Revised Pharmacophore Model for 5-HT2A Receptor Antagonists Derived from the Atypical Antipsychotic Agent Risperidone

Article abstract of DOI:10.1021/acschemneuro.8b00637

Pharmacophore models for 5-HT_2A receptor antagonists consist of two aromatic/hydrophobic regions at a given distance from a basic amine. We have previously shown that both aromatic/hydrophobic moieties are unnecessary for binding or antagonist action. Here, we deconstructed the 5-HT_2A receptor antagonist/serotonin-dopamine antipsychotic agent risperidone into smaller structural segments that were tested for 5-HT_2A receptor affinity and function. We show, again, that the entire risperidone structure is unnecessary for retention of affinity or antagonist action. Replacement of the 6-fluoro-3-(4-piperidinyl)-1,2-benz[d]isoxazole moiety by isosteric tryptamines resulted in retention of affinity and antagonist action. Additionally, 3-(4-piperidinyl)-1,2-benz[d]isoxazole (10), which represents less than half the structural features of risperidone, retains both affinity and antagonist actions. 5-HT_2A receptor homology modeling/docking studies suggest that 10 binds in a manner similar to risperidone and that there is a large cavity to accept various N₄-substituted analogues of 10 such as risperidone and related agents. Alterations of this "extended" moiety improve receptor binding and functional potency. We propose a new risperidone-based pharmacophore for 5-HT_2A receptor antagonist action.

Full text of DOI:10.1021/acschemneuro.8b00637

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Article
A revised pharmacophore model for 5-HT2A receptor antagonists
derived from the atypical antipsychotic agent risperidone
Urjita H Shah, Supriya A Gaitonde, Jose L Moreno, Richard
A. Glennon, Malgorzata Dukat, and Javier González-Maeso
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00637 • Publication Date (Web): 04 Jan 2019
Downloaded from http://pubs.acs.org on January 5, 2019
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A revised pharmacophore model for 5-HT2A receptor antagonists
derived from the atypical antipsychotic agent risperidone
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Urjita H. Shah,  Supriya A. Gaitonde,,‡,# José L. Moreno,,§ Richard A. Glennon,‡
,‡
‡*
*
Małgorzata Dukat, Javier González-Maeso
Department of Physiology and Biophysics, Virginia Commonwealth University School of
Medicine, Richmond, Virginia 23298, United States
‡Department of Medicinal Chemistry, Virginia Commonwealth University School of
Pharmacy, Richmond, Virginia 23298, United States
#Current address: Cipla Ltd. Mumbai 400013, India
§Current address: GlaxoSmithKline. Stevenage, SG1 2NY, UK
*Co-corresponding authors
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ABSTRACT
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Pharmacophore models for 5-HT2A receptor antagonists consist of two
aromatic/hydrophobic regions at a given distance from a basic amine. We have previously
shown that both aromatic/hydrophobic moieties are unnecessary for binding or antagonist
action. Here, we deconstructed the 5-HT2A receptor antagonist/serotonin-dopamine
antipsychotic agent risperidone into smaller structural segments that were tested for 5-
HT2A receptor affinity and function. We show, again, that the entire risperidone structure
is unnecessary for retention of affinity or antagonist action. Replacement of the 6-fluoro-
3-(4-piperidinyl)-1,2-benz[d]isoxazole moiety by isosteric tryptamines resulted in retention
of affinity and antagonist action. Additionally, 3-(4-piperidinyl)-1,2-benz[d]isoxazole (10),
which represents less than half the structural features of risperidone, retains both affinity
and antagonist action. 5-HT receptor homology modeling/docking studies suggest that
2A
10 binds in a manner similar to risperidone, and that there is a large cavity to accept
various N -substituted analogs of 10 such as risperidone and related agents. Alterations
4
of this “extended” moiety improve receptor binding and functional potency. We propose
a new risperidone-based pharmacophore for 5-HT receptor antagonist action.
2
A
KEYWORDS: antipsychotics, schizophrenia, serotonin 5-HT2A receptor, risperidone,
pharmacophore
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INTRODUCTION
Antipsychotic medications, including typical (i.e., first generation) agents such as
chlorpromazine and haloperidol, and atypical, or second generation, agents such as
risperidone (1) (Figure 1), are frontline treatments for many psychotic diseases including
schizophrenia and bipolar disorder.1–3 Typical antipsychotic agents act primarily via a
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dopamine D receptor antagonist mechanism whereas most atypical antipsychotic agents
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display enhanced action/potency as antagonists of serotonin (5-hydroxytryptamine, 5-
1–5
HT), specifically 5-HT2A receptors relative to dopamine D receptors. These drugs
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usually ameliorate hallucinations and delusions in patients with psychotic disorders, but
there appear to be differences between these agents in their clinical efficacy and side
effects that might be due to off-target pharmacological actions.1,5 This underscores the
need for a better understanding of the molecular structural attributes responsible for the
pharmacological interactions of atypical antipsychotics with 5-HT2A receptors, with the
ultimate goal of developing new, and more effective, treatments for psychosis and other
neuropsychiatric conditions, and agents that produce fewer undesirable dopamine-
related side effects.
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Although agents with 5-HT2A receptor antagonist action, such as risperidone (1),
ketanserin (2) (Figure 1), and others,
belong to a variety of chemical classes,
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5-HT2A
receptor
antagonist
pharmacophore models proposed over
the past 25 years are relatively similar.
That is, the models typically share two
aryl or hydrophobic moieties separated
by a given distance from each other,
and by given distances from a basic
amine (Figure 2).6–9 As a result, these
structural features are fairly common to Figure 1. Risperidone (1) and ketanserin (2).
most 5-HT2A receptor antagonists (although distances might vary from model to model
depending upon the specific antagonists examined in a given study) and are,
consequently, incorporated into new agents as a standard feature.
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Recently, we communicated that an intact risperidone (1), or bi-aryl (bi-hydrophobic)
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structure, note compounds 5 and 6, is not required for binding at 5-HT receptors. We
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Figure 2. A composite pharmacophore model for 5-HT receptor antagonists (data
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risperidone (1), 3-6 (Figure 3), not only bind at 5-HT receptors with reasonably high
2
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6-9
from references provided in the text).
affinity (K values ranging from approximately 12 to 71 nM), but retain 5-HT receptor
i
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antagonist action. Risperidone (1) was, however, the highest-affinity (K = 5.29 nM) and
i
the most potent of the antagonists examined. Nevertheless, our findings clearly
demonstrated that the entire risperidone molecule is not required either for 5-HT2A
receptor binding or 5-HT2A receptor antagonist action. Importantly, these results also
suggested that the currently accepted 5-HT receptor antagonist pharmacophore which,
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as discussed above, includes two aromatic/hydrophobic regions and a protonated amine
(
Figure 2), may no longer be valid and, hence, requires re-examination.10
Figure 3. Deconstructed analogs of risperidone (1) that we previously
10
examined and found to retain 5-HT receptor antagonist action.
2A
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To address several specific questions directly relevant to this issue, we used the atypical
antipsychotic agent risperidone (1), and the 5-HT2A receptor antagonist ketanserin (2)
(Figure 1) – ketanserin being the first and still one of the most widely used 5-HT receptor
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antagonists in molecular pharmacology and preclinical studies. We selected these two 5-
HT2A receptor antagonists because they are structurally compliant with the
pharmacophore models discussed above. They also possess some interesting structural
11
differences (“left halves”; red) as well as some bioisosteric similarities (“right halves”;
green; Figure 1).
Our first question was focused on whether the “left half”, that is, the 2-methyl-6,7,8,9-
tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one portion of risperidone (1), binds at 5-HT2A
receptors in the absence of the “right half” or 3-(4-piperidinyl)-1,2-benzisoxazole moiety.
To test this, we synthesized and examined compounds 7 and 8 (Figure 4). We also
examined whether the entire “left half” portion of risperidone is required/optimal for its
affinity/action. This was addressed by examining the pharmacology and function of
compound 9 (Figure 4), with a truncated “left-half” portion of risperidone. The common
basic amine was included in each “half”. Based on our previous findings with compounds
10
5
and 6 (see above), the next question addressed the extent to which the aryl fluoro
group of risperidone (and analogs) contributes to their pharmacological properties (i.e.,
compounds 10 and 11) (Figure 4). The 3-(4-piperidinyl)1,2-benz[d]isoxazole portion of
risperidone is structurally similar to serotonin (the endogenous 5-HT receptor orthosteric
agonist). Based on this, we decreased the aryl centroid-to-amine distance (making the
“right half” portion of risperidone more closely resemble a tryptamine moiety) to test
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whether this alteration might enhance the binding of 6, and/or if it might convert 6 to a 5-
HT receptor agonist (compound 12) (Figure 4). 3-(4-Piperidinyl)indole analogs of 5-HT
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bind at 5-HT receptors with high affinity, and are suggested to be bioisosteric with their
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corresponding tryptamine counterparts. Furthermore, given evidence that benz[d]-
isoxazoles are bioisosteric with indoles,13 it was not unreasonable to assume that 5/6
might be bioisosteric with 3-(4-piperidinyl)indoles and, thus, with a tryptamine moiety.
Considering that indole rings of tryptamines and benz[d]isoxazole rings are (bio)isosteric,
we also replaced the “right half” portion of risperidone with tryptamine analogs to test
whether these new compounds retain 5-HT receptor antagonist action (i.e., compounds
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13-16) (Figure 4).
If the “right half” portion of risperidone (1) enhances the actions (affinity/antagonist
potency) of the “left half” portion, might it do the same for ketanserin (2)? To test this, we
synthesized and examined the receptor binding and function of two hybrid molecules
where the “right” (compound 17) or “left” (compound 18) halves (Figure 4) of risperidone
were replaced with the “right” or “left” halves or ketanserin, respectively. Compounds 7-
18 were synthesized and evaluated to specifically address these questions.
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Figure 4. Deconstructed (7-11) and elaborated (12-18) analogs of risperidone (1).
Risperidone and ketanserin analogs were synthesized and tested both for 5-HT2A
receptor binding affinity and functional (i.e., agonist and antagonist) activity in living
mammalian cells stably expressing 5-HT2A receptors. Our data confirm that only about
half of the structural attributes of risperidone (1) are required in order for risperidone-
related agents to bind to 5-HT receptors with appreciable affinity and to retain functional
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antagonism. We provide here a new pharmacophore scaffold for 5-HT2A receptor
antagonism consistent with nanomolar potency, which might help with the design of
improved therapeutics for the treatment of psychosis and other neuropsychiatric
disorders.
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RESULTS AND DISCUSSION
Synthesis of deconstructed and elaborated analogs of risperidone.
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Compound 7 was prepared from 3-(2-chloroethyl)-2-methyl-6,7,8,9-tetrahydro-4H-
pyrido[1,2-a]pyrimidin-4-one (19) using a Gabriel synthesis whereas 8 was obtained by
reaction of 19 with piperidine. Compound 9 was synthesized as outlined in Scheme 1.
Intermediate 21 was synthesized using a condensation reaction between 2-bromoethyl
ether and ethyl acetoacetate (20). Cyclization of Intermediate 21 with thiourea yielded
intermediate 22. Reductive desulfurization of intermediate 22 by nickel boride resulted in
the substituted pyrimidone 23. The reaction involved the slow addition of sodium
borohydride to a solution of intermediate 22 and nickel chloride, resulting in the in situ
generation of nickel boride. Treatment of 23 with concentrated HCl, following a literature
procedure,14 provided 24. Compound 9 was synthesized from intermediate 24 by a
Finkelstein alkylation reaction.
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a
Scheme 1. Synthesis of compound 9.
O
H C
O
O
3
ii
O
CH3
O
O
i
HN
O
CH3
H C
3
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CH3
H C
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F
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CH3
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v
iv
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
0.5(COOH)2
CH3
CH3
CH3
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a
Reagents and conditions. (i) (a) NaOEt, EtOH, room temperature, 0.5 h; (b) 2-
bromoethyl ether, reflux, 18 h; (ii) thiourea, NaOEt, EtOH, reflux, 4 h. (iii) NiCl NaBH
2,
4,
MeOH, room temperature, 0.5 h; (iv) conc. HCl, 150 ºC, 3 h; (v) (a) 6-fluoro-3-(4-
piperidinyl)benz[d]isoxazole, K CO , KI, DMF, 134 °C, 18 h; (b) CHCl , (COOH) /Et O.
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Compound 12 was prepared from nipecotic acid (25) in six steps (Scheme 2). Nipecotic
acid (25) was protected with a formyl group using formic acid and acetic anhydride to
generate formic acetic anhydride in situ, which then acted as a formylating agent to yield
intermediate 26. Reaction of 26 with thionyl chloride provided intermediate 27 that was
subsequently converted to 28 via a Friedel Crafts acylation reaction with 1,3-
difluorobenzene. Reaction of intermediate 28 with hydroxylamine gave a mixture of E and
Z isomers of oxime 29. The cyclization of oxime 29 via intramolecular displacement of the
2-fluoro group to intermediate 30 was catalyzed by sodium hydride. It has been reported
for similar reactions that only the Z isomer of the oxime participates in the cyclization
reaction.11 Intermediate 30 was deprotected using concentrated HCl to afford 12.
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Compound 10 was prepared in a manner similar to that shown in Scheme 2 using
fluorobenzene, isonipecotic acid in place of difluorobenzene, nipecotic acid, respectively,
and its subsequent methylation afforded 11 as reported earlier by Strupczewski et al.11
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Scheme 2. Synthesis of analog 12.
H
H
NH
OH
i
ii
N
O
N
O
O
2
O
2
OH
O
Cl
5
6
27
iii
HO
F
N
H
F
O
H
iv
N
O
N
O
F
F
2
8
29
v
H
O
O N
O N
NH•HCl
N
vi
F
F
30
12
a
Reagents and conditions: (i) (a) HCOOH, Ac O, 65 ºC, 1 h; (b) 25, room temperature,
2
1
6 h; (ii) SOCl , room temperature, 6 h; (iii) 1,3-difluorobenzene, AlCl , reflux, 22 h;
2
3
(
iv) NH OH•HCl, NaOH/H O, EtOH, reflux, 96 h; (v) NaH, DMF, THF 75 C, 4h; (vi)
2 2
HCl (3 N), EtOH, reflux 3 h.
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1
1
1
1
1
1
1
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1
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2
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2
2
2
2
2
3
3
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3
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3
3
3
4
4
4
4
4
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4
4
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5
5
5
5
5
5
5
5
6
Compounds 13-15 were prepared by alkylation of the appropriate tryptamine with 3-(2-
chloroethyl)-2-methyl-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one (19) (Scheme
3). Although a number of different alkylation procedures were attempted, a substantial
by-product was obtained in each case when the tryptamine bore a primary amine. This
was presumed to be a di-alkylated product (e.g. 31). Using “forcing” conditions,
compound 31 was isolated and characterized.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Scheme 3. Synthesis of analogs 13-15.
O
NO2
NH2
NH
OEt
iii
NH-CH3
i
ii
N
F
N
H
N
H
N
H
F
F
F
H
32
33
13
34
35
iv
iv
14
NH2
O
O
Cl
N
N
v
N
15
N
CH3
N
H
N
N
CH3
H
19
36
31
2
a
Reagents and conditions (i) LiAlH (5 equivalents), THF, Et O, 60 ˚C, 1 h (ii) ethyl
4
2
chloroformate, Et N, CH Cl , room temperature, 3 h; (iii) (a) LiAlH (3 equivalents), THF,
3
2
2
4
reflux, 1.5 h. (b) Et O, HCl/Et O (iv) (a) 3-(2-chloroethyl)-2-methyl-6,7,8,9-tetrahydro-4H-
2
2
pyrido[1,2-a]pyrimidin-4-one (19), K CO , MeCN, reflux, 17 h; (b) CHCl , (COOH) /Et O.
2
3
3
2
2
(
v) (a) KI, K CO , MeCN, reflux, 48 h; (b) CH Cl , (COOH) /Et O.
2
3
2
2
2
2
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Compound 15 was prepared in the same manner as 13 using tryptamine as starting
material, and compound 16 was prepared in the same manner as 14 using N-
methyltryptamine as starting material.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
4
4
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4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
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5
6
7
8
9
0
1
2
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5
6
7
8
9
0
1
2
3
4
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7
8
9
0
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3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Hybrid molecule 17 was synthesized via a Finkelstein alkylation reaction of 4-(4-
fluorobenzoyl)piperidine hydrochloride with 19, and hybrid 18 was synthesized as outlined
in Scheme 4.
Scheme 4. Synthesis of compound 18.a
O
N
O
O
F
N
N
N
i
O
N
N
H
O
37
18
aReagents and conditions. (i) (a) toluene, sealed tube, 100 °C, 44 h (b) 12 N HCl.
5-HT2A Receptor Binding.
Competition binding assays were performed in membrane preparations of human
embryonic kidney (HEK293) cells stably expressing human 5-HT2A serotonin receptors.
3
Using [ H]ketanserin, binding saturation curves were generated; we first showed that this
cell line expresses 266 fmol/mg protein of 5-HT receptor (K = 3.5 nM), which is similar
2A
D
to what we have previously reported.15
The affinity of risperidone (1), ketanserin (2), and compounds 7-18 are shown in Table 1.
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1
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1
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2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 1. 5-HT2A receptor affinity (K ) and antagonist potency (IC ) of compounds 7-18
i
50
with risperidone (1) and ketanserin (2) as comparators.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
6
7
8
9
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2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Ligand
pK 
K (nM)
pIC50
S.E.M.
ND*
 IC (nM)
i
i
50
S.E.M.
7
8
<5.0
>10,000
-
-
 5,000
ND*
9
8.73 ± 0.18
6.57 ± 0.20
6.29 ± 0.18
6.55 ± 0.19
5.71 ± 0.21
7.59 ± 0.21
<5.0
1.9
8.27 ± 0.31
5.44
10
11
12
271
118
5.47 ± 0.16 3490
6.03 ± 0.21 927
5.47 ± 0.19 3363
284
13
1,935
ND*
-
1
4
5
25.8
6.91 ± 0.09
ND*
120
-
1
>10,000
237
16
6.63 ± 0.16
8.52 ± 0.11
9.44 ± 0.17
6.65 ± 0.28
7.59 ± 0.19
222
1
7
8
3.0
26.0
1
0.37 9.14 ± 0.32
0.7
21.7
32.5
Risperidone (1) 8.28 ± 0.07
Ketanserin (2) 7.81 ± 0.23
Not determined
5.3
7.77 ± 0.23
7.49 ± 0.24
15.5
*
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Risperidone (1) binds at 5-HT2A receptors with very high affinity (K = 5.3 nM; Table 1),
i
10
which is comparable to what we have previously reported. The affinity of ketanserin (2)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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3
3
3
4
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4
4
4
4
4
4
5
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5
5
5
5
5
5
5
5
6
0
1
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6
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8
9
0
1
2
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9
0
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9
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1
2
3
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
was a few-fold lower (K = 15.5 nM). Representative displacement binding curves for
i
risperidone and compounds 7 and 17 are shown in Figure 5 (See Figure S1 in Supporting
Information for displacement binding curves of the other compounds)
3
Figure 5. [ H]Ketanserin binding displacement curves for risperidone (1) (n = 2;
performed in duplicate) and compounds 7 (n = 2; performed in duplicate) and 17 (n =
3
; performed in duplicate) in membrane preparations of HEK293 cells stably expressing
-HT receptors.
5
2A
It is evident that the “left half” portion of risperidone (i.e., the primary amine 7 and its
piperidine counterpart 8) (Table 1) do not bind at 5-HT2A receptors with appreciable
affinity. In contrast, the truncated risperidone analog 9 (K = 1.9 nM) displayed twice the
i
3
affinity of risperidone at displacing [ H]ketanserin binding.
Compounds 10 and 11 (K = 271 and 118 nM, respectively), the des-F counterparts of 6
i
and 5, displayed lower affinity than their parent compounds (K = 71.4 and 12.3 nM,
i
10
respectively ), whereas compound 12 (K = 283 nM) showed lower affinity than its
i
positional isomer 6. Compound 12, with an aromatic-centroid distance similar to that of
tryptamine was also found to bind with lower affinity than compound 11 (Table 1).
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1
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2
2
2
3
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3
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4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Nevertheless, compound 12 represents a racemic mixture and, hence, the contribution of
the individual optical isomers remains to be investigated.
0
1
2
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6
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8
9
0
1
2
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9
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9
0
Tryptamine analogs 13-16 displayed a broad (>385-fold) range of affinities (K = 25.8 to
i
>
10,000 nM) for 5-HT2A receptors depending upon the nature of the basic amine and
presence or absence of an aromatic fluoro substituent. Thus, the secondary amine
tryptamine 13 (K = 1,935 nM) was of lower affinity than its N-methyl tertiary amine
i
counterpart (14, K = 25.8 nM) and here, too, removal of the fluoro group resulted in
i
substantially reduced affinity (15 and 16, K = >10,000 and 237 nM, respectively). As with
i
the 5/6 and 10/11 pairs, comparing 14 with 13, or 16 with 15, tertiary amines bind with
higher affinity than their secondary amine counterparts. Also, as with the 6/10 and 5/11
pairs, fluoro analogs 13 and 14 bind with higher affinity than their des-fluoro counterparts
15 and 16.
Compound 17 (K = 3.0 nM), where the “right half” portion of risperidone was replaced by
i
the corresponding portion of ketanserin (2) displayed higher affinity than ketanserin itself
(
K = 15.5 nM). Compound 18 (K = 0.37 nM), where the “right half” portion of ketanserin
i
i
was replaced by the corresponding portion of risperidone, displayed >10-fold higher
affinity than risperidone (1, K = 5.3 nM) and ketanserin (2, K = 15.5 nM). Thus, although
i
i
truncated pyridopyrimidine-4-one analogs 7 and 8 showed little affinity for 5-HT2A
receptors (see above), when the fluorobenzoyl portion of ketanserin is present, the
resulting compound, 17, binds with an affinity comparable to that of risperidone (1).
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Functional studies.
Analogs with 5-HT receptor affinity of K <300 nM were examined for functional action.
2A
i
The 5-HT2A receptor is a Gq/11 coupled receptor, and activation of the receptor leads to
release of calcium from endoplasmic reticulum stores (via activation of Inositol 1,4,5-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
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4
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5
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5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
5
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8
9
0
1
2
3
4
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
16
triphosphate (IP ) receptors) resulting in an increase in intracellular calcium levels. The
3
changes in intracellular calcium can be measured using a calcium-sensitive dye such as
Fura-2. Consequently, 5-HT2A receptor agonists evoke an increase in intracellular
calcium, whereas 5-HT2A receptor antagonists block the release of calcium induced by
an agonist.
When tested as potential agonists in HEK293 cells that stably express 5-HT receptors,
2A
none of the compounds (9-12, 14, 16-18) induced intracellular calcium release, which
suggests that they are devoid of agonist action at 5-HT receptors (data not shown).
2A
Assessed was whether compounds 9-12, 14, and 16-18, along with risperidone (1) and
ketanserin (2) as control, behave as 5-HT receptor antagonists. To do so, HEK293 cells
2A
stably expressing 5-HT2A receptors were sequentially exposed to the compound to be
tested and a saturating concentration of 1 µM 5-HT. This concentration of the agonist, 5-
HT, was selected based on pilot results (data not shown) as well as on previously
published studies.10 Our data reveal that all of the compounds behaved as 5-HT2A
receptor antagonists, with varying potencies (Table 1). Representative concentration
response curves for risperidone (1) and analog 17 in the presence of 1 μM 5-HT are
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1
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1
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2
2
2
2
2
2
3
3
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3
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4
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4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
shown in Figure 6 (See Figure S2 in Supporting Information for concentration response
curves of the other compounds in the presence of 1 μM 5-HT).
0
1
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8
9
0
1
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9
0
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 6. Concentration-response curves for risperidone (1) and compound 17 in the
presence of 1 μM serotonin (n = 2; performed in duplicate) in HEK293 cells stably
expressing 5-HT receptors.
2A
Risperidone is a high-affinity 5-HT2A receptor antagonist (IC50 = 21.7 nM), however,
compound 9 (IC = 5.4 nM) – a deconstructed analog of risperidone was four times more
50
potent as an antagonist as compared to risperidone. Hybrid analog 17 (IC = 26.0 nM),
50
where the “right half” portion of risperidone was replaced by the corresponding portion of
ketanserin (2) was almost as potent as ketanserin (2, IC = 32.5 nM) and risperidone
50
(
IC = 21.7 nM), and hybrid analog 18 (IC = 0.7 nM), where the “right half” portion of
50 50
ketanserin was replaced by the corresponding portion of risperidone, displayed >30-fold
higher potency than risperidone (IC50 = 21.7 nM) and ketanserin (IC50 = 32.5 nM).
Compound 16 (IC = 222 nM) is the des-fluoro counterpart of compound 14 (IC = 120
50
50
nM); removal of the fluoro group resulted in a two-fold reduction in potency.
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Homology modeling/docking.
Now that key features for the binding and antagonist action of risperidone had been
identified, homology models of the 5-HT receptor were constructed and docking studies
2
A
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
4
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4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
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4
5
6
7
8
9
0
1
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4
5
6
7
8
9
0
1
2
3
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5
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9
0
1
2
3
4
5
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8
9
0
1
2
3
4
5
6
7
8
9
0
conducted. Risperidone (1) and 10 were docked, and binding poses and interactions with
receptor amino acid residues were analyzed by docking and scoring functions.
Interactions were validated by previous site-directed mutagenesis data17–24 and quantified
by Hydropathic INTeraction (HINT) analysis.25
Because of the high sequence homology (70%) between 5-HT2A and 5-HT2B receptors,
26
the recent crystal structure of the 5-HT receptor was used as template. Risperidone
2B
(1) and the deconstructed analog 10 were energy-minimized prior to docking. Molecular
docking was conducted using the scoring function within the docking program GOLD
Suite 5.2 and the binding pocket was defined as being within a 10-Å radius surrounding
the α-carbon atom of an aspartate residue (D1553.32) in TM3 (superscript indicates the
2
7
Ballesteros-Weinstein numbering system ). Docking solutions were divided into clusters
based on the various binding poses. The clusters were analyzed and the models were
evaluated for each ligand, based on the GOLD fitness scores, and interaction of the
ammonium ion of the ligands with D1553.32. For the models selected (16 models for each
ligand), based on the fitness score and the interactions, the solutions were merged with
the models and energy-minimized using the Tripos Force field function in SYBYL-X2.1 to
optimize the interactions. These models were further re-scored by conducting a HINT
analysis. The final model for each ligand was chosen based on the most optimal (i.e.,
highest positive) HINT score.
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2
2
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4
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4
5
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5
5
5
5
5
5
5
6
The 6-fluoro-3-(4-piperidinyl)-1,2-benz[d]isoxazole (PBI; i.e., the “right half”) portion of
risperidone (Figure 1) was oriented towards TM5, reverse to a previously published
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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9
0
1
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
28
model where this moiety was oriented towards TM7. Although we observed both binding
modes, the GOLD score of the model with the fluoro atom pointing towards TM7 was
lower (51.55) compared to that of the model with the fluoro atom pointing towards TM5
(64.62). The difference in binding modes between our model and the prior literature model
might be explained by the different templates employed in the two studies. The literature
model was generated using the crystal structure of the β adrenoceptor (PBD ID: 2RH1)
2-
having 40% sequence homology with the 5-HT2A receptor, whereas our model was built
using the more recent crystal structure of the 5-HT2B receptor (PDB ID: 4IB4) with
substantially greater (70%) sequence homology. Furthermore, a risperidone/D2
dopamine receptor co-crystal structure has been solved,29 and our model is generally
consistent with the crystal structure given the caveat that it is, after all, a different receptor.
Both suggest an aspartate moiety (D3.32) is involved in an ionic interaction with the
piperidine amine. Threonine and serine residues (T3.37 and S5.46, respectively) are
conserved in both. Whereas S3.36 is present in the 5-HT2A model, the corresponding
amino acid in the crystal structure is a cysteine (C3.36), both of which can participate in
hydrogen bond formation. Other associated amino acids common both to our model and
the crystal structure include: I3.40, W6.48, F6.51, F6.52, and Y7.43. The docking pose for
risperidone in our 5-HT2A receptor model is not unlike the risperidone/D co-crystal
2
structure. As such, it would seem that risperidone binds in the same orientation (i.e.,
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direction) at our 5-HT2A receptor model as in the risperidone/D crystal structure, as
2
28
opposed to the previously suggested 5-HT receptor orientation.
2A
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
3
4
4
4
4
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4
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4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
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9
0
Risperidone was found to dock within a fairly large cavity (Figure 7); this will be described
in more detail below.
Figure 7. Putative binding mode of risperidone (1; rendered as capped sticks, magenta
carbon atoms) in the orthosteric binding site of the h5-HT2A receptor. Hydrogen bond
interaction with D1553.32 (rendered as capped sticks, slate carbon atoms) is indicated by
a yellow-dashed line. The cavity of the binding site is depicted by a slate color.
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Figure 8. Binding modes of risperidone (1; rendered as capped sticks, magenta
carbon atoms (panel A) and PBI (10; rendered as capped sticks, green carbon
atoms) (panel B) docked in a common 5-HT receptor model. The side chain of
2A
amino acids within a radius of 5 Å of 1 are rendered as capped sticks (gray carbon
atoms). Hydrogen bond interactions are indicated by red-dashed lines. A portion of a
shallow hydrophobic pocket is depicted as an atomic (or colored by atom type)
surface. For the purpose of clarity the surface of V156 is not displayed (see Figure
S3 for the full hydrophobic pocket).
Results indicated that both “halves” of intact risperidone form strong interactions with the
receptor, and that risperidone forms ionic bonds with the conserved D155 (TM3) that has
18
been reported to anchor the terminal amine of tryptamines at the receptor. Hydrophobic
interactions predominated at the “left half” of the risperidone molecule consisting of the
6
,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one ring system; however, the 4-position
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carbonyl group showed hydrogen bonding interactions both to Y370 (3.2 Å) and S131
2.7 Å) (Figure 8). Polar interactions seemed to dominate the “right half” of the molecule.
(
The piperidine nitrogen atom was within 2.8 Å of D155, and S159 appeared to form a
bifurcated hydrogen bond with the benz[d]isoxazole nitrogen atom (2.7 Å) and the ring
oxygen atom (2.8 Å) (Figure 8). The fluorinated ring was buried in a shallow hydrophobic
pocket consisting of V156, I206, and G238.
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The lower-affinity 10 appears to bind in a somewhat analogous manner to risperidone
(Figure 8). There are, however, a few differences due, perhaps, to a lack of the extended
“left-hand” portion and 4-carbonyl group of risperidone as well as a lack of the aryl fluoro
group. As a consequence, docking of 10 is slightly shifted (see Figure S3 in Supporting
Information for a more direct comparison). The piperidine nitrogen atom of 10 interacts
with D155; however, this is now a bidentate interaction (distance = 3.2 and 2.6 Å). Rather
than S159 forming a hydrogen bond with the nitrogen and oxygen atoms of the
benz[d]isoxazole ring as seen with risperidone, 10 forms bifurcated bonds with S242
(distance = 3.5 Å to the nitrogen atom, and 2.9 Å to the oxygen atom).
HINT analysis (Table 2) supported the above docking studies. For example, lacking the
extended side chain and aryl fluoro substituent of risperidone, 10 displays a lower
hydrophobic score. Conversely, because 10, but not risperidone, forms two hydrogen
bond interactions with D155, 10 displays a higher D155 HB score. HINT analysis also
supports the concept that S159, but not S242, is involved in hydrogen bond formation
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with the heteroatoms of the benz[d]isoxazole ring of risperidone, whereas the reverse is
true of 10.
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Table 2. GOLD and HINT scores for risperidone and compound 10.
Ligand
GOLD Polar Hydrophobic Total D155 S159 S242
score
HINT HB*
score
HB*
HB*
Risperidone (1) 64.62 1523 1225
PBI (10) 46.76 1862 342
1204
1463 1665
737
372
0
0
212
*HB = Hydrogen bond
The aromatic/fluoro portion of risperidone was found to interact with V156, I206, and
G238 (with a combined positive HINT score of 149). In contrast, the aryl portion of 10 only
interacted with V156 and G238 (combined HINT score of 45) but not with I206.
New pharmacophore for 5-HT2A receptor antagonists.
Compounds 10 and 11 (IC50 = 3490 nM and IC = 927 nM, respectively), the des-F
50
counterparts of 5 and 6, and analog 12 (IC50 = 3363 nM), a positional isomer of 6, all
retained 5-HT2A receptor antagonist action. Hence, although a fluoro group might
contribute, its presence is not required. Removal of the methyl group (analog 10) led to a
decrease in the potency of compound 11. Here, although a tertiary amine seems favored,
it is not a required element for antagonist action. The lowest energy conformer of 5/6 has
a calculated aromatic (i.e., benzenoid ring) centroid-to-basic-amine distance of 6.8 Å
30
whereas that of 5-HT is 6.5 Å. In an attempt to shorten this distance for 6, the position
of the basic amine was moved (i.e. 12). However, although 12 (a racemic mixture)
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displayed affinity for, and antagonist action at, 5-HT2A receptors, it bound with ~4-fold
lower affinity than analog 6. The distance of the nitrogen atom from the aromatic centroid
might be playing a role in enhancing the binding affinity of analog 6, since analog 12 binds
with ~4-fold lower affinity (if the isomers bind with equal affinity) than analog 6.
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Analog 10 – the smallest structural portion of risperidone examined in the current study –
lacks the fluoro and methyl groups of analog 6 but retains affinity for 5-HT receptors as
2A
well as 5-HT receptor antagonist action. Based on the available data, we propose a new
2A
pharmacophore for 5-HT2A receptor antagonists that is comprised of only one aromatic
center at a distance of 6.8 Å from a basic nitrogen atom, a basic protonated amine, and
hydrogen bond acceptors (i.e., the benz[d]isoxazole N and O atoms) (Figure 9). The
benz[d]isoxazole N and O atoms are at a distance of 4.9 and 6.2 Å, respectively, from the
basic amine.
O
N
HN
Figure 9. A new pharmacophore for 5-HT2A receptor antagonists based on a
risperidone scaffold.
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These findings, along with our previously published results, suggest that the “right half”
portion of risperidone (1) (Figure 1) is primarily responsible for its 5-HT receptor binding
2A
and antagonist action. The “left half”-only compounds 7 and 8 lacked appreciable ability
0
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to displace [ H]ketanserin binding in HEK293 cells stably expressing 5-HT receptors.
2A
Nevertheless, risperidone’s “left half” portion contributes to binding, since the partially
abbreviated pyrimidine analog 9 (K = 1.9 nM) displayed substantially higher affinity as
i
compared to compound 6 (K = 71.4 nM). Another unnecessary but contributing feature to
i
binding is the 6-fluoro group. The des-fluoro analogs 10 and 11 were found to bind with
about 4- and 9-fold reduced affinity relative to their corresponding fluoro counterparts 6
and 5. This might be explained by the modeling studies that suggest the fluoro substituent
interacts in a hydrophobic pocket. Incorporation of N-alkyl substituents (e.g. 11) to 10 also
resulted in enhanced affinity, and addition of both (N-alkyl substituents and aromatic
fluoro) resulted in further enhancement of compound’s affinity (i.e., 3-5, 14, 15).
Previous work from our laboratories examined the 5-HT (now 5-HT ) receptor affinity
2
2A
(
although not their antagonist action) of a series of abbreviated ketanserin analogs.31
Differing from the present study, our previous assays employed rat brain (frontal cortex)
homogenates; nevertheless, some interesting trends were noted between our present
and previous findings. Table 3 shows some simple analogs from the ketanserin (2) series
31
(i.e., 39-41) and the risperidone series (i.e., 6, 10, 8). Tertiary amines bind with higher
affinity than their corresponding secondary amines, the presence of a fluoro group
contributes to binding, and between the corresponding pairs of compounds there is a one-
order of magnitude (i.e., 6- to 12-fold) difference between the series. Although the results
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are not strictly comparable, it would seem that the benz[d]isoxazole moiety confers
greater affinity than the benzoyl moiety. Supporting this, our current data showed that
compound 18 was the highest-affinity member of the various analogs examined.
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31
Table 3. Data from a previous SAR study with potential relevance to the current studies.
a
R
X
F
H
F
K (nM)
i
39
H
430
3320
125
O
N
40
H
R
41
CH3
X
6
1
5
H
F
H
F
71
271
12
O
N
N
R
0
H
X
CH3
aData for 39-41 from Herndon et al.31 It might be noted that the
functional activity of these compounds was never investigated. Data
for 6 and 5 from Younkin et al,10 whereas that for 10 is from the
present investigation.
Our data also show that there is a positive correlation between 5-HT2A receptor affinity
(structure-affinity relationships) and antagonist potency (structure-activity relationships)
of the compounds examined (Figure 10). This further supports the concept that the affinity
of competitive G-protein receptor antagonists often dictates their functional potency in
blocking receptor-dependent function.32
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r = 0.950
10
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10
pKi
Figure 10. Relationship between the 5-HT receptor affinity and antagonist potency
2
A
for the ten compounds in Table 1 for which data were obtained.
A compound of particular interest includes the partially truncated risperidone analog 9,
3
which showed higher affinity than risperidone in displacing [ H]ketanserin binding, and
was about four times more potent than risperidone at blocking 5-HT-induced Ca2+
release. All results indicate that the entire “left half” portion of risperidone is not required
for receptor binding or antagonist potency, but that substituents appended to the “right-
hand” portion can play a role in enhancing 5-HT receptor binding affinity and functional
2
A
potency. Consistent with this concept is that the risperidone-ketanserin hybrid 17 was
nearly equipotent with risperidone and ketanserin, and that the ketanserin-risperidone
hybrid 18 was >30-fold more potent than either risperidone or ketanserin. This suggests
that the two “halves” of risperidone and ketanserin can be replaced with each other to
give relatively comparable binding and functional outcomes, and suggests that the
analogs might bind at 5-HT2A receptors in a somewhat comparable fashion. However,
further work will be needed to test whether these two hybrid compounds elicit
antipsychotic-related behavioral responses in rodent models.
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In summary, structure-activity/affinity relationship studies of risperidone at 5-HT2A
receptors suggest that the entire structure of risperidone is not required for 5-HT2A
receptor affinity or antagonist action. The fluoro group, although it contributes to binding,
also is not a required feature. The basic nature of the amine determines affinity with
tertiary amines binding with higher affinity compared to secondary amines. Additionally,
the “right half” of risperidone can be replaced with isosteric tryptamines with retention of
1
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5-HT2A receptor binding affinity and antagonist action (compound 14). The “right half” of
risperidone is sufficient for retention of 5-HT2A receptor affinity and antagonist action;
however, the “left half” bolsters binding affinity (analog 9). The introduction of amine
substituents on the “right half” of risperidone (1) enhances its binding affinity (compound
18). Based on the available data, we propose a new risperidone-based pharmacophore
for 5-HT receptor antagonists (keeping in mind that this does not preclude the possibility
2A
of other pharmacophores) that is comprised of only a single aromatic center, a basic
protonated amine, and hydrogen bond acceptors.
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