2-[(Diphenylmethyl)sulfinyl]acetamide (modafinil, (±)-1) is a unique dopamine uptake inhibitor that binds the dopamine transporter (DAT) differently than cocaine and may have potential for the treatment of psychostimulant abuse. To further investigate structural requirements for this divergent binding mode, novel thio- and sulfinylacetamide and ethanamine analogues of (±)-1 were synthesized wherein (1) the diphenyl rings were substituted with methyl, trifluoromethyl, and halogen substituents and (2) substituents were added to the terminal amide/amine nitrogen. Halogen substitution of the diphenyl rings of (±)-1 gave several amide analogues with improved binding affinity for DAT and robust selectivity over the serotonin transporter (SERT), whereas affinity improved at SERT over DAT for the p-halo-substituted amine analogues. Molecular docking studies, using a subset of analogues with DAT and SERT homology models, and functional data obtained with DAT (A480T) and SERT (T497A) mutants defined a role for TM10 in the substrate/inhibitor S1 binding sites of DAT and SERT.
2-[(Diphenylmethyl)sulfinyl]acetamide (modafinil, (±)-1) is a unique dopamine uptake inhibitor that binds the dopamine transporter (DAT) differently than cocaine and may have potential for the treatment of psychostimulant abuse. To further investigate structural requirements for this divergent binding mode, novel thio- and sulfinylacetamide and ethanamine analogues of (±)-1 were synthesized wherein (1) the diphenyl rings were substituted with methyl, trifluoromethyl, and halogen substituents and (2) substituents were added to the terminal amide/aminenitrogen. Halogen substitution of the diphenyl rings of (±)-1 gave several amide analogues with improved binding affinity for DAT and robust selectivity over the serotonin transporter (SERT), whereas affinity improved at SERT over DAT for the p-halo-substituted amine analogues. Molecular docking studies, using a subset of analogues with DAT and SERT homology models, and functional data obtained with DAT (A480T) and SERT (T497A) mutants defined a role for TM10 in the substrate/inhibitor S1 binding sites of DAT and SERT.
Inhibition
of dopamine (DA)
reuptake is proposed to be the mechanism underlying the reinforcing
effects of abused psychostimulant drugs such as cocaine and methamphetamine.
Modafinil (2-[(diphenylmethyl)sulfinyl]acetamide, (±)-1; Figure 1) is used clinically for the treatment
of sleep disorders and inhibits DA reuptake, with no evidence of abuse
liability in humans.[1,2] Recent attention has focused on
a distinctive binding mode at the dopamine transporter (DAT) to explain
this curious pharmacological profile of (±)-1 and
particularly its R-enantiomer (armodafinil, R-(−)-1).[1,3] These studies
independently demonstrated that (±)-1 binds the
DAT in a unique fashion compared to cocaine, which may be related
to its distinct behavioral profile. However, there are other reports
suggesting additional mechanisms underlying the pharmacological actions
of (±)-1 and in particular its effectiveness in
attenuating psychostimulant drug seeking in animal models.[4−9] Nevertheless, direct interaction with these other targets has not
been demonstrated.[2] One potential contribution
to the preclinical pharmacology of (±)-1 is that
it is a nonaminergic compound with limited water solubility, which
can complicate investigation due to the large concentration of drug
needed for in vitro and in vivo studies. The high doses of (±)-1 used in preclinical studies may indeed have direct or downstream
interactions with numerous targets, including histaminergic, GABAergic,
orexinergic, glutamatergic, adrenergic, and serotonergic neurons.[2,5,8] However, whether or not these
targets are related to therapeutic or behavioral outcomes remains
unknown.
Figure 1
(±)-1, its enantiomers, and the DAT-selective
amino analogue 2.
(±)-1, its enantiomers, and the DAT-selective
amino analogue 2.In a previous study, we began to explore the structure–activity
relationships (SARs) of (±)-1 analogues at the DAT,
serotonin transporter (SERT), and norepinephrine transporter (NET)
and identified one analogue, compound 2 (Figure 1), in which the terminal amide was replaced with
a 3-phenylpropyl-substituted amine group, with enhanced DAT affinity.[10] The DAT affinity for 2 was improved
by 10-fold, compared to that for (±)-1, as was its
water solubility. In addition, 2 demonstrated low micromolar
binding affinities for SERT and NET, which prompted a systematic and
comparative exploration of the SAR of the (±)-1 scaffold
at all three monoamine transporters (MATs) with a series of novel
analogues. Specifically, we wanted to further investigate the role
of the terminal amide or substituted amine functions on DAT vs SERT
and NET binding and also determine how additional diphenyl substitutions
on the sulfinylethanamine or reduced thioethanamine template affected
the binding affinities and modes. To this end, a series of thioacetamideand sulfinylacetamide analogues were prepared and compared to a set
of thioethanamine and sulfinylethanamine analogues of (±)-1.
Chemistry
Scheme 1 outlines
the synthesis of novel
thioacetamide (compounds 4a–4z) and
sulfinylacetamide (compounds 5a–5g) analogues of (±)-1. The thioacetamide analogues 4a–4z were generated via three different
synthetic routes.
Scheme 1
Synthesis of Thioacetamide and Sulfinylacetamide Analogues
of (±)-1
Reagents and conditions:
(a)
2-mercaptoacetamide or 2-mercapto-N-methylacetamide,
TFA, room temperature (60 °C for substituted phenyl analogues),
20 h (procedure A); (b) (i) thioglycolic acid, TFA, room temperature
(55–60 °C for substituted phenyl analogues), overnight;
(ii) CH3I, K2CO3, acetone, reflux,
overnight; (iii) NH4OH, NH4Cl, MeOH, 50 °C,
72 h (procedure B); (c) (i) thioglycolic acid, TFA, room temperature
(55–60 °C for substituted phenyl analogues), overnight;
(ii) CDI, THF, room temperature, 2 h; (iii) RNH2, THF,
0 °C to room temperature, overnight (procedure C); (d) H2O2 (30%), AcOH–MeOH (1:3), 40 °C, overnight.
The first route (procedure A) affords the
thioacetamides in one
step and was employed in the synthesis of compound 4a and the N-methylthioacetamides 4g–4i. As opposed to the previously reported two-step synthesis,[11] thioacetamide 4a was synthesized
in one step by coupling 2-mercaptoacetamide with diphenylmethanol
in trifluoroacetic acid (TFA) at room temperature. Similarly, N-methylthioacetamides 4g–4i were synthesized via the coupling reaction between 2-mercapto-N-methylacetamide and diphenylmethanol (for 4g) or the corresponding bis(halophenyl)methanol (for 4h and 4i) in TFA. To improve product yields, the reactions
for compounds 4h and 4i required heating
to 60 °C.
Synthesis of Thioacetamide and Sulfinylacetamide Analogues
of (±)-1
Reagents and conditions:
(a)
2-mercaptoacetamide or 2-mercapto-N-methylacetamide,
TFA, room temperature (60 °C for substituted phenyl analogues),
20 h (procedure A); (b) (i) thioglycolic acid, TFA, room temperature
(55–60 °C for substituted phenyl analogues), overnight;
(ii) CH3I, K2CO3, acetone, reflux,
overnight; (iii) NH4OH, NH4Cl, MeOH, 50 °C,
72 h (procedure B); (c) (i) thioglycolic acid, TFA, room temperature
(55–60 °C for substituted phenyl analogues), overnight;
(ii) CDI, THF, room temperature, 2 h; (iii) RNH2, THF,
0 °C to room temperature, overnight (procedure C); (d) H2O2 (30%), AcOH–MeOH (1:3), 40 °C, overnight.The second synthetic route (procedure B), used
for the synthesis
of thioacetamides 4b–4f, required
three steps, similar to previously described methods.[10] Mono- or disubstituted diphenylmethanol was coupled with
thioglycolic acid in TFA, followed by esterification with iodomethane
in acetone under reflux conditions. The resulting methyl esters were
converted into the primary amides 4b–4f through aminolysis with ammonium hydroxide in methanol.The
third route (procedure C)[10] also
involved three steps and was employed for the synthesis of N-substituted
thioacetamides4j–4z. First, diphenylmethanol
or the appropriate bis(halophenyl)methanol was coupled with thioglycolic
acid in TFA. The desired N-substituted thioacetamides4j–4z were then synthesized by coupling the carboxylic
acid to the corresponding primary amine via an in situ N,N′-carbonyldiimidazole coupling reaction.
Oxidation of the appropriate thioacetamide (4b–4g and 4x) using hydrogen peroxide (H2O2; 30%) in an acetic acid–methanol solution mixture
gave sulfinylacetamides 5a–5g.Scheme 2 outlines the synthesis of the thioethanamine
(6a–6i) and sulfinylethanamine (7a–7e) analogues of (±)-1. Thioethanamines 6a–6c were synthesized
by coupling diphenylmethanol or the appropriate bis(halophenyl)methanol
with cysteamine hydrochloride in glacial acetic acid in the presence
of the Lewis acid catalyst boron trifluoride diethyl etherate (BF3·OEt2) (procedure D).[12,13] The N-substituted thioethanamine 6d was synthesized
by a reductive amination reaction between the hydrochloride salt of
compound 6a and cyclopropanecarboxaldehyde using sodium
cyanoborohydride as the reducing agent. Similarly, N-substituted thioethanamine 6e was synthesized by coupling n-butyl bromide
to the free base of compound 6a in the presence of CsOH·H2O (Cs+ ions served as templating catalysts).[14] N-substituted thioethanamines 6f–6h were synthesized by the reduction of thioacetamides 4u–4w using alane (LiAlH4–sulfuric
acid mixture).[10] Lastly, N-substituted
thioethanamine 6i was prepared from thioacetamide 4x by reduction with borane in THF (BH3·THF).
Sulfinylethanamines7a–7e were synthesized
from the appropriate thioethanamines (6a, 6e, or 6g–6i) by oxidation of the
thioether function using either sodium periodate (NaIO4) in an ethanol–water solution (compounds 7a and 7b) or H2O2 (30%) in an acetic acid–methanol
solution (compounds 7c–7e).
Scheme 2
Synthesis
of Thioethanamine and Sulfinylethanamine Analogues of (±)-1
Reagents and conditions: (a)
cysteamine hydrochloride, BF3·OEt2, glacial
AcOH, 80–90 °C, ∼20 min (40–50 min for substituted
analogues (procedure D)); (b) (i) procedure D; (ii) cyclopropane carboxaldehyde,
NaBH3CN, MeOH, 1,2-dichloroethane, room temperature, overnight;
(c) (i) procedure D; (ii) BuBr, CsOH·H2O, 4 Å
MS, DMF, room temperature, 20 h; (d) LiAlH4, H2SO4, THF; (e) BH3·THF, THF, reflux, overnight;
(f) NaIO4, H2O, EtOH, 0 °C to room temperature,
overnight; (g) H2O2 (30%), AcOH–MeOH
(1:3), 40 °C, 24 h.
Synthesis
of Thioethanamine and Sulfinylethanamine Analogues of (±)-1
Reagents and conditions: (a)
cysteamine hydrochloride, BF3·OEt2, glacial
AcOH, 80–90 °C, ∼20 min (40–50 min for substituted
analogues (procedure D)); (b) (i) procedure D; (ii) cyclopropane carboxaldehyde,
NaBH3CN, MeOH, 1,2-dichloroethane, room temperature, overnight;
(c) (i) procedure D; (ii) BuBr, CsOH·H2O, 4 Å
MS, DMF, room temperature, 20 h; (d) LiAlH4, H2SO4, THF; (e) BH3·THF, THF, reflux, overnight;
(f) NaIO4, H2O, EtOH, 0 °C to room temperature,
overnight; (g) H2O2 (30%), AcOH–MeOH
(1:3), 40 °C, 24 h.
Results and Discussion
SARs at DAT, SERT, and NET
In a
previous study, we
showed that, in general, p-halogen substitution of
the diphenylmethyl moiety of the (±)-1 structure
gave analogues with improved binding affinities for the DAT over SERT
and NET.[10] Additionally, we confirmed that
enantioselectivity at DAT for the R- and S-enantiomers was only ∼3-fold and that replacement
of the sulfoxide (S=O) with a sulfide function may have minimal
effects on DAT binding. Importantly, we discovered that reducing the
terminal amide and appending a 3-phenylpropyl substituent resulted
in compound 2 (Figure 1), which
was identified as having higher binding affinities than the amide
analogues at all three MATs. This result was particularly encouraging,
as salts of amines present a solubility advantage over the parent
amide (±)-1. In the current study, we further explored
the effect of reducing the S=O, while adding increasingly bulky
substituents at the amidenitrogen. Additionally, we expanded the
library of amine analogues with and without the S=O motif.
Our hypothesis was that SARs within this class of (±)-1 analogues would help unravel SAR differences between the MATs and
also identify binding motifs related to the unique binding mode of
this class of DAT inhibitors.Binding affinities of all novel
(±)-1 analogues were evaluated at the DAT, SERT,
and NET in ratbrain membranes using a slightly modified version of
previously described methods[15] and are
detailed in the Experimental Methods. The
results of the in vitro assays, grouped by functionality into amides
and amines, are presented in Tables 1 and 2, respectively. All sulfinyl compounds were tested
as racemic mixtures.
Table 1
MAT Binding Data for Thio- and Sulfinylacetamide
Analoguesa
Ki [SE interval] (nM)
compd
X
Y
R
DAT
SERT
NET
(±)-1
H
S=O
H
2600 [2430– 2780]
IAb
IAb
4a
H
S
H
12400 [10800–14300]
14500 [11800–17700]
IA,b 285000 [117000–690000]
4e
3,3′-di-Cl
S
H
275 [257–295]
IA,b 808000 [706000–924000]
45400 [39600–52000]
4g
H
S
methyl
19300 [17900–20800]
IA,b 656000 [302000–1420000]
27200 [25700–28900]
4h
4,4′-di-Cl
S
methyl
4130 [3620–4710]
10700 [7310–15700]
9770 [9170–10400]
4i
4,4′-di-Br
S
methyl
3010 [2770–3260]
5720 [5320–6150]
11000 [9540–12600]
4j
H
S
allyl
8370 [6680–10500]
IA,b 303000 [267000–344000]
IA,b 171000 [88300–332000]
4k
H
S
n-propyl
20700 [20300–21100]
IA,b 419000 [240000–729000]
68000 [53200–86900]
4l
4,4′-di-F
S
n-propyl
11700 [10300–13200]
44200 [38700–50500]
59700 [51200–69600]
4m
4,4′-di-Cl
S
n-propyl
1240 [1120–1380]
10100 [8900–11400]
7540 [6830–8330]
4n
4,4′-di-Br
S
n-propyl
590 [550–632]
8900 [8150–9720]
10600 [9980–11300]
4o
H
S
cyclopropylmethyl
13600 [11900–15600]
20500 [17500–23900]
IAb
4p
4,4′-di-F
S
cyclopropylmethyl
6700 [5730–7830]
34000 [28800–40200]
57000 [51500–63000]
4q
4,4′-di-Br
S
cyclopropylmethyl
975 [852–1110]
7030 [6040–8180]
IAb
4r
H
S
n-butyl
23600 [20500–27100]
IAb
IAb
4s
4,4′-di-F
S
n-butyl
6400 [5820–7050]
25500 [23300–28000]
56100 [53900–58500]
4t
4,4′-di-Br
S
n-butyl
722 [659–792]
7090 [6990–8180]
7580 [7210–7970]
4u
H
S
3-phenylpropyl
2020 [1990–2050]
IAb
IAb
4v
4,4′-di-F
S
3-phenylpropyl
442 [385–509]
3500 [2950–4160]
IAb
4w
4,4′-di-Cl
S
3-phenylpropyl
223 [191–260]
IAb
IAb
4x
4,4′-di-Br
S
3-phenylpropyl
238 [202–280]
60700 [58400–63200]
35500 [31700–39800]
4y
H
S
4-phenylbutyl
1150 [1020–1290]
IAb
7960 [7590–8350]
4z
4,4′-di-Br
S
4-phenylbutyl
405 [348–471]
IAb
IAb
5a
4,4′-di-CH3
S=O
H
12700 [12400–13100]
IAb
IAb
5b
4,4′-di-CF3
S=O
H
35400 [34100–36700]
NTc
NTc
5c
3,3′-di-F
S=O
H
5930 [4990–7060]
IAb
IAb
5d
3,3′-di-Cl
S=O
H
881 [763–1020]
IAb
IAb
5e
H, 3-Br
S=O
H
550 [542–557]
IAb
IAb
5f
H
S=O
methyl
13100 [12600–13700]
IAb
IAb
5g
4,4′-di-Br
S=O
3-phenylpropyl
1280 [1160–1400]
892 [787–1010]
IAb
8d
4,4′-di-Cl
S
H
2200 [2060–2390]
38800 [36400–41300]
51400 [46000–57500]
Each Ki value represents
data from at least three independent experiments,
each performed in triplicate. Ki values
were analyzed by PRISM. Binding assays are described in detail in
the Experimental Methods.
IA = inactive, defined as <50%
inhibition at 100 μM; however, in some cases a Ki value could be derived and is included.
NT = not tested.
Previously reported by Cao et al.[10]
Table 2
MAT Binding
Data for Thio- and Sulfinylethanamine
Analoguesa
Ki [SE interval] (nM)
compd
X
Y
R
DAT
SERT
NET
6a
H
S
H
142 [131–155]
221 [191–257]
980 [938–1020]
6b
Cl
S
H
296 [272–323]
29.8 [28.2–31.5]
6920 [6340–7550]
6c
Br
S
H
483 [434–536]
26.1 [23.9–28.5]
8540 [7980–9130]
6d
H
S
cyclopropylmethyl
435 [406–466]
10000 [9570–10400]
17300 [15400–19400]
6e
H
S
n-butyl
310 [275–350]
5700 [5040–6440]
11500 [10700–12300]
6f
H
S
3-phenylpropyl
295 [268–325]
927 [786–1090]
5500 [5140–5880]
6g
F
S
3-phenylpropyl
114 [97.4–132]
354 [312–402]
3850 [3830–3870]
6i
Br
S
3-phenylpropyl
613 [564–667]
163 [156–170]
3160 [2950–3390]
7a
H
S=O
H
1110 [1020–1200]
3380 [2970–3820]
24500 [22800–26200]
7b
H
S=O
n-butyl
1570 [1490–1660]
63600 [56100–72200]
138000 [103000–184000]
7c
F
S=O
3-phenylpropyl
183 [140–239]
1280 [1100–1480]
3270 [3130–3420]
7d
Cl
S=O
3-phenylpropyl
645 [565–736]
553 [513–595]
5670 [5000–6420]
7e
Br
S=O
3-phenylpropyl
1270 [1130–1420]
557 [493–628]
8650 [7450–10000]
2b
H
S=O
3-phenylpropyl
192 [177–209]
987 [870–1120]
2320 [2060–2620]
Each Ki value represents
data from at least three independent experiments,
each performed in triplicate. Ki values
were analyzed by PRISM. Binding assays are described in detail in
the Experimental Methods.
Previously reported by Cao et al.[10]
In Table 1, most
of the thioacetamideand sulfinylacetamide analogues displayed micromolar
affinities at the DAT, within ±10-fold of that of (±)-1 (Ki = 2600 nM). Reducing the
S=O to the thioether 4a decreased DAT binding
by ∼5-fold, while improving SERT affinity. When the diphenyl
rings were unsubstituted, alkyl substitution of the terminal amidenitrogen decreased binding affinity at the DAT with or without the S=O motif (e.g., compounds 4a, 4g, 4j, 4k, 4o, 4r, and 5f). The exception to this trend
was observed with compounds 4u and 4y, which
displayed similar or nominally improved binding affinities (Ki = 2020 and 1150 in nM, respectively) in comparison
to (±)-1. Within each series of N-substituted thioacetamides,
binding affinity at the DAT generally increased with halogen substitution
at the para-position of the diphenylmethyl moiety
in the order H < F < Cl ≤ Br. This order
is in agreement with previously reported data[10] and applies to both the thioacetamidesand sulfinylacetamides with
or without substitution on the amidenitrogen. It has been proposed
that if a ligand can establish a halogen bond interaction with a receptor
in an optimal orientation, the Cl to Br to I substitution may result
in an increase of affinity.[16] Thus, the
order we observed might be consistent with the halogen substituent
forming a halogen bond with a polar residue of DAT. Additionally,
substitution at other positions of the diphenyl rings followed this
halogen substitution order, for example, compounds 5c–5e with halogen substituents in the meta-positions of the diphenyl rings. In general, the novel
acetamides were selective for the DAT over the SERT and NET, except
for compounds 4a and 5g, both of which displayed
roughly equal affinities at the DAT and SERT (DAT:SERT affinity ratios
of 1 and 1.4, respectively). Five amide analogues—4e, 4w, 4x, 4z, and 5e—were identified as the most DAT-selective compounds in the
series (e.g., SERT:DAT affinity ratios of >2900 for 4e and 249 for 4x, with no displacement at the SERT for 4w, 4z, or 5e). The pronounced selectivity
observed with compound 4e for DAT over SERT is remarkable,
especially in comparison to its regioisomer, compound 8(10) (Table 1), which
is only modestly selective for DAT over SERT (SERT:DAT affinity ratio
= 18).Each Ki value represents
data from at least three independent experiments,
each performed in triplicate. Ki values
were analyzed by PRISM. Binding assays are described in detail in
the Experimental Methods.IA = inactive, defined as <50%
inhibition at 100 μM; however, in some cases a Ki value could be derived and is included.NT = not tested.Previously reported by Cao et al.[10]As shown in Table 2, removal of the amidecarbonyl (C=O) function resulted in
improved affinities at the DAT, SERT, and NET (compare compounds 6a and 7a to (±)-1), with several
of the novel amino analogues having nanomolar binding affinities at
the DAT in comparison to the micromolar affinity of compound (±)-1. With the thioethanamines, in contrast to the thioacetamides,
DAT affinity generally increased with increasingly bulky substitution
on the terminal aminenitrogen for analogues with unsubstituted diphenyl
rings (see compounds 6d, 6e, and 6f). For analogues with halogen substituents on the diphenyl rings
within a particular series, DAT binding affinities generally increased
in a reverse order compared to that observed for the acetamides, viz.,
Br < Cl < F ≤ H with or without the S=O group.
Overall, compounds 6g (Ki = 114 nM) and 6a (Ki =
142 nM) displayed the highest affinities at the DAT, with each displaying
about 20-fold improved affinity compared to (±)-1. However, in terms of selectivity among the MATs, the most DAT-selective
compounds in this series are 6d, 6e, and 7b (SERT:DAT = 23, 18, and 41, respectively; NET:DAT = 40,
37, and 88, respectively). Previously, we identified only one amino
analogue of (±)-1 that was selective for the SERT
over the DAT.[10] In the series reported
herein, we identified four additional compounds—6b, 6c, 6i, and 7e—that
are SERT-selective, with compounds 6b and 6c displaying nanomolar affinities (Ki =
30 and 26 nM, respectively) at the SERT.Each Ki value represents
data from at least three independent experiments,
each performed in triplicate. Ki values
were analyzed by PRISM. Binding assays are described in detail in
the Experimental Methods.Previously reported by Cao et al.[10]
Molecular Docking and Mutagenesis
Studies
To interpret
SARs revealed by radioligand binding studies in the context of ligand–transporter
interactions, we carried out molecular docking studies with both DAT
and SERT homology models that are based on the crystal structure of
the bacterial homologue, LeuT. These studies led to the identification
of a key divergent position in transmembrane helix 10 (TM10), T497
in SERT and A480 in DAT, that appears to contribute to the DAT vs
SERT selectivity. Previously A479 and A480 of DAT were found to be
involved in the binding of benztropine (3α-(diphenylmethoxy)tropane)
and its derivatives, the atypical DAT inhibitors, many of which do
not exert cocaine-like subjective effects. In contrast, the mutation
of these two residues did not significantly affect the binding of
a cocaine analogue, WIN 35,428 (2β-carbomethoxy-3β-(4-fluorophenyl)tropane).[17] In addition, it has been reported that the covalent
modification on T497C of SERT by the cysteine-reactive MTSET (2-(trimethylammonium)ethyl
methanethiosulfonate) disrupted activity.[18]It is clear from the SARs described herein that reduction
of the amide to a secondary or primary amine significantly improves
binding affinities at all three MATs (e.g., 4a vs 6a). This effect appears to be most consistent at DAT, as
all but a few analogues in Table 2 have submicromolar
affinities. Interestingly, when the diphenyl ring system is substituted
with either p-Cl or p-Br groups,
the binding affinities at SERT are more improved than at DAT in all
cases and most dramatically with compounds 6b and 6c, which bind with Ki values
of ≤30 nM at SERT, suggesting a specific interaction at the para-position that may differ between these two transporters.
To investigate this further, we carried out molecular docking studies
with a group of representative compounds using the homology models
of DAT and SERT based on the crystal structure of LeuT[1,19] to compare the differences in their binding modes for these targets.Previously, we proposed that the sulfoxide O interacted with the
conserved Y156 in DAT.[1] Interestingly,
the residue immediately before Y156 is divergent among MATs: whereas
in DAT this residue is phenylalanine (F155), the aligned position
in SERT/NET is a tyrosine. Molecular docking studies revealed that
while both F155 in DAT and Y175 in SERT directly interact with (±)-1, this molecule differentially affects how Y156 of DAT and
Y176 of SERT are positioned when bound. Thus, the S=O is optimally
positioned to interact with Y156 of DAT but not Y176 of SERT. If the
S=O cannot properly interact with the conserved Tyr in SERT,
the S=O contributes negatively to the binding affinity, and
as a result (±)-1 has higher affinity for DAT than
SERT. Conversely, absence of the sulfoxideoxygen should increase
the affinity for SERT. Consistent with this prediction, in the presence
of the carbonyloxygen of the amide [(±)-1 vs compound 4a, Table 1], reducing the S=O
decreased the binding affinity for DAT but increased the affinity
for SERT. Nevertheless, when either of the phenyl rings was substituted
with halogens (4e vs 5d) or the terminal
amide was substituted (5f vs 4g), this trend
was not obvious, underscoring the influence of these additional substituents
on the binding mode in both DAT and SERT. Note the binding affinities
at SERT are so low for these analogues it is impossible to determine
a specific trend.By reducing the amidecarbonyl, the N becomes
positively charged,
resulting in an increase in affinity for all three MATs as described
above [compare (±)-1 to 7a]. An interpretation
is that the positive charge facilitates direct interaction between
the N and the conserved negatively charged Asp involved in the Na1
binding site for all three transporters. Additionally, the combined
effect of a global reduction of both the amidecarbonyl and sulfoxideoxygens is even higher affinities at the DAT, SERT, and NET, suggesting
that the impact of the charged N is dominant compared to removal of
the sulfoxide O, especially for DAT and SERT (compare (±)-1 to 4a vs (±)-1 to 6a].To test the hypothesis that these residues in TM10 are part
of
the primary substrate/inhibitor (S1)[1,20] binding site
and play different roles in DAT vs SERT binding for para-halogenated analogues in this series, we created two chimera mutants
in DAT and SERT in which the residues are interchanged, resulting
in DAT-A480T and SERT-T497A. The effect of the mutations on uptake
inhibition potency for compounds with a Cl substituent in the para-position were measured on intact COS-7 cells transiently
expressing WTs or the Ala- and Thr-substituted mutants (Tables 3 and 4). While this paper
was being prepared, the crystal structure of Drosophila
melanogaster DAT (dDAT) bound with the tricyclic antidepressant
nortriptyline became available.[21] The core
of the dDAT structure “closely resembles that of LeuT”,[21] which we used as the template to build the DAT
and SERT homology models for this study, and shows the aligned Ala479
of TM10 is indeed in direct contact with the edge of one of the nortriptyline
phenyl rings. Therefore, the dDAT structure supports our prediction
that this TM10 position faces the S1 binding sites of SERT and DAT.
Interestingly, the affinity of (±)-1 is increased
in DAT-A480T (∼5-fold) and perhaps slightly in the SERT-T497A
mutant, compared to those of their corresponding WTs. In addition,
whereas the affinity of the p-Cl-substituted thioacetamide 4h (a secondary amide) is significantly decreased at SERT-T497A
compared to SERT-WT (Table 4), suggesting a
direct interaction between the p-Cl and the side
chain of T497 (Figure 2), the affinity of 4h at DAT-A480T is essentially the same as that at DAT-WT,
similar to 4g, which does not possess the p-Cl substituent (Table 3). The observed affinity
is also consistent with an alternative explanation that the hydroxyl
group of the Thr497 side chain forms an intrahelical H-bond with the
protein backbone,[22] while the α-methyl
group is exposed to the binding site as a hydrophobic contact to accommodate
the halogen substitution, especially for amide analogues (e.g., 4h; Table 4).
Table 3
[3H]DA Uptake Inhibition
Potency for Selected Analogues Measured in Intact COS7 Cells Expressing
the Human DAT Wild Type or the A480T Mutanta
compd
hDAT-WT Ki [SE interval] (nM)
n
hDAT-A480T Ki [SE interval] (nM)
n
DA (KM)
1160 [980–1380]
9
1930 [1510–2480]
5
(±)-1
13000 [10000–17000]
6
3090 [2300–4200]
3
4g
5500 [4000–7600]
4
3600 [2010–6300]
3
4h
3700 [2700–5100]
5
2300 [1700–3100]
3
6a
390 [280–540]
3
720 [620–830]
4
6b
1210 [960–1510]
5
1370 [1240–1510]
3
The inhibition potency for [3H]dopamine (DA) uptake
was calculated from nonlinear regression
analysis of uptake experiments performed on COS7 cells transiently
transfected with cDNA of the human dopamine transporter (hDAT) wild
type (WT) or the Ala480 to Thr mutant (A480T). The IC50 values used in the calculation of KM and Ki were calculated from the means
of pIC50, and the indicated SE intervals were calculated
from pIC50 ± SE. Nonspecific uptake was determined
using 50 μM nomifensine.
Table 4
[3H]-5-HT Uptake Inhibition
Potency for Selected Analogues Measured in Intact COS7 Cells Expressing
the Human SERT Wild Type or the T497A Mutanta
compd
hSERT-WT Ki [SE interval] (nM)
n
hSERT-T497A Ki [SE interval] (nM)
n
5-HT (KM)
520 [360–760]
7
1090 [840–1430]
4
(±)-1
IAb
3
570000 [497000–653000]c
3
4g
IAb
3
IAb
3
4h
8300 [6000–11600]
3
27000 [14000–53000]
2
6a
690 [590–810]
4
630 [550–720]
3
6b
270 [230–330]
3
170 [91–320]
3
The inhibition potency for [3H]serotonin (5-HT) uptake
was calculated from nonlinear regression
analysis of uptake experiments performed on COS7 cells transiently
transfected with cDNA of the human serotonin transporter (hSERT) wild
type (WT) or the Thr497 to Ala mutant (T497A). The IC50 values used in the calculation of KM and Ki values were calculated from the
means of pIC50, and the indicated SE intervals were calculated
from pIC50 ± SE. Nonspecific uptake was determined
using 5 μM paroxetine.
IA = inactive, defined as <50%
inhibition at 100 μM.
According to our definition, (±)-1 would be IA.
However, we were able to determine a Ki value, and although the affinity for the T497A
mutant was very low, it was, in fact, higher than that at WT SERT,
where no Ki could be determined.
Figure 2
Docking of compound 4h in the S1
binding site of WT
SERT. Panel A is an overall view of the binding pose of compound 4h in the binding site. Panel B is a zoom-in view showing
the interaction with Thr497 from TM10. The dashed line indicates favorable
halogen bonding between 4h and the side chain OH group
of T497 in WT SERT, while a similar interaction between 4h and A497, in the mutant, is absent, resulting in a reduction in
binding affinity.
The inhibition potency for [3H]dopamine (DA) uptake
was calculated from nonlinear regression
analysis of uptake experiments performed on COS7 cells transiently
transfected with cDNA of the humandopamine transporter (hDAT) wild
type (WT) or the Ala480 to Thr mutant (A480T). The IC50 values used in the calculation of KM and Ki were calculated from the means
of pIC50, and the indicated SE intervals were calculated
from pIC50 ± SE. Nonspecific uptake was determined
using 50 μM nomifensine.The inhibition potency for [3H]serotonin (5-HT) uptake
was calculated from nonlinear regression
analysis of uptake experiments performed on COS7 cells transiently
transfected with cDNA of the humanserotonin transporter (hSERT) wild
type (WT) or the Thr497 to Ala mutant (T497A). The IC50 values used in the calculation of KM and Ki values were calculated from the
means of pIC50, and the indicated SE intervals were calculated
from pIC50 ± SE. Nonspecific uptake was determined
using 5 μM paroxetine.IA = inactive, defined as <50%
inhibition at 100 μM.According to our definition, (±)-1 would be IA.
However, we were able to determine a Ki value, and although the affinity for the T497A
mutant was very low, it was, in fact, higher than that at WT SERT,
where no Ki could be determined.Docking of compound 4h in the S1
binding site of WT
SERT. Panel A is an overall view of the binding pose of compound 4h in the binding site. Panel B is a zoom-in view showing
the interaction with Thr497 from TM10. The dashed line indicates favorable
halogen bonding between 4h and the side chain OH group
of T497 in WT SERT, while a similar interaction between 4h and A497, in the mutant, is absent, resulting in a reduction in
binding affinity.These results support
our hypothesis that halogen bond interactions
at SERT T497 affect the binding affinities of these analogues. We
also hypothesized that a change of affinity might result for the A480TDAT mutant; however, we found that the binding affinity was not affected
by this mutation. Hence, these data suggest that the binding sites
of DAT and SERT obviously have other divergences beyond this single
residue position—the ways in which the rest of the binding
sites of DAT and SERT change in response to the mutations are different—and
simply switching the residues at this position is not enough to interconvert
the specificity of the compounds. For example, in both DAT and SERT,
the affinities of 6a and 6b (both primary
amines) remain unchanged at the mutants, suggesting that the exact
configuration near the terminal nitrogen, either amide or charged
nitrogen, has a strong impact on the orientation of the diphenylmethyl
moiety in both transporters. Taken together, this residue position
of TM10 appears to be more important for binding of the amide derivatives
of (±)-1 with a p-Cl substituent
at the SERT, compared to binding at the DAT. If the amide function
is reduced to an amine, the relative importance of interaction at
these residues is diminished.Consistent with this understanding,
at the SERT, the difference
in binding affinities for halogen-substituted analogues of (±)-1 is more pronounced in compounds lacking a charged N (e.g.,
>50-fold increase in SERT affinity for amide 4i vs 4g in Table 1 and only a 9-fold increase
in SERT affinity for amine 6c vs 6a in Table 2). In both cases, improvements in DAT affinity were
diminished compared to those in SERT affinity, with only a 6-fold
improvement in DAT affinity between 4i and 4g and only a 3-fold improvement for DAT affinity between 6c and 6a. In contrast, moving the halogen substituent
to the meta-position as in compounds 4e and 5d has little if any effect on SERT binding; hence,
a decrease in or no change in binding affinity resulted compared to
those of compound 4a and (±)-1, respectively.
However, the halogen substituent in the meta-position
appears to generate new interactions that favor binding to the DAT,
further supporting the influence of other residue divergences in the
binding sites of DAT and SERT on compound affinity. Thus, we propose
that substitution at the meta-position may be more
favorable for designing DAT-over-SERT-selective analogues of (±)-1 and may warrant further exploration.
Conclusion
A series of novel thio- and sulfinylacetamide and -ethanamine analogues
of (±)-1, with or without substituents on the diphenyl
rings, were synthesized to investigate the contributions of structural
variations to selectivity across MATs. Previous SARs had suggested
that the sulfinyl (S=O) function was not critical for binding
to the DAT, but differential interactions with Y156 of DAT and Y176
of SERT may affect selectivity for SERT.[1] In addition, we showed that reduction of the amide function to the
amine not only improved water solubility, but also enhanced DAT affinity.[10] In the current study, the earlier SARs were
expanded. para- or meta-substitution
of the phenyl rings of (±)-1 with Cl or Br gave
several amide analogues with improved selectivity for DAT over SERT
and NET, whereas selectivity was improved at SERT over DAT and NET
for the amine analogues. Overall, we identified five highly DAT-selective
amide analogues (e.g., 4e, 2900-fold over SERT) and two
SERT-selective amine analogues with high affinity (Ki ≤ 30 nM). Computational modeling of DAT and SERT
led to the identification of key amino acid residues in TM10 that
form part of the S1 binding pocket in both DAT and SERT. By switching
the T497 in SERT to Ala and the A480 in DAT to Thr and then testing
a selected subgroup of analogues, the role of TM10 in DAT and SERT
binding was further defined. Moreover, we propose that this TM10 position
faces the S1 binding site and plays a role in the binding of this
class of compounds to the DAT similar to the atypical DAT inhibitors
exemplified by the benztropines, but not to cocaine.[17] Interestingly, in the benztropine class of atypical DAT
inhibitors similar observations were made in that (1) converting the
tropane aminenitrogen to an amide significantly reduced the binding
affinity (e.g., N-acetyl-3α-[bis(4′-fluorophenyl)methoxy]tropane,
DAT Ki = 2340 nM)[23] to an affinity virtually identical to that of (±)-1, and (2) the same order of halogen effect on the amine analogues
described herein on decreasing DAT affinity (F > Cl > Br) was
also
reported for the benztropines, and this is in direct opposition to
the order observed in the cocaine-like 3-phenyltropane analogues (e.g.,
WIN 35,428, 2β-carbomethoxy-3β-(4-fluorophenyl)tropane).[24] This divergence in DAT SAR between the benztropines
and 3-phenyltropane analogues formed one of the early foundations
for our hypothesis that these compounds bind differently to the DAT,
and these differences may be related to their different behavioral
profiles. Importantly, as it was shown that TM10 plays a critical
role in propagating the conformational changes of the homologous LeuT
from the S1 binding site to the intracellular gate, such divergent
interactions with TM10 are likely to have an impact on the overall
transporter conformation[25] and may contribute
to the mechanism underlying the unique pharmacology of (±)-1 and its analogues at DAT.
Experimental
Methods
Synthesis
Reaction conditions and yields were not optimized.
Anhydrous solvents were purchased from Aldrich and were used without
further purification, except for tetrahydrofuran, which was freshly
distilled from sodium benzophenone ketyl. All other chemicals and
reagents were purchased from Sigma-Aldrich Co. LLC, Combi-Blocks,
TCI America, OChem Incorporation, Acros Organics, Maybridge, and Alfa
Aesar. The diphenylmethanols (3a–c, 3e–i) were commercially available,
except 3d, which was synthesized as outlined below. Unless
otherwise stated, amine final products were converted into oxalatesalts, typically by treating the free base in 2-propanol with a 1:1
molar ratio of oxalic acid in acetone. As described, some of the oxalatesalts were recrystallized from hot methanol or a methanol–acetone
solvent mixture. Spectroscopic data and yields refer to the free base,
except for compounds 6b and 6c, which were
synthesized as the hydrochloride salts. Flash chromatography was performed
using silica gel (EMD Chemicals, Inc., 230–400 mesh, 60 Å).
Compounds 4u and 6f were purified using
a Teledyne ISCO CombiFlash Rf instrument. 1H
and 13C NMR spectra were acquired using a Varian Mercury
Plus 400 spectrometer at 400 and 100 MHz, respectively. Chemical shifts
are reported in parts per million (ppm) and referenced according to
deuterated solvent for 1H spectra (CDCl3, 7.26
ppm, or DMSO-d6, 2.50 ppm) and 13C spectra (CDCl3, 77.2 ppm, or DMSO-d6, 39.5 ppm). Gas chromatography/mass spectrometry (GC/MS)
data were acquired (where obtainable) using an Agilent Technologies
(Santa Clara, CA) 6890N gas chromatograph equipped with an HP-5MS
column (cross-linked 5% PH ME siloxane, 30 m × 0.25 mm i.d. ×
0.25 μm film thickness) and a 5973 mass-selective ion detector
in electron-impact mode. Ultrapure grade helium was used as the carrier
gas at a flow rate of 1.2 mL/min. The injection port and transfer
line temperatures were 250 and 280 °C, respectively, and the
oven temperature gradient used was as follows: the initial temperature
(100 °C) was held for 3 min, then increased to 295 °C at
15 °C/min over 13 min, and finally maintained at 295 °C
for 10 min. Combustion analysis was performed by Atlantic Microlab,
Inc. (Norcross, GA), and the results agree within ±0.5% of the
calculated values. Melting point determination was conducted using
a Thomas-Hoover melting point apparatus, and the melting points are
uncorrected. On the basis of NMR and combustion data, all final compounds
are >95% pure.
Bis(4-bromophenyl)methanol (3d)
Compound 3d was synthesized by adapting a
literature method[26] from bis(4-bromophenyl)methanone
(10.2 g, 30.0
mmol) and NaBH4 (2.55 g, 67.4 mmol) in anhydrous ethanol
(65 mL) at 0 °C under argon. The product 3d (9.8
g, 95% yield) was recovered as a white solid. Mp: 109–111 °C. 1H NMR (CDCl3): δ 7.46 (d, J = 8.6 Hz, 4H), 7.22 (d, J = 8.6 Hz, 4H), 5.76 (sd, J = 3.5 Hz, 1H), 2.21 (sd, J = 3.5 Hz,
1H). 13C NMR (CDCl3): δ 142.4, 131.9,
128.3, 121.9, 75.2.
Thioacetamides
Procedure A
A solution of 2-mercapto-N-methylacetamide (10 mmol) and diphenylmethanol, 3a,
or the appropriate substituted diphenylmethanol, 3c or 3d (10 mmol), in trifluoroacetic acid (TFA; 200 mmol) was
stirred at room temperature (60 °C for substituted analogues)
for 20 h. The solvent was removed in vacuo, and the thick oily residue
was washed with water (30 mL). After the water was decanted, a crude
solid product was isolated by addition of diisopropyl ether (20 mL)
to the oily residue and vigourous mixing. The crude solid was filtered
and purified by flash column chromatography using 5% MeOH/CH2Cl2 to give the pure, desired product.
Procedure
B
Thioacetamides 4b–4f were
synthesized[10] in three
steps. Step 1: Thioglycolic acid (1 mmol) was reacted with the appropriate
substituted diphenylmethanol, 3e–3i (1 mmol), in TFA (14 mmol) overnight at room temperature. After
solvent removal in vacuo, the residue obtained was washed with water
(5 mL) and hexanes (15 mL) to give the carboxylic acid product, which
was carried to the next step without further purification. Step 2:
The acid product (3 mmol) from step 1 was reacted with K2CO3 (4.5 mmol) and iodomethane (CH3I; 4.5 mmol)
in acetone (50 mL) overnight under reflux conditions. After solvent
removal in vacuo, the residue was suspended in water (20 mL) and extracted
with CH2Cl2 (3 × 20 mL). The combined organic
layer was dried over MgSO4 and concentrated to give the
methyl ester, which was carried to the next step without further purification.
Step 3: A mixture of the ester (3 mmol), NH4Cl (4.2 mmol),
concentrated NH4OH (28.0–30.0%, 20 mL), and MeOH
(5.7 mL) was stirred at 50 °C for 72 h. MeOH was removed in vacuo,
and the reaction mixture was diluted with water (50 mL), extracted
with ethyl acetate (3 × 50 mL), and dried over Na2SO4. The solvent was evaporated, and the recovered crude
product was purified by flash column chromatography using 1:1 ethyl
acetate/hexanes to afford the pure product.
Procedure C
Thioacetamides 4l, 4p, 4s, 4v, and 4w were synthesized
in two steps according to a published procedure,[10] while compounds 4j, 4k, 4m–4o, 4q, 4r, 4t, 4u, and 4x–4z were synthesized in two steps with slight modifications
to the published procedure in the second step. Step 1 is the same
as step 1 for procedure B. Step 2: CDI (11 mmol) was added to a solution
of the carboxylic acid product (10 mmol) from step 1 in anhydrous
THF (25 mL). The reaction mixture was stirred at room temperature
for 2 h and then cooled to 0 °C. Water (0.1–0.2 mL) was
added to the reaction mixture (to quench excess CDI), followed by
the dropwise addition of the appropriate amine (10 mmol, dissolved
in THF). The reaction mixture was left to warm to room temperature
and stir overnight. The solvent was removed under vacuum to give a
crude residue, which was dissolved in diethyl ether or ethyl acetate.
The organic solution was washed with aqueous 1.0 M HCl solution (55
mL), water (80 mL), dilute aqueous NaHCO3 solution (36
mL, 1:6 dilution of saturated NaHCO3 solution), and water
(2 × 30 mL). The organic layer was dried over MgSO4 and concentrated in vacuo to give the pure product. The bromo-substituted
analogues 4q, 4t, 4x, and 4z required further purification by flash column chromatography
as indicated.
2-(Benzhydrylthio)acetamide (4a)
Compound 4a was synthesized by stirring a
solution of 2-mercaptoacetamide
(0.63 g, 6.9 mmol; recovered from the 10% (w/v) methanol/NH3 solution) and diphenylmethanol, 3a (1.3 g, 7.1 mmol),
in TFA (11.9 g, 104 mmol) at room temperature for 4 h. The solvent
was removed in vacuo, and the brown oily residue was dissolved in
CHCl3 (30 mL) and washed with water (30 mL), followed by
a dilute NaHCO3 solution (30 mL, 1:3 dilution of saturated
NaHCO3 solution) and water (30 mL). The organic layer was
dried over MgSO4 and concentrated in vacuo. The crude product
was purified by flash chromatography using 1:1 ethyl acetate/hexanes
to give pure 4a (0.31 g, 17% yield) as a white solid.
Mp: 105–106 °C (lit.[3] 109–110
°C). 1H NMR (CDCl3): δ 7.41 (d, J = 7.6 Hz, 4H), 7.33 (t, J = 7.4 Hz, 4H),
7.25 (tt, J = 7.2, 1.4 Hz, 2H), 6.50 (br s, 1H),
5.57 (br s, 1H), 5.17 (s, 1H), 3.09 (s, 2H). 13C NMR (CDCl3): δ 171.2, 140.3, 128.9, 128.4, 127.8, 54.9, 35.7.
Anal. (C15H15NOS) C, H, N.
2-((Di-p-tolylmethyl)thio)acetamide (4b)
Compound 4b was synthesized according
to general procedure B to give 4b (450 mg, 52% yield)
as a yellow oil. 1H NMR (CDCl3): δ 7.26
−7.30 (m, 4H), 7.12 (d, J = 7.6 Hz, 4H), 6.54
(br s, 1H), 5.53 (br s, 1H), 5.11 (s, 1H), 3.07 (s, 2H), 2.31 (s,
6H). GC/MS (EI): m/z 285 (M+).
Compound 4h was synthesized
using 2-mercapto-N-methylacetamide and bis(4-chlorophenyl)methanol, 3c, at 60 °C according to general procedure A. The product 4h (2.12 g, 79% yield) was obtained as a white solid. Mp:
156–158 °C. 1H NMR (DMSO-d6): δ 7.87 (br s, 1H), 7.38–7.44 (m, 8H),
5.45 (s, 1H), 2.99 (s, 2H), 2.53 (sd, J = 4.7 Hz,
3H). 13C NMR (DMSO-d6): δ
169.3, 140.8, 132.8, 130.8, 129.6, 52.4, 35.8, 26.6. Anal. (C16H15Cl2NOS) C, H, N.
Compound 5g was synthesized
as described for 5a using compound 4x (220
mg, 0.41 mmol) to give the product 5g (100 mg, 44%) as
a colorless oil. 1H NMR (CDCl3): δ 7.51–7.55
(m, 4H), 7.16–7.31 (m, 9H), 6.69 (t, J = 5.4
Hz, 1H), 5.13 (s, 1H), 3.42 (d, J = 14.0 Hz, 1H),
3.33 (q, J = 7.0 Hz, 2H), 3.05 (d, J = 14.0 Hz, 1H), 2.67 (t, J = 7.8 Hz, 2H), 1.85–1.89
(m, 2H). 13C NMR (CDCl3): δ 163.7, 141.3, 133.4,
132.9, 132.5, 132.3, 131.3, 130.7, 128.7, 128.6, 126.3, 123.53, 123.47,
69.7, 52.4, 39.7, 33.4, 31.2. Anal. (C24H23Br2NO2S·1/2H2O) C, H, N.
Thioethanamines
Procedure D
Compounds 6a–6c were synthesized following a literature procedure.[12,13] A solution of cysteamine hydrochloride (10 mmol), diphenylmethanol, 3a, or the appropriate halogen-substituteddiphenylmethanol, 3c or 3d (10 mmol), and BF3·OEt2 (11 mmol) in glacial acetic acid (40 mL) was stirred at 90–95
°C for 20 min (40–50 min for substituted analogues). The
reaction mixture was cooled to room temperature, and diethyl ether
(200 mL) was added to precipitate a solid (the hydrochloride salt)
from the mixture. The solid was filtered and dried under vacuum for
3 days in the presence of NaOH pellets. The dried solid was dissolved
in hot ethanol and filtered and the solvent removed in vacuo. Finally,
the solid was triturated in hot (boiling) ethyl acetate to give the
pure product as the hydrochloride salt.
2-(Benzhydrylthio)ethan-1-amine
(6a)
Compound 6a was synthesized
from diphenylmethanol, 3a,
according to general procedure D to give the hydrochloride salt in
quantitative yield. The hydrochloride salt of 6a (10.1
g, 36.1 mmol) was converted to the free base by being dissolved in
saturated aqueous NaHCO3 solution (120 mL) and extracted
into CHCl3 (150 mL). The layers were separated, and the
organic layer was washed with distilled water (80 mL) and aqueous
brine solution (100 mL) and dried over MgSO4. The solvent
was evaporated in vacuo to give the free base 6a (7.90
g, 90% yield) as a yellow oil. Some of the isolated free base was
converted into the oxalate salt. Mp: 177–179 °C. 1H NMR (CDCl3): δ 7.43 (d, J = 8.0 Hz, 4H), 7.31 (t, J = 7.4 Hz, 4H), 7.22 (tt, J = 7.4, 1.5 Hz, 2H), 5.16 (s, 1H), 2.81 (t, J = 6.2 Hz,
2H), 2.51 (t, J = 6.4 Hz, 2H). 13C NMR (CDCl3): δ 141.5, 128.7, 128.4, 127.4, 54.0, 41.0, 36.7. Anal. (C15H17NS·3/4C2H2O4) C, H, N.
Compound 6d was synthesized
according to general procedure D starting with compound 6a.[27] A suspension of the hydrochloride
salt of 6a (1.0 g, 3.6 mmol) and cyclopropanecarboxaldehyde
(0.28 g, 4.0 mmol) in 1,2-dichloroethane (62 mL) was stirred at room
temperature under an argon atmosphere for 1.3 h. Sodium cyanoborohydride
(0.69 g, 11 mmol) dissolved in methanol (2.0 mL) was added to the
reaction mixture, and the mixture was stirred at room temperature
under an argon atmosphere overnight. After 19 h of reaction time,
saturated NaHCO3 solution (30 mL), distilled water (30
mL), and CH2Cl2 (15 mL) were added to the reaction
mixture, and the resulting mixure was stirred vigorously for 1 h.
The layers were separated, and the aqueous layer was washed with CH2Cl2 (3 × 25 mL). The combined CH2Cl2 extract was washed with water (50 mL), dried over
MgSO4, and concentrated in vacuo to give a crude product.
The isolated crude was purified by flash column chromatography using
an ethyl acetate/hexanes solvent gradient (from 4:1 to 1:4) to give
the free base 6d (0.50 g, 47% yield) as a yellow oil.
Some of the isolated free base was converted into the hydrochloride
salt in CHCl3 using a 1.0 M HCl in ether solution. Mp:
122–124 °C. 1H NMR (CDCl3): δ
7.42 (d, J = 7.4 Hz, 4H), 7.30 (t, J = 7.4 Hz, 4H), 7.22 (tt, J = 7.2, 1.6 Hz, 2H),
5.17 (s, 1H), 2.76 (t, J = 6.4 Hz, 2H), 2.59 (t, J = 6.6 Hz, 2H), 2.40 (d, J = 6.8 Hz, 2H),
0.81–0.97 (m, 1H), 0.44–0.48 (m, 2H), 0.09 (qd, J = 4.8, 1.2 Hz, 2H). 13C NMR (CDCl3): δ 141.6, 128.7, 128.4, 127.3, 54.7, 54.3, 48.1, 32.9, 11.4,
3.5. Anal. (C19H23NS·HCl·1/4H2O) C, H, N.
N-(2-(Benzhydrylthio)ethyl)butan-1-amine
(6e)
Compound 6e was synthesized
by adapting
a literature procedure[14] using compound 6a (general procedure D). A mixture of CsOH·H2O (0.29 g, 1.7 mmol) and activated 4 Å molecular sieves (0.52
g) in anhydrous DMF (8.3 mL, freshly distilled and stored over activated
4 Å molecular sieves) was purged of air under vacuum and flushed
with argon gas. After the mixture was stirred for 13 min, the free
base of compound 6a (0.41 g, 1.7 mmol), dissolved in
anhydrous DMF (4.0 mL), was added. The reaction mixture was stirred
under vacuum for 25 min and flushed with argon for 5 min, and n-butyl bromide (0.28 g, 2.04 mmol) was added. This was
followed by another 10 min of vacuum purging, and the reaction was
left to stir overnight at room temperature. The reaction mixture was
filtered after 20 h of reaction time, and the undissolved solids were
washed with ethyl acetate. The filtrate was evaporated in vacuo to
give a liquid residue, which was taken up in aqueous 1 M NaOH (30
mL) and extracted with ethyl acetate (2 × 25 mL). The organic
extract was washed with brine (50 mL), dried over a 1:1 Na2SO4/MgSO4 mixture, and concentrated in vacuo.
The crude product was purified by flash column chromatography using
5% diethyl ether/hexanes (with 0.5% NEt3) to give the free
base 6e (0.22 g, 44% yield) as a yellow oil. Some of
the isolated free base was converted into the oxalate salt. Mp: 209–211
°C. 1H NMR (CDCl3): δ 7.42 (d, J = 7.2 Hz, 4H), 7.30 (t, J = 7.6 Hz, 4H),
7.22 (tt, J = 7.2, 1.6 Hz, 2H), 5.17 (s, 1H), 2.74
(t, J = 6.4 Hz, 2H), 2.58 (t, J =
6.2 Hz, 2H), 2.53 (t, J = 7.2 Hz, 2H), 1.40–1.47
(m, 2H), 1.27–1.37 (m, 2H), 0.90 (t, J = 7.6
Hz, 3H). 13C NMR (CDCl3): δ 141.6, 128.7,
128.4, 127.3, 54.2, 49.3, 48.3, 32.8, 32.3, 20.6, 14.1. Anal. (C19H25NS·C2H2O4) C, H, N.
Compound 6f was synthesized
from compound 4u.[10] Briefly,
sulfuric acid (98%; 305 mg, 3.11 mmol) in THF (8.0 mL) was added dropwise
at 0 °C to LiAlH4 (227 mg, 5.99 mmol) in THF (13 mL),
and the mixture was stirred for 15 min at room temperature. Compound 4u (563 mg, 1.50 mmol) in THF (11 mL) was added dropwise to
the reduction mixture at room temperature and the resulting mixture
stirred overnight. The reaction mixture was cooled to 0 °C and
quenched with water (5.0 mL) and 10% NaOH (20 mL) successively. The
mixture was filtered, the insolubles were washed with THF, and the
filtrate was evaporated to dryness. The crude product was purified
on a Teledyne ISCO CombiFlash Rf instrument using 97:3:0.03
CHCl3/MeOH/NH4OH to give the pure product 6f (312 mg, 58%) as a yellow oil. The free base was converted
to the oxalate salt. Mp: 196–198 °C. 1H NMR
(CDCl3): δ 7.42 (d, J = 7.6 Hz,
4H), 7.27–7.32 (m, 6H), 7.16–7.23 (m, 5H), 5.16 (s,
1H), 2.73 (t, J = 6.4 Hz, 2H), 2.63 (t, J = 7.8 Hz, 2H), 2.54–2.58 (m, 4H), 1.74–1.82 (m, 2H). 13C NMR (CDCl3): δ 142.2, 141.6, 128.7, 128.53,
128.47, 128.4, 127.4, 125.9, 54.2, 49.1, 48.3, 33.7, 32.8, 31.8. Anal.
(C24H27NS · C2H2O4) C, H, N.
Compound 6g was synthesized
as described for compound 6f using compound 4v, except that the reaction mixture was stirred at room temperature
for 2 h (instead of overnight) before being quenched with water and
NaOH (15% instead of 10%).The crude product was purified by flash
column chromatography (95:5:0.5 CHCl3/MeOH/NH4OH) to give the pure product 6g (820 mg, 86.6%) as a
yellow oil. The free base was converted to the oxalate salt, which
was recrystallized from a methanol/acetone mixture. Mp: 198–200
°C. 1H NMR (CDCl3): δ 7.33–7.37
(m, 4H), 7.16–7.30 (m, 5H), 6.97–7.02 (m, 4H), 5.13
(s, 1H), 2.72 (t, J = 6.4 Hz, 2H), 2.64 (t, J = 7.8 Hz, 2H), 2.52 (m, 4H), 1.75–1.82 (m, 2H). 13C NMR (CDCl3): δ 161.9 (1JCF = 246 Hz), 142.0, 137.0 (4JCF = 3.0 Hz), 129.7 (3JCF = 8.1 Hz), 128.4, 125.8, 115.5 (2JCF = 21.4 Hz, 4C), 52.5, 48.8, 48.0, 33.6, 32.6,
31.6. Anal. (C24H25F2NS·C2H2O4) C, H, N.
Compound 6h was synthesized
as described for 6g using compound 4w (1
g, 2.2 mmol). The crude product 6h (850 mg) was obtained
as a yellow oil and carried to the next step without further purification.
Compound 6i was synthesized
by reducing compound 4x with a borane·THF reagent.[28] A solution of 1 M BH3·THF complex
(14 mL, 14.0 mmol) was added slowly (in two aliquots) to a solution
of compound 4x (1.50 g, 2.81 mmol) in freshly distilled
THF (15 mL) at 2 °C. The reaction mixture was refluxed for 16
h, cooled to 0 °C, quenched with CH3OH (30 mL), saturated
with aqueous HCl (5.0 mL of concentrated HCl (37%)), and refluxed
for another 23 h, successively. The solvent was removed in vacuo to
give a yellow, oily residue which was taken up in CHCl3 (50 mL) and washed with distilled water (2 × 50 mL). The combined
aqueous extract was back-washed with CHCl3 (3 × 30
mL) and then discarded. The combined CHCl3 extract was
washed with water (100 mL) and brine (100 mL) and concentrated in
vacuo to give the hydrochloride salt of 6i. The salt
was suspended in a small amount of water and the suspension made basic
to a pH of 13 with 10 M NaOH (20 mL). The basic solution was continuously
extracted with CHCl3 for 6 h, and the layers were separated.
Solvent was removed from the organic layer to give the crude free
base of compound 6i, which was purified by flash column
chromatography (5% MeOH/CH2Cl2). The pure product 6i (0.58 g, 40% yield) was obtained as a yellow oil and converted
to the oxalate salt. Mp: 187–189 °C. 1H NMR
(CDCl3): δ 7.43 (dt, J = 8.4, 2.3
Hz, 4H), 7.23–7.30 (m, 6H), 7.16–7.20 (m, 3H), 5.07
(s, 1H), 2.74 (t, J = 6.6 Hz, 2H), 2.64 (t, J = 7.6 Hz, 2H), 2.53–2.29 (m, 4H), 1.77–1.83
(m, 2H). 13C NMR (CDCl3): δ 142.1, 140.1,
131.9, 131.6, 130.0, 128.5, 126.0, 121.5, 53.0, 49.0, 48.3, 33.7,
32.7, 31.7. Anal. (C24H25Br2NOS·C2H2O4) C, H, N.
Sulfinylethanamines
2-(Benzhydrylsulfinyl)ethan-1-amine
(7a)
Compound 7a was synthesized
with slight modifications
to a published procedure.[29] Briefly, a
solution of sodium periodate (NaIO4; 2.25 g, 10.5 mmol)
in water (50 mL) was added in a dropwise manner to a solution of the
hydrochloride salt of compound 6a (2.80 g, 10.0 mmol)
in ethanol (150 mL) at 0 °C. The reaction was allowed to stir
and warm to room temperature for ∼20 h under an argon atmosphere.
The reaction mixture, which contained a white precipitate, was cooled
in an ice bath and filtered. The filtrate was concentrated in vacuo
to give a dark yellow, oily residue. The oily residue (the hydrochloride
salt) was dissolved in CHCl3, washed with an aqueous NaHCO3 solution (2:3 dilution in water of saturated NaHCO3 solution), distilled water, and aqueous brine, and dried over Na2SO4, successively. After filtration, solvent was
removed in vacuo to give the crude, free base of compound 7a. The crude product was purified by flash column chromatography using
a MeOH/CHCl3 (with 0.1% NH4OH) gradient (from
0% to 1% MeOH) to give pure 7a (1.12 g, 43% yield) as
a yellow oil. Some of the isolated free base was converted to the
oxalate salt. Mp: 161–163 °C. 1H NMR (CDCl3): δ 7.50 (d, J = 7.8 Hz, 2H), 7.31–7.44
(m, 8H), 4.90 (s, 1H), 3.10–3.23 (m, 2H), 2.53–2.65
(m, 2H). 13C NMR (CDCl3): δ 135.8, 135.2,
129.4, 128.9, 128.7, 128.5, 128.4, 73.1, 54.4, 36.5. Anal. (C15H17NOS·C2H2O4) C, H, N.
N-(2-(Benzhydrylsulfinyl)ethyl)butan-1-amine
(7b)
Compound 7b was synthesized
as described for 7a from compound 6e (0.070
g, 0.23 mmol) and NaIO4 (0.053 g, 0.25 mmol) in an ethanol/water
(EtOH/H2O) mixture (4.0 mL/1.2 mL, v/v). The pure free
base product 7b (0.020 g, 41% yield) was obtained as
a yellow oil after purification of the crude by flash column chromatography
using a MeOH/CHCl3 (with 0.1% NH4OH) gradient
(from 0% to 2% MeOH). The isolated free base was converted to the
oxalate salt. Mp: 162–164 °C. 1H NMR (CDCl3): δ 7.49 (d, J = 7.6 Hz, 2H), 7.30–7.44
(m, 8H), 4.92 (s, 1H), 2.99–3.13 (m, 2H), 2.65 (t, J = 5.8 Hz, 2H), 2.56 (t, J = 7.0 Hz, 2H),
1.40–1.47 (m, 2H), 1.27–1.36 (m, 2H), 0.89 (t, J = 7.4 Hz, 3H). 13C NMR (CDCl3):
δ 135.9, 135.2, 129.44, 129.42, 128.9, 128.8, 128.49, 128.44,
73.0, 51.1, 49.6, 43.6, 32.1, 20.5, 14.1. Anal. (C19H25NOS·C2H2O4·1/2H2O) C, H, N.
Compound 7d was synthesized
as described for compound 5a using 6h (850
mg, 1.97 mmol). The free base product 7d (640 mg, two
steps yield 72.6%) was obtained as a yellow oil and converted into
the oxalate salt, which was recrystallized from hot MeOH. Mp: 153–155
°C dec. 1H NMR (CDCl3): δ 7.15–7.39
(m, 13H), 4.88 (s, 1H), 2.99–3.08 (m, 2H), 2.57–2.65
(m, 6H), 1.75–1.82 (m, 2H). 13C NMR (CDCl3): δ
141.9, 134.7, 134.6, 134.0, 132.9, 130.6, 129.9, 129.6, 129.0, 128.4,
125.9, 70.6, 51.2, 49.1, 43.1, 33.5, 31.4. Anal. (C24H25Cl2NOS·C2H2O4·1/2H2O) C, H, N.
Compound 7e was synthesized
as described for compound 5a using 6i (230
mg, 0.443 mmol). The free base product 7e (130 mg, 55%
yield) was obtained as a yellow oil and converted into the oxalatesalt, which was recrystallized from hot MeOH. Mp: 161–162 °C
dec. 1H NMR (CDCl3): δ 7.45–7.53
(m, 4H), 7.15–7.32 (m, 7H), 4.83 (s, 1H), 2.99–3.08
(m, 2H), 2.57–2.65 (m, 6H), 1.75–1.82 (m, 2H). 13C NMR (CDCl3): δ 141.9, 134.5, 133.3, 132.5, 132.3,
132.0, 131.4, 130.9, 130.2, 128.4, 125.9, 122.9, 122.7, 70.7, 51.2,
49.1, 43.1, 33.5, 31.4. Anal. (C24H25Br2NOS·C2H2O4) C, H, N.
Radioligand Binding Assays
DAT Binding Assay
Striata were dissected from male
Sprague–Dawley ratbrains (supplied on ice from Bioreclamation
(Hicksville, NY), prepared by homogenizing tissues in 20 volumes (w/v)
of ice cold modified sucrose phosphate buffer (0.32 M sucrose, 7.74
mM Na2HPO4, 2.26 mM NaH2PO4, pH adjusted to 7.4) using a Brinkman Polytron (setting 6 for 20
s), and centrifuged at 30000g for 10 min at 4 °C.
The resulting pellet was resuspended in buffer, recentrifuged, and
suspended in buffer again to a concentration of 10 mg/mL, original
wet weight (OWW). Experiments were conducted in assay tubes containing
0.5 mL of sucrose phosphate buffer, 0.5 nM [3H]WIN 35,428
(Kd = 5.53, specific activity 84 Ci/mmol;
Perkin-Elmer Life Sciences, Waltham, MA), 1.0 mg of tissue OWW, and
various concentrations of inhibitor. The reaction was started with
the addition of tissue, and the tubes were incubated for 120 min on
ice. Nonspecific binding was determined using 100 μM cocaine
hydrochloride.
SERT Binding Assay
Membranes from
frozen brain stem
dissected from male Sprague–Dawley ratbrains (supplied on
ice from Bioreclamation) were homogenized in 20 volumes (w/v) of 50
mM Tris buffer (120 mM NaCl and 5 mM KCl, adjusted to pH 7.4) at 25
°C using a Brinkman Polytron (at setting 6 for 20 s). The tissue
was centrifuged at 30000g for 10 min at 4 °C.
The resulting pellet was suspended in buffer and centrifuged again.
The final pellet was resuspended in cold buffer to a concentration
of 15 mg/mL OWW. Experiments were conducted in assay tubes containing
0.5 mL of buffer, 1.4 nM [3H]citalopram (Kd = 1.94 nM, specific activity = 83 Ci/mmol; Perkin-Elmer
Life Sciences), 1.5 mg of brain stem tissue, and various concentrations
of inhibitor. The reaction was started with the addition of the tissue,
and the tubes were incubated for 60 min at room temperature. Nonspecific
binding was determined using 10 μM fluoxetine.
NET Binding
Assay
Membranes from frozen frontal cortex
dissected from male Sprague–Dawley ratbrains (supplied on
ice from Bioreclamation) were homogenized in 20 volumes (w/v) of 50
mM Tris buffer (120 mM NaCl and 5 mM KCl, adjusted to pH 7.4) at 25
°C using a Brinkman Polytron (at setting 6 for 20 s). The tissue
was centrifuged at 30000g for 10 min at 4 °C.
The resulting pellet was suspended in buffer and centrifuged again.
The final pellet was resuspended in cold buffer to a concentration
of 80 mg/mL OWW. Experiments were conducted in assay tubes containing
0.5 mL of buffer, 0.5 nM [3H]nisoxetine (Kd = 1.0 nM, specific activity 82 Ci/mmol; Perkin-Elmer
Life Sciences), 8 mg of frontal cortex tissue, and various concentrations
of inhibitor. The reaction was started with the addition of the tissue,
and the tubes were incubated for 180 min at 0–4 °C. Nonspecific
binding was determined using 1 μM desipramine.The solvent
used to dissolve the various analogues of modafinil was typically
methanol and was present at a final concentration of 5%. Extensive
studies previously in this and other laboratories determined that
methanol has no effect on binding at the DAT and SERT. However, there
is an effect of methanol on binding at the NET, and therefore, the
methanol concentration was controlled in all tubes in that assay.
When compounds were not soluble in methanol, we used either ethanol
or DMSO at final concentrations of no greater than 5% or 6%, respectively.
Previous studies found no effect of either of these solvents at these
concentrations on binding at any of the sites. For all three MAT binding
assays, incubations were terminated by rapid filtration through Whatman
GF/B filters, presoaked in 0.3% (SERT) or 0.05% (DAT, NET) polyethylenimine,
using a Brandel R48 filtering manifold (Brandel Instruments, Gaithersburg,
MD). The filters were washed twice with 5 mL of cold buffer and transferred
to scintillation vials. Cytoscint (MP Biomedicals, Solon, OH) (3.0
mL) was added, and the vials were counted the next day using a Beckman
6000 liquid scintillation counter (Beckman Coulter Instruments, Fullerton,
CA) or a Tri-Carb 2910-B liquid scintillation counter (Perkin-Elmer
Life Sciences). The Ki values for the
modafinil derivatives were obtained using nonlinear least-squares
regression (using GraphPad Prism software, GraphPad Software, Inc.,
San Diego, CA) of the displacement data, giving IC50 values,
from which affinities (Ki values) were
calculated using the Cheng–Prusoff equation.[30]
Molecular Pharmacology
Site-Directed Mutagenesis
Synthetic cDNA encoding the
humanDAT (synDAT) was subcloned into pcDNA3 (Invitrogen, Carlsbad,
CA). cDNA encoding the humanSERT (hSERT) was cloned into the pUbi1z
expression vector. Mutations herein were generated by the QuickChange
method (adapted from Stratagene, La Jolla, CA) and confirmed by restriction
enzyme mapping and DNA sequencing. Positive clones were amplified
by transformation into XL1 blue competent cells (Stratagene), positive
colony picked, and grown in LB media overnight at 37 °C in an
orbital incubator (Infors) at 200 rpm. Plasmids were harvested using
the maxi prep kit provided by Qiagen according to the manufacturer’s
manual.
Cell Culture and Transfection
COS-7 cells were grown
in Dulbecco’s modified Eagle’s medium 041 01885 supplemented
with 10% fetal calfserum, 2 mM l-glutamine, and 0.01 mg/mL
gentamicin at 37 °C in 10% CO2. Wild-type and mutant
constructs were transiently transfected into COS-7 cells with Lipo2000
(Invitrogen) according to the manufacturer’s manual using cDNA:Lipo2000
ratios of 3:6 and 2:6 for hDAT and hSERT, respectively.
[3H]Dopamine and [3H]-5-HT Uptake Experiments
Uptake
assays were performed essentially as previously described[31] using [2,5,6,7,8-3H](dihydroxyphenyl)ethylamine
([3H]DA; 94.4 Ci/mmol, Perkin-Elmer) or 5-[1,2-3H(N)]hydroxytryptamine ([3H]-5-HT; 28 Ci/mmol, Perkin-Elmer)
for hDAT- and hSERT-expressing cells, respectively. Transiently transfected
COS-7 cells were plated in 12-well (3 × 105 cells/well)
or 24-well (105 cells/well) dishes coated with polyornithine
to achieve an uptake level of no more than 10% of the total added
radioligand. The uptake assays were carried out 2 days after transfection.
Prior to the experiment, the cells were washed once in 500 μL
of uptake buffer (25 mM HEPES, 130 mM NaCl, 5.4 mM KCl, 1.2 mM CaCl2, 1.2 mM MgSO4, 1 mM l-ascorbic acid,
5 mM d-glucose, and 1 μM catechol O-methyltransferase inhibitor Ro 41-0960 (Sigma), pH 7.4) at room
temperature. All the tested ligands were solubilized in DMSO to obtain
a stock concentration of 10 mM. From here the compounds were further
diluted 10-fold in H2O, followed by consecutive dilutions
in uptake buffer. The trace amounts of DMSO (maximum 1% for the highest
added concentration) did not influence the binding affinity (Loland
et al., unpublished experiments). The unlabeled ligand (e.g., modafinil
[(±)-1] or analogues) was added to the cells in
10 concentrations from 1 nM to 0.1 mM equally distributed around the
expected IC50 value, and uptake was initiated by addition
of ∼10 nM radioligand in a final volume of 500 μL. After
3 (for the hSERT) or 5 (hDAT) min of incubation, the reaction was
stopped by rapid washing with 2 × 500 μL of ice cold uptake
buffer, lysed in 250 μL (300 μL for 12-well plates) of
1% SDS, and left for 30 min at 37 °C with gentle shaking. All
samples were transferred to 24-well counting plates, and 500 μL
(or 600 μL) of Opti-phase Hi Safe 3 scintillation fluid (Perkin-Elmer)
was added followed by counting of the plates in a Wallac Tri-Lux β-scintillation
counter (Perkin-Elmer). Nonspecific uptake was determined in the presence
of 5 μM paroxetine for hSERT-expressing cells and 50 μM
nomifensine for hDAT-expressing cells. All determinations were performed
in triplicate. Uptake data were analyzed by nonlinear regression analysis
using Prism 5.0 from GraphPad Software. The IC50 values
used in the estimation of KM for uptake
were calculated from the means of pIC50 values and the
SE intervals from the pIC50 ± SE. The Ki values were calculated from the IC50 values
using the equation Ki = IC50/(1 + (L/KM)) (L = concentration of [3H]DA or [3H]-5-HT).
Molecular Modeling
We docked the modafinil derivative
compound 4h in our LeuT-based SERT model. The preparation
and MD equilibration of the homology model of SERT were previously
described.[19] The compound was constructed
and prepared for docking using LigPrep (Schrodinger Inc., Portland,
OR). Docking of the compound was carried out with Glide (Schrodinger
Inc.). The binding modes shown in Figure 2 were
chosen on the basis of both the docking scores and the consistency
with the (±)-1 pose in the previously modeled DAT–(±)-1 complexes.[1]
Authors: Yongfang Zhao; Daniel S Terry; Lei Shi; Matthias Quick; Harel Weinstein; Scott C Blanchard; Jonathan A Javitch Journal: Nature Date: 2011-04-24 Impact factor: 49.962
Authors: Brendan J Tunstall; Chelsea P Ho; Jianjing Cao; Janaína C M Vendruscolo; Brooke E Schmeichel; Rachel D Slack; Gianluigi Tanda; Alexandra J Gadiano; Rana Rais; Barbara S Slusher; George F Koob; Amy H Newman; Leandro F Vendruscolo Journal: Neuropharmacology Date: 2017-12-05 Impact factor: 5.250
Figure 1
Scheme 1
Scheme 2
Figure 2