N-skatyltryptamines-dual 5-ht6r/d2r ligands with antipsychotic and procognitive potential

Article abstract of DOI:10.3390/molecules26154605

A series of N-skatyltryptamines was synthesized and their affinities for serotonin and dopamine receptors were determined. Compounds exhibited activity toward 5-HT_1A, 5-HT_2A, 5-HT₆, and D₂ receptors. Substitution patterns resulting in affinity/activity switches were identified and studied using homology modeling. Chosen hits were screened to determine their metabolism, permeability, hepatotoxicity, and CYP inhibition. Several D₂ receptor antagonists with additional 5-HT₆R antagonist and agonist properties were identified. The former combination resembled known antipsychotic agents, while the latter was particularly interesting due to the fact that it has not been studied before. Selective 5-HT₆R antagonists have been shown previously to produce procognitive and promnesic effects in several rodent models. Administration of 5-HT₆R agonists was more ambiguous-in naive animals, it did not alter memory or produce slight amnesic effects, while in rodent models of memory impairment, they ameliorated the condition just like antagonists. Using the identified hit compounds 15 and 18, we tried to sort out the difference between ligands exhibiting the D₂R antagonist function combined with 5-HT₆R agonism, and mixed D₂/5-HT₆R antagonists in murine models of psychosis.

Full text of DOI:10.3390/molecules26154605

molecules
Article
N-Skatyltryptamines—Dual 5-HT6R/D2R Ligands with
Antipsychotic and Procognitive Potential
Agata Hogendorf 1 , Adam S. Hogendorf 1 , Rafał Kurczab 1 , Grzegorz Satała 1, Bernadeta Szewczyk 2,
Paulina Cies´lik 2 , Gniewomir Latacz 3 , Jadwiga Handzlik 3 , Tomasz Lenda 4 , Katarzyna Kaczorowska 1
,
1
Jakub Staron´ , Ryszard Bugno 1 , Beata Duszyn´ska 1 and Andrzej J. Bojarski 1,
*
1
Department of Medicinal Chemistry, Maj Institute of Pharmacology, Polish Academy of Sciences,
12 Sme˛tna Street, 31-343 Kraków, Poland; agatah@if-pan.krakow.pl (A.H.); ahogen@if-pan.krakow.pl (A.S.H.);
kurczab@if-pan.krakow.pl (R.K.); satala@if-pan.krakow.pl (G.S.); k.kaczor@if-pan.krakow.pl (K.K.);
staron@if-pan.krakow.pl (J.S.); bugno@if-pan.krakow.pl (R.B.); duszyn@if-pan.krakow.pl (B.D.)
Department of Neurobiology, Maj Institute of Pharmacology, Polish Academy of Sciences, 12 Sme˛tna Street,
31-343 Kraków, Poland; szewczyk@if-pan.krakow.pl (B.S.); cieslik@if-pan.krakow.pl (P.C.)
Department of Technology and Biotechnology of Drugs, Jagiellonian University Medical College,
Medyczna 9, 30-688 Kraków, Poland; glatacz@cm-uj.krakow.pl (G.L.); j.handzlik@uj.edu.pl (J.H.)
Department of Neuropsychopharmacology, Maj Institute of Pharmacology, Polish Academy of Sciences,
12 Sme˛tna Street, 31-343 Kraków, Poland; lenda@if-pan.krakow.pl
2
3
4
*
Correspondence: bojarski@if-pan.krakow.pl
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Abstract: A series of N-skatyltryptamines was synthesized and their affinities for serotonin and
ꢀꢁꢂꢃꢄꢅꢆ
dopamine receptors were determined. Compounds exhibited activity toward 5-HT1A, 5-HT2A
5-HT6, and D2 receptors. Substitution patterns resulting in affinity/activity switches were identified
and studied using homology modeling. Chosen hits were screened to determine their metabolism,
permeability, hepatotoxicity, and CYP inhibition. Several D2 receptor antagonists with additional
5-HT6R antagonist and agonist properties were identified. The former combination resembled known
antipsychotic agents, while the latter was particularly interesting due to the fact that it has not been
studied before. Selective 5-HT6R antagonists have been shown previously to produce procognitive
and promnesic effects in several rodent models. Administration of 5-HT6R agonists was more
ambiguous—in naive animals, it did not alter memory or produce slight amnesic effects, while in
rodent models of memory impairment, they ameliorated the condition just like antagonists. Using
the identified hit compounds 15 and 18, we tried to sort out the difference between ligands exhibiting
the D2R antagonist function combined with 5-HT6R agonism, and mixed D2/5-HT6R antagonists in
murine models of psychosis.
,
Citation: Hogendorf, A.;
Hogendorf, A.S.; Kurczab, R.;
Satała, G.; Szewczyk, B.; Cies´lik, P.;
Latacz, G.; Handzlik, J.; Lenda, T.;
Kaczorowska, K.; et al.
N-Skatyltryptamines—Dual
5-HT6R/D2R Ligands with
Antipsychotic and Procognitive
Potential. Molecules 2021, 26, 4605.
https://doi.org/10.3390/
molecules26154605
Academic Editor: Jérôme Leprince
Received: 1 June 2021
Accepted: 26 July 2021
Published: 29 July 2021
Keywords: N-skatyltryptamine; tryptamine; D2/5-HT6R receptor agonist/antagonist; antipsychotic;
precognitive; halogen bond; serotonin dual ligands
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1. Introduction
The 5-HT6 receptor has gained the attention of the pharma industry as a putative
target for novel cognitive enhancers in dementia [1–3] as well as an important co-target
in the design of psychotropic drugs. The high affinity of several typical and atypical
antipsychotics and tricyclic antidepressants for 5-HT6, taken together with its sole CNS dis-
tribution, notably in the hippocampus, nucleus accumbens, prefrontal cortex, and striatum,
has prompted intensive research, which partially revealed the physiological function of the
receptor, in particular, its association with GABAergic and cholinergic transmission [4,5].
5-HT6R blockade has been proposed as a treatment of Alzheimer’s disease (AD)
symptoms, however, the strategy ultimately failed, with no drugs on the market so far
despite intensive clinical trial campaigns led by several companies [6–9]. Given the lack of
efficacy of selective 5-HT6R antagonists in AD, several polypharmacological approaches
have been proposed instead [10–15].
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Molecules 2021, 26, 4605. https://doi.org/10.3390/molecules26154605
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 4605
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Although 5-HT6R blockade on its own as a procognitive treatment in dementia failed,
it is still a matter of debate whether the 5-HT6R component contributes to a beneficial profile
of certain psychotropic drugs [16]. Cognitive decline has been an unresolved symptom of
schizophrenia, which prohibits the patients from a return to work and social functioning.
Co-administration of a 5-HT6 antagonist AVN-211 in patients with schizophrenia stabilized
on antipsychotic treatment has resulted in an improvement in Positive and Negative
Syndrome Scale score over the placebo group [17]. Interestingly, while only antagonists
of 5-HT6R display memory-enhancing properties per se, both agonists and antagonists
reverse the cognitive deficits in rodent models of schizophrenia [18–20].
NMDA antagonist administration is a well-established screening model of cognitive
deficits resembling the symptoms of schizophrenia [21,22]. Typically, the impairment
is induced by phencyclidine (PCP) or dizocilpine (MK-801) [23]. The clinical candidate
LU AE58054, a 5-HT6R antagonist, has been shown to reverse the cognitive impairment
induced by subchronic phencyclidine in NORT in rats [24]. Administration of MK-801
resulted in an increase in locomotion, increased pre-pulse inhibition, stereotypy, deficits in
spatial memory, and impaired novel object recognition [25]. The effects of the administra-
tion of a selective 5-HT6R antagonist and agonist on MK-801 induced memory impairment
in the NOR task have recently been published [20]. It has been shown that, counterintu-
itively, both ameliorated the MK-801 effects in NORT, which could have been related to
the restoration of physiological BDNF levels. Tryptamines represent an important group
of compounds exhibiting various biological activities, especially renowned as serotonin
receptor ligands. A SAR study of benzyl-5-methoxytryptamines, a chemotype that was
investigated due to the structural resemblance to the ultrapotent 5-HT ligands: N-benzyl-
2,5-dimethoxyphenylethylamines, was published in 2015 by Nichols et al. [26]. Compounds
were evaluated as 5-HT2AR ligands, but also screened against several other 5-HT receptors,
revealing their promiscuous nature including affinity for 5-HT6R. Surprisingly, so far, little
effort has been put to explore the chemical space of the N-arylmethyl-arylethylamines
beyond the works that focused on benzyl derivatives of tryptamines and phenylethy-
lamines as 5-HT2AR agonists. In this work, we present a part of our search for non-sulfonyl
5-HT6 receptor ligands within the aforementioned scaffold. Several mixed D2/5-HT6R
ligands exhibiting different functional profiles have been discovered, and their ADMET
properties explored.
While studying the literature, we did not come across any reports of mixed D2
antagonist–5-HT6 agonists in animal models of schizophrenia-related cognitive decline. A
compound with such a functional profile–18 was thus compared with a mixed D2/5-HT6R
antagonist in screening models.
2. Results
2.1. Chemistry
N-skatyltryptamines were synthesized using the procedure shown in Scheme 1. Sev-
eral monosubstituted aldehydes were commercially available, and the others were syn-
thesized via Vilsmeier–Haack formylation starting from appropriate monosubstituted
indoles. The aldehydes were converted into the nitriles in a two-step, one-pot process
involving reduction with NaBH4 and substitution with NaCN, supposedly going through
3-methylidene-3H-indol-1-ium species [27]. It was observed that LiAlH4 reduction of
halogenated indole-3-carbonitriles resulted in partial dehalogenation of indole. Due to
slight differences in the polarity of tryptamine and halotryptamines resulting in poor chro-
matography separation, a milder method was used instead. Indole-3-carbonitriles were
converted into the corresponding primary amines using a mixture of AlCl3 and LiAlH4.
The reductive amination with NaBH4 after imine formation allowed us to obtain the final
products of N-skatyltryptamines in good yields. The spectral data and chromatograms can
be found in Supplementary Materials.

Molecules 2021, 26, 4605
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Scheme 1. a: 1. NaBH4, HCONH2, MeOH 2. NaCN;
d: NaBH4, MeOH.
b: AlCl3, LiAlH4, Et2O;
c: POCl3, DMF;
2.2. Structure–Affinity Relationship
Compound 1, [2-(1H-indol-3-yl)ethyl][(1H-indol-3-yl)methyl]amine, exhibited high affinity
for 5-HT2A and medium for the 5-HT6 receptor (Ki 5-HT2A = 20 nM and Ki 5-HT6 = 172 nM,
Table 1–preferred substitution patterns shown in color). Modification of position 5 of the
tryptamine motif resulted in a decreased 5-HT2AR affinity (entries
was slightly increased. 5-Methoxytryptamine derivative was also bound 5-HT1AR with
considerable potency (Ki = 156 nM). Compound with the chlorine atom in R2 showed a
2–4), while 5-HT6 affinity
3
5
moderate affinity for 5-HT6R and D2R (Ki = 187 nM and 155 nM, respectively). A halogen
atom (F, Cl or Br) introduced in position R3 resulted in compounds selective toward the
5-HT6 receptor (
with a hydroxyl group or a halogen atom (F, Cl, I) gave compounds with low affinity
for 5-HT6R (entries 12). Compound 13 with a fluorine atom in R5 showed low affinity
6: Ki = 168 nM, 7: Ki = 133 nM, 8: Ki = 161 nM). Position R4 substituted
9
–
for 5-HT6R (Ki = 225 nM) and D2R (Ki = 299 nM). Substitution of R5 with a chlorine or
a bromine atom increased the affinity for 5-HT6R (14: Ki = 90 nM, 15: Ki = 102 nM); in
addition, compound 14 was also bound to D2R (Ki = 124 nM). Introducing the halogen atom
in position R6 resulted in compounds that exhibited a high affinity only for the D2 receptor
(
16: Ki = 91 nM, 17: Ki = 51 nM, 18: Ki = 55 nM). Benzyloxy substitution in R6 (19) gave
compounds with weak affinity for 5-HT6R (Ki = 303 nM). Disubstituted compounds were
synthesized combining a 5-methoxytryptamine fragment with broad serotonin receptor
affinity with an aldehyde component bearing a halogen atom that was thought to trigger
D2R antagonism, while not interfering with 5-HT6R binding. The resulting compounds
were thus substituted at both indole fragments with a methoxy at R1 and a chlorine at
either R5 or R6 and exhibited high binding affinity for 5-HT6R (Ki = 98 nM and Ki = 53 nM,
respectively) as for 5-HT1AR (Ki = 79 nM and Ki = 187 nM, respectively), but did not bind
to D2R.
2.3. Functional Assays toward 5-HT6R and D2R
For the compounds with the highest binding affinity for 5-HT6R and D2R, intrinsic
function toward 5-HT6R in agonistic and antagonistic mode and for D2R in antagonistic
mode were evaluated (Table 2). Substitution in position R6 acted as an agonism switch
for 5-HT6R, with compound 18 exhibiting EC50 5-HT6 = 77 nM. Switching from bromine to
chlorine, and substitution with a methoxy group in position 5 of the tryptamine motif in
compound 22 resulted in enhanced potency (EC50 5-HT6 = 26 nM), while the substitution
pattern was R1 = methoxy, and R6 = chlorine comparable potency (21, EC50 5-HT6 = 22 nM).
For the D2 receptor, only the antagonistic function was observed for all the tested
entries. The most efficacious were N-skatyltryptamines with halogens in positions R2 (
EC50 = 104 nM) and R5 (13 and 15, EC50 = 91 nM and EC50 = 109 nM), respectively.
5,

Molecules 2021, 26, 4605
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Table 1. Binding affinities Ki of the synthesized compounds. Substitutions, which resulted in an increased affinity were
marked in the Markush structure with arcs: 5-HT2AR—red, 5-HT6R—green, D2R—blue.
pKi [nM]
ID
R1
R2
R3
R4
R5
R6
5-HT1A
5-HT2A
5-HT6
5-HT7
D2
Trp
5-MeOTrp
5-Cl-Trp
H
MeO
Cl
H
H
H
H
H
H
-
-
-
-
-
-
-
-
-
7.16
9.05
8.80
6.51
7.35
7.50
6.95
7.92
8.42
7.22
6.32
8.80
<5.00
6.18
6.61
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
H
Me
OMe
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
F
H
H
H
H
H
H
H
H
OH
F
H
H
H
H
H
H
H
H
H
H
H
H
F
Cl
Br
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
6.82
7.16
7.81
n.d.
6.48
6.56
6.29
6.22
6.54
7.31
7.17
7.31
6.87
6.97
7.01
6.25
7.55
7.52
7.13
8.10
7.73
6.25
8.70
7.25
8.08
n.d.
6.93
7.42
7.20
7.11
6.71
6.46
6.49
6.63
6.59
7.33
6.39
6.91
6.41
6.40
6.64
7.51
7.76
6.39
7.76
7.89
7.93
7.97
7.78
7.77
7.88
7.79
6.65
7.39
7.29
7.34
7.65
8.05
7.99
7.50
7.44
7.35
7.52
8.01
8.28
7.44
5.54
6.53
7.25
n.d.
5.80
5.99
5.60
5.69
6.29
6.32
6.42
6.29
5.74
5.80
6.33
5.89
6.97
6.88
5.95
7.53
7.09
5.88
7.22
<5.00
6.49
6.61
7.81
6.53
5.93
6.52
6.12
6.07
6.68
6.68
7.52
7.91
7.54
8.04
8.29
8.26
6.39
6.63
6.81
6.30
Cl
Br
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
I
H
H
H
H
H
H
H
H
H
H
Cl
Br
OBn
Cl
H
OMe
OMe
H
Cl
n.d.—not determined.
Table 2. Determined EC50 and Kb values for the selected compounds.
5-HT6 EC50
5-HT6 Kb
D2 Kb
ID
R1
R2
R3
R4
R5
R6
[nM]
1
5
H
H
H
H
H
H
H
OMe
OMe
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
Cl
Br
H
H
H
Cl
H
H
H
H
H
Cl
Br
Cl
H
n.d.
157
n.d.
n.d.
n.d.
n.d.
77
151
136
30
112
131
n.d.
104
91
290
109
215
167
n.d.
n.d.
13
14
15
17
18
21
22
234
10,000
10,000
n.d.
22
26
n.d.–not determined.
2.4. Molecular Modeling
Functional assays showed that small structural changes caused significant differences
in intrisic function, thus the binding mode of compounds 15 and 18 was investigated to find
the plausible molecular recognition site. Structures of compounds were docked to 5-HT6R
homology models built on the β2 adrenergic receptor crystal structure as a template and to

Molecules 2021, 26, 4605
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the D2 receptor (PDB ID: 6CM4). The QPLD was used to obtain ligand–receptor complexes,
since this algorithm describes the anisotropy of the electron density of halogen atoms. In
the acquired complexes, the bromine substituent played different roles in binding to the D2
and 5-HT6 receptors. Analysis of the binding modes showed that in the D2 binding site
(Figure 1A), the bromine can be involved in the weak halogen bonds with D3.32 and Y7.43
side chains only for the 6-Br derivative. Both derivatives showed a similar binding mode
to the pose of the risperidone from the crystal structure of the D2R complex (Figure 1A).
However, the bromine in the 5-HT6R (Figure 1B) did not show any specific interactions,
and it seems that in both derivatives, bromine plays a steric function (fitting to the shape of
the binding site). These observations are in line with the in vitro data.
Figure 1. Illustration of the binding modes of selected compounds in the binding site of D2 and
5-HT6Rs. Comparison of binding modes of compound 15 (green), 18 (cyan) vs. risperidone (magenta)
in D2R (A), and 15 vs. 18 in 5-HT6R (B).
The structure–5-HT6R function relationship could not be analyzed based on the
acquired complexes. The agonist–antagonist switch observed for the 15 and 18 pair could
have been caused by the stabilization of the “flipped” ligand pose (i.e., with the skatyl
fragment pointing down to the intracellular part of the receptor), presumably resulting in
receptor antagonism. This, however, is a hypothesis that would need extensive, additional
studies to confirm.
2.5. In Vitro ADMETox Studies
2.5.1. Permeability Assay
The most common screening method to define compound permeability is the parallel
artificial membrane permeability assay (PAMPA). This test allows one to determine the
compounds’ passive penetration through the bilayer artificial membranes that mimic the
barrier between the intestine wall and blood. Caffeine was used as a reference compound
with high Pe value (Pe = 15.1
×
10−6 cm/s) and norfloxacin as a compound with very poor
14, and 17) exhibited
Pe value (Pe = 0.56 5,
×
10−6 cm/s). All tested chlorine derivatives (
medium permeability coefficients (Pe) compared to caffeine (Table 3). However, compounds
with the bromine substituent, 15 and 18, showed rather low Pe value but still higher than
norfloxacin (Pe = 2.42 × 10−6 cm/s and Pe = 3.90 × 10−6 cm/s for 15 and 18, respectively).
2.5.2. Metabolic Stability
To determine the most probable metabolite structures, compounds 5, 14, 15, 17, and 18
were incubated with MLMs (mouse liver microsomes) for prolonged time and mass spectra
of the obtained mixtures were recorded. In the next step, MetaSite 6.01 software was applied
to visualize the obtained in vitro data. In silico predictions indicated that the most probable
biotransformation for N-skatyltryptamines is hydroxylation, which was in agreement with
the in vitro experiment. The molecular masses and predicted products of metabolism

Molecules 2021, 26, 4605
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are shown in Table 4. Almost all compounds were converted into two hydroxylated
metabolites, except of compound 5, which showed only one hydroxylated metabolite.
Table 3. Permeability coefficients for the tested compounds.
PAMPA
Pe * [10 cm/s] ± SD
Compound
−6
Norfloxacin
Caffeine
0.56 ± 0.13
15.1 ± 0.40
7.09 ± 1.24
8.34 ± 1.33
2.42 ± 0.92
8.42 ± 0.27
3.90 ± 0.56
5
14
15
17
18
* tested in triplicate.
Table 4. Molecular masses of the tested compound and retention times and molecular mass of the metabolite and the most
probable metabolic pathways of the hit compounds.
Molecular Mass of the
Compound
Molecular Mass (m/z)
Retention Time (min)
Metabolic Pathway
Metabolite (m/z)
5
324.25
324.31
4.17
M1 340.26
M1 340.26
M2 340.33
M1 386.15
M2 384.09
M1 340.26
M2 340.26
M1 384.09
M2 386.08
hydroxylation
hydroxylation
hydroxylation
hydroxylation
hydroxylation
hydroxylation
hydroxylation
hydroxylation
hydroxylation
14
4.054.38
15
17
18
370.13
324.25
368.14
4.124.34
3.954.27
4.004.27
The hydroxylation of 5 was predicted to occur in position R5 (the aldehyde component
derived indole fragment). For compounds with the unsubstituted tryptamine part of the
molecule, the addition of hydroxyl group is indicated in the R2 position (Figures 2 and 3). The
remaining structures of predicted metabolites can be found in Supplementary Materials.
Figure 2. (A) Prediction of the sites of metabolism by MetaSite 6.01. Blue circle marked on the
functional group structures indicates the highest biotransformation probability. The fading red color
shows decreased probability of metabolism. (B) The most probable structure of 5 main metabolite.
2.5.3. CYP450 Inhibition
CYP2D6 inhibition is quite common for CNS drugs including antipsychotics such
as sertindole [28]. The influence of compounds
CYP3A4 and CYP2D6 were evaluated (Figure 4). In comparison to ketoconazole, which
was used as the reference inhibitor of CYP3A4, compounds 14 15 17, and 18 only slightly
inhibited the activity of this isoform. In contrast, acted as an inducer of CYP3A4. Regard-
5, 14, 15, 17, and 18 on CYP450 isoforms
,
,
5
ing CYP2D6, all the tested compounds completely inhibited the activity of this isoform,
which may result in potential drug–drug interactions.

Molecules 2021, 26, 4605
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Figure 3.
(A
) Prediction of the sites of metabolism by MetaSite 6.01. Blue circle marked on the
functional group structures indicates the highest biotransformation probability. The fading red color
shows decreased metabolism probability. (B) The most probable structure of 15 main metabolite.
Figure 4. (A) Effect of reference inhibitor ketoconazole and 5, 14, 15, 17, 18 on CYP3A4 activity.
Statistical significance (**** p < 0.0001, * p < 0.05) was analyzed by Graph Pad Prism 8.0.1 software
using one-way ANOVA and Bonferroni’s multiple comparison post-test. The compounds were
examined in triplicate. (B) Effect of reference inhibitor quinidine and 5, 14, 15, 17, and 18 on CYP2D6
activity. Statistical significance (**** p < 0.0001) was analyzed by Graph Pad Prism 8.0.1 software
using one-way ANOVA and Bonferroni’s multiple comparison post-test. The compounds were
examined in triplicate.
2.5.4. Hepatotoxicity
To investigate the hepatotoxicity of the new 5-HT6/D2 ligands, a cell-based assay
using the HepG2 line was conducted. Compounds
showed a slight antiproliferative effect, where the cell viabilities were decreased to up to
~80% of the control (Figure 5). For bromo-derivatives 15 and 18 at 1 M concentration,
a proliferative effect was observed, but it was not statistically significant. All the higher
5, 14, and 17 at 1 µM concentration
µ
concentrations of all compounds (10
pointing to hepatotoxicity.
µM, 50 µM, 100 µM) caused total cell death, clearly
2.6. In Vivo Behavioral Tests
2.6.1. MK-801-Induced Hyperactivity in Mice
Agitation, which is characteristic for schizophrenia-like behavior, can be modeled by
the administration of NMDA antagonist MK-801. The potential antipsychotic activity of
compounds 15 and 18 was thus evaluated in a MK-801-induced hyperactivity model in
mice. The administration of MK-801 (0.35 mg/kg) significantly increased the activity of the
mice compared to the control group (p < 0.05) in all doses. None of the tested compounds
reversed MK-801-elevated activity (Figure 6).

Molecules 2021, 26, 4605
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Figure 5. The effect of tested compounds (
5,
14
,
15
,
17, and 18) on the viability of the HepG2 cell
line. DMSO 1% in cell growth media (vehicle) was used as the control. Reference cytostatic drug
doxorubicin (DX, 1 M) and mitochondrial toxin CCCP were used as positive controls. Statistical
µ
significance (**** p < 0.0001, *** p < 0.001) was analyzed by Graph Pad Prism 8.0.1 software using
one-way ANOVA and Bonferroni’s multiple comparison post-test. The compounds were examined
in quadruplicate.
2.6.2. Novel Object Recognition (NOR) Test
The effect of acute treatment with compounds 15 and 18 on the cognitive function in
the novel object recognition test in mice was checked (Figure 7). Compound 15 reversed
memory impairment induced by MK-801 (0.3 mg/kg) at doses of 0.5 and 1 mg/kg (p < 0.01,
p < 0.0001), but not at 3 mg/kg. Compound 18 reversed memory impairment induced by
MK-801 (0.3 mg/kg) at all tested doses (0.1; 0.5; 1 mg/kg); p < 0.0001, p < 0.0001, p < 0.01.

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Figure 6.
(A
) Effect of compound 15 (0.5, 1, 3 mg/kg) and (
B
)
18 (0.05, 0.1, 0.5, 1, 3 mg/kg on MK-801–
induced hyperactivity in Albino Swiss mice. The test compounds were given 30 min before MK-801
administration, which was given 30 min before the test. Locomotor activity was monitored over a
60 min session immediately following an injection of MK-801. The data are presented as mean
±
SEM,
n = 5–8 mice per group. Data were analyzed with one-way ANOVA and Newman–Keuls post-hoc.
15: F(4, 29) = 5.293, p = 0.0025; 18: F(6,43) = 3.653, p = 0.005; * p < 0.05; ** p < 0.01; *** p < 0.001 vs.
NaCl + NaCl (Veh group).
Figure 7. Novel object recognition test in mice. Effectivity of 15
(A) and 18 (B). Bars represent the
means SEM, n = 5–10. Data were analyzed with one-way ANOVA and Newman–Keuls post-hoc.
±
15: F(4,33) = 18.88, p < 0.0001; 18: F(4,35) = 12.45, p < 0.0001; #### p < 0.0001 vs. Con; ** p < 0.01,
**** p < 0.0001.
2.6.3. Effect of Compound 15 and 18 on Spontaneous Activity of Mice
Compound 15 administered at the doses of 0.5, 1, and 3 mg/kg did not affect the
locomotor activity of mice (Table 5, p > 0.05). Similarly, compound 18 administered at the
doses of 0.05, 0.1, 0.5, 1, and 3 mg/kg did not influence the spontaneous locomotor activity
of mice (p < 0.05).
Mice were placed separately into activity cages for an acclimatization period of 30 min,
then they were injected i.p. with compound 15 (0.5, 1, and 3 mg/kg) or compound 18 (0.05,
0.1, 0.5, 1, and 3 mg/kg, respectively. After a further 30 min, they were injected with saline
(10 mL/kg). From this point on, the ambulation scores were measured for 60 min. The data
are presented as mean
ANOVA and Dunnett’s post-hoc. 15: F(3,16) = 1.225, p = 0.333; 18: F(5,24) = 0.791, p = 0.567.
±
SEM, n = 5 mice per group. Data were analyzed with one-way

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Table 5. Effect of compound 15 and 18 on the spontaneous activity of mice.
Compounds
Doses (mg/kg)
Ambulation Scores ± SEM
NaCl
15
15
-
0.5
1
100.0 ± 11.81
134.9 ± 11.13
124.7 ± 21.75
91.8 ± 24.61
15
3
NaCl
18
18
18
18
-
0.05
0.1
0.5
1
100.0 ± 8.95
113.6 ± 34.00
131.8 ± 19.50
116.7 ± 13.23
85.31 ± 15.86
87.79 ± 20.89
18
3
3. Discussion
There have been reports of potent 5-HT receptor ligands belonging to the class of
N-arylmethyl arylethylamines dating back to 1994 [29]. The exploration of this chemical
was fueled by the discovery of ultrapotent 5-HT2AR agonists with hallucinogenic activity:
N-benzyl[2-(2,5-dimethoxyphenyl) ethyl]amines. Despite relatively very high selectivity,
the so called NBOMe compounds also bound to 5-HT2B, 5-HT2C, 5HT6, and opioid and
histamine receptors [30]. The next important entry was the work of Nichols et al. on
N-benzyl-5-methoxytryptamines, which exhibited high affinity for 5-HT2AR as well as for
5-HT2B and a selectivity over 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, 5-HT3, 5-HT5A, and 5-HT7
receptors. Three entries: 5b, 5i, and 5l showed remarkable affinity for 5-HT6R (25, 27, and
10 nM, respectively).
In the presented work, a series of N-skatyltryptamines were synthesized and their
affinities for 5-HT1A, 5-HT2A, 5-HT6, 5-HT7, and D2 receptors were determined. The study
commenced with the synthesis of
Halogen substitution was applied to the parent compound
1
, which turned out to be a 5-HT2AR partial agonist.
in order to search for positions
1
enabling contacts with halogen bond acceptors within the receptors. Thus, selective D2R
antagonists were discovered, along with mixed 5-HT1AR, 5-HT6R, and D2R antagonists
and dual D2R, 5-HT6R ligands. 5-HT2AR affinity was observed only in the derivatives of
5-methoxytryptamine. The substitution with a halogen atom in position 6 or 7 (R5 and R6,
respectively) of the indole within the skatyl fragment dramatically enhanced D2R affinity,
while switching between the agonistic (with halogen at R6) and antagonistic (with halogen
at R5) function at 5-HT6R. Disubstituted derivatives 21 and 22 acted as potent and selective
5-HT6R agonists. The ADMET study showed that despite expectations, free tryptamine
fragments were not detected after prolonged incubation with mouse liver microsomes, with
hydroxylation being the most prevalent metabolic pathway. The PAMPA model revealed a
rather high passive permeability of the studied compounds. Although useful as molecular
probes, N-skatyltryptamines turned out to be hepatotoxic, thus not suitable to further
development as pharmaceutical drugs. The tested compounds were also strong inhibitors
of CYP2D6 isoforms, indicating possible drug–drug interactions. The pharmacological
profile of compounds 15 and 18 resembled that of atypical antipsychotics (Figure 8).
This pair of isomers differed by 5-HT6R function with 15 being an antagonist and 18
an agonist. It was thus a good opportunity to settle how a combination of D2R antagonist
and 5-HT6R agonist or antagonist differed in behavioral models of psychosis and cognitive
impairment. Contrary to antipsychotic drugs clozapine or haloperidol [32], 15 and 18
did not reverse the locomotor activity elevated by the administration of MK-801. The
exacerbation of hyperactivity at 3 mg/kg of 15 was not statistically significant. In the novel
object recognition test, compound 15 significantly reversed memory impairment after MK-
801 administration at 0.5 and 1 mg/kg, while 18 at all doses. Both compounds did not affect
spontaneous activity of mice (i.e., did not induce sedation). It is likely that at the tested
doses, the 5-HT6R mediated effect was more apparent than the D2R blockade. However, at

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the 0.3–3 mg/kg range, in the behavioral models used, we could not spot the difference
between the 5-HT6R agonist and antagonist, with both producing a procognitive effect.
Figure 8. Atypical antipsychotics and the structures of compounds 15 and 18 from this work.
Binding affinities of antipsychotic drugs and their antagonist function were taken from the work of
Dupuis et al. (2008) [31].
The observed lack of difference in the action of 15 and 18 in the MK-801 impaired
NORT may look bizarre. However, there have been reports of paradoxical actions of gluta-
matergic drugs such as different outcomes in healthy individuals and patients [33,34]. The
observed similar effects of compounds 15 and 18 in NORT could possibly be related to the
restored BDNF levels described by Rychtyk et al. [20]. The authors showed that both WAY-
181187, which is a selective 5-HT6R agonist and SB-742457, a selective antagonist, alleviated
the MK-801-induced inhibition of hippocampal BDNF signaling. There are, however, many
other possible explanations since the mechanism of 5-HT6R ligand cognition enhancement
remains elusive. Another proposed hypothesis postulates that agonists activate 5-TH6
receptors located directly on cholinergic and/or glutamatergic neurons, while antagonists
act probably on 5-HT6 receptors located on GABAergic interneurons [35].
4. Materials and Methods
4.1. Chemistry
Materials. All organic reagents were purchased from Merck and Combi-Blocks and
were used without purification. Solvents and inorganic reagents were acquired from
Chempur. The reaction progress was monitored by TLC on Merck Silica Gel 60 F 254
on aluminum plates. Column chromatography was performed on Merck Silica Gel 60
(0.063–0.200 mm; 70–230 mesh ASTM).
Analytical methods. UPLC/MS analysis was performed on a Waters TQD spectrom-
eter combined with UPLC Acquity H-Class with a PDA eLambda detector. A Waters
Acquity UPLC BEH C18 1.7
0.3 mL/min flow rate, and 1.0
µ
m 2.1
×
50 mm chromatographic column was used at 40 ◦C,
µL injection volume (the samples were dissolved in LCMS

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grade acetonitrile, typically at a concentration of 0.1–1 mg/mL prior to injection). All
mass spectra were recorded under electrospray ionization in positive mode (ESI+) and
chromatograms were recorded with UV detection in the range of 190–300 nm. The gradient
conditions used were: 80% phase A (water +0.1% formic acid) and 20% phase B (acetonitrile
+0.1% formic acid) to 100% phase B (acetonitrile +0.1% formic acid) at 3.0 min, kept for
3.5 min, then to initial conditions until 4.0 min, and kept for an additional 2.0 min. Total
time of analysis was 6.0 min.
1
Purity analysis. H and 13C NMR spectra were recorded on a Bruker Avance III HD
500 NMR spectrometer. All samples were dissolved in DMSO-d6 with TMS as the internal
standard. The spectral data of the compounds refer to their free bases or salts.
All presented compounds were of at least 95% purity as determined by LCMS. Syn-
theses and characterization details for intermediate products and final compounds as well
as the spectral data for all compounds are included in the Supplementary Materials.
Software. Marvin Sketch was used to draw the chemical structures, substructures,
and reactions, Marvin 19.8.0, ChemAxon. Instant JChem was used for structure searching
and chemical database access, Instant JChem 20.20.0, ChemAxon (www.chemaxon.com).
Mnova was used to visualize, process, analyze, and report the 1H and 13C NMR spectra.
Mendeley was used for citations (Mendeley Desktop 1.19.4, www.mendeley.com).
General procedure 1 for the synthesis of substituted N-skatyltryptamines (1–23):
To a solution of appropriate tryptamine (2.85 mmol) in 10 mL of methanol there
was added a substituted aldehyde (3 mmol) in one portion. The formation of imine was
monitored by TLC. After completion of the reaction, NaBH4 (3.3 mmol) was added in small
portions. The mixture was left overnight and then tested by TLC or LCMS. When no imine
was observed, 20 mL of water was added and the product was extracted three times with
20 mL of ethyl acetate or chloroform. The organic phases were combined, washed two
times with 20 mL of water and once with brine, dried over anhydride magnesium sulfate,
and concentrated on a rotavap. The final product was purified via flash chromatography
using ethyl acetate:methanol:triethylamine 9:1:0.03 (v/v/v).
4.2. In Vitro Pharmacology
4.2.1. Radioligand Binding Assay
Cell Culture. HEK293 cells (ATCC) with the stable expression of human serotonin
5-HT1AR, 5-HT6, and 5-HT7bR or dopamine D2LR (obtained using of Lipofectamine 2000,
Invitrogen) or CHO-K1 cells with plasmid containing the sequence coding for the human
serotonin 5-HT2A receptor (PerkinElmer) were maintained at 37 ◦C in a humidified atmo-
sphere with 5% CO2 and were grown in Dulbecco’s modified Eagle’s medium containing
10% dialyzed fetal bovine serum and 500 µg/mL G418 sulfate. For the membrane prepa-
rations, cells were subcultured into 150 cm2 cell culture flasks, grown to 90% confluence,
washed twice with phosphate buffered saline (PBS) prewarmed to 37 ◦C, pelleted by cen-
trifugation (200 g) in PBS containing 0.1 mM EDTA and 1 mM dithiothreitol, and stored
at −80 ◦C.
General. To determine affinity of all synthesized compounds for following receptors:
5-HT1A, 5-HT2A, 5-HT6, 5-HT7, and D2 radioligand binding assays were conducted. The
assays were performed via the displacement of the respective radioligands from the cloned
human receptors, all stably expressed in HEK-293 cells (except for 5-HT2A, which was
expressed in CHO cells). The experiments were conducted using 1.5 nM [3H]-8-OH-DPAT
(135.2 Ci/mmol) for 5-HT1AR, 2 nM [3H]-ketanserin (53.4 Ci/ mmol) for 5-HT2AR, 2 nM
[3H]-LSD (83.6 Ci/mmol) for 5-HT6R, 0.6 nM [3H]-5-CT (39.2 Ci/mmol) for 5-HT7R, and
[3H]-raclopride (74.4 Ci/mmol) for D2R (PerkinElmer, USA). Non-specific binding was
defined using 10 mM of 5-HT in the 5-HT1AR and 5-HT7R binding experiments, whereas
20 mM of mianserin, 10 mM of methiothepine, or 1 mM of (+)-butaclamol was used in the
5-HT2AR, 5-HT6R, and D2LR assays, respectively. Each compound was tested in triplicate
at 7–8 concentrations (10−11–10−4 M). The inhibition constants (Ki) were calculated using

Molecules 2021, 26, 4605
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the Cheng–Prusoff equation [36] and the results were expressed as the means of at least
two independent experiments.
4.2.2. D2R Functional Assay
HEK293 cell line with stable expression of human D2 (prepared with the use of
Lipofectamine 2000) was maintained at 37 ◦C in a humidified atmosphere with 5% CO2
and was grown in Dulbecco’s modified Eagle medium containing 10% dialyzed fetal bovine
serum and 500 µg/mL G418 sulfate. For functional experiments, cells were subcultured in
◦
25 cm2 flasks, grown to 90% confluence, washed twice with prewarmed to 37 C phosphate
buffered saline (PBS), and centrifuged for 5 min (160× g). The supernatant was aspirated,
and the cell pellet was resuspended in stimulation buffer (1
IBMX, 0.1% BSA).
×
HBSS, 5 mM HEPES, 0.5 mM
The functional properties of compounds were evaluated using the LANCE Ultra
cAMP Detection Kit (PerkinElmer). D2 receptors in HEK293 cells are coupled to Gi subtype
and decreased cAMP production. Cells were stimulated with 1
Each compound was tested in triplicate at eight concentrations (10−11–10−4 M).
For quantification of cAMP levels, cells (5 L) were incubated with 5 L mixture
µM of forskolin (EC90).
µ
µ
of compounds (tested ligand and forskolin with 100 nM quinpirole for antagonist bind-
ing mode) for 30 min at room temperature in 384-well white opaque microtiter plate
(PerkinElmer). After incubation, the reaction was stopped and cells were lysed by the
addition of 10 µL working solution (5 µL Eu-cAMP and 5 µL ULight-anti-cAMP). The assay
plate was incubated for 1 h at room temperature. Time-resolved fluorescence resonance
energy transfer (TR-FRET) signal was detected by an Infinite M1000 Pro (Tecan) using
instrument settings from the LANCE Ultra cAMP Detection Kit manual.
4.2.3. 5-HT6R Functional Assays
The properties of compounds to inhibit cAMP production induced by a 5-HT6R agonist
5-CT (1000 nM) was evaluated. Compounds were tested in triplicate at eight concentrations
(10−11–10−4 M). The level of cAMP was measured using frozen recombinant 1321N1
cells expressing the Human Serotonin 5-HT6R (PerkinElmer). Total cAMP was measured
using the LANCE cAMP Detection Kit (PerkinElmer), according to the manufacturer’s
instructions. For quantification of cAMP levels, 2000 cells/well (5 mL) were incubated with
a mixture of compounds (5 mL) for 30 min at room temperature in a 384-well white opaque
microtiter plate. After incubation, the reaction was stopped and cells were lysed by the
addition of 10 mL of working solution (5 mL Eu-cAMP and 5 mL ULight-anti-cAMP) for
1 h at room temperature. Time-resolved fluorescence resonance energy transfer (TR-FRET)
was detected by an Infinite M1000 Pro (Tecan) using instrument settings from the LANCE
cAMP Detection Kit manual. Kb values were calculated from the Cheng–Prusoff equation
specific for the analysis of functional inhibition curves: Kb = IC50/(1 + A/EC50) where A
represents the agonist concentration; IC50 is the concentration of antagonist producing a
50% reduction in the response to agonist; and EC50 is the agonist concentration that causes
half of the maximal response. The agonistic properties of compounds were determined
with an analogous procedure, but without stimulation with 5-CT [10,36].
4.3. Molecular Modeling
Molecular Docking. The 5-HT6R homology models built on the β2 receptor template
(PDB ID: 4LDE) were used in this study [37]. The structure of D2R in complex with
antagonist risperidone (PDB code6CM4) was retrieved from the Protein Data Bank.
The three-dimensional structures of the ligands were obtained using LigPrep and the
appropriate ionization states at pH = 7.4
±
1.0 were assigned using Epik. The Protein
Preparation Wizard was used to assign the bond orders and appropriate amino acid
ionization states and to check for steric clashes. The receptor grid was generated (OPLS3
force field) by centering the grid box with a size of 12 Å on the D3.32 side chain. Docking
was performed by the quantum-polarized ligand docking (QPLD) procedure implemented

Molecules 2021, 26, 4605
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in the Schrodinger Suite. QPLD involves the QM-derived ligand atomic charges in the
protein environment at the B3PW91 level with conjunction with the ccpVTZ basis set for Cl
and Br and the cc-pVTZ-pp basis set for I-containing ligands. Only the best ten poses per
ligand returned by the procedure were considered.
4.4. In Vitro ADMETox Studies
Materials. The following compounds: caffeine, carbonyl cyanide 3-chloropheny-
lhydrazone, doxorubicin, ketoconazole, norfloxacin and quinidine were used in this study
as the references and purchased from Sigma-Aldrich (St. Louis, MO, USA).
4.4.1. PAMPA
The permeability of compounds was determined by the Pre-coated PAMPA Plate
System Gentest
™
(Corning, Tewksbury, MA, USA) similarly to a previously described
methodology [38
,39]. Caffeine (well-permeable reference), norfloxacin (low-permeable
reference), and tested compounds were dissolved in PBS buffer (pH = 7.4) and added
to the donor wells (300 L/well) in a final concentration of 200 M. PBS in a portion of
200 L/well was added to the wells. Experiments were conducted in triplicate. Incubation
of the PAMPA Plate System took 5 h at room temperature. After that time, 50 L was
aspirated from each well and diluted with 50 L of solution of internal standard (IS).
µ
µ
µ
µ
µ
UPLCMS analyses allowed us to estimate the compounds’ concentration in the acceptor
and donor wells. The permeability coefficients were determined according to formulas
provided by the manufacturer [40].
4.4.2. Metabolic Stability
The murine liver microsomes (MLMs) were purchased from Sigma-Aldrich (St. Louis,
MO, USA). To determine metabolic pathways, the tested compounds (50
µ
M) were in-
◦
cubated with 100 mM Tris-HCl buffer at 37 C with MLMs (1 mg/mL) and NADPH
Regeneration System for 2 h. Reactions were quenched with cold, pure methanol and mix-
tures were centrifuged 14,000 g for 15 min. The supernatants were analyzed using LCMS.
The additional MS ion fragmentation of the product and the substrate were performed to
determine the most probable structure of the metabolite.
In silico prediction of metabolic biotransformations was performed by MetaSite 6.0.1
(Molecular Discovery Ltd., Hertfordshire, UK) [41]. The most probable metabolic sites of
the tested compounds were determined by a computational liver model of metabolism.
4.4.3. CYP450 Inhibition
The experiments were provided using commercially available luminescent CYP3A4
P450-Glo
The enzymatic reactions were conducted in polystyrene, flat-bottom Nunc
96-well microplates (Thermo Scientific, Waltham, MA, USA). The assays were carried out
according to the procedures provided by the manufacturer, as described previously [38 41 42].
Compounds were tested in triplicate at the final concentrations in a range from 0.01 to
25 M for both isoforms of CYP450. The references of CYP3A4 and CYP2D6 inhibitors
(ketoconazole and quinidine, respectively) were tested in a range from 0.001 to 10 M.
™
and CYP2D6 P450-Glo
™
tests purchased from Promega (Madison, WI, USA).
™
MicroWell
™
,
,
µ
µ
Tested compounds were incubated in 100 mM Tris-HCl buffer separately with CYP3A4 and
CYP2D6 membranes and the NADPH Regeneration System for 30 min at room temperature
in triplicate. The bioluminescent signal was measured after the addition of the Luciferin
Detection Reagent by using a microplate reader EnSpire PerkinElmer (Waltham, MA,
USA). Both reagents (NADPH Regeneration System and Luciferin Detection Reagent) were
purchased from Promega (Madison, WI, USA).
4.4.4. Hepatotoxicity Assay
To estimate the hepatotoxicity of compounds, the hepatoma HepG2 (ATCC® HB-
8065TM) cell line was used according to previously described protocols [39,42]. The

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CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay was obtained from
Promega (Madison, WI, USA). The compounds were tested in quadruplicate at four con-
centrations (1–100 µM) for 72 h.
4.5. Behavioral Tests
Animals. Male CD1 mice (Charles River, Germany) weighing 20–25 g at the time of
arrival were used in behavioral experiments. Animals were kept under standard laboratory
conditions (12:12 light: dark cycle, 22
±
2 ◦C) with free access to food and water. Animal
welfare has been regularly controlled by a veterinarian and animal welfare committee.
After two weeks of acclimatization and handling, the experiments began. Experimental
groups consisted of four to 10 animals, depending on the procedure. Drugs were ad-
ministered intraperitoneally (i.p.) at a volume of 10 mL/kg. Experimental assessments
were performed by an observer who was blinded to the treatment conditions. All proce-
dures were conducted in accordance with the European Communities Council Directive
of 22 September 2010 (2010/63/EU) and Polish legislation acts concerning animal exper-
imentation and were approved by the II Local Ethics Committee by the Maj Institute of
Pharmacology, Polish Academy of Sciences in Krakow (272/2019).
Drugs. MK-801 was purchased from Tocris Bioscience, Bristol, UK. MK-801 was dis-
solved in 0.9% NaCl. Compounds 15 and 18 were dissolved in 0.9% NaCl. All compounds
were administered intraperitoneally (i.p.) in a volume of 10 mL/kg. Vehicle-treated animals
received appropriate solvents. Vehicle was administered to animals in any case when drug
administration was omitted (e.g., control or MK-801-treated groups).
4.5.1. Novel Object Recognition Test
This procedure was adapted from Nilsson et al. (2007) [43] and performed as described
in a previous paper by Cieslik et al. (2018) [44]. Habituation, training, and test trials were
performed in a black plastic rectangular arena (40
×
30
×
35 cm) illuminated with a light
intensity of 335 lux. During the habituation trial (two consecutive days), each animal
was allowed to explore the arena for 10 min. The next day, during the training trial (T1),
mice were placed in the arena and presented with two identical objects (red glass cylinder;
6.5 cm in diameter and 4.5 cm high) for 5 min. After 1 h, animals were placed back into
the arena for a 5 min test trial, during which one of the previously presented familiar
objects was replaced with a novel object (a transparent glass elongated sphere-like object
with an orange cap; 5.5 cm in diameter and 8.5 cm high). Time spent exploring (i.e.,
sniffing or touching) the familiar (Tfamiliar) and novel (Tnovel) objects was measured by a
trained observer, and the recognition index [%] was calculated for each mouse [(Tnovel
−
Tfamiliar)/(Tfamiliar + Tnovel)] 100. Compounds were administered 30 min before MK-801
×
(0.3 mg/kg), which was administered 30 min before training trial.
4.5.2. MK-801-Induced Hyperactivity
The locomotor activity was recorded individually for each animal in locomotor activity
cages (according to Rorick-Kehn et al., 2007a,b) [45 46], with modifications (Wieronska et al.,
2012) [47]. The mice were placed individually into activity cages (13 23 15 cm; Opto-
,
×
×
M3; Columbus Instruments) for an acclimatization period of 30 min; then they were injected
i.p. with compound 15 (0.5, 1, 3 mg/kg) or compound 18 (0.05, 0.1, 0.5, 1, 3 mg/kg) and
placed again in the same cages. After 30 min, all of the mice were injected i.p. with MK-801
at 0.35 mg/kg and once again placed in the same cage. From then on, the ambulation
scores were counted for 60 min. All of the groups were compared with the MK-801 control
group. The experiment also included a control group treated with NaCl only.
4.5.3. Locomotor Activity of Mice
The locomotor activity was recorded individually for each animal in the OPTO-M3
locomotor activity cages described above. Each cage was surrounded with an array of
photocell beams. Interruptions of photo beams resulted in horizontal activity defined as

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ambulation scores. Mice were placed separately into activity cages for an acclimatization
period of 30 min, then were injected i.p. with compound 15 (0.5, 1, 3 mg/kg) compound 18
(0.05, 0.1, 0.5, 1, 3 mg/kg. After a further 30 min, they were injected with saline (10 mL/kg).
From this point on, the ambulation scores were measured for 60 min.
Data Analysis. The data are presented as the means
±
SEM. Statistical analysis of
the data was performed using Prism 8. One-way ANOVA, followed by the Newman–
Keuls post-hoc comparison test, was used in the analysis of the dose-dependent studies of
compounds 15 and 18. A p-value of < 0.05 was considered as statistically significant.
5. Conclusions
A further optimization of substituents might yield compounds with enhanced bind-
ing/function for the discussed receptors and more optimal profile. On the other hand,
the therapeutic potential of N-skatyltryptamines seems doubtful due to the discovered
hepatotoxicity. A clogP = 3.80 (ChemAxon) was calculated for the unsubstituted derivative
1, placing the lipophilicity at the border of the preferred range. The discovered series may
serve as a pool of new tool compounds with useful receptor profiles for CNS studies.
Supplementary Materials: The following are available online: Syntheses and characterization details
for final products, 1H and 13C NMR spectra, LC-MS spectra, conditions of radioligand binding assay,
predicted metabolites, and retention times of tested compounds and its metabolites.
Author Contributions: A.H., A.S.H., B.D. and A.J.B. designed the research. A.H., A.S.H., J.S., R.B.
and K.K. synthesized, purified, and characterized the library of compounds. G.S. and T.L. performed
binding and functional assays. B.S. and P.C. performed the in vivo experiments. G.L. and J.H.
conducted the ADMET experiments. R.K. was responsible for the in silico experiments. A.H., A.S.H.,
B.D. and A.J.B. contributed to the writing, review, and revision of the manuscript. All authors have
read and agreed to the published version of the manuscript.
Funding: This study was supported by the National Science Center within grants SONATA 2014/15/
D/NZ7/01782, OPUS13 2017/25/B/NZ7/02929 and by statutory funds from the Maj Institute of
Pharmacology Polish Academy of Sciences.
Conflicts of Interest: The authors declare that they have no known competing financial interests or
personal relationships that could have appeared to influence the work reported in this paper.
Sample Availability: Samples of compounds 1–22 are available from the authors.
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