampens the
electronic-rich ring interaction with the receptor resulting in
lower affinity.
Finally, we decided to perform a methoxy-scan on the benzyl
group while keeping the 2’-methoxy substituent resulting in the
four derivatives shown in scheme 4.
Figure 2. Structure of ligands 1b-h containing heterocycles fused to the
2
’-3’-benzylposition and their affinities for 5-HT2A/2C.
Figure 4. Structure of dimethoxy substituted ligands 1n-q and their affinities
i
for 5-HT2A/2C (pK ).
The benzofuran- and benzothiophene derivatives 1b and 1c
maintain high affinity at both 5-HT2A and 5-HT2C receptor
subtypes. Adding a second heteroatom in the 3’-position was
known to confer a modest selectivity in the case of the 2’,3’-
methylenedioxybenzyl derivative 1a, but this effect was not seen
with the benzoxazole derivative 1d. Saturation of the benzofuran
Only the 2’-3’-dimethoxy substituted derivative 1n showed
high affinity at the 5-HT2A receptor while the 2’,4’ and 2’,5’-
disubstituted derivatives 1o and 1p both displayed lower affinity.

The 2’,6’- dimethoxy compound 1q was even less potent and
exhibited a 17 fold selectivity for the 5-HT2C receptor.
response drops from 84 to 33% with a single -O- to -CH2-
substitution in the methylenedioxy moiety.
As both 1e, 1n and 1a are high-affinity compounds
confirming the spatial availability of these positions), we
decided to add two additional compounds acting as hybrids
between these compounds.
Although 1q was moderately selective for 5-HT2C in the
binding assay, this compound was considerably more potent at 5-
HT2A in the functional assay being 16 fold selective for 5-HT2A.
(
The 2’-F,3’-OMe derivative 1r was slightly less potent than
the structurally very similar 2’-OH,3’-OMe derivative 1s - with
negligible selectivity for 5-HT . 1s on the other hand proved
2
A
very selective for the 5-HT2A receptor with more than 500-fold
higher potency than at 5-HT . Interestingly, 1s showed a modest
2
C
3
2 fold selectivity for 5-HT2A in the binding assay, and this lack
of correlation between the binding and functional data mirrors
1
1
our previous study.
. Conclusion
A total of 21 N-benzylated derivatives of 4-bromo-2,5-
Figure 5. Structure of ligands 1r-u and their affinities for 5-HT2A/2C (pK
i
).
3
1
n included for comparison.
Compound 1r, where the 2’-methoxy substituent is replaced
dimethoxyphenethylamine where synthesized and affinities were
characterized using binding assays at 5-HT2A and 5-HT2C in an
attempt to uncover the structural features that govern affinity and
selectivity at these receptors. Based on the previous finding that a
methylenedioxy substitution in the 2’,3’-position of N-benzyl
phenethylamine agonists provided ligands with slight selectivity
for 5-HT , these ligands were selected in order to explore this
with a fluorine atom, had an affinity comparable to 1n but with
increased selectivity. Compound 1s with a hydroxy group in the
ortho-position gives a compound which has identical affinity for
5
-HT2A as 1e whilst being almost twice as selective.
Thus, it is clearly evident that subtle modifications in this part
2
A
of the ligands have a large impact on the affinity and selectivity.
To further probe this region of the molecules, the 3’-
methoxyderivate 1t without a hydrogen bond acceptor in the 2’-
position was evaluated and it retains a surprising level of affinity
as is the case for the 3’-hydroxyderivative 1u – both being more
potent and selective than the 2’,3’-dimethoxyderivative 1n.
These results question the previous accepted notion that a
hydrogen bond acceptor in the 2’-position is required if the
ligands are to have higher affinity for the 5-HT2A receptor.
region of the receptor-ligands interaction in further detail.
Affinity results from the binding assays show that the ligands
generally can be manipulated in this area while maintaining
affinity. Replacement of the N-benzyl moiety with a pyridine-
derivative gave compounds with lower affinity, indicating that an
electron rich aromatic system is beneficial for binding. Indeed,
ligands 1b, 1e, 1s and 1t all have pK > 9, thus being ligands with
i
some of the highest affinities reported for the 5-HT2A receptor to
date, but no clear picture has emerged yet with respect to their
structure activity relationship.
Pharmacology - functional studies
Functional characterization of selected ligands complicates the
analysis further as no clear picture yet emerges with respect to
the binding studies. Very minor manipulations lead to dramatic
effects both with respect to selectivity and intrinsic activity.
Thus, further studies are needed to understand the behavior of
Four of the compounds, 1e, 1q, 1r and 1s, were examined for
their ability to activate human 5-HT2A and 5-HT2C receptors
stably expressed in HEK-293 cells in vitro using an inositol
monophosphate assay (IP-One, Cisbio) as a measure of Gq
function (Table 1) as previously described. All four ligands
exihibited partial agonism at both receptors with maximal
responses ranging from 16-53%.
2
3
this class of ligands at the 5-HT receptors, and the compounds
2
presented herein could prove to be important tools for mapping
the N-benzyl binding pocket in the 5-HT2A receptor.
1
e proved to be more potent that 1a albeit with maximal
response on 5-HT2A in the functional assay. Also, the maximal
Table 1. Functional characterization of selected compounds at 5-HT2A and 5-HT2C (inositol monophosphate accumulation).
Ligand
pEC50 ± S.E.M.
Selectivity
Maximal response ± S.E.M. (% of 5-HT)
5
-HT2A
5-HT2C
5-HT2A/5-HT2C
5-HT2A
100
5-HT2C
100
5
-HT
6.52 ± 0.06
8.20 ± 0.03
7.98 ± 0.17
8.34 ± 0.06
5.27 ± 0.06
6.98 ± 0.10
7.47 ± 0.04
6.72 ± 0.07
7.34 ± 0.08
6.66 ± 0.06
7.49 ± 0.15
4.06 ± 0.21
6.55 ± 0.06
4.81 ± 0.11
0.63
7.2
18
DOI
87 ± 3
71 ± 6
33 ± 3
53 ± 2
22 ± 2
21 ± 2
119 ± 8
84 ± 6
52 ± 2
33 ± 3
16 ± 1
52 ± 9
a
1
a
1
e
7.1
16
1
q
1
r
2.7
457
1
s
a
Data from ref. 11.
The compounds were tested in at least three independent experiments in triplicate at each cell line.

4
. Experimental
Bethesda MD, USA. The stably expressing h5-HT - and
2A
h5HT -HEK293 cell lines were generously provided by Dr.
Anders A. Jensen.
2
C
General procedure for the synthesis of N-benzylated
phenethylamines 1b-u.
Et N (1.0 equiv.) was added to a suspension of 2-(4-bromo-2,5-
3
References and notes
dimethoxyphenyl)ethanamine hydrochloride (2C-B HCl, 1.0
mmol) and the required aldehyde (1.1 equiv.) in EtOH (10 mL)
and the reaction was stirred until formation of the imine was
complete according to TLC or GC (between 30 minutes and 3 hrs
1
2
.
.
Nichols, D. E.; Nichols, C. D. Chem. Rev. 2008, 108, 1614-1641.
Nichols, D. E. Pharmacology & Therapeutics, 2004, 101, 131-
1
81.
3
.
Weiner, D. M.; Burstein, E. S.; Nash, N.; Croston, G. E.; Currier,
E. A.; Vanover, K. E.; Harvey, S. C.; Donohue, E.; Hansen, H. C.;
Andersson, C. M. et al. J. Pharmacol. Exp. Ther. 2001, 299, 268-
depending on the aldehyde). NaBH (2.0 mmol) was then added
4
and the reaction was stirred for another 30 minutes. The reaction
mixture was concentrated under reduced pressure and the residue
was partitioned between CH Cl and water (30 mL, 1:1). The
2
76.
Karst, M.; Halpern, J. H.; Bernateck, M.; Passie, T. Cephalalgia
010, 30, 1140-1144.
4
5
.
.
2
2
2
organic layer was isolated and the aqueous layer was extracted
with CH Cl (2 × 15 mL). The combined organic extracts were
Feng, Z.; Mohapatra, S.; Klimko, P. G.; Hellberg, M. R.; May, J.
A.; Kelly, C.; Williams, G.; McLaughlin, M. A.; Sharif, N. A.
Bioorg. Med. Chem. Lett. 2007, 17, 2998-3002.
Nichols, D. E. Pharmacol. Ther. 2004, 101, 131-181 .
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Ramos, J. Exp. Brain Res. 2013, 228, 481-491.
2
2
dried over Na SO , filtered and evaporated under reduced
2
4
6
7
.
.
pressure. The residue was purified by flash chromatography
CH Cl /MeOH/NH 98:2:0.04). The purified free base was
(
2
2
3
dissolved in EtOH (2 mL) and HCl (1M in EtOH, 2 mL) was
8
.
Nau, F., Jr.; Yu, B.; Martin, D.; Nichols, C. D. PLoS One 2013, 8,
e75426.
added and the resulting solution was diluted with Et O until a
2
precipitate was formed. The crystals were collected by filtration
and dried under reduced pressure to provide the desired products.
9. (a) Nutt, D.J.; King, L.A.; Nichols, D.E. Nature Reviews
Neuroscience 2013, 14, 577–585. (b) Nutt, D. EMBO Rep. 2014,
1
5, 208-211.
Functional characterization at 5-HT2A and 5-HT2C Receptors
10. Juncosa, J.I., Jr.; Hansen, M.; Bonner, L.A.; Cueva, J. P.;
Maglathlin, R., McCorvy, J.D.; Marona-Lewicka, D.; Lill, M. A.;
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11. Hansen, M.; Phonekeo, K.; Paine, J.S.; Leth-Petersen, S.; Begtrup,
M.; Bräuner-Osborne, H.; Kristensen, J.L. ACS Chem. Neurosci.
Functional characterization was performed on HEK-293 cell lines
stably expressing human 5-HT2A and human 5-HT2C respectively
by using the IP-One assay (Cisbio, Codolet, France).
Subconfluent cells were detached from the cell culture dish by
using cell dissociation solution (Sigma-Aldrich, St. Louise, MO),
and cell suspensions of 1 x 107 cells/mL were prepared in 37°C
assay buffer (HBSS supplemented with 20 mM HEPES, 1 mM
CaCl , 1 mM MgCl , pH 7.4). Ligands were prepared in 2x final
2
014, 5, 243-249.
1
2. Thomsen, W.J.; Grottick, A.J.; Menzaghi, F.; Reyes-Saldana, H.;
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1
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1
3. Glennon, R. A.; Young, R.; Rosecrans, J. A. Eur. J. Pharmacol.
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2
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983, 91, 189-196.
concentration in ligand buffer (assay buffer supplemented with
0 mM LiCl). 5 µl/well of ligand solution and 5 µl/well of cell
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4
suspension were added to a 384-well OptiPlate (PerkinElmer,
Waltham, MA, USA), and the plate was sealed and incubated at
3
7°C for 1 hour, followed by 15 minutes incubation at room
temperature. Detection solution was prepared (IP-One Conjugate
Lysis buffer with 2.5% anti-IP1 cryptate Tb conjugate and
.5% IP1-d2 conjugate), and 10 µl/well was added to the
1
1
7. Heim, R. Ph.D. Dissertation, Freie Universität Berlin, 2003.
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Begtrup, M. et al. J. Nucl. Med. 2010, 51, 1763–70.
&
2
OptiPlate, which was then incubated for 1 hour at room
temperature away from light. The plate was read on an Envision
multilabel reader (PerkinElmer, Waltham, MA, USA), in which
the plate was excited at 340 nm and emission was measured at
1
2
2
2
2
9. Ettrup, A.; Hansen, M.; Santini, M. A.; Paine, J.; Gillings, N.;
Palner, M.; Lehel, S.; Herth, M. M.; Madsen, J.; Kristensen, J. et
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6
15 nm and 665 nm. The fluorescence resonance energy transfer
(
FRET) 665 nm/615 nm ratio is inversely proportional to the
inositol monophosphate (IP1) accumulation produced upon
activation of 5-HT2A and 5-HT . FRET ratios were converted to
2C
IP1 concentrations by interpolating values from an IP1 standard
curve generated from a calibrator provided by the manufacturer
(Cisbio, Codolet, France).
Acknowledgments
The Lundbeck Foundation, the Danish Ministry of Science,
Supplementary Material
Innovation, and Higher Education and the A. P. Møller
Foundation for the Advancement of Medical Sciences are
gratefully acknowledged for financial support. Radioligand
competition binding assay for affinity at human 5-HT2A receptors
and rat 5-HT receptors using displacement of antagonist
Supplementary data associated with this article (full
experimental detail and description of the assays) can be found,
in the online version, at: DOI: XXX
2
C
3
3
radioligands [ H]Ketanserin and [ H]Mesulergine was generously
provided by the National Institute of Mental Health's
Psychoactive Drug Screening Program, Contract # HHSN-271-
2
008-00025-C (NIMH PDSP). The NIMH PDSP is Directed by
Bryan L. Roth MD, PhD at the University of North Carolina at
Chapel Hill and Project Officer Jamie Driscol at NIMH,