5-HT2A/5-HT2C Receptor Pharmacology and Intrinsic Clearance of N-Benzylphenethylamines Modified at the Primary Site of Metabolism

Article abstract of DOI:10.1021/acschemneuro.6b00265

The toxic hallucinogen 25B-NBOMe is very rapidly degraded by human liver microsomes and has low oral bioavailability. Herein we report on the synthesis, microsomal stability, and 5-HT_2A/5-HT_2C receptor profile of novel analogues of 25B-NBOMe modified at the primary site of metabolism. Although microsomal stability could be increased while maintaining potent 5-HT₂ receptor agonist properties, all analogues had an intrinsic clearance above 1.3 L/kg/h predictive of high first-pass metabolism.

Full text of DOI:10.1021/acschemneuro.6b00265

Research Article
pubs.acs.org/chemneuro
5‑HT2A/5-HT2C Receptor Pharmacology and Intrinsic Clearance of
N‑Benzylphenethylamines Modified at the Primary Site of
Metabolism
Sebastian Leth-Petersen,†,§ Ida N. Petersen,†,§ Anders A. Jensen,† Christoffer Bundgaard,‡ Mathias Bæk,†
Jan Kehler,‡ and Jesper L. Kristensen*,†
†Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen,
Universitetsparken 2, 2100 København Ø, Denmark
‡Department of Discovery Chemistry and DMPK, H. Lundbeck A/S, Ottiliavej 9, 2500 Valby, Denmark
S
* Supporting Information
ABSTRACT: The toxic hallucinogen 25B-NBOMe is very rapidly degraded by human liver microsomes and has low oral
bioavailability. Herein we report on the synthesis, microsomal stability, and 5-HT2A/5-HT2C receptor profile of novel analogues
of 25B-NBOMe modified at the primary site of metabolism. Although microsomal stability could be increased while maintaining
potent 5-HT2 receptor agonist properties, all analogues had an intrinsic clearance above 1.3 L/kg/h predictive of high first-pass
metabolism.
KEYWORDS: Phenethylamines, hallucinogens, microsomal stability, 5-HT2A receptor agonists
he “NBOMes” constitute a novel class of phenethylamine
zyl moiety confers high binding affinity toward 5-HT2Rs and
Thallucinogens that are increasingly being used as increases the agonist potencies of the compounds at these
pharmacological and neuroimaging tools in the serotonin
field.1 In recent years, the NBOMes have also gained notoriety
within forensic science, as the recreational use of the drugs have
led to several hospitalizations and deaths in the United States
and in Western Europe, albeit the exact mechanism underlying
the toxicity of the drugs remains unresolved.2
In a previous study, the metabolism of one of these ligands
25B-NBOMe was investigated as this compound is currently
being used in its [11C]-labeled form as a PET-ligand to visualize
serotonin 2A receptors (5-HT2AR) in the brain as “CIMBI-36”
(1).3 The primary route of degradation of 1 in the body is via
demethylation of the 5′-methoxy of the phenethylamine core
(Scheme 1).4
receptors, it also increases intrinsic clearance to beyond the
human liver blood flow (1.3 L/kg/h), which is a predictor for
high first-pass metabolism and low oral bioavailability.6 With
the knowledge that 1 has a single primary metabolic soft spot in
pigs and humans, we hypothesized that structural modifications
in this part of the molecule would decrease intrinsic clearance.
To test this hypothesis we synthesized a small library of 8 25B-
NBOMe analogues altered at the position of the 5′-methoxy
(compounds 2−9 in Chart 1). These structural modifications
were made knowing that previous structure−activity relation-
ship (SAR) studies of this compound class indicate that such
modifications could be detrimental to the binding affinities of
the ligands to the 5HT2AR.7−9 In addition to assessing stability
of the compounds toward human liver microsomes (intrinsic
clearance), the binding affinities and functional properties of
We have previously investigated the human liver microsomal
stability of a series of NBOMes and found them to have very
high intrinsic clearance compared to the classic phenethylamine
hallucinogens (2C-B, 2C-I, etc.).5 Whereas the 2-methoxyben-
Chart 1. 25B-NBOMe Analogues (1−9)
Scheme 1. Metabolic “Soft Spot” of 25B-NBOMe (1)
Received: August 26, 2016
Accepted: August 26, 2016
© XXXX American Chemical Society
A
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Table 1. Intrinsic Clearance (HClInt), Binding Affinities, and Agonist Properties of Analogues 1−9 at 5-HT2A and 5-HT2C
a
Receptors
affinity (pKi)
pEC50
Rmax
compd
binding
selectivity
(2A/2C)
functional
selectivity
(2A/2C)
HClInt
(L/kg/h)
5-HT2A
5-HT2C
5-HT2A
5-HT2C
5-HT2A
R
5-HT2C
R
1 OMe
9.68 ( 0.09)
8.65 ( 0.08)
8.68 ( 0.10)
9.01 ( 0.11)
9.19 ( 0.12)
9.34 ( 0.06)
8.15 ( 0.12)
8.37 ( 0.13)
8.18 ( 0.07)
8.60 ( 0.07)
7.98 ( 0.11)
7.82 ( 0.02)
8.00 ( 0.04)
7.36 ( 0.05)
8.24 ( 0.11)
8.14 ( 0.06)
7.38 ( 0.12)
6.58 ( 0.09)
9.02 ( 0.09)
8.92 ( 0.09)
8.87 ( 0.14)
8.55 ( 0.13)
8.35 ( 0.10)
7.65 ( 0.10)
7.91 ( 0.13)
8.17 ( 0.12)
8.66 ( 0.06)
9.04 ( 0.06)
9.21 ( 0.09)
8.09 ( 0.09)
7.87 ( 0.06)
8.03 ( 0.13)
7.65 ( 0.11)
7.75 ( 0.09)
7.69 ( 0.10)
8.30 ( 0.10)
83 ( 3)
61 ( 3)
70 ( 4)
63 ( 6)
62 ( 6)
75 ( 6)
69 ( 5)
71 ( 4)
69 ( 3)
99 ( 7)
73 ( 6)
84 ( 4)
85 ( 2)
88 ( 5)
88 ( 2)
85 ( 2)
71 ( 3)
78 ( 4)
3.3
1.9
2.0
2.5
2.5
2.3
4.6
3.0
4.4
12
4.3
7.1
10
66
13
1.0
25
39
0.9
0.5
6.2
4.3
2.1
1.0
1.5
3.1
2.3
2 OH
3 H
4 F
5 Cl
6 CF3
7 I
8 OiPr
9 OCH2CH2OMe
a
Binding affinities (pKi SEM) of the analogues were determined in a [3H]CIMBI-36 binding assay using membranes from tsA201 cells transiently
expressing human 5-HT2AR and 5-HT2CR, and their agonist potencies (pEC50 SEM) and efficacies (Rmax SEM values, given in % of Rmax of 5-
HT) were determined in a Ca2+/Fluo-4 assay using stable 5-HT2AR- and 5-HT2CR-HEK293 cell lines. All pKi SEM, pEC50 SEM, and Rmax
SEM values in the table are based on at least three independent experiments.
Figure 1. cLogP and Intrinsic clearence of analogues 1−9.
the compounds at the human 5-HT2A and 5-HT2C receptors
were also determined (Table 1; see the Supporting Information
for Ki and EC50 values reported in nM).
binding affinities and functional potencies displayed by the
analogue at 5-HT2AR and 5-HT2CR. Similar modest changes in
intrinsic clearance and pharmacological properties were
observed when substituting CF3 for the fluorine of 4 (arriving
at 6). Increasing the size of the halogen of 4 even more by
substituting iodine for fluorine resulted in an analogue (7)
exhibiting higher clearance and relatively unaltered pharmaco-
logical properties compared to 4.
Keeping the 5′-alkoxy motif of 1 while increasing steric bulk
by substituting methoxy for isopropoxy (arriving at 8) had
negligible effect on intrinsic clearance, but 8 exhibited
substantially lower binding affinities and agonist potencies at
5-HT2AR and 5-HT2CR. Substituting methoxy for methox-
yethoxy (9) increased intrinsic clearance somewhat and also
decreased the binding affinities and agonist potencies at 5-
HT2AR and 5-HT2CR. The fact that in particular the binding
affinity to 5-HT2CR is decreased, results in an increased 2A/2C
binding-selectivity of 9 compared to 1 (Table 1).
Whereas the 5′-methoxy at the metabolic soft spot of 25B-
NBOMe thus apparently is not required for high agonist
potency at 5-HT2AR and 5-HT2CR, our attempts to significantly
decrease intrinsic clearance by modications of this part of the
scaffold were unsuccessful. Even though 5′-demethylation is the
preferred degradation pathway of 1, the site of oxidation
presumably changes in 2-9 retaining high intrinsic clearance
(above 1.3 L/kg/h).
RESULTS AND DISCUSSION
■
Substituting a hydroxyl for the methoxy of 1 (in effect a
demethylation, arriving at 2) decreased intrinsic clearance (1.9
L/kg/h) by approximately 40% - insufficient to significantly
remedy high first pass metabolism. Somewhat surprisingly 2
retained high binding affinity to 5-HT2AR (2.3 nM) and 5-
HT2CR (10 nM) and remained a potent partial agonist at the
receptors. The 2,5-dimethoxy motif is imperative to the 5-
HT2R affinity and potency displayed by the N-unsubstituted
“classic” phenethylamine hallucinogens,7 but in the NBOMe
class this does not appear to be the case, as omission of the
methoxy of 1 completely (arriving at 3) only reduced the 5-
HT2AR binding affinity approximately 10-fold, whereas both
agonist potencies and efficacies of the two analogues were
comparable. The intrinsic clearance of 3 (2.0 L/kg/h) was not
substantially different from that of 2. Substituting a fluorine for
the methoxy of 1 (arriving at 4) did not remedy the high
intrinsic clearance (2.5 L/kg/h), but 5-HT2AR affinity and
potency was retained whereas 5-HT2CR affinity and potency
was slightly decreased in this analogue.
Increasing the size of the halogen of 4 by substituting
chlorine for fluorine (arriving at 5) had negligible effect on
intrinsic clearance (2.5 L/kg/h) and only modest effects on the
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The fact that analogues 1−9 are characterized by cLogP
values of 4.94 0.21 on average (ChemBioDraw Ultra 14.0,
PerkinElmer) could explain their high clearance (Figure 1), as
very lipophilic compounds are known to be prime targets for
cytochrome P450 (CYP450).10
We speculated that the lipophilic nature of the compounds
possibly could target them to degradation by CYP450
regardless of blocking any specific C−H bond from
oxidation/hydroxylation. Thus, decreasing cLogP of the
compounds should, theoretically, decrease their intrinsic
clearance. The most lipophilic substituent in 1 is the bromide
in the 4′-position of the phenethylamine core, and this position
has in a previous study been found to be amenable to
substitution without concomitant loss of 5-HT2R activity.11 In
an effort to reduce cLogP of the 25B-NBOMe scaffold while
retaining 5-HT2R activity, we synthesized a small set of
analogues (Chart 2) with Br substituted for polar aliphatic
been shown to increase aqueous solubility and stability toward
liver enzymes.12 Unfortunately, the marked drop in cLogP of
10 was not accompanied by a reduction in intrinsic clearance
(2.8 L/kg/h) and 10 displayed substantially reduced binding
affinities to 5-HT2AR (18 nM) and 5-HT2CR (120 nM)
compared to 1. Increasing the ring-size of 10 by one methylene
unit (arriving at 11) increased 5-HT2AR binding affinity
substantially (1.2 nM) whereas the 5-HT2CR affinity (100
nM) was comparable to that exhibited by 10. Thus, compound
11 exhibited the desired high 5-HT2AR binding affinity and 2A/
2C-binding selectivity compared to 10, but the effect on
intrinsic clearance (13 L/kg/h) was detrimental.
Moving the oxygen in the THF-ring of 11 one atom closer to
the phenyl core (12) had little effect on 5-HT2AR and 5-HT2CR
affinity or potency, but the intrinsic clearance (17 L/kg/h) was
increased compared to 11.
The simplest open-chain aliphatic ether analogue in this
series was the “methoxy-methyl” 13 which displayed ∼10-fold
decreased binding affinities at 5-HT2AR and 5-HT2CR
compared to 1, whereas the agonist properties of the two
analogues were comparable (Table 2). Interestingly, the
intrinsic clearance of 13 (13 L/kg/h) was considerably
increased compared to 1. Extending the methoxy-methyl
group of 13 to ethoxy-methyl yielded analogue 14 characterized
by similar pharmacological properties and intrinsic clearance.
Adding “methoxymethyl” to the oxetane of 10 (arriving at 15)
did not substantially change the intrinsic clearance, but the
binding affinity of 15 at 5-HT2AR was ∼10-fold higher than that
of 10 resulting in an increased 2A/2C-binding selectivity ratio.
The lowered cLogP values of 10−15 compared to 1-9 had no
discernible effect on intrinsic clearance (Figure 2), and cLogP
thus seems to be a poor predictor of microsomal stability in the
case of the NBOMe hallucinogens.
Chart 2. 25B-NBOMe Analogues (10−15)
While our attempts to increase the microsomal stability of 1
by blocking the primary metabolic “soft spot” also were
unsuccessful, this SAR study did lead to some surprising
discoveries. Most notably, in contrast to the observed SAR for
the “classic” N-unsubstituted phenethylamine hallucinogens, a
2,5-dimethoxy pattern on the phenyl core of the NBOMes is
not required for high binding affinity and agonist potency at 5-
HT2R. Interestingly, the 5′-position tolerates a comparatively
large variety of substituents ranging from small (e.g., H) to
large (e.g., CH2CH2OMe) and from polar (e.g., OH) to apolar
(e.g., Oi-Pr).
open-chained or cyclic ethers. The resulting compounds (10−
15) have cLogP values of 3.03 0.29 on average (Chart 2),
and we measured the stability of analogues 10−15 toward
human liver microsomes and their pharmacological properties
at 5-HT2AR and 5-HT2CR (Table 2).
The oxetane structural motif has gained popularity in
medicinal chemistry research as its addition to a molecule has
Table 2. Intrinsic Clearance, Binding Affinities, and Functional Properties of Analogues 10−15 at 5-HT2A and 5-HT2C
a
Receptors
affinity (pKi)
pEC50
Rmax
binding
functional
selectivity
(2A/2C)
HClInt
selectivity
compd
5-HT2A
R
5-HT2C
R
5-HT2A
R
5-HT2C
R
5-HT2A
R
5-HT2C
R
(L/kg/h) (2A/2C)
1 OMe
9.68 ( 0.09)
7.74 ( 0.01)
8.64 ( 0.11)
8.59 ( 0.08)
8.59 ( 0.07)
8.62 ( 0.06)
8.58 ( 0.08)
8.60 ( 0.07)
6.91 ( 0.05)
7.00 ( 0.09)
6.71 ( 0.11)
7.34 ( 0.10)
7.54 ( 0.08)
6.92 ( 0.08)
9.02 ( 0.09)
8.93 ( 0.04)
8.94 ( 0.03)
8.81 ( 0.13)
9.12 ( 0.07)
9.16 ( 0.13)
9.04 ( 0.06)
9.04 ( 0.06)
8.21 ( 0.04)
7.80 ( 0.02)
8.48 ( 0.08)
8.81 ( 0.09)
8.72 ( 0.08)
8.20 ( 0.06)
83 ( 3)
85 ( 4)
79 ( 4)
85 ( 5)
67 ( 4)
72 ( 4)
68 ( 3)
99 ( 7)
86 ( 3)
85 ( 1)
99 ( 6)
88 ( 4)
85 ( 2)
83 ( 3)
3.3
2.8
12
6.7
0.9
5.2
13
10 oxetan-3-yl
11 tetrahydrofuran-3-yl
12 tetrahydrofuran-2-yl
13 CH2OMe
13
43
73
18
12
46
17
13
16
2.2
2.0
2.8
7.0
14 CH2OEt
15 (3-OMe)oxetan-3-yl
3.7
a
Binding affinities (pKi SEM) of the analogues were determined in a [3H]Cimbi-36 binding assay using membranes from tsA201 cells transiently
expressing human 5-HT2AR and 5-HT2CR and their agonist potencies (pEC50 SEM) and efficacies (Rmax SEM values, given in % of Rmax of 5-
HT) were determined in a Ca2+/Fluo-4 assay using stable 5-HT2AR- and 5-HT2CR-HEK293 cell lines. All pKi SEM, pEC50 SEM, and Rmax
SEM values in the table are based on at least three independent experiments.
C
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Figure 2. cLogP and Intrinsic clearance of 10−15.
The case of the phenol analogue 2 is particularly interesting
as it is the primary phase-I metabolite of 1 in vivo. Since 2 is a
potent 5-HT2AR/5-HT2CR agonist in its own right, it could
very feasibly play a role in the in vivo effects of 1.
CONCLUSION
■
In the present study, the SAR and metabolic properties of two
series of 5HT2AR agonists from the N-benzylphenethylamine
structural class have been delineated. In the first series of
analogues, modifications to the metabolic softspot of 1 yielded
analogues which had preserved their pharmacological profile at
5-HT2Rs, but the high intrinsic clearance characterizing the lead
compound was retained in the analogues. Thus, a different
approach was taken and less lipophilic analogueues where
pursued by replacing the bromide at the 4′-position of the
phenethylamine core of the scaffold. This series also comprised
several potent 5-HT2R agonists, but a correlation between
lipophilicity and metabolic liability was not observed. Thus, it
appears to be challenging to develop metabolically stable 5-
HT2R agonists with an N-benzylphenethylamine scaffold.
Nonetheless, important information about the SAR of this
compound class was obtained in the study, especially the fact
that the 5′-methoxy-substituent is not crucial for binding to and
activation of the 5-HT2AR as previous investigations have
suggested.
The analogues of 1 modified at the position of the 5′-
methoxy of the phenethylamine core (2−9) were designed with
optimization of intrinsic clearance as the primary goal, but at
the same time we anticipated a marked drop in agonist potency
at 5-HT2AR. Glennon et al. reported that the deletion of the 5′-
Methoxy-group in the parent phenethylamines and the
corresponding amphetamines decreased the binding affinity
for 5-HT2R 15−30-fold.9 Furthermore, Braden and Nichols
have investigated the interaction of the receptor with a variety
of agonists of the ergoline, tryptamine and phenethylamine
classes in a mutagenesis study.13 Here the 5′-methoxy group of
the phenethylamines was found to be important for 5-HT2AR
binding and activation due to its hydrogen bonding with the
Ser5.43(239) residue in the receptor, and in support of this 2-
methoxy-4-methylamphetamine and 5-ethyl-2-methoxy-4-
methylamphetamine were found to be 190-fold and 26-fold
less potent agonists at the receptor than 2,5-dimethoxy-4-
methylamphetamine, respectively.13 In another study, the
Nichols group examined the pharmacology of a variety of
primary and N-benzylated phenethylamines at 5-HT2AR by use
of mutagenesis and virtual docking.14 This study revealed that
while Ser5.43(239) is predicted to interact with the 5′-methoxy
of the N-benzylphenethylamines, the Phe6.51(339) residue is
more important for the binding affinities and agonist potencies
of this compound class, while not being essential for the
binding affinity and potency of the N-unsubstituted “classic”
phenethylamines. Moreover, a substituent (other than −H) at
the 4′-position of the phenethylamine core of the N-
benzylphenethylamines was found to be more important for
5-HT2AR binding and potency than the 5′-methoxy.14 This
observation might explain the observed tolerance to substituent
size and topology at the 5′-position of the phenethylamines in
the present study, as compared to what has previously been
reported for the “classic” phenethylamine 5-HT2AR agonists.
METHODS
■
Chemistry. Compounds (1−15) were synthesized by condensa-
tion of a suitable phenethylamine (compounds 1a−15a) with 2-
methoxybenzaldehyde, followed by sodium borohydride reduction of
the resulting imine, in one-pot fashion (Scheme 2).
a
Scheme 2. Synthesis of Compounds 1−15
a
Reaction conditions (see the Supporting Information for full
experimental details): (a) 2-methoxybenzaldehyde, N(Et)3, 3 Å
molecular sieves, EtOH, reflux; (b) NaBH4, EtOH, rt, 30−80% over
two steps.
D
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a
The phenethylamine precursors substituted at the 5′-position of the
phenyl core (1a−9a) were generally synthesized via Rieche
formylation of a suitable precursor, Henry condensation of the
resulting benzaldehyde, and DIBAL-H reduction of the resulting
nitrostyrene (Scheme 3).
Scheme 5. Synthesis of Compounds 12a−14a
a
Scheme 3. Synthesis of Compounds 1−9
a
Reaction conditions (see the Supporting Information for full
experimental details): (a) TiCl4, dichloromethyl methyl ether,
CH2Cl2, −10 to 0 °C, 30−70%; (b) propylene diammonium diacetate,
MeNO2, iPrOH, rt to reflux, 30−71%; (c) neat DIBAL-H, THF, rt to
reflux 32−80%.
a
Reaction conditions: (a) TiCl4, dichloromethyl methyl ether/4-
chlorobutanoyl chloride, CH2Cl2, −10 to 0 °C, 43−61%; (b) NaBH4,
EtOH, rt, 33−79%; (c) NaH, EtI/MeI, DMF, 0 °C to rt; (d)
hydrazine (aq.), THF, rt, 33−42% over two steps; (e) NaH, THF, −10
°C, 84%; (f) KOH, MeOH, H2O, rt, 62%.
10a, 11a, and 15a were synthesized (Scheme 4) by lithium-halogen
exchange of N,N-dibenzyl-“2C−B” followed by quench with a suitable
the transfection, the cells were harvested and scraped into ice-cold
assay buffer (50 mM Tris-HCl, 4 mM CaCl2, pH 7.4), homogenized
with a Polytron homogenizer for 10 s, and centrifuged for 20 min at
50 000g at 4 °C. Cell pellets were resuspended in fresh assay buffer,
homogenized, and centrifuged at 50 000g for another 20 min, after
which the membranes were stored at −80 °C until use.
a
Scheme 4. Synthesis of Compounds 10a, 11a, and 15a
At the day of the assay, cell membranes were resuspended in assay
buffer and incubated with [3H]Cimbi-36 (using assay concentrations
of 0.1 nM and 0.2 nM for 5-HT2AR and 5-HT2CR membranes,
respectively) and various concentrations of the test compounds.
Nonspecific binding was determined using 20 μM mianserin. The total
assay volume was 1 mL, and the reactions were incubated for 2 h at
room temperature while shaking. Whatman GF/C filters were
presoaked for 1 h in a 0.2% polyethylenimine solution, and binding
was terminated by filtration through the filters using a 48-well Brandel
cell harvester and four washes with 4 mL of ice-cold isotonic NaCl
solution. After this, the filters were dried, 3 mL of Opti-Fluor
(PerkinElmer) was added, and the amount of bound radioactivity was
determined in a scintillation counter.
Ki values for the analogues were calculated from the IC50 values
determined in the assay using the Cheng−Prusoff equation: Ki =
[IC50/(1 + [RL]/KD)], where KD and [RL] are equilibrium
dissociation constant for [3H]Cimbi-36 at the specific receptor and
the radioligand concentration used in the assay, respectively.
a
Reaction conditions: (a) n-BuLi, THF, −78 °C; (b) 3-oxetanone,
−78 °C to rt, 57% over two steps; (c) dihydrofuran-3(2H)-one, −78
°C to rt, 46% over two steps; (d) NaH, MeI, DMF, 0 °C to rt, 68%;
(e) DAST, CH2Cl2, −78 °C, 54%; (f) SiH(Et)3, TFA, DCM, 100%;
(g) Pd(OH)2, H2 (10 bar), MeOH, 50 °C, 64−78%.
Ca2+/Fluo-4 Assay. The functional properties of the analogues were
characterized at stable HEK293 cell lines stably expressing the human
5-HT2A and 5-HT2C receptors in a fluorescence-based Ca2+ imaging
assay using the fluorophore Fluo-4 essentially as previously
described.15 The cells were split into poly-D-lysine-coated black 96-
well plates with clear bottom. The following day, the culture medium
was aspirated and the cells were incubated in 50 μL of assay buffer
[Hank’s Buffered Saline Solution containing 20 mM HEPES, 1 mM
CaCl2, 1 mM MgCl2, and 2.5 mM probenecid, pH 7.4] supplemented
with 6 μM Fluo−4/AM at 37 °C for 1 h. Then the buffer was
aspirated, the cells were washed once with 100 μL assay buffer, after
which 100 μL assay buffer was added to the cells. The 96-well plate
was assayed in a FLEXStation (Molecular Devices, Crawley, UK)
measuring emission [in fluorescence units (FU)] at 525 nm caused by
excitation at 485 nm a total of 90 s before and after addition of 33.3 μL
of agonist solution in assay buffer. Experiments were performed in
duplicate at least three times for each compound at each receptor.
Concentration−response curves were constructed based on the
differences in the fluorescence units (ΔFU) between the maximal
fluorescence levels recorded before and after addition of the ligands,
and EC50 and Rmax values for the agonists were extracted from the
curve fits.
ketone. The resulting benzylic alcohol was either methylated using
strong base and MeI, removed directly in a reductive dehydroxylation
reaction or removed indirectly by conversion to the fluoride, using
DAST, followed by reductive dehalogenation using H/Pd(OH)2.
Debenzylation using H/Pd(OH)2 furnished the primary phenethyl-
amines.
12a, 13a, and 14a were synthesized (Scheme 5) by a Friedel−Crafts
type acylation of “2C−H” protected on the nitrogen as either the
phthalimide or trifluoroacetamide. The resulting carbonyl was reduced
using NaBH4, and the resulting benzylic alcohol was alkylated using
NaH as base and a suitable alkyl halide. Deprotection by either
hydrazine or KOH furnished the primary phenethylamines.
In Vitro Pharmacology. [3H]Cimbi-36 Binding Assay. The
binding affinities of the analogues were determined at membranes
from tsA201 cells transiently expressing the human 5-HT2A or 5-HT2C
receptors using [3H]Cimbi-36 as radioligand. The tsA201 cells (2 ×
106) were split into a 10 cm tissue culture plate a day before
transfection and transfected with 8 μg 5-HT2AR-pCDNA3.1 or 5-
HT2CR-pCDNA3.1 using Polyfect (Qiagen) as a DNA carrier. The day
after the transfection the culture medium was changed. 36−48 h after
Metabolic Stability. Intrinsic clearance in human liver microsmes
was determined by assessing the depletion of test compound over time
E
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as described previously.5 Test compounds were incubated at 1 μM.
Intrinsic clearance were calculated from the slope of the linear
regression of percentage of compound remaining in the incubation
against time.
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Leonhardt, S. (1992) Binding of phenylalkylamine derivatives at 5-
HT1C and 5-HT2 serotonin receptors: evidence for a lack of
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Improving the decision-making process in the structural modification
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acschemneur-
o.6b00265.
■
S
Full experimetal details (PDF)
AUTHOR INFORMATION
Corresponding Author
*Phone: +45 3533 6487. E-mail: jesper.kristensen@sund.ku.dk.
Author Contributions
S.L.P., I.N.P., and M.B. synthesized the compounds, A.J.J.
performed the in vitro pharmacology and C.B. determined the
intrinsic clearance. The manuscript was written through
contributions of all the authors who have given approval to
the final version of the manuscript.
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(15) Jensen, A. A., Plath, N., Pedersen, M. H. F., Isberg, V., Krall, J.,
Wellendorph, P., Stensbøl, T. B., Gloriam, D. E., Krogsgaard-Larsen,
P., and Frølund, B. (2013) Design, Synthesis, and Pharmacological
Characterization of N- and O-Substituted 5,6,7,8-Tetrahydro-4H-
isoxazolo[4,5-d]azepin-3-ol Analogues: Novel 5-HT2A/5-HT2C Re-
ceptor Agonists with Pro-Cognitive Properties. J. Med. Chem. 56,
1211−1227.
Author Contributions
§S.L.-P. and I.N.P. contributed equally.
Funding
The Novo Nordisk, Carlsberg and Lundbeck Foundation.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
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5-HT2AR, serotonin 2A receptor; 5-HT2CR, serotonin 2C
receptor; HClINT, human intrinsic clearence; DIBAL-H,
diisobutylaluminum hydride; DAST, diethylaminosulfur tri-
fluoride
REFERENCES
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DOI: 10.1021/acschemneuro.6b00265
ACS Chem. Neurosci. XXXX, XXX, XXX−XXX