Structure-Activity Relationships of Novel Thiazole-Based Modafinil Analogues Acting at Monoamine Transporters

Article abstract of DOI:10.1021/acs.jmedchem.9b01938

Atypical dopamine reuptake inhibitors, such as modafinil, are used for the treatment of sleeping disorders and investigated as potential therapeutics against cocaine addiction and for cognitive enhancement. Our continuous effort to find modafinil analogue

Full text of DOI:10.1021/acs.jmedchem.9b01938

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Article
Structure Activity Relationships of Novel Thiazole-Based
Modafinil Analogues Acting at Monoamine Transporters
Predrag Kalaba, Marija Ili#, Nilima Yogesh Aher, Vladimir Draga#evi#, Marcus Wieder,
Martin Zehl, Judith Wackerlig, Stanislav Beyl, Simone B. Sartori, Karl Ebner, Alexander
Roller, Natalie Lukic, Tetyana V. Beryozkina, Eduardo Rene P González, Philip John Neill,
Jawad Akbar Khan, Vasiliy A. Bakulev, Johann Jakob Leban, Steffen Hering, Christian Pifl,
Nicolas Singewald, Jana Lubec, Ernst Urban, Harald H. Sitte, Thierry Langer, and Gert Lubec
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01938 • Publication Date (Web): 16 Dec 2019
Downloaded from pubs.acs.org on December 17, 2019
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Chemistry
Lubec, Gert; Paracelsus Medizinische Privatuniversitat, Neuroscience
Laboratory
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Structure Activity Relationships of Novel
Thiazole-Based Modafinil Analogues Acting at
Monoamine Transporters
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†,*
†,*
†,*
†
†
Predrag Kalaba , Marija Ilić , Nilima Y. Aher , Vladimir Dragačević , Marcus Wieder ,
l
†
§
⁂
⁂
Martin Zehl , Judith Wackerlig , Stanislav Beyl , Simone B. Sartori , Karl Ebner , Alexander
◊
◊
‡
#
†
Roller , Natalie Lukic , Tetyana Beryozkina , Eduardo Rene Perez Gonzalez , Philip Neill ,
♦
‡
†
§
⁑
Jawad Akbar Khan , Vasiliy Bakulev , Johann Jakob Leban , Steffen Hering , Christian Pifl ,
⁂
&
†
♦
†
Nicolas Singewald , Jana Lubec , Ernst Urban , Harald H. Sitte , Thierry Langer , Gert
Lubec&
†Department of Pharmaceutical Chemistry, Faculty of Life Sciences, University of Vienna,
Althanstraße 14, 1090, Vienna, Austria
lDepartment of Analytical Chemistry, Faculty of Chemistry, University of Vienna, Währinger
Straße 38, 1090, Vienna, Austria
§Department of Pharmacology and Toxicology, Faculty of Life Sciences, University of Vienna,
Althanstraße 14, 1090, Vienna, Austria
⁂Center for Molecular Biosciences Innsbruck (CMBI), Department of Pharmacology and
Toxicology, Institute of Pharmacy, Leopold Franzens University Innsbruck, Innsbruck,
Austria
◊X-ray Structure Analysis Centre, Faculty of Chemistry, University of Vienna, Währinger
Straße 38, 1090, Vienna, Austria
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‡Ural Federal University named after the first President of Russia B. N. Yeltsin, 19 Mira st.,
Yekaterinburg 620002, Russia
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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 19060-
9
00, Presidente Prudente, SP-Brazil
♦Institute of Pharmacology, Centre of Physiology and Pharmacology, Medical University of
Vienna, 1090, Vienna, Austria
⁑Centre for Brain Research, Medical University of Vienna, Vienna, Austria.
&Paracelsus Medical University, Neuroscience Laboratory, A -5020 Salzburg
ABSTRACT
Atypical dopamine reuptake inhibitors, such as modafinil, are used for the treatment of sleeping
disorders and investigated, e.g. as potential therapeutics against cocaine addiction and for
cognitive enhancement. Our continuous effort to find modafinil analogues with higher
inhibitory activity on and selectivity towards the dopamine transporter (DAT) has previously
led to the promising thiazole-containing derivatives CE-103, CE-111, CE-123, and CE-125.
Here, we describe the synthesis and activity of a series of compounds based on these scaffolds,
which resulted in several new selective DAT inhibitors and gave valuable insights into the
structure activity relationships. Introduction of the second chiral center and subsequent chiral
separations provided all four stereoisomers, whereby S-configuration on both generally
exerting the highest activity and selectivity on DAT. The representative compound of this
series was further characterized by in silico, in vitro and in vivo studies that have demonstrated
both safety and efficacy profile of this compound class.
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INTRODUCTION
Modafinil (1) (2-[(Diphenylmethyl)sulfinyl] acetamide by IUPAC nomenclature) is an
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innovative wake-promoting agent initially marketed in France in the early 1990s for the
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treatment of narcolepsy (Figure 1). In 1998, it got approved by the United States Food and
Drug Administration (FDA) as a novel agent to treat daytime sleepiness caused by narcolepsy
2
or by shift work sleep disorder. In the US, military research in modafinil as potential
alternative to amphetamines for enhancing alertness and reducing battle fatigue is ongoing, and
its use in Air Force missions has already been approved.3,4 Along with methylphenidate, it
gained high popularity in the last decade among shift workers, students and others, who are
taking these drugs as nootropics to improve performance during the exams or on the work
place.3-5 Although the media attention has diminished lately, it was not the case with off-label
use of modafinil as cognitive-enhancing drug. Indeed, the results of two surveys recently
published in JAMA reveal high use of modafinil among German surgeons and undergraduates
5
of the University of Maryland. Due to its reported low abuse liability, modafinil has also been
suggested for the treatment of mental illnesses such as attention-deficit/hyperactivity disorder
(
ADHD).6
Modafinil is a racemic mixture of the R- and S- enantiomers in equimolar ratio (1:1),
while R-modafinil (FDA approved in June 2007) is a pure enantiomer, which displays slightly
increased pharmacological potency as a wakefulness-inducing agent compared to the racemic
mixture.7,8 The difference is more obvious in the pharmacokinetic profile as pointed out by
recent human studies which showed that R-modafinil is the metabolically more stable and
longer-acting enantiomer.9
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Despite of being brought to the market 2 decades ago, modafinil`s mode of action
remained unknown and controversially discussed for years.2,10 Studies using radioligand
binding assays (Mignot et al. 1994) as well as cell-based (Madras et al. 2006) and
synaptosome-based uptake inhibition assays (Zolkowska et al. 2009) have identified the
dopamine transporter (DAT) as a major target to which modafinil binds.12-13 It was Loland et
al., who have provided more detailed insight into modafinil`s mode of action. They confirmed
that modafinil and both of its enantiomers inhibit dopamine reuptake in a cell-based assay, with
the R- being almost threefold more potent than the S-enantiomer. In the same study, they have
also demonstrated that the binding site for modafinil and the central binding site for dopamine
overlap, and that R- and S-modafinil preferentially bind to a different DAT conformational
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isomer than cocaine. Furthermore, modafinil does not act as a substrate for DAT and does
not cause efflux of dopamine, a hallmark that clearly distinguishes it`s pharmacological profile
from those of amphetamine and amphetamine-like psychostimulants.15-17 Owing to its unique
binding mode and exclusive pharmacological profile, modafinil has been classified as an
atypical dopamine reuptake inhibitor.14,18
These findings made modafinil an interesting scaffold for medicinal chemistry, leading
to a large number of derivatives that have been synthesized and tested for their biological
activity in the recent years.6,7,19-22 In particular, much of the work in the United States has
focused on development of modafinil analogues as potential therapeutics against cocaine
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0
addiction. From this research, compound JJC8-016 (2) has emerged as an atypical dopamine
reuptake inhibitor that has no cocaine-like abuse potential (Figure 1).23,24 An independent line
of research has focused on the activity of modafinil and its analogues in animal paradigms of
working and spatial memory.25-33
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Figure 1. Structures of (±) modafinil (1), JJC8-016 (2), CE-103 (3), CE-111 (4), CE-125 (5),
and CE-123 (6).
We have previously reported a series of modafinil derivatives in which the carboxamide
moiety has been replaced with various 5- and 6-membered aromatic heterocycles.34
Compounds of this series have demonstrated (I) higher activity and better selectivity towards
DAT than modafinil when tested by reuptake inhibition assay, (II) absence of amphetamine-
and cocaine-like properties when tested in efflux and binding assays, as well as (III) absence
34
of neurotoxicity when tested in vivo. In addition, carboxamide replacement with substituted
and un-substituted thiazoles led to the discovery of compounds with an improved in vitro and
in vivo profile.30-33 For example, CE-103 (3) exerted comparable in vitro activity on DAT (IC50
=
15 µM) as modafinil, with negligible effects on serotonin and norepinephrine transporter
(
SERT and NET) and it was able to decrease working memory errors in the radial arm maze
RAM) significantly at doses of 1 mg/kg and 10 mg/kg body weight when given
(
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intraperitoneally compared to vehicle control (Figure 1). Similarly, CE-111 (4) and CE-125
5) showed higher reuptake inhibition activity on DAT (IC50 = 3.2 µM and 4.1 µM,
(
respectively) compared to modafinil and negligible activity on SERT and NET (Figure 1).
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th
th
Both compounds were able to improve the memory performance on the 8 and 9 day on the
th
th
th
RAM at 1 mg/kg body weight (i.p.) and on the 8 , 9 and 10 day at 10 mg/kg (i.p.) when
compared to the control group.31,32 Another thiazole-containing analogue, CE-123 (6) was
previously shown to inhibit dopamine reuptake (IC50 = 4.6 μM), while showing almost
negligible effects on reuptake of dopamine and serotonin in vitro. As compared to R-modafinil,
it reached higher concentrations in the brain and had a smaller elimination rate. In vivo drug
efficacy of CE-123 was demonstrated in male Sprague-Dawley rats in hole-board task, a spatial
memory paradigm. The compound was able to enhance both memory acquisition as well as
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memory retrieval. Interestingly, the S-enantiomer of CE-123 showed an almost 8-fold higher
in vitro activity on DAT than the R-enantiomer. In male rats, S-CE-123 demonstrated increased
cognitive flexibility with diminished impulsivity compared to R-modafinil as determined by an
attentional set-shifting task (ASST) and a 5-choice serial-reaction time task (5-CSRTT).35
Based on the above described results, the aim of this study was to generate a novel
series of modafinil analogues with further improved activity and selectivity towards DAT. To
that end, the structure-activity relationship (SAR) on DAT, SERT and NET was explored by
replacing the carboxamide function of modafinil with a series of substituted and un-substituted
thiazoles and by modifying the diphenylmethyl group. We also sought to explore the influence
of introducing one additional chiral center on the activity of such analogues. The four pure
stereoisomers of such an analogue were obtained, their absolute configuration was determined
by single crystal X-ray diffraction analysis, their reuptake inhibition activity was tested and the
compounds were docked within the binding pocket of human DAT homology model.
Furthermore, the compounds of this series were screened by in silico methods for their activity
on the hERG channel and the most potent DAT reuptake inhibitors were tested in vitro in order
to verify the in silico results. Finally, we have performed a pharmacokinetic study of the most
potent analogue.
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CHEMISTRY
Synthetic pathway of novel thiazole-containing modafinil analogues without
substitution on phenyl rings 12a-s is shown in Scheme 1. Starting compound, diphenylmethyl-
S-isothiuronium hydrobromide (9), was alkylated with both plain and substituted 2-, 4- or 5-
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(
chloromethyl)thiazoles to provide intermediates 11a-s in 31% to >95% yield. Intermediates
were subsequently oxidized and final sulfoxide-containing products 12a-s were obtained in
1% to >95% yield.
1
Scheme 1. Synthesis of compounds 11a-s and 12a-sa
O
S
NH2
OH
S
a
S
S
R
R
HBr
b
c
+
NH
+
Cl
R
H2N
NH2
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S
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N
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0a - 12a: R=
0b - 12b: R=
0c - 12c: R=
0d - 12d: R=
0e - 12e: R=
10f - 12f: R=
10g - 12g: R=
10h - 12h: R=
10i - 12i: R=
10j - 11j R=
1
0k - 12k: R=
N
10p - 12p: R=
10q - 12q: R=
10r - 12r: R=
10s - 12s: R=
N
N
S
S
S
S
10l - 12l: R=
N
N
O
N
S
N
S
S
10m - 12m: R=
10n - 12n: R=
10o - 12o: R=
N
S
N
S
S
S
N
N
S
S
N
N
S
Cl
Cl
N
N
S
S
S
N
S
N
N
a
Reagents and conditions: (a) 48% HBr, MeOH, reflux, 4 h; (b) MeOH, K CO , r.t. 48 h; (c)
2
3
CH COOH, 30% H O , r.t. overnight.
3
2
2
Synthesis of 2-[(9H-fluoren-9-ylsulfinyl)methyl]-1,3-thiazole (17) was achieved via a
similar synthetic route as depicted in Scheme 2. The first step involves conversion of 9-
hydroxyfluorene (13) with thiourea and 48% HBr to the corresponding thiouronium salt (14).
The second step is an alkylation reaction with 2-(chloromethyl)thiazole to give intermediate
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(
16). The final step is an oxidation reaction in CH COOH with H O that gave the final product
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2
1
7 in 67% yield.
Scheme 2. Synthesis of compounds 16 and 17a
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HBr
a
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Cl
+
+
NH2
HBr
S
H2N
NH2
13
8
14
15
16
17
a
Reagents and conditions: (a) 48% HBr, H O, 100-105℃, 1 h; (b) 1,4-dioxane, NaOH, 70℃. 5
2
h; (c) CH COOH, 30% H O , r.t. 24 h.
3
2
2
Thiazole-containing analogues with mono-substitution on one or both phenyl rings
(24a-24m) were obtained as depicted in Scheme 3. Conversion of the chloromethyl-thiazoles
1
8a-18e with thioacetic acid (19) to yield the acylated intermediates 20a-20e followed by de-
protection and isolation of the desired plane and substituted thiomethyl-thiazoles 21a-21e were
the first steps. In the subsequent reaction, 21a-21e were condensed with substituted
diphenylmethyl alcohols 22a-22m to yield intermediates 23a-23m (yield 22% up to >95%). In
the final step, oxidation of the appropriate sulfenyl moiety gave the desired products 24a-24m
in 11-92% yield.
Scheme 3. Synthesis of compounds 23a-m and 24a-ma
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3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
O
S
a
b
c
d
X
R
Cl
R
S
R
SH
X
X
SH
OH
Y
S
Y
R
R
HCl
O
Y
18
19
20
21
22
23
24
S
S
N
S
- : X= Y= p-Cl, R=
22j 24j
2
2f - 24f: X= Y= p-CH3, R=
18a, 20a, 21a: R=
22a - 24a: X= Y= p-Cl, R=
N
N
S
N
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
S
N
S
22g - 24g: X= Y= p-OCH , R=
22k - 24k: X= Y= p-F, R=
22l - 24l: X= Y= p-CH3, R=
22m - 24m: X= Y= p-CH3, R=
1
8b, 20b, 21b: R=
3
2
2
2
2
2b - 24b: X= Y= p-Cl, R=
2c - 24c: X= Y= p-CH3, R=
2d - 24d: X= Y= p-OCH3, R=
2e - 24e: X= Y= p-F, R=
N
S
N
S
S
18c, 20c, 21c: R=
N
22h - 24h: X= Y= p-Cl, R=
N
N
S
S
1
8d, 20d, 21d: R=
8e, 20e, 21e: R=
N
S
S
22i - 24i: X= p-H, Y= p-Br, R=
S
N
N
N
1
N
S
S
S
N
S
N
a
Reagents and conditions: (a) K CO , NaI, acetone, 35-40℃, 3h; (b) NaOH, EtOH, reflux, 2h;
2
3
(c) BF •Et O, CH COOH, r.t. overnight; (d) CH COOH, 30% H O , r.t. overnight.
3
2
3
3
2
2
Compounds 31c-f were obtained as depicted in Scheme 4. 4-(Chloromethyl)thiazole
18a) was reacted with thioacetic acid (19) to yield acylated compound 20a, which was
(
hydrolyzed under alkaline conditions to corresponding mercaptan 21a. Thiazol-4-
ylmethanethiol was further condensed with (4-bromophenyl)(phenyl)methanol (22i) to yield
racemic 4-({[(4-bromophenyl)(phenyl)methyl]thio}methyl)thiazole (23i) in 25% yield. In the
following step, compound 23i was separated into the enantiomers 30a and 30b using chiral
HPLC on a CHIRALPAK IA semipreparative column (10 mm diameter x 20 cm length)
(
Daicel Inc, Tokyo, Japan) and ACN as the mobile phase. Each of the enantiomers was further
oxidized to yield again racemic compounds 31a and 31b, respectively, that were finally
separated using CHIRALPACK IA semipreparative column into the individual stereoisomers
3
1c-f.
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Scheme 4. Synthesis of compounds 31c-fa
N
O
N
S
c
S
S
a
S
b
d
Cl
S
OH
SH
HS
*
S
HCl
N
N
O
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Br
Br
25
26
27
28
29
30
S
N
S
O
S
S
N
H
H
*
d
e
f
Br
0a
Br
3
31a
31c S1 (R) C1 (R)
31d S1 (S) C1 (R)
S
N
S
O
S
S
*
N
H
H
Br
0b
Br
31b
3
31e S1 (S) C1 (S)
31f S1 (R) C1 (S)
a
Reagents and conditions: (a) K CO , NaI, acetone, 35-40℃, 3h; (b) NaOH, EtOH, reflux, 2h;
2
3
(
c) BF •Et O, CH COOH, r.t. overnight; (d) chiral separations, Chiralpak IA column, isocratic
3 2 3
100% ACN as mobile phase; (e) CH COOH, H O , r.t. overnight; (f) chiral separations,
3
2
2
Chiralpak IA column, isocratic absolute EtOH as mobile phase for 31c and 31d, isocratic 80%
EtOAc 20% hexane as mobile phase for 31e and 31f.
Compounds 24h, 24k and 24l were separated into individual (less retained and more
retained) enantiomers 32a, 32b, 33a, 33b, 34a and 34b, respectively, by means of chiral-phase
high performance liquid chromatography (HPLC).
Compounds 42(a-f)-45(a-f) were obtained as depicted in Scheme 5. Thiazol-5-
ylmethanethiol (36) was reacted with para-monosubstituted diphenyl alcohols (35a – 35f) to
yield racemic monosubstituted sulfide intermediates (37a – 37f) in 22-87% yield. Each
intermediate was separated into individual enantiomers (38a-f and 39a-f) by means of chiral-
phase HPLC, that were subsequently oxidized to corresponding sulfoxide-containing
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
derivatives (40a-f and 41a-f) with 81-95 % and 76-95% yields, respectively. Racemic products
were further separated into individual enantiomers (42a-f, 43a-f, 44a-f and 45a-f). respectively
by another round of chiral-based separations.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 5. Synthetic strategy for compounds 42(a-f)-45(a-f)a
N
N
N
N
N
N
O
S
O
S
O
S
a
b
c
OH
+
HS
S
S
d
S
S
S
S
S
S
X
X
X
X
X
X
35
36
37
38
40
42
43
3
3
3
3
3
3
5a, 37a-45a: X = p-CF3
5b, 37b-45b: X = p-Cl
5c, 37c-45c: X = p-CH3
N
O
S
N
O
S
N
O
S
N
S
S
S
S
S
5d, 37d-45d: X = p-OCH3
5e, 37e-45e: X = p-Br
5f, 37f-45f: X = p-F
X
X
X
X
39
41
44
45
a
Reagents and conditions: (a) BF •Et O, CH COOH, reflux, 3h; (b) chiral-phase HPLC; (c)
3
2
3
CH COOH, H O , r.t. overnight; (d) chiral-phase HPLC.
3
2
2
.
RESULTS AND DISCSSION
SARs of racemates on reuptake inhibition of monoamine transporters
All sulfoxide-containing end products (12a-i, 11j, 12k-12s, 17, 24a-24m) were tested
for their reuptake inhibition potential on HEK293 cells with stable expression of human DAT,
NET and SERT isoforms, as described in detail under Experimental Procedures section. The
obtained values were compared to those of the parent compounds modafinil (1) (DAT IC =
5
0
34
1
1.5 ± 4.3 µM; NET IC = 284 ± 24 µM; SERT – not specific in tested concentration range)
50
and R-modafinil (DAT IC = 6.6 ± 0.8 µM; NET IC = 170 ± 90 µM; SERT IC = 232 ± 197
5
0
50
50
2
5
µM) and correlated to the chemical modifications. SAR study was aimed to elucidate the
effect of (i) replacing the carboxamide with various substituted and un-substituted thiazoles
linked via positions 2, 4 and 5 as well as of (ii) introducing substituents on one or both phenyl
rings. The results are summarized and presented in Tables 1-3.
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Heterocyclic replacement of the carboxamide moiety with unsubstituted thiazole on
position 4 yielded the compound 12a, which exerted activity (DAT IC = 6.7 ± 1.4 µM)
5
0
comparable to that of R-modafinil and almost 1-fold better than that of 1. Furthermore, this
analogue showed negligible activity on NET and SERT (Table 1). Interestingly, most of the
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
racemic compounds herein reported were found not to inhibit the uptake of [ H]-serotonin by
SERT, with an exception of weak activity of 12k (SERT IC = 201 ± 79 µM) and 24h (SERT
50
IC = 221± 13 µM).
5
0
Table 1. Inhibition of hDAT, hNET, and hSERT-mediated uptake of respective radioligands
by thiazole-containing analogues (bridged on the position 4 of thiazole)a
Analogue
Substituents
IC50 ± SD (µM)
NET
General formula
ID
4 31
5 32
X
H
H
Y
H
H
Z
n
1
1
A
H
H
B
DAT
3.25
SERT
NA
S=O
S=O
CH
3
174
cyclopropyl
4.1 ± 0.8
687.2 ± 152.8
436.1
±
129
1
2a
2e
2f
2d
2g
2i
2b
2c
1j
2h
4f
24e
4a
24c
4b
H
H
H
H
S=O
S=O
S=O
S=O
S=O
S=O
S=O
S=O
S
1
1
1
1
1
1
1
1
1
2
1
1
1
1
1
1
1
H
H
H
H
H
H
H
CH
H
H
H
H
H
H
H
H
H
H
6.66 ± 1.44
14.64 ±0.89
28.43 ± 1.93
24.30 ± 1.72
13.50 ± 2.76
3.02 ± 1.03
6.97 ± 2.30
48.09 ± 2.88
91.38 ± 2.24
61.42 ± 11.00
7.32 ± 0.04
6.76 ± 0.67
5.04 ± 0.48
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
1
CH
2
CH
3
NA
1
H
H
n-propyl
iso-propyl
n-butyl
NA
1
H
H
NA
164.4 ± 56.88
NA
1
H
H
1
H
H
Cl
1
H
H
2-thiophene
110.50 ± 6.15
NA
A
S
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
X
1
H
H
3
CH
CH
CH
H
3
3
3
B
Z
1
H
H
129.80 ± 31.91
NA
N
n
1
H
H
S=O
S=O
S=O
S=O
S=O
S=O
S=O
S=O
2
CH
F
3
3
CH
F
3
3
NA
Y
H
NA
2
Cl
CH
Cl
Cl
CH
Cl
H
107.10 ± 48.93
NA
cyclopropyl
cyclopropyl
2
14.00 ± 5.50
NA
24g OCH
4i
3
OCH
Br
3
cyclopropyl 104.70 ± 23.75
1.85 ± 1.20
NA
2
H
H
109.1 ± 15.5
1
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
IC values represent data three independent experiments, each performed in triplicate. NA -
5
0
Not active in used concentration range.
Introduction of a methyl group in position 2 yielded the previously reported compound
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4 with slightly higher activity on DAT (IC = 3.2 µM) than 12a. Substitution on the same
5
0
position with larger aliphatic groups such as ethyl, n-propyl, iso-propyl and n-butyl yielded
analogues 12e (IC = 14.6 ±0.9 µM), 12f (IC = 28.4 ± 1.9 µM), 12d (IC = 24.3 ± 1.7 µM),
5
0
50
50
and 12g (IC = 13.5 ± 2.8 µM), respectively, with decreased activity on DAT. Out of them,
5
0
only 12g exerted weak activity on NET (IC = 164 ± 57 µM). Substitution with a chlorine
5
0
atom on the same position yielded the selective DAT inhibitor 12i with an activity (IC = 3.0
5
0
± 1.0 µM) as good as that of compound 4. Introduction of a 5-membered aromatic heterocycle,
i.e. thiophene bound through position 2, gave compound 12b, with an inhibitory activity on
DAT (IC = 7.0 ± 2.3 µM) comparable to that of R-modafinil and 12a.
5
0
Further modification of 4 by introducing a second methyl group in position 5 gave
compound 12c and led to a significantly decreased activity on DAT. The same was observed
upon extension of the bridge to the sulfoxide by exchanging the methylene with an ethylene
group as in compound 12h (Table 1).
Previously, it was reported that the replacement of the sulfoxide moiety with a sulfide
function in modafinil analogues may have minor effects on DAT binding, but should lead to
increased affinities at SERT and NET.6,20 To evaluate whether our newly synthesized
compounds would fall under this concept, compound 11j was tested for monoamine
transporters reuptake inhibition activity and compared to its sulfoxide counterpart 4. While we
have indeed observed enhanced reuptake inhibition on NET, now activity was detected on
SERT and a significantly decreased inhibition activity was measured on DAT (IC = 91.4 ±
50
2.2 µM). Based on these findings, none of the other thioether intermediates were tested for
bioactivity.
1
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Compounds 5 and 12a were further modified by introducing substitutions on both para-
positions of the phenyl rings. In the case of 12a, substitution with either methyl groups or strong
electron-withdrawing groups such as fluorine and chlorine (24f, 24e, and 24a, respectively)
only had minor effects on the reuptake inhibition activity on DAT (Table 1). Different results
were obtained upon modification of 5, where introduction of chlorine to give 24b yielded an
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
-3 fold reduced but still good reuptake inhibition activity on DAT (IC = 14.0 ± 5.5 µM),
50
while electron-donating methoxy groups, as in 24g, led to a strong drop in activity (DAT IC50
=
105 ± 24 µM) and the methyl groups in 24c completely abolished the activity.
A much smaller series of compounds was synthesized based on the structure of CE-
103 (3), with the thiazole linked via position 2. Introduction of a single methyl group on
position 5 as well as of two methyl groups on positions 4 and 5 of the thiazole yielded analogues
1
2l and 12k, respectively. However, both showed decreased activity on DAT compared to the
unsubstituted compound 3 (Table 2). Analogously to 12a, substitution with methyl groups on
the para-positions of both phenyl rings (24m) led to a decrease in activity, while substitution
with chlorine atoms on the same position (24j) yielded a slightly increased activity on DAT
(
IC = 9.6 ± 1.4 µM), which is similar to those of 1 and R-modafinil. Replacement of the
50
diphenylmethyl with a fluorenyl moiety yielded analogue 17 with no activity on DAT, NET
and SERT. This clearly demonstrates that the free rotation of the phenyl groups is crucial for
activity, indicating that they need to be oriented in a non-planar configuration to fit into the
binding pocket of the monoamine transporters.
Table 2. Inhibition of hDAT, hNET, and hSERT-mediated uptake of respective radioligands
by thiazole-containing analogues (bridged on the position 2 of thiazole)a
1
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Analogue
Substituents
IC50 ± SD (µM)
NET
General formula
ID
X
H
Y
H
A
H
B
H
DAT
SERT
3 30
14.73
420.1
NA
NA
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
A
1
2l
2k
4m
H
H
H
CH
CH
H
3
3
65.59 ± 3.91
33.27 ± 1.67
29.39 ± 8.07
NA
X
O
S
N
1
H
H
CH
H
3
107.30 ± 94.18
NA
200.80 ± 79.33
NA
B
S
2
CH
3
CH
3
2
4j
Cl
Cl
H
H
9.58 ± 1.37
NA
NA
Y
O
S
N
17
-
-
-
-
NA
NA
NA
S
a
IC values represent data from three independent experiments, each performed in triplicate.
5
0
NA - Not active in used concentration range.
In the final series, analogues of CE-123 (6) bridged via position 5 of thiazole were
explored (Table 3). Introduction of methyl groups either on position 2, 4, or 2 and 4 on the
thiazole yielded analogues 12m, 12r, and 12s, respectively, which all showed strongly reduced
activity on DAT compared to 1, 6 and R-modafinil. Introduction of both, electron-withdrawing
and -donating groups on position 2 of the thiazole, as in analogues 12n, 12o, and 12q, also only
led to reduction of activity on DAT. More differentiated results were obtained with
modifications on the para-positions of both phenyl rings. Compared to 6, strong electron-
donating methoxy groups as in 24d strongly decreased the activity on DAT; methyl groups as
in 24l and F atoms as in 24k slightly reduced the activity, and strongly electron-withdrawing
groups such as Cl let to equal or slightly improved activity as shown for 24h. However, the
latter was also moderately active on NET (IC = 45.5 ± 4.2 µM) and thus less selective towards
50
DAT. Attaching the bulky adamantly group to C-2 of the thiazole was performed to probe the
1
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available space within the binding pocket, but the obtained analogue 12p showed too low
solubility to be tested in biological assays.
Table 3. Inhibition of hDAT, hNET, and hSERT-mediated uptake of respective radioligands
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
by thiazole-containing analogues (bridged on the position 5 of thiazole)a
Analogue
Substituents
IC50 ± SD (µM)
NET
General formula
ID
X
H
Y
A
H
B
H
DAT
SERT
6 33
H
H
4.61 ± 0.14
68.06 ± 18.58
44.06 ± 6.24
87.06 ± 9.33
24.32 ± 1.21
59.74 ± 8.97
NT
1
2m
H
H
CH
H
3
3
NA
NA
NA
NA
12r
H
H
CH
CH
H
3
3
1
2s
H
H
CH
Cl
NA
NA
1
2n
H
H
108.20 ± 31.01
NA
NA
A
12o
12p
12q
H
H
H
t-butyl
NA
X
N
O
B
S
H
H
H
adamantyl
NT
NT
S
H
H
H
OCH
H
3
72.48 ± 26.02
10.57 ± 0.80
6.21 ± 0.29
4.17 ± 0.69
71.59 ± 8.28
138.80 ± 22.97
215.50 ± 66.01
120.80 ± 6.58
45.49 ± 4.23
NA
NA
2
4l
CH
F
3
CH
F
3
H
NA
Y
24k
H
H
NA
2
2
4h
4d
Cl
Cl
H
H
220.9 ± 12.76
NA
OCH
3
OCH
3
H
H
a
IC values represent data from three independent experiments, each performed in triplicate.
5
0
NA - Not active in used concentration range.
In summary, substituting the carboxamide group of modafinil with various thiazole
derivatives while keeping the diphenylmethyl moiety unmodified yielded three additional
racemic compounds (12a, 12b, and 12i) that showed selective inhibitory activities towards
DAT with an IC in the range of 1-10 µM. Interestingly, while the plain thiazoles linked via
5
0
positions 2, 4 and 5 showed relatively similar activities on DAT with IC values of 14.7 µM
5
0
(
CE-103), 6.66 ± 1.44 µM (12a), and 4.61 ± 0.14 µM (CE-123), respectively, they differed
strongly upon substitution on the C-atoms of the heterocycle. While all such derivatives of CE-
03 and CE-123 showed a severely reduced activity on DAT, 12a can easily accept various
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
substituents on C-2 of thiazole to give analogs with only slightly reduced or even somewhat
improved activity (Table 1).
Based on four of the most active structures of above, namely CE-103, 12a, CE-123,
and CE-125, we further tested the effect of symmetric substitution in para-position of the two
phenyl rings. This yielded another six analogues (24a, 24e, 24f, 24h, 24j, and 24k) with IC50
values below 10 µM. Unfortunately, only a minor improvement in activity on DAT could be
obtained in this way, and even that goes in hand with decreased selectivity when taking into
account the activity on NET and SERT.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
SARs of enantiopure compounds at DAT, NET and SERT
As has been proven for modafinil, where the R-enantiomer is more active, and CE-123,
where the S-enantiomer showed an almost 8-fold higher in vitro activity, enantiopure DAT
inhibitors can be more active than the racemic mixture. Also, providing solely the more active
enantiomer as drug can reduce or abolish side-effects if these are mainly caused by the other
enantiomer. To test whether the enantiomers of our novel thiazole-based modafinil analogues
also show significant differences in their activity, racemic compounds 24h, 24k and 24l were
separated by chiral-phase HPLC. In all three cases, there was indeed a big difference, ranging
from ca. ~4-fold to ~50-fold, in the activity of the two enantiomers (Table 4). 32a, 32b and
33b could be successfully crystallized and their absolute configuration determined by single
crystal X-ray diffraction analysis, showing that at least for two more compounds of this series
the S-configuration of the sulfoxide yields the higher activity.
Table 4. Inhibition of hDAT, hNET, and hSERT-mediated uptake of respective radioligands
by single stereoisomers of thiazole-containing analogues.a, b
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7
Analogue
Substituents
Chiralitya
IC50 ± SD (µM)b
NET
General formula
ID
X
Cl
Cl
Y
Cl
Cl
S atom C atom
DAT
SERT
NA
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
X
32a
S
-
-
4.1 ± 0.6
89.8 ± 172.3
NA
N
0
O
S
3
2b
3a
3b
4a
4b
2a
R
37.8 ± 5.5
NA
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
S
3
F
F
F
F
(S)
R
x
-
-
5.3 ± 0.1
22.5 ± 2.6
6.8 ± 1.8
356.0 ± 74.1
39.5 ± 12.6
NA
85.0 ± 11.7
NA
NA
NA
3
Y
3
CH
CH
3
3
CH
CH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
3
3
x
NA
264.4 ± 49.4
NA
3
x
x
NA
4
CF
CF
CF
CF
3
x
x
NA
NA
43a
44a
45a
3
x
x
NA
NA
3
3
S
R
R
S
37.7 ± 9.9
NA
NA
NA
R
S
NA
NA
4
4
4
4
2b
3b
4b
5b
Cl
Cl
2.3 ± 0.5
33.0 ± 6.3
3.6 ±1.1
72.0 ± 4.7
NA
NA
R
S
S
NA
Cl
R
R
R
R
(S)
S
38.6 ± 7.6
36.5 ± 3.0
NA
NA
Cl
R
S
7.4 ± 1.4
6.0 ±2.0
140.0 ±12.4
NA
4
4
4
2c
3c
4c
CH
CH
CH
CH
3
3
3
3
R
(S)
R
S
19.6 ± 9.7
3.3 ± 0.9
133.6 ± 69.4
12.2 ± 7.2
36.3 ±7.8
3.8 ± 0.6
65.1 ± 3.8
1.6 ± 0.6
22.4 ± 16.8
4.2 ± 1.5
4.7 ± 2.1
2.0 ± 0.4
68.2 ± 6.8
163.7 ± 17.4
NA
201.5 ± 25.8
NA
45c
42d
NA
OCH
OCH
OCH
OCH
Br
3
3
3
3
R
R
S
NA
362.8 ± 27.2
24.7 ± 1.5
NA
4
4
4
3d
4d
5d
R
S
261.0 ± 25.6
195.8 ± 1.4
NA
R
S
S
NA
4
4
4
4
2e
3e
4e
5e
S
566.4 ± 58.4
365.1 ± 50.9
67.8 ± 9.9
22.7 ± 0.8
129.3 ± 10.2
NA
Br
R
S
S
NA
Br
R
R
S
NA
Br
R
S
29.7 ± 2.7
NA
4
2f
F
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
4
4
4
4
4
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5
5
5
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4
3f
F
F
H
H
H
H
H
H
H
R
S
S
R
R
R
R
S
27.8 ± 3.0
7.4 ± 0.6
NA
104.3 ± 7.3
230.2 ± 31.8
52.56 ± 19.55
NA
NA
NA
44f
45f
31c
F
R
R
S
8.3 ± 0.5
144.6 ± 25.6
X
Br
Br
Br
Br
1.09 ± 0.23
2.99 ± 0.15
0.65 ± 0.07
63.71 ± 6.77
58.65 ± 18.20
S
O
S
0
3
1d
1e
1f
NA
NA
NA
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
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8
9
0
1
2
3
4
5
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9
0
1
2
3
4
5
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7
8
9
0
N
3
S
73.25 ± 9.15
NA
3
R
S
Y
aConfigurations given in brackets are indirectly determined by measuring only the other
b
stereoisomer(s). IC values represent data from three independent experiments, each
5
0
performed in triplicate. NA - Not active in used concentration range.
In our previous study, we aimed to explore the influence of introducing an additional
chiral center into a thiophene-containing modafinil analogue on its activity towards monoamine
transporters. However, despite promising results for one pure stereoisomer, lack of a complete
dataset for all four separated stereoisomers due to very low solubility of some of them
3
4
prevented a fully conclusive analysis. Thus, the same strategy was applied to two of the better
soluble thiazole-containing modafinil analogues. CE-123 was modified by introduction of
either a methoxy, methyl, fluoro, chloro, bromo, or trifluromethyl-group on the para-position
of only one of the phenyl rings. The individual stereoisomers were tested and, where possible,
the absolute configuration determined (Table 4).
Again, the four stereoisomers of each of the six analogous showed remarkable
differences in both, activity and selectivity, and in every case, the most active stereoisomer was
more promising in terms of activity and selectivity than the corresponding symmetrically
modified analogues. For this set of very similar compounds, general trends are seen regarding
the effect of the configuration on both, the S- and the C-atom. Those stereoisomers shown to
have S-configuration on both atoms (S,S) showed the highest activity on DAT, followed by the
molecules with S-configuration on the sulfoxide and R-configuration on the chiral carbon (S,R).
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In all cases, the R,S-configuration gave by far the lowest activity. For optimization of the
selectivity, it is interesting that the SAR is different regarding activity on DAT, NET and
SERT. The SERT is nearly exclusively inhibited by the R,R-stereoisomers, and 43d actually
inhibits SERT more effectively than DAT. The trend for NET is much less clear, but usually
S,S-stereoisomers, which are most active on DAT, did also hit NET, but with lower IC values.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
Surprisingly, the most promising CE-123 derivative tested so far, which possesses a single Br-
atom in para-position of one of the phenyls and S,S-configuration (42e), shows neglectable
activity on NET while inhibiting DAT with an IC of only 1.6 ± 0.6 µM.
5
0
To test if these findings can be translated to the thiazole-containing analogues bridged
on position 4 of thiazole, i.e. CE-111, CE-125, and 12a, the H-atom in para-position of one of
the phenyl rings of 12a was substituted by a Br-atom. Already the racemic mixture showed
improved activity on DAT (IC = 1.85 ± 1.20 µM). Chiral HPLC separation of the racemic
5
0
thioether intermediate 30, followed by introduction of the second chiral center by oxidation of
the sulfur, and another chiral HPLC separation gave individual stereoisomers 31c-31f (Scheme
4
). The SAR regarding the configuration on the two chiral centers was nearly the same as
described above, except that here the R,R-stereoisomer was more active on DAT than the S,R-
stereoisomer. 31e, which was demonstrated by single crystal X-ray diffraction analysis to have
S,S-configuration, showed the highest activity on DAT (IC = 0.65 ± 0.07 µM) of all tested
5
0
compounds in this series, while being at the same time 100-fold less active on NET (IC = 73
5
0
± 9 µM) and exerting negligible activity on SERT (Figure 2).
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. Representative reuptake inhibition curves of compounds 31c-31f. 31c-31f
represent individual stereoisomers with significant difference in activity and selectivity
towards DAT (Table 4).
With its submicromolar activity, 31e is a 10-fold more potent DAT inhibitor compared
to R-Modafinil and almost 20-fold more active than modafinil. For these reasons, 31e was
selected for further characterization.
Molecular docking of the four stereoisomers 31c-f
An overlay of the best scored docking poses of compounds 31c-f in the binding pocket
of an hDAT homology model is shown in Figure 3. The poses are very similar with the
exception of 31d, which orientates differently in the binding pocket. The interaction patterns
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of the four stereoisomers of 31c-f are shown in Figure 4. What should be highlighted is that
the interaction between the transporter and all four ligands is mostly governed by the
interaction between primarily hydrophobic amino acids and the phenyl rings of molecules 31c-
f. The exception is the observation of an additional hydrogen bond interaction between 31d
and Ser149.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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3
3
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4
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4
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4
5
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5
5
5
5
5
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5
6
0
1
2
3
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0
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0
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1
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3
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8
9
0
In general, it can be said that from the position of the ligand in the binding pocket and
the mostly entropic governed interactions methods that incorporate entropic contributions (e.g.
relative binding free energy) might be needed for further lead development.
Figure 3. The highest scored docked poses of 31c (cyane), 31d (yellow), 31e (orange)
3
1f (pink), are shown as overlay in the binding pocket of the hDAT homology model.
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Interactions of the highest scored pose of 31c-f in the binding pocket of
hDAT. Red lines indicate hydrogen bond donor interactions, yellow highlights hydrophobic
interactions.
In silico hERG channel activity profiling
5
0
The Inte:Ligand Tox Pharmacophore DB was used for toxicity profiling of all 37
synthesized compounds, but no significant effect on hERG channel was predicted for any of
them (Figure 5).
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9
0
Figure 5. Heat map of compounds screened using the Inte:Ligand hERG eTox
pharmacophore model, showing no relevant fit of any of the compounds to the query (white
boxes).
In vitro effect of 31c-31f on action potential parameters
In order to investigate the effect of enantio-pure analogues 31c-31f on action potential
(AP) parameters, the APs of Cor.4U® hiPSC-CMs cells in the presence of 0.01, 0.1, 1 or 10
µM of 31c-31f were measured by means of a voltage‐sensitive fluorescence dye. As shown in
Figure 6, none of the tested compounds caused early after depolarizations (EADs) or changes
in action potential duration or shape even when applied at 10 µM concentration.
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5
5
5
5
6
Figure 6. Representable APs in control (black) and recorded 30 min after application of
either vehicle (0.1% of DMSO) or test compounds 31c-31f at 10 µM concentrations (red).
None of the drugs affected action potential parameters.
0
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Effect of 31e on dopamine release
In superfusion experiments on HEK293 cells stably expressing the human DAT
3
preloaded with the metabolically inert transporter substrate [ H]1-methyl-4-phenylpyridinium
3
+
(
[ H]MPP ) 3µM D-amphetamine increased tritium efflux (Figure 7, circles) whereas 10µM
1e suppressed efflux (Figure 7, squares) and blocked D-amphetamine-induced release
Figure 7 triangles). Thus, 31e did not induce reverse transport as elicited by the prototypic
releasing drug D-amphetamine, but acted as a pure uptake inhibitory drug blocking the reverse
3
(
3
+
transport induced by D-amphetamine. The inhibitory action of 31e on basal efflux of [ H]MPP
may be due to a blockade of a basal DAT-mediated efflux of the transporter substrate observed
under our experimental condition.
Figure 7. Effects of 31e and D-amphetamine, as well as their interaction, on release by
3
+
human DAT. HEK293 cells stably expressing DAT were loaded with [ H]MPP and
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superfused, and 4-min fractions of superfusate were collected. After two or three fractions of
basal efflux, cells were exposed to buffers containing 10µM 31e (triangles), 3µM D-
amphetamine (circles) or both (squares) for three or four fractions as indicated by the bars
below. Symbols represent means ± SEM of seven to eight observations of three independent
experiments. * P<0.05, **P<0.001 vs. basal efflux (mean values of fractions at 0 and 5 min)
by paired Student’s t-test.
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In vitro neurotoxicity of 31e
Compound 31e was evaluated in the neurite outgrowth assay using cryopreserved rat
cortical neurons (QBM Cell Science) and was performed by Cyprotex (Massachusetts, USA,
Study Number CYP2052 – R1). Positive control, nocodazole, had an IC of 2.22 μM for valid
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neuron count, an IC of 0.097 μM for mean neurite average length and an IC of 0.0997 μM
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for neurite total length per neuron. Negative control, chlorpromazine, had an IC of 7.99 μM
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for valid neuron count, an IC of 9.022 μM for mean neurite average length and an IC of
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13.8 μM for neurite total length per neuron. Nocodazole was positive for substantially effecting
neurite outgrowth while nominally effecting cell viability. Chlorpromazine’s effect on neurite
outgrowth was not independent of its effect on cell viability (Table 5). 31e had nominal effects
(> 100 μM) on all three endpoints when tested at top concentration of 100 µM.
Table 5. Results for Valid Neuron Count, Mean Neurite Average Length and Neurite Total
Length per Neuron
Compound
Top conc.
Valid neuron
Mean neurite avg. Neurite total length per
tested (µM)
count IC (µM)
length IC (µM)
neuron IC (µM)
50
50
50
Nocodazole
1
2.22
7.99
0.097
9.022
0.0997
13.8
Chlorpromazine
100
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concentration of compound 31e and concentration needed to exert a toxic effect. This renders
compound 31e suitable for further in vivo investigations.
Plasma and brain levels of 31e
Pharmacokinetic analysis of the most potent DAT inhibitor 31e was performed by
Evotec (Hamburg, Germany) in Sprague Dawley rats after single intraperitoneal administration
at 7.5 mg/kg. In parallel, a brain penetration assessment was performed on three satellite
groups. The results are summarized in Table 6.
Table 6. Plasma and brain levels of 31e in Sprague Dawley rats after a single intraperitoneal
administration of 7.5 mg/kg
Time (h)
Concentration of 31e (nM)
Plasma
2203
1211
89.6
SD
193
712
28.1
17.1
Brain
803
SD
131
223
25.1
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409
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BLQ*
*
BLQ – below level of quantification
As shown in Table 6, low µM levels of 31e were reached in plasma after 1 and 3 h,
which rapidly decreased thereafter. In comparison, as shown previously, ~3.9 µg/mL of R-
modafinil were detected in plasma 1 h post intraperitoneal administration (10 mg/kg body
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weight) in male Sprague Dawley rats. Furthermore, the half-life of 31e was determined to be
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.6 ± 0.4 h, and was longer as previously shown for R-modafinil (t1/2 = 0.4 ± 0.2 h) and for 6
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(t1/2 = 0.9 h). Total drug exposure over time, represented as AUC
was determined to be
(0-inf)
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843 ± 1439 and was higher than that of R-modafinil (AUC(0-inf) = 1175 ± 1803), but lower
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than that of 6 (AUC(0-inf) = 13,519) . There are several possible explanations for being so.
Firstly, the administered dose of 31e was 7.5 mg/kg body weight, i.e. 25% lower than dose for
R-Modafinil and 6. Secondly, while R-modafinil and 6 were dissolved in DMSO/Tween
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0/Saline (10:15:75), 31e was dissolved using a DMSO-free formulation (40% Kolliphor EL
in water). The necessity of DMSO-free formulations of hydrophobic substances is due to the
recent findings that even small amounts of DMSO may modulate CNS function in vitro, in vivo
and ex vivo.36, 37 In the brain, 31e reached concentration of 803 ± 103 nM at 1h, 409 ± 223 nM
at 3h, and 44.3 ± 25.1 nM at 7h. A single intraperitoneal administration of 7.5 mg/kg body
weight of 31e is thus sufficient to reach a level in the brain that is somewhat above the IC for
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DAT, but far below the IC for NET. This compound can thus be expected to exert its desired
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modafinil-like biological effects in vivo. Other pharmacokinetic parameters are available under
Supplementary Information Section.
In vivo brain microdialysis
In order to further evaluate drug induced changes in extracellular dopamine levels, i.e.
to further estimate drug efficacy in vivo, 31e was administered in two doses (1 and 10 mg/kg
bodyweight) intraperitoneally into free moving male Sprague-Dawley rats and dopamine levels
were measured in nucleus accumbens (NAcb). NAcb extracellular DA levels were stable 1 h
prior first drug injection [time effect: F(2,34) = 1.650, P = 0.207] and did not differ between
the two treatment groups [treatment effect: F(1,17) = 0.971, P = 0.338; treatment x time
interaction: F(2,34) = 0.767, P = 0.473)] reaching an average concentration of 3.5 ± 0.27
fmol/5µl microdialysate. Perfusion with aCSF containing 100 mM KCl caused a pronounced
increase in extracellular DA levels in the NAcb as compared with basaline and pre-KCl
conditions [KCl effect: F(2,34) = 19.241, P < 0.001; treatment effect: F(1,17) = 0.538,
P = 0.473; treatment x KCl interaction: F(2,34) = 0.475, P = 0.626; Figure 8A) indicating
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