Search for a 5-CT alternative. In vitro and in vivo evaluation of novel pharmacological tools: 3-(1-alkyl-1H-imidazol-5-yl)-1H-indole-5-carboxamides, low-basicity 5-HT7 receptor agonists

Article abstract of DOI:10.1039/c8md00313k

Close structural analogues of 5-carboxamidotryptamine (5-CT) based on the newly discovered indole-imidazole scaffold were synthesized and evaluated to search for a 5-HT₇ receptor agonist of higher selectivity. In vitro drug-likeness studies and in vivo pharmacological evaluation of potent and selective low-basicity 5-HT₇ receptor agonists, previously published 7 (3-(1-ethyl-1H-imidazol-5-yl)-1H-indole-5-carboxamide, AH-494) and 13 (3-(1-methyl-1H-imidazol-5-yl)-1H-indole-5-carboxamide), have supported their usefulness as pharmacological tools. Comprehensive in vitro comparison studies between 7, 13 and the commonly used 5-CT showed their very similar ADMET properties. Compound 7 at 1 mg kg^?1 reversed MK-801-induced disruption in novel object recognition in mice and alleviated stress-induced hyperthermia (SIH) at high doses. Taking into account both in vitro and in vivo data, 7 and 13 may be considered as alternatives to 5-CT as pharmacological tools with important additional benefit associated with their low-basicity: high selectivity over 5-HT_1AR.

Full text of DOI:10.1039/c8md00313k

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Hogendorf, A. Hogendorf, A. Lubelska, P. Cielik, M. Woniak, J. Wieroska, K. Kononowicz, J. Handzlik and
A. Bojarski, Med. Chem. Commun., 2018, DOI: 10.1039/C8MD00313K.
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RESEARCH ARTICLE
In search for a 5-CT alternative. In vitro and in vivo evaluation of
novel pharmacological tools: 3-(1-alkyl-1H-imidazol-5-yl)-1H-
Received 00th January 20xx,
Accepted 00th January 20xx
indole-5-carboxamides, low-basicity 5-HT receptor agonists
7
‡
a
‡,b
b
a
Gniewomir Latacz, * Adam S. Hogendorf, Agata Hogendorf, Annamaria Lubelska, Joanna M.
Wierońska, Monika Woźniak, Paulina Cieślik, Katarzyna Kieć-Kononowicz, Jadwiga Handzlik,
b
b†
b
a
a
DOI: 10.1039/x0xx00000x
b
Andrzej J. Bojarski
www.rsc.org/
Close structural analogues of 5-carboxamidotryptamine (5-CT) based on the newly discovered indole-imidazole scaffold
were synthesized and evaluated to search for a 5-HT receptor agonist of higher selectivity. In vitro drug-likeness studies
and in vivo pharmacological evaluation of potent and selective low-basicity 5-HT receptor agonists, previously published 7
3-(1-ethyl-1H-imidazol-5-yl)-1H-indole-5-carboxamide, AH-494) and 13 (3-(1-methyl-1H-imidazol-5-yl)-1H-indole-5-
7
7
(
carboxamide), have supported their usefulness as pharmacological tools. Comprehensive in vitro comparison studies
between 7, 13 and the commonly used 5-CT showed their very similar ADMET properties. Compound 7 at 1 mg/kg
reversed MK-801-induced disruption in novel object recognition in mice and alleviated stress-induced hyperthermia (SIH)
at high doses. Taking into account both in vitro and in vivo data, 7 and 13 may be considered as alternatives to 5-CT as
pharmacological tools with important additional benefit associated with their low–basicity: high selectivity over 5-HT1AR.
stimulation in adolescent rats have indicated plastic
rearrangements within forebrain networks, accounting for long-
Introduction
1
0
7
lasting behavioral changes. Pharmacological blockade of 5-HT R
The 5-HT
7 7
receptor (5-HT R), which was discovered independently
produced strong antidepressant-like response more rapidly than
by three teams in 1993, is the most recent one to be identified in
1-6
the classic antidepressant fluoxetine.
1
-6
the human serotonin system. To date, extensive studies on 5-
HT R have indicated its role in CNS development, circadian rhythm,
Great efforts have been exerted by several research groups to
7
develop new therapeutics and better molecular probes acting as
memory and learning, thermoregulation, locomotion, hormonal
regulation, hippocampus activity and mood control, as well as the
6
selective 5-HT
7
R ligands. A few 5-HT
7
R agonists have been used to
date as tool compounds (Table 1), including the long-chain
1
-6
pathophysiology of migraine, pain, depression and epilepsy.
Unfortunately, despite its possible therapeutic potential, no
selective 5-HT R drug has yet been introduced to the market.
Interestingly, the results obtained from animal models support the
possible therapeutic applications of both 5-HT R agonists and
diphenylpiperazine, e.g., LP-211 (1), and its low-weight metabolite
RA-7 (2), as well as several small molecules: pyrazoles AS-19 (3),
11
12
13
14
7
E-55888 (4) and 5-carboxamidotryptamine (5-CT) (5) (Fig. 1).
15
However, due to their poor selectivity (RA-7, 5-CT), low metabolic
stability (LP-211)
response of intracellular signaling cascades (AS-19) and low BBB
permeability (5-CT and to some extent LP-211), there is still a
16, 17
7
, failure to induce a full functional agonistic
1
antagonists. For instance, the symptoms observed in an animal
model caused by brain molecular alterations specific for Rett
18
11
7
syndrome (RTT) were relieved after seven days of 5-HT R
great demand for a “perfect” selective 5-HT
serve not only as a 5-HT R radioligand but also as a positron
emission tomography (PET) radiotracer.
serotonin 5-HT R in brain in vivo would allow direct assessments of
the involvement of this receptor in neuropsychiatric diseases and
7
agonist, which could
7
,8
7
stimulation. The selective activation of 5-HT R was also found to
7
be beneficial in an animal model of fragile X syndrome (FXS), which
15-17, 19
The ability to image
9
is one of the genetic causes of intellectual disability and autism.
7
7
Investigations examining the long-term consequences of 5-HT R
1
9
possible therapies.
The recently reported 5-(1H-indole-3-yl)-1-alkylimidazoles
represent the first examples of low-basicity scaffolds exhibiting high
2
0
affinity and selectivity for 5-HT
The hit compound AGH-107 (6) (Fig. 2) exhibited high affinity for
and intrinsic activity at the receptor (K = 6 nM, EC50 = 19 nM,
respectively). Compound 6 was also very selective over 5-HT5AR,
which is not a common feature of 5-HT R agonists (Table 1).
7
R together with agonist function.
i
7
Moreover, in drug-likeness studies in vitro, 6 was shown to be
metabolically stable after incubation with human liver microsomes
(
Clint = 6.3 ml/min/kg) and exhibited very low toxicity in human
embryonic kidney HEK-293 and hepatoma HepG2 cell lines and
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excellent water solubility. A PK experiment showed a high blood-
brain barrier permeation value of 6, consistent with its high activity
in vivo, where all three tested i.p. doses (0.5, 1 and 5 mg/kg)
reversed MK-801-induced novel object recognition (NOR)
2
0
impairment in mice. The promising results highlighted the
possible use of labeled 6 as a potential radioligand for both in vitro
and in vivo studies.
Scheme 1 Compounds 11, 12, 14 and 15 were synthesized via van
Leusen multi-component reaction, while 7 and 13 were obtained by
a controlled hydrolysis of 11 and 12, respectively.
In vitro
Radioligand displacement assays of compounds 7 and 13-15
showed, that only compounds with a carbonyl group (either ester
th
or amide) at the 5 position of indole were found to possess affinity
for the 5-HT
binding to 5-HT
slightly less potent than 7 (K
7
R (Table 1). Compound 15 exhibited only medium
R (K = 161 nM), while compound 13 was found only
= 6 and 5 nM, respectively).
7
Fig. 1 The chemical structures of 5-HT R agonists used in
7
i
neurobiological research.
i
Compounds 7 and 13 showed selectivity over 5-HT1AR in opposition
to 5-CT (25-fold and 37-fold vs no selectivity at all).
The in vitro drug-likeness studies of 7, 13 and the reference 5-
HT R molecular probe 5 included the assessment of ADME-Tox
7
parameters by using recombinant protein and enzymes (Pgp,
CYP3A4, CYP2D6), human (HLMs) rat (RLMs) and mouse (MLMs)
liver microsomes, human embryonic kidney (HEK-293),
neuroblastoma SH-SY5Y and hepatoma HepG2 eukaryotic cell
cultures and the bacterial strain Salmonella typhimurium. The
artificial membranes (PAMPA) were used for determination of
passive membrane transport of 5 and 7. The assay protocols and
Fig. 2 The chemical structures of representative 5-(1H-indole-3-yl)-
1
1
-alkylimidazoles – the structurally novel molecular probes: AGH-
07, AGH-282 and AH-494.
During this study, our attention was focused on other potent
22, 23
culture growth conditions were applied as described previously.
compounds from that series: the 1H-indole-5-carboxamides AH-494
7) (K = 5 nM, EC50 = 45 nM) and the newly obtained derivative
AGH-282 (13) (K = 6 nM) (Fig. 2), which are close structural
analogues of 5-carboxamidotryptamine (5) (Fig. 1), a molecular
The in vivo activity of CNS drugs depends on their penetration
through biological membranes. The parallel artificial membrane
(
i
24
i
permeability assay (PAMPA) is commonly used in the early
discovery and development process for the determination of
compound passive transport. The Corning® PAMPA Plate System
Gentest™ was used to determine the potential permeability of 5
and 7. The method has been shown to provide good predictability
of the membrane permeation and a high degree of correlation to
1, 21
probe commonly used in serotonergic system-related research.
Results and discussion
2
5
the data obtained from absorption in the Caco-2 cell line. The
Chemistry
–
6
calculated permeability coefficient (Pe) for 5 (Pe = 3.54 ꢀ 10
Aldehydes 8-10 were synthesized from the corresponding indoles
via Vilsmeier-Haack formylation (Scheme 1). Compounds 11 and 12
which were the intermediates for the synthesis of 7 (AGH-494) and
–6
cm/s) was slightly higher than for 7 (Pe = 2.78 ꢀ 10 cm/s) (Table
). The results were compared to the references – highly permeable
caffeine (Pe = 3.61 ꢀ 10 cm/s) and the low-permeable drug
2
–6
1
3, respectively, as well as final compounds 14 and 15 were
–6
norfloxacin (Pe = 0.95 ꢀ 10 cm/s) and indicated very good passive
synthesized via van Leusen 3-component imidazole synthesis using
transport for both 5 and 7 (Table 2).
2
0
the previously described protocol. Compounds 7 and 13 were
synthesized by a controlled hydrolysis of 11 and 12, respectively.
The potential CNS bioavailability of 5, 7 and 13 was also tested
with respect to their influence on glycoprotein P (Pgp), the main
efflux pump in both intestinal epithelium and the blood–brain
barrier (BBB). The luminescence Pgp-Glo™ Assay System
(
Promega®) based on measurements of the changes in ATP
2
6
consumed by Pgp was used. The difference in the luminescent
signal between samples treated with 100 µM of the selective Pgp
3 4
inhibitor Na VO (100% inhibition observed) and untreated samples
represented the basal Pgp ATPase activity, which was considered
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herein as a negative control. The results were juxtaposed as the % same molecule (see Fig. S4C, S7B and S10B in supplementary data).
of Pgp basal activity compared to the reference inhibitor (Na VO However, as mentioned above, the molecular mass of this
3
4
)
and stimulator (verapamil, VL) (Fig. 3). Compounds 5 and 7 showed metabolite was not determined. The UPLC-MS spectra obtained
comparable, statistically significant inhibition effects on Pgp, with a from the reaction mixtures after incubation of 7 with RLMs and
decrease in ATP consumption to ~26 and ~19% of the basal activity, MLMs revealed the presence of the same metabolite with t ~1.60
R
respectively (Fig. 3, Table 2). Despite the potentially enhanced risk min. and a molecular mass m/z of ~271 in both models (see Fig.
of drug-drug interactions (DDIs), Pgp inhibition of CNS targeting S6B, S8C, S9B and S11C in supplementary data). The molecular mass
compound 7 could exert a desirable effect by reducing its efflux of the compound
7 metabolite (+16 units) suggested that
from the BBB. No statistically significant influence of 13 on Pgp was hydroxylation had occurred, presumably at the N-ethyl substituent,
also determined.
according to the fragmentation analysis and in silico data (see Fig.
S1-S2 and S12 in supplementary data). Moreover, 7 seemed to be
slightly more stable in RLMs than in MLMs with 7% and 14%
conversion, respectively (see Fig. S6B and S9B in supplementary
data) (Table 2). The results obtained for 13 after incubation with
RLMs were similar to that for HMLs, as no metabolites were found
(
see Fig. S3C and S6C in supplementary data) (Table 2). The UPLC-
MS spectra obtained from the reaction mixture after incubation of
3 with MLMs revealed the presence of one metabolite with
1
molecular mass m/z of ~274 and with 9% conversion of 13 (see Fig.
S9C and S13 in supplementary data). According to the
fragmentation analysis and in silico data, the most probable
metabolic pathway of 13 was a double hydroxylation (see Fig. S1-S2
and S14 in supplementary data).
The cytochrome P450 isoforms 3A4 (CYP3A4),2D6 (CYP2D6) and
2
6
a
C9 (CYP2C9) are responsible for the metabolism of approximately
Fig. 3 The effect of the Pgp stimulator verapamil (VL) (200 µM), Pgp
0−70% of all marketed drugs; the influence on CYP acꢀvity may be
inhibitor Na
3
VO
4
(100 µM) and compounds 5, 7 and 13 on Pgp basal
29
source of potential dangerous DDI.
Luminescence-based
activity (Basal). Data are presented as the mean ± SD. Statistical
significance was evaluated by one-way ANOVA, followed by
Bonferroni’s comparison test (∗∗p < 0.01, ∗∗∗p < 0.001 compared
with the negative control).
TM
CYP3A4, CYP2D6 and CYP2C9 P450-Glo assays (Promega®) were
conducted to determine their activity at the presence of 5, 7 and
1
3. The assays were performed according to the manufacturer’s
22, 23
protocols as described previously.
A strong cytochrome
inhibition effect of 7 (IC50 = 0.31 µM), which is similar to the
reference inhibitor ketoconazole (IC50 = 0.14 µM), was revealed
The metabolic stability of compounds 5, 7 and 13 was evaluated
in silico using MetaSite 5.1.1 and in vitro using human, rat and
27
(
Fig.4A) (Table 2). The CYP3A4 inhibition was also observed for 13
mouse models with the respective liver microsomes, according to
2
2, 23, 28
(IC50 = 3.53 µM) (Fig. 4A) (Table 2). Surprisingly, no effect on CYP3A4
activity was observed in the presence of 5 (Fig. 4A) (Table 2). An
previously described methods and protocols.
The in silico algorithm predicted, that the most likely metabolic
site of 5 was the 2-aminoethyl chain, whereas the metabolic
transformations for 7 and 13 were expected at the N-alkyl
substituent at the imidazole ring (see Fig. S1-S2 in supplementary
data).
inhibition effects of 7 and 13 on CYP2D6 were also observed (IC50
=
7
.47 and 27.60 µM, respectively), whereas slight CYP2D6 induction
by 5 was observed at the highest doses (Fig. 4B) (Table 2). The
highest inhibition effect on CYP2C9 was observed for 13 (~40% of
the enzyme activity) only at the dose 25 µM) (Fig. 4C) (Table 2).
Thus, the N-substituted imidazole moiety of 7 and 13 is responsible
for the interaction with cytochromes, as imidazole is the common
The tested compounds were incubated with HLMs, RLMs and
MLMs for 120 min at 37°C in the presence of the NADPH
regeneration system to determine their relative in vitro metabolic
stability. UPLC analysis of the reaction mixtures collected after
reaction with HLMs showed that approximately 20% of 5 was
converted into two metabolites, M1 and M2 (see Fig. S3A in
supplementary data). However, due to the ambiguous MS spectrum
3
0
molecular feature of many CYP inhibitors.
+
of metabolite M1, only the M2 quasimolecular ion [M+H] (m/z =
2
19.11) could be interpreted (see Fig. S4C-D in supplementary data)
(
Table 2). The molecular mass of M2 (+14 units) suggests oxidation
to the carbonyl group at the 2-aminoethyl substituent as a
metabolic pathway, which was also predicted in silico (see Fig. S2 in
supplementary data). UPLC analysis of the reaction mixtures of 7
and 13 after incubation with HLMs indicated their excellent
metabolic stability, as no metabolites were present after incubation
(
(
see Fig. S3B-C in supplementary data) (Table 2). Rodent models
mouse and rat) have shown similarities in the metabolism of 5 -
only one metabolite with the same retention time (t = 1.62) was
R
found after incubation with RLMs and MLMs (see Fig. S6A and S9A
in supplementary data). Comparison of the retention time of the
metabolite obtained in rodent models with metabolite M1 (found
after incubation with HLMs, t = 1.61) indicated that it could be the
R
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Fig. 4 The effect of ketoconazole (KE), 5, 7 and 13 on CYP3A4
activity (A). The effect of quinidine (QD), 5, 7 and 13 on CYP2D6
activity (B). The effect of 5, 7 and 13 on CYP2C9 activity (C).
In vitro safety profiling is an essential component of the drug
discovery process, providing a reliable alternative to animal testing. Fig. 5 The effects of doxorubicin (DX) and 5 (A),7 (B) and 13 (C) on
The cytotoxicity, neurotoxicity, hepatotoxicity and mutagenicity of HEK-293, neuroblastoma SH-SY5Y and hepatoma HepG2 cell line
5
, 7 and 13 were tested using HEK-293, SH-SY5Y, and HepG2 viability after 72 h of incubation. The mitochondrial toxin carbonyl
eukaryotic cell lines and the bacterial strain Salmonella cyanide 3-chlorophenyl-hydrazone (CCCP) was used at 10 μM as an
typhimurium. The growth conditions and assay protocols have been additional positive control for the hepatoma HepG2 cell line.
2
2, 23
described previously.
The colorimetric standard MTS assay Statistical significance was evaluated by one-way ANOVA, followed
allowed for the determination of eukaryotic cell viability after 72 h by Bonferroni’s comparison test (∗∗p < 0.01, ∗∗∗p < 0.001
of incubation with 5, 7 or 13. A statistically significant influence on compared with the negative control).
viability was shown for 7 and 13 in one (HepG2) among the three
examined cell lines (Fig. 5). However, the decrease in HepG2
viability to ~80% was observed only for the highest dose of 100 μM the Ames microplate fluctuation protocol (MPF), as described
The mutagenicity potential of 5, 7 and 13 was evaluated using
31
2
2, 23
(
Fig. 5) (Table 2), suggesting a very weak toxicity of 7 and 13 in previously.
The Salmonella typhimurium strain TA100 was used,
comparison to the effect of the references DX and CCCP, which which enables the detection of frame shift mutations. The number
decreased the cell viability to ~40% at 1 and 10 µM, respectively of revertants observed in the control (growth medium + 1% DMSO)
(
Fig. 5). Potential hepatotoxic effects of 7 were ruled out in allowed for determination of the medium control baseline (MCB).
additional tests in which the ATP level was measured via According to the assay kit manufacturer, a 2-fold increase over the
luminescence after a short 3h exposure of the cells to 7. No MCB is considered a mutagen alert. The results were compared to
influence of 7 on cell metabolism and mitochondrial respiration was the reference mutagen nonyl-4-hydroxyquinoline-N-oxide (NQNO)
observed, even at the highest dose (100 µM, Fig. 6).
at 0.5 μM. Mutagenic potential of all compounds was not observed
(
Fig. 7).
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to improve short-term working memory – however, this effect has
3
2
also been observed for 5-HT
HT R may play a different role in the acquisition and consolidation
of recognition memory,
7
R antagonists. It is likely that the 5-
7
1
,
33
which could explain why the
administration of both agonists and antagonists can lead to
procognitive effects. The reverse dose-dependence can potentially
be attributed to the activity of the compound at the 5-HT1A receptor
(
7
or other targets), which may influence the effect of 5-HT R
activation at the highest doses. Moreover, the activity of the
compound was also evident in the SIH test, which reflects the
autonomic response to stress in animals and is one of the most
recognized tests for anxiety. However, the activity was observed
only at the highest dose (Fig. 9).
Fig. 6 The effects of doxorubicin (DX), mitochondrial toxin carbonyl
cyanide 3-chlorophenylhydrazone (CCCP) and 7 on ATP levels in
hepatoma HepG2 cells after 3 h of incubation.
Fig. 8 Reversal of MK-801 induced disruption of novel object
recognition. The results are presented as the means ± SEM. N=10.
**p<0.01. N=10. #p<0.001 and *p<0.01.
Fig. 9 The effect of 7 on stress-induced hyperthermia (SIH). The
results are presented as the means ± SEM. N=10. **p<0.01.
Fig. 7 Number of histidine prototrophy revertants of Salmonella
typhimurium strain TA100 exposed to 5 (A), 7 (B) and 13 (C) or the
reference mutagen nonyl-4-hydroxyquinoline-N-oxide (NQNO). The
dashed line marks the 2-fold increase over MCB – considered a
mutagen alert. The MCB corresponds to the mean number of
revertants in the control plus one standard deviation.
In vivo
The in vivo evaluation of the biological activity of 7 included a
novel object recognition (NOR) and stress-induced hyperthermia
(
SIH) assays. Compound 7 reversed MK-801-induced disruption in
the NOR test at relatively low doses (Fig. 8), supporting its potential
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Table 1 Pharmacological in vitro profile of 5-HT R agonists.
7
Receptor
i
Ligand/K [nM]
1
4
20
20
1
4
5-CT
5)
0.3
15
13
11
11
AGH-107
(6)
AH-494
(7)
AGH-282
(13)
5
-HT
AS-19
E-55888
LP-211
RA-7
14
15
(
5
5
5
-HT1A
-HT2A
-HT5A
3.17
89.7
N.D.
98.5
N.D.
0.6
700
>1000
N.D.
188
626
178
1571
0.6
99
1190
76
1053
123
220
>10000
>10000
N.D.
>10000
>10000
N.D
11.55
251
5012
20
>10000
>10000
N.D.
>10000
N.D.
> 1000*
1673
6
5
-HT
-HT7
6
98.41
8.11
N.D.
720
0.4
>1000
2.5
596
1.4
>10000
5
6910
6
7271
6062
5
6417
161
D
2
N.D.
N.D.
N.D.
142
N.D.
4847
>10000
5561
>10000
>10000
*
Value estimated based on the results of a screening experiment
Table 2 ADMET parameters of 5, 7 and 13.
Parameter
Units
5
7
13
NE
-6
a
a
Permeability
(10 cm/s ±SD)
3.54±8.2
2.78±9.2
a
a
a
a
Pgp
(% of basal activity at 50 μM)
(% of metabolized substrate)
(% of metabolized substrate)
(% of metabolized substrate)
(% of control ±SD at 10 μM)
(% of control ±SD at 10 μM)
26.3±12.0
19.3±12.3
60.0±16.0
c
c
Metabolism with HLMs
Metabolism with RLMs
Metabolism with MLMs
CYP3A4 activity
26
20
2
N.A.
N.A.
c
7
N.A.
14
9
a
a
95.7±1.4
2.9±0.8
29.37±2.2
a
a
a
CYP2D6 activity
108.3±2.6
43.4±0.8
55.0±9.7
a
a
a
CYP2C9 activity
HEK-293 viability
SH-SY5Y viability
HepG2 viability
(% of control ±SD at 25 μM)
(% of control ±SD at 100 μM)
(% of control ±SD at 100 μM)
(% of control ±SD at 100 μM)
(% of control ±SD at 100 μM)
78.7±1.6
104.2±15.8
37.20±21.1
b
b
b
105.3±7.0
78.7±16.8
106.7±3.5
112.2±5.2
b
b
b
b
91.0±5.7
101.3±3.8
NE
88.4±7.1
82.2±5.7
b
b
78.7±3.8
a
d
ATP level in HepG2
Ames test
101.6±2.2
N.E.
a
a
a
(No. of positive wells ±SD at 10 μM)
4.0±2.9
5.6±1.2
12.3±1.5
a
b
c
tested in triplicate
tested in quadruplicate
Not applicable – no metabolism was observed
d
N.E. – not examined
6
| J. Name., 2012, 00, 1-3
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DOI: 10.1039/C8MD00313K
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ARTICLE
Analytical methods
Experimental
UPLC/MS analysis was performed on Waters TQD spectrometer
combined with UPLC Acquity H-Class with PDA eLambda detector.
Waters Acquity UPLC BEH C18 1.7 μm 2.1x50 mm chromatographic
column was used, at 40°C, 0.300 ml/min flow rate and 1.0 μL
injection volume (the samples were dissolved in LC-MS 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 minutes, kept till 3.5
minutes, then to initial conditions until 4.0 minutes and kept for
Chemicals
The reference compounds used during in vitro study, as follows:
caffeine, norfloxacin, doxorubicin, carbonyl cyanide 3-
chlorophenylhydrazone,
hydroxyquinoline-N-oxide were provided by Sigma-Aldrich (St.
Louis, MO, USA). The references in Pgp assay: Na VO and
verapamil were provided by Promega (Madison, WI, USA).
ketoconazole,
quinidine,
nonyl-4-
3
4
All the chemicals for syntheses were supplied by the companies:
Combi-Blocks, Inc. 7949 Silverton Avenue, Suite 915 San Diego, CA
92126 USA, Sigma-Aldrich Sp. z o.o. Poznan, Poland and Chempur,
Jana Lortza 70A, 31-940 Piekary Śląskie.
1
13
additional 2.0 minutes. Total time of analysis – 6.0 minutes. H,
C
General procedures for the preparation of compounds 7-15
General procedure 1 for the synthesis of Indole-3-carboxaldehydes
NMR and 2D NMR spectra were recorded on Bruker Avance III HD
400 NMR and Bruker AVANCE 500 MHz spectrometers. All samples
were dissolved in DMSO-d with TMS as the internal standard.
6
2
0
8
-10. Aldehydes were prepared as decribed previously. The
Vilsmeier-Haack reagent was generated by the addition of 2 ml of
POCl over the course of 15 minutes to 8 ml of dry DMF cooled in an
3
Pharmacology
ice-salt bath. After the addition was complete, the ice bath was
removed and the contents of the flask were left to warm to room
temperature over approx. 30 minutes. The substituted indole (21.9
mmol) was dissolved in 10 ml of DMF and added over a period of 15
minutes to the formylating mixture. The stirring was continued for
an hour during which the flask contents were heated to 40ºC in a
hot water bath. A total of 20 ml of 5M NaOH was added, and the
Compounds 13-15 were tested in a radioligand binding assays to
determine their affinity for five receptors: 5-HT1A, 5-HT2A, 5-HT , 5-
HT and D . The assays were performed via the displacement of the
6
7
2
respective radioligands from cloned human receptors, all stably
expressed in HEK293 cells (except for 5-HT2A which was expressed
3
3
in CHO cells): [ H]-8-OH-DPAT for 5-HT1AR, [ H]-ketanserin for 5-
3
3
3
HT2AR, [ H]-LSD for 5-HT
6 7
R, [ H]-5-CT for 5-HT R and [ H]-raclopride
2
mixture was diluted with H O, quickly brought to a boil and left to
for D R. The detailed procedure can be found in supporting
2
cool slowly. The crystals were removed by filtration, washed with
cold water and vacuum dried. The products thus obtained were in
most cases sufficiently pure for the subsequent reactions The
impure aldehydes were recrystallized from ethanol-water mixtures.
information.
PAMPA
Pre-coated PAMPA Plate System Gentest™ (Corning, Tewksbury,
MA, USA) was used for permeability evaluation. All experiments
General procedure 2 for the synthesis of 1-alkylimidazoles 11, 12,
1
4, 15. Van Leusen reaction was conducted as described
2
2, 23
2
0
were performed as described before.
The calibration curves of
previously. Aromatic aldehyde (3 mmol) was mixed with amine
15 mmol) in 20 ml dry methanol. Reaction mixture was left
overnight to complete the imine formation although it can be TLC
monitored (SiO /CHCl ). Anhydrous K CO (3 mmol) and TosMIC
tosylmethylisocyanide, 3 mmol) were subsequently added. The
5
-HT R agonists 5 and 7 and reference substances were prepared
7
(
using capillary electrophoresis (CE) system P/ACE MDQ (Beckman
Coulter, Fullerton, CA, USA), controlled by 32 Karat Software
version 8.0, and equipped with diode-array detector (DAD).
2
3
2
3
(
mixture was stirred for an additional 8 hours, diluted with 50 ml
H O, and extracted three times with 20 ml ethyl acetate. The
2
Influence on Pgp activity
combined extracts were washed twice with 20 ml H
with 20 ml brine, treated with anhydrous magnesium sulfate and estimated using the luminescent Pgp-Glo Assay System (Promega,
2
O, and once The influence of 5, 7 and 13 on glycoprotein P (Pgp) activity was
TM
2
3
evaporated. The final products were purified by trituration under a Madison, WI, USA) as described previously. The reference
:1 hexane:isopropanol mixture.
inhibitor Na VO was used in 100 µM concentration and reference
2
3
4
stimulator verapamil in 200 µM, according to the manufacturer’s
recommendations. Compounds were examined at 50 and 100 µM.
All compounds were incubated with Pgp membranes for 40 minutes
at 37°C in triplicate. GraphPad Prism™ software (version 5.01, San
Diego, CA, USA) was used to calculate statistical significance.
General Procedure 3 for the Synthesis of 3-(1-alkyl-1H-imidazol-5-
yl)-1H-indole-5-carboxamides (7 and 13). Compounds were
synthesized from appropriate nitriles (11 and 12, respectively), the
cyanide group hydrolysis was accomplished according to a modified
34
procedure outlined by Agarwal et al. A suspension of the nitrile
3.4 mmol) in 1 ml MeOH and 5 ml THF was stirred in an ice-salt
(
Metabolic stability
bath for 15 minutes until 0°C was reached. Hydrogen peroxide (30%
solution, 5.4 ml) was added dropwise keeping the temperature
below 10°C. Stirring was continued for 15 minutes and sodium
hydroxide (20% solution, 5.4 ml) was added dropwise keeping
temperature below 10°C. The mixture was allowed to warm to
room temperature and stirred for 24 hours. The product was
extracted from the reaction mixture with chloroform and purified
by trituration with acetone.
Human (HLMs), rat (RLMs) and mouse liver microsomes (MLMs)
were purchased form Sigma-Aldrich (St. Louis, MO, USA). The
NADPH Regeneration System was purchased from Promega®
(
Madison, WI, USA). All experiments were performed as described
2
0, 22, 23
before.
Influence on CYP3A4 and CYP2D6 activity
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
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ARTICLE
Journal Name
The luminescent CYP3A4 P450-Glo™, CYP2D6 P450-Glo™ and The modified stress-induced hyperthermia in singly-housed mice
CYP2C9 P450-Glo™ assays and protocols were provided by
Promega® (Madison, WI, USA). All experiments were performed as Each experimental group consisted of eight to ten animals in a
2
0, 22, 23
described before.
Each compound was tested in triplicate at modified stress-induced hyperthermia (SIH) test. The animals were
the final concentrations similar forall CYP3A4, CYP2D6 and CYP2C9 housed individually in a 26 × 21 × 14 cm Macolon cage, 24 h before
assays in range from 0.01 to 25 μM. The luminescent signal was testing. The procedure for the modified stress-induced
measured by using a microplate reader EnSpire PerkinElmer hyperthermia was adapted from Van der Heyden et. al and based
3
6, 37
(
Waltham, MA, USA).
on the procedure introduced by Borsini et. al.
body temperature was measured for each mouse at t = 0 min (T
and t = +15 min (T ). Albino Swiss mice were placed into a new cage
immediately following T , with the difference in temperature
T − T ) used as the measure of stress-induced hyperthermia. The
For this assay, the
1
)
Safety
2
1
Human embryonic kidney HEK-293 cell line (ATCC® CRL−1573™) was
kindly donated by Prof. Dr. Christa Müller (Pharmaceutical Institute,
Pharmaceutical Chemistry I, University of Bonn). Hepatoma HepG2
(
2
1
pilot studies of Spooren demonstrated that a T
5 min is optimal for assay of SIH. A comparison between T in
2
1
− T interval of
38
1
1
(ATCC® HB-8065™) cell line was kindly donated by the Department
vehicle-treated mice and those administered with test compound
was used to determine whether the agent affects the body
temperature alone. The rectal temperature was measured to the
nearest 0.1 °C by a Physitemp Theralert Termometer, TH-5, (Clifton,
NJ, USA), with the Temperature sensor for mice, Type T, Copper-
Constantan Thermocouple, Braintree Scientific Inc. The lubricated
thermistor probe (2 mm diameter) was inserted 20 mm into the
rectum. The mouse was held at the base of the tail during this
determination and the thermistor probe was left in place until a
of Pharmacological Screening, Jagiellonian University Medical
College. SH-SY5Y (ATCC® CRL-2266™) cell line was purchased from
ATCC. The cell cultures’ growth conditions were applied as
described before.
Radioactive Cell Proliferation Assay (MTS) was purchased from
Promega (Madison, WI, USA). The absorbance of the samples was
2
0, 22, 23
The CellTiter 96® AQueous Non-
measured using
a microplate reader EnSpire (PerkinElmer,
Waltham, MA USA) at 490 nm. The reference compounds CCCP (10
µM) and DX (1 µM) as well as compounds 5, 7 and 13 (0.1 – 100
µM) were tested in four repetitions.
The CellTiter-Glo Luminescent Cell Viability Assay for ATP–level
assessment was purchased from Promega® (Madison, WI, USA). The
ATP level was measured as described previously after 3 h of HepG2
cells’ exposure on compound 7 tested at three concentrations (1,
constant reading was obtained for 15 s. Compound
7 was
administered at the doses 1, 5 and 15 mg/kg 60 min before test.
Neither vehicle used nor the tested doses of the compound had any
influence on the basal body temperature, which was between 36
and 37 °C, typically for Albino Swiss mice used in our laboratory.
The statistical significance was evaluated by one-way ANOVA
followed by Dunnett's post-hoc analysis.
The procedures were conducted in accordance with the European
Communities Council Directive 2010/63/EU and Polish legislation
acts concerning animal experimentation. The experiments were
approved by II Local Ethics Committee in Krakow by the Institute of
Pharmacology, Polish Academy of Sciences in Krakow (no
1
0 and 100 µM), and on the reference compounds CCCP (10 µM)
2
2, 23
and DX (1 µM).
The luminescence signal was measured with a
microplate reader EnSpire (PerkinElmer, Waltham, MA USA) in
luminescence mode. The assay was performed in quadruplicate.
The Salmonella typhimurium TA100 strain with base pair
substitution (hisG46 mutation, which target is GGG) was purchased
from Xenometrix, Allschwil, Switzerland, and has been used in
mutagenicity Ames 384 - well microtiter assay. All experiments
were performed as described before.
GraphPad Prism™ software (version 5.01, San Diego, CA, USA) was
used to calculate statistical significance in all safety experiments.
1
81/2016). All procedures were conducted according to the
guidelines of the National Institutes of Health Animal Care and Use
Committee.
2
2, 23
Conclusions
Novel Object Recognition experiment
The recently described 5-(1H-indole-3-yl)-1-alkylimidazoles are
3
5
The method was performed according to Nilsson et al. The the first examples of low-basicity agonists of the 5-HT
animals were trained and tested in a black, plastic, open field (50 × receptor. Several compounds from this group have been discovered
0 cm, 35 cm high) with the floor divided into 20-cm square as suitable molecular probe candidates, which could be used for
sections. The open field was in a dark room illuminated only by a 25 both in vitro and in vivo studies examining the physiology and
R serotonin
7
3
2
0
W bulb. On the first day (adaptation) the animals were allowed to pathophysiology of the CNS associated with 5-HT R. Indole-
7
explore the open field for 10 min. On the next day (training, T1) the imidazole derivatives 7 (previously published) and 13 are the closest
animals were administered the tested drugs, placed in the structural analogues of the commonly used molecular probe 5-CT
apparatus and allowed to explore two identical objects (cylindrical (5). However, compound 5 exhibits a multi-receptor profile, i.e.,
objects with walls painted white, 7 cm in diameter, 11 cm high) for high affinity for 5-HT
the time required to complete 15 s of exploration of either object. HT R receptors. The lack of selectivity as well as low blood-brain
For the retention trial (T2) conducted one hour later, one of the barrier permeability are the main disadvantages of 5 as a
objects presented in T1 was replaced with a novel object (a prism- pharmacological tool. The high affinity of 7 and 13 for 5-HT R as
R, 5-HT1AR, 5-HT5AR and to some extent also 5-
7
6
7
shaped object with walls painted black, 5 cm wide, 14 cm high). The well as high selectivity over the 5-HT1A receptor (25-fold and 37-
duration of exploration of each object (i.e., sitting in close proximity fold, respectively, Table 1) are the main advantages of this
to the objects or sniffing or touching them) during 5 min time was compound over 5-CT. In vitro comparison studies between 5 7 and
video recorded and measured separately by a trained observer. All 13 demonstrated their similarities (Table 2). Compounds 5 and 7
drugs were administered before the training (T1) session. MK-801 displayed very good ability to diffuse passively across biological
(
0.3 mg/kg, i.p.) was given 30 min before the session. Investigated membranes and also significantly inhibited Pgp. For compound 13
compounds were administered 60 min before MK-801.
slight, not statistically significant Pgp inhibition was observed.
Excellent metabolic stability of 5, 7, and 13 was also determined, in
8
| J. Name., 2012, 00, 1-3
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DOI: 10.1039/C8MD00313K
Journal Name
ARTICLE
particular of compound 13, for which no metabolites were 12. A. Brenchat, X. Nadal, L. Romero, S. Ovalle, A. Muro, R.
observed after incubation with HLMs and RLMs. A significant
difference between 5 and 3-(1-alkyl-1H-imidazol-5-yl)-1H-indole-5-
carboxamides was observed in the in vitro assays only in terms of
Sanchez-Arroyos, E. Portillo-Salido, M. Pujol, A. Montero, X.
Codony, J. Burgueno, D. Zamanillo, M. Hamon, R. Maldonado
and J. M. Vela, Pain, 2010, 149, 483-494.
the influence on cytochromes P-450 activity. However, inhibition 13. P. Di Pilato, M. Niso, W. Adriani, E. Romano, D. Travaglini, F.
activity of 7 and 13 was expected herein, as had been previously
Berardi, N. A. Colabufo, R. Perrone, G. Laviola, E. Lacivita and
M. Leopoldo, Rev. Neurosci., 2014, 25, 401-415.
observed for another imidazole-based 5-HT R agonist – compound
7
2
0
6.
Finally, no significant toxicity or mutagenicity was found for 5, 7 14. A. P. Bento, A. Gaulton, A. Hersey, L. J. Bellis, J. Chambers, M.
and 13.
The in vivo activity of 7 was confirmed in pharmacological
experiments in mice, providing indirect confirmation of blood-brain
Davies, F. A. Kruger, Y. Light, L. Mak, S. McGlinchey, M.
Nowotka, G. Papadatos, R. Santos and J. P. Overington, Nucleic
Acids Res., 2014, 42, D1083-D1090.
barrier permeation. Compound 7 was active in the NOR test in mice 15. A. Brenchat, L. Romero, M. García, M. Pujol, J. Burgueño, A.
at a relatively low dose of 1 mg/kg, which is in agreement with
previous results obtained for 5-CT. Moreover, its activity was also
observed in the SIH test at 15 mg/kg.
We report compounds 7 and 13 as a very promising alternatives
to 5-CT with comparable in vitro potency, very high-water solubility,
Torrens, M. Hamon, J. M. Baeyens, H. Buschmann and D.
Zamanillo, Pain, 2009, 141, 239-247.
16. E. Lacivita, S. Podlewska, L. Speranza, M. Niso, G. Satala, R.
Perrone, C. Perrone-Capano, A. J. Bojarski and M. Leopoldo,
Eur. J. Med. Chem., 2016, 120, 363-379.
39
similar in vitro ADMET properties and a desirable high selectivity 17. E. Lacivita, P. De Giorgio, D. Patarnello, M. Niso, N. A. Colabufo,
over 5-HT1A receptor. Compound 7, also confirmed in vivo activity in
Novel Object Recognition and the modified stress-induced
hyperthermia assays.
F. Berardi, R. Perrone, G. Satala, B. Duszynska, A. J. Bojarski
and M. Leopoldo, Exp. Brain Res., 2013, 230, 569-582.
18. T. M. Eriksson, S. Holst, T. L. Stan, T. Hager, B. Sjogren, S. O.
Ogren, P. Svenningsson and O. Stiedl, Neuropharmacology,
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012, 63, 1150-1160.
Conflicts of interest
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9. J. Andries, L. Lemoine, D. Le Bars, L. Zimmer and T. Billard, Eur.
J. Med. Chem., 2011, 46, 3455-3461.
0. A. S. Hogendorf, A. Hogendorf, R. Kurczab, G. Satala, T. Lenda,
M. Walczak, G. Latacz, J. Handzlik, K. Kiec-Kononowicz, J. M.
Wieronska, M.Wozniak, P. Cieslik, R. Bugno, J. Staron and A. J.
Bojarski, Sci. Rep., 2017, 7, 15.
There are no conflicts to declare.
Acknowledgements
The study was partially supported by the grant OPUS
2017/25/B/NZ7/02929 from the Polish National Science Centre
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DOI: 10.1039/C8MD00313K
ARTICLE
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1
83x79mm (150 x 150 DPI)