Heterocyclic Analogues of Modafinil as Novel, Atypical Dopamine Transporter Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.7b01313

Modafinil is a wake promoting compound with high potential for cognitive enhancement. It is targeting the dopamine transporter (DAT) with moderate selectivity, thereby leading to reuptake inhibition and increased dopamine levels in the synaptic cleft. A s

Full text of DOI:10.1021/acs.jmedchem.7b01313

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Article
Heterocyclic Analogues of Modafinil as Novel,
Atypical Dopamine Transporter Inhibitors
Predrag Kalaba, Nilima Y. Aher, Marija Ili#, Vladimir Draga#evi#, Marcus Wieder, Andras
G. Miklosi, Martin Zehl, Judith Wackerlig, Alexander Roller, Tetyana V. Beryozkina, Bojana
Radoman, Sivaprakasam R Saroja, Wolfgang Lindner, Eduardo Perez Gonzalez, Vasiliy A.
Bakulev, Johann Jakob Leban, Harald H. Sitte, Ernst Urban, Thierry Langer, and Gert Lubec
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01313 • Publication Date (Web): 01 Nov 2017
Downloaded from http://pubs.acs.org on November 3, 2017
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Heterocyclic Analogues of Modafinil as Novel,
Atypical Dopamine Transporter Inhibitors
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†,§
†,§
†
†
†
Predrag Kalaba , Nilima Y. Aher , Marija Ilić , Vladimir Dragačević , Marcus Wieder ,
†
l
†
◊
‡
Andras G. Miklosi , Martin Zehl , Judith Wackerlig , Alexander Roller , Tetyana Beryozkina ,
†
•
l
#
Bojana Radoman , Sivaprakasam R. Saroja , Wolfgang Lindner , Eduardo Perez Gonzalez ,
‡
†
♦
†
†,*
Vasiliy Bakulev , Johann Jakob Leban , Harald H. Sitte , Ernst Urban , Thierry Langer , Gert
&,*
Lubec
†
Department of Pharmaceutical Chemistry, Faculty of Life Sciences, University of Vienna,
Althanstraße 14, 1090, Vienna, Austria
‡
Ural Federal University named after the first President of Russia B. N. Yeltsin, 19 Mira st.,
Yekaterinburg 620002, Russia
l
Department of Analytical Chemistry, Faculty of Chemistry, University of Vienna, Währinger
Straße 38, 1090, Vienna, Austria
◊
Xꢀray Structure Analysis Centre, Faculty of Chemistry, University of Vienna, Währinger Straße
38, 1090, Vienna, Austria
•
Department of Pediatrics, Medical University of Vienna, 1090 Vienna, Austria;
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#
Laboratory of Fine Organic Chemistry, Department of Chemistry and Biochemistry, Faculty of
Science and Technology, University of Sao Paulo State, Roberto Simonsen 305, CEP 19060ꢀ900,
Presidente Prudente, SPꢀBrazil
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♦
Institute of Pharmacology, Centre of Physiology and Pharmacology, Medical University of
Vienna, 1090, Vienna, Austria
&
Paracelsus Medical University, Neuroscience Laboratory, A ꢀ5020 Salzburg
ABSTRACT
Modafinil is a wake promoting compound with high potential for cognitive enhancement. It is
targeting the dopamine transporter (DAT) with moderate selectivity thereby leading to reꢀuptake
inhibition and increased dopamine levels in the synaptic cleft. A series of modafinil analogs
have been reported so far, but more targetꢀspecific analogues remain to be discovered. It was the
aim of this study to synthesize and characterize such analogues and indeed, a series of
compounds were showing higher activities on the DAT and a higher selectivity towards DAT
versus serotonin and norepinephrine transporters than modafinil. This was achieved by
substituting the amide moiety by five and six membered aromatic heterocycles. In vitro studies
indicated binding to the cocaine pocket on DAT, although molecular dynamics revealed binding
different to that of cocaine. Moreover, no release of dopamine was observed ruling out
amphetamineꢀlike effects. The absence of neurotoxicity of a representative analogue may
encourage further preclinical studies of the abovementioned compounds.
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ꢀ
INTRODUCTION
Modafinil, i.e. 2ꢀ[(diphenylmethyl)sulfinyl]acetamide, (1) (Figure 1) is an FDAꢀapproved
1
, 2, 3
drug for treatment of narcolepsy and other sleep disorders
. It is a mild psychostimulantꢀlike
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agent that increases wakefulness, improves attention and enhances performance in a variety of
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, 4
cognitive tasks . It has been shown to reduce jet lag and improve mood among shift workers. It
has also been studied as an alternative to amphetamines for military usage and is currently also
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, 2
approved for Air Force missions in the US .
Figure 1. Structure of 2-[(diphenylmethyl)sulfinyl]acetamide (modafinil).
It's mode of action remained unknown for long time, despite of several years of
3
, 5
preclinical research . Using radioligand binding assays, Mignot et al. (1994) have demonstrated
that modafinil binds to the dopamine transporter (DAT) and not to any other transporters and
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receptors tested in their study . Zolkowska et al. (2009) performed an in vitro binding study and
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concluded that the DAT was the exclusive target at which modafinil displayed relevant binding .
Loland et al. (2012) showed that Rꢀ and SꢀModafinil bind to the DAT less potently than cocaine,
8
with RꢀModafinil having an in vitro profile different from cocaine .
Modafinil`s pharmacological profile can be clearly distinguished from those of other
2
, 3, 4
classical psychostimulants
. It binds to DAT in a different way as compared to cocaine, which
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may lead to its unique pharmacological profile and, unlike amphetamine, modafinil does not
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cause efflux of dopamine .
Modafinil has been intensively studied and many of its derivatives have been synthesized
and tested for biological activity. Cao et al. (2010) have synthesized a series of modafinil
analogues by adding paraꢀhaloꢀsubstituents to the aryl rings, modifying the sulfoxide function
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and replacing the primary amide group with secondary and tertiary amides and amines .
OkonulaꢀBakare et al. (2014) synthesized a series of thioacetamide and sulfinylacetamide
analogues and further investigated the role of the terminal amide or substituted amine functions
10
on DAT vs. serotonin transporter (SERT) and norepinephrine transporter (NET) binding . Cao
et al. (2016) further extended the SAR of modafinil analogues by chemical modifications of the
oxidation states of sulfoxide and the amide functional groups, by halogenating the phenyl rings,
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and/or by functionalizing the terminal nitrogen with substituted piperazines .
The aim of the current study was to generate a novel series of modafinil analogues with
higher dopamine reꢀuptake inhibition activity and higher selectivity at DAT vs. SERT and NET,
by substituting the amide moiety with 5ꢀ and 6ꢀmembered aromatic heterocycles. The most
potent inhibitors from this series were used in release studies to show that these compounds are
not causing efflux of dopamine (amphetamineꢀlike effect). Furthermore, binding studies were
carried out to determine whether these compounds are targeting the same binding pocket as
cocaine. Molecular docking was performed using a homology model of the DAT in order to
propose the binding site of two compounds that showed significant dopamineꢀreuptake inhibition
in vitro. Subsequently, molecular dynamics (MD) simulations with the highest scoring ligand
poses were performed. In order to mimic the physiological system, the DAT structural model
was placed in a double lipid membrane. The obtained binding poses for the compounds from
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molecular docking and from MD simulations were compared with already established binding
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sites for the cocaine analog CFT and mephedrone analogs . Furthermore, we aimed to
demonstrate that a selected compound from the series shows no general in vivo neurotoxicity
that may render it suitable for further preclinical development.
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ꢀ
CHEMISTRY
Synthesis
of
novel
2ꢀ[(diphenylmethyl)sulfinyl]thiadiazole
and
[
(benzhydrylsulfinyl)methyl]oxadiazole analogues (4a-e) was achieved as depicted in Scheme 1.
Nucleophilic substitution of commercially available substituted chloroꢀmethylꢀthiodiazoles and
oxadiazole analogues with diphenylmethanethiol (2) in the presence of tꢀBuOK in DMF or
diisopropylethylamine
(DIPEA)
in
1,4ꢀdioxane
provided
substituted
[(benzhydrylsulfenyl)methyl]ꢀthiodiazole and –oxadiazole intermediates (3a-e) in 35ꢀ95% yield.
The oxidation reaction in CH COOH with H O gave final products (4a-e) in 30ꢀ70% yield.
3
2
2
Substituted [(benzhydrylsulfinyl)methyl]ꢀpyridinyl, ꢀisoxazolyl, ꢀpyrimidinyl, ꢀoxadiazolyl,
and ꢀthiophenyl analogues were generated via another synthetic route depicted in Scheme 2.
Alkylation of diꢀphenylmethylꢀSꢀisothiuronium hydrobromide (5) with plain or substituted
chloroꢀmethylꢀheterocycles with K CO in MeOH provided intermediates (6a-q) in 34ꢀ97%
2
3
yield. Oxidation of the sulfenyl moiety gave the desired sulfoxideꢀcontaining analogues (7a-q) in
7ꢀ90% yield.
2
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a
Scheme 1. Synthesis of compounds 3a-e and 4a-e
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a
Reagents and conditions: (a) DMF, tꢀBuOK, r.t. overnight; (b) 1,4ꢀdioxane, DIPEA, r.t. 3 h; (c)
CH COOH, H O , r.t. 24 h.
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Scheme 2. Synthesis of compounds 6a-q and 7a-q
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a
Reagents and conditions: (a) MeOH, K CO , r.t. 48 h; (b) CH COOH, H O , r.t. overnight.
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Unꢀsubstituted and substituted 2ꢀ((benzhydrylsulfinyl)methyl)thiophene analogues were
obtained as depicted in Scheme 3. Condensation of commercially available benzhydrols (8a-f)
with thiophenꢀ2ꢀylmethanethiol (9) and BF •Et O in CH COOH provided analogues with
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sulfenyl moiety (10a-f) in 66ꢀ94% yield. As in the previous synthetic routes, oxidation of
sulfenyl intermediates with H O in CH COOH generated sulfoxide containing products (11a-f)
2
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in 19ꢀ85% yield.
a
Scheme 3. Synthesis of compounds 10a-f and 11a-f
a
Reagents and conditions: (a) BF •Et O, CH COOH, r.t. overnight; (b) CH COOH, H O , r.t.
3
2
3
3
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2
overnight.
Compound 11c was separated further into 2 mixtures 11g and 11h, both containing 2
pairs of stereoizomers. Each mixture was screened on Chiralpack IA HPLC column (Daicel Inc,
Tokyo, Japan) to separate into individual enantiomers. For compound 11g, this was achieved
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using 90% hexane 10% isopropanol as the mobile phase. Biological data is not available because
they were not soluble in stock solution. Compound 11h was separated by HPLC into compounds
11i and 11j, using 60% hexane 40% ethylacetate as the mobile phase.
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ꢀ
RESULTS AND DISCUSSION
SARs at DAT, NET and SERT. All final compounds (4a-e, 7a-q, and 11a-f) were
evaluated for upꢀtake inhibition at the monoamine transporters (DAT, SERT and NET) in
HEK293 cells stably expressing human isoforms of DAT, NET and SERT.
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a
Table 1. Upꢀtake inhibition data in DAT WT, NET WT and SERT WT
IC (µM) ± SD
50
Compound
DAT
11.5 ± 4.3
38.1 ± 2.3
107.6 ± 1.5
NA
43.7 ± 1.4
69.1 ± 4.1
NA
NET
283.6 ± 23.7
85.7 ± 33.5
145.8 ± 6.0
NA
71.0 ± 27.2
30.0 ± 8.0
NA
SERT
NA
NA
NA
NA
1
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4a
4b
4c
4d
4e
209.5 ± 13.4
NA
7a
NA
NA
NA
NA
NA
NA
NA
NA
7
7
7
b
c
d
63.5 ± 6.2
56.7 ± 0.2
3.1 ± 1.7
38.6 ± 11.3
40.4 ± 1.4
109.0 ± 14.4
90.5 ± 9.8
82.6 ± 3.9
155.7 ± 10.0
3.0 ± 0.3
29.3 ± 5.1
4.9 ± 0.3
283.6 ± 66.4
16.6 ± 0.7
12.8 ± 1.0
13.7 ± 1.8
7.2 ± 1.3
1.4 ± 0.4
4.2 ± 0.3
2.0 ± 0.5
4.0 ± 0.6
5.8 ± 1.7
3.7 ± 0.4
2.4 ± 0.8
4.1 ±0.4
NA
NA
NA
NA
NA
NA
7e
7
7
7
f
g
h
227.5 ± 47.0
767.5 ± 77.9
NA
57.6 ± 21.9
144.2 ± 33.2
188.1 ± 85.6
NA
67.2 ± 32.5
68.4 ± 33.0
NA
7i
NA
NA
NA
7
7
7
j
k
l
59.6 ± 2.7
NA
7m
7n
7o
7p
NA
44.7 ± 9.5
57.2 ± 28.1
70.9 ± 38.0
NA
NA
NA
118.4 ± 42.7
120.5 ± 58.5
NA
7q
1
1
1
1a
1b
1c
NA
NA
NA
11d
152.5 ± 15.6
112.6 ± 24.7
NA
1
1
1
1e
1f
1g
NA
NA
NA
NA
NA
NA
NA
11h
11i
1
1j
1.5 ± 0.5
NA
a
IC50 values represents data from at least three independent experiments, each performed in
triplicate. NA ꢀ Not active in used concentration range.
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The inhibition of the uptake of [ H]ꢀdopamine (for DAT), [ H]ꢀ1ꢀmethylꢀ4ꢀ
3
phenylpyridinium (for NET) and [ H]ꢀserotonin (for SERT) was measured as described in detail
in Experimental Procedures.
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Changes in DAT inhibition and selectivity as the result of modifications compared to
modafinil (1) as a parent compound were evaluated; SAR analyses explored the effect of
substituting the amide moiety with heterocycles as well as the influence of introducing of
halogen atoms and methyl groups on phenyl ring(s) in analogues containing the thiophene part.
Results are presented in Table 1.
Substitution of the amide moiety with substituted (and in some cases partially reduced)
thiadiazoles and oxadiazoles led to a decrease of DAT reꢀuptake inhibition compared to
modafinil (4a-b, 4d-e and 7c), with compound 4c being nonꢀspecific to all three transporters
under the same experimental conditions. Modifications with pyridines (7a and 7b) and 5ꢀ
cyclopropylꢀisoxazole (7e) also led to the decrease of DAT reuptake inhibition. However, when
the amide part was replaced with 2ꢀmethylpyrimidineꢀ4ꢀol (7d), an analogue with higher DAT
inhibition potency was obtained. Modifications of the substituent pattern on the pyrimidine
moiety (7f-j) led to a decrease in DAT reuptake inhibition potency.
Substitution with a 2ꢀthiophenylꢀgroup (11b) provided an analogue with higher in vitro
activity on DAT (IC50 = 1.4 µM) and higher selectivity compared with modafinil (1). An
analogue containing a 3ꢀthiophenylꢀgroup (7k) also showed higher activity on DAT (IC = 3.0
5
0
µM) than modafinil, but in contrast to 11b also displayed some affinity towards NET (IC =
5
0
5
7.6 µM). Further modifications of compounds 7k and 11b, by introduction of halogen atoms
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and methyl groups on the thiophene ring, yielded analogues 7l-q. With the exception of 7n (IC50
=
283.6 µM on DAT), those analogues showed only moderately lower activity than 7k and 11b.
Compound 11b was further modified by introducing substitutions on the phenyl rings.
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Introduction of an electronꢀdonating methyl group on a single phenyl in paraꢀ position yielded
compound 11d (IC = 2.0 µM on DAT), while introduction of a methyl group in paraꢀ position
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0
of both phenyl rings yielded compound 11f (IC = 5.8 µM on DAT). Introduction of electronꢀ
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withdrawing groups, such as chlorine and fluorine in paraꢀpositions on both phenyl rings
generated compounds 11a and 11e, respectively, with higher activity on DAT than modafinil
(
IC = 7.2 µM and 4.0 µM, respectively). Introduction of one bromine in paraꢀposition yielded
50
analogue 11c (IC = 4.2 µM on DAT).
50
Compounds 11i and 11j show better activity on DAT than modafinil (IC = 4.1 and 1.6
5
0
µM, respectively). Absolute configuration was attributed by crystallographic data and was
determined to be S,S and R,R for 11i and 11j, respectively.
Compounds 7d and 11b, are enantiomeric compounds. Separation of individual
enantiomers into Rꢀ and Sꢀform could lead to a further gain in binding to DAT.
Examination of amphetamine-like releasing properties. Amphetamines are substrates
14
of DAT which cause the release of dopamine from neuronal cells . To investigate whether our
novel dopamine reuptake inhibitors cause release of dopamine, release assays of two analogues
of choice (7d, 11b) have been performed.
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Figure 2. A release assay was performed to examine whether compounds 7d and 11b have any
ability to release the neurotransmitter from inside the cells, like amphetamine. HEKꢀDAT cells
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+
were grown on PDLꢀcoated coverslips, treated with 0.1 µM [ H]MPP (methylꢀ4ꢀ
phenylpyridinium) at 37 ºC for 20 min and washed with KHB for 40 min in superfusion
chambers. Four experiments were compared, and all four started with three fractions under
untreated conditions (baseline). The next four fractions were either again untreated or treated
with 10 µM monensin. The final five fractions were either treated with 10 µM test compound
(7d or 11b) or 10 µM Dꢀamphetamine. Nonꢀlinear regression analysis was carried out by using
GraphPad Prism 6. Values are presented as mean ± SD.
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As depicted in Figure 2, neither 10 µM 7d nor 10 µM 11b did alter [ H]MPP release
+
opposed to the positive control (10 µM Dꢀamphetamine). Elevation of intracellular Na levels
accelerates substrate efflux, hence we applied 10 µM monensin, an ionophore that promotes
+
+
electroneutral Na /H exchange, to unambiguously confirm the absence of 7d and/or 11bꢀ
induced substrate release. The observations from HEKꢀDAT cells therefore clearly indicate that
7d and 11b specifically block DAT without acting as a substrate and can be clearly distinguished
from amphetamine.
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[
H]WIN 35,428 binding assays. Dixon plots (in which the reciprocal of bound
radioactivity is plotted as a function of one inhibitors concentration at a fixed concentration of
the second inhibitor) allow reading out whether two inhibitors can occupy the same binding site
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simultaneously or whether their binding is mutually exclusive . Dixon plots showed a nearly
parallel shift for 7d in the presence of cocaine, indicating mutually exclusive binding of analogue
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7d and cocaine as shown in Figure 3A
. The same result was obtained for compound 11b as
depicted in Figure 3B.
Figure 3. Mutually exclusive binding of cocaine in the presence of analogues 7d and 11b. Intact
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HEKꢀDAT cells were incubated with the radioligand (∼ 10 nM [ H]WIN 35,428), the indicated
concentrations of 7d or 11b, and increasing concentrations of cocaine in binding buffer.
Nonspecific binding was determined in the presence of 30 µM mazindol (control). The binding
data from the competition experiments is transformed into a Dixon plot by expressing the
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reciprocal of [ H]WIN 35,428 bound (measured in femtomole) as a function of the concentration
of cocaine at a fixed concentration of 7d or 11b. Data are means ± S.D. of 3 experiments
performed in triplicate.
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Molecular docking and MD simulation. The highest scored ligand positions are shown
in Figure 4. The docked ligand positions of compound 7d and 11b are very similar to each other,
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both resemble the putative binding sites of cocaine and the cocaine analog CFT as well as the
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binding site for mephedrone analogs .
Molecular docking is a fast and often reliable tool to investigate possible ligand poses in
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the binding site of a protein, but receptor flexibility is still a challenge for docking protocols .
18, 19
This can be circumvented using MD simulations to evaluate and refine the predicted poses
.
While the simulation time of the MD simulations s sufficient to give a basis for a
validation of the ligand stability at the specific binding site, it cannot be used to validate the
binding and its affinity. Especially motion and/or rearrangement of the protein backbone happen
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on a much longer timescale and are not regarded in MD simulations at the nanosecond scale .
Visual inspection and the monitored RMSD values (provided in the supplementary
materials section) indicate that the systems were stable during the MD simulation (Figure Sꢀ1 in
Supplementary Information). The RMSD analysis shows that the ligand is stable in the binding
site, but both 7d and 11b changed their conformation in the binding site during the first 500 ps
from the initial, docked position shown in Figure 4 subplot A to the conformation shown in
Figure 4 subplot B.
The used docking protocol did not regard the dynamics of the amino acids in the binding
site ꢀ but it could still reproduce the binding position previously described for mephedrone
1
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analogs . Using MD simulations the flexibility of all simulated compounds was explicitly
regarded ꢀ and the binding position of both compounds changed significantly. Especially for a
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subsequent structureꢀbased molecular modeling approach these proposed binding poses are an
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important addition to the already established interaction patterns .
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Figure 4. For compound 7d and 11b the highest scored docking pose (A) and the final pose after
the MD simulation (B) is shown. The labeled amino acids are shown explicitly on the protein
backbone. The ligands move during the MD simulations from their initial position (A) to their
final position (B), however they were found not to leave the dopamine binding site. The
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homology model was based on the crystallized Drosophila DAT, deposited in the Protein Data
Bank with the PDBꢀID 4M48.
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Figure 5. The different interactions between parts of the ligand and amino acids are shown as
heatmap ranging from dark violet (lowest interactions), green (medium interactions) to bright
yellow (highest interactions). Rows indicate part of the ligands and columns the different amino
acids. The first letters before the column indicate the type of interaction, H for hydrophobic
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interaction, HBA for hydrogen bond acceptor, HBD for hydrogen bond donor, AR for aromatic
interaction and PI for positively ionizable interaction.
The interaction maps, shown in Figure 5, for both simulated compounds reveal that the
sulfoxide group is not involved in any interaction with the protein binding site, but interactions
are governed by hydrophobic contacts, piꢀpi and piꢀcation interactions.
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Most prominently for 7d hydrophobic contacts to Phe326 and Phe76 as well as Tyr156
are shown. Hydrogen bond interactions with Ala480, Arg85 and Asp476 occur also throughout
the simulation. In addition to the hydrophobic contact, Arg85 has a stable piꢀcation interaction
with 7d.
The interaction pattern for 11b shows hydrophobic interactions between Phe326, Phe76,
Phe320, Arg85 and Tyr156. There are no hydrogen bond interactions present during the
simulation, other than hydrophobic interactions there are weak aromatic interactions and a piꢀ
cation interaction between Arg85 and 11b. Both compounds have similar hydrophobic
interaction partners (Phe326, Phe76, Tyr156) and piꢀcation interactions with Arg85.
8
Comparing the interaction pattern to Rꢀ and Sꢀmodafinil as described by Loland et al.
some similarities and differences are striking. The reported interaction of the amide moiety of Sꢀ
and Rꢀmodafinil with Phe76 is also present during the MD simulations for 7d and 11b, but the
interaction with Ser321 is not observed. The reported interaction between Rꢀmodafinil and
Tyr156 could be observed for 11b, but not for 7d. These dissimilarities are most likely a result of
the different methods used. Loland et al. utilized inducedꢀfit docking and obtained the
interactions based on the docked pose, while herein MD simulation and analysis of the
interactions during the simulation were carried out.
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Neurotoxicity of compound 11b. In order to evaluate basic neurotoxicity, the open field,
elevated plus maze, rota rod studies and the forced swim test were carried out. Young male rats,
age 10ꢀ12 weeks were divided in two groups: 10 compound 11b treated (treated group) and 10
DMSOꢀtreated animals (control group). Rats were handled during 3 days before beginning of the
experiment. 10 mg/kg body weight of 11b was administered by intraperitoneal injection, 30
minutes before each behavioral test.
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As shown in Figure 6, there were no significant differences between treated and
untreated animals for the parameters total distance travelled, resting time, local and large
movements, average velocity, number of crossings of the center, frequency of spontaneous
changes of direction and time spent at the margin. Analogue results were obtained in the elevated
plus maze test, where no significant differences between treated and untreated rats were observed
regarding the parameters time spent in the left open arm, left closed arm, total distance travelled,
distance travelled in open and closed arms, time spend in the open and closed arm and the resting
time (Figure 7).
Likewise, the 11bꢀtreated group did not show significantly altered behavior in the rotarod
(Figure 8) and forced swim test (Figure 9) compared to the control group.
An observational battery was used to check all important neurological functions as listed
in Table 2. There were no significant differences between controls and the group treated with
11b.
Behavioral assays did not show any behavioral/toxic changes following daily
intraperitoneal administrations of 11b at an intraperitoneal dose of 10 mg/kg body weight. This
makes 11b a promising compound for further development. And indeed, first behavioral studies
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of this compound class showed cognitive enhancement that challenged synthesis and further
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development .
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Figure 6. Open Field study. Total distance travelled, resting time, local and large movements,
average velocity, number of crossings of the center, frequency of spontaneous changes of
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direction and time spent at the margin are measured as parameters between control and 11bꢀ
treated group.
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Figure 7. Elevated plus maze study. Time spent in the left open arm, left closed arm, total
distance travelled, distance travelled in open and closed arms, time spend in the open and closed
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significant difference between control and treated group.
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Figure 8. Rotarod study of 11b. Times spent on the revolving rod were measured for the
control and 11bꢀtreated group. There is no significant difference between control and treated
group.
Figure 9. Forced Swim test study. Immobility times in the water were measured. There is no
significant difference between control and treated group.
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Table 2. Results of the neurological observational battery study
Parameter
Control group
Treated group
P value (t test)
Body position
Locomotor activity
Transfer arousal
Piloerection
1.8±0.4216
2.600±0.9661
3.100±0.3162
2.100±0.3162
3.300±0.4830
3.800±0.6325
2.200±0.6325
1.400±1.350
1.200±1.033
1.900±0.3162
2.400±1.265
1.200±1.033
1.800±0.6325
3.300±0.6749
0.8000±0.7888
1.9±0.3162
2.600±1.350
3.200±0.4216
2.200±0.4216
3.400±0.6992
3.600±0.8433
1.800±0.6325
1.400±1.350
1.200±1.033
1.700±0.4830
3.000±1.700
1.400±0.9661
1.600±1.578
3.500±0.8498
0.9000±0.8756
0.5566
> 0.9999
0.5566
0.5566
0.7147
0.5566
0.1744
> 0.9999
> 0.9999
0.29
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Limb rotation
Pelvic elevation
Tail elevation
Finger approach
Touch escape
Visual placing
Grip strength
Pinna reflex
0.3833
0.6601
0.7164
0.5676
0.7915
Toe pinch
Limb tone
Tail pinch
ꢀ
CONCLUSION
In the current study, a novel series of modafinil analogues containing 5ꢀ and 6ꢀ membered
aromatic heterocycles have been synthesized. Analogues were tested for dopamine reuptake
inhibition and several compounds showed similar or improved activity and selectivity (DAT vs.
NET and SERT) compared to modafinil. Compounds 7d and 11b have been selected for further
testing. When tested in dopamine release assay, both compounds caused no efflux of dopamine
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(
amphetamineꢀlike properties). Binding studies have demonstrated that analogues 7d and 11b are
competing for binding with cocaine on DAT. This result was further supported by a docking
study of the compounds to a homology model of human DAT. While molecular docking
experiments support the finding that 7d and 11b are binding into the same putative binding
pocket of cocaine, molecular dynamics simulations indicate a different binding spot to that of
cocaine, i.e. different amino acids are involved in binding of 7d and 11b within the pocket. As
compound 11b showed the highest reuptake inhibition it was therefore the compound of choice
for neurotoxicity studies. Results indicate no general neurotoxicity of 11b. Therefore, by
introducing heterocyclic modifications to the modafinil skeleton, a novel series of atypical DAT
inhibitors has been synthetized. Several compounds showed improved activity and specificity
compared to modafinil, which together with absence of neurotoxicity for at least one tested
compound, makes them suitable for further development.
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ꢀ EXPERIMENTAL PROCEDURES
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Synthesis. H and C NMR spectra were recorded on a Bruker Avance 500 NMR
spectrometer (UltraShield) using a 5ꢀmm switchable probe (PA BBO 500SB BBFꢀHꢀDꢀ05ꢀZ,
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1H, BB = F and P ꢀ N) with z axis gradients and automatic tuning and matching accessory
1
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(Bruker BioSpin). The resonance frequency for H NMR was 500.13 MHz and for C NMR
1
25.75 MHz. All measurements were performed for a solution in fully deuterated chloroform or
DMSO at 298 K. Standard 1D and gradientꢀenhanced (ge) 2D experiments, like double quantum
filtered (DQF) COSY, NOESY, HSQC, and HMBC, were used as supplied by the manufacturer.
Chemical shifts are referenced internally to the residual, nonꢀdeuterated solvent signal for
1
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chloroform H (δ 7.26 ppm) or DMSO H (δ 2.50 ppm) and to the carbon signal of the solvent
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for chloroform C (δ 77.00 ppm) or DMSO C (δ 39.57 ppm).
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HRESIMS spectra were obtained on a maXis HD ESIꢀQqꢀTOF mass spectrometer
Bruker Daltonics, Bremen, Germany). Samples were dissolved to 20 µg/mL in MeOH and
(
directly infused into the ESI source at a flow rate of 3 µL/min with a syringe pump. The ESI ion
source was operated as follows: capillary voltage: 0.9 to 4.0 kV (individually optimized),
nebulizer: 0.4 bar (N ), dry gas flow: 4 L/min (N ), and dry temperature: 200 °C. Mass spectra
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2
2
were recorded in the range of m/z 50 – 1550 in the positiveꢀion mode. The sum formulas were
determined using Bruker Compass DataAnalysis 4.2 based on the mass accuracy (ꢁm/z ≤ 2 ppm)
and isotopic pattern matching (SmartFormula algorithm).
The purity of the compounds was determined by HPLC either on an UltiMate 3000 series
system equipped with VWD detector (Dionex/Thermo Fisher Scientific, Germering, Germany)
or on LCꢀ2010A HT Liquid Chromatograph device (Shimadzu Corporation, Tokyo, Japan).
Separation was carried out on an Acclaim 120 C18, 2.1 x 150 mm, 3 µm HPLC column (Thermo
Fisher Scientific) using LCꢀMSꢀgrade water and acetonitrile as mobile phase A and B,
respectively. The sample components were separated and eluted with a linear gradient from 10%
to 90% B in 25 min followed by an isocratic column cleaning and reꢀequilibration step. The flow
rate was 0.2 mL/min and the column oven temperature was set to 25°C. The purity was
determined from the UV chromatogram (254 nm) as the ratio of the peak area of the compound
to the total peak area (i.e., the sum of the areas of all peaks that were not present in the solvent
blank). Based on the HPLC data, all final compounds are ≥95% pure. Melting points were
measured on Leica Galen III apparatus (Leica Biosystems, Germany).
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-((Benzhydrylthio)methyl)-1,3,4-thiadiazol-2(3H)-one (3a) and 5-((Benzhydrylthio)methyl)-2-
methoxy-2,3-dihydro-1,3,4-thiadiazole (3b). To a solution of diphenylmethanethiol (1.35 g, 6.75
mmol) in DMF (15 mL) was added tꢀBuOK (1.34 g) and 2ꢀ(chloromethyl)ꢀ5ꢀmethoxyꢀ1, 3, 4ꢀ
thiadiazole (0.89, 5.4 mmol), and the reaction mixture was stirred at room temperature overnight.
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Then, saturated aqueous NH Cl solution was added and the reaction mixture was extracted with
4
EtOAc. After removal of the solvent, the residue was purified by silica gel column
chromatography (hexane: CH Cl 1:1) affording 0.20 g of compound 3b (yield 11%) and 0.60 g
2
2
of compound 3a (yield 35%).
2
-((Benzhydrylthio)methyl)-5-methyl-1,3,4-thiadiazole
(3c).
To
a
solution
of
diphenylmethanethiol (2.00 g, 10.0 mmol) in DMF (20 mL) was added tꢀBuOK (1.0 g) and 2ꢀ
(chloromethyl)ꢀ5ꢀmethylꢀ1, 3, 4ꢀthiadiazole (1.22 g, 8.2 mmol), and the reaction mixture was
stirred overnight at room temperature. Then, a saturated aqueous NH Cl solution was added and
4
the reaction mixture was extracted with EtOAc. After removal of the solvent, the residue was
purified by column chromatography on silica gel (hexane: CH Cl 1:1), to give 1.14 g of
2
2,
compound 3c (yield 44%).
5
-((Benzhydrylthio)methyl)-3-(trifluoromethyl)-1,2,4-oxadiazole (3d). To a solution of
diphenylmethanethiol (1.50 g, 7.5 mmol) in dioxane (20 mL) was added DIPEA (1.4 mL) and 5ꢀ
chloromethyl)ꢀ3ꢀ(trifluoromethyl)ꢀ1,2,4ꢀoxadiazole (1.33 g, 7.1 mmol), and the reaction
(
mixture was stirred at room temperature overnight. Then, the reaction mixture was quenched
with water and extracted with EtOAc. After removal of the solvent, the residue was purified by
flash chromatography on silica gel (CH Cl ), to give 2.47 g of compound 3d (yield >95%).
2
2
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Journal of Medicinal Chemistry
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5
-(Benzhydrylthiomethyl)-4-isopropyl-1,2,3-thiadiazole
(3e).
To
the
solution
of
diphenylmethanethiol (1.87 g, 9.30 mmol) in anhydrous 1,4ꢀdioxane (5 ml) 5ꢀ(chloromethyl)ꢀ4ꢀ
isopropylꢀ1,2,3ꢀthiadiazole (1.31 g, 7.44 mmol) and (1.54 ml) DIPEA (1.2 g, 9.30 mmol) were
added dropꢀwise. The resulting mixture was stirred at room temperature for 3 h and evaporated
in vacuum till dryness. The crude product was purified by column chromatography on silica gel
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(EtOAc:hexane, 1:20) affording 1.91 g of the colorless crystals (yield 47%).
5
-((Benzhydrylthio)methyl)-2-methoxy-2,3-dihydro-1,3,4-thiadiazole (4a). 27.6% H O (1.46
2 2
mL) was added to the solution of 3a (1.5 g) in glacial acetic acid (35 mL). The reaction mixture
was stirred at room temperature during 24 h. The solvent was evaporated in vacuum till dryness.
The residue was diluted with water, the precipitate was filtered and purified by flash
chromatography on silica gel (hexane: EtOAc, 3:1), to give 1.0 g of compound 4a, as white solid
(
[
yield 64%). Mp. 142ꢀ145ºC. Ret. time 16.96 min, purity 95.5%. HRESIMS m/z 353.0393
+
+
1
M+Na] (calcd for C H N NaO S , 353.0389, ꢁ = ꢀ1.0 ppm). H NMR (500 MHz, CDCl ) δ=
1
6
14
2
2
2
3
10.17 (br s, 1H), 7.44 – 7.39 (m, 10H), 5.10 (s, 1H), 3.89 (d, J = 15 Hz, 1H), 3.74 (d, J = 15 Hz,
13
1
H); C NMR (125 MHz, CDCl ) δ= 172.2 (C=O), 145.1 (C), 134.3 (C), 133.6 (C), 129.6
3
(
2CH), 129.5 (2CH), 128.9 (2CH + 2pꢀCH), 128.8 (2CH), 71.7 (CH), 51.5 (CH2).
5-((Benzhydrylsulfinyl)methyl)-2-methoxy-2,3-dihydro-1,3,4-thiadiazole (4b). 27.6% H O2
2
(0.07 mL) was added to the solution of 3b (6.1 mmol, 0.2 g) in glacial acetic acid (10 mL). The
reaction mixture was stirred at room temperature during 24 h. Two new compounds were
detected on TLC. The solvent was evaporated in vacuum till dryness. The residue was diluted
with water, the precipitate was filtered off and purified by column chromatography on silica gel
(CH Cl ), to give 0.07 g of compound 4b as white solid (yield 33%). Mp. 127ꢀ129ºC. Ret. time
2 2
+
+
18.61 min, purity 98.8%. HRESIMS m/z 367.0549 [M+Na] (calcd for C H N NaO S ,
17 16 2 2 2
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