A Positive Allosteric Modulator of the Serotonin 5‑HT2C Receptor for
Obesity
Javier García-Caŕceles,○,† Juan M. Decara,○,‡ Henar Vaźquez-Villa,† Ramoń Rodríguez,§

Eva Codesido,§ Jacobo Cruces,§ Jose ́ Brea,∥ María I. Loza,∥ Francisco Aleń,‡ Joaquin Botta,⊥

Peter J. McCormick,# Juan A. Ballesteros,∇ Bellinda Benhamu,́† Fernando Rodríguez de Fonseca,*,‡

and María L. Loṕez-Rodríguez*,†

†Departamento de Química Orgańica I, Universidad Complutense de Madrid, E-28040 Madrid, Spain
‡Unidad de Gestioń Clínica de Salud Mental, Instituto IBIMA, Hospital Regional Universitario, E-29010 Maĺaga, Spain
§Galchimia, E-15823 O Pino, A Coruña, Spain
∥Biofarma Research Group, USEF Screening Platform, CIMUS, USC, E-15782 Santiago de Compostela, Spain
⊥School of Pharmacy, University of East Anglia, NR4 7TJ Norwich, U.K.
#School of Veterinary Medicine, University of Surrey, GU2 7XH Guildford, U.K.
∇Vivia Biotech S.L., Parque Científico de Madrid, E-28760 Madrid, Spain

*S Supporting Information

ABSTRACT: The 5-HT2CR agonist lorcaserin, clinically
approved for the treatment of obesity, causes important side
effects mainly related to subtype selectivity. In the search for 5-
HT2CR allosteric modulators as safer antiobesity drugs, a
chemical library from Vivia Biotech was screened using
ExviTech platform. Structural modifications of identified hit
VA240 in synthesized analogues 6−41 afforded compound 11
(N-[(1-benzyl-1H-indol-3-yl)methyl]pyridin-3-amine, VA012), which exhibited dose-dependent enhancement of serotonin
efficacy, no significant off-target activities, and low binding competition with serotonin or other orthosteric ligands. PAM 11 was
very active in feeding inhibition in rodents, an effect that was not related to the activation of 5-HT2AR. A combination of 11 with
the SSRI sertraline increased the anorectic effect. Subchronic administration of 11 reduced food intake and body weight gain
without causing CNS-related malaise. The behavior of compound 11 identified in this work supports the interest of a serotonin
5-HT2CR PAM as a promising therapeutic approach for obesity.

■ INTRODUCTION

Obesity, defined as having a body mass index (weight [kg]/
height [m2]) equal to or greater than 30 kg/m2, is a major
global health issue affecting nearly one billion adults worldwide
and this number continues to rise.1 Obesity is a major risk
factor for chronic diseases, such as diabetes, cardiovascular
pathology, and certain cancers.2 Hence, it is widely believed
that obesity will become one of the leading causes of morbidity
and mortality for this and future generations. Although a
number of strategies have been used to tackle obesity, most of
the marketed medicines have been withdrawn mainly due to
safety issues,3 including CNS-related adverse effects, and the
pharmaceutical options available to obese patients are currently
limited.4,5 Therefore, there is a major need for new agents that
can provide a safe and effective mechanism for inducing weight
loss.
Among the variety of pharmacological approaches that can

be used to control body weight, the serotonin (5-hydroxytrypt-
amine, 5-HT) system has long been known to be involved in
the modulation of food intake.6−8 Accordingly, classical
antiobesity medicines such as sibutramine (1) and (dex)-

fenfluramine (2) (Figure 1A) act by increasing the level of
serotonin.6,7 The current classification of the serotonin receptor
family, which has essentially remained unchanged since
1994,9−11 includes 14 receptor subtypes classified into seven
major classes (5-HT1−7). The 5-HT2 class comprises three
GPCR members (5-HT2A, 5-HT2B, 5-HT2C) characterized by
close sequence homology and coupling primarily via Gq

proteins to intracellular signaling pathways such as phospho-
lipase C and phospholipase A.12 In particular, serotonin 2C
receptors are predominantly expressed in the CNS, where they
are widely distributed in different brain regions. There is
substantial evidence supporting that their modulation may offer
therapeutic benefit in various pathological conditions, including
schizophrenia, substance addiction, and obesity.13−15 In this
regard, the 5-HT2C receptor (5-HT2CR) is critical for the
anorectic effect of serotoninergic activation,7,13 and the satiating
effects of 2 have thus been demonstrated to be attenuated in 5-

Received: July 7, 2017
Published: November 8, 2017

Article

pubs.acs.org/jmc

© 2017 American Chemical Society 9575 DOI: 10.1021/acs.jmedchem.7b00994
J. Med. Chem. 2017, 60, 9575−9584

pubs.acs.org/jmc
http://dx.doi.org/10.1021/acs.jmedchem.7b00994


HT2CR knockout mice as well as by selective 5-HT2CR
antagonists.16,17

Stimulation of 5-HT2CRs is the most advanced approach
toward serotonin mediated control of food intake and
associated reduction in body weight, and multiple research
efforts have identified several promising 5-HT2CR ligands.18−20

In 2012, the 5-HT2CR agonist lorcaserin (3, Figure 1B) was
approved by the FDA as a first-in-class antiobesity drug.21,22

Therefore, the activation of 5-HT2CRs has been validated in the
search of therapeutic strategies for the treatment of obesity.
However, selectivity is critical because of the detrimental effects
of 5-HT2A and 5-HT2B receptor activation that are respectively
associated with potential hallucinogenic effects23 and cardiac
valvulopathy.12,24 Indeed, increased risk of cardiovascular and
cardiopulmonary diseases has led to the withdrawal of
nonselective serotoninergic drugs 1 and 2 from the EU and
the USA markets, respectively,25,26 and certain restrictions
apply to the use of compound 3 as its moderate 5-HT2BR
activity might induce major cardiac side effects.27 Clearly,
further efficacy and safety data are needed to prove the clinical
utility of 5-HT2CR ligands as antiobesity agents.
In this context, the development of 5-HT2CR agonists with

better selectivity profiles for this worldwide disease would be of
great value. This represents a significant challenge, as the
molecular determinants involved in ligand recognition by 5-
HT2R subtypes are highly conserved.11 More opportunities
may emerge from targeting an allosteric binding site as opposed
to the orthosteric site of the 5-HT2CR protein. Positive
allosteric modulators (PAMs) should elicit a more physiolog-
ically relevant enhancement of target function compared to an
orthosteric agonist.28−31 To date, PNU-69176E (4, Figure 1B)
is the only 5-HT2CR PAM described, which was identified via
screening of a chemical library of Pharmacia (now Pfizer).32,33

A derivative of this modulator (CYD-1-79, undisclosed
structure) has recently been reported to potentiate 5-HT2CR
signaling in vivo in a drug discrimination assay,34 although no
antiobesity properties have been described to our knowledge.
In the present work, we contribute to the quest for selectively

acting 5-HT2CR modulators. In the screening of a chemical
library from Vivia Biotech company using a proprietary
platform, compound 5 (VA240, Figure 2) was identified and
validated as a moderate PAM of the 5-HT2CR. Further
structural modification in synthetic analogues 6−41 (Figure
2) has led to compound 11 (VA012, N-[(1-benzyl-1H-indol-3-
yl)methyl]pyridin-3-amine) with enhanced allosteric efficacy
that was found to be very active in feeding models in rodents,
reducing both food intake and body weight gain without
inducing CNS-related malaise.

■ RESULTS AND DISCUSSION
Hit Identification: High-Throughput Screening. The

proprietary ExviTech platform developed by Vivia Biotech is a
highly sensitive method based on flow cytometry to assess
activation of GPCRs. A functional whole cell assay was used to
access activity by measuring calcium mobilization in response
to receptor activation. For screening, the tested compounds
were added to HEK-293 cells stably expressing 5-HT2CR, and
after 10 min, a 25% of the maximal effective concentration
(EC25) of serotonin was added. The resulting percentage of
cells that respond was then determined. The response for each
well (i.e., compound) was compared to control, receiving only
an EC25 concentration of serotonin, any response above this
level indicating a potential modulator. Specificity was
simultaneously analyzed by screening cell lines expressing
close family members, 5-HT2A and 5-HT2B subtypes, using
multiparametric flow cytometry and cell tracking dyes. A Vivia
Biotech chemical library of approximately 1600 compounds was
screened on ExviTech platform at a concentration of 10 μM
(Figure 2). Three compounds enhanced at ∼20% the effect
produced by serotonin in cells expressing the 5-HT2CR,
whereas no potentiation of serotonin effect was observed for
5-HT2A and 5-HT2B receptors.
The three putative modulators identified in the ExviTech

platform were validated for their ability to stimulate the 5-
HT2CR in a functional assay by determining inositol mono-
phosphate (IP) levels in cells, a well characterized signaling
pathway of receptor activation. Biological analyses were
conducted in HeLa cells stably expressing physiological levels
of the human 5-HT2CR. Cells were treated with a fixed
concentration of the tested compounds (10 μM) and increasing
concentrations of serotonin. The accumulation of IP was
quantified by homogeneous time-resolved fluorescence energy
transfer (HTRF). A 5-HT dose−response curve was obtained
in the presence of each compound, and potentiation of the 5-

Figure 1. (A) Classical serotoninergic antiobesity agents. (B) 5-
HT2CR ligands: agonist 3 clinically used for obesity and positive
allosteric modulator 4.

Figure 2. High-throughput screening of Vivia Biotech chemical library and subsequent functional validation allowed to identify hit compound 5,
which entered a medicinal chemistry program for the search of allosteric modulators of the 5-HT2CR.

Journal of Medicinal Chemistry Article

DOI: 10.1021/acs.jmedchem.7b00994
J. Med. Chem. 2017, 60, 9575−9584

9576

http://dx.doi.org/10.1021/acs.jmedchem.7b00994


HT maximal effect (Emax) was measured. Using this assay,
compound 5 (Figure 2) enhanced the IP release induced by 5-
HT, with a 20% potentiation of 5-HT Emax, which supports the
allosteric activity at the 5-HT2CR. Hence, 5 was selected as a
starting hit for the search of new synthetic modulators of the 5-
HT2CR with enhanced allosteric efficacy.
Optimization of Compound 5. Starting from the

validated hit 5, two series of analogues, 6−14 and 15−41
(Figure 2 and Tables 1 and 2), were generated by modification

of the pyrimidine or the phenyl ring attached to the indole
scaffold, respectively, to examine the effect on functional
activity and allosteric modulation at the 5-HT2CR. In
compounds 6−14 (Table 1), the phenyl ring was maintained
and different nitrogen-containing heterocycles were introduced.
These compounds were synthesized by reductive amination
starting from 1-benzyl-1H-indole-3-carbaldehyde (Scheme 1).
Derivatives 6−9 and 13 were prepared via one-pot reaction of
the aldehyde with the corresponding pyrimidin-, pyrazin-,
pyridazin-, or pyridinamine in 1,2-dichloroethane using sodium
triacetoxyborohydride as reducing agent. For compounds 10−
12 and 14, a two-step procedure was carried out. In this case,
condensation of 1-benzyl-1H-indole-3-carbaldehyde with pyr-
idazin-4-amine or the appropriate pyridinamine, catalyzed by p-
toluenesulfonic acid in refluxing toluene, afforded the
corresponding imine, which was isolated and subsequently
reduced with sodium borohydride in methanol.

Synthesized compounds 6−14 were assessed for their effect
on 5-HT-induced IP release in 5-HT2CR-HeLa cells. From the
data in Table 1, most compounds resulted in being inactive
although in general pyridyl derivatives showed potentiation of

Table 1. Effect of Identified Hit 5 and New Compounds 6-14
in 5-HT-Induced IP Production in 5-HT2CR-HeLa Cells

aPotentiation of 5-HT Emax at a fixed concentration of compound = 10
μM; values are the mean ± SEM of three independent experiments
with duplicate determinations.

Table 2. Effect of Selected Compound 11 and New
Compounds 15−41 in 5-HT-Induced IP Production in 5-
HT2CR-HeLa Cells

aPotentiation of 5-HT Emax at a fixed concentration of compound = 10
μM; values are the mean ± SEM of three independent experiments
with duplicate determinations.

Scheme 1. Synthesis of Target Compounds 6−14a

aReagents and conditions: (a) amino N-heterocycle, NaBH(OAc)3,
1,2-dichloroethane, Δ, 8−15 h, 11−21% (6−9, 13); (b) (i) amino N-
heterocycle, p-TSA, toluene, Δ, 5−18 h, (ii) NaBH4, MeOH, 0 °C to
rt, 1−2 h, 33−73% (10−12, 14).

Journal of Medicinal Chemistry Article

DOI: 10.1021/acs.jmedchem.7b00994
J. Med. Chem. 2017, 60, 9575−9584

9577

http://dx.doi.org/10.1021/acs.jmedchem.7b00994


the endogenous agonist effect. In particular, 3-pyridyl analogue
11 exhibited the highest potentiation (35% at 10 μM).
We next approached the synthesis of the second series of

proposed compounds 15−41, where the 3-pyridyl moiety of
compound 11 was maintained as N-heterocycle and the phenyl
ring was modified. In analogues 15−37 (Table 2) substituents
of different size and electronic effects (F, Cl, Br, Me, OMe, CF3,
CN) were introduced in the benzene ring. These compounds
were obtained by N-alkylation of the indole intermediate 42
with the appropriate benzyl bromide derivative using sodium
hydride as base (Scheme 2). Intermediate 42 was prepared by
reductive amination of 1H-indole-3-carbaldehyde with pyridin-
3-amine, following the two-step procedure described above.
In addition, replacement of the benzene ring with a pyridine

was considered in compounds 38−40, as well as that with a
cyclopropane ring in compound 41 (Table 2), to explore the
requirement of aromaticity at the 1-position of the indole
scaffold. In the case of pyridine-containing derivatives 38−40,
alkylation reaction of 42 with the corresponding
(bromomethyl)pyridines failed, and an alternative N-alkyla-
tion-reductive amination sequence was followed (Scheme 2).
Thus, treatment of 1H-indole-3-carbaldehyde with the
appropriate bromide derivative in the presence of cesium
carbonate yielded intermediates 43−45, which after reductive
amination with pyridin-3-amine afforded final compounds 38−
40. Similarly, cyclopropyl derivative 41 was synthesized by
reductive amination of intermediate 46, obtained by alkylation
of 1H-indole-3-carbaldehyde with (bromomethyl)cyclopropane
using sodium hydride as base (Scheme 2).
The new set of compounds 15−41 was tested in the IP

functional assay, and the data are shown in Table 2. No
substantial improvement of the allosteric potentiation was
observed for any of the aryl derivatives, which exhibited lower
values than unsubstituted analogue 11. The presence of
heterocyclic pyridine or nonaromatic cyclopropane in ana-
logues 38−41 was also less favorable than that of a phenyl ring
(Table 2). Hence, compound 11 was selected for further
pharmacological characterization as an allosteric modulator of
the 5-HT2CR.

In Vitro Characterization of Compound 11. Compound
11 was evaluated in the 5-HT-induced IP assay at different
concentrations in CHO cells stably expressing 5-HT2CR. As
shown in Figure 3A, results revealed an increase of 5-HT Emax,
reaching 35% of potentiation at 10 μM. It should be noted that
a lower concentration of 1 μM enhanced the maximal effect of
5-HT by 28%. In the concentration−response curve of the
compound over 5-HT EC70, a dose-dependent potentiation of
5-HT effect was obtained, with a high potency for compound
11 (EC50 = 16 ± 2 nM). The compound did not induce IP
release in the 5-HT2CR-CHO cells in the absence of 5-HT
(Figure 3B), so it does not present intrinsic agonist activity.
Importantly, this profile for compound 11 was distinguished
from that seen in 5-HT2AR-CHO (Figure 3C,D) and 5-HT2BR-
CHO (Figure 3E,F) cells, in which neither the compound in
the presence of 5-HT nor alone altered IP release.
In addition to the selectivity exhibited over 5-HT2 subtypes,

no significant off-target agonist or antagonist activities were
observed in a CEREP cellular GPCR profile of the compound
(no effect over 20% at 10 μM was observed in either agonist or
antagonist assays), which discarded important safety issues.
In radioligand binding competition assays, 10 μM 11 showed

low displacement of the endogenous agonist (5-HT) or other
orthosteric ligands (mesulergine and clozapine), suggesting its
binding to a different site of the receptor (Figure 3G). The
moderate affinity observed for compound 11 in the presence of
LSD may be due to the location of this orthosteric ligand close
to the extracellular domain of the receptor, where allosteric
modulators of aminergic GPCRs bind.28 This is in agreement
with the recently reported crystal structure of the closely related
5-HT2BR bound to LSD.35

Overall, the in vitro characterization of compound 11 proves
its pharmacological profile as a specific and safe PAM of the 5-
HT2CR, which supports the exploration of its activity in vivo.

In Vivo Behavior of the New 5-HT2CR PAM 11. Prior to
the evaluation of the new 5-HT2CR PAM 11 in animal models,
the in vivo brain permeability was studied (Supporting
Information, Figure S1). Results showed a ratio of concen-
trations of the compound in brain and plasma of 3.8 after 120
min when administered intraperitoneally (ip) at a dose of 10

Scheme 2. Synthesis of Target Compounds 15−41a

aReagents and conditions: (a) (i) pyridin-3-amine, p-TSA, toluene, Δ, 1−6 h, (ii) NaBH4, MeOH, 0 °C to rt, 15−60 min, 26−91%; (b) benzyl
bromide derivative, NaH, DMF, rt, 15 min, 30−87%; (c) 2-, 3-, or 4-(bromomethyl)pyridine hydrobromide, Cs2CO3, DMF, rt, 2 h, 86−94%; (d)
(bromomethyl)cyclopropane, NaH, DMF, rt, 1 h, 97%.

Journal of Medicinal Chemistry Article

DOI: 10.1021/acs.jmedchem.7b00994
J. Med. Chem. 2017, 60, 9575−9584

9578

http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.7b00994/suppl_file/jm7b00994_si_001.pdf
http://dx.doi.org/10.1021/acs.jmedchem.7b00994


mg/kg. Importantly, this ratio was constant up to 6 h. These
data prompted us to assess the behavior of 11 in animal models
of obesity.
In a first experiment, food intake was monitored upon acute

administration of studied compound or the 5-HT2CR agonist
47 (WAY 161503)36 assayed as a reference compound of 5-
HT2CR activation (Figures 4A,B). Both compounds induced a
reduction in feeding when they were acutely administered at 2
mg/kg (ip). Agonist 47 produced a reduction in food intake
that was more intense at the earlier times and almost vanished
after 4 h (Figure 4A, dose effect F (2.80) = 51.9, P < 0.01).
Notably, the newly identified allosteric modulator 11 reduced
feeding with a higher efficacy (Figure 4B, dose effect F (3.82) =
256, P < 0.001) and with a more prolonged action (time × dose
interaction F (3.82) = 5.3, P < 0.01), reaching almost 50%
reduction after 4 h at the dose of 10 mg/kg. The effects of 11
were not related to the activation of serotonin 5-HT2AR, as

revealed by the persistence of food intake inhibition despite
pretreatment with the 5-HT2AR antagonist ketanserin (Sup-
porting Information, Figure S2A). In addition, the effect of 11
was not eliminated after pretreatment with the 5-HT2CR
antagonist 48 (SB-242084, Supporting Information, Figure
S2B),37 whereas the antagonist did reduce the inhibition
produced by the 5-HT2CR agonist 47 (Supporting Information,
Figure S2C). These results suggest no direct action of PAM 11
at the orthosteric site of the receptor.
When compound 11 was administered in a restricted food

access model, it reduced both food intake (Figure 4C, F (6.98)
= 7.9, P < 0.001) and body weight gain (Figure 4E, F (6.98) =
10.7, P < 0.001). Similar results were observed after 7 days of
repeated administration of the compound in a continuous food
access model, where both daily food intake (Figure 4D, F
(6.98) = 6.2, P < 0.001) and body weight gain (Figure 4F, F
(6.98) = 3.9, P < 0.01) decreased.

Figure 3. In vitro characterization of compound 11. (A) 5-HT curves in the presence of increasing concentrations of the compound. (B) Effect of
the compound in the absence of serotonin. (C) Allosteric effect of the compound at the 5-HT2AR. (D) Agonist effect of the compound at the 5-
HT2AR. (E) Allosteric effect of the compound at the 5-HT2BR. (F) Agonist effect of the compound at the 5-HT2BR. (G) Binding displacements of
orthosteric ligands of the 5-HT2CR by 10 μM 11. Points represent the mean ± SD (vertical bars) of triplicate measurements.

Journal of Medicinal Chemistry Article

DOI: 10.1021/acs.jmedchem.7b00994
J. Med. Chem. 2017, 60, 9575−9584

9579

http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.7b00994/suppl_file/jm7b00994_si_001.pdf
http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.7b00994/suppl_file/jm7b00994_si_001.pdf
http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.7b00994/suppl_file/jm7b00994_si_001.pdf
http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.7b00994/suppl_file/jm7b00994_si_001.pdf
http://dx.doi.org/10.1021/acs.jmedchem.7b00994


To establish whether the new PAM induced side effects, we
explored the appearance of hypolocomotion, anxiety, and
conditioned taste aversion. The compound induced hypoloco-
motion in the elevated plus maze (Figure 4G, F (2.21) = 12, P
< 0.01). Anxiety cannot be discarded (Supporting Information,
Figure S3A) although the hypolocomotion made very difficult
the evaluation of this response. The hypolocomotion was also
observed in the open field test (Supporting Information, Figure
S3B). The hypolocomotor profile of PAM 11 appears as a side
effect that might limit its therapeutic utility at certain doses, as
it has also been described for 5-HT2CR agonists such as
lorcaserin in animal models of obesity or nicotine addiction.38

On the other hand, administration of compound 11 (2 mg/kg,
ip) did not result in conditioned taste aversion (Figure 4H,
treatment per saccharin interaction, F (2.58) = 26.5, P < 0.01),

indicating that the compound did not cause malaise, in contrast
to LiCl used as positive control.
As a way of demonstrating allosteric potentiation of

serotonin-induced feeding suppression by compound 11, we
tested the effects of coadministration with the selective
serotonin reuptake inhibitor (SSRI) sertraline. This anti-
depressant drug is known to induce a mild feeding suppression,
as shown in Figure 5A (F (3.112) = 29.1, P < 0.01) when
administered in a wide range of doses (0.4−10 mg/kg). In this
experiment, two different combinations of both compounds
were used: 0.5 mg/kg 11 with 2 mg/kg sertraline (Figure 5B)
and 2 mg/kg 11 with 5 mg/kg sertraline (Figure 5C). When
the extracellular concentration of serotonin increased upon
administration of sertraline, the anorectic effect of 11 was
potentiated, as expected for a positive modulator of the
receptor activity (interaction 11−sertraline F (9.111) = 4.6 P <

Figure 4. In vivo behavior of the new 5-HT2CR PAM 11. (A) The 5-HT2CR agonist 47 reduced feeding in food deprived animals. (B) In a similar
model, compound 11 has a more potent and prolonged action. (C) When administered in animals with restricted food access (4 h/day), 11 reduced
daily feeding. (D) This effect was also observed when the compound was given to animals with continuous access to food. (E,F) In both models,
food restriction and continuous access, repeated administration of 11 reduced net body weight gain. (G) Compound 11 induced hypolocomotion in
the elevated plus maze. (H) Compound 11 did not result in conditioned taste aversion. * denotes statistical significance (P < 0.01) when compared
to control. N = at least 8 animals per group.

Journal of Medicinal Chemistry Article

DOI: 10.1021/acs.jmedchem.7b00994
J. Med. Chem. 2017, 60, 9575−9584

9580

http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.7b00994/suppl_file/jm7b00994_si_001.pdf
http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.7b00994/suppl_file/jm7b00994_si_001.pdf
http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.7b00994/suppl_file/jm7b00994_si_001.pdf
http://dx.doi.org/10.1021/acs.jmedchem.7b00994


0.001). This is a very remarkable action because the prevalence
of depression associated with obesity is very high, i.e., 19% in
severe obesity.39 This combination might offer a new strategy
for feeding reduction in depressed obese people.

■ CONCLUSIONS
In this work, we have developed compound 11 (N-[(1-benzyl-
1H-indol-3-yl)methyl]pyridin-3-amine) as an allosteric modu-
lator of the serotonin 5-HT2CR that exhibits enhanced efficacy
dose-dependently, no significant off-target activities, and low
competition with the endogenous agonist or other orthosteric
ligands. The compound was found to be very active in feeding
models in rodents, reducing both food intake and body weight
gain without causing CNS-related malaise when administered
subchronically. The effects were not related to 5-HT2AR
activation, and a potentiation of the anorectic action of 11 was
observed when administered in combination with sertraline,

which suggests an allosteric modulation of serotonin effect in
vivo. These results support the interest of a 5-HT2CR PAM as a
promising therapeutic approach for obesity.

■ EXPERIMENTAL SECTION
Synthesis. Unless otherwise stated, the starting materials, reagents,

and solvents were purchased as high-grade commercial products from
Sigma-Aldrich, Acros, ABCR, Fluorochem, Scharlab, or Panreac.
Analytical thin-layer chromatography (TLC) was run on Merck silica
gel plates (Kieselgel 60 F-254), with detection by UV light (λ = 254
nm), 5% ninhydrin solution in ethanol, or 10% phosphomolybdic acid
solution in ethanol. Unless otherwise stated, products were purified by
flash chromatography using a Varian 971-FP system with cartridges of
silica gel (Varian, particle size 50 μm). All compounds were obtained
as oils, except for those whose melting points (mp) are indicated,
which were solids. Melting points (uncorrected) were determined on a
Stuart Scientific electrothermal apparatus. Infrared (IR) spectra were
measured on a Bruker Tensor 27 instrument equipped with a Specac
ATR accessory of 5200−650 cm−1 transmission range; frequencies (ν)
are expressed in cm−1. 1H, 13C, and 19F NMR spectra were recorded
on a Bruker Avance 500 MHz (1H, 500 MHz; 13C, 125 MHz) or
Bruker DPX 300 MHz (1H, 300 MHz; 13C, 75 MHz; 19F, 300 MHz)
instrument at room temperature (rt) at the Universidad Complutense
de Madrid (UCM) NMR core facility. Bruker DPX 300 MHz
equipment was used unless otherwise stated. Chemical shifts (δ) are
expressed in parts per million relative to the residual solvent peak for
1H and 13C nucleus (CDCl3: δH = 7.26, δC = 77.16; DMSO-d6: δH =
2.50, δC = 39.52) and to internal (trifluoromethyl)benzene for 19F
nucleus; coupling constants (J) are in hertz (Hz). The following
abbreviations are used to describe peak patterns when appropriate: s
(singlet), d (doublet), t (triplet), q (quartet), qt (quintet), m
(multiplet), app (apparent), and br (broad). 2D NMR experiments,
homonuclear correlation spectroscopy (H,H−COSY), heteronuclear
multiple quantum correlation (HMQC), and heteronuclear multiple
bond correlation (HMBC) of representative compounds were
acquired to assign protons and carbons of new structures. For all
final compounds, purity was determined by high-performance liquid
chromatography (HPLC) coupled to mass spectrometry (MS) using
an Agilent 1200LC-MSD VL instrument, and satisfactory chromato-
grams confirmed a purity of at least 95% for all tested compounds. LC
separation was achieved with a SunFire C18 column (3.5 μm, 2.1 mm
× 100 mm), together with a guard column (5 μm, 4.6 mm × 12.5
mm). The mobile phase consisted of A (1:1 ACN/methanol) and B (5
mM NH4OH pH 7). MS analysis was performed with an ESI source.
Spectra were acquired in positive or negative ionization mode from 80
to 800 m/z and in UV mode in the wavelength range 210−400 nm.

General Procedure for One-Pot Reductive Amination. To a
suspension of 1-benzyl-1H-indole-3-carbaldehyde (1.0 equiv) and
NaBH(OAc)3 (3.0 equiv) in anhydrous 1,2-dichloroethane (10 mL/
mmol), the corresponding amino N-heterocycle (2.5 equiv) was added
under an argon atmosphere, and the reaction was refluxed for 8−15 h.
After this time, the mixture was allowed to reach rt, and it was diluted
with DCM and washed with water. The organic layer was dried over
Na2SO4, filtered and concentrated. The crude was purified by flash
chromatography (some of them were slurred with hexane afterward)
to afford the final compounds 6−9 and 13.

N-[(1-Benzyl-1H-indol-3-yl)methyl]pyridazin-3-amine (9). Follow-
ing the general procedure for one-pot reductive amination, compound
9 was obtained from 1-benzyl-1H-indole-3-carbaldehyde (390 mg,
1.66 mmol) and pyridazin-3-amine (316 mg, 3.32 mmol) as an off-
white solid (63 mg, 12%). Chromatography, DCM/methanol 96:4;
mp, 152−153 °C; Rf, 0.27 (DCM/methanol 95:5). IR (ATR): ν 3248
(NH), 1598, 1509, 1467 (Ar). 1H NMR (CDCl3): δ 4.69 (d, J = 4.4,
2H, CH2NH), 5.19 (br s, 1H, NH), 5.30 (s, 2H, NCH2), 6.35 (d, J =
5.8, 1H, H4), 7.09−7.16 (m, 4H, H2′, H5′, H2″, H6″), 7.19−7.25 (m,
1H, H6′), 7.29−7.35 (m, 4H, H7′, 3CHPh), 7.63 (d, J = 7.7, 1H, H4′),
8.16 (d, J = 4.9, 1H, H5), 8.60 (app s, 1H, H6).

13C NMR (CDCl3): δ
37.2 (CH2NH), 50.2 (NCH2), 110.2 (C7′), 111.5 (C3′), 119.1 (C4′),
119.9 (C5′), 122.5 (C6′), 127.0 (C2″, C6″), 127.1 (C2′), 127.2 (C3a),

Figure 5. In vivo allosteric modulation of serotonin-induced feeding
suppression by compound 11. (A) Selective serotonin reuptake
inhibitor sertraline induced a mild feeding suppression in rats. (B,C)
When the extracellular concentration of serotonin increased upon
administration of sertraline, the anorectic effect of 11 was potentiated.
* denotes statistical significance (P < 0.01) when compared to control.
N = at least 8 animals per group.

Journal of Medicinal Chemistry Article

DOI: 10.1021/acs.jmedchem.7b00994
J. Med. Chem. 2017, 60, 9575−9584

9581

http://dx.doi.org/10.1021/acs.jmedchem.7b00994


127.9 (C4″), 129.0 (C3″, C5″), 137.0 (C7a), 137.3 (C1″), 155.3 (C5),
158.8 (C6), 161.8 (C3), C4 not observed. HPLC (tR, min): 17.70. MS
(ESI, m/z): calcd for C20H19N4 ([M + H]+) 315.2, found 315.2.
General Procedure for Two-Step Reductive Amination. p-

Toluenesulfonic acid (0.1 equiv) was added to a solution of 1H-indole-
3-carbaldehyde, 1-benzyl-1H-indole-3-carbaldehyde, or intermediates
43−46 (1.0 equiv) and the corresponding amino N-heterocycle (1.2
equiv) in anhydrous toluene (8 mL/mmol) under an argon
atmosphere. The reaction mixture was refluxed for 1−18 h using a
Dean−Stark equipment to remove water from the reaction medium.
Solvent was concentrated off to afford the corresponding imine, which
was used in the next step without further purification.
NaBH4 (2.5 equiv) was added portionwise to a 0 °C cooled solution

of the previous imine in methanol (8 mL/mmol). The reaction was
allowed to warm up to rt and stirred for 0.25−2 h. Then, the mixture
was poured into brine, extracted with DCM (×2), and washed with
water. The organic layer was dried over Na2SO4, filtered, and
concentrated. The crude was purified by flash chromatography (some
of them were slurred with hexane afterward) to afford intermediate 42
or final compounds 10−12, 14, and 38−41.
N-[(1-Benzyl-1H-indol-3-yl)methyl]pyridin-3-amine (11). Follow-

ing the general procedure for two-step reductive amination, compound
11 was obtained from 1-benzyl-1H-indole-3-carbaldehyde (500 mg,
2.12 mmol) and pyridin-3-amine (239 mg, 2.54 mmol) as a white solid
(488 mg, 73%). Chromatography, hexane/EtOAc from 1:1 to 3:7; mp,
159−160 °C; Rf, 0.31 (hexane/EtOAc 2:8). IR (ATR): ν 3398 (NH),
1585, 1471, 1332 (Ar). 1H NMR (CDCl3): δ 3.96 (br s, 1H, NH),
4.48 (d, J = 4.1, 2H, CH2NH), 5.30 (s, 2H, NCH2), 6.96 (ddd, J = 8.2,
2.7, 1.2, 1H, H4), 7.08−7.33 (m, 10H, H5, 4CHindole, 5CHPh), 7.67 (d,
J = 7.7, 1H, H4′), 7.99 (d, J = 4.5, 1H, H6), 8.10 (d, J = 2.5, 1H, H2).
13C NMR (CDCl3): δ 39.8 (CH2NH), 50.2 (NCH2), 110.1 (C7′),
112.3 (C3′), 118.7 (C4), 119.2 (C4′), 119.8 (C5′), 122.4 (C6′), 123.8
(C5), 127.0 (C2″,C6″), 127.1 (C2′), 127.4 (C3a), 127.9 (C4″), 128.9
(C3″, C5″), 136.3 (C2), 137.0 (C7a), 137.4 (C1″), 138.9 (C6), 144.4
(C3). HPLC (tR, min): 19.67. MS (ESI, m/z): calcd for C21H20N3 ([M
+ H]+) 314.2, found 314.4.
General Procedure for N-Alkylation Reaction. To a solution of

1H-indole-3-carbaldehyde or 42 (1.0 equiv) in anhydrous DMF (15
mL/mmol) under an argon atmosphere, sodium hydride (60%
suspension in mineral oil, 1.2 equiv), or Cs2CO3 (3.0 equiv) was
added. The mixture was stirred at rt for 15 min, and the corresponding
bromoderivative (1.2 equiv) was added. The reaction was stirred at rt
for 0.25−2 h, and then it was poured into brine and extracted with
Et2O or DCM (×2). The combined organic layers were washed with
water, dried over Na2SO4, filtered, and concentrated. DMF was
removed under high vacuum, and the residue was used in the next step
without further purification in the case of intermediates 43−46 or
purified by flash chromatography (some of them were slurred with
hexane afterward) to afford final compounds 15−37.
N-{[1-(3-Chlorobenzyl)-1H-indol-3-yl]methyl}pyridin-3-amine

(19). Following the general procedure for N-alkylation reaction,
compound 19 was obtained from 42 (260 mg, 1.16 mmol) and 1-
(bromomethyl)-3-chlorobenzene (288 mg, 1.40 mmol), using sodium
hydride, as a white solid (247 mg, 61%). Chromatography, hexane/
EtOAc from 7:3 to 3:7; mp, 136−137 °C; Rf, 0.26 (hexane/EtOAc
2:8). IR (ATR): ν 3398 (NH), 1588, 1481, 1466 (Ar). 1H NMR
(CDCl3): δ 3.99 (br s, 1H, NH), 4.50 (d, J = 2.7, 2H, CH2NH), 5.26
(s, 2H, NCH2), 6.94−6.98 (m, 2H, H4, H6″), 7.08−7.29 (m, 8H, H5,
4CHindole, 3CHPh), 7.67 (d, J = 7.7, 1H, H4′), 7.99 (dd, J = 4.6, 0.8, 1H,
H6), 8.11 (d, J = 2.8, 1H, H2).

13C NMR (CDCl3): δ 39.8 (CH2NH),
49.6 (NCH2), 110.0 (C7′), 112.8 (C3′), 118.7 (C4), 119.3 (C4′), 120.0
(C5′), 122.6 (C6′), 123.9 (C5), 125.0 (C6″), 126.9, 127.0 (C2′, C2″),
127.4 (C3a), 128.1 (C5″), 130.3 (C4″), 134.9 (C3″), 136.2 (C2), 136.9
(C7a), 139.0 (C6), 139.5 (C1″), 144.4 (C3). HPLC (tR, min): 19.77.
MS (ESI, m/z): calcd for C21H19ClN3, 348.1 ([M(35Cl) + H]+), 350.1
([M(37Cl) + H]+): found 348.2, 350.2.
Functional Activity at 5-HT2 Receptors. IP formation was

measured by using the HTRF IP-One assay kit (CisBio, France).
Assays were carried out in human 5-HT2A, 5-HT2B, and 5-HT2C
receptors expressed in CHO-K1 (5-HT2A and 5-HT2B) and HeLa-K1

(5-HT2C) cell lines. Cells were seeded (20000 cells/well for 5-HT2A
and 5-HT2C and 30000 cells/well for 5-HT2B) in a 96-well plate in 100
μL of culture medium (DMEM, 10% FBS, 4 mM L-glutamine) and
maintained at 37 °C in a 5% CO2 atmosphere for 24 h. After this time,
medium was replaced by 50 μL of kit stimulation buffer and
compounds (test compound and/or 5-HT) were added to the wells.
Cells were incubated for 20 min at 37 °C, and then the IP
accumulation was measured by using the kit reagents. HTRF was
quantified in an ultraevolution multilabel reader (Tecan M1000
Genius Pro).

Data were normalized to 5-HT maximum effect, and nonlinear
regression fitting was performed by using Prism v2.1 (GraphPad
software).

Radioligand Binding Assays at 5-HT2CR. Competition radio-
ligand binding assays were carried out by using four different
radioligands: [3H]mesulergine, [3H]serotonin, [3H]LSD, and [3H]-
clozapine. 5-HT2C−HeLa membrane suspensions (15 μg/well for
[3H]mesulergine and [3H]LSD, 60 μg/well for [3H]serotonin and 20
μg/well for [3H]clozapine) were incubated with the test compound
(10 μM) and radioligand (4 nM [3H]mesulergine, 2 nM [3H]-
serotonin, 2 nM [3H]LSD, and 15 nM [3H]clozapine) in assay buffer
(50 mM Tris-HCl, pH = 7.4). The incubation conditions were 60 min
at 27 °C for [3H]mesulergine, [3H]LSD, and [3H]clozapine and 180
min at 4 °C for [3H]serotonin. Nonspecific binding was determined in
the presence of 10 μM mianserin. After the incubation, time samples
were filtered through Multiscreen FC 96-well plates (Millipore) and
washed four times with wash buffer (50 mM Tris-HCl, pH = 6.6).
Radioactivity was detected in a Microbeta Trilux reader (PerkinElm-
er).

In Vivo Experiments. For acute experiments, male Wistar rats
(Charles Rivers Laboratories, Barcelona, Spain) weighing 200−250 g
and aged 10−12 weeks at the beginning of the experiments were used.
For subchronic experiments, male Wistar animals were made obese
after 8 weeks of treatment with a high fat diet (HFD, 60% fat diet-
D12492, Brogaarden, Gentofke, Denmark) containing 5.24 kcal/g
(20% of the metabolizable energy content was protein, 20%
carbohydrates and 60% fat). When tested for subchronic effects of
compound 11, animals had an average weight of 548 ± 20 g (see
Crespillo et al. for further details).40 Animals were housed in groups of
two in a humidity- and temperature-controlled (22 °C) vivarium on a
12:12 h light−dark cycle (lights off at 8 PM). Water and standard
laboratory chow were available ad libitum throughout the course of the
studies unless otherwise indicated (e.g., in the diet-induced obesity
procedure). All procedures were conducted in adherence to the
European Communities Council Directive (86/609/ECC) and
Spanish regulations (BOE 252/34367-91, 2005) for the use of
laboratory animals. They were reviewed and approved by the
Comisioń de Ética e Investigacioń, Hospital Carlos Haya, Maĺaga
(Spain). Procedures can be found in Gomez et al.41 All data are shown
as mean ± SEM of at least eight determinations per experimental
group. Kolmogorov−Smirnov normality tests indicated that all data
followed a Gaussian distribution (P > 0.1), so we selected a parametric
statistical test. Differences between the treatments and time of testing
(along 7-day testing) were analyzed by two-way ANOVA for repeated
measures, followed by Bonferroni post hoc test for multiple
comparisons. The level of statistical significance was set at P < 0.05.
Details on the methodology can be found on Crespillo et al.40

Acute Feeding Suppression. Adult rats were food deprived for 16 h
prior to refeeding to habituate the animals to the procedure. On the
testing day, and after food deprivation, compound 11 was ip injected
at the doses of 0.5, 2, and 10 mg/kg dissolved in sterile saline. Food
and water intake were then measured at 30, 60, 120, and 240 min after
the presentation of food. The 5-HT2CR agonist 47 (doses of 0.2 and 2
mg/kg, ip) was also tested for comparison of the acute suppression
effect. In additional experiments, animals were pretreated with the
inhibitor of the serotonin reuptake system sertraline (doses of 0.4, 2, 5,
and 10 mg/kg), the 5-HT2AR antagonist ketanserin (2 mg/kg), or the
5-HT2CR antagonist 48 (3 mg/kg), all purchased from Tocris
Cookson (Bristol, UK), that were ip injected 30 min prior to 11,
and the suppression effect was also analyzed.

Journal of Medicinal Chemistry Article

DOI: 10.1021/acs.jmedchem.7b00994
J. Med. Chem. 2017, 60, 9575−9584

9582

http://dx.doi.org/10.1021/acs.jmedchem.7b00994


Repeated Administration under Continuous Food Access. To
assess the subchronic (7-day treatment) effect of 11, free-feeding rats
were injected daily with the compound (2 mg/kg, ip) or vehicle. The
amount of standard laboratory chow (3.02 kcal/g with 30 kcal%
proteins, 55 kcal% carbohydrates, and 15 kcal% fat) eaten (kcal/kg
body weight) and the body weight gain (g) were registered daily.
Repeated Administration under Food Restriction Schedule.

Animals were presented with food for 4 h a day. Once the 4 h period
was over, food was retired until the next day. When a stable baseline of
feeding was achieved, 11 (2 mg/kg, ip) or vehicle was injected 30 min
prior to food presentation, and food and water intakes were measured
at the end of the 4 h period. The procedure was repeated for 7
consecutive days, and total food intake and body weight were
monitored.
Elevated Plus Maze. Animals naiv̈e to the maze were manipulated

for 7 days before testing. The day of the experiment, 30 min after the
ip injection of 11, animals were placed in the center of the maze, facing
an open arm. The number of entries and the % of time spent in the
exposed arms of the maze were measured. Test length was 5 min, and
animals were not retested (for details see Rodriguez de Fonseca et al.
2001).42

Open Field. Animals naiv̈e to the open field were manipulated for 7
days before testing. The day of the experiment, 30 min after the
injection of 11 (2 mg/kg, ip), animals were placed in the center of the
maze and the distance walked registered through an automated
procedure.42 Test length was 10 min, and animals were not retested.
Conditioned Taste Aversion. Rats were deprived of water for 24 h

and then accustomed to drinking from a graded bottle during a 30 min
test period for 4 days. On day 5, water was replaced with a 0.1%
saccharin solution, and 30 min later the animals received ip injections
of vehicle, 11 (2 mg/kg), or lithium chloride (0.4 M, 7.5 mL/kg).
During the following 2 days, water consumption was recorded over 30
min test periods. The animals were then presented with water or
saccharin, and drinking was finally measured.42

■ ASSOCIATED CONTENT
*S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.7b00994.

Synthesis and characterization data of compounds 6−8,
10, 12−18, and 20−42; HPLC-MS purity analyses of
final compounds 6−41; molecular formula strings for
compounds 1−48 (PDF)

■ AUTHOR INFORMATION
Corresponding Authors
*For M.L.L.-R.: phone, 34-91-3944239; fax 34-91-3944103; e-
mail, mluzlr@ucm.es.
*For F.R.F.: phone, 34-952-614-012; e-mail, fernando.
rodriguez@ibima.eu.
ORCID
Javier García-Caŕceles: 0000-0003-4614-9639
Henar Vaźquez-Villa: 0000-0001-7911-3160
Bellinda Benhamu:́ 0000-0002-0864-026X
María L. Loṕez-Rodríguez: 0000-0001-8607-1085
Author Contributions
○J.G.-C. and J.M.D. share first authorship of this work.
Notes
The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS
This work was supported by grants from MINECO (SAF2013-
48271-C2, SAF2014-57138-C2-2-R, SAF2016-78792-R, and

INNPACTO 01/12-CL-0-12-09), CAM (S2010/BMD2353),
Xunta de Galicia (GRC2014/011), Junta de Andaluciá (CTS-
8221 and CTS-433), and European Regional Development
Fund (ERDF). J.G.-C. is grateful to MINECO for a predoctoral
fellowship. We thank the NMR Core Facilities from
Universidad Complutense de Madrid.

■ ABBREVIATIONS USED

5-HT2AR, 5-HT2B receptor; 5-HT2BR, 5-HT2B receptor; 5-
HT2CR, 5-HT2C receptor; EC25, 25% of the maximal effective
concentration; Emax, maximal effect; HTRF, homogeneous
time-resolved fluorescence energy transfer; IP, inositol mono-
phosphate

■ REFERENCES
(1) Overweight and obesity. Global Health Observatory (GHO) Data;
World Health Organization: Geneva, 2017; http://www.who.int/gho/
ncd/risk_factors/overweight_text/en/ (Sep 25, 2017).
(2) Marseglia, L.; Manti, S.; D’Angelo, G.; Nicotera, A.; Parisi, E.; Di
Rosa, G.; Gitto, E.; Arrigo, T. Oxidative Stress in Obesity: A critical
component in human diseases. Int. J. Mol. Sci. 2015, 16, 378−400.
(3) Colman, E. Food and Drug Administration’s obesity drug
guidance document: a short history. Circulation 2012, 125, 2156−
2164.
(4) Rodríguez, J. E.; Campbell, K. M. Past, present, and future of
pharmacologic therapy in obesity. Prim. Care Clin. Office Pract. 2016,
43, 61−67.
(5) Narayanaswami, V.; Dwoskin, L. P. Obesity: Current and
potential pharmacotherapeutics and targets. Pharmacol. Ther. 2017,
170, 116−147.
(6) Halford, J. C. G.; Harrold, J. A.; Lawton, C. L.; Blundell, J. E.
Serotonin (5-HT) drugs: effects on appetite expression and use for the
treatment of obesity. Curr. Drug Targets 2005, 6, 201−213.
(7) Sargent, B. J.; Henderson, A. J. Targeting 5-HT receptors for the
treatment of obesity. Curr. Opin. Pharmacol. 2011, 11, 52−58.
(8) Benhamu, B.; Martin-Fontecha, M.; Vazquez-Villa, H.; Pardo, L.;
Lopez-Rodriguez, M. L. Serotonin 5-HT6 receptor antagonists for the
treatment of cognitive deficiency in Alzheimer’s disease. J. Med. Chem.
2014, 57, 7160−7181.
(9) Hoyer, D.; Clarke, D. E.; Fozard, J. R.; Hartig, P. R.; Martin, G.
R.; Mylecharane, E. J.; Saxena, P. R.; Humphrey, P. P. A., VII.
International Union of Pharmacology classification of receptors for 5-
hydroxytriptamine (serotonin). Pharmacol. Rev. 1994, 46, 157−203.
(10) Göthert, M. Serotonin discovery and stepwise disclosure of 5-
HT receptor complexity over four decades. Part I. General background
and discovery of serotonin as a basis for 5-HT receptor identification.
Pharmacol. Rep. 2013, 65, 771−786.
(11) McCorvy, J. D.; Roth, B. L. Structure and function of serotonin
G protein-coupled receptors. Pharmacol. Ther. 2015, 150, 129−142.
(12) Hoyer, D.; Hannon, J. P.; Martin, G. R. Molecular,
pharmacological and functional diversity of 5-HT receptors.
Pharmacol. Biochem. Behav. 2002, 71, 533−554.
(13) Jensen, N. H.; Cremers, T. I.; Sotty, F. Therapeutic potential of
5-HT2C receptor ligands. Sci. World J. 2010, 10, 1870−1885.
(14) Higgins, G. A.; Sellers, E. M.; Fletcher, P. J. From obesity to
substance abuse: therapeutic opportunities for 5-HT2C receptor
agonists. Trends Pharmacol. Sci. 2013, 34, 560−570.
(15) Di Giovanni, G.; De Deurwaerdere, P. New therapeutic
opportunities for 5-HT2C receptor ligands in neuropsychiatric
disorders. Pharmacol. Ther. 2016, 157, 125−162.
(16) Vickers, S. P.; Clifton, P. G.; Dourish, C. T.; Tecott, L. H.
Reduced satiating effect of d-fenfluramine in serotonin 5-HT2C
receptor mutant mice. Psychopharmacology 1999, 143, 309−314.
(17) Vickers, S. P.; Dourish, C. T.; Kennett, G. A. Evidence that
hypophagia induced by d-fenfluramine and d-norfenfluramine in the
rat is mediated by 5-HT2C receptors. Neuropharmacology 2001, 41,
200−209.

Journal of Medicinal Chemistry Article

DOI: 10.1021/acs.jmedchem.7b00994
J. Med. Chem. 2017, 60, 9575−9584

9583

http://pubs.acs.org
http://pubs.acs.org/doi/abs/10.1021/acs.jmedchem.7b00994
http://pubs.acs.org/doi/abs/10.1021/acs.jmedchem.7b00994
http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.7b00994/suppl_file/jm7b00994_si_001.pdf
mailto:mluzlr@ucm.es
mailto:fernando.rodriguez@ibima.eu
mailto:fernando.rodriguez@ibima.eu
http://orcid.org/0000-0003-4614-9639
http://orcid.org/0000-0001-7911-3160
http://orcid.org/0000-0002-0864-026X
http://orcid.org/0000-0001-8607-1085
http://www.who.int/gho/ncd/risk_factors/overweight_text/en/
http://www.who.int/gho/ncd/risk_factors/overweight_text/en/
http://dx.doi.org/10.1021/acs.jmedchem.7b00994


(18) Monck, N. J. T.; Kennett, G. A. 5-HT2C Ligands: recent
progress. Prog. Med. Chem. 2008, 46, 281−390.
(19) Garfield, A. S.; Heisler, L. K. Pharmacological targeting of the
serotonergic system for the treatment of obesity. J. Physiol. 2009, 587,
49−60.
(20) Lee, J.; Jung, M. E.; Lee, J. 5-HT2C receptor modulators: a
patent survey. Expert Opin. Ther. Pat. 2010, 20, 1429−1455.
(21) Hopkins, C. R. ACS chemical neuroscience molecule spotlight
on lorcaserin. ACS Chem. Neurosci. 2010, 1, 718−719.
(22) FY 2012 Innovative Drug Approvals; U.S. Food and Drug
Administration: Silver Spring, MD, December2012; http://www.fda.
gov/downloads/aboutfda/reportsmanualsforms/reports/ucm330859.
pdf (Sep 25, 2017).
(23) Kroeze, W. K.; Roth, B. L. The molecular biology of serotonin
receptors: therapeutic implications for the interface of mood and
psychosis. Biol. Psychiatry 1998, 44, 1128−1142.
(24) Rothman, R. B.; Baumann, M. H.; Savage, J. E.; Rauser, L.;
McBride, A.; Hufeisen, S. J.; Roth, B. L. Evidence for possible
involvement of 5-HT2B receptors in the cardiac valvulopathy
associated with fenfluramine and other serotonergic medications.
Circulation 2000, 102, 2836−2841.
(25) Fitzgerald, L. W.; Burn, T. C.; Brown, B. S.; Patterson, J. P.;
Corjay, M. H.; Valentine, P. A.; Sun, J. H.; Link, J. R.; Abbaszade, I.;
Hollis, J. M.; Largent, B. L.; Hartig, P. R.; Hollis, G. F.; Meunier, P. C.;
Robichaud, A. J.; Robertson, D. W. Possible role of valvular serotonin
5-HT2B receptors in the cardiopathy associated with fenfluramine. Mol.
Pharmacol. 2000, 57, 75−81.
(26) Astrup, A. Drug management of obesity - efficacy versus safety.
N. Engl. J. Med. 2010, 363, 288−290.
(27) DiNicolantonio, J. J.; Chatterjee, S.; O’Keefe, J. H.; Meier, P.
Lorcaserin for the treatment of obesity? A closer look at its side effects.
Open Heart 2014, 1, e000173.
(28) Conn, P. J.; Christopoulos, A.; Lindsley, C. W. Allosteric
modulators of GPCRs: a novel approach for the treatment of CNS
disorders. Nat. Rev. Drug Discov. 2009, 8, 41−54.
(29) Lane, J. R.; Abdul-Ridha, A.; Canals, M. Regulation of G
protein-coupled receptors by allosteric ligands. ACS Chem. Neurosci.
2013, 4, 527−534.
(30) Wootten, D.; Christopoulos, A.; Sexton, P. M. Emerging
paradigms in GPCR allostery: implications for drug discovery. Nat.
Rev. Drug Discov. 2013, 12, 630−644.
(31) Gentry, P. R.; Sexton, P. M.; Christopoulos, A. Novel allosteric
modulators of G protein-coupled receptors. J. Biol. Chem. 2015, 290,
19478−19488.
(32) Im, W. B.; Chio, C. L.; Alberts, G. L.; Dinh, D. M. Positive
allosteric modulator of the human 5-HT2C receptor. Mol. Pharmacol.
2003, 64, 78−84.
(33) Ding, C.; Bremer, N. M.; Smith, T. D.; Seitz, P. K.; Anastasio, N.
C.; Cunningham, K. A.; Zhou, J. Exploration of synthetic approaches
and pharmacological evaluation of PNU-69176E and its stereoisomer
as 5-HT2C receptor allosteric modulators. ACS Chem. Neurosci. 2012,
3, 538−545.
(34) McAllister, C. E.; Hartley, R. M.; Zhang, G.; Fox, R. G.;
Anastasio, N. C.; Wild, C.; Zhou, J.; Cunningham, K. A. In vivo
evaluation of novel serotonin 5-HT2C receptor positive allosteric
modulator CYD-1−79. Drug Alcohol Depend. 2015, 156, e144.
(35) Wacker, D.; Wang, S.; McCorvy, J. D.; Betz, R. M.;
Venkatakrishnan, A. J.; Levit, A.; Lansu, K.; Schools, Z. L.; Che, T.;
Nichols, D. E.; Shoichet, B. K.; Dror, R. O.; Roth, B. L. Crystal
structure of an LSD-bound human serotonin receptor. Cell 2017, 168,
377−389.
(36) Rosenzweig-Lipson, S.; Zhang, J.; Mazandarani, H.; Harrison, B.
L.; Sabb, A.; Sabalski, J.; Stack, G.; Welmaker, G.; Barrett, J. E.;
Dunlop, J. Antiobesity-like effects of the 5-HT2C receptor agonist
WAY-161503. Brain Res. 2006, 1073−1074, 240−251.
(37) Kennett, G. A.; Wood, M. D.; Bright, F.; Trail, B.; Riley, G.;
Holland, G.; Avenell, K. Y.; Stean, T.; Upton, N.; Bromidge, S.;
Forbes, I. T.; Brown, A. M.; Middlemiss, D. N.; Blackburn, T. P. SB

242084, a selective and brain penetrant 5-HT2C receptor antagonist.
Neuropharmacology 1997, 36, 609−620.
(38) Higgins, G. A.; Silenieks, L. B.; Lau, W.; de Lannoy, I. A.; Lee,
D. K.; Izhakova, J.; Coen, K.; Le, A. D.; Fletcher, P. J. Evaluation of
chemically diverse 5-HT2C receptor agonists on behaviours motivated
by food and nicotine and on side effect profiles. Psychopharmacology
2013, 226, 475−490.
(39) Dawes, A. J.; Maggard-Gibbons, M.; Maher, A. R.; Booth, M. J.;
Miake-Lye, I.; Beroes, J. M.; Shekelle, P. G. Mental health conditions
among patients seeking and undergoing bariatric surgery. A meta-
analysis. J. Am. Med. Assoc. 2016, 315, 150−163.
(40) Crespillo, A.; Alonso, M.; Vida, M.; Pavoń, F. J.; Serrano, A.;
Rivera, P.; Romero-Zerbo, Y.; Fernańdez-Llebrez, P.; Martínez, A.;
Peŕez-Valero, V.; Bermud́ez-Silva, F. J.; Suaŕez, J.; de Fonseca, F. R.
Reduction of body weight, liver steatosis and expression of stearoyl-
CoA desaturase 1 by the isoflavone daidzein in diet-induced obesity.
Br. J. Pharmacol. 2011, 164, 1899−1915.
(41) Gomez, R.; Navarro, M.; Ferrer, B.; Trigo, J. M.; Bilbao, A.; Del
Arco, I.; Cippitelli, A.; Nava, F.; Piomelli, D.; de Fonseca, F. R. A
peripheral mechanism for CB1 cannabinoid receptor-dependent
modulation of feeding. J. Neurosci. 2002, 22, 9612−9617.
(42) Rodríguez de Fonseca, F. R.; Navarro, M.; Gomez, R.; Escuredo,
L.; Nava, F.; Fu, J.; Murillo-Rodriguez, E.; Giuffrida, A.; LoVerme, J.;
Gaetani, S.; Kathuria, S.; Gall, C.; Piomelli, D. An anorexic lipid
mediator regulated by feeding. Nature 2001, 414, 209−212.

Journal of Medicinal Chemistry Article

DOI: 10.1021/acs.jmedchem.7b00994
J. Med. Chem. 2017, 60, 9575−9584

9584

http://www.fda.gov/downloads/aboutfda/reportsmanualsforms/reports/ucm330859.pdf
http://www.fda.gov/downloads/aboutfda/reportsmanualsforms/reports/ucm330859.pdf
http://www.fda.gov/downloads/aboutfda/reportsmanualsforms/reports/ucm330859.pdf
http://dx.doi.org/10.1021/acs.jmedchem.7b00994