Knowledge-Based Design of Long-Chain Arylpiperazine Derivatives Targeting Multiple Serotonin Receptors as Potential Candidates for Treatment of Autism Spectrum Disorder

Article abstract of DOI:10.1021/acschemneuro.0c00647

Autism spectrum disorder (ASD) includes a group of neurodevelopmental disorders characterized by core symptoms such as impaired social interaction and communication, repetitive and stereotyped behaviors, and restricted interests. To date, there are no effective treatments for these core symptoms. Several studies have shown that the brain serotonin (5-HT) neurotransmission system is altered in both ASD patients and animal models of the disease. Multiple pieces of evidence suggest that targeting 5-HT receptors may treat the core symptoms of ASD and associated intellectual disabilities. In fact, stimulation of the 5-HT1A receptor reduces repetitive and restricted behaviors; blockade of the 5-HT2A receptor reduces both learning deficits and repetitive behavior, and activation of the 5-HT7 receptor improves cognitive performances and reduces repetitive behavior. On such a basis, we have designed novel arylpiperazine derivatives pursuing unprecedently reported activity profiles: dual 5-HT7/5-HT1A receptor agonist properties and mixed 5-HT7 agonist/5-HT1A agonist/5-HT2A antagonist properties. Seventeen new compounds were synthesized and tested in radioligand binding assay at the target receptors. We have identified the dual 5-HT1AR/5-HT7R agonists 8c and 29 and the mixed 5-HT1AR agonist/5-HT7R agonist/5-HT2AR antagonist 20b. These compounds are metabolically stable in vitro and have suitable central nervous system druglike properties.

Full text of DOI:10.1021/acschemneuro.0c00647

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Research Article
Knowledge-Based Design of Long-Chain Arylpiperazine Derivatives
Targeting Multiple Serotonin Receptors as Potential Candidates for
Treatment of Autism Spectrum Disorder
Enza Lacivita,* Mauro Niso, Margherita Mastromarino, Andrea Garcia Silva, Cibell Resch, Andre Zeug,
María I. Loza, Marián Castro, Evgeni Ponimaskin, and Marcello Leopoldo*
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ABSTRACT: Autism spectrum disorder (ASD) includes a group of neurodevelopmental disorders characterized by core symptoms
such as impaired social interaction and communication, repetitive and stereotyped behaviors, and restricted interests. To date, there
are no effective treatments for these core symptoms. Several studies have shown that the brain serotonin (5-HT) neurotransmission
system is altered in both ASD patients and animal models of the disease. Multiple pieces of evidence suggest that targeting 5-HT
receptors may treat the core symptoms of ASD and associated intellectual disabilities. In fact, stimulation of the 5-HT1A receptor
reduces repetitive and restricted behaviors; blockade of the 5-HT2A receptor reduces both learning deficits and repetitive behavior,
and activation of the 5-HT7 receptor improves cognitive performances and reduces repetitive behavior. On such a basis, we have
designed novel arylpiperazine derivatives pursuing unprecedently reported activity profiles: dual 5-HT7/5-HT1A receptor agonist
properties and mixed 5-HT7 agonist/5-HT1A agonist/5-HT2A antagonist properties. Seventeen new compounds were synthesized
and tested in radioligand binding assay at the target receptors. We have identified the dual 5-HT1AR/5-HT7R agonists 8c and 29 and
the mixed 5-HT1AR agonist/5-HT7R agonist/5-HT2AR antagonist 20b. These compounds are metabolically stable in vitro and have
suitable central nervous system druglike properties.
KEYWORDS: 5-HT7 receptor, 5-HT1A receptor, 5-HT2A receptor, arylpiperazine, autism spectrum disorder, knowledge-based design
itory imbalance), neuroinflammation, and altered neuro-
transmitter systems.3 Serotonin (5-hydroxytryptamine, 5-HT)
exerts a very complex modulatory role in the central nervous
system (CNS) involving at least 14 diverse membrane 5-HT
receptors (5-HTRs), grouped in seven families (5-HT1−7). 5-
HT plays a crucial role in shaping neuronal circuits during
prenatal and postnatal development by promoting neurogenesis,
neuronal differentiation, axon myelination, neuropil formation,
and synaptogenesis.4,5 Accumulating findings indicate that the
INTRODUCTION
■
Autism spectrum disorder (ASD) includes a group of brain
developmental disorders characterized by core symptoms such
as impaired social interaction and communication, repetitive
and stereotyped behaviors, and restricted interests.1 In addition,
ASD patients often present a variety of additional impairments,
including intellectual disability. The frequency of ASD is
increasing, with present rates of about 1 in 100 children in
Europe and 1 in 54 in the United States.2 To date, the only drugs
approved to treat ASD-related symptoms are the atypical
antipsychotics aripiprazole and risperidone (Table 1) which are
efficacious to treat irritability, hyperactivity, and aggression but
not the core symptoms. Thus, ASD has no cure and its treatment
represents a largely unmet clinical need.3 Several mechanisms
have been implicated in ASD, such as synaptic dysfunction
(alterations in dendritic spine morphology, excitatory/inhib-
Received: October 9, 2020
Accepted: March 24, 2021
Published: April 1, 2021
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Table 1. Structural Formulas and Affinity Profiles of the Reference Compounds
a
Not Available.
brain 5-HT neurotransmission system is altered in ASD
patients6 and in rodent models of ASD.7−9 Selective serotonin
reuptake inhibitors (SSRIs) have been often prescribed to ASD
patients to treat repetitive and stereotyped behaviors as these
drugs are efficacious to treat obsessive-compulsive disorder.
However, clinical studies in ASD patients treated with SSRIs did
not give consistent results.10 In fact, some studies reported
positive effects on stereotypy and compulsions, whereas others
reported that SSRIs worsen stereotypy. In addition, side-effects
were highly prevalent probably due to the generalized elevation
of 5-HT levels that could lead to not therapeutically valid
stimulation of multiple 5-HTRs. Various studies have evidenced
that targeting certain 5-HTRs has the potential to treat the core
symptoms of ASD and associated intellectual disabilities. A
clinical trial in young children with ASD demonstrated that the
5-HT1AR partial agonist buspirone reduces repetitive and
restricted behaviors.11 Studies in rodent models of ASD have
provided further support in this direction. In fact, buspirone and
the 5-HT1AR full agonist 8-OH-DPAT enhance social
interactions.12,13 The selective 5-HT2AR antagonist M100907
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Table 2. CNS MultiParameter Optimization (CNS-MPO), Binding Affinities, Ki Ratios, and Microsomal Stability of the Target
Compounds
a
b
MS: microsomal stability (% of recovery of the parent compound after 30 min incubation with rat microsomes). Full displacement of the specific
binding was not achieved at maximum concentration assayed (100 μM); Ki value extrapolated from the analysis might not be accurately estimated;
c
Data from ref 51.
alleviates both learning deficits and repetitive behavior.14,15 The
selective 5-HT6R antagonist SLV reverses the social engagement
deficit.16 The selective 5-HT7R agonist LP-211 (Table 1)
corrects behavioral alterations in mouse models of rare
neurodevelopmental disorders. In particular, administration of
LP-211 (i) improved novel object recognition performance and
reduced stereotyped behavior in a mouse model of Fragile-X
syndrome (an X-linked disease with autistic features);17−19 (ii)
improved anxiety-related profiles, the exploratory behavior and
memory in a mouse model of Rett syndrome (a disease
characterized by intellectual disability);20−22 and (iii) normal-
ized the prepulse inhibition defects observed in a mouse model
of CDKL5 deficiency disorder (a disease characterized by severe
neurodevelopmental delay impacting cognitive, speech, and
visual function).23 Altogether, the above studies provide support
to the search of novel compounds with an appropriate
pharmacological profile at 5-HTRs in the prospect to ameliorate
the core symptoms of ASD. At present, various examples of
drugs or investigational compounds targeting multiple 5-HTRs
exist. The most notable examples are aripiprazole (dopamine
D2R/5-HT2AR/5-HT7R antagonist, 5-HT1AR partial agonist)
and risperidone (dopamine D2R/5-HT2AR/5-HT7R/5-HT1AR
antagonist) (Table 1).24 However, aripiprazole and risperidone
are not effective on the core symptoms of ASD, suggesting that
such particular activity profile is not suitable to treat the core
symptoms of ASD.3 Vortioxetine, a multimodal antidepressant
that inhibits 5-HT transporter and activates 5-HT1A and 5-HT1B
receptors, suppresses restrictive−repetitive behaviors in a rodent
model of ASD but has less efficacy as a sociability enhancer.25
These studies strongly suggest the design of new compounds
characterized by an activity profile resulting from combinations
of activities at 5-HTRs that are predicted to produce effects on
the core symptoms of ASD. The first attempt in this direction
was reported in 2015 by Canal et al., who developed the
compound (+)-5-FPT which was initially described as a high-
affinity 5-HT7R and 5-HT1AR partial agonist (Table 1). The
(+)-5-FPT potently attenuated stereotypy in three heteroge-
neous models of stereotypy in a mouse strain characterized by
repetitive behavior.26 In 2020, the same research group reported
that (+)-5-FPT was actually a 5-HT1AR/5-HT2CR agonist and 5-
HT7R antagonist.27 This compound was then studied in a
mouse model of Fragile X syndrome and found to reduce
repetitive behavior through 5-HT1AR partial agonism and to
attenuate audiogenic seizure through 5-HT2CR agonism. Thus,
the potential of concomitant activation of 5-HT1AR and 5-HT7R
was not actually explored.
Therefore, on the basis of the data discussed above, we aimed
at identifying completely new modulators of 5-HTRs for studies
in the context of ASD. In particular, we searched for (a) a dual 5-
HT7R/5-HT1AR agonist, which is predicted to increase social
interaction through the activation of 5-HT1AR and to reduce
stereotypy and improve cognition through activation of 5-
HT7R, and (b) a compound acting as 5-HT7R/5-HT1AR agonist
and 5-HT2AR antagonist, which is predicted to improve social
behavior through activation of 5-HT1AR, to reduce or eliminate
stereotyped behavior by blocking 5-HT2AR, and to improve
cognition through activation of 5-HT7R.
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a
Scheme 1. Synthesis of Target Compounds 8a−c and 9
a
Reagents and conditions: (A) NaH, Br−(CH2)n−X, anhydrous DMF, rt, 12 h, 40−70% yield; (B) 1-arylpiperazine; K2CO3, acetonitrile, reflux
overnight, 20−50% yield.
a
Scheme 2. Synthesis of Target Compounds 11a,b
a
Reagents and conditions: (A) 1-[2-(4-methoxyphenyl)phenyl]piperazine; K2CO3, acetonitrile, reflux overnight, 61% yield; (B) NaCN, anhydrous
DMF, rt, 5 h, quantitative yield; (C) Raney-nickel, H2 (4 atm), MeOH, 50% yield; (D) 2-bromoethanol, CaCO3; reflux, 7 h, 37% yield; (E) PBr3,
anhydrous toluene, reflux, 3 h, 65% yield; (F) K2CO3, acetonitrile, reflux overnight, 60% yield.
are the 5-HT1AR antagonist WAY-100635 and the 5-HT7R
agonist LP-21128 (Table 1). Instead, compounds 129 and 230
(Table 1) are dual 5-HT7R/5-HT1AR ligands, and compound
331 is a mixed 5-HT7R/5-HT1AR/5-HT2AR ligand (Table 1).
Thus, long-chain arylpiperazines are a versatile framework to
identify compounds with a diversified receptor affinity profile.
On the other hand, it appears much less trivial to identify
arylpiperazines showing the activity profile that we are pursuing
RESULTS AND DISCUSSION
■
Study Design. Long-chain arylpiperazine derivatives are
known to bind monoamine receptors, including 5-HTRs. The
general formula is Ar−piperazine−linker−terminal fragment,
and it is known that suitable modifications of Ar, linker, or
terminal fragment can lead to selective or nonselective
compounds. Two examples of selective arylpiperazine ligands
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a
Scheme 3. Preparation of Target Compounds Featuring the Coumarin Nucleus as the Terminal Fragment
a
Reagents and conditions: (A) ethyl acetoacetate; conc. H2SO4, rt, 4 h, 34% yield; (B) 1-[2-(4-methoxyphenyl)phenyl]piperazine; K2CO3,
acetonitrile, reflux overnight, 21% yield; (C) NaH, Br−(CH2)n−X, anhydrous DMF, rt, 12 h, 40−60% yield.
(i.e., dual 5-HT7R/5-HT1AR agonist, mixed 5-HT7R agonist/5-
HT1AR agonist/5-HT2AR antagonist). To fulfill our aim, we
have followed a knowledge-based design (i.e., the collection and
reuse of expert knowledge) by selecting Ar groups and terminal
fragments present in arylpiperazine-based 5-HTR agonists or
antagonists. First, we focused on the Ar group and selected the 1-
(biphenyl)piperazine moiety that is the key structural
determinant for the agonism at 5-HT7R of LP-211, TP-22,32
and BA-1018 (Table 1). Consequently, the majority of the target
compounds are 1-[2-(4-methoxyphenyl)phenyl]piperazine de-
rivatives (see Table 2). In addition, in order to identify dual 5-
HT7R/5-HT1AR ligands, the compounds 9 and 30 (Table 2),
featuring the 2-acetylphenyl group present in the dual 5-HT7R/
5-HT1AR ligand 1 (Table 1), were designed. It is worth noting
that, even if LP-211, TP-22, and BA-10 are 5-HT7R-preferring
agonists, they still show a measurable affinity for 5-HT1AR
(Table 1). Interestingly, LP-211 itself shows measurable 5-
HT2AR affinity (Table 1).28 Thus, the 1-(biphenyl)piperazine
framework appears to be compatible with 5-HT1AR and 5-
HT2AR affinity, and therefore, suitable variations of the terminal
fragments and linkers might increase 5-HT1AR and 5-HT2AR
affinity. In the case of 5-HT1AR, the terminal fragment also has a
role on the functional activity of the ligand. With this respect,
notable examples are the 2-methoxyphenyl piperazine deriva-
tives WAY-100635,33 4,34 and MMP.35 In fact, while WAY-
100635 is a 5-HT1AR antagonist, compounds 4 and MMP are
agonists. Consequently, we have selected the terminal fragments
present in the high-affinity 5-HT1AR agonists MMP (which also
shows 5-HT7R affinity in the nanomolar range) and UCM-
255036 (Table 1). As for 5-HT2AR, we selected the terminal
fragments of the antagonists risperidone, brilaroxazine,37 and
538 (Table 1). Based on the structural similarity with these
antagonists, our newly designed arylpiperazine derivatives were
expected to act as 5-HT2AR antagonist, as they display a
completely different structural motif as compared to that of 5-
HT2AR agonists.39 The fine-tuning of the affinities at the target
receptors was accomplished by incorporating 2−5 atom linkers.
In addition, the drug-likeness CNS multiparameter opti-
mization (CNS MPO) algorithm40 was applied to the designed
compounds. As shown in Table 2, 7 out of 17 compounds
present a CNS MPO desirability score higher than 4, predictive
of alignment to ADME attributes, ability to cross the blood−
brain barrier, and low safety risk. As for the remaining
compounds, 8 out 10 show CNS MPO desirability score in
the range of 3 to 4 which might still include compounds with
desirable properties.
In order to evaluate the liability of the target compounds to
metabolic degradation by first-pass oxidative metabolism, the
main cause of metabolic degradation in vivo, we screened the
metabolic stability in vitro by using rat liver microsomes.41 First,
the metabolic stability was assessed as the percentage of the
parent compound recovered after 30 min of incubation with
microsomes in the presence of an NADPH-regenerating
system.32 Subsequently, selected compounds showing recovery
higher than 20% were further characterized by evaluating half-
life (t1/2) and intrinsic clearance (CLint), which are predictive of
in vivo hepatic clearance.
Chemistry. The target compounds 8a−c and 9 bearing the
4-methyl-1,2,4-triazine-3,5(2H,4H)-dione moiety as the termi-
nal fragment were prepared according to Scheme 1. 4-Methyl-
1,2,4-triazine-3,5(2H,4H)-dione (6)42 was alkylated with the
appropriate haloalkylbromide to give the alkyl halides 7a−c,
which after nucleophilic substitution with the appropriate
arylpiperazine, afforded the desired compounds 8a−c and 9.
Scheme 2 illustrates the synthesis of target compounds 11a,b.
Compound 11a was obtained by reacting 3-(2-chloroethyl)-2-
methyl-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one
(10)43 with 1-[2-(4-methoxyphenyl)phenyl]piperazine.44
Compound 11b was obtained through a convergent synthesis
that required intermediates 13 and 16 (Scheme 2). Amine 13
was synthesized starting from alkyl chloride 10 by sequential
reaction with cyanide ion to give the nitrile 12 and reduction of
the latter with Raney nickel under hydrogen pressure. Bromide
16 was prepared from 2-(4-methoxyphenyl)aniline (14)44 and
2-bromoethanol in the presence of CaCO3 to afford the bis
alkylated product 15, which was converted into 16 with PBr3.
Condensation of the amine 13 and the bromide 16 yielded the
target compound 11b.
The target compounds featuring the coumarin nucleus as the
terminal fragment were prepared as described in Scheme 3. In
order to synthesize compound 20a from compound 19a, several
unsuccessful attempts were made to alkylate 7-hydroxy-4-
methylcoumarin (18)45 with 1-bromo-2-chloroethane. Then,
compound 19a was instead synthesized starting from 3-(2-
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a
Scheme 4. Synthetic Route to Obtain Final Compounds 26a
a
Reagents and conditions: (A) methyl 2-bromopropionate, K2CO3, acetone, reflux, 16 h, 54% yield; (B) CH3ONa, MeOH, rt, 3 h, 89% yield; (C)
1,2-dibromoethane, K2CO3, anhydrous DMF, 85 °C, 6 h, 70% yield; (D) Fe dust, AcOH, 80 °C, 1 h, 60% yield; (E) 1-[2-(4-
methoxyphenyl)phenyl]piperazine; K2CO3, acetonitrile, reflux overnight, 38% yield.
a
Scheme 5. Synthetic Route to Obtain Final Compounds 26b, 29, and 30
a
Reagents and conditions: (A) NaH, Br−(CH2)3−Cl, anhydrous DMF, rt, 24 h, 22−42% yield; (B) 1-[2-(4-methoxyphenyl)phenyl]piperazine;
K2CO3, acetonitrile, reflux overnight, 20−65% yield. (C) NaH, (R)-glycidyl nosilate, anhydrous DMF, rt, overnight, 41% yield; (D) 1-
arylpiperazine; EtOH, reflux, 4 h, 30−54% yield.
bromoethoxy)phenol (17),46 which was condensed with ethyl
acetoacetate under Pechmann conditions. Nucleophilic sub-
stitution of 19a by 1-[2-(4-methoxyphenyl)phenyl]piperazine
afforded the desired compound 20a. The analogues 20b,c were
prepared starting from phenol 18 which was alkylated with the
appropriate bromoalkylchloride to afford derivatives 19b,c.
These latter compounds were condensed with 1-[2-(4-
methoxyphenyl)phenyl]piperazine to give the desired com-
pounds 20b,c, respectively.
The final compounds 26a,b, 29, and 30 featuring the
benzoxazinone terminal fragment were prepared following two
distinct synthetic routes depending on the nature of the linker
(Schemes 4 and 5). In fact, the synthesis of the key intermediate
25a was initially pursued through alkylation of 6-hydroxy-2-
methyl-2H-benzo[b][1,4]oxazin-3(4H)-one (27) with 1-
bromo-2-chloroethane, but it was unsuccessful. Thus, an
alternative synthetic pathway was envisaged (Scheme 4): 4-
(benzyloxy)-2-nitrophenol (21)47 was alkylated with methyl 2-
bromopropanoate to afford derivative 22, which was depro-
tected under basic conditions to give phenol 23. The latter was
alkylated with 1,2-dibromoethane to give 24. Reduction of the
nitro group of 24 by iron dust in acidic conditions resulted in the
cyclization of the intermediate amine to give the key
intermediate 25a, which was condensed with 1-[2-(4-
methoxyphenyl)phenyl]piperazine to give the target compound
26a. The other target compound 26b bearing the benzoxazinone
nucleus as terminal fragment was prepared according to Scheme
5. Alkylation of phenol 2748 with 1-bromo-3-chloropropane
gave the chloroderivative 25b, which underwent nucleophilic
substitution with 1-[2-(4-methoxyphenyl)phenyl]piperazine to
give the final compound 26b. The target compounds 29 and 30
were prepared starting from phenol 27 which was alkylated with
(R)-glycidylnosilate to give oxirane 28. Ring opening of 28 with
the appropriate 1-arylpiperazine gave 29 and 30.
The target compounds bearing the tetrahydro-1H-pyrrolo-
[1,2-c]imidazole-1,3(2H)-dione as terminal fragment were
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Scheme 6. Formation of Target Compounds Bearing the Tetrahydro-1H-pyrrolo[1,2-c]imidazole-1,3(2H)-dione as Terminal
a
Fragment
a
Reagents and conditions: (A) NaH, Br−(CH2)n−Cl, anhydrous DMF, rt, 12 h, 55−75% yield; (B) 1-[2-(4-methoxyphenyl)phenyl]piperazine;
K2CO3, acetonitrile, reflux overnight, 43−74% yield.
prepared according to Scheme 6. The N-alkylation of hydantoin
derivative 3149 with the appropriate bromoalkylchloride gave
the chloroalkyl intermediates 32a,b, which were condensed with
1-[2-(4-methoxyphenyl)phenyl]piperazine to afford the desired
compounds 33a,b, respectively.
Binding Affinities to 5-HT7, 5-HT1A, 5-HT2A, and
Dopamine D2 Receptors. All the final compounds were
tested in radioligand binding assays to determine their affinity
for 5-HT7R, 5-HT1AR, and 5-HT2AR (Table 2). In addition, the
compounds were counter screened at dopamine D2 receptors,
because blockade of this receptor may cause motor dysfunc-
tions.50 The assays were performed via the displacement of the
specific binding of [3H]-5-CT (for 5-HT7R), [3H]-8-OH-DPAT
(for 5-HT1AR), [3H]ketanserin (for 5-HT2AR), and [3H]-
spiperone (for dopamine D2 receptor), at the cloned human
receptors stably expressed in HEK293 cells (5-HT7R, 5-HT1AR)
or CHO-K1 cells (5-HT2AR, D2 receptor).
the corresponding terminal fragment. Nonetheless, the
proposed structural changes led to the identification of six
compounds (8c, 11a, 20b, 26b,c, and 33b) with 5-HT1AR
affinity higher than that of LP-211.
Considering the affinity at 5-HT2AR, all the target 1-[2-(4-
methoxyphenyl)phenyl]piperazine derivatives show affinity
higher than that of LP-211, except 8a and 33a, confirming the
validity of the selection of terminal fragments present in the
reference compounds. Compounds 8a and 33a represent an
unfavorable combination of linker length and terminal fragment.
The most pronounced increase was in the case of compound
11b in which the terminal fragment is the same as in risperidone.
The 5-HT2AR affinity of the final 1-(2-acetylphenyl)piperazine
derivatives 9 and 30 was in the micromolar range, which was
similar to the affinity value of the corresponding reference
compound 1.
The D2 receptor affinities of the target 1-[2-(4-
methoxyphenyl)phenyl]piperazine derivatives were all lower
than that of LP-211, but 26c that displayed a Ki value lower than
100 nM. Most importantly, the reference compounds
risperidone (vs 11a,b), 5 (vs 20a−c), brilaroxazine (vs 26a−c,
29), and UCM-2550 (vs 33a,b) show higher D2 receptor affinity
than that of the target compounds featuring the corresponding
terminal fragment. Thus, the affinity of the target compounds at
D2 receptors seems to be determined by the 1-[2-(4-
methoxyphenyl)phenyl]piperazine moiety rather than the linker
length and the terminal fragment. As for the 1-(2-acetylphenyl)-
piperazine derivatives 9 and 30, both compounds showed high
D2 receptor affinity, being the most potent D2 receptor ligands
among the newly synthesized compounds.
Next, in order to select dual 5-HT7R/5-HT1AR or mixed 5-
HT7R/5-HT1AR/5-HT2AR ligands, we analyzed the 5-HT1AR/
5-HT7R, 5-HT2AR/5-HT7R, and 5-HT2AR/5-HT1AR Ki ratios
(Table 2). As reference values, we selected the Ki ratios of
compound 26c which, according to our recent study, behaved as
a 5-HT7R-preferring agonist in vitro and in vivo.51 Con-
sequently, to select the dual 5-HT1AR/5-HT7R ligands, we
considered compounds showing a 5-HT1AR/5-HT7R Ki ratio
lower than 9 (and greater than 0.11, i.e., the reciprocal of 9).
Compounds 8c, 29, and 30 displayed such characteristic. These
compounds were also selective over 5-HT2AR. The most
balanced 5-HT1AR/5-HT7R ligands of this set were 8c and 29,
as they showed the 5-HT7R/5-HT1AR Ki ratios closest to 1. On
the other hand, compounds 20b, 20c, 26b, and 34 displayed
mixed 5-HT7R/5-HT1AR/5-HT2AR affinity, with 20b being the
compound with the most balanced affinity profile (all three Ki
ratios close to 1).
The Ki values at 5-HT7R of the target 1-[2-(4-
methoxyphenyl)phenyl]piperazine derivatives 8a,b, 11a,b,
20a−c, 26a−c, 29, and 33a,b are in the range 6.69−91.7 nM,
indicating that the structural variations introduced to the
terminal fragment or linker of LP-211 were well tolerated. In
fact, such variations translate into small changes in affinity, the
largest variation being in the case of 33a and 33b (7-fold). Of
note, the 5-HT7R affinity values of the reference compounds
brilaroxazine (vs 26a−c, 29), UCM-2550 (vs 33a,b), risper-
idone (vs 11a,b), and MMP (vs 8a−c) fell in the same range.
Instead, the 1-(2-acetylphenyl)piperazine derivatives 9 and 30
were 50- and 11-fold less potent than the reference compound 1
at 5-HT7R.
As for the 5-HT1AR affinities, the 1-(2-acetylphenyl)-
piperazine derivatives 9 and 30 were equipotent potent to the
reference compound 1, whereas the target 1-[2-(4-
methoxyphenyl)phenyl]piperazine derivatives 8a,b, 11a,b,
20a−c, 26a−c, 29, and 33a,b showed Ki values distributed in
a wide range (3.77−1802 nM), as the result of a more
pronounced impact of the linker length. In fact, the 4-methyl-
1,2,4-triazine-3,5(2H,4H)-dione derivative 8c displays Ki = 3.77
nM, whereas its shorter homologue 8a shows a 456-fold lower 5-
HT1AR affinity. Similarly, the benzoxazinone derivative 26b
displays Ki = 23.2 nM, whereas its shorter homologue 26a shows
78-fold lower affinity. The comparison of the close analogs 26a−
c, 29, and 34 provides an interesting example of how the affinity
for 5-HT7R and 5-HT1AR can be fine-tuned by the nature of the
terminal fragment and the linker. It can be noted that the target
compounds 26a−c, 29 (vs brilaroxazine), 33a,b (vs UCM-
2550), 8a−c (vs MMP), and 20a−c (vs 5) display lower 5-
HT1AR affinity than that of the reference compounds featuring
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In Vitro Metabolic Stability. The aim of this study is to
provide the scientific community with molecules suitable for
studies in vivo. In order to predict the extent of first-pass
oxidative metabolism, the target compounds were incubated
with rat liver microsomes in the presence of an NADPH
regenerating system.41 In the initial screening phase, we assessed
the percentage of the parent compound recovered after 30 min
of incubation. The percentages of recovery of LP-211 and TP-22
were 20 and 27%, respectively,32 which represented the
reference values to compare the new compounds. As it can be
seen in Table 1, the majority of the new compounds show in
vitro stability higher than LP-211, as 11 out of 17 compounds
display a percentage of recovery >20%. Then, taking into
account the affinity, selectivity, and metabolic stability data, we
assessed the half-life and the intrinsic clearance in vitro of
compounds 8c, 20b,c, 26a,b, and 29 (Table 3). The data
fold as in the case of compound 29. Thus, the compounds listed
in Table 3 are predicted to be low-clearance compounds and
suitable for studies in vivo.52 These results provide further
support to the strategy of using structural motifs featured by
druglike compounds to obtain metabolically stable com-
pounds.40,51
The metabolic stability and the affinity data supported the
selection of the dual 5-HT7R/5-HT1AR ligands 8c and 29 and
the mixed 5-HT7R/5-HT1AR/5-HT2AR ligand 20b for further
evaluations. These compounds distinguished themselves from
the reference compounds for the binding profile at 5-HT7, 5-
HT1A, 5-HT2A, and D2 receptors. As shown in Table 1, most of
the reference compounds display an affinity for α1A
adrenoceptor, and therefore, it was not unexpected that
compounds 8c, 20b, and 29 showed α1A adrenoceptor affinity
(Ki= 23.5, 66.1, and 55.8 nM, respectively, see Supporting
Information). In fact, the search for arylpiperazine derivatives
with affinity for multiple 5-HTRs might imply that the
compounds have an affinity for other monoamine receptors
(see Supporting Information for off-target affinities of
compounds 8c, 20b, and 29). Considering that α1A
adrenoceptor activity might cause cardiovascular side-effect,
this particular off-target activity is a safety warning for future
developments of this class of compounds.
Functional Activities at 5-HT7R, 5-HT1AR, and 5-HT2AR
of Compounds 8c, 20b, and 29. To provide a functional
analysis at 5-HT7R, the cAMP response mediated by 8c, 20b, 29
and the reference 5-HT7R agonists 5-CT and LP-21129,53 was
analyzed. To this end, 5-HT7R-mCherry was coexpressed with
the FRET-based cAMP biosensor CEPAC.54 This biosensor
includes the cAMP-binding domain of the EPAC protein cloned
between mCerulean (FRET donor) and Citrine (FRET
acceptor). Upon cAMP binding, conformational changes of
the sensor occur, leading to a decrease in the FRET signal
Table 3. Half-Life (t1/2) and Intrinsic Clearance (CLint) of
Selected Compounds
compound
t1/2 (min)
CLint (μL/mg/min)
LP-21132
TP-2232
8c
20b
20c
15
45
41
39
23
49
60
63
74
58
45.9
16.1
16.9
17.7
30
14.1
11.5
11
26a
26b
26c51
29
9.4
12
3451
indicate that all the selected compounds showed higher stability
than that of LP-211, with intrinsic clearance values lower up to 5-
Figure 1. Compounds 8c, 20b, and 29 stimulate 5-HT7R-mediated cAMP production. (A) N1E cells were transfected with cAMP FRET-based
biosensor CEPAC and 5-HT7R-mCherry. Cells were stimulated with the compounds, as indicated. Mean values of the cAMP-biosensor response upon
stimulation with 8c 20b, and 29 are shown. Stimulation with LP-211 and 5-CT was used as a control. (B) Quantification of the response amplitude and
(C) response time shown as the mean SEM (3 < N < 6, in each experiment at least 20 cells were analyzed).
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Figure 2. Compounds 8c, 20b, and 29 behave as 5-HT1AR agonists in the receptor-mediated cAMP inhibition. (A) N1E cells were transfected with
cAMP FRET-based biosensor CEPAC and 5-HT1AR-mCherry. After pretreatment with 1 μM forskolin and 25 μM IBMX, cells were stimulated with
the indicated compounds. Each trace shows cAMP response at the single cell. (B) Graphs show changes of cAMP response amplitude relative to
pretreatment (mean SEM, 3 < N < 6, in each experiment at least 20 cells were analyzed).
(Figure 1A). The cAMP responses were recorded at the single-
cell level by monitoring the CEPAC fluorescence intensity ratio
of the acceptor to the donor (A/D ratio). The strength and
speed of serotonergic signaling was determined from the
amplitude and time dependence of the CEPAC fluorescence
intensity ratio.
were less effective in activation of 5-HT1AR when compared with
5-CT (Figure 2B).
Finally, compounds 8c, 20b, and 29 were investigated in
functional assays of 5-HT2AR-mediated inositol phosphate (IP)
signaling in CHO-K1 cells expressing the cloned human
receptor. The three compounds, in concentration−response
curves from 1 nM to 100 μM, fully antagonized in a
concentration-dependent manner the stimulation of IP
production elicited by 1 μM 5-HT (Figure 3). Consistently,
no agonist activity was observed for 20b at the same
concentrations in these assays (data not shown). IC50 values
were similar for the three compounds and in the nanomolar
range, consistent with their affinities at 5-HT2AR (IC50 = 595,
666, and 491 nM for 8c, 20b, and 29, respectively). The
In the absence of 5-HT7R, no cAMP response was observed
upon treatment with the ligands (data not shown). In contrast,
in cells expressing 5-HT7R, all the compounds (10 μM) were
able to increase the intracellular cAMP level, although with
different efficiencies. Statistical analysis by fitting the exper-
imental data to the single exponential revealed that 8c and 29
elicited the largest cAMP response amplitude compared with
that of 5-CT, followed by 20b (Figure 1A). Of note, response
amplitude for all compounds tested was higher than that
measured for highly selective 5-HT7R agonist LP-211 (Figure
1B). The mean response times for all compounds were higher
(i.e., slower response kinetics) than that obtained for 5-CT and
comparable with the values obtained for LP-211 (Figure 1C).
We next analyzed whether 8c, 20b, and 29 would affect the 5-
HT1AR function toward cAMP inhibition. The 5-HT1AR agonist
5-CT was used as a positive control,55 while LP-211 (selective 5-
HT7R agonist) was used as a negative control. To this end, 5-
HT1AR-mCherry was coexpressed with the CEPAC biosensor in
N1E cells. We subsequently analyzed the ability of 5-HT1AR-
mediated signaling via Gi to inhibit the forskolin (FSK)-induced
cAMP accumulation following receptor stimulation with above-
mentioned compounds (10 μM). Except for LP-211 treatment,
an increase of the A/D ratio of CEPAC was observed in all the
cases, that indicated the 5-HT1AR-mediated downregulation of
intracellular concentration of cAMP (Figure 2). Statistical
evaluation of amplitudes of the cAMP decay for 8c, 20b, and 29
revealed that, although these compounds were still able to evoke
receptor-mediated decrease of the concentration of cAMP, they
Figure 3. Functional assays of inositol phosphate (IP) signaling at
cloned human 5-HT2ARs. Concentration−response inhibition curves of
8c, 20b, 29, and risperidone (as reference 5-HT2AR antagonist) on IP
production stimulated by 1 μM 5-HT in CHO-K1 cells expressing
human 5-HT2ARs. The graph shows data (mean SEM) from one
experiment performed in duplicate.
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theoretical values. The purity of the target compounds listed in Table 2
was assessed by RP-HPLC and combustion analysis. All compounds
showed ≥95% purity. RP-HPLC analysis was performed on an Agilent
1260 Infinity Binary LC System equipped with a diode array detector
using a Phenomenex Gemini C-18 column (250 mm × 4.6 mm, 5 μm
particle size). All target compounds (Table 2) were eluted with
CH3CN/ammonium formate 50 mM pH 5, 8:2 (v/v) at a flow rate of 1
mL/min. All compounds showed ≥95% purity.
The following compounds were prepared as described in the
literature: 1-[2-(4-methoxyphenyl)phenyl]piperazine;46 1-(2-(pipera-
zin-1-yl)phenyl)ethanone;56 4-methyl-1,2,4-triazine-3,5(2H,4H)-
dione (6);43 2-(4-chlorobutyl)-4-methyl-1,2,4-triazine-3,5(2H,4H)-
dione (7c);43 3-(2-chloroethyl)-2-methyl-6,7,8,9-tetrahydro-4H-
pyrido[1,2-a]pyrimidin-4-one (10);44 4′-methoxy-[1,1′-biphenyl]-2-
amine (14);46 3-(2-bromoethoxy)phenol (17);45 7-hydroxy-4-methyl-
chromen-2-one (18);47 4-(benzyloxy)-2-nitrophenol (21);48 6-[3-[4-
[2-(4-methoxyphenyl)phenyl]piperazin-1-yl]butoxy]-2-methyl-2H-
benzo[b][1,4]oxazin-3(4H)-one (26c);51 6-hydroxy-2-methyl-2H-
benzo[b][1,4]oxazin-3(4H)-one (27);49 tetrahydro-1H-pyrrolo[1,2-
c]imidazole-1,3(2H)-dione (31);50 (R)-1-(4-chloro-2-fluorophe-
noxy)-3-[4-[2-(4-methoxyphenyl)phenyl]piperazin-1-yl]propan-2-ol
(34).51 Complete synthetic procedures and intermediated spectro-
scopic data are fully reported in the Supporting Information.
General Procedure for the Preparation of Target Com-
pounds 8a−c, 9, 11a,b, 20a−c, 26a,b, and 33a,b. A stirred mixture
of the appropriate alkylating agent (0.7 mmol), 1-[2-(4-
methoxyphenyl)phenyl]piperazine or 1(2-acetylphenyl)piperazine
(0.84 mmol), and K2CO3 (0.1 g, 0.7 mmol) in acetonitrile (20 mL)
was refluxed overnight. After cooling, the mixture was evaporated to
dryness, and H2O (20 mL) was added to the residue. The aqueous
phase was extracted with AcOEt (2 × 30 mL). The collected organic
layers were dried over Na2SO4 and evaporated under reduced pressure.
The crude residue was purified by chromatographic column as detailed
below to afford pure target compound.
2-{4-[4-[2-(4-Methoxyphenyl)phenyl]piperazin-1-yl]ethyl}-4-
methyl-1,2,4-triazine-3,5(2H,4H)-dione (8a). Eluted with CHCl3/
MeOH, 98:2. Yellow oil, 73% yield. 1H NMR (CDCl3): δ 2.43 (br s,
4H), 2.67 (t, 2H, J = 6.6 Hz), 2.80 (br s, 4H), 3.32 (s, 3H), 3.85 (s, 3H),
4.08 (t, 2H, J = 6.6 Hz), 6.91−6.93 (m, 2H), 7.00 (d, 1H, J = 7.8 Hz),
7.04 (td, 1H, J = 1.1 and 7.6 Hz), 7.21 (dd, 1H, J = 1.5 and 7.3 Hz), 7.26
(m, 1H), 7.37 (s, 1H), 7.55−7.57 (m, 2H). GC/MS m/z 422 (M+, 4),
421 (M+, 16), 281 (100). Anal. (C23H27N5O3·HCl·H2O) C, H, N.
2-{3-[4-[2-(4-Methoxyphenyl)phenyl]piperazin-1-yl]propyl}-4-
methyl-1,2,4-triazine-3,5(2H,4H)-dione (8b). Eluted with CHCl3/
EtOAc, 1:1. Brown oil, 40% yield. 1H NMR (CDCl3): δ 1.88−1.92 (m,
2H), 2.34 (br s, 4H), 2.40 (t, 2H, J = 7.0 Hz), 2.80 (br s, 4H), 3.32 (s,
3H), 3.85 (s, 3H), 4.03 (t, 2H, J = 7.0 Hz), 6.92 (d, 2H, J = 8.8 Hz),
6.98−7.05 (m, 2H), 7.02−7.07 (m, 1H), 7.19−7.22 (m, 1H), 7.36 (s,
1H), 7.56 (d, 2H, J = 8.8 Hz). GC/MS m/z 436 (M+ + 1, 20), 435 (M+,
100), 281 (70), 212 (57), 167 (34). Anal. (C24H29N5O3·HCl·H2O) C,
H, N.
reference 5-HT2AR antagonist risperidone (0.01 nM−10 μM)
showed a IC50 value of 0.81 nM (Figure 3).
Collectively, the functional activities of the selected
compounds supported the validity of the design approach. In
fact, compounds 8c, 20b, and 29 behave as 5-HT7R agonists in
the same way as LP-211−confirming that the 1-(biphenyl)-
piperazine moiety is a key structural determinant for 5-HT7R
agonism and as 5-HT2AR antagonists as various other
arylpiperazine derivatives. As for 5-HT1AR, the comparison of
the functional activities of 8c, 20b, and 29 with LP-211 suggest
that the 1-(biphenyl)piperazine moiety alone is not responsible
for the functional activity at 5-HT1AR, which, instead, is
determined also by the nature of the terminal fragment and
linker length, as already noted in the Study Design paragraph.
CONCLUSIONS
■
5-HT neurotransmission system is an active area of investigation
in ASD research. Several in vitro and in vivo studies with
selective 5-HTR agonists or antagonists have suggested that
targeting a subpopulation of the 5-HTRs might alleviate the core
symptoms of ASD. Based on the current knowledge, we aimed at
identifying a dual 5-HT7R/5-HT1AR agonist and a mixed 5-
HT7R/5-HT1AR agonist/5-HT2AR antagonist. A set of novel
arylpiperazine derivatives were designed by exploiting structural
motifs that might drive the functional activity of the target
compounds toward the desired profile (knowledge-based
design). The design strategy succeeded as we identified
compounds 8c and 29 that are 5-HT7R and 5-HT1AR preferring
agonists and compound 20b, a mixed 5-HT7R/5-HT1AR
agonist/5-HT2AR antagonist with almost identical affinity for
the three receptors. The knowledge-based design strategy had a
favorable influence on the in vitro pharmacokinetic properties of
most of the newly designed compounds. In fact, 8c, 20b, and 29
are metabolically stable in vitro and also have suitable CNS
druglike properties. Considering the complex mechanisms
underlying ASD, we believe that a polypharmacology approach
might be more suited than a single target approach. We hope
that pharmacological tools such as 8c, 20b, and 29 will
contribute to the progress of the discovery of drugs for ASD.
METHODS
■
Chemistry. Chemicals were purchased from Sigma-Aldrich, Alfa
Aesar, TCI Chemicals. Unless otherwise stated, all chemicals were used
without further purification. Thin layer chromatography (TLC) was
performed using plates from Merck (silica gel 60 F254). Column
chromatography was performed with 1:30 Merck silica gel 60 Å (63−
200 μm) as the stationary phase. Flash chromatographic separations
were performed on a Biotage SP1 purification system using flash
2-{2-[4-[2-(4-Methoxyphenyl)phenylpiperazin-1-yl]butyl}-4-
methyl-1,2,4-triazine-3,5(2H,4H)-dione (8c). Eluted with CHCl3/
MeOH, 95:5. Pale yellow oil, 30% yield. 1H NMR (CDCl3): δ 1.47 (m,
2H), 1.71−1.80 (m, 2H), 2.34 (app t, 6H), 2.85 (app t, 4H), 3.33 (s,
3H), 3.98 (t, 2H, J = 7.0 Hz), 6.91−6.95 (m, 2H), 7.00−7.07 (m, 2H),
7.29−7.24 (m, 2H), 7.37 (s, 1H), 7.55−7.59 (m, 2H). 13C NMR (500
MHz, CDCl3): δ 158.7; 155.9; 148.9; 147.7; 134.9; 134.3; 132.7; 131.5;
129.9; 128.36; 124.3; 118.9; 113.8; 124.2; 118.7; 113.8; 55.3; 52.4;
50.5; 47.7; 27.1. ESI-MS m/z 472 (M + Na)+. ESI-MS/MS m/z 323
(100). Anal. (C25H31N5O3·HCl) C, H, N.
2-[4-[4-(2-Acetylphenyl)piperazin-1-yl]butyl]-4-methyl-1,2,4-tria-
zine-3,5(2H,4H)-dione (9). Eluted with CHCl3/MeOH, 98:2. Yellow
oil, 20% yield. 1H NMR (CDCl3): δ 1.57 (m, 2H), 1.81 (m, 2H), 2.44
(t, 2H, J = 7.6 Hz), 2.60 (br s, 4H), 2.65 (s, 3H), 3.02 (app. t, 4H), 3.35
(s, 3H), 4.02 (t, 2H, J = 7.1 Hz), 7.04−7.08 (m, 2H), 7.39−7.42 (m,
3H). GC/MS m/z 386 (M+ + 1, 10), 385 (M+, 30), 237 (90), 207
(100), 161 (95). Anal. (C20H27N5O3·2HCl) C, H, N.
1
cartridges prepacked with KP-Sil 32−63 μm, 60 Å silica. H NMR
spectra were recorded on a Varian Mercury-VX spectrometer (300
MHz) or on a 500-vnmrs500 Agilent spectrometer (500 MHz). All
chemical shift values are reported in parts per million (ppm, δ). Splitting
patterns are designated as follows: app (apparent), br (broad), s
(singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets),
td (triplet of doublets). For target compounds, NMR spectra were
recorded on free bases. Recording of mass spectra was done on an
HP6890-5973 MSD gas chromatograph/mass spectrometer; only
significant m/z peaks, with their percentage of relative intensity in
parentheses, are reported. ESI-MS/MS analyses were performed with
an Agilent 1100 Series LC-MSD trap System VL workstation, mass
range 50−800 m/z, electrospray ion source in positive or negative ion
mode. All spectra were in accordance with the assigned structures.
Elemental analysis (C,H,N) of the target compounds as hydrochloride
salts were performed on a Eurovector Euro EA 3000 analyzer. Analyses
indicated by the symbols of the elements were within 0.4% of the
3-{2-[4-[2-(4-Methoxyphenyl)phenyl]piperazin-1-yl]ethyl}-2-
methyl-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one (11a).
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1
Eluted with CHCl3/MeOH, 98:2. Brown solid, 60% yield. H NMR
(CDCl3): δ 1.84−1.89 (m, 2H), 1.92−1.97 (m, 2H), 2.27 (s, 3H),
2.45−2.50 (m, 6H), 2.69−2.72 (br t, 2H), 2.85 (t, 2H, J = 6.8 Hz), 2.89
(br s, 4H), 3.85 (s, 3H), 3.91 (t, 2H, J = 6.1 Hz), 6.90−6.93 (m, 2H),
7.03−7.06 (m, 2H), 7.21 (dd, 1H, J = 1.5 and 7.3 Hz), 7.25 (td, 1H, J =
1.5 and 7.8 Hz), 7.55−7.58 (m, 2H). GC/MS m/z 458 (M+, 2), 281
(100). Anal. (C28H34N4O2·2HCl) C, H, N.
2-[2-[4-[2-(4-Methoxyphenyl)phenyl]piperazin-1-yl]ethyl]-
tetrahydro-1H-pyrrolo[1,2-c]imidazole-1,3(2H)-dione (33a). Eluted
with CHCl3/EtOAc, 1:1. Transparent oil, 74% yield. 1H NMR
(CDCl3): δ 1.64−1.74 (m, 1H), 1.98−2.07 (m, 2H), 2.17−2.25 (m,
1H), 2.40 (br s, 4H), 2.49−2.58 (m, 2H), 2.78 (br s, 4H), 3.19−3.24
(m, 1H), 3.53−3.59 (m, 2H), 3.64−3.69 (m, 1H), 3.85 (s, 3H), 4.05 (t,
1H, J = 8.3 Hz), 6.90−6.93 (m, 2H), 7.04 (td, 1H, J = 1.0 and 7.4 Hz),
7.19−7.25 (m, 2H), 7.54−7.56 (m, 2H). 13C NMR (500 MHz,
CDCl3): δ 174.2; 161.1; 158.5; 150.3; 134.7; 133.7; 131.4; 129.9;
127.9; 122.7; 118.3; 63.4; 55.7; 54.9; 53.6; 53.2; 51.1; 45.9; 36.2; 27.8;
27.0. GC/MS m/z 435 (M+ + 1, 3), 434 (M+, 13), 281 (100). Anal.
(C25H30N4O3·HCl·H2O) C, H, N.
2-[3-[4-[2-(4-Methoxyphenyl)phenyl]piperazin-1-yl]propyl]-
tetrahydro-1H-pyrrolo[1,2-c]imidazole-1,3(2H)-dione (33b). Eluted
with CHCl3/EtOAc, 1:1. Brown oil, 43% yield. 1H NMR (CDCl3): δ
1.64−1.71 (m, 2H), 1.72−1.82 (m, 2H), 2.02−2.11 (m, 2H), 2.18−
2.28 (m, 2H), 2.34−2.38 (m, 5H), 2.85 (br s, 4H), 3.19−3.27 (m, 1H),
3.48−3.52 (m, 2H), 3.62−3.70 (m, 2H), 3.85 (s, 3H), 4.02−4.07 (m,
1H), 6.92 (d, 2H, J = 8.88 Hz), 6.99−7.07 (m, 2H), 7.19−7.27 (m,
2H), 7.55 (d, 2H, J = 8.8 Hz). GC/MS m/z 449 (M+ + 1, 20), 448 (M+,
80), 281 (100), 210 (50), 70 (31). Anal. (C26H32N4O3·HCl·H2O) C,
H, N.
3-{3-[4-[2-(4-Methoxyphenyl)phenyl]piperazin-1-yl]propyl}-2-
methyl-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one (11b).
Eluted with CHCl3/EtOAc, 1:1. Brown oil, 60% yield. 1H NMR
(CDCl3): δ 1.51−1.53 (m, 2H), 1.84−1.89 (m, 2H), 1.93−1.98 (m,
2H), 2.27 (s, 3H), 2.45−2.50 (m, 6H), 2.69−2.73 (br t, 2H), 2.85 (t,
2H, J = 6.8 Hz), 2.85 (br s, 4H), 3.85 (s, 3H), 3.91 (t, 2H, J = 6.1 Hz),
6.90−6.93 (m, 2H), 7.03−7.06 (m, 2H), 7.21 (dd, 1H, J = 1.5 and 7.3
Hz), 7.25 (td, 1H, J = 1.5 and 7.8 Hz), 7.55−7.58 (m, 2H). 13C NMR
(500 MHz, CDCl3): δ 162.6; 158.4; 155.6; 134.5; 133.5; 131.3; 129.8;
129.7; 127.9; 122.6; 118.2; 113.5; 58.13; 55.2; 53.2; 50.6; 42.7; 31.4;
24.1; 22.0; 21.21; 19.25. GC/MS m/z 473 (M+ + 1, 2), 472 (M+, 9),
281 (42), 234 (100), 205 (51). Anal. (C29H36N4O2·2HCl·H2O) C, H,
N.
7-[2-[4-[2-(4-Methoxyphenyl)phenyl]piperazin-1-yl]ethoxy]-4-
methyl-2H-chromen-2-one (20a). Eluted with CHCl3/EtOAc, 1:1.
Pale yellow oil, 71% yield. 1H NMR (CDCl3): δ 2.39 (s, 3H), 2.54 (br s,
4H), 2.84 (br t, 2H), 2.91 (br s, 4H), 3.85 (s, 3H), 4.15 (br t, 2H), 6.13
(d, 1H, J = 1.5 Hz), 6.80 (d, 1H, J = 2.5 Hz), 6.86 (dd, 1H, J = 2.5 and
8.8 Hz), 6.92−6.95 (m, 2H), 7.03−7.06 (m, 2H), 7.22 (dd, 1H, J = 1.5
and 7.3 Hz), 7.24−7.27 (m, 1H), 7.48 (d, 1H, J = 8.8 Hz), 7.56−7.59
(m, 2H). ESI-MS m/z 493 (M + Na)+. ESI-MS/MS m/z 493 (89), 295
(100). Anal. (C29H30N2O4·HCl) C, H, N.
6-[3-[4-[2-(4-Methoxyphenyl)phenyl]piperazin-1-yl]propoxy]-4-
methyl-2H-chromen-2-one (20b). Eluted with CH2Cl2/EtOAc, 1:1.
Transparent oil, 15% yield. 1H NMR (CDCl3): δ 1.94−1.99 (m, 2H),
2.39 (br s, 7H), 2.50−2.52 (br t, 2H), 2.86 (br s, 4H), 3.85 (s, 3H),
4.03−4.07 (br t, 2H), 6.12 (s, 1H), 6.80−6.85 (m, 2H), 6.91−6.94 (m,
2H), 7.01−7.27 (m, 2H), 7.20−7.27 (m, 3H), 7.47 (d, 1H, J = 8.2 Hz),
7.56−7.59 (m, 2H). 13C NMR (500 MHz, CDCl3): δ 162.2; 161.5;
158.5; 155.4; 152.7; 134.7; 133.7; 131.5; 130.0; 128.1; 125.6; 122.8;
118.3; 113.7; 112.7; 112.0; 101.6; 66.9; 55.4; 55.1; 53.6; 50.9; 26.6;
18.8. ESI-MS m/z 507 (M+Na)+. ESI-MS/MS m/z 507 (100), 309
(63). Anal. (C30H32N2O4·HCl) C, H, N.
7-[4-[4-[2-(4-Methoxyphenyl)phenyl]piperazin-1-yl]butoxy]-4-
methyl-2H-1-chromen-2-one (20c). Eluted with CHCl3/MeOH,
98:2. Pale yellow oil, 36% yield. 1H NMR (CDCl3): δ 1.65−1.72 (m,
2H), 1.85−1.94 (m, 2H,) 2.38−2.43 (m + d, 9H, J = 1.1 Hz), 2.85 (app
t, 4H), 3.86 (s, 3H), 4.14 (t, 2H, J = 6.0 Hz), 6.17 (d, 1H, J = 1.1 Hz),
6.87−6.95 (m, 3H), 7.00−7.08 (m, 2H), 7.20−7.23 (m, 3H), 7.43 (d,
1H, J = 8.8 Hz), 7.55−7.60 (m, 2H). ESI-MS m/z 497 (M + H)+. ESI-
MS/MS m/z 497 (69), 335 (100). Anal. (C31H34N2O4·HCl·H2O) C,
H, N.
6-[2-[4-[2-(4-Methoxyphenyl)phenyl]piperazin-1-yl]ethoxy]-2-
methyl-2H-benzo[b][1,4]oxazin-3(4H)-one (26a). Eluted with
CHCl3/MeOH, 98:2. Transparent oil, 38% yield. 1H NMR (CDCl3):
δ 1.55 (d, 3H, J = 6.9 Hz), 2.53 (br s, 4H), 2.79−2.81 (m, 2H), 2.89 (br
t, 4 H), 3.86 (s, 3H), 4.04 (app t, 2H), 4.57 (q, 1H, J = 6.9 Hz), 6.36 (d,
1H, J = 2.9 Hz), 6.49 (dd, 1H, J = 2.9 and 8.8 Hz), 6.87 (d, 1H, J = 8.8
Hz), 6.92−6.95 (m, 2H), 7.01−7.07 (m, 2H), 7.21−7.27 (m, 2H),
7.55−7.58 (m, 2H), 8.27 (s, 1H, D2O exchanged). ESI-MS m/z 474 (M
+ H)+. ESI-MS/MS m/z 474 (76), 226 (100). Anal. (C28H31N3O4·
2HCl) C, H, N.
6-[3-[4-[2-(4-Methoxyphenyl)phenyl]piperazin-1-yl]propoxy]-2-
methyl-2H-benzo[b][1,4]oxazin-3(4H)-one (26b). Eluted with
CHCl3/MeOH, 98:2. Transparent oil, 17% yield. 1H NMR (CDCl3):
δ 1.55 (d, 3H, J = 2.5 Hz), 1.92−1.95 (m, 2H), 2.43 (br s, 4H), 2.51
(app t, 2H), 2.88 (br s, 4 H), 3.85 (s, 3H), 3.93 (t, 2H, J = 6.4 Hz), 4.57
(q, 1H, J = 6.9 Hz), 6.34 (d, 1H, J = 2.5 Hz), 6.49 (dd, 1H, J = 2.5 and
8.8 Hz), 6.86 (d, 1H, J = 8.8 Hz), 6.92−6.94 (m, 2H), 7.01−7.07 (m,
2H), 7.21−7.27 (m, 2H), 7.55−7.56 (m, 2H), 8.13 (s, 1H, D2O
exchanged). GC/MS m/z 488 (M+ + 1, 5), 487 (M+, 20), 281 (30), 194
(100), 165 (33), 91 (34). Anal. (C29H33N3O4·2HCl) C, H, N.
General Procedure for the Preparation of Compounds 29
and 30. A mixture of 1-[2-(4-methoxyphenyl)phenyl]piperazine or
1(2-acetylphenyl)piperazine (1.2 mmol) and the oxirane 28 (1.0
mmol) in ethanol (20 mL) was refluxed for 5 h. After it was cooled, the
solvent was removed in vacuo, and the crude residue was chromato-
graphed as detailed below to give desired pure compound.
6-{(2R)-2-Hydroxy-3-[4-[2-(4-methoxyphenyl)phenyl]piperazin-
1-yl]propoxy}-2-methyl-2H-benzo[b][1,4]oxazin-3(4H)-one (29).
Eluted with CHCl3/AcOEt, 1:1. White semisolid, 30% yield. 1H
NMR (CDCl3): δ 1.62 (br s, 1H, D2O exchanged), 1.47 (d, 3H, J = 6.8
Hz), 2.30−2.32 (m, 2H,), 2.39−2.45 (m, 2H), 2.48−2.54 (m, 2H),
2.79−2.83 (m, 4H), 3.78 (s, 3H), 3.82 (d, 2H, J = 4.9 Hz), 3.94−3.97
(m, 1H), 4.49 (q, 1H, J = 6.8 Hz), 6.79 (d, 1H, J = 8.8 Hz), 6.84−6.87
(m, 2H), 6.94 (dd, 1H, J = 1.1 and 8.3 Hz), 6.99 (td, 1H, J = 1.5 and 7.3
Hz), 7.14−7.20 (m, 4H), 7.48−7.51 (m, 2H), 8.35 (br, 1H, D2O
exchanged). 13C NMR (500 MHz, CDCl3): δ 168.0; 167.9; 158.7;
153.4; 147.8; 137.6; 137.5; 134.9; 132.6; 131.5; 129.8; 128.2; 127.5;
124.2; 118.7; 117.2; 114.0; 109.9; 102.6; 73.2; 70.4; 64.6; 61.0; 55.4;
54.7; 53.4; 47.8; 47.7; 16.0. ESI-MS m/z 526 (M+Na)+. ESI-MS/MS
m/z 526 (100), 347 (11). Anal. (C29H33N3O5·HCl) C, H, N.
6-[3-[4-(2-Acetylphenyl)piperazin-1-yl]-(2R)-2-hydroxypropoxy]-
2-methyl-2H-benzo[b][1,4]oxazin-3(4H)-one (30). Eluted with
1
CHCl3/MeOH, 95:5. Brown oil, 54% yield. H NMR (CDCl3): δ
1.50 (d, 3H, J = 6.9 Hz), 1.81 (br s, 1H, D2O exchanged), 2.56−2.64
(m, 4H), 2.65 (s, 3H), 2.78−2.83 (m, 2H), 3.05 (br, 4 H), 3.94−3.96
(m, 2H), 4.09−4.11 (m, 1H), 4.57 (q, 1H, 6.9 Hz), 6.44 (s, 1H), 6.52
(dd, 1H, J = 2.4 and 8.8 Hz), 6.87 (d, 1H, J = 8.8 Hz), 7.05−7.08 (m,
2H), 7.39−7.42 (m, 2H), 8.49 (br s, 1H, D2O exchanged). ESI-MS m/z
462 (M + Na)+. ESI-MS/MS m/z 462 (100). Anal. (C24H29N3O5·
2HCl) C, H, N.
Radioligand Binding Assays. Materials. Cell culture reagents
were purchased from EuroClone (Milan, Italy). G418 (Geneticin), 5-
HT, and NAN-190 were obtained from Sigma-Aldrich (Milano, Italy).
5-CT was purchased from Tocris Bioscience (Bristol, UK). [3H]-5-CT
and [3H]-8-OH-DPAT were obtained from PerkinElmer Life and
Analytical Sciences (Boston, MA, USA). MultiScreen plates with Glass
fiber filters was purchased from Merck Millipore (Billerica, MA, USA).
pcDNA3.1(+) vector containing the target 5-HT1A DNA sequence was
purchased from cDNA Resource Center (Bloomsberg, PA, USA), and
FuGENE HD Transfection Reagent was obtained from Promega
(Madison, Wisconsin, USA).
Cell Culture. HEK-293 cell line was grown in DMEM high glucose
supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/
mL penicillin, and 100 μg/mL streptomycin, in a humidified incubator
at 37 °C with a 5% CO2 atmosphere. HEK-293-5-HT7A and HEK-293-
5-HT1A transfected cell lines were grown in DMEM high glucose
supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/
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mL penicillin, 100 μg/mL streptomycin, and 0.8 μg/mL G418, in a
humidified incubator at 37 °C with a 5% CO2 atmosphere.
= 440 nm diode and 561 nm DPSS laser lines, filters = MBS 445, MBS
458/561, objective = C-Apochromat 40×/1.2W Corr. Corresponding
reference spectra for the online fingerprinting mode were obtained from
separate measurements with a single fluorophore transfection. The
semiautomatic biosensor data analysis relied on custom-written
MATLAB scripts comprising data import, preprocessing, shift
correction in the xy-plane for each time point, the exclusion of
saturated pixels from evaluation, background correction, and faint data
blurring with a kernel size of 0.5 according to Pawley.59 The pixel-based
ratio was calculated for selected regions of interest (ROIs) for
evaluation. Traces were normalized according to their mean ratio
before stimulation.
Functional Assays at 5-HT2A Receptor. The efficacy of
compounds 8c, 20b, and 29 at 5-HT2A receptor was investigated in
assays of inositol phosphate (IP) production in the CHO-K1 cell line
stably expressing the cloned human 5-HT2A receptor employed in
radioligand binding assays. Cellular IP levels were quantified by using
the homogeneous time-resolved fluorescence (HTRF)-based inositol
monophosphate kit IP-One Gq kit (Cisbio, Bioassays, Codolet, France)
following the manufacturer protocol. Cells were seeded in 96-well
plates in culture medium DMEM (Gibco, ThermoFisher Scientific,
Madrid, Spain) supplemented with 10% (v/v) dialyzed fetal bovine
serum (Sigma-Aldrich, Madrid, Spain), 100 U/mL penicillin/0.1 mg/
mL streptomycin (Sigma-Aldrich, Madrid, Spain), and 2 mM L-
glutamine (Sigma-Aldrich, Madrid, Spain)) and maintained during 24 h
at 37 °C in a 5% CO2 humidified atmosphere. Prior to the assay, cell
supernatant was removed and for assessment of possible agonist effect,
and cells were incubated with the compounds (0.1 nM−100 μM) or 5-
HT (0.1 nM−100 μM) as control agonist in stimulation buffer for 20
min at 37 °C. After this time, IP levels were quantified. For assessment
of possible antagonist effect, the compounds (0.1 nM−100 μM) were
added to the cells 10 min prior to the addition of 1 μM 5-HT, and assays
were subsequently carried out as described above. Risperidone (0.1
nM−100 μM) was used as control antagonist in these assays. In all
cases, basal IP levels were determined in control wells in the absence of
compound and agonist. Antagonist concentration−response curves
were fitted to a sigmoidal dose−response (inhibition) model (Hill slope
(nH) = 1, with best fit in comparison to sigmoidal dose−response
(variable slope) model, P < 0.05, extra sum-of-squares F test) using
Prism 6 software (GraphPad, San Diego, CA) to retrieve pIC50 (−log
IC50) values.
Stability Assays in Rat Liver Microsomes. Test compounds were
preincubated at 37 °C with rat liver microsomes (Tebu-Bio, Milan,
Italy) (1.0 mg/mL microsomal protein) at 10 μM final concentration in
100 mM potassium phosphate buffer (pH 7.4) for 10 min. Metabolic
reactions were initiated by the addition of the NADPH regenerating
system (containing 10 mM NADP, 50 mM glucose-6-phosphate, and
10 unit/mL glucose-6-phosphate dehydrogenase, final glucose-6-
phosphate dehydrogenase concentration, 1 unit/mL). Aliquots were
removed at specific time end points and immediately mixed with an
equal volume of cold acetonitrile containing the internal standard. To
assess in vitro in vitro half-life (t1/2) the aliquots were removed at 0, 5,
15, 30, 60, and 120 min. Test compound incubated with microsomes
without NADPH regenerating system was included. Quenched samples
were centrifuged at 4500 rpm for 15 min, and the supernatants were
injected for quantification analysis. Samples (100 μL) were analyzed by
using an Agilent 1260 Infinity Binary LC System equipped with a diode
array detector (Open Lab software was used to analyze the
chromatographic data) and a Phenomenex Gemini C-18 column
(250 mm × 4.6 mm, 5 μm particle size). The samples were eluted using
CH3CN/20 mM ammonium formate pH 5.5 (70:30, v/v) as eluent (1
mL/min). Concentrations were quantified by measuring the area under
the peak. The percentage of the parent compound remaining after a 30
min incubation has been calculated according to the equation
Radioligand Binding at Human Cloned 5-HT7Rs. 5-HT7R binding
was carried out as previously reported.57 The experiment was
performed in MultiScreen plates (Merck Millipore) with Glass fiber
filters (GF/C), presoaked in 0.3% PEI for 20 min. After this time, 130
μg of HEK-293-5-HT7A membranes, 1 nM [3H]-5-CT, and the test
compounds were suspended in 0.25 mL of incubation buffer (50 mM
Tris−HCl, pH 7.4, 4 mM MgCl2, 0.1% ascorbic acid, 10 μM pargyline
hydrochloride). The samples were incubated for 60 min at 37 °C. The
incubation was stopped by rapid filtration, and the filters were washed
with 3 × 0.25 mL of ice-cold buffer (50 mM TRIS-HCl, pH 7.4).
Nonspecific binding was determined in the presence of 10 μM 5-CT.
Approximately 90% of specific binding was determined under these
conditions. Concentrations required to inhibit 50% of radioligand
specific binding (IC50) were determined by using six to nine different
concentrations of the drug studied in two or three experiments with
samples in duplicate. Apparent inhibition constants (Ki) values were
determined by nonlinear curve fitting, using the Prism, version 5.0,
GraphPad software.
Radioligand Binding at Human Cloned 5-HT1AR. 5-HT1AR binding
was carried out as already reported.57 The experiment was performed in
MultiScreen plates (Merck Millipore) with Glass fiber filters (GF/C),
presoaked in 0.3% PEI for 20 min. After this time, 100 μg of HEK-293-
5-HT1A membranes, 1.5 nM [3H]-8-OH-DPAT, and the test
compound were suspended in a 0.25 mL of incubation buffer (50
mM Tris−HCl pH 7.4, 4 mM MgCl2, 0.1% ascorbic acid, 0.1 nM
EDTA, 10 μM pargyline hydrochloride). The samples were incubated
for 60 min at 25 °C. The incubation was stopped by rapid filtration, and
the filters were washed with 3 × 0.25 mL of ice-cold buffer (50 mM
TRIS-HCl, pH 7.4). Nonspecific binding was determined in the
presence of 10 μM NAN-190. Approximately 90% of specific binding
was determined under these conditions. Concentrations required to
inhibit 50% of radioligand specific binding (IC50) were determined by
using six to nine different concentrations of the test compound in two or
three experiments with samples in duplicate. Apparent inhibition
constants (Ki) values were determined by nonlinear curve fitting, using
the Prism, version 5.0, GraphPad software.
Radioligand Binding at Human Cloned Dopamine D2 and
Serotonin 5-HT2A Receptors. The affinity of the compounds for
dopamine D2 and serotonin 5-HT2A receptors was evaluated in
membrane preparations from CHO-K1 cells stably expressing the
human cloned D2S receptor or the human cloned 5-HT2A receptor,
following previously described procedures.58 Competition binding
experiments were performed using [3H]spiperone (0.2 nM; D2
receptor) or [3H]ketanserin (1 nM; 5-HT2AR) as radioligands.
Nonspecific binding was assessed in the presence of 10 μM sulpiride
(D2 receptor) or 1 μM methysergide (5-HT2AR). Haloperidol (D2
receptor) and risperidone (5-HT2AR) were included in the assays as
reference compounds. Competition binding curves constructed with 6
different concentrations of the compounds were fitted to a one-site
competition model using Prism 6 software (GraphPad, San Diego, CA,
USA), and equilibrium dissociation constant (Ki) of the compounds
was calculated according to the Cheng−Prusoff equation.
Analysis of the cAMP Response Using FRET-Based Biosensor
CEPAC. Mouse neuroblastoma N1E115 cells (American Type Culture
Collection, Manassas, USA) were seeded onto 18 mm glass coverslips
and grown in DMEM containing 10% fetal bovine serum and 1%
penicillin/streptomycin at 37 °C in a humidified atmosphere with 5%
CO2. Expression of the cAMP-biosensor CEPAC, 5-HT7R, or 5-HT1AR
was ensured by transfection (plasmid DNA to biosensor and receptor
ratio of 7:3) using Lipofectamine 2000 (Life Technologies). One day
after transfection, changes in cAMP levels upon perfusion with 10 μM
8c, 20b, 29, LP-211, or 5-CT 5-HT7R in Tyrode’s buffer (150 mM
NaCl; 5 mM KCl; 1 mM MgCl2; 2 mM CaCl2; 10 mM HEPES; pH 7.4;
adjusted osmolarity) were monitored in real-time under a Zeiss LSM
780 confocal laser-scanning microscope. A 61 cycle time series with a 10
s interval was recorded in online fingerprinting mode of the ZEN
acquisition software with the following settings: image dimension =
1024 pixels × 1024 pixels, resolution = 0.346 μm × 0.346 μm, excitation
%of parent compound remaining after 30 min
= Cparent/Ccontrol × 100
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where Cparent is ligand concentration after incubation with microsome
fraction and NADPH regenerating system and Ccontrol is ligand
concentration after incubation with microsome fraction only.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
The in vitro half-life (t1/2) was calculated using the expression t1/2
=
0.693/b, where b is the slope found in the linear fit of the natural
logarithm of the fraction remaining of the parent compound vs
incubation time.41 In vitro half-life was then used to calculate the
intrinsic plasma clearance (CLint) according to the following equation:
Funding
The present work was partially supported by Telethon
Foundation Grant GGP13145 (M.L.) and by German Research
Foundation (DFG) (grant number PO732 to E.P. and grant
number ZE994/2 to A.Z.). A.G.S., M.I.L., and M.C. acknowl-
edge support from the Spanish Ministry of Economy and
Competitiveness (MINECO) (grant number SAF2014-57138-
0.693
in vitro t1/2
1
CLint
=
×
mg/mL microsomal protein
Internal positive controls were aripiprazole (Cint = 6.93 mL/mg/min,
t
1/2 = 100 min) and LP-211 (Cint= 45.9 mL/mg/min, t1/2 = 45.9 min).
́
C2−1-R), Xunta de Galicia (Centro singular de investigacion de
Galicia accreditation 2019−2022, grant number ED431G 2019/
02), and the European Union (European Regional Develop-
ment Fund - ERDF). COST Action CA 18133 “European
Research Network on Signal Transduction − ERNEST” is
gratefully acknowledged. Funding for open access charge:
COST Action CA18133 (ERNEST).
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acschemneuro.0c00647.
■
sı
General procedures and spectroscopic data of intermedi-
ates 7a,b, 12, 13, 15, 16, 19a−c, 22, 23, 24, 25a,b, 26b,
28, and 32a,b; formula, molecular weight, and mono-
isotopic mass of the synthesized compounds; elemental
analysis of target compounds; off-target affinities of
selected target compounds; and H NMR spectra of
target compounds 8a−c, 9, 14a,b, 20a−c, 26a−c, 33a,b,
29, and 30 (PDF)
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
1
5-HT, serotonin; 5-HT1AR, serotonin 1A receptor; 5-HT2AR,
serotonin 2A receptor; 5-HT7R, serotonin 7 receptor; 8-OH-
DPAT, 8-hydroxy-2-dipropylaminotetralin; (+)-5-FTP, (+)-5-
(2′-fluorophenyl)-N,N-dimethyl-1,2,3,4-tetrahidronaphthalen-
2-amine; ADME, absorption, distribution, metabolism, and
excretion; ASD, autism spectrum disorder; cAMP, cyclic
adenosine monophosphate; CLint app, apparent intrinsic
clearance; CNS, central nervous system; FRET, fluorescence
resonance energy transfer; SAR, structure−activity relationship;
SSRI, selective serotonin reuptake inhibitor
AUTHOR INFORMATION
Corresponding Authors
■
Enza Lacivita − Dipartimento di Farmacia−Scienze del
̀
Farmaco, Universita degli Studi di Bari Aldo Moro, 70125
Bari, Italy; orcid.org/0000-0003-2443-1174;
Email: enza.lacivita@uniba.it
Marcello Leopoldo − Dipartimento di Farmacia−Scienze del
REFERENCES
■
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Farmaco, Universita degli Studi di Bari Aldo Moro, 70125
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Authors
Mauro Niso − Dipartimento di Farmacia−Scienze del
Farmaco, Universita degli Studi di Bari Aldo Moro, 70125
Bari, Italy
̀
Margherita Mastromarino − Dipartimento di
̀
Farmacia−Scienze del Farmaco, Universita degli Studi di Bari
Aldo Moro, 70125 Bari, Italy
Andrea Garcia Silva − Center for Research in Molecular
Medicine and Chronic Diseases (CIMUS), Universidade de
Santiago de Compostela, 15782 Santiago de Compostela,
Spain
Cibell Resch − Cellular Neurophysiology, Hannover Medical
School, 30625 Hannover, Germany
Andre Zeug − Cellular Neurophysiology, Hannover Medical
School, 30625 Hannover, Germany
María I. Loza − Center for Research in Molecular Medicine and
Chronic Diseases (CIMUS), Universidade de Santiago de
Compostela, 15782 Santiago de Compostela, Spain
Marián Castro − Center for Research in Molecular Medicine and
Chronic Diseases (CIMUS), Universidade de Santiago de
Compostela, 15782 Santiago de Compostela, Spain
Evgeni Ponimaskin − Cellular Neurophysiology, Hannover
Medical School, 30625 Hannover, Germany
Complete contact information is available at:
https://pubs.acs.org/10.1021/acschemneuro.0c00647
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Research Article
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