Novel Potent Dopamine-Norepinephrine and Triple Reuptake Uptake Inhibitors Based on Asymmetric Pyran Template and Their Molecular Interactions with Monoamine Transporters

Article abstract of DOI:10.1021/acschemneuro.1c00078

We have carried out a structural exploration of (2S,4R,5R)-2-(bis(4-fluorophenyl)methyl)-5-((4-methoxybenzyl)amino)tetrahydro-2H-pyran-4-ol (D-473) to investigate the influence of various functional groups on its aromatic ring, the introduction of heteroc

Full text of DOI:10.1021/acschemneuro.1c00078

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Research Article
Novel Potent Dopamine−Norepinephrine and Triple Reuptake
Uptake Inhibitors Based on Asymmetric Pyran Template and Their
Molecular Interactions with Monoamine Transporters
Soumava Santra, Sandhya Kortagere, Seenuvasan Vedachalam, Sanjib Gogoi, Tamara Antonio,
Maarten E.A. Reith, and Aloke K. Dutta*
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ABSTRACT: We have carried out a structural exploration of (2S,4R,5R)-2-(bis(4-
fluorophenyl)methyl)-5-((4-methoxybenzyl)amino)tetrahydro-2H-pyran-4-ol (D-
473) to investigate the influence of various functional groups on its aromatic ring,
the introduction of heterocyclic aromatic rings, and the alteration of the
stereochemistry of functional group on the pyran ring. The novel compounds were
tested for their affinities for the dopamine transporter (DAT), serotonin transporter
(SERT), and norepinephrine transporter (NET) in the brain by measuring their
potency in inhibiting monoamine neurotransmitter uptake. Our studies identified
some of the most potent dopamine−norepinephrine reuptake inhibitors known to-
date like D-528 and D-529. The studies also led to development of potent triple
reuptake inhibitors such as compounds D-544 and D-595. A significant influence
from the alteration of the stereochemistry of the hydroxyl group on the pyran ring of
D-473 on transporters affinities was observed indicating stereospecific preference for
interaction. The inhibitory profiles and structure−activity relationship of lead compounds were further corroborated by molecular
docking studies at the primary binding sites of monoamine transporters. The nature of interactions found computationally correlated
well with their affinities for the transporters.
KEYWORDS: Triple reuptake inhibitors, dopamine−norepinephrine reuptake inhibitor, attention-deficit hyperactivity disorder,
post-traumatic stress disorder, pharmacotherapy, homology modeling
(SSRIs).8−11 However, current antidepressant therapies are
inadequate and do not work for a significant number of people.
In addition, slow onset of action coupled with undesirable side
INTRODUCTION
■
A significant proportion of the population (about 15−20%)
suffers from debilitating neurodisorders like major depressive
effects also negatively impacts therapeutic efficacy. In the
disorder (MDD), attention-deficit hyperactivity disorder
current antidepressant therapies, dopamine is not included in
(ADHD), or post-traumatic stress disorder (PTSD) in the
spite of existing evidence pointing to the presence of a strong
United States and worldwide.1 Depression is a significant
dopaminergic component in MDD.12 The current treatment of
illness which may lead to life threatening acts and suicide.2
PTSD is limited to two FDA-approved pharmacotherapeutics
Similar to depression, PTSD is a chronic and debilitating
which are the SSRIs paroxetine and sertraline.13 It has been
illness which is caused by exposure to traumatic events.3 It has
predicted that agents potentiating all three monoaminergic
been estimated by epidemiologic studies that PTSD exists in
systems might selectively benefit PTSD patients with the
∼8% of the US population and in up to 30% of certain at-risk
dysphoric/anhedonic phenotype.14
groups.4,5 ADHD is a neurobehavioral disorder prevalent in
In the current hypothesis of ADHD, either underactive or
hyperactive dopamine−norepinephrine systems are implicated
in the more complex etiology of this disorder. Multiple drugs
children, with an occurrence ranging from 2 to 18% between 6
and 17 years of age.6 However, significant ADHD symptoms
(about >60%) also persist into adulthood.7
Modulation of levels of monoamines by different therapeutic
agents has been applied in the treatment of depression, PTSD,
and ADHD. However, due to a lack of efficacy and prevalence
of side effects in many of these drugs, there is a great unmet
need for much improved therapy. Most current antidepressants
are either serotonin−norepinephrine reuptake inhibitors
(SNRIs) or selective serotonin reuptake inhibitors
Received: February 9, 2021
Accepted: April 1, 2021
Published: April 12, 2021
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are currently used in the treatment of ADHD. Stimulant drugs
like amphetamine provide efficacy but also provide a shorter
duration of action and other side effects including abuse
potential.15,16 Current nonstimulant drugs approved so far like
the norepinephrine transporter inhibitor atomoxetine are not
very efficacious and, additionally, exhibit side effects (Figure
1).17 ADHD drugs belonging to the class of dopamine−
ADHD. Recently, a norepinephrine and dopamine preferring
triple reuptake inhibitor (TRI) EB 1020 exhibited the potential
for treatment of ADHD and is currently undergoing a clinical
trial.19,20
In our research program, we embarked on developing novel
molecules based on asymmetric pyran derivatives with the goal
to target monoamine transporters in the CNS.21−23 CNS-
active pyran compounds are relatively rare. Our extensive
structure−activity relationship (SAR) studies on the asym-
metric pyran template led to compounds with various
substitutions and inhibition profiles which included SSRIs,
TRIs, and DNRIs.21,24−27 Two orally active lead TRIs, D-473
and D-578 (Figure 1), exhibited high efficacy in the depression
and PTSD animal models.28,29 In this regard, these compounds
were more efficacious than the reference drugs. In another line
of experiments, we developed a number of potent DNRI-type
compounds which gave us an indication for the structural
requirement for such an activity in pyran derivatives.21,26 Our
proposed model from such SAR studies indicated the
importance of certain molecular features for either a DNRI
or a TRI profile.21,26 The current study is a follow up on these
earlier SAR studies in order to further map out structural
requirements for the interaction of pyran compounds with
monoamine transporters through their binding activities and
analyzing mode of interactions through molecular docking
studies at the transporters binding pockets. Our overall goal
from this SAR study is to apply knowledge gained on structural
requirements for better designing of TRIs for MDD and PTSD
and of DNRIs for ADHD.
Figure 1. Molecular structures of known DNRIs and TRIs.
norepinephrine reuptake inhibitors (DNRIs) are expected to
provide greater efficacy without introducing substantial abuse
liability like stimulants. Drugs like methylphenidate and
nomifensine are approved for ADHD treatment, but they
suffer from some limitations including side effects mostly
unrelated to the mechanism of action (Figure 1).18 This
necessitates development of more efficacious DNRIs for
Scheme 1. Reductive Amination Furnished Target Compounds
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Scheme 2. Synthesis of Aldehydes 4 and 8
Scheme 3. Synthesis of 10 (D-529) and 12 (D-530)
Reductive amination of 13 and 17 with commercially available
indole aldehyde produced D-544 and D-543. D-505 was
synthesized by following the similar synthetic pathway as
described in Scheme 3. In Scheme 5, intermediate 19, which
was synthesized by us earlier, was transformed into 20 with an
inversion of asymmetric center of the hydroxyl group.21,30 This
first involved the formation of intermediate triflate from 19
which, upon SN2 nucleophilic attack by a water molecule,
produced 20 with a hydroxyl group in the equatorial position.
Reduction of azido group to amine, followed by reductive
amination, produced the final compound 22 (D-595).
CHEMISTRY
■
Stereospecific syntheses of amines (2S,4R,5R)-5-amino-2-
(bis(4-fluorophenyl)methyl) tetrahydro-2H-pyran-4-ol 1,
(2S,4R,5R)-5-amino-2-benzhydryltetrahydro-2H-pyran-4-ol
13, and (3S,6S)-6-benzhydryltetrahydro-2H-pyran-3-amine 17
were conducted in the same way as described by us
previously.21,24 As shown in Scheme 1, reductive amination
with appropriate commercially available aldehyde in the
presence of sodium triacetoxyborohydride furnished target
compounds. However, the aldehydes 4 and 8, which are not
commercially available, were synthesized in house as described
in Scheme 2. Synthesis of 10 (D-529) and 12 (D-530) is
shown in Scheme 3. The synthesis involves reductive
amination followed by reduction of the nitro group to amine
followed by alkylation of the amine group. Scheme 4 describes
the synthesis of 14 (D-544), 16 (D-505), and 18 (D-543).
COMPUTATIONAL STUDIES
■
Human DAT and NET were homology modeled with the
crystal structure of DAT from Drosophila melanogaster (pdb
code 4XP4)31 as a structural template by the use of the
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Scheme 4. Synthesis of 14 (D-544), 16 (D-505), and 18 (D-543)
a
Scheme 5. Synthesis of 20, 21, and Final Product 22
a
Reagents and conditions: (a) Anhyd CH2Cl2, −10 °C, Py, Tf2O, 2 h. (b) H2O, 90 °C, 72 h. (c) MeOH Pd/C, H2, rt, 1 atm, overnight. (d) 4-
Methoxybenzaldehyde, 1,2-dichloroethane, MeOH, AcOH, NaCNBH3 or Na(OAc)3BH, rt.
homology modeling program Modeler (ver 9.23)32,33 as
reported previously.34 This drosophila DAT shares the
conserved 12 transmembrane domain architecture and a 55
and 60% sequence identity to DAT and NET, respectively,
enabling homology modeling of these human isoforms. The
crystal structure of human SERT has been resolved recently
and was used for the docking studies (pdb code 5I6X).35 All
structures were then minimized and subject to 3 ns long
molecular dynamics simulation to further refine the structures
and overcome any potential steric hindrances. All small
molecules were modeled using ab initio builder module and
optimized using MOPAC in the molecular operating environ-
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a
Table 1. Affinity of Drugs at DAT, SERT, and NET in Rat Brain as Determined in Uptake Assays
b
b
c
drug
DAT uptake, Ki, nM
NET uptake, Ki, nM
SERT uptake, Ki, nM
d
D-473
13.3 2.0
16.2 1.5
1092 98
1567 260
1696 246
29.8 4.1
56.3 1.1
29.6 6.9
130 18
13.2 3.5
3.23 0.99
770 100
1.30 0.22
170 48
524 132
20.8 4.6
2.14 0.58
46.7 17.0
16.2 1.5
12.2 2.4
106 17
20.0 7.2
259 77
13.6 1.6
1.72 0.48
236 25.3
71.2 9.2
320 40
367 52
144 26
339 39
4,006 839
1540 299
30.1 5.3
e
D-578
fluoxetine
e
e
desipramine
imipramine
e
D-577 (2b)
D-543 (18)
D-544 (14)
D-612 (2f)
D-559 (2e)
D-530 (12)
D-528 (2d)
D-536 (2c)
D-529 (10)
D-602 (2a)
D-505 (16)
D-595 (22)
116
9
27.2 2.9
15.3 (4) 3.3
7.94 0.66
41.3 9.7
24.5 1.2
23.5 1.9
182 50
37.3 10.0
25.9 (5) 6.7
14.6 (6) 2.9
56.6 5.2
3.92 (5) 0.71
627 31
27.9 4.9
6.74 1.80
6.29 1.52
a
b
c
Values are mean SEM of n = 3−8 independent experiments performed in triplicate. Uptake measured by [3H]DA accumulation. Uptake
d
e
measured by [3H]5-HT accumulation. Data from ref 29. Data from ref 28.
Figure 2. Crystal/homology models of DAT, NET, and SERT with D-578 docked to the respective binding pockets. (A) A representative structure
of DAT is shown with the protein in the cartoon model and colored gray, and the binding pocket is highlighted by an orange square. An expanded
view of the binding pocket of DAT (B), NET (C), and SERT (D) with residues colored red and labeled and represented as licorice sticks, and
ligand D-578 is docked and represented as licorice sticks and colored atom type (C = cyan, O = red, N = blue, F = pink, S = yellow).
ment (MOE 2018). The minimum energy conformation for
each molecule was used for docking studies. Molecular docking
was performed using the genetic algorithm based docking
program GOLD (ver 5.2)36 on all three transporters, and 20
independent conformations of each ligand were used. Based on
literature evidence, the site targeted for docking of all
compounds was the primary substrate binding site spanning
S1 and S2 sites of monoamine transporters where most other
inhibitors have been shown to have primary binding
interactions.37−39 Docked complexes were ranked using gold
score and customized ranking based on the nature and strength
of interactions within the binding pocket. The best ranked
complexes were then further refined using gradient based
energy minimizing technique adopted in MOE program. All
simulations were performed using Amber force field and
Amber partial charges as adopted in MOE. Two dimensional
interaction plots for all the molecules in with the three
transporters were generated using the ligand interactions
module of MOE.
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Figure 3. Schematic representation of D-528, D-577, D-529, D-530, and D-544 docked to DAT, NET, and SERT. The figures were generated
using the Ligand interaction module of MOE. The schematic legend below explains the type of residues and the nature of interactions represented
pictorially in the figure.
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aromatic cluster in the binding pocket with stacking
interactions with F320 and hydrophobic interactions with
F326, F155, and W84. The compound also has H-bond
interactions between the hydroxyl on the pyran ring and D79
and hydroxyl on the outer aromatic ring with M427. In
addition to these interactions, the compound is in a favorable
environment with D476, Y156, S149, and several hydrophobic
residues (Figure 3) contributing to its high affinity for DAT.
Compound D-528 also docks to NET with a similar
interaction profile. The biphenyl rings have aryl interactions
with F317 and W80, hydrophobic interactions with A477 and
V148, and hydrophilic interactions with R81. Furthermore, the
outer aromatic ring interacts with several aromatic residues
such as F72 and Y152 and a H-bond formation of the phenolic
hydroxyl group with A145. Hydroxyl group from the pyran
ring forms a H-bond with F317. Several other interactions with
F323, D75, and D473 likely contribute to its high affinity at
NET (Figure 3). At SERT, the hydroxyl group of compound
D-528 forms a H-bond with D98, and the biphenyl ring is
proximal to F335 and Y95; the outer aromatic ring interacts
with Y176 and F341 (Figure 3). These all contribute to its
modest binding activity. It is interesting to find that, when the
hydroxy and methoxy substitution pattern in compound D-528
was changed to 2b (D-577), the binding profile changed
significantly. Thus, compound D-577 with Ki values of 29, 259,
and 524 nM at DAT, SERT, and NET, respectively, showed
selective affinity toward DAT. Even so, there was some loss of
efficacy at DAT (4-fold), more so at NET (∼36 fold),
suggesting that the change in hydroxy substitution to the ortho
positions changes the optimal binding conformation of this
compound to the transporters. Despite these changes,
compound D-577 makes favorable interactions at the binding
pocket of DAT including a sandwich stacking interaction of the
biphenyl group with F320 and F326 and H-bond interaction
between the outer aromatic ring hydroxyl group with Ser149
which majorly contributes to the higher affinity for DAT
(Figure 3). At NET, compound D-577 makes an arene cation
interaction with one of the biphenyl rings, and R81 and a pyran
hydroxyl atom forms a H-bond with D75. Y152, F317, and a
few other hydrophobic residues coordinate the interactions of
the rest of the molecule (Figure 3) leading to a modest affinity
for NET which is significantly different from compound D-
528. There was not much of a change in the interaction profile
of compound D-577 with SERT (Figure 3) compared to
compound D-528. The replacement of phenyl by a substituted
pyridine moiety as shown in compound 2a (D-602) produced
a selective interaction at DAT (Ki: 23.5, 4006, and 627 nM at
DAT, SERT, and NET, respectively). Interestingly, this is the
most DAT selective molecule found in the current series of
compounds. The docking profile of compound D-602 at DAT
showed that, in addition to the interactions of the biphenyl
groups and the pyran-hydroxyl group H-bond with D79, the
substituted pyridine makes arene−hydrogen interactions with
residues V152 and G426 likely contributing to its improved
profile at DAT (Figure S1), while these interactions are not
found in NET (Figure S1) or SERT (Figure S1).
RESULTS AND DISCUSSION
■
The design of new pyran compounds in this study was guided
by SAR uncovered in our previous work. In our recent work,
we demonstrated successful development of orally active TRIs
which exhibited high efficacy in the rat animal model of
depression.28,29 Furthermore, we have recently shown that the
novel TRI D-578 exhibits greater efficacy than that of
paroxetine in normalizing traumatic-stress-induced extinction
learning in the single prolonged stress (SPS) rat model of
PTSD.28 D-578 also produces robust and efficacious
antidepressant effects in the forced swim test without
confounding locomotor effects.28 Additionally, our recent
SAR studies uncovered potent DNRI ligands with the pyran
molecular scaffold.21,26 Several molecular functional groups on
the phenyl ring of the N-benzyl moiety turned out to be crucial
for preferential interactions at DAT and NET. In the majority
of cases, these functional groups contain hydrogen bond (H-
bond) formation centers for interaction with the target
transporters.21,26 Here, as in those prior studies, the “affinity”
of a compound for a transporter is a functional property
measured by its ability to inhibit transmitter uptake by the
transporter under identical physiological conditions in assays
for DAT, SERT, and NET, allowing across-transporter
comparisons. Based on this assay, the affinity values for D-
578 were 16, 3, and 16 nM for DAT, NET, and SERT,
respectively (Table 1). Molecular docking studies showed that
the models of DAT, NET, and SERT have conserved 12 TM
domains with the substrate site formed by distinct but
overlapping subsites S1 and S2 (Figure 2A). D-578 binds to
all three transporters with strong conserved H-bond
interactions with the hydroxyl atom on the central pyran
ring (D79 in DAT, D473 in NET, and D98 in SERT), stacking
interactions with the biphenyl ring and aromatic residues such
as F320 in DAT, F317 and W80 in NET, and F335 in SERT.
The outer thiazolidium group is well coordinated in all three
transporters with stacking and aryl-cation interactions with
residues F326 and G426 in DAT, Y152, F72, and F323 and
hydrogen bond interactions with S420 in NET, Y92, and T439
in SERT (Figure 2B,C) correlating for the efficacy of D-578 at
the three transporters.
Specifically, we observed previously that bis-fluorophenyl
substitution on the pyran template contributed toward an
increase in affinity for DAT and, to some extent, for NET.29,40
In the current study, therefore, this bis-fluorophenyl template
was a major focus for exploring the effect of various functional
groups. The substitution patterns on the phenyl ring of the N-
benzyl group in the compounds shown in Schemes 1 and 3
followed the design used in our previous studies in which
hydroxyl substitution in the aromatic ring in general produces
a higher affinity for the NET, whereas a combination of
hydroxyl and methoxy groups leads to a DNRI profile.21,26 To
further understand the SAR of the molecules in the three
transporters, the molecules were docked into the three
transporters, and their molecular interactions within each
were analyzed. In the current series, the most potent DNRI
was identified as 2d (D-528) which bears hydroxyl and
methoxy substituents. The compound showed a high affinity
for DAT and NET with a weaker interaction at SERT, with Ki
values of 7.94, 14.6, and 367 nM, respectively (Table 1). Based
on its activity, this compound is one of the most potent DNRIs
known to date. Analysis of the binding mode of D-528 in DAT
shows that the biphenyl rings of the compound fit well into an
As shown in Scheme 3, in our next series, the hydroxyl group
was replaced by amine and its derivatives. Similar to a hydroxyl
group, an amine can potentially provide H-bond formation for
interacting with NET. Compounds D-529 and D-530,
representing unsubstituted and monosubstituted amines,
exhibited a strong DNRI profile; Ki values were 24.5, 339,
3.92 and 15.3, 320, and 25.9 nM for DAT, SERT, and NET,
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for D-529 and D-530, respectively (Table 1). In this regard,
compound D-529 is significantly more potent at NET
compared to D-530. This might be due to the fact that D-
529 being an unsubstituted amine can form a stronger H-bond
than monosubstituted D-530. Molecular docking studies of
compound D-529 at NET (Figure 3) show that the amine on
the outer phenyl ring forms a bifurcated H-bond interaction
with the main chain atom of A73 and side chain oxygen atoms
of D75, while the pyran-hydroxyl forms a H-bond with D473.
These strong electrostatic interactions promote a conformation
of compound D-529 in which the biphenyl groups have several
favorable interactions with W80, Y317, and Q314, while the
outer amino phenyl ring interacts with Y152 and F72 likely
contributing to the high affinity for NET. In contrast,
compound D-530, where the amine is monosubstituted,
forms a H-bond with S419, while the phenyl ring makes
stacking interactions with Y152 and F72 and hydrophobic
interactions with cluster of hydrophobic amino acids, namely,
V148, F323, V325, and A145. The biphenyl rings of compound
D-530 now form aryl interactions with F317 and favorable
hydrophobic interactions with A77, F316, and A477; mean-
while, the pyran−hydroxyl forms the conserved H-bond with
D-473, and the pyran ring interacts with W80 and R81 (Figure
3). Interestingly, the monosubstituted amine of compound D-
530 in DAT makes a H-bond interaction with S149 and several
other conserved ring interactions made by compound D-529
in DAT. In addition to these conserved interactions,
compound D-530 makes a H-bond with D79 likely correlating
for the slightly higher affinity of compound D-530 for DAT
compared to compound D-529 (Figure 3). The interactions of
compound D-529 and D-530 were very similar in SERT
correlating with their measured Ki values.
However, in the case of the dimethyl substituted amine
derivative 2c (D-536), which showed the weakest DNRI
profile (Ki: 41.3, 144, and 56.6 nM for DAT, SERT, and NET,
respectively) among the three amine derivatives, it had no H-
bond interactions from amine functionality in any of the three
transporters (see Figure S1) suggesting that this pharmaco-
phoric feature could code for the DNRI profile of the
compounds. The nonfluorinated version of compound D-529,
which is compound D-505, showed a trend in activity which
we observed earlier. Thus, the compound D-505 produced a
selective affinity for NET with a much weaker interaction at
DAT which is consistent with our proposed binding model for
our pyran derivatives indicating a role of fluorine in enhancing
interaction with DAT.26 Interestingly, the indole derivative 2f
(D-612), which can be considered to be a constrained analog
of amine D-529, turned out to be a poor DNRI compound
reflecting the importance of basicity of the N atom in the
receptor interaction. Compound 2e (D-559) which is a
carbamate derivative of D-529 displayed a profile of a TRI with
preferential interaction at DAT and NET with moderate
potency at SERT (Ki: 27, 71, and 37 nM for DAT, SERT, and
NET, respectively) with the carbamate part of the molecule
having fewer interactions at SERT than at the other two
transporters (see Figure S1).
NET followed by that for DAT (Ki: 29.6, 1.72, and 2.14 nM
for DAT, SERT, and NET, respectively). Docking results
correlate with these binding activities wherein compound D-
544 has significantly high affinity interactions with SERT
contributed by the arene−H interaction of the indole ring with
G442 and the H-bond between the indole nitrogen and T439
while maintaining the conserved aromatic interactions between
biphenyls and aromatic residues F335, F334, F341, and Y95
and a H-bond between the hydroxyl group on the pyran ring
with D98 (Figure 3). The indole ring makes similar
interactions with NET with an arene−H interaction with
G149 and a H-bond interaction with S420 (Figure 3).
Interestingly, the interaction profile of compound D-544 in
DAT is slightly shifted with the indole nitrogen making a H-
bond with the main chain of V324 and the pyran ring
substitutions making several main chain H-bond interactions
with F320, G323, and F76 (Figure 3). Modeling of the
established TRI D-578 indicates such an extensive interaction
at all three binding pockets in the monoamine transporters
(Figure 2).
Finally, in one of our goals to investigate any role of
stereochemistry of the hydroxyl group on the pyran template in
uptake activity, we synthesized compound D-595 which is a
stereoisomer of TRI D-473 reported in our earlier
publication.29 Compound D-595, where the pyran (S)-
hydroxyl group is located in an equatorial position with an
inversion of stereochemistry compared to the corresponding
axial (R)-hydroxyl group in D-473, is otherwise structurally
identical to D-473. Previously, we demonstrated the require-
ment of regioselectivity of the hydroxyl and substituted amine
groups on the pyran molecular template for affinity at
monoamine transporters.23 However, we never explored the
influence of the stereochemistry of the hydroxyl group on
activity. The uptake inhibition data indicates the overall similar
activity of D-595 to compound D-473 (Table 1), although D-
595 exhibits relatively higher potency at all three transporters
(Ki: 6, 30, and 6 nM vs 13, 46, and 13 nM for DAT, SERT, and
NET, respectively, for D-595 and D-473). The gain in potency
for DAT and NET was almost 2-fold; however, for SERT, such
a gain was smaller in magnitude. This indicated that equatorial
orientation of the hydroxyl group assisted further enhancement
of interactions with the transporters. In docking studies, we
found that this hydroxyl moiety in compound D-595 had a
bifurcated H-bond with D79 and S422 as partners, while the
hydroxyl group in D-473 maintained interactions with D79 of
DAT and while all other interactions are well conserved
between the compounds and the transporters (see Figure S2).
The additional H-bond interaction in the docking mode of
compound D-595 could be contributing to the improved
efficacy at DAT for this molecule. Similarly, the interactions of
compound D-595 and D-473 were mostly conserved in NET
and SERT.
CONCLUSION
■
Our current SAR studies identified novel potent compounds
with DNRI and TRI profiles. Compounds like D-528 and D-
529 are among some of the most potent DNRIs known to
date. Molecular docking of these molecules with homology
models for DAT and NET and with the crystal structure of
SERT was aimed at the primary substrate binding site where
inhibitors generally bind; the nature of the interactions found
computationally correlated well with their potencies. Structural
alteration of D-528 to D-577 resulted in the loss of a DNRI-
To investigate the influence of indole substitution in
nonfluorinated disubstituted and trisubstituted pyran deriva-
tives, compounds D-543 and D-544 were synthesized.
Interestingly, both compounds exhibited a TRI profile with
compound D-544 being the most potent. In fact, compound
D-544 turned out to be one of most potent TRIs known to
date. The compound showed high potency for the SERT and
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1H), 2.50 (s, 1H), 2.35 (br s, 1H), 1.64−1.77 (m, 1H), 1.38−1.48
(m, 1H).
type profile which was evident in the homology model of the
binding interaction of D-577 at the NET binding pocket.
Compounds D-544 and D-595 turned out to be the two most
potent TRIs in the current series. As shown in the molecular
computation analysis, these molecules produced extensive
interactions in their binding pocket in all three transporters.
Interestingly, compound D-595 which is a stereoisomer of D-
473 reported earlier, with the pyran hydroxyl group oriented
equatorially, exhibited enhanced activity at DAT and NET.
This was corroborated by the interactions found upon docking,
indicating an extra bifurcated interaction with the pyran
equatorial hydroxyl group of D-595 compared to D-473 at the
DAT binding sites.
13C NMR (125 MHz, CDCl3): δ 162.5, 162.4, 160.6, 160.5, 155.8,
141.5, 140,3, 137.5, 137.4, 136.9, 136.4, 129.8, 129.7, 129.6, 120.6,
119.1, 115.6, 115.4, 115.3, 115.2, 73.7, 66.6, 64.5, 56.3, 55.6, 55.1,
48.2, 33.2.
[α]25 = (−) 41.60°, c = 1 in MeOH. The product was converted
D
into the corresponding hydrochloride salt; mp = 180−185 °C. Anal.
Calcd for [C25H26 F2N2O3·H2O·2HCl] C, H, N.
Synthesis of (2S,4R,5R)-2-(Bis(4-fluorophenyl)methyl)-5-((2-hy-
droxy-4-methoxybenzyl)amino)tetrahydro-2H-pyran-4-ol, 2b (D-
577). Amine 1 (60 mg, 0.19 mmol) was reacted with 2-hydroxy-4-
methoxybenzaldehyde (34 mg, 0.21 mmol), glacial acetic acid (13 μL,
0.22 mmol), and Na(OAc)3BH (68 mg, 0.32 mmol) in a mixture of
1,2-dichloroethane (3 mL) and methanol (1 mL). The residue was
purified by gradient silica gel column chromatography using a mixture
of dichloromethane and methanol (100:1 to 6:1) to afford
corresponding compound D-577 as a colorless syrup (50 mg, 58%).
1H NMR (500 MHz, CDCl3): δ 7.26 (dd, J = 8.5, 5.5 Hz, 2H),
7.15 (dd, J = 8.5, 5.2 Hz, 2H), 6.89−7.04 (m, 4H), 6.83 (d, J = 8.2
Hz, 1H), 6.37−6.44 (m, 1H), 6.33 (dd, J = 8.2, 2.5 Hz, 1H), 4.33−
4.45 (m, 1H), 4.06 (d, J = 13.7 Hz, 1H), 4.02 (d, J = 2.1 Hz, 1H),
3.94 (dd, J = 11.9, 1.2 Hz, 1H), 3.73−3.90 (m, 3H), 3.77 (s, 3H),
2.50 (s, 1H), 1.53−1.65 (m, 1H), 1.40−1.52 (m, 1H).
EXPERIMENTAL SECTION
■
Chemistry. Reagents and solvents were obtained from commercial
suppliers and used as received unless otherwise indicated. Dry
solvents were obtained according to the standard procedures. All
reactions were performed under inert atmosphere (N2) unless
otherwise noted. Analytical silica gel-coated TLC plates (silica gel
60 F254) were purchased from EM Science and were visualized with
UV light or by treatment with either phosphomolybdic acid (PMA)
or ninhydrin. Flash chromatography was carried out on Baker Silica
Gel 40 μM. 1H NMR and 13C spectra were routinely recorded with a
Varian 400 spectrometer operating at 400 and 100 MHz, respectively.
The NMR solvent used was either CDCl3 or CD3OD as indicated.
TMS was used as an internal standard. NMR and rotation of free
bases were recorded. Salts of free bases were used for biological
characterization. Elemental analyses were performed by Atlantic
Microlab Inc. and were within 0.4% of the theoretical value. Optical
rotations were recorded on a PerkinElmer 241 polarimeter.
13C NMR (125 MHz, CDCl3): δ 162.6, 162.5, 160.6, 160.5, 159.1,
137.3, 129.8, 129.7, 129.7, 129.7, 129.1, 115.6, 115.5, 115.4, 115.2,
114.2, 105.1, 102.1, 73.6, 66.6, 64.0, 55.6, 55.2, 55.0, 49.6, 33.4.
[α]25 = (−) 53.3°, c = 1 in MeOH. The product was converted
D
into the corresponding hydrochloride salt; mp = 170−175 °C. Anal.
Calcd for [C26H27F2NO4·HCl] C, H, N.
Synthesis of (2S,4R,5R)-2-(Bis(4-fluorophenyl)methyl)-5-((4-
(dimethylamino)benzyl)amino)tetrahydro-2H-pyran-4-ol, 2c (D-
536). Amine 1 (40 mg, 0.13 mmol) was reacted with 4-
(dimethylamino)benzaldehyde (23 mg, 0.15 mmol), glacial acetic
acid (16 μL, 0.27 mmol), and NaCNBH3 (14 mg, 0.23 mmol) in a
mixture of 1,2-dichloroethane (4.5 mL) and methanol (1.5 mL). The
residue was purified by gradient silica gel column chromatography
using a mixture of dichloromethane and methanol (100:1 to 6:1) to
afford corresponding compound D-536 (50 mg, 88%) as a colorless
semisolid.
Synthesis of (2S,4R,5R)-2-(Bis(4-fluorophenyl)methyl)-5-(((5-me-
thoxypyridin-3-yl) methyl)amino)tetrahydro-2H-pyran-4-ol, 2a (D-
602). Synthesis of 5-Methoxynicotinaldehyde, 4. The synthesis is
shown in Scheme 2. The bromo compound 3 (100 mg, 0.53 mmol)
was taken in an oven-dried RB equipped with magnetic stir-bar and
i
dissolved in anhydrous THF (1 mL). Then, PrMgCl (0.3 mL) was
added at 0 °C, and the resulting mixture was stirred at room
temperature for 2 h (the solution turned into light brown beer color).
Next, DMF (0.1 mL) in anhydrous THF (0.1 mL) was added slowly.
The initially formed solid was slowly dissolved, and the solution color
turned from light brown to light yellow. After 1 h, the reaction was
cooled to 0 °C and quenched with water (2 mL). The organic layer
was separated, and the aqueous layer was washed with an additional
amount of CH2Cl2 (3 × 3 mL). The organic layers were combined,
dried over Na2SO4, and concentrated under vacuo on a rotary
evaporator. The crude product was purified via gradient silica gel
column chromatography using a mixture of hexanes and ethyl acetate
(20:1 to 1:1) to obtain the desired product 4 as a colorless syrup (45
mg, 63%).
1H NMR (500 MHz, CDCl3): δ 7.22−7.32 (m, 2H), 7.08−7.20
(m, 4H), 6.88−7.02 (m, 4H), 6.68 (d, J = 8.5 Hz, 2H), 4.32−4.44 (m,
1H), 3.84−4.03 (m, 3H), 3.78 (d, J = 12.5 Hz, 3.64 (d, J = 12.8 Hz,
1H), 2.92 (s, 6H), 2.46 (s, 1H), 1.63−1.74 (m, 1H), 1.35−1.45 (m,
1H).
13C NMR (125 MHz, CDCl3): δ 162.4, 162.3, 160.5, 160.4, 149.9,
137.7, 137.5, 129.9, 129.8, 129.7, 129.6, 129.1, 115.5, 115.3, 115.2,
115.1, 112.6, 73.5, 67.0, 64.5, 56.2, 54.7, 53.4, 50.8, 50.6, 40.7, 33.1,
29.7.
[α]25 = (−) 19.3°, c = 1 in CH2Cl2. The product was converted
D
into the corresponding hydrochloride salt; mp = 190−195 °C. Anal.
1H NMR (500 MHz, CDCl3): δ 10.09 (s, 1H), 8.65 (d, J = 0.9 Hz,
1H), 8.54 (d, J = 3.1 Hz, 1H), 7.60 (dd, J = 5.1, 1.5 Hz, 1H), 3.8 (s,
3H).
Calcd for [C27H30F2N2O2·2HCl·Et2O] C, H, N.
Synthesis of (2S,4R,5R)-2-(Bis(4-fluorophenyl)methyl)-5-((3-hy-
droxy-4-methoxybenzyl)amino) Tetrahydro-2H-pyran-4-ol, 2d (D-
528). Amine 1 (60 mg, 0.19 mmol) was reacted with 3-hydroxy-4-
methoxybenzaldehyde (34 mg, 0.23 mmol), glacial acetic acid (17 μL,
0.28 mmol), and NaCNBH3 (20 mg, 0.32 mmol) in a mixture of 1,2-
dichloroethane (4.5 mL) and methanol (1.5 mL). The residue was
purified by gradient silica gel column chromatography using a mixture
of dichloromethane and methanol (100:1 to 6:1) to afford
corresponding compound D-528 (60 mg, 70%) as a colorless syrup.
1H NMR (400 MHz, CDCl3): δ 7.17 (dd, J = 7.9, 5.6 Hz, 2H),
7.06 (dd, J = 8.2, 5.9 Hz, 2H), 6.78−6.91 (m, 4H), 6.65−6.73 (m,
2H), 5.57−6.63 (m, 1H), 4.24−4.36 (m, 1H), 3.72−3.89 (m, 6H),
3.60−3.71 (m, 2H), 3.48 (d, J = 12.6 Hz, 1H), 2.36 (s, 1H), 1.40−
1.54 (m, 1H), 1.26−1.37 (m, 1H).
13C NMR (125 MHz, CDCl3): δ 190.6, 156.2, 145.1, 144.8, 132.0,
116.3, 55.7.
(2S,4R,5R)-2-(Bis(4-fluorophenyl)methyl)-5-(((5-methoxypyridin-
3-yl)methyl)amino)tetrahydro-2H-pyran-4-ol, 2a (D-602). Amine 1
(60 mg, 0.19 mmol) was reacted with 5-methoxynicotinaldehyde (29
mg, 0.21 mmol), glacial acetic acid (13 μL, 0.22 mmol), and
Na(OAc)3BH (113 mg, 0.38 mmol) in a mixture of 1,2-dichloro-
ethane (3 mL) and methanol (1 mL). The residue was purified by
gradient silica gel column chromatography using a mixture of
dichloromethane and methanol (100:1 to 6:1) to afford correspond-
ing compound D-602 as a colorless syrup (10 mg, 23%).
1H NMR (500 MHz, CDCl3): δ 8.04−8.25 (m, 2H), 7.05−7.34
(m, 4H), 6.85−7.03 (m, 4H), 4.41 (dt, J = 10.1, 2.5 Hz, 1H), 4.00−
4.07 (m, 1H), 3.96 (dd, J = 12.2, 2.1 Hz, 1H), 3.92 (d, J = 13.7 Hz,
1H), 3.85−3.91 (m, 1H), 3.80−3.85 (m, 4H), 3.78 (d, J = 13.7 Hz,
13C NMR (100 MHz, CDCl3): δ 162.6, 162.5, 160.1, 146.4, 145.8,
137.6, 137.4, 129.8, 129.7, 129.6, 129.5, 119.5, 115.2, 115.0, 114.8,
114.7, 111.0, 73.5, 65.8, 64.0, 55.7, 54.8, 50.3, 32.7.
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Research Article
[α]25 = (−) 19.4°, c = 1 in MeOH. The product was converted
(3 mL), dichloromethane (2 mL), and methanol (1 mL) by following
procedure A. The residue was purified by gradient silica gel column
chromatography using a mixture of dichloromethane and methanol
(20:1 to 6:1) to afford corresponding compound D-612 (2S,4R,5R)-
5-(((1H-Indol-6-yl)methyl)amino)-2-(bis(4-fluorophenyl)methyl)-
tetrahydro-2H-pyran-4-ol as a white solid (yield = 67 mg, 79%).
D
into the corresponding hydrochloride salt; mp = 170−175 °C. Anal.
Calcd for [C26H27F2NO4·HCl·H2O] C, H, N.
Synthesis of tert-butyl (4-Formylphenyl)(methyl)carbamate 8.
The synthesis is shown in Scheme 2. Amine 5 (1.0 g, 6.62 mmol) was
dissolved in anhydrous THF (6 mL) and then Et3N (1.76 mL, 13.23
mmol) was added, followed by (Boc)2O (1.96 g, 9.00 mmol). The
resulting mixture was stirred at room temperature for 24 h. The
reaction was then quenched by the addition of 1 N HCl and extracted
several times with ethyl acetate. The organic layer was dried over
Na2SO4 and concentrated under vacuum. The crude product 6 was
purified by gradient column chromatography using dichloromethane
and methanol (100:1 to 10:1) to obtain the desired product 6 as a
light brown syrup (0.54 g).
[α]25 = (−) 73.6o (c = 1, MeOH).
D
1H NMR (600 MHz, CDCl3): δ 8.20 (s, 1H), 7.56 (d, J = 8.4 Hz,
1H), 7.30 (s, 1H), 7.26−7.24 (m, 3H), 7.17 (t, J = 2.4 Hz, 1H),
7.14−7.11 (m, 2H), 7.01 (dd, J = 7.8 Hz, J = 0.2 Hz, 1H), 6.97−
1
2
6.91 (m, 4H), 6.51 (bs, 1H), 4.39−4.36 (m, 1H), 4.00−3.95 (m, 2H),
3.90−3.87 (m, 2H), 3.84−3.77 (m, 2H), 2.50 (s, 1H), 1.71−1.66 (m,
1H), 1.39 (d J = 14.4 Hz, 1H). 13C NMR (125 MHz, CDCl3): δ
160.6, 158.8, 129.9, 129.8, 129.7, 129.7, 124.3, 120.6, 120.4, 115.5,
115.4, 115.2, 115.1, 110.5, 102.4, 78.0, 76.7, 73.5, 67.1, 64.6, 60.2,
56.1, 54.8, 51., 33.1. The product was converted into corresponding
hydrochloride salt. [CH26F2N2O2·HCl·1.5H2O] 165−167 °C.
(2S,4R,5R)-2-(Bis(4-fluorophenyl)methyl)-5-((4-nitrobenzyl)-
amino)tetrahydro-2H-pyran-4-ol, 9. Amine 1 (126 mg, 0.40 mmol)
was reacted with 4-nitrobenzaldehyde (37 mg, 0.40 mmol), glacial
acetic acid (25 μL, 0.41 mmol), and Na(OAc)3BH (125 mg, 0.59
mmol) in a mixture of 1,2-dichloroethane (4.5 mL) and methanol
(1.5 mL). The residue was purified by gradient silica gel column
chromatography using a mixture of dichloromethane and methanol
(100:1 to 6:1) to afford corresponding compound 9 (140 mg, 78%)
as a light yellow syrup.
1H NMR (400 MHz, CDCl3): δ 7.99 (d, J = 8.6 Hz, 2H), 6.55 (d, J
= 8.6 Hz, 2H), 2.99 (s, 3H), 1.55 (s, 9H).
13C NMR (100 MHz, CDCl3): δ 161.8, 154.0, 148.0, 132.7, 115.0,
111.1, 84.7, 29.9, 27.4.
Boc protected acid was dissolved in anhydrous THF (8 mL) and
cooled to 0 °C. Then, LiAlH4 (0.122 g, 3.23 mmol) was added in
portions, and the reaction was allowed to stir at room temperature
overnight. The reaction mixture was then cooled to 0 °C and
quenched with slow addition of methanol (5 mL) followed by
saturated NH4Cl (5 mL). The mixture was then repeatedly extracted
with ethyl acetate (10 mL × 3). The organic layer was dried on
Na2SO4 and concentrated under reduced pressure on a rotary
evaporator. The crude product was purified via gradient column
chromatography using dichloromethane and methanol (100:1 to
10:1) to afford the desired product 7 as a white solid.
1H NMR (400 MHz, CDCl3): δ 8.16 (d, 2H, J = 8.5 Hz), 7.49 (d,
2H, J = 8.5 Hz), 7.22−7.31 (m, 2H), 7.10−7.19 (m, 2H), 6.89−7.03
(m, 4H), 4.34−4.46 (m, 1H), 3.76−4.06 (m, 6H), 2.41 (s, 1H),
1.66−1.76 (m, 1H), 1.38−1.48 (m, 1H).
1H NMR (500 MHz, CDCl3): δ 7.30 (d, J = 8.2 Hz, 2H), 7.20 (d, J
= 8.2 Hz, 2H), 4.64 (s, 2H), 3.24 (s, 3H), 1.44 (s, 9H).
13C NMR (125 MHz, CDCl3): δ 154.8, 143.0, 138.0, 132.1, 127.2,
125.5, 80.3, 64.6, 28.3.
13C NMR (100 MHz, CDCl3): δ 160.3, 160.2, 148.1, 137.6, 137.3,
129.9, 129.8, 129.7, 129.6, 128.5, 123.6, 115.6, 115.4, 115.3, 115.1,
73.5, 67.3, 64.9, 56.5, 55.2, 50.7, 33.3.
(2S,4R,5R)-5-((4-Aminobenzyl)amino)-2-(bis(4-fluorophenyl)-
methyl)tetrahydro-2H-pyran-4-ol, 10 (D-529). Compound 9 (140
mg, 0.31 mmol) was dissolved in methanol (19 mL) and Pd/C (19
mg). The mixture was then stirred at room temperature under 1 atm
pressure of hydrogen for 1 h. The residue was purified by gradient
silica gel column chromatography using a mixture of dichloromethane
and methanol (100:1 to 6:1) to afford corresponding compound D-
529 (75 mg, 58%) as a yellow syrup.
The alcohol 7 (0.16 g, 0.67 mmol) was treated with MnO2 (0.23 g,
2.70 mmol) in dichloromethane (5 mL) and stirred at room
temperature for 72 h. The reaction mixture was then filtered through
a pad of Celite and concentrated under reduced pressure on a rotary
evaporator. The crude product 8 was sufficiently pure and used
directly in the following step without further purification.
1H NMR (500 MHz, CDCl3): δ 9.95 (s, 1H), 7.83 (d, J = 8.6 Hz,
2H), 7.49 (d, J = 8.6 Hz, 2H), 3.31 (s, 3H), 1.49 (s, 9H).
13C NMR (125 MHz, CDCl3): δ 191.2, 153.9, 149.2, 132.6, 130.1,
124.6, 81.4, 36.8, 28.2.
1H NMR (400 MHz, CDCl3): δ 7.22−7.34 (m, 2H), 7.04−7.21
(m, 4H), 6.76−7.03 (m, 4H), 6.59 (d, 2H, J = 7.92 Hz), 4.31−4.49
(m, 1H), 4.01−4.21 (m, 2H), 3.78−3.95 (m, 3H), 3.64−3.78 (d, J =
12.9 Hz, 1H), 2.66 (s, 1H), 1.66−1.86 (m, 1H), 1.34−1.51 (m, 1H).
13C NMR (100 MHz, CDCl3): δ 161.7, 161.6 160.2, 146.4, 137.5,
137.4, 130.1, 129.8, 129.7, 129.6, 115.6, 115.4, 115.3, 115.2, 115.1,
74.0, 65.4, 62.9, 56.5, 53.9, 50.1, 32.9.
tert-butyl (4-((((3R,4R,6S)-6-(Bis(4-fluorophenyl)methyl)-4-hy-
droxytetrahydro-2H-pyran-3-yl)amino)methyl)phenyl)(methyl)-
carbamate, 2e (D-559). Amine 1 (60 mg, 0.19 mmol) was reacted
with tert-butyl (4-formylphenyl)carbamate (49 mg, 0.21 mmol) 8,
glacial acetic acid (13 μL, 0.22 mmol), and Na(OAc)3BH (68 mg,
0.32 mmol) in a mixture of 1,2-dichloroethane (4.5 mL) and
methanol (1.5 mL). The residue was purified by gradient silica gel
column chromatography using a mixture of dichloromethane and
methanol (100:1 to 6:1) to afford corresponding compound D-559 as
a colorless syrup (60 mg, 59%).
[α]25 = (−) 34.7°, c = 1 in MeOH. The product was converted
D
into the corresponding hydrochloride salt; mp = 185−192 °C. Anal.
Calcd for [C25H26F2N2O2·2HCl·Et2O.H2O] C, H, N.
N-(4-((((3R,4R,6S)-6-(Bis(4-fluorophenyl)methyl)-4-hydroxytetra-
hydro-2H-pyran-3-yl)amino)methyl)phenyl)acetamide, 11. Amine
D-529 (60 mg, 0.19 mmol) was reacted with N-(4-formylphenyl)-
acetamide (37 mg, 0.23 mmol), glacial acetic acid (17 μL, 0.28
mmol), and NaCNBH3 (21 mg, 0.32 mmol) in a mixture of 1,2-
dichloroethane (4.5 mL) and methanol (1.5 mL). The residue was
purified by gradient silica gel column chromatography using a mixture
of dichloromethane and methanol (100:1 to 6:1) to afford
corresponding compound 11 (64 mg, 73%) as a colorless syrup.
1H NMR (500 MHz, CDCl3): δ 7.91 (s, 1H), 7.38 (d, J = Hz, 2H),
7.05−7.31 (m, 6H), 6.84−7.01 (m, 4H), 4.32−4.45 (m, 1H), 3.61−
4.08 (m, 6H), 2.48 (s, 1H), 2.08 (s, 3H), 1.61−1.71 (m, 1H), 1.36−
1.44 (m, 1H).
1H NMR (500 MHz, CDCl3): δ 7.24−7.31 (m, 2H), 7.11−7.19
(m, 2H), 6.88−7.01 (m, 4H), 4.35−4.45 (m, 1H), 3.98−4.05 (m,
1H), 3.84−3.98 (m, 3H), 3.81 (d, J = 11.9 Hz, 1H), 3.73 (d. J = 13.4
Hz, 1H), 3.22 (s, 3H), 2.50 (br s, 1H), 1.66−1.77 (m, 1H), 1.36−
1.53 (m, 10H).
13C NMR (125 MHz, CDCl3): δ 162.5, 162.4, 160.6, 160.5, 154.8,
143.0, 137.7, 137.5, 129.9, 129.8, 129.7, 129.6, 128.6, 125.5, 115.6,
115.4, 115.3, 115.2, 80.4, 73.6, 65.9, 56.4, 54.7, 50.6, 37.4, 33.1, 28.4,
15.3.
[α]25 = (−) 51.4°, c = 1 in CH2Cl2. The product was converted
D
into the corresponding hydrochloride salt; mp = 184−190 °C. Anal.
Calcd for [C31H36F2N2O4·2HCl] C, H, N.
13C NMR (125 MHz, CDCl3): δ 169.0, 162.4, 162.3, 160.5, 160.4,
137.5, 137.4, 137.1, 134.1, 129.8, 129.7, 129.6, 129.0, 127.7, 120.3,
115.5, 115.4, 115.3, 115.1, 73.7, 66.1, 63.9, 55.9, 54.8, 50.1, 32.9, 24.3.
(2S,4R,5R)-5-(((1H-Indol-6-yl)methyl)amino)-2-(bis(4-
fluorophenyl)methyl) Tetrahydro-2H-pyran-4-ol, 2f (D-612). Amine
1 (60 mg, 0.19 mmol) was reacted with 1H-indole-6-carbaldehyde
(30 mg, 0.20 mmol), glacial acetic acid (13 μL, 0.22 mmol), and
NaBH(OAc)3 (80 mg, 0.37 mmol) in a mixture of 1,2 dichloroethane
[α]25 = (−) 47.4°, c = 1 in CH2Cl2.
D
(2S,4R,5R)-2-(Bis(4-fluorophenyl)methyl)-5-((4-(ethylamino)-
benzyl)amino) Tetrahydro-2H-pyran-4-ol, 12 (D-530). Amide 11
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Research Article
(63 mg, 0.16 mmol) was dissolved in anhydrous THF (5 mL) under a
continuous flow of N2, and the colution was cooled to 0 °C. Then,
LiAlH4 (10 mg, 0.20 mmol) was added in portions, and the reaction
mixture was allowed to reach room temperature slowly; it was stirred
for 24 h. Next, the reaction was quenched by slow addition of
methanol followed by a solution of saturated NH4Cl at 0 °C. The
product was extracted with ethyl acetate and concentrated on a rotary
evaporator. The residue was purified by gradient silica gel column
chromatography using a mixture of dichloromethane and methanol
(100:1 to 6:1) to afford corresponding compound D-530 (52 mg,
85%) as a colorless semisolid.
in 1,2-dichloroethane (6 mL) using procedure A. The residue was
purified by column chromatography using 10% methanol in ethyl
acetate to afford compound D-543 (50 mg, 66%) as a thick syrup.
1
[α]25D = (−) 78.4° (c 0.5, MeOH). H NMR (400 MHz, CDCl3): δ
1.22−1.30 (m, 1H), 1.50−1.62 (m, 1H), 1.63−1.76 (m, 1H), 1.97−
2.08 (m, 1H), 2.73 (s, 1H), 3.39 (d, J = 12.0 Hz, 1H), 3.82−4.10 (m,
5H), 6.44 (s, 1H), 6.92−7.58 (m, 13H), 8.94 (s, 1H). 13C NMR (100
MHz, CDCl3): δ 24.96, 26.17, 50.35, 50.56, 56.99, 68.99, 79.45,
102.18, 112.12, 120.67, 121.07, 125.30, 126.38, 126.72, 127.80,
128.50, 128.63, 128.76, 136.34, 142.34, 142.53. The product was
converted into the corresponding oxalate salt; mp = 194−196 °C.
Anal. (C27H28N2O·C2H2O4·1.2H2O) C, H, N.
1H NMR (500 MHz, CDCl3): δ 7.21−7.34 (m, 2H), 7.10−7.20
(m, 2H), 7.08 (d. J = 8.2 Hz, 2H), 6.87−7.02 (m, 4H), 6.54 (d, J =
8.2 Hz, 2H), 4.33−4.42 (m, 1H), 3.83−3.98 (m, 3H), 3.69−3.80 (m,
2H), 3.59 (d, J = 12.8 Hz, 1H), 3.13 (dd, J = 7.0, 7.3 Hz, 2H), 2.43 (s,
1H), 1.61−1.74 (m, 1H), 1.33−1.45 (m, 1H), 1.24 (t, J = 7.3 Hz,
3H).
(2S,4S,5R)-5-Azido-2-(bis(4-fluorophenyl)methyl)tetrahydro-2H-
pyran-4-ol, 20. The azido compound 19 (196 mg, 0.57 mmol) was
dissolved in anhydrous CH2Cl2 (4 mL) under a steady flow of N2 and
cooled to −10 °C. Then, anhydrous pyridine (0.28 mL, 3.43 mmol)
was added dropwise. After the solution was stirred for 5 min, Tf2O
was added very slowly, and the resulting mixture was stirred at the
same temperature until the reaction was complete (∼3−4 h). Water
(0.22 mL, 12.22 mmol) was added to the reaction mixture, and the
resulting solution was refluxed at 90 °C for 2 h. TLC showed that the
reaction was incomplete after 2 h. Additional amount of water (1 mL)
was added, and the reaction mixture was refluxed for an additional 70
h. Next, the organic layer was separated, and the aqueous layer was
extracted with additional CH2Cl2 (3 × 5 mL). The organic layers
were combined, dried over Na2SO4, and concentrated under vacuo on
a rotary evaporator. The crude product was purified by gradient silica
gel column chromatography using a mixture of hexanes and ethyl
acetate (10:1 to 1:1) to obtain the desired product 20 as a white solid
(100 mg, 51%).
13C NMR (125 MHz, CDCl3): δ 162.5, 162.4, 160.5, 160.4, 147.6,
137.8, 137.5, 129.9, 129.8, 129.7, 129.6, 129.2, 128.4, 115.5, 115.3,
115.2, 115.1, 112.7, 73.5, 67.2, 64.8, 56.2, 54.8, 50.9, 38.5, 33.1, 14.8.
[α]25 = (−) 62.4°, c = 1 in CH2Cl2. The product was converted
D
into the corresponding hydrochloride salt; mp = 203−210 °C. Anal.
Calcd for [C27H30F2N2O2·2HCl] C, H, N.
(2S,4R,5R)-5-(((1H-Indol-6-yl)methyl)amino)-2-benzhydryltetra-
hydro-2H-pyran-4-ol, 14 (D-544). Compound 13 (50 mg, 0.18
mmol) was reacted with indole-6-caboxaldehyde (26 mg, 0.18 mmol),
glacial acetic acid (12 μL, 0.21 mmol), and NaCNBH3 (17 mg, 0.27
mmol) in 1,2-dichloroethane (6 mL) using procedure A. The residue
was purified by column chromatography using 10% methanol in ethyl
acetate to afford compound D-544 (60 mg, 82%) as a thick syrup.
1H NMR (400 MHz, CDCl3): δ 7.24−7.33 (m, 2H), 7.11−7.20
(m, 2H), 6.90−7.02 (m, 4H), 4.38 (dt, J = 10.2, 2.0 Hz, 1H), 4.01
(dd, J = 12.5, 1.9 Hz, 1H), 3.94−4.01 (m, 1H), 3.84−3.94 (m, 2H),
3.28 (d, J = 1.7 Hz, 1H), 1.96 (br s, 1H), 1.70−1.82 (m, 1H), 1.38−
1.49 (m, 1H).
1
[α]25 = (−) 59.6° (c 0.5, MeOH). H NMR (400 MHz, CDCl3): δ
D
1.34−1.42 (m, 1H), 1.68−1.78 (m, 1H), 2.52 (s, 1H), 3.68−4.10 (m,
6H), 4.48 (t, J = 9.6 Hz, 1H), 6.46 (s, 1H), 6.92−7.58 (m, 13H), 8.71
(s, 1H). 13C NMR (100 MHz, CDCl3): δ 33.40, 51.45, 56.26, 56.74,
63.90, 66.34, 74.12, 102.43, 111.64, 120.92, 125.15, 126.57, 126.79,
127.71, 128.56, 128.59, 128.64, 128.90, 136.21, 142.14, 142.24. The
product was converted into the corresponding oxalate salt; mp =
152−154 °C. Anal. (C27H28N2O2·C2H2O4·H2O) C, H, N.
13C NMR (100 MHz, CDCl3): δ 162.8, 162.7, 160.3, 160.2, 137.4,
137.3, 137.2, 137.1, 129.9, 129.8, 129.7, 129.7, 115.6, 115.4, 115.3,
115.1, 73.3, 65.9, 64.6, 59.1, 55.0, 33.1.
[α]25 = (−) 116.2°, c = 1 in CH2Cl2.
(2S,4R,5R)-2-Benzhydryl-5-(4-nitro-benzylamino)-tetrahydro-
pyran-4-ol, 15. Amine 13 (60 mg, 0.21 mmol) was reacted with 4-
nitro benzaldehyde (32 mg, 0.21 mmol), glacial acetic acid (12 μL,
0.21 mmol), and NaCNBH3 (20 mg, 0.34 mmol) in 1,2-dichloro-
ethane (6 mL) using procedure A. The crude residue was purified by
column chromatography using 60% ethyl acetate in hexane to afford
compound nitrocompound 15 (79 mg, 89%). 1H NMR (CDCl3, 400
MHz): δ 1.42 (dt, J = 14.8 Hz, 3.2 Hz, 1H), 1.64−1.76 (m, 1H), 2.40
(d, J = 2.4 Hz, 1H), 3.72−4.04 (m, 6H), 4.50 (t, J = 11.6 Hz, 1H),
7.10−7.32 (m, 10H), 7.34 (d, J = 6.4 Hz, 2H), 8.14 (d, J = 8 Hz, 2H).
(2S,4R,5R)-5-(4-Amino-benzylamino)-2-benzhydryl-tetrahydro-
pyran-4-ol, 16 (D-505). Compound 3 (60 mg, 0.143 mmol) in
methanol (10 mL) was hydrogenated (50 psi) in the presence of 10%
Pd/C (6 mg, 10 wt %) for 0.5 h. The reaction mixture was filtered
through a short bed of Celite, and the solvent was removed under
reduced pressure. The product was purified by column chromatog-
raphy using 10% methanol in ethyl acetate to afford compound D-505
D
(2S,4S,5R)-5-Amino-2-(bis(4-fluorophenyl)methyl)tetrahydro-
2H-pyran-4-ol, 21. The azide 20 (100 mg, 0.29 mmol) was dissolved
in MeOH, and Pd/C was added (40 mg). The resulting mixture was
stirred under a steady flow of H2 (1 atm) for overnight. The solution
was then filtered through whatman filter paper (grade 8), and the
filtrate was concentrated under vacuo on a rotary evaporator to obtain
the desired amine 21 as a colorless syrup (90 mg, 98%).
1H NMR (500 MHz, CDCl3): δ 7.26 (dd, J = 7.9, 5.5 Hz, 2H),
7.15 (dd, J = 8.9, 5.5 Hz, 2H), 6.87−7.03 (m, 4H), 4.37 (dt, J = 10.2,
2.1 Hz, 1H), 3.98 (dd, J = 11.9, 1.8 Hz, 1H), 3.89 (d, J = 8.9 Hz, 1H),
3.82 (d, J = 3.1 Hz, 1H), 3.60 (d, J = 11.6 Hz, 1H), 2.65 (s, 1H), 2.20
(br s, 3H), 1.61−1.79 (m, 1H), 1.33−1.48 (m, 1H).
13C NMR (125 MHz, CDCl3): δ 162.5, 162.4, 160.5, 160.4, 137.7,
137.3, 129.9, 129.8, 129.7, 129.6, 115.5, 115.4, 115.3, 115.1, 73.7,
68.7, 68.1, 55.0, 51.0, 32.6.
[α]25 = (−) 64.0°, c = 1 in CH2Cl2.
D
1
(50 mg, 90%). [α] = (−) 30.9 (c = 1, CH3OH). H NMR (CDCl3,
(2S,4S,5R)-2-(Bis(4-fluorophenyl)methyl)-5-((4-methoxybenzyl)-
amino)tetrahydro-2H-pyran-4-ol, 22 (D-595). Amine 21 (60 mg,
0.19 mmol) was reacted with 4-methoxybenzaldehyde (32 mg, 0.23
mmol), glacial acetic acid (16 μL, 0.22 mmol), and Na(OAc)3BH (73
mg, 0.34 mmol) in a mixture of 1,2-dichloroethane (3 mL) and
methanol (1 mL). The residue was purified by gradient silica gel
column chromatography using a mixture of dichloromethane and
methanol (100:1 to 6:1) to afford corresponding compound D-595 as
a light-yellow solid (45 mg, 55%).
400 MHz): δ 1.42 (dt, J = 13.6 Hz, 2.4 Hz, 1H), 1.42−1.52 (m, 1H),
2.34 (d, J = 2.4 Hz, 1H), 3.45 (d, J = 12.4 Hz, 1H), 3.61 (d, J = 12.4
Hz, 1H), 3.64 (d, J = 11.2 Hz, 1H), 3.72−3.84 (m, 3H), 3.84−4.10
(m, 3H), 4.50 (t, J = 12.0 Hz, 1H), 6.54 (d, J = 8.4 Hz, 2H), 6.94 (d, J
= 8.0 Hz, 2H), 6.98−7.06 (m, 2H), 7.08−7.18 (m, 6H), 7.22 (d, J =
7.2 Hz, 2H). 13C NMR (CDCl3, 100 MHz): δ 33.1, 50.7, 56.1, 56.7,
64.2, 66.1, 73.1, 115.7, 126.4, 126.6, 128.43, 128.46, 128.5, 128.6,
129.1, 129.6, 142.2, 145.7. The product was converted into the
corresponding mesylate salt; mp = 123−128 °C. Anal. (C25H28N2O2,
2CH3SO3H, 0.8CH2Cl2) C, H, N
1H NMR (400 MHz, CDCl3): δ 7.26 (dd, J = 9.1, 5.4 Hz, 2H),
7.21 (d, J = 8.6 Hz, 2H), 7.14 (dd, J = 8.6, 5.4 Hz, 2H), 6.89−7.01
(m, 4H), 6.85 (d, J = 9.0 Hz, 2H), 4.39 (dt, J = 10.2, 2.4 Hz, 1H),
3.97−4.04 (m, 1H), 3.86−3.94 (m, 2H), 3.72−3.86 (m, 5H), 3.66 (d,
J = 13.0 Hz, 1H), 2.78 (br s, 2H), 2.48 (d, J = 2.7 Hz, 1H), 1.61−1.75
(m, 1 H), 1.34−1.46 (m, 1H).
((3S,6S)-N-((1H-Indol-6-yl)methyl)-6-benzhydryltetrahydro-2H-
pyran-3-amine, 18 (D-543). Compound 1 (50 mg, 0.19 mmol) was
reacted with indole-6-caboxaldehyde (27 mg, 0.19 mmol), glacial
acetic acid (13 μL, 0.22 mmol), and NaCNBH3 (18 mg, 0.28 mmol)
1416
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Research Article
13C NMR (100 MHz, CDCl3): δ 162.7, 162.6, 160.3, 160.2, 158.8,
137.7, 137.4, 131.1, 129.9, 129.8, 129.7, 129.6, 129.4, 115.6, 115.4,
115.3, 115.1, 113.9, 73.6, 66.8, 64.4, 56.2, 55.2, 54.8, 50.4, 33.1.
Author Contributions
S.S. and S.K. are first authors. A.K.D. was involved with all
aspects of the studies including design of compounds and
biological (in vitro) experiments reported in the manuscript.
S.S., S.V., and S.G. were involved with synthesis of the
compounds. M.E.A.R. and T.A. screened the compounds for
the monoamine transporter. S.K. carried out molecular
modeling. A.K.D., S.K., and M.E.A.R. participated in writing
the manuscript.
[α]25 = (−) 76.0°, c = 1 in CH2Cl2. The product was converted
D
into the corresponding hydrochloride salt; mp = 195−200 °C. Anal.
Calcd for [C26H27F2N2O3·HCl·H2O] C, H, N.
Functional Transporter Assays. Inhibition by test compounds
of substrate uptake by monoamine transporters was monitored in
synaptosome-enriched fractions from rat brain as described by us
previously.21,26,41 [3H]DA ([ring 2,5,6-3H]dopamine (45.0 Ci/mmol,
PerkinElmer, Boston, MA, USA) was used for monitoring DAT (rat
striatum) and NET (rat cerebral cortex). Please note that DA is an
excellent substrate for NET21, and control experiments with a number
of test compounds did not detect significant differences between
inhibitory values obtained with [3H]dopamine and [3H]-
norepinephrine.26 [3H]5-HT ([1,2-3H]serotonin (27.9 Ci/mmol,
PerkinElmer) was the radioligand for monitoring SERT (rat cerebral
cortex). Construction of inhibition curves with multiple drug
concentrations and conversion to equilibrium dissociation constants
(Kd) was achieved as we described previously.29 For pure inhibitors in
uptake assays, the Ki value measured functionally equals the binding
Kd;42 the present work reports Ki values (Table 1).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work is supported by National Institute of Mental
Health/The National Institute of Health MH084888 (A.K.D.).
This work was partially supported by NIH grant DA019676
(M.E.A.R.) for development/maintenance of transporter
assays.
REFERENCES
■
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ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acschemneuro.1c00078.
Molecular docking of additional molecules at binding
pockets of monoamine transporters and elemental
analysis for lead molecules (PDF)
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AUTHOR INFORMATION
■
Corresponding Author
Aloke K. Dutta − Department of Pharmaceutical Sciences,
Wayne State University, Detroit, Michigan 48202, United
States; orcid.org/0000-0003-3347-0538; Phone: 1-313-
577-1064; Email: adutta@wayne.edu; Fax: 1-313-577-
2033
Authors
Soumava Santra − Department of Pharmaceutical Sciences,
Wayne State University, Detroit, Michigan 48202, United
States; orcid.org/0000-0003-4304-4030
Sandhya Kortagere − Department of Microbiology and
Immunology, Drexel University College of Medicine,
Philadelphia, Pennsylvania 19129, United States;
orcid.org/0000-0002-3532-8669
Seenuvasan Vedachalam − Department of Pharmaceutical
Sciences, Wayne State University, Detroit, Michigan 48202,
United States; orcid.org/0000-0003-2444-1903
Sanjib Gogoi − Department of Pharmaceutical Sciences,
Wayne State University, Detroit, Michigan 48202, United
States; orcid.org/0000-0002-2821-3628
Tamara Antonio − Department of Psychiatry, New York
University School of Medicine, New York 10016, United
States
Maarten E.A. Reith − Department of Psychiatry, New York
University School of Medicine, New York 10016, United
States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acschemneuro.1c00078
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