The relationship between stereochemical and both, pharmacological and ADME-Tox, properties of the potent hydantoin 5-HT7R antagonist MF-8

Article abstract of DOI:10.1016/j.bioorg.2020.104466

This study concerns synthesis and evaluation of pharmacodynamic and pharmacokinetic profile for all four stereoisomers of MF-8 (5-(4-fluorophenyl)-3-(2-hydroxy-3-(4-(2-methoxyphenyl)piperazin-1-yl)propyl)-5-methylimidazolidine-2,4-dione), the previously d

Full text of DOI:10.1016/j.bioorg.2020.104466

Bioorganic Chemistry xxx (xxxx) xxx
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
The relationship between stereochemical and both, pharmacological and
ADME-Tox, properties of the potent hydantoin 5-HT7R antagonist MF-8
,
,
,
b
c
Katarzyna Kucwaj-Brysza 1, Gniewomir Latacza 1, Sabina Podlewskaa , Ewa Zesławska ,
˙
Jarosław Handzlikd, Annamaria Lubelskaa, Grzegorz Satałab, Wojciech Niteke,
,
*
Jadwiga Handzlika
a
´
Department of Technology and Biotechnology of Drugs, Jagiellonian University Medical College, Medyczna 9, 30-688 Krakow, Poland
b
c
´
Maj Institute of Pharmacology, Polish Academy of Sciences, Smętna 12, 31-343 Krakow, Poland
˙
´
Institute of Biology, Pedagogical University of Cracow, Podchorązych 2, 30-084 Krakow, Poland
d
e
´
Faculty of Chemical Engineering and Technology, Cracow University of Technology, Warszawska 24, 31-155 Krakow, Poland
´
Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387 Krakow, Poland
A R T I C L E I N F O
A B S T R A C T
Keywords:
This study concerns synthesis and evaluation of pharmacodynamic and pharmacokinetic profile for all four
stereoisomers of MF-8 (5-(4-fluorophenyl)-3-(2-hydroxy-3-(4-(2-methoxyphenyl)piperazin-1-yl)propyl)-5-meth-
ylimidazolidine-2,4-dione), the previously described, highly potent 5-HT7R ligand with antidepressant activity
on mice. The combination of DFT calculations of 1H NMR chemical shifts with docking and dynamic simulations,
in comparison to experimental screening results, provided prediction of the configuration for one of two present
stereogenic centers. The experimental data for stereoisomers (MF-8A-MF-8D) confirmed the significant impact of
stereochemistry on both, 5-HT7R affinity and antagonistic action, with Ki and Kb values in the range of 3–366 nM
Serotonin receptors
5-HT7R ligands
Hydantoin
Arylpiperazine
Stereoisomers
DFT
Docking
and 0.024–99 μM, respectively. We also indicated the stereochemistry-dependent influence of the tested com-
ADMET
pounds on P-glycoprotein efflux, absorption in Caco-2 model, metabolic pathway as well as CYP3A4 and CYP2C9
activities.
1. Introduction
which may interact in different ways with many proteins in vivo [8–10].
Among ADME-Tox properties affected by compounds chirality, absorp-
Serotonin 5-HT7 receptor (5-HT7R) is one of the latest discovered
subtype from 14-membered serotoninergic system family and is
distributed within both central nervous system (CNS) and peripheral
tissues [1–3]. The 5-HT7R may couple to Gs protein (increase of cAMP
level) or to G12 (Rho signaling) [4]. It is attractive therapeutic target
since confirmation of its significant role in pathophysiological processes
such as depression [5], cognitive impairment, migraines. Despite the
fact that there are many approved drugs which interact with 5-HT7R,
there is no substance on pharmaceutical market which is highly selective
for this receptor [4]. Only one such compound – JNJ-18308683 has been
submitted to clinical trials and is being now evaluated in Phase 2 [6,7].
On the other hand, stereochemistry is highly important issue in
medicinal chemistry due to possible significant differences in pharma-
codynamic and pharmacokinetic properties of particular stereoisomers,
tion (the difference in binding to the efflux pumps and up-take trans-
porters), metabolism (the difference in orientation to the enzymes
reactive moiety), distribution (the difference in binding to the plasma
protein) and toxicity (the difference in binding to the hERG channels and
in interaction with CYPs) can be mentioned [10]. Especially, the
chirality may significantly influence the compounds metabolic stability,
which is an important parameter responsible for pharmacokinetic and
pharmacological properties. The enantiospecific metabolism was
determined for many drugs. For instance, the intrinsic clearance of
omeprazole metabolized by CYP3A4 to sulfone metabolite was found in
human liver microsomes as around 4-fold greater for the S- than for the
R- enantiomer [11]. The S-enantiomer of racemic anticonvulsant drug
mephenytoin is oxidized to 4′-hydroxymephenytoin by CYP2C19,
whereas the R-enantiomer is N-dealkylated by two CYPs: 2C9 and 2B6 to
* Corresponding author.
E-mail address: j.handzlik@uj.edu.pl (J. Handzlik).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2020.104466
Received 4 December 2019; Received in revised form 30 September 2020; Accepted 6 November 2020
Available online 10 November 2020
0045-2068/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: Katarzyna Kucwaj-Brysz, Bioorganic Chemistry, https://doi.org/10.1016/j.bioorg.2020.104466

K. Kucwaj-Brysz et al.
Bioorganic Chemistry xxx (xxxx) xxx
phenylethylhydantoin [12,13]. The calcium channel blocker, verapamil,
used as a racemic mixture, has shown differences between the plasma
concentrations of particular enantiomers. After oral administration, the
S-isomer has been preferentially metabolized leading to the predomi-
nance of R-verapamil in plasma [14]. The chiral antidepressant drug,
fluoxetine, is an example of the relationship between the genotype and
stereoselectivity of metabolism. In extensive metabolizers of CYP2D6,
both enantiomers are biotransformed equally to the active metabolite
norfluoxetine with similar apparent oral clearances. In CYP2D6 poor
metabolizers, however, the apparent oral clearance of R-enantiomer is
6-fold higher than that of S-fluoxetine. Moreover, the plasma concen-
tration of S-fluoxetine is 11.5-fold higher in poor metabolizers than in
the extensive ones, whereas only 2.5-fold higher in the case of the R-
isomer [15].
stereogenic centers in the structure and achiral synthesis conditions
applied in those initial studies [18], MF-8 was evaluated as a mixture of
four possible stereoisomers (MF-8rac).
Based on the very promising biological results, compound MF-8 was
selected for further investigation in term to synthesize and evaluate
both, pharmacological and ADME-Tox properties, for each stereoisomer
separately. Hence, the current studies are concentrated on the stereo-
selective synthesis, separation and both, X-ray and molecular modeling-
supported, estimation of an absolute configuration of 4 enantiopure
stereoisomers of MF-8. The obtained stereoisomers have been investi-
gated in vitro on their 5-HT7/5-HT1A affinity, 5-HT7 intrinsic activity,
passive transport through the biological membranes, absorption in
Caco-2 cell-based model, influence on P-glycoprotein (P-gp) activity,
metabolic stability, drug-drug interactions (DDI) and hepatotoxicity.
Results of corresponding studies for the previously synthesized racemic
MF-8rac [18,19,23] were used for comparison and discussion.
Recent lines of evidence have indicated a significant influence of
stereochemical properties on the compounds’ affinity towards 5-HT7R.
Among newly synthesized derivatives of pyrrolidone, enantiomers S
showed much higher potency than enantiomers R (Fig. 1) [16].
Results of the studies among the antipsychotic benzamides, i.e. N-
methylated derivatives of amisulpride (Fig. 2) have indicated a poly-
pharmacologic action of the racemate LB-102, going via 5-HT7R (anti-
depressant effect) and dopaminergic D2 and D3 receptors (antipsychotic
activity), whereas radioligand binding assays for corresponding opti-
cally pure enantiomers showed that only enantiomer R is responsible for
interaction with 5-HT7R (Fig. 2) [17]. Taking into account the afore-
mentioned importance of enantioselectivity for both pharmacological
and “drug-likeness” properties, it is obligatory to isolate enantiopure
components of active racemic mixtures in order to examine them as
early as possible in the primary levels of drug discovery process. How-
ever, it is usually a great scientific challenge due to sophisticated and
expensive stereoselective synthesis methods or even more expensive
technics of direct separation of stereoisomers. Furthermore, while the
methods of experimental assessment of enantiomeric purity are well
developed, available and reliable, a determination of absolute configu-
ration generates many problems. It is an issue strongly determined by
the structure and chemical properties of a given molecule, requiring the
use of sophisticated spectroscopic methods and/or advanced computer-
based prediction techniques, and frequently, it is even impossible to
solve without appropriate crystallographic analysis. Thus, usually
racemic mixture are under consideration in the first step of drug dis-
covery screening, and only limited “hits” with the desired pharmaco-
dynamic profile are destined to isolate/synthesize their enantiopure
components in order to re-evaluate their biological activity and ADME-
Tox properties, and thus, to eliminate the inactive, toxic and/or meta-
bolically unstable stereoisomer.
2. Results and discussions
2.1. Chemical synthesis
The particular stereoisomers of MF-8rac were synthesized via
already optimized and described 3-step synthetic pathway (Bucherer-
Berg reaction, N-alkylation and condensation with epoxide ring open-
ing) [19,20] (Scheme 1).
For N-alkylation reaction, 2-(chloromethyl)oxirane (epichlorhydrin)
with defined configuration was used. The synthesized two pairs of di-
astereoisomers were separated, after the final step using supercritical
fluid chromatography (SFC) technique, with resulting diastereoisomeric
excess 92–100% (Table 1). The optically pure stereoisomers with un-
known configuration at C5 of hydantoin were assigned with formal
signatures A-D used for further investigations.
2.2. Estimation of absolute configuration
The expensive and sophisticated methods of obtaining the stereo-
isomers (MF-8A-MF-8D) provided them in the small amount that
enabled to perform only basic spectral and chromatographic analyses,
which were not sufficient to recognize the absolute configuration of each
final product. Thus, DFT-aided molecular modeling calculations, based
on experimental crystallographic data for the racemic mixture of MF-8,
have been involved to support the 1H NMR spectral analysis in order to
estimate the most probable absolute configuration for the stereoisomers
MF-8A-MF-8D.
In this context, our previous work [18–21] concerned above 50-
membered 5-HT7R hydantoin ligands family, which has been
mentioned as recent “breakthrough in medicinal chemistry of novel and
powerful antidepressant” by Mangoni et al.[22]. The most active
member, named MF-8 (Fig. 3), showed excellent antidepressant effects
in forced swim test (FST) in mice at the dose of 5 mg/kg and very
profitable ADME-Tox profile [23]. Due to the presence of two
2.2.1. X-ray crystal structure analysis for MF-8
The chemical group of arylpiperazine 2-hydroxypropyl derivatives of
5-aryl-5-methylhydantoins is hard to obtain in basic precipitate forms,
in particular, to have crystals enough for X-ray analysis. In the case of
the stereoisomers isolated (MF-8A-MF-8D), neither the solid form of
them, nor their amount, were sufficient to perform crystallographic
studies. In contrary, our 3-year efforts allowed to obtain a crystal of
hydrochloride salt form coming from MF-8rac, appropriate for X-ray
crystallographic analysis that was a cornerstone for wider structural
consideration, carried out by the use of molecular modeling.
Results of the crystallographic studies show that the asymmetric unit
consists of one protonated MF-8 molecule, one chlorine anion and one
disordered 2-methylpropan-1-ol molecule, which comes from the sol-
vent. The molecular geometry in the crystal structure of MF-8 with the
atom numbering scheme is shown in Fig. 4.
The molecule possesses two chiral centers at C5 and C7 atoms. The
crystal structure confirms 5S, 7S configuration for one molecule and 5R,
7R for the other one related by the inversion centers. The molecule of
MF-8 is protonated at nitrogen atom (N2) by the proton transfer from
hydrochloride. The linker between hydantoin and piperazine rings
adopts extended conformation with torsion angles N3-C6-C7-C8 =
Fig. 1. Differences in 5-HT7 affinity depending on configuration among pyr-
rolidone derivatives [16].
2

K. Kucwaj-Brysz et al.
Bioorganic Chemistry xxx (xxxx) xxx
Fig. 2. The differences in pharmacodynamic profile depending on stereochemistry for N-methylated amisulpride [17].
structures containing the same linker between hydantoin and piperazine
rings [20,21]. The mutual orientation of 4-fluorophenyl substituent at
C5 and hydantoin ring is similar to another derivative containing 4-flu-
orophenyl substituent at C5 atom, for which we determined the crystal
structure earlier [21]. The angle between the planes of the aromatic ring
and the hydantoin ring is 62.9(1)◦, while in the compared compound is
62.2(1)◦. In the presented structure the piperazine ring adopts chair
conformation with equatorial location of substituents at N2 and N4. The
torsion angles C14-C13-N4-C10 and C18-C13-N4-C10 are 71.4(2)◦ and
ꢀ 110.4(2)◦, respectively, which indicate that the phenyl ring at N4 atom
is not coplanar with piperazine moiety. The angle between the planes of
aromatic and piperazine (C9, C10, C11, C12) rings is 45.8(1)◦. Similar
values have been observed in other crystal structures of hydantoin de-
rivative containing o-metoxyphenyl substituent at nitrogen atom of
piperazine ring [24] and similar angles are very often noticed in other
crystal structures, deposited in the Cambridge Structural Database, for
derivatives containing o-metoxyphenyl substituent at nitrogen atom of
piperazine ring [25].
Fig. 3. The structure of MF-8 with indicated stereogenic centers, 5-HT7 affinity
and in vivo activity.
-176.0(1)◦ and C6-C7-C8-N2 = 168.3(1)◦. The molecular structure is
stabilized by two intramolecular hydrogen bonds, namely C9-H9A⋅⋅⋅O1
and C10-H10B⋅⋅⋅O3. A similar intramolecular interaction of hydroxy
group with piperazine ring has been also observed in other crystal
–
The main intermolecular interactions are based on O H⋅⋅⋅Cl and
–
N
H⋅⋅⋅Cl hydrogen bonds (Fig. 5).
Scheme 1. The synthetic pathway for MF-8′s stereoisomers: (i) KCN, NH4(CO3)2, H2O/ethanol, 50 ◦C; yield 54% (ii) NaOH, H2O, rt, yield 61%(S)/70%(R)**; (iii)
isopropanol, reflux, yield 45%(S)/49%(R)**. The analogous approach was applied using S-epichlorhydrin to obtain pair of diastereoisomers with configuration R of
carbon linked with hydroxyl group. *configuration at C5 of hydantoin has not been determined experimentally yet. **configuration of carbon atom bound to hy-
droxyl group (C7).
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K. Kucwaj-Brysz et al.
Bioorganic Chemistry xxx (xxxx) xxx
Both hydroxy groups, from the MF-8 molecule and the solvent, are
involved in hydrogen bonds with chlorine anion. In the presented crystal
structure, it is not observed typical interaction of chlorine anion with
protonated nitrogen atom [25], but the oxygen atom of hydroxy group
from the solvent interacts with protonated nitrogen atom (N2). The ni-
trogen atom (N1) of hydantoin moiety is also engaged in hydrogen bond
with chlorine anion. Furthermore, the crystal structure is stabilized by
Table 1
The enantiopurity for isolated stereoisomers of MF-8rac.
Compound
Structure
Diastereoisomeric excess
–
MF-8rac
– – – –
C H⋅⋅⋅O, C H⋅⋅⋅Cl, C H⋅⋅⋅F and C H⋅⋅⋅π contacts.
MF-8A
MF-8B
MF-8C
MF-8D
100
92
2.2.2. DFT-aided estimation of the absolute configuration for stereoisomers
MF-8A-MF-8D
Configuration at hydroxy-substituted carbon (C7, Fig. 4) was the
only one experimental information to base on due to the specific reac-
tion mechanism of the alkylation method used. Thus, it is confirmed that
MF-8A and MF-8B are at C7-R configuration, while MF-8C and MF-8D
are at C7-S, while, the configuration at C5 was the problem to solve.
Although the properties of diastereoisomers in achiral conditions are
expected to be different, the 1H NMR spectra for all four stereoisomers
(MF-8A-MF-8D) showed high similarity (see Experimental chapter).
A slight, but relatively the most noticeable, difference could be
observed in the case of chemical shifts for proton at hydantoin N1-
nitrogen (Table S1, Supplementary) but this fragment is rather far
from both stereogenic centers and the difference more probably may
refer to a blurred detection related to the nature of the signal (broad
singlet). Thus, we tried to focus on the protons in closer surroundings of
the stereogenic center C5. In the case of chemical shifts for protons of
both, the phenyl (C24, C26, Fig. 4) and the methyl group (C21, Fig. 4),
substituted at position C5, a slight decrease was observed if comparing
MF-8A to MF-8B as well as MF-8C to MF-8D. In this context, compu-
tational DFT methods have been applied in term to predict 1H NMR
chemical shifts for each stereoisomer of MF-8 with defined absolute
configurations, i.e.: (5R,7R), (5S,7R) (5S,7R) and (5S,7S), respectively.
The stereoisomers were built on the basis of crystal structure (Fig. 4) and
100
98
Fig. 4. The molecular structure of MF-8 showing the atom numbering scheme. Displacement ellipsoids are drawn at the 30% probability level.
Fig. 5. The interactions of two MF-8 molecules with chlorine anions and 2-methylpropan-1-ol. For clarity, only carbon atoms with the higher occupancy of the
–
disordered solvent are presented. Purple dashed lines indicate hydrogen bonds, orange C H⋅⋅⋅
π
contacts. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article).
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K. Kucwaj-Brysz et al.
Bioorganic Chemistry xxx (xxxx) xxx
their geometries were optimized on the TPSSh/def2-TZVPP level of
theory. The TPSSh functional was selected among six DFT methods,
based on test calculations results (see Computational methods section
for more details). Then, M06/def2-TZVPP NMR calculations, including
solvent effects (DMSO), were performed to determine 1H NMR chemical
shifts for the MF-8 stereoisomers.
Table 2
The radioligand binding and functional tests results for MF-8rac and its
stereoisomers.
Compound
Ki ± SD [nM]
Kb ± SD [nM]
5-HT1A
5-HT7
5-HT7
MF-8rac
121 ± 17
3 ± 2
58 ± 12
In order to decode the most probable configuration, the two-step
assumption has been applied, based on the qualitative similarity be-
tween calculated and experimental NMR results (Fig. 6, Table S1,
Supplementary).
MF-8A (5R, 7R)*
MF-8B (5S, 7R)*
MF-8C (5R, 7S)*
MF-8D (5S, 7S)*
832 ± 68
3 ± 1
24 ± 7
1433 ± 219
427 ± 34
44 ± 6
26 ± 5
366 ± 53
585 ± 94
397 ± 68
99230 ± 33111
2370 ± 512
In the first step, the concordance of the order of experimental
chemical shifts (δ-values) for the Ph-3,5-H protons of considered ste-
reoisomers (B > A and C > D) with the calculated one [(5S,7R) >
(5R,7R) and (5R,7S) > (5S,7S)] was under consideration (Fig. 6a). In the
next step; the similar relationship of the calculated and experimental
δ-values for 5-CH3 protons in pairs of 7R (A,B)- and 7S (C,D)- stereo-
isomers, respectively, was analyzed (Fig. 6b). Thus, the relationship for
7R stereoisomers (A > B) was corresponding to the calculated one, as
follows: (5R,7R) > (5S,7R), while that for 7S-pair (C > D) corresponded
to (5R,7S)>(5S,7S). Both assumptions, based on this “semi-empirical”
approach, allowed us to assign the stereoisomers configuration in the
following order: (5R,7R) for MF-8A, (5S,7R) for MF-8B, (5R,7S) for MF-
8C and (5S,7S) for MF-8D. At the absence of any sufficient experimental
data, this “sequence” was established as the most probable to use for
further consideration within this study and revised by docking simula-
tion in respect to experimental receptor binding results.
*the most probable configuration not confirmed experimentally.
the functional assays were performed in which the ability to decrease
cAMP level was measured. The results correlate very well with radio-
ligand binding assays with the analogous activity increase in series MF-
8D < MF-8B < MF-8C < MF-8A with the very strong antagonistic ac-
tivity of MF-8A with Kb = 24 nM, the more profitable than the racemic
mixture (Kb = 58 nM).
2.4. In vitro ADME-Tox studies
The ADME-Tox parameters of MF-8′s stereoisomers were evaluated
by in vitro methods and compared to MF-8rac. All the applied materials
and methods were described in our previous studies [19,23,26,27]. The
results obtained for MF-8rac and stereoisomers MF-8A-MF-8D were
summarized in Table 3.
The bioavailability of MF-8rac and stereoisomers was evaluated in
following assays: parallel artificial membrane permeability assay
(PAMPA), Caco-2 cell-based absorption model and by determination of
their potential induction or inhibition activity against Pgp - the impor-
tant efflux pump which is present in both, intestinal epithelium and
blood–brain barrier (BBB).
2.3. Radioligand binding assays and functional studies
Radioligand binding assays were applied to determine the affinity
and selectivity profiles of each of the MF-8′s stereoisomers for both,
human serotonin 5-HT7bR and 5-HT1AR, which were stably expressed in
HEK-293 cells. Interestingly, there are significant differences in 5-HT7
affinity among the stereoisomers (Table 2), with the most profitable Ki
value for stereoisomer MF-8A (3 nM) and the least profitable for MF-8D
(366 nM). All the presented isomers maintained significant selectivity
over 5-HT1A receptor.
The obtained data from PAMPA were present as permeability coef-
ficient Pe calculated according formulas provided by the manufacturer.
Two references were used in this study - the antibacterial drug nor-
floxacin (NFX) with calculated very low Pe = 0.56 × 10ꢀ 6 cm/s and
highly-permeable caffeine (CFN) with Pe value estimated to 15.1 × 10ꢀ 6
cm/s. All examined stereoisomers showed good passive penetration
In order to assess the intrinsic activity of compounds MF-8A-MF-8D,
Fig. 6. The qualitative concordance between calculated and experimental chemical shifts (δ) in 1H NMR spectra for the obtained stereoisomers of MF-8; a) the
comparison for 5-Ph-3,5- protons; b) the comparison for protons of 5-CH3 group.
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K. Kucwaj-Brysz et al.
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Table 3
ADME-Tox parameters of MF-8rac and stereoisomers.
Parameter
MF-8rac
MF-8A
MF-8B
MF-8C
MF-8D
PAMPA (Pe)
6.1 ± 3.2
5.9 ± 0.4
8.0 ± 3.0
4.1 ± 0.8
8.9 ± 0.2
10ꢀ 6 cm/s ± SD
Caco-2 (Papp
)
25.6 ± 0.7
16.4 ± 0.04
19.6 ± 0.1
29.9 ± 0.1
17.50 ± 0.01
10ꢀ 6 cm/s ± SD
Pgp activity
121.5 ± 28.1
155.5 ± 14.6
166.1 ± 9.6
112.2 ± 6.7
139.7 ± 4.0
% of basal activity ± SD
metabolic pathways
in mouse
hydroxylation (M1)
demethylation (M2)
decomposition/ hydroxylation (M3)
44.2
hydroxylation (M1)
hydroxylation (M1)
demethylation (M2)
decomposition/ hydroxylation (M3)
45.4
hydroxylation (M1)
hydroxylation (M1)
mouse CLint
40.3
54.8
35.7
mL/min/kg
CYP3A4 activity*
CYP2D6 activity*
CYP2C9 activity*
HepG2 viability**
75.4 ± 2.2
98.7 ± 3.4
165.7 ± 16.1
90.6 ± 6.5
83.7 ± 5.6
90.4 ± 0.6
107.1 ± 0.5
89.2 ± 6.4
66.6 ± 3.5
93.0 ± 3.5
111.3 ± 0.8
88.3 ± 7.4
80.9 ± 2.7
95.7 ± 0.5
302.6 ± 5.4
103.1 ± 9.4
59.7 ± 1.9
97.7 ± 4.2
82.7 ± 1.6
95.0 ± 13.7
*% of control ± SD at 10 μM; ** % of control ± SD at 100 μM.
through the biological membranes in compare to the used references,
with calculated Pe values in range of 4.1–8.9 × 10ꢀ 6 cm/s (Table 3). The
lowest ability to membrane diffusion was observed for stereoisomer MF-
8C, whereas the highest for stereoisomer MF-8D. Interestingly, the
calculated for MF-8rac Pe value 6.1 × 10ꢀ 6 cm/s was the average of Pe’s
determined for all four stereoisomers (Table 3).
MF-8rac and stereoisomers were compared to the well-permeable CFN
with estimated during this study Papp value 22.04 × 10ꢀ 6 cm/s. The
obtained results differ between stereoisomers and corelate to the results
from Pgp assay. The MF-8C stereoisomer which did not show the
stimulation effect on Pgp protein activity (Fig. 7) was the highly
absorbed compound with calculated Papp = 29.9 × 10ꢀ 6 cm/s. The ste-
reoisomers MF-8A, MF-8B and MF-8D, which significantly stimulated
Pgp (Fig. 7), was absorbed in moderate way (Papp from 16.4 to 19.6 ×
10ꢀ 6 cm/s). Moreover, for MF-8rac, which also did not show the sta-
tistically significant increase in Pgp activity (Fig. 7), the high absorption
was estimated (Papp = 25.6 × 10ꢀ 6 cm/s). Interestingly, in PAMPA assay
lower ability of MF-8rac to passive transport through the cell mem-
branes was shown in compare to the well-permeable reference CFN,
whereas the results from Caco-2 assay showed similar absorption of CFN
and MF-8rac (Table 3). The literature sources indicated only passive
mechanism of CFN diffusion through biological membranes. As shown
above, some of MF-8′s stereoisomers were effluxed by Pgp and despite of
this fact, the high absorption of MF-8rac was still observed. Thus,
considering the above, the obtained data indicate the probable uptake
transporters activity involvement in MF-8rac absorption.
The influence of MF-8rac and stereoisomers on Pgp was determined
by the luminescence-based Pgp-Glo™ Assay, which measures the ATP
consumption by Pgp. The results were compared to the Pgp basal ac-
tivity, which was calculated as difference in ATP consumption between
not treated samples and treated with 100 μM of Pgp inhibitor Na3VO4.
Verapamil (VL), which stimulates significantly its activity, was used at
200 µM concentration as the Pgp substrate reference. The significant
increase in Pgp ATP consumption was determined at the presence of MF-
8A, MF-8B (p < 0.001) and MF-8D (p < 0.05), whereas for MF-8rac and
stereoisomer MF-8C no statistically significant stimulation effect was
observed (Fig. 7). Thus, all these evidences showed MF-8A, MF-8B and
MF-8D stereoisomers as the substrates of Pgp.
Caco-2, an immortal human colon carcinoma cell line, is the best-
known model of in vitro permeability assessment which enables to
determine both, the passive and active mechanism of absorption. Ac-
cording to the literature [28], the calculated in Caco-2 model perme-
ability coefficient Papp < 2 × 10ꢀ 6 cm/s indicates low permeability, Papp
from 2 × 10ꢀ 6 to 20 × 10ꢀ 6 cm/s moderate permeability, whereas
compounds with Papp > 20 × 10ꢀ 6 cm/s are considered as high
permeable. The calculated here permeability coefficient Papp values for
The metabolic stability estimation and metabolites identification of
MF-8rac and stereoisomers were performed with use of mouse liver
microsomes (MLMs) and UPLC-MS analyses. According to UPLC-MS
spectrum of MF-8rac after 2 h incubation with MLMs, this compound
was metabolized mainly into one metabolite M1 with molecular mass m/
z = 473.26 and into slight amounts of two more metabolites M2 (m/z =
443.23) and M3 (m/z = 342.06) (Fig. 8). Interestingly, the presence of
M2 and M3 was identified only in the reaction mixture with stereoiso-
mer MF-8B, whereas MF-8A, MF-8C and MF-8D were metabolized only
into M1 (Fig. 8). Thus, only MF-8B stereoisomer metabolic pathways
were found to be responsible for the presence of M2 and M3 in the MF-
8rac reaction mixture. Moreover, tR and m/z values of M1 and M2 ob-
tained here by MLMs were similar to the metabolites of MF-8rac ob-
tained in our previous studies in the presence of human liver
microsomes (HLMs) [19].
It shows, that these two metabolic pathways, identified previously as
hydroxylation (M1) and demethylation (M2) occurred in both, human
and mouse species. The molecular mass of obtained during this study by
MLMs additional metabolite M3 (m/z = 342.06) corresponds to com-
pound’s decomposition followed by double hydroxylation. The most
probable structure of M3 metabolite was proposed in Fig. 9.
Next, the intrinsic clearance (CLint) of MF-8rac and stereoisomers
was calculated by measuring of compound’s disappearance with time in
the presence of MLMs, according to proposed by Obach methods and
formulas [28]. The results have shown close for all stereoisomers CLint
values in range of 35.7 – 54.8 ml/min/kg (Table 3). Compound MF-8D
Fig. 7. The effect of Pgp stimulator Verapamil (VL), MF-8rac and stereoisomers
on Pgp basal activity. Statistical significance was evaluated by one-way
ANOVA, followed by Bonferroni’s comparison test (*p < 0.05, ***p < 0.001).
6

K. Kucwaj-Brysz et al.
Bioorganic Chemistry xxx (xxxx) xxx
Fig. 8. UPLC spectra of the reaction mixture after 120 min incubation of MF-8rac and stereoisomers with MLMs.
CYP3A4, 2D6 and 2C9 isoforms was examined at the 10 µM concen-
tration each and compared to 1 µM of appropriate strong CYP inhibitor:
ketoconazole (KE), quinidine (QD,) and sulfaphenazole (SE), respec-
tively. The statistically significant (p < 0.001) CYP3A4 inhibition effect
was observed for all tested compounds (Fig. 10a).
However, the inhibitory potential differed between tested com-
pounds. Stereoisomer MF-8D showed the highest effect (59.7% of
CYP3A4 activity) whereas MF-8A the lowest one (83.7% of CYP3A4
activity). MF-8rac inhibition of CYP3A4 was determined as the resultant
of all stereoisomers’ inhibition effects (Table 3). No significant effect of
MF-8rac and stereoisomers on CYP2D6 activity was observed (Fig. 10b,
Table 3). On the other hand, an interesting effect of tested compounds on
CYP2C9 activity was found as stereoisomer MF-8C strongly induced its
activity up to 302.6% of control activity, stereoisomer MF-8D signifi-
cantly (p < 0.05) decreased its activity to 82.7% of control, whereas no
changes in the presence of MF-8A and MF-8B were observed (Fig. 10c,
Table 3). Similarly to CYP3A4, the MF-8rac action on CYP2C9 was
estimated as a result of all the stereoisomers effects.
Fig. 9. The most probable structure of metabolite M3 (m/z = 342.06).
with the lowest CLint was determined as the most metabolically stable
stereoisomer, whereas the highest CLint was shown for stereoisomer MF-
8C. The calculated for MF-8rac CLint = 44.2 ml/min/kg was found as the
average of CLint values determined for all four stereoisomers (Table 3).
Thus, according to the classification bands for microsomal assays [29],
MF-8C showed the high clearance (>47.0 ml/min/kg), whereas MF-
8rac and stereoisomers MF-8A, MF-8B moderate ones as results for
these compounds were between 8.6 and 47 ml/min/kg.
The potential DDI predictions were performed with use of P450-
Glo™ assays, which enable to measure luminescently the respective CYP
isoform activity. The influence of MF-8rac and stereoisomers on
The hepatoma HepG2 cell culture was used to determine the hepa-
totoxicity of MF-8rac and stereoisomers. As shown in Fig. 11 no statis-
tically significant decrease in cells’ viability was observed for all
Fig. 10. The effect of the respective inhibitor, MF-8rac and stereoisomers on CYP3A4 (a), CYP2D6 (b) and CYP2C9 (c) activity. Statistical significance was evaluated
by one-way ANOVA, followed by Bonferroni’s comparison test (*p < 0.05, **p < 0.01, ***p < 0.001).
7

K. Kucwaj-Brysz et al.
Bioorganic Chemistry xxx (xxxx) xxx
Fig. 11. The effect of MF-8rac, stereoisomers and references: doxorubicin (DX,
1 µM), mitochondrial toxin carbonyl cyanide 3-chlorophenyl-hydrazone (CCCP,
10 µM) on hepatoma HepG2 cell line viability. Statistical significance was
evaluated by one-way ANOVA, followed by Bonferroni’s comparison test (***p
< 0.001).
compounds in all used concentrations after 72 h - long incubation.
These results are in accordance with our previous hepatotoxicity
examination of MF-8rac, where no significant decrease of the ATP level
in HepG2 cells was found, even at the highest used concentration 100
µM [23].
Fig. 12. a) Docking of MF-8 stereoisomers to 5-HT7R homology model; b)
ligand–protein contacts occurring in the obtained complexes.
2.5. Docking and MD simulation support for the assays in vitro
out. The changes of the set of interacting amino acids for each of the
considered diastereoisomers in time occurring during MD simulation
studies are presented in Supplementary Information (Fig. S11).
The comparison of the obtained results indicates that all of the ste-
reoisomers of MF-8 formed a strong, and conserved during the whole
simulation, interaction with D3.32 (ASP93). This interaction has been
indicated as crucial for 5-HT7R activity by many previous studies [30].
For stereoisomer MF-8D, it is also characteristic the relatively strong
interaction with CLI residues from the ELC2 (CYS162, LEU163, ILE164)
that was also not lost during the whole simulation time. The most active
MF-8A formed statistically the strongest interaction with E 7x34 (GLU
257), S 6x55 (SER 238) and F 3x28 (PHE 89).
In order to insight into the molecular interactions of stereoisomers
(MF-8A–MF-8D) with the target 5-HT7R and CYP2C9 as selected ADME-
Tox target docking and molecular dynamic (MD) simulations have been
performed. These computer-aided studies were also performed in terms
to confirm the configurations found on the basis of a comparison of
experimental affinity results to those of simulated using the compounds
with defined configurations at C5 and C7, respectively (Fig. 4).
2.5.1. Docking studies and MD simulation to 5-HT7 homology model
All the MF-8 stereoisomers were docked into 5-HT7R homology
models to analyze the differences in protein–ligand interaction. The
docking poses and, additionally, interaction matrices of ligand-receptor
contacts are presented in Fig. 12.
2.5.2. Docking studies and MD simulation to CYP2C9
In general, the most active compound (MF-8A), with Ki values to-
wards 5-HT7R of 3 nM adopted significantly different docking poses in
comparison to other stereoisomers tested. It is located deepest in the
binding site, although, similarly to MF-8B, MF-8C, and MF-8D, it still
formed interactions with amino acids from the second extracellular loop
(ECL2). MF-8A also interacted with the highest number of amino acids
from the third transmembrane helix (TM3), the lowest number of amino
acids from the 7th transmembrane helix (TM7) and did not interact with
F 6x52. All of the compounds formed charged assisted hydrogen bond
with D3.32 residue and possessed aromatic pi-pi stacking interactions
with F6.51. MF-8C, that was also quite active towards 5-HT7R (Ki = 26
nM), interacted only with one amino acid from the ECL2, while with
more amino acids from the TM7 and demonstrated different orientation
of the methoxyl group, as main differences in docking poses in com-
parison to the remaining compounds. Interestingly, although MF-8B and
MF-8D displayed significantly different affinity towards 5-HT7R (Ki =
44 nM and 366 nM, respectively), they adopted similar docking poses
with fluorine atoms pointing towards the ECL2.
Further molecular modeling studies (docking and MD simulations)
were performed in order to explain the observed dependencies between
configuration of particular MF-8 stereoisomers and their influence on
CYP2C9 activity (Fig. 10c). As a reference, a set of CYP2C9 substrates
from the ChEMBL database [31] were also docked to the respective
protein. They were selected on the basis of the following criteria:
description of activity by AC50 parameter and the reported AC50 value
below 100 nM. The docking poses obtained for particular MF-8 stereo-
isomers are presented in Fig. 13.
The main differences in docking poses that are observed between
MF-8D (depicted in green, compound inducing the CYP2C9 activity to
the least extent) and other forms of MF-8 (depicted in orange, cyan and
magenta for MF-8A, MF-8B, and MF-8C, respectively) are connected
with bend conformation of MF-8D, although the set of amino acids
interacting with particular compounds examined is similar. However,
the position of aromatic rings is significantly different for MF-8D in
comparison to other stereoisomers. The more detailed analysis of
docking results is provided by ligand interaction diagrams presented in
the Supporting Information (Fig. S12). On the other hand, MF-8C, the
As docking results do not fully explained observed relationships
between compound structure and activity, MD simulations were carried
8

K. Kucwaj-Brysz et al.
Bioorganic Chemistry xxx (xxxx) xxx
the presented data. In the light of all the herein described results and the
fact that the racemic MF-8 showed significant antidepressant effects in
mice, the corresponding pharmacological assays in vivo for particular
stereoisomers (MF-8A) would be purposeful in the close future.
4. Experimental
4.1. Chemical synthesis
1H NMR and 13C NMR spectra were recorded on a Varian Mercury VX
300 MHz PFG instrument (Varian Inc., Palo Alto, CA, USA) in DMSO‑d6
at ambient temperature using the solvent signal as an internal standard.
Data are reported using following abbreviations: s, singlet; bs, broad
singlet; d, doublet; t, triplet; def, deformated; Ph, phenyl; Pp, piperazine;
Ar, aromatic. Thin-layer chromatography (TLC) was performed on pre-
coated Merck silica gel 60 F254 aluminium sheets, and the solvent
system used was methylene chloride (DCM)/methanol (MeOH) 95:5.
The mass of compounds were recorded on a Waters ACQUITYTM UPLC
(Waters, Milford, MA, USA) coupled to a Waters TQD mass spectrometer
(electrospray ionization mode, EDI-tandem quadrupole). Retention
times (tR) are given in minutes. The UPLC/MS purity of all final com-
pounds were determined (%). The reaction conditions for Bucherer-
Berg’s condensation and N-alkylation were applied according to already
described procedure [20,21]. Physicochemical data for both oxiranes
are presented in Supplementary Information.
Fig. 13. Docking poses obtained for examined compounds to CYP2C9 – MF-8A:
orange, MF-8B: cyan, MF-8C: magenta, and MF-8D: green. (For interpretation
of the references to colour in this figure legend, the reader is referred to the web
version of this article).
least stable compound in terms of CYP2C9-mediated metabolism
adopted similar docking pose to MF-8A that was three times more stable
than MF-8C; therefore, analogously to analysis of compounds activity
towards 5-HT7R, MD simulations were carried out (Fig. S13).
Comparing results of MD simulations, especially for MF-8A and MF-
8C that adopted similar docking pose, but displayed significantly
different influence on CYP2C9 activity, revealed change in the orienta-
tion in the binding site for the former compound, resulting in making
contacts with PHE100, LEU102, ALA103, ARG108, and ALA297, which
was not observed for more stable MF-8A.
4.1.1. General procedure for condensation of oxiran with 1-(2-
methoxyphenyl)piperazine
The starting materials: 5-(4-fluorophenyl)-5-methyl-3-(((S)oxiran-2-
yl)methyl)imidazolidine-2,4-dione (3.5 mmol) and 1-(2-methox-
yphenyl)piperazine (3.0 mmol) were dissolved in isopropanol in round-
bottom flask and refluxed for 30 min. The reaction progress was moni-
tored with TLC (DCM/MeOH 95:5). After the completion of reaction, the
mixture was concentrated under reduced pressure. The crude product
was dissolved in DCM and washed with water and brine. The organic
phase was dried oved Na2SO4, filtered and concentrated under reduced
pressure. The analogous procedure were applied using 5-(4-fluo-
rophenyl)-5-methyl-3-(((R)oxiran-2-yl)methyl)imidazolidine-2,4-dione
as starting material.
3. Conclusions
Within this comprehensive study, the all four stereoisomers of pre-
viously described, highly potent 5-HT7R ligand with antidepressant ac-
tivity on mice (MF-8), were isolated and biologically evaluated in terms
of pharmacodynamic and pharmacokinetic profile. As the configuration
for one of two present chiral centers was not possible to be determined
experimentally, DFT calculations of 1H NMR chemical shifts for opti-
mized stereoisomers geometries, based on the crystal structure of MF-8,
have been applied to perform appropriate predictions, while docking
and dynamic simulations in comparison to experimental screening re-
sults, were used to confirm that prediction. The obtained experimental
data for respective stereoisomers (MF-8A-MF-8D) have indicated the
significant influence of stereochemistry on either the 5-HT7R activity or
ADME-Tox properties in vitro. The affinity for 5-HT7R differed in the
range of 3–366 nM of Ki values, with respect to configurations at both,
hydantoin position C5 or linker position C7. The docking studies showed
significantly deeper location of MF-8A (5R, 7R), the most potent ste-
reoisomer (Ki = 3 nM), in the 5-HT7R binding site in comparison to other
stereoisomers, thus elucidating its high affinity and confirming the
predicted configuration. The obtained results has also confirmed
stereochemistry-dependent influence of the tested compounds set on P-
glycoprotein efflux, absorption in Caco-2 model, metabolic pathway,
CYP3A4 and CYP2C9 activity, thus pharmacokinetic profile of racemate
is the combination of properties coming from particular stereoisomers.
The comprehensive results of ADME-Tox and radioligand binding assays
in vitro studies have demonstrated the best profile for the stereoisomer
MF-8A with estimated configuration of (5R, 7R).
The resulted two pairs of diastereoisomers were separated using
supercritical fluid chromatography (SFC) technique at Jagiellonian
Center of Innovation. The particular parameters for analytical and pre-
parative conditions are presented in Table 4. The appropriate spectral
analyses are included in Supplementary information. For both SFC
separations 100 mg of samples were used resulting with 16 mg of MF8-
A, 12 mg of MF8-B, 7 mg of MF8-C and 13 mg of MF8-D.
4.1.1.1. (R)-5-(4-fluorophenyl)-3-((R)-2-hydroxy-3-(4-(2-methox-
yphenyl)piperazin-1-yl)propyl)-5-methylimidazolidine-2,4-dione
(MF-
8A). White solid. LC/MS±: purity 100%, tR 4.18 (ESI) m/z [M + H]+
457.24. 1H NMR δ (ppm): 1.70 (s, 3H, 5-CH3), 2.28–2.50 (m, 2H, Pp-
CH2), 2.92–3.08 (m, 8H, Pp-H), 3.41 (br. s, 2H, N3-CH2), 3.78 (s, 3H,
OCH3), 3.90–4.20 (m, 1H, CHOH), 4.85 (br. s, 1H, CHOH), 6.89 (s, 2H,
5-Ph-3,5-H), 6.95 (s, 2H, 5-Ph-2,6-H), 7.20–7.26 (t def., 2H, PpPh-4,6-
H), 7.53–7.57 (m, 2H, PpPh-3,5-H), 8.96 (br.s, 1H, N1-H)
4.1.1.2. (S)-5-(4-fluorophenyl)-3-((R)-2-hydroxy-3-(4-(2-methox-
yphenyl)piperazin-1-yl)propyl)-5-methylimidazolidine-2,4-dione
(MF-
8B). White solid. LC/MS±: purity 98.94%, tR 4.15 (ESI) m/z [M + H]+
457.24. 1H NMR δ (ppm): 1.69 (s, 3H, 5-CH3), 2.28 (t def., 2H, Pp-CH2),
2.50 (br. s, 4H, Pp-2,6-H), 2.91 (br. s, 4H, Pp-3,5-H), 3.40 (br. s, 2H, N3-
CH2), 3.77 (s, 3H, OCH3), 3.91 (br. s, 1H, CHOH), 4.86 (br. s, 1H,
CHOH), 6.87 (s, 2H, 5-Ph-3,5-H), 6.93 (s, 2H, 5-Ph-2,6-H), 7.20–7.23 (t
def., 2H, PpPh-4,6-H), 7.54–7.58 (m, 2H, PpPh-3,5-H), 8.90 (br. s, 1H,
N1-H).
Although the DFT–aided simulation performed seems to correlate
well with the docking results in terms of assignment of absolute con-
figurations of chiral center at hydantoin (C5), further experimental
determination of the proposed configuration is needed to fully confirm
9

K. Kucwaj-Brysz et al.
Bioorganic Chemistry xxx (xxxx) xxx
Table 4
The parameters for analytical and preparative separation of both pairs of MF-8′s diastereoisomers (MF-8A-B and MF-8C-D).
Parameter
Analytical method
Preparative method
Equipment and
software
Acquity UPC2 Waters, PDA Detector Type HPLC 2998 800 nm,
Empower 3 Software Build 3471 SPs
Diacel Chiralpac IC 3 um 3.0 × 150 mm
2000 psi
Fully automated preparative SFC system connected with PDA detector (2998
Waters) and Mass detector (Acquity QDa). MassLynx Software V.4.1
Diacel Chiralpac IC 5 um 20.0 × 250 mm
Column
Pressure ABPR
Oven temperature
Wavelength
Solvent B
120 bar
38 ◦
C
38 ◦
C
254 nM; 3D scan
254 nM; 3D scan
MeOH + 0,5% TEA
MeOH + 0,5% TEA
Diastereoisomers MF-8A-B
Diastereoisomers MF-8C-D
Diastereoisomers MF-8A-B
Diastereoisomers MF-8C-D
Time
20 min
1.5 ml/min
85
15 min
1.5 ml/min
85
20 min
100 ml/min
85
15 min
100 ml/min
85
Flow rate
Mobile phase
% CO2
%B
15
15
15
15
4.1.1.3. (R)-5-(4-fluorophenyl)-3-((S)-2-hydroxy-3-(4-(2-methox-
yphenyl)piperazin-1-yl)propyl)-5-methylimidazolidine-2,4-dione
(10–10–10ꢀ 4 M). The inhibition constants (Ki) were calculated from the
Cheng-Prusoff equation [32]. For all the binding assays, results were
expressed as means of at least two separate experiments (SD ≤ 19%).
(MF-
8C). White solid. LC/MS±: purity 100%, tR 4.17 (ESI) m/z [M + H]+
457.24. 1H NMR δ (ppm): 1.68 (s, 3H, 5-CH3), 2.33–2.40 (m, 2H, Pp-
CH2), 2.91–3.09 (m, 8H, Pp-H), 3.40 (br. s, 2H, N3-CH2), 3.77 (s, 3H,
OCH3), 3.92 (br.s, 1H, CHOH), 4.87 (br. s, 1H, CHOH), 6.87 (s, 2H, 5-Ph-
3,5-H), 6.93 (s, 2H, 5-Ph-2,6-H), 7.21–7.26 (m, 2 h, PpPh-4,6-H),
7.52–7.56 (m, 2H, PpPh-3,5-H), 8.92 (br.s, 1H, N1-H).
4.2.2. Functional assays
The functional properties of compounds MF-8rac and MF-8A-MF-8D
on 5-HT7bR were evaluated using their ability to inhibit cAMP produc-
tion induced by 5-CT (10 nM) – a 5-HT7bR agonist, in HEK-293 cells
overexpressing 5–HT7bR. Each compound was tested in triplicate at 8
concentrations (10ꢀ 11 – 10ꢀ 4 M).
4.1.1.4. (S)-5-(4-fluorophenyl)-3-((S)-2-hydroxy-3-(4-(2-methox-
Cells (prepared with the use of Lipofectamine 2000) were main-
tained at 37 ◦C in a humidified atmosphere with 5% CO2 and were
grown in Dulbeco’s Modifier Eagle Medium containing 10% dialysed
foetal bovine serum and 500 µg/ml G418 sulphate. For functional ex-
periments, cells were subcultured in 25 cm flasks, grown to 90%
confluence, washed twice with prewarmed to 37 ◦C phosphate buffered
saline (PBS) and were centrifuged for 5 min (160 × g). The supernatant
was aspirated, the cell pellet was resuspended in stimulation buffer (1 ×
HBSS, 5 mM HEPES, 0.5 mM IBMX, 0.1% BSA). Total cAMP was
measured using the LANCE cAMP detection kit (PerkinElmer), according
to the producer’s directions. For cAMP levels quantification, cells (5 µl)
were incubated with compounds (5 µl) for 30 min at room temperature
in 384-well white opaque microtiter plate. After incubation, the reaction
was stopped and cells were lysed by the addition of 10 µl working so-
lution (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) was detected by an Infinite M1000
Pro (Tecan) using instrument settings from LANCE cAMP detection kit
manual.
yphenyl)piperazin-1-yl)propyl)-5-methylimidazolidine-2,4-dione
(MF-
8D). White solid. LC/MS±: purity 97.82%, tR 4.18 (ESI) m/z [M + H]+
457.17. 1H NMR δ (ppm): 1.67 (s, 3H, 5-CH3), 2.31–2.32 (d def., 2H, Pp-
CH2), 2.89–3.20 (m, 8H, Pp-H), 3.38–3.40 (t def., 2H, N3-CH2), 3.77 (s,
3H, OCH3), 3.90–3.96 (qu def., 1H, CHOH), 4.84–4.85 (d def., 1H,
CHOH), 6.85 (s, 2H, 5-Ph-3,5-H), 6.92 (s, 2H, 5-Ph-2,6-H), 7.20–7.24 (t
def., 2 h, PpPh-4,6-H), 7.52–7.56 (m, 2H, PpPh-3,5-H), 8.90 (br.s, 1H,
N1-H).
4.2. Receptors studies in vitro
4.2.1. Affinities for human 5-HT1A and 5-HT7b receptors
HEK-293 cells with stable expression of human 5-HT1A, 5-HT7b re-
ceptors (prepared with the use of Lipofectamine 2000) were maintained
at 37 ◦C in a humidified atmosphere with 5% CO2 and grown in Dul-
becco’s Modifier Eagle Medium containing 10% dialyzed fetal bovine
serum and 500 µg/ml G418 sulfate. For membrane preparation, cells
were subcultured in 150 cm2 flasks, grown to 90% confluence, washed
twice with phosphate buffered saline (PBS) prewarmed to 37 ◦C and
pelleted by centrifugation (200 g) in PBS containing 0.1 mM EDTA and
1 mM dithiothreitol. Prior to membrane preparation, pellets were stored
at ꢀ 80 ◦C. Cell pellets were thawed and homogenized in 10 volumes of
assay buffer using an Ultra Turrax tissue homogenizer and centrifuged
twice at 35,000 × g for 15 min at 4 ◦C, with incubation for 15 min at
37 ◦C in-between. The composition of the assay buffers was as follows:
for 5-HT1AR: 50 mM Tris HCl, 0.1 mM EDTA, 4 mM MgCl2, 10 µM
pargyline and 0.1% ascorbate and for 5- HT7bR: 50 mM Tris HCl, 4 mM
MgCl2, 10 µM pargyline and 0.1% ascorbate. All the assays were incu-
bated in a total volume of 200 µl in 96-well microtiter plates for 1 h at
37 ◦C, except those for 5-HT1AR which were incubated at room tem-
perature. The process of equilibration was terminated by rapid filtration
through Unifilter plates with a FilterMate Unifilter 96 Harvester (Per-
kinElmer, USA). The radioactivity bound to the filters was quantified on
a Microbeta TopCount instrument (PerkinElmer, USA). For competitive
inhibition studies, the assay samples contained the following as radio-
ligands (PerkinElmer, USA): 2.5 nM [3H]-8-OH-DPAT (135.2 Ci/ mmol)
for 5-HT1AR; 0.8 nM [3H]-5-CT (39.2 Ci/mmol) for 5-HT7R or 2.5 nM
[3H]-raclopride (76.0 Ci/mmol) for D2LR. Non–specific binding was
defined with 10 µM of 5-HT in 5-HT1AR and 5-HT7R binding experi-
ments. Each compound was tested in triplicate at 7 concentrations
4.3. Crystallographic studies
Single crystals suitable for an X-ray analysis were obtained from 2-
methylpropan-1-ol by slow evaporation of the solvent at room
temperature.
The intensity data for single crystal were collected using the Oxford
Diffraction SuperNova four circle diffractometer, equipped with the Mo
(0.71069 Å) K
α radiation source and graphite monochromator. The
phase problem was solved by direct methods using SIR-2014 [33] and all
non-hydrogen atoms were refined anisotropically using weighted full-
matrix least-squares on F2. Refinement and further calculations were
carried out using SHELXL [34]. The hydrogen atoms bonded to carbon
atoms were included in the structure at idealized positions and were
refined using a riding model with Uiso(H) fixed at 1.2 Ueq of C with the
exception of hydrogen atoms in methyl group for which Uiso(H) fixed at
1.5 Ueq. Hydrogen atoms attached to nitrogen and oxygen atoms were
found from the difference Fourier map and refined without any re-
straints. The molecule of 2-methylpropan-1-ol is disordered. The occu-
pancy factors of the carbon atoms were refined to be 0.62 and 0.38 for
the major and minor components, respectively. For molecular graphics
MERCURY [35] program was used.
10

K. Kucwaj-Brysz et al.
Bioorganic Chemistry xxx (xxxx) xxx
C24H31FN4O+4 Cl‾⋅C4H9OH, Mr = 567.09, crystal size = 1.17 × 0.76
× 0.07 mm3, monoclinic, space group P21/c, a = 17.1443(3) Å, b =
18.8374(3) Å, c = 9.2142(2) Å, V = 2937.2(4) Å3, Z = 4, T = 130(2)K,
40,226 reflections collected, 7109 unique reflections (Rint = 0.0390),
4.6.1.1. Caco-2 absorption assay. Caco-2 (ATCC® HTB-37™) cell line
was purchased from American Type Culture Collection (Manassas, VG,
USA). The cells were cultivated in Dulbecco’s Modified Eagle s Medium
(DMEM) supplemented with 10% fetal bovine serum (FBS) in a hu-
midified atmosphere of 5% CO2. The medium was changed every two
days and the cells were subcultured at 70–80% confluence. The Corn-
ing® 3413 Transwell® 6.5 mm polycarbonate membrane inserts with
0.4 µm pore were purchased from Sigma-Aldrich (Saint Louis, MO,
USA). The inserts were pretreated first with 50 µl of the medium for two
minutes. Next, the 200 µl of cells were seeded at 2 × 10ꢀ 4 concentration
per insert in apical compartment, whereas 600 µl was added to the
basolateral one. The plate was incubated at 37 ◦C for 14 h and the non-
adherent cells were removed. TEER (Transepithelial Electrical Resis-
tance) measurements was started from 18-day after seeding by Millicell
ERS-2 Volt-Ohm Meter (Merck Millipore, Burlington, MA, USA). The
proper monolayer integrity was determined at 20-day after seeding.
Then, the monolayer was rinsed with HBSS and the tested compounds as
well as highly permeable reference caffeine was added at 10 µM con-
centration in HBSS into the apical chambers. The 600 µl of HBSS was
added to the basolateral compartments. Lucifer yellow (5 µM) was also
added to the apical chambers as the membrane integrity marker. The
plate was placed in the orbital shaker (60 rpm) for 2 h at 37 ◦C. The
compounds’ concentrations in apical and basolateral wells were
analyzed using LC-MS method with internal standard (IS). To confirm
the membrane integrity the fluorescence was measured in basolateral
compartment by EnSpire multiplate reader (Perkin Elmer, Waltham,
MA, USA).
R1 = 0.0537, wR2 = 0.1348 [I > 2σ(I)], R1 = 0.0728, wR2 = 0.1503 [all
data].
CCDC 1958223 contains the supplementary crystallographic data.
These data can be obtained free of charge from The Cambridge Crys-
tallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
4.4. DFT calculations methodology
Geometries of the MF-8 stereoisomers were optimized with the
hybrid meta-GGA TPSSh [36] functional combined with the triple-ζ
valence def2-TZVPP basis set [37]. The starting structure of the (5R,7R)
stereoisomer (basic form) was prepared from the crystallographic data
for the cationic form of the (5R,7R) MF-8. By respective modification,
the starting structures of other stereoisomers were built. The TPSSh
functional was selected on the basis of test calculations, where the ac-
curacy of six DFT methods in predicting geometry of the basic form of
previously obtained similar compound KKB-4 (CCDC 1831620) [21]
was examined. The obtained mean unsigned errors (MUE) in bond
lengths, referred do the experimental crystal structure, were 0.0077,
0.0080, 0.0093, 0.0088, 0.0076 and 0.0083 Å for the B3LYP, B3PW91
[38], LC-ωPBE [39], PBE0 [40], TPSSh and ωB97X-D [41] functionals,
respectively. Harmonic vibrational frequencies were calculated for each
structure to confirm the potential energy minimum.
The permeability Papp were calculated according to the following
formula [54]:
Based on the previous works [42–44], the hybrid meta-GGA M06
functional [45] and the def2-TZVPP basis set were chosen to calculate
absolute 1H shielding constants for the (5R,7R), (5R,7S), (5S,7R) and
(5S,7S) stereoisomers of MF-8 using the GIAO method [46,47]. Solvent
effects (DMSO) were included by applying the polarizable continuum
model (PCM) [48]. The chemical shifts were calculated using 1H
shielding constants for DMSO as the reference (assuming the 1H NMR
shift of 2.50 ppm, according to the experimental procedure) computed
at the same level of theory. All DFT calculations were performed using
the Gaussian 16 software [49].
Papp = dc/dt*V/(A*C0)
dc/dt- the change in concentration in the receiving compartment
overtime
V- volume of the solution in the receiving compartment (mL)
A- surface area of the membrane (cm2)
C0- the initial concentration in the donor compartment (µM)
4.6.1.2. PAMPA. Pre-coated PAMPA Plate System Gentest™ purchased
from Corning (Tewksbury, MA, USA) was used for permeability evalu-
ation. The tested compounds and references solutions were prepared in
4.5. Docking and MD simulations
PBS buffer (pH = 7.4) and added to the donor wells (200 µM, 300 μl/
The three-dimensional conformations of compounds and respective
protonation states (for pH 7.4 +/- 0.0) were generated with the use of
LigPrep [50]. The docking was carried out in Glide [51] in extra preci-
sion mode. MD simulations (using Schrodinger’s Desmond software
[52]) were carried out for each of the obtained ligand-receptor com-
plexes (duration time = 100 ns for 5-HT7R and 200 ns for simulations
with CYP2C9; TIP3P [53] as solvent model and POPC (palmitoyl-oleil-
phosphatidylcoline) as membrane model used for 5-HT7R). The in-
teractions between ligands and the respective protein occurring during
whole simulations were analyzed using Simulation Interaction Diagram
from the Schrodinger Suite.
well). PBS (200
μl/well) was added to the acceptor wells. All compounds
were incubated in triplicate at room temperature for 5 h. Then, the 50
μl
was aspirated from each well and diluted next with 50 µl solution of IS.
The compounds concentrations in both acceptor and donor wells were
estimated by the UPLC-MS analyses, which were performed by UPLC/
MS Waters ACQUITY™ TQD system with the TQ Detector (Waters,
Milford, USA). The permeability coefficients (Pe, cm/s) were calculated
according described previously formulas [55].
4.6.1.3. Pgp assay. The luminescent Pgp-Glo™ Assay System was pur-
chased from Promega (Madison, WI, USA). The assay was performed in
triplicate according to the protocol provided by manufacturer. MF-8rac
4.6. In vitro ADME-Tox studies
and stereoisomers (100 μM) were incubated with Pgp membranes for 40
min at 37 ◦C. The luminescence signal was measured by microplate
reader EnSpire PerkinElmer (Waltham, MA, USA).
The ADME-Tox studies excluding Caco-2 absorption tests were car-
ried out as described previously [19,23,26,27].
4.6.1.4. Metabolic stability. The intrinsic clearance (CLint) of MF-8rac
and stereoisomers were estimated by incubation with mouse liver mi-
crosomes (MLMs) purchased from Sigma-Aldrich (St. Louis, MO, USA).
The disappearance of compounds (50 µM) in the presence of MLMs (1
mg/ml) was determined at 5, 15, 30 and 45 min of incubation in 10 mM
Tris–HCl buffer (37 ◦C). The UPLC/MS Waters ACQUITY™ TQD system
with the TQ Detector (Waters, Milford, USA) analysis with use of IS
allowed for determination of tested compound elimination. The t1/2
4.6.1. References
The following references used in ADME-Tox assays: caffeine (CFN),
carbonyl cyanide 3-chlorophenylhydrazone (CCCP), doxorubicin (DX),
ketoconazole (KE), nonyl-4-hydroxyquinoline-N-oxide (NQNO), nor-
floxacin (NFX), quinidine (QD) and sulfaphenazole (SE) were purchased
from Sigma-Aldrich (St. Louis, MO, USA). Verapamil (VL) and Na3VO4
used in Pgp activity studies were provided by Promega (Madison, WI,
USA).
11

K. Kucwaj-Brysz et al.
Bioorganic Chemistry xxx (xxxx) xxx
D. Nepomuceno, A. Ahnaou, W. Drinkenburg, W. Chai, C. Dvorak, S. Sands,
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values and CLint were calculated by protocols and formulas proposed by
Obach [29].
The metabolic pathways evaluation of MF-8rac and stereoisomers
was performed by prolonged, 120 min incubation with MLMs. The
concentration of compounds, the ingredients of the reaction mixture and
UPLC/MS analyses conditions were similar to the described above.
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CYP2D6 and CYP2C9 P450-Glo™ assays were purchased from Prom-
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manufacturer recommendations. The compounds were tested in tripli-
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purchased from Promega (Madison, WI, USA). The absorbance was
measured using a microplate reader EnSpire (PerkinElmer, Waltham,
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Declaration of Competing Interest
[18] J. Handzlik, A.J. Bojarski, G. Satała, M. Kubacka, B. Sadek, A. Ashoor, A. Siwek,
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.
´
M. Więcek, K. Kucwaj, B. Filipek, K. Kiec-Kononowicz, SAR-studies on the
importance of aromatic ring topologies in search for selective 5-HT7 receptor
ligands among phenylpiperazine hydantoin derivatives, Eur. J. Med. Chem. 78
(2014) 324–339, https://doi.org/10.1016/j.ejmech.2014.01.065.
´
[19] K. Kucwaj-Brysz, D. Warszycki, S. Podlewska, J. Witek, K. Witek, A. Gonzalez
Izquierdo, G. Satała, M.I. Loza, A. Lubelska, G. Latacz, A.J. Bojarski, M. Castro,
Acknowledgements
´
K. Kiec-Kononowicz, J. Handzlik, Rational design in search for 5-phenylhydantoin
selective 5-HT7R antagonists. Molecular modeling, synthesis and biological
evaluation, Eur. J. Med. Chem. 112 (2016) 258–269, https://doi.org/10.1016/j.
ejmech.2016.02.024.
This study was financially supported by Polish National Science
Centre (NCN) grant No UMO-2014/15/N/NZ7/03072. SP was sup-
ported by the Foundation for Polish Science (FNP) within the START
scholarship and by Polish Statutory Research Project N42/DBS/000027.
˙
[20] K. Kucwaj-Brysz, R. Kurczab, M. Jastrzębska-Więsek, E. Zesławska, G. Satała,
´
W. Nitek, A. Partyka, A. Siwek, A. Jankowska, A. Wesołowska, K. Kiec-
Kononowicz, J. Handzlik, Computer-aided insights into receptor-ligand interaction
for novel 5-arylhydantoin derivatives as serotonin 5-HT 7 receptor agents with
antidepressant activity, Eur. J. Med. Chem. 147 (2018) 102–114, https://doi.org/
10.1016/j.ejmech.2018.01.093.
Appendix A. Supplementary material
˙
´
[21] K. Kucwaj-Brysz, R. Kurczab, E. Zesławska, A. Lubelska, M.A. Marc, G. Latacz,
´
G. Satała, W. Nitek, K. Kiec-Kononowicz, J. Handzlik, The role of aryl-topology in
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2020.104466.
balancing between selective and dual 5-HT 7 R/5-HT 1A actions of 3,5-substituted
hydantoins, Med. Chem. Commun. 9 (6) (2018) 1033–1044, https://doi.org/
10.1039/c8md00168e.
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