Reformulating a Pharmacophore for 5-HT2A Serotonin Receptor Antagonists

Article abstract of DOI:10.1021/acschemneuro.6b00162

Several pharmacophore models have been proposed for 5-HT_2A serotonin receptor antagonists. These typically consist of two aromatic/hydrophobic moieties separated by a given distance from each other, and from a basic amine. Although specified distances might vary, the models are relatively similar in their general construction. Because our preliminary data indicated that two aromatic (hydrophobic) moieties might not be required for such action, we deconstructed the serotonin-dopamine antipsychotic agent risperidone (1) into four smaller structural fragments that were thoroughly examined in 5-HT_2A receptor binding and functional (i.e., two-electrode voltage clamp (TEVC) and intracellular calcium release) assays. It was apparent that truncated risperidone analogues behaved as antagonists. In particular, 6-fluoro-3-(1-methylpiperidin-4-yl)benzisoxazole (4) displayed high affinity for 5-HT_2A receptors (K_i of ca. 12 nM) relative to risperidone (K_i of ca. 5 nM) and behaved as a potent 5-HT_2A serotonin receptor antagonist. These results suggest that multiple aromatic (hydrophobic) moieties are not essential for high-affinity 5-HT_2A receptor binding and antagonist activity and that current pharmacophore models for such agents are very much in need of revision.

Full text of DOI:10.1021/acschemneuro.6b00162

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Article
Reformulating a Pharmacophore for 5-HT2A Serotonin Receptor Antagonists
Jason Younkin, Supriya A. Gaitonde, Amr Ellaithy, Rakesh H. Vekariya, Lia Baki, Jose L.
Moreno, Sneha Shah, Peter Drossopoulos, Kelsey S. Hideshima, Jose Miguel Eltit, Javier
González-Maeso, Diomedes E. Logothetis, Malgorzata Dukat, and Richard A. Glennon
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00162 • Publication Date (Web): 06 Jul 2016
Downloaded from http://pubs.acs.org on July 9, 2016
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Reformulating a Pharmacophore for 5-HT2A Serotonin Receptor
Antagonists
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Jason Younkin,1# Supriya A. Gaitonde,2# Amr Ellaithy,1# Rakesh Vekariya,2 Lia Baki,1
José L. Moreno,1 Sneha Shah,1 Peter Drossopoulos,1 Kelsey S. Hideshima,1 Jose
Miguel Eltit,1 Javier GonzálezꢀMaeso,1 Diomedes E. Logothetis,1 Malgorzata Dukat,2
Richard A. Glennon2
1Department of Physiology and Biophysics, School of Medicine, Virginia Commonwealth
University, Richmond, Virginia 23298 USA
2Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth
University, Richmond, Virginia 23298 USA
# Equal contributors
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Abstract
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Several pharmacophore models have been proposed for 5ꢀHT2A serotonin receptor
antagonists. These typically consist of two aromatic/hydrophobic moieties separated by
a given distance from each other, and from a basic amine. Although specified distances
might vary, the models are relatively similar in their general construction. Because our
preliminary data indicated that two aromatic (hydrophobic) moieties might not be
required for such action, we deconstructed the serotoninꢀdopamine antipsychotic agent
risperidone (1) into four smaller structural fragments that were thoroughly examined in
5ꢀHT2A receptor binding and functional (i.e., twoꢀelectrode voltage clamp – TEVC – and
intracellular calcium release) assays. It was apparent that truncated risperidone analogs
behaved as antagonists. In particular, 6ꢀfluoroꢀ3ꢀ(1ꢀmethylpiperidinꢀ4ꢀyl)benzisoxazole
(4) displayed high affinity for 5ꢀHT2A receptors (Ki ca 12 nM) relative to risperidone (Ki
ca 5 nM) and behaved as a potent 5ꢀHT2A serotonin receptor antagonist. These results
suggest that multiple aromatic (hydrophobic) moieties are not essential for highꢀaffinity
5ꢀHT2A receptor binding and antagonist activity and that current pharmacophore models
for such agents are very much in need of revision.
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Keywords: Risperidone, Antipsychotics, TEVC (twoꢀelectrode voltage clamp), Calcium
release, 3ꢀ(4ꢀPiperidinyl)benzisoxazoles
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Introduction
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Over the past 25 years, several pharmacophore models have been described for 5ꢀ
HT2A serotonin receptor antagonists.1ꢀ7 To some extent, the various models might reflect
the nature of the antagonists examined in the individual studies. Nevertheless, there
now seems agreement that multiple binding modes are possible for competitive 5ꢀHT2A
receptor antagonists,2,4ꢀ8 and this could account for the multiple models. Typically, the
pharmacophore models consist of two aromatic (hydrophobic) moieties separated by a
given distance from each other, and from a basic amine. Although the distances might
vary from model to model,2ꢀ5 these three features are common to nearly all the agents
examined. When the two aromatic features flank a third (i.e., central) ring, as in a
tricyclic ring system, even the foldꢀangle of the central ring is thought to play a role.9
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Two of the few 5ꢀHT2A receptor antagonists that have found clinical application are the
SDA (serotoninꢀdopamine antagonist) antipsychotic agents risperidone (1; Figure 1) and
its 9ꢀhydroxy metabolite, paliperidone. Risperidone (1) has been considered only in a
few pharmacophore studies.10,11 In one study, the authors noted that their model did not
necessarily predict structural features required for binding at 5ꢀHT2A or dopamine D2
receptors, but simply identified features important for 5ꢀHT2A/D2ꢀassociated
antipsychotic action.10 In another study, multiple pharmacophoric features for 5ꢀHT2A
antagonist action were identified including the two aromatic (hydrophobic) moieties.11
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N
O
O
5
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4
N
7
N
F
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N
CH3
9
1
Figure 1. The antipsychotic agent risperidone (1) and its ring numbering system.
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Some pharmacophore studies might have been limited by the nature of the agents
examined. Indeed, most 5ꢀHT2A receptor pharmacophore studies have examined fairly
large molecules and, as already mentioned, nearly all contained at least two aromatic
(hydrophobic) centers. The structural requirements for risperidone (1) to act as a 5ꢀHT2A
receptor antagonist have not been extensively examined. Hence, it was of interest here
to determine how much of the risperidone (1) structure is actually required in order to
retain this action. Consequently, four deconstructed analogs (i.e., partial structures) of
risperidone (1), 2ꢀ5 (Figure 2), were prepared and investigated. Compound 2 retains the
amide terminus of risperidone (1) whereas 3 possesses an amine terminus. The
terminal chain of 2/3 was truncated to the Nꢀmethylpiperidine 4 and piperidine analog 5.
N
O
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O
O
N
N
N
H3C
N
N
F
F
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4
N
N
O
O
HN
F
F
5
Figure 2. Deconstructed risperidone analogs examined in the current investigation: tertiary
amide 2, diamine 3, Nꢀmethylpiperidine 4, and piperidine 5.
Preliminary evaluation of 2ꢀ5 (at a single concentration of 10 ꢁM) revealed 5ꢀHT2A
antagonist properties. To determine if the effect involved the orthosteric binding site of
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5ꢀHT2A receptors, their binding affinity was measured. Their functional activity was then
compared with that of risperidone (1) using a twoꢀelectrode voltage clamp (TEVC)
assay as well as an intracellular calcium release assay.
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Results and Discussion
Synthesis – The deconstructed analogs of risperidone, 2ꢀ5, were synthesized as
outlined in Scheme 1. Compound 5 was prepared according to a literature procedure12
and served as a key intermediate in the preparation of 2ꢀ4. NꢀAlkylation of 5 by 4ꢀ
chloroꢀ1ꢀ(piperidinꢀ1ꢀyl)butanꢀ1ꢀone (12) utilizing a Finkelstein reaction yielded the
desired amide 2 (Scheme 1). Reduction of 2 with diborane⋅THF complex resulted in 3.
The Nꢀmethyl analog of 5, 4, was obtained using an EschweilerꢀClarke Nꢀmethylation
reaction. All compounds were prepared as water soluble hydrochloride salts.
Binding – Competition binding assays were performed in plasma membrane
preparations of human embryonic kidney (HEK293) cells transiently transfected with a
construct encoding 5ꢀHT2A receptors for determining the affinity of risperidone.
Risperidone displaced [3H]ketanserin binding (Supporting Information, Figure SIꢀ1) with
a Ki value of 5.29 nM, consistent with its previously reported high affinity for 5ꢀHT2A
receptors.13
We examined the binding affinity of the risperidone derivatives much in the same way
we did for risperidone. Compounds 2 (Ki = 39.81 nM) and 3 (Ki = 34.83 nM) retained
high binding affinity to 5ꢀHT2A receptors, even though they exhibited lower affinity (7.4ꢀ
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and 6.6ꢀfold lower affinity, respectively; Figure 3) than risperidone (1). The Nꢀ
methylpiperidine derivative (4; Ki = 12.27 nM) showed the highest binding affinity (half
that of risperidone), whereas the piperidine derivative (5; Ki = 71.41 nM) showed the
lowest binding affinity of all derivatives tested (13.5ꢀfold lower than risperidone). Data
for the binding of risperidone (Supporting Information, Figure SIꢀ1) and 2ꢀ5 are shown in
Figure 3 and are summarized in Table 1. These results suggested that all derivatives
tested bind competitively at 5ꢀHT2A receptors with relatively high affinity.
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Compound 2
Compound 3
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0
Ligand (log M)
Ligand (log M)
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Ligand (log M)
Ligand (log M)
Figure 3. Deconstructed risperidone analogs bind 5ꢀHT2A receptors with comparable high
affinities. [3H]Ketanserin binding competition curves by compounds 2ꢀ5 in HEK293 cell
membrane preparations. Data are means ± SEM (n = 4).
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Table 1. Radioligand binding assays and Ca2+ imaging results for risperidone (1) and its four
deconstructed analogs 2ꢀ5.
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5ꢀHT2A receptor binding affinity
Ca2+ imaging
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Ki (nM)
5.29
log Ki ± SEM
ꢀ8.27 ± 0.06
IC50 ±SEM (ꢁM)
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43.88 ± 1.47
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ꢀ7.40 ± 0.11
ꢀ7.45 ± 0.12
ꢀ7.91 ± 0.10
ꢀ7.14 ± 0.09
Functional assays – Ion channels can serve as sensitive reporters for G proteinꢀcoupled
receptor (GPCR) activity.14,15 We utilized the Xenopus laevis oocyte system to
heterologously express 5ꢀHT2A receptors and the G proteinꢀgated inwardly rectifying K+
(GIRK4ꢀS143T or GIRK4*) reporter, a channel activated by Gβγ associated with PTXꢀ
sensitive Gα subunits.16,17 When 1 ꢁM serotonin (5ꢀHT) was perfused in the bath in a
twoꢀelectrode voltage clamp (TEVC) experiment, two effects became apparent:
activation of a transient outwardly rectifying (larger outward than inward) current,
followed by inhibition of the inwardly rectifying (larger inward than outward) GIRK4*
current (Figure 4A). The transient current reflects activation of a calciumꢀactivated
chloride channel (ICaꢀCl) endogenous to Xenopus oocytes, providing functional evidence
that 5ꢀHT2A receptor signaling occurred (i.e. Gq activation → PLCβ1 activation →
hydrolysis of PIP2 to DAG and IP3 generation → release of Ca2+ from ER stores).e.g. 18
The ensuing inhibition of the GIRK4* current is due to phosphoinositide hydrolysis and,
thus, a decrease in the plasma membrane concentration of PIP2, as interactions of this
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and most ion channels with PI(4,5)P2 are essential to keep the channel gates open.19 In
the presence of 3 ꢁM risperidone (1), 5ꢀHTꢀmediated current inhibition was greatly
attenuated. Some ICaꢀCl could be seen only in the outward direction, while the inhibition
of the GIRK4* current was abolished (Figure 4B).
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Figure 4. Risperidone acts as an antagonist at 5ꢀHT2A receptors. (A) Representative bariumꢀ
sensitive GIRK4* inward and outward current traces obtained in response to 1 ꢁM serotonin (5ꢀ
HT) applied to oocytes expressing 5ꢀHT2A receptors. (B) Representative bariumꢀsensitive
GIRK4* inward and outward current traces obtained in response to 1 ꢁM serotonin (5ꢀHT) and 3
ꢁM risperidone concurrently applied to oocytes expressing 5ꢀHT2A receptors. (C) Summary bar
graph or (D) concentration response curve of Gq/11 activity in response to 1 ꢁM 5ꢀHT with or
without increasing concentrations of risperidone measured in oocytes (n = 7ꢀ15/condition. Data
are mean ± SEM, **p<0.01, ***p<0.001, significance compared to response to 1 ꢁM 5ꢀHT,
Dunnett’s postꢀhoc test of oneꢀway ANOVA, experiments were performed in ≥2 batches of
oocytes).
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A concentrationꢀresponse of risperidone antagonizing the action of 5ꢀHT (1 ꢁM) was
performed and the results showed significant effects at concentrations of 100 nM or
greater (Figures 4C and D). The apparent risperidone IC50 value was estimated by this
assay at 55.7 nM, ~10ꢀfold lower than its binding affinity (see Supporting Information,
Figure SIꢀ1).
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Before proceeding with similar functional characterization of antagonist action of the
deconstructed risperidone analogs, we examined their possible agonist effects. All
compounds, except compound 5, yielded significant current inhibition at concentrations
of 50 ꢁM or higher (Supporting Information, Figure SIꢀ2AꢀD). Compound 3 seemed to
cause significant current inhibition at concentrations as low as 5 ꢁM. When we
compared the effects of the risperidone derivatives at 50 ꢁM or higher in oocytes
expressing GIRK4* alone, versus GIRK4* and 5ꢀHT2A receptors together, we found that
the effects of risperidone and simplified derivatives, 2 and 3, showed no significant
differences between the two groups, suggesting that high concentrations of risperidone
and 2 and 3 inhibited the GIRK4* channel directly (Supporting Information, Figures SIꢀ
3AꢀC). Unlike compounds 1ꢀ4, compound 5 did not exhibit significant agonism at the
concentrations tested up to 50 ꢁM (data not shown). Overall, these data revealed a
limitation as to how high in concentration we could test the effects of risperidone
derivatives using GIRK4* as a reporter for 5ꢀHT2A receptor activity.
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Concentrationꢀresponse experiments aiming to antagonize 5ꢀHT responses revealed
that significant inhibition could be achieved at the highest concentrations tested (10 ꢁM)
for compounds 2, 4, and 5, while for compound 3 no significant effects could be
achieved, possibly because at as low as 5 ꢁM this compound inhibited the GIRK4*
channel directly (Figure 5B and Supporting Information, Figures SIꢀ2C and 3C).
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A
B
Compound 2 (ꢁM)
Compound 3 (ꢁM)
D
C
Compound 4 (ꢁM)
Compound 5 (ꢁM)
Figure 5. The deconstructed analogs of risperidone act as antagonists at 5ꢀHT2A receptors as
assessed by a TEVC assay. Summary bar graphs of Gq/11 activity in response to 1 ꢁM 5ꢀHT
with or without increasing concentrations of (A) compound 2 (n = 7ꢀ12/condition), (B) compound
3 (n = 7ꢀ9/condition), (C) compound 4 (n = 6ꢀ18/per condition), or (D) compound 5 (n = 7ꢀ
15/condition) measured in oocytes. Data are mean ± SEM, **p<0.01, ***p<0.001, significance
compared to response to 1 ꢁM 5ꢀHT, Dunnett’s postꢀhoc test of oneꢀway ANOVA, experiments
were performed in ≥2 batches of oocytes.
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The concentrationꢀresponse experiments in the oocyte system using GIRK4* channel
currents as the reporter of the action of risperidone analogs suggested that at least
three of the four analogs functioned as antagonists of 5ꢀHT responses. Yet, the direct
action of compounds 1ꢀ4 on the reporter channel itself limited full characterization of
these compounds.
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These results prompted us to consider a complementary functional assay to report on
the action of risperidone and its deconstructed analogs. Figure 6 shows epifluorescence
experiments in HEK 293 cells stably expressing 5ꢀHT2A receptors and using the Fura 2
dye to report changes in intracellular Ca2+ due to risperidone and its deconstructed
analogs. First we applied compounds 1ꢀ5 by themselves (up to a concentration of 100
ꢁM) and recorded no changes in intracellular Ca2+ concentration (data not shown).
Application of 1 ꢁM 5ꢀHT produced robust Ca2+ transients that could be blocked
progressively by increasing concentrations of risperidone and its derivatives (Figure 6).
Thus, based on apparent IC50 values in the [Ca2+]i assay the order of potency was as
follows: risperidone (5.59 ꢁM) > 4 (7.40 ꢁM) > 2 (16.65 ꢁM) > 5 (20.12 ꢁM) > 3 (43.88
ꢁM) (summarized in Table 1).
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Figure 6. The deconstructed analogs of risperidone act as antagonists at 5ꢀHT2A receptors as
assessed by an intracellular Ca2+ release assay A) Sample traces of calcium responses elicited
by 1 µM of 5ꢀHT in the presence of 5 ꢁM risperidone. Each line corresponds to the response of
an individual cell. BꢀF) Doseꢀresponse curves obtained with 1 µM of 5ꢀHT in the presence of
various concentrations of risperidone (B), compound 2 (C), compound 3 (D), compound 4 (E)
and compound 5 (F). Data from two independent experiments per compound and a total of 4ꢀ6
wells per compound per concentration point are expressed as mean ± SEM. The numbers
inside the graphs indicate the total number of points analyzed for each drug.
According to existing pharmacophore models, it might not have been expected that
compounds lacking two aromatic (hydrophobic) moieties would be effective 5ꢀHT2A
receptor competitive antagonists. Clearly, the entire structure of risperidone (1) is shown
here to be unnecessary for this action. For example, compounds 2, 3, and 4 bind with
only about 2ꢀ to 8ꢀfold lower affinity than 1, and are only about 2ꢀ to 3ꢀfold less potent
than risperidone as antagonists in the Ca2+ imaging assay.
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Although changes in intracellular Ca2+ proved to be a threeꢀorders of magnitude lower
sensitivity assay than the GIRK4* channel reporter, results from the two methods were
consistent in showing that risperidone (1) was the most potent antagonist, whereas
compound 3 was the least potent antagonist of 5ꢀHT responses (compare Figures 4C
and 6B versus Figures 5B and 6D). Moreover, the [Ca2+]i assay proved cleaner than the
channel reporter assay since risperidone (1) and its derivatives did not change basal
[Ca2+]i levels and allowed a complete ranking of these compounds in terms of their
functional potency.
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In summary, it would appear from the present results that truncated versions of
risperidone (1) bind with nanomolar affinity and retain 5ꢀHT2A antagonist character. In
fact, we reported some time ago that compound 5 binds only with 16ꢀfold lower affinity
than risperidone (1) at [3H]ketanserinꢀlabeled 5ꢀHT2A receptors (rat brain homogenates)
and acted as a potent 5ꢀHT2 receptor antagonist in vitro (5ꢀHTꢀinduced inositol
phosphate production) and in vivo (rats trained to discriminate the 5ꢀHT2 receptor
agonist 1ꢀ(2,5ꢀdimethoxyꢀ4ꢀmethylphenyl)ꢀ2ꢀaminopropane from vehicle) without
producing any agonist action when examined alone.13 Taken together with the present
findings, future pharmacophore models for 5ꢀHT2A receptor antagonists will need to
consider this information. Although most 5ꢀHT2A serotonin receptor antagonists
commonly possess more than a single aromatic/hydrophobic feature, this would not
appear to be an essential requirement.
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Synthesis –
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Melting points were taken on a ThomasꢀHoover melting point apparatus in glass
capillary tubes and are uncorrected. 1H NMR spectra were recorded with a Bruker ARX
400 MHz spectrometer with tetramethylsilane (TMS) as an internal standard. Peak
positions are given in parts per million (δ). Infrared spectra were obtained on a Nicolet
iS10 FTꢀIR spectrometer. Elemental analyses were performed by Atlantic Microlab Inc.
(Norcross, GA) for the indicated elements and results are within 0.4% of calculated
values. Reactions were monitored by thinꢀlayer chromatography (TLC) on silica gel
GHLF plates (250 ꢁ, 2.5 x 10 cm; Analtech Inc., Newark, DE).
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4-(4-(6-Fluorobenzisoxazol-3-yl)piperidin-1-yl)-1-(piperidin-1-yl)butan-1-one
Hydrochloride (2). Compound 12 (0.22 g, 1.14 mmol) and free base of 5 (0.25 g, 1.14
mmol) were added to a stirred solution of K2CO3 (0.31 g, 2.26 mmol) and KI (few
crystals) in anhydrous MeCN (5 mL). The reaction mixture was allowed to stir in a
screwꢀcap vial at 88 °C for 16 h. The solvent was evaporated under reduced pressure;
the residue was suspended in H2O and extracted with CHCl3 (3 x 15 mL). The
combined organic portion was washed with H2O (3 x 10 mL), dried (Na2SO4), and
evaporated under reduced pressure to yield an ivoryꢀcolored solid. The solid was
dissolved in EtOH (5 mL) and the solution was cooled to 0 °C. A saturated solution of
gaseous HCl in Et2O (2 mL) was added and the mixture was allowed to stand at room
temperature for 3 h. The solvent was evaporated to yield a white solid that was
recrystallized from EtOH to yield 0.01 g (3%) of 2 as a tanꢀcolored solid: mp 216ꢀ218 °C
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1H (DMSOꢀd6): 1.43ꢀ1.69 (m, 4H, CH2), 1.82ꢀ2.10 (m, 6H, CH2), 2.21ꢀ2.24 (m, 2H, CH2),
2.82ꢀ2.88 (m, 2H, CH2), 2.82ꢀ2.88 (m, 1H, CH), 3.23ꢀ3.26 (m, 2H, CH2), 3.40ꢀ3.52 (m,
2H, CH2), 3.63ꢀ3.66 (m, 2H, CH2), 3.98ꢀ4.02 (m, 2H, CH2), 4.48ꢀ4.51 (m, 2H, CH2),
7.29ꢀ7.38 (m, 1H, ArH), 7.70ꢀ7.76 (m, 1H, ArH), 8.05ꢀ8.18 (m, 1H, ArH), 10.17 (br s, 1H,
NH+) Anal Calcd for (C21H28FN3O2—HCl—0.5H2O) C, 60.21; H, 7.02; N, 9.77. Found: C,
60.32; H, 7.22; N, 10.03.
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6-Fluoro-3-(1-(4-piperidin-1-yl)butyl)piperidin-4-yl)benzisoxazole
Hydrochloride
(3). A 1M solution of BH3 in THF (1.41 mL) was added to a stirred solution of 2 (0.13 g,
0.35 mmol) in anhydrous THF (4 mL) at 0 °C (iceꢀbath). The reaction mixture was
allowed to warm to room temperature and stirring was continued for 10 h. The reaction
mixture was carefully quenched with 6M aqueous HCl (0.40 mL) and then heated at
reflux for 1 h. The mixture was allowed to cool to room temperature and basified with 1N
aqueous NaOH (2 mL). Water (10 mL) was added and the aqueous portion was
extracted with EtOAc (3 x 15 mL). The organic portions were combined, dried (Na2SO4),
and solvent was removed under reduced pressure to give an oily residue that was
dissolved in absolute EtOH, and HClꢀanhydrous Et2O was added to afford a solid.
Recrystallization from absolute EtOH/anhydrous Et2O gave 0.04 g (26%) of 3 as yellow
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crystals: mp 270ꢀ273 °C; HꢀNMR (DMSOꢀd6: salt) δ 1.37 (m, 1H, CH2), 1.68ꢀ1.78 (m,
10H, CH2), 2.17ꢀ2.20 (m, 2H, CH2), 2.38ꢀ2.53 (m, 2H, CH2), 2.83 (m, 2H, CH2), 3.01ꢀ
3.10 (m, 7H, CH, CH2), 3.36ꢀ3.46 (m, 1H, CH2), 3.58ꢀ3.61 (m, 2H, CH2), 7.32 (td, J = 9.1
2.0 Hz, 1H, ArH), 7.71 (dd, J = 9.0, 1.9 Hz, 1H, ArH), 8.25 (dd, J = 8.6, 5.4 Hz, 1H,
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ArH), 10.33 (br s, 1H, NH+), 11.06 (br s, 1H, NH+). Anal. Calcd for (C21H30FN3O—2HCl—
0.25H2O) C, 57.73; H, 7.50; N, 9.62. Found: C, 57.36; H, 7.21; N, 9.30.
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6-Fluoro-3-(1-methylpiperidin-4-yl)benzisoxazole Hydrochloride (4). The free base
of 6ꢀfluoroꢀ3ꢀ(piperidinꢀ4ꢀyl)benzisoxazole (5; 0.20 g, 0.90 mmol) was added to a stirred
solution of HCOOH (0.2 mL, 5.45 mmol) and HCHO (0.2 mL, 5.45 mmol) in EtOH under
an N2 atmosphere. The reaction mixture was heated at reflux for 10 h and the solvent
was evaporated to yield a white solid. The solid was dissolved in EtOH (5 mL) and
cooled to 0 °C. A saturated solution of gaseous HCl in Et2O (2 mL) was added and the
mixture was allowed to stand at room temperature for 3 h. The solvent was evaporated
and the crude solid was recrystallized from EtOH to yield 0.01 g (4%) of 4 as a white
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solid: mp 218ꢀ220 °C, H (DMSOꢀd6): 2.20ꢀ2.25 (m, 4H, CH2), 2.80 (s, 3H, CH3), 3.09ꢀ
3.19 (m, 2H, CH2), 3.53ꢀ3.56 (m, 2H, CH2), 7.33 ꢀ7.38 (m, 1H ArH), 7.73ꢀ7.75 (dd, J= 2,
8 Hz 1H, ArH), 8.15ꢀ8.18 (m, 1H, ArH), 10.64 (br s, 1H, NH+). Anal Calcd for
(C13H15FN2O—HCl—0.5H2O) C, 55.82; H, 6.13; N, 10.01. Found: C, 55.50; H, 6.08; N,
9.69.
4-Chloro-1-(piperidin-1-yl)butan-1-one (12). Piperidine (11; 1.2 mL, 11.54 mmol) was
added in a dropwise manner to a stirred solution of Et3N (1.2 mL, 11.54 mmol) in
CH2Cl2 (25 mL) and the reaction mixture was cooled to 0 °C under an N2 atmosphere. A
solution of 4ꢀchlorobutyryl chloride (1.5 mL, 11.74 mmol) in CH2Cl2 (5 mL) was added
and the reaction mixture was allowed to stir at room temperature for 75 h and washed
with H2O (2 x 25 mL). The organic portion was dried (Na2SO4) and evaporated under
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reduced pressure to yield a yellowꢀcolored oil, which was purified by vacuum distillation
to yield 1.00 g (45%) of 12 as a pale yellowꢀcolored oil: bp 103 °C at 0.02 psi. The oil
was used without further characterization in preparation of 2.
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Radioligand Binding –
Transient Transfection of HEK293 cells. Human embryonic kidney (HEK293) cells were
maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) fetal
bovine serum at 37 °C in a 5% CO2ꢀhumidified atmosphere. Transfection was
performed using Lipofectamine 200 reagent (Invitrogen) according to the manufacturer’s
instructions. The Nꢀterminally cꢀMycꢀtagged form of wild type human 5ꢀHT2A receptor
(pcDNA3.1ꢀcꢀMycꢀ5ꢀHT2A) has been described previously.21
Radioligand binding. Radioligand binding assays were performed as described
previously21 with minor modifications. Briefly, HEK293 cell pellets were homogenized
using a Teflonꢀglass grinder (10 upꢀandꢀdown strokes at 1,500 rpm) in 1 mL of binding
buffer (see below), supplemented with 0.25 M sucrose. The crude homogenate was
centrifuged at 1,000 × g for 5 min at 4 °C, and the supernatant reꢀcentrifuged at 40,000
× g for 15 min at 4 °C. The resultant pellet (P2 fraction) was washed twice in
homogenization buffer and reꢀcentrifuged in similar conditions. Aliquots were stored at
−80 °C until assay. Protein concentration was determined using the BioꢀRad protein
assay.
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[3H]Ketanserin binding was measured at equilibrium in 100ꢀµl aliquots (50 mM TrisꢀHCl,
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pH 7.4) of membrane preparations (∼ 10 µg of total protein) that were incubated at 37
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°C for 60 min. Competition curves were carried out by incubating the tested compound
(10ꢀ10 – 10ꢀ4 M; 14 concentrations) in binding buffer containing 4 nM [3H]ketanserin. Nonꢀ
specific binding was determined in the presence of 10 µM methysergide. Incubations
were terminated by dilution with 200 µl iceꢀcold incubation buffer and free ligand was
separated from bound ligand by rapid filtration under vacuum through GF/C glass fiber
filters using a microbeta filtermatꢀ96 harvester (PerkinElmer). These filters were then
rinsed twice with 200 µl iceꢀcold incubation buffer, air dried, and counted for radioactivity
by liquid scintillation spectrometry, using a MicroBeta2 detector (PerkinElmer).
Radioligand binding data were analyzed using nonꢀlinear curveꢀfitting software
(GraphPad Prism).
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Functional assays –
Expression of Recombinant Proteins in Xenopus oocytes. Oocytes were isolated and
microinjected with equal volumes (50 nl) as previously described.14 In all twoꢀelectrode
voltageꢀclamp experiments (TEVC), oocytes were injected with 2 ng of 5ꢀHT2A receptor
and 2 ng of GIRK4* and were maintained at 18 °C for 1ꢀ4 days before recording.
Two-Electrode Voltage-Clamp Recording and Analysis. Wholeꢀcell currents were
measured by conventional TEVC with a GeneClamp 500 amplifier (Axon Instruments,
Union City, CA), as previously reported.14 A highꢀpotassium (HK) solution was used to
superfuse oocytes (96 mM KCl, 1 mM NaCl, 1 mM MgCl2, 5 mM KOH/HEPES; pH 7.4)
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to obtain a reversal potential for potassium (EK) close to zero. Inwardly rectifying
potassium currents through GIRK4* were obtained by clamping the cells at ꢀ80 mV.
Basal GIRK4* currents were defined as the difference between inward currents
obtained at ꢀ80 mV in the presence of 3 mM BaCl2 in HK solution and those in the
absence of Ba2+ and measured for each trace. Current inhibition to 5ꢀHT was measured
and normalized to basal current to compensate for size variability in oocytes. Current
inhibition of risperidone derivatives was normalized to 1 ꢁM 5ꢀHT to compensate for
variability in basal currents.
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Measurement of Intracellular Ca2+. The 5ꢀHT2A receptor cDNA was subcloned into the
pcDNA5/FRT/TO plasmid and stable inducible cell lines were produced using the single
site recombination TꢀRex FlpꢀIn system as previously described.20 5ꢀHT2A receptor
expression was induced adding 1 ꢀg/mL doxycycline to the culture media at least two
days before the experiment, and cells were plated on 96ꢀwell dishes one day before the
experiment. The day of the experiment the cells were switched to serumꢀfree medium
for about 3ꢀ4 h, before being loaded with 5 ꢁm Fura2ꢀAM (Molecular Probes, OR) in
Imaging Solution (125 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 6 mM glucose,
and 25 mM Hepes/Tris, pH 7.4). Following incubation for 30 min at 37 °C, cells were
washed with Imaging Solution and kept at room temperature for about 15 min before
being placed on the stage of an epifluorescence microscope, coupled to an automatic
perfusion system and controlled by the Live Acquisition Software from Till Photonics
(see reference 20 for details). The Furaꢀ2 signal was acquired at 510 nm by switching
the excitation wavelength between 340 nm and 380 nm. Baseline was recorded for 30 s
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before perfusion of 5ꢀHT (1 µM) in the presence and absence of various concentrations
of the drugs studied for another 45 s, followed by perfusion of Imaging Solution for 30 s
to wash out the drugs. Intracellular calcium concentration was reported as fluorescence
ratio (F 340/F380) and values were normalized to the basal F340/F380 ratio level before
perfusion of the drugs. Data obtained for each drug in the presence of 5ꢀHT were further
normalized to the mean responses elicited by 5ꢀHT alone in the same experiment.
Results are expressed as mean ± SEM. Doseꢀresponse curves, curve fitting and IC50
values were obtained by analysis using GraphPad PRISM 6 software.
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Acknowlegments
The authors would like to thank Mario de la Fuente, Meng Cui, Yu Xu, and Takeharu
Kawano for their insightful discussions on the project.
Corresponding Author
Eꢀmail: glennon@vcu.edu: Mailing Address: Department of Medicinal Chemistry, School
of Pharmacy, Virginia Commonwealth University, Richmond, VA 23298, USA.
Author Contributions
SAG and RHV synthesized and characterized the target compounds and were overseen
by MD and RAG, respectively. JLM and KH performed the binding assays under the
supervision of JGꢀM. JWY, AE, SS, and PD performed the TEVC assays under the
supervision of DEL. LB and JME produced stableꢀexpressing 5HT2A receptor cell lines
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and performed the intracellular Ca2+ measurements. RAG, MD, DEL, and JGꢀM
oversaw the entire project and wrote the first draft of the manuscript. All authors had an
opportunity to contribute to the preparation of the final manuscript.
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Funding
This work was supported in part by NIH R01 HL59949ꢀ19 (DEL) and R01MH084894
(JMG).
Supporting Information Available
The Supporting Information section includes a [3H]ketanserin binding competition curve
by risperidone in HEK293 cells transfected with the 5ꢀHT2A receptor, and summary bar
graphs of Gq/11 activity in response to 5ꢀHT or test compounds. This information is
available free of charge via the Internet at http://pubs.acs.org/.
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Shim, J., Fabiato, A., MacKerell, A. D. Jr., Brezina, V., Sealfon, S. C., Filizola, M.,
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Liapakis, G., GonzalezꢀMaeso, J., Zhang, H., Cui, M., and Logothetis, D. E. (2014)
G proteinꢀcoupled receptor signaling to Kir channels in Xenopus oocytes. Curr
Pharm. Biotechnol. 15, 987–995.
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of the KACh channel, using functional homomeric mutants. J. Biol. Chem. 272,
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(2004) Characteristic interactions with phosphatidylinositol 4,5ꢀbisphosphate
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Cui, M., and Baki, L. (2015) Phosphoinositide control of membrane protein
function: a frontier led by studies on ion channels. Annu. Rev. Physiol. 77, 81–
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(2014) Electrical coupling between the human serotonin transporter and voltageꢀ
gated Ca(2+) channels. Cell Calcium 56, 25–33.
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Georgakopoulos, A., Moron, J. A., Milligan, G., LopezꢀGimenez, J. F., Robakis, N.
K., Logothetis, D. E., Meana, J. J., and GonzalezꢀMaeso, J. (2016) Allosteric
signaling through an mGlu2 and 5ꢀHT2A heteromeric receptor complex and its
potential contribution to schizophrenia. Sci. Signal. 9(410), ra5.
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Scheme 1. Synthesis of compounds 2-5.
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a Reagents and conditions: (i) (a) HCOOH, Ac2O, 65 ºC, 1 h; (b) room temperature, 16
h; (ii) (a) SOCl2, DMF, room temperature, 6 h; (b) 1,3ꢀdifluorobenzene, AlCl3, reflux, 45
h; (iii) NH2OH—HCl, NaOH/H2O, EtOH, reflux, 96 h; (iv) (a) NaH, DMF, room temperature
48 h; (v) (a) conc. HCl, EtOH, reflux, 3 h; (b) room temperature, 48 h; (vi) (a) HCOOH,
HCHO, reflux, 10 h; (b) HCl/Et2O (vii) 4ꢀchlorobutyryl chloride, Et3N, CH2Cl2, room
temperature, 75 h; (viii) K2CO3, KI, MeCN, 88 ºC, 16 h; (ix) (a) BH3—THF, reflux, 2 h; (b)
6N HCl, reflux, 1 h.
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Figure Legends
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Figure 1. The antipsychotic agent risperidone (1) and its ring numbering system.
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Figure 2. Deconstructed risperidone analogs examined in the current investigation:
tertiary amide 2, diamine 3, Nꢀmethylpiperidine 4, and piperidine 5.
Figure 3. Deconstructed risperidone analogs bind 5ꢀHT2A receptors with comparable
high affinities. [3H]Ketanserin binding competition curves by compounds 2ꢀ5 in HEK293
cell membrane preparations. Data are means ± SEM (n = 4).
Figure 4. Risperidone acts as an antagonist at 5ꢀHT2A receptors. (A) Representative
bariumꢀsensitive GIRK4* inward and outward current traces obtained in response to 1
ꢁM serotonin (5ꢀHT) applied to oocytes expressing 5ꢀHT2A receptors. (B)
Representative bariumꢀsensitive GIRK4* inward and outward current traces obtained in
response to 1 ꢁM serotonin (5ꢀHT) and 3 ꢁM risperidone concurrently applied to
oocytes expressing 5ꢀHT2A receptors. (C) Summary bar graph or (D) concentration
response curve of Gq/11 activity in response to 1 ꢁM 5ꢀHT with or without increasing
concentrations of risperidone measured in oocytes (n = 7ꢀ15/condition. Data are mean ±
SEM, **p<0.01, ***p<0.001, significance compared to response to 1 ꢁM 5ꢀHT, Dunnett’s
postꢀhoc test of oneꢀway ANOVA, experiments were performed in ≥2 batches of
oocytes).
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Figure 5. The deconstructed analogs of risperidone act as antagonists at 5ꢀHT2A
receptors as assessed by a TEVC assay. Summary bar graphs of Gq/11 activity in
response to 1 ꢁM 5ꢀHT with or without increasing concentrations of (A) compound 2 (n
= 7ꢀ12/condition), (B) compound 3 (n = 7ꢀ9/condition), (C) compound 4 (n = 6ꢀ18/per
condition), or (D) compound 5 (n = 7ꢀ15/condition) measured in oocytes. Data are mean
± SEM, **p<0.01, ***p<0.001, significance compared to response to 1 ꢁM 5ꢀHT,
Dunnett’s postꢀhoc test of oneꢀway ANOVA, experiments were performed in ≥2 batches
of oocytes.
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Figure 6. The deconstructed analogs of risperidone act as antagonists at 5ꢀHT2A
receptors as assessed by an intracellular Ca2+ release assay A) Sample traces of
calcium responses elicited by 1 µM of 5ꢀHT in the presence of 5 ꢁM risperidone. Each
line corresponds to the response of an individual cell. BꢀF) Doseꢀresponse curves
obtained with 1 µM of 5ꢀHT in the presence of various concentrations of risperidone (B),
compound 2 (C), compound 3 (D), compound 4 (E) and compound 5 (F). Data from two
independent experiments per compound and a total of 4ꢀ6 wells per compound per
concentration point are expressed as mean ± SEM. The numbers inside the graphs
indicate the total number of points analyzed for each drug.
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Figure 1
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N
O
O
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N
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F
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N
CH3
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Figure 2
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O
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H3C
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